DETERMINATION OF MORPHINE IN HUMAN URINE BY A SIMPLE REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD WITH UV DETECTION
Abstract
A simple reverse-phase high-performance liquid chromatographic method has been developed for the determination of morphine in human urine using codeine as internal standard. After solid-phase extraction (SPE), the compounds were separated on a reversed-phase Acentis Express C18 column (150mm × 4.6mm, 2.7µm) equipped with a guard column (2.7 µm, 5 mm × 4.6 mm). The channel on the UV detector was configured to acquire data at 285 nm. The mobile phase was composed of acetonitrile-sodium acetate (pH 4; 0.01M) (10:90, v/v). The flow rate was 0.6 ml/min. Injection volume was 30 ml. The run time was set at 10 min. The retention time for morphine and codeine were approximately 3.4 min and 7.2 min, respectively. The calibration curve was linear over the concentration range of 150-2000 ng/ml with good linearity. Limit of detection (LOD) and quantification (LOQ) in urine were 50 and 150 ng/ml, respectively. Recovery was greater than 85%. The inter-day precision was from 9.27% to 12.53% and the intra-day precision ranged from 1.63% to 4.58%. The inter-day accuracy ranged 81.45% to 109.88% and intra-day accuracy ranged from 81.56% to 106.69%. The stability studies showed that morphine was stable up to one month in urine. This method was applied to determine morphine concentration in human urine for toxicology analysis.
Keywords:
Morphine, HPLC, urine, method development, validationDOI
https://doi.org/10.25004/IJPSDR.2013.050103References
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17. Larry AB, Lance CP, Mike T, Cecelia Q. Improved Gas Chromatography–Mass Spectrometry Method for Simultaneous Identification and Quantification of Opiates in Urine as Propionyl and Oxime Derivatives. Clin Chem 2001; 47 (1): 127 – 29.
18. Chee GL, Wan TS. Reproducible and high-speed separation of basic drugs by capillary zone electrophoresis. J Chromatogr 1993; 612(1):172-77.
19. Bogusz MJ, Maier RD, Schiwy-Bochat KH, Kohls U. Applicability of various brands of mixed-phase extraction columns for opiate extraction from blood and serum. J Chromatogr B Biomed Appl 1996; 683(2):177-88.
20. de Zeeuw RA. Drug screening in biological fluids: the need for a systemic approach. J Chrom B 1997; 689: 71-79.
21. Bakkali A, Corta E, Ciria JI, Berrueta LA, Gallo B, Vicente F. Solid-phase extraction with liquid chromatography and ultraviolet detection for the assay of antidepressant drugs in human plasma. Talanta 1999; 49(4):773-783.
22. Wang P, Stone JA, Chen KH, Gross SF, Haller CA, Wu AH. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol 2006; 30(8):570-75.
23. Jennison TA, Wozniak E, Nelson G, Urry FM. The quantitative conversion of morphine 3-beta-D glucuronide to morphine using beta-glucuronidase obtained from Patella vulgata as compared to acid hydrolysis. J Anal Toxicol 1993; 17(4):208-10.
24. Meyer VR. Practical high-performance liquid chromatography. 4th ed. New York: John Wiley & Sons; 2004.
2. World Health Organization. Cancer Pain Relief Program. Geneva: 1986.
3. Moeller MR, Mueller C. The detection of 6-monoacetylmorphine in urine, serum and hair by GUMS and RIA. Forensic Sc Int 1995; 70: 125-33.
4. Galloway JH, Ashford M, Marsh ID, Holden M, Forrest ARW. A method for the confirmation and identification of drugs of misuse in urine using solid phase extraction and gas-liquid chromatography with mass spectrometry. J Clin Pathol 1998; 51:326-29
5. Manfio JL, Santos VJ, Lanchote VL, Santos LM, Carmona MJ, Auler JO, Brum LJ, Donaduzzi CM, Santos SR. Development and validation of an HPLC/MS/MS method for the determination of sufentanil and morphine in human plasma. J AOAC Int 2011; 94(1):136-42.
6. Hofmann U, Seefried S, Schweizer E, Ebner T, Mikus G, Eichelbaum M. Highly sensitive gas chromatographic-tandem mass spectrometric method for the determination of morphine and codeine in serum and urine in the femtomolar range. J Chromatogr B Biomed Sci Appl 1999; 727(1-2):81-88.
