Bioequivalence
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Bioequivalence Bioequivalence Presentation Transcript

  • DEVELOPMENT1 & VALIDATION OF HIGH-THROUGHPUT &ROBUST UPLC-MS/MS METHOD FOR QUANTITATION OF TERBINAFINE IN HUMAN PLASMA: ………….APPLICATION TO BIOEQUIVALENCE STUDY
  • Aim & objectives : 2 Development & Validation of High-throughput & Robust UPLC-MS/MS method for Quantitation Of Terbinafine in human plasma: Application to Bioequivalence Study.Objectives: The objective will be to achieve more selectivity, sensitivity and more rapid assay method than have been previously described. The developed method could then be applied to clinical trials to obtain accurate pharmacokinetic parameters in human plasma. To develop & validate simple and robust. To consume small amounts of solvent and biological fluid for extraction.
  • Literature review 3 A few HPLC methods have been reported so far for the determination of terbinafine in pharmaceutical preparations as well as in biological matrices . 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. Only two LCMS/ MS methods in human plasma, applied to a bioequivalence studies, have been reported.
  • Scope of application 4 latest reported LC-MS/MS METHOD: 2.5 min analytical run time………… LLOQ upto 25ng/ml………. Plasma volume 500µl which is to high......... So we develop UPLC–MS/MS method for determining Terbinafine in human plasma which having, Simple liquid–liquid extraction technique with less than 2 min analytical run time and LLOQ 15ng/ml. Method having less matrix effect, high recovery, less costly and high throughput. This method was successfully applied to a bioequivalence study of Two TER 250 mg oral tablets in 44 healthy human volunteers.
  • Presentation flow 5IntroductionBioequivalence studyMaterials & MethodsMethod ValidationResults & DiscussionConclusionReferences
  • introduction of Biopharmaceutical analysis 6 Need of Biopharmaceutical Analysis: Methods of measuring drugs in biologic media are increasingly important problems related to following studies are highly dependent on biopharmaceutical Analytic methodology. Bioavailability and bioequivalence studies New drug development Clinical pharmacokinetics and metabolism Therapeutic drug monitoring Research in basic biomedical and pharmaceutical sciences
  • Introduction- Drug profile (terbinafine) 7 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 . The empirical formula C21H25N with a molecular weight of 291.43g/mol and the following structural formula: TERBINAFINE CH3 N C(CH3)3
  • Continues….. 8 PKa of Terbinafine: 7.10 Melting Point: 195-198 °C M/A: ANTIFUNGAL Terbinafine is a highly lipophilic and fungicidal compound active against a wide range of skin pathogens. It acts by selectively inhibiting the enzyme squalene epoxidase, which is involved in the synthesis of ergosterol from squalene in the fungal cell wall.
  • Drug profile of internal standard (metoprolol) 9 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. Thus, we investigated several compounds to find a suitable IS, and chose Metoprolol as an internal standard in this study. Molecular Formula : C15H25NO3 METOPROLOL OH Molecular Weight : 267.36 NH CH3 Solubility : Methanol O CH3 OH3C
  • Bioequivalence study 10 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. The purpose of the study is that the bioavailability of the formulations under investigation, it shown to be equal. 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.
  • Study design 11 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. 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. 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.
  • Continues….. 12 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. 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.
  • Continues….. 13 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. 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.
  • Application to Bioequivalence study 14 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. 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. Subjects were informed of the aims and risks of the study by the clinical investigator. Each volunteer was judged to be in good health through medical history, physical examination and routine laboratory tests screening values were exclusion criteria.
  • Continues…… 15 The study was conducted strictly in accordance with guidelines laid down by USFDA . 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. 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.
  • Parameters measured in bioequivalence study 16 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–t and AUC0–∞ ). The maximum plasma concentration (Cmax). The time at which the maximum plasma concentration is reached (Tmax). The elimination half life (T1/2). (T1/2) was calculated by 0.692/λ. Equivalence limits currently accepted by the regulatory bodies are as follows: for the AUC„s 0.8-1.25. for Cmax 0.7-1.43 .
