MS Methods for the Analysis of Metal-Sensitive Analytes

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Improved LC/MS Methods for the Analysis of Metal-Sensitive Analytes using Medronic Acid as a Mobile Phase Additive Jordy J. Hsiao, Oscar G Potter, Te-Wei Chu, and Hongfeng Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02100 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Analytical Chemistry

Improved LC/MS Methods for the Analysis of Metal-Sensitive Analytes using Medronic Acid as a Mobile Phase Additive

Jordy J. Hsiao*, Oscar G. Potter, Te-Wei Chu, Hongfeng Yin

Agilent Technologies, Santa Clara, California 95051, United States *Corresponding Author Tel.: 1-408-553-2132 E-mail: [email protected]

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Abstract Phosphorylated compounds and organic acids with multiple carboxylate groups are commonly observed to have poor peak shapes and signal in LC/MS experiments. The poor peak shape is caused by the presence of trace metals, particularly iron, contributed from a variety of sources within the chromatographic system. To ameliorate this problem, different solvent additives were investigated to reduce the amount of metal in the flow path to achieve better analytical performance for these metal-sensitive compounds. Here, we introduce the use of a solvent additive that can significantly improve the peak shapes and signal of metal-sensitive metabolites for LC/MS analysis. Moreover, the additive is shown to be amenable for other metal-sensitive applications, such as the analysis of phosphopeptides and polar phosphorylated pesticides, where the instruments could be used in either positive or negative analysis mode.

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INTRODUCTION Liquid chromatography-mass spectrometry (LC/MS) is an important analytical technique to answer an increasing number of significant biological and clinical questions.1 These questions often require the expertise of the proteomic and metabolomic research communities.2,3 However, methods for interrogating phosphorylated analytes and metabolites are often challenging and require extensive sample preparation and enrichment steps prior to analysis.4-6 Moreover, there remain analytical challenges in resolving structural isomers, broad peak shapes, and ion suppression from sample matrices.7,8 Analysis of polar metabolites such as nucleotides and organic acids by reversed-phase chromatography is difficult because of their low retention.9 Increased retention of charged polar metabolites on reversed-phase columns could be achieved with the use of ion pairing reagents.10 However, one major concern associated with ion-paring reversed-phase (IP-RP) chromatography is that ion-pairing reagents (e.g., tributylamine, hexylamine, etc.) can never be fully washed from the LC system and the column.11-13 Moreover, trace levels of the ion-pairing reagent can change selectivity when used for non-ion-pairing applications, thus making columnto-column reproducibility a problem.14 This forces users to dedicate an entire LC system and column for ion-pairing experiments and renders them inoperable for any non-ion-pairing applications.14 Ion-pairing chromatography also requires longer column equilibration time to reestablish the ion-pair distribution after gradient elution methods.15 Alternatively, hydrophilic interaction chromatography (HILIC) is viewed as a straight forward approach to provide selective separation of both charged and uncharged polar analytes.16,17 However, poor peak shapes for charged metabolites are commonly observed in HILIC-LC/MS experiments.18-20 Poor peak shape is correlated to trace metal contamination in the LC/MS system.18 To overcome the trace metal contamination issue, the LC system can be flushed with ethylenediaminetetraacetic acid (EDTA), a strong metal chelator, or EDTA can be added to the mobile phase.18,19 EDTA and other metal chelators (e.g., citrate) have also been shown to enhance the detection of multiply phosphorylated peptides.21,22 Unfortunately, EDTA is retained on columns and causes ion suppression of co-eluting compounds.19 To limit the amount of metal-mediated effect, 10% water can be incorporated in the organic solvent mobile phase, resulting in lower quantities of iron being extracted from the LC system.23 Moreover, the use of higher pH mobile phases (e.g., pH 9.0) can limit the interaction between stainless steel (SS) surfaces and phosphorylated analytes.24 Even with this prior knowledge, there is still room for significant improvement toward optimal performance of metal-sensitive metabolites using

