CN107632105A - Ornidazole purity rubric material and preparation method and application - Google Patents

Abstract

The invention discloses a standard substance of ornidazole purity, a preparation method and application thereof. Its preparation method comprises the following steps: 1) sampling ornidazole raw material powder, adopting high performance liquid chromatography area normalization method to measure its content, obtaining the purity value of ornidazole raw material powder; 2) measuring ornidazole The mass percent of water, non-volatile impurities and organic volatile impurities in the raw material powder is calculated according to formula 1 to obtain the purity rating 1 of the ornidazole purity standard substance, which is denoted as P _HPLC-AN ; P _HPLC-AN = P ₀ ×[100%-X _w -X _n- X _v ] formula I; 3) get ornidazole raw material powder and adopt quantitative nuclear magnetic method to measure its content, obtain the purity value 2 of ornidazole purity standard substance, denoted as P _NMR ; 4) calculate the average value of P _HPLC-AN and P _NMR , obtain the purity value of ornidazole purity standard substance. The invention can be used as a reference material for the detection of drug residues in livestock and poultry products, and provides technical support and material guarantee for the establishment of a national agricultural product quality safety risk assessment and monitoring traceability system.

Description

Ornidazole purity standard substance and its preparation method and application

technical field

The invention relates to ornidazole purity standard substance and its preparation method and application, and belongs to the technical field of standard quantity transfer in chemical measurement.

Background technique

Prohibited and restricted drugs are seriously abused in the process of animal husbandry. Because of their strong toxic side effects and toxic effects, and the residues in animal tissues will be transmitted to humans through the food chain, long-term trace intake can cause various diseases. Hazard to human health. Prohibited and restricted drugs have become an important parameter in the risk assessment and monitoring activities of the agricultural product quality and safety of the Ministry of Agriculture. In order to ensure the consistency, comparability, traceability and accuracy and reliability of the measurement results, corresponding reference materials are urgently needed.

Ornidazole Chinese alias Onidazole, English name: Ornidazole, mainly used as anti-anaerobe and anti-protozoal drug. CAS: 16773-42-5. Molecular formula C ₇ H ₁₀ ClN ₃ O ₃ . The relative molecular weight is 219.63, the melting point is 86-90°C, and the boiling point is 443.2°C. It is white to slightly yellow powder, odorless, bitter in taste, easily soluble in methanol and ethanol, slightly soluble in water. Storage conditions: 2-8°C. It is the third generation of nitroimidazole derivatives. The mechanism of its antimicrobial effect may be: through the nitro group in its molecule, it is reduced to an amino group in an oxygen-free environment or through the formation of free radicals, interacting with cell components, resulting in the death of microorganisms. The molecular structural formula of ornidazole is shown in Formula 1.

Contents of the invention

The purpose of the present invention is to provide ornidazole purity reference material and its preparation method and application. The present invention can be used as reference material for the detection of drug residues in livestock and poultry products, and provides a basis for the establishment of national agricultural product quality safety risk assessment and monitoring traceability system. Technical support and material guarantee.

The preparation method of ornidazole purity standard substance provided by the invention comprises the following steps:

1) Sampling the ornidazole raw material powder, using the high performance liquid chromatography area normalization method to measure the mass percentage of ornidazole in the ornidazole raw material powder, to obtain the purity value of the ornidazole raw material powder ;

2) Determination of the moisture mass percentage content, non-volatile impurity mass percentage content and organic volatile impurity mass percentage content in the ornidazole raw material powder, combined with the described ornidazole raw material powder measured in step 1) The purity value is calculated according to the following formula I to obtain the purity value 1 of the ornidazole purity standard substance, which is denoted as P _HPLC-AN ;

P _HPLC-AN ＝P ₀ ×[100%-X _w -X _n -X _v ] formula Ⅰ;

Wherein, P ₀ is the purity fixed value of ornidazole raw material powder, _Xw is the moisture mass percentage content, _{Xn is} the non-volatile impurity mass percentage content, X _v is the organic volatile impurity mass percentage content;

3) Get the ornidazole raw material powder and adopt quantitative NMR method to measure the mass percentage of ornidazole in the ornidazole raw material powder, obtain the purity value 2 of the ornidazole purity standard substance, denoted as P _NMR ;

4) Calculate the average value of the purity rating 1 of the ornidazole purity standard substance and the purity rating 2 of the ornidazole purity standard substance to obtain the purity rating of the ornidazole purity standard substance.

In the above method, step 1) also includes the step of sampling the ornidazole raw material powder according to the requirements of JJG 1006-1994 "Technical Specifications for Primary Standard Substances" and adopting the variance analysis method for uniformity testing.

In the above-mentioned method, step 1) also includes the step of carrying out a stability test using a straight line fitting method to the ornidazole raw material powder sampling; Ask to proceed.

In the above-mentioned method, the chromatographic condition of described HPLC area normalization method is as follows:

Chromatographic column: SB-aq (250mm×4.6mm, 5.0μm); mobile phase: composed of acetonitrile and acetic acid aqueous solution, the volume ratio of acetonitrile and acetic acid aqueous solution is 13:87, the volume of acetic acid and water in acetic acid aqueous solution Number ratio: 0.001:100; flow rate: 1.0mL/min; injection volume: 20μL; UV wavelength: 312nm;

The ornidazole raw material powder is prepared as a 100-500 mg/L ornidazole methanol solution during measurement.

