‘Available online at www.sciencedirect.com Journal of Elsetroanalytcal Chemistry $93 (2006) 258-265 Journal of, Electroanalytical ‘Chemistry \www.elsoviercomvlocateeteshem Voltammetric behavior and square-wave voltammetric determination of cefotaxime in urine Mara M. Aleksié "*, Vera Kapetanovié ° * state of Physical Chemistry, Faculty of Pharmacy, Unversity of Belgrade, P.O. Box 146, Vode Stpe 450,11 O00 Belgrade, Serbia and Montenegro © Insitute of Analytical Chemistry, Faculty of Pharmacy, University of Belgrade, P.O. Bax 146, Vojeode Step 450, 11000 Belgrade, Serbia and Montenegro Revved 20 December 2005; received in tevsed form 10 May 2006; accepted 21 June 2006 Abstract ‘The voltammetric behavior of cefotaxime (CFX) is investivated by cyclic and square-wave voltammetry in BR butlers (pH 1.80-12.0} Based on the cathodic reduction peak at approximately ~0.5 V in BR bulfer (pld 2.8 and 9.25), a robust, highly reliable square-wave ‘voltammetric method (SW) was developed for determination of CFX. The linearity was achieved by SW voltammetry in 7.5% 10" M (8.52 ng mI!) to 10% 10-7 M (47 ng ml!) and from 1.2% 10-? M (S64 ng ml!) to 12x 10° M (568.8 ng ml) range at pH 2.8 with detection and quantification limits of 0.813 ng ml! and 2.71 ng mil", and 12.6 ng mt" and 42.2 ng ml‘, respectively. At pH 9.25 linear range was obtained from 4% 10M (I8.8ng ml") to 1.6% 10" M (75.2ngml-*) with detection and quantification limits of 2.947 ng mt" and 9.82 ng ml’. The method was applied for CFX determination in spiked humane urine sample. A detection and quan- tification limits of 7.2ng mI and 23.9 ngmi-' were achieved for determination of CEX in urine at pH 28 and 13.6ng ml." and 45.1 ng mi”! at pl 9.25. The comparative reference method was DPV. The proposed method was checked for determination of CFX in real human urine, and selectivity of the method over the metabolites was found to be quite sa © 2006 Elsevier BLY. All rights reserved. Keyword: Cofotaxime; Square-vave voltammetry; Urine 1. Introduction Cephalosporins, together with penicillins, represent the dominant class of antimicrobial agents, with wide broad spectrum of activity. Cefotaxime (CFX) is the third gener ation cephalosporin used against mostly respiratory and urinary infections. Chemically, it possess methoxyimino group. The pres- ence of methoxyimino group in cefotaxime molecule is very important for its chemical and electrochemical behavior. Our previous studies [1,2] concerning to electrochemical behavior of cefetamet, also the third generation cephalo- sporin resulted in the mechanistic scheme for the reduction, * Corresponding author. Tel: +381 11 59-70 379; fax: +381 11 39-74 wy, Email address: marafapbarmacy bgse-yu (M.M. Aleks) (0022-0728 se front matter © 2006 Elkevier B.V. Al rights reserved. 6:10. 1064 jeleener 2006.06011 of the O-methyloxime grouping of this antibiotic. It has, been shown that its reduction occurred in two steps, corre- sponding to a reduction to the imine and amine, respec- tively, in contrast to the majority cases where the oxime reductions take place in a single four electron step [3]. Those two two-electron processes are caused by differences, in position and rate of establishment of acid-base equilib- rium resulting in protonation of the oxime and imino grouping. Since Cefotaxime also possesses the methoxyi- mino group and additionally a good leaving group on C3 position, the reduction of 3,4-double bond is also ‘occurred, resulting in the presence of one additional catho- lic peak at more negative potential, but the main electrore- duction process takes place at methoxyimino grouping as in the case of cefetamet. Based on a good knowledge of these reduction pathways and adsorption phenomena already established, [4] the newMAM, Alesié, V. Kapetanovié {Journal of Eleetoanaltical Chemisry 593 (2006) 258.266 299 adsorptive stripping methods were developed and sug- gested for