CN113820424A - HPLC-MS/MS method for simultaneously determining concentration of 14 antidepressants in human plasma - Google Patents
HPLC-MS/MS method for simultaneously determining concentration of 14 antidepressants in human plasma Download PDFInfo
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- 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
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses an HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma, which adopts a protein precipitation method to extract an analyte in a plasma sample, an Agilent InfinityLab Poroshell 120EC-C18 chromatographic column to separate the sample, acetonitrile and water are used as mobile phases for gradient elution, and the mass spectrum condition adopts a multi-ion monitoring mode combined under an electrospray ion source positive ion mode. The total length of chromatographic separation was 4 minutes. The method of the application detects 14 antidepressantsGood linearity in blank plasma (R)2Greater than 0.99), the precision RSD between batches is 0.65-13.18%, the accuracy is 85.21-117.6%, and the matrix effect, the recovery rate, the stability, the dilution effect, the residual effect and the like all meet the requirements, thereby providing a large amount of meaningful clinical data for optimizing the clinical drug treatment scheme.
Description
Technical Field
The invention relates to the technical field of clinical blood concentration monitoring, in particular to an HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma.
Background
In recent years, with the development of psychological hygiene, the prevention and treatment of mental diseases are more and more emphasized by various social circles. However, mental diseases are extremely challenging to treat, and mainly appear in the aspects of poor medication compliance, narrow medication window, serious toxic and side effects and the like of patients in the psychiatric department. Therapeutic Drug Monitoring (TDM) plays a crucial role in the treatment of psychiatric disorders, and there is a great deal of evidence that monitoring serum or plasma drug concentrations has therapeutic and economic benefits, providing not only patient compliance, possible therapeutic response, pharmacokinetic-related data, but also valuable information for the occurrence and prevention of drug interactions and potential side effects.
High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with high specificity and high sensitivity has been widely used in clinical practice for the accurate and precise determination of drug concentration in plasma or serum. When HPLC-MS/MS is used for routine clinical TDM, at least two ion transitions are typically required for identifying each compound, and there is a significant gap in the different analyte responses in plasma (or serum). Thus, efficient simultaneous measurement of multiple compounds remains a significant challenge. In addition, although a large number of prior art analytical methods have been reported using liquid-liquid extraction (LLE) or solid-phase extraction (SPE) as a sample pretreatment method, these analytical methods are time-consuming and expensive in terms of reagent consumption. Recent reports have developed efficient and environmentally friendly sample preparation methods such as Liquid Phase Micro Extraction (LPME), Solid Phase Micro Extraction (SPME) and serial dispersion-liquid micro extraction (TDLLME) methods that provide better purification capacity but are complex and expensive and not suitable for routine clinical testing. Therefore, the applicant believes that it is necessary to establish a convenient, rapid and comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis method for determining the concentration of an antidepressant in human plasma based on the consensus guideline for neuropsychiatric drug detection for neuropsychological and pharmacogenomics treatment issued by the AGNP.
Disclosure of Invention
The invention provides an HPLC-MS/MS method for simultaneously determining the concentrations of 14 antidepressants in human plasma in order to solve the technical problems.
The application is realized by adopting the following technical scheme.
An HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma, comprising the following steps:
s1, establishing a standard curve
a. Preparation of the solution:
reference working solution: respectively dissolving sertraline hydrochloride, duloxetine hydrochloride, venlafaxine, citalopram, fluvoxamine maleate, paroxetine hydrochloride, fluoxetine hydrochloride, mianserin hydrochloride, clomipramine hydrochloride, amitriptyline hydrochloride, O-desmethylvenlafaxine, nortriptyline, norclomipramine and norfluoxetine standard substances in methanol to prepare a reference substance stock solution; mixing the 14 reference substance stock solutions, diluting with methanol to constant volume to obtain a reference substance mixed stock solution, and diluting the mixed stock solution with methanol to obtain a series of mixed reference substance working solutions;
internal standard working solution: dissolving duloxetine-d 7, venlafaxine-d 6, citalopram-d 6 and sertraline-d 3 in methanol respectively to prepare an internal standard storage solution; mixing the 4 internal standard storage solutions, and performing constant volume by using methanol to prepare a mixed internal standard working solution;
b. determination of control solutions
Adding mixed reference substance working solution and internal standard working solution with various concentrations into blank serum, uniformly mixing, adding acetonitrile, performing vortex oscillation and centrifugation, taking supernatant to a 96-well plate, and analyzing by adopting HPLC-MS/MS; measuring peak areas Ai and internal standard peak areas As of each reference, and drawing a standard curve by taking Ai/As values F As vertical coordinates and corresponding serum concentrations C of the reference As horizontal coordinates;
the chromatographic conditions were as follows:
a chromatographic column: poroshell 120EC-C18 column with specification of 2.1 × 50mm, 2.7 μm; mobile phase: phase A is 0.1% formic acid water solution, phase B is acetonitrile; the gradient elution procedure was as follows: the initial proportion is 30% B, the gradient elution is carried out for 0-4 min from 30% B to 50% B, and the chromatographic column is rebalanced before the next sample injection;
the mass spectrometry conditions were as follows:
an ion source: an electrospray ion source; ion mode: a positive ion mode; and (3) data acquisition mode: monitoring multiple ions; flow rate of drying gas: 11 L.min-1(ii) a Temperature of the drying gas: 350 ℃; atomizing gas pressure: 35 psi; capillary voltage: 4000V; the collection parameters for the 14 antidepressants are shown in Table 1
TABLE 1 analytes, parent ions, daughter ions, residence times, lysis and collision voltages and polarities
S2, treating and detecting plasma samples
Adding an internal standard working solution and acetonitrile into a serum sample, carrying out vortex oscillation and centrifugation, taking supernatant into a 96-well plate, carrying out sample injection analysis according to the method of the step S2b, and obtaining the concentration of 14 antidepressants in the plasma sample according to the standard curve of the step S2 b.
Further, in step S1a, the concentrations of the serial mixed control solutions are: mianserin: 0.04, 0.4, 0.8, 1.6, 2.4, 3.2, 4 μ g/mL; citalopram: 0.06, 0.6, 1.2, 2.4, 3.6, 4.8, 6 μ g/mL; paroxetine/duloxetine: 0.08, 0.8, 1.6, 3.2, 4.8, 6.4, 8; sertraline/amitriptyline/nortriptyline: 0.1, 1, 2, 4, 6, 8, 10 mug/mL; venlafaxine/O-desmethylvenlafaxine/fluvoxamine/clomipramine/desmethylclomipramine: 0.2, 2, 4, 8, 12, 16, 20 mug/mL; fluoxetine/norfluoxetine: 0.4, 4, 8, 16, 24, 32, 40. mu.g/mL.
Further, in step S1b, the flow rate of the column: 0.3 mL/min-1(ii) a Column temperature: 40 ℃; sample introduction amount: 5 μ L.
Further, in steps S1b and S2, vortex shaking is performed for 3-5 minutes, and centrifugation is performed at 12000-13000r/min for 10-15 minutes.
Further, in step S2, the volume ratio of the serum sample to the acetonitrile is 1: 4.
the present application has the following advantageous effects.
In order to meet the requirement of clinical routine TDM, the method establishes an HPLC-MS/MS method for simultaneously measuring 14 clinical common antidepressant drugs, considers the selectivity, precision, accuracy, stability, matrix effect, recovery rate and the like of the method, and all verification indexes meet the requirement of a biological analysis method. The method adopts simple protein precipitation to process the plasma sample, is economic and efficient, and saves labor force; a linear range meeting clinical requirements is established, and the linear range covers most clinical samples; the analysis time of a single sample is shortened to 4min by optimizing the liquid phase condition, and the clinical high-throughput detection requirement can be well met. The establishment of the method of the application provides a large amount of meaningful clinical data for optimizing clinical drug treatment protocols.
