Development of a High Performance Liquid Chromatography with Coulometric Electrochemical Detector Method for the Analysis of Morphine in Human Plasma

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Thanee Tessiri
Viroj Sumyai
Suvatana Chulavatnatol
Poj Kulvanich
Khanittha Tontisirin
Thaned Pongjanyakul
Sirikul Aumpon
Verawan Uchaipichat
Detpon Preechagoon


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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Ary K, Róna K. LC determination of morphine and morphine glucuronides in human plasma by coulometric and UV detection. J Pharm Biomed Anal 2001; 26: 179-187.

Bouquillon AI, Freeman D, Moulin DE. Simultaneous solid-phase extraction and chromatographic analysis of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochemical detection. J Chromatogr Biomed App 1992;577 (2): 354-357.

Bourget†P, Lesne-Hulin A, Quinquis-Desmaris V. Study of the bioequivalence of two controlled-release formulations of morphine. Int J Clin Pharmacol Ther 1995; 33(11): 588-594.

Drake J, Kirkpatrick CT, Aliyar CA, et al. Effect of food on the comparative pharmacokinetics of modified-release morphine tablet formulations: Oramorph SR and MST. Continous Br J Clin Pharmacol 1996; 42(5): 645-647.

Gerostamoulos J, Crump K, McIntyre IM, et al. Simultaneous determination of 6-monoacety-lmorphine, morphine and codeine in urineusing high-performance liquid chromatography with combined ultraviolet and electrochemical detection. J Chromatogr 1993; 617: 152-156.

Glare PA, Walsh TD, Pippenger EE. A simple, rapid method for the simultaneous determination of morphine and its principal metabolites in plasma using high-performance liquid chromatography and fluorometric detection. Ther Drug Monit1991; 13: 226-232.

Gourlay GK, Cherry DA, Onley MM, et al. Pharmacokinetics and pharmacodynamics of twenty-four-hourly Kapanol compared totwelve-hourly MS Contin in the treatment of severe cancer pain. Pain 1997; 69: 295-302.

Kudo K, Ishida T, Nishida N, et al. Simple and sensitive determination of free and total morphine in human liver and kidney using gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed LifeSci 2006; 830(2): 359-363.

Liaw WJ, Ho ST, Wang JJ, et al. Determination of morphine by high-performance liquid chromatography with electrochemical detection: application to human and rabbit pharmacokinetic studies. J Chromatogr B Biomed Sci App 1998; 714: 237-245.

Liu Y, Bilfinger TV, Stefano GG. A rapid and sensitive quantitation method of endogenous morphine in human plasma. Life Sci 1997; 60: 237-243.

McQuay HJ, Carroll D, Faura CC, et al. Oral morphine in cancer pain: influences on morphine and metabolite concentration. Clin Pharmacol Ther1990; 48: 236-244.

Meadway C, George S, Braithwaite R. A rapid GC-MS method for the determination of dihydrocodeine, codeine, norcodeine, morphine, normorphine and 6-MAM in urine. Forensic Sci Int 2002; 127(1-2): 136-141.

Meatherall R. GC-MS quantitation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphonein blood. J Anal Toxicol 2005; 29(5):301-308.

Meng QC, Cepeda MS, Tom Kramer, et al. High-performance liquid chromatographic determination of morphine and its 3- and6-glucuronide metabolites by two-stepsolid-phase extraction. J Chromatogr B 2000;742: 115-123.

Projean D, Minh TT, and Ducharme J. Rapid andsimple method to determine morphine and its metabolites in rat plasma by liquid chroma-tography-mass spectrometry. J Chromatogr BA nalyt Technol Biomed Life Sci 2003; 787(2):243-253.

Shoup RE. Liquid chromatography: electrochemistry. In: Horvnth, C. (ed). High-performance liquid chromatography: advances and perspectives, Vol 4, Orlando: Academic Press; 1986.91-194.

Wright AWE, Watt JA, Kenned M, et al. Quantitation of morphine, morphine-3-glucuronide, andmorphine-6-glucuronide in plasma and cerebrospinal fluid using solid-phase extraction and high-performance liquid chromatography with electrochemical detection. Ther Drug Monit1994; 16: 200-208.