Method for detecting ketamine chiral isomer in biological detection material

Abstract

The invention discloses a method for detecting ketamine chiral isomers in a biological detection material. The method comprises the following steps: 1, preparing a standard substance working solution and an internal standard working solution; 2, sample treatment: adding an internal standard working solution and methanol into a urine sample, centrifuging, filtering, and analyzing supernate; the method comprises the following steps: cleaning and drying a hair sample, cutting into pieces, adding an internal standard working solution and methanol, freezing and grinding, performing ultrasonic treatment, centrifuging, filtering, and analyzing supernate; and 3, adopting a UHPLC-MS / MS system, and carrying out sample introduction analysis. According to the method disclosed by the invention, the baseline separation of the ketamine chiral isomer is realized by combining a chiral chromatographic column with LC-MS / MS, and the effective recognition and analysis of the ketamine chiral isomer are ensured. The method is applied to analysis of ketamine chiral isomers in hair samples for the first time, and is also suitable for detection of chiral ketamine in urine; the pretreatment process is simple, sensitive and rapid, and can meet the requirements of actual case inspection.

Description

Technical Field

[0001] The present invention relates to the technical field of ketamine detection, and in particular to a method for detecting chiral isomers of ketamine in a biological sample. Background Art

[0002] Ketamine is a racemic mixture of two chiral isomers (R-ketamine and S-ketamine) and is used clinically for intravenous anesthesia. Compared with racemic ketamine, S-ketamine has stronger analgesic effects and fewer psychotomi- cal side effects. It has been used as a clinical anesthetic and analgesic in the 1990s. In 2023, Sukailang (esketamine hydrochloride nasal spray), the main ingredient of which is S-ketamine, was approved for marketing by my country's State Food and Drug Administration and used as an antidepressant. The therapeutic range of racemates and single chiral isomers may be different, so chiral analysis methods are needed to provide important information about the isomers of the drug used. Chiral analysis of ketamine used clinically can reveal whether the type of drug taken by an individual is a single chiral isomer or a racemate, which may help explain its therapeutic or toxic effects. At the same time, chiral analysis of ketamine in biological samples is of great significance for controlling the manufacture and consumption of illegal ketamine.

[0003] At present, the analytical methods for the determination of chiral ketamine in biological samples include high performance liquid chromatography (HPLC), capillary electrophoresis and supercritical fluid chromatography (SFC). However, the above methods are time-consuming, have limited separation, or have high requirements for instruments and equipment. Based on the above situation, it is necessary to develop a method for the analysis of chiral ketamine in biological samples with better separation, rapidity and simplicity. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting chiral isomers of ketamine in biological samples in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Provided is a method for detecting chiral isomers of ketamine in a biological sample, comprising the following steps:

[0007] Step 1: weigh R-ketamine and S-ketamine standard substances and dilute them with methanol to prepare standard substance stock solutions, and then prepare standard substance working solutions by stepwise dilution; weigh ketamine-d 4 Dilute with methanol to prepare ketamine-d 4 Internal standard working solution;

[0008] Step 2: Sample processing:

[0009] For urine samples, ketamine-d 4The internal standard working solution was then added with methanol, centrifuged and filtered through a membrane, and the supernatant was used for analysis;

[0010] For hair samples, the hair samples were washed, dried and chopped, and then ketamine-d was added. 4 The internal standard working solution and methanol were cryogenically ground and then sonicated, centrifuged and filtered through a membrane, and the supernatant was used for analysis;

[0011] Step 3, using UHPLC-MS / MS system for sample injection and analysis;

[0012] The chromatographic conditions are:

[0013] Chromatographic column: 3μm Cellulose-3 chiral column, 150mm×4.6mm; mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid; mobile phase B: methanol; flow rate: 0.30-0.5mL / min, A:B=20:80 isocratic elution separation constant flow elution 10-15min, column temperature set to 40-45℃, injection volume 5-8μL.

