In this paper, we investigated three ligand systems, symmetric and asymmetric pyridyl-containing tridentate ligands (L1NH2 = (bis(2-pyridylmethyl)-amino)-ethylamine, L2H = (bis(2-pyridylmethyl)-amino)-acetic acid, L3NH2 = [(6-amino-hexyl)-pyridyl-2-methyl-amino]-acetic acid) as bifunctional chelating agents for labeling biomolecules. These
ligands reacted with the precursor fac-[188Re(CO)3(H2O)3]+ and yielded the radioactive complexes fac-[188Re(CO)3L] (L = three ligands), which were identified by RP-HPLC. The corresponding stable rhenium tricarbonyl complexes (1–3) were
allowed for macroscopic identification of the radiochemical compounds. 188Re tricarbonyl complexes, with log Po/w values ranging from −1.36 to −0.32, were obtained with yields of ≥90% using ligand concentrations within the 10−6−10−4M range. Challenge studies with cysteine and histidine revealed the high stability properties of these radioactive complexes,
and biodistribution studies in normal mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion,
primarily through the renal-urinary pathway. In summary, these asymmetric and symmetric pyridyl-containing tridentate ligands
are potent bifunctional chelators for the future biomolecules labeling of fac-[188Re(CO)3(H2O)3]+.
Authors:O. N. Pozharitskaya, V. M. Kosman, M. V. Karlina, A. N. Shikov, V. G. Makarov, and G. I. Djachuk
A reliable and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection method was developed and validated for the quantification of hopantenic acid in human plasma. Hopantenic acid, with protocatechuic acid as the internal standard (IS), was extracted from plasma samples using a liquid-liquid extraction with methanol. A chromatographic separation was achieved on a Luna C18 column (4.6 mm × 150 mm, 5-μm particle size) and precolumn of the same sorbent (2.0 mm). An isocratic elution, at a flow rate of 1.0 mL min−1, was used with a mobile phase consisting of acetonitrile, water, and 0.03% trifluoroacetic acid. The UV detector was set to 205 nm. The elution times for hopantenic acid and IS were ∼4.3 and 5.4 min, respectively. The calibration curve of hopantenic acid was linear (r > 0.9994) over the range of 0.5–100 μg mL−1 in human plasma. The limit of detection and limit of quantification for hopantenic acid were 0.034 and 0.103 μg mL−1, respectively. The present method was successfully applied for the estimation of pharmacokinetic parameters of hopantenic acid following single oral administration of tablets containing 250 mg hopantenic acid to healthy volunteers. For hopantenic acid, the data showed a mean maximum plasma concentration (Cmax) of 2.32 μg mL−1, with a time to reach peak plasma concentration (tmax) of 1.56 h.
Authors:M. Elkarbane, M. Amood Al-Kamarany, H. Bouchafra, M. Azougagh, Y. Cherrah, Ph. Hubert, Y. Heyden, and A. Bouklouze
A gradient reversed phase high-performance liquid chromatography (RP-HPLC) method with ultraviolet (UV) detection to analyze hydrochlorothiazide (HCT) and valsartan (VS) simultaneously in a tablet formulation during forced degradation studies was developed. This method was validated using a novel approach, namely, the accuracy profile or total errors approach. The robustness of the method was evaluated using a Plackett-Burman design for eight factors. The algorithm of Dong was applied to determine the significant factor effects. The validation results showed that the method is precise (RSD: 1.14% for HCT and 0.43% for VS) and accurate (mean recovery: 99.90% for HCT and 99.98% for VS). On the other hand, the results of the robustness study showed that the type of column was the important factor which affects a number of responses, namely, the asymmetry factor (AF), retention time (RT), and resolution (RS). However, the assay results were not affected; therefore, the method can be considered robust. Finally, the method was applied to study the stability of HCT and VS under forced conditions. Significant results were obtained with basic hydrolysis, oxidation, and thermal stress, while the accelerated and acidic conditions did not affect the stability of HCT or VS.
