CN117890502A - Method for determining alpha-lactalbumin content in lactose - Google Patents

Method for determining alpha-lactalbumin content in lactose Download PDF

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CN117890502A
CN117890502A CN202311814622.3A CN202311814622A CN117890502A CN 117890502 A CN117890502 A CN 117890502A CN 202311814622 A CN202311814622 A CN 202311814622A CN 117890502 A CN117890502 A CN 117890502A
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solution
placing
membrane
lactose
reference substance
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陈兵
袁琪
朱子丰
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Shanghai Mingjie Pharmaceutical Technology Co ltd
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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
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • 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

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Abstract

The invention belongs to the technical field of alpha-lactalbumin, and particularly relates to a method for measuring the content of alpha-lactalbumin in lactose. The method comprises the following steps: preparing an alpha-lactalbumin solution as a reference substance stock solution I; diluting the first reference substance stock solution into a second reference substance stock solution; diluting the reference stock solution II to different final concentrations, placing in a ultrafiltration tube, centrifuging, placing on a membrane, and detecting by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry; heating lactose for dissolving, recovering to room temperature, centrifuging, placing on a membrane, dissolving, and taking the solution on the membrane as sample solution; adding a standard solution to a test sample: heating lactose for dissolving, recovering to room temperature, adding reference stock solution II, mixing, sucking the solution, centrifuging, and collecting the solution on the membrane as sample and standard solution; ultra-high performance liquid chromatography-tandem Q Exactive Plus high resolution mass spectrometry detection. The invention is simple and easy to operate and has high sensitivity.

Description

Method for determining alpha-lactalbumin content in lactose
Technical Field
The invention belongs to the technical field of alpha-lactalbumin, and particularly relates to a method for measuring the content of alpha-lactalbumin in lactose.
Background
Lactose is a pharmaceutical excipient with wide application, and can be used as excipient, diluent in oral tablet, protective agent of freeze-dried injection, etc. But at present, the Chinese pharmacopoeia and the pharmacopoeia of each country have no lactose standard for injection.
Lactose is prepared from bovine whey, wherein the main components of the whey are lactose, fat and ash, niu Ruqing are defatted, deproteinized, evaporated and concentrated, cooled, washed, crystallized and dried to obtain lactose, so that trace amounts of whey proteins including alpha-bovine whey protein, beta-bovine whey globulin, bovine serum albumin, immunoglobulin and other proteins remain in the lactose. Recently, some injections using lactose as an auxiliary material have been reported to find allergic reactions in clinical applications, and the allergen is explicitly whey protein remained in lactose.
The pharmacopoeia of each country prescribes that the residual protein in lactose is detected by a chemical method and an absorbance method, while the pharmacopoeia of the Chinese pharmacopoeia and the drug administration of Japanese detect the residual protein by the absorbance method only, but the sensitivity of the chemical examination method or the absorbance examination method is too low, and the method is only suitable for detecting the residual protein of oral lactose, and the residual protein is in microgram level for injection lactose, so that a method for detecting trace residual protein with high sensitivity is established, the anaphylactic reaction of the injection lactose is controlled, and the safety of the injection containing lactose is guaranteed. The current method for detecting beta-lactoglobulin residues is mainly an enzyme-linked immunosorbent assay. However, α -lactalbumin has not been detected by very sophisticated methods, and development of a suitable method is an urgent task.
The quantitative method of protein LC-MS has two strategies, wherein the first strategy is to directly detect the protein layer LC-MS, the second strategy is to analyze the characteristic peptide fragments corresponding to the detected protein after the protease is cut into peptide fragments, in general, the first strategy is selected to be quantitative under 10 kDa, and the second strategy is selected to be the protein with more than 10 kDa. The monoisotopic theoretical molecular weight of alpha-lactalbumin is 14168.746 Da, the peptide fragment method is mainly used for quantification in the literature, and the method is mainly applied to dairy products, but the literature for detecting the alpha-lactalbumin in lactose is very little. In the prior art, the limit level of the detection of the alpha-lactalbumin residue is high by directly carrying out the detection of the alpha-lactalbumin residue on the whole protein layer, and the detection requirement of the alpha-lactalbumin residue in lactose for injection cannot be met.
