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WO2011068654A1 - Détection de vitamine d par spectrométrie de masse avec désorption thermique par diode laser - Google Patents

Détection de vitamine d par spectrométrie de masse avec désorption thermique par diode laser Download PDF

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WO2011068654A1
WO2011068654A1 PCT/US2010/056461 US2010056461W WO2011068654A1 WO 2011068654 A1 WO2011068654 A1 WO 2011068654A1 US 2010056461 W US2010056461 W US 2010056461W WO 2011068654 A1 WO2011068654 A1 WO 2011068654A1
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ions
mass
vitamin
hydroxyvitamin
derivatized
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Nigel J. Clarke
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Quest Diagnostics Investments LLC
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Quest Diagnostics Investments LLC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • the invention relates to the detection of vitamin D metabolites.
  • the invention relates to mass spectrometry methods for detecting vitamin D metabolites ionized by a laser diode thermal desorption ionizer.
  • Vitamin D is an essential nutrient with important physiological roles in the positive
  • Vitamin D can be made de novo in the skin by exposure to sunlight or it can be absorbed from the diet.
  • vitamin D 2 ergocalciferol
  • vitamin D 3 cholecalciferol
  • Vitamin D 3 is the form synthesized de novo by animals. It is also a common supplemented added to milk products and certain food products produced in the United States. Both dietary and intrinsically synthesized vitamin D 3 must undergo metabolic activation to generate the bioactive metabolites. In humans, the initial step of vitamin D 3 activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxycholecalciferol (calcifediol; 250HD 3 ).
  • Calcifediol is the major form of Vitamin D 3 in the circulation. Circulating 250HD 3 is then converted by the kidney to form 1,25-dihydroxyvitamin D 3 (calcitriol; l,25(OH) 2 D 3 ), which is generally believed to be the metabolite of Vitamin D 3 with the highest biological activity.
  • Vitamin D 2 is derived from fungal and plant sources. Many over-the-counter dietary supplements contain ergocalciferol (vitamin D 2 ) rather than cholecalciferol (vitamin D 3 ).
  • Drisdol the only high-potency prescription form of vitamin D available in the United States, is formulated with ergocalciferol.
  • Vitamin D 2 undergoes a similar pathway of metabolic activation in humans as Vitamin D 3 , forming the metabolites 250HD 2 and l,25(OH) 2 D 2 .
  • Vitamin D 2 and vitamin D 3 have long been assumed to be biologically equivalent in humans, however recent reports suggest that there may be differences in the bioactivity and bioavailability of these two forms of vitamin D (Armas et. al., (2004) J. Clin. Endocrinol. Metab. 89:5387-5391).
  • Serum levels of 25 -hydro xyvitamin D3, 25-hydroxyvitamin D 2 , and total 25- hydroxyvitamin D are useful indexes of vitamin D nutritional status and the efficacy of certain vitamin D analogs.
  • the measurement of 250HD is commonly used in the diagnosis and management of disorders of calcium metabolism. In this respect, low levels of 250HD are indicative of vitamin D deficiency associated with diseases such as hypocalcemia,
  • hypophosphatemia secondary hyperparathyroidism, elevated alkaline phosphatase, osteomalacia in adults and rickets in children.
  • elevated levels of 250HD distinguishes this disorder from other disorders that cause hypercalcemia.
  • Measurement of l,25(OH) 2 D is also used in clinical settings, however, this test has a more limited diagnostic usefulness than measurements of 250HD.
  • Factors that contribute to limitations of the diagnostic values of l,25(OH) 2 D as an index of Vitamin D status include the precision of the endogenous regulation of renal production of the metabolite and its short half- life in circulation.
  • certain disease states can be reflected by circulating levels of l,25(OH) 2 D, for example kidney disease and kidney failure often result in low levels of l,25(OH) 2 D. Elevated levels of l,25(OH) 2 D may be indicative of excess parathyroid hormone or can be indicative of certain diseases such as sarcoidosis or certain types of lymphomas.
  • the amount of one or more vitamin D metabolites in a sample is determined by mass spectrometry methods which comprise volatizing a sample by heating the sample with a laser.
  • one or more vitamin D metabolites in the volatized sample are ionized to produce one or more ions detectable by mass spectrometry; and detecting the amount of one or more of the ions by mass spectrometry.
  • the amount of the detected ion or ions is related to the amount of one or more vitamin D metabolites in the sample.
  • the sample is not subjected to chromatography, including liquid chromatography (such as HPLC or TFLC), prior to mass spectrometry.
  • the sample may be subjected to chromatography, including liquid
  • the sample comprises a biological sample.
  • the biological sample comprises plasma or serum.
  • the sample is dried prior to heating with a laser.
  • the one or more ions detectable by mass spectrometry are ionized with atmospheric pressure chemical ionization (APCI).
  • APCI atmospheric pressure chemical ionization
  • the mass spectrometry is tandem mass spectrometry.
  • the one or more vitamin D metabolites comprises 25- hydroxyvitamin D 3 .
  • the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 383.2 ⁇ 0.5, 257.1 ⁇ 0.5, and 211.1 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to- charge ratio of 383.2 ⁇ 0.5 and one or more fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 257.1 ⁇ 0.5 and 211.1 ⁇ 0.5.
  • the one or more vitamin D metabolites comprises 25- hydroxyvitamin D 2 .
  • the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 395.3 ⁇ 0.5, 251.1 ⁇ 0.5, 209.1 ⁇ 0.5, and 179.1 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 395.3 ⁇ 0.5 and one or more fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 251.1 ⁇ 0.5, 209.1 ⁇ 0.5, and 179.1 ⁇ 0.5.
  • the one or more vitamin D metabolites comprise 25- hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 .
  • 25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 are volatilized simultaneously.
  • the one or more vitamin D metabolites comprises 1,25- dihydroxyvitamin D 2 .
  • the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 375.1 ⁇ 0.5, 393.1 ⁇ 0.5, 411.1 ⁇ 0.5, 105.3 ⁇ 0.5, 156.9 ⁇ 0.5, and 135.3 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 375.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 105.3 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 393.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 156.9 ⁇ 0.5.
