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JOURNAL OF CHROMATOGRAPHY B:

BIOMEDICAL APPLICATIONS

ELSEVIER

Journal of Chromatography B, 682 (1996) 167 172

Short communication

Simultaneous determination of phenol, cresol, xylenol isomers and naphthols in urine by capillary gas chromatography
Grakyna Bieniek
IJepartment o/' Biochemistry and Biophysics, Silesian Medical Academy. Ja~ielhmska Str. 4. 41-200 Sosnouicc. PMoml
Received 31 October 1995: revised 13 February 1996: accepted 27 February 1996

Abstract

An attempt was made to establish a method for the simultaneous determination of urinary concentrations of phenol, o-, pand m-cresols, 1- and 2-naphthol and xylenol isomers by capillary gas chromatography. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The ten substances were separated gas chromatographically using a capillary column (Ultra 2) of cross-linked 5e/c phenylmethyl silicone. Calibration graphs were linear for 5 100 p,g/ml of all the phenols determined. The corresponding detection limits for phenolic compounds varied flom 0.1 to 0.2 ,ug/ml. The relative standard deviations for samples in urine were in the range 2.6-16.6% and the accuracy was in the range 1.4-25%. Recoveries were generally over 80%.
Kevwords: Phenol: Cresol: Xylenol: Naphthol

1. Introduction

In environmental analyses, capillary gas chromatography (GC) plays a major role in the detection of urinary metabolites of toluene [1], benzene [2] and ethylbenzene [3]. It is well known that the phenols are metabolites of benzene and its alkyl derivatives I4-61. Free and conjugated phenols are also present in blood and urine. The co-occurrence of the phenols and cresols has been determined in cigarette smoke and its condensate 171. In order to analyse phenol, cresols, xylenols and naphthalenols in urine, they must first be separated from the biological carrier. This is usually accomplished by heating a urine sample with a mineral acid or with enzymes [8,9]. The transfer of phenols from the aqueous hydrolysate to an organic solvent is accomplished by extraction with a volatile organic

solvent, such as diethyl ether [9 I II, dichMromethane 1121, methanol 1131 or acetonitrile 1141. Highly purified and concentrated isolates for chromatographic analysis can be achieved by selective extraction with appropriate sorbents to yield chromatograms with minimal interferences and improved sensitivity [15]. An efficient extraction method can improve the assay precision and accuracy. The reversed-phases based on octadecylsilane sorbent have been used extensively for the trace enrichment of organics from aqueous matrices in clinical and environmental analyses [14,16]. The GC analysis of phenols with the use of capillary columns was reported previously 112,17]. Determination of the concentration of urinary phenols has been used for biological monitoring of workers exposed to phenols and aromatic hydrocarbons [10J. Urinary excretion of 2,4-xylenol 118,19] and m-cresol [7,9,20-22] was used to

0378_4347/t~6/$15.00 ~c 1996 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 4 3 4 7 ( 9 6 ) 0 0 1 0 4 - 1

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G. Bieniek / J. Chromatogr. B 682 (1996) 167 172

Table 1 Solid-phase extraction of phenols using Baker SPE octadecyl (C~x) columns. Each result is the average of three repeated measurements. The concentration of each phenol was 50 /tg/ml Analyte Recovery ( % ) R.S.D. (%) 12% Salt added Recovery (%) Phenol o-Cresol p-Cresol 2,6-Xylenol 2,5-Xylenol 2,3-Xylenol 3,5-Xylenol 3,4-Xylenol I-Naphthol 2-Naphthol Average 84 83 97 95 87 96 94 82 77 74 87 4.8 4.4 2.5 2.3 5.2 3. I 3.5 4. I 5.9 7.1 4.8 75 77 87 84 79 86 83 74 68 71 78 R.S.D. (%) 6.7 5.4 3.6 2.2 6.6 4.6 4.3 3.2 7.0 8.7 5.2

monitor human exposure to organic solvents. The present method was applied to the determination of phenols in the breathing zone air and in the urine of coke plant workers [23]. In this paper, the analytical method of separation and its validity for the determination of phenols in urine, particularly the development of solid-phase extraction (SPE) procedures, is discussed.

columns (7020) were from J.T. Baker (Phillipsburg, NJ, USA). Distilled water was used in all analyses.
2.2. Standard solutions

