Simultaneous determination of FLZ and its metabolite (M1) in human plasma and urine by UHPLC-MS/MS: Application to a pharmacokinetic study.

FLZ is a novel anti-Parkinson's disease candidate drug. The main active metabolite is FLZ O-dealkylation (M1) in preclinical studies. A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) quantitation method was developed for the simultaneous determination of FLZ and M1 with low limits of quantitation in human plasma (0.1 ng/mL) and urine (0.5 ng/mL). The plasma and urine samples were both purified by full-automatic solid phase extraction (SPE) method with ensured high extraction recovery and little matrix effect for both analytes, and then separated on a BEH C18 column (2.1 × 50 mm, 1.7 µm). Detection and quantification were performed using an electrospray ionization (ESI) source in positive mode by multiple reaction monitoring (MRM). The precursor to product ion transitions were monitored at m/z 450.3+→313.2+ for FLZ, m/z 436.3+→299.1+ for M1, m/z 462.6+→142.0+ for [D12]-FLZ (internal standard of FLZ) and m/z 447.2+→125.2+ for [D11]-M1 (internal standard of M1), respectively. This method showed good linearity, accuracy, precision and stability in the range of 0.1-100 ng/mL in plasma and 0.5-500 ng/mL in urine of two analytes. Finally, the developed method was successfully applied to a pharmacokinetic research in Chinese healthy volunteers after oral administration of FLZ tablets.

Investigation of the spectrofluorimetric behavior of azelastine and nepafenac: Determination in ophthalmic dosage forms.

The first spectrofluorimetric report investigating the fluorimetric behavior of the antihistaminic drug, azelastine (AZEL), and the non-steroidal anti-inflammatory drug, nepafenac (NEP), either in bulk or in their dosage forms, eye drops and ophthalmic suspension. After a full investigation of the factors that may influence their spectrofluorimetric behavior: pH, different organized media and organic solvents, the optimum factors were set in order to enable the analysis of each drug with maximum sensitivity. The AZEL spectrofluorimetric analysis was set at 286/364 (λex/λem) in distilled water while for NEP, the analysis was set at 228/303 (λex/λem) in methanol. The linearity range for AZEL was from 0.1 to 1.5 µg/mL while that of NEP was from 0.2 to 1.5 µg/mL. The linearity yielded good regression parameters with low LOD (0.022 and 0.032 µg/mL for AZEL and NEP, respectively) and LOQ (0.073 and 1.08 µg/mL for AZEL and NEP, respectively) when compared with those obtained from many previous spectroscopic and chromatographic reports in literature. The method was ICH validated and was applied to the analysis of AZEL and NEP with good selectivity regarding the inactive ingredients.

Variability of fluorescence spectra of coelenteramide-containing proteins as a basis for toxicity monitoring.

Nowadays, physicochemical approach to understanding toxic effects remains underdeveloped. A proper development of such mode would be concerned with simplest bioassay systems. Coelenteramide-Containing Fluorescent Proteins (CLM-CFPs) can serve as proper tools for study primary physicochemical processes in organisms under external exposures. CLM-CFPs are products of bioluminescent reactions of marine coelenterates. As opposed to Green Fluorescent Proteins, the CLM-CFPs are not widely applied in biomedical research, and their potential as colored biomarkers is undervalued now. Coelenteramide, fluorophore of CLM-CFPs, is a photochemically active molecule; it acts as a proton donor in its electron-excited states, generating several forms of different fluorescent state energy and, hence, different fluorescence color, from violet to green. Contributions of the forms to the visible fluorescence depend on the coelenteramide microenvironment in proteins. Hence, CLM-CFPs can serve as fluorescence biomarkers with color differentiation to monitor results of destructive biomolecule exposures. The paper reviews experimental and theoretical studies of spectral-luminescent and photochemical properties of CLM-CFPs, as well as their variation under different exposures - chemicals, temperature, and ionizing radiation. Application of CLM-CFPs as toxicity bioassays of a new type is justified.

Development and validation of HPLC analytical method for nepafenac in ophthalmic dosage form (suspension).

The aim of the present study was to develop and validate an analytical method for the estimation of nepafenac as a raw material as well as in dosage form (suspension) by using reverse phase high performance liquid chromatographic (RP-HPLC). The target was to obtain an easy, rapid, reproducible as well as a rugged method. The HPLC system that was used in the proposed study was LC-20AD liquid chromatograph equipped with SPD-20A UV-VIS detector. The separation was performed on C18 column which was attached with loop 20 ß l. Elution was done at ambient temperature with a mobile phase consisting of acetonitrile: Water (40: 60v/v) at a flow rate of 1ml/min and at a wavelength of 254 nm. The proposed method was validated as per the ICH guidelines. The retention time for nepafenac was 7.49 minutes (% CV=0.0076). The percentage coefficient variation (CV) of six consecutive peak areas of injections was 0.34% with tailing factor 1.76. The peak area responses were linear within the concentration range of 0.078-20.0 ßg/ml (R(2)=0.9993). The sensitivity of the method could be evaluated by limits of detection (LOD) (0.0195 ß g/ml) and limits of quantitation (LOQ) (0.039 ß g/ml). Nepafenac drug is s in its diluent that could see by intra-day (% CV =0.45-1.96) and inter-day variation (%CV=0.173-1.898%). The accuracy and recovery results of 80%, 100% and 120% were 97.40% to 102.10% with % CV of 0.3201% to 1.3496%. The robustness and ruggedness of the method are significantly broader and is reproducible. It could be used as a more convenient, efficient, easy and time saving method for the analysis of drug in raw material as well as in dosage form (ophthalmic suspension).

Determination of organic volatile impurities in nepafenac by GC method.

The methods for controlling volatile impurities, including reagent and starting material, in Nepafenac active pharmaceutical ingredient, are reported. The proposed methods were developed using gas chromatography (GC) and gas chromatography with headspace injection (GC-HS) and validated according to the requirements of the ICH (International Conference of Harmonization) validation guidelines Q2R1 and the guideline for residual solvents Q3C.

Simultaneous determination of orysastrobin and its isomers in rice using HPLC-UV and LC-MS/MS.

Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.