CN108918694B

CN108918694B - HPLC pre-column derivatization detection method for MSX residues

Info

Publication number: CN108918694B
Application number: CN201810410377.2A
Authority: CN
Inventors: 张文宇; 白羊; 李义嘉; 郭*; 章永垒; 阮卡; 葛平辉; 王明灶; 陈星�
Original assignee: Sinobioway Biomedicine Co ltd
Current assignee: Sinobioway Biomedicine Co ltd
Priority date: 2018-05-02
Filing date: 2018-05-02
Publication date: 2021-05-25
Anticipated expiration: 2038-05-02
Also published as: CN108918694A

Abstract

The invention discloses an HPLC pre-column derivatization detection method for MSX residues. Precisely absorbing a standard solution or a solution to be detected, adding the standard solution or the solution to be detected into a centrifugal tube, adding a triethylamine solution and a phenyl isothiocyanate solution, uniformly mixing, reacting at room temperature, adding n-hexane, uniformly mixing, standing, taking a lower-phase filter membrane, and filtering to obtain a derivatization standard product or a derivatization sample to be detected; respectively loading a derivatization standard substance or a derivatization sample to be detected into an HPLC column for detection; performing sample injection analysis according to the chromatographic conditions of HPLC detection, and drawing a standard curve according to different concentration gradient concentrations x and peak areas y of the standard solution; and substituting the peak area of the MSX of the solution to be detected into the obtained standard curve, thereby calculating the content of the MSX in the solution to be detected. The method has the advantages of rapidness, convenience, high accuracy, good repeatability, low cost and good applicability, can meet the detection requirements of different MSX residual quantity levels, and has great application value in the quality control of medicine research and development and production.

Description

HPLC pre-column derivatization detection method for MSX residues

Technical Field

The invention relates to a detection method in the field of biochemistry, in particular to an HPLC pre-column derivatization detection method for MSX residues.

Background

Chinese Hamster Ovary Cells (CHO) are one of the most widely used eukaryotic expression systems for foreign proteins. The CHO-GS cell system transfects a target gene of an exogenous protein and an expression vector marked by a Glutamine Synthetase (GS) gene into a CHO cell, and the GS gene and a target protein gene connected with the GS gene can be amplified together by adding an inhibitor L-Methionine Sulfoxide (MSX) of the glutamine synthetase, so that the aim of improving the expression level of the target protein gene is fulfilled, and the yield of the target protein is improved.

However, the chemical, physical and toxic properties of MSX have not been fully studied, and its impurity residues in recombinant protein products may have an impact on drug safety. According to the requirements of the guiding principle of impurity analysis of drugs of general rules 9102 of the Chinese pharmacopoeia 2015 edition, MSX belongs to process-related impurities, and the content of toxic impurities is strictly regulated by drug quality standards. However, MSX has no absorption peak in ultraviolet and lacks a corresponding color development method, so that the existing MSX residue detection method is still incomplete in research and lacks a universal detection method. High performance liquid chromatography coupled with mass spectrometry (HPLC-MS) has been reported to detect MSX content, but the method requires expensive equipment, and the cost is difficult to bear by large pharmaceutical enterprises and research institutions. Therefore, the MSX residue detection is difficult to popularize in conventional drug development and actual production of enterprises.

Disclosure of Invention

The invention aims to provide an HPLC pre-column derivatization detection method for detecting MSX residues, which is simple, reliable, good in repeatability and convenient to use in drug development and actual production.

In order to achieve the above object, the present invention provides a method for detecting HPLC pre-column derivatization of MSX residues, which is characterized in that,

sample pretreatment: precisely absorbing a standard solution or a solution to be tested, adding a triethylamine solution and a phenyl isothiocyanate solution into a centrifugal tube, uniformly mixing, reacting at room temperature, adding n-hexane, uniformly mixing, standing, taking a lower-phase filter membrane, and filtering to obtain a derivatization standard product or a derivatization sample to be tested;

and (4) HPLC detection: respectively loading a derivatization standard substance or a derivatization sample to be detected into an HPLC column for detection;

the results were obtained: performing sample injection analysis according to the chromatographic conditions of HPLC detection, and drawing a standard curve by taking different concentration gradient concentrations of the standard solution as x and the peak area as y; and substituting the MSX peak area of the solution to be detected into the obtained standard curve, thereby calculating the MSX content of the solution to be detected.

