Marco Ciulu

RP-HPLC determination of water-soluble vitamins in honey

The assessment and validation of reliable analytical methods for the determination of vitamins in sugar-based matrices (e.g. honey) are still scarcely explored fields of research. This study proposes and fully validates a simple and fast RP-HPLC method for the simultaneous determination of five water-soluble vitamins (vitamin B(2), riboflavin; vitamin B(3), nicotinic acid; vitamin B(5), pantothenic acid; vitamin B(9), folic acid; and vitamin C, ascorbic acid) in honey. The method provides low detection and quantification limits, very good linearity in a large concentration interval, very good precision, and the absence of any bias. It has been successfully applied to 28 honey samples (mainly from Sardinia, Italy) of 12 different botanical origins. While the overall amount of the analytes in the samples is quite low (always below 40 mg kg(-1)), we have observed a marked dependence of some of their concentrations (i.e. vitamin B(3) and vitamin B(5)) and the botanical origin of the honey. This insight might lead to important characterization features for this food item.

A direct RP-HPLC method for the determination of furanic aldehydes and acids in honey.

In this study 5-hydroxymethyl-2-furaldehyde (HMF), 2-furaldehyde, 3-furaldehyde, 2-furoic acid and 3-furoic acid are contemporarily determined in honey using a swift and direct RP-HPLC approach. The validation protocol was performed in terms of detection and quantification limits, precision (by repeatability and reproducibility), linearity and accuracy (by recovery tests); the acceptability of the precision and accuracy results was positively verified using Horwitzs model and AOAC guidelines, respectively. The method was tested on 18 honey samples of different ages, and botanical and geographical origin. HMF and 2-furaldehyde correlated highly with the age of the samples, whereas no correlation was observed with regards to 2-furaldehyde and 2-furoic acid. Hypotheses relating to the formation of minority furanic compounds are also proposed.

Recent Advances in the Analysis of Phenolic Compounds in Unifloral Honeys

Honey is one of the most renowned natural foods. Its composition is extremely variable, depending on its botanical and geographical origins, and the abundant presence of functional compounds has contributed to the increased worldwide interest is this foodstuff. In particular, great attention has been paid by the scientific community towards classes of compounds like phenolic compounds, due to their capability to act as markers of unifloral honey origin. In this contribution the most recent progress in the assessment of new analytical procedures aimed at the definition of the qualitative and quantitative profile of phenolic compounds of honey have been highlighted. A special emphasis has been placed on the innovative aspects concerning the extraction procedures, along with the most recent strategies proposed for the analysis of phenolic compounds. Moreover, the centrality of validation procedures has been claimed and extensively discussed in order to ensure the fitness-for-purpose of the proposed analytical methods. In addition, the exploitation of the phenolic profile as a tool for the classification of the botanical and geographical origin has been described, pointing out the usefulness of chemometrics in the interpretation of data sets originating from the analysis of polyphenols. Finally, recent results in concerning the evaluation of the antioxidant properties of unifloral honeys and the development of new analytical approaches aimed at measuring this parameter have been reviewed.

Gas chromatography analysis of major free mono- and disaccharides in milk: Method assessment, validation and application to real samples

Saccharides are functional constituents of milk. Although d-lactose represents almost the totality of the saccharides in the milk, minor species, like d-glucose, d-galactose, myo-inositol and, as a result of severe thermal treatments, monosaccharides like d-tagatose, are also detectable. Although chromatography has been the main analytical approach used for accomplishing this task, quite surprisingly a validated gas chromatographic method aimed at the simultaneous determination of these compounds is still needed. Hence, our contribution is devoted to fill this gap. After the optimization of clean-up and derivatization (conversion of saccharides in their trimethyl silyl ethers) steps, the adoption of a highly cross-linked silphenylene stationary phase permitted to obtain high resolution and a fast chromatographic run. Validation was accomplished in terms of limit of detection, limit of quantification, linearity, precision, and trueness. The accuracy of the method was successfully tested on a number of partially skimmed milk samples. Excellent limits of detection for all analytes make this method eligible, also with respect to a gas chromatographic/mass spectrometry approach, for routine analysis and quality control in the dairy industries.

HPLC determination of pantothenic acid in royal jelly

Although royal jelly constitutes one of the richest natural sources of pantothenic acid (vitamin B5), a reliable and validated chromatographic method to determine this analyte in this matrix has yet to be described in the literature. In this work we present an original RP-HPLC procedure to measure the concentration of pantothenic acid in royal jelly. A sample pre-treatment is needed to prevent the interference of high protein concentration in the matrix. The method has been validated in terms of LoD, LoQ, linearity, precision (repeatability and reproducibility) and bias. Finally, the whole procedure was tested on a number of samples of royal jelly from different origins, providing concentration values ranging from 120 ± 30 to 565 ± 40 mg kg−1 of pantothenic acid.

