Ion suppression experiments revealed that the biological matrix affected 6-TG (final concentration, 2.5 [micro]mol/L) ionization by 40.5 (3.9)%, 8-BA (final concentration 2.5 [micro]mol/L) ionization by 8.0 (1.6)%, and 6-MMP derivative (final concentration 25.0 [micro]mol/L) ionization by 8.3 (4.0)% [mean (SD) of 3 experiments on 3 different days with 5 replicates each day from a pool of erythrocyte hemolysates].
The resulting acidic extract provides the protons necessary to heat-hydrolyze the thiopurine nucleotide covalent base-ribose bond with formation of 6-TG from 6-TGNs and of 4-amino-5-(methyl)thiocarbonyl imidazole from 6-MMPN.
The kinetics of hydrolysis of thiopurine nucleotides added to erythrocytes revealed that complete conversion of 6-thioguanosine mono-, di-, and triphosphate to 6-TG and of methyl 6-thioinosine mono-, di-, and triphosphate to 6-MMP derivative was achieved during a 60-min hydrolysis at 100[degrees]C.
 Nonstandard abbreviations: 6-MP, 6-mercaptopurine; 6-TIMP, 6-thioinosine monophosphate; 6-TG, 6-thioguanine; 6-TGN, 6-thioguanine nucleotide; 6-MMP, 6-methylmercaptopurine; 6-MMPN, 6-methylmercaptopurine nucleotide; TPMT, thiopurine methyltransferase; 6-TGTP, 6-thioguanosine triphosphate; LC/MS/MS, liquid chromatography-tandem mass spectrometry; DTT, dithiothreitol; 6-TGMP, 6-thioguanosine monophosphate; 6-TGDP, 6-thioguanosine diphosphate; Me6-TIMP, methyl 6-thioinosine monophosphate; Me6-TIDP, methyl 6-thioinosine diphosphate; Me6-TfrP, methyl-6thioinosine triphosphate; 8-BA, 8-bromoadenine; UV, ultraviolet; and RFLP, restriction fragment length polymorphism.
Because the two methods use different concentrations of DTT (2 mmol/L for the Lennard procedure and 60 mmol/L for the Dervieux-Boulieu procedure), we investigated the influence of the DTT concentration on the recovery of 6-TG. For the Dervieux-Boulieu method (23), increasing the DTT concentrations from 2 to 60 mmol/L increased the recovery of 6-TG (Fig.
The method-dependent 6-TG concentration-time curves are presented in Fig.
However, more commonly, free 6-TG is determined after hydrolysis of 6-TGNs at increased temperatures and acidic pH (21-23,26,28,33).
From the results of our experiments, it seems that the procedure of Dervieux and Boulieu (23) produces more complete conversion of erythrocyte 6-TGNs to 6-TG, leading to higher measured 6-TG concentrations.
This may further contribute to incomplete hydrolysis, leading to lower measured 6-TG concentrations.