CN117554513A - HPLC-MSMS method for simultaneously measuring valproic acid, olanzapine and metabolites thereof in blood plasma - Google Patents
HPLC-MSMS method for simultaneously measuring valproic acid, olanzapine and metabolites thereof in blood plasma Download PDFInfo
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- NIJJYAXOARWZEE-UHFFFAOYSA-N Valproic acid Chemical compound CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 title claims abstract description 166
- KVWDHTXUZHCGIO-UHFFFAOYSA-N olanzapine Chemical compound C1CN(C)CCN1C1=NC2=CC=CC=C2NC2=C1C=C(C)S2 KVWDHTXUZHCGIO-UHFFFAOYSA-N 0.000 title claims abstract description 109
- 229960005017 olanzapine Drugs 0.000 title claims abstract description 95
- 229960000604 valproic acid Drugs 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 72
- 239000002207 metabolite Substances 0.000 title claims abstract description 44
- 210000002381 plasma Anatomy 0.000 title abstract description 48
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 57
- 150000002500 ions Chemical class 0.000 claims abstract description 34
- 239000012071 phase Substances 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 238000001819 mass spectrum Methods 0.000 claims abstract description 16
- 239000007791 liquid phase Substances 0.000 claims abstract description 9
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000006920 protein precipitation Effects 0.000 claims abstract description 7
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 5
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- 239000012224 working solution Substances 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 17
- KVWDHTXUZHCGIO-FIBGUPNXSA-N 4-(4-methylpiperazin-1-yl)-2-(trideuteriomethyl)-10H-thieno[2,3-b][1,5]benzodiazepine Chemical compound [2H]C([2H])([2H])c1cc2c(Nc3ccccc3N=C2N2CCN(C)CC2)s1 KVWDHTXUZHCGIO-FIBGUPNXSA-N 0.000 claims description 14
- 239000012452 mother liquor Substances 0.000 claims description 14
- 238000004458 analytical method Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 6
- GAMKTHIOQWRUNL-ZFORQUDYSA-N (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-[2-methyl-4-(4-methylpiperazin-1-yl)thieno[2,3-b][1,5]benzodiazepin-10-yl]oxane-2-carboxylic acid Chemical compound C1CN(C)CCN1C(C=1C=C(C)SC=11)=NC2=CC=CC=C2N1[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 GAMKTHIOQWRUNL-ZFORQUDYSA-N 0.000 claims description 5
- FPDIERBPQFAFSI-UHFFFAOYSA-N 2-hydroxymethylolanzapine Chemical compound C1CN(C)CCN1C1=NC2=CC=CC=C2NC2=C1C=C(CO)S2 FPDIERBPQFAFSI-UHFFFAOYSA-N 0.000 claims description 5
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
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- 239000000164 antipsychotic agent Substances 0.000 description 2
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
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- 230000007774 longterm Effects 0.000 description 2
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- 238000012544 monitoring process Methods 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 238000002552 multiple reaction monitoring Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
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- 238000011002 quantification Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
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- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- 102000040125 5-hydroxytryptamine receptor family Human genes 0.000 description 1
- 108091032151 5-hydroxytryptamine receptor family Proteins 0.000 description 1
- 208000020925 Bipolar disease Diseases 0.000 description 1
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- 208000001654 Drug Resistant Epilepsy Diseases 0.000 description 1
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- JNNOSTQEZICQQP-UHFFFAOYSA-N N-desmethylclozapine Chemical compound N=1C2=CC(Cl)=CC=C2NC2=CC=CC=C2C=1N1CCNCC1 JNNOSTQEZICQQP-UHFFFAOYSA-N 0.000 description 1
- -1 N-norolanzapine Chemical compound 0.000 description 1
- 102000034337 acetylcholine receptors Human genes 0.000 description 1
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- RMRJXGBAOAMLHD-IHFGGWKQSA-N buprenorphine Chemical compound C([C@]12[C@H]3OC=4C(O)=CC=C(C2=4)C[C@@H]2[C@]11CC[C@]3([C@H](C1)[C@](C)(O)C(C)(C)C)OC)CN2CC1CC1 RMRJXGBAOAMLHD-IHFGGWKQSA-N 0.000 description 1
- 229960001736 buprenorphine Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- HPNSNYBUADCFDR-UHFFFAOYSA-N chromafenozide Chemical compound CC1=CC(C)=CC(C(=O)N(NC(=O)C=2C(=C3CCCOC3=CC=2)C)C(C)(C)C)=C1 HPNSNYBUADCFDR-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229960004170 clozapine Drugs 0.000 description 1
- QZUDBNBUXVUHMW-UHFFFAOYSA-N clozapine Chemical compound C1CN(C)CCN1C1=NC2=CC(Cl)=CC=C2NC2=CC=CC=C12 QZUDBNBUXVUHMW-UHFFFAOYSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- 206010013663 drug dependence Diseases 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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Abstract
The invention discloses an HPLC-MSMS method for simultaneously measuring valproic acid, olanzapine and metabolites thereof in blood plasma, which comprises the following steps: s1, performing pretreatment on a plasma sample by using an acetonitrile protein precipitation method; s2, detecting a plasma sample by adopting an HPLC-MSMS method, and simultaneously carrying out qualitative and/or quantitative analysis on valproic acid, olanzapine and metabolites thereof in the plasma; wherein, the chromatographic column adopts a Agilent Poroshell EC-C18 column; adopting an electrospray ion source, wherein the mobile phase is A phase: formic acid water, phase B: acetonitrile; by optimizing conditions of liquid phase and mass spectrum, the method for simultaneously measuring valproic acid, olanzapine and metabolites thereof is realized. The method has been applied to the detection of plasma samples taken with olanzapine and valproic acid in combination and olanzapine alone, and the results obtained satisfy the linear range.
