Simultaneous determination of zidovudine and lamivudine from rat tissues by liquid chromatography/tandem mass spectrometry

A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid, placental, and fetal tissues. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C18 column (5 microm, 150 x 3.9 mm i.d). The mobile phase consisted of 30% methanol and 7.5 mM ammonium acetate (pH 6.5). The method was validated in the range of 0.05-25 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport following co-administration of these compounds at a dose of 25 mg/kg each in a pregnant rat.     

Comparison of local anesthetic-cyclodextrin non-covalent complexes using capillary electrophoresis and electrospray ionization mass spectrometry

Non-covalent complexes between three derivitized cyclodextrins (CD's) and six local anesthetics were studied using capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The CE study was performed using the complete filling technique (CFT). A comparison between the migration data from CE and ESI-MS inclusion complex peak abundances was made representing the association between local anesthetics and CD's in the solution and the gas phase, respectively. The results from this study showed comparable behavior of the complexes in the CE and mass spectrometer, indicating similarity in the parameters controlling the stability of these complexes. Therefore, the formation of specific non-covalent complexes, as shown in this study, could be used to predict the behavior of a complexing agent with a substrate in the solution phase by observing data obtained from ESI-MS.     

Quantification of the transporter substrate fexofenadine in cell lysates by liquid chromatography/tandem mass spectrometry

Drug–drug interactions at transporters present a significant and under‐investigated clinical problem. Investigations of specific transporter functions and screening for potential drug‐drug interactions, both in vitro and especially in vivo, will require validated experimental probes. Fexofenadine, an approved, well‐tolerated drug, is a promising probe for studies of membrane transporter function. Although fexofenadine pharmacokinetics are known to be controlled by transporters, the contributions of individual transporters have not been defined. We have developed a rapid, specific, and sensitive analytical method for quantitation of fexofenadine to support this work. This liquid chromatography/tandem mass spectrometry (LC/MS/MS) method quantifies fexofenadine in cell lysates from in vitro studies using cetirizine as the internal standard. Cell lysates were prepared for analysis by acetonitrile precipitation. Analytes were then separated by gradient reversed‐phase chromatography and analyzed by tandem mass spectrometry using the m/z 502.17/466.2 transition for fexofenadine and m/z 389.02/201.1 for cetirizine. The method exhibited a linear dynamic range of 1–500 ng/mL for fexofenadine in cell lysates. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5%. Intra‐ and inter‐day precision and accuracy were within the limits presented in the FDA guidelines for bioanalysis. We also will validate this method to support not only the quantification of fexofenadine, but also other probe drugs for drug–drug interaction studies. This method for quantification will facilitate the use of fexofenadine as a probe drug for characterization of transporter activity. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.     

Bio-Inspired Rhamnolipids,Cyclic Lipopeptides and a Chito-Oligosaccharide Confer Protection against Wheat Powdery Mildew and Inhibit Conidia Germination

Plant protection is mainly based on the application of synthetic pesticides to limit yield losses resulting from diseases. However, the use of more eco-friendly strategies for sustainable plant protection has become a necessity that could contribute to controlling pathogens through a direct antimicrobial effect and/or an induction of plant resistance. Three different families of natural or bioinspired compounds originated from bacterial or fungal strains have been evaluated to protect wheat against powdery mildew, caused by the biotrophic Blumeria graminis f.sp. tritici (Bgt). Thus, three bio-inspired mono-rhamnolipids (smRLs), three cyclic lipopeptides (CLPs, mycosubtilin (M), fengycin (F), surfactin (S)) applied individually and in mixtures (M + F and M + F + S), as well as a chitosan oligosaccharide (COS) BioA187 were tested against Bgt, in planta and in vitro. Only the three smRLs (Rh-Eth-C12, Rh-Est-C12 and Rh-Succ-C12), the two CLP mixtures and the BioA187 led to a partial protection of wheat against Bgt. The higher inhibitor effects on the germination of Bgt spores in vitro were observed from smRLs Rh-Eth-C12 and Rh-Succ-C12, mycosubtilin and the two CLP mixtures. Taking together, these results revealed that such molecules could constitute promising tools for a more eco-friendly agriculture.     

Pharmacokinetics,protein binding and metabolism of a quinoxaline urea analog as an NF‐κB inhibitor in mice and rats by LC‐MS/MS

13–197 is a novel NF‐κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13–197 in mice and rats. A valid, sensitive and selective LC‐MS/MS method was developed. This method was validated for the quantification of 13–197, in the range of 0.1 or 0.2‐500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13–197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12‐fold shorter T_max than in rats. Tissue concentrations were 1.3–69.2‐fold higher in mice than in rats at 72 h after intravenous administration. 13–197 is well distributed to the peripheral tissues and has relatively high tissue–plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13–197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13–197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of monosaccharides through multiple‐reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column

A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple‐reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino‐bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH₃CO₂]^? using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M–H]^?. The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N‐linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 µg of glycoprotein and 5 µL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

Micro-high-performance liquid chromatography platform for the depletion of high-abundance proteins and subsequent on-line concentration/capturing of medium and low-abundance proteins from serum. Application to profiling of protein expression in healthy and osteoarthritis sera by 2-D gel electrophoresis

In this investigation, an integrated microcolumn-based fluidic platform for the simultaneous depletion of high-abundance proteins and the subsequent on-line concentration/capturing of medium- and low-abundance proteins from human serum has been introduced. The platform consists of on-line coupling of tandem affinity micorcolumns to an RP microcolumn to achieve first the depletion of high-abundance proteins by the tandem affinity microcolumns followed by the concentration and capturing of medium- and low-abundance proteins by the RP microcolumn. The tandem affinity microcolumns are based on macroporous monoliths characterized by their relatively high permeability in pressure-driven flow while the RP microcolumn is packed with polymeric particles with an average particle diameter of 20 microm giving rise to a very little back pressure, thus allowing fast flow velocity across the coupled columns format and consequently short processing time of serum samples prior to analysis by 2-DE. The microcolumn-based fluidic platform was applied to serum samples from osteoarthritis (OA) donors before and after soy protein (SP) supplementation, and from healthy donors, and the resulting depleted serum samples from high-abundance proteins were profiled for protein expression by 2-DE. In general, the protein expression was lower in serum of the same OA patient after soy treatment than before soy treatment. Several proteins were down-regulated after soy treatment with transthyretin being the most affected by the SP supplementation. In addition, with respect to serum from healthy donors, the sera from OA patients showed difference in proteins expression.     

Simultaneous determination of zidovudine and lamivudine from rat plasma, amniotic fluid and tissues by HPLC

A sensitive HPLC method has been developed and validated for the simultaneous quantification of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid and placental and fetal tissues. Samples were processed by solid-phase extraction using C2 cartridges. Chromatography was performed using a phenyl column (5 microm, 150 x 2 mm i.d.) under a flow rate of 0.2 mL/min. The mobile phase consisted of 8% acetonitrile in 5 mM 1-heptane sulfonic acid dissolved in 30 mM ammonium formate buffer (pH 3.3). The method was validated in the range 0.25-50 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport of co-administration of these compounds in a pregnant rat.