CN117269402B - Liquid phase mass spectrum combination method for quantitatively detecting norcinnabar in human cerebrospinal fluid - Google Patents
Liquid phase mass spectrum combination method for quantitatively detecting norcinnabar in human cerebrospinal fluid Download PDFInfo
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- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The invention discloses a liquid phase mass spectrum combined method for quantitatively detecting norcinnabar in human cerebrospinal fluid, which comprises the following steps: (1) preparing a standard curve working solution; (2) preparing an internal standard working solution; (3) preparing a human cerebrospinal fluid standard curve sample; (4) Treating a human cerebrospinal fluid sample to be tested and a human cerebrospinal fluid standard curve sample; (5) performing LC-MS/MS analysis on the treated samples respectively; (6) And drawing a standard curve, and quantitatively analyzing the content of the norcinnabar in the cerebrospinal fluid sample of the human to be tested according to a standard curve method. The method is simple, quick and accurate in result, and can be used for the pharmacokinetics research of the norcinnabar.
Description
Technical Field
The invention relates to a method for quantitatively detecting concentration of nosiheptide in human cerebrospinal fluid by liquid chromatography-tandem mass spectrometry, belonging to the technical field of drug analysis.
Background
Nucina (nusinesen) was the first antisense oligonucleotide drug to treat rare spinal muscular atrophy (spinal muscular atrophy, SMA), approved by the U.S. food and drug administration (Food and Drug Administration, FDA) in 2016, and marketed in China in 2019. Norcinacalcet is injected intrathecally, bypassing the blood brain barrier and reaching the central nervous system. At present, the characteristics of Pharmacokinetics (PK) and Pharmacodynamics (PD) of the medicine in the population in China are not clear, so that quantitative bioanalytics of the medicine in the central nervous system, the circulatory system and key organ tissues are of great importance.
Antisense oligonucleotides (antisense oligonucleotide, ASO) generally refer to short-chain nucleic acids consisting of 15 to 25 nucleotides, with some chemical modifications. Currently, the types of analysis methods for ASO drugs are roughly classified into two categories:
chromatographic methods of the first type, comprising
1. High performance liquid chromatography ultraviolet/Fluorescence (high-performance liquid chromatography-UV/Fluorescence, HPLC-UV/FD);
2. capillary gel electrophoresis-ultraviolet method (capillary gel electrophoresis-UV, CGE-UV);
3. liquid chromatography-mass spectrometry (liquid chromatography tandem mass spectrometry, LC-MS/MS) and liquid chromatography-high resolution high mass accuracy mass spectrometry (liquid chromatography-high resolution accurate mass, LC-HRAM);
a second class of hybridization-based assays includes
1. Enzyme-linked immunosorbent assay based on hybridization (hybridized enzyme linked immunosorbent assay, HELISA) and hybrid-flow cytometry (hybridized flow cytometry, HFCM);
2. HPLC-FD method based on hybridization;
based on impuritiesThe analytical method of cross-linking, especially HELISA, is a common analytical method in preclinical and clinical research due to the characteristics of high sensitivity, simple sample pretreatment, high throughput and the like. However, there are also several unavoidable limitations in the HELISA method, including cross-reactivity with its metabolites (e.g., N-1, N-2, etc.), narrow dynamic range, poor tissue sample compatibility, and possible effects of the presence of anti-drug antibodies in the sample, and more importantly, the method is developed over a longer period and at a higher cost. In preclinical and clinical studies of norcinnabar, it is HELISA that is used [1] . In contrast, chromatographic methods, particularly LC-MS/MS techniques, combine high separation capacity liquid chromatography with high sensitivity and selective mass spectrometry, which have the advantage of high specificity, a broad range of quantification and simultaneous detection of multiple analytes (e.g. oligonucleotide drugs and their metabolites), in addition to the ability to accurately quantify oligonucleotides in biological samples [2] In addition, the method does not need special reagent design and synthesis, and is easier to develop, short in development period and strong in practicability.
《Results from a phase 1study of nusinersen(ISIS-SMN(Rx))in children with spinal muscular atrophy.》 [3] Reported are: the concentration of the norcinal by the original manufacturer of the norcinal is determined by using the HELISA method or the electrochemical luminescence method (ECL), and the HELISA method has the problems as above; ECL has the problem that although more sensitive and wider in detection range, it may also be affected by the cross reaction of the metabolic products of norcinnabar, the presence of anti-drug antibodies and the like, affecting the determination of the norcinnabar content [4] In addition, the method requires the synthesis of special base sequences and reagents, and has a long development period.
Chinese patent application 201910432682.6 (method for detecting midkine antisense oligonucleotide content in macaque plasma) [5] Disclosed is: taking kiwi plasma to be detected containing midkine antisense oligonucleotide, adding influenza Tide as an internal standard solution into the kiwi plasma, adding a proper amount of ammonia water solution and a phenol/dichloromethane mixed solution, and then sequentially addingPerforming vortex post centrifugation to obtain a processed blood sample; taking the supernatant of the blood sample and performing LC/MS analysis; drawing a standard curve, quantitatively analyzing the content of the midkine antisense oligonucleotide in the plasma of the macaque to be tested according to the standard curve method (see abstract part of the patent), wherein the minimum quantitative limit of the midkine antisense oligonucleotide in the plasma of the macaque is 0.18 mug/mL (see paragraph 132 of the patent specification), although the patent discloses a method for detecting the content of the midkine antisense oligonucleotide in the plasma of the macaque by adopting HPLC-MS/MS, the peptide structures of the midkine antisense oligonucleotide and the midkine antisense oligonucleotide are different, and the molecular weights are different, so the method is not applicable to the detection of the nomonade in human cerebrospinal fluid, and more importantly, the minimum quantitative limit of the method is relatively high, and the drug specification (12.3 pharmacokinetics of the American FDA (American) Neunocinnabar injection) [6] The mean terminal elimination half-life of norcinnabar in cerebrospinal fluid was estimated to be 135 to 177 days, and the mean trough concentration of norcinnabar increased cumulatively by about 1.4-3 times after multiple loading and maintenance doses and reached steady state within about 24 months. Treatment of infantile-onset spinal muscular atrophy with nusinersen, aphase 2, open-label, dose-escription study [7] Report: in clinical trials, the concentration of norcinal in cerebrospinal fluid was 3.57ng/mL at day 15 after 12mg of approval given once, and 10.9ng/mL at day 505 with multiple loading and maintenance doses [8] Report: the trough concentration of the norcinal in the cerebrospinal fluid at the steady state is 5ng/mL, so the quantitative limit of the method is higher than 0.18 mug/mL, and the method is not suitable for quantitatively detecting the content of the norcinal in the cerebrospinal fluid of a human body and is not suitable for the pharmacokinetic study of the norcinal in Chinese people.
