Spectrophotometric determination of penicillamine and carbocisteine based on formation of metal complexes

IL FARMACO 59 (2004) 493–503 www.elsevier.com/locate/farmac

Spectrophotometric determination of penicillamine and carbocisteine based on formation of metal complexes M.I. Walash *, A.M. El-Brashy, M. E.-S. Metwally, A.A. Abdelal Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt Received 14 July 2003; accepted 8 November 2003 Available online 27 March 2004

Abstract A simple spectrophotometric method was developed for the determination of penicillamine and carbocisteine. The method depends on complexation of penicillamine with Ni, Co and Pb ions in acetate buffer pH of 6.3, 6.5 and 5.3, respectively, and carbocisteine with Cu and Ni ions in borate buffer pH of 6.7; 1–70 µg/ml of these drugs could be determined by measuring the absorbance of each complex at its specific kmax. The results obtained are in good agreement with those obtained using the official methods. The proposed method was successfully applied for the determination of these compounds in their dosage forms. Also, the molar ratio and stability constant of the metal complexes were calculated and a proposal of the reaction pathway was postulated. © 2004 Elsevier SAS. All rights reserved. Keywords: Carbocisteine; Complexation; Dosage forms; Penicillamine; Spectrophometric

1. Introduction The importance of penicillamine (I) and carbocisteine (II) is due to their widespread and different pharmacological effects. Penicillamine (d-3,3 dimethyl cysteine) is used for treatment of rheumatoid arthritis and in treatment of Pb poisoning as chelating agent, it is also used for elimination of Cu in treatment of hepatolenticular degeneration (Wilson’s disease). Carbocisteine (S-carboxymethyl-L-cysteine) is a mucolytic drug used for treatment of disorders of the respiratory tract associated with excessive mucus [1,2].

The published methods reported for the determination of these drugs included titrimetry [3–5], spectrophotometry [6–10], fluorometry [11,12], electro-analysis [13–16] and chromatography [17–24]. The sulphur atom in sulphur con* Corresponding author. Fax: +20-50-247496. E-mail address: [email protected] (M.I. Walash). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2003.11.016

taining compounds is thought to be a site of chelation interaction with various metal ions [25–30]. The ability of D-penicillamine to act as chelating agent in therapeutic treatment of Wilson’s disease and mercury poison has been extensively investigated [31–39], and the stability constants for bivalent metals were reported and calculated from pH values using known mathematical relations and computer programming [40,41]. Colorimetric analysis of penicillamine based on Cu (II) complex formation was reported where the absorption maximum is measured at 522 nm (after 30 min) [42]. The aim of the present work is to study the complexation reaction of penicillamine and carbocisteine with some metal ions in acetate or borate buffer in an attempt to develop a simple and sensitive method for the determination of these drugs in pure form as well as in their dosage forms.

2. Experimental 2.1. Equipment A Shimadzu (Model 1601 PC) UV–visible spectrophotometer (Shimadzu, Kyoto, Japan) was used to measure the absorbance at 200–400 nm. Recording range is 0–2.

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2.2. Reagents and materials All reagents used were of analytical reagent grade and the water was always double distilled. Penicillamine was kindly offered by Biochemie company, Austria and carbocisteine by Amyria Pharmaceutical Industries, Egypt. The purity of these drugs was determined by applying the official methods [3]. Stock solutions of the studied drugs were prepared by dissolving 100.0 mg of the drug in distilled water (3 ml of 0.4 M NaOH was firstly added in case of carbocisteine) then completed to 100 ml with distilled water. Other concentrations were prepared by dilution with distilled water, aqueous solution of 0.005 M CuCl2 (Prolabo, France), 1% CoCl2 (Aldrich, Germany), 0.5% NiSO4 (BDH, UK), 0.5% Pb(CH3COO)2 (Aldrich, Germany), 0.4 M NaOH (BDH, UK) and 0.2 M acetic acid (Prolabo, France) were prepared.

