Supporting Information
A selective gas chromatography mass spectrometry method for ultratrace
detection of selenocyanate
Enea Pagliano*, Kelly L. LeBlanc, Zoltán Mester
National Research Council Canada, 1200 Montreal Road, K1A 0R6, Ottawa, Ontario, Canada * enea.pagliano@nrc-cnrc.gc.ca
Table of content
S1 Working condition GC-MS
S1.1 Electron ionization GC-MS settings: Select Ion Monitoring (SIM) and for Multiple Reaction Monitoring (MRM)
S1.2 Negative chemical ionization GC-MS settings: Select Ion Monitoring (SIM)
S2 Purity evaluation for KSeCN and KSe13C15N standards, determination of process yield by ICP-MS
S3 Ion chromatography ICPMS control method S4 Algal growth conditions
S5 Supporting Figures and Table
Figure S1. Variation of the EtSeCN signal intensity in function of the temperature of the
injector
Figure S2. EI GC-MS/MS product ion mass spectra at 10 eV of EtSeCN generated by
aqueous reaction between Et3OBF4 and SeCN−
Figure S3. NCI GC-MS mass spectrum of EtSeCN generated by aqueous reaction
between Et3OBF4 and SeCN−
Figure S4. Extraction efficiency of EtSeCN from aqueous to organic medium: hexane vs
chloroform
Figure S5. Repeated injections of a chloroform extract containing EtSeCN generated
from a SeCN− standard (50 ng/g Se)
Figure S6. GC-MS/MS calibration plot (MRM) and residual analysis
Figure S7. Effect of sulfate on the GC-MS/MS signal generated by analysis of 6 ng/g Se
standard.
Figure S8. IC ICP-DRC-MS chromatogram of a 10 µg Se kg-1 standard mixture containing selenite, selenate, and SeCN
Figure S9. IC ICP-DRC-MS chromatogram of a tap water sample spiked with 1 ng/g Se
(SeCN–)
Table S1. ICP-DRC-MS Operating Conditions for Anion Exchange Chromatography S6 References
S1 Working condition GC-MS
S1.1 Electron ionization GC-MS settings: Select Ion Monitoring (SIM) and for Multiple Reaction Monitoring (MRM)
For SIM and MRM acquisitions, an Agilent 7000 TripleQuad GC-MS with a CTC PAL3 autosampler was employed. A 1 μL volume of CHCl3 was injected in pulse splitless mode with an initial pressure of 30 psi for 0.4 min. The inlet temperature was optimized at 210 °C (Figure
S1) and the liner employed was a Restek 20796 (gooseneck, splitless, 2 mm ID, 6.5 x 78.5
mm). The separation was performed with a DB-5.625 column (5%-phenyl)-methylpolysiloxane: 30 m length 0.250 mm ID 0.25 μm film using the following program: 40 °C held for 2 min; 20 °C/min up to 220 °C held for 3.5 min. The GC was operated in constant flow mode with 1 mL/min He and the transfer line was set at 230 °C. The EtSeCN derivative eluted at 5.27 min (solvent delay = 4.2 min). To avoid carryover, after every injection, the syringe was rinsed 4 times with hexane and 8 times with chloroform.
The mass spectrometer was operated under standard conditions. The instrument was tuned (Agilent EI high sensitivity autotune routine) with a source temperature of 230 °C and quadrupoles temperature of 150 °C. The collision cell was run with 2.25 mL/min He (quench gas) and 1.5 mL/min N2 (collision gas).
In MRM mode (tandem mass spectrometry), collision energy of 5 eV was applied in the collision cell and the following transitions were monitored:
1. CH3CH2 80SeCN+ → H 80SeCN+, m/z 134.96 to 106.93, dwell time = 70 ms, gain factor = 5 2. CH3CH2 82SeCN+ → H 82SeCN+, m/z 136.96 to 108.93, dwell time = 70 ms, gain factor = 5 In SIM mode (single quadrupole mass spectrometry), collision energy of 0 eV was applied in the collision cell and the following m/z were monitored:
1. CH3CH2 80SeCN+, m/z 134.96, dwell time = 70 ms, gain factor = 5 2. CH3CH2 82SeCN+, m/z 136.96, dwell time = 70 ms, gain factor = 5
S1.2 Negative chemical ionization GC-MS settings: Select Ion Monitoring (SIM)
For NCI acquisitions, a Hewlett-Packard 5973 single quadrupole GC-MS with CTC CombiPAL autosampler was employed. The GC settings were those described in S1.1 for the EI experiments. The mass spectrometer was operated under standard conditions. The NCI was obtained using 99.999% ultra-pure CH4 which was supplied in the ionization chamber at 2 mL/min (40% flow). The ion source and the quadrupole were maintained at 150 °C. NCI in SIM mode (single quadrupole mass spectrometry), was operated by monitoring the following m/z: 1. 80SeCN−, m/z 105.9, dwell time = 70 ms, gain factor = 1
2. 82SeCN−, m/z 107.9, dwell time = 70 ms, gain factor = 1
S2 Purity evaluation for KSeCN and KSe13C15N standards, determination of process yield
by ICP-MS
Stock standards of approximately 1000 µg/g Se were gravimetrically prepared by dissolving KSeCN (≥99%; Sigma Aldrich, Oakville, Ontario, Canada) and KSe13C15N (97%, 99 atom % 13C, 98 atom % 15N; Sigma Aldrich) in ultrapure deionized water. Dilutions to working standards were performed gravimetrically, also in ultrapure deionized water. The total Se content in the diluted SeCN standards was calculated based on a two point standard addition using NIST 3149 (National Institute of Standards and Technology, Gaithersburg, Maryland, United States) as primary standard. Quantitation was performed on an Agilent 8800 Triple Quadrupole
Inductively-Coupled Plasma Mass Spectrometer (ICP-QQQ-MS; Agilent Technologies, Mississauga, Ontario, Canada). O2 was used as cell gas and the Se+>SeO+ transition was monitored for all isotopes (74Se, 76Se, 77Se, 78Se, 80Se, and 82Se); 80Se was used for quantitation.
