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Electrochemical Determination of Lacidipine

Article  in  Journal of AOAC International · September 1999

DOI: 10.1093/jaoac/82.5.1077

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 SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1077
DRUGS, COSMETICS, FORENSIC SCIENCES
Electrochemical Determination of Lacidipine
SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
JUAN A. SQUELLA, ANNY E. IRIBARREN, JUAN C. STURM, and LUIS J. NÚÑEZ-VERGARA
University of Chile, Chemical and Pharmaceutical Sciences Faculty, Bioelectrochemistry Laboratory, PO Box 233
Santiago 1, Chile
Lacidipine is an antihypertensive drug that is oxi-
dizable at the glassy carbon electrode. The
voltammetric oxidation of lacidipine in aqueous–al-
coholic solutions (70 + 30) produces a well-defined
voltammetric peak when subjected to a differential
pulse voltammetric experiment. This peak is due to
oxidation of the dihydropyridine ring to a pyridine
derivative. This voltammetric response result is ir-
reversible, pH dependent, and diffusion controlled.
The best resolution for the peak was obtained at
pH 6 in Britton-Robinson buffer–ethanol (70 + 30).
The peak potential at pH 6 was 800 mV against an
Ag–AgCl reference electrode. A linear relationship
between peak current and lacidipine concentration
was obtained. For analytical purposes, a calibra-
tion curve for lacidipine that covered concentra-
tions between 5 × 10–6 and 2 × 10–4M was used. De-
tection and quantitation limits were 3.52 × 10–6 and
3.78 × 10–6M, respectively. The repeatability of the
measurement was 2%. On the basis of the
voltammetric response, we also developed
high-performance liquid chromatographic methods
with electrochemical detection. For comparative
purposes, we also developed a spectrophotometric
method.
acidipine is a 1,4-dihydropyridine (1,4-DHP) deriva-
L tive (Figure 1). It is a new potent antihypertensive drug
with long-acting effect and bigger vascular selectivity
than the classic dihydropyridines such as nifedipine and re-
lated compounds (1). Clinical pharmacology of lacidipine was
evaluated by Hall et al. (2). In normotensive subjects, single
oral doses of 3–5 mg lacidipine produce a dose-related fall in
peripheral vascular resistance. Adverse events are those typi-
cally related to the vasodilatory action of lacidipine, such as
flushing and headache. Absorption, distribution, and excre-
tion of lacidipine have been investigated by Pellegatti et al.
(3). Lacidipine is extensively metabolized; no significant
amount of unchanged drug is excreted in bile or urine. The
main metabolic routes are hydrolysis of the ester moieties and
oxidation of the dihydropyridine ring to pyridine. The recom-
mended dosage of lacidipine is one 4 mg tablet given once
Received September 21, 1998. Accepted by JM March 29, 1999.
daily, preferably in the morning. Ingestion does not modify
the pharmacological activity of lacidipine. If necessary, the
dose can be increased to 6 mg.
Analytical methods for determining lacidipine in plasma
include high-performance liquid chromatography (HPLC)
with UV detection and radioimunnoassay (4, 5). To date, no
electrochemical analytical assays for determination of
lacidipine have been reported. Also, the redox behavior of
lacidipine remains unexplored. The redox behaviors of other
members of the 1,4-DHP family have been extensively re-
ported. Most studies deal with the well-defined polarographic
reduction of the nitro group in nitroaromatic substituted
1,4-DHPs (6–9). On the other hand, studies related to the an-
odic electrochemical behavior of the 1,4-DHP family are re-
stricted to 2 reports (10, 11).
The present work is devoted to the electrochemical study of
lacidipine to propose an electroanalytical method capable of de-
termining the drug in solution. We also developed spectroscopic
and chromatographic methods for comparative purposes.
Experimental
Reagents
(a) Supporting electrolyte.—Britton-Robinson buffer
(0.04M each acetic, phosphoric, and boric acid)–30% ethanol
adjusted to the desired pH with NaOH.
(b) Mobile phase.—For isocratic operation, the mobile
phase was acetonitrile–0.05M pH 6.0 phosphate buffer solu-
tion (60 + 40).
(c) Standard solutions.—Ethanolic stock solution con-
taining 1 × 10–2M lacidipine (chromatographically pure;
Glaxo-Wellcome s.p.a., Verona, Italy).
(d) Working solutions.—For voltammetric studies, dilu-
tions were made in supporting electrolyte or in mobile phase
to obtain concentrations ranging from 1 × 10–7 to 1 × 10–3M at
pH 6.0 (if not otherwise stated).
All chemicals were of analytical grade (Merck, Darmstadt,
Germany).
Apparatus
(a) Voltammetric analyzer.—Experiments were per-
formed with a Metrohm Model 693 VA-Processor, equipped
with a Model 694 VA-stand including an Ag–AgCl electrode
(6.0728.010) as reference, glassy carbon (6.1204.110) as a
working electrode, and a platinum rod (6.0343.000) as auxil-
iary electrode (Metrohm A.G., Herisau, Switzerland).
1078 SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999

