A method for detecting chiral isomers of ketamine in biological samples

Abstract

The application discloses a method for detecting chiral isomers of ketamine in biological samples, comprising the following steps: 1, preparing standard substance working solution and internal standard working solution; 2, sample processing: for urine samples, adding internal standard working solution and methanol, centrifuging, filtering, and analyzing supernatant; for hair samples, washing, drying, cutting, adding internal standard working solution and methanol, freezing, grinding, ultrasonic treatment, centrifuging, filtering, and analyzing supernatant; 3, using a UHPLC-MS / MS system for sample analysis. The method realizes baseline separation of chiral isomers of ketamine by using a chiral chromatographic column combined with LC-MS / MS, and ensures effective identification and analysis of chiral isomers of ketamine. The method is applied to analysis of chiral isomers of ketamine in hair samples for the first time, and is also suitable for detection of chiral ketamine in urine; the pre-treatment process is simple, sensitive and fast, and can meet the needs of actual case inspection.

Description

Technical Field

[0001] This invention relates to the field of ketamine detection technology, and in particular to a method for detecting chiral isomers of ketamine in biological samples. Background Technology

[0002] Ketamine is a racemic mixture of two chiral isomers (R-ketamine and S-ketamine), used clinically for intravenous anesthesia. Compared to racemic ketamine, S-ketamine has a stronger analgesic effect and fewer psychogenic side effects, and has been used as a clinical anesthetic and analgesic since the 1990s. In 2023, Sukailang (esketamine hydrochloride nasal spray), whose main component is S-ketamine, was approved for marketing in my country by the National Medical Products Administration and is used as an antidepressant. The therapeutic range of racemic and monochiral isomers may differ, so chiral analysis methods are needed to provide important information about the drug isomers used. Chiral analysis of ketamine used clinically can reveal whether an individual is taking a monochiral or racemic isomer, which may help explain its therapeutic or toxic effects. Simultaneously, chiral analysis of ketamine in biological samples is of great significance for controlling the manufacture and consumption of illicit ketamine.

[0003] Current analytical methods for determining chiral ketamine in biological samples include high-performance liquid chromatography (HPLC), capillary electrophoresis, and supercritical fluid chromatography (SFC). However, these methods are time-consuming, have limited resolution, or require sophisticated equipment. Therefore, it is necessary to develop a faster and simpler analytical method for chiral ketamine in biological samples with better resolution. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for detecting chiral isomers of ketamine in biological samples.

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

[0006] A method for detecting chiral isomers of ketamine in biological samples is provided, 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-d4 and dilute it with methanol to prepare ketamine-d4 internal standard working solution.

[0008] Step 2, Sample processing:

[0009] For urine samples, add ketamine-d4 internal standard working solution to the urine sample, then add methanol, centrifuge, filter through a filter membrane, and use the supernatant for analysis;

[0010] For hair samples, the hair samples are first cleaned, dried and then shredded. Then, ketamine-d4 internal standard working solution and methanol are added, the samples are freeze-ground and then sonicated. After centrifugation, the samples are filtered through a filter membrane, and the supernatant is used for analysis.

[0011] Step 3: Use a UHPLC-MS / MS system for sample injection and analysis;

[0012] The chromatographic conditions are as follows:

[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, isocratic elution separation A:B = 20:80, constant current elution for 10-15min, column temperature set to 40-45℃, injection volume 5-8μL.

[0014] Furthermore, the chromatographic conditions are as follows:

[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, isocratic elution separation A:B=20:80, constant flow elution for 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 8–1000 ng / mL.

[0017] Furthermore, the specific parameters in the cryogenic grinding process are as follows: temperature, -39℃; speed, 18m / s; number of cycles, 15; precooling time, 40s; grinding time, 40s; residence time, 20s.