2. E x p e r i m e n t a l
2.1. Reagents

Stock solutions of phenol, o-cresol, p-cresol, 2,3-, 2,5-, 2,6-, 3,5- and 3,4-xylenol isomers were dissolved in methanol at a concentration of 200 # g / m l . Then, they were diluted again to yield appropriate working solutions for the preparation of the calibration standards. The final concentrations of the phenolic compounds were approximately as follows: 5, 10, 20, 50 and 100 # g / m l . The solutions were kept at 4C until use.
2.3. Sample preparation

All chemicals were of reagent grade quality or better and were used as received without further purification. Phenol ( 9 9 + % ) , (CAS registry No. 108-95-2), o-cresol ( 9 9 + % ) (95-48-7), p-cresol (99+%) (106-44-5), 2,3-dimethylphenol (99%) (526-75-0), 2,5-dimethylphenol ( 9 9 + % ) (95-87-4), 2,6-dimethylphenol ( 9 9 . 8 + % ) (576-26-1), 3,4-dimethylphenol (99%) (95-65-8), 3,5-dimethylphenol ( 9 9 + % ) (108-68-9), 1-naphthol ( 9 9 + % ) (90-15-3) and 2-naphthol (98%) (135-19-3) were all obtained from Aldrich (Milwaukee, WI, USA). Concentrated hydrochloric acid was obtained from POCH (Gliwice, Poland). Acetonitrile and methanol, of gradient grade, were obtained from Riedel-de HaEn (Seelze, Germany). Bakerbond SPE octadecyl C~8

A 0.4-ml volume of concentrated hydrochloric acid was added to 1 ml of urine in a 10-ml glass tube and heated in a water bath at 95C for 90 min. After cooling to room temperature, the samples were loaded onto SPE columns.
2.4, Procedures f o r SPE

Standard samples were prepared by adding several phenols to acid-hydrolyzed urine so that the concentration of each of them was 10, 20, 50 and 100 # g / m l . The pH was adjusted to 1.8-2 with hydrochloric acid. In some cases sodium chloride was

(;. Bieniek / .I. Chromatogr. B 682 (19961 167 172

1(>9

2
|

6 3 4 S

10

3 2

Fig. 1. ('hromatogramsof (a) the urine of coke plant workers exposed to phenoliccompoundsand aromatic hydrocarbonsand ~b) a n ;.lqucotl~, standard of 2,(~-xylenol (10 #g/ml). 3,4-xylenol (10 #g/flail and other phenols (5 #g/ml). Peaks: I=phem~l: 2 :~,-crcsol: 3 /~-ct'csol: 4 =2.6 xylenol: 5=2,5-xylenol: (~: 2,3-xylenol:7=3,5-xylenol: 8:3.4-xylenol: 9 I-naphlhol alld 10::2-naphthol.

added to a concentration of 12% (w/v). A SPE column was conditioned by pumping 5 ml of methanol through the column, followed by 8 ml of distilled water. After washing, the aqueous sample was administered at a flow-rate of 1 m l / m i n by applying a pressure of 1.3-1.5 kPa. The column was washed again with 5 ml of distilled water and gently aspirated. The phenols were eluted with 1 ml of acetonitrile-methanol ( l : 1 0 v / v ) . The eluate was

then collected in a 1.8-ml GC vial. The vial was capped and kept at 0C before being analysed, to avoid evaporation. A I-#l aliquot of sample was injected for GC.
2.5. Chromctlographic conditions

The chromatographic system consisted of a Hewlett-Packard HP Model 5890 11 series with a flame-

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G. Bieniek / J. Chromatogr. B 682 (1996) 167-172


was

ionization detector (FID). A gas chromatograph and a HP 3396

2.6, G C conditions
Capillary phenylmethyl #m film column, Ultra 2 (cross-linked mm 5%

equipped with an HP 7673 autosampler/autoinjector II s e r i e s i n t e g r a t o r w a s u s e d , T h e using a phenols were separated chromatographically c a p i l l a r y c o l u m n , U l t r a 2. s i l i c o n e ) , 25 (HP m0.32 Part No. I.D., 0 . 5 2

thickness

19091B-112);

