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mass spectrometry
Wat. Res. Vol. 25, No. 3, pp. 319-324, 1991 Printed in Great Britain.All rights reserved
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NEW S T A N D A R D S FOR THE DETERMINATION OF GEOSMIN A N D METHYLISOBORNEOL IN WATER BY GAS CHROMATOGRAPHY/MASS SPECTROMETRY WOLFGANGKORTH~@, KATHLEENBOWMERJ and JOHN ELLIS2 ~) ~CSIRO Division of Water Resources, Private Mail Bag, Griffith, NSW 2680 and 2Chemistry Department, University of Wollongong, P.O. Box 1144, Wollongong NSW 2500, Australia (First received January 1990; accepted in revised form October 1990) Abstract--Deuterium labelled geosmin and methylisoborneol (MIB) have been synthesized and evaluated as internal standards in the determination of geosmin and MIB in water by dosed loop stripping followed by gas chromatography/mass spectrometry (GC/MS). The labelled standards were compared with chloroalkanes added as internal standards either at the time of sampling or immediately before closed loop stripping. When added at sampling time, the new standards enabled accurate determination of the geosmin and MIB present initially, even when the samples were analysed as much as 3 weeks later. The new standards gave better precision and accuracy than the chloroalkanes and overcame the underestimation of initial analyte concentration which usually results from losses of analyte through adsorption, volatilisation, biodegradation etc. during sample storage. Geosmin had a limit of detection of < 0. I ng/1 and 1 ng/1 was determined with a coefficient of variation (CV) of 1.2% (n = 5). MIB was determined at 1 ng/1 with a CV of 3.5% (n = 5). Key words--geosmin, methylisoborneol, odorous compounds, closed loop stripping analysis, gas chromatography/mass spectrometry, selected ion monitoring
INTRODUCTION Geosmin [la,10fl-dimethyl-9~-decalol; (1)] and MIB (( 1-R-exo )- 1,2,7,7-tetramethyl bicyclo-[2,2,1]-heptan2-ol; [2]) (Fig. 1) are secondary metabolites of certain blue-green algae and actinomycetes (Safferman et al., 1967; Medsker et al., 1968, 1969). They have been reported worldwide as imparting an earthy or musty odour to drinking water (Rosen et al., 1970; Mallevialle and Suffet, 1987). Their threshold odour concentrations are 10-20ng/1 (Wood and Snoeyink, 1977; Yasuhura and Fuwa, 1979; Persson, 1980; Bartels et al., 1989). As a consequence, only the most sensitive methods will suffice for their analysis, and current practice is to use closed loop stripping (CLSA) followed by GC/MS with selected ion monitoring (SIM) (Hwang et al., 1984; Mallevialle and Suffet, 1987; Bartels et al., 1989). Accurate quantitation requires the use of an internal standard which is added to the water prior to the closed loop stripping step. Ideally one requires a compound which has similar Henry's law behaviour to the geosmin or MIB and which resists losses through adsorption or through degradation by chemical, photochemical or biological means. An internal standard with these properties would enable accurate determination of the geosmin (or MIB) concentration present in the water at the time of stripping the sample. If this is not possible, the chosen internal standard should be lost at a similar rate to the natural odour compound. A number of compounds have been used as internal standards (Wood and Snoeyink, 1977; Hwang et al.,
1984) and a mixture of linear chloroalkanes is currently accepted as the best compromise (Barteis et al., 1989). However, it is unlikely that any other compound will behave in precisely the same way as geosmin (or MIB). Scott et al. (1988) identified some of the problems associated with chloroalkane standards and referred briefly to their substitution by labelled standards. No details were given of the positions of labelling, but when solutions containing both labelled and unlabelled geosmin were analysed, the ion intensity ratio of 114/I 12 was constant over the concentration range 0.04-1.0#g/1. Accordingly, we have synthesized deuterium labelled analogues of geosmin and MIB to compare the behaviour of these internal standards with that of the chloroalkane mix. The mass spectrum of natural geosmin gives a weak molecular ion (m/z 182; 6%) under electron impact ionization conditions, with a base peak at m / z 112. Consequently, if deuterium labelled geosmin were to be used as an internal standard, it would be highly desirable for the label to be present intact in the base peak so that maximum analytical sensitivity could be achieved. Moreover, it would be preferable to introduce the deuterium using a discrete labelled synthon, rather than by, for example, base catalysed exchange adjacent to a carbonyl group or similar reactions which might fail to give quantitative labelling and/or suffer partial loss of label through exchange at a later stage of the synthesis. For these reasons, it was decided to label the angular methyl group attached to C~0. 319
WOLFGANGKORan et al.
320
CH 3
H
: 3OH
CH3
1
2
3
Fig. 1. Structures of geosmin (1), methylisoborneol (2) and 1,10-dimethyldecal-l(9)-en-2-one (3). The mass spectral cracking p a t t e r n of M I B has been studied by M e d s k e r et al. (1969), w h o proposed m e c h a n i s m s for the f r a g m e n t a t i o n (Fig. 2). A weak molecular ion ( m / z 168, 7 % ) is p r o d u c e d u n d e r E1 conditions a n d the base peak is at m / z 95. Even t h o u g h a labelled base peak is n o t produced, the only practicable m e t h o d o f preparing labelled M I B was to react m e t h y l m a g n e s i u m iodide-d3 with d - c a m p h o r a n d to purify the MIB-d3 by the m e t h o d of W o o d a n d Snoeyink (1977).
MATERIALS AND M E T H O D S
CLSA equipment was from Brechbuehler AG, Switzerland, modified to take a tall-form bottle (380 x 70ram; 350 ml headspace). The water sample (c. 800 ml) was added to the tall-form bottle and 90 g of solid sodium chloride (previously heated to 700°C for 12 h) was added and dissolved ( < 30 s) with magnetic stirring. The stirrer bar was removed and the solution made up to 900 ml with milli-Q water. The solution was held at 25°C and stripped for 1.5 h at an air flow rate of 2 l/min. Volatile organic compounds were adsorbed by a granular activated carbon (GAC) filter held at 40°C (block temperature 50°C). At the completion of the stripping cycle, the filter was eluted sequentially with 10, 10 and 5/zl of CS2. The combined extract was stored in a 100/~1 micro vial placed in a 2 ml screw cap vial fitted with an open top cap and a Teflon septum. A stainless steel spring was used to push the micro vial up against the septum. Filters were washed with l ml portions of acetone, water (milli-Q), acetone, CS2, acetone (3 x ), methylene chloride (3 × ) and dried under vacuum.
