Method for measuring methimazole concentration in human plasma

Abstract

The invention relates to a method for determining the concentration of methimazole in human plasma, which adopts liquid chromatography-tandem mass spectrometry for determination and comprises the following steps: providing a reagent and an instrument; chromatographic conditions and mass spectrometry conditions for analysis are provided, a C18 chromatographic column is used, a 0.1% formic acid aqueous solution / acetonitrile is used for gradient elution, and an electrospray ionization source and positive ion mode is adopted for detection; the method comprises the following steps: preparing a methimazole reference substance series standard solution and a methimazole-d3 internal standard solution; the method comprises the following steps: pretreating a plasma sample by using a derivatization reagent, derivatizing to obtain a sample to be detected, and carrying out LC-MS / MS analysis; determining the linearity and range and the lowest quantitation limit when the method meets the specificity requirement; and measuring the sample and calculating the methimazole concentration in the plasma sample. The method disclosed by the invention is high in sensitivity, small in plasma sample dosage, good in reproducibility, short in analysis time and capable of realizing high-throughput analysis, and can be used for determining a large number of biological samples in methimazole bioequivalence evaluation.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a method for determining methimazole, and in particular to a method for determining the concentration of methimazole in human plasma. The method uses liquid chromatography-tandem mass spectrometry (usually referred to as LC-MS / MS or similar terms) for determination. Background Art

[0002] The methimazole (MMI) involved in the present application is 1-methylimidazole-2-thiol, with a molecular formula of C4H6N2S, a molecular weight of 114.16, and a chemical structural formula of: .

[0003] Methimazole is a white to light yellow crystalline powder with a slight peculiar smell. It is easily soluble in water, ethanol or chloroform, and slightly soluble in ether. Its melting point is 144-147°C. It is usually taken orally in tablets of 5 mg, 10 mg or 20 mg per tablet.

[0004] Methimazole is an antithyroid drug commonly used to treat hyperthyroidism and belongs to the thiourea class of drugs. Its mechanism of action is to inhibit thyroid peroxidase, thereby hindering the oxidation of iodide in the thyroid gland and the coupling of tyrosine, and hindering the synthesis of thyroxine (T4) and triiodothyronine (T3). Animal experiments have observed that it can inhibit the synthesis of antibodies by B lymphocytes, reduce the level of thyroid stimulating antibodies in the blood circulation, and restore the function of suppressive T cells to normal, thus achieving the purpose of treatment [Cooper, DS Antithyroid drugs. New England Journal of Medicin e, 352(9), 910-917].

[0005] In clinical applications, methimazole is used as a first-line treatment drug. Patients usually need to monitor thyroid hormone levels regularly during treatment to adjust the dose and ensure the safety and effectiveness of treatment [Athanasiadou I, AngelisYS, Lyris E, et al. Chemical derivatization to enhance ionization of anabolicsteroids in LC-MS for doping-control analysis. Trends in Analytical Chemistry , 2013, 42: 137-156]. In recent years, with the increasing clinical use of methimazole, monitoring of its drug concentration has become particularly important to ensure its efficacy and reduce potential side effects.

[0006] In the past, traditional HPLC, GC-MS and other methods were commonly used to detect the concentration of methimazole in human plasma [Rong Zhengxing, Xie Yifan, Chen Congying, et al. Pharmacokinetic properties and relative bioavailability of imported and domestic methimazole tablets in humans [J]. Journal of Shanghai Second Medical University, 2003, (02): 122-124; Jansson R, Lindström B, Dahlberg PA. Pharmacokinetic properties and bioavailability of methimazole. Clin Pharmacokinet, 1985,10(5):443-50; Liu Xiaoquan, Geng Fang, Wang Songlei, et al. Pharmacokinetics of methimazole in rats after transdermal administration [J]. Journal of China Pharmaceutical University, 2005, (04): 342-345]. Although these methods can achieve preliminary detection of methimazole, their sensitivity and selectivity are insufficient and they are easily interfered by other components in plasma. In addition, due to the complex metabolic process of methimazole in the human body and the influence of multiple factors on its concentration change, traditional methods are difficult to provide real-time and accurate concentration monitoring in clinical applications [Lainé, P., Bouchardon, J.-L., & De LaMarnierre, C. Evaluation of an LC-MS / MS method for the quantitation ofmethimazole in human plasma. Journal of Pharmaceutical and Biomedical Analysis, 192, 113613].

[0007] It is known that LC-MS / MS is considered the gold standard for quantitative analysis and is widely used in the determination of drugs in biological samples, especially blood / plasma. Currently, only a few papers have reported the use of LC-MS / MS to determine the concentration of methimazole in humans or animals [Ito Y, Sakai H, Nagao H, Suzuki R, Odawara M, Minato K. Development of a high-performance liquid chromatography-tandem mass spectrometric method for the determination of Methimazole in human blood matrices. J Chromatogr B Analyt Technol Biomed Life Sci. 2020;1144:122083; Fang Shaoying, Shao Huarong, Huang Yanni, et al. Pharmacokinetics of compound methimazole ointment in rats[J]. Food and Drug, 2019, 21(06):464-469; Sornsuvit C, Hongwiset D, Niamhun N, Yotsawimonwat S, Taesotikul W.Pharmacokinetics and bioequivalence of oral methimazole in healthy Thai volunteers, determined by the sensitive analytical method, LC-MS / MS. J International Journal of Advances in Science, 2017, 5(3): 67-70]. Due to the small molecular weight of methimazole (114.16) and the presence of thiol (-SH) functional groups in its molecular structure, the retention effect of methimazole in conventional C18 chromatographic columns is relatively poor. This structural feature makes it easy to elute from the chromatographic column during conventional liquid chromatography separation, thereby affecting its separation efficiency. In addition, the presence of thiol groups makes the response of methimazole in mass spectrometry relatively low. Therefore, although the sensitivity in solution samples can meet basic testing requirements, it is difficult to meet the analysis requirements of complex biological matrices, especially plasma samples. Therefore, the analytical method for methimazole needs to use derivatization or other specific technical means to improve its detection sensitivity and selectivity in biological samples. However, only one of the above-mentioned documents mentioned the impact of this aspect. The document uses a high-cost liquid-liquid extraction pretreatment technology, which is cumbersome to operate and cannot improve the throughput of biological sample analysis.

