Liang H R, Takagaki T, Foltz R L, Bennett P
Tandem Labs, A Division of NWT Inc., Salt Lake City, UT, USA.
Rapid Commun Mass Spectrom. 2005;19(16):2284-94. doi: 10.1002/rcm.2055.
Attachment of anions to sorbitol and fructose has been shown to enhance sensitivity in both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) mass spectrometry. The post-column addition of CHCl3 produced Cl-adducts of sorbitol and fructose but their signals were suppressed due to the elevated background. Different chlorinated compounds and different additive methods were systematically investigated to form more abundant Cl-adduct precursor ions and deprotonated product ions. The major causes of the high background were explored and effective methods were developed to improve the signal-to-noise ratios and reproducibility. The compositions of mobile phase, percentages of organic modifiers (MeCN, MeOH and water), columns, oven temperature, flow rates and different gradients were investigated to separate sorbitol from fructose along with their isomers including glucose, galactose, mannose, sorbose, mannitol, and dulcitol. The optimized separation was achieved on a Luna 5 mu NH2 100A column (150 x 4.6 mm) using a mobile phase containing MeCN with 0.1% of CH2Cl2 and 50% MeOH in water at a flow rate of 800 microL/min and an oven temperature of 40 degrees C using a gradient liquid chromatography (LC) system. Human nerve tissue samples were extracted by protein precipitation followed by mixed-mode solid-phase extraction. The LC/ESI-MS/MS method produced higher peak intensities than LC/APCI-MS/MS. However, there were matrix effects from extracted tissues in LC/ESI-MS/MS but not in LC/APCI-MS/MS. Consequently, APCI proved to be the more effective method of ionization. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with linear ranges of 0.2-80 ng/mg for sorbitol and 1-400 ng/mg for fructose in human nerve tissues.
已证明阴离子与山梨醇和果糖的结合可增强电喷雾电离（ESI）和大气压化学电离（APCI）质谱中的灵敏度。柱后添加CHCl₃生成了山梨醇和果糖的Cl加合物，但由于背景升高，它们的信号受到抑制。系统研究了不同的氯化化合物和不同的添加方法，以形成更丰富的Cl加合物前体离子和去质子化产物离子。探究了高背景的主要原因，并开发了有效的方法来提高信噪比和重现性。研究了流动相的组成、有机改性剂（乙腈、甲醇和水）的百分比、色谱柱、柱温、流速和不同梯度，以分离山梨醇和果糖及其异构体，包括葡萄糖、半乳糖、甘露糖、山梨糖、甘露醇和卫矛醇。在Luna 5μm NH₂ 100A色谱柱（150×4.6 mm）上实现了优化分离，使用的流动相为含有0.1%二氯甲烷的乙腈和50%甲醇的水溶液，流速为800μL/min，柱温为40℃，采用梯度液相色谱（LC）系统。人神经组织样品通过蛋白质沉淀法提取，然后进行混合模式固相萃取。LC/ESI-MS/MS方法产生的峰强度高于LC/APCI-MS/MS。然而，LC/ESI-MS/MS中存在来自提取组织的基质效应，而LC/APCI-MS/MS中不存在。因此，APCI被证明是更有效的电离方法。然后对LC/APCI-MS/MS方法进行了全面验证，并成功应用于临床样品分析。使用以山梨醇-¹³C₆和果糖-¹³C₆作为替代分析物的校准曲线测定内源性山梨醇和果糖的浓度。该方法在人神经组织中提供了出色的批内和批间精密度和准确度，山梨醇的线性范围为0.2 - 80 ng/mg，果糖的线性范围为1 - 400 ng/mg。