[0014] Further, the chromatographic conditions are:

[0015] Chromatographic column: 3μm Cellulose-3 chiral column, 150mm×4.6mm; mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid; mobile phase B: methanol; flow rate: 0.30mL / min, A:B=20:80 isocratic elution separation constant flow elution 10min, column temperature set to 40℃, autosampler temperature set to 4℃, injection volume 5μL.

[0016] Furthermore, the concentration range of the standard substance working solution is 8 to 1000 ng / mL.

[0017] Furthermore, the specific parameters in the freezing and grinding process are: temperature, -39°C; speed, 18m / s; number of cycles, 15 times; precooling time, 40s; grinding time, 40s; and residence time, 20s.

[0018] Furthermore, the supernatant needs to be filtered through a 0.22 μm polytetrafluoroethylene filter membrane.

[0019] Furthermore, the mass spectrometry conditions were: operating in electrospray positive ion and multiple reaction monitoring (MRM) mode, ion spray voltage: 5000 V; ion source temperature: 550° C.; collision gas: 9 psi; curtain gas: 35 psi; nebulizer gas: 55 psi; auxiliary gas: 55 psi.

[0020] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0021] The method of the present invention utilizes a chiral chromatographic column combined with LC-MS / MS technology to very effectively achieve baseline separation of chiral isomers of ketamine, thereby ensuring effective identification and analysis of chiral isomers of ketamine. This method is first applied to the analysis of chiral isomers of ketamine in hair samples, and is also applicable to the detection of chiral ketamine in urine; its pretreatment process is simple, sensitive and rapid, and can meet the needs of actual case inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effects of mobile phase A and mobile phase B on the chiral separation of ketamine are shown; wherein, (a) mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: methanol; (b) mobile phase A: 10mmol / L ammonium formate buffer containing 0.1% formic acid, mobile phase B: methanol; (c) mobile phase A: 10mmol / L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: methanol; (d) mobile phase A: 20mmol / L ammonium formate buffer solution containing 0.1% formic acid, mobile phase B: methanol; (e) mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: methanol; (f) mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: acetonitrile.

[0023] Figure 2 The chromatograms of blank hair sample (a) and blank urine sample (b) are shown.

[0024] Figure 3 The chromatograms of R-ketamine and S-ketamine in actual cases: actual urine sample (a) and actual hair sample (the mass fraction of R-ketamine is 437.2 pg / mg, the mass fraction of S-ketamine is 310.8 pg / mg) (b). DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0026] Example 1

[0027] This embodiment provides a method for detecting chiral isomers of ketamine in a biological sample, which specifically includes:

[0028] 1.1 Standard substances and reagents

[0029] R-ketamine and S-ketamine (1.0 mg / mL) were purchased from Shanghai Yuansi Technology Co., Ltd., and the internal standard ketamine-d 4 (1.0 mg / mL) was purchased from Cerilliant, Texas, USA. The purity of all standard substances was greater than 99%.

[0030] Chromatographic grade ammonium acetate, formic acid (content greater than 98%) and methanol were purchased from Shanghai Anpu Laboratory Technology Co., Ltd.; polytetrafluoroethylene filter membrane (0.22 μm) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0031] 1.2 Instrument conditions

[0032] The cryo-grinding instrument (BeadRuptor system) was purchased from Shanghai Jingxin Industrial Development Co., Ltd., the MiniSpin high-speed centrifuge was purchased from Eppendorf, Germany, and the BSA124S electronic balance was purchased from Beijing Sartorius Scientific Instrument Co., Ltd. Ultrapure water was purchased from Merck Q- The Element system was prepared and used (Darmstadt, Germany).

[0033] The UHPLC-MS / MS system is composed of an ACQUITYUPLC H-Class PLUS system (Waters, USA) and an API5500TRAP quadrupole-linear ion trap mass spectrometer (AB Sciex, USA). Chromatographic conditions: 3 μm Cellulose-3 chiral column (150 mm×4.6 mm). Data were processed using Analyst 1.5 software and MultiQuant 3.0.2 workstation.

