CN107478757B - Method for purifying and preparing ketamine standard substance for forensic science drug detection - Google Patents
Method for purifying and preparing ketamine standard substance for forensic science drug detection Download PDFInfo
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- CN107478757B CN107478757B CN201710316040.0A CN201710316040A CN107478757B CN 107478757 B CN107478757 B CN 107478757B CN 201710316040 A CN201710316040 A CN 201710316040A CN 107478757 B CN107478757 B CN 107478757B
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Abstract
the invention discloses a method for purifying and preparing a ketamine standard substance for forensic science drug detection, which comprises the following steps: (1) detecting the purity of ketamine in a ketamine-cured sample, and selecting the ketamine-cured sample with the ketamine mass fraction of more than or equal to 50 wt% as a raw material for purifying and preparing a ketamine standard substance; (2) preparing ketamine standard substance by using high performance liquid chromatography. The ketamine obtained by the preparation and purification method is confirmed by nuclear magnetic resonance, liquid chromatography-tandem mass spectrometry and infrared spectrum analysis, the purity of the ketamine is confirmed to be a fixed value by liquid chromatography and gas chromatography, and the chromatographic non-responsive impurities are measured; according to the development requirements of standard substances, the stability, uniformity, definite value and total uncertainty of the standard substances are estimated to meet relevant regulations and reach expected indexes.
Description
Technical Field
the invention relates to preparation of a court science drug standard. More particularly, the invention relates to a method for purifying and preparing ketamine standard substance for forensic science drug detection.
background
At present, the increasing drug problem has become a global disaster. The inundation of drugs directly harms the physical and mental health of people and brings great threat to economic development and social progress. Therefore, establishing a forensic science drug detection quantity value traceability system, improving drug component measurement technology, ensuring the reliability and comparability of measurement results, establishing sharing and mutual recognition of measurement data, providing accurate and reliable evidence for the court, and becoming a problem of general attention of drug identification organizations of all countries in the world.
The drug component measurement technology and traceability guarantee are an organic whole and are important embodiments of the core measurement capability in the field of drug component measurement in forensic science. The standard substance is a skeleton penetrating through the whole body, is a vector of a quantity value, is a key element of a drug component quantity traceability system, is an important basis for ensuring the accuracy and comparability of a measurement result in time and space, and is a fundamental guarantee for realizing effective measurement, namely accurate, comparable and traceable measurement.
the drug detection laboratories with advanced technology in Europe, America and other countries mostly adopt internationally recognized standard substances produced by Sigma and other companies, but China only can rely on imported standard substances, and the quantity is small, the price is high, and many of the standard substances cannot be provided for China. At present, the 'reference substance' used in domestic drug analysis is extremely limited in type, and corresponding technical indexes such as complete structure identification, purity determination, uniformity, stability and the like are generally lacked. These all bring certain uncertainty for the detection of the drug-related cases, and directly influence the accuracy of the quantitative result. Moreover, the shortage of domestic drug standard substances becomes a main obstacle for realizing standardization of chemical measurement methods for forensic science drug detection, and traceability and mutual recognition of measurement results in China. Therefore, the preparation of drug standard substances for forensic science drug detection becomes an urgent problem to be solved in the field of drug research in China.
The english common name for ketamine: (±) -Ketamine hydrochloride, chemical name: 2-o-chlorophenyl-2-tetraamino-cyclohexanone hydrochloride, english name: (±) -2- (2-chlorophenylyl) -2- (methylamino) cyclohexenone hydrochloride, molecular formula: C13H16ClNO HCl, molecular weight: 274.19, CA accession number: 1867-66-9, having the formula:
Physical and chemical properties: white crystalline powder, odorless. Readily soluble in water (200mg/mL), soluble in hot ethanol, and insoluble in diethyl ether and benzene. The melting point was 259-263 ℃. The aqueous solution is acidic, and the pH value is 4.0-5.5.
Disclosure of Invention
The invention aims to provide a method for purifying and preparing ketamine standard substance for forensic science drug detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
The method for purifying and preparing the ketamine standard substance for forensic science drug detection comprises the following steps: (1) detecting the purity of ketamine in a ketamine-cured sample, and selecting the ketamine-cured sample with the ketamine mass fraction of more than or equal to 50 wt% as a raw material for purifying and preparing a ketamine standard substance;
(2) Preparing ketamine standard substance by using high performance liquid chromatography.
