CN115144482A - Method for quantitatively detecting content of isopropyl-beta-D-thiogalactoside in polypeptide or protein - Google Patents
Method for quantitatively detecting content of isopropyl-beta-D-thiogalactoside in polypeptide or protein Download PDFInfo
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Abstract
The invention relates to a method for rapidly and quantitatively detecting the content of residual isopropyl-beta-D-thiogalactoside in polypeptide or protein by using a liquid chromatography-tandem mass spectrometry method. The detection method solves the problem that the prior art can not detect isopropyl-beta-D-thiogalactoside rapidly, accurately and quantitatively with low limit, can detect the residual content of IPTG in polypeptide or protein with 0.5ng/mL quantitative limit and 0.2ng/mL detection limit, and has high selectivity and low background interference of a mass spectrometer, so that the data reliability is higher and more accurate; the requirements of the drug analysis on specificity, linearity, repeatability, accuracy, quantitative limit and detection limit are met.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a liquid chromatography-mass spectrometry detection and analysis method for isopropyl-beta-D-thiogalactoside residue in polypeptide or protein.
Background
Isopropyl- β -D-thiogalactoside (IPTG), english name: isoproyl-beta-D-thiogalactopyranoside of the formula C 9 H 18 O 5 S, molecular weight 238.3.
IPTG is an allolactose mimetic, readily soluble in water, methanol, ethanol, and is an activity inducer for beta-galactosidase and beta-galactose permease. It is mainly used for selecting gene recombinants and being used as an expression inducer of an expression vector with a promoter such as lac or tac in the preparation of biochemical samples; can also be used as a molecular biological reagent which is matched with X-Gal (galactosase action substrate) for use and can carry out blue-white spot screening on the clone bacteria; in prokaryotic expression, it is a placebo inducer, and since it is not decomposed by itself, it can induce the production of the target protein continuously.
IPTG is expensive, increases process costs, and is toxic when used in excessive amounts. However, IPTG lacks a luminescent group and cannot be detected under normal UV-visible light. The conventional derivatization and fluorescence combined HPLC detection method has the problems of complicated steps, poor selectivity, high quantitative limit and the like. CN112903836A discloses a determination method for isopropyl-beta-D-thiogalactoside in-vitro cultured bear gall powder, the main component of the bear gall powder is tauroursodeoxycholic acid, which belongs to small molecular chemical drugs, and the patent method adopts a Waters Cortecs T3 chromatographic column, is expensive and is not suitable for detection of IPTG content in polypeptide and macromolecular protein.
Therefore, the development of an analysis method for detecting the IPTG content in the polypeptide or protein, which is simple and rapid, has good selectivity, low quantitative limit and lower cost, has good prospect and important significance.
Disclosure of Invention
The invention aims to provide a liquid chromatography-mass spectrometry detection and analysis method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside, which has the characteristics of specificity, linearity, repeatability, accuracy, quantitative limit and detection limit meeting the requirements of drug analysis and detection, rapidness, accuracy, high selectivity, low quantitative limit and low cost, and solves the problem that the prior art cannot perform rapid, accurate and low-limit quantitative detection.
In order to achieve the above object, the present invention provides the following technical solution, a method for quantitatively detecting the content of isopropyl- β -D-thiogalactoside, comprising: detecting the residual quantity of isopropyl-beta-D-thiogalactoside in the polypeptide or protein by a liquid chromatography tandem mass spectrometry, wherein the liquid chromatography adopts a hydrophilic interaction chromatographic column (HILIC) and a mixed solvent of an alcohol solvent and water as a mobile phase.
The hydrophilic interaction chromatographic column comprises, but is not limited to, polyamine bonded ethylene bridge hybrid silica gel chromatographic column, amido bonded ethylene bridge hybrid silica gel chromatographic column and unbonded ethylene bridge hybrid silica gel chromatographic column.
