Analyst, March 1996, Vol. 121 (351-356) 35 1

Determination of Trace Levels of Niguldipine in Urine and Blood by Adsorptive Stripping Voltammetry at the Hanging Mercury Drop Electrode

Gottfried Stubauer and Dagmar ObendorfY‘ Institut fur Analytische Chemie und Radiochemie der Universitat Innsbruck, innrain 52 a, A-6020 Innshruck, Austria

A relatively simple electroanalytical procedure for the determination of niguldipine in biological samples is described. The technique involves adsorptive accumulation of the drug at the hanging mercury drop electrode (HMDE) followed by a differential-pulse polarographic determination of the preconcentrated species. The adsorptive stripping response is evaluated with respect to various experimental conditions, such as solvent composition and pH of the supporting electrolyte, accumulation potential and accumulation time. After a simple sample preparation, the method can be used for the determination of niguldipine in blood and urine. Interfering substances are simply removed by precipitation, adding a small amount of 5% ZnS04 solution and ethanol to the urine or blood sample and centrifuging the mixture. A limit of detection of 6.7 ng per ml of urine and 41 ng per ml of blood is found with a mean recovery of 96% in urine and 71% in blood. The mean relative errors are 8.4% and 2.2%, respectively. Keywords: Niguldipine; 1,4-dihydropyridine calcium antagonist; adsorptive stripping voltammetry; urine; blood

Niguldipine (Fig. 1) belongs to the group of 4-nitrophenyl substituted 1,4-dihydropyridine calcium antagonists, which have become very important in the treatment of cardiac diseases like hypertension and angina pectoris. Recently, further activ- ities of some dihydropyridine calcium antagonists such as niguldipine have been reported; 1,2 for example, they are able to inhibit the transport of other drugs by P-glycoprotein. As a consequence these calcium antagonists seem to be useful candidates for the treatment of multi-drug resistance in cancer patients. However, unwanted side effects can be produced by the calcium channel blockers and the monitoring of drug level has become important, especially in this case where the drugs have narrow therapeutic ranges and when accurate dosing is essential to produce the desired pharmacological response with the minimum of toxic and other undesirable side effects.

CO,-CH,-CH,-CH,-N

ti Fig. 1 Structure of niguldipine.

* To whom correspondence should be addressed.

In case of the 1,4-dihydropyridine calcium antagonists, several analytical methods have been developed for the above purpose, including gas chromatography with different detection modes such as mass spectrometric detection, electron-capture detection (ECD), nitrogen or flame-ionization detection, as well as high-performance liquid chromatography with spectrophoto- metric or electrochemical detection.3.4 Electroanalytical tech- niques, especially modern pulse techniques, such as differ- ential-pulse voltammetry and square-wave voltammetry, have been used for the sensitive determination of a wide range of pharmaceuticals5 with the advantages that there is, in most instances, no need for derivatization, and that these methods are less sensitive to matrix effects than other analytical techniques. Thus, the sensitive determination of compounds, even in complex biological matrices, is possible without tedious extraction procedures being necessary before the voltammetric measurement.

Since 1,4-dihydropyridine calcium antagonists possess at least two electroactive centres, a reducible aromatic nitro-group and an oxidizable dihydropyridine ring, they can be analysed electrochemically. A few reports”* 1 concerning the electro- chemical behaviour of nifedipine and related 1,4-dihy- dropyridines have appeared in the past few years. However, no electrochemical data are available for niguldipine. The aim of the present work was the development of a sensitive electro- analytical procedure for the determination of niguldipine in complex biological media, such as blood and urine, without an extraction procedure being necessary prior to the voltammetric measurement. In our studies we investigated the redox behav- iour of niguldipine at different electrodes and in different media in order to find out the best experimental conditions for the electroanalysis of niguldipine. The results of this study, as well as the application of the elaborated procedures to the determina- tion of niguldipine in blood and urine, are presented in this paper.

