Selective and sensitive detection of chromium(VI) in waters using electrospray ionization mass spectrometry

Science and Justice 53 (2013) 293–300

Contents lists available at SciVerse ScienceDirect

Science and Justice journal homepage: www.elsevier.com/locate/scijus

Selective and sensitive detection of chromium(VI) in waters using electrospray ionization mass spectrometry Effie Weldy, Chloe Wolff, Zhixin Miao, Hao Chen ⁎ Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA

a r t i c l e

i n f o

Article history: Received 4 July 2012 Received in revised form 5 December 2012 Accepted 6 December 2012 Keywords: Chromium(VI) detection Electrospray ionization Mass spectrometry Toxicology Environmental forensics

a b s t r a c t From 2000 through 2011, there were 14 criminal cases of violations of the Clean Water Act involving the discharge of chromium, a toxic heavy metal, into drinking and surface water sources. As chromium(VI), a potential carcinogen present in the environment, represents a significant safety concern, it is currently the subject of an EPA health risk assessment. Therefore, sensitive and selective detection of this species is highly desired. This study reports the analysis of chromium(VI) in water samples by electrospray ionization mass spectrometry (ESI-MS) following its reduction and complexation with ammonium pyrrolidinedithiocarbamate (APDC). The reduction and subsequent complexation produce a characteristic [Cr(III)O]–PDC complex which can be detected as a protonated ion of m/z 507 in the positive ion mode. The detection is selective to chromium(VI) under acidic pH, even in the presence of chromium(III) and other metal ions, providing high specificity. Different water samples were examined, including deionized, tap, and river waters, and sensitive detection was achieved. In the case of deionized water, quantification over the concentration range of 3.7 to 148 ppb gave an excellent correlation coefficient of 0.9904 using the enhanced MS mode scan. Using the single-reaction monitoring (SRM) mode (monitoring the characteristic fragmentation of m/z 507 to m/z 360), the limit of detection (LOD) was found to be 0.25 ppb. The LOD of chromium(VI) for both tap and river water samples was determined to be 2.0 ppb. A preconcentration strategy using simple vacuum evaporation of the aqueous sample was shown to further improve the ESI signal by 15 fold. This method, with high sensitivity and selectivity, should provide a timely solution for the real-world analysis of toxic chromium(VI). © 2012 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Heavy metals can accumulate and persist in the environment posing a health risk to humans. Heavy metal toxicity can result in organ and nervous system damage, cancer, autoimmune diseases, and even death. Some heavy metals can also cross the placental barrier and affect fetal brain development. In infants and small children, heavy metal toxicity could lead to developmental and learning problems, arrested growth, or behavioral issues [1]. Chromium is one such heavy metal. While chromium(III) is essential for glucose metabolism, chromium(VI) is shown to be mutagenic and possibly carcinogenic if inhaled or ingested. In the body, chromium(VI) easily enters cells via sulfate channels due to its similar structure to phosphate and sulfate ions. Inside the cell, chromium(VI) is reduced to chromium(III), and studies show that this reduction reaction can potentially cause DNA damage such as single-strand breaks and base loss or alteration [2–5]. The bulk of chromium enters water supplies as a result of human activities such as leather tanning, metal plating, and textile manufacturing, with the majority being chromium(III) in the form of Cr(OH)2+ ⁎ Corresponding author at: Department of Chemistry & Biochemistry, Athens, OH 45701, USA. Tel.: + 1 740 593 0719; fax: + 1 740 597 3157. E-mail address: [email protected] (H. Chen).

or Cr(OH)2+, and chromium(VI) in the form of Cr2O72− or CrO42−, depending on the pH and concentration of the solution [6–11]. A report released in December, 2010, by the Environmental Working Group (EWG) showed the presence of chromium(VI) in the water supplies of 31 out of 35 cities tested across the United States, representing a potential hazard to public health safety [12]. The growing field of environmental forensic investigation seeks to uncover the facts behind all forms of environmental pollution including heavy metal contamination of our water. Public water supplies in the United States are safeguarded mainly by two federal laws known as the Clean Water Act (CWA), passed in 1972, and the Safe Drinking Water Act (SDWA), enacted in 1974. The SDWA directs the Environmental Protection Agency (EPA) to set quality standards for public drinking water supplies and their sources, while the CWA established rules and regulations governing the discharge of pollutants into surface waters and sets quality standards for those waters. According to the EPA website, from 2000 through 2011, there were 14 criminal cases of violations of the CWA involving the discharge of chromium, often directly into the public sewer system. Penalties for these violations ranged from probation to incarceration with total penalties exceeding $2,000,000.00. In most cases, the EPA is initially notified of a potentially hazardous discharge by companies with a National Pollution Discharge Elimination System

1355-0306/$ – see front matter © 2012 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.scijus.2012.12.003

