Simple, rapid reagent-injection spectrophotometric determination of fluorides in pharmaceutical formulations

A new simple and rapid reagent-injection method is reported for the determination of fluorides in pharmaceutical formulations. The method is based on the inhibitory effect of fluoride ions upon the Fe(III) catalytic oxidation of 2,4-diaminophenol (DAP) by H₂O₂. Free fluoride ions form stable complexes with Fe(III), thus reducing its catalytic effect. The decrease in the absorbance of the oxidation product is monitored spectrophotometrically at 500 nm. The various chemical and physical variables of the FI system were optimized and a study of interfering ions was also carried out. A linear calibration graph was obtained from 0 to 750 mg l⁻¹ for F⁻ ions. The precision was very good (s_r=0.3%) and the 3σ detection limit was satisfactory (c_L=0.471 mg l⁻¹). The sampling rate was 90 injections h⁻¹. The method has been successfully applied to the determination of F⁻ ions in pharmaceutical formulations.     

Room temperature ionic liquids and their mixtures: Potential pharmaceutical solvents

Room temperature ionic liquids (RTILs) are organic salts which are liquids at ambient temperature. Composed of relatively large asymmetric organic cations and inorganic or organic anions, they have generated interest as ‘green’ solvents. Here we report on the solvency of alkyl imidazolium salts (PF₆⁻Br⁻Cl⁻) for poorly water-soluble model drugs, albendazole and danazol, indicating their potential application as pharmaceutical solvents/cosolvents. The solubility of albendazole, for example, is increased by more than 10,000 times by 1-butyl-3-methylimidazolium hexaflourophosphate ([bmim]PF₆⁻). Ionic liquids can be water-miscible or water-immiscible. The aqueous miscibility of a poorly water-miscible RTIL such as of [bmim]PF₆⁻ can be improved by the inclusion of a second more miscible RTIL (e.g. 1-hexyl-3-methylimidazolium bromide ([hmim]Br⁻)). The extent of improvement in water miscibility was found to correlate with the hydrophilicity of the second RTIL. This ability to modulate RTILs’ aqueous miscibility increases their usefulness as pharmaceutical solvents.     

Flow-injection amperometric determination of dopamine in pharmaceuticals using a polyphenol oxidase biosensor obtained from soursop pulp

The amperometric determination of dopamine (Do) in pharmaceuticals formulations by flow injection analysis (FIA) is proposed. An enzymatically modified carbon paste electrode constituted by 25% (w/w) of polyphenol oxidase obtained from Annona muricata L. tissue, 30% (w/w) of graphite, 30% (w/w) of silicone and 15% (w/w) of 7,7,8,8 tetracyanoquinodimethane (TCNQ), was used as flow-through detector. The flow amperometric detection was carried out at a potential of 0.10 V (vs. Ag/AgCl) when an injected sample volume of 250 μl was inserted on a 0.3 M phosphate buffer carrier solution (pH 7.8) flowing at 2.5 ml/min. The developed biosensor showed good stability and reproducibility, enabling up to 500 determinations in 60 days, without considerable loss of enzymatic activity. The FIA system presented a linear response to Do concentrations in the interval from 2×10⁻² to 2×10⁻⁴ M, with relative standard deviations lower than 1.5%. The kinetic parameter K_M for the soluble and immobilized enzyme was 1.45×10⁻² and 1.91×10⁻² M, respectively. In the analyses of different commercially pharmaceutical formulations a relative deviation lower than about 3.4% was obtained.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

Flow-injection biamperometric determination of epinephrine

A flow-injection manifold is proposed for the determination of epinephrine. The experimental procedure is based on the indirect biamperometric detection of the drug by using Fe(III)-Fe(II) as an indicating redox system and a flow-through detector with two polarized Pt wire electrodes. The calibration graph is linear over the range 0.3–20 μg ml⁻¹ of epinephrine. The relative standard deviation for the determination of 10 μg ml⁻¹ of epinephrine is 1.5% (n = 25) and the sample throughput is 153 h⁻¹. The method was applied to the determination of epinephrine in two commercially available pharmaceutical preparations.     