Drawings
FIG. 1 is a chromatogram of a standard plasma sample and a blank human plasma sample according to the present invention; wherein, A: total MRM profiles of 14 analytes in blank plasma + LLOQ (top) and 4 internal standards in blank plasma (bottom); b: MRM chromatograms of 14 analytes in blank plasma spiked LLOQ (left) and blank plasma (right), respectively; c: MRM chromatograms of 4 internal standards in blank plasma spiked LLOQ (left) and blank plasma (right), respectively.
Detailed Description
1. Instruments and reagents
1.1 Main Instrument
Agilent 1260 series liquid chromatography, Agilent 6460 triple tandem quadrupole mass spectrometer.
1.2 reagents and materials
Acetonitrile: merck HPLC chromatogram pure; water: drochen distilled water; formic acid: merck HPLC chromatogram pure.
Standard and internal standard: sertraline hydrochloride (batch: 100702-201602), duloxetine hydrochloride (batch: 510144-201701), citalopram (batch: 100885-201802), fluvoxamine maleate (batch: 100792-201902), paroxetine hydrochloride (batch: 100357-201804), fluoxetine hydrochloride (batch: 100513-201602), mianserin hydrochloride (batch: 100812-200501), clomipramine hydrochloride (batch: 100843-20160518 2) and amitriptyline hydrochloride (batch: 10010084303-20160518) were purchased from China biological products assay institute. Venlafaxine (batch No. FS1603121), O-desmethylvenlafaxine (batch No. FS1606252), nortriptyline (batch No. FS1612444) were purchased from tianjina inc. Desclomipramine (batch No.: 24-IG-117-2), norfluoxetine (batch No.: 1-SGP-164-1), duloxetine-d 7 (batch No.: 27-AZC-28-1), venlafaxine-d 6 (batch No.: 5-EQJ-4-3), citalopram-d 6 (batch No.: 16-XDD-61-1) and sertraline-d 3 (batch No.: 8-MJC-182-1) were purchased from TRC, Canada.
2. Method and results
2.1 chromatographic and Mass Spectrometry conditions
2.1.1 chromatographic conditions
A chromatographic column: poroshell 120EC-C18 column (2.1X 50mm, 2.7 μm, Agilent, USA); mobile phase: phase A is 0.1% formic acid solution in water, and phase B is acetonitrile. The gradient elution procedure was as follows: the initial ratio is 30% B, and the gradient elution is carried out for 0-4 min from 30% B to 50% B, and the chromatographic column is re-balanced (2min) before the next sample injection. Flow rate: 0.3 mL/min-1(ii) a Column temperature: 40 ℃; sample introduction amount: 5 μ L.
2.1.2 Mass Spectrometry conditions
An ion source: electrospray ion source (ESI); ion mode: positive ion (positive) mode; and (3) data acquisition mode: multiple ion monitorMeasuring (MRM); flow rate of drying gas: 11 L.min-1(ii) a Temperature of the drying gas: 350 ℃; atomizing gas pressure: 35 psi; capillary voltage: 4000V. The collection parameters for the analytes are shown in table 1.
TABLE 1 analytes, parent ions, daughter ions, residence times, lysis and collision voltages and polarities
2.2 preparation of the solution
2.2.1 control and internal standard stock solutions
Control stock solutions: accurately weighing 10mg of each of sertraline hydrochloride, duloxetine hydrochloride, venlafaxine, citalopram, fluvoxamine maleate, paroxetine hydrochloride, fluoxetine hydrochloride, mianserin hydrochloride, clomipramine hydrochloride, amitriptyline hydrochloride, O-desmethylvenlafaxine, norclomipramine and norfluoxetine standard substances, placing the 10mg standard substances into a 10mL volumetric flask, adding methanol for dissolving and fixing the volume to prepare 1mg/mL standard substance mother liquor, subpackaging 1.5mL glass vials, and storing at-80 ℃.
Internal standard stock solution: duloxetine-d 7, venlafaxine-d 6, citalopram-d 6 and sertraline-d 3 are dissolved in 1mL of methanol respectively to prepare 1mg/mL of internal standard mother liquor, and the internal standard mother liquor is stored at the temperature of 80 ℃.
2.2.2 reference and internal standard working solutions
Reference working solution: precisely measuring 14 reference substance stock solutions in the same volumetric flask, and preparing reference substance mixed stock solutions by methanol constant volume, wherein the concentration of each reference substance is respectively 40 mu g/mL (mianserin), 60 mu g/mL (citalopram), 80 mu g/mL (paroxetine, duloxetine), 100 mu g/mL (sertraline, amitriptyline, nortriptyline), 200 mu g/mL (venlafaxine, O-desmethylvenlafaxine, fluvoxamine, clomipramine, norclomipramine) and 400 mu g/mL (fluoxetine, norfluoxetine). Diluting the mixed stock solution with methanol to obtain a series of mixed reference working solutions, wherein the concentrations of the mixed reference working solutions are respectively as follows: 0.04, 0.4, 0.8, 1.6, 2.4, 3.2, 4 μ g/mL (mianserin); 0.06, 0.6, 1.2, 2.4, 3.6, 4.8, 6 μ g/mL (citalopram); 0.08, 0.8, 1.6, 3.2, 4.8, 6.4, 8 (paroxetine, duloxetine); 0.1, 1, 2, 4, 6, 8, 10 μ g/mL (sertraline, amitriptyline, nortriptyline); 0.2, 2, 4, 8, 12, 16, 20 μ g/mL (venlafaxine, O-desmethylvenlafaxine, fluvoxamine, clomipramine, desmethylclomipramine); 0.4, 4, 8, 16, 24, 32, 40 μ g/mL (fluoxetine, norfluoxetine).
Internal standard working solution: precisely measuring 4 internal standard storage liquids in volumetric flasks, and preparing a mixed internal standard working solution with the methanol to be 1 mu g/mL.
2.2.3 preparation of Standard Curve and quality control sample
Putting 190 mu L of blank serum into a 1.5mL EP tube, adding 10 mu L of mixed reference substance working solution with each concentration, 10 mu L of internal standard working solution, vortexing for 5s, adding 800 mu L of acetonitrile, vortexing and shaking for 3 minutes, centrifuging for 10 minutes at 13000r/min, taking 200 mu L of supernatant into a 96-well plate, and injecting sample according to the specified sample injection amount of the method. Measuring peak area (Ai) and internal standard peak area (As) of each reference substance, drawing a standard curve by taking Ai/As value F As ordinate and corresponding serum concentration C of the reference substance As abscissa, and performing least square linear regression to obtain the standard curve of each medicine.
Precisely measuring a proper amount of reference substance working solution, placing in blank plasma, and making into Quality Control (QC) samples with low, medium and high concentration levels, wherein the three quality control samples are respectively 3 times (QC-L) of LLOQ, linear intermediate concentration (QC-M) and 75% of linear highest concentration (QC-H).
2.2.4 plasma sample treatment
Taking 200 mu L of serum sample, placing the serum sample in a 1.5mL EP tube, adding 10 mu L of internal standard working solution, adding 800 mu L of acetonitrile, carrying out vortex oscillation for 3 minutes, centrifuging for 10 minutes at 13000r/min, taking 200 mu L of supernatant to a 96-well plate, and injecting sample according to the specified sample injection amount of the method.
2.3 method validation
The method was verified according to the guidelines (draft) of the quantitative analysis method for biological samples in the pharmacopoeia 2015 edition and the verification guidelines for the biological analysis method of the U.S. Food and Drug Administration (FDA).
2.3.1 Selectivity
Blank plasma samples from 6 different sources were selected, processed and analyzed according to the protocol "2.2.4" to examine the selectivity of the protocol. For the clinical routine TDM, the effect of specific samples, such as hemolyzed samples, high lipid samples, were also included in the selective assessment.
Typical chromatograms of the standard plasma sample and the blank plasma sample are shown in fig. 1, and the experimental results show that the target compound and the internal standard interference are not detected in the blank plasma, and the peak patterns of the hemolytic sample and the high fat sample are not affected.