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LC/MS. In this study, different solvent additives were tested to enhance the performance of metal-sensitive metabolites analyzed by HILIC-LC/MS. MATERIALS AND METHODS

Chemicals and Reagents. All reagents were obtained from their respective manufacturers: acetonitrile (ACN, Chromasolv LC-MS grade), succinate (part no. S2378), malate (part no. M1000), citrate (part no. C7129), isocitrate (part no. I1252), α-ketoglutarate (part no. K3752), Lglutamine (part no. G3126), glutamate (part no. 49621), EDTA (part no. E9884), ammonium hydroxide (part no. 318612), formic acid (part no. 56302-50ml-F), AMP (part no. 01930), ADP (part no. A2754), ATP (part no. A3377), GTP (part no. G8877), NADP (part no. 481972), glucose-1-phosphate (G7018), glucose-6-phosphate (G7250), pyrophosphoric acid (part no. 433314), glufosinate (part no. 45520), glyphosate (part no. 45521), AMPA (part no. 05164), and phosphopeptide mixes (part no. MSP1L, MSP3L) were all from Sigma Aldrich (St. Louis, MO). Ammonium acetate, ACS grade, was purchased from VWR (part no. 631-61-8; Radnor, PA). Water was purified in-house using an EMD Millipore Milli-Q water purification system (Billerica, MA). The InfinityLab Poroshell 120 2.7 µm HILIC-Z columns (2.1 x 50 mm and 2.1 x 150 mm; part no. 679775-924 and 673775-924) (Agilent Technologies, Santa Clara, CA) were used for the metabolite and pesticide analysis. The AdvanceBio Peptide Mapping column (2.1x100mm; part no. 653750-902) (Agilent Technologies, Santa Clara, CA) was used for the phosphopeptide analysis in this study. The deactivator mobile phase additive (5 mM medronic acid) was obtained from Agilent (part no. 5191-4506). Instrumentation. All instrument modules were from the 1290 Infinity line from Agilent Technologies (Waldbronn, Germany): binary pumps (Model G4220A), autosampler (G4226A), and temperature controlled column compartment (Model G1316C). The mass spectrometers used included the Agilent 6545 Q-TOF, 6550 iFunnel Q-TOF, and 6490 iFunnel QQQ. Mass axis calibration was performed using a standard tuning compound mixture (Agilent, part no. G1969-85000). Hexakis(1H,1H,3H-perfluoropropoxy)phosphazene was used as a reference mass (m/z 922.0098) compound for calibration of mass spectra, which was sprayed continuously into the JetStream source from a secondary reference nebulizer at 40 psig. LC/MS Analysis of Metabolites and Polar Pesticides. Stock solutions of the analytes were made in Milli-Q purified water at 5 mg/mL. Sample solutions were made by diluting the stock to 4


