Determination of Sub-Nanomolar Levels of Low Molecular Mass

Article pubs.acs.org/ac

Determination of Sub-Nanomolar Levels of Low Molecular Mass Thiols in Natural Waters by Liquid Chromatography Tandem Mass Spectrometry after Derivatization with p‑(Hydroxymercuri) Benzoate and Online Preconcentration Van Liem-Nguyen, Sylvain Bouchet,‡ and Erik Björn* Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden S Supporting Information *

ABSTRACT: Low molecular mass (LMM) thiols is a diverse group of compounds, which play several important roles in aquatic ecosystems, even though they typically occur at low concentrations. Comprehensive studies of LMM thiols in natural waters have so far been hampered by selectivity and limit of detection constraints of previous analytical methods. Here, we describe a selective and robust method for the quantification of 16 LMM thiols in natural waters. Thiols were derivatized with 4-(hydroxymercuri)benzoate (PHMB) and preconcentrated online by solid-phase extraction (SPE) before separation by liquid chromatography and determination by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Their quantification was performed by selective reaction monitoring (SRM), while the presence of a product ion at m/z 355, specific for thiols and common for the investigated compounds, also allows to screen samples for unknown thiols by a precursor ion scan approach. The robustness of the method was validated for aqueous matrices with different pH, sulfide, and dissolved organic carbon (DOC) concentrations. The limits of detection for the thiols were in the sub-nanomolar range (0.06−0.5 nM) and the methodology allowed determination of both reduced and total thiol concentrations (using tris(2-carboxyethyl)phosphine (TCEP) as reducing agent). Six thiols (mercaptoacetic acid, cysteine, homocysteine, N-acetyl-cysteine, mercaptoethane-sulfonate, and glutathione) were detected with total concentrations of 7−153 nM in boreal lake or wetland pore waters while four thiols (mercaptoacetic acid, cysteine, homocysteine, and N-acetyl-cysteine) were detected in their reduced form at concentrations of 5−80 nM.

S

redox distribution in natural waters since disulfides have been reduced to thiols prior to analysis in most studies.11−13 Although thiols play many important biogeochemical roles in aquatic ecosystems there has been more focus on developing analytical techniques for their determination in biological samples.14−16 A comprehensive characterization of thiols at relevant concentrations (down to sub-nanomolar) in natural waters is lacking but is critical to refine our understanding of both sulfur cycling and metal speciation. Thiols are commonly derivatized before analysis to enhance their chemical stability, separation by liquid chromatography (LC) and detection by various techniques. Indeed, LC separation combined with ultraviolet/visible radiation (UV−vis) absorption16,17 or fluorescence18−22 spectroscopy, or with electrospray ionization mass spectrometry (ESI-MS),23−27 are today the most widely used techniques for the identification and quantification of thiols. Some selectivity and sensitivity issues however remain with these methods. The instrumentation for UV−vis absorption and fluorescence spectroscopy are the least complex and costly but they require thiol derivatization with luminescent active tags28,29 which in

ulfur is a versatile element that exists in multiple oxidation states among which thiol functional groups (i.e., sulfhydryls, R-SH) are some of the most reactive chemical functionality found in biomolecules and natural organic matter (NOM). Thiols can be associated with high or low molecular mass organic molecules and as reduced thiols (R-SH) or oxidized disulfides (R-S-S-R). Disulfide bonds can be reduced to the corresponding thiols through enzymatic activities, which make thiols very useful as structural elements in disulfide bridges of proteins or as part of oxidation defense systems.1,2 Living organisms produce a set of well characterized low molecular mass (LMM) thiols (e.g., cysteine, glutathione, phytochelatins) for various physiological purposes such as redox regulation and xenobiotic detoxification,3−6 which can be excreted or released upon cell lysis. In natural waters, a variety of additional thiols can be present, originating from sulfide (HS−) additions to unsaturated functional groups of NOM such as acrylates7 or from direct release by anthropogenic activities (e.g., Beiner et al.8). Reduced free thiols show a high binding affinity for class B metals, thereby controlling important processes of metals in ecosystems, such as solubility, transport, biological uptake, and reactivity.9 The concentration ratio between reduced and oxidized forms of thiols are frequently studied in biological systems,10 to infer their oxidative stress level, but very few studies have investigated this © 2014 American Chemical Society

Received: October 1, 2014 Accepted: December 17, 2014 Published: December 17, 2014 1089

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EXPERIMENTAL SECTION Chemicals and Reagents. All thiol compounds were purchased from Sigma-Aldrich; their structures and abbreviations used throughout this manuscript are given in Supporting Information Figure S-1. Tris(2-carboxyethyl)phosphine (TCEP), sodium sulfide nonahydrate (Na2S, 9 H2O) and 4(hydroxymercuri)benzoate (PHMB) were also purchased from Sigma-Aldrich. The reference humic acids (HA, CAS number 1415−93−6) and Nordic fulvic acids (FA, catalog number 1R105F) were purchased from Sigma-Aldrich and the International Humic Substance Society, respectively. Ammonium acetate was from Baker Analyzed, formic acid (FA) from Fluka, acetonitrile (ACN) and methanol (MeOH) from Merck, all of at least analytical grade. Ultrapure water (>18 MΩ.cm) was obtained through a Milli-Q Advantage A10 Ultrapure Water Purification System (Merck Millipore). Stock solutions were prepared in pure MQ water for TCEP (10 mM) and in 0.1% NH4OH (from 25% aqueous solution, Scharlau) for PHMB (5 mM), kept in the dark at +4 °C and used within 1 week. (Note that PHMB is toxic and must be handled with appropriate protections under a fume hood). Thiol standards were prepared individually with Milli-Q water deoxygenated by He purging (30 min, 200 mL min−1) (except thiosalycilic acid in 1-propanol, see below), kept at −20 °C and used within a month. Sulfide stock solutions (200 μM) were prepared in deoxygenated Milli-Q water in an alpha glovebox (Saffron Scientific Equipment Ltd., North Yorkshire, UK) under N2 atmosphere. Stock solutions of humic and fulvic materials were prepared in 0.1% NH4OH (2000 and 4000 mg L−1 for humic and fulvic, respectively) and kept in the dark at +4 °C. Thiols Reduction and Derivatization. Reduced thiols were directly derivatized with PHMB without TCEP addition but to determine total thiol concentrations, 24 μL of 10 mM TCEP were added to 10 mL samples. Vials were then shaken for 20 s and left to react for 15 min at room temperature before the addition of PHMB. The derivatization of thiols are described by the following reactions

