Shah Chintan H

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Shah Chintan H

  1. 1. DEVELOPMENT & VALIDATION OF HIGH-THROUGHPUT & ROBUST UPLC-MS/MS METHOD FOR QUANTITATION OF TERBINAFINE IN HUMAN PLASMA: <br />………………….APPLICATION TO BIOEQUIVALENCE STUDY<br />SHAH CHINTAN H.MC/2007/05DEPARTMENT OF MEDICINAL CHEMISTRYNIPER, HYDERABAD<br />1<br />
  2. 2. UNDER GUIDENCE OF<br />DR. ShivprakashRathem (M.pharm, Ph.d)<br />Chairman <br />of Synchron Research Pvt.Ltd.<br />Work Place:<br />Synchron Research Pvt.Ltd.<br />3,Chambers,near S.G.road;<br />Ahmedabad.<br />2<br />
  3. 3. Aim & objectives :<br />Development & Validation of High-throughput & Robust UPLC-MS/MS method for Quantitation Of Terbinafine in human plasma:<br /> Application to Bioequivalence Study.<br />Objectives:<br /><ul><li>The objective will be to achieve more selectivity, sensitivity and more rapid assay method than have been previously described.
  4. 4. The developed method could then be applied to clinical trials to obtain accurate pharmacokinetic parameters in human plasma.
  5. 5. To develop & validate simple and robust.
  6. 6. To consume small amounts of solvent and biological fluid for extraction.</li></ul>3<br />
  7. 7. Literature review<br /><ul><li>A fewHPLC methods have been reported so far for the determination of terbinafine in pharmaceutical preparations as well as in biological matrices .
  8. 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. 9. Only two LCMS/ MS methods in human plasma, applied to a bioequivalence studies, have been reported. </li></ul>4<br />
  10. 10. Scope of application<br /><ul><li>latest reported LC-MS/MS METHOD:
  11. 11. 2.5 min analytical run time…………
  12. 12. LLOQ upto 25ng/ml……….
  13. 13. Plasma volume 500µl which is to high.........
  14. 14. So we develop UPLC–MS/MS method for determining Terbinafine in human plasma which having,
  15. 15. Simple liquid–liquid extraction technique with less than 2 min analytical run time and LLOQ 15ng/ml.
  16. 16. Method having less matrix effect, high recovery, less costly and high throughput.
  17. 17. This method was successfully applied to a bioequivalence study of Two TER 250 mg oral tablets in 44 healthy human volunteers.</li></ul>5<br />
  18. 18. Presentation flow<br />Introduction<br />Bioequivalence study<br />Materials & Methods<br />Method Validation<br />Results & Discussion<br />Conclusion<br />References<br />6<br />
  19. 19. introduction of Biopharmaceutical analysis<br /><ul><li>Need of Biopharmaceutical Analysis:</li></ul>Methods of measuring drugs in biologic media are increasingly important problems related to following studies are highly dependent on biopharmaceutical Analytic methodology.<br /><ul><li>Bioavailability and bioequivalence studies
  20. 20. New drug development
  21. 21. Clinical pharmacokinetics and metabolism
  22. 22. Therapeutic drug monitoring
  23. 23. Research in basic biomedical and pharmaceutical sciences</li></ul>7<br />
  24. 24. Introduction- Drug profile (terbinafine)<br /><ul><li>Terbinafine (TER) is [(E)-N-(6, 6-dimethyl-2-heptene-4-ynyl)-N-methyl-1-naphthalene methanamine] synthetic allylamine antifungal compound. It is freely soluble in methanol and methylene chloride, soluble in ethanol, and slightly soluble in water .
  25. 25. The empirical formula C21H25N with a molecular weight of 291.43g/mol and the following structural formula:</li></ul>TERBINAFINE<br />8<br />
  26. 26. Continues…..<br /><ul><li>PKa of Terbinafine: 7.10
  27. 27. Melting Point: 195-198 °C
  28. 28. M/A: ANTIFUNGAL
  29. 29. Terbinafine is a highly lipophilic and fungicidal compound active against a wide range of skin pathogens.
