SIMPLE HPLC VALIDATED METHOD FOR THE DETERMINATION OF DILTIAZEM HYDROCHLORIDE IN HUMAN PLASMA
Keywords:
Diltiazem hydrochloride, HPLC, Assay validation, Stability indicating methodAbstract
Objectives: To develop and validate a high-performance liquid chromatographic method for determination of diltiazem hydrochloride (DLZ) in human plasma.
Methods: Mixture of n-hexane and 2-propanol (96:4, ratio) was added to plasma at sample preparation time followed by centrifuging the samples. The obtained upper organic layer was transferred and evaporated to dryness. The residue was reconstituted with a mobile phase and the supernatant was then injected onto the column. The mobile phase used was consisted of 0.2 M ammonium dihydrogen phosphate, acetonitrile, isopropyl alcohol and triethylamine (55:43:1.7:0.3, v/v) with pH adjusted to 4.5 using 85% phosphoric acid. The flow rate was 0.7 ml/min. UV detector set at 240 nm and samples were quantified using peak area.
Results: A well-resolved DLZ peak and free of interference from endogenous compounds in plasma with a retention time of 6.03 min was achieved. Recovery of DLZ was satisfactory (‰¥ 91.3%) over the concentration range tested 0.25 - 20 µg/ml. LOD of this assay was 0.125 µg/ml and LOQ was 0.25 µg/ml and, at this concentration, intra- and inter-day CV were 6.8 and 9.2 %, respectively. DLZ was found to be stable in plasma after storage at -80 ºC, over 90 days.
Conclusion: The HPLC method described in this article was simple, sensitive, selective, reproducible, linear, precise, accurate, stability indicating and requires only a small sample volume, lending it suitable for the determination of DLZ concentration in routine measurements for pharmacokinetic/bioavailability studies.
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References
Weir MR. Diltiazem: ten years of clinical experience in the treatment of hypertension. J Clin Pharmacol 1995;35:220-32.
Pool PE. Diltiazem Cardiovascular drug therapy. Cardiovascular Drug Therapy (2nded) 1996;931-71.
Najma SA, Saeed A, Nighat S. Pak J Pharm Sci 2007;20:279-84.
Smith DH, Neutel JM, Weber MA. Comparisons of the effects of different long-Acting Delivery Systems on the Pharmacokinetics and Pharmacodynamics of Diltiazem. Am J Hypertens 1999;2:1030-7.
Leonard RG, Talbert RL. Calcium-channel blocking agents. Clin Pharm 1982;1:17-33.
Andern L, Hoglund P, Dotevall A, Eggertsen R, Svensson A, Plson SO, et al. Diltiazem in hypertensive patients with type II diabetes mellitus. Am J Cardiol 1988;62(11):114G-20G.
Hansson L, Hedner T, Lund-Johansen P, Kjeldsen SE, LindholmLH, Syvertsen JO, Randomised trial of effects of calcium antagonists compared with diuretics and beta-blockers on cardiovascular morbidity and mortality in hypertension: the Nordic Diltiazem (NORDIL) study. Lancet 2000;356 (9227):359-65.
Kahno K, Takenchi Y, Etho A, Noda K. Pharmacokinetics and bioavailability of diltiazem(CRD-401) in dog. Arzneimittel-Forschung 1977;27 (7):1424-8.
Rovei V, Mitchard M, Morselli PL. Simple, sensitive and specific gas chromatographic method for the quantification of diltiazem in human body fluids. J Chromatogr 1977;138(2):391-8.
Clozel JP, Gaille G, Taeymans Y, Theroux P, Biron P, Besner JG. Improved gas chromatographic determination of diltiazem and desacetyldiltiazem in human plasma. J Pharm Sci 1984;73:207-9.
Sidhu AS, Kennedy JM, Deeble S. General method for the analysis of pharmaceutical dosage forms by high-performance liquid chromatography. J Chromatogr1987;391(1):233-9.
Vergese C, Smith MS, Aanonsen, L, Pritchett EL, Shaud DG. High-performance liquid chromatographic analysis of diltiazem and its metabolite in plasma. J Chromatogr 1983;272 (1):149-55.
Clozel JP, Gaille G, Taeymans Y, Theroux P, Biron P, Trudel F. High performance liquid chromatographic determination of diltiazem and six of its metabolites in human urine. J Pharm Sci 1984;73 (6):771-3.
Mohamed EA, Mohamed SS, Naji MN, Muti H. Stability indicating high performance liquid chromatographic determination of diltiazem hydrochloride. Anal Lett 1988;21 (12):2263-75.
Zhao H, Chow MS. Analysis of diltiazem and desacetyl-diltiazem in plasma using modified high-performance liquid chromatography: Improved sensitivity and reproducibility. Pharm Res 2011;6 (5):428-30.
Scully P, Meehan E, Kelly JG. High performance liquid chromatographic assay for diltiazem in small-volume blood specimens and application to pharmacokinetic studies in rats. J Chromatog A 1996;729 (1-2):297-300.
Cristensen H, Carlson E, Asberg A, Schram L, Berg KJ. A simple and sensitive high-performance liquid chromatography assay of diltiazem and main metabolites in renal transplanted patients. Clin Chim Acta 1999;283 (1-2):63-75.
Buur JL, Baynes RE, Yeatts JL, Davidson G, Defrancesco TC. Analysis of diltiazem in Lipoderm transdermal gel using reversed-phase high-performance liquid chromatography applied to homogenization and stability studies. J Pharm Biomed Anal 2005;38 (1):60-65.
Sultana N, Arayne MS, Shafi N. A validated method for the analysis of diltiazem in raw materials and pharmaceutical formulations by rp-HPLC. Pak J Pharm Sci 2007;20(4):284-90.
Vivekanand A, Chatpalliwara N, Pawan, Porwala, Neeraj U. Validated gradient stability indicating HPLC method for determining Diltiazem Hydrochloride and related substances in bulk drug and novel tablet formulation. J Pharm Anal 2012;2(3):226-37.
Espen M, Per WJ, Grethe HB, Stein B, Hege C, Hans ER, Helge R, Leon R, Gustav L. Pharmacokinetics of diltiazemHCl and its metabolites in relation to CYP2D6 genotype. Clin Pharmacol Ther 2002;(3):333-42.
Parissi-Poulou M, Ismailos G, Macheras P. Modified HPLC analysis of diltiazem in plasma for pharmacokinetic studies. Int J Pharm 1990;62 (3-2):R13-R16.
Klaas EB, Csaba H, Wayne RM, Avi N. Surface silanols in silica-bonded hydrocarbonaceous stationary phases: II. Irregular retention behavior and effect of silanol masking. J Chromatogr A1981;203:65-85.
Jeffrey SK, Stephen LM. Effects of amine modifiers on retention and peak shape in reversed-phase high-performance liquid chromatography. J Chromatogr A 1985;320:313-23.