Utility of a green fluorone-based turn-off fluorescence probe for submicromolar determination and stability testing of two macrolides. Insights into reaction thermodynamics, quenching mechanism, and identification of the oxidative degradation products by ESI+-MS

A smart, sensitive, and green turn-off fluorescence probe was developed and validated for the determination of two macrolides; spiramycin (SPM) and josamycin (JSM) at submicromolar concentration levels. The probe is based on the fluorone-based dye, eosin Y that efficiently interacts with the two compounds through ground-state ion-pair complex formation with quenching of its native fluorescence (λex/λem of 524/548 nm). The Stern-Volmer relationship confirmed the static fluorescence quenching mechanism. Furthermore, the thermodynamic parameters of the reaction were explored via the van\u27t Hoff plot. The values of the fluorescence quenching were linearly correlated to the drug concentration over the ranges of 0.12-5.93 and 0.30-9.66 μM (0.1-5.0 and 0.25-8.0 μg/mL) for SPM and JSM,respectively. Therefore, the probe was utilized for quality control of the two compounds in their tablets with mean %recoveries of 98.12±1.72 and 97.22±1.51% for SPM and JSM, respectively. Statistical analysis of the results by t- and F-tests showed excellent agreement with the results of the comparison methods. Moreover, the developed probe was applied for stability testing of the two compounds under oxidative condition along with ESI+-MS identification of the potential degradants. Besides, the greenness of the developed probe was ensured by different assessment metrics. Hence, the developed method is the first stability-indicating fluorimetric assay for the two compounds, and its chief merits include effortlessness, rapidness,sensitivity, cheapness and harmony with the green chemistry rules

The influence of pH and temperature on the stability of flutamide. An HPLC investigation and identification of the degradation product by EI+-MS

The chemical stability of flutamide (FLT) was investigated using a new validated stability-indicating HPLC method. Separation of FLT from its degradation product was achieved on a C18 column using a mobile phase of methanol-phosphate buffer (0.04 M, pH 4.0) (75:25, v/v) with UV-detection at 240 nm. The method exhibited excellent linearity for FLT over the concentration range of 0.2-25.0 μg mL-1. FLT was found to be labile to degradation in buffered, acidic and alkaline solutions. The degradation kinetics of FLT in aqueous solutions was evaluated as a function of pH and temperature. Degradation of FLT followed first-order kinetics and Arrhenius behavior over the temperature ranges of 70-100 and 60-90 °C under acidic and alkaline conditions, respectively. The pH-rate profile was studied over the pH range of 2.0-12.0 with a maximum stability at pH 3.0-5.0. The activation energies for hydrolysis of FLT were calculated as 79.4 and 52.0 kJ mol-1 at pH 0.5 (0.3 M HCl) and 12.5 (0.03 M NaOH), respectively. 4-Nitro-3-trifluoromethyl aniline was identified by mass spectrometry to be the degradation product resulting from the hydrolysis of FLT. The proposed HPLC method was validated according to ICH guidelines and applied for the quality control of FLT in commercial tablets with a mean percentage recovery of 100.09 ± 0.20%

Facile conversion of the quinone-semicarbazone chromophore of Naftazone into a fluorescent quinol-semicarbazide: kinetic study and analysis of naftazone in pharmaceuticals and human serum

Naftazone is a quinone-semi carbazone drug that possesses a strong orange color, and hence it was usually analyzed colorimetrically or by HPLC-UV. However, these methods are not sensitive enough to determine naftazone in biological samples. Naftazone lacks intrinsic fluorescence and does not possess easily derivatizable functional groups. In this contribution, we introduced the first spectrofluorimetric method for naftazone assay through reduction-elicited fluorogenic derivatization through the reduction of its quinone-semicarbazone moiety to the corresponding quinol-semicarbazide derivative by potassium borohydride as a reduction probe. The solvent-dependent fluorescence of the reaction product was studied in various protic and aprotic solvents. Eventually, the fluorescence of the reduced naftazone was measured in 2-propanol at λemission of 350 nm after excitation at λecxitation of 295 nm. The relative fluorescence intensity was linearly correlated to the drug concentration (r = 0.9995) from 10.0 to 500 ng/mL with high sensitivity, where the lower detection limit was 2.9 ng/mL. Hence, the method was effectively applied for naftazone tablets quality control with a mean %recovery of 100.3 ± 1.5, and the results agreed with those of the comparison HPLC-UV method. Furthermore, a new salting-out assisted liquid-liquid extraction (SALLE) method was established for naftazone extraction from human serum, followed by its determination using the developed reduction-based fluorogenic method. The developed SALLE method showed excellent recovery for naftazone from human serum (92.3−106.5%) with good precision (RSD ≤ 6.8%). Additionally, the reaction of naftazone with potassium borohydride was kinetically monitored, and it was found to follow pseudo-first-order kinetics with an activation energy of 43.8 kcal/mol. The developed method’s greenness was approved using three green analytical chemistry metrics

