CN107478757A - Method for preparing purified for the ketamine standard substance of forensic science illicit drugs inspection - Google Patents

Method for preparing purified for the ketamine standard substance of forensic science illicit drugs inspection Download PDF

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CN107478757A
CN107478757A CN201710316040.0A CN201710316040A CN107478757A CN 107478757 A CN107478757 A CN 107478757A CN 201710316040 A CN201710316040 A CN 201710316040A CN 107478757 A CN107478757 A CN 107478757A
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ketamine
methanol
sample
standard substance
water
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CN107478757B (en
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郑珲
高利生
郑晓雨
张春水
赵阳
常颖
贺剑锋
翟晚枫
李彭
赵彦彪
杨虹贤
刘克林
钱振华
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Institute of Forensic Science Ministry of Public Security PRC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • G01N2030/085—Preparation using an enricher using absorbing precolumn

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Abstract

The present invention discloses the method for preparing purified of the ketamine standard substance for forensic science illicit drugs inspection, comprises the following steps：(1) purity of ketamine in ketamine sample is captured in detection, the raw material captured ketamine sample and ketamine standard substance is prepared as purifying for selecting ketamine mass fraction to be more than or equal to 50wt%；(2) ketamine standard substance is prepared using high performance liquid chromatography.The present invention prepares the ketamine that purification process obtains and confirmed through nuclear magnetic resonance, Liquid Chromatography-Tandem Mass Spectrometry combination, infrared spectrum analysis, and its purity confirms definite value through liquid chromatogram, gas-chromatography, and chromatogram is measured without response impurity；Required according to Developments of certified reference samples, the estimation of its stability, uniformity, definite value, overall uncertainty meets relevant regulations, reaches expectation index.

Description

Method for preparing purified for the ketamine standard substance of forensic science illicit drugs inspection
Technical field
The present invention relates to the preparation of forensic science drugs standard items.It is used for forensic science drugs more particularly, to one kind The method for preparing purified of the ketamine standard substance of detection.
Background technology
At present, the drug issue being on the rise turns into global disaster.Drugs spread unchecked the body for directly endangering the people Heart health, and bring grave danger to economic growth and social progress.Therefore, forensic science illicit drugs inspection magnitude tracing body is established System, drugs composition measurement technique is improved, ensure the reliability and comparativity of measurement result, establish the shared and mutual of measurement data Recognize, accurately and reliably evidence is provided for court, it has also become countries in the world drugs appraisal organization question of common concern.
Drugs composition measurement technique and traceability guarantee are an organic wholes, are core measurement capabilities in section of court Learn the important embodiment of drugs ingredient amount fields of measurement.Standard substance be through in this overall skeleton, be value carrier, be The key element of drugs ingredient amount traceability system, it is the weight for ensureing measurement result accuracy over time and space and comparativity It is basic, it is to realize effectively i.e. accurate, comparable, the measurement that can trace to the source the basic assurance of measurement.
Being generally acknowledged in the world using what the companies such as Sigma produced mostly in the advanced illicit drugs inspection laboratory of the National Technicals such as America and Europe Standard substance, but the standard substance of import can only be relied in China, it is high to measure few valency, has many to be provided to China.Mesh Not only species is extremely limited " reference substance " used in preceding domestic drugs of abuse, and general lack of perfect Structural Identification, pure The corresponding technical indicators such as degree measure, uniformity and stability.These all carry out certain uncertainty to case detection band of being involved in drug traffic, Directly influence the accuracy of quantitative result.Moreover, the scarcity of domestic drugs standard substance, has become restriction China and realizes method Front yard science illicit drugs inspection chemical measurement methodological standardization, the major obstacle traced to the source and recognized each other of measurement result.Therefore, it is possible to prepare Go out the problem of having become China's drugs research field urgent need to resolve for the drugs standard substance of forensic science illicit drugs inspection.