[0008] As is well known, protein precipitation has obvious advantages in biological sample processing compared with liquid-liquid extraction. First, protein precipitation is simple and quick to operate. Usually, only a denaturant needs to be added, followed by centrifugation. The whole process takes a short time and is suitable for high-throughput analysis. Secondly, this method requires less solvent usage and can effectively reduce experimental costs. However, the prior art does not record the use of biological samples containing methimazole for LC-MS / MS determination by protein precipitation.

[0009] Therefore, establishing a sensitive and reliable bioanalytical method to accurately quantify the concentration of methimazole in plasma has become a key task for the bioequivalence evaluation of methimazole that is eagerly awaited by those skilled in the art. Summary of the invention

[0010] The object of the present invention is to provide a method for quantitatively determining the concentration of methimazole in human plasma, and it is expected that the method will show excellent effects in one or more aspects. It has been unexpectedly found that the human plasma biological sample containing methimazole is treated with a derivatization reagent NBD-Cl and then treated with a protein precipitant, and the resulting solution is subjected to LC-MS / MS analysis, which can achieve rapid detection of methimazole in human plasma and show excellent methodological performance. The present invention is completed based on such findings.

[0011] To this end, the first aspect of the present invention provides a method for determining the concentration of methimazole in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the method comprising the following steps: (1) Provide reagents and instruments Provide methimazole reference substance, methimazole-d3 reference substance, derivatization reagent, mass spectrometer, and high performance liquid chromatograph; (2) Analysis conditions Chromatographic conditions: A chromatographic column using octadecylsilane bonded silica gel as a filler; 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and the gradient elution program was: 0.1 min-35% B, 1.0 min-90% B, 1.5 min-90% B, 1.6 min-35% B, 2.6 min-stop; The flow rate was 0.5 mL / min; the injection volume was 5 µL; the column temperature was 40 °C; Mass spectrometry conditions: Electrospray ionization source (Turbo Ionspray) was used, positive ion detection was performed, the spray voltage was 5000 V, and the source temperature was 600 o C; curtain gas (CUR) was 20 psi; collision gas (CAD) was 8 psi; scanning mode was multiple reaction monitoring (MRM), and the ion reaction used for quantitative analysis was m / z 278.1→ m / z 202.1 (Methimazole-NBD, CE is 30V) and m / z 281.1→ m / z 205.1 (methimazole-d3-NBD, internal standard, CE at 30 V), scanning time was 150 msec; (3) Preparation of stock solution Preparation of methimazole reference substance series standard solution: Take the methimazole reference substance and dissolve it in acetonitrile to prepare a methimazole reference substance stock solution with a concentration of 1.0 mg / mL, and store it in a freezer at -20°C for later use; accurately pipette a certain volume of the stock solution, dilute it with 50% acetonitrile to a series of standard solutions with concentrations of 20, 40, 200, 400, 2000, 4000, 9000, and 10000 ng / mL, respectively, and store them in a refrigerator at 4°C for later use; Preparation of methimazole-d3 internal standard solution: Take the methimazole-d3 reference substance and dissolve it in acetonitrile to prepare a 1.0 mg / mL methimazole-d3 reference substance stock solution, and store it at -20°C for later use; accurately pipette a certain volume of the stock solution, dilute it with 50% acetonitrile to make an internal standard solution with a concentration of 250 ng / mL, and store it in a refrigerator at 4°C for later use; (4) Plasma sample pretreatment Take 50 μL of blank human plasma or drug-containing human plasma, add 20 μL of internal standard solution (250.0 ng / mL methimazole-d3) and 50 μL of derivatization reagent in sequence, vortex mix for 10 s, leave at room temperature for 30 min, add 200 μL of acetonitrile and vortex for 2 min, centrifuge at 3500 g for 15 min, take 40 μL of supernatant into an injection tube containing 160 μL of 50% acetonitrile aqueous solution, vortex mix, and use as the sample to be tested. Take 5 μL of the sample to be tested for LC-MS / MS analysis; (5) Method specificity Accurately take 50 µL of blank human plasma and operate according to the method in "(4) Plasma sample pretreatment" except that no internal standard solution is added. Inject 5 µL and record the chromatogram. It should show that no chromatographic peaks are detected at the retention time of methimazole and methimazole-d3; Add the internal standard solution (250.0 ng / mL methimazole-d3) to blank human plasma and follow the procedure in “(4) Plasma sample pretreatment” to inject 5 µL of the sample and record the chromatogram. It should show that no methimazole peak is detected, but a strong methimazole-d3 peak is detected. Add the methimazole standard solution at the upper limit of quantitation (ULOQ) concentration to blank human plasma and follow the procedure in “(4) Plasma sample pretreatment” to inject 5 µL of the sample and record the chromatogram. A strong methimazole peak should be detected, but no methimazole-d3 peak should be detected. Add the methimazole standard solution and internal standard solution at the lower limit of quantitation (LLOQ) concentration to blank human plasma respectively, and operate according to the method in "(4) Plasma sample pretreatment", inject 5 µL, and record the chromatogram. It should show the detection of obvious methimazole chromatographic peak and strong methimazole-d3 chromatographic peak; (6) Linear range and minimum limit of quantification Take 190 µL of blank human plasma and add 10 µL of methimazole standard series solution to prepare standard curve samples with concentrations equivalent to 1.00, 2.00, 10.0, 20.0, 100, 200, 450, and 500 ng / mL; The rest was performed as described in “(4) Plasma sample pretreatment”, 5 μL was injected, and the chromatogram was recorded; The concentration of the analyte in blank human plasma is used as the horizontal coordinate, and the peak area ratio of the analyte to the internal standard is used as the vertical coordinate. The weighted least square method is used for regression calculation. The linear regression equation obtained is the standard curve. The linearity should meet the measurement requirements. (7) Sample measurement Take 5 μL of the sample to be tested obtained by treating the drug-containing human plasma in "(4) Plasma sample pretreatment" for LC-MS / MS analysis, and calculate the concentration of the analyte methimazole in the plasma using the standard curve and the dilution factor.

