CN108226333A - Glucose degradation products detects and controls method in a kind of Multiple electrolytes injection - Google Patents
Glucose degradation products detects and controls method in a kind of Multiple electrolytes injection Download PDFInfo
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Abstract
Glucose degradation products detects and controls method in a kind of children's Multiple electrolytes injection of present invention offer, various other materials of the glucose degradation products including 5 hydroxymethylfurfurals (5 hydroxymethylfurfurale, 5 HMF), ensure the controllability of children's drug quality, so that it is guaranteed that safe medication.
Description
Technical field
The invention belongs to Pharmaceutical Analysis and production field, and in particular to a kind of electrolyte injection of children containing glucose
Middle glucose degradation products detect and control method.
Background technology
This product refer to it is a kind of at present in children's Multiple electrolytes injection of China list marketing, according to
According to its disclosed composition, pH range sets are in the range of 5.0~7.0, according to the property and industrial production of glucose
The experience of parenteral solution containing glucose, it is unstable under the conditions of higher pH or relatively low pH, particularly it is more than 5.0 in pH
Under conditions of, degradation reaction can easily occur for glucose, generate a series of catabolite, so as to decline content, so logical
The pH containing glucose injection of normal each pharmaceutical producing enterprise is controlled between 3.5~5.0.And the degradation of these glucose
Product inherently has different degrees of toxic side effect, and can further derive and form the polymer with bigger toxicity, right
The quality control of drug influences very huge.In current existing glucose degradation products determination techniques method, generally using ultraviolet
The content of spectrophotometry or high performance liquid chromatography (HPLC methods) detection 5-HMF.
Glucose degradation in secnidazole and parenteral solution is controlled to generate 5-HMF, color simultaneously for example, fourth sturdy pines employs HPLC
Spectral condition uses octadecylsilane chemically bonded silica as filler；Methanol monophosphate buffer solution (potassium dihydrogen phosphate is soluble in water,
Add people's sodium pentanesulfonate, after dissolving, with phosphoric acid tune pH value) for mobile phase, flow velocity 1.0mL.min-1；Detection wavelength:284nm.
(HPLC checks the related substance in plug gram nitre glucose injection, fourth sturdy pines etc.,《Chinese Pharmaceutical Journal》, July in 2004
The phase of volume 39 the 7th, 553-554).
Chinese patent CN106153737A discloses glucose degradation object in a kind of detection buprenorphin hydrochloride parenteral solution
Method, for detecting other glucose degradation products in addition to 5 hydroxymethyl furfural.It uses octadecylsilane chemically bonded silica
For chromatographic column carrier, mobile phase is matched by a certain percentage with organic phase and buffer salt solution, is eluted according to gradient condition, detect
Wavelength is 280-290nm, flow velocity 0.4-1.5ml/min.Grape is calculated to be not added with the principal component Self-control method of correction factor
The content of sugared degradation product.Chromatogram shows that in addition to two kinds of substances of 5 hydroxymethyl furfural and buprenorphine, also there are unknown degradations
The peak of object.
Invention content
Can be generated after glucose degradation a series of catabolite (Glucose Degradation Products,
GDPs), these GDPs include such as formaldehyde (formaldehyde), acetaldehyde (acetaldehyde), furfural (2-
Furaldehyde), 3- deoxyfructoses (3-deoxyglucosone 3-DG), 4- deoxyfructoses (4-
Deoxyglucosone 4-DG), glyoxal (glyoxal), pyroracemic aldehyde (methyl-glyoxal methylglyoxal), 5- methylols
Furfural (5-hydroxymethylfurfurale 5-HMF), glucosone (glucosone), 3,4- dihydroxy glucose
Ketone -3- alkene (3,4-dideoxyglucosone-3-ene 3,4-DGE), 3- deoxy-galactoses (3-deoxygalactosone
3-DGal), valeral, levulic acid etc..On a cellular level, aldehydes can be with interference cell signal transduction, to cell adipose membrane, cell
Albumen, mitochondrial function and RNA and DNA cause largely to destroy.In the PD liquid of heat sterilization, 3,4-DGE have maximum
Biological respinse activity.This toxicity molecule is found that wound healing can be damaged, and human leucocyte and renal epithelial cell can be caused to wither
It dies.The GDPs of high activity such as 3,4-DGE can rapidly be reacted from different molecules, and 3-DG, 5-HMF can then stay in cycle
In system.
Glucose degradation products are the precursors of glycosyl endization product, after GDPs is reached in blood, can be carried out with haemocyanin
With reference to this can cause glycosyl endization to react (AGE), and glycosyl compound can be involved in response to oxidative stress and then and cardiovascular morbidity
Related and injury of kidney.Glycosyl compound can also cause destruction and the inflammation of cell：It destroys neutrophil leucocyte, reduce cell factor point
It secretes, inhibit to microorganism phagocytosis.