Determination of 5-hydroxymethyl-2-furaldehyde in royal jelly by a rapid reversed phase HPLC method

A new method for the determination of 5-hydroxymethyl-2-furaldehyde (HMF) in royal jelly by reversed phase HPLC is presented. The method is rapid and simple and it has been validated in terms of limits of detection and quantification, linearity, precision and accuracy, in addition to being tested on twenty-five real samples supplied by a local beekeeper. The method showed good limits of detection and quantification (0.53 mg kg−1 and 1.60 mg kg−1, respectively) and an excellent linearity (R2 = 0.9999). Precision values, expressed in terms of repeatability and reproducibility (3.8% and 15.6%, respectively), are acceptable according to Horwitzs theory. Average recovery values were 98 ± 2%. Preliminary data suggest that while HMF concentration in freshly harvested royal jelly and in samples stored at 4 °C is below the quantification limit, it is possible to measure quantifiable concentrations in samples stored at room temperature.

Relationship between markers of botanical origin in nectar and honey of the strawberry tree (Arbutus unedo) throughout flowering periods in different years and in different geographical areas

The amounts of four well-recognized markers of the floral origin of strawberry tree honey (i.e., 2,5-dihydroxyphenylacetic acid, homogentisic acid; (±)-2-cis,4-trans-abscisic acid, c,t-ABA; (±)-2-trans,4-trans-abscisic acid, t,t-ABA, and 2-(1,2-dihydroxypropyl)-4,8,8-trimethyl-1-oxaspiro[2.5]oct-4-en-6-one, unedone) were measured in the nectar of the strawberry tree throughout the flowering periods of two years and at various sampling sites. The concentration of these compounds was also measured in reference to strawberry tree honey samples from the same geographical origin in order to establish correlations and verify the possibility that one (or more) of them may act as marker(s) of the purity level, i.e., of the amount of nectar by strawberry tree flowers that has been transferred to the relevant honey. The data obtained show that despite the existence of a good correlation level between the markers, none of them at the moment can be proposed as a marker of purity of this honey, due to very complex interactions between contamination by foreign nectars in strawberry tree honey and spatial (geographical origin of the samples) and temporal (year and flowering period) parameters. Among these compounds, homogentisic acid appears to be the most reliable marker of botanical origin of strawberry tree honey due to its higher concentration with respect to those measured for isomers of abscisic acid and unedone.

A Possible Freshness Marker for Royal Jelly: Formation of 5-Hydroxymethyl-2-furaldehyde as a Function of Storage Temperature and Time.

In this article we present a study of the variability of the concentration of 5-hydroxymethyl-2-furaldehyde (HMF) in natural royal jelly (RJ) as a function of its storage temperature (-18, 4, and 25 °C) and time (up to 9 months after harvesting). For this work HMF is evaluated using an RP-HPLC method we previously assessed. While all RJ samples stored at 4 and -18 °C always showed levels of HMF under the limit of detection (0.13 mg kg(-1)), samples kept at 25 °C showed an exponential increase in the concentration of HMF as a function of the storage time. This behavior and a number of desirable features of the analytical method used (ease of use in routine laboratories, availability of a complete validation protocol specifically developed for RJ, based on consolidated chemical knowledge) allow us to hypothesize the use of HMF as a possible, reliable freshness marker for RJ.

Chemometric treatment of simple physical and chemical data for the discrimination of unifloral honeys

In this work, nonspecific physico-chemical parameters were determined in 160 honey samples belonging to the four main botanical categories present in Sardinia Island, Italy (strawberry tree, thistle, asphodel and eucalyptus) in order to develop a discriminant method for determining the botanical origin of honey. All the possible combinations of the seven physico-chemical parameters (pH, free acidity, electrical conductivity, color, total phenolic compounds, FRAP activity, and DPPH activity) measured in the honey samples were evaluated by Linear Discriminant Analysis (LDA). LDA models led to the prediction of each botanical origin with a very low level of misclassification (typically less than 5%). Since very high levels of correct prediction in cross validation (98.3%) and external validation (100%) were obtained considering only four parameters (i.e. pH, acidity, conductivity and DPPH), these results might allow a fast and easy control of the botanical origin of honeys.

CHAPTER 13:Assay of B Vitamins and other Water-soluble Vitamins in Honey

Honey is not a food rich in vitamins. Only water-soluble vitamins (B group and C) are ordinarily found in this matrix, at concentration levels usually between tenths and tens of mg/kg. Beyond their negligible nutritional value, the water-soluble vitamins in honey are of interest because they can be good indicators of its origin and freshness. This chapter discusses the analytical procedures adopted in the last 70 years to determine the water-soluble vitamins in honey. Emphasis is placed on the need for an adequate validation protocol to be applied to the whole analytical procedure.