Bio-Analytical Method Validation-A Review

  

    
S.No. 
    
Validation    Parameter 
    
Minimum 
      
Experiments
    
Performance Criteria 
  
  

    
1. 
    
Selectivity
    
Matrix blanks: 6    lots, n=1 for each lot
      
Matrix blank    fortified with IS: 6 lots, n=1 for each lot
      
LLOQ Selectivity    Sample: 6 lots, fortified with analyte at LLOQ level and
      
IS. n=3 for each    lot
    
At least 5 out of    the 6 lots must meet the following criteria:
      
Response for the    analyte in matrix blanks or matrix blank fortified with IS must be ≤20% of    the mean analyte response in the acceptable LLOQ calibration standards
      
Response for IS in    matrix blanks must be ≤5% of the mean IS response in the acceptable LLOQ    calibration standards
      
At least two-thirds    of the selectivity LLOQ replicates for each lot must meet accuracy acceptance    limit, and the mean accuracy must be within ±20.0% of the nominal    concentration
  
  

    
2. 
    
Cross-analyte
      
Interference
    
Each analyte at    ULOQ evaluated separately. IS at the level of use
      
evaluated    separately
    
Interference must    be ≤ 20% of the mean analyte peak response or ≤ 5% of the mean IS response of    the acceptable LLOQ calibration Standards
  
  

    
3. 
    
Linearity
    
Minimum of 6 non zero calibration standard (CS) levels.
    
(R2) ≥0.985
  
  

    
4. 
    
Calibration
      
Standards: Accuracy
    
Injected at the    beginning and end of the analytical run
    
Minimum 6 non-zero    (or 75% of total) CS must be within ±15.0% RE of nominal (exception: LLOQ    within ±20.0 %RE)
  
  

    
5. 
    
QC Samples
      
Core Validation
    
Three concentration    levels: Low, Mid, High; n=6 at each level
    
Minimum 50% of the    QC replicates at each level and 66.7% of all QCs must be within 15.0% RE of    nominal
      
Mean inter- and    intra-assay accuracy within ±15.0% RE of nominal; Precision ≤15.0% RSD.
  
  

    
6. 
    
LLOQ Samples (Sensitivity)
    
n=6, ≥ 1 run
    
Mean accuracy    within ±20.0%RE of nominal; precision ≤20.0% RSD.
  
  

    
7. 
    
Recovery
    
Analyte at low,    medium and high levels, and IS at the level of use: pre extraction spiked    samples (n=6) are compared with mean response of post-extraction spiked    matrix samples (n=6)
    
Recovery for    analyte and IS must be relatively consistent across all QC levels.
  
  

    
8. 
    
Matrix Effect
    
Post-extraction    spiked samples (n=6, at each QC low, mid and high level) are compared with    mean response of 6 injections of analyte or IS in solvent.
    
MF will be    calculated and reported for the analyte and for the IS
  
  

    
9. 
    
Ruggedness
    
Minimum of two    variables over the course of validation (e.g. different column, instrument    and/or analyst)
    
Mean inter- &    intra-assay accuracy within ±15.0% RE of nominal;
      
precision ≤15.0%    RSD.
  

Table 1:  Overview of Validation Parameter and Stability Criteria.

1. Selectivity: It is defined as that an analytical method to differentiate and quantify the analyte in the presence of other components in the sample. For this parameter practically performed as in laboratory by selecting a minimum six set of appropriate biological matrix (plasma, urine, or other matrix). Each blank sample should be tested for interference at specific retention time and should be ensured at the lower limit of quantification (LLOQ).Chance of interfering substance in a biological matrix include endogenous matrix components, metabolites, decomposition products or other substance. For method development should be select a blank sample that ensure there is no interference in at retention time of analyte.2. Linearity: Ability of an analytical method to obtain test results which are directly proportional to the concentration of analyte in sample. In bio-analysis spiked at least five or six samples which cover entire range from lower limit to higher limit. Apart from visual observation that signal as function of the concentration special statistical calculation are recommended such as linear regression. Other parameters like slope and intercept, residual sum of squares and the and the coefficient of correlation of should be reported to better the linearity of the method.

3. Accuracy, Precision, Recovery: In Bio-analytical method development accuracy, precision and recovery is performed with a minimum of six batches with range of concentration from LOQ to highest concentration of range. Take nine samples in a one batch. Accuracy can be expressed by the following approaches, (a) Inferred from precision, linearity and specificity (b) Comparison of the results with those of a well characterized, independent procedure (c) Application to a reference material (for drug substance) (d) Recovery of drug substance spiked to placebo or drug product (e) Recovery of the impurity spiked to drug substance or drug product for impurities. The mean value should be within 15 % of the actual value except at LLOQ, where it should not deviate by more than 20%.

