Method for analyzing cathinone, methcathinone and 4-methylmethcathinone in biological sample by liquid chromatography-mass spectrometry

[0047] Example 1

[0048] The method for analyzing cathinone, methcathinone, and 4-methylmethcathinone in urine by LC/MS includes the following steps:

[0049] (1) Preparation of sample solution;

[0050] (2) Preparation of standard working fluid;

[0051] (3) Setting of testing conditions;

[0052] (4) LC/MS determination;

[0053] (5) Calculation of experimental results, among which,

[0054] Step (1): Take three blank urine samples of healthy volunteers and add cathinone, methcathinone and 4-methylmethcathinone to prepare urine sample one (the concentration of cathinone is 500ng/mL ), urine sample two (methcathinone concentration of 500ng/mL) and urine sample three (4-methylmethcathinone concentration of 500ng/mL), respectively with water according to the volume ratio of sample to water 2:1 After proportional dilution, add protein precipitant acetonitrile, add protein precipitant until no precipitation is formed in the sample, then ultrasonically shake for 15 minutes, centrifuge at 3000r/min for 10 minutes, and pass the supernatant through a gel chromatography column and gel chromatography. The column packing is biobead SX-3, 200*15mm, and the volume ratio is 1:1 ethyl acetate/cyclohexane solution, and the elution rate is 1.5mL/min. After purification by the gel chromatography column, the extract is obtained. The extract is After blowing nitrogen to near dryness, add the mobile phase (the mobile phase is the same as the mobile phase in step (3), the volume ratio of A-0.2% formic acid, B-acetonitrile and C-methanol is 60:30:10). After the volume reaches 2ml, the sample solution is obtained.

[0055] In step (2), take the standard stock solutions of cathinone, methcathinone, and 4-methylmethcathinone (methanol as solvent), respectively, and dilute them to mass concentrations of 10000, 5000, 2500, 1000, Three series of standard solutions of 500, 250, 100, 10, 1, 0.1ng/mL.

[0056] In step (3), the setting process of liquid chromatography conditions is: Chromatographic column: reverse phase C18 chromatographic column, 100mm×2.1mm, 1.8μm, mobile phase: A-0.2% formic acid (100mL formic acid aqueous solution contains 0.2mL formic acid ), B-acetonitrile, C-methanol, gradient elution, the process is shown in Table 2:

[0057] Table 2

[0058]

[0059] Flow rate: 0.4 mL/min. The retention times of cathinone, methcathinone, and 4-methylmethcathinone were 1.687min, 2.098min and 3.984min, respectively.

[0060] The setting process of mass spectrometry conditions is:

[0061] Selection of spectra conditions of cathinone, methcathinone and 4-methylmethcathinone

[0062] a. Determine the quasi-molecular ion

[0063] Scan with SCAN mode to determine the [M+H] of cathinone + 150.1, [M+H] of methcathinone + 164.2, 4-methylmethcathinone [M+H] + Is 178.2.

[0064] b. Optimize the capillary outlet voltage (Fragmentor) through selective ion scanning

[0065] Collision-induced dissociation refers to the process of transferring energy to ions through collisions with neutral molecules. Fragmentor voltage is the voltage at the outlet of the capillary, which determines whether the precursor ions can fly to the quadrupole and ensures the transmission efficiency of the precursor ions. Therefore, it is one of the important parameters that need to be optimized.

[0066] In the SIM scan mode, five Fragmentor voltages of 80, 100, 120, 140, and 200 are selected to obtain the corresponding response intensity. Considering the corresponding intensity and related parameters, the Fragmentor voltage is selected as 200.

[0067] c. Select product ions through product ion scanning and optimize collision energy (CE)

[0068] Through the first group of quadrupoles as SIM, only the parent ions of the substance to be tested are allowed to pass, and the second group of quadrupoles are used as SCAN, the best response of cathinone, methcathinone and 4-methylmethcathinone is obtained. Two strong product ions. Optimize the CE values ​​of these two product ions at the same time, select 10, 20, 30, 40, 60, 80, 100 seven CE values, and finally determine the CE values ​​of cathinone, methcathinone, and 4-methylmethcathinone See Table 1 for specific values.

[0069] d. Establish multiple reaction monitoring (MRM) methods

[0070] Multi-reaction monitoring can perform two ion selections, that is, MS1 selects a certain mass of precursor ions, and after collision and fracture with gas, MS2 selects a certain mass of product ions, which greatly improves the specificity and sensitivity of the analysis.

