CN105181823A - Method for determining content of methcathinone in sample by high performance liquid chromatography - Google Patents
Method for determining content of methcathinone in sample by high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for determining the content of methcathinone in a sample by high performance liquid chromatography. The method includes the steps of: (1) preparation of a working solution; (2) preparation of a sample solution; (3) setting of detection conditions; and (4) determination of the methcathinone content of the sample. The method provided by the invention adopts reversed-phase chromatography isocratic elution, can complete analysis within 12 minutes, has efficient and accurate detection result, can be used for determination of the methcathinone content of the sample, and provides technical support for conviction and sentencing of judicial departments.
Description
Technical field
The present invention relates to illicit drugs inspection field, criminal investigation field, particularly adopt the method for methcathinone content in high effective liquid chromatography for measuring sample.
Background technology
Methcathinone is novel drug human body being had to quite harm occurred in recent years.This harm not only affects the physiological situation of human body, also can affect the psychological activity of people, excessively also can cause death.The abuse of methcathinone not only has harm to human body, and also can cause adverse effect to the development of society.In 2007 editions " narcotics and psychotropic substances kind catalogues " that state food Drug Administration general bureau, the Ministry of Public Security, national health State Family Planning Commission issue, methcathinone is classified as class psychotropic substances and gives control.
Methcathinone systematic chemical name is: N-methcathinone, and its chemical structural formula is such as formula shown in (1):
Prior art has mensuration methcathinone sample sizes such as adopting gas chromatography/mass spectrometry method, infrared spectroscopy, high performance liquid chromatography and LC-MS technology.Wherein, in the process detected utilizing liquid phase chromatography, still there is detection time long, the technical matters that detection efficiency is low, causing the reduction of criminal investigation work efficiency.Therefore be badly in need of a kind of high-precision, and the method that in sample, methcathinone detects can be realized in shorter time.
Summary of the invention
In view of this, the invention reside in the method that a kind of precision is high, detection time is short high effective liquid chromatography for measuring methcathinone sample size is provided.
The present invention is achieved through the following technical solutions: a kind of method adopting methcathinone content in high effective liquid chromatography for measuring sample, it comprises the steps:
(1) preparation of working fluid;
(2) preparation of sample solution;
(3) setting of testing conditions;
(4) mensuration of methcathinone content in sample;
(5) experimental result calculates,
Wherein, in step (3), chromatographic column adopts anti-phase phenyl chromatographic column, and mobile phase is A-1 ~ 5mM ammonium acetate buffer: B-acetonitrile, A:B=(20 ~ 40): (60-80), and flow velocity is 1.0-1.5mL/min.
In the step (3) of said method, in step (3), the 4mM ammonium acetate buffer of A-pH=5.0: B-acetonitrile, A:B=20:80, flow velocity is 1.2mL/min.
Said method adopts inner mark method ration, selects 2-phenyl ethylamine as internal standard compound matter.
In the step (1) of said method, get methcathinone Standard Stock solutions, by mass concentration be the solution of the internal standard compound matter of 0.1mg/mL be diluted to successively methcathinone mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL, and keep the concentration of inner mark solution to be 0.01mg/mL.
In the step (2) of said method: take sample, add mobile phase and dissolve, vibration, centrifugal, get supernatant after centrifugal, add internal standard compound matter, and add mobile phase, after shaken well, namely obtain sample solution; Mobile phase is identical with the mobile phase in step (3).
Said method adopts quantified by external standard method, wherein, in step (1), gets methcathinone Standard Stock solutions, be diluted to successively mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL.
In the step (2) of said method: take sample, add mobile phase and dissolve, vibration, centrifugal, get supernatant after centrifugal, add mobile phase, after shaken well, namely obtain sample solution; Mobile phase is identical with the mobile phase in step (3).
In the step (3) of said method, chromatographic column specification is 250mm × 4.6mm, and 5 μm, column temperature is 35 DEG C, determined wavelength 254nm.
In said method, mobile phase puts into the ultrasonic 10 ~ 15min of ultrasonic cleaner before using after 0.45 μm of micro porous filtration membrane filtration, fully deviates from the gas in mobile phase.
In said method, before add mobile phase in sample, use the methcathinone in the mixed solution extraction sample of methyl phenyl ethers anisole, methylene chloride and ether, the volume ratio of methyl phenyl ethers anisole, methylene chloride and ether is 5:2:1.5, in isolated organic phase, add the hydrochloric acid that HCl massfraction is 15%, after removing organic phase residue is placed in 35 DEG C of rapid concentration instrument are concentrated into dry.
