Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chrom B )

Publisher: Elsevier


The Journal of Chromatography B. addresses advancements in and applications of analytical methodologies related to drugs, other pharmacologically active compounds, metabolites, as well as to bio-polymers such as proteins and nucleic acids. The areas considered include: clinical analysis, therapeutic drug monitoring, pharmaceutical analysis and novel approaches to sample preparation in bioanalysis; the qualitative and quantitative analysis of proteins, peptides and their post-translational modifications as well as nucleic acids; the screening and profiling of body fluids and tissues related to monitoring the level of active substances, including metabolites, biomarkers and toxicants; the comparative analysis of biological systems and proteomics and genomics including novel ways of data handling and interpretation; analytical techniques covered include the various facets of chromatography, electrophoresis and related methods, including mass spectrometry and affinity-based methodologies.

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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: An LC–MS/MS method for the determination of BMS-791325 and its metabolite in plasma.•A systematic method optimization strategy was applied during method development.•The results demonstrate that the method is accurate, precise, and reproducible.•The validated method was used to support multiple preclinical toxicokinetic studies.•The first reference for measuring the drug concentration in biological matrices.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 12/2014; 973:1-8.
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    ABSTRACT: Monomeric molecules such as amyloid-β can aggregate and transform into oligomeric and fibrous forms, which are implicated in the development and progression of Alzheimer's disease. Novel analytical techniques for the formation of oligomers are required to examine the neurotoxic amyloid-β oligomers involving fibrils. After isolating amyloid-β monomer 1-42 using a biotinylated antibody bound to membrane-immobilized avidin (immunoaffinity membrane), their masses were determined by MALDI-TOF MS. Fluorometric determination of more than 0.5μM of aggregated amyloid-β in pipette droplets was performed after aggregation and dilution of 1mM amyloid-β. Thus, large (>105nm) amyloid-β oligomers in microliter volumes of fluids were isolated using the immunoaffinity membrane and quantitatively analyzed after removal of amyloid-β monomers and small oligomers by non-denaturing electrophoresis. In addition, amyloid-β oligomers were specifically isolated from a mixture of human plasma and aggregated amyloid-β and then fluorometrically analyzed. Our results show that the combination of immunoaffinity membrane-binding and fluorescence determinations together with one drop analysis could be used to isolate and detect huge neurotoxic amyloid-β oligomers such as fibrils in plasma samples.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2014; 972C:53-57.
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    ABSTRACT: Reported homocysteine (HCY) concentrations in human serum show poor concordance amongst laboratories due to endogenous HCY in the matrices used for assay calibrators and QCs. Hence, we have developed a fully validated LC-MS/MS method for measurement of HCY concentrations in human serum samples that addresses this issue by minimising matrix effects. We used small volumes (20μL) of 2% Bovine Serum Albumin (BSA) as surrogate matrix for making calibrators and QCs with concentrations adjusted for the endogenous HCY concentration in the surrogate matrix using the method of standard additions. To aliquots (20μL) of human serum samples, calibrators or QCs, were added HCY-d4 (internal standard) and tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) as reducing agent. After protein precipitation, diluted supernatants were injected into the LC-MS/MS. Calibration curves were linear; QCs were accurate (5.6% deviation from nominal), precise (CV%≤9.6%), stable for four freeze-thaw cycles, and when stored at room temperature for 5h or at -80°C (27 days). Recoveries from QCs in surrogate matrix or pooled human serum were 91.9 and 95.9%, respectively. There was no matrix effect using 6 different individual serum samples including one that was haemolysed. Our LC-MS/MS method has satisfied all of the validation criteria of the 2012 EMA guideline.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2014; 972C:14-21.
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    ABSTRACT: Dexpramipexole (DEX) was being investigated in clinical studies for the treatment of amyotrophic lateral sclerosis (ALS). To monitor the potential chiral interconversion of dexpramipexole to pramipexole (PPX) in vivo, a highly sensitive and selective chiral LC-MS/MS assay was developed and qualified for the detection of pramipexole in the presence of dexpramipexole in human plasma. In this assay, plasma samples were extracted by protein precipitation coupled with solid phase extraction (SPE). The analyte PPX was separated from its enantiomer DEX using a chiral HPLC method. The assay was qualified with a dynamic range of 0.150-1.00ng/mL. The lower limit of quantitation (LLOQ) for PPX was 0.150ng/mL in the presence of up to 1000ng/mL of DEX. The qualified method was used to analyze plasma samples from a DEX clinical study. No PPX was detected in humans at pharmacologically significant levels after administration of dexpramipexole at single doses up to 600mg per day.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:133-140.
