Speciation of Inositol Phosphates in Lake Sediments by Ion

Chem. , 2015, 87 (5), pp 2672–2677 .... Gradient elution was used (see Table 1) with the flow rate of 200 μL min–1. ... 1, 0.00, 0. 2, 0.01, 30. ...
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Speciation of Inositol Phosphates in Lake Sediments by IonExchange Chromatography Coupled with Mass Spectrometry, Inductively Coupled Plasma Atomic Emission Spectroscopy, and NMR Spectroscopy

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Julia V. Paraskova,† Charlotte Jørgensen,‡ Kasper Reitzel,‡ Jean Pettersson,† Emil Rydin,† and Per J. R. Sjöberg*,† †

Department of Chemistry−Biomedical Centre, Uppsala University, P.O. Box 599, S-751 24 Uppsala, Sweden Department of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark

‡

ABSTRACT: A method for the detection and speciation of inositol phosphates (InsPn) in sediment samples was tested, utilizing oxalate−oxalic acid extraction followed by determination by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC−MS/MS) using electrospray ionization (ESI) in negative mode. The chromatographic separation was carried out using water and ammonium bicarbonate as mobile phase in gradient mode. Data acquisition under MS/MS was attained by multiple reaction monitoring. The technique provided a sensitive and selective detection of InsPn in sediment samples. Several forms of InsPn in the oxalate−oxalic acid extracted sediment were identified. InsP6 was the dominating form constituting 0.250 mg P/g DW (dry weight); InsP5 and InsP4 constituted 0.045 and 0.014 mg P/g DW, respectively. The detection limit of the LC−ESI-MS/MS method was 0.03 μM InsPn, which is superior to the currently used method for the identification of InsPn, 31P nuclear magnetic resonance spectroscopy (31P NMR). Additionally sample handling time was significantly reduced.

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provide a means for detection, as inositol phosphates do not contain any strongly ultraviolet-absorbing chromophores.12 The development of highly sensitive and selective detectors, such as mass spectrometers, which provide structural information on the compounds studied, has opened the possibility for the successful identification of inositol phosphates.13 In this study extraction of metal-associated InsPn, proposed as a selective method for isolating and identifying inorganically bound InsP6 in environmental samples, was combined with HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the identification and quantification of InsPn in sediment samples. The aim of this study was to develop a rapid and sensitive method for screening and quantification of InsPn using ESI-MS/MS as an alternative to 31P NMR.

nositol phosphates (InsPn) represent the dominant group of organic phosphorus (P) compounds in many soils and sediments.1,2 Despite their relative abundance compared to other organic P compounds, they are rarely considered contributors to eutrophication. InsPn are nevertheless transported and accumulated in aquatic systems, and their degradation may contribute to eutrophication effects such as the growth of cyanobacteria.2 The preferred method for the identification of InsPn in environmental samples is solution 31P nuclear magnetic resonance spectroscopy (31P NMR). The technique has been utilized for several decades, and its main advantage is the ability to detect multiple species of organic P compounds simultaneously.3,4 However, the relatively poor detection limit (0.05 mg mL−1) generally limits the use of NMR to the mere identification of InsPn. Additionally, to improve recovery and resolution of the specific compounds studied, adequate analysis requires sample preconcentration,5,6 introducing the risk of sample loss and sample degradation. Many different chromatographic techniques, such as size exclusion chromatography,7 ion-exchange chromatography,8,9 reversed-phase chromatography,10 and high-performance liquid chromatography (HPLC)11 have been used for studying InsPn. However, the biggest limitation in using chromatography has been the inability of the most-commonly used UV detectors to © XXXX American Chemical Society

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EXPERIMENTAL SECTION Samples. Experiments were performed on lake sediments from the Danish Lake Kvie, collected in May 2013 with a Kajak corer. Ten replicate sediment cores were sampled from the deepest part of the lake (2 m). The upper 2 cm of the cores

