CN115452995A - Method for testing drugs in single hair with sub-millimeter length - Google Patents
Method for testing drugs in single hair with sub-millimeter length Download PDFInfo
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- CN115452995A CN115452995A CN202211166882.XA CN202211166882A CN115452995A CN 115452995 A CN115452995 A CN 115452995A CN 202211166882 A CN202211166882 A CN 202211166882A CN 115452995 A CN115452995 A CN 115452995A
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000003814 drug Substances 0.000 title claims abstract description 22
- 229940079593 drug Drugs 0.000 title claims abstract description 18
- 238000012360 testing method Methods 0.000 title claims abstract description 7
- 239000000523 sample Substances 0.000 claims abstract description 19
- 238000000605 extraction Methods 0.000 claims abstract description 16
- 239000002390 adhesive tape Substances 0.000 claims abstract description 12
- 238000011534 incubation Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 5
- 238000011084 recovery Methods 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 239000012488 sample solution Substances 0.000 claims abstract description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 14
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical group SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000010998 test method Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 4
- 230000004888 barrier function Effects 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 2
- 231100000640 hair analysis Toxicity 0.000 description 5
- 239000012491 analyte Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000011218 segmentation Effects 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000002552 multiple reaction monitoring Methods 0.000 description 1
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 229940001470 psychoactive drug Drugs 0.000 description 1
- 239000004089 psychotropic agent Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
-
- 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
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for testing drugs in single hair with sub-millimeter length, which comprises the following steps: step one, sticking single hair on a customized adhesive tape along scales, and cutting the single hair under a table magnifier; step two, extracting the sample divided in the step one by adopting an extraction solution, wherein in order to ensure the extraction recovery rate, the extraction process comprises ultrasonic treatment and incubation, and preparing a sample solution from the extracted supernatant after extraction; and step three, performing liquid chromatography-mass spectrometry on the sample liquid obtained in the step two by using a liquid chromatograph-mass spectrometer. The invention realizes the preparation of the sub-millimeter sample of single hair, and the sample deviation is within the acceptable range. The method of the invention not only can effectively destroy the hair cuticle barrier to extract the target drugs in the hair, but also can dissolve out a large number of target detection substances with different structures in the maximum range, thereby realizing the rapid screening of various toxic (drugs) in the sample when the detection target is uncertain and the detection range is wide.
Description
Technical Field
The invention relates to the technical field of trace drug detection, in particular to a method for detecting drugs in single hair with a sub-millimeter length.
Background
Since the end of the 20 th century 90 s, hair analysis has increasingly been applied in the areas of forensic, clinical and anti-excitant agents. Compared with the traditional biological matrixes such as urine, blood and the like, the hair analysis has the outstanding advantages of noninvasive and simple sample collection, long time window for detecting toxic (medicine) substances and the like. Therefore, the hair analysis result is often used as important evidence in case investigation, and provides the reconnaissance direction and time information for the case. The previous analysis method focuses on fragment analysis of the hair clusters, and the length of 2-3 cm is mostly selected as interval segmentation. The traditional segmentation mode can only analyze the medicine taking situation in a period of time and cannot obtain exact time information. Therefore, in actual work, a method for analyzing trace amount of medicine in hair after a single medicine intake is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the method.
In order to realize the purpose, the invention adopts the technical scheme that:
the invention provides a method for testing drugs in single hair with sub-millimeter length, which comprises the following steps:
step one, sticking single hair on a customized adhesive tape along scales, and cutting the single hair under a table magnifier;
step two, extracting the sample divided in the step one by using a mixed solution of a water-soluble organic solvent and water-soluble protease as an extraction solution, wherein in order to ensure the extraction recovery rate, the extraction process comprises ultrasonic treatment and incubation, and extracting supernatant to prepare a sample solution after extraction;
and step three, performing liquid chromatography-mass spectrometry on the sample liquid obtained in the step two by using a liquid chromatograph-mass spectrometer.