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1 ng/µL (ppm) in 80:20 acetonitrile/water. Hydrophilic interaction chromatography (HILIC) was performed using a 50 mm or 150 mm x 2.1 mm i.d. InfinityLab Poroshell 120 HILIC-Z column (Agilent Technologies). A stock solution of 100 mM ammonium acetate was made by dissolving 0.1 moles of ammonium acetate in water, adjusting to pH 9.0 with ammonium hydroxide, and making the volume up to 1 L with water. Solvent A was made by mixing 100 mL of the stock solution and 900 mL of water, which yields a final concentration of 10 mM ammonium acetate (pH 9.0) in water. Solvent B was made by mixing 100 mL of the stock solution with 900 mL of ACN, which yields a final concentration of 10 mM ammonium acetate (pH 9.0) in 90% ACN. The deactivator additive (5 mM methylenediphosphonic acid) was spiked into the solvents at a final 5 µM concentration for analysis. The flow rate was 0.25 mL/min and the column temperature was set at 25ºC. A sample volume of between 0.2 - 3 µL was injected onto the column for each experiment. After loading of the sample solution, the column was held at 90% solvent B for 2 min before the gradient with solvent A was applied. The gradient elution profile was from 90 to 60% B for 10 min followed by washing with 60% B for 3 min. The column was equilibrated with 90% B for 8 min prior to subsequent analysis. Full MS (MS1) data were acquired with a mass range of 50-1000 m/z and an acquisition rate of 1 spectrum/s on the 6545 Q-TOF system. An MRM method was also set up to acquire data on the 6490 iFunnel QQQ system. The instruments were operated in negative mode for metabolite and pesticide analysis. LC/MS Analysis of Phosphorylated Peptides. The MS phosphoMix1 Light (Sigma Aldrich) and MS phosphoMix3 Light (Sigma Aldrich) were resuspended with 1% formic acid in water at 2 pmol/µL. Reversed-phase chromatography was performed using a 100 mm x 2.1 mm i.d. AdvanceBio Peptide Mapping column (Agilent Technologies). Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in ACN. The deactivator additive (5 mM methylenediphosphonic acid) was spiked into solvent A at a final 5 µM concentration for analysis. The flow rate was 0.25 mL/min and the column temperature was set at 25 ºC. 2.5 µL (5 pmol) of the phosphopeptide mix was injected onto the column for each experiment. After loading of the sample solution, the column was held at 3% B for 2 min before the gradient with solvent B was applied. The gradient elution profile was from 3 to 38% B for 10 min followed by washing with 90%B for 4 min. The column was equilibrated with 3% B for 8 min prior to subsequent analysis. MS1 data were acquired with a mass range of 400-1250 m/z and acquisition rate of 1 spectrum/s on the 6550 iFunnel Q-TOF system. The instrument was operated in positive mode for peptide analysis.

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RESULTS AND DISCUSSION Effect of Pyrophosphoric Acid and Medronic Acid on Metabolites. A compendium of organic acids and phosphorylated biomolecules were used as a standard mix to study the effect of EDTA as a metal chelator in HILIC-LC/MS. As expected, the addition of 5 µM EDTA to both A and B solvents is shown to significantly improved the peak shapes of chelating organic acids (i.e. malate, citrate, and isocitrate) and reduced the tailing of multiply phosphorylated nucleotides (i.e. ADP and ATP) (Figure 1A and Figure S1). However, the EDTA addition to the mobile phase solvents also caused a significant reduction (>50%) in the signal intensity (i.e. peak area, peak height) due to ion suppression for all tested analytes as published previously (Figure S1A and S1B).19 Therefore, experiments were conducted to identify an alternative mobile phase additive that would help achieve peak shapes with high symmetry and efficiency and have minimal ion suppression on target analytes.

Figure 1. LC/MS chromatograms of metabolites with EDTA as a mobile phase additive. (A) 15 ng of samples (3 µl) were analyzed on the HILIC-Z column with mobile phases containing 10 mM ammonium acetate, pH 9.0, with or without 5 µM EDTA in the eluent. (B) Chromatogram of EDTA (291.0837 m/z) across the gradient run is shown. The first chemical additive tested was pyrophosphoric acid. This compound was chosen because it contains two phosphate groups that could complex with trace metals and competitively remove the metals away from the target analytes. Pyrophosphoric acid was added 6


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to both A and B solvents at low micromolar concentrations (5 µM) that resulted in improved peak shapes and signal intensity for the phosphorylated nucleotides, especially the multiply phosphorylated metabolites (e.g., ADP, ATP, and GTP) (Figure 2A and Figure S1). To a lesser extent, the peak shapes of the chelating organic acids, such as malate, citrate, and isocitrate that contain two or more carboxyl groups also showed improved peak shapes but still with some tailing observed (Figure 2A and Figure S1D). Remarkably, the ion suppression effects observed with EDTA addition were not observed with pyrophosphoric acid as the additive. (Figure 2A vs. 1A). This is supported by a much lower signal detected for pyrophosphoric acid when compared to EDTA (~30x difference), which suggests that EDTA forms more gas phase ions than pyrophosphoric acid at an equimolar concentration, acting as a more potent ionization suppressor (Figure 2B vs. 1B). Although the peak shapes for the phosphorylated nucleotides were satisfactory with the pyrophosphoric acid additive, we decided to test other phosphoric acid derivatives to further improve the peak shape of the metal-sensitive metabolites.

