Frank S. Pullen

High‐performance Liquid Chromatography/NMR Spectrometry/Mass Spectrometry: Further Advances in Hyphenated Technology

The earlier use of combined liquid chromatography/NMR spectrometry/mass spectrometry (LC/NMR/MS) involved the use of a particle beam interface. This paper describes further developments of this hyphenated technology, in particular the incorporation of an electrospray interface into the LC/NMR/MS system. This improved LC/NMR/MS system was designed for the support of a combinatorial library program. The power of this technique is demonstrated in the successful structural elucidation of each compound in a mixture of commercially available peptides.

On-line liquid chromatography coupled with high field NMR and mass spectrometry (LC-NMR-MS): a new technique for drug metabolite structure elucidation

High performance liquid chromatography has been coupled simultaneously to high field NMR and MS detectors, giving UV, NMR and mass spectra for each component in a mixture, after on-line separation. This powerful new tool for the structure elucidation of components in mixtures without isolation has been successfully applied to the analysis of the metabolites of paracetamol in human urine.

Effect of increasing concentration of ammonium acetate as an additive in supercritical fluid chromatography using CO2–methanol mobile phase

The effects of increasing concentrations of ammonium acetate additive in supercritical fluid chromatography were studied on silica, 2-ethyl-pyridine and endcapped 2-ethyl-pyridine stationary phases. The study involved the addition of increasing concentrations of the ammonium acetate either in the mobile phase modifier (methanol) or in the sample solvent. The effects of ammonium acetate on retention and peak shape of the analytes were evaluated. Compounds that exhibited satisfactory chromatographic behaviour in the absence of the additive were virtually unaffected by its presence in the mobile phase or sample solvent. Nevertheless, compounds that exhibited late elution and strongly tailing peak shapes when pure methanol was used showed dramatically improved chromatographic behaviour in the presence of the additive. Shorter retention was observed not only when the modifier was introduced in the mobile phase but also when it was in the sample solvent.

Investigation of different approaches to incorporating internal standard in DBS quantitative bioanalytical workflows and their effect on nullifying hematocrit-based assay bias.

Hematocrit (HCT)-based assay bias (composed of area and recovery bias) is an important contributing factor to the barriers that currently hinder the development and acceptance of dried blood spots (DBS) as a widely used quantitative bioanalytical sampling technique for regulatory studies. This article describes the evaluation of a practical internal standard spray addition technique, used prior to LC-MS/MS analysis, which is demonstrated to nullify the effect of recovery bias. To our knowledge, this is the first time a potential solution to HCT-based recovery bias has been investigated in detail and reported in the literature. This new technique is coupled with accurate volume DBS sampling, whole-spot extraction, and automated direct elution techniques to demonstrate a workflow that both nullifies HCT-based assay bias and the additional manual extraction burden associated with DBS analysis.

Essential and trace elements content of commercial infant foods in the UK.

There is a paucity of data in respect of the nutritional quality of complementary foods for infants and young children aged between 6 and 12months. The primary objective of this study was to examine nutritive values of such complementary infant food on the UK market in order to ascertain their suitability relative to dietary guidelines for the 6-9months age group. Quantitative analyses were conducted on eight different products representing four popular brands (meat and vegetable based) currently on sale in the UK. Eight major mineral and trace elements, namely: calcium, copper, magnesium, iron, zinc, potassium, sodium and selenium were measured by ICP-OES and ICP-MS. The results of these studies were referenced to the Recommended Nutrient Intake (RNI) values for 6-9months old children, and a menu of entire daily intake of minerals and trace elements was composed taking into consideration the nutrient and energy intake from milk consumption. Based on these comparisons, all the food samples studied in this work contained less essential minerals than expected from the RNI values except for potassium in meat and vegetable based recipes. These results suggest that commercial complementary infant foods on the UK market may not contain the minimum levels of minerals required for the labelling declaration of micronutrient content (Commission Directive 2006/125/EC). This provides opportunities and scope for product optimisation to improve their nutritive value.

Putting mass spectrometry in the hands of the end user.

The ease of use of the newer liquid chromatography-mass spectrometry interfaces has made possible the automated acquisition of spectra from large batch queues of samples. This fact, combined with the realization that unit molecular mass determination was the only datum desired by a majority of drug discovery synthetic chemists, led us to develop open access mass spectrometry in the early 1990s. Open access spectrometers now scan over 100,000 samples per year from synthesis laboratories at Pfizer. Our experiences with this novel use of mass spectrometry in a large research facility are discussed and we detail some of the pitfalls we believe to be common to this approach. In addition, we offer some reflection on the cultural changes we have observed in our research environment since this experiment began.