In view of the process of glucose degradation is complicated, people are one progressive to discovery, the understanding of glucose degradation products
Up to the present process can only find and detect Partial digestion product, the toxicity, the mechanism of action to these catabolites
Understand, study at present also in progress.In the existing glucose injection listed, drug quality mark both domestic and external
Standard only controls 5 hydroxymethyl furfural to wherein catabolite, and in the catabolite of glucose, 5 hydroxymethyl furfural is neither
It is that bioactivity i.e. toxicity is maximum, nor being influenced in intermediate of degrading and glycosylation being accelerated to form glycosylation end products
Main matter, simultaneously as it is intermediate, during the production and storage of glucose preparation, 5 hydroxymethyl furfural
Secondary degradation can occur, until generation levulic acid, smaller organic acid and other organic impurities such as formic acid, therefore it is simple with
Its index as reaction glucose stability is also inappropriate.
Under normal conditions, because the degree of glucose degradation is than relatively low, to other drops in the parenteral solution containing glucose
The harm that may bring of solution product can be ignored, but in view of the special pH ranges of children's electrolyte injection sample and glucose
Stability feature, palliating degradation degree will be significantly higher than general product containing glucose infusion liquid, and this product is mainly used for infant and youngster
It is virgin.
Therefore glucose degradation products and glucose content therein are paid close attention to, ensures that its safety just has and significantly face
Bed meaning.
The present invention establishes a variety of glucose degradation products in a kind of above-mentioned electrolyte of method detection, with grape in preparation
The variation of sugared content of degradation products is investigated, evaluation preparation process, and is chosen most representative catabolite and formulated and enter product
On the one hand quality standard effectively can control and evaluate the palliating degradation degree of product, it is often more important that provide for Clinical practice and more may be used
The quality assurance leaned on.
The present invention proposes a kind of following technical solution to solve the above-mentioned problems in the prior art：
Glucose degradation products detect and control method in a kind of Multiple electrolytes injection,
Wherein glucose degradation products include but not limited to formaldehyde (formaldehyde), acetaldehyde (acetaldehyde),
Furfural (2-furaldehyde), 3- deoxyfructoses (3-deoxyglucosone or 2-keto-3-deoxyglucose
3-DG), 4- deoxyfructoses (4-deoxyglucosone 4-DG), glyoxal (glyoxal), pyroracemic aldehyde (methyl second two
Aldehyde methylglyoxal), 5 hydroxymethyl furfural (5-hydroxymethylfurfurale 5-HMF), glucosone
(glucosone), 3,4- dihydroxy grape saccharon -3- alkene (3,4-dideoxyglucosone-3-ene 3,4-DGE), 3- take off
Oxygen galactolipin (3-deoxygalactosone 3-DGal), valeral, 2-2'- hydroxyacetyls-furans (2- (2 '-
Hydroxyacetyl)-furan), 2,5- dicarboxylic acids (2,5-dicarboxylic acid), levulic acid, one in formic acid
Kind is several；
The detection method of catabolite uses high performance liquid chromatography (HPLC methods), and chromatographic condition includes：
A. using wavelength for 190~400nm ultraviolet wavelengths detection, the performance liquid chromatographic column using silane group silica gel as
Filler, the silane group silica gel are selected from C2~C18Alkyl silane bonded silica gel, cyanoalkysilane bonded silica gel and amino
One kind of silane group silica gel；
B. the mobile phase used in the high performance liquid chromatography is had by water, inorganic salt solution or aqueous solutions of organic salts and chromatography
Machine pure solvent or solution containing ion pair form according to a certain percentage, and chromatography organic solvent is alcohols or nitrile organic solvent；
A concentration of 0.01%~10%g/L of above-mentioned ion pair, containing inorganic or 0.01~1mol/L of organic salt；Above-mentioned is inorganic
Salt or organic salt are selected from phosphate or hydrophosphate, carbonate, the citrate of potassium or sodium.
C. the mobile phase is the mobile phase of gradient proportioning or isocratic mobile phase；The column temperature of the chromatographic process is
20~50 DEG C；
D. sample size：20~50 μ L.
Further, the Multiple electrolytes injection is consisting of 1000ml sodium chloride-containing 6.429g, potassium chloride
0.298g, calcium chloride (CaCl2·2H2O) 0.147g, magnesium chloride (MgCl2·6H2O) 0.203g, sodium acetate (CH3COONa·
3H2O) 4.082g and glucose (C6H12O6·H2O)10.0g。
Further, in the catabolite, α therein-dicarbonyl structure catabolite includes glyoxal
(glyoxal), pyroracemic aldehyde (methyl-glyoxal methylglyoxal), glucosone (glucosone), 3- deoxyglucoses
Aldehyde ketone (3-deoxyglucosone 3-DG), 4- deoxyfructoses (4-deoxyglucosone 4-DG), 3,4- dihydroxies
Base grape saccharon -3- alkene (3,4-dideoxyglucosone-3-ene 3,4-DGE), 3- deoxy-galactoses (3-
Deoxygalactosone3-DGal), need to be detected after derivatization reagent derives using HPLC chromatogram method.
