Method for determining rosaxostat in human plasma

Abstract

The invention discloses a method for detecting the concentration of rosaxostat in human plasma based on an LC-MS / MS technology. The method is easy and convenient to operate, simple in pretreatment and short in mass spectrum collection time (3 min), all verification indexes meet the requirements of laws and regulations, rosaxostat can be accurately quantified, laboratory operation is simplified and facilitated to the maximum extent, and clinical requirements are met.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular to a method for determining rosuvastatin in human plasma. Background Art

[0002] Roxadustat capsules are the world's first small molecule hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) drug developed to treat renal anemia. The physiological effects of hypoxia-inducible factor (HIF) not only increase the expression of erythropoietin, but also increase the expression of erythropoietin receptors and proteins that promote iron absorption and circulation. Roxadustat can inhibit prolyl hydroxylases PHD1, PHD2, and PHD3 in vitro, and can lead to rapid and reversible activation of hypoxia-inducible factor-α (HIF-α) in Hep3B cell line derivative 1G6 cells, which can induce increased erythropoietin (EPO) levels in Hep3B cells. Roxadustat inhibits prolyl hydroxylase (PH) enzymes by simulating ketoglutarate, one of the substrates of PH, affecting the role of PH enzymes in maintaining the balance between HIF generation and degradation rates, thereby achieving the purpose of correcting anemia. The launch of Roxadustat capsules provides a new treatment for patients with anemia caused by chronic kidney disease. In December 2018, the National Medical Products Administration approved the marketing of rosuvastatin capsules through the priority review and approval procedure.

[0003] At present, the common method for detecting rosuvastatin therapeutic drugs is high performance liquid chromatography, which has problems such as complex pretreatment, inaccurate qualitative and quantitative analysis, high cost, long analysis time, or high technical requirements for experimental personnel. Liquid chromatography tandem mass spectrometry (LC-MS / MS) is increasingly widely used in various fields such as medicine, food, environment, forensics, and clinical. LC-MS / MS has the characteristics of high sensitivity, strong selectivity, and good accuracy. Its application range in clinical testing far exceeds that of radioimmunoassay and chemical detection, which is unmatched by other methods. The LC-MS / MS method is used to simultaneously determine the concentration of rosuvastatin in plasma and the functional relationship between blood drug concentration and time, and to calculate pharmacokinetics and bioavailability, which is more practical. The method established in this paper can simultaneously determine rosuvastatin in plasma and can be used for pharmacokinetic and bioequivalence studies of rosuvastatin preparations. Summary of the invention

[0004] The purpose of the present invention is to provide a method for quantitatively detecting rosuvastatin in plasma using high performance liquid chromatography tandem mass spectrometry. The detection method has simple pretreatment, short mass spectrometry acquisition time, low cost, and is convenient for laboratories to conduct large-scale clinical sample detection.

[0005] The method of the present invention for quantitatively detecting rosuvastatin in plasma by using high performance liquid chromatography tandem mass spectrometry (LC-MS / MS) technology comprises the following steps:

[0006] (1) Plasma sample pretreatment

[0007] Under yellow light and room temperature, 50.0 μL of the sample to be tested was transferred to a 2 mL centrifuge tube, and 10 μL of deuterated mixed internal standard working solution was added, and 340 μL of acetonitrile solution was added. The sample was vortexed for 5 minutes, and then centrifuged at 10000 rpm and 4°C for 5 minutes. 100 μL of the supernatant was transferred to a 96-well plate with a pipette; 500 μL of acetonitrile: water (1:1) was added, vortexed for 3 minutes, and centrifuged at 4000 rpm and 4°C for 5 minutes to obtain the sample to be tested;

[0008] (2) Preparation of standard curve and quality control plasma samples

[0009] Acetonitrile: water (1:1) solution was used to prepare standard curve and quality control working solutions, wherein the working solution included rosuvastatin working solution. 10 μL of the above working solution was mixed with 490 μL of blank plasma matrix to prepare standard curve samples and quality control samples of different concentrations. The standard curve was established using the peak area ratio of rosuvastatin and the internal standard and the concentration of the standard sample to obtain the corresponding linear regression equation.

