CN115406979A - High performance liquid chromatography analysis method for residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride - Google Patents
High performance liquid chromatography analysis method for residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride Download PDFInfo
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- CN115406979A CN115406979A CN202210906696.9A CN202210906696A CN115406979A CN 115406979 A CN115406979 A CN 115406979A CN 202210906696 A CN202210906696 A CN 202210906696A CN 115406979 A CN115406979 A CN 115406979A
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 15
- 238000004458 analytical method Methods 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000002386 leaching Methods 0.000 claims abstract description 6
- 230000002441 reversible effect Effects 0.000 claims abstract description 6
- 239000012488 sample solution Substances 0.000 claims description 39
- 239000000523 sample Substances 0.000 claims description 29
- 238000001514 detection method Methods 0.000 claims description 27
- NGJUYARYEXGDNN-UHFFFAOYSA-N 1-[3-(dimethylamino)propyl]-3-ethylurea Chemical compound CCNC(=O)NCCCN(C)C NGJUYARYEXGDNN-UHFFFAOYSA-N 0.000 claims description 20
- 239000000126 substance Substances 0.000 claims description 17
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 15
- 239000012498 ultrapure water Substances 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 230000014759 maintenance of location Effects 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000012086 standard solution Substances 0.000 claims description 8
- 239000011550 stock solution Substances 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- -1 1-ethyl [3- (dimethylamino) -propyl ] group Chemical group 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010829 isocratic elution Methods 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 2
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims 1
- 239000000515 collagen sponge Substances 0.000 abstract description 12
- 239000003431 cross linking reagent Substances 0.000 abstract description 11
- 238000004811 liquid chromatography Methods 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 23
- 239000007788 liquid Substances 0.000 description 11
- 238000011084 recovery Methods 0.000 description 11
- 102000008186 Collagen Human genes 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 6
- 229920001436 collagen Polymers 0.000 description 6
- 239000012535 impurity Substances 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 150000001718 carbodiimides Chemical class 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
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- 238000006731 degradation reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
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- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 102100029469 WD repeat and HMG-box DNA-binding protein 1 Human genes 0.000 description 1
- 101710097421 WD repeat and HMG-box DNA-binding protein 1 Proteins 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000001951 dura mater Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
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- 238000010008 shearing Methods 0.000 description 1
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- 238000013112 stability test Methods 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
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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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
-
- 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
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
The invention discloses a high performance liquid chromatography analysis method of 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride, which is characterized in that a leaching liquor of a sample is subjected to quantitative analysis by a C18 reverse phase chromatographic column, a high performance liquid chromatography is matched with an ultraviolet detector and an external standard percentage method of the reverse liquid chromatography is adopted, so that the EDC residual quantity of a recombinant collagen sponge cross-linking agent is quantitatively detected.
Description
Technical Field
The invention belongs to the technical field of detection and analysis of medical instruments, and particularly relates to a high performance liquid chromatography analysis method of a cross-linking agent 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride (EDC).
Background
The carbodiimide is a compound containing a functional group-N = C = N-, the molecule of the carbodiimide is in a linear structure, and can perform a crosslinking reaction with carboxyl and amino in collagen to improve the performance of a collagen material, and the collagen sponge prepared by the crosslinking agent has a loose porous network structure, good water absorption and degradation resistance. The carbodiimide crosslinking can overcome the defects of low mechanical strength, easy enzyme degradation and the like of the collagen material, and has great potential of being applied to wound repair as a scaffold material. At present, EDC is adopted to react with substances such as polysaccharide, collagen, polypeptide, polymer and the like in a plurality of processes, and the application prospect is huge. In particular, under the rapidly developing conditions of collagen application, EDC is used to modify collagen and derive other products, such as: collagen sponge, artificial skin, dura mater, etc., but EDC may be excessive in the sample preparation process and the residue in medical devices needs to be controlled, so it is necessary to quantitatively determine the residue of crosslinking agent EDC in chemical modification.
