voltammetric behavior of mifepristone (ru-486) using square-wave and adsortive stripping-wave...

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Voltammetric Behavior of Mifepristone (RU-486) Using Square-Wave and Adsortive Stripping-Wave Techniques. Determination inUrine SamplesJ. RodrÌguez,* J. J. Berzas, G. Castanƒeda, N. RodrÌguez

Departamento de QuÌmica AnalÌtica y TecnologÌa de Alimentos, Facultad de QuÌmicas,Universidad de Castilla-La Mancha,13071 Ciudad Real, Spain*e-mail: [email protected]

Received: March 13, 2003Final version: May 6, 2003

AbstractThe behavior of Mifepristone (RU-486) was studied by square-wave technique, leading to two methods for itsdetermination in aqueous samples and urine samples at pH 2. The application of the square-wave (SW) without theadsorptive accumulation and stripping voltammetric (AdSV) show the maximum response at �0.896 V using anaccumulation potential of �0.5 V. The effect of experimental parameters that affect this determination are discussed.For the stripping technique, Mifepristone proved to be more sensitive, yielding signals four times larger than thoseobtained by applying a square-wave scan without the previous accumulation. The calibration plot to determineMifepristone was linear in the range 2.4� 10�8 and 5.4� 10�7 M by stripping mode with an accumulation time tacc of30 s. The relative standard deviation obtained for concentration levels of Mifepristone as low as 2.0� 10�7 M withsquare-wave was 1.17% (n� 10) and with stripping square-wave 2.02% (n� 10) in the same day. The two proposedmethods (SW and SWAdSV) were applied to the determination of Mifepristone in urine.

Keywords: Voltammetry, Stripping voltammetry, Square wave, Mifepristone, RU-486, Urine

1. Introducction

Mifepristone (RU-486) is a member of the 11-substitutednorsteroid family and acts as a progestin and glucocorticoidantagonist. Mifepristone is designated chemically as(11B, 17B)-11-[(4-dimethylamino))phenyl]-17-hydroxy-17-(1-propynyl)-estra-4,9-dien-3one (structural formula in Fig-ure 1). Progesterone is a critical hormone in mammalianreproduction and is essential for the maintenance ofpregnancy. Withdrawal of the influence of the hormone inthe uterus due to its competitive inhibition by Mifepristoneat the receptor site results in menstrual bleeding, disruptionof placental function and disruption of the inhibitory effectsof progesterone on the myometrial stimulatory actions ofprotaglandins. These antiprogestational activities of thedrug can result in the termination of early pregnancy.

The proposed indication is for the termination of intra-uterine pregnancy through 49 days gestational age. Theproposed recommended dosage is 600 mg Mifepristone(3� 200 mg tablets) in a single oral dose [1]. After eitheroral administration, Mifepristone is excreted in urine(approximately 10% of the administered oral dose).Termination of pregnancy for medical reasons in the

second trimester can be performed either by surgicalmethods involving cervical dilatation and uterine evacua-tion, or by medical means using prostaglandins. However,surgical procedures expose patients to the risks of cervicaldamage and uterine perforation, which increase withduration of gestation. The use of Mifepristone reduce thetime to expulsion of the fetus (around 7 hours after itsadministration ). Also the use of Mifepristone reduces theprostaglandin requirements to the fetal expulsion, as is theduration of stay in the hospital.The discovery of Mifepristone, the first antiprogesterone

molecule, was a major breakthrough in reproductivemedicine.A HPLC method [2] is described for the Mifepristone

analysis in plasma after previous extraction step withhexane: CH2Cl2 and using as mobile phase methanol/H2O/acetonitrile with measuring at 302 nm.In a theory study, adsorptive voltammetric has been

demonstrated to be a useful technique [3] for the determi-nation of Mifepristone. The information about this paper islimited because it is only available in Chinese.Fig. 1. Structural formule of Mifepristone (RU-486).