Chemiluminometric determination of propranolol in an automated multicommutated flow system

In this work, a fully automated analytical methodology for the chemiluminometric determination of propranolol hydrochloride in pharmaceutical preparations is proposed. The developed procedure was based on the oxidation of propranolol by potassium permanganate in acidic medium and was implemented in a multicommutated flow system. The combined automated actuation of syringe pumps and two-way solenoid valves, under computer control, assured a high versatility in terms of sample and reagent manipulation and an effective run-time control of all analytical parameters, including a time-based insertion that enabled a significant solutions saving and the exploitation of different strategies for sample/reagent mixing.

The calibration graph was linear over the range 20–150 mg l⁻¹ of propranolol hydrochloride with a relative standard deviation lower than 1.6% (n = 7). The results were in agreement with those obtained by the reference procedure with a relative deviation between −3.8 and 3.7% and a sampling rate of about 27 samples h⁻¹.     

Short-term regulation of the Cl-/HCO3(-) exchanger in immortalized SHR proximal tubular epithelial cells

The present study evaluated the activity of Cl⁻/HCO₃⁻ exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar–Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO₃⁻ was identical in WKY and SHR PTE cells, but V_max values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P < 0.05) increased the Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl⁻/HCO₃⁻ exchanger. The Cl⁻/HCO₃⁻ exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.     

Amperometric biosensor based on monoamine oxidase (MAO) immobilized in sol-gel film for benzydamine determination in pharmaceuticals

The development and application of a flow-through amperometric biosensor for benzydamine determination in anti-inflammatory drugs is described. The biosensor was obtained by physical entrapment of monoamine oxidase in a sol–gel film applied on platinum or carbon paste conducting support. The sol–gel membranes were prepared using an optimum concentration of 3-aminopropyltriethoxy silane, 2-(3,4-epoxycyclohexyl)ethyl-trimethoxy silane, double distilled water saturated with polyethylene glycol 6000 and HCl. The developed biosensors were incorporated in a single channel flow injection system to enable the determination of benzydamine in the concentration range of 0.05–2.5 mmol l⁻¹ (with platinum based electrode) or within 0.1–2.5 mmol l⁻¹ (carbon paste based electrode). The operational stability of the bioanalytical system developed was about 3 months permitting approximately 4700 substrate measurements. The flow injection system developed enables a sampling rate of 20–25 samples h⁻¹ and relative S.D. of results less than 4%. The analytical usefulness of the proposed procedure was evaluated through analysis of commercial pharmaceutical products containing benzydamine, available on the Portuguese market. The results obtained did not differ significantly from the values resulting from analysis of the same products by the method described in the BP Pharmacopoeia.     

Flow injection and HPLC determination of furosemide using pulsed amperometric detection at microelectrodes

The flow-injection and HPLC determination of the diuretic drug furosemide using pulsed amperometric detection (PAD) at cylindrical carbon fibre microelectrodes (CFMEs) is reported. Experimental conditions such as pH (6.5) and buffer concentration (0.05 mol l⁻¹ HPO₄²⁻/H₂PO₄⁻) were optimized using square-wave voltammetry (SWV). Repetitive flow-injection amperometric measurements at +1.25 V for furosemide showed a continuous decrease in the peak current, probably as a consequence of the microelectrode surface fouling. However, a suitable amperometric detection of furosemide was achieved using a PAD program consisting of a two-step potential waveform with alternating anodic and cathodic polarization. The anodic (detection) potential was +1.25 V (time of application 0.1 s), and the cathodic (cleaning) potential was −0.20 V (t=0.2 s). A linear calibration graph was obtained for furosemide in the 5.0×10⁻⁷–1.0×10⁻⁴ mol l⁻¹ concentration range, with a limit of detection of 1.7×10⁻⁷ mol l⁻¹. HPLC-PAD at carbon fibre microelectrodes was used for the determination of furosemide in the presence of several thiouracil drugs and oxytetracycline (OTC). The mobile phase selected was a 25:75 acetonitrile:5.0×10⁻³ mol l⁻¹ NaH₂PO₄ (pH 5.0) mixture. A linear calibration graph was obtained for furosemide in the 1–100 μM range, with a limit of detection of 0.55 μM. The usefulness of this method for the determination of furosemide in real samples was evaluated by performing the analysis of commercial milk samples spiked with furosemide at a concentration level of 4.5×10⁻⁷ mol l⁻¹ (150 ng ml⁻¹), as well as with other thiouracil drugs and OTC. A mean recovery of 95±5% furosemide was obtained.     

