Analysis of nitrated polycyclic aromatic hydrocarbons in diesel

Part A. General Topics 1992 26 (11), 2077-2087 ... Mutation Research/Environmental Mutagenesis and Related Subjects 1991 253 (2), 181-191 .... of poly...
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285

Anal. Chem. 1982, 5 4 , 265-271

Analysis of Nitrated Polycyclic Aromatic Hydrocarbons in Diesel Particulates D. Schuetrle," T. L. Rlley, and T. J. Prater Ford Motor Company, Dearborn, Michigan 48 12 1

Analytlcal Sclences Department, Engineering 13 Research Staff -Research,

T. M. Harvey and D. F. Hunt Chemistry Department, Universiw of Virglnia, Charloitesviiie, Virginia 2290 1

The analysis of nitrated polycyllc aromatic hydrocarbons (nltro-PAH) In dlesel partlculate extracts using a combinatlon of hlgh-performance llquld chromatography, gas chromatography/mass spectrometry, hlgh-resolutlon mass spectrometry, and mass spectrometry/mass spectrometry (MS/MS) technlques Is descrlbed. Trlple stage quadrupole (TSQ)-MS/MS operated In the constant neutral loss mode Is descrlbed for the rapid qualltatlve screenlng of nltro-PAH Isomer groups In solvent extracts of dlesel partlculates. Twenty such Isomer groups of nltro-PAH derlvatives were found, many of these groups containing a large number of possible compounds. Mass analyzed Ion klnetlc energy spectrometry (M 1KES)MS/MS was found to be much less selectlve than the TSQMS/MS for the analysls of nltro-PAH In unfractlonated diesel extracts. HPLC prefractlonatlon was requlred to remove Interferences In the MIKES analysls. Fused slllca capillary GC/MS uslng ontolumn lnjectlon was found to be necessary for lndentlfylng speclflc nltro-PAH Isomers and mlnlmlrlng decomposltlon. The hlghly mutagenlc 1-nltropyrene (1-NP) was the only compound found In the nltro(pyrenes and fluoranthenes) Isomer group. The MIKES and TSQ technlques gave comparable results for the quantltatlon of 1-NP.

Organic solvent extracts of diesel exhaust particulates exhibit direct-acting (Le., activation by mammalian tissue homogenate is not required) mutagenicity (1-5) using the Ames assay (6). High-performance liquid chromatography (HPLC) (1,2,4,5), thin-layer chromatography (TLC) (7,8), and liquid chromatography (9) have been used to fractionate diesel particulate extra& for subsequent mutagenicity testing. In these studies it has been shown that most of the directacting mutagenic activity is found in chemical fractions which contain compounds of moderate polarity. The polycyclic aromatic hydrocarbons (PAH) are found in a nonpolar fraction that exhibits less than 5% of the direct-acting mutagenicity for the total extract. These data are consistent with studies that show that the PAH require activation for them to be mutagenic in the Ames assay (10, 11). We have reported the identification of more than 100 PAH derivatives in moderately polar HPLC fractions of diesel particulate extracts (4, 5 ) . The PAH derivatives included substituents with hydroxy, ketone, quinone, carboxaldehyde, acid anhydride, dihydroxy, and nitro groups. Other investigators have reported the indentification of PAH derivatives in similar fractions (9, 12-14). Limited work to date has shown that the mutagenicity for most of these PAH derivatives is too low to account for the total extract mutagenicity (15). However, data from this laboratory have shown that the nitrated-PAH (nitro-PAH) compounds can make a significant contribution to the mutagenicity of diesel particulates. One of the nitro-PAH com0003-2700/82/0354-0265$01.25/0

pounds identified was 1-nitropyrene (1-NP). Our preliminary estimates of its concentration range in diesel particulate extracts together with the published value for its mutagenicity have suggested that 1-NP could account for up to 30% of the TA 98 direct-acting mutagenicity of the unfractionated extracts (4, 5, 16). It was previously reported that 1-NP was present in diesel particulate extracts as based upon GC/MS retention times, fragmentation patterns, and HRMS data ( 4 , 5 ) . It was also found that a number of other nitro-PAH may be present and that many of the nitro-PAH decompose during GC/MS analysis. Therefore, the objective of this work was to (1) develop a rapid screening technique for the analysis of isomer groups of nitro-PAH using MS/MS techniques, (2) develop GC/MS techniques for identifying specific isomers with minimal sample decomposition, and (3) examine the quantitative aspects of the MS/MS techniques. The compound 1-NP was chosen as a model compound for these studies. The biological implications of these results are explored elsewhere (17).