Experimental data were transferred to a 486 PC (Metrohm

Figure 1. Structure of lacidipine.
693 VABackup program) for analysis. The techniques used
were differential pulse voltammetry (DPV) and cyclic
voltammetry (CV).
(b) Liquid chromatograph.—The LC system consisted of
a Millipore Waters Model 600 solvent pump, Millipore Wa-
ters 996 photodiode array detector, and a Millipore Wa-
ters 464 pulsed electrochemical detector (Millipore Waters,
Milford, MA). It was interfaced with a 486 PC and Millen-
nium software for data acquisition and analysis.
A µBondapak C18 (10 µm) 3.9 × l50 mm column was used,
with the temperature control system set at 40ºC (Millipore
Waters). Flow rate was 1.5 mL/min. The applied potential of
the electrochemical detector was +1200 mV.

Figure 2. Differential pulse voltammograms of 5 × 10–5M lacidipine at different pH values. Supporting electrolyte:
Britton-Robinson buffer. Working electrode: glassy carbon.
 SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1079

Figure 3. Dependence of peak potential (Ep) and peak current (Ip) of lacidipine solutions on pH.

Figure 4. Liquid chromatograms of 5 × 10–7M lacidipine; mobile phase, acetonitrile–phosphate buffer pH 6.0, 60 + 40.
1080 SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999

Figure 5. UV spectra of 5 × 10–7M lacidipine at different pH 2.0, 4.0, 6.0, and 8.0 in Britton-Robinson buffer.

Figure 6. Oxidation pathway of lacidipine.

 SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1081
Table 1. Calibration curves for lacidipine obtained from different techniques
Technique Calibration curve
Differential pulse voltammetry Ip = 36350C + 0.09
UV spectrophotometry A = 672882C + 0.009
HPLC with electrochemical detection Area = 5.89 × 1011C + 21425
11
HPLC with UV detection Area = 6.43 × 10 C – 15050
(c) UV–Vis spectrophotometer.—Unicam UV-3 com-
puter-controlled double-beam spectrophotometer (ATI
Unicam, Cambridge, UK) with Vision software.
Results and Discussion
In an aqueous ethanol solution (70 + 30), lacidipine can be
oxidized at the glassy carbon electrode over a broad range of
pH. Oxidation produces only one anodic peak, which is better
resolved by using DPV than by other voltammetric modes.
Figure 2 shows the differential pulse voltammograms for
lacidipine at different pH values.
The pH behavior was studied in the pH range 2–14. The
peak potential versus pH plots (Figure 3) show 3 linear seg-
ments with breaks at approximately pH 5 and pH 10. The first
linear segment until pH 5 is slightly pH dependent, the next
segment between pH 5 and pH 10 has a slope of –35 mV/pH,
and the third segment between pH 10 and pH 14 has a slope of
–66 mV/pH. The insert in Figure 3 shows the peak current (Ip)
versus pH behavior. Ip is not markedly affected by pH
changes between pH 2 and pH 7 and between pH 10 to pH 14.
In the pH 7–10 zone, Ip is strongly pH dependent. Higher Ip
values were obtained in the acid zone than in the alkaline zone.
The behavior in pH-independent zones suggests that the limit-
ing current is controlled by the diffusion of the electroactive
species to the electrode surface (diffusion-controlled limiting
current).
CV experiments show only one irreversible anodic peak up
to scan rates of 5 V/s for lacidipine solutions. The irreversible
peak shows a current function value (Ip/V1/2) constant with
scan rate. Consequently, Ip shows a linear relation with the
square root of the scan rate. These results indicate that the
electrochemical process is controlled by the diffusion of the
electroactive species to the electrode surface.
Table 2. Repeatability and limits of measurements by different techniques
Measurement DPV
Detection limit, M 3.52 × 10–6 4.19 × 10–7
–6
Quantitation limit, M 3.78 × 10 4.28 × 10
Repeatability, CV%a 2.0
a
Coefficient of variation, calculated from 10 independent runs.
Regression coefficient Range, M
0.9970 5 × 10–6–2.0 × 10–4
0.9998 3 × 10–7–1.2 × 10–6
0.9960 1 × 10–7–1.2 × 10–6
0.9920 1 × 10–7–1.2 × 10–6
From the chemical structure shown in Figure 1, the most
likely electrooxidizable group is the 1,4-DHP ring. To support
this assumption, we compared the anodic peak obtained from
lacidipine with those obtained from very related compounds
(11). The similar responses indicated that the peak is due to
oxidation of the 1,4-DHP ring to give the pyridine derivative
according to Figure 4. Both reactions in Figure 4 correlate
with the results shown in the insert in Figure 3. At pH <10, the
electroactive species is the dihydropyridine moiety, but at pH
>10, it is the anionic form of this moiety.
According to the results, DPV can be used for quantitation
of lacidipine. Britton-Robinson buffer (pH 6.0)–ethanol (70 +
30) was selected for analytical purposes. Under this condition,
a linear dependence between Ip and lacidipine concentration
exists. For quantitation, the calibration plot method was used.
The calibration curve obeys the following equation:
Ip, µA = 36350.8C [M] + 0.09
(obtained from 10 points between 5 × 10–6 and 2 × 10–4M; re-
gression coefficient = 0.9970). The repeatability of 10 inde-
pendent measurements of a 1 × 10–5M lacidipine solution was
2%. Detection and quantitation limits were 3.52 × 10–6 and
3.78 × 10–6M, respectively. Although there are no readily
available dosage forms for this new drug, we tested the main
excipients used in the manufacture of pharmaceutical forms of
lacidipine described by some European laboratories, such as
lactose, magnesium stearate, starch, and talc. These excipients
did not interfere with the voltammetric analysis of lacidipine.
Consequently, separation steps are not required.
To compare different techniques and to better understand
the solution chemistry of lacidipine, a UV–Vis spectrophoto-
metric study also was completed. UV spectra at different pHs
(Figure 5) reveal 3 absorption signals, at 242, 286, and
Technique
UV HPLC with EC HPLC with UV
2.92 × 10–8 3.55 × 10–9
–7 –8
4.07 × 10 7.90 × 10–9
0.4 3.1 1.8
1082 SQUELLA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
375 nm. All absorption bands are pH independent. The band at
242 nm at pH 6 was selected for analytical purposes.
According to the results obtained with electrochemical and
spectroscopic techniques, it is possible to apply these tech-
niques to the quantitative analysis of lacidipine. Conse-
quently, an HPLC method using electrochemical or spectro-
photometric detection will be also possible. With the selected
experimental conditions, a peak with a retention time of
4.7 min was obtained (Figure 6). This peak shows a linear de-
pendence between the peak area and the lacidipine concentra-
tion, both for the electrochemical (1200 mV) and the UV–Vis
(242 nm) detectors.
A comparison of the calibration curves obtained from all
the developed methods is shown in Table 1. The repeatability
of the measurements and both detection and quantitation lim-
its are shown in Table 2.
The DPV determination developed represents a good ana-
lytical alternative because the method is easy, selective, and
adequately accurate and precise. The principal advantage of
the proposed voltammetric determination over HPLC meth-
ods is that the excipients do not interfere in the analysis. Con-
sequently, neither separation nor extraction procedures are re-
quired. With HPLC methods, excipients must be separated
previously to protect the column. Compared with other tech-
niques, the DPV method is cheap and the measurement is not
time consuming. The present voltammetric method is recom-
mended for quantitative determination of lacidipine in dosage
forms.
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Acknowledgments
We thank FONDECYT project No. 8970023 and DID
University of Chile for support.
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