CN107478757B - Purification and preparation method of ketamine standard substance for forensic drug detection - Google Patents

Abstract

The invention discloses a method for purifying and preparing a ketamine standard substance used in forensic drug detection, comprising the following steps: (1) detecting the purity of ketamine in a seized ketamine sample, and selecting a seized ketamine sample whose mass fraction of ketamine is greater than or equal to 50 wt % as the purified preparation The raw material of ketamine standard substance; (2) utilize high performance liquid chromatography to prepare ketamine standard substance. The ketamine obtained by the preparation and purification method of the present invention is confirmed by nuclear magnetic resonance, liquid chromatography-tandem mass spectrometry, and infrared spectrum analysis, and its purity is determined by liquid chromatography and gas chromatography, and the chromatographic non-responsive impurities are measured; according to the standard The material development requirements, its stability, uniformity, definite value, and estimation of total uncertainty all meet the relevant regulations and meet the expected indicators.

Description

Purification and preparation method of ketamine standard substance for forensic drug detection

technical field

The invention relates to the preparation of forensic science drug standard products. More specifically, it relates to a method for purifying and preparing ketamine standard substances used in forensic drug detection.

Background technique

At present, the increasingly serious drug problem has become a global disaster. The proliferation of drugs directly endangers people's physical and mental health, and poses a huge threat to economic development and social progress. Therefore, establishing a forensic scientific drug detection quantity traceability system, improving the measurement technology of drug components, ensuring the reliability and comparability of measurement results, establishing the sharing and mutual recognition of measurement data, and providing accurate and reliable evidence for courts has become a national standard in the world. A general concern of drug identification agencies.

Drug composition measurement technology and traceability guarantee are an organic whole, which is an important embodiment of core measurement capabilities in the field of forensic drug composition measurement. The reference material is the skeleton that runs through the whole, the carrier of the quantity value, the key element of the traceability system of drug ingredients, the important basis for ensuring the accuracy and comparability of the measurement results in time and space, and the realization of effective measurement that is The fundamental guarantee for accurate, comparable and traceable measurements.

Most of the advanced drug testing laboratories in Europe and the United States use internationally recognized reference materials produced by companies such as Sigma, but in my country we can only rely on imported reference materials, which are small in quantity and high in price, and many of them cannot be provided to China. At present, the types of "reference substances" used in domestic drug analysis are not only extremely limited, but also generally lack corresponding technical indicators such as perfect structure identification, purity determination, uniformity and stability. All of these bring certain uncertainties to the detection of drug-related cases and directly affect the accuracy of quantitative results. Moreover, the lack of domestic drug standard substances has become a major obstacle restricting my country from realizing the standardization of forensic drug detection chemical measurement methods, traceability and mutual recognition of measurement results. Therefore, the ability to prepare drug reference substances for forensic drug detection has become an urgent problem in the field of drug research in my country.

Ketamine’s English common name: (±)-Ketamine hydrochloride, chemical name: 2-o-chlorophenyl-2-tetraamino-cyclohexanone hydrochloride, English name: (±)-2-(2-Chlorophenyl)- 2-(methylamino)cyclohexanone hydrochloride, molecular formula: C ₁₃ H ₁₆ ClNO·HCl, molecular weight: 274.19, CA registration number: 1867-66-9, structural formula:

Physical and chemical properties: white crystalline powder, odorless. Soluble in water (200mg/mL), soluble in hot ethanol, insoluble in ether and benzene. The melting point is 259-263°C. The aqueous solution is acidic, with a pH value of 4.0-5.5.

Contents of the invention

An object of the present invention is to provide a method for purifying and preparing ketamine standard substances used in forensic drug testing.

To achieve the above object, the present invention adopts the following technical solutions:

The method for purifying and preparing a ketamine standard substance for forensic drug detection comprises the following steps: (1) detecting the purity of ketamine in a seized ketamine sample, and selecting a seized ketamine sample whose mass fraction of ketamine is greater than or equal to 50wt% as a purified ketamine standard substance raw materials;

(2) Preparation of ketamine standard substance by high performance liquid chromatography.

