A method for simultaneous detection of multiple statins and their active metabolites

Abstract

The present invention discloses a method for simultaneously detecting multiple statins and their active metabolites, including: step one, establishing a standard curve of a target statin and its active metabolite; step two, pre-treating a blood sample to be tested to obtain a sample to be tested; step three, detecting the sample to be tested by UPLC-MS / MS method, and using the standard curve prepared in step one, quantifying the target statin and its active metabolite in the sample to be tested. The detection method of the present invention can simultaneously detect 11 different polarities and different types of statins and their metabolites, and the detection time can be controlled within 5.5 minutes, which greatly shortens the detection time of statins and their metabolites, and only two internal standard compounds are needed to meet the detection requirements, greatly saving the detection cost.

Description

Technical Field

[0001] The present invention relates to the field of drug detection, and more specifically, to a method for simultaneously detecting a plurality of statins and their active metabolites. Background Art

[0002] Statins are widely used in the clinic to treat hyperlipidemia, and have significant therapeutic effects on primary hyperlipidemia and mixed dyslipidemia. Statins can delay the aggregation and precipitation of arterial plaques in the blood vessel walls, reduce the risk of myocardial infarction and stroke, and are an important tool for primary and secondary prevention of cardiovascular disease. Long-term and high-intensity use of statins often causes serious adverse reactions such as abnormal liver function, muscle pain, and even rhabdomyolysis. Previous studies have found that the efficacy and adverse reactions of drugs are related to the amount of drug exposure in the body. Therefore, it is particularly important to clarify the concentration of drugs in the blood to avoid adverse reactions and evaluate the efficacy of drugs in real time.

[0003] At present, the drug concentration detection method of a single statin has been reported, such as in CN107144648A, CN111983113A, and CN102384954A. However, with the increase in clinical combined medication and drug adjustment, a single drug concentration detection cannot timely evaluate the patient's treatment status. Summary of the invention

[0004] The purpose of the present invention is to provide a method for simultaneously detecting multiple statins and their active metabolites, which meets the clinical demand for detecting combined use of statins.

[0005] In order to achieve the purpose and other advantages of the present invention, a method for simultaneously detecting multiple statins and their active metabolites is provided, comprising:

[0006] Step 1: Establish a standard curve of the target statin and its active metabolites;

[0007] Step 2, pre-treating the blood sample to be tested to obtain the sample to be tested;

[0008] Step 3: Detect the sample to be tested by UPLC-MS / MS method, and use the standard curve prepared in step 1 to quantify the target statin and its active metabolites in the sample to be tested.

[0009] Preferably, in the method for simultaneously detecting multiple statins and their active metabolites, the target statins and their active metabolites include: atorvastatin, 2-hydroxy atorvastatin, 4-hydroxy atorvastatin, rosuvastatin, pravastatin, pitavastatin, fluvastatin, simvastatin, simvastatin acid, lovastatin, and lovastatin acid.

[0010] Preferably, in the method for simultaneously detecting multiple statins and their active metabolites, the pretreatment comprises: after centrifuging the whole blood sample, taking the upper plasma, adding a precipitant containing an internal standard compound, shaking and centrifuging again, and taking the supernatant to obtain the sample to be tested, wherein the volume ratio of the upper plasma to the precipitant is 1:2-5, for example, 1:3, 1:4, etc., the precipitant is 0.05%-0.5% formic acid methanol solution, for example, 0.1% formic acid methanol solution, 0.2% formic acid methanol solution, 0.3% formic acid methanol solution, 0.4% formic acid methanol solution, etc., and the pH value of the precipitant is 2.5-3.5, for example, 3.0.

[0011] Preferably, in the method for simultaneously detecting multiple statins and their active metabolites, the volume ratio of the upper plasma to the precipitant is 1:3, and the precipitant is 0.1% formic acid methanol solution.

[0012] Preferably, in the method for simultaneously detecting multiple statins and their active metabolites, the internal standard compounds are atorvastatin-d5 and rosuvastatin-d6.

[0013] Preferably, the method for simultaneously detecting multiple statins and their active metabolites is performed by using UPLC-MS / MS method.

