Determination of the anticancer drug 5-fluorouracil added to blood serum by liquid chromatography with anodic amperometric detection

Analytica ChimicaActa, 207 (1988) 183-194

183

Elsevier Science Publishers B.V., Amsterdam-- Printed in The Netherlands

D E T E R M I N A T I O N OF T H E A N T I C A N C E R D R U G 5 - F L U O R O U R A C I L A D D E D TO B L O O D S E R U M B Y L I Q U I D CHROMATOGRAPHY WITH ANODIC AMPEROMETRIC DETECTION

T.R.I. CATALDI,A. GUERRIERI, F. PALMISANO* and P.G. ZAMBONIN Laboratorio di Chimica Analitica, Dipartimento di Chimica dell, Universita, Via G. Amendola 173, 70126 Bari (Italy)

(Received 18th August 1987)

SUMMARY Liquidchromatographywith on-lineanodic amperometricdetection is used after a liquid/solid extraction step for the determination of the anticancer drug 5-fluorouraciladded to blood serum. A detectionlimit of 15 ng ml- 1can be achieved.The anodic electrochemicalbehaviourof the drug at a mercury electrode, which is the basis of the detection principle, is also described. Anodic amperometric detection at a mercury electrode is compared with detection at a glassy carbon electrode in terms of sensitivity, linearity and selectivity.

5-Fluorouracil is an antineoplastic agent which acts as an antimetabolite to uracil and blocks the conversion of deoxyuridylic acid to thymidylic acid by the cellular enzyme thymidylate synthetase. It is used primarily for the treatment of solid tumors of breast, colon and rectum, often in combination with other chemotherapeutic agents designed to be synergic in cytotoxicity while minimizing the toxic effects of 5-fluorouracil, which may be severe and sometimes fatal. Although 5-fluorouracil exerts its anticancer activity following intracellular metabolic activation to 5-fluorodeoxyuridine monophosphate, the measurement of free serum concentration of 5-fluorouracil remains the most reasonable clinical and pharmacological approach for studying individual variations in metabolism and response. Several procedures have been devised for plasm a / s e r u m drug determination. Microbiological assays [ 1 ] are generally highly sensitive but their accuracy at low concentrations is questionable [2] because of interference effects arising from 5-fluorouracil metabolites. Gas chromatography with mass spectrometric detection [3,4] offers the lowest limits of detection. Both nitrogen-phosphorus [5 ] and electron-capture [6 ] detectors offer a convenient sensitivity whereas flame-ionization detection [7] is only applicable to plasma levels exceeding 200 ~g ml-1. Liquid chromatography with

0003-2670/88/$03.50

© 1988 Elsevier Science Publishers B.V.

184

ultraviolet detection [8-12 ] is certainly the most widely used technique for drug detection in body fluids. Fluorescence detection is also possible [13] following a pre-column derivatization step with 4-bromomethyl-7-methoxycoumarin. Liquid chromatography~electrochemical detection (LC/EC) has not yet been proposed probably because of lack of information about the anodic electrochemical behaviour of 5-fluorouracil in particular and of pyrimidines in general. The electrochemical behaviour of pyrimidine derivatives and the polarographic reductive behaviour of 5-halouracils have been extensively reviewed by Dryhurst [ 14 ]. In contrast, there are only a few reports on the electrochemical oxidation of pyrimidines. In 1984, it was observed [ 15 ] that 5-fluoro- and 5-bromo-uracil could be oxidized at a glassy carbon electrode and the possibility of L C / E C detection in serum was suggested. Palecek et al. [16] investigated the reaction of a number of purines and pyrimidines at a mercury electrode and noted that 5-halouracils, in borate buffer, gave a response in the cathodic stripping region. The use of cathodic stripping voltammetry for determination of 5-fluorouracil in serum has been recently attempted [17]. The method has, however, certain drawbacks such as a narrow linear range, potential interferences from endogenous co-extracted components a n d / o r metabolites, the absence of an internal standard, and inadequate sensitivity for extended pharmacokinetic studies. The feasibility of differential-pulse amperometry and cathodic stripping voltammetry in flowing solution was exploited by Bouzid and Macdonald [18] for determinations of 5-fluorouracil, but only in synthetic samples. Adsorptive stripping voltammetry of 5-fluorouracil was described by Wang et al. [ 19 ] but applicability to real samples remains still to be demonstrated. In the authors' laboratory, it has been demonstrated that some purine [20] and pyrimidine [21 ] derivatives (denoted below as L n- ) yield anodic waves caused by the formation of sparingly soluble mercury compounds according to overall electrode reactions of the kind H g + 2L n- ~-HgL~ (l-n) + + 2e 2 H g + 2 L ' - ~Hg2L~ (l-n)+ + 2e and that these reactions can provide the basis for liquid chromatography with on-line anodic amperometric detection. Recently [22 ], it was shown that this method is also applicable to pterine derivatives such as amethopterin, folic and folinic acids. In the present paper, the anodic electrochemical behaviour of 5-fluoro- and 5-bromo-uracil on mercury and an LC/EC method for the determination of 5fluorouracil in human serum are described. Anodic amperometric detection at a mercury electrode is compared to amperometric detection at a glassy carbon electrode in terms of sensitivity, selectivity and linear range. A solid/liquid

