Method for extracting and detecting small molecular substances of medicines in nails or toenails

Abstract

The invention discloses a method for extracting and detecting small molecular substances in fingernails or toenails, which comprises the following steps: collecting fingernail or toenail samples to be detected, and carrying out coarse treatment to obtain sample powder to be detected; mixing to-be-detected sample powder with the extracting solution, and reacting to obtain a to-be-detected solution, or mixing to-be-detected sample powder with an enzyme lysis solution, reacting, and adding a stop solution to obtain the to-be-detected solution; and finally, detecting the to-be-detected solution by adopting the immunochromatography reagent strip. The invention provides a method for performing splitting decomposition extraction and detection by utilizing an extracting solution (chemical formula) or enzyme splitting solution (biological formula) method aiming at the detection of small drug molecules in a nail (toe) sample, and the extracting solution or the enzyme splitting solution can efficiently split keratin and stably preserve split small drug molecule substances and the like. The method provided by the invention is not limited to fingernail (toe) samples, and is widely applicable to various keratosis detection materials, such as hair and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay detection technology, and particularly relates to a method for extracting and detecting a drug small molecule substance in a nail or a toe nail. BACKGROUND

[0002] The nail (toe nail) is divided into nail plate, nail bed, nail fold, nail groove, nail root, nail epithelium, and nail hypodermis. The nail plate is equivalent to the stratum corneum of the skin, and the nail fold is the part of the skin bending into the nail groove. The nail bed is composed of the auxiliary layer, the basal layer and the reticular layer of the dermis. It is directly fused with the periosteum of the phalanx. The posterior nail fold covers the nail root and migrates to the nail epithelium. The nail bed is covered by the nail hypodermis. The nail bed and the nail fold do not participate in the growth of the nail plate. The growth of the nail plate is the proliferation, keratinization and migration of the nail matrix cells at the nail root to the front. However, the nail bed controls the growth of the nail plate in a certain shape, and the damage of the nail bed will cause the abnormal growth of the nail plate. The nail bed and the nail root have rich blood vessels, which provide rich nutrients for the nail regeneration.

[0003] The growth rate of the nail (toe nail) is related to the length of the distal phalanx (the outermost phalanx). Therefore, the nail of the human index finger grows faster than that of the little finger. The growth rate of the nail is four times faster than that of the toe nail. In humans, the average growth rate of the nail is about 3.5 mm per month, which is about half of the growth rate of the toe nail, about 1.6 mm per month. It takes 3 to 6 months for the nail to grow completely, while it takes 12 to 18 months for the toe nail to grow completely. The actual growth rate depends on age, gender, season, exercise level, diet and genetic factors.

[0004] During the growth of the nail (toe nail), substances enter the nail (toe nail) through the diffusion of blood supply. However, unlike hair, the nail (toe nail) grows in two directions: about 80% of the nail (toe nail) is produced in the hair matrix, and the other 20% of the nail (toe nail) is formed on the nail bed, which helps the thickness of the nail (toe nail) to grow from the proximal end to the distal end. The third combined path, similar to hair, is through external contamination by sweat.

[0005] Compared with traditional matrices such as blood and urine, keratinized matrices such as hair and nail have unique advantages such as wide detection window, convenient acquisition of detection materials, non-invasive, convenient storage and transportation, long detection time window, etc. The keratinized matrices can be preserved for a longer time after death, which will help to collect more information about the drug use and disease of the identified person in some forensic scenes (such as the case where blood, urine and other tissues of the identified person cannot be collected).

[0006] The patent CN202010954796.X previously applied by the applicant provides a method for collecting and detecting the abuse of drugs in the nail or toe nail sample, which still has certain limitations, such as not enough mildness of pure chemical reaction, and the detection effect of lysis needs to be further improved. SUMMARY

[0007] The present application aims at the deficiencies of the prior art, and provides a method for detecting drug small molecule substances in nails.

