Diazocoupling reaction for the spectrophotometric determination of physiologically active catecholamines in bulk and pharmaceutical preparations

A simple and sensitive spectrophotometric method for the determination of four catecholamines viz., adrenaline bitartarate (ABT), methyldopa (MDP), dopamine hydrochloride (DPH), and levodopa (LDP), in both pure form and in pharmaceutical formulations is described. The method is based on the reaction of diazotized sulphanillic acid (DSA) with catecholamines in a basic medium to yield orange-red colored products having absorption maxima at 507 nm for MDP and at 475 nm for others analytes. The colored species obeyed Beer’s law in the range of 1–27, 0.5–17, 0.6–15, and 1.5–19.2 μg/mL for ABT, MDP, DPH, and LDP, respectively. The molar absorptivity values as obtained from Beer’s law data were found to be 0.812 × 10⁴, 0.947 × 10⁴, 0.927 × 10⁴, and 0.709 × 10⁴ L/(mol cm), while Sandell’s sensitivity values were observed to be 412.03, 25.15, 20.44, and 27.81 ng/cm² for ABT, MDP, DPH, and LDP, respectively. Common excipients did not interfere with the proposed method. The results of the proposed methods compare favorably with those of official methods. The proposed method offers a simplicity, reliability, rapidity, and accuracy compared to the existing methods. The text was submitted by the author in English.     

Determination of chlorite in drinking water by on-line coupling of capillary isotachophoresis and capillary zone electrophoresis

An isotachophoresis (ITP)–capillary zone electrophoresis (CZE) combination was used for the determination of chlorite in drinking waters. No sample preparation is needed and no interfering by other anions in tap water was observed. The reached limits of detection with conductivity detector were 0.012–0.017 mg l⁻¹. By four-fold sample loading with a 30 μl valve, 0.005 mg l⁻¹ of chlorite was determined with R.S.D.=3.3%. The concentrations of 0.05 and 0.20 mg l⁻¹ were measured with R.S.D. of 2.2 and 2.7%, respectively. The recoveries of chlorite from drinking water were 96–106% in the range of 0.02–0.20 mg l⁻¹. The R.S.D. values of migration times (inter-day) were up to 1.3%. The time for analysis is about 15 min.     

Cellulose based macromolecular chelator having pyrocatechol as an anchored ligand: synthesis and applications as metal extractant prior to their determination by flame atomic absorption spectrometry

Pyrocatechol is immobilized on cellulose via ---NH---CH₂---CH₂---NH---SO₂---C₆H₄---N=N--- linker and the resulting macromolecular chelator characterized by IR, TGA, CPMAS ¹³C NMR and elemental analyses. It has been used for enrichment of Cu(II), Zn(II), Fe(III), Ni(II), Co(II), Cd(II) and Pb(II) prior to their determination by flame atomic absorption spectrometry (FAAS). The pH ranges for quantitative sorption (98.0–99.4%) are 4.0–7.0, 5.0–6.0, 3.0–4.0, 5.0–7.0, 5.0–8.0, 7.0–8.0 and 4.0–5.0, respectively. The desorption was found quantitative with 0.5 mol dm⁻³ HCl/HNO₃ (for Pb). The sorption capacity of the matrix for the seven metal ions has been found in the range 85.3–186.2 μmol g⁻¹. The optimum flow rate of metal ion solution for quantitative sorption of metal onto pyrocatechol functionalized cellulose as determined by column method, is 2–6 cm³ min⁻¹, whereas for desorption it is 2–4 cm³ min⁻¹. The tolerance limits for NaCl, NaBr, NaI, NaNO₃, Na₂SO₄, Na₃PO_4, humic acid, EDTA, ascorbic acid, citric acid, sodium tartrate, Ca(II) and Mg(II) in the sorption of all the seven metal ions are reported. Ascorbic acid is tolerable up to 0.8 mmol dm⁻³ with Cu and Pb where as sodium tartrate does not interfere up to 0.6 mmol dm⁻³ with Pb. There is no interference of NaBr, NaCl and NaNO₃ up to a concentration of 0.5 mol dm⁻³, in the sorption of Cu(II), Cd(II) and Fe(III) on to the chelating cellulose matrix The preconcentration factors are between 75 and 300 and t_1/2 values ≤5 min for all the metal ions. Simultaneous sorption of Cu, Zn, Ni and Co is possible at pH 5.0 if their total concentration does not exceed lowest sorption capacity. The present matrix coupled with FAAS has been used to enrich and determine the seven metal ions in river and tap water samples (relative standard deviation (R.S.D.) 1.05–7.20%) and synthetic certified water sample SLRS-4 (NRC, Canada) with R.S.D. 2.03%. The cobalt present in pharmaceutical vitamin tablets was also preconcentrated on the modified cellulose and determined by FAAS (R.S.D. 1.87%).     

