Determination of hydroxycamptothecin and its relat

2022-08-11
  • Detail

Determination of hydroxycamptothecin and its related substances by high performance capillary electrophoresis Abstract: Objective To establish a method for the determination of hydroxycamptothecin and its related substances by high performance capillary electrophoresis. Methods 35mmol/l borax solution (pH 7.5) was used as the running buffer; The unpainted fused silica capillary column is 57cm ×μ 75m 徂 and the effective length is 50cm; The injection pressure is 5psi, the injection time is 3S; The operating voltage is 18kV; The separation temperature is 20 ℃: the detection wavelength is 254nrno, and topotecan is the internal standard. Determination of hydroxycamptotheca acuminata. Results the determined experimental conditions can satisfactorily separate hydroxycamptothecin and its related substances. Conclusion this method is simple, efficient and can effectively control the quality of hydroxy camptothecin

key words: high performance capillary electrophoresis hydroxycamptothecin related substances

hydroxycamptothecin is a trace alkaloid isolated from the seeds of Camptotheca acuminata, a Chinese unique Davidia tree. It is the compound with the strongest anti-cancer effect among more than 20 monomers obtained from Camptotheca acuminata. The anticancer mechanism of this product is different from other anticancer drugs. It interferes with DNA replication by selectively inhibiting topoisomerase I (topp1), and promotes the death of cancer cells, so as to achieve the purpose of anticancer. It has the characteristics of high efficiency, low toxicity and broad-spectrum anticancer. It is used in the treatment of bladder cancer, rectal cancer, liver cancer, gastric cancer, ovarian cancer, leukemia and head and neck tumors. The difference between camptothecin and hydroxycamptothecin is that hydrogen rather than hydroxyl is connected to the carbon at the 1o position. It is a related substance of hydroxycamptothecin, and its toxicity is greater than that of hydroxycamptothecin. The content of hydroxycamptotheca acuminata raw materials and their preparations can be determined by tightening the screws after padding. The method is usually UV spectrophotometry. The specificity of this method is not strong, and the content determination results are often high. Therefore, people have successively used high performance liquid chromatography and high performance capillary zone electrophoresis to determine its content, so that its quality has been effectively controlled. In this paper, 10 hydroxy_ Using dimethylaminomethylene camptothecin as the internal standard, the content of hydroxycamptothecin and its related substances was determined by high performance capillary electrophoresis

1instruments and drug testing

1.1 instruments: American Beckman P/ACE 5010 capillary electrophoresis instrument, fixed wavelength ultraviolet detector and P/ACE data workstation; Hebei Yongnian optical fiber factory uncoated fused silica capillary, 57cmx75 μ M I.D, the effective length is 50cm; Japan sibata Su - 6The ultrasonic cleaner

1.2 reagents and drugs: hydroxycamptothecin and camptothecin reference substance are provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and topotecan hydrochloride reference substance is provided by Chongqing runkang Pharmaceutical Co., Ltd; Hydroxycamptothecin API (batch 01, 02 and O3) and its crude products were provided by a pharmaceutical factory; All reagents used are domestic analytical pure reagents; The test water is triple distilled water

2 experimental method

2.1 preparation of solution

2.1.1 preparation of reference stock solution: accurately weigh an appropriate amount of hydroxycamptothecin reference, dissolve it with methanol and make a solution containing 25mg/l as the reference stock solution

2.1.2 preparation of internal standard stock solution: accurately weigh an appropriate amount of topotecan hydrochloride reference substance, dissolve it with methanol and prepare a solution containing 25mg/l as internal standard stock solution

2.1.3 preparation of test solution

2.13.1 determination of content: accurately weigh an appropriate amount of hydroxycamptothecin API, dissolve it with methanol and make a solution containing 25mg/l, accurately measure 5ml, place it in a 25ml measuring bottle, add 5.0ml of internal standard stock solution, add methanol to the scale, shake well, and use it as the test solution for determination of content

