David P.
Bishop
a,
Dominic J.
Hare
abc,
Adrian
de Grazia
a,
Fred
Fryer
d and
Philip A.
Doble
*a
aElemental Bio-imaging Facility, University of Technology Sydney, Broadway, New South Wales, Australia. E-mail: philip.doble@uts.edu.au
bThe Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia
cExposure Biology Laboratory, Frank R. Lautenberg Environmental Health Sciences Laboratory, Department of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, USA
dAgilent Technologies, Mulgrave, Victoria, Australia
First published on 11th May 2015
A rapid method was developed for the determination of monobutyltin, dibutyltin, tributyltin, monophenyltin, diphenyltin, and triphenyltin by liquid chromatography-isotope dilution-inductively coupled plasma-mass spectrometry in sediments and drinking water. All six species were eluted in less than 6.5 minutes with a binary gradient. Offline solid phase extraction was used to pre-concentrate the organotin compounds for quantification employing two calibration procedures; external standard calibration and isotopic dilution. The external standard calibration approach yielded detection limits in the range of 1.5 to 25.6 ng L−1. The method was linear over four orders of magnitude with regression coefficients greater than 0.99 and a peak area repeatability less than 4.5% RSD (n = 7) for all compounds. The isotopic dilution method was three times more sensitive with detection limits in the range of 0.5–1.2 ng L−1. Recoveries for the external calibration method were from 33–68% with % RSDs of 5.7–12.7%. The isotopic dilution method had recoveries of 70–114% with % RSDs of 1.2–2.9%. The methods were applied to sediments sampled from the Cooks River in Sydney. The isotopic dilution method provided a viable alternative to the more common analysis by gas chromatography-inductively coupled plasma-mass spectrometry for contaminated sediment without the requirement of sample derivatisation.
Trialkyltin compounds are extremely toxic biocides that are deliberately and directly released into the environment.3 Trialkyltins, and in particular the tributyl-substituted species (TBT), are the active agents in marine antifouling paints. Their use has been regulated by the International Maritime Organisation (IMO) since 19904 due to their extreme toxicity toward aquatic organisms. Organotin compounds are classed as persistent pollutants and remain in coastal waters for many years.5 Organotin compounds are also known to leach from sediments into surrounding water, thereby becoming a steady source of pollution.6 The Australian Drinking Water Guideline is set at a maximum level of 1 μg L−1 for TBT by the National Health and Medical Research Council and the Agriculture and Resource Management Council of Australia and New Zealand, while the Australian Water Quality Guideline for Fresh and Marine Waters of the Australia and New Zealand Environment and Conservation Council set a limit for TBT at 0.004 μg L−1 for a 99% protection level of species in marine waters. The recommended sediment quality guideline for TBT is 5 μg Sn kg−1 normalised to 1% total organic carbon.7 TPhTs are also potent marine toxins and have been shown to be a causal agent in the mutation of Chinese sturgeon.8 TPhT is not readily detected in water and sediment, but through biomagnification can be found at detectable levels in marine organisms.9
Traditional approaches for the speciation of organotin compounds involve separation by gas chromatography (GC) with various detection methods including flame ionisation detection (FID), flame photometric detection (FPD) and inductively coupled plasma – mass spectrometry (ICP-MS). Organotin compounds with one to three substituents are polar and non-volatile due to their ionic properties,3 and require derivatisation before analysis by GC. Three common derivatisation methods are normally applied to GC-based analysis of organotin compounds: hydride generation; alkylation by Grignard reagents; and ethylation by sodium tetraethylborate (NaBEt4).10 Hydride generation and NaBEt4 ethylation are particularly useful for aqueous matrices, where direct derivatisation of the sample can be performed. NaBEt4 ethylation is a relatively simple procedure that is principally governed by three factors: acidity of the solution, NaBEt4 concentration and the time of reaction. However, differing degrees of ethylation may be observed depending on the organotin species present. Ethyl- and butyl-substituted tins are both more efficiently derivatised at high pH, whereas methyltin ethylation is preferential at low pH. Methyl- and ethyltin compounds have improved yield with larger concentrations of reagent, yet ethylation of butyltin compounds occurs independent of NaBEt4 concentration.11 Additionally, NaBEt4 decomposes rapidly in air and light and is extremely flammable.12 Derivatisation of organotins in sediments by NaBEt4 requires prior extraction13 unless a large amount of reagent is used to compensate for the side reactions with metals and other matrix components.10
Liquid chromatography (LC-) ICP-MS is an alternative approach for the speciation of organotin compounds, and is an attractive option as derivatisation is not required and the sample preparation procedures are comparatively minimal. Various LC-ICP-MS methods have been used for speciation analysis including micellar,14 ion-pairing2,15–17 and reverse phase6,18–21 separation mechanisms.
