CN110531020A - The measuring method of fructose limit in a kind of compound electrolyte glucose injection - Google Patents
The measuring method of fructose limit in a kind of compound electrolyte glucose injection Download PDFInfo
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- CN110531020A CN110531020A CN201910937881.2A CN201910937881A CN110531020A CN 110531020 A CN110531020 A CN 110531020A CN 201910937881 A CN201910937881 A CN 201910937881A CN 110531020 A CN110531020 A CN 110531020A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8836—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
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Abstract
The invention discloses a kind of measuring method of fructose limit in compound electrolyte glucose injection, include the following steps: that (1) prepares reference substance solution: weighing fructose standard items and add ultrapure water dissolution, dilution, obtain reference substance stock solution；It pipettes reference substance stock solution and adds ultrapure water dilution, constant volume, obtain reference substance solution；(2) it prepares test solution: measuring compound electrolyte glucose injection and ultrapure water is added to dilute, it shakes up, up to test solution, the concentration of fructose is no more than 2 percent of concentration of glucose in test solution in reference substance solution in the step (1)；(3) reference substance solution, test solution chromatographic peak area are detected and read by high performance liquid chromatograph, are calculated in conjunction with reference substance solution concentration using external standard method and are obtained fructose concentration.Measuring method accuracy of the present invention is high, easy to operate.
Description
Technical field
The present invention relates to fructose concentration measuring method, in particular to fructose limits in a kind of compound electrolyte glucose injection
The measuring method of degree.
Background technique
Glucose solution is unstable under alkaline condition, when glucose is in dilute alkaline solution, when room temperature is longer
Between store after will occur Partial Conversion, generate fructose and mannose, as the further carbochain of redox reaction cracks and occur
It decomposes.It is found by the strong Degrading experiment of preparation, destroys the item (with heating) in acid destruction, alkalinity destruction and Strong oxdiative
Under part, fructose content is obviously increased in preparation, it is therefore necessary to be limited the potential related substance fructose in preparation
Degree control.There has been no the concentration of fructose in accurate, stable, quick method detection compound glucose preparation at present.
Summary of the invention
Goal of the invention: it is an object of the present invention to provide a kind of precision height, compound electrolyte glucose injection easy to operate
The measuring method of middle fructose limit.
Technical solution: the present invention provides a kind of measuring method of fructose limit in compound electrolyte glucose injection, packet
Include following steps:
(1) it prepares reference substance solution: weighing fructose standard items and add ultrapure water dissolution, dilution, obtain reference substance stock solution；It pipettes
Reference substance stock solution adds ultrapure water dilution, constant volume, obtains reference substance solution；
(2) it prepares test solution: measuring compound electrolyte glucose injection and ultrapure water is added to dilute, shake up to get confession
Test sample solution, the concentration of fructose is no more than hundred of concentration of glucose in test solution in reference substance solution in the step (1)
/ bis-；
(3) reference substance solution, test solution chromatographic peak area are detected and read by high performance liquid chromatograph, in conjunction with right
It is calculated according to product solution concentration external standard method and obtains fructose concentration.
Further, the parameter setting that high performance liquid chromatograph detects in the step (3) is as follows:
Chromatographic column: full multi-hole blangel particle is bonded non-crosslinked NH2 stationary phase；Flow velocity: 1.2-1.8mL/min；Column temperature: 35-
45℃；Sample volume: 10 μ L；Mobile phase: acetonitrile-water；Detector: EFI fog detector；Detector temperature: 50 DEG C.
Further, the parameter setting that high performance liquid chromatograph detects in the step (3) is as follows:
Chromatographic column: Shodex Asahipak NH2P-504E；Flow velocity: 1.6mL/min；Column temperature: 45 DEG C；Sample volume: 10 μ
L；Mobile phase: acetonitrile-water；Detector: EFI fog detector；Detector temperature: 50 DEG C.
Further, high performance liquid chromatograph is attached to EFI fog detector in the step (3).
Further, the volume ratio of the mobile phase acetonitrile-water is 70: 30-90: 10.
Further, the volume ratio of the mobile phase acetonitrile-water is 80: 20.
Further, the volume ratio of the mobile phase acetonitrile-water is 85:15.
Further, pass through 0.22 μm of membrane filtration before the test solution detection.