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    ABSTRACT: The simultaneous analysis of free-form and conjugated flavonoids in the same sample is difficult but necessary to properly estimate their bioavailability. A method was developed to optimise the extraction of both free and conjugated forms of catechins and metabolites in a biological sample following the consumption of green tea. A double-blind randomised controlled trial was performed in which 26 volunteers consumed daily green tea and vitamin C supplements and 24 consumed a placebo for 3 months. Urine was collected for 24h at 4 separate time points (pre- and post-consumption) to confirm compliance to the supplementation and to distinguish between placebo and supplementation consumption. The urine was assessed for both free and conjugated metabolites of green tea using LC-MS(2) analysis, after a combination extraction method, which involved an ethyl acetate extraction followed by an acetonitrile protein precipitation. The combination method resulted in a good recovery of EC-O-sulphate (91±7%), EGC-O-glucuronide (94±6%), EC (95±6%), EGC (111±5%) and ethyl gallate (74±3%). A potential total of 55 catechin metabolites were investigated, and of these, 26 conjugated (with methyl, glucuronide or sulphate groups) and 3 free-form (unconjugated) compounds were identified in urine following green tea consumption. The majority of EC and EGC conjugates significantly increased post-consumption of green tea in comparison to baseline (pre-supplementation) samples. The conjugated metabolites associated with the highest peak areas were O-methyl-EC-O-sulphate and the valerolactones M6/M6'-O-sulphate. In line with previous studies, EC and EGC were only identified as conjugated derivatives, and EGCG and ECG were not found as mono-conjugated or free-forms. In summary, the method reported here provides a good recovery of catechin compounds and is appropriate for use in the assessment of flavonoid bioavailability, particularly for biological tissues that may contain endogenous deconjugating enzymes.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 972C:29-37.
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    ABSTRACT: A new process was developed for the selective extraction and pre-concentration of amitriptyline (AT) from human plasma using nano-sized molecularly imprinted polymer (MIP) with ultrasound-assisted extraction (UAE). The nano-sized AT imprinted polymer particles were synthesized using suspension polymerization in silicon oil and characterized by Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscope (SEM) methods. With the application of optimized values, linearity values in the ranges of 20-200μgmL(-1) and 35-200μgmL(-1) were obtained for AT with the correlation of determination values (r(2)) 0.998 and 0.995 in water and plasma, respectively. The limits of detections (S/N=3) for AT were found to be 0.7 and 1.2μgmL(-1) in water and plasma, respectively. The enrichment factors of AT in water and plasma were 52 and 40, respectively. The inter-day precisions (%) were in the range of 5.8-9.2%. Relative recovery rates ranged from 82.4% to 92.3%. The method was successfully applied to determine AT in the human plasma samples.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 972C:6-13.
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    ABSTRACT: A new method based on liquid chromatography-tandem time-of-flight mass spectrometry was developed to identify the metabolites in rat urine after oral administration of YiGan San (YGS). Eighteen prototype compounds and four metabolites named 11-hydroxyhirsuteine, 19-carbonylhirsutine, 19-carbonyl-dihydrocorynantheine, and 18-hydroxy-geissoschizine methyl ether were identified. Subsequently, a method of high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry was established for pharmacokinetic study of YGS in rat plasma. The concentration-time curves of four prototype compounds, senkyunolide I, ajmalicine, isocorynoxeine and rhynchophylline were constructed after an oral (9.1g YGS per kilogram of body weight) administration in rats. Method validation revealed excellent linearity over the range 220.00-0.55, 220.00-0.55, 21.40-0.05, and 19.80-0.05ng/mL for the four prototype compounds respectively. The stabilities results indicate that all of the analytes were stable in rat plasma in the autosampler for 24h, under freeze/thaw cycles (4 times in 24h), and at -20°C for one week. Residual analysis, heteroskedasticity test, and goodness-of-fit test were also performed to determine the accuracy of the linear regression method. The pharmacokinetic parameters were obtained. Four hours after administration, compound 11-hydroxyhirsuteine can be detected in rat plasma. Compared with purified ligustilide, YGS required a slightly longer period to reach maximum concentration (Cmax) in rat plasma.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 972C:22-28.
