CN104422607A

CN104422607A - Hair pretreatment method

Info

Publication number: CN104422607A
Application number: CN201310398947.8A
Authority: CN
Inventors: 高颖; 魏建良
Original assignee: HANGZHOU YOUMADA BIOTECHNOLOGY Co Ltd
Current assignee: HANGZHOU YOUMADA BIOTECHNOLOGY Co Ltd
Priority date: 2013-09-05
Filing date: 2013-09-05
Publication date: 2015-03-18

Abstract

The invention discloses a hair pretreatment method belonging to the technical field of biological chemistry. The method comprises the following steps: cleaning hair by using a cleaning agent, and airing; crushing hairs into fragments, and finally, adding the hair fragments into digestive fluid, putting the digestive fluid into a water bath at the temperature of 60-100 DEG C for 5 minutes to 2 hours so as to hydrolyze the hairs, standing and cooling the digestive fluid to room temperature after the hydrolysis is finished, and detecting supernatant. According to the method disclosed by the invention, the hairs serve as a detection material, are easy to sample, non-traumatic and convenient to preserve and can be repeatedly sampled, the steps of a conventional hair pretreatment method are simplified and optimized, various components in the used digestive fluid do not influence with one another, the detection effect is good, the detection accuracy is high, the false positive condition is difficultly caused, and the misjudgment condition is avoided as much as possible in the detection process.

Description

A kind of pre-treating method of hair

Technical field

The invention belongs to technological field of biochemistry, be specifically related to a kind of hair pre-treating method being applicable to detect drug abuse in hair.

Background technology

At present, the drug issue be on the rise has become global disaster.The physical and mental health of spreading unchecked the directly harm people of drugs, and bring huge threat to economic growth and social progress.Statistics according to the United Nations shows, the annual drug trade volume in the whole world reaches more than 5,000 hundred million dollars, the scope that drugs spread has expanded to more than 200 countries and regions of five continents, and the number of various drugs is sucked up to more than 200,000,000 in the whole world, and wherein the person between twenty and fifty of 17 ~ 35 one full year of life account for 78%.On the other hand, along with countries in the world, particularly the developed country such as European Union, Japan, U.S. was increasingly strict to the requirement of drug residue of food in recent years, along with the raising of instrument and equipment detectability and detection technique level, also more and more stricter to the requirement of the medicine maximum residue limit(MRL) of animal.

Drug abuse generally refers to the medical application and social regulation and use any one medicine having run counter to and generally acknowledged.This use self-administration often, thus all can cause certain infringement to the health of medication person and society.Generally common drug abuse refers to: narcotics, as opiates, cocaines, cannabis etc.; Psychotropic substances, comprise central depressant, as hypnotic sedative agent, also have central stimulant, as caffeine, also have psychedelic in addition, as mescaline, LSD etc.; Microbiotic medicine, as penicillin, gentamicin etc.; β class excitant medicine, as common clenbuterol hydrochloride etc.; And anabolic hormone and cortin etc.

Conventional biological material such as blood, urine, bile etc. do not have a kind of medication information providing long-range as hair.Body fluid (mainly referring to urine and blood) is still drug abuse and checks good biological material, but the time limit of body fluid Chinese traditional medicine is short, sample is not easily preserved, and can only provide the information of individual short-period used medicine; And hair is drawn materials easily, without wound, easy to carry, to preserve easily for a long time, and medicine in hair, metabolism more slowly can long-term existence, therefore hair analysis than bulk liquid analysis in qualification drug abuse is more practical, convenient.Over 10 years, the develop rapidly of hair analysis technology, due to the advantage of the uniqueness that hair sample itself has, in people and animal hair, the detection of drug abuse has started to receive increasing concern.The forensic chemistry laboratory of a lot of country, hair analysis has become the routine operation that drug abuse detects, and has obtained the admitting of court, adopts.

The pre-treatment of hair is generally divided into two steps: cleaning hair; The digestion of hair, extraction.

The cleaning of hair: hair contacts with external environment, vulnerable to pollution.With regard to external contamination, hair can be divided into 3 regions: easily contact region, half contact region and non-contactable region.Easy contact region refers to the surperficial outermost layer of hair, is easily stained with the dirt in environment, even the powder of medicine, and this district adopts some anhydrous organic solvents to clean; Half contact region refers to the region that pollutant not easily enters and need can enter as an aqueous solution, and this district needs the washing such as use water or hydrophilic organic solvent; Relief area is maximum region, usually accounts for about 90% of hair structure, and be the main region that medicine is combined with hair, cleaning solvent can not enter this district.The digestion of hair, extraction: medicine solidifies in the denatured keratin of hair, first must digest and make it be that free state extracts again.Its surface area can be increased after hair shreds or grinds, be conducive to the release of medicine.Conventional digestion method has basic hydrolysis, acid hydrolysis, enzyme hydrolysis, methyl alcohol ultrasonic.But existing extracting method, as acid, alkali hydrolysis method, often hydrolysising condition is fiercer, is not suitable for the sample preparation of the unstable compounds such as monoacetylmorphine; Enzyme hydrolysis method is applied widely, and release efficiency is high, and shortcoming is that cost is too expensive; The methyl alcohol ultrasonic method scope of application is comparatively wide, but insoluble drug release not exclusively and impurity is more.Therefore, owing to lacking a kind of hair pre-treating method applied widely, quick, easy, efficient and don't that have an impact to compound, hair analysis work still rests on laboratory level, does not see actual product for many years.

