Method for detecting residual content of triethanolamine by ion exchange-high performance liquid chromatography

Abstract

The invention relates to the field of medicine detection, and particularly provides a method for detecting the residual content of triethanolamine by ion exchange-high performance liquid chromatography, and the chromatographic conditions in the method are as follows: a chromatographic column adopts a bonded cation exchange medium sulfonic group or carboxyl as a filler; a mobile phase is a phosphate buffer solution, and isocratic elution is carried out. According to the method disclosed by the invention, the triethanolamine does not need to be subjected to pre-column derivatization, and macromolecular protein and triethanolamine separation development (direct flow-through of macromolecular protein) is also not needed, so that the problem that the macromolecular protein is precipitated and removed to detect the triethanolamine when the gas chromatography is used is solved; and the problem that other detector modules such as a mass spectrum detector, an evaporative light detector and an electric conductivity detector need to be equipped and maintained is also solved. The method has the advantages of simple operation, accurate result, small interference and high sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a quantitative detection method for triethanolamine by using an ion exchange-high performance liquid chromatography method. Background Art

[0002] Triethanolamine (TEA), also known as trihydroxytriethylamine or tri(2-hydroxyethyl)amine, is a chemical raw material with a wide range of uses. In the protein drug purification process of this study, triethanolamine solution will be used as a buffer system, so it is very necessary to control the residual content of triethanolamine in protein drugs in this study.

[0003] At present, the detection methods of triethanolamine are mainly gas chromatography, liquid chromatography, ion chromatography or mass spectrometry. In the prior art, capillary gas chromatography was used to establish a method for determining triethanolamine residues in the pharmaceutical excipient povidone K90, and the detection limit of the method was 12.1 μg / mL. Yang Xiao et al. used the HPLC-ELSD method to detect triethanolamine mono-, di-, and triester quaternary ammonium salts. The method established a linear calibration curve of the natural logarithm of the mass concentration of each ammonium salt of triethanolamine and the natural logarithm of the sum of its chromatographic peak area. According to the chromatographic peak area, the percentage of triethanolamine mono-, di-, and triester quaternary ammonium salts in industrial samples can be calculated. Liquid chromatography-tandem mass spectrometry was used to determine the migration amount of triethanolamine in food contact materials. The HPLC-ELSD method was used to determine the content of triethanolamine in triethanolamine cream using an evaporative light scattering detector. The cream was subjected to pre-treatment such as extraction ice bath before content detection, and the methodology was verified. The content of triethanolamine in imported and exported cosmetics was determined using ion chromatography-conductivity detection. The method optimized the mobile phase, extraction time, etc., and triethanolamine was determined in commercially available imported and exported cosmetics.

[0004] The above methods are mainly for the detection of triethanolamine content or the trace detection of triethanolamine in samples, and have achieved good results, but the operation process is generally cumbersome, and the instrument configuration requirements are higher than those of common liquid phase instruments.

[0005] Therefore, it is necessary to develop a more efficient method for detecting triethanolamine content. Summary of the invention

[0006] In view of the above technical status, the present invention provides an ion exchange-high performance liquid chromatography detection method for the residual content of triethanolamine in protein drugs. The chromatographic conditions in the detection method include:

[0007] The chromatographic column uses a chromatographic column with a bonded cation exchange medium sulfonic acid group or carboxyl group as filler; the mobile phase is a phosphate buffer solution with a pH of 6.5 to 7.5; isocratic elution is performed;

[0008] In the present invention, as one of the embodiments, the chromatographic column adopts CaptoTM HiRes S 5 / 50 with a specification of 5mm×50mm.

[0009] In the present invention, as one of the embodiments, the mobile phase is a 1 mM phosphate buffer solution with a pH value of 6.5 to 7.5, and more preferably, a pH value of 7.35.

[0010] In the present invention, as one of the implementation schemes, the detection wavelength is 210-220 nm, preferably 215 nm.

[0011] In the present invention, as one of the embodiments, the flow rate is 0.9 to 1.1 mL / min, preferably 1.0 mL / min.

[0012] In the present invention, as one of the embodiments, the column temperature is 28°C to 32°C, preferably 30°C.

[0013] In the present invention, as one of the embodiments, the injection volume is 10 to 20 μL; preferably 10 μL;

[0014] In the present invention, as one of the embodiments, the detector includes but is not limited to an ultraviolet detector.