  • Materials & methods 17 Materials: Terbinafine working standard Metoprolol working standard Acetonitrile (HPLC grade) Methanol (HPLC grade) Milli-Q/HPLC water Ammonium acetate (LR grade) Formic acid (GR grade) Tert- butyl methyl ether (HPLC grade) N-hexane (HPLC grade) Human plasma
  • 18Column and mobile phase selection 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. But among these best suitable column was Acquity UPLC and best mobile phase composition was ammonium acetate buffer (10mM): acetonitrile(15:85).
  • General chromatographic condition Matrix Human 19plasma Sample volume 0.500 µI Injection volume 2 µl Extraction method Liquid-liquid extraction Internal standard Metoprolol Detection Q3 (Quattro premierXE, Micromass) m/z ratio 292.37> 92.90 Weighing factor 1/x Column Acquity UPLC, Symmetry; C18, 2.1x50mm, 2.5µm Mobile phase Buffer(pH 5 ±0.05): ACN (15:85)v/v Flow Rate 0.3 mL/minute Ion Mode Positive Mode Column temperature 40 ±5ºC Sample cooler temperature 20 ±5ºC Retention time Terbinafine 1.8 min; Metoprolol 0.7 min Run time 2.0 min
  • Mass tune parameters Parameter 20 Drug ISTD Capillary Voltage (KV) 3.5 3.5 Source Temperature 120°C 120°CDesolvation Temperature 450°C 450°C Cone Voltage (V) 26 33 LM Resolution-1 14.5 14.5 HM Resolution-1 14.5 14.5 Collision Energy (eV) 18 18 Entrance 2 2 Exit 2 2 LM Resolution-2 14.5 14.5 HM Resolution-2 14.5 14.5 Detection 292.37> 92.90 268.33>115.90 Dwell Time (Second) 0.200 0.200 Desolvation Gas Flow 500 500
  • Preparation of calibration standards & quality control samples 21CC ID Final conc. QC ID Final conc. (ng/ mL) (ng/ml)STD-1 (Cmax x4 /100%) 1595.53 H.Q.C (70 to 90%) 1236.51STD-2 (90%) 1435.97 M.Q.C (30 to 50%) 726.04STD-3 (75%) 1195.17 L.Q.C (LLOQ x 3) 43.56STD-4 (50%) 796.65 LOQQC 15.92STD-5 (15%) 238.99STD-6 (5%) 79.58STD-7 (LOQ x 2) 31.83STD-8 (LOQ) 15.92 Minimum r2 value > 0.98
  • Trials for selection of method 22 Three trials used for sample extraction methods: SPE (Solid phase extraction): Conditioning » washing » loading of sample » eluting. LLE (Liquid-liquid extraction): Sampling »adding solvent/ solvent mixture » vortex » centrifuge » evaporate to dryness » reconstitute. Protein precipitation : Sampling + ppt. solvent. In these methods, LLE method is less costly, simple, rapid and having less interference of endogenous materials. so, we selected LLE method.
  • Finally Selected Method (LLE): 23 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. 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. The organic layer was separated into clean evaporation tubes and evaporated to dryness under N2 at 45°C. 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. All prepared samples were kept in an auto sampler at 10± 5°C until injection.
  • Method validation 24 Method validation was performed according to the USFDA guidance for industrial bio analytical method validation. PARAMETERS: Selectivity Sensitivity Linearity Accuracy and precision(INTRA-DAY/INTER-DAY) Recovery Matrix effect LLOQ LOD Reinjection /Reproducibility
  • 25 STABILITY STUDIES: Auto sampler stability Short-term stock solution stability Bench top stability Freeze-thaw stability Long term stability Blood stability 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.).