661

Electroanalysis 2004, 16, No. 8 ¹ 2004 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim DOI: 10.1002/elan.200302865

Gu et al. [4, 5] has described two different biosensors forMifepristone determination by using modified metallic orcarbon paste electrodes. These were applied to tablets withsatisfactory results.This paper shows thatMifepristone is a drug,which suffers

a strong absorption process onto a mercury electrode. Byusing this phenomenon and by accumulation of this com-pound at hanging mercury dropping electrode (HMDE)prior to square-wave voltammetric measurment, highersensitivites can be readily achieved. The influence of severalexperimental (pH, ionic strength) and instrumental (stepincrement, pulse amplitude, frequency, drop size) variableson the square-wave and on the stripping square-waveresponse has been evaluated. The proposed method wasapplied in the determination of Mifepristone in humanurine.

2. Experimental

2.1. Materials

All solvents and reagents were of analytical reagent gradeunless indicatedotherwise. Solutions for voltammetric studywere made with deionized water (Milli-Q quality). Stocksolutions of 0.012 MHClO4 were prepared using suprapuregrade reagent.Mifepristone was obtained from Sigma. Standard solu-

tions (144.3 mg/L) were prepared in ethanol and stored at4 �C. Diluted solutions of Mifepristone were prepared dailyin ethanol.

2.2. Apparatus

Voltammograms were obtained with a Princeton AppliedResearch (PAR) (Princeton, NJ) Model 384B polarograph-ic analyzer combined with a PAR Model 303a hangingmercury drop electrode (HMDE) using an Ag/AgClreference electrode, A PAR Model 305 magnetic stirrer, aHP computer, using 384 software, and a HP 510 printer.Cyclic voltammograms were obtained with a PARModel

264A polarographic analyzer/stripping voltammeter com-bined with the PAR 303A stand and a Yokogawa 3022 A4X-Y recorder. A crison micropH 2002 was used for the pHmeasurements.

2.3. Procedure

For voltammetric experiments, 10 mL of perchloric acid(12 mM) was purged with oxygen-free nitrogen for 10 min(and for 2 min in subsequent runs). The required accumu-lation potential (Eacc) of �0.5 V was then applied to theelectrode for a selected accumulation time (tacc� 30 s) whilethe solution was stirred at 600 rpm and after a 30 s restperiod a square-wave scan started in the negative direction(stripping square-wave method).

3. Results and Discussion

Thepreconcentration ofMifepristone at theHMDEand theapplication of a subsequent differential pulse voltammetric(DPV) scan in the negative direction gave rise to a strippingreduction peak (Ep��0.776 V) at pH 2.0 (HClO4) (Fig-ure 2). As can be seen in Figure 2, a high response isobtained only after 10 s of accumulation time for an assayedconcentration of 2.0� 10�7 M of the drug. On the contrary,the solution phase response (direct response, tacc� 0 s)shows a very small reduction peak as expected for such aslow concentration. These facts indicate that the Mifepri-stone undergoes an adsorptive preconcentration at themercury electrode before its reduction process.In Figure 2 it can be readily seen that a square-wave form

applied to the stripping of the adsorbate yields highanalytical signals (the more sensitive reduction peak wasplaced at Ep��0.896 V, Ip� 479 nA), compared withapplying a differential pulse wave form (Ep��0.776 V, Ip� 66 nA) after an accumulation step of 10 s. It has beenreported by different authors [6 ± 9] that an analyticallyuseful polarographic response for steroids containing aconjugated carbonyl group at the ring is seen. The reductionprocess probably occurring at the �C�O bound of theestradien ring.The nature of the electrochemical reduction process was

also studiedby cyclic voltammetry (CV). Figure 3 shows twodifferent cyclic voltammograms for a 5.04� 10�6 M solutionof Mifepristone without accumulation and after a step of10 s of accumulation time (scan rate 100 mV/s). The

Fig. 2. Differential pulse (DP) and square-wave (SW) adsorptivestripping voltammograms from a solution containing 2.0� 10�7 Mof Mifepristone at pH 2. Accumulation time: a) 0 s and b) 10 s.Experimental conditions: (DP) accumulation potential �0.5 V,equilibration time 10 s, scan rate 10 mV s�1, pulse amplitude�40 mV, scan increment 6 mV and step/drop 0.6 s; (SW) accumu-lation potential �0.5 V, equilibration time 10 s, frequency 100 Hz,pulse amplitude �40 mV and scan increment 10 mV.