Experimental Apparatus

Differential-pulse voltammetry and adsorptive stripping experi- ments were carried out on a PAR 264A (EG&G Princeton Applied Research) stripping analyser. Cyclic voltammetry was performed with a HEKA potentiostat/galvanostat PG 28 system. Voltammograms were recorded on a Philips PM-8 133 X-Y recorder. For reductions a PAR 303A static mercury drop working electrode, an Ag/AgCl (saturated KCl) reference electrode and a platinum wire auxiliary electrode were used. Oxidations and cyclovoltammetric measurements were per- formed in a conventional three-electrode cell, consisting of a rotating-disc working electrode with different disc materials (Pt, C, Au) (diameter 3 mm, Metrohm Herisau, Switzerland), a glassy carbon counter electrode and a silver/silver chloride

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352 Analyst, March 1996, Vol. 121

(KCl, Friscolyt, Ingold GmbH, S teinbach, Germany) reference electrode. The cell was covered with aluminium foil to prevent decomposition of the drug in daylight. Magnetic stirring (reductions) or rotating of the electrode (oxidations) was employed during the preconcentration period. All measure- ments were performed at room temperature.

Reagents Niguldipine was kindly provided by the Institut fur Biomedizi- nische Pharmakologie (University of Innsbruck). Stock solu- tions of niguldipine (2.6 X 10-3 mol 1-1 in 50% MeOH) were stored for 1 week in the dark under refrigeration to avoid decomposition. For internal standard additions the stock solution was diluted daily with the corresponding supporting electrolyte (buffer-methanol mixture) to give a niguldipine standard solution of about 1 X 10-6 mol 1-1 concentration.

The supporting electrolytes were phosphate buffer solutions of different pH and a final ionic strength of 0.05 mol 1-1. The composition of the buffer solutions was calculated with Acid- Base (a Journal of Chemical Education) software and the preparation was accomplished by dissolving the appropriate amount of phosphate salts, Na3P04. 1 2H20, NaH2P04-2H20 and Na2HP04 (Fluka, Buchs, Switzerland and Merck, Darm- stadt, Germany, high purity) in purified water. The pH value of the final solutions was controlled with a glass electrode and a calibrated pH-meter. A small amount of methanol or ethanol (Fluka, puriss. p.a.) was added to the supporting electrolyte whenever modification of the adsorption properties of niguldi- pine was intended. Urine and blood samples were obtained from healthy volunteers. Blood samples were heparinized to prevent coagulation. All samples were kept in the dark at 4 "C.

Procedures

Adsorptive stripping diflerential-pulse voltammetry A 5 ml volume of buffer and 5 ml of purified water (or a mixture of water and organic solvent) were added to the polarographic cell (final ionic strength, 0.05 mol 1-1) and de-aerated by passage of nitrogen or argon for 8 min. A preconcentration potential (-0.4 V) was applied for a selected period of time while the solution was stirred. After a 15 s rest a differential- pulse scan (pulse amplitude 50 mV, scan rate 5 mV s-') towards more negative potential values was started in order to obtain the stripping voltammograms. Before adding a specific amount of sample to the solution a stripping voltammogram of the blank supporting electrolyte was recorded under the same conditions. Determination of the niguldipine concentration was accomplished by the method of internal standard additions.

Adsorptive stripping voltammetry in urine

A mixture of 0.5 ml of urine and 0.5 ml of ethanol was adjusted to pH 1 1 by addition of 0.1 ml of 1 moll-' NaOH. After having added 0.5 ml of 5% ZnS04 the solution was centrifuged for 10 min at 13 000 rpm in a high speed centrifuge. An aliquot (1 ml) of the clear solution was added to a mixture of 6 ml of phosphate buffer, pH 12, and 1 ml of ethanol. After the solution had been de-aerated for at least 8 min an accumulation potential of -0.58 V was applied for 120 s, and after a 15 s rest a differential pulse scan towards more negative potential values was applied. The determination of niguldipine in urine was accomplished by adding various amounts of niguldipine standard solution to the voltammetric cell (internal standard additions).

Adsorptive stripping voltammetry in blood

A 0.5 ml volume of ethanol and 0.3 ml of 5% ZnS04 were added to 0.3 ml blood and the mixture was centrifuged for 10 min at

13 000 rpm in a high speed centrifuge. An aliquot (0.9 ml) of the clear solution was added to a mixture of 6 ml of phosphate buffer (pH 1 1.7, ionic strength 0.1 mol 1-1) and 1 ml of ethanol and the pH was adjusted to about 12 by addition of 40 p1 of 2 moll-' NaOH. The solution was de-aerated for 8 min and, after a preconcentration period of 30 s at -0.50 V, the adsorptive stripping voltammogram was recorded.