As an embodiment of the present invention, the method comprises the steps of:
1) Preparing isopropyl-beta-D-thiogalactoside standard solutions with different concentrations, performing liquid chromatography tandem mass spectrometry detection, and drawing a standard curve;
2) Preparing a polypeptide or protein sample solution, and performing liquid chromatography tandem mass spectrometry detection;
3) And (2) obtaining the concentration of isopropyl-beta-D-thiogalactoside in the polypeptide or protein sample solution by contrasting the standard curve obtained in the step 1), thereby realizing the quantitative detection of the isopropyl-beta-D-thiogalactoside.
The polypeptide of the invention refers to a peptide with the number of amino acid residues below 50; the protein of the invention refers to a peptide with more than 50 amino acid residues.
As an embodiment of the present invention, the polypeptide or protein includes, but is not limited to, insulin and its analogs, glucagon-like peptide and its analogs, enzymes, and the like, which can be prepared by recombinant biotechnology or fermentation technology, and related products such as drugs, prodrugs, pharmaceutical intermediates or reagents, and the like. The polypeptide or protein can be prepared into a polypeptide/protein crude product by gene cloning, strain screening, strain culture, fermentation, extraction and other processes, and then the polypeptide/protein crude product is prepared by further purification processes (such as extraction, crystallization, filtration, chromatographic separation and the like) to obtain a polypeptide/protein fine product or a polypeptide/protein pure product.
As an embodiment of the invention, the insulin and the analogues thereof comprise insulin analogues such as animal insulin, human insulin, insulin aspart, insulin glargine, insulin deglutamide, insulin lispro, insulin detemir and the like; the glucagon-like peptides and analogs thereof include homologous peptides derived from a preproglucagon gene, insulinotropic peptides and analogs and derivatives thereof, wherein the peptides derived from the preproglucagon gene include glucagon, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 1 (GLP-2), and Oxyntomodulin (OXM). Wherein the glucagon-like peptide 1 comprises liraglutide, linagliptin, semaglutide, exenatide, benaglutide, dulaglutide and other medicaments.
As an embodiment of the present invention, the hydrophilic chromatographic column is a polyamine-bonded ethylene bridge hybrid silica gel chromatographic column, and more preferably, the stationary phase used is a polyethyleneimine-bonded silica gel stationary phase, and compared with a Waters Cortecs T3 chromatographic column (an inverse C18 chromatographic column), the polyethyleneimine-bonded silica gel stationary phase has a good retention effect on a strong polar compound (such as IPTG) with poor retention capability relative to C18, and polypeptides or proteins and the like are substances with smaller polarity, and a peak is generated after a substance with smaller polarity (such as polypeptides or proteins) and a peak is generated after a substance with larger polarity (such as IPTG) are present on a HILIC chromatographic column (a hydrophilic interaction chromatographic column), so that the polypeptides or proteins and IPTG can be effectively separated, and the advantages of low price, reduction of interference caused by the polypeptides or proteins to detection and the like are provided.
As an embodiment of the present invention, the alcohol solvent in the mobile phase is methanol or ethanol; methanol is preferred, because the methanol has high cost performance, strong universality, small system pressure and sharper detection peak type. The water in the mobile phase is purified water.
As an embodiment of the invention, the mobile phase consisting of methanol and water or the mobile phase consisting of ethanol and water is subjected to gradient elution, so that better peak shape can be obtained and matrix interference can be reduced.
As a specific embodiment of the present invention, the gradient elution has a volume ratio of methanol to water of 5 to 90:95-10 or the volume ratio of ethanol to water is 5-90:95-10.
As a more specific embodiment of the present invention, the gradient elution has a volume ratio of methanol to water of 5 to 90:95-10 or the volume ratio of ethanol to water is 5-90:95-10, the flow rate of the mobile phase is 0.10-0.35mL/min, more specifically 0.2mL/min; the column temperature is 25-35 deg.C, more particularly 30 deg.C.
As one embodiment of the invention, an isopropyl-beta-D-thiogalactoside standard solution and a polypeptide or protein sample solution are prepared by using an acidic solution as a diluent.
