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Use of Ion Chromatography/Mass Spectrometry for Targeted Metabolite Profiling of Polar Organic Acids Chris Petucci, Andrew Zelenin, Jeffrey A. Culver, Meghan Gabriel, Ken Kirkbride, Terri T. Christison, and Stephen J. Gardell Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03435 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Analytical Chemistry

Use of Ion Chromatography/Mass Spectrometry for Targeted Metabolite Profiling of Polar Organic Acids

Chris Petucci*,1,2, Andrew Zelenin1,2, Jeffrey A. Culver1,2, Meghan Gabriel1, Ken Kirkbride3, Terri T. Christison3, and Stephen J. Gardell1,2

1

Sanford Burnham Prebys Medical Discovery Institute, 6400 Sanger Road, Orlando, FL 32827, 2 Southeast Center for Integrated Metabolomics, Clinical and Translational Sciences Institute, University of Florida, 2004 Mowry Road, Gainesville, FL 32610, and 3Thermo Fisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94088

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ACS Paragon Plus Environment


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ABSTRACT Organic acids (OAs) serve as metabolites that play pivotal roles in a host of different metabolic and regulatory pathways. The polar nature of many OAs poses a challenge to their measurement using widely-practiced analytical methods. In this study, a targeted metabolomics method was developed using ion chromatography/triple quadrupole mass spectrometry (IC/MS) to quantitate 28 polar OAs with limits of quantitation ranging from 0.25-50 µM. The inter-day assay precisions ranged from 1-19%, with accuracies ranging from 82 to 115%. The IC/MS assay was used to quantitate OAs in quadriceps muscle from sedentary mice compared to fatigued mice subjected to either a low intensity, long duration (LILD) or high intensity, short duration (HISD) forced treadmill regimen. Among the OAs examined, significant differences were detected for hippuric acid, malic acid, fumaric acid, and 2-ketoglutaric acid between the sedentary and fatigued mice. In conclusion, the IC/MS method enabled the separation and quantitative survey of a broad range of polar OAs that are difficult to analyze by chromatographic techniques.

INTRODUCTION The human metabolome is composed of hundreds of polar metabolites that play pivotal roles in numerous metabolic and regulatory pathways. This pool of metabolites is often perturbed in diseases such as cancer,1 diabetes, 2 and cardiovascular disease.3 These polar metabolites include organic acids (OAs), pyridine nucleotides, and the phosphate sugars of the glycolysis/pentose-phosphate pathways. Measuring these metabolites in biological extracts can be challenging in order to retain and separate these species. 2


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Quantification of ionic or polar metabolites has been achieved using C18 reversed phase chromatography,1-3 and in some cases derivatization was performed to enhance the retention of these species.4-6 Complementary chromatographic techniques such as capillary electrophoresis (CE),7 ion-pairing,8 HILIC, 9-11 and porous graphitic carbon (Hypercarb™)12-14 stationary phases have been successfully used for metabolomics applications to resolve several classes of polar metabolites including short chain acylcarnitines, pyridine nucleotides, phosphate sugars, and amino acids. In recent years, ion chromatography (IC) has been shown to highly retain several polar metabolites for metabolomics studies.18-20 This work prompted us to pursue IC as an additional complementary technique to achieve the retention and resolution of a broad range of polar OAs and quantitate them by mass spectrometry. IC has been used widely for trace level analyses of ions in the environmental15 and food industry,16-17 but it is largely untested for separating ionic and polar compounds in metabolomics research. A few applications have coupled IC to mass spectrometry (MS) for global profiling or quantitation of phosphate sugars, OAs, and nucleotides in biological samples.18-20 Herein, we showed IC/MS to be a highly selective and sensitive complementary approach to other modes of chromatography for quantitating a broad range of 28 polar, low molecular weight OAs. Our method was applied to the analysis of OAs in quadriceps muscle from sedentary mice compared to fatigued mice that underwent low intensity, long duration (LILD) or high intensity, short duration (HISD) forced treadmill tests. Overall, the IC/MS method provided for the high retention and quantitation of polar OAs in mouse quadriceps.