[0012] According to the method of the present invention, for example, the mass spectrometer is an API 4500 tandem mass spectrometer produced by AB Sciex.

[0013] According to the method of the present invention, for example, it is equipped with a Turbo Ionspray ionization source and an Analyst 1.6.3 data processing system.

[0014] According to the method of the present invention, for example, the high performance liquid chromatograph is a Shimadzu LC-20AD high performance liquid chromatography system.

[0015] According to the method of the present invention, wherein, for example, the high performance liquid chromatograph comprises a binary gradient pump, a degasser and an automatic sample injector.

[0016] According to the method of the present invention, for example, the particle size of the chromatographic column filler is 2.6 µm, and for example, the specification of the chromatographic column is 30×4.6 mm.

[0017] According to the method of the present invention, wherein, for example, the chromatographic column is Accucore TM C 18 Column, for example, an inline filter is set before the chromatographic column as a guard column.

[0018] According to the method of the present invention, for example, in the gradient elution program, mass spectrometry detection can be cut in at 0.8-1.8 min.

[0019] According to the method of the present invention, the derivatization agent is selected from fluorescamine, dansyl chloride, n-(9-fluorenylmethoxycarbonyloxy)succinimide, and NBD-Cl.

[0020] According to the method of the present invention, the derivatization agent is NBD-Cl.

[0021] According to the method of the present invention, the derivatization reagent in the step "(4) plasma sample pretreatment" is prepared using acetonitrile as solvent.

[0022] According to the method of the present invention, the derivatization reagent in the step "(4) plasma sample pretreatment" is a 5 mg / mL NBD-Cl acetonitrile solution prepared with acetonitrile as solvent.

[0023] According to the method of the present invention, the drug-containing human plasma is the plasma collected from a subject who has taken methimazole.

[0024] According to the method of the present invention, the r value of the standard curve, ie the regression equation, is greater than 0.99, for example, greater than 0.995.

[0025] Furthermore, the second aspect of the present invention provides a method for evaluating the human bioequivalence of an oral methimazole preparation, the method comprising the following steps: determining the methimazole concentration in human plasma collected from an oral test preparation or an oral reference preparation using methimazole using the method described in any one of the first aspects of the present invention, and then comparing the average blood drug concentration-time curves of the test preparation and the reference preparation based on the concentration results, thereby evaluating the human bioequivalence of the test preparation and the reference preparation.

[0026] In the method steps described in the present invention, although the specific steps described therein are different from the steps described in the examples in the specific implementation section below in some details or language descriptions, those skilled in the art can fully summarize the above-mentioned method steps based on the detailed disclosure of the full text of the present invention.

[0027] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not conflict. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical features in other embodiments, as long as they do not conflict. The present invention is further described below.

[0028] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.

[0029] The liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established in the present invention is used to determine the concentration of methimazole in human plasma, and can be used for the bioequivalence evaluation of methimazole oral preparations.

[0030] The method according to the present invention also has the features as described above and below in any embodiment of the present invention.

[0031] In the present invention, NBD-Cl is used to derivatize plasma samples, and the methimazole-NBD generated after derivatization is used as the analyte for detection. The established method has been verified to meet the relevant requirements for biological sample analysis [Pharmacopoeia of the People's Republic of China 2020 edition: Part IV 9012 "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples"], and the verified method has been successfully applied to the bioequivalence evaluation of methimazole in healthy Chinese subjects.

[0032] The method of the present invention exhibits many effects. For example, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established by the present invention determines the concentration of methimazole in human plasma. The method can be applied to the bioequivalence evaluation of methimazole preparations. The sample processing of the method of the present invention adopts a derivatization method and a protein precipitation method to improve the sensitivity and accuracy of the determination. After the plasma sample is derivatized with NBD-Cl for 30 minutes, the concentration of methimazole in the plasma is measured by a Thermo Accucore TM C 18 (2.4µm, 30mm×4.6mm ID) was used as the analytical column, and 0.1% formic acid aqueous solution / acetonitrile was used as the mobile phase for gradient elution at a flow rate of 0.5mL / min. ESI source was used for positive ion detection under multiple reaction monitoring (MRM) mode, and the ion reaction used for quantitative analysis was m / z 278.1→202.1 (methimazole-NBD) and m / z 281.1→205.1 (methimazole-d3-NBD, internal standard).