some cases are not fully specific toward thiol compounds. UV− vis absorption detection lacks both sensitivity and selectivity since many organic compounds absorb radiation in the UV range. Fluorescence is more sensitive and selective than UV−vis absorption but it may be affected by autoquenching (radiation self-absorption) and interferences from NOM, as well. Inductively coupled plasma mass spectrometry (ICP-MS) enables thiol quantification with low limits of detection (LODs) after tagging with heteroatoms.23 Still, all these detection techniques rely only on chromatographic retention time to identify individual thiol compounds and the risks of misidentification and quantification problems are therefore relatively large since many thiols have similar structure. ESIMS potentially provides a higher reliability because analyte identification is ensured by compound specific mass spectra including transitions from induced dissociations while low LODs can be achieved as well. Seiwert et al.24 reported a method to determine thiols in urine samples by a tandem mass spectrometry (MS/MS) method using N-(2-ferrocenethyl)maleimide as a probe with LODs ranging from 30 to 110 nM. The compound 4(hydroxymercuri)benzoate (PHMB) is also known to be an efficient probe due to its high selectivity for thiol groups, short reaction time and stability of the complexes formed. The reaction between PHMB and reduced thiol groups is normally complete in less than 90 s and thiol−PHMB complexes are stable at least 1 day at room temperature and up to 3 months at −20 °C.25,30 Recently, Rao et al.25 and Bakirdere et al.23 achieved LODs of 1− 32 nM for 6 different thiols in yeast extracts following derivatization with PHMB and LC-ESI-MS analysis with an Orbitrap instrument operated in positive ionization mode and a resolving power setting of 100 000. However, because of the reactive nature of LMM thiols, the steady state concentration of thiols in natural waters are often in the low nanomole range13 and the matrix composition of natural waters presents some additional challenges that have not been addressed in previous works. For example, natural waters contain various amounts of dissolved sulfide and organic compounds that may affect the thiol derivatization reaction and analyte recovery of preconcentration procedures if used. Additionally, only few previous methods have concerned determination of reduced thiols in such samples, but have only included total thiol concentrations by using a reducing agent. Tris(2-carboxyethyl)phosphine (TCEP) has been commonly employed for this purpose due to its high efficiency and stability in a wide pH range.25,31 In this work, we have developed a novel methodology for the determination of a comparatively large number of thiols (16 compounds) at relevant concentration levels in natural waters. We obtained excellent instrument signal-to-noise ratio and selectivity using regular tandem MS instrumentation which are more commonly available than high resolution instrumentation. This was accomplished by using triple quadrupole MS in negative ESI mode and a selective reaction monitoring (SRM) methodology based on each thiol fragmentation pathway. Combined with online preconcentration by solid phase extraction (SPE) and separation by reversed-phase LC this method offered sub nM LODs for thiol compounds, which is an improvement compared to previous MS based methods and comparable to the lowest LODs reported up to date with fluorescence detection. The method allowed to determine both reduced and total thiols in various types of natural waters and also to screen for unknown thiols.

RSH + HOHgC6H4COOH → RSHgC6H4COOH + H 2O

(1)

RS− + HOHgC6H4COO− → RSHgC6H4COO− + OH−

(2)

Reaction 1 takes place in acidic while reaction 2 occurs in alkaline media23,32 The optimization of the PHMB to TCEP concentration ratio was carried out by keeping the TCEP concentration constant at 24 μM, 4.8 μM of thiols (300 nM each of 16 thiols), while 4.8− 38.4 μM of PHMB were subsequently added to the vials in both Milli-Q water and in the presence of 20 mg L−1 dissolved organic carbon (DOC) matrices, which were again shaken for 20 s and left 5 min to react. The optimization of the PHMB to thiol molar ratio was done by keeping constant the concentration of thiols (300 nM each) while varying the PHMB and TCEP concentrations from 3.9 to 117 μM in the case of pure Milli-Q water solutions or from 3.9 μM to 273 μM in the presence of 20 mg L−1 DOC. Sulfide interferences were investigated by varying the concentration of dissolved sulfides from 0 to 50 μM while keeping constant the concentrations of thiol (300 nM each, total 4.8 μM for 16 thiols), TCEP (24 μM), and PHMB (24 μM). Analyte recoveries of the online SPE method was determined for 1090