  30. 30. It acts by selectively inhibiting the enzyme squaleneepoxidase, which is involved in the synthesis of ergosterol from squalene in the fungal cell wall. </li></ul>9<br />
  31. 31. Drug profile of internal standard (metoprolol)<br /><ul><li>Stable labelled isotope internal standards are the first choice, they are not economical and deuterium-labelled compounds may sometimes demonstrate unexpected behaviour, such as different retention times or recoveries, than the analyte.
  32. 32. Thus, we investigated several compounds to find a suitable IS, and chose Metoprolol as an internal standard in this study.
  33. 33. Molecular Formula : C15H25NO3METOPROLOL</li></ul>Molecular Weight : 267.36 <br /> Solubility : Methanol<br />10<br />
  34. 34. Bioequivalence study<br /><ul><li>Nowadays bioequivalence studies are a pivotal part of registration dossiers. These studies measure the bioavailability of two (or more) formulations of the same active ingredient.
  35. 35. The purpose of the study is that the bioavailability of the formulations under investigation, it shown to be equal.
  36. 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.</li></ul>11<br />
  37. 37. Study design<br /><ul><li>Basically two types of designs are possible, that is the parallel and cross-over design. The major difference between these designs is the way they deal with inter-subject variability.
  38. 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. 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. </li></ul>12<br />
  40. 40. Continues…..<br /><ul><li>The most common design for a cross-over trial is the well known AB/BA trial. It tells that one splits the entire sample of subjects randomly into two groups. Group 1 will receive the drug or formulations (frequently called the treatments) in the order A-B or reference-test and the other group in the order B-A or test-reference. These two orders are called the sequences, so any two-formulation trial is a two-sequence trial.
  41. 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.</li></ul>13<br />
  42. 42. Continues…..<br /><ul><li>One of the problems with any AB/BA trial is carry-over, if Carry-over is present when the effects of the drugs in period 1 are still noticeable in period 2. For a bioequivalence study this would be the case if the first plasma level before administration of the drug in the second period is not 0. If that is the case the washout between the two periods was not sufficiently long.
  43. 43. The study must have 1] been a single dose study, </li></ul>2] been in healthy normal volunteers, <br />3] not been comparing an endogenous <br /> substance, <br /> 4] had an adequate washout and <br />5] used an appropriate <br />design, analysis and <br /> equivalence must be present.<br />14<br />
  44. 44. Application to Bioequivalence study<br /><ul><li>The proposed analytical method was applied to a bioequivalence study of two Terbinafine formulations under fasting condition in healthy volunteers of 37±7 years in age and 54±6 kg in weight.
  45. 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. 46. Subjects were informed of the aims and risks of the study by the clinical investigator.
  47. 47. Each volunteer was judged to be in good health through medical history, physical examination and routine laboratory tests screening values were exclusion criteria.</li></ul>15<br />
  48. 48. Continues……<br /><ul><li>The study was conducted strictly in accordance with guidelines laid down by USFDA .
  49. 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. 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. </li></ul>16<br />
  51. 51. Parameters measured in bioequivalence study<br /><ul><li>The Area Under the Plasma concentration-time curve (AUC), because it describes the total number of molecules present in plasma, thereby informing the researchers on the extent of the release…..(AUC0–tand AUC0–∞ ).
  52. 52. The maximum plasma concentration (Cmax).
  53. 53. The time at which the maximum plasma concentration is reached (Tmax).
  54. 54. The elimination half life (T1/2). (T1/2)was calculated by 0.692/λ.
  55. 55. Equivalencelimits currently accepted by the regulatory bodies are as follows:</li></ul> for the AUC‘s 0.8-1.25.<br /> for Cmax 0.7-1.43 .<br /> <br />17<br />
  56. 56. Materials & methods<br /><ul><li>Materials:
  57. 57. Terbinafine working standard
  58. 58. Metoprolol working standard
  59. 59. Acetonitrile (HPLC grade)
  60. 60. Methanol (HPLC grade)
  61. 61. Milli-Q/HPLC water
  62. 62. Ammonium acetate (LR grade)
  63. 63. Formic acid (GR grade)
  64. 64. Tert- butyl methyl ether (HPLC grade)
  65. 65. N-hexane (HPLC grade)
  66. 66. Human plasma</li></ul>18<br />
  67. 67. Column and mobile phase selection<br /><ul><li>Here, we tried different columns like Sunfire symmetry, Acquity UPLC, Kromasil, Phenomenax etc. and also different mobile phase composition like water- methanol, water-acetonitrile, Phosphate buffer- acetonitrile,0.1% formic acid in water buffer- acetonitrile, ammonium acetate buffer- acetonitrile, ammonium acetate and buffer- acetonitrile- methanol.