In the present invention, HPLC: Shimadzu LC-20A system.

In the above-mentioned method, the quantitative NMR method adopts the internal standard method, and the purity rating 2 of the ornidazole purity standard substance is calculated according to the following formula II:

In formula II: P _NMR is the purity of the analyte measured by nuclear magnetic resonance;

I _x is the integrated area of the designated peak of the ornidazole raw material powder;

I _std is the integrated area of the specified peak of the internal standard;

n _std is the number of nuclei group nuclei of the specified peak of the internal standard;

n _x is the number of nuclei group nuclei of the designated peak of the ornidazole raw material powder;

M _x —the molecular weight of the ornidazole raw material powder;

M _{std —the} molecular weight of the internal standard;

m _std — the mass of the added internal standard;

m _x —the sample weight of the ornidazole raw material powder;

P _std — the purity of the internal standard.

In the present invention, the internal standard substance is benzoic acid.

In above-mentioned method, adopt Karl Fischer method to measure the moisture content in described ornidazole raw material powder;

The mass percentage content of non-volatile impurities in the ornidazole raw material powder is determined by microwave digestion-inductively coupled plasma mass spectrometry, microwave digestion-inductively coupled plasma emission spectrometry and ion chromatography; the microwave digestion-inductively coupled Plasma mass spectrometry measures the content of at least one of As, Se, Cd, Mn, Sn, Li, Cr, Cs, Ni, Pb, Sr, Rb, Ti and Ba in the non-volatile impurities; the microwave digestion- Inductively coupled plasma emission spectrometry measures the content of at least one of Cu, Ca, Mg, K, Na, P, Mn, Mo, Zn, Fe and Co in the non-volatile impurities; Chloride ion, nitrate ion and sulfate ion content;

The mass percentage content of the organic volatile impurities in the ornidazole raw material powder was determined by headspace gas chromatography.

In the above-mentioned method, step 2) also includes the step of performing uncertainty analysis on the determination of the ornidazole purity standard substance.

The present invention also provides the ornidazole purity standard substance with definite value obtained by the above method.

In the present invention, the purity of the mass percent content of the ornidazole purity standard substance is rated at 99.9%.

The present invention further provides the application of the ornidazole purity standard substance in any one of the following 1)-6):

1) Used as a reference material for the detection of ornidazole components in veterinary drugs and/or feed products in the field of agricultural product production and circulation;

2) Detection of ornidazole drug residues in livestock and poultry products in the food field;

3) Testing laboratory quality control;

4) Calibrate the analytical instrument as a reference material;

5) Validation and evaluation of analytical methods;

6) Assign a value to a given material.

The present invention has the following advantages:

Candidates for this reference material are prepared by ordering corresponding commercially available standard products, drying them under reduced pressure according to their physical and chemical properties, shaking them well, and subpackaging under dry conditions after qualitative and preliminary analysis meet the requirements. Using liquid chromatography-area normalization method (UV detector) and quantitative nuclear magnetic method to determine the purity of standard substances, by using preparation methods, measurement methods and measuring instruments that meet the requirements of metrology characteristics , to ensure the traceability of the characteristic value of the standard substance.

Description of drawings

Fig. 1 is the 6-month stability monitoring result of the ornidazole purity standard substance of the present invention.

Fig. 2 is the short-term stability monitoring result of acyclovir of the present invention, wherein Fig. 2 (a) is the short-term stability result under 20 ℃ of conditions, Fig. 2 (b) is the short-term stability result under 40 ℃ of conditions, Fig. 2 (c ) is the short-term stability result at 60°C.

Fig. 3 is the chromatogram of ornidazole under different concentration conditions of the present invention, wherein Fig. 3 (a) is the chromatogram of the ornidazole methanol solution of 100mg/L, and Fig. 3 (b) is the ornidazole methanol solution of 500mg/L .

Fig. 4 is the chromatogram of methanol blank and ornidazole candidate sample of the present invention, wherein Fig. 4(a) is the chromatogram of methanol blank, and Fig. 4(b) is the chromatogram of ornidazole candidate sample.

Fig. 5 is the measurement of non-volatile impurity content, and wherein Fig. 5 (a) is the standard curve of chloride ion, Fig. 5 (b) is the standard curve of nitrate, Fig. 5 (c) is the standard curve of sulfate ion.

Fig. 6 is the ion chromatogram of ornidazole (2000mg/L).

Fig. 7 is the determination of volatile impurity content in ornidazole, wherein Fig. 7 (a) is the headspace chromatogram of ornidazole, Fig. 7 (b) is the headspace chromatogram of air blank, and Fig. 7 (c) is ornidazole Headspace gas chromatogram of azole compared with single standard.

Fig. 8 is the quantitative NMR spectrum of ornidazole, and its Fig. 8 (a) is the quantitative nuclear magnetic spectrum ( ¹ H-NMR spectrum) of ornidazole, and Fig. 8 (b) is the quantitative nuclear magnetic spectrum of benzoic acid standard substance ( ¹ H-NMR spectrum), Figure 8(c) is the quantitative NMR spectrum ( ¹ H-NMR spectrum) after ornidazole and internal standard are mixed.