185

extraction step as an alternative for the usual liquid/liquid extraction procedure is also presented. EXPERIMENTAL

Chemicals 5-Fluorouracil, 5-bromouracil, 5-fluorodeoxyuridine and 5-fluorodeoxyuridine monophosphate (Sigma) were used as received. Stock solutions were prepared in methanol and stored in the dark at 4 ° C. More diluted solutions were prepared, when necessary, by dilution with mobile phase. The strong anion-exchange resin used for solid/liquid extraction was Duolite-101D (100-200 mesh; Rohm & Haas). The extraction microcolumns were prepared by slurry-packing the resin over a length of 2.5-3 cm in polypropylene tubes (3-ml capacity) fitted with a 20-pro pore polyethylene flit at the bottom. All solvents used were HPLC grade (J.T. Baker); the other chemicals were analytical reagent grade (Carlo Erba, Milan). Borate buffer pH 8.7 was prepared by mixing 0.05 M H3BO3 with 0.05 M NaOH. Buffers used in the mobile phase were filtered through a 0.45-/~m membrane (Gelman Sciences, Ann Arbor, MI).

Apparatus For the voltammetric experiments, a PAR 174A polarographic analyzer (E.G. & G. Princeton Applied Research) was coupled to a PAR model 303 static mercury drop electrode (SMDE) and an X-Y-t recorder. For cyclic voltammetric and chronoamperometric experiments, the same apparatus was used with the SMDE in the "hanging-mercury-drop" mode. In some experiments, a conventional dropping mercury electrode (DME) was used. Chromatographic apparatus and electrochemical detectors have been described elsewhere [20-22]. Unless otherwise specified, the following chromatographic conditions were used: RP18 column 5-gm packing (250× 4.6 mm); mobile phase, 0.05 M phosphate buffer pH 6.5/methanol (98: 2 v/v) at a flow rate of I ml rain-l; injection volume 20 pl; ambient temperature.

Sample treatment Liquid~liquid extraction. To 1 ml of serum, add the internal standard (5bromouracil), I ml of saturated ammonium sulphate solution and mix briefly in a vortex-mixer. Extract twice with 3 ml of diethyl ether/isopropanol (80: 20) mixture, centrifuging briefly to assist phase separation. Evaporate the combined organic phases to dryness and reconstitute the residue with mobile phase. The reconstitution volume ranged from 0.1 to i ml, as necessary. Solidfliquid extraction. Pipet I ml of serum, 100 ttl of internal standard, 5 ml of 0.025 M carbonate buffer pH 10 into a centrifuge tube and mix for I rain in a vortex-mixer. Transfer the sample quantitatively (two successive washings

186

with 1 ml of the buffer) to the extraction microcolumn, which has been previously conditioned with two bed volumes of 1 M HC1 in ethanol and washed with 5 bed volumes of water to remove the excess of acid. Wash the column with 10 ml of water followed by 10 ml of methanol. Elute successively with 1 and 4 ml of 0.3 M acetic acid in methanol, discarding the first 1 ml of eluate. Evaporate the eluate to dryness and reconstitute the residue in the mobile phase (reconstitution volumes as above). RESULTS AND DISCUSSION

Polarographic behaviour Normal pulse polarograms (SMDE) of 5-fluorouracil dissolved in a 0.05 M borate buffer pH 8.7 are shown in Fig. 1A. There are four significant features: (a) the presence of a pre-wave caused by adsorption of the reaction product; (b) a wave caused by the formation of a mercury compound, the height of which increases on increasing the concentration of 5-fluorouracil; (c) a cathodic shift of the waves on increasing the 5-fluorouracil concentration; and (d) a cathodic shift of the background (mercury dissolution). Figure 1B shows a sampled DC polarogram obtained at the SMDE; the prewave is still clearly evident while the main anodic wave loses the expected