  • Results & discussion 26 A Representative Regression Analysis of a Calibration curve of Terbinafine - LinearityCompound name: TerbinafineCorrelation coefficient: r = 0.998306, r^2 = 0.996615Calibration curve: 0.00272614 * x + 0.00680601Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x^2, Axis trans: None 4.00 3.00Response 2.00 1.00 0.00 ng/µl 0 200 400 600 800 1000 1200 1400
  • 27MV_148 Sm ooth(Mn,3x2) MRM of 2 channels ,ES+AQ STD FS-03 AQ STD FS-03 292.37 > 92.9 Terbinafine 4.059e+006100 1.79 473674.31 % 42499.41 0 m inMV_148 Sm ooth(Mn,3x4) MRM of 2 channels ,ES+AQ STD FS-03 AQ STD FS-03 268.33 > 115.9 ISTD;0.75;166975.06;49958.02 1.237e+006100 % 0 m in 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50MV_013 Sm ooth(Mn,3x2) MRM of 2 channels ,ES+Specificity-K30090 Specificity-K30090 292.37 > 92.9 0.01 1.41 1.49 2.26 2.308e+003100 0.25 0.44 0.56 0.87 1.03 1.13 1.68 1.83 % 0 m inMV_013 Sm ooth(Mn,3x4) MRM of 2 channels ,ES+Specificity-K30090 Specificity-K30090 268.33 > 115.9 0.16 0.49 2.962e+002100 0.76 1.15 1.70 2.20 2.32 1.01 1.36 1.52 1.92 % 0 m in 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50
  • A Representative Chromatograph of LLOQ & HQC 28MV_152 Sm ooth(Mn,3x2) MRM of 2 channels ,ES+STD 8 BS-01 STD 8 BS-01 292.37 > 92.9 Terbinafine 3.983e+004100 1.79 4111.85 % 362.94 0 m inMV_152 Sm ooth(Mn,3x4) MRM of 2 channels ,ES+STD 8 BS-01 STD 8 BS-01 268.33 > 115.9 ISTD;0.75;83226.54;16888.04 6.229e+005100 % 0 m in 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50MV_160 Sm ooth(Mn,3x2) MRM of 2 channels ,ES+HQC BS-01 HQC BS-01 292.37 > 92.9 Terbinafine 2.556e+006100 1.80 290219.72 % 13666.40 0 m inMV_160 Sm ooth(Mn,3x4) MRM of 2 channels ,ES+HQC BS-01 HQC BS-01 268.33 > 115.9 ISTD;0.75;81945.05;27743.38 6.119e+005100 % 0 m in 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50
  • BACK CALCULATED CALIBRATION CURVE CONCENTRATIONS- SENSITIVITY 29 Back calculated calibration curve concentrations for Terbinafine in human Plasma Curve parameter summary Standard ID STD 1 STD 2 STD 3 STD 4 STD 5 STD 6 STD 7 STD 8 R² Slope InterceptNominal (ng/mL) 1595.527 1435.974 1196.167 796.647 238.994 79.585 31.834 15.917 1356.198 1220.578 1016.742 677.150 203.145 67.647 27.059 12.734 Range 1834.856 1651.370 1375.592 916.144 274.843 91.523 36.609 19.100 P&A I 1543.362 1361.017 1163.833 775.477 257.257 86.271 31.717 15.626 0.9966 0.0027 0.0068 P&A II 1487.835 1338.031 1204.180 815.769 254.835 83.193 32.216 15.621 0.9969 0.0037 0.0087 P&A III 1512.698 1280.026 1152.599 823.897 263.082 85.926 32.050 15.527 0.9938 0.0030 0.0116 Mean 1514.632 1326.358 1173.537 805.048 258.391 85.130 31.994 15.591 ± S.D 27.814 41.738 27.125 25.929 4.239 1.686 0.254 0.056 % CV 1.8 3.1 2.3 3.2 1.6 2.0 0.8 0.4 % Nominal 94.9 92.4 98.1 101.1 108.1 107.0 100.5 98.0 Acceptance criteria: % CV ≤ 15% ( ≤ 20 % for LLOQ) % Nominal ± 15 % ( ± 20 % for LLOQ)
