CN113325103A - Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair - Google Patents
Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair Download PDFInfo
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- VTLYEMHGPMGUOT-UHFFFAOYSA-N Koumine Natural products C12=NC3=CC=CC=C3C31CC1C4COC2CC4C3(C=C)CN1C VTLYEMHGPMGUOT-UHFFFAOYSA-N 0.000 description 3
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- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
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- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
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Images
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
-
- 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
-
- 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
Abstract
The invention provides an analysis method for simultaneously measuring gelsemine, gelsemine and gelsemine in hair, which comprises the following steps: preparing a standard product stock solution, a standard product working solution and an internal standard solution in sequence; step two, sample treatment; and step three, LC-MS/MS analysis. The invention firstly carries out the development and verification of the analysis method of gelsemine, gelsemine and gelsemine in hair. The method is simple, convenient and quick, the hair is extracted in one step, the hair consumption is less and only 10mg is needed, the target object in the hair is extracted by wet grinding the extraction solution at low temperature, the grinding time is 10min, the chromatographic running time is short (8min), and the sensitivity meets the daily inspection case requirement.
Description
Technical Field
The invention relates to the technical field of gelsemium alkaloid detection, in particular to an analysis method for simultaneously determining gelsemium, gelseminum and gelsemine in hair.
Background
Gelsemium elegans, which is commonly known as a lot in folk, is a whole plant of wild evergreen winding vine (Gelsemium elegans Benth) of the genus calamus of the family premaceae, and is mainly distributed in Guizhou, Guangdong, Fujian, Yunnan, Guangxi and other provinces in southeast Asia and south China. The whole herb is extremely toxic, and the toxicity of the herb is mainly from indole or oxindole alkaloids. The toxic components mainly comprise Gelsemine (Koumine), Gelsemine (Gelsemine), gelsoline (Gelsenicine) and the like, wherein the Gelsemine is the most abundant gelsoline alkaloid, the gelsoline is the gelsoline, and the gelsoline is the gelsoline with the highest toxicity. The gelsemium and other benign herbs are often confused due to their morphologies, which results in mistaking and poisoning, so the detection of gelsemine, koumine and gelsemine in biological samples such as blood, urine and hair is the key to the determination of gelsemium poisoning or the identification of related case events.
The concentrations of gelsemine, gelsemine and gelsemine were rarely reported in blood samples, but hardly in other biological samples such as urine or hair. In some cases of poisoning, many poisoners die after many days of rescue and fail to take blood, urine, etc. in time, at which time hair analysis can provide valuable evidence. Hair analysis has been widely used in forensic and clinical toxicology. Compared with the traditional biological detection materials (such as blood and urine), the hair has the advantages of long detection time, capability of reflecting the drug use condition for a long time (several months), and the like, and the sample is easy to obtain and store, so that no research report on analyzing and determining the gelsemium alkaloid in the hair is known.
Disclosure of Invention
The invention aims to provide an analysis method for simultaneously measuring gelsemine, gelsemine and gelsemine in hair aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the analysis method for simultaneously measuring gelsemine, gelsemine and gelsemine in hair is characterized by comprising the following steps of:
preparing a standard product stock solution, a standard product working solution and an internal standard solution in sequence;
cleaning the blank hair sample and the hair sample to be detected with ultrapure water and acetone respectively, collecting the last acetone cleaning solution for blow-drying, re-dissolving with methanol respectively after blow-drying for removing external pollution factors, and drying the hair sample at room temperature; cutting the dried hair sample to be detected into small sections, precisely weighing, placing in a grinding tube, adding grinding beads, the internal standard solution and methanol, and grinding by using a freeze grinder; then centrifuging, and filtering the obtained supernatant through a 0.22 mu m microporous filter membrane to obtain a sample solution to be detected;
cutting the dried blank hair sample into small sections, precisely weighing, respectively placing into a plurality of grinding tubes, respectively adding a proper amount of the standard substance working solution to obtain a series of blank hair samples with different mass concentrations, adding grinding beads, and grinding by using a freezing grinder; then centrifuging, and filtering the obtained supernatant through a 0.22 mu m microporous filter membrane to obtain a series of blank hair sample solutions;
step three, respectively injecting sample solution to be detected and a series of blank hair sample solutions, performing LC-MS/MS analysis,
wherein, the chromatographic conditions are as follows: restekalure PFP propyl column, 100mm × 2.1mm, 5 μm; the mobile phase A is 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, and the mobile phase B is methanol; the flow rate is 0.3-0.5 mL/min, and the ratio of mobile phase A: b is 30-40: eluting at constant current of 60-70 ℃ for 8-12 min, wherein the column temperature is room temperature; the autosampler was maintained at 4 ℃;
the mass spectrum conditions are as follows: detecting positive ion mode of electrospray ionization mass spectrum, monitoring multiple ion modes, and collecting and analyzing data by adopting an analysis software 1.5(Waters) and a MultiQuant 3.0.2 workstation; the ion spray voltage of the mass spectrometer is 5500V, the ion source temperature is 550 ℃, the air curtain gas is 30psi, and the atomization gas (GS1) and the auxiliary gas (GS2) are 50 psi.