Spectrophotometric Determination of Certain Vicinal Dihydroxybenzene Derivatives with Sodium Bismuthate

A simple and accurate spectrophotometric method is developed for the determination of certain vicinal dihydroxybenzene derivatives, namely, dopamine (DPH), levodopa (LD), methyldopa (MD) and pyrocatechol (PC) using sodium bismuthate in an acid medium. The method is based on the formation of a yellow colored product by the oxidation of vicinal dihydroxybenzene derivatives using sodium bismuthate. Beer's law was obeyed over the concentration range of 8–130 g/mL with the maximum absorption at 422–429 nm. The molar absorptivity and Sandell's sensitivity as evaluated from Beer's law data were found to be in the range of 1.02–1.34 × 10^–3L mol^–1cm^–2and 108–185 ng/cm²respectively. The proposed method has been successfully applied for the determination of DPH, LD and MD in injections and tablets of pharmaceutical preparations. The reliability of the methods was established by replicate determinations with the reported and official method.     

Flow-injection determination of phenylephrine hydrochloride in pharmaceutical dosage forms with on-line solid-phase extraction and spectrophotometric detection

A flow injection method is proposed for the determination of phenylephrine hydrochloride in pharmaceutical dosage forms. The method involves the use of on-line solid-phase extraction by means of a microcolumn containing Dowex 50W X8 ion-exchange resin for the separation of the analyte prior to colour development and spectrophotometric detection in the visible region.

The influence of preconcentration flow, preconcentration pH and elution volume was studied.

The method exhibits appropriate linearity (r² = 0.9999) which was proved statistically by means of the “F”-test. When applied to commercial samples containing several active ingredients and excipients, a significant reduction of interferences was found. Accuracy, evaluated by means of the spike recovery method was in the range 99.7–100.8%, with precision (R.S.D., %) better than 1%.

In order to achieve the automation the system was controlled from a notebook computer by means of a program written in QuickBASIC language. Under these conditions, a sampling frequency of 40 samples per hour could be attained.     

Cost-effective flow injection spectrophotometric assay of iron content in pharmaceutical preparations using salicylate reagent

A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyretic powder, which is a cheap and easily available reagent, is proposed. A red complex was continuously monitored by a laboratory-made green LED colorimeter. A linear calibration was obtained in the range of 1–20 mg Fe l⁻¹ with a detection limit of 0.5 mg Fe l⁻¹ and R.S.D.s of 1.4–5.4% (n=3, for 1–20 mg Fe l⁻¹). The new procedure was applied to assay iron contents in pharmaceutical preparations. The results were in good agreement with those of the USP standard method.     

Spectrophotometric methods for the determination of certain catecholamine derivatives in pharmaceutical preparations

Two simple, rapid and sensitive spectrophotometric methods for the determination of catecholamine derivatives (pyrocatechol, dopamine, levodopa and methyldopa) are developed. The first method involves the oxidation of o-dihydroxybenzene derivatives by N-bromosuccinimide followed by oxidative coupling with isoniazid leading to the formation of a red-coloured products of maximum absorbance (lambda(max)=480-490 nm). The second method is based on the formation of green to blue complex (lambda(max)=635-660 nm) between o- dihydroxybenzene derivatives and sodium nitroprusside in the presence of hydroxylamine hydrochloride. All measurements of the two procedures are carried out in an alkaline medium at room temperature. The two methods are successfully applied for the determination of dopamine hydrochloride, levodopa and methyldopa in injections and tablets of pharmaceutical preparation. The common excipients used as additives in pharmaceuticals do not interfere in the proposed methods. The reliability of these methods are established by parallel determination with the reported and official methods.     