Received: September 5, 2014 Accepted: February 4, 2015

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DOI: 10.1021/ac5033484 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry were pooled and air-dried at ambient temperature (22 °C) for 7 days prior to analyses. Chemicals. All chemicals were of analytical grade (SigmaAldrich, Germany), unless otherwise specified. Dilution was performed with Milli-Q (MQ) water (Millipore, Bedford, MA). In-house standard solutions were prepared from the solution of phytic acid (∼40% in H2O), phytic acid sodium salt hydrate from rice, and the dipotassium salt of myo-inositol hexakis(dihydrogen phosphate). The calibration of the inductively coupled plasma atomic emission spectrometer (ICP-AES) was done with solutions prepared from single-element P stock (Spectrascan, Teknolab AB, Kungsbacka, Sweden). Extraction of Inositol Phosphates from Sediment. Sediment samples were extracted with ammonium oxalate− oxalic acid after Jørgensen et al.14 A brief description of the method follows. Air-dried sediment was extracted in darkness with 0.2 M oxalate−oxalic acid (pH 3.0) for 2 h. An aliquot of the extract was filtered through a 0.20 μm RC membrane syringe filter (Thermo Scientific, Waltham, MA) and stored at 4 °C for analysis by HPLC−MS/MS. The iron content in the remaining extract was reduced by shaking the sample with Fluka Amberlite IR120 cation-exchange resin (pH 10.0) for 1 h, and the pH in the supernatant was further increased to >12 to standardize to routine 31P NMR measurement protocols.15 The solution was subsequently frozen and lyophilized. The lyophilized extracts were redissolved in 0.3 mL of deuterium oxide (D2O) and 2.7 mL of solution containing 1 M sodium hydroxide (NaOH) and 0.1 M ethylenediaminetetraacetic acid (EDTA), centrifuged at 3300g for 10 min to remove particulates, and immediately analyzed by solution 31P NMR spectroscopy. Preparation of In-House Standards. In-house standard solutions were prepared by hydrolyzing three varieties of phytate to produce a mixture of myo-inositol phosphate isomers: mono- (InsP1), bis- (InsP2), tris- (InsP3), tetrakis(InsP4), pentakis- (InsP5), and hexakisphosphate (InsP6) by modifying a published method.16 The available phytate salts and solution were dissolved in 2.5 mL of 3.2 M acetic acid, heated for at least an hour, dried under a stream of nitrogen, and redissolved in 1 mL of MQ-water. The distribution of the different InsPn (InsP1−InsP6) in the processed mixture thus depended on the temperature and the length of the procedure (combinations tried were 100 °C for 60 min, 120 °C for 60 and 90 min, and 150 °C for 60 min). The goal was to achieve a mixture of the higher inositols (InsP4, InsP5, and InsP6) as they were expected to have higher relevance in environmental samples. Characterization of the In-House Standards. The total concentration of P in the prepared InsPn standards was determined by direct infusion experiments with ICP-AES. Subsequently, the characterization of InsPn in the in-house standard was performed by HPLC−ICP-AES. The distribution of the InsPn in the standards was calculated by integrating peak areas in the obtained chromatogram with Origin 9.0 (OriginLab, Northampton, MA) and relating them to the total integrated area. InsPn standards with concentrations ranging from 0.3 to 46 μM were tested to ensure that the relative response (peak area vs total peak area) was independent of concentration. HPLC. The chromatographic system used was 1260 Infinity (Agilent Technologies, Waldbronn, Germany) with binary pumps, an autosampler, and a column oven. The dwell volume in the pump was reduced by disconnecting the mixer and pulse

damper. The column used was a weak anion-exchange column, Hypersil GOLD AX (100 mm × 2.1 mm, particle size 3 μm) from Thermo Scientific. The mobile phases for the chromatographic separation were A, MQ-water, and B, 400 mM aqueous (NH4)HCO3 (pH = 9). Gradient elution was used (see Table 1) with the flow rate of 200 μL min−1. Table 1. Gradient Used for the Elution and Separation of Inositol Phosphates in the In-House Standard and Extract Solutions step

time [min]

mobile phase B [%]

