Impurities Studies Of Chemical Drugs Based On HPLC And HPLC-MS

Impurities in drugs refer to any substances which can decrease the drug purity, including organic and inorganic impurities and residual solvents. Inorganic impurities and residual solvents have already been fully studied. The analyses on such impurities are also developed with nice versatility and thorough control standards. Compared to inorganic impurities and residual solvents, the studies and controls of organic impurities are more complex for the separation, source analysis and limit of the impurities. All those studies need a large amount of painstaking works related to the structure, productive technology and storing stability of the drug. Although the technical guiding principle on related substances has been published in2005, the studies and the control of related substances are important and difficult during new chemical drugs developments and technique evaluations, which are also the weak spots in domestic drug studies. Therefore, impurities studies are significant for the national drug standards improvement.HPLC is widely used in the related substance research, especially for the same series of products and related substances separation, because of its high sensitivity, good reproducibility and durability. LC-MS also plays an important role in the qualification of related substances and quantification of trace impurities. In this thesis, A HPLC method was established for related substances determination in medroxyprogesterone acetate at first. Then impurities in aminocaproic acid injection were studied by HPLC and HPLC-QTOF MS. In the last two chapters, the investigation on impurities of qualitative and quantitative analysis by HPLC-QTOF MS in epinephrine and enalapril maleate and its intermediate were developed, respectively.1. Investigation on impurities of medroxyprogesterone acetate by HPLCA HPLC method was established for related substances determination in medroxyprogesterone acetate. Medroxyprogesterone acetate bulk drug and its tablet have been collected in China Pharmacopoeia2010edition. The impurities analytic methods in China Pharmacopoeia2010edition and European Pharmacopoeia7.0were compared. Method in European Pharmacopoeia7.0showed better specificity and resolution between impurities’peaks. The result indicated that impurity H and D were the main impurities in tablets. And impurity H and D were generated from synthesis process and storage, respectively. Simple system applicability test method was also developed. Detecting the relative retention time between medroxyprogesterone acetate and megestrol acetate can ensure the system accuracy and the correct result without system applicability reference substance of European Pharmacopoeia7.0.As impurity F (4,5-dihydrogen medroxyprogesterone acetate) cannot be detected in the HPLC system above, another HPLC method was established for it. Under the optimized condition, standard curve of F was linear in the range of2～50μg/ml (r=0.9999). The recoveries of bulk drug and tablet were both99.2%. RSDs were0.9%(n=6) and1.0%(n=4), respectively, with the LOD of2.0μg/ml. For the aim of the quantification of F, the relative calibration factor and relative retention time between medroxyprogesterone acetate and F have been also measured. As a result, the problem of lack of F reference substance could be solved.2. Investigation on impurities of aminocaproic acid injection by HPLC and HPLC-QTOF MSImpurities in aminocaproic acid injection were studied and HPLC method was established. Under the optimized HPLC condition, standard curve of aminocaproic acid was linear in the range of10.12-1012μg/ml (r=1.0000). LOD was2.5μg/ml. Two major impurities were analyzed by HPLC-QTOF MS and proposed as caprolactam and aminocaproic acid dipolymer. One of these impurities was confirmed as caprolactam by reference substance. Quantitative analysis on caprolactam reference substance showed good linear (r=0.9999) in0.1006～2.013μg/ml with0.04μg/ml LOD. The recovery of caprolactam was102.0%(RSD1.0%, n=6). Response factor of aminocaproic acid dipolymer was studied initially. The relative calibration factor of aminocaproic acid dipolymer to aminocaproic acid was0.061.3. HPLC-QTOF MS determination of ethyl p-toluenesulfonate in enalapril maleate and intermediateAlkylation of methylmethane sulfonate and methanesulfonic acid ethyl ester can cause mutagenic effect, carcinogenic effect and teratogenic effect. So alkyl esters of low molecular weight sulfonic acid such as p-toluenesulfonic acid could have the same toxic effects so they are toxic impurities. A method of HPLC-QTOF MS was developed for the quantitative analysis of ethyl p-toluenesulfonate in the enalapril maleate and its intermediate (N-[1-(s)-Ethoxycarbonyl-3-phenylpropyl]-1-alanine). Standard curve was linear in the range of0.2005-5.0127μg/ml (r~2=0.9912). The values of limit of quantification and limit of detection were0.2μg/ml and0.1μg/ml, respectively. The RSD for the intra-and inter-day was less than6.7%(n=9). The recoveries of ethyl p-toluenesulfonate in the enalapril maleate and its intermediate were102.61%(RSD7.0%, n=9),90.80%(RSD7.2%, n=9), respectively.4. HPLC-QTOF MS determination of related substances in epinephrine and epinephrine hydrochloride injectionEpinephrine bulk drug and its injection were studied by HPLC-QTOF MS and the related substances were identidied under the MS and MS/MS spectral. There are three impurities in bulk drug and injection, named adrenalone,(lR)-1-(3,4-dihydroxyphenyl)-2-(methylamino) ethanesulphonic acid and4-[2-(butan-2-ylamino)-1-hydroxyethyl]phenol, respectively. QTOF MS not only gives accurate molecular weights, but also obtains MS/MS spectal of interested compoumd through collision. Combine these imformation, it is more reasonabal to identified the unknow compounds.The article perfected the studies on impurities of medroxyprogesterone acetate and aminocaproic acid injection, developed the testing methods, and improved the drug quality standards to ensure the medication safety. During the research, HPLC-MS was applied in the qualitative and quantitative analysis of epinephrine and epinephrine hydrochloride injection, enalapril maleate and intermediate and layed the foundation of further impurities studies.