Gas chromatography was performed using a Varian 3300 gas chromatograph with He carrier gas (0.75 ml/min) and a Hewlett-Packard fused silica capillary column (25m, 0.20 mm i.d., coated with 0.30/zm crosslinked methyl silicone). A 1.0 #1 sample was injected and the column held at 25°C for 1.5 min with the splitter off for 1 min. The splitter was then turned on and the column heated at 10°C/min to 80°C, then 5°C/min to 250°C and held at 250°C for 5 min. Splitter gas flow rate was 50 ml/min and both injector and interface were at 260°C. For concentrations < I ng/1, three consecutive injections of 2#1 of extract were made per analysis. Cumulative transfer to the column was achieved without band broadening by holding the oven at 25°C until all three injections were made. After 5 min, for each of the first two injections, the splitter was opened for 20 s and then closed again prior to the next injection. For the third injection the splitter remained open after 5 min and the oven was ramped as described above. The eluate from the GC reported to a Hewlett-Packard 5970 mass selective detector, using electron impact ionization (70 eV) and operated in the SIM mode. The ions monitored and those used for quantitation were as follows:
Compound 1-Chlorodecane MIB Geosmin MIB-d 3 Geosmin-d 3
91 95 112
168 112,182 171 115,185
Ions monitored (m/z) A B 43,91,93 95, 107, 135 111,112,125
A = l-chlorodecane as internal standard. B = MIB-d3 and geosmin-d3 as internal standard.
H--C3H60
m/z 168
(m/z) used for quantitation A B Ions
*
m/z 110
- CH3
m/z 95
m/z 153 Fig. 2. Fragmentation of methylisoborneol under electron impact ionization.
168 112,182 171 115,185
Determination of geosmin and methylisoborneol
321
When comparing relative areas of peaks from labelled and unlabelled compounds, computed corrections were applied where necessary for contributions of isotope satellites in the unlabelled compound to the labelled peak being monitored, and vice versa. For example, when using the base peaks 112/115 for quantitation of geosmin the followingequations were required to be solved simultaneously to obtain corrected ion area values for the two masses:
The MIB-d3 was prepared by reaction of CD3MgI with (+)-camphor. Unreacted camphor was removed by reaction with hydroxylamine, and the MIB-d 3 purified by chromatography on silica gel and sublimation (Wood and Snoeyink, 1977). The mass spectrum showed the expected molecular ion at m/z 171 (6%) (168 in MIB), but the base peak area l12obS= area ll2corr+ (% area 1121x area llScor~) remained at m/z 95, as in unlabelled MIB. Hence, area 115ohS= area 115corr+ (% area 115ulx area 112corr) when using MIB-da as an internal standard, selected ion monitoring had to utilise the molecular ion at obs = observed; corr = corrected m/z 171, rather than the base peak. The salted CLSA procedure employed was based % area 112j= peak area at mass 112, in labelled geosmin, expressed as a percentage of its peak area at mass 115 on that of Hwang et al. (1984). The water sample was % area 115urn= peak area at mass 115, in unlabelled geos- kept at 25°C and stripped for 1.5 h. However, sodium min, expressed as a percentage of its peak area at mass 112. chloride was substituted for sodium sulphate. Equimolar solutions of the authentic MIB and geosmin Depending on source, when the latter is heated to were added to equimolar solutions of the corresponding 700°C to remove organic impurities, it tends to cake labelled compounds to establish the purity of the latter. and thus is slow to dissolve in water. Addition of Chemicals used were sourced as follows: 1-chloro-3-pen- sodium chloride had a marked effect on the recovery tanone, cyclohexene oxide, methylmagnesium iodide-d3, of MIB and geosmin and a lesser effect on the 1-chlorooctane, l-chlorodecane, (+)-camphor (Aldrich); 1-chlorodecane. The identical stripping behaviour of 1-chlorododecane, carbon disulphide (Merck); ( - ) methylisoborneol, racemic geosmin (Wako); acetone, MIB vs MIB-d3 and geosmin vs geosmin-d3 was dichloromethane, sodium chloride (Ajax). confirmed by stripping solutions containing both the labelled and unlabelled compounds. Hwang et al. (1984) observed that stripping efficiency increased RESULTS AND DISCUSSION for additions of Na2SO4 up to 100g/l. We found a The most recent synthesis of geosmin was reported similar trend for NaCI concentrations up to by Gosselin et al. (1989), who obtained racemic 100-150g/1. The improved stripping efficiency was geosmin in about 40% yield from the attributed by Hwang et al. (1984) to reduced dimethyldecalenone [3] (Fig. 1), which can be pre- solubility of the organic solutes and formation of pared by condensation of 1-chloro-3-pentanone and smaller air bubbles by the sparging gas when salt 2-methylcyclohexanone (Zoretic et al., 1975). For the was added. Table 1 shows the comparative behaviour of MIBpresent purpose, labelled 2-methylcyclohexanone was prepared by reaction of CD3MgI and cyclohexene d3 and l-chlorodecane internal standards for different oxide, followed by oxidation of the 2-methylcyclo- initial concentrations of MIB (and of internal stanhexanol-d3 to 2-methylcyclohexanone-d3. The mass dard). The CLSA results using the 1-chlorodecane spectra and N M R spectra of the labelled compound standard were calculated using the procedure of [3] confirmed that the methyl group on C10 had been Mallevialle and Suffet (1987). A response factor (RF) labelled quantitatively with three deuterium atoms. for MIB was calculated from the CLSA of one water The geosmin-d3 was shown to retain this label and, sample containing known concentrations of MIB and 1-chlorodecane calibration standard. This response most importantly, the base peak of the mass spectrum was now at m/z 115 instead of m/z 112. The base factor was then used to calculate the MIB concenpeak can be accounted for by the ion radical in Fig. 3. tration of the unknown sample (u) for a further four Proof that this ion is derived from ring B was CLSA runs, using the formula obtained by preparing geosmin-d5 labelled with two area MIBu x amount (IS)u further deuterium atoms (one at C i and the other at MIBu (rig/l) = RF (MIB) x area (IS)u C2). This gave a molecular ion at m/z 187, while the base peak remained at m/z 115. Details of the seven where step synthesis will be reported elsewhere. area MIBu = peak area of the ion used for quantiration (m/z 95) and area (IS)u = peak area of m/z 91 (see list above).
I
÷1 H m/z 115
Fig. 3. Base peak of geosmin under electron impact ionization.
In the case of the labelled internal standard, the response factor for MIB-d3 was the same as that for unlabelled MIB. The labelled internal standard procedure gave better precision than the l-chlorodecane in all cases except for the 0.2 ng/1 concentration, where the molecular ion intensity was too low to give an acceptable
WOLFGANG KORTH et al.
322
Table 1. Precision of measurement of MIB by GC/MS (SIM) using MIB-d3 or 1-chlorodeeane as internal standard (a) MIB-d3 internal standard: m/z 168/171
Concn present
Conch of int. std.