1 2 3 4 6 7

0.00 0.01 20.00 23.00 23.01 48.00

0 30 100 100 0 0

The injected sample size was 20 μL. Total run time was 48 min, where 22 min were used for the separation and 26 min for washing and equilibrating the column between the runs. ICP-AES. Spectro Ciros CCD ICP-AES (Spectro Analytical Instruments, Kleve, Germany) equipped with a modified Lichte nebulizer and a cyclonic spray chamber (Precision Glassblowing, Centennial, CO) was used for total phosphorus determination. The chromatographic system was connected to the ICP through a poly(ether ether ketone) (PEEK) capillary which was fitted directly onto the nebulizer. The flow from the column was split 1:1 prior to the connection with the ICP to reduce carbon load to the plasma. Operating conditions used were as follows: plasma power 1.25 kW, coolant flow 14.00 L min−1, auxiliary flow 0.90 L min−1, nebulizer flow 0.90 L min−1. Smart Analyzer 2.1 (Spectro Analytical Instruments, Kleve, Germany) was used to collect transient signal for 25 min with an integration time of 3 s for the atomic P lines at 177.495 and 213.618 nm. The instrument was calibrated with a set of standard solutions ranging from 0 to 1 ppm P, prepared in 160 mM aqueous (NH4)HCO3. The effect of (NH4)HCO3 on the response was first tested on 10 standard solutions with addition of (NH4)HCO3 ranging from 0 to 400 mM. ESI-MS. The chromatographic system was coupled with a 3200 Q Trap LC−MS/MS system (AB Sciex, Concord, ON, Canada). The flow from the column (200 μL min−1) was mixed in a T-junction with a flow of methanol (200 μL min−1) resulting in a total flow of 400 μL min−1 prior to entering the interface (Figure 1). The ESI source was operated in negative ion mode with ion spray voltage set to −2.6 kV, curtain gas flow to 10 psi, nebulizer gas flow to 60 psi, turbo gas flow to 60 psi, and temperature to 700 °C. Optimal conditions for the MS/MS

Figure 1. Schematic setup of the HPLC−MS system. In the HPLC− ICP-AES setup, the line to waste was used as a split and pump 2 disconnected. B

DOI: 10.1021/ac5033484 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Heating a solution of the dipotassium salt of phytic acid for 90 min at 120 °C gave suitable results (Figure 2).

detection were screened through direct infusion experiments. The selected flow for infusion experiments was 10 μL min−1 (ramping of collision energy, declustering potential). Each InsPn was identified through multiple reaction monitoring (MRM) (Table 2). Table 2. Specifications for Multiple Reaction Monitoring of the Precursor and Product Ions of Inositol Phosphates (InsPn) analyte

precursor ion m/z

product ion m/z

collision energy V

InsP1

259 259 339 339 418 418 499 499 579 579 659 659

97.0 78.8 241.0 159.0 321.0 158.9 400.9 421.0 480.8 401.0 560.9 580.8

−35 −40 −30 −35 −20 −30 −30 −40 −35 −40 −40 −35

InsP2 InsP3 InsP4 InsP5 InsP6

31 P NMR. 31P NMR spectra were recorded at 80.9 MHz on a Varian 200 MHz NMR spectrometer at ambient temperature using a 45° observe pulse, 5 s acquisition time, and a relaxation delay of 15 s, acquiring ca. 16 000 transients. Chemical shifts were indirectly referenced to external 85% H3PO4 via the lock resonance, and the InsP6 signals were identified by comparison with literature findings.15 Peak integration was performed with MestReNova (Mestrelab Research, Santiago de Compostela, Spain). Quantification of the InsP6 was based on the total P concentration in the oxalate−oxalic acid extract and the relative area of the InsP6 signals.

Figure 2. ESI-MS chromatogram of the autoclaved in-house inositol phosphate standard. Each line is a signal for multiple reaction monitoring of a different pair of a precursor and a product ion (see Table 2).

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As can be seen in Figure 2, HPLC with MS/MS detection provided good selectivity for the different InsPn forms. To minimize the likelihood of false identification each InsPn was identified by two MRM transitions, based on neutral-loss of HPO3 (80 Da) or H3PO4 (98 Da). The two MRM transitions corresponding to a specific InsPn resulted in the overlapping of two chromatographic peaks, which was used as a confirmation that the monitored signals came from the intact InsPn and not from an InsPn that has undergone fragmentation in the ion source. For example, for InsP6 eluting at around 20 min (panel 6, Figure 2), the MRM transitions corresponding to InsP5 could clearly be seen (panel 5, Figure 2). However, the chromatographic profiles overlap perfectly, indicating that up-front fragmentation occurs to some extent. The need for high selectivity, provided by the combination of HPLC and MS/MS, is illustrated very well by the signals obtained around 13 min. The signals originate from two intact InsPn, InsP4 and InsP5, as two pairs of separated MRM peak profiles can be observed. An additional advantage of using HPLC−ESI-MS/MS for the identification of standard constituents is that potential impurities can be disregarded, as they are excluded from the monitored signals. So far it has been demonstrated that HPLC−ESI-MS/MS can provide high selectivity, but calibration and quantification at this stage could not be done because the standard composition (concentration of different InsPn in the in-house standard) and the relative response factors of InsP1−InsP6 were unknown. Equation 1 shows the relationship between the response, RInsPn,ESI, and the concentration of the different InsPn forms,