EXPERIMENTAL SECTION Particulate Collection and Extraction. Four light-duty diesel exhaust particulate samples designated as NI-1, OP-1, OL-1, and PG-1 were collected on T60A20V and TX40HI20-WW Pallflex filters (Teflon-impregnated glass fiber filters) using a dilution tube and a chassis dynamometer test facility (18). The filter samples were Soxhlet extracted with dichloromethane (DCM) (18). The NI-1 extract was provided by R. Bradow and S. Tejada of the U.S.Environmental Protection Agency. Extracts were stored in DCM (4mg/mL) at -80 "C until used. Analytical procedures were performed in subdued room incadescent light to avoid possible photochemical degradation. Solvents and Standards. All solvents were "distilled in gla,d"' UV grade (certified phthalate and particulate free), from Burdick and Jackson. Synthesis for qualitative MS analysis was conveniently accomplished by placing approximately 500 ng of the parent PAH on the mass spectrometer direct insertion probe (glass) and exposing the probe to concentrated nitrogen dioxide and nitric acid fumes for a few seconds. Larger quantities of standards for qualitative GC/MS analysis were prepared by coating the inside surface of a 25-mL Erlenmeyer flask with approximately 1 mg of the parent PAH and injecting nitrogen dioxide and nitric acid fumes into the flask for a few seconds. We also followed the procedures described in the literature (19,22). Room-temperature nitration of fluoranthene gives the %nitro isomer (mp 159-160 "C) as a major product, the 8-nitro isomer (mp 158-160 "C)as a minor product, and the 1-nitro (mp 151-153 "C) and 7-nitro (mp 144 "C) isomers as trace products (19,ZO). Further nitration gives predominantly 3,9-dinitrofluoranthene (mp 275 "C) (21). The 2-ring position is nonreactive and not nitrated directly. A seven-stepsynthesiswas necessary to produce 2-nitrofluoranthene (19). Nitration of pyrene initially yields the 1-isomer (mp 153 "C) (22) and further substitution gives rise to mixtures of the dinitro, trinitro, and tetranitro species (23). The 2- and 4-ring positions are nonreactive and not nitrated directly (24). The 1-NP as supplied by C. King, Michigan Cancer, was found to be contaminated with 1.6% dinitropyrenes. Purified 0 1982 American Chemical Society

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ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982

isomers of dinitropyreneswere supplied by R. Mermelstein,Xerox Corp. High-Performance Liquid Chromatography. Fractionation of the diesel particulate extracts and purification of nitro-PAH standards for mass spectrometry were performed by using HPLC techniques described previously (4,5) with some minor modifications. Semipreparative HPLC was undertaken on a 7.8 mm i.d. by 30 cm Microporasil(l0 pm) normal phase column. Up to 20 mg of extract (=20 mg/mL) in DCM could be injected onto the column. The solvent program consisted of operating isocratidy for the f i t 10 min with a 1% DCM in n-hexane mixture, programming at a 10% DCM/min gradient to 100% DCM in 10 min, and then operating isocratically with 100% DCM until the yz peak was eluted (see Figure 1). A 7.0 mL/min solvent flow rate was maintained throughout the program. Fractions separated by HPLC were collected manually in amber glass vials equipped with Teflon-linedcaps. In samples separated for HRMS analysis, the y1 peak was subfractionated into the ylr and yly fractions illustrated in Figure 1. High-Resolution Mass Spectrometry. HRMS measurements were made on a VG Micromass ZAB-2Fmass spectrometer interfaced to a Finnigan INCOS 2000 data system. The mass spectrometer was operated at a resolution between 11000 and 15OOO in the electron impact (EI) mode at an acceleratingvoltage of 8 kV. Source temperature was maintained at 240 "C and electron energy at 70 eV. The magnet was exponentially downscanned from 400 to 60 amu every 8 s. Samples were injected into the mass spectrometer using a direct insertion probe fitted with a gold probe tip. Gas Chromatography/Mass Spectrometry. GC/MS analyses were performed on a VG-Micromass MM-16 mass spectrometer interfaced to a Finnigan INCOS Model 2000 data system. Separations were performed on both packed and fused silica capillary columns. The mass spectrometer was operated at a resolution of approximately 1000. All measurements were made in the E1 mode at 70 eV. Packed column analyses were performed on a Varian Model 1400 GC interfaced to the mass spectrometer via a VG single-stage glass jet separator. Separationswere performed on a 180 cm long, 2 mm i.d. glass column packed with 1% SP-2250on 80-120 mesh Supelcoport. The column was programmed from 80 to 320 "C at 4 "C/min. The mass spectrometer source and GC interface were maintained at 230 and 280 "C, respectively. The mass spectrometer was exponentially down-scanned from 350 to 100 amu every 1.7 s. Capillary column analyses were performed on a Perkin-Elmer Model 910 gas chromatographequipped with a fused silica SE-54 WCOT column, 30 m X 0.25 mm i.d. The column was interfaced directly to the mass spectrometer source. Temperature programming was from 80 to 270 OC at 8 "C/min and the column flow rate was 0.9 mL/min. The mass spectrometer source and GC interface were maintaned at 240 and 270 "C, respectively. The mass spectrometer was exponentially downscanned from 350 to 100 amu every 2 s. Sample introduction was either splitless injection or direct on-column injection. The latter technique was accomplished by using a modified injector (J and W, Rancho Cordova, CA). Mass Analyzed Ion Kinetic Energy Spectrometry. MIKES analyses were performed on a VG Micromass ZAB-2F mass spectrometer interfaced to a Finnigan INCOS 2000 data system. All experiments were conducted at an accelerating voltage of 8 kV and a magnetic sector resolution of approximately2000 (10% valley). Intermediate and collector slits were adjusted to provide 90 and 100% transmission, respectively. Electron impact (EI) and chemical ionization (CI) measurements were made at an electron energy of 70 and 50 eV, respectively. Trap current in the E1 mode and emission current in the CI mode were maintained at 100 and 200 FA, respectively. Methane reagent gas was optimized at an estimated source pressure between 0.7 and 1.0 torr during negative and positive ion CI measurements. Source temperature was maintained between 180 and 190 OC. Helium collision gas pressure in the second field-free region was maintained a t 1.3 X 10-7 torr to provide maximum daughter ion transmission. The INCOS data system was used to acquire energy spectra and control electric sector voltage scanning. Typical voltage scans were from 500 to 9000 eV every 2.5 s. All samples

