Abstract:
Sample clean-up with the protein precipitation solvent trichloroacetic acid (TCA), combined with a stable isotope labeled internal standard, is widely used for the analysis of endogenous and exogenous compounds in serum and plasma with liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the application of an assay for methylmalonic acid (MMA), used for routine analysis in patient care, negative long-term side effects of TCA on assay performance were observed. Step-by-step extensive troubleshooting disclosed the limitations of using TCA in MS. After running over 2000 samples with the MMA assay over a course of one year, a black coating formed between the probe and the heater that was traced to the use of TCA. The MMA assay used a C18 column with an isocratic eluent of 95% water (0.1% formic acid) as starting condition, on which TCA was more retained than MMA. Next, concentrations of 2.2% TCA in the prepared serum or plasma sample caused a drop in spray voltage during ionization into the MS. This was caused by the strong acid properties of TCA, resulting in current loss of the spray voltage between the heated electrospray ionization (HESI) needle and the union holder, which had also a grounding function. Replacing the original metal HESI needle with a custom made fussed silica HESI needle or detaching the union from the union holder, eliminated the effect of the drop in spray voltage. In conclusion, TCA can seriously affect the long-term robustness by affecting the source of the MS. We recommend the use of a very low sample injection volume, and/or shifting the mobile phase to waste when TCA is eluting, when using TCA in LC-MS/MS analysis.
摘要:
用蛋白质沉淀溶剂三氯乙酸(TCA)净化样品,结合稳定同位素标记的内标,广泛用于通过液相色谱-串联质谱法(LC-MS/MS)分析血清和血浆中的内源性和外源性化合物。在应用甲基丙二酸(MMA)测定法期间,用于患者护理中的常规分析,观察到TCA对测定性能的长期负面副作用。逐步广泛的故障排除揭示了在MS中使用TCA的局限性。在一年的过程中使用MMA测定运行了2000多个样品之后,在探针和加热器之间形成的黑色涂层,可追溯到使用TCA。MMA分析使用C18柱,以95%水(0.1%甲酸)的等度洗脱液作为起始条件,TCA比MMA保留更多。接下来,制备的血清或血浆样品中2.2%的TCA浓度导致在电离到MS期间喷雾电压下降。这是由TCA的强酸特性引起的,导致加热的电喷雾电离(HESI)针和接头支架之间的喷雾电压电流损失,它也有接地功能。用定制的杂化二氧化硅HESI针更换原始金属HESI针或将接头从接头固定器上拆下,消除了喷雾电压下降的影响。总之,TCA会通过影响MS的来源而严重影响长期稳健性。我们建议使用非常低的样品进样量,和/或在TCA洗脱时将流动相转移至废物,当在LC-MS/MS分析中使用TCA时。