Chemical Hazards in the Workplace - ACS Publications - American

26 High Performance Liquid Chromatographic Determination of Aromatic Amines in Body Fluids and Commercial Dyes P. J. M. VANTULDER, C. C. HOWARD, and R. M. RIGGIN

Downloaded by UNIV OF ARIZONA on January 7, 2013 | http://pubs.acs.org Publication Date: April 2, 1981 | doi: 10.1021/bk-1981-0149.ch026

Battelle-Columbus Laboratories, 505 King Avenue, Columbus, OH 43201

This paper focuses primarily on the application of High Performance Liquid Chromatography (HPLC) with electrochemical detection (EC) for the determination of aromatic amines. A l l of the studies described in this paper were conducted in the HPLC f a c i l i t y of Battelle Columbus Laboratories during the past two years. Much of the work is only cursory in nature (due to time and funding limitations) but as a whole the data presented are useful in projecting the range of applicability of HPLC/EC. The operational characteristics of HPLC/EC have been reviewed (1) and w i l l only be described briefly in this paper. HPLC/EC, for the purposes of this paper, refer to the coupling of a HPLC with a thin-layer flow-through electrochemical c e l l , as shown in Figure 1. The working electrode (in most cases glassy carbon or carbon paste) i s held at a fixed potential relative to the reference electrode. The counter or auxiliary electrode (in this case a platinum tube through which the column eluent flows) c a r r i e s the r e q u i r e d counter c u r r e n t . When an e l e c t r o c h e m i c a l l y a c t i v e component e l u t e s from the column i t exchanges e l e c t r o n s w i t h the working e l e c t r o d e s u r f a c e , causing a current t o f l o w . T h i s current i s converted to a v o l t a g e and recorded on a s t r i p chart recorder as a f u n c t i o n of time, g i v i n g r i s e to a chromatogram. Since c u r r e n t s can be measured very s e n s i t i v e l y using modern o p e r a t i o n a l a m p l i f i e r s the e l e c t r o c h e m i c a l d e t e c t o r can be extremely s e n s i t i v e (e.g. 1-10 picograms i n j e c t e d ) f o r e l e c t r o a c t i v e compounds. A t y p i c a l e l e c t r o c h e m i c a l r e a c t i o n f o r an aromatic amine (benzidine) i s shown i n F i g u r e 2. Most of the HPLC/EC work t o date has been conducted u s i n g carbon working e l e c t r o d e s and thus the o x i d a t i v e mode of the d e t e c t o r has been e x p l o i t e d (e.g. f o r benzidine) t o the g r e a t e s t extent. However, platinum and mercury have been used s u c c e s s f u l l y f o r e l e c t r o r e d u c i b l e species such as metal ions C?/and parathionC?). One o f the most important f e a t u r e s of HPLC/EC i s the e f f e c t of e l e c t r o d e p o t e n t i a l on d e t e c t o r response. F i g u r e 3 i l l u s t r a t e s t h i s f e a t u r e wherein the response o f the d e t e c t o r as a 0097-6156/81/0149-0413$05.00/0 © 1981 American Chemical Society

In Chemical Hazards in the Workplace; Choudhary, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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(•-REFERENCE ^ELECTRODE

I

^

AUXILIARY EL


FROM COLUMN

^WORKING

Figure 1.

Ε

Schematic of HPLC/EC cell

HeN^OWOhNH,

Figure 2.

HN=
2H

+

: NH

1.2

Figure 3. Hypothetical HPLC/EC de tector potential/response curve

Detector Potential, volts


26.

VAN TULDER E T A L .

Aromatic Amines in Body Fluids

415

f u n c t i o n of working e l e c t r o d e p o t e n t i a l f o r two components, A and B, i s p l o t t e d . Component A g i v e s v i r t u a l l y no response at 0.3 v o l t s but i t s response i n c r e a s e s as the p o t e n t i a l i s increased and reaches a maximum at 0.6 v o l t s . Component B, however, s t i l l gives no response at 0.6 v o l t s and r e q u i r e s a p o t e n t i a l of 0.9 v o l t s to g i v e maximum response. Therefore one could analyze f o r A u s i n g a d e t e c t o r p o t e n t i a l of 0.6 v o l t s w i t h v i r t u a l l y no i n t e r f e r e n c e from B, even i f the two components are not separated chromatographically.


