University Campus Bio-Medico, Via Álvaro del Portillo 21, 00128 Rome, Italy.
J Chromatogr A. 2011 Sep 16;1218(37):6476-82. doi: 10.1016/j.chroma.2011.07.036. Epub 2011 Jul 23.
The combined use of the state-of-the-art hybrid mass spectrometers together with high efficient liquid chromatography could surely be a useful tool for such a challenging task, as phospholipids (PLs) analysis. In this research, we used hydrophilic interaction liquid chromatography (150 mm×2.1 mm I.D., 2.7 μm d.p. partially porous column) to achieve the separation of major PLs classes in cow's and donkey's milk samples. Solid-phase extraction (SPE) was performed in order to pre-concentrate minor PLs from non polar lipids (triacylglycerols) and the recovery for the extraction method was assayed on a milk sample, fortified with 5 μg/mL of SM pure standard, and analyzed in triplicate. A value of 89.99% was calculated, with a coefficient of variation (CV%) of 1.93. A 70-min long stepwise gradient of water/acetonitrile afforded baseline separation of PLs classes, at 50 μL/min flow rate. Accurate detection by an ion trap-time of flight (IT-TOF) mass spectrometer (in both positive and negative ionization mode) allowed to fully characterize the distinctive phospholipid profile and fatty acid composition of cow's and donkey's milk, the latter being analyzed for the first time. Evaporative light scattering detection was further employed to attain the quantitative evaluation of major PLs classes identified, by the external calibration method using reference material solutions in the 5-200 μg/mL concentration range. Major difference between the two analyzed samples consisted in the total PLs amount, which in cow's milk was determined as over 20-fold higher than the donkey's.
将最先进的混合质谱仪与高效液相色谱法相结合，肯定可以成为分析磷脂（PLs）等具有挑战性任务的有用工具。在这项研究中，我们使用亲水相互作用液相色谱（150mm×2.1mm ID，2.7μm d.p.部分多孔柱）来实现牛和驴乳样品中主要 PLs 类别的分离。固相萃取（SPE）用于从非极性脂质（三酰基甘油）中预浓缩少量 PLs，并在牛奶样品中测定提取方法的回收率，该样品用 5μg/mL 的 SM 纯标准进行强化，并进行三倍分析。计算出回收率为 89.99%，变异系数（CV%）为 1.93。在 50μL/min 的流速下，水/乙腈的 70 分钟逐步梯度可实现 PLs 类别的基线分离。离子阱飞行时间（IT-TOF）质谱仪（正、负离子模式）的准确检测允许充分表征牛和驴乳的独特磷脂谱和脂肪酸组成，这是首次对驴乳进行分析。蒸发光散射检测进一步用于通过外部校准方法，使用 5-200μg/mL 浓度范围内的参考物质溶液对鉴定的主要 PLs 类别的定量评估。两种分析样品之间的主要区别在于总 PLs 含量，牛乳中的总 PLs 含量是驴乳的 20 多倍。