The perceived disadvantages of LC-ICP-MS are poorer sensitivities and inferior peak capacities compared to GC separation. A typical run time for LC-ICP-MS speciation of organotins in a complex matrix is 20 minutes for six species.6 Further, a higher number of organotin species can be resolved by GC than standard-bore LC.18 Leaching of inorganic and organic tin from the LC column has been reported.3 and metal free LC systems have previously been recommended.20 Regardless, both recent improvements in LC technology and the ease of sample preparation has allowed LC-ICP-MS to remain a viable option for organotin speciation, particularly using isotope dilution as a definitive quantitative methods.22
Isotope dilution (ID) is regarded as the optimal method for trace element analysis. The International Bureau of Weights and Measures classifies isotope dilution mass spectrometry as a primary ratio method of the highest metrological quality.23 ID has been shown to be the method best suited to the certification and characterisation of reference materials.24 The precision of butyltin quantification is increased by one order of magnitude when ID is used, as opposed to standard addition and external calibration.25,26 Spiking of the isotope dilution standard with simultaneous sample equilibration and extraction is now routine.12,27,28 Monperrus et al.29 demonstrated that a seven-minute simultaneous spike equilibration and sample extraction was as effective as a twelve-hour spike equilibration time before extraction, thus greatly enhancing the capacity for ID to be integrated in high-throughput workflows.
The aim of this work was to develop a rapid separation and quantification of the six main organotin species of environmental concern by LC-ICP-MS: monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), monophenyltin (MPhT), diphenyltin (DPhT), and triphenyltin (TPhT). Off line solid phase extraction was employed to meet the environmental and drinking water detection limits. External standard calibration and isotopic dilution methods of quantification were compared. The method described here has the potential to dramatically increase sample throughput in high volume laboratories using a relatively simple and robust quantitative LC-ICP-MS approach.
119Sn-enriched butyltin mix (ISC Science) | PACS-2 (NRCC) | |
---|---|---|
MBT | 0.110 ± 0.005 | — |
DBT | 0.691 ± 0.009 | 1047 ± 64 |
TBT | 1.046 ± 0.020 | 890 ± 105 |
LC: Agilent Technologies 1200SL; 1290 Infinity | ICP-MS: Agilent Technologies 7500cx | ||
---|---|---|---|
Column | Zorbax XDB Eclipse C18, 50 × 2.1 mm, 1.8 μm particle size | RF power | 1600 W |
Flow rate | 0.5 mL min−1 | Carrier gas flow rate | 0.7 L min−1 |
Injection volume | 5 μL | Auxiliary gas flow rate | 0.2 L min−1 |
Mobile phase A | 94% H2O, 6% acetic acid, 0.1% triethylamine, 0.0625% tropolone | Option gas flow rate | 0.2 L min−1 (w/0.04 L min−1 O2) |
Mobile phase B | 100% acetonitrile | Sample and skimmer cones | Pt |
Gradient | 0–5 s, 45% A-55% B | Monitored masses | 119, 120 |
Temperature | Ambient |
Sediment extraction was based on modification of the procedure by Ruiz Encinar et al.12 The ID standard was diluted 1:10, and 100 μL of the 119Sn butyltin enriched spike was added to the sediment sample (ca. 0.1 g) which was immediately extracted with 2 mL of 75:25 CH3COOH:MeOH. The mixture was extracted in an ultrasonic bath for 30 minutes at approximately 40 °C. The sample was filtered before analysis.
