Determination of Metronidazole, Itraconazole and Hydroxy-Itraconazole in Insect-repelling Powder by HPLC-MS / MS

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for determining metronidazole, itraconazole, and hydroxyitraconazole in anthelmintic powder by using HPLC-MS / MS method. The present invention optimizes chromatographic conditions and mass spectrometry conditions, performs qualitative analysis of metronidazole, itraconazole, and hydroxyitraconazole according to the retention time and mass spectrometry information of the sample, and performs quantitative analysis according to the ratio of the peak area to the internal standard peak area. The method has the advantages of high efficiency, sensitivity, good reproducibility, high recovery rate, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining metronidazole, itraconazole and hydroxyitraconazole in anthelmintic powder by utilizing an HPLC-MS / MS method. Background Art

[0002] The anthelmintic powder is a brown powder with a fragrant smell, bitter and astringent taste. Its main ingredients include lice, quisqualis, betel nut, mugwort, thunder ball, black plum, rhubarb, stemona, costus root, etc. It can be used to kill the adults, larvae and eggs of various nematodes, trematodes, tapeworms and whipworms in livestock and poultry (chickens, ducks, geese, pigeons, quails, etc.), prevent and control diseases caused by parasitic insects such as mites, ticks and lice in livestock and poultry; promote weight gain, improve production performance, and improve feed conversion rate.

[0003] Metronidazole is an antibiotic and antiprotozoal agent, mainly used to treat or prevent systemic or local infections caused by anaerobic bacteria. Itraconazole is mainly used for systemic infections caused by deep fungi, such as blastomycosis, histoplasmosis, paracoccidioidomycosis, chromomycosis, sporotrichosis, coccidioidomycosis, etc. It can also be used for candidiasis and aspergillosis. Hydroxyitraconazole is a metabolite impurity of itraconazole.

[0004] Therefore, adding a certain dose of metronidazole and itraconazole to the anthelmintic powder for combined use can improve the inhibition of bacterial reproduction in poultry and enhance the anthelmintic effect. However, the anthelmintic powder is prepared from multiple Chinese medicines with complex ingredients, making it difficult to separate and detect the metronidazole and itraconazole therein, and itraconazole is easily metabolized to produce hydroxyitraconazole impurities. How to accurately control the quality of metronidazole, itraconazole, and the impurity hydroxyitraconazole in the anthelmintic powder is a difficult problem.

[0005] Adding anthelmintics such as metronidazole and itraconazole can enhance the efficacy of anthelmintic powder. Veterinary drug products have good efficacy, but when the drugs enter the animal body, veterinary drug residues accumulate in the liver, kidney or muscle tissue. When veterinary antibiotics enter the animal body, drug residues can be produced. When people eat "antibiotic foods" with veterinary drug residues, they will cause allergic reactions, urticaria, liver and kidney failure and other side effects. In severe cases, food poisoning and shock will occur. Second, after being eaten by people, they have carcinogenic, teratogenic and mutagenic effects on the human body. Third, when people eat "antibiotic foods" with drug-resistant genes, "super bacteria" can pass their drug-resistant genes to other bacteria in the human intestine, causing them to mutate into drug-resistant bacteria. When these drug-resistant strains have chromosomal mutations, they can pass their drug-resistant genes vertically to the next generation and become naturally resistant bacteria. The drug-resistant genes of these bacteria can also be transmitted between different strains, causing bacteria to have cross-resistance to many antibiotics. This not only increases the difficulty of preventing and treating human diseases, but also makes antibiotic treatment ineffective, leading to human deaths and endangering human health. Summary of the invention

[0006] In order to improve the therapeutic effect, metronidazole and itraconazole are added to the anthelmintic powder for combined use. How to accurately control the quality of metronidazole, itraconazole and impurity hydroxyitraconazole in the anthelmintic powder is a technical difficulty. Since the main ingredients of the anthelmintic powder are lice, quisqualis, betel nut, convolvulus, thunder ball, black plum, rhubarb, stemona, costus root, etc., the ingredients are complex, so separation and detection require exploratory methods. The present invention provides a detection method for metronidazole, itraconazole and hydroxyitraconazole in the anthelmintic powder, which solves the above problems.