RESULTS AND DISCUSSION In this study a rapid method for screening and quantification of InsPn in sediment samples was successfully developed. Sample preparation was limited to extraction and filtration, the identification was based on making an in-house standards, and quantification was done by HPLC−ESI-MS/MS. Compared to 31P NMR, which has been extensively used for the direct identification of organic P in environmental samples for decades,3 the method is faster and more sensitive. 31P NMR analysis has so far only been able to identify the isomers of InsP6,17 whereas, to our knowledge, no reports on the lower order InsPn isomers exists, probably due to the relatively low sensitivity of the 31P NMR technique. Therefore, the use of HPLC−ESI-MS/MS provides a new and sensitive tool for the identification and quantification of both higher and lower orders of InsPn in environmental samples. Preparation, Characterization, and Quantification of In-House Standards. The work with in-house standards was primarily undertaken because of the high cost of commercially purified InsPn. However, it has been shown that different isomers of InsPn can easily be produced by hydrolyzing phytic acid. Previously described methods have produced a mixture of InsPn via autoclaving, heating in acid, or enzymatic treatment.11,16,18,19 The methods involving increased temperatures produced a more evenly distributed range of InsPn with a better yield of InsP4 and InsP5 and was therefore adopted in this study, albeit with modifications and significantly scaled down. C

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Analytical Chemistry [InsPn], in the in-house standard, where RfInsPn,ESI represents the response factors in ESI. RInsPn,ESI = RfInsPn,ESI[InsPn]

Table 3. Identified Peaks in the In-House Inositol Phosphate Standard peak

(1)

1 2 3 4 5 6

It is recognized that the response in ESI is compounddependent, thus greatly influenced by compound identity and therefore difficult to predict accurately. To overcome this problem the HPLC was hyphenated with ICP-AES as a detector allowing the characterization of the distribution and concentration of the different InsPn forms in the in-house standard. This was based on the assumption that the ICP provides uniform response (RP,ICP) for different P forms (eq 2), meaning that the response factors in the ICP are constant regardless of the origin of P.

RP,ICP = RfP,ICP[P]

RInsPn,ICP = RfP,ICPn[InsPn]

(4)

area [a.u.]

% of total area

phosphate InsP3 InsP4 InsP4 + InsP5 InsP5 InsP6 rest

5.4 8.8 11.1 13.6 17.1 20.9

0.0195 0.0020 0.0130 0.0166 0.0612 0.1792 0.0169 0.3065

6.4 0.6 4.4 5.4 20.0 58.5 4.7 100

chromatogram was identified via HPLC−MS as orthophosphate. The signals for InsP1 and InsP2 were too low for individual characterization and have been characterized as rest. Additionally the fourth peak has been identified as a combination of InsP4 and InsP5, based on the HPLC−MS results (Figure 2). After the relative ICP responses for the different InsPn and the total P concentration in the InsPn standards has been determined, the HPLC−ESI-MS/MS calibration curves could be constructed utilizing eqs 1−4. It is important to point out the peak identified as a combination of InsP4 and InsP5 could not be used for calibration purposes as the selectivity of the HPLC−ICP-AES measurement was not sufficient. The nonoverlapping peaks were used for the calculation of InsP4 and InsP5 response factors in the ESI-MS/MS, and total peak areas were used for quantification. The in-house standard used in this study was prepared from a potassium salt of InsP6 in MQ water and additionally tested in a matrix of ammonium bicarbonate. The only source of P in the standard thus came from the phosphate groups attached to the inositol. It was therefore possible, once characterized, to use the in-house standard for quantification of InsPn in other samples. The in-house InsPn standard was diluted to five different P concentrations, ranging from 0.3 to 46 μM, and the resulting solutions were analyzed with HPLC−ESI-MS/MS. Calibration curves in which peak areas for InsP3−InsP6 were related to concentrations were used for quantification. Linearity was achieved for all the InsPn in the studied range. Although regression coefficients were above 0.99 for all the InsPn, the slopes, i.e., the response factors, of the lines for the lower InsPn were much higher (Table 4), confirming

where RfP,ICP is the response factor in the ICP and [P] is the P concentration. The assumption of structure-independent response in the ICP is not uncommon and has previously been used for the quantification of peptides and proteins, using inorganic elemental standards.20−22 Furthermore, this assumption is supported by experimental data. The total-P measurement of the in-house standard performed with ICP-AES agrees well (within 3%) with the theoretical value for the P-level based on the amount of IP-salt used for preparing the standard. By using P standards with known P concentration and measuring the ICP response during a continuous infusion experiment, the response factors in the ICP (RInsPn,ICP) can be obtained, i.e., the ICP can be calibrated. Additional experiments with standards of constant P concentration and increasing concentrations of (NH4)HCO3 were performed to ascertain that the ICP response was unaffected by increasing concentrations of salt. The concentration of the different InsPn forms in the in-house standard, [InsPn], could then be determined by measuring the response from an HPLC−ICP experiment and utilizing eqs 3 and 4. (3)

time [min]

total

(2)

