CN119780301B - Peptide map detection and analysis method for thrombopoietin peptoid - Google Patents
Peptide map detection and analysis method for thrombopoietin peptoidInfo
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Abstract
本发明属于药物分析技术领域，具体涉及一种血小板生成素拟肽的肽图检测分析方法，包括以下步骤：（1）血小板生成素拟肽通过超滤离心进行缓冲体系置换；（2）采用胰蛋白酶对血小板生成素拟肽进行酶解；（3）反相高效液相色谱法对酶解的肽段进行检测分析。本发明的有益效果：本发明中通过超滤管进行样品浓缩与缓冲液置换，且酶解后的样品纯度高，可直接用反相高效液相色谱进行检测，从而该血小板生成素拟肽的肽图检测分析方法的操作简便，提高了血小板生成素拟肽类药物的检测效率；本发明中通过控制色谱条件，提高高效液相色谱检测的准确性和精确度，能够实现血小板生成素拟肽类药物的质量分析和控制。
The present invention belongs to the technical field of drug analysis, and specifically relates to a peptide mapping detection and analysis method for thrombopoietin mimetic peptides, comprising the following steps: (1) thrombopoietin mimetic peptides are subjected to buffer system replacement by ultrafiltration centrifugation; (2) thrombopoietin mimetic peptides are enzymatically hydrolyzed by trypsin; and (3) the enzymatically hydrolyzed peptide segments are detected and analyzed by reversed-phase high-performance liquid chromatography. Beneficial effects of the present invention include: in the present invention, sample concentration and buffer replacement are performed by ultrafiltration tubes, and the sample after enzymatic hydrolysis has high purity and can be directly detected by reversed-phase high-performance liquid chromatography, thereby simplifying the operation of the peptide mapping detection and analysis method for thrombopoietin mimetic peptides and improving the detection efficiency of thrombopoietin mimetic peptide drugs; in the present invention, the accuracy and precision of high-performance liquid chromatography detection are improved by controlling chromatographic conditions, thereby enabling quality analysis and control of thrombopoietin mimetic peptide drugs.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a peptide map detection and analysis method of thrombopoietin peptidomimetic.
Background
The thrombopoietin peptidomimetic is a Thrombopoietin (TPO) receptor agonist, and through specific binding with TPO receptor, activates cell pathways, stimulates proliferation, differentiation and maturation of megakaryocytes, promotes the generation of platelets, can remarkably improve the level of platelets and reduce the bleeding risk.
The peptide map is characterized in that according to the amino acid composition characteristics of the protein, specific proteolytic enzyme acts on specific sites to crack the protein polypeptide into small fragments, and a characteristic fingerprint map is formed by a certain separation detection method (such as high performance liquid chromatography). With the continuous development of the biopharmaceutical field, peptide map detection has become a key method in the process of representing a bioprotein drug, and high-specificity representation of the bioprotein structure is ensured. In the conventional recombinant protein peptide map detection method at present, a buffer system is replaced by a membrane dialysis method, so that the time cost is high, the operation is complex, and more impurities are introduced in the dialysis process. Therefore, how to design a peptide map detection method of thrombopoietin peptidomimetic can simply and efficiently obtain a proteolytic sample with higher purity, and save time and cost, and the method is a problem to be solved urgently.
Disclosure of Invention
The present invention provides a peptide map detection and analysis method for thrombopoietin peptidomimetic peptide, which aims at the problems described above.
The invention relates to a peptide map detection and analysis method of thrombopoietin peptoid, which comprises the following steps:
(1) The thrombopoietin peptidomimetic is subjected to buffer system replacement by ultrafiltration centrifugation;
(2) Carrying out enzymolysis on the thrombopoietin peptoid by adopting trypsin;
(3) And detecting and analyzing the enzymatic hydrolysis peptide fragments by using a reversed-phase high performance liquid chromatography.