determination of cephalosporin antibiotics in biological fluids [5.6], Modern voltammetric techniques, such as stripping voltammetry, dealt with the analytical determination of cefotaxime, begun with papers of Ogo- reve and coworkers [7,8]. The linear curves were obtained from micromolar down to nanomolar range, by applying the accumulation time of S-14 min. The electrochemical methods, more or less sensitive were reported for determi- nation of CFX at glassy carbon specially activated or mod- ified (with multi wall carbon nanotubes) or mixed carbon paste electrodes [9,10]. Generally, a long time of accumula- tion in stripping techniques and less sensitivity obtained at solid electrodes (2% 10-*-10~* M), led us to idea to find out the appropriate method for cefotaxime determination, taking into consideration the general knowledge of cepha- losporins electrochemical behavior and its acid base equi- librium, recently established (11) Some available other methods recently found in the lit- erature for determination of CFX in biological fluids or pharmaceutical products are based on the HPLC, FIA, CE [12-16] techniques. However, no literature data for square-wave voltamme- try (SWV) for determination of CFX in biological fluids were found ‘Therefore, this work presents an electroanalytical meth- odology for determination of CEX in spiked urine human samples, simple, fast and sufficiently sensitive. The data were compared with DPV method as a reference one. The selectivity over the main metabolite, desacetylcefotaxime (DCFX) was checked in real urine sample both with SWV and HPLC methods 2. Experimental 2.1, Reagents and solutions CEX was from Sigma and all chemicals were of analyti- cal grade quality. A stock solution (So) of 1x 10° M of CFX was prepared in redistilled water and stored in free- zer. More diltted solutions were prepared daily from stock solution (So). Desacetylcefotaxime (DCFX) was prepared by alkaline hydrolysis procedure [17] Tolycar (injections) was from Jugoremedija, (Zrenjanin, SCG), Britton-Robinson buffer solution, used as supporting electrolyte was prepared in the usual way [18]. The mobile phase in HPLC was consisted of a mixture of 0.007 M_orthophosphorie acid (Carlo Erba, Milan, Italy) and acetonitrile (Lab Scan, Ireland) (85:15 v/v) pumped at a flow rate of 1.0 ml/min. Before use the mobile phase was degassed and vacuum filtered through 0.45 im nylon membranes (Alltech Associates Inc., Belgium). Water for chromatography was obtained from a Milli pore System Simplicity 185 purification system, ‘The human urine samples were collected daily from at least two healthy individuals and a pool of these was used. Urine stock solution (Uo) was prepared from protein-free urine as 10% solution. ‘The real urine sample was obtained from the patient after the cefotaxime administration, 2.2. Instrumentation ‘The Voltammetric measurements were performed with an Amel 433-4 computerized polarographic analyzer. Three-electrode system was employed: hanging mercury dropping electrode (HMDE), Ag/AgC! reference electrode and a Pt-auxiliary electrode, When DPV mode was used the following parameters were applied: pulse repetition 100 ms, pulse amplitude 25 mV, pulse width 20mV and sean speed 100 mV s~! ‘A Hewlett-Packard HP 1100 (Palo Alto, CA, USA) chromatographic system equipped with HP 1100 binary pump and HP 1100 UV-Vis Detector was used. Sample injection was made through Rheodyne injector valve with 4 20 jl sample loop. The isoeratic chromatographic method used a CjgXTerra™ (150 x 3.9 mm, 5 um) column (Waters, USA). Column’s temperature was adjusted to 25°C. The detection of all analytes was performed at 262 nm. Data was acquired with ChemStation software from HP. A cen- trifuge Tehtnica {Zelezniki) LC-320 was used. A Radiometer pH meter, PHM 220, with appropriate standard butfer solutions was used, 23. Procedures 2.3.1. Procedure for CV ~ pH investigations In