172 LOW LEVEL ORGANIC CONTAMINATION OF WATER Anal. Proc., Vol. 21

Low Level Organic Contamination of Water: Its Measurement and Significance The following is a collective summary of the introductory address and three of the papers presented at a Joint Symposium organised by the Environment Group of the Industrial Division and the Analytical Division held on October 19th, 1983, at the Scientific Societies Lecture Theatre, London, W.l. Conventional summaries of the other three papers, by Dr. D. E. Games, Dr. G. W. Aherne and Messrs. S. P. Scott and R. J. Vincent, follow the collective summary.

Summary

M. L. Richardson 6 Birch Drive, Maple Cross, Rickmansworth, Hertfordshire, WD3 2UL

Dr. P. A. Gilbert for the Environment Group introduced the Chairman, Professor J. W. Bridges (University of Surrey). Professor Bridges indicated that the attendance of over 100 showed the considerable importance being placed on the measurement and significance of organic micro- pollutants. He stated that over 3000 organic compounds had been reported as being measured in various water samples, and these could originate from natural sources, industrial plants, agricultural practice, water and sewage treatment, water pipes, domestic discharges and transport. The matter was complicated by the fact that chemicals released to the aquatic environment might be converted by abiotic or biotic processes to other products.

The major concerns related to the presence of micro-contaminants in water included: sensory (i .e. , manifested in taste, odour, etc.); toxicological (involves lifetime exposure to all members of the population); and emotive (belief in the concept of pure/wholesome water). Possible human intake or exposure could arise from the ingestion of food and water (2-3 1 d-1, there is no basis for calculating the volume from processed food), uptake through bathing and washing and medical treatment, e.g., dialysis, infusion, etc.

Sources of information in assessing toxic hazards included animal and in vitro toxicological tests on individual chemicals in water fractions, epidemiological findings and structural relationships to known toxic chemicals. There were particular problems in interpreting epidemiological data including the need to ascertain the adequacy of the control group, the amounts of exposure levels in retrospective studies (usually not known), the fact that exposure was invariably to many chemicals, a number of

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May, 1984 LOW LEVEL ORGANIC CONTAMINATION OF WATER 173 which would not have been identified, while others would often show greatly changing levels over the period of the study, the fact that it was very difficult to assess the life style contribution to any disease and that criteria for the identification of disease and efficiency in detection change with time.

There were also potential problems in the use of animal and/or in vitro toxicological data for assessing potential hazards because the doses used were often much higher than the levels to which man was likely to be exposed and hence conditions were quite different, the test species may not have shown human responses and the tests themselves may not have been able to assess variable response because of the range of population exposed, e.g., the age, sex, disease or genetic variability of the exposed population, and the fact that the chemical in question would be present in admixture and additive, synergistic and antagonistic effects might need to be considered.

In discussing the World Health Organisation guidelines for drinking water Professor Bridges considered that these were related more to the ability of the analyst than to the chemicals which might actually be present. He also indicated the difference in levels in the WHO guidelines when compared with workplace exposure.

The first lecture by Dr. A. Waggott and Mr. K . Connor of the Water Research Centre, Stevenage, was entitled “Analysis of Sulphonic Acids and other Ionic Organic Compounds using Reversed-phase HPLC.” They indicated that the determination of ionic organic compounds in aqueous samples by reversed-phase liquid chromatography posed problems not encountered with more non-polar organic compounds, e.g., polynuclear aromatic hydrocarbons.

The ionisation of sulphonated organic compounds had to be suppressed by the use of ion-pairing reagents,l by rendering the eluted solvent acidic or by the use of more polar reversed-phase packings.2 These techniques for ionic suppression could themselves be a problem, particularly when an in situ pre-concentration of the organic compound was required prior to HPLC separation.* Dr. Waggott gave examples of reversed-phase HPLC in the analysis of fluorescent whitening agents and certain Acid Blue dyestuffs in water samples.

In concluding he mentioned the potential advantages of microbore columns , multi-dimensional work and post column reaction detection systems, particularly those using atomic-spectroscopic detection.

Dr. E . Longstaff (ICI Toxicology Laboratory) and Dr. J. R. Lawrence (ICI Brixham Laboratory) spoke on “The Application of Mutagenicity Tests to the Determination of Water Quality.’’

The chemical industry was aware that many of its raw materials, by-products and products reached the aquatic environment by various routes, It was concerned to play its part in ensuring that the highest standards were maintained in the quality of our daily water supply and to co-operate in the identification and removal of possible causes of cancer. Certain in vitro tests were rapid and inexpensive methods of identifying possible mutagens. The relevance of the Ames test in testing water supplies was then discussed, together with its value as a research tool and/or as a routine screen in this context.

Stress was laid on the need to allocate resources to have the maximum beneficial effect on human health and to avoid the possibility of generating alarm through inadequate interpretation of test results. They emphasized that any environment was potentially dangerous. There was a requirement for a high quality water supply. Tests needed to be cost effective for the identification of cancer inducing compounds, which in turn would require removal to ensure safety. In vitro tests needed to be accurate, comparable and applicable. They were currently research tools and hence they should not be embodied into water quality criteria.

Mr. M . L. Richardson and Miss J . M . Bowron (Thames Water), in the final paper, described a predictive technique “Catchment Quality Control-an Alternative Approach.”