The method for purifying and preparing the ketamine standard substance for forensic science drug detection comprises the following steps in step (1):
(1.1) preparation of sample solution: dissolving a 1.0mg ketamine sample in 1mL methanol to prepare a 1.0mg/mL ketamine sample solution for qualitative and quantitative analysis, and filtering the sample solution through a 0.45-micrometer microporous filter membrane before use;
(1.2) determination of liquid chromatography conditions: the chromatographic column is a Shim-pack HRC-ODS column, 250mm multiplied by 4.6mm I.D., 5 μm; the mobile phase is methanol V: v0.05% trifluoroacetic acid/water (33-35) (65-67), isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 1.0 mL/min; the column temperature is 35 ℃;
(1.3) calculating a regression equation of the standard curve and determining a linear range: diluting ketamine standard stock solution with chromatographic pure methanol, precisely preparing ketamine reference substance solution with the concentrations of 0.5, 1, 5, 10, 50, 100, 500 and 1000 mug/mL respectively, determining according to the reversed phase chromatographic condition in the step (1.2), repeating each concentration for 3 times, calculating by using an average value, recording the area of the ketamine chromatographic peak, drawing by using the sample injection concentration of the reference substance as a horizontal coordinate, the concentration as mug/mL and the chromatographic peak area value as a vertical coordinate, and calculating a regression equation of a standard curve; the peak area is plotted against concentration, and the regression equation for the standard curve is:
Y＝3×10X+92617，R＝0.9999，
ketamine has good linear relation in the range of 0.5-1000 mug/mL;
(1.4) analyzing and measuring a ketamine sample solution with the concentration of 1.0mg/mL according to the reversed phase chromatography method in the step (1.2), repeating the measurement for 3 times, recording the area of the ketamine peak, calculating the average value of the area of the ketamine peak, and calculating the content of the ketamine in the sample according to an external standard method of the area of the peak.
The method for purifying and preparing the ketamine standard substance for forensic science drug detection comprises the following steps in step (2):
(2.1) preparation of sample solution: 3.369g of ketamine sample is firstly dissolved in 10mL of methanol, the solution is centrifuged after being fully dissolved, the precipitate is removed by filtration, the organic phase is taken out, the solution is centrifugally concentrated to dryness, water is added to the solution to a constant volume of 10mL, a ketamine sample solution is prepared for separating and preparing a ketamine standard substance by preparative high performance liquid chromatography, and the sample solution is filtered by a 0.45-micrometer microporous filter membrane before use;
(2.2) determining liquid chromatography conditions:
(2.2.1) the column was a Shim-pack VP-ODS preparative column (250 mm. times.20 mm I.D., 15 μm); the mobile phase is methanol V: mixing methanol and water at a ratio of 30:70, degassing, and eluting at equal rate; ultraviolet detection wavelength is 210 nm; the flow rate is 8 mL/min; loading 700 μ L; the column temperature is room temperature; after 20 minutes of operation the column was washed with 100% methanol; or
(2.2.2) the column was a Shim-pack VP-ODS preparative column (250 mm. times.20 mm I.D., 15 μm); the mobile phase is methanol V: v0.05% trifluoroacetic acid/water 20:80, isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 8 mL/min; the loading amount is 500 mu L; the column temperature is room temperature;
(2.3) under the chromatographic condition in the step (2.2.2), removing methanol from the collected fractions by rotary evaporation, adjusting the pH of the residual aqueous solution to 11, extracting with chromatographic pure chloroform, combining organic phases, carrying out centrifugal concentration until white precipitates appear, injecting water into the white precipitates, slowly adding 0.1N hydrochloric acid until the pH of the aqueous solution is 5-6, removing the organic phase, and carrying out freeze drying on the aqueous phase to obtain ketamine hydrochloride crystals.
the invention has the following beneficial effects:
In the ketamine crystal obtained by the purification method, the purity of the ketamine is calculated to be more than or equal to 99.1 wt% according to a high performance liquid chromatography peak area normalization method.