Description
Technical Field
The invention relates to the technical field of detection methods, in particular to an HPLC-MSMS method for simultaneously measuring valproic acid, olanzapine and metabolites thereof in blood plasma.
Background
Studies show that olanzapine is five times before the use frequency of antipsychotics, and compared with the traditional antipsychotics, the novel atypical antipsychotics have better curative effects and lighter adverse reactions. The application of novel atypical antipsychotics such as olanzapine to combination with other drugs has been approved by the FDA today with the aim of improving the efficacy and reducing adverse effects [2].
Olanzapine is antagonized by its binding to dopamine receptors, serotonin receptors and cholinergic receptors [3], and is used clinically mainly for the treatment of schizophrenia or affective disorders, and sometimes for the treatment of depression. Valproic acid increases gamma aminobutyric acid energy activity by affecting centrally inhibitory neurotransmitters [4], and is used clinically primarily for the treatment of epilepsy and mania. The combination of olanzapine and valproic acid is commonly used for the treatment of refractory epilepsy, bipolar disorders [5], etc.
According to the research of Pu Qixia [6] and the like on the combined administration of olanzapine and valproic acid, the combined administration of the olanzapine and valproic acid can enhance the curative effect and reduce adverse reaction. However, most mental patients need to take the medicine for a long time, and the olanzapine and valproic acid have the problems of narrow treatment window range, close effective concentration to toxic concentration, large individual medicine taking difference, multiple adverse reactions and the like [7], and meanwhile, the valproic acid also has influence on the metabolism of olanzapine in the medicine taking process, so that the medicine taking process is troublesome. Therefore, the monitoring of the blood concentration of the psychotropic drugs has important clinical significance for the reasonable medication safety of patients.
References referred to in the background art:
[1] wang Jun, liu Xiuping, zhao Xiaoyan, et al. Psychiatric clinic one day mental medicine use case investigation [ J ]. Chinese journal of drug dependence, 2015,24 (04): 276-279.
[2] Jiang Lixin, chen Yongxin, zhang Ruiling. Psychiatric hospitalization patients 964 psychotic time point surveys [ J ]. Occupation and health, 2014,30 (15): 2198-2200.
[3] Buprenorphine, liu Shuai, miao Liyan. Comparison of olanzapine tablet dissolution from different manufacturers [ J ]. Anti-infective pharmaceutical 2012,9 (04): 290-292.
[4] Zhang Yongjiang, xue Ranran, wang Xubo, et al, development of studies on the treatment of bipolar disorders with sodium valproate [ J ]. Modern pharmaceutical community, 2017,15 (19): 20-22.
[5] Lin Xiaoyong, hao Jierong, duchesner sodium valproate in combination with olanzapine has been studied for blood concentration and efficacy in the treatment of bipolar affective disorder in adolescents [ J ]. Extra-Chinese medical research 2021,19 (25): 65-67.
[6] Pu Qixia, wu Haibo, huang Xiong, et al, olanzapine combined with sodium valproate control analysis of refractory schizophrenia [ J ]. Modern biomedical advances, 2013,13 (18): 3503-3506.
[7] Peng Qingping, song Cangsang, li Xingde, et al, valproic acid therapeutic drug monitoring study progression [ J ]. Drug evaluation study, 2021,44 (05): 1111-1116.
Disclosure of Invention
In view of the above, the present invention aims to provide a rapid, convenient and cost-effective HPLC-MS/MS method for determining valproic acid, olanzapine and metabolites thereof in plasma, which is applied to detect plasma samples taking olanzapine and valproic acid in combination and olanzapine alone, and the results obtained by the method satisfy the linear range.
The adopted technical scheme is as follows:
an HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and metabolites thereof in plasma comprising the steps of:
s1, performing pretreatment on a plasma sample by using an acetonitrile protein precipitation method;
s2, detecting a plasma sample by adopting an HPLC-MSMS method, and simultaneously carrying out qualitative and/or quantitative analysis on valproic acid, olanzapine and metabolites thereof in the plasma;
wherein, the chromatographic column adopts a Agilent Poroshell EC-C18 column; adopting an electrospray ion source, wherein the mobile phase is A phase: formic acid water, phase B: acetonitrile; by optimizing conditions of liquid phase and mass spectrum, the method for simultaneously measuring valproic acid, olanzapine and metabolites thereof is realized.