[0015] In the present invention, as one of the embodiments, the ion exchange-high performance liquid chromatography detection method for the residual content of triethanolamine in the protein drug comprises:

[0016] (1) Preparation of standard solution: weigh triethanolamine standard, dissolve it in ultrapure water, and adjust the pH of the standard solution to 6.8±0.2 with dilute hydrochloric acid; then use mobile phase to dilute the standard solution into a series of concentration solutions (at least 5) as the working standard for this external calibration method;

[0017] (2) Preparation of working curve of external standard method of standard solution: using high performance liquid chromatograph to absorb the working standard sample of step (1) respectively, collecting UV spectrum, obtaining liquid chromatograms of triethanolamine standard solutions of different concentrations, taking the concentration of triethanolamine standard solution as the abscissa and the peak area of ​​the standard solution as the ordinate, performing linear regression fitting, obtaining the working curve of external standard method, which is used for the determination of triethanolamine content;

[0018] (3) Preparation of test solution: Add the test sample into the injection bottle and prepare for testing.

[0019] (4) Detection:

[0020] The detector is an ultraviolet detector;

[0021] Flow rate: 1.0 mL / min;

[0022] Column temperature: 30°C;

[0023] Detection wavelength: 215nm;

[0024] Injection volume: 10 μL;

[0025] Running time: 40 minutes

[0026] Chromatographic column selection: CaptoTM HiRes S 5 / 50 prepacked column;

[0027] Mobile phase selection: 1 mM phosphate buffer solution, pH 7.35;

[0028] Perform isocratic elution;

[0029] (5) Quantitative detection:

[0030] The test sample was aspirated for determination using a high performance liquid chromatograph, and the triethanolamine peak area in the test sample was substituted into the external standard method working curve to quantitatively detect and obtain the triethanolamine content in the protein sample.

[0031] The invention utilizes a high performance liquid chromatograph to absorb a test sample for determination, substitutes the triethanolamine peak area in the test sample into the external standard method working curve, and quantitatively detects and obtains the triethanolamine content in the protein sample.

[0032] The mobile phase and triethanolamine standard solution pH in the method of the present invention are adjusted to about 6.5-7.5, and the pH is higher than the isoelectric point of the protein drug. The protein drug exists in the form of negatively charged ions in this pH range, so there is no binding with the cation exchange chromatography column, and flow-through occurs after injection, so there is no need to perform gradient elution of the protein sample and triethanolamine, and separation is naturally obtained. The pH of this mobile phase and standard solution can change with the isoelectric point of the protein drug to be tested. The pH of the triethanolamine aqueous solution is about 10.5-11, and generally, protein drugs with isoelectric points lower than this value can be applied to this method.

[0033] The ion exchange high performance liquid chromatography method of the present invention does not require pre-column derivatization of triethanolamine, nor does it require separation and development of macromolecular proteins and triethanolamine (macromolecular proteins directly flow through), and also solves the problem of precipitating and removing macromolecular proteins to detect triethanolamine when using gas chromatography, thereby providing a simpler and more accurate detection method for quality control of trace triethanolamine residues in protein drugs.

[0034] The advantages of the present invention are: (1) It is easy to operate. Before the sample is tested, there is no need for pre-treatment such as triethanolamine derivatization treatment (RP-HPLC method) or protein macromolecule precipitation and filtration treatment. At the same time, the damage to triethanolamine caused by the pre-treatment of the test is eliminated, and the test result is more accurate. (2) A widely used ultraviolet detector is used for detection, and there is no need to use detectors with limited popularity and use range such as evaporative light detector (HPLC-ELSD method) and mass spectrometer detector (LC-MS method). (3) The method has high sensitivity, and the detection limit of triethanolamine is as low as 1 μg / mL. The test result is accurate, and the recovery rate of triethanolamine addition detection is between 93.39% and 105.32%.

[0035] The present invention uses high performance liquid chromatography to achieve quality control of the residual content of triethanolamine, which makes up for the current lack of a technical gap in directly performing ultraviolet detector-high performance liquid chromatography detection on the substance (without derivatization), and can replace gas chromatography and reversed-phase high performance liquid chromatography after derivatization to control the quality of triethanolamine content, achieving more convenient quality control and meeting detection requirements. The present invention can effectively separate protein samples from triethanolamine peaks, and the interference of protein on triethanolamine detection can be avoided without prior separation. The ion exchange liquid chromatography that allows the protein sample to flow directly through also simplifies the cumbersome steps of developing a gradient elution separation method for reversed-phase isochromatography.