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    ABSTRACT: A detailed study of individual Aβ-interacting proteins has always been a difficult task because Aβ has a wide range of molecular weights and can easily form aggregates. In this study, we established a novel method for isolating Aβ-interacting proteins by utilizing regular comb polymer immobilized on Sepharose CL-4B. To achieve site-directed ligation of Aβ, a cysteine residue was added at the N-terminus of Aβ. Asp and Asp12, which have 2 and 13 carboxyl groups, respectively, were selected as the carriers for the regular comb polymer. Firstly, the N-termini of Asp and Asp12 were immobilized on Sepharose CL-4B. Next, modified Aβ molecules were coupled to the carboxyl groups of Asp and Asp12 using bromoethylamine as a spacer. To obtain homogeneous comb polymer, the efficiency of the reaction was controlled during the synthesis process. Thioflavin T staining indicated that homogeneous Aβ was achieved. The prepared Aβ-adsorbents were used to isolate Aβ-interacting proteins from mice brain extracts. The results showed that the adsorption capacity of the Aβ-adsorbents for proteins in mice brain extracts increased with the ages of the animals. SDS-PAGE analysis of the Aβ-interacting proteins showed that many kinds of brain proteins were selectively adsorbed by the Aβ adsorbents, and the levels of some of these proteins varied with the ages of the animals. The results indicated that Aβ-interacting proteins could be successfully obtained through the use of immobilized comb polymer. Similar method could also be used to isolate other amyloid-interacting proteins.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:94-98.
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    ABSTRACT: A sensitive and selective high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the simultaneous determination of kudinoside A, kudinoside D and kudinoside F in human plasma has been firstly developed. Samples were prepared after protein precipitation and analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. Negative electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (35:65) at the flow rate of 0.3mL/min. The analytes and internal standard Ginsenoside Rb1 were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 2.5-1000.0ng/mL. The lower limit of quantification (LLOQ) was 2.5ng/mL. The intra-and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.4%. The accuracy determined at three concentrations was within ±4.9% in terms of relative error. The total run time was 7.0min. This assay offers advantages in terms of expediency, and suitability for the analysis of kudinoside A, kudinoside D and kudinoside F in various biological fluids.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 972C:1-5.
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    ABSTRACT: Deng-yan granule, consisting of Herba Erigerontis Breviscapi, Rhizoma Corydalis Yanhusuo and Radix Astragali Mongolici, is a widely used Traditional Chinese Medicine preparation for treatment of coronary heart disease. Scutellarin and tetrahydropalmatine are main active constituents in Herba Erigerontis Breviscapi and Rhizoma Corydalis Yanhusuo, and have been used as marker components for quality control of Deng-yan preparations. In order to make good and rational use of Deng-yan granule in the future, a rapid, sensitive and high throughput ultra-fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) method was developed for the simultaneous determination of scutellarin and tetrahydropalmatine in rat plasma using rutin as internal standard (IS). The plasma samples were extracted by liquid-liquid extraction with ethyl acetate after acidification and separated on a Shim-pack XR-ODS C18 column (75mm×3.0mm, 2.2μm) with a mobile phase consisting of methanol-0.1% formic acid water (50:50, v/v) at a flow rate of 0.4mL/min. Mass spectrometric detection was conducted on an API 3200 QTRAP mass spectrometry equipped with electrospray ionization source in positive ionization mode. Quantification was performed using multiple reaction monitoring (MRM) by monitoring the fragmentation of m/z 463.2→287.1 for scutellarin, m/z 356.1→192.1 for tetrahydropalmatine and m/z 611.2→303.2 for IS, respectively. The linear range was 10-5000ng/mL for both scutellarin and tetrahydropalmatine with lower limit of quantitation (LLOQ) of 10ng/mL. The intra- and inter-day precisions were below 12.2% for scutellarin and below 9.7% for tetrahydropalmatine in terms of relative standard deviation (RSD), and the accuracy was within ±9.1% for scutellarin and within ±11.2% for tetrahydropalmatine in terms of relative error (RE). Extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of scutellarin and tetrahydropalmatine after oral administration of Deng-yan granule to rats.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:126-132.