As an embodiment of the present invention, the acidic solution includes one or more of an aqueous hydrochloric acid solution, an aqueous phosphoric acid solution, or acetic acid. The concentration of the acidic solution may be 0.001 to 0.1mol/L, preferably 0.01mol/L. Formic acid and IPTG will form [ M + CHOO ] in the ion source] - Ions are added to reduce IPTG mass spectrum response, and the method is not suitable for serving as an acidic additive.
As an embodiment of the present invention, the polypeptide or protein sample may optionally be subjected to a purification treatment as necessary before the polypeptide or protein sample solution is prepared. Purification methods include, but are not limited to, extraction, filtration, crystallization, or chromatographic separation, among others.
In one embodiment of the invention, the mass spectrum in the liquid chromatography tandem mass spectrometry adopts a triple quadrupole mass spectrometer as a detector, and Multiple Reaction Monitoring (MRM) is carried out in an electrospray ionization (ESI) negative ion mode, wherein the ion pair comprises 237.1/160.7, 237.1/100.8, and preferably 237.1/160.7.
Compared with the prior art, the analysis and detection method has the advantages that: a novel method for liquid chromatography-tandem mass spectrometry is developed, a mixed solvent composed of alcohols and water is used as a mobile phase, a polyamine bonded ethylene bridge hybrid silica gel chromatographic column, an amido bonded ethylene bridge hybrid silica gel chromatographic column and an unbonded ethylene bridge hybrid silica gel chromatographic column are adopted as hydrophilic action chromatographic columns, and if polyethyleneimine is used as a stationary phase, the interference of substrates such as polypeptide, inorganic salt and the like on IPTG detection can be avoided; the mobile phase used in the analysis method has common composition, simple preparation, trouble and labor saving, and the price of the polyethyleneimine stationary phase is relatively lower and is only half of that of a Waters Cortecs T3 chromatographic column, so that the cost can be greatly saved; the analysis time is short, and the time is saved. Meanwhile, the liquid chromatogram is matched with a triple quadrupole mass spectrum, so that the residual content of IPTG in the polypeptide or protein can be detected at a quantitative limit of 0.5ng/mL and a detection limit of 0.2ng/mL, and the mass spectrometer has high selectivity and low background interference, so that the data reliability is higher and more accurate; the method is quick, simple, convenient, good in specificity and high in sensitivity, and is suitable for popularization and application.
Drawings
FIG. 1 is a 100ng/mL IPTG XIC standard mass spectrum obtained according to example 1.
FIG. 2 is a linear curve obtained according to example 1.
FIG. 3 is a XIC mass spectrum of a refined sample solution of insulin aspart obtained in example 1.
FIG. 4 is a XIC mass spectrum of a competitive labeling sample solution of insulin aspart obtained in example 1.
FIG. 5 is a mass spectrum of XIC of crude insulin aspart sample obtained in example 2.
FIG. 6 is a mass spectrum of a top-quality insulin aspart sample XIC obtained in example 2.
FIG. 7 is the XIC mass spectra of crude and fine deglutaric insulin samples obtained in example 3.
FIG. 8 is a mass spectrum of a refined deglutaric insulin sample XIC obtained in example 3.
Figure 9 is a mass spectrum of XIC of a crude semaglutide sample obtained according to example 4.
Figure 10 is a mass spectrum of a top quality semaglutide sample XIC obtained according to example 4.
FIG. 11 is a mass spectrum of a crude liraglutide sample XIC obtained in example 5.
Fig. 12 is a mass spectrum of a fine liraglutide sample XIC obtained in example 5.
FIG. 13 shows a spectrum of XIC mass spectrum obtained in comparative example 1 and detected by IPTG separation using C8 column.
FIG. 14 shows a spectrum of XIC mass spectrum obtained in comparative example 1 and detected by IPTG separation using C18 column.
Figure 15 is a methanol isocratic elution IPTG XIC mass spectrum obtained according to comparative example 2 (50).
Detailed Description
Raw materials and reagents:
the isopropyl thiogalactoside (> 99%) used in the examples was from Kaisen Lai technology Inc.