Recovery experiments

In order to carry out recovery experiments a known amount of niguldipine was added to the urine or blood sample prior to the sample preparation. Then the sample pretreatment was per- formed. With an aliquot of the pretreated sample the adsorptive stripping voltammetric procedure was continued as described before. The determination of the niguldipine content in the spiked biological sample was accomplished by adding increas- ing amounts of niguldipine standard solution to the voltam- metric cell.

Results and Discussion No previous electrochemical data were available concerning the redox mechanism of niguldipine. Therefore, several measure- ments with different electrochemical techniques (cyclovoltam- metry, chronoamperometry and differential-pulse voltammetry) were performed using various supporting electrolytes (non- aqueous and aqueous solvents) and working electrodes (Pt, GC, Au, Hg) in order to obtain such information. These studies revealed a good agreement with the redox mechanism postu- lated for similar compounds and other 1,4-dihydropyri- dines6.l*.13 and suggested that niguldipine can be determined electrochemically, either by reduction of the aromatic nitro- group or by oxidation of the dihydropyridine ring. Although niguldipine contains an oxidizable piperidine group, no oxida- tion peak that might be attributed to the oxidation of this group could be detected within the potential window accessible, either in non-aqueous solvents like acetonitrile or in buffer solu- tions.

Further studies revealed that niguldipine adsorbs readily at electrode surfaces and that a considerable increase in sensitivity can be gained by adsorptive accumulation at the electrode surface prior to the voltammetric measurement. Based on these results oxidative as well as reductive adsorptive stripping voltammetric procedures could be developed for the determi- nation of niguldipine. However, compared with reductive measurements at the hanging mercury drop electrode (HMDE), oxidation on a glassy carbon electrode proved to be less sensitive and much more care had to be taken as to surface renewal and reproducibility of the individual measurements. Moreover, the direct determination of niguldipine in biological samples, such as urine or blood, was not possible by oxidative adsorptive stripping voltammetry without extracting the drug from the biological sample. Determination of niguldipine in urine and blood by oxidative adsorptive stripping voltammetry was possible after the sample preparation described in this paper had been applied. However, the sensitivity (limit of detection) was not satisfactory compared with the sensitivity of reductive measurements of niguldipine. Only relatively high concentra- tions of niguldipine (about 5 pg of niguldipine per ml of urine) could be determined. Probably a different sample preparation prior to oxidative measurements would give better results. However, since reduction at the HMDE seemed to be sufficiently sensitive for the determination of niguldipine in urine and blood, our interest was focused on the optimization of the reductive adsorptive stripping procedure. The results of the

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reductive measurements at the HMDE will be discussed in this paper.

Reduction of Niguldipine at the HMDE The reductive determination of niguldipine was performed at a mercury electrode in phosphate buffers of different pH using differential-pulse polarography . Only one irreversible reduction peak between -0.50 (pH 6) and -0.76 V (pH 12) was observed that corresponded to the irreversible 4-electron reduction of the aromatic nitro-group. The peak potential shifted linearly to more negative values with increasing pH [Fig. 2(a)] following the equation: E , (V) = -0.34 V -0.035 pH. This indicates that a transfer of protons is coupled to the reduction process and a higher proton concentration facilitates the reduction process.

The peak height depends strongly on the pH of the supporting electrolyte, increasing with increasing pH [Fig. 2(b)]. The best sensitivity was obtained in alkaline buffer solutions (pH 10-12) so that for the rest of the experiments basic buffer solutions were preferred. The spontaneous adsorption of niguldipine at the mercury electrode can be used as an effective preconcentration step prior to the voltammetric measurement of niguldipine. Various experimental factors influencing the adsorption pro- cess, like pH and composition of the supporting electrolyte, accumulation potential and preconcentration time, were investi- gated in order to optimize the accumulation procedure and thus the sensitivity of the adsorptive stripping voltammetric measurement of niguldipine.