294

E. Weldy et al. / Science and Justice 53 (2013) 293–300

(cydta=trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid) after reducing chromium(VI) to chromium(III) using ascorbic acid followed by complexation with cydta ligands. The reported limit of detection (LOD) for chromium(VI) was 10 ppb using negative ion ESI-MS [27]. This article describes for the first time the detection of chromium(VI) in water using ESI-MS in the positive ion mode, in conjunction with derivatization using ammonium pyrrolidinedithiocarbamate (APDC; see its structure in panel a of Scheme 1). APDC is known to reduce chromium(VI) and form a complex with the resulting chromium(III) to yield [Cr(III)O]–PDC complex (see the complex structure in panel b of Scheme 1), a protocol widely employed previously using spectroscopic methods for the detection of chromium(VI) [14–20]. The rationale of this work is twofold; first, it is known that the positive ion mode of ESI-MS usually has a higher sensitivity than the negative ion mode. Second, the [Cr(III)O]–PDC complex resulting from the reduction and complexation of chromium(VI) with APDC can be detected as a characteristic positively charged ion (m/z 507), the protonated [Cr(III)O]–PDC complex, using (+)-ESI-MS. The detection of m/z 507 for chromium(VI) can avoid the possible interference from chromium(III) and other metal ions, as demonstrated in this paper. The LOD for chromium(VI) in water using the single-reaction monitoring (SRM) mode can be down to the sub-ppb level, and preconcentration using simple vacuum evaporation of the sample solution may further enhance the detection signal. This highly sensitive and selective method for chromium(VI) analysis would be of great value in practical environmental and forensic applications.

(NPDES) permit. This permit is required for all businesses and municipals discharging pollutants into surface waters. Periodic sampling by permit holders and the reporting of results to the EPA is mandatory. Determining the source of the discharge can be difficult, and involves taking note of the businesses in the area and determining which ones make use of the pollutant in question. Then, physical inspections of possible discharge points may reveal staining or illegal drainage connections. Additionally, a review of company records can reveal poor recordkeeping or lapsed or missing permits, and current and former employees may also be interviewed. Sampling of drains and sewers can then confirm the point of discharge. The EPA began an in-depth health assessment of chromium(VI) in 2008, initially intending to post their finding in 2011. However, the EPA has decided to extend the assessment thereby postponing their conclusions until late 2014 or early 2015. The current EPA regulation regarding chromium levels in drinking water was introduced in 1991 with the maximum contamination level set at 100 ppb total chromium. Since chromium(III) can be oxidized to chromium(VI) under certain environmental conditions, the EPA assumes all chromium detected to be chromium(VI) [13]. Research on the detection of chromium(VI) in the past involve extractions such as cloud point extraction (CPE) [14–16], vesicular liquid coacervate extraction (VLCE) [17], high-performance liquid chromatography (HPLC) [18], or dispersive liquid–liquid microextraction (DLLME) [19,20], and then analysis by inductively coupled plasma mass spectrometry (ICP-MS) [17,18], electrothermal atomic absorption spectrometry (ETAAS, also known as graphite furnace atomic absorption spectrometry (GFAAS)) [18,19], or flame atomic absorption spectrometry (FAAS) [14,16,17,20]. Although some of these methods gain success in detection, they require large volumes of samples (25 to 30 mL), and interference from other metals can be an important issue. Thus, it is often necessary to use a chelator, ethylenediaminetetraacetic acid (EDTA), to remove the unwanted metals from the reaction prior to analysis. Metal ions on the surface of the glassware used could also interfere with analysis and were removed by overnight soaking of glassware in HNO3 [19], which can be time-consuming and laborious. Therefore it is necessary to develop a simple, sensitive, and selective method for the detection of chromium(VI) in environmental samples. Electrospray ionization (ESI) is a soft ionization method [21] whereby the analyte solution is introduced into the ionization source and nebulized into charged microdroplets under high voltage. The charged droplets undergo desolvation to eventually form dry gaseous analyte ions which enter into the vacuum chamber of a mass spectrometer for mass analysis [22–25]. Electrospray ionization-mass spectrometry (ESI-MS) is a proven technique for trace analysis of both small organic compounds and large biological molecules in solution with high sensitivity and selectivity. The trace level detection and quantitative analysis of chromium(VI) has been carried out by ESI-MS in the negative ion mode [26–29]. For instance, Tsunoda's group measured chromium(VI) by ESI-MS based on monitoring ions HCrO4− (m/z 117) and [CrIII(cydta)]− (m/z 394)

a)

2. Material and methods 2.1. Reagents The 1000 ppm chromium(VI) standard, as potassium dichromate K2CrO7 (MW 294 Da), was purchased from Ricca Chemical Company (Arlington, TX) and the chromium(VI) sample solutions used in the experiments were prepared via dilution from this stock solution. The chromium(III) standard, as chromium(III) nitrate nonahydrate [Cr(H2O)6](NO3)3·3H2O (MW 400 Da), ammonium pyrrolidinedithiocarbamate (APDC, MW 164 Da), and lead nitrate were purchased from Sigma-Aldrich (St. Louis, MO). Cadmium nitrate and zinc nitrate were purchased from Spectrum Chemicals (Gardena, CA). Glacial acetic acid, barium sulfate, ferric nitrate, and the MRFA peptide (Met-Arg-Phe-Ala, MW 523 Da) were purchased from Fisher Chemicals (Fairlawn, NJ) and the HPLC grade methanol and ammonium hydroxide were purchased from GFS Chemicals (Columbus, OH). Deionized water was obtained using a Nanopure Diamond Purification System from Barnstead International (Dubuque, IA). The tap water was from the city water supply (Athens, OH), and the river water was taken from the Hocking River (Athens, OH). River water was filtered through Whatman Grade 1 filter paper to remove large particulates.

c)

b) N

N

S S

-

S

+ NH4 S

N S

S

S

O

S Cr

N

Cr

N

S

S

S

S

S

S N

N

Scheme 1. Molecular structures of a) APDC, b) [Cr(III)O]–PDC complex and c) Cr(III)–PDC complex. Note that the two structures shown in b) and c) differ by one oxygen atom.

