Department of Pharmaceutical Sciences, Faculty of Pharmacy, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
J Chromatogr A. 2012 Jan 13;1220:92-100. doi: 10.1016/j.chroma.2011.11.060. Epub 2011 Dec 7.
The use of 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD) as a pre-column derivatization reagent for HPLC (high performance liquid chromatography) analysis of octopamine (oct) and tyramine (tyr) is proposed. The compound reacts under mild conditions (2 min at ambient temperature) with primary amino groups. The derivatization conditions were optimized by considering different parameters (temperature, time and reagent concentration). The synthesized oct and tyr adducts were characterized by (1)H NMR (nuclear magnetic resonance), ESI-MS (electrospray ionization mass spectrometry), IR (infrared) and UV (ultraviolet). Derivative chromatographic separations were performed on a Sinergy Hydro-RP column (150 mm × 4.6 mm i.d.) using a mobile phase consisting of methanol and triethylammonium phosphate buffer (pH 3; 10mM) at varying composition gradient elution and at a flow rate of 0.8 mL/min. Detection was set at λ=320 nm. The obtained results were compared with those achieved by a validated direct HPLC method with detection at λ=275 nm using a Sinergy Polar-RP column (250 mm × 3 mm i.d.) by isocratic elution conditions with a mobile phase consisting of methanol/acetonitrile/sodium pentanesulphonate (SPS; pH 3; 10mM), 7.5:7.5:85 (v/v/v) at a flow rate of 0.3 mL/min. Derivatization method sensitivity proved to be ten times higher than direct method. Limit of detection of oct and tyr was 0.010 and 0.008 μg/mL, respectively. The reliability of the pre-column method was satisfactory also in terms of linearity (from 0.028 to 1.255 and 0.024 to 1.244 μg/mL for oct and tyr, respectively), precision (relative standard deviation ≤2, without significant differences between intra-day and inter-day data) and recovery (from 98.9 to 101.2%). The proposed method showed to be suitable for a reliable determination of oct and tyr traces in commercially available phytoproducts using the instrumentation usually present in any analytical laboratory.
提议使用 2,5-二甲基-1H-吡咯-3,4-二醛（DPD）作为高效液相色谱（HPLC）分析章鱼胺（oct）和酪胺（tyr）的柱前衍生化试剂。该化合物在温和条件下（环境温度下 2 分钟）与伯氨基反应。通过考虑不同的参数（温度、时间和试剂浓度）对衍生化条件进行了优化。合成的 oct 和 tyr 加合物通过（1）H NMR（核磁共振）、ESI-MS（电喷雾电离质谱）、IR（红外）和 UV（紫外）进行了表征。衍生色谱分离在 Sinergy Hydro-RP 柱（150mm×4.6mmid）上进行，流动相由甲醇和三乙铵磷酸盐缓冲液（pH3；10mM）组成，采用不同组成的梯度洗脱和 0.8mL/min 的流速。检测波长设置为 λ=320nm。所得结果与使用 Sinergy Polar-RP 柱（250mm×3mmid）通过等度洗脱条件，流动相由甲醇/乙腈/戊烷磺酸钠（SPS；pH3；10mM），7.5:7.5:85（v/v/v）在 0.3mL/min 的流速下获得的经验证的直接 HPLC 方法在 λ=275nm 下检测的结果进行了比较。衍生化方法的灵敏度比直接方法高十倍。oct 和 tyr 的检出限分别为 0.010 和 0.008μg/mL。柱前方法的可靠性也令人满意，表现在线性度（oct 和 tyr 分别为 0.028 至 1.255 和 0.024 至 1.244μg/mL）、精密度（相对标准偏差≤2%，日内和日间数据无显著差异）和回收率（98.9%至 101.2%）。该方法适用于使用任何分析实验室通常都有的仪器在市售植物制品中可靠地测定 oct 和 tyr 痕量。