The invention has the beneficial effects as follows:
1) in prior art, because methcathinone is difficult to be separated in chromatogram detects with jute alkali, in order to ensure obtaining good degree of separation, the mode reducing flow velocity can only be taked, improve degree of separation, improve peak shape, cause the detection time of methcathinone usually more than 20 minutes, and inventor is found by lot of experiments, it is improper that the principal element causing detection time in prior art long is that acid modification agent in mobile phase is selected, organic phase ratio can not be too high, by screening a large amount of modifier, inventor finds when using acidic ammonium acetate damping fluid, can by increasing organic phase ratio, greatly shorten detection time, the high separation of test substance methcathinone and related impurities jute alkali can also be guaranteed simultaneously, the technique effects such as high theoretical cam curve, proved by test, the detection time of methcathinone can be foreshortened to about 12 minutes by the present invention, the half of prior art used time nearly, greatly improve detection efficiency, for effectively carrying out of criminal investigation work in reality provides powerful guarantee.
2) present invention employs inner mark method ration, compared to quantified by external standard method of the prior art, because internal standard method avoids because the accidental error that effect causes discriminated against by the consistance of sample introduction and sample, thus, its analytical precision is higher, a kind of more satisfactory quantitative analysis method, Plays working curve R of the present invention
2reach 1; Wherein, the determination of internal standard compound matter kind is one of difficult point marked in setting up in analytical approach, internal standard compound kind will directly affect degree of accuracy, the accuracy of testing result, interior mark selection is improper will directly cause detection to realize, inventor passes through lot of experiments, determine that 2-phenyl ethylamine is as internal standard compound matter, itself and methcathinone degree of separation well, achieve the accurate quantification of methcathinone.
Accompanying drawing explanation
Fig. 1 is the liquid chromatogram of methcathinone and synthesis precursor ephedrine thereof, 1-methcathinone, 2-jute alkali;
Fig. 2 is methcathinone and interior target liquid chromatogram, 1-methcathinone, mark in 2-;
Fig. 3 is the Internal standard working curve method of methcathinone;
The ultra-violet absorption spectrum of Fig. 4 methcathinone;
Fig. 5 water-methanol (90:10) chromatograms;
First acid aqueous solution – acetonitrile (95:5) chromatograms of Fig. 6 pH=3.5;
First acid aqueous solution – acetonitrile (95:5) chromatograms of Fig. 7 pH=4;
Figure 80 .05% trifluoroacetic acid is as the chromatograms of mobile phase;
The chromatograms of Figure 91 50mm phenyl post;
Mixed the marking on a map of Figure 10 Cathinone, ephedrine, methcathinone and 4-methyl methcathinone;
Figure 11 trifluoroacetic acid is as the liquid chromatogram of mobile phase;
The each buffer salinity of Figure 12 A-Figure 12 C and post imitate curve.
Embodiment
Embodiment 1: inner mark method ration detects
The present invention adopts the method for methcathinone content in high effective liquid chromatography for measuring sample, comprises the steps:
(1) preparation of working fluid: get methcathinone Standard Stock solutions, by mass concentration be the solution of the internal standard compound matter 2-phenyl ethylamine of 0.1mg/mL be diluted to successively methcathinone mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL, and keep the concentration of inner mark solution to be 0.01mg/mL.
(2) preparation of sample solution: take sample sample and be about 45mg (the sample sample of the present embodiment uses methcathinone standard items to mix with other material to prepare afterwards, methcathinone content is 51.32wt%), add 20mL mobile phase with bottleneck pipettor to extract, vibrate 10 minutes, centrifugal 5 minutes, centrifugal rear 1mL pipettor Aspirate supernatant 1mL, add about 0.1mg internal standard compound matter 2-phenyl ethylamine, and add 10mL mobile phase with bottleneck pipettor, get 1.5mL after shaken well and fill auto injection bottle, namely obtain sample solution;
(3) setting of testing conditions: adopt Agilent
sB-Phenyl chromatographic column, specification is 250mm × 4.6mm, 5 μm, the 4mM ammonium acetate buffer of chromatographic condition: A-pH=5.0: in B-acetonitrile=20:80[the present invention, ammonium acetate buffer calculates with the concentration of acetate, and 4mM is 4mmol/L, the ratio of ammonium acetate buffer solution and acetonitrile is volume ratio], flow velocity 1.2mL/min, column temperature 35 DEG C, determined wavelength 254nm;
(4) mensuration of methcathinone content in sample, extracting sample solution 5 μ L sample detection;
(5) experimental result calculates, and utilize inner mark method ration to calculate, namely utilize standard working curve, in the methcathinone sample to be measured finally obtained, the mass content of methcathinone is: 51.22wt%.
