CN105181823B - A kind of method of methcathinone content in use high effective liquid chromatography for measuring sample - Google Patents
A kind of method of methcathinone content in use high effective liquid chromatography for measuring sample Download PDFInfo
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Abstract
The method that the present invention discloses methcathinone content in a kind of use high effective liquid chromatography for measuring sample, comprises the following steps：(1) preparation of working solution；(2) preparation of sample solution；(3) setting of testing conditions；(4) measure of methcathinone sample size.The present invention uses reverse-phase chromatography isocratic elution, and analysis was completed in 12 minutes；It has efficient, accurate Detection results, can be used for the measure of methcathinone sample size, for judicial department's conviction and sentence provides technical support.
Description
Technical field
The present invention relates to criminal investigation field illicit drugs inspection field, more particularly to using first in high effective liquid chromatography for measuring sample
The method of Cathinone content.
Background technology
Methcathinone is that what is occurred in recent years have the novel drug of suitable harm to human body.This harm not only influences human body
Physiological situation, can also influence the psychological activity of people, excessively also result in death.The abuse of methcathinone not only has danger to human body
Evil, and adverse effect will also result in social development.In state food Drug Administration general bureau, the Ministry of Public Security, national health meter
2007 editions of raw committee's issue《Narcotics and psychotropic substances kind catalogue》In, methcathinone is classified as a class psychotropic substances and is given
Control.
Methcathinone systematic chemical name is：N- methcathinones, shown in its chemical structural formula such as formula (1)：
Prior art has using gas chromatography/mass spectrometry method, infrared spectroscopy, high performance liquid chromatography and liquid
Matter GC-MS etc. determines methcathinone sample size.Wherein, during being detected using liquid chromatography, however it remains inspection
The survey time is long, the low technical problem of detection efficiency, causes the reduction of criminal investigation operating efficiency.Therefore urgent need is a kind of high-precision, and
The method of methcathinone detection during sample can be realized in shorter time.
The content of the invention
In view of this, the invention reside in a kind of high effective liquid chromatography for measuring first that precision is high, detection time is short of offer
The method of Cathinone sample size.
The present invention is achieved through the following technical solutions：One kind is using first cassie in high effective liquid chromatography for measuring sample
The method of ketone content, it comprises the following steps：
(1) preparation of working solution；
(2) preparation of sample solution；
(3) setting of testing conditions；
(4) in sample methcathinone content measure；
(5) experimental result is calculated,
Wherein, in step (3), chromatographic column uses anti-phase phenyl chromatographic column, and mobile phase is A-1~5mM ammonium acetate buffers：
B- acetonitriles, A:B=(20~40):(60-80), flow velocity is 1.0-1.5mL/min.
In the step of above method (3), in step (3), the 4mM ammonium acetate buffers of A-pH=5.0：B- acetonitriles, A:B=
20:80, flow velocity is 1.2mL/min.
The above method uses inner mark method ration, and selection 2- phenyl ethylamines are used as internal standard substance.
In the step of above method (1), methcathinone Standard Stock solutions are taken, with the internal standard that mass concentration is 0.1mg/mL
The solution of material 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 of above method (2)：Sample is weighed, flowing phased soln is added, vibration, centrifugation takes supernatant after centrifugation,
Internal standard substance is added, and adds mobile phase, sample solution is obtained after shaken well；Mobile phase in mobile phase and step (3)
It is identical.
The above method uses quantified by external standard method, wherein, in step (1), methcathinone Standard Stock solutions are taken, dilute successively
Be respectively 0.5 into mass concentration, 0.1,0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL.
In the step of above method (2)：Sample is weighed, flowing phased soln is added, vibration, centrifugation takes supernatant after centrifugation,
Mobile phase is added, sample solution is obtained after shaken well；Mobile phase is identical with the mobile phase in step (3).
Chromatographic column specification is 250mm × 4.6mm in the step of above method (3), and 5 μm, column temperature is 35 DEG C, Detection wavelength
254nm。
In the above method, mobile phase is put into ultrasound in ultrasonic cleaner using preceding after 0.45 μm of micro porous filtration membrane filtration
Gas in 10~15min, fully abjection mobile phase.
In the above method, before adding mobile phase in sample, the mixing using methyl phenyl ethers anisole, dichloromethane and ether is molten
Methcathinone in liquid extraction sample, the volume ratio of methyl phenyl ethers anisole, dichloromethane and ether is 5:2:1.5, it is organic to what is isolated
The hydrochloric acid that HCl mass fractions are 15% is added in phase, residue is placed in concentration on 35 DEG C of rapid concentration instrument after removing organic phase
It is extremely dry.
