Determination of lorazepam in human urine by adsorptive stripping voltammetry

MICROCHEMICAL

JOURNAL

Determination

41, 1&21 (19%)

of Lorazepam in Human Urine by Adsorptive Stripping Voltammetry

ANTONIO ZAPARDIEL, **I Josh ANTONIO PBRJSZ LOPEZ,* ESPERANZABERMEJO,* LUCAS HERNANDEZ,* AND MARIAJOSB VALENCIANot *Department

of Chemistry,

Autonoma University, 28049 Madrid, Madrid, Spain

Received July 31, 1989; accepted September

Spain, and tRetesa,

13, 1989

The interfacial and redox behavior of lorazepam at the hanging mercury drop electrode was studied by adsorptive stripping voltammetry. Both linear and differential pulse scan modes were used to record the stripping curves. A critical evaluation of adsorptive accumulation in stripping analysis in which differential pulse voltammetry was used for the measurement step is described. Generally applicable conditions for the method are 0.02 M B&ton-Robinson buffer at pH 2.0 with a -0.39 V accumulation potential. The effect of various urine components on the voltammetric response was also studied, and preliminary separation of the drug was found necessary to avoid interference caused by albumin and uric acid. The applicability to human urine analysis is described. The detection limit was 15 ng lorazepam/ml urine (25 s accumulation time) and the mean standard deviation was lower than 3.1% for 316 ng ml-’ samples (n = 5 and 25 s accumulation time), with a mean RUSS,Inc. recovery of 99%. Q 1990Ademic

INTRODUCTION

Benzodiazepines are the most important group within ansyolitic drugs and, although their effect is primarily ansyolitic-sedative, they are also active as hypnotic, myorelaxant, and anticonvulsive agents. Not all of the more than 2000 compounds included in the group have therapeutic uses, but they all share a number of similar actions, differing, however, in their potency and special characteristics. As far as the relationship between pharmacological action and chemical structure is concerned, the presence of the 5-phenyl ring has proved essential in the ansyolitic effect. Ortho substitutions enhance ansyolitic potency, whereas substitutions with meta or para groups lower it. On the other hand, addition of radicals in C-7 is of great interest, since a higher ability to accept electrons on the part of the substituent means a corresponding increase in activity. Substitutions in the sixth, eighth, and ninth positions cause a decrease in activity. Metabolization of benzodiazepines is achieved mainly through the liver, although some of them are metabolized in the stomach or the small intestine. Metabolic decomposition starts in the diazepinic nucleus, giving off innumerable metabolites, although some benzodiazepines undergo little or no metabolization, * To whom correspondence

should be addressed.

10 0026-265X&O $1.50 Copyright 0 1990 by Academic Press, Inc. Au rights of rcproductio” in any form reserved.

