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