Repost: Analysis Methods for Related Substances in Chemical Drugs

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　　Analysis Methods for Related Substances in Chemical Drugs Abstract: Objective: To summarize and review the quantitative analysis methods used for detecting related substances in drugs via HPLC, to outline their respective advantages and disadvantages, and to provide specific examples. Detecting related substances, performing qualitative and quantitative analysis on them, and establishing relevant quality control standards are important aspects in ensuring the safety and efficacy of drugs. When using HPLC for the quantitative analysis of drug-related substances, it is necessary to select an appropriate quantitative analysis method based on the specific circumstances. 　　Keywords: Chemical drugs; Related substances; Analysis methods. Related substances in chemical drugs have a significant impact on the purity of medications; they can reduce the efficacy of these drugs, and may even cause harm to the human body or lead to serious adverse reactions. The related substances in chemical drugs are mostly organic compounds, which originate primarily from intermediates in the drug synthesis process. 　　1 Analysis of related substances in acetaminophen by HPLC 1.1 Preparation of instruments and reagents The main instruments required for HPLC analysis include an HPLC system, an electronic balance, and pH test strips. The reagents needed are methanol, triethylamine, ultrapure water, and disodium dihydrogen phosphate dihydrate. 　　1.2 Solution preparation: Accurately weigh 50 mg of the paracetamol sample and place it in a 10 mL volumetric flask. Then dissolve it in anhydrous methanol and dilute to the mark to prepare a solution sample with a concentration of 5 mg/mL, which will be used as the test solution. The solution should be prepared just before use; samples that have been stored for a long time cannot be used. Additionally, 5 mg of 4-N-demethylanilin was accurately weighed and placed in a 200 mL volumetric flask; it was then diluted to the mark with methanol and mixed thoroughly to produce a solution sample with a concentration of 2.5 μg/mL, which was used as the control solution. 1.3 Chromatography conditions: The chromatography column used was of the ODSC184.6*150 mm, 5 μm type. The detection wavelength on the HPLC instrument was set at 254 nm. The ratio of methanol to phosphate buffer (1000 mL of disodium hydrogen phosphate at a concentration of 6.0 g/L, with 1 mL of triethylamine added, and the pH adjusted to 7 using concentrated sodium hydroxide) in the mobile phase was 25:75. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. 1.4 Injection: Under the aforementioned chromatography conditions, 10 μL of each of the test solution and the control solution were injected into the HPLC instrument as specified by pharmacopoeial requirements. By comparing the resulting chromatograms, it was determined that 4-N-demethylanilin appeared around 6 minutes after injection. This allowed for the identification of the types of impurities present in the anilin sample; moreover, the peak corresponding to 4-N-demethylanilin was clearly identified. 　　Unknown impurity 1 and unknown impurity 2 are also present in the color spectrum. Based on the related substances formed during the synthesis of acetaminophen, it can be determined that these two unknown impurities are likely to be formamidopyrine and aminopyrine, respectively. Under unchanged HPLC operating conditions, chromatographic analysis was performed on the synthetic intermediates formamidyl antipyrin and aminopyrin; thereafter, appropriate amounts of these two substances were added to the analgin tablet samples, resulting in chromatograms of the two reference substances. Comparative analysis of the chromatograms showed that the retention times of the two unknown impurities were exactly identical to those of formamidyl antipyrin and aminopyrin. It was evident that after adding the two reference substances, the chromatographic peaks corresponding to the original two impurities increased significantly in height. Based on these two points, it can be determined that the two unknown impurities in An’an Naijin are formamidopyrin and aminopyrin respectively. 　　2 Analysis of related substances in citidine disodium trisodium chloride injection by UPLC method 2.1 Preparation of instruments and reagents The main instruments required for UPLC analysis are: a UPLC system and an electronic balance. The reagents needed include citric acid, sodium citrate, tetraethylammonium bromide, as well as a 10% solution of tetraethylammonium hydroxide, along with ultrapure water. 　　2.2 Solution preparation: Acidic degradation solution: Add 1 mL of 2 mol/L HCl solution to 10 mL of citidine disodium triphosphate and sodium chloride injection, place it in boiling water for 2 hours, remove it, cool it down, adjust the pH to 6.5, and then add 10 mL of water. 　　The solution is destroyed by alkali: 10 mL of Cytidine Diphosphate Disodium Sodium Chloride Injection is mixed with 1 mL of 3 mol/L NaOH solution, then left in boiling water for 2 hours. After removal and cooling, the pH value is adjusted to 6.5, followed by the addition of 10 mL of water. 　　Oxidative damage solution: 10 mL of citidine disodium triphosphate and sodium chloride injection is mixed with 1 mL of 30% hydrogen peroxide solution; it is then placed in boiling water for 2 hours. After removal from the heat and cooling, the pH value is adjusted to 6.5, followed by the addition of 10 mL of water. 　　Oxidative degradation of the blank solution: Take 1 mL of 30% hydrogen peroxide solution, add 10 mL of water, place it in boiling water for 2 hours, remove it and cool it down, then adjust the pH to 6.5, and add another 10 mL of water. 　　High-temperature degradation solution: 10 mL of Cytidine Diphosphate Disodium and Sodium Chloride Injection, placed in boiling water for 4 hours; after removal and cooling, 10 mL of water is added. 　　2.3 Chromatography conditions: The chromatographic column used is of the HSS T3 type, with dimensions of 50mm*2.1nm and a particle size of 1.8um. The detection wavelength in the UPLC instrument is set at 280nm. The mobile phase consists of citrate buffer; 9.76g of citric acid and 1.03g of sodium citrate are added to a 1000mL container. First, 900mL of water is added to dissolve these substances, after which the pH value is adjusted to 3.0. Then, 2.5mL of 10% tetrabutylammonium hydroxide is added, and finally, water is added to reach the desired volume. The flow rate is 1.0mL/min, the sample volume injected is 10ul, and the column temperature is 25°C.

