Simple and sensitive liquid chromatography-tandem mass spectrometry methods for quantification of paraquat in plasma and urine: Application to experimental and clinical toxicological studies

Publication year: 2011Source: Journal of Chromatography B, Available online 10 September 2011Klintean Wunnapuk, Gregory A. Medley, Xin Liu, Jeffrey E. Grice, Sudheera Jayasinghe, …Simple, sensitive and specific liquid chromatography-tandem mass …

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 September 2011

Klintean Wunnapuk, Gregory A. Medley, Xin Liu, Jeffrey E. Grice, Sudheera Jayasinghe, ...

Simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for quantification of paraquat (PQ) in plasma and urine. Plasma and urine sample preparation were carried out by one-step protein precipitation using cold acetronitrile (-20 to -10 °C). After centrifugation, an aliquot of 10 μl of supernatant was injected into a Kinetex™ hydrophilic interaction chromatography (HILIC) column with a KrudKatcher™ Ultra in-line filter. The chromatographic separation was achieved using the mobile phase mixture of 250 mM ammonium formate (with 0.8% aqueous formic acid) in water and acetonitrile at a flow rate of 0.3 mL/min. Detection was performed using an API2000 triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear over the concentration range of 10 to 5,000 ng/mL, with an LLOQ of 10 ng/mL. The inter- and intra-day precision (% R.S.D.) were <8.5% and 6.4% for plasma and urine, respectively with the accuracies (%) within the range of 95.1 to 102.8%. PQ in plasma and urine samples was stable when stored at -70 °C for three freeze-thaw cycles. The methods were successfully applied to determine PQ concentration in rat and human samples.

Highlights

► Simple sample preparation with a one-step protein precipitation ► Analytical separation was achieved by using the HILIC silica column and ion-pair reagents are not required for the method ► The LLOQ of the method was 10 ng/ml in both plasma and urine samples with acceptable precision and accuracy ► Method was successfully applied in clinical and animal studies


Quantitative urine amino acid analysis using liquid chromatography tandem mass spectrometry and aTRAQreagents

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2695-2703Patrice K. Held, Laura White, Marzia PasqualiIon-exchange chromatography (IEC) is the most widely used method for amino acid analysis …

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2695-2703

Patrice K. Held, Laura White, Marzia Pasquali

Ion-exchange chromatography (IEC) is the most widely used method for amino acid analysis in physiological fluids because it provides excellent separation and reproducibility, with minimal sample preparation. The disadvantage, however, is the long analysis time needed to chromatographically resolve all the amino acids. To overcome this limitation, we evaluated a novel liquid chromatography tandem mass spectrometry (LC–MS/MS) method, which utilizes aTRAQreagents, for amino acid analysis in urine. aTRAQreagents tag the primary and secondary amino groups of amino acids. Internal standards for each amino acid are also labeled with a modified aTRAQtag and are used for quantification. Separation and identification of the amino acids is achieved by liquid chromatography tandem mass spectrometry using retention times and mass transitions, unique to each amino acid, as identifiers. The run time, injection-to-injection, is 25 min, with all amino acids eluting within the first 12 min. This method has a limit of quantification (LOQ) of 1 μmol/L, and is linear up to 1000 μmol/L for most amino acids. The Coefficient of Variation (CV) was less than 20% for all amino acids throughout the linear range. Method comparison demonstrated concordance between IEC and LC–MS/MS and clinical performance was assessed by analysis of samples from patients with known conditions affecting urinary amino acid excretion. Reference intervals established for this method were also concordant with reference intervals obtained with IEC. Overall, aTRAQreagents used in conjunction with LC–MS/MS should be considered a comparable alternative to IEC. The most attractive features of this methodology are the decreased run time and increased specificity.

Simultaneous quantification of VX and its toxic metabolite in blood and plasma samples and its application forin vivoandin vitrotoxicological studies

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2704-2713Georg Reiter, John Mikler, Ira Hill, Kendal Weatherby, Horst Thiermann, …The present study was initiated to develop a sensitive an…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2704-2713

Georg Reiter, John Mikler, Ira Hill, Kendal Weatherby, Horst Thiermann, ...

