Development of Novel Microwell-Based Spectrofluorimetry and High-Performance Liquid Chromatography with Fluorescence Detection Methods and High Throughput for Quantitation of Alectinib in Bulk Powder and Urine Samples.

Background and Objectives: This study presents the development and validation of the 96-microwell-based spectrofluorimetric (MW-SFL) and high performance liquid chromatography (HPLC) with fluorescence detection (HPLC-FD) methods for the quantitation of alectinib (ALC) in its bulk powder form and in urine samples. Materials and Methods: The MW-SFL was based on the enhancement of the native fluorescence of ALC by the formation of micelles with the surfactant cremophor RH 40 (Cr RH 40) in aqueous media. The MW-SFL was executed in a 96-microwell plate and the relative fluorescence intensity (RFI) was recorded by utilizing a fluorescence plate reader at 450 nm after excitation at 280 nm. The HPLC-FD involved the chromatographic separation of ALC and ponatinib (PTB), as an internal standard (IS), on a C18 column and a mobile phase composed of methanol:potassium dihydrogen phosphate pH 7 (80:20, v/v) at a flow rate of 2 mL min-1. The eluted ALC and PTB were detected by utilizing a fluorescence detector set at 365 nm for excitation and 450 nm for emission. Results: Validation of the MW-SFL and HPLC-FD analytical methods was carried out in accordance with the recommendations issued by the International Council for Harmonization (ICH) for the process of validating analytical procedures. Both methods were efficaciously applied for ALC quantitation in its bulk form as well as in spiked urine; the mean recovery values were ≥86.90 and 95.45% for the MW-SFL and HPLC-FD methods, respectively. Conclusions: Both methodologies are valuable for routine use in quality control (QC) laboratories for determination of ALC in pure powder form and in human urine samples.

Novel spectrofluorimetric determination of brigatinib in bulk powder and human urine samples via ion-pair complex formation using eosin Y.

The developed spectrofluorimetric method was successfully applied to the analysis of brigatinib (BRG) in its bulk powder form, and human urine sample. It is based on the investigation of the fluorescence spectrum behavior of the BRG-eosin Y complex. The relative fluorescence intensity (RFI) was recorded at 560 nm after excitation at 480 nm. The principle of the proposed method was thoroughly explained. All experimental parameters affecting method development were optimized. Moreover, the obtained results were fully discussed and statistically analyzed. The molar ratio method was applied to study the stoichiometric relationship between BRG and eosin Y complex. The method revealed a ratio of 1:3 for BRG-eosin Y afforded the highest RFI. The developed method was validated over the concentration range of 62.5-4000 ng mL-1. The results were compared positively with the reported method.

Derivative emission spectrofluorimetry for the simultaneous determination of guaifenesin and phenylephrine hydrochloride in pharmaceutical tablets.

Rapid, simple and sensitive derivative emission spectrofluorimetric methods have been developed for the simultaneous analysis of binary mixtures of guaifenesin (GUA) and phenylephrine hydrochloride (PHE). The methods are based upon measurement of the native fluorescence intensity of the two drugs at λex = 275 nm in methanolic solutions, followed by differentiation using first (D1) and second (D2) derivative techniques. The derivative fluorescence intensity-concentration plots were rectilinear over a range of 0.1-2 µg/mL for both GUA and PHE. The limits of detection were 0.027 (D1, GUA), 0.025 (D2, GUA), 0.031 (D1, PHE) and 0.033 (D2, PHE) µg/mL and limits of quantitation were 0.089 (D1, GUA), 0.083 (D2, GUA), 0.095 (D1, PHE) and 0.097 (D2, PHE) µg/mL. The proposed derivative emission spectrofluorimetric methods (D1 and D2) were successfully applied for the determination of the two compounds in binary mixtures and tablets with high precision and accuracy. The proposed methods were fully validated as per ICH guidelines.

Novel stereoselective high-performance liquid chromatographic method for simultaneous determination of guaifenesin and ketorolac enantiomers in human plasma.

A novel method was developed for the simultaneous determination of guaifenesin (GUA) and ketorolac tromethamine (KET) enantiomers in plasma samples. Since GUA probably increases the absorption of coadministered drugs (e.g., KET), it would be extremely important to monitor KET plasma levels for the purpose of dose adjustment with a subsequent decrease in the side effects. Enantiomeric resolution was achieved on a polysaccharide-based chiral stationary phase, amylose-2, as a chiral selector under the normal phase (NP) mode and using ornidazole (ORN) as internal standard. This innovative method has the advantage of the ease and reliability of sample preparation for plasma samples. Sample clean-up was based on simply using methanol for protein precipitation followed by direct extraction of drug residues using ethanol. Both GUA and KET enantiomers were separated using an isocratic mobile phase composed of hexane/isopropanol/trifluoroacetic acid, 85:15:0.05 v/v/v. Peak area ratios were linear over the range 0.05-20 µg/mL for the four enantiomers S (+) GUA, R (-) GUA, R (+) KET, and S (-) KET. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines in terms of system suitability, specificity, accuracy, precision, robustness, and solution stability. Finally, this procedure was innovative to apply the rationale of developing a chiral high-performance liquid chromatography (HPLC) procedure for the simultaneous quantitative analysis of drug isomers in clinical samples.