Further, in S1, the specific steps of pretreatment of the plasma sample by acetonitrile protein precipitation method are as follows: precisely sucking 200 mu L of plasma sample into a 1.5mL centrifuge tube, adding 10 mu L of olanzapine internal standard working solution and 10 mu L of valproic acid internal standard working solution, adding 800 mu L of acetonitrile solution into the mixed solution, carrying out vortex oscillation for 3min, carrying out 12700r/min centrifugation for 10min, and taking 200 mu L of supernatant into a 96-well plate.
Further, in S2, the chromatographic conditions are: the chromatographic column adopts Agilent Poroshell EC-C18 column with 2.1mm×50mm,2.7 μm, and the chromatographic mobile phase is phase A: formic acid water with the volume concentration of 0.1%; and B phase: acetonitrile; the flow rate is 0.4mL/min; the column temperature is maintained at 35+/-2 ℃; the amount of one sample was 5. Mu.L.
Further, the mass spectrum condition is that an electrospray ion source is adopted for mass spectrum, the mass spectrum is in a positive ion mode within 0-2min, and the mass spectrum is in a negative ion mode from 2min to the end of detection; the flow rate of the drying gas is 11L/min; the temperature of the drying gas is 350 ℃; the atomization air pressure is 35Psi; setting the capillary voltage to 4000V as positive ion mode; the negative ion mode was set to 3500V.
Further, the valproic acid, olanzapine and metabolites thereof were the following 5 analytes: valproic acid, olanzapine, 2-hydroxymethyl olanzapine, olanzapine-10N-glucuronide, N-norolanzapine.
Further, the olanzapine internal standard working solution is prepared by the following method: 1mg of olanzapine-D3 is precisely weighed, 1mL of methanol is added to prepare 1mg/mL of olanzapine-D3 mother liquor, 10 mu L of olanzapine-D3 mother liquor is taken in a 10mL volumetric flask, methanol is added to fix the volume, and the olanzapine-D3 internal standard working solution with the concentration of 1000ng/mL is prepared.
Further, the valproic acid internal standard working solution is prepared by the following method: precisely weighing 100mg of valproic acid-D15 in a 10mL volumetric flask, adding methanol to fix the volume to prepare 10mg/mL valproic acid-D15 mother liquor, taking 1mL of valproic acid-D15 mother liquor in another 10mL volumetric flask, adding methanol to fix the volume to prepare the valproic acid-D15 internal standard working solution with the concentration of 1000 mug/mL.
Further, in S2, olanzapine and its metabolites peak at 0.4-0.6min, while valproic acid peak at 3.1-3.2 min; the method comprises the steps of carrying out sectional collection on a mass spectrum, collecting olanzapine and metabolites and internal standards thereof in 0-2min by using a positive ion mode, and collecting valproic acid and internal standards thereof in 2-4.5min, so that the method realizes that the positive ion and negative ion collection modes are simultaneously used in the same analysis method, and simultaneously determines valproic acid, olanzapine and metabolites thereof.
The invention has the beneficial effects that:
the total duration of the method is 4.5min, and the olanzapine, the olanzapine metabolite and the valproic acid have good linear relation in a standard curve (r 2 is more than or equal to 0.99). The precision and accuracy LLOQ quality control products in the batch and between the batch are all within the specified range. The matrix effect, the recovery rate and the dilution effect all meet the detection requirements. Stability under 5 different conditions was within the detection range. The peak pattern of each detected object is good, and endogenous substances in blank plasma do not interfere with detection. No significant residue was observed in this process.
The invention establishes an HPLC-MS/MS method for rapidly, conveniently and cost-effectively measuring valproic acid, olanzapine and metabolites thereof in blood plasma. The method has been applied to the detection of plasma samples taken with olanzapine and valproic acid in combination and olanzapine alone, and the results obtained satisfy the linear range.
Drawings
FIG. 1 is a calibration graph of 5 analytes.
FIG. 2 shows the TIC (I) of LLOQ in 5 analytes and 2 internal standard blank plasmas and the TIC (II) of blank plasmas.
FIG. 3 is an extraction chromatogram of 5 analytes and 2 internal standard blank plasma spiked with LLOQ (I) and blank plasma (II).
Detailed Description
The present invention will be described in detail by way of specific examples, but the purpose and purpose of these exemplary embodiments are merely to illustrate the present invention, and do not constitute any limitation to the actual scope of the present invention in any way.
The HPLC-MSMS method for simultaneously measuring valproic acid, olanzapine and metabolites thereof in blood plasma comprises the following steps:
s1, performing pretreatment on a plasma sample by using an acetonitrile protein precipitation method;
s2, detecting a plasma sample by adopting an HPLC-MSMS method, and simultaneously carrying out qualitative and/or quantitative analysis on valproic acid, olanzapine and metabolites thereof in the plasma;
wherein, the chromatographic column adopts a Agilent Poroshell EC-C18 column; adopting an electrospray ion source, wherein the mobile phase is A phase: formic acid water, phase B: acetonitrile; by optimizing conditions of liquid phase and mass spectrum, the method for simultaneously measuring valproic acid, olanzapine and metabolites thereof is realized.
