Journal of Chromatography B, 879 (2011) 17–24 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Determination of dexamethasone and dexamethasone sodium phosphate in human plasma and cochlear perilymph by liquid chromatography/tandem mass spectrometry Mei Zhang a,∗ , Grant A. Moore b , Berit P. Jensen a , Evan J. Begg a , Philip A. Bird c a Department of Medicine, University of Otago-Christchurch, Christchurch, New Zealand b Toxicology, Canterbury Health Laboratories, Christchurch, New Zealand c Department of Otolaryngology, Head and Neck Surgery, Christchurch Hospital, Christchurch, New Zealand article info Article history: Received 25 June 2010 Accepted 2 November 2010 Available online 10 November 2010 Keywords: Dexamethasone Dexamethasone sodium phosphate Cochlear perilymph Plasma LC–MS/MS abstract A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was developed for the determination of dexamethasone (Dex) and dexamethasone sodium phosphate (Dex SP) in plasma and human cochlear perilymph. After proteins were precipitated with a mixture of acetoni- trile and methanol, Dex, Dex SP and flumethasone, the internal standard, were resolved on a C18 column using gradient elution of 5 mM ammonium acetate and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.5–500 g/L (r > 0.99), bias was <±10%, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.5 g/L for both Dex and Dex SP. The assay has been used successfully in a clinical pharmacokinetics study of Dex and Dex SP in cochlear perilymph and plasma. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Systemic administration of glucocorticoids has been the tradi- tional method of treatment of a number of inner ear conditions including autoimmune inner ear disease, acute postmeningitic labyrinthitis, idiopathic sudden sensorineural hearing loss, and Cogan’s syndrome. Recently intratympanic (IT) delivery of glu- cocorticoids has become an accepted approach for treatment of some of these conditions. The advantages of IT administration include reducing systemic drug concentrations and side effects, and delivering higher doses to the inner ear when compared with systemic administration [1]. Dexamethasone (Dex) is a glu- cocorticoid that is used for IT delivery, and because of low water-solubility administered in the form of the water-soluble ester prodrug dexamethasone sodium phosphate (Dex SP). This prodrug is hydrolysed rapidly by phosphatases to its active form free Dex [2]. To date, no study has been published looking at the concentrations of Dex and Dex SP in cochlear perilymph and plasma after IT treatment versus IV treatment in humans. ∗ Corresponding author at: Clinical Pharmacology, Department of Medicine, Uni- versity of Otago-Christchurch, PO Box 4345, Christchurch, New Zealand. Tel.: +64 3 364 0640x89746; fax: +64 3 364 1003. E-mail address: mei.zhang@cdhb.govt.nz (M. Zhang). Pharmacokinetic studies of Dex and Dex SP in cochlear peri- lymph and plasma require a highly sensitive method to analyse the concentrations of Dex and Dex SP because of the extremely small volume of cochlear perilymph samples (∼20 l) available from patients during cochlear implantation, and the low Dex plasma concentration from the low therapeutic dose given by IT route. HPLC with tandem mass spectrometric detection (LC–MS/MS) has been demonstrated to provide high sensitivity for the quantitative determination of drugs and metabolites in biological fluids. Some LC–MS/MS methods for measuring Dex in biological samples have been reported [3–6], in which a relatively large volume of sample was required (>200 l). For Dex SP analysis, there is one published HPLC–UV–MS method that has been used to report the analysis of Dex SP in cochlear perilymph of guinea pigs [7], in which HPLC with UV detection was used for Dex SP quantification and MS for Dex SP identification. The limit of detection was 100 g/L. Two LC–MS/MS methods have been reported for the simultaneous determination of Dex and Dex SP in plasma or connective tissue for pharmacoki- netics studies [8,9]. The method for plasma [8] required 100 l of plasma, and protein precipitation by acetonitrile was used for sample preparation. The method for connective tissue [9] required 50 mg of tissue, which was homogenized with water. After centrifu- gation, the supernatant was filtered and the filtrate was injected into LC–MS/MS. Both methods were sensitive (0.5 ng/ml in plasma and 1 ng/g in connective tissue) but details of the methodology and validation were lacking. 1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2010.11.003