Statistics and Validation Data

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HPLC Method Validation

Statistics and Validation Data

Fundamentals of Statistics Application of Statistics to Validation Data Statistical Power Practical Guidance

HPLC Method Validation

Fundamental of Statistics
Two types of statistical parameters and procedures
- Descriptive statistics *used to describe, organize, summarize, or visually display data *example: mean, range, std. deviation, graphs - Inferential statistics *used to make prediction and decisions *based on probability *examples: t-test, F-test, confidence intervals, analysis of variance (ANOVA)
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Fundamental of Statistics
The most commonly used inferential procedures are based on the assumption that the data are a random sampling from a normal distribution
-all sample within the population have an equal chance of being selected -the distribution of all samples (results) is continuous, symmetrical, and the mean=median=mode

If we were to measure the entire population, we would know the true population mean and standard deviation. In practice, we only measure a small sample of the entire population.
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Example Data Set

ASSAY(%)
96.75 96.81 96.79 96.65 96.81 96.84 96.41 96.42 96.46 96.93 96.43 96.17 96.43 95.94 95.6 97.49 96.36 95.99
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Summary
Mean Std.dev. Variance Minimum Maximum

Statistics
96.49 0.4308 0.1856 95.6 97.49

Confidence Interval
A confidence interval is a range of values that has a certain probability of including the true population parameter

Population mean=sample mean(reliability term) (std error)

Confidence Interval=mean t(s) n
where t = t value for the appropriate level of confidence(i.e. 95%) and n-1 degree of freedom n = number of measurements s = standard deviation
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Confidence Interval
Using the example data set:
Confidence Interval = mean t(s) n =96.49(2.11)(0.4308) 18 =96.490.214

=96.28-96.70
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F-Test
The F-Test is used to compare the precision of two sets of data (from two methods, operators, or laboratories).

Remember the assumptions and hypothesis -random sampling from a normal distribution -hypothesis: no difference between the variances -95% confidence level is considered significant

HPLC Method Validation

F-Test
Calculation the test statistic:

Fcalc = s12
s2
2

Look up the appropriate F value from the F table If Fcalc>Ftable, then reject the hypothesis If Fcalc<Ftable, then accept the hypothesis
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F-test
Using the first two columns of data from the
example data set

Fcalc =s12
s22 Fcalc =0.26932 0.078082

Fcalc =11.89

Ftable =7.2,therefore reject the null hypothesis

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t-Test
The t-test is used to compare the means of two sets of data (from two methods, operator, or laboratories), or to compare a measured value with a true value.

Remember the assumptions and hypothesis

-random sampling from a normal distribution -hypothesis: no difference between the means -95% confidence level is considered significant
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t-Test
Or comparing a measured value with a
true value:

tcalc=X-0 S n
Look up the appropriate value from the t table -If tcalc>ttable, then reject the hypothesis -If tcalc<ttable, then accept the hypothesis
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ANOVA
Analysis of variance is an expansion of the t-test to more than two variables. It is useful in identifying the amount of error attributed to each variable.

Although useful in evaluating validations data, it is not required.

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Validation Data Requirements

Specificity
-non-numerical data (chromatograms, spectra) -comparison of responses from samples with and

without analyte present

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Validation Data Requirements

Linearity
- Visual inspection of standard plot - Correlation coefficient

- y-intercept
- slope - residual sum of squares

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Validation Data Requirements

Accuracy -direct comparison of your test results with the known results -direct comparison of your test results with the results from a second method -comparison of your spiked recovery results to 100% recovery

-infer accuracy from precision, linearity, and specificity

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Evaluation of Recovery Results

% Recovery 99.2 99.3 99.4 Mean Std.dev. 95%C.I T-table value=4.3 99.3 0.1 99.3+/-0.25 T-calc=12.1 % Recovery 98.9 99.3 99.7 99.3 0.4 99.3+/-0.99 T-calc=3.0
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ICH Data Requirements

Precision

-standard deviation
-relative standard deviation (coefficient of variation) -confidence interval

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Precision Data
Test Results
96.75 96.81

Mean
Std.Dev. RSD

96.74% 0.07808 0.08% Mean0.08

96.79
96.65 96.81 96.64

Conf.Inter.

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ICH Data Requirements

Detection and quantitation limits

-report calculation or estimated values, based on a

suitable number of anaysis Robustness -comparison of numerical and/or non-numerical results (chromatograms) for standard and varied

method conditions
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