662 J. RodrÌguez et al.

Electroanalysis 2004, 16, No. 8 ¹ 2004 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

reduction process (Ep��0.850 V) was not accompanied byanodic waves, which indicates that the redox reactions aretotally irreversible. When the potential was scanned atincreasing rates from 10 to 100 mV/s, under the sameexperimental conditions, a linear relationship was observedbetween thepeak intensity Ip (nA) and the scan rateVb (mV/s), demonstrating that the phenomenon is adsorption-controlled.

Ip� (60.27� 7.01)� (1.16� 0.02) Vb

(r2� 0.998, n� 9, tacc� 0 s)

Apart from this increase in the analytical response caused bythe use of higher scan rates, the significant improvement insensitivity achieved by employing a square-wave scan can bedescribed in terms of the characteristic parameters govern-ing this technique.The influence of pH on the voltammetric response of

Mifepristone was studied. In order to establish a suitablepH, a range of values was examined between pH 1.0 and 7.2.We checked for higher values of pH 2.0, the solution ofMifepristone shows a instability with the time. For thisreason, a value of 2.0 (HClO4) was considered suitable andthe methods were developed at this pH value.

3.1. Square-Wave Voltammetry SWV

The checked SW parameters were the ionic strength of thesupporting electrolyte, the initial potential, the pulse height,the frequency and the pulse increment. These parametersare interrelated and have a combined effect on the responsebut only the general trends will be examined.

The ionic strength of the supporting electrolyte (HClO4-NaClO4) was studied in the range from 0.01 to 0.14 M for a2� 10�7 M of Mifepristone solution. A low ionic strength(0.01 M) was seen to yield the best and large response asexpected in adsorptive phenomena. The peak potential (Ep)stayed constant in all this experiment. The lowest ionicstrength was very effective for the adsorptive accumulationof theMifepristone on the electrode as described byWang inthe behavior of riboflavine in a medium of 1 mM NaOH[10].In order to find the optimal conditions, experiments were

carried out for different pulse amplitudes (10 ± 60 mV)showing that there is a magnification of peak intensity whenthe pulse amplitude increased from 10 to 40 mV. For thelarger pulse, the peak-width at half-height increased and anamplitude of 40 mV was adopted as optimum.The influence of the scan rate on the analytical signal was

studied varying �s from 2 to 10 mV for a given samplingpulse (40 mV) an a frequency (100 Hz) constant. A linearrelationship between peak intensity, Ip, and �s was found.Due to an increase in the scan increment resulting in biggerpeaks, the best signals were achieved at 10 mV.The dependence of the peak intensitywith the frequency f

(Hz) follows the equation:

Ip� (9.73� 5.64)� (0.59� 0.07)f r2� 0.998

This study was done for a constant scan increment of 6 mV,and the results, as expected, showed a linear relationshipbetween peak intensity and frequency for the whole rangestudied (10 ± 120 Hz). A value of 120 Hz was chosen assatisfactory.The square-wave signal increased with the electrode area.

