Penicillamine determinant

However, in these studies, no serious attempts were made ot assess the antibodies specific for penamaldate and penicillamine determinants, which are precisely those which could be expected. Benzylpenicilloic acid, when applied to guinea pig skin, seems able through whatever immunochemical mechanism to induce contact hypersensitivity (Levine 1960 a). 

D-Penicillamine may arise directly from penicilloic acid or through formation of a penicillamine-cysteine mixed disulfide, following a penamaldate rearrangement (Levine 1960 c Yemal et al. 1978). This reaction also occurs with functional derivatives of the alpha-carboxylic group of penicilloic acid (Schneider et al. 1973). The penicillamine determinant may therefore arise in vivo from any penicilloyl conjugate. 

The British Pharmacopoeia [2] describes a potentiometric titration method for the determination of penicillamine as the pure drug substance. The method is performed by dissolving 100 mg of the substance in 30 mL of anhydrous acetic acid. The mixture is titrated with 0.1 M perchloric acid, and the endpoint determined potentiometrically. Each milliliter of 0.1 M perchloric acid is equivalent to 14.92 mg of C6HnN02S. 

The British Pharmacopoeia [2] also describes a method for determination of penicillamine tablets. Weigh and powder 20 tablets. Dissolve a quantity of the powder containing 100 mg of penicillamine as completely as possible in 50 mL of water and filter. Add 5 mL of 1 M sodium hydroxide to the filtrate and 0.2 mL of a 0.1% (w/v) solution of dithizone in 96% ethanol. Titrate with 0.02 M mercury(II) nitrate VS each mL of 0.02 M mercury(II) nitrate VS is equivalent to 5.968 mg of C6HnN02S. 

Vollmer et al. [4] compared the Hg(II) acetate method described in the Code of Federal Regulations for the determination of penicillamine in bulk drug and formulations with (i) a nonaqueous lithium methoxide titration, (ii) a nonaqueous HCLO4 titration, and (iii) a colorimetric method with hydroxylamine. Method (ii) was unsatisfactory for bulk determinations. Method (i) was less precise than the Hg(II) acetate method, but gave satisfactory results for bulk drug and capsule samples. Method (iii) was the only method that gave satisfactory results in the presence of EDTA. 

Lopez-Fonseca et al. [11] discussed the theory of reverse pulse polarography and the technique was applied in the determination of penicillamine electrochemically coated on a dropping-mercury electrode. Using long drop times and short pulses, the drug can be determined at levels as low as 50 nM in the presence of Cu(II), and the technique compares well with normal-pulse and differential-pulse polarography. 

Besada [12] described a spectrophotometric method for determination of penicillamine by reaction with nitrite and Co(II). Penicillamine is first treated with 1 M NaN02 (to convert the amino-group into a hydroxy-group), then with 0.1 M CoCl2, and finally the absorbance of the brownish-yellow complex obtained is measured at 250 nm. The process is carried out in 50% aqueous ethanol, and the pH is adjusted to 5.4— 6.5 for maximum absorbance. The calibration graph is linear over the concentration range of 0.25-2.5 mg per 50 mL, and the mean recovery (n = 3) of added drug is 99.7%. Cystine, cysteine, methionine, and other amino adds do not interfere. 

Besada et al. [13] described spectrophotometric methods for determination of penicillamine in pure and dosage forms. Penicillamine was measured spectrophoto-metrically in 0.1 M HC1 (at 195 nm) or in 0.1 M NaOH (at 238 nm). Both methods gave recoveries of 100% with good precision. For determination of the drug in tablets, in the presence of excipients, ground samples were extracted with each of these solvents and the difference in absorbance at 238 nm between the two solutions were measured. The recovery of the drug from commercial tablets was 99.8%, with a coefficient of variation of 0.38%. The three methods were suitable for 4—130 ppm of the drug. 

Walash et al. [14] described a kinetic spectrophotometric method for determination of several sulfur containing compounds including penicillamine. The method is based on the catalytic effect on the reaction between sodium azide and iodine in aqueous solution, and entails measuring the decrease in the absorbance of iodine at 348 nm by a fixed time method. Regression analysis of the Beer s law plot showed a linear graph over the range of 0.01 0.1 pg/mL for penicillamine with a detection limit of 0.0094 pg/mL. 