The present study was initiated to develop a sensitive and highly selective method for the simultaneous quantification of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) and its toxic metabolite (EA-2192) in blood and plasma samplesin vivoandin vitro. For the quantitative detection of VX and EA-2192 the resolution was realized on a HYPERCARB HPLC phase. A specific procedure was developed to isolate both toxic analytes from blood and plasma samples. The limit of detection was 0.1 pg/ml and the absolute recovery of the overall sample preparation procedure was 74% for VX and 69% for EA-2192. After intravenous and percutaneous administration of a supralethal doses of VX in anaesthetised swine both VX and EA-2192 could be quantified over 540 min following exposure. This study is the first to verify thein vivoformation of the toxic metabolite EA-2192 after poisoning with the nerve agent VX. Further toxicokinetic and therapeutic studies are required in order to determine the impact of EA-2192 on the treatment of acute VX poisoning.

Ultra-performance liquid chromatography–tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2714-2719Yanwen Liu, Ronghua Zhu, Huande Li, Miao Yan, Yanqing LeiA selective, simple and efficient method-ultra-performance liquid chromatog…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2714-2719

Yanwen Liu, Ronghua Zhu, Huande Li, Miao Yan, Yanqing Lei

A selective, simple and efficient method-ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C18column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48–496.4 ng/ml for strychnine and 2.64–528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucinein vivo.

Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2720-2725Jianxing Zhao, Hong Chen, Peihua Ni, Bingxin Xu, Xuemei Luo, …A high performance liquid chromatography method with ultraviolet and…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2720-2725

Jianxing Zhao, Hong Chen, Peihua Ni, Bingxin Xu, Xuemei Luo, ...

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze–thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1 mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r) > 0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0–106.4%; Kyn, 97.9–106.9%; Kyna, 98.5–105.6%; Trp, 96.7–105.2% and 5-HIAA, 96.1–99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.

Chromatographic Methods for the Determination of Therapeutic Oligonucleotides

Publication year: 2011Source: Journal of Chromatography B, Available online 10 September 2011A. Cary McGinnis, Buyun Chen, Michael BartlettBoth DNA and RNA are being explored for their therapeutic potential against a wide range of diseases. As these…

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 September 2011

A. Cary McGinnis, Buyun Chen, Michael Bartlett

Both DNA and RNA are being explored for their therapeutic potential against a wide range of diseases. As these new drugs emerge, new demands arise for the analysis and quantitation of these biomolecules. Pharmacokinetic and pharmacodynamic analysis requirements for drug approval place enormous challenges on the methods for analyzing these therapeutics. This review will focus on bioanalytical methods for DNA antisense and aptamers as well as small-interfering RNA (siRNA) therapeutics. Chromatography methods employing ultraviolet (UV), fluorescence and mass spectrometric (MS) detection along with Matrix-Assisted Laser Desorption/Ionization (MALDI) will be covered. Sample preparation from biological matrices will be reviewed as well as metabolite analysis and identification. All of these techniques are important contributions toward oligonucleotide therapeutic development. They will also be important in microRNA (miRNA biomarker discovery and RNomics in general, as more non-coding RNAs are inevitably discovered.

A quantitative LC–MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2726-2732Mark S. Lowenthal, Hugo Gasca-Aragon, John E. Schiel, Nathan G. Dodder, David M. BunkA mass spectrometry-based antibody selection pr…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2726-2732

Mark S. Lowenthal, Hugo Gasca-Aragon, John E. Schiel, Nathan G. Dodder, David M. Bunk

A mass spectrometry-based antibody selection procedure was developed to evaluate optimal ‘capture’ monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC–MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (Kd) were determined for ‘bound mAb–antigen’ vs. ‘unbound antigen’ using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal ‘capture’ mAbs were determined through evaluation of relativeKdconstants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalentKdconstants as determined using NIST Standard Reference Material (SRM) 2921 – Human Cardiac Troponin Complex. This ID LC–MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification.