The english common name of ketamine：(±)-Ketamine hydrochloride, chemical name：2- Chloro-O-Phenyls- 2- tetraminos-cyclohexanone hydrochloride, English name：(±)-2-(2-Chlorophenyl)-2-(methylamino) Cyclohexanone hydrochloride, molecular formula：C13H16ClNOHCl, molecular weight：274.19 CA registration numbers： 1867-66-9, structural formula are：
Physicochemical property：White crystalline powder, it is odorless.(200mg/mL) soluble in water, is dissolved in hot ethanol, insoluble in ether And benzene.Fusing point is 259-263 DEG C.The aqueous solution is in acidity, pH value 4.0~5.5.
The content of the invention
It is an object of the present invention to provide a kind of the pure of ketamine standard substance for forensic science illicit drugs inspection Change preparation method.
To reach above-mentioned purpose, the present invention uses following technical proposals：
For the method for preparing purified of the ketamine standard substance of forensic science illicit drugs inspection, comprise the following steps：(1) examine The purity for capturing ketamine in ketamine sample is surveyed, selects ketamine mass fraction to capture ketamine more than or equal to 50wt% Sample prepares the raw material of ketamine standard substance as purifying；
(2) ketamine standard substance is prepared using high performance liquid chromatography.
The method for preparing purified of the above-mentioned ketamine standard substance for forensic science illicit drugs inspection, in step (1), bag Include following steps：
(1.1) preparation of sample solution：1.0mg ketamine samples are dissolved in 1mL methanol, are made into 1.0mg/mL ketamine samples Product solution is used for Qualitative and quantitative analysis, passes through 0.45 μm of filtering with microporous membrane using preceding sample solution；
(1.2) liquid phase chromatogram condition is determined：Chromatographic column is Shim-pack HRC-ODS posts, 250mm × 4.6mm I.D., 5 μm；Mobile phase is VMethanol：V0.05% trifluoroacetic acid/water=(33-35):(65-67), isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 1.0mL/min；35 DEG C of column temperature；
(1.3) calculate the regression equation of standard curve and determine the range of linearity：Ketamine standard is diluted with Chromatographic Pure Methanol Storing solution, precision are configured to the ketamine reference substance solution that concentration is respectively 0.5,1,5,10,50,100,500,1000 μ g/mL, Determined by the reverse-phase chromatography condition in step (1.2), each concentration is repeated 3 times, and with mean value calculation, records ketamine chromatographic peak Area, using the sample introduction concentration of reference substance as abscissa, concentration is μ g/mL, and chromatographic peak area value is mapped for ordinate, and calculates mark The regression equation of directrix curve；Concentration is mapped with peak area, the regression equation of standard curve is：
Y=3 × 107X+92617, R2=0.9999,
Ketamine linear relationship in the range of 0.5--1000 μ g/mL is good；
(1.4) it is molten to press the ketamine sample that the reverse-phase chromatography method analysis measure concentration in step (1.2) is 1.0mg/mL Liquid, replication 3 times, ketamine peak area is recorded, calculates its average value, the ketamine in sample is calculated by peak area external standard method Content.
The method for preparing purified of the above-mentioned ketamine standard substance for forensic science illicit drugs inspection, in step (2), bag Include following steps：
(2.1) preparation of sample solution：3.369g ketamine samples are first dissolved in 10mL methanol, are fully centrifuged after dissolving, Filter off except precipitation, organic phase are taken out, centrifugal concentrating adds water to be settled to 10mL to doing, and is made into ketamine sample solution and is used to make Standby type high performance liquid chromatography separation prepares ketamine standard items, passes through 0.45 μm of filtering with microporous membrane using preceding sample solution；
(2.2) liquid phase chromatogram condition is determined：
(2.2.1) chromatographic column is that Shim-pack VP-ODS prepare post (250mm × 20mm I.D., 15 μm)；Mobile phase is VMethanol：VWater=30:70, first alcohol and water uses after fully mixing degassing, isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 8mL/ min；The μ L of applied sample amount 700；Column temperature is room temperature；Chromatographic column is rinsed with 100% methanol after running 20 minutes；Or
(2.2.2) chromatographic column is that Shim-pack VP-ODS prepare post (250mm × 20mm I.D., 15 μm)；Mobile phase is VMethanol：V0.05% trifluoroacetic acid/water=20:80, isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 8mL/min；The μ L of applied sample amount 500；Column temperature For room temperature；
(2.3) under the chromatographic condition in using step (2.2.2), by the cut being collected into after revolving removes methanol, Adjust remaining aqueous solution pH to 11, with chromatographically pure chloroform recovery, merge organic phase, centrifugal concentrating to there is white precipitate, to Water is wherein injected, is slowly added to 0.1N hydrochloric acid to aqueous pH to 5-6, wherein organic phase is removed, can be obtained after aqueous phase freeze-drying Obtain ketalar crystal.