Therefore, there is a need to develop a novel method which is suitable for not only proteins of 10 kDa or more but also for detecting the residual of α -lactalbumin at the intact protein level with a low limit level.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a method for determining the alpha-lactalbumin content of lactose which is simple and easy to implement and has a high sensitivity.
To achieve the above and other related objects, the present invention provides the following technical solutions:
A method for determining the alpha-lactalbumin content in lactose based on ultrafiltration pretreatment-ultra-high performance liquid chromatography-tandem Q Exactive Plus high resolution mass spectrometry comprising the steps of:
Preparing a control stock solution: weighing and dissolving alpha-lactalbumin to obtain alpha-lactalbumin solution, and obtaining reference substance stock solution I; diluting the first reference substance stock solution into a second reference substance stock solution, and diluting the second reference substance stock solution into a third reference substance stock solution;
It should be noted that: the method of the invention can quantify the whole protein: the protein quantification can be performed by using characteristic peptide fragment quantification and complete protein quantification, the conventional protein quantification scheme is based on a characteristic peptide fragment method, the sample needs to be subjected to pretreatment of denaturation, reduction, alkylation, enzyme digestion and acidification, the pretreatment is complex, and the time consumption is long (generally 16-24 h).
Preparing a linear verification solution: and (3) diluting the reference substance stock solution II with different volumes to different final concentrations, respectively precisely sucking the solution, placing the solution into a ultrafiltration tube, centrifuging, placing the solution on a membrane, taking the solution on the membrane to obtain a linear verification solution, detecting the linear verification solution by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry method, drawing a standard curve by taking the mass spectrum peak area as an ordinate and the concentration as an abscissa, and obtaining a regression equation.
In one embodiment of the invention, accuracy and repeatability tests are performed:
Preparing a test solution: weighing lactose, placing into a centrifuge tube, heating for dissolving, recovering to room temperature, sucking the solution, placing into a ultrafiltration tube, centrifuging, placing onto a membrane, dissolving, and taking the solution on the membrane as a sample solution;
Adding a standard solution to a test sample: weighing lactose, placing into a centrifuge tube, heating for dissolving, returning to room temperature, adding the reference substance stock solution II, uniformly mixing, absorbing the solution, placing into a ultrafiltration tube, centrifuging, placing onto a membrane, and taking the solution on the membrane to obtain a sample adding standard solution;
detecting the sample solution and the sample labeling solution by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry, and calculating the result according to a single-point contrast method.
By adopting the technical scheme, the sample pretreatment method can bring high sensitivity to ultrafiltration: since lactose is the main component of the sample, the amount of protein in lactose is very trace, if a limit of 0.15 ng/mg (protein/lactose mass ratio) is detected, no pretreatment is usually performed, and a large amount of lactose interference is caused, so that the direct detection is very difficult to meet the requirement.
Further, the centrifugal parameter is 10000-17000 g, the centrifugal is carried out for 10-30 min, and the heating temperature is 50-100 ℃.
Further, the specific steps may be as follows:
Preparing a control stock solution: weighing and dissolving alpha-lactalbumin to obtain alpha-lactalbumin solution, and obtaining reference substance stock solution I; diluting the first reference substance stock solution into a second reference substance stock solution with the concentration of 5 mu g/mL, and diluting the second reference substance stock solution into a third reference substance stock solution with the concentration of 0.2 mu g/mL;
Preparing a linear verification solution: diluting the reference substance stock solution II with different volumes by using ultrapure water to obtain final concentrations of 60 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL and 400 ng/mL, respectively precisely sucking 200 mu L of the solution into a ultrafiltration tube, centrifuging 13000 g for 25: 25 min, adding pure water to a membrane, blowing and dissolving by a liquid mover, taking the solution on the membrane to obtain a linear verification solution, detecting the linear verification solution by using an ultra-high performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry, taking the mass spectrum peak area as an ordinate and the concentration as an abscissa, drawing a standard curve and obtaining a regression equation;
in the accuracy and repeatability test:
Preparing a test solution: weighing lactose, placing into a centrifuge tube, adding ultrapure water, heating to dissolve at 80 ℃, recovering to room temperature, sucking the solution, placing into a ultrafiltration tube, centrifuging 13000 g to 25: 25 min, adding pure water, placing onto a membrane, blowing by a pipette to dissolve, taking the solution on the membrane to obtain a solution serving as a sample, and preparing multiple parts in parallel;
Preparing a sample adding and marking solution: weighing lactose, placing into a centrifuge tube, adding ultrapure water, heating to dissolve at 80 ℃, recovering to room temperature, adding the reference substance stock solution II, uniformly mixing, absorbing the solution, placing into a ultrafiltration tube, centrifuging 13000 g for 25: 25 min, adding pure water, placing onto a membrane, blowing by a liquid transfer device to dissolve, taking the solution on the membrane to obtain the sample-adding standard solution, and preparing multiple parts in parallel.