The beneficial effects of the invention are as follows：
1) it is good in order to ensure obtaining in the prior art, because methcathinone is difficult to separate with jute alkali in chromatogram detection
Good separating degree, can only take the mode for reducing flow velocity, improve separating degree, improve peak shape, cause the detection time of methcathinone
Generally more than 20 minutes；And inventor is had found by lot of experiments, the principal element for causing detection time in the prior art long
It is the improper of acid modification agent selection in mobile phase, organic Phase Proportion can not be too high, by screening a large amount of modifying agent, inventor
It was found that when using acidic ammonium acetate buffer solution, detection time can be greatly shortened by increasing organic Phase Proportion, while can also
The technique effects such as high separation, the theoretical cam curve high of test substance methcathinone and related impurities jute alkali are ensured, by examination
Checking is bright, and the detection time of methcathinone can be foreshortened to 12 minutes or so by the present invention, nearly the half of prior art used time,
Detection efficiency is greatly improved, for effective development of criminal investigation work in practice 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 is avoided
Because the uniformity and sample of sample introduction discriminate against accidental error caused by effect, thus, its analytical precision is higher, is one
Plant more satisfactory quantitative analysis method, Plays working curve R of the present invention2Reach 1；Wherein, the determination of internal standard substance species
It is one of difficult point for setting up in internal standard analysis method, internal standard species will directly affect the accuracy of testing result, accuracy, interior
Mark selection is improper to be will result directly in detection and cannot realize, inventor determines 2- phenyl ethylamines as internal standard substance by lot of experiments,
It is good with methcathinone separating degree, realizes the accurate quantification of methcathinone.
Brief description of the drawings
Fig. 1 is the liquid chromatogram of methcathinone and its synthesis precursor ephedrine, 1- methcathinones, 2- jute alkali；
Fig. 2 is methcathinone and interior target liquid chromatogram, 1- methcathinones, 2- internal standards；
Fig. 3 is the Internal standard working curve method of methcathinone；
The ultra-violet absorption spectrum of Fig. 4 methcathinones；
Fig. 5 water-methanols (90:10) chromatograms；
Aqueous formic acid-the acetonitrile (95 of Fig. 6 pH=3.5:5) chromatograms；
Aqueous formic acid-the acetonitrile (95 of Fig. 7 pH=4:5) chromatograms；
Chromatograms of the trifluoroacetic acids of Fig. 8 0.05% as mobile phase；
The chromatograms of Fig. 9 150mm phenyl posts；
Mixed the marking on a map of Figure 10 Cathinones, ephedrine, methcathinone and 4- methyl methcathinones；
Liquid chromatogram of Figure 11 trifluoroacetic acids as mobile phase；
Figure 12 each buffer salinities of A- Figure 12 C imitate curve with post.
Specific embodiment
Embodiment 1：Inner mark method ration is detected
The present invention is comprised the following steps using the method for methcathinone content in high effective liquid chromatography for measuring sample：
(1) preparation of working solution：Methcathinone Standard Stock solutions are taken, with the internal standard substance that mass concentration is 0.1mg/mL
The solution of 2- phenyl ethylamines 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：(the sample sample of the present embodiment is to use methcathinone to weigh sample sample about 45mg
What standard items were prepared after mixing with other materials, methcathinone content is 51.32wt%), add 20mL to flow with bottleneck pipettor
It is dynamic mutually to extract, vibrate 10 minutes, it is centrifuged 5 minutes, 1mL pipettor Aspirate supernatant 1mL are used after centrifugation, add about 0.1mg internal standards
Material 2- phenyl ethylamines, and 10mL mobile phases are added with bottleneck pipettor, 1.5mL dress auto injection bottles are taken after shaken well, obtain final product
To sample solution；
(3) setting of testing conditions：Using AgilentSB-Phenyl chromatographic columns, specification be 250mm ×
4.6mm, 5 μm, chromatographic condition：The 4mM ammonium acetate buffers of A-pH=5.0：B- acetonitrile=20:80【In the present invention, ammonium acetate delays
Fliud flushing is calculated with the concentration of acetate, and 4mM is 4mmol/L, and ammonium acetate buffer solution is volume ratio with the ratio of acetonitrile】,
Flow velocity 1.2mL/min, 35 DEG C of column temperature, Detection wavelength 254nm；
(4) in sample methcathinone content measure, take the μ L sample detections of sample solution 5；
(5) experimental result is calculated, and is calculated using inner mark method ration, i.e., using standard working curve, what is finally given is to be measured
The mass content of methcathinone is in methcathinone sample：51.22wt%.
Wherein, mobile phase using it is preceding be put into after 0.45 μm of micro porous filtration membrane filtration ultrasound 10 in ultrasonic cleaner~
Gas in 15min, fully abjection mobile phase.