[0010] (3) Liquid chromatography-mass spectrometry detection

[0011] ① Liquid chromatography conditions: Chromatographic column: ACE EXCEL 5C18 (50*2.1mm);

[0012] Mobile phase: aqueous phase: 0.1% formic acid in water, organic phase: 0.1% formic acid in acetonitrile, needle washing solution: methanol: water (1:9);

[0013] Column temperature: 35°C; Autosampler temperature: 4°C;

[0014] Aqueous phase and organic phase were used as mixed mobile phase for gradient elution. The collection time for each sample was 3 min, the injection volume was 2 mL, the flow rate was 0.4 mL / min, and the mobile phase gradient elution parameters were as follows: 0 min, 48% organic phase; 1.8 min, 95% organic phase; 2.3 min, 95% organic phase; 2.31 min, 48% organic phase; 3 min, stop.

[0015] ②Mass spectrometry conditions: Under the electrospray ionization detection mode, the mass spectrometry scanning mode of selected reaction monitoring was adopted, in which rosuvastatin was in positive ion mode, the ionization voltage was 3500 V, the sheath gas was 60 Arb, the auxiliary gas (AuxGas) was 15 Arb, the sweep gas was 2 Arb, the ion transfer tube temperature (Ion Transfer Tube Temp) was 380°C, the evaporator temperature (Vaporizer Temp) was 350°C, the cycle time (Cycle Time) was 0.8 sec, and the ion pairs were: m / z 353.2→278.17 (rosuvastatin), m / z 358.18→283.13 (rosuvastatin-d5).

[0016] (4) Determination of the concentration of each substance in the plasma to be tested

[0017] The plasma to be tested is prepared according to the sample preparation method of step (1), and the sample to be tested is tested by liquid chromatography-mass spectrometry detection according to step (3). The peak area ratio of rosuvastatin and each internal standard is substituted into the constructed standard curve to calculate the concentration of rosuvastatin in the plasma to be tested.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects:

[0019] The present invention selects formic acid acetonitrile as the organic phase and 0.1% formic acid water as the aqueous phase instead of using an aqueous solution with added buffer salt as the mobile phase, thereby avoiding the damage of the buffer salt to the chromatographic column and the influence on the response of the compound.

[0020] The use of buffer salts can easily cause the buffer salts to precipitate in the chromatographic column and block the pores between the plug plate and the bonded phase particles. Buffer salts are usually soluble in water but difficult to dissolve in organic solvents. When using a mobile phase containing buffer salts (especially when the water used as the mobile phase is a saturated buffer salt solution) for analysis, if the proportion of water in the mobile phase used to preserve the chromatographic column before analysis is relatively small and is not rinsed off first, and the organic solvent content in the mobile phase used for the next sample is large, and the water contained in the proportion is not enough to dissolve the buffer salts, the buffer salts will precipitate and settle on the chromatographic column. Before using buffer salts as mobile phases, the chromatographic column needs to be rinsed with a mobile phase without buffer salts until the baseline is stable. If there is a high concentration of buffer salts in the mobile phase, since the salts are composed of anions and cations, their ability to gain or lose charge in ESI is often stronger than that of the analyte, so they will gain / lose charge first, thus competing with the analyte, reducing the ionization efficiency of the analyte, causing ion suppression, and reducing the response of the analyte. Since rosuvastatin is sensitive to acid, a simpler mobile phase was considered. Therefore, adding formic acid to acetonitrile can improve the compound tailing and provide a higher response compared to using pure acetonitrile as the organic phase. It can also avoid the damage of the buffer salt to the chromatographic column and the effect on the compound response.