The ketamine obtained by the preparation and purification method is confirmed by nuclear magnetic resonance, liquid chromatography-tandem mass spectrometry and infrared spectrum analysis, the purity of the ketamine is confirmed to be a fixed value by liquid chromatography and gas chromatography, and the chromatographic non-responsive impurities are measured; according to the development requirements of standard substances, the stability, uniformity, definite value and total uncertainty of the standard substances are estimated to meet relevant regulations and reach expected indexes.
The preparation and purification method can provide ketamine standard substances with accurate and traceable values for judicial appraisal departments in China, fills in the blank of drug standard substances in the field of forensic science in China, improves the analysis and measurement quality, improves the accuracy of quantitative results and furthest ensures the effectiveness of the measurement results. The method is beneficial to establishing a forensic science drug detection quantity value traceability system, is beneficial to realizing the standardization of a domestic forensic science drug detection chemical measurement method, and realizes the reliability, effectiveness and mutual recognition of measurement results.
the method overcomes the defects of low purity, poor stability, poor uniformity, complex preparation process and the like of a ketamine sample prepared by a purification method in the prior art, and provides a preparation method of a ketamine standard substance which is high in purity, stability and recovery rate and is convenient for large-scale production and is obtained by a high performance liquid chromatography separation method.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1-1 ultraviolet spectrum of impurity 1; FIGS. 1-2 are UV spectrograms of ketamine; FIGS. 1-3 UV spectrograms of impurity 2;
FIG. 2A reverse phase HPLC chromatogram of a ketamine sample; acetonitrile-phosphate buffer (25: 75 by volume);
FIG. 3A reverse phase HPLC chromatogram of a ketamine sample; methanol-0.05% TFA water (35: 65 by volume);
FIG. 4A reverse phase HPLC chromatogram of a ketamine sample; acetonitrile-0.05% TFA water (33: 67 vol.);
Figure 5 ketamine sample prep HPLC chromatogram (methanol: 0.05% TFA/water 20:80 by volume);
Fig. 6 reverse phase HPLC chromatogram of ketamine (methanol: 0.05% TFA/water 33:67 by volume);
FIG. 7A liquid chromatogram of ketamine preparation is methanol/water (40: 60 volume ratio), 10mL/min, 210 nm;
FIG. 8 preparation of liquid chromatogram of ketamine with methanol/water (volume ratio of 40: 60), 8mL/min, 210 nm;
FIG. 9A liquid chromatogram of ketamine preparation is methanol/water (30: 70 volume ratio), 10 mL/min;
FIG. 10 preparation of ketamine A liquid chromatogram of methanol/water (30: 70 volume ratio), 8 mL/min;
Fig. 11 reverse phase HPLC chromatogram of ketamine (methanol: 0.05% TFA/water 35:65 by volume);
FIG. 12 flow chart of a purification preparation method of ketamine standard substance;
figure 13 hydrogen spectrum of ketamine; figure 14 carbon spectrum of ketamine;
FIG. 15 mass spectrum of ketamine hydrochloride sample; FIG. 16 Infrared spectrum of ketamine hydrochloride;
Figure 17 gas mass spectrum of ketamine hydrochloride; FIG. 18 GC/MS total ion flow graph of ketamine
FIG. 19 hydroximine mass spectrum.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
In the embodiment, the sample of the ketamine crystal remaining after case identification is mainly used for optimizing and screening the experimental conditions for preparing ketamine by separation and purification through preparative liquid chromatography.
instrument, reagent and material
1.1 Main Instrument
Analytical high performance liquid chromatograph (shimadzu, japan), comprising: LC-20AD high-pressure infusion pump; SIL-10A autosampler; an SPD-20A diode array detector; CTO-20A column incubator.
A preparative high performance liquid chromatograph (Agilent) comprising: G1361A high-pressure infusion pump; G2260A autosampler; a G1315D diode array detector; G1364B automated fraction collector.
BUCHI rotary evaporator (BUCHI corporation, japan); KQ3200 type ultrasonic cleaner (kunshan ultrasonic instrument ltd); flying pigeon brand TDL-40B desk centrifuge (Shanghai' an pavilion scientific instrument factory); XS105Dual Range electronic balance (Mettler TOLEDO, Switzerland).
