Method for detecting multiple drugs in hair

Abstract

The invention discloses a method for detecting multiple drugs in hair, which comprises the following steps: S1, preparing a drug stock solution by using methanol, respectively diluting the drug stock solution into intermediate solutions with different concentrations, and respectively adding quantitative intermediate solutions into an equal amount of blank hair extracting solution to obtain labeled hair extracting solutions with different concentration grades; s2, performing mass spectrometric detection after solid-phase extraction, and performing linear regression on the standard concentration by using the signal intensity of a quantitative ion mass spectrum peak to construct a standard curve; s3, taking to-be-detected hair, cutting the to-be-detected hair into pieces, adding the to-be-detected hair pieces into the mixed solution, grinding the to-be-detected hair pieces into powder to obtain dispersion liquid, standing the dispersion liquid, taking supernate, and filtering the supernate to obtain a detected material hair extracting solution; s4, performing solid-phase extraction on the detected hair extracting solution, collecting eluent, and performing mass spectrometric detection to obtain quantitative ion peak signal intensity of the drug analyte; and S5, comparing with a standard curve, and calculating the concentration of the drug in the hair sample. According to the invention, on-site rapid screening is realized, and relatively high detection accuracy and reliability are also realized.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology in biological matrices. Specifically, it relates to a method for detecting multiple drugs in hair. Background Technology

[0002] In recent years, new psychoactive substances have emerged in an endless stream, posing a huge threat to human health and social stability. Up-to-date drug detection methods are essential for combating the proliferation of new drugs and psychoactive substances, and the demand for rapid on-site screening of drug users is also increasing.

[0003] However, existing drug detection methods based on biological fluids such as urine and blood largely rely on laboratory analysis, which has limitations such as complex pretreatment, long detection cycles, and inability to operate outside of fixed laboratories. These methods struggle to meet the urgent needs for timeliness and convenience in on-site operations. This contradiction is particularly pronounced in trace drug detection, where complex biological matrices severely interfere with the accuracy and reliability of the detection. Therefore, developing a detection technology that can be used in the field environment to achieve rapid and accurate screening of multiple drugs in biological fluids has become an urgent need in drug control practice. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for detecting multiple drugs in hair, which is simple to operate and enables rapid on-site screening, while also achieving relatively high detection accuracy and reliability.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] A method for detecting multiple drugs in hair includes the following steps:

[0007] S1. After preparing the drug stock solution with methanol, the drug stock solution is then diluted with methanol to obtain intermediate solutions of different concentrations. A certain amount of the drug stock solution is added to an equal amount of blank hair extract to obtain spiked hair extracts of different concentration levels.

[0008] S2. Hair extracts with different concentration levels were subjected to solid-phase extraction and then mass spectrometry detection. The signal intensity of the quantitative ion mass spectrometry peak was used to perform linear regression on the spike concentration to construct a standard curve.

[0009] S3. The hair sample to be tested is shredded and added to a mixture of methanol and water. It is then ground into a powder to obtain a dispersion. After the dispersion has settled, the supernatant is collected and filtered through an organic microporous membrane to obtain the hair extract.

[0010] S4. Solid-phase extraction is performed on the hair extract of the sample. The solid-phase extraction uses a mixed solution of ammonia and methanol as the eluent. After collecting the eluent, mass spectrometry is performed to obtain the signal intensity of the quantitative ion mass spectrometry peak of the drug analyte.

[0011] S5. Compare the signal intensity of the quantitative ion mass spectrometry peak of the drug analyte with the standard curve to calculate the concentration of the drug in the hair sample to be tested.

[0012] This application describes a process where hair is shredded and ground into powder in a methanol-water mixture to obtain a dispersion. After standing, the dispersion is filtered through an organic microporous membrane and eluted using a mixture of ammonia and methanol (5:95 volume ratio) as the eluent and MCX as the packing material for solid-phase extraction, achieving efficient recovery of the target analyte. These purification steps effectively remove interference from complex matrices such as lipids and proteins in the hair, significantly reducing the ion inhibition effect that may occur during ionization, improving ionization efficiency, and thus enhancing the mass spectrometry signal intensity. This allows for preliminary quantification without the need for internal standards, enabling rapid preliminary screening of hair samples while achieving relatively high accuracy and reliability in detecting multiple drugs in hair. Furthermore, the synergistic effect of these processing steps fundamentally solves the technical challenge of efficient release and purification of drug molecules from the dense hair keratin matrix, laying the foundation for subsequent efficient ionization.

