WO2005063962A1 - System for comminuting, extracting and detecting analytes in sold biological samples - Google Patents

Abstract

The invention relates to reagents, methods, devices and systems for comminuting, extracting and detecting drug, pesticide, herbicide and steroid analytes in solid biological samples originating e.g. from man, domestic animals, plants, amphibians, insects and reptiles.

Description

SYSTEM FOR COMMINUTING, EXTRACTING AND DETECTING ANALYTES IN SOLID BIOLOGICAL SAMPLES

Field of the Invention The invention relates to rapid analyte detection systems and devices for use in the field, on-site at a manufacturing or agricultural processing facility, or in a test laboratory; for identifying compounds of interest in solid biological samples; and, for processing a solid biological sample for detection by comminuting, extracting and detecting. The processes are applicable to a wide range of solid biological samples including e.g. human and animal hair, feathers, nails and hoofs, hide, muscle, insects and plants, as well as, dried fluid samples.

This application is a Continuation-In-Part of Provisional U.S. Patent Application Serial No. 60/532,765, filed December 24, 2003. BACKGROUND OF THE INVENTION

Solid biological samples containing analytes of interest pose significant difficulties in analysis, particularly if the solids are not amenable to dissolution in aqueous or organic solvents. In the case of samples which cannot be made to dissolve, impenetrability to reagent solutions often makes analyte detection highly problematic. In forensic sciences often the best preserved and least contaminated biological samples are solid samples, e.g., dried fluids, eyelashes, nail clippings, hair, insects that have ingested biological samples, beaks, claws, feathers, scales, skin, muscle and plant material. There are significant problems encountered in the art when working with such solid samples and significant need for improvements. Testing hair samples for drugs of abuse has provided prototypic examples of the multiple problems that may be encountered working with solid biological samples.

Efficient rapid extraction of analytes from human hair could allow routine testing for both the presence and the duration of drug abuse, e.g. in workers in the transportation industry or in athletes using drugs or steroids. Similarly, extraction of analytes from animal hair could allow routine testing of foods for the presence and duration of exposure to pesticides, steroids, nicotine, cotinine, ETG (ethylglucuronide), herbicides, lead, mercury, carcinogens and the like. However, present methods are commonly neither rapid nor routine. Baumgartner and co-workers (1) reported in 1979 the use of laborious overnight chemical and enzymatic methods to isolate drug analytes from human hair samples for subsequent radioimmunoassay. These methods are still, by in large, in use today in present day laboratory practice, e.g., extraction of drugs involving prolonged incubation in combinations of acids and/or bases, enzymes (e.g. U.S. Patent Serial Nos. 6,582,924; 6,350,582; 6,022,693; 5,466,579; 5,324,642) or organic solvents. Present day hair testing methods are labor intensive and commonly require technical expertise and training that is only available in a test laboratory. Due to confounding variables, extraction of drugs from different hair samples often suffers from poor reproducibility, i.e., both with respect to the ability to uniformly isolate analytes from duplicate samples (intra-assay variability); and, with respect to inter-assay variability in detection and quantification of drugs and metabolites. Harsh treatment conditions can also result in conversion of drug metabolites such as 6-monoacetylmorphine, i.e., a judicial marker confirming drug use, into the parent compounds such as morphine which could constitute environmental contaminants in hair. Increased drug abuse in North America has been associated with criminal activities, health problems, newborn addiction, lost worker productivity and staggeringly high medical costs. Currently of greatest concern are opiates (heroin, morphine, codeine), cocaine, marijuana, MDMA (Ecstasy), phencyclidine, amphetamine and methamphetamine. In a legal setting, the probative advantages of hair testing may often depend critically on the methods used to assess how a drug analyte became associated with a hair sample, i.e., environmental contamination or metabolic incorporation. Current methods attempt to discriminate between the latter two possibilities by: (i) extensive tedious washing procedures designed to theoretically remove drug that is "passively associated" with hair; and/or (ii) use of kinetic measurements to assess how rapidly drug is released from hair samples under different conditions, i.e., assuming that environmentally associated drugs will be released more rapidly. Commercial test laboratories engaged in hair drug testing commonly use different protocols for rinsing and standards do not presently exist for assessing the effectiveness of different decontamination procedures. The unfortunate repercussion has been that doubts have been raised in Court and in the scientific literature about the absolute ability of hair testing to discriminate between environmental contamination and metabolic incorporation. While discussions have considered the possible impact on isolation of differences in hair porosity, type of hair (kinky vs. straight), amount of environmental drug exposure, effects of hair conditioners, shampoos, dyes, chemical treatments and the like, methodological solutions have often been elusive. In one attempted solution, kinetic and mathematical assessments of the concentrations of analytes in wash fluids; the assessment of a "curvature ratio" and the calculation of an "extended wash ratio"; in combination with a hair porosity test using methylene blue (e.g., DuPont and Baumgartner, 6), have all been brought to bear in an attempt to solve the problem. Unfortunately, since the chemical basis of drug association with hair is (at present) mostly unknown, optimizing the conditions for routine, uniform and standardized commercial use has mostly been on a trial-and-error basis. Improvements in methodology would potentially bring great benefit to humanity. Attempting to understand how drugs might associate with hair, Kidwell and Blank (2) suggested predominant interactions of drug cationic residues with protein anionic side chain residues in acidic amino acids (e.g. Asp and Glu). Cone and Joseph (3) suggested a possible association of certain drugs with melanin hair pigment. In 1994, Morrison et al. (4) studied possible mechanisms for association of cocaine and benzoylecgonine (BZE) with hair suggesting to the authors in 1998 (5) a possible cationic interaction of these drugs with hair. Laboratory-based methods require shipment of samples to a test laboratory. Rapid on-site screening assays at manufacturing and processing plants could significantly decrease the number of samples that need to be submitted to test laboratories, i.e., offering considerable cost- and time-saving advantages. As hair testing has shown, there are needs for rapid methods, reagents, kits and mechanical devices that improve the detection of analytes in solid biological specimens. There is also need for methods and devices that are more easily adapted to use in the field or in an on-site testing environment at a manufacturing or processing facility. There are also needs for methods that would increase the speed of sample preparation, extraction and detection, as well as, those that would decrease intra- and inter-assay variability and improve reproducibility. Solving these problems in the art could open up opportunities for testing a wide range of solid biological samples, e.g. in forensics, veterinary and human medicine, and agricultural processing. In addition, there are significant problems often encountered in field collection and preservation of agricultural and patients samples, e.g., samples collected in Africa or third world countries. In many cases these latter problems might be alleviated by purposefully drying the test sample if methods were available for routine processing of the resultant solid samples. Objects of the invention provide methods for rapid uniform preparation and testing of solid biological samples for the presence or amount of analytes.

SUMMARY OF THE INVENTION The invention relates to methods, reagents, kits, devices and automated systems useful for processing, extraction and rapid detection of analytes in solid biological samples. In different objects, reagents, methods and kits are provided that are useful in either a portable on-site analyte detection device, or alternatively, in a laboratory-based detection device. In other objects, the methods steps relate to washing, comminuting, extracting and detecting the presence or amount of one or more analytes in solid biological samples. The instant methods and kits have the advantage of enabling rapid processing, extraction and detection of analytes, such as drugs of abuse, in a manner that also achieves uniformity and reproducibility. In presently preferred objects, the reagents, methods and kits relate to comminuting devices that enable rapid washing, comminuting and extracting of drugs of abuse from uniformly prepared fine powdered human hair samples. In another object, comminuting of human hair to particles having a size of less than about 250 microns (250 μm) is accomplished in as little as about 10 seconds to about 2 minutes, and the particles so obtained enable extraction of drugs of abuse in about 10 minutes to about 30 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the present invention will now be described in detail with reference to the accompanying figure in which: FIGURE 1 is a schematic representation of a device of the present invention for comminuting a solid biological sample, wherein the following numbers relate to subcomponents of the device as follows: namely, 1. Single-use sample collection device; 2. single-use comminuting device; 3. single-use extraction vessel; 4. filtered outlet of extraction vessel; 5. multi-port selector valve; 6. heating block; 7. sonicating probe; and, 8. device enclosure. DETAILED DESCRIPTION OF THE INVENTION

The invention provide methods, kits and instrumentation for solving these problems in the art. In one presently preferred embodiment, comminuting a solid biological sample, (e.g. human hair, nail clippings, eyelashes, animal fur, hoof clippings, feathers, insects, plants and the like), offers the user several unexpected theoretical advantages, i.e., as follows: namely, (a) analytes previously assumed to be electrostatically and/or covalently bound within the matrix of solid fibrous biological samples such as hair and nails are more rapidly extracted from comminuted biological samples than would be expected, i.e., making it likely that some chemical interactions are disrupted, hydrolyzed, reduced and/or chemically-, physically- or enzymatically-degraded as the samples are subjected to shear forces and local micro-environmental changes during the comminuting process; (b) analytes extractable by incubation in an alkaline or methanolic-HCl solutions, are more rapidly and more uniformly extracted from comminuted fibrous biological samples, i.e., making it likely that the comminuted particles have undergone a change in inter- or intra-fiber cross-linking interactions; (c) enzymatic digestion of eratinized hair and nail samples occurs much more rapidly with finely comminuted sample than expected from data generated with either coarsely comminuted or cut samples, i.e., making it likely that fiber structure within the comminuted fibrous sample is altered to a more loose-fibrillar-form that allows better access to bulky enzyme proteins; and, (d) extraction of relatively intractable firmly-bound analytes, such as metabolites of drugs of abuse, is accomplished in one-tenth the time from a finely comminuted human samples of hair than from a coarsely comminuted or cut haif samples. In other embodiments, the invention provides portable and laboratory sample processing, analyte extraction and detection systems and devices. Analytes may be extracted from a human or animal sample, including, but not limited to, hair, feather, nail, hoof, fur, skin, muscle and the like. These and other aspects of the invention are detailed below and illustrated in the accompanying EXAMPLES section which follows.

Abbreviations used herein include the following, namely, amphetamine (AM), methamphetamine (MA), cocaine (COC), benzoylecgonine (BZE), cocaethylene (COCA), gas chromatography (GC), mass spectrometry (MS), gas chromatography in coupled detection with mass spectrometry (GC/MS), methylenedioxyamphetamine (MDA), 6-monoacetylmorphine (MAM), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA), nicotine (NIC), heroine (HER), morphine (MOR), codeine (COD), Δ-9-tetrahydrocannabinol (Δ-THC), Δ-9- tetrahydrocannabinol-9-carboxylic acid (Δ-THC-acid), di-hydroxy- tetrahydrocannabinol (DH-THC), phencyclidine (PCP), pesticide (as defined below; P), herbicide (as defined below, H); TEA, triethylamine, DEA, diethylamine, MeOH, methanol, DCM, dichloromethane, DCE, dichloroethane, BA, n-butylamine.

Terms used herein are intended to have meaning as follows: namely,

"Analyte" is used herein to refer to a compound present in a biological sample whose determination is of interest to a user of the instant methods. Representative examples of analytes include intact and degraded naturally occurring and non-natural chemical analytes, drug analytes, pesticides, herbicides, nicotine, cotinine, ETG (ethylglucuronide), herbicides, lead, mercury, steroids, their metabolites, glucuronides, oxidation products, modified adducts and the like;

"Assay Formats", when used in the context of detecting an analyte according to an embodiment of the invention, is intended to mean the process of steps by which an analyte in a solid biological test sample is comminuted, extracted and detected. Representative assay formats include chromatographic, e.g., gas chromatography (GC), mass spectrometric (MS), high performance liquid chromatography (HPLC), immunoassays and combinations thereof, e.g., Tandem GC/MS.

"Background", when used in the context of an assay according to an embodiment of the invention, is intended to mean the uncertainty occasioned by substances which may interfere with the proper performance of the assay when they are present in the assay. Representative examples of substances which may so interfere (i.e., interfering substances, confounding substances, and the like) in an assay include materials present in biological samples such as oligosaccharide cell wall components of bacteria, fungi, plants, insects and aquatic animals and plants; enzyme inhibitors; free radical reactive compounds; endogenous peroxides; spontaneously fluorescent and spectroscopically active compounds; and the like. Background may result in an increase in the percentage of false-positive or false-negative test results or in a decreased degree of confidence in a test result;

"Biological sample" is intended to mean a solid sample obtained from a living or dead organism, e.g., a mammal, fish, bird, reptile, amphibian, marsupial, insect, plant and the like, as well as, materials contaminated with samples from living or dead organisms such as clothing, soil and contaminants in air and water. Biological samples include e.g. tissue products such as hair, nail clippings, hoof clippings, dry tears, dry saliva and dry mucous; as well as, dry biological fluids, tissue sections, solid biological materials carried on clothing or in the air or in water (e.g., animal dander) and collectable there from e.g., by filtration, centrifugation, and the like. Representative solid biological fluids, e.g., useful in forensic analysis, include , e.g. dry urine, dry blood, dry plasma, dry serum, dry cerebrospinal fluid, dry semen, dry lung lavage fluid, dry feces, dry sputum, dry mucus, filtered air and water carrying biological materials and the like. Representative biological samples also include processed and un-processed foodstuffs, e.g., meats, fishes, dairy products, cereal grains and the like. Biological samples also include test samples from agricultural processing facilities, e.g., restaurants, slaughter-houses, cold storage facilities, supermarket packaging and the like. Biological samples may also include dried tissues and bodily fluids (i.e., samples purposefully dried for testing). Representative uses of the instant methods include e.g. detecting the presence or severity of drug abuse in test subjects; the presence or extent of antibiotic or pesticide contamination in animal carcasses and packing plants; as well as, the presence or severity of contamination of cereal grain products. Thus, embodiments of the invention provide methods useful in testing a variety of different types of solid biological samples for the simultaneous presence or amount of one or more analytes. In one present preferred embodiment, the sample is selected from human and animal hair, nails and skin; and animal hoofs, feathers and hide. In one presently most preferred embodiment, samples are human and animal hair;

"Capture reagent", when used in reference to an immunoassay, is intended to mean an LBP bound to, or bindable to, a solid phase. In the latter case, the subject capture reagent may consist of a binding solution which when added to a solid phase in the presence of the LBP will result in LBP binding to the solid phase; or alternatively, an LBP that has been modified so as to promote its binding to a solid phase. LBP may be immobilized on a solid phase e.g. by attachment through electrostatic forces, van Der Waals forces, hydrophobic forces, covalent chemical bonds, and the like;

"Cascade assay formats" is intended to mean detecting a drug analyte compound through a process: wherein, a first signal generating compound (i.e., SGC #1) produces a product that can be utilized by a second SGC #2 to produce a product which e.g. can be utilized by a third SGC #3. The subject cascade of products from SGC #1-3 results in amplification which results in a greater overall signal that could be achieved by any single SGC;