Result shows that methcathinone tailing factor 1.586, theoretical cam curve is 13726, and methcathinone peak shape is preferable, and
Ephedrine can be completely separable, and liquid chromatogram is as shown in Figure 1.
Embodiment 2：Quantified by external standard method is detected
(1) preparation of working solution；Take methcathinone Standard Stock solutions, be diluted to successively mass concentration be respectively 0.5,
0.1st, 0.05,0.01,0.005,0.001, the series standard solution of 0.0005mg/mL.
(2) preparation of sample solution：(the sample sample of the present embodiment is to use methcathinone to weigh sample sample about 45mg
What standard items were prepared after mixing with other materials, methcathinone content is 51.32wt%), add 20mL to flow with bottleneck pipettor
It is dynamic mutually to extract, vibrate 10 minutes, it is centrifuged 5 minutes, 1mL pipettor Aspirate supernatant 1mL are used after centrifugation, added with bottleneck pipettor
10mL mobile phases, take 1.5mL dress auto injection bottles after shaken well, that is, obtain sample solution；
(3) setting of testing conditions：Using AgilentSB-Phenyl chromatographic columns, specification be 250mm ×
4.6mm, 5 μm, chromatographic condition：The 4mM ammonium acetate buffers of A-pH=5.0：B- acetonitrile=20:80, flow velocity 1.2mL/min, post
35 DEG C of temperature, Detection wavelength 254nm；
(4) in sample methcathinone content measure, take the μ L sample detections of sample solution 5；
(5) experimental result is calculated, and is calculated using quantified by external standard method, i.e., using standard working curve, what is finally given is to be measured
The mass content of methcathinone is in methcathinone sample：51.01wt%.
Wherein, mobile phase using it is preceding be put into after 0.45 μm of micro porous filtration membrane filtration ultrasound 10 in ultrasonic cleaner~
Gas in 15min, fully abjection mobile phase.
Embodiment 3
The present embodiment is that sample and sample pretreatment process are different from the difference of embodiment 1：Specimen in use is first cassie
Ketone content is the blood 45mg of 51.32wt%, that is, take blank blood, adds methcathinone standard items；To addition benzene first in sample
The mixed solution 17mL of ether, dichloromethane and ether, the methcathinone in extraction sample, methyl phenyl ethers anisole, dichloromethane and ether
Volume ratio is 5:2:1.5, to the hydrochloric acid 10mL that HCl mass fractions are 15% is added in the organic phase isolated, remove organic phase
Residue is placed on 35 DEG C of rapid concentration instrument is afterwards concentrated to dryness, add 20mL mobile phases to extract with bottleneck pipettor, vibration 10
Minute, it is centrifuged 5 minutes, 1mL pipettor Aspirate supernatant 1mL are used after centrifugation, about 0.1mg internal standard substance 2- phenyl ethylamines are added, and
10mL mobile phases are added with bottleneck pipettor, 1.5mL dress auto injection bottles are taken after shaken well, that is, obtain sample solution.This reality
Apply example and finally give the mass content of methcathinone in sample and be：50.96wt%.
Embodiment 4
The present embodiment is that sample and sample pretreatment process are different from the difference of embodiment 2：Specimen in use is first cassie
Ketone content is the blood of 51.32wt%, that is, take blank blood, adds methcathinone standard items.To addition methyl phenyl ethers anisole, two in sample
The mixed solution 17mL of chloromethanes and ether, the methcathinone in extraction sample, the volume ratio of methyl phenyl ethers anisole, dichloromethane and ether
It is 5:2:1.5, to the hydrochloric acid 10mL that HCl mass fractions are 15% is added in the organic phase isolated, will be remained after removing organic phase
Excess is placed on 35 DEG C of rapid concentration instrument and is concentrated to dryness, and adds 20mL mobile phases to extract with bottleneck pipettor, vibrates 10 minutes, from
The heart 5 minutes, uses 1mL pipettor Aspirate supernatant 1mL after centrifugation, 10mL mobile phases are added with bottleneck pipettor, after shaken well
1.5mL dress auto injection bottles are taken, that is, obtains sample solution.The present embodiment finally gives the mass content of methcathinone in sample
For：50.44wt%.
Checking example 1
(1) 2- phenyl ethylamines are selected as the process of internal standard substance.
Compound concentration is respectively the methcathinone of 0.1mg/mL and the inner mark solution of 0.1mg/mL, using the color in embodiment
Spectral condition, the liquid chromatogram of acquisition is as shown in Figure 2, it is seen that both have preferable separating degree.
