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2Hazardous Materials Laboratory, California Department of Health Services, ... decomposition product could usually be identified ... applications of L...
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Chapter 15

Particle Beam Liquid Chromatography/Mass Spectrometry of Phenols and Their Sulfate and Glucuronide Conjugates 1

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F. Reber Brown and William M. Draper 1

California Public Health Foundation, 2151 Berkeley Way, Berkeley, CA 94704 Hazardous Materials Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704

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The particle beam-liquid chromatography-mass spectrometry (PB-LC-MS) of phenols (phenol, 4nitrophenol, and 1-naphthol) and their glucuronide and sulfate conjugates in electron impact (EI) and positive chemical ionization (PCI) is described. The compounds were separated on a strong anion exchange (SAX) HPLC column with a pH 4.5 ammonium formate-acetonitrile mobile phase. Molecular ions were not obtained for any of the conjugate structures due to decomposition, but the phenols were detected in a l l cases as theM or [M+H] ion. The phenol formed from each conjugate as a decomposition product could usually be identified by computerized library search. In SIM mode, limits of detection ranged from 0.25 ng for 4nitrophenyl glucuronide to 51 ng for phenol. +

+

In t h e modern a n a l y t i c a l l a b o r a t o r y , gas chromatography-mass s p e c t r o m e t r y (GC-MS) i s a v i t a l t o o l i n the c h a r a c t e r i z a t i o n and i d e n t i f i c a t i o n o f unknowns. The advantages o f GC-MS a r e accuracy i n q u a n t i t a t i o n , low d e t e c t i o n l i m i t s , t e n t a t i v e i d e n t i f i c a t i o n of unknowns by s p e c t r a l l i b r a r y s e a r c h , and a h i g h degree o f r e l i a b i l i t y and v e r s a t i l i t y ( 1 ) . Of a l l t h e c h e m i c a l s known, however, o n l y a f r a c t i o n (-20%) a r e amenable t o a n a l y s i s by GC. The r e m a i n i n g compounds, because o f t h e i r h i g h m o l e c u l a r weight, thermal i n s t a b i l i t y , o r i o n i c and/or p o l a r c h a r a c t e r , a r e not s u i t e d t o d i r e c t GC d e t e r m i n a t i o n . Some o f these compounds can be a n a l y z e d by GC a f t e r d e r i v a t i z a t i o n t o v o l a t i l e s p e c i e s . D e r i v a t i z a t i o n , however, r e q u i r e s e x t r a sample h a n d l i n g , may g i v e low o r v a r i a b l e r e c o v e r i e s , and o f t e n r e q u i r e s t h e use o f hazardous r e a g e n t s . I n

0097-6156/90/0420-0232$06.00/0 © 1990 American Chemical Society Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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15. BROWN & DRAPER