"Chemical analytes" is intended to mean compounds which are not commonly useful as drug substances, but which may instead constitute health risks for man, domestic animals and the environment. The subject chemical analyte may be associated with hair either metabolically, e.g., as a result of ingestion or inhalation, or passively, e.g., as a result of environmental contamination of the hair. Representative examples of the subject chemical analytes include the following: namely, pesticides; herbicides; pollutants; carcinogens; toxic chemicals; environmental microbial toxins such as present in contaminated grains; industrial organic chemicals and plastic polymers comprising risks to human health; organic compounds in animal feeds such as may indicate potential consumption of animal body parts including brain; and the like; "Combined simultaneous assay format" is intended to mean simultaneous detection of more than one drug analyte in a single assay format, e.g., simultaneous detection of two drug analytes by detecting the binding of two different SGC compounds. In one representative example, the two different SGC compounds used for simultaneous detection preferably have different fluorescence excitation- and emission-profiles. Preferably, the subject excitation- and emission-profiles are only partially overlapping and most preferably the subject profiles are non-overlapping;

"Comminute" and "Comminuting", as used herein refers to the instant process by which a solid biological sample is reduced to small fine cutting of particles, preferably having the consistency of a powder or a dust, i.e., referred to as a "Comminuted" sample. Most preferably, the latter particles are tubular, round or elliptical and have an average diameter, (i.e., as expressed mathematically diameter = (length/width)/2), that is in the range of about 100 μm to about 2000 μm, preferably about 80 μm to about 500 μm, yet more preferably 80 μm to about 400 μm and most preferably about 80 μm to about 200 μm. The process of comminuting a sample may include subcomponent steps such as macerating, drying, lyophilizing, physically shearing, cutting and mincing, as well as, steps designed to soften and breakdown the component tissue constituents in a biological sample. Smaller particle sizes than 50 μm pose potential problems in (a) sample handling; (b) removal of solid residue after extraction, e.g. through filters or screens; and, (c) opportunities for air-borne cross-contamination of samples, as well as, on-site facilities and laboratory workspace. Presently preferred processes for comminuting a biological sample are disclosed further below. Other representative mechanical systems for comminuting a biological sample to achieve a desired particle size and uniform consistency include pulverizing methods utilizing blades, augers, gears, as well as, other non-mechanical systems such as electronic discharge and laser flash disintegration. Representative examples of subcomponent macerating steps include: e.g., addition of an acidic or a basic solution (e.g. pH 2-3 or pH 10-12, respectively); addition of an enzyme (e.g. mucosidase, protease, glycosidase); addition of an organic solvent; freezing (e.g. -20°C to -70°C) and thawing (e.g.20°C to 37°C); addition of detergent (e.g. TWEEN, CHAPS); application of heat (e.g. 65°C/30 minutes); physical homogenizing e.g. Dounce; crushing a biological sample frozen in dry-ice acetone (-20°C to -40°C) or liquid nitrogen (-70°C to -120°C) with a mortar and pestle; and/or, sonication (e.g. probe or water bath). Preferably, in the case of addition of acidic or basic solutions incubation times are about 2 minutes to about 30 minutes at temperatures that are in the range of 20°C to 65°C, preferably 25°C to 45°C, and the solution that is added is about 2 M to about 5 M of a strong base solution, e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide, barium hydroxide, calcium hydroxide, potassium carbonate, sodium carbonate, potassium acetate and sodium barbital and like. Preferably, the pH of the chemical base solution is greater than about pH 9 and most preferably greater than about pH 10;

"Confirming assay", used interchangeably herein with "Confirmation Assay", means an Assay Format that is performed on a comminuted biological test sample to determine the nature and amount of an analyte therein. Commonly, a Confirming Assay is capable of identifying the molecular species of a drug or its metabolite by virtue of recognizing both a chromatographic elution behavior and the types of molecular ions derived by fragmenting the analyte. Representative confirming assays include GC/MS, Tandem GC/MS, ion trap GC/MS methodologies, Tandem GC/MS, Tandem LC/MS and the like. Embodiments of the invention provide methods, reagents, kits and devices for conducting Screening Assays to decrease the number of Confirming Assay that must be performed. Commonly, in a probative setting, a positive result is recorded in a Screening Assay is confirmed in a Confirming Assay;

"Detect reagent" is intended to mean a commercial reagent suitable for use in a test assay for identifying an analyte in a biological sample. For example, in an immunoassay which is a "Screening Assay", (i.e., defined below), the subject LBP-Conjugate may be provided in a reagent suitable for binding an analyte under conditions maximizing the specific binding interactions between the LBP and the analyte while also minimizing cross-reactivity with any unrelated plant, mammalian, insect or avian material that may be present in the biological sample. The subject Detect Reagent may be packaged in one container, e.g., just an LBP-Conjugate, or in several separate containers, e.g. commonly containing: (i) an LBP-Conjugate; (ii) one or more buffers, additives, excipients and the like for stabilizing and preserving the subject LBP-Conjugate during storage; (iii) an assay buffer containing one or more substances for promoting the binding activity of the subject LBP-Conjugate to an analyte in a test assay; (iv) a signal generating reagent e.g. a substrate for an enzyme; and, (v) one or more reaction vessels in which to conduct an assay. In an illustrative example of a GC/MS "Confirmation Assay", (defined below), the subject commercial Detect Reagent package may commonly contain, e.g. (i) a column for cleaning the sample prior to GC, (illustrated further in the EXAMPLES section, which follows); (ii) one or more derivatizing agents for modifying the analyte to insure GC mobility and MS detection; (iii) one or more organic solvents useful in derivatizing or GC/MS; and, (iv) one or more containers for collecting or derivatizing the analyte in a test sample;

"Detect", when used herein to refer to the process of identifying an analyte in a comminuted or extracted biological sample, means that the analyte is so identified in an assay process that is analyte-specific, i.e., as set forth further below in regard "Specificity", "Sensitivity" and "Background". Representative non-limiting examples of detection devices and assay include those based on changes recorded in optical systems such as fluorescence, chemiluminescence, absorbance, Raman or reflection, light scattering, refractive index, radiation, turbidity, color, size spotting or area spotting; changes recorded in electrochemical systems such as amperometric, ptoentiometric, conductometric methods; changes recorded in coupled mechano- chemical systems; changes recorded in thermal systems such as measuring conductivity and flame ionization; and, changes in chemo-magnetic systems, i.e., as set forth further below;

"Diagnostic reagent" is intended to mean a reagent suitable for use in a test assay for identifying an analyte in a biological sample derived from man and domestic and zoo animals. Many of the subject reagents find uses in reaching informed medical decisions about human therapy and are regulated as medical devices by the United States Food and Drug Administration (FDA); or alternatively, the test assays may find uses in veterinary medicine and be regulated by the United States Department of Agriculture. The instant diagnostic reagent commonly contains a "Detect Reagent";

"Drug Analyte" is used herein to refer to a chemical compound present in a biological sample whose determination is of interest to a user of the instant methods, wherein the instant chemical compound, or one or more of its metabolites, produces or alleviates at least one biological response in a mammal. Representative biological responses include those manifest in neurological symptoms such as anxiety, pain, stress, euphoria, well-being; performance enhancement; as well as, biological responses to foreign invaders such as microbes. Representative examples of chemical compounds so capable include pharmaceutical drug compounds, e.g., as set forth in the US Pharmacopia and Physicians Desk Reference, but also other compounds such as those forth in the Merck Index. Representative drug analytes include therapeutic pharmacological drugs used in man and domestic animals, e.g. antibiotics used in veterinary medicine and aqua culture; drugs of abuse such as morphine, opium, cocaine, codeine, amphetamine, methamphetamine, MDMA, THC, β-agonists; steroids abused by athletes; as well as, the metabolites of these drugs. The subject "drug analytes" are distinguished in definition, herein, from potentially toxic chemical compounds, carcinogens and the like which are referred to as "chemical analytes";

"Drug test assay" is intended to mean an assay format performed on a test sample using a US FDA regulated, or unregulated, device for the purpose of detecting the possible presence or amount of a drug analyte in the test sample. The subject test procedure may e.g., be performed either (a) "On-Site", i.e., at the employer facility, a collection facility, doctor's office, a manufacturing or processing facility; or alternatively, (b) in a test laboratory. Representative examples of the subject drug test assay include a variety of enzyme-linked immunoassay "Screening Assay", (defined below), devices and "Confirmation Assays", (defined below). Data relating to the technical performance of a laboratory test, Detect Reagent, Diagnostic Reagent, manufactured diagnostic test device, or other assay methodology, may be collected, stored and used to make calculations such as those known to those skilled in the diagnostic arts, e.g., accuracy, precision, specificity, sensitivity, "false positive" and "false negative" rates, and the like;

"Endogenous drug analyte" is used herein interchangeably with "metabolϊcally incorporated" to mean that the subject drug analyte compound is incorporated within a synthesized cellular product of an organism as a result of purposeful exposure e.g. therapeutic use or drug abuse. Representative cellular products so associated with drug analyte include hair keratin matrices, the exfoliative skin keratinous matrices, finger and toe nails, mucus secretions, sputum, biological fluids and the like. Representative routes by which the "purposeful exposure" may occur include administration via oral, trans-mucosal-, trans-dermal-, intra-ocular-, intra-nasal-, intravenous-, subcutaneous-, intradermal-, intramuscular- and intrathecal-routes;

"Exogenous drug analyte" is used interchangeably with "environmental contamination" intended to mean drag analyte that is not the result of a "purposeful exposure", e.g., passive association as a result of an accidental environmental exposure;

"Extraction" is intended to mean the process of drawing out, isolating, separating and/or collecting, (e.g. onto a solid phase or into a fluid filtrate, a gas or a vapor), one or more endogenous analytes from a comminuted biological sample. Representative examples of extraction solutions are provided below and in the EXAMPLES section, below, including e.g. conditions suitable for extraction using organic solvents, acidic solutions, alkaline solutions, enzyme solutions, detergent solutions and supercritical fluid solutions;

"Extraction vessel" is intended to mean a fluid reservoir capable of retaining both a test sample and an extraction fluid, i.e., under optional temperature and pressure. Representative temperature and pressure controlled extraction vessels are known in the art, e.g. the "Guard Cartridge" device manufactured by UpChurch Scientific or the Hitachi-cup manufactured by Hitachi. For extraction of the analyte into organic solutions or supercritical fluids, preferably the extraction device comprises both of an inlet port and an exit port, both of which ports may be optionally fitted with one or more static or dynamic pressure regulators, flow regulators and/or flow restrictors (e.g., pressure relief and flow controlling valves). Preferably, the extraction vessel is capable of retaining a relatively small volume of an extraction fluid, in this case about 1 mL to about 7 mL, most preferably about 1 mL to about 5 mL. Preferably, the instant extraction vessel also comprises a hair collection device as set forth supra.Most preferably, the instant collection device, comminuting device and extraction vessel comprise a single use disposable cartridge;

"Herbicide" is intended to mean a compound toxic for a plant. Representative herbicides include e.g. alachlor, bromacil, hexazinone, metolachlor, metribuzin and the like; "Hair collection device" is intended to mean a device for securely collecting and retaining within a chamber a sample of hair from a test subject. Manual devices may be used e.g. including scissors, latex gloves and a 12x75 mm tube for retaining the hair. Automated devices may be operated mechanically or electronically to: (i) remove a hair test sample from a test subject; (ii) retain the hair test sample within a chamber; (iii) accomplish each of said removal and retention processes without an operator physically touching the hair test sample. Preferably, the subject hair collection device comprises a device such as that disclosed by certain of the inventors in U.S. Patent Serial No. 6,478,750. Most preferably, the subject device is ueful as both a hair collection device, a comminuting device andor an extraction vessel, as set forth further below;

"Immunoassay device" is intended to mean a machine for conducted an automated assay on a comminuted biological test sample by at least: (i) extracting an analyte from the comminuted test sample; and (ii) detecting the analyte using an immunoassay format, above;

"Immunoassay Format" is intended to mean an assay for detecting an analyte in a comminuted biological test sample, which uses for detection a LBP-Conjugate, i.e., a ligand binding partner conjugated with a signal generating compound as defined supra. Representative immunoassay formats include "simultaneous" ligand-binding assay methods, also referred to by those in the art as "homogeneous" assay formats, wherein the step of analyte binding to a ligand binding partner is effective, i.e., simultaneously, to accomplish signal generation. Representative assay formats also include those referred to in the art as "heterogeneous" ligand-binding assays, wherein one or more steps for separating a "bound"-analyte from a "free"-analyte is required prior to the generation of a signal. In other representative assay formats, "competition binding" methods have a step in which analyte is added to compete with the binding of a labeled ligand to an LBP, i.e., also known to those in the art as an "indirect" assay format. In other representative "non-competitive" or "direct" assay formats, binding of an analyte to a LBP is detected by adding a second LBP#2-Conjugate, e.g., an antibody directed to an antibody. Representative methods for separating "bound" analyte, ligand or LBP from "free" include filtration, column separation , magnetic separation, electrophoretic separation, as well as, attaching one or more of the reactants to a solid phase. Representative ligand-binding assay methods for detecting a signal generating compound commonly include: using an enzyme that converts a substrate to a visually or spectrophotometrically identifiable product, or alternatively, exciting a fluorescent SGC-compound that is coupled to an LBP so that a detectable signal is emitted, e.g. at a different wavelength such as in a fluorimetric analysis. In a first illustrative immunoassay format, a ligand -binding solid-phase assay format involves the following steps: namely, (a) "capturing" an analyte present in a comminuted biological sample, e.g. by binding to a LBP that is attached electrostatically (or covalently) to a solid phase; and then, (b) detecting the bound analyte by reacting it with an LBP-Conjugate; and,

(c) introducing an excitation wavelength, adding a substrate or otherwise modifying conditions so that a detectable signal is generated by the SGC;

"Kit", when used in the context of assay components that are packaged together for use according in one or more embodiments of the invention, is intended to mean a container containing 2 or more assay components selected from among the following: namely, (i) instructions for collecting and comminuting a solid biological sample according to the methods of the instant invention; (ii) a single use sample collection device (set forth below); (iii) a single-use comminuting device (set forth below); (iv) one or more extraction vessels (set forth below); (v) a container having an extraction fluid; (vi) a container having one or more LBP-Conjugates; (vii) a container having a substrate for an enzyme SGC; (viii) containers having a Capture Reagent, a Detect Reagent or a Diagnostic Reagent; (ix) a container having a Solid Phase; (x) containers having one or more positive or negative controls; (xi) containers having or one or more assay calibrators e.g. useful for determining specificity, sensitivity or precision of the assay; (xii) a machine packet; (xiii) a container having a reagent useful in gas chromatography or mass spectrometry; instructions for conducting a Confirming Assay; and, (xiv) instructions for the user to determine that the testing procedure has been performed properly. Preferably, the instant kit contains 3 or more assay components and most preferably the instant kit contains 4 or more assay components;