DETERMINATION

OF LORAZEPAM

IN

HUMAN

URINE

11

mostly due to the fact that they are metabolites of other, more complex benzodiazepines, as is the case with lorazepam. The elimination rate depends on the characteristics of each compound and its metabolites; elimination takes place mainly in conjugate form via the kidney, although in some instances a small fraction is eliminated through biliar secretion or in the feces. Development of new, quicker, and more sensitive methods for determination of such substances in biological fluids has become an important need with the increase in the number of acute intoxications (both intentional and accidental) which call for forensic analysis, the growing demand for a better regulation of dosage, and, in the end, the desire to gain a better understanding of the action of these compounds on biological matrices. Adsorptive stripping voltammetry is among the most sensitive analytical techniques used and has been applied in a great variety of analytical determinations. The technique allows determination of substances, both organic and inorganic, which show a high level of superficial activity (I-7). Lorazepam (7-chloro-5-(2chlorophenyl)-I ,3-dihydro-3-hydroxy-2H-1,4benzodiazepin-Zone) is a benzodiazepine with a wide market distribution, short action, and no active metabolite. Maximum concentration time after oral ingestion is 1-2 h with the onset of action after 15-45 min. Conjugation is the main elimination route and mean plasmatic life is 5-15 h. Spectrophotometric and fluorometric studies (8, 9) were carried out to analyze acid-base characteristics and determination of lorazepam. Coulometry and DC polarography (10-13) reveal that the reduction process in an acid medium involves a four-electron exchange which corresponds both to the reduction of the 4,5azomethine group and the breakdown of the bond between the carbon 3 and the hydroxyl in the diazepinic ring. DP polarography (23-26) has also been applied to the identification and determination of the amount of lorazepam found in the urine of lorazepam-treated patients, once the appropriate extraction procedure had been carried out. Electrochemical studies on the oxidation process were conducted using a glassycarbon electrode (17). Gas chromatography coupled with mass spectrometry (18) and HPLC (19) have all been used for determination of lorazepam in plasma and urine. Adsorptive stripping voltammetry has been used in the study of lorazepam-like drugs, giving detection limits between 10T9 and IO-” M (20-25). The present paper studies in close detail lorazepam behavior at the hanging mercury drop electrode (HMDE) using adsorptive stripping voltammetry. Both linear and differential scanning are applied in the stripping step and a method for determination of lorazepam in human urine by differential pulse scan mode is readied. EXPERIMENTAL Apparatus

A Metrohm 646 VA processor in conjunction with a 647 VA stand was used. A multimode mercury drop electrode (Metrohm 6.1246.020) served as the worl&g

12

ZAPARDIEL

ET AL.

electrode in the HMDE mode with a medium surface area of 0.60 mm2. The reference electrode was Ag/AgCl/3 M KC1 and the auxiliary electrode was a glassy carbon rod. The solution was stirred in the stand cell with a built-in rotor. Reagents Stock solutions 1.03 x 10e3 M of pure lorazepam (Roche) were prepared by dissolving the compound in methanol. The solution was stored in the dark under refrigeration to minimize the risk of decomposition. Dilute solutions were prepared from the stock solutions daily. The supporting electrolyte was 0.02 M B&ton-Robinson buffer at pH 2.0. All chemicals used were of analytical reagent grade E. Merck. Aqueous solutions were prepared in purified water (Milli Q and Milli Ro, Millipore). Procedures Adsorptive stripping voltammetry. Pure nitrogen (99.999%) is passed through 25 ml of solution placed in the polarographic cell for 10 min in the initial cycle and for 30 s in each successive cycle. A mercury drop is formed and, while being stirred at 1920 rpm, an accumulation potential (usually -0.390 V) is applied for a time which depends on the concentration of the solution being used (90 s accumulation time for a 7.94 X lo-* M). Next there is a rest step when the stirring is stopped but potential continues to be applied for 10 s. Finally, in the measuring step, a sweep toward more negative potentials is carried out by linear (scan rate 40 mV s-l) or differential pulse scanning (scan rate 20 mV s- ’ and pulse amplitude 70 mV) in order to determine the amount of accumulated drug. Treatment of urine samples. Urine (2.0 ml) containing 100-1000 ng lorazepam, 1 ml of 0.04 M boric-borate buffer, pH 9.2, and 3 ml of diethyl ether is placed in a decanting funnel, shaken for 2 min, and allowed to rest for 5 min. The organic phase is removed to a centrifugal tube and evaporated by passing nitrogen. The residue is then dissolved in 200 ~1 methanol and diluted to 25.0 ml with 0.02 M Britton-Robinson buffer at pH 2.0. The resulting solution is transferred to the polarographic cell for analysis by adsorptive stripping voltammetry. RESULTS AND DISCUSSION

Study of Lorazepam Adsorption The electrochemical studies conducted prove the existence of adsorption processes on the electrode surface prior to molecule reduction. Figure 1 shows electrocapillary curves with and without lorazepam and proves the existence of adsorption. The molecule adsorbed seems to be a neuter form. Successive cyclic voltammograms on the same mercury drop are shown in Fig. 2, after a 90-s accumulation of lorazepam. A noticeable peak (Zp = 32.9 nA; Ep = -0.668 V) is obtained in the first reduction scan (curve 1); reduction product reoxidation does not occur over the same potential range which means it is an irreversible process. In successive scans (curves 2-4) there is a remarkable de-