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EXPERIMENTAL SECTION Chemicals. A total of 37 unlabeled OAs were generously provided by Thermo Scientific (San Jose, CA; Supporting Information, Table S-1). Seventeen isotopically labeled OAs were generously provided by Cambridge Isotope Laboratories, Inc. (Cambridge, MA; Supporting Information, Table S-1). Acetonitrile, methanol, ethanol, ethyl acetate, formic acid, hydrochloric acid, sodium hydroxide, ammonium formate, and O-benzylhydroxylamine were purchased from Sigma-Aldrich (St. Louis, MO). Preparation of Standards. Individual stock solutions of OAs were prepared on ice in deionized H2O, 50% ethanol, 100% ethanol, 150 mM sodium hydroxide, or 0.1 M hydrochloric acid with concentrations ranging from 25 mM to 1000 mM. Aliquots (10 µL to 50 µL) of these individual stocks were combined and diluted to 1 mL with deionized H2O to prepare a calibration stock mixture. This calibration stock mixture was serially diluted using deionized H2O to prepare working solutions of 0.25, 0.5, 1.25, 2.5, 12.5, 50, 125, 250, 500, and 1250 µM for most OAs. For 2-ketoglutaric acid, the working solutions were 0.1, 0.2, 0.5, 1, 5, 20, 50, 100, 200, and 500 µM. Working solutions for 3-hydroxybutyric acid were 0.1, 0.2, 5, 10, 50, 200, 500, 100, 2000, and 5000 µM. Lactic acid working solutions were 5, 10, 25, 50, 250, 1000, 2500, 5000, 10000, and 25000 µM. Standard curves were prepared by spiking 10 µL of each working solution into separate 40 µL aliquots of 50:50 acetonitrile/0.3% formic acid along with 10 µL spikes of the internal standard (IS) mixture (prepared as describe below). For most OAs, the resultant calibration curves had final spiked concentrations of 0.05, 0.1, 0.25, 0.5, 2.5, 10, 25, 50, 100, and 250 µM (Supporting Information, Table S-2). Exceptions were the calibration curves for 2ketoglutaric acid (0.02, 0.04, 0.1, 0.2, 1, 4, 10, 20, 40, and 100 µM), 3-hydroxybutyric acid 4


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(0.02, 0.04, 1, 2, 10, 40, 100, 200, 400, and 1000 µM), and lactic acid (1, 2, 5, 10, 50, 200, 500, 1000, 2000, and 5000 µM). Individual stocks of 17 isotopically labeled OAs were prepared on ice in deionized H2O, 50% ethanol, 150 mM sodium hydroxide, or 50% methanol with stock concentrations ranging from 10 mM to 125 mM (Supporting Information, Table S-1). Aliquots (10 µL to 100 µL) of these individual stock solutions were combined and diluted up to 1 mL with deionized H2O to prepare an IS mixture. This IS mixture was diluted by a factor of 4 with H2O resulting in concentrations of 50 µM to 625 µM. A 10 µL aliquot of this diluted IS mixture was spiked into each calibration and study sample resulting in final IS concentrations ranging from 10 µM to 125 µM. In addition, surrogate ISs were used to quantitate several organic acids that did not have a commercially available IS (Table S-1) in order to achieve linear data. Keto-acids in calibration samples were stabilized by derivatization with 50 µL of 0.2 M O-benzylhydroxylamine, dissolved in 50:50 methanol/200 mM ammonium formate at pH 5, for 10 min at room temperature. After derivatization, 100 µL of deionized H2O was added to each sample followed by 1000 µL of ethyl acetate. The vials were vortexed for 30 sec and centrifuged at 18,000 x g for 5 min at 10 oC. A 900 µL aliquot of the top ethyl acetate layer from each vial was dried down under nitrogen in a 96-well plate. Then, samples were reconstituted in 100 µL of deionized H2O and transferred to glass vials for injection onto the IC/MS. Mouse Exercise Protocols. All animal studies and experimental procedures were performed according to protocols approved by the Sanford Burnham Prebys Medical Discovery InstituteLake Nona Institutional Animal Care and Use Committee. Mice (C57BL/6) at 13 weeks of age were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were subjected to a low 5



