Bisp Marianne R, Bor Mustafa Vakur, Heinsvig Else-Marie, Kall Morten A, Nexø Ebba
Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, DK-8000, Denmark.
Anal Biochem. 2002 Jun 1;305(1):82-9. doi: 10.1006/abio.2002.5638.
Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.
维生素B6的边缘性缺乏最近被认为与心血管疾病有关。因此，人们对在常规检验医学中定量检测这种维生素的合适且可靠的方法越来越感兴趣。我们开发了一种基于高效液相色谱（HPLC）的方法，该方法能够定量检测维生素B6的几种形式：吡哆醛5'-磷酸酯（PLP）、吡哆醛（PL）、吡哆胺5'-磷酸酯（PMP）、吡哆醇（PN）、吡哆胺（PM）以及降解产物4-吡哆酸（4-PA）。分离是使用C18（ODS）分析柱和离子对反相色谱法完成的。维生素B6的几种形式在含有1-辛烷磺酸和三乙胺（pH 2.16）的磷酸钾缓冲液中，用乙腈梯度（0.5 - 15%）洗脱。在使用含有1 g/L亚硫酸氢钠的磷酸盐缓冲液进行柱后衍生化后，用荧光检测器（激发波长328 nm，发射波长393 nm）测定这些维生素形式的浓度。通过在3个月的时间内分析6个浓度相关的血浆样本以及2个对照样本（未加标和加标了维生素形式的样本）来评估该检测方法的性能。在46分钟的分析运行中，HPLC方法能够从血浆中的所有其他化合物中鉴别出PLP、4-PA、PM、PL、PN和PMP。不精密度和平均值（以nmol/L为单位，括号内给出）分别为：PLP（未加标和加标样本）9 - 8%（41 - 65）、4-PA为12 - 7%（18 - 40）、PL为67 - 28%（4 - 19）、PN为15%（21）、PM为10%（27）、PMP为27%（17）。未加标血浆中存在的所有三种维生素B6形式（PLP、4-PA和PL）在（nM）8 - 60（4-PA）、1 - 19（PL）和11 - 99（PLP）范围内均表现出良好的线性关系。总之，我们报告了一种基于HPLC的方法，该方法能够分离并检测纳摩尔量的六种维生素B6形式，并证明该方法适用于人血浆中PLP和4-PA的常规定量检测。