S i g n i f i c a n c e of Determining Aromatic Amines Aromatic amines as a compound c l a s s account f o r many of the known carcinogenic organic chemicals. Perhaps the most w i d e l y p u b l i c i z e d compounds are b e n z i d i n e and 3 , 3 - d i c h l o r o b e n z i d i n e (DCB)(4,5)which u n t i l r e c e n t l y were w i d e l y used i n the manuf a c t u r e of d y e s t u f f s . Another w i d e l y p u b l i c i z e d suspected carcinogenic aromatic amine i s 4-methoxy-m-phenylenediamine (MMPDA) (A) which i s used as an i n g r e d i e n t i n many permanent h a i r dye f o r m u l a t i o n s . 4 , 4 - m e t h y l e n e b i s ( 2 - c h l o r o a n i l i n e ) o r MB0CAÇ7)and 4,4-methylenedianiline (MDA) are w i d e l y used as c u r i n g agents i n polyurethane r e s i n s and are s a i d to be c a r cinogenic. Various halogenated and n i t r a t e d a n i l i n e d e r i v a t i v e s are used i n the manufacture of d y e s t u f f s and carbamate and urea based p e s t i c i d e s . Many of these compounds are suspected to be carcinogenic. Since workers can be exposed to these compounds d u r i n g t h e i r manufacture and use, i t i s important to have r e l i a b l e a n a l y t i c a l methods f o r determining the degree of exposure through body f l u i d a n a l y s i s . A d d i t i o n a l l y , s i n c e these compounds can be present at s i g n i f i c a n t l e v e l s i n commercial products (derived from them) i t i s d e s i r a b l e to monitor t h e i r l e v e l i n such products (e.g. d y e s t u f f s ) as w e l l . Furthermore, many of the commercial products can be metabolized to the o r i g i n a l chemical (e.g. benzidine based dyes can be metabolized to b e n z i d i n e ) making i t d e s i r a b l e to monitor the body f l u i d s of workers exposed t o the commercial products. f

f

A p p l i c a b i l i t y Of HPLC/EC To The Determination Of Aromatic Amines In order to examine the range of aromatic amines which could p o s s i b l y be determined by HPLC/EC, c y c l i c voltammograms were run on a l a r g e number of aromatic amines. I n t h i s study a B i o a n a l y t i c a l Systems DCV-3 p o t e n t i o s t a t (designed f o r use i n c y c l i c voltammetry) and a Hewlett-Packard Model 7004B XYrecorder were used. The working e l e c t r o d e was g l a s s y carbon (Model 9333 from P r i n c e t o n A p p l i e d Research). A Ag/AgCl reference e l e c t r o d e and platinum a u x i l i a r y e l e c t r o d e were used. A scan range of 0-1.5 v o l t s v s . Ag/AgCl a t 250 mV/second was