(1) |
Triethylamine was also added to mobile phase A to manipulate the selectivity of TBT.20 Acetic acid was added to maintain low pH and manipulate selectivity by complexation. Acetic acid also reduced the adsorption of organotins on the column17 by minimising interactions with the stationary phase,31 significantly reducing peak tailing of di-substituted organotins.20 Increasing acetic acid concentration also reduces the retention time of all organotins.32 In contrast to previous reports,33,34 plasma instability and baseline drift were not observed with this mobile phase composition. The system was very sensitive to small changes in organic modifier concentrations. Preliminary isocratic experiments showed that MBT and MPhT were resolved with a mobile phase of up to 45% ACN. An isocratic separation with 45% acetonitrile was greater than 10 minutes for all compounds. At 55% ACN, DBT, TBT, DPhT and TPhT were resolved within 6 minutes, though MBT and MPhT co-eluted. Therefore, elution with a 45% B-55% B step gradient over 5 seconds from the point of injection was employed.
Fig. 1 shows the separation of the six target organotin species in a 500 μg L−1 standard solution. Complete separation was achieved in under 7 minutes, less than half the time required for methods reported by Chiron et al.6 and Inagaki et al.5 Total separation times were similar to that obtained by GC-ICP-MS.18 However, the re-equilibration time of 2 minutes before sequential runs is less than the time typically required by GC-ICP-MS between injections and temperature ramps.
Fig. 1 Chromatogram of a 500 μg L−1 mixed standard (5 μL injection volume). Peak order: 1- MPhT, 2- MBT, 3- TBT, 4- DBT, 5- TPhT, 6- DPhT. |
LOD (μg L−1) | Peak area RSD (%) n = 7 | Linearity (1 μg L−1 to 1000 μg L−1) | Retention time RSD (%), n = 7 | |
---|---|---|---|---|
MBT | 5 | 4.4 | 0.9994 | 0.47 |
DBT | 3.5 | 2.2 | 0.9992 | 0.40 |
TBT | 5 | 2.5 | 0.9996 | 0.33 |
MPhT | 5 | 2.4 | 0.9995 | 0.38 |
DPhT | 1.6 | 1.5 | 0.9996 | 0.43 |
TPhT | 3.7 | 2.6 | 0.9999 | 0.47 |
Preliminary off-line preconcentration experiments indicated that 100 mL of a standard 1 μg L−1 organotin solution could be loaded onto the SPE cartridges without breakthrough. A number of elution solvents were trialled to minimise the volume of the SPE eluent for complete recovery of the organotins. Elution of the organotins from the SPE cartridge with solvents that contained greater than 70% ACN produced a negative system peak that interfered with MBT. This interference peak was due to suppression of the background signal from the introduction of the SPE elution solvent to the plasma.35 The optimal elution solvent was 20:10:70 CH3COOH:H2O:ACN and 0.1% TEA and 0.0625% tropolone. All target compounds were eluted from the SPE cartridge with 4 mL of this solution.
Fig. 2 shows a chromatogram of a 1 μg L−1 standard with a 1:25 pre-concentration factor. The peak at 2.5 minutes was a system peak generated from enhancement of the Sn background due to the rapid gradient changing the organic load in the plasma.36 The effect of the ACN gradient on the background Sn intensity is dependent on the lens conditions. Brown et al.37 observed a similar peculiarity in the development of a LC-ICP-MS speciation method for Pb. They experienced baseline suppression at the start of a gradient before a rise and fall similar to that observed here. The ICP-MS lenses were optimised for Sn with a solution containing 50% ACN. The gradient was from 45–55% ACN explaining a rise in the baseline from 45–50% and a fall after 50–55%. This peak was present in the blanks and did not represent an unknown organic Sn species.
