CN106198775A - A kind of method measuring chloromethanes in gemcitabine hydrochloride, ethyl chloride - Google Patents
A kind of method measuring chloromethanes in gemcitabine hydrochloride, ethyl chloride Download PDFInfo
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- CN106198775A CN106198775A CN201610470655.4A CN201610470655A CN106198775A CN 106198775 A CN106198775 A CN 106198775A CN 201610470655 A CN201610470655 A CN 201610470655A CN 106198775 A CN106198775 A CN 106198775A
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Abstract
The invention discloses and a kind of measure the method for chloromethanes, ethyl chloride in gemcitabine hydrochloride crude drug, it comprises the steps: that the chromatographic column of gas chromatograph is DB 5 capillary tube, and carrier gas is nitrogen, and detector is ECD, and input mode is headspace sampling.Draw in reference substance solution, need testing solution, blank solution each 1ml top set empty bottle, seal, hatching, take upper gas 100 l injection gas chromatography instrument, record chromatogram.The range of linearity of chloromethanes is 0.1010 0.6059 g/ml, and the range of linearity of ethyl chloride is 0.1290 0.7742 g/ml.Chloromethanes, the residual of ethyl chloride in present invention application gas chromatography determination gemcitabine hydrochloride crude drug, separation efficiency is high, speed is fast, detection sensitivity is high in analysis, by chloromethanes, the residual quantity of ethyl chloride in detection gemcitabine hydrochloride crude drug, it is possible to more preferably control the quality of gemcitabine hydrochloride.
Description
Technical field
The present invention relates to analysis technical field, be specifically related to chloromethanes in a kind of gas Chromatographic Determination gemcitabine hydrochloride,
The method of ethyl chloride.
Background technology
Gemcitabine hydrochloride is a kind of Difluoronucleosides class antimetabolite cancer therapy drug destroying cellular replication, is mainly used in
(1) advanced pancreatic cancer, advanced Non-small cell lung, Limited-stage or transitivity bladder cancer and the first-line treatment of metastatic breast cancer；
(2) second line treatment of advanced ovarian cancer；(3) lower rectal cancer of early cervical carcinoma.Gemcitabine hydrochloride antitumor spectra is wide, to other
Solid tumor includes mesothelioma, the esophageal carcinoma, gastric cancer and colorectal cancer, and hepatocarcinoma, cancer of biliary duct, nasopharyngeal carcinoma, tumor of testis, lymphoma and
Incidence cancer etc. all has certain curative effect.
Gemcitabine hydrochloride production process can use methanol, ethanol as solvent, owing to hydrochloric acid and methanol, ethanol can produce
Raw nucleophilic substitution generates chloromethanes, ethyl chloride.Chloromethanes, ethyl chloride belong to halogenated alkane, have genotoxicity, need for this
Its residual quantity is controlled, it is achieved chloromethanes, the separation detection of ethyl chloride, its quality control aspect is had important showing
Sincere justice.
Summary of the invention
It is an object of the invention to set up and a kind of measure the method for chloromethanes, ethyl chloride in gemcitabine hydrochloride raw material, permissible
Preferably control the chloromethanes of residual, ethyl chloride in gemcitabine hydrochloride crude drug, can be the most former to gemcitabine hydrochloride
In material medicine, gene poison impurity that may be present detects.
The technical scheme is that, chloromethanes, the side of ethyl chloride in gas Chromatographic Determination gemcitabine hydrochloride crude drug
Method, it comprises the steps:
Prepared by contrast solution: take chloromethanes and ethyl chloride is appropriate, accurately weighed, dissolves with DMSO and makes in every 1ml respectively containing about 0.6
μ g chloromethanes, 0.6 μ g ethyl chloride are as contrast solution；
Prepared by need testing solution: this product is appropriate, adds DMSO lysate and makes the solution of every 1ml hydrochloric gemcitabine 20mg, makees
For need testing solution；
The preparation of blank solution: DMSO；
Chromatographic column is: DB-5 quartz capillary column；
Chromatographic condition: column temperature is initially 40 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 10
Minute；Rise to 40 DEG C with the speed of intensification per minute 50 DEG C again, keep 5 minutes；Injector temperature: 200 DEG C；Detector: ECD；Inspection
Survey device temperature: 250 DEG C；Carrier gas: N2；Flow velocity: 0.8ml/min
Head space condition: furnace temp: 45 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 40min, sample size 100 l.
