JOURNAL OF MASS SPECTROMETRY, VOL. 30, 1034-1040 (1995)

Application of Reversed Phase Liquid Chromatography with Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry to the Determination of Polycyclic Aromatic Sulfur Heterocycles in Environmental Samples?

Darren Thomas, Sheila M. Crain and P. Greig Sim! National Research Council Canada, Institute for Marine Biosciences, 141 1 Oxford Street, Halifax, Nova Scotia, B3H 321, Canada

Frank M. Benoit Environmental Health Centre, Health Canada, Tunney's Pasture, Ottawa, Ontario, KIA OL2, Canada

Reversed-phase liquid chromatography (RPLC) was combined with atmospheric pressure chemical ionization mass spectrometry (APCI-MS), via a heated pneumatic nebulizer interface, for the determination of the polycyclic aromatic sulfur heterocycle (PASH) content of samples obtained by the fractionation of an extract of a pond sediment contaminated by coke-oven residues. Some of the samples produced by the fractionation procedure con- tained large amounts of other polycyclic aromatic compounds (PACs) which co-eluted with the compounds of interest, making it difficult to obtain mass spectra suitable for compound identification and verification. Therefore, the use of tandem mass spectrometry (MS/MS), as a selective method for the identification of target analytes in complex matrices, was investigated. Initially, PASH standards were injected into the mass spectrometric system by flow injection and their collisionally induced dissociation mass spectra recorded. From these results, it was possible to select ions suitable for selected reaction monitoring (SRM) experiments on both the PASH standards (to estab- lish detection limits and also retention times which could be used to identify these compounds) and the fractions (to establish the possible presence of the selected PASHs in the fractions). The RPLCSRM experiments led to a tentative identification of some of the PASH standards in the fractions. However, the use of multiple reaction monitoring experiments allowed the positive identification of dibenzothiophene, phenanthrol4,5-bcdl thiophene, phenanthro[3,4-b] thiophene and benzo [ bl naphthol 2,341 thiophene in the fractions, along with several of their isomers. Quantification of the PASH standards by RPLC-SRM in the extracts found them to be present at high levels.

INTRODUCTION

The identification and quantification of polycyclic aro- matic sulfur heterocyclics (PASHs) in a complex environmental mixture of polycycli'c aromatic com- pounds (PACs) is usually a difficult and laborious task, involving extensive fractionation and preconcentration steps to isolate a PASH fraction suitable for analysis. A variety of techniques have been employed to this end, including chemical transformations,'*2 designed either to concentrate selectively those oxygen- and sulfur- containing compounds capable of oxidation to quin- ones or sulfones, or to effect sielective chemical reduction, followed by tandem mass spectrometry3 (MS/MS). A variety of column chromatographic tech- niques have also been which generally employ some form of ligand-exchange chromatography. Thus, Nishioka et aL4 have used palladium chloride adsorbed on silica gel as the column material, and this

t NRCC No. 38073. 3 Author to whom correspondence should be: addressed.

work was furthered by Andersson,' who studied the retention properties of a variety of PASHs on this material. Other workers have used silver nitrate coated silica gel columns6-' or adsorbents containing

to the same end. In all these techniques, one is obliged to perform either a lengthy chemical manipulation of the sample or multiple column chro- matographic separations. In the former case detection is necessarily restricted to those compounds that exhibit the chosen chemical behavior, whereas in the latter case only those compounds that have the desired com- bination of absorption characteristics will be concen- trated. Depending on the nature of the original sample, these steps may or may not selectively concentrate all the PASHs into the final extract subjected to instrumen- tal analysis.

A variety of more selective instrumental approaches have also been developed. The earliest such method appears to be that of Nishioka et al.," who employed gas chromatography (GC) with a flame photometric detector for the selective detection of sulfur compounds in heavy oils. A more recently developed GC technique, employing an atomic emission detector for the selective

@ 1076-5 174/95/071034-07 Crown Copyright (Canada)

Received 21 September 1994 Accepted 20 March 1995

ANALYSIS OF OLIGONUCLEOTIDES BY ES/MS/MS 1035

detection of sulfur-containing compounds, has also been applied to the problem." Also, the selective detection of sulfur-containing compounds by GC with a chemical reduction interface interposed between the GC oven and a mass spectrometer has been dem~nstrated.'~ In all of these techniques, the analysis is perforce restricted to only those compounds capable of elution from the GC column. Recent GC column developments have extended the accessible molecular mass range upward from N 300, but high-performance liquid chromatog- raphy (HPLC) still has a considerable advantage over GC for the analysis of higher molecular mass com- pounds. We were interested in exploring the possibilities of developing selective HPLC/MS techniques for these compounds.

