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1855
ANALYST, DECEMBER 1992, VOL. 117
Determination of Thiocyanate in Human Saliva and Urine by Ion Chromatography
Published on 01 January 1992. Downloaded by University of Michigan Library on 29/10/2014 13:03:35.
Yoshimasa Michigami, Kanae Fujii, Kazumasa Ueda and Yoshikazu Yamamoto Department of Chemistry and Chemical Engineering, Faculty of Technology, Kanazawa University, 2-40 Kodatsuno, Kanazawa, 920, Japan
A simple ion-chromatographic method has been developed for the determination of trace amounts of thiocyanate in human saliva and urine. Thiocyanate separation and detection were carried out on an ODs column coated with cetyldimethylamine and by an ultraviolet detector, respectively. Citrate solution (1 mmol l-1) was used as the mobile phase. Thiocyanate was clearly separated from many organic and inorganic anions found in saliva and urine samples. The analytical results obtained by the proposed method agreed with those of the FeS+-thiocyanate spectrophotometric method. Thiocyanate concentrations in the saliva and urine of smokers were found t o be significantly higher than those of non-smokers. Keywords: Thiocyanate; urine; saliva; ion chromatography; cetyldimethylamine-coated column
Thiocyanate is usually present in low concentrations in biological fluids (e.g., serum, saliva and urine). High concentrations of thiocyanate, which is a major metabolite of cyanide, in biological fluids arise from tobacco smoke. The harmfulness of tobacco smoke is well known, and thiocyanate concentrations in the biological fluids of smokers are several times higher than those of non-smokers. Many methods have been reported for the determination of thiocyanate in biological samples. These are the spectrophotometric methods based on the Konig reaction172 and on the reaction with iron(iii) ,3,4 continuous-flow analyses based on the Konig and iron(n1) reaction,5 on use of iron(ni)-chloramine-Ts and of 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol and dichromate,7 gas chromatography,8>9high-performance liquid chromatography involving detection of the red complex formed with iron(iii)10 and fluorescence detection based on the Konig reaction" or a redox reaction with cerium(iv),l2 atomic absorption spectrometry based on use of the thiourea-copper complex13 and linear-sweep polarography.14 Many of these methods are laborious to perform and involve use of harmful reagents. Whereas a few papers15 have been published on the determination of thiocyanate in biological samples by ion chromatography, very few reports have appeared concerning thiocyanate in urine samples. Thiocyanate ions tend to be strongly retained on the column in most instances because of the hydrophobic interaction between the resin and thiocyanate ion in ion chromatography with hydrophobic ion-exchange resin,16 and the determination of thiocyanate in urine by ion chromatography with a hydrophilic resin suffers from interference from organic anions in urine. However, these impediments were overcome by using an ODS column coated with cetyldimethylamine. Although most inorganic and organic anions had weak retention on the column, thiocyanate was retained by an ion-exchange reaction. Thiocyanate was thus separated from other anions on the chromatogram and thereby determined. This paper describes a simple and rapid method for the determination of thiocyanate in urine and saliva by ion chromatography with use of an ODS column coated with cetyldimethylamine.
Tosoh) and a pen recorder (YEW Type 3066, Yokogawa, Tokyo, Japan). The operating conditions are listed in Table 1. All the chemicals were of analytical-reagent grade, and the solutions used were prepared with distilled, de-ionized water. Standard thiocyanate solution (1 mg ml-1) was prepared from potassium thiocyanate and was standardized by titration with silver nitrate solution. The eluent of 1 mmol 1-1 citrate was adjusted to pH 7.0 with dilute sodium hydroxide solution and filtered through a 0.45 pm membrane filter before use. Dynamically coated columns were prepared from columns (50 x 4.6 mm i.d.) packed with ODS resin (Capcell Pack C18, AG120, 5 ym particle size, Shiseido, Tokyo, Japan). The coating procedure was similar to that used by Duval and Fritz17 and Mullins.18 The column was coated with about 70 ml of 0.01 mol 1-1 cetyldimethylamine in 0.001 mol 1-1 hydrochloric acid containing 10% methanol at a flow rate of 0.5 ml min-1 and then conditioned with the eluent before testing. The ion-exchange capacity of the coated column was about 0.04 mequiv per column and the number of theoretical plates in the column was >lo00 for thiocyanate. The coated column allowed the analysis of ab0u.t 60 samples before the peak tailing was observed.
Procedure
Centrifuge a'fresh urine or saliva sample diluted five times with distilled, de-ionized water and transfer 1 or 2 ml, respectively, of the supernatant phase into a 10 ml calibrated flask. Dilute to volume with distilled, de-ionized water and inject 100 yl of the diluted solution onto the column through a 0.45 pm membrane filter. Repeat each measurement three times. Measure the peak height of thiocyanate on the chromatogram and calculate the concentration of thiocyanate by use of a calibration graph that is constructed daily.