At present, a method for detecting residual carbodiimide in polysaccharide protein vaccines is established in literature, and the method uses a liquid chromatography and mass spectrometry (LC-MC) technology to carry out quantitative analysis on carbodiimide urea derivatives. However, the cost of liquid chromatography-mass spectrometry is high, and not all enterprises and researchers can use the liquid chromatography-mass spectrometry generally, so that the application is greatly limited. The invention patent application No. 201510239250.5 discloses a method for detecting EDC residue by using HPLC-ELSD, the detection object is EDC, and a high performance liquid chromatograph and an evaporative light scattering detector are used, but the method mentioned in the patent is not suitable for a sample processed in an aqueous solution, because EDC is converted into EDU in the aqueous solution and is irreversible.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography analysis method of residual cross-linking agent 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride (EDC), which can detect the residual cross-linking agent EDC in a recombinant collagen sponge through a high performance liquid chromatograph ultraviolet detector (HPLC-UV), thereby ensuring the quality of the recombinant collagen sponge.
The high performance liquid chromatography analysis method of the residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride comprises the following steps:
(1) Preparation of sample solution: adding a substance to be detected into ultrapure water, leaching at 40-60 ℃, hydrolyzing residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride into 1- [3- (dimethylamino) propyl ] -3-ethylurea, and filtering with a 0.22 mu m water film to obtain a sample solution; the hydrolysis reaction process is as follows:
wherein R is 1 represents-CH 2 -CH 3 (ethyl), R 2 Represents- (CH) 2 ) 3 -N-(CH 3 ) 2 (dimethylaminopropyl);
(2) Preparing a 1- [3- (dimethylamino) propyl ] -3-ethylurea standard solution;
(3) Detecting the 1- [3- (dimethylamino) propyl ] -3-ethylurea standard substance solution by adopting a high performance liquid chromatography and an ultraviolet detector, and recording the peak area under the same retention time; the chromatographic conditions are as follows: the chromatographic column adopts a C18 reverse phase column, and the mobile phase is a mobile phase A and a mobile phase B in a volume ratio of 99:1 to 80:20, wherein the mobile phase A is an aqueous solution containing 0.1mM 1-sodium heptanesulfonate and 0.04% triethylamine by volume concentration, the pH value of the aqueous solution is adjusted to 3 by phosphoric acid, the mobile phase B is acetonitrile, and the elution mode is isocratic elution; drawing a standard curve according to the detection result of the standard solution by taking the concentration of the 1- [3- (dimethylamino) propyl ] -3-ethylurea as a vertical coordinate and the peak area as a horizontal coordinate to obtain a standard curve equation;
(4) Detecting the sample solution in the step (1) according to the conditions in the step (3), substituting the sample solution into the standard curve equation obtained in the step (3) to calculate the concentration of the 1- [3- (dimethylamino) propyl ] -3-ethylurea in the sample solution according to the formula: EDC residual quantity = EDU concentration x sample solution total volume/173 x 191/substance to be measured x 100%, EDC residual quantity in the substance to be measured is calculated, EDC is 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride, EDU is 1- [3- (dimethylamino) propyl ] -3-ethylurea.
In the step (2), preferably, 10mg of 1- [3- (dimethylamino) propyl ] -3-ethylurea standard substance is weighed, placed in a 100mL volumetric flask, added with ultrapure water to scale marks, and shaken to be fully dissolved to obtain 100 mu g/mL of 1- [3- (dimethylamino) propyl ] -3-ethylurea stock solution; the stock solution of 1- [3- (dimethylamino) propyl ] -3-ethylurea was diluted with ultrapure water to give 3. Mu.g/mL, 6. Mu.g/mL, 12. Mu.g/mL, 24. Mu.g/mL, and 48. Mu.g/mL solutions of 1- [3- (dimethylamino) propyl ] -3-ethylurea as a standard.