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

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

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

[0021] The method of this invention utilizes a chiral chromatographic column combined with LC-MS / MS technology to achieve baseline separation of chiral isomers of ketamine very effectively, ensuring the efficient identification and analysis of chiral isomers of ketamine. This method is the first to be 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, meeting the needs of practical case examination. Attached Figure Description

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

[0023] Figure 2 Chromatograms of blank hair sample (a) and blank urine sample (b).

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

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0026] Example 1

[0027] This embodiment provides a method for detecting chiral isomers of ketamine in biological samples, specifically including:

[0028] 1.1 Standard substances and reagents

[0029] R-ketamine and S-ketamine (1.0 mg / mL) were purchased from Shanghai Yuansheng Technology Co., Ltd., and the internal standard ketamine-d4 (1.0 mg / mL) was purchased from Cerilliant, Texas Instruments, USA. The purity of all standards was greater than 99%.

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

[0031] 1.2 Instrument Conditions

[0032] The BeadRuptor cryogenic grinder was purchased from Shanghai Jingxin Industrial Development Co., Ltd., the MiniSpin high-speed centrifuge from Eppendorf GmbH, Germany, and the BSA124S electronic balance from Sartorius Scientific Instruments Co., Ltd., Beijing. Ultrapure water was supplied by Merck Q- The Element system was prepared using (Darmstadt, Germany).

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

[0034] 1.3 Solution Preparation

[0035] Accurately measure appropriate amounts of two standard substances (R-ketamine and S-ketamine), dilute with methanol to a final volume of 5 μg / mL to prepare standard stock solutions. These stock solutions are then serially diluted with methanol to prepare working solutions with concentrations of 1000, 500, 400, 200, 100, 50, 20, 10, and 8 ng / mL. Ketamine-d4 internal standard working solutions with concentrations of 100 ng / mL and 40 ng / mL are prepared in methanol. All solutions are stored at -20°C for future use.

[0036] Mobile phase A (20 mmol / L ammonium acetate buffer solution containing 0.1% formic acid): Weigh 0.77 g of ammonium acetate and place it in a 500 mL volumetric flask. Dissolve it in some ultrapure water, then add 0.5 mL of 98% formic acid solution. Dilute with ultrapure water and bring the volume to 500 mL. Mix 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 ketamine-d4 internal standard working solution (100 ng / mL) to 50 μL of urine, followed by 950 μL of methanol. Centrifuge the sample at 12100 × g for 5 min, filter the supernatant through a polytetrafluoroethylene (PTFE) membrane, and use the supernatant for instrument analysis.

[0041] 1.4.2 Hair

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

[0043] Hair samples were washed twice each with water and acetone, and then air-dried at room temperature. The hair was then shredded into 2-3 mm pieces, thoroughly mixed, and 20 mg of hair was weighed into a 2 mL grinding tube containing ceramic grinding beads. 10 μL of ketamine-d4 (40 ng / mL) internal standard working solution and 390 μL of methanol were added. The hair sample was ground using a cryogenic grinder with the following parameters: temperature -39℃; speed 18 m / s; number of cycles 15; pre-cooling time 40 s; grinding time 40 s; residence time 20 s. After grinding, the sample was sonicated for 10 min, centrifuged at 12100 × g for 5 min, and the supernatant was filtered through a polytetrafluoroethylene (PTFE) membrane for instrument analysis.

[0044] 1.5 Instrument Conditions

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

[0046] The above chromatographic conditions were obtained through the following optimization:

[0047] use A 5 μm Cellulose-3 chiral column (250 mm × 4.6 mm) was used to separate R-ketamine and S-ketamine in a mobile phase of 20 mmol / L ammonium acetate buffer (mobile phase A) containing 0.1% formic acid and acetonitrile (mobile phase B), with a retention time of approximately 12 min. Using a shorter column of the same type (150 mm) could shorten the retention time by 6 min, but the separation effect was poor; therefore, the mobile phase was optimized. Adding formic acid to water slightly enhanced the ketamine response. Adding either ammonium acetate or ammonium formate improved the peak shape, but ammonium acetate showed a better improvement. The effect of different concentrations of ammonium acetate was studied, and it was found that higher concentrations of ammonium acetate resulted in better peak shapes. Comparing the effects of the organic phases methanol and acetonitrile, it was found that ketamine had a better response when using methanol. Therefore, methanol was chosen as the organic phase. Figure 1 Optimizing column temperature can improve the symmetry of chromatographic peaks. The peak shape is optimal at a column temperature of 40℃, and the resolution improves to 1.11. Simultaneously, increasing the column temperature lowers the column pressure, which helps extend the column's lifespan. Therefore, choosing... Ketamine enantiomers were separated at 40 °C by isocratic elution with 20% ammonium acetate buffer (20 mmol / L) 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 ionization (ESI) positive ion and multiple reaction monitoring (MRM) modes. The optimized mass spectrometry conditions were 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 this invention was validated according to the international standard (ANSI / ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology). Evaluation parameters mainly included 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] Selective testing was performed on blank urine and hair samples from 10 healthy volunteers to ensure that there was no interference from other substances during the peak elution time of the target component.

[0056] By comparing the chromatograms of various compounds in 10 blank urine and hair samples, it was found that endogenous substances did not interfere with the target analyte and internal standard. The chromatograms are shown below. Figure 2 As shown.

[0057] 2.2 LOD and LOQ

[0058] LOD refers to the lowest detectable concentration of an analyte in urine and hair. It should have a signal-to-noise ratio (S / N) ≥ 3, and the retention time, peak shape, and ion abundance ratio should be consistent with the standard. LOQ is the lowest concentration point within the linear range that meets the requirement of S / N ≥ 10, and the precision and accuracy should be 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 Linear

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

[0062] This invention uses a least-squares linear regression model for calculation. The calibration curves for R-ketamine and S-ketamine in urine range from 2 to 500 ng / mL, while the linear range for both substances in hair is 5 to 1000 pg / mg. A weighted (1 / x) linear regression was used, with a correlation coefficient r > 0.995.

[0063] 2.4 Precision and Accuracy

[0064] Precision and accuracy were evaluated for four quality control (QC) samples at four concentrations: LOQ, low, medium, and high. Three replicates were performed for each concentration over five consecutive days. Precision of the analytical method describes the closeness of repeated individual measurements of urine and hair samples, 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, the CV (%) should be less than 20%, and the bias should be within ±20%. For other concentrations of QC samples, the 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%–4.5% and 0.6%–2.5%, respectively, and the inter-day precision (%CV) was 3.5%–10.3% and 6.5%–8.5%, respectively. In hair, the intra-day precision (%CV) of R-ketamine and S-ketamine was 2.4%–3.9% and 1.0%–5.3%, respectively, and the inter-day precision (%CV) was 4.3%–5.9% and 3.7%–7.3%, respectively. The accuracy of the two substances in urine and hair was -11.5%–7.8% and -9.9%–9.1%, respectively. The data showed good precision and accuracy, meeting the requirements of international standards. The linearity, accuracy, and precision of this method are shown in Table 2.

[0066] Table 2

[0067]

[0068]

[0069] 2.5 Residual Effects

[0070] A blank sample was analyzed immediately after the sample at the highest concentration point of the calibration curve to evaluate the delayed effect. The determination was repeated three times.

[0071] No residues were found in the blank urine and hair samples after analysis of urine samples spiked at 500 ng / mL and hair samples spiked at 1000 pg / mg.