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by direct infusion of a 0.2 mM thiol−PHMB solution (25 μL min−1) mixed in 200 μL min−1 of 0.1% formic acid (FA)/MeOH (50/50 v/v). Optimum tube lens and collision energy voltages were thereafter set up individually for each thiol for the SRM method, while compromise values of these two parameters were used when screening samples for unknown thiols using the precursor ion scan method. The structures of the major common product ions were further investigated for two representative thiols (Cys and SUC, diluted in 0.1% FA, ACN) with a LTQ Orbitrap XL equipped with a chip-based nanoelectrospray (Thermo Fisher Scientific, San Jose, CA, USA) and operated at a resolving power of 100 000. Figures of Merit Determinations. The recovery of the online SPE procedure was calculated by comparing the analyte signal intensities without (i.e., direct injection onto the analytical column) and with SPE and correcting for differences in injection volume and analyte concentrations. The thiol−PHMB complexes recoveries of the online SPE in DOC matrices were expressed relative to the Milli-Q water matrix. Three sets of freshly prepared standards were used to investigate linearity and establish calibration curves per concentration basis. The LODs were calculated as 3 times the standard deviation (SD) of blank areas (n = 11) divided by the sensitivity on a peak area per concentration basis. The method’s repeatability was evaluated with triplicate injections of standards or samples. The stability over time of thiol−PHMB complexes was investigated by adding 300 nM of each standard to 500 mL of a natural water sample containing 20 mg L−1 DOC. After homogenization and derivatization, it was split to different types of containers (polypropylene or FEP Teflon) and storage temperature (+4 or −20 °C), and sub samples were regularly analyzed during 40 days. In this case, the instrument drift in sensitivity was corrected by analyzing a set of freshly prepared thiol standards (300 nM). Field Sampling, Sample Processing, and Preservation. Pore waters and surface waters were sampled at three boreal wetland sites: Sjöarödd (SRD) (63°57′37″N 20°40′24″E), Kroksjön (KSN) (63°57′8″N 20°38′13″E), Storkälsmyran (SKM) (63°56′52″N 20°38′48″E), and one lake, Ä ngessjön (64°2′56″N 20°50′17″E), in the north of Sweden during November 2013. These sites were previously characterized for ancillary chemistry, including pH, DOC, and sulfides.33,34 Surface water samples were collected from the lake and wetland streams while pore water samples were extracted from wetland soils at 5−20 cm depth, after discarding the uppermost grass layer, and from the lake sediment at 1−10 cm depth after removing the oxidized top sediment layer (1 cm). Samples were kept overnight in the dark at +4 °C in tight containers. Surface waters were only filtered while sediment and wetland pore waters were first extracted and then filtered, using a funnel filter (0.2 μm, 500 mL, Sarstedt Inc., Newton, USA) under low pressure and N2 atmosphere in the glovebox. Separate subsamples were collected for pH, DOC and sulfide measurements. The reduced and total thiols concentrations were determined in two separate subsamples (10 mL each) derivatized without or with prior TCEP reduction, respectively. The amount of added PHMB and TCEP was estimated based on previous data for DOC and sulfide concentrations at these sites; however, the concentrations of DOC and sulfide were remeasured afterward. The sulfide concentrations were determined by the methylene blue method35 using UV−vis absorption spectrophotometry (GBC 920 UV−vis, 1 cm quartz cuvette). The DOC concentrations were measured using a TOC-VCPH instrument from Shimadzu.

different pH and sample matrices. Humic and fulvic certified materials or natural water samples were diluted with Milli-Q water to obtain DOC concentrations of 20, 40, or 80 mg L−1, then 300 nM of each thiol was added to the solutions followed by TCEP and PHMB additions. Solid-Phase Extraction, Liquid Chromatography, and Mass Spectrometry Operation Procedures. The separation of the thiol−PHMB complexes was evaluated on three reversedphase (RP) columns: an Agilent Zorbax SB-C8 (2.1 × 100 mm, 3.5 μm) fitted with an Ultra C8 guard column (10 × 2.1 mm, 5 μm, Restek); an Inertsil Phenyl column (2.1 × 150 mm, 5 μm); and a Merck Purospher Star C18 column (2.1 × 150 mm, 3 μm). The instrumentation setup consisted of a PAL HTC autosampler (CTC Analytics AG, Zwingen, Switzerland) with a cooled tray (+ 5 °C), a Surveyor and Accela LC-pump (Thermo Fisher Scientific, San Jose, CA, USA) dedicated to the online SPE cartridge (Waters, Oasis HLB, 2.1 × 20 mm, 15 μm) and analytical LC columns, respectively and a TSQ Quantum Ultra electrospray ionization triple quadrupole mass spectrometer instrument (Thermo Fisher Scientific, San Jose, CA, USA). Peek tubing (Ø 0.13 mm, Restek) was used to connect the different instrumental parts. The preconcentration of thiol−PHMB complexes was achieved by loading 1 mL of sample by a stainless steel injection loop onto the online SPE cartridge and a switching-column array made up of a 6-port and a 10-port switching valve manufactured by Valco Instruments Co. Memory effects from previous injections were minimized by adequate washing (syringe, loop, SPE cartridge) after each injection (Supporting Information Table S-1) and was quantified for both calibration standards and natural water matrices by Milli-Q water injections. The elution gradients used for the analytical columns with and without SPE are shown in Supporting Information Table S-2. The general operating conditions for the ESI-MS instruments are given in Table 1. The fragmentation of each thiol−PHMB complex was first studied with a TSQ Quantum Ultra instrument Table 1. Operating Parameters for the Triple Quadrupole and Orbitrap ESI-MS Instruments ESI-MS HPLC column SPE cartridge injection volumes mobile phase ion source mode sheath/auxiliary gas flow collision gas electrospray voltage capillary/vaporizer temperature scan range HR-ESI-MS ion source spraying nozzles mode electrospray voltage capillary temperature capillary voltage scan range resolving power

Thermo Scientific TSQ Quantum Ultra Agilent Zorbax SB-C8 (2.1 × 100 mm, 3.5 μm) Oasis HLB column (2.1 × 20 mm, 15 μm) 10 μL or 1 mL 0.1% FA in water/MeOH (10−90%) heated electrospray ionization negative/positive 60/25 (arbitrary units) 1.5 mL min−1 (argon) 3.5 kV 325/225 °C 100−1500 m/z Thermo Scientific LTQ Orbitrap XL nanoelectrospray triversa NanoMAte 4.1 μM negative/positive 4.5 kV 320 °C 42 V 100−1000 m/z 100 000 or 60 000 1091

