Pharmacokinetic study of granisetron in human plasma measured by UPLC-MS/MS and its use in healthy Chinese subjects.

In this study, a rapid, simple and sensitive UPLC-MS/MS method was established for the quantification of granisetron in human plasma for the prevention of vomiting after radiotherapy and chemotherapy. The precipitated proteins were extracted and gradient eluted on a ZORBAX Eclipse Plus C18 column (2.1×50mm, 1.8µm) to achieve ideal chromatographic separation. Multiple reaction mode (MRM) was performed using a Turboion Spray API5500 mass spectrometer equipped with Electron Spray Ionization (ESI). For method validation, good linearity was observed for each analyte of interest in the validation concentration range of 0.05 to 20.0ng/mL. The CV% of inter-batch and intra-batch precision were in the range of -3.6% to 4.7% and the precision of both inter-batch and intra-batch was ≤15.0%. In addition, the method had the advantage of a low matrix effect. In human plasma, all analytes remained stable for 2 hours when kept at room temperature; samples were stable within the autosampler (5°C) for 141 h after preparation and after four freeze-thaw cycles at -20°C and -70°C for 48 days. The UPLC-MS method that had been validated was later utilized for the pharmacokinetic investigation of granisetron hydrochloride tablets in orally administered doses to healthy Chinese volunteers, both before and after meals.

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.

Theoretical evaluation of antiemetic effects of 5-HT3 receptor antagonists for prevention of vomiting induced by cisplatin.

5-HT(3) receptor antagonists are widely used as antiemetic agents in clinical setting, of which palonosetron, with a long elimination half life (t(1/2)), has recently become available. It is important to evaluate the concentration of serotonin when investigating the antiemetic effects of 5-HT(3) receptor antagonists, as those effects are not based solely on the t(1/2) value. We theoretically evaluated the antiemetic effects of three 5-HT(3) receptor antagonists (granisetron, azasetron, palonosetron) on cisplatin-induced nausea and vomiting by estimating the time course of the 5-HT(3) receptor occupancy of serotonin. We estimated the 5-HT(3) receptor occupancy of serotonin in the small intestine, based on the time course of plasma concentration of each 5-HT(3) receptor antagonist and the time course of concentration of serotonin near the 5-HT(3) receptor in the small intestine after administration of cisplatin. The antiemetic effect of each 5-HT(3) receptor antagonist was evaluated based on the normal level of 5-HT(3) receptor occupancy of serotonin. Our results suggest that an adequate antiemetic effect will be provided when a dose of 75 mg/m(2) of cisplatin is given to patients along with any single administration of granisetron, azasetron, or palonosetron at a usual dose. On the other hand, the 5-HT(3) receptor occupancy of serotonin was found to be significantly lower than normal for several days after administration of palonosetron, as compared to granisetron and azasetron, indicating that constipation may be induced. Our results show that granisetron, azasetron, and palonosetron each have an adequate antiemetic effect after administration of 75 mg/m(2) of cisplatin.

A high-performance liquid chromatography-tandem mass spectrometry method coupled with protein precipitation for determination of granisetron in human plasma and its application to a comparative pharmacokinetic study.

A rapid, simple and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been developed for the determination of granisetron in human plasma. Plasma samples were pre-purified by protein precipitation procedure. The chromatographic separation was achieved with Synergi Polar-RP (75 × 2 mm, 4 µm) column using a mixture of 5 mm pH4.0 ammonium formate and methanol (300:316, v/v) under isocratic conditions at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The analysis time was about 2.5 min. The method was fully validated over the concentration range 0.1-10 ng/mL. The lower limit of quantification was 0.1 ng/mL. Inter- and intra-batch precision was <6.1% and the accuracy was within 95.6-100.0%. The mean extraction recovery was 96.3%. Selectivity, matrix effect and stability were also validated. The method was applied to the comparative pharmacokinetic study of granisetron in Chinese healthy subjects.

Simultaneous Determination of Co-administrated Deflazacort, Aprepitant and Granisetron in Dosage Forms and Spiked Human Plasma by RP-HPLC/PAD.

A selective reversed phase high performance liquid chromatography/photodiode array detector (RP-HPLC/PAD) method has been developed for simultaneous determination of the three co-administrated deflazacort, aprepitant and granisetron drugs used with chemotherapy. The three cited drugs have been chromatographed on C18 column using a mobile phase consisting of acetonitrile-0.2% v/v triethylamine (80:20 v/v, pH of 6.6 ± 0.05) with isocratic elution and monitored by photodiode array at 220 nm. International conference on harmonization (ICH) guidelines were followed to validate the developed method. Successful application of the developed method was assessed by the simultaneous determination of the studied drugs in pure forms, dosage forms and plasma samples in the ranges of 0.2-20, 0.4-40 and 0.2-20 µg/mL for deflazacort, aprepitant and granisetron, respectively.

