Anal. Chem. 1998, 70, 4864-4867
Refinement of the Borohydride Reduction Method for Trace Analysis of Dissolved and Particulate Dimethyl Sulfoxide in Marine Water Samples Rafel Simo´,*,† Gillian Malin, and Peter S. Liss
School of Environmental Sciences, University of East Anglia, NR4 7TJ Norwich, United Kingdom
A recently reported borohydride reduction method for the trace determination of aqueous dimethyl sulfoxide (DMSO) was adapted for use with a different sample preparation and analytical system, and the adaptation and optimization steps that we followed gave us further insight into the method. Increasing the proportion of reducing agent was critical. A number of compounds with potential for analytical interference were tested, but all proved negative. Water blanks were problematic, with substantial DMSO contamination observed in all but very recently purified water. Preliminary comparison with the highly specific and precise enzyme-linked method gave very good agreement. When DMSO analysis was done sequentially after analysis of dimethyl sulfide and alkali hydrolysis for dimethylsulfoniopropionate, we found that the DMSO concentration was not affected by increasing the length of the hydrolysis step. This allows storage and/or transport of hydrolyzed samples in gastight containers. The adapted method was applied to the novel determination of DMSO on glass fiber filters, and this revealed a significant pool of particulate DMSO in marine particles. Recent interest in dimethyl sulfoxide (DMSO) in the marine environment stems from its widespread occurrence in nature and its potential role in the biogeochemical cycle of dimethyl sulfide (DMS), a key species in the global sulfur cycle and the precursor of climatically active sulfur aerosols in the atmosphere.1 However, relatively few measurements of DMSO levels in natural waters have been made to date,2,3 essentially because of the scarcity of sufficiently sensitive and selective analytical procedures. During the past few years, five methods for trace analysis of aqueous DMSO have been reported. All involve gas chromatography, either via direct injection of the water aliquot4,5 or via reduction † Current address: Institut de Cie ` ncies del Mar, CSIC, Pg. Joan de Borbo´ s/n, 08039-Barcelona, Catalonia, Spain. (fax) 34 93 2217340; (e-mail) rsimo@ icm.csic.es. (1) Hatton, A. D.; Malin, G.; Turner, S. M.; Liss, P. S. In Biological and Environmental Chemistry of DMSP and Related Sulfonium Compounds; Kiene, R. P., et al., Eds.; Plenum Press: New York, 1996; pp 405-412. (2) Lee, P. A.; de Mora, S. J. In Biological and Environmental Chemistry of DMSP and Related Sulfonium Compounds; Kiene, R. P., et al., Eds.; Plenum Press: New York, 1996; pp 391-404. (3) Simo´, R. J. Chromatogr., A 1998, 807, 151-164. (4) de Mora, S. J.; Lee, P.; Shooter, D.; Eschenbruch, R. Am. J. Enol. Vitic. 1993, 44, 327-332. (5) de Mora, S. J.; Lee, P.; Grout, A.; Schall, C.; Heumann, K. Antarctic Sci. 1996, 8, 15-22.
4864 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
and subsequent determination of the evolved DMS.6-9 Simo´ et al.9 developed a borohydride reduction method which is relatively simple and performs well at nanomolar concentration levels. When used as part of a sequential protocol, this technique allows analysis of a suite of methylated sulfur compounds, e.g., DMS, methanethiol, dimethylsulfonipropionate (DMSP), and DMSO, in the same water sample.9 The method has been applied successfully in a number of field studies (refs 10 and 11 and Simo´, unpublished work). In this paper, we report on refinements to the borohydride reduction method for DMSO analysis which resulted from adapting the technique for a different sample preparation and GC analytical system to that described by Simo´ et al.9 New insight into the method has been gained, including the need to adjust the proportion of reductant specificity, blank troubleshooting, sample storage, and the first-ever application of the method to analysis of particulate DMSO (DMSOp). This information should be useful for those intending to analyze aqueous DMSO by reduction methods. EXPERIMENTAL SECTION Standards and Reagents. Standard solutions of DMSO were prepared by weighing DMSO (99.5%, Sigma, UK) into a volumetric flask and making successive dilutions in Milli-Q water. The stock and the working standards were stored at 4 °C after slight acidification with HCl. DMSP-HCl was obtained from Research Plus (Bayonne, NJ), and standard solutions were prepared as for DMSO. Sodium borohydride (98%, Aldrich Chemical Co., Milwaukee, WI) was used in the form of 0.045- and 0.1-g pellets. Hydrochloric acid (7% w/w) and a 10 M solution of sodium hydroxide in Milli-Q water were used for acidification and alkali hydrolysis. Analytical Setting. The system for purging and cryotrapping DMS was identical to that described by Turner et al.12,13 and used by Hatton et al.8 The analytical system consisted of a Varian 3300 gas chromatograph fitted with a dual-flame photometric detector (6) Ridgeway, R.; Thornton, D.; Bandy, A. J. Atmos. Chem. 1992, 14, 53-60. (7) Kiene, R. P.; Gerard, G. Mar. Chem. 1994, 47, 1-12. (8) Hatton, A. D.; Malin, G.; McEwan, A. G.; Liss, P. S. Anal. Chem. 1994, 66, 4093-4096. (9) Simo´, R.; Grimalt, J. O.; Albaige´s, J. Anal. Chem. 1996, 68, 1493-1498. (10) Simo´, R.; Grimalt, J. O.; Pedro´s-Alio´, C.; Albaige´s, J. Mar. Ecol. Prog. Ser. 1995, 127, 291-299. (11) Simo´, R.; Grimalt, J. O.; Albaige´s, J. Deep-Sea Res. II 1997, 44, 929-950. (12) Turner, S. M.; Malin, G.; Bagander, L. E.; Leck, C. Mar. Chem. 1990, 29, 47-62. 10.1021/ac980345o CCC: $15.00
© 1998 American Chemical Society Published on Web 10/10/1998
and a Chromosil 330 column.12 The GC was operated isothermally at 40 °C, and the DMS retention time was ∼2 min. The detector output was monitored on a Hewlett-Packard 3394 reporting integrator. Procedures. The analytical protocol used was slightly different from that reported in detail in ref 9, in that it was adapted to incorporate some of the procedures from refs 8 and 12. Determinations of DMS, DMSPd, DMSPp, and DMSO were performed sequentially on the same water aliquot. In brief, 5-50mL samples were taken with syringe and filtered through a GF/F filter while being injected into the purge tube. To minimize cell damage during filtration, a very gentle positive pressure was applied by hand, so that filtration of 25-50 mL of seawater took 1-1.5 min, depending on particulate matter concentration. Volatiles were stripped from the sample with nitrogen at a flow rate of 60 mL min-1 for 15 min and cryotrapped in a Teflon loop held at -150 °C. Previous work8 had shown that these sparging flow rates and times were enough to remove >95% of DMS. At the end of the purge, the cryotrap was immersed in hot water at ∼80 °C and injected onto the GC column via a six-port switching valve. Following DMS analysis, the sample was transferred to a glass vial that was filled to the brim with Milli-Q water and 2 mL of 10 M NaOH, crimped with a Teflon-faced seal, and kept in the dark for 6-24 h to allow DMSPd hydrolysis. After that time, the water was injected again into the purge tube, and the DMS resulting from the DMSPd hydrolysis was analyzed as described before. Finally, the alkaline solution was adjusted to a pH of ∼5 (as measured with a pH paper tape) with HCl, and 100 mg of NaBH4 was added to the purge tube to reduce the DMSOd into DMS. During the first 3 min following NaBH4 addition, the flow rate was reduced to ∼10 mL min-1, after which time the sample was purged for 12 min at 60 mL min-1. Then, the flow rate was lowered again, and 0.7 mL of 7% HCl was injected into the solution. After 1 min, the purge rate was returned to 60 mL min-1 for 15 min and the DMS generated from the reduction of DMSO was analyzed as before. The filter used for the initial filtration of the sample was quickly placed in a glass vial, which was filled to the brim with Milli-Q water and 1 mL of 10 M NaOH, crimp-sealed, and allowed to hydrolyze for 12-24 h in the dark. The resulting solution was withdrawn and treated as described above for DMSPp and DMSOp determination. The major differences between this procedure and that reported previously9 are the quantity of borohydride used (see Results and Discussion below) and that the neutralization between DMSP hydrolysis and DMSO reduction is done after, rather than before, purging of the DMS from DMSP. This reduces the possibility of losses of DMS and eliminates the need to neutralize the sample via injection through the crimp seal. The determination of DMSOp is a novel aspect of the new protocol. With respect to safety, apart from the usual caution necessary for work with concentrated acid and base, the only procedure to take into consideration is the mixing of borohydride and acidified water. The mixture produces gaseous hydrogen, and therefore, borohydride reduction of DMSO should not be done in sealed vessels where pressure can build up. Calibration. Calibration curves for the quantification of DMSO were produced with working standards of DMSO. Alter-
natively, curves generated from the hydrolysis of DMSP were also used. Calibration with DMSO requires making a number of reduction runs, which take ∼30 min each, whereas alkaline hydrolysis of each DMSP standard takes ∼15 min. Simultaneous calibrations with DMSO and DMSP showed that both techniques give rise to quantitative results (as previously observed by Hatton et al.8) and agree within analytical precision. RESULTS AND DISCUSSION Reduction Conditions. We found that the key change needed to adapt the borohydride reduction method developed by Simo´ et al.9 to the analytical setup at UEA was an increase of the amount of NaBH4 used. Initial tests with 45 mg of NaBH4 gave low and scattered reduction yields (71 ( 19%, n ) 15). Varying the amount of HCl added after borohydride or increasing the reaction time did not improve the yields significantly. Only use of 100 mg of NaBH4 yielded the close to quantitative results (99 ( 11%, n ) 12) observed previously. We suggest that difference is likely to be related to the higher efficiency of the present purge system. Dissolution of NaBH4 in the slightly acidic solution gives rise to boranes and hydride. Boranes are volatile, and hydride is transformed to purgeable H2 by reaction with H3O+. With increased purge efficiency, the volatile forms of the reducing agents are sparged more rapidly, which may decrease probability of the reaction and lead to lower reduction yields. Increasing the supply of reagent alleviates this problem. Blanks. Owing to the ubiquitous occurrence of DMSO, a great deal of care has to be taken to obtain acceptable blanks. Using their titanium chloride reduction method, Kiene and Gerard7 described severe blank problems associated with glassware that was not scrupulously cleaned and heated in a muffle furnace prior to use. Hatton et al.8 detected erratic background DMSO levels when analyzing the Milli-Q water used for standards and reagents using DMSO reductase. They also found very high DMSO levels in commercially available purified water. Simo´ et al.9 reported sporadic, although very low, concentrations of DMSO after borohydride reduction of Milli-Q water controls. In the present study, blank tests revealed low background levels in fresh Milli-Q water produced from a well-maintained purification system. A small DMS signal, corresponding to 5-25 pmol, usually appeared after treatment of blank controls with NaBH4/HCl. These blank concentrations tended to increase, though somewhat erratically, in stored water. High DMSO concentration blanks (>50 nM) were obtained during a period when one of the cartridges in the Milli-Q water system was inoperative. Blanks returned to low levels when the cartridge was replaced. As a general rule, Milli-Q grade water (>18 MΩ C m) is appropriate if the water is used fresh. Interferences. When Andreae first developed the borohydride reduction method for DMSO analysis 18 years ago,14 he tested a number of sulfur biosynthates that could potentially cause interference with the specificity of NaBH4 reduction of DMSO. Among the substances tested, only DMSP gave a positive reaction.14 While modifying Andreae’s method, Simo´ et al.9 observed quantitative borohydride-induced DMS production from DMSO, but on the assumption that only DMSP would interfere (13) Turner, S. M.; Malin, G.; Liss, P. S.; Harbour, D. S.; Holligan, P. M. Limnol. Oceanogr. 1988, 33, 364-375. (14) Andreae, M. O. Anal. Chem. 1980, 52, 150-153.
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Table 1. Interference Tests with Various Sulfur Compounds To Check for Possible DMS Production on Reduction of DMSO with NaBH4/HCl substance tested dimethyl sulfone methionine methionine sulfoxide dimethylsulfoniopropionate S-methylmethionine a
amount (nmol) 2.72 3.44 14.53 0.1-2 15.02
Table 2. Effect of 48-h Dark Alkaline Hydrolysis on Recovery of DMSP and DMSO hydrolysis time
DMS yield nda nd nd quantitative nd
nd, not detected.
with DMSO determination, no further tests with additional compounds were performed. In the present study, we investigated potential interference with dimethyl sulfone, methionine, methionine sulfoxide, DMSP, and S-methylmethionine (Table 1). Only DMSP gave rise to DMS by reduction with NaBH4/HCl, thus confirming the validity of the previous assumption. Using a single-flame FPD for DMS detection, Simo´ et al. observed two peaks of boranes eluting at lower retention times than DMS.9 Chromatographic conditions were adjusted to obtain baseline resolution of interfering peaks and DMS.9 Boranes were not observed in the dual-flame FPD used in this study. This is probably because of the higher specificity of the dual-flame detector in that the sulfur signal is not as prone to overlap with borane chemiluminescence. Does DMSP Hydrolysis Influence DMSO Recovery? When the borohydride reduction method is used as part of a sequential protocol involving initial purge of volatile sulfur compounds and hydrolysis of DMSP, a detailed speciation of methylated sulfur compounds in a single water sample aliquot is possible.3,9 However, the reliability of the DMSO concentrations obtained is dependent upon the assumption that DMSO is neither produced nor consumed during alkali hydrolysis of DMSP. Previously no such cross-effects were observed with a reaction time of 6 h with NaOH for DMSP hydrolysis prior to DMSO reduction.9 However, DMSO recoveries after longer hydrolysis times were not tested. Therefore, the possibility of sample storage in alkali over time scales of days without reducing the accuracy of DMSP and DMSO determinations remained open. Tests have now been performed with hydrolysis times of 48 h. Standard DMSP + DMSO + NaOH mixtures were stored in the dark in crimp-sealed vials with minimal headspace. No significant effects were observed on either DMSP or DMSO concentrations (Table 2). These results suggest that sample aliquots filtered and purged of volatiles can be stored and/or transported over few days while alkaline hydrolysis takes place. Such storage during hydrolysis at room temperature is both time-saving and convenient for field studies and sample transportation. Other techniques reported for storage of DMSO solutions include acidification, freezing, or both.3,7,9 Borohydride vs Enzymatic Reduction. Five methods for trace analysis of DMSO have been developed in the past few years, but no intercalibration or comparison between techniques has been attempted so far. Here we present the results of a preliminary comparison of the borohydride method with the accurate and highly specific, enzyme-linked method developed by Hatton et al.8 Standard solutions of DMSO were analyzed using 4866 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
0h std mixture concns 7.0 nM DMSP, 7.5 nM DMSO
48 h
DMSP DMSO DMSP DMSO 6.6 6.5
6.7 7.1
6.5 6.7
6.8 7.3
mean yield, % 31.9 nM DMSP, 15.3 nM DMSO
6.5 93 29.9 29.3
6.9 92 16.0 15.2
6.6 95 29.6 30.4
7.1 94 15.7 13.7
mean yield, %
29.6 93
15.6 102
30.0 94
14.7 96
Figure 1. Measured vs expected DMSO concentrations in standard solutions as determined with the borohydride (filled circles) and the enzyme-linked (open circles) reduction methods. Quantification was made relative to a DMSP calibration curve. Error bars indicate the mean precision of each method (11 and 2% coefficients of variation, respectively). Notice that both trend lines agree well within precision range and give a slope that is close to 1; i.e., in both cases, the reduction yield is close to 100%.
