Anal. Chem. 1989, 61 233-236 I
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High-Performance Liquid Chromatography-Fluorometric Determination of Selenium Based on Selenotrisulfide Formation Reaction Terumichi Nakagawa,* Eiji Aoyama, Noriko Hasegawa, Nobuhiro Kobayashi, and Hisashi Tanaka Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto-shi, 606 Japan
A new hlgh-performance llquld chromatography-fluorometrlc method has been developed for selectlve determlnatlon of selenium( IV). The method Involves precolumn reaction of selenium( I V ) wlth penlclllamlne (Pen) to produce stable selenotrlsutflde (Pen-SSeS-Pen) and subsequent derlvatlzatlon to a fluorophore by reactlon wlth 7-fluoro-4-nltrobenz2,1,3-oxadlazole. The fluorophore was separated by reversed-phase HPLC and selenium content was determlned by fluorometrlc detectlon. The callbratlon plots showed a llnear relatlonshlp In the range of 10-2000 ppb of selenium( I V ) wlth a detectlon llmlt of 5 ppb (slgnal to noise ratio ( S I N ) > 2). The method could determlne total content of selenium In blologlcal and envlronmental samples after dlgestlon of the samples and reduction of selenlum(V1) to selenium( IV). The results from standard samples Indicated satlsfactory agreement wlth those obtalned by other establlshed methods and certtfled values wlth good reproduclblllty. Thls method Is as sensltlve as, but simpler In Operation than, conventlonal fluorometry uslng dlamlnonaphthalene.
INTRODUCTION Selenium is an element known to have both essential ( I ) and toxic activities in man. The dietary deficiency and excess intake of selenium are known to associate with various disorders in man such as Keshan disease and selenosis. The essentiality of selenium has been recognized by the fact that the element is involved at the active site of glutathione peroxidase. Recent studies (2-4) have suggested that selenium plays a preventive role in the etiology of cancer. One of difficulties in exploring these versatile activities of selenium arises from a relatively narrow gap between essential and toxic levels in addition to its low concentration in living things and in the environment. Therefore, the biological and environmental investigations of selenium demand a sensitive and reliable m e t h d for the determination of low levels of selenium. Fluorometry (5, 6), atomic absorption spectrometry (7,€9, inductively coupled plasma emission spectrometry (9),neutron activation analysis (IO),and gas chromatography (11) have so far been appreciated for this purpose. The fluorometric method based on the formation of piazselenol from selenium(1V) and 2,3-diaminonaphthalene (DAN) (5) has been widely used owing to its low detection limit. However it needs complicated procedures to cleanup the reagent and to extract the fluorophore, and further purification of the fluorophore was needed for precise determination of selenium in biological sample (6, 12). It has been known that selenium(IV) reacts selectively with various thiols in accord with the following equation to form selenotrisulfides (STS) involving -S-Se-S- linkage (13),which is generally unstable: 4RSH H2Se03 RSSeSR RSSR + 3H20 In the preceding papers (14, 15), we reported that selenotrisulfide formed from penicillamine (Pen) was exceptionally stable and that the reaction proceeded in acid solution where
+
-
+
most of metal ions do not form chelates with penicillamine. These findings prompted us to apply the reaction to the selective determination of selenium(1V). Although UV absorption of penicillamine selenotrisulfide (PenSTS) enabled a parts-per-million level of selenium to be determined, the limit of detection could be lowered to a parts-per-billion level by conversion of PenSTS to a fluorophore. Thus, to develop a new method that allows assay of a low level of selenium with easy operation, this study aims to find the optimum reaction conditions for the quantitative formation of PenSTS and subsequent derivatization of PenSTS to a fluorophore using 7-fluoro-4-nitrobenz-2,1,3-oxadiazole (NBD-F), a labeling reagent for amino groups (16,17). The HPLC conditions for separation and detection of the fluorophore were also investigated. The applicability of the method is demonstrated by comparing the results for contents of selenium in standard samples with the certified values and those obtained by other established methods. The recovery of a known amount of selenium(1V) spiked to digested solution of standard sample was obtained to investigate the interferences by coexisting substances in the sample.
