Analysis of naltrexone and its metabolite 6-beta-naltrexol in serum with high-performance liquid chromatography
© Heinälä et al.; licensee BioMed Central Ltd. 2012
Received: 3 January 2012
Accepted: 2 August 2012
Published: 15 August 2012
Naltrexone has been proven to be an effective treatment option for the treatment of alcohol dependency. In this article we introduce a reliable and simple method developed for the simultaneous determination of naltrexone and 6-β-naltrexol in human serum by using high-performance liquid chromatography (HPLC).
Liquid-liquid extraction with butyl acetate from basic solutions (pH 9) was chosen for extraction with nalorphine as an internal standard (IS). Analytes were back-extracted from organic solvent into perchloric acid. The acid extract was chromatographed by HPLC with a reverse-phase ODS-column and electrochemical detector. The mobile phase was a NaH2PO4-solution with acetonitrile as an organic modifier and octanesulphonic acid and tetraethylammonium hydrogen sulphate as ion-pair reagents. The recovery of the extraction method was 48% for naltrexone and 75% for 6-β-naltrexol. The limit of quantification was 5.0 ng/ml for naltrexone and 1.0 ng/ml for 6-β-naltrexol. The analysed concentrations of naltrexone differed from the theoretic concentrations by 0.7 to 2.3% and those of 6-β-naltrexol by 2.6%. The relative standard deviation of within-day assay was from 0.9 to 5.7% for naltrexone and from 0.8 to 4.2% for 6-β-naltrexol; for the between-day assay it was 5.7% and 4.2%, respectively.
Our results indicate that the developed method is suitable for determination of naltrexone and 6-β-naltrexol in human serum.
KeywordsNaltrexone 6-β-naltrexol High-performance liquid chromatography Alcoholism
Several methods for therapeutic drug monitoring of naltrexone and 6-β-naltrexol have been developed over the years. These include gas chromatography with electron capture detection (GC-ECD) [13, 16], high-performance liquid chromatography with electrochemical detection (HPLC-EC) [17, 18], gas chromatography–mass spectro-metry (GC-MS) [19, 20], and gas chromatography-tandem mass spectrometry (GC-MS-MS) . Recently Brünen et al.  introduced two novel methods, HPLC/UV (high-performance liquid chromatography and UV spectrophotometric detection) and LC-MS/MS (liquid-chromatographic method coupled with tandem mass spectrometry), for the determination of naltrexone and 6-β-naltrexol. The methods developed by Brünen et al.  have several benefits, such as better automatization and higher sensitivity, as compared to other methods developed for the simultaneous quantitative determination of naltrexone and 6-β-naltrexol in biological fluids.
With therapeutic drug monitoring the treatment outcomes of drugs can be significantly improved. It has been suggested that therapeutic drug monitoring should be a standard protocol while using naltrexone for the treatment of substance abuse . Thus we aimed to develop even more efficient analysis method for determi-nation of naltrexone and 6-β-naltrexol in human serum than has been earlier developed. To achieve this goal we used liquid-liquid extraction and subsequent analysis by HPLC with electroche-mical detection for determination of naltrexone and 6-β-naltrexol in human serum.
Materials and methods
The naltrexone (Dupont Merck) and 6-β-naltrexol (NIDA) stock solutions were prepared in methanol to give concentrations of 1 mg/ml of free base. These solutions were used to prepare calibration curves covering the range from 0 to 1000 ng/ml. All calibration standards, blanks and quality control samples, were prepared in drug-free cattle serum. All buffers were prepared with deionized water.
Samples from 87 (23 women and 64 men) alcoholics (DSM-IV) were taken. The alcoholics were participating in a double blind, placebo-controlled, prospective clinical trial evaluating naltrexone in the treatment of alcohol dependence. The main inclusion criteria were: age between 18–65, alcohol dependency (DSM-IV and ICD-10). Naltrexone (naltrexone HCl, ReVia®) was taken daily 50 mg orally every morning for a three-month period. The blood sample was collected approximately 2–4 h after the medication consumption. Fasting serum samples were collected by vein puncture at weeks 0, 2, and 8, and allowed to clot, and centrifuged (1000 g, 10 min, at room temperature). All samples were stored at −70°C until analysis.
1 ml of serum was extracted for all standard, quality control, and unknown clinical samples. The pH of the sample was adjusted to pH 9 by 0.5 M Na2HPO4⋅2H2O. The sample was extracted with 5 ml of butyl acetate with nalorphine (1 μg / 100 ml) as the internal standard. After vortex-mixing (30 s), the sample was centrifuged, and the upper butyl acetate layer was transferred to a clean test tube and back-extracted into 150 μl of 0.1 M HClO4. After vortex-mixing (30 s) and centrifugation, the upper butyl acetate phase was thrown out. The acid phase was transferred to the autosampler vial, and 25 μl of the acid phase was injected into the HPLC.