Figure 9 is a flow chart of source analysis and assessment of uncertainty in the determination of ornidazole purity reference materials.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

In the following examples, the standard substance candidate ornidazole was purchased from TCI Company, the batch number is Lot SFHEL, the label purity is 99.9% (HPLC) and 99.9% (Nonaqueous Titration), and the packaging specification of the candidate is 25g. After the preliminary confirmation of the qualitative and purity respectively, the liquid chromatography (ultraviolet detector, UV) was used to carry out the preliminary inspection of the homogeneity of the standard substance. After drying under reduced pressure, the well-mixed ornidazole candidate was divided into 5.0mL clean In vials, each package is 100mg, 436 bottles in total. Store at low temperature (4°C), away from light.

Embodiment 1, the definite value of ornidazole purity standard substance

The definite value step of ornidazole purity standard substance of the present invention is as follows:

1. Uniformity test

According to the requirements of JJG 1006-1994 "Technical Specifications for Primary Reference Materials", the homogeneity test was carried out for the standard materials. The present invention adopts the variance analysis method to statistically check the uniformity of the ornidazole purity standard substance, randomly extracts 15 packages from the subpackaged ornidazole, each package is taken in parallel three times, and the weight-volume method is used to prepare 100mg/L methanol solution, Using the conditions when the value was fixed, liquid chromatography-area normalization method inspection, the obtained uniformity data and statistical results are shown in Table 1.

Table 1 ornidazole purity standard substance homogeneity inspection result (%)

According to the data in Table 1, the data of the homogeneity test were statistically analyzed, and the results The standard deviation s _H for homogeneity is calculated as follows:

2. Stability inspection

(1) Long-term stability study

According to the requirements in JJG1006-1994 "Technical Specifications for Primary Standard Substances", the stability of standard substances shall be investigated in accordance with the principle of density first and sparseness later. The present invention began in February 2016, and carried out stability investigations at the 0th, 1st, 3rd, and 6th months by liquid chromatography. Two packages were drawn each time, and the solution was prepared by the gravimetric-volume method, and each package was tested 3 times in parallel. The stability test results are shown in Table 2 and Figure 1. It can be seen from Table 2 and Figure 1 that the purity standard substance is stable for six months under the condition of 4°C dark storage.

Table 2 ornidazole purity standard substance long-term stability monitoring result (%)

(2) Short-term stability investigation

The samples were stored in incubators at 20°C, 40°C, and 60°C (simulating transportation conditions), and the stability monitoring was carried out on the 1st, 3rd, 5th, 7th, and 9th day respectively. The detection method was the same as the long-term stability monitoring. The trend analysis was used to investigate the data, and the results are shown in Table 3 and Figure 2. The results in Table 3 and Figure 2 show that the standard substance can guarantee a stable purity value within 9 days under the transportation conditions of 60°C and below.

Table 3 ornidazole purity standard substance short-term stability investigation result (%)

In summary, the ciprofloxacin standard substance has good long-term stability for 6 months, and its characteristic values are stable under the simulated transportation temperature of 20°C, 40°C, and 60°C, and the transportation time of 9 days.

3. Determination of standard substances

(1) Purity determination: liquid chromatography-area normalization method

In accordance with the "National Technical Specifications for Primary Standard Substances", two methods of liquid chromatography-area normalization method and NMR quantitative method are used to determine the value of ornidazole standard substance.

The area normalization method includes the determination of the main component content of ornidazole by HPLC-UV method, the determination of moisture content in the candidate by Karl Fischer Coulomb method, and the determination of non-volatile impurity content by ICP-MS and ion chromatography , Determination of volatile impurity content by headspace gas chromatography. The NMR quantitative method uses benzoic acid as the internal standard, and selects the appropriate resonance peak internal standard for quantification. The source analysis and systematic assessment of the uncertainty of ornidazole purity reference materials were carried out.

Prepare 100mg/L and 500mg/L ornidazole methanol solutions, and inject samples under the same chromatographic conditions (see the following ornidazole liquid chromatography determination conditions for details). As shown in Figure 3, by comparing Figure 3, it is found that the signal-to-noise ratio of impurities is improved under high concentration conditions, and there are more impurity responses, and the maximum concentration of ornidazole in methanol solution is 500mg/L. Therefore, 500 mg/L ornidazole methanol solution was selected for further optimization of chromatographic conditions.

Ornidazole liquid chromatography determination conditions are as follows:

Chromatographic column: Agilent ZORBAX SB-aq (250mm×4.6mm, 5.0μm); composed of acetonitrile and acetic acid aqueous solution, the volume fraction ratio of acetonitrile and acetic acid aqueous solution is 13:87, the volume fraction of acetic acid and water in acetic acid aqueous solution The ratio is 0.001:100; flow rate: 1.0mL/min; injection volume: 20μL; UV wavelength: 312nm; HPLC: Shimadzu LC-20A system.

As shown in Figure 4, the main components and impurities were determined by comparing the chromatograms of the methanol blank and the ornidazole candidate sample.

7 packaging units were selected for the experiment, and the sampling volume of each packaging was 25 mg, which was prepared into a 50 mL solution with methanol at a concentration of 500 mg/mL. The purity of the main component of ornidazole was measured under optimized liquid chromatography conditions, and the average value was measured three times. The results are shown in Table 4.