Further, the derivatization reagent include o-phenylenediamine, 2,3- diaminonaphthalenes, 2,3- diaminonaphthalenes it is similar
Object, 1,2- diamino -4,5- dimethoxy-benzene (1,2-diamino-4,5-dimethoxybenzene dihydrochoride
DDB), 1,2- diamino -4,5- methylenedioxy benzenes (1,2-diamino-4,5-methylenedioxybenzene
Dihydrochoride DMB), o-phenylenediamine, 2,4 dinitrophenyl hydrazine, alkyl hydrazine.
Further, in the catabolite, wherein, α-it is preferable to use 2,3- for dicarbonyl structure catabolite derivative reagent
Diaminonaphthalene, 1,2- diamino -4,5- dimethoxys-benzene or o-phenylenediamine are as derivative reagent；Formaldehyde, acetaldehyde use 2,4- bis-
Nitrophenyl hydrazine derives as derivative reagent；Then it is measured using HPLC chromatogram method.
Further, specific derivatization conditions are：Electrolyte is configured to：0.1g/ml~0.001g/
ml；Derivatization reaction temperature：- 10 DEG C~100 DEG C；Derivative reagent is a concentration of：0.01%~1%；The derivative time is：1~10h；Spread out
The reactant doses ratio of raw reagent and glucose in parenteral solution is：1:0.00001~0.00001:1；Derivative pH ranging from 2.5
~7.5, derivatization reaction is carried out in the dark.
Further, the 5 hydroxymethyl furfural in the catabolite, furfural can be measured directly using HPLC chromatogram method.
In a specific embodiment, the detection and control of glucose degradation products in the Multiple electrolytes injection
Method processed, the specific steps are：
1) analyte derivative：Take derivatization reagent and electrolyte injection sample, vortex mixing, room temperature avoid light place 1h；
2) derivatization sample is detected using HPLC methods, specific chromatographic condition is：It is bonded with octadecylsilane
Silica gel is filler；Elution is to carry out gradient elution, Detection wavelength 320nm with the mobile phase that water and acetonitrile form, and flow velocity is
1ml/min, 30 DEG C of column temperature, 20 μ l of sample size；Specific elution program is as shown in the table：
| Min | Acetonitrile | Water |
| 0 | 18 | 82 |
| 4 | 30 | 70 |
| 10 | 25 | 75 |
| 15 | 18 | 82 |
Further, the actual conditions of the analyte derivative are：By 0.6ml 0.4% (weight) o-phenylenediamine aqueous solution
With 1ml electrolyte injection sample vortex mixings, tune pH value to 7.0, room temperature avoid light place 1h.
The technique effect that the present invention obtains has：
Glucose degradation products detect and control method in electrolyte injection provided by the invention, can accurately, side
Just other catabolites including 5-HMF are detected, ensure the controllability of children's pharmaceutical properties, so that it is guaranteed that safety is used
Medicine.
Description of the drawings
Fig. 1 is the chromatogram of 1 reference substance solution of embodiment；
Fig. 2 is the chromatogram of 1 test solution of embodiment；
Fig. 3 is the chromatogram of 3 reference substance solution of embodiment；
Fig. 4 is the chromatogram of 3 reference substance solution of embodiment；
Fig. 5 is the chromatogram of 5 reference substance solution of embodiment；
Fig. 6 is the chromatogram of 5 test solution of embodiment.
Specific embodiment
For preferably the principle of the present invention and scheme, to various substances related to the technical solution of the present invention and preparation process
Middle various parameters are made to be explained in detailed below.More specific technical solution, reference can be made to embodiment scheme.
<Multiple electrolytes injection>
The country has listed Multiple electrolytes injection and few, the currently preferred compound for child infusion at present
Electrolyte balance solution is based on the basis of the external children's electrolyte balance solution developed, and the one kind for copying research and development is novel
Children's compound electrolyte infusion kind.
This product kind belongs to electrolyte injection, and compositing formula is as follows：
1000ml sodium chloride-containing 6.429g, potassium chloride 0.298g, calcium chloride (CaCl2·2H2O) 0.147g, magnesium chloride
(MgCl2·6H2O) 0.203g, sodium acetate (CH3COONa·3H2O) 4.082g and glucose (C6H12O6·H2O)10.0g。
According to the property of glucose and the experience of industrial production glucose injection, in higher pH or relatively low pH items
Unstable under part, particularly under conditions of pH is more than 5.0, degradation reaction can easily occur for glucose, generate a series of
Catabolite, so as to decline content, so the pH of usually glucose injection is controlled between 3.5~5.0.
