Requirement of assay using Single point Calibration
• Linear response function OK
• Negligible constant systemic error
CL is not significantly different to zero, or •
Negligible constant systemic error CL is not significantly different to zero, or
Intercept 2 from target compound
• Homogeneity of variance
J. Ermer P. Nethercote, Validation in PharmaceuticaAnalysis, Wiley-VCH, 2015.
Confidence range of intercept:
Qxx Xc
N 1
. S
. t
a =
C
2 y
2 -
N P,
table L
+ ±
Detection limit DL, Quantification limit QL:
• Impurities
• Degradation products
• Cleaning validation
• Heavy metals
• Heavy metals
•
Toxic metabolites
• Pesticides
• Waste products
• Bio-analytical methods
• Residuals
Detection Limit Quantitation Limit
Detection limit DL defined as the lowest concentration of an analyte that can be detected
under the analytical condition to be used, but cannot be measured quantitatively.
Quantitation limit QL is the lowest concentration that can be determined with
acceptable accuracy and precision under the analytical conditions. Generally QL can be
estimated as 3 times of DL.
DL and QL for instrumental chromatographic analytical methods can be defined in the term of the
signal-noise ratio 2-31 for DL and 101 for QL
By constructing a linear regression of relative low concentrations of analyte, accurate value of DL
can be calculated, in this case DL = Xp.
We recommend using of 5-10 relatively low
concentrations of analyte Funk’s Method, 1992.
Determination of DL using method of Funk et al DL = XaXp: QL = 3DL; X2 and X1 highest and lowest concentrations of standards should be as low as possible
For determination of DL of toxic Compounds heavy Metals, pesticide:
Accuracy Trueness and Precision Accuracy Trueness and Precision
Evaluation of Accuracy
Accuracy or trueness of an analytical method is given by the
extend by which the value obtained deviates from the true value. Firstly accuracy can be determined by
analyzing a sample with known concentration
and comparing between the measured and true value.
The second approach is by
comparing test results obtained from new
method with results from the existing method that known to be accurate. The third and fourth approaches are based on the percent recovery of
known
analyte spiked into blank matrices or products. The last technique is known as standard addition method.
For spiked samples into blank matrices, it is recommended to prepare sample in 5 different concentrations in the level of
80-120 of the target
concentration. For standard addition method, the spiking concentrations are
30-60 of
the label claimed
Cited from : Indrayanto, G Yuwono, M. 2003, in : Cazes, J. Ed. Encyclopedia of Chromatography Marcel Dekker, Supplement
ll1.workcast.net10311...MelissaHannaBrown_QbD_SepSci.pdf 2014
Must be as as possible
E. Rozet, Ph. Hubert, Presentation University de Liege, Erasme, January 2012
Accuracy is combination of “trueness” and “precision”
Ermer McB Miller2005 Method Validation in Pharmaceutical Analysis Wiley-VCH
PRECISION
For general rule, the standard deviation of the method should be lower than 16 of the specification range or RSD value was not more than 2 .
For method validation purposes, precision is determined by multiple application of the complete analytical procedure on
one homogenous real sample.
According to ICH, both repeatability and intermediate precision should be According to ICH, both repeatability and intermediate precision should be
tested
Repeatability defined as precision under the same conditions i.e. same analyst, equipment, reagents time and columnTLC-Plate.
Intermediate precision is performed by repeatability testing on the different combinations of analyst, equipment, reagents and time within one
laboratory. It is recommended to do 6-10 measurements on each of repeatability study
Cited from : Indrayanto, G Yuwono, M. 2002, Encyclopedia of Chromatography Marcel Dekker, Taylor Francis.
Cited from: J. Ermer J. H. McB. Miller, Method validation in Pharmaceutical Analysis, Willey-VCH 2005
RSD maximum for System Precision SST replicates injections
RSD = RSD =
Ermer. J Nethercote, P Eds, Method validation In Pharmaceutical Analysis, Wiely-VCH, 2015
B = upper limit minus 100 t double sides
Evaluation of the Precision by one-way ANOVA 2 levels of precisions: Sr and SR
If within-condition variance repeatability = S
r 2
, and between-condition variance is expressed as
S
2
S
B 2
Intermediate precision variance is
S
R 2
= S
r 2
+ S
B 2
Cited by G. Indrayanto In Profile of Drugs Substances, Excipients and Related Methodology, Vol 37, Elsevier 2012
Cited from: Ermer et al. 2005 Method validation in Pharmaceutical Analysis, Wiley-V
USP: Accuracy: 1 recovery, and 2 evaluating linearity of estimated and actual concentrations = recovery curve
Determination of Accuracy: 1Recovery of LM preparation. 2 Standard addition 3 Comparing with valid method
M. Yuwono G. Indrayanto 2005, Validation method of Chromatography Methods of Analysis, Profiles of Drugs Substances, Excipients and Related Methodology, Vol. 32,
Elsevier Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo, Toronto, pp. 243-258
OK
Recovery CURVE
Line 2: constant systematic error Line 3: constant and proportional
systematic error
Line 4: proportional systematic error
Evaluation of Accuracy:
Recovery CURVE Funk’s et al., 1992 :
To prove whether systematic errors did not occur, a linear regression of recovery curve of X
f concentration of the analyte measured by the propose method against X
c nominal concentration of the analyte should be constructed
X f
= a f
+ b f
. X c
and the confidence range Cr of the intercept {VBa f
} and slope {VBb f
} from the recovery curves were calculated for p = 0.05
recovery curves were calculated for p = 0.05
Qxx Xc
N 1
. S
. t
a =
a Cr
2 yf
table f
f
+ ±
Qxx .S
t b
= b
Cr
yf table
f f
±
2 -
N ]
X -
b +
a -
[X =
S
2 i
f f
if yf
∑
t = Student-t-factor f = N-2, P = 95 .
Acceptance criteria According to AOAC 2013
TruenessAccuracy
Precision
E. Rozet, Ph. Hubert, Presentation University de Liege, Erasme, January 2012, reproduced with permission
Range: 97- 103
98 ± 2
E. Rozet, Ph. Hubert, Presentation University de Liege, Erasme, January 2012, reproduced with permission
98 ± 2 96 – 100