[0033] Due to the small molecular weight of methimazole (114.17), and the unstable functional group thiol contained in the structure, the retention effect of methimazole in the conventional C18 chromatographic column is poor, and the response in the mass spectrometry is low, and the sensitivity can only meet the test requirements of the solution sample, but cannot meet the test requirements of the plasma sample. When testing the analyte, the present invention first performs NBD-Cl derivatization treatment on the analyte, and the methimazole-NBD generated after derivatization is used as the analyte for detection. The linear range of the analytical method is 1.00~500ng / mL, and the intra-batch and inter-batch precision (CV) are less than 15%, and the accuracy (RE) is between -4.7% and 1.0%.

[0034] In general, the LC-MS / MS method established in the present invention has good sensitivity and accuracy, can be effectively applied to the concentration determination of methimazole in human plasma samples, and provides a reliable experimental basis for clinical diagnosis and treatment monitoring of thyroid-related diseases. The required sample volume is small, the analysis time is short, and it is suitable for human bioequivalence studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : MS / MS spectra of methimazole-NBD (A) and internal standard methimazole-d3-NBD (B).

[0036] Figure 2 : Chromatogram of LC-MS / MS determination of methimazole (I) and internal standard (II) in human plasma (blank human plasma sample).

[0037] Figure 3 : Chromatogram of LC-MS / MS determination of methimazole (I) and internal standard (II) in human plasma (blank human plasma sample with only internal standard added).

[0038] Figure 4 : Chromatogram of LC-MS / MS determination of methimazole (I) and internal standard (II) in human plasma (blank human plasma sample with only upper limit of quantitation (ULOQ) methimazole added, without internal standard).

[0039] Figure 5 : Chromatogram of LC-MS / MS determination of methimazole (I) and internal standard (II) in human plasma (blank human plasma sample with added lower limit of quantification (LLOQ) methimazole and internal standard).

[0040] Figure 6 : Chromatogram of methimazole (I) and internal standard (II) in human plasma determined by LC-MS / MS (plasma samples of volunteers who took methimazole orally and added internal standard).

[0041] Figure 7 :The average blood concentration-time curves of methimazole test preparation (Test, T) and reference preparation (Reference, R) after oral administration on an empty stomach in healthy subjects DETAILED DESCRIPTION

[0042] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible herein. The following examples further illustrate the present invention, rather than limiting the present invention.

[0043] Example 1: Determination of methimazole concentration in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS) 1. Reagents and instruments Methimazole reference substance was purchased from QCC Company of the United States, methimazole-d3 reference substance was purchased from TLC Company of Canada, NBD-Cl was purchased from Tokyo Chemical Industry Co., Ltd., chromatographic grade methanol and acetonitrile were purchased from Fisher Company of the United States; Wahaha purified water was purchased from Hangzhou Wahaha Group Co., Ltd.

[0044] API 4500 tandem mass spectrometer (equipped with Turbo Ionspray ionization source and Analyst 1.6.3 data processing system) from AB Sciex, USA; LC-20AD high performance liquid chromatography system (including binary gradient pump, degasser and autosampler) from Shimadzu, Japan.

[0045] 2. Analysis conditions Chromatographic conditions: The chromatographic column is Accucore TM C 18 column (2.6 µm, 30 × 4.6 mm, Thermo, USA); the guard column was an inline filter; 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and the gradient elution program was: 0.1 min-35% B, 1.0 min-90% B, 1.5 min-90% B, 1.6 min-35% B, 2.6 min-stop (0.8-1.8 min cut into mass spectrometry); The flow rate was 0.5 mL / min; the injection volume was 5 µL; and the column temperature was 40 °C.

[0046] Mass spectrometry conditions: Electrospray ionization source (Turbo Ionspray) was used, positive ion detection was performed, the spray voltage was 5000 V, and the source temperature was 600 o C; curtain gas (CUR) was 20 psi; collision gas (CAD) was 8 psi; scanning mode was multiple reaction monitoring (MRM), and the ion reaction used for quantitative analysis was m / z278.1→ m / z 202.1 (Methimazole-NBD, CE is 30V) and m / z 281.1→ m / z 205.1 (methimazole-d3-NBD, internal standard, CE is 30V), the scanning time is 150msec; the secondary full scan mass spectrum is as follows Figure 1 shown.

[0047] 3. Preparation of stock solution Preparation of methimazole reference substance series solutions: Accurately weigh the methimazole reference substance, correct the weighing factor, add an appropriate volume of acetonitrile to prepare a stock solution with a concentration of 1.0 mg / mL, and store it in a freezer at -20°C; Accurately pipette a certain volume of the stock solution and dilute it with 50% acetonitrile to make a series of standard solutions with concentrations of 20, 40, 200, 400, 2000, 4000, 9000, and 10000 ng / mL, respectively, and store them in a refrigerator at 4°C for future use.

[0048] Preparation of methimazole-d3 internal standard solution: One bottle of methimazole-d3 reference substance (1 mg) was corrected for the weighing factor, and an appropriate volume of acetonitrile was added to completely dissolve it to prepare a 1.0 mg / mL methimazole-d3 stock solution, which was stored frozen at -20°C; Accurately pipette a certain volume of the stock solution, dilute it with 50% acetonitrile to make an internal standard solution with a concentration of 250 ng / mL, and store it in a refrigerator at 4°C for later use.

[0049] 4. Plasma sample pretreatment Take 50 μL of blank human plasma or drug-containing human plasma sample, add 20 μL of internal standard solution (250.0 ng / mL methimazole-d3) and 50 μL of NBD-Cl derivatization reagent (5 mg / mL NBD-Cl acetonitrile solution) in sequence, vortex mix for 10 s, let stand at room temperature for 30 min, add 200 μL acetonitrile and vortex for 2 min, centrifuge at 3500g for 15 min, take 40 μL of supernatant into an injection tube containing 160 μL of 50% acetonitrile aqueous solution, vortex mix and wait for injection analysis, and aspirate 5 μL for LC-MS / MS analysis.

