CN116953131A - Glyceraldehyde detection method and application thereof - Google Patents
Glyceraldehyde detection method and application thereof Download PDFInfo
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- CN116953131A CN116953131A CN202210399247.XA CN202210399247A CN116953131A CN 116953131 A CN116953131 A CN 116953131A CN 202210399247 A CN202210399247 A CN 202210399247A CN 116953131 A CN116953131 A CN 116953131A
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- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 title claims abstract description 84
- 238000001514 detection method Methods 0.000 title abstract description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 94
- 238000000034 method Methods 0.000 claims abstract description 41
- 238000001212 derivatisation Methods 0.000 claims abstract description 40
- 239000005715 Fructose Substances 0.000 claims abstract description 32
- 229930091371 Fructose Natural products 0.000 claims abstract description 32
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 32
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000013558 reference substance Substances 0.000 claims abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 11
- 239000012488 sample solution Substances 0.000 claims abstract description 11
- 230000015556 catabolic process Effects 0.000 claims abstract description 7
- 238000006731 degradation reaction Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 62
- 239000000523 sample Substances 0.000 claims description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 239000011550 stock solution Substances 0.000 claims description 27
- 238000012360 testing method Methods 0.000 claims description 20
- 239000003814 drug Substances 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 239000007924 injection Substances 0.000 claims description 16
- 238000002347 injection Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 229940079593 drug Drugs 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 239000008354 sodium chloride injection Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000013074 reference sample Substances 0.000 claims description 4
- 239000007810 chemical reaction solvent Substances 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- 238000007781 pre-processing Methods 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 238000000825 ultraviolet detection Methods 0.000 claims description 2
- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 239000000377 silicon dioxide Substances 0.000 claims 1
- 238000010129 solution processing Methods 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 9
- 238000010521 absorption reaction Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000012795 verification Methods 0.000 abstract description 3
- 230000014759 maintenance of location Effects 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 26
- 235000011187 glycerol Nutrition 0.000 description 24
- 238000001816 cooling Methods 0.000 description 11
- 239000002904 solvent Substances 0.000 description 10
- 238000007865 diluting Methods 0.000 description 9
- 238000001914 filtration Methods 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 7
- 238000005303 weighing Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000012494 forced degradation Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 4
- 238000011835 investigation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 150000002373 hemiacetals Chemical class 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine group Chemical group NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- 150000002429 hydrazines Chemical class 0.000 description 1
- 150000007857 hydrazones Chemical group 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- -1 saccharide compounds Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a detection method of glyceraldehyde, which is a degradation impurity of fructose and glycerol. The invention establishes a simple and universal derivatization HPLV-UV method for measuring glyceraldehyde generated by degradation of glycerol or fructose based on the characteristic that the glyceraldehyde has ultraviolet absorption after being reacted with a proper derivatization reagent. According to the detection method, 2, 4-dinitrophenylhydrazine or the same type of derivatization reagent is used for derivatizing glyceraldehyde in the sample solution and the reference substance solution to form a product which is absorbed in an ultraviolet light region, and compared with glyceraldehyde, the polarity of the product is reduced, the retention of the product on a chromatographic column is enhanced, and the quantitative analysis of the glyceraldehyde is realized. Proved by methodological verification, the method has good specificity and sensitivity.
Description
Technical Field
The invention belongs to the field of medicine analysis and detection, in particular relates to a method for measuring glyceraldehyde by derivatization, and particularly relates to a method for detecting glyceraldehyde in injection containing glycerol and/or fructose and medicines in other dosage forms by a derivatization HPLC-UV method.
Background
Glycerol is widely used as an auxiliary material in medicines; in addition, glycerol and fructose belong to saccharide compounds, so that the safety of human bodies is high, and clinically, high-concentration solution is used as a dehydration medicine, such as glycerol fructose sodium chloride injection. According to the production conditions of domestic injection and the related legal requirements, high-temperature sterilization is mostly adopted in the production process of injection. The inventor finds that glycerin and fructose can be degraded to generate glyceraldehyde under the conditions of high temperature and the like, so that related medicines possibly contain glyceraldehyde impurities.
Impurities in a drug refer to all other chemicals than the target compound introduced or generated during the production, storage or use of the drug. Impurities in the medicament directly affect the curative effect of the medicament and may cause other adverse toxic and side effects, and the impurities must be controlled.