"Ligand Binding Partner", abbreviated LBP, as used herein refers to a compound capable of binding to an analyte. Representative examples of LBP include antibodies, lectins, receptors, inactive enzymes and the like, as well as, enzymatic and non-enzymatic degradation fragments thereof and portions such as may be expressed as recombinant proteins and peptides. The subject analyte is capable of filling a three-dimensional space in binding site of a LBP so that electrostatic repulsive forces are minimized, electrostatic attractive forces are maximized, and hydrophobic and hydrogen bonding forces are maximized. Analytes bind to LBP in a specific and saturable manner, and binding affinities may be measured according to ligand binding assays known to those skilled in the art;

"Machine packet" is used interchangeably with "equipment packet" to mean a container restraining one or more solid or liquid reagents and allowing those reagents to be released by the machine into in a Screening Assay or Confirmation Assay, i.e., the machine packet engaged in a test device has conductive channels for establishing communication with fluid processing systems in the test device. Representative examples of machine packets include the following: namely, (i) in-line cartridges, columns and the like, wherein fluid communication is e.g. through the two opposing ends or sides of the cylindrical or disc-shaped cartridge or column; (ii) cartridges insertable within an extraction chamber (defined below), wherein fluid communication is e.g. through the walls and/or ends of the cartridge; (iii) tea-bag-like porous packages, wherein the subject fluid communication is through the porous walls of the packages; (iv) semi-permeable tea-bags, wherein exposure to the supercritical fluid changes the porosity of the walls of the package; (v) capsules, wherein the contact with the subject fluid communication solubilizes and/or dissolves the wall of the capsule to release the subject reagents; (vi) tablets or pellets, wherein contact with the subject fluid dissolves the tablet or pellet; and the like;

"Matrix-associated", when used in regard to an analyte that is bound in the structure of multi-chain polypeptide having a fibrous structure as a result of inter-chain cross- linking bonds, is used interchangeably with "metabolically incorporated", "physiologically incorporated" and "endogenous drug analyte" to mean that the subject analyte is located within the fiber structure. Representative of fibrous multi-chain polypeptide structures include chitin matrices in shell fish and insects; proteoglycan matrices in plants; actin filament networks in muscle meats; and, keratin matrices in hair, fur, nails, hoofs, exfoliated skin and the like; "Mixed assay format" is intended to mean that in a first step of the assay the molecule capable of capturing a analyte compound is different than the molecule capable of detecting the captured analyte in a second step, e.g., non-antibody LBP#1 in the first step, (e.g., a receptor fragment) and an anti-LBP#l antibody (e.g., anti-receptor antibody) in the second step;

"On-Site", when used herein with regard to the location in which an assay is performed, is intended to mean a location that is not a test laboratory and preferably the assay performed at this location is a screening assay, as defined supra. Representative examples of on-site locations include employer sites, factories, manufacturing plants, food processing plants and slaughter houses, private schools, penal institutions, doctor's offices and the like;

"Pesticide" is intended to mean a chemical analyte toxic for an insect. Representative examples of the subject pesticides include the following: namely, organochlorine compounds (OCCs) including chlorophenols,, polychlorinated biphenyl compounds (PCB), sulfonylureas, and the like such as, DDT, atrazine, dieldrin, carbofuran, 2,4- (dichlorophenoxy)acetic acid (2,4-D), 2,4-(dichlorophenoxy)butanoic acid (2,4-DB), 2,4-dichlorophenol (2,4-DCP), 4-nonylphenol (4-NP), chlorophos, methamidophos, dichlrovos, methamidophos, mevinphos, acephate, tetrahydrophthalimide, pentachlorobenzyne, o-phenylphennol, omethoate, propoxur, diphenylamine, chloropropham, trifluralin, phorate, hexachlorobenzyne, dicloran, dimethoate, carbofuran, atrazine, quintozene, lindane, terbufos, diazinon, chlorothalonil, disulfoton, phosphamidon, vinclozolin, parathion-methyl, carbaryl, malathion, chloropyrifos, aldrin, dacthal, parathion, dicofol, captan, methidathion, disulfoton sulfone, endosulfan, fenamiphos, myclobutanil, endosulfan, ethion, propargite, iprodione, phosmet, methoxychlor, phosalone, azinphos-methyl, permethrin, cyfluthrin, cypermethrin, fenvalerate, antrhacene, chrysene and, where applicable, their glucosides and metabolites such as l,l-dichloro-2,2-di(4-chlorophenyl)ethylene (DDE), l,l,l-trichloro-2,2-bis(4-chloroρhenyl)ethane, heptachlor, chlorpyrifos, chlordane, endrin, dichloran, tecnazene, diazinon and the like;

"Processing the Extract", when used in the context of an extraction solution, is intended to mean that the subject extraction solution containing an analyte of interest is altered physically or chemically to allow the subsequent detection of the analyte. Representative examples of processing extracts are as follows: namely, (a) bringing an organic solvent extract solution to dryness, e.g., to allow resuspension of the analyte in an aqueous-based detection system or to change solvents; (b) bringing the pH of an acidic or alkaline solution to a neutral pH, e.g., to prevent destruction of the analyte or of a subsequent immunoassay reagent in a detection assay; (c) inhibiting an enzyme activity in an enzyme solution, e.g. by adjusting the pH or adding an enzyme inhibitor; (d) diluting a detergent solution, e.g., so that the detergent will not interfere with a detection assay; and, (e) bringing a supercritical fluid solution to room temperature and pressure in a manner effective to collect an analyte in the supercritical fluid extract to be adsorbed onto a solid phase;

"Screening assay" is used to mean an assay procedure that is performed on a biological specimen to determine the presence or amount of a Drug Analyte (supra). Representative screening assays include a variety of immunoassays as set forth supra. Commonly, screening assays are rapid an will not have the specificity, sensitivity, precision and probative value of a Confirming Assay;

"Sensitivity", when used in the context of an assay according to an embodiment of the invention, is intended to mean that the subject assay is capable of identifying at an "indicated" percentage those samples which contain an analyte from within a panel of samples containing both positive test samples and negative control test samples lacking the analyte. Preferably the subject "indicated" sensitivity is greater than 85% and most preferably greater than 90%;

"Signal generating compound", abbreviated SGC, is intended to mean a molecule that (a) can be linked to a Ligand Binding Partner, e.g. using a chemical linking method as disclosed further below, to form a "Ligand Binding Partner-Conjugate", i.e., abbreviated LBP-Conjugate; and further, (b) the SGC is capable, when in the LBP- Conjugate, of producing a chemical or physical reaction product which is detectable in an assay according to the instant disclosure. Representative examples of signals produced by SGC include reaction products formed as precipitates, fluorescent signals, compounds having color, compounds having metallic tags capable of generating electrochemical signals and the like, i.e., as set forth in "Detect", supra. Representative SGC include e.g., bioluminescent compounds such as luciferase; fluorophores; bioluminescent and chemiluminescent compounds; radioisotopes such as ^,251, ¹³¹1, ¹⁴C, ¹³C, ⁵¹Cr and ³H; enzymes; binding proteins such as biotin, avidin and streptavidin; magnetic particles; chemically reactive compounds such as colored stains; labeled-oligonucleotides; and, molecular probes such as commercially available from Research Organics, Inc and Molecular Probes, Inc. Representative fluorophores include fluorescein isothiocyanate, succinyl fluorescein, rhodamine B, lissamine, 9,10- diphenlyanthracene, perylene, rubrene, pyrene and fluorescent derivatives thereof such as isocyanate, isothiocyanate, acid chloride or sulfonyl chloride, umbelliferone, rare earth chelates of lanthanides such as Europium (Eu) and the like. Representative SGC useful in an LBP-conjugate include enzymes in: IUB Class 1, especially 1.1.1 and 1.6 (e.g., alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and the like); IUB Class 1.11.1 (e.g., catalase, peroxidase, amino acid oxidase, galactose oxidase, glucose oxidase, ascorbate oxidase, diaphorase, urease and the like); IUB Class 2, especially 2.7 and 2.7.1 (e.g., hexokinase and the like); IUB Class 3, especially 3.2.1 and 3.1.3 (e.g., alpha amylase, cellulase, β-galacturonidase, amyloglucosidase, β-glucuronidase, alkaline phosphatase, acid phosphatase and the like); IUB Class 4 (e.g., lyases); IUB Class 5 especially 5.3 and 5.4 (e.g., phosphoglucose isomerase, trios phosphatase isomerase, phosphoglucose mutase and the like.) The subject SGC enzymes may either be coupled directly to the Ligand Binding Partner, e.g. an antibody or catalytically inactive enzyme; or alternatively, may be coupled to a second binding partner, e.g. an antibody to an antibody, that is used in combination with the LBP, e.g., a different LBP (i.e., LBP #2; as disclosed further, below.) The subject SGC share the common property of allowing detection and/or quantification of the analyte in the biological sample. Preferably, the subject signal generating compounds are detectable using a visual method, a spectrophotometric method, an electrical method (e.g., a change in conductance, impedance, resistance and the like), a fluorescent detection method, a turbimetric method, a light scattering method; and/or a chromatographic or mass spectrometric method. Representative methods for covalently linking SGC to LBP include those using hetero-bifunctional cross-linking reagents that are reactive with carbonyl, aldehyde, carboxyl, amino, disulfide and thiol groups of amino acids, e.g., carbodiimide, N-hydroxy succinimide, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), maleimide, succinimidyl-pyridylthioproprionate, m-maleimidobenzoyl N-hydroxysuccinimide ester, succinimidyl pyridylthiopropionate and the like. Methods for linking SGC fluorophores to LBP include, e.g., encouraging electrostatic interactions of the subject LBP with the fluorophore by placing the LBP in a buffer having a pH below its isoelectric point (e.g., pH 2.5-3). Methods suitable for linking phycobiliproteins to LBP are also disclosed in Stryer et al. U.S. Patent Serial No. 5,055,556. The instant LBP-SGC conjugates (supra) are preferably prepared for commercial distribution as detect reagents, preferably by solubilizing them in one or more buffer solutions and then dispensing them into reagent bottles, packages and the like; or, alternatively, by lyophilizing them and dispensing them as powders into reagent containers and packets. The subject commercial LBP-SGC preparations may include additives such as buffers, stabilizers, emulsifiers, detergents and the like for the purpose of e.g. preserving the activity of the instant LBP-conjugate during storage; or, for promoting the binding activity of the instant LBP for an analyte in an immunoassay. Examples of agents that may be added to the commercial preparations also include those designed to promote binding interactions including additives that (a) decrease total fluid volume in an assay such as polyethylene glycol and sucrose; and, agents that promote interactions by providing an electrostatic surface in solution, e.g. dextran, polystyrene beads, polyacrylate beads, and the like;

"Solid phase", as used herein, is intended to mean a surface on which, or in which, an assay may be conducted to detect an analyte. For immunoassays, solid phase surfaces commonly comprise one or more reactants such as e.g. LBP, SGC, LBP-Conjugates and the like which may be passively dried onto the surface of the solid phase and/or attached electrostatically, hydrophobically, or covalently. For example, coating a polystyrene (e.g., 96-well Dynatech-Immulon II, Nunc, or similar plates) with a ligand binding polypeptide is accomplished at a concentration of about 1 mgml in a carbonate buffer at pH 8 for about 16 hours results in binding of about 2 to about 2000 ng/well to the solid phase. Representative solid phases for immunoassays include e.g.: nylon 6; nylon 66; polystyrene; latex beads; magnetic beads; glass beads; polyethylene; polypropylene; polybutylene; butadiene-styrene copolymers; silastic rubber; polyesters; polyamides; cellulose and derivatives; acrylates; methaciylates; polyvinyl; vinyl chloride; polyvinyl chloride; polyvinyl fluoride; copolymers of polystyrene; silica gel; silica wafers glass; agarose; dextrans; liposomes; insoluble protein metals; and, nitrocellulose. Representative solid phases for immunoassays include those formed as beads, tubes, strips, disks, filter papers, plates and the like. Solid phase filters may serve to a capture comminuted biological sample, e.g. as a filtrate, or by entrapment, or by covalently-binding LBP onto the filter. According to certain embodiments of the invention, a solid phase capture reagent for distribution to a user may consist of a solid phase (supra) coated with a "capture reagent" (below), and packaged (e.g., under a nitrogen atmosphere) to preserve and/or maximize binding of the capture reagent to analyte in a comminuted biological sample. For GC/MS assays, solid phases in which "trapping" or detection may be accomplished include commercial silica based columns, i.e., as set forth further in the EXAMPLES section which follows;

"Specificity", when used in the context of an assay according to an embodiment of the invention, is intended to mean that the subject assay, as performed according to the steps of the invention, is capable of properly identifying an "indicated" percentage of test samples containing analyte from within a panel of test samples (e.g., a panel of 100 samples) that contain and do not contain the analyte. The subject panel of samples all contain one or more analytes, e.g., positive test samples and negative control samples relating to testing for a drug of abuse. Preferably the subject "indicated" specificity is greater than 85%, e.g., the assay is capable of indicating that more than 85 of the 100 samples contain one or more analytes; and most preferably, the subject assay has an indicated specificity that is greater than 90%;

"Steroid" is intended to mean a compound having a tetracyclic (cyclopentanophenanthrene) nucleus. Representative examples of the subject steroids include aldosterone, androsterone, cholecalciferol, testosterone, nortestosterone, methyl-testosterone, cortisol, cortisone, ergocalciferol, estradiol, estrone, lanosterol, progesterone and their metabolites, glucuronides and the like; "Substantially purified" when used herein to refer to a preparation that contains an analyte extracted from a comminuted biological sample, means that the analyte is enriched greater than about 10-fold to about 25-fold, preferably greater than about 26-fold to about 50-fold and most preferably greater than about 100-fold from the levels present in the biological sample;

"Test sample" is intended to mean a portion of a biological sample, e.g. a blood sample collected from a test subject, a weighed amount of a cereal grain, a measured amount of soil, a weighed amount of hair and the like. While presently preferred embodiments are directed toward biological samples which are fibrous keratin matrices, in alternative embodiments, it is anticipated that the methods of the invention will find use with a variety of biological samples, as set forth above;

"Test subject" is used interchangeably with "subject" to mean humans; domestic and zoo animals; plant species; amphibians; and, aquatic organisms including e.g. marine plants, crustaceans, cartilagenous and non-cartilagenous fishes and mammals;

"Uniform assay format" is intended to mean that the molecule capable of capturing (e.g., LBP #1) an analyte, e.g., on a solid phase, and the molecule capable of detecting the captured analyte (i.e., LBP #2) are the same, e.g., LBP#1 and LBP#2 are the same. It is intended within this definition that different forms of the antibody and non-antibody LBP (as defined by LBP#1, LBP#2, and LBP#3, supra) may be used for the capture reagent and the detect reagent, i.e., receptor polypeptide as LBP#1 and an antibody fragment-SGC as LBP#2;

"Validating assay" is used to mean an assay procedure performed on a biological sample for the purpose of detecting the possible adulteration of that particular biological test sample. The subject test procedure may, e.g., be performed on-site or in a test laboratory. Representative validation assays for urine include those testing for the concentration of a normal constituent, e.g., creatinine or specific gravity, to determine whether a test sample has been diluted, as well as, assays specifically designed to detect adulterants.