Variation of the preconcentration potential had only a small influence on the peak current intensity if the preconcentration step was carried out in closed circuit at an accumulation potential between -0.20 and -0.50 V. With a preconcentration

Y

a -0.4

80

60

a 5 40

20

0

4 6 7 8 9 10 11 12

PH

. pH1 0.5

50 100 150 200 250 300

Concentration of niguldipinehg ml-’

Fig. 2 Dependence of (a) the reduction peak potential (E,) and (h) the reduction peak current (i,) on the pH of the supporting electrolyte. Differential-pulse polarography; niguldipine concentration = 47-282 ng ml-1 (7.7 X 10-8-4.6 X 10-7 moll-I).

time of 30 s and varying the accumulation potential in steps of -0.10 V, the current magnitudes measured for a solution containing 47 ng ml-1 niguldipine (7.7 X 10-8 mol 1-l) were 4.0,5.0,7.5 and 5.5 nA, respectively. A peak current maximum was obtained at a preconcentration potential of -0.40 V; therefore this potential was chosen as the accumulation potential for all further measurements. The accumulation time, however, considerably influences the stripping peak current. In pure buffer solutions (pH 12) containing 73 ng ml-l (1.2 X 10-7 mol 1-1) of niguldipine, the peak current first increases with increasing accumulation time, reaching a maximum at about 60 s. A further increase in the accumulation time leads to a decrease in the peak height, probably due to saturation of the electrode surface. Using a preconcentration time of 30 s a linear calibration plot in a concentration range between 9.4 ng ml-l (1.5 X 10-8) and 73 ng ml-’(1.2 X 10-7 moll-1) was obtained following the equation: i, (nA) = -2.47 + 0.76 C (ng ml-l) (correlation coefficient = 0.9993). A detection limit (3s) of 2.7 ng ml-1 (4.7 X 10-9 mol 1-1) could be calculated.

Previous studies revealed378 that nitrophenyl-substituted 1,4-dihydropyridine calcium channel blockers are very sensi- tive to daylight. Because the influence of light results in a change in the redox properties of the compound the extent of the photodegradation can be followed electrochemically via a decrease in the reduction peak current and appearance of new reduction waves at a more positive potential. Niguldipine seems to be less light-sensitive, as no degradation could be observed after exposure to daylight for 3 h. Thus, no special precautions are necessary during the voltammetric measurement. However, another effect was noted, especially when very low niguldipine concentrations were measured in pure buffer solutions. Within the first 15 min a decrease in the peak current occurred during repeated measurements, leading to deviations from linearity of the calibration plot. This effect was most probably due to adsorption of niguldipine on the glass wall of the voltammetric cell and could be prevented by using buffer-methanol mixtures instead of pure buffer solutions as supporting electrolyte and by performing the analysis as quickly as possible.

A second effect of adding methanol to the supporting electrolyte (Fig. 3) was a further increase in the stripping peak current with increasing amounts of methanol and thus an increase in sensitivity. A peak current maximum was reached with a supporting electrolyte containing 30% of methanol (or ethanol).

Fig. 4 shows the dependence of the peak current on the preconcentration time for different solution conditions. Com- pared with measurements in pure buffer solutions, a higher peak current is obtained in buffer-methanol mixtures at lower

1601 140

120 1 p 100

80

60 c I I I I I I

0 10 20 30 40

Methanol in the voltammetric solution (%)

Fig. 3 Dependence of the reduction peak current on the composition of the supporting electrolyte. Adsorptive stripping voltammetry at the HMDE: E,, = -0.40 V , tac, = 30 s; niguldipine concentration = 73 ng ml-1 (1.2 x rnol 1-I).

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354 Analyst, March 1996, Vol. 121

niguldipine concentrations (15 ng ml-1 = 2.5 X 10-8 mol 1-1) even if differential-pulse polarography at the HMDE without accumulation prior to the voltammetric measurement is per- formed.

Moreover, the adsorptive stripping voltammetric determi- nation of low niguldipine concentrations in a buffer-methanol mixture permits longer preconcentration times before surface saturation is reached. For a 15 ng ml-1 niguldipine solution (2.5 X 10-8 mol 1-1) a linear increase in the peak height with accumulation time is observed up to 160 s, with a slope of 0.54 nA s-1. Thus, a further increase in sensitivity can be gained. The ultimate choice of preconcentration times will, however, depend on the concentration range studied.

When using an optimized adsorptive stripping procedure in a supporting electrolyte consisting of buffer-methanol (7 : 3 v/v), very low niguldipine concentrations could be determined. In the range between 1.4 (2.3 X 10-9moll-1) and 15 ng ml-1 (2.5 X 10-8 mol 1-l) a linear calibration plot was obtained, following the equation ip (nA) = -7.26 + 5.8 C (ng ml-l) with a correlation coefficient of 0.9992. The lowest concentration measured was 1.4 ng ml-1 (2.3 X 10-9 mol 1-I), with an accumulation time of 120 s at a preconcentration potential of -0.4 V. A detection limit (3s) of 0.7 ng ml-l (1.1 X 10-9 mol 1-1) was calculated. Precision was determined by 5 successive measurements of solutions containing different amounts of niguldipine (15 and 73 ng ml-I) using preconcen- tration times of 120 and 30 s at -0.4 V. The relative standard deviation was found to be lower than 3.0% and the relative standard error of the mean14 (sJn”2) was lower than 1.0%.