2.3.2 Linear Range
6 independent mixed standard curves were prepared for evaluating the linearity of the method using mixed blank plasma as the matrix, and the samples were processed according to the method under "2.2.4". A calibration curve for each analyte was generated by plotting the peak area ratio (analyte/ISTD) against the theoretical concentration, using the weighting factor "1/X2"calculate quadratic regression equation.
Internal standard, linear range, linear equation, correlation coefficient (R) of 14 antidepressants2) And retention times are shown in table 2. As can be seen from Table 2, the correlation coefficient R of all standard curves prepared in the blank plasma2And the standard curve range of all analytes covers the treatment reference range, so that the requirement of routine clinical TDM can be met.
TABLE 214 Linear Range, Linear equation and Retention time for antidepressants
2.3.3 precision and accuracy
Mixing blank plasma as a substrate, preparing mixed standard low, medium and high concentration quality control samples (QC-L, QC-M and QC-H) and LLOQ, respectively 6 parts, processing the samples according to the method under the item of '2.2.4' in the same day and different days, and calculating the concentration by using a following standard curve to evaluate the precision and accuracy in day and day.
The precision and accuracy data are shown in table 3, and the experimental results show that the precision of LLOQ in day and day is less than 20% for all compounds, the other QC is less than 15%, and the accuracy is between 85.51% and 114.77%. The method meets the guideline requirements, and shows that the method is accurate and precise.
TABLE 3 precision and accuracy
2.3.4 matrix Effect and recovery
Matrix Effects (ME) and Extraction Recovery (ER) were evaluated at three levels (QC-L, QC-M and QC-H) in blank plasma samples. Three sets of samples were prepared, 6 samples for each set, as follows:
1) adding standard analyte into blank plasma sample, and processing the sample into group A according to the method under item "2.2.4";
2) after processing blank plasma according to the method under the item of '2.2.4', adding quality control substances to obtain group B;
3) preparing quality control products with the same concentration by using the initial proportion of the mobile phase, and directly injecting the sample to form a group C.
The calculation formulas of the matrix effect and the recovery rate are respectively as follows: ME ═ B/C100%, ER ═ a/B100%, where the matrix effect was calculated by internal standard normalization.
The results of the matrix effect and recovery are shown in table 4. The experimental result shows that the 14 antidepressants measured by the protein precipitation method have relatively high and stable recovery rate, and the recovery rate range is 79.02-107.12%. The recovery of all analytes and the Coefficient of Variation (CV) for the normalized matrix effect were < 15%, meeting the guidelines.
2.3.5 stability Studies
The stability of the low, medium and high concentration quality control products under four conditions is respectively inspected by combining the practical condition of sample treatment in the laboratory: the stability after preparation is that the tea is stored for 24 hours at room temperature (20-25 ℃), the long-term stability of being stored for 30 days at 20 ℃, the freeze-thaw stability of being repeatedly frozen and thawed for three times at 80 ℃, and the stability after preparation of being stored for 24 hours at room temperature after extraction.
The stability results are shown in table 4, and the experimental results show that under four conditions, the stability is in the range of 86.01% -113.47%, and the RSD of all the examined batches is not higher than 12.34%, which meets the requirements of the guidelines.
TABLE 4 matrix Effect, recovery and stability
2.3.6 dilution effect and residual effect
Residual effects were examined by immediately sampling blank samples after the upper limit of quantitation (ULOQ) samples, and the dilution integrity was assessed by 10-fold dilution of high concentration samples (approximately 5-fold of ULOQ).
The precision (CV%) of the dilution integrity is within 7.52 percent, the accuracy is 95.43-108.67 percent, and the requirements of the guideline are met. In addition, no significant residual effects were observed in the present process.
