Establishing Reference Intervals And Developing A Functional Lc-Ms/Ms Assay For Aspirin And Metabolites

Aspirin is one of the oldest, and most commonly used drugs. Low dose aspirin (75 mg) is recommended as an antiplatelet agent for secondary prevention in cardiovascular disease (CVD), and in high-risk pregnancies for the prevention of pre-eclampsia (PE). Aspirin prevents platelet aggregation by inhibiting the cyclooxygenase (COX-1) enzyme at low doses through acetylation of a serine amino acid, thus blocking the production of downstream prostaglandins including thromboxane A2 (TXA2). Aspirin is rapidly metabolised to salicylic acid (SA) and subsequently to conjugated glucuronides and glycine derivatives. There is significant inter-individual variation in aspirin metabolism depending on sex, ethnicity, and diet. Additionally, studies have reported that up to 65% of the population are categorised as aspirin resistant due to suboptimal platelet inhibition. Previous studies have also shown that up to 30% of patients are non-adherent to their medications using chemical adherence testing (CAT). Yet there are a lack of studies which have reported on non-adherence to aspirin in the general population using CAT due to the absence of reference intervals (RIs). RIs for aspirin metabolites in the blood are required for interpretation of CAT results as these metabolites are also derived from dietary intake. Therefore, baseline levels need to be determined for the purpose of distinguishing between dietary salicylates and salicylate metabolites of aspirin. Moreover, current assays for functional testing of aspirin are burdened by poor reproducibility. Therefore, new robust assays which test for adequate inhibition of the COX-1 enzyme by aspirin are required.The aim of this thesis was to use liquid chromatography tandem mass spectrometry (LC-MS/MS) to develop an assay to quantify levels of major metabolites of aspirin including salicylic acid (SA) in plasma. The assay was analytically validated according to the American National Standards Institute-American Standards Board (ANSI-ASB) guidelines. Calibration curves, bias, and precision met the acceptable criteria for all metabolites bar aspirin.RIs required for interpreting the levels found in patients were established in two cohorts. The first cohort was from the study titled “Evaluation of Trimethylamine-N-oxide (TMAO) levels as a risk factor for coronary artery disease (CAD) in South Asians vs. Caucasians” containing a nearly equal number of females and males in addition to Caucasian and South Asian individuals with CAD. The second cohort was the Pre-Eclampsia And Chronic Hypertension, rEnal and SLE (PEACHES) cohort, consisting of pregnant women at high risk of developing PE. The 97.5th percentiles were higher in females within the TMAO cohort compared to the males (97.5th centile being 8481 ng/mL in females compared to 7651 ng/mL in males prescribed aspirin). Additionally, non-pregnant females had significantly higher levels than pregnant females when the TMAO percentiles were compared with the PEACHES percentiles. The 97.5th centile in the TMAO cohort females prescribed aspirin was 8481 ng/mL compared to 458 ng/mL in those prescribed aspirin in the PEACHES cohort (p