Table 4 ornidazole main component area normalization method content determination result (%)

(2) Determination of impurity content

1) Determination of moisture content

For the 7 packaging units selected in the experiment for definite value, the METTLER TOLEDO DL39 series METTLERTOLEDO Stromboli instrument was used to measure the moisture according to the national standard (GB/T606 Chemical Reagent Moisture Determination General Method Karl-Fisher method), and the average value was measured three times. The result As shown in Table 5.

Table 5 ornidazole moisture content measurement result (%)

2) Determination of non-volatile impurity content

2)-1 Inductively coupled plasma mass spectrometry

The present invention uses microwave digestion-inductively coupled plasma mass spectrometry (Microwave-assisted ICP-MS) to detect As, Se, Cd, Mn, Sn, Li, Cr, Cs, Ni, Pb, Sr in ornidazole purity standard substances , Rb, Ti, Ba content, the specific experimental process is as follows.

Experimental equipment and reagents:

Inductively coupled plasma mass spectrometer (ICP-MS) Thermo Fisher Co., Ltd. ICP-MS X series 2, 1/10,000 analytical balance, purity>99.99% high-purity argon, CEM microwave digestion instrument (MARS SYSTEM), Ultrapure water, nitric acid (excellent grade), hydrogen peroxide (excellent grade), As, Se, Cd, Mn, Mo, Co, Cu, Cs, Ni, Pb, Sr, Rb, Ti, Ba mixed standard solution (10mg /L).

experimental method:

Establishment of the standard working curve: The standard working curve is prepared by configuring different concentrations of mixed standard solutions (0.01ppm, 0.1ppm, 1.0ppm, 10.0ppm, 50.0ppm).

Internal standard solution (In 114): use ultrapure water as the medium, and prepare a solution with a concentration of 1 μg/L.

Sample pretreatment: microwave digestion. Weigh 0.100g of a uniform dry sample, accurate to 0.0001g, add 5mL of nitric acid to a 55mL digestion tube, soak overnight, add 1mL of hydrogen peroxide, cover the sample-dissolving cap, place it in a high-pressure tank, and then put it into a microwave sample-dissolving device , set the digestion program of the microwave system, and start to digest the sample. After the digestion is complete, take out the inner tank, place it on an acid flusher, heat at 100°C to flush out the acid until 1mL of the solution remains, remove it and cool it to room temperature, transfer it to a 10mL volumetric flask with water, dilute to volume, and mix well for later use. At the same time, make a reagent blank. Instrument conditions: atomizer flow rate 1.02L/min; purging times 45 times; auxiliary gas flow rate 0.80L/min; cooling gas flow rate 13L/min.

2)-2 Inductively Coupled Plasma Emission Spectrometry

The present invention uses microwave digestion-inductively coupled plasma emission spectrometry (Microwave-assisted ICP-AES) to detect Cu, Ca, Mg, K, Na, P, Mn, Mo, Zn, Fe, Co content, the experimental procedure is as follows.

Experimental instruments and reagents: Inductively coupled plasma emission spectrometer (ICP-AES) ICP-5000 from Shanghai Concentrating Technology Instrument Co., Ltd., one ten-thousandth analytical balance, high-purity argon gas with a purity of >99.99%, air compressor, circulating cooling water, CEM microwave digestion instrument (MARS SYSTEM), ultrapure water, nitric acid (excellent grade), hydrogen peroxide (excellent grade), Cu, Ca, Mg, K, Na, P, Mn, Mo, Zn, Fe, Co standard solutions (100mg/L)

experimental method:

Establishment of the standard working curve: The standard working curve is prepared by configuring different concentrations of mixed standard solutions (0.1ppm, 1.0ppm, 5.0ppm, 10.0ppm).

Sample pretreatment: microwave digestion. Weigh 0.100g of a uniform dry sample, accurate to 0.0001g, add 5mL of nitric acid to a 55mL digestion tube, soak overnight, add 1mL of hydrogen peroxide, cover the sample-dissolving cap, place it in a high-pressure tank, and then put it into a microwave sample-dissolving device , set the digestion program of the microwave system, and start to digest the sample. After the digestion is complete, take out the inner tank, place it on an acid flusher, heat at 100°C to flush out the acid until 1mL of the solution remains, remove it and cool it to room temperature, transfer it to a 10mL volumetric flask with water, dilute to volume, and mix well for later use. At the same time, make a reagent blank. Instrument conditions: horizontal observation mode; flushing pump speed 100rpm; RF power: 1150w; analysis pump speed 50rmp; atomizer flow rate 0.6SLM; analysis time 25s; auxiliary gas flow rate 1.0SLM; cooling gas flow rate 12SLM.

The contents of each element in ornidazole measured by the above two methods are shown in Table 6.

Non-volatile inorganic element impurity determination results in table 6 ornidazole purity standard substance

2)-3 ion chromatography

Instruments and reagents:

ICS-3000 ion chromatograph (American Dionex company), equipped with EG40 eluent automatic generator, conductivity detector and Chromeleon 6.80 chromatographic workstation; Ionpac AS11-HC separation column (250mm×4mm); IonpacAG11-HC protection Column (50mm×4mm); ASRS-ULTRA anion suppressor; OnGuard RP column (Dionex, USA), 0.45 filter membrane (Milipore); ultrapure water system (Pine-Tree); KQ100-DE ultrasonic cleaner (Kunshan City Ultrasonic Instrument Co., Ltd.).