Finally, the established method is used for evaluating the drug concentration of the clinical sample and verifying the practicability of the test method.
The specific implementation is as follows:
2 materials and methods
2.1 reagents
The reagents used in this study are shown in Table 2-1
TABLE 2-1 reagent information Table
2.2 instruments
TABLE 2-2 Instrument information Table
2.3 chromatography and Mass Spectrometry conditions
2.3.1 chromatographic conditions
The chromatographic column was a Agilent Poroshell EC-C18 column (2.1 mm. Times.50 mm,2.7 μm). The chromatographic mobile phase is phase A: 0.1vol% formic acid water; and B phase: acetonitrile. The flow rate is 0.4mL/min; the column temperature is maintained at about 35 ℃ (+ -2 ℃); the amount of one sample was 5. Mu.L.
The gradient elution procedure for the column is shown in tables 2-3 and the ratio of phase A to phase B varies during the reaction run.
TABLE 2-3 gradient elution program table
2.3.2 Mass Spectrometry conditions
The mass spectrum adopts an electrospray ion source (ESI source), MRM is in a positive ion mode within 0-2min, and the mass spectrum is in a negative ion mode from 2min to the end of detection. The flow rate of the drying gas is 11L/min; the temperature of the drying gas is 350 ℃; the atomization air pressure is 35Psi; setting the capillary voltage to 4000V as positive ion mode; the negative ion mode was set to 3500V. The parameters for 5 analytes and 2 internal standards olanzapine-D3 and valproic acid-D15 are shown in tables 2-4.
Table 2-4 5 analytes and related parameters for 2 internal standards olanzapine-D3 and valproic acid-D15
2.4 preparation of solutions
2.4.1 working solution for reference substance
Accurately weighing proper amounts of olanzapine and N-norolanzapine, respectively using methanol to fix volume, preparing mother liquor with concentration of 10mg/mL, and preserving at-20 ℃. Precisely measuring olanzapine, N-norolanzapine, olanzapine-10N-glucuronide, 2-hydroxymethyl olanzapine mother liquor 4, 400 and 400 mu L, placing into the same 10mL volumetric flask, and preserving at-20deg.C with methanol constant volume as mixed standard solution with concentration of 4 mu g/mL. Respectively measuring 50, 25, 20, 10 and 10 mu L of standard substance solutions of olanzapine and olanzapine metabolite mixture precisely, placing the standard substance solutions in different 1.5mL EP pipes, and respectively and sequentially adding 50, 75, 140, 150, 310, 630 and 1270 mu L of methanol to prepare standard curve standard substance solutions. The olanzapine working solution has a mass concentration range of 31.25-4000ng/mL.
Accurately measuring a proper amount of valproic acid solution in a volumetric flask, adding methanol to fix the volume of 10mg/mL of mother solution, and preserving at-20 ℃. And precisely measuring a proper amount of valproic acid mother solution, adding a standard substance solution with the constant volume of 4mg/mL of methanol, and gradually diluting to prepare a valproic acid standard curve reference substance solution. The mass concentration of the valproic acid working solution is 31.25-4000 mug/mL.
2.4.2 preparation of quality control product working solution
The high concentration quality control (QC-H) is 75vol% of the highest concentration point of the standard curve, the medium concentration quality control (QC-M) is 20vol% of the high concentration quality control, and the low concentration quality control (QC-L) is 20vol% of the medium concentration quality control (QC-M).
Respectively precisely measuring 150, 30 and 15 mu L of olanzapine standard substance solution, respectively placing the solution in a 1.5mL EP tube, respectively adding 50, 170 and 485 mu L of methanol to prepare high-concentration, medium-concentration and low-concentration quality control sample working solutions, wherein the concentration of the final olanzapine quality control sample working solution is 3000ng/mL of high-concentration quality control (QC-H), 600ng/mL of medium-concentration quality control (QC-M) and 120ng/mL of low-concentration quality control (QC-L).
Precisely measuring 375 mu L of the valproic acid standard solution in a 1.5mL EP tube, adding 125 mu L of methanol solution, and uniformly mixing to obtain a high-concentration quality control sample working solution; precisely measuring 100 mu L of the high-concentration quality control sample working solution in another 1.5mL EP tube, adding 400 mu L of methanol solution, and uniformly mixing to obtain the medium-concentration quality control sample working solution; precisely measuring 100 mu L of the medium-concentration quality control sample working solution in another 1.5mL EP tube, adding 400 mu L of methanol solution, and uniformly mixing to obtain the low-concentration quality control sample working solution. The final valproic acid quality control working solution concentration is 3000 mug/mL of high-concentration quality control (QC-H), 600 mug/mL of medium-concentration quality control (QC-M) and 120 mug/mL of low-concentration quality control (QC-L).