As the noise level that accompanied the use of a largerelectrode surface was not significant, the remainder of thestudy was carried out using the largest electrode area(0.025 cm2) that provided the most sensitive signal and thebest signal-to-noise ratio.From these studies, the optimal conditions for the

determination of Mifepristone were established as dropsize� 0.025 cm2 (large), �E� 40 mV, f� 120 Hz, �s�10 mV, Eacc��0.5 V.Voltammograms at different concentrations of Mifepri-

stone were recorded using the optimal conditions. From thisexperiment, the linearity range was tested between 2.4�10�8 and 5.4� 10�7M and the regression line, calculatedusing the least-squares method, was:

I� (15.25� 9.65)� (6.51� 108� 3.67� 107)Cr2� 0.997 n� 8

whereC is the concentration (molL�1) ofMifepristone and Iis intensity peak (in nA corresponding reduction process).The relative standard deviations obtained for concentra-

tion levels of Mifepristone as low as 2.0� 10�7 M withsquare-wave was 1.17% (n� 10) in the same day.

Fig. 3. Cyclic voltammogram obtained for a 5.04� 10�7 M Mife-pristone solution at pH 2. Accumulation time: a) 0 s and b) 10 s.Experimental conditions: accumulation potential �0.5 V, equili-bration time 10 s, scan rate 100 mV s�1, scan increment 10 mVandstep/drop 0.1 s.

663Determination in Urine Samples


3.2. Square-Wave Adsorptive Stripping Voltammetry(SWAdSV)

Considering the larger response obtained in the stripping ofMifepristone by SWAdSVwith respect to SWV (Figure 2) itwas expected that the SWAdSV might be successfullyapplied to the determination of this drug. Consequently, theparameters governing this voltammetricmodewere studied.Several preconcentration-stripping voltammograms were

recorded for accumulation potentials varying from �0.5 to�0.70 v and a tacc of 30 s. Experiments showed a big intensityfor the application of an accumulation potential of �0.5 v,for this reason, it was chosen as optimal accumulationpotential.Further experiments were made varying the pulse ampli-

tude (10 ± 60 mV), scan increment (2 ± 10 mV), frequency(10 ± 120 Hz) and drop size (0.010 ± 0.025 cm2), in order tofind the best analytical signal. The optimal values wereselected as 40 mV, 6 mV, 120 Hz and the larger size of theelectrode, respectively.Different voltammograms with increasing accumulation

times were recorded for a solution containing 2.0� 10�7 Mof Mifepristone using the select conditions. The resultingpeaks showed (Fig. 4) a linear relationship between peakintensity and accumulation time up to 90 s for the studiedsolution. Due to the saturation of the electrode area, thepeak intensity started to decrease but with a smaller slope.Thus, when a certain coverage of the electrode is reached,interactions among the molecules in the adsorbed statebecome noticeable. Calibration plot was constructed using30 s accumulation time.

Voltammograms at different concentrations of Mifepri-stone were recorded using the optimal conditions. From thisexperiment, the linearity range was tested between 2.4�10�8 and 5.4� 10�7 M and the regression line, calculatedusing the least-squares method, was:

I� (13.67� 63.29)� (3.15� 109� 2.48� 108)Cr2� 0.998 n� 7

whereC is the concentration (molL�1) ofMifepristone and Iis intensity peak (in nA corresponding reduction process).The relative standard deviations obtained for concentra-

tion levels of Mifepristone as low as 2.0� 10�7 M withsquare wave was 2.02% (n� 10) in the same day.

3.3. Determination of Mifepristone in Urine

The present optimized procedures were also successfullyapplied for the determination of Mifepristone spiked tourine. 50 �L of centrifuged urine was added to 10 mL ofperchloric acid (12 mM) over the voltammetric cell.Three samples urine were selected from a nonpregnant

woman and two pregnant women at different gestationweeks (18 and 20 weeks respectively), with the object toevaluate the possible differences between them.The linearity of the voltammetric response at two

accumulation times (0 and 30 s) were checked in threedifferent human urines spiked with Mifepristone. Theregression lines, calculated using the least-squares method,for a nonpregnant woman were:

SWI� (�46.46�11.51)� (9.27�108�3.11�107)C r2�0.999;n� 6 (1.44� 10�7 ± 6.46� 10�7 M) (1)

SWAdSVI� (�22.98�31.78)� (3.58�109�1.09�108)C r2�0.998;n� 10 (6.22� 10�8 ± 3.13� 10�7 M) (2)