The efficiency of the derivatization process was determined using a similar ICP-QQQ-MS method, but with the addition of an internal standard (5 ppb In and Rh in 2% HNO3) to the sample, added via “T” connection just prior to the nebulizer to account for potential differences caused by the slight variation in the matrix between samples. Briefly, a 1 mL volume SeCN– standard (750 ng/g Se) was derivatized with triethyloxonium tetrafluoroborate and resulting EtSeCN was extracted in chloroform. The amount of residual un-extracted Se was quantified in the aqueous phase by ICP-QQQ-MS after a 1:30 dilution. Less than 6.5% of the total initial Se was found in the aqueous phase demonstrating that the overall efficiency of the derivatization/extraction procedure is higher than 90%.
S3 Ion chromatography ICPMS control method
Analysis was performed on a Thermo Scientific Dionex ICS 5000+ High Performance Ion Chromatograph (Sunnyvale, California, United States) equipped with Thermo Scientific Dionex AS16 guard (4 x 50 mm) and analytical (4 x 250 mm) anion exchange columns. Separation was conducted at 1.5 mL/min using a step gradient consisting of 17.5 mM NaOH from 0-4 minutes, 100 mM from 4-12 minutes, and 17.5 mM from 12-15 minutes, all of which contained 2% methanol to increase ICP-MS sensitivity to selenium. The injection volume was 500 µL and the eluent passed through a 4 mm AERS suppressor operating at 250 mA before being directed through a glass nebulizer into a cyclonic spray chamber attached to a Perkin Elmer Elan DRC II inductively coupled plasma mass spectrometer (ICP-DRC-MS; Thornhill, Ontario, Canada). The H2 reaction gas was used to reduce polyatomic interferences on selenium isotopes – 78Se, 80Se, and 82Se were monitored; 78Se was used for quantitation as there was a fairly high background on 80Se, even in DRC mode. Operating conditions are listed in Table S1. Peaks from the resulting chromatograms were manually integrated using the software program OpenChrom (Lablicate, GmbH, Hamburg, Germany), and quantitation of SeCN was based on external calibration using dilutions prepared from a SeCN standard of established concentration (see Section S2). A sample chromatogram can be seen in Figure S8.
S4 Algal growth conditions
An axenic stock culture of C. vulgaris (CPCC 90) was purchased from the Canadian Phycological Culture Collection (University of Waterloo, Ontario, Canada) and was grown roughly following the methods of Wallschlӓger and coworkers.1,2 Subcultures were grown in sterile 10% Bold’s Basal Medium which contained, per litre: 17.5 mg KH2PO4, 2.5 mg CaCl2∙2H2O, 7.5 mg MgSO4∙7H2O, 25 mg NaNO3, 7.5 mg K2HPO4, 2.5 mg NaCl, 1 mg Na2EDTA∙2H2O, 0.62 mg KOH, 0.498 mg FeSO4∙7H2O, 0.286 mg H3BO3, 0.986 mg MnCl2∙4H2O, 22.2 µg ZnSO4∙7H2O, 39 µg NaMoO4∙5H2O, 7.9 µg CuSO4∙5H2O, and 4.9 µg Co(NO3)2∙6H2O. 100 mL cultures were grown in 250 mL flasks, capped with sterile foam stoppers, in an incubation chamber held at 22°C, on an orbital shaker operating at 120 rpm, under a 12 hour / 12 hour light / dark cycle. Exposure cultures were prepared by inoculating selenium-containing media with a 2 mL aliquot of a subculture on day 3 of growth (in the exponential growth phase). Selenium was added to the media, after autoclaving, to achieve concentrations of 10 and 25 µg Se L-1 as Na
2SeO4 (SeO42- in solution). Additionally, some cultures were supplemented with nitrogen at 5x the base amount, added as KNO3. Cultures
were sampled on days 5 and 11, when an aliquot of the culture was filtered through a 0.2 µm syringe filter.