Carbon film resistor electrode for amperometric determination of acetaminophen in pharmaceutical formulations

Flow injection analysis (FIA) with amperometric detection was employed for acetaminophen quantification in pharmaceutical formulations using a carbon film resistor electrode. This sensor exhibited sharp and reproducible current peaks for acetaminophen without chemical modification of its surface. A wide linear working range (8.0 × 10⁻⁷ to 5.0 × 10⁻⁴ mol L⁻¹) in phosphate buffer solution as well as high sensitivity (0.143 A mol⁻¹ L cm⁻²) and low submicromolar detection limit (1.36 × 10⁻⁷ mol L⁻¹) were achieved. The repeatability (R.S.D. for 10 successive injections of 5.0 × 10⁻⁶ and 5.0 × 10⁻⁵ mol L⁻¹ acetaminophen solutions) was 3.1 and 1.3%, respectively, without any memory effect between injections. The new procedure was applied to the analyses of commercial pharmaceutical products and the results were in good agreement with those obtained utilizing a spectrophotometric method. Consequently, this amperometric method has been shown to be very suitable for quality control analyses and other applications with similar requirements.     

Fluorimetric determination of some antibiotics in raw material and dosage forms through ternary complex formation with terbium (Tb(3+))

A highly sensitive and specific fluorimetric method was developed for the determination of cefazolin sodium I, cefoperazone sodium II, ceftriaxone sodium III, and cefixime IV. The proposed method involves the formation of ternary complex with Tb³⁺ in the presence of Tris buffer. The quenching of the terbium fluorescence due to the complex formation was quantitative for the four studied drugs. The effect of pH, concentration of Tris buffer and terbium were studied. The formation of the complex was highly dependent on the pH. The optimum pH was found to be pH 8 for cefazolin sodium I, ceftriaxone sodium III, cefixime IV and pH 10 for cefoperazone sodium II. The optimum concentration for Tb³⁺ was found 1 ml of 10⁻⁴ M solution and for Tris buffer 1 ml of the prepared solution. Under the described conditions, the proposed method was applicable over the concentration range 8.79×10⁻⁶–7.91×10⁻⁵, 9.7×10⁻⁶–4.49×10⁻5, 6.10×10⁻⁶–2.50×10⁻⁵, and 4.92×10⁻⁶–2.95×10⁻⁵ mol with mean percentage accuracy of 99.79±0.24, 98.97±1.25, 100.05±0.79, and 100.15±0.54 for I, II, III, and IV, respectively. The proposed method was applied successfully for the determination of studied drugs in bulk powder and in pharmaceutical formulations. The results obtained by applying the described method were statistically analyzed and compared with those obtained by applying the official method. The proposed method was used as stability indicating method for the determination of the studied drugs in the presence of their degradation products.     

Fluorimetric assay of rufloxacin in serum and in pharmaceutical formulations

A simple and rapid fluorimetric method for the determination of 9-fluoro-10-[N-(4′-methyl)piperazinyl]-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-d,e][1–4]benzothyazin-6-carboxylic acid hydrochloride (MF 934), in serum and in pharmaceutical formulations, has been developed based on its strong fluorescence, in 0.1 N H₂SO₄, at 526 nm (excitation wavelength at 340 nm). The procedure which involves the direct dilution of the sample requires only a few minutes and the sample volume is only 20–100 μl of serum, depending on the drug concentration. Tedious sample preparation procedures such as extraction, deproteinization, or centrifugation are not necessary. The minimum concentration that can be detected is 0.3 ng ml⁻¹, the standard curve in 0.1 N H₂SO₄ was found to be linear from 0.005 to 1.5 μg ml⁻¹ and from 0.01 to 0.07 g in plasma after dilution with 0.1 N H₂SO₄.     