Further, the preparation process of the standard stock solution comprises the following steps: precisely weighing gelsemine, gelsemine and gelsemine standard substances, respectively dissolving with methanol to obtain standard stock solution with mass concentration of 1.0mg/mL, sealing, and storing in refrigerator at-20 deg.C; the preparation process of the standard working solution comprises the following steps: and taking a proper amount of the standard substance stock solution, and gradually diluting with methanol to obtain standard substance working solutions with the concentrations of 10000, 5000, 1000, 100 and 10 ng/mL.
Further, the preparation process of the internal standard solution is as follows: precisely sucking 10 mu L of ketamine-d 4 standard solution, and adding 990 mu L of methanol to dilute the solution to prepare an internal standard substance stock solution; taking a proper amount of the internal standard substance stock solution, and diluting with methanol to obtain an internal standard solution with the concentration of 10 ng/mL.
Further, the hair sample after drying was cut into 2 to 3mm pieces, 10mg of the hair sample after cutting was precisely weighed and placed in a 2mL grinding tube, and an appropriate amount of grinding beads, 50. mu.L of the internal standard solution and 450. mu.L of methanol were added and ground using a cryo-grinder.
Further, the parameters of the cryo-mill were set as follows: speed, 2500 rpm; running times, 15 times; the interval time was 60s and the polishing was carried out at a rate of 18 m/s.
Further, the conditions of the centrifugation are: centrifuge at 9700 Xg for 3 min.
Further, the chromatographic conditions are: restekalure PFP propyl column, 100mm × 2.1mm, 5 μm; the mobile phase A is 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, and the mobile phase B is methanol; flow rate 0.3mL/min, mobile phase a: b is 30: eluting at constant flow of 70 deg.C for 8min, and keeping the column temperature at room temperature; the autosampler was maintained at 4 ℃.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention firstly carries out the development and verification of the analysis method of gelsemine, gelsemine and gelsemine in hair. The method is simple, convenient and quick, the hair is extracted in one step, the hair consumption is less and only 10mg is needed, the target object in the hair is extracted by wet grinding the extraction solution at low temperature, the grinding time is 10min, the chromatographic running time is short (8min), and the sensitivity meets the daily inspection case requirement.
Drawings
FIG. 1 is a chromatogram of gelsemium elegans at a LOQ mass concentration in hair;
FIG. 2 is a chromatogram of gelsemine at a LOQ mass concentration in hair;
FIG. 3 is a chromatogram of loprazine at a LOQ mass concentration in hair;
fig. 4 is a chromatogram of blank hair.
Detailed Description
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The reagents and instruments adopted by the invention are as follows:
(1) standards, reagents and samples
Gelsemium (purity 99.56%), gelsemine (purity 98.36%), and gelsemine (purity 99.53%) standard control powders were purchased from Dormant Biotech, Inc. Internal standard ketamine-d 4 standard solution (1.0mg/mL) was purchased from Cerilliant, usa.
