Second Derivative Spectrophotometry Research Articles

Production of phenylalanine-reduced soymilk for phenylketonuria patients

Phenylketonuria (PKU) is a hereditary disease caused by the deficiency of phenylalanine (Phe) hydroxylase enzyme or its cofactor tetrahydrobiopterin. Treatment involves a Phe-restricted diet, although food options are limited. Soymilk, derived from soybeans, is a functional food with nutritional benefits. In this study, soymilk was produced and hydrolyzed with protease of Aspergillus oryzae and papain, and then activated carbon was used to remove Phe for PKU patients. The second-derivative spectrophotometry method was used to determine Phe content in soymilk. The results showed no significant difference in dry matter, fat, and crude fiber between soymilk and Phe-extracted soymilk with respect to the control group (P < 0.05). Soymilk’s protein content was calculated as 2.74% (w/w) and that of Phe-reduced soymilk as 1.94% (w/w). Similarly, consecutive Phe content was 40.53 mg/L and 5.09 mg/L. After hydrolization, the calculated Phe removal rate was 87.44% (w/w), and the protein content was reduced by 29.19% (w/w). The study demonstrates that Phe-reduced soymilk is suitable for PKU patients, thus reducing the need for imported products and offering new market opportunities.

Smart green spectrophotometric assay of the ternary mixture of drotaverine, caffeine and paracetamol in their pharmaceutical dosage form

Three green and facile spectrophotometric methods were developed for the assay of Petro® components; drotaverine HCl (DRT), caffeine (CAFF), and paracetamol (PAR). The three methods depend on measuring the absorbance of the studied drugs through their ethanolic solution. The first derivative spectrophotometry (FDS) at (Δλ = 10) were good parameters for DRT and CAFF resolution; DRT and CAFF could be well calibrated using FDS at 320 and 285 nm, respectively. PAR could be estimated at 308 nm utilizing the second derivative spectrophotometry (SDS). Method II relies on the double divisor ratio derivative spectroscopy (DDRDS). The first derivative was applied on each drug where they would be assayed at 309, 288, and 255 nm for DRT, CAFF, and PAR, respectively. Method III depends on the mean centering (MCR) technique. DRT, CAFF, and PAR could be determined at 309, 214, and 248 nm, respectively. The concentrations were rectilinear in the ranges of 2–20 µg/mL for DRT, 1.5–15 µg/mL for CAFF, and 2–40 µg/mL for PAR in double devisor and mean centering but PAR from 5 to 40 µg/mL in derivative method. Method validation was performed according to ICH guidelines assured by the agreement with the comparison method. In addition, greenness assessment of the proposed methods was investigated. The application of the proposed method was extended to analyse tablet dosage form and performing invitro dissolution testing.

First- and Second-Derivative Spectrophotometry for Simultaneous Determination of lorazepam and clonazepam in pharmaceutical formulations.

First- and Second-Derivative Spectrophotometry for Simultaneous Determination of lorazepam and clonazepam in pharmaceutical formulations.

Second-derivative spectrophotometry for the analysis of simvastatin in polymeric nanocapsules

Conventional spectrophotometry methods are very susceptible to the presence of interferences in complex mixtures such as nanoparticules, requiring prior treatment or extraction of the analyte, and not always providing an adequate response. Derivative spectrophotometry method is capable to eliminate its interference; it is an alternative method for drugs determination in complex matrices. This work investigated the utility of derivate spectrophotometry in assay of simvastatin in polymeric nanocapsules (SIVNC). Shimadzu® UV-1650 double-beam spectrophotometer with 1.0 cm quartz cells was used in this study. The second-order deriva­tive spectrum was obtained employing Δλ=20,000 nm and scaling factor=9.0. The determinations were made at 239 nm (2D239) by zero-crossing method. 2D239 method was validated employing the parameters: specificity, linearity, robustness, precision and accuracy. Results: The specificity test showed there was no interference of constituents commonly found in SIVNC formulation in 2D239. The standard curve showed a correlation coefficient of 0.994. The robustness was evaluated by small changes in the conditions of sample analysis and however, no significant changes were observed regarding drug quantitation. The precision was demonstrated by relative standard deviation (RSD) of intra-day (RSD=1.61-3.76) and inter-day studies (RSD=2.32). The recovery test resulted in an average of 100.66%, which confirmed the accuracy of the method. The procedure was simple and rapid; therefore this technique offers an alternative for determination of SIVNC without interferences.

