Table 1.

Comparison of methods used to evaluate the main analytical validation parameters (linearity, sensititivity, accuracy, precision, recovery and matrix effect) as well as the corresponding acceptance criteria reported in the guidelines typically followed for bioanalytical methods validation developed for drugs quantitation in biological fluids and tissues.

Guideline		Linearity	Sensitivity	Accuracy	Precision	Recovery	Matrix effect
FDA	Method	Analyze a blank (no analyte, no IS), a zero calibrator (blank plus IS), and at least six, non-zero calibrator levels covering the quantitation range, including LLOQ in every run.	The lowest non-zero standard on the calibration curve defines the sensitivity (LLOQ).	Accuracy should be established by analysis of replicate concentrations with at least three independent runs, four QC levels per run (LLOQ, L, M, H QC), and ≥ five replicates per QC level.	Precision should be established with at least three independent runs, four QC levels per run (LLOQ, L, M, H QC), and ≥ five replicates per QC level.	Extracted samples at L, M, and H QC concentrations versus extracts of blanks spiked with the analyte post extraction (at L, M, and H)	Compare calibration curves in multiple sources of the biological matrix against a calibration curve in the matrix for parallelism (serial dilution of incurred samples) and nonspecific binding.
	Acceptance criteria	Non-zero calibrators should be ±15% of nominal (theoretical) concentrations, except at LLOQ where the calibrator should be ±20% of the nominal concentrations in each run.	The analyte response at the LLOQ should be ≥ five times the analyte response of the zero calibrator.	±15% of nominal concentrations; except ±20% at LLOQ.	±15% CV, except ±20% CV at LLOQ	Recovery of analyte and IS need not be 100%, but it should consistent, precise and reproducible	/
EMA	Method	A minimum of six calibration concentration levels should be used, in addition to the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS). Each calibration standard can be analysed in replicate.	Not described	Accuracy should be assessed on samples spiked with known amounts of the analyte (a minimum of 4 QC levels). The QC samples are analysed against the calibration curve, and the obtained concentrations are compared with the nominal value.	Precision should be assessed on minimum of five samples per concentration level at LLOQ, L, M and H QC samples in a single run (within- run precision) or in at least three runs analysed on at least two different days should be evaluated (between-run precision).	Not described	For each analyte and the IS, the matrix factor (MF) should be calculated for 6 lots of blank matrix, by the ratio of the peak area in the presence of matrix (measured by analysing blank matrix spiked after extraction with analyte), to the peak area in absence of matrix (pure solution of the analyte).
	Acceptance criteria	The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%	/	The mean concentration should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value.	The within-run and between-run CV value should not exceed 15% for the QC samples, except for the LLOQ which should not exceed 20%.	/	The CV of the IS-normalized MF calculated from the 6 lots of matrix should not be greater than 15%.
ICH	Method	A calibration curve should be generated with a blank sample, a zero sample (blank sample spiked with IS), and at least 6 concentration levels of calibration standards, including the LLOQ and the ULOQ.	Not described	Within-run accuracy should be evaluated by analysing at least 5 replicates at 4 QC concentration level in each analytical run. Between-run accuracy should be evaluated by analysing each QC concentration level in at least 3 analytical runs over at least two days.	Within-run precision should be evaluated by analysing at least 5 replicates at 4 QC concentration level in each analytical run. Between-run precision should be evaluated by analysing each QC concentration level in at least 3 analytical runs over at least two days.	Recovery is determined by comparing the analyte response in a biological sample that is spiked with the analyte and processed, with the response in a biological blank sample that is processed and then spiked with the analyte (L, M and H QC levels).	The matrix effect should be evaluated by analysing at least 3 replicates of L and H QCs, each prepared using matrix from at least 6 different sources/lots.
	Acceptance criteria	The accuracy of the back-calculated concentrations of each calibration standard should be within ±20% of the nominal concentration at the LLOQ and within ±15% at all the other levels.	/	The overall accuracy at each concentration level should be within ±15% of the nominal concentration, except at the LLOQ, where it should be within ±20%.	The precision (%CV) of the concentrations determined at each level should not exceed 15%, except at the LLOQ, where it should not exceed 20%.	Recovery of the analyte does not need to be 100%, but the extent of recovery of an analyte and of the IS (if used) should be consistent.	The accuracy should be within ±15% of the nominal concentration and the precision (%CV) should not be greater than 15% in all individual matrix sources/lots.
European Commission Decision 2002/657/EC	Method	At least five levels (including zero) should be used in the construction of the curve	Not described	Accuracy is determined by evaluating the trueness and the precision of the method. Trueness is evaluated by analysing six replicates of the CRM. Then, divide the detected mean concentration by the certified value (measured as concentration) and multiply by 100, to express the trueness as a percentage. If no CRM is available, instead of trueness, the recovery can be determined.	Fortify matrix with the analyte (to calculate repeatability) or CMR (for reproducibility) to yield concentrations equivalent to 1, 1,5 and 2 times the minimum required performance limit or 0,5, 1 and 1,5 times the permitted limit. At each level the analysis should be performed with at least six replicates analysed in repeatability conditions or reproducibility conditions.	Select 18 aliquots of a blank material and fortify six aliquots at each of 1, 1,5 and 2 times the minimum required performance limit or 0,5, 1 and 1,5 times the permitted limit. Then, analyze the samples and calculate the concentration present in each sample to determine recovery as: % Recovery = 100 × measured content/fortification level.	Not described
	Acceptance criteria	/	/	/	The CV under reproducibility conditions, shall not exceed the level calculated by the Horwitz Equation, or one half and two thirds (repeatability)	/	/