[0034] 1.3 Solution preparation

[0035] Accurately measure the two standard substances (R-ketamine and S-ketamine), dilute with methanol and make the volume to 5 μg / mL to prepare the standard substance stock solution, and use methanol to dilute step by step to make the standard substance working solution with concentrations of 1000, 500, 400, 200, 100, 50, 20, 10, and 8 ng / mL. Prepare ketamine-d in methanol with concentrations of 100 ng / mL and 40 ng / mL. 4 Internal standard working solution. All solutions were stored in a refrigerator at -20°C until use.

[0036] Mobile phase A (20 mmol / L ammonium acetate buffer solution containing 0.1% formic acid): Weigh 0.77 g of ammonium acetate, place it in a 500 mL volumetric flask, dissolve it with some ultrapure water, then add 0.5 mL of 98% formic acid solution, dilute it with ultrapure water and make up to 500 mL, mix it thoroughly before use.

[0037] 1.4 Sample collection and preparation

[0038] 1.4.1 Urine

[0039] Blank urine samples were provided by healthy volunteers in the laboratory.

[0040] Add 10 μL of 100 ng / mL ketamine-d to 50 μL of urine. 4 Internal standard working solution, then 950 μL methanol was added. The sample was centrifuged at 12100 × g for 5 min, and the supernatant was filtered through a polytetrafluoroethylene filter membrane for instrument analysis.

[0041] 1.4.2 Hair

[0042] Blank hair samples were provided by healthy volunteers in the laboratory.

[0043] The hair was washed twice with water and twice with acetone, and then dried at room temperature. The hair was cut into pieces of 2-3 mm, mixed evenly, and 20 mg of hair was weighed and placed in a 2 mL grinding tube containing ceramic grinding beads. 10 μL of ketamine-d 4 (40ng / mL) internal standard working solution and 390μL methanol. The hair samples were ground using a cryo-grinding instrument under the following parameter settings: temperature, -39℃; speed, 18m / s; number of cycles, 15 times; precooling time, 40s; grinding time, 40s; residence time, 20s. After grinding, the sample was ultrasonically treated for 10min, centrifuged at 12100×g for 5min, and the supernatant was filtered through a polytetrafluoroethylene filter membrane for instrument analysis.

[0044] 1.5 Instrument conditions

[0045] Chromatographic column: 3μm Cellulose-3 chiral column (150mm×4.6mm); mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid; mobile phase B: methanol; flow rate: 0.30mL / min, A:B=20:80 isocratic elution separation constant flow elution 10min, column temperature set to 40℃, autosampler temperature set to 4℃, injection volume was 5μL.

[0046] The above chromatographic conditions were obtained by optimizing the following:

[0047] use A 5μm Cellulose-3 chiral column (250mm×4.6mm) was used to separate R-ketamine and S-ketamine in a 20mmol / L ammonium acetate buffer (mobile phase A) containing 0.1% formic acid and acetonitrile (mobile phase B) mobile phase, with a retention time of approximately 12min. Using a short column of the same type (150mm) can shorten the retention time by 6min, but the separation effect is poor, so the mobile phase was optimized. Adding formic acid to water can slightly enhance the response of ketamine. Adding ammonium acetate or ammonium formate can improve the peak shape, but ammonium acetate has a better improvement effect. The effects of different concentrations of ammonium acetate were studied, and it was found that higher concentrations of ammonium acetate would have better peak shapes. Comparing the effects of methanol and acetonitrile in the organic phase, it was found that ketamine had a better response when methanol was used. Therefore, methanol was selected as the organic phase ( Figure 1 ). Optimizing the column temperature can improve the symmetry of the chromatographic peak. When the column temperature is 40°C, the peak shape is optimal and the separation degree is increased to 1.11. At the same time, the increase in column temperature will reduce the column pressure, which is beneficial to extend the life of the chromatographic column. Therefore, choose Ketamine enantiomers were separated on a 3 μm Cellulose-3 chiral column (150 mm × 4.6 mm) at 40°C by isocratic elution with 20% 20 mmol / L ammonium acetate buffer containing 0.1% formic acid (mobile phase A) and 80% methanol (mobile phase B) at a flow rate of 0.30 mL / min.

