Method for determining the concentration of methimazole in human plasma

Abstract

The present application relates to the method for determining the concentration of methimazole in human plasma, which uses liquid chromatography-tandem mass spectrometry for determination, comprising the following steps: providing reagents and instruments; providing chromatographic conditions and mass spectrometric conditions for analysis, using C18 chromatographic column, gradient elution with 0.1% formic acid aqueous solution / acetonitrile, using electrospray ionization source, positive ion mode detection; preparing methimazole control product series standard solution and methimazole-d3 internal standard solution; pretreating and derivatizing the plasma sample with derivatizing reagent to obtain the sample to be tested, performing LC-MS / MS analysis; determining that the method meets the specificity requirements, determining the linearity and range, and the minimum limit of quantification; determining the sample and calculating the concentration of methimazole in the plasma sample. The method has high sensitivity, small plasma sample consumption, good reproducibility, short analysis time, can realize high-throughput analysis, and can be used for the determination of a large number of biological samples in methimazole bioequivalence evaluation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for determining methimazole, and more particularly to a method for determining the concentration of methimazole in human plasma. This method uses liquid chromatography-tandem mass spectrometry (commonly referred to as LC-MS / MS or similar terms) for determination. Background Technology

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

[0003] Methimazole is a white to pale yellow crystalline powder with a slight characteristic odor; it is readily soluble in water, ethanol, or chloroform, and slightly soluble in ether; its melting point is 144–147°C. Clinically, it is commonly administered orally in tablets of 5 mg, 10 mg, or 20 mg strength.

[0004] Methimazole is a commonly used antithyroid drug for treating hyperthyroidism, belonging to the thionamide class of drugs. Its mechanism of action is to inhibit thyroid peroxidase, thereby hindering the oxidation of iodides accumulated in the thyroid gland and the coupling of tyrosine, thus inhibiting the synthesis of thyroxine (T4) and triiodothyronine (T3). Animal experiments have shown that it can inhibit the synthesis of antibodies by B lymphocytes, reduce the level of thyroid-stimulating antibodies in the bloodstream, and restore the function of suppressor T cells to normal, achieving the therapeutic goal. [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. Patients typically need to have their thyroid hormone levels monitored regularly during treatment to adjust the dosage and ensure the safety and effectiveness of the treatment [Athanasiadou I, AngelisYS, Lyris E, et al. Chemical derivatization to enhance ionization of anabolic steroids 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 its drug concentration has become particularly important to ensure its efficacy and reduce potential side effects.

[0006] In the past, traditional methods such as HPLC and GC-MS were commonly used to detect the concentration of methimazole in human plasma [Rong Zhengxing, Xie Yifan, Chen Congying, et al. Pharmacokinetics and relative bioavailability of imported and domestically produced 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 administered transdermally in rats[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 with by other components in the plasma. In addition, due to the complex metabolic process of methimazole in the human body, its concentration changes are affected by a variety of factors, and 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 of methimazole in human plasma]. Journal of Pharmaceutical and Biomedical Analysis, 192, 113613).

[0007] LC-MS / MS is widely used as the gold standard for quantitative analysis in biological samples, especially for the determination of drugs in blood / plasma. However, 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 Sci2020;1144:122083;Fang Shaoying, Shao Huarong, Huang Yanni, et al. 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 its small molecular weight (114.16) and the presence of thiol (-SH) functional groups in its molecular structure, methimazole exhibits relatively poor retention in conventional C18 columns. This structural characteristic makes it prone to elution from the column during conventional liquid chromatography, thus affecting its separation efficiency. Furthermore, the presence of thiol groups results in a relatively low response of methimazole in mass spectrometry analysis. Therefore, while its sensitivity in solution samples may meet basic testing requirements, it is insufficient for analyzing complex biological matrices, especially plasma samples. Consequently, analytical methods for methimazole require derivatization or other specific techniques to improve its detection sensitivity and selectivity in biological samples. However, only one of the aforementioned studies mentions this impact, employing a costly liquid-liquid extraction pretreatment technique, which is cumbersome and fails to improve throughput in biological sample analysis.