(2) foundation of Internal standard working curve method
Experimental technique：Methcathinone Standard Stock solutions are taken, it is dilute successively with the inner mark solution that mass concentration is 0.1mg/mL
Be interpreted into 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
The concentration of inner mark solution is kept to be 0.01mg/mL.The μ L sample introductions of titer 5 of various concentrations, the peak face of recording responses are taken respectively
Product.Each concentration sample introduction 3 times, and average value A and mass concentration ratio c (mg/mL) to 3 peak area ratios linearly returned
Return.
Test result indicate that, the range of linearity of methcathinone is 0.005-0.5mg/mL, and linear equation is A=0.6664c-
0.0172, R2=1, Fig. 3 are the Internal standard working curve method of methcathinone.
Checking example 2
The foundation of external standard working curve
Take methcathinone Standard Stock solutions, be diluted to successively mass concentration be respectively 0.5,0.1,0.05,0.01,
0.005th, 0.001, the series standard solution of 0.0005mg/mL.The μ L sample introductions of titer 5 of various concentrations, recording responses are taken respectively
Peak area.Each concentration sample introduction 3 times, and average value A and mass concentration c (mg/mL) to 3 peak areas linearly returned
Return.
Test result indicate that, the range of linearity of methcathinone is 0.005-0.5mg/mL, and linear equation is A=6 × 106c+
11143, R2=0.9999, detection is limited to 0.2 μ g/mL (S/N >=3).
Comparative example
Investigate influence of the acid modification agent to experimental result in the prior art
(1) influence of the phosphoric acid to experimental result is investigated
Mobile phase：A-0.5% phosphate aqueous solutions；B- acetonitriles.
Conclusion：Mobile phase is done with phosphoric acid, with the reduction of organic Phase Proportion, hangover is serious, and ephedrine gets bad.
(2) influence of the acetic acid to experimental result is investigated
Mobile phase：A-0.5% acetic acid aqueous solutions；B- acetonitriles.
Conclusion：The peak shape of methcathinone preferably, but can not be separated with ephedrine.
(3) influence of the trifluoroacetic acid to experimental result is investigated
Mobile phase：The aqueous solution of A- trifluoroacetic acids (pH=3.5)；B- acetonitriles.
Conclusion：Mobile phase is done with the aqueous solution of trifluoroacetic acid (pH=3.5), preferable peak shape, but reason are obtained during low flow velocity
It is relatively low by the number of plates；High flow rate time-varying is into bimodal.
It can be seen that, existing acid modification agent cannot meet the good detection requirement of high flow rate, theoretical cam curve high, peak shape.
The optimum choice experiment of testing conditions
The ultra-violet absorption spectrum of 1.1 methcathinones
As can be seen from Fig. 4, a length of 254nm of the maximum absorption wave of methcathinone, therefore selection 254nm is as main quantitative ripple
It is long.Due to its only one of which absworption peak, therefore in the absence of auxiliary quantitative wavelength.
The selection of 1.2 liquid-phase conditions
Instrument：Shimadzu supper-fast liquid chromatogram (UFLC) instrument, including LC-20AD types pump, SIL-20AC types injector, SPD-
M20A types detector, CTD-20AC types column oven, data processing software LC solution.
1.2.1 the influence of alcohol, water system to experimental result is investigated
Chromatographic condition：XR-ODS chromatographic columns (75mm × 2.0mm, 2.2 μm)；Column temperature：35℃；Detection wavelength 254nm；Flowing
Phase：Water-methanol (90:10), flow velocity 0.2mL/min；Sample size：10μL.
Conclusion：As can be seen from Fig. 5, alcohol, water system are not suitable for the analysis of methcathinone, and peak shape is in huge steamed bun peak.
1.2.2 influence of different types of acid to experimental result is investigated
Chromatographic condition：XR-ODS chromatographic columns (75mm × 2.0mm, 2.2 μm)；Column temperature：35℃；Detection wavelength 254nm.
The species for investigating acid includes formic acid and trifluoroacetic acid.
1.2.2.1 influence of the different formic acid ratios to experimental result is investigated
Mobile phase：A-0.1% aqueous formic acids；B- acetonitriles
Influence of the different mobile phase ratios of table 1. to experimental result
Conclusion：Several mobile phase ratios are converted, experimental result is undesirable, and peak shape is bad, and retention time is too short.
1.2.2.2 influence of the different pH value formic acid to experimental result is investigated
(1) influence of the formic acid solution of pH=3 to experimental result
Mobile phase：The aqueous formic acid of A-pH=3；B- acetonitriles.
Influence of the different mobile phase ratios of table 2. to experimental result
Conclusion：Peak shape is bad, and retention time is too short.
(2) influence of the formic acid solution of pH=3.5 to experimental result
Mobile phase：The aqueous formic acid of A-pH=3.5, B- acetonitriles, A:B=95:5.
Conclusion：As shown in fig. 6, chromatographic peak trails, appearance time is too fast, the appearance before solvent peak.