electrochemical cell 13.5 ml of BR buffer of different pHs was transferred, degassed for 10 min with high purity nitrogen and 1.5 ml of CFX stock solution (S5) was added to make its final concentration of Lx 10~* M. The solution ‘was purged for another 3 min and eycle voltammograms were recorded 2.3.2. Procedure for making calibration graphs An aliquot of 15 ml of supporting electrolyte (only BR. 0 0.75 ml of Uo and 14.25 ml BR) solution was introduced into electrochemical cell and degassed with pure nitrogen for 10min. A selected accumulation potential was then applied to a mercury drop, for a selected accumulation pet= iod, while the solution was stirred at 300 rpm, The stirring, was then stopped, and after a 10° rest period, a DP or a square-wave voltammetrie stripping was applied in the neg- ative direction over the range of 0.0 V to -1.6V vs. Ag/ AgCI. Afier the background voltammogram was recorded, the aliquot of the CFX was introduced into cell and the voltammogram was then recorded at a new mercury drop. 2.3.3. Procedure for human urine analysis Concentrated urine sample (4.0:ml) was treated with 0.3ml of methanol as urine protein precipitating agent.20 MEM, Aleks, V. Kapetanové | Jounal of Electroanalstical Chemsory 503 (2006) Alter the vortex during ~30 s, the precipitated protein was, separated out by centrifugation for 25min at 3900 rpm. ‘The clear supernatant layer was used in further procedure as protein-free human urine. 1.0 ml of this protein-free urine was diluted with redistilled water up to 10 ml (Uo), and filtered through the 0.2 jm membrane filters. The same procedure was applied for the real urine sample, 2.3.3.1. Procedure for human urine analysis in SWV and DPV methods. The volume of 0.75 ml of Uo was diluted with BR bulfer of selected pH up to 15 ml. This solution ‘was the supporting electrolyte for calibration and determi- nation of CFX in urine. When the accumulation time was completed, the stirring was stopped and after a rest period a square-wave ot DP voltammogram was recorded follow- ing the optimized conditions, Aliquots of the CFX containing between 30 and 100 yl were added to a cell containing deoxygenated supporting electrolyte, When the accumulation was completed quanti- fication was performed by standard addition method under the optimized conditions. 2.3.3.2. Procedure for human urine analysis in HPLC ‘method. Urine samples which were initially at a volume ratio 1:10 and filtered using 0.2m filters were subse- quently diluted ten times. Aliquots from these samples were diluted with BR bulfer pH 2.8, injected into the HPLC, analyzed and chromatographed under the condition given above 3. Results and diseussion 3.1, Influence of pH of the supporting electrolyte CFX chemically is 5-thia-I-azabieyclo[4.2.0joct-2-cne-2- carboxylic acid, 3-[(acetyloxy)methyl}7-[(2Z){2-amino-4- thiazolyl) (methoxyiminojacetyl] amino}-8-ox0-, (6R,7R)- (8C), (Scheme 1). The voltammetric behavior of CFX has been examined in the pH range 1.8-12.0 by using CV and DPV. Some, representative, cyclic voltammograms of 1x10-*M CFX in BR buffer solutions of pHs 2.75, 6.00, 9.00 and 11.00 are presented in Fig. I(a) and (b). In an acid medium (curve 1), one well formed peak (I) at 0.5 V and the other one, not well developed (V) at more negative potential (1.0 V) are evident. Those two peaks He hn 0 Fy mit e CH;0 oe ono Scheme 2s. 3 08. « as as } eM Fig. 1. The representative CV cures of 1 10" M of CFX in BR bul: (a) curve 1, pH 2.75; curve 2, pH 60 and (b) curve 3, pH 90; curve 4, pH TO, Sean rate 100 mV 5! are due to four electron reduction of the methoxyimino group, and two electron reduction of the unsaturated ‘C=C bond at C-3 position, respectively. In the pH range from 5.5 to 10 (curves 2 and 3), peak I splits into peaks Il and III, while peak V disappears In the alkaline medium, pH > 10, peak IV appears at negative potential E~ ~1.5 V, as a result of the reduction of