[0007] The basic principle of the technical solution of the present invention is: using high performance liquid chromatography-mass spectrometry technology, using liquid chromatography as a separation system and mass spectrometry as a detection system. The sample is separated from the mobile phase in the mass spectrometry part, and after being ionized, the mass analyzer of the mass spectrometry separates the ion fragments according to the mass number, and the mass spectrum is obtained by the detector. The qualitative analysis of metronidazole, itraconazole and hydroxyitraconazole is carried out according to the retention time and ion abundance ratio, and the quantitative analysis of metronidazole, itraconazole and hydroxyitraconazole is carried out by the internal standard method.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] The present invention provides a method for detecting metronidazole, itraconazole and hydroxyitraconazole in anthelmintic powder, comprising the following steps:

[0010] (1) Pretreatment: Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale;

[0011] (2) Preparation of standard curve: Take appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile-0.1% formic acid solution to prepare a series of concentration standard solutions;

[0012] (3) High performance liquid chromatography-mass spectrometry.

[0013] Furthermore, the method is:

[0014] (1) Pretreatment: Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale;

[0015] (2) Preparation of standard curve: Take appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile-0.1% formic acid solution (10:90) to prepare a series of concentration standard solutions;

[0016] (3) High performance liquid chromatography-mass spectrometry determination:

[0017] The HPLC conditions are:

[0018] Chromatographic column: C18 column; Mobile phase A: acetonitrile-chloride solution; Mobile phase B: 0.05-0.5% formic acid aqueous solution; Column temperature: 35-45°C; Flow rate: 0.1-0.5 ml·min-1; Elution mode: gradient elution;

[0019] The mass spectrometry conditions are:

[0020] Ionization mode: electrospray ionization (ESI+); drying gas temperature: 320-380°C; drying gas flow rate: 10.0 l / min; collision gas: 40 psi; capillary voltage: 4 kV; scanning mode: positive ion scanning; detection mode: multiple reaction monitoring.

[0021] Furthermore, the mobile phase A is: acetonitrile-potassium chloride or acetonitrile-sodium chloride solution, and the volume ratio of acetonitrile to chloride is 10:1-3.

[0022] Furthermore, the pH of the potassium chloride or sodium chloride is 3.0-4.0.

[0023] It is worth mentioning that the inventors tried various solvents such as acetonitrile and acetonitrile-formic acid solution as mobile phase A, but the separation was not ideal. Finally, they unexpectedly found that adding a certain proportion of chloride to mobile phase A and adjusting the pH value significantly improved the effect.

[0024] Further, the gradient elution procedure is:

[0025]

[0026] Preferably, the gradient elution procedure is:

[0027]

[0028] Furthermore, the chromatographic column is Agilent ZORBAX Eclipse Plus C18 (3.0×100 mm, 1.8 μm) or Waters BEH C18 (2.1 mm×100 mm, 1.7 μm), preferably Agilent ZORBAX Eclipse Plus C18 (3.0×100 mm, 1.8 μm).

[0029] The standard curve is prepared with a series of standard solutions with the following concentrations: the internal standard concentration of metronidazole is 10 ng / mL, the external standard solution concentrations are: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, the internal standard concentration of itraconazole and hydroxyitraconazole is 50 ng / mL, the external standard solution concentrations are: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, for liquid chromatography-tandem mass spectrometry determination.

[0030] The injection volume during detection was 10 μl.