  • ACCURACY & PRECISION 30 Intra-day and Inter-day Precision & Accuracy for Terbinafine in human Plasma H QC M QC L QC Nominal concentration(ng/ml) 1312.145 734.801 44.088Upper limit 1508.967 845.021 50.701Lower limit 1115.323 624.581 37.475 P&A I P&A II P&A III P&A I P&A II P&A III P&A I P&A II P&A III 1296.644 1209.171 1211.743 784.648 722.852 737.658 48.203 44.213 44.708 1284.567 1189.750 1255.359 786.163 715.786 771.130 49.019 45.394 45.991 1270.637 1219.779 1221.620 740.848 781.918 748.529 46.883 45.048 45.034 1230.314 1199.136 1266.205 751.143 689.092 729.660 44.061 41.247 43.952 1217.894 1169.371 1218.618 716.192 676.164 748.579 44.282 41.265 45.801 1191.553 1142.248 1222.798 692.644 698.074 779.779 45.061 44.758 48.719 Mean 1248.602 1188.243 1232.724 745.273 713.981 752.556 46.252 43.654 45.701 ± SD 41.519 28.373 22.335 37.127 37.417 19.310 2.096 1.898 1.655 % CV 3.3 2.4 1.8 5.0 5.2 2.6 4.5 4.3 3.6% Nominal 95.2 90.6 93.9 101.4 97.2 102.4 104.9 99.0 103.7 Inter-day (Global Data) Mean 1223.189 737.270 45.202 ± SD 39.769 34.976 2.116 % CV 3.3 4.7 4.7% Nominal 93.2 100.3 102.5Acceptance Criteria: % CV ≤ 15% , % Nominal ± 15%
  • recovery 31 Observed recovery of Terbinafine in human plasma H QC M QC L QC Sr. No. Aq Drug Area Plasma Drug Area Aq Drug Area Plasma Drug Area Aq Drug Area Plasma Drug Area 1 421191.219 290219.719 243559.078 175485.016 16700.244 11369.333 2 419504.125 291795.313 245428.016 177559.188 16511.398 11525.202 3 422869.906 294490.688 243815.875 173163.266 16894.643 11586.529 4 422561.844 286986.344 246656.516 171331.719 16722.109 11075.873 5 422879.000 281461.500 245197.438 170673.813 16675.064 11056.864 6 425736.188 285047.406 243402.453 166565.469 16608.121 11337.260 Mean 422457.047 288333.495 244676.563 172463.079 16685.263 11325.177% Recovery 68.3 70.5 67.9 Observed recovery of Metoprolol (ISTD) in human plasma H QC M QC L QC Sr. No. Aq ISTD Area Plasma ISTD Area Aq ISTD Area Plasma ISTD Area Aq ISTD Area Plasma ISTD Area 1 170275.406 81945.055 157707.672 81778.203 162895.953 82259.352 2 169434.672 83163.031 161290.328 82585.820 160177.641 82064.992 3 163860.547 84849.531 161501.656 85451.039 162179.781 86071.148 4 163097.203 85391.938 157602.469 83392.305 159686.094 87264.625 5 166623.063 84600.289 154672.656 87111.977 163532.375 86704.109 6 164176.438 87568.258 162024.406 87894.977 164626.781 87446.945 Mean 166244.555 84586.350 159133.198 84702.387 162183.104 85301.862% Recovery 50.9 53.2 52.6
  • STABILITIES PARAMETERS: Stss stability & auto sampler stability 32 Short Term Stock Solution Stability for Terbinafine and ISTD Area response of Drug Area response of ISTD Sr. No. 0.0 Hours 05:53 Hours Sr.No. 0.0 Hours 05.39 Hours 1 494137.000 493664.719 1 152183.219 154145.859 2 494503.125 495642.344 2 156211.391 159190.438 3 493187.094 502107.156 3 156946.828 161370.891 4 491781.469 501911.406 4 153198.500 167676.625 Mean 493402.172 498331.406 Mean 154634.985 160595.953 ± SD 1214.497 4323.645 ± SD 2302.486 5607.008 % CV 0.2 0.9 % CV 1.5 3.5 % of Mean ratios 101.0 % of