Analysis ofPhenols and Their Conjugates

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c o n t r a s t , v e r y few compounds a r e known which cannot be a n a l y z e d d i r e c t l y by some form o f HPLC. The major l i m i t a t i o n o f HPLC has been t h e l a c k o f a d e t e c t o r w i t h u n i f o r m l y h i g h s e n s i t i v i t y . With t h i s i n mind, a n a l y t i c a l chemists have been w o r k i n g f o r decades t o couple l i q u i d chromatographs t o mass s p e c t r o m e t e r s , a c o m b i n a t i o n t h a t would t h e o r e t i c a l l y a l l o w d e t e r m i n a t i o n o f t h e r e m a i n i n g 80% o f known s u b s t a n c e s . S e v e r a l i n t e r f a c e s have been developed f o r c o u p l i n g HPLC's and mass s p e c t r o m e t e r s , i n c l u d i n g t h e moving b e l t , d i r e c t l i q u i d i n t r o d u c t i o n , and atmospheric p r e s s u r e i o n i z a t i o n d e s i g n s . Some of these i n t e r f a c e s have been reviewed i n the r e c e n t l i t e r a t u r e (2-5) and d i s c u s s e d i n t h i s symposium. The two i n t e r f a c e s showing t h e g r e a t e s t p o t e n t i a l f o r r o u t i n e l a b o r a t o r y use a r e the thermospray (TSP) and t h e "monodisperse a e r o s o l g e n e r a t o r f o r i n t r o d u c t i o n o f chromatography" (MAGIC) t o mass s p e c t r o m e t r y , a l s o known as a p a r t i c l e beam (PB) i n t e r f a c e . Thermospray LC-MS was developed by V e s t a l and co-workers and has been t h o r o u g h l y reviewed (2,3,6). The PB i n t e r f a c e ( F i g u r e 1 ) , developed by Browner and W i l l o u g h b y ( 7 ) , f u n c t i o n s as f o l l o w s : The LC e l u e n t passes through a s i l i c a c a p i l l a r y i n a c o n c e n t r i c pneumatic n e b u l i z e r . The n e b u l i z e r c r e a t e s an a e r o s o l of a p p r o x i m a t e l y u n i f o r m l y s i z e d d r o p l e t s , which passes i n t o a heated d e s o l v a t i o n chamber h e l d a t near atmospheric p r e s s u r e . M o b i l e phase evaporates from t h e d r o p l e t s , f o r m i n g a m i x t u r e o f mobile phase vapor and a n a l y t e - c o n t a i n i n g p a r t i c l e s which then pass through a n o z z l e i n t o a two-stage momentum s e p a r a t o r . The momentum s e p a r a t o r pumps away t h e lower momentum s o l v e n t vapor and s e l e c t i v e l y t r a n s m i t s t h e h i g h e r momentum a n a l y t e p a r t i c l e s to the MS source ( 8 ) . The PB i n t e r f a c e accommodates common r e v e r s e and normal phase chromatographic mobile phases a t f l o w r a t e s up to 0.5 ml/min, and i s m e c h a n i c a l l y s i m p l e , rugged, and easy t o o p e r a t e . U n l i k e t h e TSP i n t e r f a c e , b u f f e r i o n s a r e not r e q u i r e d t o e f f e c t i o n i z a t i o n , which i s i n s t e a d a c c o m p l i s h e d by EI o r CI i n a c o n v e n t i o n a l MS source ( 3 , 8 ) . Thermospray LC-MS a l l o w s h i g h e r f l o w r a t e s , but r e q u i r e s a s p e c i a l s o u r c e , and g e n e r a l l y r e q u i r e s a v o l a t i l e b u f f e r t o i o n i z e n e u t r a l molecules. Thermospray LC-MS p r o v i d e s mostly m o l e c u l a r weight i n f o r m a t i o n , whereas PB-LC-MS p r o v i d e s EI as w e l l as CI s p e c t r a . The l o n g term o b j e c t i v e o f our work i s t o examine t h e a p p l i c a t i o n s o f LC-MS i n measuring human exposure t o t o x i c substances. S p e c i f i c a l l y , we a r e i n v e s t i g a t i n g t h e d i r e c t measurement o f p o l a r and i o n i c m e t a b o l i t e s o f t o x i c compounds i n the u r i n e . Common mammalian m e t a b o l i c r o u t e s i n c l u d e c o n j u g a t i o n w i t h g l u c u r o n i d e o r s u l f a t e m o i e t i e s ( 9 ) , and such c o n j u g a t e s a r e d i f f i c u l t t o a n a l y z e by GC w i t h o u t e x t e n s i v e sample p r e p a r a t i o n (10,1_1). The model compounds chosen f o r t h i s s t u d y and t h e i r t y p i c a l p a r e n t compounds a r e shown i n Table I . The s p e c i f i c o b j e c t i v e s o f t h i s study were t o : (1) determine t h e g e n e r a l o p e r a t i n g c h a r a c t e r i s t i c s o f t h e PB i n t e r f a c e ; (2) i n v e s t i g a t e t h e ease o f a d a p t i n g an e x i s t i n g LC-UV method t o PB-LC-MS; (3) determine t h e c a p a b i l i t y o f PB-LC-MS f o r the i d e n t i f i c a t i o n o f p o l a r and i o n i c s u b s t a n c e s .

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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SPECTROMETRY

Table I.

Model Compounds I n v e s t i g a t e d by PB-LC-MS P a r e n t Compound/ Exposure Marker P h e n o l , phenyl g l u c u r o n i d e . p h e n y l s u l f a t e Benzene 4-Nitrophenol, 4-nitrophenyl glucuronide, 4-nitrophenyl sulfate Parathion 1-Naphthol, 1-naphthyl g l u c u r o n i d e , 1-naphthyl s u l f a t e Carbaryl Compound