Crude products and fine products of polypeptide or protein raw material medicine samples used in the embodiment are provided by Nanjing Hanxin medicine science and technology Limited company, and strains are screened mainly through gene cloning; culturing, fermenting and extracting strains to obtain insulin or an analogue precursor thereof; then, a crude product is prepared by the processes of renaturation, enzyme digestion, primary purification and the like, and a fine product or a pure product is prepared by the processes of further purification and/or crystallization. For example, the preparation process of insulin aspart and insulin deguelin can refer to PCT patent WO2020051812 already disclosed by the company, the preparation process of liraglutide can refer to Chinese patent CN201910515446.0 already disclosed by the company, the preparation process of semaglutide can refer to Chinese patent CN202111664146.2 already applied by the company, and the whole content can be directly introduced into the invention.
The equipment or other reagents/materials used in the examples are commercially available.
Example 1
The method provided by the embodiment is subjected to methodology verification of specificity, linearity, repeatability, accuracy, quantitative limit and detection limit performance parameters, and the verification takes insulin aspart competitive products as samples.
The experimental conditions are as follows:
equipment: exionLC AD liquid phase and SCIEX 6500+ triple quadrupole mass spectrometer (SCIEX, USA), equipped with Iondrive Turbo V ion source.
A chromatographic column: polyethyleneimine is used as a stationary phase, and the thickness of the polyethyleneimine is 150mm multiplied by 2.1mm,3 mu m.
The mobile phase A is methanol, the mobile phase B is purified water, and gradient elution is carried out.
The gradient is shown in table 1 below:
TABLE 1 mobile phase gradiometer
| Time (min) | A | B％ | |
| 0 | 5 | 95 | |
| 1.0 | 5 | 95 | |
| 1.5 | 90 | 10 | |
| 5.5 | 90 | 10 | |
| 6.0 | 5 | 95 | |
| 10.0 | 5 | 95 |
The invention adopts the mobile phase composed of methanol and water or the mobile phase composed of ethanol and water to carry out gradient elution and relatively isocratic elution, thereby obtaining better peak shape and reducing matrix interference.
The flow rate was 0.2mL/min, the column temperature was 30 ℃ and the amount of sample was 5. Mu.L.
The ion pair parameters are shown in table 2 below:
TABLE 2 ion-pair parameter table
The ion source parameters are shown in table 3 below:
TABLE 3 ion Source parameters
| Parameter(s) | Numerical value |
| Air curtain gas (CUR) | 25psi |
| Collision gas (CAD) | 8 |
| Ionization voltage (IS) | -4500V |
| Temperature (TEM) | 450℃ |
| Spray mist (GS 1) | 40psi |
| Auxiliary heating gas (GS 2) | 60psi |
(1) Specificity
Respectively and precisely absorbing 5 mu L of refined insulin aspart sample (4 mg/mL), purified water, 0.01mol/L hydrochloric acid aqueous solution and IPTG standard solution (4 ng/mL, 0.01mol/L hydrochloric acid aqueous solution is used as a diluent), injecting the mixture into a liquid chromatograph-mass spectrometer for determination, and the result shows that the refined insulin aspart sample, the purified water and the 0.01mol/L hydrochloric acid aqueous solution have no interference on IPTG detection and have good specificity.
(2) Linearity
Preparing a standard solution: dissolving and diluting IPTG (isopropyl-beta-thiogalactoside) by taking 0.01mol/L hydrochloric acid aqueous solution as a diluent, preparing standard solutions of 0.2, 0.5, 2, 5, 20, 50 and 100ng/mL respectively, injecting the standard solutions into a liquid chromatograph-mass spectrometer in sequence for detection, detecting each concentration for three times in parallel, recording the mass spectrum of the standard solution XIC (extracted ion current) at each concentration (wherein the mass spectrum of the 100ng/mL IPTG XIC is shown in figure 1), and fitting a standard curve according to the concentration-peak area (shown in figure 2). The respective test data are shown in tables 4 and 5:
TABLE 4 results of the Linear test-1
TABLE 5 Linear test results-2
Taking the concentration x as an abscissa and the peak area y as an ordinate, performing linear fitting, wherein the weight W =1/x 2 Obtaining a standard linear equation: y =2.58e +003x +658 (R = 0.9969), the results show that good performance is achieved in the range of 0.2ng/mL-100ng/mLA linear relationship.
