In the present study, hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) combined with tandem mass spectrometric detection (MS/MS) were evaluated and compared for the determination of donepezil, cetirizine and loratadine in human plasma, in terms of sensitivity and sample preparation procedure. A retention study for the above compounds of various polarities was performed, using both C(18) and silica columns, with several aqueous-organic mobile phase ratios, in order to investigate their retention mechanism profile under HILIC and RPLC. Both chromatographic conditions were compared for chromatographic analysis of plasma samples processed with a liquid-liquid extraction (LLE) method for donepezil determination, resulting in significantly higher sensitivity under HILIC. Furthermore, HILIC and RPLC were compared for direct injection, and novel methods including LLE, solid-phase extraction and protein precipitation protocols were developed. Direct injection technique significantly reduced sample preparation time, increasing at the same time method sensitivity. The current study contributes to broadening the range of analyzable compounds by HILIC-MS/MS to molecules of medium polarity.

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An automated, sensitive and high-throughput liquid chromatographic/electrospray tandem mass spectrometric (LC-MS/MS) assay was developed for the simultaneous determination of losartan (LOS), its major circulating metabolite EXP-3174 and hydrochlorothiazide (HCTZ) in human plasma. LOS and HCTZ coexist in the same drug formulation, and this is the first method that enables the simultaneous determination of both drugs along with the active metabolite of LOS. Since these drugs have different physicochemical properties, the employment of a liquid-liquid extraction (LLE) protocol was precluded. A fully automated solid-phase extraction (SPE) protocol, based on 96-well format plates, was used to isolate these compounds and furosemide (internal standard, IS) from plasma. Washing and elution steps were amended accordingly in order to minimize any matrix effect from components of the plasma without reducing the elution of the molecules of interest. The compounds were eluted from a C18 column and detected with an API 3000 triple-quadrupole mass spectrometer using negative electrospray ionization and multiple reaction monitoring (MRM). The assay was linear over the range 1.00-400 ng/mL for LOS and EXP-3174 and 0.500-200 ng/mL for HCTZ, respectively, when 200 microl of plasma was used in the extraction. The overall intra- and interassay variations were within acceptance limits. The analysis time for each sample was 4 min, and more than 300 samples could be analyzed in one day by running the system overnight. The assay was simple, highly sensitive, selective, precise, fast, and it enables the reliable determination of LOS, EXP-3174 and HCTZ in pharmacokinetic or bioequivalence studies after per os administration of a single tablet containing both drugs.

A semi-automated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of the antifungal drug itraconazole (ITZ) and its coactive metabolite hydroxyitraconazole (OH-ITZ) in human plasma. The plasma samples underwent liquid-liquid extraction (LLE) in 2.2 mL 96 deepwell plates. ITZ, OH-ITZ and the internal standard (IS) R51012 were extracted from plasma, using a mixture of acetonitrile (ACN) and methyl t-butyl ether (MTBE) as the organic solvent. This specific mixture, due to its composition, had a significant impact on the performance of the assay. All liquid transfer steps, including preparation of calibration standards and quality control samples as well as the addition of the IS, were performed automatically using robotic liquid handling workstations for parallel sample processing. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analytes and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by combined reversed phase LC/MS/MS, with positive ion electrospray ionization and a TurboIonSpray interface, using multiple reactions monitoring (MRM). The method was shown to be sensitive and specific to both ITZ and OH-ITZ, it revealed excellent linearity for the range of concentrations 2-500 ng mL(-1) for ITZ and 4-1000 ng mL(-1) for OH-ITZ, it was very accurate and it gave very good inter- and intra-day precisions. The proposed high-throughput method was employed in a bioequivalence study after per os administration of two 100 mg tablets of ITZ, and it allowed this study to be completed in under four days.