Evaporation of the eluent was trialled to further improve detection limits. 250 mL of a 25 ng L−1 standard mix of the target compounds were loaded onto the SPE cartridges. The target compounds were eluted from the SPE cartridges with 80:20 ACN:CH3COOH and 0.1% TEA and 0.0625% tropolone before evaporation to dryness. The increase in ACN concentration resulted in elution of all compounds within a volume of 2 mL. The samples were then reconstituted in 250 μL of 70:20:10 ACN:CH3COOH:H2O water and 0.1% TEA and 0.0625% tropolone before injection.
The analytical performance of the pre-concentration method is detailed in Table 4. The pre-concentration factors for the SPE cartridges ranged from 24 to 32, corresponding to 96–130% recoveries. The pre-concentration factors for the combination of SPE and evaporation ranged from 325 to 677, corresponding to 33–67% recoveries. The % RSDs for these recoveries were 5.7–8.8%, indicating that the method was reproducible. The detection limits for all compounds, calculated as three times the signal-to-noise ratio of a 25 ng L−1 standard, ranged from 1.5 to 25.6 ng L−1. These detection limits are comparable to other methods that have been reported by LC-ICP-MS. Yang et al.17 reported detection limits of 28 ng L−1 and 33 ng L−1 for TPhT and TBT, respectively. Fairman et al.16 reported approximately 2 ng L−1 for both TPhT and TBT. Ugarte et al.38 used SPME-HPLC-ICP-MS for the speciation of tri-substituted organotin compounds reporting detection limits of 11 ng L−1 and 185 ng L−1 for TPhT and TBT, respectively. The 45 minute SPME extraction procedure is significantly longer than aqueous ethylation for GC-ICP-MS detection.
Solid phase extraction | Solid phase extraction and evaporation | |||||||
---|---|---|---|---|---|---|---|---|
CF | % Recovery | DLa (ng L−1) | % RSD | CF | % Recovery | DLa (ng L−1) | % RSDb | |
a Determined as 3:1 signal to noise ratio. Reported as the concentration of total organotin compound. b n = 5, reported on evaporated pre-concentration factor. | ||||||||
MBT | 32 | 128 | 100 | 6.9 | 433 | 43 | 1.5 | 5.7 |
DBT | 17 | 68 | 100 | 6.7 | 509 | 51 | 3.2 | 9.4 |
TBT | 27 | 108 | 150 | 9.1 | 477 | 48 | 3.7 | 6.0 |
MPhT | 28 | 112 | 330 | 5.1 | 476 | 48 | 1.8 | 12.7 |
DPhT | 29 | 116 | 110 | 8.1 | 677 | 68 | 2.0 | 11.5 |
TPhT | 24 | 96 | 180 | 12.5 | 325 | 33 | 25.6 | 5.7 |
CF | % Recovery | DL (ng L−1) | % RSD | |
---|---|---|---|---|
MBT | 18 | 72 | 0.5 | 2.9 |
DBT | 27 | 108 | 1.1 | 1.2 |
TBT | 29 | 114 | 1.2 | 2.4 |
The LC-ID-ICP-MS method was applied to PACS-2 certified reference material (see Fig. 3). DBT and TBT were in good agreement with the certified values (see Table 6). MBT is not certified in PACS-2 but is known to be present.