Beneficial effects of the present invention are as follows：
The present invention is prepared in the ketamine crystal that purification process obtains, according to high performance liquid chromatography areas of peak normalization method meter Calculate ketamine purity and be more than or equal to 99.1wt%.
The present invention prepares ketamine that purification process obtains through nuclear magnetic resonance, Liquid Chromatography-tandem Mass, infrared light Spectrum is analyzed to identify, and its purity confirms definite value through liquid chromatogram, gas-chromatography, and chromatogram is measured without response impurity；According to Developments of certified reference samples requirement, the estimation of its stability, uniformity, definite value, overall uncertainty meet relevant regulations, reach expected Index.
The present invention prepare purification process can be provided for judicial expertise department of China value accurately, the ketamine mark that can trace to the source Quasi- material, China's forensic science field drugs standard substance blank is filled up, to improve analysis measurement quality, improves quantitative result The degree of accuracy, farthest ensure the validity of measurement result.Help to establish forensic science illicit drugs inspection magnitude tracing system, Domestic forensic science illicit drugs inspection chemical measurement methodological standardization is advantageously implemented, realizes the reliable, effective and mutual of measurement result Recognize.
Overcome prior art and prepare existing for the ketamine sample that purification process obtains that purity is low, stability is poor, uniform Property poor, preparation process complexity the defects of, there is provided one kind using high performance liquid chromatography separation method obtain high-purity, high stability, Rate of recovery height, the ketamine standard substance preparation method for being easy to large-scale production.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
The ultraviolet spectrogram of Fig. 1-1 impurity 1；The ultraviolet spectrogram of Fig. 1-2 ketamines；The ultraviolet spectra of Fig. 1-3 impurity 2 Figure；
Fig. 2 ketamine sample reverse-phase HPLC chromatography figures；(the two volume ratio is 25 to acetonitrile-phosphate buffer:75)；
Fig. 3 ketamine sample reverse-phase HPLC chromatography figures；(the two volume ratio is 35 to methanol -0.05%TFA water:65)；
Fig. 4 ketamine sample reverse-phase HPLC chromatography figures；(the two volume ratio is 33 to acetonitrile -0.05%TFA water:67)；
Fig. 5 ketamine sample preparation HPLC chromatogram (methanol：0.05%TFA/ water=20:80, volume ratio)；
Reverse-phase HPLC chromatography figure (the methanol of Fig. 6 ketamines：0.05%TFA/ water=33:67, volume ratio)；
(the two volume ratio is 40 to Fig. 7 ketamine preparative liquid chromatography figures methanol/water：60), 10mL/min, 210nm；
(the two volume ratio is 40 to Fig. 8 ketamine preparative liquid chromatography figures methanol/water：60), 8mL/min, 210nm；
(the two volume ratio is 30 to Fig. 9 ketamine preparative liquid chromatography figures methanol/water：70), 10mL/min；
(the two volume ratio is 30 to Figure 10 ketamine preparative liquid chromatography figures methanol/water：70), 8mL/min；
Reverse-phase HPLC chromatography figure (the methanol of Figure 11 ketamines：0.05%TFA/ water=35:65, volume ratio)；
The method for preparing purified flow chart of Figure 12 ketamine standard substances；
The hydrogen spectrogram of Figure 13 ketamines；The carbon spectrogram of Figure 14 ketamines；
Figure 15 ketalar sample mass spectrums；The infrared spectrum of Figure 16 ketalars；
The makings spectrogram of Figure 17 ketalars；The GC/MS total ion current figures of Figure 18 ketamines
Figure 19 hydroxyl imines mass spectrograms.
