Validation of liquid chromatography and liquid chromatography/tandem mass spectrometry methods for the determination of etoricoxib in pharmaceutical formulations

Reversed-phase liquid chromatography (LC) and LC/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of etoricoxib in pharmaceutical dosage forms. The LC method was performed by reversed-phase chromatography on a Synergi fusion C18 column (150 x 4.6 mm id) maintained at ambient temperature. The mobile phase consisted of 0.01 M phosphoric acid, pH 3.0-acetonitrile (62 + 38, v/v) at a flow rate of 1.0 mL/min, and photodiode array detection at 234 nm was used. The chromatographic separation was obtained within 7.0 min, and calibration curves were linear in the concentration range of 0.02-150 microg/mL. The LC/MS/MS method was performed on a Luna C18 column (50 x 3.0 mm id). The mobile phase consisted of acetonitrile-water (95 + 5)-0.1% acetic acid (90 + 10, v/v). Detection was performed by positive electrospray ionization in the multiple reaction monitoring mode, monitoring the transitions 359.3 > 280.0 and 332.0 > 95.0 for etoricoxib and piroxicam (internal standard), respectively. The chromatographic separation was obtained within 2.0 min, and calibration curves were linear in the concentration range of 1-5000 ng/mL. Validation parameters, such as specificity, linearity, precision, accuracy, and robustness, were evaluated, which gave results within the acceptable range for both methods. Moreover, the proposed methods were successfully applied for routine quality control analysis of pharmaceutical products and showed significant correlation (r = 0.9999) of the results.     

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Ezetimibe in Human Plasma and Pharmaceutical Formulations

An analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed and validated for the determination of ezetimibe in human plasma. Ezetimibe and etoricoxib (internal standard) were extracted from the plasma by liquid-liquid extraction and separated on a C₁₈ analytical column (50 × 3.0 mm I.D.) with acetonitrile:water (85:15, v/v) as mobile phase. Detection was carried out by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The chromatographic separation was obtained within 2.0 min and was linear in the concentration range of 0.25–20ng mL⁻¹ for free ezetimibe and of 1–300ng mL⁻¹ for total ezetimibe. The mean extraction recoveries for free and total ezetimibe from plasma were 96.14 and 64.11%, respectively. Method validation investigated parameters such as linearity, precision, accuracy, specificity and stability, giving results within the acceptable range. The proposed method was successfully applied to the quantitation of ezetimibe and its glucuronide in human plasma to support clinical and pharmacokinetic studies. Moreover, the method was used for the quality control analysis of pharmaceutical dosage forms. Revised: 4 January and 3 February 2006     

Determination of rupatadine in pharmaceutical formulations by a validated stability-indicating MEKC method

A stability-indicating MEKC was developed and validated for the analysis of rupatadine in tablet dosage forms, using nimesulide as internal standard. The MEKC method was performed on a fused-silica capillary (50 microm id; effective length, 40 cm). The BGE consisted of 15 mM borate buffer and 25 mM anionic detergent SDS solution at pH 10. The capillary temperature was maintained at 35 degrees C and the applied voltage was 25 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 5 s, with detection by photodiode array detector set at 205 nm. The method was linear in the range of 0.5-150 microg/mL (r2=0.9996). The specificity and stability-indicating capability of the method were proven through degradation studies inclusive by MS, and showing also that there was no interference of the excipients and no increase of the cytotoxicity. The accuracy was 99.98% with bias lower than 1.06%. The LOD and LOQ were 0.1 and 0.5 microg/mL, respectively. The proposed method was successfully applied for the quantitative analysis of rupatadine in pharmaceutical formulations, and the results were compared to a validated RP-LC method, showing non-significant difference (p>0.05).     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min^?1 and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL^?1 (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL^?1, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.     

Validation of the anti-factor Xa assay for the potency assessment of enoxaparin in pharmaceutical formulations

Enoxaparin is a low-molecular weight heparin used clinically for the prevention and treatment of venous and arterial thrombosis. An anti-factor Xa assay was used to evaluate the potency of the final drug preparation. Method validation investigated parameters such as the range, linearity (r2 = 0.9971), precision, accuracy, and robustness; the biological assay incorporated a chromogenic endpoint and detection at 405 nm. The method yielded good results with a quantitation limit of 0.037 IU/mL and a detection limit of 0.011 IU/mL. The results demonstrated the validity of the anti-factor Xa assay for the determination of enoxaparin.     

Imaging cytoplasmic cAMP in mouse brainstem neurons

Background  

cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca²⁺ levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before.     

Stable isotopic studies of earthworm feeding ecology in tropical ecosystems of Puerto Rico

Feeding strategies of earthworms and their influence on soil processes are often inferred from morphological, behavioral and physiological traits. We used (13)C and (15)N natural abundance in earthworms, soils and plants to explore patterns of resource utilization by different species of earthworms in three tropical ecosystems in Puerto Rico. In a high altitude dwarf forest, native earthworms Trigaster longissimus and Estherella sp. showed less (15)N enrichment ((15)N = 3-6 per thousand) than exotic Pontoscolex corethrurus ((15)N =7-9 per thousand) indicating different food sources or stronger isotopic discrimination by the latter. Conversely, in a lower altitude tabonuco forest, Estherella sp. and P. corethrurus overlapped completely in (15)N enrichment ((15)N = 6-9 per thousand), suggesting the potential for interspecific competition for N resources. A tabonuco forest converted to pasture contained only P. corethrurus which were less enriched in (15)N than those in the forest sites, but more highly enriched in (13)C suggesting assimilation of C from the predominant C(4) grass. These results support the utility of stable isotopes to delineate resource partitioning and potential competitive interactions among earthworm species. Copyright 1999 John Wiley & Sons, Ltd.     

Validation of a stability-indicating RP-LC method for the assessment of recombinant human interleukin-11 and its correlation with bioassay

A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11), based on the ICH guidelines. The method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 25°C. The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using a photodiode array (PDA) detection at 214 nm. Separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1-200 μg/mL (r(2) = 0.9995). Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p > 0.05). The method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay.     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min⁻¹ and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL⁻¹ (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL⁻¹, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.