Wherein, mobile phase puts into the ultrasonic 10 ~ 15min of ultrasonic cleaner before using after 0.45 μm of micro porous filtration membrane filtration, fully deviates from the gas in mobile phase.
Result shows, methcathinone tailing factor 1.586, and theoretical cam curve is 13726, and methcathinone peak shape is better, and ephedrine can separate completely, and liquid chromatogram as shown in Figure 1.
Embodiment 2: quantified by external standard method detects
(1) preparation of working fluid; Get methcathinone Standard Stock solutions, be diluted to successively mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL.
(2) preparation of sample solution: take sample sample and be about 45mg (the sample sample of the present embodiment uses methcathinone standard items to mix with other material to prepare afterwards, methcathinone content is 51.32wt%), add 20mL mobile phase with bottleneck pipettor to extract, vibrate 10 minutes, centrifugal 5 minutes, centrifugal rear 1mL pipettor Aspirate supernatant 1mL, added 10mL mobile phase with bottleneck pipettor, get 1.5mL after shaken well and fill auto injection bottle, namely obtain sample solution;
(3) setting of testing conditions: adopt Agilent
sB-Phenyl chromatographic column, specification is 250mm × 4.6mm, 5 μm, the 4mM ammonium acetate buffer of chromatographic condition: A-pH=5.0: B-acetonitrile=20:80, flow velocity 1.2mL/min, column temperature 35 DEG C, determined wavelength 254nm;
(4) mensuration of methcathinone content in sample, extracting sample solution 5 μ L sample detection;
(5) experimental result calculates, and utilize quantified by external standard method to calculate, namely utilize standard working curve, in the methcathinone sample to be measured finally obtained, the mass content of methcathinone is: 51.01wt%.
Wherein, mobile phase puts into the ultrasonic 10 ~ 15min of ultrasonic cleaner before using after 0.45 μm of micro porous filtration membrane filtration, fully deviates from the gas in mobile phase.
Embodiment 3
The present embodiment and the difference of embodiment 1 be sample and sample pretreatment process different: the blood 45mg of specimen in use to be methcathinone content be 51.32wt%, namely get blank blood, add methcathinone standard items, methyl phenyl ethers anisole is added in sample, the mixed solution 17mL of methylene chloride and ether, methcathinone in extraction sample, methyl phenyl ethers anisole, the volume ratio of methylene chloride and ether is 5:2:1.5, the hydrochloric acid 10mL that HCl massfraction is 15% is added in isolated organic phase, after removing organic phase residue is placed in 35 DEG C of rapid concentration instrument are concentrated into dry, add 20mL mobile phase with bottleneck pipettor to extract, vibrate 10 minutes, centrifugal 5 minutes, centrifugal rear 1mL pipettor Aspirate supernatant 1mL, add about 0.1mg internal standard compound matter 2-phenyl ethylamine, and add 10mL mobile phase with bottleneck pipettor, get 1.5mL after shaken well and fill auto injection bottle, namely sample solution is obtained.The mass content that the present embodiment finally obtains methcathinone in sample is: 50.96wt%.
Embodiment 4
The present embodiment and the difference of embodiment 2 be sample and sample pretreatment process different: the blood of specimen in use to be methcathinone content be 51.32wt%, namely get blank blood, add methcathinone standard items.Methyl phenyl ethers anisole is added in sample, the mixed solution 17mL of methylene chloride and ether, methcathinone in extraction sample, methyl phenyl ethers anisole, the volume ratio of methylene chloride and ether is 5:2:1.5, the hydrochloric acid 10mL that HCl massfraction is 15% is added in isolated organic phase, after removing organic phase residue is placed in 35 DEG C of rapid concentration instrument are concentrated into dry, add 20mL mobile phase with bottleneck pipettor to extract, vibrate 10 minutes, centrifugal 5 minutes, centrifugal rear 1mL pipettor Aspirate supernatant 1mL, 10mL mobile phase is added with bottleneck pipettor, get 1.5mL after shaken well and fill auto injection bottle, namely sample solution is obtained.The mass content that the present embodiment finally obtains methcathinone in sample is: 50.44wt%.