EXPERIMENTAL SECTION Reagents. Penicillamine selenotrisulfide (PenSTS) used as standard was synthesized from Ppenicillamine and selenious acid according to the method reported previously (14). 7-Fluoro-4nitrobenz-2,1,3-oxadiazole(NBD-F) was purchased from Dojin Laboratories Co. (Kumamoto,Japan). D-Penicillamine, selenious acid, and other reagents were commercial products of analytical grade. Ethanolic solution of NBD-F was freshly prepared and kept ice cold to avoid degradation, and an aqueous solution of D-penicillamine (0.3 mg/mL) containing 0.2 M disodium EDTA was daily prepared. The standard solution of selenium(1V) was prepared by diluting a commercial standard solution for atomic absorption spectrometry containing loo0 ppm selenium(IV) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) with 0.5 M HC1. Deionized distilled water was further purified with a Milli-Q four houses water purification system. Good's buffer solution (pH 7.5) was prepared by titration of 0.8 M 3-(N-morpholino)propanesulfonic acid (MOPS) solution with 5 M NaOH solution. Borate buffer solutions (pH 7.0-9.0) were prepared by titration of 0.2 M boric acid with 5 M NaOH. A standard sample of lobster (TORT-1) was supplied by the National Research Council of Canada (NRC). Standard samples of river sediment (SRM-1645), oyster tissue (SRM-1566) and wheat flour (SRM-1567) were supplied by the National Bureau of Standards (NBS). High-Performance Liquid Chromatography. A Twincle pump (JASCO, Tokyo, Japan) equipped with a Model 7125 sample injector (Rheodyne, Cotati, CA) was used. A column packed with Capcell Pak C18 (250 mm X 4.6 mm i.d., Shiseido Co., Tokyo, Japan) was used at 40 "C. A mixture of acetonitrile, water, and phosphoric acid (400/600/1, v/v/v) containiig 10 mM lithium sulfate (pH 2.6) was used as a mobile phase at a flow rate 1.0 mL/min. A Shimadzu RF-500 spectrofluorometer equipped with a 12-fiLflow cell was employed for the detection with an excitation wavelength of 470 nm and an emission wavelength of 530 nm. The chromatogram and the peak area were recorded and processed on a Shimadzu C-R3A Chromatopak. Recommended Procedure for Determination of Selenium(1V). A 0.2-mL portion of sample solution (0.5 M hydrochloric acid solutions containing selenium(IV))and 0.1 mL of an aqueous
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989
solution of D-peniCillamine (0.3 mg/mL) containing 0.2 M disodium ethylenediaminetetraacetate (EDTA) were mixed in a capped glass tube. The mixture was kept at 80 "C for 7 min and then cooled in ice water. At this moment, EDTA might deposit as a white precipitate in the solution, but it caused no trouble for the following procedure. The solution was adjusted to pH 7.5 by adding an appropriate volume of 5 N NaOH and 0.3 mL of MOPS buffer solution. Then 0.1 mL of the solution was transferred to another tube covered with aluminum foil and added with 0.1 mL of NBD-F solution (3.0 mg/mL in ethanol). The tube was capped and kept at 70 "C for 1.5 min. After the mixture was cooled on ice for 0.5 min, 0.05 mL of 2.4% hydrochloric acid was added to the reaction mixture to stop the reaction. A 25-pL portion of the solution was immediately injected to HPLC. Optimization of Reaction Conditions. To investigate pH dependence of fluorophore formation reaction, 0.1 mL of standard solution of PenSTS (400 nM) was mixed with 0.2 mL of NBD-F (1.5 mg/mL) solution and the mixture was kept at 70 "C for 1 min at various pH values. The pH values were controlled by addition of 0.1 mL of 0.2 M borate buffer solutions of pH ' 7.0, 7.25, 7.5, 7.75, 8.0, and 9.0. Then the reaction was