Validasi Metode Analisis

Prof. Dr.rer.nat. Mochammad Yuwono, MS.

Bag. Kimia Farmasi/Unit Layanan Pengujian
Fakultas Farmasi Universitas Airlangga
U Perlindungan Konsumen

Era Global
Quality (AFTA)

cGMP
GMP
ISO Industri Farmasi
1702
5 (Dept. QA)
9000
9001 TQM
9002
 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 Akurasi tinggi Akurasi tinggi Akurasi rendah

Presisi tinggi Presisi tinggi Presisi 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 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
Hal-hal yang terkait sebelum
melakukan validasi metode
Instrumen
• 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 Suitability

Validation

Calibration
Pump Injector
HPLC
Detector Data System

Analyst Method

Sample
 Performance verification of HPLC
Module Performance attributes General Frequency
Expectation
Pump Flowrate accuracy ±2% 6 months
Gradient accuracy ±1% 6 months
Pressure test No leak 6 months
Injector Precision 1% RSD 6 months
Linearity r > 0.999 12 months
Carry over < 1% 6 months

Detector Wavelength accuracy ±2nm 6 months

Linearity of response r > 0.999 12 months
Noise and drift Noise: 10-5 AU 12 months
Drift: 10-4 AU/h

Column Temperature accuracy ±2% 6 months

compartment
 Langkah-langkah Validasi Metode
1. Bentuk tim terpadu dan tentukan
tanggungjawab masing-masing individu
2. Definisikan tujuan dan ruang lingkup
3. Tentukan macam pendekatan, jenis metode
dan karakteristik kinerja analitik yang terkait
4. Siapkan SOP validasi
5. Tentukan “acceptance criteria”
6. Tuliskan metode uji
7. Lakukan percobaan pre-validation
8. Pelaksanaan validasi dan evaluasi hasil
9. Pembuatan laporan
10. Penyimpanan arsip
 Analytical Method
Development

Optimization

Pre-Validation

Revalidation Validation

Implementation
 Acuan Validasi Metode
• ICH Guidelines
– Q2A, Text on Validation of Analytical
procedures (March 1995)
– Q2B, Validation of Analytical Procedures:
Methodology (May 1997)
• USP
– Validation of Compendial Methods
 Validasi Metode
• Single Laboratory method
• Fully Validated metehod
(melibatkan berbagai laboratorium)
 Parameter Validasi:
USP and ICH
Accuracy
Precision
Limit of Detection
Method Limit of Quantitation
Validation
Specificity
Linearity and Range
Ruggedness/Robustness
System Suitability
 USP 26/ NF 21, 2003
Categories of Analysis
I. Quantitation of major components of bulk drug
substances/active ingredients
II. Determination of impurities/degradation
products
A. Quantitative B. Limit Test
III. Determination of performance characteristics
(dissolution test, drug release etc.)
IV. Identification test
USP Data Elements Required For Assay
Validation
Assay Category 2
Analytical
Performanc Assay Assay
e Category 1 Quantitative Limit Tests Category 3
Parameter

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.
Ruggednes
Category 1: Quantitation of major components or active ingredients
Yes Yes Yes Yes
s Category 2: Determination of impurities or degradation products
Category 3: Determination of performance characteristics
 ICH Validation Characteristics vs.
Type of Analytical Procedure
Impurity testing
Type of
Analytical Identificatio
Assay
n Quantitative Limit Tests
Procedure

Accuracy No Yes No Yes

Precision

No Yes No Yes
Repeatability
Interm.
No Yes No Yes
Prec.
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity No Yes No Yes
 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)
 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 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
 Peak purity/identity testing (W.F. Kartinasari, H. Chufianti, G. Indrayanto,
J. Liq. Chromatogr. & RT, 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, 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
 Spot Identity test: TLC

• Scan spektra in situ UV/Vis analit

dan zat standar 
Evaluasi korelasinya  r > 0.999
 [mv]

[nm]

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
studies
– Bandingkan profil sebelum dan
sesudahnya
 Forced Degradation Studies

• Heat  • Temperatur (50 - 60 oC)

• Humidity  • Kelembaban (70 to
• Acid Hydrolysis 
80%)
• Base Hydrolysis 
• HCl 0.1 N
• Oxidation 
• NaOH 0.1 N
• Light 
• H2O2 (3 to 30%)
 Liniearitas
• Melalui analisis statistik Linear Regression (y
= mx + b)
• Correlation Coefficient, y-intercept (b), slope
(m), residual sum of squares
• Disarankan menggunakan minimum 5 macam
konsentrasi analit
– Dilakukan terhadap sampel yang independen,
bukan dari sampel hasil pengenceran
 Persamaan matematik
untuk evaluasi Linieritas
S xo
VXO  100%
X
S 
 i i
y  ˆ
y  2

S XO  Y where SY  , yˆ  a  bxi
b N 2

Y  Y 
2
1
X P  2 S XO .ttable .  1  2P with
N b .Q XX
1 X2
YP  a  SY . ttable . 1
N Q XX
2
Q XX   X i
2

1
 X  i , t  student t factor for f  N - 2
N
and p  0.05
(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’ test
• Residual test
• ANOVA-linearity testing
• RSD of the Plot of response factor Vs.
concentration
• Xp value- Funk’s et al.
• r value (cannot be used alone)
• Homogeneity of the linear-curve
Uji Homogenitas kurva kalibrasi
Jangan menggunakan hanya ‘Correlation coefficient (r)
untuk menguji linieritas, kecuali jika r > 0.999’

• 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: 281-285
• G. 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)
 Rentang (Range)