Embodiment
In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings It is bright.Similar part is indicated with identical reference in accompanying drawing.It will be appreciated by those skilled in the art that institute is specific below The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.
The present embodiment is mainly using remaining ketamine crystal sample after case identification, to utilizing preparative liquid chromatography Isolate and purify and prepare the experiment condition of ketamine screening is optimized.
First, instrument, reagent and material
1.1 key instrument
Analytic type high performance liquid chromatograph (Japanese Shimadzu), including：LC-20AD high pressure pumps；SIL-10A auto injections Device；SPD-20A PDADs；CTO-20A column ovens.
Preparative high performance liquid chromatography instrument (Agilent), including：G1361A high pressure pumps；G2260A automatic samplers； G1315D PDADs；G1364B automatic fraction collectors.
BUCHI rotary evaporators (Japanese BUCHI companies)；(city of Kunshan's ultrasonic instrument has KQ3200 types ultrasonic cleaner Limit company)；Flying pigeon board TDL-40B desk centrifuges (Anting Scientific Instrument Factory, Shanghai)；XS105Dual Range electronic balances (METTLER TOLEDO companies of Switzerland).
1.2 main agents and material
Methanol (chromatographically pure, Fisher Scientific companies of the U.S.), (chromatographically pure, Chinese lark prestige are public for trifluoroacetic acid Department), ultra-pure water (purifies, French Millipore companies) through Millipore ultrapure water production systems.Ketamine 1mg/mL standards Solution (Chinese lark prestige company)；Ketamine sample (white powder) is captured and is applied in this research by case.
It is prepared by the purifying for the 2nd, capturing the purity testing of ketamine and ketamine standard substance in ketamine sample
The preparation of 2.1 sample solutions
Analytic type：1.0mg ketamine samples are dissolved in 1mL methanol, be made into 1.0mg/mL ketamines sample solution be used for it is fixed Property and quantitative analysis, pass through 0.45 μm of filtering with microporous membrane using preceding sample solution.
Preparative sample solution one：3.369g ketamine samples are first dissolved in 10mL methanol, are fully centrifuged, are filtered after dissolving Precipitation is removed, organic phase is taken out, and centrifugal concentrating adds water to be settled to 10mL, be made into ketamine sample solution and be used for preparative to doing High performance liquid chromatography separation prepares ketamine standard items.Pass through 0.45 μm of filtering with microporous membrane using preceding sample solution.
Preparative sample solution two：Weigh ketamine sample 3368.51mg (equivalent to pure ketamine 1999.548mg) in In 50mL volumetric flasks, water is added to be settled to scale.The sample aqueous solution that ketamine content is 40mg/mL is configured to, for preparative High performance liquid chromatography separation prepares ketamine standard items, passes through 0.22um mixing membrane filtrations using preceding.
2.2 liquid phase chromatogram condition
2.2.1 RPLC (RP-HPLC) analysis method:
Chromatographic condition one：Chromatographic column is Shim-pack HRC-ODS posts (250mm × 4.6mm I.D., 5 μm)；Mobile phase For methanol：0.05% trifluoroacetic acid/water=35:65 (volume ratio, 0.05% trifluoroacetic acid/water refer to trifluoroacetic acid volume fraction Trifluoroacetic acid aqueous solution for 0.05%), isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 1.0mL/min；35 DEG C of column temperature.