S5 Supporting Figures and Table 100 150 200 250 300 1 1.4 1.8 2.2 2.6 3
Injection temperature (°C)
Relative signal (/10 5)Figure S1. Variation of the EtSeCN signal intensity in function of the temperature of the injector
(three replicate injections).
4 x10 0 0.25 0.5 0.75 1 1.25 1.5 1.75
+EI Product Ion:1 (5.089-5.105 min, 3 Scans) CID@10.0 (134.9 -> **) PI-10.D 107.0 79.9 95.6 135.2 51.4 Counts vs. Mass-to-Charge (m/z) 40 60 80 100 120 140 4 x10 0 0.2 0.4 0.6 0.8 1 1.2
+EI Product Ion:2 (5.093-5.110 min, 3 Scans) CID@10.0 (106.9 -> **) PI-10.D
106.9 79.9 91.0 65.4 Counts vs. Mass-to-Charge (m/z) 40 60 80 100 120 140
Figure S2. EI GC-MS/MS product ion mass spectra at 10 eV of EtSeCN generated by aqueous
reaction between Et3OBF4 and SeCN−. Precursor ion: 134.9 (right), 106.9 (left). 5 x10 0 0.5 1 1.5 2
- Scan (4.971-5.014 min, 10 Scans) SECN-1.D Subtract
105.8 103.8 107.8 101.8 99.8 110.8 119.7 94.7 Counts vs. Mass-to-Charge (m/z) 95 100 105 110 115 120
Figure S3. NCI GC-MS mass spectrum of EtSeCN generated by aqueous reaction between
3 x10 0.2 0.4 0.6 0.8 1 1.2 1.4
+EI MRM CID@5.0 (135.0 -> 106.9) eC-1.D
Counts vs. Acquisition Time (min)
5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4 5.45 5.5 5.55
Figure S4. Extraction efficiency of EtSeCN from aqueous to organic medium: hexane (red) vs
chloroform (blue). The chloroform is five times more efficient extracting EtSeCN.
3 x10 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 +EI MRM CID@5.0 (135.0 -> 106.9) C-R5.D
Counts vs. Acquisition Time (min)
5.21 5.22 5.23 5.24 5.25 5.26 5.27 5.28 5.29 5.3 5.31 5.32 5.33
Figure S5. Repeated injections of a chloroform extract containing EtSeCN generated from a
0 2 4 6 8 10 12 0 400 800 1200 1600
SeCN– mass fraction (ng/g Se)
SeCN – MRM sign al 0 20 40 60 80 100 120 0 3750 7500 11250 15000
SeCN– mass fraction (ng/g Se)
SeCN – MRM sign al 0 20 40 60 80 100 120 -2 -1 0 1 2
SeCN– mass fraction (ng/g Se)
Residual Average = -1.78 10
-15 Standard deviation = 0.72
Figure S6. GC-MS/MS calibration plot (MRM) and residual analysis.
-0.5 0 0.5 1 1.5 2 2.5 230 240 250 260 270 280 290 300 SO42- concentration (%) Relative signal
Figure S7. Effect of sulfate on the GC-MS/MS signal generated by analysis of 6 ng/g Se
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0 10000 20000 30000 40000 50000 60000 70000 Time (min) 78Se Sign al Intensity
Figure S8. IC ICP-DRC-MS chromatogram of a 10 µg Se kg-1 standard mixture containing selenite (5.7 min), selenate (6.7 min), and SeCN (11.7 min).
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Blank SeCN Spike Time (minutes) 78Se SIgnal (cps)
Figure S9. IC ICP-DRC-MS chromatogram of a tap water sample spiked with 1 ng/g Se (SeCN– ). By interaction with the tap water matrix, the SeCN– was fully converted into Se(IV).
Table S1. ICP-DRC-MS Operating Conditions for Anion Exchange Chromatography
Parameter Value
Nebulizer Gas Flow 0.95 L/min
Auxiliary Gas Flow 1.2 L/min
Plasma Gas Flow 15.0 L/min
Lens Voltage 14.25 V
ICP RF Power 1350 W
Cell Gas Flow 3 mL/min
RPq 0.25
Dwell Time, Per Isotope 50 ms
S6 References
1. Simmons, D.B.D.; Wallschläger, D. Release of Reduced Inorganic Selenium Species into Waters by the Green Fresh Water Algae Chlorella vulgaris. Environ. Sci. Technol.
2011, 45, 2165-2171. DOI: 10.1021/es103337p
2. LeBlanc, K.L.; Smith, M.S.; Wallschläger, D. Production and Release of Selenocyanate by Different Green Freshwater Algae in Environmental and Laboratory Samples. Environ. Sci. Technol. 2012, 46, 5867-5875. DOI: 10.1021/es203904e