(3) influence of the formic acid solution of pH=4 to experimental result
Mobile phase：The aqueous formic acid of A-pH=4, B- acetonitriles, A:B=95:5.
Conclusion：As shown in fig. 7, chromatographic peak trails, appearance time is too fast, the appearance before solvent peak.
(4) influence of the formic acid solution of pH=4.5 to experimental result
Mobile phase：The aqueous formic acid of A-pH=4.5, B- acetonitriles.
Influence of the different mobile phase ratios of table 3. to experimental result
Conclusion：The acidity of mobile phase is weaker, and the retention time of methcathinone is shorter, and peak shape has hangover.
1.2.2.2 influence of the trifluoroacetic acid to experimental result is investigated
Mobile phase：A-0.05% trifluoroacetic acid aqueous solutions；B- acetonitriles；A:B=90:10.
Conclusion：As shown in figure 8, using trifluoroacetic acid, peak shape is greatly improved, but chromatographic peak trails, and needs further excellent
Change condition.
1.2.3 influence of the buffer salt to experimental result is investigated
Chromatographic condition：XR-ODS chromatographic columns (75mm × 2.0mm, 2.2 μm)；Column temperature：35℃；Detection wavelength 254nm；Flowing
Phase：1mM ammonium acetates (pH=4.5)-acetonitrile, flow velocity 0.2mL/min；Sample size：10μL.
Influence of the different mobile phase ratios of table 4. to experimental result
Conclusion：Peak shape is undesirable, and with the increase of organic Phase Proportion, appearance time is too fast, and mobile phase is done with ammonium acetate
Relatively satisfactory experimental result is not obtained.
1.2.4 influence of the chromatographic column to experimental result is investigated
By above-mentioned experiment, how undesirable the chromatographic peak of resulting methcathinone is, and peak shape and retention time are all in the presence of scarce
Fall into.In addition, under these experimental conditions, methcathinone and its synthesis precursor ephedrine can not be kept completely separate, therefore from
Whether pillar should be changed from the point of view of chromatographic column.
Chromatographic condition：Column temperature：35℃；Detection wavelength 254nm；Sample size：10μL.
1.2.4.1 influence of the different size phenyl post to experimental result
(1) chromatographic column：AgilentSB-Phenyl chromatographic columns (100mm × 2.1mm, 1.8 μm)
Mobile phase：The trifluoroacetic acid aqueous solution of A-pH=3.5；B- acetonitriles.
Influence of the different mobile phase conditions of table 5. to experimental result
Conclusion：Using the pillar of 100mm, methcathinone peak shape hangover, and ephedrine is not completely separated.
(2) chromatographic column：AgilentSB-Phenyl chromatographic columns (150mm × 4.6mm, 5 μm).
Mobile phase：The trifluoroacetic acid aqueous solution of A-pH=3.5；B- acetonitriles.Flow velocity：0.2mL/min.
Conclusion：As shown in figure 9, with the pillar of 150mm, methcathinone and ephedrine are inseparable.
(3) chromatographic column：AgilentSB-Phenyl chromatographic columns (250mm × 4.6mm, 5 μm)
Mobile phase：The trifluoroacetic acid aqueous solution of A-pH=3.5；B- acetonitriles.Flow velocity：0.2mL/min.
Conclusion：As shown in Figure 10, using the pillar of 250mm, 4 standard specimens obtain good peak shape, and Cathinone, ephedrine
Preferably separated with methcathinone.Therefore the final choice chromatographic column carries out follow-up test.
1.2.4.2 influence of the variety classes acid to experimental result is investigated
(1) influence of the phosphoric acid to experimental result is investigated
Mobile phase：A-0.5% phosphate aqueous solutions；B- acetonitriles.
Influence of the different mobile phase conditions of table 6. to experimental result
Conclusion：Mobile phase is done with phosphoric acid, with the reduction of organic Phase Proportion, hangover is serious, and ephedrine gets bad.
(2) influence of the acetic acid to experimental result is investigated
Mobile phase：A-0.5% acetic acid aqueous solutions；B- acetonitriles.
Conclusion：The peak shape of methcathinone preferably, but can not be separated with ephedrine.
(3) influence of the trifluoroacetic acid to experimental result is investigated
Mobile phase：The aqueous solution of A- trifluoroacetic acids (pH=3.5)；B- acetonitriles.
Conclusion：As shown in figure 11, mobile phase is done with the aqueous solution of trifluoroacetic acid (pH=3.5), is obtained during low flow velocity preferably
Peak shape, but theoretical cam curve is relatively low；High flow rate time-varying is into bimodal.
