Development and validation of a sensitive liquid chromatographic-tandem mass spectrometric method for the simultaneous analysis of granisetron and 7-hydroxy granisetron in human plasma and urine samples: application in a clinical pharmacokinetic study in pregnant subject.

A liquid chromatography-tandem mass spectrometric method for the quantification of granisetron and its major metabolite, 7-hydroxy granisetron in human plasma and urine samples was developed and validated. Respective stable isotopically labeled granisetron and 7-hydroxy granisetron were used as internal standards (IS). Chromatography was performed using an Xselect HSS T3 analytical column with a mobile phase of 20% acetonitrile in water (containing 0.2 mM ammonium formate and 0.14% formic acid, pH 4) delivered in an isocratic mode. Tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring was used for quantification. The standard curves were linear in the concentration ranges of 0.5-100 ng/mL for granisetron and 0.1-100 ng/mL for 7-hydroxy granisetron in human plasma samples, and 2-2000 ng/mL for granisetron and 2-1000 ng/mL for 7-hydroxy granisetron in human urine samples, respectively. The accuracies were >85% and the precision as determined by the coefficient of variations was <10%. No significant matrix effects were observed for granisetron or 7-hydroxy granisetron in either plasma or urine samples. Granisetron was stable under various storage and experimental conditions. This validated method was successfully applied to a pharmacokinetic study after intravenous administration of 1 mg granisetron to a pregnant subject.

Structural basis of ligand recognition in 5-HT3 receptors.

The 5-HT(3) receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT(3) receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation-π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT(3) receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT(3) receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT(3) receptor.

Toward biophysical probes for the 5-HT3 receptor: structure-activity relationship study of granisetron derivatives.

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices.

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LC-MS-MS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3-8.7%) and accuracy (93-105%) across the calibration range of 0.1 to 50 ng/ml using an 80 microliters plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LC-MS-MS is applicable for the quantification of other pharmaceuticals in pharmacokinetic studies in animals and humans which require high sensitivity.

Simultaneous determination of granisetron and its 7-hydroxy metabolite in human plasma by reversed-phase high-performance liquid chromatography utilizing fluorescence and electrochemical detection.

A highly sensitive and selective high-performance liquid chromatographic method was developed for the determination of granisetron and its active metabolite, 7-hydroxygranisetron (7OH-G) in human plasma. Granisetron is a selective 5-hydroxytryptamine receptor antagonist used in the treatment of cytotoxic drug-induced emesis. The method involves isolation of granisetron, 7OH-G and the internal standards from plasma by solid-phase extraction prior to reversed-phase ion-pair chromatographic separation on an octyl silica column with subsequent quantification of analytes simultaneously either with electrochemical (7OH-G) or fluorescence (granisetron) detectors which are placed in series. The recovery of granisetron and 7OH-G from human plasma was quantitative. Using 1 ml of plasma, the limits of quantification for granisetron and 7OH-G were 0.1 and 0.25 ng/ml, respectively. Linear responses in analyte/internal standard peak-area ratios were observed for analyte concentrations ranging from 0.1 to 50 ng/ml plasma. Precision and accuracy were within 13% across the calibration range for both granisetron and 7OH-G. The method was sufficiently sensitive, accurate and precise to support pharmacokinetic studies for granisetron and 7OH-G, in both normal and patient populations.

Simultaneous determination of granisetron and 7-hydroxygranisetron in human plasma by high-performance liquid chromatography with fluorescence detection.

A highly sensitive high-performance liquid chromatographic method for the determination of granisetron, a novel antiemetic agent for cancer therapy, and its pharmacologically active and major metabolite in human plasma is described. Methylation of the metabolite with trimethylsilyldiazomethane was employed for sample pretreatment. As a result of this treatment, the originally non-fluorescent metabolite could be detected simultaneously with granisetron using fluorescence detection. Both the compounds and the internal standard added were isocratically well resolved from endogenous peaks in plasma on an ODS column. The concentrations of the compounds were proportional to the peak-area ratios over the practical determination ranges of 0.2-100 ng/ml for granisetron and 0.1-50 ng/ml for the metabolite. In tests, reproducibilities for granisetron at 1 ng/ml and the metabolite at 0.5 ng/ml as determined by the relative standard deviation (n = 3) were less than 3.98 and 7.23%, respectively, in a single run.