Further, in the sample pretreatment step, the standard solution or the solution to be measured: triethylamine solution: phenyl isothiocyanate solution: the volume usage ratio of n-hexane (1-3): (0.5-2): (0.5-2): (2-5); preferably, the volume ratio of the dosage is 2: 1: 1: 4.

further, in the sample pretreatment step, the reaction time is 0.5-1.5h at room temperature; preferably, the reaction time is 1h at room temperature.

Further, in the sample pretreatment step, n-hexane is added and uniformly mixed, and the n-hexane is added for vortex oscillation; preferably, the vortex oscillation time is 5-15 min; more preferably, the time of vortex oscillation is 10 min.

Further, in the sample pretreatment step, standing time is 20-40 min; preferably, the time of standing is 30 min.

Furthermore, in the sample pretreatment step, a filter membrane is a 0.45 μm filter membrane.

Further, the HPLC conditions are that mobile phase A and mobile phase B are adopted as mobile phase; wherein the mobile phase A is a mixed solution of 0.1mol/L sodium acetate and acetonitrile with pH of 6.5, and the mobile phase B is an acetonitrile water solution with the volume percent of 80; preferably, the mobile phase A is a mixed solution of 93 parts by volume of pH6.5, 0.1mol/L sodium acetate and 7 parts by volume of acetonitrile;

further, the conditions of the HPLC were such that elution was started with 100% by volume of mobile phase a; start changing to 85 vol% mobile phase a +15 vol% mobile phase B at 15 min; start changing to 76 vol% mobile phase a +24 vol% mobile phase B at 18 min; change to 60 volume% mobile phase a +40 volume% mobile phase B starting at 25 minutes; a change to 60 volume% mobile phase a +40 volume% mobile phase B was initiated at 30 minutes; start to change to 100 volume% mobile phase B at 30.01 min; the start of 40.01 minutes was changed to 100 volume% mobile phase a.

Further, the HPLC conditions are that a DIONEX Ultimate 3000 high performance liquid chromatograph is adopted;

preferably, the chromatographic column used is a reverse phase C18 chromatographic column; more preferably Sepax AAA, with a specification of 4.6mm x 250mm x 5 μm;

optionally, the column temperature is 36 ℃.

Further, the HPLC condition is that the flow rate of the mobile phase is 1.0 mL/min;

optionally, the sample size is 100 μ L;

optionally, the UV detection wavelength is 254 nm.

For MSX, no published literature data indicates that derivatization is suitable for MSX or with suitable derivatizing agents. According to the invention, through research on the chemical structure of MSX, the chemical composition of MSX is considered to comprise two parts, namely an amino acid similar structure and a sulfoxide imine structure, wherein the amino acid similar structure has the potential of being used as a derivatization target. Phenyl isothiocyanate is a broad-spectrum derivatization reagent capable of reacting with amino acid and imino acid, and phenyl thiocyanate-amino acid derivatives generated by the reaction of phenyl isothiocyanate and free amino acid can be detected under the ultraviolet 254 nm. Therefore, the invention creatively uses phenyl isothiocyanate to perform derivatization on MSX, uses a general High Performance Liquid Chromatography (HPLC) to match with an ultraviolet detector to detect the MSX, and uses a commonly used reversed phase C18 chromatographic column for detecting small molecular compounds in a matching way, and has simple operation and good separation degree, thereby leading the MSX residue to be rapidly and accurately quantitatively detected and obtaining unexpected technical effects.

The invention has the following beneficial effects:

(1) the detection method provided by the invention is simple to operate, is rapid and convenient, has high accuracy and good repeatability, can quantitatively judge the MSX residual quantity of the sample to be detected, and has great application value in the quality control of medicine research and development and production.

(2) The pre-column derivatization and detection time of the sample to be detected are short, a large batch of samples can be detected quickly, the detection speed is high, the cost is low, and the applicability is good.

(3) The detection accuracy is high, the MSX has good linear relation in the range of 1.5625 mu mol/L-50 mu mol/L, and the detection requirements of different MSX residual quantity levels can be met.

(4) The method provided by the invention has high universality, the required instruments, derivatization reagents and the like are all conventional configurations of medical enterprises and research institutions, the operation is simple, the cost is low, and the method is suitable for large-scale popularization and application.

Drawings

FIG. 1 is a HPLC detection profile of 1.5625. mu. mol/L standard solution. 1 is the MSX peak.