Checking example 1
(1) select 2-phenyl ethylamine as the process of internal standard compound matter.
Compound concentration is respectively the methcathinone of 0.1mg/mL and the inner mark solution of 0.1mg/mL, adopts the chromatographic condition in embodiment, and as shown in Figure 2, visible both have good degree of separation to the liquid chromatogram of acquisition.
(2) foundation of Internal standard working curve method
Experimental technique: get methcathinone Standard Stock solutions, with the inner mark solution that mass concentration is 0.1mg/mL be diluted to successively mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL, and keep the concentration of inner mark solution to be 0.01mg/mL.Get the titer 5 μ L sample introduction of variable concentrations respectively, the peak area of recording responses.Each concentration sample introduction 3 times, and linear regression is carried out to the mean value A of 3 peak area ratios and mass concentration ratio c (mg/mL).
Experimental result shows, the range of linearity of methcathinone is 0.005-0.5mg/mL, the Internal standard working curve method of linear equation to be A=0.6664c-0.0172, R2=1, Fig. 3 be methcathinone.
Checking example 2
The foundation of external standard working curve
Get methcathinone Standard Stock solutions, be diluted to successively mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL.Get the titer 5 μ L sample introduction of variable concentrations respectively, the peak area of recording responses.Each concentration sample introduction 3 times, and linear regression is carried out to the mean value A of 3 peak areas and mass concentration c (mg/mL).
Experimental result shows, the range of linearity of methcathinone is 0.005-0.5mg/mL, and linear equation is A=6 × 10
6c+11143, R
2=0.9999, detect and be limited to 0.2 μ g/mL (S/N>=3).
Comparative example
In investigation prior art, acid modification agent is on the impact of experimental result
(1) phosphoric acid is investigated on the impact of experimental result
Mobile phase: A-0.5% phosphate aqueous solution; B-acetonitrile.
Conclusion: do mobile phase with phosphoric acid, along with organic phase ratio reduces, hangover is serious, and ephedrine is got bad.
(2) acetic acid is investigated on the impact of experimental result
Mobile phase: A-0.5% acetic acid aqueous solution; B-acetonitrile.
Conclusion: the peak shape of methcathinone is better, but can not separate with ephedrine.
(3) trifluoroacetic acid is investigated on the impact of experimental result
Mobile phase: the aqueous solution of A-trifluoroacetic acid (pH=3.5); B-acetonitrile.
Conclusion: do mobile phase by the aqueous solution of trifluoroacetic acid (pH=3.5), obtain good peak shape during low flow velocity, but theoretical cam curve is lower; Become bimodal during high flow rate.
Visible, existing acid modification agent cannot meet high flow rate, high theoretical cam curve, testing requirement that peak shape is good.
The optimum choice experiment of testing conditions
The ultra-violet absorption spectrum of 1.1 methcathinones
As can be seen from Fig. 4, the maximum absorption wavelength of methcathinone is 254nm, therefore selects 254nm as main quantitative wavelength.Because it only has an absorption peak, therefore there is not auxiliary quantitative wavelength.
The selection of 1.2 liquid-phase conditions
Instrument: the supper-fast liquid chromatography of Shimadzu (UFLC) instrument, comprises LC-20AD type pump, SIL-20AC type injector, SPD-M20A type detecting device, CTD-20AC type column oven, data processing software LCsolution.
1.2.1 alcohol, water system is investigated on the impact of experimental result
Chromatographic condition: XR-ODS chromatographic column (75mm × 2.0mm, 2.2 μm); Column temperature: 35 DEG C; Determined wavelength 254nm; Mobile phase: water-methanol (90:10), flow velocity 0.2mL/min; Sample size: 10 μ L.
Conclusion: as can be seen from Fig. 5, alcohol, water system are not suitable for the analysis of methcathinone, and peak shape is huge steamed bun peak.
1.2.2 the impact of different types of acid on experimental result is investigated
Chromatographic condition: XR-ODS chromatographic column (75mm × 2.0mm, 2.2 μm); Column temperature: 35 DEG C; Determined wavelength 254nm.
The kind investigating acid comprises formic acid and trifluoroacetic acid.
1.2.2.1 different formic acid ratio is investigated on the impact of experimental result
Mobile phase: A-0.1% aqueous formic acid; B-acetonitrile
The different mobile phase ratio of table 1. is on the impact of experimental result
Conclusion: convert several mobile phase ratio, experimental result is all undesirable, and peak shape is bad, and retention time is too short.