DETERMINATION

OF LORAZEPAM

IN HUMAN

URINE

13

2.3In

f 2.2 i= 2.1 -

0.0

- 0.5

Potential,V FIG. 1. Electrocapillary curves of 0.02 MB&ton-Robinson of 1.12 x 10v4 M lorazepam (2).

- 1.0 (v. SCE) buffer at pH 2.0 (1) and in the presence

crease in reduction peak intensity (approx 83%, which implies a quick desorption of the drug from the electrode surface). The scan rate effect on peak intensity and peak potential was studied by linear scan (in the range l&60 mV s- ‘) with a drug-saturated electrode surface; linear

-U.JY

V

FIG. 2. Successive cyclic voltammograms for 4.86 X lo-’ Mlorazepam in 0.02 M Britton-Robinson buffer at pH 2.0 (90 s at -0.39 V). Scan rate 40 mV s-l. Drop size 0.60 mm2 and stirring speed 1920 rpm (for details see text).

14

ZAPARDIEL

ET AL.

variations in the peak intensity logarithm versus the scan rate logarithm, according to the formulation log Zp = -0.61 + 0.83 log v (r = 0.9990), were obtained for 7.80 x lo-* M lorazepam solutions (with a -0.390 V potential and a 90 s accumulation time). For ideal redox processes where the substance is saturated on the electrode surface, the value of the slope should be 1.O. Peak potential shifts toward negative values as scan rate increases, from -0.637 V (10 mV s-l) to -0.674 V (60 mV s- ‘), which implies an irreversible electrodic reaction. Linear scan studies on the influence of lorazepam concentration on peak intensity and fraction of covered surface (0) using solutions of the drug (from 3.92 x lo-’ to 1.24 x 10m6M with a 90-s accumulation period) show a tendency to a stabilization of intensity from 9.00 x 10d7 M onward and a linear dependence up to 3.70 x lop7 M. The decrease in the ratio peak intensity concentration proves the process to be limited by the reactant adsorption. There is evidence that adsorption follows a Frumkin-type isotherm (26). Effects of Working Electrolyte and pH The possible influence of the supporting electrolyte on both the adsorption process and the voltammetric response was studied by using different electrolytes (B&ton-Robinson, phosphate, borate, acetate and ammoniacal buffers, ammonia chloride, sulfuric, perchloric, and hydrochloric acids) in concentrations ranging from 0.003 to 0.23 M at pH from 0 to 11. Figures 3 and 4 show the influence of pH and ionic strength on peak intensity obtained with the different electrolytes and different accumulation times. As the ionic strength of the medium decreases, there is a corresponding increase in the signal. The lowest electrolyte concentrations, however, usually cause the response to be of low reproducibility. The intluence of pH with a 90-s accumulation time for the most appropriate electrolytes is shown in Fig. 5. The highest intensities are reached using an acid pH, 2.0 being the most adequate. At pH > 6.0, two reduction peaks are observed (Britton-Robinson buffer 0.02 M, pH 7.0, Epl = - 1.000 V and Ep, = - 1.170 V). The first peak gradually diminishes in intensity as pH increases, until it becomes imperceptible at alkaline pH. The intensity of the second peak, on the other hand, is very low and remains essentially unaltered. After considering the higher response, the lower residual current, the presence of buffer peaks similar to those of the drug, and the reproducibility of the response, a 0.02 M Britton-Robinson buffer with pH 2.0 was chosen as the most appropriate for the study. Figure 5 also shows the influence of pH on peak potential; for the first peak three zones of linear variations can be established with intersections at pH 2.0 and pH 54. The first is near pK value (8-9). The mechanism of lorazepam reduction involves protons, the slope of the first variation zone being exactly one-half that of the second. The potential of the second peak, on the other hand, remains virtually constant with the increase in pH. Main Features of the Voltammograms The voltammetric signals obtained with linear and differential pulse scanning

