Development of a GC-MS/MS organic acid profiling method for abalone samples: challenges and future prospects

Abstract

South African abalone (H.midae) locally known as Perlamoen in South Africa is a marine mollusc that is found on the South African shore-line. Due to overfishing and the high commercial value coupled to this marine mollusc, wild abalone stocks are on a decrease. Abalone farming plays and integral role in the aquaculture industry as it allows for additional resources to be produced, it also takes the pressure of wild abalone stocks. Even though there are many research areas fulfilled in terms of the optimal farming and environmental factors for cultured abalone, there can still be additional factors on a biochemical level which could influence the period abalone takes to reach market size. Previous studies performed by our group, hypothesised that there is an absence of organic acids during hypoxia, therefore it can be hypothesised that abalone do not rely heavily on mitochondrial oxidative phosphorylation for energy production but on alternative energy pathways. Metabolomics is an emerging field that allows for the use of multiple analytical platforms to detect and quantify small molecules such as organic acids. This study aimed to develop a GC-MS/MS organic acid profiling method for abalone samples. To test the best approach, two GC-MS/MS were developed, one used Chemical ionisation (CI) and the other Electron impact (EI). The CI MRM method provided reasonable linearity across the concentration range with most organic acids giving a R2 > 0.9. However, certain organic acids such as Succinate, Malate, Lactate and Oxaloacetate show a decline in their curves, which could possibly be due to poor ionisation from CI, poor extraction, or compound degradation. The determined LOD and LOQs (from the curves) are relatively high, which highlight the issue of sensitivity with this method and CI in general. Overall, the EI method gave unsatisfactory response over the concentration range with less compounds having a R2 > 0.9. Organic acids such as 3-Hydroxybutyric acid, Pyruvate and Lactate also showed low precision at the different concentrations. One of the reasons for this is the low specificity of the EI quantitative transitions which detect inferring compounds / peaks leading to inaccurate peak areas for each concentration. It can be concluded that the method performance of the GC-MS/MS method fitted with CI is more satisfactory than EI, but day-to-day challenges with the CI source hinders further optimisation and use in the routine lab. Future prospects include using alternative derivatisation methods that could improve EI MRM detection and linearity of organic acids and should be considered in future studies. The use of LC-MS/MS can also be explored for the detection and quantification of organic acids in abalone, especially as derivatives which could bridge the negative ionisation problem.