M.L. Constanzer

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC-MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm x 50 mm, 3 microm) using a mobile phase of ACN/H(2)O (80/20, v/v) containing 10 mM NH(4)Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408-->235 for sitagliptin and m/z 412-->239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 microL of plasma is processed. The linear calibration range is 1-1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n=6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection.

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 A), 150x4.6 mm C8 analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor-->product ion combinations of m/z 1093.7-->1033.6 and 1094.7-->1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-1000 ng/ml using ISP, and 2.5-500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.

Effect of Impaired Renal Function and Haemodialysis on the Pharmacokinetics of Aprepitant

AbstractBackground: The neurokinin NK1-receptor antagonist aprepitant has demonstrated efficacy in preventing highly emetogenic chemotherapy-induced nausea and vomiting.n Objective: The objective of the present study was to investigate the effects of impaired renal function on the pharmacokinetics and safety of aprepitant.n Subjects and methods: A total of 32 patients and healthy subjects were evaluated in this open-label, two-part study. Pharmacokinetic parameters after a single oral dose of aprepitant 240mg were measured in eight patients with end-stage renal disease (ESRD) requiring haemodialysis, eight patients with severe renal insufficiency (SRI [24-hour creatinine clearance <30 mL/min/1.73m2]) and 16 healthy and age-, sex- and weight-matched subjects (controls).n Results: In ESRD patients, the area under the plasma concentration-time curve (AUC) from 0 to 48 hours (AUC48) for aprepitant was on average approximately 36% lower than that observed in control subjects (ratio [ESRD patients/healthy controls] of geometric means = 0.64), but the 90% confidence interval 0.52, 0.78 for the ratio of true mean AUC48 fell within the predefined target interval of 0.5, 2.0. Also in ESRD patients, there was no statistically or clinically significant difference in unbound aprepitant AUC (which may be more clinically relevant than total aprepitant AUC) when compared with healthy control subjects. Aprepitant pharmacokinetic parameters in ESRD patients were clinically similar when haemodialysis was initiated at 4 hours or 48 hours after aprepitant administration. In SRI patients, the ratio (SRI patients/healthy controls) of aprepitant AUC from zero to infinity (AUC∞) geometric mean value was 0.79 with a 90% confidence interval of 0.56, 1.10. On average, in SRI patients the principal aprepitant pharmacokinetic parameters (AUC∞, initial maximum plasma concentration [Cmax], time to initial Cmax, and apparent elimination half-life) were not statistically different from those obtained in healthy control subjects. Aprepitant was generally well tolerated in both ESRD and SRI patients.n Conclusion: The results of this study demonstrate that ESRD, haemodialysis and SRI have no clinically meaningful effect on aprepitant pharmacokinetics. Therefore, no dosage adjustment of aprepitant is warranted in SRI or ESRD patients.

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of a novel 4-aza-steroidal inhibitor of 5 alpha-reductase in human plasma has been developed. The assay is based on a single solid-phase extraction and an efficient HPLC separation on two analytical columns in series. The assay has been fully validated and used to support Phase II and III clinical pharmacokinetic studies. The lowest limit of quantification was found to be at 1 ng/ml and allowed pharmacokinetic evaluation of the drug at doses down to 5 mg.

Bioavailability of Alendronate and Vitamin D3 in an Alendronate/Vitamin D3 Combination Tablet

These studies were designed to demonstrate that the alendronate (ALN) component of an ALN/vitamin D3 combination tablet was bioequivalent to the 70‐mg ALN tablet and that the pharmacokinetic parameters of vitamin D3 were similar with or without ALN. These were open‐label, randomized, 2‐part, 2‐period, crossover studies. In part I, participants received either a single combination tablet or ALN 70 mg. In part II, participants received either a single combination tablet or vitamin D3 alone. Results from part I showed that the geometric mean ratio (GMR) for total urinary excretion of ALN for both studies fell within the prespecified bioequivalence bounds. Results from part II showed that the pharmacokinetic profiles of vitamin D3 with or without ALN were also similar. The combination tablets are bioequivalent to the ALN 70‐mg tablet with respect to ALN bioavailability. The bioavailability of vitamin D3 is similar in the combination tablets and when administered alone. No serious adverse experiences were reported.

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid-liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50-2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.

Amiloride: Biological Fluid Analysis by Reverse-Phase HPLC

Amiloride is an effective anti-kaliuretic-diuretic which has been used clinically since 1965. Because of a lack of sensitivity in reported analytical methods, useful pharmacokinetic data have been sparse. This publication describes a sensitive (1 ng/ml), rapid, and reproducible reverse-phase HPLC technique for the quantitative measurement of amiloride levels in both serum and urine specimens. Sample clean-up is based on the adsorption of amiloride to commercially available silica cartridges and its selective elution with perchloric acid. The eluted drug is further resolved from endogenous interferences on a Waters C-18 µBondapak analytical column with a mobile phase consisting of methanol:sodium perchlorate (0.1 M, pH 4.0) 40:60 v/v and fluorescence detection (λex = 368 nm, λem = 417 nm). This method has been applied to the analysis of human serum and urine samples.

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor.

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.

Determination of 2,3-dihydro-6-[3-(2-hydroxymethyl)-phenyl-2-propenyl]-5-benzofuranol in plasma using liquid chromatography with electrochemical detection

A reversed-phase column liquid chromatographic (LC) method with electrochemical detection (ED) is described for the quantification of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (compound 1), a new locally active dual inhibitor of leukotriene and prostaglandin synthesis, in plasma. After a single liquid-liquid extraction of the biological specimen, the extract was analyzed using a liquid chromatograph with an amperometric detector set at an oxidation potential of +0.55 V. The resulting chromatograms are free from endogenous interference and the limit of detection is 0.2 ng/ml. Several other analogous dihydrobenzofuranols were shown to be electrochemically active, permitting their determination using LC with ED. The described analytical method has been fully validated in the concentration range 0.5-20 ng/ml of plasma and utilized in the analysis of plasma samples from human clinical studies. The analytical methodology has also been adapted for analysis of compound 1 in human skin blister fluid after topical administration of 1.