[0031] Preferably, the method comprises the following steps:

[0032] (1) Pretreatment: Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale;

[0033] (2) Preparation of standard curve: Take appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile-0.1% formic acid solution (10:90) to prepare a series of standard solutions: the internal standard concentration of metronidazole is 10 ng / mL, and the series of external standard solutions are: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL; the internal standard concentration of itraconazole and hydroxyitraconazole is 50 ng / mL, and the series of external standard solutions are: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, for liquid chromatography-tandem mass spectrometry determination;

[0034] (3) High performance liquid chromatography-mass spectrometry determination:

[0035] The HPLC conditions are:

[0036] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (3.0×100mm, 1.8μm); mobile phase A: acetonitrile and potassium chloride solution, potassium chloride solution pH=3.5, acetonitrile and potassium chloride solution volume ratio of 5:1; mobile phase B: 0.05-0.5% formic acid aqueous solution; column temperature: 40°C; flow rate: 0.3ml·min-1; elution mode is gradient elution:

[0037]

[0038] The mass spectrometry conditions are:

[0039] Ionization mode: electrospray ionization (ESI+); drying gas temperature: 320-380°C; drying gas flow rate: 10.0 l / min; collision gas: 40 psi; capillary voltage: 4 kV; scanning mode: positive ion scanning; detection mode: multiple reaction monitoring.

[0040] The injection volume was 10 μl.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The invention provides a method for detecting metronidazole, itraconazole and hydroxyitraconazole in anthelmintic powder, adopting high performance liquid chromatography-mass spectrometry technology, performing qualitative analysis of metronidazole, itraconazole and hydroxyitraconazole according to the retention time and mass spectrum information of the sample, and performing quantitative analysis according to the ratio of their peak area to the internal standard peak area. The method has the advantages of high speed, sensitivity, good reproducibility, high recovery rate and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 :Metronidazole, itraconazole, hydroxyitraconazole standard spectra

[0044] Figure 2 : Blank sample spectrum

[0045] Figure 3 : Spectrum of samples with added concentrations of metronidazole 0.2 mg / kg, itraconazole and hydroxy-itraconazole 1.0 mg / kg

[0046] Figure 4 : Sample spectra with added concentrations of metronidazole: 0.4 mg / kg, itraconazole and hydroxy-itraconazole at 2.0 mg / kg

[0047] Figure 5 : Sample spectra with added concentrations of metronidazole: 1.0 mg / kg, itraconazole and hydroxy-itraconazole at 5.0 mg / kg

[0048] Figure 6 :Standard curve of hydroxyitraconazole

[0049] Figure 7 :Standard curve of metronidazole

[0050] Figure 8 :Standard curve of itraconazole DETAILED DESCRIPTION

[0051] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0052] 1. Instruments and Equipment

[0053] Liquid chromatography tandem mass spectrometer: electrospray ionization source (ESI + ).

[0054] Analytical balance: sensitivity 0.00001g, sensitivity 0.0001g.

[0055] Ultrasonic cleaner.

[0056] Centrifuge.

[0057] 2. Experimental reagents, materials and solution preparation

[0058] Reagent materials:

[0059] Water: in accordance with the requirements of GB / T 6682 Grade 1 water, acetonitrile (CH3CN), formic acid (HCOOH).

[0060] Organic microporous filter membrane: 0.22μm.

[0061] Filter paper: quantitative filter paper, medium speed, 12.5cm.

[0062] Unless otherwise specified, all reagents used were of chromatographic grade.

[0063] Solution preparation:

[0064] Formic acid-water solution: acid-water (1+999, volume ratio): absorb 1.0 mL of formic acid, dilute to 1000 mL with water, filter through a 0.22 μm microporous membrane, and set aside.

[0065] Acetonitrile-formic acid aqueous solution (1+9, volume ratio): measure 50 mL of acetonitrile (4.2) and 450 mL of 0.1% formic acid aqueous solution, mix well and set aside.

[0066] Mobile phase B: 0.1% formic acid in water

[0067] Mobile phase A:

[0068] Acetonitrile-water solution (2+3, volume ratio): measure 40 mL of acetonitrile (4.2) and 60 mL of water (4.4), mix well and set aside.

[0069] 8% potassium chloride or sodium chloride solution, pH = 3.0-4.0 for use.

