title: Development and validation of a bioanalytical method for the simultaneous determination of heroin, its main metabolites, naloxone and naltrexone by LC-MS/MS in human plasma samples: Application to a clinical trial of oral administration of a heroin/naloxone formulation.
abstract: A bioanalytical method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for simultaneous quantification of heroin, its main metabolites and naloxone. In addition, naltrexone was detected qualitatively. This method was used to analyse human plasma samples from a clinical trial after oral administration of a heroin/naloxone formulation in healthy volunteers. O-methylcodeine was used as an internal standard. Samples were kept in an ice-bath during their processing to minimize the degradation of heroin. A short methodology based on protein precipitation with methanol was used for sample preparation. After protein precipitation, only the addition of a formic acid solution was needed to elute heroin, 6-monoacetylmorphine, morphine, naloxone and naltrexone. Morphine metabolites were evaporated to dryness and reconstituted in a formic acid solution. Chromatographic separation was achieved at 35 °C on an X-Bridge Phenyl column (150 × 4.6 mm, 5 μm) using a gradient elution with a mobile phase of ammonium formate buffer at pH 3.0 and formic acid in acetonitrile. The run time was 8 min. The analytes were monitored using a triple quadrupole mass spectrometer with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The method was found to be linear in a concentration range of 10-2000 ng/mL for M3G and 10-1000 ng/mL for the rest of compounds. Quality controls showed accurate values between -3.6% and 4.0% and intra- and inter-day precisions were below 11.5% for all analytes. The overall recoveries were approximately 100% for all analytes including the internal standard. A rapid, specific, precise and simple method was developed for the determination of heroin, its metabolites, naloxone and naltrexone in human plasma. This method was successfully applied to a clinical trial in 12 healthy volunteers.
authors.affiliation: NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, Faculty of Pharmacy, Vitoria-Gasteiz, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Paseo de la Universidad, 7, 01006, Vitoria-Gasteiz, Spain. Electronic address: raquel.moreno@ehu.es.
authors.forename: Raquel
authors.initials: R
authors.lastname: Moreno-Vicente
authors.affiliation: Pharmaceutical Development Department, Tecnalia Research & Innovation Foundation, Vitoria-Gasteiz, Spain. Electronic address: zurine.fernandez@tecnalia.com.
authors.forename: Zuriñe
authors.initials: Z
authors.lastname: Fernández-Nieva
authors.affiliation: Pharmaceutical Development Department, Tecnalia Research & Innovation Foundation, Vitoria-Gasteiz, Spain. Electronic address: arantza.navarro@tecnalia.com.
authors.forename: Arantza
authors.initials: A
authors.lastname: Navarro
authors.affiliation: One-Dose Pharma, Barcelona, Spain. Electronic address: igasconcrespi@gmail.com.
authors.forename: Irene
authors.initials: I
authors.lastname: Gascón-Crespí
authors.affiliation: Human Pharmacology and Clinical Neurosciences Research Group, Hospital del Mar Medical Research Institute-IMIM, Parc de Salut Mar, Universidad Autónoma de Barcelona, Barcelona, Spain. Electronic address: mfarre@imim.es.
authors.forename: Magí
authors.initials: M
authors.lastname: Farré-Albaladejo
authors.affiliation: NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, Faculty of Pharmacy, Vitoria-Gasteiz, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Paseo de la Universidad, 7, 01006, Vitoria-Gasteiz, Spain. Electronic address: manoli.igartua@ehu.es.
authors.forename: Manuela
authors.initials: M
authors.lastname: Igartua
authors.affiliation: NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, Faculty of Pharmacy, Vitoria-Gasteiz, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Paseo de la Universidad, 7, 01006, Vitoria-Gasteiz, Spain. Electronic address: rosa.hernandez@ehu.es.
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Hernández
authors.affiliation: NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, Faculty of Pharmacy, Vitoria-Gasteiz, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Paseo de la Universidad, 7, 01006, Vitoria-Gasteiz, Spain. Electronic address: joseluis.pedraz@ehu.es.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Pedraz
mesh_terms: D000284:Administration, Oral; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D003061:Codeine / Q000032:analysis; D005561:Formates / Q000737:chemistry; D064368:Healthy Volunteers; D003932:Heroin / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D009020:Morphine / Q000737:chemistry; D009270:Naloxone / Q000097:blood*; D009271:Naltrexone / Q000097:blood*; D011786:Quality Control; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005561:Formates; C030544:formic acid; D009270:Naloxone; D009271:Naltrexone; D003932:Heroin; D009020:Morphine; D003061:Codeine
keywords: Heroin; LC–MS/MS; Pharmacokinetic; Plasma; Validation