Authors:Magdalena Bartnik, Kazimierz Głowniak, and Alicja Gromek
Flavonoid glycosides from the aerial parts (leaves, flowers, and fruits) of
) have been analyzed by thin-layer chromatography (TLC) with densitometry and by reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array detection (DAD). Solid-phase extraction (SPE) was used to isolate the flavonoid fraction from methanolic extracts of different parts of the plant. TLC separation of the compounds was performed on silica gel plates with two different mobile phases, ethyl acetate-methyl ethyl ketone-formic acid-water, 5 + 3 + 1 + 1 (
), and ethyl acetate-formic acid-water, 9 + 1 + 1 (
). HPLC separation was performed on a C
analytical column (250 mm × 4 mm i.d., 5-μm particles) with 45% methanol in water containing 1% (
) acetic acid as mobile phase. Rutoside, isoquercitrin, isorhamnetin-3-glycoside, isorhamnetin-3-rutinoside, and quercetin, as the free aglycone, were detected and identified in the extracts. All the compounds investigated had flavonol-like structures and are chemotaxonomically important in plants of the genus
. This is the first time isoquercitrin and isorhamnetin-3-glucoside have been identified in
Bieb. The fruits of this plant have also been investigated for the presence of flavonoids, for the first time.
Authors:M. Karwowski, M. Masson, M. Lenzi, A. Scheer, and C. Haminiuk
This study assessed the rheological behaviour, physical stability, and the phenolic compounds of Eugenia pyriformis Cambess. (uvaia) and Eugenia uniflora L. (pitanga) fruits. The effect of temperature on the rheological behaviour of the fruit was evaluated (10–60 °C) by means of non-oscillatory rheological analysis in a rheometer. The whole samples (purée) exhibited shear-thinning behaviour (n<1), whereas the centrifuged samples exhibited Newtonian behaviour (n=1). The Arrhenius equation accurately described the effect of temperature on the apparent viscosity of the fruit. The stability study was performed by means of sedimentation and turbidity testing. The stabilisation of purées occurred on the second (Eugenia pyriformis Cambess.) and on the third (Eugenia uniflora L.) day of storage. The results demonstrated that turbidity increased with an increase in homogenization velocity. The phenolic compounds of the fruit were identified and quantified by reverse-phase high-performance liquid chromatography (RP-HPLC). The samples presented significant differences in the concentration of phenolic compounds. Among the phenolic acids identified, gallic acid was the one with higher concentration in both fruit assayed. Comparing the fruits, it was observed that Eugenia pyriformis presented higher content of flavonoids (5-fold) and Eugenia uniflora presented higher content of phenolic acids (8.5-fold), especially gallic acid. Myricetin and quercetin were the main flavonoids determined in Eugenia pyriformis.
Authors:Azza Aziz Moustafa, Hesham Salem, Maha Hegazy, and Omnia A. Mahmoud
Simple, accurate, precise, sensitive, and validated high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)–densitometric methods were developed for the simultaneous determination of carbinoxamine (CAR), pholcodine (PHL), and ephedrine (EPH) in antitussive syrup. In method A, reversed-phase (RP)-HPLC analysis was performed on an Inertsil CN-3 column (250 mm × 4.6 mm, 5 μm), using a mobile phase consisting of acetonitrile–distilled water (pH 3.5) using orthophosphoric acid in the ratio 70:30 (v/v) and flow rate of 1.5 mL min−1. Quantitation was achieved with ultraviolet (UV) detection at 220 nm. In method B, TLC analysis was carried out on an aluminum-backed sheet of silica gel 60 F254 layer using chloroform–propanol–ammonia (6:4:0.1, v/v) as the mobile. Quantification was carried out with UV detection at 245 nm. The validation of the proposed methods was applied according to the International Conference on Harmonization (ICH) guidelines. The suggested methods were successfully applied for the determination of the cited drugs in bulk powder and commercial dosage form.