Amarnth and Amamth [15] described a specific method for the determination of penicillamine and cysteine. Treatment with 1,1-thiocarbonyl diimidazole converts penicillamine to 5,5-dimethyl-2-thioxothiazolidine-4-carboxylic add. The detection limit is 2 pmol of the drug per injection, and the detector response is linear up to 1 nmol. 

Al-Majed [16] described a simple colorimetric method for the determination of (iq-penicillamine in pure form and in pharmaceutical formulations that is based on coupling between (/))-penicillamine and 2,6-dichloroquinone-4-chlorimide (DCQ) in... 

Raggi et al. [21] described a spectrophotometric analysis method with ammonium tetrachloropalladate for penicillamine in pharmaceutical formulations. An aqueous solution of penicillamine (298 pg/mL) was treated with 1.5 mL of 20 mM (NH4)2PdCl4 in 1 M HC1. The mixture was diluted to 10 mL, and the absorbance measured at 403 nm after 20 min. The method has a recovery of 98.8%, and was used to determine penicillamine in aqueous extracts of capsules. 

A specific and sensitive fluorimetric method was proposed by Al-Majed for the determination of (7))-penicillamine in its pure state and in its dosage forms [24], The method is based on the coupling between (/))-penicillamine and 4-fluoro-7-nitroben-zo-2-oxa-1,3-diazole, and analysis of the fluorescent product was measured at an excitation wavelength of 465 nm and an emission wavelength of 530 nm. The fluorescence intensity was found to be a linear function of the drug concentration over the range of 0.6-3 pg/mL, and the detection limit was 2 ng/mL (13 nM). 

Al-Ghannam et al. [25] described a simple fluorimetric procedure for determination of three pharmaceutical compounds containing thiol groups, including penicillamine. In this method, the drugs are treated with 1,2-naphthoquinone-4-sulfonic acid. The later compound is reduced to l,2-dihydroxynaphthalene-4-sulfonic acid, which is measured fluorimetrically (excitation = 318 nm, emission = 480 nm). The method is sensitive to 0.5 1.5 pg/mL, with a detection limit of 0.05 pg/mL (S/N = 2). 

Perez Ruiz et al. [26] determined penicillamine and tiopronin in pharmaceutical preparations by flow injection fluorimetry. The procedure is based on the oxidation of these drugs by thallium(III), whereupon the fluorescence of T1(T) produced in the oxidation of penicillamine is monitored using excitation at 227 nm and emission at 419 nm. A linear calibration graph for penicillamine was obtained between 3 x 10-7 and 8 x 10 5 6 M. 

Martens et al. [28] reported a system for resolving the optical isomers, and determining the enantiomeric purity of ( )-penicillamine by thin-layer chromatography. 

Favaro and Fiorani [34] used an electrode, prepared by doping conductive C cement with 5% cobalt phthalocyanine, in LC systems to detect the pharmaceutical thiols, captopril, thiopronine, and penicillamine. FIA determinations were performed with pH 2 phosphate buffer as the carrier stream (1 mL/min), an injection volume of 20 pL, and an applied potential of 0.6 V versus Ag/AgCl (stainless steel counter electrode). Calibration curves were developed for 5-100 pM of each analyte, and the dynamic linear range was up to approximately 20 pM. The detection limits were 76, 73, and 88 nM for captopril, thiopronine, and penicillamine, respectively. LC determinations were performed using a 5-pm Bio-Sil C18 HL 90-5S column (15 cm x 4.6 mm i.d.) with 1 mM sodium 1-octanesulfonate in 0.01 M phosphate buffer/acetonitrile as the mobile phase (1 mL/min) and gradient elution from 9 1 (held for 5 min) to 7 3 (held for 10 min) in 5 min. The working electrode was maintained at 0.6 V versus Ag/AgCl, and the injection volume was 20 pL. For thiopronine, penicillamine, and captopril, the retention times were 3.1, 5.0, and 11.3 min, and the detection limits were 0.71, 1.0, and 2.5 pM, respectively. 