[P] = n[InsPn]

name

where n is the number of phosphate groups in the respective InsPn. Transient signal data (HPLC−ICP-AES) for the total P response was collected, and individual peaks were identified and related to each other (Figure 3, Table 3). Peak 1 in the

Table 4. ESI-MS/MS Calibration Curves for Inositol Phosphates Ranging from 0.3 to 46 μM InsPn analyte

slope

R2

InsP3 InsP4 InsP5 InsP6

102.4 68.6 43.6 22.4

0.9976 0.9986 0.9987 0.9990

that the response in the ESI is highly dependent on the analyte. Furthermore, an interesting observation was that the response factor was inversely proportional to the number of phosphate groups in the respective InsPn. It can be concluded that the number of phosphate groups in the different InsPn influences the response in the ESI probably due to differences in solvation, i.e., molecules with increasing numbers of phosphate groups have the added difficulty transitioning from aqueous to gas phase. However, it should be pointed out that the absolute response is influenced by the number of instrument parameters,

Figure 3. Phosphorus signal of the autoclaved in-house inositol phosphate standard, detected with HPLC−ICP-AES. D

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Analytical Chemistry such as composition of the mobile phase, collision energy, and MRM transition identity. Sediment Samples. The general procedure for isolating organic P in environmental samples is by extraction of the sample in an alkaline solution containing high concentrations of Na+. This is usually a problem for ESI-MS as different adduct ions, both with analytes and solvents, can be formed, resulting in more complex and noisy spectra. The use of an oxalate extraction combined with 31P NMR has been proposed as a selective method for isolating and identifying inorganically bound InsP6 in environmental samples.14 Extraction with acidified ammonium oxalate in darkness assumes the extraction of amorphous metal oxides of aluminum (Al) and iron (Fe).23 InsPn are often stabilized in the soil by those oxides,15,24,25 which means that an oxalate extraction indirectly selectively enables the extraction of InsPn. It has been shown that in mineral soils the majority of InsP6 is bound to the oxalateextractable amorphous Fe and Al hydroxides. Therefore, oxalate−oxalic acid was used to extract InsPn from the sediment matrix to prevent the addition of Na+ ions to the sample. With HPLC−ESI-MS/MS several forms of InsPn were identified in the oxalate−oxalic acid extracted sediment (Figure 5), which was not possible with 31P NMR. InsP6 was, as expected, the dominating form of InsPn constituting 0.250 mg/g DW (dry weight). Additionally, InsP5 and InsP4 constituted 0.045 and 0.014 mg/g DW, respectively. The results are in good agreement with a previous study that investigated the occurrence of orthophosphate monoesters in lake sediment using solution 31P NMR spectroscopy.1 In Lake Kvie the combined amount of myo-InsP6 and scyllo-InsP6 extracted with the standard EDTA−NaOH method was 0.26 mg/g DW. In this study, 31P NMR identified and quantified InsP6 in the oxalate extract at 0.076 mg/g DW, which was 3 times lower than with the HPLC−ESI-MS/MS method. The discrepancy can, however, be explained by precipitation when the pH value was increased to 10 during the resin treatment. It is necessary to point out that although HPLC combined with ICP-AES was used in this project for standard characterization it clearly lacks the selectivity of the HPLC−MS/MS for quantification of InsPn in sediment samples. An HPLC−ICPAES experiment on a sediment extract (Figure 4) showed that the signals for all lower InsPn were too low to be distinguished from each other. Additionally, a number of other peaks not observed during standard analysis were detected. The broadest peak, which could be contributed to InsP6, is the only InsPn

peak that could be identified. However, when HPLC−ESI-MS/ MS was employed, signals for all InsPn can be identified and all higher InsPn can be quantified (Figure 5).

Figure 5. HPLC−ESI-MS/MS chromatogram of the sediment sample from Lake Kvie.

