A kit for detecting psychotropic drugs in hair and its use

Abstract

The present invention provides a kind of kit and its use for detecting psychotropic drugs in hair, the kit includes hair cleanser and hair digester, the hair cleanser includes Tween-20 and TXR-1, and the hair digester includes dithiothreitol, tris (2-carboxyethyl) phosphine hydrochloride and CHAPS. The hair sample is pre-treated with the kit provided by the present invention, and the pollutants attached to the hair sample can be effectively removed, the operating steps are simplified, the accuracy and stability of the test results are improved, and the sample extract can still be accurately detected after being stored for a certain period of time. In addition, the psychotropic drugs in the sample extract are detected in combination with quantum dot immunofluorescence chromatography, and a variety of new psychotropic drugs can be fully extracted and accurately detected, which greatly improves the sensitivity and specificity of the detection method, and is conducive to rapid screening on site.

Description

Technical Field

[0001] The present invention belongs to the technical field of identification, and in particular relates to a kit for detecting psychotropic drugs in hair and a use thereof. Background Art

[0002] The abuse of psychotropic drugs seriously endangers social security and human life and health. After the human body takes such drugs, the drugs and their metabolites will mainly remain in the blood, urine, hair, sweat, feces, nails, etc. The presence of psychotropic drugs in the test sample can reflect whether the user has taken psychotropic drugs. At present, the test samples for drug abuse are often based on urine, saliva, blood, and hair. The psychotropic drugs remaining in hair samples can exist for at least 3 months, which can reflect whether the user of psychotropic drugs has taken psychotropic drugs in the past 3 months or even half a year. In addition, hair samples are easy to collect and preserve, and are currently commonly used samples for testing.

[0003] Hair is rich in keratin, and the ingested drugs are embedded in keratin. However, hair is in contact with the external environment and is easily contaminated by the external environment. In addition, the test subjects often perm and dye their hair, and the hair dyes will also affect the test results. The existing detection technology is prone to false negatives or false positives. Patent document CN112525641B discloses a method for preparing a test solution for psychotropic drugs in hair, which includes multiple steps such as hair washing, softening, and cracking. The formula of the softening solution and the cracking solution involves multiple substances, and the cracking process requires grinding to help cracking, and the steps are complicated. At the same time, the test drug is a traditional psychotropic drug, and the detection effect for new psychotropic drugs is unknown. Patent document CN108844922B discloses a rapid detection method for psychotropic drugs in hair, which uses enzymatic hydrolysis to extract psychotropic drugs in hair, and then combines colloidal gold method and SPR to detect target substances. The enzymatic hydrolysis method has limited digestion effect on hair, which makes the release efficiency of the psychotropic drug substances to be tested in the hair low.

[0004] Therefore, there is an urgent need to develop a simpler, faster and more effective reagent and method for extracting and detecting psychotropic drugs in hair, especially for the detection of some new psychotropic drugs, which can be more accurate and sensitive, while ensuring the stability and repeatability of the detection and extending the storage time of the samples to be tested. Summary of the Invention

[0005] To address the challenges of the existing technology, the present invention provides a kit for detecting psychotropic drugs in hair and its use. By improving the hair cleaning and digestion reagents, the kit allows for the complete extraction of psychotropic drugs from hair in a two-step process, which is then combined with immunochromatographic analysis to detect the test sample. The kit provided by the present invention demonstrates comparable accuracy and stability in the detection of both traditional and novel psychotropic drugs. It also effectively eliminates interference from factors such as hair oil, hair dyes, and external contamination, simplifying the procedure and facilitating practical operation and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a kit for extracting psychotropic drugs from hair, the kit comprising a cleaning agent and a digesting agent, the cleaning agent comprising Tween-20 and TXR-1, and the digesting agent comprising dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and CHAPS.

[0008] Currently, various methods for detecting psychotropic drugs in hair generally involve multiple reagents, steps, and processes. These methods are cumbersome and complex, and their accuracy and sensitivity for processing hair samples and extracting and detecting new psychotropic drugs in hair are limited, resulting in high false positive and false negative rates. Therefore, the present invention makes innovative improvements to hair cleaning and digestion reagents for the extraction and detection of new psychotropic drugs in hair. A hair cleaning agent prepared using a combination of Tween-20 and TXR-1 effectively removes contaminants such as grease and hair dye from the hair surface and, to a certain extent, disrupts the hair's keratin structure, facilitating subsequent digestion and extraction with a digestion agent. A hair digestion agent prepared using a combination of DTT, TCEP, and CHAPS effectively improves the accuracy of test results, reduces the false negative rate, and enhances the sensitivity and stability of the detection method. It can also accurately detect hair samples containing stubborn contaminants such as hair dye and bleach.

[0009] Tween-20, a mild surfactant, primarily removes hydrophilic contaminants (such as sweat, saliva, and environmental dust) without damaging hair keratin structure. TXR-1, a next-generation sustainable detergent with excellent biocompatibility, effectively removes oil, sebaceous gland secretions, and bound contaminants from hair. It also penetrates the hair medulla to release embedded lipid-soluble psychotropic substances, such as synthetic cannabinoids and fentanyl analogs. When these two are used together to clean hair samples, Tween-20 removes hydrophilic contaminants while TXR-1 dissolves deep-seated lipids, achieving a balanced cleaning power and compatibility, making them suitable for most psychotropic drug testing.

[0010] In some embodiments, the present invention verifies the necessity of combining Tween-20 and TXR-1 to prepare a hair cleanser. The results show that when Tween-20 or TXR-1 is used in combination with other surfactants such as SDS or CHAPS, the detection results have a high false negative rate, low method sensitivity, poor stability, and poor cleaning and detection sensitivity for hair dye samples containing toluene-2,5-diamine and resorcinol. The reason may be that only the combined cleanser of Tween-20 and TXR-1 can effectively remove contaminants attached to the hair samples, reduce interference from other factors, significantly promote the subsequent digestion and extraction efficiency, and improve the accuracy of detection.