Fig. 3 Chromatogram of PACS-2 certified reference sediment. Peak order: (1) MBT, (2) TBT, (3) DBT, * ionic Sn. |
MPhT | MBT | TBT | DBT | TPhT | DPhT | |
---|---|---|---|---|---|---|
a Not detected. b Not available. | ||||||
PACS-2 (ID) | nda | 405 ± 13 | 952 ± 15 | 1044 ± 25 | nd | nd |
PACS-2 (external calibration) | nd | 259 ± 20 | 889 ± 48 | 790 ± 38 | nd | nd |
PACS-2 certified | nab | na | 890 ± 105 | 1047 ± 64 | na | na |
Cooks River (ID) | nd | 771 ± 110 | 2113 ± 205 | 1356 ± 288 | nd | nd |
Cooks River (external calibration) | nd | 727 ± 172 | 1369 ± 71 | 1410 ± 235 | nd | nd |
Fig. 4 Chromatogram of sediment sample from Cooks River in Sydney, Australia. Peak order: (1) MBT, (2) TBT, (3) DBT, * ionic Sn. |
The results shown in Table 5 demonstrate the difficulty of analysing organotins. Extraction of organotins is complex as there are strong interactions with sediment matrices requiring mild conditions to preserve the chemical integrity of the analytes.41 Numerous approaches to the extraction of organotins have been reviewed.42 Many different sediment extraction procedures have been evaluated and were appropriate for the sediment analysed. Abalos et al.43 identified a toluene:acetic acid mixture to yield the highest extraction efficiency while minimising degradation during extraction. They achieved accuracy of 82% and 92% of DBT and TBT in PACS-1 CRM and 70% and 90% of DBT and TBT in CRM-462. Concentrated HBr and tropolone was critical for the extraction of the more polar organotin compounds in sediments collected off the Huelva coast in the southwest of Spain.44 Sediment extraction was based on modification of the procedure by Ruiz Encinar et al.,12 who obtained an extraction yield for DBT and TBT within the certified values for PACS-2. MBT was strongly bound to the matrix and required harsher extraction techniques to recover it quantitatively. Ultrasonic, mechanical, and microwave extractions were compared and all resulted in high extraction efficiencies for MBT, short extraction times and no degradation products.
Extraction optimisation for new samples is less arduous with ID analysis. ID has many advantages over classical calibration procedures such as external calibration and standard addition. These include results not being affected by instrumental instability or matrix effects, and once equilibration has been achieved loss of sample will have no influence on the final result. The same extraction procedure was applied to PACS-2 and the sediment from Cooks River. With external calibration PACS-2 shows good agreement for TBT with the certified value while DBT is underestimated and MBT is lower than the value obtained by isotope dilution. The Cooks River sample shows good agreement for MBT and DBT with the values obtained by ID while TBT is underestimated. Every sediment sample will have a different composition leading to different interactions between the analytes and the sample matrix. The assumption that if an extraction procedure is effective for the CRM it should be able to be applied successfully to the sample is not applicable for organotin speciation. Isotope dilution eliminated the uncertainties due to the extraction procedure and matrix interactions on-column. External calibration relies on complete extraction of the analytes. Isotopic dilution relies on equilibration of the spike after extraction of the natural sample from the matrix is complete. This compensates for incomplete extraction and differences in matrices.
The method described here has several advantages over GC-ICP-MS. The need for derivatisation is removed, eliminating the requirement for the use of hazardous chemicals in ethylation. NaBEt4 is a pyrophoric, unstable compound that is aggressive to the front end of the GC column, leading to faster degradation and reduced column stability over time. The SPE-LC-ICP-MS procedure is shorter than GC-based methods, even when taking into account improvements in GC technology that have improved sample throughput. Sample derivatisation and ethylation can be performed in a similar timeframe, though a standard GC-ICP-MS run is 15 minutes (versus our described 8 minute LC runtime), and an additional 1–3 minutes for inter-sample cooldown make our LC method capable of approximately twice the throughput. Though detection limits using LC-ICP-MS are still an order of magnitude higher than GC,45 this method meets mandated environmental detection limits and is a suitable, rapid screening method that can process large sample volumes in a short timeframe.
This journal is © The Royal Society of Chemistry 2015 |