In the present work, we adopted an approach that is a combination of preconcentration of the target frac- tion, selective detection and (if desired) quantification. A sequence of column chromatographic steps, which selec- tively isolate the polycyclic aromatic hydrocarbons (PAHs) and PASHs from the oxygen- and nitrogen- containing polycyclic aromatic compounds (PACS),'~ was used to produce the analytical sample, which was then subjected to reversed-phase chromatographic/ tandem mass spectrometric (RPLC/MS/MS) analysis. It was possible to identify sensitively and selectively some of the PASHs present, despite the relatively more abun- dant PAHs present in the final sample.

~

Table 1. Structures and molecular masses of the PASH stan- dards used in the experiments

PASH

Dibenzothiophene (A)

Benzo[b]naphtho[2,3-d]thiophe;ie (B)

Benzo[b]naphtho[l,2-d]thiophene (C)

Phenanthro[4,5-bcd]thiophene (D)

Phenanthro[4,3-b]thiophene (E)

Phenanthro[S,lO-blthiophene (F)

EXPERIMENTAL Phenanthro[3,4-b]thiophene ( G )

PASH standards (see Table 1 for structures) were pro- vided by Aldrich (Milwaukee, WI, USA) and the NCI Chemical Carcinogen Repository (Kansas City, MO, USA), and were dissolved in acetonitrile- dichloromethane (1 : 1). Contaminated pond sediment from the Sydney Tar Ponds (Sydney, Nova Scotia, Canada) was fractionated as follows: the sample was dissolved in a small volume of chloroform-cyclohexane (1 : 1) and applied to the top of a short (5 cm) column of silica gel, which had been treated with an ammoniacal copper solution to produce an immobilized metal ion adsorbent. This column was eluted with successive 10 ml portions of the same solvent mixture, to produce 11 fractions, of which fractions 1-2, 6-7 and 9-11 exhibited relatively intense color, the remaining frac- tions being almost colorless. This procedure is described in more detail elsewhere. l4 The fractions produced were then dissolved in acetonitrile-dichloromethane (1 : 1). The RPLC and RPLC/MS experiments were performed using a Hewlett-Packard (Palo Alto, CA, USA) 1090 Series I1 liquid chromatographic system. Volumes of 5 p1 of each fraction were injected on to a Vydac 201TP52 C,, reversed-phase column (250 mm x 2.1 mm id. with 5 pm particle size, obtained from Keystone Scientific, Bellefonte, PA, USA) and eluted with the mobile phase gradient described in Table 2 (unless stated otherwise) at a flow rate of 200 p1 min-'. Detection in the first instance was performed using a Hewlett-Packard 1040 diode-array detector (DAD) set at a wavelength of 254 nm. For these DAD experiments the HPLC system was controlled and data were collected and processed by

Structure and molecular mass

234

Hewlett-Packard 3365 ChemStation software installed on a Hewlett-Packard Vectra 486/33U computer.

The RPLC/MS experiments were carried out using a SCIEX (Thornhill, Ontario, Canada) API I11 triple quadrupole mass spectrometer equipped with an APCI source. The sample was injected on to the column and the eluent from the LC conveyed into the ion source of the mass spectrometer through a heated pneumatic nebulizer interface. Medical-grade air was used as both nebulizer gas and make-up gas. The interface was oper- ated at 400°C, with a nebulizer pressure of 100 psi and

Table 2. RPLC mobile pbase gradient employed for the separation of PASH standards and fractions obtained by the fractionation of a pond sediment contaminated by coke-oven residues'

Time (min) Water (% ) Acetonitrile (%) Dichloromethane (YO)

0 40 60 0 45 0 100 0 55 0 100 0 105 0 0 100 120 0 0 1 00

"The PASH standards all eluted in the 045 min portion of the mobile Dhase aradient.

