Milovanović Vesna, Cirić Biljana, Milenković Jasna, Kilibarda Vesna, Curcić Marijana, Vucinić Slavica, Antonijević Biljana
Agencija za hemikalije Republike Srbije, Beograd, Srbija.
Vojnosanit Pregl. 2012 Feb;69(2):141-6. doi: 10.2298/vsp1202141m.
BACKGROUND/AIM: 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.
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.
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.
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.
背景/目的：在毒理学中，唾液是用于确定药物滥用情况的一种替代样本。因此，本研究的目的是优化一种用于唾液样本制备的方法，以通过液相色谱 - 质谱联用（LC/MS）测定海洛因代谢物、吗啡和6 - 单乙酰吗啡（6 - mam）以及可待因，并将该方法应用于从患者采集的唾液样本。
采用氯仿和异丙醇（9 : 1；v/v）混合液对吗啡、可待因和6 - mam进行液/液萃取来制备唾液样本。提取物通过HPLC/MS技术进行分析：分离柱为Waters Spherisorb 5微米，ODS2，4.6×100毫米；流动相：醋酸铵：乙腈（80 : 20；v/v），流动相流速0.3毫升/分钟；质量检测范围：100 - 400 m/z。进行概率水平为0.05的回归和相关性分析。在通过试纸条检测尿液中含有“阿片类药物”的患者唾液样本中测定吗啡、可待因和6 - mam的浓度。
每种分析物质在0.1至1毫克/升的浓度范围内进行校准，相关系数R2>0.99。我们分别获得了吗啡、可待因和6 - mam的校准曲线：y = 385531x + 14584；y = 398036x + 31542；以及y = 524162x - 27105。吗啡和可待因测定的回收率为99%，而6 - mam的回收率为94%。所提出方法的检测限和定量限分别为0.01毫克/升和0.05毫克/升。海洛因使用者唾液中吗啡浓度在0.54至5.82毫克/升之间，可待因浓度在0.05至5.33之间，6 - mam浓度在0.01至0.68毫克/升之间。可待因和6 - mam浓度之间存在统计学上的显著相关性。
所提出的用于唾液中吗啡、可待因和6 - mam测定的HPLC/MS方法准确、简单、廉价，适用于海洛因滥用的常规分析和监测。