stopped by cooling on ice and adding 0.1 mL of 2.4% HCl. The yield of NBD derivative of PenSTS was determined from the fluorescence intensity (peak area) measured by fluorometric HPLC as described above. To investigate the effect of temperature on the reaction, 0.1 mL of a standard solution of PenSTS (400 nM) and 0.2 mL of NBD-F solution (3.0 mg/mL) were mixed in 0.1 mL of 0.8 M MOPS buffer solution (pH 7.5) and kept at various temperatures (55, 60, 65, 70 "C) for 1, 1.5, 2, or 2.5 min. The reaction was stopped and time courses for the formation of fluorophore were determined by fluorometric-HPLC as described above. To investigate the effect of concentration of NBD-F, standard solutions of PenSTS, were reacted with various concentrations of NBD-F solutions (1.5, 2.25, 3.0 mg/mL) at 70 "C at pH 7.5 (controlled by addition of 0.1 mL of 0.8 M MOPS buffer solution), and time courses of formation of fluorophore were obtained in the same way as described above. A 0.2-mL portion of 0.5 M HCl or 0.1 M phosphate buffer solution (pH 7.0) containing 1.3 pM selenium(1V) and penicillamine solution (2.0 mM in water) were mixed and kept at 80 "C. Time courses for yields of PenSTS were obtained by determination of PenSTS in the mixture by fluorometric-HPLC after derivatization of PenSTS to the fluorophore according to recommended procedure described above. The yield of PenSTS was calculated from the calibration line obtained from standard PenSTS. Digestion of Standard Sample. The digestion of sample was achieved according to the following procedure, which was the modification of the method previously reported (18). About 100-200 mg of each standard sample (NRC lobster, NBS wheat flour, NBS oyster, NBS River Sediment) supplied in powdered state was accurately weighed and digested with 1.6 mL of a mixture of nitric acid, perchloric acid, and sulfuric acid (10/5/1, v/v/v) in a tightly stoppered poly(tetrafluoroethy1ene) (PTFE) vessel. The digestion and evaporation were carried out by heating the mixture at about 130-150 "C under a hood until white fume of the perchloric acid appeared. Hydrochloric acid (0.5 mL) was added to the digested solution and heated at 100 "C for 40 rnin in a tightly capped vial to reduce selenium(V1) to selenium(1V). These procedures enabled total contents of selenium in the samples to be converted t o selenium(1V). RESULTS AND DISCUSSION Optimization of Reaction Conditions. NBD-F, fluorescamine, and o-phthaladehyde, which are the labeling reagent for amino group, were tested for derivatization of penicillamine selenotrisulfide to fluorophore, and the optimum reaction condition for each reagent was investigated in preliminary study. Among the reagents NBD-F was chosen for the present purpose because it reacted more readily with PenSTS, producing the fluorophore with stronger fluorescence intensity than the others. The reaction conditions for the derivatization of penicillamine selenotrisulfide to fluorophore by reaction with NBD-F
I
2
0
i
I
I
7 0
8.0
9 0
I'
tI
Figure 1. Effect of pH on formation of NBD derivatlve of PenSTS. Relative fluorescence intensity presents the percent ratio of HPLC peak
area of NBD-PenSTS at each measured point to the maximum of those. Detailed conditions are described in the Experimental Section.
: > Y
;'
100
50 3
~~
Y
4
cz
0
Figure 2. Time courses of derivatization of PenSTS with NBDF at various NBD-F concentrations. Final concentrations of NBDF in reaction mixture were 1.5 (0),1.13 (e),and 0.75 (0)mg/mL. Relative fluorescence intensity was obtained as described in Figure 1. Detailed conditions are described in the Experimental Section.
