Recent Developments of Chemical Validation Method,

and its application in the Pharmaceutical Industries*

Gunawan Indrayanto
Faculty of Pharmacy, Surabaya University, Surabaya 60293,
E-mail: gunawanindrayanto@yahoo.com

Presentasi ini untuk Pelatihan yang diselenggarakan oleh oleh HISFARIN Jatim,
Presentasi ini bukan suatu Publikasi dan tidak untuk maksud2 komersial,
Tidak dapat diperbanyak tanpa ijin dari Penulis dan Hisfarin Jatim
25 September 2021
-----------------------------------------------------------------------------------------------------------------------
* Bioanalytical and chemometrics were not included.
 Results of Analysis of
Products/samples and
QC samples
QC samples
reject QC samples OK
Discard Conclusion:
data Products: OK / Reject
Research: Reliable data
System Suitability check
Calibrated Software
IQ, OQ, PQ are valid
Calibrated Instruments
Products/samples Validation/verification
+ QC samples method
Using validated
Methods
Authentic
standard/Target
Routine Analytical Method compound must be
stable in the
selected solvent
Sampling methods
Qualified Person
 QTTP Drugs, cosmetics, foods, Herbs

and Evaluations

or re-validation

Amandine DISPAS, Cédric HUBERT, Philippe HUBERT,, What constitutes a quality paper in drug analysis?, Talanta Open,
2021, https://doi.org/10.1016/j.talo.2021.100054. Modified
Specification of the
Producer/ Research

Quality Target Product

Profile (QTPP) or
Research’s target

Analytical
Target Profile
(ATP)

TMU : target measurement uncertainty

M. Argentine et al. USP PF 43 (6) 2018
 Step taken for validation Process

• Analytical goal: ATP

• Method selection (Official, Published/new)
• Optimization
• Stability testing (SST and pre-validation)
• Testing validation parameters
• Validation report
• Transfer of Method (if any, e.g., R&D to QC, Industry A to B)
• SOP of the method for routine analysis
 Pre-Validation: Stability testing
• Scope:
Usage of autosampler for overnight runs with samples
in solution for hours in laboratory environment raises
concern about the stability of samples.
e.g., degradation by hydrolysis, photolysis, adhesion to
glassware, etc.,
• Recommendations:
Generate data to support standard and sample solution
stability under normal lab conditions for duration of
test procedure, e.g., 24 hours, 3-5 hours for TLC Profile
Acceptance Criteria
Maximal peak area difference is ± 2 %, compare to the
fresh solution
No new Peaks and the analyte peak is still pure
Reproduced with Permission from Abadi Nusa Jakarta (Camag’s authorized dealer)
Reproduced with Permission from Abadi Nusa Jakarta (Camag’s authorized dealer)
Pre-Validation: Stability data of reference compound and sample
should be reported. Example: HPLC-DAD method
(Unpublished data)
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>

Validation parameters: 〈1226〉 VERIFICATION OF COMPENDIAL PROCEDURES:

1. Accuracy Verification process of compendial test procedure
2. Precision is the assessment whether the procedure can be
used for its intended application, under actual
3. Specificity (!!) conditions of use for a specified drug substance
4. Detection limit and/or drug product/matrix
5. Quantitation limit Examples are checking the SST, specificity or recovery for
6. Linearity/Range evaluating the matrix effects of the product (not all parameters
should be evaluated); SST were described in the monograph of
7. Robustness each of compound
Example of
Permitted adjustment
of HPLC’s method

Verification of procedure

SST, Specification/Selectivity, Accuracy

Method OK/NOT OK
FI VI, 2020 <931>, USP 44-NF39 <621>
Massart’s Golden rules of Validation method
(Cited by Gonzales & Herrador, Trend in Analytical Chemistry 26 (3)227-238 ,2007)
 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>.
 The target peak must be proved regarding its identity and purity