2.4.3 internal Standard working fluid
1mg of olanzapine-D3 is precisely weighed, 1mL of methanol is added to prepare 1mg/mL of olanzapine-D3 mother liquor, 10 mu L of olanzapine-D3 mother liquor is taken in a 10mL volumetric flask, methanol is added to fix the volume, and the olanzapine-D3 internal standard working solution with the concentration of 1000ng/mL is prepared.
Precisely weighing 100mg of valproic acid-D15 in a 10mL volumetric flask, adding methanol to fix the volume to prepare 10mg/mL valproic acid-D15 mother liquor, taking 1mL of valproic acid-D15 mother liquor in another 10mL volumetric flask, adding methanol to fix the volume to prepare the valproic acid-D15 internal standard working solution with the concentration of 1000 mug/mL.
2.5 sample pretreatment method
2.5.1 pretreatment method for standard curve sample and quality control sample
The pretreatment method of the standard curve sample comprises the following steps: 180 mu L of blank plasma is placed in a 1.5mL centrifuge tube, 10 mu L of serial working solutions containing standard curve concentration of each object to be detected and 10 mu L of olanzapine and valproic acid internal standard working solution are precisely measured respectively, 800 mu L of acetonitrile solution is added into the mixed solution, vortex oscillation is carried out for 3min,12700r/min is carried out for 10min, and 200 mu L of supernatant is taken and analyzed in a 96-well plate.
The pretreatment method of the quality control sample comprises the following steps: 180 mu L of blank plasma is placed in a 1.5mL centrifuge tube, 10 mu L of serial working solutions containing the quality control concentration of each object to be detected and 10 mu L of olanzapine and valproic acid internal standard working solution are precisely measured respectively, 800 mu L of acetonitrile solution is added into the mixed solution, vortex oscillation is carried out for 3min,12700r/min is carried out for 10min, and 200 mu L of supernatant is taken and analyzed in a 96-well plate.
Standard curve concentration points and quality control concentrations of olanzapine and its metabolites and valproic acid are shown in tables 2-5.
TABLE 2-5 standard curve concentration points and quality control product concentrations of olanzapine and its metabolites and valproic acid
The standard curves for olanzapine and its metabolites and valproic acid (i.e., 5 analytes) are shown in figure 1.
2.5.2 plasma sample pretreatment method
200. Mu.L of plasma sample is precisely sucked into a 1.5mL centrifuge tube, 10. Mu.L of olanzapine internal standard working solution and 10. Mu.L of valproic acid internal standard working solution are added, 800. Mu.L of acetonitrile solution is added into the mixed solution, vortex oscillation is carried out for 3min,12700r/min is carried out for 10min, and 200. Mu.L of supernatant is taken out for analysis in a 96-well plate.
2.6 method validation of valproic acid and olanzapine
The methodology of the invention verifies the guidelines of biological sample quantitative analysis method and chemical drug clinical pharmacokinetics study technology in the 2020 edition of Chinese pharmacopoeia [8] The specified method design requirements are analyzed. The methodological verification of the invention comprises: linear range and lower limit of quantification, accuracy and precision, stability (including stability at room temperature for 6 hours, stability after 48 hours preparation at room temperature, repeated freeze-thaw stability after repeated 3 times of freezing at-20 ℃ and repeated freeze-thaw cycles at-20 ℃ and long-term stability after preservation at-20 ℃ and-80 ℃) selectivity, matrix effect, extraction recovery rate, dilution effect and residual effect.
2.6.1 Linear Range and lower quantitative limit
8 concentration standard curve samples were prepared, and samples were introduced after "2.5.1 standard curve samples and quality control sample preparation and pretreatment methods" were run in order of concentration from low to high, and the linearity of the method was evaluated using independent calibration curves in 6 groups of plasma treated for three days. Calibration curves for analytes were generated by plotting the peak area ratio (analyte/internal standard) against the theoretical concentration. The regression equation is calculated using the weighting factor "1/X2".
8 parallel standard samples are prepared, S/N is required to be more than or equal to 10, RSD is required to be less than or equal to 20% as the lowest concentration of a standard curve, and LLOQ is not qualified within the range.
2.6.2 accuracy and precision
Accuracy refers to the proximity of the detection result of the method used to the concentration of the analyte marker using the formula: (measured value/true value) ×100%. Precision refers to the proximity of the results obtained by repeated analyte determinations. LLOQ, QC-L, QC-M, QC-H were prepared from blank plasma samples, 5 parallel samples were made for each concentration level, three batches were measured consecutively, 3 days apart, the same procedure was used, and the accuracy and precision within and between the batches were calculated from the standard curve of the current batch.
For QC-L, QC-M, QC-H quality control samples, the precision of the RSD in the batch and the RSD among the batches cannot exceed 15%, and the accuracy average value is within +/-15% of the standard value; the precision of LLOQ in batch and rsD among batches must not exceed 20%, and the average accuracy value should be within +/-20% of the standard value.