[0013] First, wet milling significantly increases the sample's specific surface area, thoroughly disrupting the hair structure and allowing embedded drug molecules to dissolve fully—that is, efficiently extracting them into the solvent—thus significantly increasing the number of ions in the final analyte. Furthermore, wet milling prevents degradation of the target analyte due to overheating. Second, subsequent filtration, using MCX as a packing material, and solid-phase extraction elution with a mixture of ammonia and methanol selectively enrich the target drug, optimizing drug recovery. Finally, when the purified and enriched analyte enters the mass spectrometer, its ionization efficiency is greatly enhanced due to matrix purification, resulting in a stronger and more stable mass spectrometry signal. Together, these techniques enable rapid on-site screening while achieving relatively high accuracy and reliability in detecting multiple drugs in hair.

[0014] In the above-mentioned method for detecting multiple drugs in hair, in step S1, the concentration of the drug stock solution for methamphetamine, 3,4-methylenedioxymethamphetamine, ketamine, norketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one is 5 mg·mL. -1 Mixed storage solution;

[0015] In S1, the preparation method of blank hair extract is as follows: the blank hair that tested negative is washed and dried with distilled water and methanol in sequence to remove some contaminants on the surface of the hair. The blank hair is then cut into small pieces and added to a mixed solution of methanol and water. The mixture is then ground into powder to obtain a dispersion. After the dispersion is allowed to stand, the supernatant is taken and filtered through an organic microporous membrane to obtain the blank hair extract.

[0016] In the above-mentioned method for detecting multiple drugs in hair, in step S3, the volume ratio of methanol to water in the mixed solution is 1:1, which can efficiently extract multiple target substances that are hydrophilic, lipophilic, and amphoteric.

[0017] In the above-mentioned method for detecting multiple drugs in hair, in step S3, the hair to be tested is washed sequentially with distilled water and methanol before being dried, and then cut into pieces.

[0018] In the above-described method for detecting multiple drugs in hair, in steps S2 and S5, the signal intensity of the quantitative ion mass spectrometry peak is the average of multiple consecutive signal intensities of the corresponding quantitative ion mass spectrometry peak. Preferably, the signal intensity is the average of six consecutive signal intensities of the corresponding ion mass spectrometry peak to reduce error.

[0019] In the above-mentioned method for detecting multiple drugs in hair, in step S4, mass spectrometry analysis revealed the following quantitative ions: methamphetamine (m / z 119), 3,4-methylenedioxymethamphetamine (m / z 163), ketamine (m / z 207), norketamine (m / z 207), etomidate (m / z 141), isopramidine (m / z 155), and 2-(ethylamino)-2-phenylcyclohexane-1-one (m / z 173).

[0020] In the above-mentioned method for detecting multiple drugs in hair, S4, solid-phase extraction is performed using a pipette equipped with solid-phase extraction packing material. The extraction method using a pipette is as follows: the pipette is immersed in the hair extract solution, and aspiration and discharge are repeated several times; then it is rinsed with methanol, and then eluted repeatedly with a mixed solution containing ammonia and methanol as the eluent. The supernatant is then used for detection.

[0021] The aforementioned method for detecting multiple drugs in hair utilizes MCX solid-phase extraction packing material in the pipette tip. MCX is a mixed-mode cation exchange packing material with dual retention properties of reverse-phase and sulfonic acid group cation exchange, achieving dual retention of drug molecules in the hair extract and resulting in a high extraction recovery rate, thus facilitating excellent purification effects. A mixed solution of ammonia and methanol is used as the eluent, with a volume ratio of ammonia to methanol of 5:95 and an ammonia concentration of 25-28 wt%. Methanol effectively disrupts the reverse-phase adsorption between the polymer backbone of the packing material and the target analyte. The use of an alkaline solution containing ammonia is to utilize its high pH value to neutralize the charge of the alkaline analyte and to utilize ammonium ions as competing ions, thereby efficiently, cleanly, and selectively disrupting cation exchange and achieving efficient recovery of the target analyte.

