The ion suppression effect on the ESI response was evaluated according to
the following procedure [44]: nine drug-free urine samples from different
sources were extracted as described previously. The extracts were then fortified
with all drugs at a concentration of 50 ng/mL. Three reference solutions in pH
3.0 ammonium formate buffer were also fortified with all drugs to the same
nominal concentration. The reconstituted extracts and the reference solutions
were injected into the LC–MS/MS system. Peak areas obtained from the
extracts were compared with the corresponding peak areas produced by the
reference solutions. The ion suppression percentage was calculated as follows:
(mean peak area ‘‘reconstituted extract in urine’’ mean peak area ‘‘reference
solution’’) 100/mean peak area ‘‘reference solution’’.
Relative intensities of the detected ions were expressed as a percentage of
the intensity of the quantitation trace (relative ion intensities = intensity of
secondary trace 100/intensity of quantitation trace). The maximum permitted
tolerance for relative ion intensities is: 20% (relative ion intensities > 50%),
25% (relative ion intensities > 20–50%), 30% (relative ion intensities
> 10–20%), 50% (relative ion intensities 10%).
Urine samples above the upper limit of quantitation (ULOQ) were diluted
with drug-free urine.