Methanol and acetonitrile (HPLC) were purchased from Sigma-Aldrich (st. louis, MO, USA), acetone (analytical grade) was purchased from shanghai linkun chemical reagents ltd (99.5%, shanghai, china); ammonium formate (HPLC,. gtoreq.97.0%), ammonium acetate (HPLC,. gtoreq.98.0%), formic acid (HPLC,. gtoreq.98.0%) were purchased from CNW Technologies (Duesseldorf, Germany). Deionized water was prepared from a Milli-Q (Millipore, MA, USA) water purification system. PTFE filters (0.22 μm) were purchased from Chemicals, Inc., national drug group. Blank hair was provided by healthy volunteers.
(2) Conditions of the apparatus
The vortex mixer (XW-80A) was purchased from Shanghai Huxi analytical instruments, Inc., the cryo-milling apparatus was purchased from Shanghai Jingxin Industrial development, Inc., the MiniSpin high-speed centrifuge was purchased from Eppendorf, Germany, the Milli-Q ultrapure water preparation system was purchased from Millipore, USA, and the BSA124S electronic balance was purchased from Sadoris scientific instruments, Beijing, Inc.
API 4000QTRAP triple quadrupole linear ion trap mass spectrometer (Applied Biosystems, USA). AcquisytTM Ultra Performance LC Ultra high pressure liquid chromatograph (Waters corporation, USA). Chromatographic conditions are as follows: the liquid chromatography column was a Restek Allure PFP propyl column (100 mm. times.2.1 mm, 5 μm) preceded by an Agilent guard column Zorbax C8 (12.5. times.2.1 mm, 5 μm). Data analysis was performed using Analyst 1.5 software and multisquant 3.0.2 workstation.
Example 1
This example provides an analytical method for the simultaneous determination of gelsemine, gelsemine and gelsemine in hair, and a methodological validation.
1.1 solution preparation
Stock standard (1.0 mg/mL): precisely weighing 5.0mg of each of gelsemine, gelsemine and gelsemine standard substances, respectively placing in 5mL volumetric flasks, adding methanol to dissolve and fix volume to scale, shaking up to obtain standard substance stock solution with mass concentration of 1.0mg/mL, sealing, and storing in a refrigerator at-20 deg.C for use.
Standard working solution: taking a proper amount of standard substance stock solution, and gradually diluting with methanol to obtain working solutions with concentrations of 10000, 5000, 1000, 100 and 10 ng/mL.
Mobile phase a [20mmol/L ammonium acetate buffer solution (containing 0.1% formic acid) ]: weighing 0.77g of ammonium acetate, placing the ammonium acetate in a 500mL volumetric flask, adding ultrapure water for dissolving, then adding 0.5mL of 98% formic acid solution, finally using ultrapure water for constant volume to 500mL, and fully and uniformly mixing.
Internal standard solution (10 ng/mL): a stock solution (10. mu.g/mL) of the internal standard substance was prepared by precisely pipetting 10. mu.L of ketamine-d 4 standard solution (100. mu.g/mL) and diluting with 990. mu.L of methanol. Taking a proper amount of internal standard substance stock solution, and diluting with methanol to obtain an internal standard solution with the concentration of 10 ng/mL.
1.2 sample pretreatment
Respectively placing a blank hair sample and a hair sample to be detected in test tubes with plugs, respectively and sequentially cleaning with ultrapure water for 3 times and acetone for 3 times, collecting acetone cleaning liquid (used for blow-drying redissolution to see whether external pollution exists), redissolving with methanol after blow-drying to remove external pollution factors, and drying at room temperature.
The dried hair sample to be tested is cut into 2-3mm small sections, 10mg is precisely weighed and placed in a 2mL grinding tube, and an appropriate amount of grinding beads, 50 muL internal standard solution (10ng/mL) and 450 muL methanol are added. The hair was ground using a cryo-grinder (parameters set as follows: speed, 2500 rpm; number of runs, 15 times; interval time 60s and grinding at a speed of 18 m/s). Then centrifuging at 9700 Xg for 3min, filtering the supernatant with 0.22 μm microporous membrane to obtain sample solution to be tested, and analyzing with 10 μ L sample injection.