(3) Repeatability of
Taking the same refined insulin aspart sample, adding a proper amount of IPTG (isopropyl-beta-D-thiogalactoside) to prepare a sample with the concentration of 4mg/mL (containing IPTG4 ng/mL), preparing 6 parts in parallel, measuring, and calculating the concentration, wherein the result is shown in a table 6.
TABLE 6 results of the repeatability tests
According to the fourth general rule 9101 of the Chinese pharmacopoeia 2020, the repeatability limit is 8% when the content of the component to be measured in the sample is 1 ppm. The RSD verified at this time is 3.44%, so the repeatability of the method is good.
(4) Accuracy of
Preparing a sample solution: weighing a proper amount of refined insulin aspart samples, taking 0.01mol/L hydrochloric acid aqueous solution as a diluent to prepare sample solution with the concentration of 4mg/mL, and injecting the sample solution into a liquid chromatograph-mass spectrometer for detection once.
Preparing a standard sample solution: weighing a proper amount of refined aspart insulin samples and IPTG, taking 0.01mol/L hydrochloric acid aqueous solution as a diluent, respectively preparing aspart insulin standard-adding sample solutions with the concentrations of 1ng/mL, 4ng/mL and 10ng/mL IPTG, preparing three parts of standard-adding sample solutions with each concentration in parallel, and calculating the standard-adding recovery rate.
The mass spectrum of the sample solution for detecting the 10ng/mL IPTG XIC of the insulin aspart is shown in a figure 3, the mass spectrum of the sample solution for detecting the 10ng/mL IPTG XIC containing insulin aspart is shown in a figure 4, and the test results are shown in a table 7:
TABLE 7 accuracy test results
According to the fourth general rule 9101 of China pharmacopoeia 2020, when the content of the component to be detected in the sample is 1ppm, the recovery rate limit is 75-120%; when the content of the component to be detected in the sample is 10ppm, the recovery rate limit is 80-115%, so that the recovery rate of the method meets the requirement.
(5) Quantitative limit and detection limit
Taking a proper amount of refined insulin aspart samples and IPTG, configuring a sample concentration of 4mg/mL with a quantitative Limit (LOQ) sample of IPTG 0.5ng/mL and a sample concentration of detection Limit (LOD) sample of IPTG 0.2ng/mL, and preparing 3 parts in parallel; 0.01mol/L hydrochloric acid aqueous solution is used as a solvent blank; the refined aspart insulin sample solution without IPTG was used as a sample blank, and the blank was separately injected into a liquid chromatograph-mass spectrometer for detection, and the recovery rate and the signal-to-noise ratio (S/N) were calculated, and the results are shown in tables 8 and 9.
TABLE 8 limit of quantitation (LOQ) test results
TABLE 9 Limit of detection (LOD) test results
As a result: the signal-to-noise ratio of IPTG with the concentration of 0.5ng/mL is more than 10, and the recovery rate is in the range of 70-125 percent, which can be used as the limit of the method; the signal-to-noise ratio of IPTG with the concentration of 0.2ng/mL is more than 3, has obvious difference with solvent blank and sample blank, and can be used as the detection limit of the method.
Example 2 (application of the detection method to insulin aspart)
The IPTG content in the crude insulin aspart product and the refined insulin aspart product is detected, and whether the IPTG inducer added in the fermentation process of the insulin aspart can be effectively removed by the purification process of the insulin aspart is investigated.
The experimental conditions are as follows: the equipment, chromatographic column, mobile phase and set ion pair parameters and ion source parameters used were the same as in example 1.
Preparing a standard solution: dissolving and diluting IPTG with 0.01mol/L hydrochloric acid aqueous solution as a diluent to prepare 0.2, 0.5, 2, 5, 20, 50 and 100ng/mL standard solutions, sequentially injecting the standard solutions into a liquid chromatograph-mass spectrometer for detection, detecting each concentration once, and drawing a standard curve.