E. Weldy et al. / Science and Justice 53 (2013) 293–300

295

117 HCrO 4-

117 HCrO 4-

b)

a) 1.5e4

2.0e5

- OH -

Intensity, cps

Intensity, cps

1.5e5 Cr2O 72-

1.0e5

CrO 3

-

HCrO 4- + H 2O

5.0e4

135

1.0e4 CrO 3- 100

5000.0

HCr 2O 7-

100 108

217

0.0

0.0

60 80 100 120 140 160 180 200 220 240 260 280 300 320

80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

m/z

m/z

Fig. 1. (a) Negative ion ESI-MS spectrum of 5 ppm Cr(VI) solution in MeOH/H2O/NH4OH (50:50:0.5 by volume, pH 10.8); (b) CID MS/MS spectrum of HCrO4− (m/z 117).

2.2. Apparatus All the experiments employed a hybrid triple-quadrupole–linear ion trap mass spectrometer Qtrap 2000 (Applied Biosystems/MDS SCIEX, Concord, Canada), with a TurboIonSpray ionization interface. Sample injection flow rate was 10 μL/min. Nitrogen was used as the carrier and curtain gas at 20 and 10 psi, respectively, and as the collision gas for collision induced dissociation (CID). The spray voltages applied for electrospray were 5 kV for the positive ion mode and −5 kV for the negative ion mode. The declustering potential was set at 15 V, and the collision energy was 10 eV. Analyst software (version 1.4.2, Applied Biosystems/MDS SCIEX) was used for data acquisition. An Eppendorf Vacufuge Plus model 5301 was used for sample evaporation. 2.3. Method To obtain the MS and MS/MS data of the [Cr(III)O]–PDC complex, 1 mL of a 4.5 ppm chromium(VI) solution was prepared in deionized water. The pH was adjusted to ~3.5 by adding two drops of acetic acid and 50 μL of 200 mM APDC in water. Following the derivatization procedure outlined elsewhere [15,20], the tube was placed in a 45 °C water bath for 15 min to effect complexation. Subsequently, a 10 μL aliquot of the resulting solution was diluted to 500 μL using MeOH/H2O/HOAc (50:50:1 by volume) to obtain an APDC-derivatized chromium(VI) sample with the final concentration of 0.075 ppm for ESI-MS analysis. The selectivity of the chromium(VI) detection in the presence of chromium(III) under different pH values was examined through

a)

360

4.2e5

391

several experiments. First, two 1 mL solutions were prepared, each containing 4 ppm chromium(VI) and chromium(III). The pH of the first solution was adjusted to ~3.5 with two drops of HOAc and the pH of the second solution was adjusted to ~8.9 with one drop of NH3·H2O. Then 200 μL of 5 mM APDC was added to each solution for derivatization. Both the acidic and basic solutions were heated in water baths at 45 °C and 85 °C, respectively, for 15 min. A 20 μL aliquot of each solution was diluted to 1000 μL with the MeOH/H2O/HOAc solvent and analyzed using the enhanced MS mode. For comparison, a solution was prepared containing only 4 ppm chromium(III) and derivatized by APDC for ESI-MS following the same experimental conditions as mentioned above (derivatization pH: 3.5; heated at 45 °C for 15 min). Similarly, to investigate the interference of other metals on the detection of chromium(VI), a solution containing 4 ppm chromium(VI), barium(II), cadmium(II), iron(III), lead(II), and zinc(II) was derivatized in the same way (derivatization pH: 3.5; heated at 45 °C for 15 min) prior to ESI-MS analysis. For the calibration curve measurements, a 1 mL solution of 9.6 ppm chromium(VI) was prepared in deionized water and the pH was adjusted to ~ 3.5. Then, 125 μL of 5 mM APDC was added and the solution was heated. Six samples ranging in concentration from 3.7 to 148 ppb were prepared by diluting aliquots to 500 μL with the MeOH/H2O/HOAc solvent. After the addition of 0.5 μM of MRFA as an internal standard, the samples were analyzed using the enhanced MS mode. Also, single-reaction monitoring (SRM) scan mode was used to evaluate the LOD for chromium(VI) assay, in which four APDC-derivatized samples of 0.125 to 1.0 ppb were used.

b)

100

3.8e5 344

S

1600

2.2e5

510

515

[[Cr(III)O ]-PDC+H+]

1.8e5

Intensity, cps

Intensity, cps

0 505

2.6e5

507

1.4e5

1400

S S

6.0e4

HN

491

350

370

390

410

430

m/z

450

470

490

N

S

[[Cr(III)O ]-PDC+H+] -163 Da

S HO

400

[Cr(III) -PDC+H+]

2.0e4

SH

S

1000 800

507

-147 Da S

S

N

S

344

Cr

N

1200

O

600

1.0e5

0.0

N

1800

50

3.4e5 3.0e5

360

2000

200 510

530

0 200

240

280

320

360

400

440

480

520

m/z

Fig. 2. (a) Positive ion ESI-MS spectrum of APDC-derivatized Cr(VI) sample; the inset shows the actual (solid line) and simulated (discrete line) isotopic distribution of m/z 507; and (b) CID MS/MS spectrum of m/z 507.