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used. The g l a s s y carbon e l e c t r o d e was p o l i s h e d a f t e r each scan, u s i n g f i n e g r i t alumina and emery c l o t h . The compounds were prepared as 100 yg/mL s o l u t i o n s i n 50% a c e t o n i t r i l e / 50% 0.2 M sodium acetate b u f f e r , pH 4.7. The data f o r the aromatic amines s t u d i e d are given i n Table I . A l l data are reported as Ep (peak p o t e n t i a l s ) from the c y c l i c voltammograms. The lower the peak p o t e n t i a l f o r o x i d a t i o n of a compound, the more s p e c i f i c a l l y i t can be determined ( s i n c e a lower d e t e c t o r v o l t a g e can be used). Therefore compounds such as MMPDA, MDA, DCB, MBOCA, and benzidine ( i . e . aromatic diamines) can be more r e a d i l y determined by HPLC/EC than are the a n i l i n e d e r i v a t i v e s . T h i s does not imply that HPLC/EC i s not u s e f u l for the determination of a n i l i n e s . I n f a c t , a recent paper\°/ i l l u s t r a t e s the usefulness of HPLC/EC f o r the determination of halogenated a n i l i n e s i n u r i n e . The data i n Table I shows that a d d i t i o n of a n i t r o group to a n i l i n e increases the peak p o t e n t i a l ^200-250 mV whereas a d d i t i o n o f a halogen has very l i t t l e a f f e c t . Therefore HPLC/EC should be more u s e f u l f o r determining a n i l i n e and i t s halogen s u b s t i t u t e d d e r i v a t i v e s , than f o r n i t r o s u b s t i t u t e d a n i l i n e s . The data given i n Table I I support t h i s c o n c l u s i o n . Chromatographic parameters used t o o b t a i n the data i n Table I I were as f o l l o w s : Column - L i c h r o s o r b RP-2, 5 ym p a r t i c l e diameter, 25 cm long χ 0.46 cm ID Mobile Phase - 40% methanol/60% 0.2 M sodium acetate b u f f e r , pH 4.7 Flow Rate - 1 mL/min Detectors • UV-LDC UV I I I @ 254 nm, 0.016 AUFS • E C - B i o a n a l y t i c a l Systems LC-2A, @ 1.0 v o l t v s . Ag/AgCl, equipped w i t h a g l a s s y carbon working electrode. The d e t e c t i o n l i m i t f o r each compound i s d e f i n e d as the q u a n t i t y which g i v e s a s i g n a l to noise r a t i o of 5. These data i l l u s t r a t e that a t a d e t e c t o r p o t e n t i a l of 1 V s e n s i t i v i t y f o r halogenated a n i l i n e s i s approximately 100-1000 X greater than f o r t h e i r n i t r o - s u b s t i t u t e d c o u n t e r p a r t s . The s e n s i t i v i t y f o r n i t r o - s u b s t i t u t e d a n i l i n e s can be increased by r a i s i n g the d e t e c t o r p o t e n t i a l , but only a t the expense of d e t e c t o r s e l e c t i v i t y . I t has been our experience that d e t e c t o r v o l t a g e s greater than 1.1-1.2 V are of l i t t l e value due to a h i g h background s i g n a l and n o n - s e l e c t i v i t y . In summary, the data given i n t h i s s e c t i o n show that HPLC/EC technique should be u s e f u l f o r the determination of aromatic diamines and halogenated a n i l i n e s but i s of l i t t l e value f o r determining n i t r o - s u b s t i t u t e d a n i l i n e s .



26.

All


TABLE I . PEAK POTENTIALS FOR AROMATIC AMINES

Compounds

Peak P o t e n t i a l (mV)

f

4 , 4 - M e t h y l e n e d i a n i l i n e (MDA) 1


4,4 -Methylenebis(2-chloroaniline)

830 (MBOCA)

870

Benzidine

550

3,3'-Dichlorobenzidine (DCB)

650

4-Methoxy-m-phenylenediamine (MMPDA)

370

Aniline

950

2- C h l o r o a n i l i n e

1000


1030


900

3,4-Dichloroaniline 2,4,5-Trichloroaniline

980 1000

4-Bromoaniline

900

2-N i t r o a n i l i n e

1200


1080


1180

2,4-Dinitroaniline

1400

2-Chloro-4-nitroaniline

1150


1180

2,6-Dichloro-4-nitroaniline

1380

2-Chloro-4,6-dinitroaniline

1380

2,6-Dibromo-4-nitroaniline

1180

2-Bromo-4,6-dinitroaniline

1330

2-Bromo-6-chloro-4-nitroaniline

1150


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TABLE I I . ESTIMATED HPLC DETECTION LIMITS FOR SELECTED ANILINES


Compound

Retention Time (min)

Est: Detection Limits (Nanograms I n j e c t e d ) EC UV 0.1

Aniline

5.2

2

4-Nitroaniline

7.0

2

3-Nitroaniline

7.0

0.5

5

2,4-Dinitroaniline

8.9

0.3

100

2-Chloroaniline

9.0

5

0.2

4-Chloroaniline

9.0

2

0.5

2-Nitroaniline

9.1

4

3-Chloroaniline

9.5

4

0.5

4-Bromoaniline

10.0

4

0.5

2-Chloro-4,6-dinitroaniline

10.8

5

100


11.5

5

100

2-Bromo-4,6-dinitroaniline

12.0

5

100


16.2

4

100

3,4-Dichloroaniline

16.5

4

2

2,6-Dichloro-4-nitroaniline

17.8

6

100

2,6-Dibromo-4-nitroaniline

23.0

8

100

2,4,5-Trichloroaniline

38.0

6

5

10

100


26.