<Glucose degradation products>
A series of catabolite (Glucose can be generated in technical solution of the present invention, after glucose degradation
Degradation Products, GDPs), these GDPs include such as formaldehyde (formaldehyde), acetaldehyde
(acetaldehyde), furfural (2-furaldehyde), 3- deoxyfructoses (3-deoxyglucosone 3-DG), 4-
Deoxyfructose (4-deoxyglucosone 4-DG), glyoxal (glyoxal), pyroracemic aldehyde (methyl-glyoxal
Methylglyoxal), 5 hydroxymethyl furfural (5-hydroxymethylfurfurale 5-HMF), glucosone
(glucosone), 3,4- dihydroxy grape saccharon -3- alkene (3,4-dideoxyglucosone-3-ene 3,4-DGE), 3- take off
Oxygen galactolipin (3-deoxygalactosone3-DGal), valeral, levulic acid etc..On a cellular level, aldehydes can interfere
Cellular signal transduction causes largely to destroy to cell adipose membrane, cell protein, mitochondrial function and RNA and DNA.It is heating
In the PD liquid of sterilizing, 3,4-DGE have maximum biological respinse activity.This toxicity molecule is found that wound healing can be damaged, can
Lead to human leucocyte and renal epithelial cell apoptosis.The GDPs of high activity such as 3,4-DGE can be carried out rapidly from different molecules
Reaction, and 3-DG, 5-HMF can be then stayed in the circulatory system.
Main glucose degradation products and its structural formula, such as 1 depicted of table.
Table 1
Glucose degradation products are the precursors of glycosyl endization product, after GDPs is reached in blood, can be carried out with haemocyanin
With reference to this can cause glycosyl endization to react (AGE), and glycosyl compound can be involved in response to oxidative stress and then and cardiovascular morbidity
Related and injury of kidney.Glycosyl compound can also cause destruction and the inflammation of cell：It destroys neutrophil leucocyte, reduce cell factor point
It secretes, inhibit phagocytosis to microorganism.
In view of the process of glucose degradation is complicated, people are one progressive to discovery, the understanding of glucose degradation products
Up to the present process can only find and detect Partial digestion product, the toxicity, the mechanism of action to these catabolites
Understand, study at present also in progress.It is existing listed containing in glucose injection, drug quality both domestic and external
Standard only controls 5 hydroxymethyl furfural mostly to wherein catabolite, and in the catabolite of glucose, 5- methylol chaffs
Aldehyde neither bioactivity, that is, toxicity is maximum, nor influence and accelerate to glycosylate to form advanced glycosylation endproduct in intermediate of degrade
The main matter of product, simultaneously as it is intermediate, during the production and storage of glucose preparation, 5- methylols
Secondary degradation can also occur for furfural, until smaller organic acid and other organic impurities such as generation levulic acid, formic acid, therefore it is single
It is pure using its index as reaction glucose stability be also inappropriate.
Below with reference to the composition of embodiment the present invention will be described in detail technical solution, but it is not limited to following embodiment party
Formula.
<Embodiment>
The HPLC of 1 glucose degradation products of embodiment -3-DG, glyoxal and pyroracemic aldehyde is measured
1) prepared by reference substance solution：Prepare deoxyfructose containing 3-, glyoxal and a concentration of 20 μ g/ml of pyroracemic aldehyde
Solution, precision measure 1.0ml, are respectively placed in 10ml measuring bottles, add water to scale, shake up spare；Take reference substance solution 1ml (3-
Deoxyfructose, glyoxal and a concentration of 2 μ g/ml of pyroracemic aldehyde), add water 1ml, then add in o-phenylenediamine solution (4mg/
Ml) 1.2ml, mixing adjust pH value to be filtered after room temperature avoid light place 1h to 7.0 to obtain the final product.
2) prepared by test solution：Children to be detected are taken with the electrolyte injection sample 1ml containing glucose, the adjacent benzene of addition
Diamine solution (4mg/ml) 0.6ml, mixing adjust pH value to be filtered after 1h is placed at room temperature for 7.0 to obtain the final product.
3) HPLC is detected：Chromatographic column：Agilent ZORBAX SB-C18,5 μm, 4.6 × 250mm；Elution is with water and second
The mobile phase of nitrile composition carries out gradient elution, Detection wavelength 320nm, flow velocity 1ml/min, 30 DEG C of column temperature, 20 μ l of sample size；
Specific elution program is as shown in the table：
| Min | Acetonitrile | Water |
| 0 | 18 | 82 |
| 4 | 30 | 70 |
| 10 | 25 | 75 |
| 15 | 18 | 82 |
4) measuring method：Reference substance solution and each 20 μ l of test solution are drawn respectively, injects high performance liquid chromatograph, are surveyed
It is fixed, HPLC-UV detection (Fig. 1 and Fig. 2) is obtained, measures the peak area of ingredient to be measured in reference substance solution and test solution, in addition
Mark method calculates the concrete content of 3- deoxyfructoses, glyoxal and pyroracemic aldehyde.