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RESULTS AND DISCUSSION Fragmentation of the Thiol−PHMB Complexes and Selection of Product Ions. The fragmentation of each thiol− PHMB complex was first studied by MS/MS in both positive and negative electrospray ionization mode and a mass scan ranging from 100 to 1000 m/z. The spectra of representative thiol− PHMB complexes are given in Supporting Information Figure S2−4. The 3 major product ions associated with each complex are presented in Table 2 with their optimal tube lens voltage,

product ion, while the second and third product ions were used for confirmation (Table 2). We used high resolution mass spectrometry (LTQ Orbitrap XL operated at a resolving power of 100 000 or 60 000) to identify the chemical structure of the most important fragments. Accurate mass measurements of the m/z 355 fragment produced by two representative thiols (Cys and SUC) agreed within 5 ppm with the theoretical mass (354.9738 u) of a complex between the PHMB and a sulfur atom, i.e. C7H5O2Hg−S (Supporting Information Figure S-5). In the same way, the other fragments were identified as C7H5O2Hg (323 m/z), C6H4Hg−S (311 m/z) and C7H5O2 (121 m/z), C9H9O2Hg−S (383 m/z), C5H7O3N2 (143 m/z) (Supporting Information Figure S-6). Therefore, the fragments at m/z 355 and 311 are ideal product ions since the Hg−S bond is preserved, which reinforce the confidence in identification of thiol compounds. It represents a strength of this methodology compared to unspecific detectors, such as electrochemical or UV−vis absorption or fluorescence spectroscopy detection, when screening and quantifying thiols in natural samples containing complex mixtures of organic molecules. Previous ESI based methods for thiol analysis have utilized positive ionization mode and have not reported the generation of fragments specific for thiol compounds as observed with negative ionization mode in our study. Vichi et al.36 used an HR Orbitrap instrument and observed the generation of a common fragment for volatile thiols in virgin olive oil derivatized with ebselen (2phenyl-1, 2-benzisoselenazol-3(2H)-one) via cleavage of the Se− S bond, leaving the derivatizing probe as the common fragment. Reversed-Phase LC Separation of the Various Thiol− PHMB Complexes. Three reversed-phase columns (phenyl, C18, and C8) and two organic solvents (MeOH and ACN) were evaluated to separate the derivatized thiols. The separation of the various thiol−PHMB was overall not satisfactory when using the phenyl column with a 10 mM ammonium acetate mobile phase at pH 5.5 (data not shown). Especially, the TCEP-PHMB complex (m/z 571) could not be separated from the PHMB complexes with Cys, Cyst, and CysGly, suppressing their ionization, and this column was thus discarded. With the C8 and C18 columns the separation of these complexes from the TCEP-PHMB was improved considerably. Overall the separation of thiol−PHMB complexes was not significantly improved when using the C18 compared to the C8 column (data not shown), and the C8 column was chosen for further method optimization due to its lower backpressure and faster re-equilibration. For the C8 column, 0.1% FA in H2O combined with 0.1% FA in MeOH performed better to resolve the complexes (Figure 1) than the same mobile phase using ACN. Two elution gradients differing in flow rate and MeOH content were optimized in order to obtain the best separations and peak shapes with and without the use of SPE (Supporting Information Table S-2). Optimization of the Online SPE Preconcentration Method. The Oasis HLB SPE cartridge employed in this method contains phenyl and 2-pyrrolidone as functional groups to offer both hydrophobic and hydrophilic interactions. The use of SPE preconcentration significantly reduced the intensity of the TCEP-PHMB peak compared to direct injection to the C8 analytical column, further minimizing potential ionization suppression problems for closely eluting compounds. This is likely due to its dissociation or poor retention on the SPE stationary phase. The preconcentration efficiencies were evaluated for thiol standards with respect to pH and increasing concentrations of DOC. For standards in Milli-Q water and 20 mg L−1 of DOC, the highest analytes recovery was found at the

Table 2. Product Ions Obtained from Each Thiol−PHMB Complex with the Triple Quadrupole Mass Spectrometer Instrument (Thermo Scientific TSQ Quantum Ultra)a precursor mass (m/z)

tube lens (V)

ETH

398.8

−136.9

Cyst

399.7

104.3

MAC

412.8

−107.8

2-MPA

426.9

−111.5

3-MPA

426.9

−111.5

Glyc

428.8

−133.6

Cys

442.0

−111.4

Hcys

455.8

−116.0

SULF

462.8

−117.1

Pen

469.7

−101.8

SUC

470.8

−91.8

NACCys

483.9

−106.3

CysGly

498.9

−114.6

NACPen

511.9

−99.3

GluCys

570.9

−103.1

GSH

627.8

−115.1

thiols

product ions (m/z) (relative intensity (%) and optimum collision energy (V) are given in parentheses) 355.0 (100%, 14), 323.1 (78%, 12), 108.3 (50%, 28) 383.0 (100%, 10), 323.0 (49%, 17), 340.1 (24%, 10) 121.1 (100%, 24), 323.1 (18%, 14), 367.1 (15%, 6) 121.1 (100%, 22), 355.0 (21%, 23), 166.1 (13%, 23) 121.1 (100%, 22), 355.0 (21%, 23), 166.1 (13%, 23) 323.1 (100%, 13), 279.2 (14%, 24), 355.0 (11%, 19) 355.0 (100%, 17), 234.2 (5%, 46), 109.1 (5%, 44) 355.0 (100%, 18), 311.1 (7%, 30), 354.0 (2%, 14) 355.0 (100%, 25), 311.1 (8%, 33), 109.2 (5%, 44) 355.0 (100%, 17), 311.1 (6%, 33), 109.2 (4%, 37) 355.0 (100%, 21), 311.1 (13%, 32), 453.2 (10%, 12) 355.0 (100%, 18), 109.2 (4%, 42), 354.0 (3%, 17) 355.0 (100%, 21), 311.1 (13%, 30), 109.2 (3%, 40) 355.0 (100%, 18), 311.1 (6%, 36), 109.2 (3%, 47) 355.0 (100%, 23), 442.1 (45%, 14), 553.2 (13%, 14) 143.1 (100%, 27), 355.0 (58%, 25), 610.4 (6%, 16)

a

The product ions displayed in bold font were used for quantification and the other product ions for confirmation.