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    ABSTRACT: An ultra high-pressure liquid chromatography/mass spectrometry (UHPLC/MS) separation and analysis method has been devised for open access analysis of synthetic reactions used in the production of DNA-encoded chemical libraries. The aqueous mobile phase is 100mM hexafluoroisopropanol and 8.6mM triethylamine; the organic mobile phase is methanol. The UHPLC separation uses a C18 OST column (50mm×2.1mm×1.7μm) at 60°C, with a flow rate of 0.6mL/min. Gradient concentration is from 10 to 40% B in 1.0min, increasing to 95% B at 1.2min. Cycle time was about 5min. This method provides a detection limit of a 20-mer oligonucleotide by mass spectrometry of better than 1pmol on-column. Linear UV response for 20-mer extends from 2 to 200pmol/μL in concentration, same-day relative average deviations are less than 5% and bias (observed minus expected) is less than 10%. Deconvoluted mass spectra are generated for components in the predicted mass range using a maximum entropy algorithm. Mass accuracy of deconvoluted spectra is typically 20ppm or better for isotopomers of oligonucleotides up to 7000Da.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:120-125.
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    ABSTRACT: Methyl-tert-butyl ether (MTBE) is widely used as an antiknock additive for increasing octane number of gasoline. Recently, the in vivo studies demonstrated that MTBE has genotoxic potential and able to form adducts with DNA. In the work, the interactions of MTBE with calf thymus DNA (ct-DNA) and the Na(+) form of G-quadruplex DNA (wtTel22) were studied by using of head space-solid phase microextraction technique coupled to gas chromatography. The binding equilibrium constants were measured through the equilibriums of a four phase system. In addition, the MTBE Henry's law constants for two different buffers in the temperature range of 283-303K were measured. Thermodynamic studies revealed that the complexation of MTBE to both DNAs is enthalpy favored and entropy disfavored. The thermodynamic results revealed that MTBE may have interaction with ct-DNA via the minor groove of DNA. Also, MTBE may be complexed into the basket of G-quadruplex structure. In addition, the low difference in the binding constants of MTBE for both different DNA targets may confirm that MTBE is poorly selective for different conformations of DNA.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:112-119.
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    ABSTRACT: A rapid and sensitive ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ/MS) method was developed for simultaneous quantification of Akebia saponin D (ASD) and its five metabolites in intestinal mixtures of bacteria from human feces. After protein precipitation, the analytes and internal standard (IS), glycyrrhetinic acid, were determined in selected ion recording (SIR) mode with negative ion ESI source. Chromatographic separation was carried out on an ACQUITY UPLC™ BEH C18 column (100mm×2.1mm, 1.7μm) using gradient elution. The mobile phase consisted of solvents A (acetonitrile) and B (0.1% aqueous formic acid) at the flow rate of 0.4mL/min. Each sample was chromatographed within 10.5min including equilibration time. The linearity ranged from 0.1 to 100μg/mL for ASD, and 2-1000ng/mL for five metabolites, Dipsacus saponin A (M1), HN-saponin F (M2), hederagenin-28-O-β-d-glucopyranoside (M3), Akebia saponin PA (M4), hederagenin (M5). The limits of detection (LOD) were 0.41, 0.59, 0.61, 0.55, 0.52 and 0.31ng/mL for ASD, M1, M2, M3, M4 and M5, respectively. The intra- and inter-day precision was all within 11.1% and accuracy ranged from -8.33% to 12.47%. The conversion rate of five metabolites was 41.21% in 24h. The method was validated and successfully applied to quantification of ASD and its five metabolites in human intestinal bacteria.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:81-88.
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    ABSTRACT: An ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of zopiclone and zolpidem in whole blood, for use in cases with suspected driving under influence of drugs (DUID) and autopsy cases. Sample preparation was performed with liquid-liquid extraction (LLE) using ethyl acetate/n-heptane (80:20, v/v) and 0.1mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7μm), with a flow rate of 0.5mL/min, 1μL injected and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode. The run time of the method was 4.5min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R(2) values above 0.999 for both compounds. Intermediate precision and accuracies (bias) were 2.4-12.9% RSD and from -5.9 to 6.8%, respectively. Recoveries of the compounds were ≥70%. The lower limit of quantification (LLOQ) for zopiclone was 0.50nmol/L (0.19ng/mL) or 0.05pg injected on column, and 3.5nmol/mL (1.10ng/mL) for zolpidem, or 0.27pg injected on column. The limit of detection (LOD) was 0.2nmol/L (0.08ng/mL) for zopiclone and 0.3nmol/L (0.09ng/mL) for zolpidem. Matrix effects (ME) were between 108 and 115% when calculated against IS. A comparison with former confirmation LC-MS method at the Norwegian Institute of Public Health, Division of Forensic Medicine (NIPH) was performed during method validation. Good correlation was seen for both compounds. The method has been running on a routine basis for two years, and has proven to be very robust and reliable with satisfactory long term precision and bias and with results for external quality samples corresponding well to consensus mean or median. Zopiclone and zolpidem concentrations in post mortem and ante mortem cases were reported. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs introduced in the Norwegian Road Traffic Act Law from 2012.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:72-80.