A

B

c I O.2#A

o,

I

o2

,

oo,

,

P O T E N T I A L ~ V vs. Ag/A~Cl

Fig. 1. (A) Normal-pulse polarograms at a SMDE for 5-fluorouracil (FU) dissolved in a 0.05 M borate buffer pH 8.7. Curves: (a) supporting electrolyte; (b) 0.48 mM FU; (c) 1.9 mM FU. Potential scan rate 2 mV s-l; drop time 1 s; drop area 0.016 cm 2. (B) Sampled DC polarogram (SMDE) for 0.77 mM FU dissolved in a 0.05 M borate buffer pH 8.7. Drop time 1 s; scan rate 5 mV s-l; drop area 0.016 cm2.

187

lITC

[

+0i2

l

If

1/~A

•

I

-0.2 ;

~ i

la

Ha T

I

M

E

POTENTIA

L/V

vs. A g / A g C I

Fig. 2. Current/time curves recorded on single mercury drops at a conventional D M E for 0.77 m M FU in a 0.05 M borate buffer p H 8.7. Drop time 5 s; applied potential +0.152 V vs. Ag/AgC1. Fig. 3. Cyclic voltammogram of 5-fluorouracil in 0.05 M borate buffer p H 8.7 at a H M D E for 0.77 m M FU. Scan rate 50 mV s -1, electrode area 0.016 cm 2.

sigmoidal shape and a needle-like peak appears. Such unusual behaviour could be rationalized assuming film formation (caused by an insoluble electrode reaction product) that subsequently causes inhibition of the forward electrode reaction. Direct evidence for inhibition of the electrode process by the reaction product can be derived from examination of current/time curves during the lifetime of a single drop (conventional DME) as shown in Fig. 2. In potential regions where films of the reaction product inhibit the electrode process, the curves deviate markedly from the behaviour expected for a simple diffusioncontrolled process. In the case of the SMDE, the effect of electrode filming is more pronounced because the drop is quite instantaneously formed (ca. 100 ms) and then remains static. It is just the opposite situation, e.g., high rate of surface area formation, which can minimize the extent of surface coverage during drop life [23]. The absence of such anomalies in normal pulse polarograms at SMDE should not be surprising on considering the short duration (50 ms) of the potential pulses driving the electrode process. Figure 3 shows a representative cyclic voltammogram of 5-fluorouracil displaying the same general features of the polarograms discussed above. Peaks Ia/Ic fit the diagnostic criteria for adsorption peaks (e.g., peak current linearly dependent on scan rate) while peaks I I J I I c display the typical shape expected for the formation of an insoluble film and for the dissolution (stripping) of the

188 1.0

'3¢~•• <> •

1

4~A

- 0,5

C, + 0.14

T

I

M

E

POTENTIAL

• +0.22

+0.18

/ V vs.

Ag/AgCI

Fig. 4. Potentiostatic current/time transient obtained on the rising portion of peak II, in Fig. 3 for 0.77 mM FU. Supporting electrolyte 0.05 M borate buffer pH 8.7; applied potential + 0.110 V vs. Ag/AgC1. Fig. 5. Hydrodynamic voltammograms generated at a mercury electrode: (•) 5-fluorouracil; (~) 5-bromouracil (BrU). Chromatographic conditions: see Experimental; injected quantity 100 ng and 200 ng for FU and BrU, respectively;injection volume 20/~l;drop time, I s. surface film, respectively. Further evidence for the formation of an electrode film is given by potentiostatic i/t transients. While purely continuously decreasing transients were obtained at the potential of peak Ia, current maxima (see Fig. 4) characteristic of a nucleation and growth process were observed at the potential of peak II a. Essentially similar results were observed for 5-bromouracil, the main difference being somewhat altered adsorption characteristics of the electrode reaction product and the presence of tensammetric peaks, probably originating from some reorientation of the adsorbed layer. A more complete electrochemical characterization of the mercury/5-fluorouracil system (adsorption parameters, nucleation mechanism, stoichiometry of the reaction product) will be given elsewhere. 5-Fluorodeoxyuridine and 5-fluorodeoxyuridine-5-monophosphate were found to be electroinactive on both glassy carbon and mercury electrodes. Inactivity of these nucleotides may be due to the presence of a bulky sugar residue in the molecule or, as suggested by Palecek [24] for uridine and deoxyuridine on mercury electrode, to N (1) substitution.