The standard samples of sodium nitrite and hexanoic acid and other organic acid salts were high-purity reagents from Sigma Company, and the solutions used were all prepared with ultrapure water with a resistivity of 18.2MΩ·cm.

Chromatographic conditions:

Chromatographic column: IonPac AS11-HC separation column (250mm x4mm), IonPac AG11-HC guard column (50mm x 4mm); mobile phase: 15mM KOH isocratic elution for 18min; flow rate: 1.0ml/min; suppressor regeneration mode : Additional hydroelectric suppression; conductivity detector detection; injection volume: 25 μL. Quantified by peak area.

Experimental results:

Prepare the standard curve of chloride ion, nitrate ion and sulfate ion, the standard curve is shown in Figure 5, and the measurement data are shown in Table 7.

Table 7 Ion Chromatography Standard Solution Preparation Concentration and Measurement Results

Standard solution concentration/ppm Chloride peak area Nitrate peak area Sulfate peak area 0.1 0.036 0.019 0.0852 0.5 0.0984 0.0581 0.1374 1 0.1743 0.1134 0.2018 5 0.9316 0.6301 0.8015 10 1.9381 1.3298 1.7156 20 3.9721 2.7854 3.4592

The ion chromatogram of ornidazole (2000mg/L) is as shown in Figure 6, and integral result calculates that chloride ion content is 0.00601% in the pure product of ornidazole, and nitrate ion is below detection limit, and sulfate ion content is 0.000341%.

In summary, the content of non-volatile impurities in ornidazole is 0.00935%.

3) Determination of volatile impurities content

During the synthesis of ornidazole, organic solvents are inevitably used. In order to verify whether there is organic solvent residue in the pure product, headspace gas chromatography was used in the experiment to detect the organic volatile components in the pure product of ornidazole.

Main instruments and reagents: Agilent 6890 gas chromatograph, hydrogen flame ionization detector (FID) (Agilent, USA), Agilent 1888 headspace autosampler (Agilent, USA), 10mL headspace bottle (Agilent, USA) , methanol, ethanol, propanol, isopropanol, acetonitrile, n-hexane, ethyl acetate, dichloromethane standard material (National Institute of Metrology).

Instrument working conditions: chromatographic column: DB-624 (60m×0.25mm, 1.40μm); carrier gas flow rate: hydrogen 40ml/min, air 400ml/min; temperature programming conditions: initial 45°C for 5min, at a rate of 7°C per minute The temperature was raised to 120°C at a rate of 15°C per minute to 230°C and kept for 15 minutes. Injection volume: 1mL, split ratio: splitless.

Heating box: 70°C; Loop: 90°C; Transfer line: 110°C

Experimental procedure: Dissolve 10 μL standard substances of methanol, ethanol, propanol, isopropanol, acetonitrile, n-hexane, ethyl acetate, and dichloromethane solutions in 990 μL of pure water and accurately record their masses, and place them in headspace bottles Immediately seal the prepared single standard with a capper and wait for determination. Weigh 50.00mg (accurate to 0.01mg) of ornidazole in 10mL headspace vials and immediately seal them with a capper, pending determination.

The samples and single standards were measured under the above experimental conditions, and the experimental results are shown in Table 8 and Figure 7.

Table 8 Quality and Content Record Form of Single Standard

According to the retention time and the comparison with the single standard and the air blank, the ornidazole candidate contains two volatile impurities, methanol and acetonitrile, and the content of methanol and acetonitrile is determined by the relationship between the peak area and the content. The results are shown in Table 9. As shown, the test results show that the volatile impurities in ornidazole are methanol and acetonitrile, but the content is very low, which has little influence on the determination result of the purity of ornidazole, so it can be ignored.

Volatile impurities and their content in table 9 ornidazole

4) Determination result of ornidazole purity standard substance

The purity determination formula of liquid chromatography-area normalization method (HPLC-AN) is as follows:

P _HPLC-AN = P ₀ ×[100%-X _w -X _n -X _v ] × 100%.

Wherein, P ₀ is the purity value of ornidazole raw material powder, X _w is the water content, X _n is the content of non-volatile impurities, and X _v is the content of volatile impurities.

The liquid chromatography-area normalization method value results of the ornidazole candidates after 7 bottles of subpackage are shown in Table 10.

Table 10 ornidazole area normalization method value result (%)

(2) Purity fixed value: quantitative NMR method

NMR instrument: NMR model: AvanceⅢ400MHz, 1H NMR measurement parameters: excitation pulse angle 90°, sampling time 3.9846s, scan width: 8223.43Hz, relaxation delay 60s, cumulative sampling 16 times, probe temperature 293.4K, bias frequency: 2464.5137Hz, receiving gain 64.00, pulse sequence zg30; electronic balance: UMX2, Swiss MettlerToledo company (graduation value: 0.1 μ g); deuterated reagent: deuterated methanol (US sigma company); NMR internal standard: benzoic acid (US NIST standard material, SRM-350b, mass fraction is 99.9978%±0.0044%).

