Quantification of ketone bodies in saliva using gas chromatography-mass spectrometry

Type 1 Diabetes Mellitus is an autoimmune disease where pancreatic b-cells within the Islets of Langerhans are destroyed. A complication associated with type 1 diabetes mellitus is diabetic ketoacidosis, and if left untreated can result in coma or death. There are commercial methods for individuals to analyze ketone body concentrations within urine and blood, but these are invasive, expensive, and aren’t always accurate. Therefore, saliva should be examined as a potential alternative to the current commercial methods on the market. This experiment aimed to quantify the amount of beta-hydroxybutyrate and acetoacetate, in the saliva, blood, and urine samples using GC-MS by inducing ketosis in consenting participants. The participants followed a ketogenic diet for four days, and their biological samples were obtained before and after. The blood samples were centrifuged to isolate the plasma and deproteinated. All sample matrices were evaporated, the ketone bodies were derivatized using BSTFA + 1% TMCS at 80°C for 1.5 h, and the headspace was analyzed. The participants followed an Atkins diet to induce ketosis and the amount of beta-hydroxybutyrate was able to be quantified for each participant. The urine samples had high concentrations of beta hydroxybutyrate, which could be a result of the acidic urine increasing the derivatization efficiency. There was very little beta-hydroxybutyrate detected within saliva. Since there was detection, this could potentially be used as a method to quantify ketone bodies upon further method development. No acetoacetate was detected in any of the samples. Future work should further optimize the methodology to detect acetoacetate, and the addition of recovery and internal standards to the samples to ensure all the analyte is derivatized and to determine if there is any variation between runs.