1.2 Primary reagents and materials
methanol (chromatographically pure, Fisher Scientific, usa), trifluoroacetic acid (chromatographically pure, warfarin, china), ultrapure water (purified by Millipore ultrapure water preparation system, Millipore, france). Ketamine 1mg/mL standard solution (carbofuran, china); ketamine samples (white powder) were collected from the cases and used in this study.
Secondly, the purity of the ketamine in the ketamine sample is measured, and the ketamine standard substance is purified and prepared
2.1 preparation of sample solutions
analytical type: a1.0 mg sample of ketamine was dissolved in 1mL of methanol to make a 1.0mg/mL sample solution of ketamine for qualitative and quantitative analysis, and the sample solution was filtered through a 0.45 μm microporous membrane before use.
preparation of a prototype sample solution one: 3.369g of ketamine sample is firstly dissolved in 10mL of methanol, and then is centrifuged after being fully dissolved, the precipitate is removed by filtration, the organic phase is taken out, is centrifuged and concentrated to be dry, and is added with water to be constant volume to 10mL to prepare ketamine sample solution for preparing ketamine standard substance by preparative high performance liquid chromatography separation. The sample solutions were filtered through 0.45 μm microporous membrane before use.
Preparation type sample solution two: weighing 3368.51mg of ketamine sample (equivalent to 1999.548mg of pure chloramine ketone) in a 50mL volumetric flask, and adding water to the volume to reach the scale mark. Preparing a sample aqueous solution with ketamine content of 40mg/mL, separating by preparative high performance liquid chromatography to prepare a ketamine standard substance, and filtering by a 0.22um mixed membrane before use.
2.2 liquid chromatography conditions
2.2.1 reversed-phase high-performance liquid chromatography (RP-HPLC) analytical method:
The chromatographic conditions are as follows: the column was a Shim-pack HRC-ODS column (250 mm. times.4.6 mm I.D., 5 μm); the mobile phase is methanol: 0.05% trifluoroacetic acid/water 35:65 (volume ratio, 0.05% trifluoroacetic acid/water means 0.05% trifluoroacetic acid in water by volume fraction, isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 1.0 mL/min; the column temperature was 35 ℃.
And (2) chromatographic conditions II: the column was a Shim-pack HRC-ODS column (250 mm. times.4.6 mm I.D., 5 μm); the mobile phase is methanol: 0.05% trifluoroacetic acid/water 33:67 (volume ratio, 0.05% trifluoroacetic acid/water means 0.05% trifluoroacetic acid in water by volume fraction, and isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 1.0 mL/min; the column temperature was 35 ℃.
2.2.2 reverse phase high Performance liquid chromatography (RP-HPLC) preparation:
the chromatographic conditions are as follows: the chromatographic column is a Shim-pack VP-ODS preparative column (250 mm. times.20 mm I.D., 15 μm); the mobile phase is methanol: water (30: 70 by volume), and isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 8 mL/min; loading 700 μ L; the column temperature was room temperature.
And (2) chromatographic conditions II: the chromatographic column is a Shim-pack VP-ODS preparative column (250 mm. times.20 mm I.D., 15 μm); the mobile phase is methanol: 0.05% trifluoroacetic acid/water 20:80 (vol/vol), isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 8 mL/min; the loading amount is 500 mu L; the column temperature was room temperature.
2.3 optimization of HPLC analysis conditions
2.3.1 selection of chromatography columns
The reverse phase chromatography column used in this experiment was a Shim-pack HRC-ODS column (250 mm. times.4.6 mm I.D., 5 μm).
2.3.2 selection of detection wavelength
by combining the ultraviolet absorption graphs of ketamine hydrochloride and impurities (as shown in figures 1-1, 1-2 and 1-3), the response differences of main components and impurities at the detection wavelengths of 210nm, 220nm, 230nm, 240nm and 254nm are compared, the baseline noise, the detection sensitivity and the result stability data are comprehensively compared and analyzed, and 210nm is determined to be selected as a fixed value and the detection wavelength.
