Development of an Acid Hydrolysis Method for the Quantification of Total Urinary Opiates by LC-MS/MS

Alison Armitage

Alison Armitage and Craig Webster

Direct measurement of urinary opiates using tandem mass spectrometry is limited since it cannot detect conjugated forms of the drugs; consequently such direct methods would underestimate opiate concentrations. The aim of this project is to develop an electrospray tandem mass spectrometry method for the quantification of urinary total opiates, including conjugated forms. The approach is based on acid hydrolysis, followed by solvent extraction, to release free opiates from the conjugated forms. A standard urine specimen was prepared by spiking with opiates at concentrations corresponding to the UK workplace testing thresholds. 5 mL aliquots of this urine standard were mixed with morphine-D6 deuterated internal standard, followed by 1 mL of concentrated HCl. The samples were heated at 100 ºC for 30 minutes. After cooling, the samples were neutralised using potassium hydroxide. The opiates were extracted by the additions of 2g of a mixture of NaCl, NaHCO3 and Na2CO3 (5:5:1) and 2 mL of an extraction solvent containing dichloromethane, dichloroethane, heptane and propan-2-ol. After mixing and centrifugation, the top layer was removed and then evaporated to dryness. The samples were reconstituted with mobile phase (4 mmol/L ammonium acetate in 5% methanol). 5 μL were injected to an API 3200 mass spectrometer through an Onyx Monolithic €œSol-Gel€ C8 column. Preliminary results suggest that this approach of acid hydrolysis with solvent extraction prior to mass spectrometry could be used to quantify total opiates.