Library Dissertation Showcase

Investigating the effect of storage temperature on acetylsalicylic acid concentration in blood samples using high-performance liquid chromatography

  • Year of Publication:
  • 2022

Aspirin (ASA) is one of the most widely used medications in the world ā€“ utilised for its analgesic, antipyretic and anti-inflammatory effects. Following administration, ASA is metabolically converted by hydrolysis to salicylic acid (SA) by carboxylesterase enzymes.

This research aimed to study the hydrolysis of ASA to SA in whole blood samples by HPLC. It was hypothesised that ASA would hydrolyse to SA at a faster rate at higher temperatures. Further, ASA was hypothesised to be relatively stable at both fridge and freezer temperature. Determination and quantitation of ASA and SA along with further compounds or metabolites by HPLC has been demonstrated in biological matrices such as serum and plasma but rarely in whole blood. These methods also did not introduce an independent variable like temperature to study its effect on the rate of hydrolysis and stability of ASA.

During this research, blood samples were spiked with 400 mg/L-1 of ASA (mild poisoning level) and 50 mg/L-1 internal standard (o-Toluic acid) to measure PAR during analysis and stored at one of four temperatures. Following storage, the compounds were extracted from blood via liquid-liquid extraction using ethyl acetate and hydrochloric acid then analysed using HPLC.

Results were quantified using calibration curves made up in blood. Results from this research supported the main hypothesis wherein rate of hydrolysis increased as storage temperature increased. At the higher temperatures, all ASA had been hydrolysed to SA within 1 day. 55 mg Lāˆ’1 ASA remained in the fridge temperature sample after 1 day whereas 441 mg Lāˆ’1 remained after freezer storage. 298 mg Lāˆ’1 of SA had been produced in the fridge condition whereas a not detectable value was produced in the freezer condition. Thus ASA was stable only at freezer temperature. Based on the results, blood samples containing ASA should be stored at freezer temperature in order to prevent hydrolysis to SA. Hydrolysis could also be reduced further through the addition of esterase inhibitor which would likely be included in reallife toxicological samples.

Further work could include repeats to improve the statistical power of results. Unassigned chromatographic peaks could also be investigated by analysing standard solutions of other metabolites such as salicyluric acid and gentisic acid or analysis by LC-MS to identify molecular weight of peaks. There is also the potential to adapt the extraction method to reduce the risk of further hydrolysis prior to analysis e.g. solid phase extraction.

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