[0008] As is well known, protein precipitation has significant advantages over liquid-liquid extraction in the processing of biological samples. Firstly, protein precipitation is simple and quick, typically requiring only the addition of a denaturing agent followed by centrifugation; the entire process is relatively short, making it suitable for high-throughput analysis. Secondly, this method requires less solvent, effectively reducing experimental costs. However, current techniques do not document the use of protein precipitation to process biological samples containing methimazole for LC-MS / MS determination.

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

[0010] The purpose of this invention is to provide a method for the quantitative determination of methimazole concentration in human plasma, with the expectation that this method will exhibit one or more superior effects. It has been unexpectedly discovered that treating human plasma biosamples containing methimazole with the derivatization reagent NBD-Cl followed by treatment with a protein precipitant, and then analyzing the resulting solution using LC-MS / MS, enables rapid detection of methimazole in human plasma and exhibits excellent methodological performance. This invention is based on this discovery.

[0011] Therefore, 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 We provide methimazole reference standards, methimazole-d3 reference standards, derivatization reagents, mass spectrometers, and high-performance liquid chromatographs. (2) Analysis conditions Chromatographic conditions: Chromatographic columns using octadecylsilane-bonded silica gel as the packing material; Using 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, 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; and the column temperature was 40 °C. Mass spectrometry conditions: Electrospray ionization (Turbo Ionspray) source was used for detection in a positive ion mode; the spray voltage was 5000V; and the source temperature was 600℃. o C; Curtain gas (CUR) 20 psi; Collision gas (CAD) 8 psi; Scanning mode: Multiple reaction monitoring (MRM); Ion reactions used for quantitative analysis: 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 30V), scan time 150msec; (3) Preparation of stock solution Preparation of a series of methimazole reference standard solutions: Dissolve methimazole reference standard in acetonitrile to prepare a methimazole reference standard stock solution with a concentration of 1.0 mg / mL, and store it frozen at -20℃ for later use; accurately transfer a certain volume of the stock solution and dilute it with 50% acetonitrile to prepare a series of standard solutions with concentrations of 20, 40, 200, 400, 2000, 4000, 9000, and 10000 ng / mL, and store them in a refrigerator at 4℃ for later use; Preparation of methimazole-d3 internal standard solution: Dissolve methimazole-d3 reference standard in acetonitrile to prepare a 1.0 mg / mL methimazole-d3 reference standard stock solution, and store it frozen at -20℃ for later use; accurately transfer a certain volume of the stock solution and dilute it with 50% acetonitrile to prepare a 250 ng / mL internal standard solution, and store it in a refrigerator at 4℃ 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, vortex mix for 10 s, let stand 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 a sample tube containing 160 μL of 50% acetonitrile aqueous solution, vortex mix well, and take 5 μL of the sample to be tested for LC-MS / MS analysis. (5) Method specificity Take 50µL of blank human plasma precisely. Except for the addition of internal standard solution, follow the procedure under “(4) Plasma Sample Pretreatment”. Inject 5µL and record the chromatogram. It should show that no chromatographic peaks were detected at the retention times of methimazole and methimazole-d3. Add the internal standard solution (250.0 ng / mL methimazole-d3) to blank human plasma, and follow the procedure under “(4) Plasma Sample Pretreatment”. Inject 5 µL and record the chromatogram. It should show that no methimazole chromatogram peak was detected, but a strong methimazole-d3 chromatogram peak was detected. Add the methimazole standard solution at the upper limit of quantification (ULOQ) concentration to blank human plasma, and operate according to the method under "(4) Plasma sample pretreatment". Inject 5µL and record the chromatogram. It should show a strong methimazole chromatographic peak, but no methimazole-d3 chromatographic 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 under "(4) Plasma sample pretreatment". Inject 5µL and record the chromatogram. It should show a clear methimazole chromatographic peak and a strong methimazole-d3 chromatographic peak. (6) Linear range and limit of quantitation Take 190µL of blank human plasma and add 10µL of methimazole standard series solutions to prepare standard curve samples with concentrations of 1.00, 2.00, 10.0, 20.0, 100, 200, 450, and 500 ng / mL. For the rest, follow the procedure under “(4) Plasma Sample Pretreatment”, inject 5µL of sample, and record the chromatogram; Using the concentration of the analyte in blank human plasma as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis, a regression calculation was performed using the weighted least squares method. The resulting linear regression equation is the standard curve, and the linearity should meet the measurement requirements. (7) Sample determination Take 5 μL of the sample to be tested obtained by treating human plasma containing the drug in “(4) Plasma Sample Pretreatment” and perform LC-MS / MS analysis. Calculate the concentration of the analyte methimazole in the plasma using the standard curve and dilution factor.

