Simultaneous Determination and Quantitation of Diosmetin and Hesperetin in Human Plasma by Liquid Chromatographic Mass Spectrometry With an Application to Pharmacokinetic Studies.

Among the secondary metabolites which are widely distributed in plants and foods in plant origin flavonoids is important one. Flavonoids have antioxidant activities as free radical scavenging action. They also have anti-inflammatory, antiulcer and anti-carcinogenic activities. Diosmin and hesperidin, the metabolites of which are diosmetin and hesperitin respectively are considered in the present study. Diosmetin has anticancer, antioxidant and blood lipid lowering activities. It also enhances venous tone and microcirculation and by reducing systemic oxidative stress it protects capillaries. Hesperitin also has antioxidant, anti-inflammatory, blood lipid and cholesterol lowering, anti-carcinogenic activities. In the present study efforts were given to develop and validate a bioanalytical method for simultaneous estimation of diosmetin and hesperitin in human plasma by liquid chromatography electron spray ionization mass spectrometry with an application to the analysis of plasma samples obtained from the comparative pharmacokinetic studies on healthy human volunteers under the framework of bioequivalence study. The developed method for simultaneous determination and quantification of diosmetin and hesperitin in human plasma was validated as per the US-FDA guidelines. The validation parameters found within the specified regulatory limit, hence acceptable. The present method also has a short run time (6.0 min) and easy extraction process. The developed method was found to be simple, specific, highly selective, sensitive and reproducible. This was applied for the analysis of the volunteer plasma samples. On the basis of comparison of the AUC0-t, the relative bioavailability of the test preparation was found 100.94 and 95.09% for diosmetin and hesperitin respectively of that of the reference preparation.

LC-MS/MS determination of 4-hydroxynimesulide, an active metabolite of nimesulide and application to bioequivalence study in Indian subjects.

A simple and highly sensitive bioanalytical method was developed and validated for simultaneous quantification of nimesulide (NSD) and its active metabolite 4-hydroxy-nimesulide (M1) in human plasma by liquid chromatography-tandem mass spectrometer (LC-MS/MS) and applied in a bioequivalence study performed on Indian subjects. The bioanalytical method was carried out by LC-MS/MS with celecoxib (CXB) as an internal standard (IS) using liquid-liquid extraction technique. The chromatographic separation was performed on a reversed-phase Agilent eclipse plus C18 (75 mm × 4.6 mm, particle size 3.5 µm) column with a mobile phase of acetronitrile and water containing 5 mM ammonium formate (9:1, v/v). Method validation and clinical sample were analysed as per USFDA and EMA guidelines and results met the acceptance criteria. The lower limit of quantitation of NSD and M1 was found 10 ng/mL with a large linearity range from 10 to 6000 ng/mL for both NSD and M1 using only 100 µL of plasma and reported no matrix effect. The multiple reaction monitoring transitions of m/z 307.20 → 229.20, m/z 323.00 → 245.00 and m/z 380.20 → 316.20 were used to measure NSD, M1 and CXB (IS), respectively. The assay method was successfully applied for the simultaneous quantification of both NSD and M1 in plasma samples after oral administration of nimesulide 100 mg tablet in healthy human subjects.

LC-MS/MS assay for quantitation of enalapril and enalaprilat in plasma for bioequivalence study in Indian subjects.

BACKGROUND: Enalapril (EPL) is an angiotensin-converting enzyme inhibitor for the treatment of hypertension and chronic heart failure. Enalaprilat (EPLT) is an active metabolite that contributes to the overall activity of EPL. AIM: To quantitate EPL along with its metabolite EPLT using LC-MS/MS, a bioanalytical method was developed and validated with tolbutamide in human plasma using a protein precipitation technique. RESULTS: The sensitive and selective method has an LLOQ of 1 ng/ml with a linearity range of 1-500 ng/ml for both EPL and EPLT using 300 µl of plasma without any matrix effect. CONCLUSION: Linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples after oral administration of 20 mg of EPL maleate in healthy volunteers demonstrate applicability to bioavailability/bioequivalence studies.

Development and Validation of an LC-MS/MS-ESI Method for Comparative Pharmacokinetic Study of Ciprofloxacin in Healthy Male Subjects.

A sensitive, specific and reproducible liquid chromatography coupled to tandem mass spectrometric method was developed and validated for the estimation of ciprofloxacin, an extensively used second-generation quinolone antibiotics, in human plasma. A liquid-liquid extraction of ciprofloxacin and the internal standard, ofloxacin, has been approached from the biological matrix using chloroform. Chromatographic separation was achieved in positive ion modes, isocratically on a 3.5 µm C18 analytical column (75 mm×4.6 mm, i.d.) with 0.2% formic acid solution in water: methanol (10:90, v/v) as mobile phase, at a flow rate of 0.5 mL.min-1. The MS/MS ion transitions were monitored as 332.0→231.3 for ciprofloxacin and 362.2→261.0 for IS. The method showed good linearity in the range of 0.01-5.00 µg.mL-1 (r2 >0.99) with a good precision (3.37-12.60%) and accuracy (87.25-114%). At the same time, ciprofloxacin was found to be stable during stability studies viz. bench-top, auto-sampler, freeze-thaw cycle and long-term. The developed and validated method was successfully applied to measure plasma ciprofloxacin concentrations in a single dose bioequivalence study.

Drug release, preclinical and clinical pharmacokinetics relationships of alginate pellets prepared by melt technology.

INTRODUCTION: Alginate pellets prepared by the aqueous agglomeration technique experience fast drug dissolution due to the porous pre-formed calcium alginate microstructure. OBJECTIVE: This study investigated in vitro drug release, preclinical and clinical pharmacokinetics relationships of intestinal-specific calcium acetate-alginate pellets against calcium-free and calcium carbonate-alginate pellets. METHOD: Alginate pellets were prepared by solvent-free melt pelletization instead of aqueous agglomeration technique using chlorpheniramine maleate as model drug. RESULTS: A fast in situ calcium acetate dissolution in pellets resulted in rapid pellet breakup, soluble Ca(2+) crosslinking of alginate fragments and drug dissolution retardation at pH 1.2, which were not found in other pellet types. The preclinical drug absorption rate was lower with calcium acetate loaded than calcium-free alginate pellets. In human subjects, however, the extent and the rate of drug absorption were higher from calcium acetate-loaded pellets than calcium-free alginate pellets. The fine, dispersible and weakly gastric mucoadhesive calcium alginate pellets underwent fast human gastrointestinal transit. They released the drug at a greater rate than calcium-free pellets in the intestine, thereby promoting drug bioavailability. CONCLUSION: Calcium acetate was required as a disintegrant more than as a crosslinking agent clinically to promote pellet fragmentation, fast gastrointestinal transit and drug release in intestinal medium, and intestinal-specific drug bioavailability.