高效液相色谱法测定乙醛的优化方法。
An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography.
机构信息
Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
出版信息
Alcohol Clin Exp Res. 2012 Mar;36(3):398-405. doi: 10.1111/j.1530-0277.2011.01612.x. Epub 2011 Sep 6.
BACKGROUND
Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high-performance liquid chromatography (RP-HPLC).
METHODS
We have developed an optimized method for the measurement of acetaldehyde by RP-HPLC in hepatoma cell culture medium, blood, and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent, time, and temperature. Extraction methods of the acetaldehyde-hydrazone (AcH-DNP) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison with AcH-DNP standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol.
RESULTS
Derivatization of acetaldehyde was performed at pH 4.0 with an 80-fold molar excess of DNPH. The reaction was completed in 40 minutes at ambient temperature, and the product was stable for 2 days. A clear separation of AcH-DNP from DNPH was obtained with a new 11-minute chromatography program. Acetaldehyde detection was linear up to 80 μM. The recovery of acetaldehyde was >88% in culture media and >78% in plasma. We quantitatively determined the ethanol-derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 μM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 μl) plasma sampling.
CONCLUSIONS
An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP-HPLC separation of AcH-DNP. The method has an extended linear range, is reproducible and applicable to small-volume sampling of culture media and biological fluids.
背景
乙醛主要由肝内的乙醇脱氢酶在乙醇代谢过程中产生，并通过醛脱氢酶迅速氧化为乙酸盐。在生物基质中循环乙醛水平的评估是通过顶空气相色谱法和反相高效液相色谱法（RP-HPLC）进行的。
方法
我们开发了一种优化的方法，用于通过 RP-HPLC 在肝癌细胞培养液、血液和血浆中测量乙醛。在样品去蛋白后，用 2,4-二硝基苯肼（DNPH）衍生化乙醛。优化了 pH 值、衍生化试剂用量、时间和温度。进行了 AcH-DNP 稳定衍生物的提取方法和产物稳定性研究。通过与 AcH-DNP 标准品比较保留时间，使用新的色谱梯度程序鉴定乙醛，并在存在和不存在乙醇的情况下，根据外部参考标准和标准添加校准曲线进行定量。
结果
在 pH 4.0 下用 80 倍摩尔过量的 DNPH 进行乙醛衍生化。在室温下 40 分钟内完成反应，产物在 2 天内稳定。通过新的 11 分钟色谱程序可清晰分离 AcH-DNP 与 DNPH。AcH-DNP 的检测线性范围高达 80 μM。在培养基中乙醛的回收率>88%，在血浆中>78%。我们使用该方法定量测定肝癌细胞、大鼠血液和血浆中的乙醇衍生乙醛，检测限约为 3 μM。该方法在小体积（70 μl）血浆采样中，日内精度<9%，日间精度<15%。
结论
使用 DNPH 衍生化，随后通过短 RP-HPLC 分离 AcH-DNP，开发了一种用于生物系统中乙醛定量测定的优化方法。该方法线性范围扩展，重现性好，适用于培养基和生物液的小体积采样。
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