This project involved the separation of twenty Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) from equine urine, by Solid Phase Extraction (SPE) and detection by electron impact GCMS. Liquid-Liquid Extraction (LLE) has been used in the past but it does have disadvantages. LLE techniques involve large volumes of solvent that may be toxic and harmful, optimum conditions are not always attainable due to formation of emulsions, very polar substances may not extract well, and in particular it is labour intensive. By using SPE, the isolation method can be automated which will increase reproducibility of the extraction and also reduce labour intensity.
Before loading the NSAID spiked urine sample, the column is conditioned with methanol and then a buffer at pH 3.0. Isolute-Confirm HCX SPE columns were used (strong cation exchange/C8 mixed-mode, standard 130mg column) to extract the acidic drugs from 3mL of the spiked urine (adjusted to pH 3.0) loaded onto the column, followed by a wash with pH 3.0 buffer solution. The column is dried (centrifuged) and a fraction eluted with 5% acetone in dichloromethane collected.
The SPE eluate is then methylated (dimethyl sulphate, K2CO3 in acetone at 60°C for 1 hour). Methylation is necessary prior to GCMS analysis to convert the acidic target drugs to their methyl esters. Twenty drugs, namely, Ibufenac, Probenecid, Fenoprofen, Diflunisal, Naproxen, Indomethacin, Ethacrynic Acid, Diclofenac, Flufenamic Acid, Flunixin, Oxyphenbutazone, Phenylbutazone, Tolfenamic Acid, Meclofenamic Acid, Ketorolac, Butibufen, Mefenamic Acid, Ibuprofen, Tiaprofenic Acid and Carprofen were analysed on a Hewlett Packard (HP) 5890 Series 2 Gas Chromatogram and a HP 5989A Mass Spectrometer with a 12m column (HP Ultra 1-Crosslinked Methyl Silicone Gum, 0.2mm ID) and characterised by EI MS.
Recovery efficiency was assessed by comparisons with control samples which underwent identical workup but were kept in an aqueous matrix.