Chiral derivation detection method for methylamphetamine and amphetamine enantiomer

Abstract

The invention relates to the technical field of drug analysis, in particular to a chiral derivatization detection method for methylamphetamine and amphetamine enantiomers, which at least comprises the following steps: (1) adding an internal standard solution, a buffer solution and a derivatization reagent into a sample to be detected for derivatization reaction; (2) after the derivatization reaction, adding an extracting agent for liquid-liquid extraction, blow-drying an organic phase after liquid-liquid extraction, and redissolving with a redissolving solvent to obtain a redissolving solution; and (3) carrying out liquid chromatography-high resolution mass spectrometry detection on the redissolved solution, and carrying out qualitative and semi-quantitative determination according to the detection result, the method is used for detecting enantiomers of methamphetamine and amphetamine in a complex biological sample, and the method has the advantages of good separation degree, good repeatability, high sensitivity and high detection sensitivity, and can be used for detecting the enantiomers of methamphetamine and amphetamine in the complex biological sample. The method has good practicability in the aspects of judicial expertise and stimulant screening.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, in particular to a chiral derivatization detection method for methamphetamine and amphetamine enantiomers. Background Art

[0002] Methamphetamine (MAMP), also known as methamphetamine, is an amphetamine chemical substance. It is a psychoactive substance regulated by my country and a Class S6 stimulant on the banned list issued by the World Anti-Doping Agency. As a psychoactive drug, methamphetamine seriously affects human health and public safety. At the same time, as a stimulant drug banned by athletes, the illegal use of methamphetamine also affects the health of athletes and the fairness of sports. Therefore, it is very necessary to establish a high-sensitivity detection method for MAMP and its metabolites in human samples.

[0003] The chemical structure of MAMP contains a chiral carbon atom, so there is a pair of enantiomers, namely S-(+)-methamphetamine and R-(-)-methamphetamine. S-(+)-MAMP is the main component of the addictive drug "ice". At the beginning of this century, the MAMP seized by the Chinese police was mainly S-type, but as the crackdown continued to increase, R-type MAMP gradually appeared and its proportion increased rapidly. Amphetamine (AMP), as the main metabolite of methamphetamine, also exists in R-type and S-type. R-type and S-type methamphetamine have similar physical and chemical properties, but their pharmacological and toxicological effects are very different. Therefore, it is extremely important to establish a chiral separation detection method for MAMP and its metabolite AMP. When used as a stimulant, according to the description in the banned stimulant list, S-type MAMP belongs to the S6a non-specific stimulant, while R-type MAMP belongs to the S6b specific stimulant. When there is a violation of the use of this type of stimulant, there is a significant difference in the severity of the punishment for the two. Therefore, from the perspective of doping screening, it is also urgent to establish chiral separation and qualitative detection methods for MAMPs and their metabolites in human samples.

[0004] In recent years, researchers have mostly used chromatographic columns filled with chiral stationary phases to perform chiral separation of MAMPs and their analogs and metabolites. For example, a Chinese patent (publication number CN 108181385 A) discloses a method for simultaneously detecting the enantiomers of methamphetamine and amphetamine in liquid biological samples. This method uses the stereochemical difference between the enantiomers and the chiral chromatographic column for separation, and the sample processing is simple. The disadvantage is that the chiral recognition ability of this method is relatively specific, the sample types analyzed by the same chromatographic column are relatively limited, the versatility is not great, and it is relatively expensive. The technology based on chiral derivatization has also begun to be applied to the chiral separation of MAMPs and related substances by virtue of its good separation ability and good compatibility with conventional chromatographic conditions. For human biological samples commonly used in forensic identification and doping detection, the matrix components are very complex, and there are often a large number of high-concentration amine structural analogs, which are easy to interfere with the test results. At the same time, the addition of excessive derivatization reagents and buffer solutions also further increases the complexity of the test samples, affecting the accuracy and sensitivity of the test. Therefore, it is very necessary to establish a chiral derivatization detection method with good repeatability, accuracy and sensitivity. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and the method is used for the detection of methamphetamine and amphetamine enantiomers in complex biological samples. The method not only has good separation, but also has good repeatability and high sensitivity, and has good practicality in forensic identification and stimulant screening.