[0011] Furthermore, the concentration of the Tween-20 is 0.005%-0.01% (v / v), and the concentration of the TXR-1 is 0.01%-0.05% (v / v).

[0012] Furthermore, the concentration of the dithiothreitol is 0.015 g / 100 mL to 2.0 g / 100 mL, the concentration of the tris(2-carboxyethyl)phosphine hydrochloride is 0.025 g / 100 mL to 3.0 g / 100 mL, and the concentration of the CHAPS is 0.03 g / 100 mL to 3.0 g / 100 mL.

[0013] During the digestion of hair samples, dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) act as reducing agents, breaking disulfide bonds in hair keratin, opening up the tight structure of hair fibers and releasing embedded drug molecules. CHAPS, a zwitterionic surfactant, acts simultaneously with its hydrophilic (sulfonic acid) and hydrophobic (cholic acid) ends to dissolve the hair lipid layer and membrane proteins, disrupting the hydrophobic barrier on the hair surface and enhancing the penetration of the reducing agents (DTT and TCEP) into the internal structure. In some embodiments, the present invention verifies the necessity of using a combination of DTT, TCEP, and CHAPS to digest hair samples. The results show that compared with using a single reducing agent or CHAPS, the combination of DTT and CHAPS or TCEP and CHAPS can increase the release of psychotropic drugs in hair samples, but compared with the use of all three combinations, the effect is limited. The use of two reducing agents, DTT and TCEP, combined with CHAPS can synergistically increase drug release, effectively remove interferences, and improve the accuracy and stability of detection. It has the same detection sensitivity for hair samples using toluene-2,5-diamine and resorcinol hair dyes.

[0014] On the other hand, the present invention provides a kit for detecting psychotropic drugs in hair, the kit comprising a cleaning agent, a digestive agent and a test paper, the cleaning agent comprising Tween-20 and TXR-1, and the digestive agent comprising dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride and CHAPS.

[0015] Furthermore, the concentration of the Tween-20 is 0.005%-0.01% (v / v), and the concentration of the TXR-1 is 0.01%-0.05% (v / v).

[0016] Furthermore, the concentration of the dithiothreitol is 0.015 g / 100 mL to 2.0 g / 100 mL, the concentration of the tris(2-carboxyethyl)phosphine hydrochloride is 0.025 g / 100 mL to 3.0 g / 100 mL, and the concentration of the CHAPS is 0.03 g / 100 mL to 3.0 g / 100 mL.

[0017] In another aspect, the present invention provides a method for extracting psychotropic drugs from hair, using the kit as described in any one of the above items to extract psychotropic drugs from hair.

[0018] Furthermore, the method comprises the following steps:

[0019] (1) Hair cleaning: Use a cleaning agent to clean the hair after cutting it into pieces;

[0020] (2) Hair digestion and extraction: The cleaned hair is digested and extracted using a digestive agent for 5-15 minutes to obtain a sample extract.

[0021] The hair cleanser and digestion reagent pretreatment method provided by the present invention does not require physical means such as heating and ultrasound, greatly simplifies the operating steps, and effectively removes pollutants such as oil and hair dye from different hair samples, promotes the release of psychotropic drugs, and improves the accuracy of detection results and the stability of the method.

[0022] In another aspect, a method for determining psychotropic drugs in hair comprises the following steps:

[0023] (1) Extracting psychotropic drugs from hair using the kit described above;

[0024] (2) Detection was performed using immunochromatographic assay.

[0025] In some embodiments, the immunochromatography method in step (2) specifically employs quantum dot immunofluorescence chromatography based on the competitive principle. Quantum dot immunofluorescence chromatography is a rapid detection method that combines the high fluorescence properties of quantum dots (QDs) with immunochromatography technology. Quantum dots are used to label antibodies or antigens, and through chromatography, a test line (T line) and a quality control line (C line) are formed on a test strip. The target psychotropic drug molecule is quantitatively or qualitatively analyzed using the fluorescent signal. Compared to ordinary fluorescent dyes, quantum dots have a wide excitation spectrum and a narrow, symmetrically distributed emission spectrum. Furthermore, quantum dots have high fluorescence intensity, good stability, and enhanced biocompatibility.

[0026] In some embodiments, the principle of competitive immunochromatography involves labeling a certain amount of target drug with quantum dots and immobilizing them on a conjugate pad. The T-line and C-line are sprayed with an antibody (primary antibody) that specifically binds to the target drug and an "anti-antibody" against species-specific IgG, respectively. For negative samples, the quantum dot-labeled antigen flows laterally with the sample, binding to the primary antibody at the T-line and the secondary antibody at the C-line, resulting in fluorescence signals on both the T-line and the C-line. For positive samples, the unlabeled target antigen in the sample and the quantum dot-labeled target antigen on the conjugate pad compete for binding to the primary antibody at the T-line. The more target antigen in the sample, the fewer quantum dots bind to the T-line, resulting in a weaker fluorescence signal. Excess quantum dots bind to the secondary antibody at the C-line, resulting in fluorescence only on the C-line. The test samples are then analyzed separately according to this detection principle.

[0027] Furthermore, the hair sample is hair, sweat hair, armpit hair or pubic hair of a person taking psychotropic drugs.

[0028] Research conducted in the present invention has demonstrated that the hair cleaning reagent and hair digestion reagent provided by the present invention can achieve equally excellent extraction effects when used to treat a variety of samples with keratinized structures, such as human hair, sweat hair, armpit hair, or pubic hair. Furthermore, for hair samples containing stubborn interfering substances such as bleach and hair dye, the cleaning and digestion reagents provided by the present invention can also effectively remove the interfering substances, achieving excellent extraction and detection effects.

[0029] Furthermore, the psychotropic drugs include any one or more of fentanyl analogues, synthetic cannabinoids, synthetic cathinones, etomidate, and GHB.

