Analisis Sediaan Farmasi Henry K.S.,M.Si.Apt,
Jadwal Kuliah Minggu
Bahan kajian
I
Validasi metode analisis
II
Validasi metode analisis
III
IV
V
VI
Pemisahan komponen aktif dari bahan pembantu sediaan obat, serta pemilihan pemilihan metode analisis Analisis kuantitatif sediaan obat monokomponen (padat & semi padat) secara spektrofotometri, spektrofluorometri dan elektrokimia Analisis kuantitatif sediaan obat monokomponen (cair dan steril) secara spektrofotometri, spektrofluorometri dan elektrokimia Analisis kuantitatif sediaan obat multikomponen (padat & semi padat) secara spektrofotometri dan spektrofluorometri
VII
Analisis kuantitatif sediaan obat multikomponen (Cair dan Steril) secara spektrofotometri dan spektrofluorometri
VIII
Penentuan dan Identifikasi permasalahan dalam hal analisis
IX
Analisis kuantitatif sediaan obat multikomponen secara KCKT
X
Analisis kuantitatif sediaan obat multikomponen secara KLT KLT dan KG Analisis Sediaan Kosmetika Analisa Sediaan Makanan dan minuman
XI XII XIII XIV
Analisa dalam Sampel Biologis Studi kasus
Daftar Pustaka: • United States Pharmacopoeia • Horwitz, W., and Latimer, G.W., 2005, Official Methods of Analysis, AOAC International, Maryland • Robert V Smith, et. Al, Textbook of Biopharmaceutic Analysis,1981. Analysis,1981. • Cahyadi W. , 2006, Analisi 2006, Analisis s dan Aspek Kesehatan Kesehatan Bahan Tambahan Pangan, Pangan, Bumi Aksara: Jakarta • Rohman A., dan I.G. Gandjar, 2007, Metode Kromatografi untuk Analisis Makanan, Makanan , Pustaka Pelajar: Yogyakarta • Mitra S., 2003, Sample Preparation Techniques in Analytical Analytical Chemistry , John Willey & Sons: New Jersey
Analisis Sediaan Farmasi Analisis = ????? ????? Sediaan Farmasi = ???? C – GMP........ Perlu adanya Validasi
Validation Proses penilaian terhadap parameter analitik tertentu, berdasarkan percobaan laboratorium, untuk membuktikan bahwa parameter tersebut memenuhi syarat untuk tujuan penggunaannya
The Validation Process
Hardware
Method Validation
Validation Software
System Suitability
Validation (4M)
• Man • Machine • Material • Method
Qualification • Qualification is a subset of the validation process that verifies module and system performance prior to the instrument being placed on-line. • If the instrument is not qualified prior to use and a problem is encountered, the source of the problem will be difficult to identify.
The Validation Timeline
Vendor’s Site
User’s Site
User’s Site
Structural
Qualification
Calibration
and
IQ
and
Software Qualification
OQ
Maintenance,
PQ
System Suitability
Before Purchase
Before Use
After Use
Installation Qualification (IQ) • The IQ process can be divided into two steps: preinstallation and physical installation. • During the preinstallation, all the information pertinent to the proper installation, operation, and maintenance of the instrument is reviewed. • Site requirements and the receipt of all of the parts, pieces, manuals, etc., necessary to perform the installation are confirmed.
Installation Qualification (IQ) • During the physical installation, serial numbers are recorded, and all of the fluidic, electrical, and communication connections are made for components in the system. • Documentation describing how the instrument was installed, who performed the installation, and other miscellaneous details should be archived.
Operational Qualification (OQ) • The OQ process ensures that the specific modules of the system are operating according to the defined specifications for accuracy, linearity and precision. • This process may be as simple as verifying the module’s self diagnostic routines, or it may be performed in more depth by running specific tests, for example, to verify detector wavelength accuracy, flow rate, or injector precision.
Performance Qualification (PQ) • The PQ process verifies system performance. • PQ testing is conducted under actual running conditions across the anticipated working range. • In practice, however, OQ and PQ are frequently performed together, particularly for linearity and precision (repeatability) tests, which can be conducted more easily at the system level. • For HPLC, the PQ test should use a method with a well-characterized analyte mixture, column, and mobile phase. A system suitability must be performed. • Proper documentation supporting the PQ process should be archived.
Validation • Process Validation – Prospective Validation – Ongoing Validation – Re-Validation: After change, Periodic, – Retrospective Validation
• Analytical Method Validation – Specificity, Linearity, Precision, Accuracy/Recovery, Ruggedness
• Cleaning Validation • Utility System Validation • Computer Validation
What is not Analytical Method Validation? • 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.