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

[0013] According to the method of the present invention, it is, for example, 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, the high-performance liquid chromatograph includes, for example, a binary gradient pump, a degasser, and an autosampler.

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

[0017] According to the method of the present invention, the chromatographic column is, for example, Accucore. TM C 18 For example, a line filter can be placed in front of a chromatography column as a guard column.

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

[0019] According to the method of the present invention, the derivatizing reagent is selected from fluorescein, dansinyl chloride, n-(9-fluorenylmethoxycarbonyloxy)succinimide, and NBD-Cl.

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

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

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

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

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

[0025] Furthermore, a second aspect of the present invention provides a method for evaluating the human bioequivalence of oral methimazole formulations. The method includes the following steps: measuring the methimazole concentration in human plasma collected after administration of an oral test formulation or an oral reference formulation of methimazole using the method described in any one of the first aspects of the present invention; and comparing the mean blood concentration-time curves of the test formulation and the reference formulation based on the concentration measurement results to evaluate the human bioequivalence of the test formulation and the reference formulation.

[0026] Although the specific steps described in the method steps of this invention differ in some details or language from the steps described in the examples in the detailed embodiments section below, those skilled in the art can fully summarize the above-described method steps based on the detailed disclosure of the entire invention.

[0027] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not contradict each other. Furthermore, any technical feature in any embodiment of any aspect of the present invention can be applied to the same technical feature in other embodiments, as long as they do not contradict each other. The present invention will now be further described.

[0028] All references cited in this invention are incorporated herein by reference in their entirety, and in the event of any inconsistency between the meanings expressed in these references and those expressed herein, the meanings expressed herein shall prevail. Furthermore, the various terms and phrases used in this invention have their general meanings known to those skilled in the art; however, this invention still seeks to provide a more detailed explanation and interpretation of these terms and phrases, and in the event of any inconsistency between the mentioned terms and their known meanings and those expressed herein, the meanings expressed herein shall prevail.

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

[0030] The method according to the invention also has features as described in any embodiment of the invention in the context of this invention.

[0031] In this invention, plasma samples are derivatized using NBD-Cl, and the resulting methimazole-NBD 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 "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples"]. The validated method has been successfully applied to the bioequivalence evaluation of methimazole in healthy subjects in China.

[0032] The method of this invention exhibits numerous advantages. For example, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established in this invention for determining the concentration of methimazole in human plasma can be applied to the bioequivalence evaluation of methimazole preparations. The sample processing method of this invention employs derivatization and protein precipitation to improve the sensitivity and accuracy of the assay. After derivatization with NBD-Cl for 30 min, plasma samples are then analyzed using Thermo Accucore. TM C 18 The analytical column was 2.4 µm, 30 mm × 4.6 mm ID. Gradient elution was performed using a 0.1% formic acid aqueous solution / acetonitrile mobile phase at a flow rate of 0.5 mL / min. Positive ion detection was performed using an ESI source in multiple reaction monitoring (MRM) mode. 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 its small molecular weight (114.17) and the presence of an unstable thiol functional group in its structure, methimazole exhibits poor retention on conventional C18 columns and low response in mass spectrometry. Its sensitivity is only sufficient for testing solution samples, not plasma samples. This invention addresses this issue by first derivatizing the analyte with NBD-Cl, using the resulting methimazole-NBD as the analyte for detection. The linear range of this analytical method is 1.00–500 ng / mL, with intra- and inter-batch precision (CV) both less than 15%, and accuracy (RE) between -4.7% and 1.0%.

[0034] Overall, the LC-MS / MS method established in this invention has good sensitivity and accuracy, and can be effectively applied to the determination of methimazole concentration in human plasma samples, providing reliable experimental evidence for the clinical diagnosis and treatment monitoring of thyroid-related diseases. It requires a small sample volume and has a short analysis time, making it suitable for human bioequivalence studies. Attached Figure Description

[0035] Figure 1 Secondary mass spectra of methimazole-NBD (A) and internal standard methimazole-d3-NBD (B).