Sample introduction: take contrast solution, need testing solution, each 1ml of blank solution, put in 10ml ml headspace bottle respectively, seals, puts into
Head space heater, headspace sampling record chromatogram；
Calculate the equation of linear regression of value and the respective peaks area value of reference substance solution concentration, correlation coefficient and should be not less than
0.99, reference substance solution peak shape is symmetrical, theoretical cam curve in terms of chloromethanes more than 2000；If any chlorine in need testing solution chromatogram
Methane, ethyl chloride chromatographic peak, should be consistent with chloromethanes in contrast solution, ethyl chloride chromatographic peak retention time, blank solution chromatograph
Figure, occurs without the peak identical with chloromethanes, ethyl chloride reference substance, i.e. blank solution is noiseless；
Chloromethanes, ethyl chloride must not be crossed in every gram of gemcitabine hydrochloride raw material 30ug respectively, and the concentration range of chloromethanes is
0.1010 ~ 0.6059 g/ml, the concentration range of ethyl chloride is 0.1290 ~ 0.7742 g/ml.
Accompanying drawing illustrates: Fig. 1 detector condition gropes chromatogram；
Fig. 2 input mode gropes chromatogram；
Fig. 3 selects the comparative selection figure of chromatographic column；
The eliminating chromatogram of Fig. 4 blank interference；
The establishment chromatogram of Fig. 5 chromatographic column；
The screening chromatogram of Fig. 6 chromatographic column；
The linear regression graph of Fig. 7 chloromethanes；
The linear regression graph of Fig. 8 ethyl chloride；
Fig. 9 detector temperature change test result comparison diagram；
Figure 10 initiates column temperature change test result comparison diagram；
Figure 11 holding temperature change test result comparison diagram；
Figure 12 sample size change test result comparison diagram；
Figure 13 change in flow test result comparison diagram；
Figure 14 temperature retention time change test result comparison diagram；
Figure 15 split ratio change test result comparison diagram.
Present disclosure is described in further detail by form more by the following examples, but should not be interpreted as this with regard to this
It is only limitted to following example in inventing above-mentioned subject area.Without departing under the present invention above-mentioned technology premise, general according to this area
The corresponding amendment replaced or change that logical technological know-how and customary means are made, is included within the scope of the present invention.
Embodiment 1 test method is screened: the condition of detector is groped
Instrument: gas chromatograph TrermoscientificTrace1300, Trermoscientific chromatographic work station；
Reagent: chloromethanes, ethyl chloride, DMSO；
Chromatographic column: DB-624 quartz capillary column, 30m × 0.530mm × 3.00 m.
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 7 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Temperature: 200 DEG C；Detector: FID；Detector temperature: 250 DEG C；Carrier gas: N2；Flow velocity: 0.8ml/min shunts: 16ml/min.
Head space condition: furnace temp: 45 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 40min, sample size 100 l.
The preparation of test solution:
0.01N chloromethanes solution, takes 1.0Mol/L chloromethanes and inhales 1ml to 100ml, to obtain final product；
0.02N ethyl chloride stock solution, takes 2.0Mol/L ethyl chloride and inhales 1ml to 100ml, to obtain final product；
Blank solution, DMSO；
Sample 10%, sample 100mg adds 1mlDMSO.
Result is shown in Fig. 1.
Conclusion: using fid detector detection under this chromatographic condition, the appearance time of chloromethanes is 6.867min, chloroethene
The appearance time of alkane be 8.248min, DMSO appearance time be about 11min, chromatogram has DMSO chromatographic peak, and other
Unknown solvent peak occurs, the appearance of these chromatographic peaks can disturb the detection of chloromethanes, ethyl chloride.
Embodiment 2 test method is screened: groping of input mode
Instrument: gas chromatograph TrermoscientificTrace1300, Trermoscientific chromatographic work station
Chromatographic column: DB-624 quartz capillary column, 30m × 0.530mm × 3.00 m
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 10 minutes, rises to 220 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Mode: Liquid sample introduction；Flow velocity: 0.8ml/min；Sample size: 1 l；Injector temperature: 200 DEG C；
Detector: ECD；Detector temperature: 250 DEG C；Carrier gas: N2；Split ratio: 1:20.
The preparation of reference substance storing solution: precision measures the chloromethanes methanol solution 0.5ml of 5mg/ml, in 25ml measuring bottle,
Add methanol constant volume to scale, as reference substance storing solution.
The preparation of contrast solution: precision measures reference substance storing solution 5ml, in 10ml measuring bottle, adds methanol constant volume to scale,
Make containing about 0.05mg chloromethanes solution in every 1ml, as contrast solution.
The preparation of test solution: precision weighs this product 100mg, in 2ml measuring bottle, adds methanol constant volume to scale, makes every
Sample containing about 50mg in 1ml, as need testing solution.
