title: Validation of high-performance liquid chromatography assay for quantification of formoterol in urine samples after inhalation using UV detection technique.
abstract: A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb (250 mm x 4.6 mm x 5 microm) analytical column maintained at 30 degrees C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r2=0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n=6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n=10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.
authors.affiliation: School of Pharmacy and Institute of Pharmaceutical Innovation, Bradford, BD7 1DP, UK.
authors.forename: D K
authors.initials: DK
authors.lastname: Nadarassan
authors.forename: H
authors.initials: H
authors.lastname: Chrystyn
authors.forename: B J
authors.initials: BJ
authors.lastname: Clark
authors.forename: K H
authors.initials: KH
authors.lastname: Assi
mesh_terms: D000280:Administration, Inhalation; D000319:Adrenergic beta-Antagonists / Q000008:administration & dosage / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004983:Ethanolamines / Q000008:administration & dosage / Q000652:urine*; D000068759:Formoterol Fumarate; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: D000319:Adrenergic beta-Antagonists; D004983:Ethanolamines; D000068759:Formoterol Fumarate