[0030] In some embodiments, the research results of the present invention demonstrate that the kit and extraction and detection methods provided by the present invention have significantly excellent technical effects for the extraction and detection of new psychotropic drugs in hair, including but not limited to fentanyl analogs, synthetic cannabinoids, synthetic cathinones, etomidate, and GHB. These methods improve the accuracy and stability of psychotropic drug detection, increase detection efficiency in practical applications, and are suitable for rapid screening at sites such as traffic police drug enforcement and hospital diagnosis.

[0031] In another aspect, the present invention provides a use of a composition for preparing a reagent for extracting psychotropic drugs from hair, wherein the composition comprises dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride and CHAPS.

[0032] In another aspect, the present invention provides a use of a composition for preparing a reagent for improving the sensitivity and stability of detecting psychotropic drugs in hair, wherein the composition comprises dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride and CHAPS.

[0033] In another aspect, the present invention provides a use of a composition for preparing a reagent for improving the accuracy of detecting psychotropic drugs in dyed hair, wherein the composition comprises dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride and CHAPS.

[0034] In some embodiments, the present invention has experimentally demonstrated that treating hair samples stained with toluene-2,5-diamine and resorcinol with a digestion reagent prepared with dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, and CHAPS can significantly improve the accuracy and sensitivity of the detection method.

[0035] The present invention has the following beneficial effects:

[0036] 1. The hair cleaning reagent formula has been optimized. The combination of Tween-20 and TXR-1 is used to clean hair samples, effectively removing interfering substances such as oil and hair dye from the hair, thereby improving the accuracy of the test results.

[0037] 2. A combination of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and CHAPS is used as a hair digestion reagent. The synergistic effect of the three substances effectively destroys the hair keratin structure and protein, fully releasing the psychotropic drug molecules in the hair sample. No physical means such as heating and ultrasound are required, which simplifies the operation steps, improves the extraction efficiency and the accuracy of the test results.

[0038] 3. The hair cleanser and digestive agent provided by the present invention are combined with quantum dot immunofluorescence chromatography to detect psychotropic drugs in sample extracts. The advantages of high fluorescence intensity and good stability of quantum dots are utilized to further improve the detection efficiency. It is suitable for rapid screening at the scene of drug enforcement by traffic police and emergency diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the test strip structure, where serial number 1 represents the sample pad, serial number 2 represents the conjugate pad, serial number 3 represents the detection pad, serial number 4 represents the absorbent pad, serial number 5 represents the supporting base, serial number 31 represents the test line (T line), serial number 32 represents the quality control line (C line), and black dots represent marking materials.

[0040] Figure 2 Schematic diagram of the 4PL curve of fentanyl.

[0041] Figure 3 Schematic diagram of the 4PL curve of 5F-ADB. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to embodiments. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0043] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0044] Example 1: Kit and method for determining psychotropic drugs in hair provided by the present invention

[0045] The kit provided in this embodiment includes a cleaning agent, a digestion agent and a test strip.

[0046] 1. Reagent Preparation

[0047] (1) Preparation of cleaning agent

[0048] Take about 80 mL of ultrapure water into a beaker, slowly add 50 μL of TXR-1 (purchased from Croda Pharmaceuticals) while stirring; add 10 μL of Tween-20 (purchased from Sigma) and continue stirring until completely transparent; add more ultrapure water to 100 mL, mix well, and check the pH is 7.0.

[0049] (2) Preparation of digestive agents

[0050] Weigh 0.03-3.0 g CHAPS (preferably 1.0 g in this example, purchased from Sangon Biotech Co., Ltd.) and add it to 80 mL of 50 mM Tris-HCl buffer (pH 7.0). Stir until completely transparent. Weigh 0.025 g-3.0 g TCEP-HCl (preferably 2.5 g in this example, purchased from Sangon Biotech Co., Ltd.) and add it to the above solution, stirring to dissolve. Weigh 0.015-2.0 g DTT (preferably 2.0 g in this example, purchased from Sigma) and add it to the solution, stirring to dissolve in the dark. Add more buffer to 100 mL and mix thoroughly to obtain a hair digester with a DTT concentration of 2.0 g / 100 mL, a TCEP concentration of 2.5 g / 100 mL, and a CHAPS concentration of 1.0 g / 100 mL. Sterilize the solution by filtering through a 0.22 μm filter membrane. Aliquot into light-proof EP tubes and store at -20°C.

[0051] (3) Test strip preparation

[0052] like Figure 1 As shown, the test strip 10 includes a sample pad 1, a conjugation pad 2, a detection pad 3, and a water absorbent pad 4. The sample pad 1, conjugation pad 2, detection pad 3, and water absorbent pad 4 are overlapped with each other in sequence and adhered to a supporting base plate 5.

[0053] Preparation method of absorbent pad 4: Place the filter paper in a 50°C oven to dry for later use.

[0054] Preparation method of conjugate pad 2: For the conjugate pad for fentanyl analog detection, quantum dot-labeled fentanyl antigen analogs (Fentanyl-BSA-QDs) were diluted with coating diluent and sprayed on the glass fiber, and then dried in a 37°C drying oven overnight. For the conjugate pad for synthetic cannabinoid (5F-ADB) detection, quantum dot-labeled 5F-ADB antigen analogs (5F-ADB-OVA-QDs) were diluted with coating diluent and sprayed on the glass fiber, and then dried in a 37°C drying oven overnight.

[0055] Test pad 3 is equipped with test lines and control lines. For fentanyl analogue detection, test line 31 (T line) is coated with anti-fentanyl monoclonal antibody (Anti-Fentanyl mAb), and control line 32 (C line) is coated with goat anti-mouse IgG antibody. For synthetic cannabinoid detection, test line 31 (T line) is coated with anti-5F-ADB monoclonal antibody (Anti-5F-ADB mAb), and control line 32 (C line) is coated with goat anti-rabbit IgG antibody. The test pad is made of nitrocellulose membrane.