FIG. 2 is an HPLC detection profile of 3.1250. mu. mol/L standard solution. 1 is the MSX peak.

FIG. 3 is an HPLC detection profile of 6.2500. mu. mol/L standard solution. 1 is the MSX peak.

FIG. 4 is an HPLC detection profile of 12.5000. mu. mol/L standard solution. 1 is the MSX peak.

FIG. 5 is an HPLC detection profile of 25.0000. mu. mol/L standard solution. 1 is the MSX peak.

FIG. 6 is an HPLC detection profile of 37.5000. mu. mol/L standard solution. 1 is the MSX peak.

FIG. 7 is an HPLC detection profile of 50.0000. mu. mol/L standard solution. 1 is the MSX peak.

Fig. 8 is a MSX standard graph. The x-axis is the MSX concentration (. mu. mol/L) and the y-axis is the peak area (mAU. min.).

FIG. 9 is a detection profile after water derivatization.

FIG. 10 is a HPLC detection profile of a mixed sample containing 12.5. mu. mol/L MSX and 12.5. mu. mol/L MET. 1 is the MSX peak and 2 is the MET peak.

FIG. 11 is a HPLC detection spectrum of a sample to be detected.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1:

1. preparation of Standard solutions

Preparing 50 mu mol/L MSX standard stock solution: 0.9012g of MSX were weighed out and dissolved in 0.1mol/L sodium acetate (pH 6.5): acetonitrile 93:7(V/V) solution, to volume of 1L.

Preparation of 25. mu. mol/L MSX standard solution: add an equal volume of 0.1mol/L sodium acetate (pH6.5) to the above 50. mu. mol/L MSX standard stock: and (3) acetonitrile 93:7 (V/V).

Preparing standard solutions with different concentration gradients: respectively sucking 1000, 750, 500, 250, 125, 62.5 and 31.25 mu L of 50 mu mol/L MSX standard stock solution, respectively adding 0, 250, 500, 750, 875, 937.5 and 968.75 mu L of 0.1mol/L sodium acetate (pH 6.5): the volume of the acetonitrile 93:7(V/V) solution was adjusted to 1mL, and concentration gradient standard solutions having concentrations of 50, 37.5, 25, 12.5, 6.25, 3.125, and 1.5625 μmol/L were obtained. Standard solution concentration gradient formulation is given in Table 1.

TABLE 1 preparation table of standard solutions with different concentration gradients

2. Preparation of derivatization reagents

Triethylamine solution: 14mL of triethylamine and 86mL of acetonitrile were mixed well.

Phenyl isothiocyanate solution: a12.5. mu.L/mL acetonitrile solution of phenylisothiocyanate was prepared.

3. Pre-column derivatization method

Precisely sucking 200 μ L of standard solution or solution to be tested (CHO cell recombinant human nerve growth factor stock solution, lot number L01, not known as biological medicine Co., Ltd.) and adding into 1.5mL centrifuge tube, adding 100 μ L of the above triethylamine solution and 100 μ L of the above phenyl isothiocyanate solution, mixing, reacting at room temperature for 1h, adding 400 μ L of n-hexane, and vortexing and oscillating for 10 min. Standing for 30min, and filtering the lower phase with 0.45 μm filter membrane to obtain derivatized sample to be detected.

The preparation method of the CHO cell recombinant human nerve growth factor stock solution comprises the following steps: the recombinant human nerve growth factor encoding DNA sequence (mature peptide, 118 amino acids, GenBank No. V01511) containing mature 118 amino acids and expression vector of Glutamine Synthetase (GS) gene marker are transfected into Chinese hamster ovary cells (CHO cells), CHO cell seed cells are cultured in CD FortiCHO medium (cargo number: A1148301, GIBCO company of America) for 10 days, then recombinant human nerve growth factor CHO cell fermentation liquor is taken, centrifuged at 4000rpm for 5min, and supernatant is taken for purification, wherein the purification method is shown in Chinese patent application file CN106478801A, and CHO cell recombinant human nerve growth factor stock solution is obtained by purification according to the method of example 1.

4. HPLC-UV detection conditions

The detection conditions are as follows:

high performance liquid chromatograph: a DIONEX UltiMate 3000 hplc chromatograph (including online degasser, quaternary pump, autosampler, column oven, configured DAD detector Chromeleon 7 workstation).