Chromatographic condition two：Chromatographic column is Shim-pack HRC-ODS posts (250mm × 4.6mm I.D., 5 μm)；Mobile phase For methanol：0.05% trifluoroacetic acid/water=33:67 (volume ratio, 0.05% trifluoroacetic acid/water refer to trifluoroacetic acid volume fraction Trifluoroacetic acid aqueous solution for 0.05%), isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 1.0mL/min；35 DEG C of column temperature.
2.2.2 RPLC (RP-HPLC) preparation method:
Chromatographic condition one：Chromatographic column is that Shim-pack VP-ODS prepare post (250mm × 20mm I.D., 15 μm)；Flowing It is mutually methanol：Water=30:70 (volume ratios), isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 8mL/min；The μ of applied sample amount 700 L；Column temperature is room temperature.
Chromatographic condition two：Chromatographic column is that Shim-pack VP-ODS prepare post (250mm × 20mm I.D., 15 μm)；Flowing It is mutually methanol：0.05% trifluoroacetic acid/water=20:80 (volume ratios), isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 8mL/ min；The μ L of applied sample amount 500；Column temperature is room temperature.
The optimization of 2.3 efficient liquid phase chromatographic analysis conditions
2.3.1 the selection of chromatographic column
The reverse-phase chromatographic column that this experiment uses is Shim-pack HRC-ODS posts (250mm × 4.6mm I.D., 5 μm).
2.3.2 the selection of Detection wavelength
With reference to the UV absorption figure of ketalar and impurity (as shown in Fig. 1-1, Fig. 1-2 and Fig. 1-3), detection is compared Wavelength is 210nm, 220nm, and principal component and impurity response difference under 230nm, 240nm, 254nm, comprehensive comparative analysis baseline are made an uproar Sound, detection sensitivity and result stability data, it is determined that selection 210nm is as definite value and Detection wavelength.
2.3.3 the selection of flow visualizing
On the selection of reverse-phase chromatography flow visualizing, experiment compares several different mobile phase acetonitrile-phosphate and delayed Fliud flushing, methanol-phosphate buffer, the acetonitrile -0.05%TFA aqueous solution【The 0.05%TFA aqueous solution refers to trifluoroacetic acid volume integral Number is 0.05% trifluoroacetic acid aqueous solution】, methanol -0.05%TFA the aqueous solution【The 0.05%TFA aqueous solution refers to trifluoroacetic acid Volume fraction is 0.05% trifluoroacetic acid aqueous solution】And methanol-water solution is to the chromatographic isolation degree of ketamine and impurity, peak shape With the influence of retention time etc..As a result show, use acetonitrile-phosphate buffer (volume ratio 25:75), methanol -0.05% TFA water (volume ratio 35:And acetonitrile -0.05%TFA water (volume ratios 33 65)：67), the ketamine component in sample with Impurity component therein can be separated preferably, and it is small to trail, and appearance time is fast, and ketamine in methanol-water solution Chromatographic peak seriously trails, therefore the system is not suitable for separating each component in ketamine sample.Experiment is considered slow The service life of chromatographic column can be shortened by rushing salt, and acetonitrile toxicity is big and increases experimentation cost, therefore considers selective flow phase System is that (the two volume ratio is 35 to methanol -0.05%TFA water:65) system.The liquid phase of ketamine sample under each flow visualizing Chromatogram is as shown in Fig. 2, Fig. 3 and Fig. 4.
2.3.4 standard curve and linear relationship
With chromatogram methanol dilution ketamine standard reserving solution, precision be configured to concentration be respectively 0.5,1,5,10,50,100, 500th, 1000 μ g/mL ketamine reference substance solution, determined by reverse-phase chromatography condition, each concentration is repeated 3 times, in terms of average value Calculate, record ketamine chromatographic peak area, with the sample introduction concentration (μ g/mL) of reference substance for abscissa, chromatographic peak area value is sat to be vertical Figure is denoted as, and calculates the regression equation of standard curve.