[0022] The above-mentioned method for detecting multiple drugs in hair uses a pipette extraction method as follows: a pipette with solid phase extraction packing is immersed in the hair extraction, and aspiration and discharge are repeated at least 9 times, with each aspiration / discharge time being 8-12 seconds; then it is rinsed with methanol, and then a mixed solution of ammonia and methanol is used as the elution solution, and the elution is repeated 2-5 times, with each elution time being 8-12 seconds; finally, the supernatant is used for detection.

[0023] In the above-mentioned method for detecting multiple drugs in hair, S4 uses a miniature mass spectrometer for mass spectrometry detection, which is easy to carry.

[0024] The above-mentioned method for detecting multiple drugs in hair uses paper spray ionization injection for mass spectrometry.

[0025] In the above-mentioned method for detecting multiple drugs in hair, the spray voltage for ionization injection is 4200 V for methamphetamine, 4200 V for 3,4-methylenedioxymethamphetamine, 4100 V for ketamine, 4100 V for norketamine, 4400 V for etomidate, 4400 V for isopramidine, and 4100 V for 2-(ethylamino)-2-phenylcyclohexane-1-one.

[0026] In the above-mentioned method for detecting multiple drugs in hair, the ISO1 energy for methamphetamine is 7V, the ISO1 energy for 3,4-methylenedioxymethamphetamine is 7V, the ISO1 energy for ketamine is 8V, the ISO1 energy for norketamine is 10V, the ISO1 energy for etomidate is 9V, the ISO1 energy for isopramidine is 9V, and the ISO1 energy for 2-(ethylamino)-2-phenylcyclohexane-1-one is 10V.

[0027] The ISO2 energy of methamphetamine is 2.5 V, the ISO2 energy of 3,4-methylenedioxymethamphetamine is 3 V, the ISO2 energy of ketamine is 3.5 V, the ISO2 energy of norketamine is 3.5 V, the ISO2 energy of etomidate is 3 V, the ISO2 energy of isopramipex is 4 V, and the ISO2 energy of 2-(ethylamino)-2-phenylcyclohexyl-1-one is 4 V.

[0028] The CID energy of methamphetamine is 1 V, the CID energy of 3,4-methylenedioxymethamphetamine is 3.75 V, the CID energy of ketamine is 1.75 V, the CID energy of norketamine is 2.25 V, the CID energy of etomidate is 1.25 V, the CID energy of isopramipexole is 1.25 V, and the CID energy of 2-(ethylamino)-2-phenylcyclohexyl-1-one is 1.5 V.

[0029] The technical solution of the present invention achieves the following beneficial technical effects:

[0030] I. This application obtains a dispersion by grinding shredded hair into powder in a methanol-water mixture. After the dispersion is allowed to stand, it is filtered through an organic microporous membrane and eluted using a mixture of ammonia and methanol (volume ratio of ammonia to methanol 5:95) as the eluent and MCX as the packing material for solid-phase extraction, achieving efficient recovery of the target analyte. These purification steps effectively remove interference from complex matrices such as lipids and proteins in the hair, significantly reducing the ion inhibition effect that may occur during ionization, improving ionization efficiency, and thus enhancing the mass spectrometry signal intensity. This allows for preliminary quantification without the need for internal standards, enabling rapid preliminary screening of hair samples while achieving relatively high accuracy and reliability in detecting multiple drugs in hair. Furthermore, the synergistic effect of these processing steps fundamentally solves the technical challenge of efficient release and purification of drug molecules from the dense hair keratin matrix, laying the foundation for subsequent efficient ionization.

[0031] II. This application utilizes wet grinding to significantly increase the specific surface area of ​​the sample, thoroughly disrupting the hair structure and allowing embedded drug molecules to fully dissolve, i.e., efficiently extracting them into the solvent, thereby significantly increasing the number of ions in the final analyte. Furthermore, wet grinding prevents the target analyte from degrading due to overheating. Subsequent filtration, using MCX as a packing material, and solid-phase extraction elution with a mixed solution of ammonia and methanol selectively enrich the target drug, optimizing drug recovery. Finally, when the purified and enriched analyte enters the mass spectrometer, its ionization efficiency is greatly enhanced due to the purified matrix, resulting in a stronger and more stable mass spectrometry signal. Ultimately, this approach achieves rapid on-site screening while maintaining relatively high accuracy and reliability in detecting multiple drugs in hair.

