(M1330-06-34) Use of Derivatization for a Sensitive and Selective Liquid Chromatography–Mass Spectrometry Method for Quantification of Acetaldehyde in Plasma

Purpose: Acetaldehyde, a volatile compound, serves as a biomarker for certain diseases, including lung cancer and diabetes. Additionally, it is a significant metabolite of ethanol, undergoing oxidation by alcohol dehydrogenase and therefore useful to monitor concentration levels in biological fluids such as blood or plasma. In spite of the low molecular weight and volatile properties of acetaldehyde, it can be quantified by GC-MS however, this detection technique often leads to poor selectivity and low signal strength. Direct detection with LC-MS is also unreliable due to suppression affects and poor volatilization and ionization. Derivatization coupled with LC-MS was therefore utilized to significantly increase selectivity and sensitivity. This sensitive and selective liquid chromatography–mass spectrometry method was qualified for the quantification of acetaldehyde in the rat plasma and shown to be reliable and robust for the determination of the pharmacokinetics of acetaldehyde in rat plasma.

Methods: Rat blood samples, post-ethanol challenge, were collected and then treated at the preclinical testing facility to generate the samples. Acetaldehyde was extracted from the plasma after addition of a stable-isotope labeled internal standard by a protein precipitation method followed by derivatization and evaporation. The evaporated samples were then reconstituted with water and acidic methanol, then injected directly onto a reverse phase C18 column using an HPLC gradient elution and detected on an ESI-MS triple quadrupole mass spectrometer (AB Sciex API6500⁺). Due to interferences from endogenous plasma acetaldehyde and background in the reagents, the concentrations of spiked calibration standards and quality control samples were adjusted by the acetaldehyde level determined in zero standards. A set of rat plasma samples were evaporated to remove the endogenous acetaldehyde from plasma and reconstituted as reagent background samples. This background samples were processed in each run to correct the back-calculated sample concentration. In this method, the lower quantitation limit (LQL) achieved was 50.0 ng/mL compared to minimized reagent background. The quantification range used in this work was from 50.0 to 1000.0 ng/mL acetaldehyde in rat plasma.

Results: This method for quantification of acetaldehyde in rat plasma was fully qualified and successfully assessed for sensitivity, matrix selectivity, matrix effect, short-term stability and processing sample stability, and with reliable inter and intra batch precision and accuracy results. This method was successfully applied to analyze acetaldehyde in actual rat study samples with good inter and intra batch precision and accuracy results, leading to a high analytical run acceptance rate. The two study runs have an overall % CV of 6.5 and its mean %Bias was at ±6.4 or better for each of the quality control concentration levels. The background of reagent in two runs were almost same (40.5 ng/mL and 41.0 ng/mL). This demonstrates the method is reproducible, robust, and reliable and can be used for reliable characterization of the pharmacokinetic profile of acetaldehyde in rat plasma.

Conclusion: A sensitive and selective HPLC mass spectrometric method was developed and qualified to determine acetaldehyde in rat plasma at a relatively low quantification limit of 50.0 ng/mL. This method has been applied for routine pharmacokinetic bioanalysis study with good throughput and better selectivity.

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Acknowledgements: This work has been supported by Sophrosyne Pharmaceuticals Limited without conflict of interest.