Further, the external standard method calculation formula is as follows:
In formula, Ci: fructose concentration (mg/ml) in test solution；
Cs: reference substance solution concentration (mg/ml)；
C0: concentration of glucose (mg/ml) in test solution；
Ai: test solution chromatographic peak area (pA*min)；
As: reference substance solution chromatographic peak area (pA*min)；
W (%): fructose content in preparation
EFI fog detector (CAD) belongs to novel universal type detector.When being combined with high performance liquid chromatography (HPLC), base
In the principle of atomization detector, eluent forms particles of solute after the effect of nitrogen is atomized, after evaporation tube is dry with
Charging nitrogen gas collision, so that analyte particles surface is positively charged, finally by the electricity on electrometer measurement analyte particles surface
Lotus amount eventually becomes the foundation of determining material concentration so that chromatographic peak area is related to the surface electrically charged amount of institute.CAD detector
Sensitivity with higher can be detected the compound of ng rank, and be combined with liquid chromatographic separation system, take into account reproducibility
With stability, accurately quantitatively or semi-quantitatively analyzed to be that major part is non-volatile with semi-volatile organic matter progress.CAD's
Response is not influenced by compound structure, can detecte the substances without UV absorption such as fructose, the glucose that UV can not be detected.
The utility model has the advantages that detection accuracy of the present invention is high, easy to operate, can be used for compound electrolyte glucose MG3 injection
Potential related substance fructose carry out limit handling.The chromatographic column of non-crosslinked NH2 stationary phase is bonded using full multi-hole blangel particle,
The influence of potassium in compound preparation, sodium, calcium salt plasma can be excluded, and under conditions of without gradient elution, is realized
Principal component (glucose) and related substance (fructose) in preparation efficiently separate.HPLC combination EFI fog detector (CAD), can
Effectively fructose of the detection without UV absorption.
Detailed description of the invention
Fig. 1 is the specificity map of 1 blank solvent of embodiment；
Fig. 2 is the specificity map of 1 reference substance solution of embodiment；
Fig. 3 is the specificity map of 1 test solution of embodiment；
Fig. 4 is the specificity map of 2 blank solvent of embodiment；
Fig. 5 is the specificity map of 2 reference substance solution of embodiment；
Fig. 6 is the specificity map of 2 test solution of embodiment；
Fig. 7 is the durability map of embodiment 31.2ml/min flow velocity；
Fig. 8 is the durability map of embodiment 31.8ml/min flow velocity；
Fig. 9 is the durability map of 35 DEG C of 3 column temperature of embodiment；
Figure 10 is the durability map of 45 DEG C of 3 column temperature of embodiment；
Figure 11 is 3 acetonitrile of embodiment: the durability map of water (85:15).
Specific embodiment
Embodiment 1
1, instrument and reagent
3000 high performance liquid chromatograph of Thermo-Ultimate (subsidiary EFI fog detector), chromatographic column is
ShodexAsahipak NH2P-50 4E。
(lot number: 201711070, Shi Yao groups are holy for fructose reference substance (lot number: 100231-201606, Zhong Jian institute) glucose
Avenge glucose Co., Ltd)；
Sodium chloride (lot number: 20170229, the diligent medicine company in Jiangsu Province)；
Potassium chloride (lot number: 20170713-1, the diligent medicine company in Jiangsu Province)；
Sodium lactate (lot number: 180102, Fu Yue Industrial Co., Ltd.s)；
Compound electrolyte glucose MG3 injection (lot number: 8J74F11, Otsuka Pharmaceutical (China) Co., Ltd.)；
Acetonitrile (lot number: 191994, Thermo Fisher).
