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Abstract Introduction
The ratio of nicotinamide adenine dinucleotide (NADH) and oxidized nicotinamide adenine dinucleotide (NAD+) may be used to evaluate the energy metabolism. NADH/NAD+ ratio can be related to the phosphorylation state of the cell, which is defined as the ratio of adenosine triphosphate (ATP) to the sum of adenosine diphosphate (ADP) and inorganic phosphate (Pi). Direct measurements are complicated, however; NADH/NAD+ ratio can be estimated using the beta-hydroxybutyrate (BHB) to acetoacetate (AcAc) ratio in the mitochondria. The BHB/AcAc ratio (ketone body ratio) mirrors the hepatic mitochondrial redox state, which may serve as an indicator of mitochondrial dysfunction or disease.
Due to the stability of AcAc it is challenging to accurately measure the ketone body ratio in a clinical environment in addition to sensitivity limitations. Here, we present a novel liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) based assay that measures BHB, AcAc, and their ratio, in addition to the BHB isomers alpha-hydroxybutyrate (AHB), gamma-hydroxybutyrate (GHB), and beta-hydroxyisobutyrate (BHIB). This highly sensitive and robust assay is thoroughly validated for both human serum and plasma; moreover, stability concerns have been investigated and addressed.
Methods
Serum or plasma samples were collected, placed on ice, and spun down within 2H, followed by storage in -80C freezer until analysis. Sample preparation includes a protein precipitation on ice, whereafter all five analytes are separated using a 6.5 min reversed phased LC method (Waters Acquity) followed by electrospray ionization and MS/MS analysis (Waters TQ-S). Data was analyzed and curated with Ascent V4 (Indigo) using validated customized rules to reduce processing times and human errors.
Results
All five analyte peaks were adequately separated chromatographically or held unique MS/MS transitions; for example, isomers AHB and GHB shared the same MS/MS transition, however, their LC peaks were baseline resolved at 1.77 and 1.67 min, respectively. A thorough validation was performed starting with the analytical measurement range, a span of three orders of magnitude for all analytes was established, creating a high sensitivity assay. Linearity was determined as following: 0.0025-1.5 mM (AcAc and AHB), 0.0050-1.5 mM (BHB and BHIB), and 0.0025-1.2 mM (GHB), in addition to a dilution up to 50x is permitted. The accuracy of the assay was surveyed by spike and recovery, results were obtained from ten random patient samples; an average of 99.9% (AcAc), 102.7% (BHB), 95.3% (AHB), 85.7% (GHB), and 87.5% (BHIB) was measured. Additionally, at least 40 samples were assayed for BHB using two independent assays, a mean bias of 0.01 mM and a Pearson’s R=0.996 were reported. Other analytes were not tested due to unavailability of matching reference tests. Moreover, a 20-day precision study was completed for each analyte at three levels determining the inter-assay (day), intra-assay (replicate), and intra-assay (injection) coefficient of variation (CV); all the %CVs were reported at or below 6.3%. The analytical specificity was determined by surveying a significant presence of 16 common therapeutic drugs, unconjugated bilirubin, conjugated bilirubin, hemoglobin, triglycerides, and high protein levels for a low- and high-level ketone body samples. No significant interferences were observed, all five analytes reported a %Bias < 10% for the individually surveyed interferences; except, high levels of triglycerides and protein reported a %Bias < 15%.
To accommodate multiple sample types, a specimen compatibility test was performed, heparinized plasma, red top serum, and gold top serum show a bias of <15% for all analytes, purple EDTA specimen were not accepted due to a larger bias for multiple analytes. To diagnose patients, new reference ranges have been established using approximately 120 reference samples.
Finally, due to the stability concerns of AcAc an extensive stability study was executed for all analytes, which showed that specimen are stable up to 2H on ice after draw, followed by spin down and storage at -80C. Post-plasma/serum separation, samples are stable up to 6H at room temperature, 1 day at 5C, 3 days at -20C, or at least 90 days at -80C. Additionally, none of the analytes showed a bias >20% after 5 freeze/thaw cycles, however, AHB showed positive trending 1 day. In the situation of a repeat injection, stability of the sample extracts were evaluated, extracts were stored at 5C or -20C, increased bias was observed at day two and immediately at day 1, respectively.
Conclusion
A rapid and robust LC-MS/MS based clinical assay was developed to quantify the ketone bodies AcAc and BHB, to three other isomers (i.e., AHB, GHB, and BHIB), as well as the establishment of clear standard operating procedures through an extensive validation and stress testing.
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= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
