CN115628951A - Human hair lysate for trace drug detection and application thereof - Google Patents

Abstract

The invention discloses a human hair lysate for trace drug detection and application thereof, belonging to the technical field of drug detection and comprising the following components: 0.8-1.5mg/mL protease, 0.5-1.2% acetic acid, 2.6-7.8mg/mL sodium bisulfite, 2.9-4.1mg/mL sodium chloride, 12.5-20mg/mL tris (2-carboxyethyl) phosphine, 0.5-1.5% isopropanol, 0.5-1.5% polyethylene glycol and pH4-7 weak acid buffer solution, wherein the percentage is volume percentage.

Description

Human hair lysate for trace drug detection and application thereof

Technical Field

The invention relates to the technical field of drug detection, in particular to a human hair lysate for trace drug detection and application thereof.

Background

According to statistics, the number of people taking novel drugs in China increases by about 30% every year, wherein teenagers account for 86%. The abuse of novel drugs causes serious individual harm and social harm, and in order to effectively attack novel drug crimes and closely match the drug prohibition requirements of law enforcement departments such as public security, the analysts establish a series of accurate, rapid and high-sensitivity methods for qualitatively and quantitatively analyzing and detecting the novel drugs. The hair is used as a special carrier of the drug, has incomparable advantages of other test materials, is easy to obtain materials, convenient to carry and easy to store for a long time, and the drug is metabolized in the hair slowly and can exist for a long time, so that the analysis of the drug in the hair becomes a research hotspot of court science and clinical analysis and plays an increasingly important role. The drugs in the hair are mainly located in the cuticle and medulla layers of the hair and are bound with proteins, so that a hair sample needs to be pretreated before the drugs in the hair are detected, the drugs are released from the hair, and grease, sweat and various pollutants outside the hair are eliminated.

In the prior art, methods for hair treatment are divided into an acid treatment method and an alkali treatment method, for example, a rapid hair lysis solution disclosed in patent CN 110208072B, which consists of alkaline protease, thioglycolic acid, glycine, tris hydrochloride buffer, sodium chloride-sodium hydroxide buffer, polyethylene glycol octyl phenyl ether and dithiothreitol, wherein the alkaline protease is used for hydrolyzing protein peptide bonds under alkaline conditions, decomposing keratin and releasing drug small molecules; thioglycolic acid and dithiothreitol are used as reducing agents to promote the activity of keratinase; glycine, tris hydrochloride buffer solution, sodium chloride and sodium hydroxide buffer solution and the like provide a proper cracking environment; polyoxyethylene octyl phenyl ether is used to solubilize lipids to increase the permeability of cell membranes to antibodies. When a mercapto group-containing compound is used as a reducing agent, the mercapto group-containing reducing agent has a certain toxicity, and is accompanied by an odor, and the monothiol group-containing reducing agent generally needs to be in excess in order to ensure the destruction degree of disulfide bonds, and a dimercapto reducing compound such as Dithiothreitol (DTT) has a good reducing effect but has a disadvantage of being difficult to store in a solution, and in order to ensure the effect, DTT is preferably prepared as it is. Under acidic conditions, the commonly used cleaved and reduced product is a phosphorus-containing compound, such as Tris (2-carboxyethyl) phosphine (TECP), which is slightly soluble in water, easy to store and can be operated without a protective gas), has a stronger reducing power for disulfide bonds than DTT, and has a wide pH tolerance range. Patent CN 101979428B discloses a preparation method and use of an animal hair dissolving agent and a keratin solution, which uses tris (2-carboxyethyl) phosphine hydrochloride, tris (2-cyanoethyl) phosphine or tri-n-butylphosphine as main components for separating keratin.

Under the acidic or neutral condition, the medicine release is incomplete, while the content of the poison and the metabolite thereof in the hair is very low, only in ng/mg level, and the detection accuracy is affected by the incomplete medicine release.

Disclosure of Invention

In view of the technical defects, the invention aims to provide a human hair lysate for trace drug detection, which is characterized by comprising the following components: 0.8-1.5mg/mL protease, 0.5-1.2% acetic acid, 2.6-7.8mg/mL sodium bisulfite, 2.9-4.1mg/mL sodium chloride, 12.5-20mg/mL tris (2-carboxyethyl) phosphine, 0.5-1.5% isopropanol, 0.5-1.5% polyethylene glycol and pH4-7 weakly acidic buffer, wherein the% is volume percent.