[0021] The pretreatment method of rosuvastatin plasma sample of the present invention adopts the precipitation protein method, which is easy to operate, highly sensitive, and has a short single sample analysis time, which greatly reduces the cost of consumables, shortens the operation time, and reduces the analysis cost. The quantitative results of using stable isotope internal standard are accurate, the mass spectrometry acquisition time is short (3min), and all verification items meet regulatory requirements, can accurately quantify rosuvastatin, simplify and facilitate laboratory operations to the greatest extent, and meet clinical needs. We evaluated the effects of methanol and acetonitrile on matrix effect and recovery rate, and the results showed that acetonitrile, as a precipitant, has advantages over methanol in terms of extraction recovery rate and matrix effect, so acetonitrile was finally selected as the final precipitant. Under the conditions of the present invention, rosuvastatin can be accurately quantified, can be used for clinical pharmacokinetic research, and provides a stable, reliable and simple detection method for corresponding drug concentration monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the standard curve of rosuvastatin;

[0023] Figure 2 This is the liquid chromatogram of roxadustat with 0.1% formic acid water as the aqueous phase in Example 1;

[0024] Figure 3 This is the liquid chromatogram of roxadustat in Comparative Example 1 with 5 mM ammonium acetate aqueous solution as the aqueous phase. DETAILED DESCRIPTION

[0025] The following is a detailed description of the specific implementation mode by way of examples, which further describes the above content of the present invention in detail. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0027] 1. Main instruments

[0028] Thermo TSQ Altis triple quadrupole mass spectrometer, equipped with Vanquish UHPLC ultra-high performance liquid phase system (Thermo); XPR2 one-millionth balance (Mettler, Switzerland); one-tenth balance (Shuangjie Electronics); Milli-Q water purifier (Merck Chemical); Centrifuge 5810R centrifuge (Eppendorf, Hettich); adjustable pipettes (Eppendorf 2-20μL, 10-100μL, 20-200μL, 100-1000μL); 96-well plates, sample bottles, etc.

[0029] 2. Blank Matrix, Reagents and Reference Substances

[0030] The blank matrix used in the present invention comes from Hunan Yiyang Kangya Hospital and Changsha Taihe Hospital; acetonitrile (HPLC, Merck), water is homemade ultrapure water, methanol (HPLC, Merck), formic acid (HPLC, aladdin); rosuvastatin (RS22060101-1, content 99.8%), rosuvastatin-d5 (TLC, 3860-052A3, content 96.1%).

[0031] Example 1: Experimental process

[0032] 1. Plasma Sample Pretreatment

[0033] Pipette 50.0 μL of plasma sample (blank sample and internal standard blank sample plus 50.0 μL of blank plasma) into a 96-well plate, add 10.0 μL of IS-C (500 ng / mL) solution (blank sample adds 10.0 μL of acetonitrile: water (1:1)); add 340 μL of acetonitrile solution respectively, vortex the sample for 5 minutes, centrifuge at 4000 rpm and 4°C for 5 minutes, use a pipetting workstation to transfer 100 μL of supernatant to a 96-well plate; add 500 μL of acetonitrile: water (1:1) to mix evenly, vortex for 5 minutes, centrifuge at 4000 rpm and 4°C for 5 minutes, and analyze by LC-MS / MS.

[0034] 2. Preparation of standard curve working solution and quality control working solution

[0035] (1) Preparation of rosuvastatin stock solution and working solution:

[0036] ①Rosuvastatin stock solution SS-A

[0037] Accurately weigh the rosuvastatin reference substance, dissolve it in an appropriate amount of DMSO, and prepare a stock solution SS-A with a concentration of 2.00 mg / mL (using a correction factor).

[0038] ②Rosuvastatin stock solution B (SS-B)

[0039] Take 1.60 mL of SS-A and add it to 1.60 mL of acetonitrile: water (1:1) solution, mix well to prepare stock solution SS-B (1.00 mg / mL).

[0040] ③Rosuvastatin stock solution C (SS-C)

[0041] Take 0.100 mL of SS-B and add it to 1.90 mL of acetonitrile: water (1:1) solution, mix well to prepare the stock solution SS-C (50000 ng / mL).