[0014] Chromatographic conditions: Column: ACQUITY UPLC TM BEH C18 column 50mm×2.1mm 1.7μm; flow rate: 0.2ml / min; injection volume: 1μl; autosampler temperature: 10℃; column temperature: 40℃; mobile phase: A: 0.1% formic acid-10mmol ammonium acetate aqueous solution, B: acetonitrile; gradient elution: 0-3.0min, 55% A, 45% B; 3.0-5.0min, 55%-90% A, the rest is B; 5.0-5.5min, 90-55% A, the rest is B;

[0015] Mass spectrometry conditions: ionization mode: electrospray ionization, positive and negative ion switching mode; mass spectrometry scanning mode: multiple reaction monitoring; capillary voltage: 3.50 kV; source and desolvation temperatures were set at 150 and 350 °C, respectively; purge gas flow rate: 1000 l / h; collision gas flow rate: 0.13 ml / min.

[0016] The present invention has at least the following beneficial effects:

[0017] First, the blood sample pretreatment method of the present invention can be directly injected for detection after treatment without the need for dilution with a diluent, and the injection volume is only 1 μl. The blood sample collection and loading can be controlled within 10 minutes, which greatly saves the pretreatment time of the blood sample.

[0018] Second, the detection method of the present invention requires less blood sample, and only 100ul of whole blood plasma sample is needed to meet the detection requirements.

[0019] Third, the detection method of the present invention uses a specific chromatographic column and cooperates with specific detection conditions to simultaneously detect 11 statins and their metabolites of different polarities and types. The detection time can be controlled within 5.5 minutes, which greatly shortens the detection time of statins and their metabolites. Only two internal standard compounds are needed to meet the detection requirements, greatly saving the detection cost.

[0020] Fourthly, the standard curve range of the detection method of the present invention can completely cover the therapeutic concentration ranges and warning concentrations of 11 statins and their metabolites, thus avoiding as much as possible the problem that actual clinical samples may exceed the detection linear range.

[0021] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0023] It should be noted that the experimental methods described in the following examples are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

[0024] Embodiment 1:

[0025] Establish standard curves for target statins and their active metabolites

[0026] 1. Prepare standard solution

[0027] Accurately weigh a certain amount of target statin and its active metabolite standard, dissolve them in methanol to the stock solution concentration, see the table below, and store at -80℃ for later use. Accurately pipette a certain amount of each standard stock solution, mix them, and dilute them with blank plasma to obtain at least 3 different concentrations of standard sample solutions.

[0028]

[0029] 2. Prepare internal standard compound stock solution

[0030] Accurately weigh a certain amount of atorvastatin-d5 and rosuvastatin-d6 standards and dissolve them in methanol to a stock solution concentration of 2000 ng / ml.

[0031] 3. Prepare standard test solution

[0032] The standard sample solution was pre-treated. Specifically, the standard sample solution was centrifuged at 3000 rpm and 4°C for 3 minutes, 100 μl of the supernatant was taken, 300 μl of the precipitant containing the internal standard compound was added, and then centrifuged for 5 minutes, and the supernatant was taken to obtain the standard test solution. Among them, the internal standard compounds were atorvastatin-d5 and rosuvastatin-d6, the final concentration of the internal standard compound was 20 ng / ml, the precipitant was 0.1% formic acid methanol solution, and the precipitant was adjusted to pH 3.0 using 0.1 mol / L disodium hydrogen phosphate solution.

[0033] 4. Detection

[0034] 1 μl of standard test solution was loaded and tested by UPLC-MS / MS. Testing equipment: Waters I-CLASS UPLC chromatograph, T-QS triple quadrupole mass spectrometer, Waters, USA; Chromatographic conditions: Chromatographic column: ACQUITYUPLC TM BEH C18 column (50mm×2.1mm, 1.7μm); flow rate: 0.2ml / min; injection volume: 1μl; autosampler temperature: 10℃; column temperature: 40℃; mobile phase: A: 0.1% formic acid-10mmol ammonium acetate aqueous solution, B: acetonitrile; gradient elution: 0-3.0min, 55% A, 45% B; 3.0-5.0min, 55%-90% A, the rest is B; 5.0-5.5min, 90-55% A, the rest is B; mass spectrometry conditions: ionization mode: electrospray ionization, positive and negative ion switching mode (ESI+-ESI-); mass spectrometry scanning mode: multiple reaction monitoring (MRM); capillary voltage is 3.50kV; source and desolvation temperatures are set at 150 and 350℃ respectively; purge gas (nitrogen) flow rate is 1000l / h; collision gas (argon) flow rate is 0.13ml / min; among them, atorvastatin, rosuvastatin, 2-hydroxy atorvastatin, 4-hydroxy atorvastatin, pitavastatin are in positive ion scanning mode; fluvastatin, pravastatin, simvastatin, lovastatin, simvastatin acid, lovastatin acid are in negative ion scanning mode. The detection result is the ratio of the response value of the target compound (target statin and its active metabolite) to the response value of the internal standard compound.