Embodiments of the invention provide methods, reagents, kits and devices for routine and uniform detection of analytes in solid biological samples. The instant methods involve steps of comminuting solid samples to a powder-like consistency having uniform preferred particle sizes, (set forth supra); and, combined with one or more method steps having improved extraction conditions and solutions tailor-made for these powders to increase extraction rates and preserve chemical structure of analytes and metabolites. Overall, the instant methods, reagents, kits and devices provide for the first time systems for routine and uniform on-site testing of solid biological samples in Screening Assays, as well as, improved laboratory Confirming Assay test methods that offer significant advantages of speed, reproducibility and uniformity with increased specificity, sensitivity and precision. Advantages provided by the instant invention also include cost-savings, speed, ease of sample handling and reduced technical manipulation. Embodiments of the invention provide methods whereby solid biological samples are reduced to powders in comminuting steps that require just 2 minutes to 5 minutes; and, analytes, including drug metabolites, that are trapped in the comminuted samples are extractable within about 10 minutes to about 30 minutes. In other embodiments, the invention provides advantages of single-use device containers for collecting, comminuting and extracting solid biological samples. The instant device containers, methods and reagents constitute components of kits which offer needed advantages of uniformity and standardization, i.e., both in Screening and Confirming Assays. Test Sample: For human hair, the sample size typically ranges from 8 milligrams (mg) to 20 mg. After comminuting, the volume of the extract solution into which analyte is extracted is preferably in the range of about 0.2 mL to about 0.5 mL. Sample Preparation: Embodiments of the invention also provide methods for detecting analytes in fluid biological samples that are purposefully dried to produce solid biological samples. The instant methods offer advantages of improved sample handling e.g. for field and on-site workers who may not have easy access to a test laboratory or refrigerated conditions for sample storage. In these settings, the instant methods are particularly useful for improving detection of "stable analytes", i.e.., analytes that remain unchanged as after drying. Examples of fluid biological samples that may be comminuted after drying include blood, serum, plasma, cerebrospinal fluid, mucus, semen and the like. Sample preparation to produce a solid biological sample prior comminuting may include e.g. drying at room temperature; treatments such as freeze- drying (lyophilization); depositing an extracted analyte onto a solid phase, e.g. by evaporating at atmospheric temperature and pressure a supercritical C0₂ fluid extract; or, where the analyte is stable and does not degrade, drying with heat at ambient environmental conditions. Extraction Solutions and Conditions: In other embodiments, methods are provided for rapid extraction of analytes from comminuted biological extracts by using extraction solutions selected from the group consisting e.g. of acidic and basic solutions, enzyme solutions, detergent solutions, organic solvent solutions and supercritical fluid solutions such as C0₂ , i.e., as set forth in Applicants co-pending U.S. Patent Application Serial No. 60/381,109 which is incorporated herein in its entirety. Preferable temperature and pressure conditions for the instant method step of acidic, basic and organic extraction are selected from the following: namely, (i) atmospheric pressure; (ii) for alkaline extraction, a temperature of about 45°C to about 90°C; preferably, about 65°C to about 88°C and most preferably about 80°C to about 88°C; (iii) for organic solvent extraction, about 60°C to about 65°C; and, (iv) with extraction for a time of about 10 minutes to about 120 minutes, preferably about 10 minutes to about 60 minutes and most preferably about 10 minutes to about 30 minutes. During the extracting step, the extraction solution may optionally be subject to sonication and/or mechanical agitation, e.g. in a vortex mixer or with a motor driven stirrer. Accordingly, nonlimiting examples of aqueous bases useful in extraction solutions include sodium hydroxide (NaOH) and potassium hydroxide (KOH). Nonlimiting examples of acids useful in extraction solutions include phosphoric acid (H₃P0₄), hydrochloric acid (HCl),sulfuric acid (H₂S0 ), and nitric acid (HN0₃). Nonlimiting examples of organic solvents or solvent mixtures useful in extraction solutions include methanol (MeOH), ethanol, isopropanol, ethyl acetate, mixtures of such solvents. In a presently preferred embodiment, the extraction solution comprises both an organic solution and an acidic or basic solution, e.g. a methanol solution mixed with either hydrochloric acid or acetic acid. Following extraction of analyte from the comminuted biological sample, the analyte in the extract solution may be "captured" (supra) or "trapped" (below) onto a solid phase, or alternatively, the extract solution may be "processed" to allow detection, e.g., an organic solution such as methanol may be brought to dryness or an alkaline or basic solution may be brought to a neutral pH. In optional embodiments, sonication during extraction has the advantage of lowering the temperature and decreasing the time required to extract analytes from comminuted biological sample. Ultrasonic energy may be applied e.g. either using a probe or water bath sonicator. Devices and Methods of Their Construction and Use: One embodiment of the invention is schematically depicted in FIG. 1, and the following description is made with reference to that figure. The invention, however, is not limited by the embodiments shown therein and the skilled artisan will appreciate modifications which may be made staying within the spirit and scope of the invention. In the embodiment schematically depicted in FIG. 1, a device for high-throughput of collecting, washing, comminuting, extracting and detecting of analytes in solid biological samples is depicted wherein multiple flow paths allow processing and transfer of biological samples, washing solutions, extraction solutions and, optionally, detection reagents. The instant device preferably provides an internal, tamper-proof, closed loop system for stepwise washing, comminuting and extracting of biological samples in one or more device containers, i.e., a collection vessel, a comminuting chamber, an extraction vessel and a detection chamber. The subject device container(s) is in a fluid connection with a reservoir for a wash solution; a reservoir for an extraction solution; a reservoir for processing the extraction solution, e.g. to bring the pH to neutrality; and, a chamber for analyte detection. In one presently most preferred embodiment, the instant automated device method involves the following steps: namely, the biological sample containing an analyte is collected in a single-use collection device; the collection device is engaged into a single-use comminuting device where (a) a screen with small particle-sized holes is located in the chamber of the collection device; (b) the screen separates the biological sample from a cutting foil in the comminuting device; (c) engaging the collection device into the comminuting device brings the chamber of the collection device into an interruptable fluid communication with a washing reservoir, an extraction reservoir, an optional reservoir for processing the extraction solution and an optional detection device chamber; the biological sample in the collection device chamber is subject to washing, i.e., by opening the fluid communication to wash solution in the wash reservoir; the washed biological sample is subject to comminuting e.g. by bringing the biological sample into contact with the cutting foil through the screen, the resultant particulate test sample is brought into fluid communication with an extract solution under conditions and for a time sufficient to enable extraction of the analyte; the extract solution is then optionally brought into fluid communication with an extract processing solution, e.g., to neutralize an acidic or basic extract solution; and, finally, the extract solution is brought into fluid communication with a detection vessel located within a detection device. Referring to FIG. 1, in one embodiment, a sample, e.g. hair, is collected from a test subject into a single-use sample collection device 1 (e.g. a cartridge) which is interfaced either manually or using automation with comminuting device 2. Sample collection cartridge 1 is preferably a cylindrical shape, with end-caps on the inlet and outlet sides to allow secure storage of the sample until processing. Alternatively, the sample is collected directly into, or by, the comminuting device, thereby eliminating the need for a separate collection device. The collection device may be fabricated from such non-limiting examples as metals, plastics, ceramics or combinations or coatings thereof. When processing is to occur, the sample is either removed from collection device 1 and manually placed into a comminuting vessel, e.g., a single-use "comminuting cup", or preferably, collection device 1 links with comminuting device 2, e.g. by engaging collecting device 1 into a lock in comminuting device 2. In the latter embodiments, linking comminuting device 2 with collection vessel 1 allows comminuting of the biological test sample without operator handling. Non-limiting examples for moving the collected biological test sample from collection device 1 into comminuting device 2 include uses of mechanical agitators, vacuum sources, pressurized air, fluids or gas and mechanical transfer such as accomplished using a plunger or other direct-displacement means. Non-limiting examples of disposable sample collection cartridges are described by certain of the Applicants in United States Provisional Patent Application No. 60/526,403 (filed on December 1, 2003), United States Patent No. 6,478,750, and U.S. Provisional Patent Application Serial No. 60/526,404, (also filed on December 1, 2003), the disclosures of which are incorporated herein in their entirety. Comminuting device 2 preferably is a single-use device having a disposable ram and screen, (described further below), assembled in a unit that directly engages a disposable sample collection device 1. The disposable sample collection device 1, in turn, preferably serves as both a washing, a comminuting and an extracting vessel. Alternatively, a chamber in sample collection device 1 may be manually, or using automation or mechanics, opened upon engaging with comminuting device 2; and, as a result, the biological test sample in the chamber may be released into a comminuting vessel 3. In one presently preferred manual embodiment, a hair test sample is manually loaded, e.g. with forceps, from a single-use collection device 1 into the single-use comminuting vessel 3. In the latter embodiment, comminuting vessel 3 also serves as a sample processing vessel for washing and comminuting the sample. Comminuting vessel 3 has a screen at outlet 4 ("screen 4"). In one presently preferred automated embodiment, comminuting vessel 3 is also a collection device, washing vessel and extraction vessel. Preferably, comminuting vessel 3 is formed into an open conical shaped vessel having a filtered outlet screen orifice 4. Screen 4 allows charging and releasing liquid and air from and into the sample in vessel 3 while also retaining the sample. In a first embodiment, comminuting vessel 3 is loaded manually into comminuting device 2; a comminuting device motor is manually activated; and, the resultant comminuted sample is manually transferred into extraction vessel 3 using, as aids, gravity and mechanical agitation. In a second embodiment, after activating the comminuting device motor automation engages a downstream vacuum source that is effect to both pull the sample into the comminuting device and pull the comminuted particulate sample into comminuting cup 3. Additional aspects of comminuting devices are disclosed below. The extraction vessel 3 is fabricated from a such non-limiting material examples as metals such as aluminum, plastics such as polypropylene, glass, ceramics or combinations of coatings thereof and other materials or combination of materials that transmit the sonic energy and meet chemical and thermal requirements while minimizing static electricity. In presently preferred embodiments, the vessel volume typically ranges from about 0.5 mL to about 10 mL in size, and most preferably about 0.5 mL to about 5 mL and is sample and process dependent. In certain optional embodiments, vessel 3 may incorporate a conical shaped end with a filter or screen 4. In other optional embodiments, a chamber in vessel 3 is, prior to use, loaded with a breakable ampoule containing an extraction solution; or, with one or more other reagents such as may be contained within packets such as a filter-bags or may be included in the chamber as freeze-dried reagents. In the case of filter-bags containing dried reagents when an extraction solution enters the chamber the reagents are designed to be solubilized and/or resuspended. In the case of the ampoule or packet, breakage of the ampoule or packet is designed to release a solution that may, in turn, be used to dissolve freeze-dried reagents in the chamber into the extraction solution. Embodiments of the invention provide comminuting devices with different alternative methods for retaining and moving different types of samples and fluids. Representative examples for moving comminuted particulate test samples include vacuum, positive pressure and mechanical agitation and gravity. Representative examples for retaining comminuted particulate test samples include electrostatic adhesion to tacky coatings, electrostatic retention via static electricity discharge systems such as generated in by a de-Gaussier ion implantation generator. Where buildup of static electricity is detrimental, alternative embodiments provide electric grounds within the comminuting vessel and/or use the Gaussier ion generator as a source of positive ions to neutralize the static electricity building-up in the system or device during use. Where producing a charge on a particle is useful in moving the particles e.g. within an electric field, embodiments of the invention provide methods for "charging" particles so that they can be moved. Where producing a magnetic field on a particle is useful in moving the particles e.g. within an electromagnetic field, embodiments of the invention provide methods for adsorbing magnetic particles to the comminuted test sample particle, e.g. through electrostatic interactions, particle coating applied in solutions and the like. In one alternative embodiment, a comminuted particulate test sample is delivered in measured amounts into extraction vessel 3, the extraction solution, in turn, is delivered in measured amounts into the test sample through multi-port selector valve 5. Measured amounts of extraction solution are delivered e.g. a metering pump, a syringe pump, a syringe, a sample loop or another fluid movement and metering system known in the art. Alternative Delivery Method for Extraction Solution and Additives: The extract solution may also be constructed in a stepwise manner e.g. by delivering a liquid into an extraction chamber followed by breaking a reagent pack or ampoule (supra) within the chamber. Alternative methods and devices for metered addition of extract solution, releasing reagents or extract solution by puncturing or breaking a vessel, or reconstituting dried reagents or solutions are known to those skilled in the art. In one presently preferred embodiment, the extract solution is included in an ampoule or other reagent containing pack which is placed or embedded in a single-use hair collection device 1 which also serves as comminuting vessel 3 and an extraction vessel. In this object, after comminuting the biological sample, breakage of the ampoule initiates extraction of analyte. Any additional additives that may be desirable in preparing the sample for extraction, extraction- or detection processes may optionally be included in the hair collection device, comminuting or extracting vessels, e.g., as coated filters, or additional ampoules or surface coatings or powders. Breakage of the ampoules or reconstitution of embedded reagents within the extraction chamber may be accomplished e.g. by pressure differential, by heating to dissolve, by flow of organic solvent (dissolving the ampoule), by charging a liquid or by puncturing. In a presently preferred embodiment, the extraction solution is acidified methanol, i.e., methanol and HC1 in a volume percentage ratio of 99.5% methanol to 0.5% HC1, and in this embodiment electronic pipettes, e.g. an electronic Rainin pipette, or alternatively, a liquid pump and sample-loop, are used to charge the extraction vessel with 500 μl of the extraction solution. In a second presently preferred embodiment, the extraction solution is an alkaline solution and an electronic pipette is used to charge 200 μL of 0.5 Molar (M) Sodium Hydroxide (NaOH) or Potassium hydroxide (KOH) into the extraction chamber. In other embodiments, alternative reagent delivery methods are provided, such as reagent coated filters placed in the chamber; impeded ampoules that break or dissolve upon initiation of some process step; and pressure, heat, flow or puncture-sensitive storage devices. In one presently preferred embodiment where analytes are detected using an immunoassay device, electronic Rainin pipettes or a liquid pump and sample loop are used to deliver 500 μL of a commercially available buffer to the extract. Components for Modifying Extraction Conditions: In optional embodiments, heat may be applied to the extraction vessel during the extraction process, e.g., using a temperature controlled oven, an electric heating probe or an electric heating bath. Alternative nonlimiting heating methods include temperature controlled heat blocks, chemical or radiant heat as are known in the art. In other optional embodiments, sonication may be used to improve analyte extraction from a comminuted biological sample. In this object, after the extraction solution is delivered into the extraction chamber, sonication 7 is activated. Ultrasound energy can be applied e.g. using an internal probe sonicator and/or an external source such as a water bath sonicator that uses an intermediary vessel to transmit sonic energy through the wall of the extraction vessel. In a presently preferred embodiment using an acidifϊed-methanol extraction solution, analytes are preferably extracted at a temperature of about 60°C to about 65°C. In one embodiment, an organic extraction solution of acidified methanolic is employed with sonification and analytes in a comminuted biological sample are extracted without heat in about thirty (30) minutes. After comminuting and extracting the test sample, if the extract solution is in a form suitable for analysis in a detect assay format, it is delivered through the multi-port valve 5 into a collection vessel or detection chamber in a detection device e.g. a chamber in an automated immunoassay device or a trap in a GC/MS automated device. If the extract solution requires "processing" (supra) before it is suitable for use in a detect assay format, then additional processing solutions may be delivered through multi-port valve 5, e.g., processing solutions designed to neutralize the pH of an acidic or basic extract solution. Alternatively, where the extract solution is an organic solution that can be evaporated using a combination of heat and a flow of nitrogen gas, provision is made for charging gas into the extraction vessel while heating the chamber. Delivery into a Detection Chamber or Vessel: When extraction is complete, (and/or processing of the extraction solution is complete), embodiments of the invention provide for purification to remove residual solids, e.g., by filtering the extract through screen 4 and/or by passing the extract solution through an optional in-line filter as it is delivered into a detection chamber or vessel. In different optional embodiments, detection vessels are machine reservoirs e.g. capable of introducing the extracted sample into an automated immunoassay machine or GC/MS device; in manual alternatives, the detection vessels are test tubes, vials, ampoules, microtiter test wells, dipstick immunoassay sample pads and the like; or in measured detection systems, the detection vessel may be a syringe, pipette or pipette-tip filter or sample loop. In one presently preferred embodiment, the processed extract solution is collected and stored in a self-contained reservoir for later analysis in the detection step. Analyte Detection: As set forth further below, the invention incorporates a variety of detection process that are analyte-specific, including e.g. such non-limiting examples as those based on (a) optical systems such as fluorescence, chemiluminescence, absorbance, Raman or reflection, light scattering, refractive index, radiation, turbidity, color, size spotting or area spotting; (b) electrochemical measurements that are amperometric, ptoentiometric, conductometric; (c) chemico-mechanical coupled reactions; (d) measurements of thermal conductivity, flame ionization and the like; (e) measurements in chemical and magnetic systems; (f) measurements of chromatographic elution rates e.g. in HPLC or GC; (g) measurements of molecular ions e.g. in MS. Examples of immunoassay detection methods, include enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemilluminescent immunoassays, charge-coupled electrical immunoassays and the like. Devices for automated immunoassays and automated GC/MS sample processing are known to those in the art. The one presently preferred embodiments, detection of analytes in a Screening