Further, in the step one, the customization precision adopted by the customized adhesive tape is 0.1-1.0mm.
Further preferably, in the first step, the customization precision of the customized adhesive tape is 0.4mm.
Further, in the first step, the magnification of the desk magnifier is 30 ×.
A large number of experiments show that the method in the first step can accurately and conveniently divide and collect single hair in a submillimeter level. Other sample preparation methods are adopted, and certain problems exist in the aspects of actual operation, sample stability, collection and the like. The present invention is not restricted to the length of the sample, and in embodiments, the sample may be 0.1 to 1mm. The material and type of the customized adhesive tape are not strictly limited, and in the implementation process, the customized adhesive tape can be transparent adhesive tape, non-transparent adhesive tape, paper material, plastic material and the like.
Further, in the second step, the water-soluble organic solvent is an EM solution formed by mixing methanol, acetonitrile and 2mM ammonium formate; the water-soluble protease is dithiothreitol DTT.
A great deal of research shows that compared with other various organic reagents, the invention adopts EM-DTT solvent to obtain high extraction recovery rate and has obvious advantages. With EM-DTT solutions, the target drug is extracted from the hair by disrupting the hair cuticle barrier.
Further preferably, the volume ratio of methanol, acetonitrile, 2mM ammonium formate in said EM solution is 25:25:50 (v/v/v).
Further preferably, in the second step, the concentration of dithiothreitol in the EM-DTT solution is 1-100mg/mL.
Further, in the second step, the volume of the extraction solution is 25 to 50 μ L. More preferably 25. Mu.L.
Further, in the second step, the temperature of the ultrasonic wave is 15-35 ℃, the time is 30-60min, and more preferably 60min; the incubation time is 10-20h, more preferably 20h.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention realizes the preparation of the sub-millimeter sample of single hair, and the sample deviation is within the acceptable range.
The method of the invention extracts the sample by using the mixed solution of the water-soluble organic solvent and the water-soluble protease and then extracts the target drug by using the technologies of ultrasound, incubation and the like, thereby not only effectively destroying the cuticle barrier of the hair to extract the target drug in the hair, but also dissolving out a large amount of target detection substances with different structures in the maximum range, thereby realizing the rapid screening of various toxic (drugs) substances in the sample when the detection target is uncertain and the detection range is wide.
The preparation method of the single sub-millimeter hair sample has excellent selectivity, linear correlation, precision and accuracy.
Drawings
FIG. 1 shows the analysis results of 42 common psychotropic drugs in the present invention.
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.
Example 1
A method of testing a drug in individual sub-millimeter length hair comprising the steps of:
1. sample preparation
A single hair sample (root + shaft) was wiped across with acetone. Custom scotch tape was nailed to the foam board with the adhesive facing up. And (3) sticking a single hair on a transparent adhesive tape with the precision of 0.4mm along the scales. The swatches with glued hair were fixed on clean white printing paper. And dissected under a bench magnifier (30 ×) with surgical scissors. And precisely measuring the hair section by adopting an electronic differential ruler with the precision of 0.01 mm. The hair segment length was determined to be 0.4 ± 0.02mm (n = 50). Individual 0.4mm hair pieces were placed in 200 μ L tubes.
Medicine extraction from single hair of 2.0.4mm
A200. Mu.L tube was filled with 25. Mu.L of an EM-DTT solution containing an internal standard (the volume ratio of methanol to acetonitrile to 2mM ammonium formate in the EM solution was 25 (v/v/v), the concentration of dithiothreitol in the EM-DTT solution was 10 mg/mL), and after 1h of sonication at room temperature, incubation was carried out for 20h. The supernatant was transferred to an autosampler vial and 10. Mu.L was injected into the UPLC/MS-MS system.