[0071] The optimized ion source parameters are: drying gas temperature: 350℃, drying gas flow rate: 10L/min, atomizing gas pressure: 25psi.

[0072] Step (4): The sample solution is detected according to the above detection conditions.

[0073] Step (5): After calculation, the concentration of cathinone in urine sample 1 is 482ng/mL, the concentration of dimethylcathinone in urine sample is 476ng/mL, and the concentration of 3-methylmethcathinone in urine sample is 486ng/mL.

[0074] Example 2

[0075] The LC/MS method for analyzing cathinone, methcathinone, and 4-methylmethcathinone in blood includes the following steps:

[0076] (1) Preparation of sample solution;

[0077] (2) Preparation of standard working fluid;

[0078] (3) Setting of testing conditions;

[0079] (4) LC/MS determination;

[0080] (5) Calculation of experimental results, among which,

[0081] Step (1): Take three blank blood samples of healthy volunteers and add cathinone, methcathinone and 4-methylmethcathinone to prepare urine sample one (the concentration of cathinone is 500ng/mL) , Urine sample two (methcathinone concentration of 500ng/mL) and urine sample three (4-methylmethcathinone concentration of 500ng/mL), respectively diluted with water at a volume ratio of 1:1, Add the protein precipitant acetonitrile, and add the protein precipitant until no precipitation is generated in the sample. Then, after ultrasonic vibration for 15 minutes, centrifuge at 2000r/min for 20 minutes at low speed. The supernatants are passed through the gel chromatography column. The gel chromatography column packing is biobeadSX. -3,200*15mm, eluted with a 1:1 ethyl acetate/cyclohexane solution with a volume ratio of 1.5mL/min. After purification by a gel chromatography column, the extract is obtained, and the extract is blown to near dryness by nitrogen Then, add the mobile phase (the mobile phase is the same as the mobile phase in step (3), the volume ratio of A-0.2% formic acid, B-acetonitrile and C-methanol is 60:30:10) and dilute to 2ml. Get the sample solution.

[0082] In step (2), take the standard stock solutions of cathinone, methcathinone, and 4-methylmethcathinone (methanol as solvent), respectively, and dilute them to mass concentrations of 10000, 5000, 2500, 1000, Three series of standard solutions of 500, 250, 100, 10, 1, 0.1ng/mL.

[0083] In step (3), the process of setting the liquid chromatography conditions is the same as in Example 1. The results show that the retention times of cathinone, methcathinone, and 4-methylmethcathinone are: 1.690min, 2.091, respectively min, 3.995min.

[0084] The setting process of mass spectrometry conditions is the same as in Example 1.

[0085] Step (4): The sample solution is detected according to the above detection conditions.

[0086] Step (5): It is calculated that the concentration of cathinone in blood sample 1 is 452ng/mL, the concentration of dimethylcathinone in blood sample is 443ng/mL, and the concentration of trimethylcathinone in blood sample is 451ng/mL .

[0087] Example 3

[0088] The method of LC/MS analysis of cathinone, methcathinone, 4-methylmethcathinone and their common adulterants in blood includes the following steps:

[0089] (1) Preparation of sample solution;

[0090] (2) Preparation of standard working fluid;

[0091] (3) Setting of testing conditions;

[0092] (4) LC/MS determination;

[0093] (5) Calculation of experimental results, among which,

[0094] The common adulterants are norephedrine, amphetamine, amphetamine-d8, methamphetamine, methamphetamine-d8, nicotine, ephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, coffee Inine, ketamine, dolantin, tramadol, methorphan, nitroazepam, diazepam, morphine, estazolam, codeine, cocaine, alprazolam, methadone, cannabidiol, thebaine, tetrahydrocannabis Phenols, cannabidiol, monoacetylmorphine, fentanyl, papaverine, acetylcodeine, triazolam, heroin, narcotine.

[0095] Step (1): Take blank blood from healthy volunteers, add methcathinone to prepare blood sample one (methcathinone concentration is 500ng/mL), use 17mL of a mixed solution of anisole, dichloromethane and ether, Extract the methcathinone in the sample, the volume ratio of anisole, dichloromethane and ether is 5:2:1.5, add 10mL of hydrochloric acid with 15% HCl mass fraction to the separated organic phase, and remove the organic phase Place the residue on a rapid concentrator at 35°C and concentrate to dryness, and add the mobile phase (the mobile phase is the same as the mobile phase in step (3), and the volume ratio of A-0.2% formic acid, B-acetonitrile and C-methanol is 60:30:10) After the volume is adjusted to 2ml, the sample solution is obtained.

