Fulvia Caretti

Evaluation of a method based on liquid chromatography–diode array detector–tandem mass spectrometry for a rapid and comprehensive characterization of the fat-soluble vitamin and carotenoid profile of selected plant foods

The feasibility of using reversed-phase liquid chromatography/diode array/tandem mass spectrometry (LC-DAD-MS/MS) for a rapid and comprehensive profiling of fat soluble vitamins and pigments in some foods of plant origin (maize flour, green and golden kiwi) was evaluated. The instrumental approach was planned for obtaining two main outcomes within the same chromatographic run: (i) the quantitative analysis of ten target analytes, whose standards are commercially available; (ii) the screening of pigments occurring in the selected matrices. The quantitative analysis was performed simultaneously for four carotenoids (lutein, zeaxanthin, β-cryptoxanthin, and β-carotene) and six compounds with fat-soluble activity (α-tocopherol, δ-tocopherol, γ-tocopherol, ergocalciferol, phylloquinone and menaquinone-4), separated on a C30 reversed-phase column and detected by atmospheric pressure chemical ionization (APCI) tandem mass spectrometry, operating in Selected Reaction Monitoring (SRM) mode. Extraction procedure was based on matrix solid-phase dispersion with recoveries of all compounds under study exceeding 78 and 60% from maize flour and kiwi, respectively. The method intra-day precision ranged between 3 and 7%, while the inter-day one was below 12%. The mild isolation conditions precluded artefacts creation, such as cis-isomerization phenomena for carotenoids. During the quantitative LC-SRM determination of the ten target analytes, the identification power of the diode array detector joined to that of the triple quadrupole (QqQ) allowed the tentatively identification of several pigments (chlorophylls and carotenoids), without the aid of standards, on the basis of: (i) the UV-vis spectra recorded in the range of 200-700nm; (ii) the expected retention time; (iii) the two SRM transitions, chosen for the target carotenoids but also common to many of isomeric carotenoids occurring in the selected foods.

Comprehensive profiling of carotenoids and fat-soluble vitamins in milk from different animal species by LC-DAD-MS/MS hyphenation.

This paper describes a novel and efficient analytical method to define the profile of fat-soluble micronutrients in milk from different animal species. Overnight cold saponification was optimized as a simultaneous extraction procedure. Analytes were separated by nonaqueous reversed-phase (NARP) chromatography: carotenoids on a C(30) column and fat-soluble vitamins on a tandem C(18) column system. Besides 12 target analytes for which standards are available (all-trans-lutein, all-trans-zeaxanthin, all-trans-β-cryptoxanthin, all-trans-β-carotene, all-trans-retinol, α-tocopherol, γ-tocopherol, δ-tocopherol, ergocalciferol, cholecalciferol, phylloquinone, and menaquinone-4), the DAD-MS combined detection allowed the provisional identification of other carotenoids on the basis of the expected retention times, the absorbance spectra, and the mass spectrometric data. Retinol and α-tocopherol were the most abundant fat-soluble micronutrients and the only ones found in donkeys milk along with γ-tocopherol. Ewes milk also proved to be a good source of vitamin K vitamers. Bovine milk showed a large variety of carotenoids that were absent in milk samples from other species with the only exception of all-trans-lutein and all-trans-zeaxanthin.

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and 1H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.

Evaluation of oxidized buckypaper as material for the solid phase extraction of cobalamins from milk: Its efficacy as individual and support sorbent of a hydrophilic-lipophilic balance copolymer.

This work describes a new analytical method for the determination of four cobalamins (adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) in cows milk. The extraction procedure is fast and based on dilution/protein precipitation of a milk sample with 50mM sodium acetate buffer (pH 4.6), followed by solid phase extraction (SPE) of the filtered supernatant. Relative recoveries higher than 60% have been obtained for all the cobalamins by combining two different types of sorbents in the same SPE cartridge: two disks of buckypaper (BP), a nanoporous felt composed of oxidized multiwalled carbon nanotubes (MWCNTs), separated by a Teflon frit from OASIS HLB (500mg), a hydrophilic-lipophilic balance copolymer. Before its use as sorbent, BP was characterized in terms of porosity, permeability, surface area, specific adsorption capacity and tested for a potential reuse after adequate chemical regeneration. The analysis of the extracts was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) on an analytical C18 column in less than 10min. After validation, the method was applied to the determination of the natural content of the four B12 homologues in cows milk samples, providing data lacking in the literature.