[0070] The volume ratio of acetonitrile-water solution: 8% potassium chloride or sodium chloride solution (pH=3.0-4.0) is 5:1.

[0071] Standard substances: Metronidazole (CAS: 443-48-1, purity > 99%), Itraconazole (CAS: 84625-61-6, purity > 98%) and Hydroxyitraconazole (CAS: 112559-91-8, purity > 98%), Metronidazole-d4, Itraconazole-d5, Hydroxyitraconazole-d6.

[0072] External standard stock solution: Accurately weigh the converted amount of metronidazole, itraconazole and hydroxyitraconazole standard substances to an accuracy of 0.0001g, and prepare 200μg / mL standard stock solution with acetonitrile (stored at -20℃ away from light, valid for 3 months).

[0073] Internal standard stock solution: Accurately weigh the converted amount of metronidazole, itraconazole and hydroxyitraconazole internal standard substances to an accuracy of 0.0001g, and prepare them into 100μg / mL standard stock solution with acetonitrile (stored at -20℃ away from light, valid for 3 months).

[0074] Metronidazole, itraconazole and hydroxyitraconazole external standard intermediate solution: Accurately pipette 0.5 mL of 200 μg / mL metronidazole, itraconazole and hydroxyitraconazole standard stock solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, mix well, and the concentration is 1.0 μg / mL. Store in a refrigerator at 4°C away from light, with a shelf life of 1 month.

[0075] Metronidazole, itraconazole and hydroxyitraconazole internal standard intermediate solution: Accurately pipette 1.0 mL of 100 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard stock solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, mix well, and the concentration is 1.0 μg / mL. Store in a refrigerator at 4°C away from light, with a shelf life of 1 month.

[0076] 3. Experimental data processing

[0077] The internal standard method is used for quantification, and the residual amount of the substance to be tested in the sample is calculated using chromatography data processing software or according to formula (1). The blank value must be subtracted from the calculation result.

[0078]

[0079] Where:

[0080] X——Residue of the substance to be tested in the sample, μg / kg;

[0081] C——the concentration of the analyte obtained from the standard curve, μg / L;

[0082] A i ——The peak area of ​​the analyte in the sample solution;

[0083] A' is ——Peak area of ​​internal standard in standard solution;

[0084] C s ——Concentration of internal standard in standard solution, μg / L;

[0085] C is ——Concentration of internal standard in the sample solution, μg / L;

[0086] A is ——Peak area of ​​internal standard in sample solution;

[0087] A s ——Peak area of ​​the analyte in the standard solution;

[0088] C' is ——Concentration of internal standard in quasi-solution, μg / L;

[0089] V——final fixed volume of the sample, mL;

[0090] m——mass of the test material, g;

[0091] f——dilution multiple.

[0092] Take two samples of each sample for parallel measurement, and take the arithmetic mean of the two parallel measurement results as the measurement result. The result shall be rounded to three significant figures.

[0093] Example 1 HPLC-MS / MS method for the detection of metronidazole, itraconazole and hydroxyitraconazole in anthelmintic powder 1. Sample pretreatment and extraction

[0094] Accurately weigh 1.0g (accurate to 0.001g) of the sample to be tested into a 100mL volumetric flask, add the corresponding internal standards of the three compounds respectively, add 50mL of 20% acetonitrile solution for ultrasonic extraction for 15min, accurately make up to 100mL, add mobile phase B to dilute to the scale. Take the upper filter membrane to filter, and the filtrate is to be tested. At the same time, perform a sample blank test according to the above steps.

[0095] 2. Preparation of Standard Curve

[0096] Accurately measure an appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile-0.1% formic acid solution (10:90) to prepare a series of concentration standard solutions. The internal standard concentration of metronidazole is 10 ng / mL, and the concentration series of external standard solutions are: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL. The internal standard concentration of itraconazole and hydroxyitraconazole is 50 ng / mL, and the concentration series of external standard solutions are: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL for liquid chromatography-tandem mass spectrometry determination. The ratio of the measured characteristic ion peak area to the internal standard characteristic ion peak area was used as the ordinate and the corresponding standard working solution concentration was used as the abscissa to draw a standard curve and calculate the regression equation and correlation coefficient.

