Validasi Metode Analisis Prof. Dr.rer.nat. Mochammad Yuwono, MS.Bag. Kimia Farmasi/Unit Layanan Pengujian Fakultas Farmasi Universitas Airlangga

Era Global(AFTA)ISO17025900090019002TQMUU Perlindungan KonsumenIndustri Farmasi(Dept. QA)GMPcGMPQuality

Validasi Metode AnalisisSerangkaian percobaan laboratoriumuntuk menunjukkan bahwa metode yang dipakai telah memenuhi beberapa persyaratan yang telah ditetapkan lebih dahulu

Merupakan sub bagian dari validasi proses

DefinitionValidation 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 MetodeAkurasi rendah Presisi tinggi Akurasi tinggi Presisi tinggi Akurasi tinggi Presisi rendahAkurasi rendah Presisi rendah

Kenapa perlu validasi metode?- Agar dihasilkan data yang akurat, ajeg dan terpercaya- 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 analisisSamplingSample PreparationAnalysisData Evaluation & Reporting

Verifikasi vs. ValidasiCompendial vs. Non-compendial MethodsCompendial methods VerifikasiSasaran Prosedur analisis resmi (misal Farmakope)Non-compendial methods Validasi Sasaran Prosedur analisis alternatif

Hal-hal yang terkait sebelum melakukan validasi metodeInstrumen kualifikasi dan kalibrasi Bahan-bahan Ketersediaan Reference Standards, Reagents, blanko (plasebo) Analis Kualifikasi (Pendidikan, Pelatihan dan Pengalaman) Dokumen Dukungan pustaka tentang Prosedur analisis, sifat fisika, sifat kimia dsb.; Protokol Validasi

System SuitabilitySampleValidationMethodAnalystCalibrationPumpDetectorInjectorData SystemHPLC

Performance verification of HPLC

ModulePerformance attributesGeneral ExpectationFrequencyPumpFlowrate accuracyGradient accuracyPressure test2%1%No leak6 months6 months6 monthsInjectorPrecisionLinearityCarry over1% RSDr > 0.999< 1%6 months12 months6 monthsDetectorWavelength accuracyLinearity of responseNoise and drift2nmr > 0.999Noise: 10-5 AUDrift: 10-4 AU/h 6 months12 months12 monthsColumn compartmentTemperature accuracy2%6 months

Langkah-langkah Validasi Metode1. Bentuk tim terpadu dan tentukan tanggungjawab masing-masing individu2. Definisikan tujuan dan ruang lingkup 3. Tentukan macam pendekatan, jenis metode dan karakteristik kinerja analitik yang terkait4. Siapkan SOP validasi 5. Tentukan acceptance criteria 6. Tuliskan metode uji 7. Lakukan percobaan pre-validation 8. Pelaksanaan validasi dan evaluasi hasil9. Pembuatan laporan 10. Penyimpanan arsip

Analytical MethodOptimizationValidationImplementationDevelopmentRevalidationPre-Validation

Acuan Validasi MetodeICH Guidelines Q2A, Text on Validation of Analytical procedures (March 1995) Q2B, Validation of Analytical Procedures: Methodology (May 1997)USP Validation of Compendial Methods

Validasi MetodeSingle Laboratory methodFully Validated metehod (melibatkan berbagai laboratorium)

Parameter Validasi:USP and ICH

MethodValidationAccuracyPrecisionLimit of DetectionLimit of QuantitationLinearity and RangeSpecificitySystem SuitabilityRuggedness/Robustness

USP 26/ NF 21, 2003 Categories of Analysis

Quantitation of major components of bulk drug substances/active ingredientsDetermination of impurities/degradation products A. Quantitative B. Limit TestDetermination of performance characteristics (dissolution test, drug release etc.)Identification test

USP Data Elements Required For Assay Validation* May be required, depending on the nature of the specific test.Category 1: Quantitation of major components or active ingredientsCategory 2: Determination of impurities or degradation productsCategory 3: Determination of performance characteristics

Analytical PerformanceParameterAssay Category 1Assay Category 2Assay Category 3QuantitativeLimit TestsAccuracyYesYes**PrecisionYesYesNoYesSpecificityYesYesYes*LODNoNoYes*LOQNoYesNo*LinearityYesYesNo*RangeYesYes**RuggednessYesYesYesYes

ICH Validation Characteristics vs. Type of Analytical Procedure

Type of Analytical ProcedureIdentificationImpurity testingAssayQuantitativeLimit TestsAccuracyNoYesNoYesPrecision RepeatabilityNoYesNoYes Interm. Prec.NoYesNoYesSpecificityYesYesYesYesLODNoNoYesNoLOQNoYesNoNoLinearityNoYesNoYesRangeNoYesNoYes

Spesifisitas: ICH/USPDilakukan untuk tes identifikasi dan penentuan kontaminan (impurities).Prosedur yang dipakai tergantung metode analisis yang digunakan.