[0006] The present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, which at least comprises the following steps:

[0007] (1) adding an internal standard solution, a buffer solution and a derivatization reagent to the sample to be tested to perform a derivatization reaction;

[0008] (2) adding an extractant after the derivatization reaction to perform liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction, and then re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution;

[0009] (3) The reconstituted solution is subjected to liquid chromatography-high resolution mass spectrometry detection, and qualitative and semi-quantitative determination is performed based on the detection results.

[0010] As a preferred technical solution, the sample to be tested includes human blood, urine, saliva and sweat.

[0011] As a preferred technical solution, the pH of the buffer solution is 7-12, preferably 8.0-9.0.

[0012] As a preferred technical solution, the buffer solution is a sodium bicarbonate buffer solution, and the concentration of the sodium bicarbonate buffer solution is 0.5-21 mol / L, preferably 1±0.1 mol / L.

[0013] As a preferred technical solution, the preparation method of the internal standard solution is: dilute the (±)-D9-Methamphetamine (D9-MAMP) and (±)-D10-Amphetamine-(D10-AMP) standard substances with a solvent to a mixed solution with a concentration of 200-300 ng / mL, which is the internal standard solution.

[0014] Preferably, the solvent is methanol.

[0015] As a preferred technical solution, the derivatization reagent is Marfey (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) solution.

[0016] Preferably, the concentration of the Marfey solution is 3-7 mg / mL, preferably 4-6 mg / mL, and most preferably 5±0.5 mg / mL.

[0017] Preferably, the solvent of the Marfey solution is DMSO (dimethyl sulfoxide).

[0018] As a preferred technical solution, the amount of the sample to be tested is 50-200 μL, the amount of the internal standard solution is 10-30 μL, the amount of the buffer solution is 20-60 μL, and the volume ratio of the derivatization reagent to the sample to be tested is 1:(0.8-1.5).

[0019] As a preferred technical solution, the temperature of the derivatization reaction is 40-60°C, and the derivatization time is 0.5-2h. Preferably, the temperature of the derivatization reaction is 45-55°C, and the derivatization time is 1-2h.

[0020] As a preferred technical solution, the extractant is an alcohol ether extractant, preferably methyl tert-butyl ether.

[0021] As a preferred technical solution, the amount of the extractant added is 0.8-1.5 mL.

[0022] As a preferred technical solution, the liquid-liquid extraction specifically comprises: vortexing the reaction system with the extractant and then centrifuging it to achieve stratification to obtain the organic phase.

[0023] As a preferred technical solution, the re-dissolution solvent is methanol aqueous solution.

[0024] Preferably, the mass proportion of methanol in the methanol aqueous solution is 50-65%.

[0025] As a preferred technical solution, the liquid chromatography separation conditions in the liquid chromatography-high resolution mass spectrometry detection are: using a reverse phase C18 column, the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is an aqueous solution of ammonium formate with a concentration of 2-6 mM, mobile phase B is methanol, a gradient elution program is used, the flow rate is 0.25±0.02 mL / min, the column temperature is 40±2°C, and the injection volume is 2-10 μL.

[0026] As a preferred technical solution, the reverse phase C18 column is Waters ACQUITY UPLC BEH C18, with a specification of 100 mm*2.1 mm and a particle size of 1.7 μm.

[0027] As a preferred technical solution, the gradient elution program is shown in Table 1.

[0028] Table 1

[0029] Time (minutes) A％ B％ 0 50％ 50％ 10 30％ 70％ 15 30％ 70％ 15.1 0％ 100 16 0％ 100 16.1 50％ 50 21 50％ 50

[0030] As a preferred technical solution, the high-resolution mass spectrometry detection conditions in the liquid chromatography-high-resolution mass spectrometry detection are as follows: the mass spectrometer is a quadrupole electrostatic field orbital trap mass spectrometer, an electrospray ion source (ESI source) negative ion mode is adopted, the ion source voltage is 2.8KV-3.0KV, the sheath gas pressure is 32-38Arb, the auxiliary gas pressure is 12-18Arb, the auxiliary gas temperature is 300-380°C, the capillary temperature is 300-330°C, and the parallel reaction monitoring mode (PRM) is adopted for data acquisition.