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Experimental S e c t i o n C h e m i c a l s . Sources o f the s t a n d a r d s used a r e d e s c r i b e d elsewhere (12»13). S t a n d a r d s o l u t i o n s o f the model compounds were prepared i n e i t h e r d i s t i l l e d d e i o n i z e d water o r p e s t i c i d e grade a c e t o n i t r i l e . The chromatographic mobile phase was a 2:3 (v/v) m i x t u r e o f a c e t o n i t r i l e and ammonium formate b u f f e r p r e p a r e d by a d j u s t i n g a 0.05 M s o l u t i o n o f f o r m i c a c i d t o pH 4.5 w i t h c o n c e n t r a t e d ammonia. Chromatographic System. The chromatographic system used to d e v e l o p the SAX-HPLC-UV method was d e s c r i b e d p r e v i o u s l y ( 1 2 ) . The chromatograph used f o r PB-LC-MS was a H e w l e t t - P a c k a r d Model 1090 equipped w i t h a t e r n a r y s o l v e n t d e l i v e r y system, a v a r i a b l e volume (0.1 - 25uL) sample i n j e c t o r , an auto sampler, and a column bypass v a l v e . A H e w l e t t - P a c k a r d Model 59980A P a r t i c l e Beam I n t e r f a c e was used. Mass Spectrometer. The mass s p e c t r o m e t e r was a H e w l e t t - P a c k a r d 5988A quadrupole mass s p e c t r o m e t e r w i t h a d u a l EI/CI source and p o s i t i v e and n e g a t i v e i o n d e t e c t i o n . The system was c o n t r o l l e d by a H e w l e t t - P a c k a r d 1000 computer. The mass s p e c t r o m e t e r was p e r i o d i c a l l y tuned manually u s i n g p e r f l u o r o t r i b u t y l a m i n e (PFTBA) on i o n s m/z 69, m/z 214, and m/z 502 i n EI and PCI modes, and on i o n s m/z 245, m/z 414, and m/z 633 i n NCI mode. System performance was monitored d a i l y by i n j e c t i n g 10 uL o f a 10 ng/uL s o l u t i o n o f c a f f e i n e i n t o a c e t o n i t r i l e u s i n g f l o w i n j e c t i o n a n a l y s i s (FIA - no column i n l i n e ) mode. The f l o w r a t e was 0.4 mL/min. F i v e r e p l i c a t e i n j e c t i o n s were made, and the b a s e l i n e and mean s i g n a l - t o - n o i s e r a t i o (S/N) were determined. Losses i n b a s e l i n e s i g n a l o r S/N were i n d i c a t i v e o f problems such as c l o g g i n g o r c o n t a m i n a t i o n o f the skimmer cones i n the momentum s e p a r a t o r , c o n t a m i n a t i o n o f t h e mass s p e c t r o m e t e r f i l a m e n t o r s o u r c e , o r e l e c t r o n i c d r i f t i n the mass s p e c t r o m e t e r . P o s i t i o n i n g o f t h e c a p i l l a r y t i p i n the n e b u l i z e r o r i f i c e was c r i t i c a l t o o p t i m i z i n g the system response. In order to determine the optimum t i p p o s i t i o n , the auto sampler was s e t f o r 16 i n j e c t i o n s 18 sec a p a r t , and the c a p i l l a r y was f u l l y extended. A f t e r each i n j e c t i o n , the n e b u l i z e r was r o t a t e d one u n i t (10 u n i t s p e r r o t a t i o n ) . The optimum p o s i t i o n was t h a t which gave maximum i o n i n t e n s i t y . The mass s p e c t r o m e t e r was o p e r a t e d i n EI mode, w i t h s i n g l e i o n m o n i t o r i n g (SIM) o f m/z 194 w i t h a d w e l l time o f 900 ms.

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Flow I n j e c t i o n A n a l y s i s o f Phenols and C o n j u g a t e s . In o r d e r t o determine t h e mode o f i o n i z a t i o n g i v i n g maximum s e n s i t i v i t y , FIA was used t o o b t a i n mass s p e c t r a o f t h e i n d i v i d u a l s t a n d a r d s o f the phenols and t h e i r c o n j u g a t e s i n b o t h a c e t o n i t r i l e and a c e t o n i t r i l e - p H 4.5 ammonium formate ( 2 : 3 , v/v) u s i n g E I , methane PCI and NCI, and i s o b u t a n e PCI and NCI. O p e r a t i n g c o n d i t i o n s were: f l o w r a t e , 0.4 mL/min; d e s o l v a t i o n chamber temperature, 45°C; n e b u l i z e r p r e s s u r e , 40 p s i ; s o u r c e t e m p e r a t u r e , 250°C. In EI mode, t h e scan range was m/z 50 to m/z 500 w i t h a scan c y c l e time o f 1.9 sec. I n methane PCI and NCI, t h e scan range was m/z 90 t o m/z 500 w i t h a scan c y c l e time o f 1.7 s e c and t h e source p r e s s u r e was 2 x 10~ t o r r . I n i s o b u t a n e PCI and NCI, t h e scan range was from m/z 100 t o m/z 500 w i t h a scan c y c l e o f 1.7 sec and a s o u r c e p r e s s u r e o f 7 x 1 0 ~ t o r r was used. A l a r g e i n c r e a s e i n background p r e c l u d e d s c a n n i n g any lower than m/z 90 or m/z 100 i n methane o r i s o b u t a n e CI modes, r e s p e c t i v e l y . The e m i s s i o n c u r r e n t was 332 uA, arid the e l e c t r o n energy was 70 v o l t s f o r EI mode and 200 v o l t s f o r CI mode.

