Application Note 47

Organic Acid Quantitation in Mouse Muscle by Ion Chromatography-Mass Spectrometry with Isotopically Labeled Standards

Christopher Petucci,^†* Andrew Zelenin, Steven J. Gardell

Metabolomics Core Facility, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL USA
Southeast Center for Integrated Metabolomics, University of Florida, Gainesville, FL USA

Andrew J. Percy,^† Krista Backiel

Cambridge Isotope Laboratories, Inc., Tewksbury, MA USA

† These authors contributed equally. 
*For further information on original content, please refer to reference 14 (PMID: 27782384). Corresponding author C. Petucci (cpetucci@sbpdiscovery.org).

Highlights

• New IC-MS method developed for organic acid detection and quantification
• Stable isotope-labeled organic acids employed for improved precision and accuracy
• 28 polar, low molecular weight organic acids quantified in mouse muscle
• Statistically significant differences in levels of organic acids found in fatigued vs. sedentary mice

Introduction

Organic acids (OAs) are important metabolites that play an essential role in an array of energy metabolism pathways (e.g., glycolysis and tricarboxylic acid cycle).^1,2 In addition, short chained OAs are emerging as important regulators of host immune responses and transcriptional regulation.^3,4 Their significance to cellular metabolism is heightened by their association with diseases, such as cancer and diabetes.^5-7 As a result, research has been focused on quantifying OAs in various biological samples (e.g., urine,⁸ plasma,⁹ serum¹⁰). In these studies, measurements of OAs were accomplished by liquid chromatography (LC) or capillary electrophoresis (CE) coupled to mass spectrometry (MS).^11,12 The commonly utilized modes of chromatography include reversed-phase (with C₁₈ bonded silica), ion pair, and hydrophilic interactions. Despite that, the efficiency of separating polar OAs with these techniques can be challenging. An attractive complementary technique for untargeted metabolomics of polar metabolites is ion chromatography (IC)-MS.¹³

In this note, a targeted IC-MS method using stable isotope-labeled standards (SIS) was used to quantify a panel of polar OAs in mouse muscle.¹⁴ The SIS OAs served as internal standards for enhanced precision and accuracy of OA measurements. Statistically significant quantitative differences were observed for four OAs in the quadricep muscle of sedentary and fatigued mice. Overall, this study demonstrated the ability of IC-MS with stable isotope-labeled OAs to separate and quantify a collection of low molecular weight polar metabolites that are difficult to analyze by other techniques.

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