Figure 2. Chromatograms of metabolites with pyrophosphoric acid as the mobile phase additive. (A) 15 ng of samples (3 µL) were analyzed on the HILIC-Z column with mobile phases containing 10 mM ammonium acetate, pH 9.0, with or without 5 µM pyrophosphoric acid in the eluent. (B) Chromatogram of pyrophosphoric acid (176.9364 m/z) across the gradient run is shown. Another candidate mobile phase additive was medronic acid, also known as methylenediphosphonic acid. Medronic acid has two phosphate groups flanking a central carbon atom instead of an oxygen atom like pyrophosphoric acid, which may provide stronger

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chemical stability. Therefore, medronic acid was added to the mobile phase solvents to determine whether it could improve the performance of metal-sensitive analytes. As shown in Figure 3A, addition of medronic acid to the mobile phase solvents at 5 µM concentration significantly improved the peak shape and signal intensity for all target analytes (Figure S1). Specifically, sugar phosphates and phosphorylated nucleotides showed sharpened peak shape and increased signal intensity. Moreover, the medronic acid addition resulted in minimal or no tailing observed and increased signal intensity for the organic acids (Figure S1). Importantly, medronic acid yielded better peak shape with reduced USP tailing factor for malate and citrate/isocitrate than pyrophosphoric acid, and thus functions as a better mobile phase additive (Figure S1).

Figure 3. Chromatograms of metabolites with medronic acid as a mobile phase additive. (A) 15 ng of samples (3 µL) were analyzed on the HILIC-Z column with mobile phases containing 10 mM ammonium acetate, pH 9.0, with or without 5 µM medronic acid in the eluent. (B) Chromatogram of medronic acid (174.9567 m/z) across the gradient run is shown.

Next, we sought to determine the optimal medronic acid concentration in the mobile phase solvents for metabolite analysis by LC/MS. To test this, the metabolite standard mix was analyzed with mobile phase solvents containing 0.5, 1, 2.5, 5, and 10 µM of medronic acid. The results showed that 2.5 – 5 µM of the additive gave the best performance without sacrificing sensitivity caused by ion suppression (Figure 4 and Figure S2B). At 0.5 – 1 µM concentration, the performance of a few analytes started to deteriorate relative to the 2.5 – 5 µM additive concentration range. For example, ADP splits into two peaks and malate suffers from noticeable 8


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peak tailing. At 10 µM concentration, ion suppression was noticeable across all analytes tested (Figure S2A and S2B), which suggests that at high enough concentration, the medronic acid additive could still impose ion suppressive effects on target analytes (Figure 4).

Figure 4. Optimizing medronic acid concentration as a mobile phase additive with metabolite standards. 1 ng (0.2 µL) of the sample was analyzed on the HILIC-Z column in stainless steel hardware. The connection capillaries were PEEK-lined with a 1290 Binary Pump coupled to a 6490 iFunnel QQQ. The mobile phases were switched after 5 consecutive runs. Since ion-pairing reagents are known to retain on LC systems and analytical columns, we wanted to determine whether medronic acid also exhibited similar characteristics. To test this, a LC system that had been continuously operated with mobile phase solvents containing 5 µM of medronic acid was switched to mobile phase solvents without the additive. Specifically, after 3 runs (75 min) with medronic acid in the mobile phase solvents, the LC instrument was left overnight, and 3 more runs (75 min) were conducted. Mobile phase solvents without medronic acid were then switched onto the LC instrument, and 5 runs (125 min) were conducted. In the absence of the mobile phase additive, the medronic acid level decreased more than 50% within the first analytical run (4x105 vs. 1.2x105 counts). By the 5th analytical run without the mobile phase additive, the level precipitously decreased from 4x105 to 3x103 counts

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(Figure 5). Thus, in contrast to ion-pairing reagents, medronic acid does not persist on an analytical column or LC/MS instrument.