In the step (3), the C18 reverse phase column is an octadecylsilane chemically bonded silica column
AQ-C18 WeLch with specification of 4.6mm multiplied by 250mm and filling aperture diameter
The particle size was 5 μm.
In the step (3), the sample injection volume is preferably 10 to 20 μ L, the flow rate is 0.8 to 1.2mL/min, the column temperature is 25 to 40 ℃, and the detection wavelength is 200 to 210nm.
In the step (3), it is further preferable that the mobile phase equilibrium column is used for 120 to 150min before the sample injection of the chromatographic column.
In the step (3), it is further preferable that the mobile phase is a volume ratio of the mobile phase a to the mobile phase B of 97:3 to 95:5, and (b) a mixed solution.
Compared with the prior art, the invention has the following beneficial effects:
(1) The detection time is short, the use of organic phases is reduced, the cost of enterprises is saved, and the pollution to the environment is reduced; in addition, when sample introduction is carried out, a mixed mobile phase is used, so that baseline fluctuation is reduced;
(2) The EDU penetrates through the C18 column without being reserved due to strong polarity, the ultraviolet absorption of the EDU at 200-210 nm is weak, the reservation of the EDU on the C18 column is prolonged through reagents such as 1-sodium heptanesulfonate, triethylamine and phosphoric acid, so that the main peak of the EDU reaches reasonable reservation time, the absorption of the EDU on a chromatographic column is enhanced, the influence of other impurities on the EDU is reduced, the detection limit is more sensitive, and the level of nanogram is reached; the precision is good (the repeatability RSD percent is less than 1.97 percent), the linear relation is good, when the detection limit concentration is 0.44 percent of the predetermined limit value of the EDC impurity of the recombinant collagen sponge cross-linking agent, the signal-to-noise ratio is 3.71, the recovery rate of each concentration is over 94 percent, the recovery rate is good, the accuracy is high, and the durability is good (the RSD percent is less than 2 percent);
(3) Compared with a liquid chromatography-mass spectrometry (LC-MS) and a high performance liquid chromatograph combined evaporation light scattering detector (HPLC-ELSD), the method is cheaper and more universal, so that the enterprise cost is reduced, the operation is simple, the detection limit of the method reaches 40ng/mL by adjusting related reagents in a mobile phase, the EDC is converted into the EDU and has irreversibility, the result of directly detecting the EDU is more reliable and accurate, the stability is better, the high-throughput detection of enterprise production can be met, and the method is also suitable for detection of a laboratory for research and development.
Drawings
FIG. 1 is a standard graph of EDU.
FIG. 2 is a detection limit chromatogram of flow versus EDU of the present invention.
FIG. 3 is a detection limit chromatogram of Normal flow versus EDU.
FIG. 4 is a high performance liquid chromatogram of an EDU stock solution.
FIG. 5 is a high performance liquid chromatogram of a freshly prepared aqueous solution of EDC.
FIG. 6 is a high performance liquid chromatogram of an aqueous EDC solution after standing for 7 days.
FIG. 7 is a high performance liquid chromatogram of the recombinant collagen-like sponge leaching solution.
Detailed Description
The invention will be elucidated below with reference to the drawings and examples, without the scope of protection of the invention being limited to these examples.
Example 1
1. Preparation of sample solution: weighing 100mg of recombinant collagen sponge, shearing the recombinant collagen sponge by using scissors, placing the cut recombinant collagen sponge into a centrifugal tube, adding 2mL of ultrapure water, sealing the centrifugal tube by using a sealing film, and leaching for 72 hours in a 50 ℃ water bath kettle to convert the residual cross-linking agent EDC in the recombinant collagen sponge into EDU in the ultrapure water; and then filtering with a filter membrane of 0.22 mu m to obtain a recombinant collagen sponge leaching solution, namely a sample solution.