Application number 202180016257.4, use of liquid chromatography and Mass Spectrometry for characterization of oligonucleotides [9] Disclosed is: the present disclosure provides oligonucleotides of interest that have been characterized using liquid chromatography and mass spectrometry methods described herein and including the oligonucleotides of interestCompositions of a population of oligonucleotides (see paragraph 85 of this patent specification), in some embodiments, the oligonucleotide of interest is a therapeutic oligonucleotide (see paragraph 86 of this patent specification), and the present disclosure provides pharmaceutical compositions comprising the oligonucleotide of interest, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of the oligonucleotides in the composition are the oligonucleotide of interest (see paragraph 212 of this patent specification). This patent is used for characterization analysis, qualitatively identifying the sequence or modification of oligonucleotides, and the like, and this method can only detect oligonucleotides in a single component solution, and cannot extract enrichment and detection oligonucleotides from complex human cerebrospinal fluid samples.
《HYBRIDIZATION LC-MS/MS:AN ALTERNATIVE BIOANALYTICAL METHOD FOR ANTI-SENSE OLIGONUCLEOTIDE QUANTITATION IN PLASMA AND TISSUE SAMPLES》 [10] Reported are: quantitative bioassays of plasma and tissue samples are required to study the pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides. To overcome the inherent shortcomings of conventional Ligand Binding Assays (LBAs) and LC-MS/MS methods in terms of specificity, sensitivity and throughput, an alternative bioassay method was developed that combines oligonucleotide hybridization with LC-MS/MS technology. Target ASO is extracted from the biological sample by hybridization with biotinylated sense strand oligonucleotides coupled to streptavidin magnetic beads. The typical challenges of sensitivity degradation in conventional ion-paired LC-MS/MS were first overcome using ion-paired chromatography and tandem mass spectrometry (MS/MS) methods that demonstrate high sensitivity (0.5 ng/mL when 100 μl of plasma is used), high specificity, wide linear range, fully automated and universal application in ternary pump systems in tests using multiple ASOs. Due to high specificity, the quantification of various biological matrixes is realized by using a calibration standard in blood plasma, and the efficiency and consistency are greatly improved. Another major advantage is the ability to simultaneously quantify ASO metabolites. Hybrid LC-MS/MS is considered an improved alternative to the quantification of ASOs and metabolites in plasma and tissue samples, showing replacement of traditional LBA and LC-MS +.The great potential of the MS method, which is a hybridization-based HPLC-FD method in which the structure, modification and molecular weight of the analyte are differentiated from that of norcinal, has problems in that if the method is used to detect norcinal in cerebrospinal fluid, a base pairing reagent for norcinal is required, a great deal of effort is required to optimize the pairing reagent for optimal length and optimal sequence, and the cost is high, and the operation steps are complicated.
Chinese patent application No. 202211619155.4 (method for detecting oligonucleotide compound concentration by liquid-phase mass spectrometry) [11] Disclosed is: the invention discloses a liquid phase mass spectrum combined detection method of oligonucleotide compound concentration, which is used for the semi-quantitative and quantitative biological sample analysis and detection of oligonucleotide compounds; the mobile phase of the liquid phase part uses hexafluoroisopropanol and tertiary amine buffer salt system to meet the detection work of most oligonucleotides, so that the problem that the traditional mobile phase cannot be reserved in a chromatographic column due to the fact that an oligonucleotide compound cannot be made by introducing ion pair solvent components is solved, meanwhile, the problem of large interference of other traditional methods is solved by utilizing a mass spectrum screening type acquisition mode, the stability and sensitivity of the liquid phase method of the oligonucleotide compound are well improved, and the problem of optimizing mass spectrum ion pairs and ion source conditions of the oligonucleotide compound with multiple charges by using the liquid phase mass spectrum method is effectively solved through computer assistance. The structure, modification and molecular weight of the analyte in the patent are different from that of the norcinnabar, different structures, modifications and analysis methods are different, the method is not necessarily suitable for detecting the norcinnabar in human cerebrospinal fluid, and the minimum concentration of a standard curve detection sample of the method is 47.5ng/ml, so the quantitative limit of the method in the patent is higher, and the method is not suitable for detecting the content of the norcinnabar in human cerebrospinal fluid for the same reason.
《Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy》 [12] A method for determining the profile of a metabolite of norcinnabar is reported, in which the sample is processed by liquid-liquid extraction (LLE) followed by Solid Phase Extraction (SPE)The additional purification effect is optimal, and then the high-resolution mass spectrum Q-TOF-MS is used for identifying the moxifloxacin metabolite graph, and the research belongs to qualitative research and non-quantitative research.
《Improvement of serum sample preparation and chromatographic analysis of nusinersen used for the treatment of spinal muscular atrophy》 [13] A methodological optimisation of the extraction of the norcinnabar metabolites from the serum matrix and optimisation of chromatographic methods, and identification of the metabolites, is reported, sample processing using liquid-liquid extraction (LLE); dispersed solid phase extraction (spe); sample processing methods based on hybridization extraction, analytical methods include UHPLC-UV, UHPLC-Q-TOF-MS, which are qualitative studies, not quantitative studies.
In summary, no LC-MS/MS quantitative analysis method specially used for detecting the norcinal in human cerebrospinal fluid has high accuracy, strong specificity, high sensitivity and relatively simple method at present, so that the method is suitable for further researching the Pharmacokinetic (PK) and Pharmacodynamics (PD) characteristics of the norcinal in the crowd in China.
Reference is made to:
[1]R.S.Finkel,C.A.Chiriboga,J.Vajsar,et al.,Treatment of infantile-onset spinal muscular atrophy with nusinersen:a phase 2,open-label,dose-escalation study,Lancet.388(2016)3017-3026.