Preparing a sample solution: taking 0.01mol/L hydrochloric acid aqueous solution as a diluent, precisely weighing crude insulin aspart (2 batches) and refined insulin aspart (2 batches) respectively to prepare four sample solutions with the concentration of 4mg/mL, and injecting the four sample solutions into a liquid chromatograph-mass spectrometer for detection. And (3) calculating the IPTG concentration in the sample by comparing with the obtained standard curve, and calculating the IPTG content according to the concentration (4 mg/mL) of insulin aspart, wherein the specific result is shown in a table 10, the XIC mass spectrum of the crude insulin aspart is shown in a figure 5, and the XIC mass spectrum of the refined insulin aspart is shown in a figure 6.
TABLE 10 IPTG content detection results in crude and fine insulin aspart samples
The result shows that the analysis method can simply and rapidly detect the IPTG content in insulin aspart with lower quantitative limit and detection limit, and the selected mass spectrometer has the characteristics of high selectivity and low background interference, so that the data reliability is higher and more accurate. Wherein the IPTG concentration in the crude insulin aspart products of the two batches is 0.32ng/mL or 0.481ng/mL respectively, and IPTG residue is not detected in the refined insulin aspart products of the two batches, so that the effectiveness of the purification process is proved, and the impurity requirement of the obtained product meets the pharmacopoeia standards of various countries.
Example 3 (application of the detection method to insulin deglutamide)
In the embodiment, the IPTG content in the degummed insulin crude product and the degummed insulin refined product is detected, and whether the IPTG inducer added in the degummed insulin fermentation process can be effectively removed by the purification process of the degummed insulin is examined.
The experimental conditions are as follows: the equipment, chromatographic column, mobile phase and set ion pair parameters and ion source parameters used were the same as in example 1.
The standard curve equation fitting method is referred to example 1.
Preparing a sample solution: taking 0.01mol/L hydrochloric acid aqueous solution as a diluent, precisely weighing the deglutaric insulin crude product (2 batches) and the deglutaric insulin fine product (2 batches) respectively to prepare four sample solutions with the concentration of 4mg/mL, and injecting the four sample solutions into a liquid chromatograph-mass spectrometer for detection. And calculating IPTG concentration in the sample according to the obtained standard curve, and calculating IPTG content according to degummed insulin concentration (4 mg/mL), wherein specific results are shown in a table 11, an XIC mass spectrum of a degummed insulin crude product is shown in a figure 7, and an XIC mass spectrum of a degummed insulin refined product is shown in a figure 8.
TABLE 11 IPTG content detection results in degu insulin crude and fine samples
And (4) conclusion: the analysis method can simply and quickly detect the IPTG content in the insulin deglutaric product with lower quantitative limit and detection limit. Wherein the IPTG concentration in the two batches of deglutated insulin crude products is 0.288ng/mL or 0.360ng/mL respectively, and IPTG residue is not detected in the two batches of deglutated insulin refined products, thereby proving the effectiveness of the purification process.
Example 4 application of the detection method to semaglutide
In the example, the IPTG content of the crude semaglutide and the fine semaglutide is detected, and whether the purification process of the semaglutide can effectively remove the IPTG inducer added in the fermentation process of the semaglutide is investigated.
The experimental conditions are as follows: the equipment, chromatographic column, mobile phase and set ion pair parameters and ion source parameters used were the same as in example 1.
The standard curve equation fitting method is referred to example 1.
Preparing a sample solution: taking 0.01mol/L hydrochloric acid aqueous solution as a diluent, respectively and precisely weighing a crude semaglutide product (2 batches) and a fine semaglutide product (2 batches), preparing four sample solutions with the concentration of 4mg/mL, and injecting the four sample solutions into a liquid chromatograph-mass spectrometer for detection. And (3) calculating the IPTG concentration in the sample by comparing with the obtained standard curve, and calculating the IPTG content according to the concentration (4 mg/mL) of the semaglutide, wherein the specific result is shown in a table 12, the XIC mass spectrum of the crude semaglutide is shown in a figure 9, and the XIC mass spectrum of the refined semaglutide is shown in a figure 10.