the unprotonated form of the methoxyimino group [1]. The effect of the pH on the peak current and potential is presented in Fig. 2. The dependence of the peak I current ¥s. pH shows maximum at pH 2.8, The negative shift of the peak I potential towards more negative values with increasing the pH indicates that protonation step precedes the electron transfer [19]. The nature of the reduction pro- cess was studied by following the effect of the scan rate on. the peak current. Both i, vs. v/? and i vs. v dependencies gave non-linear plots, indicating that the diflusion-con- trolled process is strongly influenced by the adsorption. In the pH range from 5.5 to 10 where the splitting occurs (peaks IT and IID, both peak currents are pH dependent, and the obtained curves show the similar shape with max- imum at pH 9.25. Peak potential of both peaks shiftsMAM, Alehsié, V. Kapetaovié {Journal of Eleetoanaltcal Chemisry 593 (2006) 258.266 261 18 10 o) Ep Pee ee eee © pH Fig. 2. The influence of the pH on: (a) peak current and (b) peak potential response for 1.10" M of CFX in BR baler pH 2-12, towards more negative values with the increase of the pH. Compared to peak I, the AE,/ApH for those two peaks smaller. This indicates the slower protonation process at pH > 5.5, which is the main reason for the spliting (1) Since pH does not influence both current and potential of peak IV (Fig. 2) this confirms that the reducible specie in this case is the unprotonated form of the methoxyimino group [1] Reduction of the unsaturated C=C bond in the C-3 position is presented with peak V, whose peak current and potential are pH dependent (Fig. 2), and the effect of the scan rate shows the usual behavior for the diffusion controlled process. The irreversibility of all reduction processes was con- firmed at HMDE, since no anodic peak was observed in whole pH range. Since the best shape and sensitivity of the peak current ‘was obtained at pH 2.8 for peak I, and at 9.25 for both peaks IT and III, those pH values are the most convenient for further analytical determination, Due to the negative reduction potential of peaks IV and V,, those peaks were not suitable for analytical purposes. 3.2, Adsorptive character of the drug Adsorptive character of the drug was confirmed by recording the eyelic voltammograms of 2x 10° M CFX at pH 28 (Fig. 3a)) and 9.25 (Fig. 3(b)) without precon- centration, fgg =0, and after preconcentration of 30s and then its repetitive cycle at the same mercury drop. ‘Afier preconcentration of the drug onto the electrode sur face, higher peak current was achieved, whereas the second cyele at the same mercury drop showed lower peak inten- sity due to the desorption of the CFX from the mercury surface. According to Fig. 3, itis evident that after the accumu lation at both pH values (2.8 and 9.25) current increase is, obtained at reduction potential of 0.5 V. Concerning, the acid medium, it is in the agreement with results obtained without the preconcentration. At pH 9.25, how- ever, besides peaks II and III (Fig. 1) new peak at 0.5 V arises after the CEX adsorption at the mercury surface. Investigating the effect of increasing CFX concentration 06 420 «0 $00 o (mv) Fig. 3. Cyeie voltammograms of (a) pH 28, and (b) pH 9.25. 1: BR buffer, 2: 210M. CFX without preconcentration fue =0; 3: 2x 10-°M CEX after preconcentration of 30:4: its repetitive yee at the sume mercury deop (#= 100 mV and Fux =-0220, MM, Aleks, V. Kapetanoé | Jounal of Electroanalytical Chemisory $03 (2006 ‘on those three peak currents it was concluded that peaks I and III, although showing higher currents, do not increase linearly, On the contrary, the new peak at —0.5 V showed linear increase with the CFX concentration, and therefore ‘was selected for further analytical work. 3.2.1. Choice of the applied mode (waveform) In order to achieve the high sensitivity for monitoring of the accumulated drug, the cathoding