We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.

Background Growth hormones are widely available on the Internet for those who want to enhance their physical performance and improve body satisfaction. Illegitimate websites market somatropin injections without medical prescription and encourage misuse. Customers potentially put their health at risk when purchasing parenteral medications online. Objective The objective of our study was to evaluate the online market of no-prescription somatropin products and to analyse and document Internet pharmacy characteristics, distribution and pharmaceutical quality. Setting Websites indexed in Google promoting somatropin for sale direct to patients. Method Websites promoting the sale of growth hormone products were identified and analysed from June to August 2014. Internet vendor sites were evaluated to identify possible patient and medication safety concerns. Website characteristics, delivery time, storage conditions, packaging and attached product information were assessed. Investigation of the somatropin content was achieved using capillary electrophoresis with UV detection and electrospray ionization mass spectrometry. Main outcome measure Accessibility and quality of somatropin injections. Results Seventeen individual Internet vendor websites distributed somatropin products directly to patients, majority (94%) did not require a valid medical prescription before dispensing the products. Majority (70%) of Internet pharmacies displayed no medical information and none (0%) of the vendors displayed any regulatory body logo. All online samples had significantly (p < 0.001) lower somatropin concentration than labelled. Conclusion Our results clearly illustrate that prescription only biologic drugs are widely available online and can be easily accessed by anyone. Unprofessional distribution and handling is likely to cause degradation and possible patient safety concerns.

Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument. Charge-promoted consecutive and competitive eliminations of the acyl chains and cross-ring cleavages of the sugar residues were observed. The A-type fragment ion series and the complementary X-type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non-reducing and the reducing ends, respectively, of the non-phosphorylated lipid A backbone. Reversed-phase liquid chromatography in combination with negative-ion electrospray ionization quadrupole time-of-flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non-phosphorylated lipid A. As a standard, the hexa-acylated ion at m/z 1636 with the Escherichia coli-type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non-phosphorylated hexa- and several hepta-, penta-, and tetra-acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter-ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4' phosphate group) were simultaneously present in the sample. Continuous series of high-abundance B-type and low-abundance Y-type inter-ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative-ion mode, they were very similar in the positive-ion mode. The complementary use of positive-ion and negative-ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1-monophosphorylated lipid A species.

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