Evaluation of new micro solid-phase extraction cartridges for on-column derivatisation reactions

A novel on-column derivatisation technique using micro solid-phase extraction (mu-SPE) cartridges has been evaluated and applied to the derivatisation of short-chain fatty acids in olive oil. The results show that mu-SPE is a good candidate for on-column derivatisation, giving good results with only a few microlitres of sample

Sensitive and quantitative determination of short-chain fatty acids in human serum using liquid chromatography mass spectrometry

Short-chain fatty acids (SCFAs) are increasingly being monitored to elucidate the link between gut health and disease. These metabolites are routinely measured in faeces, but their determination in serum is more challenging due to their low concentrations. A method for the determination of eight SCFAs in serum is described here. High-resolution mass spectrometry and gas chromatography were used to identify the presence of isomeric interferences, which were then overcome through a combination of chromatographic separation and judicious choice of MS fragment ion. The SCFAs were derivatised to form 3-nitrophenylhydrazones before being separated on a reversed-phase column and then detected using liquid chromatography tandem mass spectrometry (LC-QQQ-MS). The LODs and LOQs of SCFAs using this method were in the range 1 to 7 ng mL−1 and 3 to 19 ng mL−1, respectively. The recovery of the SCFAs in serum ranged from 94 to 114% over the three concentration ranges tested

Identifying H–N proximities in solid-state NMR using14N overtone irradiation under fast MAS

Two NMR methods for the identification of 1 H- 14N proximities in solid powder samples are considered. Both approaches involve the irradiation of the 14N overtone transition (Δm = 2) and observation of 1 H signals under fast MAS and at an intermediate magnetic field strength (11.7 T). First, the recently reported 1 H- 14NOT HMQC experiment is developed into a generally-applicable, broadband version using frequency-swept WURST pulses capable of exciting the full range of 14NOT signals. We demonstrate the validity of this approach on various samples, also showing that standard pulses can be used to selectively excite individual nitrogen sites and provide more accurate indirect 14NOT powder patterns from which 14N NMR parameters can be extracted if required. As a second approach, we present some preliminary results demonstrating the feasibility of 1 H- 14NOT overtone rotary recoupling (ORR) under fast MAS. Dephasing fractions of up to 0.3 have been obtained using a basic TRAPDORstyle pulse sequence, a simple approach that shows much potential for optimisation. WURST pulses are also shown to provide improvements in the recoupling bandwidth. Crucially, for both of these methods our experimental results and exact numerical simulations confirm that the +2ωr overtone sideband should be irradiated for optimal experimental efficiency

Off-line two-dimensional liquid chromatography for metabolomics: an example using Agaricus bisporus mushrooms exposed to UV irradiation

It has previously been shown that irradiation with UV light increases the vitamin D content of certain mushroom species, but the effect on other nutrients is unknown, and is difficult to assess due to the complexity of the sample matrix. Here, an offline reversed phase × reversed phase two-dimensional liquid chromatography methodology was developed and applied to Agaricus bisporus mushrooms in order to demonstrate the potential of the technique and assess the effect of UV irradiation on the mushroom's metabolic profile. The method allowed the detection of 158 peaks in a single analytical run. A total of 51 compounds including sugars, amino acids, organic and fatty acids and phenolic compounds were identified using certified reference standards. After irradiation of the mushrooms with UV for 30 s the number of peaks detected decreased from 158 to 150; 47 compounds increased in concentration while 72 substances decreased. This is the first time that two-dimensional liquid chromatography has been carried out for the metabolomic analysis of mushrooms. The data provide an overview of the gain/loss of nutritional value of the mushrooms following UV irradiation and demonstrate that the increased peak capacity and separation space of two-dimensional liquid chromatography has great potential in metabolomics

Combining computational and experimental approaches to select chromophores to enable the detection of fatty acids via HPLC

This paper outlines a protocol, which combines quantum mechanics calculations and experimental synthesis, to enable systematic selection of suitable chromophores based on their stability of fluorescence and efficiency of the chemical reaction. The experimental conditions were optimised for the esterification of fatty acids with the most effective chromophore, 5-(4-(diphenylamine)phenylthiopheny-2-yl)methanol

Multi-dimensional liquid chromatography and metabolomics, are two dimensions better than one?

While data processing methods in metabolomic studies often work with 'n' number of dimensions, analytical techniques, with the notable exception of NMR, have mostly stuck only to one. Peak overlap continues to be a problem and there is an ever-present demand to maximize the number of metabolites that can be separated and identified in a single run. One method that might help to overcome these issues is multidimensional liquid chromatography, which uses two columns of different phases. A sequential collection of aliquots is made from the first column and reinjected onto a second, and the resulting data are then plotted in 2D or 3D space. The total peak capacity of such a system is the combined peak capacities of each column. The 'offline' version of this technique, using a fraction collector, was introduced over 30 years ago but with recent advances in instrumentation and software, particularly the 'online' approach using automated switching valves, has led to increasing interest in the technique. Both offline and online methods can be carried out as a comprehensive procedure, or via 'heart-cutting', in which only specific peaks are analysed in the second dimension. Past applications include proteomics, natural product chemistry, forensic science and pharmaceutical analysis. These successes are likely to be built on in the future as new column chemistries and bio-informatic approaches are developed. In this review an overview of the theory of twodimensional liquid chromatography is presented, its potential in the field of metabolomics is assessed and predictions for future research directions are made

Combining computational and experimental approaches to select chromophores to enable the detection of fatty acids: Via HPLC

This paper outlines a protocol, which combines quantum mechanics calculations and experimental synthesis, to enable systematic selection of suitable chromophores based on their stability of fluorescence and efficiency of the chemical reaction. The experimental conditions were optimised for the esterification of fatty acids with the most effective chromophore, 5-(4-(diphenylamine)phenylthiopheny-2-yl)methanol

Infrared heating of cereals

Infrared heating is a technology that has many applications in post-harvest cereal production. Through its heating and drying effects, infrared radiation is today commonly applied to cereals to increase their shelf-life, and improve their safety by destroying spores, bacteria, or fungus as well as deactivating enzymes that would otherwise cause rancidity or off-flavours. The use of infrared (IR) has therefore improved much of the logistics of bringing cereals safely from the field to our table. While cereals are consumed as a staple food due to their high starch and protein content and ability to meet our daily energy demand, they also contain a plethora of bioactive compounds. The latter depending on their composition and concentration can have either health-promoting effects, such as anti-inflammatory, or may cause unwanted effects, such as reduction of digestibility of cereals. Interestingly, in many cases IR heating alters the biochemical compositions of cereals affecting starch, protein, lipid, phytochemical, and bioactive content as well as physical properties such as taste, odour, and texture. Therefore, for the cereal industry it is crucial IR strategies are optimised for the desired health benefits. The aim of this chapter is to summarise and discuss the effect of IR irradiation on the nutritional, functional, and biological characteristics of cereals