Quantitative liquid chromatographic analysis of anthracyclines in biological fluids

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2471-2486Kristof E. Maudens, Christophe P. Stove, Willy E. LambertAnthracyclines are amongst the most widely used drugs in oncology, being part …

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2471-2486

Kristof E. Maudens, Christophe P. Stove, Willy E. Lambert

Anthracyclines are amongst the most widely used drugs in oncology, being part of the treatment regimen in most patients receiving systemic chemotherapy. This review provides a comprehensive summary of the sample preparation techniques and chromatographic methods that have been developed during the last two decades for the analysis of the 4 most administered anthracyclines, doxorubicin, epirubicin, daunorubicin and idarubicin in plasma, serum, saliva or urine, within the context of clinical and pharmacokinetic studies or for assessing occupational exposure. Following deproteinization, liquid–liquid extraction, solid phase extraction or a combination of these techniques, the vast majority of methods utilizes reversed-phase C18 stationary phases for liquid chromatographic separation, followed by fluorescence detection, or, more recently, tandem mass spectrometric detection. Some pros and cons of the different techniques are addressed, in addition to potential pitfalls that may be encountered in the analysis of this class of compounds.

Simultaneous determination of six phthalate esters in bottled milks using ultrasound-assisted dispersive liquid–liquid microextraction coupled with gas chromatography

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2507-2512Hongyuan Yan, Xiaoling Cheng, Baomi LiuA new method was developed for the simultaneous determination of six phthalate esters in bottled…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2507-2512

Hongyuan Yan, Xiaoling Cheng, Baomi Liu

A new method was developed for the simultaneous determination of six phthalate esters in bottled milks using ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME) followed by gas chromatography–flame ionization detection (GC–FID). 0.8 mL of methanol (dispersant) and 40 μL of CCl4(extractant) were injected into 8.0 mL of milk solution and then emulsified the mixture by ultrasound for 2.0 min to form the cloudy solution. Under the optimum condition, the enrichment factors of the analytes ranged from 220 to 270 fold and the recovery ranged from 93.2% to 105.7%. Good linearity was observed for all analytes in a range of 0.8–51 ng gwith the correlation coefficient (r) ≥0.9992. The limits of detection (LODs) based on signal to noise of 3 were 0.64–0.79 ng g. The repeatability evaluated as intra-day and inter-day precision (relative standard deviation, RSD) were less than 4.0% (n = 5). The presented UA-DLLME–GC–FID method was successfully applied to determine the six phthalate esters in different bottled milk products.

Quantitative analysis ofmyo-inositol in urine, blood and nutritional supplements by high-performance liquid chromatography tandem mass spectrometry

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2759-2763Kit-Yi Leung, Kevin Mills, Katie A. Burren, Andrew J. Copp, Nicholas D.E. GreeneMyo-inositol plays key physiological functions, nece…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2759-2763

Kit-Yi Leung, Kevin Mills, Katie A. Burren, Andrew J. Copp, Nicholas D.E. Greene

Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression ofmyo-inositol signals, for example in blood or urine samples. We describe an HPLC–MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection ofmyo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed.

Conformational changes and bioactivity of lysozyme on binding to and desorption from magnetite nanoparticles

Publication year: 2011Source: Journal of Chromatography B, Available online 10 September 2011Jun Sun, Rongrong Xu, Yanjun YangA fundamental understanding of the conformational behaviors of lysozyme during the process of adsorption and desorption has…

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 September 2011

Jun Sun, Rongrong Xu, Yanjun Yang

A fundamental understanding of the conformational behaviors of lysozyme during the process of adsorption and desorption has been studied using spectrophotometric techniques, and interpreted in terms of the secondary structures in this work. FTIR data show an increase inα-helix andβ-sheet content when lysozyme interaction with magnetite nanoparticles (Fe3O4(PEG + CM-CTS) NPs) which indicates that the lysozyme would adopt a more compact conformation state. The mechanism of fluorescence quenching of lysozyme by magnetite nanoparticles is due to the formation of lysozyme–nanoparticles complex. High desorption of lysozyme from Fe3O4(PEG + CM-CTS) NPs were achieved using phosphate buffer solution (PBS) (20 mM, pH 5.0, 0.2 M NaCl), PBS (20 mM, pH 5.0, 0.5 M NaCl) and acetic acid (0.2 M, pH 4.0) as eluents. The alterations of lysozyme secondary structure on desorption from nanoparticles were confirmed by circular dichroism and fluorescence spectroscopy. Lysozymes desorbed by PBS (20 mM, pH 5.0, 0.2 M NaCl) and PBS (20 mM, pH 5.0, 0.5 M NaCl) retain high fraction of its native structure with negligible effect on its activity, and about 92.4% and 89.5% activity were retained upon desorption from nanoparticles, however, lysozyme desorbed by acetic acid (0.2 M, pH 4.0) solution showed significant conformational changes. The stability of NPs-conjugated protein and retention of higher activity may find useful applications in biotechnology ranging from enzyme immobilization to protein purification.