  • a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 375.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 105.3 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 411.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 135.3 ⁇ 0.5.
  • the one or more vitamin D metabolites comprises 1,25- dihydroxyvitamin D 3 .
  • the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 363.1 ⁇ 0.5, 381.1 ⁇ 0.5, 399.1 ⁇ 0.5, 156.8 ⁇ 0.5, 157.0 ⁇ 0.5, and 158.8 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 363.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 156.8 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 381.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 157.0 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 399.1 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 158.8 ⁇ 0.5.
  • the one or more vitamin D metabolites comprise 1,25- dihydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 2 .
  • 1,25- dihydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 2 are volatilized simultaneously.
  • the one or more vitamin D metabolites are derivatized with a Cookson-type reagent prior to volatilization of the sample to generate one or more derivatized vitamin D metabolites.
  • the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise one, two, three, or four vitamin D metabolites selected from the group consisting of 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3; 1,25- dihydroxyvitamin D 2 , and 1,25-dihydroxyvitamin D 3 .
  • the one or more vitamin D metabolites derivatized with a Cookson-type reagent are 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3, 1,25-dihydroxyvitamin D 2 , and 1,25-dihydroxyvitamin D 3 .
  • the one or more derivatized vitamin D metabolites comprise one, two, three, or four derivatized vitamin D metabolites selected from the group consisting of 4-phenyl- l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D 2 , 4-phenyl-l,2,4-triazoline- 3,5-dione (PTAD) derivatized 25-hydroxyvitamin D 3 , 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D 2 , and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D 3 .
  • PTAD 4-phenyl- l,2,4-triazoline-3,5-dione
  • the one or more derivatized vitamin D metabolites are 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25- hydroxyvitamin D 2 , 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D 3 , 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D 2 , and 4- phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D 3 .
  • PTAD 4-phenyl-l,2,4-triazoline-3,5-dione
  • the one or more derivatized vitamin D metabolites may be volatized at the same time.
  • the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 558.3 ⁇ 0.5 and 298.1 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 558.3 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 298.1 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 570.3 ⁇ 0.5 and 298.1 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 570.3 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 298.1 ⁇ 0.5.
  • the one or more vitamin D metabolites derivatized with a Cookson-type reagent are selected from the group consisting of 25-hydroxyvitamin D 2 , 25- hydroxyvitamin D 3 , 1,25-dihydroxyvitamin D 2 , and 1,25-dihydroxyvitamin D 3 .
  • the Cookson-type reagent is 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD).
  • the one or more derivatized vitamin D metabolites are selected from the group consisting of PTAD derivatized 25-hydroxyvitamin D 2 , PTAD derivatized 25- hydroxyvitamin D 3 , PTAD derivatized 1,25-dihydroxyvitamin D 2 , and PTAD derivatized 1,25- dihydroxyvitamin D 3 .
  • the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to- charge ratio of 550.4 ⁇ 0.5, 568.4 ⁇ 0.5, 586.4 ⁇ 0.5, 227.9 ⁇ 0.5, 298.0 ⁇ 0.5, and 314.2 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 550.4 ⁇ 0.5 and a fragment ion with a mass-to- charge ratio of 227.9 ⁇ 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 568.4 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 298.0 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 586.4 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 314.2 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to- charge ratio of 538.4 ⁇ 0.5, 556.4 ⁇ 0.5, 574.4 ⁇ 0.5, 278.1 ⁇ 0.5, 298.0 ⁇ 0.5, and 313.0 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 538.4 ⁇ 0.5 and a fragment ion with a mass-to- charge ratio of 278.1 ⁇ 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 556.4 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 298.0 ⁇ 0.5.
  • the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 574.4 ⁇ 0.5 and a fragment ion with a mass-to-charge ratio of 313.0 ⁇ 0.5.
  • the one or more vitamin D metabolites comprise 1,25- dihydroxyvitamin D 2 and 1,25-dihydroxyvitamin D3.
  • the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D 2 and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D3.
  • PTAD derivatized 1,25-dihydroxyvitamin D 2 and PTAD derivatized 1,25-dihydroxyvitamin D3 are volatilized simultaneously.
  • mass spectrometry is performed in positive ion mode.
  • mass spectrometry is performed in negative ion mode.
  • the preferred ionization technique used in methods described herein is a laser diode thermal desorption ionization technique.
  • one or more separately detectable internal standards is provided in the sample, the amount of which is also determined in the sample.
  • all or a portion of both the analyte of interest and the one or more internal standards is ionized to produce a plurality of ions detectable in a mass spectrometer, and one or more ions produced from each are detected by mass spectrometry.
  • exemplary compounds suitable for use as internal standards include 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- 3 ⁇ 4 and 25- hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 3 ⁇ 4.
  • ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 401.3 ⁇ 0.5, 251.1 ⁇ 0.5, 209.1 ⁇ 0.5, and 179.1 ⁇ 0.5.
  • a 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion has m/z of 401.3 ⁇ 0.5, and one or more fragment ions are selected from the group of ions with m/z of 251.1 ⁇ 0.5, 209.1 ⁇ 0.5, and 179.1 ⁇ 0.5.
  • ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 389.3 ⁇ 0.5, 257.1 ⁇ 0.5, and 211.1 ⁇ 0.5.
  • a 25-hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 3 ⁇ 4 precursor ion has m/z of 389.3 ⁇ 0.5, and one or more fragment ions are selected from the group of ions with m/z of 257.1 ⁇ 0.5 and 211.1 ⁇ 0.5.
  • exemplary compounds suitable for use as internal standards may include vitamin D analogues that are not isotopic variants of 25-hydroxyvitamin D 2 or 25-hydroxyvitamin D 3 .
  • the presence or amount of ions generated from the analyte of interest may be related to the presence of amount of analyte of interest in the sample.
  • the amount of the one or more vitamin D metabolite ion or ions may be determined by comparison to one or more external reference standards.
  • Exemplary external reference standards include blank plasma or serum spiked with 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 , 25-hydroxyvitamin D 3 , and 25- hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 2 H 6 -
  • vitamin D metabolite refers to any vitamin D analog or any chemical species related to vitamin D.