Figure 5. Unlike ion-pairing reagents, medronic acid does not contaminate the LC/MS system. After 3 runs (75 min) with medronic acid in the mobile phases, the LC instrument was left overnight, and 3 more runs (75 min) were conducted. The mobile phases were then switched to mobile phases without medronic acid, and 5 runs (125 min) were conducted. Chromatograms of medronic acid (174.9567 m/z) in negative analysis mode are shown. HILIC is often perceived to have reproducibility issues, 25 which could be partly attributed to interactions with trace metals contaminants within the LC and chromatography system. To demonstrate the reproducibility of this method, analytical runs with metabolite standards were conducted across 3 different days with mobile phase solvents containing medronic acid or in the absence of the additive as control. As shown in Figure S3, the metabolites standards exhibited poor reproducibility in peak area over the 3-day experiment with relative standard deviation (RSD) values greater than 30% for all tested analytes in the control runs. In contrast, the tested analytes yielded higher peak area reproducibility with less than 10% RSD for all tested analytes in the analytical runs using mobile phase solvents containing 5 µM medronic acid (Figure S3B).

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Interestingly, the retention time at maximum height was highly reproducible for both experimental conditions, as the RSD values were less than 1% (Figure S3C). To expand on the reproducibility study, samples spiked with increasing concentrations of urea and NaCl (up to 160 mM urea and 80 mM NaCl) intended to mimic urine samples were analyzed with the HILICLC/MS method (Figure S4). The results showed that this method maintained retention time reproducibility with minimal ion suppression detected for the target sugar phosphate isomers (i.e. glucose-1-phosphate and glucose-6-phosphate) and nucleotides (e.g., AMP, ADP, ATP, GTP, and NADP) in high salt samples (Figure S4). These results conclusively showed that the use of medronic acid as a mobile phase additive improves the reproducibility of HILIC-LC/MS experiments. Effect of Medronic Acid on Phosphorylated Polar Pesticides. Because routine pesticide testing of food and water has become increasingly critical to ensure the health and safety of consumers,

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we decided to expand the use of the additive to polar pesticides containing

phosphate groups. To determine the metal-sensitivity of the polar pesticides, glufosinate, glyphosate, and aminomethylphosphonic acid (AMPA) were analyzed with HILIC-Z stationary phase packed in either stainless steel (SS) or PEEK-lined SS hardware (Figure 6). These results showed that phosphate containing pesticides such as glyphosate and AMPA are sensitive to metals in the sample flow path. This is evident as decreased signal and poor peak shape was observed for glyphosate and AMPA analyzed on the SS column (Figure 6). In contrast, the PEEK-lined SS column yielded much better peak shape and intensity than the SS column (Figure 6). For glufosinate, which does not contain a phosphate group, the signal and peak shape was generally not affected by column hardware (Figure 6).

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Figure 6. 1 ng (1 µL, 1 ppm) of pesticides were analyzed to determine the effects of SS hardware compared to PEEK-lined SS hardware without any additives on the 6490 iFunnel QQQ instrument. Next, the pesticides were analyzed on a PEEK-lined SS column to test whether the addition of medronic acid to the mobile phase solvents could improve the performance of these analytes (Figure 7).

As expected, for glyphosate and AMPA, the peak shape and signal

strength improved with the addition of 5 µM medronic acid to the mobile phase solvents (Figure 7). Interestingly, we observed a slight ion suppression to the glufosinate signal. This indicated that 5 µM medronic acid in the mobile phase solvent can cause ion suppression of glufosinate, and suggests a further optimization of the medronic acid concentration is needed for users interested in targeting all 3 pesticides in a single LC/MS run.

