Colorimetric determination of carbidopa in anti-Parkinson drugs based on 4-hydroxy-3-methoxybenzaldazine formation by reaction with vanillin

Abstract

In this paper is reported the selective colorimetric detection and quantification of carbidopa, an inhibitor of aromatic amino acid decarboxylase, in the co-presence of levodopa as dopamine precursor in pharmaceutical formulations for the treatment of Parkinson's disease. The method is based on the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of vanillin, a natural flavoring agent, in acidified alcoholic solution. The yellow color development (λ_max ~ 420 nm) due to the formation of 4-hydroxy-3-methoxybenzaldazine (HMOB) was observed for carbidopa only, whereas levodopa, lacking the hydrazine group, did not color the solution, as expected. The calibration curves for two tablet formulations of levodopa in combination with carbidopa (4:1) were superimposable with levodopa/carbidopa (4:1), as well as carbidopa alone, in standard solution, i.e., the excipients and additives did not interfere with carbidopa determination, corresponding to a mean recovery about 105%. The linear dynamic range was between 5.00 and 50.0 mg L^-1 with very good reproducibility within this range (CV_av% about 3-4%) and very good sensitivity, with limits of quantification of about 1 mg L^-1. The colorimetric method developed here is very simple, inexpensive, and effective for drug estimation and quality control of pharmaceutical formulations.

Keywords: Anti-Parkinson drugs; Benzaldazine; Carbidopa; Colorimetry; Levodopa; Vanillin.

Fig. 1

Scheme of condensation reactions of vanillin with carbidopa and levodopa. Carbidopa hydrazide functional group produces a hydrazone that further reacts with an excess of vanillin to form the 4-hydroxy-3-methoxybenzaldazine (HMOB), with a maximum visible absorbance about 420 nm (yellow). Levodopa amine group produces an imine that cannot generate the azine, leaving the solution uncolored at acidic pH

Fig. 2

Influence of solvent composition and pH on color development. All the solutions contain 50.0 mg L⁻¹ (0.221 mM) of CD and 10.0 mM vanillin reacted for 4 h at 70 °C. (A) Absorbance spectra in acetonitrile (MeCN, red line), methanol (MeOH, red dashes), ethanol (EtOH, black line), water (H₂O, blue dashes), dimethyl sulfoxide (DMSO, blue dots). (B) Absorbance spectra for different EtOH:H₂O compositions, from neat water (H₂O, blue dashes) to neat ethanol (EtOH, black line). (C) Absorbance spectra in EtOH:H₂O (1:1), containing HCl 5 mM (blue line), 50 mM (black line), or 500 mM (red line)

Fig. 3

Influence of vanillin concentration, temperature, and heating time on color development. Absorbance at 415 nm for CD 50.0 mg L⁻¹ samples dissolved in EtOH/H₂O 1:1 with HCl 0.500 M. (A) Vanillin 0.00, 1.25, 2.50, 5.00, 10.0, 20.0 mM after 4 h at 70 °C. (B) Vanillin 10.0 mM at 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C after 4 h. (C) Vanillin 10.0 mM at 70 °C after 15, 30, 60, 120, 240 min in presence of carbidopa (CD, black circles), levodopa (LD, white circles), LD + CD 4:1 (half black circles), brand drug (blue circles), and generic drug (red circles). Each point represents the mean ± SD of 4 replicates

Fig. 4

Colorimetric calibration plots. Absorbance values at 415 nm for sample solutions in EtOH/H₂O 1:1 with HCl 0.500 M and vanillin 10.0 mM after 4 h. Carbidopa (CD, black circles), levodopa (LD, white circles), LD + CD 4:1 (half black circles), brand drug (blue circles), and generic drug (red circles). The explored concentration range was between 5.00 and 50.0 mg L⁻¹ for CD, and between 20.0 and 200 mg L⁻¹ for LD alone or in combination with CD. Each point represents the mean ± SD of 4 replicates. Data were fitted according to linear Eq. 1