Table 2 Precision and accuracy of the assay of phenolic compounds Compound Concentration added ( # g / m l ) 10 20 50 100 10 20 50 100 I0 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 I0 20 50 100 Concentration (mean-+S.D.) ( # g / m l ) 8.31 +0.59 16.23 + I.I 2 42.62-+2.64 86.31 +-4.31 8.88-+0.43 17.87-+0.97 48.32 -+2.28 96.76-+2.63 8.26 + 0.79 17.80+0.83 41.02 + 2.26 87.82 + 3.05 8.87+0.80 18.64 + 1.29 47.52 -+2.81 98.58 +4.02 8.46 + 0.61 17.44-+0.92 42.25-+2.41 86.17-+2.23 9.49+0.44 19.05-+ 1.09 45.77-+ 1.98 94.03 + 3.29 8.56-+ 1.34 16.88-+ 1.22 42.66+-2.73 86.28 -+4.26 9.42-+0.67 20.04 + I. I 1 4 7 . 8 2+ - 1.31 95.73 + 4.06 7.82-+0.69 15.03 + 1.26 39.73-+2.85 82.26-+ 3.03 7.70-+ 1.28 15.14 + 1.87 37.40-+3.05 81.44+5.03 R.S.D." (%) 7.1 6.9 6.2 5.0 5.0 5.4 4.6 2.8 9.6 4.7 5.5 3.5 9.0 6.9 5.9 4. I 7.2 5.2 5.7 2.6 4.6 5.7 4.3 3.5 15.6 7.2 6.4 4.9 6.8 5.5 2.7 4.2 8.8 8.4 7.2 3.7 16.6 12.4 8.2 6.2 Accuracy h (~) - 16.9 - 18.8 14.8 13.7 - l 1.2 10.6 - 3.4 -3.2 17.4 11.0 18.0 12.2 - 11.3 -8.0 - 4.9 - 1.4 - 15.4 - 12.8 15.5 13.8 -5. I -4.7 -8.5 6.0 - 14.4 - 15.6 - 14.7 13.7 -6.8 0.2 -4.4 4.2 --21.8 24.8 -20.5 17.7 -23.3 24.3 25.2 - 18.6 Recovery (%) 83 81 85 86 88 89 96 96 83 89 82 87 88 93 95 98 84 87 84 86 94 95 9I 94 86 84 85 86 94 100 95 95 78 75 79 82 77 76 74 81

Phenol

p-Cresol

o-Cresol

2,3-Xylenol

2,5-Xylenol

2,6-Xylenol

3,4-Xylenol

3,5-Xylenol

I-Naphthol

2-Naphthol

Analyses were carried out under the experimental conditions described in Section 2. " Relative standard deviation. b Defined as the percentage deviation between the average concentration obtained from the experiment and the theoretical concentration.

G. Bieniek / .I. Chromatogr. B 682 (1996) 107 172

171

injector temperature, 270C; detector temperature, 280C: oven temperature, 50C for 1 min, then increased by 5C/min to 90C, by 2C/min up to 104C and then by 10C/min to 250C; final temperature, 250C for 1 rain: carrier gas, helium at a flow-rate of 2.5 ml/min; injection volume, 1 p,h splitless time, I rain: split ratio, 1:30.

3. Results and discussion


(',s cohmms were chosen fl)r reversed-phase chromatography with non-polar bonded sorbents, to extract the phenols. Earlier investigations revealed that higher recoveries were obtained by adding salt to the aqueous samples [14,24]. In this particular case, addition of sodium chloride resulted in recoveries thai were about 9% lower than those with no salt (Table I). The results obtained in these investigations are in excellent agreement with the previously published data by Schmidt et al. [131. Separation of all phenols was achieved using the Ultra 2 column. Representative chromatograms are shown in Fig. I. Chromatogram (a) was obtained by analysis of the hydrolysed urine sample (1 ml) collected from the workers exposed to phenolic compounds and aromatic hydrocarbons. Chromatogram (b) presents results of the analysis of an aqueous solution. The peaks of interest were well separated from potential interferences. Fig. l a shows a typical gas chromatogranl of the phenols isolated fl'om an acetonitrile concentrate of hydrolysed urine. Phenol, cresols and xylenol isomers were the main