2.3. Procedures 2.3.1. Procedure for penicillamine A sample aliquot containing 1–70 µg/ml of the drug was transferred to a 10-ml volumetric flask and 1.5 ml of NiSO4 (1 ml of Pb(CH3COO)2 or 1 ml of CoCl2 solutions) was added followed by 5 ml of acetate buffer. The absorbance was measured against a reagent blank prepared simultaneously at the specific kmax (Table 1). 2.3.2. Procedure for carbocisteine A sample aliquot containing 4–70 µg/ml of the drug was transferred to a 10-ml volumetric flask and 2 ml of CuCl2 or 1 ml of NiSO4 solutions were added followed by 5 ml of

borate buffer. The absorbance was measured against a reagent blank prepared simultaneously at the specific kmax (Table 1). 2.3.3. Procedure for the dosage forms An accurately weighed quantity of the mixed contents of 10 capsules or an accurately measured volume of the syrup equivalent to 100 mg of the drug was transferred into a 100-ml volumetric flask and the volume was made up to the mark with distilled water (3 ml of 0.4 M NaOH solution was firstly added in case of Rhinathiol® syrup). The contents of the flask were sonicated for 5 min and filtered, if necessary. The above procedure was then followed. The nominal content was calculated either from a previously plotted calibration graph or using the corresponding regression equation.

3. Results and discussion Penicillamine and carbocisteine possess a sulphur atom, which reacts with metal ions forming stable complexes. Trials were adopted to determine these pharmaceutical drugs by complexation with a large number of metal ions. Only Co+2, Ni+2 and Pb+2 reacted with penicillamine, Cu+2 and Ni+2 reacted with carbocisteine. These ions gave high sensitivity while Zn+2 and Mn+2 gave very low sensitivity. All used metals for penicillamine were proved to be equally satisfactory but complexation with Ni+2 metal was with more sensitivity, detection limit, quantification limit and A% of the formed complex than Co+2 and Pb+2 which were nearly the same in detection limit and quantification limit but Co+2 with more sensitivity and A% than Pb+2. Also, Pb+2 is not preferred in use because it is considered one of the hazard metals. For carbocisteine, complexation with Ni+2 was with more sensitivity, detection limit, quantification limit and A% of the formed complex than Cu+2.

Table 1 Collective data of the studied compounds by complexometry

Optimum pH Volume of metal solution Wavelength (kmax) (nm) Concentration range (µg/ml) A% Regression equation *Y = a + bX Correlation coefficient Detection limit (µg/ml) Molar ratio (drug/metal) *

Penicillamine NiSO4 6.3 Acetate buffer 1.5 ml of (0.5%)

CoCl2 6.5

Pb(CH3COO)2 5.3

1 ml of (1%)

1 ml of (0.5%)

Carbocisteine CuCl2 6.7 Borate buffer 2 ml of (0.005 M)

270 1–20

291 2–20

267 2–25

280 5–70

521.5 Y = 2.158 × 10–3 + 0.0522X 0.9999 0.07 2:1

363.5 Y = –1.79 × 10–3 + 0.03445X 0.9999 0.14 2:1

344 106.6 Y = –2.09 × 10–3 + 0.03445X Y = –1.69 × 0–3 + 0.0106X

146.4 Y = –7.29 × 10–4 + 0.01465X

0.9999 0.11 2:1

0.9999 0.21 1:1

Y: absorbance; a: intercept; X: Concentration (µg/ml); b: slope.

0.9999 0.53 1:1

NiSO4 6.7 1 ml of (0.5%) 252 4–50

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3.1. Optimization of the reaction conditions 3.1.1. Spectra Penicillamine has maximum absorbance at 215 nm in acetate buffer, on addition of NiSO4, CoCl2 and Pb(CH3COO)2, three maxima are obtained at 270, 291 and

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267 nm for these metal ions, respectively (Figs. 1–3). On the other hand, carbocisteine absorbs at maximum 213 nm in borate buffer, on addition of CuCl2 and NiSO4 the maximum wavelength is shifted to 280 and 251 nm for these two metal ions, respectively (Figs. 4 and 5).