¹ H-NMR test conditions:

The excitation pulse angle is 30 degrees, the sampling time is 3.9846s, the sweep width is 8223.43Hz, the relaxation delay is 60s, the cumulative sampling is 128 times, the probe temperature is 293.4K, the bias frequency is 2464.5137Hz, the receiving gain is 64.00, and the pulse sequence is zg30. Before sampling, it goes through automatic tuning and automatic shimming, then manual tuning and shimming, and finally automatically adjusts the gain. The spectral width is 20.00ppm, the center of the spectrum is located at 8.0ppm, and the longitudinal relaxation is 8.5s. After sampling, deuterated methanol was used for calibration. The window function (exponential multiplication) is used for optimization, and the line width factor is 0.1.

Sample preparation: take out the ornidazole sample stored at 4° C., place it at room temperature, and raise the temperature of the sample to room temperature. An electronic balance with an accuracy of 0.01 mg was used to accurately weigh 20 mg of ornidazole and 10 mg of internal standard benzoic acid (internal standard) into the same glass vial, and then dissolve it in deuterated methanol (0.75 ml). Ultrasound was used for 15 minutes to promote its complete dissolution, and finally it was transferred to a (5mm/20cm) NMR tube. According to the above steps, the same 7 deuterated solution samples sealed in NMR tubes were obtained, which were directly used for the determination of hydrogen spectra.

Repeated measurement: take the same deuterated solution of ornidazole sealed in an NMR tube, and measure 3 times continuously under the above conditions.

Data processing: use the American data processing software ACD Lab to process the original spectrogram through Fourier transform, baseline calibration, automatic phase and manual phase adjustment to obtain the spectrogram.

Quantitative NMR measurement results: It has been verified by experiments that the molecular structure of ornidazole is suitable for quantification of purity by quantitative NMR method. According to the position of its characteristic peak, this experiment selects the US NIST standard substance benzoic acid as the internal standard.

Since the H of ornidazole in the low-field area is on the heterocycle, it will not interfere with the H of the benzene ring on the benzoic acid, and the hydrogen in the low-field area is very characteristic, so the low-field area is selected to be located at δ=7.87 One of the CH characteristic hydrogens was used as a calibration, and the 2,6-position hydrogen δ=8.01 of the benzene ring on the internal standard benzoic acid was selected as a calibration. The hydrogens of these two compounds are very close in the nuclear magnetic resonance spectrum, and the magnetic field is quite uniform in this small range, so their accumulated integral areas are used to determine their content. The quantitative nuclear magnetic spectrum of standard substance benzoic acid and the quantitative nuclear magnetic spectrum of ornidazole (internal standard is standard substance benzoic acid) are as shown in Figure 8.

Confirmed by the method, the molecular structure of ornidazole is suitable for quantification by nuclear magnetic resonance method, and the purity standard substance benzoic acid can be used as the internal standard substance, and the purity calculation formula is as follows:

In the formula: P _NMR —the purity of the analyte measured by nuclear magnetic resonance;

I _x —integrated area of the specified peak of the sample;

I _{std —integrated} area of internal standard specified peak;

n _std — the number of nuclei group nuclei of the specified peak of the internal standard;

n _x - the number of nuclei group nuclei of the specified peak of the sample;

M _x —molecular weight of the sample;

M _{std —the} molecular weight of the internal standard;

m _std — the mass of the added internal standard;

m _x - the weighing amount of the sample;

P _std — the purity of the internal standard.

The quantitative NMR value results of ornidazole are shown in Table 11.

Table 11 ornidazole quantitative nuclear magnetic method (qNMR) fixed value result (%)

Purity rating results:

The results of the purity determination take the average value of the measurement results of two different principle methods, and the results are as follows:

In summary, the value determination result of ornidazole purity standard substance is 99.9%.

4. Uncertainty evaluation

The uncertainty of the ornidazole purity reference material consists of three parts: the uncertainty introduced by the inhomogeneity of the standard material; the uncertainty introduced by the instability of the standard material; the uncertainty introduced by the process of determining the value of the standard material.

The source analysis and evaluation process of ornidazole purity reference material uncertainty is shown in Figure 9.

(1) Uncertainty introduced by uniformity

Homogeneity was assessed by one-way analysis of variance. Then the standard deviation s _H of the uniformity can be calculated by the formula:

In the formula: s _H is the standard deviation of uniformity; is the variance between groups; is the variance within the group; n is the number of measurements within the group.

In this case, s _H is equal to the uncertainty component u _bb caused by the inhomogeneity between bottles, that is, the formula u _bb =s _H =0.0096%

(2) Uncertainty introduced by stability

According to the data in Table 2, the uncertainty u _lts introduced by the long-term stability with a valid period of 6 months is calculated as follows:

u _lts =s(b ₁ )·X=0.000894%×6=0.00408%≈0.00537%

According to the data in Table 3, the uncertainty u _sts introduced by the short-term stability is calculated as follows:

At 20°C: u _sts1 =s(b ₁ )·X=0.00315%×9=0.028%

At 40°C: u _sts1 =s(b ₁ )·X=0.00278%×9=0.025%

At 60°C: u _sts2 =s(b ₁ )·X=0.00014%×9=0.001%

so:

(3) Uncertainty introduced by fixed value

1) Uncertainty introduced by the area normalization method

The calculation formula of the uncertainty u(P _HPLC-AN ) of the area normalization method is as follows:

Among them, u(P _{HPLC-AN, rel} ) is the uncertainty of area normalization method, u _rel (P ₀ ) is the uncertainty of liquid chromatography, u(X _w ) is the uncertainty of moisture measurement , u(X _n ) is the measurement uncertainty of non-volatile impurities, and u(X _v ) is the measurement uncertainty of volatile impurities.