collision energy, and respective abundances. The absolute intensities of the precursor ions were higher in positive compare to negative ionization mode; however, most of the complexes did not produce detectable or useful product ions in positive mode and noise levels were relatively high. In negative ionization mode, the absolute intensities were lower but the signal-to-noise ratios were improved considerably for the precursor and product ions for all thiols except Cyst, which contains only an amino functional group. Interestingly, the complexes’ fragmentation often led to the same product ions and the most recurrent ones were found at m/z 355, 323, 311, and 121. Especially, 14 of the 16 thiol−PHMB complexes showed a major product ion at m/z 355 and it was the dominant one for 10 of them. On the basis of their fragmentation patterns, the quantification of the thiol−PHMB complexes was thereafter performed by SRM using the main 1092

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PHMB/TCEP ratio up to a ratio of about 1 while the signal intensities decreased for half the complexes at further increased ratio, in both Milli-Q and natural waters (Supporting Information Figure S-9, 10). Therefore, a PHMB to TCEP ratio of 1 was used thereafter. For the optimization of PHMB to thiol ratio, in both Milli-Q water and in the presence of 20 mg L−1 DOC, maximum signal intensities for all 16 thiols were achieved when the ratio of PHMB to thiol reached 3. The intensities of most thiol−PHMB complexes did not vary much when the ratio of PHMB to thiol was increased further up to the maximum investigated ratio (30 times in Milli-Q water and 70 times in 20 mg L−1 DOC) (Supporting Information Figure S-11, 12). It demonstrated that a large excess of PHMB can be used without causing interferences. Concerning the effect of free dissolved sulfides, the signal intensities of 5 thiols (Cys, HCys, CysGly, Glyc, and ETH) significantly decreased when the sulfide concentration was gradually increased from 0 to 20 μM such that the molar ratio of PHMB to sulfide approached 1, and the signal intensities for all of the thiols were drastically reduced when the sulfide concentration increased to 50 μM and thus exceeded the PHMB concentration (Supporting Information Figure S-13). Studies on the chemical speciation of sulfur in various extracted humic and organic soils by sulfur X-ray absorption near edge structure (S-XANES) spectroscopy have shown that the amount of thiol groups were relatively constant at 0.15% of total carbon on a mass basis.37−39 On the other hand, a study based on an equilibrium dialysis ligand exchange (EDLE) method have reported 0.014% thiol groups of DOC on a mass basis.40 Considering this variability, the concentration of thiol groups in samples containing 15−150 mg L−1 DOC could range from 66 nM (15 mg L−1 DOC and 0.014% of DOC) to maximum 5.8 μM (150 mg L−1 DOC and 0.15% of DOC). As the molar ratio of PHMB to the sum of total thiols and sulfides should be at least 3, the appropriate PHMB concentration used for derivatization depends on the thiol and sulfide concentrations in the sample. Consequently, for oxic samples containing low sulfide concentrations such as surface, stream, and soil pore waters, a concentration of 15−50 μM of PHMB is recommended but for suboxic/anoxic samples exhibiting high sulfide contents, such as pore waters of marine sediments, a more careful adjustment of the PHMB concentration, according to sulfide concentration is required. Figures of Merit. The recoveries for the thiol−PHMB complexes when using the SPE preconcentration procedure compared to direct injection to the LC column was in average 74 ± 7% (range 65−88%) for a Milli-Q water matrix. Also relative recoveries were determined in the presence of DOC matrices compared to Milli-Q water when using the SPE procedure. These relative recoveries were on average 84 ± 14% and 90 ± 15% (range 70−110%) for a mixture of humic reference materials (humic + fulvic acids) and a natural water sample, respectively, both containing 20 mg L−1 DOC (Supporting Information Table S-3). It should be observed that in these experiments, prederivatized thiol−PHMB complexes were added to the DOC matrix solutions to evaluate specifically the effect of DOC on the SPE preconcentration and LC-ESI-MS/MS analysis. The relative recoveries decreased when the concentration of DOC increased to 40 and 80 mg L−1 and was on average 60 ± 17% and 48 ± 28%, respectively (range 18−102%). The deteriorated recoveries at increased DOC concentration are likely caused by decreased retention efficiency of the thiol− PHMB complexes on the SPE phase because of a competition for

Figure 1. Typical LC-ESI-MS/MS chromatogram obtained for the 16 thiol−PHMB complexes (300 nM each) and the TCEP-PHMB complex using an Agilent Zorbax SB-C8 (2.1 × 100 mm, 3.5 μm) reversed-phase column fitted with an Ultra C8 guard column (10 × 2.1 mm, 5 μm), after online preconcentration by SPE (1 mL).

range of pH from 2.5 to 5.0 and it decreased when pH was increased further (Supporting Information Figure S-7, 8). This pattern of analyte recovery suggests that the complexes are mainly retained by hydrophobic rather than hydrophilic interactions. Indeed, the pKa of the carboxylic group range from 1.7 to 5.4 for the thiols, while it is 3.9 for PHMB. As the pH is increased, the complexes became more charged, and thus hydrophilic, which likely decreased their retention. Optimization of the Thiol Derivatization Procedure. Natural waters contain reduced sulfur species that might compete with LMM thiols for PHMB, such as free sulfide and thiol groups associated with DOC. The optimization of the PHMB to TCEP, and PHMB to thiol molar ratios were evaluated with standards in both Milli-Q and a natural water containing 20 mg L−1 DOC and for varying sulfide concentrations. For the optimization of PHMB to TCEP molar ratio, both the peak shape and intensity of thiol−PHMB complexes improved with 1093