DETERMINATION

OF LORAZEPAM

IN HUMAN

URINE

15

I

I

50

100 Accumulation

150 time,

200 s

FIG. 3. Influence of pH on the voltammogram peak current for 7.94 x lo-‘M

lorazepam at different accumulation times. Electrolytes: (1) 0.10 M hydrochloric acid; (2-6) 0.02 MB&ton-Robinson buffers at pH 2.0, 3.0, 4.0, 5.0, and 6.0, respectively. Accumulation potential -0.45 V, pulse amplitude 50 mV, and scan rate 20 mV SC’. Other conditions were as in Fig. 2.

and different accumulation times are shown in Fig. 6. For 7.94 X 10e8 A4 solutions of the drug, the differential pulse scan mode gives a single peak which originates at 31.4 nA intensity with a 90-s accumulation time and -0.640 V potential, whereas an intensity of 5.4 nA is obtained for the same accumulation time and a -0.655 V potential if the linear mode is used. In general, the response obtained by differential pulse scanning is 5.8 times higher than that obtained with the linear mode. In both techniques voltammograms obtained after an accumulation period are ostensibly higher than those obtained without it (369% in DP and 222% in linear for 90 s). Evaluation of Experimental and Instrumental Parameters Accumulation potential. The values of peak potential (-0.640 V in 0.02 M Britton-Robinson buffer at pH 2.0) and half-peak width (WI,, = 38 mV) are independent of accumulation potential. Figure 7 shows lorazepam stripping peak intensity as a function of accumulation potential. Peak intensity can be seen to remain virtually constant despite variations in accumulation potential. The accumulation potential adopted for the analytical process -0.390 V. It has been observed that the optimum accumulation potential that favours the adsorption

16

ZAPARDIEL

I

50

ET AL.

I

100

I

150

Accumulation time, s FIG. 4. Influence of ionic strength on the peak current for 7.94 x lo-’ M lorazepam at Were accumulation times. (l-4) pH 2.0 Britton-Robinson buffers 0.02,0.03,0.05, and 0.06 M, respective1 (5-10) pH 3.8 acetate buffers 0.02, 0.05, 0.09, 0.14, 0.18, and 0.23 M, respectively. Other conditio were as in Fig. 3.

phenomenon is a potential between 240 and 300 mV more positive than the pes potential of the reduction process of the drug. Accumulation time. The values of peak potential and half-peak width are dl pendent on accumulation time. Peak intensity increases linearly with increasing (Zp (nA) = 7.9 + 0.48 f,; DP mode) in the range O-30 s (lorazepam 7.94 x loA4) but remains almost unchanged between 90 and 150 s, as shown in Fig. 7. Tl most adequate accumulation time for lorazepam concentrations similar to the: indicated above was judged to be 90 s for both measuring modes. It has also been established that the optimum accumulation time rises for lows concentrations of the drug and decreases for higher ones, a fact which is direct1 related to the saturation of the electrode surface. The rest time, or the time elapsed between the end of the accumulation step an the beginning of the scanning, used for eliminating convection phenomena an homogenizing the amount of substance adsorbed on the electrode surface, hi practically no influence on the height of the response peak. After experimentatic with times from 0 to 30 s, which gave relative typical variations between 0.9 ar 2.5% in the reproducibility of the measurements, a 10-s rest time was chosen fc the analytical procedure. Electrode size. Influence of this parameter was studied at 20 mV s-* scan rat

DETERMINATION

OF LORAZEPAM

IN

HUMAN

URINE

17

FIG. 5. Variations of (A, B) peak potential and (C, D, E) peak current for 7.94 x 10v8 Mlorazepam in the most appropriate electrolytes: (A, B, C) 0.02 MB&ton-Robinson buffers; (D) 0.100,0.010, and 0.003 M hydrochloric acid. (E) 0.023 M perchloric acid. Accumulation time 90 s. Other conditions were as in Fig. 3.

