Rodríguez Patiño Gabriela, Castillo Rodríguez Miriam Aide, Ramírez Bribiesca Jacinto Efren, Ramírez Noguera Patricia, Gonsebatt Bonaparte María Eugenia, López-Arellano Raquel
National Autonomous University of Mexico, Cuautitlán, Multidisciplinary Research Unit, Cuautitlán Izcalli, Edo. Mexico, Mexico, CP, 54714.
Postgraduate College, Livestock Program, Montecillo, Texcoco, Edo. Mexico, Mexico, CP, 56230.
Rapid Commun Mass Spectrom. 2018 Oct 15;32(19):1675-1682. doi: 10.1002/rcm.8224.
Isoprostane 8-iso-PGF2α is a biomarker of lipid peroxidation in cell membranes. The method developed to measure plasma total levels (esterified + free) of 8-iso-PGF2α must be reproducible and be able to reduce the use of solvents in solid phase extraction. It should be useful to evaluate oxidative stress due to the excess of free radicals that are generated by some disorder or disease.
The method was developed using solid-phase microextraction with Oasis®MAX μElution plates and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). Electrospray ionization was performed in the negative mode (ESI-); the multiple reaction monitoring mode (MRM) was used. The development of the method included the optimization of the chromatographic conditions to achieve the separation of PGF2α and 8-iso-PGF2α as well as the optimization of the microextraction conditions of the analyte of interest in ovine and goat plasma.
The developed method was validated with a calibration curve of plasma samples fortified with standards at five concentration levels in the range 49-639 pg/mL. The average recovery was 89% with a standard deviation of 10.73%. The inter-day precision was evaluated, obtaining a coefficient of variance (CV) less than 15%. The limit of quantification was 20 pg/mL and the limit of detection was 10 pg/mL. 8-iso-PGF2a was determined in the plasma of 14 sheep and 20 goats of 5 months of age and 6 goats of 24 months of age. The concentrations found were 50-300 pg/mL.
The method developed is precise, accurate and reliable with low reagent consumption compared with conventional solid-phase extraction. The analysis time was decreased because, with the use of the microextraction plate, the step of the evaporation and reconstitution of the analyte was avoided. The method is applicable to quantify the plasma total levels (esterified + free) of 8-iso-PGF2α.
异前列腺素8-异前列腺素F2α是细胞膜脂质过氧化的生物标志物。所开发的用于测量8-异前列腺素F2α血浆总水平（酯化+游离）的方法必须具有可重复性，并且能够减少固相萃取中溶剂的使用。它对于评估某些疾病或病症产生的过量自由基所导致的氧化应激应该是有用的。
该方法采用Oasis®MAX μElution板固相微萃取和超高效液相色谱/串联质谱（UPLC/MS/MS）开发。电喷雾电离在负离子模式（ESI-）下进行；采用多反应监测模式（MRM）。该方法的开发包括优化色谱条件以实现前列腺素F2α和8-异前列腺素F2α的分离，以及优化绵羊和山羊血浆中目标分析物的微萃取条件。
所开发的方法通过在49-639 pg/mL范围内的五个浓度水平用标准品强化的血浆样品校准曲线进行验证。平均回收率为89%，标准偏差为10.73%。评估了日间精密度，变异系数（CV）小于15%。定量限为20 pg/mL，检测限为10 pg/mL。在14只5月龄绵羊、20只5月龄山羊和6只24月龄山羊的血浆中测定了8-异前列腺素F2α。所测得的浓度为50-300 pg/mL。
所开发的方法精确、准确且可靠，与传统固相萃取相比试剂消耗低。由于使用了微萃取板，避免了分析物蒸发和重构步骤，分析时间缩短。该方法适用于定量8-异前列腺素F2α的血浆总水平（酯化+游离）。