2.4 Sample injection: Under the aforementioned chromatography conditions, 10ul of each solution was taken and injected into the UPLC instrument in accordance with pharmacopeial requirements. The resulting chromatograms are as follows: The retention time of the main component, disodium cytidine triphosphate sodium chloride, is 14.7 minutes. After treatment with acids, bases, high temperatures, and oxidation, the various related substances can be properly separated from the main peak. Based on specificity tests, it was determined that these related substances are disodium cytidine monophosphate and disodium cytidine diphosphate.

3 Conclusion: As people’s living standards improve and their awareness of health increases, the requirements for the quality of drugs also become higher. Therefore, it is necessary to strengthen the identification and analysis of various components in chemical drugs in order to control their concentrations and improve the quality of such drugs. As effective methods for identifying impurities in chemical drugs, HPLC and UPLC should be better understood and mastered to improve the efficiency of impurity analysis. 　　References: Huang Hongshen. Detection of related substances in acetaminophen. Northern Pharmacy, 2011, (02): 7-8. Li Tao, Hu Changqin, Bi Kaishun. Optimization of HPLC analysis method for lovastatin related substances. Journal of Pharmaceutical Analysis, 2011, (09): 1707-1714. Lei Yongsheng, Song Liming, Jiang Qingfeng. Application of liquid chromatography-mass spectrometry in the analysis of related substances in drugs. Modern Instruments, 2011, (04): 9-13+8. Zhang Tong, Zhou Changming, Han Nanyin. Determination of related substances and content in citicidine disodium triphosphate injection by HPLC. Journal of Pharmaceutical Analysis, 2013, (01): 154-158.　　 Luis P.Backfill. A Theory of Distribution Camel Structure. Press of California University, 2001, 21.　　 Louis Stem and Ambary. Marketing Channels. 5th ed. Upper Saddle River: Prentice Hall, 2003, 14-15.　　 Coughlin, Anent, Erin Anderson, Louis W. Stefan, and El-Ambary. Marketing Channels (6th ed.). Beijing: Tsinghua University Press, 2001, 20.　 Article source: http://www.yinghuiyun.com/fanwen/fen*huaxue/