Non-extraction batchwise and FIA determination of cationic and nonionic surfactants using Cu(II)-chromazurol S-surfactant complexes

Batchwise and FIA determinations have been developed for cationic and nonionic surfactants, based on the formation of ternary Cu(II)-chromazurol S-surfactant compounds. Optimum reaction conditions have been found (pH 8.0, lambda=590 and 630 nm, respectively). For the batchwise measurement, the molar absorption coefficient values epsilon(590)=5.1-5.7x10(4) l mol(-1) cm(-1) for cationic surfactants, epsilon(630)=0.7-1.5x10(4) l mol(-1) cm(-1) for nonionic surfactants. A factorial design has been carried out to determine the optimum flow conditions. Calibration curves were constructed and statistically evaluated for both the batchwise and FIA determination. For example, the linear concentration ranges for batch determination of the cationic surfactant cetyltrimethylammonium bromide and nonionic surfactant Marlophen NP 10 are 0-15 mug ml(-1) (R=0.9996, R.S.D.=6.62-0.64%) and 13-53 mug ml(-1) (R=0.9993, R.S.D.=4.48-1.40%), respectively; the respective detection limits are 0.02 and 4.0 mug ml(-1). For FIA determination of the same surfactants, the linear concentration ranges are 0-13 mug ml(-1) (R=0.9995, R.S.D.=4.44-0.49%) and 66-397 mug ml(-1) (R=0.9994, R.S.D.=8.92-1.12%), respectively, detection limits are 0.08 and 38 mug ml(-1), respectively.     

Enantioseparation and quality control of biperiden in pharmaceutical formulations by capillary electrophoresis

An original capillary electrophoretic method has been developed and applied for the enantioselective analysis of the antiparkinson drug biperiden in pharmaceutical formulations, using a modified cyclodextrin as the chiral selector. Baseline enantioseparation of the racemic compound was achieved in less than 7 min using an uncoated fused silica capillary (50 μm i.d. and 48.5, 40.0 cm, total and effective length, respectively), filled with a background electrolyte consisting of a 50 mM phosphate buffer at pH 3.5 supplemented with 3% (w/v) β-cyclodextrin sulphate and applying a voltage of 20 kV, reversed polarity. Samples were injected by pressure (50 mbar, 90 s) at the cathodic end of the capillary and detection wavelength was 195 nm (bandwidth: 10 nm). A simple and fast pre-treatment procedure allowed the complete extraction of the drug from commercial formulations (sustained release tablets and ampoules for injections) without any interference from the matrix. Good linearity was found in the 1–50 μg/mL concentration range; the limit of quantitation was 1 μg/mL and the limit of detection was 0.4 μg/mL. Precision and accuracy were good, with R.S.D. values always lower than 2.8% and a mean recovery value of 101.1%. The method was suitable for the quality control of biperiden in commercial formulations.     

Chemiluminescence determination of sodium 2-mercaptoethane sulfonate by flow injection analysis using cerium(IV) sensitized by quinine

A flow injection method with chemiluminescence (CL) detection is proposed for the determination of sodium 2-mercaptoethane sulfonate (MESNA), a drug often used to reduce the urotoxic effects of antineoplastic alkylating agents. The procedure is based on the reaction of the thiol with Ce(IV) in a sulfuric acid medium and measurement of the CL intensity produced by quinine used as a sensitizer. The optimum conditions for CL emission were investigated. Using the CL peak height as the analytical parameter, MESNA was determined over the concentration range 0.29–2.21 ng (1.97–9.85 μg·l⁻¹) with a detection limit of 0.21 ng (1.38 μg·l⁻¹) and a relative standard deviation (R.S.D.) of 4.1%. The method was applied satisfactorily to the determination of MESNA in pharmaceutical preparations with percentages of recovery between 94 and 105.     

Determination of trace metal ions by AAS in natural water samples after preconcentration of pyrocatechol violet complexes on an activated carbon column

A simple preconcentration method is described for the determination of Cu, Mn, Co, Cd, Pb, Ni and Cr in water samples by flame AAS. Trace metal ions in water were sorbed as pyrocatechol violet complexes on activated carbon column at the pH range of 4–8, then eluted with 1 M HNO₃ in acetone. The effect of major cations and anions of the natural water samples on the sorption of metal ions has been also investigated. The concentration of the metal ions detected after preconcentration was in agreement with the added amount. The present method was found to be applicable to the preconcentration of Cu, Mn, Co, Cd, Pb, Ni and Cr in natural water samples with good results such as R.S.D. from 3 to 8% (N=10) and detection limits under 70 ng l⁻¹.     