[0032] Third, this invention combines pretreatment to significantly increase the signal value of the target analyte with solid-phase extraction elution, optimized detection parameters of a micro mass spectrometer, and paper spray ionization technology. This enables a complete analysis process from hair sample to quantitative result within 5 minutes. While ensuring high transmission and ionization efficiency, it can detect multiple drugs in hair in a single step, including methamphetamine, 3,4-methylenedioxymethamphetamine, ketamine, norketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one. This allows for rapid and continuous detection and screening of multiple drugs, as well as quantitative concentration detection of drugs. It meets the stringent timeliness requirements of rapid on-site detection while relatively ensuring accuracy, reliability, and stability, playing an important role in the investigation and crackdown on drug crimes.

[0033] IV. This method achieves high specificity for the target analyte, effectively eliminating interference from other substances. The target analyte exhibits a good linear relationship with the detection signal over a wide concentration range, and this linear relationship demonstrates high stability. The LOQ of this method for drugs in hair is ≥ 5 ng·mL. -1 LOD ≥ 1 ng·mL -1 Each drug ranges from 5 to 500 ng / mL −1 Within the linear range, the linear relationship is good (r 2 (≥0.99), with a recovery rate of 85%-115% and RSD<15%. Attached Figure Description

[0034] Figure 1 A shows the mass spectra of seven drugs in MS mode; Figure 1 B is methamphetamine. Figure 1 E represents norketamine. Figure 1 F represents the MS of etomidate. 2 picture;

[0035] Figure 2 C is 3,4-methylenedioxymethylamphetamine. Figure 2 D represents ketamine. Figure 2 G represents isopramipexyl ester. Figure 2 H represents the MS concentration of 2-(ethylamino)-2-phenylcyclohexane-1-one. 2 picture;

[0036] Figure 3 A represents 50 ng / mL of solid-phase extraction solution from Example 1. -1 Injection signal diagram of methamphetamine hair sample in 4-paper spray ionization mode. Figure 3 B represents 50 ng / mL of Comparative Example 1 without solid-phase extraction. -1 A graph showing the injection signal of a methamphetamine hair sample in paper spray ionization mode. Detailed Implementation

[0037] Drugs tested: methamphetamine, 3,4-methylenedioxymethamphetamine, ketamine, norketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one.

[0038] Materials used: Blank hair was provided by 10 adult volunteers who were in good health, had no history of drug use, and had no abnormal laboratory test results. The blank hair was mixed and stored at room temperature.

[0039] A mass spectrometry method for the detection of multiple drugs in hair by solid-phase extraction, comprising the following steps:

[0040] S1. Preparation of standard solutions:

[0041] The concentrations of methamphetamine, 3,4-methylenedioxymethamphetamine, ketamine, norketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one were prepared in methanol at 5 mg·mL⁻¹. -1 The mixed stock solution is stored at -20°C.

[0042] The mixed stock solution was diluted to concentrations of 0.5, 1, 2.5, 5, 10, 25, and 100 μg / mL, respectively. -1 The intermediate solution was then added in batches of 990 μL to prepare seven concentration levels (5, 10, 25, 50, 100, 250, and 500 ng·mL). -1 Spiked hair extract was used as a calibration solution.

[0043] The preparation method of blank hair extract is as follows: After washing and drying the blank hair sample that tested negative with distilled water and methanol in sequence, 20 mg of blank hair was cut into small pieces and added to a mixed solution of methanol and water in 1 mL. The mixture was then ground into a powdered dispersion. After the dispersion was allowed to stand, the supernatant was collected and filtered through an organic microporous membrane to obtain the blank hair extract.

[0044] Solutions containing 8, 20, 200, and 400 ng·mL were prepared using the same method as the calibration solutions. -1 Hair extract standards for various drugs were used as quality control samples for method validation. Calibration solutions can also be used as quality control samples for method validation.

[0045] S2. Constructing the standard curve:

[0046] After solid-phase extraction of spiked hair extracts with different spiking concentrations, the hair extracts of seven analytes at different concentrations were detected by mass spectrometry. A standard curve was constructed by linear regression of the spiking concentration with the average value of the mass spectrum signal intensity of six consecutive quantitative ion mass spectrometry peaks.