using the drop size possible with our equipment (0.20-0.60 mm’). The values of Ep and W,,*are independent of the size of the electrode. There is a linear relationship between the Zp and the electrode surface (slope 55.6 nA mm-‘; 7.94 x lo-* M lorazepam; AE = 50 mV; 1920rpm; 90 s), indicating that the compound deposited remains on the surface and does not diffuse into the electrode (27). A 0.60 ? 0.01 mm2 electrode area was adopted for all further work. Stirring and potential scan rates. Stirring rate, studied within the 1220 to 2620 rpm range possible in the equipment, has no significant effect on the response signal. An intermediate rate was chosen (1920 rpm) to avoid turbulence which could affect the mercury drop. The influence of potential scan rate on the stripping peak was studied. Ep shifts toward more negative values and WCj2rises erratically as potential scan rate increases. Peak intensity also elevates linearly with increased potential scan rate (slope 1.4 nA s mV-‘, 7.94 x lo-* M lorazepam, accumulation time 90 s) in the differential pulse mode with a pulse amplitude of 50 mV. A stripping scan rate of 20 mV s- ’ was chosen for the analytical procedure. An increase in peak intensity has been observed to occur with the elevation of pulse amplitude (slope 0.74 nA mV-‘, 7.94 x 10v8 M lorazepam, 90 s) up to 75

18

ZAPARDIEL

ET AL.

A

-a39 v

FIG. 6. Adsorptive stripping voltammograms for 7.94 X 10-s M lorazepam in 0.02 M BrittonRobinson buffer at pH 2.0 for different accumulation times. (A) Diierential pulse voltammograms (scan rate 20 mV s - r and pulse amplitude 50 mV). (B) Linear scan voltammograms (scan rate 40 mV s-r). Accumulation time 90 s. Other conditions were as in Fig. 2. Accumulation

potential,

V (A)

-0.50 I

-0.40 I

-0.30 I

1

50

I

100

1

150

Accumulation time, s (B,C) FIG. 7. Variations of peak current of 7.94 x 1Om8M lorazepam with (A) accumulation potential and (B) accumulation time in differential pulse mode (scan rate 20 mV s-’ and pulse amplitude 50 mV) and (C) in linear scan mode (scan rate 40 mV s-r). Britton-Robinson (0.02 M) buffer at pH 2.0, accumulation potential -0.39 V. Other conditions were as in Fig. 2.

DETERMINATION

OF LORAZEPAM TABLE

IN HUMAN

19

URINE

1

Characteristics of the Analytical Determination of Lorazepam in 0.02 Britton-Robinson Buffer at pH 2.0 Calibration functions

Ip = a + time(s) 0

30 60 90 120 150 180

4m

0.37 -0.29 1.06

-0.24 -0.25 -0.86 - 0.42

bC

b(nA 1 mol - ‘) 1.91 x 4.01 x 5.78 x 7.17 x 7.96 x 8.41 x 8.86 x

108 108 108

108 lo8 lo8 108

r

0.9993 0.9998 0.9994 0.9997 0.9998 0.9996 0.9992

Detection limit (30) (mol 1- ‘)

Determination limit (loo) (mol 1- ‘)

3.7 x 10-g 1.8 x lo-’ 1.2 x 10-g 9.8 x lo-lo 8.8 x 10-l’ 8.3 x lo-”