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A

5

PB-HPLC-MS o f Phenols and Conjugates. The SAX chromatographic s e p a r a t i o n o f p h e n o l s , s u l f a t e s , and g l u c u r o n i d e s i s d e s c r i b e d i n d e t a i l elsewhere ( 1 2 ) . B r i e f l y , a 25 cm x 4.6 mm SAX column ( S u p e l c o , B e l l e f o n t e , PA) i s used w i t h an a c e t o n i t r i l e - p H 4.5 ammonium formate b u f f e r (2:3) m o b i l e phase a t a f l o w r a t e o f 1.5 mL/min. Under t h e s e c o n d i t i o n s , t h e n i n e model compounds were r e s o l v e d t o b a s e l i n e i n l e s s than 20 minutes. For PB-HLC-MS, t h e f l o w r a t e was reduced from 1.5 mL/min t o 0.4 mL/min, and t h e n e b u l i z e r p r e s s u r e was s e t t o 50 p s i . MS parameters were: i o n i z a t i o n mode, i s o b u t a n e PCI; f i l a m e n t c u r r e n t , 300 uA; e l e c t r o n energy, 200 V; source p r e s s u r e , 8 x l 0 ~ t o r r ; source temperature, 250°C. Standards o f a p p r o x i m a t e l y 12, 30, 60, 250, and 500 ng/uL were r u n i n SIM mode. Ions o f m/z 95.0, m/z 139.8, and m/z 144.9 were m o n i t o r e d f o r 600 msec each. These i o n s a r e c h a r a c t e r i s t i c i o n s f o r phenol and i t s c o n j u g a t e s , 4 - n i t r o p h e n o l and i t s c o n j u g a t e s , and 1 -naphthol and i t s c o n j u g a t e s , r e s p e c t i v e l y . 5

R e s u l t s and D i s c u s s i o n N e b u l i z e r O p t i m i z a t i o n . The optimum p o s i t i o n o f t h e c a p i l l a r y t i p i n the n e b u l i z e r was determined f o r s o l v e n t c o m p o s i t i o n s o f 100%, 80%, 60%, 40%, and 20% a c e t o n i t r i l e i n pH 4.5 ammonium formate b u f f e r . T y p i c a l r e s u l t s ( F i g u r e 2) show two maxima o c c u r r i n g a t s e t t i n g s o f 4 and 13. F o r 100% a c e t o n i t r i l e , t h e maximum a t 4 i s g r e a t e r than t h a t a t 14. In the a c e t o n i t r i l e b u f f e r systems, t h e maxima a t 13 a r e s l i g h t l y g r e a t e r , but at t h i s s e t t i n g the c a p i l l a r y t i p i s i n s i d e the n e b u l i z e r causing s p a t t e r i n g o f t h e s o l v e n t and e r r a t i c b a s e l i n e s h i f t s . For t h i s reason, the n e b u l i z e r s e t t i n g o f 4 was used r o u t i n e l y and proved s t a b l e i n day t o day o p e r a t i o n . Flow I n j e c t i o n A n a l y s i s o f Phenols and C o n j u g a t e s . A summary o f the EI s p e c t r a o f t h e phenols and t h e i r c o n j u g a t e s o b t a i n e d i n FIA mode i s found i n Table I I . I n e x p l i c a b l y , phenol was not

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Two-Stage Momentum Separator ^

^kimmer Cones

Fused Silica Capillary

Transfer Tube

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LC Effluent - >

Concentric Pneumatic Nebulizer Rough Pumps for Momentum Separator F i g u r e 1.

Diagram o f p a r t i c l e beam i n t e r f a c e .

300

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Nebulizer Position F i g u r e 2.

Mass s p e c t r a l response v s n e b u l i z e r s e t t i n g as a

function of solvent composition. (M

), 80% a c e t o n i t r i l e ;

acetonitrile;

( Q

), 20%

(B

(

B

), 100%

acetonitrile;

), 6 0 % a c e t o n i t r i l e ;

( B ) , 40%

acetonitrile.

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

15.

BROWN & DRAPER

Analysis of Phenols and Their Conjugates

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Table I I .

