HPLC-SEC detection method for content of deoxyribonucleic acid in androstenedione prepared by fermentation method

Abstract

The invention belongs to the field of analysis and detection, and particularly relates to an HPLC-SEC detection method for detecting the content of deoxyribonucleic acid in androstenedione prepared by a fermentation method, the method is rapid, simple and convenient, the androstenedione and the deoxyribonucleic acid can be effectively separated, meanwhile, the low-concentration deoxyribonucleic acid can be effectively detected and accurately quantified, and the detection accuracy is high. And more reliable guarantee is provided for product quality and medication safety.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a HPLC-SEC detection method for the deoxyribonucleic acid content in androstenedione prepared by a fermentation method. Background Art

[0002] Deoxyribonucleic acid (DNA) is a long-chain polymer whose basic unit is called nucleotide, in which deoxyribose and phosphate are connected by ester bonds to form the backbone of the long DNA chain. Deoxyribonucleic acid (DNA) contamination is a problem that needs to be paid attention to and solved in the production process of raw materials. It will have a serious impact on the quality and safety of drugs, induce allergic reactions or infections, affect the efficacy of drugs and cause side effects.

[0003] Androstendione, referred to as 4-AD, chemical name is androsten-4-ene-3,17-dione, molecular formula: C19H26O2, molecular weight: 286.4, structural formula as shown in Formula I. Androstendione is the main raw material for the production of danazol API, which is sold on the market. Androstendione is obtained by fermentation of plant sterols. In the process of producing androstendione by fermentation, DNA contamination comes from multiple sources. For example, plant extracts are used as starting materials in fermentation. These materials themselves contain a large amount of DNA. If these raw materials are not fully cleaned, disinfected or purified, exogenous DNA may be brought into the fermentation process, causing DNA contamination; and in the fermentation process, bacteria are used for fermentation. If the DNA carried by the bacteria itself is not cleaned, it will remain in the final product, causing androstendione contamination; in addition, the pollution of fermentation equipment and production environment may bring exogenous DNA into the fermentation process, causing DNA contamination. In order to reduce and avoid DNA contamination in androstenedione produced by fermentation process, it is necessary to take effective measures to control and manage the above sources of contamination and establish a complete quality control system to monitor and detect the residual DNA content in the fermentation process to ensure the quality and safety of androstenedione.

[0004]

[0005] The detection methods for residual DNA content mainly include: ultraviolet spectrophotometry, polymerase chain reaction (PCR), gene sequencing, electrophoresis, bioassay, etc.; ultraviolet spectrophotometry has low sensitivity in detecting DNA content; PCR has high sensitivity and specificity, but cannot accurately quantify; gene sequencing has high resolution and high accuracy, but the cost is high; electrophoresis requires certain experimental skills and experience; bioassay has the characteristics of simplicity, rapidity, sensitivity, etc., but requires the selection of appropriate microbial species and culture conditions. In addition, the molecular weight of deoxyribonucleic acid (DNA) is very large, and separation using a chromatographic column with ordinary silane bonded silica as a filler (such as C8, C18 column, etc.) will slow down its migration speed in the mobile phase, resulting in a long separation time. In order to solve this problem, the present invention selects a molecular exclusion chromatography column as a separation chromatographic column. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a high performance liquid phase-size exclusion chromatography (HPLC-SEC) detection method for the deoxyribonucleic acid (DNA) content in androstenedione, so as to provide a more reliable guarantee for the quality of the product and the safety of medication.

[0007] The technical scheme of the present invention is as follows: a method for detecting the content of deoxyribonucleic acid in androstenedione, which adopts a high performance liquid phase-molecular exclusion chromatography detection method, a chromatographic column with high-purity spherical silica gel particles with a narrow particle size distribution as a filler, and a mobile phase is prepared by the following method: 6.05 g of tris(hydroxymethyl)aminomethane is dissolved in water and diluted to 500 ml, 420 ml of 0.1 mol / L hydrochloric acid and 30 ml of tetrahydrofuran are added, and the mixture is diluted to 1000 ml with water and filtered.

[0008] In some embodiments, the chromatographic column is Sepax SRT-10SEC-1000 with a specification of 7.8*300mm 5μm.

[0009] In some embodiments, the column temperature of the chromatographic column is 30-50°C, preferably 40°C.

[0010] In some embodiments, the injection volume is 50 μl.

[0011] In some embodiments, the flow rate of the mobile phase is 1 ml to 1.5 ml / min, preferably 1 ml / min.