SpesifisitasMampu membedakan senyawa dan derivat/metabolitnyaDigunakan plasebo ditambah derivat/metabolit atau sampel ditambah analit

Spesifisitas: Jika tersedia zat hasil degradasiSuntikkan 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)

Peak Purity test: HPLC

Spektra UV/Vis analit dan zat standar (authentic reference material) dengan diode array detector overlay Evaluasi korelasinya (r, MF, FTIR, MS)

Pengukuran spektra pada upslope, apex dan down slope Peak harus pure

Match FactorMF = 1000 r = 1 100% pure peakMF > 990 pureMF < 900 not pure900 < MF < 950 contaminated

Pure and Impure HPLC peaksPeak purity tests can also be evaluated with The 3D-spectra of Photodiode array detectors Mass spectrometry

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, v/v as mobile phase, with flow rate of 0.7 mL min-1. (A) HPLC chromatogram at 254 nm, (B). Contour plot of the HPLC chromatogram from 210 400 nm, (C) UV spectrum of flunarizine dihydrochloride peak Peak purity/identity testing (W.F. Kartinasari, H. Chufianti, G. Indrayanto, J. Liq. Chromatogr. & RT, 2003 (7) : 1059-1067

Spot Identity test: TLC

Scan spektra in situ UV/Vis analit dan zat standar Evaluasi korelasinya r > 0.999

Densitograms ( = 260 nm) obtained from: (1) solution of standard mometasone furoate, (2) extract from excipients of laboratory-made cream, (3) extract of laboratory-made cream, (4) solution of nipagin, (5) solution of nipasol, (6) extract of commercial ointment-1, (7) extract of commercial lotion, (8) extract of commercial cream-1, (9) extract of commercial ointment-2 and (10) extract of commercial cream-2. Peak identities: (A) mometasone furoate, (B) nipagin and nipasol, (C) unknown.

Wulandari, L, Tan, KS., Indrayanto,G. (2003), J. Liq. Chromatogr. & RT,26, 109-117

Specificity:Jika tidak tersedia zat hasil degradasi (Degradants)Lakukan forced degradation studiesBandingkan profil sebelum dan sesudahnya

Forced Degradation Studies Heat Humidity Acid Hydrolysis Base Hydrolysis Oxidation Light Temperatur (50 - 60 oC)Kelembaban (70 to 80%)HCl 0.1 NNaOH 0.1 NH2O2 (3 to 30%)UV/Vis

LiniearitasMelalui analisis statistik Linear Regression (y = mx + b)Correlation Coefficient, y-intercept (b), slope (m), residual sum of squaresDisarankan menggunakan minimum 5 macam konsentrasi analitDilakukan terhadap sampel yang independen, bukan dari sampel hasil pengenceran

Persamaan matematik untuk evaluasi Linieritas(Cited from : Indrayanto, G & Yuwono, M. (2003), in: Cazes, J., Ed. Encyclopedia of Chromatography (Marcel Dekker), Supplement

Evaluasi terhadap Linieritas Relative process standard deviation (Vxo)Mandel testResidual testANOVA-linearity testingRSD of the Plot of response factor Vs. concentrationXp value- Funks et al. r value (cannot be used alone)Homogeneity of the linear-curve

Uji Homogenitas kurva kalibrasi

Analytical Methods Committee, Analyst (1988), 113: 1469-1471.W. Horwitz, Referee (1995), December, 2.J. van Loco; M. Elskens; C. Croux; H. Beernaert. Accred. Qual. Assur (2002), 7: 281-285G. Indrayanto; M. Yuwono, in: J. Cazes (ed), Encyclopedia of Chromatography (Supp.), Marcel Dekker, Inc, New York, NY 10016, 2003.M. Yuwono, G. Indrayanto, Validation of Chromatographic Method of Analysis in Brittain (Ed) Profiles of Drug, Excipient and Related Methodology, Academic Press-Elsevier, Vol 32, 2005 (in Press) Jangan menggunakan hanya Correlation coefficient (r) untuk menguji linieritas, kecuali jika r > 0.999

Rentang (Range)Interval antara konsentrasi terbesar dan terkecil dari analit dalam sampelMemenuhi syarat dalam hal presisi, akurasi dan linieritas

Minimum Specified Range:For Drug Substance & Drug product Assay80 to 120% of test ConcentrationFor Content Uniformity Assay70 to 130% of test ConcentrationFor Dissolution Test Method+/- 20% over entire Specification RangeFor Impurity AssaysFrom Reporting Level to 120% of Impurity Specification for Impurity Assays

AccuracyThe extend by which the value deviates from the true valueAnalyzing samples with known concentration and comparing between measured and true valuesComparing the results obtained from new method with known to be accuratePercent recovery

Evaluation of accuracy testing

KURVA AKURASI menurut Funk et. al.