[0056] During the test, a sample treated with hair cleansing and hair digestion solutions is dripped onto the sample pad of the test strip. The target antigen fentanyl or 5F-ADB and quantum dot-labeled fentanyl antigen analogs (Fentanyl-BSA-QDs) or 5F-ADB antigen analogs (5F-ADB-OVA-QDs) in the sample flow chromatographically along the nitrocellulose membrane (test pad) toward the absorbent pad. At the test line, the target antigen competes with the antibody specifically binding to the target antigen, forming a "target antigen-target antigen-specific antibody" complex. When passing the control line (line C), the target antigen binds to the coated IgG antibody, forming the final reaction complex for quality control. The fluorescence signal is read using a portable fluorescence reader (Suzhou Hemai Precision Instrument Co., Ltd., model FIC-H2), analyzed, and displayed.

[0057] 2. Hair Pretreatment

[0058] (1) Hair washing

[0059] Take about 5 mg of hair sample, cut the hair to 0.5-1 cm with medical scissors, and then wash the hair 3-5 times alternately with hair cleanser and pure water, vortexing for 3-5 minutes each time.

[0060] (2) Hair digestion

[0061] After step (1), the washed hair was immersed in the hair digester provided by the present invention for 5-10 minutes. The hair:digester ratio was 1 mg:100 μL. The hair was vortexed three times during the digestion period. After the digestion was completed, the hair test solution was obtained.

[0062] 3. Determination of psychotropic drugs in hair

[0063] (1) Establishment of standard curve

[0064] Blank samples (hair from individuals with no history of psychotropic drug use) were pre-treated with the cleaning agent and digestive agent provided in this embodiment to obtain sample extracts. Different concentrations of the psychotropic drug fentanyl and synthetic cannabinoid (5F-ADB) were added to the extracts to prepare eight concentrations of standard solutions (S1-S8), as shown in Table 1:

[0065] Table 1 Standard concentrations of fentanyl and 5F-ADB

[0066]

[0067] The test strips prepared in this example were used in a quantum dot competitive immunochromatographic assay to measure the eight different concentrations of fentanyl and 5F-ADB standard samples listed in Table 1. Each concentration was tested three times, and the mean fluorescence signal was calculated. Using the dilution concentration as the independent variable and the mean fluorescence signal as the dependent variable, a blank sample was used for background matrix calibration. A fitted four-parameter logistic regression (4PL) curve was obtained. The 4PL curve for fentanyl is: ,like Figure 2 ; The 4PL curve of 5F-ADB is: ,like Figure 3 shown.

[0068] (2) Accuracy verification

[0069] According to industry standard methods, 36 positive hair samples at a concentration of 2 ng / mL for fentanyl and 40 positive hair samples at a concentration of 1 ng / mL for synthetic cannabinoids were prepared. 50 mg of hair (negative samples) was collected from 50 individuals with no history of psychotropic drug use, and 5 mg of hair samples were taken from each individual. The specific steps for preparing positive hair samples are as follows:

[0070] S1: A blank hair sample was washed in deionized water, dried at a constant temperature of 25°C, and then reacted in an ethanol solution of 0.5 wt% dimercaptoethanol and sodium sulfite. During the reaction, five samples were taken out every hour for tensile testing until the average elongation at break of the blank hair sample reached ≥20%. The hair sample was then removed, washed with deionized water, and dried at a constant temperature of 60°C to obtain the pretreated hair sample.

[0071] S2: 400 mg of pretreated hair sample was soaked in 250 ml of 10 ppm fentanyl aqueous solution for 10 h, then taken out and dried at a constant temperature of 50°C to obtain the soaked hair sample;

[0072] S3: The soaked hair sample was placed in a 3wt% peracetic acid aqueous solution for reaction. During the reaction, three samples were taken out every 5 minutes for tensile testing until the average elongation at break of the hair sample was ≤5%. The hair sample was then taken out, rinsed with deionized water, and dried at a constant temperature of 50°C to obtain fentanyl-positive hair. After testing, the fentanyl content in the positive hair was 2 ng / mL.

[0073] The preparation steps for a positive hair sample with a synthetic cannabinoid concentration of 1 ng / mL were essentially the same as above. Sample pretreatment and quantum dot competitive immunochromatography were performed using the kit and test strips provided in this example. The control line (C line) and the test line (T line) were observed for clarity and tailing. The test results were statistically analyzed based on the display of the C line and T line, as shown in Table 2.

[0074] Table 2 Hair test results

[0075]

[0076] The test results showed that among the 36 fentanyl-positive hair samples, only one had a clear test line, resulting in a positive detection rate of 97.22%. For the 40 synthetic cannabinoid-positive hair samples, the test lines disappeared, and synthetic cannabinoids were detected in all samples, resulting in a positive detection rate of 100%. No psychotropic drugs were detected in any of the 50 negative hair samples. These test results demonstrate that the method for detecting psychotropic drugs in hair provided by the present invention is highly efficient and accurate for the detection of novel psychotropic drugs such as fentanyl and 5F-ADB. The positive detection rate for 5F-ADB can reach as high as 100%, demonstrating increased accuracy and facilitating applications in psychotropic drug detection and rapid on-site screening.

[0077] (3) Sensitivity verification

[0078] Two batches I and II of the kit for detecting fentanyl analogs and synthetic cannabinoids, 10 samples each, were taken and operated according to the procedures of this example. Fentanyl and 5F-ADB standard S3 were repeatedly tested, with each sample tested three times. The fluorescence signal values ​​(T / C values) were substituted into the above standard curves to determine the concentrations. The results are shown in Table 3.

[0079] Table 3 Sensitivity detection of the kit provided by the present invention

[0080]

[0081] As shown in the results in Table 3, when different batches of the kit of the present invention were used to detect the same fentanyl standard, the detection value was basically consistent with its detection limit (1 ng / ml). When detecting the 5F-ADB standard, the detection value reached its detection limit (0.5 ng / ml), indicating that the detection kit provided by the present invention has high sensitivity, especially when used for 5F-ADB detection.