[2]A.Liu,M.Cheng,Y.Zhou,et al.,Bioanalysis of Oligonucleotide by LC-MS:Effects of Ion Pairing Regents and Recent Advances in Ion-Pairing-Free Analytical Strategies,Int.J.Mol.Sci.23(2022)
[3]Chiriboga CA,Swoboda KJ,Darras BT,Iannaccone ST,Montes J,De Vivo DC,Norris DA,Bennett CF,Bishop KM.Results from a phase 1study of nusinersen(ISIS-SMN(Rx))in children with spinal muscular atrophy.Neurology.2016Mar 8；86(10):890-7.doi:10.1212/WNL.0000000000002445.Epub 2016Feb 10.PMID:26865511；PMCID:PMC4782111.
[4]Norris DA,Post N,Yu RZ,Greenlee S,Wang Y.Bioanalysis considerations on the pharmacokinetic evaluation of antisense therapeutics.Bioanalysis.2019Nov；11(21):1909-1912.doi:10.4155/bio-2019-0194.Epub 2019Oct 25.PMID:31648523.
[5] che Jinjing, original plum Zhu Xiaoyu, etc. A method for detecting midkine antisense oligonucleotide content in macaque plasma is 201910432682[ P ] [2023-10-07].
[6] Inonocinal injection medicine instruction book of American FDA official gazebo
https://www.drugfuture.com/fda-ndc/label.aspx
[7]Finkel RS,Chiriboga CA,Vajsar J,Day JW,Montes J,De Vivo DC,Yamashita M,Rigo F,Hung G,Schneider E,Norris DA,Xia S,Bennett CF,BishopKM.Treatment of infantile-onset spinal muscular atrophy with nusinersen:a phase 2,open-label,dose-escalation study.Lancet.2016Dec 17；388(10063):3017-3026.doi:10.1016/S0140-6736(16)31408-8.Epub 2016Dec 7.PMID:27939059.
[8]D.MacCannell,Z.Berger,L.East,et al.,Population pharmacokinetics-based recommendations for a singledelayed or missed dose of nusinersen,Neuromuscul.Disord.31(2021)310-318.
[9] Yellow name Haibo, xu Xiaobin, et al liquid chromatography and mass spectrometry were used to characterize the use of oligonucleotides CN202180016257.4[2023-10-07].
[10]Li,P.,Gong,Y.,Kim,J.,Liu,X.,&Rooney,M.(2020).Hybridization lc-ms/ms:an alternative bioanalytical method for anti-sense oligonucleotide quantitation in plasma and tissue samples.Analytical Chemistry,2020Aug 4；92(15):10548-10559.
[11] A method for detecting the concentration of oligonucleotide compound by liquid-phase mass spectrum combination is 202211619155[ P ] [2023-10-30].
[12]Buszewski B.Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy[J].International Journal of Molecular Sciences,2022,23.DOI:10.3390/ijms231710166.
[13]Studzińska S,Szymarek J,M.Improvement of serum sample preparation and chromatographic analysis of nusinersen used for the treatment of spinal muscular atrophy.Talanta.2023Sep 6；267:125173.doi:10.1016/j.talanta.2023.125173.Epub ahead of print.PMID:37690419.
Disclosure of Invention
Problems to be solved by the invention:
in order to solve the problems in the prior art, the invention establishes a method for quantitatively detecting the concentration of the norcinal in human cerebrospinal fluid by HPLC-MS/MS, the method has high specificity, and parameters such as accuracy, precision, sample freeze thawing stability and the like all meet detection requirements, thereby being suitable for being used as the pharmacokinetic study of the norcinal.
In order to achieve the above purpose, the specific technical scheme of the invention is as follows:
1. detection method and step
1. Preparing standard curve working solution:
(1) Preparing standard curve working solution stock solution
Precisely weighing the norcinal, placing the norcinal into a low-adsorption plastic bottle, adding a proper amount of 90% dimethyl sulfoxide solution to prepare a 1.00mg/mL norcinal standard curve working solution stock solution, and placing the norcinal stock solution into a refrigerator at the temperature of minus 10 ℃ to minus 30 ℃ for standby;
(2) Preparing standard curve working solution
The standard curve working solution of the norcinnabar is prepared in a low-adsorption plastic bottle, and the stock solution of the standard curve working solution of the norcinnabar is diluted by adding methanol-plasma-water solution (10:0.5:90, v/v/v) to prepare the standard curve working solution with the concentration of 100, 200, 400, 1000, 4000, 8000, 16000 and 20000 ng/mL.
2. Preparing an internal standard working solution:
(1) Preparing internal standard working solution stock solution
Taking glipizide, adding a proper amount of 90% dimethyl sulfoxide solution to prepare an internal standard working solution stock solution with the glipizide concentration of 1.00mg/mL, and storing in a refrigerator at-10 to-30 ℃ for later use;
(2) Preparing an internal standard working solution
Diluting an internal standard working solution stock solution with a methanol-water solution (5:95, v/v) to prepare an internal standard working solution with the glipizide concentration of 500 ng/mL;
3. preparing human cerebrospinal fluid standard curve samples:
vortex mixing human cerebrospinal fluid and standard curve working solution in a proper proportion under the condition of wet ice to prepare a human cerebrospinal fluid standard curve sample, wherein the concentration of the norcinnabar in the human cerebrospinal fluid standard curve sample is 2.00, 4.00, 8.00, 20.0, 80.0, 160, 320 and 400ng/mL respectively;
4. treatment of a human cerebrospinal fluid sample to be tested and a human cerebrospinal fluid standard curve sample:
respectively taking 50 mu L of human cerebrospinal fluid sample to be tested and 50 mu L of human cerebrospinal fluid standard curve sample, respectively adding 20 mu L of internal standard working solution under the condition of wet ice, then adding 50 mu L of methanol aqueous solution (2:98, v:v) containing 0.5% triethylamine for dilution, carrying out vortex oscillation for 5min, centrifuging 3500g for 1min at 4 ℃, and respectively taking the supernatant, thus obtaining the treated sample.
