Gustavo D. Mendes

Ticlopidine quantification in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific analytical method was developed and validated to quantify gabapentin in human plasma using acetaminophen as an internal standard. The method employs a single plasma protein precipitation. The analytes are chromatographed on a C4 reversed-phase chromatographic column and analyzed by mass spectrometry in the multiple reaction monitoring (MRM) mode. The method has a chromatographic run time of 4 min and a linear calibration curve over the range 50-10 000 ng x ml(-1) (r > 0.999). The between-run precision, based on the relative standard deviation for replicate quality controls, was < or = 4.8 % (200 ng x ml(-1)), 6.0% (1000 ng x ml(-1)) and 4.4% (5000 ng x ml(-1)). The between-run accuracy was +/-2.6, 4.4 and 0.5% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two gabentin capsule formulations (Progresse from Biosintética, Brazil, as a test formulation, and Neurotin from Parke-Davis, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 300 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Progresse/Neurotin was 87.9-115.6% for AUC(0-36 h) and 88.6-111.7% for Cmax. Since both 90% CI for AUC(0-36 h) and Cmax were included in the 80-125% interval proposed by the US Food and Drug Administration, Progresse was considered bioequivalent to Neurotin according to both the rate and extent of absorption.

Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry.

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats

OBJECTIVE in this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. MATERIALS AND METHODS oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. RESULTS in NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. CONCLUSIONS in addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0min at a flow-rate of 1.5 mL min⁻¹ into a Phenomenex Gemini® C18, 5 μm analytical column (150 × 4.6 mm i.d.). The calibration curve was linear over the range from 0.2 to 200 ng mL⁻¹ for dextromethorphan and doxylamine and 0.05 to 10 ng mL⁻¹ for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study.

Comparative bioavailability of three ibuprofen formulations in healthy human volunteers

OBJECTIVE To assess the bioequivalence of three ibuprofen formulations (Test formulation: ibuprofen (400 mg capsule) manufactured by Cardinal Health Brasil 402 Ltda. (Sorocaba, Brazil) and licensed to Boehringer Ingelheim do Brasil Quim. e Farm. Ltda. (SA poundo Paulo, Brazil); Reference formulation (1): ibuprofen (AdvilA(R); 2 A 200 mg coated tablet) from Wyeth-Whitehall Ltda. (Itapevi, Brazil); Reference formulation (2): ibuprofen (AliviumA; 8 ml A 50 mg/ml solution) from Schering Plough S.A. (Rio de Janeiro, Brazil)) in 24 healthy volunteers of both sexes. METHODS The study was conducted using an open, randomized, three-period crossover design with at least 5-day washout interval. Plasma samples were obtained over a 24-h period. Plasma ibuprofen concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with negative ion electrospray ionization using multiple reaction monitoring (MRM). The following pharmacokinetic parameters were obtained from the ibuprofen plasma concentration vs. time curves: AUC(last), AUC(trunctmax), AUC(inf) and C(max). RESULTS The limit of quantification for ibuprofen was 0.1 microg A ml(1). The geometric mean with corresponding 90% confidence interval (CI) for Test/Reference (1) percent ratios were 114.24% (90% CI = 105.67, 123.50%) for C(max), 98.97% (90% CI = 94.69, 103.44%) for AUC(last) and 99.40% (90% CI = 95.21, 103.78%) for AUC(inf). The geometric mean and respective 90% confidence interval (CI) for Test/Reference (2) percent ratios were 108.38% (90% CI = 100.19, 117.25%) for C(max), 100.79% (90% CI = 96.39, 105.40%) for AUC(last) and 101.26% (90% CI = 96.94, 105.77%) for AUC(inf); t(max) for the 400 mg Test capsule was shorter than that for the 2 A 200 mg Reference (1) tablets (p < 0.002). CONCLUSION Since the 90% CI for AUC(last), AUC(inf) and Cmax ratios were within the 80 - 125% interval proposed by the US FDA, it was concluded that ibuprofen formulation manufactured by Cardinal Health Brasil 402 Ltda. and licensed to Boehringer Ingelheim do Brasil Quim. e Farm. Ltda. is bioequivalent to the AdvilA and AliviumA formulations with regard to both the rate and the extent of absorption.

Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a diethyl-ether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v)+0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 µm analytical column, (150 mm x 4.6 mm I.D.). The method had a chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 > 0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine + cobamamide (4 mg + 1 mg) tablet formulations (Cobactin® [cyproheptadine + cobamamide] test formulation supplied from Zambon Laboratórios Farmacêuticos Ltda. and Cobavital® from Solvay Farma (standard reference formulation)). A single 4 mg + 1 mg [cyproheptadine + cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin®) is bioequivalent to the Cobavital® formulation for both the rate and the extent of absorption of cyproheptadine.

Ciprofibrate quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry for pharmacokinetic studies.