In addition, glyceraldehyde has a terminal aldehyde group in its structure, and cannot form a cyclic structure of a hemiacetal, which belongs to a mutation-causing warning structure, meaning that glyceraldehyde may be genotoxic. Based on the information, it is necessary to establish a glyceraldehyde detection method with strong specificity and high sensitivity.
High Performance Liquid Chromatography (HPLC) is one of the most commonly used instruments in drug analysis, while Ultraviolet (UV) is the most commonly used detector in high performance liquid chromatography. Glyceraldehyde has a simple structure and lacks a chromophoric group, so that ultraviolet absorption is weak; and the polarity is large, and the retention on the reversed phase chromatographic column is weak, so that the detection is difficult by adopting a conventional HPLC-UV instrument. In the medicine, the detection matrix is more complex due to the fact that the medicine contains more components such as main components, main component degradation impurities, auxiliary materials and the like, and the glyceraldehyde detection difficulty is increased. In medicines containing glycerol and/or fructose, no detection method of glyceraldehyde is reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discloses a glyceraldehyde detection method which can be used for detecting glyceraldehyde in medicines containing glycerol and/or fructose, such as glycerol injection, fructose injection, glycerol fructose sodium chloride injection and the like.
The detection method of the invention uses an external standard method to quantify, and adopts glyceraldehyde reference substance to detect glyceraldehyde in the test sample. The method mainly comprises derivatization pretreatment and high performance liquid chromatograph detection, and qualitative or quantitative detection of glyceraldehyde is realized through detection of a derivatization product.
The specific technical scheme of the invention is as follows:
the derivatization pretreatment method comprises the following steps:
(1) Preparing glyceraldehyde reference substance aqueous solution, and preprocessing the solution to obtain reference substance stock solution; preparing a test sample aqueous solution serving as a test sample stock solution;
(2) Respectively transferring a proper amount of sample stock solution and a proper amount of reference sample stock solution, placing the sample stock solution and the reference sample stock solution into respective reaction systems, adding a derivatization reagent, adding a pH regulator and a reaction solvent, and reacting for 1-3 hours at 40-70 ℃ to obtain a sample solution and a reference solution.
Through the above reaction, glyceraldehyde forms a hydrazone structure corresponding to the derivatizing agent:
in the step (1), the pretreatment of the reference stock solution is that the prepared glyceraldehyde aqueous solution is placed in an environment of 50-100 ℃ for 0.5-5 hours to ensure that glyceraldehyde is fully dissolved in water, so that the subsequent derivatization reaction can be completely carried out. The environment temperature can be maintained by dry heating or wet heating, preferably wet heating, and preferably water bath heating. Further, the treatment temperature of the glyceraldehyde solution is preferably 60 to 70 ℃.
And (2) the derivatization reagent adopts hydrazine compounds containing strong ultraviolet absorption groups such as benzene rings, naphthalene rings and the like, the reaction system adopts acetonitrile-water solution, and a proper pH regulator is added.
Further, the derivatizing agent is preferably 2, 4-dinitrophenylhydrazine, wherein the concentration of the derivatizing agent is 2 to 10mg/ml.
Further, an acidic pH regulator is added to make the pH environment of the derivatization reaction acidic, preferably hydrochloric acid solution, further preferably 1mol/L hydrochloric acid solution, and 30-300. Mu.l of the solution is added.
Further, the temperature of the derivatization reaction is selected to be 40-70 ℃, and preferably 50-60 ℃; the reaction time is 0.5 to 5 hours, preferably 1 hour.
The HPLC-UV detection method belongs to reversed phase chromatography, and the used high performance liquid chromatograph is required to be provided with an on-line vacuum degasser, a double pump or quaternary pump, an automatic sampler, a column temperature box, an ultraviolet detector or a diode array detector. The chromatographic column takes octadecylsilane chemically bonded silica gel as a filler; phosphate buffer solution or water is used as a mobile phase A, acetonitrile or acetonitrile-water is used as a mobile phase B, and the elution mode is isocratic or gradient elution; the column temperature is 25-40 ℃; the flow rate is 1.0-2.0 ml/min, and the sample injection amount is 5-30 μl.
Further, the detection wavelength is selected from 300 to 400nm, preferably 360 to 380nm, and more preferably 365nm.