metabolites in the urine of workers employed in the distillation of the carbolic oil [6,23]. The precisitm and accuracy were evaluated by using the samples spiked at concentrations of 10, 20, 50 and 100 # g / m l . The accuracy was expressed as the percentage deviation between the mean concentration value of the six samples and the theoretical concentration. The samples were extracted and subjected to GC analysis. Each concentration was calculated on the basis of peak areas with respect to the calibration graphs. The integrator programme (external standard) was employed. The precision and accuracy of the proposed method are shown in Table 2. A linear relationship was found between the peak areas and the concentrations of phenols lk~r each measurement consisting of six samples spiked at levels of 5, 10. 20, 50 and 100 # g / m l . The parameters of the calibration graphs are given m Table 3. The detection limit was defined as the lowest concentration of each of the phenolic compounds resuiting f r o m a signal-to-noise ratio t)l 3. In our case, the detection limits lot phenolic compounds ~aried from 0.1 to 0.2 # g / m l . The temperature application programme presented in this paper can be used in the deternfination of phenols in the extracts of either urine samples or aqueous solutions as well as during the analysis of air 1231.

4. Conclusions
An assay involving solid-phase extraction with non-polar bonded octadecyl (C3s) sorbents and simultaneous determination of phenols by GC analysis was described. The proposed method may be useful for environmental and toxicoh)gical stndies of phenols as well as of aqueous solutions and air.

Table 3 Equations o f linear calibration graphs Compound Phenol o-Cresol p-Cresol 2,(~ Xylcnol 2,5-Xylenol 2,3 Xylcnol 3,5 Xylenol 3,4-Xyleno[ I -Naphlhol 2 Naphlhol
' v

Equation" v v v v v \' v v vv1653.55x 3 6 2 . 8 3 + 1260.56.'~ 1869.01.~ 418.95 4- 1566.86x 1642.19v 1810.17x 170.91 filSI4.5Z,~ 1660.04x 1753.04x 265(1.03+ 1464.97x

re 0.}94 0.992 0.')94 0.992 0.995 0.994 0.995 0.992 0.994 0.993

Acknowledgments
I am grateful to Professor T. Wilczok for general supervision of the experimental work. 1 would like to thank the Managing Board of the Zabrze Cokery Plant for their kind permission to continue my research. 1 am grateful to L. Swi~ttkowska for her help with statistical analyses.

peak area (arbitrary units): x = c o n c e n t r a t i o n (mg/I).

172

G. Bieniek

J. Chromatogr, B 682 (1996) 167-172 [12] P. Stommel, G. Mi.iller, W. Stiicker, C. Verkoyen, S. Sch6bel and K. Norpoth, Carcinogenesis, I0 (1989) 279. [13] L. Schmidt, J. Sun and J.S. Fritz, J. Chromatogr., 641 (1993) 57. [14] B. R6ssner and G. Schwedt, Fresenius' Z. Anal. Chem., 315 (1983) 610. [15] K.C. Van Home (Editor), Sorbent Extraction Technology, Analytichem International, Harbor City, CA, 1985, p. 110. [16] K.G. Furton and J. Rein, Anal. Chim. Acta, 236 (1990) 99. [171 Y. Hoshika and N. Murayama, Analyst, 108 (1983) 984. [18] V. Riihim/iki, K. Savolainen, P. Pffiffli, K. Pekari, H.W. Sippel and A. Laine, Arch. Toxicol., 49 (1982) 253. [19] M. Yashiki, T. Kojima, T. Miyazaki, F. Chikasue and M. Ohtani, Forensic. Sci. Int., 47 (1990) 21. [20] W. Woiwode and K. Drysch, Br. J. Ind. Med., 38 (1981) 194. [21] J. Meulenbelt, G. de-Groot and T.J. Savelkoul, Br. J. Ind. Med., 47 (1990) 417. [22] A. Brega, E Prandini, C. Amaglio and E. Pafumi, J. Chromatogr., 535 (1990) 31 I. [23] G. Bieniek, Am. J. Ind. Med., submitted for publication. [24] C.G. Markell and D.F. Hagen, Proceedings of the 7th Annual Waste Testing and Quality Assurance Symposium, Washington, DC, July 8-12, 1991,Vnl. II, EPA, Washington, DC, p. 27.

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