Application of Adsorptive Stnpping Voltammetry at the HMDE to the Determination of Niguldipine in Urine and Blood Niguldipine can be determined directly in biological fluids, such as urine and blood, by reductive adsorptive stripping voltammetry at the HMDE. The measurements were performed in 6 ml of phosphate buffer (pH 12bmethanol (7 : 3 v/v) containing 0.5 ml of blood. The stripping peak attributed to the reduction of the nitro-group of niguldipine appeared at -0.70 V and increased with increasing accumulation time, especially at relatively low concentrations. For example, a preconcentration time of 60 s in a solution containing 0.66 pg ml-l of niguldipine (1.1 X 10-6 moll-1) led to an increase in peak height of 200% compared with a measurement performed without preconcen- tration, whereas increase of preconcentration time in a more concentrated solution (4.2 pg ml-1 = 6.9 X 10-6 mol 1-l) had

100

80

p 60 \ .P

40

20

I I I I I I I I

0 20 40 60 80 100 120 140 160 Accumulation time/s

Fig. 4 Influence of the accumulation time on the stripping peak current intensity. Adsorptive stripping measurements in different supporting electrolytes and different concentrations: E,,, = -0.40 V; U, buffer (pH 12): MeOH = 7 : 3 (c = 15 ng ml-1); 7 , pure buffer (pH 12) (c = 44 ng ml-I).

almost no influence on the peak height. However, whenever adsorptive stripping voltammetry was performed directly in biological samples without sample preparation, a second interfering peak appeared at about -0.62 V, in blood as well as in urine, that was attributed to adsorbing substances, such as proteins, competing with niguldipine for the adsorption sites at the electrode surface. Any attempt to suppress this interfering peak selectively by variation of the experimental parameters, such as accumulation potential and time, and composition of the supporting electrolyte, failed. As a consequence, only compar- atively high niguldipine concentrations could be determined by differential-pulse polarography without preconcentration. Pre- cision of these measurements, as well as the results of recovery experiments, were not satisfactory.

Therefore, a sample preparation had to be found that was both quick and easy to perform and would permit the selective and sensitive determination of niguldipine by adsorptive stripping voltammetry in blood and urine. We tried several procedures that are usually applied to eliminate the influence of interfering substances, such as proteins, in complex biological matrices. These experiments revealed that the interfering peak, occurring during the determination of niguldipine by adsorptive stripping voltammetry in blood and urine, can be suppressed by a very simple sample preparation that can be applied in a similar manner to blood as well as to urine samples. Addition of a 5% ZnS04 solution and ethanol to the spiked sample leads to the formation of a precipitate, which can be separated from the solution by centrifugation. The amount of niguldipine in the centrifugate was determined by highly sensitive adsorptive stripping voltammetry. The extent of the suppression of the interfering peak and, as a consequence, the increase in sensitivity of the adsorptive stripping voltammetric determina- tion of niguldipine, depended on the amount of ZnS04 and ethanol added to the spiked blood or urine sample. Only a small amount of ethanol (between 30% and 50% v/v) had to be added to prevent coprecipitation of niguldipine and ZnS04 concentra- tions higher than 40% v/v led to a decrease of the voltammetric response.

For measurements of niguldipine in urine the best results with regard to sensitivity and recovery were obtained if the sample preparation was performed with solutions containing equal volumes of urine, 5% ZnS04 and ethanol, adjusting the pH to 11 before centrifugation. In the adsorptive stripping voltammetry experiment the accumulation potential had almost no influence on the stripping peak height in the range between -0.45 and -0.60 V. In general, for determinations of niguldipine in urine an accumulation potential of -0.58 V was chosen. The height of the stripping peak current depends on the accumulation time applied (Fig. 5), especially at low niguldipine concentrations ( e .g . , 5.7 ng ml-1 = 9.4 x 10-9 mol 1-I), increasing linearly with increasing accumulation time up to 120 s. Thus, a considerable increase in sensitivity can be gained by using higher accumulation times for the determination of low niguldipine concentrations.