[0036] The method of the present invention is simple to operate, has accurate results, and has the characteristics of low interference and high sensitivity. The method of the present invention has good specificity, linearity, accuracy and precision, and can accurately determine the residual content of triethanolamine in protein drugs. It is suitable for online monitoring of the residual amount of triethanolamine in the production process of protein drugs, and provides a more accurate and reliable detection method for quality monitoring of triethanolamine in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Gas chromatograms of triethanolamine at concentrations of 10000 μg / mL, 1000 μg / mL and 100 μg / mL in the comparative example;

[0038] Figure 2 : The ultraviolet absorption spectrum scanning diagram of triethanolamine and ultrapure water in Screening Example 1;

[0039] Figure 3 : HPLC chromatogram of triethanolamine when detecting with different pH mobile phases in Screening Example 2;

[0040] Figure 4 : HPLC chromatogram of triethanolamine when detecting with mobile phase of different salt concentrations in Screening Example 3;

[0041] Figure 5 : HPLC chromatogram of triethanolamine when detecting with different pH mobile phases in Screening Example 4;

[0042] Figure 6 : Working curve diagram of triethanolamine standard substance in Example 1;

[0043] Figure 7 : Chromatogram of the test solution in Example 1;

[0044] Figure 8 : Chromatogram of the test sample spiked solution in Example 1;

[0045] Fig. 9 : Specificity experiment chromatogram in Example 2;

[0046] Fig.10 : Linear range experimental chromatogram in Example 3;

[0047] Fig.11 : Chromatogram of accuracy experiment in Example 4;

[0048] Fig.12 : Precision experiment chromatogram in Example 5. DETAILED DESCRIPTION

[0049] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0050] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0051] The comparative example compares the detection results of the gas chromatography method with the method of the present invention, and shows that the method of the present invention is easy to operate and has a significantly higher detection sensitivity than the gas phase method. This application has tested the key chromatographic condition scheme, such as the research and experiments of screening examples 2 to 4 below, and investigated the measurement effect under each key detection condition, and based on the characteristics of protein drugs and triethanolamine, investigated the absorption spectrum of triethanolamine, determined the wavelength range suitable for this detection of ultraviolet absorption, and developed a high-performance liquid chromatography method suitable for the detection of triethanolamine residual content in protein drugs.

[0052] Comparative Example 1 Detection of triethanolamine residue in the test sample by gas chromatography

[0053] Instrument and testing conditions:

[0054] Instrument: Gas chromatograph;

[0055] Heating program: starting temperature at 200 °C, hold for 2 min, increase to 280 °C at a rate of 10 °C / min, hold for 5 min;

[0056] The injection port temperature was 280°C; no pulse; split ratio was 1:1;

[0057] The detector is a hydrogen flame detector, and the detector temperature is 300°C;

[0058] Hydrogen flow rate: 40 mL / min; air flow rate: 300 mL / min; nitrogen flow rate: 50 mL / min; column flow rate: 3 mL / min.

[0059] Preparation of test solution:

[0060] Triethanolamine solution: Dilute the triethanolamine standard to 10000 μg / mL, 1000 μg / mL, and 100 μg / mL using ultrapure water.

[0061] Test solution: Take 100 μL of the test sample and add 900 μL of 3M KCl to precipitate the protein. After precipitation, centrifuge and take the supernatant and add it to the injection bottle for testing.

[0062] The above test samples were directly injected with an injection volume of 1 μL.

[0063] Results: The detection spectra of 10000μg / mL, 1000μg / mL and 100μg / mL triethanolamine solutions are shown in Figure 1 , 100μg / mL triethanolamine has almost no peak, indicating that the detection limit of the method is greater than 100μg / mL, which is far from the detection limit of 1μg / mL. Therefore, the sensitivity of this method does not meet expectations and is therefore not adopted.

[0064] Screening Example 1 Investigation of the UV Absorption Spectrum of Triethanolamine

[0065] Instrument and testing conditions:

[0066] Instrument: UV spectrophotometer;

[0067] Detection mode: spectral;

[0068] Determination method: absorbance value;

[0069] Wavelength range: 185nm~500nm;

[0070] Preparation of detection solution: Accurately weigh an appropriate amount of triethanolamine standard into a beaker, add ultrapure water to dissolve, adjust the pH to 6.8±0.2 with dilute hydrochloric acid, transfer to a volumetric flask, add ultrapure water several times to thoroughly rinse the beaker, transfer all the cleaning solution to the volumetric flask, add water to the volumetric flask to the fixed volume, shake well, and obtain.