[0008] A method for detecting drug small molecule substances in nails, comprising the following steps:

[0009] Step (1), collecting a nail sample to be tested, and coarsely processing to obtain a sample powder to be tested;

[0010] Step (2), mixing the sample powder to be tested with an extraction solution, and obtaining a test solution after reaction; wherein the formula of the extraction solution is: Tris-HCl 0.05-0.15M, 0.5-1.5g / L of a thiol compound, 2.5-5g / L of urea, 1-5g / L of sodium lauryl polyoxyethylene ether sulfate, and the rest is pure water;

[0011] Or, mixing the sample powder to be tested with an enzyme lysis solution, adding a termination solution after reaction, and obtaining a test solution; wherein the formula of the enzyme lysis solution is: Tris-HCl 0.05-0.1M, 5-10mg / ml of protease, 3-5mg / ml of NaCl, 0.5%-3% of TritonX-100, 0.02% of preservative, and the rest is pure water;

[0012] Step (3), detecting the test solution by using an immunochromatography reagent strip.

[0013] As a preferred, the coarse processing in step (1) comprises: placing the nail sample to be tested in liquid nitrogen for processing, and then grinding, or polishing by a grinding pen, to obtain the sample powder to be tested.

[0014] As a preferred, in step (1), the processing time in liquid nitrogen is 1-3min, and the grinding processing time is 1.5-3min.

[0015] As a preferred, in step (2), the mass of the powder sample to be tested and the volume of the extraction solution are in a ratio of 30-100mg:0.5-1mL.

[0016] As a preferred, in step (2), the mass of the powder sample to be tested, the volume of the enzyme lysis solution, and the volume of the termination solution are in a ratio of 20-100mg:1mL:75-110μL.

[0017] As a preferred, in step (2), the pH of the extraction solution is 8-9.5.

[0018] As a preferred, in step (2), the thiol compound can be selected from one or more of mercaptoacetic acid, mercaptoethanol, DTT (dithiothreitol), and GSH (glutathione); and the combination use has a better lysis effect.

[0019] As preferred, in step (2), the protease in the enzyme lysate can be selected from alkaline protease or keratinase.

[0020] As preferred, in step (2), the preservative in the enzyme lysate can be selected from proclin300 or NaN3.

[0021] As preferred, in step (2), the reaction time of the sample powder in the extraction solution is 10-20 min.

[0022] As preferred, in step (2), after the sample powder is added to the enzyme lysate, it is mixed and placed for 5-15 min, and the reaction is terminated by adding a termination solution.

[0023] As preferred, in step (2), after the sample powder is added to the extraction solution or the enzyme lysate, it can be treated in a water bath for 5-15 min, more preferably 5-10 min; if the enzyme lysate is added and treated in a water bath, it can be selected whether to add a termination solution.

[0024] As preferred, in step (2), the termination solution is prepared by mixing 0.01-0.03 mol / L HCl, 0.02-0.04% (volume fraction) NaN3, and pure water.

[0025] As preferred, in step (2), the water bath temperature is 45-90℃.

[0026] Compared with hair, the matrix of the nail is harder and more complex in composition, so a more severe method (grinding after liquid nitrogen freezing, sulfhydryl compounds with higher protein lysis efficiency, water bath assistance, etc.) is selected in the sample pretreatment process to destroy the cuticle of the nail and release the small molecule drug substances therein. The two sample collection and rough processing methods do not need to be one-to-one corresponding to the sample lysis method.

[0027] Tris-HCl is the basic buffer system in the extraction solution, which provides the basic environment required for nail lysis; sulfhydryl compounds and urea are used to destroy the keratin structure, wherein the sulfhydryl compounds can be selected from one or more of mercaptoacetic acid, mercaptoethanol, DTT, and GSH; mercaptoacetic acid and mercaptoethanol have better lysis effect than their salts (sodium mercaptoacetate); DTT and urea are used as protein denaturants to help destroy the nail structure; the sulfhydryl group on the cysteine of GSH is an active group, which is easy to combine with certain drugs, toxins, etc., so as to bring the small molecule drug substances into the extraction solution and improve the lysis efficiency; sodium lauryl polyoxyethylene ether sulfate is used as a surfactant to reduce the surface tension of the organic matter that is insoluble or difficult to dissolve in water, so as to achieve the purpose of solubilization.

