Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska University Hospital, Stockholm, Sweden.
J Sep Sci. 2012 Feb;35(3):367-75. doi: 10.1002/jssc.201100739.
The aim of this work was to synthesize morphine-3-O-sulfate and morphine-6-O-sulfate for use as reference substances, and to determine the sulfate conjugates as possible heroin and morphine metabolites in plasma and urine by a validated LC-MS/MS method. Morphine-6-O-sulfate and morphine-3-O-sulfate were prepared as dihydrates from morphine hydrochloride, in overall yields of 41 and 39% with product purities of >99.5% and >98%, respectively. For bioanalysis, the chromatographic system consisted of a reversed-phase column and gradient elution. The tandem mass spectrometer was operated in the positive electrospray mode using selected reaction monitoring, of transition m/z 366.15 to 286.40. The measuring range was 5-500 ng/mL for morphine-3-O-sulfate and 4.5-454 ng/mL for morphine-6-O-sulfate in plasma. In urine, the measuring range was 50-5000 ng/mL for morphine-3-O-sulfate and 45.4-4544 ng/mL for morphine-6-O-sulfate. The intra-assay and total imprecision (coefficient of variation) was below 11% for both analytes in urine and plasma. Quantifiable levels of morphine-3-O-sulfate in authentic urine and plasma samples were found. Only one authentic urine sample contained a detectable level of morphine-6-O-sulfate, while no detectable morphine-6-O-sulfate was found in plasma samples.
本工作旨在合成吗啡-3-O-硫酸盐和吗啡-6-O-硫酸盐，作为参考物质，并通过验证的 LC-MS/MS 方法将硫酸结合物确定为可能的海洛因和吗啡代谢物在血浆和尿液中。吗啡-6-O-硫酸盐和吗啡-3-O-硫酸盐分别由盐酸吗啡二水合物制备，总收率分别为 41%和 39%，产物纯度均>99.5%和>98%。对于生物分析，色谱系统由反相柱和梯度洗脱组成。串联质谱仪在正电喷雾模式下操作，使用选择反应监测，转换 m/z 366.15 到 286.40。吗啡-3-O-硫酸盐在血浆中的测量范围为 5-500ng/mL，吗啡-6-O-硫酸盐的测量范围为 4.5-454ng/mL。在尿液中，吗啡-3-O-硫酸盐的测量范围为 50-5000ng/mL，吗啡-6-O-硫酸盐的测量范围为 45.4-4544ng/mL。两种分析物在尿液和血浆中的日内和总精密度（变异系数）均低于 11%。在真实尿液和血浆样本中发现了可定量水平的吗啡-3-O-硫酸盐。只有一个真实的尿液样本中含有可检测水平的吗啡-6-O-硫酸盐，而在血浆样本中未发现可检测的吗啡-6-O-硫酸盐。