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

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

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

[0039] Figure 5 Chromatograms of methimazole (I) and internal standard (II) in human plasma determined by LC-MS / MS (blank human plasma sample with lower limit of quantitation (LLOQ) methimazole and internal standard added).

[0040] Figure 6 Chromatograms of methimazole (I) and internal standard (II) in human plasma determined by LC-MS / MS (plasma samples from volunteers who had been given oral methimazole and had internal standard added).

[0041] Figure 7 Mean plasma concentration-time curves of methimazole after fasting oral administration of the test formulation (Test, T) and the reference formulation (Reference, R) to healthy subjects. Detailed Implementation

[0042] The invention will be further described through the following embodiments; however, the scope of the invention is not limited to the embodiments described below. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The invention provides a general and / or specific description of the materials and methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, the invention is still described in as much detail as possible herein. The following embodiments further illustrate the invention, but are not intended to limit it.

[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 standard was purchased from QCC, USA; methimazole-d3 reference standard was purchased from TLC, Canada; NBD-Cl was purchased from Tokyo Chemical Industry Co., Ltd.; chromatographic grade methanol and acetonitrile were purchased from Fisher Scientific, USA; Wahaha purified water was purchased from Hangzhou Wahaha Group Co., Ltd.

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

[0045] 2. Analysis conditions Chromatographic conditions: The chromatographic column is Accucore. TM C 18 Column (2.6µm, 30×4.6mm, Thermo, USA); guard column is an in-line filter; Using 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, 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-in 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 (Turbo Ionspray) source was used for detection in a positive ion mode; the spray voltage was 5000V; and the source temperature was 600℃. o C; Curtain gas (CUR) 20 psi; Collision gas (CAD) 8 psi; Scanning mode: Multiple reaction monitoring (MRM); Ion reactions used for quantitative analysis: 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 30V), scan time 150 msec; secondary full scan mass spectrum as shown. Figure 1 As shown.

[0047] 3. Preparation of stock solution Preparation of a series of methimazole reference standard solutions: Accurately weigh methimazole reference standard, correct for 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 frozen at -20℃. Accurately transfer a certain volume of the stock solution and dilute it with 50% acetonitrile to prepare a series of standard solutions with concentrations of 20, 40, 200, 400, 2000, 4000, 9000, and 10000 ng / mL, respectively. Store the solutions at 4°C for later use.

[0048] Preparation of methimazole-d3 internal standard solution: One vial (1 mg) of methimazole-d3 reference standard was dissolved in an appropriate volume of acetonitrile after weighing factor correction to prepare a 1.0 mg / mL methimazole-d3 stock solution, which was then stored frozen at -20°C. Accurately transfer a certain volume of the stock solution, dilute it with 50% acetonitrile to prepare an internal standard solution with a concentration of 250 ng / mL, and store it in a refrigerator at 4℃ 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) sequentially, vortex mix for 10 s, let stand 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 a sample tube containing 160 μL of 50% acetonitrile aqueous solution, vortex mix well and wait for injection analysis, and take 5 μL for LC-MS / MS analysis.

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

[0051] 5. Method specificity (1) Accurately take 50µL of blank human plasma. Except for the absence of internal standard solution, follow the procedure under "4. Plasma Sample Pretreatment". Inject 5µL and record the chromatogram. Figure 2 As shown in the figure, no chromatographic peak was detected in channel I for the determination of methimazole, while channel II for the determination of methimazole-d3 showed a weak peak of endogenous plasma substances at 1.61 min.

[0052] (2) Add the internal standard solution (250.0 ng / mL methimazole-d3) to blank human plasma, and proceed as above, recording the chromatogram. Figure 3 As shown in the figure, no chromatographic peak was detected in channel I for methimazole determination, while channel II for methimazole-d3 determination showed a strong methimazole-d3 chromatographic peak at 1.36 min, and a small similar peak at approximately rt=1.6 min. Figure 2 The peak of endogenous substances in plasma of type II.

[0053] (3) Add the methimazole standard solution at the upper limit of quantitation (ULOQ) concentration to blank human plasma, without adding an internal standard, and proceed as described above. Record the chromatogram. Figure 4 As shown in the figure, the I channel for measuring methimazole showed a strong methimazole chromatographic peak at 1.38 min, while the II channel for measuring methimazole-d3 did not detect a methimazole peak and showed small peaks of plasma endogenous substances 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, respectively, and perform the same procedure as above. Record the chromatograms, as follows: Figure 5 As shown in the figure, channel I for methimazole determination shows a distinct methimazole chromatographic peak at 1.38 min, while channel II for methimazole-d3 determination shows a strong methimazole-d3 chromatographic peak at 1.36 min, with a small peak of endogenous plasma substances appearing at approximately 1.6 min. It should be noted that the above description is for convenience only. Essentially, rt=1.38 min represents the methimazole-NBD peak, and rt=1.36 min represents the methimazole-d3-NBD peak. However, when calculating plasma drug concentrations, the methimazole concentration is used, not the methimazole-NBD concentration. Unless otherwise specified, this meaning applies throughout this invention.

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

[0056] 6. Linear range and minimum limit of quantitation Take 190µL of blank human plasma and add 10µL of methimazole standard series solutions to prepare standard curve samples with concentrations of 1.00, 2.00, 10.0, 20.0, 100, 200, 450, and 500 ng / mL. For the rest, follow the procedure under "4. Plasma Sample Pretreatment", inject 5µL, and record the chromatogram; Plotting the concentration of the analyte in blank human plasma on the x-axis and the peak area ratio of the analyte to the internal standard on the y-axis, a weighted least squares regression was performed. The resulting linear regression equation is the standard curve: y = 0.00911x + 0.0000211 (r = 0.9989). According to the standard curve, the linear range of the methimazole plasma concentration assay method is 1.00~500 ng / mL, which can meet the requirements for quantitative detection of methimazole in human plasma for bioequivalence evaluation of different formulations or the same formulation 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 examine the precision and accuracy of the blank human plasma. Take 200µL of blank human plasma and follow the procedure under "4. Plasma Sample Pretreatment". The concentration obtained is the concentration of methimazole in the blank human plasma. Use this concentration as the lower limit of quantification (QC) of the true matrix in the linear range. 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 QC sample concentrations 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 LC-MS / MS method for determining methimazole in blank human plasma (n=18) .

[0059] 8. Extraction recovery rate and matrix effect Take 3 blank human plasma samples, and after following the procedures in "4. Plasma Sample Pretreatment", directly perform LC-MS / MS determination to obtain the baseline value (average peak area) of the blank human plasma. Calculate the extraction recovery rate of the analyte in the real matrix by using the ratio of the peak area measured from the low, medium and high concentration human plasma QC samples to the peak area of ​​the blank human plasma after pretreatment with the corresponding concentration solution (peak area is reduced by the baseline value). The extraction and recovery rate of the analyte in the blank human plasma was calculated by comparing the peak area measured from the blank human plasma QC samples at low, medium and high concentrations with the peak area of ​​the blank human plasma after pretreatment with the corresponding concentration solution. The recoveries of real human plasma matrix at three concentrations (low, medium, and high, 3.00, 150, and 400 ng / mL), after internal standard correction, were 99.3±4.9%, 100.0±1.2%, and 106.7±2.5%, respectively. The matrix effect of the real matrix was calculated by pretreating blank human plasma from six subjects and adding low and high concentrations of QC solution. The peak area of ​​the blank human plasma after subtracting the baseline value was compared with that of the corresponding concentration of solution. The matrix effect of the blank human plasma was also calculated by pretreating one batch of blank human plasma and adding low and high concentrations of QC solution. The coefficient of variation (CV) of the matrix effect factor of the internal standard normalized matrix at two concentrations of methimazole (3.00 and 400 ng / mL) in the real human plasma matrix was 4.4% and 1.9%, respectively, indicating that the matrix effect could be ignored under the analytical conditions selected in this experiment.