237

PB E l e c t r o n Impact Mass S p e c t r a f o r Phenols and Conjugates [ M a s s ( r e l a t i v e i n t e n s i t y ) ] Compound Acetonitrile 2:3 A c e t o n i t r i l e Buffer Phenol Not d e t e c t e d Not d e t e c t e d Phenyl g l u c u r o n i d e 173(11),114(17),94a 115(9),94a(100),77b (100),85(12),77b(16) (13) ,71(21),66(24), 73(20),71(35),66(11) 64(21),55(11) 65c(11) Phenyl s u l f a t e 94a(100),79(10),72(10) , 116(38),115(100),109 66(21),65c(20),55(19) (17),73(25),72(24), 69(15),65c(13),56 (16),55(24),54(38), 52(22) 4 -Nitrophenol 139d(60),109e(34),98f 109e(67),93f(18),81 (37),80(20),65g(100), (21),81(23),65g(100), 53(47),52(20) 63(27),53(23) 109h(78),93i(23),81 4-Nitrophenyl 139a(84),109h(50) 93i glucuronide (30) ,65j(100),63(33) (24) ,81(28),73(21),64 53(54).51(32) (100),63(32),53(35) 4-Nitrophenyl 109h(64),93i(21),81 139a(73),109h(40),93i sulfate (31) , 8 0 ( 2 2 ) , 6 5 j ( 1 0 0 ) , (25) , 8 1 ( 2 8 ) , 6 5 j ( 1 0 0 ) , 64(29),63(33),53(53), 64(26),63(26),53(29) 52(29) 1 Naphthol 115k(83),77(83),61 144d(81),116(35),115k (64),55(54),54(100), (100),89(18),65(18),63 52(50) (28),57(13) 1 -Naphthyl I44a(100) ,116(42) ,1.15c 116(45),115c(100),89 glucuronide (100),89(14),72(13), (14) ,72(11),63(16), 66(19) 57(14) 1-Naphthyl 144a(100),116(41),115c 116(39),115c(100),89 sulfate (15) ,64(23),63(20) (93),89(15),72(13), 66(20) f

a [aglycone]^ b [aglycone-OH]+ c [aglcyone-CH0]+ d [M] +

e [M-N0]+ f [M-N0 ] + g [M+H-N0 -CH0]+ h [aglycone-N0]+ 2

2

i [ a g l y c o n e - N0 ]+ j [aglycone+H-N0 -CHO]+ k [M-CH0]+

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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2

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SPECTROMETRY

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d e t e c t e d , w h i l e 4 - n i t r o p h e n o l and 1-naphthol both gave prominent molecular ions. M o l e c u l a r i o n s f o r the c o n j u g a t e s were not d e t e c t e d and i n each case o n l y the aglycone was p r e s e n t . T h i s was a t t r i b u t e d t o e i t h e r h y d r o l y s i s i n the d e s o l v a t i o n chamber o r thermal d e c o m p o s i t i o n i n the MS s o u r c e . The r e s u l t s o f the l i b r a r y s e a r c h f o r these compounds a r e summarized i n T a b l e I I I . G e n e r a l l y , the phenols were c o r r e c t l y identified. The c o n j u g a t e s were not p r e s e n t i n the mass s p e c t r a l library. However, they were c o n s i s t e n t l y i d e n t i f i e d as t h e i r aglycone o r phenol d e c o m p o s i t i o n p r o d u c t .

T a b l e I I I . I d e n t i f i c a t i o n o f Phenols and Phenol Conjugates by Mass S p e c t r a l L i b r a r y Search Acetonitrile 2:3 A c e t o n i t r i l e Buffer Hit* Probability Probability Hit* Match Match .4 Phenyl g l u c u r o n i d e 1 59 ..3 Phenyl S u l f a t e l 73 4 -Nitrophenol 1 89 .1 85 4-Nitrophenyl glucuronide l 92 l 96 4-Nitrophenyl s u l f a t e l 96 I 91 ..4 1 Naphthol 2 64 .1 -Naphthyl g l u c u r o n i d e 1 96 l 89 1-Naphthyl s u l f a t e I I 89 95 1

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P o s i t i o n out o f f i r s t f i v e c h o i c e s w i t h 1 i n d i c a t i n g best match. ^ Q u a l i t y o f match w i t h 99 i n d i c a t i n g h i g h e s t q u a l i t y Not i d e n t i f i e d among f i r s t f i v e responses, but a g l y c o n e i n library. No r e t r i e v a l s from d a t a base I d e n t i f i e d as aglycone