The above-mentioned purification preparation method for the ketamine standard substance used for forensic science drug detection, in step (1), comprises the following steps:

(1.1) Preparation of sample solution: 1.0mg ketamine sample was dissolved in 1mL methanol to prepare 1.0mg/mL ketamine sample solution for qualitative and quantitative analysis. Before use, the sample solution was filtered through a 0.45 μm microporous membrane;

(1.2) Determine liquid chromatography condition: chromatographic column is Shim-pack HRC-ODS post, 250mm * 4.6mm ID, 5 μ m; Mobile phase is V _methanol : V _{0.05% trifluoroacetic acid/water} =(33-35):( 65-67), isocratic elution; UV detection wavelength 210nm; flow rate 1.0mL/min; column temperature 35°C;

(1.3) Calculate the regression equation of the standard curve and determine the linear range: dilute the ketamine standard stock solution with chromatographically pure methanol, and precisely prepare ketamine controls with concentrations of 0.5, 1, 5, 10, 50, 100, 500, and 1000 μg/mL Product solution, measured by the reversed-phase chromatographic conditions in step (1.2), each concentration was repeated 3 times, calculated with the average value, and recorded the ketamine chromatographic peak area, taking the injection concentration of the reference substance as the abscissa, and the concentration is μg/mL , the chromatographic peak area value is plotted on the ordinate, and calculates the regression equation of the standard curve; plots the concentration with the peak area, and the regression equation of the standard curve is:

Y=3×10 ⁷ X+92617, R ² =0.9999,

Ketamine has a good linear relationship in the range of 0.5--1000μg/mL;

(1.4) Analyze and measure the ketamine sample solution whose concentration is 1.0 mg/mL according to the reverse phase chromatography method in step (1.2), repeat the measurement 3 times, record the peak area of ketamine, calculate its average value, and calculate the sample by the peak area external standard method ketamine content in .

The above-mentioned purification preparation method for the ketamine standard substance used for forensic science drug detection, in step (2), comprises the following steps:

(2.1) Preparation of sample solution: Dissolve 3.369g of ketamine sample in 10mL of methanol first, centrifuge after fully dissolving, filter to remove precipitate, take out the organic phase, concentrate to dryness by centrifugation, add water to make up to 10mL, and prepare ketamine sample solution for use in Preparative high-performance liquid chromatography was used to separate and prepare ketamine standard products, and the sample solution was filtered through a 0.45 μm microporous membrane before use;

(2.2) Determine liquid chromatography conditions:

(2.2.1) The chromatographic column is a Shim-pack VP-ODS preparative column (250mm×20mm ID, 15μm); the mobile phase is V _methanol : V _water = 30:70, methanol and water are fully mixed and degassed before use, etc. Ultraviolet detection wavelength 210nm; flow rate 8mL/min; sample volume 700μL; column temperature at room temperature; wash the column with 100% methanol after running for 20 minutes; or

(2.2.2) The chromatographic column is a Shim-pack VP-ODS preparative column (250mm×20mm ID, 15 μm); the mobile phase is V _methanol : V _{0.05% trifluoroacetic acid/water} =20:80, isocratic elution; UV The detection wavelength is 210nm; the flow rate is 8mL/min; the sample volume is 500μL; the column temperature is room temperature;

(2.3) Under the chromatographic conditions in the step (2.2.2), after the collected fractions are removed by rotary evaporation, the pH of the remaining aqueous solution is adjusted to 11, extracted with chromatographically pure chloroform, the organic phases are combined, and concentrated by centrifugation to A white precipitate appears, inject water into it, slowly add 0.1N hydrochloric acid until the pH of the aqueous solution reaches 5-6, remove the organic phase, and freeze-dry the aqueous phase to obtain ketamine hydrochloride crystals.

The beneficial effects of the present invention are as follows:

In the ketamine crystals obtained by the preparation and purification method of the present invention, the purity of ketamine calculated according to the peak area normalization method of high performance liquid chromatography is greater than or equal to 99.1 wt%.

The ketamine obtained by the preparation and purification method of the present invention is confirmed by nuclear magnetic resonance, liquid chromatography-tandem mass spectrometry, and infrared spectrum analysis, and its purity is determined by liquid chromatography and gas chromatography, and the chromatographic non-responsive impurities are measured; The material development requirements, its stability, uniformity, definite value, and estimation of total uncertainty all meet the relevant regulations and meet the expected indicators.

