My thesis presentation

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

4. The developed method could then be applied to clinical trials to obtain accurate pharmacokinetic parameters in human plasma.

5. To develop & validate simple and robust.

6. To consume small amounts of solvent and biological fluid for extraction.3

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

11. 2.5 min analytical run time…………

12. LLOQ upto 25ng/ml……….

13. Plasma volume 500µl which is to high.........

14. So we develop UPLC–MS/MS method for determining Terbinafine in human plasma which having,

15. Simple liquid–liquid extraction technique with less than 2 min analytical run time and LLOQ 15ng/ml.

16. Method having less matrix effect, high recovery, less costly and high throughput.

17. This method was successfully applied to a bioequivalence study of Two TER 250 mg oral tablets in 44 healthy human volunteers.5

18. Presentation flow Introduction Bioequivalence study Materials & Methods Method Validation Results & Discussion Conclusion References 6

20. New drug development

21. Clinical pharmacokinetics and metabolism

22. Therapeutic drug monitoring

23. Research in basic biomedical and pharmaceutical sciences7

25. The empirical formula C21H25N with a molecular weight of 291.43g/mol and the following structural formula:TERBINAFINE 8

27. Melting Point: 195-198 °C

28. M/A: ANTIFUNGAL

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

32. Thus, we investigated several compounds to find a suitable IS, and chose Metoprolol as an internal standard in this study.

33. Molecular Formula : C15H25NO3METOPROLOLMolecular Weight : 267.36 Solubility : Methanol 10

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

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

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

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

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.

46. Subjects were informed of the aims and risks of the study by the clinical investigator.

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

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

52. The maximum plasma concentration (Cmax).

53. The time at which the maximum plasma concentration is reached (Tmax).

54. The elimination half life (T1/2). (T1/2)was calculated by 0.692/λ.

55. Equivalencelimits currently accepted by the regulatory bodies are as follows: for the AUC‘s 0.8-1.25. for Cmax 0.7-1.43 . 17

57. Terbinafine working standard

58. Metoprolol working standard

59. Acetonitrile (HPLC grade)

60. Methanol (HPLC grade)

61. Milli-Q/HPLC water

62. Ammonium acetate (LR grade)

63. Formic acid (GR grade)

64. Tert- butyl methyl ether (HPLC grade)

65. N-hexane (HPLC grade)

66. Human plasma18

68. But among these best suitable column was Acquity UPLC and best mobile phase composition was ammonium acetate buffer (10mM): acetonitrile(15:85). 19

70. Sample volume 0.500 µI

71. Injection volume 2 µl

72. Extraction method Liquid-liquid extraction

73. Internal standard Metoprolol

74. Detection Q3 (Quattro premierXE, Micromass)

75. m/z ratio 292.37> 92.90

76. Weighing factor 1/x

78. Flow Rate 0.3 mL/minute

79. Ion Mode Positive Mode

80. Column temperature 40 ±5ºC

81. Sample cooler temperature 20 ±5ºC

82. Retention time Terbinafine 1.8 min; Metoprolol 0.7 min

83. Run time 2.0 min20

84. Mass tune parameters 21

85. Preparation of calibration standards & quality control samples 22

87. SPE (Solid phase extraction): Conditioning » washing » loading of sample » eluting.

88. LLE (Liquid-liquid extraction): Sampling »adding solvent/ solvent mixture » vortex » centrifuge » evaporate to dryness » reconstitute.

89. Protein precipitation : Sampling + ppt. solvent.

90. In these methods, LLE method is less costly, simple, rapid and having less interference of endogenous materials. so, we selected LLE method. 23

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.

95. All prepared samples were kept in an auto sampler at 10± 5°C until injection.24

97. PARAMETERS:

98. Selectivity

99. Sensitivity

100. Linearity

101. Accuracy and precision(INTRA-DAY/INTER-DAY)

102. Recovery

103. Matrix effect

104. LLOQ

105. LOD

106. Reinjection /Reproducibility25

108. Auto sampler stability

109. Short-term stock solution stability

110. Bench top stability

111. Freeze-thaw stability

112. Long term stability

113. Blood stability

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

116. A Representative Chromatograph of Aqueous Standard & Specificity 28

117. A Representative Chromatograph of LLOQ & HQC 29

118. BACK CALCULATED CALIBRATION CURVE CONCENTRATIONS- SENSITIVITY 30

119. ACCURACY & PRECISION 31

120. recovery 32

121. STABILITIES PARAMETERS:Stss stability & auto sampler stability 33

122. Freeze thaw stability & bench top stability 34

123. Matrix effect & reinjection reproducibility 35

124. LIMIT OF QUANTIFICATION & Limit of Detection 36

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

128. Mean Plasma Concentration versus Time profile Curve of Both Test & Reference Drugs 40

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

135. Bonate P, Howard D “Pharmacokinetics in Drug development” AAPS Press Year 2004 Vol.2 Page No.105, 127-229.

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.

140. 35. H.P.Rang, M.M.Dale, J.M.Ritter, P.K.Moore, “Pharmacology” Fifth Edition, Page No.394- 403.

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.

142. Wikipedia free encyclopedia, Metoprolol; http://en.wikipedia.org/wiki/Metoprolol.

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

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

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