  • Vitamin D metabolites may include analogs of, or a chemical species related to, vitamin D 2 or vitamin D 3 .
  • Vitamin D metabolites may be found in the circulation of an animal and/or may be generated by a biological organism, such as an animal, or by biotransformation of vitamin D 2 or vitamin D 3 .
  • Vitamin D metabolites may be metabolites of naturally occurring forms of vitamin D or may be metabolites of synthetic vitamin D analogs.
  • a vitamin D metabolite is one or more compounds selected from the group consisting of 25-hydroxyvitamin D 3 , 25-hydroxyvitamin D 2 , 1,25- dihydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 2 .
  • a derivatizing agent is an agent that may be reacted with another substance to derivatize the substance.
  • PTAD 4-phenyl-l,2,4-triazoline-3,5-dione
  • PTAD is a derivatizing reagent that may be reacted with a vitamin D metabolite to form a PTAD-derivatized vitamin D metabolite.
  • the names of derivatized forms of vitamin D metabolites include an indication as to the nature of derivatization.
  • the PTAD derivative of 25- hydroxyvitamin D 2 is indicated as PTAD derivatized 25- hydroxyvitamin D 2 , PTAD-25- hydroxyvitamin D 2 , or PTAD-250HD 2 .
  • a "Cookson-type derivatizing agent” is a 4-substituted 1,2,4-triazoline- 3,5-dione compound.
  • Exemplary Cookson-type derivatizing agents include 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4- dihydroquinoxalyl)ethyl]- 1 ,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)- 1 ,2,4- triazoline-3,5- dione (NPTAD), and 4-ferrocenylmethyl-l,2,4-triazoline-3,5-dione (FMTAD). Additionally, isotopically labeled variants of Cookson-type derivatizing agents may be used in
  • the C 6 -PTAD isotopic variant is 6 mass units heavier than normal PTAD and may be used in some embodiments.
  • Derivatization of vitamin D metabolites by Cookson-type reagents can be conducted by any appropriate method. See, e.g., Yeung B, et al, J Chromatogr. 1993, 645(1): 115-23; Higashi T, et al, Steroids. 2000, 65(5):281-94; Higashi T, et al, Biol Pharm Bull. 2001, 24(7):738-43; Higashi T, et al, J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T, et al, Anal. Biochanal Chem, 2008, 391 :229-38; and Aronov, et al, Anal Bioanal Chem, 2008, 391 : 1917-30.
  • purification does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest.
  • Purification of the sample by various means may allow relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, is entirely removed by purification.
  • sample refers to any sample that may contain an analyte of interest.
  • body fluid means any fluid that can be isolated from the body of an individual.
  • body fluid may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like.
  • the sample comprises a body fluid sample; preferably plasma or serum.
  • solid phase extraction refers to a process in which a chemical mixture is separated into components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) for a solid through or around which the solution is passed (i.e., solid phase).
  • a solution i.e., mobile phase
  • the solid phase may be retained by the solid phase resulting in a purification of the analyte in the mobile phase.
  • the analyte may be retained by the solid phase, allowing undesired components of the mobile phase to pass through or around the solid phase.
  • a second mobile phase is then used to elute the retained analyte off of the solid phase for further processing or analysis.
  • SPE including utilization of a turbulent flow liquid chromatography (TFLC) column as an extraction column, may operate via a unitary or mixed mode mechanism.
  • Mixed mode mechanisms utilize ion exchange and hydrophobic retention in the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention; or may exhibit column exhibit strong cation exchange and hydrophobic retention.
  • chromatography refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.
  • liquid chromatography means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • separation techniques which employ "liquid chromatography” include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes known as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).
  • RPLC reverse phase liquid chromatography
  • HPLC high performance liquid chromatography
  • TFLC turbulent flow liquid chromatography
  • an SPE column may be used in combination with an LC column. For example, a sample may be purified with a TFLC extraction column, followed by additional purification with a HPLC analytical column.
  • HPLC high performance liquid chromatography
  • HPLC high pressure liquid chromatography
  • TFLC turbulent flow liquid chromatography
  • TFLC turbulent flow liquid chromatography
  • TFLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also, U.S. Patents No. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC.
  • laminar flow When fluid flows slowly and smoothly, the flow is called “laminar flow”. For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the motion of the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary "outruns” that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it flows in eddies and whirls (or vortices), with more "drag" than when the flow is laminar.
  • GC gas chromatography
  • the terms “on-line” and “inline”, for example as used in “on-line automated fashion” or “on-line extraction” refers to a procedure performed without the need for operator intervention.
  • the term “off-line” as used herein refers to a procedure requiring manual intervention of an operator.
  • MS mass spectrometry
  • MS refers to an analytical technique to identify compounds by their mass.
  • MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z”.
  • MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means.
  • a “mass spectrometer” generally includes an ionizer and an ion detector.
  • one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).
  • m mass
  • z charge
  • the term "operating in negative ion mode” refers to those mass spectrometry methods where negative ions are generated and detected.
  • the term "operating in positive ion mode” as used herein, refers to those mass spectrometry methods where positive ions are generated and detected.
  • the term "ionization” or “ionizing” refers to the process of generating an ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.
  • the term "desorption” refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase.
  • Laser diode thermal desorption (“LDTD") is a technique wherein a sample containing the analyte is thermally desorbed into the gas phase by a laser pulse.
  • the laser is directed to the back of a specially made 96-well plate with a metal base, wherein the laser pulse heats the base and the heat causes the analyte to transfer into the gas phase.
  • the analyte may be pre-dried onto the base of the plate prior to heating with the laser, or may be in solution at the beginning of the heating process.
  • LDTD ionization technique refers to a method for ionizing an analyte (e.g., a vitamin D metabolite) in which LDTD is used to volatilize an analyte, which is then ionized by any suitable technique.
  • the volatilized sample is ionized via atmospheric pressure chemical ionization; that is, the volatilized analyte is drawn past a corona discharge and ionized before being drawn into the mass spectrometer.
  • An exemplary ionization source comprising LDTD is described in detail in US Patent No. 7,321,116 (filed 5/20/05), incorporated herein by reference in its entirety.