In one embodiment of the present invention, the conditions of the ultra performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry include:
Chromatographic conditions: the chromatographic column is MAbPacTM RP chromatographic columns, 2.1 mm multiplied by 50 mm and 4 mu m;
Mobile phase a is an aqueous solution containing 0.1% formic acid;
mobile phase B is an acetonitrile solution containing 0.1% formic acid;
The flow rate is 0.3 mL.min -1; the column temperature is 80 ℃; the sample injection amount is 10 mu L;
Gradient elution conditions: 0-15 min, wherein the volume percentage of the mobile phase B is changed from 28% to 35%; 15-17 min, wherein the volume percentage of the mobile phase B is changed from 35% to 80%; 17-17.5 min, the volume percentage of the mobile phase B is changed from 80% to 28%; 17.5-20 min, and the volume percentage of the mobile phase B is 28%.
Further, the conditions of the ultra performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry include: mass spectrometry conditions: and quantitatively adopting a SIM mode, and setting the target m/z as 1576.20.
By adopting the technical scheme, the specific parameters of the ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry set by the invention bring high specificity and high sensitivity: the high-resolution mass spectrum has high resolution, is used for quantifying ions from target proteins, can eliminate interference of small molecular weight difference, has high sensitivity, and can reach ng/ml level.
In an embodiment of the present invention, the method further includes the following steps:
And (3) carrying out specificity verification: preparing a specificity verification solution: the specificity verification solution comprises blank solution, reference substance solution and labeled test sample solution;
taking ultrapure water as a blank solution;
placing the reference substance stock solution III in a ultrafiltration tube, centrifuging, placing on a membrane, dissolving, and taking the solution on the membrane as a reference substance solution;
weighing lactose, placing the lactose into a centrifuge tube, heating and dissolving, returning to room temperature, adding the reference substance stock solution II, uniformly mixing, absorbing and uniformly mixing the solution, placing the solution into a ultrafiltration tube, centrifuging, placing the solution on a membrane, dissolving, and taking the solution on the membrane as a solution for adding a standard sample;
And taking 3 specificity verification solutions, respectively carrying out sample injection analysis according to an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry, and superposing the extracted ion flow diagrams of the 3 specificity verification solutions on the same abscissa.
Further, the centrifugation parameter is 10000-17000 g and centrifugation is carried out for 10-30 min.
Further, the specific steps may be as follows:
In the specificity verification: placing the reference substance stock solution III in a ultrafiltration tube, centrifuging 13000 g to 25: 25 min, adding pure water onto the membrane, blowing by a liquid transfer device to dissolve, and taking the solution on the membrane as reference substance solution;
Weighing lactose, placing into a centrifuge tube, adding ultrapure water, heating to dissolve at 80 ℃, returning to room temperature, adding the second reference substance stock solution, uniformly mixing, sucking the uniformly mixed solution, placing into a ultrafiltration tube, centrifuging 13000 g for 25: 25 min, adding pure water onto a membrane, blowing with a liquid transfer device to dissolve, and taking the solution on the membrane as a solution for adding a standard sample.
In an embodiment of the present invention, the method further includes the following steps:
solution stability detection was performed: and (3) respectively adding the reference substance solution and the sample to be tested into the standard solution, and detecting by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry method.
Further, specific parameters may be as follows:
detection conditions in performing the solution stability detection: the detection time is 0-24.6 h, and the detection temperature is 2-8 ℃.