[0071] Take an appropriate amount of the above triethanolamine standard solution, adjust it to zero with water in the wavelength range of 185nm to 500nm, use water as blank, and measure the absorption spectrum of the triethanolamine solution in the wavelength range of 185nm to 500nm.

[0072] result:

[0073] The UV scanning spectrum shows that the UV absorption of triethanolamine mainly exists before 230nm. The UV detector of high performance liquid chromatography generally uses a wavelength range of more than 200nm. When triethanolamine is in the range of 200nm to 230nm, the absorbance gradually decreases with the increase of wavelength. Water has basically no absorption at wavelengths above 210nm. Considering comprehensively, this method uses 210 to 220nm for detection, and the detection wavelength is preferably 215nm (see attached Figure 2 Note: Black: UV spectrum scan of triethanolamine standard solution (pH 6.8); Red: UV spectrum scan of ultrapure water).

[0074] Screening Example 2 Investigation of Ion Exchange Chromatographic Column

[0075] Instrument and testing conditions:

[0076] Instrument: High performance liquid chromatography

[0077] Detector: UV detector;

[0078] Column selection: Thermo ProPac TM WCX-10, 4×250 mm weak cation exchange column;

[0079] Flow rate: 1.0 mL / min;

[0080] Column temperature: 30°C;

[0081] Detection wavelength: 215nm;

[0082] Injection volume: 10 μL;

[0083] Running time: 50 minutes;

[0084] Mobile phase selection: 1 mM phosphate buffer solution, pH 6.0, 7.0, 7.5.

[0085] The preparation of the detection solution is the same as that of Screening Example 1. 10 μL of the detection solution is injected for detection and the chromatogram is recorded.

[0086] Results: When the above-mentioned different pH mobile phases were used for detection, the retention time of triethanolamine ranged from about 5.2min to 9.5min (see Appendix Figure 3 Note: blue: pH 6.0; black: pH 7.5; purple: pH 7.0), the retention time is short and the distance from the peak of the test sample is close. The expected target was not achieved.

[0087] Screening Example 3 Investigation of Mobile Phase Salt Concentration

[0088] Instrument and testing conditions:

[0089] Chromatographic column selection: CaptoTM HiRes S 5 / 50 prepacked column;

[0090] Mobile phase selection: 0.5 mM phosphate buffer solution, pH 7.2, 1 mM phosphate buffer solution, pH 7.2;

[0091] Other conditions are the same as those in Screening Example 2

[0092] Results: When the mobile phase was 0.5 mM phosphate buffer solution, the retention time of the triethanolamine peak was greater than 60 min, and the retention time of the triethanolamine peak was about 24 min when the mobile phase was 1 mM phosphate buffer solution (see Appendix Figure 4 Note: Black: 1 mM phosphate; Red: 0.5 mM phosphate). To save detection time, the final optimal mobile phase is 1 mM phosphate buffer solution.

[0093] Screening Example 4: Investigation of Mobile Phase pH

[0094] Instrument and testing conditions:

[0095] Chromatographic column selection: CaptoTM HiRes S 5 / 50 prepacked column;

[0096] Mobile phase selection: 1 mM phosphate buffer solution, pH 7.0, 7.3, 7.5;

[0097] Other conditions are the same as those in Screening Example 2

[0098] Results: When the mobile phases with different pH values ​​were used for detection, the retention time of triethanolamine decreased with the increase of mobile phase pH, and the retention time ranged from about 14 min to 35 min (see Appendix). Figure 5 Note: Red: pH 7.5; Blue: pH 7.3; Black: pH 7.0). The final optimal mobile phase was 1 mM phosphate buffer solution, pH 7.3 (retention time about 27 min).

