Validasi Metode AnalisisProf. Dr.rer.nat. Mochammad Yuwono, MS. Bag. Kimia Farmasi/Unit Layanan Pengujian Fakultas Farmasi Universitas Airlangga UU Perlindungan Konsumen Quality Era Global (AFTA) cGMP GMP ISO 1702 5 9000 9001 9002 Industri Farmasi (Dept. QA) TQM Validasi Metode Analisis Serangkaian percobaan laboratorium untuk menunjukkan bahwa metode yang dipakai telah memenuhi beberapa persyaratan yang telah ditetapkan lebih dahulu • Merupakan sub bagian dari validasi proses Definition Validation of an analytical method is primarily concerned with: – the identification of the sources of potential errors – quantification of the potential errors in the method. . An Assay Validation describes in mathematical and quantifiable terms the performance characteristics of an assay. Akurasi dan Presisi Metode Akurasi rendah Presisi tinggi Akurasi tinggi Presisi tinggi Akurasi tinggi Presisi rendah Akurasi rendah Presisi rendah . Agar dihasilkan data yang akurat. ajeg dan terpercaya .Kenapa perlu validasi metode? .Memenuhi persyaratan cGMP GLP (Good Laboratory Practices) GCP (Good Clinical Practices) ISO 17025 European Norm (EN) FDA (Food and Drug Administration) EPA (Environmental Protection Agency) . Validasi Metode melibatkan keseluruhan prosedur analisis Sampling Sample Preparation Analysis Data Evaluation & Reporting . Verifikasi vs. Validasi • Compendial vs. Non-compendial Methods – Compendial methods Verifikasi • Sasaran Prosedur analisis resmi (misal Farmakope) – Non-compendial methods Validasi • Sasaran Prosedur analisis alternatif . sifat fisika. Reagents..Hal-hal yang terkait sebelum melakukan validasi metode Instrumen • kualifikasi dan kalibrasi Bahan-bahan • Ketersediaan Reference Standards. Pelatihan dan Pengalaman) Dokumen • Dukungan pustaka tentang Prosedur analisis. blanko (plasebo) Analis • Kualifikasi (Pendidikan. sifat kimia dsb. Protokol Validasi . System Suitability Validation Calibration Pump Detector HPLC Analyst Injector Data System Method Sample . Performance verification of HPLC Module Performance attributes General Expectation Frequency Pump Flowrate accuracy Gradient accuracy Pressure test ±2% ±1% No leak 6 months 6 months 6 months Injector Precision Linearity Carry over 1% RSD r > 0.999 < 1% 6 months 12 months 6 months Detector Wavelength accuracy Linearity of response Noise and drift ±2nm r > 0.999 Noise: 10-5 AU Drift: 10-4 AU/h 6 months 12 months 12 months Column compartment Temperature accuracy ±2% 6 months . Tentukan macam pendekatan. Definisikan tujuan dan ruang lingkup 3. jenis metode dan karakteristik kinerja analitik yang terkait 4. Pelaksanaan validasi dan evaluasi hasil 9. Tuliskan metode uji 7. Bentuk tim terpadu dan tentukan tanggungjawab masing-masing individu 2.Langkah-langkah Validasi Metode 1. Siapkan SOP validasi 5. Tentukan “acceptance criteria” 6. Pembuatan laporan 10. Lakukan percobaan pre-validation 8. Penyimpanan arsip . Analytical Method Development Optimization Pre-Validation Revalidation Validation Implementation . Validation of Analytical Procedures: Methodology (May 1997) • USP – Validation of Compendial Methods . Text on Validation of Analytical procedures (March 1995) – Q2B.Acuan Validasi Metode • ICH Guidelines – Q2A. Validasi Metode • Single Laboratory method • Fully Validated metehod (melibatkan berbagai laboratorium) . Parameter Validasi: USP and ICH Accuracy Precision Limit of Detection Method Validation Limit of Quantitation Specificity Linearity and Range Ruggedness/Robustness System Suitability . Determination of impurities/degradation products A. 2003 Categories of Analysis I. drug release etc.USP 26/ NF 21. Identification test . Quantitation of major components of bulk drug substances/active ingredients II. Quantitative