Gotti et al. [42] reported an analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis. Dispersions of the drug (0.4 mg/mL for the determination of (/q-penicillaminc in water containing 0.03% of the internal standard, S -met hy I - r-cystei ne, were injected at 5 kPa for 10 seconds into the capillary (48.5 cm x 50 pm i.d., 40 cm to detector). Electrophoresis was carried out at 15 °C and 30 kV, with a pH 2.5 buffer of 50 mM potassium phosphate and detection at 200 rnn. Calibration graphs were linear for 0.2-0.6 pg/mL (detection limit = 90 pM). For a more sensitive determination of penicillamine, or for the separation of its enantiomers, a derivative was prepared. Solutions (0.5 mL, final concentration 20 pg/mL) in 10 mM phosphate buffer (pH 8) were mixed with 1 mL of methanolic 0.015% 1,1 -[ethylidenebis-(sulfonyl)]bis-benzene and, after 2 min, with 0.5 mL of pH 2.5 phosphate buffer. An internal standard (0.03% tryptophan, 0.15 mL) was added and aliquots were injected. With the same pH 2.5 buffer and detection at 220 nm, calibration graphs were linear for 9.3-37.2 pg/mL, with a detection limit of 2.5 pM. For the determination of small amounts of (L)-penicillamine impurity, the final analyte concentration was 75 pg/mL, the pH 2.5 buffer contained 5 mM beta-cyclodextrin and 30 mM (+)-camphor-10-sulfonic acid, with a voltage of 20 kV, and detection at 220 nm. Calibration graphs were linear for 0.5-2% of the toxic (L)-enantiomer, with a detection limit of 0.3%. 

Russell and Rabenstein [43] described a speciation and quantitation method for underivatized and derivatized penicillamine, and its disulfide, by capillary electrophoresis. Penicillamine and penicillamine disulfide were determined by capillary electrophoresis on a capillary (24 cm x 25 pm i.d. or 50 cm x 50 pm i.d. for underivatized thiols) with detection at 357 nm (200 nm for underivatized thiols). The run buffer solution was 0.1 M phosphate (pH 2.3). Detection limits were 20-90 pM without derivatization, and 5-50 pM after derivatization. Calibration graphs were linear from 1 pM to 5 mM thiols. 

Zhang et al. [44] determined penicillamine via a FIA system. A 50 pL sample was injected into an aqueous carrier stream of a flow injection manifold and mixed with... 

Garcia et al. [45] determined penicillamine in pharmaceutical preparations by FIA. Powdered tablets were dissolved in water, and the solution was filtered. Portions (70 pL) of the filtrate were injected into a carrier stream of water that merged with a stream of 1 mM PdCl2 in 1 M HC1 for determination of penicillamine. The mixture was passed though a reaction coil (180 cm long) and the absorbance was measured at 400 nm. Flow rates were 1.2 and 2.2 mL/min for the determination of penicillamine, the calibration graphs were linear for 0.01-0.7 mM, and the relative standard deviation (n = 10) for 0.17 mM analyte was 0.8%. The method was sufficiently selective, and there were no significant differences between the labeled contents and the obtained results. 

Vinas et al. [46] also determined penicillamine by chemiluminescence - flow injection analysis. The sample was dissolved in water, and a portion of resulting solution was introduced into an FIA system consisting of 5 mM luminol in 0.1 M KOH-boric acid buffer (pH 10.4), 50 pM Cu(II), and 10 mM H202 eluted at 7.2 mL/min. Chemiluminescent detection was used, the calibration graphs were linear from 0.1 to 10 mM of penicillamine, and the coefficients of variation were from 1.2% and 2.1%i. 

Vinas et al. [47] determined penicillamine routinely by using batch procedures and FIA. A capsule was dissolved in water, diluted to 250 mL, and a suitable portion of the solution treated with 1 mM Co(II) solution (2.5 mL) and 2 M ammonium acetate (2.5 mL). The mixture was diluted to 25 mL and the absorbance of the yellow complex was determined at 360 nm. Calibration graphs were linear for 0.02-0.3 mM of penicillamine. The method was modified for flow injection analysis using peak-height or peak-width methods, but in both cases the flow rates were maintained at 3.3 mL/min. For the peak-height technique, calibration graphs were linear for 0.1-2 mM, and the sampling frequency was 150 samples per hour. For the peak-width method, the response was linear for 50 pM to 0.1 M, and this method was particularly useful for routine determinations. 

FIA determinations of penicillamine was performed by Favaro and Fiorani [48] with phosphate buffer (pH 2) as the carrier stream (eluted at 1 mL/min), an injection volume of 20 pL, and an applied potential of 0.6 V versus Ag/AgCl (stainless steel counter electrode). The calibration curves were presented for 5-100 pM of the analyte, the dynamic linear range was up to approximately 20 pM, and the detection limits was 88 nM for penicillamine. 

Zhang et al. [49] determined penicillamine in urine by the coupled technique of chemiluminescence detection and liquid chromatography. The urine sample was adjusted to pH 2 with 2 M H2S04 and centrifuged at 3000 rpm for 5 min. A 12 mL... 