Precisely weighing the dried blank hair sample, cutting into 2-3mm segments, precisely weighing, respectively placing into a plurality of grinding tubes, respectively adding appropriate amount of standard working solution to obtain series blank hair samples with different mass concentrations, adding grinding beads, and grinding with a refrigerated grinding apparatus (parameters are set as follows: speed, 2500 rpm; operation times, 15 times; interval time is 60s, and grinding at 18 m/s). Then centrifuging at 9700 Xg for 3min, filtering the obtained supernatant with 0.22 μm microporous membrane to obtain a series of blank hair sample solutions, and analyzing with 10 μ L sample injection respectively.
1.3 apparatus conditions
A chromatographic column: the column was a restallure PFP propyl column (100mm × 2.1mm, 5 μm); mobile phase A: 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: methanol; flow rate: 0.3mL/min, A: b is 30: eluting at constant flow of 70 deg.C for 8min, and keeping the column temperature at room temperature; the autosampler was maintained at 4 ℃.
The detection system is positive ion mode detection by electrospray ionization mass spectrometry (Applied Biosystems/MDS SCIEX, Toronto, Canada), multi-ion mode monitoring, data collection and analysis using an analysis software 1.5(Waters) and MultiQuant 3.0.2 workstation. The ion spray voltage of the mass spectrometer is 5500V, the ion source temperature is 550 ℃, the air curtain gas is 30psi, and the atomization gas (GS1) and the auxiliary gas (GS2) are 50 psi. After direct sample injection, the parent ions and the daughter ions, and the Declustering Potential (DP) and the Collision Energy (CE) are screened to obtain the maximum ion strength, and the Collision dissociation energy is kept stable. Fragment ions, declustering voltage and collision energy of the target compound and the internal standard compound are sequentially established by respectively and directly injecting the target analytes by mass spectrometry (table 1), so that the fragment ions have maximum response values, and each compound is calculated by two fragment ions.
Table 1 mass spectral parameters, retention times and ion ratios of the compounds
Quantitation of ion pairs.
1.4 methodological validation
The methodology was validated according to The Society of hair analysis (The Society of hair Testing, SoHT) guidelines and international guidelines. The evaluation parameters mainly include selectivity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, linearity, recovery rate, matrix effect, and the like.
1.4.1 Selectivity
Blank hair samples from 8 healthy volunteers were collected for selective testing to ensure that there was no interference from other substances during the peak time of the target component. In addition, possible interferences caused by the same drugs were investigated.
As a result, as shown in fig. 1 to 4, when the chromatograms of the respective compounds in the blank hair samples were compared, it was confirmed that the endogenous substances were not interfering with the target substance and the internal standard. The peak-out time of each compound is 2.69-4.50 min.
1.4.2 LOD and LLOQ
And taking the mass concentration when the signal-to-noise ratio S/N is larger than or equal to 3 as a detection limit, taking the mass concentration when the signal-to-noise ratio S/N is larger than or equal to 10 as a quantification limit, and taking the quantification limit as the minimum concentration of a linear range to carry out experimental investigation on the precision and accuracy of the hair sample at the LOQ position.
And (3) respectively inspecting a series of blank hair sample solutions, selecting a solution which meets the signal to noise ratio S/N of more than or equal to 3 as a detection limit, selecting a solution which meets the signal to noise ratio S/N of more than or equal to 10 as a quantification limit, and controlling the accuracy and precision of the hair sample at the LOQ position within +/-20%. The results of the experiments are shown in tables 2 and 3 below. Experimental results show that LOD of gelsemium, gelsemine and gelsemine is 0.005ng/mg, 0.002ng/mg and 0.001ng/mg respectively; LOQ of gelsemine, gelsemine and gelsemine is 0.01ng/mg, 0.005ng/mg and 0.002ng/mg respectively.
TABLE 2 regression equation, Linear Range, correlation coefficient, LOD and LOQ for each target in hair
TABLE 3 precision and accuracy of targets in hair
1.4.3 Linearity
Taking the mass concentration of the target in the hair as an abscissa and the peak area ratio of the target to an internal standard as an ordinate, performing regression calculation by using a weighted (W is 1/x) least square method to obtain a linear equation, and calculating a correlation coefficient (R)2). The linear equation for each compound was calculated from 7 points with the lowest concentration being LOQ (S/N.gtoreq.10) and ensuring that the concentration of the actual sample falls within the linear range.