2.4 clinical applications
After verification, the method is successfully used for monitoring clinical conventional therapeutic drugs, 674 samples are measured, and the sample amount and the actually measured blood concentration range of each monitored drug are shown in table 5. The long-term clinical practice shows that the method is stable, economical and efficient. The reference range of treatment and the concentration of vigilance for clinical use were established according to the third edition of the AGNP guidelines. Escitalopram is a dextrorotatory isomer of citalopram and cannot be distinguished in the method, so the escitalopram and citalopram are subjected to statistical analysis together. The antidepressant drug samples tested in this application are mostly from outpatient patients, and the first three drugs tested in the samples were sertraline, (escitalopram) and duloxetine, respectively. According to the requirements of the AGNP guidelines, the effective plasma concentrations of venlafaxine, fluoxetine, clomipramine, and amitriptyline are the sum of the parent drug and its active metabolites. To date, all test samples were in the linear range and no dilution of the treated samples was found.
TABLE 5 plasma samples of patients in the plasma range of 14 antidepressants
The statistical result is the sum of samples of citalopram (7 cases) and escitalopram (134 cases)
Effective blood concentration of # venlafaxine, fluoxetine, clomipramine and amitriptyline is the sum of the parent drug and the active metabolite thereof
3. Conclusion
The method uses simple protein precipitation pretreatment to establish a comprehensive, stable and efficient HPLC-MS/MS method for simultaneously measuring 14 antidepressants, and is used for routine clinical TDM monitoring. The varieties monitored by the method comprise AGNP guide recommended application TDM grade: the method is characterized in that 5 types of medicines (amitriptyline, nortriptyline, clomipramine, norclomipramine and citalopram) are recommended in the I grade (strong recommendation), 5 types of medicines (duloxetine, escitalopram, fluvoxamine, sertraline and venlafaxine) are recommended in the II grade (strong recommendation), 5 types of medicines (mianserin, paroxetine, norvenlafaxine, fluoxetine and norfluoxetine) are recommended in the III grade (useful), and most antidepressants on the market in China are basically contained in selected medicines.
Tricyclic antidepressants such as amitriptyline, nortriptyline, clomipramine and norclomipramine, although less commonly used in clinical applications (even without clinical samples in past clinical applications), have a toxic concentration very close to the therapeutic concentration range of such drugs, which means that south strongly recommends TDM drugs, which are retained in the assay for toxic sample screening.
Compared with SPE, LLE or other pretreatment methods, the method selects a simple, economic and rapid protein precipitation method in the aspect of selecting the sample pretreatment method. Protein precipitation can remove proteins and other potential interferents, is convenient and quick, and is particularly suitable for high-throughput biological analysis such as conventional TDM. In addition to this, the protein precipitation method minimizes the ion suppression or ion enhancement of the matrix and protects the column.
The method of the application fully combines the recommended range of the guidelines and the requirements of clinical samples on the setting of the linear range of each drug, and the linear range comprises the effective blood concentration range and the laboratory warning concentration of all drugs. Considering that toxic screening and the like may occur in clinical application to cause the plasma drug concentration of a sample to be super-linear, the dilution effect is considered in the methodological verification. In clinical applications to date, no super-linear samples were found.
In the aspect of stability investigation, the stability is investigated by combining the practical situation of the laboratory, and the four stability conditions are respectively as follows: storing at room temperature for 24h, extracting the sample, placing in a sample tray for 24h, repeatedly freezing and thawing at-80 ℃ for three times, and storing at-20 ℃ for 30 days, which is suitable for routine TDM operation flow in laboratory.
To ensure the accuracy of the analysis method, in addition to the laboratory routine configuration of the satellite quality control samples, the laboratory was also conducted with multiple units and institutions for laboratory quality assessment while participating in and passing the LGC therapeutic drug monitoring capacity test (LGC is the institute for chemical and biological assay specified in the uk nations).