2. Preparing an EDU standard solution: 10mg of EDU standard was weighed into a 100mL volumetric flask, ultrapure water was added to the scale line, and the mixture was shaken to dissolve it sufficiently, to obtain a 100. Mu.g/mL EDU stock solution. The EDU stock solution was diluted 0, 2, 4, 8, and 16 times with ultrapure water to obtain EDU standard solutions at concentrations of 3. Mu.g/mL, 6. Mu.g/mL, 12. Mu.g/mL, 24. Mu.g/mL, and 48. Mu.g/mL, respectively.
In addition, weighing 10mg of EDC, placing the EDC in a 100mL volumetric flask, adding ultrapure water to scale marks, uniformly mixing, and placing for 7 days for later use; before detection, 10mg of EDC is weighed and placed in a 100mL volumetric flask, ultrapure water is added to scale marks, and the mixture is uniformly mixed for later use.
3. Detecting the EDU standard substance solution by adopting an angiont Infinity 1260 II high performance liquid chromatograph in combination with an ultraviolet detector, and recording the peak area under the same retention time; the chromatographic conditions are as follows: the chromatographic column adopts octadecylsilane bonded silica gel column (C18 reversed phase column)
AQ-C18 Welch with specification of 4.6mm x 250mm and filler aperture diameter
The particle size is 5 μm, the volume ratio of the mobile phase A to the mobile phase B is 97:3, the mobile phase A is an aqueous solution containing 0.1mM 1-sodium heptanesulfonate and 0.04% triethylamine by volume, the pH value is adjusted to 3 by phosphoric acid, and the mobile phase B isAcetonitrile (Fisher Chemical, chromatographic grade), wherein a chromatographic column is subjected to a mobile phase for 150min before sample injection, the sample injection volume is 20 muL, the flow rate is 1mL/min, the column temperature is 30 ℃, the detection wavelength is 201nm, the detector is an ultraviolet detector, and the elution mode is isocratic elution. And (3) drawing a standard curve according to the detection result of the standard solution by taking the concentration y of the EDU as a vertical coordinate and the peak area x as a horizontal coordinate to obtain a standard curve equation: y =0.039x +0.4939 (see fig. 1), R 2 =0.9995, the linear relationship is good. The reason is that the retention of EDU on the C18 column is prolonged by reagents such as 1-sodium heptanesulfonate, triethylamine, phosphoric acid and the like, so that the main peak reaches reasonable retention time, the absorption of the EDU on a chromatographic column is enhanced, and the influence of other impurities on the EDU is weakened. When the detection limit concentration is 0.44% of the specified limit value of the EDC impurity of the recombinant collagen sponge cross-linking agent, the signal-to-noise ratio is 3.71, the detection limit reaches 40ng/mL, namely 40ng/mL 20 mu L =800pg, and the chromatogram is shown in FIG. 2.
When the mobile phase was replaced with ultrapure water containing trifluoroacetic acid at a volume concentration of 0.1% (mobile phase a) and acetonitrile containing trifluoroacetic acid at a volume concentration of 0.1% (mobile phase B) in a volume ratio of 97:3 (common mobile phase), the result shows that the detection limit is in microgram scale, and other components have great influence on the main peak, and the chromatogram is shown in fig. 3.
The EDU stock solution, the EDC aqueous solution left to stand for 7 days, and the freshly prepared EDC aqueous solution were each diluted with ultrapure water to 9.05. Mu.g/mL and detected as described above. And comparing by using a high performance liquid chromatogram map 4-6, and obtaining the EDU as a final product.
4. The sample solution in step 1 was assayed under the conditions of step 3, and the chromatogram thereof is shown in FIG. 7. Substituting the peak area into the standard curve equation obtained in the step 3 to calculate the concentration of the 1-ethyl [3- (dimethylamino) -propyl ] carbodiimide hydrochloride in the sample solution according to the formula: EDC residual amount = EDU concentration × total volume of sample solution/173 × 191/substance to be measured × 100%. The result shows that the residual amount of EDC in the recombinant collagen-like sponge is less than 0.02%, namely the residual amount of EDC is less than 9 mu g/mL.