For quantification of InsPn in real samples it was assumed that the response factor in ESI-MS/MS was the same for all InsPn with the same number of phosphate groups. For example, the total areas for peaks at 14 and 16−18 min, respectively (Figure 4), were used for the quantification of InsP5. The assumption was validated by calculating the peak area for the overlapping peak, InsP4 + InsP5 (Figure 3), by combining the peak areas obtained from the ESI-MS/MS (Figure 2) using the response factor calculated for the separated InsP4 and InsP5. Compared to the 31P NMR spectrum (Figure 6) of the lake sediment, the sensitivity of the LC−ESI-MS/MS was superior

Figure 6. 31P NMR spectrum of a sediment sample from Lake Kvie: peak 1, orthophosphate; peaks 2 and 3, myo-InsP6; peak 4, scyllo-InsP6.

identifying several different forms of InsPn. However, by using 31P NMR it was possible to identify two isomers of InsP6 (scyllo and myo). It should be mentioned here that the inhouse standards that were prepared for the HPLC−ESI-MS/ MS experiments were the isomers of myo-InsP6. These are also the fractions which were quantified. Instrumentation/Hyphenation. Several challenges were met due to the need of hyphenating HPLC with both MS and ICP. While the preferred mobile phase for MS would need to include significant levels of volatile organic solvent to facilitate sample ionization in ESI, the ICP instrument required keeping

Figure 4. Phosphorus signal of the sediment sample from Lake Kvie, detected with HPLC−ICP-AES. E

DOI: 10.1021/ac5033484 Anal. Chem. XXXX, XXX, XXX−XXX

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organic solvents to a minimum, because it has been shown to cool the plasma and decrease sensitivity and stability.26 In some screening experiments performed for this study, mobile phase containing methanol interfered with the plasma formation, extinguishing it during the run. Fortunately the separation of InsPn could be performed equally well with and without the presence of organic modifiers. When the HPLC was coupled with the MS the outlet of the column was connected to the ESI interface through a T-junction, with an additional pump providing an equal flow of methanol. When the HPLC was coupled with the ICP, a T-junction instead split the flow from the column, with half going directly into the nebulizer and half to waste, subsequently reducing the flow rate and carbon load from the ammonium bicarbonate in the mobile phase. The relatively high level of salt in the mobile phase decreased performance of the MS instrument because it tended to build up on the spray capillary tip. The MS signals obtained after separation were significantly reduced if the HPLC−MS was run continuously for more than 10 h or if the system was in standby mode for more than a few hours. Since removing the capillary for cleaning required a cool-down and shut-down of the system, an automated wash procedure was implemented. During the equilibration phase, the nebulizer gas flow was stopped for 3 min and the turbo gas flow was reduced to 20 psi to discontinue spray formation. However, the flow from the HPLC system was not shut off. As a result, water droplets formed at the tip of the capillary, effectively washing any buildup salts from the separation, while the column equilibrated. The optimization of the spray potential had to be executed with great care as signal stability was strongly affected by the applied potential. If the electrical field was too strong very unstable MRM signals were observed, especially if salt residues were present at the spray tip. Carr et al. showed that most buffers affect signals and detection limits in ICP-AES only minimally.26 To confirm this, the effect of ammonium bicarbonate on the ICP-AES signal in this study was investigated by measuring P standards with salt concentrations ranging from 20 to 400 mM. Signal variation for 10 consecutive measurements was under 3%, and there was no correlation between salt content and signal strength. The use of pure aqueous mobile phase, though advantageous for HPLC−ICP analysis, had a negative effect on the column’s performance. The ability of the anion-exchange phase, a polymeric amine ligand bonded to highly pure base-deactivated silica, to retain the analytes started to deteriorate after the column has been used for more than 8 h. The column could be regenerated by filling it with pure ethanol, which was also the preferred storage medium. However, that required a longer wait during a run set. An alternative regeneration step that could easily be automated and which was adopted in this study was one or several consecutive injections of 100 μL of an ethanol/ MQ mix (v/v 50:50), containing 1% formic acid.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46-18-471 3685. Fax: +46-18-471 3692. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by Stiftelsen Lantbruksforskning, SLF (Swedish Farmers’ Foundation for Agricultural Research) and Villum Kann Rasmussen Centre of Excellence: Centre for Lake Restoration (CLEAR).

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REFERENCES

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CONCLUSIONS

The current method is advantageous due to the shortened sample preparation time and minimal sample handling between extraction and measurement as well as providing detailed information on the presence of the different InsPn forms in the sample. For future work it is important to assess whether the extraction and the quantification methods can be applied to other matrixes. F

DOI: 10.1021/ac5033484 Anal. Chem. XXXX, XXX, XXX−XXX