0

10

2 0 30

40

50

60

TIME (MIN)-

Figure 1. HPLC fractionation profilesfor diesel particulate samples NI-1, OL-1, and OP-1, utilizing a normal phase Blosil A slllca column with fluorescencedetection, X, = 313 nm, X, 1 418 nm. The elutlon times of some standards are shown in the figure.

were introduced with a direct insertion probe fitted with either a solid gold probe tip or a cupped Pyrex probe tip. Triple Stage Quadrupole Mass Spectrometry. The triple stage quadrupole (TSQ) mass spectrometer has been described previously (26). All experiments were conducted using positive ion chemical ionization with methane reagent gas at a 0.4 torr source pressure. Source emimion current and electron energy were maintained at 400 LA and 100 eV, respectively. Both the first and third quadrupoles were operated at unit mass resolution. Nitrogen collision gas pressure in the second quadrupole was torr. Source temperature optimized at approximately 5 X was maintained at 145 OC. Samples were introduced with a direct insertion probe fitted with a Pyrex cup tip. The probe was temperature programmed from 30 to 350 OC at 30-40 OC/min. In TSQ collisionally activated dissociation (TSQ-CAD)studies, the first quadrupole was set manually to transmit a parent ion of interest and the third quadrupole was scanned repetitivelyfrom 100 to 350 amu every 1.15 s to collect daughter ion spectra. The second quadrupole functioned as a collision cell. It was operated in the rf voltage mode only and transmitted ions of all m/z. TSQ constant neutral loss studies were also undertaken by using the second quadrupole as a collision cell. The first and third quadrupoles were scanned in parallel with a 17 amu mass deficit (to represent loss of OH) from 100 (83) to 350 (333) amu every 1.15 8. Under these scanning conditions, only ions which experience the loss of a 17 amu neutral fragment when collisionally dissociated in the second quadrupole were detected. RESULTS AND DISCUSSION Identification of Nitro-PAH. HPLC Fractionation. Figure 1shows the elution profile for three diesel particulate extracts using the analytical HPLC procedure. The regions of the chromatogram which elute nonpolar and moderately polar compounds are designated as (a1, (YZ, P) and (n, yz), respectively. The HPLC elution times of several nitro-PAH standard compounds were determined and some of these are shown in Figure 1. It was found that mononitro-PAH (2-5 rings) and dinitro-PAH (2 rings) elute in the y1 fraction. Larger dinitro-PAH (4 rings) were found to elute in the yz fraction. Over 100 PAH derivatives have been identified ( 4 , 5 )in the y1 and yz fractions. Because of the complex composition of

ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982

287

Table I. Mass Spectra for Nitro-PAH Identified in Sample OL-1 by HRMS mass (re1 abund) (MY (M - NO)+ (1

formula

compounds ylx

Fraction

nitro(anthracenes and phenanthrenes) nitro( methylfluorenes) nitro( pyrenes and fluoranthenes)

223.064 225.080 247.064

CMH9N02

Cl,HllN02 Cl, H, NO2 yly

nitrofluorenones nitrohydrox yfluorenes nitro(methy1anthracenes and methylphenanthrenes) nitro(anthrones and phenanthrones)

re1 concn b

n.d. n.d. 217.062 (1.04)

0.84 0.35 0.61

Fraction C,,H,NO, Cl,H,NO, C,,HllNO,

225.043 n.d. 1.00 n.d. 0.75 227.058 207.077 (0.85) 0.61 237.079 n.d. 0.42 C1+H9N03 239.058 a Masses measured to within i: 1 0 ppm at 1 2 000 resolution (10% valley). Relative abundance of fragments given with respect to molecular ion. Relative concentration with respect to nitrofluorenones as based upon the integrated ion current Ion not sufficiently resolved from other ions mesent or not found. for Mt. these fractions, attempts to characterize the nitro-PAH content by HPLC alone were not possible. The semipreparative HPLC procedure was used primarily to isolate fractions containing nitro-PAH for MS/MS analysis. HRMS Analysis. HRMS analysis and the exact mass information which it provides was used as a screening technique for determining the possible presence of nitro-PAH compounds in HPLC-fractionated diesel extracts (25). Only a few nitro-PAH were tentatively identified (Table I) in the y l x and yly HPLC fractions (Figure 1) by this technique. All identifications are based on the exact mass of molecular ions. In two cases this molecular ion formation was supported by the presence of exact masses correspondingto the (M - NO)+ ion which is a predominant electron impact fragment of nitro-PAH (9). The (M)+/(M- NO)+ ion abundance ratio of the compound(s)tentatively identified to be the nitro (pyrenes and fluoranthenes) isomer group was found to be 0.96 which agrees closely with the abundance ratio of 1.00 measured for a 1-nitropyrene standard. Standards of nitro(methy1anthracenes and methylphenanthrenes) were not available for comparison purposes. The fragmentation of 1-NP was found to be dependent on the temperature of the MS source. Therefore, mass spectra of standards and unknowns should be compared under identical conditions. The identification of fragment ions characteristic of other nitro-PAH compounds was complicated by the presence of oxygenated PAH which gave fragments of similar mass. In conclusion, the HRMS provided some information on the presence of isomer groups of nitro-PAH, but specific isomer determination was not possible and data interpretation was difficult because of the complexity of the data. GC/MS Analysis. GC/MS was used to determine the presence of isomer groups and identify isomers of nitro-PAH tentatively identified by HRMS. Initial GC/MS separations were done by packed column on samples prefractionated by HPLC. As described previously ( 4 , 5 ) the resulting total ion chromatograms were quite complex. However, the use of reconstructed mass chromatograms was quite definitive in confirming the presence of expected nitro-PAH groups, Isomer groups of nitro(anthracenes and phenanthrenes), nitrofluorenes, nitro(methy1anthracenes and methylphenanthrenes), and nitro(methy1pyrenes) were found to be present using this technique. At least three weaknesses were associated with the packed column GC/MS method. First, some discrimination against high boiling compounds was exhibited by the single-stage jet separator and sample losses were encountered on the injector. Second, as illustrated in Figure 2A, the packed column chromatographicresolution was sufficient to resolve the 1-NP from ita isomer 3-nitrofluoranthene (3-NF),but 1-NP was not separated from the 8-nitrofluoranthene(8-NF)isomer. Third,

A. PACKED COLUMN

E. CAPILLARY COLUMN

L NF+I-NP STD

I-,7-NF

1

1

I -NP STD

k-" A

A

LL NI- I

EXTRACT

SCAN 4 0 0 SCAN 4 0 0

TLyG 37.20

EXTRACT

450 450

m m

880 880

BOO BOO

42.00

46.40

2e:m

30 00

Figure 2. GC/MS mass chromatograms of the m Ir 247 ion generated from 1-NF, 7-NF, 3-NF, E-NF, and 1-NP standards and a HPLC fractionation NI-1 extract: (A) 180 cm 1 % SP2250 packed column separation, (B) 30 m SE-54 capillary column separation.