1.2.2.2 different pH value formic acid is investigated on the impact of experimental result
(1) formic acid solution of pH=3 is on the impact of experimental result
The aqueous formic acid of mobile phase: A-pH=3; B-acetonitrile.
The different mobile phase ratio of table 2. is on the impact of experimental result
Conclusion: peak shape is bad, retention time is too short.
(2) formic acid solution of pH=3.5 is on the impact of experimental result
The aqueous formic acid of mobile phase: A-pH=3.5, B-acetonitrile, A:B=95:5.
Conclusion: as shown in Figure 6, chromatographic peak trails, and appearance time is too fast, before solvent peak, go out peak.
(3) formic acid solution of pH=4 is on the impact of experimental result
The aqueous formic acid of mobile phase: A-pH=4, B-acetonitrile, A:B=95:5.
Conclusion: as shown in Figure 7, chromatographic peak trails, and appearance time is too fast, before solvent peak, go out peak.
(4) formic acid solution of pH=4.5 is on the impact of experimental result
The aqueous formic acid of mobile phase: A-pH=4.5, B-acetonitrile.
The different mobile phase ratio of table 3. is on the impact of experimental result
Conclusion: the acidity of mobile phase is more weak, the retention time of methcathinone is shorter, and peak shape all has hangover.
1.2.2.2 trifluoroacetic acid is investigated on the impact of experimental result
Mobile phase: A-0.05% trifluoroacetic acid aqueous solution; B-acetonitrile; A:B=90:10.
Conclusion: as shown in Figure 8, use trifluoroacetic acid, peak shape is greatly improved, but chromatographic peak hangover, need further optimal conditions.
1.2.3 investigate buffer salt to the impact of experimental result
Chromatographic condition: XR-ODS chromatographic column (75mm × 2.0mm, 2.2 μm); Column temperature: 35 DEG C; Determined wavelength 254nm; Mobile phase: 1mM ammonium acetate (pH=4.5)-acetonitrile, flow velocity 0.2mL/min; Sample size: 10 μ L.
The different mobile phase ratio of table 4. is on the impact of experimental result
Conclusion: peak shape is all undesirable, along with the increase of organic phase ratio, appearance time is too fast, does with ammonium acetate the experimental result that mobile phase is comparatively satisfied with.
1.2.4 chromatographic column is investigated on the impact of experimental result
By above-mentioned experiment, how undesirable the chromatographic peak of the methcathinone obtained is, peak shape and retention time all existing defects.In addition, under these experimental conditions, methcathinone all can not be separated completely with its synthesis precursor ephedrine, therefore considers whether should change pillar from the angle of chromatographic column.
Chromatographic condition: column temperature: 35 DEG C; Determined wavelength 254nm; Sample size: 10 μ L.
1.2.4.1 different size phenyl post is on the impact of experimental result
(1) chromatographic column: Agilent
sB-Phenyl chromatographic column (100mm × 2.1mm, 1.8 μm)
The trifluoroacetic acid aqueous solution of mobile phase: A-pH=3.5; B-acetonitrile.
The different mobile phase condition of table 5. is on the impact of experimental result
Conclusion: the pillar using 100mm, methcathinone peak shape is trailed, and can not be separated completely with ephedrine.
(2) chromatographic column: Agilent
sB-Phenyl chromatographic column (150mm × 4.6mm, 5 μm).
The trifluoroacetic acid aqueous solution of mobile phase: A-pH=3.5; B-acetonitrile.Flow velocity: 0.2mL/min.
Conclusion: as shown in Figure 9, with the pillar of 150mm, methcathinone and ephedrine inseparable.
(3) chromatographic column: Agilent
sB-Phenyl chromatographic column (250mm × 4.6mm, 5 μm)
The trifluoroacetic acid aqueous solution of mobile phase: A-pH=3.5; B-acetonitrile.Flow velocity: 0.2mL/min.
Conclusion: as shown in Figure 10, use the pillar of 250mm, 4 standard specimens obtain good peak shape, and Cathinone, ephedrine are separated preferably with methcathinone.Therefore this chromatographic column is finally selected to carry out follow-up test.
1.2.4.2 the impact of variety classes acid on experimental result is investigated
(1) phosphoric acid is investigated on the impact of experimental result
Mobile phase: A-0.5% phosphate aqueous solution; B-acetonitrile.