2, method
The preparation of 2.1 reference substance solutions
Precision weighs fructose reference substance 20mg, sets in 10ml measuring bottle, adds ultrapure water to make to dissolve and be diluted to scale, shakes up,
Up to the reference substance stock solution of 2mg/ml；Precision pipettes reference substance stock solution 0.5ml, sets in 5ml measuring bottle, and ultrapure water dilution is fixed
Hold the reference substance solution to get 200 μ g/ml；
The preparation of 2.2 test solutions
Precision measures compound electrolyte glucose MG3 injection 2ml, sets in 10ml measuring bottle, ultrapure water is added to be diluted to scale,
It shakes up to get test solution；
2.3 linear preparations
Precision pipettes reference substance stock solution 0.1ml, 0.25ml, 0.4ml, 0.6ml, 0.75ml in 5ml measuring bottle respectively, adds
Ultrapure water dilutes and is settled to scale, shake up to get.Linear solvent shown in table 1 is made:
1 fructose content list of table
2.4 chromatographic condition
Flow velocity: 1.6mL/min, 45 DEG C of column temperature, 10 μ L of sample volume, mobile phase is acetonitrile: water (80:20), detector temperature:
50℃。
3. result
3.1 system suitability
Appropriate fructose, glucose control product mixed solution are taken, by machine testing on chromatographic condition under " 2.4 " item, reference substance is mixed
It closes fructose chromatographic peak tailing factor in solution and is not more than 2.0, fructose and glucose separating degree are greater than 2.0.
3.2 specificity
The test solution prepared under appropriate ultrapure water, reference substance solution, " 2.2 " item is taken, by chromatographic condition under " 2.4 " item
Upper machine testing, the result is shown in Figure 1-3, analysis shows remaining ingredient is noiseless to the detection of fructose in blank solvent, test sample.
3.3 filter membrane adsorption tests
Take the reference substance solution prepared under appropriate " 2.1 " item, through 0.22 μm of miillpore filter abandon respectively primary filtrate 0ml,
0.25ml, 0.5ml take subsequent filtrate as solution to be measured；The reference substance solution about 1.5mL prepared under " 2.1 " item is separately taken,
10000rpm is centrifuged 5min, takes supernatant as contrast solution.By machine testing on chromatographic condition under " 2.4 " item, testing result (see
Table 2) show sample introduction after abandoning primary filtrate, the peak area rate of recovery is within the scope of 98%-102%, it was demonstrated that the filter membrane is to it without suction
Attached, rear proposed adoption abandons primary filtrate 0.5ml sample introduction.Furthermore to fructose without suction-operated, cross 0.22 μm of filter membrane does not influence to contain the filter membrane
It is fixed to measure.
2 filter membrane adsorption test result list of table
| Group | Peak area mAU*min | The rate of recovery (%) |
| Filter membrane absorption control | 4.543 | 100.00 |
| Filter membrane adsorbs 0ml | 4.483 | 98.68 |
| Filter membrane adsorbs 0.25ml | 4.507 | 99.21 |
| Filter membrane adsorbs 0.5mi | 4.549 | 100.13 |
3.4 linear relationship
Precision measures the 10 μ l of linear solvent under " 2.3 " item, by machine testing on chromatographic condition under " 2.4 " item, records peak
Area using peak area as ordinate, draws standard curve, carries out linear regression using reference substance concentration as abscissa.
Fructose is good with peak area linear relationship within the scope of 40~300 μ g/ml, regression equation Y=0.0313x+
0.9197 (r=0.9991)；
3.5 accuracy test
Glucose feed medicine 1g, sodium lactate 22.4mg, sodium chloride 17.5mg, potassium chloride 15.0mg are weighed according to recipe quantity,
Into 10ml measuring bottle, with ultrapure water dissolution and constant volume, it is prepared into the preparation stock solution that concentration of glucose is 100mg/ml.Take 1ml
Preparation stock solution, 0.5ml fructose reference substance stock solution (2mg/ml), set in 5ml measuring bottle, and ultrapure water is diluted to scale, shakes up, i.e.,
.It is parallel to prepare 6 parts, every part of 2 needle of parallel sample introduction measurement.It the results are shown in Table 3, fructose in compound electrolyte glucose MG3 injection
Average recovery rate be 90.74%, RSD 4%.Meet Chinese Pharmacopoeia 2015 editions regulations, this law has certain accuracy.
3 experimental result list of table
3.6 precision test
Precision takes same reference substance solution, continuous sample introduction 6 times, measures peak area.Calculating fructose RSD is 1.0%.As a result table
Bright, this method precision is good.
3.7 detection limited amount limits
It takes reference substance solution to dilute, by machine testing on chromatographic condition under " 2.4 " item, records signal-to-noise ratio, measure determining for fructose
Amount is limited to 40 μ g/ml, and detection is limited to 20 μ g/ml.