  • selective ion monitoring is a detection mode for a mass spectrometric instrument in which only ions within a relatively narrow mass range, typically about one mass unit, are detected.
  • multiple reaction mode is a detection mode for a tandem mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.
  • the term "lower limit of quantification”, “lower limit of quantitation” or “LLOQ” refers to the point where measurements become quantitatively meaningful.
  • the analyte response at this LOQ is identifiable, discrete and reproducible with a relative standard deviation (RSD %) of less than 20% and an accuracy of 85% to 115%.
  • limit of detection is the point at which the measured value is larger than the uncertainty associated with its measurement.
  • the LOD is defined as three times the RSD of the mean at the zero concentration.
  • an “amount" of an analyte in a body fluid sample refers generally to an absolute value reflecting the mass of the analyte detectable in volume of sample. However, an amount also contemplates a relative amount in comparison to another analyte amount. For example, an amount of an analyte in a sample can be an amount which is greater than a control or normal level of the analyte normally present in the sample.
  • Figures 1 A-D show exemplary chromatograms for 25-hydroxyvitamin D 2 , 25- hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 (internal standard), 25-hydroxyvitamin D 3 , and 25-hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 (internal standard), respectively. Details are discussed in Example 3.
  • Figure 2 shows a plot of analytical results for calibrator solutions of various
  • Figure 3 shows a plot of analytical results for calibrator solutions of various
  • Figure 4A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 2 ions.
  • Figure 4B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D 2 precursor ion with m/z of about 395.2. Details are described in Example 5.
  • Figure 5 A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 2 -[6, 19, 19]- ⁇ 3 ions.
  • Figure 5B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25- hydroxyvitamin D 2 -[6, 19, 19]- H 3 precursor ion with m/z of about 398.2. Details are described in Example 5.
  • Figure 6A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 ions.
  • Figure 6B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 401.2. Details are described in Example 5.
  • Figure 7A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 3 ions.
  • Figure 7B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D 3 precursor ion with m/z of about 383.2. Details are described in Example 5.
  • Figure 8A shows an exemplary Ql scan spectrum (covering the m/z range of about 350
  • Figure 8B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25- hydroxyvitamin D 3 -[6, 19, 19]- H 3 precursor ion with m/z of about 386.2. Details are described in Example 5.
  • Figure 9A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 ions.
  • Figure 9B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25-hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 389.2. Details are described in Example 5.
  • Figure 10A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamin D 2 ions.
  • Figure 10B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 - hydroxyvitamin D 2 precursor ion with m/z of about 570.3. Details are described in Example 6.
  • Figure 11A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D 2 -[6, 19, 19]- ⁇ 3 ions.
  • Figure 1 IB shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD- 25 -hydroxyvitamm D 2 -[6, 19, 19]- H 3 precursor ion with m/z of about 573.3. Details are described in Example 6.
  • Figure 12A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D 2 -[26, 26, 26, 27, 27, 27]- 2 H 6 ions.
  • Figure 12B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 -hydroxyvitamm D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 576.3. Details are described in Example 6.
  • Figure 13A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D 3 ions.
  • Figure 13B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 - hydroxyvitamm D 3 precursor ion with m/z of about 558.3. Details are described in Example 6.
  • Figure 14A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D 3 -[6, 19, 19]- ⁇ 3 ions.
  • Figure 14B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD- 25 -hydroxyvitamm D 3 -[6, 19, 19]- H 3 precursor ion with m/z of about 561.3. Details are described in Example 6.
  • Figure 15A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D 3 -[26, 26, 26, 27, 27, 27]- 2 H 6 ions.
  • Figure 15B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 -hydroxyvitamm D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 564.3. Details are described in Example 6.
  • Figure 16A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D 2 ions.
  • Figure 16B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 2 precursor ion with m/z of about 375.1.
  • Figure 16C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 2 precursor ion with m/z of about 393.1.
  • Figure 16D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 2 precursor ion with m/z of about 411.1. Details are described in Example 7.
  • Figure 17A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 ions.
  • Figure 17B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 381.1.
  • Figure 17C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 399.1.
  • Figure 17D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- 2 H 6 precursor ion with m/z of about 417.1. Details are described in Example 7.
  • Figure 18A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D 3 ions.
  • Figure 18B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 precursor ion with m/z of about 363.1.
  • Figure 18C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 precursor ion with m/z of about 381.1
  • Figure 18D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 precursor ion with m/z of about 399.1. Details are described in Example 7.
  • Figure 19A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- ⁇ 3 ions.
  • Figure 19B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25- dihydroxyvitamin D 3 -[6, 19, 19]- H3 precursor ion with m/z of about 366.1.
  • Figure 19C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- H 3 precursor ion with m/z of about 384.1.
  • Figure 19D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for 2
  • Figure 20A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 ions.
  • Figure 20B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 369.1.
  • Figure 20C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 387.1.
  • Figure 20D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 2 H 6 precursor ion with m/z of about 405.1. Details are described in Example 7.
  • Figure 21 A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD- 1,25-dihydroxyvitamin D 2 ions.
  • Figure 21B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1,25- dihydroxyvitamin D 2 precursor ion with m/z of about 550.4.
  • Figure 21C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 2 precursor ion with m/z of about 568.4.
  • Figure 21D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 2 precursor ion with m/z of about 586.4. Details are described in Example 8.
  • Figure 22A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- 2 H 6 ions.
  • Figure 22B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 556.4.
  • Figure 22C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 574.4
  • Figure 22D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 592.4. Details are described in Example 8.
  • Figure 23 A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-hydroxyvitamin D 3 ions.
  • Figure 23 B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1,25- dihydroxyvitamin D 3 precursor ion with m/z of about 538.4.
  • Figure 23C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 3 precursor ion with m/z of about 556.4.
  • Figure 23D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D 3 precursor ion with m/z of about 574.4. Details are described in Example 8.
  • Figure 24A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-dihydroxyvitamin D 3 -[6, 19, 19]- ⁇ 3 ions.
  • Figure 24B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- H 3 precursor ion with m/z of about 541.4.