[0031] As a preferred technical solution, in the parallel reaction monitoring mode, the normalized fragmentation energy (NCE) of the secondary mass spectrometer is set to 30-35, preferably 35.

[0032] At present, the chiral derivatization detection method of methamphetamine and amphetamine enantiomers is usually added after derivatization reaction, and concentrated / diluted directly on the machine for detection, but the addition of hydrochloric acid causes the salt concentration in the system to be too high, and the mass spectrometry signal noise is significantly increased, and the detection sensitivity is not ideal. Based on this, the present invention combines the liquid-liquid extraction technology by optimizing the chiral derivatization reaction, and in the face of biological samples with very complex matrix components, the interference caused by the presence of a large number of highly concentrated amine structural analogs to the detection result can be effectively overcome, and it is not only possible to achieve the distinction between methamphetamine and propylaniline enantiomers, but also the sample can be purified simultaneously to improve the detection sensitivity.

[0033] Specifically, in order to obtain the best separation and detection effect, in the process of method optimization of the present invention, a pH regulator is added after the derivatization reaction is completed to adjust the pH of the system before subsequent liquid-liquid extraction is performed, including adding hydrochloric acid solution and saturated sodium carbonate solution to the system after the derivatization reaction is completed and then performing liquid-liquid extraction. However, the liquid-liquid extraction efficiency after adding hydrochloric acid is low and the repeatability is poor. Adding a saturated sodium carbonate solution will also cause the extraction efficiency to deteriorate. Therefore, the present invention determines that after the derivatization reaction is completed, the pH regulator is no longer added, and the derivatization reaction is directly terminated by liquid-liquid extraction, thereby achieving the enrichment and purification of methamphetamine and amphetamine at the same time.

[0034] Furthermore, the present invention significantly increases the mass spectrometric response of methamphetamine and amphetamine by controlling the amount of derivatization reagent added and the temperature and time of the derivatization reaction, while controlling the derivatization reaction to proceed rapidly at a mild temperature with good robustness.

[0035] Furthermore, the present invention controls the PRM monitoring parameters of the analyte and the internal standard, the normalized fragmentation energy (NCE) of the secondary mass spectrometer is 30-35, and the mass spectrometric response of the main fragment ions of methamphetamine and amphetamine is the highest, thereby obtaining the best detection sensitivity.

[0036] In addition, the present invention uses octadecylsilane bonded silica gel as the chromatographic stationary phase, which has the advantage of low price compared to the chiral crown ether silica gel stationary phase (the chromatographic column is SupelcoAstec ChirobioticTMV2 chiral column, 2.1mm×250mm, 5μm), and effectively reduces the cost.

[0037] In summary, the present invention realizes chiral separation through the chemical reaction of Marfey reagent with enantiomeric amino or imino groups. The method has high sensitivity, good accuracy, good durability, and convenient operation. It can be further extended to the chiral separation of emerging new amine stimulants and psychoactive drugs, and has good practical significance in judicial detection and stimulant screening.

[0038] Beneficial Effects

[0039] 1. The present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, which is used for the detection of methamphetamine and amphetamine enantiomers in complex biological samples. The method not only has good separation, but also has good repeatability and high sensitivity, and has good practicality in forensic identification and stimulant screening.

[0040] 2. The present invention combines the optimization of chiral derivatization reaction and liquid-liquid extraction technology. When faced with biological samples with very complex matrix components, it can effectively overcome the interference of a large number of high-concentration amine structural analogs on the detection results. It can not only distinguish between methamphetamine and propylaniline enantiomers, but also purify the samples and improve the detection sensitivity.

[0041] 3. The present invention determines that after the derivatization reaction is completed, no pH regulator is added, and the derivatization reaction is terminated directly by liquid-liquid extraction, thereby achieving the enrichment and purification of methamphetamine and amphetamine at the same time.

[0042] 4. The present invention significantly increases the mass spectrometric response of methamphetamine and amphetamine by controlling the amount of derivatization reagent added and the temperature and time of the derivatization reaction, while controlling the derivatization reaction to proceed rapidly at a mild temperature with good robustness.