Peak identity/purity testing Peaks should be well separated

using Densitometry or DAD
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.
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
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
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
Standard

Rts of standard and sample,

must be always identical, at
Sample different conditions

Detector ELSD
Non-specific detector
P. Li et al., Molecules
2019, 24, 323;
doi:10.3390/molecules2
4020323
 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/applicati
ons/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-MS

https://www.fda.gov/media
/96499/download
 Linearity and Range
USP 44-NF 39, 2021 <1225>, Farmakope Indonesia VI 2020 <1381>:
Definition: The linearity of an analytical procedure is its ability to elicit test results that are directly, or by a
well-defined mathematical transformation, proportional to the concentration of analyte in samples within a
given range.
The correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares should be
submitted. Acceptance criteria: r2, must > 0.995 for Category I assays, and > 0.99 for Category II quantitative
tests (MS <736>, MIR <854>, UV/VIS <857>, AAS <852>).
ICH recommends that, for the establishment of linearity, a minimum of five concentrations normally be used.
Concentrations below the defined concentration range may introduce an error due to poor signal-to-noise ratio,
and concentrations exceeding the defined concentration range may introduce an error due to multiple
scattering.
The range of an analytical procedure is the interval between the upper and lower levels of analyte (including
these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and
linearity using the procedure as written.
Eurachem 2014: Range : 6-10 concentrations spiked into matrix/blank. Evaluations using r2, y observed
compared to y calculated for each of x (=back calculation), random distribution of residuals/; It cannot be
performed by dilution of stock solution.
– The calibration spots/standards should be independent of each other,
therefore spots should be applied from different, independently
weighed solutions – not by diluting from one and the same stock solution.

– As a consequence, calibration spots should be applied using

uniform volume of solutions, but different concentrations, to
avoid the different band-broadening effects of the different
amounts of solution per spot or band

Analogy : HPLC, GC, CE

K. Forenczi-Fodor, B. Renger, Z. Fegh, Journal of Planar Chromatography 23 (2010) 3, 173–

179 DOI: 10.1556/JPC.23.2010.3.1 173 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
 General USP, SNI ISO/IEC 17025
G. Indrayanto (2018), in: H.R.
ISO 8466-1, 1990 Funk 1994 Brittain, Ed. Profile of Drug
Substances, Excipients and
CAMAG, TLC Scanner
Related Methodology, Vol 43,
ISO 8466-1 Funk 1994 Elsevier, 359-387

Funk 1994 AOAC 2013 appendix k (2013):

Eurachem 2014 Use “r “ as a test for linear test
is incorrect.
Araujo 2009
Important references: 1. Analytical
SNI/ISO/IEC 17025 Methods Committee, Analyst
(1988), 113: 1469-147; 2. J. van
ISO 8466-1 Funk 1994 Loco; M. Elskens; C. Croux;
H. Beernaert. Accred. Qual. Assur
(2002), 7: 281-285; 3. P. Araujo, J.
Chromatogr. B. (2009) , 877, 2224-
SNI ISO 17025: Laboratory control standard should be 100 +/- 5%; intercept <MDL 2234
Why did BSN/SNI ISO 17025, 2017 not apply Vx0? Vx0 was described by ISO 8466-1
:
 Y = a + bx

Parameters
linear of USP 44
Sy = residual sum pf square
and FI VI:
r, a, b, Sy
Back calculation method for testing the linearity

Y original

BSN: ISO/IEC 17025-2017:

Y calculated Maximum difference of X original
and X calculated is 5 %

Eurachem 2014:
Comparing Y original and Y calculated

(This method is applied in Bioanalytical

X Original X Calculated Method of Analysis: FDA 2019, ICH-M10
2019)
 USP Medicines Compendium 2013, General information <10> (March 2015: not available anymore)