The methodological study of the HPLC detections of 2 3-DG of embodiment, glyoxal and pyroracemic aldehyde
1) detection limit and quantitative limit
According to the method for embodiment 1, the signal-to-noise ratio for quantitatively diluting each dirt solution to each impurity peaks is about 3, calculates to obtain 3-
Deoxyfructose detectable concentration is 0.2 μ g/ml, 20 μ l of sample size, and detection is limited to 4ng；Glyoxal detectable concentration is 0.2 μ
G/mL, 15 μ l of sample size, detection are limited to 3ng；Pyroracemic aldehyde detectable concentration is 0.2 μ g/mL, 20 μ l of sample size, and detection is limited to 4ng.
2) linear and range
According to the method for embodiment 1, deoxyfructose containing 3-, glyoxal and a concentration of 20 μ g/ml of pyroracemic aldehyde are prepared
Solution, precision measure 0.25,0.5,1.0,2.0,4.0ml, be respectively placed in 10ml measuring bottles, add water to scale, shake up；Separately take
The solution 1ml of a concentration of 2 μ g/ml, is placed in 10ml measuring bottles, adds water to scale, shake up derivatization under linear test item.It takes above-mentioned
Solution sample introduction, 20 μ l of sampling volume record chromatogram.With impurity concentration (μ g/ml) for X-axis, absorption peak is y-axis, is made linear
It returns, it is as a result as follows：
1 3- deoxyfructose standard curve result of the tests of table
2 glyoxal standard curve result of the test of table
3 pyroracemic aldehyde standard curve result of the test of table
Conclusion：3- deoxyfructoses concentration linear relationship in the range of 0-100 μ g/ml is good.Glyoxal and acetone
Aldehyde concentration linear relationship in the range of 0-20 μ g/ml is good.
3) recovery test
3.1 method
(1) supplementary material storing solution：Weigh sodium chloride about 12.858g, potassium chloride about 596mg, calcium chloride (CaCl2·2H2O)
About 294mg, magnesium chloride (MgCl2·6H2O) about 406mg, sodium acetate (CH3COONa·3H2O) about 816.4mg DEXTROSE ANHYDROUSs are about
20g until in 100ml measuring bottles, adds appropriate amount of water to make dissolving, adds water to scale, shake up to get；
(2) 3 concentration test solutions：Respectively it is accurate measure impurity storing solution (3- deoxyfructoses, glyoxal and
20 μ g/ml of pyroracemic aldehyde) 1.0,2.0,4.0ml put in 3 10ml measuring bottles, add water to scale, shake up.Above-mentioned 3 concentration is taken respectively
Solution 1ml, add in supplementary material storing solution 1ml, add in o-phenylenediamine solution (4mg/ml) 1.2ml, mixing, in being placed at room temperature for 1h
After filter, as low, medium and high concentration test solution.Each concentration prepares 3 parts of solution with method.Separately take reference substance solution (3-
2 μ g/ml of deoxyfructose, glyoxal and pyroracemic aldehyde) 1ml, add water 1ml, operated with method, as reference substance solution.
3.2 measuring method：Above-mentioned solution sample introduction is taken, 20 μ l of sampling volume record chromatogram, as a result see figure.By linear test
Standard curve calculates sodium acetate content, and calculates the rate of recovery.
3.3 results and conclusion
Determination of recovery rates the results are shown in Table.
4 3- deoxyfructose recovery test results of table
5 glyoxal recovery test result of table
6 pyroracemic aldehyde recovery test result of table
Conclusion：This law measures 3- deoxyfructoses, glyoxal and the pyroracemic aldehyde rate of recovery and meets the requirements, accuracy compared with
It is high.
4) precision test
It is 4.1 repeated
Precision measures impurity storing solution (20 μ g/ml of 3- deoxyfructoses, glyoxal and pyroracemic aldehyde) 2.0ml and puts 10ml
In measuring bottle, scale is added water to, is shaken up.1ml is taken, adds in supplementary material storing solution 1ml, adds in o-phenylenediamine solution (4mg/ml)
1.2ml, mixing filter after 1h is placed at room temperature for, and 6 parts of solution are prepared as repetitive test test solution with method.It is another to take pair
According to product solution (2 μ g/ml of 3- deoxyfructoses, glyoxal and pyroracemic aldehyde) 1ml, add water 1ml, operated with method, as control
Product solution.
Repeated measurement result is shown in Table.
The repeated testing result of table 7
Conclusion：It is good that this law measures 3- deoxyfructoses, glyoxal and pyroracemic aldehyde repeatability.
5) derivatising condition is investigated
Reference substance original solution prepared by Example 1, is handled according to different derivatising conditions, passes through HPLC respectively
The detection for detecting each standard items is limited (referring to item 1), and actual conditions are：
| O-phenylenediamine concentration | PH value of reaction system | |
| 1 | 2mg/ml | 8.0 |
| 2 | 4mg/ml | 7.0 |
| 3 | 6mg/ml | 6.5 |
Concrete outcome is as follows：
| Detection limit | 3- deoxyfructoses | Glyoxal | Pyroracemic aldehyde |
| 1 | 24ng | 24ng | 12ng |
| 2 | 4ng | 3ng | 4ng |
| 3 | 12ng | 12ng | 16ng |
3 glucose degradation products of embodiment -5 hydroxymethyl furfural, furfural, levulic acid measure
1) prepared by reference substance solution：Precision is prepared containing 5 hydroxymethyl furfural and each 2 μ g/ml of furfural, 200 μ g/ of levulic acid
The solution of ml.