  • Figure 24C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- H 3 precursor ion with m/z of about 559.4.
  • Figure 24D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- H 3 precursor ion with m/z of about 577.4. Details are described in Example 8.
  • Figure 25 A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 2 H 6 ions.
  • Figure 25B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 544.4.
  • Figure 25C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 562.4.
  • Figure 25D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 precursor ion with m/z of about 580.4. Details are described in Example 8. DETAILED DESCRIPTION OF THE INVENTION
  • Methods are described for measuring the amount of one or more vitamin D metabolites in a sample. More specifically, mass spectrometric methods are described for ionizing one or more vitamin D metabolites and detecting ion thereby produced by mass spectrometry. These methods may include purifying one or more vitamin D metabolites in the sample prior to ionization and mass spectrometry. However, the methods may be performed without purifying the sample with chromatography. In some embodiments, the methods of the invention may be used to detect two or more vitamin D metabolites in a single assay. Preferred embodiments are particularly well suited for application in large clinical laboratories for automated vitamin D metabolite quantification.
  • the vitamin D metabolites may be derivatized prior to mass spectrometric analysis. Derivatization of vitamin D metabolites is known in the art and described, for example, by Aronov, et al, Anal Bioanal Chem, 2008, 391 : 1917-30; Higashi, et al, Anal. Bioanal Chem, 2008, 391 :229-238; and Yeung, et al, J. Chromatog, 1993, 645: 115- 23. Derivatization may occur at any point prior to thermal desorption by LDTD, and the ions monitored are selected depending on the derivatization agent utilized.
  • mass spectrometric detection of 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-OH vitamin D 2 and 25-OH vitamin D 3 may be conducted by monitoring mass transitions (in a MS/MS technique) from 570.3 ⁇ 0.5 to 298.1 ⁇ 0.5 and 558.3 ⁇ 0.5 to 298.1 ⁇ 0.5, respectively.
  • PTAD 4-phenyl-l,2,4-triazoline-3,5-dione
  • Suitable test samples for use in methods of the present invention include any test sample that may contain the analyte of interest.
  • a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc.
  • samples are obtained from a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc.
  • kits for a vitamin D metabolite quantitation assay may include a kit comprising the compositions provided herein.
  • kits may include packaging material and measured amounts of an isotopically labeled internal standard, in amounts sufficient for at least one assay.
  • the kit may include a derivatizing agent, preferably PTAD, in a quantity sufficient for at least one assay.
  • the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for use in a vitamin D metabolite quantitation assay.
  • Quality control (QC) pools having known concentrations are preferably prepared using a matrix similar to the intended sample matrix.
  • Calibration pools for use in embodiments of the present invention may be prepared using a matrix similar to the intended sample matrix, or may be prepared from a vitamin D free matrix containing albumin and phosphate buffered saline solution.
  • vitamin D metabolites may be enriched relative to one or more other components in the sample (e.g. protein) by various methods known in the art, including for example, solid phase extraction (SPE), LC, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including ethyl acetate or methanol extraction, and the use of chaotropic agents or any combination of the above or the like.
  • SPE solid phase extraction
  • LC filtration, centrifugation, thin layer chromatography
  • electrophoresis including capillary electrophoresis
  • affinity separations including immunoaffinity separations
  • extraction methods including ethyl acetate or methanol extraction
  • chaotropic agents any combination of the above or the like.
  • chromatography including LC, is not used.
  • Protein precipitation is one method of preparing a test sample, especially a biological sample, such as serum or plasma.
  • a biological sample such as serum or plasma.
  • Protein purification methods are well known in the art, for example, Poison et ah, Journal of Chromatography B 2003, 785:263-275, describes protein precipitation techniques suitable for use in methods of the present invention.
  • precipitation may be used to remove most of the protein from the sample leaving vitamin D metabolites in the supernatant.
  • the samples may be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively the samples may be filtered to remove precipitated proteins.
  • the resultant supernatant or filtrate may then be applied directly to mass spectrometry analysis; or alternatively to liquid chromatography and subsequent mass spectrometry analysis.
  • liquid-liquid extraction methods including ethyl acetate or methanol extraction are used to extract vitamin D metabolites from the a sample.
  • sample such as between 10 and 250 ⁇ , such as between 10 and 100 ⁇ , such as about 50 ⁇
  • the quantity of extraction solvent is commensurate with sample volume and may vary depending on the extraction solvent used, but is preferably between about 50 and 1000 ⁇ , such as about 200 ⁇ .
  • the sample/solvent mixtures are mixed and centrifuged, and a portion of the supernatant or organic phase (depending on solvent used) is drawn off for further analysis.
  • Solvent may be removed from the drawn off portion, for example under a nitrogen flow, and the residue reconstituted in a different solvent such as methanol. A portion of the resulting solution is then spotted in a well of a LDTD plate.
  • a LDTD well plate is similar to a standard 96-well plate, but has a metal coating at least on the bottom of the plate at each well.
  • Vitamin D metabolites present in the LDTD well plate are then ionized by an ionization technique comprising LDTD.
  • LDTD is described in great detail in U.S. Patent No. 7,321,116 (filed May 20, 2005).
  • thermal desorption is induced indirectly by a laser beam.
  • a laser is directed to the metal coating on the bottom of the plate beneath a well containing a spotted sample.
  • Vitamin D metabolites in the spotted sample desorb from the well and are directed past a corona discharge, creating vitamin D metabolite ions.
  • Ions of multiple vitamin D metabolites e.g., 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3
  • Samples prepared for LDTD according to this method can be thermally desorbed without a support matrix, and ionization achieved without introduction of a liquid mobile phase into the system.
  • the LDTD method described herein differs from MALDI and SELDI, even though all three methods employ a laser.
  • Vitamin D metabolites may be ionized in positive or negative mode. In preferred embodiments, vitamin D metabolites are ionized in positive mode.
  • mass spectrometry techniques generally, after the sample has been ionized, the positively or negatively charged ions thereby created may be analyzed to determine a mass-to- charge ratio.
  • Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion traps analyzers, and time-of-flight analyzers. Exemplary ion trap methods are described in Bartolucci, et ⁇ , Rapid Commun. Mass Spectrom. 2000, 14:967-73.