Preferably, the following components are included: 0.9-1.1mg/mL protease, 0.7-1.0% acetic acid, 3.6-4.2mg/mL sodium bisulfite, 3.5-4.1mg/mL sodium chloride, 16.6-18.7mg/mL tris (2-carboxyethyl) phosphine, 1.1-1.5% isopropanol, 1.1-1.5% polyethylene glycol and pH4-7 weak acidic buffer, wherein the% is volume percent. Preferably, the weakly acidic buffer solution with the pH value of 4-7 is at least one of 0.01-1mol/L phosphate buffer solution, citric acid buffer solution, acetic acid buffer solution and disodium hydrogen phosphate-citric acid buffer solution, the buffer solution is used for catalyzing and decomposing keratin, a proper environment is provided for hair surface treatment, hair surface grease decomposition is facilitated under an acidic condition, and the phenomenon that a drug entering a lysate is combined with protein again is effectively avoided under the acidic condition.

Preferably, the polyethylene glycol is PEG-200 or PEG-400 or a mixture of the two.

In view of the technical shortcomings, the invention also aims to provide an application of human hair lysate for trace drug detection, which is characterized in that: the lysate is used for cracking hair and releasing drug components in the hair.

Preferably, the hair is a hair within 3cm in length from the hair growth end.

Preferably, the hair is hair taken from the human body.

Preferably, the amount of lysate is 150 ± 10 μ L per mg of hair.

Preferably, the lysis mixture obtained by lysing the hair with the lysis solution is adjusted to a pH of 8 to 9 before the test is performed.

The invention has the beneficial effects that: 1.2, the invention takes tri (2-carboxyethyl) phosphine as a reducing agent, is easy to store, and the disulfide bonds between the tri (2-carboxyethyl) phosphine keratins are broken to promote the decomposition of the keratin; 3. isopropanol and polyethylene glycol are dissolved in water, and are added, so that the components of the composition are in a uniform state, the overall polarity of the solution is changed, the interaction force between tris (2-carboxyethyl) phosphine and keratin is increased, and the disulfide bond in the keratin is favorably broken; 4. except gamma-hydroxybutyric acid, the common drugs such as 6 acetylmorphine, morphine, methamphetamine, amphetamine, 3,4 methylenedioxyamphetamine, 3,4 methylenedioxymethamphetamine, ketamine, norketamine, gamma-hydroxybutyric acid and the like are low in water solubility or insoluble in water, isopropanol and polyethylene glycol are added to change the medium environment, reduce the interaction force between drug molecules and keratin, promote the drug molecules to be released into a lysate, promote the drug to be released from the keratin, and improve the release amount of the drug, so that the subsequent detection accuracy is improved; 5. acetic acid is added to soften the hair scales on the hair surface, so that the subsequent solvent can permeate the hair scales, and the permeation speed of isopropanol and polyethylene glycol is accelerated.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1:

sample acquisition: collecting hair from the root of the hair of a person to be tested, obtaining a hair sample, and keeping the hair within 3cm from the hair growing end by taking the hair growing end as a reference.

Sample a: the person to be tested is a voluntary drug-dropping person, the sample is obtained 101mg, the sample hair is cut into thin sections by scissors, four parts are equally divided and respectively marked as D-A, T-A1, T-A2 and T-A3, and each part is about 5mg;

sample B: the person to be tested is a voluntary drug-dropping person, 133mg of sample is obtained, the sample hair is cut into thin sections by scissors, four parts are equally divided and are respectively marked as D-B, T-B1, T-B2 and T-B3, and each part is about 5mg;

sample C: the person to be tested is a voluntary drug-dropping person, samples are obtained 97mg, sample hair is cut into thin sections by scissors, four parts are equally divided and respectively marked as D-C, T-C1, T-C2 and T-C3, and each part is about 5mg;

wherein, the samples D-A, D-B and D-C adopt a commercial cracking agent which takes DTT as a main reduction component of a disulfide bond to respectively detect the samples D-A, D-B and D-C, taking the operation steps of the samples D-A as an example, the steps are as follows:

step one, adding commercially available DTT (diethylenetriamine pentaacetic acid) serving as a main reducing component of a disulfide bond and alkaline protease serving as a cracking agent of a hydrolysis component into a 1.5mL centrifugal tube, wherein the volume of the cracking agent is about 750 mu L;

step two, transferring the pre-obtained and cut hair sample D-A into a centrifuge tube;

step three, carrying out ultrasonic water bath, setting the temperature to be 25 ℃, and setting the time to be 6 minutes;

and step four, taking out the sample, standing for 1 minute, sucking the upper liquid, adding the upper liquid into a sample adding area of a sensing chip of the detector, adding 4 drops of sample adding amount, about 80 mu L of sample adding amount, waiting for about 6-10 minutes, inserting the sample adding area into a full biological sample Drug analyzer for detection, and recording a detection result, wherein the model of the full biological sample Drug analyzer is Drug Test 5900H.

The operations of the samples D-B and D-C are the same as the steps, and the detection results are recorded respectively after the detection is finished.

Example 2:

the hair lysate is prepared according to the proportion, in the embodiment, the proportion of each component is as follows: 1.1mg/mL protease, 1.0% acetic acid, 4.2mg/mL sodium bisulfite, 4.1mg/mL sodium chloride, 18.7mg/mL tris (2-carboxyethyl) phosphine, 1.5% isopropanol, 1.5% polyethylene glycol 200, acetic acid buffer ph =6, where% is volume percent.

Samples T-A1, T-B1 and T-C1 were each detected using the cleavage agent of this example, and the sample T-A₁ The operation steps are as follows:

step one, adding the lysis solution in the embodiment into a 1.5mL centrifuge tube, wherein the lysis solution is about 750 mu L;

step two, transferring the pre-obtained and cut hair sample T-A1 into a centrifuge tube;

step three, carrying out ultrasonic water bath, setting the temperature to be 30-35 ℃ and the time to be 10 minutes;

step four, adjusting the pH value of the hair drug release mixed solution to 8-9 by using a sodium hydroxide solution;

and step five, taking out the sample, standing for 1 minute, sucking the upper liquid, adding the upper liquid into a sample adding area of a sensing chip of the detector, adding 4 drops of sample adding amount, about 80 mu L, waiting for about 6-10 minutes, inserting the whole biological sample drug analyzer for detection, and recording the detection result.

The operation of the samples T-B1 and T-C1 is the same as the steps, and the detection results are recorded respectively after the detection is finished. Example 3:

the hair lysate is prepared in proportion, in the embodiment, the proportion of each component is as follows: 1.0mg/mL protease, 0.7% acetic acid, 3.9mg/mL sodium bisulfite, 4.1mg/mL sodium chloride, 17.5mg/mL tris (2-carboxyethyl) phosphine, 1.3% isopropanol, 1.3% 1 mixture of polyethylene glycol 200 and polyethylene glycol 400, pH4-7 weak acidic buffer, wherein% is volume percent.

Samples T-A2, T-B2 and T-C2 were each assayed using the cleavage agent of this example, and sample T-A was used₁ The operation steps are as follows:

step one, adding the lysis solution in the embodiment into a 1.5mL centrifuge tube, wherein the lysis solution is about 750 mu L;

step two, transferring the pre-obtained and cut hair sample T-A2 into a centrifuge tube;

step three, carrying out ultrasonic water bath, setting the temperature to be 35 ℃, and setting the time to be 15 minutes;

step four, adjusting the pH value of the hair drug release mixed solution to 8-9 by using a sodium hydroxide solution;

and step five, taking out the sample, standing for 1 minute, sucking the upper layer liquid, adding the upper layer liquid into a sample adding area of a sensing chip of the detector, adding 4 drops of sample adding amount, about 80 mu L of sample adding amount, waiting for about 6-10 minutes, inserting the whole biological sample drug analyzer for detection, and recording the detection result.

The operation of the samples T-B2 and T-C2 is the same as the steps, and the detection results are recorded respectively after the detection is finished. Example 4:

the hair lysate is prepared according to the proportion, in the embodiment, the proportion of each component is as follows: 1.1mg/mL protease, 4.2mg/mL sodium bisulfite, 4.1mg/mL sodium chloride, 18.7mg/mL tris (2-carboxyethyl) phosphine, 3mg/mL urea, pH =6 acetate buffer, wherein% is volume percent.