The preparation and purification method of the present invention can provide accurate and traceable ketamine standard substances for judicial appraisal departments in my country, fill in the blank of drug standard substances in the field of forensic science in my country, improve the quality of analysis and measurement, improve the accuracy of quantitative results, and maximize the Guarantee the validity of the measurement results. It is helpful to establish a traceability system for forensic science drug detection quantity, is conducive to the standardization of domestic forensic science drug detection chemical measurement methods, and realizes the reliability, effectiveness and mutual recognition of measurement results.

It overcomes the defects of low purity, poor stability, poor uniformity, and complicated preparation process of the ketamine sample obtained by the preparation and purification method of the prior art, and provides a high-purity, high-stability, high-recovery method that utilizes high-performance liquid chromatography separation method to obtain The preparation method of the ketamine standard substance is high and convenient for large-scale production.

Description of drawings

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings.

Figure 1-1 UV spectrum of impurity 1; Figure 1-2 UV spectrum of ketamine; Figure 1-3 UV spectrum of impurity 2;

Fig. 2 ketamine sample reversed-phase HPLC chromatogram; Acetonitrile-phosphate buffer saline (both volume ratios are 25:75);

Fig. 3 ketamine sample reverse-phase HPLC chromatogram; Methanol-0.05% TFA water (both volume ratios are 35:65);

Fig. 4 ketamine sample reverse-phase HPLC chromatogram; Acetonitrile-0.05% TFA water (both volume ratios are 33:67);

Fig. 5 Ketamine sample preparation HPLC chromatogram (methanol: 0.05% TFA/water=20:80, volume ratio);

The reverse phase HPLC chromatogram of Fig. 6 ketamine (methanol:0.05%TFA/water=33:67, volume ratio);

Figure 7 Ketamine preparation liquid chromatogram Methanol/water (both volume ratio is 40:60), 10mL/min, 210nm;

Figure 8 Ketamine preparation liquid chromatogram Methanol/water (both volume ratio is 40:60), 8mL/min, 210nm;

Figure 9 Ketamine preparation liquid chromatogram Methanol/water (both volume ratio is 30:70), 10mL/min;

Figure 10 Ketamine preparation liquid chromatogram Methanol/water (both volume ratio is 30:70), 8mL/min;

The reverse phase HPLC chromatogram of Fig. 11 ketamine (methanol: 0.05% TFA/water=35:65, volume ratio);

The flow chart of the purification preparation method of Fig. 12 ketamine standard substance;

The hydrogen spectrogram of Fig. 13 ketamine; The carbon spectrogram of Fig. 14 ketamine;

Fig. 15 ketamine hydrochloride sample mass spectrogram; Fig. 16 the infrared spectrum of ketamine hydrochloride;

The gas spectrogram of Fig. 17 ketamine hydrochloride; The GC/MS total ion chromatogram of Fig. 18 ketamine

Figure 19 Hydroxylimine mass spectrum.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

This example mainly utilizes the remaining ketamine crystal samples after case identification to optimize and screen the experimental conditions for separation and purification of ketamine by preparative liquid chromatography.

1. Instruments, reagents and materials

1.1 Main Instruments

Analytical high-performance liquid chromatography (Shimadzu, Japan), including: LC-20AD high-pressure infusion pump; SIL-10A autosampler; SPD-20A diode array detector; CTO-20A column thermostat.

Preparative high-performance liquid chromatography (Agilent), including: G1361A high-pressure infusion pump; G2260A automatic sampler; G1315D diode array detector; G1364B automatic fraction collector.

BUCHI rotary evaporator (Japan BUCHI company); KQ3200 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Feige brand TDL-40B desktop centrifuge (Shanghai Anting Scientific Instrument Factory); XS105Dual Range electronic balance (Switzerland METTLER TOLEDO company).

1.2 Main reagents and materials

Methanol (chromatographically pure, Fisher Scientific, USA), trifluoroacetic acid (chromatographically pure, China Bailingwei Company), ultrapure water (purified by Millipore ultrapure water preparation system, Millipore, France). Ketamine 1mg/mL standard solution (China Bailingwei Company); ketamine sample (white powder) was seized by the case and used in this study.

2. Determination of the purity of ketamine in the seized ketamine samples and purification and preparation of ketamine standard substances

2.1 Preparation of sample solution

Analytical type: Dissolve 1.0mg ketamine sample in 1mL methanol to prepare 1.0mg/mL ketamine sample solution for qualitative and quantitative analysis. Before use, the sample solution is filtered through a 0.45μm microporous membrane.