[0048] The mass spectrometer was operated in electrospray positive ionization and multiple reaction monitoring (MRM) mode. The mass spectrometry conditions were optimized as follows: ion spray voltage: 5000 V; ion source temperature: 550 °C; collision gas: 9 psi; curtain gas: 35 psi; nebulizer gas: 55 psi; auxiliary gas: 55 psi. The precursor ions, product ions, retention times, declustering voltages, and collision energies of the analytes are shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] Example 2

[0053] The detection method of the present invention was validated according to international standards (ANSI / ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology). The evaluation parameters mainly include selectivity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, residual effect, matrix effect, extraction recovery, stability and dilution reliability.

[0054] 2.1 Selectivity

[0055] Blank urine and hair samples from 10 healthy volunteers were selectively tested to ensure that there was no interference from other substances within the peak time of the target component.

[0056] By comparing the chromatograms of each compound in 10 blank urine and hair samples, it was found that the endogenous substances had no interference with the target compound and the internal standard. The chromatograms are as follows: Figure 2 shown.

[0057] 2.2LOD and LOQ

[0058] LOD refers to the lowest concentration of analyte added in urine and hair that can be detected. The signal-to-noise ratio (S / N) should be ≥3, and the retention time, peak shape and ion abundance ratio should be consistent with the standard substance. LOQ is the lowest concentration point in the linear range that meets S / N ≥10, and the precision and accuracy are within ±20%.

[0059] The results are as follows: In urine samples, the LOD of R-ketamine and S-ketamine was 1 ng / mL and the LOQ was 2 ng / mL. In hair samples, the LOD of R-ketamine and S-ketamine was 4 pg / mg and the LOQ was 5 pg / mg.

[0060] 2.3 Linearity

[0061] The calibration model is used to describe the correlation between the signal response (ratio of the peak area of ​​the analyte to the internal standard) and the concentration of the analyte in the sample, and at least six different non-zero concentration points should be used to evaluate the calibration model. The spike concentrations in urine were 2, 5, 10, 50, 100, 200, 400, and 500 ng / mL, and the spike concentrations in hair were 5, 10, 50, 100, 200, 500, and 1000 pg / mg.

[0062] The present invention uses the least square linear regression model for calculation. The calibration curves of R-ketamine and S-ketamine in urine are in the range of 2-500 ng / mL, and the linear range of the two substances in hair is 5-1000 pg / mg. Weighted (1 / x) linear regression is used, and the correlation coefficient r>0.995.

[0063] 2.4 Precision and Accuracy

[0064] Precision and accuracy were evaluated for quality control (QC) samples at LOQ, low, medium and high concentrations, with 3 replicates for each concentration, and the measurements were performed for 5 consecutive days. The precision of the analytical method describes the closeness of repeated individual measurements of samples in urine and hair, expressed as the coefficient of variation (CV). Accuracy is the difference between the test result value and the added concentration value, expressed as bias. For LOQ samples, CV (%) should be less than 20%, and the bias should be within ±20%. For other concentrations of QC samples, CV (%) should be less than 15%, and the bias should be within ±15%.

[0065] In urine, the intra-day precision (%CV) of R-ketamine and S-ketamine was 1.4% to 4.5% and 0.6% to 2.5%, respectively, and the inter-day precision (%CV) of R-ketamine and S-ketamine was 3.5% to 10.3% and 6.5% to 8.5%, respectively. In hair, the intra-day precision (%CV) of R-ketamine and S-ketamine was 2.4% to 3.9% and 1.0% to 5.3%, respectively, and the inter-day precision (%CV) of R-ketamine and S-ketamine was 4.3% to 5.9% and 3.7% to 7.3%, respectively. The accuracy of the two substances in urine and hair was -11.5% to 7.8% and -9.9% to 9.1%, respectively. The data had good precision and accuracy, meeting the requirements of international guidelines. The linearity, accuracy and precision of the method are shown in Table 2.