5. The treated samples were subjected to LC-MS/MS analysis, respectively:
the chromatographic conditions were as follows:
chromatographic column: waters Xbridge C18,3.5μm，4.6×50mm；
mobile phase a: an aqueous solution containing 0.5% triethylamine and 0.5% hexafluoroisopropanol;
mobile phase B: an acetonitrile solution containing 0.5% triethylamine and 0.5% hexafluoroisopropanol;
strong needle washing liquid: 0.1% triethylamine is dissolved in a methanol-acetonitrile-isopropanol-water mixed solution, wherein the volume ratio of the methanol-acetonitrile-isopropanol-water mixed solution is 1:1:1:1, methanol, acetonitrile, isopropanol and water;
weak needle washing liquid: 10% methanol aqueous solution, wherein the 10% methanol aqueous solution is prepared from the following components in percentage by volume: 90 methanol and water;
flow rate: 0.800mL/min;
sample injection amount: 20. Mu.L;
the elution mode is gradient elution, and the gradient elution program is as follows:
0 to 0.01min, wherein the phase A is 95 percent and the phase B is 5 percent;
0.01-2.00 min, the A phase is reduced from 95% to 65%, and the B phase is increased from 5% to 35%;
2.00-3.00 min, the A phase is reduced from 65% to 5%, and the B phase is increased from 35% to 95%;
3.00 to 3.80 minutes, wherein the phase A is 5 percent and the phase B is 95 percent;
3.80 to 3.85min, the A phase is increased from 5 percent to 95 percent, and the B phase is reduced from 95 percent to 5 percent;
3.85 to 4.20min, wherein the phase A is 95 percent and the phase B is 5 percent;
4.20 to 4.25 minutes, the A phase is reduced from 95 percent to 5 percent, and the B phase is increased from 5 percent to 95 percent;
4.25 to 4.60 minutes, wherein the phase A is 5 percent and the phase B is 95 percent;
4.60 to 4.65 minutes, the A phase is increased from 5 percent to 95 percent, and the B phase is reduced from 95 percent to 5 percent;
4.65 to 5.00min, wherein the phase A is 95 percent and the phase B is 5 percent;
5.00-5.05 min, the A phase is reduced from 95% to 5%, and the B phase is increased from 5% to 95%;
5.05 to 5.25min, wherein the phase A is 5 percent and the phase B is 95 percent;
5.25 to 5.30 minutes, the A phase is increased from 5 percent to 95 percent, and the B phase is reduced from 95 percent to 5 percent;
the mass spectrometry conditions were as follows:
high-purity nitrogen is used as gas curtain gas and collision gas, ESI source anions are scanned, and a multi-ion reaction monitoring scanning mode is adopted;
mass spectrometry parameters:
collision gas: high; air curtain gas: 55psi; ion source gas 1:50psi; ion source gas 2:50psi; ion spray voltage: -4500V; temperature: 550 ℃; mass spectrum acquisition duration: 6.00min;
parameters of the analyte during LC-MS/MS analysis:
the monitoring ion pair of the Norcinacalcet is m/z 890.300- & gt m/z393.400; the monitored ion pair for glipizide was m/z444.400 →m/z170.200.
6. Drawing a standard curve, and quantitatively analyzing the content of the norcinnabar in the cerebrospinal fluid sample of the human to be tested according to the standard curve method:
taking the chromatographic peak-to-peak area ratio of the norcinnabar and the glipizide in the human cerebrospinal fluid standard curve sample as an ordinate, and taking the concentration of the human cerebrospinal fluid standard curve sample as an abscissa, and performing linear regression to obtain a standard curve; substituting the chromatographic peak-to-peak area ratio of the norcinnabar and the glipizide in the cerebrospinal fluid sample of the human to be tested into a standard curve for calculation, and obtaining the content of the norcinnabar in the cerebrospinal fluid sample of the human to be tested.
2. Preparing a quality control sample:
1. preparing a quality control sample working solution:
preparing a quality control sample working solution in a low-adsorption plastic bottle, taking a norcinnating standard curve working solution stock solution, and adding a methanol-plasma-water solution (10:0.5:90, v/v/v) to dilute to prepare the quality control sample working solution with the concentration of 100, 300, 3000, 6000 and 15000 ng/mL;
2. preparing quality control samples
Human cerebrospinal fluid is mixed with a working solution of a quality control sample under the condition of wet ice to prepare the quality control samples with the concentration of 2.00, 6.00, 60.0, 120 and 300ng/mL of the norcinal.
3. Method verification of the above detection method
1. Specialization of
Taking normal human blank cerebrospinal fluid, norcinnabar+human cerebrospinal fluid (the concentration of the norcinnabar is 2.00 ng/mL) and glipizide+human cerebrospinal fluid (the concentration of the glipizide is 500 ng/mL), and operating according to the methods under the items of 'one, detection methods and 4 and 5' in the steps respectively.
The specificity results show that: with the method of the present application, endogenous substances in cerebrospinal fluid do not interfere with the determination of the norcinnabar and internal standard.
2. Linear range and minimum quantitative limit
Taking prepared human cerebrospinal fluid standard curve sample, respectively operating according to "one, detection method and 4, 5 and 6" methods in steps, and establishing working curve by Watson LIMSTM 7.5SP1 (Th)ermo Scientific inc.) quantitative analysis and using Linear, weighting factor=1/x 2 The model was fitted linearly.
Linear range results: the linear range of the detection method is 2.00-400ng/mL, and the lowest quantification limit is 2.00ng/mL.
3. Accuracy and precision
And respectively taking quality control samples prepared by 'two quality control sample preparation', wherein the concentration of the quality control samples is 2.00, 6.00, 60.0, 120 and 300ng/mL, 6 samples are taken for each concentration, the operation is carried out according to a 'one, detection method and the methods under the items 4, 5 and 6' in the steps, the continuous measurement is carried out for 3 days, the measured concentration of the quality control samples is calculated according to the working curve of the standard product on the same day, and the accuracy and the precision of the measurement method are obtained by comparing the measured concentration with the prepared concentration.
Accuracy and precision results: the accuracy and precision deviation and the variation coefficient in the detection method are respectively between 0 and 10.0 percent and between 1.8 and 9.6 percent; the accuracy, precision deviation and variation coefficient between batches are respectively 3.3-5.7 percent and 3.0-6.3 percent; it is stated that the accuracy and precision within and between batches of the methods of the present application are satisfactory.
4. Sample stability
Preparing a quality control sample according to the 'preparation of the quality control sample II' and the 'preparation of the quality control sample', wherein the concentration of the quality control sample is 6.00 ng/mL and 300ng/mL respectively, and examining the short-term stability (room temperature and 19 h), the freeze-thawing stability (-80 ℃ for 3 times of freeze-thawing cycles), the long-term stability (-80 ℃ for 28 days) and the sample stability (sample to be detected, 4 ℃ and 143 h) of the quality control sample in an automatic sampler
Quality control sample stability results: the sample of the norcinal cerebrospinal fluid can be stably placed for 19 hours at room temperature, is stable after 3 times of freeze thawing cycles from-80 ℃ to room temperature, can be stably stored for at least 28 days at-80 ℃, and is stable after being placed for 143 hours in an automatic sampler (4 ℃).