It should be noted that: when the existing mass spectrometry is used for detecting alpha-lactalbumin in lactose, the interference of lactose is very serious when the trace (such as 0.3 ng/mg) limit is achieved, and the mass spectrometry cannot obtain the signal of the alpha-lactalbumin, so that the existing mass spectrometry is low in mass spectrometry sensitivity for detecting the alpha-lactalbumin, and the linear range is 50 mug/ml. In addition, ultrafiltration generally requires multiple solution substitutions, resulting in large protein losses, and for small volumes of sample (< 0.5 mL), there is no good method for metering the volume in the prior art, resulting in poor batch-to-batch reproducibility. For the defects and the technical problems existing in the prior art, the ultrafiltration centrifuge tube for ultrafiltration concentration enrichment is selected as a flat-bottom ultrafiltration tube, the molecular weight cut-off is 3 kDa, the centrifugal force is 13000 g, the centrifugal force is carried out until all liquid is separated to dryness, pure water with the same volume is used for redissolution, and other parameters of the invention are matched, so that the interference of lactose in the lactose alpha-lactalbumin detection process by a mass spectrometry method is solved; the ultrafiltration tube is centrifuged once, so that the loss of samples subjected to repeated solution replacement is reduced, and the centrifugation is carried out until the samples are dried, so that the volume of the solution between the samples is consistent after the re-dissolution, and the problems of ultrafiltration protein loss and poor reproducibility can be solved.
The beneficial effects of the invention are as follows:
the method is based on ultra-filtration pretreatment-ultra-high performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry, and comprises the steps of treating a sample before ultra-filtration and detecting by using the ultra-high performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry.
First, the sample pretreatment by ultrafiltration according to the invention can bring high sensitivity: since lactose is the main component of the sample, the amount of protein in lactose is very trace, if a limit of 0.15 ng/mg (protein/lactose mass ratio) is detected, no pretreatment is usually performed, and a large amount of lactose interference is caused, so that the direct detection is very difficult to meet the requirement.
Secondly, the specific parameters of the ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry set by the invention bring high specificity and high sensitivity: the high-resolution mass spectrum has high resolution, is used for quantifying ions from target proteins, can eliminate interference of small molecular weight difference, has high sensitivity, and can reach ng/ml level. The protein concentration of the method is 60-400 ng/mL, and the linear relation is good. The method can meet the detection of trace alpha-lactalbumin in lactose, the lactose is used as an auxiliary material in an injection preparation, the trace protein is required to be detected, and in addition, compared with the prior art, the sensitivity of the method can be improved by 1000 times, and the LOD of the method can reach 0.15 ppm.
Third, the method of the invention allows quantification of intact proteins: the protein quantification can be performed by using characteristic peptide fragment quantification and complete protein quantification, the conventional protein quantification scheme is based on a characteristic peptide fragment method, the sample needs to be subjected to pretreatment of denaturation, reduction, alkylation, enzyme digestion and acidification, the pretreatment is complex, the time consumption is long (generally 16-24 h), the pretreatment is simple based on complete protein quantification, and the experimental period is short.
In conclusion, the invention establishes a method for measuring the content of alpha-lactalbumin in lactose by adopting ultrafiltration pretreatment-ultra-high performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry. The method is simple and easy to implement, high in sensitivity, accurate in quantification and good in precision, can meet the detection requirement of the alpha-lactalbumin in lactose samples, and provides a powerful and effective technical means for detecting the content of the alpha-lactalbumin in the pharmaceutical excipients.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
In the drawings:
FIG. 1 is a first-order mass spectrum of the α -lactalbumin of the present invention;
FIG. 2 is a superposition of specificity experiments of the present invention; wherein, the marks in the figures are as follows: 1. a control solution; 2. adding a standard solution into a test sample; 3. blank solution
Fig. 3 is a linear diagram of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise indicated, all reagents used in the examples were readily available from commercial companies or from home-made sources.
Wherein, the manufacturers of partial materials are as follows:
1. Sample and main reagent:
Lactose, purchased from aledine, i.e., lactose sample;
alpha-lactalbumin, sigma in the united states;
Ultrafiltration tube, flat bottom ultrafiltration tube, company PALL, U.S.
2. Instrument consumable
Vanquish Flex ultra high performance liquid chromatograph, Q Exactive Plus ultra high resolution mass spectrometer, MAbPac TM RP column (2.1 mm X50 mm, 4 μm), all of which are ThermoFisher, inc. of America.
In addition, the "membrane" in the present invention is an ultrafiltration membrane in an ultrafiltration tube.