GABAergic synapses supramolecular organization and biochemical regulation

Extraneurally released gamma-aminobutyric acid (GABA) interacts with specific recognition sites associated with proteins located in postsynaptic neuronal membranes that function as chloride (Cl⁻)ionophores. As a result of the interaction between GABA and the recognition sites, C1⁻ ionophores are opened causing an influx or an efflux of Cl⁻, depencling on the values of the Cl⁻ equilibrium potential and of the membrane potential. Hyperpolarization or depolari/ation will result from inward or outward Cl⁻ fluxes, respectively. Independently of the change in conductivity elicited by GABA, this amino acid transmitter will reduce the effectiveness of the sodium ion (Na ⁺) excitatory potential. In attempts to elucidate the molecular mechanism, whereby benzodiazepines facilitate the action of GABA on membrane conductance without changing the activity of Cl⁻ or other ionophore, a basic protein (GABA-modulin, GM) has been isolated from rat brain which is similar in structure to the small molecular weight myelin basic protein, found in rodent brain. While GABA-modulin is located in synaptosomes, the small molecular weight myelin basic protein is located in the myelin fraction: more important, GABA-modulin inhibited the high affinity bincling of GABA to crude synaptic membranes while the basic myelin protein did not. Also, amino acid composition and molecular weight differentiate the two proteins. The GABA-modulin can be phosphorylated with different stoichiometry by cyclic AMP-dependent protein kinase (4 mol PO₄⁻³) or Ca²⁺-dependent protein kinase (1 mol PO₄⁻³). Only cyclic AMP-dependent phosphorylation inhibited the action of GABA-modulin on GABA bincling.     

Cathodic adsorptive stripping voltammetric determination of the anti-inflammatory drug indomethacin

Sensitive voltammetric methods have been developed for the determination of the anti-inflammatory, anti-pyretic and analgesic drug indomethacin sodium. The methods are based on the controlled adsorptive preconcentration of the drug on a hanging mercury drop electrode (HMDE), followed by tracing the voltammogram in a cathodic potential scan. The modes used are cyclic voltammetry (CV), cathodic stripping voltammetry (CSV) and differential pulse stripping voltammetry (DPSV). Amounts as low as 10 nM (10 ng ml⁻¹) (60 s preconcentration) by CSV and 0.5 μM (190 ng ml⁻¹) (300 s) by DPSV can be determined accurately. The R.S.D. at the 1×10⁻⁶ M level is 1.4%. The interference of some metal ions and the application of the method to analysis of urine, plasma and pharmaceutical formulations are described.     

2,6-Dichloroquinone chlorimide and 7,7,8,8-tetracyanoquinodimethane reagents for the spectrophotometric determination of salbutamol in pure and dosage forms

A simple, rapid and sensitive spectrophotometric method for the determination of sulbutamol in pure form and in different pharmaceutical preparations has been developed. The charge transfer (CT) reaction between salbutamol as electron donor and 2,6-dichloroquinone chlorimide (DCQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a π-electron acceptor have been spectrophotometrically studied. The optimum experimental conditions for these CT reactions have been studied carefully. Beer's law is obeyed over the concentration range of 1.0–30.0 μg ml⁻¹ and 2.0–20.0 μg ml⁻¹ for salbutamol using DCQ and TCNQ, respectively. For more accurate results, Ringbom optimum concentration range is calculated and found to be 10.0 to 30.0 and 8.0 to 20 μg ml⁻¹ for salbutamol using DCQ and TCNQ, respectively. The Sandell sensitivity is found to be 0.011 and 0.010 g cm⁻² for salbutamol using DCQ and TCNQ, respectively, which indicate the high sensitivity of the proposed methods. Relative standard deviations (R.S.D.) of 0.27 to 0.68% and 0.20 to 1.40% (n=5) were obtained for five replicates of salbutamol using DCQ and TCNQ, respectively. The results obtained by the two reagents are comparable with those obtained by British pharmacopoeia assay for the determination of salbutamol in raw materials and in pharmaceutical preparations.     