the E1 fragmentation pattern for all three of these compounds was too similar to allow them to be accurately differentiated by MS techniques alone. In order to overcome these weaknesses, we developed a capillary GC/MS method. Direct coupling of a SE-54 fused silica capillary column to the MS source eliminated the need for a jet separator and reduced sample losses. Both splitless and direct on-column injection procedures were attempted with the capillary column. The on-column procedure provided better sensitivity and discriminated much less against high boiling compounds;however, it was very easy to overload the column by this technique. Thus, the maximum quantity of extract used for injection was 20-30 pg. A detection limit of about 1.0 ng was observed for 1-NP using the on-column injection procedure. Figure 2B shows the GC resolution of the 1-NP from the other chemically possible isomers 3-NF, 8-NF, 1-NF, and 7-NF. All these isomers gave somewhat similar fragmentation patterns. The 2-NF, 2-NP, and 4-NP isomers would not be present since the only means of producing these isomers is through a complex indirect chemical route and not a direct nitration (19-23). The unequivocal identification of the 1-NP in these samples was based upon the fragmentation pattern and a nearly exact vatch of retention times (to within 4 s)

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ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982

13.4pg EXTRACT(N1-I) W

u a 0

z

3

m

< A

lop9 EXTRACT (OP-I)

4 3 ng I-NITROPYRENE

(8000)

(7000)

I

250

230

210

(60001

(5000) I

(40001 I

190 170 150 MASS (ENEROY,sV)

130

(3200)ENERQYW

110

MASS

Flgure 3. MIKES spectrum of m l z 247 obtained under E1 conditions: 13.4 pg of unfractlonatedNI-1 extract: p y, HPLC fractions of 10 pg of sample OP-1; 43 ng of 1-NP standard.

+

for the 1-NP in the standard and samples. The standard addition of 1-NP did not change the shape or position of the 1-NP mass chromatogram for m/z 247 in the samples. Recovery studies which compared the amount of 1-NP injected to the amount detected indicated that, with respect to analysis by direct insertion probe, essentially no losses of 1-NP were experienced with the direct on-column capillary technique, whereas approximately 30-40% losses were experienced with the splitless injection capillary and packed column-jet separator sample introduction techniques. Sample recoveries of other nitro-PAH compounds such as dinitropyrenes were as low as 5%. MS/MS Analysis. The objective of this study was to develop a rapid and selective technique for the direct insertion probe analysis of nitro-PAH in diesel particulate extracb using MIKES and TSQ techniques. MIKES Analysis. The selectivity of the MIKES technique for nitro-PAH in diesel extracts was compared by use of positive ion electron impact (EI), positive ion chemical ionization with methane (PICI), and negative ion chemical ionization with methane (NICI) source ionization processes. This selectivity study was conducted by using 1-NP as a model compound. In the EL and NICI-MIKES techniques the molecular ion of 1-NP (mlz 247) was selectively focused through the magnetic sector a t a resolution of approximately 2000. Collision-activated dissociation took place in the second field free intersector region. The resulting daughter ion spectra were recorded by repetitively scanning the electric sector. The observed resolution of the MIKES spectra was approximately 75. The PICI-MIKES technique was identical except that the pseudo-molecular ion mlz 248 was examined. Figure 3 compares the EI-MIKES spectra of unfractionated NI-1 extract, the ylr HPLC fraction of sample OP-1, and 43 ng of 1-NP standard. The peaks in the energy spectra are fairly broad with an average half-height peak width of 3-5 amu. In addition, there is some high-frequency noise superimposed on the peaks. The MIKES spectrum for the NI-1 sample was similar to that of the 1-NP standard (relative abundances of major peaks are nearly the same) suggesting good selectivity for this compound. However, several interference peaks were observed in the OP-1 spectrum. A deconvolution algorithm was used to help resolve the components present in the OP-1 spectrum for the m/z 201 peak. At least three peaks were resolved. Similar interference problems were encountered for the OP-1 and PG-1 samples when PICI-MIKES was attempted. NICI appeared to be the most selective ionization process

Flgure 4. NICI-MIKES spectrum of m l r 247 for (A) 30 ng of 1-NP standard: (B) p y, HPLC fractions obtained from 28 pg of sample OP-1; and (C) p y, HPLC fractions obtained from 6.6 pg of sample Pol.