Sample-adding recovery sample solution preparation: precision weighs sample 100mg, in 2ml measuring bottle, adds 100 l reference substance storages
Standby liquid, adds methanol constant volume to scale, as the sample solution of the response rate 100%.
Take contrast solution, test solution, sample-adding recovery sample solution draw 1 l sample introduction respectively, record chromatogram.
Having solvent peak at 7.8min, chloromethanes goes out peak at 5.8min.
Conclusion: under the process conditions, uses Liquid sample introduction that the mensuration of the recovery test of chloromethanes is had interference, causes
Chloromethanes response value does not has headspace gas sample introduction high.Result is shown in Fig. 2.
Embodiment 3 test method is screened: the condition of temperature programming is groped
Instrument and reagent:
Instrument: gas chromatograph TrermoscientificTrace1300, Trermoscientific chromatographic work station
Chromatographic column: DB-624 quartz capillary column, 30m × 0.530mm × 3.00 m,
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 7 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Temperature: 200 DEG C；Detector: ECD；Detector temperature: 250 DEG C；Carrier gas: N2；Flow velocity: 0.8ml/min shunts: 16ml/min
Head space condition: furnace temp: 45 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 40min, sample size 100 l
0.01N chloromethanes solution, 0.02N ethyl chloride stock solution, DMSO；
2% sample, takes 20mg sample and adds DMSO dissolving to 1ml；
Take above-mentioned solution, respectively sample introduction, record chromatogram
Conclusion: the appearance time of chloromethanes is 9.240min under this chromatographic condition, the big chromatographic peak of sample introduction concentration trails.
The appearance time of ethyl chloride is 11.170min, and the big chromatographic peak of sample introduction concentration trails, and ethyl chloride peak occurs in program
After intensification, Chromatogram Baseline is uneven.DMSO has chromatographic peak to occur at 7.253min.
The sample of 10% had peak to occur at 6.578 minutes.Owing to equilibration time is short, initial baseline is uneven.
Embodiment 4 test method is screened: the alternative condition of chromatographic column is groped
Instrument: gas chromatograph (TrermoscientificTrace1300) Trermoscientific chromatographic work station
Chromatographic column: DB-624 quartz capillary column, 30m × 0.530mm × 3.00 m
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 10 minutes, rises to 220 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Mode: headspace sampling；Flow rate of carrier gas: 0.8ml/min；Sample size: 100 l；Injector temperature: 200 DEG C；Detector: ECD；Detection
Device temperature: 250 DEG C；Carrier gas: N2；Split ratio: 1:20；Head space furnace temp: 45 DEG C；Temperature retention time: 30min.
Result is shown in Fig. 3, has air peak at 7.2min, 9.3min, and chloromethanes goes out peak at 9.3min.
Conclusion: under the process conditions, uses DB-624 quartz capillary column chloromethanes peak and air peak to have interference.
Embodiment 5 test method is screened: the alternative condition of chromatographic column is groped
Instrument: gas chromatograph (TrermoscientificTrace1300) Trermoscientific chromatographic work station
Chromatographic column: DB-WAX quartz capillary column, 30m × 0.530mm × 3.00 m
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 10 minutes, rises to 220 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Mode: headspace sampling；Flow rate of carrier gas: 0.8ml/min；Sample size: 100 l；Injector temperature: 200 DEG C；Detector: ECD；Detection
Device temperature: 250 DEG C；Carrier gas: N2；Split ratio: 1:20；Head space furnace temp: 45 DEG C；Temperature retention time: 30min.
The preparation of reference substance storing solution: precision measures the chloromethanes methanol solution 0.5ml of 5mg/ml, in 25ml measuring bottle,
Add methanol constant volume to scale, as reference substance storing solution.
The preparation of contrast solution: precision measures reference substance storing solution 5ml, in 10ml measuring bottle, adds methanol constant volume to scale,
Make containing about 0.05mg chloromethanes solution in every 1ml, as contrast solution.
The preparation of test solution: precision weighs this product 100mg, in ml headspace bottle, adds the methanol of 2ml, makes in every 1ml
Containing about the sample of 50mg, as need testing solution.
Sample-adding recovery sample solution preparation: precision weighs this product 100mg, in ml headspace bottle, adds the contrast solution of 2ml,
Sample solution as the response rate 100%.
Take contrast solution, test solution, sample-adding recovery sample solution sample introduction respectively, record chromatogram.
Having solvent peak at 7.07min, 9.14min, chloromethanes goes out peak at 8.80min.
