Measurement of Serum Free Vitamin D Concentrations: Importance, Challenges, and the Emerging Role of Mass Spectrometry.

Background: Serum total 25-hydroxyvitamin D [25(OH)D] concentration is the most widely used clinical biomarker for vitamin D status. Under certain physiological and pathological conditions, however, total 25(OH)D may not always be the best index for vitamin D status. Instead, the nonprotein-bound (free) fraction of total 25(OH)D has been suggested as a more appropriate marker in certain clinical situations.

An Overview of Different Vitamin D Compounds in the Setting of Adiposity.

A large body of research shows an association between higher body weight and low vitamin D status, as assessed using serum 25-hydroxyvitamin D concentrations. Vitamin D can be metabolised in adipose tissue and has been reported to influence gene expression and modulate inflammation and adipose tissue metabolism in vitro. However, the exact metabolism of vitamin D in adipose tissue is currently unknown.

2-fluoro-1-methylpyridinium p-toluene sulfonate: a new LC-MS/MS derivatization reagent for vitamin D metabolites.

Vitamin D analysis by MS faces several analytical challenges, including inefficient ionization, nonspecific fragmentation, interferences from epimers, isomers, and isobars, as well as very low concentration levels. In this study, we used 2-fluoro-1-methylpyridinium (FMP) p-toluene sulfonate for derivatization of vitamin D3 metabolites to increase detection sensitivity and allow for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for measurement of five vitamin D3 metabolites in human serum.

Comparing derivatization reagents for quantitative LC-MS/MS analysis of a variety of vitamin D metabolites.

The present study systematically compares the sensitivity and selectivity of the analysis of multiple vitamin D metabolites after chemical derivatization using different reagents for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Generally, chemical derivatization is applied to vitamin D metabolites to increase the ionization efficiency, which is particularly important for very low abundant metabolites. Derivatization can also improve the selectivity of the LC separation.

Stability of sample extracts of vitamin D metabolites after chemical derivatization for LC-MS/MS analysis.

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used to determine vitamin D metabolites in biological samples. The ionization efficiencies of these metabolites, however, are poor under electrospray ionization conditions. Moreover, the chromatographic separation of multiple vitamin D metabolites and their epimers can be challenging.

Sample preparation techniques for extraction of vitamin D metabolites from non-conventional biological sample matrices prior to LC-MS/MS analysis.

The determination of vitamin D metabolites as status marker or for diagnostic purposes is almost entirely conducted from blood serum or plasma. Other biological matrices, however, have also interested researchers, for two main reasons: (1) alternative matrices may allow non-invasive sampling, permit easier sample transfer and require less demanding storage conditions; and (2) the levels of vitamin D metabolites in other body compartments may further aid the understanding of vitamin D metabolism and function. Thus, the development of reliable and efficient sample preparation protocols for sample matrices other than serum/plasma, which will remove potential interferences and selectively extract the targeted metabolites, is of great importance.

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the "gold standard" method and integration into clinical practice.

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front- and back-end of MS-based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple-quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs.

GC-MS analysis of underivatised new psychoactive substances in whole blood and urine.

Herein a method was develop and validated for the detection and quantification of five new psychoactive substances (NPS) belonging to three categories: synthetic cathinones (mephedrone, 3,4-MDPV), opioids (AH-7921) and cannabinoids (JWH-018, AM-2201) by EI GC-MS. Target analytes were quantified in whole blood; in urine the same compounds plus methylone were detected. Liquid-liquid extraction by MTBE - butyl acetate (1:1, v/v) in blood and butyl acetate in urine was applied for the recovery of analytes, while no derivatization was necessary for their sensitive detection and quantification.

Α Simple Method for the Determination of Lacosamide in Blood by GC-MS.

Lacosamide is a functionalized amino acid with antiepileptic function. Therapeutic drug monitoring (TDM) in patients for lacosamide is critical as it allows clinicians to control epileptic seizures. A single liquid-liquid extraction step was applied for the extraction of lacosamide from whole blood samples which were thereafter analyzed by GC-MS.

C-PAmP: large scale analysis and database construction containing high scoring computationally predicted antimicrobial peptides for all the available plant species.

Background: Antimicrobial peptides are a promising alternative to conventional antibiotics. Plants are an important source of such peptides; their pharmacological properties are known since antiquity. Access to relevant information, however, is not straightforward, as there are practically no major repositories of experimentally validated and/or predicted plant antimicrobial peptides.

PepServe: a web server for peptide analysis, clustering and visualization.

Peptides, either as protein fragments or as naturally occurring entities are characterized by their sequence and function features. Many times the researchers need to massively manage peptide lists concerning protein identification, biomarker discovery, bioactivity, immune response or other functionalities. We present a web server that manages peptide lists in terms of feature analysis as well as interactive clustering and visualization of the given peptides.

A two-dimensional proteomic profile of Tetrahymena thermophila whole cell lysate.

Tetrahymena thermophila is a unicellular eukaryotic model organism used for a variety of biochemical, molecular and biological studies. According to its macronucleus genome sequence, it is expected to contain more than 27,000 protein-coding genes, although only a small proportion of them have information published specifically about them. Here, we present a reference map for whole cell lysate of T.

UniMaP: finding unique mass and peptide signatures in the human proteome.

Unlabelled: The uniqueness of a measured molecular mass or peptide sequence plays a very important role in the fields of protein identification and peptide/protein-biomarker investigation. We present a publicly available web application that offers information concerning the uniqueness of one or more molecular masses and one or more peptide sequences in the human proteome. When a sequence is found to be unique in humans, the application is able to search across all species querying whether this sequence is unique, not only in humans but also in other species found in the Swiss-Prot Database.

Peptide Finder: mapping measured molecular masses to peptides and proteins.

Unlabelled: The identification of unknown amino acid sequences of peptides as well as protein identification is of great significance in proteomics. Here, we present a publicly available web application that facilitates a high resolution mapping of measured molecular masses to peptides and proteins, irrespectively of the enzyme/digestion method used. Furthermore, multi-filtering may be applied in terms of measured mass tolerance, molecular mass and isoelectric point range as well as pattern matching to refine the results.

The role of aging, high fat diet and blue light exposure in an experimental mouse model for basal laminar deposit formation.

We sought to investigate the role of aging as a susceptibility factor for the capacity of dietary fat intake to increase the development of subretinal deposits. Mice of various ages (2, 9 and 16 months) were fed a normal diet or a diet high in saturated and unsaturated fats for a total four and a half months. Some eyes were also exposed to non-phototoxic levels of blue-green light.