The precision of an analytical method expressed the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple of a single homogeneous volume of biological matrix. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentration is recommended .The precision determined at each level should not exceed 15 % of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20 % of the CV. Precision is further subdivided into within –run, intrabatch precision or repeatability.

The recovery of an analyte in bio-analytical method is the detector response obtained from an amount of the analyte added to and extracted from biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to extraction efficiency of an analytical method within the limits of variability. Analyte recovery need not be 100% but the extent of recovery of an analyte and of the internal standard should be consistent, precise and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (Low, Medium and High).

4. Limit of Detection (LOD): The lowest concentration of sample that can be differentiating signal from background noise. Use a standard solution of analyte and signal to noise ratio not less than 3.0. The lowest concentration of sample which can be quantitavely determined with suitable precision and accuracy. Various approaches are applied for the determinations of LOD, (a) Visual definition (b) calculation from single to noise ratio (c) calculation from the standard deviation of the blank (d) calculation from the calibration line at low concentration.

5. Limit of Quantification (LOQ): It is the lowest concentration of analyte at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met.

6. Calibration Curve: A standard curve or calibration curve is a relationship between instrument response and analyte concentration. A calibration curve covers a range from lower concentration to higher concentration with eight different concentration values. Calibration curve should be prepared same matrix as used in bio-analysis of drug. The number of standard for the calibration curve will be a function of the anticipated range of analytical values and the nature of the analyte response. Concentration of standard should be chosen on the basis of concentration range on particular study. A calibration curve should consist of blank sample (matrix sample without internal standard), zero sample (matrix with internal standard) and six to eight non-zero samples covering the expected range including LLOQ.

LOWER LIMIT OF QUANTIFICATION (LLOQ): For preparation of calibration curve LLOQ standard analyte response must be five times response compared to blank response. Analyte response should be identifiable and reproducible with a precision of 20% and accuracy 80-120%. Standard curve response of the standard must be met the following, that LLOQ deviation not more than 20% and other standard deviation not more than 15 %. A minimum of eighty percent of sample must within the acceptance criteria then the standard curve will be passing.

7. Stability of spiked sample: Drug stability in biological fluid is related to the chemical properties of analyte, the storage condition, the matrix and the container system. Stability procedure should evaluate the stability of the analyte during sample collection and handling. Stability study performed by the following parameters –Freeze and thaw stability, Short term temperature stability, Long term stability, Stock solution stability and Post –preparative stability [29] (Table 2).

  

    
S.No. 
    
Validation    Parameter 
    
Minimum 
      
Experiments
    
Performance Criteria 
  
  

    
1. 
    
Stock Solution
    
n ≥6; long term at    typical storage conditions; bench top at conditions representing typical    processing conditions for ≥6 hours.
    
Precision of area    response or relative response must be ≤15.0% RSD; RD within 7.0% for    analytes, 20.0% for internal standards
  
  

    
2. 
    
Bench top
    
≥ 4 hours n ≥6 at    QC Low and High levels
    
Mean accuracy    within ±15.0% RE of nominal; precision ≤15.0% RSD
  
  

    
3. 
    
Freeze/Thaw
    
3 freeze/thaw cycles n ≥6 at QC Low and High levels
    
Mean accuracy within ±15.0% RE of nominal; precision ≤15.0% RSD
  
  

    
4. 
    
Long term
    
n ≥ 6 at QC Low and    High levels at -10 to -30°C or -50 to -90°C for at least 1 and 4 months
    
Mean accuracy    within ±15.0% RE of nominal; precision ≤15.0% RSD
  
  

    
5. 
    
Reinjection
      
Reproducibility
    
Calibrations    standards (CS) and QCs (n=6 at each level) reinjected from an acceptable    validation batch run, maintained at autosampler temperature for ≥72 hours.
    
Mean accuracy    within ±15.0% RE of nominal; precision ≤15.0% RSD; calculated
      
using calibration    standards from re-injected run
  
  

    
6. 
    
Extract Stability
    
Stored extracts at    QC Low, Mid, and High levels (n=6) maintained at autosampler temperature for    ≥72 hours.
    
Mean accuracy    within ±15.0% RE of nominal; precision ≤15.0% RSD; calculated using freshly    extracted curves or back calculated using the original curves from the batch    the aged extracts were originally extracted and injected
  

Table 2:  Overview of Validation Stability and Criteria.