Analisis Sediaan Farmasi

Henry K.S.,M.Si.Apt,
03/17/2020 1
Jadwal Kuliah
Minggu Bahan kajian
I
Validasi metode analisis

II Validasi metode analisis

Pemisahan komponen aktif dari bahan pembantu sediaan obat, serta pemilihan metode
III
analisis
Analisis kuantitatif sediaan obat monokomponen (padat & semi padat) secara
IV spektrofotometri, spektrofluorometri dan elektrokimia
Analisis kuantitatif sediaan obat monokomponen (cair dan steril) secara spektrofotometri,
V
spektrofluorometri dan elektrokimia
Analisis kuantitatif sediaan obat multikomponen (padat & semi padat) secara
VI spektrofotometri dan spektrofluorometri
Analisis kuantitatif sediaan obat multikomponen (Cair dan Steril) secara spektrofotometri
VII
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 dan KG
XI Analisis Sediaan Kosmetika
XII Analisa Sediaan Makanan dan minuman
XIII Analisa dalam Sampel Biologis
XIV Studi kasus
03/17/2020 2
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

03/17/2020 4
Validation

Proses penilaian terhadap parameter

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

03/17/2020 5
The Validation Process

Hardware Method
Validation

Validation

System
Software Suitability
Validation (4M)

• Man
• Machine
• Material
• Method

7
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 User’s

Site Site
Structural Qualification Calibration
and IQ and
Software OQ Maintenance,
Qualification 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

14
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.

15
HPLC Detector calibration

• Wavelength Accuracy
• Linear Range
• Noise Level

• Drift

16
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%

17
Method Life Cycle

Validation

Development Optimization

18
Verification vs. Validation

• Compendial vs. Non-compendial Methods

– Compendial methods-Verification

– Non-compendial methods-Validation

19
Today’s Validation Requirements

ICH/USP

GMPs
(legal) FDA

20
GMP Validation Parameters

• Accuracy
• Specificity
• Sensitivity
• Reproducibility

21
FDA Validation Parameters

• Accuracy
• Precision
• Linearity
(& Range)
• Specificity
(& Determination Limit)
• Recovery
• Ruggedness

1987 FDA Guidelines

22
ICH/USP Validation Requirements &
Parameters

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
• Limit of Quantitation 23
USP Data Elements Required For Assay
Validation

Assay Category 2
Analytical
Assay Assay
Performance Category 1 Category 3
Parameter Quantitative 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.

24
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

25
ICH Validation Characteristics vs. Type of
Analytical Procedure
Impurity testing
Type of
Analytical Identification Assay
Procedure Quantitative Limit Tests

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

26
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

03/17/2020 28
 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

1. Hasil analisis absah/valid, dapat

dipercaya dan dapat dipertanggung
jawabkan secara ilmiah
2. Hasil analisis dapat menunjukkan
kesesuaian dengan tujuan pengujian

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Accuracy vs precision

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03/17/2020 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
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

03/17/2020 42
 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 Recovery Acceptance
mg/ml % detected (%) Criteria

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

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
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

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

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
 Bgm menentukan selektifitas?

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03/17/2020 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
Cara penetapan

• Ditetapkan terhadap minimum

konsentrasi pada rentang minimum 50 %
- 150 % dari kadar analit
• Dihitung regresi liniernya dan didapat
persamaan regresi: Y = a + bx

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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

2009 74
Analytical Method Development

LOD, LOQ and Signal to Noise Ratio (SNR)

LOQ

Signal to Noise = 10:1

LOD
Signal to Noise = 3:1
Noise
• Berdasarkan kurva kalibrasi analit
Menurut Miller:

LOD = 3.SY/X + yb yb = intersep

LOQ = 10.SY/X +yb

Sy/ x   i
( y  ˆ
y ) 2
/ N 2

03/17/2020 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

03/17/2020 77
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

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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

• 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

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 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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