[0099] Example 1 HPLC detection method

[0100] The HPLC detection method for determining the residual content of triethanolamine in protein drugs comprises the following steps: respectively taking blank samples, standard solution, and test solution for injection, performing detection according to high performance liquid chromatography conditions, and recording chromatograms;

[0101] The chromatographic conditions are: a chromatographic column with a cation exchange medium bonded with a sulfonic acid group or a carboxyl group as filler; a mobile phase of a phosphate buffer solution; isocratic elution;

[0102] Detector: UV detector;

[0103] Flow rate: 1.0 mL / min;

[0104] Column temperature: 30°C;

[0105] Detection wavelength: 215nm;

[0106] Injection volume: 10 μL;

[0107] Running time: 40 minutes

[0108] Chromatographic column selection: CaptoTM HiRes S 5 / 50 prepacked column;

[0109] Mobile phase selection: 1 mM phosphate buffer solution, pH 7.35.

[0110] The triethanolamine standard solution is prepared by the following process: accurately weigh an appropriate amount of triethanolamine standard into a beaker, add ultrapure water to dissolve, adjust the pH to 6.8±0.2 with dilute hydrochloric acid, transfer to a volumetric flask, add ultrapure water several times to thoroughly wash the beaker, and transfer all the washing solution to the volumetric flask, add water to the volumetric flask to make up the volume, shake well, and obtain; triethanolamine concentration: 1000μg / mL.

[0111] Take 1000 μg / mL triethanolamine standard solution and prepare standard curve samples STD1 to STD5 according to Table 1.

[0112] Table 1

[0113]

[0114] Take the above series of concentrations of STD1 to STD5 standard solutions, inject and test, and record the chromatogram. Integrate the triethanolamine peak, and the test data is shown in Table 2.

[0115] The concentration of STD1 to STD5 standard solutions (μg / mL) was used as the abscissa and the peak area of ​​triethanolamine in the standard solutions was used as the ordinate to perform linear regression fitting and obtain the external standard method working curve (see Appendix Figure 6 ).

[0116] The experimental result shows that the working curve of the triethanolamine standard with a concentration between 20 and 80 μg / mL is y=1397.51x-2693.65 (linear fitting coefficient R 2 is 0.9996), where y is the peak area of ​​triethanolamine and x is the concentration of triethanolamine (in μg / mL).

[0117] Table 2 Linear relationship test data

[0118]

[0119] Preparation of test solution: Take an appropriate amount of the protein sample to be tested.

[0120] Preparation of test sample spike solution: Take 240 μL of the protein sample to be tested, add 10 μL of 1000 μg / mL triethanolamine standard solution, mix well, and wait for detection.

[0121] Use the HPLC autosampler to inject 10 μL of the above spiked solution or the test solution, and record the chromatogram. Integrate the triethanolamine peak, and no triethanolamine peak is found in the test solution spectrum (or below the detection limit of this method). Substitute the triethanolamine peak area y1 in the test solution into the above linear regression equation to calculate the triethanolamine concentration x1 (μg / mL) in the test solution. Parallel determination was performed 3 times, and the triethanolamine content in the test solution was 38.527μg / mL, 39.091μg / mL, and 41.078μg / mL, respectively. The average spike recovery rate was between 96.32% and 102.70%, and the recovery rate was good.

[0122] From the results obtained, the chromatogram of the test solution (see Appendix Figure 7 ), and the triethanolamine detection values ​​in the spiked solution of the test sample were between 95% and 105% of the added amount, indicating that this method is accurate and reliable for the detection of triethanolamine residues in the test sample, and the triethanolamine content in the test sample is below the method detection limit. The results of the spiked solution of the test sample are shown in the attached Figure 8 The peak shape of triethanolamine is good, the theoretical plate number is 3000, and the separation degree with the test sample flow-through peak is good, which does not interfere with its content determination.

[0123] Example 2 Specificity Experiment

[0124] Blank sample: ultrapure water, mobile phase.

[0125] Standard solution: Take 50 μL of 1000 μg / mL triethanolamine standard solution, add 450 μL of mobile phase, and mix well; take 200 μL of the above diluted sample, add 300 μL of mobile phase, and mix well. The final concentration of triethanolamine is 40 μg / mL. Take an appropriate amount into the injection bottle for detection.

[0126] Test sample solution 1: Take an appropriate amount of test sample 1 into the injection bottle for testing.

[0127] Test solution 2: Take an appropriate amount of test sample 2 into the injection bottle for testing.

[0128] Test sample spike solution: Take 240 μL of the protein sample to be tested (test sample 1), add 10 μL of 1000 μg / mL triethanolamine standard solution, mix well, prepare 3 portions in parallel, and take appropriate amount into the injection bottle for testing.