[0028] The enzyme lysis solution also takes Tris-HCl as a basic buffer system, the biological protease has high specificity and catalytic efficiency, can accelerate the hydrolysis reaction of the protein under mild conditions; can retain most of the information in the sample, while reducing non-specific binding and interference.

[0029] The present application has the following beneficial effects:

[0030] The present application provides a method for lysis extraction and detection by using an extraction solution (chemical formula) or an enzyme lysis solution (biological formula) for the detection of drug small molecules in nail samples, the extraction solution or the enzyme lysis solution can efficiently lyse keratin, stably store the lysis drug small molecule substances, etc.; compared with the chemical extraction method, the enzyme lysis reaction is more gentle and efficient, the water bath can improve the lysis efficiency and terminate the reaction, and the termination solution can be added to terminate the reaction, the method can retain most of the information in the sample, while reducing non-specific binding and interference.

[0031] In addition, for the pretreatment process of the nail sample, the present application selects liquid nitrogen freezing and grinding or polishing the pen to obtain a powder sample with finer powder, a thiol compound with higher lysis protein efficiency, and a more violent method of water bath assistance to destroy the cuticle of the nail and release the drug small molecule substances therein, which is a more targeted and efficient extraction and detection method.

[0032] The method of the present application is not limited to nail samples, but can also be widely used in various keratinized materials, such as hair, etc. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with the specific embodiments.

[0034] The following examples use human nails as detection samples to detect nicotine metabolite cotinine in the nails.

[0035] Example 1

[0036] Thirty non-smoking people and twenty smoking people were recruited, and ten finger-shaped nail fragments were obtained from each person using a nail clipper, and were placed in a steel pipe containing stainless steel beads. After the steel pipe was treated with liquid nitrogen for 3 minutes, the treated sample powder was obtained by a frozen grinder for 1.5 minutes.

[0037] 30mg of the treated powder sample was added to 0.5ml of the extraction solution, mixed and stood for 10min to prepare a detection solution; the extraction solution formula is: Tris-HCl 0.1M, mercaptoethanol 0.25g / L, mercaptoacetic acid 0.5g / L, DTT 0.3g / L, urea 3g / L, sodium lauryl polyoxyethylene ether sulfate 1g / L, and the rest is pure water, pH is 9.0.

[0038] The sample to be tested is added to the immunochromatographic test strip, and the timing is started at the same time. Whether the quality control line and the detection line are clear and whether there is tailing is observed. The timing is ended after 5 minutes, and the results are analyzed.

[0039] The detection results are taken as the reference. According to the detection results, the positive coincidence rate, the negative coincidence rate, and the total sample coincidence rate are 95.0%, 100%, and 98.0%, respectively.

[0040] Table 130 nail detection results of 30 non-smoking people and 20 smoking people

[0041]

[0042] Comparative Example 1 (the same nail sample as Example 1, and the difference from Example 1 is that the thiol compound is sodium thioglycolate)

[0043] 30 mg of the treated powder sample is added to 0.5 ml of the extraction solution, mixed and left for 10 minutes to prepare the sample to be tested. The extraction solution formula is: Tris-HCl 0.1M, sodium thioglycolate 0.75g / L, DTT 0.3g / L, urea 3g / L, sodium lauryl polyoxyethylene ether sulfate 1g / L, and the rest is pure water, with a pH of 9.0.

[0044] The sample to be tested is added to the immunochromatographic test strip, and the timing is started at the same time. Whether the quality control line and the detection line are clear and whether there is tailing is observed. The timing is ended after 5 minutes, and the results are analyzed.

[0045] The detection results are taken as the reference. According to the detection results, the positive coincidence rate, the negative coincidence rate, and the total sample coincidence rate are 85.0%, 100%, and 94.0%, respectively.