1036 D. THOMAS ET AL.

Table 3. Time periods and fragmentation transitions moni- tored for the SRM and MRM experiments used in the study

Time period (min)

A" 0-1 3.00 13.00-1 5.80 1 5 . 8 ~ 8.20 18.20-21.40

21.40-23.60

B" 0-1 3.00

Compounds monitored

A D G E, F, C

B

A

13.00-15.80 D

15.80-18.20 G

18.20-21.40 E, F, C

21.40-23.60 B

Precursor ion

184 208 234 234

234

184

208

234

234

234

Fragment ion@) ( m p )

139 163

202 189 202

152 139 89 176 164 163 202 189 165 208 202 189 232 202 189

i 89

Dwell time (ms)

200 200 200 200

200

100

100

1 00

100

100

' A = S R M ; B = M R M .

a make-up gas flow rate of 1-2.5 1 miri-'. The corona discharge needle was maintained at a current-controlled 3 pA discharge. For the collisionally induced disso- ciation (CID) mass spectrometric experiments, argon was used as the collision gas, in a high-pressure colli- sion cell,' with a (laboratory-frame) collision energy of 45 eV and a collision gas thickness of 350 x 1013 atoms cn-'. Mass spectrometer control, data acquisition and data processing were accomplished using a Macintosh Quadra 950 microcomputer. The qiuadrupole mass spectrometer was operated using dwell times of 5 and 6 ms per m/z for the full-scan studies of the complex frac- tions and the standard solution, respectively, and 100 and 200 ms u-' for the multiple reaction monitoring (MRM) and selected reaction monitoririg (SRM) experi- ments (the full details of the SRM and MRM experi- ments are shown in Table 3 and will also be commented upon in the Results section). These latter experiments were conducted in such a way that the transitions associated with the compounds of interest were moni- tored for the times necessary for the compounds to elute from the column.

RESULTS AND DISCUSSION

The fractionation pr~cedure '~ produced 1 1 fractions which were profiled initially by RPLC with DAD to determine the best chromatographic resolution and analysis time, and subsequently by MS to provide selec- tivity and specificity in the identification of the PACs. The results obtained from these experiments showed that each individual fraction contained markedly differ- ent compound compositions. Figure 1(A) shows the

0 4 . . . I . . . , . , . ,

ICa 1 I

0 0 20 40 60 80 100 120

Time (minutes)

Figure 1. (A) Total-ion chromatogram (m/z 150-650) obtained for fraction 1 of the tar pond sediment by RPLC/APCI-MS. (B) Reconstructed ion chromatogram for m/z 208 obtained by RPLC/ APCI-MS for fraction 1 of the tar pond sediment. Inset: mass spectrum acquired from the time position in the chromatogram at which phenanthro[4,5-bcd]thiophene (molecular mass 208) would be expected to elute.

total-ion chromatogram (TIC) obtained by RPLC/ APCI-MS for the first fraction obtained by the fraction- ation of the tar pond sediment. It can be seen that the fraction is composed of a large number of compounds that elute mainly in the acetonitrile-rich portion of the mobile phase gradient. The reconstructed ion chro- matogram (RIC) for m/z 208 [Fig. l(B)] demonstrates that there are many compounds present which yield ions at this m/z value. The inset of Fig. 1(B) shows the mass spectrum acquired from the time position in the chromatogram in which phenanthro[4,5-bcd-Jthiophene (molecular mass 208) would be expected to appear (see Fig. 3, peak 2). This mass spectrum contains a number of additional peaks which mask the presence of the m/z 208 peak, thereby making the identification and verifi- cation of phenanthro[4,5-bcdlthiophene impossible from these data alone. Similar results were found when mass spectra were acquired at the time periods in the chromatogram in which the other PASH standards would be expected to elute.

It has been found16." that the responses of different PACs in APCI-MS vary widely when the sample is introduced under supercritical fluid chromatographic (SFC) conditions. For example, the mass spectra of the lower molecular mass PAHs are characterized by the formation of both the molecular ion species M" and the protonated molecules [M + HI', and the higher molecular mass PAHs (> 200) typically form only protonated molecules within an APCI environment.