[0082] (4) Consistency verification

[0083] In order to further verify the high sensitivity and high accuracy of the kit and method provided by the present invention, this example further prepared a fentanyl-positive hair sample with a concentration of 2 ng / ml and a synthetic cannabinoid-positive hair sample with a concentration of 1 ng / ml, using the same preparation steps as those in step 3 (2) of this example. The kit and detection method provided by this example were then used to conduct comparative tests with the liquid chromatography-tandem mass spectrometry method (LC-MS / MS) in the forensic identification technical specification "Liquid chromatography-tandem mass spectrometry method for 15 drugs and metabolites in hair," and the results are shown in Table 4.

[0084] Table 4 Comparison of the kit provided in this example and the mass spectrometry detection results

[0085]

[0086] As can be seen from the results in Table 4, for hair samples from people taking two psychotropic drugs, the detection values ​​of the kit provided by the present invention are basically consistent with the LC-MS / MS detection results of the gold standard, indicating that the kit and detection method provided by the present invention can achieve the same sensitivity and accuracy as LC-MS / MS.

[0087] (5) Repeatability and stability verification

[0088] In practical applications, the reagents or methods for extracting and detecting hair samples often need to have good repeatability and long-term stability to ensure that hair samples containing psychotropic drugs can still be accurately retested after being stored for a certain period of time. Therefore, the present invention also further verifies the stability of the detection method.

[0089] Referring to the preparation steps in step 3 (2) of this embodiment, one fentanyl-positive hair sample with a concentration of 2 ng / ml and one synthetic cannabinoid-positive hair sample with a concentration of 1.5 ng / ml were prepared and tested 20 times using the kit and assay method provided by the present invention. The fluorescent signal value of each test was recorded using a portable fluorescence reader. The corresponding psychotropic drug concentration was calculated based on the standard curve. The mean value M and standard deviation SD of the 20 measurement results were calculated, and the coefficient of variation CV was calculated. The test results are detailed in Table 5.

[0090] Table 5 Results of inter-batch difference test

[0091]

[0092] As shown in Table 5, the kit and method provided by the present invention were used to process and detect hair samples containing different new psychotropic drugs, and the coefficient of variation was within 5%, indicating good stability.

[0093] The kit prepared by the present invention was used to measure the fentanyl-positive hair samples with a concentration of 2 ng / ml and the synthetic cannabinoid-positive hair samples with a concentration of 1.5 ng / ml after being stored for 1, 3, 6, 12, and 24 months, respectively. The results are shown in Table 6.

[0094] Table 6 Stability test results

[0095]

[0096] As shown in the results in Table 6, after the kit provided by the present invention was stored for a certain period of time, the detection value of the same hair sample did not drop significantly, indicating that the detection kit and method provided by the present invention have good long-term stability.

[0097] (6) Specificity verification

[0098] In order to further verify that the kit provided by the present invention can be used to specifically detect hair samples containing fentanyl analogs and synthetic cannabinoids, referring to the positive hair sample preparation steps in step 3 (2) of this embodiment, positive hair samples of fentanyl, synthetic cannabinoids, morphine, ketamine, cocaine, methamphetamine, methcathinone, pseudoephedrine, diazepam, phenobarbital, methadone, tramadol, ranitidine, gatifloxacin and procaine at a concentration of 1 ng / mL were prepared, and the above positive hair samples were treated with the kit provided by this embodiment to obtain sample extracts. The above sample extracts were all tested by test strips containing antibodies to fentanyl and 5F-ADB, and compared with the LC-MS / MS method test results in the forensic identification technical specification "Liquid chromatography-tandem mass spectrometry method for 15 drugs and metabolites in hair". The results are shown in Table 7.

[0099] Table 7 Specificity verification results

[0100]

[0101] As shown in Table 7, the kit provided by the present invention can be used for the specific detection of two types of psychotropic drugs: fentanyl analogs and synthetic cannabinoids. Morphine, ketamine, cocaine, methamphetamine, methcathinone, pseudoephedrine, diazepam, phenobarbital, methadone, tramadol, ranitidine, gatifloxacin, and procaine do not cross-react with this product.

[0102] Example 2: Screening of hair cleansing agents

[0103] In order to verify the necessity of using a combination of Tween-20 and TXR-1 as a cleaning agent to improve the accuracy, sensitivity and stability of psychotropic drug detection in hair, the present invention also evaluated the differences in the effects of several commonly used reagents for hair cleaning.

[0104] 1. Accuracy Verification

[0105] First, referring to the positive hair sample preparation steps in step 3 (2) of Example 1, 40 synthetic cannabinoid-positive hair samples with a concentration of 1.5 ng / mL were prepared. Then, the 40 positive hair samples were cleaned with the five hair cleaning reagents shown in Table 8. The same hair digestion reagent and immunochromatographic method as in Example 1 were used for drug extraction and detection. The results are shown in Table 9.

[0106] Table 8 Formulas of 5 hair cleansers

[0107]

[0108] Table 9 Effects of different cleaning agents on the accuracy of test results

[0109]

[0110] As shown in Table 9, even after washing the same hair sample with different cleaning agents and then using the same digestion, extraction, and testing methods, significant differences in test accuracy were observed. The combination of 0.1% Tween-20 and 0.5% TXR-1 accurately detected all positive samples, achieving a 100% positive detection rate. Washing with either Tween-20 or TXR-1 alone resulted in a high false-negative rate, while combined washing with Tween-20 and CHAPS, or SDS and TXR-1, also resulted in failure to detect all positive samples, resulting in negative results. Similarly, the present invention employed the aforementioned three cleaning agents, followed by digestion and testing, on the hair of individuals who had taken fentanyl analogs, yielding consistent results.

[0111] 2. Sensitivity Verification

[0112] Similarly, referring to the preparation steps of the positive hair samples in step 3 (2) of Example 1, one fentanyl-positive hair sample with a concentration of 2 ng / mL and one synthetic cannabinoid-positive hair sample with a concentration of 1.5 ng / mL were prepared, and each was cleaned with the cleaning agents shown in Table 8. The remaining reagents and steps were the same as the kit and detection method provided in Example 1. At the same time, the same samples were extracted and detected using LC-MS / MS in accordance with the forensic identification technical specification "Liquid chromatography-tandem mass spectrometry test method for 15 drugs and metabolites in hair". The results are shown in Table 10.