[0129] The detection conditions were the same as those in Example 1. 10 μL of the blank sample, standard solution, test solution 1, test solution 2 and test spiked solution were injected respectively using an automatic sampler of a high performance liquid chromatograph, and the chromatograms were recorded.

[0130] Results: The blank sample ultrapure water and mobile phase had no interference near the retention time of triethanolamine;

[0131] There was no interference between samples 1 and 2 near the retention time of triethanolamine; the separation between sample 1 and triethanolamine in the sample spiked solution was between 19.064 and 20.815, which was much greater than 1.5, with good separation and method specificity meeting the requirements. The specific results are shown in Table 3 and Appendix Fig. 9 Note: Blue: ultrapure water; Red: mobile phase; Black: test solution 1; Purple: test solution 2; Green: test solution spiked with the test sample; Brown: standard solution.

[0132] Table 3 Specificity test results

[0133] Sample name Peak separation of sample 1 and triethanolamine Test sample spike solution -40μg / mL-1 20.815 Test sample spike solution - 40 μg / mL - 2 19.064 Test sample spike solution - 40 μg / mL - 3 20.135

[0134] Example 3 Linear range experiment

[0135] Linear range sample preparation: Take the 1000 μg / mL triethanolamine standard solution in Example 1, and prepare linear range samples STD2 to STD7 according to Table 4. At the same time, the 1000 μg / mL triethanolamine standard solution is used as the linear range sample STD1.

[0136] Table 4

[0137]

[0138] The detection conditions were the same as those in Example 1. 10 μL of the STD1 to STD7 standard solutions of the above series of concentrations were injected using an automatic sampler of a high performance liquid chromatograph, and the chromatograms were recorded.

[0139] The triethanolamine peak was integrated, and the concentration of STD1 to STD7 standard solutions (μg / mL) was used as the abscissa, and the triethanolamine peak area in the standard solution was used as the ordinate, and a linear regression fitting was performed to obtain a linear curve (see Appendix Fig.10 ).

[0140] The experimental results show that the working curve of the triethanolamine standard with a concentration between 5 and 1000 μg / mL is y=2252.03x+10164.0 (linear fitting coefficient R 2 is 0.9999), where y is the peak area of ​​triethanolamine and x is the concentration of triethanolamine (in μg / mL).

[0141] Results: When the concentration of triethanolamine was in the range of 5 μg / mL to 1000 μg / mL, the linear correlation coefficient R between the concentration and the peak area standard curve was 2 The value was 0.9999, which met the requirement of ≥0.98, and the method had good linearity. This showed that the method could accurately detect triethanolamine concentration in the range of 5 μg / mL to 1000 μg / mL.

[0142] Example 4 Accuracy Experiment

[0143] Test sample solution: Take an appropriate amount of the test sample into the injection bottle for testing.

[0144] Accuracy sample preparation: Take the test solution and 1000μg / mL triethanolamine standard solution, prepare high, medium and low concentrations of triethanolamine spiked test solution according to Table 5, and prepare 3 copies of each concentration solution in parallel.

[0145] Table 5

[0146]

[0147] The detection conditions were the same as those in Example 1. The test solution and 9 high, medium and low concentration accuracy samples were injected with 10 μL each using an automatic sampler of a high performance liquid chromatograph, and the chromatograms were recorded.

[0148] At the same time, a series of concentration standard solutions were prepared and injected according to Example 1, and a working curve was drawn to obtain a linear regression equation. The peak area of ​​the accuracy sample triethanolamine was substituted into the linear regression equation to calculate the triethanolamine concentration in the accuracy sample. The recovery rate of each solution and the RSD of the recovery rates of 9 solutions were calculated according to the following formula.

[0149] Recovery rate (%) = detected value / theoretical value * 100%

[0150] Results: The recovery rates of high, medium and low concentration accuracy samples ranged from 93.39% to 105.32%, meeting the requirement of 90% to 110% recovery rate; the RSD of the recovery rate was 4.41%, meeting the requirement of ≤5.0%. The method had good accuracy. The specific results are shown in Table 6 and Appendix Fig.11 .

[0151] Table 6 Accuracy experimental results

[0152]

[0153]

[0154] Example 5 Precision Experiment

[0155] Test sample spike solution: Take 240 μL of the protein sample to be tested, add 10 μL of 1000 μg / mL triethanolamine standard solution, mix well, and the final concentration of triethanolamine is 40 μg / mL. Prepare 6 portions of the above solution in parallel, and take appropriate amounts into the injection bottle for testing.