Therefore a kind of simple and quick efficient hair pre-treating method of invention, and corresponding detection means has very important realistic meaning.

Summary of the invention

The present invention is directed to current hair pre-treating method narrow application range, complicated, the problem of poor efficiency, proposes a kind of pre-treating method of hair.

A pre-treating method for hair, carries out in accordance with the following steps:

(1) hair cleaning: with detersive cleaning, dry;

(2) hair is pulverized: pulverized by hair as fragment;

(3) hair digestion: by step (2) hair fragment, add in digestive juice, 60-100 DEG C of water-bath 5min-2h, makes hair be hydrolyzed, leaves standstill above-mentioned digestive juice after being hydrolyzed and is cooled to room temperature, get supernatant and detect.

The component of described digestive juice is: the PBS damping fluid of 0.5-5% volume parts, 0.1-10mmol/L calcium acetate, 1-20mmol/L dithiothreitol (DTT), 0.1-2mmol/L lauryl sodium sulfate and 1-10mg/ml Proteinase K, and surplus is deionized water.

The pH of described PBS damping fluid is 7.2.

The method pulverized of described hair is for shred with scissors or bowl mill grinds.

Described detersive be one in water, methyl alcohol, lauryl sodium sulfate and more than.

Beneficial effect of the present invention: the present invention take hair as sample, not only sampling easily, can repeated sampling, atraumatic, preservation convenient, the most important thing is that hair can constantly grow, can dynamic continuously for a long time drug abuse be monitored.The step of the present invention to conventional hair pre-treating method is simplified, is optimized, because the digestive juice hydrolysis hair used can not bring impurity or the accessory substance of impact detection into, therefore hair sample can be directly used in detection after digestion, dry up without the need to further solvent or the step such as extraction, not only make pretreatment process simplify, save the time, and decrease the loss of leaching process Chinese traditional medicine, and avoid using the nitrogen needed for extracting to blow and extraction equipment, therefore significantly reduce cost.In the middle of hair, more than 90% is keratin, and by three-dimensional structure that the disulfide bond of cystine, hydrogen bond, sat linkage and other crosslink bond effect height of formation are crosslinked between keratin molecule.Therefore opened disulfide bond improves the key of digestive efficiency.Disulfide bond is opened by the present invention's application reductive agent, and to destroy between keratin firmly covalent structure, then by the effect of proteinase, keratin of degrading further, makes the drug molecule of solidification in keratin free out.Various component in the digestive juice that the present invention uses, can not have an impact each other, therefore directly adopt single stage method, digestive juice be joined in hair the reaction that is hydrolyzed, whole operating process is easy, quick, save time and save trouble.Selected reagent is by gentle hydrolysis hair, and the concentration that corresponding reagent uses can not have an impact to method detections such as immunoassays, Detection results is good, and detection accuracy is high, not easily there is false positive situation to occur, the generation of erroneous judgement situation can be avoided as far as possible in testing process.

Accompanying drawing explanation

Fig. 1 is pig hair pre-treatment effect comparison diagram (, for before dissolving, B is rear for dissolving for A).

Fig. 2 is human hair's pre-treatment effect comparison diagram (, for before dissolving, B is rear for dissolving for A).

Fig. 3 is the testing result of Clenbuterol in pig galley proof product.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described.

The pre-treating method of embodiment 1 pig galley proof product

By the pig hair collected respectively with 0.1% lauryl sodium sulfate and water clean, at room temperature dry, then shred into the fragment of 2mm.Take 10mg pig hair in EP pipe, add digestive juice 500ul and (include the PBS damping fluid of 0.5% volume fraction, 1mmol/L calcium acetate, 5mmol/L lauryl sodium sulfate, 10mmol/L dithiothreitol (DTT), 5mg/ml Proteinase K, surplus is deionized water), 65 DEG C of water-baths, within every 10 minutes, vortex oscillation (also can carry out water-bath in ultrasonic, accelerate dissolution time) once, after 45 minutes, pig hair all dissolves (in ultrasonic 30 minutes), and solution of pig hair situation is as shown in Figure 1.