adsorptive stripping, voltammograms of Sx 10-7 M CFX were recorded in BR buffer (pH 2.8 and 9.25) at an accumulation potential of —0.2 V, followed by preconcentration of 1205s, using wave- forms: linear sweep (LS), differential pulse (DP) and square-wave (SW) as shown in Fig, 4. The signal intensity ‘of SW adsorptive stripping voltammetry (SWAdSV) was found to be 8.5 and 17 times higher than those of the DPAGS and LSAdS voltammetry, respectively. Well defined and much larger analytical signal obtained by SWV related to DPV after an accumulation step was the reason that the former was chosen as a preferable tech- nique in further work: 3.2.2. Optimization of the SW parameters Several experimental parameters were examined in developing a suitable analytical procedure for the determi- nation of CFX, such as frequency (/), scan increment (As) and pulse amplitude (Ey). The study was done with 1x 10-7 M CEX in BR buffer (pH 2.8). At a constant sean increment As =4 mV and a pulse amplitude of 50 mV, the peak current intensity increased linearly over the frequency range 10-100 Hz, following the relationship ip (WA) = 0.06013/ (Hz) — 0.446 (r = 0.99913), n = 5. The frequency ‘of 100 Hz was chosen as the best one. a ‘ a * = z : 2 2 lira 3 ean res ad ew) el aeealtz aes aaa iste alee cera mnt (= 125M, As=SmV, Bow 1208 at Baa = ~02V. 25m) after the preconcentration of 266 At frequency of 100 Hz, and pulse amplitude of S0 mV, the peak current intensity increased linearly with the scan increment up to 4mV following the relationship i, (uA) =0.4126As (mY) +5451. (r= 0.9844), n=4. The scan increment of 4 mV was further applied. The increase of the wave amplitude increases the current intensity and the linearity was achieved up to 50 mV. This value was chosen as the optimal one for further analytical purposes Besides those three parameters, the influence of drop size, stirring rate and rest time on the peak current were also investigated. The chosen working conditions were: drop size of 60 au, stirring rate of 300 rpm and rest time of 10's. 3.2.3. Effect of accumulation potential and time To determine the optimal accumulation potential the potential range of 0.0 to —1.0V was examined for 1x 107 M CEX in BR buffer at different pH in the range from 2 to 3, and from 8.5 to 9.5 after the preconcentration period of 60 s. The best results were recorded at pH 2.8, and pH =9.25 both at deposition potential Eycc— — 100mV (Fig. 5). The dependence of peak current on accumulation time was studied at wo concentrations levels of C 1x 10°7M and 110M, The peak current increased with increasing accumulation time. The adsorptive satura- tion was achieved for periods longer than 30's, in the case of lower and 20 s for higher concentrations. Hence, an accu- mulation time of 30 s and 20s were chosen to evaluate the best conditions for the method proposed. 3.3. Analytical parameters The calibration curves were constructed for CFX in BR buffer and in the urine sample, both at pH 2.8 and 9.25, under the optimal chosen conditions. Concentration range ‘and regression equations are presented in Table 1. As seen, ‘a decrease in the slope of the calibration graph for the CPX at pH 9.25 (compared to pH 2.8) is obtained. A decrease in the slope was also obtained in the presence of human urine, comparing with the calibration obtained for pure CFX, especially in the acid medium, The limits of detection (LOD) and quantification (LOQ) were calculated using the relation 3$Da/b and 10SDa/b for LOD and LOQ, respectively, where SDa is the standard deviation of the intercept and b is the slope of the calibra- tion curve. The LOD and LOQ values, summarized in Table 1, indi- cate the higher sensitivity of the proposed procedure in acid medium, both for CFX in BR and in urine samples, The comparison of the obtained LOD (0.8 ng ml-') and LOQ (2.7 ng mI!) values using electroanalytical procedure with, those reported in the