[0047] S3. Sample processing:

[0048] The hair sample to be tested was first washed and dried with distilled water and methanol in sequence, then cut into 1 mm pieces. 20 mg of the hair sample was weighed and added to 1 mL of a mixed solution of methanol and water (the volume ratio of methanol to water in the mixed solution was 1:1). The hair sample was ground into powder to obtain a dispersion. After the dispersion was allowed to stand for 2 min, the supernatant was taken and filtered through an organic microporous membrane to obtain the hair extract of the sample.

[0049] S4. Sample extraction and detection:

[0050] The hair extract was subjected to solid-phase extraction and then detected by mass spectrometry. The average signal intensity of six consecutive quantitative ion mass spectrometry peaks of the drug analyte was used as the detection intensity.

[0051] Solid-phase extraction uses a pipette with solid-phase extraction packing material for extraction, which is more effective in removing impurities from hair. The interference of the hair matrix in the extracted solution is greatly reduced, which significantly reduces the ion suppression effect that may occur on the target during ionization, resulting in a significant increase in the signal value of the target.

[0052] The pipette tip can be filled with C18, HLB, or MCX packing material for solid-phase extraction. In this embodiment, an MCX pipette tip is used for solid-phase extraction, with a mixed solution of ammonia and methanol (ammonia to methanol volume ratio of 5:95, ammonia concentration of 28 wt%) as the eluent, resulting in a high extraction recovery rate. The solid-phase extraction process is as follows:

[0053] Connect the MCX pipette tip to the pipette and immerse the tip in 1 mL of hair extract sample. Repeat aspiration and expulsion 9 times, each aspiration / expulsion lasting 10 seconds. Rinse the loaded pipette tip with 300 μL of methanol to remove impurities. Then, use 100 μL of a mixture of 5% ammonia and methanol (5% ammonia and 95% methanol) as the eluent to elute the drug from the hair extract 3 times, each eluent lasting 10 seconds. The eluent is used for detection, requiring a total of 3 minutes. This procedure results in a stronger drug signal in subsequent detection processes, ensuring the recovery rate of the target compound.

[0054] The sample introduction method is paper spray ionization. Paper spray ionization is less susceptible to ion suppression by the matrix and can be used for the detection of samples with complex matrices.

[0055] The specific procedure is as follows: Use a pipette to draw 10 μL of the extracted sample and add it to the inlet at the front of the paper spray test kit of the miniature mass spectrometer. Wait about 2 minutes for it to dry, then add 50 μL of methanol to the back of the paper spray test kit, and it is ready for detection. Under high pressure, the sample that has been initially separated on the paper chromatography is sprayed out along the capillary and ionized at the tip of the capillary.

[0056] S5. Quantitative analysis of samples:

[0057] The concentrations of seven drugs in hair samples were calculated by comparing the average signal intensity of six consecutive quantitative ion mass spectrometry peaks of the drug analytes with the standard curve.

[0058] Use Microsoft Excel 2019 to calculate the peak area, analytical mean, standard deviation, and significance level of the injected mass spectrometer bees.

[0059] In this application, multiple injection sequences are set for paper spray ionization injection, with different energies in each sequence. The optimal spray voltage, ISO1 and ISO2, and CID for different drugs are shown in Table 1.

[0060] Table 1. Mass spectrometry detection parameters for different drugs

[0061]

[0062] The hair standard solution was analyzed by mass spectrometry according to the method in Example 1, and the seven drugs in the hair matrix were obtained by MS analysis. 2 Image, as shown Figure 1 As shown.

[0063] Figure 1 In this study, the concentrations of methamphetamine, 3,4-methylenedioxymethamphetamine, norketamine, ketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one were all 100 ng·mL. -1 .

[0064] Figure 1 A is the mass spectrum in MS mode. For trace detection, MS... 1 The diagram is not very useful for reference. Figure 1 A, which does not show the parent ion of the target compound, can serve as a marker for matrix identification.

