CN115452995B - Method for inspecting medicine in single sub-millimeter length hair - Google Patents

Method for inspecting medicine in single sub-millimeter length hair Download PDF

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CN115452995B
CN115452995B CN202211166882.XA CN202211166882A CN115452995B CN 115452995 B CN115452995 B CN 115452995B CN 202211166882 A CN202211166882 A CN 202211166882A CN 115452995 B CN115452995 B CN 115452995B
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hair
sample
extraction
liquid
solution
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CN115452995A (en
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徐多麒
沈敏
向平
严慧
纪佼佼
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Academy Of Forensic Science
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Academy Of Forensic Science
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/62Detectors specially adapted therefor
    • G01N30/72Mass spectrometers

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Analytical Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

The invention discloses a method for detecting medicines in single sub-millimeter-length hair, which comprises the following steps: step one, sticking single hair on a customized adhesive tape along scales, and dividing under a desk magnifying glass; extracting the sample after the segmentation in the step one by adopting an extraction solution, wherein the extraction process comprises ultrasonic and incubation, and extracting supernatant liquid is prepared into a sample liquid after the extraction to ensure the extraction recovery rate; and thirdly, performing liquid chromatography-mass spectrometry on the sample liquid obtained in the second step by using a liquid chromatograph-mass spectrometer. The invention realizes the preparation of a sub-millimeter sample of a single piece of hair, and the sample deviation is within an acceptable range. The method of the invention not only can effectively destroy the barrier of the hair cuticle to extract target drugs in the hair, but also can dissolve out a large amount of target detection substances with different structures in the maximum range, thereby realizing the rapid screening of various toxic (drug) substances in the sample when the detection targets are uncertain and the detection range is wide.

