Assays - Formoterol

Ultra-sensitive determination of Formoterol in human serum by high performance liquid chromatography and electrospray tandem mass spectrometry

Author: D.G. Mascher, K. Zech, R. Nave, K.M. Kubesch, H.J. Mascher
Publisher: J Chromatogr. B 2006, 830, 25-34

An analytical method was developed and validated to determine Formoterol in human serum in the range from 0.40 to 100.24 pg/ml by high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) due to the lack of efficient methods to determine very low levels of Formoterol in serum and plasma. Serum was diluted by water and mixed with the internal standard (d6-Formoterol). Formoterol and internal standard were extracted using a cation-exchange solid phase column (SCX-3). After eliminating endogenous serum constituents through washing steps with water and methanol, elution took place using methanol/ammonia. After evaporation of the elution liquid the residue was redissolved and analyzed by HPLC-MS/MS with electrospray ionisation (ESI) in positive mode. A gradient between 10 mM ammonium formate and acetonitrile was used. The inter-batch precision of the calibration standards range from 1.55 % to 9.01 %. The inter-batch accuracy of the calibration standards ranged from 93.37 % to 107.30 %. The lower limit of quantitation (LLOQ, 0.40 ng/ml) had a precision of 19.67 % and an accuracy of 96.8 %. Comparable results were obtained for quality control samples. Stability in human serum was given over three freeze/thaw cycles and 2 h at room temperature. Formoterol in human serum was stable for at least 6 months below -20°C. This method has been used widely for quantifying Formoterol after inhalation of 9 - 36 µg of the drug by volunteers. A cross validation with human plasma versus serum was performed after this method was successfully validated in human serum.