2) test solution：Children to be detected are with the electrolyte injection sample 1ml containing glucose.
3) HPLC is detected：
Mobile phase：0.015mol/L sodium dihydrogen phosphates (with phosphoric acid tune pH2.60)：Acetonitrile (90:10 volume ratios)
Detection wavelength：283nm
Flow velocity：0.8ml/min
Column temperature：30℃
Sample size：20μl
4) measuring method：Reference substance solution and each 20 μ l of test solution are drawn respectively, injects high performance liquid chromatograph, are surveyed
It is fixed, HPLC-UV detection (Fig. 3 and Fig. 4) is obtained, measures the peak area of ingredient to be measured in reference substance solution and test solution, in addition
Mark method calculates the concrete content of 5 hydroxymethyl furfural, furfural, levulic acid.
The methodological study that 45 hydroxymethyl furfural of embodiment, furfural, levulic acid HPLC are detected
1) detection limit and quantitative limit
According to the method for embodiment 3, the signal-to-noise ratio for quantitatively diluting each dirt solution to each impurity peaks is about 3, calculates to obtain 5-
Hydroxymethylfurfural detectable concentration is 0.02 μ g/ml, 25 μ l of sample size, and detection is limited to 0.5ng；Furfural detectable concentration is 0.02 μ g/
ML, 25 μ l of sample size, detection are limited to 0.5ng；Levulic acid detectable concentration is 20 μ g/mL, 20 μ l of sample size, and detection is limited to 0.4 μ
g。
2) linear and range
Impurity reference substance solution (containing 5 hydroxymethyl furfural and each 20 μ g/ml of furfural, levulic acid 2mg/ml) is prepared, it is accurate
Measurement 0.1,0.2,0.5,1.0,2.0,4.0,5.0ml, are respectively placed in 10ml measuring bottles, add water to scale, shake up, as linear
Measure serial solution.Above-mentioned solution sample introduction is taken, 20 μ l of sampling volume record chromatogram, as a result see figure.With impurity concentration (μ g/
Ml it is) X-axis, absorption peak is y-axis, makees linear regression, as a result as follows：
Table 85- hydroxymethylfurfural standard curve result of the tests
9 furfural standard curve result of the test of table
10 levulic acid standard curve result of the test of table
Conclusion：5 hydroxymethyl furfural and furfural content linear relationship in the range of 0.2-20 μ g/ml are good.Levulic acid is dense
Degree linear relationship in the range of 20-2000 μ g/ml is good.
3) recovery test
3.1 method
(1) supplementary material storing solution：Weigh sodium chloride about 12.858g, potassium chloride about 596mg, calcium chloride (CaCl22H2O)
About 294mg, magnesium chloride (MgCl26H2O) about 406mg, sodium acetate (C2H3NaO53H2O) about 816.4mg DEXTROSE ANHYDROUSs
About 20g until in 100ml measuring bottles, adds appropriate amount of water to make dissolving, adds water to scale, shake up to get；
(2) 3 concentration test solutions：Precision measures impurity storing solution (the 2 μ g/ml containing 5 hydroxymethyl furfural and furfural, second
200 μ g/ml of acyl propionic acid) 5.0ml put in 10ml measuring bottles, and supplementary material storing solution is added to shake up to scale, prepare 3 parts of solution with method, make
For low concentration test solution；Precision measures impurity storing solution (the 20 μ g/ml containing 5 hydroxymethyl furfural and furfural, levulic acid
2000 μ g/ml) 10.0ml put in 50ml measuring bottles, and precision measures 5.0ml and puts in 10ml measuring bottles, adds supplementary material storing solution to scale,
It shakes up, 3 parts of solution is prepared with method, as middle concentration test solution；Precision measure impurity storing solution (containing 5 hydroxymethyl furfural and
20 μ g/ml of furfural, 2000 μ g/ml of levulic acid) 10.0ml put in 25ml measuring bottles, and precision measures 5.0ml and puts in 10ml measuring bottles, adds
Supplementary material storing solution shakes up to scale, 3 parts of solution is prepared with method, as high concentration test solution；Separately take the chaff of methylol containing 5-
2 μ g/ml of aldehyde and furfural, the solution of 200 μ g/ml of levulic acid is as reference substance solution.
3.2 measuring method：Above-mentioned solution sample introduction is taken, 20 μ l of sampling volume record chromatogram, as a result see figure.
3.3 results and conclusion
Determination of recovery rates the results are shown in Table.