[0035] The concentration of the target compound is used as the horizontal axis, and the ratio of the response value of the target compound to the response value of the internal standard compound is used as the vertical axis to make a standard curve, as shown in the table below.

[0036]

[0037]

[0038] It can be seen that within the above concentration range, the linearity of the target statin and its active metabolites is good, which can be used to determine the content of the target statin and its active metabolites in whole blood samples, and the quantitative range can fully meet the content requirements of the target statin and its active metabolites in clinical samples.

[0039] The mass spectrometry detection parameters and retention times of the target statins and their active metabolites are shown in the table below.

[0040]

[0041] It can be seen that in the above detection method, the target statins and their active metabolites can all be eluted and successfully separated, and the target statins and their active metabolites will not interfere with each other, so accurate measurement can be achieved.

[0042] Embodiment 2:

[0043] Precision, accuracy and recovery test

[0044] 1. Prepare standard quality control samples

[0045] The standard stock solution prepared in Example 1 was diluted using blank plasma to obtain standard quality control samples of different concentrations, as shown in the table below.

[0046]

[0047]

[0048] 2. Preparation of plasma quality control samples

[0049] The target statins and their active metabolites corresponding to the standard quality control concentration groups H, M and L were mixed to obtain three different concentrations of plasma quality control sample solutions.

[0050] 3. Prepare the plasma quality control sample test solution by referring to the pretreatment method in Example 1.

[0051] 4. Perform detection according to the detection method in Example 1.

[0052] The precision, accuracy and recovery of plasma quality control samples are shown in the table below.

[0053]

[0054] It can be seen that the above detection method has good accuracy in plasma quality control samples, with absolute recoveries higher than 85%, high precision, and RSD between -15 and 15, which meets the detection requirements of various indicators.

[0055] Comparative Example 1:

[0056] A certain amount of standard stock solutions of 11 target statins and their active metabolites were precisely drawn and mixed, and diluted with blank plasma to obtain plasma sample solution, and plasma sample test solution was prepared according to the pretreatment method in Example 1, and detection was performed according to the detection method in Example 1. During the pretreatment, methanol, 0.05% formic acid methanol solution, 0.1% formic acid methanol solution, 0.2% formic acid methanol solution and 0.5% formic acid methanol solution were used as precipitants, respectively.

[0057] The average absolute recoveries of each target statin and its active metabolites were calculated and the results are shown in the table below.

[0058]

[0059] It can be seen that adding 0.05%-0.5% formic acid to methanol can help improve the recovery rate of each target statin and its active metabolites. Among them, adding 0.1% formic acid to methanol can most significantly improve the recovery rate of each target statin and its active metabolites.

[0060] Comparative Example 2:

[0061] The standard quality control samples of atorvastatin and rosuvastatin were mixed according to groups H, M and L, respectively, to obtain three test samples of different concentrations, and the test samples were pre-treated to prepare test sample test solutions, wherein the pre-treatment was carried out in two groups, the first group was treated with reference to the pre-treatment method in Example 1, that is, the internal standard compound was first added to the precipitant and mixed, and then the precipitant containing the internal standard compound was added to the supernatant, and the second group first added the same amount of internal standard compound as the first group to the supernatant, and then added the same amount of precipitant as the first group. The concentrations of atorvastatin and rosuvastatin in the test samples were detected with reference to the detection method in Example 1 and the intra-day precision was calculated, and the results are shown in the following table.

[0062]

[0063]

[0064] It can be seen that adding the internal standard compound to the precipitant and mixing it, and then adding the precipitant containing the internal standard compound to the supernatant, the day-to-day precision of the analyte is higher, which is more helpful for routine detection. In addition, adding the internal standard compound to the precipitant and mixing it first can also reduce the sampling time during operation, reduce operational errors, improve detection efficiency, and improve reproducibility and stability.

