Simultaneous Quantification of Related Substances of Perindopril Tert-Butylamine Using a Novel Stability Indicating Liquid Chromatographic Method Zolta´ n-Istva´ n Szabo´ 1 *, Zenko´´-Zsuzsa´nna Re´ ti 2 ,La´szlo´ Gagyi 2 , Erika Lilla Kis 2 and Emese Sipos 1 1 Department of Pharmaceutical Industry and Biotechnology, Faculty of Pharmacy, University of Medicine and Pharmacy of Tıˆrgu Mures¸ , Gheorghe Marinescu 38, Tıˆrgu Mures¸ RO-540139, Romania, and 2 Vim Spectrum, Sos. Sighisoarei 409, Corunca, Romania *Author to whom correspondence should be addressed. Email: szabo.zoltan@umftgm.ro Received 14 August 2014; revised 26 November 2014 A novel stability indicating gradient reverse-phased high-performance liquid chromatographic method has been developed for the quantifi- cation of impurities of perindopril tert-butylamine (PER) in pharma- ceutical dosage form. Separation of the active substance and its known (Impurities B, C, D, E, F) and unknown impurities was achieved on a BDS Hypersil C18 column (250 mm 3 4.6 mm, 5 mm), thermostat- ed at 708C, using a mobile phase comprised of aqueous solution of sodium 1-heptanesulfonate adjusted to pH 2 with perchloric acid and acetonitrile. The flow rate was maintained at 1.5 mL min 21 , injection volume of 20 mL was utilized and detection of analytes was performed at 215 nm. The developed method was validated in accordance with current ICH Guidelines for all suggested parameters, including forced degradation studies and proved to be linear, accurate, precise and suit- able for the impurity testing of PER, being subsequently applied during on-going stability studies of a newly developed generic formulation. Introduction Perindopril is a member of the angiotensin-converting enzyme (ACE) inhibitors class, which prevent conversion of angiotensin I to angiotensin II resulting in a reduction of angiotensin II in the plasma and increasing vasodilator bradykinin level, being suc- cessfully utilized in numerous cardiovascular diseases, including treatment of hypertension, heart failure and ischemic heart dis- ease (1, 2). It acts as a prodrug, the ester being hydrolyzed to form the diacid perindoprilat, a potent, long-acting metabolite (3 – 5). Oral administration of perindopril is generally via its tert- butylamine salt, however, in some countries from climatic zones III and IV, perindopril arginine is also available, which is therapeutically equivalent to the previous, but more stable (6). ACE inhibitors in general are relatively unstable and can under- go degradation via hydrolysis of the side chain ester group, intra- molecular cyclization, isomerization at chiral carbon atoms and oxidation. In the case of PER, main degradation pathways include hydrolysis of the ester group, especially in alkaline media, to form the diacid derivative, perindoprilat (Impurity B) and intramolec- ular cyclization, under excessive heat, to form the diketopipera- zine derivate (Impurity F). Other degradants include Impurity C and D, which are epimers, formed after further hydrolytic degra- dation of Impurity F (5, 7, 8). Along with the mentioned degrada- tion products, perindopril isopropyl ester (Impurity E) is also listed as specified impurity in perindopril erbumine tablets monograph of the British Pharmacopoeia (BP) (9). Chemical structure of PER and related substances are presented in Figure 1. A literature survey reveals several methods for quantitative de- termination of degradation products of PER in pharmaceutical dosage forms. The official BP monograph proposes a 65-min gra- dient RP-HPLC method, using water adjusted to pH 2.5 with per- chloric acid as mobile phase A and 0.03% solution of perchloric acid in acetonitrile as mobile phase B (9). Apart from its time con- suming nature, the method did not succeed in providing ade- quate peak shape in the case of Impurity E. Medenica et al.(5) described a simple isocratic HPLC method for the simultaneous determination of PER and its impurities: perindoprilat (Impurity B), Y31 (Impurity F), Y32 (Impurity C) and Y33 (Impurity D). Unfortunately, when employing the pro- posed method, insufficient retention of Impurity B was observed along with inadequate resolution between Impurities C, D and PER. Moreover, the method was not validated for the quantitative analysis of Impurity E and the stability-indicating nature of the technique has not been demonstrated, failing to meet current regulatory requirements. The same problems arose in the case of the microemulsion liquid chromatographic method described by members of the same research group (10). Zaazaa et al. reported the development and validation of two stability-indicating methods (SIMs) for the impurity testing of amlodipine besylate and perindopril arginine in combined dos- age forms (11). However, both TLC-densitometric and RP-HPLC methods were validated only for the quantitative deter- mination of perindoprilat (Impurity B), but not the other speci- fied impurities. When developing SIMs, a clear definition needs to be made be- tween specific and selective SIMs. As highlighted by Bakshi and Singh (12), specific SIMs are able to unequivocally determine and quantify the active pharmaceutical ingredient (API) of inter- est in the presence of degradation products and excipients. Selective SIMs, however, can be used to quantify both the API and its degradation products in the presence of excipients and possible additives. Thus, it is able to separate the API and all deg- radation products and it is also specific, i.e., can be used to quan- titatively measure them. Apart from the selective SIMs, a number of specific SIMs have been developed for the quantitative deter- mination of PER, in the presence of excipients and degradation products (13 – 16). However, in our case, where the quantitative assessment of PER impurities was of primary importance, these methods cannot be used successfully. As it can be seen, none of the methods described in the liter- ature deals with the quantitative assessment of all impurities specified in the official monograph, while successfully discrimi- nating between them and unknown degradation products # The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com Journal of Chromatographic Science 2015;53:424– 430 doi:10.1093/chromsci/bmu223 Advance Access publication January 22, 2015 Article