2.6.3 stability
The invention mainly detects the stability of a sample under 5 different conditions, namely the stability at room temperature for six hours, the stability after preparation at room temperature for forty-eight hours, the repeated freeze-thawing stability after repeated three times of freezing at-20 ℃ and thawing cycle at-20 ℃, the long-term stability after preservation at-20 ℃ and-80 ℃ for 30 days, wherein QC-L, QC-M, QC-H is respectively prepared under each condition, and 5 parts of QC-L, QC-M, QC-H are prepared at each concentration level in parallel. Analysis was performed immediately after treatment and after the storage conditions detected. A standard curve is prepared for each detection, a quality control sample is analyzed according to the prepared standard curve, the obtained result is compared with a marked value, and the concentration mean value of each determination condition is not more than 15% from the marked value.
2.6.4 Selectivity
Six blank substrate samples of different sources (batches) without valproic acid, olanzapine and olanzapine metabolites and internal standard were taken and evaluated with LLOQ samples prepared from the corresponding substrates. The analysis and the interference were performed according to the "2.5.2 plasma sample pretreatment method".
The detected peak area of the to-be-detected object is smaller than or equal to 20.0% of the peak area of the to-be-detected object in the LLOQ sample prepared by the corresponding matrix, and the peak area of the internal standard detector is smaller than or equal to 5.0% of the peak area of the LLOQ sample prepared by the corresponding matrix, so that the interference is considered to have no significant influence on the experiment, and the interference experiment meets the methodological verification requirement.
2.6.5 matric effect and extraction recovery
Preparing QC-L, QC-M, QC-H respectively, preparing 6 samples of quality control samples with each concentration level in parallel by adopting the same method, treating the samples according to a standard curve sample of 2.5.1 and a preparation and pretreatment method of the quality control samples, and then injecting the samples, wherein the recorded peak area is A; taking blank plasma, adding reference substance working solution, preparing QC-L, QC-M, QC-H, preparing 6 parts of quality control products with the concentration level of each concentration in parallel by adopting the same method, carrying out sample injection analysis, and recording that the peak area is B; taking the reference substance working solution with the same concentration level, directly injecting sample without treatment, and recording the peak area as C.
The calculation formula is as follows:
the RSD of the internal table normalized matrix effect and extraction recovery calculated from 6 replicates was not greater than 15%.
2.6.6 dilution Effect and residual Effect
The sample of 5 of the highest point concentration of the standard curve is diluted by 10 times by blank plasma, and the dilution effect is evaluated by sample injection analysis after the sample is processed under the item of 'preparation and pretreatment methods of the standard curve sample and the quality control sample', namely, 2.5.1 standard curve sample. The precision and accuracy of the diluted sample are within +/-15% of the marked concentration, and RSD is less than or equal to 15%.
Immediately after the sample at the highest point of the standard curve was sampled, a blank sample was sampled to determine the peak areas of the analyte and internal standard for evaluation of residual effects. The peak area of the analyte and its internal standard in the blank sample should not exceed 20% of LLOQ.
2.6.7 clinical application
The HPLC-MS/MS method was used to determine the drug concentration in the collected plasma samples according to the "2.5.2 plasma sample pretreatment method".
3 results
The invention establishes an HPLC-MS/MS method for measuring valproic acid, olanzapine and metabolites thereof in blood plasma.
The development steps are as follows: searching mass spectrum parameters of related objects to be detected by utilizing a laboratory liquid chromatography-mass spectrometer, optimizing chromatographic conditions, and perfecting a detection method; verifying the established method according to the related requirements of methodology verification and daily verification actual conditions; clinical sample drug concentrations were tested using established methods to assess the utility of the method.
3.1 liquid phase and Mass Spectrometry Condition optimization
Valproic acid and olanzapine are detected by respectively using a negative ion mode and a positive ion mode of an ESI source, if two compounds are detected simultaneously, the two compounds are required to be separated by optimizing chromatographic conditions, and the positive ion mode and the negative ion mode are used simultaneously, so that the optimization of conditions of liquid phase and mass spectrum is particularly important; we optimized the liquid phase conditions as follows: optimizing a liquid phase elution program to make olanzapine and metabolites thereof show peaks at 0.4-0.6min, and valproic acid shows peaks at 3.1-3.2 min; by carrying out sectional collection on a mass spectrum, collecting olanzapine and metabolites and internal standards thereof in 0-2min (without 0 value) and collecting valproic acid and internal standards thereof in 2-4.5min (without 2 value), the method realizes that the positive and negative ion collection modes are simultaneously used in the same analysis method, and simultaneously measuring valproic acid, olanzapine and metabolites thereof, and provides technical support for discussing interaction of valproic acid and olanzapine.
3.2 results of methodological validation
3.2.1 Linear Range and lower quantitative limit
Olanzapine and olanzapine metabolites and valproic acid have a good linear relationship over a linear range (r 2> 0.99). The linear regression equations for olanzapine and olanzapine metabolites and valproic acid are shown in Table 3-1.