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LILD or HISD motorized treadmill protocol. The LILD group of mice ran for an average of 1.5 hr on the treadmill at 0o grade at 10 m/min for 10 min, with an increase to 20 m/min until exhaustion. The HISD group of mice ran for an average of 34 min on the treadmill at 0o grade at 10 m/min for 10 min with an increase in speed of 3 m/min every 4 min until exhaustion. Tissue Harvesting. Between 1230 and 1330 hr, mice were subjected to an intraperitoneal (IP) injection of sodium pentobarbital (200 mg/kg, Premier Pharmacy Labs Inc.). The quadricep muscles were harvested via blunt dissection, quickly rinsed in saline, depleted of excess moisture by blotting in sterile gauze, placed in 1.5 ml tubes, and flash frozen immediately using liquid nitrogen. Processing of Mouse Muscle. Frozen mouse quadriceps were lyophilized using a Labconco Freezone 4.5 benchtop lyophilizer. The tissues were powdered in CK28-R tubes (2 mL capacity) containing ceramic beads using a Precellys Evolution high efficiency homogenizer with a Cryolys cooling unit (Bertin Corp., Rockville, MD). The settings were 7200 rpm for 2 min at 6 x 20 sec cycles with 15 sec pauses between cycles. Approximately 5 mg of powder was weighed into a separate CK28-R tube and homogenized in 500 µL of 50:50 acetonitrile/0.3% formic acid using the Precellys Evolution at 6800 rpm (2 min at 6 x 20 sec cycles with 15 sec pauses between cycles) at 5-10 oC. OAs were extracted from 50 µL aliquots of homogenates in the same way as the standard calibration curves to prepare samples for injection onto the IC/MS. Ion Chromatography. A Dionex ICS-5000 was interfaced to a Thermo Quantiva TSQ triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). The OA-containing samples were injected onto a Dionex IonPac™ AS-11-HC-4 µm 2 x 250 mm column with a Dionex IonPac AG11-HC-4 µm guard column. OAs were eluted with a KOH step gradient (Dionex 6


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EGC 500 KOH cartridge) beginning at 5-10 mM KOH over 3 min, 10-20 mM KOH from 3-5 min, 20-50 mM KOH from 5-8 min, 50-80 mM KOH from 8-10 min, 80-100 mM KOH from 1011 min, held at 100 mM KOH from 11-17 min, and re-equilibrated at 5 mM KOH at 17.1-19.1 min at a flow rate of 0.35 mL/min and 35 oC. The flow from the anion exchange column was directed to a Dionex AERS 500 2 mm electrolytically regenerated suppressor at 20 oC. The suppressor was regenerated by deionized water through the anion regeneration pump at a rate of 600 µL/min. The suppressor removed potassium cations and neutralized the KOH eluent at pH 14 to pH 7 before reaching the mass spectrometer. A post column pump introduced methanol at a flow of 0.06 mL/min into the eluent through a low dead volume mixing tee to assist in desolvation of the eluent by electrospray ionization. Mass Spectrometry. A Thermo Quantiva TSQ triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA) was operated in negative ion mode using electrospray ionization with an ESI capillary voltage of 2500 V. The ion transfer tube and vaporizer temperature were 338 o

C. The ESI source gases were set to 42 for sheath gas, 12 for auxiliary gas, and 1 for sweep gas.

Single reaction monitoring (SRM) was used with a resolution of 0.7 Da, cycle time of 0.8 sec, and argon collision gas of 1.5 mTorr for the generation of product ions of each parent ion OA. Optimization of the collision induced dissociation parameters was accomplished by infusion of solutions of standard OAs into the ESI source using Thermo Scientific tuning software. The SRM transitions for the OAs are given in Supporting Information, Table S-1.

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RESULTS AND DISCUSSION Assay Validation. The IC/MS method was validated by running calibration curves in duplicate on 3 separate days. The raw data was acquired and processed by TraceFinder 3.2. Twenty eight of 37 tested OAs yielded calibration curves that met the criteria for bioanalytical method validation. The organic acids that could not be quantitated were glyceric acid, oxalic acid, 2ketobutyric acid, cysteic acid, homogentisic acid, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, and indole-3-acetic acid. The absence of signal or insufficient sensitivity for several medium polar to non-polar OAs is presumably due to either their low ionization efficiency or precipitation in the anion suppressor when the KOH eluent was neutralized to pH 7. Future studies will be aimed at introducing methanol in the KOH eluent to solubilize and elute medium to non-polar OAs. The isobaric metabolites examined in this study were 2-hydroxybutyric acid, 3hydroxyisobutyric acid, 3-hydroxybutyric acid, citric acid, isocitric acid, methylmalonic acid, and succinic acid. All of these metabolites were able to be distinguished by either unique daughter ions (Table S-1) or by being chromatographically resolved (e.g., citrate and isocitrate). Calibration curves were fitted with a linear or a quadratic curve with 1/X or 1/X2 weighting with R2 values of 0.99 or greater. The interday accuracy of the measured concentration of each calibration standard was between 82 and 109% (Supporting Information, Table S-2). The interday precisions, expressed as the coefficient of variation ranged from 0.8 to 19.4% (Supporting Information, Table S-2). Recovery and Matrix Effect. The recoveries and matrix effects of OAs were determined by spiking 10 µL of the IS mixture into aliquots of mouse quadriceps homogenate. The percent 8