[0060] 9. Stability The stability of human plasma real matrix samples at two concentrations of methimazole (3.00 and 400 ng / mL) was investigated. The stability results of the methimazole real matrix samples are as follows: Stable after 22 hours at room temperature (RE between -9.0% and -1.7%) After being stored at -60°C or below for 48 hours, the system is stable after 5 freeze-thaw cycles (RE between 5.3% and 10.0%). The treated samples were stable after 71 hours at room temperature (RE between -4.0% and 4.0%). Stable for 71 days after long-term storage at -60℃ (RE between 4.0% and 10.7%). Stable for 71 days at -20℃ (RE between 5.5% and 16.0%) Whole blood is stable after being left 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 levels) were prepared simultaneously, with three replicates for each group. One group of samples underwent derivatization (derivatized group), and the other group did not undergo derivatization (non-derivatized group). The derivatization efficiency was calculated using the following formula: Derivatization efficiency = (average peak area of ​​underivatized analyte prototypes - average peak area of ​​derivatized analyte prototypes) / average peak area of ​​underivatized analyte prototypes × 100%.

[0062] The derivatization efficiency of the method in this embodiment was measured to be 99.9%, which can significantly improve the sensitivity of methimazole in blank human plasma.

[0063] From the above text Figures 2-5 The results show that methimazole-NBD achieved excellent retention under the conditions of this embodiment, with a prolonged retention time. The retention time of methimazole on the C18 column was extremely short, almost non-existent, to a suitable retention time for methimazole-NBD. As is well known, if the retention time is too short, the mass spectrometric response of the analyte is more easily interfered with by endogenous substances in the plasma. As shown in the figure, the peak of endogenous substances in the plasma at approximately rt=1.6 min does not interfere with the analyte at rt=1.38 min.

[0064] In addition, by Figures 2-5 The results show that methimazole and methimazole-d3 exhibit significant and strong chromatographic peaks with very high peak heights and areas, indicating a high mass spectrometry response value. This ultimately improves the detection sensitivity and enables the determination of low concentrations of plasma samples.

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

[0066] The test was conducted according to Example 1, except that dansinyl chloride was used as the derivatizing reagent. After adjusting various derivatization conditions, the derivatization efficiency ranged from 74% to 81%, with the highest not exceeding 81%. This was compared to... Figure 5 The peak heights of the internal standard in the chromatograms obtained by Part II were all below 7.6e4.

[0067] The test was conducted according to Example 1, except that n-(9-fluorenylmethoxycarbonyloxy)succinimide was used as the derivatizing 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 obtained by II was in the range of 3.6e4 to 4.3e4, and no cases exceeding 4.5e4 were obtained. In addition, the chromatographic retention time shifted to about 1.04 min, which indicates that the chromatographic retention effect of this derivative is poor and the chromatographic determination is easily interfered with by endogenous substances.

[0068] The test was conducted according to Example 1, except that methanol or ethanol was used as the solvent to prepare the NBD-Cl derivatization reagent. The derivatization efficiencies were 95.3% and 91.2%, respectively, which were significantly lower than those using acetonitrile as the solvent.

[0069] Furthermore, in additional experiments, the inventors compared the ionization degree of methimazole with different mobile phases and found that acetonitrile significantly promoted the ionization of methimazole-NBD, improving the mass spectrometry response. Ultimately, we chose 0.1% formic acid-water-acetonitrile as the mobile phase for gradient elution, obtaining good chromatographic peak shapes, with an analysis time of 2.6 min for each sample. However, when acetonitrile was replaced with methanol in the chromatographic conditions, compared to... Figure 5 The chromatogram obtained in Part II showed an internal standard peak height of approximately 7.3e4 and an analyte retention time of approximately 1.16 min, with a significantly lower mass spectrometric response and a significantly earlier protection time. For example, in an additional experiment, when the chromatographic conditions were changed to the following gradient elution program: A was 5 mM ammonium acetate containing 0.1% formic acid aqueous solution, B was methanol, 0.1 min - 10% B, 0.5 min - 80% B, 1.2 min - 80% B, 1.4 min - 100% B, 2.6 min - stop (0.8-1.8 min cut-in to mass spectrometry), compared to... Figure 5 The peak height of the internal standard in the chromatogram obtained by Part II was approximately 7.1e4, and the retention time of the analyte was approximately 1.12 min.

[0070] Example 3: Application of the Method The LC-MS / MS method established in Example 1 of this invention has undergone detailed methodological validation as described above and complies with the relevant regulations for biological sample determination [Pharmacopoeia of the People's Republic of China 2020 Edition: Part IV 9012 "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples"]. Then, using the method of Example 1, this invention conducted a bioequivalence study on the fasting oral administration of methimazole test formulation (Test, tablet, 10 mg / tablet) and reference formulation (Reference, tablet, 10 mg / tablet, Thyrozol) to healthy subjects, and determined the plasma drug concentration before and after the subjects took the drug (20 mg).