- Untuk mengetahui ada atau tidaknya kesalahan sistematis, Dibuat kurva regresi antara Xf (konsentrasi analit hasil analisi) terhadap Xc (konsentrasi nominal analit) Xf = af + bf. Xc Dihitung confidence range (Cr) dari intercept {VB(af)} dan slope {VB(bf)} dari recovery Pada p = 0.05 t = Student-t-factor f = N-2, P = 95 %.

M. Yuwono & G. Indrayanto, Validation method of Chromatography Methods of Analysis, Profiles of Drugs Substances, Excipients and Related Methodology, Vol. 32, Elsevier Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo, Toronto. In Press (2005)

Determination of Accuracy-testing according to USP It is recommended, that accuracy testing should be assessed using minimum of NINE determination over minimum three concentration levels (3 x three replicates)Our recommendation: Using 80, 90, 100, 110, 120 % of targeted concentration in duplicate (n = 5 x2 = 10)

Table 2 Results from determination of the accuracy-studies of the laboratory-made (LM) and commercial preparations

a % of label claimb Xf and Xc are, respectively, the measured and nominal concentration of the analyte (g mL-1; injection volume 20 L)c For p = 0.05; d n = 10; e n = 3

A. D. Lestari, A, T. Prasetyo, T. Palupi, E. Umayah, M. Yuwono , G. Indrayanto, HPLC determination of piracetam, and its validation, J. Liq. Chromatogr. & RT., 28, 1407-1416, 2005

SampleAmount founda (Mean SD)eAmount addeda% Recovery(Mean SD)Recovery curvebVB(af)cVB9bf)CLM-tablet--99. 6 1.5 dXf = 17.695 + 0.965 Xc17.695 48.2420.965 0.078CT-198.77 0.2434.22100.38 0.17 e---CT-2101.49 0.7033.0798.77 0.51e---

Analyte recovery at different concentrationAOAC manual for the Peer-Verified Methods program

Analyte Ingred. (%)Analyte ratioUnitMean recovery (%)1001100 %98-102 1010-110 %98-102 110-21%97-103 0.110-30.1%95-1050.0110-4100 ppm90-1070.00110-510 ppm80-1100.000110-61 ppm80-1100.0000110-7100 ppb80-1100.00000110-810 ppb60-1150.000000110-91 ppb40-120

PresisiKedekatan dari suatu seri pengukuran yang diperoleh dari sampel yang homogenDalam bentuk RSDMeliputi: - Repeatability - Intermediate Precision

RepeatabilityKondisi sama pada interval waktu yang singkatDisebut juga Intra-assay precision

Intermediate Precisionwithin-laboratory variations.Berbeda hari, analis, instrument dllDinyatakan dalam SD, RSD (CV)Sebagai bagian Ruggedness menurut USPTergantung kondisi lingkungan tempat metode dipakai

Repeatability test at two different labs. Reproducibility

Recommendation for precision studies:Using three different levels i.e. 80, 100 and 120 % of targeted concentrationEach was evaluated six replicatesThe study was performed in three different time and performed with different analyst

Minimal samples for evaluation: 3 x 6 x 3 =54 samples

Results from evaluation of Precision of Laboratory-made Tabletsa Evaluated on one plate by one analyst (repeatability)bEach measurement was performed by a different analyst on the different plates and days within one laboratorycMeasurement was performed in the different laboratory, using TLC Scanner III equipped with CATS software version 4.06, 1998 (Camag)d Not determinedE. Sumalik., H.B. Tampubolon, M. Yuwono, G. Indrayanto (2005), Densitometry determination of desloratadine in tablets, and validation of the method J. Planar Chromatography, 18, 19-22.

Measurement RSD Value (%, n = 6)a Desloratadine4.0 mg tablet -1 Desloratadine5.0 mg tablet -1 Desloratadine6.0 mg tablet -1 1b 0.87 1.55 1.58 2b 1.03 0.73 0.413b 0.76 0.45 1.404cNd dNd d 1.49

Evaluation of Precision-Testing RSD < 2 % [P. A. D. Edwardson et al. 1999, J. Pharm. Biomed. Anal. (8):931; G. Indrayanto & M. Yuwono, 2003, Encyclopedia of Chromatography, Supp., Marcel Dekker, New York] SD < 1/6 Specification range (USL LSL) [J. Ermer, 2001, J. Pharm. Biomed. Anal. 24: 755-767] RSD < x 100 %[S. Kromidas, 1999,

Validierung in der Analytik, Willley-VCH, 1999]David-, Grubss-, Dixon-, Neumann- Test should be OK

Analyte concentration versus precisionAOAC manual for the Peer-Verified Methods program

Analyte %Analyte ratioUnitRSD (%)1001100 %1.3 1010-110 %2.7 110-21 %2.8 0.110-30.1%3.70.0110-4100 ppm5.30.00110-510 ppm7.30.000110-61 ppm110.0000110-7100 ppb150.00000110-810 ppb210.000000110-91 ppb30

Lowest amount of analyte in a sample that can be detected but not necessarily quantitated.Estimated by Signal to Noise Ratio of 3:1.Detection Limit (DL)Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision.Estimated by Signal to Noise Ratio of 10:1.