2.3.3 selection of mobile phase systems
With respect to the selection of the reverse phase chromatography mobile phase system, several different mobile phases of acetonitrile-phosphate buffer, methanol-phosphate buffer, acetonitrile-0.05% TFA in water [ 0.05% TFA in water means trifluoroacetic acid in water with a trifluoroacetic acid volume fraction of 0.05% ], methanol-0.05% TFA in water [ 0.05% TFA in water means trifluoroacetic acid in water with a trifluoroacetic acid volume fraction of 0.05% ], and methanol-water system were compared for their effects on the degree of chromatographic separation, peak shape, retention time, and the like of ketamine and impurities. The results show that with acetonitrile-phosphate buffer (25: 75 by volume), methanol-0.05% TFA water (35: 65 by volume) and acetonitrile-0.05% TFA water (33: 67 by volume), the ketamine component in the sample can be well separated from the impurity components in the sample, and the tail is small, the peak time is fast, while the ketamine chromatographic peak in the methanol-water system is severely tail, so the system is not suitable for separating the components in the ketamine sample. The experiments considered that the buffer salt shortened the service life of the chromatographic column, the acetonitrile was toxic and the cost of the experiments was increased, so the mobile phase system was selected as methanol-0.05% TFA water (35: 65 by volume). The liquid chromatogram of ketamine sample for each mobile phase system is shown in fig. 2, 3 and 4.
2.3.4 Standard Curve and Linear relationship
The method comprises the steps of diluting a ketamine standard stock solution with chromatographic methanol, precisely preparing a ketamine reference substance solution with the concentration of 0.5, 1, 5, 10, 50, 100, 500 and 1000 mu g/mL, respectively, measuring according to a reverse phase chromatographic condition, repeating each concentration for 3 times, calculating by using an average value, recording the chromatographic peak area of the ketamine, drawing by using the sample injection concentration (mu g/mL) of the reference substance as an abscissa and the chromatographic peak area value as an ordinate, and calculating a regression equation of a standard curve.
TABLE 1 concentration and Peak area of Ketamine
the peak area is plotted against concentration, and the regression equation for the standard curve is:
Y＝3×10X+92617，R＝0.9999……………………
indicating that ketamine is in good linearity in the range of 0.5-1000. mu.g/mL.
2.3.5 measurement of Ketamine content in samples
and analyzing the ketamine sample solution with the measured concentration of 1.0mg/mL by using a reverse phase chromatography method, repeatedly measuring for 3 times, recording the area of the ketamine peak, calculating the average value of the area of the ketamine peak, and calculating the content of the ketamine in the sample by using an external standard method of the area of the peak.
TABLE 2 measurement of ketamine content in samples
As can be seen from Table 2, the content of ketamine hydrochloride in the ketamine sample solution of 1.0mg/mL is 0.5936mg/mL by calculation from the standard curve equation by the peak area external standard method, and the purity of ketamine hydrochloride in the ketamine sample is determined to be 59.36 wt%.
2.3.6 optimization of preparation conditions of high performance liquid chromatography
The method comprises the steps of separating a ketamine component in a ketamine sample by using a methanol-TFA water system (using a preparative sample solution II and a chromatographic condition II), wherein tests show that the impurity component and the ketamine component are overlapped by using the same flow phase ratio as that in an analysis condition, so that the impurity component cannot be separated from the ketamine component, and finally the ketamine and other components achieve a better separation effect by adjusting the methanol ratio, and a prepared liquid phase spectrogram is shown in figure 5. The collected fractions were further purified: the ketamine sample is in the form of hydrochloride, and after separation of the preparation liquid phase, the fraction is subjected to centrifugal concentration or rotary evaporation to remove methanol, but the residual trifluoroacetic acid in the fraction cannot be removed, and further purification treatment is required to obtain the final preparation product ketamine hydrochloride. Removing methanol by rotary evaporation, adjusting the pH value of the residual water solution to 11, repeatedly extracting with chromatographic pure chloroform for multiple times, combining organic phases, centrifuging and concentrating until white precipitates appear, injecting water into the organic phases, slowly adding 0.1N hydrochloric acid until the pH value of the water solution is 5-6, removing the organic phases, and freeze-drying the water phase to obtain ketamine hydrochloride crystals. The HPLC chromatogram thereof is shown in FIG. 6. The percentage content of ketamine is 99.72 wt% determined by HPLC analysis and peak area normalization.