The concentration and peak area of the ketamine of table 1
Concentration is mapped with peak area, the regression equation of standard curve is：
Y=3 × 107X+92617, R2=0.9999 ... ... ... ...
Show that ketamine linear relationship in the range of 0.5--1000 μ g/mL is good.
2.3.5 in sample ketamine content measure
The ketamine sample solution that measure concentration is 1.0mg/mL, replication 3 times, record are analyzed by reverse-phase chromatography method Ketamine peak area, its average value is calculated, the ketamine content in sample is calculated by peak area external standard method.
The measurement result of ketamine content in the sample of table 2
As shown in Table 2,1.0mg/mL ketamine sample is calculated by calibration curve equation by peak area external standard method The content of ketalar is 0.5936mg/mL in solution, and then the purity for trying to achieve ketalar in ketamine sample is 59.36wt%.
2.3.6 the optimization of high performance liquid chromatography preparation condition
Using methanol-TFA aqueous systems (preparative sample solution two is used to separate the ketamine component in ketamine sample With chromatographic condition two), experiment shows to use makes impurity component and ketamine component with identical proportion of mobile phase in analysis condition Be overlapped mutually, impurity component is separated with ketamine component, by the regulation to methanol ratio, final ketamine and its His component has reached preferable separating effect, and it is as shown in Figure 5 to prepare liquid chromatography(LC figure.The cut being collected into is further purified：Chlorine Ketamine is existing in the form of hydrochloride in amine ketone sample, passes through centrifugal concentrating or rotation through preparing liquid phase separation tails Methanol is removed after evaporation, but the trifluoroacetic acid remained in cut can not be removed, final chlorine product ketone is prepared to obtain Hydrochloride, further purification process need to be passed through.After revolving removes methanol, remaining aqueous pH is adjusted to 11, with chromatographically pure Chloroform repeatedly extracts repeatedly, and after merging organic phase, centrifugal concentrating injects moisture, be slowly added to thereto to there is white precipitate 0.1N hydrochloric acid is 5-6 to aqueous pH, removes wherein organic phase, ketalar crystal can be obtained after aqueous phase freeze-drying.Its HPLC chromatogram is as shown in Figure 6.Analyzed through HPLC, areas of peak normalization method measure percentage composition, ketamine content 99.72wt%.
Secondarily purified process of the ketamine standard substance in cut is prepared above with methanol-trifluoroacetic acid aqueous systems separation In, it is necessary to adjust alkalescence condition to be carried with organic solvent is counter after being rotated to cut again, component carries out acidified to obtain chloramines Keto hydrochloride, operating process is complicated, and the chance for introducing impurity increases, therefore, further preparation condition is improved, and attempts Ketamine component is prepared using methanol-water solution separation (using preparative sample solution one and chromatographic condition one).
Experiment proves, is not suitable for analyzing ketamine sample using methanol-water solution, but simultaneously it has also been found that making in the system For mobile phase when whole component can be eluted out, therefore attempted methanol-water solution prepare liquid phase on to ketamine Separating effect.From experimental result, when flowing phase composition is methanol：Water (volume ratio 40：60), flow velocity 10mL/min When, ketamine can be completely separable with impurity component behind, but with the impurity before it while appearance；Further reduce stream Speed, flowing phase composition is constant, is separated under the conditions of 8mL/min, and the separate condition of ketamine and impurity before it does not obtain To improvement.Preparative liquid chromatography figure is shown in Fig. 7 and Fig. 8.
Methanol ratio in mobile phase is reduced, when flowing phase composition is methanol/water (volume ratio 30：70), flow velocity 10mL/ Min can obtain preferable separating effect.Ketamine reaches with the impurity component before and after it preferably to be separated.In view of flow velocity Chromatogram column pressure increases when high, therefore effectively to extend using life of chromatographic column, flow velocity is reduced, is 8mL/min bars in flow velocity Tested under part, still obtain preferable separating effect, the time that each group distributes chromatographic column adds about 6min.The final choosing of experiment It is methanol to determine separation condition：Water (volume ratio 30：70), flow velocity 8mL/min.Preparative liquid chromatography figure is shown in Fig. 9-Figure 10.