Concentration found
(ng/l)
(ng/l)
(ng/l)
1000 100 10.0 1.00 0.200
930 93.0 9.30 0.930 0.186
999 100.5 10.42 1.10 ND
1065 100.2 10.50 1.18 ND
1051 100.3 10.40 1.08 ND
CV Mean
1040 100.2 10.79 1.10 ND
1034 99.9 10.97 1.09 ND?
(b)l-Chlorodeeane internal standard: m/z 95 (MIB)/91 (chlorodecane) Concn Conch present int. std Concentration found (ng/I) (rig/l) RF (rig/l) 1000 930 1.39 968 991 949 928 100 93.0 1.56 89.3 101.0 84.9 82.6 10 9.30 1.48 11.88 11.96 12.05 12.51 1 0.930 1.91 0.97 1.07 1.00 0.93 0.1 0.186 3.07 0.19 0.21 0.35 0.32 *S/N = 5 : 1. tND = not detected.
signal. However, using the multiple injection technique, good results were o b t a i n e d at the 1 ng/1 concentration, which is below the threshold o d o u r c o n c e n t r a t i o n for MIB. Table 2 shows the c o m p a r a t i v e b e h a v i o u r of geosmin-d3 a n d 1-chlorodecane as internal standards for different initial c o n c e n t r a t i o n s of geosmin (and of internal standard). The required response factors were determined as for MIB. Using the respective base peaks ( m / z 112 a n d 115), the geosmin-d3 internal s t a n d a r d gave better accuracy a n d precision t h a n the l-chlorodecane over the whole concent r a t i o n range (0.2-1000ng/1). A (visual) signal to noise ratio (S/N) > 30:1 was o b t a i n e d readily, even for c o n c e n t r a t i o n s o f 0.2 ng/l. Using the molecular ion, S/N was 2:1 at 0.2 ng/1 a n d 8:1 at 1 ng/l. F o r all
(%)
1038 100.2 10.6 1.11
2.1 0.2 2.3 3.5*
Mean 959 89.4 12.1 0.99 0.27
CV (%) 2.4 8.0 2.0 5.2 25.5
concentrations, the precision using the base peak was better t h a n using the molecular ion. Table 3 shows the effect of storage o n the concentrations of M I B , geosmin a n d l-chlorodecane which h a d been added to a surface water sample to give c o n c e n t r a t i o n s of 1000, 980, 927 ng/1, respectively. The water sample was then divided into two portions, the first of which was dosed with MIB-d3 (900 ng/l) a n d geosmin-d3 (1020 rig/l). Each of these p o r t i o n s was then divided into a set of sub-samples (of varying volume depending on the intended storage time). Each sub-sample was stored in a completely filled, sealed c o n t a i n e r a n d the separate containers analyzed after varying times up to 3 weeks. This procedure was followed to avoid the loss of volatile solute into head space which m a y occur w h e n sequential aliquots are
Table 2. Precision of measurement of geosmin by GC/MS (SIM) using geosmin-d3 or l-chlorodecane as internal standard (a) Geosmin-dj internal standard: m/z Conch Conch of present int. std (ng/1) (ng/I) 980 1018 982 (978) 98.0 101.8 95.7 (95.7) 9.80 10.18 10.15 (10.47) 0.980 1.018 0.975 (I.06) 0.196 0.204 0.219 (0.248) (b) l-Chlorodecane internal Concn Cohen of present int. std (rig/l) (ng/l) 980 930 98.0 93.0 9.80 9.30 0.980 0.930 0.196 0.186
112/115. Numbers in parentheses were determined using m/z 182/185 Concentration found CV (rig/l) Mean (%) 988 989 986 988 987 0.3 (969) (985) (975) (949) (971) (0.6) 97.0 96.0 96.3 95.6 96.1 0.6 (99.2) (97.2) (98.0) (96.6) (97.3) (1.4) 10.14 10.09 10.05 9.89 10.1 1.1 (9.93) (10.59) (10.68) (9.69) (10.3) (4.2) 0.986 0.981 0.965 0.957 0.973 1.2 (1.10) (1.04) (1.02) (1.01) (1.03) (3.3) 0.192 0.232 0.193 0.216 0.210 8.3 (0.265) (0.304) (0.250) (0.229) (0.259) (10.8)
standard: m/z 112 (geosmin)/91 (1-chlorodecane) RF 1.81 2.09 1.87 2.12 2.63
911 92.6 11.1 1.05 0.19
Concentration found (ng/l) 957 949 98.8 91.1 11.7 11.3 0.98 1.03 0.25 0.21
Mean 880 94.6 11.2
924 94.3 11.3
1.04
1.03
0.27
0.23
CV (%) 3.2 3.0 2.5 2.8 13.7
Determination of geosmin and methylisoborneol
323
Table 3. Effect of storage on the concentrationof MIB, geosminand l-chlorodecane* in surfacewater determinedusingMIB-d3 and geosmin-d3as internalstandards Apparent concn? Cohen found:l: (ng/l) (ng/l) Time Temp. (days) MIB Geosmin MIB Oeosmin 1-Chlorodecane 0.1 997 1000 949 949 165 5°C 2 1006 974 951 875 93 4 1006 980 912 797 23 8 1022 965 889 656 4 15 988 949 851 475 -22 993 956 808 252 -0.1 997 1000 949 949 165 20~C 1 985 975 943 603 -3 1006 958 853 509 -7 989 974 823 36 -14 992 979 783 29 -21 967 945 714 1 -*Initialconcentrations(ng/l):MIB 1000,geosmin980, l-chlorodecane927. tDetermined by addingMIB-d3and geosmin-d3 at the time of dosing the water with MIB, geosminand l-chlorodecane. :~Determinedby addingMIB-daand geosmin-d3 at the time of stripping.