Why did USP-NF still used

R-square????
(<MS <736>, MIR <854>,
R-square ca. 0.999 – 0.99, % error could be ca. 1-4 % UV/VIS <857>) etc.
Calculation of Xp value is very essential;
Xp =DL; 3 DL = QL (Funk's method 1994)

https://github.com/blue-wind-25/VMA-
Solutions/releases/download/v1.4.3a/vma-
1.4.3a.jar
G. Indrayanto (2018), Validation of
Chromatographic Analysis: Application
for Drugs that derived from Herbs, in:
H.R. Brittain, Ed. Profile of Drug
Substances, Excipients and Related
Methodology, Vol 43, Elsevier, 359-387
 Accuracy and Precision according USP 44-NF 39, 2021 <1225>
Definition of Accuracy: The accuracy of an analytical procedure is the closeness of test results obtained by that
procedure to the true value. The accuracy of an analytical procedure should be established across its range.
According to the International Organization for Standardization (ISO), “accuracy” has a different meaning. In
ISO, accuracy combines the concepts of unbiasedness (termed “trueness”) and precision.]
Determination: In the case of the assay of a drug substance, accuracy may be determined (1) by application of the
analytical procedure to an analyte of known purity (e.g. a Reference Standard) or (2) by comparison of the results
of the procedure with those of a second, well-characterized procedure, the accuracy of which has been stated or
defined. (3) Accuracy may be determined by application of the analytical procedure to synthetic mixtures of the
drug product components to which known amounts of analyte have been added within the range of the
procedure. Accuracy should be assessed using a minimum of nine determinations over a minimum of three
concentration levels, covering the specified range (i.e., three concentrations and three replicates of each
concentration).
Assessment of accuracy can be accomplished in a variety of ways, including (1) evaluating the recovery of the
analyte (percent recovery) across the range of the assay, or (2) evaluating the linearity of the relationship
between estimated and actual concentrations. The statistically preferred criterion is that the confidence interval
for the slope be contained in an interval around 1.0, or alternatively, that the slope be close to 1.0.

Outlier test should be performed according to USP 44, 2021 <1010> and <111> : Dixon Test or Grubbs Test
Definition of Precision according USP 44-NF 39, 2021 <1225> : The precision of an analytical procedure is the
degree of agreement among individual test results when the procedure is applied repeatedly to multiple
samplings of a homogeneous sample. The precision of an analytical procedure is usually expressed as the
standard deviation or relative standard deviation (coefficient of variation) of a series of measurements.
Precision may be a measure of either the degree of reproducibility or of repeatability of the analytical procedure
under normal operating conditions. In this context, reproducibility refers to the use of the analytical procedure
in different laboratories, as in a collaborative study. Intermediate precision (also known as ruggedness)
expresses within-laboratory variation, as on different days, or with different analysts or equipment within the
same laboratory. Repeatability refers to the use of the analytical procedure within a laboratory over a short
period of time using the same analyst with the same equipment.
Determination: The precision of an analytical procedure is determined by assaying a sufficient number of
aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or
relative standard deviation (coefficient of variation). Assays in this context are independent analyses of
samples that have been carried through the complete analytical procedure from sample preparation to final
test result. Acceptance criteria: Repeatability (R)/Intermediate precision (IP): < 1%; Accuracy: drug substances
(DS) 98-102%, Products (P): 95-105% (MS <736>); UV/VIS <857>:R <1%, IP <2%, accuracy =MS. MIR see <854>
The ICH documents recommend that repeatability should be assessed using a minimum of nine determinations
covering the specified range for the procedure (i.e., three concentrations and three replicates of each
concentration) or using a minimum of six determinations at 100% of the test concentration
Farmakope Indonesia VI, 2020 <1381>: Penentuan Akurasi Metoda Analisa

Eurachem guide 2014: % recovery = % Bias; Accuracy = trueness

Cited from: J. Ermer in : J.Ermer & P. Nethercote Eds., Method
validation in Pharmaceutical Analysis, Willey-VCH (2015), p 85.
 Calculation of Intermediate Precision (SR) according to Ermer (2015)