[0065] MS of methamphetamine 2 As shown in the picture Figure 1As shown in Figure B, after ionization, a precursor ion is formed at m / z 150. Following fragmentation, several fragment ion peaks are generated, with noticeable responses observed at m / z 119 and m / z 91. Other observable responses are also observed at m / z 91, m / z 93, m / z 107, and m / z 133. Among these, the fragment ion at m / z 119 is the methamphetamine precursor ion [M+H]. + The ion at m / z 91 is formed by the removal of a methylamine ion, while the ion at m / z 91 is a methamphetamine ion resulting from β-CC fragmentation. Other peaks appearing in the mass spectrum are suspected to be impurity peaks, possibly originating from impurities with the same mass number (m / z 150) in the hair that have been fragmented.

[0066] MS of 3,4-methylenedioxymethylamphetamine 2 As shown in the picture Figure 2 As shown in Figure C, the ion peak (m / z 194) fragments to produce fragments at m / z 163, m / z 135, m / z 133, m / z 105, m / z 152, and m / z 175. Fragmentation pathway analysis indicates that ions m / z 163, m / z 133, and m / z 135 originate from the fragmentation of 3,4-methylenedioxymethylamphetamine, with m / z 163 derived from the 3,4-methylenedioxymethylamphetamine parent ion [M+H]. + Formed after the removal of one methylamine molecule; m / z 135 is due to [M+H] + The formation of m / z 133 is attributed to the ring-opening reaction of the methylenedioxane ring in product ion m / z 163 and the loss of aH₂C=O. Product ions m / z 105, m / z 152, and m / z 175 are presumed to be interfering ions.

[0067] MS of ketamine 2 As shown in the picture Figure 2 As shown in Figure D, there are few interfering peaks. Besides the ion peaks at m / z 220, m / z 207, and m / z 179 generated from the fragmentation of the ketamine precursor ion at m / z 238, there are only two impurity ion peaks at m / z 152 and m / z 182, and their responses are both lower than the target ion. The daughter ion at m / z 220 is generated from the ketamine precursor ion [M+H]. + It is formed by the removal of a water molecule; the daughter ion m / z 207 is the ketamine parent ion [M+H]. + The formation of daughter ion m / z 179 is achieved by removing a methylamine group from daughter ion m / z 207, which is then further formed by removing a CO group.

[0068] MS of norketamine 2 As shown in the picture Figure 1As shown in Figure E, numerous impurity ions were observed. Besides the peaks at m / z 207, m / z 179, and m / z 125 generated by the fragmentation of the norketamine precursor ion (m / z 224), the response at m / z 163 is likely an interfering signal. However, these interfering signals are far from the fragment ions and do not affect the detection of the target compound. The m / z 207 ion is generated by the norketamine precursor ion [M+H]. + It is formed by removing an NH2 group, and then removing a CO group to form a daughter ion with m / z 179. Further removing a C4H6 group will form a daughter ion with m / z 125.

[0069] MS of etomidate 2 As shown in the picture Figure 1 As shown in Figure F, numerous impurity examples were observed. Besides the ion peak at m / z 141 generated by the fragmentation of the etomidate precursor ion (m / z 245), the corresponding signals at m / z 188, m / z 213, and m / z 241 are likely interfering signals. However, these interfering signals are far from the fragment ions and do not affect the detection of the target compound. The m / z 141 ion is generated by the etomidate precursor ion [M+H]. + It is formed after the removal of ethylphenyl group.

[0070] MS of isopramipexole 2 As shown in the picture Figure 2 As shown in G, numerous impurity examples were observed. Besides the ion peaks at m / z 155 and m / z 113 generated by the fragmentation of the isopramipexyl ion (m / z 259), the corresponding peaks at m / z 203, m / z 227, and m / z 241 are likely interfering signals. However, these interfering signals are far from the fragment ions and do not affect the detection of the target compound. The m / z 155 ion is generated by the isopramipexyl ion [M+H]. + It is formed by removing ethylphenyl group, and then removing a C3H6 group to form daughter ion m / z113.

[0071] MS of 2-(ethylamino)-2-phenylcyclohexane-1-one 2 As shown in the picture Figure 2 As shown in Figure H, numerous impurity examples were observed. Besides the ion peak at m / z 173 generated by the fragmentation of the 2-(ethylamino)-2-phenylcyclohexane-1-one parent ion (m / z 218), the corresponding signals at m / z 88, m / z 105, and m / z 203 are likely interfering signals. However, these interfering signals are far from the fragment ions and do not affect the detection of the target compound. The m / z 173 ion is generated by the isopramester parent ion [M+H]. + It is formed by removing a C2H8N group.