[0096] Take two blank blood samples of healthy volunteers and add cathinone and 4-methylmethcathinone respectively to prepare blood sample two (the cathinone concentration is 500ng/mL) and blood sample three (4-methylmethcathinone). The concentration of citrinone is 500ng/mL), and 25 mL of a mixed solution of toluene, anisole, dichloromethane and ether are used to extract cathinone and 4-methylmethcathinone, toluene and anisole in the sample. The volume ratio of dichloromethane and ether is 6:3:2:1.5. Add 20 mL of hydrochloric acid with 20% HCl mass fraction to the separated organic phase. After removing the organic phase, place the residue on a rapid concentrator at 35°C. Concentrate to dryness, add the mobile phase (the mobile phase is the same as the mobile phase in step (3), the volume ratio of A-0.2% formic acid, B-acetonitrile and C-methanol is 60:30:10) after constant volume, Get the sample solution.

[0097] In step (2), take the standard stock solutions of cathinone, methcathinone, and 4-methylmethcathinone, and dilute them to mass concentrations of 10000, 5000, 2500, 1000, 500, 250, 100 respectively. , 10, 1, and 0.1ng/mL three series of standard solutions.

[0098] In step (3), the setting process of liquid chromatography and mass spectrometry is the same as in Example 1;

[0099] Among them, for cathinone, methcathinone, 4-methylmethcathinone and common adulterants, by selecting parent ions and product ions, optimizing Fragmentor voltage and CE value, obtaining the optimal MRM parameters, and then establishing a mass spectrum library, As shown in Table 1:

[0100] Table 1

[0101]

[0102]

[0103]

[0104]

[0105] The optimized ion source parameters are: drying gas temperature: 350℃, drying gas flow rate: 10L/min, atomizing gas pressure: 25psi.

[0106] Step (4): The sample solution is detected according to the above detection conditions.

[0107] Step (5): After calculation, the concentration of cathinone in blood sample 1 is 488ng/mL, the concentration of dimethylcathinone in blood sample is 463ng/mL and the concentration of tri4-methylcathinone in blood sample is 471ng/mL .

[0108] Verification example

[0109] Drawing of external standard working curve

[0110] (1) Take the standard stock solution of cathinone and dilute sequentially into a series of standard solutions with mass concentrations of 10000, 5000, 2500, 1000, 500, 250, 100, 10, 1, 0.1 ng/mL. Take 3 μL of standard solutions of different concentrations for injection, and select the transition of 150.1→117 to record the peak area of ​​the response. Inject 3 times for each concentration, and perform linear regression on the average value A of the 3 peak areas and the mass concentration c (ng/mL), and the regression equation of cathinone is A=43.771c+636.96, r 2 =0.9999, the linear range is 10-1000ng/mL, the detection limit is 0.1ng/mL (S/N≥3), and the system deviations are all below 12%, the standard working curve is as follows figure 1 Shown.

[0111] (2) Take the standard stock solution of methcathinone and dilute sequentially into a series of standard solutions with mass concentrations of 10000, 5000, 2500, 1000, 500, 250, 100, 10, 1, and 0.1 ng/mL. Take 3μL of standard solutions of different concentrations for injection, and select the transition from 164.2→131.1 to record the peak area of ​​the response. Inject 3 times for each concentration, and perform linear regression on the average value A of the 3 times peak area and the mass concentration c (ng/mL), and the regression equation of methcathinone is A=1054.9c-73.697, r 2 =0.9999, the linear range is 1-1000ng/mL, the detection limit is 0.04ng/mL (S/N≥3), and the system deviations are all below 10%, the standard working curve is as follows figure 2 Shown.

[0112] (3) Take the 4-MMC standard stock solution and dilute sequentially into a series of standard solutions with mass concentrations of 10000, 5000, 2500, 1000, 500, 250, 100, 10, 1, and 0.1 ng/mL. Take 3μL of standard solutions of different concentrations for injection, and select the transition from 178.2→160.2 to record the peak area of ​​the response. Inject 3 times for each concentration, and perform linear regression on the average value A of the 3 peak areas and the mass concentration c (ng/mL), and the regression equation of 4-MMC is A=108.59c+1079.3, r 2 =0.9998, the linear range is 10-1000ng/mL, the detection limit is 0.1ng/mL (S/N≥3), and the system deviations are all below 11%, the standard working curve is as follows image 3 Shown.