Fluorimetric quantification of brimonidine tartrate in eye drops.

A simple and sensitive spectrofluorimetric method has been developed for the estimation of brimonidine tartrate in pure and eye drops. Linearity was obeyed in the range of 0.2-3.0 ΅g/ml in dimethyl formamide as solvent at an emission wavelength (λem) of 530 nm after excitation wavelength (λex) of 389 nm with good correlation coefficient of 0.998. The limit of detection and limit of quantification for this method were 22.0 and 72.0 ng/ml, respectively. The developed method was statistically validated as per International Conference on Harmonisation guidelines. The percentage relative standard deviation values were found to be less than 2 for accuracy and precision studies. The results obtained were in good agreement with the labelled amounts of the marketed formulations. The proposed method was effectively applied to routine quality control analysis of brimonidine tartrate in their eye drops.

Mechanisms involved in the regulation of bovine pulmonary vascular tone by the 5-HT1B receptor.

BACKGROUND AND PURPOSE: 5-HT(1B) receptors may have a role in pulmonary hypertension. Their relationship with the activity of BK(Ca,) a T-type voltage-operated calcium channel (VOCC) and cyclic nucleotide-mediated relaxation was examined. EXPERIMENTAL APPROACH: Ring segments of bovine pulmonary arteries were mounted in organ baths in modified Krebs-Henseleit buffer (37 degrees C) under a tension of 20 mN and gassed with 95% O(2)/5% CO(2). Isometric recordings were made using Chart 5 software. KEY RESULTS: Contractile responses to 5-HT (10 nM-300 microM) were inhibited similarly by the 5-HT(1B) receptor antagonist SB216641 (100 nM) and the T-type VOCC blockers mibefradil (10 microM) and NNC550396 (10 microM) with no additive effect between SB216641 and mibefradil. Inhibition by SB216641 was prevented by the potassium channel blocker, charybdotoxin (100 nM). 5-HT(1B) receptor activation and charybdotoxin produced a mibefradil-sensitive potentiation of responses to U46619. Bradykinin (0.1 nM-30 microM), sodium nitroprusside (0.01 nM-3 microM), zaprinast (1 nM-3 microM), isoprenaline (0.1 nM-10 microM) and rolipram (1 nM-3 microM) produced 50% relaxation of arteries constricted with 5-HT (1-3 microM) or U46619 (30-50 nM) in the presence of 5-HT(1B) receptor activation, but full relaxation of arteries constricted with U46619, the 5-HT(2A) receptor agonist 2,5 dimethoxy-4 iodoamphetamine (1 microM) or 5-HT in the presence of 5-HT(1B) receptor antagonism. Enhanced relaxation of 5-HT-constricted arteries by cGMP-dependent pathways, seen in the presence of the 5-HT(1B) receptor antagonist, was reversed by charybdotoxin whereas cAMP-dependent relaxation was only partly reversed by charybdotoxin. CONCLUSIONS AND IMPLICATIONS: 5-HT(1B) receptors couple to inhibition of BK(Ca), thus increasing tissue sensitivity to contractile agonists by activating a T-type VOCC and impairing cGMP-mediated relaxation. Impaired cAMP-mediated relaxation was only partly mediated by inhibition of BK(Ca).

Mechanisms of U46619- and 5-HT-induced contraction of bovine pulmonary arteries: role of chloride ions.

BACKGROUND AND PURPOSE: Thromboxane A(2) and 5-hydroxytryptamine (5-HT) are implicated in pulmonary hypertension. The involvement of chloride, voltage-operated calcium channels (VOCCs), store-operated calcium channels (SOCCs) and the Rho kinase in the contractile response of bovine pulmonary arteries (BPA) to the thromboxane A(2) mimetic U46619 and 5-HT was investigated. EXPERIMENTAL APPROACH: Endothelium-intact ring segments of BPA were mounted in Krebs/Henseleit buffer (37 degrees C) under a tension of 2g and gassed with 95%O(2)/5%CO(2). KEY RESULTS: Depletion or removal of extracellular chloride, inhibition of chloride and SOCC, Na:K:2Cl, Cl/HCO(3), Rho kinase inhibited contractions to U46619. Combining Rho kinase inhibition and chloride channel blockade (with NPPB) almost abolished the contractions to U46619. In contrast 5-HT-induced contraction was inhibited by verapamil and mibefradil. Depletion of stored calcium with caffeine almost abolished the response to U46619 but not 5-HT. The contraction by the sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibitor CPA was abolished by SOCC and chloride channel blockade (with NPPB) and by chloride depletion. CONCLUSIONS AND IMPLICATIONS: This study suggests that the contractile response of BPA to U46619 involves Rho kinase together with a chloride-sensitive mechanism, which does not involve VOCC but may have a role in calcium release and calcium entry via SOCC. In contrast contraction of the BPA by 5-HT appears to involve verapamil- and mibefradil-sensitive VOCC. This study may indicate that the use of calcium channel blockers in the management of pulmonary hypertension may not always be effective and that Rho kinase and chloride channels may be targets for the development of new therapies.

Validated HPLC method for the determination of tinidazole in human serum and its application in a clinical pharmacokinetic study.

A high performance liquid chromatographic (HPLC) method for the determination of tinidazole in human serum using metronidazole as internal standard (IS) is described. Protein precipitation is used for the preparation of sample. Mobile phase consisting of 0.002 M phosphate buffer, methanol and acetonitrile mixture (85:7.5:7.5/v/v/v) was used at a flow rate of 1 ml/min on a C18 column. The eluate was monitored using an UV/Vis detector set at 320 nm. Ratio of peak area of analyte to IS was used for quantification of serum samples. The absolute recovery was greater than 95% over a concentration range of 0.5 to 30 micrograms/ml and the limit of quantitation was 0.05 microgram/ml. The intra-day relative standard deviation (RSD) measured at 0.5, 5, 15 and 30 micrograms/ml ranged from 0.36 to 6.14%. The inter-day RSD ranged from 1.14 to 4.21%. The method is simple, sensitive and has been successfully used in a pharmacokinetic study conducted in healthy human volunteers.