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

[0072] Figure 7The invention provides mean plasma concentration-time curves after subjects orally administered the test formulation (Test, self-made, n=20) and the reference formulation (Reference, n=20) on an empty stomach in this bioequivalence trial using the method of Example 1 of the present invention. The results in the figure show that the mean plasma concentration-time curves of the test formulation and the reference formulation are basically consistent.

[0073] Industrial applicability of the method The LC-MS / MS method established in this invention has undergone methodological validation and complies with the relevant regulations for biological sample determination [Pharmacopoeia of the People's Republic of China 2020 Edition: Part IV 9012 "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples"]. This method has been successfully applied to the analysis and detection of methimazole tablets in bioequivalence test samples under fasting and postprandial conditions. Because methimazole contains functional groups such as thiol groups, exhibiting significant polarity, it faces insufficient sensitivity in routine detection. Therefore, this invention employs chemical derivatization technology to pretreat plasma samples, thereby improving the detection sensitivity and selectivity of the target analyte. Sample derivatization enhances the retention and response signal of methimazole, enabling more accurate and stable results in complex biological samples.

[0074] This study aimed to determine the concentration of methimazole in human plasma. However, methimazole, as a thiourea antithyroid drug, is highly reactive, and its thiol group in its structure results in high polarity, leading to poor retention on a conventional reversed-phase C18 column. This characteristic may stem from the weak interaction of the methimazole molecule with the C18 column. On the one hand, its small molecular weight limits its adsorption and retention capacity on the column; on the other hand, the interaction between the thiol functional group and the stationary phase is not significant, resulting in a short retention time. While the sensitivity in solution samples is sufficient for basic testing requirements, the low response of methimazole is more pronounced in complex biological matrices, especially plasma samples, due to the presence of numerous endogenous substances and other interfering factors, making it difficult to meet analytical requirements. To improve the detection sensitivity and selectivity of methimazole in biological samples, derivatization or other specific techniques are necessary. Given the aforementioned problems, this study used NBD-Cl as a derivatization reagent to perform protein precipitation and derivatization treatment on plasma samples, and analyzed the derivatized samples by LC-MS / MS. The analysis results showed that methimazole did not elute in the m / z 115.0→m / z 88.0 ion pairs, while methimazole-NBD successfully elutes in the m / z 278.1→m / z 202.1 ion pairs, and the derivative's retention time was later than that of the underivative. To comprehensively evaluate the stability of the derivatized product, its performance under light-protected, light-illuminated, 4℃, and -20℃ conditions was further investigated. The experimental results showed that the methimazole-NBD derivative maintained good stability under all the above conditions. Most importantly, the derivatization efficiency was as high as 99.9%. This high derivatization efficiency fully demonstrates 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 analyses.

[0075] Methimazole tablets are rapidly absorbed orally but eliminated slowly in vivo, exhibiting significant individual variability. To meet the needs of methimazole bioequivalence evaluation, this study designed a plasma sample pretreatment step combining protein precipitation and derivatization, suitable for evaluating methimazole bioequivalence. The LC-MS / MS method established in this study is highly sensitive, requires small plasma sample volumes, has good reproducibility, and short analysis time, enabling high-throughput analysis. It has been successfully used for the determination of large numbers of biological samples in methimazole bioequivalence evaluation.