2.4 batch program
Due to by experiment investigation, (He of preparative sample solution one is used under the conditions of used preparative liquid chromatography Chromatographic condition one), ketamine component just can be eluted out from preparing in post in 20min, but impurity appearance below makes too late Disengaging time extends.Again because the flow visualizing of preparative separation is made up of first alcohol and water, when water mixes with methanol, will go out Existing exothermic phenomenon, mixed solvent volume reduce, while have a large amount of bubbles to discharge.Therefore, experiment is using entering first alcohol and water before pump (volume ratio 30:70) mix in proportion, fully mix degassing after use, preparation method run 20 minutes after with 100% methanol Chromatographic column is rinsed, makes to remain in the impurity component in post and is eluted rapidly.The batch program of foundation is that following three steps are pressed Order repeatedly circulation, i.e.,：Methanol rinses chromatographic column first, flows the chromatographic column that balances each other afterwards, then runs preparation method.
Ketamine is existing in the form of hydrochloride, through being still chlorine in preparation liquid phase separation tails in ketamine sample Amine keto hydrochloride, further purification process need to be passed through.The methanol content in cut is removed after revolving, is freeze-dried and true Ketalar crystal can be obtained after sky dehydration.Its HPLC chromatogram is as shown in figure 11.
The structural identification of the 2.5 ketamine standard substances being prepared
2.5.1 nuclear magnetic resonance spectroscopy
In order to determine the structure of the compound obtained by preparation liquid phase separation, nuclear magnetic resonance spectroscopy is carried out, spectrogram is shown in figure 13-14。
Solvent：D2O
Position Chemical shift13C Chemical shift1H Hydrogen spectrum is split point
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 method
Under ESI positive ion modes, voltage 80V is crushed, impact energy 8eV, the mass spectrogram of sample is as shown in figure 15.From mass spectrum Following message can be obtained in figure：
Quasi-molecular ion peak [M+H]+m/z238.20, differ 1 with the relative molecular mass 237.72g/mol of ketamine, Thus the detection molecular weight of sample and being consistent for ketamine.
2.5.3 infra-red sepectrometry
The infrared light of ketamine is determined on AVATAR 330FT-IR infrared spectrometers (Thermo Nicolet companies) Spectrum, such as Figure 16 are as a result consistent with NIST spectrograms storehouse Plays spectrogram.
2.5.4 gas chromatography-mass spectrography
The standard spectrogram of the spectrogram (Figure 17) that gas chromatography-mass spectrography obtains and ketamine is consistent.
Due to a larger impurity in gas chromatographic analysis be present, its content is 0.4%.Therefore gas phase color is utilized Spectrum-mass spectrography is analyzed it.Mass spectrogram analysis (Figure 18) according to measured by instrument, major impurity are hydroxyl imines.Hydroxyl Imines is mainly used as medicine intermediate, is the important intermediate for synthesizing ketamine, coffee color and cream powder.In 2008 It is put into " regulation on Management of Drug-Making Chemicals ".CAS 90717-16-1.
The purity testing result of 2.6 gained ketamine standard substances
Analyzed through HPLC, GC and QNMR method definite value, the purity of the ketalar standard substance of preparation is 99.47wt%, expanded uncertainty are 0.62% (k=2).Have good uniformity, stability at least more than 1 year.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description To make other changes in different forms, all embodiments can not be exhaustive here, it is every to belong to this hair Row of the obvious changes or variations that bright technical scheme is extended out still in protection scope of the present invention.

Claims (3)

1. the method for preparing purified of the ketamine standard substance for forensic science illicit drugs inspection, it is characterised in that including as follows Step：(1) purity of ketamine in ketamine sample is captured in detection, selects ketamine mass fraction more than or equal to 50wt%'s Capture the raw material that ketamine sample prepares ketamine standard substance as purifying；
(2) ketamine standard substance is prepared using high performance liquid chromatography.