[0065] Comparative Example 3:

[0066] The preparation method of whole blood quality control sample is the same as that of Example 2, except that:

[0067] The pretreatment method is as follows: take 200ul of whole blood quality control sample, add 100ul of 0.1mol / L ammonium acetate solution (PH=4) and 1ml of ethyl acetate, shake for 5min, centrifuge at 14000rpm for 15min, take the upper organic phase, vacuum concentrate at 40℃ for 30min, add 200ul of methanol, vortex for 3min to re-dissolve, centrifuge at 14000rpm for 5min, and the injection volume is 10ul.

[0068] Testing equipment: Waters I-CLASS UPLC chromatograph, T-QS triple quadrupole mass spectrometer, Waters, USA. Chromatographic column: ACQUITY UPLC TM BEH C18 column (50mm×2.1mm, 1.7μm); flow rate: 0.2ml / min; injection volume: 10μl; autosampler temperature: 10℃; column temperature: 40℃; mobile phase: A: 0.2% formic acid aqueous solution, B: methanol; gradient elution: 0-3.0min, 35% A; 3.0-3.5min, 5% A; 3.5-4min, 5% A; 4.0-6.0min, 35% A. Mass spectrometry conditions: ionization mode: electrospray ionization, positive ion mode (ESI+); mass spectrometry scanning mode: multiple reaction monitoring (MRM); capillary voltage of 3.50kV; source and desolvation temperatures were set at 150 and 550℃, respectively; purge gas (nitrogen) flow rate was 1000l / h; collision gas (argon) flow rate was 0.13ml / min.

[0069] The detection method provided in Comparative Example 3 requires longer sample pretreatment time and analysis time, but the recovery rate is lower, and the target statin and its active metabolites are lost more during the pretreatment process. If the content of the target statin and its active metabolites in the patient sample is low, the sample pretreatment method and mass spectrometry detection method in Comparative Example 3 will not be able to accurately quantify. When testing batch samples, the accuracy of all substances to be tested cannot be guaranteed.

[0070] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the method of the present invention for simultaneously detecting multiple statins and their active metabolites are obvious to those skilled in the art.

[0071] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for simultaneously detecting multiple statins and their active metabolites, characterized in that: include: Step 1: Establish a standard curve of the target statin and its active metabolites; Step 2, pre-treating the blood sample to be tested to obtain the sample to be tested; Step 3: Detecting the sample by UPLC-MS / MS method, and using the standard curve prepared in step 1 to quantify the target statin and its active metabolites in the sample; The target statins and their active metabolites include: atorvastatin, 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, rosuvastatin, pravastatin, pitavastatin, fluvastatin, simvastatin, simvastatin acid, lovastatin and lovastatin acid; The pretreatment comprises: after centrifuging the whole blood sample, taking the upper plasma, adding a precipitant containing an internal standard compound, shaking and centrifuging again, and taking the supernatant to obtain the sample to be tested, wherein the volume ratio of the upper plasma to the precipitant is 1:2-5, the precipitant is a 0.05%-0.5% formic acid methanol solution, and the pH value of the precipitant is 2.5-3.5; When UPLC-MS / MS method is used for detection, Chromatographic conditions: Column: ACQUITY UPLC TM BEH C18 column 50 mm×2.1 mm 1.7 μm; flow rate: 0.2 ml / min; injection volume: 1 μl; autosampler temperature: 10°C; column temperature: 40°C; mobile phase: A: 0.1% formic acid-10 mmol ammonium acetate aqueous solution, B: acetonitrile; gradient elution: 0-3.0 min, 55% A; 3.0-5.0 min, 55%-90% A; 5.0-5.5 min, 90-55% A; Mass spectrometry conditions: ionization mode: electrospray ionization, positive and negative ion switching mode; mass spectrometry scanning mode: multiple reaction monitoring; capillary voltage: 3.50 kV; source and desolvation temperatures were set at 150 and 350 °C, respectively; purge gas flow rate: 1000 l / h; collision gas flow rate: 0.13 ml / min.

2. The method for simultaneously detecting multiple statins and their active metabolites according to claim 1, characterized in that: The volume ratio of the upper plasma to the precipitant is 1:3, and the precipitant is 0.1% formic acid methanol solution.

3. The method for simultaneously detecting multiple statins and their active metabolites according to claim 1, characterized in that: The internal standard compounds are atorvastatin-d5 and rosuvastatin-d6.

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