TABLE 12 IPTG content detection results in crude and fine sergelutin samples
And (4) conclusion: the analysis method can simply and quickly detect the IPTG content in the semaglutide product by using lower quantitative limit and detection limit. Wherein the IPTG concentration in the two batches of crude semaglutide is 0.392ng/mL or 0.265ng/mL respectively, and no IPTG residue is detected in the two batches of refined semaglutide, thus proving the effectiveness of the purification process.
Example 5 application of the detection method to Liraglutide
In the example, the IPTG content in the crude liraglutide product and the refined liraglutide product is detected, and whether the purification process of the liraglutide can effectively remove the IPTG inducer added in the fermentation process of the liraglutide is examined.
The experimental conditions are as follows: the equipment, chromatographic column, mobile phase and set ion pair parameters and ion source parameters used were the same as in example 1.
The standard curve equation fitting method is referred to example 1.
Preparing a sample solution: taking 0.01mol/L hydrochloric acid aqueous solution as a diluent, precisely weighing the crude liraglutide (2 batches) and the refined liraglutide (2 batches) respectively to prepare four sample solutions with the concentration of 4mg/mL, and injecting the four sample solutions into a liquid chromatograph-mass spectrometer for detection. And (3) calculating the IPTG concentration in the sample by comparing the obtained standard curve, and calculating the IPTG content according to the concentration (4 mg/mL) of the liraglutide, wherein the specific result is shown in Table 13, the XIC mass spectrum of the crude liraglutide is shown in figure 11, and the XIC mass spectrum of the refined liraglutide is shown in figure 12.
TABLE 13 IPTG content detection results in crude and fine liraglutide samples
And (4) conclusion: the analysis method can simply and quickly detect the IPTG content in the liraglutide product by using lower quantitative limit and detection limit. Wherein the IPTG concentration in the two batches of crude liraglutide products is 0.422ng/mL or 0.378ng/mL respectively, and IPTG residue is not detected in the two batches of refined liraglutide products, thereby proving the effectiveness of the purification process.
Example 6 replacement with amido-bonded ethylene bridged hybrid silica stationary phase
The amido bonded ethylene bridge hybrid silica gel stationary phase is used for replacing the polyethyleneimine bonded silica gel stationary phase in the example 1, the liquid phase mass spectrum parameters of other mobile phases are the same as those in the example 1, the standard solution preparation and the sample solution preparation are the same as those in the example 2, and the IPTG content in the crude insulin aspart product and the refined insulin aspart product is detected, wherein the specific results are shown in a table 14.
TABLE 14 results of IPTG content detection in insulin aspart after stationary phase replacement
And (4) conclusion: the analytical method of the invention is replaced by the amido bonded ethylene bridge hybrid silica gel stationary phase, and can also simply and rapidly detect the IPTG content in the insulin aspart product by using lower quantitative limit and detection limit. Wherein the IPTG concentration in the crude insulin aspart products of the two batches is 0.545ng/mL and 0.508ng/mL respectively, and IPTG residue is not detected in the refined insulin aspart products of the two batches, so that the effectiveness of the purification process is proved, and the impurity requirement of the obtained product meets the pharmacopoeia standards of various countries.
Example 7 (use of a mixed solvent of ethanol and water as a mobile phase)
A mixed solvent mobile phase of ethanol and water is used for replacing a methanol and water mobile phase system in the embodiment 1, liquid phase mass spectrum parameters of other mobile phases are the same as those in the embodiment 1, standard solution preparation and sample solution preparation are the same as those in the embodiment 2, and IPTG content in crude insulin aspart and refined insulin aspart is detected, and specific results are shown in a table 15.
TABLE 15 results of IPTG content detection in insulin aspart after mobile phase replacement
And (4) conclusion: according to the analysis method disclosed by the invention, the mixed solvent of ethanol and water is used as a mobile phase, the IPTG content in the insulin aspart product can be simply and quickly detected at a lower quantitative limit and a lower detection limit, and although the detected peak shape is not sharp as the peak shape detected by using a methanol and water mixed system as the mobile phase, the judgment of a final result is not influenced. Wherein the IPTG concentrations in the crude insulin aspart products of the two batches are 0.553ng/mL and 0.527ng/mL respectively, and IPTG residues are not detected in the refined insulin aspart products of the two batches, so that the effectiveness of the purification process is proved, and the impurity requirements of the obtained product meet the pharmacopoeia standards of various countries.