Hydrodynamic voltammetry Although slightly alkaline buffers are best suited for electrochemical studies, they cannot be used for reversed phase chromatographic work for stability reasons. Therefore hydrodynamic voltammograms of 5-fluoro- and 5-bromouracil were generated at lower p H values in order to obtain some indication about the optimum working potential for H P L C detection purposes. It was

189 observed that lowering the pH of the supporting electrolyte caused a shift of the wave towards anodic potentials with loss of resolution from background (mercury oxidation). A pH value around 6.5 offered the best compromise between chemical stability of the reversed-phase column, control of ionization equilibria and detectability requirements. A solution consisting of 0.05 M phosphate buffer pH 6.5 containing a small amount of methanol as organic modifier was then used as the mobile phase. The resulting hydrodynamic voltammograms (SMDE) are presented in Fig. 5; these suggest a detection potential of + 0.21 V vs. Ag/AgC1. Similar voltammograms were generated on glassy carbon; + 1.2 V vs. Ag/AgC1 was then the most appropriate detection potential. Because the present detection method can be thought of as a derivatization reaction in which the derivatizing agent is electrogenerated in situ (i.e., at the electrode surface), the influence of flow rate on the current response was evaluated in order to ascertain whether or not the kinetics of the mercury complex formation will limit the values of flow rates that can be used. Large volumes (1 ml) of mobile phase containing 5-fluorouracil or ascorbic acid were injected into a flow-injection system (0.05 M phosphate buffer pH 6.5 as carrier) and the resulting current maxima (/max) at different flow rates V (ml min -1 ) were measured. Ascorbic acid, which is anodically oxidized at a mercury electrode to dehydroascorbic acid, was taken as a model compound to test the hydrodynamic characteristics of the polarographic detector. A plot of log/max VS. log V for ascorbic acid was linear (slope 0.5 ) over the investigated range (log Vvarying from - 0 . 8 to + 0.6). In contrast, a similar plot for 5-fluorouracil showed marked negative deviations from linearity for flow rates higher than about 1 ml min-1, indicating the presence of some "kinetic" effects. Similar effects were earlier observed by Kok et al. [ 25,26 ] who found that the current response of a copper anode towards complexing agents, such as amino acids, varied markedly with the nature of the amino acid itself. The decrease in current response observed, at increasing flow rates, for some amino acids such as alanine and proline was rationalized in terms of a slow complexing reaction between the amino acid and Cu(II) ions.

Comparison of detectors The polarographic detector was operated in the sampled DC mode with a drop time of I s. A medium drop size (estimated electrode area 0.016 cm 2) gave the best signal-to-noise ratio. The voltammetric detector (thin-layer configuration with a 3-mm diameter glassy carbon working electrode and a 0.13-mm thick PTFE gasket) was operated in the DC mode. The signals from both detectors were passed through a filter with a time constant of 0.3 s. Some data (obtained for standard solutions of 5-fluorouracil) on the performance of both detectors are shown in Table 1. The glassy carbon detector does not show a substantially better performance, as one might expect. Detec-

190 TABLE 1

Comparison of the performance of LC/EC and LC with amperometric detection. All data refer to 5-fluorouracil dissolvedin mobile phase~ Detector

Sensitivity (nAng -1)

Detection limit

Linear range (mol 1-1)

(S/N=3)

Peak height reproducibilityb

(%)

(ng) Glassy carbon (thinlayer) DMEc

1.20

0.65

2.5 X 10-7-2.5 X 10-4

1.2

1.15

2.5

10-6-10 -4

1.1

aColumn (250x4.6 mm) with 5-#m ODS packing; flow rate, I ml rain- 1; injection volume, 20 ttl. b60 ng injected (n--5). c With a wall-jet configuration. tion limits for this detector are, by a factor of ca. 4, lower than those of the D M E detector, but in real samples chemical noise can often be more important than instrumental noise so that the higher detection limits of the D M E detector could be overcompensated by its higher selectivity (see later). It should be noted that in the case of amperometric detection at the mercury electrode, doubled chromatographic peaks were observed for 5-fluorouracil when it was injected in amounts about three times the upper limit of the linear range. This anomalous behaviour is not of chromatographic origin (a change in the detector type produced "normal" peaks) b u t can be ascribed to the abovementioned inhibition of the forward electrode reaction by the surface film, which is particularly pronounced when a large amount of 5-fluorouracil is injected.