withdrawn from a single container (Cline and Severin, 1989). A set of containers, one from each portion, was stored at both 20 and 5°C. All containers were stored in the dark. Each sub-sample was made up to 900 ml in the stripping bottle, to which 90 g of NaCI had been added. For each time/temperature storage condition, three sub-samples with and three sub-samples without initial addition of labelled internal standards were analysed. Apparent concentrations of MIB and geosmin were determined using the corresponding labelled internal standards (added initially) while the concentrations of MIB, geosmin and 1-chlorodecane remaining at the end of each storage interval were determined by adding a known mass of MIB-d3 and geosmin-d3 to the water immediately before stripping. For storage times < 1 week, the sub-samples were treated with mercuric chloride at the selected sampling time to suppress biological degradation during the course of replicate analyses. For samples stored at 20°C the loss of geosmin was very rapid while the MIB concentration declined more slowly; in 3 days the losses were 94 and 15%, respectively. The loss of l-chlorodecane was even more rapid; after 1 day it was no longer detectable. The initial loss of l-chlorodecane is probably due to adsorption; the water was very turbid (70 NTU). For samples stored at 5°C, the rate of loss of geosmin, MIB and l-chlorodecane was much lower (see Table 3). When mercuric chloride (40 mg/l) was added at the outset, loss of MIB and geosmin was suppressed. The presence of sunlight did not alter this result. This suggests that MIB and geosmin are lost mainly by biological pathways rather than by photochemical means. The greater resistance of MIB to biological degradation has been noted previously (Krasner, 1988). The most important result from the storage trial was that the samples dosed with MIB-d3 and geosmin-d3 still indicated the correct initial concentration of MIB and geosmin, even when < 10% of the
original geosmin and MIB remained in solution. By contrast, the chloroalkane was lost rapidly and, at 20°C, all chloroalkane had disappeared after 1 day. This is a major advantage of the labelled internal standards: for our surface water, assuming an initial concentration of about 50 ng/1 of MIB or geosmin, the labelled internal standards may be added at the time of sampling and provided the sample is analysed within 2 weeks (for samples stored at room temperature) or 3--4 weeks (for samples stored under refrigeration) accurate results for the initial concentrations can still be obtained. This obviates the need for toxic preservatives such as mercuric chloride and eliminates the health and waste disposal problems which it poses. A further advantage of the labelled standards is that they are insensitive to losses by volatilization, adsorption etc. since their behaviour is virtually identical with the unlabelled analogues. Thus, when a small air leak was induced in the stripping apparatus, a circumstance which would normally force the abandonment of an experimental run, 100ng/1 of MIB was determined as 103 ng/l (CV = 2.3%) and 100ng/1 of geosmin was determined as 99.5ng/1 ( C V = 2 . 1 % ) using the base peak or 99.9ng/1 (CV = 3.4%) using the molecular ion. This is because the labelled and unlabelled components undergo proportional losses. Similarly, when labelled internal standards are used, the accuracy of determination is not sensitive to variations in stripping time or water temperature. This is in contrast to the behaviour of 1-chlorodecane. Response factors, determined using 1-chlorodecane, vary with stripping time, because geosmin and MIB do not strip at the same rate as the l-chlorodecane (Hwang et aL, 1984). Hence it is essential to use a fixed stripping time and bath temperature when using l-chlorodecane as internal standard. The detection limit for the procedure may be lowered by concentrating the carbon disulphide extract under a stream of nitrogen although the gain
324
WOLFGANGKORTrIet al.
in sensitivity is somewhat less than would be expected from the reduction in volume because some of the analyte is lost as well as the solvent. However, because the labelled and unlabelled components undergo proportional losses, accurate determination of the original concentrations of MIB and geosmin is obtained. For routine use, solvent removal or oncolumn injection is preferable to the triple injection technique described earlier. In our case, the triple injection technique was necessary because on-column injection was not available and the differential losses of 1-chlorodecane, geosmin and MIB precluded solvent removal as an option. An additional advantage of the labelled standards relates to the filters used to adsorb the volatile organics. Hwang et al. (1984) have shown that the absolute recoveries of odorants vary with filter resistance (as measured by flow rate) in a different manner compared with the 1-chlorodecane internal standard. As a result, it is necessary to use filters which are matched for flow resistance for each set of standards and samples. Because filter flow rate tends to decline with repeated use, it is necessary to re-calibrate frequently (Hwang et al., 1984). These precautions
are unnecessary when using labelled internal standards. For example, when MIB was determined using MIB-da as internal standard, changing the stripping time from 1 to 1.5 to 2 h had no effect on the precision or accuracy of the determination. It has been shown previously that the relative intensities of the MIB fragment ions can vary quite markedly with the cleanliness of the mass spectrometer source (APHA, 1985). With a clean source, the base peak is m / z 95, but with a dirty source it is m / z 107. This is a further reason why frequent calibration is needed when calculating response factors based on these ions using l-chlorodecane as internal standard. By contrast, because the molecular ion ratio of labelled to unlabelled compound is independant of changes in their mass spectral fragment peak intensities, the current procedure is unaffected by this problem. CONCLUSIONS
The deuterium labelled standards offer many advantages compared with conventional internal standards used for CLSA/GC/MS determination of MIB and geosmin in natural waters. They offer high accuracy and precision down to concentrations below the threshold odour concentrations. They may be added at the time of sampling and so compensate for losses of analyte by physical, chemical and biological processes during sample storage. They also compensate for variations in sparging rate and other parameters of the CLSA procedure. Much time is saved by not having to determine response factors regularly and to analyse unknowns with and without spikes where an accurate determination of concentration is required.
The method could also be used with advantage to determine MIB and geosmin in foods and beverages. Acknowledgements--W. K. wishes to thank Edward G.
Means (Laboratory Manager) and Stuart Krasner (Senior Research Chemist), Water Quality Division, The Metropolitan Water District of Southern California and Professor F. Juttner and K. Wurster (Institfit fiir Chemischepttanzenphysiologie, University of Tubingen) for helpful discussions. REFERENCES
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G. and Bjorseth A.), pp. 2-13. Kluwer Academic Publishers, London. Wood N. F. and Snoeyink V. L. (1977) 2-Methylisoborneol, improved synthesis and a quantitative gas chromatographic method for trace concentrations producing odor in water. J. Chromat. 132, 405-420. Yasuhara A. and Fuwa K. (1979) Determination of geosmin in water by computer-controlled mass fragmentography. J. Chromat. 172, 453-456. Zoretic P. A., Branchaud B. and Maestrone T. (1975) Robinson annelations with a/~-chloroketone in the presence of an acid. Tetrahed. Lett. 527-528.