[0156] The detection conditions were the same as those in Example 1. 10 μL of the sample spiked solution was injected using an automatic sampler of a high performance liquid chromatograph, and the chromatogram was recorded.

[0157] At the same time, a series of concentration standard solutions were prepared and injected according to Example 1, and a working curve was drawn to obtain a linear regression equation. The triethanolamine peak area of ​​the test sample spiked solution was substituted into the linear regression equation to calculate the triethanolamine concentration in the test sample spiked solution. The RSD of the triethanolamine concentration test results was calculated for 6 times.

[0158] Replace person B and repeat the above steps to calculate the RSD of person B's 6 triethanolamine concentration test results and the RSD of a total of 12 triethanolamine concentration test results by person A and person B.

[0159] Results: Personnel A and Personnel B measured 6 spiked samples in parallel on different dates using different instruments and different batches of chromatographic columns, and calculated the triethanolamine concentration detection value of each solution. The RSD values ​​of triethanolamine concentration of 6 spiked samples independently obtained by Personnel A and Personnel B were 2.19% and 4.38%, respectively, meeting the requirement of ≤5.0%, indicating that the repeatability of the method was good; the RSD value of triethanolamine concentration of 12 spiked samples obtained by Personnel A and Personnel B was 4.06%, meeting the requirement of ≤5.0%, indicating that the intermediate precision of the method was good, which comprehensively indicated that the precision of the method was good. The specific results are shown in Table 7 and Appendix Fig.12 .

[0160] Table 7 Accuracy experimental results

[0161]

[0162]

Claims

1. A method for detecting the residual content of triethanolamine by ion exchange-high performance liquid chromatography, characterized in that: The chromatographic conditions of the method include: The chromatographic column uses a bonded cation exchange medium sulfonic acid group or carboxyl group as filler; The mobile phase was phosphate buffer solution with a pH of 6.5 to 7.5; Perform isocratic elution.

2. The detection method according to claim 1, characterized in that: The chromatographic column adopts CaptoTM HiRes S 5 / 50, and the specification is 5mm×50mm.

3. The detection method according to claim 1, characterized in that: The mobile phase is 1 mM phosphate buffer solution with a pH of 7.

35.

4. The detection method according to claim 1, characterized in that: The detection wavelength is 210-220 nm, preferably 215 nm.

5. The detection method according to claim 1, characterized in that: The flow rate is 0.9-1.1 mL / min, preferably 1.0 mL / min.

6. The detection method according to claim 1, characterized in that: The column temperature is 28°C to 32°C, preferably 30°C.

7. The detection method according to claim 1, characterized in that: The injection volume is 10 to 20 μL, preferably 10 μL.

8. The detection method according to claim 1, characterized in that: The detection method further comprises: (1) Preparation of standard solution: weigh triethanolamine standard, dissolve it in ultrapure water, and adjust the pH of the standard solution to 6.8±0.2 with dilute hydrochloric acid; then use mobile phase to dilute the standard solution into a series of concentration solutions (at least 5) as the working standard for this external calibration method; (2) Preparation of working curve of external standard method of standard solution: using high performance liquid chromatograph to absorb the working standard sample of step (1) respectively, collecting UV spectrum, obtaining liquid chromatograms of triethanolamine standard solutions of different concentrations, taking the concentration of triethanolamine standard solution as the abscissa and the peak area of ​​the standard solution as the ordinate, performing linear regression fitting, obtaining the working curve of external standard method, which is used for the determination of triethanolamine content; (3) Preparation of test solution: Add the test sample into the injection bottle and prepare for testing; (4) Detection: The detector is an ultraviolet detector; Flow rate: 1.0 mL / min; Column temperature: 30°C; Detection wavelength: 215nm; Injection volume: 10 μL; Running time: 40 minutes Chromatographic column selection: CaptoTM HiRes S 5 / 50 prepacked column; Mobile phase selection: 1 mM phosphate buffer solution, pH 7.35; Perform isocratic elution; (5) Quantitative detection: The test sample was aspirated for determination using a high performance liquid chromatograph, and the triethanolamine peak area in the test sample was substituted into the external standard method working curve to quantitatively detect the triethanolamine content in the protein sample.

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