An improved LC-MS/MS method for quantitation of indapamide in whole blood: application for a bioequivalence study.

An improved LC-MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide-d3 was used as internal standard (IS) and liquid-liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP-column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 µL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 → m/z 188.9 and m/z 367.0 → m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25-50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration-time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratórios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption.

Determination of 3-α-hydroxytibolone in human plasma by LC-MS/MS: application for a pharmacokinetic study after administration of a tibolone formulation.

A new method was developed for the quantitation of 3-α-hydroxy tibolone, in human plasma, after oral administration of a tablet formulation containing tibolone (2.5 mg). 3-α-Hydroxy tibolone was extracted by a liquid-liquid procedure, using cyproterone acetate as internal standard and chlorobutane as extraction solvent. After extraction, samples were submitted to a derivatization step with p-toluenesulfonyl isocyanate. A mobile phase consisting of acetonitrile and water (72:28 v/v) was used and chromatographic separation was achieved using Agilent XDB C18 column (100 × 4.6 mm i.d.; 5 µm particle size), at 40°C. Mass spectrometric detection was performed using atmospheric pressure chemical ionization in negative mode for 3-α-hydroxy tibolone and in positive mode for cyproterone acetate. The fragmentation transitions were m/z 510.2 → m/z 170.1 and m/z 417.0 → m/z 357.1 for 3-α-hydroxy tibolone and cyproterone acetate, respectively. Calibration curves were constructed over the range 100-30,000 pg/mL and the method was shown to be specific, precise and accurate, with a mean recovery rate of 94.2% for 3-α-hydroxy tibolone. No matrix effect or carry-over was detected in the samples. The validated method was applied in a pharmacokinetic study with a tibolone formulation in healthy female volunteers.

A rapid and sensitive HPLC-APCI-MS/MS method determination of fluticasone in human plasma: application for a bioequivalency study in nasal spray formulations.

A sensitive method for the determination of fluticasone in plasma was developed using high performance liquid chromatography with tandem mass spectrometric detection, whereas beclomethasone was used as internal standard. The analytes were extracted with a simple liquid-liquid extraction from the plasma samples and separated on an ACE C(18) 50 × 4.6 mm i.d.; 5 µm particle size column with a mobile phase consisting of acetonitrile - 0.01% formic acid (48:52, v/v) at a flow rate of 1 ml/min. Detection was achieved by an Applied Biosystems API 5000 mass spectrometer (LC-MS/MS) set at unit resolution in the multiple reaction monitoring mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for fluticasone propionate was 85%, with a lower limit of quantification set at 2 pg/mL. The validated analytical method was applied to a bioequivalence study of fluticasone propionate administered by nasal spray formulations in human volunteers.

Quantitation of glucosamine sulfate in plasma by HPLC-MS/MS after administration of powder for oral solution formulation.

A rapid method for the quantification of glucosamine in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The sample preparation includes a simple deproteinization step, using D-[1-¹³C] glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2 mM ammonium acetate containing 0.025% formic acid (80:20). Selected reaction monitoring was performed using the transitions m/z 180.1 → m/z 72.1 and m/z 181.0 → m/z 74.6 to quantify glucosamine and internal standard, respectively. The method was validated and proved to be linear, accurate and precise over the range 50-5000 ng/mL of glucosamine. Recovery rates higher than 90% were obtained for both glucosamine and internal standard. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after oral administration of a powder for oral solution formulation containing glucosamine sulfate.

Development and validation of a high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma.

A sensitive and fast high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma was developed and validated. A simple protein precipitation step with perchloric acid was used for the cleanup of plasma, and methyldopa was added as an internal standard. The analyses were carried out using an ACE C(18) column (50 × 4.6 mm i.d.; 5 µm particle size) and a mobile phase consisting of 0.2% formic acid and acetonitrile (90:10). The triple-quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 198.1 → m/z 107.0, m/z 227.2 → m/z 181.0, and m/z 212.1 → m/z 139.2 for levodopa, carbidopa, and methyldopa, respectively. The method was validated and proved to be linear, accurate, and precise over the range 50-5000 ng/mL for levodopa and 3-600 ng/mL for carbidopa. The proposed method was successfully applied in a pharmacokinetic study with a levodopa/carbidopa tablet formulation in healthy volunteers.

Determination of triamcinolone in human plasma by a sensitive HPLC-ESI-MS/MS method: application for a pharmacokinetic study using nasal spray formulation.

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantitation of triamcinolone in human plasma after nasal spray application was developed and validated. Betamethasone was used as internal standard (IS). The analytes were extracted by a liquid-liquid procedure and separated on a Zorbax Eclipse XDB C(18) column with a mobile phase composed of 2 mM aqueous ammonium acetate pH 3.2 and acetonitrile (55:45). Selected reaction monitoring was performed using the transitions m/z 435 → 415 and m/z 393 → 373 to quantify triamcinolone acetonide and betamethasone, respectively. Calibration curve was constructed over the range of 20-2000 pg/ml for triamcinolone acetonide. The lower limit of quantitation was 20 pg/ml. The mean RSD values were 4.6% and 5.7% for the intra-run and inter-run precision, respectively. The mean accuracy value was 98.5% and a recovery rate corresponding to 97.5% was achieved. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of triamcinolone acetonide in healthy volunteers, in a pharmacokinetic study with nasal spray formulation.