Compared with the prior art, the beneficial effect of this application lies in:
the invention adopts the LC-MS/MS method to quantitatively detect the concentration of the norcinnabar in the cerebrospinal fluid of the human body, has simple and quick measurement and accurate result, and is suitable for being applied to the pharmacokinetics research of the norcinnabar.
1. The method has high selectivity by adopting a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method, can effectively reduce background interference, can still achieve high sensitivity on complex cerebrospinal fluid samples, and has the advantages of high resolution, wide quantitative range, quick scanning and the like; the method can effectively improve the detection sensitivity, the minimum quantitative limit can reach 2.00ng/mL, the analysis time is short, and the required sample amount is small;
2. the method can avoid the defects of the traditional HELISA method, such as cross reactivity with the norcinnabar metabolites (such as N-1, N-2 and the like), narrow dynamic range, poor tissue sample compatibility and possible influence of the existence of anti-drug antibodies in the sample, and more importantly, the HELISA method has longer development period and high cost; the method can avoid the influence of the cross reaction of the metabolic products of the norcinnabar, the existence of anti-drug antibodies and the like in the ECL method, and the determination of the norcinnabar drug is influenced.
3. The methods of the present application are relatively simpler compared to hybridization-based HPLC-FD methods.
Drawings
FIG. 1, nocina Q1 scan;
FIG. 2, nocina Q2 scan;
FIG. 3, internal standard glipizide Q1 scan;
fig. 4, internal standard glipizide Q2 scan;
fig. 5, blank human cerebrospinal fluid anion scanning chromatograms, wherein,
a is the negative ion scanning chromatogram of human cerebrospinal fluid and blank human cerebrospinal fluid in norcinnabar,
b is negative ion scanning chromatogram of human cerebrospinal fluid and blank human cerebrospinal fluid in glipizide;
fig. 6, human cerebrospinal fluid + norcinnabar and human cerebrospinal fluid + internal standard glipizide chromatograms, wherein,
a is blank human cerebrospinal fluid+Nocina chromatograms,
b is a blank human cerebrospinal fluid+glipizide chromatogram;
FIG. 7 Nocinal Standard Curve in human cerebrospinal fluid
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
All materials, reagents, etc. in the examples described below are commercially available unless otherwise specified.
Example 1, establishing a liquid Mass Spectrometry combination method for quantitatively detecting Norcinal in human cerebrospinal fluid
1. Experimental materials
1.1 pharmaceutical products and reagents
Norcinnabar (nusinesen) control standard: the sequence of the MINGKANGDE New drug is synthesized by Tianjin drug Limited company: 5'-TCACTTTCATAATGCTGG-3' (SEQ ID No. 1), purity: 98.37%, lot number: ET58940-8-P1, and preserving at a temperature of less than or equal to 4 ℃.
Glipizide control standard: purity of the product purchased from Shanghai screening Biotech Co., ltd.): 99.99%, lot number: 23Z294-D1, and preserving at 0-8 ℃.
Normal human cerebrospinal fluid sample: purchased in Innovative Research (united states), product number: IRHUCSF 1ML-40812-39, stored: -20 ℃.
Normal human blank K2EDTA plasma (plasma for short): purchased from Changshatai and Hospital phase I clinical institute, -80℃for storage.
Methanol, acetonitrile, dimethyl sulfoxide and isopropanol used in the test: purchased from Thermo Fisher Scientific (U.S.), wherein methanol, acetonitrile, dimethyl sulfoxide are chromatographically pure and isopropanol is of LC/MS grade.
Hexafluoroisopropanol, triethylamine: purchased from Sigma-Aldrich, USA, all HPLC grade.
1.2 instruments
The ∈liquid phase system comprises a vacuum degasser, a quaternary pump, a sample injector and a column temperature box
2. Experimental method and procedure
2.1 preparing a standard curve working solution and a quality control sample working solution
Precisely weighing a norcinal reference substance, placing the norcinal reference substance in a low-adsorption plastic bottle, adding a proper amount of 90% dimethyl sulfoxide solution to prepare a 1.00mg/mL norcinal standard curve working solution stock solution, and placing the norcinal standard curve working solution stock solution in a refrigerator at the temperature of minus 10 ℃ to minus 30 ℃ for later use;
the standard curve of the norcinnabar and the quality control sample working solution are prepared in a low-adsorption plastic bottle.
Taking a norcinal standard curve working solution stock solution, adding a methanol-plasma-water solution (10/0.5/90, v/v/v) to dilute and prepare standard curve working solutions with the concentrations of 100, 200, 400, 1000, 4000, 8000, 16000 and 20000ng/mL respectively;
and diluting the norcinal standard curve stock solution (1.00 mg/mL) with a methanol-plasma-water (10/0.5/90, v/v/v) solution to prepare a quality control sample working solution with the concentration of 100, 300, 3000, 6000 and 15000 ng/mL.
2.2 preparing an internal Standard working solution
Taking glipizide reference standard substance, adding a proper amount of 90% dimethyl sulfoxide solution to prepare internal standard working solution stock solution with the glipizide concentration of 1.00mg/mL, and storing in a refrigerator at-10 to-30 ℃.
And diluting the internal standard working solution stock solution with a methanol aqueous solution (5:95, v/v) to prepare an internal standard working solution with the concentration of 500ng/mL, and placing and storing the internal standard working solution in a refrigerator at the temperature of minus 10 ℃ to minus 30 ℃.
2.3 preparing human cerebrospinal fluid standard curve sample and quality control sample
Human cerebrospinal fluid standard curve samples were prepared using human cerebrospinal fluid and standard curve working solutions in 2.1 in a suitable ratio by vortexing under wet ice conditions, the concentration of norcinal in the human cerebrospinal fluid standard curve samples being 2.00, 4.00, 8.00, 20.0, 80.0, 160, 320, 400ng/mL, respectively.
The quality control samples were formulated using human cerebrospinal fluid vortex mixed with 2.1 quality control sample working solution in appropriate proportions under wet ice conditions, with the concentration of norcinal in the human cerebrospinal fluid quality control samples being 2.00 (LLOQ), 6.00 (LQC), 60.0 (MQC-1), 120 (MQC-2), 300ng/mL (HQC), respectively.
Wherein LLOQ designates a lower limit concentration, LQC designates low concentration quality control, MQC designates medium concentration quality control, and HQC designates high concentration quality control.
