Research Article Hydrophilic stationary phases: A practical approach for the co-analysis of compounds with varying polarity in biological matrices The aim was to simultaneously extract, separate and detect not only the opioid methadone and its primary metabolites (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline), but also creatinine from urine, plasma and fingerprints. Creatinine is highly polar and analysis by RP chromatography using conventional stationary phases such as silica-bonded C8 and C18 is unsuitable. Hydro- philic stationary phases are increasingly being applied for the analysis of highly polar analytes, this chemistry being investigated as a suitable alternative. A hydrophilic inter- action liquid chromatography phase column successfully retained creatinine and permitted the co-analysis of methadone and its metabolites by LC-MS/MS. Prior to analysis, an extraction protocol for urine and plasma was required but for fingerprint deposits this was not necessary. Alteration of sample pH, necessary to extract methadone, and its metabolites led to difficulties associated with the extraction of creatinine. This problem was addressed by first performing an SPE incorporating a hydrophilic interaction phase to extract creatinine, and the eluent then combined with the opioid extract from a mixed-mode cation exchange phase. The assay for creatinine, methadone and its primary phase I metabolites met validation criteria. LC-MS/MS analysis of creatinine and drug compounds together offers considerable advantages over traditional approaches that necessitate the quantification of creatinine using spectrophotometric approaches. Keywords: Creatinine / HILIC / LC-MS/MS / Opioids / SPE DOI 10.1002/jssc.200900727 1 Introduction Ongoing research comparing the distribution of drugs in an alternative biological matrix (sweat), using the opioid methadone (MTD) as a model drug, necessitated the quantification of the drug and its major metabolites in blood and urine. MTD is usually prescribed as a therapy to heroin addicts, the drug being administered daily to achieve a steady-state concentration in plasma/serum [1]. In sweat and urine its concentration will fluctuate, nonetheless, due to considerable variation in excretory volume. To compen- sate for the varying degrees of dilution of the target analytes between biological matrices, creatinine was also targeted. The objective was to develop an SPE and LC-MS/MS method that could quantify these analytes in the same assay, which presented a challenge as creatinine is not retained on conventional RP materials (C18 and C8). The use of mixed- mode cation-exchange (MCX) did not address the problem, as efficient elution could not be achieved using an aqueous solution of sodium chloride, and using a dilute ammonium hydroxide solution was ineffective as basic conditions cause the breaking of the ring and dehydration resulting in conversion to creatine. To overcome this problem, the application of hydrophilic interaction liquid chromatogra- phy (HILIC) was investigated, together with HILIC SPE, for sample clean-up. The major metabolites of MTD present in blood and urine are 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) (structures are shown as part of Fig. 1). Creatinine is a metabolic product of creatine (N-methyl-N-guanylgly- cine) and it is also produced non-enzymatically by sponta- neous cyclisation of creatine in urine under acidic conditions (urine typically has a pH around 6, although its reference interval extends from 4.5 to 8.2 under extreme Edward Goucher 1 Andrew Kicman 1 Kim Wolff 2 Norman Smith 1 Sue Jickells 1,Ã 1 Department of Forensic Science & Drug Monitoring, Pharmaceutical Science Research Division, King’s College London, London, UK 2 Insititute of Psychiatry, King’s College London, London, UK Received November 9, 2009 Revised December 9, 2009 Accepted December 23, 2009 Ã Current address: University of East Anglia, Norwich, UK Abbreviations: EDDP, 2-ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine; EMDP, 2-ethyl-5-methyl-3,3-diphenyl- 1-pyrroline; HILIC, hydrophilic interaction liquid chromatography; MCX, mixed-mode cation exchange; MTD, methadone; QC, quality control; RE, relative error; SRM, selective reaction monitoring Correspondence: Edward Goucher, Department of Forensic Science & Drug Monitoring, Pharmaceutical Science Research Division, King’s College London, London, SE1 9NH, UK E-mail: edward.goucher@kcl.ac.uk Fax:144-2078-484980 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2010, 33, 955–965 955