Table 1 Operating conditions
Experimental Apparatus and Reagents The ion-chromatographic equipment consisted of a pump (CCPD, Tosoh, Tokyo, Japan), a variable-wavelength ultraviolet detector (UV-8011or UV-SOOO,Tosoh), an injector (Rheodyne, Cotati, CA, USA), a column oven (CO-SOOO,
Column Column temperature Detection wavelength Flow rate Sample loop Eluent
ODS column dynamically coated with cetyldimeth ylamine 35 "C 210 nm 1.0 ml min-1 loo pl 1mmol l-1 citrate (pH 7.0)
View Article Online
1856
ANALYST, DECEMBER 1992, VOL. 117
20
-
15
-
.-C
E
\
.-E 4-
C
.-
CI
a
Published on 01 January 1992. Downloaded by University of Michigan Library on 29/10/2014 13:03:35.
c1
8 0
I
I
1.o
2.0
-
51
Eluent concentration/mmol 1-1
Fig. 1 Effect of eluent concentration. Other chromatographic conditions as in Table 1
1.1
10
I
1
6.0
7.0
I
8.0 Eluent pH
I
I
9.0
Fig. 3 Effect of eluent pH. Other chromatographic conditions as in Table 1
1 01
-3.0 -2.7 Log ([cit.rate]/mol1-1)
-3.3
Fig. 2 Graph of log k‘ against log([citrate]/mol matographic conditions as in Table 1
1-1).
Other chro-
Results and Discussion Choice of Coating Reagent The ODS columns coated with cetylamine, cetyldimethylamine, cetyltrimethylammonium bromide or cetylethyldimethylammonium bromide were examined with respect to their retention times of thiocyanate. The columns coated with quaternary ammonium salts, such as cetyltrimethylammonium bromide and cetylethyldimethylammonium bromide , strongly retained thiocyanate and so the elution of thiocyanate was difficult. However, weak retention of thiocyanate was observed for the column coated with cetylamine and favourable retention of thiocyanate was found for the column coated with cetyldimethylamine. Effect of Citrate Concentration The effect of the citrate concentration on the retention time of thiocyanate was examined in the concentration range 0.051.50 mmol 1-1 at pH 7.0. Fig. 1shows the results obtained. The retention time of thiocyanate decreased with increasing citrate concentration, decreasing very gradually at more than 0.8 mmol 1-1 citrate, while the peak for thiocyanate became sharper with increasing citrate concentration. An eluent citrate concentration of 1 mmol 1-1 afforded good repeatability. A basic equation,4 eqn. (l),has been published for ion chromatography in terms of the capacity factor (k‘) and the concentration of the eluent ion (E), where all other chromatographic conditions are constant except for the eluent concentration: log k’ = -a/b log ( E ) + constant
(1)
I
I
I
200
220
240
I
Wavelengthlnm
Fig. 4 Effect of detection wavelength. Other chromatographic conditions as in Table 1
where a and b are the charges on the sample ion and the eluent ion, respectively. Fig. 2 shows the plot of log k’ versus log (citrate concentration); the slope was 0.30. The charges on the citrate and thiocyanate ions under the chromatographic conditions described above are about 3 and 1, respectively. The theoretical slope (-alb) was then 0.33, which agrees approximately with the experimental value (0.30). Other non-coated ODS columns did not retain thiocyanate. Therefore, thiocyanate was mainly retained on the column by the ion-exchange reaction; little effect of the hydrophobic interaction was observed with this system. Effect of Eluent pH The effect of eluent pH on the retention time of thiocyanate was examined in the pH range 5.0-9.0 The results obtained are shown in Fig. 3. The retention time of thiocyanate decreased with increasing eluent pH. An eluent pH of 7.0 was used, showing good repeatability and sensitivity for thiocyanate. Detection Wavelength The relative absorbances of thiocyanate (10 pg ml-1) in the wavelength range 200-240 nm are shown in Fig. 4. The relative absorbances of thiocyanate were almost constant in the wavelength range 205-220 nm. Hence, 210 nm was used as an optimum detection wavelength. Retention Times of Various Anions The retention times of various inorganic and organic anions were examined, and these are reported in Table 2. The retention times of all the anions studied were shorter than that
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1857
ANALYST, DECEMBER 1992, VOL. 117
Published on 01 January 1992. Downloaded by University of Michigan Library on 29/10/2014 13:03:35.
Table 2 Retention times of various anions Anion Acetate Ascorbate Aspartate Bromide Chlorate Chloride Fluoride Formate Glutamate Guanidoacetate Hippurate Iodide * Negative peak.
