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Analisis Sediaan Farmasi

Henry K.S.,M.Si.Apt,
9/23/2013 1

Jadwal Kuliah
Minggu I II III Bahan kajian

Validasi metode analisis Validasi metode analisis Pemisahan komponen aktif dari bahan pembantu sediaan obat, serta 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 Analisis kuantitatif sediaan obat multikomponen (Cair dan Steril) secara spektrofotometri dan spektrofluorometri





Penentuan dan Identifikasi permasalahan dalam hal analisis Analisis kuantitatif sediaan obat multikomponen secara KCKT Analisis kuantitatif sediaan obat multikomponen secara KLT dan KG Analisis Sediaan Kosmetika Analisa Sediaan Makanan dan minuman 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. Cahyadi W. , 2006, Analisis dan Aspek Kesehatan Bahan Tambahan Pangan, Bumi Aksara: Jakarta Rohman A., dan I.G. Gandjar, 2007, Metode Kromatografi untuk Analisis Makanan, Pustaka Pelajar: Yogyakarta Mitra S., 2003, Sample Preparation Techniques in Analytical Chemistry, John Willey & Sons: New Jersey
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Analisis Sediaan Farmasi

Analisis = ????? Sediaan Farmasi = ???? C GMP........ Perlu adanya Validasi


Proses penilaian terhadap parameter analitik tertentu, berdasarkan percobaan laboratorium, untuk membuktikan bahwa parameter tersebut memenuhi syarat untuk tujuan penggunaannya


The Validation Process


Method Validation

System Suitability

Validation (4M)

Man Machine Material Method

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

Vendors Site
Structural and Software Qualification

Users Site
Qualification IQ OQ PQ

Users Site
Calibration and Maintenance, System

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 modules 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.

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?

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





Verification vs. Validation

Compendial vs. Non-compendial Methods
Compendial methods-Verification Non-compendial methods-Validation


Todays Validation Requirements


GMPs (legal)



GMP Validation Parameters

Accuracy Specificity Sensitivity Reproducibility


FDA Validation Parameters

Precision Linearity

(& Range)
Specificity (& Determination Limit) Recovery Ruggedness
1987 FDA Guidelines

ICH/USP Validation Requirements & Parameters


International Conference on Harmonization

Specificity Accuracy Precision

Specificity Linearity Range Accuracy Precision Repeatability

Linearity and Range

Limit of Detection

Limit of Quantitation


Intermediate Precision
Reproducibility Limit of Detection

Limit of Quantitation

USP Data Elements Required For Assay Validation

Analytical Performance Parameter Accuracy Precision Specificity LOD Assay Category 1 Yes Yes Yes No Assay Category 2 Quantitative Yes Yes Yes No Limit Tests * No Yes Yes Assay Category 3 * Yes * *

Linearity Range Ruggedness

Yes Yes Yes

Yes Yes Yes

No * Yes

* * Yes

* 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

Category 4: Identification test


ICH Validation Characteristics vs. Type of Analytical Procedure

Type of Analytical Procedure Accuracy Precision Repeatability Interm. Prec. Specificity LOD LOQ No No Yes No No Yes Yes Yes No Yes No No Yes Yes No Yes Yes Yes No No Impurity testing Identification No


Limit Tests

Assay Yes






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.

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)



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?

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.

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

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.

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

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 mg/ml 0.280 0.320 0.360 0.380 0.400 0.420 0.440 0.480 0.520 % 70 80 90 95 100 105 110 120 130 Area detected 1473566 1677013 1904848 1905862 2091215 2180374 2293647 2518976 2670144 Recovery (%) 99.32 99.48 100.94 100.51 100.06 100.03 100.07 101.01 98.99 98.0102.0 % <2% Acceptance Criteria

Mean (recovery) : 100.04 Standard deviation : 0.699 Relative standard deviation (RSD) : 0.699 %

Table 1: Acceptable Recovery Percentages
Analyte (%) 100 10 1 Unit 100% 10% 1% Mean Recovery (%) 98-102 98-102 97-103

0.01 0.001 0.0001 0.00001 0.000001 0.0000001

100 ppm 10 ppm 1 ppm 100 ppb 10 ppb 1 ppb

90-107 80-110 80-110 80-110 60-115 40-120

Source: AOAC (2002). AOAC Requirements for Single Laboratory Validation of Chemical Methods. DRAFT 2002-11-07, \AOACI\eCam\SingleLab_Validation_47.doc.

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 measurements.

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 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.

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. 1 2 3 4 5 6 7 Concentration (mg/ml) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 : : : Area detected 1902803 1928083 1911457 1915897 1913312 1897702 1907019 1910896 9841.78 0.515 % Acceptance criteria: Relative standard deviation (RSD): not more than 2 %

Mean Standard deviation Relative standard deviation (RSD)

Secara umum: - RSD < 1.0 % (Bahan baku obat) - RSD < 2.0 % (Sediaan obat) - RSD < 5.0 % (Cemaran/impurity)




Table 1: Acceptable Recovery Percentages

Analyte (%) 100 10 1 0.1 0.01 0.001 0.0001 0.000001 Unit 100% 10% 1% 0.1% 100 ppm 10 ppm (g/g) 1 ppm 10 ppb (g/kg) RSD (%) 1 1.5 2 3 4 6 8 15

AOAC (2002). AOAC Requirements for Single Laboratory Validation of Chemical Methods. DRAFT 2002-11-07, \AOACI\eCam\Single-Lab_Validation_47.doc.

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.

Ability of an assay to elicit a direct and

response to changes in analyte concentration.


Linearity Should be Evaluated

By Visual Inspection of plot of signals vs. analyte

By Appropriate statistical methods
Linear Regression (y = mx + b) Correlation Coefficient, y-intercept (b), slope (m)

Acceptance criteria: Linear regression r2 > 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



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 its there vs Know how much is there

Detection limit

Is any of it present?

Is it there?

How much of it is present???

Quantitation limit

How much of it is there?

Method Validation- LOD and LOQ

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)

Signal to Noise = 10:1

LOD Noise

Signal to Noise = 3:1

Berdasarkan kurva kalibrasi analit Menurut Miller: LOD = 3.SY/X + yb LOQ = 10.SY/X +yb
Sy/ x
2 ( y y ) / N 2 i

yb = intersep



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



Small changes do not affect the parameters of the assay

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



Contoh variasi parameter MA yang umum: Stabilitas larutan sampel Waktu / lamanya ekstraksi Contoh variasi yang lazim dalam kromatografi cair: Pengaruh variasi pH & komposisi fase gerak Pengaruh perbedaan kolom (lot/merk) Pengaruh suhu kolom Pengaruh laju alir fase gerak
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