Fig. 1. Spectra of: (A) Penicillamine (20 µg/ml) in acetate buffer (pH = 6.3). (B) NiSO4 (1.5 ml of 0.5%) in acetate buffer (pH = 6.3). (C) Complex of penicillamine (20 µg/ml) with NiSO4 (1.5 ml of 0.5%) in acetate buffer (pH = 6.3) at 270 nm. (D) Complex of penicillamine disulphide (20 µg/ml) with NiSO4 (1.5 ml of 0.5%) in acetate buffer (pH = 6.3).

Fig. 2. Spectra of: (A) Penicillamine (20 µg/ml) in acetate buffer (pH = 6.5). (B) CoCl2 (1 ml of 1%) in acetate buffer (pH = 6.5). (C) Complex of penicillamine (20 µg/ml) with CoCl2 (1 ml of 1%) in acetate buffer (pH = 6.5) at 291 nm. (D) Complex of penicillamine disulphide (20 µg/ml) with CoCl2 (1 ml of 1%) in acetate buffer (pH = 6.5).

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Fig. 3. Spectra of: (A) Penicillamine (20 µg/ml) in acetate buffer (pH = 5.3). (B) Pb(CH3COO)2 (1 ml of 0.5%) in acetate buffer (pH = 5.3). (C) Complex of penicillamine (20 µg/ml) with Pb(CH3COO)2 (1 ml of 0.5%) in acetate buffer (pH = 5.3) at 267 nm. (D) Complex of penicillamine disulphide (20 µg/ml) with Pb(CH3COO)2 (1 ml of 0.5%) in acetate buffer (pH = 5.3).

Fig. 4. Spectra of: (A) Carbocisteine (50 µg/ml) in borate buffer (pH = 6.7). (B) CuCL2 (2 ml of 0.005 M) in borate buffer (pH = 6.7). (C) Complex of carbocisteine (50 µg/ml) with CuCL2 (2 ml of 0.005 M) in borate buffer (pH = 6.7) at 280 nm.

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Fig. 5. Spectra of: (A) Carbocisteine (50 µg/ml) in borate buffer (pH = 6.7). (B) NiSO4 (1 ml of 0.5%) in borate buffer (pH = 6.7). (C) Complex of carbocisteine (50 µg/ml) with NiSO4 (1 ml of 0.5%) in borate buffer (pH = 6.7) at 252 nm.

3.1.2. Effect of pH The reaction between each of penicillamine and carbocisteine and the studied metal ions was performed in different media. In acid medium, low sensitivity is noticed, while in alkaline medium, the metal ions are precipitated as hydroxides. Acetate pH (2.5–6.3) and borate pH (6.5–7.5) buffers are the best media for the reaction (Figs. 6 and 7). The absorbance of the complexes increases up to 5 ml of each buffer, after that no further increase. 3.1.3. Effect of metal ion concentration The required amount of metal ions for maximum absorbance, besides the optimum pH, is summarized in Table 1.

Fig. 6. Effect of pH on complexation of (20 µg/ml) penicillamine with 1 ml 1% CoCl2, 1.5 ml 0.5% NiSO4 and 1 ml 0.5% Pb(CH3COO)2, respectively.

The required volume of metals was 1.5 ml of NiSO4, 1 ml of CoCl2 and 1 ml of Pb(CH3COO)2 solutions for penicillamine and for carbocisteine were 2 ml of CuCl2 and 1 ml of NiSO4 solutions. 3.1.4. Effect of time on the formation and stability of the formed complexes The effect of time on the absorbance of drug metal complex was investigated. It was found that the complex formation was instantaneous and the formed complex was stable for more than 1 h.

Fig. 7. Effect of pH on complexation of (50 µg/ml) carbocisteine with 2 ml 0.005 M CuCl2 and 1 ml 0.5% NiSO4.

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Fig. 8. Calibration curve of penicillamine with 1% CoCl2, 0.5% NiSO4 and 0.5% Pb(CH3COO)2.

