Determination of theophylline in rabbit plasma by triple quadrupole LC/MS

Aim and Background: A simple, sensitive and rapid method was developed for quantitation of theophylline in rabbit plasma utilizing Triple Quadrupole LC/MS. Materials and Methods: An aliquot of 0.1 mL of plasma sample was extracted with ethyl acetate using Heidolph Vortex. The chromatographic separation was performed by using HyPURITY ADVANCE™ C18 Column (3 × 50 mm) with a mobile phase of 80% methanol and 20% 2 mM ammonium acetate buffer followed by MS/MS detection. The analyte was quantitated in positive ionization mode. Multiple reaction monitoring (MRM) using the transition m/z 181.1→124.2 and m/z 180.2→110.1 was performed to quantify theophylline with internal standard (IS, Phenacetin), respectively. The method had a total chromatographic runtime of 3 min and linear calibration curves over the concentration range of 50.418-5062.063 ng/mL. The lower limit of quantification (LLOQ) was 50.418 ng/mL Sodium heparin (3.50%) used as an anticoagulant to prepare rabbit plasma and samples were maintained at 10°C in the auto sampler during the assay period. Inter and intraday batch precision and accuracy of the method were determined by using six quality control samples. Results: Average accuracies for the assay were 89 to 106%, inter and intra-day coefficients variation (CV) of < 9% and the recovery is 39.30% for theophylline and 57.00% for Phenacetin. Conclusion: Currently we are extensively using this method in our laboratory for quantitative analysis of theophylline in rabbit plasma samples and proved to be simple, accurate and precise.