[0046] Table 230 nail detection results of 30 non-smoking people and 20 smoking people

[0047]

[0048] Example 2: GSH is combined with other substances in the extraction solution containing thiol compounds

[0049] 30 mg of the treated powder sample is added to 0.5 ml of the extraction solution, mixed and left for 10 minutes to prepare the sample to be tested. The extraction solution formula is: Tris-HCl 0.1M, mercaptoethanol 0.25g / L, mercaptoacetic acid 0.5g / L, DTT 0.3g / L, GSH 0.5g / L, urea 3g / L, sodium lauryl polyoxyethylene ether sulfate 1g / L, and the rest is pure water, with a pH of 9.0.

[0050] The sample to be tested liquid is added to the immunochromatography test strip, and the timing starts at the same time. Whether the quality control line and the detection line are clear or not, and whether there is tailing or not, the timing is ended at 5 minutes, and the results are analyzed.

[0051] The GC / MS detection results are taken as the reference. From the detection results, the positive coincidence rate, the negative coincidence rate, and the total sample coincidence rate are 100.0%, 100%, and 100.0%, respectively.

[0052] Table 3: 30 non-smoking people and 20 smoking people nail detection results

[0053]

[0054]

[0055] Example 3: Sampling method using a grinding pen

[0056] 30 non-smoking people and 20 smoking people are recruited, and a grinding dust collection pen with a storage cavity is used to collect samples from their nails. The grinding head grinds the nails, and the dust collection cavity absorbs the dust at the same time. The dust enters the storage cavity, and the sample powder to be tested is collected from the storage cavity.

[0057] 30 mg of the treated powder sample is added to 0.5 ml of the extraction solution, mixed and placed for 10 minutes to prepare the sample to be tested. The extraction solution formula is: Tris-HCl 0.1M, mercaptoethanol 0.25g / L, mercaptoacetic acid 0.5g / L, DTT 0.3g / L, urea 3g / L, sodium lauryl polyoxyethylene ether sulfate 1g / L, and the rest is pure water, pH 9.0.

[0058] The sample to be tested liquid is added to the immunochromatography test strip, and the timing starts at the same time. Whether the quality control line and the detection line are clear or not, and whether there is tailing or not, the timing is ended at 5 minutes, and the results are analyzed.

[0059] The GC / MS detection results are taken as the reference. From the detection results, the positive coincidence rate, the negative coincidence rate, and the total sample coincidence rate are 95.0%, 100%, and 98.0%, respectively.

[0060] Table 4: 30 non-smoking people and 20 smoking people nail detection results

[0061]

[0062] Example 4: Lysis method using enzyme lysis solution

[0063] Take 30 mg of the treated powder sample and add it to 1 ml of enzyme lysate, mix well, stand for 10 min, then add 80 μL of termination solution to prepare the sample to be tested; the enzyme lysate formula is: Tris-HCl 0.1 M, keratinase 10 mg / ml, NaCl 3 mg / ml, Triton X-100 1.5%, preservative 0.02%, the rest is pure water, pH is 9.0; the termination solution formula is HCl 0.01-0.03 mol / L, NaN3 0.02-0.04%, the rest is pure water.

[0064] Add the sample to be tested to the immunochromatography test strip, start timing at the same time, observe whether the quality control line and the detection line are clear and whether there is tailing, and end timing at 5 min and analyze the results.

[0065] Take the GC / MS detection results as the reference. From the detection results: the positive coincidence rate, the negative coincidence rate, and the total sample coincidence rate are 95.0%, 100%, and 98.0%, respectively.

[0066] Table 5 Nail detection results of 30 non-smoking people and 20 smoking people

[0067]

[0068] Comparative Example 2 (the same nail sample as in Example 1, different from Example 2 in extraction time and temperature)

[0069] Take 30 mg of the treated powder sample and add it to 0.5 ml of extraction solution, mix well, stand for 3, 5, 8, 10, and 15 min to prepare samples to be tested 1, 2, 3, 4, and 5; the extraction solution formula is: Tris-HCl 0.1 M, mercaptoethanol 0.25 g / L, mercaptoacetic acid 0.5 g / L, DTT 0.3 g / L, GSH 0.5 g / L, urea 3 g / L, sodium lauryl polyoxyethylene ether sulfate 1 g / L, the rest is pure water, pH is 9.0.