The different mobile phase condition of table 6. is on the impact of experimental result
Conclusion: do mobile phase with phosphoric acid, along with organic phase ratio reduces, hangover is serious, and ephedrine is got bad.
(2) acetic acid is investigated on the impact of experimental result
Mobile phase: A-0.5% acetic acid aqueous solution; B-acetonitrile.
Conclusion: the peak shape of methcathinone is better, but can not separate with ephedrine.
(3) trifluoroacetic acid is investigated on the impact of experimental result
Mobile phase: the aqueous solution of A-trifluoroacetic acid (pH=3.5); B-acetonitrile.
Conclusion: as shown in figure 11, does mobile phase by the aqueous solution of trifluoroacetic acid (pH=3.5), obtain good peak shape, but theoretical cam curve is lower during low flow velocity; Become bimodal during high flow rate.
1.2.4.3 buffer salt is investigated on the impact of experimental result
Owing to using the phenyl post of 250mm instead, again investigate ammonium acetate buffer solution to the impact of experimental result.
(1) impact of buffer salt on experimental result of different pH value is investigated
A. mobile phase A is the 1mM ammonium acetate buffer of pH3.5, and B is acetonitrile.
Investigate the impact of different mobile phase ratio on experimental result, the index of investigation comprises retention time, tailing factor and theoretical cam curve.
The impact of different mobile phase ratio on experimental result investigated by table 7.
Conclusion: organic phase ratio from 10% to 40% time, retention time reduces successively, and afterwards along with the increase of organic phase ratio, retention time increases, and reason may be the change of solution ph because mobile phase ratio difference causes; Tailing factor is minimum when organic phase ratio is 30%, increases gradually afterwards, but when organic phase ratio reaches 90%, drops to 0.637 suddenly; Theoretical cam curve increases with the increase of organic phase concentration, when reaching 85% to organic phase ratio, reaches maximum, diminishes again afterwards.
B. mobile phase A is the 1mM ammonium acetate buffer of pH4.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 8.
Conclusion: along with the increase of pH value, the retention time of each mobile phase ratio all increases.With the increase of organic phase ratio, retention time first reduces rear increase; But tailing factor reduces with the increase of organic phase ratio, minimum when organic phase ratio is 85%; Theoretical cam curve is also increase with the increase of mobile phase ratio, maximum when organic phase ratio is 85%, reduces suddenly again afterwards.But under this pH value condition, methcathinone and synthesis precursor ephedrine thereof can not be separated completely.
C. mobile phase A is the 1mM ammonium acetate buffer of pH4.5, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 9.
Conclusion: along with the increase of pH value, the retention time of each mobile phase ratio all increases.With the increase of organic phase ratio, retention time first reduces rear increase; But tailing factor reduces with the increase of organic phase ratio, minimum when organic phase ratio is 85%; Theoretical cam curve is also increase with the increase of mobile phase ratio, maximum when organic phase ratio is 85%, reduces suddenly again afterwards.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 10.
Conclusion: under this pH value condition, along with flow velocity increases, retention time reduces, and the tailing factor of methcathinone and ephedrine all reduces, but theoretical cam curve also decreases.Degree of separation only reaches when flow velocity is 1.4 and is 1.532 to the maximum, and methcathinone and synthesis precursor ephedrine thereof can not be separated completely.
D. mobile phase A is the 1mM ammonium acetate buffer of pH5.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 11.
Conclusion: along with the increase of pH value, the retention time of each mobile phase ratio all increases.With the increase of organic phase ratio, retention time first reduces rear increase; But tailing factor reduces with the increase of organic phase ratio, minimumly when organic phase ratio is 80% to increase subsequently; Theoretical cam curve is also increase with the increase of mobile phase ratio, maximum when organic phase ratio is 80%, reduces suddenly again afterwards.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 12.
Conclusion: under this pH value condition, along with flow velocity increases, retention time reduces, and the tailing factor change of methcathinone and ephedrine is not obvious, but theoretical cam curve decreases.Degree of separation is better, under each flow velocity, all reach more than 4.5, and methcathinone and synthesis precursor ephedrine thereof can be separated completely.Thus pH value is 5.0 is good pH value.