2.4 treatment of human cerebrospinal fluid sample to be measured, human cerebrospinal fluid standard curve sample and human cerebrospinal fluid quality control sample:
respectively taking 50 mu L of human cerebrospinal fluid sample to be tested, 50 mu L of human cerebrospinal fluid standard curve sample and 50 mu L of human cerebrospinal fluid quality control sample, respectively adding 20 mu L of internal standard working solution under the condition of wet ice, then adding 50 mu L of methanol aqueous solution (2:98, v:v) containing 0.5% triethylamine for dilution, carrying out vortex oscillation for 5min, centrifuging 3500g for 1min at 4 ℃, and respectively taking the supernatant, thus obtaining the treated sample.
2.5 LC-MS/MS analysis of the treated samples respectively
2.5.1 chromatographic conditions:
chromatographic column: waters Xbridge C18,3.5μm，4.6×50mm；
mobile phase
Mobile phase a: an aqueous solution containing 0.5% triethylamine and 0.5% hexafluoroisopropanol;
mobile phase B: an acetonitrile solution containing 0.5% triethylamine and 0.5% hexafluoroisopropanol;
needle washing liquid
Strong needle washing liquid: 0.1% triethylamine in methanol/acetonitrile/isopropanol/water (1/1/1/1; v/v/v/v);
weak needle washing liquid: 10% aqueous methanol (10/90, v/v);
flow rate: 0.800mL/min;
sample injection amount: 20. Mu.L;
gradient elution, elution procedure:
the percentages of the mobile phase A and the mobile phase B are volume percentages
2.5.2 Mass Spectrometry conditions
High-purity nitrogen is used as Curtain gas, collision gas ESI source anions are scanned, the MRM scanning mode is adopted, and mass spectrum conditions are as follows:
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2.5.3 test parameters are as follows:
first and second level scans of norcinazon and internal standard glipizide are shown in fig. 1-4. Fig. 1: a Q1 scan of norcinal; fig. 2: a Q2 scan of norcinal; fig. 3: an internal standard glipizide Q1 scan; fig. 4: and (5) scanning an internal standard glipizide Q2.
2.6 drawing a standard curve, and quantitatively analyzing the content of the norcinnabar in the cerebrospinal fluid sample of the human to be tested according to the standard curve method:
taking the chromatographic peak-to-peak area ratio of the norcinnabar and the glipizide in the human cerebrospinal fluid standard curve sample as an ordinate, and taking the concentration of the human cerebrospinal fluid standard curve sample as an abscissa, and performing linear regression to obtain a standard curve; substituting the chromatographic peak-to-peak area ratio of the norcinnabar and the glipizide in the cerebrospinal fluid sample of the human to be tested into a standard curve for calculation, and obtaining the content of the norcinnabar in the cerebrospinal fluid sample of the human to be tested.
3. Methodological verification
3.1 specificity
Taking normal human blank cerebrospinal fluid, and respectively carrying out LC-MS/MS analysis under-item operation according to the treatment of a human cerebrospinal fluid sample to be detected, a human cerebrospinal fluid standard curve sample and a human cerebrospinal fluid quality control sample and the sample treated by 2.5 to obtain a chromatogram of the blank human cerebrospinal fluid sample; then, respectively diluting a certain concentration of norcinnabar solution and an internal standard solution with blank cerebrospinal fluid of a normal person, and operating according to the same method to obtain chromatograms of human cerebrospinal fluid+norcinnabar (2.00 ng/mL), human cerebrospinal fluid+glipizide (500 ng/mL), wherein the chromatograms are shown in fig. 5-6, fig. 5 is a blank cerebrospinal fluid anion scanning chromatogram, A is a blank cerebrospinal fluid anion scanning chromatogram in human cerebrospinal fluid+norcinnabar, and B is a blank cerebrospinal fluid anion scanning chromatogram in human cerebrospinal fluid+glipizide; FIG. 6 is a blank human cerebrospinal fluid+norcinnabar and blank human cerebrospinal fluid+glipizide chromatogram, wherein A is a blank human cerebrospinal fluid+norcinnabar chromatogram; b is blank human cerebrospinal fluid+glipizide chromatogram.
The special test results show that: in the method, endogenous substances in cerebrospinal fluid do not interfere with the determination of the norcinnabar and internal standard.
3.2 Linear Range and lower quantitative Limit
Respectively taking prepared human cerebrospinal fluid standard curve samples, respectively performing LC-MS/MS analysis on the samples according to the treatment of the human cerebrospinal fluid sample to be detected 2.4, the human cerebrospinal fluid standard curve sample and the human cerebrospinal fluid quality control sample and the treatment of the samples after the treatment of 2.5, establishing a working curve, quantitatively analyzing the working curve by using Watson LIMSTM 7.5SP1 (Thermo Scientific Inc.), and adopting Linear, weighting factor=1/x 2 The model was fitted linearly. The human cerebrospinal fluid standard curve of norcinnabar is shown in FIG. 7, and the linear range of norcinnabar in human cerebrospinal fluid is 2.00-400ng/mL. If the lowest concentration of the standard curve is taken as the lowest detection limit, the lowest quantitative limit of the norcinnabar in the cerebrospinal fluid is 2.00ng/mL.
3.3 accuracy and precision
And respectively taking 2.3 prepared quality control samples, wherein 5 concentrations are respectively 2.00, 6.00, 60.0, 120 and 300ng/mL, 6 samples are taken for each concentration, the samples are respectively processed according to a human cerebrospinal fluid sample to be detected, a human cerebrospinal fluid standard curve sample and a human cerebrospinal fluid quality control sample, the samples after 2.5 treatment are respectively subjected to LC-MS/MS analysis, the standard curve is drawn by 2.6, the quantitative analysis of the norcinal content in the human cerebrospinal fluid sample to be detected is carried out according to a standard curve method, the analysis is carried out, the continuous measurement is carried out for 3 days, the measured concentration of the QC sample is respectively calculated according to a standard working curve of the current day, the comparison of the prepared concentration is carried out, and the accuracy and the precision of the measurement method are obtained, and the result is shown in Table 1.
TABLE 1 accuracy and precision of the LC-MS/MS determination method of Norcina in human cerebrospinal fluid samples
a: mean is the average concentration, represented by the 3 significant digits;
b: bias is Bias, bias (%) = [ (average detected concentration-theoretical concentration)/(theoretical concentration) ]×100 (reserved to the decimal place later);
c: CV is the relative standard deviation, relative standard deviation (%) = standard deviation/mean x 100 (reserved to the decimal point one digit later).
