Henry K.S.,M.Si.Apt,
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Jadwal Kuliah
Minggu Bahan kajian
I Validasi metode analisis
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Analisis Sediaan Farmasi
Analisis = ?????
Sediaan Farmasi = ????
C – GMP........ Perlu adanya Validasi
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Validation
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The Validation Process
Hardware Method
Validation
Validation
System
Software Suitability
Validation (4M)
• Man
• Machine
• Material
• Method
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Qualification
and IQ and
Software OQ Maintenance,
Qualification System
PQ
Suitability
• Calibration
The Process of Performing Tests on Individual
System Components to Ensure Proper function
• System Suitability
Test to verify the proper functioning of the operating
system, i.e., the electronics, the equipment, the
specimens and the analytical operations.
15
HPLC Detector calibration
• Wavelength Accuracy
• Linear Range
• Noise Level
• Drift
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Typical System Suitability Test
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Method Life Cycle
Validation
Development Optimization
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Verification vs. Validation
– Non-compendial methods-Validation
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Today’s Validation Requirements
ICH/USP
GMPs
(legal) FDA
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GMP Validation Parameters
• Accuracy
• Specificity
• Sensitivity
• Reproducibility
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FDA Validation Parameters
• Accuracy
• Precision
• Linearity
(& Range)
• Specificity
(& Determination Limit)
• Recovery
• Ruggedness
ICH
USP International Conference on Harmonization
Specificity • Specificity
Linearity and Range • Linearity
Accuracy • Range
Precision • Accuracy
Limit of Detection • Precision
Limit of Quantitation – Repeatability
Ruggedness – Intermediate
Robustness Precision
– Reproducibility
• Limit of Detection
23 • Limit of Quantitation
USP Data Elements Required For Assay
Validation
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ICH Validation Characteristics vs. Type of
Analytical Procedure
Type of Impurity testing
Analytical Identificatio
Assay
n Quantitative Limit Tests
Procedure
Accuracy No Yes No Yes
Precision
Repeatability 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
Range No Yes No Yes
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Method Validation for USP
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2. Terhadap MA yang dibuat dari
modifikasi metode resmi (standard
yang telah dipublikasi secara
internasional, regional atau nasional;
jurnal ilmiah yang relevan)
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TUJUAN
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Accuracy vs precision
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Accuracy vs precision
What you
would like
to see!
Accuracy vs precision
• Poor accuracy
• Good precision
Accuracy vs precision
• Poor precision
• Good accuracy
Accuracy vs precision
What would you
call this?
• Totally hopeless!
• Poor precision
• Poor accuracy
So what definitions do these
concepts lead us to in the
context of assay validation?
ACCURACY (1)
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Analytical Method Development
• Accuracy: Application of the method to synthetic mixtures of the
drug product components to which known quantities of the
analyte have been added
• Recovery reduced
by ~10 – 15%
From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam,
Y.C. Lee and Xue-Ming Zhang, ISBN 0-471-25953-5, Wiley & Sons
Recommended Data
• Taken from:
ASEAN Operational Manual for
Implementation of GMP ed. 2000 p.405
• Nine solutions containing different
concentrations of ketotifen fumarate
reference standard added to ketotifen
tablet batch no. 2506VAMG were
assayed
Example (continued):
Conc. of ketotifen fumarate Area Recovery Acceptance
detected (%) Criteria
mg/ml %
0.280 70 1473566 99.32
0.320 80 1677013 99.48
0.360 90 1904848 100.94
0.380 95 1905862 100.51
0.400 100 2091215 100.06
0.420 105 2180374 100.03
0.440 110 2293647 100.07
0.480 120 2518976 101.01
0.520 130 2670144 98.99
Mean (recovery) : 100.04 98.0–102.0 %
Standard deviation : 0.699
Relative standard deviation (RSD) : 0.699 % <2%
Accuracy
Table 1: Acceptable Recovery Percentages
Analyte (%) Unit Mean Recovery (%)
100 100% 98-102
10 10% 98-102
1 1% 97-103
0.1 0.1% 95-105
0.01 100 ppm 90-107
0.001 10 ppm 80-110
0.0001 1 ppm 80-110
0.00001 100 ppb 80-110
0.000001 10 ppb 60-115
0.0000001 1 ppb 40-120
• Taken from:
ASEAN Operational Manual for
Implementation of GMP ed. 2000 p.403
• The active ingredient, ketotifen
fumarate,
in tablets (batch no. 2506VAMG) was
assayed seven times using HPLC and
the reference standard
Example (continued)
Sample no. Concentration (mg/ml) Area detected
1 0.4 1902803
2 0.4 1928083
3 0.4 1911457
4 0.4 1915897
5 0.4 1913312
6 0.4 1897702
7 0.4 1907019
Mean : 1910896
Standard deviation : 9841.78
Relative standard deviation (RSD) : 0.515 %
Acceptance criteria:
Relative standard deviation (RSD): not more than 2 %
Kriteria
Secara umum:
- RSD < 1.0 % (Bahan baku obat)
- RSD < 2.0 % (Sediaan obat)
- RSD < 5.0 % (Cemaran/impurity)
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Precision
Table 1: Acceptable Recovery Percentages
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Specificity: Impurities Assay
• Chromatographic Methods
– Demonstrate Resolution
• Impurities/Degradants Available
– Spike with impurities/degradants
– Show resolution and a lack of interference
• Impurities/Degradants Not Available
– Stress Samples
– For assay, Stressed and Unstressed Samples should be
compared.
– For impurity test, impurity profiles should be compared.
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Forced Degradation Studies
• Temperature (50-60℃)
• Humidity (70-80%)
• Acid Hydrolysis (0.1 N HCl)
• Base Hydrolysis (0.1 N NaOH)
• Oxidation (3-30%)
• Light (UV/Vis)
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Bgm menentukan selektifitas?
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Examples of pure and impure HPLC peaks
• Ability of an assay
to elicit a direct and
proportional
response to
changes in analyte
concentration.
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Linearity Should be Evaluated
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Method Validation- Linearity
Cara penetapan
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RANGE
Is it there?
Quantitation limit
How much of it is present???
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Analytical Method Development
LOQ
LOD
Signal to Noise = 3:1
Noise
• Berdasarkan kurva kalibrasi analit
Menurut Miller:
Sy/ x i
( y ˆ
y ) 2
/ N 2
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RUGGEDNESS
Definisi :
Derajat reprodusibilitas hasil uji dari sampel yang
sama di bawah kondisi normal, dengan
parameter penetapan berbeda, seperti lab,
analis, alat, lot pereaksi, hari, waktu & suhu
penetapan yang berbeda. Jadi merupakan
ukuran reprodusibilitas hasil uji di bawah
kondisi normal dari lab ke lab dan dari analis
ke analis
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Cara penetapan
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Robustness
Small changes do not affect
the parameters of the
assay
ROBUSTNESS
Definisi :
Ukuran kemampuan MA untuk tidak
terpengaruh oleh perubahan / variasi
kecil dari parameter MA yang sengaja
dibuat dan memberikan indikasi
kehandalan dalam penggunaan normal
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Cara penetapan
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Contoh variasi parameter MA yang umum:
• Stabilitas larutan sampel
• Waktu / lamanya ekstraksi
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