ANALYSIS OF OLIGONUCLEOTIDES BY ES/MS/MS 1037

202

189

150 175

200

i

234

200 225 250 m/Z

Figure 2. CID mass spectrum obtained by recording the fragment ions resulting from the molecular ion (m/z 234) for benzo[b]naphtho[2, 3-dlthiophene by flow injection mass spectrometry (argon collision gas with a collision energy of 45 eV and a collision gas thickness of 350 x atoms

100

s d 25

'B -

I

t 4 .5 .6

0 10 20 30 40

Time (minutes)

Figure 3. (A) Total-ion chromatogram (m/z 150-250) obtained by RPLC/APCI-MS for a mixture of standards: (1) dibenzothio- phene; (2) phenanthro[4,5-bcd]thiophene; (3) phenanthro[3,4- blthiophene; (4, 5, 6) phenanthro[4,3-b]thiophene, phenanthro [9,1O-b] thiophene and benzo [b] naphtho[l,2-d]thiop- hene; (7) benzo[b]naphtho[2,3-d]thiophene. Inset: mass spec- trum obtained for dibenzothiophene (RMM 184). ( 8 ) RPLC/APCI-SRM trace obtained for the PASH standards [SRM conditions shown in Table 3(A)]

The molecular masses of the PASH standards used in this study ranged from 184-234, and the mass spectra of the PASHs under RPLC/APCI-MS conditions were similar to those exhibited by low molecular mass PAHs under SFC/MS conditions. That is, the mass spectra obtained were characterized by formation of base-peak molecular ion species and lower abundance protonated molecules (see Table 4, column 1).

In order to determine the fragment ions that could be monitored using RPLC with SRM and MRM for both the PASH standards and the fractions, the PASH stan- dards were analysed by flow injection and their CID mass spectra recorded. It was found that greater MS/MS sensitivity was afforded when transitions from the molecular ion species were monitored as opposed to transitions from the protonated molecules. The result shown in Fig. 2 corresponds to the CID mass spectrum obtained by recording the fragment ions resulting from MS/MS analysis of the molecular ion (m/z 234) of benzo[b]naphtho[2,3-dlthiophene (0.1 mg ml-', 4 pl injected). The mass spectrum is characterized by the loss of H', H,, S, HS', H,S and HCS' resulting in ions at m/z 233, 232, 202, 201, 200 and 189, respectively. The fragment ion resulting from the loss of S was found to be the base-peak in this instance [that this fragment ion did indeed arise from the unexpected loss of a bare S atom was confirmed by CID mass spectrometric experi- ments using electron impact ionization and linked-scan techniques (data not shown here)]. Similar results char- acterized the other CID mass spectra obtained for the other PASH standards, with the base-peak fragment ion resulting from either the loss of S, H, or HCS (see Table 4, column 2).

Figure 3(A) shows the TIC obtained for the RPLC/ APCI-MS of the PASH standard mixture (0.01 mg ml- ' of each), and the insert shows the mass spectrum

1038 D. THOMAS ET AL.

T i e (rnin).

~~ _ _ _ ~ ~~~ ~ _ _ _ _

Table 4. Column 1 = relative intensity of M": [M + HI + obtained for the standards by APCI-MS, column 2 = precursor ion a d product ions (with relative abundance > 10% of the base peak) obtained for the standards by CID-MS and column 3 = detection limits (and SRM tramition monitored) for the standards

Compound

1 2 3 Detection limit Relative intensity Praeursor Fragment ions

M+' : [M + H I + ion m/z (+relative abundance > 10%) (+SRM transition. m/z)

Di benzothiophene 100:16

Benzo[b]naphtho[2,3-d]thiophene 100:98 (A)

(B)

Benzo[b] naphtho [l,Z-b]thiophene 100:22 (C)

Phenanthro[4,5-bcd]thiophene 100 75

Phenanthro[4.3-b]thiophene 100:87 (D)

( E)

Phenanthro[9,1 0-b] thiophene 100:74 ( F)

Phenanthro[3,4-6] thiophene 100:67 (GI

184

234

69 (16), 89 (36). 134 (31),

163 (ll), 187 (11). 189 (74).