Conclusion: under the process conditions, uses DB-WAX quartz capillary column chloromethanes peak and air peak separating degree to reach not
To requiring.
Embodiment 6 test method is screened: the eliminating of blank interference
Instrument and reagent:
Instrument: gas chromatograph TrermoscientificTrace1300, Trermoscientific chromatographic work station
Chromatographic column: DB-624 quartz capillary column, 30m × 0.530mm × 3.00 m, USD592144H
Chromatographic condition:
Chromatographic condition: column temperature is initially 40 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 10
Minute；It is being down to 40 DEG C with the speed of intensification per minute 50 DEG C, is keeping 5 minutes injector temperature: 200 DEG C；Detector: ECD；Detection
Device temperature: 250 DEG C；Carrier gas: N2；Flow velocity: 0.8ml/min shunts: 16ml/min.
Head space condition: furnace temp: 45 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 40min, sample size 100 l.
Take empty bottle and filled the sample bottle of nitrogen, containing the difference sample introduction of 0.01mol/L sodium hydrate aqueous solution ml headspace bottle, note
Record chromatogram, result is shown in Fig. 4.
Conclusion: the appearance time of empty bottle is 7.102min under this chromatographic condition, has small peak to occur at 9.133.
Filled the sample bottle 7.110min of nitrogen going out peak area substantially time of diminishing at peak is still to also have at 9.433min
Small peak occurs.Judge the oxygen interference during chromatographic peak is air at 7.110min.
Go out peak containing 0.01mol/L sodium hydrate aqueous solution 9.4min, reduce and judge at 9.4min as carbon dioxide.
Embodiment 7 test method is screened: the establishment of chromatographic column
Instrument and reagent:
Instrument: gas chromatograph TrermoscientificTrace1300, Trermoscientific chromatographic work station
Chromatographic column: DB-5 quartz capillary column, 30m × 0.530mm × 3.00 m, USE381032H
Chromatographic condition:
Chromatographic condition: column temperature is initially 40 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 10
Minute；It is being down to 40 DEG C with the speed of intensification per minute 50 DEG C, is keeping 5 minutes injector temperature: 200 DEG C；Detector: ECD；Detection
Device temperature: 250 DEG C；Carrier gas: N2；Flow velocity: 0.8ml/min shunts: 16ml/min
Head space condition: furnace temp: 45 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 40min, sample size 100 l.
Take DMSO, contrast solution containing 0.0001Mol/L chloromethanes and 0.0001Mol/L ethyl chloride solution is respectively put into
Ml headspace bottle sample introduction record chromatogram respectively, result is shown in Fig. 5.
Conclusion:
The appearance time of blank solvent is 6.850min, and 7.822min.
In contrast solution, the appearance time of air is 6.850min, and when going out peak of 7.818min, chloromethanes and ethyl chloride
Between be 8.543min, and 11.080min, the detection of chloromethanes and ethyl chloride is not interfered with by air
The screening of embodiment 8 chromatographic column
Instrument: gas chromatograph TrermoscientificTrace1300, Trermoscientific chromatographic work station
Chromatographic column: DB-5 quartz capillary column, 30m × 0.530mm × 3.00 m
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 10 minutes, rises to 220 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Mode: headspace sampling；Flow rate of carrier gas: 0.8ml/min；Sample size: 100 l；Injector temperature: 200 DEG C；Detector: ECD；Detection
Device temperature: 250 DEG C；Carrier gas: N2；Split ratio: 1:20；Head space furnace temp: 45 DEG C；Temperature retention time: 30min.
Result is shown in Fig. 6, has air peak at 7.1min, 8.5min, and chloromethanes goes out peak at 8.7min.
Conclusion: under the process conditions, uses DB-5 quartz capillary column chloromethanes peak and air peak to separate.
The specificity of embodiment 9 method
Instrument: gas chromatograph (TrermoscientificTrace1300) Trermoscientific chromatographic work station
Chromatographic column: DB-5 quartz capillary column, 30m × 0.530mm × 3.00 m
Chromatographic condition:
Column temperature is initially 40 DEG C, keeps 10 minutes, rises to 220 DEG C with the speed of intensification per minute 50 DEG C, keeps 25 minutes；Sample introduction
Mode: headspace sampling；Flow rate of carrier gas: 0.8ml/min；Sample size: 100 l；Injector temperature: 200 DEG C；Detector: ECD；Detection
Device temperature: 250 DEG C；Carrier gas: N2；Split ratio: 1:20.