Description

Method for inspecting medicine in single sub-millimeter length hair
Technical Field
The invention relates to the technical field of trace drug detection, in particular to a method for detecting a drug in a single sub-millimeter length hair.
Background
Since the end of the 90 s of the 20 th century, hair analysis has been increasingly used in forensic, clinical and anti-contrastive fields. Compared with traditional biological matrixes such as urine, blood and the like, the hair analysis has the outstanding advantages of noninvasive and simple sample collection, long toxic (drug) detection time window and the like. Therefore, the hair analysis result is often used as important evidence in case investigation to provide the case with reconnaissance direction and time information. The prior analysis method focuses on fragment analysis of the hair, and the length of 2-3 cm is used as interval segmentation. The traditional segmentation mode can only analyze the medicine taking condition in a period of time, and cannot obtain exact time information. Therefore, in practical work, there is an urgent need for a method of analyzing trace amounts of drugs in hair after a single intake of the drugs.
Disclosure of Invention
The present invention aims to address the deficiencies in the prior art and provides for a method.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a method for detecting medicines in single sub-millimeter length hair, which comprises the following steps:
step one, sticking single hair on a customized adhesive tape along scales, and dividing under a desk magnifying glass;
step two, extracting the sample after the segmentation in the step one by taking a mixed solution of a water-soluble organic solvent and a water-soluble protease as an extraction solution, wherein the extraction process comprises ultrasonic and incubation, and extracting supernatant liquid after extraction to prepare a sample liquid;
and thirdly, performing liquid chromatography-mass spectrometry on the sample liquid obtained in the second step by using a liquid chromatograph-mass spectrometer.
Further, in the first step, the customizing precision of the customizing adhesive tape is 0.1-1.0mm.
Further preferably, in the first step, the customizing accuracy of the customizing adhesive tape is 0.4mm.
Further, in the first step, the magnification of the desk magnifier is 30×.
According to the invention, a large number of experiments show that the method in the step one can accurately and conveniently divide and collect single hair at the sub-millimeter level. Other sample preparation methods are adopted, so that certain difficulties exist in the aspects of actual operation, sample stability, collection and the like. The present invention is not limited to a strict limitation on the sample length, and in an embodiment, the sample may be 0.1 to 1mm. The material and the category 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.
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.
According to the invention, a large number of researches show that compared with other various organic reagents, the EM-DTT solvent can be adopted to obtain high extraction recovery rate, and the advantages are obvious. With EM-DTT solutions, the target drug in the hair is extracted by disrupting the hair cuticle barrier.
Further preferably, the volume ratio of methanol, acetonitrile and 2mM ammonium formate in the EM solution is 25:25:50 (v/v/v).
Further preferably, in step two, 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 25-50. Mu.L. More preferably 25. Mu.L.
Further, in the second step, the temperature of the ultrasound is 15-35 ℃, and the time is 30-60min, more preferably 60min; the incubation time is 10-20 hours, more preferably 20 hours.
Compared with the prior art, the invention has the following technical effects:
the invention realizes the preparation of a sub-millimeter sample of a single piece of hair, and the sample deviation is within an acceptable range.
According to the method, after the sample is extracted by adopting the mixed solution of the water-soluble organic solvent and the water-soluble protease, the target medicine is extracted by utilizing the technologies of ultrasound, incubation and the like, so that not only can the barrier of the hair cuticle be effectively destroyed to extract the target medicine in the hair, but also a large amount of target detection substances with different structures can be dissolved out in the maximum range, and the rapid screening of various toxic (medicine) substances in the sample can be realized 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 results of 42 common psychotropic drugs according to the present invention.
Detailed Description
The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Example 1
A method of inspecting a single sub-millimeter length of hair for a drug, comprising the steps of:
1. sample preparation
A single hair sample (root + shaft) was taken and rubbed laterally with acetone. The customized scotch tape was attached to the foam board with the tack facing upward. A single hair was stuck along the scale to a transparent adhesive tape with an accuracy of 0.4mm. The hair-stuck sample was fixed on a clean white printing paper. And divided with surgical scissors under a desk magnifier (30×). An electronic differential ruler with the precision of 0.01mm is adopted to accurately measure the hair segment. The hair segment length was determined to be 0.4±0.02mm (n=50). A single 0.4mm hair segment was placed in a 200. Mu.L tube.
2.0.4mm extraction of the drug from the individual hairs
A200. Mu.L tube was filled with 25. Mu.L of an EM-DTT solution containing an internal standard (volume ratio of methanol, acetonitrile, 2mM ammonium formate in the EM solution: 25:25:50 (v/v), and the concentration of dithiothreitol in the EM-DTT solution: 10 mg/mL), and after 1 hour of ultrasonic treatment at room temperature, incubated for 20 hours. The supernatant was transferred to an autosampler and 10. Mu.L was injected into the UPLC/MS-MS system.
3. Liquid chromatography-mass spectrometry analysis by using liquid chromatography-mass spectrometer
1) Liquid phase process
The liquid chromatography column was 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 was as shown in table 1. The flow rate is 0.2mL/min, the sample injection amount is 10 mu L, and the total sampling time is 12min.
TABLE 1 gradient of chromatographic mobile phases
2) Mass spectrometry method
Electrospray ionization-positive ion mode (ESI+), multiple reaction monitoring mode (MRM) detection was used. The operating parameters were as follows: ion Spray Voltage (ISV): 5500V; ion source Temperature (TEM): 450 ℃; air curtain gas: 40psi; atomizing gas (GS 1) and auxiliary gas (GS 2): 35psi (1 psi. Apprxeq. 6.9 kPa). LC/MS-MS parameters are shown in table 2.
TABLE 2 analyte and internal Standard MS/MS parameters
3) Method verification
In addition, the present invention also implements a series of experiments to verify the methods of the present invention, which essentially include verifying the selectivity, linear correlation, and quantitative limits of the methods of the present invention, respectively, as described below:
no interference of endogenous compounds was observed during the retention time of analyte and ISs. All analytes had a good linear relationship (R > 0.99). The LOD ranges from 0.1 to 10pg/mm and the LLOQ ranges from 0.5 to 20pg/mm. 26 analytes (about 62%) of the 42 analytes covered by the method had LLOQ of 5pg/mm or less and 8 analytes (20%) of 1pg/mm or less. Regression equations, LOD, LLOQ and correlation coefficients for each analyte are shown in Table 3.
TABLE 3 regression equation, correlation coefficient, LOD and LLOQ for 42 psychoactive substances and metabolites in hair
The foregoing is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the embodiments and scope of the present invention, and it should be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the teachings and illustrations of the present invention, and that such variations are intended to be included within the scope of the present invention.

Claims (4)

1. A method of inspecting a single sub-millimeter length of hair for a drug, comprising the steps of:
step one, sticking single hair on a customized adhesive tape along scales, and dividing under a desk magnifying glass; the customizing precision of the customizing adhesive tape is 0.4mm;
step two, extracting the sample after the segmentation in the step one by taking a mixed solution of a water-soluble organic solvent and a water-soluble protease as an extraction solution, wherein the extraction process comprises ultrasonic and incubation, and extracting supernatant liquid after extraction to prepare a sample liquid;
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; the volume ratio of methanol, acetonitrile and 2mM ammonium formate in the EM solution is 25:25:50 (v/v/v); the concentration of dithiothreitol in the EM-DTT solution is 1-100mg/mL;
and thirdly, performing liquid chromatography-mass spectrometry on the sample liquid obtained in the second step by using a liquid chromatograph-mass spectrometer.
2. The method of claim 1, wherein in step one, the magnification of the bench magnifier is 30 x.
3. The method of inspecting a medicament in a single sub-millimeter length hair according to claim 1, wherein in step two, the volume of the extraction solution is 25-50 μl.
4. The method for inspecting a medicament in a single sub-millimeter length of hair according to claim 1, wherein in step two, the temperature of the ultrasound is 15-35 ℃ for 30-60min; the incubation time is 10-20h.
CN202211166882.XA 2022-09-23 2022-09-23 Method for inspecting medicine in single sub-millimeter length hair Active CN115452995B (en)