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Figure 7. Chromatograms of polar pesticides with medronic acid as a mobile phase additive. 1 ng of pesticides were analyzed on the HILIC-Z column in PEEK-lined SS hardware. Black chromatogram indicates runs without medronic acid addition. Red chromatograms indicate runs with medronic acid addition. Runs were repeated 5 times on the 6490 iFunnel QQQ instrument. Effect of Medronic Acid on Phosphorylated Peptides. The application of the medronic acid additive was also extended to phosphopeptide analysis in positive mode by LC/MS. A phosphorylated standard peptide mix, consisting of singly, doubly, triply, and tetraphosphorylated peptides, was subjected to RP chromatography coupled to LC/MS analysis. In this analysis, the results clearly demonstrated that addition of medronic acid to the mobile phase solvent promoted the detection of the phosphorylated peptides that had either low or undetectable signal in the absence of the additive (Figure 8). This finding represents a significant advancement in the phosphoproteomic field as enrichment steps and fractionation strategies are generally required for the detection and identification of phosphorylated peptides in the proteome.4

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Figure 8. Chromatograms of phosphopeptides with medronic acid as a mobile phase additive. 5 pmol (2.5 µl) of phosphopeptide mix was analyzed on the AdvanceBio Peptide Mapping column with water and acetonitrile containing 0.1% formic acid as mobile phases in positive analysis mode. Black chromatograms indicate runs without medronic acid addition. Red chromatograms indicate runs with medronic acid addition. Runs were repeated 5 times. Red text for the peptide sequences indicates the phosphorylated residues. CONCLUSION In this study, we have developed chromatographic conditions that can significantly improve the performance of negatively charged molecules with minimal or no signal suppression observed. This is achieved through the addition of medronic acid at low micromolar concentrations to the mobile phase solvents for LC/MS analysis. More specifically, medronic acid addition to the mobile phase solvents improved peak shape and signal strength for a wide variety of anionic molecules including organic acids, nucleotides, sugar phosphates, phosphopeptides, and phosphorylated polar pesticides. Unlike EDTA or ion-pairing reagents, the medronic acid additive is readily cleared from the LC/MS system and does not show a residual signal after a few chromatographic runs. Moreover, medronic acid enhanced the reproducibility of analytical runs with samples containing high salt that mimics urine samples. Lastly, the medronic acid additive may be used in both positive and negative analysis modes by LC/MS. Pyrophosphoric acid was also tested as a mobile phase additive. However, it was well established that pyrophosphoric acid hydrolyzes over time into orthophosphoric acid.27 This makes it less suitable as an additive compared to medronic acid, since the degradation of pyrophosphoric acid solutions over time may lead to inconsistent results. Furthermore, medronic acid was observed to give improved enhancement of analyte signals compared to an equimolar concentration of pyrophosphoric acid in the mobile phase solvents. The use of medronic acid with low pH mobile phase solvents for HILIC-LC/MS experiments was also investigated (Figures S5 and S6), but in general most target analytes containing multiple phosphate and carboxyl groups performed better with high pH mobile phase solvents. It’s important to be aware that not all analytes will ionize with the high pH mobile phase solvents. For example, polyamines (e.g., spermine, spermidine, and putrescine) would only ionize with low pH mobile phase solvents and could only be detected with the mass spectrometer set in positive analysis mode. 28

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The use of medronic

acid as a mobile phase additive

will facilitate the

identification/quantification of metal-sensitive analytes and help achieve lower detection limits. We envision medronic acid could also be utilized in additional applications targeting metalsensitive analytes such as aqueous normal phase on silica hydride, ion-pairing reversed-phase, ion exchange chromatography (IEX), or liquid chromatography-inductively coupled plasmamass spectrometry (LC-ICP-MS). Acknowledgment The authors wish to thank Dr. Gregory O. Staples for the insightful scientific discussions and editing of the manuscript.

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MS Methods for the Analysis of Metal-Sensitive Analytes

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