Take DMSO1ml and put into sample introduction after the heating of head space heating furnace, as blank, record chromatogram.Result shows solvent
Chloromethanes, ethyl chloride are measured noiseless by DMSO.Each component qualitative: take respectively containing chloromethanes 1.5 μ g/ml, ethyl chloride 1.8 μ
The DMSO solution of g/ml, respectively takes 1ml sealing and puts into sample introduction after the heating of head space heating furnace, record chromatogram.When each solvent peak retains
Between be shown in Table 1.
The retention time of each solvent of table 1
With test result evaluation under system suitability item, taking the chromatogram of standard solution, each residual solvent peak sequence is followed successively by
Its retention time of air, chloromethanes, ethyl chloride and relative retention time ratio (RRT) data are shown in Table 2.
The retention time of each solvent in table 2 standard solution
Conclusion: the method is because separating degree is all higher than 1.5 between adjacent component, and adjacent peak all can be kept completely separate, and specificity is good.
Embodiment 10 sample introduction precision detects
Chromatographic condition, with embodiment 9, takes sample introduction after the heating of standard solution 1ml head space heating furnace, record chromatogram to 35min, with
Upper each solvent goes out peak completely, and peak sequence is followed successively by chloromethanes, ethyl chloride.Standard solution continuous sample introduction 5 times, calculates each sample introduction
The peak area of each solvent, tries to achieve relative standard deviation (should be not more than 10.0%).
Table 3 precision test
Conclusion: test shows that this chromatographic system precision is good.Data are shown in Table 3.
Embodiment 11 linear relationship is investigated
Chromatographic condition is with embodiment 9, and precision measures chloromethanes, ethyl chloride, with DMSO, is prepared as chloride methane concentration and is respectively
0.1010、0.2020、0.3029、0.4039、0.5049、0.6059µg/ml；Chloroethane concentration is respectively 0.1290,0.2581,
0.3871, the reference substance solution of 0.5162,0.6452,0.7742 g/ml.Each precision is noted after measuring the heating of 1ml head space heating furnace
Enter chromatograph of liquid, record chromatogram, measure peak area, the results are shown in Table 4, table 5.With peak area value (A), concentration (C) is carried out
Linear regression, obtains a straight line, sees Fig. 7, Fig. 8.
Table 4 chloromethanes Linear regression data
Table 5 ethyl chloride Linear regression data
The regression equation y=0.1015x+0.0031, r=0.9960 of chloromethanes
The regression equation y=0.1158x+0.0053, r=0.9977 of ethyl chloride
Conclusion: result above shows, chloromethanes is in the range of 0.1010 ~ 0.6059 g/ml, and ethyl chloride is 0.1290 ~ 0.7742
In the range of g/ml, sample introduction concentration and peak area value have good linear relationship.
Embodiment 12 detection limit and quantitative limit
Chromatographic condition compares with embodiment 9, the signal that the signal measured by known low concentrations sample and blank sample are measured,
With corresponding concentration during S/N ≈ 3 for detection limit concentration, the detection limit concentration obtaining chloromethanes is 0.0101 μ g/ml, ethyl chloride
Detection limit concentration is 0.0043 μ g/ml.
The signal that the signal measured by known low concentrations sample and blank sample are measured compares, with S/N ≈ 10 phase
The concentration answered is quantitative limit concentration, and the quantitative limit concentration obtaining chloromethanes is 0.0549 μ g/ml, and the quantitative limit concentration of ethyl chloride is
0.0129μg/ml。
Conclusion: chloromethanes quantitative limit concentration is 0.0549 μ g/ml, ethyl chloride quantitative limit concentration is 0.0129 μ g/ml.
Embodiment 13 replica test
Press text method, be measured with 5 parts of a collection of gemcitabine hydrochloride sample (lot number 20130202), try to achieve relative standard
Deviation, the results are shown in Table 6.
Table 6 repeatability is tested
Conclusion: result of the test shows that this method repeatability is good.
Embodiment 14 replica test recovery test
Chromatographic condition, with embodiment 9, presses chloromethanes, the amount of ethyl chloride 80%, 100%, 120% respectively, adds gemcitabine hydrochloride same
One batch sample (lot number 20130202), in measuring bottle, is prepared by the preparation method of text need testing solution and above-mentioned chromatographic condition is surveyed
Fixed.It is calculated as follows the response rate, the results are shown in Table 7, table 8.
Table 7 chloromethanes recovery test
Table 8 ethyl chloride recovery test
Conclusion: test shows that the accuracy of this method is good.