Table 115- hydroxymethylfurfural recovery test results
12 furfural recovery test result of table
13 levulic acid recovery test result of table
Conclusion：This law measures 5 hydroxymethyl furfural, furfural, the levulic acid rate of recovery and meets the requirements, and accuracy is higher.
4) it is repeated
Precision measures impurity storing solution (the 20 μ g/ml containing 5 hydroxymethyl furfural and furfural, 2000 μ g/ml of levulic acid)
10.0ml is put in 50ml measuring bottles, and precision measures 5.0ml and puts in 10ml measuring bottles, and supplementary material storing solution is added to shake up, match with method to scale
6 parts of solution processed, as repetitive test test solution；Separately take containing 2 μ g/ml of 5 hydroxymethyl furfural and furfural, levulic acid 200
The solution of μ g/ml is as reference substance solution.Above-mentioned solution sample introduction is taken, 20 μ l of sampling volume record chromatogram, as a result see figure.
Repeated measurement result is shown in Table.
The repeated testing result of table 14
Conclusion：It is good that this law measures 5 hydroxymethyl furfural, furfural, levulic acid repeatability.
5 glucose degradation products of embodiment-formaldehyde, Iso-propyl alcohol
Derivative reagent：0.3%2,4- dinitrophenylhydrazine acetonitrile solutions：2,4 dinitrophenyl hydrazine 0.15g is claimed to put 50ml browns
In measuring bottle, appropriate acetonitrile is added to make dissolving, adds in 1mol/LHCl 0.5ml, add acetonitrile to scale, shake up to get.
Deriving method：Sample 5ml is taken, adds 0.3%2,4- dinitrophenylhydrazine acetonitrile solution 1ml, acetonitrile 4ml is added in, shakes up,
Sealing, 60 DEG C are protected from light 1h.
Chromatographic condition：Chromatographic column：Agilent ZORBAX SB-C18, 5 μm, 4.6 × 250mm
Mobile phase and retention time：
Detection wavelength：360nm
Flow velocity：1.0ml/min
Column temperature：40℃
Sample size：20μl
Specific step of the determination step as described in specific embodiment is operated, and obtains the chromatogram such as Figures 5 and 6.
The study on the stability of 6 children's electrolyte injection of embodiment
Correlated samples stability is investigated according to the stability guideline of 2010 editions two annex of Chinese Pharmacopoeia, is investigated respectively
25 DEG C place 12 months, 45 DEG C and relative humidity 75% under the conditions of place the medicine stability of 6 months, concrete outcome is such as
Under：
The above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be to the present invention
Embodiment restriction.For those of ordinary skill in the art, it can also make on the basis of the above description
Other various forms of variations or variation.There is no necessity and possibility to exhaust all the enbodiments.It is all the present invention
All any modification, equivalent and improvement made within spirit and principle etc., should be included in the protection of the claims in the present invention
Within the scope of.
Claims (9)
1. glucose degradation products detect and control method in a kind of Multiple electrolytes injection, it is characterized in that：
Wherein glucose degradation products include but not limited to formaldehyde (formaldehyde), acetaldehyde (acetaldehyde), furfural
(2-furaldehyde), 3- deoxyfructoses (3-deoxyglucosone or 2-keto-3-deoxyglucose 3-
DG), 4- deoxyfructoses (4-deoxyglucosone 4-DG), glyoxal (glyoxal), pyroracemic aldehyde (methyl-glyoxal
Methylglyoxal), 5 hydroxymethyl furfural (5-hydroxymethylfurfurale 5-HMF), glucosone
(glucosone), 3,4- dihydroxy grape saccharon -3- alkene (3,4-dideoxyglucosone-3-ene 3,4-DGE), 3- take off
Oxygen galactolipin (3-deoxygalactosone 3-DGal), valeral, 2-2'- hydroxyacetyls-furans (2- (2 '-
Hydroxyacetyl)-furan), 2,5- dicarboxylic acids (2,5-dicarboxylic acid), levulic acid, one in formic acid
Kind is several；
The detection method of catabolite uses high performance liquid chromatography (HPLC methods), and chromatographic condition includes：
A. it is detected using wavelength for 190~400nm ultraviolet wavelengths, the performance liquid chromatographic column is using silane group silica gel as filling
Agent, the silane group silica gel are selected from C2~C18Alkyl silane bonded silica gel, cyanoalkysilane bonded silica gel and amino silane
One kind of bonded silica gel；
B. the mobile phase used in the high performance liquid chromatography is organic pure by water, inorganic salt solution or aqueous solutions of organic salts and chromatography
Solvent or solution containing ion pair form according to a certain percentage, and chromatography organic solvent is alcohols or nitrile organic solvent；
A concentration of 0.01%~10%g/L of above-mentioned ion pair, containing inorganic or 0.01~1mol/L of organic salt；Above-mentioned inorganic salts or
Organic salt is selected from phosphate or hydrophosphate, carbonate, the citrate of potassium or sodium.