Test is accomplished using an immunoassay format such as ELISA (Enzyme-Linked Immunosorbent Assay) and those test samples which give a positive test result are subject to an additional Confirming Test wherein the detection system of choice is Tandem GC/MS. In other embodiments, detection of a signal generated by a Signal Generating

Compound is accomplished using spectrometers and spectrophotometers employing Charged Coupled Devices (CCDs). Alternative non-limiting embodiments of the invention employ photodiodes, photomultiplier tubes and other photon detection-devices. In yet other embodiments, detection is accomplished by applying excitation energy to the SGC using e.g. a Light Emitting Diode (LED). Representative examples of LEDs include lasers, infrared (TR) devices and flash lamps. In yet other embodiments, detection of a signal generated by an SGC is accomplished using a microfluidic card(s), vessel(s) and/or chip(s) in which electrical signal generation is linked to one or more chemical reactions/reactants produced by the SGC. The instant microfluidic device(s) have desirable properties, including requirements for relatively small volumes or mass of reagents or additives to detect analyte in a test sample. In a presently preferred embodiment, a microfluidic device(s) is utilized for detection of analyte in a comminuted biological sample, i.e., either analyte in whole particles or in particles where analytes have been extracted into a solution. In yet other embodiments, detection of a signal generated by an SGC is accomplished by an electrical measurement, e.g. measuring an electrical change in capacitance, resistance change, voltage or current such as may be generated in a biosensor chip following binding of analyte to an LBP. Device Electronic Data Handling: In currently most preferred embodiments, the methods of the invention employ device(s) that comprise electronic information processing systems for recording and retaining digital data, including the data of the following illustrative types: namely, (a) identity of a test sample; (b) conditions of comminuting, extracting and detecting for each test sample; (c) sample integrity during processing, i.e., lack of possible contamination with other samples; (d) tracking information to identify the location of the sample in the comminuting, extracting and detecting process. Information useful in the digital electronic processing may involve e.g. bar-coding, data logging, data collection, transmission, analysis, calibration, traceability and control; and, verification of the identity of the test subject, e.g., digitized copies of identifying documents such as a driver's license, a valid government issued document, a passport, or a fingerprint verification from a previous test session. Extract Trapping: A variety of different traps may be employed to collect the instant drug and chemical analytes from the extract solutions prior to use in a detect assay format. For example, organic and aqueous solutions may be brought into contact with solid phase traps include silica, cellulose, anion and cation exchange resins, octyldecylsilane (ODS), Tenax, Porapak-Q, silica bonded with diol, florisil, basic and neutral alumina, charcoal, a solid phase such as a filter; or, onto a surface such as glass beads or a polymer; and the like. Immunoassay Methods: Embodiments of the invention provide instructions, reagents, method steps and kits for preparing and extracting comminuted solid biological samples to obtained Test Samples that are useful in immunoassay formats for identifying analytes. Representative assay formats useful for detecting analytes include enzyme-linked solid-phase absorbent assays (e.g., ELISA), radiolabeled binding assays (e.g., RIA), fluorescence binding assays (e.g., FIA), time-resolved fluorescence assays (e.g., TRF), as well as, sandwich- and enzyme-cascade assay formats. Illustrative methods, as may be adaptable from the immunoassay art for use in the subject assays include: homogeneous assay formats; heterogeneous assay formats; competitive assay formats; non-competitive assay formats, enzyme-linked solid phase assay formats, fluorescence assay formats, time resolved fluorescence assay formats, bioluminescent assay formats and the like. The instant assay methods include those having a step effective to simultaneously accomplish binding and signal generation as an analyte binds to a LBP, i.e., a "simultaneous" or "homogeneous" assay format. The instant methods also include "heterogeneous" drug-binding assay formats, including one or more steps for separating a "bound" from a free analyte and then generating a signal. The instant methods also include those having a step in which analyte is added to compete with the binding of a labeled ligand to an LBP, i.e., a competitive binding (or indirect) assay format , or alternatively, in which binding of an analyte to a LBP is detected by adding a second LBP having signal generating compound, i.e., a non-competitive (or direct) assay format. Illustrative methods for separating "bound" analyte, ligand or LBP from "free" include filtration, column separation, magnetic separation , as well as, attaching one or more of the reactants to a solid phase . Illustrative assay methods for detecting a signal generating compound commonly include: using an enzyme as a SGC that converts a substrate to a visually or spectrophotometrically identifiable product), or alternatively, exciting a fluorescent SGC coupled to an SBP so that a detectable signal is emitted, e.g. at a different wavelength, e.g. fluorimetric analysis (as set forth further, supra). Commonly, coating a solid-phase e.g. polystyrene in a 96-well Dynatech-Immulon II, Nunc, or similar plate with an LBP may be accomplished at a concentration of about 1 mg/ml in a carbonate buffer at pH 8 for about 16 hours results in binding of about 20 to about 150 μg/well to the solid phase. Immunoassay Kits: Embodiments of the invention also provide kits useful for identifying analytes in comminuted biological test samples. A representative kit contains the following: namely, one or more reagent packages at least one of which contains a LBP-conjugate; an assay buffer; an optional solid-phase assay surface, e.g., a tray, a vessel, or dipstick; a set of instructions; and one or more optional assay calibrators or reference compounds (e.g., a positive and negative control). In one presently preferred embodiment, a kit contains reagent packages containing: (i) one or more optional pre-treatment solutions for reducing interfering substances, e.g. a mucosidase, a detergent such as TWEEN-20, a DNAase, a collagenase and/or a protease; (ii) one or more reference calibrator solutions (e.g., one or more drug-analyte-derivatives); (iii) one or more optional modifier solutions suitable for use in SFE; (iv) one or more LBP-conjugates (e.g., LBP-FITC, LBP-enzyme and the like); (v) one ore more optional assay buffers and/or wash buffers (e.g., assay buffer containing PEG and/or detergents that promote binding between LBP and a drug analyte compound); (vi) one or more optional enzyme substrate solutions; (vii) one or more optional blocking buffers for reducing nonspecific background (e.g., solutions containing BSA or milk proteins); and (viii) one or more optional solid phase reaction surfaces upon which, or in which, the assay may be conducted (e.g., microtiter plates, dipsticks, strips, and the like.) Preparation ofLBP-Conjugates: In certain other embodiments, the invention provides diagnostic reagents containing LBP that are detect reagents specific for derivatized drug analytes. The instant detect reagents contain one or more signal generating compounds conjugated to an LBP. Representative methods for covalently linking SGC to LBP include those using hetero-bifunctional cross-linking reagents that are reactive with carbonyl, aldehyde, carboxyl, amino, disulfide and thiol groups of amino acids, e.g., carbodiimide, N-hydroxy succinimide, N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), maleimide, succinimidyl-pyridylthioproprionate, m- maleimidobenzoyl N-hydroxysuccinimide ester, succinimidyl pyridylthiopropionate, and the like. Methods for linking SGC fluorophores to LBP include, e.g., encouraging electrostatic interactions of the subject LBP with the fluorophore by placing the LBP in a buffer having a pH below its isoelectric point (e.g., Ph 2.5-3). Methods suitable for linking phycobiliproteins to LBP are disclosed in Stryer et al. U.S. (Patent Serial No. 5,055,556). Optional Packaging Strategies: The instant LBP-SGC conjugates (supra), Diagnostic Reagents, Detect Reagents, substrate solutions for an enzyme SGC and the like, may be prepared for commercial distribution as e.g. powders or solutions in ampoules, vials, single use foil packages, break and release dipsticks and the like.

Reagent powders may be made suitable for use in assays by including one or more buffer dried solutions, additives, stabilizers and the like. Solutions may be dispensed into reagent bottles, packages and the like which may subsequently be lyophilized to obtain dried powders. Packages may include optional additives (e.g., stabilizers), emulsifiers (e.g., detergents), and the like for preserving the activity of the instant LBP-conjugate during storage; or, for promoting the binding activity of the instant LBP for a drug analyte in the instant assays. Examples of agents that may be used to promote the subject binding interactions include additives that decrease total fluid volume in an assay (e.g., polyethylene glycol, sucrose and the like); and, agents that promote interactions by provide an electrostatic surface in solution (e.g., dextran, polystyrene beads, polyacrylate beads, and the like.) Comminuting Device: Certain of the Applicants are co-inventors in a comminuting device disclosed in a US Patent Application 10/537,402, incorporated herein by reference in its entirety. Briefly, the subject device employs a ram to drive a solid biological sample through a cutting screen where a cutting-foil shears the sample into pieces of uniform length; the resultant cut biological solid preferably has a powder like consistency; and, the particles are collectable into an extraction chamber e.g. by gravity and mechanical agitation, by vacuum suction, or by positive pressure. The subject device incorporates the following general features: namely, (a) The sharpness of the edges of the cutting-foil is enhanced by grinding or lapping or etching to achieve a ridge-height less than the diameter of the thickness of the hair strands (about 80μm to about lOOμm/ 0.002-0.004 inch), i.e., a thickness of approximately 0.002-0.0025 inch; (b) Orientation of human hair at a comminuting disk surface is flat or generally coplanar once the ram is installed into the comminuting vessel, pushing the sample into the bottom where it is in contact with the disk surface; and, (c) The comminuting screen has apertures of about 0.025 inch. Using this instant device a typical 20mg sample of hair preferably requires about