Embodiment 2
1, instrument and reagent
3000 high performance liquid chromatograph of Thermo-Ultimate (subsidiary EFI fog detector), chromatographic column Shodex
Asahipak NH2P-504E。
Fructose reference substance (lot number: 100231-201606, Zhong Jian institute)
Glucose (lot number: 201711070, Shi Yao Sheng Xue glucose Co., Ltds, group)；
Sodium chloride (lot number: 20170229, the diligent medicine company in Jiangsu Province)；
Potassium chloride (lot number: 20170713-1, the diligent medicine company in Jiangsu Province)；
Sodium lactate (lot number: 180102, Fu Yue Industrial Co., Ltd.s)；
Calcium chloride (lot number: 03170509, Tianjin sea light medicine company)；
Compound Sodium Lactate and Glucose Injection (lot number: 9D72F1, Otsuka Pharmaceutical (China) Co., Ltd.)
Acetonitrile (lot number: 191994, Thermo Fisher)
2, method
The preparation of 2.1 reference substance solutions
Precision weighs fructose reference substance 20mg, sets in 10ml measuring bottle, adds ultrapure water to make to dissolve and be diluted to scale, shakes up,
Up to the reference substance stock solution of 2mg/ml；Precision pipettes reference substance stock solution 0.5ml, sets in 5ml measuring bottle, and ultrapure water dilution is fixed
Hold the reference substance solution to get 200 μ g/ml
The preparation of 2.2 test solutions
Precision measures Compound Sodium Lactate and Glucose Injection 2ml, sets in 5ml volumetric flask, ultrapure water is added to be diluted to scale, shake
It is even to get test solution；
2.3 chromatographic condition
Flow velocity: 1.6mL/min, 45 DEG C of column temperature, 10 μ L of sample volume, mobile phase is acetonitrile: water (80:20), detector temperature:
50℃。
3. result
3.1 specificity
The test solution prepared under ultrapure water, reference substance solution, " 2.2 " item is taken, by machine on chromatographic condition under " 2.4 " item
Detection, is as a result shown in Fig. 4-6, shows that blank solvent, remaining ingredient is noiseless to the detection of fructose in test sample.
3.2 accuracy test
Glucose feed medicine 1g, sodium lactate 62mg, sodium chloride 120mg, potassium chloride 6mg, calcium chloride are weighed according to recipe quantity
4mg, with ultrapure water dissolution and constant volume, is prepared into the preparation stock solution that concentration of glucose is 100mg/ml into 10ml measuring bottle.It takes
1ml preparation stock solution, 0.5ml fructose reference substance stock solution (2mg/ml), set in 5ml measuring bottle, ultrapure water is diluted to scale, shakes
It is even to get.It is parallel to prepare 6 parts, every part of 2 needle of parallel sample introduction measurement.It the results are shown in Table 4, fruit in Compound Sodium Lactate and Glucose Injection
The average recovery rate of sugar is 92.22%, RSD 7%.Meet Chinese Pharmacopoeia 2015 editions regulations, this law has certain accuracy.
4 accuracy test result list of table
Embodiment 3
Durability detection: fructose, glucose control product mixed solution are taken, is adjusted according to chromatographic condition under " 2.4 " item, color
Spectral condition is respectively flow velocity 1.2ml/min, 1.8ml/min；35 DEG C of column temperature, 45 DEG C；Mobile phase: acetonitrile: machine examination on water (85:15)
It surveys, as a result sees Fig. 7-11, within the scope of the chromatographic condition of adjustment, fructose chromatographic peak tailing factor is little in reference substance mixed solution
In 2.0, fructose and glucose separating degree are greater than 2.0, are applicable to detect fructose.
4, conclusion
For 4.1 glucose as main formulation ingredients in compound glucose injection, quality control is extremely crucial.U.S.'s medicine
Allusion quotation (the 40th edition), European Pharmacopoeia (9.0 editions) are all made of high performance liquid chromatography-differential pulse polarograpll method (HPLC-RI) to glucose
Related substance (fructose etc.) in bulk pharmaceutical chemicals has carried out limit handling.By the strong degradation experiment of compound glucose injection it is found that
Fructose is the important potential related substance of glucose.But due to complicated component in compound preparation, especially various salt ions pair
The analysis of the carbohydrates such as glucose, fructose generates interference, therefore the mass analysis method of bulk pharmaceutical chemicals is not suitable for compound glucose system
The analysis of agent.It is developed based on this present invention a kind of using NH2 column combination high performance liquid chromatography-EFI fog detector (HPLC-
CAD detection method) realizes limit that is accurate, convenient, steadily measuring fructose in compound electrolyte glucose injection,
It lays the foundation to promote the quality standard of product.