[0072] Based on the above analysis, it can be seen that the target ions in the hair after solid-phase extraction with a gun tip can all be detected by MS. 2 The target ions were easily identified in the image, and the interference of impurity ions on the target ions was minimal, demonstrating the feasibility of this method for analyzing seven drugs in hair.

[0073] The fragment ions with the strongest response for each drug were used as the quantitative ions in this experiment. The quantitative ions for different drugs are shown in Table 1.

[0074] Linear regression analysis of the average signal intensity and concentration of six consecutive quantitative ion mass spectrometry peaks showed that the seven drugs exhibited good linearity within their respective linear ranges (R0). 2 >0.99), gun tip solid phase extraction and paper spray-micro mass spectrometry showed good quantitative performance for seven drugs in hair, and the specific values ​​are detailed in Table 2.

[0075] Table 2. Linearity, Limit of Detection, and Limit of Quantification for Seven Drugs

[0076]

[0077] The sensitivity of this method is calculated based on a signal-to-noise ratio of LOD ≥3 and a signal-to-noise ratio of LOQ ≥10.

[0078] As shown in Table 2, the LODs of methamphetamine, 3,4-methylenedioxymethamphetamine, norketamine, ketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one ranged from 1 to 3 ng·mL. -1 Between, LOQ≥5ng·mL -1 The results showed that tip solid-phase extraction and paper spray-micromass spectrometry had high sensitivity for detecting seven drugs in hair.

[0079] Quality control testing:

[0080] Before hair sample testing, a quality control sample is injected, and the actual concentration is compared with the test concentration. The comparison deviation must not exceed 15%. If the deviation exceeds 15%, the testing method, instrument performance, and quality control sample must be checked. Spike concentrations of 8, 20, 200, and 400 ng / mL are used. -1 The recovery and precision of the quality control samples were tested using the pipette tip solid phase extraction and paper spray-micro mass spectrometry methods. Each sample group was injected 6 times, and the results are shown in Table 3.

[0081] Table 3. Stability and recovery rate of seven drugs

[0082]

[0083] This indicates that the recovery rates of the seven drugs in their respective quality control analyses were good, generally ranging from 91% to 115%, with good stability and an RSD of <15%. The intra-day and inter-day precision were high, with good stability and an RSD of <10%, which can meet the needs of daily monitoring.

[0084] In summary, this method is simple to operate, highly sensitive, and has sufficient quantitative capability to achieve rapid on-site detection of seven drugs in hair.

[0085] Comparative Example 1:

[0086] Comparative Example 1 is basically the same as Example 1, except that the hair sample of methamphetamine was not subjected to solid phase extraction.

[0087] The hair samples from Example 1 and Comparative Example 1 were subjected to mass spectrometry analysis, and the signal values ​​of the detected target substances were compared. The detection results are as follows: Figure 3 As shown, Figure 3 A represents Instance 1 and Figure 3 B is Comparative Example 1. The comparison shows that after solid-phase extraction, the signal intensity of methamphetamine sample loaded via paper spray ionization increased from 81069 to 1332803. Therefore, after solid-phase extraction of hair samples using a spray nozzle, the detection performance of paper spray ionization improved by approximately 16.4 times.

[0088] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for detecting multiple drugs in hair, characterized in that, Includes the following steps: S1. After preparing the drug stock solution with methanol, the drug stock solution is then diluted with methanol to obtain intermediate solutions of different concentrations. A certain amount of the intermediate solution is added to an equal amount of blank hair extract to obtain spiked hair extracts of different concentration levels. S2. Hair extracts with different concentration levels were subjected to solid-phase extraction and then mass spectrometry detection. The signal intensity of the quantitative ion mass spectrometry peak was used to perform linear regression on the spike concentration to construct a standard curve. S3. The hair sample to be tested is shredded and added to a mixture of methanol and water. It is then ground into a powder to obtain a dispersion. After the dispersion has settled, the supernatant is collected and filtered through an organic microporous membrane to obtain the hair extract. S4. Solid-phase extraction is performed on the hair extract of the sample. The solid-phase extraction uses a mixed solution of ammonia and methanol as the eluent. After collecting the eluent, mass spectrometry is performed to obtain the signal intensity of the quantitative ion mass spectrometry peak of the drug analyte. S5. Compare the signal intensity of the quantitative ion mass spectrometry peak of the drug analyte with the standard curve to calculate the concentration of the drug in the hair sample to be tested.