[0043] 5. In the present invention, by controlling the PRM monitoring parameters of the analyte and the internal standard, the normalized fragmentation energy (NCE) of the secondary mass spectrometer is 30-35, and the mass spectrometric response of the main fragment ions of methamphetamine and amphetamine is the highest, thereby obtaining the best detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The mass spectrometry response results of R-MAMP, S-MAMP, R-AMP and S-AMP under the methods of Examples 1, 2, 3 and 4 of the present invention are compared. Examples 1, 2, 3 and 4 correspond to Figure 1 C, A, B, D.

[0045] Figure 2 The mass spectrometry response results of R-MAMP, S-MAMP, R-AMP and S-AMP under the methods of Examples 5, 6 and 7 of the present invention are compared. Examples 5, 6 and 7 correspond to Figure 2 C, B, A in Chinese.

[0046] Figure 3 This is a comparison chart of the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP under the methods of Examples 8, 9, 10, and 11 of the present invention. Examples 8, 9, 10, and 11 correspond to Figure 3 C, A, B, D.

[0047] Figure 4 The chromatogram is of methamphetamine and amphetamine after chiral derivatization under the method of Example 1.

[0048] Figure 5 This is the secondary mass spectrum of methamphetamine and amphetamine after chiral derivatization under the method of Example 1. DETAILED DESCRIPTION

[0049] Example 1

[0050] Embodiment 1 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, comprising the following steps:

[0051] (1) adding an internal standard solution, a buffer solution and a derivatization reagent to the sample to be tested to perform a derivatization reaction;

[0052] (2) after the derivatization reaction, cooling to 25° C., adding an extractant to perform liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction, and then re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution;

[0053] (3) The reconstituted solution is subjected to liquid chromatography-high resolution mass spectrometry detection, and qualitative and semi-quantitative determination is performed based on the detection results.

[0054] The sample to be tested is human urine.

[0055] The buffer solution is a sodium bicarbonate buffer solution, and the concentration of the sodium bicarbonate buffer solution is 1±0.1 mol / L.

[0056] The preparation method of the internal standard solution is: dilute (±)-D9-Methamphetamine (D9-MAMP) and (±)-D10-Amphetamine-(D10-AMP) standard substances with a solvent to obtain a mixed solution with a concentration of 250 ng / mL, which is the internal standard solution.

[0057] The solvent is methanol.

[0058] The derivatization reagent is Marfey (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) solution.

[0059] The concentration of the Marfey solution is 5±0.5 mg / mL.

[0060] The solvent of the Marfey solution is DMSO (dimethyl sulfoxide).

[0061] The amount of the sample to be tested is 100 μL, the amount of the buffer solution is 40 μL, the amount of the internal standard solution is 20 μL, and the volume ratio of the derivatization reagent to the sample to be tested is 1:0.8.

[0062] The temperature of the derivatization reaction is 45° C., and the derivatization time is 1 h.

[0063] The extractant is methyl tert-butyl ether.

[0064] The amount of the extractant added was 1.0 mL.

[0065] The liquid-liquid extraction is specifically as follows: the reaction system with the extractant added is vortexed for 3 minutes and then centrifuged at 12000 rpm for 5 minutes to achieve stratification to obtain 850 μL of an organic phase.

[0066] The re-dissolution solvent is methanol-water solution.

[0067] The weight percentage of methanol in the methanol aqueous solution is 60%.

[0068] The amount of the methanol aqueous solution used is 100 μL.

[0069] The liquid chromatography separation conditions in the liquid chromatography-high-resolution mass spectrometry detection are as follows: a reverse phase C18 column is used, the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is a 5 mM ammonium formate aqueous solution, mobile phase B is methanol, a gradient elution program is used, the flow rate is 0.25±0.02 mL / min, the column temperature is 40±2°C, and the injection volume is 5 μL.

[0070] The reverse phase C18 column is Waters ACQUITY UPLC BEH C18, with specifications of 100 mm*2.1 mm and a particle size of 1.7 μm.

[0071] The gradient elution program is shown in Table 1.