[0050] In this article, unless otherwise specified, the term "drug-containing human plasma" refers to plasma samples containing methimazole collected from healthy volunteers or patients after oral administration of methimazole; in this article, unless otherwise specified, the term "blank human plasma samples" refers to plasma samples collected from healthy volunteers or patients who have not taken methimazole.

[0051] 5. Method Specificity (1) Accurately take 50 µL of blank human plasma and follow the procedure in “4. Plasma sample pretreatment” except that no internal standard solution is added. Inject 5 µL of the sample and record the chromatogram. Figure 2 As shown in the figure, no chromatographic peak was detected in channel I for measuring methimazole, and a weak plasma endogenous substance peak was shown at 1.61 min in channel II for measuring methimazole-d3.

[0052] (2) Add the internal standard solution (250.0 ng / mL methimazole-d3) to blank human plasma and perform the same operation as above. Record the chromatogram. Figure 3 As shown in the figure, no chromatographic peak was detected in the I channel for the determination of methimazole, and a strong methimazole-d3 chromatographic peak was shown in the II channel for the determination of methimazole-d3 at 1.36 min, and a small similar peak was shown at about rt = 1.6 min. Figure 2 II is the plasma endogenous substance peak.

[0053] (3) Add the ULOQ concentration of methimazole standard solution to blank human plasma without adding internal standard and perform the same operation as above. Record the chromatogram. Figure 4 As shown in the figure, channel I for determining methimazole shows a strong methimazole chromatographic peak at 1.38 min, channel II for determining methimazole-d3 does not detect a methimazole peak, and shows tiny plasma endogenous substance peaks at rt=0.82 min and rt=1.63 min.

[0054] (4) Add the methimazole standard solution and internal standard solution at the lower limit of quantitation (LLOQ) concentration to blank human plasma, and perform the same operation as above. Record the chromatogram, as shown in Figure 2. Figure 5 As shown in the figure, the I channel for measuring methimazole shows an obvious methimazole chromatographic peak at 1.38min, and the II channel for measuring methimazole-d3 shows a strong methimazole-d3 chromatographic peak at 1.36min, and a small plasma endogenous substance peak is presented at about 1.6min. It should be noted that the above description is only for convenience. In essence, rt=1.38min is the methimazole-NBD peak, and rt=1.36min is the methimazole-d3-NBD peak. However, when calculating the plasma drug concentration result, it is the methimazole concentration rather than the methimazole-NBD concentration. If not otherwise specified, the present invention has this meaning.

[0055] The above results show that blank human plasma does not interfere with the determination of the analyte and the internal standard, and the analyte and the internal standard do not interfere with each other.

[0056] 6. Linear range and minimum limit of quantification Take 190 µL of blank human plasma and add 10 µL of methimazole standard series solution to prepare standard curve samples with concentrations equivalent to 1.00, 2.00, 10.0, 20.0, 100, 200, 450, and 500 ng / mL; The rest was performed as in "4. Plasma sample pretreatment", 5 µL was injected, and the chromatogram was recorded; The concentration of the analyte in blank human plasma is used as the horizontal coordinate, and the peak area ratio of the analyte to the internal standard is used as the vertical coordinate. The weighted least square method is used for regression calculation. The linear regression equation obtained is the standard curve. The standard curve is: y=0.00911x+0.0000211(r=0.9989). According to the standard curve, the linear range of the methimazole plasma concentration determination method is 1.00~500ng / mL, which can meet the requirements for quantitative detection of methimazole in human plasma for bioequivalence evaluation of different preparations or the same preparation under fasting and postprandial conditions.

[0057] 7. Precision and Accuracy Quality control (QC) samples of 1.00, 3.00, 30.0, 150, and 400 ng / mL prepared from blank human plasma were used to investigate the precision and accuracy of blank human plasma; Take 200 µL of blank human plasma and perform the operation under "4. Plasma sample pretreatment" to obtain the concentration of methimazole in blank human plasma. This concentration is used as the true matrix quantitative lower limit QC sample concentration in the linear range, and then add methimazole quality control solution to prepare the true matrix QC sample; Six samples were analyzed for each concentration, and three analytical batches were measured simultaneously with the standard curve. The concentrations of QC samples of blank human plasma and real matrix were calculated from the standard curve. The precision (CV) and accuracy (RE) of blank human plasma and real matrix were calculated. The results are shown in Table 1.

[0058] Table 1: Precision and accuracy of methimazole in blank human plasma determined by LC-MS / MS (n=18) .

[0059] 8. Extraction recovery and matrix effect Take 3 portions of blank human plasma, perform LC-MS / MS determination directly after following the procedures in “4. Plasma sample pretreatment” to obtain the blank human plasma baseline value (peak area average), and calculate the extraction recovery of the analyte in the real matrix by using the ratio of the peak area measured by the human plasma QC samples at low, medium and high concentrations to the peak area of ​​blank human plasma after pretreatment with solutions of corresponding concentrations (the peak areas are all deducted from the baseline values); The extraction recovery of the analyte in blank human plasma was calculated by the ratio of the peak area measured by the blank human plasma QC samples with low, medium and high concentrations to the peak area of ​​the blank human plasma after pretreatment and adding the corresponding concentration solution; The extraction recoveries of the real human plasma matrix at three concentrations (low, medium, and high (3.00, 150, and 400 ng / mL)) were 99.3±4.9%, 100.0±1.2%, and 106.7±2.5%, respectively, after internal standard correction; Blank human plasma from 6 subjects was pretreated and then added with low and high concentrations of QC solutions. The matrix effect of the real matrix was calculated by the ratio of the peak area of ​​the solution with the corresponding concentration after deducting the corresponding blank human plasma baseline value. The matrix effect of blank human plasma was calculated by the ratio of the peak area of ​​the solution with low and high concentrations of QC solutions added after pretreatment with one batch of blank human plasma. The coefficient of variation (CV) of the internal standard normalized matrix effect factor of methimazole at low and high concentrations (3.00 and 400 ng / mL) in the real matrix of human plasma was 4.4% and 1.9%, indicating that under the analytical conditions selected in this experiment, the influence of the matrix effect on the determination can be ignored.