were optimized with respect to pH, temperature, reaction time, and concentration of NBD-F. The result for the pH dependence is shown in Figure 1. The fluorescence intensity reached a maximum level in a relatively narrow pH range between 7.25 and 7.75, which is a little lower than that for amino acids (17). The rapid decrease in the fluorescence intensity a t higher pH (>8.0) is possibly due to cleavage of -S-Se-S- linkage or hydrolysis of the fluorophore. Figure 2 shows the effect of concentration of NBD-F on the yield of NBD derivative of PenSTS a t 70 "C, indicating that the reaction proceeded faster with increasing concentration of NBD-F, and a maximum yield was obtained by reaction with NBD-F of final concentration a t 1.5 mg/mL for 1.5 min. Reaction temperature was optimized from the results shown in Figure 3; the reaction was accelerated at the elevated temperature up to 70 "C where the maximum fluorescence intensity was attained by the reaction for 1.5 min. Much higher temperature was impractical because of evaporation of ethanol used as solvent of NBD-F solution. The effects of pH and reaction time on the formation of penicillamine selenotrisulfide were investigated by reaction of standard selenium(1V) with a large excess of penicillamine. The use of excess penicillamine could afford the rapid and complete reaction with wide concentration range of selenium(1V). Figure 4 shows the time courses of the reaction at 80 "C at different pH values. The reaction in HCl solution proceeded faster than in neutral phosphate buffer solution and was completed within 4 min, where the yield of penicillamine selenotrisulfide reached almost 100% of theoretical. A slight lowering of the yield of penicillamine selenotrisulfide by reaction in phosphate buffer solution may be due to cleavage of -S-SeS- linkage at higher pH, although PenSTS is much more stable in neutral pH than selenotrisulfides
ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989 Table
235
I. Contents of Selenium in S t a n d a r d Samples ( p g / g , Mean f S t a n d a r d Deviation) present methodb (recovery)d
1.44f 0.02
1.68 f 0.02
2.10 f 0.10
1.92 f 0.10
1.1f 0.2
1.0 f 0.0
1.0 i 0.08
6.88 f 0.47
6.50 f 0.10
6.25 f 0.55
1.60 f 0.10 (1.1) 2.02 f 0.07 (0.95) 1.08 f 0.10 (1.0) 6.71 f 0.33 (1.0)
HG-AAS"
certified value
sample NBS-SRM-1645 (river sediment) NBS-SRM-1566 (oyster tissue) NBS-SRM-1567 (wheat flour) NRC-TORT-1 (lobster)
(1.5)O 2.1
" Cited from ref 19, n = 3. * n = 4.
: o 0 .l 5
DAN-fluorometry with tellurium coprecipitationa
* 0.5
Uncertified reference value. Mean value, n = 3.
3
I
I
I
I
1.5 2.0 Reaction Time Cminl
1.0
2.5
Flgure 3. Time courses of derivatiration of PenSTS with NBD-F at varlous temperatures. Reaction temperatures were 55 (A),60 (a), 65 (O),and 70 O C (0). Relative fluorescence Intensity was obtained as described In the legend of Figure 1. Detailed conditions are described in the Experimental Section.
,
..
I
0
2
I
I
I
I
6 8 R e a c t i o n TimeCminl
10
12
4
I
Flguro 4. Time courses of the yield of PenSTS in 0.33 M HCI solution (0)and in pH 7.0 phosphate buffer solution (0). Yield of PenSTS presents percent yield to theoretical. Detailed condltions are described in the Experimental Section.
formed from other thiols such as cysteine and glutathione (15) which can exist only in a strongly acid solution. This is why PenSTS was employed in the present method. On the basis of the above results, the recommended procedure for the determination of selenium(1V) was established as described in the Experimental Section. High-Performance Liquid Chromatography. The reaction solution to be applied to HPLC separation contained several substances that responded to the fluorescence detection, These are the NBD derivative of excess penicillamine, penicillamine disulfide, and reagent blank including hydrolyzed product of NBD-F. The separation of NBD derivative of penicillamine selenotrisulfide from these substances was examined by selection of stationary phase and mobile phase conditions. Among the reversed-phase columns tested, Nucleosil5C18 (M. Nagel, West Germany) and Capcell Pak C18 (Shiseido Co., Tokyo, Japan) exhibited symmetric peak and base-line separation. The separation and peak symmetry were
Flgure 5. Chromatograms of (A) selenium(1V) reacted with penicillamine followed by NBD derlvatlzation, (B) standard PenSTS reacted with NBDF, (C) penicillamine (not containing selenium) with NBDF, (D) buffer solution and NBD-F (reagent blank).