[0066] Table 2

[0067]

[0068]

[0069] 2.5 Residual Effect

[0070] Analyze the blank sample immediately after the sample with the highest concentration in the calibration curve to evaluate the delay effect. Repeat the measurement three times.

[0071] After analyzing urine samples spiked at 500 ng / mL and hair samples spiked at 1000 pg / mg, there was no residue in the blank urine and hair samples.

[0072] 2.6 Matrix Effect and Extraction Recovery

[0073] The matrix effect and extraction recovery were calculated according to the method proposed by Matuszewski et al. Seven blank hair and urine samples were used to evaluate the matrix effect and extraction recovery at low concentrations (5 ng / mL in urine and 10 pg / mg in hair) and high concentrations (400 ng / mL in urine and 1000 pg / mg in hair). Three groups of samples were prepared: Group A was the peak area of ​​the standard substance solution at a certain concentration, Group B was the peak area of ​​the standard substance with the corresponding concentration added after the blank hair and urine samples were extracted, and Group C was the peak area of ​​the standard substance with the corresponding concentration added before the blank hair and urine samples were extracted. The ratio of B / A represents the matrix effect, and the ratio of C / B represents the extraction recovery.

[0074] The extraction recovery and matrix effect of the two substances in urine were 71.4% to 89.3% and 84.2% to 126.2%, respectively, and the extraction recovery and matrix effect in hair were 98.3% to 102.9% and 59.8% to 86.9%, respectively. The coefficient of variation of matrix effect and extraction recovery was within 15%. After correction with internal standard, the matrix effect of the two analytes was 90.0% to 127.4% (%CV was 2.6% to 12.5%). The matrix effect and extraction recovery results are shown in Table 3.

[0075] Table 3

[0076]

[0077]

[0078] 2.7 Stability

[0079] The stability of urine samples was evaluated for three parallel samples at low and high concentrations. Specifically, it includes autosampler stability, freeze-thaw cycle stability and long-term stability. Three urine samples of two concentrations were prepared and placed in a 4°C autosampler for 24 hours to evaluate the stability of the treated samples. Freeze-thaw cycle stability was evaluated by freezing three quality control samples of each concentration at -20°C for 24 hours, thawing them at room temperature for 24 hours, and repeating three freeze-thaw cycles. After storage at -20°C for one month, long-term stability was evaluated after pre-treatment of three parallel urine samples containing two concentrations. In order to evaluate the stability of substances in the treated hair samples, the precision and accuracy data of three parallel samples of two concentrations were analyzed after storage in an autosampler at 4°C for 24 hours.

[0080] Urine samples were stored in an autosampler at 4°C for 24 hours, after three freeze-thaw cycles, and in a -20°C refrigerator for four weeks. The accuracy was -12.8% to 3.2%, -11.1% to 9.8%, and -1.9% to 11.5%, respectively, and the precision was 0.9% to 9.6%, 0.4% to 7.7%, and 1.0% to 4.8%, respectively. This indicates that R-ketamine and S-ketamine are stable in urine samples stored in an autosampler at 4°C for 24 hours, in a -20°C refrigerator for four weeks, and after three freeze-thaw cycles.

[0081] The accuracy of hair samples stored in an autosampler at 4°C for 24 h was -13.6% to 9.0%, and the precision was 2.4% to 9.5%, which indicates that hair samples can be stably placed in an autosampler at 4°C for 24 h.

[0082] 2.8 Dilution Reliability

[0083] The 1000 ng / mL urine sample and the 10 ng / mg hair sample were diluted 50 times using blank matrix, and three parallel samples were prepared for each sample for analysis. The precision and accuracy deviations were within ±15%, indicating that the dilution reliability was acceptable.