Validated spectrophotometric methods for determination of Alendronate sodium in tablets through nucleophilic aromatic substitution reactions

<p>Abstract</p> <p>Background</p> <p>Alendronate (ALD) is a member of the bisphosphonate family which is used for the treatment of osteoporosis, bone metastasis, Paget's disease, hypocalcaemia associated with malignancy and other conditions that feature bone fragility. ALD is a non-chromophoric compound so its determination by conventional spectrophotometric methods is not possible. So two derivatization reactions were proposed for determination of ALD through the reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and 2,4-dinitrofluorobenzene (DNFB) as chromogenic derivatizing reagents.</p> <p>Results</p> <p>Three simple and sensitive spectrophotometric methods are described for the determination of ALD. Method I is based on the reaction of ALD with NBD-Cl. Method II involved heat-catalyzed derivatization of ALD with DNFB, while, Method III is based on micellar-catalyzed reaction of the studied drug with DNFB at room temperature. The reactions products were measured at 472, 378 and 374 nm, for methods I, II and III, respectively. Beer's law was obeyed over the concentration ranges of 1.0-20.0, 4.0-40.0 and 1.5-30.0 μg/mL with lower limits of detection of 0.09, 1.06 and 0.06 μg/mL for Methods I, II and III, respectively. The proposed methods were applied for quantitation of the studied drug in its pure form with mean percentage recoveries of 100.47 ± 1.12, 100.17 ± 1.21 and 99.23 ± 1.26 for Methods I, II and III, respectively. Moreover the proposed methods were successfully applied for determination of ALD in different tablets. Proposals of the reactions pathways have been postulated.</p> <p>Conclusion</p> <p>The proposed spectrophotometric methods provided sensitive, specific and inexpensive analytical procedures for determination of the non-chromophoric drug alendronate either per se or in its tablet dosage forms without interference from common excipients.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-25-i3.gif"/></display-formula></p

Stability Study of the Antihistamine Drug Azelastine HCl along with a Kinetic Investigation and the Identification of New Degradation Products

The first stability-indicating HPLC method was developed and validated for azelastine HCl (AZL). The separation of AZL from its degradation products was achieved on a C18 column using acetonitrile-0.04 M phosphate buffer of pH 3.5 (32:68, v/v) as a mobile phase with UV-detection at 210 nm and naftazone as an internal standard. The method was rectilinear over the range of 0.2 - 20.0 μg mL-1 with a detection limit of 7.05 ng mL-1. The degradation behavior of AZL was studied under different ICH-recommended stress conditions along with a kinetic investigation; also, degradation products were identified by mass spectrometry. The method was applied for the quality control and stability assessment of AZL in eye drops and nasal spray. The obtained results were favorably compared with those obtained by a comparison method

Simultaneous HPLC Determination of Chlordiazepoxide and Mebeverine HCl in the Presence of Their Degradation Products and Impurities

A simple, rapid, and sensitive RP-HPLC method was developed and validated for the simultaneous determination of chlordiazepoxide (CDO) and mebeverine HCl (MBV) in the presence of CDO impurity (2-amino-5-chlorobenzophenone, ACB) and MBV degradation product (veratric acid, VER). Separation was achieved within 9 min on a BDS Hypersil phenyl column (4.5 mm × 250 mm, 5 µm particle size) using a mobile phase consisting of acetonitrile: 0.1 M potassium dihydrogen phosphate: triethylamine (35 : 65 : 0.2, v/v/v) in an isocratic mode at a flow rate of 1 mL/min. The pH of the mobile phase was adjusted to 4.5 with orthophosphoric acid and UV detection was set at 260 nm. A complete validation procedure was conducted. The proposed method exhibited excellent linearity over the concentration ranges of 1.0–100.0, 10.0–200.0, 2.0–40.0, and 2.0–40.0 µg/mL for CDO, MBV, VER, and ACB, respectively. The proposed method was applied for the simultaneous determination of CDO and MBV in their coformulated tablets with mean percentage recoveries of 99.75 ± 0.62 and 98.61 ± 0.38, respectively. The results of the proposed method were favorably compared with those of a comparison HPLC method using Student t-test and the variance ratio F-test. The chemical structure of MBV degradation product was ascertained by mass spectrometry and IR studies