Embodiment 15 ruggedness detector temperature changes
Detector temperature, with embodiment 9, is changed to by chromatographic condition: detector temperature change 1:240 DEG C, head space detector temperature
Change 2:260 DEG C, original head space detector temperature be: 250 DEG C, test result is shown in Fig. 9, table 9.
Table 9 detector temperature change test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that detector temperature at 240 DEG C～
In 260 DEG C of allowed bands, change separates not impact to residual solvent.
Embodiment 16 ruggedness initiates column temperature change
Initial column temperature, with embodiment 9, is changed to by chromatographic condition: initial column temperature change 1:35 DEG C, initial column temperature change 2:45 DEG C,
Original initial column temperature: 40 DEG C, test result is shown in Figure 10, table 10.
Table 10 initiates column temperature change test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that chromatographic column initial temperature is at 35 DEG C
～change separates not impact to residual solvent in 45 DEG C of allowed bands.
Embodiment 17 ruggedness holding temperature changes
Head space heater holding temperature, with embodiment 9, is changed to by chromatographic condition: holding temperature change 1:43 DEG C, holding temperature
Change 2:47 DEG C, original thermophore temperature be: 45 DEG C, test result is shown in Figure 11, table 11.
Table 11 holding temperature change test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that holding temperature is at 43 DEG C～45 DEG C
In allowed band, change separates not impact to residual solvent.
Embodiment 18 ruggedness sample amount changes
Sample size, with embodiment 9, is changed to by chromatographic condition: sample size change 1:200 μ l, sample size change 2:300 μ l, original
Sample size is: 100 μ l, and test result is shown in Figure 12, table 12.
Table 12 sample size change test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that sample size is at 100 μ l～300 μ l
In allowed band, change separates not impact to residual solvent.
Embodiment 19 ruggedness change in flow
Change in flow, with embodiment 9, is by chromatographic condition: change in flow 1:0.7ml/min, change in flow 2:0.9ml/min, former
Beginning flow velocity is: 0.8ml/min, and test result is shown in Figure 13, table 13.
Table 13 change in flow test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that flow velocity at 0.7ml/min～
In 0.9ml/min allowed band, change separates not impact to residual solvent.
Embodiment 20 ruggedness temperature retention time changes
Temperature retention time, with embodiment 9, is changed to by chromatographic condition: temperature retention time changes 1:10 minute, temperature retention time change 2:20
Minute, original temperature retention time: 40 minutes, test result was shown in Figure 14, table 14.
Table 14 temperature retention time change test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that during temperature retention time at 10 minutes～
In 40 minutes allowed bands, change separates not impact to residual solvent.
Embodiment 21 ruggedness split ratio changes
Split ratio, with embodiment 9, is changed to by chromatographic condition: split ratio change 1:1:15, split ratio change 2:1:25, original point
Flow ratio is: 1:20, and test result is shown in Figure 15, table 15.
Table 15 split ratio change test result contrast table
Conclusion: measure by under above-mentioned chromatographic condition, all can reach required separating effect, it is seen that split ratio at 250 DEG C of 1:15～
In 1:25 allowed band, change separates not impact to residual solvent.
Chloromethanes and ethyl chloride content measuring in embodiment 22 gemcitabine hydrochloride
Chromatographic condition with embodiment 9 weigh respectively lot number be 20150111,20150206,20150818,20151116,
20151216, the gemcitabine hydrochloride sample of 20160118 lot numbers, sample introduction test result is shown in Table 16.
Table 16 sample introduction test result
Remarks: reagent chloromethanes is ladder uncommon love chemical conversion LOT:2RYZE-MO, ethyl chloride is ladder uncommon love chemical conversion LOT:MZYGG-CE,
DMSO is ScharlauLOT:14398313).