Preparative sample solution 1: Dissolve 3.369g ketamine sample in 10mL methanol first, centrifuge after fully dissolving, filter to remove precipitate, take out the organic phase, concentrate to dryness by centrifugation, add water to make up to 10mL, and prepare ketamine sample solution for preparative sample solution Preparation of Ketamine Standards by High Performance Liquid Chromatography. The sample solution was filtered through a 0.45 μm microporous membrane before use.

Preparative sample solution 2: Weigh 3368.51mg of ketamine sample (equivalent to 1999.548mg of pure ketamine) into a 50mL volumetric flask, add water to make up to the mark. Prepare a sample aqueous solution with a ketamine content of 40mg/mL, which is used for preparative high-performance liquid chromatography to separate and prepare ketamine standard products, and is filtered through a 0.22um mixed membrane before use.

2.2 Liquid chromatography conditions

2.2.1 Reversed phase high performance liquid chromatography (RP-HPLC) analysis method:

Chromatographic condition one: the chromatographic column is a Shim-pack HRC-ODS column (250mm×4.6mm I.D., 5μm); the mobile phase is methanol: 0.05% trifluoroacetic acid/water=35:65 (volume ratio, 0.05% trifluoroacetic acid/ Water refers to a trifluoroacetic acid aqueous solution with a volume fraction of 0.05% trifluoroacetic acid), isocratic elution; ultraviolet detection wavelength 210nm; flow rate 1.0mL/min; column temperature 35°C.

Chromatographic condition two: the chromatographic column is a Shim-pack HRC-ODS column (250mm×4.6mm I.D., 5μm); the mobile phase is methanol: 0.05% trifluoroacetic acid/water=33:67 (volume ratio, 0.05% trifluoroacetic acid/ Water refers to a trifluoroacetic acid aqueous solution with a volume fraction of 0.05% trifluoroacetic acid), isocratic elution; ultraviolet detection wavelength 210nm; flow rate 1.0mL/min; column temperature 35°C.

2.2.2 Reversed phase high performance liquid chromatography (RP-HPLC) preparation method:

Chromatographic condition 1: Chromatographic column is Shim-pack VP-ODS preparative column (250mm×20mm I.D., 15μm); mobile phase is methanol: water = 30:70 (volume ratio), isocratic elution; UV detection wavelength 210nm; flow rate 8mL/min; sample volume 700μL; column temperature is room temperature.

Chromatographic condition two: the chromatographic column is a Shim-pack VP-ODS preparative column (250mm×20mm I.D., 15μm); the mobile phase is methanol: 0.05% trifluoroacetic acid/water = 20:80 (volume ratio), isocratic elution; The ultraviolet detection wavelength is 210nm; the flow rate is 8mL/min; the sample volume is 500μL; the column temperature is room temperature.

2.3 Optimization of HPLC analysis conditions

2.3.1 Selection of chromatographic column

The reverse-phase chromatographic column used in this experiment is Shim-pack HRC-ODS column (250mm×4.6mm I.D., 5μm).

2.3.2 Selection of detection wavelength

Combined with the ultraviolet absorption diagrams of ketamine hydrochloride and impurities (as shown in Figure 1-1, Figure 1-2 and Figure 1-3), the difference in response between the main component and the impurity at the detection wavelengths of 210nm, 220nm, 230nm, 240nm, and 254nm was compared , comprehensively compared and analyzed the baseline noise, detection sensitivity and result stability data, and determined to select 210nm as the fixed value and detection wavelength.

2.3.3 Selection of mobile phase system

Regarding the selection of the reversed-phase chromatography mobile phase system, the test compared several different mobile phases: acetonitrile-phosphate buffer saline, methanol-phosphate buffer, acetonitrile-0.05% TFA aqueous solution [0.05% TFA aqueous solution refers to the volume of trifluoroacetic acid Chromatographic separation of ketamine and impurities in 0.05% trifluoroacetic acid aqueous solution], methanol-0.05% TFA aqueous solution [0.05% TFA aqueous solution refers to trifluoroacetic acid aqueous solution with a volume fraction of 0.05% trifluoroacetic acid] and methanol-water system Influence of degree, peak shape and retention time etc. The results showed that the sample The ketamine component and the impurity components in it can be well separated, and the tailing is small, and the peak time is fast, but the ketamine chromatographic peak in the methanol-water system is severely tailed, so this system is not suitable for ketamine samples. The components in are separated. The test considers that the buffer salt will shorten the service life of the chromatographic column, and the toxicity of acetonitrile will increase the cost of the test. Therefore, the mobile phase system is selected as methanol-0.05% TFA water (the volume ratio of the two is 35:65) system. The liquid chromatograms of ketamine samples under various mobile phase systems are shown in Figure 2, Figure 3 and Figure 4.