(2) impact of different buffer salinity on experimental result is investigated
A. mobile phase A is the 2mM ammonium acetate buffer of pH5.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 13.
Conclusion: the ammonium acetate buffer being 1mM with the concentration of same pH compares, and when organic phase ratio is 10% and 20%, retention time slightly increases, but organic phase ratio reaches after 30%, and retention time obviously reduces; Little than with organic phase ratio of tailing factor; Theoretical cam curve is all be significantly increased before 70% in organic phase ratio, slightly reduces after 80%.Comprehensive each factor analysis, when buffer concentration is 2mM, when peak parameter is better than 1mM.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 14.
Conclusion: under this buffer salinity condition, along with flow velocity increases, retention time reduces, and reduces when the tailing factor of methcathinone and ephedrine is 1mM than buffer salinity, and closer to 1, peak shape is better, but theoretical cam curve decreases.Methcathinone and synthesis precursor ephedrine thereof can be separated completely.
B. mobile phase A is the 3mM ammonium acetate buffer of pH5.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 15.
Conclusion: the ammonium acetate buffer being 1mM, 2mM with the concentration of same pH compares, and when organic phase ratio is 10%, retention time slightly increases, but organic phase ratio reaches after 20%, and retention time obviously reduces; Little than with organic phase ratio of tailing factor; Theoretical cam curve is all be significantly increased before 70% in organic phase ratio, slightly reduces after 80%.Comprehensive each factor analysis, when buffer concentration is 3mM, when peak parameter is better than 1mM and 2mM.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 16.
Conclusion: under this buffer salinity condition, along with flow velocity increases, retention time reduces, and increase when the tailing factor of methcathinone and ephedrine is 1mM, 2mM than buffer salinity, theoretical cam curve also decreases.Though methcathinone and synthesis precursor ephedrine thereof can be separated completely, peak parameter and buffer salinity are that 2mM does not have advantage.
C. mobile phase A is the 4mM ammonium acetate buffer of pH5.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 17.
Conclusion: the ammonium acetate buffer being 1mM-3mM with the concentration of same pH compares, and retention time all slightly reduces; Tailing factor compares no significant difference with same organic phase ratio; Theoretical cam curve obviously increases, and when organic phase ratio reaches 80%, the number of plates is the highest.Comprehensive each factor analysis, when buffer concentration is 4mM, when peak parameter is better than 1mM to 3mM.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 18.
Conclusion: under this buffer salinity condition, along with flow velocity increases, retention time reduces; The tailing factor of methcathinone and ephedrine remains on about 1.5, not obvious with change in flow; Though theoretical cam curve increases with flow velocity and reduces, all keep high value.Methcathinone and synthesis precursor ephedrine thereof can be separated completely, and this buffer salinity is a better concentration.
D. mobile phase A is the 5mM ammonium acetate buffer of pH5.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 19.
Conclusion: the ammonium acetate buffer being 4mM with the concentration of same pH compares, retention time is slightly increased before 40% in organic phase ratio, reduces afterwards; Tailing factor and same organic phase ratio more slightly reduce but difference is little; Theoretical cam curve increases, and when organic phase ratio reaches 80%, the number of plates is the highest.Comprehensive each factor analysis, when buffer concentration is 5mM, peak parameter compares no significant difference with 4mM.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 20.
Conclusion: under this buffer salinity condition, along with flow velocity increases, retention time reduces; The tailing factor of methcathinone and ephedrine remains on about 1.3, not obvious with change in flow; Though theoretical cam curve increases with flow velocity and reduces, all keep high value.Methcathinone and synthesis precursor ephedrine thereof can be separated completely, and this buffer salinity is a better concentration.
E. mobile phase A is the 10mM ammonium acetate buffer of pH5.0, and B is acetonitrile.
The impact of different mobile phase ratio on experimental result investigated by table 21.
Conclusion: the ammonium acetate buffer being 5mM with the concentration of same pH compares, and retention time is in obvious reduction; Tailing factor and same organic phase ratio more slightly reduce but difference is little; Theoretical cam curve increases, and when organic phase ratio reaches 80%, the number of plates is the highest.Comprehensive each factor analysis, when buffer concentration is 10mM, peak parameter compares no significant difference with 4mM, 5mM.Consider retention time, tailing factor and theoretical cam curve 3 factor, select organic phase ratio to be the condition of 80%, investigate flow velocity to the impact of experimental result.
The impact of different in flow rate on experimental result investigated by table 22.