Quantification of ondansetron, granisetron and tropisetron in goat plasma using hydrophilic interaction liquid chromatography-solid phase extraction coupled with hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry.

An assay method to quantify ondansetron (OND), granisetron (GRA) and tropisetron (TRO) in goat plasma has been successfully developed and validated. This method procedure for the analysis of OND, GRA and TRO was involved of extracting samples with hydrophilic interaction liquid chromatography (HILIC) solid phase extraction (SPE) and determination by liquid chromatography coupled to tandem mass spectroscopy. An SPE method for the simultaneous extraction of OND, GRA and TRO with high efficiency and selectivity was developed. Prior to HPLC-MS/MS analysis, most of the sources of interference present in the supernatant after protein precipitation of plasma proteins was efficiently removed from the samples by the HILIC SPE treatment. For the quantification of OND, GRA and TRO in the samples, tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring was used. The calibration curve was performed in the range of 0.2-20 ng/mL for the target OND, GRA and TRO in goat plasma samples. The precision of the intra- and inter-day assay for OND, GRA and TRO were 1.84-6.23% and 3.89-5.31%, 2.63-6.29% and 3.76-5.31%, 1.99-5.67% and 2.64-4.70%, respectively. The accuracy of the intra- and inter-day assay for OND, GRA and TRO were 89.15-97.39% and 89.46-95.17%, 91.08-100.82% and 91.24-99.47%, 92.30-100.74% and 94.21-97.90%, respectively. For the determination of OND, GRA and TRO in plasma samples, no significant matrix effects were observed. The mean absolute recoveries were 103-150%, 115-121%, and 98-141% for OND, GRA and TRO, respectively. Furthermore, the mean process efficiency values of silica SPE were 98-135%, 92-124%, and 72-109% for OND, GRA and TRO, respectively.

Analgesic effect of acetaminophen in humans: first evidence of a central serotonergic mechanism.

OBJECTIVES: Preclinical studies have suggested that the mechanism of the analgesic action of acetaminophen (INN, paracetamol) is linked to the serotonergic system and that it is inhibited by tropisetron, a 5-hydroxytryptamine type 3 antagonist. The aim of this study was to confirm these findings in humans. METHODS: Twenty-six rapid metabolizers of tropisetron were included in this double-blind crossover study. After ethical approval, at weekly intervals, the subjects took a single oral dose of 1 g acetaminophen combined with either intravenous tropisetron (5 mg), granisetron (3 mg), or placebo (saline solution). For each session, the analgesic effect of acetaminophen was assessed by use of a pain self-evaluation instrument, the Pain Matcher. The pain detection threshold was determined 5 times over the period of the 4 postdosing hours. The area under the curve (0-4 hours) (mean +/- SD) of acetaminophen/tropisetron and the area under the curve of acetaminophen/granisetron were compared with the effect of acetaminophen/placebo. Blood samples for acetaminophen concentration measurements were taken to evaluate a pharmacokinetic interaction. RESULTS: The analgesic effect of acetaminophen/placebo (expressed as the area under the curve of the percentage of the individual pain score reported at baseline along time [% x min]) (2145 +/- 2901 % x min) was totally inhibited by both tropisetron (89 +/- 1747 % x min, P = .007) and granisetron (45 +/- 2020 % x min, P = .002). Acetaminophen concentration was not significantly different when associated with tropisetron (P = .919) or granisetron (P = .309). CONCLUSION: These results clearly show for the first time in humans that the coadministration of tropisetron or granisetron with acetaminophen completely blocks the analgesic effect of acetaminophen. They support the hypothesis that the mechanism of the analgesic action of acetaminophen might involve the serotonergic system. Furthermore, they demonstrate a pharmacodynamic interaction between these 2 types of drugs, which are frequently coadministered, especially in cancer patients.

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study.

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study.

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.

Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging.

Serotonin-gated ionotropic 5-HT3 receptors are the major pharmacological targets for antiemetic compounds. Furthermore, they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5-HT3 receptors might alleviate symptoms of other neurological disorders. Highly selective, high-affinity antagonists, such as granisetron (Kytril) and palonosetron (Aloxi), belong to a family of drugs (the "setrons") that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo, we report the synthesis of 8-fluoropalonosetron (15) that has a binding affinity (Ki = 0.26 ± 0.05 nM) similar to the parent drug (Ki = 0.21 ± 0.03 nM). We radiolabeled 15 by nucleophilic 18F-fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1-(methyl-11C)-N-granisetron ([11C]2) through N-alkylation with [11C]CH3I, respectively. Both compounds [18F]15 (chemical and radiochemical purity >95%, specific activity 41 GBq/µmol) and [11C]2 (chemical and radiochemical purity ≥99%, specific activity 170 GBq/µmol) were evaluated for their utility as positron emission tomography (PET) probes. Using mouse and rat brain slices, in vitro autoradiography with both [18F]15 and [11C]2 revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5-HT3 receptors at significant levels. Subsequent PET experiments suggested that [18F]15 and [11C]2 are of limited utility for the PET imaging of brain 5-HT3 receptors in vivo.