Catchment Quality Control (CQC) was a means of predicting the presence, fate and significance of organic chemicals that might be present in river waters which were subsequently abstracted for potable supply purposes. It was complementary to analytical chemistry, because it was realised that GC - MS surveys of rivers and potable waters would only detect approximately 20% of compounds. More specialised, and often very time-consuming techniques had to be developed for the majority of compounds. CQC ascertained most of the compounds being discharged by major industrial and commercial premises, and predicted the concentrations at the relevant water abstraction points. Detailed consideration was given to those compounds on which satisfactory data on degradability and innocuousness were not readily available.3

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174 LOW LEVEL ORGANIC CONTAMINATION OF WATER Anal. Proc., Vol. 21 Both published and manufacturers’ data were studied with the aim of assessing the long-term public

health risks of each compound. In many instances, it was deemed necessary to consider metabolism or degradation products, or even the results of chemical interactions.

The meeting concluded with a very lively discussion in which the importance of the significance and environmental impact on determined and predicted low level organic contaminants in water was stressed.

References 1. Concerted Action Analysis of Organic Micropollutants in Water. Activity Report of the Commission of the

European Communities, COST Project 64b BIS covering the period October 1978-December 1981, Volume 2, OMP/29/82-XIIlENVl17l82, 1982.

Concerted Action Analysis of Organic Micropollutants in Water. Activity Report of the Commission of the European Communities, COST Project 64b B/S, meeting of September 19-21, 1983, in the press.

Richardson, M. L., and Bowron, J. W., “Catchment Quality Control,” Notes on Water Research No. 32, Water Research Centre, Medmenham, 1983.

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3.

Application of LC - MS to the Analysis of Water

David E. Games

M. Glenys Foster and 0. Meresz Ontario Ministry of the Environment, P . 0. Box 213, Rexdale, Ontario, M9W 5L1 , Canada

Combined gas chromatography - mass spectrometry (GC - MS) is used extensively for the analysis of organic compounds in water. The technique provides a sensitive and specific method for the qualitative and quantitative analysis of organic compounds that are volatile and thermally stable. However, it has been estimated that only between 10 and 20% of organics present in water are amenable to study by this approach, because of their low volatility and/or thermal instability. High-performance liquid chromatography (HPLC) has become the method of choice for the analysis of compounds of this type. The technique also has advantages for the study of many classes of compound which are amenable to GC because, by use of trace enrichment techniques, such as pre-column concentration, water samples can be directly analysed without the necessity of using extraction techniques.

A combined system for high-performance liquid chromatography/mass spectrometry (LC - MS) should enable the types of water analysis currently undertaken by GC - MS to be extended to a wider range of compounds. An additional benefit is the provision of a more universal detection system for the liquid chromatograph.

It is not the purpose of this paper to review systems for LC - MS. The reader is referred to recent review articles on the subject.’-13 A brief description of the three most widely used systems for LC - MS will be given and areas of application relevant to water analysis will be described. This will be followed by a brief description of our studies in this area.

Department of Chemistry, University College, P . 0. Box 78, Cardijf, CFl 1XL

Systems for LC - MS The simplest approach to LC-MS consists of feeding a portion of the eluent from the liquid

chromatograph into the ion source of a mass spectrometer configured for chemical ionization (CI) mass spectrometry. Solvent mediated CI mass spectra are produced. If conventional LC columns are used the system has limited sensitivity. However, the use of microbore LC enables all of the eluent to be fed into the mass spectrometer ion source, resulting in excellent sensitivity.14 Systems of this type are commercially available from Nermagls and Hewlett-Packard16 and simple systems can be readily constructed in one’s own laboratory. This type of system is commonly referred to as direct liquid introduction. A related system is available from Kratos Analytical Instruments. l7

A second approach involves the removal of solvent using a continuously moving belt. The LC eluent is fed on to the belt and solvent is removed by use of an infrared heater and two vacuum locks. The residua1 solute is flash vaporized into the ion source of a mass spectrometer, where conventional electron impact (EI) and chemical ionization (CI) mass spectra can be obtained. Systems of this type are available from Finnigan MAT18319 and VG Analytical.20

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Recently, a third system has been developed which shows considerable potential for the analysis of compounds that are difficult to handle by conventional mass spectroscopic techniques.2l The system is referred to as thermospray ionization. A buffered mobile phase is passed through a stainless-steel capillary at flow-rates between 0.5 and 2 ml min-1. The end of the capillary is electrically heated. A supersonic jet of vapour containing solute ions is produced which traverses the ion source of a mass spectrometer and enters into a 1 cm diameter pumping line, which is connected to a mechanical vacuum pump. A conical exit aperture is sited at right angles to the jet and ions are sampled through it into a quadrupole mass analyser. Impressive LC - MS data have been obtained with the system on a range of compounds which have proved difficult to handle with other systems. Currently, the system is available from Vestec and Finnigan MAT.22

Applications of LC - MS to the Analysis of Water Samples In the early stages of development of LC - MS, studies were mainly confined to elucidating the types

of compound that were amenable to study by the various types of interface. Most studies have been of a qualitative nature, although it has been shown that the three types of interface referred to earlier are capable of providing quantitative data. More recently, real applications using LC - MS to identify organic compounds in water have appeared.