Example 1
A method for determining the alpha-lactalbumin content of lactose, in this example the desired solution is prepared comprising the steps of:
1. Preparing a solution:
1.1 preparation of control stock:
precisely weighing 2mg of alpha-lactalbumin, adding ultrapure water 2mL for dissolution, transferring to a 10 mL measuring flask for constant volume, and obtaining a reference substance stock solution I of 0.2 mg/mL; transferring the control stock solution of 0.125 mL to a 5mL measuring flask to fix the volume to obtain a control stock solution II of 5 mug/mL; taking 40 mu L of reference substance stock solution II to a 1.5 mL centrifuge tube, adding 960 mu L of ultrapure water, and uniformly mixing to obtain 0.2 mu g/mL of reference substance stock solution III.
1.2 Preparation of a specificity verification solution
The specific verification solution is a blank solution, a reference substance solution and a marked test substance solution, which are 3 kinds.
Wherein,
① Taking ultrapure water as a blank solution;
② 200 mu L of reference substance stock solution III (the protein concentration of the reference substance stock solution III is 0.2 mu g/mL) is taken and placed in a 3 kD ultrafiltration tube, 13000 and g are centrifuged to 25 and min, 200 mu L of pure water is added and placed on a membrane, a liquid transfer device is used for blowing and dissolving, and the solution on the membrane is taken as a reference substance solution;
③ Precisely weighing lactose about 100 mg, placing into a 1.5 mL centrifuge tube, adding ultrapure water 0.48 and mL, heating and dissolving at 80 ℃, recovering to room temperature, adding 20 mu L of reference substance stock solution II (the protein concentration of the reference substance stock solution II is 5 mu g/mL), mixing, precisely sucking 200 mu L of the solution, placing into a 3 KD ultrafilter tube, 13000 g centrifuging to 25 min, adding 200 mu L of pure water, placing onto a membrane, blowing and dissolving by a liquid-transfering device, and taking the solution on the membrane as the solution for adding the standard test sample.
1.3 Linear verification solution, quantitative Limit solution, and detection Limit solution
And (3) diluting the reference substance stock solution II with different volumes by using ultrapure water to obtain final concentrations of 60, 100, 200, 300 and 400 ng/mL, precisely sucking 200 mu L of the solution, placing the solution into a 3KD ultrafilter tube, centrifuging 13000 g for 25 and min, adding 200 mu L of pure water onto a membrane, blowing and dissolving by a liquid transfer device, and taking the solution on the membrane to obtain the linear verification solution. Wherein 60 ng/mL of the linear verification solution is also the quantitative limiting solution. And (5) transferring 50 mu L of quantitative limiting solution, placing the solution into a 1.5 mL centrifuge tube, adding 50 mu L of ultrapure water, and uniformly mixing to obtain the detection limiting solution.
1.4 Verification of accuracy and repeatability solutions
Test solution: precisely weighing lactose about 100 mg, placing in 1.5 mL centrifuge tube, adding ultrapure water 0.5 mL, heating at 80deg.C for dissolving, and recovering to room temperature. Precisely sucking 200 μl of the above solution, placing in a 3KD ultrafilter tube, centrifuging 13000 g for 25 min, adding 200 μl of pure water, placing on a membrane, blowing with a pipette for dissolving, and collecting the solution on the membrane to obtain the solution as the sample. 2 parts were prepared in parallel.
Adding a standard solution to a test sample: precisely weighing lactose about 100 mg, placing in 1.5 mL centrifuge tube, adding ultrapure water 0.48 and mL, heating to 80deg.C for dissolving, recovering to room temperature, adding control stock solution 2 (5 μg/mL) 20 μl, and mixing. Precisely sucking 200 mu L of the solution, placing the solution in a 3 KD ultrafiltration tube, centrifuging the solution in 13000 g for 25 min, adding 200 mu L of pure water, placing the solution on a membrane, blowing and dissolving the solution by a liquid transfer device, and taking the solution on the membrane to obtain the sample adding standard solution. 6 parts were prepared in parallel.