+ +

IA)

AM

AMs17

El (TSQ)

SOURCE

AM-I7 IAr

QUAD-I COLLISION CELL(Nd

Flgure 5. (A) Schematic represented of TSQ constant neutral loss scanning technique and associated ion processes used for the analysis of nitro-PAH. (8) Schematic representation of MIKES instrumentation.

for nitro-PAH. Figure 4A shows the NICI-MIKES daughter ion spectra of 30 ng of 1-NP standard. Similar spectra for HPLC prefractionated samples of OP-1 and PG-1 are shown in Figure 4B,C. The major daughter ion in these spectra is m/z 216 which corresponds to the (M - NOH)- ion. The sample spectra are quite similar to the standard except for the presence of a minor m / z 232 ion of unknown origin. The three MIKES techniques provided comparable detection limits at about the l ng level for l-NP. This limit appeared to be more a function of background interference level, i.e., selectivity, than absolute instrumental sensitivity. TSQ Analysis. The TSQ mass spectrometer was found to be a valuable screening tool for nitro-PAH compounds when operated in the constant neutral loss mode. Figure 5A schematically illustrates the TSQ instrument and the scanning functions and ion reactions associated with the constant neutral loss mode of operation. The instrument used for the MIKES analysis is shown in Figure 5B for comparison. Positive ion methane chemical ionization (PICI) conditions were used to form nitro-PAH pseudo-molecular ions (M + 1) and to reduce interferences found in the E1 analysis. Under the conditions of the analysis, nitro-PAH compounds predominantly eliminated the neutral fragment OH(M + 1- 17) during collisionally activated dissociation in the second quadrupole. The fiist and third quadrupoles were repetitively scanned at the same rate with a mass differential of 17 amu. In this manner, only those compounds which lose 17 amu in

ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982

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Table 11. Nitro-PAH Tenatively Identified in Whole Diesel Particulate Extracts by TSQ Constant Neutral Loss Analysis mass ( r n / z ) re1 abund (%) Xa X + 16 X - 2ge OP-1 NI-1 OL-1’ nitro-PAH derivatives nitroacenaphthylenes nitro(acenaphthenes, biphenyls) nitronaphthaquinones nitrodihy droxynaphthalenes nitrofluorenes nitro( methylacenaphthenes, methylbiphenyls) nitro(trimethylnaphthalenes) nitro(naphtha1ic acid) nitro(anthracenes and phenanthrenes) nitro( fluorenones and methylfluorenes) nitro( methylanthracenes and methylphenanthrenes) nitro(anthrones and phenanthrones) nitro(pyrenes and fluoranthenes) nitro( dimethylanthracenes and dimethylphenanthrenes) nitro( methylpyrenes and methylfluoranthenes) nitro( pyrones and fluoranthones) nitro(pyrene and flu0ranthene)quinones nitro( dimethylphenanthrene and dimethylanthracene) carboxaldehydes nitro( methylbenzo[a]anthracenes, methylchrysenes, and methyltriphenylenes) nitro(benzo[u]pyrenes, benzo[e]pyrenes, and perylenes)

181 183 187 189 195 197

197 199 203 205 211 e 21 3

152 154 158 160 166 168

20 26 28 20 28 28

15 25 13 15 20 32

4 18 21 16 12 23

199 201 207 209 221

21 5 21 7 223 225 237

170 172 178 180 192

18 18 22 24 13

12 15 58 12 20

13 16 23 15 16

223 231 235

239 e 247 251 e

194 202 206

12 7 8

12 100 12

12 13 14

24 5

261 f

21 6

5

18

8

24 7 261 263

263 277 279

21 8 23 2 234

8 5 6

9 3 5

8 4 5

27 1

287

24 2

5

5

3

281

297

252

3

10

4

epf

epf

Mass of parent nitro-PAH derivative ( X + 16). a Masses as measured (X); see Figure 7 for spectrum of sample NI-1. Relative abundance of nitro-PAH with respect to nitro(pyrenes and Mass of unsubstituted parent-PAH ( X - 29). fluoranthenes) in sample NI-1(abundance value of 100%). e Nitro-PAH found in the ylx and y l y fractions of sample ,OL-1 by HRMS (see Table 11). f Nitro-PAH found in the ylX and yly fractions by GC/MS. g Both isomer groups detected by HRMS. the collision cell, like nitro-PAH, are detected. Figure 6A gives the constant neutral loss spectrum for the direct insertion probe analysis of 46 ng of a 1-NP standard. The only peaks observed were m / z 231 and m / z 232 (a 13C isotope of m/z 231). Figure 6B shows the constant neutral loss spectrum for the direct insertion probe analysis of unfractionated sample NI-1. Addition of 16 amu to the observed ions will give the molecular weight of the corresponding nitro-PAH. The subtraction of 29 amu from the observed ion (M + 1- 46, loss of NOz) will yield the molecular weight of the parent PAH species. Table I1 summarizes the masses observed for this spectrum and for spectra s i m i i l y obtained from whole extra& of OP-1 and OL-1 samples. The percent relative abundance of each nitro-PAH with respect to the nitro(pyrene and fluoranthene) isomer in sample NI-1 is given. These relative abundance values give an estimate for the relative concentrations of each nitro-PAH isomer group assuming each compound produces the same relative intensity of (M + 1 - 17) ion. It must be emphasized that the TSQ constant neutral loss screening technique only monitors an ion reaction characteristic of nitro-PAH compounds but does not confirm their presence. The (M + 1 - 17) ion for several other PAH derivatives was examined. It was found that PAH carboxaldehydes, quinones, alcohols and carboxylic acids would not cause a detectable interference level when present in the extracts at concentrations more than 200 times that of the nitro-PAH. Amine-PAH do give (M + 1- 17) ions but the abundance of this ion was found to be at least 50 times in lower abundance than that of the corresponding nitro-PAH. Higher pressures (> 0.4 torr) in the collision cell reduced the selectivity of the technique for nitro-PAH in the presence of amine-PAH. A more extensive examination of the selectivity of the TSQ mass spectrometer for nitro-PAH was conducted with 1-NP