10 seconds to about 5 minutes, more preferably about 10 seconds to about 2 minutes, and most preferably about 10 seconds to about 60 seconds of comminuting to reach a consistency suitable for extraction and detection. Methods for an On-Site Assay Device: In other embodiments, the invention provides methods enabling on-site screening assays for analytes in solid biological specimens. The instant methods involve the following steps: namely, (a) collecting a biological sample from a test subject; (b) preparing the collected biological sample for comminuting and extracting, e.g. by washing to remove exogenous matter; (c) comminuting the sample (supra); (d) bringing an extraction solution into contact with the comminuted sample under conditions suitable for extracting the analyte; (e) optionally, where the extract solution does not have chemical properties suitable for detection of the analyte, processing the extract solution to bring it into condition for detection of the analyte; and, (f) detecting the analyte in the processed extract solution. Representative examples of comminuting methods are disclosed above and are illustrated in the Examples section, below. Representative examples of extraction solutions are disclosed above and illustrated in the Examples section, below. Representative examples of methods for processing extract solutions are disclosed above and illustrated in the Examples section, below. Representative examples of methods useful in detecting an analyte in a processed extract solution are disclosed above and illustrated in the Examples section, below. The biological sample is collected into a collection vessel; the step of comminuting the biological sample is conducted in a comminuting vessel; the step of extracting the comminuted biological sample is conducted in an extraction vessel; the step of processing the extract solution is conducted in the extraction vessel; and, the step of detecting the analyte in the extracted comminuted biological sample is conducted in a "detection vessel". Preferably, the steps of collecting and comminuting are both conducted within the same vessel, e.g. the collection vessel; and more preferably, the steps of collecting, comminuting and extracting are all conducted in the same vessel. In certain presently preferred embodiment, a collection vessel is engagable into a comminuting device and is also in fluid communication with reservoirs containing extraction solution and optional "extract processing solutions", (e.g. to neutralize acidic or basic extract solutions). Thus, in the most presently most preferred embodiment, all of the steps of collecting, comminuting, extracting and optionally processing the extract are accomplished in the same vessel and the detection step is conducted in a separate vessel. Hair Samples. Assays according to the instant methods are performed using hair samples comprising about 1 mg to about 50 mg by weight, preferably about 10 mg to about 20 mg and preferably about 5 mg to about 10 mg. Preferably, the hair sample comprises a group of hair fibers cut from a test subject to an approximate length of 1.5 inches and having a weight of approximately 20 mg, which, when the hair is taken from the head, represents about 90 days of potential drug use. It is presently most preferred that about 10-20 hairs comprise a test sample. In certain optional embodiments, the hair sample is loaded into a comminuting device in fluid connection with a one or more option extraction vessels and detection devices (supra); in one presently preferred embodiment, the extraction vessel comprises a hair collection device (supra). Advantages: Embodiments the invention offer e.g. the following advantages: namely, improved extraction efficiency, increased sample throughput, decreased sample handling and pre-treatment, decreased method development and operator time, cost- savings, time-savings, lower solvent consumption, minimal hazardous waste generation, and simplification of post-extraction concentration steps, all of which advantages are highly useful both in the test laboratory where high throughput may be desired; also, in on-site locations in manufacturing and processing facility, as well as, in field work. EXAMPLE 1 Feasibility Testing: Wettability of Hair Clippings Hair is representative of many fibrillar solid biological samples, in that it is composed of a complex network consisting of different types of intramolecularly cross- linked keratin fibers; with endogenously associated cellular glycoproteins, extracellular glycoconjugates, lipids, waxes and oils; with possible exogenously associated cosmetic products and hair treatments including emulsion-based conditioners containing animal and plant oils and waxes; and, with also the possibility for environmental contaminants. In order to extract or detect analytes of interest in a hair sample using an aqueous based assay format, the hair sample must be wet-able ("wetable"), i.e., meaning that any hydrophobic surface waxes, oils or lipids must not inhibit penetration of aqueous solvents (and reagents) to the level of the analyte. This property of wetting-ability is particularly important where the analyte is metabolically incorporated into the hair matrix. Testing hair for drugs of abuse was first disclosed by Baumgartner and coworkers in the 1970s. Therefore, it seemed likely that some individual skilled in the art would surely have attempted to use finely powdered hair samples. Since the inventors were unable to find such reference in the scientific and patent literature, it was initially considered likely that such an approach had been tried but did not work. For instance, the possible problems that might be encountered using particulate samples, include the following: namely, fine particles have a large surface area might trap significant amounts of air at their surface; or, the particles might be hydrophobic, i.e., limiting the access of aqueous solvents to the analytes; or, analytes might be destroyed in the powdering process and/or rendered non-extractable; or, hair matrix powdered in this manner might release potential interfering substances making it difficult to detect analytes using either immunoassays or GC/MS approaches. However, the possibility was also considered that perhaps the proper conditions had not previously been achieved for processing hair and fibrillar solid biological samples into particulate samples. Therefore, experiments were first conducted to determine the wetting-ability ("wettability") of fine ground hair. For these studies, facial and head hair was collected from normal human test subjects and ground into a powder by pushing the hair through the screen in a Remington model electric shaver. The resultant powder was collected and added to a test solution in a 2 ml screw capped glass vial: i.e., 1 ml of distilled water, or 1 ml of sodium phosphate (0.10 M) buffered 0.15 M saline, pH 7.4 (PBS), or 1 ml of PBS containing 0.05% TWEEN-80, or 1 ml of methanol. After capping the vial and briefly shaking the vial, the wettability of the hair powder was visually assessed. In different test subjects, approximately 10-35% of the total added hair particles were non-wettable in the distilled water and PBS, i.e., floating on the surface of the aqueous solution while the wetable particles rapidly settled to the bottom of the vial. Addition of the TWEEN-80 detergent to PBS did not markedly improve the wettability of test samples. However, in organic solvent, i.e., methanol, virtually all particles were wetable with less than 3% floating and most rapidly settling to the bottom of the vial. Considered this relatively poor penetration of aqueous solvents into hair, it was considered not-surprising that existing hair test assay formats using aqueous extraction solvents require lengthy extraction times. For example, in existing assays using cut hair samples a test sample may be incubated in an aqueous-based solution with enzymes and/or acids or bases for about 2-3 hrs., or even overnight. Considering that organic solvents seemed to satisfactorily resolve the problems in the tests, it was considered likely that the poor wettability was due to hydrophobic hair waxes, oils and the like. Unfortunately, many organic solvents denature the proteins commonly used in immunoassay formats, i.e., LBP-Conjugates, signal generating compounds such as enzymes. Also, since organic solvents are not environmentally friendly or easily amenable to use in a non-laboratory environment, conditions were sought that would allow routine and uniform extraction of analytes from comminuted solid biological samples.

EXAMPLE 2 Extraction of Analytes from Hair Clippings Cutting hair into fine particles by shearing could theoretically result in heating, denaturing of components in the hair matrix, releasing of potential interfering substance from the matrix and/or destroying, or rendering non-extractable analytes of interest. Considering the results recorded in EXAMPLE 1, above, it was hoped that the 65-90% wetable particles might be extractable and liberate sufficient drug analyte into an aqueous solution to enable detection in an immunoassay. To test the extractability of drug analytes from fine powdered hair, known drug user hair samples, (i.e., from cocaine-users) were pushed into the screens of different commercial shavers; the resultant powders were collected and added to PBS; after vigorous mixing on a vortex mixer the samples were subjected to gentle mixing for 15-30 minutes on a tilt-table platform. After removing the hair powder by filtration, the resultant solution was tested in a competitive binding immunoassay specific for cocaine. Remarkably, not only were cocaine drug analytes detectable, (i.e., no apparent interfering substances were released that interfered with the antibody or analyte in the competition assay format), but the immediate first impression was that, given the strength of the colorimetric signal generated, more analyte might be extracted in a shorter period of time from the drug user hair than obtained using conventional longer incubations with cut hair samples. Experiments were therefore conducted to further examine, confirm and extend these observations. EXAMPLE 3 Characterization of Particulate Hair Samples Nitrogen Frozen Mortar and Pestle Sizes: Normal hair samples selected for normal and fine consistency were placed in a mortar then covered with liquid nitrogen. The sample was manually powdered with a pestle for 5 minutes or 10 minutes. The samples were subject to scanning electron microscopy in a Cambridge StereoScan

Scanning Electron Microscope at a beam voltage of 10 kV and at magnifications of 50X, 100X, 500X and 1500X. Hair particles were roughly cylindrical and relatively uniformly sized with diameters and lengths of particles determined as follows: namely, (a) for normal consistency hair, the diameters (hair shaft) were in the range of 80 microns to 100 microns (micrometer; μm); (b) after 5 minutes particles had lengths in the range of 400 μm to 2000 μm; and (c) after 10 minutes particles had lengths of less than about 500 μm.

Shaver Particle Sizes: Normal human hair samples selected for normal and fine consistency were powdered by pushing the hair into different commercial shavers. The resultant particles were visibly larger than those produced by mortar, pestle and liquid nitrogen (supra).

First generation Comminuting Device - Particle Sizes: Normal hair samples selected for rough and fine consistency were subject to mechanical shearing in the comminuting device, (described supra), i.e., shearing the samples between a rotating ram and the cutting-foil through a screen, i.e., the rotating ram alternately applied and released pressure continuously for 1.5 to 3 minutes. Vacuum, agitation and gravity were used to transfer the comminuted sample into a collecting vessel. The resultant samples were subject to scanning electron microscopy in a Cambridge StereoScan Scanning Electron Microscope at a beam voltage of 10 kV and at magnifications of 50X, 100X, 500X and 1500X. Unexpectedly, and perhaps advantageously, hair particles appeared to be 'ripped' along the length of the shaft and were relatively uniformly sized with diameters and lengths of particles determined as follows: namely, (a) for normal consistency hair, the diameters (hair shaft) were in the range of 80 microns to 100 microns (micrometer; μm); (b) rough and fine hair particles had lengths in the range of about 200 μm to about 500 μm. Combination of First Generation Comminuting Device with Paper Filter -

Particle Sizes: Normal hair samples selected for normal and fine consistency were subject to mechanical shearing in the First Generation Comminuting Device, i.e., as set forth above. To test the efficacy of a selective screen, the comminuted samples were collected through a 70 μm filter into a first collection vessel and the particles too large to move through the filter screen were collected into a second collection vessel. The sample filtrate in the first collection vessel was subject to scanning electron microscopy under the same conditions set forth above. Interesting, the filtration appeared highly selective as no portion, of any whole, hair strands were visible and the sample consisted entirely of relatively uniformly sized particles with diameters and lengths determined as follows: namely, (a) for normal consistency hair, the diameters (hair shaft) were in the range of 80 microns to 100 microns (micrometer; μm); and, (b) the lengths of the hair particles in the 70 μm filtrate were less than 100 μm. Thus, where it is not feasible to produce shaver- type metal screens with 70μm holes, or where shearing times would be inordinately long because of difficulties in getting fibers to fit through small holes, then instead downstream filtration of a comminuted biological sample is shown here to be a highly effective means for obtain uniform small particle sizes from which analytes are extracted rapidly and in a uniform manner. Similarly, the importance of screen size and ridge height on the cutting-foil of the comminuting device were demonstrated, (see above, "Comminuting Device"), as without uniform hole sizes in screens and low ridge height uniform particles are not obtained. Comminuting Device Particle Sizes: Improvements were made in design and fabrication of a comminuting device, i.e., as disclosed by certain of the Applicants in co- pending U.S. Patent Application serial number 10/537,402. Considering the results and experience gained ( e.g. above), improvements were made to the screen and cutting-foil and rather than applying intermittent pressure, the ram was modified to apply a constant pressure for less than about 1 minute in order to push samples through a cutting screen. A gravity-mechanical agitating system was used to collect the resultant particles. Using this device, normal hair samples selected from normal and fine consistency were collected, i.e., without filtration, and subject scanning electron microscopy as set forth above. Results were recorded as follows: namely, (a) for normal consistency hair, the diameters (hair shaft) were again about 80 μm to about 100 μm (supra); and, particles had average lengths of about 100 μm, wherein the small number of particles having lengths greater than about 200 μm appeared to have split and/or broken shafts; and, (b) for finer hair, the average length was about 200 μm. In regard to the comminuting device disclosed above, the hair shaft thickness of about 80 μm to about 100 μm (0.0020-0.0025 inch) was used to set the thickness of the cutting screen, i.e., 0.025 inch, as well as, the ridge height of the cutting foil, i.e., ten fold greater than the diameter of the hair shafts at about 0.025 inch. The modified comminuting device processed hair into a fine powder of uniform particles in about 10 seconds to about 2 minutes. Experiments were next conducted to determine whether analytes of interest could be extracted from these uniform particles of a comminuted solid biological sample, i.e., human hair.

EXAMPLE 4 Comminuted Samples: Extraction and Extract Processing Methods A. Screening Test Methods: Samples of hair were collected from test subjects weighing approximately 100 mg. The samples were either collected from below the crown of the head, or if head hair was insufficient, axillary or chest hair was collected and 20-50 mg was sent to an outside lab for parallel testing. Screening assays were conducted to identify drugs of abuse analytes in comminuted hair samples, i.e., prepared according to EXAMPLE 3 "Comminuting Device Particle Sizes". Briefly, to remove exogenous environmental contaminants, hair was first subject to a vigorous washing procedure in which the hair test sample was soaked in 12x75 mm borosilicate glass tubes, or for THC silanized tubes, for about 2 minutes each in methanol (MeOH), then deionized water and then for a second time in MeOH. Following washing, the test sample was dried. The dried washed test sample of hair was next loaded into a sample cup for comminuting. So that sample weight could be determined and routine conditions of uniform analysis applied, the comminuted sample was collected by mechanical agitation into a pre-weighed (tared) extraction tube. Sample weight was adjusted to ensure that a minimum of 10 mg of test sample was present in the extraction tube. Extraction was initiated by adding either 1 mL of methanolic:HCl, i.e., MeOH mixed with HC1 in the volume percent (v/v%) ratios of 99.5 parts of methanol and 0.5 parts of HC1; or alternatively for acid sensitive analytes, 1 mL of 99:5:0.5 MeOH:water, i.e., MeOH 99.5 volumes and deionized water 0.5 volumes. The extraction process was accomplished by incubating the extraction tubes at about 60°C to about 63°C in a heated block for about 30 minutes. Since the comminuted hair particles had settled to the bottom of the extraction tube during the incubation, the extract solution containing extracted analyte was carefully removed with a pipette and brought to dryness under a stream of nitrogen gas in a sand bath at a temperature of about 40°C to about 60°C. After cooling, extracted analytes were resuspended with Vortex mixing in 500 μl of 46mM phosphate buffer, pH 6.95-7.05. The analytes extracted from the comminuted biological samples were detected at room temperature (68-74°F) using commercially available competition immunoassay ELISA assay formats for THC, Methamphetamine, Opiates, Cocaine and PCP, i.e., in 8 well strips, with low and high controls and calibrators following the manufacturers instructions (International Diagnostic Systems, (IDS), St. Joseph, MI). After stopping the LBP-Conjugate enzyme reaction with its substrate (3,3',5,5'-Tetramethylbenzidine, TMB) by addition to each well of 100 μl 3N H₂S0₄, the signal generated in each well was determined spectrophotometrically at 450 nm wavelength with 620 nm set to remove any possible background absorbance. As an additional negative control, known normal/negative hair samples were run in tandem through the comminuting, extracting and detecting steps. B. Confirmation Test Methods: In the event that a sample was found to be positive in a screening assay (supra), the stored non-comminuted hair test sample from above, was retrieved from storage at room temperature and about 27 mg of hair was weighed for subsequent confirmation testing. Briefly, the steps of comminuting, extracting and detecting were performed as follows: namely, Bl. Washing: Test samples were first washed twice by placing them into a 12x75 mm glass tube and adding 1 mL of methanol each of two times for about 2 minutes each. After the first 2 minute wash, the methanol wash from one sample was collected and saved as a control. Next, the samples were washed twice, i.e., two times in 1 mL of deionized water, for about 2 minutes. Each of the water washes was removed and discarded. Finally 1 mL of methanol was again added for about 2 minutes. After discarding the final methanol wash, the hair sample was air dried. B2. Comminuting: A weighed amount of test sample hair was subject to comminuting, as set forth in regard to the Screening Assay Methods above, and about 10 mg to about 20 mg of comminuted hair was collected by mechanical agitation and gravity into a pre-weighed (tared) culture tube. B3A. Extracting: For Test Samples Containing Endogenous Opiates and PCP: Extraction of analytes in the test sample was initiated by adding 3.0 mL of methanol and incubating at 60-65°C for 2hours. After incubation, 50 μl of 1% HCl-Methanol, (i.e., 1 volume HCl to 99 volumes of methanol) was added. During incubation the hair particles had settled and the resultant analytes in the extract solution were carefully removed with a pipette and placed into an assay tube. Extracted analytes in each of the assay tubes were brought to dryness under a stream of nitrogen gas at about 40°C to about 60°C. For test samples containing opiates, the dried extract residue was brought back into solution by adding 1.0 mL of 100 mM Acetate buffer, pH 4.5; and, for test samples containing PCP, the dried extract residue was brought back into solution by adding 1.0 mL of 100 mM phosphate buffer, pH 6. B3B. Exlractins For Test Samples Containing Endogenous Methamphetamine and Cocaine: Extraction of analytes in the test sample was initiated by adding 1 L of 0.1N HCl and incubating at 60-65°C for 2hours. After incubation, the hair particles had settled and the resultant analytes in the extract solution were carefully removed with a pipette and placed into an assay tube. To neutralize the acidic extract solution, 1.0 mL of 46 mM phosphate buffer, pH 6.0 and 0.7 mL of 0.1 M NaOH were added to each assay tube. After checking the pH to ensure a pH of about 6.95 to about 7.05, an additional 3 mL of 100 mM sodium phosphate buffer, pH 6.0 was added to each assay tube. The pH of the resultant solutions was slightly acidic and ready for solid phase trapping of cocaine and/or methamphetamine analytes. B4. Trapping and Substantial Purification of Analyte on a Solid Phase: For GC/MS analysis in Confirming Assays, Commercial Clean Screen® columns were used to trap analytes from the extracts of comminuted hair samples. Subsequent elution of the extracted analytes resulted in substantially pure solutions suitable for analysis by GC/MS. Briefly, columns were conditioned for analyte trapping by washing as follows: namely, first, with about 4 mL of dichloromethane:isopropanol:ammonium hydroxide at ratios of 70:26:4 % v/v/v, having a pH of about 11 to about 12; second, with about 3 mL of absolute methanol; and, third with about 3 mL of deionized water. For trapping extracted cocaine, PCP and amphetamine analytes, the final column conditioning wash was with 1 mL 100 mM sodium phosphate buffer, pH 6.0; and, for opiates the final wash was with 100 mM sodium acetate buffer, pH 4.5. Extracted analytes were trapped on the conditioned columns by pouring each different extracted test sample into the reservoir atop a properly conditioned column, i.e., at a flow rate of about 1 mL/minute to about 2 mL/minute; and, washing the column with about 2 mL to about 3 mL of deionized water. For test extracts containing cocaine, the second column wash was with about