Overall variance SR2 = Sr2 + SB2

If within-condition variance or intra-serial variance = Sr2 , and between-

condition variance or variance run or inter-variance is expressed as (SB2)
Sr = repeatability
SR = intermediate precision
=
According to USP 42/43, 2019/2020
<1010> (March 1, 2019- October 31,
2020 : (can be calculated using ANOVA), or using our self develop software:
https://github.com/blue-wind-25/VMA-Solutions/releases/download/v1.4.3a/vma-1.4.3a.jar
Cited from: Ermer et al. (2005) Method validation in Pharmaceutical Analysis, Wiley-VCH

Intra-serial variance = Sr2

Inter-variance = SB2
Overall variance = SR2
Result Details

Source SS df MS

Between-
7.3565 3 2.4522
treatments

Within-
9.5364 20 0.4768
treatments

Total 16.893 23

SB2 = (2.4522-0.4768) : 6 = 0.3292

Intermediate precision = Sr2 + SB2 =
V(0.4768 + 0.3292) = V0.806= 0.8977=0.90
Repeatability = V0.4768 = 0.69
 Recovery CURVE OK Determination of accuracy according
USP(40- 44)-NF (35-39),<1225>
( = Farmakope Indonesia V/VI, 2014/2020)
1. Percent recovery
2. evaluating the linearity of the
relationship between estimated and
actual concentrations = constructing
https://github.co “recovery curve”
m/blue-wind-
25/VMA- Acceptance criteria (2):
Solutions/releases 2a. slope is not significantly
/download/v1.4.3 different from 1 (one)
a/vma-1.4.3a.jar 2b. intercept is not significantly
different from 0(zero)

Line 2: constant systematic error

Line 3: constant and proportional systematic
error
M. Yuwono and G. Indrayanto (2005), Validation of Chromatographic
Method of analysis, in: (Brittain, H Ed.) Profile of Drug Substances, Line 4: proportional systematic error
Excipients and Related Methodology Vol. 32, Elsevier, 243-258
Acceptance criteria for accuracy (% Recovery) and precision were described by
ATP, based on the specification range, which is described by the official
Guidance (Current Pharmacopoeia, BPOM, AOAC, FDA or other official
institution), or self determined by the researcher.
Evaluation of Accuracy and Precision according to
USP44-NF39, 2021 <1210>

1. “Separated” evaluation of Accuracy and Precision

Calculation of confidence of bias and standard deviation

2. “Combined evaluation of accuracy and precision:

Calculation of prediction interval (PI) and Tolerance Interval
 Probability inside the specification limit
Probability OOS:

Total variances of production processes USP 43/44-NF 38/39 <1033>

SD of result of analysis by QC
Prob(OOS) = 2 · Φ(−3 · 0.94)%

Prob(OOS) = 2 · Φ(−3 · Cp) %

Φ represents the standard

normal cumulative distribution
Total variances of method
function (Z value)
of analysis
Cp = (Upper-lower)/6SD
Online calculation p from z value
:https://www.socscistatistics.com/pvalues/n
ormaldistribution.aspx
= USP Compendium <10>, 2013 Can be applied for Validation Process of a certain product
Acceptance criteria of ATP for Validation

Acceptance criteria of Accuracy, that

described by ATP = specification
range (SR) of product described by
Pharmacopoeia minus RSD of
Process)

e.g. SR product 5%: 95-105 %

e.g. total RSD process: 2 %
SR ATP for accuracy = 5 -2 = 3 %
(NOT 5 %)

During Process Validations,

“Total Process/production
Variance”
should be determined
Evaluation of Accuracy and Precision according to USP44-NF39, 2021 <1210>,

1. “Separated” evaluation of Accuracy and Precision

B must be included in specification range of ATP,

Accuracy: Confidence interval of bias = B =
e.g. – 5 % to + 5 %, or -3 % to + 3 %