Simple and Selective Determination of Zr (IV) with 1,4-dichloro-2, 5-dihydroxyquinone in a Micellar Solution of Cetylpyridenium Chloride by Zero and Second-Derivative Spectrophotometry

Simple and Selective Determination of Zr (IV) with 1,4-dichloro-2, 5-dihydroxyquinone in a Micellar Solution of Cetylpyridenium Chloride by Zero and Second-Derivative Spectrophotometry

Modeling and optimization of simultaneous removal of ternary dyes onto copper sulfide nanoparticles loaded on activated carbon using second-derivative spectrophotometry

In this work, a response surface methodology (RSM) and artificial neural network (ANN) were used to study the ultrasound-assisted simultaneous removal of ternary toxic dyes onto copper sulfide nanoparticles loaded on activated carbon (CuS–NP–AC), while dyes contentment analysis were undertaken by derivative spectrophotometry. The influence of process variables (adsorbent mass, sonication time, MG, DB and MB concentration dyes on adsorption was investigated by central composite rotatable design (CCRD) of RSM. The experimental data obtained through CCRD was used to train the ANN model. The results reveal satisfactory results of empirical models (p < 0.0001) for fitting to the experimental data. According to constructed the response model the maximum removal efficiency close to 100% for all dyes at following optimum value of operating variables: 10 mg/L of MG, DB and MB, 6 min sonication time: and 0.02 g CuS–NP–AC, at pH 8.0 was achieved. The ANN based on the Levenberg–Marquardt algorithm (LMA) composed of linear transfer function (purelin) at output layer and tangent sigmoid transfer function (tansig) at hidden layer with 5, 8 and 9 neurons for MG, DB and MB, respectively, give best operation conditions for good prediction of behavior adsorption system. The maximum adsorption capacity of CuS–NP–AC according to Langmuir isotherm model was 263.2, 243.9 and 204.1 mg/g for MG, DB and MB, respectively. The pseudo-second-order model extensively with high ability is able to predict behavior of dyes adsorption onto adsorbent.

Partitioning of organophosphorus pesticides into phosphatidylcholine small unilamellar vesicles studied by second-derivative spectrophotometry

In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome–water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol–water system (R2=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome–water system is more effective for modeling the quantitative structure–activity relationship than that for the n-octanol–water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes.

CO-DETERMINATION OF TWO ANTIPARASITICS DRUGS BY DERIVATIVE SPECTROPHOTOMETRY AND ITS PHOTODEGRADATION STUDIES

This work proposes a method for the co-determination of praziquantel and fenbendazole in veterinary pharmaceutical formulations (VPF) by second-derivative spectrophotometry at 226.4 nm (Zero-crossing method) and 324.6 nm (Graphic method), respectively. The excipients commonly used in VPF do not interfere. To obtain the optimal determination conditions, studies of solvent and light effects, were performed. The results of the light effects show that the expose of both drugs to indirect light and darkness does not produce any chemical change, since their zero-order spectra remained unchanged during the experiment. However, the exposition to the direct light only affects the fenbendazole. The zero order spectrum of FBZ in EtOH-HCl, present two broad bands (200 to 240 nm and 260 to 330 nm), these signals disappear and appear others totally different, at 230 nm and other with two peaks (285 and 295 nm). The FT-IR spectrum shows characteristics signals of the FBZ structure as the amide-NH group (3336.3 cm-1), this signal disappear indicating the bond break of the amide group caused by the exposition to sunlight for a time. These spectral changes evidenced a structural transformation as a result of photochemical decomposition in acid medium.

Comparison of second-derivative spectrophotometry and HPLC for determination of amygdalin in wild apricot kernels

Amygdalin is a cyanogenic glucoside found in apricot kernels. To facilitate the exploitation and use of wild apricot resources, it is necessary to be able to rapidly and efficiently determine the amygdalin levels within their kernels. To do so, this paper compares two methods, one based on second-derivative UV spectrophotometry and the other on high- performance reversed-phase liquid chromatography (HPLC). The second-derivative spectrophotometry method effectively eliminates background noise and is straightforward to perform while retaining the accuracy of the HPLC method. It is likely to be a useful tool for the large-scale determination of amygdalin concentrations in wild apricot kernels and press cake.