[0012] In some embodiments, the method uses an ultraviolet detector, and its detection wavelength is 210nm to 280nm, preferably 260nm.

[0013] In some typical embodiments, the present invention provides a method for detecting the content of deoxyribonucleic acid in androstenedione, comprising the following steps:

[0014] 1) Test solution: Accurately weigh 50 mg of androstenedione sample, accurately add 5 ml of chloroform to dissolve, accurately add 5 ml of mobile phase and shake, let it stand and take the upper solution.

[0015] 2) Control solution: Take 12.5 mg of deoxyribonucleic acid, place it in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well; accurately measure 1 ml of the above solution, place it in a 50 ml volumetric flask, add mobile phase to dilute to the scale.

[0016] 3) Accurately measure 50 μl of the test solution and the control solution, respectively, and inject them into the liquid chromatograph, record the chromatogram, and calculate the content of deoxyribonucleic acid by peak area according to the external standard method;

[0017] The chromatographic conditions are as follows: the chromatographic column is Sepax SRT-10SEC-1000; the specification is 7.8*300mm 5μm;

[0018] The mobile phase was prepared as follows: 6.05 g of tris(hydroxymethyl)aminomethane was dissolved in water and diluted to 500 ml, 420 ml of 0.1 mol / L hydrochloric acid and 30 ml of tetrahydrofuran were added, and the mixture was diluted to 1000 ml with water and filtered;

[0019] The column temperature was 40°C; the injection volume was 50 μl; the flow rate was 1.0 ml / min; and the detection was carried out using an ultraviolet detector with a wavelength of 260 nm.

[0020] The method for detecting the content of deoxyribonucleic acid in androstenedione provided by the invention is used for detecting deoxyribonucleic acid in the process of preparing androstenedione by fermentation technology.

[0021] The beneficial effects of the present invention are as follows: a high performance liquid phase-size exclusion chromatography (HPLC-SEC) detection method for the deoxyribonucleic acid (DNA) content in androstenedione, which is rapid and simple, can effectively separate androstenedione from deoxyribonucleic acid, and can also effectively detect low concentrations of deoxyribonucleic acid and can accurately quantify, thereby providing more reliable guarantees for product quality and drug safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Typical chromatogram of blank solvent (using the chromatographic conditions of the present invention)

[0023] Figure 2 Typical chromatogram of the control solution (using the chromatographic conditions of the present invention)

[0024] Figure 3 Typical chromatogram of the test solution (using the chromatographic conditions of the present invention)

[0025] Figure 4 Linear test graph of the method of the present invention (using the chromatographic conditions of the present invention)

[0026] Figure 5 Typical chromatogram of the test solution (using the chromatographic conditions in patent CN110487923B)

[0027] Figure 6 Typical chromatogram of low concentration DNA solution (using the chromatographic conditions of the present invention)

[0028] Figure 7 Typical chromatogram of low-concentration DNA solution (using the chromatographic conditions in patent CN110487923B) DETAILED DESCRIPTION

[0029] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0030] Example 1

[0031] 1. HPLC-SEC chromatographic conditions

[0032] Chromatographic column: Use high-purity spherical silica gel particles with narrow particle size distribution as filler (Sepax SRT-10SEC-10007.8*300mm 5μm or chromatographic column with equivalent performance);

[0033] Mobile phase: Dissolve 6.05 g of tris(hydroxymethyl)aminomethane in water and dilute to 500 ml, add 420 ml of 0.1 mol / L hydrochloric acid and 30 ml of tetrahydrofuran, dilute to 1000 ml with water, and filter;

[0034] Column temperature: 40°C;

[0035] Injection volume: 50 μl;

[0036] Flow rate: 1.0ml / min;

[0037] Detector: UV detector;

[0038] Wavelength: 260nm

[0039] Running time: 30 minutes

[0040] 2. Implementation of sample testing

[0041] Test solution: Accurately weigh 50 mg of androstenedione sample, accurately add 5 ml of chloroform to dissolve, accurately add 5 ml of mobile phase and shake, let it stand and take the upper solution.

[0042] Control solution: Take 12.5 mg of deoxyribonucleic acid, place it in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well; accurately measure 1 ml of the above solution, place it in a 50 ml volumetric flask, add mobile phase to dilute to the scale.

