(T1530-09-51) Novel HILIC-LC-MS Method for Quantification of Ketone Bodies (beta-hydroxybutyrate and acetoacetate) in Human Plasma

Purpose: Ketone bodies (beta-hydroxybutyrate, acetoacetate, and acetone) play an important role in the general energetics of the body, ensuring uninterrupted energy supply to vital organs such as the brain, heart, and kidney in extreme cases of starvation. Recently, it has been shown that increased amounts of circulating ketone bodies in the blood play an important role in minimizing the damage to cells in cases such as traumatic brain injury, stroke or heart attack. Unfortunately, there is no easy way to measure the concentration of these molecules in the blood. The existing methods to detect ketone bodies suffer from various disadvantages such as low sensitivity and higher error margin (as in the case of enzymatic home test kits), pre-processing of samples (derivatization before analysis), and need for specialized equipment (such as GC-MS). The aim of the work presented here is to develop and partially validate a HILIC-LC-MS assay to detect and quantify ketone bodies (beta-hydroxybutyrate and acetoacetate) in human plasma in a range covering physiologic and potential therapeutic concentrations.

Methods: While various mobile phases and additives were investigated, the best results were obtained using methanol and 0.1% v/v formic acid in water in a ratio of 1:4 respectively, in an isocratic fashion. The column used was an Agela Technologies Venusil HILIC (amide) column (150x4.6 mm) with a particle size of 5 µm. The flow rate of the mobile phase was 1ml/min; the autosampler temperature was kept at 4oC while the column was kept at room temperature (Waters 2695). The injection volume of the analyte was 10µL. Detection (Waters QDa) was performed with a cone voltage of positive or negative 15 V  and a source temperature of 120oC. Beta-hydroxybutyrate was detected at an m/z of 103.00 in negative ion mode while acetoacetate was detected as a sodium adduct at an m/z of 125.00 in positive ion mode. Ethyl lactate was used as an internal standard and was detected as a sodium adduct at an m/z of 141.00 in positive ion mode. Various stock solutions (starting from 100mM) of both ketone bodies were made in distilled water and human plasma was spiked to achieve desired concentrations such that the total dilution of plasma was less than 10% v/v. The plasma proteins were precipitated with 4 volumes of ice-cold methanol (containing 300µM ethyl lactate as internal standard) and centrifuged at 4oC for 10 mins at 27,237 RCF. The method was evaluated for its linearity, accuracy, and precision at three QC levels- 50µM (LQC), 700µM (MQC), 2500µM (HQC) for Acetoacetate, and 150µM (LQC), 1250µM (MQC), and 5000uM (HQC) for beta-hydroxybutyrate.

Results: It was found that while a linear fit gave the best fit for beta-hydroxybutyrate (50-6000µM), a quadratic fit was preferred for acetoacetate (50-4000µM). The R² value for both fits was >0.99. The precision and accuracy of the method were calculated within acceptable limits at all QC levels ( < 15%). The retention times for beta-hydroxybutyrate, acetoacetate, and ethyl lactate were found to be 2.50, 2.34, and 5.51 minutes respectively.

Conclusion: A novel HILIC-LC-MS method was developed and partially validated for the bioanalysis of ketone bodies- beta-hydroxybutyrate and acetoacetate in human plasma. The range of the method was found to be large enough to quantify these biomolecules at their physiologic and potential therapeutic levels, without the need for excessive dilutions.

References:

Table of validation parameters for assay



Standard curves for beta-hydroxybutyrate and acetoacetate along with equations of curve