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.

The effect of repeat dosing with cimetidine on the pharmacokinetics of intravenous granisetron in healthy volunteers.

The primary route of elimination of granisetron is by oxidative hepatic metabolism, thus its pharmacokinetic profile may be altered by co-administration of other drugs that inhibit or induce hepatic drug metabolizing enzymes. This open-label study investigated the effect of inhibition of cimetidine, a potent inhibitor of CYP1A2, CYP2D6 and CYP3A4, on the pharmacokinetic profile of intravenous granisetron in healthy male volunteers. Subjects (n = 12; 18-60 years) received granisetron (40 microg kg(-1)) infused over 3 min, six days before and on the eighth day of dosing with cimetidine (200 mg, four times a day). Blood samples were taken for pharmacokinetic analysis at intervals over 48 h following the administration of each dose of granisetron. Clinical chemistry, haematology and urinalysis were performed before, and 24 h after, each infusion. Electrocardiogram (ECG), resting blood pressure (BP) and pulse were monitored. There were no significant changes in the ECG, lead II trace or ECG time intervals, pulse or blood pressure on each study day. Minor falls in pulse rate and BP (likely to be related to recumbent posture) were seen during both granisetron dosing days, lasting 2 h after each infusion. No significant changes were apparent in the clinical chemistry, haematology or urinalysis measurements following granisetron dosing. No pharmacokinetic parameters measured after cimetidine administration were significantly different from those taken before. Adverse events were mild-to-moderate in severity and were similar to those reported in other studies with granisetron. The pharmacokinetics of granisetron, when administered as a single dose, appeared to be unaltered by cimetidine, an inhibitor of multiple hepatic enzymes (CYP1A2, CYP2D6 and CYP3A4). Granisetron was equally well tolerated before and after repeated dosing with cimetidine.

Provesicular granisetron hydrochloride buccal formulations: in vitro evaluation and preliminary investigation of in vivo performance.

Granisetron hydrochloride (granisetron) is a potent antiemetic that has been proven to be effective in acute and delayed emesis in cancer chemotherapy. Granisetron suffers from reduced oral bioavailability (≈60%) due to hepatic metabolism. In this study the combined advantage of provesicular carriers and buccal drug delivery has been explored aiming to sustain effect and improve bioavailability of granisetron via development of granisetron provesicular buccoadhesive tablets with suitable quality characteristics (hardness, drug content, in vitro release pattern, exvivo bioadhesion and in vivo bioadhesion behavior). Composition of the reconstituted niosomes from different prepared provesicular carriers regarding type of surfactant used and cholesterol concentration significantly affected both entrapment efficiency (%EE) and vesicle size. Span 80 proniosome-derived niosomes exhibited higher encapsulation efficiency and smaller particle size than those derived from span 20. Also, the effect of %EE and bioadhesive polymer type on in vitro drug release and in vivo performance of buccoadhesive tablets was investigated. Based on achievement of required in vitro release pattern (20-30% at 2h, 40-65% at 6h and 80-95% at 12h), in vivo swelling behavior, and in vivo adhesion time (>14 h) granisetron formulation (F19, 1.4 mg) comprising HPMC:carbopol 974P (7:3) and maltodextrin coated with the vesicular precursors span 80 and cholesterol (9:1) was chosen for in vivo study. In vivo pharmacokinetic study revealed higher bioavailability of buccal formulation relative to conventional oral formulation of granisetron (AUC0-∞ is 89.97 and 38.18 ng h/ml for buccal and oral formulation, respectively). A significantly lower and delayed Cmax (12.09±4.47 ng/ml, at 8h) was observed after buccal application compared to conventional oral tablet (31.66±10.15 ng/ml, at 0.5 h). The prepared provesicular buccoadhesive tablet of granisetron (F19) might help bypass hepatic first-pass metabolism and improve bioavailability of granisetron with the possibility of reducing reported daily dose (2mg) and reducing dosing frequency.

Impact of plasma and whole-blood anticoagulant counter ion choice on drug stability and matrix effects during bioanalysis.