  • the ions may be detected using several detection modes. For example, selected ions may be detected, i.e. using a selective ion monitoring mode (SIM), or alternatively, mass transitions resulting from collision induced dissociation or neutral loss may be monitored, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
  • MRM multiple reaction monitoring
  • SRM selected reaction monitoring
  • the mass-to-charge ratio is determined using a quadrupole analyzer.
  • ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge ratio.
  • the voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected.
  • quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.
  • a precursor ion also called a parent ion
  • the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure.
  • fragment ions also called daughter ions or product ions
  • the MS/MS technique may provide an extremely powerful analytical tool.
  • the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.
  • Alternate modes of operating a tandem mass spectrometric instrument include product ion scanning and precursor ion scanning. For a description of these modes of operation, see, e.g., E. Michael Thurman, et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez -Alba, ed., Elsevier 2005) (387).
  • the results of an analyte assay may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, external standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, one or more internal standards are used to generate standard curves for calculating the quantity of a vitamin D metabolite.
  • isotopically labeled vitamin D 2 e.g., 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 , or 25-hydroxyvitamin D 2 -[6, 19, 19]- H 3
  • isotopically labeled vitamin D 2 e.g., 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 , or 25-hydroxyvitamin D 2 -[6, 19, 19]- H 3
  • isotopically labeled vitamin D 2 e.g., 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 , or 25-hydroxyvitamin D 2 -[6, 19, 19]- H 3
  • Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.
  • an "isotopic label” produces a mass shift in the labeled molecule relative to the unlabeled molecule when analyzed by mass spectrometric techniques.
  • suitable labels include deuterium ( H), C, and N.
  • 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 has a mass about six units higher than 25-hydroxyvitamin D 2 .
  • the isotopic label can be incorporated at one or more positions in the molecule and one or more kinds of isotopic labels can be used on the same isotopically labeled molecule.
  • One or more steps of the methods may be performed using automated machines.
  • one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed in an on-line fashion.
  • collision activated dissociation CAD
  • precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as "unimolecular decomposition.”
  • Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.
  • vitamin D metabolite ions are in a gaseous state and an inert collision gas is argon or nitrogen; preferably argon.
  • vitamin D metabolites in a sample are detected and/or quantified using MS/MS as follows.
  • the vitamin D metabolites are purified in a sample by liquid-liquid extraction, with the a portion of the resulting solution spotted in a LDTD well.
  • the spotted sample is heated indirectly with a laser which results in thermal desorption of vitamin D metabolites, and the desorbed vitamin D metabolites then pass a corona discharge and are ionized before being introduced into a triple quadrupole MS instrument.
  • Quadrupoles 1 and 3 are mass filters, allowing selection of ions (i.e., selection of "precursor” and “fragment” ions in Ql and Q3, respectively) based on their mass-to-charge ratio (m/z).
  • Quadrupole 2 is a collision cell, where precursor ions are fragmented.
  • the vitamin D metabolite ions e.g. precursor ions
  • the first quadrupole of the mass spectrometer (Ql) selects for molecules with the mass-to-charge ratios of a vitamin D metabolite.
  • Precursor ions with the correct mass/charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated.
  • Precursor ions entering Q2 collide with neutral gas molecules, preferably argon, and fragment.
  • the fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions of a vitamin D metabolite are selected while other ions are eliminated.
  • Q3 quadrupole 3
  • the methods may involve MS/MS performed in either positive or negative ion mode; preferably positive ion mode.
  • MS/MS performed in either positive or negative ion mode; preferably positive ion mode.
  • one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of a vitamin D metabolite that may be used for selection in quadrupole 3 (Q3).
  • Q3 quadrupole 3
  • the areas under the peaks corresponding to particular ions, or the amplitude of such peaks, may be measured and correlated to the amount of the analyte of interest.
  • the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of vitamin D metabolite.
  • the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard.
  • multiple vitamin D metabolites may be determined
  • the amounts of 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 may be determined by mass spectrometric analysis of the ions formed from the volatilization of a single sample.
  • Calibrant solutions were prepared with 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 in a vitamin D metabolite free matrix containing albumin and phosphate buffered saline. Concentration of the calibrant solutions ranged from 4 to 128 ng/mL for each analyte.
  • Serum samples were obtained from Golden West Biologicals (Temecula, CA) and were certified free of common human pathogens (SP1060-VD (defibrinated, delipidized, charcoal stripped and solvent extracted serum) and SP1010 (defibrinated and delipidized, Lots
  • MS/MS was performed using a Thermo Finnigan TSQ Vantage MS/MS system (Thermo Electron Corporation).
  • the ionization source was a Laser Diode Thermal Desorption (LDTD) source from Phytronix Technologies (Quebec, Qc, Canada). Laser intensity varied from 10% to 60% of maximum output during development experiments, and was set to about 25% maximum output for quantitative analysis.
  • LDTD Laser Diode Thermal Desorption
  • Exemplary chromatograms for the four analytes shown in Table 1 are found in Figures 1 A-D. During the three minutes of data collection shown in the chromatograms, five different wells were lased/ionized under different laser intensity settings to find the optimal laser intensity setting for the desired analytes.
  • Example 5 Exemplary spectra from LDTD-MS/MS analysis of 25-hydroxyvitamin D? and 25- hydroxyvitamin D 3
  • Exemplary Ql scan spectra from the analysis of samples containing 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 2 -[6, 19, 19]- 2 H 3 , and 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 are shown in Figures 4A, 5A, and 6A, respectively. These spectra were collected by scanning Ql across a m/z range of about 350 to 450.
  • An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D 2 is fragmenting a precursor ion with a m/z of about 395.2 to a product ion with a m/z of about 208.8.
  • An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D 2 -[6, 19, 19]- H 3 is fragmenting a precursor ion with a m/z of about 398.2 to a product ion with a m/z of about 211.9.
  • a preferred MRM transition for the quantitation of 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 is fragmenting a precursor ion with a m z of about 401.2 to a product ion with a m/z of about 269.2.