HPLC Detector calibration
• Wavelength Accuracy • Linear Range • Noise Level • Drift
Typical System Suitability Test
• Minimum Resolution of 3.0 between the analyte peak and internal standard peaks • Relative Standard Deviation of replicate standard injections of not more than 2.0%
Method Life Cycle Validation
Development
Optimization
Verification vs. Validation • Compendial vs. Non-compendial Methods – Compendial methods-Verification – Non-compendial methods-Validation
Today’s Validation Requirements
ICH/USP
GMPs (legal)
FDA
GMP Validation Parameters • Accuracy • Specificity • Sensitivity • Reproducibility
FDA Validation Parameters • Accuracy • Precision • Linearity (& Range) • Specificity (& Determination Limit) • Recovery • Ruggedness 1987 FDA Guidelines
ICH/USP Validation Requirements & Parameters ICH
USP
I nter national Conf er ence on H armoni zation
Specificity
•
Specificity
Linearity and Range
•
Linearity
Accuracy
•
Range
Precision
•
Accuracy
Limit of Detection
•
Precision
Limit of Quantitation
– Repeatability
Ruggedness
– Intermediate Precision
Robustness
– Reproducibility •
Limit of Detection
USP Data Elements Required For Assay Validation
* May be required, depending on the nature of the specific test.
USP Categories • Category 1: Quantitation of major components or active ingredients • Category 2: Determination of impurities or degradation products • Category 3: Determination of performance characteristics • Category 4: Identification test
ICH Validation Characteristics vs. Type of Analytical Procedure
Method Validation for USP • Method validation, according to the United States Pharmacopeia (USP), is performed to ensure that an analytical methodology is accurate, specific, reproducible, and rugged over the specified range that an analyte will be analyzed. • Method validation provides an assurance of reliability during normal use and is sometime described as the proces of providing documented evidence that the method does what it is intended to do.
KAPAN DILAKUKAN? 1. Pengembangan metode analisis (MA) yang telah ada misalnya untuk: - Matriks sampel yang spesifik - Memperbaiki “Analytical Performance” MA dengan adanya instrument atau teknik baru - MA yang terlalu mahal, memakan banyak waktu & energi - MA alternatif untuk konfirmasi
2. Terhadap MA yang dibuat dari modifikasi metode resmi (standard yang telah dipublikasi secara internasional, regional atau nasional; jurnal ilmiah yang relevan)
TUJUAN 1. Hasil analisis absah/valid, dapat dipercaya dan dapat dipertanggung jawabkan secara ilmiah 2. Hasil analisis dapat menunjukkan kesesuaian dengan tujuan pengujian
Accuracy vs precision
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) • The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness.
ACCURACY (2) Assay of Drug Substance: a) application of an analytical procedure to an analyte of known purity (e.g. reference material); b) comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure, the accuracy of which is stated and/or defined (independent procedure) c) accuracy may be inferred once precision, linearity and specificity have been established
ACCURACY (3) Assay of Drug Product: a) application of the analytical procedure to synthetic mixtures of the drug product components to which known quantities of the drug substance to be analysed have been added; b) in cases where it is impossible to obtain samples of all drug product components, it may be acceptable either to: – add known quantities of the analyte to the drug product or – to compare the results obtained from a second, well characterized procedure, the accuracy of which is stated and/or defined (independent procedure)
c) accuracy may be inferred once precision, linearity and specificity have been established.
ACCURACY (4) Impurities (Quantitation): • Accuracy should be assessed on samples (drug substance/drug product) spiked with known amounts of impurities. • In cases where it is impossible to obtain samples of certain impurities and/or degradation products, it is considered acceptable to compare results obtained by an independent procedure. • It should be clear how the individual or total impurities are to be determined e.g., weight/weight or area percent, in all cases with respect to the major analyte.
The Matrix Effect • The matrix effect problem occurs when the unknown sample contains many impurities. • If impurities present in the unknown interact with the analyte to change the instrumental response or themselves produce an instrumental response, then a calibration curve based on pure analyte samples will give an incorrect determination
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 • Accuracy should be assessed using a min.
of 9 determinations over a min. of 3 concentration levels covering the specified range (e.g. 3 concentrations/3 replicates each of the total analytical procedure). • Accuracy should be reported as: – % recovery by the assay of known added amount of analyte in the sample or as – the difference between the mean and the accepted true value together with the confidence intervals
Example: • 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 detected
Recovery (%)
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
Standard deviation
: 0.699
Relative standard deviation (RSD) : 0.699 %
Acceptance Criteria
98.0 – 102.0 % <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
Source: AOAC (2002). AOAC Requirements for Single Laboratory Validation of Chemical Methods. DRAFT 2002-11-07, \AOACI\eCam\SingleLab_Validation_47.doc. http://www.aoac.org/Ag_Materials/additives/aoac_slv.pdf
PRECISION • The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. • Precision may be considered at three levels: – – –
repeatability, intermediate precision and reproducibility.
• Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution. • The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of
Repeatability (1) • Repeatability expresses the precision under the same operating conditions over a short interval of time. • Repeatability is also termed intra-assay precision.
Repeatability (2) • Repeatability should be assessed using: a) a minimum of 9 determinations covering the specified range for the procedure (e.g. 3 concentrations/3 replicates each) or b) a minimum of 6 determinations at 100% of the test concentration.
Intermediate precision • Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. • The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. • The applicant should establish the effects of random events on the precision of the analytical procedure. • Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. The use of an experimental design (matrix) is encouraged.
Reproducibility • Reproducibility is assessed by means of an inter-laboratory trial. • Reproducibility should be considered in case of the standardization of an analytical procedure, for instance, for inclusion of procedures in pharmacopoeias.
Recommended Data • The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated.
Example • 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)
Precision Table 1: Acceptable Recovery Percentages Analyte (%)
Unit
RSD (%)
100
100%
1
10
10%
1.5
1
1%
2
0.1
0.1%
3
0.01
100 ppm
4
0.001
10 ppm (μg/g)
6
0.0001
1 ppm
8
0.000001
10 ppb (μg/kg)
15
AOAC (2002). AOAC Requirements for Single Laboratory Validation of Chemical Methods. DRAFT 2002-11-07, \AOACI\eCam\Single-Lab_Validation_47.doc. http://www.aoac.org/Ag_Materials/additives/aoac_slv.pdf.
Specificity/Selectivity • Ability of an analytical method to measure the analyte free from interference due to other components. • Selectivity describes the ability of an analytical method to differentiate various substances in a sample
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.
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)
Intent is to create 10 to 30 % Degradation
Bgm menentukan selektifitas?
Examples of pure and impure HPLC peaks
Source: LabCompliance (2007). Validation of Analytical Methods and Procedures: Tutorial. http://www.labcompliance.com/tutorial/methods/default.aspx?sm=d_d
Linearity • Ability of an assay to elicit a direct and proportional response to changes in analyte concentration.
Linearity Should be Evaluated
• By Visual Inspection of plot of signals vs. analyte concentration • By Appropriate statistical methods – Linear Regression (y = mx + b) – Correlation Coefficient, y-intercept (b), slope (m)
• Acceptance criteria: Linear regression r 2 > 0.99
Requires a minimum of 5 concentration levels
Method Validation- Linearity
Cara penetapan • Ditetapkan terhadap minimum konsentrasi pada rentang minimum 50 % - 150 % dari kadar analit • Dihitung regresi liniernya dan didapat persamaan regresi: Y = a + bx
RANGE • The specified range is normally derived from linearity studies and depends on the intended application of the procedure. • It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure.
Minimum Specified Ranges • for the assay of a drug substance or a finished (drug) product: normally from 80 - 120 % of the test concentration • for content uniformity, covering a minimum of 70 - 130 % of the test concentration • for dissolution testing: +/-20 % over the specified range; e.g., if the specifications for a controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24 hours, the validated range would be 0-110% of the label claim
Detection limit vs Quantitation limit
‘Know that it’s there’
vs ‘Know how much is there’
Detection limit (means)
Is any of it present?
Is it there?
Quantitation limit How much of it is present???
How much of it is there?
Method Validation- LOD and LOQ Sensitivity • Limit of detection (LOD) – “the lowest content that can be measured with reasonable statistical certainty.”
• Limit of quantitative measurement (LOQ) – “the lowest concentration of an analyte that can be determined with acceptable precision (repeatability) and accuracy under the stated conditions of the test.”
• How low can you go?
LOD and LOQ 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
Analytical Method Development LOD, LOQ and Signal to Noise Ratio (SNR) LOQ
Signal to Noise = 10:1
Signal to Noise = 3:1
LOD Noise
• Berdasarkan kurva kalibrasi analit Menurut Miller: LOD = 3.SY/X + yb
yb = intersep
LOQ = 10.SY/X +yb S y / x
9/23/2013
2 ( ) / N 2 y y i ˆ
76
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
Cara penetapan • Sampel dianalisis dari lot sampel homogen, oleh analis berbeda dalam lab berbeda, menggunakan kondisi operasional & lingkungan berbeda tetapi masih dalam spesifikasi yang dipersyaratkan • Ruggedness ditetapkan sebagai fungsi dari variabel penetapan • Ukuran ruggedness MA didapat dari membandingkan reprodusibilitas ini dengan penetapan presisi di bawah kondisi normal
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
Cara penetapan • Dilakukan selama pengembangan MA dan tergantung pada tipe prosedur MA • Bila pengukuran peka terhadap variasi kondisi analitis, maka kondisi analitis tersebut harus dikendalikan • Pada evaluasi robustness, harus ditetapkan parameter kesesuaian sistem (mis: resolusi) untuk menjamin validitas MA tetap terpelihara ketika digunakan