BACKGROUND: Anticoagulants are used to prevent coagulation in blood samples. The plasma pH may change with a different counter ion and anticoagulant; thus, it is essential to determine effects on drug stability and the matrix effect during the bioanalytical method development. RESULTS: Cross-validation of multiple compounds between different counter ions was performed and no impact from the counter ion nature was demonstrated. Moreover, plasma stabilities and matrix effects for both fluconazole and granisetron were investigated thoroughly in numerous counter ions/anticoagulants (K(3)ethylenediaminetetraacetic acid [K(3)EDTA], K(2)EDTA, NaEDTA, NaHeparin and LiHeparin). Sirolimus, a large cyclic molecule, was also tested in different whole-blood EDTA counter ions. Results showed percentage deviation less than 8.5% and percentage cross-validation less than 8.4%. CONCLUSION: None of the compounds tested had an impact on the matrix stabilities and matrix effect.

Randomized crossover pharmacokinetic evaluation of subcutaneous versus intravenous granisetron in cancer patients treated with platinum-based chemotherapy.

BACKGROUND: 5-HT3-receptor antagonists are one of the mainstays of antiemetic treatment, and they are administered either i.v. or orally. Nevertheless, sometimes neither administration route is feasible, such as in patients unable to admit oral intake managed in an outpatient setting. Our objective was to evaluate the bioavailability of s.c. granisetron. PATIENTS AND METHODS: Patients receiving platinum-based chemotherapy were randomized to receive 3 mg of granisetron either s.c. or i.v. in a crossover manner during two cycles. Blood and urine samples were collected after each cycle. Pharmacokinetic parameters observed with each administration route were compared by analysis of variance. RESULTS: From May to November 2005, 31 patients were included and 25 were evaluable. Subcutaneous granisetron resulted in a 27% higher area under the concentration-time curve for 0-12 hours (AUC(0-12h)) and higher levels at 12 hours, with similar values for AUC(0-24h). The maximum concentration was lower with the s.c. than with the i.v. route and was observed 30 minutes following s.c. administration. CONCLUSION: Granisetron administered s.c. achieves complete bioavailability. This is the first study that shows that s.c. granisetron might be a valid alternative to i.v. delivery. Further trials to confirm clinical equivalence are warranted. This new route of administration might be especially relevant for outpatient management of emesis in cancer patients.

Quantification of granisetron in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry.

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of granisetron in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/138 for granisetron and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for granisetron in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

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.

Simultaneous measurement of blood and brain microdialysates of granisetron in rat by high-performance liquid chromatography with fluorescence detection.

Simultaneous microdialysis probes in the blood and brain and sensitive high-performance liquid chromatography with fluorescence detection were used to examine the granisetron concentration in the jugular vein and frontal cortex of rats after drug administration. Two microdialysis probes were inserted into the right jugular vein and frontal cortex of male Sprague-Dawley rats to which granisetron (6 mg/kg, i.v.) had been administered. Dialysates were automatically collected using a microfraction collector. Samples were eluted with a mobile phase containing 25 mM acetate buffer (pH 4.8)-acetonitrile (72:28, v/v). Excitation and emission wavelengths were set at 305 and 360 nm, respectively, on a scanning fluorescence detector. The limit of quantification for granisetron was 0.5 ng/ml. The in vitro recovery of granisetron was 29.7+/-1.2% (n=6) for the jugular vein microdialysis probe and 6.1+/-0.5% (n=6) for the frontal cortex microdialysis probe. The increasing brain/blood concentration ratio of granisetron suggests that granisetron penetrates the blood-brain barrier.

High-performance liquid chromatographic determination of granisetron in human plasma.

This paper describes a high-performance liquid chromatographic method (HPLC) with fluorometric detection for the analysis of granisetron in plasma. The detection is performed at 305 nm for excitation and 365 nm for emission. The method involves sample clean-up by liquid-liquid extraction. N-(1-Naphthyl) ethylenediamine dihydrochloride is used as internal standard. Toluene and phosphate buffer were added to 0.5 ml of plasma added to the internal standard. After extraction, the organic layer was separated and then evaporated to dryness. The residue was reconstituted in eluent mixture. An aliquot was injected onto the HPLC column, Spherisorb CN, equilibrated with an eluent mixture constituted by acetonitrile-phosphate buffer (pH 4.5) (15:85). The proposed technique is reproducible, selective, reliable, and sensitive. Linear detector responses were observed for the calibration curve standards in the range of 0.50 to 100 ng/ml. Extraction recovery from plasma proved to be more than 90%. Precision expressed as C.V. was in the range 2 to 8%. As low as 0.3 ng of granisetron per ml of plasma can be measured with good accuracy. The method has been validated, and stability tests under various conditions have been performed. Its sensitivity is adequate for pharmacokinetic studies.

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.