Comparative example 1
The chromatographic column of example 1 (using polyethyleneimine as stationary phase, thermo Hypersil GOLD HILIC 150mm × 2.1mm,3 μm) was replaced by a Waters CORTECS C8 chromatographic column (100 mm × 2.1mm,1.6 μm) and a Waters ACQUITY UPLC BEH C18 chromatographic column (100 mm × 2.1mm,1.7 μm), respectively, other equipment and parameters were the same as those of example 1, and other standard solution preparation and sample solution preparation methods were the same as those of example 2, so that IPTG content in crude insulin aspart was detected, and the results are shown in FIGS. 13 and 14, which indicate that IPTG has poor separation effect on both C8 and C18 chromatographic columns, disordered mass spectrum signals, high baseline, and inaccurate quantification. The reason for the analysis may be that IPTG belongs to a hydrophilic substance, is easily dissolved in water and methanol, has short retention time in chromatographic columns of C8 and C18, is not easy to be effectively separated from a large amount of polar substances and organic salts in a matrix, seriously interferes mass spectrum signals, and cannot be accurately and quantitatively detected.
Comparative example 2
Methanol and water were used in a 50: gradient elution is carried out at a volume ratio of 50, other equipment and parameters are the same as those in example 1, reference example 2 is prepared by other standard solutions, and the IPTG content in the standard solution is detected, and the result is shown in figure 15, which shows that methanol and water (50) isocratic elution can cause phenomena of asymmetric peak shape, serious tailing, unreasonable quantitative detection and the like.
Claims (10)
1. A method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside is characterized by comprising the following steps: detecting the residual quantity of isopropyl-beta-D-thiogalactoside in polypeptide or protein by a liquid chromatography tandem mass spectrometry, wherein the liquid chromatography adopts a hydrophilic action chromatographic column and a mixed solvent of an alcohol solvent and water as a mobile phase.
2. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 1, characterized in that it comprises the following steps:
1) Preparing isopropyl-beta-D-thiogalactoside standard solutions with different concentrations, performing liquid chromatography tandem mass spectrometry detection, and drawing a standard curve;
2) Preparing a polypeptide or protein sample solution, and performing liquid chromatography tandem mass spectrometry detection;
3) And (2) obtaining the concentration of isopropyl-beta-D-thiogalactoside in the polypeptide or protein sample solution by contrasting the standard curve obtained in the step 1), thereby realizing the quantitative detection of the isopropyl-beta-D-thiogalactoside.
3. The method for quantitatively detecting the content of isopropyl- β -D-thiogalactoside according to any one of claims 1 or 2, wherein the polypeptide or protein comprises insulin and its analogs, glucagon-like peptide and its analogs.
4. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 1, wherein the stationary phase used in the hydrophilic interaction chromatographic column is a polyethyleneimine bonded silica gel stationary phase.
5. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 1, wherein the alcohol solvent in the mobile phase is methanol or ethanol.
6. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 5, wherein the mobile phase consisting of methanol and water or the mobile phase consisting of ethanol and water is subjected to gradient elution.
7. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 6, wherein the gradient elution has a volume ratio of methanol or ethanol to water of 5-90:95-10.
8. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 2, wherein the standard solution of the step 1) and the sample solution of the step 2) are prepared by using an acidic solution as a diluent.
9. The method for quantitatively detecting the content of isopropyl-beta-D-thiogalactoside according to claim 6, wherein the acidic solution comprises one or more of aqueous hydrochloric acid, aqueous phosphoric acid, formic acid or acetic acid.
10. The method for quantitatively detecting the content of isopropyl- β -D-thiogalactoside according to claim 2, wherein the polypeptide or protein sample of step 2) is subjected to a purification treatment.
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