Both s i g n a l - t o - n o i s e and a b s o l u t e s i g n a l i n t e n s i t y were t h e c r i t e r i a used t o s e l e c t the mode o f i o n i z a t i o n f o r LC-MS (Table IV). Except f o r the s u l f a t e s and 4 - n i t r o p h e n o l i n PCI, t h e r e i s a decrease i n S/N when the s o l v e n t i s changed from a c e t o n i t r i l e to a c e t o n i t r i l e - b u f f e r . The o p p o s i t e i s observed f o r t h e s u l f a t e s and 4 - n i t r o p h e n o l . I n most c a s e s , t h e r e i s a p a r a l l e l l o s s o f s i g n a l i n t e n s i t y when g o i n g from a c e t o n i t r i l e t o acetonitrile-buffer. The d e l e t e r i o u s e f f e c t s o f the b u f f e r on S/N i s l e a s t pronounced i n PCI. With a c e t o n i t r i l e - a m m o n i u m formate mobile phase, i s o b u t a n e PCI p r o v i d e s optimum s e n s i t i v i t y when compared t o methane PCI o r E I . I n both methane and i s o b u t a n e NCI, peak b r o a d e n i n g was u n a c c e p t a b l y l a r g e , and NCI s p e c t r a were d i f f i c u l t t o i n t e r p r e t . Based on t h e s e c o n s i d e r a t i o n s , i s o b u t a n e PCI was chosen f o r LC-MS s t u d i e s . The i s o b u t a n e PCI s p e c t r a o b t a i n e d i n FIA mode a r e summarized i n Table V. 4 - N i t r o p h e n o l and 1-naphthol were d e t e c t e d as p r o t o n a t e d m o l e c u l a r i o n s ; phenol was not d e t e c t e d as i t s p r o t o n a t e d m o l e c u l a r i o n i s below the scan range used.

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Table IV.

S i g n a l / n o i s e R a t i o and Peak H e i g h t s of Phenols and Conjugates Compound (ng used) 2:3 A c e t o n i t r i l e Acetonitr.i l e Ionization Buffer Mode S/N Peak S/N Peak Ratio Height Ratio Height . 4 .4 .4 Phenol (973) EI Me-PCI 39 0.48 0.49 5 . .5 _5 ..ft iBu-PCI Phenyl g l u c u r o n i d e 39 350 41 0.54 EI (954) Me-PCI 170 190 51 11 _ 5 _5 iBu-PCI Phenyl s u l f a t e (964) 0. 12 EI 68 0.89 11 Me-PCI 30 0.63 43 3.3 _5 5 f> .. 5 iBu-PCI 4 - N l t r o p h e n o l (909) EI 68 1.8 340 23 30 Me-PCI 240 180 73 iBu-PCI 72 240 98 150 125 4-Ni t r o p h e n y l 330 19 2.7 EI g l u c u r o n i d e (911) 66 10 82 140 Me-PCI 49 iBu PCJ 140 133 90 4-Ni t r o p h e n y l 8.8 250 3.1 EI 160 s u l f a t e (964) 13 MePCI 9.4 120 69 43 iBu-PCI 69 24 20 I-Naphthol (1091) 0.049 5.3 7.7 EI 210 83 MePCI 10 2.3 160 4.0 iBu P C i 13 88 17.1 1-Naphthyl 20 230 3.8 EI 290 g l u c u r o n i d e (954) 7.6 79 Me-PCI 83 20 100 4.0 37 iBu-PCI 110 1-Naphthyl s u l f a t e 42 2.7 3.3 EI 38 (1027) Me-PCI 50 4.8 69 7.8 20 iBu PCI 38 9.3 94 1

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Peak Height / 1 0 Methane p o s i t i v e chemical i o n i z a t i o n Isobutane p o s i t i v e c h e m i c a l i o n i z a t i o n Not a n a l y z e d - below scan range Not d e t e c t e d

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A g a i n , none o f the c o n j u g a t e d s p e c i e s gave m o l e c u l a r i o n s , and as b e f o r e , t h e aglycone i s d e t e c t e d as t h e [M+H] " s p e c i e s . The l a c k of m o l e c u l a r s p e c i e s f o r any o f the c o n j u g a t e s i n CI mode p r o v i d e s a d d i t i o n a l evidence f o r d e c o m p o s i t i o n . 4

Table V.

PB P o s i t i v e Chemical and Phenol Conjugates

Compound

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4-Nitrophenol 4-Nitrophenyl 4-Nitrophenyl

glucuronide sulfate

1-Naphthol 1-Naphthyl g l u c u r o n i d e 1-Naphthyl s u l f a t e a [M+H]+ b [M+H-N0]>

I o n i z a t i o n Mass S p e c t r a o f Phenols [ M a s s ( R e l a t i v e Abundance)] 2:3 A c e t o n i t r i l e Acetonitrile Buffer 140a(100),110b(29), 181(11),140a(100) 109(16) 140c(100),124(11) 181(10),140c(100) 168(31),140c(100), 181(10),146(16), 124(14)108(17) 140c(100),115(11) 146(ll)145a(100) 145a(100) 144(24) 146(100),145e(100) 146(12),145c(100) 146(10),145e(100) 145c(26),140(100) 124(10),115d(43) c [aglycone]* d [aglycone CH0]f