[0072] 2.6 Matrix effect and extraction recovery

[0073] 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 matrix effect and extraction recovery at low concentrations (5 ng / mL in urine, 10 pg / mg in hair) and high concentrations (400 ng / mL in urine, 1000 pg / mg in hair). Three groups of samples were prepared: Group A consisted of peak areas of a standard solution of a certain concentration; Group B consisted of peak areas of blank hair and urine samples after extraction with the corresponding concentration of standard solution added; and Group C consisted of peak areas of blank hair and urine samples before extraction with the corresponding concentration of standard solution added. The ratio of B / A represents the matrix effect, and the ratio of C / B represents the extraction recovery.

[0074] The recoveries and matrix effects of the two substances in urine were 71.4%–89.3% and 84.2%–126.2%, respectively, while those in hair were 98.3%–102.9% and 59.8%–86.9%, respectively. The coefficients of variation for both matrix effect and extraction recovery were within 15%. After correction with internal standards, the matrix effects of the two analytes were 90.0%–127.4% (%CV 2.6%–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 using three parallel samples at low and high concentrations. Specifically, this included autosampler stability, freeze-thaw cycle stability, and long-term stability. Three urine samples of each concentration were prepared and placed in an autosampler at 4°C for 24 hours to assess the stability of the treated samples. Freeze-thaw cycle stability was evaluated by placing three quality control samples of each concentration at -20°C for 24 hours, then thawing them at room temperature for 24 hours, repeating this cycle three times. After storage at -20°C for one month, long-term stability was assessed by pretreatment of three parallel urine samples of each concentration. To assess the stability of substances in treated hair samples, precision and accuracy data for three parallel samples of each concentration were analyzed after storage in an autosampler at 4°C for 24 hours.

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

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

[0082] 2.8 Dilution Reliability

[0083] Urine samples of 1000 ng / mL and hair samples of 10 ng / mg were diluted 50-fold using a blank matrix, and three parallel samples were prepared for each type of 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. The results showed that the precision (%CV) of R-ketamine and S-ketamine in urine was 2.1% and 2.3%, respectively, with accuracies of 12.1% and 11.8%. In hair, the precision (%CV) of R-ketamine and S-ketamine was 4.5% and 3.9%, respectively, with accuracies of 2.0% and 0.8%. The accuracy and precision values ​​of the diluted samples were all within 15%.

[0085] Example 3

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

[0087] Example 4

[0088] After methodological validation, this method was applied to the analysis of chiral ketamine in 45 real hair samples. The results showed that all 45 hair samples contained both R-ketamine and S-ketamine, with mass fractions 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 positive sample is shown below. Figure 3 As shown in (b).

[0089] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting chiral isomers of ketamine in biological samples, characterized in that, Includes the following steps: 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-d4 and dilute it with methanol to prepare ketamine-d4 internal standard working solution. Step 2, Sample processing: For urine samples, add ketamine-d4 internal standard working solution to the urine sample, then add methanol, centrifuge, filter through a filter membrane, and use the supernatant for analysis; For hair samples, the hair samples were first cleaned, dried, and then shredded. Then, ketamine-d4 internal standard working solution and methanol were added, followed by cryogenic grinding and ultrasonic treatment. After centrifugation, the samples were filtered through a filter membrane, and the supernatant was used for analysis. The specific parameters for the cryogenic grinding process were: temperature, -39℃; speed, 18m / s; number of cycles, 15; pre-cooling time, 40s; grinding time, 40s; residence time, 20s. Step 3: Use a UHPLC-MS / MS system for sample injection and analysis; The chromatographic conditions are as follows: Column: Lux ® 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, isocratic elution separation A:B=20:80, constant flow elution for 10min, column temperature set to 40℃, autosampler temperature set to 4℃, injection volume: 5μL; The mass spectrometry conditions were as follows: operation in electrospray positive ion and multiple reaction monitoring (MRM) mode, ion spray voltage: 5000V; ion source temperature: 550℃; collision gas: 9psi; curtain gas: 35psi; nebulizer gas: 55psi; auxiliary gas: 55psi.

2. 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 8~1000ng / mL.

3. 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 0.22μm polytetrafluoroethylene filter membrane.

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