[0113] Table 10 Hair test results of different cleaning agents

[0114]

[0115] As shown in Table 10, using either Tween-20 or TXR-1 as a single cleaning agent (cleaners 4 and 5) resulted in significantly lower drug concentrations detected after digestion than those detected by mass spectrometry. Psychotropic drug concentrations detected in hair samples cleaned with cleaning agents 2 or 3 were also significantly lower than those detected by the LC-MS / MS method. Only hair samples treated with cleaning agent 1, prepared with 0.01% Tween-20 and 0.05% TXR-1, achieved consistent or higher values ​​using both the test kit and test strips than those detected by mass spectrometry. This may be due to the fact that the combination of one surfactant and the surfactants in the other two cleaning agents is too mild, failing to effectively disrupt the protein and keratin structure in the hair, or may contain residual contaminants that interfere with subsequent detection, resulting in significantly lower test results and reduced sensitivity.

[0116] 3. Stability Verification

[0117] Furthermore, the present invention evaluated the differences in the stability of the test kits prepared using the five cleaning agents shown in Table 8. After collecting, cleaning, and digesting a hair sample positive for synthetic cannabinoids at a concentration of 1.5 ng / mL using the test kits prepared using the five cleaning agents, the concentration of 5F-ADB in the sample extract was determined by a quantum dot competitive immunochromatographic assay using the test strips prepared in Example 1. The five test kits were then stored for 1, 3, 6, 12, and 24 months, and the 5F-ADB concentration in the same sample was tested again. The measurement results are shown in Table 11.

[0118] Table 11 Comparison of the stability of cleaning agents with different components

[0119]

[0120] The results in Table 11 demonstrate that different cleaning agents significantly affect the extraction and detection of drugs in hair samples. Comparison of the initial drug concentrations under the three cleaning conditions reveals that cleaning agents ②, ④, and ⑤ produced significantly lower 5F-ADB concentrations than the other two. Furthermore, after 1, 3, 6, 12, and 24 months of storage, only the kit prepared with cleaning agent ① showed no significant change in the detection value of the same sample extract, still detecting 5F-ADB levels close to the initial level. However, the kits containing cleaning agents ② through ⑤ all showed a significant decrease in concentration after several months of storage. This may be due to the inability of the active ingredients in the other reagents to fully function after a certain period of storage, or to contamination of the reagents over time, which in turn affects detection accuracy. The combination of Tween-20 and TXR-1 avoids these issues. TXR-1 fully dissolves lipids and membrane proteins, while Tween-20 gently removes contaminants. The synergistic effect of these two agents promotes drug release and reduces contamination during use and storage, improving the long-term stability of the assay.

[0121] 4. Testing of hair dye samples

[0122] In addition, considering that in actual situations, some people who take psychotropic drugs will dye their hair, and bleaching agents, hair dyes, etc. pose great challenges to the full extraction of drugs from the hair and will have a significant impact on the test results, the present invention further compares the differences in the effects of the five cleaning agents shown in Table 8 on the hair samples after dyeing and the hair samples without dyeing. The fentanyl-positive hair samples with a concentration of 2 ng / mL and the synthetic cannabinoid-positive samples with a concentration of 1.5 ng / mL prepared according to the preparation steps of the positive hair samples in step 3 (2) of Example 1 were dyed respectively. The hair dye used a hair dye containing substances such as toluene-2,5-diamine and resorcinol (purchased from Suzhou Shangmei International Cosmetics Co., Ltd., L'Oreal, National Makeup Special No. 20231478) for hair dyeing and color development. The samples after dyeing were subjected to the same pretreatment method and immunochromatographic test as in Example 1, except that the kits prepared with the five cleaning agents shown in Table 8 were used. The hair dye samples were all subjected to and compared with the LC-MS / MS test results in the forensic identification technical specification "Liquid chromatography-tandem mass spectrometry test method for 15 drugs and metabolites in hair", see Table 12.

[0123] Table 12 Detection sensitivity of different cleaning agents for dyed hair samples

[0124]

[0125] As shown in Table 12, only the kit containing detergent ① achieved the same detection level as LC-MS / MS for the extraction and detection of two psychotropic drugs in dyed hair samples. Fentanyl and 5F-ADB concentrations in samples treated with detergents ② through ⑤ were significantly lower than those detected by LC-MS / MS. In particular, the fentanyl levels detected in hair samples from individuals who had taken fentanyl analogs using the kits containing detergents ④ and ⑤ fell below the detection limit, resulting in false negative results. This suggests that different detergent compositions can have significant differences in the effectiveness of hair samples previously exposed to hair dye, significantly affecting the accuracy and sensitivity of the test results. This may be due to the limited effectiveness of a single surfactant for the removal of stubborn contaminants such as hair dye. Furthermore, the combination of Tween-20 and CHAPS in detergent ② and the combination of SDS and TXR-1 in detergent ③ were unable to completely remove the contaminants. Hair dye residues impaired the adequate release of psychotropic drug molecules and the sensitivity of subsequent immunoassays. Only the cleanser ① prepared with Tween-20 and TXR-1 can effectively remove the hair dye from the hair and fully release the drug in the hair sample, achieving the same detection accuracy and sensitivity as the LC-MS / MS method. The treatment and detection effects on hair samples containing 5F-ADB are even better. Therefore, the combination of Tween-20 and TXR-1 is preferred for preparing the hair cleanser.

[0126] Example 3: Screening of hair digestive agents

[0127] 1. Accuracy and Sensitivity Verification

[0128] In hair detection methods for psychotropic drugs, hair washing during pretreatment is crucial for more thorough digestion and extraction. The effectiveness of the hair digestion reagent during the extraction process is crucial. Therefore, the present invention further demonstrates the necessity of using a combination of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and CHAPS as a hair digestion reagent.