In the step (1), the sample and the reference substance are diluted to 1.0mg/ml respectively by using an ammonium bicarbonate solution with the mass percent concentration of 1% to obtain a sample diluent and a reference substance diluent;
Taking a sample diluent, a reference diluent and a blank diluent, respectively adding the sample diluent, the reference diluent and the blank diluent into a 10kD ultrafiltration tube, centrifuging for 30min, discarding the lower filtrate after centrifuging, adding an ammonium bicarbonate solution with the mass percent concentration of 1% into the 10kD ultrafiltration tube, centrifuging for 30min, repeating the centrifuging operation twice, flushing filter membranes on two sides of the ultrafiltration tube with the ammonium bicarbonate solution with the mass percent concentration of 1%, and collecting washing liquid in an ultrafiltration sleeve to obtain a sample solution, a reference solution and a blank solution.
In the step (2), a sample solution, a reference substance solution and a blank solution in an ultrafiltration sleeve are taken, trypsin solution with the mass concentration of 0.5mg/ml is respectively added to carry out enzymolysis reaction at 37 ℃, acetic acid solution with the mass percent concentration of 50% is respectively added to stop enzyme digestion after enzyme digestion is finished, and supernatant fluid is collected after centrifugation, so that the sample supernatant fluid, the reference substance supernatant fluid and the blank supernatant fluid are obtained.
Preferably, the volume ratio of the sample solution, the reference solution, the blank solution and the trypsin solution is 10:1, and the volume ratio of the sample solution, the reference solution, the blank solution and the acetic acid solution is 10:1.
Preferably, the step (3) comprises sample configuration, reversed-phase high performance liquid chromatography detection and experimental result analysis, wherein in the reversed-phase high performance liquid chromatography detection, the chromatographic conditions are as follows:
Chromatographic column, kromasil 300-5-C18 chromatographic column, 4.6X105 mm,5 μm;
a protective column, namely Kromasil 300-5-C18 3.0-4.6mm guard starter kit;
Mobile phase A, 0.1% trifluoroacetic acid-water solution;
Mobile phase B, 0.1% trifluoroacetic acid-acetonitrile solution;
Mobile phase C, 70% acetonitrile-water solution;
the detection wavelength is 214nm;
The sample injection volume is 50 μl;
the flow rate is 1.0ml/min;
Column temperature is 35 ℃;
Sample tray temperature 4 ℃;
the elution mode is gradient elution.
Preferably, the gradient elution is performed by the following steps:
。
Preferably, in the sample preparation, a blank supernatant is taken as an enzyme digestion blank solution, 3 parallel samples are prepared from a control supernatant and marked as an enzyme digestion control solution 1, an enzyme digestion control solution 2 and an enzyme digestion control solution 3, a test supernatant is taken as an enzyme digestion test solution, and the control supernatant and the test supernatant with the same volume are taken and evenly mixed to be used as an enzyme digestion control-test 1:1 mixed solution.
Preferably, in the reversed-phase high performance liquid chromatography detection, the sample injection sequence is a digestion blank solution, a digestion reference substance solution 1, a digestion reference substance solution 2, a digestion reference substance solution 3, a digestion test substance solution and a digestion reference substance-test substance 1:1 mixed solution in sequence.
Preferably, in the reversed-phase high performance liquid chromatography detection, the integration parameters are:
the integration algorithm is Apextrack, the peak detection is carried out, the integration interval is 4.5-40, the peak width is 10s, the threshold value is 300, the peak integration is that the peak starting point% is 0.5%, the peak end point% is 0.5%, the minimum height is 0 mu V, and the minimum area is 300000.
Preferably, in the analysis of the experimental result, the identification peak comprises an identification peak 1, an identification peak 2, an identification peak 3 and an identification peak 4, and the relative retention time of the identification peak 1, the identification peak 2, the identification peak 3 and the identification peak 4 is 18.750 min+/-0.5 min, 22.897 +/-0.5 min, 25.069 +/-0.5 min and 27.100+/-0.5 min respectively;
The experiment establishment standard is that 1) no obvious new peak appears in the peptide map result, the judgment standard of the obvious new peak is that the total peak area of the new peak area in the peptide map is more than or equal to 0.5 percent, the peak area can be detected in the peptide map of the mixed solution of the reference substance=1:1 to be tested, and the peak area is 50+/-20 percent of the peptide map of the solution to be tested, and 2) the peak shapes of all the identification peaks of the enzyme digestion reference substance solution 1, the enzyme digestion reference substance solution 2 and the enzyme digestion reference substance solution 3 are consistent;
The detection result judging standard comprises 1) enzyme cutting reference substance solution 1, enzyme cutting reference substance solution 2, enzyme cutting reference substance solution 3, enzyme cutting test substance solution and enzyme cutting reference substance-sample 1:1 mixed solution, wherein the peak shapes of the identification peaks are consistent, 2) the identification peak 4 is taken as an internal standard peak, and the relative peak area ratio of the identification peaks of the test substance is required to be met, namely, the relative peak area ratio of the identification peaks of the test substance to the identification peaks of the reference substance is required to be 100+/-15%;
Calculation formula of relative peak area:
Wherein Area n is the peak Area of the n-th peptide fragment peak in the sample, n=1, 2,3.
