Gioia M G, Andreatta P, Boschetti S, Gatti R
Dipartimento di Scienze Farmaceutiche, Alma Mater Studiorum - Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
J Pharm Biomed Anal. 2008 Sep 29;48(2):331-9. doi: 10.1016/j.jpba.2008.01.026. Epub 2008 Jan 19.
A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10min at ambient temperature) in the dark in sodium acetate buffer (80mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150mmx4.6mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was < or =1.40% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all compounds (99.7-101.8%) with R.S.D. ranging from 0.56 to 1.82%. The limits of quantitation were about 40, 50 and 140pmol for acetaminophen, AA and DHA, respectively. The DHA impurity values found in dosage forms were < or =0.2% of AA.
本文提出并验证了一种反相离子对液相色谱法（RP-LC），用于测定脱氢抗坏血酸（DHA）、抗坏血酸（AA）以及药物中所含的对乙酰氨基酚。AA和对乙酰氨基酚可直接进行分析，而DHA需用4,5-二甲基-1,2-苯二胺（DMPD）进行柱前衍生化后再测定。衍生化反应在温和条件下（室温10分钟）于暗处，在含有作为金属螯合剂的EDTA的醋酸钠缓冲液（80mM；pH 3.7）溶液中进行。色谱分离在Phenomenex Synergi 4u hydro-RP（150mm×4.6mm）柱上，于等度洗脱条件下进行，流动相中使用十六烷基三甲基溴化铵（CTAB）作为离子对试剂。每种化合物均呈现线性响应。日内精密度（相对标准偏差）≤1.40%，日内和日间数据无显著差异。回收率研究表明，所有化合物的回收率良好（99.7 - 101.8%），相对标准偏差范围为0.56%至1.82%。对乙酰氨基酚、AA和DHA的定量限分别约为40、50和140pmol。在剂型中发现的DHA杂质值≤AA的0.2%。