Spectroscopic Studies on the Interaction of Mefloquine with Phosphatidylcholine- Phosphatidylserine Bilayer Vesicle and Bovine Serum Albumin

To assess the interaction of mefloquine (MQ) with biomembranes and serum albumin, the partitioning of MQ into phosphatidylcholine-phosphatidylserine small unilamellar vesicles (PC-PS SUV) and its binding with bovine serum albumin (BSA) were examined using various spectroscopic methods. The partition coefficients (Kps) of MQ, determined by the second-derivative spectrophotometric method, increased in accordance with the PS content in PC-PS SUV: the Kp values for PC-PS SUV with PS contents of 10 mol% were about 2.5 times that for PC SUV. The zeta potential values of PC-PS SUV fell from -10 mV at a PS content of 0 mol% to -42 mV at PS contents of 10 mol%, and were found to depend to some extent on the Kp values. Meanwhile, the binding constant (logK) and the number of binding sites (n) for the binding of MQ to BSA were determined using a fluorescence quenching method. These values were significantly reduced by the presence of Cl– ions, from 5.55 to 4.66 for logK and from 1.30 to 1.11 for n. To examine the binding site of MQ on BSA, a 19F nuclear magnetic resonance (NMR) spectroscopic study was performed using typical competing ligands bound to BSA. The results revealed that whereas most of the MQ binds nonspecifically to BSA, MQ also partially binds to Site II. Finally, these results showed that the major interactions between MQ and PC-PS SUV or BSA were essentially different; the partitioning of MQ into PC-PS SUV was electrostatic and the binding of MQ to BSA was hydrophobic. Keywords: Binding constant, Bovine serum albumin, Fluorescence quenching, 19F nuclear magnetic resonance, Liposome, Mefloquine, Partition coefficient, Phosphatidylcholine, Phosphatidylserine, Second-derivative spectrophotometry, Zeta potential

Second-derivative spectrophotometric assay of pseudoephedrine, ibuprofen and loratadine in pharmaceuticals.

In this paper the second-derivative spectrophotometric method for the simultaneous determination of pseudoephedrine (CAS 90-82-4) in the combinations with ibuprofen (CAS 15687-27-1) and loratadine (CAS 79794-75-5) is described. Optimal conditions for the quantitative analysis of coated tablets (Test I) and tablets (Test II) were settled. The second-derivative order of the spectra in ethanol with the wavelength modulation was used. For the quantitative assay for all of the investigated substances in the laboratory mixture or in respective pharmaceutical dosage forms the "zero-crossing" technique was applied. The authors propose this second-derivative spectrophotometry for a rapid, simple, precise and accurate determination of the mentioned tests or a corresponding multicomponent mixture.

Simultaneous determination of Cu 2+, Zn 2+, Cd 2+, Hg 2+ and Pb 2+ by using second-derivative spectrophotometry method

A new method of simultaneous determination of Cu 2+, Zn 2+, Cd 2+, Hg 2+ and Pb 2+ is proposed here by using the second-derivative spectrophotometry method. In pH = 10.35 Borax–NaOH buffer, using meso-tetra (3-methoxyl-4-hydroxylphenyl) porphyrin ([T-(3-MO-4-HP)P]) as chromomeric reagent, micelle solution was formed after Tween-80 surfactant was added into the solution containing Cu 2+, Zn 2+, Cd 2+, Hg 2+ and Pb 2+ ions. The original absorption spectrum of the above complexes was obtained after heating in the boiling water for 25 min. The second-derivative absorption peaks of five metal–porphyrin complexes can be separated from the original absorption spectrum by using chemometric tool. In this way, Cu 2+, Zn 2+, Cd 2+, Hg 2+ and Pb 2+ ions can be determined simultaneously. Under the optimal conditions, the linear ranges of the calibration curve were 0–0.60, 0–0.60, 0–0.40, 0–0.80 and 0–0.48 μg mL −1 for Cu 2+, Zn 2+, Cd 2+, Hg 2+ and Pb 2+, respectively. The molar absorptivity of these color systems were 1.38 × 10 5, 1.01 × 10 5, 3.24 × 10 5, 1.07 × 10 5 and 1.29 × 10 5 L mol −1 cm −1. The method developed in this paper has advantages in selectivity, sensitivity, operation and can effectively resolve spectra overlapping problem. This method has been applied to determine the real samples with satisfactory results.

Serum paraquat concentration detected by spectrophotometry in patients with paraquat poisoning.