Aromatic

Amines

in Body Fluids

419

Determination Of Benzidine And DCB I n Commercial Dyes


Two experiments were conducted t o study the u t i l i t y of HPLC i n determining aromatic amines i n commercial dyes. I n the f i r s t experiment a b e n z i d i n e based dye, D i r e c t Blue 6, and a commercial h a i r dye f o r m u l a t i o n c o n t a i n i n g D i r e c t Blue 6 were analyzed f o r b e n z i d i n e . I n the second experiment a DCB based pigment ( d i a r y l i d e yellow) was analyzed f o r r e s i d u a l DCB. Determination of Benzidine i n H a i r Dyes. Experiments were conducted to determine whether o r not b e n z i d i n e could be detected i n benzidine based dyes and/or h a i r dye products c o n t a i n i n g benzidine based dyes. The b e n z i d i n e based dye D i r e c t Blue 6 was obtained commercially as were two shades of a p a r t i c u l a r h a i r dye product. One shade, "Lucky Copper", contained D i r e c t Blue 6 whereas another shade, " S i l v e r L i n i n g " , d i d not c o n t a i n any benzidine based dyes. The h a i r dye f o r m u l a t i o n s were e x t r a c t e d u s i n g the f o l l o w i n g procedure: 1.

Adjust 100 pL of the h a i r dye formula t o pH 7 w i t h phosphate b u f f e r and add 200 mL o f d i s t i l l e d water. 2. E x t r a c t w i t h 2 χ 100 mL of chloroform. C e n t r i f u g e f o r 10 minutes a t approximately 1000 χ g t o separate the chloroform. 3. Wash the chloroform l a y e r s w i t h 40 mL o f d i s t i l l e d water. 4. Add 40 mL of methanol t o the chloroform and concentrate to 1 mL. 5. Add 10 mL of chloroform, wash w i t h 3 mL of water and then back e x t r a c t w i t h 2 mL o f 0.01 M HC1. 6. Assay e x t r a c t f o r benzidine by HPLC w i t h e l e c t r o c h e m i c a l detection. The HPLC c o n d i t i o n s used were as f o l l o w s : Column - L i c h r o s o r b RP-2, 5 \im p a r t i c l e diameter, 25 cm long χ 0.46 cm ID s t a i n l e s s s t e e l Mobile Phase - 50% a c e t o n i t r i l e / 5 0 % 0.2 M sodium a c e t a t e b u f f e r , pH 4.7 Flow Rate - 0.8 mL/min I n j e c t i o n Volume - 50 \ïL Detector - E l e c t r o c h e m i c a l (LC-2A B i o a n a l y t i c a l Systems) equipped w i t h t h i n - l a y e r g l a s s y carbon e l e c t r o d e operated a t 0.9 V. The D i r e c t Blue 6 was d i s s o l v e d i n pH 7 phosphate b u f f e r (0.5 gram i n 20 mL) and e x t r a c t e d i n a s i m i l a r manner.