B. Limit Test III.) IV. Determination of performance characteristics (dissolution test. USP Data Elements Required For Assay Validation Analytical Performanc e Parameter Assay Category 2 Assay Category 1 Quantitative Limit Tests Assay Category 3 Accuracy Yes Yes * * Precision Yes Yes No Yes Specificity Yes Yes Yes * LOD No No Yes * LOQ No Yes No * Linearity Yes Yes No * Range Yes Yes * * * May be required, depending on the nature of the specific test. Category 1: Quantitation of major components or active ingredients Ruggednes Yes Yes Yes s Category 2: Determination of impurities or degradation products Category 3: Determination of performance characteristics Yes ICH Validation Characteristics vs. Type of Analytical Procedure Type of Analytical Procedure Impurity testing Identificatio n Quantitative Limit Tests No Yes No Yes No Yes No Yes Interm. Prec. No Yes No Yes Specificity Yes Yes Yes Yes LOD No No Yes No LOQ No Yes No No Linearity No Yes No Yes Accuracy Assay Precision Repeatability Spesifisitas: ICH/USP • Dilakukan untuk tes identifikasi dan penentuan kontaminan (impurities). • Prosedur yang dipakai tergantung metode analisis yang digunakan. Spesifisitas • Mampu membedakan senyawa dan derivat/metabolitnya • Digunakan plasebo ditambah derivat/metabolit atau sampel ditambah analit . Spesifisitas: Jika tersedia zat hasil degradasi Suntikkan atau totolkan: • Blanko/Placebo (Sampel minus analit) • Zat Hasil degradasi (zat/produk) • Zat yang memiliki struktur mirip (Related Substances) • Peak/noda harus terpisah dengan sempurna Kromatografi Resolusi (Rs 1.2 – 1.5) . apex dan down slope” • Peak harus “pure” . MF. FTIR.Peak Purity test: HPLC • Spektra UV/Vis analit dan zat standar (authentic reference material) dengan diode array detector overlay Evaluasi korelasinya (r. MS) • Pengukuran spektra pada “upslope. Match Factor • • • • MF = 1000 r = 1 100% pure peak MF > 990 pure MF < 900 not pure 900 < MF < 950 contaminated . Pure and Impure HPLC peaks • Peak purity tests can also be evaluated with – The 3D-spectra of Photodiode array detectors – Mass spectrometry . . . H. Kartinasari. (A) HPLC chromatogram at 254 nm. Liq. (C) UV spectrum of flunarizine dihydrochloride peak . Indrayanto. Chufianti. Contour plot of the HPLC chromatogram from 210 – 400 nm. (B).Peak purity/identity testing (W.7 mL min-1. v/v as mobile phase. 2003 (7) : 1059-1067 A typical of HPLC chromatogram of flunarizine dihydrochloride (Sigma) using LiChrospher 100 RP-18 (stationary phase) and a mixture of methanol – ion pair solution 8 + 2. Chromatogr. G. & RT. J.F. with flow rate of 0. 999 .Spot Identity test: TLC • Scan spektra in situ UV/Vis analit dan zat standar Evaluasi korelasinya r > 0. Peak identities: (A) mometasone furoate. Wulandari. Indrayanto. Tan. (6) extract of commercial ointment-1. (4) solution of nipagin. (9) extract of commercial ointment-2 and (10) extract of commercial cream-2.G. KS. (B) nipagin and nipasol. Chromatogr.[mv] [nm] Densitograms ( = 260 nm) obtained from: (1) solution of standard mometasone furoate. (2003). (3) extract of laboratory-made cream. J. L. (C) unknown. (7) extract of commercial lotion. (8) extract of commercial cream-1.. (5) solution of nipasol. & RT. (2) extract from excipients of laboratory-made cream.26. 109-117 . Liq. Specificity: Jika tidak tersedia zat hasil degradasi (Degradants) – Lakukan forced degradation studies – Bandingkan profil sebelum dan sesudahnya . Forced Degradation Studies • Heat • Temperatur (50 .60 oC) • Humidity • Kelembaban (70 to • Acid Hydrolysis • Base Hydrolysis • Oxidation • Light 80%) • HCl 0.1 N • H2O2 (3 to 30%) .1 N • NaOH 0. . residual sum of squares • Disarankan menggunakan minimum 5 macam konsentrasi analit – Dilakukan terhadap sampel yang independen. slope (m).Liniearitas • Melalui analisis statistik Linear Regression (y = mx + b) • Correlation Coefficient. bukan dari sampel hasil pengenceran . y-intercept (b). yˆ a bxi 2 with 1 X2 YP a SY . 