[0084] Urine and hair samples were diluted 50-fold to assess dilution integrity, and the results showed that the precision (%CV) of R-ketamine and S-ketamine in urine was 2.1% and 2.3%, respectively, and the accuracy was 12.1% and 11.8%, respectively. In hair, the precision (%CV) of R-ketamine and S-ketamine was 4.5% and 3.9%, respectively, and the accuracy was 2.0% and 0.8%, respectively. The accuracy and precision values ​​of the diluted samples were within 15%.

[0085] Example 3

[0086] After methodological validation, the method was applied to the analysis of chiral isomers of ketamine in urine in an actual case. The results showed that only S-ketamine was present in urine with a mass concentration of 2005 ng / mL. Figure 3 (a) shown.

[0087] Example 4

[0088] After methodological validation, the method was applied to the analysis of chiral ketamine in 45 real hair samples. The results showed that all 45 hair samples contained R-ketamine and S-ketamine, with the mass fractions of R-ketamine and S-ketamine ranging from 30 to 52666 pg / mg (median 751 pg / mg) and 31 to 43234 pg / mg (median 815 pg / mg), respectively. The chromatogram of one of the positive samples is shown in the figure below. Figure 3 (b) as shown.

[0089] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting chiral isomers of ketamine in biological samples, characterized in that: The steps include: Step 1, weigh R-ketamine and S-ketamine standard substances and dilute them with methanol to prepare standard substance stock solutions, and prepare standard substance working solutions by stepwise dilution; weigh ketamine-d4 and dilute it with methanol to prepare ketamine-d4 internal standard working solution; Step 2: Sample processing: For urine samples, ketamine-d4 internal standard working solution was added to the urine sample, followed by methanol, centrifugation and filtration through a membrane, and the supernatant was used for analysis; For hair samples, the hair samples were first washed, dried and chopped, then ketamine-d4 internal standard working solution and methanol were added, and then cryo-grinded and ultrasonicated, centrifuged and filtered through a filter membrane, and the supernatant was used for analysis; Step 3, using UHPLC-MS / MS system for sample injection and analysis; The chromatographic conditions are: Chromatographic column: 3μm Cellulose-3 chiral column, 150mm×4.6mm; mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid; mobile phase B: methanol; flow rate: 0.30-0.5mL / min, A:B=20:80 isocratic elution separation constant flow elution 10-15min, column temperature set to 40-45℃, injection volume 5-8μL.

2. The method for detecting chiral isomers of ketamine in biological samples according to claim 1, characterized in that: The chromatographic conditions are: Chromatographic column: 3μm Cellulose-3 chiral column, 150mm×4.6mm; mobile phase A: 20mmol / L ammonium acetate buffer solution containing 0.1% formic acid; mobile phase B: methanol; flow rate: 0.30mL / min, A:B=20:80 isocratic elution separation constant flow elution 10min, column temperature set to 40℃, autosampler temperature set to 4℃, injection volume 5μL.

3. The method for detecting chiral isomers of ketamine in biological samples according to claim 1, characterized in that: The concentration range of the standard substance working solution is 8-1000 ng / mL.

4. The method for detecting chiral isomers of ketamine in biological samples according to claim 1, characterized in that: The specific parameters in the freezing and grinding process are: temperature, -39°C; speed, 18 m / s; number of cycles, 15 times; precooling time, 40 s; grinding time, 40 s; and residence time, 20 s.

5. The method for detecting chiral isomers of ketamine in biological samples according to claim 1, characterized in that: The supernatant needs to be filtered through a 0.22 μm polytetrafluoroethylene filter membrane.

6. The method for detecting chiral isomers of ketamine in biological samples according to claim 1, characterized in that: The mass spectrometry conditions were as follows: operating in electrospray positive ion and multiple reaction monitoring (MRM) mode, ion spray voltage: 5000 V; ion source temperature: 550 °C; Collision gas: 9psi; Air curtain gas: 35psi; Atomizing gas: 55psi; Auxiliary gas: 55psi.

Images