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Analytical Chemistry

matrix to LODs was investigated by comparing signal-to-noise ratio (S/N) between Milli-Q water and the 20 mg L−1 DOC matrix (and 30 nM of each thiol). The results showed that the difference in S/N between the two matrices was less than 20% for all thiols except ETH and CysGly, for which the S/N was reduced 2 and 3 times, respectively, in the 20 mg L−1 DOC compare to the Milli-Q water matrix. Using negative ESI mode and SRM without the SPE preconcentration, we achieved LODs comparable to the methods using high resolution Orbitrap instruments23,25 and those typically reported with fluorescence detection.19,22,41,42 In combination with online SPE preconcentration our method gained LODs comparable to the lowest ones reported with fluorescence detection,21,43 in the sub-nanomolar range. Because of poorer selectivity, the fluorescence methods are however more prone to deteriorated LODs for complex sample matrices21 and interference effects between different thiols.19 It is well-known that reduced thiol groups are highly reactive and must be stabilized to ensure preservation before analysis.15 Our investigations using a natural water sample matrix demonstrated that derivatized thiols can be stored in propylene or Teflon containers at room temperature for a full working day (data not shown), at +4 °C for at least up to 10 days, and in Teflon containers at −20 °C for at least 40 days for all thiols (Supporting Information Figure S-16). Determination of LMM Thiols in Natural Water Samples. We applied the optimized method to determine the concentrations of reduced and total thiols in various pristine surface, stream and pore waters of a freshwater lake and boreal wetlands (Table 4). From the 16 selected thiols, 6 were detected at the sites investigated, five being of direct biological origin (Cys, HCys, NACCys, GSH, and SULF) and one (MAC) being of indirect biological origin, that is, addition of sulfides to unsaturated organic matter. Reduced thiols were not detected in the lake surface or the wetland stream waters; however their concentrations ranged from 5.2 to 80 nM in pore waters, and the sum of reduced thiols ranged from 26 to 110 nM. On the contrary, oxidized thiols were present in all samples and total (oxidized + reduced) concentrations of individual thiols ranged from 6.2 to 150 nM. Cys and MAC were detected in most samples while HCys, SULF, NACCys, and GSH were mainly present in pore waters. The sums of the total thiol concentrations were highest in the wetland pore waters (170−410 nM), intermediate in the wetland stream waters (99−120 nM) and lowest in the lake (12−63 nM). These concentration levels were in good agreement with previous studies where total LMM thiol concentrations, but not reduced LMM thiols, were quantified. Zhang et al.13 reported concentrations of various thiols (Cys, MAC, 3-MPA, NACCys and GSH) up to 250 nM in overlying waters and sediment pore waters from Canadian wetlands using LC with fluorescence spectroscopy detection. Dryden et al.12 studied the seasonal variations of thiol concentrations in the water column of a polluted estuary and detected Cys, GSH, SUC, MAC, ETH and 3-MPA, ranging from below LOD to 168 nM using LC with UV/vis absorption spectroscopy detection. To our knowledge, this study is the first time that SULF and HCys are detected in natural waters. Screening for Unknown Thiols Using a Precursor Ion Scan Method. The specific fragmentation pathways of the thiol−PHMB complexes in negative ESI mode offer an opportunity to screen samples for unknown thiols by performing precursor ion scan while targeting the 355 or 311 m/z fragments. We exemplified the potential of such an approach by adding two

adsorption sites, and ionization suppression in the ESI source. These results suggest that water samples containing more than 20 mg L−1 DOC should be diluted to maintain high analyte recoveries and/or calibration techniques such as standard addition or isotope dilution should be applied to correct for such losses. Calibration curves associated with the two different methods, i.e. with and without SPE preconcentration, are given in Supporting Information Figure S-14, 15. Significant differences in the slope were observed for the complexes, ranging from 0.3 × 106−3 × 107 arbitrary unit peak area units per μM (without SPE). The calibration curves remained linear up to the highest concentrations investigated, that is, 20 and 0.5 μM without and with SPE, respectively. It is not expected that the concentrations of individual thiols would frequently exceed 20 μM in natural water samples.12 The method reproducibility and LODs of the various thiols are given in Table 3. The relative standard Table 3. Limits of Detection (LODs) and Analytical Reproducibility (Given as 1 Relative Standard Deviation, RSD) Achieved for Each Thiol with Optimized LC-ESI-MS/ MS Conditions without and with Solid Phase Extraction (SPE)a without SPE

with SPE

thiols

LODs (nM)

RSD (%)

LODs (nM)

RSD (%)

ETH MAC 2-MPA 3-MPA Glyc Cys Hcys SULF Pen SUC NACCys CysGly NACPen GluCys GSH Cyst

5.4 2.6 0.8 1.0 6.2 1.4 3.2 0.7 1.6 0.8 1.3 2.3 0.6 3.1 3.6 3.3

1.7 1.1 8.5 9.1 2.1 3.3 6.5 8.3 4.0 1.7 3.9 6.2 8.2 5.6 8.6 0.7

0.5 0.4 0.2 0.1 0.3 0.1 0.2 0.3 0.2 0.1 0.2 0.1 0.06 0.2 0.2 0.1

10.8 13.3 13.8 13.2 3.4 12.0 7.0 9.4 12.8 1.9 5.8 7.3 11.7 8.1 5.7 9.1

Min Max average

0.6 6.2 2.5

0.7 9.1 5.0

0.06 0.5 0.2

1.9 13.8 9.2

a

The LODs were determined as 3σ of 11 blank replicates.The RSDs were established with 5000 and 100 nM standard solutions in Milli-Q water without SPE and with SPE pre-concentration, respectively.