Mean ratios 103.9Acceptance criteria :% of Mean ratios 90 % - 110 % Autosample r / We t extract Stability for Te rbinafine in human plasma Fre shly Inje cte d Afte r 47:30 Hrs L QC H QC L QC H QC Sr. No. Nominal Conce ntration(ng/mL) Nominal Conce ntration (ng/mL) 44.088 1312.145 44.088 1312.145 1 43.768 1275.200 51.380 1351.889 2 44.326 1236.528 47.918 1379.008 3 46.073 1263.705 50.620 1401.761 4 49.583 1293.190 51.859 1412.937 M e an 45.938 1267.156 50.444 1386.399 ± SD 2.621 23.752 1.760 26.993 %CV 5.7 1.9 3.5 1.9 % of M e an ratios 109.8 109.4Acce ptance crite ria : % M e an ratios 90% - 110%
  • Freeze thaw stability & bench top stability 33 Freeze T haw Stability for T erbinafine in human plasma Freshly Injected A fter 3 Cycles L QC H QC L QC H QC Sr. No. Nominal Concentration (ng/m L) Nominal Concentration (ng/m L) 44.088 1312.145 44.088 1312.145 1 49.152 1184.764 48.363 1243.275 2 45.418 1189.865 49.408 1261.610 3 46.993 1250.392 52.942 1376.460 4 44.450 1188.012 46.312 1283.317 Mean 46.503 1203.258 49.256 1291.166 ±SD 2.053 31.493 2.773 59.171 %CV 4.4 2.6 5.6 4.6 % of Mean ratios 105.9 107.3Acceptance Criteria: % of mean ratios 90% - 110% Bench Top Stability for Terbinafine in human plasma Freshly Injected After 4:49 hours L QC H QC L QC H QC Sr. No. Nominal Concentration (ng/mL) Nominal Concentration (ng/mL) 44.088 1312.145 44.088 1312.145 1 44.919 1190.989 46.641 1203.575 2 44.979 1211.968 47.376 1233.521 3 46.380 1172.823 46.420 1251.024 4 44.855 1169.857 45.606 1207.376 Mean 45.283 1186.409 46.511 1223.874 ±SD 0.733 19.432 0.729 22.468 %CV 1.6 1.6 1.6 1.8 % of Mean ratios 102.7 103.2Acceptance Criteria: % of mean ratios 90% - 110%
  • Matrix effect & reinjection reproducibility Matrix Effect 34 for Terbinafine and ISTD in human plasma Sr. No. Drug Area ISTD Area Ratio of Drug/ISTD 1 4315.825 82725.516 0.052 2 4285.869 84228.656 0.051 3 4284.799 82394.680 0.052 4 4268.056 83748.758 0.051 5 4284.604 83212.945 0.051 6 4270.618 83743.492 0.051 Mean 0.051 ±SD 0.001 % CV 1.1 Acceptance Criteria: % CV ≤ 20% Reinjection Reproducibility for Terbinafine in human plasma Freshly Injected Re Injected L QC H QC L QC H QC Sr. No. Nominal Concentration (ng/mL) Nominal Concentration (ng/mL) 44.088 1312.145 44.088 1312.145 1 48.203 1296.644 46.179 1219.405 2 49.019 1284.567 47.357 1223.081 3 46.883 1270.637 48.036 1265.528 Mean 48.035 1283.949 47.191 1236.005 ±SD 1.078 13.014 0.940 25.634 % CV 2.2 1.0 2.0 2.1 % of Mean ratios 98.2 96.3Acceptance Criteria: % of mean ratios 90% - 110%
  • LIMIT OF QUANTIFICATION & Limit of Detection 35 Limit of Qua ntifica tion for Te rbina fine in huma n pla sma LLOQ ULOQ Sr. No. Nomina l conce ntra tion (ng/ml) 15.917 1595.527 1 15.764 1478.191 2 15.347 1466.952 3 15.425 1514.500 4 15.458 1553.139 5 15.983 1501.326 6 15.855 1550.375 Me a n 15.639 1510.747 ± SD 0.262 35.912 % CV 1.7 2.4 %Nomina l 98.3 94.7Acce pta nce crite ria :For ULOQPre cision: The % CV s hould be ≤ 15%Accura cy: The me a n va lue s hould be w ithin 85% - 115%For LLOQPre cision: The % CV s hould be ≤ 20%Accura cy: The me a n va lue s hould be w ithin 80% - 120% Limit of Detection for Terbinafine Sr. No. Sample ID Peak Area S/N Ratio 1 LLOQ 1/2 3069.583 316.654 2 LLOQ 1/4 1546.045 104.398 3 LLOQ 1/8 800.601 32.289Acceptance Criteria: S/N Ratio ≥ 5