Detailed studies of the LC - MS behaviour of a group of 19 carbamate pesticides using a moving belt interface have been reported.23 It was shown that compounds of this type could be quantified down to the low nanogram level. However, the studies were conducted on a prototype system and recent developments should yield improved data. Other studies have confirmed the findings in this investigation and have shown that the technique can be used for the analysis of carbamate and urea pesticides in crop residues at the p.p.m. leve1,24 and that aldicarb, aldicarb sulphoxide and aldicarb sulphone can be analysed in well water samples down to the p.p.b. leve1.25 Other classes of potential environmental contaminants which have been studied with moving belt systems include perchloro cage pesticides,26 chloropropham and its metabolites,27 chlorophenolszg and polychlorinated biphenyls and their metabolites.29 Interfaces of the direct liquid introduction type have been used to study the LC - MS of organophosphorus pesticides ,30 triazine31 and phenylurea herbicides in river water samples.32

The applications described so far are of the target analysis type, where a specific compound or group of compounds is being sought. This type of approach means that many potentially hazardous compounds can be overlooked. LC - MS has the ability to be used for wider types of investigation. The usefulness of a moving belt interface for analysis of water samples in this context has been assessed.33 Because of the high chemical background and the system being unable to handle aqueous mobile phase systems without splitting off some of the mobile phase, it was concluded that the approach has limited suitability for compounds present in low concentrations. The use of microbore LC34 and spray deposition35 with interfaces of this type overcome some of the problems encountered in this study. Studies of tannery effluents using a moving belt system illustrate the merits of the approach.36 Twenty different compounds were identified by LC - MS and one of the major components was found to be binaphthyl sulphone, which was not identified in gas-chromatographic studies of the same sample.

Studies of Test Well Samples from a Landfill Site by LC - MS We have been investigating the usefulness of LC - MS, together with other mass spectral techniques,

for the analysis of extracts of test well samples from landfill sites. Extracts are studied by LC - MS and capillary GC-MS, using both EI and CI mass spectrometry. In addition, relative molecular mass profiling of the extracts is carried out by use of field desorption and desorption chemical ionization mass spectrometry. The latter measurements serve as a check for loss or decomposition of sample during a GC - MS or LC - MS study.

Fig. 1 shows the computer reconstructed total ion current trace obtained under EI LC-MS conditions, using a moving belt interface, from a basic fraction of one of our extracts. Examination of the EI and CI mass spectral data enables most of the major components and many of the minor components present in the sample to be identified. The main components are substituted benzothiazoles, together with aromatic amines and chlorinated phenols. These last compounds, because of the complexity of the sample matrix, were carried over into the base fraction in our extraction procedure. Comparison of the GC - MS and LC - MS data showed that the two techniques were mutually complementary. Low volatility thermally labile compounds not found by GC - MS were found using LC-MS and some compounds of high volatility were observed by W - M S but not by

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176 LOW LEVEL ORGANIC CONTAMINATION OF WATER Anal. Proc., Vol. 21

0 10 20 30 Time/m in

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Fig. 1. Computer reconstructed total ion current trace obtained from the base fraction of an extract from a test well sample using EI LC - MS with a moving belt interface. A 200 x 5 mm column packed with 5 pm Hypersil ODS was used with a mobile phase initially of acetonitrile -water (1 + 1) changed to (8 + 2) after 12 min. A mobile phase flow-rate of 1 ml min- was used, approximately 10% of which was fed onto the LC - MS interface.

LC - MS. In addition, the differences in chromatographic behaviour enabled better identifications to be made of multi-component chromatographic peaks, as compounds not resolved by capillary GC - MS were resolved by LC - MS and vice versa.

Recently, we have found37 that use of microbore LC - MS enables considerable advantages to be gained in studies of this type, particularly when reverse-phase LC is being performed. All of the eluent from the liquid chromatograph can be handled by the LC - MS interface, the sensitivity in terms of sample amount injected on-column is improved and high percentage aqueous mobile phases are more readily handled.

Conclusions

LC-MS can add a new dimension to studies of water samples, enabling low volatility and/or thermally labile organic compounds to be more readily identified. The choice of interface depends on the class of compound and type of investigation being pursued. The advent of interfaces of the thermospray type of LC-MS enables the range of compounds that can be studied to be extended to ionic compounds, which present difficulties in analysis by other types of interface.

We thank the SERC and Royal Society for assistance in the purchase of mass spectral and chromatographic equipment.

1. 2.

3. 4. 5. 6. 7. 8.

9.

References Arpino, P. J., and Guiochon, G., Anal. Chem., l979,51,682A. Arpino, P. J., in Vickrey, T. M., Editor. “Liquid Chromatography Detectors,” Marcel Dekker, New York,

McFadden, W. H., J. Chromatogr. Sci., 1979, 17,2. McFadden, W. H. , J . Chromatogr. Sci., 1980, 18, 9. McFadden, W. H., Anal. Proc., 1982, 19, 258. Games, D. E., Anal. Proc., 1980, 17,110,332. Games, D. E., Biomed. Mass Spectrom., 1981, 8, 454. Games, D. E., in Morris, H. R., Editor, “Soft Ionization Biological Mass Spectrometry,” Heyden, London,

Games, D. E., in Giddings, J. C., Grushka, E., Gazes, J., and Brown, P. R., Editors, “Advances in

1983, p. 243.

1981, p. 54.

Chromatography,” Volume 21, Marcel Dekker, New York, 1983, p. 1.

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18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31. 32. 33.

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35.

36. 37.

1984 LOW LEVEL ORGANIC CONTAMINATION OF WATER 177 Knox, J. H., Anal. Proc., 1982, 19, 166. Levsen, K., in Bjgrseth, A., and Angeletti, G., Editors, “Analysis of Organic Micropollutants in Water,”

D. Reidel, Dordrecht, 1982, p. 149. Curry, Z. F., J. Liq. Chromatogr., 1982, S(Sup. 2), 257. Willoughby, R. C., and Browner, R. F., in Lawrence, J. F., Editor, “Trace Analysis,” Volume 2, Academic

Henion, J. D., and Maylin, G. A., Biomed. Mass Spectrom., 1980, 7 , 115. Arpino, P. J. , Bounine, J. P., Dedieu, M., and Guiochon, G., J . Chromatogr., 1983, 271, 43. Melera, A., Adv. Mass Spectrom., 1980, 8B, 1597. Chapman, J. R., Harden, E. H., Evans, S., and Moore, L. E., Znt. J. Mass Spectrom. Zon. Phys., 1983,46,

McFadden, W. H., Schwartz, H. L., and Evans, S., J. Chromatogr., 1976, 122,389. Dobberstein, P., Korte, E., Meyerhoff, G., and Pesch, R., Int. J. Mass Spectrom. Zon Phys., 1983,46,185. Millington, D. S., Yorke, D. A., and Burns, P., Adv. Mass Spectrom., 1980, SB, 1819. Blakely, C. R., and Vestal, M. L., Anal. Chem., 1983, 55,750. McFadden, W. H., Spectra, 1983, 9, 23. Wright, L. H., J. Chromatogr. Sci., 1982, 20, 1. Cairns, T., Siegmund, E. G., and Doose, G. M., Biomed. Mass Spectrom., 1983, 10, 24. Wright, L. H., Jackson, M. D., and Lewis, R. G., Bull. Environm. Contam. Toxicol., 1982,28,740. Cairns, T., Siegmund, E. G., and Doose, G. M., Anal. Chem., 1982,54, 953. Games, D. E., and Weerasinghe, N. C. A., J. Chromatogr., Sci., 1980, 18, 106. Wright, L. H., Edgerton, T. R., Arbes, S. J., Jr., and Lores, E. M., Biomed. Mass Spectrom., 1981,8,47S. Dymerski, P., Kennedy, M., and Kaminsky, L., in Hertz, H. S . , and Cheder, S. N., Editors, “Trace

Organic Analysis: A New Frontier in Analytical Chemistry,” N.B.S., Washington, DC, USA, 1979, p. 685.

Press, New York, 1982, p. 69.

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Parker, C. E., Haney, C. A., Harvan, D. J., and Hass, J. R., J. Chromatogr., 1982,242,77. Parker, C . E., Haney, C. A., and Hass, J. R., J. Chromaiogr., 1982, 237, 233. Levsen, K., Schafer, K. H., and Freudenthal, J., J. Chromatogr., 1983, 271, 51. Schauenberg, H., Schlitt, H., and Knoppel, H., in Bjgrseth, A., and Angeletti, G., Editors, “Analysis of

Alcock, N. J., Corbelli, L., Games, D. E., Lant, M. S., and Westwood, S. A., Biomed. Mass. Spectrom.,

Hayes, M. J., Lankmayer, E. P., Vouros, P., Karger, B. L., and McGuire, J. M., Anal. Chem., 1983,55,

Thruston, A. D., Jr., and McGuire, J . M., Biomed. Mass Spectrom., 1981,8,47. Foster, M. G., Meresz, O., Games, D. E.: Lant, M. S., and Westwood, S. A., Biomed. Mass Spectrom.,

Organic Micropollutants in Water,” D. Reidel, Dordrecht, 1982, p. 193.

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1983, 10, 338.

Use and Significance of lmmunoassays in the Analysis of Water

G. W. Aherne Department of Biochemistry, University of Surrey, Guildford, Surrey

The first radioimmunoassay (RIA) was described in 19601 for the measurement of insulin in human plasma. Since then the technique has been widely applied, not only in clinical chemistry, but also in such diverse fields of study as endocrinology, immunology and pharmacology, as well as in forensic, veterinary and food sciences. Although RIA has been the most widely used technique, other types of immunoassay , e.g. , enzyme immunoassay (EIA) and fluoroimmunoassay (FIA) have become popular and can be readily adopted in laboratories without existing facilities and the expertise often required for radioimmunoassay.

In spite of their attributes of high sensitivity and specificity, immunoassays have not yet been widely applied to the analysis of water contaminants. Immunoassays are attractive, cost effective alternatives to more conventional techniques, such as gas chromatography, high-performance liquid chromato- graphy and mass spectrometry. Also, they are useful for screening large numbers of samples prior to analysis by more sophisticated and expensive techniques. Few problems should be encountered in adapting existing immunoassays, primarily designed for other purposes, to the analysis of water.

Principles of Radioimmunoassay RIA depends on the competition of an antigen and a fixed amount of a radiolabelled form of that

antigen for a limited number of antibody binding sites. At the end of a period of incubation the

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antibody bound fraction and the unbound fraction are separated from each other in a procedure known as phase separation. Methods which exploit physical, chemical or immunological differences between the two fractions can be used. The most commonly used phase separation agents are dextran coated charcoal (absorbs free fraction), ammonium sulphate (precipitates antibody bound fraction) and double antibody immunological precipitation by a second antibody specific for the immunoglobin of the first antibody. Many assays now utilise solid-phase techniques, so that phase separation can be achieved by simple centrifugation and washing steps.

The distribution of radioactivity between the bound and free fractions is related to the amount of standard antigen added and then unknown concentrations of antigen can be determined. The same principles apply to other forms of immunoassay, where the radiolabel is replaced by, for example, an enzyme or fluorimetrically labelled antigen, the distribution of which is measured following phase separation using appropriate instrumentation. More detailed information on the principles of immunoassays can be obtained from some excellent reviews.2-3

Two key reagents are thus required in order to set up an immunoassay: a suitable antibody to the analyte and a labelled form of that analyte.

Antibody Antisera to a wide range of substances are now available commercially or can often be obtained from

various laboratories with supplies in excess of their needs. If a source of antibodies is not readily available, antisera can be produced in one of a number of laboratory animal species using suitable immunisation schedules. Molecules of less than 5 000 relative molecular mass are not normally immunogenic and must first be conjugated to a carrier protein such as bovine serum albumin, ovalbumin or y-globulin. For use in immunoassays antibodies should be of high avidity and exhibit the desired specificity.

The specificity of an antiserum depends on a number of factors, e .g . , the purity of the immunising preparation with polypeptide hormones or the chemical nature of the hapten - protein conjugate with small molecules, as well as the individual response of each immunised animal. Absolute specificity for an analyte is difficult to achieve as most antisera cross-react, to some extent, with closely related compounds. The usefulness of each antiserum should be carefully assessed for each particular application and results obtained interpreted in the light of the cross-reactivity data. Antisera that exhibit a low degree of cross-reactivity with a closely related compound, for example, metabolite, can often be used successfully as the cross reactant is likely to be bound less avidly than the analyte. This is particularly so if the cross reactant is present in much lower concentrations than the analyte. If necessary, absolute specificity can be achieved by including a simple extraction step prior to assay.

Many highly cross reacting antisera can be used in screening procedures, e . g . , drug abuse screening programmes, and such antisera have been used successfully in combined HPLC - RIA techniques where the immunoassay is used as an extremely sensitive detection system.

Antisera raised by conventional means, i e . , by active immunisation of experimental animals, are polyclonal in nature and are a mixture of antibodies with heterogeneous specificity and avidity. In contrast, monoclonal antibodies raised using hybridoma techniques are homogeneous because the specificity is restricted to a single antigenic determinant. Although monoclonal antibodies are proving to be of enormous value in the analysis of complex systems and as diagnostic tools, their use in routine immunoassays has not yet been properly evaluated.

Labels The sensitivity of a particular immunoassay depends, to a large extent, on the type and amount of

label used. Exquisitely low assay sensitivity can be achieved using high specific activity labels. Tritium and 1251 have been the most commonly used radiolabels. Tritiated labels are generally stable, and the incorporation of the radioisotope causes little change in immunoreactivity , but such labels are not always available and have the great practical disadvantage that they require liquid scintillation counting. Iodine-125 labelled compounds have been widely used and although they are easily counted and are of high specific activity, their use is associated with a number of disadvantages. The half-life of 1251 is only 60 d and new batches of label must be prepared at frequent intervals. The incorporation of an iodine molecule, especially into small molecules, can cause dramatic changes in immunoreactivity . Many small molecules, which cannot be directly radioiodinated, can be “tagged” with a tyrosine or histidine containing residue, but this often reduces (or abolishes) antibody recognition and hence assay sensitivity.

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Other types of label have been introduced to overcome the disadvantages associated with the use ot radiolabels, e.g., disposal of radioactive waste and the potential health hazard of frequent radioiodination procedures. EIA is directly related to RIA in that the radiolabel is substituted by an enzyme label, and following phase separation the amount of enzyme in either the free or bound fraction is quantitated in the appropriate way. Other forms of EIA have also been developed. Enzyme-linked immunosorbent assay (ELISA) techniques4 have been widely applied to a range of substances and many kits are now available commercially. Enzyme assays are particularly suited to laboratories which are not already committed to RIA. They may also be suitable for field work in that a qualitative result as shown by a change of colour can be observed without sophisticated equipment.

Alternative forms of label have been investigated. Fluorimetric labelling is relatively inexpensive, results in stable products and can be applied widely in immunoassays. FIA techniques are similar to those used in conventional RIA but homogeneous assays, requiring no separation phase, have been described. In fluorescence quenching methods, for example, the fluorescence of the label is quenched when bound to an antibody.5 FIA techniques have not yet fulfilled their promise in clinical chemistry because in many instances endogeneous fluorescence from serum components has reduced the sensitivity of the assay. This limitation may not apply in the analysis of water. Chemiluminescent immunoassays (CLIA) are currently being evaluated and have the potential to be extremely sensitive.6

Application of Immunoassays to Water Analysis Immunoassays, especially RIA and CLIA techniques, are capable of detecting concentrations of

analytes as low as 10-10 M . Such techniques are therefore ideally suited to the analysis of microcontaminants in water where concentrations of analytes are expected to be very low (<< 1 pg 1-l). In clinical chemistry the sample volume is often a critical factor, but no such limitations exist in the analysis of water. The limit of detection of a particular assay can be increased by sample concentration prior to assay. Sample concentration can be easily achieved by evaporation or lyophilisation, followed by re-dissolution in assay buffer. Most assays are tolerant of changes in ionic strength and are probably less affected by such changes than by changes in protein concentration (which often occur in the clinical situation).

Immunoassays for methotrexate, a potent anti-cancer drug, progesterone, norethisterone and ethinyl oestradiol (oral contraceptives), have been successfully adapted to the analysis of water. A concentration by lyophilisation step was included in the assays with resultant detection limits of between 5 and 10 ng 1-1.7

References

1. 2.

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4. 5. 6. 7.

Yalow, R. S., and Berson, S. A. , J. Clin. Invest., 1960, 39, 1157. Ekins, R., in Voller, A. , Bartlett, A., and Bidwell, D., Editors, “Immunoassays for the ~ O S , ” MTP Press

Bolton, A. E., in Voller, A . , Bartlett, A., and Bidwell, D., Editors, “Immunoassays for the ~ O S , ” MTP Press

Voller, A., Bartlett, A., and Bidwell, D. E., I . Clin. Pathol., 1978, 31, 507. Shaw, E. J., Watson, R. A. A., Landon, J . , and Smith, D. S., J. Clin. Pathol., 1977,30, 526. Whitehead, T. P., Kricka, L. J., Carter, T. J. N., and Thorpe, G. H. G., Clin. Chem., 1979, 25, 1531. Aherne, G. W., and English, J. , to be published.

Ltd., Lancaster, 1981, p. 5.

Ltd., Lancaster, 1981, p. 69.

The Application of Pre-concentration and GC - MS Techniques to the Analysis of Water Sam ples-Pro blems and Sign if icance S. P. Scott, N. Sutherland and R. J. Vincent Water Services, Thames Water Authority, Directorate of Scientific Services, New River Head Laboratories, 177 Rosebery Avenue, London, E C l R 4TP There is an ever-increasing awareness of low-level contamination of water by organic compounds. Recent review papers’%* set out an impressive array of information derived from analytical studies. The technique of combined gas chromatography - mass spectrometry (GC - MS) has played a very important part in facilitating this increase; several hundred compounds have been tentatively identified in drinking waters with the list extended by several hundred more when raw and waste waters are considered.3

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180 LOW LEVEL ORGANIC CONTAMINATION OF WATER Anal. Proc., Vol. 21

Thames Water has had its own GC - MS facility since 1981. The emphasis of the work undertaken so far has been in the building up of an essentially qualitative picture of the trace organics present in drinking water derived from river sources. Well over one hundred different organic compounds have been tentatively identified, generally at concentrations roughly in the range up to 0.1 pg 1-1.

The limitations of GC - MS must be recognised. They are: first, only compounds that can be extracted by some means from water samples ready for injection into the GC can be studied; and secondly, the scope of the analysis is restricted to those compounds which are sufficiently volatile, or which can be made so by derivatization, to pass through the GC column. This presentation deals with aspects of the first of these limitations.

Methods In our laboratories, three distinct methods of extraction are employed, These are: liquid - liquid

extraction; resin adsorption; and closed loop stripping analysis (CLSA). For liquid - liquid extraction the solvent is dichloromethane (DCM), which has the advantages of relatively low toxicity and non-inflammability to add to its properties as a relatively non-polar solvent capable of extracting a wide range of organic compounds from water. Two-litre water samples are collected in glass bottles fitted with PTFE screw caps that have been cleaned with chromic acid. To the sample in its bottle is added 100 ml of DCM and the mixture is stirred magnetically overnight. A “cocktail” of several deuterium- labelled surrogate standards4 is added prior to extraction in order to assess the efficiency of the process. At the end of extraction the DCM is separated from the water prior to concentration.

Resin adsorption consists in passing the water sample through a specially selected and prepared bed of resin that is capable of adsorbing a wide range of organic compounds from the aqueous phase. This technique came into widespread use as a result of the activities of Junk et al.5 and has usually emloyed XAD-2, XAD-4 or XAD-8 macroreticular resins, made of a styrene - divinylbenzene copolymer. Typically, a sample volume of between 2 and 20 1 is passed through a resin bed of about 20 ml volume contained in a 15 mm diameter chromatography column. We have found it preferable to suck the water through by means of a reciprocating pump at a rate of 20 ml min-1 in the interests of good, reproducible recoveries and of avoiding (by virtue of the suction) any possible contamination of the water sample before extraction.

Adsorbed organic compounds are then eluted from the resin bed with two 15 ml and one 30 ml portions of diethyl ether. The eluates are combined prior to concentration. As in liquid - liquid extraction, a cocktail of deuterium labelled standards is added to the water sample before extraction to give a concentration of approximately 250 ng 1-1 of each compound.

Concentration of the extract, whether from liquid - liquid or resin adsorption extractions, is carried out in a Kuderna-Danish evaporator apparatus fitted with a three-ball Snyder column to minimise the loss of volatile components, after drying the extract, first by freezing out the bulk of the water and then by passing the extract through a small column filled with anhydrous sodium sulphate (previously heated to 400 “C in order to remove any organic impurities). The extract is reduced in volume to about 2 ml (contained in the detachable graduated tube of the apparatus) and then further reduced in volume to 0.5 ml under a stream of dry nitrogen.

The final extract, corresponding to a four thousand-fold concentration of a 2-1 sample, is examined by GC - MS using a VG Micromass 7035 mass spectrometer interfaced to a Dani 3800 capillary gas chromatograph. On-column injection of one or two rnicrolitres of extract to a 50 m fused silica column coated with OV-1 is employed and the mass spectrometer is operated at low resolution using 70 eV electron impact ionisation for most purposes. A typical GC - MS run will occupy about 1 h, involving a GC temperature programme from 25 to 250 “C at 4 “C min-1 and an MS cycle time of 1.4 s.

A third technique for the extraction and concentration of the more volatile organic compounds that is enjoying increased popularity is closed loop stripping analysis (CLSA). This technique was pioneered by Grob6 and has the attraction of combining extraction and concentration in one operation. Its basis is the purging of volatile organic compounds from, typically, 1 1 of sample by recirculating the headspace above the sample through a small trap containing 1-5 mg of granular activated carbon. The organic compounds are adsorbed thereon and can be eluted with about 20 pl of carbon disulphide to yield, ready for injection to the GC - MS, a concentrate representing a concentration factor of 40 000 for the compounds that are amenable to purging and adsorption. The advantages of speed and freedom from the possible contamination offered by this technique are to an extent offset by its restricted scope .7

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May, 1984 LOW LEVEL ORGANIC CONTAMINATION OF WATER 181

Problems Having outlined the techniques of extraction and concentration, we can now report some of the

problems that we have encountered. Inherent in the resin adsorption technique is the need to obtain resin free from organic contaminants.

Some suppliers offer specially-purified grades of XAD resins and elaborate procedures for resin clean-up in the laboratory are described in the literature5 involving sequential Soxhlet extraction with a variety of solvents. Notwithstanding the application of these techniques, it became apparent that a large part of the total ion current (TIC) response in samples analysed using resin adsorption was due to artefact formation. Fig. 1 shows TIC traces from: (a), an ether eluate of a resin bed left steeping in about 20 ml of high-purity water overnight; (b), a concentrate of the ether used for elution; and (c) an ether eluate of a resin bed through which had been passed 20 1 of high purity water. It can be seen that many of the peaks in trace (a ) are present at much the same level in trace (b) and that trace (c) shows very little enhancement of the response in trace (a ) even though about one thousand times more water is involved. This led us to conclude that a very significant contribution to over-all artefact levels arises from the mere interaction of water and resin if, as directed in the literature procedure,S the resin bed is prepared as a methanol slurry and used immediately thereafter for water extraction. Elution of the bed with diethyl ether immediately before water extraction much reduces the artefact contribution from the resin; it is thought that the heat of solution of methanol in water causes resin bead rupture, thus releasing artefacts.8

lo(

101

+4

E 1

C 0

Q

.- - w

I-" I

101

4 1 200 Scan number

Fig. 1 . XAD 4/XAD 8 resin adsorption: TIC traces of diethyl ether eluates (after concentration). (a ) , Resin bed steeped in about 20 ml of high purity water; ( b ) , diethyl ether blank; (c ) , 20 1 of high purity water passed through resin bed.

We have found it necessary to re-distil diethyl ether immediately before use and, moreover, to analyse the concentrate with a minimum of delay. Diethyl ether left standing for more than a few days will form compounds which obscure the extract chromatogram and which have mass spectra similar to

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182 LOW LEVEL ORGANIC CONTAMINATION OF WATER Anal. Proc., Vol. 21 that of ethyl acetate. An ion at mlz 61 is characteristic of these interfering compounds. Fig. 2 shows: (a) , the TIC trace from a resin adsorption concentrate left standing for 2 months; and ( b ) , a mass chromatogram for mlz 61 run on the same concentrate. Several of the large peaks in the TIC are thus shown to be associated with the ether impurities.

L 200 400 600 800 1000 1200 Scan number

0:1 4:47 9:318 14:14 18:57 23:49 28:24 Retention time/min

Fig. 2. (a), TIC trace of diethyl ether eluate (after concentration) stored for about 2 months; (b), rnlz 61 mass chromatogram of the same, to show ether impurity peaks.

With careful attention to detail, these problems with the resin adsorption technique can be contained within acceptable limits. However, it was largely problems such as these that prompted a change to liquid - liquid extraction for most routine purposes, although this technique was found not to be entirely trouble-free.

It was found that all TIC traces, irrespective of the nature of the sample, were characterised by a complicated array of peaks eluting between 12 and 20 min into the run. Their mass spectra showed them to be alkyl benzenes, with chain lengths of up to four carbons, and various alkane fragments. These are characteristic of petroleum. The problem was found not to occur when samples were extracted in more rural surroundings, leading us to accept this state of affairs as a consequence of our urban situation. Re-distillation of the solvent immediately prior to use tended, if anything, to exacerbate the problem so we now rely upon commercially-available glass-distilled dichloromethane without further “purification.”

A more serious problem was encountered when samples of treated water containing a chlorine residual of about 0.4 mg 1-1 were analysed. TIC chromatograms showed large peaks in the early part of the run. Some were attributable to the solvent blank, but others gave mass spectra characteristic of halogenated methyl butenes (with mlz 69 a prominent ion) and of haloethers. Such compounds, if present in treated water, would be a cause for some concern on account of their toxicity.

Further investigation showed that these compounds were absent from the chromatogram when the samples were treated with sodium thiosulphate prior to extraction in order to destroy residual chlorine. A chloromethyl butene spiked into a thiosulphate-treated sample was, however, recovered, thus removing any anxiety that dechlorination was also destroying these halogenated organic compounds.

It appeared that reaction between residual chlorine in the sample and dichloromethane (or impurities therein) was responsible for the formation of these compounds. In order to verify this possibility, closed loop stripping analysis (CLSA) was employed on the same treated water sample with and without prior dechlorination. None of the compounds were found in either sample. However, if 1 ml of dichloromethane was added to the residual chlorine-containing sample (1 1) prior to CLSA

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183 May, 1984 LOW LEVEL ORGANIC CONTAMINATION OF WATER

extraction, a TIC chromatogram very similar to that obtained with DCM extraction of a residual-containing sample was produced, characterised again by the halogenated methylbutenes. Fig. 3 summarises these findings.

I *-.

2 3

C

- 100 c

I-

0 0 200 280 360 440' 5:6 6:12 7:18 8:30 9:3510:4011:49 13:2 14:915:13 16:18

Scan number Retention time/min

Fig. 3. (a), TIC trace of a DCM extract of a water sample containing residual chlorine; ( h ) , TIC trace of a CLSA extract of the same sample; (c), TIC trace of a CLSA extract of the same sample with 1 ml 1-1 of DCM added.

TABLE I CHEMICALS CONSIDERED TO BE ARTEFACTS OF THE METHOD OF SEPARATION - ANALYSIS

Chemical Methyl dichloroethanoate 2,3-Dichloro-2-methylbutane Diethenylbenzene

Do d e c a n e Ethenyiethylbenzene 2-Ethoxypropane

3-Ethyl-4-methyl- furan-2,5-dione

Ethylnaphthalenes

Hexane Hex-1-ene 1-Methyl-1H-indene Met hy lnap ht halenes 2-Methylpentane l,l,l-Trichloro-

propan-2-one

I ,1,3-Trichloro- propan-2-one

Undecane

Thought to be derived from: ? Predicted to be noxious ? Predicted to be noxious XAD resin Used as a cross linking

agent for vinyl polymers XAD resin XAD resin Propan-2-one or diethyl ether Tobacco smoke

Remarks

XAD resin or tobacco smoke Diethyl ether Diethyl ether XAD resin XAD resin Diethyl ether May arise from chlorination of propan-2-one added during spiking with deuterated standards May arise from chlorination of propan-2-one added during spiking with deuterated standards XAD resin

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184 EQUIPMENT NEWS Anal. Proc., Vol. 21

Discussion These experiences all illustrate the hazards from artefacts inherent in the analysis of trace organic

compounds in water samples. A further illustration can be drawn from the outcome of the scrutiny of the results of an early GC - MS analysis (using resin adsorption) by Thames Water’s Catchment Quality unit; out of 107 compounds tentatively identified in a treated water sample, 16 were considered, upon investigation of the chemical and toxicological literature, to be artefacts of the analysis. Table I summarises this investigation.

We offer the following advice as a result of our experiences: ensure the highest quality of reagents; always run a method blank; be aware of the sample’s history (e.g. , its origins, method of collection and storage and its extraction); consider alternative extraction methods as an aid to the confirmation of validity; assess the environmental significance of the compounds tentatively identified and be alert to the possibility of artefacts in the method.

The authors acknowledge the assistance and encouragement of Mr. M. L. Richardson (Scientist, Catchment Quality, Thames Water) and of the advice of the Water Research Centre, Stevenage and Medmenham laboratories. This summary is published with the permission of Dr. M. C . Dart, Director of Scientific Services, Thames Water.

1. 2. 3. 4. 5 .

6. 7.

8.

References Bedding, N. D., McIntyre, A. E., Perry, R., and Lester, J. N., Sci. Total Envirun., 1982, 25, 143. Bedding, N. D., McIntyre, A. E., Perry, R., and Lester, J. N., Sci. Total Environ., 1983, 26, 225. Kraybill, H. F., J . Am. Water Works Assoc., 1981,73, 370. James, H. A., Fielding, M., Gibson, T. M., and Steel, C. P., Adv. Mass Spectrom., 1980,8, 1429. Junk, G. A., Richard, J . J., Grieser, M. D., Witziak, D., Witziak, J. L., Arguello, M. D., Vick, R., Svec,

Grob, K., J. Chrumatogr., 1973, 84,255. Melton, R. G., Coleman, W. E., Slater, R. W., Kopfler, F. C., Allen, W. K., Avrand, T. K., Mitchell,

D. E., Voto, S. J., Lucas, S. V., and Watson, S. C., in Keith, L. H., Editor, “Advances in the Identification and Analysis of Organic Pollutants in Water,” Volume 2, Ann Arbor Science, Ann Arbor, MI, USA, 1981, chapter 36.

H. J., Fritz, J. S., and Calder, G. V., J. Chrumafugr., 1974, 99, 745.

Water Research Centre, Medmenham Laboratory, private communication.

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