Development and validation of a highly sensitive and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: Application to a bioequivalence study

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 868; no. 1; pp. 70 - 76
• Main Authors: Bharathi, D. Vijaya, Hotha, Kishore Kumar, Sagar, P.V.Vidya, Kumar, Sanagapati Sirish, Reddy, Pandu Ranga, Naidu, A., Mullangi, Ramesh
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.06.2008; Elsevier Science
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Liquid - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; Human plasma; Humans; Hydroxyitraconazole; Itraconazole; Itraconazole - analogs & derivatives; Itraconazole - blood; Itraconazole - pharmacokinetics; LC–MS/MS; Medical sciences; Method validation; Pharmacokinetics; Pharmacology. Drug treatments; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods; Therapeutic Equivalency; Human plasma; Method validation; Humans; Hydroxyitraconazole; Pharmacokinetics; LC–MS/MS; Itraconazole; Human; Validation; Biological fluid; Chemical analysis; Bioequivalence; Liquid chromatography; Electrospray; Blood plasma; LC-MS/MS; Antifungal agent; Simultaneous measurement; Triazole derivatives; Development; Quantitative analysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2008.04.029

A highly sensitive and specific LC–MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 μL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C 18 (50 mm × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5–263 ng/mL ( r > 0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.