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (5)
1. An HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma, which is characterized in that: the method comprises the following steps:
s1, establishing a standard curve
a. Preparation of the solution:
reference working solution: respectively dissolving sertraline hydrochloride, duloxetine hydrochloride, venlafaxine, citalopram, fluvoxamine maleate, paroxetine hydrochloride, fluoxetine hydrochloride, mianserin hydrochloride, clomipramine hydrochloride, amitriptyline hydrochloride, O-desmethylvenlafaxine, nortriptyline, norclomipramine and norfluoxetine standard substances in methanol to prepare a reference substance stock solution; mixing the 14 reference substance stock solutions, diluting with methanol to constant volume to obtain a reference substance mixed stock solution, and diluting the mixed stock solution with methanol to obtain a series of mixed reference substance working solutions;
internal standard working solution: dissolving duloxetine-d 7, venlafaxine-d 6, citalopram-d 6 and sertraline-d 3 in methanol respectively to prepare an internal standard storage solution; mixing the 4 internal standard storage solutions, and performing constant volume by using methanol to prepare a mixed internal standard working solution;
b. determination of control solutions
Adding mixed reference substance working solution and internal standard working solution with various concentrations into blank serum, uniformly mixing, adding acetonitrile, performing vortex oscillation and centrifugation, taking supernatant to a 96-well plate, and analyzing by adopting HPLC-MS/MS; measuring peak areas Ai and internal standard peak areas As of each reference, and drawing a standard curve by taking Ai/As values F As vertical coordinates and corresponding serum concentrations C of the reference As horizontal coordinates;
the chromatographic conditions were as follows:
a chromatographic column: poroshell 120EC-C18 column with specification of 2.1 × 50mm, 2.7 μm; mobile phase: phase A is 0.1% formic acid water solution, phase B is acetonitrile; the gradient elution procedure was as follows: the initial proportion is 30% B, the gradient elution is carried out for 0-4 min from 30% B to 50% B, and the chromatographic column is rebalanced before the next sample injection;
the mass spectrometry conditions were as follows:
an ion source: an electrospray ion source; ion mode: a positive ion mode; and (3) data acquisition mode: monitoring multiple ions; flow rate of drying gas: 11 L.min-1(ii) a Temperature of the drying gas: 350 ℃; atomizing gas pressure: 35 psi; capillary voltage: 4000V; the collection parameters for the 14 antidepressants are shown in Table 1
TABLE 1 analytes, parent ions, daughter ions, residence times, lysis and collision voltages and polarities
S2, treating and detecting plasma samples
Adding an internal standard working solution and acetonitrile into a serum sample, carrying out vortex oscillation and centrifugation, taking supernatant into a 96-well plate, carrying out sample injection analysis according to the method of the step S2b, and obtaining the concentration of 14 antidepressants in the plasma sample according to the standard curve of the step S2 b.
2. The HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma according to claim 1, wherein: in step S1a, the concentrations of the serial mixed reference working solutions are: mianserin: 0.04, 0.4, 0.8, 1.6, 2.4, 3.2, 4 μ g/mL; citalopram: 0.06, 0.6, 1.2, 2.4, 3.6, 4.8, 6 μ g/mL; paroxetine/duloxetine: 0.08, 0.8, 1.6, 3.2, 4.8, 6.4, 8; sertraline/amitriptyline/nortriptyline: 0.1, 1, 2, 4, 6, 8, 10 mug/mL; venlafaxine/O-desmethylvenlafaxine/fluvoxamine/clomipramine/desmethylclomipramine: 0.2, 2, 4, 8, 12, 16, 20 mug/mL; fluoxetine/norfluoxetine: 0.4, 4, 8, 16, 24, 32, 40. mu.g/mL.
3. Root of herbaceous plantThe HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma according to claim 1, wherein: in step S1b, flow rate of the column: 0.3 mL/min-1(ii) a Column temperature: 40 ℃; sample introduction amount: 5 μ L.
4. The HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma according to claim 1, wherein: in steps S1b and S2, vortex shaking is carried out for 3-5 minutes, and centrifugation is carried out for 10-15 minutes at 12000-13000 r/min.
5. The HPLC-MS/MS method for simultaneously determining the concentration of 14 antidepressants in human plasma according to claim 1, wherein: in step S2, the volume ratio of the serum sample to acetonitrile is 1: 4.
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Application publication date: 20211221 |