In order to prove the beneficial effects of the invention, the method of the invention is subjected to repeatability, stability, standard addition recovery rate and sample detection experiments, and the specific experiments are as follows:
1. repeatability
Weighing a plurality of recombinant collagen-like sponges, preparing a sample solution according to the method in the example 1, feeding 20 mu l of the sample solution, repeating the sample feeding for 6 times, detecting according to the chromatographic conditions in the example 1, and recording the peak areas under the same retention time, wherein the results are shown in the table 1.
TABLE 1 repeatability results
| Name(s) | Main peak retention time | Peak area | Peak height | Tailing factor | Number of theoretical plate |
| Sample solution-1 | 7.299 | 166.147 | 18.872 | 1.14 | 17018.9 |
| Sample solution-2 | 7.294 | 171.659 | 18.988 | 1.14 | 16743.1 |
| Sample solution-3 | 7.285 | 170.430 | 19.181 | 1.14 | 16901.59 |
| Sample solution-4 | 7.279 | 171.238 | 19.279 | 1.14 | 16946.11 |
| Sample solution-5 | 7.274 | 175.433 | 19.549 | 1.18 | 16865.19 |
| Sample solution-6 | 7.27 | 174.895 | 19.604 | 1.16 | 16865.65 |
| Standard deviation SD | 0.011 | 3.37 | 0.294 | -- | -- |
| Arithmetic mean Xbar | 7.284 | 171.633 | 19.246 | -- | -- |
| Relative standard deviation RSD (%) | 0.16 | 1.96 | 1.53 | -- | -- |
The data prove that the high performance liquid chromatograph has good precision.
2. Stability of
Sampling the sample solution, standing at room temperature for 0h, 4h, 24h, 48h and 96h, detecting the stability of the poplar sample solution for 0h, 4h, 24h, 48h and 96h, precisely sucking 20 mu L of the sample solution, injecting the sample solution into a high performance liquid chromatograph, detecting according to the chromatographic conditions of example 1, recording the peak area under the same retention time, and detecting results are shown in Table 2.
TABLE 2 stability test results
| Name (R) | Peak area | Peak height | Number of theoretical plate |
| Sample solution 0h | 433.006 | 48.204 | 16803.10 |
| Sample solution 0h | 430.951 | 48.074 | 16834.84 |
| Sample solution 4h | 438.734 | 49.327 | 16947.52 |
| Sample solution 4h | 432.476 | 49.332 | 16997.93 |
| Sample solution 24h | 452.562 | 50.691 | 16908.13 |
| Sample solution 24h | 455.001 | 50.762 | 16860.88 |
| Sample solution for 48h | 448.592 | 50.529 | 16651.98 |
| Sample solution for 48h | 446.718 | 50.537 | 16671.07 |
| Sample solution 96h | 437.212 | 46.049 | 15683.01 |
| Sample solution 96h | 438.526 | 46.251 | 15746.76 |
| Standard deviation SD | 8.70 | -- | -- |
| Arithmetic mean Xbar | 441.378 | -- | -- |
| Relative standard deviation RSD (%) | 1.97 | -- | -- |
The data prove that the sample solution has stable properties after being placed at room temperature for 96h, the placing time has no influence on EDU impurities in the sample within 96h, and the irreversibility of EDC converted into EDU is proved.
3. Recovery rate of added standard
Weighing a plurality of recombinant collagen-like sponges, and preparing a test solution according to the method in the embodiment 1; precisely weighing EDU 0.2mg, placing in a 10mL volumetric flask, performing constant volume to scale mark with ultrapure water, and mixing to obtain a control solution. The sample solution was added to the sample solution and the control solutions of different volumes were prepared to prepare a sample solution to be recovered, and the detection was carried out under the chromatographic conditions of example 1. The sample recovery rate was calculated according to the following formula: the sample recovery rate = recovery% = (C-A)/B100%, wherein A is the measured component content in the test sample solution; b is the amount of the reference substance; c is an observed value. The results are shown in Table 3.