literature [10] confirms the higher sen- sitivity of the proposed method. These values confirmed the sensitivity of the proposed method for determination of CPX. The repeatability of the proposed method was deter- mined for CFX concentration of 5x 10-7 M (pH 2.8 andMAM, Alesié, V. Kapetanovié {Journal of Eleetoanaltical Chemisry 593 (2006) 258.266 oe S} Fig, 5, Dependence ofthe SWAdSV peak curren of 1x 10-7 M CEX on pH ebtained at diferent accumulation potentials: (a) in acid solutions pl 2-3: (b) in alkaline solutions pH 85-95 (= 100 Hz As=4mnV, Egy = SOV, f= 605) 9.25) in the presence of the urine by performing 10 indepen- dent measurements. The relative standard deviation (RSD) for CFX in the presence of urine was found to be 1.4% (pH 2.8) and 1.6% (9.25). The precision of the SWAdSV method in urine sample was checked for CFX concentration of 100 ng mI! and 250 ng mI~' both at pH 2.8 and 9.25, Five determinations were performed in all cases, and the RSD were 2.1% and 1.5% (pH 2.8) and 2.3 % and 1.6% (pH 9.25) for CFX of 100 ng mi-! and 250 ng ml-', respectively. Since the main active metabolite of CFX is desacetilcefotaxime (DCFX), the real urine sample was taken into the consideration. The real urine sample was obtained from the patients after the administration of Tolycar injections. After the urine sample preparation (given in Section 2.3.3.) the SW vol- tammetry and HPLC investigations were performed, simul- taneously and the results obtained are given in Fig. 6(a) and (b). Curve | (Fig. 6(a)) represents the real urine sample obtained 60 min after administration, with two peaks at Ey 0.330 V and —0.620 V. The peak at ~0.620 V was already established as a consequence of the CFX reduction, and the first peak was supposed to be from its active metabolite DCPX, since its increase was obtained by addi- tion of the DCFX. After the DCFX addition (curve 2) Table | ‘of CFX in BR bulfer and urine samples £00 (M) W_LOD aM) SD UAy R Regresion equa Linear concentration range (M) g Sex 10 807% 2.00% 10-8 TISx10" Dea 10-8 62710 1 Is 7 's P= 0776-4 5278 % 10x (00326 + 2.682 10s P= 0052543035 12% 0-712% 10 —05s 28 925 a 133.10 73x10 ea 5.00% 0 a E 2 q 2 5 z 2 Urine samples 9.6% s 8 & 26264 MLM, Aleks, V. Kapetanoré | Jounal of Eletroanalytical Chemisory 93 (2006) 258-266 peak at -0.33 V increased significantly (92%), but at the ‘same time peak at 0.620 V increased about 14%. This is, expected, since after the recommended procedure of alka- line hydrolysis (17), less than 20% of CFX remains non- hydrolized. These findings are checked by HPLC method from the same solutions, simultaneously, and shown in Fig. 6(b). The first chromatogram was obtained from real urine sample 60min after the drug administration, and the second one after the addition of CFX and DCFX. Peaks of CFX and DCFX are well separated The results obtained by SW voltammetry with real urine sample show good selectivity of the proposed method for the CFX determination over its active metabolite DCFX, since the peaks of CFX and DCFX are well separated, and there is no chanee for DCEX to superpose to analyti- cal signal of CFX at the chosen potential. This is confirmed by the HPLC method. 48 2 3 10 ° 0 200 400 600-800 1000” -1200 @ E (mv) The robustness [20] of the method proposed was exam- ined by evaluating the influence of small variations of some most important operational parameters such as pH, accu- mulation potential, accumulation time, wave amplitude, ‘wave increment, pulse period, drop size, and stirring speed. ‘on the recovery of the CFX concentration of 1x 10-! M, Difference between the recovery obtained under the chosen experimental conditions and the recovery obtained within the studied range of variation of the operational parame- ters was between 1.6% and 2.4%, The most pronounced influence was observed with the change of wave amplitude and pH, 2.4% and 2.1%, respectively. Since these variables, did not significantly effect the determination of CFX, the proposed procedure can be considered robust. 