[0060] 9. Stability The stability of methimazole real plasma matrix samples (3.00 and 400 ng / mL) with low and high concentrations (3.00 and 400 ng / mL) was investigated. The stability results of methimazole real matrix samples are as follows: Stable at room temperature for 22 hours (RE between -9.0% and -1.7%) Stable after 5 freeze-thaw cycles at -60°C or below for 48 hours (RE between 5.3% and 10.0%) After treatment, the sample was placed at room temperature for 71 hours and was stable (RE was between -4.0% and 4.0%). -60℃ long-term storage for 71 days, stable (RE between 4.0% and 10.7%), -20℃ long-term storage for 71 days is stable (RE is between 5.5% and 16.0%). Whole blood is stable at room temperature for 2 hours (RE is between -3.4% and 0.6%).

[0061] 10. Derivatization efficiency Two groups of methimazole samples (methimazole working solution ULOQ concentration level) were prepared at the same time, each group of samples had 3 replicates, one group of samples was derivatized (derivatized group), and one group of samples was not derivatized (underivatized group). The derivatization efficiency was calculated according to the following formula: Derivatization efficiency = (mean peak area of ​​the original analyte in the underivatized group - mean peak area of ​​the original analyte in the derivatized group) / mean peak area of ​​the original analyte in the underivatized group × 100%.

[0062] It has been determined that the derivatization efficiency of the method of this embodiment reaches 99.9%, which can significantly improve the sensitivity of methimazole in blank human plasma.

[0063] From the above Figure 2~Figure 5 It can be seen from the results that methimazole-NBD obtains excellent retention effect under the conditions of this embodiment, and the retention time is extended from the extremely short retention time of methimazole on the C18 chromatographic column, which is almost not retained, to the appropriate retention time of methimazole-NBD. It is well known that if the retention time is too short, the mass spectrometric response of the analyte is more susceptible to interference from plasma endogenous substances. As shown in the figure, the plasma endogenous substance peak presented at about rt=1.6min does not interfere with the analyte at rt=1.38min.

[0064] In addition, by Figure 2~Figure 5 The results show that methimazole and methimazole-d3 present significant and strong chromatographic peaks, and the peak height and peak area of ​​the chromatographic peaks are very high, which indicates that the mass spectrometry response value is quite high, which ultimately improves the detection sensitivity and can complete the determination of low-concentration samples in plasma samples.

[0065] Example 2: Determination of methimazole concentration in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS) The test was performed with reference to Example 1, except that fluorescent amine was used as the derivatization reagent. After adjusting various derivatization conditions, the derivatization efficiency was between 63% and 68%, and the highest did not exceed 70%, indicating that the derivatization efficiency was too low.

[0066] The test was carried out with reference to Example 1, except that dansyl chloride was used as the derivatization reagent. After adjusting various derivatization conditions, the derivatization efficiency was between 74% and 81%, and the highest was no more than 81%. Figure 5 The peak heights of the internal standard substances in the chromatograms measured by II were all lower than 7.6e4.

[0067] The test was carried out in accordance with Example 1, except that n-(9-fluorenylmethoxycarbonyloxy)succinimide was used as the derivatization reagent. After adjusting various derivatization conditions, the derivatization efficiency was between 86% and 93%. Figure 5 The peak height of the internal standard in the chromatogram measured by II was in the range of 3.6e4~4.3e4, and did not exceed 4.5e4; in addition, the chromatographic retention time was moved forward to about 1.04min, which indicated that the chromatographic retention effect of this derivative was poor and the chromatographic determination was easily interfered by endogenous substances.

[0068] The test was carried out with reference to Example 1, except that methanol or ethanol was used as the solvent to prepare the NBD-Cl derivatization reagent, and the derivatization efficiency was 95.3% and 91.2% respectively, which was obviously lower than that using acetonitrile as the solvent.

[0069] In addition, the inventors also compared the ionization degree of methimazole with different mobile phases in additional experiments and found that acetonitrile can significantly promote the ionization of methimazole-NBD and improve the mass spectrometry response. Finally, we chose 0.1% formic acid water-acetonitrile as the mobile phase for gradient elution, and obtained a good chromatographic peak shape. The analysis time for each sample was 2.6 minutes. When acetonitrile was replaced with methanol in the chromatographic conditions, compared with Figure 5 The peak height of the internal standard in the chromatogram measured by II is about 7.3e4, and the retention time of the analyte is about 1.16min. The mass spectrometer response is significantly lower, and the protection time is significantly moved forward. For example, in an additional experiment, when the chromatographic conditions are changed to the following gradient elution program: A is 5mM ammonium acetate containing 0.1% formic acid in water, B is methanol, 0.1min-10%B, 0.5min-80%B, 1.2min-80%B, 1.4min-100%B, 2.6min-stop (0.8-1.8min cut into the mass spectrometer), compared with Figure 5 The peak height of the internal standard in the chromatogram measured by II was about 7.1e4, and the retention time of the analyte was about 1.12min.