2 mL of 0:1 M HCl; for PCP and amphetamine the second column wash was about 1 mL to about 3 mL of 0.1 M acetic acid; and, for opiates the second column wash was about

3 mL of 0.1 M sodium acetate buffer, pH 4.5. After a third column wash with about

3 mL of absolute methanol the substantially purified trapped sample analytes were eluted by adding 3 mL of dichloromethane:isopropanol:ammonium hydroxide in ratios of

70:26:4 % v/v/v. The resulting eluates were brought to dryness using one of two different methods: namely, for amphetamines, opiates and cocaine the column eluate was brought to dryness under a flow of nitrogen gas at about 35 C to about 45°C; and, for PCP, the column eluate was dried under nitrogen gas at about 35 C to about 45°C until about 0.5 L remained at which time 0.5 mL of 4% formic acid in methanol was added and the diluted column eluate was then brought to dryness. In preparation for GC/MS each dried sample was resuspended in either: (a) 50 μL of acetonitrile, i.e., for PCP, or (b) 50 μL of PFPOH, i.e., for cocaine, or (c) 50 μL of MSTFA + 1% TM i.e., for opiates, or (d) 50 μL of HFAA and 50 μL Ethyl Acetate, i.e., for amphetamines. With some modifications, remaining preparation for derivatization and GC/MS detection were accomplished in a manner that has become somewhat routine in the art of testing for drugs of abuse.

EXAMPLE 5 Comminuted Samples: Extraction Efficiency Comparisons A. In-House Side-by-Side Comparison of Cut and Comminuted Hair Test Samples: Codeine, Morphine and Heroine Metabolites: Side-by-side comparisons were made to evaluate the relative efficiency of extracting analytes, using traditional methods of cutting hair test samples into small pieces versus comminuting the hair test samples as described in EXAMPLES 3, "Comminuting Device Particle Sizes", above. Two different mixtures of drug-user hairs that had different levels of morphine (MOR), codeine (COD) and 6-monoacetylmorphine (MAM), i.e., a metabolite of heroine, were subject to careful cutting or comminuting. In an attempt to mimic a real-world testing environment, samples were run blind and initial screening-type assays, i.e., according to EXAMPLE 4, were conducted under identical conditions with cut and comminuted samples using a 30 minute extraction period. The samples that were determined to be positive in the screening assay were then subject to GC/MS confirmation testing, i.e., as set forth in EXAMPLE 4, wherein samples were extracted for 2 hours and the absolute amount of analyte was quantified. The results presented in TABLE 1, below, show that in 2 hours more analytes were extracted from comminuted mixed hair test samples than from cut mixed hair samples: i.e., 163% and 183% more codeine, 164% and 167% more morphine and 80% or 95% more methamphetamine.

* Abbreviations: COD, codeine; MOR, morphine; MAM, 6-monoacetylmorphine; %, is percentage difference in concentration extracted (pg/mg hair) as calculated: % = (Process #2-Process #l)/(Process #1) x 100%. B. Independent Comparison of Cut and In-House Comminuted Hair Test Samples: Cocaine: Samples of a confirmed cocaine-user hair were next submitted to an independent test laboratory for cut hair processing ("Cut"), extraction and GC/MS quantification ("Confirm") of cocaine and benzyolyecgonine; and, in parallel, the same samples were comminuted and extracted for analytes and metabolites at 0.5 hrs., 2 hrs. and 24 hrs. of extraction, i.e., as set forth above in EXAMPLE 4. For purposes of determining the uniformity of the processing, in both cases the samples were processed six times and the results averaged for the data presented in TABLE 2, below. Absolute levels of cocaine extracted using the instant methods in 30 minutes were about 85% of the total extracted in 24 hours by the independent commercial laboratory, i.e., using cut hair samples. Similarly, the levels of analytes extracted at 30 minutes using the instant methods were 3.48-fold greater than recorded in the independent laboratory at 30 minutes; and, even at 12 hours extraction the levels extracted using the instant methods were still 1.28-fold higher than those obtained in the independent laboratory. Importantly, absolute levels of the cocaine metabolite benzoylecgonine that was extracted using the instant methods in 30 minutes were about 80% of those extracted in 24 hours in the independent commercial laboratory; levels extracted at 30 minutes using the instant methods were 3.8-fold greater than in the independent laboratory; and, even at 12 hours extraction the levels extracted using the instant methods were 1.11 -fold higher than those obtained in the independent laboratory. Ability to extract benzoylecgonine is a important because this compound is a cocaine metabolite that confirms drug use.

TABLE 2 Comparison of the Results Obtained Using Instant Methods to a Present-Da Commercial Method

* nd, not determined; %, percent of highest value extracted. C. Relative Extraction Rates for Cut and Comminuted Hair Samples: Cocaine and Metabolites: Side-by-side comparisons were made to evaluate the relative efficiency of extracting analytes using (a) traditional methods of clipping hair test samples into small pieces versus b) comminuting the hair test samples as described in EXAMPLES 3, "Comminuting Device Particle Sizes", above. Confirmed cocaine-user hair was subject either to cutting or comminuting and then both sets of test samples were extracted for either 30 minutes or 2 hours, i.e., as set forth in EXAMPLE 4. The resulting extracts were tested in-house, i.e., using the GC/MS Confirmation Assay set forth in EXAMPLE 5, above, or were sent to an independent test laboratory where the analyte levels were determined by GC/MS. The results presented in TABLE 2, below, show that more than 94% of the cocaine analyte extractable from the hair sample at 2 hours in the independent laboratory was extracted in-house from comminuted hair samples in about 30 minutes. Importantly, metabolic markers of drug use were also rapidly extracted with levels in 30 minutes extraction of comminuted hair reaching greater than 85% for cocaethylene and greater than 92% for benzylecgonine. Clearly, for this analyte even shorter extraction times seem highly likely. Significant differences were also noted in extractability at 30 minutes from cut hair when compared with comminuted hair, in this case, particularly for the important metabolic markers where extraction from cut hair samples was less than 78% for cocaethylene and benzyoylecgonine, as compared with an average of 91% and 94%, respectively, for these drug analytes in comminuted samples (TABLE 3). Since metabolic markers are present, and/or extractable, at lower and variable levels than cocaine analytes, small differences in extractability can easily mean the difference between reaching a cut-off value necessary for positive detection of a drug-user and reporting a negative assay result.

*Average extracted analyte in 2 hrs. from comminuted hair test sample. EXAMPLE 6 Comminuted Samples: Extraction Uniformity: Comparisons

Uniformity of Analyte Extraction from Comminuted and Cut Hair Test Samples: The uniformity of extraction of analytes from comminuted and cut hair test samples was determined by the methods set forth in EXAMPLE 4-5, supra, i.e., using same In-House processing methods and the same confirmed cocaine-user hair sample. For these studies the comminuting and extracting processes (#l-#6, TABLE 6) were run at least six independent times and the analyte levels in the extracts were determined by In-House Confirmation Assay (supra; "GC/MS"). The different processes used in this study were as set forth in TABLE 5, below.

The results presented in TABLE 6 show relative standard deviations (RSD) for cocaine extracted from comminuted hair samples at about 26-34% RSD at 30 min. extraction decreasing to about 3-15% RSD at 2 hrs. extraction; as compared with lower levels of extraction from cut hair samples at 2 hr., but 2% RSD. Unexpectedly, deviations observed between assays on cut hair samples at 25 hrs. were highest, i.e., 31% RSD in the 6 replicate assays. These findings suggest that slower extraction over longer periods of time may result in less uniformity in the assay results obtained, i.e., perhaps as a result of degradation of cocaine analytes during the longer incubations. Relative interassay standard deviations for cocaine metabolites were considerably lower, i.e., at RSDs of just 5% to 13% for comminuted hair samples. Once again, the relative interassay standard deviation for benzoylecgonine and cocaethylene were highest in assays where samples were extracted for 25 hrs., i.e., 10% and 16%, respectively.

*Avg., average of n=6 replicate processes; Sx, standard deviation; RSD, relative standard deviation calculated as a percent of the average value, i.e., %=(Sx/Avg.) EXAMPLE 7 Comminuted Samples: Performance Comparisons

Independent Out-of-House Comparison: Parallel comparisons were made of the relative extraction efficiency of analytes using traditional methods of clipping hair test samples versus comminuting the hair samples. For these studies, hair samples were collected from 16 suspected prison drug users and split for testing, i.e., either in a commercial laboratory using hair cutting to generate small pieces for extraction, or alternatively, versus the instant in-house methods of comminuting, extracting and GC/MS Confirmation Assay detecting described in EXAMPLE 4, above. Of the 16 test samples, 6 were subsequently confirmed as being from cocaine drug-users. In addition to the difference in sample cutting or comminuting, the commercial testing laboratory also employed a 2 hour extraction method in 0.1 N HCl, while the In-House Evaluation used the 30 minute extraction and Confirmation Assay detection methods described in EXAMPLE 4. The results presented in TABLE 7, below, show that in 30 minutes an average of more than 2.76-fold more cocaine analytes were extracted from prison drug- user hair that was comminuted and assayed as set forth in EXAMPLE 4 (Process #1) than those cocaine analytes extracted in 2 hours from cut hair samples (Process #2); i.e., n=5; range 1.56-fold more to 5.76-fold.. Most significantly, the increase in sensitivity of the assay allowed detection of a prison drug user who was missed by the commercial test laboratory, i.e., a prisoner confirmed as being positive for cocaine use in the instant assay who tested negative in the commercial assay (Test subject #6, TABLE 7). Interestingly, extracting comminuted hair samples for 2 hours (Process #3), i.e., rather than 30 minutes (Process #1), did not appear to gain any additional advantage over the commercial assay in respect to extraction of cocaine analytes, but did apparently increase the levels of detectable BZE metabolite in sample samples having lower levels of analyte. Since the presence of BZE metabolite is a relatively certain indication of drug-user status, the latter changes in extraction levels at 2 hours are likely highly significant in everyday use.

* Abbreviations: COC, cocaine; BZE benzoylecgonine metabolite of cocaine confirming a drug-user status; %, is percentage difference in concentration extracted (ng/mg hair) as calculated: % = (Process #2 COC- Process #1 COC)/(Process #1 COC) x 100%. EXAMPLE 9 Comminuted Extracts: Immunoassay Detection Screening Assay in Combination with Confirmation Assay Sixty seven (67) hair samples, consisting of 53 samples of head hair, 10 samples of underarm hair and 4 samples of chest hair, were subject to testing, (according to the methods set forth in EXAMPLE 4-6), as follows: namely, (a) all test samples of hair were comminuted, extracted for 30 minutes and tested in the Screening Assay, i.e., using the immunoassay format (EXAMPLE 4, supra); and, (b) those samples testing positive in the Screening Assay were subject to testing both: (i) in the in-house GC/MS confirmation assay (EXAMPLE 4), i.e., using comminuted samples and a 2 hour extraction period; and for comparison, (ii) in a commercial laboratory that uses cut samples and a 2-3 hour (or overnight) extraction period in an acidic extraction solution. Of the 67 samples so tested, 32 were determined to produce a positive result in the screening immunoassay; of these 32, the in-house GC/MS confirmation assay indicated that 17 were positive; and, all of the 17 positive were also shown to be positive by the commercial laboratory. Thus, the screening assay which is completed in less than about 70 minutes, (i.e., less than 2 minutes comminuting, 30 minutes extraction and approximately 15 minutes to detect signal in the immunoassay), successfully eliminated half of the test samples as being negative. For the 32 samples determined to be positive in the screening assay, it was subsequently shown in the in-house and commercial GC MS confirmation assays that 15 were in fact negative, i.e., a false positive rate of about 47% which is highly desirable in a screening assay where a confirming assay is also mandatory and where it is desirable that the sensitivity of the assay be such that no possible drug-user escapes detection. Importantly, overall agreement in the identification of true positive test samples was 17/17 (100%) between the in-house confirmation test and the commercial test laboratory.