Claims

1. A method for determining the concentration of methimazole in human plasma using liquid chromatography-tandem mass spectrometry, comprising the following steps: (1) Provide reagents and instruments The system provides methimazole reference standard, methimazole-d3 reference standard, derivatization reagent, mass spectrometer, and high-performance liquid chromatograph. The derivatization reagent is selected from fluorescent amine, danshen acyl chloride, and n-(9-fluorenylmethoxycarbonyloxy)succinimide. (2) Analysis conditions Chromatographic conditions: Chromatographic columns using octadecylsilane-bonded silica gel as the packing material; Using 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, 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; and the column temperature was 40 °C. Mass spectrometry conditions: Electrospray ionization source was used for detection in a positive ion mode; the spray voltage was 5000V; and the source temperature was 600℃. o C; curtain gas 20 psi; collision gas 8 psi; scanning mode: multiple reaction monitoring; ionic reaction used for quantitative analysis: methimazole-NBD with CE 30V. m / z 278.1→ m / z 202.1, Internal standard methimazole-d3-NBD with CE 30V as... m / z 281.1→ m / z 205.1, scan time 150msec; (3) Preparation of stock solution Preparation of a series of methimazole reference standard solutions: Dissolve methimazole reference standard in acetonitrile to prepare a methimazole reference standard stock solution with a concentration of 1.0 mg / mL, and store it frozen at -20℃ for later use; accurately transfer a certain volume of the stock solution and dilute it with 50% acetonitrile to prepare a series of standard solutions with concentrations of 20, 40, 200, 400, 2000, 4000, 9000, and 10000 ng / mL, and store them in a refrigerator at 4℃ for later use; Preparation of methimazole-d3 internal standard solution: Dissolve methimazole-d3 reference standard in acetonitrile to prepare a 1.0 mg / mL methimazole-d3 reference standard stock solution, and store it frozen at -20℃ for later use; accurately transfer a certain volume of the stock solution and dilute it with 50% acetonitrile to prepare a 250 ng / mL internal standard solution, and store it in a refrigerator at 4℃ for later use. (4) Plasma sample pretreatment Take 50 μL of blank human plasma or drug-containing human plasma, add 20 μL of 250.0 ng / mL methimazole-d3 internal standard solution and 50 μL of derivatization reagent, vortex mix for 10 s, let stand 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 a sample tube containing 160 μL of 50% acetonitrile aqueous solution, vortex mix well, and take 5 μL of the sample to be tested for LC-MS / MS analysis. (5) Method specificity Take 50µL of blank human plasma precisely. Except for the addition of internal standard solution, follow the procedure under "(4) Plasma Sample Pretreatment". Inject 5µL and record the chromatogram. It should show that no chromatographic peaks were detected at the retention times of methimazole and methimazole-d3. Add the 250.0 ng / mL methimazole-d3 internal standard solution to blank human plasma, and operate according to the method under "(4) Plasma Sample Pretreatment". Inject 5 µL and record the chromatogram. It should show that no methimazole chromatogram peak was detected, but a strong methimazole-d3 chromatogram peak was detected. Add the methimazole standard solution with the upper limit of quantitative concentration to blank human plasma, and operate according to the method under "(4) Plasma sample pretreatment". Inject 5µL and record the chromatogram. It should show a strong methimazole chromatographic peak, but no methimazole-d3 chromatographic peak should be detected. Add the methimazole standard solution and internal standard solution with the lower limit of quantitation concentration to blank human plasma respectively, and operate according to the method under "(4) Plasma sample pretreatment". Inject 5µL and record the chromatogram. It should show the detection of obvious methimazole chromatographic peak and strong methimazole-d3 chromatographic peak. (6) Linear range and limit of quantitation Take 190µL of blank human plasma and add 10µL of methimazole standard series solutions to prepare standard curve samples with concentrations of 1.00, 2.00, 10.0, 20.0, 100, 200, 450, and 500 ng / mL. For the rest, follow the procedure under "(4) Plasma Sample Pretreatment", inject 5µL of sample, and record the chromatogram; Using the concentration of the analyte in blank human plasma as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis, a regression calculation was performed using the weighted least squares method. The resulting linear regression equation is the standard curve, and the linearity should meet the measurement requirements. (7) Sample determination Take 5 μL of the sample to be tested obtained by treating human plasma containing the drug in "(4) Plasma Sample Pretreatment" and perform LC-MS / MS analysis. Calculate the concentration of the analyte methimazole in the plasma using the standard curve and 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. The method according to claim 1, wherein the high performance liquid chromatograph is an LC-20AD 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 particle size of the chromatographic column packing is 2.6µm and the column size is 30×4.6mm.

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

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

8. A method for evaluating the bioequivalence of oral methimazole formulations in humans, the method comprising the following steps: determining the concentration of methimazole in human plasma collected after administration of an oral test formulation or an oral reference formulation of methimazole using the method described in any one of claims 1-7; and comparing the mean plasma concentration-time curves of the test formulation and the reference formulation based on the concentration determination results to evaluate the human bioequivalence of the test formulation and the reference formulation.

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