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recoveries were calculated from the mean response ratio of the pre-extraction spiked, OA samples (unlabeled OA/isotopically labeled OA) over the mean response ratio of the postextraction spiked OA samples multiplied by 100. Recoveries ranged from 13 to 107% (Supporting Information, Table S-3). The low recoveries for certain highly polar OAs, like citrate at 13%, resulted from using liquid-liquid extraction to obtain a cleaner extract rather than extraction by protein precipitation with methanol. However, liquid-liquid extraction was preferred to avoid unnecessarily contaminating the anion exchange column and anion suppressor with phospholipids and other species. Even though the recoveries for certain OAs were low, the signals for low recovery OAs like citric acid were adequate enough (i.e., low calibrator 10,000 area counts or greater; high calibrator > 100e6 counts) to achieve the required assay sensitivity. Matrix effects were determined similarly to recoveries by spiking ISs of OAs into aliquots of homogenates of mouse quadriceps muscle. The matrix effect was calculated by taking the mean post-spike analyte response ratio divided by the mean analyte response ratio of neat samples subtracted by a factor of 1 and then multiplied by 100. The matrix effects ranged from 2 to 37% revealing that there was an enhancement in the ionization of the OAs in the negative ionization mode (Supporting Information, Table S-3). Stability of OAs in Muscle Homogenates Subjected to Freeze Thaw Cycles. Three freezethaw cycles were performed on aliquots of pooled, stored muscle homogenates to assess the stability of OAs. The reproducibility of the values derived for each of the freeze-thaw samples mostly ranged from 92 to 110% (Supporting Information, Table S-3) with the exception of the keto-acids, pyruvic acid (85%) and 2-ketoglutaric acid (80%). The decreased stability of the keto-acids with repetitive freeze-thaws indicates that these OAs should be determined 9



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immediately after preparation of homogenates. Alternatively, if homogenates are stored for future analysis, they should be spiked with ISs to track the stability of the keto-acids. IC/MS Quantitation of OAs in Mouse Muscle. The IC/MS method was used to quantitate OA differences in quadricep muscles from sedentary mice compared to fatigued mice subjected to either a LILD or HISD forced treadmill exercise regimen (n = 5, each group). The HISD and LILD treadmill runs are similar to a power and an endurance performance test, respectively. Both tests culminate in exhaustion and cessation of running. However, disparate mechanisms for fatigue are undoubtedly in play.21, 22 During high intensity exercise, there is a greater reliance on ATP production by glycolysis, fueled by glucose and glycogen, and creatine phosphate stores. Use of ATP from these non-mitochondrial sources for muscle contraction is linked to the accumulation of inorganic phosphate and protons with the latter eliciting cellular acidosis. On the other hand, the ATP requirement during low intensity exercise is largely met by mitochondrial (oxidative) respiration. Under these circumstances, muscle fatigue during prolonged exercise has been linked to glycogen depletion and hypoglycemia. Narrow Gaussian OA peaks with a very clean baseline free of extraneous chemical noise were obtained for a biological extract of mouse quadriceps separated by IC/MS (Fig. 1). OAs eluted during IC in the following sequential order: monocarboxylic acids (e.g., lactic acid), dicarboxylic acids (e.g., succinic acid) and lastly tricarboxylic acids (e.g., citric acid). Eleven of 28 OAs were above the lower limit of quantification in the mouse quadricep muscle homogenate. The data revealed statistically significant differences in the levels of select OAs in quadricep muscles from sedentary, LILD, and HISD mice (n = 5/group, Fig. 2). The levels of hippuric acid in LILD mice (0.08 ± 0.04 nmol/mg dry powder, p-value = 0.023) were decreased 10