2.3.4 Standard curve and linear relationship

Dilute the ketamine standard stock solution with chromatographic methanol, and precisely prepare ketamine reference substance solutions with concentrations of 0.5, 1, 5, 10, 50, 100, 500, and 1000 μg/mL, and measure according to reversed-phase chromatography conditions, and repeat 3 times for each concentration. Times, calculated as the average value, record the ketamine chromatographic peak area, take the injection concentration (μg/mL) of the reference substance as the abscissa, and the chromatographic peak area value as the ordinate to plot, and calculate the regression equation of the standard curve.

The concentration and peak area of table 1 ketamine

The peak area is plotted against the concentration, and the regression equation of the standard curve is:

Y=3×10 ⁷ X+92617, R ² =0.9999……………………

It shows that ketamine has a good linear relationship in the range of 0.5--1000μg/mL.

2.3.5 Determination of ketamine content in samples

Analyze and measure the ketamine sample solution with a concentration of 1.0 mg/mL according to the reversed-phase chromatography method, repeat the measurement 3 times, record the peak area of ketamine, calculate its average value, and calculate the content of ketamine in the sample according to the external standard method of peak area.

The assay result of ketamine content in the sample of table 2

It can be seen from Table 2 that the content of ketamine hydrochloride in the ketamine sample solution of 1.0mg/mL is calculated as 0.5936mg/mL by the standard curve equation through the peak area external standard method, and then the purity of ketamine hydrochloride in the ketamine sample is obtained as 59.36wt%. .

2.3.6 Optimization of HPLC preparation conditions

The methanol-TFA water system is used to separate the ketamine components in the ketamine sample (preparative sample solution two and chromatographic condition two), and the test shows that the impurity components and the ketamine components are superimposed on each other by using the same mobile phase ratio as in the analysis conditions, The impurity components could not be separated from the ketamine components, and by adjusting the proportion of methanol, a better separation effect was finally achieved between ketamine and other components, and the prepared liquid chromatogram is shown in Figure 5. Further purification of the collected fractions: Ketamine exists in the form of hydrochloride in the ketamine sample. After the separation of the liquid phase, the fractions are centrifugally concentrated or rotary evaporated to remove methanol, but the residual trifluoroacetic acid in the fractions cannot be removed. , in order to obtain the final preparation product ketamine hydrochloride, further purification treatment is required. After the methanol was removed by rotary evaporation, the pH of the remaining water was adjusted to 11, and the chromatographically pure chloroform was repeatedly extracted. After the organic phase was combined, it was concentrated by centrifugation until a white precipitate appeared, and water was injected into it, and 0.1N hydrochloric acid was slowly added to the water. The pH of the solution is 5-6, the organic phase is removed, and the water phase is freeze-dried to obtain ketamine hydrochloride crystals. Its HPLC chromatogram is shown in Figure 6. After HPLC analysis, the percentage content was determined by the peak area normalization method, and the ketamine content was 99.72wt%.

In the secondary purification process of the distillate, the distillate needs to be rotary-evaporated and then adjusted to alkaline conditions, back-extracted with an organic solvent, and the components are hydrochloricized to obtain ketamine salt. salt, the operation process is complicated, and the chance of introducing impurities increases. Therefore, the preparation conditions are further improved, and the methanol-water system is used to separate and prepare ketamine components (using preparative sample solution 1 and chromatographic condition 1).

Tests have proved that the methanol-water system is not suitable for analyzing ketamine samples, but it is also found that all components can be eluted when the system is used as the mobile phase. separation effect. As can be seen from the experimental results, when the mobile phase is composed of methanol: water (volume ratio is 40:60), and when the flow rate is 10mL/min, ketamine can be completely separated from the impurity components behind it, but peaks simultaneously with the impurities in front of it; further The flow rate was reduced, the composition of the mobile phase remained unchanged, and the separation was carried out under the condition of 8mL/min, and the separation status of ketamine and its previous impurities was not improved. The preparative liquid chromatograms are shown in Figure 7 and Figure 8.

