单糖是维持生物体正常生理功能所必需的。在疾病状态下,体内代谢紊乱将不可避免地影响单糖的水平,这带来了单糖作为某些疾病的生物标志物的可能性。在这项研究中,开发并验证了一种同时测定10种单糖(葡萄糖,半乳糖,甘露糖,鼠李糖,岩藻糖,木糖,艾杜糖醛酸,葡萄糖醛酸,使用液相色谱-串联质谱法在SD大鼠血浆中的N-乙酰半乳糖胺和N-乙酰葡糖胺)。该方法使用1-苯基-3-甲基-5-吡唑啉酮(PMP)作为衍生试剂,大大提高了单糖的色谱保留率和电离效率。血浆样品蛋白沉淀后,将单糖和同位素内标衍生化,并进行液-液萃取以去除多余的PMP。为了实现几种异构体的基线分离,所得衍生物在Bridged乙基杂化物(BEH)苯基柱上使用梯度洗脱进行色谱分离,总运行时间为8分钟。鼠李糖在0.0100-5.00μg/mL范围内线性关系良好,岩藻糖0.0500-25.0μg/mL,木糖,艾杜糖醛酸,葡萄糖醛酸,N-乙酰半乳糖胺和N-乙酰葡糖胺,对于半乳糖,1.00-500μg/mL,甘露糖10.0-5000μg/mL,和50.0-25,000μg/mL的葡萄糖。并且替代基质样品和血浆样品的准确性和精密度验证满足所要求的标准。该方法已成功用于研究肝功能不全对大鼠单糖水平的影响。发现肝损伤大鼠血浆中葡萄糖醛酸的浓度异常升高。
Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100-5.00 μg/mL for rhamnose, 0.0500-25.0 μg/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00-500 μg/mL for galactose, 10.0-5000 μg/mL for mannose, and 50.0-25,000 μg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury.