Template to calculations of Analytical method validation

To realize calculations with this template, you just must to fill the spaces in yellow, the other cells have to be kept in blank

DATA TO VARIANCE HOMOGENEITY TEST Hypothesis test to variance homogeneity Test cochran

Amount*Area o
Concentration% Amount(mg) Peak Area or Absorbance Amount2 Area2
Absorbance Response Factor Average Response Standard Ho: The variances are not homogeneous
Variance S2
(y/x) Factor per level deviation (s)
X Y X2 Y2 XY Ho: The variances are not homogeneous
80 0.00 0 0 #DIV/0! Criterio de aceptacion
80 0.00 0 0 #DIV/0! #DIV/0! #DIV/0! #DIV/0!
If Gexp<Gtab the variances are hogeneous and reject Ho
80 0.00 0 0 #DIV/0!
100 0.00 0 0 #DIV/0! Conclusion
100 0.00 0 0 #DIV/0! #DIV/0! #DIV/0! #DIV/0!
#DIV/0!
100 0.00 0 0 #DIV/0!
120 0.00 0 0 #DIV/0!
120 0.00 0 0 #DIV/0! #DIV/0! #DIV/0! #DIV/0!
120 0.00 0 0 #DIV/0!
Summations 0 0 0 0 0 s2 maxima #DIV/0!
Square of summations 0 0 0 0 0 Σs2 #DIV/0!
Slope(b) #VALUE! Intercept(a) #VALUE! k 3.00
r #VALUE! r2 #VALUE! S2x,y #VALUE! n 3.00
n 0 n-2 -2 Gexp #DIV/0!
Deviation STD(Sb) #DIV/0! Gtab 0.87
Deviation STD(Sa) #DIV/0!
Ssexplained #VALUE! SStotal #DIV/0! SSunexplained #DIV/0!

f(x) = NaN x + NaN

R² = 0
mg added Vs Area #VALUE!

mg added Vs Area Linear (mg added Vs Area)

12.00

10.00

8.00

6.00
Area

4.00

2.00

0.00
Mean square of error
0.00 2.00 4.00 6.00 8.00 10.00 12.00
regression/Mean
squere of error
mg added residual
n-1 n-2
SSExplained/
freedom degrees of
regrssion
Ftab(α ,n-2; n-1)

Variance Analysis (ANOVA)

Mean square of error

Variation source Sum of square (SS) Freedom degrees Fcalculated F Tab
(MSE)

Regresión Lineal Explicada 1.00 #VALUE! #VALUE! Err:502

Residual Inexplicada -2.00 #DIV/0!
Total Total -1.00
alfa 0.05 Ssunexplained/
freedom degrees of
residual
Hypothesis test Acceptance criterion and results

Regression test
Ho:Amount in mg and area are not related linearly
H1:Amount in mg and area are related linearly If Fcalculated > Ftab Ho is rejected and
there is linear relation between variables
#VALUE!
Confidence interval to slope
Ho:b=0
H1:b different at zero If tcalculated >ttabulado Ho is rejected
alfa 0.05 so the slope doesn´t include zero
tcalculated #VALUE! values and the method responses
Confidence interval Err:502 Superior to changes in concentration
Err:502 Inferior
ttab Err:502 #VALUE!
Confidence interval to intercept
Ho:The intercept doesn´t include zero
H1:The intercept include zero

alfa 0.05 If tcalculated>ttab Ho is rejected and the intercept includes zero

tcalculated Err:502 and the method is proportional
ttab Err:502 Err:502

Confidence interval Err:502 Superior

Err:502 Inferior

Evaluation of Method exactness and repeatability - repeatability - Analist 1

Concentration% Amount (mg) %found Recovered Amount (mg) %Recovery

80.00 0.00 0.00 #DIV/0!

80.00 0.00 0.00 #DIV/0!
80.00 0.00 0.00 #DIV/0!
AVERAGE(x) 0.00 #DIV/0! 0.00
DEV.STD(s) #DIV/0!
%RSD #DIV/0!
100.00 0.00 0.00 #DIV/0!
100.00 0.00 0.00 #DIV/0!
100.00 0.00 0.00 #DIV/0!
AVERAGE(x) 0.00 #DIV/0! 0.00
DEV.STD(s) #DIV/0!
%RSD #DIV/0!
120.00 0.00 0.00 #DIV/0!
120.00 0.00 0.00 #DIV/0!
120.00 0.00 0.00 #DIV/0!
AVERAGE(x) 0.00 #DIV/0! 0.00
DEV.STD(s) #DIV/0!
%RSD #DIV/0!