[0042] According to Table 1 below, acetonitrile: water (1:1) was used as solvent to prepare the standard curve working solution and the quality control working solution. The standard curve working solution and the quality control working solution came from different stock solutions.

[0043] Table 1: Standard curve working solution and quality control working solution

[0044]

[0045] 3. Preparation of internal standard stock solution and working solution:

[0046] Take rosuvastatin-d5 reference substance, dissolve it with an appropriate amount of DMSO, and prepare an internal standard stock solution IS-A with a concentration of 2.00 mg / mL (using a correction factor).

[0047] Take 0.0100 mL of IS-A, add it to 0.99 mL of acetonitrile: water (1:1) solution, mix well to prepare the internal standard stock solution IS-B (20.0 μg / mL).

[0048] Take 0.100 mL of IS-B and add it to 3.90 mL of acetonitrile: water (1:1) solution, mix well to prepare the internal standard working solution IS-C (500 ng / mL).

[0049] 4. Preparation of standard curve plasma samples and quality control plasma samples:

[0050] Take 10 μL of the above working solution and mix with 490 μL of blank plasma matrix to prepare standard curve plasma samples and quality control plasma samples of different concentrations. The quantitative range of rosuvastatin is 5.00 ng / mL to 8000 ng / mL, and the standard curve consists of 8 standard samples with concentrations of 5.00 ng / mL (LLOQ level), 10.0 ng / mL, 90.0 ng / mL, 800 ng / mL, 1800 ng / mL, 4500 ng / mL, 7000 ng / mL, and 8000 ng / mL (ULOQ level). The concentrations of quality control samples were as follows: lower limit of quantification (LLOQ, 5.00 ng / mL), low concentration (LQC, 15.0 ng / mL), geometric mid-concentration (GMQC, 300 ng / mL), mid-concentration (MQC, 3200 ng / mL), and high concentration (HQC, 6000 ng / mL). As shown in Table 2 below:

[0051] Table 2: Standard curve plasma samples and quality control plasma samples

[0052]

[0053] 4.HPLC-MS / MS detection

[0054] (1) Liquid chromatography conditions

[0055] Mobile phase (aqueous phase: 0.1% formic acid in water, organic phase: 0.1% formic acid in acetonitrile, needle washing solution: methanol: water (1:9));

[0056] Chromatographic column: ACE EXCEL 5C18 (50*2.1mm);

[0057] Column temperature: 35°C; Autosampler temperature: 4°C;

[0058] Aqueous phase and organic phase were used as mixed mobile phase for gradient elution. The elution parameters are shown in Table 3. The collection time for each sample was 3 min, the injection volume was 2 mL, and the flow rate was 0.4 mL / min.

[0059] Table 3: Mobile phase gradient elution parameters

[0060] Time (min) 0 1.8 2.3 2.31 3.0 %Organic phase 48 95 95 48 stop

[0061] The liquid chromatogram of rosuvastatin with 0.1% formic acid water as the aqueous phase was obtained, as shown in Figure 2 shown.

[0062] (2) Mass spectrometry conditions

[0063] In the electrospray ionization detection mode, the mass spectrometry scanning mode of selected reaction monitoring was adopted, and the positive ion mode was used for rosuvastatin. The corresponding mass spectrometry parameters of the analyte and the internal standard are shown in Table 4.

[0064] Table 4: Mass spectrometry parameters

[0065]

[0066] 6. Determination of the concentration of each substance in the plasma to be tested:

[0067] After the plasma to be tested is pretreated, the sample to be tested is tested by liquid chromatography-mass spectrometry, and the peak area ratio of rosuvastatin and the internal standard and the concentration of the standard sample are used to establish a standard curve to obtain the corresponding linear regression equation, such as Figure 1 As shown, the peak area ratio of roxadustat and each internal standard was substituted into the constructed standard curve to calculate the concentration of roxadustat in the plasma to be tested.