7. Ruiz-Colón K, Martínez MA, Silva-Torres LA, Chavez-Arias C, Meléndez-Negrón M, Conte-Miller MS, Bloom-Oquendo J. Simultaneous determination of xylazine, free morphine, codeine, 6-acetylmorphine, cocaine and benzoylecgonine in postmortem blood by UPLC-MS-MS. J Anal Toxicol 2012; 36(5):319-26.
8. van der Slooten EP, van der Helm HJ. Comparison of the EMIT (enzyme multiplied immunoassay technique) opiate assay and a gas-chromatographic--mass-spectrometric determination of morphine and codeine in urine. Clin Chem 1976; 22(7):1110-11.
9. Wasels R, Belleville F. Gas chromatographic-mass spectrometric procedures used for the identification and determination of morphine, codeine and 6-monoacetylmorphine. J Chromatogr A 1994; 674(1-2): 225-34.
10. Schönberg L, Grobosch T, Lampe D, Kloft C. New screening method for basic compounds in urine by on-line extraction–high-performance liquid chromatography with photodiode-array detection. J Chrom A 2006; 1134 (1–2): 177–85.
11. Szkutnik-Fiedler D, Grześkowiak E, Gaca M, Borowicz M. HPLC-UV determination of morphine in human plasma and its application to the clinical study. Acta Pol Pharm 2011; 68(4):473-79.
12. Ferrara SD, Tedeschi L, Frison G, Castagna F. Solid-Phase Extraction and HPLC-UV Confirmation of Drugs of Abuse in Urine. J Anal Toxicol 1992; 16 (4): 217-22.
13. Colin H, Guiochon G. Introduction to reversed-phase high-performance liquid chromatography. J Chrom 1977; 141: 289-312.
14. Snyder LR, Glajch JL, Kirkland JJ. Practical HPLC method development. New York: John Wiley & Sons; 1988.
15. CDER. Guidance for industry: bioanalytical method validation, Centre for Drug Evaluation and Research, Food and Drug Administration; 2001. p 1-11.
16. Ruzilawati AB, Suhaimi AW, Imran A, Ismail Z, Gan SH. Method development and validation of repaglinide in human plasma by HPLC and its application to pharmacokinetics study. J Pharm Biomed Anal 2007; 43(5): 1831-35.
17. Larry AB, Lance CP, Mike T, Cecelia Q. Improved Gas Chromatography–Mass Spectrometry Method for Simultaneous Identification and Quantification of Opiates in Urine as Propionyl and Oxime Derivatives. Clin Chem 2001; 47 (1): 127 – 29.
18. Chee GL, Wan TS. Reproducible and high-speed separation of basic drugs by capillary zone electrophoresis. J Chromatogr 1993; 612(1):172-77.
19. Bogusz MJ, Maier RD, Schiwy-Bochat KH, Kohls U. Applicability of various brands of mixed-phase extraction columns for opiate extraction from blood and serum. J Chromatogr B Biomed Appl 1996; 683(2):177-88.
20. de Zeeuw RA. Drug screening in biological fluids: the need for a systemic approach. J Chrom B 1997; 689: 71-79.
21. Bakkali A, Corta E, Ciria JI, Berrueta LA, Gallo B, Vicente F. Solid-phase extraction with liquid chromatography and ultraviolet detection for the assay of antidepressant drugs in human plasma. Talanta 1999; 49(4):773-783.
22. Wang P, Stone JA, Chen KH, Gross SF, Haller CA, Wu AH. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol 2006; 30(8):570-75.
23. Jennison TA, Wozniak E, Nelson G, Urry FM. The quantitative conversion of morphine 3-beta-D glucuronide to morphine using beta-glucuronidase obtained from Patella vulgata as compared to acid hydrolysis. J Anal Toxicol 1993; 17(4):208-10.
24. Meyer VR. Practical high-performance liquid chromatography. 4th ed. New York: John Wiley & Sons; 2004.
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01-01-2013
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“DETERMINATION OF MORPHINE IN HUMAN URINE BY A SIMPLE REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD WITH UV DETECTION”. International Journal of Pharmaceutical Sciences and Drug Research, vol. 5, no. 1, Jan. 2013, pp. 18-22, https://doi.org/10.25004/IJPSDR.2013.050103.
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How to Cite
“DETERMINATION OF MORPHINE IN HUMAN URINE BY A SIMPLE REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD WITH UV DETECTION”. International Journal of Pharmaceutical Sciences and Drug Research, vol. 5, no. 1, Jan. 2013, pp. 18-22, https://doi.org/10.25004/IJPSDR.2013.050103.