[0097] 3.HPLC-MS / MS detection:

[0098] HPLC reference conditions:

[0099] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (3.0 × 100 mm, 1.8 μm), 100 mm × 3.0 mm, particle size 1.8 μm;

[0100] Mobile phase A: acetonitrile solution: 8% potassium chloride solution (pH = 3.5), volume ratio of 5:1;

[0101] Mobile phase B: 0.1% formic acid in water;

[0102] Flow rate: 0.3 mL / min;

[0103] Injection volume: 10 μL;

[0104] Column temperature: 40℃.

[0105] Liquid chromatography gradient elution conditions:

[0106]

[0107] Mass spectrometry reference conditions:

[0108] a) Ionization method: electrospray ionization (ESI + );

[0109] b) Drying gas temperature: 350°C;

[0110] c) Drying gas flow rate: 10.0L / min;

[0111] d) Collision gas: 40 psi;

[0112] e) Capillary voltage: 4kV;

[0113] f) Scanning mode: positive ion scanning;

[0114] g) Detection method: multiple reaction monitoring (MRM);

[0115] The qualitative and quantitative ion pairs, fragmentor voltages and collision energies of metronidazole, itraconazole and hydroxyitraconazole are shown in Table 1.

[0116] Table 1 Qualitative ion pairs, quantitative ion pairs, fragmentor voltages and collision energies

[0117]

[0118]

[0119] A. Qualitative determination

[0120] The retention time of the sample solution chromatogram, the retention time of each chromatographic peak characteristic ion and the corresponding standard solution, and the characteristic ion of each chromatographic peak are compared to determine the qualitative. If the deviation of the retention time of the sample solution and the standard solution of equivalent concentration is within ±2.5%; the relative deviation of the relative ion abundance ratio of the characteristic ion in the sample solution and the relative ion abundance ratio of the standard solution of equivalent concentration does not exceed the provisions of Table 2, it can be determined that metronidazole, itraconazole and hydroxyitraconazole are present in the sample. Results are shown in Figure 1-8 The metronidazole and itraconazole / hydroxyitraconazole in the samples met the requirements.

[0121] Table 2 Maximum allowable deviation of relative ion abundance for qualitative confirmation

[0122] Relative ion abundance, % ＞50 >20 to 50 >10 to 20 ≤10 Permissible relative deviation, % ±20 ±25 ±30 ±50

[0123] B. Quantitative determination

[0124] Take 10 μL of sample solution and standard solution respectively and inject them into liquid chromatography-tandem mass spectrometer, measure under the above chromatography-mass spectrometry conditions, record characteristic ion mass chromatogram, and quantify by internal standard method. Figure 1-8 .

[0125] Example 1 Methodological Study

[0126] Precision and sensitivity investigation

[0127] The quantitative limits of this method are: 0.2 mg / kg for metronidazole, 1.0 mg / kg for itraconazole and hydroxyitraconazole.

[0128] Example 1 Recovery and precision of the detection method

[0129] The spiked recovery test of the insect repellent was carried out by repeating three independent measurements according to the sample preparation method and under the same chromatographic conditions. The recovery rate and relative standard deviation of each added level were calculated. The relative standard deviation of the results should not exceed 10% of the arithmetic mean.