2 ˆ y y i i N 2 Y Y 1 1 2P N b . (2003). J. ttable .Persamaan matematik untuk evaluasi Linieritas VXO S XO S xo 100% X S Y where SY b X P 2 S XO . Ed. Encyclopedia of Chromatography (Marcel Dekker).QXX .2 and p 0. t student t factor for f N . Supplement .. G & Yuwono. in: Cazes. M.ttable .05 (Cited from : Indrayanto. 1 N QXX QXX X i 2 1 N X i 2 . Funk’s et al.Evaluasi terhadap Linieritas • • • • • Relative process standard deviation (Vxo) Mandel’ test Residual test ANOVA-linearity testing RSD of the Plot of response factor Vs. • r value (cannot be used alone) • Homogeneity of the linear-curve . concentration • Xp value. . Uji Homogenitas kurva kalibrasi . 113: 1469-1471. Horwitz. Excipient and Related Methodology. Indrayanto.Jangan menggunakan hanya ‘Correlation coefficient (r) untuk menguji linieritas. 7: 281-285 G.999’ • • • • • Analytical Methods Committee. Academic Press-Elsevier. in: J. J. Encyclopedia of Chromatography (Supp. December. Referee (1995). Yuwono. Yuwono. Analyst (1988). Qual. M. Vol 32. W. Croux. Inc. 2003. Cazes (ed). kecuali jika r > 0. Assur (2002). G. Accred. C. H. van Loco. Indrayanto. NY 10016. Validation of Chromatographic Method of Analysis in Brittain (Ed) Profiles of Drug. M. 2005 (in Press) . Elskens. Beernaert. 2.). New York. M. Marcel Dekker. akurasi dan linieritas .Rentang (Range) • Interval antara konsentrasi terbesar dan terkecil dari analit dalam sampel • Memenuhi syarat dalam hal presisi. Minimum Specified Range: • For Drug Substance & Drug product Assay – 80 to 120% of test Concentration • For Content Uniformity Assay – 70 to 130% of test Concentration • For Dissolution Test Method – +/.20% over entire Specification Range • For Impurity Assays – From Reporting Level to 120% of Impurity Specification for Impurity Assays . Accuracy • The extend by which the value deviates from the true value • Analyzing samples with known concentration and comparing between measured and true values • Comparing the results obtained from new method with known to be accurate • Percent recovery . 105 % (at Industry) of expected value(s) Accuracy using Standard addition method If A concentration after addition of analyte.Evaluation of accuracy testing Found / measured value % Recovery (Absolute) x 100 % No min al / True value Requirement : 98 .102 % or 95 . B concentration of original sample before addition % Recovery A B X 100 % Amount of addition % Recovery A X 100 % B Amount of addition . P = 95 %. Dibuat kurva regresi antara Xf (konsentrasi analit hasil analisi) terhadap Xc (konsentrasi nominal analit) Xf = af + bf. al. Syf .Untuk mengetahui ada atau tidaknya kesalahan sistematis. ttable. N Qxx t = Student-t-factor f = N-2.KURVA AKURASI menurut Funk et. .05 Cr bf = bf 1 Xc 2 Cr af = af ttable .Syf Qxx Syf = 2 [X if (a f + b f X i )] N-2 . Xc -Dihitung confidence range (Cr) dari intercept {VB(af)} dan slope {VB(bf)} dari recovery Pada p = 0. In Press (2005) . 32. San Diego. New York.M. Profiles of Drugs Substances. Sydney. Toronto. Validation method of Chromatography Methods of Analysis. London. Boston. Yuwono & G. Excipients and Related Methodology. Vol. Elsevier Academic Press. Tokyo. Indrayanto. 110. 100.Determination of Accuracy-testing according to USP • It is recommended. 90. 120 % of targeted concentration in duplicate (n = 5 x2 = 10) . that accuracy testing should be assessed using minimum of NINE determination over minimum three concentration levels (3 x three replicates) Our recommendation: Using 80. 