Area Internal standard is the peak Area of the internal standard peak.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) According to the invention, the sample concentration and buffer replacement are carried out through the ultrafiltration tube, and the purity of the sample after enzymolysis is high, and the sample can be directly detected by reverse-phase high performance liquid chromatography, so that the operation of the peptide map detection and analysis method of the thrombopoietin peptoid is simple and convenient, and the detection efficiency of the thrombopoietin peptoid medicine is improved;
(2) According to the invention, by controlling chromatographic conditions, the accuracy and precision of high performance liquid chromatography detection are improved, and the quality analysis and control of thrombopoietin peptidomimetic medicaments can be realized;
(3) In the invention, three enzyme digestion reference substance solutions are established, and the establishment of a detection experiment is ensured through a system applicability test, so that the accuracy and the nature of thrombopoietin peptidomimetic are ensured.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly introduced,
FIG. 1 is a peptide map of the enzyme-digested blank solution provided in example 1;
FIG. 2 is an overlapping peptide view of the enzyme digestion control solutions 1,2, 3 provided in example 1;
FIG. 3 is a peptide map of the enzyme-cleaved sample solution provided in example 1;
FIG. 4 is a peptide map of the enzyme-cleaved control-test sample 1:1 mixed solution provided in example 1;
FIG. 5 shows the enzyme-cleaved reference substance 1, enzyme-cleaved reference substance solution 2, enzyme-cleaved reference substance solution 3, and enzyme-cleaved reference substance obtained in example 1 overlapping peptide patterns of enzyme cutting test sample solution and enzyme cutting reference substance-test sample 1:1 mixed solution.
Detailed Description
In order that the above objects, features and advantages of the invention may be more clearly understood, a further description of the invention will be provided with reference to the following examples.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein, and therefore the present invention is not limited to the specific embodiments of the disclosure that follow.
Example 1
A method for detecting and analyzing a peptide map of a thrombopoietin peptidomimetic, comprising the steps of:
(1) Solution preparation:
1% ammonium bicarbonate solution 1g ammonium bicarbonate is weighed, dissolved in ultrapure water and then fixed to 100ml.
A trypsin solution of 0.5mg/ml was prepared by taking 20. Mu.g of trypsin and adding 40. Mu.l of trypsin buffer to dilute to 0.5mg/ml.
Preparing a blank buffer solution, namely weighing 1g of polysorbate 20, adding purified water for dissolution, then fixing the volume to 100ml to obtain polysorbate 20 solution, weighing 8g of mannitol, 4g of sucrose and 0.32g of histidine, adjusting the pH to 5.0 by using dilute hydrochloric acid, adding water for dissolution, adding 0.8ml of the prepared polysorbate 20 solution, uniformly mixing, fixing the volume to 200ml, and filtering by using a 0.22um filter membrane to obtain the blank buffer solution.
(2) Sample solution dilution:
diluting the sample and the reference substance to 1.0mg/ml with 1% ammonium bicarbonate solution to obtain sample diluent and reference substance diluent, and diluting the blank buffer solution with 1% ammonium bicarbonate solution to obtain blank diluent.