  • BIOEQUIVALENCE STUDY PARAMETERS & CHROMATOGRAM 36 PK Parameters of Terbinafine in Human Plasma (Test A) Test- A Cmax (ng/ml) tmax (h) AUC(0-t) AUC(0-∞) (ng*h/mL) t1/2 (ng*h/mL) (h) Mean 1045.15 1.85 5353.77 6069.17 23.04 Geo. Mean 939.83 1.71 4516.30 4982.52 12.81 Median 931.00 1.50 4568.49 4907.65 9.04 Minimum 251.54 1.00 1010.09 1108.11 2.31 Maximum 2461.51 4.00 16849.30 21039.00 91.37 Range 2209.97 3.00 15839.21 19930.89 89.06 PK S.D. Parameters of Terbinafine in Human Plasma (Ref- B) 485.55 0.76 3307.12 4090.03 24.87 Ref- B Cmax (ng/ml) tmax (h) AUC(0-t) AUC(0-∞) (ng*h/mL) t1/2 (ng*h/mL) (h) Mean 1033.72 1.65 5031.48 5843.08 22.34 Geo. Mean 938.76 1.52 4344.52 4876.70 13.28 Median 915.45 1.50 4360.79 4334.08 10.13 Minimum 213.40 1.00 902.87 919.13 0.59 Maximum 1980.77 4.00 15228.70 18992.80 87.80 Range 1767.37 3.00 14325.83 18073.67 87.21 S.D. 433.55 0.74 2742.61 3602.75 21.79
  • Comparison of Bioequivalence Parameters of Both Test & Reference Drugs 37 Parameters Test Reference (mean±S.D) (mean±S.D) 6069.17±4090.03 5843.08±3602.75 AUC0–∞ (ng h/mL) 5353.77±3307.12 5031.48±2742.61 AUC0–t (ng h/mL) 23.04±24.87 22.34±21.79 Terminal half-life (h) 1045.15±485.55 1033.72±433.55 Cmax (ng/mL) 1.85±0.76 1.65±0.74 Tmax(h)AUC0–t= The area under the plasma concentration–time curve from time zero to last sampling timeAUC0–∞= The area under the plasma concentration–time curve from time zero to infinity.Cmax = Maximum plasma concentrationTmax = Maximum time to reach Cmax
  • Comparisin of mean plasma concentration & time profile of Test & reference drugs of 250 mg dose tabletsTime in hours Test A 38 Reference B 0 1.286 0.646 0.5 286.882 399.101 1 745.861 837.743 1.5 875.068 903.518 2 844.197 835.952 2.5 762.016 716.767 3 651.566 601.015 3.5 545.499 486.478 4 476.225 399.212 5 303.932 256.572 6 236.017 194.110 7 176.576 150.221 9 115.395 107.900 12 71.337 66.239 16 41.862 37.673 24 31.198 26.394 48 13.250 12.812 72 8.300 8.166 96 4.949 7.564 120 3.241 1.424 144 3.283 2.148
  • Mean Plasma Concentration versus Time profile Curve of Both Test & Reference Drugs 39 Linear Scale Test A 1000 Reference B 900Mean Plasma Concentrations (ng/mL) 800 700 600 500 400 300 200 100 0 0 15 30 45 60 75 90 105 120 135 150 Time (hrs.)
  • conclusion 40 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. 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. 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. The precision and accuracy for calibration and QC samples were well within the acceptable limits. 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.
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