TABLE 3-1 Linear regression equation for olanzapine and olanzapine metabolite and valproic acid
3.2.2 accuracy and precision
The results show that the in-batch precision RSD of valproic acid, olanzapine and the metabolite of olanzapine measured by the method is LLOQ <20%, QC-L, QC-M, QC-H <15%; the accuracy of LLOQ in batch is 80.91% -115.81%, and the accuracy of QC-L, QC-M, QC-H in batch is 85.33% -114.32%. The precision between batches of RSD is LLOQ <20%, QC-L, QC-M, QC-H <15%; LLOQ batch-to-batch accuracy is 92.78% -108.97% and QC-L, QC-M, QC-H batch-to-batch accuracy is 87.67% -112.16%. The in-batch and inter-batch accuracy and precision of olanzapine and olanzapine metabolites and valproic acid are shown in tables 3-2.
TABLE 3-2 intra-and inter-batch accuracy and precision of olanzapine metabolites and valproic acid
3.2.3 stability
The results are shown in the following table, the stability of each test article is 85.39% -114.97% and RSD is in the range of 1.42% -14.86%. Stability of olanzapine and olanzapine metabolites and valproic acid are shown in tables 3-3.
TABLE 3-3 stability of olanzapine and olanzapine metabolites and valproic acid
3.2.4 Selectivity
Chromatograms of the LLOQ samples, blank plasma samples, of 5 analytes and 2 internal standards are shown in fig. 2 and 3. The peak pattern of each detected object is good, and endogenous substances in blank plasma do not interfere with detection.
FIG. 2 shows TIC (I) for LLOQ in 5 analytes and 2 internal standard blank plasmas and TIC (II) for blank plasmas;
FIG. 3 is an extraction chromatogram of 5 analytes and 2 internal standard blank plasma spiked with LLOQ (I) and blank plasma (II).
3.2.5 matrix effect and extraction recovery
The method verifies that the matrix effect of internal standard normalization is 85.48-110.90%, and the RSD is 0.54-13.56%; the recovery rate is 85.26% -102.83%, and the RSD is 1.81% -10.83%. The matrix effects and recovery of olanzapine and olanzapine metabolites are shown in tables 3-4.
Tables 3-4 matrix effects and recovery of olanzapine and olanzapine metabolites
3.2.6 dilution Effect and residual Effect
The RSD of the dilution effect is <15% with an accuracy in the range 102.38% -114.02%. No significant residue was observed in this process.
3.3 detection results of clinical samples
After verification, the method is used for combined administration of 66 parts of olanzapine and valproic acid, 19 parts of olanzapine are independently used, the detection results are shown in tables 3-5, and the obtained results are in a linear range, so that the method can be used for clinical blood concentration detection.
Tables 3-5 clinical sample test result ranges
Discussion 4
The HPLC-MS/MS method for measuring valproic acid, olanzapine and metabolites thereof in blood plasma is fast, convenient and cost-saving, and the separation and positive and negative ion mode switching analysis of valproic acid, olanzapine and metabolites thereof in 4.5min are realized by optimizing the conditions of liquid phase and mass spectrum, so that method support is provided for clinical discussion of interactions of olanzapine and valproic acid.
At the same time, the invention aims at the metabolite 2-hydroxymethyl olanzapine of the olanzapine at present [9] olanzapine-10N-glucuronide, N-norolanzapine [10-12] The invention fills up the blank of researching olanzapine metabolites including 2-hydroxymethyl olanzapine, olanzapine-10N-glucuronide and N-norolanzapine.
Conclusion 5
In summary, the invention establishes an HPLC-MS/MS method for measuring valproic acid, olanzapine and metabolites thereof in blood plasma, and performs methodological verification, and the obtained results meet the requirements. The method adopts acetonitrile protein precipitation for treatment, and is simple and quick to operate; simultaneously, by optimizing chromatographic conditions and using an electrospray ion source (ESI source), a multi-reaction monitoring (MRM) mode is used for simultaneously measuring positive and negative ion modes, valproic acid, olanzapine and olanzapine metabolites are separated and analyzed within 4.5min, and the rapid detection requirement of clinical samples is met.
The method has been applied to the detection of plasma samples taken with olanzapine and valproic acid in combination and olanzapine alone, and the results obtained satisfy the linear range.
And (2) the following steps: references referred to in the detailed description:
[8] chemical drug clinical pharmacokinetics study technical guidelines [ J ].
[9]Saar E,Gerostamoulos D,Drummer O H,et al.Identification of2-hydroxymethyl-olanzapine as a novel degradation product of olanzapine[J].Forensic Sci Int,2012,220(1-3).
[10] Li Wenbiao, yimin, wang Chuanyue, et al, high performance liquid chromatography of clozapine, norclozapine, olanzapine, electrochemical detection characteristics research [ J ] chromatography, 2000 (06): 550-553.
[11] Ma Botao liquid chromatography-tandem mass spectrometry detection method of olanzapine and N-norolanzapine in plasma establishes analysis of influence factors of plasma concentration [ D ]. University of Beijing medical department, 2021.
