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. Hydrophilic stationary phases are increasingly being applied for the analysis of highly polar analytes, this chemistry being investigated as a suitable alternative. A hydrophilic interaction 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.