Linear data and a correlation coefficient R are obtained by examining 9 blank hair sample solutions with mass concentrations of 0.002, 0.005, 0.01, 0.02, 0.03, 0.5, 1, 5 and 10ng/mg, and performing regression operation on the peak area ratio and the concentration of the target compound and the internal standard according to a weighted (1/x) least square method2. The results show that the individual compounds in the hair samples are well linear in the corresponding concentration ranges, R2All are greater than 0.99, the linear data meet the requirements, and the results are shown in Table 2.
1.4.4 precision and accuracy
And (3) carrying out precision and accuracy examination on the LOQ, low, medium and high concentrations, adding a proper amount of mixed reference substance solution into blank hair to obtain quality control samples, and continuously measuring for 4 days by 6 samples of each concentration. Sample pretreatment is carried out according to the method under the item '1.2', sample injection analysis is carried out according to the method under the item '1.3', and the precision and accuracy in the day are calculated. Accuracy is expressed in bias by calculating the concentration linearly and comparing the percentage between the linearly calculated value and the added concentration. Precision is expressed as Relative Standard Deviation (RSD). The measurement is carried out continuously for 4 days and is simultaneously carried out with the standard curve, the concentration of the quality control sample is calculated according to the standard curve of the day, and the precision during the day is calculated. Precision the variation of the experiment repeated on the same day and on different days was examined.
The precision and accuracy of the LOQ, low, medium and high concentrations were measured for four consecutive days with six samples per concentration. The results show that the precision of each compound in the day is 1.8-10.8%, and the precision in the day is 1.9-9.6%. The accuracy ranges within and during the day are-3.6-8.8% and-2.9% -4.5%, respectively. The data prove that the method has good accuracy and precision on the target compound and meets the requirements. The precision and accuracy results are shown in table 3.
1.4.5 matrix Effect and recovery
The extraction recovery and matrix effect were calculated according to the method proposed by Matuszewski et al, which divided the samples into three groups. Group I: adding mixed reference solution with certain concentration into 8 parts of hair from different sources before extraction according to the method under the item 1.2; and (II) group: extracting 8 parts of hair from different sources by the method under the item 1.3, and adding a mixed reference substance solution with corresponding concentration; group III: preparing a mixed reference substance solution with corresponding concentration. Blank hairs from different sources are added into a proper amount of the mixed reference substance solution to obtain quality control samples with mass concentrations of 0.03ng/mg and 10ng/mg (low concentration), and each concentration is 8 samples. The sample was analyzed by the method under "1.3" and the peak area (A) was recorded. The extraction recovery rate is AI/AII, and the matrix effect is AII/AIII.
The results show that the extraction recovery rate of each target substance ranges from 79% to 83%, the matrix effect ranges from 74% to 87%, the Relative Standard Deviation (RSD) of the matrix effect of the hairs from different sources is less than or equal to 14%, the matrix effect of the internal standard (0.01ng/mg) is 80%, the extraction recovery rate is 77%, and the effects of the extraction recovery rate and the matrix effect are compensated. The matrix effect and extraction recovery results are listed in table 4.