8-Hydroxyquinoline anchored to silica gel via new moderate size linker: synthesis and applications as a metal ion collector for their flame atomic absorption spectrometric determination

The silica gel modified with (3-aminopropyl-triethoxysilane) was reacted with 5-formyl-8-hydroxyquinoline (FHOQ_x) to anchor 8-quinolinol ligand on the silica gel. It was characterised with cross polarisation magic angle spinning (CPMAS) NMR and diffuse reflectance infrared Fourier transformation (DRIFT) spectroscopy and used for the preconcentration of Cu(II), Pb(II), Ni(II), Fe(III), Cd(II), Zn(II) and Co(II) prior to their determination by flame atomic absorption spectrometry. The surface area of the modified silica gel has been found to be 227 m² g⁻¹ and the two pKa values as 3.8 and 8.0. The optimum pH ranges for quantitative sorption are 4.0–7.0, 4.5–7.0, 3.0–6.0, 5.0–8.0, 5.0–8.0, 5.0–8.0 and 4.0–7.0 for Cu, Pb, Fe, Zn, Co, Ni and Cd, respectively. All the metals can be desorbed with 2.5 mol l⁻¹ HCl or HNO₃. The sorption capacity for these metal ions is in range of 92–448.0 μmol g⁻¹ and follows the order Cd3, NaCl, NaBr, Na₂SO₄ and Na₃PO₄, glycine, sodium citrate, EDTA, humic acid and cations Ca(II), Mg(II), Mn(II) and Cr(III) in the sorption of all the seven metal ions are reported. The preconcentration factors are 150, 250, 200, 300, 250, 300 and 200 for Cd, Co, Zn, Cu, Pb, Fe and Ni, respectively and t_1/2 values <1 min except for Ni. The 95% extraction by batch method takes ≤25 min. The simultaneous enrichment and determination of all the metals are possible if the total load of the metal ions is less than sorption capacity. In river water samples all these metal ions were enriched with the present ligand anchored silica gel and determined with flame atomic absorption spectrometer (R.S.D.≤6.4%). Cobalt contents of pharmaceutical samples (vitamin tablet) were preconcentrated with the present chelating silica gel and estimated by flame AAS, with R.S.D.1.4%. The results are in the good agreement with the certified value, 1.99 μg g⁻¹ of the tablets. Iron and copper in certified reference materials (synthetic) SLRS-4 and SLEW-3 have been enriched with the modified silica gel and estimated with R.S.D.<5%.     

Simultaneous determination of catecholamines by ion chromatography with direct conductivity detection

A simple method for simultaneous determination of three catecholamines using ion chromatography (IC) with direct conductivity detection (CD) based on the ionization of catecholamines in acidic medium without chemical suppression is developed in the present paper. The method could be used for the determination of these catecholamines in pharmaceutical preparations for the purpose of drug quality control. The recovery of catecholamines was more than 97% (n=3) and the relative standard deviation (R.S.D.) (n=11) was less than 2.1%. In a single chromatographic run, norepinephrine (NE), epinephrine (E) and dopamine (DA) can be determined in less than 10 min. The detection limits were found to be 0.001 μg/ml for NE, 0.01 μg/ml for E and DA respectively. Linear ranges were 0.01–50 μg/ml for NE (r²=0.9998), 0.1–50 μg/ml for E (r²=0.9995) and DA (r²=0.9999), respectively.     

Reactions of synthetic phenolic antioxidants with electrogenerated titrants and their analytical applications

Synthetic phenolic compounds (pyrogallol, catechol, hydroquinone, and their derivatives bearing heterocyclic fragments) react with electrogenerated titrants, halogens, and ferricyanide(III) ions. Stoichiometric coefficients of reactions are found. It is shown that the use of ferricyanide(III) ions as a titrant and a one-electron oxidant for the determination of this class of antioxidants offers advantages in comparison with titrants-halogens. The found amounts of pyrogallol, pyrocatechol, and hydroquinone derivatives in model solutions with the RSD 1–5% are fractions of milligrams. It is found that, in the series of the studied synthetic phenolic compounds, pyrocatechol derivatives possess the maximum antioxidant capacity (AOC).     