(3) Ultrafiltration liquid exchange:
Taking 400 mu l of each of the sample diluent, the reference diluent and the blank diluent, respectively adding the sample diluent, the reference diluent and the blank diluent into a 10kD ultrafiltration tube, centrifuging at 4 ℃ for 30min, centrifuging, discarding the lower filtrate, adding 200 mu l of 1% ammonium bicarbonate solution into the ultrafiltration tube, centrifuging at 4 ℃ for 30min, repeating the centrifugation for two times, taking 200 mu l of 1% ammonium bicarbonate solution into the ultrafiltration tube, flushing the filter membrane of the ultrafiltration tube by a pipettor for 10 times (5 times on each of two side filter membranes), then pouring the ultrafiltration tube into another centrifugation sleeve, centrifuging at 4 ℃ for 3min, taking 1% ammonium bicarbonate solution into the ultrafiltration tube of the previous step twice, taking 100 mu l of each time, flushing the filter membrane of the ultrafiltration tube for 10 times (5 times on each of two side filter membranes), and collecting the washing solution into the ultrafiltration sleeve to obtain the sample solution, the reference solution and the blank solution.
(4) The trypsin carries out enzymolysis on the thrombopoietin peptoid:
Taking 400 mu l of each of the sample solution, the reference substance solution and the blank solution in the ultrafiltration sleeve, respectively adding 40 mu l of trypsin solution with the mass concentration of 0.5mg/ml, carrying out water bath for 18h at 37 ℃, respectively adding 40 mu l of acetic acid solution with the mass percent concentration of 50% after enzyme cutting, stopping enzyme cutting, carrying out centrifugation for 10min at 4 ℃, and collecting supernatant to obtain sample supernatant, reference substance supernatant and blank supernatant.
(5) Sample configuration:
Taking 200 mu l of blank supernatant as enzyme cutting blank solution, taking 110 mu l of each control supernatant to prepare 3 parallel samples, marking the parallel samples as enzyme cutting control solution 1, enzyme cutting control solution 2 and enzyme cutting control solution 3, taking 200 mu l of test supernatant as enzyme cutting test solution, taking 100 mu l of each control supernatant and each test supernatant, and uniformly mixing the two solutions to obtain an enzyme cutting control-test 1:1 mixed solution.
(6) Mobile phase configuration of high performance liquid chromatography:
Mobile phase a 2.0ml tfa was removed and added to 2000ml ultra pure water, after mixing well, the ultrasound was degassed for 20min.
Mobile phase B1.0 ml tfa was removed and added to 1000ml acetonitrile (chromatographic grade), mixed well and then degassed by ultrasound for 20min.
Mobile phase C350 ml acetonitrile (chromatographic grade) and 150ml ultra-pure water are measured and mixed evenly, and then ultrasonic degassing is carried out for 20min.
(7) Balancing and flushing before high performance liquid chromatography sample injection:
After washing the tubes with mobile phase A, B, C and washing the sample injection que, the column was installed at low flow rate according to the flow direction marked on the column, 10 column volumes were washed with 100% mobile phase B at a flow rate of 1.0ml/min, 10 column volumes were washed with 100% mobile phase a at a flow rate of 1.0m/min, and finally 20 column volumes were equilibrated with 98% mobile phase a and 2% mobile phase B at a flow rate of 1.0 ml/min.
(8) The chromatographic conditions of the high performance liquid chromatography are as follows:
the model of the high performance liquid chromatograph is Waters e2695;
Chromatographic column, kromasil 300-5-C18 chromatographic column, 4.6X105 mm,5 μm;
a protective column, namely Kromasil 300-5-C18 3.0-4.6mm guard starter kit;
Mobile phase A, 0.1% trifluoroacetic acid-water solution;
Mobile phase B, 0.1% trifluoroacetic acid-acetonitrile solution;
Mobile phase C, 70% acetonitrile-water solution;
the detection wavelength is 214nm;
The sample injection volume is 50 μl;
the flow rate is 1.0ml/min;
Column temperature is 35 ℃;
Sample tray temperature 4 ℃;
the elution mode is gradient elution.
The gradient elution procedure was:
。
(9) High performance liquid chromatography compiled batch analysis table:
Inputting sample bottle number, sample name, sample volume and sample sequence, wherein the sample sequence comprises a needle mobile phase (mobile phase A: mobile phase B=98:2), a needle enzyme cutting blank solution, a needle enzyme cutting reference substance solution 1, a needle enzyme cutting reference substance solution 2, a needle enzyme cutting reference substance solution 3, a needle enzyme cutting test sample solution and a needle enzyme cutting reference substance-test sample 1:1 mixed solution.