2.1.3.2 determination of related substances: accurately weigh an appropriate amount of hydroxycamptothecin API (crude product), dissolve it with methanol and make a solution containing 100mg/l, accurately measure 5ml, put it into a 25ml measuring bottle, add methanol to 10 ℃, shake it well, and use it as the test solution for determination of related substances

2.2 electrophoresis conditions: take 35mmol/l borax aqueous solution (adjust the pH value to 7.5 with dilute hydrochloric acid) as the running buffer; The operating voltage is 18kV; The detection wavelength is 254nm; The working temperature is 20 ℃; The injection pressure is 0.5psi and the injection time is 3S. After each startup, wash the capillary with lmol/l sodium hydroxide solution and water for 2min in turn, and wash the capillary with buffer solution for 2min between two injections, so as to obtain better reproducibility. Each of the above solutions needs to pass 0.45 before use μ M microporous membrane filtration and ultrasonic degassing

3 validation and results of the experimental method

3.1 linear relationship and sensitivity

3.1.1 linear test: accurately measure LML, 2ml, 3ml, 5ml, 10ml and 15ml of the reference stock solution, place them in 25ml volumetric flasks respectively, add 5.0ml of internal standard stock solution respectively, dilute them to the scale with methanol, shake them up, and determine them according to the electrophoresis conditions under item 2.2. Taking the ratio of the peak area of hydroxycamptothecin and topotecan hydrochloride as the ordinate (y) and the concentration of hydroxycamptothecin as the abscissa (x), draw the standard curve. After regression analysis, the linear growth equation is: y=1.1 83x+1.43x.r=0.9991. The results show that in L μ In the range of g/ml, the ratio of peak area of hydroxycamptothecin and topotecan hydrochloride showed a good linear relationship with the concentration of hydroxycamptothecin

3.1.2 sensitivity test: accurately measure LML of the reference stock solution and dilute it with methanol to a solution containing hydroxycamptothecin concentration corresponding to the electrophoretic peak when the signal-to-noise ratio is 3. The results showed that the lowest detectable concentration was 0.5 μ g/ml 。

3.2 precision and stability

3.2.1 precision test: accurately measure 2m], 5m], and 10ml of the test solution with the concentration of 25 mg/ml respectively, place them in 25ml measuring bottles, add 5.0ml of internal standard stock solution respectively, and dilute them to the scale with methanol at the same time, shake them well, and use them for standby. Prepare the standard curve according to the method under 3.1.1, and then determine according to the electrophoresis conditions under 2.2. Replace the ratio of the peak area of the measured test solution and internal standard solution with the standard curve to obtain the actual content of the test solution. The tested solution with the same concentration is measured five times a day for a total of five days, and the results shown in Table 1 are obtained

3.2.2 stability test: the concentration used for precision measurement precision test is 5 μ G/ml of the test solution was placed in the dark at room temperature for 0, 1, 3, 5, 8h, and then determined according to the law. The results showed that the concentration of this solution was 4.892 ± 0.02531 within 5h μ G/ml, RSD is 2.68%

33 recovery experiment: accurately measure 2ml of the test solution with a concentration of 25mg/ml, put it into a 25ml measuring bottle, add 5.0ml of internal standard stock solution, dilute it to the scale with methanol, shake it well, and use it as the test solution. Prepare the standard curve according to the method under 3.1.1, then determine the above test solution according to the electrophoresis conditions under 2.2, and replace the ratio of the peak area of the measured test solution and internal standard solution with the standard curve to obtain the actual content of the test solution. Accurately measure 2ml of the test solution with measured content respectively, put it into different 25ml measuring bottles, add 5.0ml of internal standard stock solution

and 2.0, 5.0 and 10.0ml of control stock solution respectively, dilute it to the scale with methanol, shake it up, measure it according to law, and calculate the recovery rate. Each concentration was measured three times

3.4.1 determination results of crude products: take the test solution prepared from crude hydroxycamptothecin for the determination of related substances, and determine it according to the electrophoresis conditions under item 2.2. Adjust the detection sensitivity appropriately so that the peaks of related substances can be displayed, and record the electrophoretic spectrum to twice the retention time of the principal component peak. Its typical h reason: see Figure 2 for PCE map. It can be seen from this figure that the method established in this paper can achieve baseline separation of hydroxycamptothecin and its related substances