Claims (4)
1. chloromethanes, the method for ethyl chloride in gas Chromatographic Determination gemcitabine hydrochloride crude drug, it is characterised in that it include as
Lower step:
Prepared by contrast solution: take chloromethanes and ethyl chloride is appropriate, accurately weighed, dissolves with DMSO and makes in every 1ml respectively containing about 0.6
μ g chloromethanes, 0.6 μ g ethyl chloride are as contrast solution；
Prepared by need testing solution: this product is appropriate, adds DMSO lysate and makes the solution of every 1ml hydrochloric gemcitabine 20mg, makees
For need testing solution；
The preparation of blank solution: DMSO；
Chromatographic column is: DB-5 quartz capillary column；
Chromatographic condition: column temperature is initially 35 ~ 45 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, protects
Hold 10 minutes；Rise to 40 DEG C with the speed of intensification per minute 50 DEG C again, keep 5 minutes；Injector temperature: 200 DEG C；Detector:
ECD；Detector temperature: 240 ~ 260 DEG C；Carrier gas: N2；Flow velocity: 0.7 ~ 0.9ml/min；
Head space condition: furnace temp: 43 ~ 47 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 10 ~ 40min, sample size 100 ~
300µl；
Sample introduction: take contrast solution, need testing solution, each 1ml of blank solution, put in 10ml ml headspace bottle respectively, seals, puts into head space
Heater, headspace sampling record chromatogram；
Calculating the value of reference substance solution concentration and the equation of linear regression of respective peaks area value, correlation coefficient should be not less than 0.99,
Reference substance solution peak shape is symmetrical, theoretical cam curve in terms of chloromethanes more than 2000；In need testing solution chromatogram if any chloromethanes,
Ethyl chloride chromatographic peak, should be consistent with chloromethanes in contrast solution, ethyl chloride chromatographic peak retention time, blank solution chromatogram, nothing
The peak identical with chloromethanes, ethyl chloride reference substance occurs, i.e. blank solution is noiseless；
Chloromethanes, ethyl chloride must not be crossed in every gram of gemcitabine hydrochloride raw material 30ug respectively, and the concentration range of chloromethanes is
0.1010 ~ 0.6059 g/ml, the concentration range of ethyl chloride is 0.1290 ~ 0.7742 g/ml.
2. chloromethanes, the method for ethyl chloride, its feature in gas Chromatographic Determination gemcitabine hydrochloride crude drug described in claim 1
Being, it comprises the steps:
Prepared by contrast solution: take chloromethanes and ethyl chloride is appropriate, accurately weighed, dissolves with DMSO and makes in every 1ml respectively containing about 0.6
μ g chloromethanes, 0.6 μ g ethyl chloride are as contrast solution；
Prepared by need testing solution: this product is appropriate, adds DMSO lysate and makes the solution of every 1ml hydrochloric gemcitabine 20mg, makees
For need testing solution；
The preparation of blank solution: DMSO；
Chromatographic column is: DB-5 quartz capillary column；
Chromatographic condition: column temperature is initially 40 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 10
Minute；Rise to 40 DEG C with the speed of intensification per minute 50 DEG C again, keep 5 minutes；Injector temperature: 200 DEG C；Detector: ECD；Inspection
Survey device temperature: 250 DEG C；Carrier gas: N2；Flow velocity: 0.8ml/min；
Head space condition: furnace temp: 45 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 40min, sample size 100 l；
Sample introduction: take contrast solution, need testing solution, each 1ml of blank solution, put in 10ml ml headspace bottle respectively, seals, puts into head space
Heater, headspace sampling record chromatogram；
Calculating the value of reference substance solution concentration and the equation of linear regression of respective peaks area value, correlation coefficient should be not less than 0.99,
Reference substance solution peak shape is symmetrical, theoretical cam curve in terms of chloromethanes more than 2000；In need testing solution chromatogram if any chloromethanes,
Ethyl chloride chromatographic peak, should be consistent with chloromethanes in contrast solution, ethyl chloride chromatographic peak retention time, blank solution chromatogram, nothing
The peak identical with chloromethanes, ethyl chloride reference substance occurs, i.e. blank solution is noiseless；
Chloromethanes, ethyl chloride must not be crossed in every gram of gemcitabine hydrochloride raw material 30ug respectively, and the concentration range of chloromethanes is
0.1010 ~ 0.6059 g/ml, the concentration range of ethyl chloride is 0.1290 ~ 0.7742 g/ml.