0043-1354/91 $3.00+0.00 Copyright© 1991 PergamonPress pie
NEW S T A N D A R D S FOR THE DETERMINATION OF GEOSMIN A N D METHYLISOBORNEOL IN WATER BY GAS CHROMATOGRAPHY/MASS SPECTROMETRY WOLFGANGKORTH~@, KATHLEENBOWMERJ and JOHN ELLIS2 ~) ~CSIRO Division of Water Resources, Private Mail Bag, Griffith, NSW 2680 and 2Chemistry Department, University of Wollongong, P.O. Box 1144, Wollongong NSW 2500, Australia (First received January 1990; accepted in revised form October 1990) Abstract--Deuterium labelled geosmin and methylisoborneol (MIB) have been synthesized and evaluated as internal standards in the determination of geosmin and MIB in water by dosed loop stripping followed by gas chromatography/mass spectrometry (GC/MS). The labelled standards were compared with chloroalkanes added as internal standards either at the time of sampling or immediately before closed loop stripping. When added at sampling time, the new standards enabled accurate determination of the geosmin and MIB present initially, even when the samples were analysed as much as 3 weeks later. The new standards gave better precision and accuracy than the chloroalkanes and overcame the underestimation of initial analyte concentration which usually results from losses of analyte through adsorption, volatilisation, biodegradation etc. during sample storage. Geosmin had a limit of detection of < 0. I ng/1 and 1 ng/1 was determined with a coefficient of variation (CV) of 1.2% (n = 5). MIB was determined at 1 ng/1 with a CV of 3.5% (n = 5). Key words--geosmin, methylisoborneol, odorous compounds, closed loop stripping analysis, gas chromatography/mass spectrometry, selected ion monitoring
INTRODUCTION Geosmin [la,10fl-dimethyl-9~-decalol; (1)] and MIB (( 1-R-exo )- 1,2,7,7-tetramethyl bicyclo-[2,2,1]-heptan2-ol; [2]) (Fig. 1) are secondary metabolites of certain blue-green algae and actinomycetes (Safferman et al., 1967; Medsker et al., 1968, 1969). They have been reported worldwide as imparting an earthy or musty odour to drinking water (Rosen et al., 1970; Mallevialle and Suffet, 1987). Their threshold odour concentrations are 10-20ng/1 (Wood and Snoeyink, 1977; Yasuhura and Fuwa, 1979; Persson, 1980; Bartels et al., 1989). As a consequence, only the most sensitive methods will suffice for their analysis, and current practice is to use closed loop stripping (CLSA) followed by GC/MS with selected ion monitoring (SIM) (Hwang et al., 1984; Mallevialle and Suffet, 1987; Bartels et al., 1989). Accurate quantitation requires the use of an internal standard which is added to the water prior to the closed loop stripping step. Ideally one requires a compound which has similar Henry's law behaviour to the geosmin or MIB and which resists losses through adsorption or through degradation by chemical, photochemical or biological means. An internal standard with these properties would enable accurate determination of the geosmin (or MIB) concentration present in the water at the time of stripping the sample. If this is not possible, the chosen internal standard should be lost at a similar rate to the natural odour compound. A number of compounds have been used as internal standards (Wood and Snoeyink, 1977; Hwang et al.,
1984) and a mixture of linear chloroalkanes is currently accepted as the best compromise (Barteis et al., 1989). However, it is unlikely that any other compound will behave in precisely the same way as geosmin (or MIB). Scott et al. (1988) identified some of the problems associated with chloroalkane standards and referred briefly to their substitution by labelled standards. No details were given of the positions of labelling, but when solutions containing both labelled and unlabelled geosmin were analysed, the ion intensity ratio of 114/I 12 was constant over the concentration range 0.04-1.0#g/1. Accordingly, we have synthesized deuterium labelled analogues of geosmin and MIB to compare the behaviour of these internal standards with that of the chloroalkane mix. The mass spectrum of natural geosmin gives a weak molecular ion (m/z 182; 6%) under electron impact ionization conditions, with a base peak at m / z 112. Consequently, if deuterium labelled geosmin were to be used as an internal standard, it would be highly desirable for the label to be present intact in the base peak so that maximum analytical sensitivity could be achieved. Moreover, it would be preferable to introduce the deuterium using a discrete labelled synthon, rather than by, for example, base catalysed exchange adjacent to a carbonyl group or similar reactions which might fail to give quantitative labelling and/or suffer partial loss of label through exchange at a later stage of the synthesis. For these reasons, it was decided to label the angular methyl group attached to C~0. 319
WOLFGANGKORan et al.
320
CH 3
H
: 3OH
CH3
1
2
3
Fig. 1. Structures of geosmin (1), methylisoborneol (2) and 1,10-dimethyldecal-l(9)-en-2-one (3). The mass spectral cracking p a t t e r n of M I B has been studied by M e d s k e r et al. (1969), w h o proposed m e c h a n i s m s for the f r a g m e n t a t i o n (Fig. 2). A weak molecular ion ( m / z 168, 7 % ) is p r o d u c e d u n d e r E1 conditions a n d the base peak is at m / z 95. Even t h o u g h a labelled base peak is n o t produced, the only practicable m e t h o d o f preparing labelled M I B was to react m e t h y l m a g n e s i u m iodide-d3 with d - c a m p h o r a n d to purify the MIB-d3 by the m e t h o d of W o o d a n d Snoeyink (1977).
MATERIALS AND M E T H O D S
CLSA equipment was from Brechbuehler AG, Switzerland, modified to take a tall-form bottle (380 x 70ram; 350 ml headspace). The water sample (c. 800 ml) was added to the tall-form bottle and 90 g of solid sodium chloride (previously heated to 700°C for 12 h) was added and dissolved ( < 30 s) with magnetic stirring. The stirrer bar was removed and the solution made up to 900 ml with milli-Q water. The solution was held at 25°C and stripped for 1.5 h at an air flow rate of 2 l/min. Volatile organic compounds were adsorbed by a granular activated carbon (GAC) filter held at 40°C (block temperature 50°C). At the completion of the stripping cycle, the filter was eluted sequentially with 10, 10 and 5/zl of CS2. The combined extract was stored in a 100/~1 micro vial placed in a 2 ml screw cap vial fitted with an open top cap and a Teflon septum. A stainless steel spring was used to push the micro vial up against the septum. Filters were washed with l ml portions of acetone, water (milli-Q), acetone, CS2, acetone (3 x ), methylene chloride (3 × ) and dried under vacuum.
Gas chromatography was performed using a Varian 3300 gas chromatograph with He carrier gas (0.75 ml/min) and a Hewlett-Packard fused silica capillary column (25m, 0.20 mm i.d., coated with 0.30/zm crosslinked methyl silicone). A 1.0 #1 sample was injected and the column held at 25°C for 1.5 min with the splitter off for 1 min. The splitter was then turned on and the column heated at 10°C/min to 80°C, then 5°C/min to 250°C and held at 250°C for 5 min. Splitter gas flow rate was 50 ml/min and both injector and interface were at 260°C. For concentrations < I ng/1, three consecutive injections of 2#1 of extract were made per analysis. Cumulative transfer to the column was achieved without band broadening by holding the oven at 25°C until all three injections were made. After 5 min, for each of the first two injections, the splitter was opened for 20 s and then closed again prior to the next injection. For the third injection the splitter remained open after 5 min and the oven was ramped as described above. The eluate from the GC reported to a Hewlett-Packard 5970 mass selective detector, using electron impact ionization (70 eV) and operated in the SIM mode. The ions monitored and those used for quantitation were as follows:
Compound 1-Chlorodecane MIB Geosmin MIB-d 3 Geosmin-d 3
91 95 112
168 112,182 171 115,185
Ions monitored (m/z) A B 43,91,93 95, 107, 135 111,112,125
A = l-chlorodecane as internal standard. B = MIB-d3 and geosmin-d3 as internal standard.