3.4.2 determination results of related substances: take the test solution prepared from hydroxycamptothecin API for the determination of related substances, and determine it according to the electrophoresis conditions under item 2.2. Adjust the detection sensitivity appropriately, so that the peaks of related substances can be displayed, and record the electrophoresis spectrum to twice the retention time of the principal component peak. The content of related substances was calculated by peak area normalization method

4 determination of experimental method

4.1 determination of detection wavelength: the instrument only has filters with four wavelengths of 200, 214, 254 and 280nm. In order to improve the detection sensitivity, the detection wavelength is selected as 214nm in this experiment. The experiment proved that the determination of hydroxycamptothecin and its related substances was not interfered at this wavelength

4.2 determination of buffer

4.2.1 composition of buffer: in this experiment, the separation effects of carbonate buffer system, borate buffer system, phosphate buffer system and phosphate borate buffer system on hydroxycamptothecin and its related substances were studied. The results showed that both carbonate and phosphate buffer systems could not achieve the expected separation effect; Borate and phosphate borate buffer systems can make hydroxycamptothecin and its seven related substances get baseline separation under optimized conditions, but phosphate borate buffer system will increase the current of the system, which is reflected in the electrophoretic spectrum, which is to widen the spectral peak and reduce the number of theoretical plates. Therefore, the borate buffer system is used as the background electrolyte in this experiment

4.2.2 buffer concentration: experiments show that the buffer concentration within a certain range (mmol/l) has a great impact on the separation effect. Increasing the concentration will prolong the separation time, which is conducive to the separation. Reflected on the electrophoretic spectrum, it is to increase the number of spectral peaks. On the contrary, the separation effect is reduced and the number of spectral peaks is reduced. Further increasing the concentration of buffer will increase Joule heat, which is unfavorable for separation. After experiments, the concentration of the selected buffer is 35 mmol/L

4.23 pH value of buffer solution: the separation mode of alkaloids is usually capillary zone electrophoresis. Under the appropriate pH buffer solution conditions, the alkaloids are partially positively charged in the buffer solution, and the separation is realized based on the different mass charge ratio. Therefore, the influence of pH4.0 ~ 9.5 buffer system on the separation effect was investigated in this experiment. The results showed that under the pH value of acid I, the migration time of substances was prolonged and the spectral peak was broadened; Under the condition of strong alkali production, the electroosmosis increases, and the material migration speed is too fast, which affects the separation effect. After experiments, the pH value of the selected buffer is 7.5. Under this condition, hydroxycamptothecin and its related substances can achieve baseline separation, and the number of common peaks is the largest

4.3 determination of separation voltage: in capillary zone electrophoresis, the voltage increases, the electroosmotic migration speed increases, and the component migration time shortens. However, if the voltage is too high, it will produce too much Joule heat, which will significantly expand the zone, reduce the column efficiency and separation efficiency, and it is also easy to form bubbles in the capillary, making the separation impossible. Therefore, the determination of separation voltage is very important. Through experiments, the separation voltage is selected as 18kV. Under this condition, the separation efficiency is higher and the analysis time is shorter

44 determination of solvent: hydroxycamptothecin is insoluble in water and slightly soluble in organic solvents such as methanol. In addition, methanol can improve the peak shape in the electrophoresis system. Therefore, methanol is selected as the solvent to dissolve the sample

4.5 others: hydroxycamptothecin is easy to decompose when exposed to light, so it is necessary to avoid light during the experiment. From the stability test results, it can be seen that the content has a downward trend with the increase of storage time. Therefore, the determination should be completed within 5 days after the preparation of the sample and reference solution

5 summary

in this experiment, capillary zone electrophoresis was used to determine hydroxycamptothecin API, which has the characteristics of fast and high separation efficiency, and can be used for the quality control of the drug

references

[1] Chen Xin

Copyright © 2011 JIN SHI