Potentiometric determination of acetylsalicylic acid by sequential injection analysis (SIA) using a tubular salicylate-selective electrode

This paper deals with the development of an automated procedure for formulation assays and dissolution tests based on a sequential injection analysis (SIA) system involving an ion-selective electrode as sensing device. Construction of a tubular salicylate (Sal) selective electrode suitable for potentiometric determination of acetylsalicylic acid (Asa) in pharmaceutical formulations is described. The flow-through electrode is formed by a PVC membrane containing 29.2% (w/w) PVC, 5.8% (w/w) tetraoctylammonium salicylate (ionic sensor), 58.5% o-nitrophenyloctylether (plasticizer) and 6.5% (w/w) p-tert-octylphenol (stabilising additive which increases electrode selectivity). The calibration range is 0.05–10 mM Sal, the limit of detection (LOD) is 0.05 mM Sal, the slope is 56.0 mV per decade at 22°C. The R.S.D. is 0.20% (15 readings) when determining 2.5 mM Sal in standard solution. The electrode is used for sensing Asa after its on-line chemical hydrolysis to Sal in a SIA system. The sampling rate is 6 h⁻¹ but for the dissolution tests the frequency is increased to 20 h⁻¹. The SIA set-up is employed for the assay of Asa in plain tablets, composed tablets and effervescent tablets and for performing dissolution tests of normal and sustained release tablets. Results obtained by this technique compare well with those required by the US Pharmacopoeia XXIV.     

Electrochemical determination of Cephalothin antibiotic by adsorptive stripping voltammetric technique

A sensitive and reliable stripping voltammetric method was developed to determine Cephalothin antibiotic drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode and then a negative sweep was initiated, which yield a well defined cathodic peak at −625 mV versus Ag/AgCl reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, drug concentration, scan rate, convection rate and working electrode area. The monitored adsorptive current was directly proportional to the concentration of Cephalothin and it shows a linear response in the range from 4×10⁻⁷ to 1.2×10⁻⁶ mol l⁻¹ (correlation coefficient=0.9995) and the detection limit (S/N=3) is 3.3×10⁻⁹ mol l⁻¹ at an accumulation time of 3 min. The developed AdSV procedure shows a good reproducibility, the relative standard deviation R.S.D.% (n=10) at a concentration level of 5×10⁻⁷ mol l⁻¹ was 0.94%. Possible interferences by other pharmaceutical drugs and surfactants have been also evaluated. The applicability of this approach was illustrated by the determination of Cephalothin in pharmaceutical preparation and biological fluids such as serum and urine.     

Direct scavenging of nitric oxide and superoxide by green tea

In the present study, we investigated the free radical scavenging effects of green tea extract and green tea tannin mixture and its components using a nitric oxide (NO) and superoxide (O₂⁻) generating system in vitro. Green tea extract showed direct scavenging activity against NO and O₂⁻ and green tea tannin mixture, at the same concentration, showed high scavenging activity. Comparison of the activities of seven pure compounds isolated from green tea tannin mixture showed that (−)-epigallocatechin 3-O-gallate (EGCg), (−)-gallocatechin 3-O-gallate (GCg) and (−)-epicatechin 3-O-gallate (ECg) had higher scavenging activities than (−)-epigallocatechin (EGC), (+)-gallocatechin (GC), (−)-epicatechin (EC) and (+)-catechin (C), showing the importance of the structure of flavan-3-ol linked to gallic acid for this activity. Among the gallate-free tannins, EGC and GC were more effective O₂⁻ scavengers than EC and C, indicating the O-trihydroxy structure in the B ring is an important determinant of such activity. However, this structure did not affect the NO scavenging activity. These findings confirm that green tea tannin has excellent antioxidant properties, which may be involved in the beneficial effect of this compound.     

Determination of bopindolol using the flow injection technique coupled with solid phase extraction

In the proposed procedure, the determination of bopindolol using a flow injection analysis (FIA) technique, with spectrophotometric detection at 635 nm, is described. The method is based on the production of a green, water-soluble complex with ferric ions in acid medium. The automated lab-made FIA system was used for the direct determination of bopindolol in tablets. Bopindolol was adsorbed onto the solid phase in a mini-column, which was integrated directly into the flow system. The positive feature of the use of solid phase extraction (SPE) was the pre-concentration of bopindolol (seven times). The sample throughput was 50 samples per hour. Using the SPE method, bopindolol was determined with a linear range from 125 to 1000 μg ml⁻¹ (Relative standard deviation (R.S.D.)=1.87%), with a detection limit (3σ) of 70 μg ml⁻¹. The method was applied to the determination of bopindolol in Sandonorm® tablets. The results obtained were compared with a conventional HPLC method, both analytical techniques were in good agreement.