3.3.1. Analysis of human urine The proposed method was applied to the determination of CFX in spiked human urine using the standard addition, method. Since no response of blank diluted urine was obtained at working potential (Fig. 7, curve 1), the determi- nation of low concentration of CFX in complex urine matrix was possible, At Fig. 7, curves 2-7 denote increasing concentrations of spiked CFX. As a reference method the DPAdSV was used from the same solutions, As a result of those simultaneous determi- nations, the concentration range, linearity of the calibra- tion curves including the SD, SD values of the slope and intercept, LOD and LOQ are summarized in Table 2. The higher slopes of the calibration curves obtained by SWAASY indicate its better sensitivity. The results for CFX determination in urine samples by applying both methods are shown in Table 3. The recovery values obtained with SWAdS voltammetry showed the bet- ter accuracy compared to DPV. Hua) ° 2 7 o min Fig. 6, Results obtained in real urine sample (60 min after administra tion}, at pH 2.8 by: (a) SWAUSV ~ 1 rea urine sample; 2: rea urine sumple after addition of 1% 10°°M DCFX (f= 100 Hz, as= 4m, Foy = SOmV, Eye= 0.1 ¥) (b) HPLC ~ 1: real urine simple; 2: real unie sample after adition of 1 10-* M of CFX and DCFX. 2 oa a8 oe 0 ew Fig. 7. Square-wave adsorptive stripping voltammograms obtained for the increasing CFX coneenteations at pH 28, J: blank urine, sample (0.75 ml Uo. in 15m BR butler: 22 1x10-7M; 3: 2x 10-7M: 4 Bx 107M; 5: 410" Ms 6 SX1D-TM; 7 6x 10-7 M (f= 1001, Ay=4 MV, Fy = 5OMV, Fue = OAV, fee = 308)Table 2 ial parameters for DPAGSY determination of CFX in BR bufler and urine samples st MAM, Aleksié, V. Kapetanovié {Journal of Electr oanal tical Chemisry 593 (2006) 258-266 265 109 (MH) N_LOD iM SD (uA) 0.983 00153, S Regression equation » (nA). (M) Linear concentration range (M) 12x10 i 5 x10 243% 10 29x10 64% 10°% Dot 10° 4 5 5 6 0.9950 0.0049 908 0.990 14x10" 346x108 126x10° 377x10 007s ons nos, nis 9.26% 10% 335 eee oe z x10" U<107-$% 107 055 28 928 £ 15x10 Lax 10% Vct0™?-6% 107 6.0114 Urine samples (0.00137 928 ‘Standard deviation of the intercept; Sy sanded dev tion ofthe slope Table 3 Results obtained by application ofthe proposed methods in urine samples Method pil Spiked Foune RSD (7) Gm) oem!) SWAGSV 28 5D 31 00 495 925 6s 9s 250 245 10) DPAdSV 28 5D 4“ 450 so si6 314 925 6 468 250 240 4d 4. Conclusion ‘The SWAS voltammetry on the mercury drop electrode can be used for determination of CFX at trace levels due to the low detection limit at nano-levels. The sensitivity of the method is increased by effectively accumulated CFX from aqueous solution or urine samples on the mereury surface. ‘The method proposed is fast, precise and simple to per- form. Selectivity of the method related to the main active metabolite DCFX was checked in real urine sample, The real urine sample preparation was the same as the procedure applied in the method proposed for spiked urine. The inter- ferences from urine matrix do not interfere with determina- tion of CFX, so dilution of the urine sample was quite satisfactory and there was no need for further extraction procedures. Therefore, the method can be suggested as a 200d alternative for the routine control in biological fluids. Acknowledgement This work was supported by Ministry for Science and Environmental Protection of Republic of Serbia, Project No, 142071 References [1] V. Kapetanovié, M. Aleksié, P. Zaman, J. Eeetroanal. Chem. 507 (2001) 263-269, PIM. Aleksic, V. Kapetanovig, P. Zuman, Collet. Czech, Chem. Commun. 69 (2008) 1429-1442, [B) P. Zaman, V. Kapetanovie, M. Aleks, Amal, Lett 33 (2000) 2821 2857, [4] M.Ereeg V. Kapetanovig, D.Suzajvié, D. Dumanovié, Microchem. 4.57 (1987) 73. [9] M. 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