[0130] Table 3 Recovery and precision of metronidazole, itraconazole and hydroxyitraconazole in chondroitin powder

[0131]

[0132]

[0133] Example 2 HPLC-MS / MS method for detecting metronidazole, itraconazole and hydroxyitraconazole in anthelmintic powder

[0134] 1. Sample Pretreatment

[0135] Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale;

[0136] 2. Preparation of standard curve

[0137] Accurately measure an appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile-0.1% formic acid solution (10:90) to prepare a series of concentration standard solutions. The internal standard concentration of metronidazole is 10 ng / mL, and the concentration series of external standard solutions are: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL. The internal standard concentration of itraconazole and hydroxyitraconazole is 50 ng / mL, and the concentration series of external standard solutions are: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL for liquid chromatography-tandem mass spectrometry determination. The ratio of the measured characteristic ion peak area to the internal standard characteristic ion peak area was used as the ordinate and the corresponding standard working solution concentration was used as the abscissa to draw a standard curve and calculate the regression equation and correlation coefficient.

[0138] 3. High performance liquid chromatography-mass spectrometry determination:

[0139] The HPLC conditions are:

[0140] Chromatographic column: Waters BEH C18 (2.1 mm × 100 mm, 1.7 μm);

[0141] Mobile phase A: acetonitrile solution: 5% sodium chloride solution (pH = 3.0), volume ratio of 10:1;

[0142] Mobile phase B: 0.05% formic acid in water;

[0143] Column temperature: 35°C;

[0144] Flow rate: 0.5 ml min -1 ;

[0145] Gradient elution:

[0146]

[0147]

[0148] Mass spectrometry reference conditions:

[0149] a) Ionization mode: electrospray ionization (ESI+);

[0150] b) Drying gas temperature: 350°C;

[0151] c) Drying gas flow rate: 10.0L / min;

[0152] d) Collision gas: 40 psi;

[0153] e) Capillary voltage: 4kV;

[0154] f) Scanning mode: positive ion scanning;

[0155] g) Detection method: multiple reaction monitoring (MRM);

[0156] Example 3 Detection of Metronidazole, Itraconazole and Hydroxy-Itraconazole in Insect-Changing Powder by HPLC-MS / MS

[0157] 1. Sample Pretreatment

[0158] Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale;

[0159] 2. Preparation of standard curve

[0160] Accurately measure an appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile-0.1% formic acid solution (10:90) to prepare a series of concentration standard solutions. The internal standard concentration of metronidazole is 10 ng / mL, and the concentration series of external standard solutions are: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL. The internal standard concentration of itraconazole and hydroxyitraconazole is 50 ng / mL, and the concentration series of external standard solutions are: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL for liquid chromatography-tandem mass spectrometry determination. The ratio of the measured characteristic ion peak area to the internal standard characteristic ion peak area was used as the ordinate and the corresponding standard working solution concentration was used as the abscissa to draw a standard curve and calculate the regression equation and correlation coefficient.

[0161] 3. High performance liquid chromatography-mass spectrometry determination:

[0162] The HPLC conditions are:

[0163] Chromatographic column: Waters BEH C18 (2.1 mm × 100 mm, 1.7 μm);

[0164] Mobile phase A: acetonitrile solution: 10% potassium chloride solution (pH = 4.0), volume ratio of 10:3;

[0165] Mobile phase B: 0.5% formic acid in water;

[0166] Column temperature: 45°C;

[0167] Flow rate: 0.1 ml min -1 ;

[0168] Gradient elution;

[0169]

[0170] Mass spectrometry reference conditions:

[0171] a) Ionization mode: electrospray ionization (ESI+);

[0172] b) Drying gas temperature: 350°C;

[0173] c) Drying gas flow rate: 10.0L / min;

[0174] d) Collision gas: 40 psi;

[0175] e) Capillary voltage: 4kV;

[0176] f) Scanning mode: positive ion scanning;

[0177] g) Detection method: multiple reaction monitoring (MRM);

[0178] The results showed that the HPLC-MS / MS method of Examples 2 and 3 was used to detect metronidazole, itraconazole and hydroxyitraconazole in the anthelmintic powder, and the detection effect was equivalent to that of Example 1.