Example 2
The method for determining the content of alpha-lactalbumin in lactose further comprises the following steps:
Setting chromatographic-mass spectrometry conditions:
Chromatographic conditions: the chromatographic column is MAbPac TM RP chromatographic column (2.1 mm multiplied by 50 mm, 4 mu m);
Mobile phase a is an aqueous solution containing 0.1% formic acid;
mobile phase B is an acetonitrile solution containing 0.1% formic acid;
The flow rate is 0.3 mL.min -1, and the column temperature is 80 ℃; the sample injection amount is 10 mu L;
Gradient elution conditions: 0-15 min,28% B to 35% B; 15-17 min,35% B to 80% B; 17-17.5 min,80% B- > 28% B; 17.5-20 min,28% B.
Mass spectrometry conditions: positive ion t-SIM scan mode, target ions: 1576.20 Primary Mass Spectrometry resolution 70000, microscan 1, isolation Window 4 m/z. And (3) data processing: and carrying out data processing by using chameleon software.
Example 3
The method for determining the alpha-lactalbumin content of lactose, unlike example 1, further comprises the steps of:
2. Detection of
2.1 Target m/z selection for alpha-lactalbumin quantification
A first-order mass spectrum of the alpha-lactalbumin reference substance acquired by Q Exactive Plus high-resolution mass spectrum is shown as figure 1, the molecular weight of the monoisotopic theory of the protein is 14168.746 Da, the protein is multi-charged in the electrospray ionization process, the charge valence state is distributed in 5-14, the valence state with the highest intensity is 9+, and the highest m/z is 1576.20. Therefore, the SIM mode is quantitatively employed, setting the target m/z to 1576.20.
2.2 Methodological verification
2.2.1 Specificity
The 3 specificity verification solutions of 1.2 in example 1 were taken and analyzed according to the chromatograph-mass spectrum conditions of example 2, respectively, and the extracted ion flowsheet of the 3 solutions was superimposed on the same abscissa, as shown in fig. 2. The result shows that at the retention time of 7.0 min of alpha-lactalbumin (namely the reference substance solution), the blank solution is free from interference, other peaks in the solution of the standard sample are free from interference at the target peak, and the method has good specificity.
2.2.2 Linearity and range
Taking the linear verification solution of 1.3 in the example 1, carrying out sample injection analysis according to the chromatographic-mass spectrometry condition of the example 2, drawing a standard curve by taking the mass spectrometry peak area as an ordinate and the concentration as an abscissa, and obtaining a regression equation. The results are shown in fig. 3, and the regression equation is y=718.8x-16289, and the regression coefficient R 2 is 0.992. The method shows that the protein concentration is 60-400 ng/mL, and the linear relation is good.
2.2.3 Limit of detection and limit of quantification
The linear verification solution of 1.3 in the example 1, the quantitative limit solution and the detection limit solution of the detection limit solution are respectively taken, and the detection is carried out according to the method of the example 2, and the result shows that when the concentration of the detection limit solution is 30 ng/mL, the signal to noise ratio S/N is 21; when the concentration of the quantitative limiting solution is 60 ng/mL, the signal to noise ratio S/N is 58, which shows that the method has good sensitivity.
2.2.4 Accuracy and repeatability
Taking the accuracy and repeatability verification solution of 1.4 in the embodiment 1, detecting according to the method of the embodiment 2, calculating according to a single-point contrast method, and detecting no sample solution in the accuracy result; 6 parts of sample addition standard solution (Re-100% -1-Re-100% -6) has a recovery rate of 82% -117%, and 6 parts of solution has a recovery rate of RSD of 12%, and the following table 1 is referred to.
TABLE 1
2.2.5 Solution stability
The control solution in the specificity verification solution prepared in 1.2 and the test sample addition solution in the accuracy and repeatability verification solution in 1.4 are respectively taken and detected according to the method of the example 2. At the temperature of 2-8 ℃, the ratio of the concentration value of the reference substance solution at each time point in 24.6 h to the concentration value of 0h is 84% -98%, which indicates that the reference substance solution is kept stable at least in 24.6 h under the condition. At the temperature of 2-8 ℃, the ratio of the concentration value of the sample adding and marking solution at each time point in 20.8 h to the concentration value of 0h is 94% -98%, which indicates that the sample adding and marking solution is at least kept stable in 20.8 h under the condition.