Paraquat (PQ) is a world-wide used herbicide and also a type of common poison for suicide and accidental poisoning. Numerous studies have proved that the concentration of serum PQ plays an important role in prognosis. Spectrophotometry, including common spectrophotometry and second-derivative spectrophotometry, is commonly used for PQ detection in primary hospitals. So far, lack of systematic research on the reliability of the method and the correlation between clinical features of patients with PQ poisoning and the test results has restricted the clinical use of spectrophotometry. This study aimed to evaluate the reliability and value of spectrophotometry in detecting the concentration of serum PQ. The wavelengths for detecting the concentration of serum PQ by common and second-derivative spectrophotometry were determined. Second-derivative spectrophotometry was applied to detect the concentration of serum PQ. The linear range and precision for detection of PQ concentration by this method were confirmed. The concentration of serum PQ shown by second-derivative spectrophotometry and HPLC were compared in 8 patients with PQ poisoning. Altogether 21 patients with acute poisoning 4 hours after PQ ingestion treated in the period of October 2008 to September 2010 were retrospectively reviewed. The patients were divided into higher and lower than 1.8 μg/mL groups based on their concentrations of serum PQ measured by second-derivative spectrophotometry on admission. The severity of clinical manifestations between the two groups were analyzed with Student's t test or Fisher's exact test. The absorption peak of 257 nm could not be found when common spectrophotometry was used to detect the PQ concentration in serum. The calibration curve in the 0.4-8.0 μg/mL range for PQ concentration shown by second-derivative spectrophotometry obeyed Beer's law with r=0.996. The average recovery rates of PQ were within a range of 95.0% to 99.5%, relative standard deviation (RSD) was within 1.35% to 5.41% (n=6), and the lower detection limit was 0.05 μg/mL. The PQ concentrations in serum of 8 patients with PQ poisoning shown by second-derivative spectrophotometry were consistent with the quantitative determinations by HPLC (r=0.995, P<0.0001). The survival rate was 22.2% in patients whose PQ concentration in serum was more than 1.8 μg/mL, and the incidences of acidosis, oliguria and pneumomediastinum in these patients were 55.6%, 55.6% and 77.8%, respectively. These clinical manifestations were different significantly from those of the patients whose PQ concentration in serum was less than 1.8 μg/mL (P<0.05). For common spectrophotometry, the wavelength at 257 nm was not suitable for detecting serum PQ as no absorbance was shown. Second-derivative spectrophotometry was reliable for detecting serum paraquat concentration. Serum PQ concentration detected by second-derivative spectrophotometry could be used to predict the severity of clinical manifestations of patients with PQ poisoning, and PQ content higher than 1.8 μg/mL 4 hours after ingestion could be an important predictive factor for poor prognosis.

Distribution of Chloroaluminum Phthalocyanine in a Lipid Nano-Emulsion as Studied by Second-Derivative Spectrophotometry

Focusing on the development of a nanometer scale carrier in the drug-delivery-system for cancer therapy, we prepared a lipid nano-emulsion (LNE) from a lipid mixture of soybean oil (SO), phosphatidylcholine (PC) and sodium palmitate as a vehicle for chloroaluminum phthalocyanine (ClAlPC), a photosensitizer used for the photodynamic treatment of cancer. To elucidate its distribution in our LNE formulation, we proposed a modified three-phase (SO core, PC monolayer and water phase) model using the molar partition coefficients (Kps) of ClAlPC in LNE/water and PC small unilamellar vesicle (PC SUV)/water systems. In the presence of LNE or PC SUV, a spectrophotometric absorption maximum due to monomeric ClAlPC was observed, but was absent in buffered solution because of its property of selfaggregation in aqueous media, showing that the partitioned ClAlPC exists as a monomer. The Kp values of ClAlPC were calculated using the intensity of the absorption maximum in the second derivative spectra. Although the Kp values showed ClAlPC concentration dependence due to the association of ClAlPC in the water phase, they were on the order of 104 for the concentrations of 1 – 5 μM ClAlPC, indicating that our LNE having a lipid concentration of 72 mM can encapsulate 95 – 99% of the spiked ClAlPC. Furthermore, most of ClAlPC was found to be located in the PC monolayer surrounding the SO droplets in the LNE. These results indicate that the PC monolayer not only acts as a water-oil interface, but also stores monomeric ClAlPC in our LNE effectively. Keywords: Lipid nano-emulsion, Chloroaluminum phthalocyanine, Second-derivative spectrophotometry, Liposome, Threephase model, Distribution, EPR, lipid emulsion, LE, phosphatidylcholine, LNE, ClAlPC, photodynamic therapy, high-density lipoprotein, DMSO, dynamic light scattering, DLS, submicron particle analyzer, phosphovanadomolybdate method, spectrophotometer, Hitachi U-3310, Savitzky-Golay method, photosensitizer

Simultaneous Determination of Albendazole and Praziquantel by Second Derivative Spectrophotometry and Multivariated Calibration Methods in Veterinary Pharmaceutical Formulation

The simultaneous determination of albendazole (ABZ) and praziquantel (PZQ) was performed by different mathematical approaches: second derivative spectrophotometry (SDS), classical least squares, regression of partial least squares and principal components regression based on spectral data of drugs dissolved in methanol-hydrochloric acid solution. The detection limits for multivariate calibrations were determined by creating a surrogate variable signal. SDS presented the best analytical features. The recoveries of ABZ and PZQ from the synthetic samples were near to 100 +/- 5%. The methods were applied in veterinary pharmaceutical formulation whose mass ratio ABZ:PZQ is 10:1; the results obtained were according to nominal content.

Partitioning of Anti-inflammatory Steroid Drugs into Phosphatidylcholine and Phosphatidylcholine-Cholesterol Small Unilamellar Vesicles as Studied by Second-Derivative Spectrophotometry

The partition coefficients (Kps) of six anti-inflammatory steroid drugs, dexamethasone (DMS), betamethasone (BMS), triamcinolone acetonide (TCLA), fluocinolone acetonide (FCLA), betamethasone 17,21-dipropionate (BMSDP), and clobetasole propionate (CBSP), for phosphatidylcholine (PC), and PC-cholesterol small unilamellar vesicles (SUVs) were determined by a second-derivative spectrophotometric method. The Kp values were obtained with a relative standard deviation of below 10% and the following order was observed: BMS< or =DMS<TCLA<FCLA<<BMSDP<CBSP. BMSDP which has a structure that the two hydroxyl groups of BMS are esterified with propionic acid showed a largely enhanced Kp value of 10.5 times that of BMS. Further, replacement of a propionate group in BMSDP with a chlorine atom resulted in the highest Kp value (CBSP) within the drugs examined, i.e., the Kp value of CBSP was 1.2 times that of BMSDP. The presence of 30 mol% cholesterol in the SUV bilayers reduced these Kp values to approximately 35-50% of those values for the PC SUVs, although the order of the Kp values remained unchanged. The order of the Kp values agreed with that of the reported dermatological therapeutic potency of these drugs, although the order of their log P values for n-octanol/water systems showed a discrepancy. Our results indicate that the potency of steroid drugs in dermatological treatments depends to some extent on the Kp values of the drug, that is, the affinity of steroid drugs for PC bilayers influences their clinical potency, since potency is related to transdermal absorption.

Estimation of Atorvastatin Calcium and Fenofibrate in Tablets by Derivative Spectrophotometry and Liquid Chromatography

Two simple and accurate methods to determine atorvastatin calcium (ATO) and fenofibrate (FEN) in combined dosage forms were developed using second-derivative spectrophotometry and reversed-phase liquid chromatography (LC). ATO and FEN in combined preparations (tablets) were quantitated using the second-derivative responses at 245.64 nm for ATO and 289.56 nm for FEN in spectra of their solution in methanol. The calibration curves were linear [correlation coefficient (r) = 0.9992 for ATO and 0.9995 for FEN] in the concentration range of 3-15 microg/mL for ATO and FEN. In the LC method, analysis was performed on a Hypersil ODS-C18 column (250 mm x 4.6 mm id, 5 microm particle size) in the isocratic mode using the mobile phase methanol-water (90 + 10, v/v), adjusted to pH 5.5 with orthophosphoric acid, at a flow rate of 1 mL/min. Measurement was made at a wavelength of 246.72 nm. Both drugs were well resolved on the stationary phase, and the retention times were 1.95 min for ATO and 5.50 min for FEN. The calibration curves were linear (r = 0.9985 for ATO and 0.9976 for FEN) in the concentration range of 3-15 microg/mL for ATO and FEN. Both methods were validated, and the results were compared statistically. They were found to be accurate, precise, and specific. The methods were successfully applied to the estimation of ATO and FEN in combined tablet formulations.

