Koskinen M, Elo H, Lukkari P, Riekkola M L
Department of Pharmacy, University of Helsinki, Finland.
J Chromatogr B Biomed Appl. 1996 Oct 11;685(1):141-9. doi: 10.1016/0378-4347(96)00126-0.
A reversed-phase (C18) HPLC method with diode-array detection was developed for the separation and determination of methylglyoxal bis(amidinohydrazone) (mitoguazone) and seven closely related aliphatic analogs thereof, namely the bis(amidinohydrazones) of glyoxal, dimethylglyoxal, ethylmethylglyoxal, methylpropylglyoxal, butylmethylglyoxal, diethylglyoxal and dipropylglyoxal. The mobile phase consisted of a non-linear binary gradient of methanol and 0.03 M aqueous sodium acetate buffer (pH 4.3). Good separation of the eight congeners was achieved. On increasing the methanol content of the eluent, the bis(amidinohydrazones) eluted in the order of increasing number of carbon atoms in the side-chains. The method was also applied to the quantitative analysis of the compounds in aqueous solution and, combined with ultrafiltration, for the separation of the eight congeners in spiked human blood serum. A separate simplified method for the quantitative determination of each of the compounds in spiked human blood serum samples was also developed. The methods developed made for the first time possible the simultaneous HPLC analysis of more than one bis(amidinohydrazones). The results obtained indicate that the bis(amidinohydrazones) studied obviously have a distinct tendency to form ion associates with acetate ions and probably also other carboxylate ions in aqueous solution. This aspect may be of biochemical significance, especially concerning the intracellular binding of the compounds. Each one of the compounds studied invariably gave rise to one peak only, this result supporting the theory that the conventional synthesis of each of the compounds gives rise to one geometrical isomer only. This result is completely in agreement with the results of previous proton and carbon NMR spectroscopic as well as X-ray diffraction studies.
建立了一种采用二极管阵列检测的反相（C18）高效液相色谱法，用于分离和测定丙酮醛双脒腙（米托胍腙）及其七种结构相近的脂肪族类似物，即乙二醛、二甲基乙二醛、乙基甲基乙二醛、甲基丙基乙二醛、丁基甲基乙二醛、二乙基乙二醛和二丙基乙二醛的双脒腙。流动相由甲醇和0.03M醋酸钠水溶液缓冲液（pH 4.3）组成的非线性二元梯度洗脱液构成。实现了这八种同系物的良好分离。随着洗脱液中甲醇含量的增加，双脒腙按照侧链中碳原子数增加的顺序洗脱。该方法还应用于水溶液中化合物的定量分析，并结合超滤用于加标人血清中八种同系物的分离。还开发了一种单独的简化方法，用于定量测定加标人血清样品中的每种化合物。所开发的方法首次实现了对多种双脒腙的同时高效液相色谱分析。所得结果表明，所研究的双脒腙在水溶液中明显有与醋酸根离子以及可能还有其他羧酸根离子形成离子缔合物的明显趋势。这一方面可能具有生化意义，特别是在化合物的细胞内结合方面。所研究的每种化合物都只产生一个峰，这一结果支持了每种化合物的常规合成仅产生一种几何异构体的理论。这一结果与先前的质子和碳核磁共振光谱以及X射线衍射研究结果完全一致。