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    ABSTRACT: Rebaudioside A (RA) and stevioside (SS) are the primary effective glycoside components in Stevia Rebaudiana. The RA glycoside is sweeter, and it tastes similarly to sucrose. Because extracts with a high RA content can be used as natural sweeteners for food additives approved by the FAO and FDA, RA should generate high market demand. In this study, an efficient method for separating RA was established based on the synergistic multi-hydrogen bonding interaction between a polymeric adsorbent and the RA glycoside. To overcome the destruction of the hydrophobic affinity required for the selective adsorption of RA, an innovative non-aqueous environment was established for adsorption and separation. To this end, an initial polymeric adsorbent composed of a glycidyl methacrylate and trimethylolpropane trimethacrylate (GMA-co-TMPTMA) copolymer matrix was synthesized, and polyethylene polyamine was employed as a functional reagent designed to react with the epoxy group on GME-co-TMPTMA to form a highly selective macroporous adsorbent. The effects of the different functional reagents and the solvent polarity on the adsorption selectivity for RA and SS, respectively, were investigated. Matching the structure of the polyethylene polyamine and sugar ligand on the glycoside molecule was essential in ensuring that the maximum synergistic interaction between adsorbent and adsorbate would be achieved. Moreover, the hydrogen-bonding force was observed to increase when the polarity of the adsorption solvent decreased. Therefore, among the synthesized macroporous polymeric adsorbents, the GTN4 adsorbent-bonding tetraethylenepentamine functional group provided the best separation in an n-butyl alcohol solution. Under the optimized gradient elution conditions, RA and SS can be effectively separated, and the contents of RA and SS increased from 33.5% and 51.5% in the initial crude extract to 95.4% and 78.2% after separation, respectively. Compared to conventional methods, the adsorption-desorption process is more advanced due to its procedural simplicity, low cost and adaptability for industrial production.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 971C:141-149.
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    ABSTRACT: The present study describes a novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous estimation of ramipril (RAM) and hydrochlorothiazide (HCTZ) in human plasma using liquid-liquid extraction technique. This method made use of electrospray ionization in positive mode for RAM and in negative mode for HCTZ using triple quadrupole mass spectrometry where carbamazepine was used as an internal standard (IS). Analytes were recovered by methyl tertiary butyl ether:dichloromethane (85:15) subsequently separated on an Enable C18 G column (150mm×4.6mm, 5μm) using methanol:0.1% formic acid in water (85:15) as a mobile phase, at a flow rate of 0.5mL/min. Quantification of RAM, HCTZ and IS was performed using multi-reaction monitoring mode (MRM) where transition of m/z 417.2→234.1 (RAM) and 237.0→194.0 (IS) in positive mode and 296.1→205.0 for HCTZ in negative mode. The calibration curve was linear (r(2)>0.99) over the concentration range of 2-170ng/mL for RAM and 8-680ng/mL for HCTZ. The intra-day and inter-day precisions were <15% and the accuracy was all within ±15% (at LLOQ level ±20%). Additionally, the LC-MS/MS method was fully validated for all the other parameters such as selectivity, matrix effect, recovery and stability as well. In conclusion, the findings of the present study revealed the selectivity and sensitivity of this method for the simultaneous estimation of RAM and HCTZ in human plasma.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 970C:53-59.
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    ABSTRACT: We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 970C:60-67.
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    ABSTRACT: Accurate measurement of aldosterone is important for the standardized testing of primary hyperaldosteronism. Commercial immunoassays show substantial between-method variations resulting in significant clinical consequences. We developed a specific two dimensional (2D)-LC-MS/MS method for measuring aldosterone in human serum and plasma and compared it with three commercial immunoassays and an LC-MS/MS method.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 970C:102-107.
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    ABSTRACT: Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC-MS/MS method for quantitation of mithramycin in human plasma was developed. Solid-phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ∼55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1×50mm, 1.7μm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5→268.9 and 1181.5→269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5-500ng/mL and proved to be linear (r(2)>0.996), accurate (≤10% deviation), and precise (CV<15%). Mithramycin was stable in plasma at room temperature for 24h, as well as through three freeze-thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2014; 970C:95-101.