1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations

Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, 1H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that 1H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d6. Unexpected or unwanted extract constituents were also easily identified in the 1H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of 1H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that 1H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts

A novel ABO allele with a 21-bp duplication identified in two unrelated European individuals with weak A expression

Objectives: To carry out genetic and serological analyses of a Swiss blood donor and a Danish patient carrying an aberrant ABO phenotype with weak A expression. Background: ABO is the most clinically important blood group system but also one of the most complex. The system antigens are determined by carbohydrate structures generated by A and B glycosyltransferases encoded by the ABO gene. Genetic variants of ABO may encode a glycosyltransferase with reduced activity, leading to weak expression of A antigen. Methods: Samples from two individuals were examined using genetic testing and extended immunohaematological evaluation, including standard serological methods, flow cytometry and analysis of plasma glycosyltransferase activity. Results: Both individuals were serologically determined to be AweakB. Genetic testing revealed that both were heterozygous for a novel ABO*A1.01-like allele with an in-frame duplication of 21 nucleotides in exon 7 (c.543_563dup), leading to the insertion of seven amino acids (QDVSMRR). Flow cytometric testing of native red blood cells (RBCs) showed very weak A antigen expression. This was in accordance with the enzyme activity test. Conclusion: In summary, we describe a novel A allele with a duplication of 21 nucleotides in exon 7 that significantly decreases the enzyme activity and leads to very weak expression of A antigen. (200 words)