Development of a High Performance Liquid Chromatography with Coulometric Electrochemical Detector Method for the Analysis of Morphine in Human Plasma
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Abstract
A HPLC method using reversed-phase chromatography coupled with a coulometric electrochemical detector (ECD) was developed for the determination of morphine in human plasma. Hydromorphone was selected as an internal standard. The compounds were extracted using solid-phase extraction with C₁₈ cartridges and separated on a reversed-phase C¹⁸ column with the mobile phase consisting of 69% v/v phosphate buffer pH 2.2 (5 mM sodium phosphate monobasic and 0.7 mM sodium dodecyl sulfate) and 31% v/v acetonitrile. The working electrode of the ECD was set at +450 mV for analytical purposes. Under this condition, the limit of detection for morphine was 0.33 ng/mL, whereas the limit of quantitation was 1.88 ng/mL. The percent recovery, intra-day and inter-day variation of morphine determinations were in the percentage range of 85.07-93.41, 2.86-6.41 and 3.76-11.39, respectively. The frozen samples kept at -20°C proved to be stable for at least one month prior to extraction. Also, the repeated use of cartridges for extraction was found to generate reliable results.
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