4.2, since fructose is without the functional group with UV absorption, can not pass through common HPLC- UV detection
Method (HPLC-UV) is detected.Therefore the present invention selects EFI fog detector to replace conventional UV detector.Electron spray detection
Device (CAD) belongs to novel universal type detector.With high sensitivity, reproducible, signal response is consistent, dynamic monitoring range is wide
Etc. advantages, accepted extensively by pharmacy corporation.Electron spray detection technique is ultraviolet and mass detector strong supplement, can be practical
Applied to any non-volatile or semi-volatile compounds detections.
4.3 use the chromatographic column that full multi-hole blangel particle is bonded non-crosslinked NH2 stationary phase, and the chromatographic column is to glucide
With preferable separating effect, analysis detection can be carried out to various saccharides simultaneously, be widely used in carbohydrate in food and detect.Due to
The detectable any ingredient being eluted out from chromatographic column of EFI fog detector (CAD), influences greatly baseline, and selects herein poly-
It closes object nh 2 column Shodex Asahipak NH2P-504E filler to be lost seldom, therefore is compared to silica gel nh 2 column, baseline is more
Add stabilization.In addition, passing through analysis of experiments it is found that the chromatographic column can effectively exclude the interference of salt ion in compound preparation, realize
The efficiently separating of ingredient to be measured, convenient for the analysis detection to fructose concentration in preparation.
Claims (9)
1. the measuring method of fructose limit in a kind of compound electrolyte glucose injection, characterized by the following steps:
(1) it prepares reference substance solution: weighing fructose standard items and add ultrapure water dissolution, dilution, obtain reference substance stock solution；Pipette control
Product stock solution adds ultrapure water dilution, constant volume, obtains reference substance solution；
(2) it prepares test solution: measuring compound electrolyte glucose injection and ultrapure water is added to dilute, shake up to get test sample
Solution, the concentration of fructose is no more than percent of concentration of glucose in test solution in reference substance solution in the step (1)
Two；
(3) reference substance solution, test solution chromatographic peak area are detected and read by high performance liquid chromatograph, in conjunction with reference substance
Solution concentration is calculated using external standard method and obtains fructose concentration.
2. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 1
In: the parameter setting that high performance liquid chromatograph detects in the step (3) is as follows:
Chromatographic column: full multi-hole blangel particle is bonded non-crosslinked NH2 stationary phase；Flow velocity: 1.2-1.8mL/min；Column temperature: 35-45 DEG C；
Sample volume: 10 μ L；Mobile phase: acetonitrile-water；Detector: EFI fog detector；Detector temperature: 50 DEG C.
3. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 2
In: the parameter setting that high performance liquid chromatograph detects in the step (3) is as follows:
Chromatographic column: Shodex Asahipak NH2P-50 4E；Flow velocity: 1.6mL/min；Column temperature: 45 DEG C；Sample volume: 10 μ L；Stream
Dynamic phase: acetonitrile-water；Detector: EFI fog detector；Detector temperature: 50 DEG C.
4. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 2
In: high performance liquid chromatograph is attached to EFI fog detector in the step (3).
5. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 2
In: the volume ratio of the mobile phase acetonitrile-water is 70: 30-90: 10.
6. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 5
In: the volume ratio of the mobile phase acetonitrile-water is 80: 20.
7. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 5
In: the volume ratio of the mobile phase acetonitrile-water is 85: 15.
8. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 1
In: pass through 0.22 μm of membrane filtration before the test solution detection.
9. the measuring method of fructose limit, feature exist in compound electrolyte glucose injection according to claim 1
In: external standard method calculation formula is as follows in the step (3):
In formula, Ci: fructose concentration (mg/ml) in test solution；Cs: reference substance solution concentration (mg/ml)；
Ai: test solution chromatographic peak area (pA*min)；As: reference substance solution chromatographic peak area (pA*min).
W (%): fructose content in preparation.
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Application publication date: 20191203 |