Claims

1. A method for detecting metronidazole, itraconazole and hydroxyitraconazole in anthelmintic powder, characterized in that: The method comprises the following steps: (1) Pretreatment: Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale; (2) Preparation of standard curve: Take appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile and 0.1% formic acid solution in a volume ratio of 10:90 to prepare a series of concentration standard solutions; (3) High performance liquid chromatography-mass spectrometry determination: The HPLC conditions are: Chromatographic column: C18 column; mobile phase A: acetonitrile aqueous solution-potassium chloride in a volume ratio of 10:1-3 or acetonitrile aqueous solution-sodium chloride in a volume ratio of 10:1-3, the volume ratio of acetonitrile to water is 2:3, and the pH of potassium chloride or sodium chloride is 3.0-4.0; mobile phase B: 0.05-0.5% formic acid aqueous solution; column temperature: 35-45°C; flow rate: 0.1-0.5ml·min -1 ; Elution mode: gradient elution; The gradient elution procedure is: The mass spectrometry conditions are: Ionization mode: electrospray ionization; Drying gas temperature: 320-380°C; Drying gas flow rate: 10.0L / min; Collision gas: 40psi; Capillary voltage: 4kV; Scanning mode: positive ion scanning; Detection mode: multiple reaction monitoring.

2. The method according to claim 1, characterized in that The gradient elution procedure is: 。 3. The method according to claim 1, characterized in that The chromatographic column is Agilent ZORBAX EclipsePlus C18, 3.0×100 mm, 1.8 μm or Waters BEH C18, 2.1 mm×100 mm, 1.7 μm.

4. The method according to claim 3, characterized in that The chromatographic column is Agilent ZORBAX EclipsePlus C18, 3.0×100 mm, 1.8 μm.

5. The method according to claim 1, characterized in that The standard curve is prepared with a series of standard solutions having the following concentrations: an internal standard concentration of metronidazole of 10 ng / mL, and a series of external standard solutions having the following concentrations: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL; an internal standard concentration of itraconazole and hydroxyitraconazole of 50 ng / mL, and a series of external standard solutions having the following concentrations: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, and 100 ng / mL, for liquid chromatography-tandem mass spectrometry determination.

6. The method according to claim 1, characterized in that The injection volume was 10 μl.

7. The method according to claim 1, characterized in that The method comprises the following steps: (1) Pretreatment: Weigh the sample to be tested into a volumetric flask, add metronidazole, itraconazole, and hydroxyitraconazole corresponding internal standards, add 20% acetonitrile solution for ultrasonic extraction, and add mobile phase B to dilute to the scale; (2) Preparation of standard curve: Take appropriate amount of 1.0 μg / mL metronidazole, itraconazole and hydroxyitraconazole internal standard working solution and 1.0 μg / mL external standard working solution, dissolve and dilute with acetonitrile and 0.1% formic acid solution in a volume ratio of 10:90 to prepare a series of standard solutions: the internal standard concentration of metronidazole is 10 ng / mL, and the series concentrations of external standard solutions are: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL; the internal standard concentration of itraconazole and hydroxyitraconazole is 50 ng / mL, and the series concentrations of external standard solutions are: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, for liquid chromatography-tandem mass spectrometry determination; (3) High performance liquid chromatography-mass spectrometry determination: The HPLC conditions are: Chromatographic column: Agilent ZORBAX Eclipse Plus C18, 3.0×100mm, 1.8μm; mobile phase A: acetonitrile aqueous solution-potassium chloride solution, potassium chloride solution pH=3.5, the volume ratio of acetonitrile aqueous solution to potassium chloride solution is 5:1; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 2:3, mobile phase B: 0.05-0.5% formic acid aqueous solution; column temperature: 40°C; Flow rate: 0.3 ml min -1 ; The elution method is gradient elution: The mass spectrometry conditions are: Ionization mode: electrospray ionization; Drying gas temperature: 320-380℃; Drying gas flow rate: 10.0L / min; Collision gas: 40psi; Capillary voltage: 4kV; Scanning mode: positive ion scanning; Detection mode: multiple reaction monitoring; The injection volume was 10 μl.

Images