[0043] Accurately measure 50 μl of the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0044] 3. Calculation of deoxyribonucleic acid (DNA) content

[0045] Calculated by external standard method using peak area, the calculation formula is as follows:

[0046]

[0047] Where:

[0048] f is the average correction factor;

[0049] W 对照品 is the sample weight of the reference substance, mg;

[0050] A 对照品 is the main peak area of ​​the control solution;

[0051] V 对照品 Dilution multiple of the reference substance;

[0052]

[0053] Where:

[0054] faverage is the average correction factor;

[0055] W 供试品 is the sample weight of the test product, mg;

[0056] A 供试品 is the main peak area of ​​the test solution;

[0057] V 供试品 is the dilution multiple of the test product;

[0058] That is to say, the content of deoxyribonucleic acid (DNA) in androstenedione is obtained.

[0059] Test Case

[0060] 1. Exclusivity

[0061] 1.1 Solution preparation

[0062] Blank: mobile phase.

[0063] Control solution: Accurately weigh 13.15 mg of deoxyribonucleic acid, place it in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well; Accurately measure 1 ml of the above solution, place it in a 50 ml volumetric flask, add mobile phase to dilute to the scale.

[0064] Sample solution: Accurately weigh 50.23 mg of androstenedione from batch C01-20121806, accurately add 5 ml of chloroform to dissolve, accurately add 5 ml of mobile phase and shake, let stand and take the upper solution.

[0065] Mixed solution: Accurately weigh 51.17 mg of androstenedione from batch C01-20121806, accurately add 5 ml of chloroform to dissolve, accurately add 5 ml of control solution and shake, let stand and take the upper solution.

[0066] 1.2 Determination method

[0067] Accurately measure 50 μl of the above solution, operate according to the above HPLC-SEC analysis conditions, and record the chromatogram. Measure the control solution for 5 consecutive injections, and the RSD value of the DNA peak area should not be greater than 2.0%.

[0068] 1.3. Measurement effect

[0069] Table 1 System suitability solutions

[0070]

[0071] Table 2 Positioning solution

[0072]

[0073] Conclusion: The blank has no interference with the determination; the mean peak area of ​​the control solution was 267285 after 5 consecutive injections, and the RSD value was 0.54%, which was less than 2.0%. The retention time of DNA in the mixed solution was consistent with that in the control solution, and the peak area of ​​DNA in the mixed solution was basically consistent with that in the control solution, indicating that the method has good specificity.

[0074] 2. Linear

[0075] 2.1 Solution preparation

[0076] Blank: mobile phase.

[0077] Linear stock solution: Accurately weigh 13.15 mg of deoxyribonucleic acid, place in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to scale, shake well. Accurately measure 1 ml of the above solution, place in a 25 ml volumetric flask, add mobile phase to dilute to scale.

[0078] Linear solution 1: Accurately measure 2 ml of linear stock solution, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well. Linear solution 2: Accurately measure 3 ml of linear stock solution, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well. Linear solution 3: Accurately measure 4 ml of linear stock solution, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well. Linear solution 4: Accurately measure 5 ml of linear stock solution, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well. Linear solution 5: Accurately measure 6 ml of linear stock solution, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well.

[0079] 2.2 Determination method

[0080] Accurately measure 50 μl of the above solution, operate according to the above HPLC-SEC analysis conditions, and record the chromatogram. The linear coefficient r>0.99.

[0081] 2.3. Measurement effect

[0082] Table 3 DNA linearity test results

[0083]

[0084] The linearity is as follows Figure 4 :

[0085] Conclusion: In the concentration range of 0.002003mg / ml to 0.006010mg / ml, the linear equation is y=42,722,729.347x+47,014.867, R 2 =0.996, r=0.997, which are greater than 0.99, indicating a good linear relationship.

[0086] 3. Recovery rate

[0087] 3.1 Solution preparation

[0088] Blank: mobile phase.

[0089] Control solution: Accurately weigh 13.15 mg and 11.54 mg of deoxyribonucleic acid, place them in 50 ml volumetric flasks, add mobile phase to dissolve and dilute to scale, shake well; accurately measure 1 ml of the above solution, place it in a 50 ml volumetric flask, add mobile phase to dilute to scale. This is used as control solution 1 and control solution 2.

[0090] Sample solution: Accurately weigh 49.78 mg of androstenedione from batch C01-20121806, accurately add 5 ml of chloroform to dissolve, accurately add 5 ml of mobile phase and shake, let stand and take the upper solution.

[0091] Recovery rate stock solution: same as linear stock solution, sample weight 11.54 mg.