296

E. Weldy et al. / Science and Justice 53 (2013) 293–300

a) Cr(III) only, pH 3.5

b) Cr(VI) and Cr(III)

391

2.2e5 2.0e5 1.8e5 1.6e5

Intensity, cps

Intensity, cps

1.4e5 1.2e5 1.0e5 8.0e4 6.0e4

344

4.0e4 491

2.0e4 0.0 340

360

380

400

420

440

460

480

500

520

344 1.4e6 1.3e6 1.2e6 1.1e6 9.5e5 360 8.5e5 7.5e5 6.5e5 5.5e5 4.5e5 3.5e5 2.5e5 1.5e5 5.0e4 0.0 340 360

pH 3.5

391

507

491 380

400

c) Cr(VI) and Cr(III) pH 8.9

Intensity, cps

Intensity, cps

344 491

360 360

507 380

400

440

460

480

500

520

d) Cr(VI), Ba(II),Ca(II), Fe(III), Pb(II) and Zn(II); pH 3.5

391 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0 340

420

m/z

m/z

420

440

460

480

500

520

m/z

1.25e6 1.15e6 1.05e6 9.50e5 8.50e5 7.50e5 6.50e5 5.50e5 4.50e5 3.50e5 344 2.50e5 360 1.50e5 5.00e4 0.0 340 360

391

507 491 380

400

420

440

460

480

500

520

m/z

Fig. 3. Positive ion ESI-MS spectra of (a) Cr(III) at pH 3.5; (b) a mixture of Cr(VI) and Cr(III) at pH 3.5; (c) a mixture of Cr(VI) and Cr(III) at pH 8.9; and (d) a mixture of Cr(VI), Ba(II), Ca(II), Fe(III), Pb(II), and Zn(II) at pH 3.5.

The detection of chromium(VI) in tap and river waters was investigated. In the experiment, samples were prepared by doping tap and river waters with a small volume of the 1000 ppm chromium(VI) standard stock solution. After pH adjustment to ~ 3.5, 50 μL of 5 mM APDC was added to each and the solutions were heated. Aliquots of 20 μL were diluted to 1000 μL with the MeOH/H2O/HOAc solvent and analyzed using enhanced MS mode. To determine the LODs, samples in low ppb concentrations were prepared, derivatized and then analyzed using the SRM scan mode. To demonstrate that simple vacuum evaporation could serve as a preconcentration method to enhance the detection signal, a 750 μL 0.14 ppm chromium(VI) sample in deionized water was derivatized with APDC at pH 3.5 and then diluted with 750 μL MeOH to obtain a 0.05 ppm APDC-derivatized chromium(VI) solution. One milliliter of the sample was evaporated to dryness, reconstituted with 20 μL of MeOH/H2O/HOAc solvent (enrichment factor of 50 fold) and then analyzed using the enhanced MS mode. The remaining 500 μL of the sample solution was also analyzed using the enhanced MS mode for comparison.

pH= 3.2), neutral (50:50 MeOH/H2O, pH=6.2) and basic conditions (50:50:0.5 MeOH/H2O/NH4OH, pH= 10.8). Ions of HCrO4− (m/z 117) and [HCrO4− + H2O] (m/z 135) were detected under all pH conditions, which is in agreement with a previous report in literature [28]. Fig. 1a shows the negative ion mode ESI-MS spectrum of a 5 ppm Cr(VI) solution in MeOH/H2O/NH4OH (50:50:0.5 by volume) at pH 10.8 (using the enhanced MS scan mode), in which the ions Cr2O72−, HCrO4−, [HCrO4− + H2O], and HCr2O7− appear at m/z 108, m/z 117, m/z 135 and m/z 217, respectively. HCrO4− (m/z 117) is the most abundant ion detected. Upon CID, this ion dissociates into CrO3− (m/z 100) by loss of OH• (Fig. 1b), confirming its structure. The ion CrO3− (m/z 100) seen in Fig. 1a might result from in-source ion dissociation of HCrO4− (m/z 117). Among samples tested at different pH values, the basic pH chromium(VI) sample gave the highest signal intensity of HCrO4− (m/z 117). Thus, the LOD for chromium(VI), based on monitoring HCrO4− under the basic pH condition scan mode, was determined to be 10 ppb using the enhanced MS mode, which is in agreement with that of a previous negative ESI-MS study mentioned before [27].

