title: Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of nortriptyline and 10-hydroxynortriptyline in human plasma: application to a human pharmacokinetic study.
abstract: A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10-hydroxynortriptyline (OH-NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract NTP, OH-NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH-NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C(18) column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH-NTP. A linear response function was established for the range of concentrations 1.09-30.0 ng/mL (r > 0.998) for both NTP and OH-NTP. The intra- and inter-day precision values for NTP and OH-NTP met the acceptance as per FDA guidelines. NTP and OH-NTP were stable in a battery of stability studies, i.e. bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in humans.
authors.affiliation: Bioanalytical Department, Integrated Product Development, Dr Reddy's Laboratories Ltd, Bachupalli, Hyderabad-500 072, India.
authors.forename: Kishore Kumar
authors.initials: KK
authors.lastname: Hotha
authors.forename: L K
authors.initials: LK
authors.lastname: Ravindranath
authors.forename: K N Jaya
authors.initials: KN
authors.lastname: Veera
authors.forename: K Kishore
authors.initials: KK
authors.lastname: Kumar
authors.forename: Y Ramachandra
authors.initials: YR
authors.lastname: Reddy
authors.forename: B
authors.initials: B
authors.lastname: Jagadeesh
authors.forename: D V Narayana
authors.initials: DV
authors.lastname: Rao
authors.forename: D Vijaya
authors.initials: DV
authors.lastname: Bharathi
authors.forename: Ramesh
authors.initials: R
authors.lastname: Mullangi
mesh_terms: D019540:Area Under Curve; D002220:Carbamazepine / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D008297:Male; D009661:Nortriptyline / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C013567:10-hydroxynortriptyline; D002220:Carbamazepine; D009661:Nortriptyline