7.9 x 1o-‘o

1.2 x 5.8 x 4.0 x 3.2 x 2.9 x 2.8 x 2.6 x

10-S lo-’ 10-g lo-’ 1O-9 10-g lo-’

mV, remaining constant from that value upward. Wl12varies from 32 to 48 mV when pulse amplitudes from 25 to 125 mV are applied. A 70 mV pulse amplitude giving a 38 mV half-peak width was finally chosen since a better sensitivity/ resolution ratio was obtained in the peak. Concentration Linear plots of peak current with lorazepam concentrations in the range of 8.00 lop9 to 1.00 x 10m7M have been observed. Table 1 is a summary of the characteristics of the analytical determination of lorazepam in 0.02 M BrittonRobinson buffer at pH 2.0. Accuracy and precision were evaluated by measuring 10 lorazepam solutions of concentration between 1.60 x lo-* and 9.50 x 10m8M after a 90-s accumulation time; the relative error was found to be lower than 1.3% and the mean standard deviation lower than 3.2%. Reproducibility is directly dependent on the automatic control of the equipment used, basically the electrode and the stirring instrument. x

Urine Components Differential pulse scanning was applied to determine the influence of several urine constituents on the voltammetric response. The following is a list of those constituents selected, together with an indication of the normal concentrations:* uric acid (60 ppm), albumin (6.4 ppm), urea (2400 ppm), glucose (8 ppm), phosphate (72 ppm), potassium (80-312 ppm), and chloride (312-712 ppm). The presence of 2.5 ppm of albumin and 17 ppm of uric acid causes a 50% reduction in the voltammetric response obtained and an almost complete elimination of the response in ordinary concentrations. Urea and glucose at the levels ordinarily found in urine originate a slight diminution in the signal (98 and 93%, respectively). The remaining constituents do not affect the signal at the usual concentrations. * Values for normal content are calculated with 2.0 ml of urine in 25.0 ml of solution.

20

ZAPARDIEL

ET AL.

Analytical Application The existence on the electrode of adsorption processes of uric acid and albumin, which enter into competition with the drug and even prevent it from being adsorbed, makes a previous separation of these components necessary in order to determine lorazepam in urine. An extraction procedure was devised after experimenting with several organic solvents (toluene, dichloromethane, chloroform, ethyl acetate, diethyl ether), different buffers, and different conditions. The most effective way of determining lorazepam by adsorptive stripping voltammetry, and therefore the one we recommend, has been summarized under Procedures. Comparison of the calibration plots obtained after extracting urine and then adding lorazepam in the range 50-570 ng ml-’ of urine (blank assay) with those obtained after extracting lorazepam added to the urine show a recovery of 94 to 104%. Figure 8 shows the voltammograms obtained for lorazepam amounts between 63 and 570 ng ml- ’ of urine for 10-s accumulation time and the calibration plots for a O- to 20-s accumulation time. Table 2 summarizes the characteristics of lorazepam determination in urine. After analyzing a series of five solutions of the drug in amounts of 63-570 ng ml-’ of urine, after having carried out the extraction and stripping process described above, at 10-s accumulation time, the relative error was found to be lower than 4.5% and the mean standard deviation lower than 5.5%.

L

‘: (0

O.lV

Concentration, ngl ml urine FIG. 8. Differential pulse stripping peaks for diierent concentrations of lorazepam in urine: (1) 0, (2) 63, (3) 190, (4) 316, (5) 442, (6) 569 ng of lorazepam per milliliter of urine. Accumulation time 10 s. Calibration plots for determination of lorazepam in urine for (A) 0, (B) 10, (C) 20, and (D) 25 s of accumulation time. Pulse amplitude 70 mV. Other conditions were as in Fig. 2.

DETERMINATION

OF LORAZEPAM TABLE

IN HUMAN

21

URINE

2

Results Obtained in the Determination of Lorazepam in Urine at -0.3% V: Accumulation Potential

Accumulation time(s)

Calibrationfunctions Ip = a + bC ab-4)

0 10

-0.60

20 25

-0.30 -0.45

0.34

&Angg’ml)

r

0.042

0.997

i:E

0.99997 0.9990 0.9990

0.079

Concentration range (ng in-’ urine)

Detection limit (3~) (ng ml- i urine)

Determination limit (loa) (ng ml-’ urine)

90-7cnl

27 18

91

IS 15

Yf 48

606oo xuioo 504m

ACKNOWLEDGMENT The authors thank CAICYT for the financial support awarded to this project (No. 2077-83).