TABLE 3 results of recovery with addition of standard
Note: 2.4 in control 2.4-1 means that 2.4mL of 20. Mu.g/mL control solution was taken, added with water to a volume of 10mL and mixed, and-1 represents replicate 1. 2.4 in the sample + pair 2.4-1-1 means 2.4mL of a control solution of 20. Mu.g/mL, 3mL of a test solution was added, and the volume was further adjusted to 10mL, -1-1 means a parallel sample 1 of the group 1 sample, 3.6 in the sample + pair 3.6-2-2 means 3.6mL of a control solution of 20. Mu.g/mL, 3mL of a test solution was added, and the volume was further adjusted to 10mL, -2-2 means a parallel sample 2 of the group 2 sample, and the other means are the same as described above.
As can be seen from Table 3, when the EDU content is between 0.1% and 1%, the recovery rate limit is between 90% and 108%, the recovery rate of high and low concentrations in actual detection is 95%, and the sample recovery rate is good.
4. Sample detection
Weighing three continuous batches of recombinant collagen-like sponge samples, preparing sample solutions according to the method in the embodiment 1, detecting according to the chromatographic conditions in the embodiment 1, and recording peak areas under the same retention time, wherein the detection results are shown in table 4.
TABLE 4 results of sample testing
| Name (R) | Main peak retention time | Peak area | Peak height | Tailing factor | Number of theoretical plate | Content% |
| 15-1 | 7.114 | 145.555 | 16.780 | 1.18 | 16664.16 | 0.014％ |
| 15-2 | 7.113 | 144.908 | 16.760 | 1.19 | 16698.24 | 0.013％ |
| 15-3 | 7.111 | 144.621 | 16.737 | 1.18 | 16695.04 | 0.013％ |
| 29-1 | 7.108 | 124.032 | 14.490 | 1.15 | 16977.49 | 0.012％ |
| 29-2 | 7.106 | 125.145 | 14.527 | 1.16 | 16786.46 | 0.012％ |
| 29-3 | 7.103 | 123.961 | 14.508 | 1.15 | 16993.17 | 0.012％ |
| 05-1 | 7.099 | 202.664 | 23.514 | 1.20 | 16644.25 | 0.018％ |
| 05-2 | 7.096 | 202.349 | 23.568 | 1.18 | 16741.64 | 0.018％ |
| 05-3 | 7.093 | 203.013 | 23.609 | 1.20 | 16726.95 | 0.018％ |
Note: in the table 15, 29, 05 are three batches of samples, the following-1, -2, -3 represent three replicates of each batch.
The content values of the detection results are all lower than the limit value, and the results are qualified.
The above examples are only methods for detecting the residual amount of the crosslinking agent EDC in the recombinant collagen-like sponge, but are not limited to the recombinant collagen-like sponge, and the methods are applicable to the detection of the crosslinking agent EDC in other substances.
Claims (6)
1. A method for high performance liquid chromatography analysis of residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride, characterized in that the method comprises the steps of:
(1) Preparation of sample solution: adding a substance to be detected into ultrapure water, leaching at 40-60 ℃, hydrolyzing residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride into 1- [3- (dimethylamino) propyl ] -3-ethylurea, and filtering with a 0.22 mu m water film to obtain a sample solution;
(2) Preparing a 1- [3- (dimethylamino) propyl ] -3-ethylurea standard solution;
(3) Detecting the 1- [3- (dimethylamino) propyl ] -3-ethylurea standard substance solution by adopting a high performance liquid chromatography and an ultraviolet detector, and recording the peak area under the same retention time; the chromatographic conditions are as follows: the chromatographic column adopts a C18 reverse phase column, and the mobile phase is a mobile phase A and a mobile phase B with the volume ratio of 99:1 to 80:20, wherein the mobile phase A is an aqueous solution containing 0.1mM 1-sodium heptanesulfonate and 0.04% triethylamine by volume concentration, the pH value of the aqueous solution is adjusted to 3 by using phosphoric acid, the mobile phase B is acetonitrile, and the elution mode is isocratic elution; drawing a standard curve according to the detection result of the standard solution by taking the concentration of 1- [3- (dimethylamino) propyl ] -3-ethylurea as a vertical coordinate and the peak area as a horizontal coordinate to obtain a standard curve equation;
(4) Detecting the sample solution in the step (1) according to the conditions in the step (3), substituting the sample solution into the standard curve equation obtained in the step (3) to calculate the concentration of the 1- [3- (dimethylamino) propyl ] -3-ethylurea in the sample solution according to the formula: EDC residual quantity = EDU concentration x sample solution total volume/173 x 191/test substance weight x 100%, EDC residual quantity in the test substance is calculated, EDC is 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride, EDU is 1- [3- (dimethylamino) propyl ] -3-ethylurea.