3. Liquid chromatography-mass spectrometry with LC-MS
1) Liquid phase process
The liquid chromatographic column is a RestekAllure PFPP pentafluorophenyl column (100X 2.1mm,5 μm); mobile phase a was 20mmol/L ammonium acetate in water containing 0.1% formic acid and B was acetonitrile and the gradient elution is shown in table 1. The flow rate is 0.2mL/min, the sample volume is 10 muL, and the total sampling time is 12min.
TABLE 1 chromatographic mobile phase gradient
2) Mass spectrometry method
Detection was performed in electrospray ionization-positive ion mode (ESI +), multiple reaction monitoring mode (MRM). The operating parameters were as follows: ion Spray Voltage (ISV): 5500V; ion source Temperature (TEM): at 450 ℃; air curtain air: 40psi; atomizing gas (GS 1), auxiliary gas (GS 2): 35psi (1 psi ≈ 6.9 kPa). The LC/MS-MS parameters are shown in Table 2.
TABLE 2 analyte and internal standard MS/MS parameters
3) Method verification
In addition, the invention also implements a series of tests to verify the method of the invention, which mainly comprises verifying the selectivity, the linear correlation and the quantitative limits of the method of the invention as follows:
no interference of endogenous compounds is observed during the retention time of the analyte and ISs. All analytes had a good linear relationship (R > 0.99). LOD ranges from 0.1-10pg/mm and LLOQ ranges from 0.5-20pg/mm. The method covers 26 (about 62%) of the 42 analytes with LLOQ < 5pg/mm and 8 (20%) analytes < 1pg/mm. The regression equation, LOD, LLOQ and correlation coefficients for each analyte are shown in Table 3.
TABLE 3 regression equation, correlation coefficient, LOD and LLOQ of 42 psychoactive substances and metabolites in hair
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 (9)
1. A method of testing a drug in individual sub-millimeter length hair comprising the steps of:
step one, sticking single hair on a customized adhesive tape along scales, and cutting the single hair under a table magnifier;
step two, extracting the sample divided in the step one by using a mixed solution of a water-soluble organic solvent and water-soluble protease as an extraction solution, wherein in order to ensure the extraction recovery rate, the extraction process comprises ultrasonic treatment and incubation, and extracting supernatant to prepare a sample solution after extraction;
and step three, performing liquid chromatography-mass spectrometry on the sample liquid obtained in the step two by using a liquid chromatograph-mass spectrometer.
2. The method of claim 1, wherein in step one, the custom adhesive tape is applied with a custom precision of 0.1-1.0mm.
3. The method of claim 2, wherein in step one, the custom adhesive tape is applied with a custom precision of 0.4mm.
4. The method of claim 1, wherein in step one, the desktop magnifier magnification is 30 x.
5. The method for testing drugs in individual sub-millimeter length hair according to claim 1, wherein in the second step, the water-soluble organic solvent is an EM solution mixed by methanol, acetonitrile and 2mM ammonium formate; the water-soluble protease is dithiothreitol DTT.
6. Method for testing drugs in individual sub-millimeter length hair according to claim 5, characterized in that the volume ratio of methanol, acetonitrile, 2mM ammonium formate in the EM solution is 25:25:50 (v/v/v).
7. The method of claim 5, wherein in step two, the concentration of dithiothreitol in the EM-DTT solution is 1-100mg/mL.
8. The method of claim 1, wherein in step two, the volume of the extraction solution is 25-50 μ L.
9. The method of claim 1, wherein the drug is detected in individual sub-millimeter length hair,
in the second step, the temperature of the ultrasonic is 15-35 ℃, and the time is 30-60min; the incubation time is 10-20h.
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|---|---|---|---|
| CN202211166882.XA CN115452995B (en) | 2022-09-23 | 2022-09-23 | Method for inspecting medicine in single sub-millimeter length hair |
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| CN202211166882.XA CN115452995B (en) | 2022-09-23 | 2022-09-23 | Method for inspecting medicine in single sub-millimeter length hair |
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| CN115452995B CN115452995B (en) | 2024-01-19 |
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| CN115452995B (en) | 2024-01-19 |
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