500 pg (1 84 + 1 39)

240 pg (234 + 189) 139 (loo), 152 (32), 170 (44)

190 (11). 200 (25), 201 (21), 202 (100). 220 (10). 232 (69). 233 (24)

190 (15). 200 (24). 202 (36). 205 (10). 218 (15), 232 (loo), 233 (30)

162 (12), 163 (100). 164 (26), 180 (24). 181 (11). 194 (30)

163 (14), 165 (11). 187 (14), 188 (13). 189 (loo), 190 (11). 202 (36). 208 (24), 220 (10). 232 (51), 233 (16)

200 (31), 201 (18), 202 (100). 220 (19), 232 (73). 233 (27)

187 (18). 188 (12). 189 (loo), 190 (24). 200 (24), 201 (19), 202 (64). 220 (16). 232 (38). 233 (14)

234 163 (12), 188 (15), 189 (50). 550 pg (234 + 189)

208

234

200 pg (208 + 163)

260 pg (234 + 189)

234 152 (21), 163 (19), 189 (77), 330 pg (234 + 202)

190 pg (234 + 189) 234

obtained for dibenzothiophene (peak 1) indicating the formation of M+' and [M + HI+ ions. It was not pos- sible to obtain baseline separation for all of the stan- dards used in these experiments using the mobile phase gradient indicated in Table 3. Figure 3(B) shows the RPLC/APCI-SRM chromatogram obtained for the PASH standards. For this particular experiment, five time periods corresponding to the elution time of the particular PASH standards were employed [see Table 3(A)] utilizing different SRM transitions of precursor ions (M +*), each fragmenting to its base-peak fragment ion.

RPLC/MS/MS detection limits, using SRM, were determined for each of the standards used in these experiments. Using an isocratic mobile phase (100% acetonitrile), duplicate injections of each PASH stan- dard were made into the RPLC/MS/lMS system at 2 min intervals, starting with the lowest concentration used. In this way the instrumental detection limit (signal-to-noise ratio = 2, peak-height definition) was determined, and the linearity of the calibration graph was measured using the mean peak areas. Figure 4(A) shows the calibration graph obtained far the mean peak areas for phenanthro[4,5-bcJJthiophe1;1e (SRM of m/z 208 fragmenting to m/z 169, using a 200 ms dwell). The inset shows the expansion of the low-picogram region, indicating an instrumental detection llimit of 200 pg. The response is seen to be linear (T = 0.999) over four orders of magnitude. Similar results were found for the for the other PASH standards, with detection limits in the range 200-600 pg injected on-column (see Table 4, column 3). Figure 4(B) shows the raw data obtained for phenanthro[4,5-bcdJthiophene, and the inset shows the peak-area as a response of an increase in concentration

Amount 0

Oe+00 I

0 20M)oo 400000 600000

Amount (pg) yaw

100 1 B I

0.15

2 0.10

i 2 0.05

0.00

ANALYSIS OF OLIGONUCLEOTIDES BY ES/MS/MS 1039

of standard at the lower picogram level. It should be noted that the peak heights in this region do not increase significantly, but the peak widths broaden, since the increase in standard concentration injected on to the column in this range was achieved by increases in the volumes of standard solution injected.

The sediment fractions were analysed by RPLC/ APCI-SRM [see Table 3(A)], and the results obtained from these experiments showed that the PASH com- pounds were exclusively contained in the first two frac- tions. It was possible to tentatively identify some of the PASH standards in the fractions, but compound confir- mation was accomplished by RPLC/APCI-MRM using the conditions stipulated in Table 3(B), which moni- tored fragmentations of each of the precursor ions to form three of its higher abundance fragment ions. The transitions chosen included, where possible, those that corresponded to the loss of a sulfur-containing frag- ment, as it was felt that such transitions would be more diagnostic of PASHs. Although the greatest sensitivity in MRM experiments would come from monitoring transitions from any chosen precursor ion to its three highest abundance fragments, it was felt that any loss in sensitivity from the selections employed would be more than offset by the gain in selectivity. The results obtained are shown in Figs 5(A) and (B), which show the RPLC/APCI-MRM results obtained for the first two fractions of the tar pond sediment. These results allowed the positive identification of dibenzothiophene, phenanthro[4,5-bcdlthiophene, phenanthro[3,4-b]thio- phene and benzo[b]naphtho[2,3-d]thiophene in the fractions, along with several isomers of these standards. Although the LC/MRM chromatogram indicates that others of the available PASH standards may also be present, it was not possible to confirm their presence positively because they all yielded similar tandem mass spectra and they could not be resolved under the RPLC conditions employed.