Determination of selenium, zinc and cadmium in antidandruff shampoos by atomic spectrometry after microwave assisted sample digestion

Microwave assisted pre-treatments for atomic spectrometric determination (inductive coupled plasma-optical emission spectrometry, ICP-OES or flame atomic absorption spectrometry, FAAS) of metallic elements, usually present in antidandruff shampoos, are proposed. They are based on the digestion of the sample with HNO₃ into a closed reactor, which is irradiated at 800 W for a few minutes. Selenium was determined by ICP-OES. The limit of detection was 0.11 mg l⁻¹; the relative standard deviation (R.S.D.) for the selenium content in the samples was in the 0.6–3.6% range. The results obtained were in agreement with the label contents and the recovery of the proposed method was in the 100–106% range. Zinc and cadmium were determined by FAAS. The limit of detection for zinc determination was 0.078 mg l⁻¹; the R.S.D. for zinc contents was in the 0.8–8.6% range. A limit of detection of 0.09 mg l⁻¹ was obtained for cadmium determination; the R.S.D. for cadmium contents was in the 0.7–2.7% range. The determinations were performed after two different sample mineralization pre-treatments — dry ashing (in an electric furnace) and wet mineralization (in a microwave oven). Both methodologies provided comparable results for zinc and cadmium determination in shampoos. The proposed microwave assisted digestion procedures allow a precise and accurate determination of selenium, zinc and cadmium in commercial antidandruff shampoos, and the sample pre-treatment is less time-consuming than the classic methods.     

Spectrophotometric determination of some histamine H1-antagonists drugs in their pharmaceutical preparations

Two rapid, simple and sensitive extractive specrophotometric methods has been developed for the determination of three histamine H1-antagonists drugs, e.g., chlorphenoxamine hydrochloride (CPX), diphenhydramine hydrochloride (DPH) and clemastine (CMT) in bulk and in their pharmaceutical formulations. The first method depend upon the reaction of molybdenum(V) thiocyanate ions (Method A) with the cited drugs to form stable ion-pair complexes which extractable with methylene chloride, the orange red color complex was determined colorimetrically at lambda(max) 470nm. The second method is based on the formation of an ion-association complex with alizarin red S as chromogenic reagents in acidic medium (Method B), which is extracted into chloroform. The complexes have a maximum absorbance at 425 and 426nm for (DPH or CMT) and CPX, respectively. Regression analysis of Beer-Lambert plots showed a good correlation in the concentration ranges of 5.0-40 and 5-70microgmL(-1) for molybdenum(V) thiocyanate (Method A) and alizarin red S (Method B), respectively. For more accurate analysis, Ringbom optimum concentration ranges were calculated. The molar absorptivity, Sandell sensitivity, detection and quantification limits were calculated. Applications of the procedure to the analysis of various pharmaceutical preparations gave reproducible and accurate results. Further, the validity of the procedure was confirmed by applying the standard addition technique and the results obtained in good agreement well with those obtained by the official method.     

大口径毛细管气相色谱法直接测定复方氨酚苯海拉明片中的4种组分

建立了同时测定复方氨酚苯海拉明片中对乙酰氨基酚(PAR)、咖啡因(CAF)、盐酸苯海拉明(DPH)和盐酸麻黄碱(EPD)的大口径毛细管气相色谱法。无需衍生化处理,直接进样,在HP-1大口径毛细管色谱柱(10 m×0.53 mm,2.65 μm)上,上述4种药物在3 min内完全分离。PAR、CAF、DPH和EPD 4种药物的质量浓度分别为50~2400,10~500,10~500和10~500 mg/L时,药物与内标的浓度比和它们的峰面积比呈良好的线性关系(线性相关系数均高于0.999),各组分的平均回收率为98.5%~101.1%,日内相对标准偏差均小于2.2%,最低检测限为1~30 μg/L。该法快速、灵敏、准确,可为复方氨酚苯海拉明片的质量控制提供科学依据。     

Spectrophotometric study of the reaction mechanism between DDQ Pi- and iodine sigma-acceptors and chloroquine and pyrimethamine drugs and their determination in pure and in dosage forms

