Extraction protocol optimized and applied for betaine determination in beetroot pulp, peel and juice.
Betaine was expected to be retained on the stationary phase due to polar interactions, e. In order to improve peak symmetry and to increase sensitivity, a buffer was introduced in the mobile phase. Acetonitrile-rich mobile phase, as known, are beneficial for MS sensitivity.
Despite the slight peak width broadening observed with acid eluents, acid conditions were preferred to enhance MS sensitivity in the positive ion mode. On this basis, the transition Since it is well established that the MRM sensitivity depends drastically on the tuning of instrument parameters [ 16 ], an optimization of collision energy CoE , fragmentor voltage FV and temperature of the source Temp was performed by means of a CCD.
The response variable was the betaine MRM peak area. It is worth to be mentioned it is well known that the expected concentrations of betaine in beetroots samples are not in trace levels. Main and quadratic effects of the variables considered were calculated with Yates algorithm and R-chemometric software. A graphical representation of the b i and b ii coefficients is represented in Fig. As shown, the linear term X 2 FV suggests that this parameter strongly influences the response.
In detail the increase of FV causes a decrease of the response. On the contrary, the increase of the temperature of source leads to a higher response. The CoE parameter only slightly influences the sensitivity for betaine. However, all the quadratic terms from 4 to 6, corresponding to b ii coefficients have high values, meaning that the dependence of the response on parameters is not linear, as confirmed by the response surfaces graphs Fig. Lines in Fig. As shown, experimental conditions that lead to the highest peak area can be easily derived. There were found to be 0.
It should be remarked that very few approaches for determination of betaine by detectors different from MS are available. Shin et al.
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In order to extract betaine from beetroot samples and to isolate the molecule from interfering species, an extraction procedure was optimized comparing the performance of different approaches. In previous studies showing the determination of betaine in numerous food and vegetable matrices, solid samples were simply mixed with water, homogenized and centrifuged to recover the aqueous supernatant. The aqueous supernatant was then extracted with dichloromethane which removes hydrophobic compounds without removing betaine. Also, in case of liquid samples, they were shaken with an equal volume of dichloromethane, centrifuged and the resulting aqueous layer directly employed for analysis [ 17 , 21 ].
However, these procedures are not selective towards betaine, and high hydrophilic and polar compounds are dissolved in the aqueous phase which is injected and analysed. As well, described by Wruss et al. All the above-mentioned compounds are possible interfering species that should be removed and, therefore, the optimization of purification method to isolate betaine is discussed in the next paragraphs. As previously mentioned, betaine could be present in both liquid juice and solid peel and pulp matrices. For the latter, extraction and clean-up steps are necessary, while for liquid samples, both the steps could be done within the same technique.
The analysis of the washing solutions, before the elution step, showed no evidence of betaine. Since we observed that when using pure silica cartridge, betaine has to be dissolved in methanol; otherwise, interactions with sorbent are weak and low recoveries are obtained, this solvent was used during the loading step.
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Aromatic sulfonic acid groups present in the SCX interact with the positively charged amino group of betaine by electrostatic interactions. Consequently, eluents that can give rise to competitive interaction were evaluated. The expected interaction between the silica sorbent and betaine is based on polarity and adsorption on hydrogen bonding. Since the procedure for extraction and recovery of betaine appeared to be very easy, cheap and effective, the same adsorbent and elution conditions were selected for MEPS technique as well. However, the enrichment factor is not as high as in other methods [ 22 ].
Recovery yields obtained for each tested substrate.
For activation and recovery procedures, see text. In order to evaluate the efficiency of the SPE technique on real matrices, the procedure was used for the determination of betaine in beetroot juice. The study has been performed on both the red and golden beetroot juices. Recoveries were Lower recovery values, compared to standard solution samples, could be ascribed to competition effects that occurred between the matrix of the beetroot juice and the silica sorbent.
In Fig. Full scan Fig. The chromatograms obtained both in full scan and MRM detection are overlaid in Fig. Analysis of the eluted fraction after SPE extraction of betaine from Beta vulgaris golden juice.
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Betaine full-scan spectrum and product ion spectrum a and b , respectively , and chromatogram overlay of the betaine signal obtained both in MRM and full-scan mode c. Since it was not possible to evaluate the extraction efficiency on a blank solid sample, tests were performed comparing betaine concentration in pulp and peel of red and golden beetroots with the concentration of spiked samples.
For both pulp and peel matrices, high recoveries from To the best of our knowledge, this is the first time that an ASE procedure is proposed for the extraction of betaine from beetroots. Once optimized, the procedure for the extraction of betaine from both the solid and the liquid parts of the beetroots, a quantitative determination of this compound was performed in real samples. In literature, many studies determine the content of betaine in foods, especially in sugar beet and molasses, but this is the first time in which betaine content is specifically quantified for each portion of B.
It can be observed that B. Comparison of the betaine concentration level in all the three portions juice, peel and pulp of Beta vulgaris red and golden. Data have been compared with determinations already performed on sugar beets and on different species of the same family of B. It is interesting to highlight that B. As an example, betaine concentration is typically about 0. The extraction procedures optimized in the previous paragraphs allow us to extract betaine from all portions of B.
However, for solid samples as peel and pulp , this extraction is accomplished by two different, consecutives techniques: ASE for extraction and SPE for isolation of betaine. The QuEChERS technique, developed for the extraction of pesticides from food [ 24 ], with extensive applications event to other analytes and matrices [ 25 , 26 ] is an easy and fast procedure, since it is based on liquid extraction of target analytes, assisted by hand shaking and followed by a dispersive-SPE d-SPE clean-up step, using selective adsorbents.
A modified QuEChERS procedure was here tested on the peel of golden beetroot: this portion was chosen since, according to the previous results, it has the highest concentration of betaine. In spite of acetonitrile, used in the traditional procedure, methanol was selected as the extraction solvent, due to the high affinity towards betaine observed in the extraction techniques previously commented. Magnesium sulphate and sodium chloride salts were added in order to enhance the salting-out effect, promoting a better extraction of betaine.
PSA is necessary to remove co-extracted sugars and weak organic acids, while GCB is necessary to remove dyes and aromatic interferences.
It should be mentioned that the replacement of GCB with a C18 resin was not effective in removing co-extracted dyes and additional purification would be necessary, losing the intrinsic advantages of QuEChERS technique. In conclusion, the method detection limits MDLs obtained for betaine extraction from B. Victor R. He is a leading expert on nutritional and food sciences and has a long standing interest in how food, food quality, nutrition and diet affect wellbeing and health.
He has lectured nationally and internationally. To his credit, Professor Preedy has over publications, which includes peer-reviewed manuscripts based on original research, abstracts and symposium presentations, reviews, books and volumes. Language of text : English. Language of summary : English.
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