In the above secondary purification process of the fraction, the fraction needs to be subjected to rotary evaporation and then alkaline condition adjustment, and then is subjected to back extraction by using an organic solvent, the components are subjected to salination to obtain ketamine hydrochloride, the operation process is complex, and the opportunity of introducing impurities is increased, so that the preparation condition is further improved, and the preparation of the ketamine component by using the methanol-water system (adopting a preparation type sample solution I and a chromatographic condition I) is attempted.
Experiments prove that the methanol-water system is not suitable for analyzing ketamine samples, but all components can be eluted when the system is used as a mobile phase, so that the separation effect of the methanol-water system on the ketamine on a preparation liquid phase is tried. From the experimental results, when the mobile phase composition is methanol: water (40: 60 by volume) at a flow rate of 10mL/min, ketamine is completely separated from its following impurity components but peaks simultaneously with its preceding impurities; the flow rate was further reduced, the mobile phase composition was unchanged, the separation was performed at 8mL/min, and the separation of ketamine from its preceding impurities was not improved. The liquid chromatogram is prepared as shown in FIGS. 7 and 8.
The ratio of methanol in the mobile phase is reduced, and when the mobile phase consists of methanol/water (the volume ratio is 30:70), the flow rate is 10mL/min, and a better separation effect can be obtained. Ketamine and its impurity components before and after reach better separation. Considering that the pressure of the chromatographic column is increased when the flow rate is high, the flow rate is reduced to effectively prolong the service life of the chromatographic column, and the experiment is carried out under the condition that the flow rate is 8mL/min, so that a better separation effect is still obtained, and the time for each component to flow out of the chromatographic column is increased by about 6 min. The separation conditions finally selected for the experiment were methanol: water (volume ratio 30:70) at a flow rate of 8 mL/min. The prepared liquid chromatogram is shown in FIGS. 9-10.
2.4 batch procedure
Due to experimental investigation, the ketamine component elutes from the preparative column within 20min under the preparative liquid chromatography conditions employed (preparative sample solution one and chromatography condition one), but the later impurity peaks too late for the separation time to be extended. And because the prepared and separated mobile phase system consists of methanol and water, when the water is mixed with the methanol, the heat release phenomenon occurs, the volume of the mixed solvent is reduced, and simultaneously, a large amount of bubbles are released. Therefore, the test adopts the steps of mixing methanol and water (the volume ratio is 30:70) in proportion before pumping, fully mixing and degassing for use, and washing the chromatographic column by 100 percent methanol after the preparation method is operated for 20 minutes so that impurity components remained in the chromatographic column are quickly eluted. The established batch processing program is formed by the following three steps which are circulated for a plurality of times in sequence, namely: the column was first washed with methanol, followed by a mobile phase equilibration column, and the preparative run was then run.
The ketamine in the ketamine sample exists in the form of hydrochloride, and after the ketamine sample is separated by the preparation liquid phase, the fraction is still ketamine hydrochloride, and further purification treatment is required. Removing methanol components in the distillate after rotary evaporation, and obtaining ketamine hydrochloride crystals after freeze drying and vacuum dehydration. The HPLC chromatogram thereof is shown in FIG. 11.
2.5 Structure confirmation of ketamine Standard substance prepared
2.5.1 NMR analysis
in order to determine the structure of the compound obtained by preparative liquid phase separation, NMR analysis was performed and the spectra are shown in FIGS. 13 to 14.
Solvent: D2O
| Position of | 13chemical shift 13C | 1Chemical shift 1H | Hydrogen spectrum 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 | s |
2.5.2 liquid chromatography-tandem Mass Spectrometry combination
in the ESI positive ion mode, the fragmentation voltage is 80V, the collision energy is 8eV, and the mass spectrum of the sample is shown in FIG. 15. From the mass spectra the following information can be obtained:
The M/z238.20 of the excimer peak [ M + H ] + was different from the relative molecular mass 237.72g/mol of ketamine by 1, so that the molecular weight of the test sample corresponded to that of ketamine.
2.5.3 Infrared Spectroscopy
the infrared spectrum of ketamine was measured on an AVATAR 330FT-IR spectrometer (Thermo Nicolet corporation) as shown in FIG. 16 and was consistent with the standard spectra in the NIST spectrum library.
