Penyesuaian, Modifikasi dan Perubahan

Metoda Analisa menggunakan HPLC

Gunawan Indrayanto

Fakultas Farmasi, Universitas Surabaya

gunawanindrayanto@yahoo.com

Presentasi ini untuk tujuan Pelatihan di PPPOMN, BPOM Jakarta.

Dipakai untuk Kalangan Sendiri, dan bukan suatu Publikasi,
dan bukan untuk maksud2 Komersial
October 4, 2021
 Results of Analysis of
Products/samples and Products/samples Validation/verification
QC samples + QC samples method

Using validated
QC samples Methods
reject QC samples OK

Discard
data
Authentic
Conclusion: standard/Target
Products: OK / Reject Routine Analytical Method compound must be
Research: Reliable data stable in the
selected solvent

System Suitability check Sampling methods

Calibrated Software
Qualified Person
IQ, OQ, PQ are valid
Calibrated Instruments
 USP 44 –NF 39, 2021 <1225> VALIDATION OF COMPENDIAL PROCEDURES
Validation of an analytical procedure is the process by which it is
established, by laboratory studies, that the performance characteristics of the
procedure meet the requirements for the intended analytical applications
<1226> Users of analytical methods, which described in USP–NF, are not required to
validate the accuracy and reliability of these methods, but merely verify their
suitability under actual conditions; permitted modification for chromatography
<621> 〈1226〉 VERIFICATION OF COMPENDIAL PROCEDURES:

Validation parameters: Verification process of compendial test procedure is

1. Accuracy the assessment whether the procedure can be used
2. Precision for its intended application, under actual conditions of
3. Specificity (!!) use for a specified drug substance and/or drug
product/matrix
4. Detection limit
Examples are checking the SST, specificity or recovery
5. Quantitation limit
for evaluating the matrix effects of the product (not all
6. Linearity/Range
parameters should be evaluated); SST were described
7. Robustness in the monograph of each of compound
FI VI<,2020
<1381>

<1382>
USP 44 – NF 39 <1058>, 2021

Verification

Calibration

IQ, OQ, PQ
Quality assurance for analytical chemistry

Commercial Laboratory

IQC (internal quality control):

For QC and Research

According to USP 44-NF39 <1210>, 2021,

Uncertainty was estimated by CI/PI/TI

I. Taverniers et al., Trends Anal. Chem.,

23, 535–552, 2004, modified
Guideline for validation method of analysis
• ICH Q2 R1 (2005)
• Eurachem Guide 2014
• AOAC Internationals, appendix k (dietary supplements &
botanical, 2013); f ( Standard methods performances, 2016);
appendix e (Laboratory Quality Assurance, 2005), etc.
• BSN SNI ISO 17025-2017
• Indonesian Pharmacopeia VI, 2020 <1381>
• British Pharmacopeia 2020
• USP 44 –NF 39, 2021 <1225>, <1210>, <1010>, <1224>, <1226>,
<1039> (multivariate)
• Etc.
Validasi Metoda:
Selektivitas, Linearitas, Range, Akurasi, Presisi (intra-, inter day),
LOD, LOQ, Robustness

atau
Verifikasi Metoda
SST, Selektivitas, Akurasi ..
 Lihat masing2 monograph

https://www.agilent.com/cs/library/eseminars/Public/Validated%20HPLC%20Methods.p
df
SST
Untuk Kromatografi:

https://www.agile
nt.com/cs/library/
whitepaper/public/
5991-8285EN.pdf

?
Menghitung harga RSD maksimal pada penyuntikan
ulang bila tidak tercantum pada monograp masing2

FI VI, 2020 <931>

https://www.agilent.com/cs/library/eseminars/Public/Validated%20HPLC%20Methods.pdf
Contoh Prosedur
Analisa di
FI VI, 2020
https://www.chromatographyonline.com/view/method-adjustment-usp-way
FI VI <931>
FI VI <931>
FI VI <931>
FI VI, 2020 <931>
Jka ukuran partikel diubah, laju alir memerlukan penyesuaian, karena kolom
dengan partikel kecil membutuhkan kecepatan linear yang lebih tinggi untuk
kinerja yang sama (yang ditunjukkan dengan pengurangan jumlah lempeng.
Perubahan laju alir baik untuk perubahan diameter kolom dan ukuran partikel
dapat dihitung dengan rumus

F1 dan F2 masing masing adalah laju alir; dc1 dan dc2 masing masing adalah
diameter kolom; dan dp1 dan dp2 adalah ukuran partikel pada kondisi awal dan
setelah modifikasi,