A Hewlett-Packard 1090 Series II high-performance liquid chromatography with an Esa Coulochem 5100A electrochemical coulometric detector (potentials: Detector 1 + 0.2 V, Detector 2 + 0.5 V) was used. The mobile phase was acetonitrile-potassium dihydrogen phosphate (19 mM) (10:45, v/v) with 1-octanesulphonic acid (5 mM) and tetraethylammonium hydrogen sulphate (5 mM) as ion-pair reagents. Chromatographic sepa-ration was achieved using an ODS Hypersil reverse-phase column (Hewlett Packard) with a length of 125 mm, an i.d. of 4 mm, and a particle size of 5 μm. The mobile phase was pumped at a flow-rate of 1.2 ml/min.
The identification of drugs was based on retention times, which were checked with calibration standards before each run. An internal standard method with linear one-point calibration based on peak heights was used for quantification. Reliability of the method was tested for linearity, accuracy, and within-day and between-day assay precision. The suitability for determination of naltrexone and its major metabolite was tested by analysing serum samples from patients participated in alcoholism treatment with naltrexone.
Results and discussion
The linearity range, mean correlation coefficient (r 2 ), and limit of quantification(LQ) with RSD-value for naltrexone and 6-β-naltrexol
Linearity range (ng/ml)
Average concentrations found, accuracy and within-day assay precision for naltrexone and 6-β-naltrexol
Concentration added (ng/ml)
Concentration found (ng/ml)
Between-day precision of the assay was determined by analysing the same serum standard 15 times in 30 days. A serum standard, spiked with 100 ng/ml of naltrexone and 6-β-naltrexol, was stored at −20°C. The average concentrations found were 98.8 and 98.9 ng/ml, and between-day assay precision was 5.7% and 4.2% for naltrexone and 6-β-naltrexol, respectively. Small changes in laboratory conditions (room temperature, atmospheric moisture) had no effect on the results. Retention times seemed to increase slightly because of contamination within the chromatographic system. This had, however, no effect on results because the retention times were checked with calibration standards before each determination. Although the extraction recovery was relatively low, the accuracy and the precision were good. The specific stability validation was not performed, but the between-day precision showed that the serum standard samples were stable for 30 days at −20°C. The clinical serum samples were stored at −70°C.
On the basis of validation results, the developed method was found suitable for determination of naltrexone and 6-β-naltrexol in human serum. The within-day and between-day assay precision was acceptable for all tested concentration levels. The developed method was used for determination of naltrexone and 6-β-naltrexol in serum samples from patients participating in naltrexone treatment of alcoholism. Analysed serum samples were collected after two and eight weeks of continuous naltrexone treatment. Concentrations of naltrexone and its major metabolite in serum samples varied greatly between patients. Naltrexone concentrations in serum were 0–70 ng/ml after two weeks and 0–19 ng/ml after eight weeks of continuous naltrexone treatment. 6-β-naltrexol concentrations were 15–136 ng/ml and 3–163 ng/ml after two and eight weeks, respectively. 6-β-naltrexol seemed to be the more reliable compound to monitor because some unidentified compound was found to interfere with the quantification of naltrexone. The laboratory verification demonstrated the tests were accurate in showing the levels of naltrexone and naltrexol in serum samples, but high variability was seen in the results from patients reporting that they were taking naltrexone. The reasons for the variability in practice remain to be determined but could have an influence on the efficacy of the treatment.
High-performance liquid chromatography
Gas chromatography with electron capture detection
High-performance liquid chromatography with electrochemical detection
Gas chromatography-mass spectrometry
Gas chromatography-tandem mass spectrometry
Relative standard deviations.
- Litten RZ, Allen JP: Advances in development of medications for alcoholism treatment. Psychopharmacology. 1998, 139: 20-33. 10.1007/s002130050686.PubMedView Article
- Volpicelli JR, Alterman AI, Hayashida M, O’Brien CP: Naltrexone in the treatment of alcohol dependence. Arch Gen Psychiatry. 1992, 49: 876-879. 10.1001/archpsyc.1992.01820110040006.PubMedView Article
- O’Malley SS: Opioid antagonists in the treatment of alcohol dependence: Clinical efficacy and presentation of relapse. Alcohol Alcohol. 1996, 1: 77-81.View Article
- Anton RF, O’Malley SS, Ciraulo DC, Cisler RA, Couper D, Donovan DM, Gastfriend DR, Hosking JD, Johnson BA, LoCastro JS, Longabaugh R, Mason BJ, Mattson ME, Miller WR, Pettinati HM, Randall CL, Swift R, Weiss RD, Williams LD, Zweben AZ, for the COMBINE Study Research Group: Combined pharmacotherapies and behavioral interventions for alcohol dependence: The COMBINE Study: A Randomized Controlled Trial. JAMA. 2006, 295: 2003-2017. 10.1001/jama.295.17.2003.PubMedView Article
- Renault PF: Treatment of heroin-dependent persons with antagonists: Current status. Bull Narc. 1978, 30: 21-29.PubMed
- Krupitsky E, Zvartau E, Masalov D, Tsoy M, Burakov A, Egorova V, Didenko T, Romanova T, Ivanova E, Bespalov A, Verbitskaya EV, Neznanov NG, Grinenko AY, O'Brien CP, Woody GE: Naltrexone with or without fluoxetine for preventing relapse to heroin addiction in St. Petersburg, Russia. J Subst Abuse Treat. 2006, 31: 319-328. 10.1016/j.jsat.2006.05.005.PubMedView Article
- Crabtree BL: Review of naltrexone, a long-acting opiate antagonist. Clinical Pharmacology. 1984, 3: 273-280.
