title: Development of a validated LC-MS/MS method for determination of doxofylline on rat dried blood spots and urine: application to pharmacokinetics.
abstract: A rapid and highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of doxofylline on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse phase C18 column (250 mm × 4.6 mm, 5 μm), using 20 mM ammonium acetate (pH adjusted to 3.5 with trifluoroacetic acid) and acetonitrile (75:25 v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 267 and m/z 195 for doxofylline and caffeine used as internal standard respectively. The calibration curve showed a good linearity in the concentration range of 1-5000 ng/mL. The effect of hematocrit on extraction of doxofylline from DBS was evaluated. The mean recoveries of doxofylline from DBS and urine were 93.46 and 89.86% respectively. The intra and inter-day precisions were less than 4.28% in DBS as well as urine. The limit of detection and quantification were 0.24 and 0.84 ng/mL in DBS and 0.28 and 1.00 ng/mL in urine samples respectively. The method was validated as per ICH guidelines and successfully applied to a pharmacokinetic study of doxofylline in rats.
authors.affiliation: Analytical Chemistry Division, Discovery Laboratory, Tarnaka, Hyderabad 500 607, India. Electronic address: rnrao@iict.res.in.
authors.forename: R Nageswara
authors.initials: RN
authors.lastname: Rao
authors.forename: K Guru
authors.initials: KG
authors.lastname: Prasad
authors.forename: Ch Gangu
authors.initials: ChG
authors.lastname: Naidu
authors.forename: Shaik
authors.initials: S
authors.lastname: Saida
authors.forename: Sachin B
authors.initials: SB
authors.lastname: Agwane
mesh_terms: D000818:Animals; D001993:Bronchodilator Agents / Q000097:blood* / Q000493:pharmacokinetics; D002853:Chromatography, Liquid / Q000379:methods*; D057230:Limit of Detection; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013806:Theophylline / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics
chemical_list: D001993:Bronchodilator Agents; D013806:Theophylline; C029797:doxofylline