Paulo Alexandre Rebelo Galvinas

Budesonide quantification by HPLC coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometry. Application to a comparative systemic bioavailability of two budesonide formulations in healthy volunteers

In the present study, a novel, fast, sensitive and robust method to quantify budesonide in human plasma using 3-keto-desogestrel as the internal standard (IS) is described. The analyte and the IS were extracted from human plasma by liquid-liquid extraction (LLE) using ether. Extracted samples were analyzed by high performance liquid chromatography coupled to Atmospheric pressure photoionization tandem mass spectrometry (HPLC-APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 μm analytical column. The temperature of the autosampler was kept at 6 °C and the run time was 4.00 min. A linear calibration curve over the range 7.5-1000 pg ml⁻¹ was obtained and the lowest concentration quantified was 7.5 pg ml⁻¹, demonstrating acceptable accuracy and precision. This analytical method was applied in a relative bioavailability study in order to compare a test budesonide 64 μg/dose nasal spray formulation vs. a reference 64 μg/dose nasal spray formulation (Budecort Aqua) in 48 volunteers of both sexes. The study was conducted in an open randomized two-period crossover design and with a one-week washout period. Plasma samples were obtained over a 14 h interval. Since the 90% CI for both C(max), AUC(last) and AUC(0-inf) were within the 80-125% interval proposed by the Food and Drug Administration and ANVISA, it was concluded that budesonide 64 μg/dose nasal spray was bioequivalent to Budecort Acqua® 64 μg/dose nasal spray, according to both the rate and extent of absorption.

Determination of chlorpheniramine in human plasma by HPLC‐ESI‐MS/MS: application to a dexchlorpheniramine comparative bioavailability study

In the present study a fast, sensitive and robust validated method to quantify chlorpheniramine in human plasma using brompheniramine as internal standard (IS) is described. The analyte and the IS were extracted from plasma by LLE (diethyl ether-dichloromethane, 80:20, v/v) and analyzed by HPLC-ESI-MS/MS. Chromatographic separation was performed using a gradient of methanol from 35 to 90% with 2.5 mm NH(4)OH on a Gemini Phenomenex C(8) 5 microm column (50 x 4.6 mm i.d.) in 5.0 min/run. The method fitted to a linear calibration curve (0.05-10 ng/mL, R > 0.9991). The precision (%CV) and accuracy ranged, respectively: intra-batch from 1.5 to 6.8% and 99.1 to 106.6%, and inter-batch from 2.4 to 9.0%, and 99.9 to 103.1%. The validated bioanalytical procedure was used to assess the comparative bioavailability in healthy volunteers of two dexchlorpheniramine 2.0 mg tablet formulations (test dexchlorpheniramine, Eurofarma, and reference Celestamine, Schering-Plough). The study was conducted using an open, randomized, two-period crossover design with a 2 week washout interval. Since the 90% confidence interval for C(max) and AUC ratios were all within the 80-125% interval proposed by ANVISA and FDA, it was concluded that test and reference formulations are bioequivalent concerning the rate and the extent of absorption.

Comparative bioavailability of two oral formulations of clozapine in steady state administered in schizophrenic volunteers under individualized dose regime.

In the present study, a novel, fast, sensitive and robust method to quantify clozapine in human plasma using quetiapine as the internal standard (IS) is described. The analyte and the IS were extracted from plasma using a single protein precipitation extraction technique with methanol and analyzed by high performance liquid chromatography coupled to the electrospray ionization tandem mass spectrometric (HPLC-ESI-MS/MS). The method was linear over the range 20 to 1500 ng.mL-1. The intra-assay precisions ranged from 3.8 to 5.9%, while inter-assay precisions ranged from 4.2 to 6.0%. The intra-assay accuracies ranged from 99.3 to 107.5%, while the inter-assay accuracies ranged from 98.9 to 101.7%. This method agrees with the requirements proposed by the US Food and Drug Administration of high sensitivity, specificity and high sample throughput and was used to evaluate the pharmacokinetic profiles and bioequivalence of the two clozapine formulations in twenty six schizophrenic patients affected by refractory schizophrenia under steady-state conditions. During the hospitalization period the patients received the 100 mg clozapine formulation tablets corresponding to the same dose they were using 14 days before hospitalization. The clozapine pharmacokinetic did not differ significantly after administration of both test and the reference formulations. The Tmax and T1/2 for the test formulation were 2.26 and 10.92 h, respectively. In addition, the Tmax and T1/2 for the reference formulation were 2.44 and 11.08 h, respectively. The 90% confidence interval of the mean ratio of lnAUC0-t was within 0.80-1.25 range which indicates that the test formulation was bioequivalent to the reference formulation when orally administered to schizophrenic patients regarding both the rate and extent of absorption.