TABLE 4 extraction recovery and matrix Effect of each target in Hair
Application example
After methodological verification, the method of the invention is applied to the actual case of death due to mistaking gelsemium poisoning. The length of hair of a victim is about 10cm, and the hair sample to be tested is divided into 10 fragments of S1(0-1cm), S2(1-2cm), S3(2-3cm), S4(3-4cm), S5(4-5cm), S6(5-6cm), S7(6-7cm), S8(7-8cm), S9(8-9cm) and S9(9-10cm) for analysis and measurement. The results showed that koumine, gelsemine and gelsemine were detected in all hair sections S6(5-6cm), S7(6-7cm), S8(7-8cm), with the highest concentrations being S7(6-7cm) and the gelsemine, gelsemine and gelsemine concentrations being 29.4, 17.6 and 4.7pg/mg, respectively. The results showed that the victims took gelsemium several months ago.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (7)
1. An analysis method for simultaneously measuring gelsemine, gelsemine and gelsemine in hair is characterized by comprising the following steps:
preparing a standard product stock solution, a standard product working solution and an internal standard solution in sequence;
cleaning the blank hair sample and the hair sample to be detected with ultrapure water and acetone respectively, collecting the last acetone cleaning solution, drying, and redissolving with methanol to remove external pollution factors; the hair is dried at room temperature; cutting the dried hair sample to be detected into small sections, precisely weighing, placing in a grinding tube, adding grinding beads, the internal standard solution and methanol, and grinding by using a freeze grinder; centrifuging, and filtering the obtained supernatant through a microporous filter membrane to obtain a sample solution to be detected;
cutting the dried blank hair sample into small sections, precisely weighing, respectively placing into a plurality of grinding tubes, respectively adding a proper amount of the standard substance working solution to obtain a series of blank hair samples with different mass concentrations, adding grinding beads, and grinding by using a freezing grinder; centrifuging, and filtering the obtained supernatant with microporous membrane to obtain a series of blank hair sample solutions;
step three, respectively injecting sample solution to be detected and a series of blank hair sample solutions, performing LC-MS/MS analysis,
wherein, the chromatographic conditions are as follows: restek Allure PFP propyl column, 100mm × 2.1mm, 5 μm; the mobile phase A is 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, and the mobile phase B is methanol; the flow rate is 0.3-0.5 mL/min, and the ratio of mobile phase A: b is 30-40: eluting at constant current of 60-70 ℃ for 8-12 min, wherein the column temperature is room temperature; the autosampler was maintained at 4 ℃;
the mass spectrum conditions are as follows: detecting positive ion mode of electrospray ionization mass spectrum, monitoring multiple ion modes, and collecting and analyzing data by adopting an analysis software 1.5(Waters) and a MultiQuant 3.0.2 workstation; the ion spray voltage of the mass spectrometer is 5500V, the ion source temperature is 550 ℃, the air curtain gas is 30psi, and the atomization gas (GS1) and the auxiliary gas (GS2) are 50 psi.
2. The method of claim 1, wherein the standard stock solution is prepared by: precisely weighing gelsemine, gelsemine and gelsemine standard substances, respectively dissolving with methanol to obtain standard stock solution with mass concentration of 1.0mg/mL, sealing, and storing in refrigerator at-20 deg.C; the preparation process of the standard working solution comprises the following steps: and taking a proper amount of the standard substance stock solution, and gradually diluting with methanol to obtain standard substance working solutions with the concentrations of 10000, 5000, 1000, 100 and 10 ng/mL.
3. The method according to claim 1, wherein the internal standard solution is prepared by the following process: precisely sucking 10 mu L of ketamine-d 4 standard solution, and adding 990 mu L of methanol to dilute the solution to prepare an internal standard substance stock solution; taking a proper amount of the internal standard substance stock solution, and diluting with methanol to obtain an internal standard solution with the concentration of 10 ng/mL.
4. The method according to claim 1, wherein the dried hair sample is cut into 2-3mm pieces, 10mg of the cut hair sample is precisely weighed and placed in a 2mL grinding tube, and a suitable amount of grinding beads, 50 μ L of the internal standard solution and 450 μ L of methanol are added and ground using a cryo-grinder.
5. The method of claim 1, wherein the cryo-mill parameters are set as follows: speed, 2500 rpm; running times, 15 times; the interval time was 60s and the polishing was carried out at a rate of 18 m/s.
6. The method of claim 1, wherein the conditions of the centrifugation are: centrifuge at 9700 Xg for 3 min.
7. The method of claim 1, wherein the chromatographic conditions are: restek Allure PFP propyl column, 100mm × 2.1mm, 5 μm; the mobile phase A is 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, and the mobile phase B is methanol; flow rate 0.3mL/min, mobile phase a: b is 30: eluting at constant flow of 70 deg.C for 8min, and keeping the column temperature at room temperature; the autosampler was maintained at 4 ℃.
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