Improvement and Application HPLC-UV Method for detecting of N-drugs in Pharmaceutical Formulations

(Safanah M. Alkulaib, Ahmed O. Alnajjar and Esam M. Bakir)

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Abstract

The ultraviolet-visible high-performance liquid chromatography (HPLC–UV) coupled method was developed to detect five pharmaceutical compounds: indapamide (INDP), clomipramine (CMI), promethazine HCl (PMH), lisinopril (LSP) and trifluoperazine HCl (TFPH). The mobile phase consisted of 70% acetonitrile and 30% water, ensuring specific retention times (tR) for each compound. Analytical validation parameters were calculated from a calibration curve, including sensitivity, limit of detection (LOD) and linearity of the method. The LOD values of N-drugs were calculated as 6.24 μM for INDP, 2.19 for CMI, 10.57 for PMH, 6.68 for LSP and 1.25 for TFPH. Peak deconvolution or spectral deconvolution of standard solutions of drug mixtures was used to separate overlapping peaks in a dataset and extract information about individuals. Chromatographic separation of the standard mixture of pure compounds achieved a good resolution (R_s), ensuring the effective separation of the mixture components. However, other mixtures did not achieve satisfactory resolution due to retention time interference. Overall, the HPLC–UV method demonstrated good sensitivity and selectivity for the detection of the chosen pharmaceutical compounds. 
KEYWORDS
Chromatogram, deconvolution, limit of detection, pharmaceutical analysis, retention time, sensitivity