Quantification of chlordesmethyldiazepam by liquid chromatography–tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC-MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid-liquid extraction (diethyl-ether/hexane, 80/20, v/v) procedure. The LC-MS/MS method on a RP-C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5-50 ng/mL (R > 0.999). The between-run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification-LLOQ (0.500 ng/mL). The between-run accuracies were 0.1, -1.5, -2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam -- test, Eurofarma Lab. Ltda and Olcadil -- reference, Novartis Biociências S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUC0-inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA.

Cyproterone acetate quantification in human plasma by high-performance liquid chromatography coupled to atmospheric pressure photoionization tandem mass spectrometry. Application to a comparative pharmacokinetics study.

A specific, fast and sensitive high performance liquid chromatography (HPLC) coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometric (LC-MS/MS) assay was developed for the determination of cyproterone (CYP) acetate (CAS 427-51-0) in human plasma. The retention times were 3.26 and 2.90 min for CYP acetate and its internal standard (I. S.) finasteride (FIN), respectively. The overall mean recovery, using liquid/liquid extraction, was found to be 109.0, 107.7 and 100.3%, for low, medium and high concentrations, respectively. Calibration curves were linear in the concentration range of 0.1-50.0 ng/ml, and the lower limit of quantification (LLOQ) was 0.1 ng/ml. The LLOQ, 0.1 ng/ml, was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 2.2 to 5.55%, while Inter-batch accuracy ranged from 95.5 to 100.0%. Intra-batch precision ranged from 1.8 to 5.6%, while Intra-batch accuracy ranged from 92.0 to 99.4% at concentrations of 0.3 ng/ml, 20.0 and 40.0 ng/ml. The developed method was applied to a bioequivalenc study of CYP acetate in a group of 44 female volunteers at a single oral dose of a 2 mg tablet, in a combination of ethinylestradiol/CYP acetate (0.25/2 mg). The plasma concentration of CYP acetate did not differ significantly after administration of both formulations (test formulation and the reference one). The geometric mean and respective 90% CI of CYP acetate test/reference percent ratios were 90.66% (84.39-97.40%) for Cmax and 96.20% (90.45-102.33%) for AUC0-t.

A fast, sensitive and simple method for mirtazapine quantification in human plasma by HPLC-ESI-MS/MS. Application to a comparative bioavailability study.

In the present study a simple, fast, sensitive and robust method to quantify mirtazapine in human plasma using quetiapine as the internal standard (IS) is described. The analyte and the IS were extracted from human plasma by a simple protein precipitation with methanol and were analyzed by high-performance liquid chromatography coupled to an electrospray tandem triple quadrupole mass spectrometer (HPLC-ESI-MS/MS). Chromatography was performed isocratically on a C(18), 5 µm analytical column and the run time was 1.8 min. The lower limit of quantitation was 0.5 ng/mL and a linear calibration curve over the range 0.5-150 ng/mL was obtained, showing acceptable accuracy and precision. This analytical method was applied in a relative bioavailability study in order to compare a test mirtazapine 30 mg single-dose formulation vs a reference formulation in 31 volunteers of both sexes. The study was conducted in an open randomized two-period crossover design and with a 14 day washout period. Since the 90% confidence interval for C(max) , AUC(last) and AUC(0-inf) were within the 80-125% interval proposed by the Food and Drug Administration and ANVISA (Brazilian Health Surveillance Agency), it was concluded that mirtazapine 30 mg/dose is bioequivalent to the reference formulation, according to both the rate and extent of absorption.