Rapid, simultaneous and nanomolar determination of pyroglutamic acid and cis-/trans-urocanic acid in human stratum corneum by hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization tandem mass spectrometry
A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0-250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3-102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.
Available from: Ted M. Lakowski
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ABSTRACT: Protein arginine methylation is one of the epigenetic modifications to proteins that is studied in yeast and is known to be involved in a number of human diseases. All eukaryotes produce Nη-monomethylarginine (ηMMA), asymmetric Nη1, Nη1-dimethylarginine (aDMA), and most produce symmetric Nη1, Nη2-dimethylarginine (sDMA) on proteins, but only yeast produce Nδ-monomethylarginine (δMMA). It has proven difficult to differentiate among all of these methylarginines using mass spectrometry. Accordingly, we demonstrated that the two forms of MMA have indistinguishable primary product ion spectra. However, the secondary product ion spectra of δMMA and ηMMA exhibited distinct patterns of ions. Using incorporation of deuterated methyl-groups in yeast, we determined which secondary product ions were methylated and their structures. Utilizing distinct secondary product ions, a triple quadrupole multiple reaction monitoring cubed (MRM(3)) assay was developed to measure δMMA, ηMMA, sDMA and aDMA derived from hydrolyzed protein. As a proof-of-concept, δMMA and ηMMA were measured using the MRM(3) method in wild type and mutant strains of Saccharomyces cerevisiae and compared to the total MMA measured using an existing assay. The MRM(3) assay represents the only method to directly quantify δMMA and the only method to simultaneously quantify all yeast methylarginines.
Journal of proteomics 01/2013; 80. DOI:10.1016/j.jprot.2013.01.003 · 3.89 Impact Factor
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ABSTRACT: Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. In addition to the well-known flavonol glycosides and terpene lactones, G. biloba leaves are also rich in amino acids. To determine the content of free amino acids, a reliable method has been established by using hydrophilic interaction ultra-HPLC coupled with ESI-MS. 20 free amino acids were simultaneously determined without derivatization in 12 min. The proposed method was fully validated in terms of linearity, sensitivity, repeatability, as well as recovery. Furthermore, the principal component analysis was applied to different G. biloba leaves collected in November (after fruit harvest season), which revealed that the samples from different production areas exhibited regional disparity in different clusters in accordance with their various hydrophilic interaction chromatograms coupled with mass profiles. The established approach could be helpful for evaluation of the potential values as dietary supplements and the quality control of G. biloba leaves, which might also be utilized for the investigation of other medicinal herbs containing amino acids.
Journal of Separation Science 09/2013; 36(17):2878-87. DOI:10.1002/jssc.201201045 · 2.74 Impact Factor
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ABSTRACT: This paper reports a new class of pencil graphite electrode duly modified with a molecularly imprinted metallo-polymer for enantio-selective sensing of D-/L-pyroglutamic acid, in aqueous and real samples. Herein, specific binding sites of D- and L-isomers were created in their respective three-dimensional motifs of highly conducting imprinted film. This was developed through the electropolymerization of copper(II)-5-methyl-thiophene-2-carboxylic acid complex, in the presence of analyte (template:monomer molar ratio 1:2). The detection of isomers could be feasible at operating conditions (pH, deposition potential, deposition time, etc.) of differential pulse anodic stripping voltammetry [aqueous sample, linear range 2.8-170.0 ngmL(-1), limit of detection, 0.77 ngmL(-1) (S/N=3)]. The proposed sensor was also validated with dilute real samples (urine, cerebrospinal fluid and blood plasma). Although several chronic diseases (metabolic acidosis, 5-oxoprolinuria, etc.) are known to be manifested at hyper-concentrations of L-pyroglutamic acid (D-isomer is biologically inactive), the sample dilution by several fold, which mitigates biological matrix effect, is necessarily required. This warrants a highly sensitive probe of analysis within the linear quantitation range 1.3-180.0 ng mL(-1), without any cross-reactivity and false-positives. The endogenous concentrations of real samples could simply be obtained by multiplying the concentration of dilute sample with respective dilution factor.
Sensors and Actuators B Chemical 09/2013; 186:407-416. DOI:10.1016/j.snb.2013.06.041 · 4.10 Impact Factor
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