A chromatographic column: sepax AAA with specification of 4.6mm × 250mm × 5 μm, i.e. inner diameter 4.6mm, column length 250mm, and filler particle size 5 μm;

temperature of the column: 36 ℃;

mobile phase: mobile phase a was 0.1mol/L sodium acetate (ph6.5) -acetonitrile 93:7(V/V), mobile phase B was 80% (V/V) acetonitrile in water;

elution procedure: the gradient elution ratio of the mobile phase A and the mobile phase B is shown in the table 2;

TABLE 2 gradient elution volume ratio table for mobile phase A and mobile phase B

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
15	85	15
			18	76	24
25	60	40
			30	60	40
30.01	0	100
			40	0	100
40.01	100	0
			45	100	0

Flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 100 mu L of the solution;

UV detection wavelength: 254 nm;

5. preparation of Standard Curve

After derivatization is carried out on standard solutions with different concentration gradients according to the derivatization conditions, sample injection analysis is carried out according to the chromatographic conditions, and a standard is drawn by taking the concentration as x and the peak area as yThe results are shown in Table 4. The HPLC detection superposition spectrum of the standard solution with different concentration gradients is shown in figures 1-7, and the standard curve is shown in figure 8. In FIGS. 1 to 7, 1 is the MSX peak, and the peak heights are 50, 37.5, 25, 12.5, 6.25, 3.125 and 1.5625. mu. mol/L MSX standard solutions from high to low, respectively. As can be seen from Table 3 and FIGS. 1-2, MSX has a good linear relationship in the range of 1.5625-50 μmol/L, the regression equation is that y is 0.3339x-0.4705, R²＝0.9986。

TABLE 3 Standard Curve test results table

Standard solution numbering	Concentration (μmol/L)	Peak area (mAU. min)
			7	1.5625	0.2300
6	3.1250	0.5837
			5	6.2500	1.6480
4	12.5000	3.6126
			3	25.0000	7.4376
2	37.5000	12.3519
			1	50.0000	16.2277

6. Specificity experiments

A mixed solution containing 12.5. mu. mol/L of MSX and 12.5. mu. mol/L L-Methionine (MET) was prepared by adding 100. mu.L of 25. mu. mol/L of the 25. mu. mol/L MSX standard solution prepared above to 100. mu.L of 25. mu. mol/L-Methionine (MET) aqueous solution. The aqueous solution and the mixed solution obtained above were each 200. mu.L, derivatized under the above derivatization conditions, and then measured under the above detection conditions. The detection map is shown in FIGS. 9 and 10. In FIG. 10, 1 is the MSX peak and 2 is the MET peak.

As can be seen from fig. 9 and 10, the water-derivatized sample had no absorption peak at the MSX position; the retention time of the MSX is 13.747min, the retention time of the MET is 24.803min, the separation degree between the two is 13.24, and the MET does not influence the detection of the MSX residual quantity, which indicates that the specificity of the method meets the requirement.

7. Experiment of accuracy

Respectively adding 80, 100 and 120 mu L of 50 mu mol/L MSX standard stock solution into 50 mu L of sample solution to be detected, and then supplementing 0.1mol/L sodium acetate (pH6.5): acetonitrile 93:7(V/V) to 200 μ L, sample solutions to be tested containing 20, 25, 30 μmol/L MSX standards were prepared, derivatization was performed under the above derivatization conditions, and the results of 3 samples to be tested were measured under the above test conditions, and the recovery rate and relative standard deviation RSD were calculated, and the results are shown in table 5. As can be seen from Table 5, the RSD at all three concentration levels is less than 2%, indicating that the method is highly accurate.

Table 4 accuracy test results table

8. Intermediate precision experiment

Taking 25 mu mol/L MSX standard solution, after derivatization, respectively standing for 0 and 24 hours, respectively determining the results according to the detection conditions, recording the peak area, and calculating the relative standard deviation RSD, wherein the results are shown in Table 5. As can be seen from Table 5, the RSD detected at different times is 0.720%, which is less than 2%, indicating that the intermediate precision of the method is high.

TABLE 5 intermediate precision test results table

9. Repeatability test

Taking 80, 100 and 120 mu L of 50 mu mol/L MSX standard stock solution respectively, and supplementing 0.1mol/L sodium acetate (pH6.5): after derivatization, 20, 25 and 30 μmol/L MSX standard solutions were prepared from 93:7(V/V) acetonitrile to 200 μ L, and the results of 3 samples were measured under the above-described detection conditions, and the average peak area, standard deviation and relative standard deviation were calculated, and the results are shown in table 6. RSD of three concentration levels is less than 2%, which shows that the method has good repeatability.