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F i g u r e 4 shows the r e s u l t s f o r the D i r e c t Blue 6 when i t was e x t r a c t e d unspiked and a f t e r s p i k i n g w i t h 1 ppm (500 ng per 0.5 gram of dye). A peak f o r benzidine corresponding to appro ximately 400 ppb was noted i n the unspiked e x t r a c t e d (a process blank showed no response). Figure 5 shows the r e s u l t s f o r the h a i r dye f o r m u l a t i o n c o n t a i n i n g D i r e c t Blue 6 (Lucky Copper) and F i g u r e 6 shows the r e s u l t s f o r a second shade of the same product, not c o n t a i n i n g any benzidine based dyes. The Lucky Copper e x h i b i t s a response corresponding to approximately 2 ppb benzidine (Figure 5) whereas S i l v e r L i n i n g shows no such response (benzidine l e s s than 0.2 ppb). Recoveries ranged from 50-80% f o r the samples spiked before p r o c e s s i n g . The work i l l u s t r a t e s the a b i l i t y of HPLC/EC to detect t r a c e q u a n t i t i e s of benzidine i n h a i r dye products, although p o s i t i v e r e s u l t s should be v e r i f i e d by GC-MS or other s p e c t r o s c o p i c means to be c e r t a i n of the component i d e n t i t y . U n f o r t u n a t e l y , the s e n s i t i v i t i e s of such techniques are g e n e r a l l y much poorer than HPLC/EC, thus making absolute c o n f i r m a t i o n i m p r a c t i c a l . Determination Of R e s i d u a l DCB In D i a r y l i d e Yellow. D i a r y l i d e y e l l o w i s a w i d e l y used pigment d e r i v e d from DCB. The purpose of t h i s study was to determine r e s i d u a l DCB i n a l o t of the commercial pigment being used i n animal feeding experiments. The chromatographic c o n d i t i o n s used were the same as those given i n the previous s e c t i o n . The e x t r a c t i o n procedure was as f o l l o w s : F i v e grams of the dye was S o x h l e t - e x t r a c t e d w i t h 200 mL of methylene c h l o r i d e (MeCl2) f o r 48 hours. The MeCl2 e x t r a c t and 100 mL of 1M H2SO4 were combined i n a beaker and s t i r r e d f o r 10 minutes. The H2SO4 l a y e r was decanted and the MeCl2 f r a c t i o n e x t r a c t e d once again w i t h 100 mL of 1M H2SO4. A c i d f r a c t i o n s were combined and n e u t r a l i z e d to pH 7 w i t h 0.4 Μ Ν3βΡ04 and 1M NaOH i n an i c e bath. The n e u t r a l i z e d f r a c t i o n was e x t r a c t e d 2 times w i t h 50 mL CHC1- (preserved w i t h ethanol) and washed w i t h 15 mL of H2O. F i f t e e n m i l l i l i t e r s of methanol were added to the e x t r a c t and i t was concentrated to 5 mL on a r o t a r y evaporatory (room temperature), then to 0.2 mL w i t h a v o r t e x evaporator (40° C). The e x t r a c t was then d i l u t e d w i t h 0.5 mL w i t h 0.2 M sodium acetate b u f f e r , pH 4.7 and analyzed by HPLC. Chromatographic c o n d i t i o n s were the same as f o r the determination of benzidine i n h a i r dye f o r m u l a t i o n s . For the p a r t i c u l a r l o t of d i a r y l i d e y e l l o w s t u d i e d ^46 yg/kg of DCB was found. In an attempt to confirm the i d e n t i t y of the chromatographic peak, i t s response as w e l l as the response f o r the authentic DCB standard was determined at s e v e r a l d i f f e r e n t e l e c t r o d e p o t e n t i a l s . These data, shown i n F i g u r e 7, i l l u s t r a t e the a b i l i t y of HPLC/EC to y i e l d q u a l i t a t i v e as w e l l as q u a n t i t a t i v e i n f o r m a t i o n f o r unknown components.





All

V 5nA

A (Unspiked)

Β (Spiked with I ppm of Benzidine)

Figure 4. Chromatogram for Direct Blue 6 extract

CD

β

J

A (Unspiked)

5nA

Β (Spiked with 5 ppb of Benzidine)

Figure 5. Chromatogram for extract of commercial hair dye formulation contain ing Direct Blue 6



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5nA

Figure 6. Chromatogram for extract of commercial hair dye formulation not con taining benzidine-based dyes

Figure 7. Detector potential/response curve for (Φ) DCB and (X) component extracted from diarylide yellow (offset 1 cm for clarity)

A (Unspiked)

0.4

Β (Spiked with 2ppb of Benzidine)

0.5 0.6 0.7 0.8 0.9 Detector Potential, volts vs Ag/AgCl


1.0

26.



423

Determination Of Aromatic Amines In Urine In t h i s s e c t i o n we w i l l d i s c u s s p r e l i m i n a r y work done a t B a t t e l l e Columbus L a b o r a t o r i e s wherein HPLC/EC was used t o determine MMPDA, DCB, and MBOCA i n u r i n e . Other workers have demonstrated i t s usefulness f o r determining halogenated a n i l i n e s ( A ) a n d benzidine i n urine.


Determination of DCB i n Rat U r i n e . I n t h i s study a method f o r the determination of DCB i n r a t u r i n e was developed. The sample e x t r a c t i o n procedure employed was as f o l l o w s : 1. 2. 3. 4. 5. 6. 7. 8.

P l a c e 2 mL of u r i n e i n a 15 mL c e n t r i f u g e tube. Adjust the u r i n e t o pH 7 w i t h 0.1 M HC1 o r 0.1 M NaOH. E x t r a c t 3 times w i t h 2 mL of benzene, c e n t r i f u g i n g between each e x t r a c t i o n . Combine benzene f r a c t i o n s and wash w i t h 1 mL of d i s t i l l e d water ( d i s c a r d aqueous l a y e r ) . Add 1 mL of methanol t o the benzene e x t r a c t . Concentracte t o 0.5 mL on v o r t e x evaporator (40° C). Add 1 mL methanol and concentrate t o 0.2 mL. D i l u t e t o 0.5 mL w i t h 0.2 M sodium acetate b u f f e r , pH 4.7, mix w e l l , and analyze by HPLC.