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by 68% compared to the sedentary control mice (0.26 ± 0.13 nmol/mg). Also, the levels of hippuric acid in the LILD mice decreased by 59% compared to HISD mice (0.20 ± 0.07 nmol/mg, p-value = 0.017). Malic acid was 74% higher in LILD mice (2.32 ± 0.40 nmol/mg, pvalue = 0.001) compared to sedentary mice (1.34 ± 0.21 nmol/mg). HISD mice (2.19 ± 0.63 nmol/mg, p-value = 0.021) had a 64% increase in malic acid compared to sedentary mice. There was a 107% increase in the levels of fumaric acid in LILD mice (0.43 ± 0.08 nmol/mg, p-value = 0.018) compared to the sedentary mice (0.21 ± 0.08 nmol/mg). In addition, the levels of 2ketoglutaric acid were 204% higher in the LILD mice (0.41 ± 0.17 nmol/mg, p-value = 0.014) compared to the sedentary mice (0.13 ± 0.11 nmol/mg). LILD mice exhibited a significant 282% increase in 2-ketoglutaric acid compared to the HISD mice (0.11 ± 0.07 nmol/mg, p-value = 0.006). Overall, the IC/MS method yielded highly reproducible data which allowed us to discern significant changes in the levels of OAs in quadricep muscle from sedentary mice compared to fatigued mice that were subjected to two different forced exercise challenges. It is interesting that the level of 2-ketoglutarate is specifically increased in the LILD test. The blood level of 2-ketoglutarate, a product of glutamate metabolism, was shown to be elevated for long periods of time in human subjects engaged in intense “spinning exercise” for 60 min.23

CONCLUSIONS This pilot study showed that IC when coupled to MS is a complementary separation method to HPLC (i.e., HILIC and ion-pairing HPLC) for retaining and quantitating highly polar OAs. The main advantage of IC is that it separated highly polar OAs that cannot be adequately 11



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retained by reversed phase chromatography. One shortcoming of IC is that it cannot be used to analyze non-polar OAs. Overall, IC/MS has great potential for quantifying numerous other classes of polar compounds and providing a uniquely-powerful platform for targeted metabolomics.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: Multiple reaction monitoring parameters for the quantitation of organic acids by IC/MS; Inter-day precisions and accuracies of organic acids quantitated by IC/MS; Recoveries, matrix effects, and freeze/thaw stabilities of select organic acids (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: (407) 745-2000. Fax: (407) 745-2001.

ACKNOWLEDGEMENTS The Southeast Center for Integrated Metabolomics (SECIM) is supported by NIHU24 DK097209. We thank Thermo Scientific (San Jose, CA) for providing the Dionex ICS-5000+ Reagent Free HPIC System and unlabeled OAs used in this study. We also thank Cambridge Isotope Laboratories (Cambridge, MA) for providing the isotopically labelled OAs used in this study. 12


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Figure Captions Figure 1. Total ion chromatograms for the IC/MS separation of OAs in mouse quadriceps muscle. The sequential elution positions of the OAs were 1) lactate 2) pantothenate 3) 3hydroxyisobutyrate 4) 2-hydroxybutyrate 5) hippurate 6) succinate 7) malate 8) fumarate 9) pyruvate 10) citrate 11) 2-ketoglutarate (~14.5 and 16.5 min.).

Figure 2. A) Histogram plots showing the different levels of OAs quantitated in the mouse quadriceps muscle of sedentary, LILD, and HISD mice by IC/MS (3-isoHBA is 3hydroxyisobutyric acid and 2-HBA is 2-hydroxybutyric acid) B) Histogram plot of the levels of lactate in the quadriceps muscle of sedentary, LILD, and HISD mice C) Histogram plot of the levels of pantothenate in the quadriceps muscle of sedentary, LILD, and HISD mice. An asterisk denotes p < 0.05.

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te

la ct at e

nmol/mg dry powder

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17



For Table of Contents Only (Top: TIFF graphic; Bottom: Alternative PowerPoint graphic that has a higher resolution than the TIFF graphic)

100 Relative Abundance

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citrate 50

isocitrate 0 10.0

11.0

12.0

13.0

14.0

Time (min)

18


15.0

Mass Spectrometry for Targeted

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