[0070] Example 3: Application of the method The LC-MS / MS method established in Example 1 of the present invention has been verified by the detailed methodology described above and complies with the relevant provisions for biological sample determination [Pharmacopoeia of the People's Republic of China 2020 Edition: Part IV 9012 "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples"]. Then, the present invention uses the method of Example 1 to conduct a bioequivalence test of the test preparation (Test, tablets, 10 mg / tablet) and the reference preparation (Reference, tablets, 10 mg / tablet, Thyrozol) of methimazole taken orally on an empty stomach in healthy subjects, and the plasma drug concentration of the subjects before and after taking the drug (20 mg) is determined.

[0071] In this bioequivalence study, the concentration of methimazole in the plasma collected from a volunteer 2 hours after oral administration of methimazole was as shown in the chromatogram. Figure 6 As shown in the figure, channel I for measuring methimazole shows a strong methimazole chromatographic peak at 1.34 min, channel II for measuring methimazole-d3 shows a strong methimazole-d3 chromatographic peak at 1.32 min, and the concentration of methimazole in the plasma is calculated to be 191 ng / mL using the standard curve.

[0072] Figure 7The present invention provides a graph of the average blood drug concentration-time curves of the test preparation (Test, homemade, n=20) and the reference preparation (Reference, n=20) after the subjects orally took the test preparation (Test, homemade, n=20) on an empty stomach in the bioequivalence test using the method of Example 1 of the present invention. From the results in the graph, it can be seen that the average blood drug concentration-time curves of the test preparation and the reference preparation are basically consistent.

[0073] Industrial applicability of the method The LC-MS / MS method established in the present invention has been methodologically validated and complies with the relevant provisions for the determination of biological samples [Pharmacopoeia of the People's Republic of China 2020 Edition: Part IV 9012 "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples"]. This method has been successfully applied to the analysis and detection of bioequivalence test samples of methimazole tablets under fasting and postprandial conditions. Since the methimazole molecule contains functional groups such as thiol and exhibits a large polarity, it faces the problem of insufficient sensitivity in conventional detection. Therefore, the present invention adopts chemical derivatization technology to pre-treat the plasma sample to improve the detection sensitivity and selectivity of the target analyte. Through sample derivatization, the retention and response signal of methimazole are enhanced, so that more accurate and stable results can be obtained in complex biological samples.

[0074] This study aims to determine the concentration of methimazole in human plasma. However, as a thiourea antithyroid drug, methimazole is active in nature. The thiol group in its structure makes it highly polar and poorly retained on a conventional reversed-phase C18 column. This characteristic may be due to the weak interaction of the methimazole molecular structure on the C18 column. On the one hand, its small molecular weight limits the adsorption and retention capacity on the column. On the other hand, the thiol functional group does not interact significantly with the stationary phase of the column, resulting in a short retention time on the column. In solution samples, although the sensitivity can meet the basic testing requirements, in complex biological matrices, especially plasma samples, due to the presence of a large number of endogenous substances and other interfering factors, the low response problem of methimazole is more prominent and difficult to meet the analytical requirements. In order to improve the detection sensitivity and selectivity of methimazole in biological samples, it is necessary to use derivatization or other specific technical means. In view of the above problems, this study used NBD-Cl as a derivatization reagent, protein precipitation and derivatization treatment were performed on plasma samples, and the derivatized samples were analyzed by LC-MS / MS. The analysis results showed that methimazole did not elute in the m / z115.0→m / z88.0 ion pair, while methimazole-NBD successfully elutes in the m / z278.1→m / z202.1 ion pair, and the derivative has a later retention time than the non-derivative. In order to comprehensively evaluate the stability of the derivatized product, its conditions under light protection, light, 4°C and -20°C were further investigated. The experimental results showed that the methimazole-NBD derivative maintained good stability under the above conditions, and more importantly, the derivatization efficiency was as high as 99.9%. This high derivatization efficiency fully confirms the high stability and effectiveness of the derivatization process, greatly improving the detection sensitivity of methimazole in blank human plasma, making it easier to detect and accurately quantify in subsequent analysis.

[0075] Methimazole tablets are quickly absorbed orally, but eliminated slowly from the body, with large individual differences. To meet the needs of methimazole bioequivalence evaluation, this study designed a plasma sample pretreatment step of protein precipitation plus derivatization, which is suitable for the evaluation of methimazole bioequivalence. The LC-MS / MS method established in this study has high sensitivity, small amount of plasma sample, good reproducibility, short analysis time, and can achieve high-throughput analysis. It has been successfully used for the determination of a large number of biological samples in the evaluation of methimazole bioequivalence.