2.2.6 Sample detection
2 Parts of each of 3 lactose samples were taken, and test solutions were prepared according to the method of 1.4 verification of accuracy and repeatability in example 1, and the measurement was performed under the chromatographic mass spectrometry conditions in example 2, and no alpha-lactalbumin was detected in each of the 3 lactose samples.
In conclusion, the invention establishes a method for measuring the content of alpha-lactalbumin in lactose by adopting ultrafiltration pretreatment-ultra-high performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry. The method is simple and easy to implement, high in sensitivity, accurate in quantification and good in precision, can meet the detection requirement of the alpha-lactalbumin in lactose samples, and provides a powerful and effective technical means for detecting the content of the alpha-lactalbumin in the pharmaceutical excipients.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for determining the alpha-lactalbumin content of lactose, which method is based on ultrafiltration pretreatment-ultra-high performance liquid chromatography-tandem Q Exactive Plus high resolution mass spectrometry, comprising the steps of:
Preparing a control stock solution: weighing and dissolving alpha-lactalbumin to obtain alpha-lactalbumin solution, and obtaining reference substance stock solution I; diluting the first reference substance stock solution into a second reference substance stock solution, and diluting the second reference substance stock solution into a third reference substance stock solution;
Preparing a linear verification solution: taking the reference substance stock solution II with different volumes, diluting to different final concentrations, respectively precisely sucking the solution, placing the solution into a ultrafiltration tube, centrifuging, placing the solution on a membrane, taking the solution on the membrane to obtain a linear verification solution, detecting the linear verification solution by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry, taking the mass spectrum peak area as an ordinate and the concentration as an abscissa, drawing a standard curve and obtaining a regression equation;
accuracy and repeatability tests were performed:
Preparing a test solution: weighing lactose, placing into a centrifuge tube, heating for dissolving, recovering to room temperature, sucking the solution, placing into a ultrafiltration tube, centrifuging, placing onto a membrane, dissolving, and taking the solution on the membrane as a sample solution;
Adding a standard solution to a test sample: weighing lactose, placing into a centrifuge tube, heating for dissolving, returning to room temperature, adding the reference substance stock solution II, uniformly mixing, absorbing the solution, placing into a ultrafiltration tube, centrifuging, placing onto a membrane, and taking the solution on the membrane to obtain a sample adding standard solution;
detecting the sample solution and the sample labeling solution by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry, and calculating the result according to a single-point contrast method.
2. The method for determining the alpha-lactalbumin content of lactose as claimed in claim 1, wherein,
Preparing a control stock solution:
Weighing alpha-lactalbumin, adding ultrapure water for dissolution, transferring into a measuring flask for constant volume to obtain a reference substance stock solution I of 0.2 mg/mL; transferring the control stock solution to a measuring flask for constant volume to obtain a control stock solution II of 5 mug/mL; adding the second reference substance stock solution into a centrifuge tube, adding ultrapure water, and uniformly mixing to obtain a third reference substance stock solution of 0.2 mug/mL;
preparing a linear verification solution: diluting the reference substance stock solution II with different volumes by using ultrapure water to obtain final concentrations of 60 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL and 400 ng/mL, respectively precisely sucking 200 mu L of the solution into a 3KD ultrafiltration tube, centrifuging 13000 g to 25 min, adding pure water to a membrane, blowing and dissolving by a liquid transfer device, taking the solution on the membrane to obtain a linear verification solution, detecting the linear verification solution by using an ultra-high performance liquid chromatography-tandem Q Exactive Plus high-resolution mass spectrometry, taking the mass spectrum peak area as an ordinate and the concentration as an abscissa, drawing a standard curve and obtaining a regression equation;
Preparing a test solution: weighing lactose, placing into a centrifuge tube, adding ultrapure water, heating to dissolve at 80 ℃, recovering to room temperature, sucking the solution, placing into a ultrafiltration tube, centrifuging 13000 g to 25: 25 min, adding pure water, placing onto a membrane, blowing by a pipette to dissolve, taking the solution on the membrane to obtain a solution serving as a sample, and preparing multiple parts in parallel;
Preparing a sample adding and marking solution: weighing lactose, placing into a centrifuge tube, adding ultrapure water, heating to dissolve at 80 ℃, recovering to room temperature, adding the reference substance stock solution II, uniformly mixing, absorbing the solution, placing into a ultrafiltration tube, centrifuging 13000 g for 25: 25 min, adding pure water, placing onto a membrane, blowing by a liquid transfer device to dissolve, taking the solution on the membrane to obtain the sample-adding standard solution, and preparing multiple parts in parallel.