further improved by addition of lithium sulfate in the mobile phase. The acidic mobile phase of pH 2.6 containing phosphoric acid resulted in the increased retention and enhanced stability of the fluorophore compared with the mobile phase of neutral pH. Addition of organic modifier decreased the retention time and increased the fluorescence intensity. Thus, 40 % acetonitrile was selected as a compromising condition. When the standard solution of selenium(1V) was analyzed according to the recommended procedure, HPLC separation of the reaction solution exhibited the chromatogram as shown in Figure 5A. When selenite was absent from the solution, the chromatogram (Figure 5C) indicated disappearance of the peak corresponding to the asterisked peak in Figure 5A. The retention time of the asterisked peak agreed exactly with that found in the reaction of standard PenSTS with NBD-F (Figure 5B). The chromatogram of hydrolyzed NBD-F (Figure 5D) exhibited only a major peak with retention time shorter than 3 min. Therefore, the asterisked peak in Figure 5A was ascribed to NBD derivative of PenSTS. It was suggested from the preliminary studies of fast-atom bombardment mass spectrometry that each of two amino groups in PenSTS molecule reacted with NBD-F, although exact structure was not obtained. The quantitation of selenium(IV) was achieved with the area of the asterisked peak in Figure 5A. Calibration Line, Detection Limit, and Precision. The standard solutions of selenium(1V) ranging from 10 to 2000 ppb were treated by the recommended procedure described in the Experimental Section, and the calibration line was obtained as the plots of peak area versus concentration of selenium(1V). The results showed a linear relationship with a correlation coefficient of 0.999. The detection limit calculated for the signal to noise ration >2 was about 0.2 pmol of selenium per injection, which corresponded to 5 ppb selenium(1V) in standard solution. This detection limit is sufficient for the determination of selenium in biological and environmental samples. The precision of the method evaluated from five determinations of 100 ppb selenium(1V) showed the
Anal. Chem. 1989, 61, 236-240
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relative standard deviation to be 2.34%. Determination of Selenium in Standard Samples. The results for contents of selenium in standard samples determined by the present method are shown in Table I, which refers to the certified values and the results obtained by DAN fluorometry with tellurium coprecipitation (19) and by atomic absorption spectrometry with hydride generation (HG-AAS) (19). These standard samples contained some coexistent metals with higher contents than selenium. Since the formation of PenSTS proceeds in strongly acid solution, only a limited sort of metal ions such as copper, which is known to form stable chelate with penicillamine, may interfere with the selenotrisulfide formation reaction. Such ions and other unknown substances may still cause deposit of precipitates during derivatization procedure, lowering of recovery and poor separation of fluorophore on HPLC. To eliminate these fears, we added EDTA to the penicillamine solution and used a large excess of penicillamine for the formation of penicillamine selenotrisulfide. When 5 pg of Se(1V) was spiked to the digested solution of each standard sample, 95-110% of the spiked Se(1V) was recovered as shown in Table I. Thus, it is concluded that no significant interferences with the determination of selenium were not observed, though the standard samples used in this work contained about 2-70-fold higher amount of copper than selenium. Student's paired t tests (p < 5%) were carried out with respect to the selenium contents (pg/g) given as the certified value and obtained by the present method and by the previously established method. The results indicated there was no significant difference, except for the pair of the results for SRM-1645 obtained by the present method and by HG-AAS. The present method, though it takes about 21 min for a HPLC analysis, allows determination of a low level of selenium in a small sample size with easy operation. The applicability
of the present method can be expanded to include the samples with much lower contents of selenium and different sorts of interfering substances, when it is combined with the selective preconcentration method of selenium using anion exchange resin loaded with bismuthiol-I1 sulfonate, which the authors developed (20).
LITERATURE CITED (1) Schwartr, K.; Foitz, C. M. J . Am. Chem. SOC. 1957,79, 3292. (2) Das, N. P.; Ma, C. W.; Salman, Y. M. Blol. Trace Elem. Res. 1988,
IO, 215. (3) Schrauzer, G. N.; White, D. A.; Schneider, C. J. Bloinorg. Chem.