Reduce the proportion of methanol in the mobile phase. When the mobile phase is composed of methanol/water (volume ratio 30:70), a better separation effect can be obtained at a flow rate of 10mL/min. Ketamine and its impurity components before and after achieved good separation. Considering that the pressure of the chromatographic column increases when the flow rate is high, in order to effectively prolong the service life of the chromatographic column, the flow rate is reduced. In the experiment at a flow rate of 8mL/min, a good separation effect is still obtained. The time for each component to flow out of the chromatographic column Added about 6 minutes. The final selected separation condition of the experiment was methanol: water (volume ratio 30:70), flow rate 8mL/min. The preparative liquid chromatograms are shown in Figure 9-Figure 10.

2.4 Batch processing program

Due to the experimental investigation, under the adopted preparative liquid chromatographic conditions (using preparative sample solution one and chromatographic condition one), the ketamine component can be eluted from the preparative column within 20min, but the impurities behind come out of the peak Too late prolongs the separation time. And because the mobile phase system for preparing separation is composed of methanol and water, when water and methanol are mixed, exothermic phenomenon will occur, the volume of the mixed solvent will decrease, and a large number of bubbles will be released simultaneously. Therefore, before the test, methanol and water (volume ratio is 30:70) were mixed in proportion before entering the pump, and used after fully mixing and degassing. After the preparation method was run for 20 minutes, the chromatographic column was rinsed with 100% methanol to make the residue in the column Impurities are quickly eluted. The batch procedure was established as multiple cycles of the following three steps in sequence, first flushing the column with methanol, then equilibrating the column with the mobile phase, and then running the preparative method.

Ketamine in the ketamine sample exists in the form of hydrochloride, and the distillate after liquid phase separation is still ketamine hydrochloride, which needs to be further purified. Ketamine hydrochloride crystals can be obtained after rotary evaporation to remove the methanol component in the fraction, freeze-drying and vacuum dehydration. Its HPLC chromatogram is shown in Figure 11.

2.5 Confirmation of the structure of the prepared ketamine standard substance

2.5.1 NMR analysis

In order to determine the structure of the compound obtained by the preparative liquid phase separation, nuclear magnetic resonance analysis was carried out, and the spectra are shown in Figures 13-14.

Solvent: _D2O

Location Chemical shift¹³C Chemical shift¹H hydrogen splitting 1 72.629 2 211.455 3 39.535 2.63; 2.59 m; m 4 30.140 2.13; 1.77 m; m 5 36.109 3.33; 1.92 m; m 6 21.177 1.89; 1.75 m; m 7 127.238 8 134.161 9 132.014 7.61 m 10 128.470 7.61 m 11 132.768 7.61 m 12 131.821 7.86 dd 13 26.841 2.41 the s

2.5.2 Liquid chromatography-tandem mass spectrometry

In the ESI positive ion mode, the fragmentation voltage is 80V, and the collision energy is 8eV. The mass spectrum of the sample is shown in FIG. 15 . The following information can be obtained from the mass spectrum:

The m/z of the quasi-molecular ion peak [M+H]+ is 238.20, and the relative molecular mass of ketamine is 237.72g/mol. The difference is 1, so the molecular weight of the detected sample is consistent with that of ketamine.

2.5.3 Infrared spectroscopy

The infrared spectrum of ketamine was measured on the AVATAR 330FT-IR infrared spectrometer (Thermo Nicolet Company), as shown in Figure 16, and the result was consistent with the standard spectrum in the NIST spectral library.

2.5.4 Gas chromatography-mass spectrometry

The spectrogram obtained by gas chromatography-mass spectrometry ( FIG. 17 ) is consistent with the standard spectrogram of ketamine.

Due to the presence of a larger impurity in gas chromatographic analysis, its content was 0.4%. Therefore, it was analyzed by gas chromatography-mass spectrometry. According to the mass spectrogram analysis (Figure 18) measured by the instrument, the main impurity is hydroxylimine. Hydroxyimine is mainly used as a pharmaceutical intermediate, an important intermediate for the synthesis of ketamine, in the form of brown and milky white powder. In 2008, it was included in the "Regulations on the Administration of Precursor Chemicals". CAS number 90717-16-1.

