analisis sediaan farmasi
TRANSCRIPT
04/17/2023 1
Analisis Sediaan Farmasi
Henry K.S.,M.Si.Apt,
04/17/2023 2
Jadwal KuliahMinggu Bahan kajian
I Validasi metode analisis
II Validasi metode analisis
IIIPemisahan komponen aktif dari bahan pembantu sediaan obat, serta pemilihan metode analisis
IVAnalisis kuantitatif sediaan obat monokomponen (padat & semi padat) secara spektrofotometri, spektrofluorometri dan elektrokimia
VAnalisis kuantitatif sediaan obat monokomponen (cair dan steril) secara spektrofotometri, spektrofluorometri dan elektrokimia
VIAnalisis kuantitatif sediaan obat multikomponen (padat & semi padat) secara spektrofotometri dan spektrofluorometri
VIIAnalisis 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 KCKTX Analisis kuantitatif sediaan obat multikomponen secara KLT dan KGXI Analisis Sediaan KosmetikaXII Analisa Sediaan Makanan dan minumanXIII Analisa dalam Sampel BiologisXIV Studi kasus
04/17/2023 3
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
04/17/2023 4
Analisis Sediaan Farmasi
Analisis = ?????
Sediaan Farmasi = ????
C – GMP........ Perlu adanya Validasi
04/17/2023 5
Validation
Proses penilaian terhadap parameter analitik tertentu, berdasarkan percobaan laboratorium, untuk membuktikan bahwa parameter tersebut memenuhi syarat untuk tujuan penggunaannya
The Validation Process
Validation
Method Validation
System Suitability
Hardware
Software
7
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
and
Software Qualification
Qualification
IQ
OQ
PQ
Calibration
and
Maintenance,
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.
• 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.
Installation Qualification (IQ)
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.
14
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
15
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.
16
HPLC Detector calibration
• Wavelength Accuracy
• Linear Range
• Noise Level
• Drift
17
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%
18
Method Life Cycle
Validation
Development Optimization
19
Verification vs. Validation
• Compendial vs. Non-compendial Methods
– Compendial methods-Verification
– Non-compendial methods-Validation
20
Today’s Validation Requirements
ICH/USP
GMPs(legal) FDA
21
GMP Validation Parameters
• Accuracy
• Specificity
• Sensitivity
• Reproducibility
22
FDA Validation Parameters
• Accuracy• Precision • Linearity
(& Range)• Specificity
(& Determination Limit)• Recovery• Ruggedness
1987 FDA Guidelines
23
ICH/USP Validation Requirements & Parameters
• Specificity• Linearity• Range• Accuracy• Precision
– Repeatability– Intermediate
Precision– Reproducibility
• Limit of Detection• Limit of Quantitation
ICH International Conference on Harmonization
Specificity Linearity and Range
Accuracy Precision
Limit of Detection Limit of Quantitation
Ruggedness Robustness
USP
24
USP Data Elements Required For Assay Validation
Analytical PerformanceParameter
Assay Category
1
Assay Category 2Assay
Category 3
Quantita-tive Limit Tests
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 * *
Ruggedness Yes Yes Yes Yes* May be required, depending on the nature of the specific test.
25
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
26
ICH Validation Characteristics vs. Type of Analytical Procedure
Type of Ana-lytical
Procedure
Identifica-tion
Impurity testing
AssayQuantita-tive Limit Tests
Accuracy No Yes No Yes
Precision
Repeata-bility 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
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.
04/17/2023 28
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
04/17/2023 29
2. Terhadap MA yang dibuat dari modifikasi metode resmi (standard yang telah dipublikasi secara internasional, regional atau nasional; jurnal ilmiah yang relevan)
04/17/2023 30
TUJUAN
1. Hasil analisis absah/valid, dapat dipercaya dan dapat dipertanggung jawabkan secara ilmiah
2. Hasil analisis dapat menunjukkan kesesuaian dengan tujuan pengujian
04/17/2023 31
Accuracy vs precision
04/17/2023 32
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
• Totally hopeless!
• Poor precision• Poor accuracy
What would you call this?
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.
04/17/2023 42
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 reducedby ~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
detectedRecovery
(%)Acceptance
Criteriamg/ml %
0.2800.3200.3600.3800.4000.4200.4400.4800.520
70809095
100105110120130
147356616770131904848190586220912152180374229364725189762670144
99.3299.48
100.94100.51100.06100.03100.07101.0198.99
Mean (recovery) : 100.04Standard deviation : 0.699Relative standard deviation (RSD) : 0.699 %
98.0–102.0 %
< 2 %
Accuracy
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
Table 1: Acceptable Recovery Percentages
Source: 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.
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 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
• 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
1234567
0.40.40.40.40.40.40.4
1902803192808319114571915897191331218977021907019
Mean : 1910896Standard deviation : 9841.78Relative standard deviation (RSD) : 0.515 %
Acceptance criteria:
Relative standard deviation (RSD): not more than 2 %
04/17/2023 56
Kriteria
Secara umum:
- RSD < 1.0 % (Bahan baku obat)
- RSD < 2.0 % (Sediaan obat)
- RSD < 5.0 % (Cemaran/impurity)
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
Table 1: Acceptable Recovery Percentages
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.
Precision
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
58
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.
59
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
60
04/17/2023 61
Bgm menentukan selektifitas?
04/17/2023 62
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.
64
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 r2 > 0.99
Requires a minimum of 5 concentration levels
65
Method Validation- Linearity
04/17/2023 67
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 limitHow 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 canbe measured with reasonable statistical certainty.”
• Limit of quantitative measurement (LOQ) – “the lowestconcentration of an analyte that can be determined withacceptable precision (repeatability) and accuracy under
the stated conditions of the test.”
• How low can you go?
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
74
LOD and LOQ Estimated by
2009
Analytical Method Development
Noise
LOD Signal to Noise = 3:1
LOQ
Signal to Noise = 10:1
LOD, LOQ and Signal to Noise Ratio (SNR)
04/17/2023 76
• Berdasarkan kurva kalibrasi analit
Menurut Miller:
LOD = 3.SY/X + yb yb = intersep
LOQ = 10.SY/X +yb
2/)ˆ( 2/ NyyS ixy
04/17/2023 77
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
04/17/2023 78
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
04/17/2023 80
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
04/17/2023 81
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
04/17/2023 82
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