Analytical study for the charge-transfer complexes of rosuvastatin calcium with π-acceptors.

Studies were carried out to investigate the charge-transfer (CT) reaction of ROS-Ca, as a n-electron donor with various p-acceptors: tetracyanoethylene, p-chloranilic acid, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, 2,3,5,6-tetrabromo-1,4-benzoquinone, 1,3,5-trinitrobenzene, 2,3,5,6-tetrachloro-1,4-benzoquinone, 7,7,8,8-tetracyano-quinodimethane, and 2,4,7-trinitro-9-fluorenone. Different colored CT complexes were obtained. The reaction mechanism and site of interaction were determined by ultraviolet-visible spectrophotometric techniques and computational molecular modeling. The formation of the colored complexes was utilized in the development of simple, rapid and accurate spectrophotometric methods for the determination of ROS-Ca. Under the optimum reaction conditions, linear relationships with good correlation coefficients (0.9984-0.9995) were found between the absorbances and the concentrations of ROS-Ca in the range of 2-200 mg mL⁻¹. The limits of detection ranged from 0.41 to 12.24 mg mL⁻¹. No interference could be observed from the additives commonly present in the tablets or from the drugs that are co-formulated with ROS-Ca in its combined formulations. The methods were successfully applied to the analysis of tablets with good accuracy and precision; the recovery percentages ranged from 99.54-100.46 ± 1.58-1.82%. The results were compared favorably with the reported method. The proposed methods are practical and valuable for routine application in quality control laboratories for determination of ROS-Ca in its bulk form and tablets.

Capillary electrophoretic determination of antimigraine formulations containing caffeine, ergotamine, paracetamol and domperidone or metoclopramide.

A novel, fast, sensitive and specific technique using capillary electrophoresis coupled to a diode array detector has been developed for the separation and simultaneous determination of two antimigraine mixtures in tablet formulation. The two combinations are ergotamine tartrate (ERG), caffeine (CAF) and paracetamol (PAR) with either domperidone (DOM), combination (I) or metoclopramide (MET), combination (II). The proposed method utilized a fused silica capillary (55 cm × 75 µm i.d.) and background electrolyte composed of phosphate buffer (25 mM, pH 9.8). The separation was achieved at 20 KV applied voltage and at 25°C. The described method was linear over the range of 1-80 and 2-100 µg/mL for CAF and MET, respectively, and 1-80 µg/mL for DOM, ERG and PAR. Intra-day and inter-day relative standard deviation (n = 5) was ≤1.10%. The limits of detection of CAF and PAR were 0.20 and 0.10 µg/mL, respectively, and 0.50 µg/mL for MET, DOM and ERG. Other aspects of analytical validation were also evaluated. The proposed method was successfully applied to the analysis of the two combinations in their tablets. Therefore, the proposed method is suitable for the routine control of these ingredients in multicomponent dosage forms.

A validated capillary electrophoresis method for simultaneous determination of ezetimibe and atorvastatin in pharmaceutical formulations.

A simple, precise, and sensitive capillary electrophoresis technique coupled with a diode array detector has been developed for the separation and simultaneous determination of ezetimibe and atorvastatin in pharmaceutical formulations. Separation of both ezetimibe and atorvastatin was achieved utilizing fused silica capillary (58 cm × 75 µm ID) and background electrolyte solution that consisted of phosphate buffer (2.5 mM, pH 6.7): methanol (70:30 v/v). The proposed method was validated by testing its specificity, linearity, precision, accuracy, recovery, and detection limit/quantitation limit values. The method was linear over the range 2.5-50 µg/ml for ezetimibe (r = 0.9992) and 1-100 µg/ml for atorvastatin (r = 0.9999). Within-day and between-day RSD for ezetimibe and atorvastatin were ⩽5.6% and ⩽2.9%, respectively. The detection limit was 0.07 µg/ml for ezetimibe and 0.06 µg/ml for atorvastatin. The validated method was successfully employed for the determination of ezetimibe and atorvastatin in tablets with no interfering peaks from common pharmaceutical excipients. The percentage recoveries of the two drugs from their tablets were 99.80 ± 1.76 and 100.19 ± 1.83, respectively.