  • Product ion scans in Figures 4B, 5B, and 6B several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 4B, 5B, and 6B to replace or augment the exemplary fragment ions. Table 4.
  • An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D 3 is fragmenting a precursor ion with a m/z of about 383.2 to a product ion with a m/z of about 186.9.
  • An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D 3 -[6, 19, 19]- H 3 is fragmenting a precursor ion with a m/z of about 386.2 to a product ion with a m/z of about 256.8.
  • An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- 3 ⁇ 4-PTAD is fragmenting a precursor ion with a m/z of about 389.2 to a product ion with a m/z of about 210.0.
  • a precursor ion with a m/z of about 389.2 to a product ion with a m/z of about 210.0.
  • Additional product ions may be selected from those indicated in Figures 7B, 8B, and 9B to replace or augment the exemplary fragment ions. Table 5.
  • Example 6 Exemplary spectra from LDTD-MS/MS analysis of PTAD derivatized 25- hydroxyvitamin D? and 25-hydroxyvitamin D 3
  • [6, 19, 19]- H 3 , and 25-hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the procedure outlined above in Examples 2 and 3.
  • Exemplary Ql scan spectra from the analysis of samples containing PTAD-25- hydroxyvitamin D 2 , PTAD-25 -hydroxyvitamin D 2 -[6, 19, 19]- 2 H 3 , and PTAD-25- hydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- H 6 are shown in Figures 10A, 11A, and 12A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.
  • An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D 2 is fragmenting a precursor ion with a m/z of about 570.3 to a product ion with a m/z of about 298.0.
  • An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D 2 -[6, 19, 19]- H 3 is fragmenting a precursor ion with a m/z of about 573.3 to a product ion with a m/z of about 301.0.
  • An exemplary MRM transition for the quantitation of PTAD-25-hydroxyvitamin D 2 -[26, 26, 27, 27, 27]- 3 ⁇ 4 is fragmenting a precursor ion with a m/z of about 576.3 to a product ion with a m/z of about 298.0.
  • a precursor ion with a m/z of about 576.3 to a product ion with a m/z of about 298.0.
  • additional product ions may be selected from those indicated in Figures 10B, 1 IB, and 12B to replace or augment the exemplary fragment ions.
  • Exemplary Ql scan spectra from the analysis of PTAD-25 -hydroxyvitamin D 3 , PT AD- 25 -hydroxyvitamin D 3 -[6, 19, 19]- 2 H 3 , and PTAD-25 -hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 3 ⁇ 4 are shown in Figures 13 A, 14 A, and 15 A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.
  • An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D 3 is fragmenting a precursor ion with a m/z of about 558.3 to a product ion with a m/z of about 298.1.
  • An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D 3 -[6, 19, 19]- H 3 is fragmenting a precursor ion with a m/z of about 561.3 to a product ion with a m/z of about 300.9.
  • An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 is fragmenting a precursor ion with a m/z of about 564.3 to a product ion with a m/z of about 298.0.
  • a precursor ion with a m/z of about 564.3 is fragmenting a precursor ion with a m/z of about 564.3 to a product ion with a m/z of about 298.0.
  • additional product ions may be selected from those indicated in Figures 13B, 14B, and 15B to replace or augment the preferred fragment ions.
  • Example 7 Exemplary spectra from LDTD-MS/MS analysis of 1,25-dihydroxyvitamin D? and 1,25-dihydroxyvitamin D 3
  • Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D 2 include fragmenting a precursor ion with a m/z of about 375.1 to a product ion with a m/z of about 105.3; fragmenting a precursor ion with a m/z of about 393.1 to a product ion with a m/z of about 156.9; and fragmenting a precursor ion with a m/z of about 411.1 to a product ion with a m/z of about 135.3.
  • Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D 2 -[26, 26, 27, 27, 27]- H 6 include fragmenting a precursor ion with a m/z of about 381.1 to a product ion with a m/z of about 104.9; fragmenting a precursor ion with a m/z of about 399.1 to a product ion with a m/z of about 156.6; and fragmenting a precursor ion with a m/z of about 417.1 to a product ion with a m/z of about 134.9.
  • Exemplary Ql scan spectra from the analysis of 1,25-dihydroxyvitamin D3, 1,25- dihydroxyvitamin D 3 -[6, 19, 19]- 2 H 3 , and 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 2 H 6 are shown in Figures 18 A, 19A, and 20A, respectively. These spectra were collected by scanning Ql across a m/z range of about 340 to 440.
  • Exemplary product ion scans generated from three different precursor ions for each of 1,25-dihydroxyvitamin D 3 , 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- H 3 , and 1,25- dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 are presented in Figures 18B-D, 19B-D, and 20B-D, respectively.
  • the precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 9.
  • Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D 3 include fragmenting a precursor ion with a m/z of about 363.1 to a product ion with a m/z of about 156.8; fragmenting a precursor ion with a m/z of about 381.1 to a product ion with a m/z of about 157.0; and fragmenting a precursor ion with a m/z of about 399.1 to a product ion with a m/z of about 158.8.
  • Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- H 3 include fragmenting a precursor ion with a m/z of about 366.1 to a product ion with a m/z of about 158.8; fragmenting a precursor ion with a m/z of about 384.1 to a product ion with a m/z of about 160.2; and fragmenting a precursor ion with a m/z of about 402.1 to a product ion with a m/z of about 162.0.
  • Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D 3 -[26, 26, 27, 27, 27]- 3 ⁇ 4 include fragmenting a precursor ion with a m/z of about 369.1 to a product ion with a m/z of about 156.9.9; fragmenting a precursor ion with a m/z of about 387.1 to a product ion with a m/z of about 156.8; and fragmenting a precursor ion with a m/z of about 405.1 to a product ion with a m/z of about 158.8.
  • Example 8 Exemplary spectra from LDTD-MS/MS analysis of PTAD derivatized 1,25- dihydroxyvitamin D? and 1,25-dihydroxyvitamin D 3
  • PTAD derivatives of 1,25-dihydroxyvitamin D 2 , 1,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- 2 H 6 , 1,25-dihydroxyvitamin D 3 , 1,25-dihydroxyvitamin D 3 -[6, 19, 19]- 2 H 3 , and 1,25- dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- ⁇ 6 were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the procedure outlined above in Examples 2 and 3.