Further, a GL Science InertSustain C column was selected for the column, water and acetonitrile were selected for the mobile phase, the column temperature was 30 ℃, the flow rate was 1.5ml/min, and elution was performed using the following gradient procedure:
TABLE 1 elution gradient table
And calculating the glyceraldehyde content in the test sample by adopting an external standard method. Glyceraldehyde has 1 chiral center and its configuration is divided into D-type and L-type. In the method, glyceraldehyde of two configurations shows peaks after empirical biochemical treatment, and the content of glyceraldehyde in a test sample is calculated according to the sum of peak areas of two derivatives.
The beneficial effects of the invention are that
The invention establishes a simple and universal derivatization HPLC-UV method for measuring the content of glyceraldehyde based on that a derivatization reagent containing chromophore and hydrazine group in the structure can generate a derivatization product with ultraviolet absorption after reacting with aldehyde group in glyceraldehyde.
The result of methodology verification shows that glycerol and fructose and other degradation impurities of both can not interfere with analysis, and the method has good specificity. In addition, the detection sensitivity of the method is high, and the quantitative limit can reach 0.1534 mug/ml; the linear relation is good (r > 0.995); RSD for reproducibility and intermediate precision were 0.44% and 1.3%, respectively; average recovery is between 96.9-100.9% (RSD < 2.0%), no significant matrix interference; and the derivative product has good stability within 24 hours. The method can be used for detecting glyceraldehyde impurities in the medicines containing glycerol and/or fructose, thereby further ensuring the product quality of the related medicines and the medication safety of patients.
Drawings
In example 1 of FIG. 1, the glycerol was subjected to forced degradation to derivatize the chromatogram of the solution.
FIG. 2 is a chromatogram of a derivatizing solution after forced degradation of fructose in example 1.
FIG. 3 shows the chromatogram of derivatization solutions of derivatization reagents, glyceraldehyde, glycerol, and fructose under the chromatographic conditions described in the present invention.
FIG. 4 is a graph of the ultraviolet absorption spectrum of glyceraldehyde-derived products under chromatographic conditions according to the invention.
FIG. 5 is a graph of glyceraldehyde concentration versus peak area.
Detailed description of the preferred embodiments
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific examples and with reference to the data. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art.
1.1 instruments
The instrumentation used in the present method is shown in the following table:
table 2 instrument information
| Name of the name | Manufacturer (S) | Model number |
| Electronic analytical balance (0.01 mg) | Sidoris (Sidoris) | BT125D |
| Electronic analytical balance (0.1 mg) | Sidoris (Sidoris) | BSA124S |
| Electronic analytical balance (0.001 mg) | Sidoris (Sidoris) | MSE3.6P-0CE-DM |
| Electronic analytical balance (0.01 mg) | Sidoris (Sidoris) | BT25S |
| High performance liquid chromatograph | Shimadzu (Shimadzu) | LC-2030C 3D, DAD detector, quaternary pump |
| High performance liquid chromatograph | Shimadzu (Shimadzu) | LC-2030C 3D, UV detector, quaternary pump |
| High performance liquid chromatograph | Agilent | Agilent 1260UV detector, quaternary pump |
| Medicine strong light irradiation test box | Chongqing immortal | SHH-300GD-2 |
| Vertical pressure steam sterilizer | Shanghai Boxun (Shanghai Boxun) | YXQ-LS-50SⅡ |
| Digital display constant temperature water bath kettle | Changzhou national flower | HH-4 |
| Ultrapure water instrument | Sichuan Unpu | UPR-Ⅱ-5T |
| Ultrasonic cleaner | Kunshan Hechuang | KH-250B |
1.2 reagents
The reagents used in the present invention are shown in the following table:
TABLE 3 reagent information table
1.3 preparation of solutions
(1) Solvent: weighing 60ml of acetonitrile, adding into 40ml of water, and shaking uniformly to obtain the finished product.
(2) 1mol/L hydrochloric acid: taking 90ml of hydrochloric acid, adding a proper amount of water to form 1000ml, and shaking uniformly to obtain the product.
(3) Derivatizing agent: about 250mg of 2, 4-dinitrophenylhydrazine is taken, precisely weighed, placed in a 50ml measuring flask, added with acetonitrile and ultrasonically dissolved and diluted to a scale, and uniformly shaken to obtain the product (about 5 mg/ml).
(4) Glyceraldehyde stock solution: taking glyceraldehyde reference substance about 10mg, precisely weighing, placing into a 100ml measuring flask, diluting with water to scale, shaking, placing into 50deg.C water bath for 1 hr, taking out, and cooling to obtain (about 0.1 mg/ml).