2. The method for detecting multiple drugs in hair according to claim 1, characterized in that, In S1, the drug stock solution contains methamphetamine, 3,4-methylenedioxymethamphetamine, ketamine, norketamine, etomidate, isopramipexole, and 2-(ethylamino)-2-phenylcyclohexane-1-one, each at a concentration of 5 mg / mL. -1 Mixed storage solution; In S1, the preparation method of blank hair extract is as follows: the blank hair that tested negative is washed and dried sequentially with distilled water and methanol, the blank hair is cut into pieces and added to a mixed solution of methanol and water, and ground into powder to obtain a dispersion; after the dispersion is allowed to stand, the supernatant is taken and filtered through an organic microporous membrane to obtain the blank hair extract.

3. The method for detecting multiple drugs in hair according to claim 1, characterized in that, In S3, the volume ratio of methanol to water in the mixed solution is 1:

1.

4. The method for detecting multiple drugs in hair according to claim 1, characterized in that, In S4, mass spectrometry analysis revealed the following quantitative ions: methamphetamine (m / z 119), 3,4-methylenedioxymethamphetamine (m / z 163), ketamine (m / z 207), norketamine (m / z 207), etomidate (m / z 141), isopramazoride (m / z 155), and 2-(ethylamino)-2-phenylcyclohexane-1-one (m / z 173).

5. The method for detecting multiple drugs in hair according to claim 1, characterized in that, In S4, solid-phase extraction is performed using a pipette packed with solid-phase extraction packing material. The extraction method using a pipette is as follows: the pipette is immersed in the hair extract of the sample, and the aspiration and discharge are repeated several times; then it is rinsed with methanol, and then the mixture of ammonia and methanol is used as the eluent for repeated elution. The supernatant is then used for detection.

6. The method for detecting multiple drugs in hair according to claim 5, characterized in that, The pipette tip uses MCX solid-phase extraction packing material; the volume ratio of ammonia to methanol in the mixed solution of ammonia and methanol is 5:95, and the concentration of ammonia is 25-28 wt%.

7. The method for detecting multiple drugs in hair according to claim 1, characterized in that, In S4, a miniature mass spectrometer is used for mass spectrometry detection.

8. The method for detecting multiple drugs in hair according to claim 7, characterized in that, For mass spectrometry detection, paper spray ionization is used for sample introduction.

9. The method for detecting multiple drugs in hair according to claim 8, characterized in that, During ionization injection, the spray voltage for methamphetamine was 4200 V, for 3,4-methylenedioxymethamphetamine it was 4200 V, for ketamine it was 4100 V, for norketamine it was 4100 V, for etomidate it was 4400 V, for isopramipex ester it was 4400 V, and for 2-(ethylamino)-2-phenylcyclohexyl-1-one it was 4100 V.

10. The method for detecting multiple drugs in hair according to claim 8, characterized in that, During ionization injection, the ISO1 energy for methamphetamine was 7 V, for 3,4-methylenedioxymethamphetamine it was 7 V, for ketamine it was 8 V, for norketamine it was 10 V, for etomidate it was 9 V, for isopramipex ester it was 9 V, and for 2-(ethylamino)-2-phenylcyclohexane-1-one it was 10 V. The ISO2 energy of methamphetamine is 2.5 V, the ISO2 energy of 3,4-methylenedioxymethamphetamine is 3 V, the ISO2 energy of ketamine is 3.5 V, the ISO2 energy of norketamine is 3.5 V, the ISO2 energy of etomidate is 3 V, the ISO2 energy of isopramipex is 4 V, and the ISO2 energy of 2-(ethylamino)-2-phenylcyclohexyl-1-one is 4 V. The CID energy of methamphetamine is 1 V, the CID energy of 3,4-methylenedioxymethamphetamine is 3.75 V, the CID energy of ketamine is 1.75 V, the CID energy of norketamine is 2.25 V, the CID energy of etomidate is 1.25 V, the CID energy of isopramipexole is 1.25 V, and the CID energy of 2-(ethylamino)-2-phenylcyclohexyl-1-one is 1.5 V.

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