HPLC analysis of salicyluric acid as a marker of low dose aspirin consumption
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Background . A low dose aspirin regimen (81 mg/day) is now commonly prescribed for the prevention of occlusive cardiovascular disease in both symptomatic and asymptomatic patients and for patients suffering from chronic inflammatory diseases. The effects of aspirin in combination with other drugs in the prevention of CVD and chronic inflammatory diseases are being currently studied. Clinical trials are also underway to study pregnancy outcomes in women on low dose aspirin therapy who have suffered miscarriages due to pre-eclampsia or pregnancy induced hypertension. It is thus necessary to develop a fast and non-invasive monitoring tool to measure compliance to aspirin therapy. This would allow validation of therapeutic studies and detection of non-compliance in clinical trials. The goal of this study was to first develop and validate a non-invasive measurement of the major urinary aspirin metabolite salicyluric acid (SUA) by reverse phase - high pressure liquid chromatography (RP-HPLC) and then to determine the utility of urinary SUA as a marker of compliance to a low dose aspirin regimen in human volunteers. Methods . A direct RP-HPLC analysis of urinary SUA was developed and validated for linearity, reproducibility, limit of detection and recovery. In the background/acute phase of this study, urine specimens from two volunteers were analyzed during consecutive days where no aspirin was ingested or after the consumption of salicylate containing foods. The volunteers were not on any aspirin therapy and had not ingested the drug in more than 10 days. The mean plus three standard deviations of the average SUA concentrations from these measurements were taken as the upper threshold limit for background urinary SUA levels. Urinary SUA levels were then measured in response to low-dose aspirin. In the chronic phase of this study, the threshold parameters from the background study were applied to longitudinal samples which were collected from a single volunteer who consumed a daily low dose of aspirin over 6 days. Results . The HPLC assay developed was linear from its limit of quantitation of 0.84 μg/mL to greater than 350 μg/mL. The imprecision was estimated to be <5% CV for both within and between-runs and the recovery estimate was between 96% and 103%. SUA was detectable in all background samples and the threshold value (background mean +3SD) was 10.250 mg/mL. In response to chronic low dose aspirin, urinary SUA levels rose sharply within 2 hours of the dose however, fell below background threshold levels within 18 hours; prior to the next daily dose. Conclusion . Urinary SUA can be easily measured by HPLC with strong performance characteristics. However, during chronic low-dose aspirin therapy, SUA is quickly cleared to levels indistinguishable from background levels and steady state increases in urinary SUA are not achieved. These results indicate that urinary SUA is not a useful marker for assessing compliance to low-dose aspirin therapy.