[12] Shao Gongxia and Mo Changliang measuring olanzapine, clozapine and N-desmethylclozapine concentrations [ J ] in human serum by column-switching HPLC, northern pharmaceutical, 2016,13 (06): 1-3.
And (2) the following steps: abbreviation, english holonomy and Chinese holonomy
HPLC-MS/MS High performance liquid chromatography-Tandem mass spectrometry high performance liquid chromatography-tandem mass spectrometry
ESI Electron spray ionization electrospray ion source
LLOQ Lower limit of quantification minimum quantitative lower limit
QC Quality control quality control sample
SD Relative standard deviation relative standard deviation
MRM Multiple reaction monitoring multiple reaction monitoring
RT Ret time retention time
RSD Relative standard deviatio relative standard deviation
CE Collision energy collision Voltage
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (8)
1. An HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and metabolites thereof in plasma comprising the steps of:
s1, performing pretreatment on a plasma sample by using an acetonitrile protein precipitation method;
s2, detecting a plasma sample by adopting an HPLC-MSMS method, and simultaneously carrying out qualitative and/or quantitative analysis on valproic acid, olanzapine and metabolites thereof in the plasma;
wherein, the chromatographic column adopts a Agilent Poroshell EC-C18 column; adopting an electrospray ion source, wherein the mobile phase is A phase: formic acid water, phase B: acetonitrile; by optimizing conditions of liquid phase and mass spectrum, the method for simultaneously measuring valproic acid, olanzapine and metabolites thereof is realized.
2. The method for simultaneous determination of valproic acid, olanzapine and metabolites thereof in plasma by HPLC-MSMS according to claim 1, wherein in S1, the specific step of pretreatment of the plasma sample by acetonitrile protein precipitation is: precisely sucking 200 mu L of plasma sample into a 1.5mL centrifuge tube, adding 10 mu L of olanzapine internal standard working solution and 10 mu L of valproic acid internal standard working solution, adding 800 mu L of acetonitrile solution into the mixed solution, carrying out vortex oscillation for 3min, carrying out 12700r/min centrifugation for 10min, and taking 200 mu L of supernatant into a 96-well plate.
3. The HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and metabolites thereof in plasma according to claim 1, wherein in S2 the chromatographic conditions are: the chromatographic column adopts Agilent Poroshell EC-C18 column with 2.1mm×50mm,2.7 μm, and the chromatographic mobile phase is phase A: formic acid water with the volume concentration of 0.1%; and B phase: acetonitrile; the flow rate is 0.4mL/min; the column temperature is maintained at 35+/-2 ℃; the amount of one sample was 5. Mu.L.
4. The method for simultaneous determination of valproic acid, olanzapine and metabolites thereof in plasma by HPLC-MSMS according to claim 1, wherein the mass spectrometry conditions are electrospray ion source for mass spectrometry, positive ion mode in 0-2min, negative ion mode from 2min to end of detection; the flow rate of the drying gas is 11L/min; the temperature of the drying gas is 350 ℃; the atomization air pressure is 35Psi; setting the capillary voltage to 4000V as positive ion mode; the negative ion mode was set to 3500V.
5. The HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and metabolites thereof in plasma according to claim 1, wherein the valproic acid, olanzapine and metabolites thereof are the following 5 analytes: valproic acid, olanzapine, 2-hydroxymethyl olanzapine, olanzapine-10N-glucuronide, N-norolanzapine.
6. HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and its metabolites in plasma according to claim 2, characterized in that said olanzapine internal standard working solution is prepared by the following method: 1mg of olanzapine-D3 is precisely weighed, 1mL of methanol is added to prepare 1mg/mL of olanzapine-D3 mother liquor, 10 mu L of olanzapine-D3 mother liquor is taken in a 10mL volumetric flask, methanol is added to fix the volume, and the olanzapine-D3 internal standard working solution with the concentration of 1000ng/mL is prepared.
7. HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and their metabolites in plasma according to claim 2, characterized in that the valproic acid internal standard working solution is prepared by the following method: precisely weighing 100mg of valproic acid-D15 in a 10mL volumetric flask, adding methanol to fix the volume to prepare 10mg/mL valproic acid-D15 mother liquor, taking 1mL of valproic acid-D15 mother liquor in another 10mL volumetric flask, adding methanol to fix the volume to prepare the valproic acid-D15 internal standard working solution with the concentration of 1000 mug/mL.
8. HPLC-MSMS method for simultaneous determination of valproic acid, olanzapine and its metabolites in plasma according to claim 1, characterized in that in S2 olanzapine and its metabolites are allowed to peak at 0.4-0.6min and valproic acid is allowed to peak at 3.1-3.2 min; the method comprises the steps of carrying out sectional collection on a mass spectrum, collecting olanzapine and metabolites and internal standards thereof in 0-2min by using a positive ion mode, and collecting valproic acid and internal standards thereof in 2-4.5min, so that the method realizes that the positive ion and negative ion collection modes are simultaneously used in the same analysis method, and simultaneously determines valproic acid, olanzapine and metabolites thereof.
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