Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample. Copyright ? 2012 John Wiley & Sons, Ltd.     

Porphyrinogen fragmentation profiles by ultra-high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

An ultra-high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry system is described for the separation and characterisation of uroporphyrinogen, heptacarboxylic acid porphyrinogen, hexacarboxylic acid porphyrinogen, pentacarboxylic acid porphyrinogen and coproporphyrinogen. The separation was carried out on a 100 mm × 2.1 mm Thermo-Hypersil BDS column (2.4 μm average particle size) by gradient elution with a mixture of acetonitrile, methanol and 1 mol/L aqueous ammonium acetate buffer, pH 5.16, as eluent. The fragmentation pattern of each compound was established by collision-induced dissociation tandem mass spectrometry. The most characteristic fragmentation was ring opening at one of the four methylene bridges of the protonated porphyrinogen molecule followed by further cleavages of methylene bridges linking the four pyrrole rings at various points to give product ions with methylenepyrrolenine, methylene-dipyrrolenine and methylene-tripyrrolenine structures.     

Analysis of furanocoumarins from Yemenite Dorstenia species by liquid chromatography/electrospray tandem mass spectrometry

A series of prevailing prenylated furanocoumarins from leaves of Dorstenia gigas and Dorstenia foetida (Moraceae) were investigated by liquid chromatography/electrospray tandem mass spectrometry. The mass spectral behavior of the furanocoumarins under positive ion electrospray conditions is discussed using both an ion trap and a triple quadrupole system. It is demonstrated that both methods represent valuable tools not only for the rapid classification of this type of compounds, but also with respect to their substitution pattern.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI-HR-MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague-Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid-phase extraction and then subjected to LC/HR-MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O-sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC-MS/MS and MS(n) experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways.     

Screening of glycoside isomers in P. scrophulariiflora using ionic liquid-based ultrasonic-assisted extraction and ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

A powerful ionic liquid-based ultrasonic-assisted extraction (ILUAE) method combined with ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOFMS(n) ) was employed in the rapid simultaneous screening of iridoid glycosides, phenylethanoid glycosides, and cucurbitacin glycosides from P. scrophulariiflora. The ILUAE procedure was optimized over several ultrasonic parameters, including the ultrasonic power, concentration of the ionic liquid, and solid-liquid ratio. A comparison with conventional heat-reflux extraction and regular UAE demonstrated that the optimized approach yielded a high extraction efficiency (Picroside I, 2.84%; Picroside II, 3.57%; 6-O-E-feruloyl catalpol, 2.20%) within a short extraction time of 30?min. Negative ion mode ESI-QTOFMS(2) analysis of the fragmentation reactions of the [M-H](-) ions was conducted to characterize the diagnostic ions related to the glycosyl moieties, aglycone units, and the type and substituted position of the ester groups. Interestingly, the positional isomers of the iridoid glycosides could be easily discriminated based on the characteristic ions. A total of 15 glycosides, including three groups of iridoid glycoside isomers and two groups of phenylethanoid glycoside isomers, were conveniently identified within 13.5?min. Moreover, 6'-O-vanilloyl catalpol was identified in P. scrophulariiflora for the first time. The method developed here was further validated by measuring the recovery, correlation coefficient (R(2) ), and reproducibility (RSD, n?=?5) of three iridoid glycosides: 89.60%-109.02%, 0.9991-0.9998, and 0.93%-1.44%, respectively. This study demonstrated the capabilities of ILUAE combined with UPLC/ESI-QTOFMS(n) for the rapid screening of glycosides in P. scrophulariiflora. This method offers an approach to similar studies on other natural plants.     

A discharge adaptor interface for use in liquid chromatography/mass spectrometry

A discharge adaptor, composed of a metal casing and platinum (Pt) wire needle, was directly attached to an electrospray ionization (ESI) probe tip, to transform the ionization into atmospheric pressure chemical ionization (APCI). Six generic drugs were analyzed with the developed discharge adaptor (DA) and two commercial interfaces. The DA interface produced more intense radical anions, [M]˙?, and less sodium adduct ions, [M + Na]?, than the ESI interface, whereas almost the same molecular ions were detected as the APCI interface. The effects of solvent and desolvation gas flow in the DA interface were similar to those in the ESI interface, but differed from those in the APCI interface. Better sensitivity of the tested drugs was obtained relative to the commercial APCI interface. For human plasma samples, the DA interface also demonstrated good tolerance to plasma matrices, linearity from 5 or 20 to 500 ng/mL (r2 > 0.99) and ruggedness.     

Analysis of 30 synthetic cannabinoids in serum by liquid chromatography-electrospray ionization tandem mass spectrometry after liquid-liquid extraction

The analysis of synthetic cannabinoids in human matrices is of particular importance in the fields of forensic and clinical toxicology since cannabis users partly shift to the consumption of 'herbal mixtures' as a legal alternative to cannabis products in order to circumvent drug testing. However, comprehensive methods covering the majority of synthetic cannabinoids already identified on the drug market are still lacking. In this article, we present a fully validated method for the analysis of 30 synthetic cannabinoids in human serum utilizing liquid-liquid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry. The method proved to be suitable for the quantification of 27 substances. The limits of detection ranged from 0.01 to 2.0 ng/mL, whereas the lower limits of quantification were in the range from 0.1 to 2.0 ng/mL. The presented method was successfully applied to 833 authentic serum samples during routine analysis between August 2011 and January 2012. A total of 227 (27%) samples was tested positive for at least one of the following synthetic cannabinoids: JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-210, JWH-307, AM-2201 and RCS-4. The most prevalent compounds in positive samples were JWH-210 (80%), JWH-122 (63%) as well as AM-2201 (29%). Median serum concentrations were all below 1.0 ng/mL. These findings demonstrate a significant shift of the market of synthetic cannabinoids towards substances featuring a higher CB(1) binding affinity and clearly emphasize that the analysis of synthetic cannabinoids in serum or blood samples requires highly sensitive analytical methods covering a wide spectrum of substances.     

Determination of bakkenolide A in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A sensitive rapid analytical method was established and validated to determine the bakkenolide A (BA) in rat plasma. This method was further applied to assess the pharmacokinetics of BA in rats receiving a single dose of BA. Liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode was used in the method, and costundide was used as internal standard. A simple protein precipitation based on methanol was employed. The combination of a simple sample cleanup and short chromatographic running time (2.4?min) increased the throughput of the method substantially. The method was validated over the range of 1-1000?ng/mL with a correlation coefficient?>?0.99. The lower limit of quantification was 1?ng/mL for BA in plasma. Intra- and inter-day accuracies for BA were 93-112% and 103-104%, respectively, and the inter-day precision was less than 15%. After a single oral dose of 20?mg/kg of BA, the mean peak plasma concentration (C(max) ) of BA was 234.7?±?161?ng/mL at 0.25?h. The area under the plasma concentration-time curve (AUC(0-24 h) ) was 535.8?±?223.7?h·ng/mL, and the elimination half-life (T(1/2) ) was 5.0?±?0.36?h. In case of intravenous administration of BA at a dosage of 2?mg/kg, the area under the plasma concentration-time curve (AUC(0-24 h) ) was 342?±?98 h?ng/mL, and the elimination half-life (T(1/2) ) was 5.8?±?0.7?h. Based on the results, the oral bioavailability of BA in rats at 20?mg/kg is 15.7%. Copyright ? 2012 John Wiley & Sons, Ltd.