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References

Agrawal, G.P. (2021). Validated stability indicating method for determination of indapamide in pharmaceutical formulation. Research Journal of Pharmacy and Technology, 14(6), 3347–52. DOI: 10.52711/0974-360X.2021.00582
Ahuja, S. and Dong, M. (2005). Handbook of Pharmaceutical Analysis by HPLC. Elsevier.
Alkulaib, S.M., Bakir, E.M. and Alnajjar, A.O. (2024). Fluorometric detection of five nitrogen-based pharmaceuticals based on ion-pairing association with EY: DFT calculations. Chemistry, 6(5), 981–92. DOI: 10.3390/chemistry6050057
Beasley, C.A., Shaw, J., Zhao, Z. and Reed, R.A. (2005). Development and validation of a stability indicating HPLC method for determination of lisinopril, lisinopril degradation product and parabens in the lisinopril extemporaneous formulation. Journal of pharmaceutical and biomedical analysis, 37(3), 559–67. DOI: 10.1016/j.jpba.2004.11.021
Bernard, P., Dufresne-Favetta, C., Favetta, P., Do, Q.T., Himbert, F., Zubrzycki, S. and Lugnier, C. (2008). Application of drug repositioning strategy to TOFISOPAM. Current Medicinal Chemistry, 15(30), 3196–203. DOI: 10.2174/092986708786848488
Chaudhary, A.B.,  Raval,  R.J., Vaghela, K. and Patel,  E. (2016). Development and validation of analytical method for simultaneous estimation of chlordiazepoxide, trifluoperazine hydrochloride and trihexyphenidyl hydrochloride in tablet dosage form. International Bulletin of Drug Research, 6(10), 1–6. 
Goa, K.L., Haria, M. and Wilde, M.I. (1997). Lisinopril: A review of its pharmacology and use in the management of the complications of diabetes mellitus. Drugs, 53(6), 1081–105. DOI: 10.2165/00003495-199753060-00010
Hang, T.J.,  Zhao, W., Liu, J.,  Song,  M.,  Xie, Y.,  Zhang,  Z. and Zhang,  Y. (2006). A selective HPLC method for the determination of indapamide in human whole blood: Application to a bioequivalence study in Chinese volunteers. Journal of Pharmaceutical and Biomedical Analysis, 40(1), 202–5. DOI: 10.1016/j.jpba.2005.06.035
Jennings, W., Mittlefehldt, E. and Stremple, P. (1997). Analytical Gas Chromatography. San Diego: Academic Press.
Mohiuddin, I., Bhogal, S., Grover, A., Malik, A.K. and Aulakh, J.S. (2021). Simultaneous determination of amitriptyline, nortriptyline and clomipramine in aqueous samples using selective multi-template molecularly imprinted polymers. Environmental Nanotechnology, Monitoring and Management, 16(n/a), 100527. DOI:.org/10.1016/j.enmm.2021.100527
Mostafavi, S.A., Tahvilian, R., Poudeh, M.D. and Rafeepour, Z. (2010). A simple sample preparation with HPLC-UV Method for estimation of clomipramine from plasma. Iranian Journal of Pharmaceutical Research: IJPR, 9(3), 243. 
Özsar, S.A. and Altınöz, S. (2024). Developing and validation of a high-performance liquid chromatography method for the determination of combined perindopril, indapamide and amlodipine from pharmaceutical preparations. Hacettepe University Journal of the Faculty of Pharmacy, 44(4), 306–17. DOI:10.52794/hujpharm.1445884 
Palakeeti, B., Rao, P.N. and Chinta,  J.P. (2021). Development of new stability indicating UPLC-UV method for the extraction and quantification of perindopril and indapamide from human plasma. Future Journal of Pharmaceutical Sciences, 7(n/a), 1–9. DOI: 10.1186/s43094-021-00220-8
Pawar, V.T., More, H.N. and Bhatia, M.S. (2021). Development and validation of RP-HPLC method for the determination of lisinopril and amlodipine in bulk and multicomponent pharmaceutical cardiovascular dosage form. NVEO-NATURAL VOLATILES and ESSENTIAL OILS Journal| NVEO, 8(4), 9441–51.
Pippalla, S., Nekkalapudi, A.R. and Komreddy, V.R. (2024). A validated stability‐indicating reversed‐phase‐UPLC method for simultaneous estimation of promethazine hydrochloride, methylparaben, propylparaben and sodium benzoate assay of cough suppressant and antihistamine liquid oral dosage forms. Biomedical Chromatography, 38(9), e5944. DOI: 10.1002/bmc.5944
Pirola, R., Mundo, E., Bellodi, L. and Bareggi, S. (2002). Simultaneous determination of clomipramine and its desmethyl and hydroxy metabolites in plasma of patients by high-performance liquid chromatography after solid-phase extraction. Journal of Chromatography B, 772(2), 205–10. DOI: 10.1016/s1570-0232(02)00089-2 
Rao, T.N. (2018). Validation of analytical methods. Calibration and Validation of Analytical Methods—A Sampling of Current Approaches, n/a(n/a)131–41. 
Robards, K. and Ryan, D. (2021). Principles and Practice of Modern Chromatographic Methods. London, UK: Academic Press.
Shearer, C.M., and Miller, S.M. (1976). Promethazine hydrochloride. In Analytical Profiles of Drug Substances. London, UK: Academic Press.
Shetti, P. and Venkatachalam, A. (2010). Stability indicating HPLC method for simultaneous quantification of trihexyphenidyl hydrochloride, trifluoperazine hydrochloride and chlorpromazine hydrochloride from tablet formulation. E-Journal of Chemistry, 7(1), S299–S313. DOI: 10.1155/2010/529386
Thomas, J.R. (1985). A review of 10 years of experience with indapamide as an antihypertensive agent. Hypertension, 7(6) 2, II152. DOI: 10.1161/01.hyp.7.6_pt_2.ii152 
Thumma, S., Zhang, S.Q. and Repka, M. (2008). Development and validation of a HPLC method for the analysis of promethazine hydrochloride in hot-melt extruded dosage forms. Die Pharmazie-An International Journal of Pharmaceutical Sciences, 63(8), 562–7. DOI:10.1691/ph.2008.08.8022
UNODC "United Nations Office on Drugs, Crime". Laboratory and Scientific Section. (2009). Guidance for the Validation of Analytical Methodology and Calibration of Equipment Used for Testing of Illicit Drugs in Seized Materials and Biological Specimens: A Commitment to Quality and Continuous Improvement. United Nations Publications.
Van-Scheyen, J.D. and Van-Kammen, D.P. (1979). Clomipramine-induced mania in unipolar depression. Archives of General Psychiatry, 36(5), 560–5. DOI: 10.1001/archpsyc.1979.01780050070008
Vardanyan, R. and Hruby, V. (2006). Synthesis of Essential Drugs. Elsevier.
Verfaillie, D., Li, J., Van Droogenbroeck, B., Pannecoucque, J., Tavernier, G., Van Royen, G. and Wouters, A.G. (2024). Genetic and environmental variation in protein composition of Belgian soy determined with a novel size-exclusion chromatography method. Journal of Food Composition and Analysis, 130(n/a), 106187. DOI: 10.1016/j.jfca.2024.106187 
Vijayavaani, C., Pavankumar, K., Yogesh, N., Hossain, A. and Hossain, A. (2024). Analytical method development and validation for the estimation of clomipramine HCL in API form and marketed pharmaceutical dosage form by reverse phase-high performance liquid chromatography. International Journal of Multidisciplinary Research and Growth Evaluation, 5(3), 467–75.