1.2.4.3 influence of the buffer salt to experimental result is investigated
Due to using the phenyl post of 250mm instead, influence of the ammonium acetate buffer solution to experimental result is investigated again.
(1) influence of the buffer salt of different pH value to experimental result is investigated
A. mobile phase A is the 1mM ammonium acetate buffers of pH3.5, and B is acetonitrile.
Investigate influence of the different mobile phase ratios to experimental result, the index of investigation include retention time, tailing factor and
Theoretical cam curve.
Table 7. investigates influence of the different mobile phase ratios to experimental result
Conclusion：When organic Phase Proportion is from 10% to 40%, retention time is reduced successively, afterwards with the increasing of organic Phase Proportion
Plus, retention time increases, and reason is probably due to the change of the different caused solution ph of mobile phase ratio；Tailing factor is having
It is minimum when machine Phase Proportion is 30%, gradually increase afterwards, but when organic Phase Proportion reaches 90%, suddenly drop to 0.637；It is theoretical
The number of plates increases with the increase of organic phase concentration, when reaching 85% to organic Phase Proportion, reaches maximum, diminishes again afterwards.
B. mobile phase A is the 1mM ammonium acetate buffers of pH4.0, and B is acetonitrile.
Table 8. investigates influence of the different mobile phase ratios to experimental result
Conclusion：With the increase of pH value, the retention time of each mobile phase ratio increases.With the increase of organic Phase Proportion,
Retention time first reduces and increases afterwards；But tailing factor reduces with the increase of organic Phase Proportion, when organic Phase Proportion is 85%
It is minimum；Theoretical cam curve is also to increase with the increase of mobile phase ratio, maximum when organic Phase Proportion is 85%, again prominent afterwards
So reduce.But under the pH value condition, methcathinone and its synthesis precursor ephedrine are not completely separated.
C. mobile phase A is the 1mM ammonium acetate buffers of pH4.5, and B is acetonitrile.
Table 9. investigates influence of the different mobile phase ratios to experimental result
Conclusion：With the increase of pH value, the retention time of each mobile phase ratio increases.With the increase of organic Phase Proportion,
Retention time first reduces and increases afterwards；But tailing factor reduces with the increase of organic Phase Proportion, when organic Phase Proportion is 85%
It is minimum；Theoretical cam curve is also to increase with the increase of mobile phase ratio, maximum when organic Phase Proportion is 85%, again prominent afterwards
So reduce.Consider retention time, tailing factor and the factor of theoretical cam curve 3, select the condition that organic Phase Proportion is 80%,
Investigate influence of the flow velocity to experimental result.
Table 10. investigates the influence to experimental result different in flow rate
Conclusion：Under the pH value condition, as flow velocity increases, retention time reduces, the hangover of methcathinone and ephedrine
The factor reduces, but theoretical cam curve is also decreased.Separating degree only reaches when flow velocity is 1.4 and is 1.532, first card to the maximum
Western ketone and its synthesis precursor ephedrine are not completely separated.
D. mobile phase A is the 1mM ammonium acetate buffers of pH5.0, and B is acetonitrile.
Table 11. investigates influence of the different mobile phase ratios to experimental result
Conclusion：With the increase of pH value, the retention time of each mobile phase ratio increases.With the increase of organic Phase Proportion,
Retention time first reduces and increases afterwards；But tailing factor reduces with the increase of organic Phase Proportion, when organic Phase Proportion is 80%
Minimum then increase；Theoretical cam curve is also to increase with the increase of mobile phase ratio, maximum when organic Phase Proportion is 80%,
Reduce suddenly again afterwards.Consider retention time, tailing factor and the factor of theoretical cam curve 3, the organic Phase Proportion of selection is
80% condition, investigates influence of the flow velocity to experimental result.
Table 12. investigates the influence to experimental result different in flow rate
Conclusion：Under the pH value condition, as flow velocity increases, retention time reduces, the hangover of methcathinone and ephedrine
Factor variations are not obvious, but theoretical cam curve is decreased.Separating degree preferably, reaches more than 4.5, first cassie under each flow velocity
Ketone and its synthesis precursor ephedrine can be kept completely separate.Thus pH value is 5.0 to be preferable pH value.
(2) influence of the different buffer salinities to experimental result is investigated
A. mobile phase A is the 2mM ammonium acetate buffers of pH5.0, and B is acetonitrile.
Table 13. investigates influence of the different mobile phase ratios to experimental result
Conclusion：Compare with the ammonium acetate buffer that the concentration of same pH is 1mM, organic Phase Proportion is 10% and 20%
When, retention time is increased slightly, but organic Phase Proportion is reached after 30%, and retention time is obviously reduced；Tailing factor ratio is same to be had
Machine Phase Proportion it is small；Theoretical cam curve was significantly increased before organic Phase Proportion is 70%, was slightly reduced after 80%.