As can be seen from table 1:
the accuracy, precision deviation and variation coefficient in the norcinal sample are respectively 0-10.0 percent and 1.8-9.6 percent, which indicates that the method for detecting the norcinal in the human cerebrospinal fluid accords with the rule of the Chinese pharmacopoeia of 2020 edition;
the accuracy, precision deviation and variation coefficient of human cerebrospinal fluid sample Nocinal batches are respectively 3.3% -5.7%, and 3.0% -6.3%, which shows that the accuracy and precision of detecting Nocinal in human cerebrospinal fluid in the method of the application are in accordance with the rule of Chinese pharmacopoeia of 2020 edition.
3.4 sample stability
The quality control samples with low and high concentrations in human cerebrospinal fluid are respectively prepared according to the method under the item of '2.3 preparation of human cerebrospinal fluid standard curve sample and quality control sample', the concentration of the cerebrospinal fluid quality control sample is respectively 6.00 (LQC) and 300ng/mL (HQC), each concentration is 3 parts, and the stability result of the norcinnabar in the cerebrospinal fluid is shown in Table 2.
TABLE 2 stability results of human cerebrospinal fluid Nocinal
a: mean is the average concentration, represented by the 3 significant digits;
b: bias is Bias, bias (%) = [ (average detected concentration-theoretical concentration)/(theoretical concentration) ]×100 (reserved to the decimal place later);
c: CV is the relative standard deviation, relative standard deviation (%) = standard deviation/mean x 100 (reserved to the decimal point one digit later).
As can be seen from table 2: the sample of the norcinal cerebrospinal fluid can be stably placed for 19 hours at room temperature, kept stable after 3 freeze thawing cycles from-80 ℃ to room temperature, and can be stably stored for at least 28 days at-80 ℃, and in addition, the treated sample is kept stable after being placed for 143 hours in an autosampler (4 ℃).
Compared with a plasma sample, the cerebrospinal fluid has less protein content and simpler components, so that the cerebrospinal fluid sample treatment process has only a dilution step and no protein precipitation process, and the recovery rate verification is not needed; the plasma is normal human plasma, hemolytic plasma and high-fat plasma, and the matrix effect is required to be examined, and the cerebrospinal fluid is different from the plasma, so that the matrix effect is not examined.
4. Conclusion of the test
The research uses glipizide as an internal standard, establishes and verifies a quantitative detection method of the norcinnabar LC-MS/MS in human cerebrospinal fluid, and the verification result of the method shows that endogenous substances in the human cerebrospinal fluid do not interfere with the determination of the norcinnabar and the internal standard. The minimum quantitative limit of the norcinal in the human cerebrospinal fluid is 2.00ng/mL, the linear range is 2.00-400ng/mL, the deviation and the variation coefficient of the accuracy and the precision in the batch are respectively 0-10.0%, 1.8-9.6%, and the deviation and the variation coefficient of the accuracy and the precision between the batches are respectively 3.3-5.7% and 3.0-6.3%. The sample of the norcinal cerebrospinal fluid can be stably placed for 19 hours at room temperature, kept stable after 3 freeze thawing cycles from-80 ℃ to room temperature, and can be stably stored for at least 28 days at-80 ℃, and in addition, the treated sample is kept stable after being placed for 143 hours in an autosampler (4 ℃).
The LC-MS/MS method established by the test can sensitively, accurately, rapidly and quantitatively detect the norcinal content in the cerebrospinal fluid of the human, and the parameters such as the specificity, the linear range, the quantitative lower limit, the accuracy, the precision, the sample stability and the like all meet the requirements of the pharmacopoeia of 2020 edition.
Method optimization in example 2, example 1
1. Screening and optimization of chromatography columns
The column was run on a model Acquity UPLC BEH C (1.0X100 mm,1.7 μm, waters), but the column Waters Xbridge C18 in example 1,3.5 μm, 4.6X50 mm works best, which can withstand highly alkaline mobile phases, resulting in increased column durability.
2. Screening and optimization of mobile phases
After the ion pair and the ion pair concentration are optimized, the peak type is optimal after the mobile phase is finally determined and 0.5 percent of hexafluoroisopropanol and 0.5 percent of triethylamine ion pair reagent are added.
3. Screening and optimization of needle washing liquid
After the needle washing liquid is optimized, the strong needle washing liquid is finally determined to be 0.1 percent triethylamine in methanol/acetonitrile/isopropanol/water (1/1/1; v/v/v/v) and the weak needle washing liquid is determined to be 10 percent methanol water solution (10/90, v/v), so that the residue can be reduced.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. A method for quantitatively detecting the content of norcinnabar in human cerebrospinal fluid, comprising the steps of:
(1) Preparing standard curve working solution:
precisely weighing a norcinal reference substance, placing the norcinal reference substance in a low-adsorption plastic bottle, adding a proper amount of 90% dimethyl sulfoxide solution to prepare a 1.00mg/mL norcinal standard curve working solution stock solution, and placing the norcinal standard curve working solution stock solution in a refrigerator at the temperature of minus 10 ℃ to minus 30 ℃ for later use; preparing a standard curve working solution of the norcinnabar in a low-adsorption plastic bottle, and diluting a stock solution of the standard curve working solution of the norcinnabar with a methanol-plasma-water solution to prepare standard curve working solutions with the concentrations of 100, 200, 400, 1000, 4000, 8000, 16000 and 20000ng/mL respectively, wherein the methanol-plasma-water solution is prepared by the following steps: 0.5:90, methanol, plasma and water;
(2) Preparing an internal standard working solution:
taking a glipizide reference standard substance, adding a proper amount of 90% dimethyl sulfoxide aqueous solution to prepare an internal standard working solution stock solution with the glipizide concentration of 1.00mg/mL, and storing in a refrigerator at the temperature of minus 10 ℃ to minus 30 ℃ for standby; diluting an internal standard working solution stock solution with a 5% methanol aqueous solution to prepare an internal standard working solution with the glipizide concentration of 500 ng/mL; the 5% methanol aqueous solution is prepared from methanol and water in a volume ratio of 5:95;