[0129] Referring to the preparation steps of the positive hair samples in step 3 (2) of Example 1, fentanyl-positive hair samples with a concentration of 2 ng / mL and synthetic cannabinoid-positive samples with a concentration of 1.5 ng / mL were prepared. The hair pretreatment method and quantum dot competitive immunochromatography test basically the same as in Example 1 were used to detect whether 40 synthetic cannabinoid-positive hair samples contained 5F-ADB. The difference was that the digestants used were the hair digestants shown in Table 13. The test results were then statistically analyzed, as shown in Table 13.

[0130] Table 13 Effects of different digestants on the accuracy of test results

[0131]

[0132] The results showed that when either DTT or TCEP was used alone to digest and extract hair samples, the number of positive samples detected was significantly reduced, with some false-negative results and an increased false-negative rate. However, when CHAPS (a zwitterionic surfactant) was used alone, the positive detection rate was significantly reduced, while the false-negative rate was significantly increased. Combining DTT with TCEP (digestant ④), DTT with CHAPS (digestant ⑤), or TCEP with CHAPS (digestant ⑥) increased the positive detection rate, but false-negative results still occurred. This suggests that combining two reducing agents, or a reducing agent with a surfactant, can improve detection accuracy, but the effect is limited. Comparing the results with digestants ⑦ and ⑧, it was found that the combination of DTT, TCEP, and CHAPS accurately detected synthetic cannabinoids in the hair of individuals who had taken the drug, achieving a 100% positive detection rate, while the combination of SDS, TCEP, and CHAPS still had some false-negative results. Similarly, the accuracy trends of the results obtained using the eight digestants listed in Table 13 for extracting hair samples from individuals who had taken fentanyl analogs were consistent with the above. In summary, only by using the combination of DTT, TCEP and CHAPS to prepare the digestive agent can the detection accuracy of psychotropic drugs in hair be effectively improved and the false negative rate be reduced.

[0133] Furthermore, for one hair sample positive for fentanyl at a concentration of 2 ng / mL and one sample positive for synthetic cannabinoids at a concentration of 1.5 ng / mL, kits prepared with the eight digestive agents shown in Table 13 were used to extract psychotropic drugs from the hair samples. The remaining reagents and steps were the same as the kit and detection method provided in Example 1. The same samples were also tested using the LC-MS / MS method in the forensic identification technical specification "Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of 15 Drugs and Metabolites in Hair". The results are shown in Table 14.

[0134] Table 14 Detection sensitivity of different digestants

[0135]

[0136] As shown in Table 14, the effects of different digestion reagents differed significantly. Compared with the LC-MS / MS results, the results of hair samples treated with digestion reagent ⑦, prepared with 2.0 g / mL DTT, 2.5 g / mL TCEP, and 1 g / mL CHAPS, using the kit and test strips were consistent with the LC-MS / MS values. However, the detection values ​​of a single reducing reagent or surfactant (digestion reagents ① to ③) were significantly lower than the LC-MS / MS values. In particular, the detection results of fentanyl and 5F-ADB in the sample extracts of samples treated with CHAPS alone were all false negatives. Combining DTT with TCEP or DTT / TCEP with CHAPS (digestants 4-6) can help improve detection sensitivity and accuracy, but the effect is limited and still cannot achieve the same sensitivity as LC-MS / MS. Similarly, digestant 8, which combines SDS, TCEP, and CHAPS, also yielded inferior results compared to digestant 7 and LC-MS / MS. This may be because CHAPS disrupts hydrophobic interactions, helping to release drug molecules embedded in the hair medulla. DTT and TCEP reduce disulfide bonds (—S—S—) in keratin, opening the tight structure of the hair fiber and releasing deeply bound psychotropic drugs. Simultaneously, DTT, TCEP, and CHAPS act synergistically, targeting both hydrophilic and hydrophobic regions of the hair, effectively disrupting the keratin structure and promoting drug release. Other digestants cannot achieve the same effect. Digestant 7 showed higher sensitivity for 5F-ADB in the digestion and detection of hair samples from individuals taking two psychotropic drugs. Therefore, only a digestion reagent composed of DTT, TCEP, and CHAPS can effectively improve the sensitivity and accuracy of the detection method.

[0137] 2. Stability Verification and Testing of Hair Dye Samples

[0138] Similarly, the present invention also investigated the long-term stability of the kits prepared with the eight digestive agents in Table 13, as well as the differences in the effects of the kits used for the extraction and detection of hair dye samples. Referring to the preparation steps of the positive hair sample in step 3 (2) of Example 1, fentanyl-positive hair samples with a concentration of 2 ng / mL and synthetic cannabinoid-positive samples with a concentration of 1.5 ng / mL were prepared. The hair pretreatment and immunochromatography methods were basically the same as those in Example 1, wherein the digestive agents used were the eight digestive agents in Table 13, and the LC-MS / MS method of the forensic identification technical specification "Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of 15 Drugs and Metabolites in Hair" was used for extraction and detection.

[0139] In the stability assessment experiment, the same synthetic cannabinoid-positive hair sample was collected, cleaned, and tested using kits prepared with eight digestive agents. The 5F-ADB content in the same sample was then measured again using the eight kits after storage for 1, 3, 6, 12, and 24 months. The results are shown in Table 15.

[0140] In the experiment to evaluate the extraction effect of dyed hair samples, hair dye containing substances such as toluene-2,5-diamine and resorcinol (purchased from Suzhou Shangmei International Cosmetics Co., Ltd., L'Oreal, National Makeup Special No. 20231478) was used to treat fentanyl-positive hair samples and synthetic cannabinoid-positive samples for dyeing. Eight digestive agents were used to clean, digest and test the two positive samples respectively. The results are shown in Table 16.