[0068] Example 2: Methodological Investigation

[0069] (1) Accuracy and precision test: 10 μL of the corresponding working solution was mixed with 490 μL of blank matrix to prepare four different concentrations of quality control samples: LLOQ, LQC, MQC, and HQC. Six replicates were performed for each concentration. The freshly prepared standard curve was used for determination. The results showed that the intra-batch accuracy deviation of the four concentrations of rosuvastatin quality control samples: high, medium, geometric medium, and low was between -6.00% and 3.67%, and the intra-batch precision was between 0.83% and 3.49%. The inter-batch accuracy deviation was between -4.00% and 3.00%, and the inter-batch precision was between 1.86% and 2.96%. The accuracy and precision of rosuvastatin met the corresponding acceptance criteria.

[0070] (2) Matrix effect: Blank samples were prepared from blank human plasma from at least 6 different sources. After pretreatment, a certain amount of the analyte and internal standard were added to make their final concentrations consistent with the injection concentrations of LQC and HQC, respectively. At the same time, pure solutions containing a certain amount of the analyte and internal standard were prepared, and their final concentrations were consistent with the injection concentrations of LQC and HQC, respectively. Each concentration was measured in parallel for at least 6 copies. For each source matrix, the matrix factor of each analyte and internal standard should be calculated by calculating the peak area in the presence of the matrix (measured by adding the analyte and internal standard after pretreatment of blank human plasma) and the average value of the corresponding peak area of ​​the sample without matrix (pure solution of the analyte and internal standard). Further, the matrix factor normalized by the internal standard was calculated by dividing the matrix factor of the analyte by the matrix factor of the internal standard. The results showed that the matrix effect factors of rosuvastatin normalized by the internal standard were 1.00 and 1.00, respectively, and the %CV were 0.56 and 1.38, respectively.

[0071] (3) Extraction recovery test: 6 replicates of LQC, MQC, HQC and 18 mixed blank plasma samples were processed together. After processing, the solution containing the analyte and internal standard was added to the blank sample so that its final concentration was consistent with the injection concentration of the high, medium and low quality control samples. The recovery was evaluated by comparing the response value of the analyte or internal standard in a single quality control sample with the average response value of the blank sample to which the analyte and internal standard were added after pretreatment. The results showed that the average extraction recoveries of the three quality control samples of high, medium and low concentrations of rosuvastatin were 84.47%, 88.44% and 88.35%, and the average extraction recovery of the internal standard was 94.64%, indicating that rosuvastatin had sufficient and relatively uniform extraction recovery within the quantitative range of this method, and the internal standard had a higher recovery.

[0072] (4) Stability test: The stability test examined the stability samples after being placed under different conditions (pretreatment, preparation, freeze-thaw cycle, long-term stability, whole blood) for a period of time. The deviation between the stability samples and the theoretical concentration was quantified using a freshly prepared standard curve. The results showed that the rosuvastatin matrix samples remained stable after being placed under different conditions for a period of time.

[0073] Comparative Example

[0074] The concentration of roxadustat in plasma was detected according to the method of Example 1, which differed from Example 1 only in that the aqueous phase in the liquid chromatography conditions was a 5 mM ammonium acetate aqueous solution, and a liquid chromatogram was obtained, as shown in FIG. Figure 3 As shown. Figure 2 and Figure 3 It can be seen from the comparison that when 0.1% formic acid water is used as the water phase, the response of samples with the same concentration is higher compared with 5 mM ammonium acetate aqueous solution as the water phase. In the figure, when 50 ng of sample is injected, the peak area of ​​5 mM ammonium acetate aqueous solution is 4241, and the peak area of ​​0.1% formic acid water is 6324. When 0.1% formic acid water is used as the water phase, the chromatographic peak shape is better and smoother.