A rapid, sensitive and specific method for quantifying ciprofibrate in human plasma using bezafibrate as the internal standard (IS) is described. The sample was acidified prior extraction with formic acid (88%). The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (diethyl ether/dichloromethane 70/30 (v/v)). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed using Genesis C18 4 μm analytical column (4.6 × 150 mm i.d.) and a mobile phase consisting of acetonitrile/water (70/30, v/v) and 1mM acetic acid. The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 0.1-60 μg/mL (r>0.99). The limit of quantification was 0.1 μg/mL. The intra- and interday accuracy and precision values of the assay were less than 13.5%. The stability tests indicated no significant degradation. The recovery of ciprofibrate was 81.2%, 73.3% and 76.2% for the 0.3, 5.0 and 48.0 ng/mL standard concentrations, respectively. For ciprofibrate, the optimized parameters of the declustering potential, collision energy and collision exit potential were -51 V, -16 eV and -5 V, respectively. The method was also validated without the use of the internal standard. This HPLC-MS/MS procedure was used to assess the bioequivalence of two ciprofibrate 100mg tablet formulations in healthy volunteers of both sexes. The following pharmacokinetic parameters were obtained from the ciprofibrate plasma concentration vs. time curves: AUC(last), AUC(0-168 h), C(max) and T(max). The geometric mean with corresponding 90% confidence interval (CI) for test/reference percent ratios were 93.80% (90% CI=88.16-99.79%) for C(max,) 98.31% (90% CI=94.91-101.83%) for AUC(last) and 97.67% (90% CI=94.45-101.01%) for AUC(0-168 h). Since the 90% CI for AUC(last), AUC(0-168 h) and C(max) ratios were within the 80-125% interval proposed by the US FDA, it was concluded that ciprofibrate (Lipless 100mg tablet) formulation manufactured by Biolab Sanus Farmacêutica Ltda. is bioequivalent to the Oroxadin (100 mg tablet) formulation for both the rate and the extent of absorption.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice.

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF. In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NOx- levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite Nomega-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the Nomega-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with l-NAME yields low serum Nomega-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO.

PHARMACOKINETICS AND PHARMACODYNAMICS OF A NITRIC OXIDE-RELEASING DERIVATIVE OF ENALAPRIL IN MALE BEAGLES

1 Pharmacological compounds that release nitric oxide (NO) have been useful tools in the evaluation of the broad role of NO in physiopathology and therapeutics. The present study compared the pharmacokinetics and pharmacodynamics of enalapril and an NO‐releasing enalapril molecule (NCX899) in conscious male beagles. The effects of both enalapril and NCX899 in the arterial hypertension and bradycardia induced by acute NO inhibition in anaesthetized dogs were also investigated. 2 Dogs received either NCX899 (4 µmol/kg, i.v.) or enalapril (4 µmol/kg, i.v.), after which plasma concentrations of the analytes and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). 3 In the NCX899 group, the area under the time‐course curve (AUC0−24h) was 29.18 ± 4.72, 229.37 ± 51.32 and 5159.23 ± 514.88 µg·h/L for the analytes nitro‐enalapril, enalapril and enalaprilat, respectively. In the enalapril group, the AUC0−24h was 704.53 ± 158.86 and 4149.27 ± 847.30 µg·h/L for the analytes enalapril and enalaprilat, respectively. Statistical analysis of data from both groups showed a significant difference for the analyte enalapril, but not for enalaprilat. Moreover, NCX899 and enalapril were equally effective in inhibiting the activity of serum angiotensin‐converting enzyme. 4 In anaesthetized dogs, i.v. administration of the NO synthase (NOS) inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME; 0.1–10 mg/kg) significantly elevated arterial blood pressure, with concomitant bradycardia. The compound NCX899 significantly attenuated both arterial hypertension and bradycardia, whereas enalapril had no significant effect. 5 In conclusion, the present results showed that the NO‐releasing derivative of enalapril NCX899 presents a pharmacokinetic/pharmacodynamic relationship similar to its parent compound enalapril. Moreover, NCX899 (but not enalapril) was effective in protecting against the cardiovascular changes induced by acute NOS inhibition.

Comparative bioavailability of betahistine tablet formulations administered in healthy subjects.

OBJECTIVE To assess the comparative bioavailability of two formulations (16 mg tablet) of betahistine (CAS 5579-84-0) in healthy volunteers of both sexes. METHODS The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Plasma samples were obtained for up to 36 h post dose. Plasma 2-pyridylacetic acid concentrations were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). From the 2-pyridylacetic acid plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained for AUCIast and Cmax. RESULTS The limit of quantification was 4 ng/mL for plasma 2-pyridylacetic acid analysis. The geometric mean and 90% confidence interval (CI) of test/reference percent ratios were: 98.94% (92.21%-106.16%) for Cmax, 95.42% (91.74%-99.25%) for AUClast. CONCLUSION Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% interval proposed by the US Food and Drug Administration Agency, it was concluded that the test formulation is bioequivalent to the reference for both the rate and the extent of absorption.