Concentration (%) mg added mg Recovered

80.00 0.00 0.00
80.00 0.00 0.00
80.00 0.00 0.00
100.00 0.00 0.00
100.00 0.00 0.00
100.00 0.00 0.00
120.00 0.00 0.00
120.00 0.00 0.00
120.00 0.00 0.00

f(x) = NaN x + NaN

R² = 0 mg ADDED VS mg RECOVERED

1.00
0.90
0.80
mg RECOVERED

0.70
0.60
mg ADDED VS mg RECOVERED
0.50
Linear (mg ADDED VS mg RECOVERED)
0.40
0.30
0.20
0.10
0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

mg ADDED

HIPOTHESIS TEST
Ho: The method isn´t accurate
H1: The method is accurate
CONCENTRATION LEVEL (%) t CALCULATED PER LEVEL
alfa 0.05 80 #DIV/0!
n 3.00 100 #DIV/0!
n-2 1.00 120 #DIV/0!
ttab 12.7
CONCENTRATION LEVEL (%) ACCEPTANCE CRITERION RESULTS

80 #DIV/0!
If tcalculated<ttab so Ho is rejected and the method is
100 accurate #DIV/0!

120 #DIV/0!

PRECISIÓN - REPEATABILITY - ANALIST1

Concentration(%) %RSD Acceptance criterion Resultados

80 #DIV/0! Not more than 2,0% #DIV/0!
100 #DIV/0! Not more than 2,0% #DIV/0!
120 #DIV/0! Not more than 2,0% #DIV/0!

Acceptance criterion %RSD

not more than 2.0

Evaluation of Method exactness and repeatability - repeatability - Analist 2

Concentration% Amount (mg) %found Recovered Amount (mg) %Recovery

80.00 0.00 0.00 #DIV/0!

80.00 0.00 0.00 #DIV/0!
80.00 0.00 0.00 #DIV/0!
AVERAGE(x) 0.00 #DIV/0! 0.00
DEV.STD(s) #DIV/0!
%RSD #DIV/0!
100.00 0.00 0.00 #DIV/0!
100.00 0.00 0.00 #DIV/0!
100.00 0.00 0.00 #DIV/0!
AVERAGE(x) 0.00 #DIV/0! 0.00
DEV.STD(s) #DIV/0!
%RSD #DIV/0!
120.00 0.00 0.00 #DIV/0!
120.00 0.00 0.00 #DIV/0!
120.00 0.00 0.00 #DIV/0!
AVERAGE(x) 0.00 #DIV/0! 0.00
DEV.STD(s) #DIV/0!
%RSD #DIV/0!

Intermediate precision calculations - MEAN COMPARISON TO DETERMINATION OF ANALISTS 1 Y 2 AT DIFFERENT CONCENTRATION LEVELS

Estándar deviation Estándar deviation

Concentration(%) Variance (s2)Analist1 Variance (s2)Analist2 n Analist 1 (n1) n Analist 2 (n2) Mean Analist 1 Mean Analist 2 tcalculated ttab Conclusions
(s)Analist 1 (s)Analist 2
80 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 0 0 #DIV/0! #DIV/0! #DIV/0! Err:502 #DIV/0!
100 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 0 0 #DIV/0! #DIV/0! #DIV/0! Err:502 #DIV/0!
120 #DIV/0! #DIV/0! #DIV/0! #DIV/0! 0 0 #DIV/0! #DIV/0! #DIV/0! Err:502 #DIV/0!
Variance pooled (Sp) Freedom degrees
#DIV/0! -2.00

HYPOTHESIS TEST

Ho: µ1 = µ2
Ha: µ1 ≠ µ2
If tcalculated > ttab (1-α/2) o tcalculated < - ttab (1-α/2), Ho is rejected y the means of both analist are significatly different and the method is not precise

If tcalculated < ttab (1-α/2) o tcalculated > - ttab (1-α/2), Ho is not rejected y the means of both analist are not significatly different and the method is precise

SELECTIVIY OF METHOD
NOMINAL CONCENTRATION(mg/mL):
MUESTRA RETENTION TIME(min) RESOLUTION %RECOVERED
Placebo condiciones Normales N/A
Placebo condiciones Normales N/A
Placebo Fotólisis N/A
Placebo Fotólisis N/A
Placebo Termólisis N/A
Placebo Termólisis N/A
Placebo Degradación ácida N/A
Placebo Degradación ácida N/A
Placebo Degradación alcalina N/A
Placebo Degradación alcalina N/A
Placebo Oxidación N/A
Placebo Oxidación N/A
Condiciones Normales
Condiciones Normales
Fotólisis
Fotólisis
Térmolisis
Térmolisis
Degradación ácida
Degradación ácida
Degradación alcalina
Degradación alcalina
Oxidación
Oxidación

Evaluation of method robutness

Influence of different variables over the recovery of Active pharmaceutical ingredient (API)

Assay 1 2 3 4 5 6 7 8
FACTOR
A/a A A A A a a a a
B/b B B b b B B b b
C/c C c C c C c C c
D/d D D d d d d D D
E/e E e E e e E e E
F/f F f f F F f f F
G/g G g g G g G G g
Result (%Recovery)
s t u v w x y z

Variation A= 0.00
Variation B= 0.00
Variation C= 0.00
Variation D= 0.00
Variation E= 0.00
Variation F= 0.00
Variation G= 0.00