  68. 68. But among these best suitable column was Acquity UPLC and best mobile phase composition was ammonium acetate buffer (10mM): acetonitrile(15:85). </li></ul>19<br />
  69. 69. General chromatographic condition<br /><ul><li>Matrix Human plasma
  70. 70. Sample volume 0.500 µI
  71. 71. Injection volume 2 µl
  72. 72. Extraction method Liquid-liquid extraction
  73. 73. Internal standard Metoprolol
  74. 74. Detection Q3 (Quattro premierXE, Micromass)
  75. 75. m/z ratio 292.37> 92.90
  76. 76. Weighing factor 1/x
  77. 77. Column Acquity UPLC, Symmetry;</li></ul> C18, 2.1x50mm, 2.5µm<br /><ul><li>Mobile phase Buffer(pH 5 ±0.05): ACN (15:85)v/v
  78. 78. Flow Rate 0.3 mL/minute
  79. 79. Ion Mode Positive Mode
  80. 80. Column temperature 40 ±5ºC
  81. 81. Sample cooler temperature 20 ±5ºC
  82. 82. Retention time Terbinafine 1.8 min; Metoprolol 0.7 min
  83. 83. Run time 2.0 min</li></ul>20<br />
  84. 84. Mass tune parameters<br />21<br />
  85. 85. Preparation of calibration standards & quality control samples <br />22<br />
  86. 86. Trials for selection of method<br /><ul><li> Three trials used for sample extraction methods:
  87. 87. SPE (Solid phase extraction): Conditioning » washing » loading of sample » eluting.
  88. 88. LLE (Liquid-liquid extraction): Sampling »adding solvent/ solvent mixture » vortex » centrifuge » evaporate to dryness » reconstitute.
  89. 89. Protein precipitation : Sampling + ppt. solvent.
  90. 90. In these methods, LLE method is less costly, simple, rapid and having less interference of endogenous materials. so, we selected LLE method. </li></ul>23<br />
  91. 91. Finally Selected Method (LLE):<br /><ul><li>A 0.490-mL aliquot of plasma sample was spiked with 25 μL of IS (metoprolol, 10 µg/mL) in 10 mL glass tubes and vortexed for 1 min.
  92. 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. 93. The organic layer was separated into clean evaporation tubes and evaporated to dryness under N2 at 45°C.
  94. 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. 95. All prepared samples were kept in an auto sampler at 10± 5°C until injection.</li></ul>24<br />
  96. 96. Method validation<br /><ul><li>Method validation was performed according to the USFDA guidance for industrial bio analytical method validation.
