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Dushyant Patel LC-MS/MS Method

S.K.P.C.P.E.R., Kherva 43 M .Pharm. Thesis

¾ A calibration standard should be prepared in the same biological matrix same as that of

the study samples, by spiking with known concentrations of the analyte.

¾ A calibration curve should be comprised of a “blank matrix” (matrix processed without

analyte and internal standard), a “zero standard” (blank matrix processed only with

internal standard) and six or more calibration standards covering the expected range,

including the LLOQ and ULOQ.

¾ LLOQ should cover at least 4-5 half-life of the reported Cmax.

¾ ULOQ should cover the expected Cmax value. When no reference is available, it should

be decided on basis of development studies results.

¾ Calibration curve must contain minimum six calibration standards. Selection of

calibration standard expressed as multiple of LLOQ and percent of ULOQ are as follows:

TABLE NO: 4.10 SELECTION OF CALIBRATION STANDARD

A 8 Standard

Calibration Curve

CODE A 9 Standard Calibration

Curve

LLOQ Standard 1 – CS-1 LLOQ

5% ULOQ or 2 X LLOQ Standard 2 – CS-2 2 X LLOQ

10% ULOQ Standard 3 – CS-3 2% to 5% ULOQ

20% ULOQ Standard 4 – CS-4 10% ULOQ

40% ULOQ Standard 5 – CS-5 20% ULOQ

60% ULOQ Standard 6 – CS-6 40% ULOQ

80% ULOQ Standard 7 – CS-7 60% ULOQ

100% ULOQ Standard 8 – CS-8 80% ULOQ

Standard 9 – CS-9 100% ULOQ

Procedure:

¾ LLOQ and ULOQ calibration standards in duplicate (add suffix “D” in sample name of

duplicate standard) were prepared, so that if first does not meet acceptance criteria, then

only second standard of the same concentration level should be used in regression

analysis. If both are within acceptance criteria consider the first standard for regression

analysis.

¾ Blank matrix, zero standard and calibration standards were processed and analyzed as per

the procedure described in sample preparation section.

¾ Summary of the curve fitting parameters and of back-calculated concentration at each

standard level from each calibration curve were prepared.

¾ % nominal concentration of back-calculated value at each calibration level to determine

accuracy at each calibration level was calculated.

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