The samples T-A3, T-B3 and T-C3 are respectively detected by adopting the cracking agent in the embodiment, taking the operation steps of the sample T-A3 as an example, the steps are as follows:

step one, adding the lysis solution in the embodiment into a 1.5mL centrifuge tube, wherein the lysis solution is about 750 mu L;

step two, transferring the pre-obtained and cut hair sample T-A3 into a centrifuge tube;

step three, performing ultrasonic water bath, setting the temperature to be 30-35 ℃, and setting the time to be 10 minutes;

step four, adjusting the pH value of the hair drug release mixed solution to 8-9 by using a sodium hydroxide solution;

and step five, taking out the sample, standing for 1 minute, sucking the upper liquid, adding the upper liquid into a sample adding area of a sensing chip of the detector, adding 4 drops of sample adding amount, about 80 mu L, waiting for about 6-10 minutes, inserting the whole biological sample drug analyzer for detection, and recording the detection result.

The operation of the samples T-B3 and T-C3 is the same as the steps, and the detection results are recorded respectively after the detection is finished.

The detection results are as follows:

table 1: sample detection condition

Compared with the existing phosphorus-containing decomposition liquid, the pyrolysis liquid simplifies the processing steps, shortens the processing time, greatly improves the processing efficiency of the pyrolysis liquid, is easy to store, prolongs the storage life of the pyrolysis liquid, adopts water-soluble reagents as medium reagents, is easy to carry out subsequent processing, dissolves isopropanol and polyethylene glycol in water, adds isopropanol and polyethylene glycol, enables the components of the composition to be in a uniform state, changes the overall polarity of the solution medium, increases the interaction force between tris (2-carboxyethyl) phosphine and keratin, and is beneficial to the breakage of disulfide bonds in the keratin; the acetic acid is a solvent commonly used in chemical reaction, and is matched with the isopropanol and the polyethylene glycol to change the medium environment, reduce the interaction force between drug molecules and keratin, promote the drug molecules to be released into a lysate, promote the drug to be released from the keratin, improve the release amount of a drug, enable the drug to be easily detected, and increase the subsequent detection accuracy.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (8)

1. A human hair lysate for trace drug detection is characterized by comprising the following components: 0.8-1.5mg/mL protease, 0.5-1.2% acetic acid, 2.6-7.8mg/mL sodium bisulfite, 2.9-4.1mg/mL sodium chloride, 12.5-20mg/mL tris (2-carboxyethyl) phosphine, 0.5-1.5% isopropanol, 0.5-1.5% polyethylene glycol and pH4-7 weakly acidic buffer, wherein the% is volume percent.

2. The human hair lysate for trace drug detection according to claim 1, comprising the following components: 0.9-1.1mg/mL protease, 0.7-1.0% acetic acid, 3.6-4.2mg/mL sodium bisulfite, 3.5-4.1mg/mL sodium chloride, 16.6-18.7mg/mL tris (2-carboxyethyl) phosphine, 1.1-1.5% isopropanol, 1.1-1.5% polyethylene glycol and pH4-7 weakly acidic buffer, wherein the% is volume percent.

3. The human hair lysate for trace drug detection according to claim 1 or 2, wherein the pH4-7 weak acid buffer is at least one of a phosphate buffer, a citric acid buffer, an acetic acid buffer, and a disodium hydrogen phosphate-citric acid buffer of 0.01-1 mol/L.

4. A human hair lysate used in the detection of trace drugs according to claim 1 or 2, characterised in that the polyethylene glycol is PEG-200 or PEG-400 or a mixture of both.

5. The use of a human hair lysate for trace drug detection according to claim 1 or 2, wherein the lysate is used to lyse hair, resulting in the release of drug components from hair.

6. Use of a human hair lysate according to claim 5, wherein the hair is a hair having a length of less than 3cm from the hair growth end.

7. Use of a human hair lysate according to claim 5, wherein the hair is human hair.

8. Use of a human hair lysate according to claim 5, in which the lysate is used in an amount of 150 ± 10 μ L per mg of hair.