(10) High performance liquid chromatography sample injection and washing:
100% mobile phase C flushed the column at a flow rate of 1.0ml/min for 30min.
(11) Integration parameters of high performance liquid chromatography:
the integration algorithm is Apextrack, the peak detection, the integration interval is 4.5-40, the peak width is 10, the threshold value is 300, the peak integration is that the peak starting point% is 0.5%, the peak end point% is 0.5%, the minimum height is 0 mu V, and the minimum area is 300000.
(12) Analysis of experimental results:
The peptide diagram of the enzyme cutting blank solution is shown in figure 1, the overlapping peptide diagram of enzyme cutting reference substance solutions 1,2 and 3 is shown in figure 2, the chromatographic peak information is shown in table 1, the peptide diagram of enzyme cutting test substance solution is shown in figure 3, the peptide diagram of enzyme cutting reference substance-test substance 1:1 mixed solution is shown in figure 4, the overlapping peptide diagrams of enzyme cutting reference substance solution 1, enzyme cutting reference substance solution 2, enzyme cutting reference substance solution 3, enzyme cutting test substance solution and enzyme cutting reference substance-test substance 1:1 mixed solution are shown in figure 5, the chromatographic peak information is shown in table 2, and the relative peak area ratio result of each identification peak of the enzyme cutting test substance solution is shown in table 3.
TABLE 1 chromatographic peak results of overlapping peptide maps for the cleavage reference solutions 1,2,3
TABLE 2 chromatographic peak results of overlapping peptide patterns of the digested control, test and control-test 1:1 mixed solution
TABLE 3 results of relative peak area ratio of the identified peaks of the enzyme-digested sample solution
From fig. 1-5 and tables 1-3, the peak types of the identification peaks in the peptide graph are consistent, and the relative peak area ratio of the identification peaks without obvious new peaks is 100+/-15%, which shows that the batch of samples meets the detection standard.
The present invention is not limited to the above-mentioned embodiments, and any equivalent embodiments which can be changed or modified by the technical content disclosed above can be applied to other fields, but any simple modification and equivalent changes to the above-mentioned embodiments according to the technical substance of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (9)
1. A peptide map detection and analysis method of thrombopoietin peptoid is characterized by comprising the following steps of (1) replacing the thrombopoietin peptoid by a buffer system through ultrafiltration and centrifugation, (2) carrying out enzymolysis on the thrombopoietin peptoid by trypsin, and (3) carrying out detection and analysis on an enzymolysis peptide segment by reverse-phase high performance liquid chromatography;
The step (3) comprises sample preparation, reversed-phase high performance liquid chromatography detection and experimental result analysis, wherein in the reversed-phase high performance liquid chromatography detection, the chromatographic conditions are as follows:
Chromatographic column, kromasil 300-5-C18 chromatographic column, 4.6X105 mm,5 μm;
a protective column, namely Kromasil 300-5-C18 3.0-4.6mm guard starter kit;
Mobile phase A, 0.1% trifluoroacetic acid-water solution;
Mobile phase B, 0.1% trifluoroacetic acid-acetonitrile solution;
Mobile phase C, 70% acetonitrile-water solution;
the detection wavelength is 214nm, and the elution mode is gradient elution;
the gradient elution procedure was:
In the analysis of the experimental results, the identification peaks comprise an identification peak 1, an identification peak 2, an identification peak 3 and an identification peak 4, and the retention time of the identification peak 1, the identification peak 2, the identification peak 3 and the identification peak 4 is 18.750 min+/-0.5 min, 22.897 +/-0.5 min, 25.069 +/-0.5 min and 27.100+/-0.5 min respectively.
2. The method for detecting and analyzing the peptide map of the thrombopoietin peptidomimetic according to claim 1, wherein in the step (1), a sample and a reference substance are diluted to 1.0mg/ml by using an ammonium bicarbonate solution with the mass percent concentration of 1% respectively to obtain a sample diluent and a reference substance diluent, a proper amount of blank buffer solution is taken, the same multiple as the sample diluent is diluted by using the ammonium bicarbonate solution with the mass percent of 1%, the blank diluent is taken, the sample diluent, the reference substance diluent and the blank diluent are respectively added into a 10kD ultrafilter tube, the 10kD ultrafilter tube is centrifuged for 30min, the lower filtrate is centrifuged, the ammonium bicarbonate solution with the mass percent concentration of 1% is added into the 10kD ultrafilter tube, the centrifugation operation is repeated for two times, and filter membranes on both sides of the ultrafilter tube are washed by using the ammonium bicarbonate solution with the mass percent concentration of 1%, and washing solutions are collected in the ultrafilter tube, so as to obtain the sample solution, the reference substance solution and the blank solution.