3. Results and discussion

3.2. Detection of Cr(VI) using (+)-ESI-MS

3.1. Detection of Cr(VI) using (−)-ESI-MS

3.2.1. Identification and confirmation of the formation of [Cr(III)O]–PDC complex This study mainly focused on the detection of chromium(VI) using ESI-MS in the positive ion mode in conjunction with APDC derivatization.

Direct analysis of chromium(VI) was first carried out using ESI-MS in the negative ion mode under acidic (50:50:1 MeOH/H2O/HOAc,

E. Weldy et al. / Science and Justice 53 (2013) 293–300

a) 2.0

pyrrolidinedithiocarbamic acid (PDC, 147 Da) and one ligand of hydroxyl pyrrolidinedithiocarbamate (163 Da), respectively (Fig. 2b), consistent with its structure. The CID of m/z 491 also revealed a fragmentation ion at m/z 344 by loss of one ligand of PDC (147 Da). The fragment ions of m/z 360 and 344 are also observed in the ESI-MS spectrum, probably due to the in-source dissociation of their precursor ions. The base peak, m/z 391, is from the background and can be seen in all mass spectra.

1.6

[360] / [524]

297

R² = 0.9972 1.2 0.8 0.4 0.0 0.00

50.00

100.00

150.00

Concentration of Cr (VI) (ppb)

b) 1.2 [507] / [524]

1.0 R² = 0.9904

0.8 0.6 0.4 0.2 0.0 0.00

50.00

100.00

150.00

Concentration of Cr (VI) (ppb)

c) 60

1.0 ppb

SRM monitoring

50

Intensity, cps

0.5 ppb 40 30

0.125 ppb

0.25 ppb

20 10 0 5

10

15

20

25

30

35

40

45

50

55

Time, min Fig. 4. Calibration curves: (a) the plot of the relative ion intensity of m/z 360 to m/z 524 vs. Cr(VI) sample concentration and (b) the plot of the relative ion intensity of m/z 507 to m/z 524 vs. Cr(VI) sample concentration; (c) SRM spectrum for the monitoring of dissociation of m/z 507 into m/z 360 for Cr(VI) sample solutions in different concentrations.

The rationale as mentioned above is that (+)-ESI-MS mode might provide higher sensitivity and the derivatization with APDC might enhance the detection selectivity. A 0.075 ppm APDC-derivatized chromium(VI) sample was analyzed using the enhanced MS mode to obtain the ESI-MS spectrum. The ESI-MS spectrum shown in Fig. 2a clearly shows the protonated [Cr(III)O]–PDC complex at m/z 507. There is also a smaller peak at m/z 491 which represents the Cr(III)–PDC complex (the structure shown in panel c of Scheme 1), but clearly the vast majority of chromium(VI), after reduction to chromium(III), binds to form the [Cr(III)O]–PDC complex. These two complexes, [Cr(III)O]–PDC and Cr(III)–PDC, only differ by an oxygen atom in structure, and possibly result from the APDC complexation after the reduction of chromium(VI) to CrO+ and Cr3+, respectively. The inset in Fig. 2a compares a simulated isotopic peak distribution of the [Cr(III)O]–PDC complex ion (discrete line) to the observed isotopic peak distribution (solid line). Except for the isotopic peak at m/z 506 which may result from background, the distribution profiles are almost identical. Upon CID, the ion of m/z 507 dissociates into m/z 360 and m/z 344 by losses of one ligand of

3.2.2. Selectivity The optimal derivatization conditions and the selectivity of this method for chromium(VI) over chromium(III) and other common metal ions were also examined. Fig. 3a displays the ESI-MS spectrum of a solution containing only 0.06 ppm chromium(III) that was derivatized by APDC at pH 3.5. A very small peak can be seen at m/z 491, corresponding to the protonated Cr(III)–PDC complex. The low peak intensity suggests that binding of chromium(III) with APDC is inefficient in acidic solution. This is in line with literature reports [14–20] as the chromium(III) cation is strongly hydrated under acidic conditions. Notably, no peak is seen at m/z 507, indicating that the peak of m/z 507 observed in Fig. 2a is unique to chromium(VI) and cannot be formed through the complexation of chromium(III) with APDC. Fig. 3b and c shows the ESI-MS spectra of mixtures of chromium(VI) and chromium(III) (0.06 ppm each) that were subjected to APDC derivatization under acidic (pH 3.5) and basic (pH 8.9) conditions, respectively. The peak intensity of m/z 507 in Fig. 3b (7.9e5, arbitrary units) is higher by more than one order of magnitude than that of m/z 507 in Fig. 3c (6.0e4) and that of m/z 491 in Fig. 3b (4.2e4). It is thereby clear that APDC derivatization under acidic condition is favorable to the formation of the characteristic complex [Cr(III)O]–PDC from chromium(VI) and is selective to chromium(VI) rather than chromium(III), which is again consistent with previous reports [14–20]. As mentioned previously, interference from other metals has commonly been a problem associated with selective chromium(VI) detection [14–20]. To test the selectivity of APDC in the presence of other metals, five common metal salts, barium(II), cadmium(II), iron(III), lead(II), and zinc(II), were added to the chromium(VI) solution and underwent APDC derivationization. All the metals, with the exception of zinc, were present in concentrations higher than the maximum contamination levels set by the US EPA [13]. Fig. 3d presents the ESI-MS spectrum of the resulted sample solution (final concentration of each metal: 0.06 ppm). Again, the peak corresponding to the protonated [Cr(III)O]–PDC complex arising from chromium(VI) is clearly seen at m/z 507, while no peaks corresponding to the complex ions of APDC with any of the five other metal ions were observed. This emphasizes that, under the derivatization conditions used, APDC derivatization is selective to chromium(VI). In order to prevent occurrence of random errors or possible sample carryover issues, all the selectivity comparison experiments mentioned above were repeated three times. The obtained mass spectra shown in Fig. 3 were reproducible, confirming that the detection of chromium(VI) based on the formation of the characteristic ion at m/z 507 has high specificity. 3.2.3. Quantification To validate this method, calibration curves, seen in Fig. 4, were created to determine a possible linear relationship between the observed intensity of the peak at m/z 507 and the concentration of chromium(VI). Six solutions ranging in concentration from 3.7 to 148 ppb, with MRFA peptide (MW 523) as an added internal standard, were analyzed by ESI-MS following APDC derivatization at pH 3.5. The relative peak intensity of m/z 360 (Fig. 4a) or m/z 507 (Fig. 4b) to the protonated MRFA (m/z 524) was plotted against the concentration of chromium(VI). The correlation coefficients were 0.9972 and 0.9904, respectively, with a LOD of 3.7 ppb with a signal-to-noise ratio of 3:1. Indeed, as expected, the LOD