Conclusion: under this buffer salinity condition, along with flow velocity increases, retention time reduces; The tailing factor of methcathinone and ephedrine remains on about 1.5, compares tailing factor become large with 5mM; Though theoretical cam curve increases with flow velocity and reduces, all keep high value.Methcathinone and synthesis precursor ephedrine thereof can be separated completely, but degree of separation all obviously reduces.We draw curve that each buffer salinity and post imitate (data in table 23, Figure 12 A-C), and increase with buffer salinity, retention time obviously reduces; Though tailing factor changes, non-linear, rule is not obvious; Theoretical cam curve also becomes curvilinear motion, but buffer salinity increases, and the degree of separation of methcathinone and ephedrine reduces.Comprehensive each influence factor, the final 4mM that selects is best buffer salinity.
The each buffer salinity of table 23. and post imitate parameter
(3) impact of different mobile phase ratio on experimental result is investigated
Owing to have selected the 4mM ammonium acetate buffer that mobile phase A is pH5.0, B is acetonitrile, as can be seen from the data of table 17, mobile phase A: during B=80:20, theoretical cam curve reaches maximum, and tailing factor is minimum simultaneously, and the optimal flow Phase Proportion thus selected is 80:20.
(4) different in flow rate is investigated on the impact of experimental result
In mobile phase A: under B=80:20 condition, as can be seen from the data of table 18, along with flow velocity increases, retention time reduces; The tailing factor of methcathinone and ephedrine remains on about 1.5, not obvious with change in flow; Though theoretical cam curve increases with flow velocity and reduces, all keep high value.Consider the impact of analysis time on experiment, the final optimum flow rate selected is 1.2mL/min.
Claims (10)
1. adopt a method for methcathinone content in high effective liquid chromatography for measuring sample, it comprises the steps:
(1) preparation of working fluid;
(2) preparation of sample solution;
(3) setting of testing conditions;
(4) mensuration of methcathinone content in sample;
(5) experimental result calculates,
Wherein, in step (3), chromatographic column adopts anti-phase phenyl chromatographic column, and mobile phase is A-1 ~ 5mM ammonium acetate buffer: B-acetonitrile and/or methyl alcohol, A:B=(20 ~ 40): (60-80), and flow velocity is 1.0-1.5mL/min.
2. method according to claim 1, is characterized in that: in step (3), the 4mM ammonium acetate buffer of A-pH=5.0: B-acetonitrile, A:B=20:80, and flow velocity is 1.2mL/min.
3. method according to claim 1, is characterized in that: adopt inner mark method ration, selects 2-phenyl ethylamine as internal standard compound matter.
4. method according to claim 3, it is characterized in that: in step (1), get methcathinone Standard Stock solutions, by mass concentration be the solution of the internal standard compound matter of 0.1mg/mL be diluted to successively methcathinone mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL, and keep the concentration of inner mark solution to be 0.01mg/mL.
5. method according to claim 3, is characterized in that: in step (2): take sample, adds mobile phase and dissolves, vibration, centrifugal, get supernatant after centrifugal, add internal standard compound matter, and add mobile phase, after shaken well, namely obtain sample solution; Mobile phase is identical with the mobile phase in step (3).
6. method according to claim 1, it is characterized in that: adopt quantified by external standard method, wherein, in step (1), get methcathinone Standard Stock solutions, be diluted to successively mass concentration be respectively 0.5,0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL.
7. method according to claim 6, is characterized in that: in step (2): take sample, adds mobile phase and dissolves, vibration, centrifugal, gets supernatant, adds mobile phase, namely obtain sample solution after shaken well after centrifugal; Mobile phase is identical with the mobile phase in step (3).
8. the method according to claim 1 to 7 any one, is characterized in that: in step (3), and chromatographic column specification is 250mm × 4.6mm, 5 μm, and column temperature is 35 DEG C, determined wavelength 254nm.
9. the method according to claim 1 to 7 any one, is characterized in that, mobile phase puts into the ultrasonic 10 ~ 15min of ultrasonic cleaner before using after 0.45 μm of micro porous filtration membrane filtration, fully deviates from the gas in mobile phase.
10. the method according to claim 5 or 7 any one, it is characterized in that, before add mobile phase in sample, use the methcathinone in the mixed solution extraction sample of methyl phenyl ethers anisole, methylene chloride and ether, the volume ratio of methyl phenyl ethers anisole, methylene chloride and ether is 5:2:1.5, in isolated organic phase, add the hydrochloric acid that HCl massfraction is 15%, after removing organic phase residue is placed in 35 DEG C of rapid concentration instrument are concentrated into dry.
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