3. chloromethanes, the method for ethyl chloride, its feature in gas Chromatographic Determination gemcitabine hydrochloride crude drug described in claim 1
Being, it comprises the steps:
Prepared by contrast solution: take chloromethanes and ethyl chloride is appropriate, accurately weighed, dissolves with DMSO and makes in every 1ml respectively containing about 0.6
μ g chloromethanes, 0.6 μ g ethyl chloride are as contrast solution；
Prepared by need testing solution: this product is appropriate, adds DMSO lysate and makes the solution of every 1ml hydrochloric gemcitabine 20mg, makees
For need testing solution；
The preparation of blank solution: DMSO；
Chromatographic column is: DB-5 quartz capillary column；
Chromatographic condition: column temperature is initially 35 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 10
Minute；Rise to 40 DEG C with the speed of intensification per minute 50 DEG C again, keep 5 minutes；Injector temperature: 200 DEG C；Detector: ECD；Inspection
Survey device temperature: 240 DEG C；Carrier gas: N2；Flow velocity: 0.7ml/min；
Head space condition: furnace temp: 43 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 10min, sample size 200 l；
Sample introduction: take contrast solution, need testing solution, each 1ml of blank solution, put in 10ml ml headspace bottle respectively, seals, puts into head space
Heater, headspace sampling record chromatogram；
Calculating the value of reference substance solution concentration and the equation of linear regression of respective peaks area value, correlation coefficient should be not less than 0.99,
Reference substance solution peak shape is symmetrical, theoretical cam curve in terms of chloromethanes more than 2000；In need testing solution chromatogram if any chloromethanes,
Ethyl chloride chromatographic peak, should be consistent with chloromethanes in contrast solution, ethyl chloride chromatographic peak retention time, blank solution chromatogram, nothing
The peak identical with chloromethanes, ethyl chloride reference substance occurs, i.e. blank solution is noiseless；
Chloromethanes, ethyl chloride must not be crossed in every gram of gemcitabine hydrochloride raw material 30ug respectively, and the concentration range of chloromethanes is
0.1010 ~ 0.6059 g/ml, the concentration range of ethyl chloride is 0.1290 ~ 0.7742 g/ml.
4. chloromethanes, the method for ethyl chloride, its feature in gas Chromatographic Determination gemcitabine hydrochloride crude drug described in claim 1
Being, it comprises the steps:
Prepared by contrast solution: take chloromethanes and ethyl chloride is appropriate, accurately weighed, dissolves with DMSO and makes in every 1ml respectively containing about 0.6
μ g chloromethanes, 0.6 μ g ethyl chloride are as contrast solution；
Prepared by need testing solution: this product is appropriate, adds DMSO lysate and makes the solution of every 1ml hydrochloric gemcitabine 20mg, makees
For need testing solution；
The preparation of blank solution: DMSO；
Chromatographic column is: DB-5 quartz capillary column；
Chromatographic condition: column temperature is initially 45 DEG C, keeps 12 minutes, rises to 250 DEG C with the speed of intensification per minute 50 DEG C, keeps 10
Minute；Rise to 40 DEG C with the speed of intensification per minute 50 DEG C again, keep 5 minutes；Injector temperature: 200 DEG C；Detector: ECD；Inspection
Survey device temperature: 260 DEG C；Carrier gas: N2；Flow velocity: 0.9ml/min；
Head space condition: furnace temp: 47 DEG C；Sampling probe temperature: 55 DEG C, temperature retention time 20min, sample size 300 l；
Sample introduction: take contrast solution, need testing solution, each 1ml of blank solution, put in 10ml ml headspace bottle respectively, seals, puts into head space
Heater, headspace sampling record chromatogram；
Calculating the value of reference substance solution concentration and the equation of linear regression of respective peaks area value, correlation coefficient should be not less than 0.99,
Reference substance solution peak shape is symmetrical, theoretical cam curve in terms of chloromethanes more than 2000；In need testing solution chromatogram if any chloromethanes,
Ethyl chloride chromatographic peak, should be consistent with chloromethanes in contrast solution, ethyl chloride chromatographic peak retention time, blank solution chromatogram, nothing
The peak identical with chloromethanes, ethyl chloride reference substance occurs, i.e. blank solution is noiseless；
Chloromethanes, ethyl chloride must not be crossed in every gram of gemcitabine hydrochloride raw material 30ug respectively, and the concentration range of chloromethanes is
0.1010 ~ 0.6059 g/ml, the concentration range of ethyl chloride is 0.1290 ~ 0.7742 g/ml.
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| CN113203802A (en) * | 2021-03-25 | 2021-08-03 | 中化蓝天霍尼韦尔新材料有限公司 | Method for determining content of organic components in pentachloropropane synthesis process |
| CN115876902A (en) * | 2021-09-29 | 2023-03-31 | 江苏正大清江制药有限公司 | Method for determining potential genotoxic impurities chloromethane and chloroethane in clohexine dry suspension |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111707747A (en) * | 2020-06-19 | 2020-09-25 | 江西省药品检验检测研究院 | Method for detecting mesylate genotoxic impurities in gemcitabine hydrochloride by GC-MS/MS (gas chromatography-Mass spectrometer/Mass spectrometer) method |
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| CN113203802A (en) * | 2021-03-25 | 2021-08-03 | 中化蓝天霍尼韦尔新材料有限公司 | Method for determining content of organic components in pentachloropropane synthesis process |
| CN115876902A (en) * | 2021-09-29 | 2023-03-31 | 江苏正大清江制药有限公司 | Method for determining potential genotoxic impurities chloromethane and chloroethane in clohexine dry suspension |
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