The HPLC c o n d i t i o n s used were the same as described f o r the commercial h a i r dye experiments. F i g u r e 8 shows t y p i c a l chromatograms f o r spiked and unspiked r a t u r i n e . Recovery was found to be 50, 55, and 35% f o r three r e p l i c a t e samples spiked a t the 2 yg/L l e v e l , which we have found to be t y p i c a l recovery values f o r t r a c e l e v e l s of DCB i n aqueous media (e.g. i n d u s t r i a l wastewater). Determination Of MMPDA In Human U r i n e . MMPDA i s w i d e l y used i n permanent h a i r dye f o r m u l a t i o n s and has been shown t o be c a r c i n o g e n i c i n rodents . A q u e s t i o n e x i s t s as whether or not MMPDA can be absorbed through the s c a l p to enter the blood stream of persons u s i n g the dye. I n order t o answer t h i s q u e s t i o n a s e n s i t i v e , r e l i a b l e a n a l y t i c a l procedure f o r MMPDA i n u r i n e i s needed. For t h i s reason we decided t o i n v e s t i g a t e the usefulness of HPLC/EC i n t h i s problem. The e x t r a c t i o n procedure developed was as f o l l o w s : 1. 2. 3.

4.

Adjust 2 mL of u r i n e t o pH 7 w i t h 0.1 M HC1 o r 0.1 M NaOH. E x t r a c t twice w i t h 4 mL of MeCl2 i n a 15 mL screw capped c e n t r i f u g e tube. Combine the organic e x t r a c t s and wash w i t h 1 mL o f 0.01 M ammonium c i t r a t e , pH 6.9. D i s c a r d aqueous layer. E x t r a c t organic l a y e r w i t h 1 mL of 0.05 M HC1. Analyze


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IN T H E W O R K P L A C E

a c i d e x t r a c t by HPLC. The chromatographic c o n d i t i o n s were as f o l l o w s : Column - Dupont Zorbax ODS, 5 urn p a r t i c l e diameter, 25 cm long χ 0.46 cm ID Mobile Phase - 30% methanol/70% 0.5 M ammonium c i t r a t e b u f f e r , pH 5 Flow Rate - 0.7 mL/min Detector Voltage - 0.9 V.


F i g u r e 9 shows a chromatogram f o r human u r i n e spiked a t 50 yg/L. Recovery was found to be 58% a t t h i s spike l e v e l . Determination Of MBOCA i n Human U r i n e . MBOCA i s commer c i a l l y important as a c u r i n g agent f o r polyurethanes and epoxy r e s i n systems. Since MBOCA was found to be c a r c i n o g e n i c i n animals and i s a suspected human carcinogen'—' , i t i s important to have a r e l i a b l e method a v a i l a b l e f o r the d e t e r mination of MBOCA i n the u r i n e of those workers who a r e p o t e n t i a l l y exposed to t h i s compound. I n a NIOSH p u b l i c a t i o n ^ a GC method was described f o r the determination o f MBOCA i n u r i n e . However, s i n c e HPLC does not r e q u i r e d e r i v a t i z a t i o n and a lower d e t e c t i o n l i m i t was expected, the GC method was modified to be performed by HPLC/EC. I n order to be able to compare both methods, we used the same e x t r a c t i o n procedure. The e x t r a c t i o n and sample p r e p a r a t i o n procedures are as f o l l o w s : 1.

S t a b i l i z e u r i n e by adding 2 mL of an aqueous 30% c i t i c a c i d s o l u t i o n t o the b o t t l e before c o l l e c t i o n . 2. P l a c e 50 mL of s t a b l i z e d u r i n e i n a 125 mL separatory funnel and spike the u r i n e w i t h 1 mL of MBOCA standard s o l u t i o n i n methanol. S w i r l to mix and l e t stand 5 minutes. 3. Add 10 mL of e t h a n o l , shake t o mix and l e t stand 5 minutes. 4. Add 5 mL of an aqueous 10% sodium bicarbonate s o l u t i o n , shake to mix (vent CO2 through stopcock) and t e s t pH w i t h testpaper. I f not a l k a l i n e (pH 7.5-8) add stepwise a d d i t i o n a l 1 mL p o r t i o n s u n t i l a l k a l i n e . 5. Add 50 mL of d i e t h y l ether and shake u n i n t e r r u p t e d l y f o r two minutes, v e n t i n g p e r i o d i c a l l y . 6. L e t s o l u t i o n s stand f o r 5 minutes, d r a i n o f f the lower u r i n e l a y e r and d i s c a r d . 7. Add 5 mL of the 10% bicarbonate s o l u t i o n t o the separatory f u n n e l and shake f o r 10 seconds. 8. A f t e r 5 minutes d r a i n o f f the bottom l a y e r . 9. P l a c e the ether e x t r a c t i n a 100 mL round bottom f l a s k and evaporate t o approximately 5 mL on a r o t a r y evaporator a t ambient temperature.