[0072] Table 1

[0073] Time (minutes) A％ B％ 0 50％ 50％ 10 30％ 70％ 15 30％ 70％ 15.1 0％ 100 16 0％ 100 16.1 50％ 50 21 50％ 50

[0074] The high-resolution mass spectrometry detection conditions in the liquid chromatography-high-resolution mass spectrometry detection are as follows: the mass spectrometry quadrupole electrostatic field orbital trap mass spectrometry adopts an electrospray ionization source (ESI source) negative ion mode, an ion source voltage of 2.8KVKV, a sheath gas pressure of 35Arb, an auxiliary gas pressure of 15Arb, an auxiliary gas temperature of 350°C, a capillary temperature of 320°C, and parallel reaction monitoring mode (PRM) is used for data acquisition.

[0075] In the parallel reaction monitoring mode, the normalized fragmentation energy (NCE) of the secondary mass spectrometer was set to 35, and the PRM monitoring parameters of the analyte and the internal standard were shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079] The mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP under the method of Example 1 are shown in Figure 1 , the chromatograms of methamphetamine and amphetamine after chiral derivatization are shown in Figure 4, it can be seen that both pairs of enantiomers have been baseline separated; the secondary mass spectra of methamphetamine and amphetamine after chiral derivatization are shown in Figure 5 , it can be seen that the similarity of the secondary mass spectra of the enantiomers is very high.

[0080] Example 2

[0081] Example 2 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 1, except that step (2) is replaced by: adding 80 μL of 1 mol / L hydrochloric acid solution after the derivatization reaction, then cooling to 25°C and adding an extractant for liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction and re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution, and the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 1 .

[0082] Example 3

[0083] Example 3 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 1, except that step (2) is replaced by: adding 40 μL of 1 mol / L hydrochloric acid solution after the derivatization reaction, then cooling to 25°C and adding an extractant for liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction and re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution, and the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 1 .

[0084] Example 4

[0085] Example 4 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers. The specific implementation method is the same as that of Example 1, except that step (2) is replaced by: adding 40 μL of a saturated sodium carbonate solution after the derivatization reaction, then cooling to 25° C. and adding an extractant for liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction and re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution, and the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 1 .

[0086] Example 5

[0087] Example 5 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 1, except that step (2) is replaced by: adding 80 μL of 1 mol / L hydrochloric acid solution after the derivatization reaction, then cooling to 25°C and adding an extractant for liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction and re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution, and at the same time changing the chromatographic injection volume to 3 μL, and the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 2 .

[0088] Example 6

[0089] Example 6 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 5, except that the volume ratio of the derivatization reagent to the sample to be tested is 1:0.6, and the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 2 .

[0090] Example 7

[0091] Example 7 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 5, except that the volume ratio of the derivatization reagent to the sample to be tested is 1:0.4, and the mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 2 .

[0092] Example 8

[0093] Example 8 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers. The specific implementation method is the same as that of Example 1, except that the liquid-liquid extraction in step (2) is changed to adding 40 μL 1M HCl solution and 100 μL water, which are fully mixed. The mass spectrometry response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 3 .

[0094] Example 9

[0095] Example 9 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 8, except that the mass spectrum fragmentation energy (NCE) is set to 25, and the mass spectrum response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 3 .

[0096] Example 10

[0097] Example 10 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 8, except that the mass spectrum fragmentation energy (NCE) is set to 30, and the mass spectrum response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 3 .

[0098] Embodiment 11

[0099] Example 11 of the present invention provides a chiral derivatization detection method for methamphetamine and amphetamine enantiomers, and its specific implementation method is the same as that of Example 8, except that the mass spectrum fragmentation energy (NCE) is set to 40, and the mass spectrum response results of R-MAMP, S-MAMP, R-AMP, and S-AMP are shown in Figure 3 .

[0100] Method validation

[0101] Validation Example 1 provides the selectivity, detection limit, repeatability and spike recovery of the method.

[0102] (1) Selective specific test methods:

[0103] 10 blank human urine samples of different pH, specific gravity and gender were selected (see Table 3 for blank urine sample information) and tested and analyzed according to the method provided in Example 1. No suspicious signals were detected, indicating that the selectivity was qualified.