1.4 derivatization procedure
Precisely measuring 2ml of a sample to be measured, placing the sample into a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, diluting to a scale by adding a solvent, shaking uniformly, immediately placing the sample into a water bath at 50 ℃ for reaction for 60 minutes, cooling the sample in an ice bath, taking out the sample, and filtering the sample to obtain a sample solution.
Precisely measuring 2ml of glyceraldehyde stock solution, placing into a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, diluting to scale with solvent, shaking, immediately placing into a water bath at 50deg.C for reaction for 60 min, cooling in ice bath, and filtering to obtain reference solution.
Accurately taking 2ml of water, placing in a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, diluting to scale with solvent, shaking, immediately placing in a water bath at 50deg.C for reacting for 60 min, cooling in ice bath, and filtering to obtain blank solution.
1.5 chromatographic conditions
Chromatographic column: octadecyl bonded silica gel as filler (GL Sciences Inc Inert Sustain C column, 4.6X105 mm,5 μm or equivalent);
mobile phase a: water;
mobile phase B: acetonitrile;
flow rate: 1.5ml/min;
column temperature: 30 ℃;
detection wavelength: 365nm;
sample injection amount: 10 μl;
elution was performed according to the following table gradient:
TABLE 4 elution gradient Table
EXAMPLE 1 degradation of Glycerol and fructose to give glyceraldehyde
And preparing glycerol stock solution and fructose stock solution from glycerol and fructose respectively, and performing forced degradation experiments, wherein the forced degradation experiments comprise strong acid damage, strong alkaline damage, high temperature damage, oxidation damage and illumination damage, and the specific conditions are shown in the table below.
TABLE 5 forced degradation method
And (3) respectively derivatizing the destroyed solutions, and detecting the glyceraldehyde content in each solution by an external standard method, wherein the experimental results are shown in the following table.
TABLE 6 degradation of Glycerol and fructose to give glyceraldehyde
The results in the table above show that the glyceraldehyde content of glycerol is significantly increased under oxidizing conditions; fructose generates more glyceraldehyde under high temperature and alkali damage conditions, and in addition, fructose generates little glyceraldehyde under strong acid conditions.
EXAMPLE 2 Effect of pretreatment of glyceraldehyde stock solution on derivatization effect
Glyceraldehyde stock solutions were prepared separately, treated and derivatised under different conditions and the results are shown in the following table:
TABLE 7 derivatization Condition investigation 1
Note that: the derivatization level is the ratio of the sum of the peak areas of the solution to the sum of the peak areas of the glyceraldehyde solution with the same concentration after complete derivatization.
As is evident from the results of the above table, the glyceraldehyde stock solution was derivatized for 5 hours at room temperature without special treatment, and the degree of derivatization was only 43%, and the reaction was substantially complete at 50℃for 5 hours. And the derivatization efficiency can be greatly increased even if the glyceraldehyde stock solution is left to stand under the low-temperature condition after being treated for a long time.
Based on the above results, the effect of the standing time at 50℃on the level of derivatization was examined with the same glyceraldehyde solution, and the results are shown in the following table. The glyceraldehyde stock solution is placed for 0.5 to 5 hours at 50 ℃ so as to ensure the completion of the subsequent derivatization reaction.
TABLE 8 derivatization Condition investigation 2
Example 3 selection of derivatization temperature and time
The glyceraldehyde solution and the sample solution are respectively derivatized for 1 hour at different temperatures, and the result shows that the derivatization level of the glyceraldehyde solution and the sample solution reaches more than 90% within 1 hour at the temperature of 40-70 ℃. The results of derivatization reaction of glyceraldehyde solution at 50 ℃ for 1-5 hours are not obviously different.
TABLE 9 derivatization Condition investigation 3
Note that: the sample solutions in the above table refer to sample solutions prepared from glycerol fructose sodium chloride injection.
Example 4 methodological verification and application
4.1 specificity
Accurately taking 2ml of water, placing in a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, diluting to scale with solvent, shaking, immediately placing in a water bath at 50deg.C for reaction for 60 min, cooling in ice bath, filtering, and analyzing by sample injection.
Taking about 10mg of glyceraldehyde reference substance, precisely weighing, placing into a 100ml measuring flask, adding water to dilute to a scale, shaking uniformly, placing into a water bath at 50 ℃ for 1 hour, taking out, cooling, precisely weighing 2ml, placing into a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 mu L of 1mol/L hydrochloric acid solution, adding solvent to dilute to the scale, shaking uniformly, immediately placing into a water bath at 50 ℃ to react for 60 minutes, cooling in an ice bath, filtering, and carrying out sample injection analysis.