[0141] Table 15 Effects of different digestants on sample stability

[0142]

[0143] As shown in Table 15, only the kit prepared with a combined digestant of DTT, TCEP, and CHAPS (digestant ⑦) was able to accurately detect psychotropic drug levels in samples after a certain period of storage. When using either component alone (digestants ①–③) or a combination of both (digestants ④–⑥), the initial 5F-ADB content extracted from hair samples was low, resulting in false-negative results for the kits prepared with CHAPS. Furthermore, the detectable 5F-ADB content of all six kits decreased significantly after storage, resulting in false-negative results for positive samples after 12 months. While digestant ⑧ improved drug extraction efficiency, the detectable drug content in samples continued to decrease after a period of storage. This may be due to degradation or volatilization of digestants ①–⑥ and ⑧ over time, or increased contamination with repeated use. The combined digestant of DTT, TCEP, and CHAPS effectively mitigates the volatilization of the reagents themselves, resisting contamination and maintaining accuracy and stability over a certain period of time. After retesting and verifying the hair samples of people who took fentanyl analogs using 8 digestive agents, the results were the same as above, indicating that the digestive agent prepared by the combination of DTT, TCEP and CHAPS can effectively improve the stability of the detection method for new psychotropic drugs.

[0144] Table 16 Detection sensitivity of different digestants for hair dye samples

[0145]

[0146] The test results in Table 16 show significant differences in the results obtained when treating dyed hair samples with different digestants. Only the digestants prepared with DTT, TCEP, and CHAPS demonstrated excellent sensitivity for the extraction and detection of both psychotropic drugs in hair samples, achieving detection accuracy consistent with mass spectrometry. This indicates that the combination of DTT, TCEP, and CHAPS effectively removes contaminants adhering to hair and fully releases the psychotropic drugs from hair. Digestants 1-6 detected significantly lower levels of drugs in dyed hair samples than the mass spectrometry results, indicating that the use of a single reducing agent or surfactant, or a combination of any two, is ineffective in eliminating the effects of residual bleach, hair dye, and other contaminants, resulting in decreased detection sensitivity. Digestant 8 also failed to address the effects of residual hair dye, resulting in lower drug levels. Digestant 7, used to treat dyed hair samples from individuals taking fentanyl analogs and synthetic cannabinoids, achieved detection values ​​close to those detected by mass spectrometry, with the highest sensitivity for 5F-ADB. In summary, the combination of DTT, TCEP and CHAPS is preferably used to prepare a hair digester, which helps to improve the accuracy and sensitivity of drug extraction, detection in hair samples containing pollutants such as hair dyes.

[0147] The present invention initially added a digestant to the cleaned hair sample and placed it in a water bath at 55°C to 65°C for heating and digestion. However, it was found that the detection results of hair samples positive for different psychotropic drugs could not always reach the level consistent with mass spectrometry detection, and were significantly lower than the mass spectrometry results. Therefore, a method without water bath heating was further adopted, and it was found that the detection level was significantly improved. Therefore, in order to verify the necessity of not heating to aid digestion during the pre-treatment of the sample in the kit provided by the present invention, the present invention further prepared 20 synthetic cannabinoid-positive hair samples (with a concentration of 1.5 ng / mL) with reference to the positive hair sample preparation step in Example 1 (2), used the kit provided in Example 1 for pre-treatment, and measured the 5F-ADB content by immunochromatographic test using test strips. The difference was that the digestion process used heated digestion or non-heated digestion, and the heating condition was a water bath at 55°C to 65°C. At the same time, the results were compared with the detection results of the LC-MS / MS method in the forensic identification technical specification "Liquid chromatography-tandem mass spectrometry test method for 15 drugs and metabolites in hair". The results are shown in Table 17.

[0148] Table 17 5F-ADB content under different digestion conditions

[0149]

[0150] The data in Table 17 demonstrate that, for the same sample, the psychotropic drug detection values ​​obtained by digesting hair using the kit provided by the present invention without heating were nearly identical to those detected by mass spectrometry. However, after heating, the drug content detected in the extract was significantly lower than the mass spectrometry value. This is likely due to the excessively intense digestion process after heating, which resulted in drug volatilization and loss in the sample, reducing the detected content. Therefore, only by using a non-heating digestion method can the digestion reagents in the kit function optimally, extracting the maximum drug content and thus improving the accuracy of the detection method.

[0151] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A kit for extracting psychotropic drugs from hair, characterized in that: The kit comprises a cleaning agent and a digestion agent, wherein the cleaning agent comprises Tween-20 and detergent TXR-1, and the digestion agent comprises dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride and surfactant CHAPS.

2. The kit according to claim 1, wherein The concentration of the Tween-20 is 0.005%-0.01% v / v, and the concentration of the detergent TXR-1 is 0.01%-0.05% v / v.

3. The kit according to claim 1, wherein The concentration of the dithiothreitol is 0.015 g / 100 mL to 2.0 g / 100 mL, the concentration of the tris(2-carboxyethyl)phosphine hydrochloride is 0.025 g / 100 mL to 3.0 g / 100 mL, and the concentration of the surfactant CHAPS is 0.03 g / 100 mL to 3.0 g / 100 mL.

4. A kit for detecting psychotropic drugs in hair, characterized in that: The kit comprises a cleaning agent, a digestion agent and a test strip, wherein the cleaning agent comprises Tween-20 and a detergent TXR-1, and the digestion agent comprises dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride and a surfactant CHAPS.

5. A method for extracting psychotropic drugs from hair, characterized in that: Psychotropic drugs in hair are extracted using the kit according to any one of claims 1 to 3.

6. The method according to claim 5, wherein The steps include: (1) Hair cleaning: Use a cleaning agent to clean the hair after cutting it into pieces; (2) Hair digestion and extraction: The cleaned hair is digested and extracted using a digestive agent for 5-15 minutes to obtain a sample extract.

7. A method for determining psychotropic drugs in hair, characterized in that: The steps include: (1) Extracting psychotropic drugs from hair using the kit according to any one of claims 1 to 3; (2) Detection was performed using immunochromatographic assay.

8. The method according to claim 7, wherein The hair sample may be the head hair, sweat hair, armpit hair or pubic hair of a person taking psychotropic drugs.

9. The method according to claim 7, wherein The psychotropic drugs include any one or more of fentanyl, synthetic cannabinoids, synthetic cathinones, etomidate, and GHB.

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