[0070] Take 30 mg of the treated powder sample and add it to 0.5 ml of extraction solution, mix well, stand for 3, 5, 8, 10, and 15 min to prepare samples to be tested 1, 2, 3, 4, and 5; the extraction solution formula is: Tris-HCl 0.1 M, mercaptoethanol 0.25 g / L, mercaptoacetic acid 0.5 g / L, DTT 0.3 g / L, GSH 0.5 g / L, urea 3 g / L, sodium lauryl polyoxyethylene ether sulfate 1 g / L, the rest is pure water, pH is 9.0.

[0071] Add the sample to be tested to the immunochromatography test strip, start timing at the same time, observe whether the quality control line and the detection line are clear and whether there is tailing, and end timing at 5 min and analyze the results.

[0072] The results of GC / MS detection were used as a reference. The results showed that water bath heating can promote the release of small molecules, shorten the cracking period and accelerate the experimental process.

[0073] Table 630 nail detection results of 60 non-smoking people and 20 smokers

[0074]

[0075]

Claims

1. A method for extracting and detecting a small molecule drug substance in a nail, characterized by, The extraction detection method comprises the following steps: Step (1), collecting the nail sample to be tested, and coarsely processing to obtain a sample powder to be tested; Step (2), mixing the sample powder to be tested with an extraction solution, and obtaining a test solution after reaction; wherein the formula of the extraction solution is: Tris-HCl 0.05-0.15M, 0.5-1.5g / L of a thiol compound, 2.5-5g / L of urea, 1-5g / L of sodium lauryl polyoxyethylene ether sulfate, and the rest is pure water; Step (3), detecting the test solution by using an immunochromatography reagent strip.

2. The method for extracting and detecting a drug small molecule substance in a nail according to claim 1, characterized in that, Step (1) comprises: placing the nail sample to be tested in liquid nitrogen for processing and then grinding, or polishing by using a grinding pen, to obtain the sample powder to be tested.

3. The method according to claim 1, wherein the method is characterized by, In step (2), the mass of the powder sample to be tested and the volume of the extraction solution are in a ratio of 30-100mg: 0.5-1mL.

4. The method for extracting and detecting a drug small molecule substance in a nail according to claim 1, characterized in that, In step (2), the thiol compound includes one or more of mercaptoacetic acid, mercaptoethanol, dithiothreitol, and glutathione.

5. The method of claim 1, wherein the method is characterized by, Step (2) can also be: mixing the sample powder to be tested with an enzyme lysis solution, adding a termination solution after reaction, and obtaining a test solution; wherein the formula of the enzyme lysis solution is: Tris-HCl 0.05-0.1M, 5-10mg / ml of protease, 3-5mg / ml of NaCl, 0.5%-3% of Triton X-100 by volume fraction, 0.02% of preservative by volume fraction, and the rest is pure water.

6. The method for extracting and detecting a drug small molecule substance in a nail according to claim 5, characterized in that, In step (2), the mass of the powder sample to be tested, the volume of the enzyme lysis solution, and the volume of the termination solution are in a ratio of 20-100mg: 1mL: 75-110μL.

7. The method according to claim 5, wherein the drug small molecule substance is extracted from the nail. In step (2), the protease in the enzyme lysis solution is alkaline protease or keratinase.

8. The method according to claim 5, wherein the drug small molecule substance is extracted from the nail. In step (2), the formula of the termination solution is: 0.01-0.03mol / L of HCl, 0.02-0.04% of NaN3 by volume fraction, and the rest is pure water.

9. The method according to claim 5, wherein the drug small molecule substance is extracted from the nail. In step (2), the reaction time of the sample powder to be tested in the extraction solution is 10-20min; and the reaction time of the sample powder to be tested after being added into the enzyme lysis solution is 5-15min.

10. The method according to claim 1 or 5, wherein In step (2), water bath heating is performed for 5-15min during the reaction.

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