PB-LC-MS o f Phenols and Conjugates. The t o t a l i o n c u r r e n t chromatogram and e x t r a c t e d i o n p r o f i l e s f o r m/z 95.0, m/z 139.8, and m/z 144.9 a r e shown i n F i g u r e 3. F o r t h i s chromatogram, a p p r o x i m a t e l y 600 ng o f each compound was i n j e c t e d . I n e x p l i c a b l y , phenyl g l u c u r o n i d e was not d e t e c t e d . The o t h e r peaks showed t h e expected r e t e n t i o n time and e l u t l o n o r d e r (13). Chromatographic e f f i c i e n c i e s f o r PB-LC-MS v a r i e d from 550 t h e o r e t i c a l p l a t e s f o r phenol t o 8700 t h e o r e t i c a l p l a t e s f o r 4nitrophenyl sulfate. U s i n g the HPLC-UV method, t h e chromatographic e f f i c i e n c y was somewhat b e t t e r , 4300 t h e o r e t i c a l p l a t e s f o r phenol and 16000 f o r 4 - n i t r o p h e n y l s u l f a t e . The manufacturer c l a i m s an e f f i c i e n c y o f 7900 t h e o r e t i c a l p l a t e s f o r t h i s column. Thus, t h e r e i s some added band broadening i n t h e PB-LC-MS system. A c a l i b r a t i o n curve f o r 1 -naphthyl g l u c u r o n i d e i s shown i n F i g u r e 4. F o r t h i s d a t a , the best f i t s t r a i g h t l i n e has a c o r r e l a t i o n c o e f f i c i e n t o f 0.989. When f i t t e d t o a q u a d r a t i c e q u a t i o n the c o r r e l a t i o n c o e f f i c i e n t was 1.00. Q u a d r a t i c c a l i b r a t i o n curves were a l s o seen f o r t h e o t h e r compounds i n t h i s s t u d y , and t h i s i s c o n s i s t e n t w i t h another r e p o r t i n t h i s symposium (Brown, M. A., e t a l . t h i s volume). Instrument d e t e c t i o n l i m i t s were e s t i m a t e d from t h e l i n e a r c a l i b r a t i o n curve u s i n g two times t h e average peak h e i g h t o f t h e b a s e l i n e n o i s e f o r each i o n monitored as t h e minimum d e t e c t a b l e signal. E s t i m a t e d i n s t r u m e n t l i m i t s o f d e t e c t i o n (ng) a r e : phenol, 51; 4 - n i t r o p h e n o l , 3.6; 1 n a p h t h o l , 165; 4 - n i t r o p h e n y l g l u c u r o n i d e , 0.25; 1-naphthyl g l u c u r o n i d e , 5.3; phenyl s u l f a t e , 7.7; 4 n i t r o p h e n y l s u l f a t e , 0.29; 1-naphthyl s u l f a t e , 3.1. F o r the UV d e t e c t o r a t 254 nm, t h e c o r r e s p o n d i n g l i m i t s o f d e t e c t i o n (ng) were: p h e n o l , 5.0; 4 - n i t r o p h e n o l , 8.2; 1-naphthol, 4.0; 4-

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F i g u r e 3. T o t a l i o n c u r r e n t chromatogram and e x t r a c t e d i o n p r o f i l e s f o r i o n s o f m/z 95.0, m/z 139.8, and m/z 144.9. Peaks a r e i d e n t i f i e d as phenol ( A ) , phenyl s u l f a t e ( B ) , 4n i t r o p h e n o l ( C ) , 4 - n i t r o p h e n y l g l u c u r o n i d e (I)), 4 - n i t r o p h e n y l s u l f a t e ( E ) , 1-naphthol ( F ) , l ~ n a p h t h y l g l u c u r o n i d e (G), and 1-naphthyl s u l f a t e (H).

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200

Concentration (ng/uL)

F i g u r e 4. C a l i b r a t i o n curve f o r 1-naphthyI g l u c u r o n i d e showing l i n e a r and q u a d r a t i c c a l i b r a t i o n c u r v e s .

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n i t r o p h e n y l g l u c u r o n i d e , 10; 1-naphthyl g l u c u r o n i d e , 14; phenyl s u l f a t e , 85; 4 - n i t r o p h e n y l s u l f a t e , 2.8; 1-naphthyl s u l f a t e , 7.0.