- O’Malley S, Jaffe AJ, Chang G, Schottenfeld RS, Meyer RE, Rounsaville B: Naltrexone and coping skills therapy for alcohol dependence. Arch Gen Psychiatry. 1992, 49: 881-887. 10.1001/archpsyc.1992.01820110045007.PubMedView Article
- Volpicelli JR, Rhines KC, Rhines JS, Volpicelli LA, Alterman AI, O’Brien CP: Naltrexone and alcohol dependence: role of subject compliance. Arch Gen Psychiatry. 1997, 54: 737-742. 10.1001/archpsyc.1997.01830200071010.PubMedView Article
- Therapeutic Drugs 2: Therapeutic Drugs 2. 1993, Churchill Livingstone, London
- Wall ME, Brine DR, Perez-Reyes M: The metabolism of naltrexone in man. Naltrexone Research Monograph. 1980, 28: 105-131.
- Wall ME, Brine DR, Perez-Reyes M: Metabolism and disposition of naltrexone in man after oral and intravenous administration. Drug Metab Dispos. 1981, 9: 369-375.PubMed
- Verebey K, De Pace A, Jukofsky D, Volavka JV, Mule SJ: Quantitative determination of 2-hydroxy-3-methoxy-6-beta-naltrexol (HMN), naltrexone, and 6-beta-naltrexol in human plasma, red blood cells, saliva and urine by gas liquid chromatography. J Anal Toxicol. 1980, 4: 33-37.PubMedView Article
- McCaul ME, Wand GS, Sullivan J, Mummford G, Quigley J: Beta-naltrexol level predicts alcohol relapse. Alcohol Clin Exp Res. 1997, 21: 32A-
- Bertolotti M, Ferrari A, Vitale G, Stefani M, Trenti T, Loria P: Effect of liver cirrhosis on the systemic availability of naltrexone in humans. J Hepatol. 1997, 27: 505-511. 10.1016/S0168-8278(97)80355-8.PubMedView Article
- Reuning RH, Ashcraft SB, Morrison BE: An electron-capture gas chromatographic assay for naltrexone in biological fluids. Naltrexone Research Monograph. 1980, 28: 25-35.
- Davidson AF, Emm TA, Pieniaszek HJ: Determination of naltrexone and its major metabolite, 6-β-naltrexol, in human plasma using liquid chromatography with electrochemical detection. J Pharm Biomed Anal. 1996, 14: 1717-1725. 10.1016/0731-7085(96)01794-3.PubMedView Article
- Zuccaro P, Altieri I, Betto P, Pacifici R, Ricciarello G: Determination of naltrexone and 6-β -naltrexol in plasma by high-performance liquid chromatography with coulometric detection. J Chromatogr. 1991, 567: 485-490. 10.1016/0378-4347(91)80156-7.PubMedView Article
- Monti KM, Foltz RL, Chinn DM: Analysis of naltrexone and 6-β-naltrexol in plasma and urine by gas chromatography/negative ion chemical ionization mass spectrometry. J Anal Toxicol. 1991, 15: 136-140.PubMedView Article
- Huang W, Moody DE, Foltz RL: Determination of naltrexone and 6-β-naltrexol in human plasma by solid-phase extraction and gas chromatography- negative ion chemical ionization-mass spectrometry. J Anal Toxicol. 1997, 21: 252-257.PubMedView Article
- Nelson CC, Fraser MD, Wilfahrt JK, Foltz RL: Gas chromatography/ tandem mass spectrometry measurement of Δ9-tetrahydrocannabinol, naltrexone and their active metabolites in plasma. Ther Drug Monit. 1993, 15: 557-562. 10.1097/00007691-199312000-00019.PubMedView Article
- Brünen S, Krüger R, Finger S, Korf F, Kiefer F, Wiedemann K, Lackner KJ, Hiemke C: Determination of naltrexone and 6beta-naltrexol in human blood: comparison of high-performance liquid chromatography with spectrophotometric and tandem-mass-spectrometric detection. Anal Bioanal Chem. 2010, 396 (3): 1249-1257. 10.1007/s00216-009-3301-z.PubMedView Article
- Brünen S, Vincent PD, Baumann P, Hiemke C, Havemann-Reinecke U: Therapeutic drug monitoring for drugs used in the treatment of substance-related disorders: literature review using a therapeutic drug monitoring appropriateness rating scale. Ther Drug Monit. 2011, 33 (5): 561-572.PubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.