1)-1 Measurement uncertainty of liquid chromatography

The uncertainty u ₁ introduced by the repeatability of the liquid chromatography value measurement is calculated from the standard deviation of the measurement, u ₁ = 0.021%;

The uncertainty introduced by the response difference of each component at different detection wavelengths:

In the formula, B _imaxλ is the maximum percentage content of impurity component i under the five detection wavelengths;

B _{i fixed value λ} is the percentage content of impurity component i at the fixed value wavelength;

u _2-i is the uncertainty component of impurity component i.

The response and uncertainty evaluation results of each wavelength are shown in Table 8.

1)-2 Uncertainty of moisture introduction

The uncertainty component introduced by the balance sample is very small, so the main source of uncertainty in the moisture measurement is the repeatability u _A of the measurement, the measurement result X _w of the moisture is 0.0131%, and the standard deviation is 0.001%, so the uncertainty introduced by the moisture measurement is not Degree of certainty: u(X _W )=0.001%.

1)-3 Uncertainty introduced by other impurities

The uncertainty component introduced by the balance sample is very small, so the main source of uncertainty in the measurement of non-volatile impurities is the repeatability u _A of the measurement, and the measurement result X _n of non-volatile impurities is 0.00935%. Therefore, it is considered that the non-volatile impurities Uncertainty of impurity determination: u(X _n )=0.00935%.

1)-4 Relative uncertainty of area normalization method:

1) Absolute uncertainty of -5 area normalization method:

u _HPLC-AN = 99.93% × u _{HPLC-AN, rel} = 0.027%

2) Uncertainty introduced by quantitative NMR method

The purity measurement uncertainty formula is obtained from the NMR quantitative formula:

The sources of NMR quantitative uncertainty include measurement uncertainty, molecular weight uncertainty, weighing uncertainty, and internal standard purity uncertainty.

Uncertainty u(I _x /I _std ) of the integral area measurement: the standard uncertainty of the integral area measurement results is evaluated by Class A, expressed by the standard deviation of the NMR method measurement results, and the uncertainty is 0.072%.

Internal standard substance ethyl paraben and sample balance uncertainty u(m _std ) and u(m _x ): the minimum division of the weighing balance is 0.01mg, and the weighing sample size is 20mg and 10mg, so the balance The weighing uncertainty is:

Internal standard purity uncertainty u(P _std ): The purity uncertainty of internal standard benzoic acid is obtained from the standard substance certificate, and the relative expanded uncertainty of the purity value is 0.0044% (k=2), so its relative standard The uncertainty is 0.0022%.

Uncertainty of molecular weight u(M _x ) and u(M _std ): Calculation formula of standard uncertainty of relative molecular weight of ornidazole and internal standard benzoic acid:

In the formula: N _j —atomic number of element j; u _j —uncertainty of relative atomic mass of element j.

According to the IUPAC International Atomic Scale, the relative atomic masses and uncertainties of the four atoms are C: 12.0107±0.0008; H: 1.00794±0.00007; O: 15.9994±0.0003; N: 14.00674±0.00007; Cl: 35.4527±0.0009.

For each element, its standard uncertainty is converted from the reference uncertainty according to the uniform distribution.

The molecular formula of ornidazole is C ₇ H ₁₀ ClN ₃ O ₃ , the relative molecular weight is 219.63, and its standard uncertainty is:

The relative standard uncertainty of the relative molecular mass of ornidazole:

u(M _x )/M _x =0.0033/219.63=1.5×10 ⁻⁵

Similarly, the internal standard benzoic acid (C ₇ H ₆ O ₂ ), with a relative molecular weight of 122.12, has a relative standard uncertainty of:

u(M _std )/M _std =2.7×10 ^-5 .

Therefore, the synthetic standard uncertainty of NMR quantitative method purity measurement is:

Therefore, the fixed value uncertainty is calculated as follows:

Uncertainty of standard substance: The uncertainty of the value determination result of ornidazole standard substance is composed of three parts: the uncertainty caused by the method of determination, the inhomogeneity of the standard substance and the instability. The formula for calculating the overall uncertainty of the reference material is as follows:

The expanded uncertainty is: U _CRM =k×u _CRM =0.4% (k=2).

In summary, the values and uncertainties of ornidazole purity standard substances are shown in Table 12.

Purity, uncertainty of table 12 ornidazole purity standard substance

Element Quantity/% Uncertainty/%(k=2) Ornidazole purity 99.9 0.4

For the determination of drug residues in exported chicken, in addition to the national standard method for the detection of ornidazole in exported chicken, it is also necessary to use ornidazole standard substances. Prepare the standard solution by the volumetric method and draw the working curve. At the same time, the sample to be tested and the standard working solution obtained after sample processing are detected by liquid chromatography or liquid chromatography mass spectrometry, and the content of ornidazole in the sample to be tested is calculated by the peak area. , so that the content in the sample to be analyzed can be directly traced to the SI unit through this purity standard substance, which increases the accuracy, comparability and traceability of the data.