Wakabayashi et al. [51] determined penicillamine in serum by HPLC. Serum (0.1 mL) was vortex-mixed for 30 s with 50 pL of 0.1% EDTA and 0.2 mL of 10% TCA. The solution was centrifuged at 1500 x g and filtered. A 5 pL portion was analyzed on a Shodex C18 column (15 cm x 4.6 mm i.d.), using a mobile phase of 19 1 methanolic 0.05 M phosphate buffer (pH 2.8) containing 1 mM sodium octylsulfate and 10 pM EDTA. Liver or kidney samples were similarly extracted, and the extracts were cleaned up on a Bond-Elut cartridge prior to HPLC analysis. Detection was effected with an Eicom WE-3G graphite electrode maintained at +0.9 V versus Ag/AgCl. The calibration graph was linear up to 500 ng, and the detection limits were 20 pg. For 1 pg of penicillamine added to serum, liver, or kidney, the respective relative standard deviations (n = 5) were 3.6, 5.1, and 4.4%. 

Rabenstein and Yamashita [52] determined penicillamine and its symmetrical and mixed disulfides by HPLC in biological fluids. Plasma and urine were deproteinized with trichloroacetic acid, and HPLC was performed on a column (25 cm x 4.6 mm) or Biophase ODS (5 pm) with a mobile phase comprising 0.1 M phosphate buffer (pH 3) and 0.34 mM Na octylsulfate at 1 mL/min. Detection was with a dual Hg-Au amalgam electrode versus a Ag-AgCl reference electrode. (z>)-penicillamine and homocysteine were determined at the downstream electrode at +0.15 V, and homocystine, penicillamine-homocysteine, and penicillamine disulfides were first reduced... 

Webb et al. [56] determined free penicillamine in the plasma of rheumatoid arthritis patients. Plasma ultrafiltrate was mixed with trichloroacetic acid and 4-aminobenzoic acid as internal standards, and HPLC mobile phase to determine total reduced penicillamine. Plasma was vortexed with trichloroacetic acid, the precipitated protein was removed after 15 min by centrifuging, and the supernatant solution was filtered and mixed with 4-aminobenzoic acid. In each instance, a 50-pL portion of solution was analyzed on a 25-cm column of Spherisorb-NH2 (5 pm) at 25 °C, with an electrochemical detector having dual porous graphite electrodes set at... 

Marnela et al. [57] used an amino acid analyzer using fluorescence detection to determine penicillamine in urine. Urine is analyzed on a Kontron Chromakon 500 amino acid analyzer containing a column (20 cm x 3.2 mm) of AS70 resin in the Li (I) form. Buffers containing LiOH, citric acid, methanol, HC1, and Brij 35 at pH 2.60, 3.20, and 3.60 are used as mobile phases (0.4 mL/h). The fluorescence reagent is prepared by the method of Benson and Hare. Detection is at 450 nm (excitation at 350 nm). The analyte response is linear from 0.025 to 10 mM, with a limit of detection of 25 pM. 

Mann and Mitchell [58] described a simple colorimetric method for estimation of (-D)-penicillamine in plasma. Blood containing 2-50 pg of penicillamine was mixed with 0.1 M EDTA solution in tromethamine buffer solution. 0.1 mL of this solution was adjusted to pH 7.4 and centrifuged. To a portion of the plasma was added 3 M HCL, the mixture was freeze-dried, and a suspension of the residue in ethanol was centrifuged. The supernatant liquid was mixed with tromethamine buffer solution (pH 8.2) and 10 mM 5,5 -dithiobis-(2-nitrobenzoic acid) in phosphate buffer solution (pH 7.0), the mixture was shaken with ethyl ether, and the absorbance of the separated aqueous layer was measured at 412 nm. The mean recovery was 60% (four determinations), and the calibration graph was linear for the cited range. 

Usof et al. [59] utilized the derivatizing agent NPM and reversed-phase HPLC as a method for analysis of (/))-pcnicillamine. The relative standard deviation for within-precision and between-precision of 500 nM standard (z>)-penicillamine were 2.27% and 2.23%, respectively. Female Sprague-Dawley rats were given 1 g/kg (/+penicillamine I.P., and the amounts of (iff-penicillamine in the samples were subsequently determined. The assay is rapid, sensitive, and reproducible for determining it in biological samples. 

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