By using an accumulation time of 120 s and an accumulation potential of -0.58 V a linear dependence of the stripping peak current on the niguldipine concentration could be obtained within the concentration range of 212 ng-3.7 pg of niguldipine per ml of urine (3.5 X 10-7-6.1 X moll-’). The resulting calibration plot was adjusted to the equation i, (nA) = - 13.1 + 3.87 C (nmol 1-1) with a correlation coefficient of 0.9992.

Fig. 6 shows an example of a recovery experiment for niguldipine in urine in the concentration range of 144 ng-1.19 pg ml-1 urine (2.36 x 10-7-1.96 X 10-6 mol 1-l). The resulting linear calibration plot followed the equation i, (nA) = 0.08 + 3.49 C (nmol 1-1) (correlation coefficient = 0.9999). Recovery experiments were performed by repeated measure- ments ( n = 8) of spiked urine samples that contained niguldipine concentrations between 37 and 265 ng ml-1 of

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urine (for procedure see Experimental). The mean recovery in the range mentioned above was 96% with a mean relative error of 8.4%. The precision of the measurements was calculated from the regression lines of the recovery experiments according to reference 14 (page 1 19 ff) and reference 15 (pages 19 and 117 ff) using the formula for the standard deviation, s,E of the extrapolated x-value (xE)

c

where b is the slope of the regression line and n is the number of points in the standard additions experiment: X and are the mean values of all xi and yi, respectively. The relative

standard deviation in % is given by

For the determination of niguldipine in urine in the concentration range mentioned above ( n = 6-8) the relative

SXE VxE = T . 100. X

I I I I I I

0 20 40 60 80 100 120

Accumulation time/s

Fig. 5 Influence of accumulation time on the stripping peak current for the determination of niguldipine in urine by adsorptive stripping voltammetry: solution consisting of 6 ml phosphate buffer (pH 12), 1 ml ethanol and 1 ml pretreated spiked urine sample: E,,, = -0.58 V; niguldipine concentration = 5.7 ng ml-1 (9.4 x 10-9 mol 1-1).

I l l ,

-0.6 -0.7 -0.8 4 . 9 V Fig. 6 Recovery experiment of niguldipine in urine sample. Adsorptive stripping voltammograms (E,,, = -0.58 V, t,,, = 120 s): 1, blank supporting electrolyte [6 ml phosphate buffer (pH 12, I = 0.1 mol l-'), 1 ml ethanol]; 2, after addition of 1 ml pretreated, spiked urine sample; 3-6, successive addition of 10 pl of a 1.35 X 10- moll-' niguldipine standard (82-328 ng).

standard deviation was less than 3.2%. The detection limit (estimated as the concentration corresponding to a signal-to- noise ratio of 3) was 6.7 ng per ml of urine.

The determination of niguldipine in blood proved to be more difficult than the determination in urine, probably due to the higher protein content in blood compared with urine. The best results with respect to sensitivity and recovery were obtained when a similar pretreatment as described for the urine samples was applied. A mixture of 5% ZnS04+ethanol+blood in a volume ratio of (3 + 5 + 3 v/v) and adjustment of the pH of the final solution to 11-12 proved to be most efficient for the suppression of the interfering peak in the subsequent adsorptive stripping voltammogram.

As in the case of urine the accumulation potential had almost no influence on the stripping peak height, whereas increasing the accumulation time had a remarkable effect on the sensitivity of the measurement (Fig. 7). An increase in the peak current was observed for increasing preconcentration times up to 100 s. However, most determinations of niguldipine in blood were performed with an accumulation time of 30 s at a preconcentra- tion potential of -0.50 V.

Fig. 8 shows a recovery experiment for the determination of niguldipine in blood with an optimized procedure within the concentration range of 477 ng-2.19 pg per ml of blood (7.8 X 10-7-3.5 x 10-6 moll-'). The stripping peak current depends linearly on the niguldipine concentration according to the equation i, (nA) = 0.09 + 1.14 C (nmol 1-1) (correlation coefficient = 0.9994). Recovery experiments were performed by repeated measurements ( n = 4) of blood samples containing different amounts of niguldipine (in the range from 164 to 200 ng per ml of blood). The mean recovery in the range mentioned above was 71%, with a mean relative error of 2.2%. Preci- sion'4.15 was determined in the same manner as described for the urine samples ( n = 6). The relative standard deviation in the range mentioned above was less than 2.9%. The limit of detection (estimated as the concentration corresponding to a signal to noise ratio of 3) was 41 ng of niguldipine per ml of blood.