[0104] Table 3

[0105] serial number gender proportion pH Characteristics 1 female 1.006 6.861 Light yellow, clear 2 male 1.007 6.15 Light yellow, clear 3 female 1.007 5.916 Medium yellow, clear 4 male 1.012 7.987 Light yellow, a small amount of precipitation 5 female 1.012 7.186 Light yellow, clear 6 male 1.015 7.041 Medium yellow, clear 7 female 1.015 6.874 Medium yellow, clear 8 male 1.018 5.087 Medium yellow, clear 9 female 1.021 5.745 Dark yellow, clear 10 male 1.022 6.143 Dark yellow, clear

[0106] (2) Specific test method for detection limit:

[0107] In doping detection, the minimum reporting limit of MAMP and its metabolites is 50 ng / mL, and the detection limit should be lower than this concentration. The 10 blank urine samples in Table 2 were used to prepare spiked samples with concentrations of 50 ng / mL, 25 ng / mL, 12.5 ng / mL and 5 ng / mL, respectively, and the method provided in Example 1 of the present invention was used for detection. All 10 urine samples were detected at a concentration of 5 ng / mL, so the detection limits of MAMP and AMP were both 5 ng / mL.

[0108] (3) Precision, deviation and system residual verification:

[0109] A blank urine matrix was spiked with 50 ng / mL, repeated six times, and the RSD value was calculated to obtain the method precision. The results are shown in Table 4. A single calibration point with a concentration of 60 ng / mL was prepared, and 10 blank urine samples in Table 2 were respectively prepared with spiked samples with a concentration of 50 ng / mL for semi-quantitative detection. The deviation (spiking recovery rate) of the 10 spiked samples was calculated, and the results are shown in Table 4. Spiked samples with a concentration of 200 ng / mL and two blank samples were prepared for continuous detection to examine the system residue ("√" indicates no system residue). The results are shown in Table 4.

[0110] Table 4

[0111]

[0112] Verified by the above methods, the detection limit of this method is as low as 10ng / mL, and it has good selectivity, which meets the daily needs of doping detection and the needs of judicial detection, and has good practical value.

Claims

1. A chiral derivatization detection method for methamphetamine and amphetamine enantiomers, It is characterized in that At least the following steps are included: (1) adding an internal standard solution, a buffer solution and a derivatization reagent to the sample to be tested to perform a derivatization reaction; (2) adding an extractant after the derivatization reaction to perform liquid-liquid extraction, drying the organic phase after the liquid-liquid extraction, and then re-dissolving it with a re-dissolving solvent to obtain a re-dissolved solution; (3) The reconstituted solution is subjected to liquid chromatography-high resolution mass spectrometry detection, and qualitative and semi-quantitative determination is performed based on the detection results.

2. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The pH of the buffer solution is 7-12.

3. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The amount of the sample to be tested is 50-200 μL, the amount of the internal standard solution is 10-30 μL, the amount of the buffer solution is 20-60 μL, and the volume ratio of the derivatization reagent to the sample to be tested is 1:(0.8-1.5).

4. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The temperature of the derivatization reaction is 40-60° C., and the derivatization time is 0.5-2 h.

5. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The extractant is an alcohol ether extractant.

6. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The amount of the extractant added is 0.8-1.5 mL.

7. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The liquid-liquid extraction specifically comprises: vortexing the reaction system with the extractant and then centrifuging it to achieve stratification to obtain the organic phase.

8. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The liquid chromatography separation conditions in the liquid chromatography-high resolution mass spectrometry detection are as follows: a reverse phase C18 column is used, the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is an aqueous solution of ammonium formate with a concentration of 2-6 mM, mobile phase B is methanol, a gradient elution program is used, the flow rate is 0.25±0.02 mL / min, the column temperature is 40±2°C, and the injection volume is 2-10 μL.

9. The chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 1, It is characterized in that The high-resolution mass spectrometry detection conditions in the liquid chromatography-high-resolution mass spectrometry detection are as follows: the mass spectrometer is a quadrupole electrostatic field orbital trap mass spectrometer, an electrospray ion source negative ion mode is used, the ion source voltage is 2.8KV-3.0KV, the sheath gas pressure is 32-38Arb, the auxiliary gas pressure is 12-18Arb, the auxiliary gas temperature is 300-380°C, the capillary temperature is 300-330°C, and data collection is performed in parallel reaction monitoring mode.

10. A chiral derivatization detection method for methamphetamine and amphetamine enantiomers according to claim 9, It is characterized in that The normalized fragmentation energy of the secondary mass spectrometer was set to 30-35 in parallel reaction monitoring mode.

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