Pregnant woman A (18 weeks)

SWI� (�137.60�35.56)� (1.15�109�6.19�107)C r2�0.996;n� 9 (2.45� 10�7 ± 6.83� 10�7 M) (3)

SWAdSVI� (�26.14�25.03)� (2.88�109�1.30�108)C r2�0.996;n� 11 (6.22� 10�8 ± 3.27� 10�7 M) (4)

Pregnant woman A (20 weeks)

SWI� (�98.20�72.44)� (1.10�109�1.55�108)C r2�0.994;n� 5 (2.45� 10�7 ± 6.75� 10�7 M) (5)

SWAdSVI� (�16.97�58.02)� (2.25�109�2.77�108)C r2�0.995;n� 8 (6.24� 10�8 ± 3.24� 10�7 M) (6)

Fig. 4. Effect of the accumulation time on the SW adsorptivestripping signal for a solution containing 2.0� 10�7 M of Mifepri-stone (pH 2). Accumulation time: a) 0 s; b) 180 s. Experimentalconditions: accumulation potential �0.5 V, equilibration time 10 s,frequency 100 Hz, pulse amplitude �40 mV and scan increment10 mV.



Where I represents the peak intensity (in nA), C theconcentration united (in Molar) of Mifepristone and r2

denotes determination coefficient.An analysis of variance (ANOVA) test was performed to

compare the different regression lines obtained, to deter-mine whether the data could be combined to enableestimation of appropriate quantities by use of a compre-hensive regression line [11, 12]. The analysis of variancevalues are show in Table 1. If the experimental value of F isless than the theoretical value there are no significantdifferences between variances whereas if Ftheor is less thanthe experimental value of F there are significant differencesbetween variances. The t-test was carried out to compare thedifferent slopes of the calibration plots. If the experimentalvalue is less than the theoretical value there are nosignificance between slopes.As can be seen in Table 1, significant differences were

found between the calibration plots performed in womanand pregnant woman (18 weeks) and between the regres-sion lines performed in pregnant women urine by SWAdSVtechniquebut no significant differenceswere foundbetweenthe two pregnant women urine by SW technique, butsignificant differences were found by stripping techniques.This fact could be due to changes in thematrix composition.(change of ionic strength).

As consequence of the previous studies, the determina-tion of Mifepristone can not be performed by directmeasurement from the calibration plot as it changes infunction of the woman . For this reason, in all cases, thestandard addition method must be used.In order to test the accuracy of the proposed method,

several aliquots of Mifepristone standard solutions wereadded into urine samples in different proportion. Thevoltammogramswere recorded using the optimal conditionsfor their analytical determination. In all cases the standardaddition method was used for the drug determination.Figure 5 shows the square-wave voltammograms corre-sponding to 2.15� 10�7 M of Mifepristone in urine at pH 2and different accumulation times a) 0 s; b) 30 s. As can be

Fig. 5. Square-wave voltammograms corresponding to 2.15� 10�7 M of Mifepristone in urine at pH 2 and different accumulation timesa) 0 s; b) 30 s. Experimental conditions: accumulation potential �0.5 V, equilibration time 10 s, frequency 120 Hz, pulse amplitude�40 mV and scan increment 10 and 6 mV for 0 and 30 s, respectively.

Table 1. Analysis of Variance (ANOVA).

Comparison Regression Line Fexp Ftheor texp ttheor

SW (Pregnant and nonpregnant) 10.650 6.260 6.797 2.586SWS (Pregnant and nonpregnant) 3.648 3.370 5.212 2.420SW (at different gestation weeks) 1.080 5.410 1.697 2.310SWS (at different gestation weeks) 1.350 3.860 6.434 2.180

Table 2. Recoveries in urine samples.

Sample Mifepristone [a] Recovery(%) SW

Recovery(%) SWAdSV

S1 1.25� 10�7 92 88S2 1.84� 10�7 76 108S3 2.15� 10�7 46 96S4 3.04� 10�7 57 107

[a] Concentration (mol L�1) of Mifepristone in the voltammentric cell.