3. The method for detecting and analyzing the peptide map of the thrombopoietin peptidomimetic according to claim 2, wherein in the step (2), a sample solution, a reference solution and a blank solution in an ultrafiltration sleeve are taken, respectively, trypsin solution with the mass concentration of 0.5mg/ml is added for enzymolysis reaction at 37 ℃, after the enzyme digestion is finished, acetic acid solution with the mass percent concentration of 50% is added for stopping the enzyme digestion, and supernatant is collected after centrifugation, so that the sample supernatant, the reference supernatant and the blank supernatant are obtained.
4. The method for detecting and analyzing the peptide map of thrombopoietin peptidomimetic according to claim 3, wherein the volume ratio of the sample solution, the control solution, the blank solution and the trypsin solution is 10:1, and the volume ratio of the sample solution, the control solution, the blank solution and the acetic acid solution is 10:1.
5. The method for detecting and analyzing the peptide map of thrombopoietin peptidomimetic according to claim 3, wherein in the reversed-phase high performance liquid chromatography detection, the sample injection volume is 50. Mu.l, the flow rate is 1.0ml/min, the column temperature is 35 ℃, and the sample tray temperature is 4 ℃.
6. The method for detecting and analyzing the peptide map of the thrombopoietin peptidomimetic according to claim 5, wherein in the preparation of the sample, blank supernatant is taken as enzyme digestion blank solution, 3 parallel samples are prepared from control supernatant and marked as enzyme digestion control solution 1, enzyme digestion control solution 2 and enzyme digestion control solution 3, test supernatant is taken as enzyme digestion test solution, and the control supernatant and the test supernatant with the same volume are uniformly mixed to be used as enzyme digestion control-test 1:1 mixed solution.
7. The method for detecting and analyzing the peptide map of thrombopoietin peptidomimetic according to claim 6, wherein in the reversed-phase high performance liquid chromatography detection, the sample injection sequence is an enzyme digestion blank solution, an enzyme digestion reference solution 1, an enzyme digestion reference solution 2, an enzyme digestion reference solution 3, an enzyme digestion test solution and an enzyme digestion reference-test 1:1 mixed solution in sequence.
8. The method according to claim 5, wherein the integration parameters in the reversed-phase HPLC detection are Apextrack as an integration algorithm, 4.5-40 as an integration interval, 10s as a peak width, 300 as a threshold, 0.5 as a peak integral as a peak start point% and 0.5 as a peak end point%, 0 μV as a minimum height, and 300000 as a minimum area.
9. The method for peptide map detection and analysis of thrombopoietin peptidomimetic according to claim 6, wherein,
The experiment establishment standard is that 1) no obvious new peak appears in the peptide map result, the judgment standard of the obvious new peak is that the total peak area of the new peak area in the peptide map is more than or equal to 0.5 percent, the peak area can be detected in the peptide map of the mixed solution of the reference substance=1:1 to be tested, and the peak area is 50+/-20 percent of the peptide map of the solution to be tested, and 2) the peak shapes of all the identification peaks of the enzyme digestion reference substance solution 1, the enzyme digestion reference substance solution 2 and the enzyme digestion reference substance solution 3 are consistent;
The detection result judging standard comprises 1) the peak shapes of the identification peaks of the enzyme digestion reference substance solution 1, the enzyme digestion reference substance solution 2, the enzyme digestion reference substance solution 3, the enzyme digestion test substance solution and the enzyme digestion reference substance-test substance 1:1 mixed solution are consistent, 2) the identification peak 4 is taken as an internal standard peak, the relative peak area ratio of each identification peak needs to meet the following formula:
RArea= Wherein Area n is the peak Area of the nth peptide fragment peak in the sample, n=1, 2, 3.;
Area Internal standard is the peak Area of the internal standard peak.
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