‘ 50 O o00 l

A‘

a

231

207

I

50.0

60

120

140

160

I80 2W m /z

220

240

260

280

500

Flgure 6. TSQ constant neutral loss spectrum of (A) 46 ng of 1-NP standard (composlte average of 90 scans) and (B) 20 bg of unfractionated NI-1 extract (composite average of 90 scans; ions wlth an abundance below 10% are not shown).

as a model compound. In this study the TSQ mass spectrometer was operated in the collisionally activated decomposition mode (TSQ-CAD)using PIC1 source conditions. In the TSQ-CAD mode, the first quadrupole was used to selectively focus the pseudomolecular ion of 1-NP (mlz 248), the second quadrupole was used as a collision chamber, and the third quadrupole was scanned repetitively to collect the CAD spectra of the m/z 248 parent ion. Figure 7A,B compares the TSQ-CAD spectra of 46 ng of 1-NP standard and 54 bg of unfractionated NI-1 diesel particulates extract, respectively. The mlz 2311248 abundance ratio was found to be 0.41 and 0.63 for the sample and standard, respectively. This suggests the possibility of another ion at mlz 248 in addition to the Cl6HIoNO2ion from 1-NP. However, it is difficult to determine the origin of this ion since

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ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982 1000-

c

Table 111. Quantitation of 1-Nitropyrene Using Coupled Mass Spectrometry Techniques CAD technique

500

I

-

ion a sam- instru- ioniz- -___ ple ment ation P D TSQ MIKES OL-1 TSQ OP-1 TSQ MIKES MIKES PG-1 MIKES NI-1

2lri

'"O'OI

PICI E1 PICI PICI E1 NICI NICI

248 247 248 248 247 247 247

extract, fig

231

20.0

201 231 231 201 216 216

13.4 50.0 50.0

concn, ppm 2280k 2080 f 204 k 77*

230 220 30 15

10.0 4105 2 ~ 3 . 0 ~ 5 5 k 11 6.6b 150 30

a P represents parent ion used for CAD analysis; D represents daughter ion used for quantitation. Sample fractionated by HPLC prior to analvsis.

L

IW

m/z

140

I80

m /z

220

260

Figure 7. TSQ-CAD spectra for m l z 248 obtained from the positive ion methane chemical ionization of (A) 46 ng of 1-NP standard (composite average of 60 scans), (B) 54 fig of unfractionated NI-1 extract (composite average of 130 scans), (C) 25 pg of unfractionatedOL-1 extract (composite average of 160 scans, and (D) 44 fig of unfractionated OP-1 extract (composite average of 180 scans).

there are only minor peaks in the sample spectrum at m/z 204,205,220, and 230. The TSQ-CAD spectrum of sample OL-1 (Figure 7C) shows the definite presence of another compound or compounds in addition to 1-NP. HRMS analysis of sample OL-1 showed that the major ions present at m / z 247 were Cl7Hl1O2(m/z 247.086), ClsHlg (m/z247.149)) and C16HgN02(m/z 247.063). From these data and the presence of the additional peaks in the CAD spectrum, it is postulated that one of the interfering ions could be accounted for by a substituted dihydropyrene carboxylic acid. PICI ionization of compound I (see below) would produce the ion shown in 11. Loss of the substituent X would result in 111. Major fragments from the TSQ-CAD analysis of I11 would be expected to yield m/z 230 (M - OH2), m / z 220 (M - CO), m / z 203 (M - C02H), m / z 202 (M C02H2),and m/z 201 (M - C02H3)ions. These are the major ions found in the spectrum shown in Figure 7C. I

rn/z(247+X)

m

II

m/z ( 2 4 S + X )

mh(248)