  • Exemplary product ion scans generated from three different precursor ions for each of PTAD-l,25-dihydroxyvitamin D 2 and PTAD-l,25-dihydroxyvitamin D 2 -[26, 26, 26, 27, 27, 27]- 3 ⁇ 4 are presented in Figures 21B-D, and 22B-D, respectively.
  • the precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 10.
  • Exemplary MRM transitions for the quantitation of PTAD-l,25-dihydroxyvitamin D 2 include fragmenting a precursor ion with a m/z of about 550.4 to a product ion with a m/z of about 277.9; fragmenting a precursor ion with a m/z of about 568.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 586.4 to a product ion with a m/z of about 314.2.
  • Exemplary MRM transitions for the quantitation of PTAD-1,25- dihydroxyvitamin D 2 -[26, 26, 27, 27, 27]- H 6 include fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 278.1; fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 298.1; and fragmenting a precursor ion with a m/z of about 592.4 to a product ion with a m/z of about 313.9.
  • Exemplary Ql scan spectra from the analysis of PTAD-1,25 -hydroxyvitamin D3, PTAD- 1,25 -dihydroxyvitamin D 3 -[6, 19, 19]- 2 H 3 , and PTAD-1,25 -dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- 2 H 6 are shown in Figures 23 A, 24A, and 25 A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.
  • Exemplary product ion scans generated from three different precursor ions for each of PTAD-l,25-hydroxyvitamin D 3 , PTAD-l,25-dihydroxyvitamin D 3 -[6, 19, 19]- 2 H 3 , and PTAD- 1,25-dihydroxyvitamin D 3 -[26, 26, 26, 27, 27, 27]- H 6 are presented in Figures 23B-D, 24A-D, and 25B-D, respectively.
  • the precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 11.
  • Exemplary MRM transitions for the quantitation of PTAD- 1,25 -hydroxyvitamin D 3 include fragmenting a precursor ion with a m/z of about 538.4 to a product ion with a m/z of about 278.1 ; fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 313.0.
  • Exemplary MRM transitions for the quantitation of PTAD- 1,25- dihydroxyvitamin D 3 -[6, 19, 19]- H 3 include fragmenting a precursor ion with a m/z of about 541.4 to a product ion with a m/z of about 280.9; fragmenting a precursor ion with a m/z of about 559.4 to a product ion with a m/z of about 301.1; and fragmenting a precursor ion with a m/z of about 577.4 to a product ion with a m/z of about 317.3.
  • Exemplary MRM transitions for the quantitation of PTAD- 1,25 -dihydroxyvitamin D 2 -[26, 26, 27, 27, 27]- H 6 include fragmenting a precursor ion with a m/z of about 544.4 to a product ion with a m/z of about 278.0; fragmenting a precursor ion with a m/z of about 562.4 to a product ion with a m/z of about 298.2; and fragmenting a precursor ion with a m/z of about 580.4 to a product ion with a m/z of about 314.0.

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Abstract

L'invention porte sur des procédés de détermination de la quantité d'un ou de plusieurs métabolites de la vitamine D dans un échantillon à l'aide d'une spectrométrie de masse. De manière générale, les procédés comportent la volatilisation d'un échantillon par le chauffage au moyen d'un laser, l'ionisation d'un ou de plusieurs métabolites de la vitamine D dans l'échantillon volatilisé, et la détection de la quantité d'un ou de plusieurs ions par spectrométrie de masse. Les procédés peuvent être utilisés pour détecter deux ou plus de deux métabolites de la vitamine D dans une seule analyse.
PCT/US2010/056461 2009-12-03 2010-11-12 Détection de vitamine d par spectrométrie de masse avec désorption thermique par diode laser Ceased WO2011068654A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140625A1 (fr) * 2013-03-15 2014-09-18 Micromass Uk Limited Syntonisation automatisée pour imagerie ionique à désorption/ionisation laser assistée par matrice
WO2018198043A1 (fr) * 2017-04-28 2018-11-01 Waters Technologies Corporation Procédés de quantification de vitamine d totale
CN111936851A (zh) * 2018-01-29 2020-11-13 Dh科技发展私人贸易有限公司 用于检测维生素d代谢物的方法和系统

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Publication number Priority date Publication date Assignee Title
WO1995033279A1 (fr) * 1994-05-31 1995-12-07 University Of Warwick Spectrometre de masse tandem
US20060228809A1 (en) * 2005-04-06 2006-10-12 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
WO2008097246A2 (fr) * 2006-05-24 2008-08-14 Swce Système et procédé d'extraction et de détection
US20090137056A1 (en) * 2007-11-28 2009-05-28 Brett Holmquist Methods for detecting dihydroxyvitamin d metabolites by mass spectrometry

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995033279A1 (fr) * 1994-05-31 1995-12-07 University Of Warwick Spectrometre de masse tandem
US20060228809A1 (en) * 2005-04-06 2006-10-12 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites by mass spectrometry
WO2008097246A2 (fr) * 2006-05-24 2008-08-14 Swce Système et procédé d'extraction et de détection
US20090137056A1 (en) * 2007-11-28 2009-05-28 Brett Holmquist Methods for detecting dihydroxyvitamin d metabolites by mass spectrometry

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140625A1 (fr) * 2013-03-15 2014-09-18 Micromass Uk Limited Syntonisation automatisée pour imagerie ionique à désorption/ionisation laser assistée par matrice
US9673029B2 (en) 2013-03-15 2017-06-06 Micromass Uk Limited Automated tuning for MALDI ion imaging
WO2018198043A1 (fr) * 2017-04-28 2018-11-01 Waters Technologies Corporation Procédés de quantification de vitamine d totale
CN111936851A (zh) * 2018-01-29 2020-11-13 Dh科技发展私人贸易有限公司 用于检测维生素d代谢物的方法和系统
EP3746781A4 (fr) * 2018-01-29 2021-11-24 DH Technologies Development Pte. Ltd. Procédés et systèmes de détection de métabolites de vitamine d

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