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Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103207242A (en) * 2012-01-11 2013-07-17 司法部司法鉴定科学技术研究所 Method for rapidly identifying monoacetylmorphine and morphine in hair
CN103592400A (en) * 2013-09-24 2014-02-19 广州正孚检测技术有限公司 Extraction and detection method of opioid matters in hair
CN108287206A (en) * 2018-03-29 2018-07-17 河北医科大学 Barbiturate quantitative detecting method and application in a kind of hair
CN109142576A (en) * 2018-09-03 2019-01-04 江西省农业科学院农产品质量安全与标准研究所 The detection method of roxithromycin residual quantity in a kind of animal hair
CN109507355A (en) * 2019-01-17 2019-03-22 浙江工业大学 Method for determining content of syphilis and ecstasy in human hair by flash evaporation-gas chromatography-mass spectrometry
CN109507354A (en) * 2019-01-17 2019-03-22 浙江工业大学 Method for determining content of K powder in human hair by flash evaporation-gas chromatography-mass spectrometry
CN111208247A (en) * 2020-01-08 2020-05-29 浙江工业大学 Method for the determination of γ-hydroxybutyric acid in human hair by on-line heat-assisted methylation-gas chromatography-mass spectrometry
CN111487336A (en) * 2020-04-13 2020-08-04 司法鉴定科学研究院 Method for analyzing 37 fentanyl novel psychoactive substances in hair
CN111656165A (en) * 2017-11-28 2020-09-11 吾爱吾身研究有限公司 Method for analyzing hair by polarized light
CN111983222A (en) * 2020-07-08 2020-11-24 北京金鹏达科技发展有限公司 Drug extracting solution in hair, extracting method and application
CN112525641A (en) * 2020-12-03 2021-03-19 杭州奥泰生物技术股份有限公司 Preparation method of liquid for testing mental drugs in hair
CN112630363A (en) * 2020-12-31 2021-04-09 四川大学华西医院 MXene biological response characteristic metabonomics analysis method
CN113092615A (en) * 2021-04-01 2021-07-09 四川大学华西医院 Method for detecting antiepileptic drug and metabolite thereof in hair by liquid chromatography-mass spectrometry
CN113325103A (en) * 2021-05-28 2021-08-31 司法鉴定科学研究院 Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair
CN114544824A (en) * 2020-11-25 2022-05-27 中国科学院大连化学物理研究所 Pretreatment method for hair-stem proteome sample

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103207242A (en) * 2012-01-11 2013-07-17 司法部司法鉴定科学技术研究所 Method for rapidly identifying monoacetylmorphine and morphine in hair
CN103592400A (en) * 2013-09-24 2014-02-19 广州正孚检测技术有限公司 Extraction and detection method of opioid matters in hair
CN111656165A (en) * 2017-11-28 2020-09-11 吾爱吾身研究有限公司 Method for analyzing hair by polarized light
CN108287206A (en) * 2018-03-29 2018-07-17 河北医科大学 Barbiturate quantitative detecting method and application in a kind of hair
CN109142576A (en) * 2018-09-03 2019-01-04 江西省农业科学院农产品质量安全与标准研究所 The detection method of roxithromycin residual quantity in a kind of animal hair
CN109507355A (en) * 2019-01-17 2019-03-22 浙江工业大学 Method for determining content of syphilis and ecstasy in human hair by flash evaporation-gas chromatography-mass spectrometry
CN109507354A (en) * 2019-01-17 2019-03-22 浙江工业大学 Method for determining content of K powder in human hair by flash evaporation-gas chromatography-mass spectrometry
CN111208247A (en) * 2020-01-08 2020-05-29 浙江工业大学 Method for the determination of γ-hydroxybutyric acid in human hair by on-line heat-assisted methylation-gas chromatography-mass spectrometry
CN111487336A (en) * 2020-04-13 2020-08-04 司法鉴定科学研究院 Method for analyzing 37 fentanyl novel psychoactive substances in hair
CN111983222A (en) * 2020-07-08 2020-11-24 北京金鹏达科技发展有限公司 Drug extracting solution in hair, extracting method and application
CN114544824A (en) * 2020-11-25 2022-05-27 中国科学院大连化学物理研究所 Pretreatment method for hair-stem proteome sample
CN112525641A (en) * 2020-12-03 2021-03-19 杭州奥泰生物技术股份有限公司 Preparation method of liquid for testing mental drugs in hair
CN112630363A (en) * 2020-12-31 2021-04-09 四川大学华西医院 MXene biological response characteristic metabonomics analysis method
CN113092615A (en) * 2021-04-01 2021-07-09 四川大学华西医院 Method for detecting antiepileptic drug and metabolite thereof in hair by liquid chromatography-mass spectrometry
CN113325103A (en) * 2021-05-28 2021-08-31 司法鉴定科学研究院 Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair

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