U must be fulfilled the requirement of the

Precision: Confidence range of SD (S) = specification of ATP,
e.g. < 2 %
Example:
ATP : True value = 1000 mg/g; -15 < B < 15 mg/g; U < 20 mg/L
 ATP:
Bias < 3%
Range:± 3%
97- 103 %;
CV <2 %

Mean : 98.0 %
CV : 1.9 % (seems OK)

98.0 ± 1.9 % =
96.1 –99.9 % (NOT OK)

E. Rozet, Ph. Hubert, Presentation University de Liege, Erasme, January 2012,

reproduced with permission
 Evaluation of Accuracy and Precision according to USP 43/44, 2020/2021 <1210>

2. “Combined evaluation” of Accuracy and Precision

Using Prediction Interval (PI) =

Using Tolerance Interval (TI) =

Combined evaluation of
accuracy and Precision is
better compared to
separated evaluations
 Summary of Combined evaluation of Accuracy and Precision

Traditional : Mean ± (R)SD (NOT recommended)

Confidence interval USP 41-44 <1010>

(CI)

http://graphpad.com/quickcalcs/CImean1/?Format=SD
TI>PI>CI
Prediction interval USP 44 <1210>,
(PI)

http://statpages.org/tolintvl.html
Tolerance Interval
(TI) USP 41-44 <1210>

Reportable value of analysis Y should be “Mean ± CI/PI/TI” instead of “Mean ± (R)SD”,

must be included in predetermined specification range ; uncertainty is estimated by CI/PI/TI
 Prediction interval:
984.1 to 1001.5 mg/g
Tolerance interval:

https://statpages.info/tolintvl.html
Any result of analysis must be included in the specification range of the ATP

ATP: (e.g., 95 -105%)

G. Indrayanto (2018), Validation of Chromatographic Analysis: Application for Drugs that derived
from Herbs, in: H.R. Brittain, Ed. Profile of Drug Substances, Excipients and Related Methodology,
Vol 43, Elsevier, 359-387
 Estimation of uncertainty according
to BSN: SNI ISO /IEC 17025, 2017

S = Intermediate Precision
B = bias = trueness

Identical to : ML Jane Weitzel, Juris Meija, David LeBlond, Steven Walfishd, STIMULI TO THE
REVISION PROCESS, USP Forum 44 (1), 2019
 Detection Limit and Quantitation Limit according USP 44 NF39, 2021 <1225>
Definition of detection limit (DL); It is the lowest amount of analyte in a sample that can be
detected, but not necessarily quantitated, under the stated experimental conditions
Determination: DL is generally determined by the analysis of samples with known
concentrations of analyte and by establishing the minimum level at which the analyte can
be reliably detected. Typically, acceptable signal to- noise ratios are 2:1 or 3:1.
Definition of the quantitation limit (QL): It is a characteristic of quantitative assays for
low levels of compounds in sample matrices, such as impurities in bulk drug substances
and degradation products in finished pharmaceuticals. It is the lowest amount of analyte
in a sample that can be determined with acceptable Precision and Accuracy under the
stated experimental conditions
Determination: the QL is generally determined by the analysis of samples
with known concentrations of analyte and by establishing the minimum level at which the
analyte can be determined with acceptable Accuracy and Precision. A typically acceptable
signal- to-noise ratio is 10:1
Detection limit (DL), Quantification limit (QL),
must be determined for :
•Impurities
•Degradation products
•Cleaning validation
•Heavy metals
•Toxic metabolites
•Pesticides
•Bio-analytical methods
•Residual solvents
USP 434 –NF 39 <621> (2021)

Peak = signal = S

Noise =N

n x peak with at half height (DL 3, QL 10)

Positive by Chromatographic method means:
1. Have identical Rt/Rrt/Rf/Rrf to authentic standard
2. S/N of target peak must be at least 3
3. Identity test (DAD, MS): pass
4. Purity test: pass