298

E. Weldy et al. / Science and Justice 53 (2013) 293–300

a) Cr(VI) in tap water

b) Cr(VI) in river water 391

391

Intensity, cps

Intensity, cps

7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 344 360 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 0.0 350 370

507

491

390

410

430

450

470

490

510

530

6.5e5 6.0e5 360 5.5e5 5.0e5 344 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 0.0 350 370

507

491

390

410

m/z

430

450

470

490

510

530

m/z

Fig. 5. Positive ion ESI-MS spectra of APDC-derivatized Cr(VI) in (a) tap and (b) river waters.

for chromium(VI) measured by (+)-ESI-MS is lower than that measured by (−)-ESI-MS (10 ppb), using the enhanced MS scan mode. The detection sensitivity can be further enhanced using single reaction monitoring (SRM) mode, based on the monitoring of the dissociation of the protonated molecular ion (m/z 507) to its fragment ion (m/z 360). Four APDC-derivatized chromium(VI) solutions ranging in concentration from 0.125 to 1.0 ppb were analyzed using the SRM scan mode. As seen in Fig. 4c, the LOD was found to be 0.25 ppb with a signal-to-noise ratio of 3:1. It can be seen that LOD can be significantly lowered using SRM rather than the enhanced MS mode.

(theoretical value corresponding to a 50-fold enrichment factor), and (b) the unevaporated sample at the original concentration of 0.05 ppm. The peak intensity of m/z 507 was increased by 15-fold, from 1.4e3 prior to evaporation to 2.1e4 after evaporation (the evaporated and unevaporated samples were analyzed under the same experimental conditions). Although there is a slight loss of analyte, most likely from retention on the walls of the Eppendorf tube used for preconcentration, it is clear that preconcentration by evaporation is a viable method that produces a sufficient increase in peak intensity.

3.2.4. Applications Tap and river water containing 0.12 ppm chromium(VI) were analyzed following APDC derivatization, and the acquired spectra are shown in Fig. 5a and b, respectively. Both m/z 360 and 507 were clearly detected in the two practical samples. The LOD in tap and river waters was also investigated and found to be 2.0 ppb using the SRM scan mode, with a signal-to-noise ratio of 3:1, approximately one order of magnitude higher than that in deionized water, which might be ascribed to the possible matrix effect from the real water samples.

3.2.6. Comparison with other methods Table 1 presents a comparison of ESI-MS to the other methods previously mentioned for the detection of Cr(VI). ESI-MS offers excellent sensitivity with only three methods, CPE-ICP-MS, DLLME-ETAAS, and DLLME-FAAS, [16,19,20] reporting lower limits of detection. Only one paper, presenting RPHPLC–GFAAS and RPHPLC–ICP-MS results, reported run times, with those times being more than three times longer than that of ESI-MS. A comparison of complexation times shows only one method, DLLME-ETAAS [19], offering a complexation time only slightly shorter than that of ESI-MS. Two methods, VLCE-FAAS and CPE-ICP-MS [16,17], record complexation times of 45 min and 2 h, respectively. Five of the methods, CPE-FAAS, CPE-ICP-MS, VLCE-FAAS, DLLME-ETAAS, and DLLME-FAAS [15–17,19,20], report a calibration curve linearity only slightly higher than that of ESI-MS, with CPE-FAAS [14] presenting a much lower linearity. CPE-FAAS, VLCE-FAAS, RPHPLC–GFAAS, RPHPLC–

3.2.5. Preconcentration using vacuum evaporation As chromium(VI) occurs in aqueous solution, preconcentration of the species by evaporation of the sample was attempted to improve the detection sensitivity. Fig. 6 presents the ESI-MS spectra from (a) the preconcentrated sample at the final concentration of 2.5 ppm

b) Before preconcentration

a) After preconcentration 360

507

Intensity, cps

Intensity, cps

2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 0.0 340

360

380

400

420

m/z

440

460

480

500

520

2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 0.0 340

360 507

360

380

400

420

440

460

m/z

Fig. 6. ESI-MS spectra of the APDC-derivatized Cr(VI) sample (a) after preconcentration and (b) before preconcentration.