Recovery The widely used liquid/liquid extraction with diethyl ether/isopropanol (8 + 2 ) mixture was tested also in the present work. The average recovery was found to be around 55% with a relative standard deviation of 4% ( n - - 5 at 10 /~g ml-1). A solid-phase extraction procedure was devised in order to improve the efficiency of the extraction step with the possibility of simultaneously processing several samples by means of solid-phase extraction equipment which is commercially available. The procedure described gave a recovery of 92.5 + 2.8% (n = 10 at 10/~g m l - 1) which did not significantly change on varying the amount of 5-fluorouracil added to serum.

Analytical applications Pooled human-serum samples spiked with 5-fluorouracil were analyzed. Figure 6 shows typical chromatograms obtained with amperometric detection for a serum sample extracted by the liquid/liquid procedure. As can be seen, this

191

-(a) :•

"

:

F

U

"

::L:

. :

:

:

:=

7.

-+..

a2-__

:

-::7 : ::

~72 :z

~-

-

: ._::= =...

k-

:~i:-

--5i

C--

....

r

=

,:

.----~

:EEg

77!

-:-~-277--

: : 1 : = : ~ : = = = = = : ~

!= -I

I

=i

i

, -

= :~

÷ :: : : - L

:5 : - : > : :

. . . . . .

~0

7 ~7-

A. i:_: ~ t i re_e-:;Cm{p)

0

10

2O

J

f

T

I

M

0

10 I

20 I

E / rain

Fig. 6. Liquid chromatograms with amperometric detection of spiked and blank serum samples. (A) Sample spiked with 1 #g m[ -~ FU and extracted with diethyl ether/isopropanol; 250X4.6 mm column of ODS (10-/~m irregular packing) with 0.025 M phosphate buffer pH 6.5/methanol (98/2 v/v) as mobile phase at 1.5 ml min -~ and amperometric detection at the DME held at + 0.21 V vs. Ag/AgC1 in sampled DC mode; IS is the internal standard; the lower part is the blank chromatogram. (B) Sample spiked with 0.2/~g ml- ~ FU and extracted by the solid/liquid extraction procedure; column as for (A) but with a 5-/~m ODS packing and 0.05 M phosphate buffer pH 6.5/methanol (98/2 v/v) as mobile phase at 1 ml min-L (C) Glassy-carbon electrode; sample spiked with 0.6 #g m l - ~FU and extracted as in (B); chromatographic conditions as in (B) except for a phosphate/methanol ratio of 96/4 (v/v); applied potential, + 1.2 V vs. Ag/AgC1.

extraction method coupled to LC with amperometric detection gave a highly selective response towards 5-fluorouracil and its internal standard 5bromouracil.

192 TABLE 2 Detection limits of selected procedures for the determination of 5-fluorouracil in serum Method

Detection limit (ng m1-1)

Note

Biological assay GC/electron capture GC/NP GC/MS

10 20 150 1 0.39

LC/UV

100 50

Capillary column Selected ion monitoring Negative-ion chemical ionization of pentafluorobenzil derivatives Reversed-phase LC on ODS column Solvent extraction followed by silica-gel chromatography cleanup; HPLC separation on a Phenyl column

Proposed method

Ref.

1 6 7 3 4 9 10

15

The solid-phase extraction procedure gave an improved recovery for 5-fluorouracil, but at the same time, led to the co-extraction of some polarographically active interferences which, however, were completely chromatographically resolved from the two uracils (see Fig. 6B). Non-interfering strong negative peaks, probably arising from double-layer capacity depression by major components in the sample extract, were also observed. The small insert in Fig. 6B represents the blank chromatogram showing the absence of sample interferents at the retention times of the two uracils. The voltammetric responses on spiked and blank serum samples are shown in Fig. 6C. As can be seen, an interfering peak arising from an unknown endogenous component is coeluted with 5-fluorouracil, thus preventing accurate quantitation of the drug at low concentration and substantially nullifying the lower detection limit potentially offered by glassy carbon voltammetric detection. Interference by sample components is of no concern in the case of detection at a mercury electrode. Absolute detection limits on serum extracts are essentially similar to those evaluated on drug standard solutions. On considering the ten-fold preconcentration factor of the extraction procedure and a 20-/ll injection volume, a serum concentration of 15 ng m l - 1 5-fluorouracil can be measured. Detection limits of some of the presently available assays for 5fluorouracil are compared in Table 2. G C / M S is the only technique offering detection limits considerably lower than LC with amperometric detection but at the expense of much greater instrumental complexity and cost. Conclusions The present work demonstrates the potential of LC with anodic amperometric detection for the determination of 5-fluorouracil in serum samples.