3.2. Applications By adjusting the optimum conditions required for the reaction between carbocisteine and penicillamine with the studied metal ions, rectilinear calibration graphs are obtained in the concentration ranges given in Table 1. The limits of detection as well as regression equations and molar ratios are tabulated in Table 1. Calibration graphs are shown in Figs. 8 and 9. The % recoveries of these studied drugs compared with those obtained by the official methods [3] are

given in Tables 2–4. The latter method recommends nonaqueous titration. Statistical analysis was conducted between the results of proposed and official methods [3] by calculating Student’s t-test and variance ratio F-test [43]. As shown in Tables 2–4, there is no significant difference between the two procedures regarding accuracy and precision. The proposed method was successfully applied for the determination of the studied drugs in their different dosage forms, as shown in Table 5, compared with the results obtained by the reference methods [6,9]. The latter methods recommended spec-

Fig. 9. Calibration curve of carbocisteine with 0.005 M CuCl2 and 0.5% NiSO4. Table 2 Determination of carbocisteine by CuCl2 Compound

Carbocisteine

Mean ± S.D. t-test F-test a b

Proposed method Taken (µg/ml) Found (µg/ml) 70 70.25 60 60.07 50 50.44 40 40.25 30 30.07 20 20.16 10 9.88 5 5.06 100.38 ± 0.731

% Recovery a 100.36 100.12 100.88 100.62 100.23 100.80 98.80 101.2

Official method [3] Taken (mg) % Recovery a

100 150 200

100.36 99.16 99.46

99.66 ± 0.624 0.836 2.69

(2.262) b (19.36)

Each result is the average of three separate experiments. The value between brackets are the tabulated student’s t-test and variance test (at P = 0.05) [43].

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Table 3 Determination of penicillamine and carbocisteine by NiSO4 Compound Taken (µg/ml) 20 15 12 10 5 2 1

1-Penicillamine

Mean ± S.D. t-test F-test 2-Carbocisteine

50 40 30 20 10 5 4

Proposed method Found (µg/ml) 20.02 15.00 11.96 10.00 5.01 1.995 1.02 100.22 ± 0.737

50.02 40.05 30.02 19.91 10.02 4.96 4.07

Mean ± S.D. t-test F-test

% Recovery a 100.10 100.00 99.67 100.00 100.20 99.75 101.84

Taken (mg)

0.248 1.86 100.04 100.13 100.07 99.55 100.20 99.29 101.93 100.17 ± 0.845 1.15 1.83

(2.306) b (5.14)

Official method [3] % Recovery a

100 150 200

101.49 99.50 100.l75

100.58 ± 1.006

100 150 200

100.36 99.16 99.46

99.66 ± 0.624 (2.306) b (19.33)

For footnote see Table 2.

trophotometric methods for determination of penicillamine and carbocisteine in their dosage forms, and also by calculating Student’s t-test and variance ratio F-test [43]. As shown in Table 5, there is no significant difference between the two procedures regarding accuracy and precision.

of the parent compound at its kmax (215 nm) was observed, with no absorbance at the kmax of the complexes (270 and 291 nm). It is thus concluded that the method is stability indicating and selective for penicillamine in partially degraded solutions. 3.4. Mechanism of the reaction

3.3. Stability indication of the method Penicillamine disulphide is the major degradation product of penicillamine and is frequently found mixed with the latter as a known impurity [3]. Penicillamine disulphide was tested with the studied metal ions. No observed changes in the absorption spectrum of the compound were noticed using Pb+2. In case of Ni+2 and Co+2, an increase in the absorbance

The stoichiometry of the reaction is studied adopting the molar ratio method [44], Job’s method of continuous variation [45] and limiting logarithmic method [46]. The ratio of penicillamine to reagent is found to be 2:1 to Co+2, Ni+2 and Pb+2. The ratio of carbocisteine is found to be 1:1 to Cu+2 and Ni+2. This ratio agreed with that reported for penicillamine and carbocisteine with metal [47–51]. The molar ratio is shown in Figs. 10–13.

Table 4 Determination of penicillamine by CoCl2 and Pb-acetate Compound

Penicillamine

Proposed method CoCl2 Taken (µg/ml) Found (µg/ml) 20 20.03 15 14.95 12 11.95 10 10.05 8 8.02 5 5.03 2 1.97

Mean ± S.D. t-test F-test For footnote see Table 2.