480

500

520

E. Weldy et al. / Science and Justice 53 (2013) 293–300

299

Table 1 Comparison of this proposed ESI-MS method with other reported methods. Reference #

Method

Cr(VI) LOD (ppb)

Runtime

Complexation time

Calibration curve linearity (R2)

Interferences

ESI-MS CPE-FAAS

0.20 0.60

3 min

[14]

15 min 15 min

0.9972/0.9904 0.669

N Y

[15]

CPE-FAAS

0.65

a

15 min

0.9992

Y

[16] [17]

CPE-ICP-MS VLCE-FAAS

0.01 1.90

a

2h 45 min

0.9998 0.9993

N Y

12+ min 10+ min

a

a

a

a

a

10 min 15 min

0.9991 0.9996

Y Y N Y

[18] [18] [19] [20]

RPHPLC–GFAAS RPHPLC–ICP-MS DLLME-ETAAS DLLME-FAAS

0.60 0.20 0.07 0.07

a

a

a

Note

Cobalt, iron, and nickel significantly suppress the signal — EDTA used; Triton-X114 suppressed the signal at high concentrations Cobalt, iron, and nickel significantly suppress the signal — EDTA used Cobalt, iron, and nickel significantly suppress the signal — EDTA used Cr-carbides Ar-carbides Cobalt, iron, and nickel significantly suppress the signal — EDTA used

CPE-FAAS; cloud point extraction-flame atomic absorption spectrometry. CPE-ICP-MS; cloud point extraction-inductively coupled plasma-mass spectrometry. VLCE-FAAS; vesicular liquid coacervate extraction-flame atomic absorption spectrometry. RPHPLC–GFAAS; reverse phase high performance liquid chromatography–graphite furnace atomic absorption spectrometry. RPHPLC–ICP-MS; reverse phase high performance liquid chromatography–inductively coupled plasma-mass spectrometry. DLLME-ETAAS; dispersive liquid–liquid microextraction-electrothermal atomic absorption spectrometry. DLLME-FAAS; dispersive liquid–liquid microextraction-flame atomic absorption spectrometry. a Data not given.

ICP-MS, and DLLME-FAAS [14–16,18,20], all report interferences from metals, an issue not greatly effecting ESI-MS. This comparison clearly demonstrates that ESI-MS offers an impressive LOD, short complexation and run times, and high linearity. 4. Conclusions This paper presented a novel, selective and sensitive method for the qualification and quantification of chromium(VI) in different waters using ESI-MS in the positive ion mode. It is clear that the chromium(VI) present in the water forms the characteristic [Cr(III) O]–PDC complex with APDC and the detection of the protonated complex at m/z 507 is highly selective to chromium(VI). We have shown that this method does not suffer interference from the presence of other metal ions, which is a common problem associated with other methods. Preconcentration of aqueous chromium(VI) sample via simple evaporation could further improve the sensitivity of this method. It is our belief that the method presented here would benefit the environmental forensic investigator in the specific and selective identification and quantitation of chromium(VI), a heavy metal which is toxic at extremely low concentrations. Acknowledgments This research was supported by NSF (CHE-0911160 and CHE1149367). References [1] In: C.D. Klaassen (Ed.), Casarett and Doull's Toxicology: The Basic Science of Poisons, McGraw-Hill, New York, 1996. [2] K. Salnikow, A. Zhitkovich, Genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium, Chemical Research in Toxicology 21 (2008) 28–44. [3] F.L. Petrilli, S. De Flora, Toxicity and mutagenicity of hexavalent chromium on Salmonella typhimurium, Applied and Environmental Microbiology 33 (1977) 805–809. [4] H. Dai, J. Liu, L.H. Malkas, J. Catalano, S. Alagharu, R.J. Hickey, Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome, Toxicology and Applied Pharmacology 236 (2009) 154–165. [5] H. Xie, A.L. Holmes, J.L. Young, Q. Qin, K. Joyce, S.C. Pelsue, C. Peng, S.S. Wise, A.S. Jeevarajan, W.R. Wallace, D. Hammond, J.P. Wise Sr., Zinc chromate induces chromosome instability and DNA double strand breaks in human lung cells, Toxicology and Applied Pharmacology 234 (2009) 293–299.