And respectively adding water into proper amounts of glycerol and fructose to prepare a 100mg/ml glycerol solution and a 50mg/ml fructose solution, respectively precisely weighing 2ml, placing into a 20ml measuring flask, adding 2ml of a 2, 4-dinitrophenylhydrazine solution and 50 mu L of a 1mol/L hydrochloric acid solution, adding a solvent to dilute to scale, shaking uniformly, immediately placing into a water bath at 50 ℃ to react for 60 minutes, cooling in an ice bath, taking out, filtering, and carrying out sample injection analysis.
The chromatogram is shown in figure 3; the ultraviolet absorption chromatogram of glyceraldehyde derivative is shown in figure 4. In the chromatogram, glyceraldehyde is derivatized to generate double peaks, which are completely separated from derivatization reagent peaks, glycerol and other chromatographic peaks introduced by fructose, and the glycerol, the fructose and other impurities do not interfere with measurement of glyceraldehyde, so that the specificity of the method is good.
4.2 linearity
Preparing glyceraldehyde stock solution with concentration of 0.2mg/ml, respectively transferring proper amount of the stock solution, diluting with water to obtain glyceraldehyde series solutions of 0.01, 0.025, 0.05, 0.08, 0.10, 0.12 and 0.15mg/ml, performing derivatization according to a law, and respectively injecting samples. And (3) taking glyceraldehyde concentration as an abscissa and the sum of peak areas of the derivative products as an ordinate, and carrying out a linear regression equation on the sum (A) of peak areas of the derivative products by using glyceraldehyde solution concentration C (mug/ml) according to a least square method. As a result, glyceraldehyde was found to have a good linear relationship in the range of 0.01 to 0.15mg/ml, and the correlation coefficient r was more than 0.995. The results are shown in FIG. 5.
4.3 sensitivity
Stepwise diluting glyceraldehyde stock solution, performing derivatization according to a law and sample injection analysis, determining peak height of glyceraldehyde derivative chromatographic peak and baseline noise near the peak, and calculating detection limit according to signal-to-noise ratio (S/N) not lower than 3:1; calculating a quantitative limit according to the signal to noise ratio (S/N) not lower than 10:1, continuously sampling the solution with the quantitative limit concentration for 6 times, and continuously sampling the solution with the detection limit concentration for 3 times.
The results showed that the detection limit of glyceraldehyde was 0.0377. Mu.g/ml and the quantitative limit was 0.2622. Mu.g/ml.
4.4 precision test
Preparing a reference substance solution, a test substance solution and a test substance standard adding solution respectively, calculating the content of glyceraldehyde in the test substance standard adding solution according to an external standard method, examining the recovery rate of the glyceraldehyde added in the test substance standard adding solution, and calculating the relative standard deviation of each measurement result of 6. Two persons detect by using different chromatographs on different days respectively.
The recovery rate of 6 parts of personnel A is 99.3-100.9%, and the RSD is 0.6%; the recovery rate of 6 parts of personnel B is 100.5-103.8%, and the RSD is 1.2%; the RSD of 12 recovery of both persons was 1.3%. The method has good precision.
4.5 accuracy
Glyceraldehyde solutions of 0.025 mg/ml, 0.05 mg/ml, 0.1mg/ml and 0.12mg/ml are prepared respectively, the glyceraldehyde solutions are added into a test sample for derivatization to prepare test sample standard adding solutions with different concentration levels, the background quantity in the test sample solutions measured in parallel is combined, the sample adding recovery rate of glyceraldehyde is inspected, and each concentration is inspected to be 3 parts in parallel.
Recovery = (measured amount in test sample addition-background amount in test sample solution)/addition amount.
The experimental results show that: the recovery rate of glyceraldehyde is between 96.9% and 100.9%, the average value of the recovery rate of 12 parts of solution is 99.3%, and the RSD is 1.6%, so that the method has good accuracy.
4.6 solution stability
And (3) taking glyceraldehyde stock solution, preparing a reference substance solution by derivatization according to a method, placing the reference substance solution in an automatic sampler for different times, and repeatedly sampling, and examining the stability of the reference substance solution according to the change condition of the area of a derivatization peak.