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Conclusions Flow i n j e c t i o n a n a l y s i s w i t h t h e PB i n t e r f a c e p r o v i d e s an e f f e c t i v e means f o r i n t r o d u c i n g p o l a r compounds t o t h e EI source and can be viewed as an a l t e r n a t i v e t o s o l i d probe i n t r o d u c t i o n . I t a l s o i s a u s e f u l means f o r e s t i m a t i n g the r e l a t i v e s e n s i t i v i t y i n d i f f e r e n t PB-LC-MS mobile phases and i o n i z a t i o n modes. While EI g i v e s s p e c t r a t h a t can be matched w i t h o n l i n e d a t a bases, i t was found t o be t h e l e a s t s e n s i t i v e o p e r a t i n g mode. CI r e s t r i c t s the mass s c a n n i n g range t o g r e a t e r than m/z 100, and may contaminate t h e i o n source more r a p i d l y , but g i v e s b e t t e r s e n s i t i v i t y and p r o v i d e s m o l e c u l a r weight i n f o r m a t i o n . A d a p t i n g HPLC UV s e p a r a t i o n s t o PB-LC-MS i s s t r a i g h t f o r w a r d as l o n g as f l o w r a t e l i m i t a t i o n s a r e c o n s i d e r e d and v o l a t i l e b u f f e r s a r e s e l e c t e d where needed. The PB-MS d e t e c t o r does n o t cause e x c e s s i v e peak broadening except i n n e g a t i v e i o n mode. While g l u c u r o n i d e and s u l f a t e m o l e c u l a r i o n s p e c i e s a r e n o t seen i n the PB mass s p e c t r a , the sSAX-LC s e p a r a t i o n groups t h e compounds by c o n j u g a t e c l a s s . Phenols e l u t e f i r s t , f o l l o w e d by g l u c u r o n i d e s , and then s u l f a t e s . Thus, t e n t a t i v e i d e n t i f i c a t i o n of unknown m e t a b o l i t e s i s p o s s i b l e based upon i d e n t i f i c a t i o n o f the aglycone and t h e r e t e n t i o n time window. CA Regis t r y Numbers P h e n o l , [108 95-2]; phenyl s u l f a t e potassium s a l t , [1733-88-6]; phenyl g l u c u r o n i d e , [17685 05 1 ] ; 4 - n i t r o p h e n o l , [100-02 7 ] ; 4rii t r o p h e n y l s u l f a t e potassium s a l t , [6217-68-1]; 4 - n i t r o p h e n y l g l u c u r o n i d e , [10344-94-2]; 1-naphthol, [90-15-3]; 1-naphthyl s u l f a t e potassium s a l t , [6295 74 5 ] ; 1-naphthyl g l u c u r o n i d e , [17238-47-0]. Acknowledgement s T h i s work was c a r r i e d out a t t h e C a l i f o r n i a Department o f H e a l t h S e r v i c e s Hazardous M a t e r i a l s L a b o r a t o r y , and was funded by Superfund P r o j e c t No. ES 04705 from t h e N a t i o n a l I n s t i t u t e o f Environmental H e a l t h S c i e n c e s o f the N a t i o n a l I n s t i t u t e s o f Health.

Literature Cited 1. 2. 3. 4. 5. 6.

Fenselau, C. Anal. Chem. 1977, 49, 563A. Arpino, Patrick J.; Guiochon, Georges. Anal. Chem. 1979, 51, 682A. Covey, Thomas R.; Lee, Edgar D.; Bruins, Andries P.; Henion, Jack D. Anal. Chem. 1986, 58, 1451A. Bruins, A. P. J . Chromatogr. 1985, 323, 99. Arpino, Patrick J . J . Chromatogr. 1985, 323, 3. Garteiz, D. A.; Vestal, M. L. Liq. Chromatogr. 1985, 3.

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244 7. 8. 9.

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10.

11.

12. 13.

LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626-31. Browner, R. F.; Winkler, P. C.; Perkins, D. D; Abbey, L. E. Microchemical Journal 1986, 34, 15-24. Klaassen, C. D.; Amdur, M. O.; Doull, J., Eds. Toxicology: The Basic Science of Poisons; Macmillan Publishing, New York, 1986. Bakke, J. E. in Bound and Conjugated Pesticide Residues; Kaufman, D. D.; Still, G. G.; Paulson, G. D.; Bandal, S. K., Eds. ACS Symposium Series No. 29; American Chemical Society: Washington, D.C., 1976; pp. 55-67. Paulson, G. D. in Bound and Conjugated Pesticide Residues; Kaufman, D. D.; Still, G. G.; Paulson, G. D.; Bandal, S. K., Eds. ACS Symposium Series No. 29; American Chemical Society: Washington, D.C., 1976; pp. 86-102. Brown, F. R.; Draper, W. M. J. Chromatogr. 1989, 479, 441444. Draper, W. M.; Brown, F. R.; Miille, M. J.; Bethem, R. Biomed. Environ. Mass Spectrom. 1989, 18, 767-774.

RECEIVED October 6, 1989

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.