[6] M. Miró, J.M. Estela, V. Cerdá, Application of flowing stream techniques to water analysis part III. Metal ions: alkaline and alkaline-earth metals, elemental and harmful transition metals, and multielemental analysis, Talanta 63 (2004) 201–223. [7] H. Sereshti, V. Khojeh, S. Samadi, Optimization of dispersive liquid–liquid microextraction coupled with inductively coupled plasma-optical emission spectrometry with the aid of experimental design for simultaneous determination of heavy metals in natural waters, Talanta 83 (2011) 885–890. [8] L. Macháčková, M. Žemberyová, Utilisation of solid phase extraction procedures for preconcentration and determination of vanadium and chromium in various types of water samples by atomic absorption spectrometry, International Journal of Environmental Analytical Chemistry 89 (2009) 705–715. [9] H. Lai, L.S. McNeill, Chromium redox chemistry in drinking water systems, Journal of Environmental Engineering 132 (2006) 842–851. [10] S. Thomyasirigul, H. Fukuda, J. Hasegawa, Y. Oguri, C. Ratantamskul, Application of ion exchange paper to preconcentration of chromium (III) and chromium (VI) in water for PIXE analysis, International Journal of PIXE 18 (2008) 13–19. [11] N.N. Greenwood, A. Earnshaw, Chemistry of the Elements, second ed., Elsevier, Oxford, Boston, 1997. [12] R. Sutton, Chromium 6 in US Tap Water, Environmental Working Group, 2010. [13] US EPA, Water home, water.epa.com. [14] E.K. Paleologos, C.D. Stalikas, M.I. Karayannis, An optimised single-reagent method for the speciation of chromium by flame atomic absorption spectrometry based on surfactant micelle-mediated methodology, Analyst 126 (2001) 389–393. [15] E.K. Paleologos, C.D. Stalikas, S.M. Tzouwara-Karayanni, G.A. Pilidis, M.I. Karayannis, Micelle-mediated methodology for speciation of chromium by flame atomic absorption spectrometry, Journal of Analytical Atomic Spectrometry 15 (2000) 287–291. [16] N.N. Meeravali, S. Jiang, A novel cloud point extraction approach using cationic surfactant for the separation and preconcentration of chromium species in natural water prior to ICP-DRC-MS determination, Talanta 80 (2009) 173–178. [17] G.Z. Tsogas, D.L. Giokas, A.G. Vlessidis, N.P. Evmiridis, A single-reagent method for the speciation of chromium in natural waters by flame atomic absorption spectrometry based on vesicular liquid coacervate extraction, Spectrochimica Acta Part B 59 (2004) 957–965. [18] C.M. Andrle, N. Jakubowski, J.A.C. Broekaert, Speciation of chromium using reversed phase-high performance liquid chromatography coupled to different spectrometric detection methods, Spectrochimica Acta Part B 52 (1997) 189–200. [19] H. Chen, P. Du, J. Chen, S. Hu, S. Li, H. Liu, Separation and preconcentration system based on ultrasonic probe-assisted ionic liquid dispersive liquid–liquid microextraction for determination trace amount of chromium(VI) by electrothermal atomic absorption spectrometry, Talanta 81 (2010) 176–179. [20] P. Hemmatkhah, A. Bidari, S. Jafarvand, M.R.M. Hosseini, Y. Assadi, Speciation of chromium in water samples using dispersive liquid–liquid microextraction and flame atomic absorption spectrometry, Microchimica Acta 166 (2009) 69–75. [21] J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse, Electrospray ionization for mass spectrometry of large biomolecules, Science 246 (1989) 64–71. [22] A. El-Aneed, A. Cohen, J. Banoub, Mass spectrometry, review of the basics: electrospray, MALDI, and commonly used mass analyzers, Applied Spectroscopy Reviews 44 (2009) 210–230. [23] B.C. Laughlin, C.C. Mulligan, R.G. Cooks, Atmospheric pressure ionization in a miniature mass spectrometer, Analytical Chemistry 77 (2005) 2928–2939.

300

E. Weldy et al. / Science and Justice 53 (2013) 293–300

[24] M. Volný, W.T. Elam, B.D. Ratner, F. Tureček, Preparative soft and reactive landing of gas-phase ions on plasma-treated metal surfaces, Analytical Chemistry 77 (2005) 4846–4853. [25] X. Shi, J. Ren, J.H. Parks, Aldehyde complexes with protonated peptides in gas phase, The Journal of Physical Chemistry. B 115 (2011) 11183–11192. [26] A.B. Gwizdala, S.K. Johnson, S. Mollah, R.S. Houk, Speciation of chromium(VI) and chromium(III) using pneumatically assisted electrospray mass spectrometry, Journal of Analytical Atomic Spectrometry 12 (1997) 503–506. [27] H. Hotta, K. Yata, K.F. Bin Kamarudin, S. Kurihara, K. Tsunoda, N. Fukumoto, I. Kojima, S. Kinugasa, Determination of chromium(III), chromium(VI) and total chromium in

chromate and trivalent chromium conversion coatings by electrospray ionization mass spectrometry, Talanta 88 (2012) 533–536. [28] I.I. Stewart, G. Horlick, Investigations into chromium speciation by electrospray mass spectrometry, Journal of Analytical Atomic Spectrometry 11 (1996) 1203–1214. [29] A.M. Zawadzka, R.L. Crawford, A.J. Paszczynski, Pyridine-2,6-bis(thiocarboxylic acid) produced by pseudomonas stutzeri KC reduces chromium(VI) and precipitates mercury, cadmium, lead and arsenic, BioMetals 20 (2007) 145–158.