As a result, the peak area of the glyceraldehyde derivative was changed to a degree of <.+ -. 0.5% at room temperature within 24 hours, the RSD of the peak area was 0.15%, and the glyceraldehyde derivative was excellent in stability.
Example 5
Accurately taking 2ml of water, placing in a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, diluting to scale with solvent, shaking, immediately placing in a water bath at 50deg.C for reacting for 60 min, cooling in ice bath, and filtering to obtain blank solution.
Preparing 0.1mg/ml glyceraldehyde stock solution according to a method, precisely weighing 2ml, placing into a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, adding a solvent to dilute to a scale, shaking uniformly, immediately placing into a water bath at 50 ℃ for reacting for 60 minutes, placing into an ice bath for cooling, filtering, and taking the mixture as a reference substance solution for sample injection analysis according to the method.
Precisely measuring 2ml of a sample to be measured, placing the sample into a 20ml measuring flask, adding 2ml of 2, 4-dinitrophenylhydrazine solution and 50 μl of 1mol/L hydrochloric acid solution, diluting to a scale by adding a solvent, shaking uniformly, immediately placing the sample into a water bath at 50 ℃ for reaction for 60 minutes, cooling the sample in an ice bath, taking out the sample, filtering the sample, and taking the sample as a sample solution according to a law.
The glyceraldehyde content was calculated as peak area according to the external standard method. 1 batch of samples of glycerin fructose sodium chloride injection of two manufacturers and two specifications are taken for measurement.
TABLE 10 detection results of glyceraldehyde in 4 batches of sodium glycerfructosyl chloride injection by the method of the invention
| Sample of | Manufacturer 1-250ml specification | Manufacturer 1-500ml specification | Manufacturer 2-250ml specification | Manufacturer 2-500ml specification |
| Detection result | 34.50μg/ml | 34.05μg/ml | 17.09μg/ml | 18.82μg/ml |
Claims (10)
1. A method for determining glyceraldehyde produced by degradation of glycerol or fructose by a derivatizing HPLC-UV method, comprising the steps of:
(1) Preparing glyceraldehyde water solution, and preprocessing the solution to obtain a reference stock solution; preparing a test sample aqueous solution serving as a test sample stock solution;
(2) Respectively transferring a proper amount of sample stock solution and a proper amount of reference sample stock solution, placing the sample stock solution and the reference sample stock solution into respective reaction systems, adding a derivatization reagent, adding a pH regulator and a reaction solvent, and reacting for 1-3 hours at 40-70 ℃ to obtain a sample solution and a reference solution;
(3) Detecting related derivatization products of the reference substance solution and the test substance solution by adopting an HPLC-UV method, and calculating by adopting an external standard method.
2. The method according to claim 1, wherein the test sample is a liquid pharmaceutical product containing glycerol and/or fructose.
3. The method according to claim 2, characterized in that the liquid drug containing glycerol and/or fructose is glycerol injection, fructose injection, glycerol fructose injection or glycerol fructose sodium chloride injection.
4. The method according to claim 1, wherein the preprocessing is: the glyceraldehyde water solution is placed for 0.5 to 5 hours at the temperature of 50 to 100 ℃.
5. The method according to claim 1, characterized in that the derivatizing agent is a compound containing a stronger chromophore and a hydrazino group.
6. The method of claim 5 wherein the derivatizing agent is 2, 4-dinitrophenylhydrazine.
7. The method according to claim 5, wherein the step (2) is carried out at 50 to 60℃for 1 hour using acetonitrile-water as a reaction solvent.
8. The method according to claim 1, wherein in the step (3), the HPLC-UV method uses octadecylsilane chemically bonded silica as a filler; phosphate buffer solution or water is used as a mobile phase A, acetonitrile or acetonitrile-water is used as a mobile phase B, and the elution mode is isocratic or gradient elution; the column temperature is 25-40 ℃; the flow rate is 1.0-2.0 ml/min.
9. The method of claim 8, wherein the HPLC-UV method has an ultraviolet detection wavelength of 300-400 nm.
10. The method according to claim 8, characterized in that the gradient elution method is as follows:
0-20 min, the volume percentage of the mobile phase A is reduced from 87% to 70%;
20-21 min, the volume percentage of the mobile phase A is reduced from 70% to 20%;
21-25 min, keeping the volume percentage of the mobile phase A to be 20%;
25-26 min, the volume percentage of the mobile phase A is increased from 20% to 87%;
and (3) keeping the volume percentage of the mobile phase A at 87% for 27-35 min.
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