Validation by Type of Method - Analytical Validation - page 145

to remove excess protein and other potential interferences can be as much work to
develop as the chromatographic method. Automation of both sample preparation and
analysis is common.
The development and use of a bioanalytical method can be divided into three parts:
1. Reference standard preparation
2. Method development and validation
3. Application of the validated method to routine drug analyses
7.5.1 r eference S tAndArd P rePArAtIon
Reference standards are necessary for quantitation of the analyte in a biological
matrix. They are used to generate standard curves, to check method performance,
quality control, QC, or samples. Reference standards can be one of three types: (1)
standards whose purity is certified by a recognized organization (e.g., USP compen-
dial standards), (2) reference standards obtained from another commercial source
(e.g., a company in the business of the sales of general or specialty chemicals), and
(3) custom-synthesized standards. Whenever possible, the reference standard should
be identical to the analyte, or at least an established chemical form (e.g., free acid or
base, or salt). In each case, the purity of the standards must be demonstrated through
appropriate documentation, usually in the form of a certificate of analysis (CoA).
Supporting documentation such as the lot number, expiration date, and evidence of
identity and purity should be kept with other method data for regulatory inspection.
Compounds used for internal standards (often, isotopically labeled drug) must have
similar data to support purity.
7.5.2 b IoAnAlytIcAl m ethod d eveloPment And v AlIdAtIon
The key bioanalytical performance parameters that must be validated for each ana-
lyte of interest in the matrix include accuracy, precision, selectivity, range, reproduc-
ibility, and stability. In practice, to develop the method and validate the method, four
areas are investigated:
1. Selectivity
2. Accuracy, precision, and recovery
3. Calibration/standard curve
4. Bioanalytical sample stability
From each of these investigations, data is gathered to support the remaining
parameters.
7.5.2.1 selectivity
The selectivity of a bioanalytical method shows that the analyte can be accurately mea-
sured in the presence of potential interferences from other components in the sample
(including the sample matrix). Interferences can take the form of endogenous matrix
components (proteins, lipids, etc.), metabolites, degradation products, concomitant