Claims

1. A method for determining the concentration of methimazole in human plasma using liquid chromatography-tandem mass spectrometry, the method comprising the following steps: (1) Provide reagents and instruments Provide methimazole reference substance, methimazole-d3 reference substance, derivatization reagent, mass spectrometer, and high performance liquid chromatograph; (2) Analysis conditions Chromatographic conditions: A chromatographic column using octadecylsilane bonded silica gel as a filler; 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and the gradient elution program was: 0.1 min-35% B, 1.0 min-90% B, 1.5 min-90% B, 1.6 min-35% B, 2.6 min-stop; The flow rate was 0.5 mL / min; the injection volume was 5 µL; the column temperature was 40 °C; Mass spectrometry conditions: Electrospray ionization source was used, positive ion detection was used; the spray voltage was 5000 V; the source temperature was 600 o C; curtain gas was 20 psi; collision gas was 8 psi; scanning mode was multiple reaction monitoring, and the ion reaction used for quantitative analysis was: methimazole-NBD with CE 30 V m / z 278.1→ m / z 202.1、Internal standard methimazole-d3-NBD is CE 30V m / z 281.1→ m / z 205.1, scanning time is 150msec; (3) Preparation of stock solution Preparation of methimazole reference substance series standard solution: Take the methimazole reference substance and dissolve it in acetonitrile to prepare a methimazole reference substance stock solution with a concentration of 1.0 mg / mL, and store it in a freezer at -20°C for later use; accurately pipette a certain volume of the stock solution, dilute it with 50% acetonitrile to a series of standard solutions with concentrations of 20, 40, 200, 400, 2000, 4000, 9000, and 10000 ng / mL, respectively, and store them in a refrigerator at 4°C for later use; Preparation of methimazole-d3 internal standard solution: Take the methimazole-d3 reference substance and dissolve it in acetonitrile to prepare a 1.0 mg / mL methimazole-d3 reference substance stock solution, and store it at -20°C for later use; accurately pipette a certain volume of the stock solution, dilute it with 50% acetonitrile to make an internal standard solution with a concentration of 250 ng / mL, and store it in a refrigerator at 4°C for later use; (4) Plasma sample pretreatment Take 50 μL of blank human plasma or drug-containing human plasma sample, add 20 μL of 250.0 ng / mL methimazole-d3 internal standard solution and 50 μL of derivatization reagent in sequence, vortex mix for 10 seconds, leave at room temperature for 30 minutes, add 200 μL of acetonitrile and vortex for 2 minutes, centrifuge at 3500 g for 15 minutes, take 40 μL of supernatant into an injection tube containing 160 μL of 50% acetonitrile aqueous solution, vortex mix, and use as the sample to be tested. Take 5 μL of the sample to be tested for LC-MS / MS analysis; (5) Method specificity Accurately take 50 µL of blank human plasma and operate according to the method in "(4) Plasma sample pretreatment" except that no internal standard solution is added. Inject 5 µL and record the chromatogram. It should show that no chromatographic peaks are detected at the retention time of methimazole and methimazole-d3; Add 250.0 ng / mL methimazole-d3 internal standard solution to blank human plasma and follow the procedure in "(4) Plasma sample pretreatment" to inject 5 µL of the sample and record the chromatogram. It should show that no methimazole peak is detected, but a strong methimazole-d3 peak is detected. Add the methimazole standard solution at the upper limit of quantitative concentration to blank human plasma and operate according to the method in "(4) Plasma sample pretreatment". Inject 5 µL and record the chromatogram. A strong methimazole peak should be detected, but no methimazole-d3 peak should be detected. Add the methimazole standard solution and internal standard solution at the lower limit of quantitation concentration to blank human plasma respectively, and operate according to the method in "(4) Plasma sample pretreatment", inject 5 µL of the sample, and record the chromatogram. It should show the detection of an obvious methimazole chromatographic peak and a strong methimazole-d3 chromatographic peak; (6) Linear range and minimum limit of quantification Take 190 µL of blank human plasma and add 10 µL of methimazole standard series solution to prepare standard curve samples with concentrations equivalent to 1.00, 2.00, 10.0, 20.0, 100, 200, 450, and 500 ng / mL; The rest was performed as described in "(4) Plasma sample pretreatment", 5 µL of sample was injected, and the chromatogram was recorded; The concentration of the analyte in blank human plasma is used as the horizontal coordinate, and the peak area ratio of the analyte to the internal standard is used as the vertical coordinate. The weighted least square method is used for regression calculation. The linear regression equation obtained is the standard curve. The linearity should meet the measurement requirements. (7) Sample measurement Take 5 μL of the sample to be tested obtained by treating the drug-containing human plasma in "(4) Plasma sample pretreatment" for LC-MS / MS analysis, and calculate the concentration of the analyte methimazole in the plasma using the standard curve and the dilution factor.

2. The method according to claim 1, wherein the mass spectrometer is an API 4500 tandem mass spectrometer equipped with a TurboIonspray ionization source and an Analyst 1.6.3 data processing system.

3. according to the method for claim 1, wherein the high performance liquid chromatograph is a LC-20AD type high performance liquid chromatography system.

4. The method according to claim 3, wherein the high performance liquid chromatograph comprises a binary gradient pump, a degasser and an autosampler.

5. The method according to claim 1, wherein the chromatographic column filler has a particle size of 2.6 μm and the chromatographic column has a specification of 30×4.6 mm.

6. The method according to claim 1, wherein the chromatographic column is Accucore TM C 18 A line filter is set in front of the chromatographic column as a guard column.

7. The method according to claim 1, wherein in the gradient elution program, mass spectrometry detection is cut in at 0.8-1.8 min.

8. The method according to claim 1, wherein the derivatization agent is selected from fluorescamine, dansyl chloride, n-(9-fluorenylmethoxycarbonyloxy)succinimide, and NBD-Cl.

9. The method according to claim 1, wherein the derivatization reagent is a solution of NBD-Cl prepared with acetonitrile as solvent at a concentration of 5 mg / mL.

10. A method for evaluating the bioequivalence of an oral methimazole preparation in humans, the method comprising the following steps: using the method according to any one of claims 1 to 9 to determine the methimazole concentration in human plasma collected from humans after taking an oral test preparation or an oral reference preparation of methimazole, and then comparing the average blood drug concentration-time curves of the test preparation and the reference preparation based on the concentration results, thereby evaluating the bioequivalence of the test preparation and the reference preparation in humans.

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