REFERENCES 1. 2. 3. 4. 5.

Kalvoda, R.; Kopanica, M. Pure Appl. Chem., 1989,61, 97-l 12. Smyth, W. F. CRC Crit. Rev. Anal. Chem., 1987, 18, 155-208. Wang, J. ht. Lab., 1985, 72, 68-76. Kalvoda, R. Anal. Chim. Acta, 1982, 138, 11-18. Smyth, W. F. Adsorptive stripping voltammetry of selected molecules. In Electrochemistry, Sensors and Analysis (M. R. Smyth and J. G. Vos, Eds.), pp. 28-35. Elsevier, Amsterdam, 1986. 6. Smyth, W. F.; Tution, P. Quim. Anal., 1987,6, 37748. 7. Hemandez, L.; Zapardiel, A.; Perez Lopez, J. A.; Bermejo, E.; Lancha, A. M. Qut’m. Znd., 1988, 34, 140-145. 8. Barret, J.; Smyth, W. F.; Davidson, J. E. J. Pharm. Pharmacol., 1973, 25, 387-393. 9. Rodriguez, J.; Hemandez, P.; Hemandez, L. Analyst (London), 1987, 112, 79-82. 10. Ellaithy, M. H.; Volke, J.; Hlavaty, J. Collect. Czech. Commun., 1976, 41, 3014-3026. II. Clifford, J. M.; Smyth, W. F. Z. Anal. Chem., 1972, 264, 149-153. 12. Goldsmyth, J. A.; Jenkins, H. A.; Grant, J.; Smyth, W. F. Anal. Chim. Acta, 1973,66,427-434. 13. Oelschlager, H.; Sengun, F. I. Chem. Ber., 1975, 108, 3303-3308. 14. Clifford, J. M.; Smyth, W. F. Analyst (London), 1974, 99, 241-244. 15. Smyth, W. F.; Smyth, M. R.; Groves, J. A.; Tan, S. B. Analyst (London), 1978, 103, 497-508. 16. De Silva, J. A. F.; Berkersky, I.; Brooks, M. A. J. Pharm. Sci., 1974, 63, 1943-1945. 17. Smyth, W. F.; Ivaska, A. Analyst (London), 1985, 110, 1377-1379. 18. Higuchi, S.; Urobe, H.; Shiobare, Y. J. Chromatogr., 1979, 164, 55-61. 19. Marin, S.; Font, G.; Maties, J. Farmacia Clin., 1985, 2, 218-222. 20. Kalvoda, R. Anal. Chim. Acta, 1984, 162, 197-205. 21. Hemandez, L.; Zapardiel, A.; Perez Lopez, J. A.; Rodriguez, V. Determination of benzodiazepines by adsorptive stripping voltammetry. In Electrochemistry, Sensors and Analysis (M. R. Smyth and J. G. Vos, Eds.), pp. 385-390. Elsevier, Amsterdam, 1986. 22. Hemandez, L.; Zapardiel, A.; Perez Lopez, J. A.; Bermejo, E. Analyst (London), 1987, 112, 1149-1153. 23. Hemandez, L.; Zapardiel, A.; Perez Lopez, J. A.; Bermejo, E. Talanta, 1988, 35, 287-292. 24. Zapardiel, A.; Perez Lopez, J. A.; Bermejo, E.; Hemandez, L. Fresenius Z. Anal. Chem., 1988, 330,707-710. 25. Hemandez, L.; Zapardiel, A.; Perez Lopez, J. A.; Bermejo, E. J. Electroanal. Chem., 1988,255, 85-95. 26. Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals and Applications, pp. 516-518. Wiley, New York, 1980. 27. Perchard, J. P.; Buret, M.; Molina, R. J. Electroanal. Chem., 1%7, 14, 57-74.