1.88 1.93

Official method [3] % Recovery a 100.15 99.67 99.58 100.50 100.25 100.60 98.50 99.89 ± 0.725 (2.306) b (5.14)

Pb(CH3COO)2 Taken (µg/ml) 25 20 15 10 5 2

Found (µg/ml) 25.02 20.06 14.84 10.02 5.05 2.01

% Recovery a 100.08 100.30 98.93 100.20 101.00 100.50

1.038 1.71

100.22 ± 0.640 (3.365) b (5.79)

Taken (mg)

% Recovery a

100 150 200

101.49 99.50 100.75

100.58 ± 1.006

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Table 5 Determination of penicillamine and carbocisteine in their dosage forms by complexometry CuCl2 Taken Rec. (µg/ml) %* 1-Artamine® capsule (250 mg of penicillamine/capsule) Mean ± S.D. t-test F-test 2-Rhinathiol® Syrup (2 mg carbocisteine/100 ml)

Mean ± S.D. t-test F-test

50 40 30

100.56 101.08 99.44

100.36 ± 0.84 0.823 (2.447)** 2.007 (19.25)

Proposed methods NiSO4 CoCL2 Taken Rec. Take n Rec. µg/ml %* (µg/ml) %* 20 99.04 20 99.42 15 99.22 15 100.40

Pb-acetate Taken Rec. (µg/ml) %* 3 99.00(9) 8 100.25

Reference methods Taken Rec. %* µg/ml 20 18.75 15 99.84

12

12

12

98.49 99.27 ± 0.546 0.945 (2.776)** 1.467 (19.00) 50 100 40 101.45 30 99.50

12

98.67 98.92 ± 0.381 1.313 (2.776)** 3.010 (19.00)

100.33 99.5 ± 0.868 0.625 (2.776)** 1.724 (19.00)

50 40 30 20 10

100.63 ± 1.01 0.934 (2.447)** 1.388 (19.25)

99.23 100.35 ± 0.661

101.02(6) 98.93 98.57 100.00 101.20 99.95 ± 1.19

• The results are the average of 6–separate determinations (1) Biochemie compagny, Austria. (2) Amyria-Pharmaceutical Compagny, Egypt.

3.5. The Proposal mechanism may be as follows and 3.6. Stability constants of the complexes

Fig. 10. Mole ratio of penicillamine and metal ions (6.7 × 10–4 M for penicillamine, NiSO4, CoCl2, Pb(CH3COO)2).

The stability constants of the complexes are calculated A⁄Am n n according to the equation [52]: Kf = n−1 C n 关 共 1−A 兲⁄Am 兴 where A and Am are absorbance and maximum absorbance obtained from Job’s continuous variation curves; n: ratio between drug to metal; C: molar concentration of the drug; Kf: stability formation of the complex. The stability constant of penicillamine with the used metal ions is 2.1 × 107, 4.2 × 107 and 1.5 × 108 for Ni+2, Co+2 and Pb+2 ions, respectively. The stability constants of carbocisteine are 1.2 × 105 and 5.6 × 105 with Cu+2 and Ni+2, respectively.

4. Conclusion

Fig. 11. Mole ratio of carbocisteine and metal ions (5.58 × 10–4 M for each carbocisteine, NiSO4 and CuCl2).

The proposed method is simple, accurate, precise, sensitive, very rapid, low cost and relatively selective compared to the official method. Furthermore, the proposed method does not require elaboration of procedures which usually associated with chromatography methods. The proposed method could be applied successfully for determination of penicillamine and carbocisteine in pure form as well as different dosage forms. Moreover, small amounts of the studied compounds could be determined compared with the official methods [3], which required at least 100 mg to be determined by non-aqueous titration.

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Fig. 12. Continuous variation graph for penicillamine with (A) Co+2 (B) Pb+2 (C)Ni+ (6.7 × 10–4 M for penicillamine and every metal ion).

Fig. 13. Continuous variation graph for carbocisteine with (A) Cu+2 (B) Ni+2 (5.58 × 10–4 M for carbocisteine and every metal ion).

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