Having confirmed the presence of the aforementioned PASH standards in the tar pond extract, the levels of PASH standards contained in the fractions were deter- mined. A 5 g amount of the tar pond sediment was ini- tially subjected to the 'fractionation pro~edure '~ and, after completion of the fractionation steps, the fractions were dissolved in 1 ml of acetonitrile-dichloromethane (1 : 1) prior to LC/MS analysis. The quantitative results obtained for the PASH standards by RPLC-SRM in the tar pond fractions are shown in Table 5. These data were generated by estimating the amounts of PASH in each fraction from the calibration graphs obtained by the RPLC/SRM experiments on the PASH standards. These results show that the PASH standards are present at extremely high levels in the fractions (an

0 10 20 30 40

Time (minutes)

Figure 5. RPLC/APCI-MRM traces obtained for (A) fraction 1 and (6) fraction 2 of the tar pond sediment showing the positive identification of dibenzothiophene, phenanthro[4,5-bcd]thio- phene, phenanthro[3,4-b] thiophene and benzo[b]naphtho[?,3-d] thiophene.

observation which is qualitatively supported by the high signal-to-noise ratio obtained for the standards in the fractions by the RPLC/MRM experiments). However, this method of quantification is incapable of estimating the fractional recoveries of the target analytes from the raw sediment into the final extracts, and in the absence of suitable surrogate internal stan- dards the quantitative levels in the original sediment can only be estimated as lower limits. In the present context, a 'surrogate internal standard' is deemed to be a compound that may be added to the raw sample and be expected to exhibit, as closely as possible, the same behavior as the target compounds during the extrac- tion, clean-up, fractionation and chromatographic analysis steps, yet be readily distinguishable from the analytds) of interest. It is usual in our laboratory to employ isotopically labeled variants of the compounds of interest, since these generally meet all the criteria listed above. In the analysis of samples for PAHs, for example, we use a solution of perdeuterated PAHs as

Table 5. Levels of dibenzothiophene, pheoanthro [ 4,5-bcd] thiophene, phenanthro[ 3,Qbl thiophene and benzol6 1 naphtho [ 2,344 thiophene found in fractions 1 and 2 of the tar pond extract

Compound Amount in first Amount in second Tot81 amount in

fraction fraction sediment

Dibenzothiophene (A) Phenanthro[4,5-b]thiophene (D)

Benzo[b]naphtho[2,3-d]thiophene (6)

1.3 pg pi-' = 260 pg g-' 85 ng pi-' = 17 ng g-'

68 ng pi-' = 14 ng g-'

98 ng pi-' = 19 pg g-' 5 ng pi-' = 1 ng g-'

12 ng pi-' = 2 ng g-'

279 pg g-' 18 ng g-'

16 ng g-' Phenanthro[3,4-b]thiophene ( G ) 23 pg PI-' = 45 pg g-' 20 pg PI-' I 4 pg g-' 49 pg g-'

1040 D. THOMAS ET AL.

surrogates. However, in the present work, no such iso- topically labeled analogues were available to us. Given these cautions, it is still possible to obtain an estimate of the lower limits of some of the target co'mpounds in the sample. Since 1 ml of each fraction solution represented the content of 5 g of sediment, a PASH concentration in the final solution analysed of x ng p1-' corresponds to a lower limit of 0 . 2 ~ pg g-' in the original sediment. These values range from 279 pg g-' for dibenzothio- phene to 49 pg g- ' for phenanthro[:3,4-b]thiophene (Table 5).

CONCLUSION

fraction consisting largely of PAHs. The sensitivity of the technique developed here, employing multiple reac- tion monitoring for a high degree of specificity, was more than adequate to detect and determine the thio- phenic compounds in the mixture.

The Sydney tar ponds, being the product of approx- imately 80 years of uncontrolled discharge for a metal- lurgical coking operation are the most heavily PAC contaminated sediments in Canada. A large-scale clean- up program is under way, and the principal remediation measure is incineration of this air-dried sediment. The sulfur compounds examined here are present at rela- tively high levels, and the incineration procedure may have to be adjusted to ensure the destruction of these extremely carcinogenic compounds.

This work has demonstrated the application of CID mass spectrometry in combination with reversed-phase liquid chromatography to the problem of identifying (and quantifying where possible) minor components in a This work was f u n d 4 by the Panel for Energy Research and complex mixture of PACs. Despite ext(Xlsive fractions- tion, the sulfur compounds of interest are present in a

opment under OERD project 57118. The authors thank Dr J. Curtis for carrying out the electron impact mass spectrometric experiments.

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