Highlights

► ► Fundamental understanding of the conformational behavior of lysozyme during the process of adsorption/desorption has been studied using spectrophotometric techniques. ► On the surface of magnetite nanoparticles, the lysozyme would adopt a more compact conformation state. ► The mechanism of fluorescence quenching of lysozyme by magnetite nanoparticles is due to the formation of lysozyme–nanoparticles complex. ► The alterations of lysozyme secondary structure on desorption from nanoparticles were confirmed by circular dichroism and fluorescence spectroscopy. ► Fe3O4(PEG + CM-CTS) NPs is capable of preserving the structure and bioactivity of lysozyme.


Simultaneous free and glycosylated pyridinium crosslink determination in urine: Validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2764-2771Elena Monticelli, Caroline Stéphanie Aman, Maria Letizia Costa, Paola Rota, Daniela Bogdan, …Pyridinoline (Pyr), deoxypyridinolin…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2764-2771

Elena Monticelli, Caroline Stéphanie Aman, Maria Letizia Costa, Paola Rota, Daniela Bogdan, ...

Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl–galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free + bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n = 20, aged 27–41) and girls (n = 20, aged 5–10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p < 0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (<LLOQ, <21.20 pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.

REVERSED PHASE LIQUID CHROMATOGRAPHY METHOD WITH FLUORESCENCE DETECTION OF GEMIFLOXACIN IN RAT PLASMA AND ITS APPLICATION TO THE PHARMACOKINETIC STUDY

Publication year: 2011Source: Journal of Chromatography B, Available online 10 September 2011Moacir Kaiser, Lauren D. Grünspan, Teresa Dalla Costa, Leandro TassoA simple, accurate and precise high-performance liquid chromatographic method with flu…

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 September 2011

Moacir Kaiser, Lauren D. Grünspan, Teresa Dalla Costa, Leandro Tasso

A simple, accurate and precise high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin (GEM) in rat plasma using furosemide as internal standard (I.S.). Plasma samples were pretreated by direct deproteinization and all samples and standard solutions were chromatographed at 45 °C using triethylamine solution (0.5% v/v, pH 3.0 ± 0.1), methanol and acetonitrile (63:30:7, v/v/v) as the mobile phase. Chromatographic resolution was achieved using a RP-C18column (Atlantis, Waters, 150 mm x 4.6 mm, 5 μm) at a flow rate of 1.0 mLminand an injection volume of 30 μL. The analytes were measured by fluorescence detection with excitation and emission wavelengths of 344 nm and 399 nm, respectively. The retention times for GEM and I.S. were approximately 7.5 and 12.6 min, respectively. The lower limit of quantitation (LLOQ) was 20 ngmLand the calibration curves were linear over a concentration range of 20 - 5000 ngmL. The intra and inter-day precision, expressed by relative standard deviation (R.S.D.) were lower than 6.24% and 4.49%, respectively. The accuracy ranged from 91.3% to 112% and from 98.8% to 106% for the lower and upper limit of quantitation of the calibration curve, respectively. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. No interferences from endogenous substances were found. The recovery of GEM and I.S. from plasma was greater than 90%. Drug stability in plasma was shown at room temperature for 4 h, after three freeze-thaw cycles for 24 h, in freezer at -80 °C for 60 days, and in the autosampler after processing for 12 h. The utility of the assay was confirmed by the successfully analysis of plasma samples from GEM pharmacokinetics studies in the rats after intravenous administration.

Comprehensive analysis of the intracellular metabolism of antiretroviral nucleosides and nucleotides using liquid chromatography–tandem mass spectrometry and method improvement by using ultra performance liquid chromatography

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2772-2782Leon Coulier, Henk Gerritsen, Jeroen J.A. van Kampen, Mariska L. Reedijk, Theo M. Luider, …Nucleoside reverse transcriptase inhibi…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2772-2782

Leon Coulier, Henk Gerritsen, Jeroen J.A. van Kampen, Mariska L. Reedijk, Theo M. Luider, ...

Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1 × 10cells. The platform consists of two liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC–MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC–MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1 nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%.

Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC-MS/MS

Publication year: 2011Source: Journal of Chromatography B, Available online 10 September 2011Hanna Amelina, Marcus O.D. Sjödin, Jonas Bergquist, Susana CristobalAging is a complex multifactorial phenomenon, which is believed to result from the acc…

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 September 2011

Hanna Amelina, Marcus O.D. Sjödin, Jonas Bergquist, Susana Cristobal

Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC-MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p < 0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisomal β-oxidation, detoxification of xenobiotics and production of reactive oxygen species (ROS). Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.

One single HPLC-PDA/(-)ESI-MS/MS analysis to simultaneously determine 30 components of the aqueous extract ofRabdosia rubescens

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2783-2793Jingcheng Tang, Ming Zhao, Yuji Wang, Guifeng Kang, Jianhui Wu, …In China the leaves ofRabdosia rubescenshave been cooked in water…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 26, 15 September 2011, Pages 2783-2793

Jingcheng Tang, Ming Zhao, Yuji Wang, Guifeng Kang, Jianhui Wu, ...

In China the leaves ofRabdosia rubescenshave been cooked in water and widely drank to treat inflammatory and pain related diseases. To explore the components that were possibly absorbed by people the aqueous extract of the leaves was prepared, and one single HPLC-PDA/(-)ESI-MS/MS analysis was developed to simultaneously determine the components. Using the HPLC-PDA analysis 39 peaks were found in the aqueous extract, while using the (-)ESI-MS/MS analysis we were able to identify 30 peaks represented components, including 5 nucleic acids, 21 phenolic acids and 4 diterpenoids. On mouse models thein vivoanti-inflammation and analgesic actions demonstrate that 0.32 g/kg of the aqueous extract of the leaves ofRabdosia rubescenscan effectively inhibit the inflammation-induced chronic pain.

Development of a semi-automated LC/MS/MS method for the simultaneous quantitation of 14,15-epoxyeicosatrienoic acid, 14,15-dihydroxyeicosatrienoic acid, leukotoxin and leukotoxin diol in human plasma as biomarkers of soluble epoxide hydrolase activityin vivo

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2487-2493Penny Zhu, Brian Peck, Hermes Licea-Perez, James F. Callahan, Catherine Booth-GentheSubstrates and products of soluble epoxide hydrol…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2487-2493

Penny Zhu, Brian Peck, Hermes Licea-Perez, James F. Callahan, Catherine Booth-Genthe

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography–mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid–liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05–50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.

Capillary electrophoresis coupled with mass spectrometry for the evaluation of substance P enzymatic degradation by SaOS-2 human osteosarcoma

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2501-2506Antonella Cavazza, Claudio Corradini, Mario Marini, Luigi Giorgio Roda, Angela ValentiA new analytical method for the detection and t…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2501-2506

Antonella Cavazza, Claudio Corradini, Mario Marini, Luigi Giorgio Roda, Angela Valenti

A new analytical method for the detection and the quantitative evaluation of the undecapeptide substance P by capillary electrophoresis coupled with ion trap mass spectrometry (CE–MS) by a co-axial sheath liquid interface has been developed. Conditions of analysis employed an acidic buffer and a 60 cm fused silica capillary installed by overcoming the UV window position, thus allowing to perform the analysis in a brief time. The method has been applied to the evaluation of substance P enzymatic hydrolysis during incubation with the human osteosarcoma SaOS-2 cell line. The analysis of amino acids derived from the cleavage of substance P has been also carried out simultaneously under the same electrophoretic conditions allowing the description of a kinetic of amino acid formation, parallel with substance P disappearance. The amounts of intact substance P and of free amino acids were monitored along 600 s of incubation time. A steady decrease of substance P as function of reaction time was observed. Peptide's half-life was found to be about 4.3 s, indicating an extremely fast hydrolysis in the presence of the SaOS-2 cells. Proline, phenilalanine and methionine were the predominant free amino acids recorded. Obtained results lead to hypothesize the occurrence of endopeptidases activity, followed by aminopeptidases responsible for the release of free amino acids originated after primary bond cleavage.