The pre-treating method of embodiment 2 human hair sample

The hair collecting people is used washed with methanol respectively, blots with thieving paper, then shred into the fragment of 2mm.Take 2mg hair in EP pipe, add digestive juice 150ul and (include the PBS damping fluid of 0.1% volume fraction, 1mmol/L calcium acetate, 5mmol/L lauryl sodium sulfate, 10mmol/L dithiothreitol (DTT), 15mg/ml Proteinase K, surplus is deionized water), 65 DEG C of water-baths, within every 10 minutes, vortex oscillation (is had ready conditions and can be carried out water-bath in ultrasonic, accelerate dissolution time) once, after 35 minutes, hair all dissolves (in ultrasonic 25 minutes), and hair dissolves situation as shown in Figure 2.

Embodiment 3 detects the pre-treating method of Clenbuterol in pig galley proof product

In the on-the-spot urine examination examination in slaughterhouse, find that 3 pig Clenbuterols are positive, and select 10 negative pigs immediately, collect the pig hair of these 13 pigs, by these pig hairs respectively with 0.1% lauryl sodium sulfate and water clean, at room temperature dry, then shred into the fragment of 2mm.Take 10mg pig hair in EP pipe, add digestive juice 500ul and (include the PBS damping fluid of 0.1% volume fraction, 1mmol/L calcium acetate, 5mmol/L lauryl sodium sulfate, 5mmol/L dithiothreitol (DTT), 5mg/ml Proteinase K, surplus is deionized water), 65 DEG C of water-baths 20 minutes, every 5 minutes vortex oscillation, then be placed in boiling water water-bath 5 minutes, terminate rear leaving standstill and be cooled to room temperature, get 100ul supernatant and detect.Clenbuterol detection reagent kit is purchased from DNA Sci-tech Co., Ltd., and commodity are called Clenbuterol one-step ELISA kit, carry out operation detection by kit instructions, and testing result as shown in Figure 3.

Embodiment 4 detects the pre-treating method of morphine in human hair's sample

Positive hair sample is provided by certain narcotic house, and negative sample obtains from ordinary people, and checks the morphine positive and negative situation by urine examination.The hair collecting people is used washed with methanol respectively, blots with thieving paper, then shred into the fragment of 2mm.Take 2mg hair in EP pipe, add digestive juice 150ul and (include the PBS damping fluid of 0.1% volume fraction, 1mmol/L calcium acetate, 2mmol/L lauryl sodium sulfate, 5mmol/L dithiothreitol (DTT), 10mg/ml Proteinase K, surplus is deionized water), 65 DEG C of water-baths 20 minutes, every 5 minutes vortex oscillation, then be placed in boiling water water-bath 5 minutes, terminate rear leaving standstill and be cooled to room temperature, get 100ul supernatant and detect.Morphine test card is purchased from Biosynex Deutschland GmbH, and trade name Drugs ofAbuse Rapid Test Device, carry out operation detection by test card instructions, testing result is as shown in table 1.

Table 1

Numbering	Sex	Age	Drug abuse kind	Hair sample testing result
					1	Man	43	Heroin	Positive
2	Man	33	Heroin	Positive
					3	Man	47	Heroin	Positive
4	Man	30	Heroin	Positive
					5	Man	53	Heroin	Positive
6	Man	37	Heroin	Positive
					7	Man	41	Heroin	Positive
8	Man	35	Heroin	Positive
					9	Man	37	Heroin	Positive
10	Man	37	Heroin	Positive
					11	Man	31	Heroin	Positive
12	Man	35	Heroin	Positive
					13	Man	50	Heroin	Positive
14	Man	30	Heroin	Positive
					15	Man	31	Heroin	Positive
16	Man	33	Heroin	Positive
					17	Man	37	Heroin	Positive
18	Man	39	Heroin	Positive
					19	Man	34	Heroin	Positive
20	Man	49	Heroin	Positive

A	Man	34	Nothing	Negative
					B	Man	19	Nothing	Negative
C	Man	43	Nothing	Negative
					D	Man	29	Nothing	Negative
E	Man	25	Nothing	Negative

Testing result shows, and negative sample (A-E) does not all detect heroin and remains, and positive (1-20) all detects heroin, proves to can be used for detecting by the hair of the method process.

Claims

1. a pre-treating method for hair, is characterized in that, carries out in accordance with the following steps:

(1) hair cleaning: with detersive cleaning, dry;

(2) hair is pulverized: pulverized by hair as fragment;

2. the pre-treating method of a kind of hair according to claim 1, it is characterized in that, the component of described digestive juice is: the PBS damping fluid of 0.5-5% volume parts, 0.1-10mmol/L calcium acetate, 1-20mmol/L dithiothreitol (DTT), 0.1-2mmol/L lauryl sodium sulfate and 1-10mg/ml Proteinase K, surplus is deionized water.

3. the pre-treating method of a kind of hair according to claim 2, it is characterized in that, the pH of described PBS damping fluid is 7.2.

4. the pre-treating method of a kind of hair according to claim 1, is characterized in that, the method pulverized of described hair is for shred with scissors or bowl mill grinds.

5. the pre-treating method of a kind of hair according to claim 1, is characterized in that, described detersive be one in water, methyl alcohol, lauryl sodium sulfate and more than.