[0075] In summary, the quantitative results of the present invention are accurate, and all verification items meet regulatory requirements. Under the conditions of the present invention, rosuvastatin can be accurately quantified and can be used for clinical pharmacokinetic studies, providing a stable, reliable and simple detection method for corresponding drug concentration monitoring.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining rosuvastatin in human plasma, characterized in that: The steps include: The pretreated plasma samples were detected by high performance liquid phase tandem mass spectrometry, and the isotope internal standard method was used for quantification. The concentration of roxadustat in the plasma samples of the standard curve was used as the horizontal coordinate, and the ratio of the peak area of ​​roxadustat to the peak area of ​​the internal standard was used as the vertical coordinate. The standard curve equation was established, and the ratio of the peak area of ​​roxadustat in the plasma samples to the peak area of ​​the internal standard was substituted into the standard curve equation to calculate the concentration of roxadustat in the test plasma.

2. The method according to claim 1, characterized in that The liquid chromatography conditions of the high performance liquid phase tandem mass spectrometry are: Chromatographic column: ACE EXCEL 5C18 (50*2.1mm); Mobile phase: aqueous phase: 0.1% acetic acid in water, organic phase: 0.1% formic acid in acetonitrile, needle washing solution: methanol: water (1:9); Column temperature: 35°C; Autosampler temperature: 4°C; Aqueous phase and organic phase were used as mixed mobile phase for gradient elution. The collection time for each sample was 3 min, the injection volume was 2 mL, the flow rate was 0.4 mL / min, and the mobile phase gradient elution parameters were as follows: 0 min, 48% organic phase; 1.8 min, 95% organic phase; 2.3 min, 95% organic phase; 2.31 min, 48% organic phase; 3 min, stop.

3. The method according to claim 1, characterized in that The mass spectrometry conditions of the high performance liquid phase tandem mass spectrometry are: in the electrospray ionization detection mode, a mass spectrometry scanning mode of selected reaction monitoring is adopted, wherein rosuvastatin adopts a positive ion mode, ionization voltage: 3500V, sheath gas: 60Arb, auxiliary gas: 15Arb, tail gas: 2Arb, ion transfer tube temperature: 380°C, evaporator temperature: 350°C, cycle time: 0.8sec, and the ion pairs are: m / z 353.2→278.17 (rosuvastatin), m / z 358.18→283.13 (rosuvastatin-d5).

4. The method according to claim 1, characterized in that: The specific steps of the plasma sample pretreatment are: remove 50.0 μL of plasma sample, add 10 μL of deuterated mixed internal standard working solution, add 340 μL of acetonitrile, vortex the sample for 5 minutes, centrifuge at 10000 rpm and 4°C for 5 minutes, and remove 100 μL of supernatant with a pipette; add 500 μL of acetonitrile: water (1:1) solution for re-dissolution, vortex for 3 minutes, and centrifuge at 4000 rpm and 4°C for 5 minutes to obtain the sample to be tested.

5. The method according to claim 4, wherein the internal standard working solution preparation process is as follows: using dimethyl sulfoxide (DMSO) to dissolve the rosuvastatin-d5 reference substance to prepare an internal standard stock solution IS-A with a concentration of 2.00 mg / mL, taking the internal standard stock solution IS-A and adding it to an acetonitrile: water (1:1) solution, mixing evenly to prepare an internal standard stock solution IS-B with a concentration of 20.0 μg / mL, taking the internal standard stock solution IS-B and adding it to an acetonitrile: water (1:1) solution, mixing evenly to prepare an internal standard working solution IS-C with a concentration of 500 ng / mL.

6. The method according to claim 1, characterized in that The preparation process of the standard curve is as follows: using dimethyl sulfoxide (DMSO) to dissolve rosuvastatin, using acetonitrile: water (1:1) solution as a solvent, preparing standard curve working solutions and quality control working solutions of multiple concentrations, adding the standard curve working solutions and quality control working solutions of multiple concentrations into blank plasma matrix and mixing them, preparing standard curve plasma samples and quality control plasma samples of multiple concentrations, using the peak area ratio of rosuvastatin and internal standard and the concentration of the standard sample to establish a standard curve, and obtaining a corresponding linear regression equation.

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