Claims (10)

1. the preparation method of Ornidazole purity rubric material, comprises the following steps：

1) Ornidazole material powder is sampled, the Ornidazole material powder is determined using high performance liquid chromatography area normalization method The weight/mass percentage composition of middle Ornidazole, obtain the purity definite value of the Ornidazole material powder；

2) biodiversity percentage composition in the Ornidazole material powder, non-volatile impurities weight/mass percentage composition and organic are determined Volatile impurity weight/mass percentage composition, with reference to the purity definite value of the Ornidazole material powder determined in step 1), according to such as Lower formula I calculates, and obtains the purity definite value 1 of Ornidazole purity rubric material, is designated as P_HPLC-AN；

P_HPLC-AN=P₀× [100%-X_w-X_n-X_v] formula I；

Wherein, P₀For the purity definite value of Ornidazole material powder, X_wFor biodiversity percentage composition, X_nFor non-volatile impurities matter Measure percentage composition, X_vFor organic volatile impurities weight/mass percentage composition；

3) the Ornidazole material powder is taken to determine the quality of Ornidazole in the Ornidazole material powder using nuclear-magnetism method is quantified Percentage composition, the purity definite value 2 of Ornidazole purity rubric material is obtained, is designated as P_NMR；

4) the purity definite value 1 of the Ornidazole purity rubric material and the purity definite value of the Ornidazole purity rubric material are calculated 2 average value, that is, obtain the purity definite value of Ornidazole purity rubric material.

2. according to the method for claim 1, it is characterised in that：Also include taking the Ornidazole material powder in step 1) Sample uses JJG 1006-1994《Primary standard material technical specification》Requirement uniformity testing is carried out using method of analysis of variance Step.

3. method according to claim 1 or 2, it is characterised in that：Also include in step 1) to the Ornidazole raw material powder The step of end sampling carries out stability test using line fitting approach.

4. according to the method any one of claim 1-3, it is characterised in that：The high performance liquid chromatography area normalization The chromatographic condition of method is as follows：

Chromatographic column：SB-aq(250mm×4.6mm,5.0μm)；Mobile phase：It is made up of acetonitrile and acetic acid aqueous solution, acetonitrile and acetic acid The volume parts ratio of the aqueous solution is 13:87, the volume parts ratio of acetic acid and water in acetic acid aqueous solution is 0.001:100；Sample introduction Amount：20μL；UV wavelength：312nm；

100~500mg/L Ornidazole methanol solution is formulated as during the Ornidazole material powder measure.

5. according to the method any one of claim 1-4, it is characterised in that：The quantitative nuclear-magnetism method uses internal standard method, The purity definite value 2 of the Ornidazole purity rubric material is calculated according to equation below II：

In formula II：P_NMRFor the purity of the measured object measured using nuclear magnetic resonance method；

I_xThe integral area at peak is specified for the Ornidazole material powder；

I_stdThe integral area at peak is specified for internal standard compound；

n_stdThe nuclear colony core number at peak is specified for internal standard compound；

n_xThe nuclear colony core number at peak is specified for the Ornidazole material powder；

M_xThe molecular weight of-Ornidazole the material powder；

M_stdThe molecular weight of-internal standard compound；

m_stdThe quality of the internal standard compound of-addition；

m_xThe sample weighting amount of-Ornidazole the material powder；

P_stdThe purity of-internal standard compound.

6. according to the method any one of claim 1-5, it is characterised in that：The nitre difficult to understand is determined using Karl_Fischer method Moisture in azoles material powder；

Non-volatile impurities weight/mass percentage composition uses micro-wave digestion-inductively coupled plasma in the Ornidazole material powder Mass spectrography, micro-wave digestion-inductively coupled plasma emission spectrography and ion-chromatographic determination；Micro-wave digestion-the inductance As, Se, Cd, Mn, Sn, Li, Cr, Cs, Ni, Pb, Sr, Rb, Ti in coupled plasma mass measure non-volatile impurities With at least one of Ba content；The micro-wave digestion-inductively coupled plasma emission spectrography measure is described non-volatile At least one of Cu, Ca, Mg, K, Na, P, Mn, Mo, Zn, Fe and Co in impurity content；The ion-chromatographic determination chlorine The content of ion, nitrate ion and sulfate ion；

Organic volatile impurities weight/mass percentage composition described in the Ornidazole material powder is determined using headspace gas chromatography.

7. according to the method any one of claim 1-6, it is characterised in that：Also include in step 2) to Ornidazole purity The definite value of standard substance carries out the step of analysis on Uncertainty.

8. the Ornidazole purity rubric material for the definite value that the method any one of claim 1-7 obtains.

9. Ornidazole purity rubric material according to claim 8, it is characterised in that：The Ornidazole purity rubric material The purity definite value of weight/mass percentage composition is 99.9%.

10. the Ornidazole purity rubric material of claim 8 or 9 is following 1) -6) application in any one：

1) it is applied in agricultural product production field of circulation herbal medicine and/or feed product Ornidazole into go-on-go as standard substance Survey；

2) Ornidazole medicament residue detects in animal products in field of food；

3) testing laboratory's quality control；

4) analytical instrument is calibrated as standard substance；

5) analysis method confirms evaluation；

6) to giving material assignment.