  97. 97. PARAMETERS:
  98. 98. Selectivity
  99. 99. Sensitivity
  100. 100. Linearity
  101. 101. Accuracy and precision(INTRA-DAY/INTER-DAY)
  102. 102. Recovery
  103. 103. Matrix effect
  104. 104. LLOQ
  105. 105. LOD
  106. 106. Reinjection /Reproducibility</li></ul>25<br />
  107. 107. <ul><li>STABILITY STUDIES:
  108. 108. Auto sampler stability
  109. 109. Short-term stock solution stability
  110. 110. Bench top stability
  111. 111. Freeze-thaw stability
  112. 112. Long term stability
  113. 113. Blood stability
  114. 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.).</li></ul>26<br />
  115. 115. Results & discussion<br /><ul><li>A Representative Regression Analysis of a Calibration curve of Terbinafine- Linearity</li></ul>27<br />
  116. 116. A Representative Chromatograph of Aqueous Standard & Specificity<br />28<br />
  117. 117. A Representative Chromatograph of LLOQ & HQC<br />29<br />
  118. 118. BACK CALCULATED CALIBRATION CURVE CONCENTRATIONS- SENSITIVITY<br />30<br />
  119. 119. ACCURACY & PRECISION<br />31<br />
  120. 120. recovery<br />32<br />
  121. 121. STABILITIES PARAMETERS:Stss stability & auto sampler stability<br />33<br />
  122. 122. Freeze thaw stability & bench top stability<br />34<br />
  123. 123. Matrix effect & reinjection reproducibility<br />35<br />
  124. 124. LIMIT OF QUANTIFICATION & Limit of Detection<br />36<br />
  125. 125. BIOEQUIVALENCE STUDY PARAMETERS & CHROMATOGRAM<br />PK Parameters of Terbinafine in Human Plasma (Test A)<br /> PK Parameters of Terbinafine in Human Plasma (Ref- B)<br />37<br />
  126. 126. Comparison of Bioequivalence Parameters of Both Test & Reference Drugs<br />38<br />AUC0–t= The area under the plasma concentration–time curve from time zero to last <br />sampling time<br />AUC0–∞= The area under the plasma concentration–time curve from time zero to infinity.<br />Cmax = Maximum plasma concentration<br />Tmax = Maximum time to reach Cmax<br />
  127. 127. Comparisin of mean plasma concentration & time profile of Test & reference drugs of 250 mg dose tablets<br />39<br />
  128. 128. Mean Plasma Concentration versus Time profile Curve of Both Test & Reference Drugs<br />40<br />
  129. 129. conclusion<br /><ul><li>We reported method development and validation of a rapid (high-throughput), selective and sensitive UPLC–MS/MS method with liquid–liquid extraction for the determination of TER in human plasma, over a concentration range of 15.91–1595 ng/mL.
  130. 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. 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. 132. The precision and accuracy for calibration and QC samples were well within the acceptable limits.
  133. 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.</li></ul>41<br />
  134. 134. references<br /><ul><li>Chung S, Liu P “Design and Analysis of Bioavailability and Bioequivalence studies” Marcel Dekker Inc., Year 2000, Edition 2nd, Page No.8-33.
  135. 135. Bonate P, Howard D “Pharmacokinetics in Drug development” AAPS Press Year 2004 Vol.2 Page No.105, 127-229.
  136. 136. Bruce et. al.,1996, Method development; view point and discussion, </li></ul>http://linkinghub.elsevier.com/retrieve/pii/S0381530400544088 .<br /><ul><li>The definitions of LOQ & LOD that we are used provided by Dadger et.al.1995, </li></ul>http://linkinghub.elsevier.com/retrieve/pii/S0308814208107942.<br /><ul><li>International Conference on Harmonisation of technical requirement for the registration of Pharmaceutical for human use, Validation of analytical procedure, ICH-Q2B.10.
  137. 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.
  138. 138. Fundamental Validation pareameters for Bioanalytical method development </li></ul> &validation, Green et .al.1998, Hartmann et. al.,1998, <br />http://linkinghub.elsevier.com/retrieve/pii/S03447146010009267. <br /><ul><li>Stability parameters for Method Validation, degradation and criteria, Witling et. al.,1998, </li></ul>http://linkinghub.elsevier.com/retrieve/pii/S0331468873002788.<br />42<br />
  139. 139. <ul><li>Wikipedia free encyclopedia, Terbinafine; http://en.wikipedia.org/wiki/Terbinafine.
  140. 140. 35. H.P.Rang, M.M.Dale, J.M.Ritter, P.K.Moore, “Pharmacology” Fifth Edition, Page No.394- 403.
  141. 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. 142. Wikipedia free encyclopedia, Metoprolol; http://en.wikipedia.org/wiki/Metoprolol.
  143. 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.</li></ul>43<br />
  144. 144. <ul><li>DenouelJ, Keller HP, Schaub P, Delaborde C and Humbert H. Determination of terbinafine and its desmethyl metabolite in human plasma by high-performance iquid chromatography. Journal of Chromatography B 1995; 663: 353.
  145. 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. 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. 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. 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.</li></ul>44<br />
  149. 149. 45<br />
  150. 150. 46<br />

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