H--C3H60
m/z 168
(m/z) used for quantitation A B Ions
*
m/z 110
- CH3
m/z 95
m/z 153 Fig. 2. Fragmentation of methylisoborneol under electron impact ionization.
168 112,182 171 115,185
Determination of geosmin and methylisoborneol
321
When comparing relative areas of peaks from labelled and unlabelled compounds, computed corrections were applied where necessary for contributions of isotope satellites in the unlabelled compound to the labelled peak being monitored, and vice versa. For example, when using the base peaks 112/115 for quantitation of geosmin the followingequations were required to be solved simultaneously to obtain corrected ion area values for the two masses:
The MIB-d3 was prepared by reaction of CD3MgI with (+)-camphor. Unreacted camphor was removed by reaction with hydroxylamine, and the MIB-d 3 purified by chromatography on silica gel and sublimation (Wood and Snoeyink, 1977). The mass spectrum showed the expected molecular ion at m/z 171 (6%) (168 in MIB), but the base peak area l12obS= area ll2corr+ (% area 1121x area llScor~) remained at m/z 95, as in unlabelled MIB. Hence, area 115ohS= area 115corr+ (% area 115ulx area 112corr) when using MIB-da as an internal standard, selected ion monitoring had to utilise the molecular ion at obs = observed; corr = corrected m/z 171, rather than the base peak. The salted CLSA procedure employed was based % area 112j= peak area at mass 112, in labelled geosmin, expressed as a percentage of its peak area at mass 115 on that of Hwang et al. (1984). The water sample was % area 115urn= peak area at mass 115, in unlabelled geos- kept at 25°C and stripped for 1.5 h. However, sodium min, expressed as a percentage of its peak area at mass 112. chloride was substituted for sodium sulphate. Equimolar solutions of the authentic MIB and geosmin Depending on source, when the latter is heated to were added to equimolar solutions of the corresponding 700°C to remove organic impurities, it tends to cake labelled compounds to establish the purity of the latter. and thus is slow to dissolve in water. Addition of Chemicals used were sourced as follows: 1-chloro-3-pen- sodium chloride had a marked effect on the recovery tanone, cyclohexene oxide, methylmagnesium iodide-d3, of MIB and geosmin and a lesser effect on the 1-chlorooctane, l-chlorodecane, (+)-camphor (Aldrich); 1-chlorodecane. The identical stripping behaviour of 1-chlorododecane, carbon disulphide (Merck); ( - ) methylisoborneol, racemic geosmin (Wako); acetone, MIB vs MIB-d3 and geosmin vs geosmin-d3 was dichloromethane, sodium chloride (Ajax). confirmed by stripping solutions containing both the labelled and unlabelled compounds. Hwang et al. (1984) observed that stripping efficiency increased RESULTS AND DISCUSSION for additions of Na2SO4 up to 100g/l. We found a The most recent synthesis of geosmin was reported similar trend for NaCI concentrations up to by Gosselin et al. (1989), who obtained racemic 100-150g/1. The improved stripping efficiency was geosmin in about 40% yield from the attributed by Hwang et al. (1984) to reduced dimethyldecalenone [3] (Fig. 1), which can be pre- solubility of the organic solutes and formation of pared by condensation of 1-chloro-3-pentanone and smaller air bubbles by the sparging gas when salt 2-methylcyclohexanone (Zoretic et al., 1975). For the was added. Table 1 shows the comparative behaviour of MIBpresent purpose, labelled 2-methylcyclohexanone was prepared by reaction of CD3MgI and cyclohexene d3 and l-chlorodecane internal standards for different oxide, followed by oxidation of the 2-methylcyclo- initial concentrations of MIB (and of internal stanhexanol-d3 to 2-methylcyclohexanone-d3. The mass dard). The CLSA results using the 1-chlorodecane spectra and N M R spectra of the labelled compound standard were calculated using the procedure of [3] confirmed that the methyl group on C10 had been Mallevialle and Suffet (1987). A response factor (RF) labelled quantitatively with three deuterium atoms. for MIB was calculated from the CLSA of one water The geosmin-d3 was shown to retain this label and, sample containing known concentrations of MIB and 1-chlorodecane calibration standard. This response most importantly, the base peak of the mass spectrum was now at m/z 115 instead of m/z 112. The base factor was then used to calculate the MIB concenpeak can be accounted for by the ion radical in Fig. 3. tration of the unknown sample (u) for a further four Proof that this ion is derived from ring B was CLSA runs, using the formula obtained by preparing geosmin-d5 labelled with two area MIBu x amount (IS)u further deuterium atoms (one at C i and the other at MIBu (rig/l) = RF (MIB) x area (IS)u C2). This gave a molecular ion at m/z 187, while the base peak remained at m/z 115. Details of the seven where step synthesis will be reported elsewhere. area MIBu = peak area of the ion used for quantiration (m/z 95) and area (IS)u = peak area of m/z 91 (see list above).
I
÷1 H m/z 115
Fig. 3. Base peak of geosmin under electron impact ionization.
In the case of the labelled internal standard, the response factor for MIB-d3 was the same as that for unlabelled MIB. The labelled internal standard procedure gave better precision than the l-chlorodecane in all cases except for the 0.2 ng/1 concentration, where the molecular ion intensity was too low to give an acceptable
WOLFGANG KORTH et al.
322
Table 1. Precision of measurement of MIB by GC/MS (SIM) using MIB-d3 or 1-chlorodeeane as internal standard (a) MIB-d3 internal standard: m/z 168/171
Concn present
Conch of int. std.
Concentration found
(ng/l)
(ng/l)
(ng/l)
1000 100 10.0 1.00 0.200
930 93.0 9.30 0.930 0.186
999 100.5 10.42 1.10 ND
1065 100.2 10.50 1.18 ND
1051 100.3 10.40 1.08 ND
CV Mean
1040 100.2 10.79 1.10 ND
1034 99.9 10.97 1.09 ND?