Comprehensive each factor analysis, when buffer concentration is 2mM, when peak parameter is better than 1mM.Consider retention time, tailing factor and reason
By the factor of the number of plates 3, the condition that organic Phase Proportion is 80% is selected, investigate influence of the flow velocity to experimental result.
Table 14. investigates the influence to experimental result different in flow rate
Conclusion：Under the conditions of the buffer salinity, with flow velocity increase, retention time reduction, methcathinone and ephedrine
Tailing factor reduce when being 1mM than buffer salinity, closer to 1, preferably, but theoretical cam curve is decreased peak shape.First card
Western ketone and its synthesis precursor ephedrine can be kept completely separate.
B. mobile phase A is the 3mM ammonium acetate buffers of pH5.0, and B is acetonitrile.
Table 15. investigates influence of the different mobile phase ratios to experimental result
Conclusion：Compare with the ammonium acetate buffer that the concentration of same pH is 1mM, 2mM, when organic Phase Proportion is 10%,
Retention time is increased slightly, but organic Phase Proportion is reached after 20%, and retention time is obviously reduced；The same organic phase of tailing factor ratio
Ratio it is small；Theoretical cam curve was significantly increased before organic Phase Proportion is 70%, was slightly reduced after 80%.It is comprehensive
Each factor analysis, when buffer concentration is 3mM, when peak parameter is better than 1mM and 2mM.Consider retention time, tailing factor and reason
By the factor of the number of plates 3, the condition that organic Phase Proportion is 80% is selected, investigate influence of the flow velocity to experimental result.
Table 16. investigates the influence to experimental result different in flow rate
Conclusion：Under the conditions of the buffer salinity, with flow velocity increase, retention time reduction, methcathinone and ephedrine
Tailing factor increase when being 1mM, 2mM than buffer salinity, theoretical cam curve also decreases.Before methcathinone and its synthesis
Though body ephedrine can be kept completely separate, peak parameter is with buffer salinity for 2mM compares without advantage.
C. mobile phase A is the 4mM ammonium acetate buffers of pH5.0, and B is acetonitrile.
Table 17. investigates influence of the different mobile phase ratios to experimental result
Conclusion：Compare with the ammonium acetate buffer that the concentration of same pH is 1mM-3mM, retention time is slightly reduced；
Tailing factor and the comparing no significant difference with organic Phase Proportion；Theoretical cam curve substantially increases, and is reached in organic Phase Proportion
Number of plates highest when 80%.Comprehensive each factor analysis, when buffer concentration is 4mM, when peak parameter is better than 1mM to 3mM.Consider
Retention time, tailing factor and the factor of theoretical cam curve 3, select the condition that organic Phase Proportion is 80%, investigate flow velocity to experiment
The influence of result.
Table 18. investigates the influence to experimental result different in flow rate
Conclusion：Under the conditions of the buffer salinity, as flow velocity increases, retention time reduces；Methcathinone and ephedrine
Tailing factor be maintained at 1.5 or so, it is unobvious with change in flow；Though theoretical cam curve increases and reduces with flow velocity, protect
Hold high value.Methcathinone and its synthesis precursor ephedrine can be kept completely separate, and the buffer salinity is a preferable concentration.
D. mobile phase A is the 5mM ammonium acetate buffers of pH5.0, and B is acetonitrile.
Table 19. investigates influence of the different mobile phase ratios to experimental result
Conclusion：Compare with the ammonium acetate buffer that the concentration of same pH is 4mM, retention time is in organic Phase Proportion
It was increased slightly before 40%, is reduced afterwards；Tailing factor with organic Phase Proportion relatively slightly reduce but difference is little；It is theoretical
The number of plates increases, the number of plates highest when organic Phase Proportion reaches 80%.Comprehensive each factor analysis, when buffer concentration is 5mM
Peak parameter compares no significant difference with 4mM.Consider retention time, tailing factor and the factor of theoretical cam curve 3, select organic phase ratio
Example is 80% condition, investigates influence of the flow velocity to experimental result.
Table 20. investigates the influence to experimental result different in flow rate
Conclusion：Under the conditions of the buffer salinity, as flow velocity increases, retention time reduces；Methcathinone and ephedrine
Tailing factor be maintained at 1.3 or so, it is unobvious with change in flow；Though theoretical cam curve increases and reduces with flow velocity, protect
Hold high value.Methcathinone and its synthesis precursor ephedrine can be kept completely separate, and the buffer salinity is a preferable concentration.
