title: [Determination of morphine, codeine and 6-monoacetylmorphine in saliva of substance-abuse patients using HPLC/MS methods].
abstract: OBJECTIVE
  Saliva represents an alternative specimen for substances abuse determination in toxicology. Hence, the aim of this study was to optimize a method for saliva specimen preparation for heroin metabolites, morphine and 6-monoacetylmorphine (6-mam), and codeine determination by liquid chromatography-mass spectrometry (LC/MS), and to apply this method on saliva samples taken from the patients.
  
  
  METHODS
  Saliva specimen was prepared using liqiud/liquid extraction of morphine, codeine and 6-mam by mixture of chloroform and isopropanol (9 : 1; v/v). Extracts were analysed by HPLC/MS technique: separation column Waters Spherisorb 5 microm, ODS2, 4.6 x 100 mm; mobile phase: ammonium acetate : acetonitile (80 : 20; v/v), mobile phase flow rate 0.3 mL/min; mass detection range: 100-400 m/z. Regression and correlation analyses were performed with the probalility level of 0.05. Concentrations of morphine, codeine and 6-mam were determined in saliva samples of the patients with "opiates" in urine identified by the test strips.
  
  
  RESULTS
  Calibration for each analysed substance was done in the concentration range from 0.1 to 1 mg/L and the coefficient of correlation was R2 > 0.99. We obtained following calibration curves: y = 385531x + 14584; y = 398036x + 31542; and y = 524162x - 27105, for morphine, codeine and 6-mam, respectively. Recovery for morphine and codeine determination was 99%, while for 6-mam it was 94%. Limits of detection and quantification of a proposed method were 0.01 mg/L and 0.05 mg/L, respectively. Concentration of morphine in the saliva of the heroin users ranged between 0.54 and 5.82 mg/L, concentration of codeine between 0.05 and 5.33, and 6-mam between 0.01 and 0.68 mg/L. A statistically significant correlation between codeine and 6-mam concentrations was obtained.
  
  
  CONCLUSIONS
  A proposed HPLC/MS method for morphine, codeine and 6-mam determination in saliva is accurate, simple, cheap and suitable for routine analysis and monitoring of heroin abuse.
authors.affiliation: Agencija za hemikalije Republike Srbije, Beograd, Srbija. vesna.milovanovic@gmail.com
authors.forename: Vesna
authors.initials: V
authors.lastname: Milovanović
authors.forename: Biljana
authors.initials: B
authors.lastname: Cirić
authors.forename: Jasna
authors.initials: J
authors.lastname: Milenković
authors.forename: Vesna
authors.initials: V
authors.lastname: Kilibarda
authors.forename: Marijana
authors.initials: M
authors.lastname: Curcić
authors.forename: Slavica
authors.initials: S
authors.lastname: Vucinić
authors.forename: Biljana
authors.initials: B
authors.lastname: Antonijević
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003061:Codeine / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D009020:Morphine / Q000032:analysis*; D009022:Morphine Derivatives / Q000032:analysis*; D012463:Saliva / Q000737:chemistry*; D015813:Substance Abuse Detection
chemical_list: D009022:Morphine Derivatives; D009020:Morphine; C026979:6-O-monoacetylmorphine; D003061:Codeine