(b)l-Chlorodeeane internal standard: m/z 95 (MIB)/91 (chlorodecane) Concn Conch present int. std Concentration found (ng/I) (rig/l) RF (rig/l) 1000 930 1.39 968 991 949 928 100 93.0 1.56 89.3 101.0 84.9 82.6 10 9.30 1.48 11.88 11.96 12.05 12.51 1 0.930 1.91 0.97 1.07 1.00 0.93 0.1 0.186 3.07 0.19 0.21 0.35 0.32 *S/N = 5 : 1. tND = not detected.
signal. However, using the multiple injection technique, good results were o b t a i n e d at the 1 ng/1 concentration, which is below the threshold o d o u r c o n c e n t r a t i o n for MIB. Table 2 shows the c o m p a r a t i v e b e h a v i o u r of geosmin-d3 a n d 1-chlorodecane as internal standards for different initial c o n c e n t r a t i o n s of geosmin (and of internal standard). The required response factors were determined as for MIB. Using the respective base peaks ( m / z 112 a n d 115), the geosmin-d3 internal s t a n d a r d gave better accuracy a n d precision t h a n the l-chlorodecane over the whole concent r a t i o n range (0.2-1000ng/1). A (visual) signal to noise ratio (S/N) > 30:1 was o b t a i n e d readily, even for c o n c e n t r a t i o n s o f 0.2 ng/l. Using the molecular ion, S/N was 2:1 at 0.2 ng/1 a n d 8:1 at 1 ng/l. F o r all
(%)
1038 100.2 10.6 1.11
2.1 0.2 2.3 3.5*
Mean 959 89.4 12.1 0.99 0.27
CV (%) 2.4 8.0 2.0 5.2 25.5
concentrations, the precision using the base peak was better t h a n using the molecular ion. Table 3 shows the effect of storage o n the concentrations of M I B , geosmin a n d l-chlorodecane which h a d been added to a surface water sample to give c o n c e n t r a t i o n s of 1000, 980, 927 ng/1, respectively. The water sample was then divided into two portions, the first of which was dosed with MIB-d3 (900 ng/l) a n d geosmin-d3 (1020 rig/l). Each of these p o r t i o n s was then divided into a set of sub-samples (of varying volume depending on the intended storage time). Each sub-sample was stored in a completely filled, sealed c o n t a i n e r a n d the separate containers analyzed after varying times up to 3 weeks. This procedure was followed to avoid the loss of volatile solute into head space which m a y occur w h e n sequential aliquots are
Table 2. Precision of measurement of geosmin by GC/MS (SIM) using geosmin-d3 or l-chlorodecane as internal standard (a) Geosmin-dj internal standard: m/z Conch Conch of present int. std (ng/1) (ng/I) 980 1018 982 (978) 98.0 101.8 95.7 (95.7) 9.80 10.18 10.15 (10.47) 0.980 1.018 0.975 (I.06) 0.196 0.204 0.219 (0.248) (b) l-Chlorodecane internal Concn Cohen of present int. std (rig/l) (ng/l) 980 930 98.0 93.0 9.80 9.30 0.980 0.930 0.196 0.186
112/115. Numbers in parentheses were determined using m/z 182/185 Concentration found CV (rig/l) Mean (%) 988 989 986 988 987 0.3 (969) (985) (975) (949) (971) (0.6) 97.0 96.0 96.3 95.6 96.1 0.6 (99.2) (97.2) (98.0) (96.6) (97.3) (1.4) 10.14 10.09 10.05 9.89 10.1 1.1 (9.93) (10.59) (10.68) (9.69) (10.3) (4.2) 0.986 0.981 0.965 0.957 0.973 1.2 (1.10) (1.04) (1.02) (1.01) (1.03) (3.3) 0.192 0.232 0.193 0.216 0.210 8.3 (0.265) (0.304) (0.250) (0.229) (0.259) (10.8)
standard: m/z 112 (geosmin)/91 (1-chlorodecane) RF 1.81 2.09 1.87 2.12 2.63
911 92.6 11.1 1.05 0.19
Concentration found (ng/l) 957 949 98.8 91.1 11.7 11.3 0.98 1.03 0.25 0.21
Mean 880 94.6 11.2
924 94.3 11.3
1.04
1.03
0.27
0.23
CV (%) 3.2 3.0 2.5 2.8 13.7
Determination of geosmin and methylisoborneol
323
Table 3. Effect of storage on the concentrationof MIB, geosminand l-chlorodecane* in surfacewater determinedusingMIB-d3 and geosmin-d3as internalstandards Apparent concn? Cohen found:l: (ng/l) (ng/l) Time Temp. (days) MIB Geosmin MIB Oeosmin 1-Chlorodecane 0.1 997 1000 949 949 165 5°C 2 1006 974 951 875 93 4 1006 980 912 797 23 8 1022 965 889 656 4 15 988 949 851 475 -22 993 956 808 252 -0.1 997 1000 949 949 165 20~C 1 985 975 943 603 -3 1006 958 853 509 -7 989 974 823 36 -14 992 979 783 29 -21 967 945 714 1 -*Initialconcentrations(ng/l):MIB 1000,geosmin980, l-chlorodecane927. tDetermined by addingMIB-d3and geosmin-d3 at the time of dosing the water with MIB, geosminand l-chlorodecane. :~Determinedby addingMIB-daand geosmin-d3 at the time of stripping.
withdrawn from a single container (Cline and Severin, 1989). A set of containers, one from each portion, was stored at both 20 and 5°C. All containers were stored in the dark. Each sub-sample was made up to 900 ml in the stripping bottle, to which 90 g of NaCI had been added. For each time/temperature storage condition, three sub-samples with and three sub-samples without initial addition of labelled internal standards were analysed. Apparent concentrations of MIB and geosmin were determined using the corresponding labelled internal standards (added initially) while the concentrations of MIB, geosmin and 1-chlorodecane remaining at the end of each storage interval were determined by adding a known mass of MIB-d3 and geosmin-d3 to the water immediately before stripping. For storage times < 1 week, the sub-samples were treated with mercuric chloride at the selected sampling time to suppress biological degradation during the course of replicate analyses. For samples stored at 20°C the loss of geosmin was very rapid while the MIB concentration declined more slowly; in 3 days the losses were 94 and 15%, respectively. The loss of l-chlorodecane was even more rapid; after 1 day it was no longer detectable. The initial loss of l-chlorodecane is probably due to adsorption; the water was very turbid (70 NTU). For samples stored at 5°C, the rate of loss of geosmin, MIB and l-chlorodecane was much lower (see Table 3). When mercuric chloride (40 mg/l) was added at the outset, loss of MIB and geosmin was suppressed. The presence of sunlight did not alter this result. This suggests that MIB and geosmin are lost mainly by biological pathways rather than by photochemical means. The greater resistance of MIB to biological degradation has been noted previously (Krasner, 1988). The most important result from the storage trial was that the samples dosed with MIB-d3 and geosmin-d3 still indicated the correct initial concentration of MIB and geosmin, even when < 10% of the
original geosmin and MIB remained in solution. By contrast, the chloroalkane was lost rapidly and, at 20°C, all chloroalkane had disappeared after 1 day. This is a major advantage of the labelled internal standards: for our surface water, assuming an initial concentration of about 50 ng/1 of MIB or geosmin, the labelled internal standards may be added at the time of sampling and provided the sample is analysed within 2 weeks (for samples stored at room temperature) or 3--4 weeks (for samples stored under refrigeration) accurate results for the initial concentrations can still be obtained. This obviates the need for toxic preservatives such as mercuric chloride and eliminates the health and waste disposal problems which it poses. A further advantage of the labelled standards is that they are insensitive to losses by volatilization, adsorption etc. since their behaviour is virtually identical with the unlabelled analogues. Thus, when a small air leak was induced in the stripping apparatus, a circumstance which would normally force the abandonment of an experimental run, 100ng/1 of MIB was determined as 103 ng/l (CV = 2.3%) and 100ng/1 of geosmin was determined as 99.5ng/1 ( C V = 2 . 1 % ) using the base peak or 99.9ng/1 (CV = 3.4%) using the molecular ion. This is because the labelled and unlabelled components undergo proportional losses. Similarly, when labelled internal standards are used, the accuracy of determination is not sensitive to variations in stripping time or water temperature. This is in contrast to the behaviour of 1-chlorodecane. Response factors, determined using 1-chlorodecane, vary with stripping time, because geosmin and MIB do not strip at the same rate as the l-chlorodecane (Hwang et aL, 1984). Hence it is essential to use a fixed stripping time and bath temperature when using l-chlorodecane as internal standard. The detection limit for the procedure may be lowered by concentrating the carbon disulphide extract under a stream of nitrogen although the gain
324
WOLFGANGKORTrIet al.