Simultaneous Determination of Hyoscine Butylbromide and Ketoprofen in Pharmaceutical Preparations by Spectrophotometric and Liquid Chromatographic Methods

A binary mixture of hyoscine butylbromide and ketoprofen was determined by 4 different methods. The first involved determination of hyoscine butylbromide and ketoprofen using the ratio-spectra first-derivative spectrophotometric technique at 211 and 234 nm over the concentration ranges of 2-14 and 5-45 microg/mL with mean accuracies 99.84 +/-0.92 and 99.98+/- 0.64%, respectively. The second method utilized second-derivative spectrophotometry over the concentration ranges of 2-14 and 5-35 microg/mL with mean accuracies 99.32+/- 1.06 and 99.55+/-1.15%, respectively. The third method was based on the resolution of the 2 components by bivariate calibration depending on a simple and rapid mathematical algorithm and quantitative evaluation of the absorbances at 206 and 254 nm over concentration ranges of 2-16 and 5-35 microg/mL; mean accuracies of 100.21+/-1.30 and 100.19 +/-1.07% were obtained for hyoscine butylbromide and ketoprofen, respectively. The fourth method was reversed-phase liquid chromatography using 0.05 M ammonium dihydrogen phosphate-acetonitrile-methanol (20 + 30 + 6, v/v) as the mobile phase with ultraviolet detection at 220 nm over concentration ranges of 1-90 and 5-70 microg/mL; mean accuracies were 99.92+/-1.02 and 99.61+/- 0.98%, respectively. The suggested procedures were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical preparations. The methods retained their accuracy and precision when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the manufacturer's method.

Effects of Inorganic Ions on the Binding of Triflupromazine and Chlorpromazine to Bovine Serum Albumin Studied by Spectrometric Methods

The effects of inorganic salts, NaCl, NaBr, NaI, Na2SO4, KCl, KBr, KI, on the binding constants (Ks) of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine, to bovine serum albumin (BSA) were examined by using second-derivative spectrophotometry. All of the salts examined, with the exception of Na2SO4, decreased the K values significantly, depending on the concentration of the salt, e.g., the decrease in the K values of both drugs were about 40% for 0.1 M NaCl. The results obtained with Na2SO4 indicated that neither Na+ nor SO4(2-) had any affect on the binding of the phenothiazines to BSA. Based on the Na2SO4 results and the finding that the effect of each potassium salt on binding was quite similar to that of the corresponding sodium salt, the effects of these halogen salts can be considered to be derived from their anions, although the phenothiazines are positively charged at pH 7.4. The effectiveness of the anions was determined to occur in the following order: I->>Br->Cl-; these results coincided with the published order of the binding affinity of these anions to albumin. The 19F-NMR spectra of TFZ in the presence of each of these halogen salts revealed a concentration-dependent decrease in the intensity of the signal at 13.8 ppm that had previously been assigned to the TFZ bound to Site II. Consequently, the effects of these anions on the binding of positively charged phenothiazine drugs are thought to be local steric effects caused by the binding of these anions to Site II.

Estimation of Pioglitazone Hydrochloride and Metformin Hydrochloride in Tablets by Derivative Spectrophotometry and Liquid Chromatographic Methods

Two simple and accurate methods of analysis to determine pioglitazone hydrochloride (PIO) and mefformin hydrochloride (MET) in combined dosage forms were developed using second-derivative spectrophotometry and reversed-phase liquid chromatography (LC). PIO and MET in combined preparations (tablets) were quantified using the second-derivative responses at 227.55 nm for PIO and 257.25 nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile (30 + 70). The calibration curves were linear [correlation coefficient (r) = 0.9984 for PIO and 0.9986 for MET] in the concentration range of 8-40 microg/mL for PIO and 4-12 microg/mL for MET. In the LC method, analysis was performed on a Hypersil ODS-C18 column with 5 microm particle size using the mobile phase acetonitrile-water-acetic acid (75 + 25 + 0.3), adjusted to pH 5.5 with liquor ammonia, at a flow rate of 0.5 mL/min. Measurement was made at a wavelength of 230 nm. Both the drugs were well resolved on the stationary phase, and the retention times were 8.5 min for PIO and 16.0 min for MET. The calibration curves were linear (r = 0.9933 for PIO and 0.9958 for MET) in the concentration range of 4-20 microg/mL for PIO and MET. Both methods were validated, and the results were compared statistically. They were found to be accurate, precise, and specific. The methods were successfully applied to the estimation of PIO and MET in combined tablet formulations.