2.6 The purity measurement result of gained ketamine standard substance

The purity of the prepared ketamine hydrochloride standard substance was 99.47wt% and the expanded uncertainty was 0.62% (k=2) through HPLC, GC and QNMR methods. The uniformity is good, and the stability is at least 1 year.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those of ordinary skill in the art can also make It is impossible to exhaustively list all the implementation modes here, and any obvious changes or changes derived from the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims (1)

1. The method for purifying and preparing ketamine standard substance for forensic science drug detection, characterized in that it comprises the following steps: (1) detecting the purity of ketamine in the seized ketamine sample, selecting the seized ketamine sample whose mass fraction of ketamine is greater than or equal to 50wt% As a raw material for purification and preparation of ketamine standard substances; (2) Utilize high performance liquid chromatography to prepare ketamine standard substance; In step (1), include the following steps: (1.1) Preparation of sample solution: 1.0mg ketamine sample was dissolved in 1mL methanol to prepare 1.0mg/mL ketamine sample solution for qualitative and quantitative analysis. Before use, the sample solution was filtered through a 0.45 μm microporous membrane; (1.2) Determine liquid chromatography condition: chromatographic column is Shim-pack HRC-ODS post, 250mm * 4.6mm ID, 5 μ m; Mobile phase is V _methanol : V _{0.05% trifluoroacetic acid/water} =(33-35):( 65-67), isocratic elution; UV detection wavelength 210nm; flow rate 1.0mL/min; column temperature 35°C; (1.3) Calculate the regression equation of the standard curve and determine the linear range: dilute the ketamine standard stock solution with chromatographically pure methanol, and precisely prepare ketamine controls with concentrations of 0.5, 1, 5, 10, 50, 100, 500, and 1000 μg/mL Sample solution, measured by the reversed-phase chromatographic conditions in step (1.2), each concentration was repeated 3 times, calculated as the average value, and recorded the ketamine chromatographic peak area, taking the injection concentration of the reference substance as the abscissa, and the concentration unit is μg/ mL, the chromatographic peak area value is plotted on the ordinate, and the regression equation of the standard curve is calculated; the peak area is plotted against the concentration, and the regression equation of the standard curve is: Y=3×10 ⁷ X+92617, R ² =0.9999, Ketamine has a good linear relationship in the range of 0.5--1000μg/mL; (1.4) Analyze and measure the ketamine sample solution whose concentration is 1.0 mg/mL according to the reverse phase chromatography method in step (1.2), repeat the measurement 3 times, record the peak area of ketamine, calculate its average value, and calculate the sample by the peak area external standard method ketamine content in In step (2), comprise the following steps: (2.1) Preparation of sample solution: Dissolve 3.369g of ketamine sample in 10mL of methanol first, centrifuge after fully dissolving, filter to remove precipitate, take out the organic phase, concentrate to dryness by centrifugation, add water to make up to 10mL, and prepare ketamine sample solution for use in Preparative high-performance liquid chromatography was used to separate and prepare ketamine standard products, and the sample solution was filtered through a 0.45 μm microporous membrane before use; (2.2) Determine liquid chromatography conditions: (2.2.1) Chromatographic column is Shim-pack VP-ODS preparative column, 250mm×20mm ID, 15μm; mobile phase is V _methanol : V _water = 30:70, methanol and water are fully mixed and degassed before use, isocratic Elution; UV detection wavelength 210nm; flow rate 8mL/min; sample volume 700μL; column temperature at room temperature; wash the column with 100% methanol after running for 20 minutes; or (2.2.2) Chromatographic column is Shim-pack VP-ODS preparative column, 250mm×20mm ID, 15μm; mobile phase is V _methanol : V _{0.05% trifluoroacetic acid/water} =20:80, isocratic elution; UV detection Wavelength 210nm; flow rate 8mL/min; sample volume 500μL; column temperature is room temperature; (2.3) Under the chromatographic conditions in the step (2.2.2), after the collected fractions are removed by rotary evaporation, the pH of the remaining aqueous solution is adjusted to 11, extracted with chromatographically pure chloroform, the organic phases are combined, and concentrated by centrifugation to A white precipitate appears, inject water into it, slowly add 0.1N hydrochloric acid until the pH of the aqueous solution reaches 5-6, remove the organic phase, and freeze-dry the aqueous phase to obtain ketamine hydrochloride crystals.