in sensitivity is somewhat less than would be expected from the reduction in volume because some of the analyte is lost as well as the solvent. However, because the labelled and unlabelled components undergo proportional losses, accurate determination of the original concentrations of MIB and geosmin is obtained. For routine use, solvent removal or oncolumn injection is preferable to the triple injection technique described earlier. In our case, the triple injection technique was necessary because on-column injection was not available and the differential losses of 1-chlorodecane, geosmin and MIB precluded solvent removal as an option. An additional advantage of the labelled standards relates to the filters used to adsorb the volatile organics. Hwang et al. (1984) have shown that the absolute recoveries of odorants vary with filter resistance (as measured by flow rate) in a different manner compared with the 1-chlorodecane internal standard. As a result, it is necessary to use filters which are matched for flow resistance for each set of standards and samples. Because filter flow rate tends to decline with repeated use, it is necessary to re-calibrate frequently (Hwang et al., 1984). These precautions
are unnecessary when using labelled internal standards. For example, when MIB was determined using MIB-da as internal standard, changing the stripping time from 1 to 1.5 to 2 h had no effect on the precision or accuracy of the determination. It has been shown previously that the relative intensities of the MIB fragment ions can vary quite markedly with the cleanliness of the mass spectrometer source (APHA, 1985). With a clean source, the base peak is m / z 95, but with a dirty source it is m / z 107. This is a further reason why frequent calibration is needed when calculating response factors based on these ions using l-chlorodecane as internal standard. By contrast, because the molecular ion ratio of labelled to unlabelled compound is independant of changes in their mass spectral fragment peak intensities, the current procedure is unaffected by this problem. CONCLUSIONS
The deuterium labelled standards offer many advantages compared with conventional internal standards used for CLSA/GC/MS determination of MIB and geosmin in natural waters. They offer high accuracy and precision down to concentrations below the threshold odour concentrations. They may be added at the time of sampling and so compensate for losses of analyte by physical, chemical and biological processes during sample storage. They also compensate for variations in sparging rate and other parameters of the CLSA procedure. Much time is saved by not having to determine response factors regularly and to analyse unknowns with and without spikes where an accurate determination of concentration is required.
The method could also be used with advantage to determine MIB and geosmin in foods and beverages. Acknowledgements--W. K. wishes to thank Edward G.
Means (Laboratory Manager) and Stuart Krasner (Senior Research Chemist), Water Quality Division, The Metropolitan Water District of Southern California and Professor F. Juttner and K. Wurster (Institfit fiir Chemischepttanzenphysiologie, University of Tubingen) for helpful discussions. REFERENCES
APHA (1985) Standard Methods for the Examination of Water and Wastewater, 16th edition. American Public Health Association, Washington, D.C. Bartels J. H. M., Brady B. M. and Suffet I. H. (Eds) (1989) Taste and odor in drinking water supplies. Combined final report, Phase I and II. American Water Works Association, Denver, CO 80235. Cline S. M. and Severin B. F. (1989) Volatile organic losses from a composite water sampler. Wat. Res. 23, 407-412. Gosselin P., Joulain D., Laurin P. and Rouessac F. (1989) Synthesis of earthy-mouldy smelling compounds--l. Stereoselective synthesis of ( + / - ) geosmin. Tetrahed. Lett. 30, 2775-2778. Hwang C. J., Krasner S. W., McGuire M. J., Moylan M. S. and Dale M. S. (1984) Determination of subnanogram per liter levels of earthy-musty odorants in water by the salted closed-loop stripping method. Envir. Sci. Technol. 18, 535-539. Krasner S. (1988) Personal communication. MallevialleJ. and Suffet I. H. (Eds) (1987) Identification and treatment of tastes and odors in drinking water. American Water Works Association, Denver, CO 80235. Medsker L. L., Jenkins D. and Thomas J. F. (1968) Odorous compounds in natural waters. An earthy smelling compound associated with blue-green algae and actinomycetes. Envir. Sci, Technol. 2, 461-464. Medsker L. L., Jenkins D., Thomas J. F. and Koch C. (1969) Odorous compounds in natural waters. 2-Exohydroxy-2-methylbornane,the major odorous compound produced by several actinomycetes.Envir. Sci. Technol. 3, 476--477. Persson P. E. (1980) Sensory properties and analysis of two muddy odour compounds, geosmin and 2-methylisoborneol, in water and fish. Wat. Res. 14, 1113-1118. Rosen A. A., Mashni C. I, and Safferman R. S. (1970) Recent developments in the chemistry of odour in water: the cause of earthy/musty odour. Wat. Treat. Exam. 19, 106-119. Safferman R. S., Rosen A. A., Mashni C. I. and Morris M. E. (1967) Earthy-smelling substance from a bluegreen alga. Envir. Sci. Technol. 1, 429-430. Scott S. P., Keeling R. L., James H. A., Waggott A. and Whittle P. (1988) The use of low cost mass spectrometers for the analysis of organic micropollutants in water. In Organic Micropollutants in the Aquatic Environment-Proc. 5th European Symposium, 1987 (Edited by Angeletti
G. and Bjorseth A.), pp. 2-13. Kluwer Academic Publishers, London. Wood N. F. and Snoeyink V. L. (1977) 2-Methylisoborneol, improved synthesis and a quantitative gas chromatographic method for trace concentrations producing odor in water. J. Chromat. 132, 405-420. Yasuhara A. and Fuwa K. (1979) Determination of geosmin in water by computer-controlled mass fragmentography. J. Chromat. 172, 453-456. Zoretic P. A., Branchaud B. and Maestrone T. (1975) Robinson annelations with a/~-chloroketone in the presence of an acid. Tetrahed. Lett. 527-528.