Retention time/min 1.7* 3.4 4.0 1.9 3.4*
1855
ANALYST, DECEMBER 1992, VOL. 117
Determination of Thiocyanate in Human Saliva and Urine by Ion Chromatography
Published on 01 January 1992. Downloaded by University of Michigan Library on 29/10/2014 13:03:35.
Yoshimasa Michigami, Kanae Fujii, Kazumasa Ueda and Yoshikazu Yamamoto Department of Chemistry and Chemical Engineering, Faculty of Technology, Kanazawa University, 2-40 Kodatsuno, Kanazawa, 920, Japan
A simple ion-chromatographic method has been developed for the determination of trace amounts of thiocyanate in human saliva and urine. Thiocyanate separation and detection were carried out on an ODs column coated with cetyldimethylamine and by an ultraviolet detector, respectively. Citrate solution (1 mmol l-1) was used as the mobile phase. Thiocyanate was clearly separated from many organic and inorganic anions found in saliva and urine samples. The analytical results obtained by the proposed method agreed with those of the FeS+-thiocyanate spectrophotometric method. Thiocyanate concentrations in the saliva and urine of smokers were found t o be significantly higher than those of non-smokers. Keywords: Thiocyanate; urine; saliva; ion chromatography; cetyldimethylamine-coated column
Thiocyanate is usually present in low concentrations in biological fluids (e.g., serum, saliva and urine). High concentrations of thiocyanate, which is a major metabolite of cyanide, in biological fluids arise from tobacco smoke. The harmfulness of tobacco smoke is well known, and thiocyanate concentrations in the biological fluids of smokers are several times higher than those of non-smokers. Many methods have been reported for the determination of thiocyanate in biological samples. These are the spectrophotometric methods based on the Konig reaction172 and on the reaction with iron(iii) ,3,4 continuous-flow analyses based on the Konig and iron(n1) reaction,5 on use of iron(ni)-chloramine-Ts and of 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol and dichromate,7 gas chromatography,8>9high-performance liquid chromatography involving detection of the red complex formed with iron(iii)10 and fluorescence detection based on the Konig reaction" or a redox reaction with cerium(iv),l2 atomic absorption spectrometry based on use of the thiourea-copper complex13 and linear-sweep polarography.14 Many of these methods are laborious to perform and involve use of harmful reagents. Whereas a few papers15 have been published on the determination of thiocyanate in biological samples by ion chromatography, very few reports have appeared concerning thiocyanate in urine samples. Thiocyanate ions tend to be strongly retained on the column in most instances because of the hydrophobic interaction between the resin and thiocyanate ion in ion chromatography with hydrophobic ion-exchange resin,16 and the determination of thiocyanate in urine by ion chromatography with a hydrophilic resin suffers from interference from organic anions in urine. However, these impediments were overcome by using an ODS column coated with cetyldimethylamine. Although most inorganic and organic anions had weak retention on the column, thiocyanate was retained by an ion-exchange reaction. Thiocyanate was thus separated from other anions on the chromatogram and thereby determined. This paper describes a simple and rapid method for the determination of thiocyanate in urine and saliva by ion chromatography with use of an ODS column coated with cetyldimethylamine.
Tosoh) and a pen recorder (YEW Type 3066, Yokogawa, Tokyo, Japan). The operating conditions are listed in Table 1. All the chemicals were of analytical-reagent grade, and the solutions used were prepared with distilled, de-ionized water. Standard thiocyanate solution (1 mg ml-1) was prepared from potassium thiocyanate and was standardized by titration with silver nitrate solution. The eluent of 1 mmol 1-1 citrate was adjusted to pH 7.0 with dilute sodium hydroxide solution and filtered through a 0.45 pm membrane filter before use. Dynamically coated columns were prepared from columns (50 x 4.6 mm i.d.) packed with ODS resin (Capcell Pack C18, AG120, 5 ym particle size, Shiseido, Tokyo, Japan). The coating procedure was similar to that used by Duval and Fritz17 and Mullins.18 The column was coated with about 70 ml of 0.01 mol 1-1 cetyldimethylamine in 0.001 mol 1-1 hydrochloric acid containing 10% methanol at a flow rate of 0.5 ml min-1 and then conditioned with the eluent before testing. The ion-exchange capacity of the coated column was about 0.04 mequiv per column and the number of theoretical plates in the column was >lo00 for thiocyanate. The coated column allowed the analysis of ab0u.t 60 samples before the peak tailing was observed.
Procedure
Centrifuge a'fresh urine or saliva sample diluted five times with distilled, de-ionized water and transfer 1 or 2 ml, respectively, of the supernatant phase into a 10 ml calibrated flask. Dilute to volume with distilled, de-ionized water and inject 100 yl of the diluted solution onto the column through a 0.45 pm membrane filter. Repeat each measurement three times. Measure the peak height of thiocyanate on the chromatogram and calculate the concentration of thiocyanate by use of a calibration graph that is constructed daily.