deviation (RSD) of standard triplicate injections was always better than 14% and averaged 5.0% and 9.2% without and with SPE, respectively. For the complexes added to the natural water sample (containing 20 mg L−1 DOC), the reproducibility ranged from 4 to 14% and averaged 9% with SPE preconcentration. The LODs without and with SPE preconcentration ranged from 0.6 to 6.2 nM and 0.06 to 0.5 nM, respectively. The LODs were thus improved on average by a factor of 13 when using the SPE procedure. This improvement was lower than expected from the theoretical preconcentration factor (i.e., 100), which is explained by nonquantitative recovery of the complexes and a rise in noise levels due to preconcentration of other organic molecules than the thiol−PHMB complexes by the SPE. The effect of sample 1094

DOI: 10.1021/ac503679y Anal. Chem. 2015, 87, 1089−1096

Article

T 140 ± 12 31 ± 1 7.1 ± 0.5 9.3 ± 1 13 ± 2 200

3.89 ± 0.02 86.3 ± 5.3 R T 73 ± 6 26 ± 2 99 3.97 ± 0.03 49.1 ± 3.5 R T 94 ± 2 23 ± 2 120

R 80 ± 8 28 ± 3 5.2 ± 0.4 110

KSN

3.77 ± 0.04 1.16 ± 0.02 63.2 ± 5.5

3.86 ± 0.07 0.47 ± 0.05 34.0 ± 3.6 R T 100 ± 9 11 ± 2 35 ± 3 15 ± 1 36 ± 2 26 170

SKM

3.91 ± 0.04 2.83 ± 0.12 151.2 ± 11.3 R T 130 ± 7 31 ± 3 150 ± 10 6.4 ± 0.5 13 ± 0.6 18 ± 2 25 ± 1 88 ± 9 49 410

contrasting “unknown” thiols to a natural water sample containing 20 mg L−1 DOC. These two compounds (3mercaptopropansulfonate and thiosalicylic acid) were not included in the previous methodological developments and represent hydrophilic and hydrophobic thiols. Two peaks for m/ z 355 were clearly detected at 11.7 and 20.9 min, respectively, for which the precursor ion masses matched the corresponding thiol−PHMB complexes for these two compounds (Supporting Information Figure S-17). This proof-of-concept experiment demonstrates the potential of such a methodology to screen for unknown thiols by targeting these fragments. For real samples, once the precursor masses and retention times of unknown thiols are determined, their structures can be elucidated by analyzing the sample again either by product ion scans for precursor masses of interest by tandem MS and/or by accurate mass determination by high resolution MS measurements. We estimated the LOD for the precursor ion scan method to be approximately 10 nM for 4 representative thiols (Cys, Pen, NACCys, and NACPen, Supporting Information Figure S-18), using the 3σ criterion of blank measurements.

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CONCLUSIONS A novel and robust method for the determination of 16 LMM thiols in natural water samples with LODs among the lowest up to date was successfully developed. The unique fragmentation pathway of thiol−PHMB complexes in negative electrospray ionization mode provides excellent selectivity also for complex sample matrices and allow to screen samples for unknown thiol compounds by using a precursor ion scan methodology targeting a common PHMB-thiol product ion at m/z 355. The method was successfully applied for the determination of LMM thiols in different natural water samples and 6 thiols were found with concentrations ranging from 6 to 153 nM. The methodology brings an opportunity to a more comprehensive understanding about the processes of LMM thiols in aquatic ecosystem, including the binding affinity of class B metals to thiols.

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4.16 ± 0.02 37.4 ± 2.3 T 80 ± 4 29 ± 1 6.2 ± 0.5 120

soil pore water

SRD SKM KSN

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ‡

- represents not detected concentrations.

S.B.: IPREM-LCABIE, CNRS UMR 5254, Université de Pau et des Pays de l’Adour, Technopole Helioparc, 2, avenue du Président Angot 64053 PAU cedex 09.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financial supported by the Kempe Foundations (SMK-2745, SMK-2840), the JC Kempe Memorial Scholarship Foundation, and Umeå University. Jerker Fick and Richard Lindberg, Umeå University, are greatly acknowledged for technical support on the TSQ Quantum Ultra instrument. The Swedish Metabolomics Centre (www. swedishmetabolomicscentre.se) is acknowledged for the help with the Orbitrap MS analysis.

a

7.26 ± 0.04 0.33 ± 0.05 22.6 ± 3.2 R T 23 ± 3 35 ± 4 11 ± 2 14 ± 1 8.3 ± 0.7 6.3 ± 0.2 34 63 6.55 ± 0.02 13.3 ± 2.1 R T 12 ± 1 12 pH sulfide (μM) DOC (mg L−1) thiols (nM) MAC Cys Hcys SULF NACCys GSH Sum

sediment pore water surface water

ASSOCIATED CONTENT

S Supporting Information *

R -

SRD

stream water

wetlands lake

Table 4. Determined pH, Concentrations of Dissolved Sulfides, DOC, and Reduced (R) and Total (T) Thiols in a Freshwater Lake (Ä ngessjön) and Three Boreal Wetland Sites Sjöarödd (SRD), Kroksjön (KSN), Storkälsmyran (SKM) Located in Northern Swedena

Analytical Chemistry

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(38) Skyllberg, U.; Bloom, P. R.; Qian, J.; Lin, C. M.; Bleam, W. F. Environl. Sci. Technol. 2006, 40, 4174−4180. (39) Skyllberg, U. J. Geophys. Res.: Biogeosci. 2008, 113, No. G00C03. (40) Haitzer, M.; Aiken, G. R.; Ryan, J. N. Environ. Sci. Technol. 2002, 36, 3564−3570. (41) Ivanov, A.; Nazimov, I.; Baratova, L. J. Chromatogr. A 2000, 870, 433−442. (42) Sakhi, A. K.; Blomhoff, R.; Gundersen, T. E. J. Chromatogr. A 2007, 1142, 178−184. (43) Tang, D.; Hung, C.-C.; Warnken, K. W.; Santschi, P. H. Limnol. Oceanogr 2000, 45, 1289−1297.