193

Practical difficulties in operating polarographic detectors probably remains the most significant drawback for routine application of the proposed method. Probably DC amperometric detection on a solid-state detector such as an amalgamated gold electrode would be more attractive to use but the irreproducibility of the electrode response (somewhat understated in the literature) caused by electrode fouling may represent a significant limitation. Pulsed-amperometric detection techniques capable of an electrochemical cleaning of the electrode surface would solve, in theory, the above problem. Work in this direction is in progress in this laboratory. Financial assistance from "Ministero della Pubblica Istruzione" is gratefully acknowledged. This work will form part of the PhD thesis of A. Guerrieri.

REFERENCES 1 E.R. Garrett, G.H. Hurst and J.R. Green, J. Pharm. Sci., 66 (1977) 1422. 2 N. Kawabata, S. Sugiyama, T. Kuwarmura, Y. Odaka and T. Satoh, J. Pharm. Sci., 72 (1983) 1162. 3 C. Aubert, J.P. Sommadossi, P. Coassolo, J.P. Cano and J.P. Rigault, Biomed. Mass Spectrom., 9 (1982) 336. 4 R.M. Kok, A.P.J.M. De Jong, C.J. Van Groeningen, G.J. Peters and J. Lankelma, J. Chromatogr., 343 (1985) 303. 5 E.A. DeBruijn, O. Driessen, N. Van den Bosch, E. van Strijen, P.H.Th.J. Slee, A.T. Van Oosterom and U.R. Tjaden, J. Chromatogr., 278 (1983) 283. 6 H.W. Van den Berg, R.F. Murphy, R. Hunter and D.T. Elmore, J. Chromatogr., 145 (1978) 311. 7 J.J. Windheuser, J.L. Sutter and E. Auen, J. Pharm. Sci., 61 (1972) 301. 8 P.T. Stetson, V.A. Shukla and W.D. Ensminger, J. Chromatogr., 344 (1985) 385. 9 M.W. DiGregorio, W.M. Holleran, C.C. Benz and E.C. Caduan, Anal. Lett., 18(B1) (1985) 51. 10 L.S. Shaaf, D.G. Ferry, C.T. Hung, D.G. Perrier and I.R. Edwards, J. Chromatogr., 342 (1985) 303. 11 D.C. Sampson, R.M. Fox, M.H.N. Tattersall and W.J. Hensley, Ann. Clin. Biochem., 19 (1982) 125. 12 G.J. Peters, I. Kraal, E. Laurensse, A. Leyva and H.M. Pinedo, J. Chromatogr., 307 (1984) 464. 13 M. Iwamoto, S. Yoshida and S. Hirose, J. Chromatogr., 310 (1984) 151. 14 G. Dryhurst, Electrochemistry of Biological Molecules, Academic, London, 1977, Chap. IV. 15 F. Palmisano and P.G. Zambonin, Ann. Chim. (Rome), 74 (1984) 633. 16 E. Palecek, F. Jelen, M.A. Hung and J. Lasowsky, Bioelectrochem. Bioenerg., 8 (1981) 62. 17 A.J. Miranda Ordieres, M.J. Garcia Gutierrez, A. Costa Garcia, P. Tunon Blanco and W. Franklin Smyth, Analyst, 112 (1987) 243. 18 B. Bouzid and A.M.G. Macdonald, Anal. Proc., 23 (1986) 295. 19 J. Wang, M. Shan Lin and V. Villa, Analyst, 112 (1987) 247. 20 F. Palmisano, E. Desimoni and P.G. Zambonin, J. Chromatogr., 205 (1984) 306. 21 F. Palmisano, T.R.I. Cataldi and P.G. Zambonin, J. Chromatogr., 344 (1985) 249. 22 A. Guerrieri and F. Palmisano, Anal. Chem., 59 (1987) 2127.

194 23 24 25 26

D.R. Canterford, A.S. Buchanan and A.M. Bond, Anal. Chem., 45 (1973) 1327. E. Palecek, Anal. Chim. Acta, 174 (1985) 103. W.Th. Kok, U.A.Th. Brinkman and R.W. Frei, J. Chromatogr., 256 (1983) 17. W.Th. Kok, H.B. Hanekamp, P. Bos and R.W. Frei, Anal. Chim. Acta, 142 (1982) 31