The TSQ-CAD spectrum of sample OP-1 (Figure 7D) indicates that interferences may pose a significant problem for the quantitative analysis of 1-NP. The presence of an interference such as the substituted dihydropyrene carboxylic acid is apparent in this spectrum. As mentioned earlier, another possible interference may arise from an analogue of a CI9Hl9ion. It is possible that the remainder of the interfering peaks was due to the presence of this species. It can be concluded from the foregoing discussion that considerable care must be exercised in assessing the selectivity of the TSQ instrument. The most suitable ion for the quantitation of 1-NP in the sample analyzed would appear to be m/z 231. It is possible that a minor interference may arise from the loss of OH from the ion shown in 111. However, loss of OH from the c/o 'OH2

functionality is expected to be a relatively minor process compared to the loss of OH2 (m/z 230). This was demon-

strated by TSQ-CAD analysis for the model compound 7benz[a]anthracene carboxylic acid. The m / z 230 ion was in low abundance in all the samples analyzed for 1-NP by TSQ-CAD. Quantitation of 1-NP.Quantitative determinations of 1-NP in extracts of particulate from four different diesel engines were made using TSQ-CAD and MIKES techniques. Table 111summarizes the results of these analyses. Quantitative MIKES analysis of 1-NP in the NI-1 sample was accomplished under E1 conditions. The MIKES analyses of samples OP-1 and PG-1 took advantage of the increased selectivity of the NICI mode of ionization. The TSQ mass spectrometer was operated in the CAD mode under PICI conditions during all analyses. Complete daughter ion spectra were collected by both instruments. The daughter ions chosen specifically for quantitation purposes are described in Table

111. Both standard addition and external standardization procedures were used for the quantitation of 1-NP. An excellent linear relationship between added nitropyrene and instrument response was observe with both the TSQ and EI-MIKES techniques. The NICI-MIKES technique exhibited a nonlinear response for 1-NP at levels below 10 ng, suggesting either minor, reproducible losses during sample introduction or sample matrix effects. As previously discussed, capillary GC/MS analysis was used to confirm that 1-NP was the only molecular weight 247 nitro-PAH present in the diesel samples. Therefore, MS/MS quantitation of the nitro(pyrene and fluoranthene) isomer group can be assumed to represent the 1-NP isomer. For sample NI-1, there was excellent quantitative agreement between the TSQ (2280 f 230 ppm) and EI-MIKES (2080 f 220 ppm) techniques. TSQ analysis of sample OL-1 indicated a 204 ppm level of 1-NP. MIKES analysis was not performed on this sample. However, the 204 ppm level determined by TSQ is in reasonable agreement with the value of 140 ppm previously estimated for this sample using GC/MS techniques (9). Quantitative analysis of sample OP-1 was much more difficult since this sample contained a relatively low concentration of 1-NP and a large amount of hydrocarbon contamination. As described in the qualitative analysis section, the EI-MIKES and PICI-MIKES techniques lacked the specificity required to quantitate 1-NP in this sample. At least three components were found to be present at m/z 201 in the EIMIKES spectrum. Deconvolution techniques used to resolve the contribution of 1-NP to this ion suggested a concentration of 1105 ppm. SemipreparativeHPLC fractionation was used to remove nonpolar and highly polar contaminants from the sample and analysis by the more selective NICI-MIKES technique yielded a 1-NP concentration of 55 f 11ppm. The

ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982

TSQ analysis of the unfractionated OP-1 sample indicated a 1-Nl?level of 77 f 15 ppm which agrees reasonably well with the MIKES results. The primary advantages of the MS/MS techniques we have developed are their use as rapid qualitative and semiquantitative procedures for the analysis of isomer groups of nitro-PAH in complex environmental samples. However, we were not able to determine specific isomers using MS/MS. Identification of specific isomers can be accomplished using fused silica capillary column GC/MS.

ACKNOWLEDGMENT Thanks are due F. S.-C. Lee, S. Levine, M. Paputa, and L. Skewes for the HPLC, analytical and preparative fractionations, to R. Gorse for the collecting the OP-1 and PG-1 particulate samples, and to F. Ferris for preparing some of the standards. D.F.H. gratefully acknowledges support from the U.S. Environmental Protection Agency.

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RECEIVED for review March 25,1981. Accepted October 16, 1981. Presented a t the Fifth International Symposium on Polynuclear Aromatic Hydrocarbons, Columbus, OH, Oct 28, 1980.