2.5.4 gas chromatography-Mass Spectrometry
the spectrum obtained by gas chromatography-mass spectrometry (fig. 17) was consistent with the standard spectrum of ketamine.
The content was 0.4% due to the presence of a larger impurity in the gas chromatography analysis. It was therefore analyzed by gas chromatography-mass spectrometry. According to the mass spectrometric analysis measured by the instrument (fig. 18), the main impurity is hydroxyimine. The hydroxyimine is mainly used as a medical intermediate, is an important intermediate for synthesizing ketamine, and is coffee and milk white powder. In 2008, the drug-making chemical management regulation is listed. CAS number 90717-16-1.
2.6 purity determination of Ketamine Standard substance
The purity of the prepared ketamine hydrochloride standard substance was 99.47 wt% and the extended uncertainty was 0.62% (k ═ 2) by HPLC, GC and QNMR method quantitative analysis. Good uniformity and stability for at least 1 year.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (1)
1. the method for purifying and preparing the ketamine standard substance for forensic science drug detection is characterized by comprising the following steps of: (1) detecting the purity of ketamine in a ketamine-cured sample, and selecting the ketamine-cured sample with the ketamine mass fraction of more than or equal to 50 wt% as a raw material for purifying and preparing a ketamine standard substance;
(2) Preparing a ketamine standard substance by using high performance liquid chromatography;
In the step (1), the method comprises the following steps:
(1.1) preparation of sample solution: dissolving a 1.0mg ketamine sample in 1mL methanol to prepare a 1.0mg/mL ketamine sample solution for qualitative and quantitative analysis, and filtering the sample solution through a 0.45-micrometer microporous filter membrane before use;
(1.2) determination of liquid chromatography conditions: the chromatographic column is a Shim-pack HRC-ODS column, 250mm multiplied by 4.6mm I.D., 5 μm; the mobile phase is methanol V: v0.05% trifluoroacetic acid/water (33-35) (65-67), isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 1.0 mL/min; the column temperature is 35 ℃;
(1.3) calculating a regression equation of the standard curve and determining a linear range: diluting ketamine standard stock solution with chromatographic pure methanol, precisely preparing ketamine reference substance solution with the concentrations of 0.5, 1, 5, 10, 50, 100, 500 and 1000 mug/mL respectively, determining according to the reversed-phase chromatographic condition in the step (1.2), repeating each concentration for 3 times, calculating by using an average value, recording the area of the ketamine chromatographic peak, taking the sample injection concentration of the reference substance as a horizontal coordinate, the concentration unit as mug/mL and the chromatographic peak area value as a vertical coordinate, and calculating a plotting equation of a standard curve; the peak area is plotted against concentration, and the regression equation for the standard curve is:
Y＝3×10X+92617，R＝0.9999，
Ketamine has good linear relation in the range of 0.5-1000 mug/mL;
(1.4) analyzing and measuring a ketamine sample solution with the concentration of 1.0mg/mL according to the reversed phase chromatography method in the step (1.2), repeating the measurement for 3 times, recording the area of the ketamine peak, calculating the average value of the ketamine peak, and calculating the content of the ketamine in the sample according to an external standard method of the area of the peak;
In the step (2), the method comprises the following steps:
(2.1) preparation of sample solution: 3.369g of ketamine sample is firstly dissolved in 10mL of methanol, the solution is centrifuged after being fully dissolved, the precipitate is removed by filtration, the organic phase is taken out, the solution is centrifugally concentrated to dryness, water is added to the solution to a constant volume of 10mL, a ketamine sample solution is prepared for separating and preparing a ketamine standard substance by preparative high performance liquid chromatography, and the sample solution is filtered by a 0.45-micrometer microporous filter membrane before use;
