Determination of theophylline by HPLC and GC-IDMS, the effect of chemically similar xanthine derivatives on the specificity of the method and the possibility of paracetamol as interfering substance.
The aim of this study was to develop and compare high-performance liquid chromatography (HPLC) and gas chromatography coupled with isotope dilution-mass spectrometry (GC-IDMS) methods with a common extraction procedure for the determination of substituted xanthines in biological matrices such as serum and urine. For HPLC both isocratic and gradient methods were evaluated. Difficulties occurred in separation of all 6 xanthines of interest - uric acid, theobromine, theophylline, paraxanthine, caffeine and 1,3-dimethyl-7-(2-hydroxyethyl) xanthine as internal standard. In addition, paracetamol was seen to interfere at higher concentrations, which meant that a system had to be developed to separate all 7 components of interest. The final solution chosen consisted of precipitation of serum samples with 6 mol/l trichloroacetic acid followed by neutralisation with 3 mol/l KOH and chromatography on a 150 x 4.6 mm Nautilus C-18 column (Macherey & Nagel) using an isocratic elution consisting of 0.02 mol/l acetate-phosphate buffer, pH 3.0 containing 9.6% v/v acetonitrile and monitoring at 273 +/- 7 nm. Comparisons with GC-IDMS and FPIA were acceptable. Run times of 10 minutes were possible. An additional "safe time" of 5 minutes was allowed to elute any substances with similar absorption maxima which were sometimes present in commercial control sera. Precision of the method was 1.64% (intra-assay) and 2.87% (inter-assay) at 4.1 mg/l and 1.51% respectively 2.15% at 25 mg/l including extraction and measurement steps. Recovery was between 86 and 101% between 1.25 and 100 mg/l and peak time deviations for all 7 components between 0.07% and 0.34% (coefficient of variation) in 7 consecutive measurements.
Kress
Michael
M
Institute for Standardisation and Documentation, Medical Laboratory Düsseldorf, Germany.
Meissner
Dieane
D
Kaiser
Patricia
P
Hanke
Rainer
R
Wood
William Graham
WG
D000082:Acetaminophen; D002138:Calibration; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods / Q000592:standards; D006801:Humans; D007118:Immunoassay; D012680:Sensitivity and Specificity; D013806:Theophylline / Q000032:analysis / Q000302:isolation & purification*; D014238:Trichloroacetic Acid; D014970:Xanthines / Q000032:analysis / Q000302:isolation & purification
D014970:Xanthines; D000082:Acetaminophen; D014238:Trichloroacetic Acid; D013806:Theophylline