1977, 7 , 23. (4) Greeder, G. A.; Milner, J. A. Science 1980,209, 825. (5) Hiraki, K.; Yoshii, 0.; Hirayama, H.; Nlshlkawa, Y.; Shlgematsu, T. Bunseki Kagaku 1973,2 2 , 713. (6) Shlbata, Y.; Morita, M.; Fuwa, K. Anal. Chem. 1084,5 6 , 1527. (7) Narasaki, H.; Ikeda, M. Anal. Chem. 1984,56, 2059. (8) Campbell, A. D. Pure Appl. Chem. 1984,56. 645. (9) Kimberly, M. M.; Paschal, D. C. Anal. Chlm. Acte 1985, 174, 203. (10) Maenhaut, W.; Dereu, L.; Tomza, U. Anal. Chim. Acta 1982, 136, 301. (1 1) Dllli, S.;Sutikno, I.J . Chromatogr. 1884,300, 265. (12) Tamarl, Y.; Ohmori, S.;Hiraki, K. Clin. Chem. 1986,32, 1464. (13) Ganther, H. E. Biochemistry l988?7 , 2899. (14) Nakagawa, T.; Hasegawa, Y.; Yamaguchi, Y.; Chikuma, M.; Sakurai, H.; Nakayama, M.; Tanaka, H. Biochem. Biophys. Res. Common. 1988, 135, 183. (15) Nakagawa, T.; Aoyama, E.; Kobayashi, N.; Tanaka, H.; Chlkuma. M.; Sakurai, H.; Nakayama, M. Biochem. Blophys . Res. Commun. 150, 1149. (16) Imai, K.; Watanabe, Y. Anal. Chim. Acta 1881. 130, 377. (17) Watanabe, Y.; Imai, K. J . Chromatogr. 1882,239, 723. (18) Itoh, K.; Nakayama. M.; Chikuma, M.; Tanaka, H. Fresenius' 2.Anal. Cbem. 1086,325, 539. (19) Itoh, K.; Chikuma, M.; Tanaka, H. Fresenius' 2.Anal. Chem. 1988, 330, 800. (20) Nakayama, M.; Tanaka, T.; Tanaka. M.; Chikuma, M.; Itoh. K.; Sakurai, H.; Tanaka, H.; Nakagawa, T. Tahnta 1987,34, 435.
RECEIVED for review December 31,1987. Resubmitted August 16, 1988. Accepted October 31, 1988.
Thermal Gradient Liquid Chromatography: Application to Selective Element Detection by Inductively Coupled Plasma Atomic Emission Spectrometry Wilton R. Biggs* and John C. Fetzer Chevron Research Company, Richmond, California 94802-0627
The use of temperature gradlents to achleve reversed-phase llquld chromatographlc Separations for systems detected by plasma technlques is demonstrated. Aqueous (detecting S) and nonaqueous (detectlng SI) systems are studied. The technique poves usekd for soMng the general eluHon problem while anowlng detectlon on an elemental basis by Inductively coupled plasma atomic emlsslon spectrometry ( ICP-AES).
INTRODUCTION This work describes the use of temperature programming in high-performance liquid chromatographic (HPLC) separations detected by inductively coupled plasma atomic emission spectrometry (ICP-AES). Although temperature programming is a widely used technique in gas chromatography, it has only recently begun to receive attention as a 0003-2700/89/0361-0236$01.50/0
means of controlling liquid chromatographic retention. Several workers (1-10) have demonstrated successful temperature-programmed separations, and the theory of temperature effects in HPLC has been developed (11,12),as well as important selectivity considerations (13) and a systematic optimization strategy (14). This work directly impacts efforts to employ plasma emission devices as specific element detectors in liquid chromatography because temperature programming allows the matching of operational requirements for optimum performance of each component in a coupled HPLC/ICP system. Specific element detection of HPLC separations offers several potential advantages; among these are the simplification of complex sample matrices, the use of elemental response factors which are independent of the chemical form of the element, and the detection of compounds having no molecular property which can be suitably exploited for de0 1989 American Chemical Society