Probing noncovalent protein–ligand interactions of the cgmp-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog β,γ-imidoadenosine 5′-triphosphate (AMPPNP) and Mn2+ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg2+ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl2 to the spray solution. This is in agreement with binding of AMPPNP/Mn2+ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the β and γ phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substrate-docking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP)4(AMPPNP/Mn2+)2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3–4 phosphates per subunit.

Using density functional theory to rationalise the mass spectral fragmentation of maraviroc and its metabolites

Tandem mass spectrometry (MS/MS) is widely used for the identification of metabolites at all stages of the pharmaceutical discovery and development process. The assignment of ions in the product ion spectra can be time-consuming and hence delay feedback of results that may influence the direction of a project. A deeper understanding of the processes involved in generation of the product ions formed via collision-induced dissociation may allow development of chemically intelligent software to aid spectral interpretation. Current commercially available spectral interpretation software takes a mainly arithmetical approach resulting in extensive lists of numerically plausible ions, many of which may not be chemically feasible. In this study, high-resolution MS/MS spectra were obtained for maraviroc and two of its synthetic metabolites, and structures for the product ions proposed. Density functional theory (DFT) based on in silico modelling was undertaken to investigate whether the fragmentation observed was potentially a result of bond lengthening (and hence weakening) as a consequence of protonation of the molecule at the most thermodynamically stable site(s). It was determined that for all three compounds, where the product ions resulted from simple bond cleavages (not rearrangements), the bonds that cleaved had been calculated to elongate after protonation. It was also noted that the protonated molecule may represent a mixture of singly charged protonated species and that the most basic sites in the molecule may not necessarily be the most thermodynamically stable for protonation.

Elemental content of commercial ‘ready to-feed’ poultry and fish based infant foods in the UK

The study reported herein was conducted in order to establish the concentration of 20 essential and non-essential elements in a representative range of commercial infant foods in the UK targeted for infants aged between 6-12 months. The primary objective of this study was to examine the nutritive values and safety of such complementary infant foods on the UK market in relation to dietary and safety guidelines. Quantitative analyses were conducted on eight different products representing four popular brands (poultry and fish based) of ready to-feed infant foods currently on sale in the UK. Six essential elements, namely: calcium, iron, magnesium, potassium, sodium and zinc were determined by ICP-OES. The concentrations of six essential trace elements (selenium, molybdenum, cobalt, copper, chromium, manganese) and eight non-essential, potentially toxic, elements (arsenic, barium, nickel, cadmium, antimony, lead, mercury, aluminium) were determined by ICP-MS due to the higher sensitivity required. The total daily intakes of essential and trace elements from the consumption of such products were then estimated, based on the results of this study, and were referenced to the Recommended Nutrient Intake (RNI) values and safety guidelines for 6-9 months old children. Based on these comparisons the concentration of essential, except for potassium, and trace elements were found to be inadequate in meeting the RNI. In terms of the risk of exposure to toxicity, the concentration of toxic elements in ready to feed products analysed in this study, were not considered to be of concern. These results suggest that commercial complementary infant foods on the UK market may not contain minimum levels of minerals required for labelling declaration of micronutrient content (Commission Directive 2006/125/EC). This provides opportunities and scope for product optimisation to improve their nutritive value.

Automated high resolution mass spectrometry for the synthetic chemist.

Exact mass measurement at high resolution is an important tool alongside other spectroscopic methods to help confirm the structure of a novel compound prepared by the synthetic chemist. Exact mass measurement is used in the pharmaceutical industry to confirm the expected empirical formula of a product when problems have been experienced using elemental analysis. Because of the amount of manual intervention necessary when acquiring exact mass measurements, especially when using probe ionization techniques such as fast atom bombardment ionization or electron ionization, this method has been seen to be time consuming and labor intensive for the mass spectrometrist. An automated high resolution mass spectrometric method has been developed at Pfizer Central Research which has streamlined exact mass measurement. The method, which uses electrospray ionization on a double focusing mass spectrometer, is described. The samples are analyzed using a flow injection technique, with sodiated polyethylene glycol present in the mobile phase to provide mass reference peaks. The data are acquired and processed using a macro developed “in house.” This automated technique can process 15–20 samples an hour including data processing and report generation, using very small amounts of compound (∼25 µg), but more importantly it can be left to run unattended overnight. This allows the instrument to be used for more complex experiments during the day when it is important to have a mass spectrometrist present. The results presented here demonstrate that this method gives exact mass measurements within an acceptable limit of 5 ppm, and the variation on one sample, injected 10 times, is not excessively high (−1.8 to +1.6 mDa).