Quantifying endogenous and tracer-derived ketone bodies using a dual-label UHPLC-MS/MS method.

Area of Science:

• Metabolic biochemistry
• Analytical chemistry
• Biomarker quantification

Background:

• Ketone bodies, acetoacetate (AcAc) and β-hydroxybutyrate (βOHB), are crucial for energy metabolism during glucose scarcity.
• Quantifying ketone bodies in metabolic tracing requires precise measurement of labeled and unlabeled pools.
• Current UHPLC-MS/MS methods face limitations with ¹³C-labeled standards and AcAc instability.

Purpose of the Study:

• To develop a novel, stable isotope-labeled internal standard approach for simultaneous quantification of labeled and unlabeled ketone bodies.
• To overcome challenges associated with AcAc instability and the lack of suitable standards in tracer studies.
• To optimize a UHPLC-MS/MS method for high-throughput, accurate ketone body analysis.

Main Methods:

• Synthesized deuterated acetoacetate (d₃-AcAc) and [3,4,4,4-d₄]βOHB as internal standards.
• Optimized base-catalyzed hydrolysis for d₃-AcAc synthesis, confirming purity via ¹H NMR.
• Validated the stability of d₃-AcAc in biological matrices and compared the method's performance against conventional techniques.

Main Results:

• Achieved high purity (99.2 ± 0.2%) for synthesized d₃-AcAc.
• Demonstrated stability of d₃-AcAc in extraction buffer and serum for at least 5 hours.
• The novel method showed superior precision, accuracy, and ease of use compared to traditional approaches.

Conclusions:

• The developed method using d₃-AcAc and [3,4,4,4-d₄]βOHB offers a robust solution for ketone body quantification.
• This approach significantly enhances metabolic tracing capabilities for ketone bodies.
• Enables rapid and accurate investigation of ketone body metabolism in diverse physiological and pathological states.