Quantitation Limit (QL)

Detection Limit (DL) and Quantitation Limit (QL) Estimated byBased in Visual Evaluations- Used for non-instrumental methodsBased on Signal-to Noise-Ratio- 3:1 for Detection Limit- 10:1 for Quantitation LimitBased on Standard Deviation of the Response and the Slope

Based on Signal-to Noise-Ratio

Definition: Capacity to remain unaffected by small variations in method parametersDetermination: Comparison results under differing conditions with precision under normal conditionsVariations may include: stability of analytical solution, variation of pH in a mobile phase, different column (lot/supplier), temperature, flow rate. Robustness

Robustness Variations All Assays

HPLC Assays

GC Assays-Sample Prep Manipulation-Extraction Time-Mobile Phase Composition-Different Columns-Temperature-Flow Rate-Different Columns-Temperature-Flow Rate

InInjection temperature Column temperature Detection temperatureFor temperature program initial temperature final temperature slope of the temperature gradient

GCFlow rate of the gas For flow programinitial flow final flowslope of the flow gradient

Column factor batch of stationary phase manufacturer of the columnSplit flowType of liner

Robustness-Mobile Phase Change

MeOH/WaterRetentionTime 1RetentionTime 2Resolution75:2511.9416.417.3980:208.4711.176.1785:157.8110.185.93

TLCEluent composition pH of the mobile phaseTemperatureDevelopment distanceSpot shapeSpot sizeBatch of the platesVolume of sampleDrying conditions (temperature, time)Y. Vander Heyden et al. / J. Pharm. Biomed. Anal. 24 (2001) 723 - 753

Re-ValidationWhen sponsors make changes in the analytical procedure, drug substance, drug product, the changes, may necessitate revalidation of the analytical procedures.The degree of revalidation depends on the nature of the change.FDA intends to provide guidance in the future on post-approval changes in analytical procedures.

ICH/USP System SuitabilityICHDefinition: evaluation of equipment, electronic, analytical operations and samples as a wholeDetermination: repeatability, tailing factor (T), capacity factor (k), resolution (R), and theoretical Plates (N)USP 23 System Suitability Requirements

Parameters RecommendationsK In general k 2.0R R > 2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc..)T T 2N In general N > 2000Repeatability RSD 2.0% (n 5)

UJI KESESUAIAN SISTENSYSTEM SUITABILITY TESTSBagian integral prosedur analisisParameter SST Capacity Factor ( k' )Precision/Injection Repeatability ( RSD )Relative Retention ()Resolution ( Rs )Tailing Factor ( T )Theoretical Plate Number ( N )

SYSTEM SUITABILITY PARAMETERSCapacity Factors ( k' ) k' = () / k'Precision/Injection Repeatability ( RSD ) RSDRelative Retention ( ) = k'1 / k'2Resolution ( Rs ) Tailing Factor ( T ) T = x / TTheoretical Plate Number ( N ) N =(/)2 = / N

References ICH Q2AICH Q2BMichael E. Swatrz and Ira S. Krull, Analytical method development and validation. Mrcel Dekker, Inc. New York, 1997.USP 26 http://www.waters.comLudwig Huber, Validation and Qualification in Analytical Laboratories, Interpharm Press Inc. Buffalo Grove, Illinois, 1999 G. Indrayanto; M. Yuwono, in: J. Cazes (ed), Validation of TLC Analysis, Encyclopedia of Chromatography (Supp.), Marcel Dekker, Inc, New York, NY 10016, 2003. M. Yuwono, G. Indrayanto, Validation of Chromatographic Method of Analysis in Brittain (Ed) Profiles of Drug, Excipient and Related Methodology, Academic Press-Elsevier, Vol 32, 2005 (in Press)

**3*The relationship between accuracy and precision can be represented by arrows being shot at a target.

The first small target at the top shows the arrows have landed indiscriminately. This is neither accurate nor precise.

The second target on the left shows the arrows have grouped together nicely but are not on the bullseye. This is precise but inaccurate. This is sometimes called analytical bias and sometimes a correction factor can be applied.

The third, small target shows the arrows AVERAGE is on the bullseye, but the precision is unacceptable.

The fourth, large target shows the arrows are all clustered on or in the bullseye; this shows accuracy and precision.

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