Improved Analytical Sensitivity and Chromatographic Peak Shape for the Quantification of TCA Cycle Analytes in Human Plasma using the ACQUITY Premier System Solution

The analysis of TCA cycle and related metabolites in human plasma is challenging due to their small, polar nature with many isobars, wide range of concentrations in human plasma, and finally, metal sensitivity. To address retention and selectivity, the analytical method applies mixed-mode anion exchange chromatography with a simple reversed-phase like mobile phase. Tandem quadrupole mass spectrometry offers sensitive detection, while MaxPeak High Performance Surfaces mitigate interactions with metal surfaces present in the UPLC and column. The result is a simple and sensitive analytical method fit to quantify organic acid metabolites in plasma. 

Benefits

• A fast and simple method for the quantification of TCA cycle and other metabolites in human plasma without complicated mobile phases
• Improved analytical sensitivity and peak shape by eliminating metal-analyte interactions using MaxPeak High Performance Surfaces Technology in the ACQUITY Premier System Solution  

The Tricarboxylic acid cycle (TCA cycle) is a series of enzyme-mediated chemical reactions of substrates arising from the breakdown of carbohydrates, fats, and proteins.¹ The TCA cycle a tightly regulated pathway that is used for both anabolic and catabolic cellular processes.¹ Further, metabolites arising from its reactions have been shown to facilitate cell signaling through the mitochondria.² The ultimate fate of metabolites from the TCA cycle put it in a central role of cell homeostasis, and the monitoring of its constituents and products can give insights to various diseases. 

The measurement of compounds such as those of the TCA cycle can be troublesome. Aside from their low molecular weight coupled with the complexity of matrix extracts as well as stability issues, the interaction of compounds with carboxylic and phosphoric acid moieties with metal is a known complication causing loss of analytical sensitivity and poor peak shape.^3-6 The uncertainty in measurement accompanying, such secondary interactions, can be detrimental to the analysis. Methods of attenuation include the addition of chelators in the mobile phase or in the sample diluent, however such mobile phase modifiers could contribute to ion suppression or changes in chromatography leading to the remediation of one problem, but the creation of another.^7-10 To address the metal interactions, Waters has developed MaxPeak High Performance Surfaces (HPS) Technologies.¹¹ By utilizing this technology with LC components as well as column hardware, we are able to mitigate analyte interactions with the metal surfaces and eliminate the need for additional mobile phase additives.  

Stock Solutions Preparation

Analyte stock solutions were prepared individually at 50 mM free acid in H₂O and subsequently combined at 2.5 mM in H₂O for the working stock solution. Stable labelled internal standards (Cambridge Isotope Laboratories) were each prepared at 1 mM free acid in 50%ACN/50%H₂O. A working internal standard mix of 38 µm for cis-aconitic acid-C13, 2-hydroxyglutaric acid-C13, fumaric acid-C13, malic acid-C13, succinic acid-C13, and 120 µm for citric acid-C13 was then prepared in H₂O.  

Plasma Extraction

Four individual donor female healthy plasma samples as well as breast cancer positive samples were purchased from BioIVT (Westbury, NY). Following defrosting on ice, 25 µL of each plasma sample was added to a new 1.5 mL microcentrifuge tube followed by 5 µL of working internal standard mix. 75 µL of cold methanol was added and the samples vortex mixed for 1 minute. The samples were centrifuged at 21,130 rcf and 4 °C for 10 minutes. Following, 75 µL of supernatant was transferred to a new microcentrifuge tube and dried in a centrifugal evaporator under vacuum without heating for 1.5 hours. Samples were immediately reconstituted with 75 µL of H₂O and placed at 20 °C for 10 minutes. Reconstituted samples were then centrifuged as above, and samples transferred to new silanized vials for analysis. Plasma samples were injected in triplicate. 

Calibration Curve Preparation

Calibration curves in H₂O were prepared by serial dilution from the working stock solution to make stock standards of 2.5 mM, 1.25 mM, 500 µM, 250 µM, 125 µM, 50 µm, 25 µM, 12.5 µM, 5 µM, 2.5 µM, 1.25 µM, and 0.50 µM. Five microliters of each standard as well as 5 µL of the internal standard mixture was added to 15 µL of water in a silanized vial. This represents calibration solutions of 500, 250, 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, and 0.1 µM respectively in the same initial volume as plasma, 25 µL. 50 µL of water was then added to make a final volume of 75 µL, matching the sample preparation and dilution procedure for the plasma samples. Thus, the actual concentration of the calibration curve as measured on the mass spectrometer was 167, 83.3, 33.3, 16.7, 8.33, 3.33, 1.67, 0.833, 0.333, 0.167, 0.083, and 0.033 µM. Calibration standards were injected in duplicate. 

Mobile Phase Preparation

The mobile phases were prepared using a volumetric flask and formic acid accurately measured from ampules every three days to ensure method reproducibility. 

Measurement of the TCA and related components is achieved by a variety of LC-MS or GC-MS methods. Previously, we developed an LC-MS assay for the separation of TCA components in human urine by mixed-mode anion exchange chromatography on a standard CSH Phenyl-Hexyl Column and LC system.¹² Here we expand on that method by incorporating the ACQUITY Premier System and ACQUITY Premier CSH Phenyl-Hexyl Column. Both the ACQUITY Premier System and the ACQUITY Premier Column contain MaxPeak HPS Technologies to mitigate metal-analyte interactions. 

