title: Ultra-sensitive determination of Formoterol in human serum by high performance liquid chromatography and electrospray tandem mass spectrometry.
abstract: 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 ranged 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 pg/mL) had a precision of 19.67% and an accuracy of 96.78%. Comparable results were obtained for quality control samples. Stability in human serum was given over three freeze/thaw cycles and 2h at room temperature. Formoterol in human serum was stable for at least 6 months below -20 degrees C. This method has been used widely for quantifying Formoterol after inhalation of 9-36 microg of the drug by volunteers. A cross validation with human plasma versus serum was performed after this method was successfully validated in human serum.
authors.affiliation: Pharm-analyt Labor GmbH, Baden, Austria.
authors.forename: Daniel Gerhard
authors.initials: DG
authors.lastname: Mascher
authors.forename: Karl
authors.initials: K
authors.lastname: Zech
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Nave
authors.forename: Klaus Michael
authors.initials: KM
authors.lastname: Kubesch
authors.forename: Hermann Josef
authors.initials: HJ
authors.lastname: Mascher
mesh_terms: D000318:Adrenergic beta-Agonists / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004983:Ethanolamines / Q000097:blood* / Q000493:pharmacokinetics; D000068759:Formoterol Fumarate; D006801:Humans; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000318:Adrenergic beta-Agonists; D004983:Ethanolamines; D000068759:Formoterol Fumarate