Analysis of thrombin‐antithrombin complex formation using microchip electrophoresis and mass spectrometry

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin‐antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.     

Evading metal adduct formation during desorption‐ionization mass spectrometry

An investigation into the propensity of metal adduct formation in the recently developed Desorption Ionization by Charge Exchange (DICE) mass spectrometric technique has demonstrated that this method could be utilized to minimize spectral complications caused by metal adducts. For example, peaks for sodium and other metal adducts were not observed in the mass spectra acquired by the ambient‐pressure DICE technique from samples deposited on a solid surface, even after the salt content of samples was deliberately increased. A mass spectrum recorded from a urine sample by this technique showed peaks only for the proton adducts of urea and creatinine. This technique employs a nebulized spray of charged toluene droplets for analyte desorption. Because of the non‐polar nature of the spray reagent, it neither contains any appreciable amount of cations nor provides any favored ‘pickup’ of metal cations from the sample matrix. Consequently, peaks for metal adducts that are commonly observed with other desorption techniques are minimal or absent in the spectra recorded by the DICE method. Copyright © 2010 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of THC, 11‐hydroxy‐THC and 11‐nor‐9‐carboxy‐THC in whole blood by ultra‐performance liquid chromatography/tandem mass spectrometry

A qualitative and quantitative analytical method was developed for the simultaneous determination of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (11‐OH‐THC) and l1‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THC‐COOH) in whole blood. The samples were prepared by solid‐phase extraction followed by ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis using positive ion electrospray ionization and multiple reaction monitoring. The chromatographic separation was performed with an Acquity UPLC® HSS T3 (50 × 2.1 mm i.d., 1.8 µm) reversed‐phase column using a methanol/2 mM ammonium formate (formic acid 0.1%) gradient in a total run time of 9.5 min. MS/MS detection was achieved with two precursor‐product ion transitions per substance. The method was fully validated, including selectivity and capacity of identification, according to the identification criteria (two transitions per substance, signal‐to‐noise ratio, relative retention time and ion ratio) without the presence of interferences, limit of detection (0.2 µg/L for THC and 0.5 µg/L for 11‐OH‐THC and THC‐COOH), limit of quantitation (0.5 µg/L for all cannabinoids), recovery (53–115%), carryover, matrix effect (34‐43%), linearity (0.5‐100 µg/L), intra‐assay precision (CV < 10% for the relative peak area ratios and <0.1% for the relative retention time), inter‐assay accuracy (mean relative error <10%) and precision (CV <11%). The method has already been successfully used in proficiency tests and subsequently applied to authentic samples in routine forensic analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Oxidation‐assisted structural elucidation of compounds containing a tertiary amine side chain using liquid chromatography mass spectrometry

A novel analytical technique for the structural elucidation of compounds bearing a tertiary amine side chain via “in vial” instantaneous oxidation and liquid chromatography mass spectrometry (LC‐MS) was developed. A series of lidocaine homologs and benzimidazole derivatives with a major/single amine representative base peak in both their EI‐MS and ESI‐MS/MS spectra were subjected to oxidation by a 0.1% solution of hydrogen peroxide (including several ¹⁶O/¹⁸O exchange experiments), followed by LC‐ESI‐MS/MS analysis. The N‐oxide counterparts promoted extensive fragmentation with complete coverage of all parts of the molecule, enabling detailed structural elucidation and unambiguous identification of the unoxidized analytes at low nanogram per milliliter levels.     

Analysis of hydroxamate siderophores in soil solution using liquid chromatography with mass spectrometry and tandem mass spectrometry with on‐line sample preconcentration

A liquid chromatography with electrospray ionization mass spectrometry method was developed to quantitatively and qualitatively analyze 13 hydroxamate siderophores (ferrichrome, ferrirubin, ferrirhodin, ferrichrysin, ferricrocin, ferrioxamine B, D₁, E and G, neocoprogen I and II, coprogen and triacetylfusarinine C). Samples were preconcentrated on‐line by a switch‐valve setup prior to analyte separation on a Kinetex C₁₈ column. Gradient elution was performed using a mixture of an ammonium formate buffer and acetonitrile. Total analysis time including column conditioning was 20.5 min. Analytes were fragmented by applying collision‐induced dissociation, enabling structural identification by tandem mass spectrometry. Limit of detection values for the selected ion monitoring method ranged from 71 pM to 1.5 nM with corresponding values of two to nine times higher for the multiple reaction monitoring method. The liquid chromatography with mass spectrometry method resulted in a robust and sensitive quantification of hydroxamate siderophores as indicated by retention time stability, linearity, sensitivity, precision and recovery. The analytical error of the methods, assessed through random‐order, duplicate analysis of soil samples extracted with a mixture of 10 mM phosphate buffer and methanol, appears negligible in relation to between‐sample variations.     

Rapid analysis of long‐chain glycolipids in heterocystous cyanobacteria using high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Under nitrogen‐depleted conditions, N₂‐fixing cyanobacteria of the order Nostocales and Stigonematales differentiate vegetative cells into heterocysts. The cell envelope of these specialized cells contains unique glycolipids, consisting of a sugar moiety glycosidically bound to long‐chain diols, triols and hydroxyketones. Only few reports have been published on these glycolipids in cultured cyanobacteria and none has reported them in natural environments. Here we show that heterocyst glycolipids can be rapidly and sensitively analyzed using high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS²). Positive ion mass spectra of the glycolipids consisted of protonated molecules and diagnostic product ions, indicating losses of sugar groups as well as hydroxyl and carbonyl functionalities from an alkyl chain. Using this method, heterocyst glycolipids were for the first time identified in a natural ecosystem, i.e., a microbial mat from the North Sea barrier island Schiermonnikoog, The Netherlands. This technique will facilitate the quick screening of cyanobacterial cultures and natural environments for the presence of heterocyst glycolipids, which may aid in assessing the role of heterocystous cyanobacteria in the global nitrogen cycle. Copyright © 2009 John Wiley & Sons, Ltd.