C. the mobile phase is the mobile phase of gradient proportioning or isocratic mobile phase；The column temperature of the chromatographic process for 20~
50℃；
D. sample size：10~100 μ L.
2. glucose degradation products detects and controls method, spy in Multiple electrolytes injection as described in claim 1
Sign is, the Multiple electrolytes injection is consisting of 1000ml sodium chloride-containing 6.429g, potassium chloride 0.298g, calcium chloride
(CaCl2·2H2O) 0.147g, magnesium chloride (MgCl2·6H2O) 0.203g, sodium acetate (CH3COONa·3H2O) 4.082g and Portugal
Grape sugar (C6H12O6·H2O)10.0g。
3. glucose degradation products detect and control method in Multiple electrolytes injection as claimed in claim 1 or 2,
It is characterized in that, in the catabolite, the catabolite of α-dicarbonyl structure therein includes glyoxal (glyoxal), acetone
Aldehyde (methyl-glyoxal methylglyoxal), glucosone (glucosone), 3- deoxyfructoses (3-
Deoxyglucosone 3-DG), 4- deoxyfructoses (4-deoxyglucosone 4-DG), 3,4- dihydroxy glucose
Ketone -3- alkene (3,4-dideoxyglucosone-3-ene 3,4-DGE), 3- deoxy-galactoses (3-deoxygalactosone
3-DGal), it need to be detected after derivatization reagent derives using HPLC chromatogram method.
4. glucose degradation products detects and controls method, spy in Multiple electrolytes injection as claimed in claim 3
Sign is, the derivatization reagent includes o-phenylenediamine, 2,3- diaminonaphthalenes, 2,3- diaminonaphthalenes analog, 1,2- bis-
Amino -4,5- dimethoxys-benzene (1,2-diamino-4,5-dimethoxybenzene dihydrochoride DDB), 1,
2- diamino -4,5- methylenedioxy benzenes (1,2-diamino-4,5-methylenedioxybenzene
Dihydrochoride DMB), 2,4 dinitrophenyl hydrazine, alkyl hydrazine.
5. glucose degradation products detects and controls method, spy in Multiple electrolytes injection as claimed in claim 4
Sign is, in the catabolite, wherein, the catabolite derivative reagent of α-dicarbonyl structure is it is preferable to use o-phenylenediamine, and 2,
3- diaminonaphthalenes, 1,2- diamino -4,5- dimethoxys-benzene are as derivative reagent；Formaldehyde, acetaldehyde use 2,4- dinitrobenzenes
Hydrazine, alkyl hydrazine are measured after deriving using HPLC chromatogram method.
6. such as the detection and control of glucose degradation products in a kind of Multiple electrolytes injection of claim 3-5 any one of them
Method processed, which is characterized in that specifically derivatization conditions are：Electrolyte is configured to：0.1g/ml~0.001g/
ml；Derivatization reaction temperature：- 10 DEG C~100 DEG C；Derivative reagent is a concentration of：0.01%~1%；The derivative time is：1~10h；Spread out
The reactant doses ratio of raw reagent and glucose in parenteral solution is：1:0.00001~0.00001:1；Derivative pH ranging from 2.5
~7.5, derivatization reaction is carried out in the dark.
7. glucose degradation products detect and control method in Multiple electrolytes injection as claimed in claim 1 or 2,
It is characterized in that, the 5 hydroxymethyl furfural, furfural in the catabolite can be measured directly using HPLC chromatogram method.
8. glucose degradation products detect and control method in the Multiple electrolytes injection as described in claim 1-7,
It is characterized in that, the specific steps are：
1) analyte derivative：Take derivatization reagent and electrolyte injection sample, vortex mixing, room temperature avoid light place 1h；
2) derivatization sample is detected using HPLC methods, specific chromatographic condition is：With octadecylsilane chemically bonded silica
For filler；Elution is to carry out gradient elution, Detection wavelength 320nm, flow velocity 1ml/ with the mobile phase that water and acetonitrile form
Min, 30 DEG C of column temperature, 20 μ l of sample size；Specific elution program is as shown in the table：
9. glucose degradation products detects and controls method, spy in Multiple electrolytes injection as claimed in claim 8
Sign is that the actual conditions of the analyte derivative are：By (volume ratio) the o-phenylenediamine aqueous solutions of 0.6ml 0.4% and 1ml electricity
Solve matter injection liquid samples vortex mixing, tune pH value to 7.0, room temperature avoid light place 1h.
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| CN112268972A (en) * | 2020-10-21 | 2021-01-26 | 青岛力腾医药科技有限公司 | Method for detecting glucose degradation product in compound electrolyte injection |
| CN113686995A (en) * | 2021-09-14 | 2021-11-23 | 西安乐析医疗科技有限公司 | Method for measuring 3-DG and 3,4-DGE in peritoneal dialysis solution or preparation containing glucose |
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| CN115453025A (en) * | 2022-09-30 | 2022-12-09 | 成都施贝康生物医药科技有限公司 | Method for detecting related substances of chloral hydrate |
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