EXAMPLE 9 Comminuted Extracts: Other Alternative GC/MS Methods. Analytes extracted into organic solvents such as methanol are amenable to derivatization procedures useful in Tandem GC/MS assay formats. Certain of the inventors have disclosed methods for supercritical fluid extraction (SFE), online derivatization and Tandem GC/MS detection of analytes from biological samples in co-pending U.S. Patent Application Serial No. 60/381,109, incorporated herein by reference in its entirety. Briefly, supercritical C0₂ is useful for extracting analytes from within comminuted biological samples. Analytes are collected from the extracts off-line, i.e., through a restrictor allowing controlled decompression into a liquid such as water or an organic solvent; or, onto the surface of an inert solid phase or through a solid phase such as a filter; or, onto a surface such as glass beads or a polymer such as may subsequently be useful in performing an immunoassay (e.g., as set forth supra). Controlled decompression with simultaneous pressurized solvent trapping is also envisaged, i.e., by pressurizing a solvent stream into the decompressing supercritical extract. Alternatively, in-line collection methods may also be employed including collection of derivatized and non-derivatized drug analytes and chemical analytes from SFE fluids into column matrices such as commonly used in GC/MS, e.g. SPE cartridges and the like. Collection is preferably accomplished by adjusting the temperature and pressure of the restrictor, preferably, to about 40°C to about 50°C, and any extract collection vials, preferably, to about 4°C to about 10°C, so as to minimize clogging of the restrictor, as well as, decreasing potential evaporative and aerosol losses analytes. In situations where solid phase trapping is advantageous, analytes may subsequently be eluted from the trap for use in analytical assays such as immunoassays and GC/MS. Representative examples of the subject solid phase traps include silica, cellulose, anion and cation exchange resins, octyldecylsilane (ODS), Tenax, Porapak-Q, silica bonded with diol, florisil, basic and neutral alumina, charcoal and the like. Preferably, the subject solid phase traps are contained within about a 1 ml volume collector cartridge. Representative examples of solvents that may be used to elute analytes from solid phase traps include acetone, methanol, ethyl acetate, acetonitrile and the like. Elution is not absolutely required since it is envisaged that certain analytical assays, e.g., immunoassays, may be conducted either in-line through the trap; or alternatively, on one or more surfaces within the trap; or, on solid phase samples removed from the trap for analysis. In one presently preferred embodiment a trap housing is a clear plastic polymer or glass collector cartridge and the subject analytical assay is conducted by visually inspecting the cartridge for the presence of a detectable signal product from a signal generating compound housed within the trap solid phase material. For purposes of protecting the subject glass or plastic against temperature and pressure, it may optionally be contained within a stainless steel sleeve housing.

Citations

1. Baumgartner, A.M., Jones, P.F., Baumgartner, W.A., Black, C.T. 1979. J. Nucl. Med. 20: 748-752. 2. Kidwell, D. A.; Blank, D. L. In Drug Testing in Hair, Kintz, P., ed.; CRC Press, Inc.: Boca Raton, FL, 1996, Chapter 2

3 Cone,EJ.; Josephs,R.D. 1996. In: Drug Testing in Hair. Ed. KintzJP. CRC Press, Boca Raton, FL.

4. Morrison.J.F.; MacCrehan,W.A. 1994. Poster F-16 Proceedings 5th International Symposium on Supercritical Fluid Chromatography, Baltimore, MD 5. Morrison, J.F.; Chesler, S.N.; Yoo, W.J.; Selavka, CM. . 1998. Anal. Chem. 70, 163-167.

6. DuPont, R.L. and Baumgartner, W.A. 1995. Forensic Sci. Int. 70 (1-3): 63-76.

Claims

WE CLAIM:

1. In a method for detecting an analyte in a solid biological sample, the step of comminuting the biological sample to obtain a particle size in the range of 100 μm to 2000 μm, wherein the analyte is selected from the group comprising a drug analyte, a chemical analyte, a pesticide, a herbicide and a steroid; and, the solid biological sample is selected from the group comprising a hair sample, a nail sample, a talon sample, a beak sample, a hoof sample, a feather sample, a scale sample, a tear sample, a saliva sample, a dry sweat sample, a dry mucous sample, a tissue sample, a sample of clothing, a filtered sample of air or water containing organic material from a living or dead animal, a dry urine sample, a dry blood sample, a dry plasma sample, a dry serum sample, a dry cerebrospinal fluid sample, a dry semen sample, a dry lung lavage fluid sample, a dry feces sample, a dry sputum sample, an insect sample and a plant tissue sample.

2. The method of Claim 1, wherein the particle size is selected from within the range of 80 μm to 500 μm.

3. The method of Claim 2, wherein the particle size is selected from within the range of 80 μm to 400 μm.

4. The method of Claim 3, wherein the particle size is selected from within the range of 80 μm to 200 μm.

5. The method of Claim 1, wherein the drug analyte comprises a drug of abuse selected from the group comprising morphine, opium, cocaine, codeine, amphetamine, methamphetamine, MDMA, THC, β-agonists, steroids and their metabolites, cotinine, ETG, and nicotine.

6. The method of Claim 1, wherein the pesticide comprises a compound selected from the group comprising organochlorine compounds (OCCs) including chlorophenols,, polychlorinated biphenyl compounds (PCB), sulfonylureas, and the like such as, DDT, atrazine, dieldrin, carbofuran, 2,4-(dichlorophenoxy)acetic acid (2,4-D), 2,4- (dichlorophenoxy)butanoic acid (2,4-DB), 2,4-dichlorophenol (2,4-DCP), 4-nonylphenol (4-NP), chlorophos, methamidophos, dichlrovos, methamidophos, mevinphos, acephate, tetrahydrophthalimide, pentachlorobenzyne, o-phenylphennol, omethoate, propoxur, diphenylamine, chloropropham, trifluralin, phorate, hexachlorobenzyne, dicloran, dimethoate, carbofuran, atrazine, quintozene, lindane, terbufos, diazinon, chlorothalonil, disulfoton, phosphamidon, vinclozolin, parathion-methyl, carbaryl, malathion, chloropyrifos, aldrin, dacthal, parathion, dicofol, captan, methidathion, disulfoton sulfone, endosulfan, fenamiphos, myclobutanil, endosulfan, ethion, propargite, iprodione, phosmet, methoxychlor, phosalone, azinphos-methyl, permethrin, cyfluthrin, cypermethrin, fenvalerate, antrhacene, chrysene and their glucosides and metabolites.

7. The method of Claim 1, wherein the herbicide comprises a compound selected from the group comprising alachlor, bromacil, hexazinone, metolachlor and metribuzin.

8. The method of Claim 1, wherein the steroid comprises a compound selected from the group comprising aldosterone, androsterone, cholecalciferol, testosterone, nortestosterone, methyl-testosterone, cortisol, cortisone, ergocalciferol, esfradiol, estrone, lanosterol, progesterone and their metabolites and glucuronides.

9. The method of Claim 1, comprising the additional step of extracting the analyte from the comminuted biological sample into an extraction solution.

10. The method of Claim 9, wherein the extraction solution is selected from the group comprising an alkaline extraction solution, an acidic extraction solution, an organic extraction solution, an enzyme solution, a detergent solution and a supercritical fluid solution.

11. The method of Claim 10, wherein the alkaline extraction solution comprises potassium hydroxide at a pH selected from within the range of pH 11 to pH 12 and the extraction of the analyte is accomplished in about 30 minutes at a temperature selected from within the range of 80°C to 88°C.

12. The method of Claim 10, wherein the organic extraction solution comprises methanolic HCl and the extraction of the analyte is accomplished with sonication in about 30 minutes at a temperature selected from within the range of 60°C to 65°C.

13. The method of Claim 1, wherein the method of detecting the analyte comprises an assay selected from group comprising an immunoassay, a gas chromatographic assay, an HPLC assay and a mass spectrometric assay.

14. The method of Claim 13, wherein the immunoassay method of detecting the analyte comprises a homogeneous immunoassay format, a heterogeneous imunoassay format, a competition assay format, a simultaneous immunoassay format, an indirect immunoassay format, a uniform assay format, a mixed assay format, a combined simultaneous assay format and a cascade immunoassay format.

15. The method of Claim 14, wherein the immunoassay method further comprises an enzyme-linked immunoassay, a radioimmunoassay, a fluorescent immunoassay, a chemilluminescent immunoassay, an electrical capacitance change immunoassay, an electrical resistance change immunoassay, an electrical voltage change immunoassay and an electrical current change immunoassay.

16. A method for detecting an analyte in a solid biological sample, comprising the steps of: comminuting the solid biological sample to obtain a particle size selected from within the range of 100 μm to 2000 μm; extracting greater than 60% of the analyte from the comminuted biological sample in less than 60 minutes; and, processing the extract solution in a manner effective to detect the analyte in the extract solution.

17. The method of Claim 16, wherein the extraction step further comprises contacting the comminuted biological sample with an extraction solution selected from the group comprising an organic solvent, an acidic solution, an alkaline solution, an enzyme solution, a detergent solution and a supercritical fluid solution.

18. The method of Claim 17, wherein the extraction solution comprises an organic solvent and the method comprises the processing step of taking to dryness the organic extract solution prior to detecting the analyte.

19. The method of Claim 16, wherein the biological sample comprises human hair, the sample size is less than 20 mg, the comminuting step is completed in less than

2 minutes, the extract solution is an acidic organic solution, the extract volume is less than 5 ml and the extracting step is completed in less than 30 minutes.

20. A kit for comminuting, extracting and detecting an analyte in a solid biological sample comprising: (a) a single-use device selected from the group comprising a hair collection device, a sample comminuting cup and an extraction vessel; and, (b) one or more kit components selected from the group consisting of a container having an extraction fluid; a container having one or more Ligand Binding Partners; a container having a Signal Generating Compound; a container having a Ligand Binding Partner-Conjugate; a container having a Capture Reagent; a container having a Detect Reagent; a container having a Solid Phase; a container having Diagnostic Reagent; a machine packet; a container having a reagent useful in gas chromatography or mass spectrometry; a container having a control analyte; instructions for conducting an Assay Format to detect one or more analytes in a comminuted biological sample; and, instructions and control reagents for assessing the Specificity, Sensitivity or Precision of an assay conducted on a comminuted biological sample.

21. An on-site screening assay method for preparing a solid biological sample from a test subject, extracting an analyte into an extraction solution in a comminuting vessel and detecting the analyte in the extraction solution, comprising the steps of: collecting the biological sample from the test subject; washing the biological sample for a time and under conditions effective to remove environmental contaminants; comminuting the biological sample for a time and under conditions effective to produce a powder; extracting the comminuted biological sample in an extraction solution in a manner effective to liberate the analyte; processing the extract solution in a manner effective to enable detection of the analyte; and, detecting the analyte in the processed extract solution, wherein the steps of collecting, comminuting, extracting and processing are all conducted in the comminuting vessel, the detecting step is conducted outside the comminuting vessel and the screening assay is suitable for on-site use.

22. An improved method for testing a hair sample to detect an analyte in a hair sample, comprising the step of comminuting the hair sample for less than 5 minutes and extracting the comminuted hair sample for less than 60 minutes.

23. The improved method of claim 22, wherein the comminuting step is conducted for less than 2 minutes and the extraction step is conducted for less than 30 minutes.

24. A device for comminuting a solid biological sample to allow detection of an analyte in the solid biological sample in a detection assay, comprising a processing vessel with a proximal screen, a ram, a cutting-foil comprising a plurality of ridges effective to shear the solid biological sample and a distal test sample cup, wherein: the hair sample is retained within' the processing vessel; the ram is effective to push the hair sample in the processing vessel through the screen and engage the plurality of ridges in the cutting-foil thereby shearing the solid biological sample and producing a plurality of sample particles; the sample particles are retained in the distal sample cup; the screen comprises a plurality of holes each having a diameter of about 0.025 inches and the cutting-foil ridges comprise a ridge thickness selected from with the range of 0.0020 inches to 0.0025 inches; and, the device is capable of comminuting the biological sample into particles having a size of about 80μm to about 2000μm in less than 5 minutes and retaining the samples in the distal test sample cup in a manner suitable for collection by an operator; and, in a form suitable for detection of the analyte in the detection assay.

25. The device of Claim 24, wherein the detection assay is selected from the group consisting of an immunoassay, an HPLC assay, an atomic mass spectral assay and a GC/MS assay.

26. An automated method for processing a solid biological sample containing an analyte of interest to collect an extract that is suitable for detection of the analyte, comprising the steps of: collecting a biological sample into a chamber of a collection device having a screen with a plurality of particle-sized holes; engaging the collection device into a comminuting device comprising a cutting foil having a plurality of cutting ridges, wherein the screen in the collection device separates the biological sample in the collection device chamber from the cutting foil in the comminuting device and engaging the collection device into the comminuting device brings the chamber of the collection device into an interruptable fluid communication with a washing reservoir, an extraction reservoir, an extract processing reservoir and an analyte detection chamber; washing the biological sample in the collection device chamber by opening the fluid communication to a wash solution in the wash reservoir; comminuting the washed biological sample by bringing the biological sample into contact with the ridges of the cutting foil through the screen; extracting the resultant particle-sized test sample by bringing it into fluid communication with an extract solution in the extraction reservoir under conditions and for a time sufficient to enable extraction of the analyte; processing the extract solution in a manner effective to allow detection of the analyte by bringing the extract solution into fluid communication with an processing solution in the processing reservoir; and, collecting the processed extract into the analyte detection chamber by bringing the processed extract solution into fluid communication with the analyte detection chamber.

27. The method of Claim 26, wherein the plurality of particle-sized holes in the screen comprise holes having a diameter of about 0.025 inches.

28. The method of Claim 26, wherein the plurality of ridges in the cutting foil comprise ridges having a ridge height of about 0.025 inches.

29. The method of Claim 26, wherein the analyte detection chamber comprises a vessel selected from the group consisting of a test tube, a vial, a cuvette, a well in a microtiter assay plate and an immunoassay dipstick.

30. The method of Claim 26, wherein the analyte detection chamber comprises a chamber in a detection device comprising an automated immunoassay analyzer.

1. The method of Claim 26, wherein the analyte detection chamber comprises an analyte trap for a gas chromatography or a mass spectrometric analyzer.