Conclusions The present results show that adsorptive stripping voltammetry at the HMDE is a very powerful technique for the determination of niguldipine in low concentrations, even in complex bio- logical matrices. The sensitivity is significantly enhanced by adsorption of the drug on the electrode surface and, after careful choice of the operating parameters, extremely low detection

180 -

160 -

5 140- 1

120 - 100-

80 -

.-a

60 I 1 I 1 1 0 20 40 60 80 100

Accumulation time/s

Fig. 7 Influence of accumulation time on the stripping peak current for the determination of niguldipine in blood by adsorptive stripping voltammetry. Solution consisting of 6 ml phosphate buffer (pH 12, I = 0.1 moll-'), 1 ml ethanol and 0.9 ml pretreated, spiked blood sample; E,,, = -0.50 V; niguldipine concentration = 64 ng ml-l (1.0 X mol 1-I).

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limits can be reached. Compared with other techniques the method is cheap and the measurement is not time consuming, leading to results that are adequately accurate and precise.

The authors thank Professor Dr. H. Glossmann and Doz. Dr. J. Striessnig (Institut fur Biomedizinische Pharmakologie, Uni- versity of Innsbruck) for providing 1,4-dihydropyridine calcium

6 5 4 3

2

2

I 1 I I

-0.5 -0.6 -0.7 -0.8 -0.9 v Fig. 8 Recovery experiment of niguldipine in blood sample. Adsorptive stripping voltammograms (E,,, = -0.50 V, t,,, = 30 s): 1, blank supporting electrolyte [6 ml phosphate buffer (pH 12, I = 0.1 moll-]), 1 ml ethanol]; 2, repeated measurements after addition of 0.90 ml pretreated, spiked blood sample; 3-6, successive addition of 30 p1 of a 5.4 X moll-' niguldipine standard solution (99-396 ng).

antagonists. Financial support by the Osterreichische Nationalbank (project No. 4778) is gratefully acknowledged.

References 1

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Weaver, J. L., Szabo, G., Jr., Pine, P. S., Gottesmann, M. M., Goldenberg, S., and Aszalos, A., Int. J . Cancer, 1993, 54,456. Gietzen, K., Bai, G., and Abdallah, F., Med. Sci. Res., 1990, 18, 627. Ahnhoff, M., and Persson, B. A., J. Chromatogr., 1990,531, 181, and references cited therein. Telting-Diaz, M., Kelly, M. T., Chi Hua, and Smyth, M. R., J . Pharm. Biomed. Anal., 1991, 9, 889. Bersier, P. M., and Bersier, J., in Comprehensive Analytical Chemistry, ed. Smyth, M. R., and Vos, J. G., Elsevier, Amsterdam, New York, 1992, vol. XXVII, pp. 159465. Ludvik, J., Volke, J., and Klima, J., Electrochim. Acta, 1987, 32, 1063. Baumane, L., Stradins, J., Gavars, R., and Duburs, G., Electrochim. Acta, 1992, 37, 2599. Squella, J. A., and Nufiez-Vergara, L.-J., Bioelectrochem. Bioenerg., 1990, 23, 161. Barrio Diez-Caballero, R. J., Lopez de la Torre, L., Arranz Valentin, J. F., and Arranz Garcia, A., Talanta, 1989, 36, 501. Wang, J., Deshmukh, B. K., and Bonakdar, M.,Anal. Lett., 1985,18, 1087. Alvarez-Lueje, A., Nufiez-Vergara, L.-J., and Squella, J. A., Electro- analysis (N.Y. ) , 1994, 6, 259, and references cited therein. Fry, A. J., Synthetic Organic Electrochemistry, Wiley, New York, USA, 1989, p. 189. Obendorf, D., and Stubauer, G.,J. Pharm. Biomed. Anal., 1995, in the press. Miller, J. C., and Miller, J. N., Statistics for Analytical Chemistry, Ellis Horwood Series, PTR Prentice Hall, New York, London, 1993, pp. 41 and 119 ff. Funk, W., Dammann, V., and Donnevert, G., Qualitutssicherung in der Analytischen Chemie, VCH, Weinheim, Germany, 1992.

Paper 51051 80K Received August 3,1995

Accepted November 13, I995

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