TABLE 6 repeatability test results table

10. Detection of MSX residues in a sample to be tested

And (3) taking the sample liquid to be detected after derivatization in the step (3), detecting according to the same detection conditions, calculating the peak area of MSX in the sample to be detected, substituting the peak area into the obtained standard curve, and calculating the content of MSX in the sample to be detected. The results are shown in FIG. 11. As can be seen from FIG. 11, the HPLC detection spectrum of the sample to be detected does not produce a peak at the MSX position, which indicates that the MSX residue in the CHO cell recombinant human nerve growth factor stock solution is lower than the detection limit, and the safety is ensured.

The protection scope of the present invention is not limited to the detection of the MSX residue in the CHO cell recombinant human nerve growth factor stock solution, and for other types of samples (such as CHO cell recombinant protein, stock solution and preparation of antibody, etc.), the skilled person can select the corresponding pretreatment mode to obtain the sample to be tested, and the chromatographic conditions and the standard curve in the embodiment are also applicable.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary, preferred embodiments are not to be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.

Claims

1. An HPLC pre-column derivatization detection method of MSX residue is characterized in that,

sample pretreatment: precisely absorbing a standard solution or a solution to be detected, adding a triethylamine solution and a phenyl isothiocyanate solution into a centrifugal tube, uniformly mixing, reacting at room temperature for 0.5-1.5h, adding n-hexane, uniformly mixing, standing for 20-40min, taking a lower phase of a 0.45-micrometer filter membrane, and filtering to obtain a derivatization standard product or a derivatization sample to be detected; wherein the solution to be detected is CHO cell recombinant human nerve growth factor stock solution, antibody stock solution and preparation; standard solution or test solution: triethylamine solution: phenyl isothiocyanate solution: the volume usage ratio of n-hexane (1-3): (0.5-2): (0.5-2): (2-5);

and (4) HPLC detection: respectively loading a derivatization standard substance or a derivatization sample to be detected into an HPLC column for detection; the HPLC conditions are that a Sepax AAA reversed phase C18 chromatographic column with the specification of 4.6mm multiplied by 250mm multiplied by 5 mu m is adopted, the temperature of the chromatographic column is 36 ℃, and a mobile phase A and a mobile phase B are adopted as mobile phases; wherein mobile phase a is 0.1mol/L sodium acetate-acetonitrile 93:7(V/V), pH =6.5, mobile phase B is 80 volume% acetonitrile in water; elution was started with 100% by volume of mobile phase a; start changing to 85 vol% mobile phase a +15 vol% mobile phase B at 15 min; start changing to 76 vol% mobile phase a +24 vol% mobile phase B at 18 min; change to 60 volume% mobile phase a +40 volume% mobile phase B starting at 25 minutes; a change to 60 volume% mobile phase a +40 volume% mobile phase B was initiated at 30 minutes; start to change to 100 volume% mobile phase B at 30.01 min; start to change to 100 volume% mobile phase a at 40.01 min; the flow rate of the mobile phase is 1.0 mL/min; the sample size is 100 muL; the UV detection wavelength is 254 nm;

the results were obtained: analyzing by sample injection according to the above chromatographic conditions of HPLC detection, with different concentration gradient concentrations of standard solution asxThe peak area isyDrawing a standard curve; and substituting the MSX peak area of the solution to be detected into the obtained standard curve, thereby calculating the MSX content of the solution to be detected.

2. The method for detecting the HPLC pre-column derivatization of MSX residues according to claim 1, wherein in the sample pretreatment step, the ratio of standard solution or solution to be detected: triethylamine solution: phenyl isothiocyanate solution: the volume usage ratio of n-hexane is 2: 1: 1: 4.

3. the method for detecting the HPLC pre-column derivatization of MSX residues according to claim 1, wherein the reaction time at room temperature in the sample pretreatment step is 1 h.

4. The method for detecting HPLC pre-column derivatization of MSX residues of claim 1, wherein in the sample pretreatment step, n-hexane is added and mixed uniformly, and the n-hexane is added and vortexed for 10 min.

5. The method for detecting HPLC pre-column derivatization of MSX residues according to claim 1, wherein the standing time in the sample pretreatment step is 30 min.