Table 1 Operating conditions
Experimental Apparatus and Reagents The ion-chromatographic equipment consisted of a pump (CCPD, Tosoh, Tokyo, Japan), a variable-wavelength ultraviolet detector (UV-8011or UV-SOOO,Tosoh), an injector (Rheodyne, Cotati, CA, USA), a column oven (CO-SOOO,
Column Column temperature Detection wavelength Flow rate Sample loop Eluent
ODS column dynamically coated with cetyldimeth ylamine 35 "C 210 nm 1.0 ml min-1 loo pl 1mmol l-1 citrate (pH 7.0)
View Article Online
1856
ANALYST, DECEMBER 1992, VOL. 117
20
-
15
-
.-C
E
\
.-E 4-
C
.-
CI
a
Published on 01 January 1992. Downloaded by University of Michigan Library on 29/10/2014 13:03:35.
c1
8 0
I
I
1.o
2.0
-
51
Eluent concentration/mmol 1-1
Fig. 1 Effect of eluent concentration. Other chromatographic conditions as in Table 1
1.1
10
I
1
6.0
7.0
I
8.0 Eluent pH
I
I
9.0
Fig. 3 Effect of eluent pH. Other chromatographic conditions as in Table 1
1 01
-3.0 -2.7 Log ([cit.rate]/mol1-1)
-3.3
Fig. 2 Graph of log k‘ against log([citrate]/mol matographic conditions as in Table 1
1-1).
Other chro-
Results and Discussion Choice of Coating Reagent The ODS columns coated with cetylamine, cetyldimethylamine, cetyltrimethylammonium bromide or cetylethyldimethylammonium bromide were examined with respect to their retention times of thiocyanate. The columns coated with quaternary ammonium salts, such as cetyltrimethylammonium bromide and cetylethyldimethylammonium bromide , strongly retained thiocyanate and so the elution of thiocyanate was difficult. However, weak retention of thiocyanate was observed for the column coated with cetylamine and favourable retention of thiocyanate was found for the column coated with cetyldimethylamine. Effect of Citrate Concentration The effect of the citrate concentration on the retention time of thiocyanate was examined in the concentration range 0.051.50 mmol 1-1 at pH 7.0. Fig. 1shows the results obtained. The retention time of thiocyanate decreased with increasing citrate concentration, decreasing very gradually at more than 0.8 mmol 1-1 citrate, while the peak for thiocyanate became sharper with increasing citrate concentration. An eluent citrate concentration of 1 mmol 1-1 afforded good repeatability. A basic equation,4 eqn. (l),has been published for ion chromatography in terms of the capacity factor (k‘) and the concentration of the eluent ion (E), where all other chromatographic conditions are constant except for the eluent concentration: log k’ = -a/b log ( E ) + constant
(1)
I
I
I
200
220
240
I
Wavelengthlnm
Fig. 4 Effect of detection wavelength. Other chromatographic conditions as in Table 1
where a and b are the charges on the sample ion and the eluent ion, respectively. Fig. 2 shows the plot of log k’ versus log (citrate concentration); the slope was 0.30. The charges on the citrate and thiocyanate ions under the chromatographic conditions described above are about 3 and 1, respectively. The theoretical slope (-alb) was then 0.33, which agrees approximately with the experimental value (0.30). Other non-coated ODS columns did not retain thiocyanate. Therefore, thiocyanate was mainly retained on the column by the ion-exchange reaction; little effect of the hydrophobic interaction was observed with this system. Effect of Eluent pH The effect of eluent pH on the retention time of thiocyanate was examined in the pH range 5.0-9.0 The results obtained are shown in Fig. 3. The retention time of thiocyanate decreased with increasing eluent pH. An eluent pH of 7.0 was used, showing good repeatability and sensitivity for thiocyanate. Detection Wavelength The relative absorbances of thiocyanate (10 pg ml-1) in the wavelength range 200-240 nm are shown in Fig. 4. The relative absorbances of thiocyanate were almost constant in the wavelength range 205-220 nm. Hence, 210 nm was used as an optimum detection wavelength. Retention Times of Various Anions The retention times of various inorganic and organic anions were examined, and these are reported in Table 2. The retention times of all the anions studied were shorter than that
View Article Online
1857
ANALYST, DECEMBER 1992, VOL. 117
Published on 01 January 1992. Downloaded by University of Michigan Library on 29/10/2014 13:03:35.
Table 2 Retention times of various anions Anion Acetate Ascorbate Aspartate Bromide Chlorate Chloride Fluoride Formate Glutamate Guanidoacetate Hippurate Iodide * Negative peak.
Retention time/min 1.7* 3.4 4.0 1.9 3.4*