E. mobile phase A is the 10mM ammonium acetate buffers of pH5.0, and B is acetonitrile.
Table 21. investigates influence of the different mobile phase ratios to experimental result
Conclusion：Compare with the ammonium acetate buffer that the concentration of same pH is 5mM, retention time is being obviously reduced；Hangover
The factor with organic Phase Proportion relatively slightly reduce but difference is little；Theoretical cam curve increases, and is reached in organic Phase Proportion
Number of plates highest when 80%.Comprehensive each factor analysis, peak parameter compares without substantially poor with 4mM, 5mM when buffer concentration is 10mM
It is different.Consider retention time, tailing factor and the factor of theoretical cam curve 3, select the condition that organic Phase Proportion is 80%, investigate flow velocity
Influence to experimental result.
Table 22. investigates the influence to experimental result different in flow rate
Conclusion：Under the conditions of the buffer salinity, as flow velocity increases, retention time reduces；Methcathinone and ephedrine
Tailing factor be maintained at 1.5 or so, tailing factor is compared with 5mM and becomes big；Though theoretical cam curve increases and reduces with flow velocity,
Keep high value.Methcathinone and its synthesis precursor ephedrine can be kept completely separate, but separating degree is significantly reduced.We paint
The curve (data are shown in Table 23, Figure 12 A-C) of each buffer salinity and post effect is made, is increased with buffer salinity, retention time is obvious
Reduce；Though tailing factor is changed, non-linear, rule is not obvious；Theoretical cam curve is also into curvilinear motion, but buffer salt is dense
Degree increases, and methcathinone is reduced with the separating degree of ephedrine.Comprehensive each influence factor, final choice 4mM is optimal buffer salt
Concentration.
Each buffer salinity of table 23. imitates parameter with post
(3) influence of the different mobile phase ratios to experimental result is investigated
Due to having selected for the 4mM ammonium acetate buffers that mobile phase A is pH5.0, B is acetonitrile, from the data of table 17
As can be seen that mobile phase A：B=80:When 20, theoretical cam curve reaches maximum, while tailing factor is minimum, thus selection is most
Good mobile phase ratio is 80:20.
(4) influence to experimental result different in flow rate is investigated
In mobile phase A：B=80:Under the conditions of 20, as can be seen that with flow velocity increase, retention time from the data of table 18
Reduce；The tailing factor of methcathinone and ephedrine is maintained at 1.5 or so, unobvious with change in flow；Though theoretical cam curve with
Flow velocity increases and reduces, but keeps high value.Consider influence of the analysis time to testing, the optimum flow rate of final choice is
1.2mL/min。
Claims (3)
1. in a kind of use high effective liquid chromatography for measuring sample methcathinone content method, it comprises the following steps：
(1) preparation of working solution；The preparation of the working solution, using inner mark method ration, selection 2- phenyl ethylamines are used as internal standard substance；
In step (1), methcathinone Standard Stock solutions are taken, diluted successively with the solution of the internal standard substance that mass concentration is 0.1mg/mL
Be respectively 0.5 into methcathinone mass concentration, 0.1,0.05,0.01,0.005,0.001, the series standard of 0.0005mg/mL it is molten
Liquid, and keep the concentration of inner mark solution to be 0.01mg/mL；
(2) preparation of sample solution；In step (2)：Sample is weighed, flowing phased soln is added, vibration, centrifugation takes after centrifugation
Supernatant, adds internal standard substance, and adds mobile phase, and sample solution is obtained after shaken well；In mobile phase and step (3)
Mobile phase is identical；
(3) setting of testing conditions；In step (3), chromatographic column specification is 250mm × 4.6mm, and 5 μm, column temperature is 35 DEG C, detection
Wavelength 254nm；
(4) in sample methcathinone content measure；
(5) experimental result is calculated,
Wherein, in step (3), chromatographic column uses anti-phase phenyl chromatographic column, and mobile phase is A-1~5mM ammonium acetate buffers：B- second
Nitrile, A:B=(20~40):(60-80), flow velocity is 1.0-1.5mL/min；Mobile phase uses preceding through 0.45 μm of micropore filtering film mistake
10~15min of ultrasound in ultrasonic cleaner is put into after filter, fully the gas in abjection mobile phase.
2. method according to claim 1, it is characterised in that：In step (3), the 4mM ammonium acetate buffers of A-pH=5.0
Liquid：B- acetonitriles, A:B=20:80, flow velocity is 1.2mL/min.
3. method according to claim 1, it is characterised in that before adding mobile phase in sample, using methyl phenyl ethers anisole,
The mixed solution of dichloromethane and ether extracts the methcathinone in sample, and the volume ratio of methyl phenyl ethers anisole, dichloromethane and ether is
5:2:1.5, to the hydrochloric acid that HCl mass fractions are 15% is added in the organic phase isolated, residue is put after removing organic phase
It is concentrated to dryness on 35 DEG C of rapid concentration instrument.
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