Jika perubahan partikel dari >3-µm menjadi <3-µm dalam pemisahan isokratik,
tambahan peningkatan kecepatan linier (dengan menyesuaikan laju alir
diperbolehkan, jika efisiensi kolom (N) idak turun lebih dari 20 %
FI VI <931>
 Free software untuk Penyesuaian /Pengaturan Metoda HPLC

https://appslab.thermofisher.com/lc-method-transfer-calculator
https://ispso.unige.ch/labs/fanal/hplc_calculator:en
Plate Number Pressure
 Dapat verifikasi,
Bila perbedaan
kondisi
Masih
diperkenankan
Farmakope

https://www.waters.com/webassets/cms/library/docs/720004313en.pf
Modifikasi Metoda HPLC:

Definisi: Bila kondisi HPLC diubah diluar persyaratan yang

diperkenakan oleh Farmakope.
Bila modifikasi dilakukan, Validasi Lengkap harus dilakukan,
kalau diperlukan harus dilakukan optimasi Metoda sebelum
melakukan Metoda Validasi Lengkap.

Pergantian (Perubahan ) Metoda:

Metoda HPLC diganti karena alasan tertentu misal dengan
Metoda HPLC ke metoda GC; Optimasi Metoda dan Validasi
lengkap harus dilakukan.
The solvent-selectivity triangle approach to method development.
Snyder’s solvent characterization scheme
Webinar September 2, 2020:
Strategies for Improving Resolution in Chromatography - APAC
Pada modifikasi Metoda atau Perubahan metoda,
harus dilakukan Validasi lengkap

Yang Paling penting adalah

Uji Selektivitas atau Spesifisitas Metoda

(kalau metoda tidak selektip/spesifik parameter yang lain

tidak perlu dikerjakan)
 Selectivity and Specificity:
Eurachem Guide (2014) : Analytical selectivity relates to “the extent to which the method can be used to determine
particular analytes in mixtures or matrices without interferences from other components of similar behavior
Methods: The chromatography could be repeated using a column of different polarity, employing a different separation
principle to establish whether the signal and the signal generated by the RM still appear at the same time. Where a peak is
due to more than one compound, a different polarity column may be a good way of separating the compounds. In many
cases modern mass spectrometric instruments can offer a high selectivity, e.g. gas or liquid chromatography with mass
spectrometric detection.
USP 44-NF39, 2021 <1225>: Specificity defined as the ability to assess unequivocally the analyte in the presence of
components such as impurities, degradation products, and matrix components. Lack of specificity of an individual
analytical procedure may be compensated by other supporting analytical procedures.
Methods: spiking the drug substance or product with appropriate levels of impurities or excipients and demonstrating that
the assay result is unaffected by the presence of these extraneous materials
If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results
of samples containing impurities or degradation products to a second well-characterized procedure (e.g., a pharmacopeial
or other validated procedure). These comparisons should include samples stored under relevant stress conditions (e.g.,
light, heat, humidity, acid/base hydrolysis, and oxidation). Peak purity tests (e.g., using diode array or mass spectrometry)
may be useful to show that the analyte chromatographic peak is not attributable to more than one component
Eurachem, IUPAC, AOAC have preferred the term “selectivity”, reserving “specificity” for those procedures that
are completely selective. FI VI, 2020 <1381> identical to USP <1225>.
Farmakope Indonesia VI, 2020 <1381>:
Penentuan Selektivitas = USP 44-2021;
Idem ISO Q2 (R1):
Selectivity should be evaluated using
authentic standard, possible degradation
products and Impurities

If are not available, stress conditions

testing must be performed
 R & D/QC Lab. at Pharmaceutical Industry

Nice if
related
compounds,
possible
degradation B
products, A
known Standard
impurities
can be
tested

Densitograms ( = 260 nm) obtained from: (1) solution of standard mometasone furoate, (2) extract
from excipients of laboratory-made cream, (3) extract of laboratory-made cream, (4) solution of
nipagin, (5) solution of nipasol, (6) extract of commercial ointment-1, (7) extract of commercial lotion,
(8) extract of commercial cream-1, (9) extract of commercial ointment-2 and (10) extract of commercial
cream-2. Peak identities: (A) mometasone furoate, (B) nipagin and nipasol, (C) unknown.

Wulandari, L, Tan, KS., Indrayanto,G. (2003), J. Liq. Chromatogr. R & T, 26, 109-117
 The target peak must be proved regarding its identity and purity

Peaks should be well separated

Identical RTs do not mean

identical compounds,

One Peak does not mean

a single compound

Recommended Rs > 1.75

(F.Melianita, J. Witha, S.Arifin, WF.Karina, G. Indrayanto (2009).