2. according to the purifying preparation side of the ketamine standard substance for forensic science illicit drugs inspection described in claim 1 Method, it is characterised in that in step (1), comprise the following steps：
(1.1) preparation of sample solution：1.0mg ketamine samples are dissolved in 1mL methanol, and it is molten to be made into 1.0mg/mL ketamine samples Liquid is used for Qualitative and quantitative analysis, passes through 0.45 μm of filtering with microporous membrane using preceding sample solution；
(1.2) liquid phase chromatogram condition is determined：Chromatographic column is Shim-pack HRC-ODS posts, 250mm × 4.6mm I.D., 5 μm； Mobile phase is VMethanol：V0.05% trifluoroacetic acid/water=(33-35):(65-67), isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 1.0mL/ min；35 DEG C of column temperature；
(1.3) calculate the regression equation of standard curve and determine the range of linearity：Ketamine standard inventory is diluted with Chromatographic Pure Methanol Liquid, precision is configured to the ketamine reference substance solution that concentration is respectively 0.5,1,5,10,50,100,500,1000 μ g/mL, by step Suddenly the reverse-phase chromatography condition measure in (1.2), each concentration are repeated 3 times, with mean value calculation, record ketamine chromatographic peak face Product, using the sample introduction concentration of reference substance as abscissa, concentration unit is μ g/mL, and chromatographic peak area value is mapped for ordinate, and is calculated The regression equation of standard curve；Concentration is mapped with peak area, the regression equation of standard curve is：
Y=3 × 107X+92617, R2=0.9999,
Ketamine linear relationship in the range of 0.5--1000 μ g/mL is good；
(1.4) the ketamine sample solution that the reverse-phase chromatography method analysis measure concentration in step (1.2) is 1.0mg/mL, weight are pressed Repetition measurement is fixed 3 times, records ketamine peak area, calculates its average value, and the ketamine content in sample is calculated by peak area external standard method.
3. according to the purifying preparation side of the ketamine standard substance for forensic science illicit drugs inspection described in claim 1 Method, it is characterised in that in step (2), comprise the following steps：
(2.1) preparation of sample solution：3.369g ketamine samples are first dissolved in 10mL methanol, are fully centrifuged, are filtered after dissolving Precipitation is removed, organic phase is taken out, and centrifugal concentrating adds water to be settled to 10mL, be made into ketamine sample solution and be used for preparative to doing High performance liquid chromatography separation prepares ketamine standard items, passes through 0.45 μm of filtering with microporous membrane using preceding sample solution；
(2.2) liquid phase chromatogram condition is determined：
(2.2.1) chromatographic column is that Shim-pack VP-ODS prepare post, 250mm × 20mm I.D., 15 μm；Mobile phase is VMethanol： VWater=30:70, first alcohol and water uses after fully mixing degassing, isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 8mL/min； The μ L of applied sample amount 700；Column temperature is room temperature；Chromatographic column is rinsed with 100% methanol after running 20 minutes；Or
(2.2.2) chromatographic column is that Shim-pack VP-ODS prepare post, 250mm × 20mm I.D., 15 μm；Mobile phase is VMethanol： V0.05% trifluoroacetic acid/water=20:80, isocratic elution；Ultraviolet detection wavelength 210nm；Flow velocity 8mL/min；The μ L of applied sample amount 500；Column temperature is room Temperature；
(2.3) under the chromatographic condition in using step (2.2.2), by the cut being collected into after revolving removes methanol, regulation Remaining aqueous solution pH to 11, with chromatographically pure chloroform recovery, merge organic phase, centrifugal concentrating is to there is white precipitate, thereto Water is injected, is slowly added to 0.1N hydrochloric acid to aqueous pH to 5-6, wherein organic phase is removed, salt can be obtained after aqueous phase freeze-drying Sour ketamine crystal.
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