Negative means concentration < detection limit

DL depends on the instrument
Determination of DL (LOD) according to USP 43, 2020 <1210>: identical to Xp
(Funk’s method 1994)

Signal-to-noise ratios
(<1225>), can be used to
estimate LOD and LOQ.
 USP 43/44-NF38/39 2020/2021 <1210>

LOD USP 43 = 0.0033

(Y = 0.000235 + 0.3032X
LOQ can be calculated
by replacing LOD (Funk, = Xp) = 0.0032141
(Y = 0.00023474 + 0.30319 X)

with 10.
Determination of Sample’s DL and QL according to VICH GL 49 (R), FDA 2015

https://www.fda.gov/media/78356/download
Determination of “cutoff value” according to Eurachem Guide 2014

G. Indrayanto
(2018), Validation
of
Chromatographic
MPL
Analysis:
DL Application for
Drugs that derived
Cutoff value from Herbs, in:
H.R. Brittain, Ed.
Profile of Drug
Substances,
Excipients and
Related
Methodology, Vol
43, Elsevier, 359-
387
 For proving that the used validated method is still valid for routine application at QC Lab. Eurachem Guide
2014/2016, FDA/ORA Laboratory Manual 5.9, 2019 recommended the application of “Internal quality control”
Internal QC refers to procedures undertaken by laboratory staff for the continuous monitoring of operations
and measurement results in order to decide whether results are reliable enough to be released [18, 75].
This includes replicate analysis of stable test samples, blanks, standard solutions or materials similar to
those used for the calibration, spiked samples, blind samples and QC samples [76]

QC samples are typical samples which over a given period are sufficiently stable and homogeneous to give
the same result (subject to random variation in the performance of the method), and available in sufficient
quantities to allow repeat analysis over time. QC samples were generally made by R&D.

It is widely accepted that for routine analysis, a level of internal QC of 5 % is reasonable, i.e. 1 in every 20
samples analyzed should be a QC sample.
Limits are set for the values on the chart (conventionally ‘warning limits’ are set ±2s about the mean value,
and ‘action limits’ are set at ±3s about the mean value). Mean and S can be obtained or estimated from
previous validation data of IP, but it will be nice after one year or when enough results have been collected.

USP43-NF38 <1010> applied a control charts to monitor the performance of manufacturing processes and
analytical procedures. (do not mention QC samples)
 + 3 SD or or PI or TI or U

+ 2 SD or CI

Cp and Cpk can be also applied:

But it is too simple….
Concentrations

James N Miller and Jane N Miller

Statistic and Chemometrics for
Analytical Chemistry, Pearson Educated
Limited, 2005, p 80, 2005 (modified)
Number of QC samples or samples
 Determination of Upper control limit (UCL) and
Lower Control Limit (LCU) according to USP 44-NF39 <1010., 2021
1. Using process SD : Process Mean ± 3 × Process Standard Deviation

Process SD =
d2 = 1.128
2. Using Moving Range Statistic (MR)
USP 44-NF 39 <1010>, 2021
USP44-NF39 <1010>, 2021
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.
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>
 SCENARIO 2: VARIATION ACROSS TEST SAMPLES USP 44 –NF39, 2020 <1010.
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, 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>

USP 43, 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 Ha1 : μN − μO < d: 100.08 - 99.85 = 0.23 (<1), and

not mean “equality.”
(100.8 # 99.85) Ha2 : μN − μO > −d: 100.08 - 99.85 = 0.23 (> -1)