26.


A (Unspiked)

Aromatic

Β (Spiked with 2ppb of 3,3'-Dfchforobenzidine)

1

Amines

in Body

Figure 8.

Fluids

425

Determination of DCB in rat urine

5nA Figure 9.

Determination of MMPDA in spiked human urine


426

CHEMICAL

10.

11.


12.

H A Z A R D S IN

THE

WORKPLACE

Transfer the 5 mL of e x t r a c t q u a n t i t a t i v e l y to a 15 mL c o n i c a l screw-capped c e n t r i f u g e tube. Rinse the f l a s k w i t h two p o r t i o n s of 2 mL of ether and combine w i t h the o r i g i n a l e x t r a c t . Evaporate the e x t r a c t to 0.2 mL using a g e n t l e stream of n i t r o g e n . Add mobile phase (see below) to 1 mL and analyze by HPLC.

The chromatographic c o n d i t i o n s were the same as f o r the determination of benzidine i n h a i r dye f o r m u l a t i o n s , except f o r the flow r a t e , which was 1 mL/min. F i g u r e 10 shows t y p i c a l chromatograms f o r spiked and unspiked u r i n e . P r e l i m i n a r y r e s u l t s i n d i c a t e that the d e t e c t i o n l i m i t f o r the HPLC/EC method w i l l probably be i n the order of 1-10 ppb. which i s s u b s t a n t i a l l y lower than the 40 ppb d e t e c t i o n l i m i t ( J ) f o r the GC method.

00 < ο ο m 2

50nA

Figure 10.

Determination of MBOCA in spiked human urine

A (Unspiked)

B (Spiked with 100 ppb of MBOCA)


26.

VAN T U L D E R E T A L .

Aromatic

Amines

in Body Fluids

Ml


LITERATURE CITED 1.

Kissinger, P. T., "Amperornetrie and Coulometric Detectors for High-Performance Liquid Chromatography", Anal. Chem., 1977, 49, 447A-456A.

2.

Johnson, D. C. and Larochelle, J., "Forced-Flow Liquid Chromatography with a Courlometric Detector", Talanta, 1973, 20, 959-971. Pergamon Press, Great Britian.

3.

Stillman, R. and Ma, T. S., "Application of High-Speed Liquid Chromatography to Organic Microanalysis II. Separa tion and Polarographic Detection of Pesticides, Vitamins, and Analgesics", Mikrochimica Acta [Wien], 1974, 641-648.

4.

Haley, T. J., "Benzidine Revisited: A Review of the Literature and Problems Associated with the Use of Benzidine and i t s Congeners", C l i n . Toxicol., 1975, 8 (1), 13-42.

5.

Price, J. Μ., "Benign and Malignant Tumors in the Urinary Bladder"; Medical Examination Publishing C o . , Inc. Flushing, New York, 1971, p. 264.

6.

"2,4-Diaminoanisole (4-Methoxy-m-Phenylenediamine) in Hair and Fur Dyes", DHEW (NIOSH) Publication No. 78-111, 1978.

7.

"Special Hazard Review with Control Recommendations for 4,4'-Methylenebis(2-chloroaniline)", DHEW (NIOSH) Publication No. 70-188, 1978.

8.

Lores, Ε. Μ., B r i s t o l , D. W., and Moseman, R. F., "Deter mination of Halogenated Anilines and Related Compounds by HPLC with Electrochemical and UV Detection", J. Chromatogr. S c i . , 1978, 16, 358-362.

9.

Rice, R. L., and Kissinger, P. T., "Determination of Benzidine and its Acetylated Metabolites in Urine by Liquid Chromatography", J. Anal. T o x i c o l . , 1979, 3, 64-66.

RECEIVED October 27,

1980.


Chemical Hazards in the Workplace - ACS Publications - American

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