1. DEVELOPMENT & VALIDATION OF HIGH-THROUGHPUT & ROBUST UPLC-MS/MS METHOD FOR QUANTITATION OF TERBINAFINE IN HUMAN PLASMA: ………………….APPLICATION TO BIOEQUIVALENCE STUDY SHAH CHINTAN H.MC/2007/05DEPARTMENT OF MEDICINAL CHEMISTRYNIPER, HYDERABAD 1
2. UNDER GUIDENCE OF DR. ShivprakashRathem (M.pharm, Ph.d) Chairman of Synchron Research Pvt.Ltd. Work Place: Synchron Research Pvt.Ltd. 3,Chambers,near S.G.road; Ahmedabad. 2
3.
4. The developed method could then be applied to clinical trials to obtain accurate pharmacokinetic parameters in human plasma.
6. To consume small amounts of solvent and biological fluid for extraction.3
7.
8. Further- more, the latest methods have used liquid chromatography coupled to a mass spectrometry detector for the determination of terbinafine in human hair or tandem mass spectrometry detector in human and minipig plasma.
9. Only two LCMS/ MS methods in human plasma, applied to a bioequivalence studies, have been reported. 4
29. Terbinafine is a highly lipophilic and fungicidal compound active against a wide range of skin pathogens.
30. It acts by selectively inhibiting the enzyme squaleneepoxidase, which is involved in the synthesis of ergosterol from squalene in the fungal cell wall. 9
31.
32. Thus, we investigated several compounds to find a suitable IS, and chose Metoprolol as an internal standard in this study.
35. The purpose of the study is that the bioavailability of the formulations under investigation, it shown to be equal.
36. Based on that conclusion, one may subsequently claim that the therapeutic quality of these formulations is identical. The latter means that both the beneficial and side effects are identical and hence the formulations are truly interchangeable.11
37.
38. Inter-subject variability is a measure of the differences between subjects. On the other hand intra-subject variability is a measure of the differences within subjects. Both types of variability are present in each trial, but in the cross-over design the inter-subject variability is eliminated.
39. The subject functions as his or hers own control and a difference between formulations within one person is only influenced by the (non)random within variability. This makes the cross-over design much more efficient in terms of sample size. One should remember than sometimes the intra-subject variability is very high and in these cases the advantage of a cross-over design rapidly fades away. This happens with so called highly variable drugs. 12
40.
41. At the same time any two-formulation trial is also a two-period trial. In the first period 50% of the volunteers receive A or reference and 50% B or test. In the second period the order is reversed of course. The periods and the sequences are not supposed to exert an influence on the measured parameters like the AUC, T1/2 or any other one. When a significant period or sequence effect is noted, the study can be invalid.13
42.
43. The study must have 1] been a single dose study, 2] been in healthy normal volunteers, 3] not been comparing an endogenous substance, 4] had an adequate washout and 5] used an appropriate design, analysis and equivalence must be present. 14
44.
45. The design of study comprised of a randomized, open label, single dose, two treatments, two periods, two sequence crossover bioequivalence study of 250 mg TER formulation in 44 healthy Indian volunteers.
47. Each volunteer was judged to be in good health through medical history, physical examination and routine laboratory tests screening values were exclusion criteria.15
48.
49. Blood samples were obtained following oral administration of 250mg of TER tablet into K3EDTA vacutainer solution as an anticoagulant at predose, 0.5h, 1h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 5 h, 6 h, 7 h, 9 h, 12 h, 16 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h.
50. Plasma was harvested by centrifuging the blood using an eppendorf centrifuge 5810R (Eppendorf, Germany) at 3000 rpm for 5 min and stored frozen at −20±5˚C until analysis. 16
68. But among these best suitable column was Acquity UPLC and best mobile phase composition was ammonium acetate buffer (10mM): acetonitrile(15:85). 19
90. In these methods, LLE method is less costly, simple, rapid and having less interference of endogenous materials. so, we selected LLE method. 23
91.
92. To this hexane: t-butyl methyl ether (TBME) (80:20) was added (4.5 mL), and the mixture was vortexed for 5 min and centrifuged at 2000 rpm for 5 min.
93. The organic layer was separated into clean evaporation tubes and evaporated to dryness under N2 at 45°C.
94. The residue was reconstituted with 500 µL of acetonitrile: ammonium acetate (70:30) mobile phase solution and injected 2 μL injection into the LC–MS/MS system.
114. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (i.e., ±15% S.D.) and precision (i.e., 15% R.S.D.).26
125. BIOEQUIVALENCE STUDY PARAMETERS & CHROMATOGRAM PK Parameters of Terbinafine in Human Plasma (Test A) PK Parameters of Terbinafine in Human Plasma (Ref- B) 37
126. Comparison of Bioequivalence Parameters of Both Test & Reference Drugs 38 AUC0–t= The area under the plasma concentration–time curve from time zero to last sampling time AUC0–∞= The area under the plasma concentration–time curve from time zero to infinity. Cmax = Maximum plasma concentration Tmax = Maximum time to reach Cmax
127. Comparisin of mean plasma concentration & time profile of Test & reference drugs of 250 mg dose tablets 39
130. This method required only 500 µL of a biological sample and owing to the simple sample preparation and short run time (2 min) and also only 2 µl injection volume; it allows high sample throughput with fast analysis and less saturation of column.
131. We achieved a lower LLOQ (15 ng/ml) and shorter retention times (1.8 min. for TER, 0.7 min for IS) than previous reports.
132. The precision and accuracy for calibration and QC samples were well within the acceptable limits.
133. This method was sensitive enough to monitor Terbinafine plasma concentrations up to 144 h after dosing and provided us with a successful application in pharmacokinetics and bioequivalence study.41
134.
135. Bonate P, Howard D “Pharmacokinetics in Drug development” AAPS Press Year 2004 Vol.2 Page No.105, 127-229.
136.
137. FDA. Guidance for Industry: Bioavailability Studies for Orally Administered Drug- Products -General Considerations. US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER): Washington, DC, 2000.
141. Gokhale VM and Kulkarni VM. Understanding the antifungal activity of terbinafine analogues using quantitative structure-activity relationship (QSAR) models. Bioorganic and Medicinal Chemistry, 2000; 8: 2487.
143. "Effect of metoprolol in chronic heart failure: Metoprolol Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF)". Lancet 353 (9169): 2001–7. June 12 1999.43
144.
145. Zehender H, Denouel J, Roy M, Le Saux L and Schaub P. Simultaneous determination of terbinafine (Lamisil) and five metabolites in human plasma and urine by high-performance liquid chromatography using on-line solid-phase extraction. Journal of Chromatography B 1995; 664: 347.
146. Majumdar TK, Bakhtiar R, Melamed D and Tse FLS. Determination of terbinafine (Lamisil®) in human hair by microbore liquid chromatography/ tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2000; 14: 1214.
147. De Oliveira CH, Barrientos-Astigarraga RE, De MoraesMO,Bezerra FAF, De Moraes MEA and De Nucci G. Terbinafine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry: Application to a bioequivalence study. Theraputic Drug Monitoring ; 2001; 23: 709.
148. Yannis L. Loukas, ConstantinosApostolou, ConstantinosKousoulos, Georgia Tsatsou and YannisDotsikas .An improved high-throughput liquid chromatographic/tandem mass spectrometric method for terbinafine quantification in human plasma, using automated liquid–liquid extraction based on 96-well format plates Biomed. Chromatogr. 21: 201–208 (2007). Voltammetric determination of terbinafine in biological fluid, Bioelectrochemistry (2008); 107–1155 December 2007.44