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
USP 44-NF39, 2021 <1225>: Robustness
Definition:
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small but deliberate variations in procedural parameters listed in
the procedure documentation and provides an indication of its suitability
during normal usage. Robustness may be determined during development of
the analytical procedure.
Typical variations are the stability of analytical solutions, different equipment,
and different analysts. In the case of liquid chromatography, typical variations
are the pH of the mobile phase, the mobile phase composition, different lots or
suppliers of columns, the temperature, and the flow rate. In the case of gas
chromatography, typical variations are different lots or suppliers of columns,
the temperature, and the flow rate.
(Unaffected means result of analysis using certain factors still in the
pre-determined specification range)
USP 44-NF39 <1092> 2021: Validation of Dissolution procedures
The dissolution step is the release of the drug substance in the dissolution
medium and sampling.
The amount of drug substance dissolved during the dissolution procedure
are termed the “analytical finish”. Validation of the suitability of the
analytical finish will evaluate the attributes, linearity and range, precision,
specificity, accuracy/recovery, robustness, and stability of the sample and
standard solutions.
Validation of the dissolution step will include evaluation of precision and
robustness of the dissolution sample preparation.
Validation of the analytical finish is performed either using a standard
solution or spiked placebo or by the method of standard addition
USP 44-NF39 <1092> 2021: Validation of Dissolution procedures
Specificity/Placebo Interference: It is necessary to demonstrate that the results are not
unduly affected by dissolution medium blank, placebo constituents, other active drug
substances, or potential degradation products from the dissolved drug substance in the
dissolution medium. The placebo consists of all the excipients and coatings, with inks and
capsule shells included if appropriate, without the drug substance. Placebo interference can
be evaluated by using a spiked placebo that is prepared by weighing samples of the placebo
blend, dissolving or dispersing them in dissolution medium at concentrations that that
would be encountered during testing, and adding a known amount of the drug in solution.
The blank is the dissolution medium without dissolved sample, and it is treated in the same
manner as the sample. The effect of the absorbance of the blank at the analytical
wavelength should be evaluated. In most cases, the absorbance of the dissolution medium
blank may not exceed 1% of the standard solution at the concentration used for analysis.
Values >1% should be evaluated on a case-by-case basis. If the placebo interference exceeds
2%, modification of the method may be necessary. Possible modifications include choosing
another wavelength, subtracting baseline using a longer wavelength, transforming
absorbance values (e.g., first derivative), and using an alternative analytical technique such
as HPLC
 USP 44-NF39 <1092> /FI VI <1353>: Validation of Dissolution procedure
Acceptance criteria:
Linearity : a square of the correlation coefficient (r2 ≥ 0.98) demonstrates linearity. In
addition, the y-intercept must not be importantly different from zero (NOT significance
difference from zero)

The measured recovery is typically 95%–105% of the amount added. Bracketing or

matrixing of multiple strengths may be useful. (special case for delayed-release of dosage
forms were also described)

Repeatability : RSD <2 %: for Analytical Finish

Intermediate Precision: The mean value for dissolution results between any two
conditions, using the same dosage strength, does not exceed an absolute 10% at time
points with <85% dissolved, and does not exceed 5% for time points > 85%: Differents
days, analysts, Equipment's
 Conclusions and recommendations:
1. The reliability of any result of chemical analysis are depended on some factors i.e. (1) qualification and
motivation of the analysis, (2) validity of all used equipment's and software’s, (3) the availability of
authentic chemical standards and (4) validity of all used methods.

2. All methods should be first validated using the newest available guidance’s (e.g., USP 44-NF39, 2021,
Eurachem 2014-2019 etc.), before can be applied at the QC laboratory/Research; Many methods were
described for determining the Accuracy and Precision, the best method need to be investigated and
determined; r cannot be applied as a sole parameter for linearity.

3. The validity of the reportable results of a QC laboratory should be confirmed by analyzing QC samples
(Internal QC); if the QC samples yielded out of specification, all results of analysis must be rejected,
and the method should be evaluated, to decide whether a revalidation is needed.

4. It is recommended to report the results of analysis as “ Mean ± CI/PI/TI” instead of “Mean ± (R)SD”;
According to USP uncertainty can be estimated by using CI/PI/CI.

5. The application of significance test is not recommended, for comparison of two or more procedures
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