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

• Published in: Biomedical chromatography 2010-10, Vol.24 (10), p.1113-1119
• Main Authors: Hotha, Kishore Kumar, Ravindranath, L. K., Veera, K. N. Jaya, Kumar, K. Kishore, Reddy, Y. Ramachandra, Jagadeesh, B., Rao, D. V. Narayana, Bharathi, D. Vijaya, Mullangi, Ramesh
• Format: Article
• Language: English
• Subjects: 10-hydroxynortriptyline; Area Under Curve; Carbamazepine - analysis; Carbamazepine - chemistry; Chromatography, Liquid - methods; Drug Stability; human plasma; Humans; LC-MS/MS; Linear Models; Male; method validation; nortriptyline; Nortriptyline - analogs & derivatives; Nortriptyline - blood; Nortriptyline - chemistry; Nortriptyline - pharmacokinetics; pharmacokinetics; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.1413

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 C18 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. Copyright © 2010 John Wiley & Sons, Ltd.