Gerd Vanhoenacker

Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry

A lipidomics strategy, combining high resolution reversed-phase liquid chromatography (RPLC) with high resolution quadrupole time-of-flight mass spectrometry (QqTOF), is described. The method has carefully been assessed in both a qualitative and a quantitative fashion utilizing human blood plasma. The inherent low technical variability associated with the lipidomics method allows to measure 65% of the features with an intensity RSD value below 10%. Blood plasma lipid spike-in experiments demonstrate that relative concentration differences smaller than 25% can readily be revealed by means of a t-test. Utilizing an advanced identification strategy, it is shown that the detected features mainly originate from (lyso-)phospholipids, sphingolipids, mono-, di- and triacylglycerols and cholesterol esters. The high resolution offered by the up-front RPLC step further allows to discriminate various isomeric species associated with the different lipid classes. The added value of utilizing a Jetstream electrospray ionization (ESI) source over a regular ESI source in lipidomics is for the first time demonstrated. In addition, the application of ultra high performance LC (UHPLC) up to 1200bar to extend the peak capacity or increase productivity is discussed.

The acetonitrile shortage: Is reversed HILIC with water an alternative for the analysis of highly polar ionizable solutes?

In hydrophilic interaction chromatography (HILIC), best results are obtained with high concentrations of ACN. In the framework of green chromatography and the present shortage and very high price of this hazardous solvent, reversing the stationary phase to apolar and the mobile phase to aqueous can be of interest for several applications. The features of the aqueous RP technique called per aqueous LC (PALC) are illustrated with the analysis of catecholamines, nucleobases, acids, and amino acids. The ca. three-fold higher viscosity of water compared to ACN has consequences on the shape of the Van Deemter plot. For dopamine (N = 26.450 on a 25 cm x 4.6 mm id, 5 microm bare silica column), a reduced plate height of 1.9 at an u(opt) of 0.3 mm/s was calculated. The plate number, however, strongly depends on pH and ionic strength. As in RP separations, retention is shortened by adding an organic modifier. In the framework of green chromatography, the biodegradable ethanol was used. On the other hand, retention increased by lengthening the carbon chain of ion-pair reagents supporting the RP mechanism as well.

Recent applications of capillary electrochromatography.

A review is presented of the most important recent applications of capillary electrochromatography (CEC) for the analysis of acidic, basic, and neutral compounds, of biomolecules, environmental substances, natural products, pharmaceuticals, and chiral compounds. Packed‐column CEC (packed‐CEC), open‐tubular (OT‐CEC), as well as pressure‐assisted CEC (pseudo‐CEC) are hereby considered. Papers published between July 1999 and April 2001 were taken into account. Applications before July 1999 have been reviewed in Electrophoresis 1999, 20, 3027 135 135–3065.

Comparison of high-performance liquid chromatography - Mass spectroscopy and capillary electrophoresis - Mass spectroscopy for the analysis of phenolic compounds in diethyl ether extracts of red wines.

SummaryReversed-phase high-performance liquid chromatography-mass spectroscopy (HPLC-MS) and capillary electrophoresis-mass spectroscopy (CE-MS) have been compared for the analysis of phenolic compounds in diethyl ether extracts of red wines. MS was performed in the electrospray negative-ionization mode. Despite the much higher separation efficiency of CE compared with LC, LC-MS furnishes far superior information for elucidation of the structure of the constituents. LC-MS enabled the identification of twenty-four compounds whereas only thirteen were characterized by CE-MS.

Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and 2D retention time opening interesting perspectives for use in QA/QC testing.

Analysis of benzodiazepines in dynamically coated capillaries by CE-DAD, CE-MS and CE-MS2.

The applicability of a low pH volatile electrolyte for fast analysis of benzodiazepines with CE-MS was investigated. The electrolyte is based on a commercially available CEofix buffer system that produces a substantial and highly reproducible electroosmotic flow through a dynamic double coating principle. The system was first evaluated with a mixture of benzodiazepine standards in CE-DAD and the electrolyte composition was further optimized for CE-MS. The LOD for the six selected benzodiazepines with CE-MS was ca. 100 ppb, except for diazepam, for which the LOD was lower than 50 ppb. RSDs varied from 0.51 to 1.02% (n = 7) for migration times and from 4.75 to 11.80% (n = 7) for peak areas. The method was successfully applied to the analysis of a spiked urine sample after solid-phase extraction (SPE). CE-MS2 was performed on a standard mixture.

Determination of arylamines and aminopyridines in pharmaceutical products using in-situ derivatization and liquid chromatography-mass spectrometry

Arylamines and aminopyridines form a class of potentially genotoxic impurities (PGIs) that can be present at trace levels in active pharmaceutical ingredients (APIs). A generic method was developed that allows the analysis of a selected set of these solutes at sub-ppm level relative to the drug substance. A highly concentrated solution of the pharmaceutical compound is analyzed by LC-MS using a single quadrupole mass spectrometer in the selected ion monitoring (SIM) mode. Since a number of target compounds show little or no retention in the reversed-phase LC setup, a fast and simple derivatization procedure using hexylchloroformate was applied. The amide derivatives of the PGI result in a higher molecular weight (more specific ion for SIM) and better chromatographic behavior. The methodology, consisting of a dual run on respectively a non-derivatized and a derivatized sample, was validated and applied to a selection of pharmaceutical substances. The method was found to be sufficiently sensitive and robust and is applicable in a QA/QC environment.

Chemotaxonomic features associated with flavonoids of cannabinoid-free cannabis (Cannabis sativa subsp sativa L.) in relation to hops (Humulus lupulus L.).

The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-g-D-glucuronide (3), and apigenin-7-O-g-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.

Single‐run capillary electrochromatographic analysis of hop acids and prenylated hop flavonoids

The characteristic bitter taste of pale beers is due to the presence of hop-derived substances, the so-called iso-aacids, which are formed from the hop a-acids during the boiling of sweet wort with hops (Humulus lupulus L.) in the brewery [1]. The analysis of hop acids, which include, in addition to a-acids, also b-acids (Figure 1) is important to assess the quality of hops and hop products. Often, prenylated flavonoids interfere with the separation of aand bacids. The interest in prenylated flavonoids pertains to their bioactivities and a particular constituent, 8-prenylnaringenin, has been identified as the most potent phytoestrogen known to date [2, 3].

Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine

Antibody-drug conjugates might be the magic bullets referred to by Paul Ehrlich over 100 years ago. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry to its limits. The use of multiple heart-cutting (mLC-LC) and comprehensive (LC×LC) multidimensional LC in combination with high resolution mass spectrometry for the characterization of the lysine conjugated antibody-drug conjugate ado-trastuzumab emtansine, commercialized as Kadcyla, is presented. By combining protein and peptide measurements, attributes such as drug loading, drug distribution and drug conjugation sites can be assessed in an elegant manner.