Department of Pharmacy and Biotechnology, Alma Mater Studiorum - Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
J Chromatogr A. 2013 Jul 12;1298:95-102. doi: 10.1016/j.chroma.2013.05.026. Epub 2013 May 18.
A RP-HPLC method with pre-column derivatization was developed and validated for the simultaneous quantification of carnosine (Carn), acetylcarnitine taurinate (AC-Tau), asparagine (Asn), potassium aspartate (Asp) and for the determination of phosphoserine (p-Ser) in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,4-dinitrofluorobenzene (DNFB) both in standard and placebo solutions. The reaction was carried out for 20 min at 70 °C in alkaline medium (pH10) for p-Ser analysis, whereas for 60 min in the case of Carn, AC-Tau, Asn and Asp analysis. The adducts have been separated on a Discovery RP Amide C16 (250 mm×4.6mm, i.d.) column using a mobile phase consisting of acetonitrile (ACN) and triethylammonium (TEA) phosphate buffer (pH 3, 0.05 M) under gradient elution conditions at a flow-rate of 0.8 mL/min. Detection was set at λ=360 nm. The validation parameters such as linearity, sensitivity, accuracy, precision and specificity were found to be highly satisfactory. Linear responses were observed by placebo solutions (determination coefficient ≤0.9996). Intra-day precision (relative standard deviation, RSD) was ≤1.06% for corrected peak area and ≤0.99% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all examined compounds (from 97.7% to 101.5%) with RSD ranging from 0.5% to 1.3%). The high stability of derivatized compound solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of a large number of samples and consecutive chromatographic analyses by the use of an autosampler. The developed method can be considered suitable for the quality control of new and commercial products.
建立了一种反相高效液相色谱法，并用柱前衍生法对新的和商业的食品补充剂中的肌肽（Carn）、乙酰肉碱牛磺酸（AC-Tau）、天冬酰胺（Asn）、天门冬氨酸钾（Asp）和磷酸丝氨酸（p-Ser）进行同时定量分析和测定。通过研究 2,4-二硝基氟苯（DNFB）与标准溶液和安慰剂溶液中的氨基酸衍生化反应，评估了复杂基质的影响。对于 p-Ser 的分析，衍生化反应在 70°C 的碱性介质（pH10）中进行 20 分钟，而对于 Carn、AC-Tau、Asn 和 Asp 的分析则进行 60 分钟。在梯度洗脱条件下，使用乙腈（ACN）和三乙铵（TEA）磷酸盐缓冲液（pH3，0.05 M）作为流动相，在 Discovery RP Amide C16（250mm×4.6mm，内径）柱上分离加合物，流速为 0.8 mL/min。检测波长设定为 λ=360nm。验证参数如线性、灵敏度、准确性、精密度和特异性都非常令人满意。通过安慰剂溶液观察到线性响应（决定系数≤0.9996）。日内精密度（相对标准偏差，RSD）对于校正后的峰面积≤1.06%，对于保留时间（tR）≤0.99%，且日内和日间数据之间无显著差异。回收研究表明，所有被测化合物的回收率均较好（97.7%至 101.5%），RSD 范围为 0.5%至 1.3%。衍生化合物溶液在室温下的高稳定性是该方法的一个明显优势，允许使用自动进样器同时制备大量样品并连续进行色谱分析。所建立的方法可用于新的和商业产品的质量控制。