REFERENCES

(1) Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711− 760. (2) Gilbert, H. F. Methods Enzymol. 1984, 107, 330. (3) Dickinson, D. A.; Forman, H. J. Ann. N.Y. Acad. Sci. 2002, 973, 488−504. (4) Gulati, P.; Klöhn, P. C.; Krug, H.; Göttlicher, M.; Markova, B.; Böhmer, F. D.; Herrlich, P. IUBMB Life 2001, 52, 25−28. (5) Moran, L. K.; Gutteridge, J.; Quinlan, G. J. Curr. Med. Chem. 2001, 8, 763−772. (6) Moriarty-Craige, S. E.; Jones, D. P. Annu. Rev. Nutr. 2004, 24, 481− 509. (7) Vairavamurthy, A.; Mopper, K. Nature 1987, 329, 623−625. (8) Beiner, K.; Popp, P.; Wennrich, R. S. J. Chromatogr. A 2002, 968, 171−176. (9) Boulegue, J.; Lord, C. J., III; Church, T. M. Geochim. Cosmochim. Acta 1982, 46, 453−464. (10) Hansen, R. E.; Roth, D.; Winther, J. R. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 422−427. (11) Hu, H.; Mylon, S. E.; Benoit, G. Limnol. Oceanogr 2006, 51, 2763−2774. (12) Dryden, C. L.; Gordon, A. S.; Donat, J. R. Mar. Chem. 2007, 103, 276−288. (13) Zhang, J.; Wang, F.; House, J. D.; Page, B. Limnol. Oceanogr 2004, 49, 2276−2286. (14) Toyo’oka, T. J. Chromatogr. B 2009, 877, 3318−3330. (15) Hansen, R. E.; Winther, J. R. Anal. Biochem. 2009, 394, 147−158. (16) Kuśmierek, K.; Chwatko, G.; Głowacki, R.; Kubalczyk, P.; Bald, E. J. Chromatogr. B 2011, 879, 1290−1307. (17) Vairavamurthy, A.; Mopper, K. Anal. Chim. Acta 1990, 236, 363− 370. (18) Toyo’oka, T.; Imai, K. J. Chromatogr. A 1983, 282, 495−500. (19) Tang, D.; Wen, L.-S.; Santschi, P. H. Anal. Chim. Acta 2000, 408, 299−307. (20) McMenamin, M. E.; Himmelfarb, J.; Nolin, T. D. J. Chromatogr. B 2009, 877, 3274−3281. (21) Guo, X. F.; Zhu, H.; Wang, H.; Zhang, H. S. J. Sep. Sci. 2013, 36, 658−664. (22) Isokawa, M.; Funatsu, T.; Tsunoda, M. Analyst 2013, 138, 3802− 3808. (23) Bakirdere, S.; Bramanti, E.; D’ulivo, A.; Ataman, O. Y.; Mester, Z. Anal. Chim. Acta 2010, 680, 41−47. (24) Seiwert, B.; Karst, U. Anal. Chem. 2007, 79, 7131−7138. (25) Rao, Y.; Xiang, B.; Bramanti, E.; D’Ulivo, A.; Mester, Z. J. Agric. Food. Chem. 2010, 58, 1462−1468. (26) Huang, Y. Q.; Ruan, G. D.; Liu, J. Q.; Gao, Q.; Feng, Y. Q. Anal. Biochem. 2011, 416, 159−166. (27) Rao, Y. L.; McCooeye, M.; Mester, Z. Anal. Chim. Acta 2012, 721, 129−136. (28) Kusmierek, K.; Chwatko, G.; Glowacki, R.; Kubalczyk, P.; Bald, E. J. Chromatogr. B 2011, 879, 1290−1307. (29) Peng, H. J.; Chen, W. X.; Cheng, Y. F.; Hakuna, L.; Strongin, R.; Wang, B. H. Sensors 2012, 12, 15907−15946. (30) Bramanti, E.; Vecoli, C.; Neglia, D.; Pellegrini, M. P.; Raspi, G.; Barsacchi, R. Clin. Chem. 2005, 51, 1007−1013. (31) Krijt, J.; Vacková, M.; Kožich, V. Clin. Chem. 2001, 47, 1821− 1828. (32) Bramanti, E.; D’Ulivo, L.; Lomonte, C.; Onor, M.; Zamboni, R.; Raspi, G.; D’Ulivo, A. Anal. Chim. Acta 2006, 579, 38−46. (33) Tjerngren, I.; Karlsson, T.; Bjorn, E.; Skyllberg, U. Biogeochemistry 2012, 108, 335−350. (34) Skyllberg, U.; Karlsson, A.; Fredriksson, I.; Björn, E.; Meili, M. Geochim. Cosmochim. Acta 2009, 73, 1236. (35) Cline, J. D. Limnol. Oceanogr. 1969, 14, 454−458. (36) Vichi, S.; Cortes-Francisco, N.; Caixach, J. J. Chromatogr. A 2013, 1318, 180−188. (37) Qian, J.; Skyllberg, U.; Frech, W.; Bleam, W. F.; Bloom, P. R.; Petit, P. E. Geochim. Cosmochim. Acta 2002, 66, 3873−3885. 1096

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