2. The method for high performance liquid chromatography analysis of residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride according to claim 1, characterized in that: weighing 10mg of 1- [3- (dimethylamino) propyl ] -3-ethylurea standard substance in the step (2), placing the standard substance in a 100mL volumetric flask, adding ultrapure water to scale marks, and shaking to fully dissolve the standard substance to obtain 100 mu g/mL of 1- [3- (dimethylamino) propyl ] -3-ethylurea stock solution; the stock solution of 1- [3- (dimethylamino) propyl ] -3-ethylurea was diluted with ultrapure water to 3. Mu.g/mL, 6. Mu.g/mL, 12. Mu.g/mL, 24. Mu.g/mL, and 48. Mu.g/mL of a standard solution of 1- [3- (dimethylamino) propyl ] -3-ethylurea, respectively.
3. Root of herbaceous plantsResidual 1-ethyl [3- (dimethylamino) -propyl ] group according to claim 1]-a method for high performance liquid chromatography of carbodiimide hydrochloride, characterized in that: in the step (3), the C18 reverse phase column is an octadecylsilane bonded silica gel column
AQ-C18 WeLch with specification of 4.6mm multiplied by 250mm and filling aperture diameter
The particle size was 5 μm.
4. The method for high performance liquid chromatography analysis of residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride according to claim 1, wherein: in the step (3), the sample injection volume is 10-20 mu L, the flow rate is 0.8-1.2 mL/min, the column temperature is 25-40 ℃, and the detection wavelength is 200-210 nm.
5. The method for high performance liquid chromatography analysis of residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride according to claim 1, wherein: in the step (3), a mobile phase equilibrium column is required for 120-150 min before the sample injection of the chromatographic column.
6. The method for high performance liquid chromatography analysis of residual 1-ethyl [3- (dimethylamino) -propyl ] -carbodiimide hydrochloride according to claim 1, wherein: in the step (3), the volume ratio of the mobile phase A to the mobile phase B is 97:3 to 95:5, and (b) a mixed solution.
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| CN104833753A (en) * | 2015-05-12 | 2015-08-12 | 苏州景卓生物技术有限公司 | HPLC-ELSD detection method for EDC residue |
| CN106153748A (en) * | 2015-03-31 | 2016-11-23 | 深圳翰宇药业股份有限公司 | A kind of method of N, N-DIC in detection polypeptide |
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| CN106153748A (en) * | 2015-03-31 | 2016-11-23 | 深圳翰宇药业股份有限公司 | A kind of method of N, N-DIC in detection polypeptide |
| CN104833753A (en) * | 2015-05-12 | 2015-08-12 | 苏州景卓生物技术有限公司 | HPLC-ELSD detection method for EDC residue |
Non-Patent Citations (1)
| Title |
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| Q. PAULA LEI等: "Quantification of residual EDU (N-ethyl-N_-(dimethylaminopropyl) carbodiimide (EDC) hydrolyzed urea derivative) and other residual by LC–MS/MS", JOURNAL OF CHROMATOGRAPHY B, pages 103 - 112 * |
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