665Determination in Urine Samples


seen awell-defined voltammetric peakwas obtained in bothcases.The results obtained for the determination of Mifepri-

stone in urine samples are given in Table 2. As can be seen,for the drug determination, better recoveries were obtainedby square-wave adsortive stripping voltammetry thansquare-wave voltammetry. The worst recoveries obtainedin urine samples (by SW method) may be due to thepresence of electrochemical active compounds in the urinebut they have no adsorbtion properties on the electrodesurface [13]. For this reason, the results obtained bySWAdSVare always satisfactory.

4. Conclusions

This work shows that the Mifepristone concentration inhuman urine can be determine by using voltammetrictechniques on the basis of their reduction process corre-sponding to the keto group (�C�O ) present in the ring overthe hanging mercury drop electrode. This adsorptivebehavior provides a useful tool for their detection andquantification at low levels of concentration in biologicalfluids.This technique could be used for monitoring the levels of

Mifepristone in clinical samples due to the dangerous effectof very high concentrations of this hormone or prostaglan-dins used in the abortion hospitalary process such as uterinerupture, excessive bleeding with the need of transfusion,high decrease of hemoglobin.It is obvious that the Mifeprsitone can bring the develop-

ing countries a tremendous improvement in abortiveprocedures, provided the therapy is used where surgicalfacilities exists but are over-stretched by the enormousdemand.

5. Acknowledgements

The authors are also grateful to the DGES of theMinisteriode Educacio¬n y Ciencia for the financial support (ProjectBQU 2001-1190).

6. References

[1] M. D. Mognilewsky, D. Philibert. Biochemical Profile ofMifepristone in the Antipropestin Steroid RU 486 and HumanFertility Control (Ed.: E. E. Baulien, S. J. Segal) PlenumPress, New York 1985, pp. 87 ± 97.

[2] C. Gao, Z. G. Zhao, Y. Wang, S. P. Ren, Yaowu-Fenxi-Zazhi2000, 20, 119.

[3] Y. H. Zeng, G. R. Zhang, Fenxi-Huaxue 1997, 25, 1278.[4] J. Z. Xu, J. J. Zhu, Y. L. Zhu, K.Gu, H. Y. Chen, Anal. Lett.

2001, 34, 503.[5] J. Z. Xu, J. J. Zhu, Y. L. Zhu, K.Gu, Fresenius× J. Anal. Chem.

2000, 368, 832.[6] H. S. de Boer, J. den Hartigh, H. Ploegmakers, W. van Oort,Anal. Chim. Acta 1978, 102, 141.

[7] B. Z. Chowdhry, in Polarography of Molecules of BiologicalSignificance (Ed.: W. F. Smyth), Academic Press, London1979, pp. 173.

[8] M. Bond, I. D. Heritage, M. H. Briggs, Anal. Chim. Acta1981, 127, 135.

[9] J. J. Berzas, J. RodrÌguez, G. Castanƒeda, Electroanalysis 1999,11, 268.

[10] J. Wang, D. B. Luo, P. A. M. Farias, J. S. Magmoud, Anal.Chem. 1985, 57, 158.

[11] P. D. Lark, B. R. Craven, R. C. L. Bosworth, The Handling ofChemical Data, Pergamon Press, Oxford 1968, ch. 4,pp. 136 ± 206.

[12] D. L. Massart, B. G. M. Vandeginste, S. N. Deming, L.Kaufmann, In: Chemometrics: A Textbook, 1st ed. (Eds.:B. G. M. Vandeginste, L. Kaufmann), Oxford UniversityPress, Oxford 1988.

[13] J. Wang, in Electroanalytical Chemistry, Vol. 16 (Ed: A. J.Bard), Marcell Dekker, New York 1989, p. 39.



voltammetric behavior of mifepristone (ru-486) using square-wave and adsortive stripping-wave...

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