J. Liq. Chromatogr. R & T, 32, 567-577)
Peak’s purity evaluation using diode array (DAD) detector (HPLC) or Densitometer (TLC-Scanner)

All recent HPLC/TLC equipment's

have been completed by
software’s, which can perform
these evaluations (identity &
purity testing)
Different compounds maybe have
similar/identical UV/Vis's spectra:
MS is a better detector,
but MS/MS is the best

M. Yuwono and G. Indrayanto (2005), Validation of Chromatographic

Method of analysis, in (Brittain, H. Ed) Profile of Drug Substances, Excipients and
Related Methodology Vol 32, Elsevier, 243-258.
Stress conditions experiments
(for Drugs):
The identity and purity of peaks A and B
must be confirmed (DAD or MS)

If we have no impurities and/or degradation

products;
we should do stressed forced experiments to
prove the selectivity Identity and purity
check
of peaks A and B must be performed

D. Widiretani, I. Luailia, G. Indrayanto,

J. Planar Chromatogr. 26 (2013) , 37-42
The purity of the peak can be calculated
by comparing the MS of the u, a and d
of the TIC peak.

M.W. Dong, Modern HPLC

for Practicing Scientist, Wiley, 2006
Detector ELSD: Non-specific detector

Standar
d

Sample Rts of standard and

sample,
must be always
identical, at
different conditions

P. Li et al.,
Molecules 2019,
24, 323;
doi:10.3390/mole
cules24020323
 Determination of Xanthorrhizol in the rhizomes of Curcuma xanthorrhiza by GC FID

Xanthorrhizol ?

Should tried
3 different conditions
For FID, RID, ELSD
(= Blind methods)

(F. Melinata & G. Indrayanto (2006), Unpublished

work)
 Rs should be not less 1.75

according USP Medicines Compendium, 2013)

 USP 44-NF39, 2021 <1469>

Untuk zat tertentu harus menggunakan LC-MS atau LC-MS/MS

 Analysis using (LC) MS/MS triple Quad

For confirmation of the identity of a peak

using MS/MS, it needs to have at least
2 specific daughter ions from a selected
molecular or precursor ions; in this
example m/z 210 breaks into m/z 191
and 158 , these ion-fragments will be
used as qualifier- and quantification-ion.

210 to 191 AND 158 = MRM (Multiple

Reaction monitoring)
210 to 191 OR 156 = SRM (Selected Reaction
Monitoring)

Every known compound has specific

molecular-, qualifier- and quantification-ion.
Searchable by Google and/or
Cited from Agilent LCMSD triple Quad training, Singapore, May 2007
other MS data bases
Example of specific MRM for
pharmaceutical compounds

https://www.agilent.com/cs/library/applicat
ions/5989-9665EN.pdf
Confirmation of peak identity of the target analyte using identification point (IP) system according to Commission Decision 2002/657/EC

The identity of a peak can be confirmed, if the peak has IP at least =4,
and the ratio of the two daughter ions fulfill Table 12.3; e.g., for LC-
MS, it needs 4 specific fragments (rel. intensities at least 10 %), but
for LC-MS/MS, it needs only 1 precursor (IP =1) and 2 daughter ions
(IP=1.5), total IP = 1 + 2x1.5 = 4; LC-HR-MS/MS: I precursor (IP=2) + 1
daughter (IP =2.5) =4.
Quantitative analysis using LC-MS needs an internal standard (IS);
The ratio of the chromatographic retention time of the analyte to
that of the internal standard, i.e. the relative retention time of the
analyte, shall correspond to that of the calibration solution at a
tolerance of ± 0,5 % for GC and ± 2,5 % for LC

For completed discussion:

G. Indrayanto (2012) Validation of Analytical
Methods-Updated 2011
In: (HR Brittain, Ed., Profile of Drug
Substances, Excipients
and Related Methodology, Elsevier, 439-463
 Qualitative analysis using LC-HR-MS

https://www.fda.gov/media
/96499/download

IP : 2x2 = 4 2x 2.5=5 2+2.5 = 4.5

+ Mass accuracy (HR MS)

+ EIC of precursors and daughter ions
should be overlap each others
TYPES OF TRANSFERS OF ANALYTICAL PROCEDURES: USP 43/44-NF38/39 <1224>, 2021
Comparative Testing (comparison of proposed method and validated method)
Comparative testing requires the analysis of a predetermined number of samples of the same lot by both the
sending and the receiving units. Other approaches may be valid, e.g., if the receiving unit meets a
predetermined acceptance criterion for the recovery of an impurity in a spiked product. Such analysis is based
on a preapproved transfer protocol that stipulates the details of the procedure, the samples that will be used,
and the predetermined acceptance criteria, including acceptable variability. Meeting the predetermined
acceptance criteria is necessary to assure that the receiving unit is qualified to run the procedure.
(See USP 44-NF39 <1010>, 2021; t-test and ANOVA cannot be applied)
Covalidation

Between Two or More Laboratories. The laboratory that performs the validation of an analytical procedure is
qualified to run the procedure. The transferring unit can involve the receiving unit in an interlaboratory co-
validation, including them as a part of the validation team at the transferring unit and thereby obtaining data
for the assessment of reproducibility. This assessment is made using a preapproved transfer or validation
protocol that provides the details of the procedure, the samples to be used, and the predetermined acceptance
criteria. The general chapter Validation of Compendial Procedures <1225> provides useful guidance about
which characteristics are appropriate for testing.
Revalidation

Revalidation or partial revalidation is another acceptable approach for transfer of a validated procedure. Those
characteristics described in <1225>, which are anticipated to be affected by the transfer, should be addressed.