Rapid isolation procedure for Δ9-tetrahydrocannabinolic acid A (THCA) fromCannabis sativausing two flash chromatography systems

Publication year: 2011Source: Journal of Chromatography B, Available online 10 September 2011Ariane Wohlfarth, Hellmut Mahler, Volker AuwärterTwo isolation procedures for Δ9-tetrahydrocannabinolic acid A (THCA), the biogenetic precursor in the bio…

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 September 2011

Ariane Wohlfarth, Hellmut Mahler, Volker Auwärter

Two isolation procedures for Δ9-tetrahydrocannabinolic acid A (THCA), the biogenetic precursor in the biosynthesis of the psychoactive Δ9-tetrahydrocannabinol (THC) in the cannabis plant, are presented. Two flash chromatography systems that can be used independently from each other were developed to separate THCA from other compounds of a crude cannabis extract. In both systems UV absorption at 209 and 270 nm was monitored. Purity was finally determined by HPLC-DAD, NMR and GC-MS analysis with a focus on the impurity THC.System 1 consisted of a normal phase silica column (120 g) as well as cyclohexane and acetone - both spiked with the modifier pyridine - as mobile phases. Gradient elution was performed over 15 minutes. After the chromatographic run the fractions containing THCA fractions were pooled, extracted with hydrochloric acid to eliminate pyridine and evaporated to dryness. Loading 1800 mg cannabis extract yielded 623 mg THCA with a purity of 99.8% and a THC concentration of 0.09%.System 2 was based on a reversed-phase C18 column (150 g) combined with 0.1% formic acid and methanol as mobile phases. A very flat gradient was set over 20 minutes. After pooling the THCA-containing fractions methanol was removed in a rotary evaporator. THCA was re-extracted from the remaining aqueous phase with methyltert-butyl ether. The organic phase was finally evaporated under high vacuum conditions. Loading 300 mg cannabis extract yielded 51 mg THCA with a purity of 98.8% and a THC concentration of 0.67%.

Highlights

► Tetrahydrocannabinolic acid A is the biogenetic precursor of THC in the Cannabis plant. ► We present an isolation procedure for THCA with two flash chromatography systems. ► System 1 runs on normal phase, system 2 on reversed phase columns. ► Purity was tested with HPLC-DAD and GC-MS analysis, identity confirmed with NMR. ► The methods are rapid, do not use harmful solvents and yield purity grades > 98.8%.


A rapid and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of sibiricaxanthone F in rats

Publication year: 2011Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2513-2518Xiaojuan Yang, Guanshen Zhou, Pingping Zou, Ying Ning, Ke Zan, …A simple, rapid and sensitive liquid chromatography–tandem mass s…

Publication year: 2011
Source: Journal of Chromatography B, Volume 879, Issue 25, 1 September 2011, Pages 2513-2518

Xiaojuan Yang, Guanshen Zhou, Pingping Zou, Ying Ning, Ke Zan, ...

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantifying sibiricaxanthone F (SF) in rat plasma following oral and intravenous dosings. After addition of the internal standard (IS) kaempferol and the antioxidant, 20% ascorbic acid, plasma samples were precipitated with acetonitrile and separated on an Aglient Zorbax XDB-C18column (50 mm × 4.6 mm I.D., 2.1 μm) with gradient acetonitrile and water (both containing 0.01% formic acid) as the mobile phase. The detection was performed on a Sciex API 4000 LC–MS/MS with electrospray ionization (ESI) inlet in the negative multiple reaction monitoring (MRM) mode. Good linearity was achieved over the concentration range of 0.5–500.0 ng/mL (r > 0.996). Intra- and inter-day precisions were less than 7.60%, and accuracy ranged from 97.18% to 99.84%. The lower limit of quantification for SF was 0.5 ng/mL, and analytes were stable under various conditions (during freeze-thaw, at room temperature and under deep-freeze conditions). This validated method was successfully applied to the preliminary pharmacokinetic study of SF in rats for the first time, and the absolute bioavailability of SF was found to be only 0.22 ± 0.15%.