Lazzarino Giacomo, Longo Salvatore, Amorini Angela Maria, Di Pietro Valentina, D'Urso Serafina, Lazzarino Giuseppe, Belli Antonio, Tavazzi Barbara
Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Largo F. Vito 1, 00168 Rome, Italy.
Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Largo F. Vito 1, 00168 Rome, Italy; LTA-Biotech srl, Viale Don Orione, 3D, 95047 Paternò, CT, Italy.
J Chromatogr A. 2017 Dec 8;1527:43-52. doi: 10.1016/j.chroma.2017.10.053. Epub 2017 Oct 24.
Fat-soluble vitamins and antioxidants are of relevance in health and disease. Current methods to extract these compounds from biological fluids mainly need use of multi-steps and multi organic solvents. They are time-consuming and difficult to apply to treat simultaneously large sample number. We here describe a single-step, one solvent extraction of fat-soluble vitamins and antioxidants from biological fluids, and the chromatographic separation of all-trans-retinoic acid, 25-hydroxycholecalciferol, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8'-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, phylloquinone, lycopene, α-carotene, β-carotene and coenzyme Q. Extraction is obtained by adding one volume of biological fluid to two acetonitrile volumes, vortexing for 60s and incubating for 60min at 37°C under agitation. HPLC separation occurs in 30min using Hypersil C18, 100×4.6mm, 5μm particle size column, gradient from 70% methanol+30% HO to 100% acetonitrile, flow rate of 1.0ml/min and 37°C column temperature. Compounds are revealed using highly sensitive UV-VIS diode array detector. The HPLC method suitability was assessed in terms of sensitivity, reproducibility and recovery. Using the present extraction and chromatographic conditions we obtained values of the fat-soluble vitamins and antioxidants in serum from 50 healthy controls similar to those found in literature. Additionally, the profile of these compounds was also measured in seminal plasma from 20 healthy fertile donors. Results indicate that this simple, rapid and low cost sample processing is suitable to extract fat-soluble vitamins and antioxidants from biological fluids and can be applied in clinical and nutritional studies.
脂溶性维生素和抗氧化剂与健康和疾病相关。目前从生物流体中提取这些化合物的方法主要需要使用多步和多种有机溶剂。它们耗时且难以应用于同时处理大量样本。我们在此描述了一种从生物流体中一步提取脂溶性维生素和抗氧化剂的方法，以及全反式视黄酸、25-羟基胆钙化醇、全反式视黄醇、虾青素、叶黄素、玉米黄质、反式-β-阿朴-8'-胡萝卜醛、γ-生育酚、β-隐黄质、α-生育酚、叶绿醌、番茄红素、α-胡萝卜素、β-胡萝卜素和辅酶Q的色谱分离。提取方法是将一体积的生物流体加入两体积的乙腈中，涡旋60秒，并在37°C搅拌下孵育60分钟。使用粒径为5μm、规格为100×4.6mm的Hypersil C18柱，在30分钟内进行HPLC分离，梯度从70%甲醇+30%水到100%乙腈，流速为1.0ml/min，柱温为37°C。使用高灵敏度紫外可见二极管阵列检测器检测化合物。从灵敏度、重现性和回收率方面评估了HPLC方法的适用性。使用目前的提取和色谱条件，我们获得了50名健康对照者血清中脂溶性维生素和抗氧化剂的值，与文献中报道的值相似。此外，还测量了20名健康有生育能力供体的精浆中这些化合物的谱图。结果表明，这种简单、快速且低成本的样品处理方法适用于从生物流体中提取脂溶性维生素和抗氧化剂，可应用于临床和营养研究。