1407-1416. Liq. Yuwono . respectively. and its validation.965 0.38 0.078 CT-1 98.242 0.49 0. injection volume 20 L) For p = 0. Palupi.695 48. HPLC determination of piracetam. T.77 0. the measured and nominal concentration of the analyte (g mL-1. Indrayanto. G. 6 1.05. A. Prasetyo.24 34.77 0.695 + 0. Umayah. d n = 10.07 101. D.. E.965 Xc 17. e n = 3 A. & RT.22 100. T.17 e - - - CT-2claim 33. 2005 - .51e % of label Xf and Xc are. Chromatogr. 28.Table 2 Results from determination of the accuracy-studies of the laboratory-made (LM) and commercial preparations a b c Sample Amount founda (Mean SD)e Amount addeda % Recovery (Mean SD) Recovery curveb VB(af)c VB9bf)C LM-tablet - - 99.70 98. J. M. Lestari.5 d Xf = 17. 1 10-3 0.00001 10-7 100 ppb 80-110 0. (%) Analyte ratio Unit Mean recovery (%) 100 1 100 % 98-102 ≥ 10 10-1 10 % 98-102 ≥1 10-2 1% 97-103 ≥ 0.001 10-5 10 ppm 80-110 0.1% 95-105 0.0000001 10-9 1 ppb 40-120 AOAC manual for the Peer-Verified Methods program .Analyte recovery at different concentration Analyte Ingred.000001 10-8 10 ppb 60-115 0.0001 10-6 1 ppm 80-110 0.01 10-4 100 ppm 90-107 0. Presisi • Kedekatan dari suatu seri pengukuran yang diperoleh dari sampel yang homogen • Dalam bentuk RSD • Meliputi: .Repeatability .Intermediate Precision . Repeatability • Kondisi sama pada interval waktu yang singkat • Disebut juga Intra-assay precision . analis. instrument dll lingkungan tempat metode Dinyatakan dalam SD.Intermediate Precision “within-laboratory variations”. Tergantung kondisi Berbeda hari. RSD (CV) dipakai Sebagai bagian Ruggedness menurut USP . .Reproducibility • Repeatability test at two different labs. 80.Recommendation for precision studies: • Using three different levels i. 100 and 120 % of targeted concentration • Each was evaluated six replicates • The study was performed in three different time and performed with different analyst Minimal samples for evaluation: 3 x 6 x 3 = 54 samples .e. Indrayanto (2005). Sumalik.55 1.87 1.0 mg tablet -1 1b 0.45 Nd d 1.06. 1998 (Camag) d Not determined a E. Planar Chromatography. n = 6)a Measurement Desloratadine 4. Densitometry determination of desloratadine in tablets. using TLC Scanner III equipped with CATS software version 4. 18.41 3b 0.Results from evaluation of Precision of Laboratory-made Tablets RSD Value (%. .03 0. Tampubolon.. M. 19-22. and validation of the method J.0 mg tablet -1 Desloratadine 6.73 0.0 mg tablet -1 Desloratadine 5.58 2b 1. G. H.B.76 4c Nd d 0.49 Evaluated on one plate by one analyst (repeatability) b Each measurement was performed by a different analyst on the different plates and days within one laboratory c Measurement was performed in the different laboratory. Yuwono.40 1. 2001. 1999. Biomed. Anal. Pharm. Indrayanto & M. Pharm. Edwardson et al.. Biomed. Anal. New York] SD < 1/6 Specification range (USL – LSL) [J. Neumann. J. Ermer. 1999] •David-. (8):931. G. Marcel Dekker. Validierung in der Analytik. Kromidas.Test should be OK . Supp. D.Evaluation of Precision-Testing RSD < 2 % [P. Willley-VCH. Grubss-. Encyclopedia of Chromatography. 24: 755-767] RSD < (USL LSL) x n 4 x1. Yuwono. J. 1999. 2003. Dixon-. A.96 xMean x 100 %[S. 0001 10-6 1 ppm 11 0.0000001 10-9 1 ppb 30 AOAC manual for the Peer-Verified Methods program .3 0.3 ≥ 10 10-1 10 % 2.3 0.001 10-5 10 ppm 7.1 10-3 0.7 ≥1 10-2 1% 2.01 10-4 100 ppm 5.7 0.000001 10-8 10 ppb 21 0.00001 10-7 100 ppb 15 0.Analyte concentration versus precision Analyte % Analyte ratio Unit RSD (%) 100 1 100 % 1.1% 3.8 ≥ 0. . Quantitation Limit (QL) • Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision.Detection Limit (DL) • Lowest amount of analyte in a sample that can be detected but not necessarily quantitated. • Estimated by Signal to Noise Ratio of 10:1. • Estimated by Signal to Noise Ratio of 3:1. Based on Signal-to Noise-Ratio . Based in Visual Evaluations . Based on Standard Deviation of the Response and the Slope .Detection Limit (DL) and Quantitation Limit (QL) Estimated by 1.Used for non-instrumental methods 2.10:1 for Quantitation Limit 3.3:1 for Detection Limit . Based on Signal-to Noise-Ratio . Robustness • Definition: Capacity to remain unaffected by small variations in method parameters • Determination: Comparison results under differing conditions with precision under normal conditions – Variations may include: stability of analytical solution. flow rate. different column (lot/supplier). temperature. . variation of pH in a mobile phase. Robustness Variations All Assays -Sample Prep Manipulation -Extraction Time HPLC Assays -Mobile Phase Composition -Different Columns -Temperature -Flow Rate GC Assays -Different Columns -Temperature -Flow Rate . . GC Injection temperature For temperature program initial temperature final temperature slope of the temperature gradient Flow – rate of the gas For flow – program initial flow final flow slope of the flow gradient Column temperature Detection temperature In Column factor Split flow Type of liner batch of stationary phase manufacturer of the column . 39 80:20 8.41 7.17 85:15 7.17 6.18 5.81 10.93 .47 11.Robustness-Mobile Phase Change Retentio MeOH/ Retention Resolutio n n Water Time 1 Time 2 75:25 11.94 16. Pharm. / J. 24 (2001) 723 . Vander Heyden et al.TLC Eluent composition pH of the mobile phase Temperature Development distance Spot shape Spot size Batch of the plates Volume of sample Drying conditions (temperature.753 . time) Y. Anal. Biomed. ” .” • “The degree of revalidation depends on the nature of the change. drug substance.Re-Validation • “When sponsors make changes in the analytical procedure. may necessitate revalidation of the analytical procedures.” • “FDA intends to provide guidance in the future on post-approval changes in analytical procedures. the changes. drug product. . impurity. capacity factor (k’). resolution (R).) repeatability. analytical operations and samples as a whole – Determination: • USP 23 <621> – System Suitability Requirements Parameter s K’ In general k’ ≥ 2. etc…. internal STD. between the peak of interest and the closest potential interferent (degradant. electronic. tailing factor (T). excipient.0 R R > 2. and theoretical Plates (N) Recommendations T N Repeatabilit T≤2 In general N > 2000 .ICH/USP System Suitability • ICH – Definition: evaluation of equipment. UJI KESESUAIAN SISTEN SYSTEM SUITABILITY TESTS • Bagian integral prosedur analisis • Parameter SST – Capacity Factor ( k' ) – Precision/Injection Repeatability ( RSD ) – Relative Retention (α) – Resolution ( Rs ) – Tailing Factor ( T ) – Theoretical Plate Number ( N ) . SYSTEM SUITABILITY PARAMETERS Capacity Factors ( k' ) k' = ( ｔＲ－ｔ ０ ) / ｔ０ k' ＞２ Precision/Injection Repeatability ( RSD ) ｎ≧５ RSD≦ １％ Relative Retention (α ) α = k' 1 / k' 2 Resolution ( Rs ) Tailing Factor ( T ) T = Ｗ x / ２ｆ T≦ ２ Theoretical Plate Number ( N ) N = １６ ( ｔ Ｒ / ｔ Ｗ ) 2 = Ｌ / Ｈ N ＞２０００ . 2003. Yuwono. Swatrz and Ira S. M. Inc. G. New York. NY 10016. Analytical method development and validation. Indrayanto. USP 26 http://www. Mrcel Dekker. 1999 G. Excipient and Related Methodology. Validation of TLC Analysis. Vol 32. Encyclopedia of Chromatography (Supp. Krull. Yuwono. M.com Ludwig Huber.References • • • • • • • • ICH Q2A ICH Q2B Michael E. Marcel Dekker. Inc. Validation and Qualification in Analytical Laboratories. 1997. New York. Cazes (ed). in: J. Buffalo Grove. Indrayanto. Academic Press-Elsevier. Illinois.). 2005 (in Press) . Validation of Chromatographic Method of Analysis in Brittain (Ed) Profiles of Drug.waters. Interpharm Press Inc.