Two simple and accurate spectrophotometric methods are presented for the determination of anti-malarial drugs, chloroquine phosphate (CQP) and pyrimethamine (PYM), in pure and in different pharmaceutical preparations. The charge transphere (CT) reactions between CQP and PYM as electron donors and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) pi-acceptor and iodine sigma-acceptor reagents to give highly coloured complex species have been spectrophotometrically studied. The optimum experimental conditions have been studied carefully. Beer' law is obeyed over the concentration range of 1.0-15 microg ml(-1) for CQP and 1.0-40 microg ml(-1) for PYM using I(2) and at 5.0-53 microg ml(-1) for CQP and 1.0-46 microg ml(-1) for PYM using DDQ reagents, respectively. For more accurate results, Ringbom optimum concentration range is calculated and found to be 10-53 and 8-46 microg ml(-1) for CQP and PYM using DDQ, respectively and 5-15 and 8-40 microg ml(-1) for CQP and PYM using iodine, respectively. The Sandell sensitivity is found to be 0.038 and 0.046 g cm(-2) for DDQ method and 0.0078 and 0.056 g cm(-2) for I(2) method for CQP and PYM, respectively which indicates the high sensitivity of both methods. Standard deviation (S.D.=0.012-0.014 and 0.013-0.015) and relative standard deviation (R.S.D.=0.09-1.4 and 1.3-1.5%) (n=5) for DDQ and I(2) methods respectively, refer to the high accuracy and precision of the proposed methods. These results are also confirmed by between day precision of percent recovery of 99-100.6%, and 98-101% for CQP and PYM by DDQ method and 99-102% and 99.2-101.4% for CQP and PYM by I(2) method respectively. These data are comparable to those obtained by British and American pharmacopoeias assay for the determination of CQP and PYM in raw materials and in pharmaceutical preparations.     

Spectrophotometric study of the reaction mechanism between DDQ as pi-acceptor and potassium iodate and flucloxacillin and dicloxacillin drugs and their determination in pure and in dosage forms

Two simple and accurate spectrophotometric methods are presented for the determination of beta-lactam drugs, flucloxacillin (Fluclox) and dicloxacillin (Diclox), in pure and in different pharmaceutical preparations. The charge transfer (CT) reactions between Fluclox and Diclox as electron donors and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) pi-acceptor and potassium iodate via oxidation reduction reaction where the highly coloured complex species or the liberated iodine have been spectrophotometrically studied. The optimum experimental conditions have been studied carefully. Beer's law is obeyed over the concentration range of 2-450 microg ml(-1) for Fluclox and 10-450 microg ml(-1) for Diclox using DDQ reagent and at 50-550 microg ml(-1) for Fluclox and 50-560 microg ml(-1) for Diclox using iodate method, respectively. For more accurate results, Ringbom optimum concentration range is calculated and found to be 6-450 and 15-450 microg ml(-1) for Fluclox and Diclox using DDQ, respectively, and 65-550 and 63-560 microg ml(-1) for Fluclox and Diclox using iodine, respectively. The Sandell sensitivity is found to be 0.018 and 0.011 microg cm(-2) for DDQ method and 0.013 and 0.011 microg cm(-2) for iodate method for Fluclox and Diclox, respectively, which indicates the high sensitivity of both methods. Standard deviation (S.D.=0.01-0.80 and 0.07-0.98) and relative standard deviation (R.S.D.=0.13-0.44 and 0.11-0.82%) (n=5) for DDQ and iodate methods, respectively, refer to the high accuracy and precision of the proposed methods. These results are also confirmed by between-day precision of percent recovery of 99.87-100.2 and 99.90-100% for Fluclox and Diclox by DDQ method and 99.88-100.1 and 99.30-100.2% for Fluclox and Diclox by iodate method, respectively. These data are comparable to those obtained by British and American pharmacopoeias assay for the determination of Fluclox and Diclox in raw materials and in pharmaceutical preparations.     

Penicillamine determination using a tyrosinase micro-rotating biosensor

Tyrosinase [EC 1.14.18.1], immobilized on a rotating disk, catalyzed the oxidation of catechols to o-benzoquinone, whose back electrochemical reduction was detected on glassy carbon electrode surface at −150 mV versus Ag/AgCl/NaCl 3 M. Thus, when penicillamine (PA) was added to the solution, this thiol-containing compound participate in Michael type addition reactions with o-benzoquinone to form the corresponding thioquinone derivatives, decreasing the reduction current obtained proportionally to the increase of its concentration. This method could be used for sensitive determination of PA in drug and human synthetic serum samples. A linear range of 0.02–80 μM (r = 0.999) was obtained for amperometric determination of PA in buffered pH 7.0 solutions (0.1 M phosphate buffer). The biosensor has a reasonable reproducibility (R.S.D. < 4.0%) and a very stable amperometric response toward this compound (more than 1 month).