Transfer prosedur Analisa Tidak ada di FI, VI, 2020

Transfer Metoda Analisa: Metoda komparasi
1. Dari Lab R & D ke Lab QC
2. Dari Lab Riset Komersial ke Lab Pengguna
3. Dari Pabrik 1 ke Pabrik 2

Metoda Komparasi Juga digunakan untuk

mengevaluasi metoda baru dengan metoda yang
sudah tervalidasi (Sesuai aturan FI VI, 2020 <1381>
dan USP44-NF39, 2021)
Relative newest development:
• For comparing 2 Analytical methods, (new, proposed) it is
recommended to apply methods of USP44-NF39 <1010>.
• Significance test (t-test, ANOVA), is not recommended, just
report the mean value +/- CI
(Gandivea, S., Replication: Do not trust your p-value, be it
small or large, J Physiol 599.6 (2021) pp 1719–1721), and
related references.
SNI ISO/IEC 17025, 2017 p 103: Comparing the results of intra-
laboratory (different analyst) using t-test or ANOVA (as IQC)
Gandivea S., Replication: Do not
trust yourp-value, be it small or
large, J Physiol 599.6 (2021) pp
1719–1721
 Comparison 2 procedures according USP 44-NF39, 2021 <1010>

Scenario 1: for comparative testing according to <1225> and <1224>

using homogenous test materials, comparing new procedure (N) and old procedure
(O), By mathematical calculation, it was recommended number of replication is at
least nN = nO =18 to get power of analysis (1-β) is 80 % (β is Type II error); if n = 23,
power is 90 %.

must be < ± d, d is maximum permitted difference between N and O

USP 44, 2021 <1010>
USP 44-NF39, 2021 <1010>
Ratio of precision U must be: , and U=

recommended: d maximum is 1, and k maximum is 2, to get maximal OOS 1 %

Detail mathematical calculations were described by USP 43-NF38 <1010>
USP 44-NF39, 2021 <1010>
 USP 44 –NF39, 2020 <1010.

SCENARIO 2: VARIATION ACROSS TEST SAMPLES

It is often desirable to compare procedures across manufactured lots or use
different manufactured levels of an analyte. This is important if the study
objective is to ensure the range of the procedure in the new laboratory, or
when the procedure is intended to measure degraded samples

By mathematical calculation, it was recommended number of replication is at

least n =18 to get power of analysis (1-β) is 80 % (β is Type II error); if n = 23,
power is 90 %.

If
The 90% confidence interval on the difference in means for a paired design used
to test equivalence of means with the data

(must be < ± d)
USP 44-NF39, 2021 <1010>
USP 44, 2021 <1010>
 If data of SO is available U can be calculated (from old procedure)

σ = S = IP
U=
U must be < k
If data of SO is not available U can be calculated

U=

USP 44-NF39, 2020, <1010>

 TOST : Two one side-test according to USP 44-NF39, 2021 <1010>
Ho : |μN − μO | ≥ d ; Ha : |μN − μO | < d Accuracy: d maximal =1

When the null is rejected, we conclude that the two procedures are equivalent in their means.
“equivalence” does
not mean
Ha1 : μN − μO < d: 100.08 - 99.85 = 0.23 (<1), and
“equality.” Ha2 : μN − μO > −d: 100.08 - 99.85 = 0.23 (> -1)
(100.8 # 99.85)
Precision (K maximal = 2) If N= 99.85, O= 100.85
Ha1: 99.85-100.08 = -0.23 (<1)
Ha2: 99.85-100.08 = -0.23 > -1

V (0.214) = 0.462; V (159) = 0.398

0.462/0.398 = 1.16
 Software untuk menghitung
parameter Validasi metoda,
kecuali Metoda Transfer
(masih
dalam pengembangan)

Free download

https://github.com/blue-wind-25/VMA-Solutions/releases/tag/1.5.1
Ada Pertanyaan Bapak Ibu?
 Thank you very much!
Have a Great Day!!

Jika bapak ibu, ada masalah tentang bagaimana cara untuk Penyesuaian,
Modifikasi atau Perubahan Metoda dengan HPLC, silahkan kontak, kami
akan merasa terhormat, kalau dapat berdiskusi untuk menyelesaikannya