3. The method for determining the content of alpha-lactalbumin in lactose according to claim 1, wherein the centrifugation parameter is 10000-17000 g, the centrifugation is performed for 10-30 min, and the heating temperature is 50-100 ℃.
4. The method for determining the alpha-lactalbumin content of lactose according to claim 1, wherein the conditions of the ultra-high performance liquid chromatography-tandem Q Exactive Plus high resolution mass spectrometry comprise:
chromatographic conditions: the chromatographic column is MAbPac TM RP chromatographic column, 2.1 mm multiplied by 50 mm and 4 mu m;
Mobile phase a is an aqueous solution containing 0.1% formic acid;
mobile phase B is an acetonitrile solution containing 0.1% formic acid;
The flow rate is 0.3 mL.min -1; the column temperature is 80 ℃; the sample injection amount is 10 mu L;
Gradient elution conditions: 0-15 min, wherein the volume percentage of the mobile phase B is changed from 28% to 35%; 15-17 min, wherein the volume percentage of the mobile phase B is changed from 35% to 80%; 17-17.5 min, the volume percentage of the mobile phase B is changed from 80% to 28%; 17.5-20 min, and the volume percentage of the mobile phase B is 28%.
5. The method for determining the alpha-lactalbumin content of lactose according to claim 1, wherein the conditions of the ultra-high performance liquid chromatography-tandem Q Exactive Plus high resolution mass spectrometry comprise:
Mass spectrometry conditions: and quantitatively adopting a SIM mode, and setting the target m/z as 1576.20.
6. The method for determining the alpha-lactalbumin content of lactose according to claim 1, further comprising the steps of:
And (3) carrying out specificity verification: preparing a specificity verification solution: the specificity verification solution comprises blank solution, reference substance solution and labeled test sample solution;
taking ultrapure water as a blank solution;
placing the reference substance stock solution III in a ultrafiltration tube, centrifuging, placing on a membrane, dissolving, and taking the solution on the membrane as a reference substance solution;
weighing lactose, placing the lactose into a centrifuge tube, heating and dissolving, returning to room temperature, adding the reference substance stock solution II, uniformly mixing, absorbing and uniformly mixing the solution, placing the solution into a ultrafiltration tube, centrifuging, placing the solution on a membrane, dissolving, and taking the solution on the membrane as a solution for adding a standard sample;
And taking 3 specificity verification solutions, respectively carrying out sample injection analysis according to an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry, and superposing the extracted ion flow diagrams of the 3 specificity verification solutions on the same abscissa.
7. The method for determining the content of alpha-lactalbumin in lactose according to claim 6, wherein the third reference stock solution is placed in a ultrafiltration tube, the method comprises the steps of centrifuging 13000 g to 25 min, placing pure water on a membrane, blowing and dissolving by a liquid transfer device, and taking the solution on the membrane as a reference solution;
Weighing lactose, placing into a centrifuge tube, adding ultrapure water, heating to dissolve at 80 ℃, returning to room temperature, adding the second reference substance stock solution, uniformly mixing, sucking the uniformly mixed solution, placing into a ultrafiltration tube, centrifuging 13000 g for 25: 25 min, adding pure water onto a membrane, blowing with a liquid transfer device to dissolve, and taking the solution on the membrane as a solution for adding a standard sample.
8. The method for determining the content of alpha-lactalbumin in lactose according to claim 6, wherein the centrifugation parameter is 10000-17000 g for 10-30 min.
9. The method for determining the alpha-lactalbumin content of lactose according to claim 1, further comprising the steps of:
solution stability detection was performed: and (3) respectively adding the reference substance solution and the sample to be tested into the standard solution, and detecting by using an ultra-high performance liquid chromatography-serial Q Exactive Plus high-resolution mass spectrometry method.
10. The method for determining the alpha-lactalbumin content of lactose according to claim 9, wherein the detection conditions are as follows: the detection time is 0-24.6 h, and the detection temperature is 2-8 ℃.
CN202311814622.3A 2023-12-27 2023-12-27 Method for determining alpha-lactalbumin content in lactose Pending CN117890502A (en)

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