We assessed the ion suppression (27) caused by the biological matrix by comparing the ion counts after injection of 6-TG, 8-BA, and 6-MMP derivative added to water (final concentrations of 2.5, 10, and 25 [micro]mol/L, respectively) with the ion counts of 6-TG, 8-BA, and 6-MMP derivative (at the same final concentration) added to blank acidic extracts (after acid hydrolysis) from a pool of erythrocytes for 3 consecutive days with 5 replicates each day.
After hydrolysis and neutralization with 3.4 mol/L NaOH and 2 mol/L Tris base, 6-TG and 6-MMP derivative were extracted with 0.3 g/L phenylmercuric chloride in methylene chloride.
A chromatogram of blank erythrocytes vs erythrocytes supplemented with 6-TG and 6-MMP and subjected to the sample treatment procedure is presented in Fig.
The limits of detection, defined as 5 times the signalto-noise ratio, were 0.10 [micro]mol/L of erythrocytes for 6-TG and 0.50 [micro]mol/L of erythrocytes for the 6-MMP derivative.
The conversion of 6-TG and 6-MMP nucleoside mono-,di-,and triphosphates to 6-TG and 6-MMP derivative was complete after a 60-min acid hydrolysis at 100[degrees]C (Fig.
The quality-control samples for both methods [in-house drug-free erythrocyte lysates to which 6-TG (100 and 700 pmol/0.8 X [10.sup.9] erythrocytes) had been added] were analyzed in each run.
The detection limit for 6-TG was calculated for both methods on the basis of a signal-to-noise ratio of 3.
In a second set of experiments, we investigated the effect of several DTT concentrations (2, 5, 10, 25, and 60 mmol/L) on 6-TG recovery with both methods, using pooled erythrocytes from patients on azathiprine therapy.
Our observed extraction efficiency for 6-TG with the Lennard procedure was ~40% compared with the 64% reported by Lennard (21).