Electron-rich moieties such as carboxylate and phosphate can act as metal chelators and cause unwanted interactions with metal surfaces and may ultimately result in a reduction of the amount of analyte measured by the detector. Benefits of the MaxPeak Premier Technology in this application include an increase in the peak area as well as improved peak shape for metal sensitive compounds. Figure 1 shows chromatograms of isocitric and citric acids, malic acid, and 3-phosphoglyceric acid from extracted plasma samples as analyzed on an ACQUITY Premier System with ACQUITY Premier Column compared with a standard UPLC and standard column. It is immediately evident of the increased peak area for isocitric and citric acids, as well as 3-phosphoglyceric acid, and the improved peak shape of malic acid. Isocitric acid and citric acid peak area increased 41x and 5x respectively while 3-phosphoglyceric acid topped >100x. Malic acid had a 58% reduction in peak tailing at 10% height. To determine the impact of MaxPeak Premier Technology on system and column readiness, injections of extracted plasma were performed with new columns on each system. Using citric acid as an example, peak areas in plasma were >2 orders of magnitude on the first injection (Figure 2) demonstrating that the ACQUITY Premier UPLC and Column gives superior analytical sensitivity and requires less equilibration of sample matrix before analysis. 

Calibration curves were performed for the analytes in water, using isotopically labeled internal standards where possible and linear regression with 1/x weighting. The calibration results are summarized in Table 2. Back-calculated concentrations of the analytes extracted from plasma were performed using the internal standard area ratio from the solvent standards (Table 3, Figure 3). A TargetLynx quantification example for citric acid is shown in Figure 4. Overlays of the analytes from each of the four healthy control as well as breast cancer positive extracted plasma samples depict the range of concentrations detected with good retention time alignment (Figure 5). 

In this work, we have demonstrated that the analysis of the TCA cycle and related metabolites in human plasma can be achieved with great analytical sensitivity when incorporating MaxPeak HPS Technology into the liquid chromatograph as well as the analytical column. The ACQUITY Premier System Solution mitigates analyte interactions with metal to improve peak shape as well as analytical sensitivity without full system passivation with strong acids or chelating additives in the mobile phase. The simple mixed-mode anion exchange separation using the ACQUITY Premier CSH Phenyl-Hexyl Column allows for fast separations and easy adaptation.   

1. Murray RK, Bender DA, et al. Harper's Illustrated Biochemistry, 28th ed. New York: McGraw-Hill, 2009. Chapters 16–18. Pages 131–156. 
2. Martínez-Reyes I, Chandel NS. Mitochondrial TCA Cycle Metabolites Control Physiology and Disease. Nat Commun 2020, 11 (1). 
3. Siegel D, Permentier H, Reijngoud D, Bischoff R. Chemical and Technical Challenges in the Analysis of Central Carbon Metabolites by Liquid-Chromatography Mass Spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2014, 966, 21–33. 
4. Gil A, Siegel D, Permentier H, Reijngoud D, Dekker F, Bischoff R. Stability of Energy Metabolites-An Often Overlooked Issue in Metabolomics Studies: A review. Electrophoresis 2015, 36 (18), 2156–2169. 
5. Abrahamson HB, Rezvani AB, Brushmiller JG. Photochemical and Spectroscopic Studies of Complexes, of Iron(III) with Citric Acid and Other Carboxylic Acids. Inorg Chim Acta 1994, 226 (1–2), 117–127. 
6. Sakamaki H, Uchida T, Lim L W, Takeuchi T. Evaluation of Column Hardware on Liquid Chromatography-Mass Spectrometry of Phosphorylated Compounds. J Chromatogr A 2015, 1381, 125–131. 
7. Siegel D, Permentier H, Bischoff R. Controlling Detrimental Effects of Metal Cations in the Quantification of Energy Metabolites via Ultrahigh Pressure-Liquid Chromatography Electrospray Tandem Mass Spectrometry by Employing Acetylacetone as a Volatile Eluent Modifier. J Chromatogr A 2013, 1294, 87–97. 
8. Roberts D, Ruane R, Wilson I. Picolinic Acid a Mobile Phase Additive for Improved Chromatography. Journal of Chromatography 1989, 471, 437–441. 
9. Hsiao JJ, Potter OG, Chu TW, Yin H. Improved LC-MS Methods for the Analysis of Metal-Sensitive Analytes Using Medronic Acid as a Mobile Phase Additive. Anal Chem 2018, 90 (15), 9457–9464. 
10. Myint K, Uehara T, Aoshima K, Oda Y. Polar Anionic Metabolome Analysis by Nano-LC-MS with Metal Chelating Agent. Analytical Chemistry 2009, 81, 7766–7772. 
11. Lauber M, Walter TH, Boissel C, Gilar M, Smith K, Birdsall R, Rainville P, Belanger J, Wyndham K. Low Adsorption UPLC Columns Based on MaxPeak High Performance Surfaces; Waters White Paper 720006930EN. 2020.
12. Smith KM, Plumb R, Rainville PD. Separation and Detection of TCA Cycle Metabolites and Related Compounds in Human Urine by UPLC-MS/MS. Waters Technology Brief 720006463EN. 2019.