Occurrence of non-steroidal anti-inflammatory drugs in surface waters of Central Italy by liquid chromatography–tandem mass spectrometry

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered to be the cure-all of any pain: from headache and cold symptoms to toothaches and even labour pain. Their wide use in human medicine has been favoured by the possibility to purchase them without medical prescription, their low cost and by the absence of opioid-like side effects. At the same time, their administration to domestic and food-producing animals has become a common practice to improve their well-being. Therefore, human and veterinary applications are the main sources of NSAIDs in the environment and the major pathways are excretion and discharge through sewage treatment plants. Although their environmental occurrence is well-known, there is a lack of data regarding their levels in surface waters of Central Italy. In this study, a monitoring campaign was arranged in some of the most important rivers and lakes of Central Italy, characterised by a different anthropic impact, in spring–summer and autumn–winter 2012. A broad range of NSAIDs for human and animal use was analysed through a reliable analytical method based on liquid chromatography–tandem mass spectrometry, appropriately developed and validated. Results have shown the constant presence of all drugs commonly used in human medicine with a composition that mirrored the incidence of seasonal diseases quite well. A veterinary drug (flunixin) was found in Bracciano Lake (Rome district), an important tourist attraction surrounded by farmlands. Salicylic acid is a phytohormone and this explains its presence in all the analyzed samples. All the results collected during the extensive survey have proved that Central Italy is aligned with the rest of Europe since its natural waters have shown low levels of contamination (ng L−1) but with a chronic input.

Analysis of vitamins by liquid chromatography

Abstract High-performance liquid chromatography (HPLC) has become the technique of choice to perform an accurate determination of water-soluble and fat-soluble vitamins and provitamin A carotenoids in foods, especially for routine work. An overview of main HPLC methods for the individual and simultaneous vitamin analysis in foods is here presented. All precautions indispensable for handling these analytes, easily susceptible to degradation, are described as well as problems connected with the sample preparation, chromatographic separation and detection. Difficulties related to the development of multivitamin methods are also assessed and the potentiality of the latest extraction and chromatographic detection techniques are highlighted.

Residue analysis of thyreostats in baby foods via matrix solid phase dispersion and liquid chromatography – dual-polarity electrospray – tandem mass spectrometry

ABSTRACT This paper describes a rapid method for confirming residues of thyreostats in meat-based baby foods by using liquid chromatography – dual polarity electrospray – tandem mass spectrometry (LC-ES(±)-MS/MS). Six thioureylenes, belonging to the group of thiouracil and imidazole, were selected for this work: thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU), phenylthiouracil (PhTU), mercaptobenzimidazole (MBI) and tapazole (TAP). The amphoteric nature of these compounds allows their electrospray detection in both positive and negative ionisation. Nevertheless, MS detection is not favoured by their low molecular weights, while their chromatographic retention is also thwarted by their high polarity. A pentafluorophenyl (PFP) core-shell phase column was selected to avoid peak asymmetry or peak splitting, and a dual-polarity ionisation method was optimised to obtain a sensitivity as high as possible. The method was validated according to the Commission Decision 657/2002/EC. A simple and fast procedure based on matrix solid phase dispersion (MSPD) was optimised to extract analytes from baby foods with recoveries exceeding 82%. Limit of decision (CCα) and detection capability (CCβ) were lower than the permissible maximum concentration (10 ng g−1). The validated method was then applied to assess the potential occurrence of the six selected thyreostats in nine commercial products. All the samples were found free of contamination.

Chapter 16:LC-DAD-tandem MS Analysis of Retinoids and Carotenoids: Applications to Bovine Milk

Milk is the most complete single food in the human diet which constitutes the only source of feeding for newborns and which continues to be consumed as a nutritional supplement during juvenile and adult life. Nutrients of this complex physiological fluid are distributed in three chemical phases: lip...

Subcritical water extraction of thyreostats from bovine muscle followed by liquid chromatography-tandem mass spectrometry

ABSTRACT Thyreostats can be used fraudulently to promote a rapid increase in weight of breeding animals at low cost. Their severe toxicological effects impose the development of reliable analytical methods to be used in monitoring plans. This work describes an alternative approach to isolate residues of thiouracil, methyl-thiouracil, propyl-thiouracil, phenyl-thiouracil, tapazole and mercaptobenzimidazole from bovine muscle tissue. The developed procedure is based on the following three steps: i) matrix solid-phase dispersion with C18 for the preliminary sample preparation; ii) subcritical water extraction (SWE) at 160°C and 100 bar; iii) clean-up on an Oasis HLB cartridge. The quantitative determination was performed by LC-MS/MS in dual polarity ionization by using internal standardization. The SWE-LC-MS/MS method was validated according to the identification criteria of the Commission decision 2002/657/EC. The relative recoveries ranged from 72 to 97%; within-lab reproducibility was less than 18%. The decision limit and the detection capability of all analytes were below the recommended concentration, set at 10 µg kg−1, but the validation results demonstrated that this method could only be applied for screening of thiouracil and methyl-thiouracil. Besides the analytical advantages related to the use of water as solvent extraction, the procedure allowed significant removal of lipids, whose detrimental effects on instrumentation and MS sensitivity are well-known. Graphical Abstract

Chapter 19 – Analysis of vitamins by liquid chromatography

Liquid chromatography (LC) is the technique of choice to perform the individual and simultaneous analysis of water- and fat-soluble vitamins and carotenoids, especially in complex matrices such as foods. This chapter offers a comprehensive overview of the main LC methods, describes the precautions necessary for handling these problematic substances in each analytical phase, and examines the difficulties connected with the sample preparation, chromatographic separation, and detection. Each section also affords insights on last generation chromatographic materials, innovative methodological approaches, and trends emerged during the last years in this research field.