(2.2) determining liquid chromatography conditions:
(2.2.1) the column was a Shim-pack VP-ODS preparative column, 250mm × 20mm I.D., 15 μm; the mobile phase is methanol V: mixing methanol and water at a ratio of 30:70, degassing, and eluting at equal rate; ultraviolet detection wavelength is 210 nm; the flow rate is 8 mL/min; loading 700 μ L; the column temperature is room temperature; after 20 minutes of operation the column was washed with 100% methanol; or
(2.2.2) the column was a Shim-pack VP-ODS preparative column, 250mm × 20mm I.D., 15 μm; the mobile phase is methanol V: v0.05% trifluoroacetic acid/water 20:80, isocratic elution; ultraviolet detection wavelength is 210 nm; the flow rate is 8 mL/min; the loading amount is 500 mu L; the column temperature is room temperature;
(2.3) under the chromatographic condition in the step (2.2.2), removing methanol from the collected fractions by rotary evaporation, adjusting the pH of the residual aqueous solution to 11, extracting with chromatographic pure chloroform, combining organic phases, carrying out centrifugal concentration until white precipitates appear, injecting water into the white precipitates, slowly adding 0.1N hydrochloric acid until the pH of the aqueous solution is 5-6, removing the organic phase, and carrying out freeze drying on the aqueous phase to obtain ketamine hydrochloride crystals.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808846A (en) * | 2014-02-20 | 2014-05-21 | 福建国际旅行卫生保健中心 | Series quadrupole-rod gas-chromatographic mass spectrometry detection method for 35 toxic medicaments in urine |
| CN103823008A (en) * | 2014-03-14 | 2014-05-28 | 北京市疾病预防控制中心 | Method for detecting unknown poison by establishing liquid chromatography-mass spectrometry database |
| CN103896898A (en) * | 2014-03-07 | 2014-07-02 | 中国计量科学研究院 | Naringenin standard substance as well as preparation and application thereof |
| CN105717240A (en) * | 2016-03-24 | 2016-06-29 | 中国标准化研究院 | Method for preparing Alitame standard substance |
| CN106442034A (en) * | 2016-03-24 | 2017-02-22 | 中国标准化研究院 | 2, 4-dichlorophenoxyacetic acid standard substance preparation method and application thereof |
| JP2019066360A (en) * | 2017-10-02 | 2019-04-25 | 国立研究開発法人農業・食品産業技術総合研究機構 | Analysis method and analysis system of procyanidin group |
-
2017
- 2017-05-08 CN CN201710316040.0A patent/CN107478757B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808846A (en) * | 2014-02-20 | 2014-05-21 | 福建国际旅行卫生保健中心 | Series quadrupole-rod gas-chromatographic mass spectrometry detection method for 35 toxic medicaments in urine |
| CN103896898A (en) * | 2014-03-07 | 2014-07-02 | 中国计量科学研究院 | Naringenin standard substance as well as preparation and application thereof |
| CN103823008A (en) * | 2014-03-14 | 2014-05-28 | 北京市疾病预防控制中心 | Method for detecting unknown poison by establishing liquid chromatography-mass spectrometry database |
| CN105717240A (en) * | 2016-03-24 | 2016-06-29 | 中国标准化研究院 | Method for preparing Alitame standard substance |
| CN106442034A (en) * | 2016-03-24 | 2017-02-22 | 中国标准化研究院 | 2, 4-dichlorophenoxyacetic acid standard substance preparation method and application thereof |
| JP2019066360A (en) * | 2017-10-02 | 2019-04-25 | 国立研究開発法人農業・食品産業技術総合研究機構 | Analysis method and analysis system of procyanidin group |
Non-Patent Citations (6)
| Title |
|---|
| Determination of ketamine and norketamine enantiomers in plasma by solid-phase extraction and high-performance liquid chromatography;Jan-Olof Svensson 等;《Journal of Chromatography B》;19960412;第678卷(第2期);第373-376 * |
| Se＇bastien Bolze 等.HPLC determination of ketamine, norketamine,and dehydronorketamine in plasma with a high-purity reversed-phase sorbent.《Clinical Chemistry》.1998,第44卷(第3期), * |
| Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection;Frank Niedorf 等;《Journal of Chromatography B》;20030705;第791卷(第1-2期);第421-426页 * |
| 二乙酰吗啡盐酸盐标准物质的研制(1)——均匀性和稳定性检验;冯超 等;《刑事技术》;20131231(第4期);第20-24页 * |
| 人血中级胺酮的高效液相色谱法测定;陈弘博 等;《中国医院药学杂志》;19911231;第11卷(第10期);第439-440页 * |
| 高效液相色谱法测定血中氯胺酮浓度的改进;张金玲;《中国医院药学杂志》;20001231;第20卷(第1期);第11-12页 * |
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