[0092] Low concentration solution of androstenedione: accurately weigh 50 mg of androstenedione from batch C01-20121806, accurately add 5 ml of chloroform to dissolve, accurately add 2 ml of recovery rate reserve solution and 3 ml of mobile phase, shake, let stand and take the upper layer solution. (Prepare 3 portions in the same way, inject 1 needle into each portion)

[0093] Medium concentration solution of androstenedione: accurately weigh 50 mg of androstenedione from batch C01-20121806, accurately add 5 ml of chloroform to dissolve, accurately add 2.5 ml of recovery rate reserve solution and 2.5 ml of mobile phase, shake, let stand, and take the upper layer of solution. (Prepare 3 portions in the same way, and inject 1 needle into each portion)

[0094] High concentration solution of androstenedione: accurately weigh 50 mg of androstenedione from batch C01-20121806, accurately add 5 ml of chloroform to dissolve, accurately add 3 ml of recovery rate reserve solution and 2 ml of mobile phase, shake, let stand and take the upper layer solution. (Prepare 3 portions in the same way, inject 1 needle into each portion)

[0095] 3.2 Determination method

[0096] Accurately measure 50 μl of the above solution, operate according to the above HPLC-SEC analysis conditions, and record the chromatogram. The recovery rate at each concentration should be between 90.0 and 110.0%, and the RSD value of the recovery rate of 9 samples should not be greater than 10.0%.

[0097] 3.3. Measurement effect

[0098] Table 4 DNA recovery test results

[0099]

[0100] Conclusion: The recovery rate of DNA at each concentration was in the range of 90.0% to 110.0%, with an average recovery rate of 106.61% and an RSD value of 6.02%, which was less than 10.0%, indicating that the method had good accuracy.

[0101] 4. Limit of Quantitation and Limit of Detection

[0102] 4.1 Solution preparation

[0103] Blank solution: mobile phase.

[0104] Quantitation limit stock solution: Take the linear 1 solution under the linear item as the quantitation limit stock solution.

[0105] Quantitation limit solution: Accurately measure 2 ml of quantitation limit stock solution, place it in a 10 ml volumetric flask, add mobile phase to dilute to the scale, and shake well.

[0106] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, add the mobile phase to dilute to the scale, and shake well.

[0107] 4.2 Determination method

[0108] Accurately measure 50 μl of the above solution, operate according to the above HPLC-SEC analysis conditions, and record the chromatogram. Measure the quantitative limit solution for 5 consecutive injections, and the RSD value of the peak area should not be greater than 5.0%.

[0109] 4.3. Measurement effect

[0110] Table 5 DNA quantification limit determination results

[0111]

[0112]

[0113] Table 6 DNA detection limit determination results

[0114]

[0115] Conclusion: The detection limit of DNA is 0.0002105mg / ml, equivalent to 0.0021% of the sample; the quantitative limit of DNA is 0.000421mg / ml, equivalent to 0.0042% of the sample; the RSD value of the peak area of ​​5 consecutive needles of the quantitative limit solution is 1.75%, less than 5.0%; the RSD value of the retention time is 0.20%, less than 2.0%. The above results show that this method has high detection sensitivity and is suitable for the detection of DNA content in androstenedione.

[0116] Comparative Example

[0117] 1. Chromatographic conditions in patent CN111812230A:

[0118] The mobile phase is a mixture of sodium phosphate and sodium chloride, and the pH range of the mobile phase is 7-8; the isocratic elution method is adopted, the flow rate is 0.3-0.6ml / min; the column temperature is 20-30℃; the detection wavelength is 260nm;

[0119] 2. Chromatographic conditions in patent CN109765289A:

[0120] The mobile phase was 10 mM Tirs (pH=7.4), prepared with ultrapure water (18 MΩ / cm resistivity), the flow rate was 1 mL / min, the injection volume was 50 μL; and the detection wavelength was 260 nm.

[0121] The chromatographic column was a PEG-20M stainless steel column (4×2 mm). The column temperature was programmed to start at 210°C and maintain for 3 min, then increase to 280°C at a rate of 30°C / min and maintain for 10 min.

[0122] After testing, the above two chromatographic conditions are not applicable to the test sample of the present invention. These conditions cannot exclude the influence of androstenedione and other impurities contained in androstenedione on DNA detection, which will interfere with the detection.