either NaBH4/HCl or a mixture of DMSO reductase plus EDTA and flavin mononucleotide. In both cases, the DMS signal obtained was quantified with calibration curves generated with DMSP standard solutions. As shown in Figure 1, both reduction techniques gave close to quantitative yields and were in good agreement within analytical precision. Hence, we can conclude that the two methods offer similar analytical performance. However, it should be noted that the enzymatic method, as reported by Hatton et al.,8 gives better precision (2%). It also has the advantage of offering faster results, since it has no interference other than DMS. Its major limitation is that at present the DMSOreductase enzyme is not commercially available. All the available methods for DMSO analyses have been reviewed recently by Simo´.3 Particulate DMSO. Biologically mediated DMS oxidation and DMSO reduction pathways are known (reviews in refs 15-
17); hence we anticipated that DMSO might be found in the particulate phase of natural waters, in living organisms, and/or in detritus. The borohydride reduction method detailed here was applied to the first-ever determination of DMSO in particulates retained on GF/F filters after seawater filtration. North Sea water samples were collected 6 miles off Great Yarmouth (Norfolk, U.K.) in June-August 1996 and processed as described in the Experimental Section. Using the same criteria as those applied for DMSPp analysis, the DMSO fraction retained by the GF/F filter was attributed to particulate DMSO (DMSOp). In waters with chlorophyll concentrations in the range 1.3-2.7 µg L-1, DMSOp concentration was 3-4 nM, that is, of the same order as dissolved DMSO (DMSOd, 2-5 nM), DMS (1-2 nM) DMSP (DMSPd, 4 nM), and particulate DMSP (DMSPp, 5-7 nM). A higher concentration of 16 nM was found during an intense bloom of the alga Phaeocystis sp. (chlorophyll, 13.3 µg L-1; DMSPp, 340 nM).18 Although the accuracy of the DMSOp determination cannot be fully assessed since it is not possible to make particulate DMSO standards, the coefficients of variation of duplicates ranged 4-20% (mean, 11%, n ) 12), which is the same range as for DMSOd. Complete blank runs including GF/F filters gave no detectable DMS signal. The discovery of the occurrence of a particulate pool of DMSO in seawater is of significance for sulfur biogeochemistry and DMS cycling in the marine environment.18 Storage tests for DMSOp and DMSPp in filter samples were performed. Replicate aliquots of two nutrient-enriched coastal (15) Taylor, B. F.; Kiene, R. P. In Biogenic Sulfur in the Environment; Saltzman, E. S., Cooper, W. J., Eds.; ACS Symposium Series 393; American Chemial Society: Washington, 1989; pp 202-221. (16) Taylor, B. F. In Biogeochemistry of Global Change: Radiatively Active Trace Gases; Oremland, R., Ed.; Chapman and Hall: New York, 1993; pp 745781. (17) Kiene, R. P. Mitt. Int. Ver. Limnol. 1996, 25, 137-151. (18) Simo´, R.; Hatton, A. D.; Malin, G.; Liss, P. S. Mar. Ecol. Prog. Ser. 1998, 167, 291-296.
seawater samples, with concentrations as high as 120-860 nM DMSPp and 25-30 nM DMSOp, were filtered through GF/F using a vacuum pump (e50 mmHg). Half of the replicates were immediately frozen at -70 °C for 2-3 h and then stored at -20 °C for 2 weeks. The remaining replicates were immediately placed in glass vials with Teflon-faced screw caps, which were completely filled with Milli-Q water and 1 mL of NaOH, 10 M. The vials were stored at room temperature in the dark over the same two-week period. The frozen replicates gave DMSPp concentrations that were consistently 9-11% lower than the hydrolyzed samples, i.e., a percentage that is close to the 10% maximum error inherent in the method. DMSOp did not appear to be greatly affected by the choice of storage method, since both agreed within 2-4%. Previously, we had observed significant DMSPp losses in filter samples stored frozen at -20 °C (data not shown). These losses may be due to DMSP-lyase activity on the filters during the period necessary for the samples to reach the final storage temperature of -20 °C. We suggest that faster freezing at a temperature e-70 °C is necessary if samples are to be stored frozen prior to DMSPp and DMSOp analysis. ACKNOWLEDGMENT Thanks are due to Angela Hatton for the enzymatic reduction calibration data, and to Jonathan Baker for field sampling assistance. This work was done while R.S. was working at the University of East Anglia with funding from the Royal Society exchange fellowship scheme. The work of G.M. and P.S.L. is a contribution to the European Union ELOISE Program (ELOISE No. 032) in the framework of the ESCAPE project carried out under Contract MAS3-CT96-0050. Received for review March 27, 1998. Accepted August 18, 1998. AC980345O
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