• Interval antara konsentrasi terbesar

dan terkecil dari analit dalam sampel
• Memenuhi syarat dalam hal presisi,
akurasi dan linieritas
 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
 Evaluation of accuracy
testing
Found / measured  value
% Recovery (Absolute)  x 100 %
No min al / True  value
Requirement : 98 - 102 % or 95 - 105 % (at Industry) of expected value(s)

Accuracy using Standard addition method

If A  concentration after addition of analyte,
B  concentration of original sample before addition

A B
% Recovery  X 100 %
Amount of addition

A
% Recovery  X 100 %
B  Amount of addition
 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
ttable.Syf
Cr bf = bf 
Qxx
1 Xc 2
Cr af = af  ttable . Syf . 
N Qxx
Syf =
 [Xif - (af + bf - Xi )]2

N-2

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

Sample Amount founda Amount % Recovery Recovery curveb VB(af)c VB9bf)C

(Mean  SD)e addeda (Mean  SD)
LM-tablet - - 99. 6  1.5 d Xf = 17.695 + 0.965 17.695  0.965  0.078
Xc 48.242

CT-1 98.77  0.24 34.22 100.38  0.17 e - - -

a
% of CT-2
label claim 101.49  0.70 33.07 98.77  0.51e - - -
b
Xf and Xc are, respectively, the measured and nominal concentration of the analyte (g mL ; injection volume 20 L)
-1

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
 Analyte recovery at different concentration
Analyte Ingred. Analyte Mean recovery
Unit
(%) ratio (%)
100 1 100 % 98-102
≥ 10 10-1 10 % 98-102
≥1 10-2 1% 97-103
≥ 0.1 10-3 0.1% 95-105
0.01 10-4 100 ppm 90-107
0.001 10-5 10 ppm 80-110
0.0001 10-6 1 ppm 80-110
0.00001 10-7 100 ppb 80-110
0.000001 10-8 10 ppb 60-115
0.0000001 10-9 1 ppb 40-120

AOAC manual for the Peer-Verified Methods program

 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
 Intermediate
Precision
“within-laboratory variations”. Tergantung kondisi

Berbeda hari, analis, instrument dll lingkungan tempat metode

Dinyatakan dalam SD, RSD (CV) dipakai

Sebagai bagian Ruggedness

menurut USP
 Reproducibility

• Repeatability test at two different labs.

 Recommendation for precision studies:

• Using three different levels i.e. 80, 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
 Results from evaluation of Precision of Laboratory-made Tablets

RSD Value (%, n = 6)a

Desloratadine Desloratadine Desloratadine
Measurement 4.0 mg tablet 5.0 mg tablet 6.0 mg tablet
-1 -1 -1

1b 0.87 1.55 1.58

2b 1.03 0.73 0.41
3b 0.76 0.45 1.40
4c Nd d Nd d 1.49

a
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, using TLC Scanner III
equipped with CATS software version 4.06, 1998 (Camag)
d
Not determined

E. 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.
 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 < (USL  LSL) x n x 100 %[S. Kromidas, 1999,

4 x1.96 xMean
Validierung in der Analytik, Willley-VCH, 1999]

•David-, Grubss-, Dixon-, Neumann- Test should

be OK
 Analyte concentration versus
precision
Analyte % Analyte ratio Unit RSD (%)
100 1 100 % 1.3
≥ 10 10-1 10 % 2.7
≥1 10-2 1% 2.8
≥ 0.1 10-3 0.1% 3.7
0.01 10-4 100 ppm 5.3
0.001 10-5 10 ppm 7.3
0.0001 10-6 1 ppm 11
0.00001 10-7 100 ppb 15
0.000001 10-8 10 ppb 21
0.0000001 10-9 1 ppb 30
AOAC manual for the Peer-Verified Methods program
 Detection Limit Quantitation Limit
(DL) (QL)

• Lowest amount of • Lowest amount of

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

1. Based in Visual Evaluations

- Used for non-instrumental methods
2. Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
3. Based on Standard Deviation of the Response
and the Slope
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,

variation of pH in a mobile phase, different column
(lot/supplier), temperature, flow rate.
 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
batch of stationary phase
manufacturer of the
column

Split flow
Type of liner
 Robustness-Mobile Phase
Change
Retentio
MeOH/ Retention Resolutio
n
Water Time 1 n
Time 2
75:25 11.94 16.41 7.39

80:20 8.47 11.17 6.17

85:15 7.81 10.18 5.93

 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, time)

Y. Vander Heyden et al. / J. Pharm. Biomed. Anal. 24 (2001) 723 - 753

 Re-Validation
• “When 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 Suitability
• ICH
• USP 23 <621>
– Definition: evaluation of
equipment, electronic,
analytical operations and
samples as a whole
– Determination:
repeatability, tailing
factor (T), capacity factor
(k’), resolution (R), and
theoretical Plates (N)
Repeatabilit
– System Suitability
Requirements
Parameter
Recommendations
s
K’ In general k’ ≥ 2.0
R > 2, between the peak
of interest and the
closest potential
R
interferent (degradant,
internal STD, impurity,
excipient, etc…..)
T T≤2
N In general N > 2000
 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
Capacity Factors ( k' )
PARAMETERS
 k' = ( ｔＲ－ｔ０ ) / 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 ( ｔＲ / ｔＷ )2 = Ｌ / Ｈ N ＞２０００
 References
• ICH Q2A
• ICH Q2B
• Michael E. Swatrz and Ira S. Krull, Analytical method
development and validation. Mrcel Dekker, Inc. New York, 1997.
• USP 26
• http://www.waters.com
• Ludwig 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)