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The % CV for area ratio (analyte/ ISTD for both TMD, GBP) of high standard solution should be less than 4.
The percentage of interference due to exogenous and endogenous components at retention times of analytes and ISTD was evaluated by processing eight different lots of blank plasma along with each two lots of hemolytic and lipemic plasma.
The matrix effect was evaluated at LQC, HQC levels by calculating matrix factor of analyte and ISTD. Later ISTD normalized matrix factor was calculated by using matrix factor of analyte and ISTD.
To evaluate true effect of matrix on recovery of analyte and ISTD (absolute recovery-AR), the response of extracted samples was also compared with aqueous samples.
Three calibration curves were generated by plotting the area ratios (analyte response/ ISTD response) on y-axis and concentration on x-axis.
Selected-ion monitoring was used, and quantitation was based on the area ratios of the native and ISTD molecular ions at m/z 176 and 179, respectively.
Quantitation in these assays is based on the ratio of the area counts from Ion A (177[right arrow]80) to that of the ISTD ion (Ion B, 180[right arrow]80), whereas the confirmation ratio is determined from the ratio of Ion C (177[right arrow]98) to Ion A.
Preliminary evaluations showed good agreement between recoveries estimated from the ISTD area count for each sample normalized to the mean ISTD area count for that day's standards vs recoveries estimated by adding 5-methylcotinine as an additional external standard.
In all cases, native cotinine losses during sample preparation are associated with compensatory losses of the ISTD and thus should not alter the sample quantitation on the basis of the ratio of the two analytes.
(2) Nonstandard abbreviations: ETS, environmental tobacco smoke; NHANES, National Health and Nutrition Examination Survey; APCI MS/MS, atmospheric pressure chemical ionization tandem mass spectrometry; MRM, multiple reaction monitoring; GC, gas chromatography; LOD, limit of detection; EIA, enzyme-linked immunoassay; ISTD, internal standard.