[0123] 3. Chromatographic conditions in patent CN110487923B:

[0124] The mobile phase was 0.3 M ammonium chloride containing 10 mM Tris-HCl and 1 mM EDTA, and the pH of the mobile phase was 7.5 with an ammonia solution. The loading volume was 10 μl, the flow rate was 0.5 ml / min, the column temperature was room temperature, and the detection wavelength was 260 nm.

[0125] Because the objects of detection and analysis are different, the present invention detects the DNA content in androstenedione, and androstenedione is produced by a fermentation process. The samples obtained by this process are relatively complex, and the impurities contained therein will interfere with the detection, and the matrix effect is large; while the existing patent detects the plasmid DNA content, the sample is single and clean, and the matrix effect is very small. The present invention adds tetrahydrofuran as an additive mainly to eliminate this matrix effect. The chromatogram of the test solution of the present invention is tested using the chromatographic conditions of patent CN110487923B as shown in Figure 5 As shown, it can be seen from the results that the method for detecting DNA of the present invention can effectively eliminate the matrix effect and reduce the interference of other components in androstenedione on DNA detection, while the chromatographic conditions of patent CN110487923B cannot eliminate this interference, multiple peaks are piled up, the separation is unqualified, and DNA cannot be accurately quantified.

[0126] Secondly, the present invention detects the residual DNA content in androstenedione, which is very low; while the existing patent detects the plasmid DNA content, which is relatively high, so a more sensitive method is required to detect the residual DNA content in androstenedione. Therefore, the present invention adopts a higher and uncommon 40°C column temperature to improve the sensitivity of the method. The chromatogram of the test solution of the present invention tested using the chromatographic conditions of the present invention is as follows: Figure 6 As shown; the chromatogram of the test solution of the present invention is tested using the chromatographic conditions of patent CN110487923B as shown Figure 7 As shown: From the results, it can be seen that the method for detecting DNA of the present invention has higher sensitivity and can effectively detect the content of low-concentration DNA with a signal-to-noise ratio greater than 10, while the chromatographic conditions of patent CN110487923B cannot effectively detect the content of low-concentration DNA with a signal-to-noise ratio less than 10.

Claims

1. A method for detecting the content of deoxyribonucleic acid in androstenedione, the method adopts a high performance liquid phase-molecular exclusion chromatography detection method, a chromatographic column with high purity spherical silica gel particles with a narrow particle size distribution as a filler, and the mobile phase is prepared by the following method: 6.05g of tris(hydroxymethyl)aminomethane is dissolved in water and diluted to 500ml, 420ml of 0.1mol / L hydrochloric acid and 30ml of tetrahydrofuran are added, and the mixture is diluted to 1000ml with water and filtered.

2. The detection method according to claim 1, characterized in that: The chromatographic column is Sepax SRT-10SEC-1000 with specifications of 7.8*300mm 5μm.

3. The detection method according to claim 1, characterized in that: The column temperature of the chromatographic column is 30-50°C, and the column temperature is preferably 40°C.

4. The detection method according to claim 1, characterized in that: The injection volume was 50 μl.

5. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 1 ml to 1.5 ml / min, preferably 1 ml / min.

6. The detection method according to claim 1, characterized in that: The method adopts an ultraviolet detector, and its detection wavelength is 210nm to 280nm, preferably 260nm.

7. A method for detecting the content of deoxyribonucleic acid in androstenedione, comprising the following steps: 1) Test solution: accurately weigh 50 mg of androstenedione sample, accurately add 5 ml of chloroform to dissolve, accurately add 5 ml of mobile phase and shake, let stand and take the upper solution; 2) Control solution: Take 12.5 mg of deoxyribonucleic acid, place it in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to the mark, shake well; accurately measure 1 ml of the above solution, place it in a 50 ml volumetric flask, add mobile phase to dilute to the mark; 3) Accurately measure 50 μl of the test solution and the control solution, respectively, and inject them into the liquid chromatograph, record the chromatogram, and calculate the content of deoxyribonucleic acid by peak area according to the external standard method; The chromatographic conditions are as follows: the chromatographic column is Sepax SRT-10SEC-1000; the specification is 7.8*300mm 5μm; The mobile phase was prepared as follows: 6.05 g of tris(hydroxymethyl)aminomethane was dissolved in water and diluted to 500 ml, 420 ml of 0.1 mol / L hydrochloric acid and 30 ml of tetrahydrofuran were added, and the mixture was diluted to 1000 ml with water and filtered; The column temperature was 40°C; the injection volume was 50 μl; the flow rate was 1.0 ml / min; and the detection was carried out using an ultraviolet detector with a wavelength of 260 nm.

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