A stability-indicating gradient reverse-phase liquid chromatographic method was developed for the quantitative determination of process-related impurities and forced degradation products of oxcarbazepine in pharmaceutical formulation. The method was developed by using Inertsil cyano (250 × 4.6 mm) 5 μm column with mobile phase containing a gradient mixture of solvent A (0.01 M sodium dihydrogen phosphate, pH adjusted to 2.7 with orthophosphoric acid and acetonitrile in the ratio of 80:20 v/v) and B (50:40:10 v/v/v mixture of acetonitrile, water, and methanol). The flow rate of mobile phase was 1.0 mL min−1. Column temperature was maintained at 25°C and detection wavelength at 220 nm. Developed reverse-phase high-performance liquid chromatography (RP-HPLC) method can adequately separate and quantitate five impurities of oxcarbazepine, namely imp-A, imp-B, imp-C, imp-D, and imp-E. Oxcarbazepine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Oxcarbazepine was found to degrade significantly in acid, base, and oxidative stress conditions. The degradation products were well resolved from oxcarbazepine and its impurities. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness.
Authors:A. Azeem, Z. Iqbal, F. Ahmad Ahmad, R. Khar Khar, and S. Talegaonkar
An accurate, sensitive, precise, rapid, and isocratic reversed phase HPLC (RP-HPLC) method for analysis of ropinirole in the bulk drug and in pharmaceutical preparations has been developed and validated. The best separation was achieved on a 250 mm × 4.6 mm i.d, 5-μm particle, C18 reversed-phase column with methanol-0.05 m ammonium acetate buffer (pH 7) 80:20 (v/v) as mobile phase, at a flow rate of 1 mL min–1. UV detection was performed at 250 nm. The method was linear over the concentration range 0.2–100 μg mL–1 (r = 0.9998), with limits of detection and quantitation of 0.061 and 0.184 μg mL–1, respectively. The drug was subjected to oxidation, hydrolysis, photolysis, and heat as stress conditions. Degradation products resulting from the stress did not interfere with detection and assay of ropinirole and thus the method can be regarded as stability-indicating. The method can be used for quality-control assay of ropinirole.
Authors:J. Zirojevic, Z. Jovic, A. Djurdjevic, A. Ciric, and P. Djurdjevic
A simple ion chromatographic (IC) method was developed and validated for simultaneous or individual determination of zoledronic, alendronic, pamidronic acids and their related substances in pharmaceutical formulation. The analytes were separated on Waters IC-Pak Anion HR analytical column with a nitric acid (3 mM) without any other additives, as mobile phase at a flow rate of 1.0 mL min−1. Inverse UV detection was used at 240 nm. Important chromatographic factors that influence the chromatography responses were screened by 11/12 Pluckett–Burman design and their interaction were assayed by 23 full factorial design. The RP-HPLC method was optimized with the aid of LC-Simulator® (ACD Labs, Toronto, Ontario, Canada) software. Validated method was successfully used for quantitative analysis of PAMIFOS®, concentrate for infusion (Habitfarm AD, Ivanjica, Serbia), ZOMETA®, powder for infusion (Novartis Pharma, Stein AG, Switzerland) and BONAP® tablets (Hemofarm, Vrsac, Serbia). Total chromatographic analysis time per sample was approximately 6 min, which represents significant improvement over existing methods. Validation studies revealed that the method is specific, rapid, reliable, and reproducible. Calibration plots were linear over the concentration ranges 20–120 μg mL−1 and 0.1–2 μg mL−1 for bisphosponates and their related substances, respectively. The LODs were 8.7, 4.7, 2.5, 0.026 and 0.011 μg mL−1 for alendronate, pamidronate, zoledronate, phosphoric acid and phosphorous acid, respectively.
A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for simultaneous analysis of three triterpene acids (corosolic, oleanolic, and ursolic acids) in extracts from inflorescences, leaves, and fruits of Prunus serotina Ehrh. (American black cherry). Separation of the acids was accomplished on a C18 column (5 μm, 250 mm × 4.6 mm i.d.) and recorded at 210 nm. The greatest resolution was achieved with 90:10 (υ/υ) methanol-1% aqueous orthophosphoric acid as mobile phase at a flow rate of 0.6 mL min−1. The correlation coefficients for all the calibration plots (r > 0.998) showed linearity good over the range tested. The relative standard deviation of the method was less than 3.3% for intra and inter-day assays, and average recovery was between 95.9 and 100.9%. Sensitivity was high; detection limits were between 0.034 and 0.067 μg mL−1. Total amounts of triterpene acids were 0.451–0.928, 0.031, and 0.911–1.455% in the inflorescences, fruits, and leaves of P. serotina, depending on the time of harvesting.