(3) Preparing human cerebrospinal fluid standard curve samples:
preparing a human cerebrospinal fluid standard curve sample by mixing human cerebrospinal fluid and the standard curve working solution in the step (1) in a proper proportion under a wet ice condition, wherein the concentration of the norcinal in the human cerebrospinal fluid standard curve sample is 2.00, 4.00, 8.00, 20.0, 80.0, 160, 320 and 400ng/mL respectively;
(4) Treatment of a human cerebrospinal fluid sample to be tested and a human cerebrospinal fluid standard curve sample:
respectively taking 50 mu L of a human cerebrospinal fluid sample to be tested and 50 mu L of a human cerebrospinal fluid standard curve sample prepared in the step (3), respectively adding 20 mu L of an internal standard working solution under the condition of wet ice, then adding 50 mu L of a methanol aqueous solution containing 0.5% of triethylamine for dilution, carrying out vortex oscillation for 5min, centrifuging 3500g for 1min at 4 ℃, respectively taking the supernatant, namely a treated sample, and preparing the methanol aqueous solution containing 0.5% of triethylamine: 5mL of triethylamine is measured, and the volume ratio is 2:98 methanol and water to 1000mL;
(5) The treated samples were subjected to LC-MS/MS analysis, respectively:
the chromatographic conditions for the LC-MS/MS analysis are as follows:
chromatographic column: waters Xbridge C18,3.5μm，4.6×50mm；
mobile phase a: an aqueous solution containing 0.5% triethylamine and 0.5% hexafluoroisopropanol;
mobile phase B: an acetonitrile solution containing 0.5% triethylamine and 0.5% hexafluoroisopropanol;
strong needle washing liquid: 0.1% triethylamine is dissolved in a methanol-acetonitrile-isopropanol-water mixed solution, wherein the volume ratio of the methanol-acetonitrile-isopropanol-water mixed solution is 1:1:1:1, methanol, acetonitrile, isopropanol and water;
weak needle washing liquid: 10% methanol aqueous solution, wherein the 10% methanol aqueous solution is prepared from the following components in percentage by volume: 90 methanol and water;
flow rate: 0.800mL/min;
sample injection amount: 20. Mu.L;
the elution mode is gradient elution, and the gradient elution program is as follows:
0 to 0.01min, wherein the phase A is 95 percent and the phase B is 5 percent;
0.01-2.00 min, the A phase is reduced from 95% to 65%, and the B phase is increased from 5% to 35%;
2.00-3.00 min, the A phase is reduced from 65% to 5%, and the B phase is increased from 35% to 95%;
3.00 to 3.80 minutes, wherein the phase A is 5 percent and the phase B is 95 percent;
3.80 to 3.85min, the A phase is increased from 5 percent to 95 percent, and the B phase is reduced from 95 percent to 5 percent;
3.85 to 4.20min, wherein the phase A is 95 percent and the phase B is 5 percent;
4.20 to 4.25 minutes, the A phase is reduced from 95 percent to 5 percent, and the B phase is increased from 5 percent to 95 percent;
4.25 to 4.60 minutes, wherein the phase A is 5 percent and the phase B is 95 percent;
4.60 to 4.65 minutes, the A phase is increased from 5 percent to 95 percent, and the B phase is reduced from 95 percent to 5 percent;
4.65 to 5.00min, wherein the phase A is 95 percent and the phase B is 5 percent;
5.00-5.05 min, the A phase is reduced from 95% to 5%, and the B phase is increased from 5% to 95%;
5.05 to 5.25min, wherein the phase A is 5 percent and the phase B is 95 percent;
5.25 to 5.30 minutes, the A phase is increased from 5 percent to 95 percent, and the B phase is reduced from 95 percent to 5 percent;
the mass spectrometry conditions for the LC-MS/MS analysis are as follows:
high-purity nitrogen is used as gas curtain gas and collision gas, ESI source anions are scanned, and a multi-ion reaction monitoring scanning mode is adopted;
mass spectrometry parameters:
collision gas: high; air curtain gas: 55psi; ion source gas 1:50psi; ion source gas 2:50psi; ion spray voltage: -4500V; temperature: 550 ℃; mass spectrum acquisition duration: 6.00min;
(6) Drawing a standard curve, and quantitatively analyzing the content of the norcinal in the cerebrospinal fluid sample of the human to be tested according to a standard curve method:
taking the chromatographic peak-to-peak area ratio of the norcinnabar and the glipizide in the human cerebrospinal fluid standard curve sample as an ordinate, and taking the concentration of the human cerebrospinal fluid standard curve sample as an abscissa, and carrying out linear regression to obtain a standard curve; substituting the chromatographic peak-to-peak area ratio of the norcinnabar and the glipizide in the cerebrospinal fluid sample of the human to be tested into the standard curve for calculation, thus obtaining the content of the norcinnabar in the cerebrospinal fluid sample of the human to be tested.
2. The method of claim 1, wherein during LC-MS/MS analysis, the monitored ion pair of norcinnabar is m/z890.300 →m/z393.400; the monitored ion pair of glipizide is m/z 444.400- > m/z170.200.
3. The method of claim 1, wherein the method has a linear range of 2.00ng/mL to 400ng/mL.
4. The method of claim 1, wherein the minimum quantification limit of the method is 2.00ng/mL.
5. The method of claim 1, wherein: the step (3) further comprises the preparation of a quality control sample, wherein the preparation process of the quality control sample comprises the following steps:
preparing a quality control sample working solution in a low-adsorption plastic bottle, taking the norcinnabar standard curve working solution stock solution, and adding a methanol-plasma-water solution to dilute the stock solution to prepare the quality control sample working solution with the concentration of 100, 300, 3000, 6000 and 15000ng/mL respectively, wherein the methanol-plasma-water solution is prepared by the following steps of: 0.5:90, methanol, plasma and water;
and mixing human cerebrospinal fluid with the working solution of the quality control sample under the condition of wet ice to prepare the quality control samples with the concentration of the norcinal sodium at 2.00, 6.00, 60.0, 120 and 300ng/mL respectively.
6. The method of claim 5, wherein after the quality control sample is processed in step (4), LC-MS/MS analysis is performed in step (5), and the established method is verified by the chromatographic peak-to-peak area ratio of norcinnabar and glipizide and the concentration data thereof in the quality control sample.
7. The method of claim 5, wherein the quality control sample having a concentration of 6.00 ng/mL of norcinnabar is stable for 19 hours at room temperature, stable after 3 freeze-thaw cycles from-80 ℃ to room temperature, and stable for at least 28 days at-80 ℃; the treated sample remained stable after being placed in an autosampler at 4 ℃ for 143 h.
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