Simultaneous Determination of in Total Seventeen Opium Alkaloids and Opioids in Blood and Urine by Fast Hplc-dad-fl Detection, after Solid-phase Extraction

A fast liquid chromatographic method with tandem diode array-fluorescence detection for the simultaneous determination of in total seventeen opium alkaloids and opioids is presented. Blank blood and urine samples (1 mL) were spiked with different concentrations of a standard mixture, as well as with the internal standard, butorphanol (200 ng/mL). After solid-phase extraction, based on weak cation exchange (CBA SPE columns), the extracts were examined by HPLC-DAD-FL. By using a ‘high-speed’ phenyl column (53 x 7.0 mm I.D., particle size 3μm) eluted with a gradient system (A : water/methanol (90 :10, v/v), B : methanol, both containing 25mM triethylammoniumformate (pHA = 4.5)) all compounds could be baseline separated within 12 minutes. Subsequently, the method was validated. With diode-array detection the following validation parameters were obtained, LOD: from 1.62 to 52.50 ng/mL; LOQ: 5.40 to 105.30 ng/mL; within-day reproducibility: CV% < 10.80 for all compounds at LOQ. For fluorescence detection these parameters ranged from 1.08 to 9.65 ng/mL for LOD, 4.99 to 31.86 ng/mL for LOQ, and CV% < 6.33 for all compounds at LOQ for within-day reproducibility. In addition accuracy (at 500ng/mL, DAD: CV% < 9.60, FL: CV% < 13.85), selectivity and regression analysis (using seven calibrator samples, ranging from 1ng/mL up to 1μg/mL, R > 0.95 for all compounds) also proved to fulfil analytical standard criteria. Finally, the method was successfully applied to real-time forensic cases in an effort to test the applicability of the analysis.