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Abstract

An interlaboratory comparison was done for the analysis of carotenoids in freeze-dried mango. The study was performed from July to September 2018. Mango fruit was freeze-dried, homogenized, and packaged under vacuum conditions in portions of 6 g (test sample). Two test samples were sent to the participating laboratories for analysis. Laboratory results were rated using Z-scores in accordance with ISO 13528 and ISO 17043. The standard deviation for proficiency assessment (also called target standard deviation) was determined using a modified Horwitz function and varied between 10 and 25%, depending on the analyte. Out of 14 laboratories from 10 different countries, 9 laboratories (64%) obtained a satisfactory performance (Z ≤ 2) for the analysis of β-carotene. While for 7 laboratories that analyzed α-carotene, (9Z)-β-carotene, β-cryptoxanthin, and zeaxanthin, 4 laboratories (57%) obtained a satisfactory performance. However, only 2 laboratories out of 7 (29%) obtained a satisfactory performance for lutein. Based on the comparability of the analytical results, this study concludes that freeze-dried mango pulp can be used as a reference material for the analysis of α and β-carotene, (9Z)-β-carotene, β-cryptoxanthin, and zeaxanthin by applying different analytical procedures for their extraction and quantification.

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For implementation of food and feed legislation, there is a strong need for development and harmonisation of reliable, validated and if possible, robust and simple analytical methods. In addition, precise methods used for measuring the exposure of humans to certain types of food contaminants and residues (natural, man-made or produced during technological treatment) such as, e.g. mycotoxins, acrylamide, pesticides and allergens have to be available, in order to compare results derived from monitoring studies. Methods should be validated (in-house or in a collaborative trial) according to harmonised protocols and good laboratory practice must be in place in order to be compliant with internationally harmonised standards. The way in which this is implemented depends strongly on the analyte, interference within the food matrix and other requirements that need to be met. Food and feed certified reference materials, when matrix matched and containing the appropriate concentration of the certified substance, are an extremely useful tool in validation of measurements.
Article
The method for separation and quantitative determination of the main carotenoids in food by high-performance liquid chromatography (HPLC) was in-house validated. Tomato (Lycopersicon esculentum M.) as food matrix was used to demonstrate its linearity, repeatability, intermediate precision, detection and quantification limits, sensitivity and bias. In addition, stability of carotenoids was studied in function of temperature and time. Method accuracy was quantified through measurement uncertainties estimate based on this validation study. Furthermore, a study was conducted to evaluate variability coming from location in an experimental field composed by 12 subfields. The use of two metal free reverse phase columns and an organic mobile phase based on acetonitrile, methanol and dichloromethane enabled the separation of the six target compounds (trans-α-carotene, trans-β-carotene, β-cryptoxanthin, all-lycopene, lutein, zeaxanthin) within a 30min run; detection at 450nm and external calibration allowed the quantification of the analytes. Carotenoids concentration and measurement uncertainty, in mg/100g, in tomato varieties "lido" and "for salad" were, respectively, 1.0±0.14 and 0.39±0.056 for trans-β-carotene, 8±2.0 and 2.3±0.57 for all-lycopene and 0.10±0.017 and 0.08±0.015 for lutein; trans-α-carotene, β-cryptoxanthin and zeaxanthin were not detected in both varieties (detection limits, in μg/100g, 0.81, 0.57 and 0.77, respectively). For β-carotene and lutein, uncertainty associated with the entire process including small-scale within-region variation was statistically different, at a significance level of 5%, from measurement uncertainty (which includes sampling in the laboratory). Copyright © 2007 Elsevier Ltd. All rights reserved.
Article
The efficiency of C30 stationary phases in the separation of carotenes and diverse hydroxycarotenoids has been the subject of several studies. However, little is known concerning their ability to resolve epoxycarotenoids isomers, whose study is of great importance due to the functions they serve and the information they can reveal concerning the processing of foods. We have concluded that C30 columns provide an excellent separation of structural, geometrical and optical isomers of epoxycarotenoids and that the presence of 5,8-epoxide groups leads to a better shape recognition, to the extent that over 10 geometrical-optical isomers of 5,8-epoxycarotenoids have been separated. Additionally, it was observed that these carotenoids elute later than their 5,6-epoxide counterparts, albeit the latter have a longer chromophore.
Article
Several factors render carotenoid determination inherently difficult. Thus, in spite of advances in analytical instrumentation, discrepancies in quantitative results on carotenoids can be encountered in the international literature. A good part of the errors comes from the pre-chromatographic steps such as: sampling scheme that does not yield samples representative of the food lots under investigation; sample preparation which does not maintain representativity and guarantee homogeneity of the analytical sample; incomplete extraction; physical losses of carotenoids during the various steps, especially during partition or washing and by adsorption to glass walls of containers; isomerization and oxidation of carotenoids during analysis. On the other hand, although currently considered the method of choice for carotenoids, high performance liquid chromatography (HPLC) is subject to various sources of errors, such as: incompatibility of the injection solvent and the mobile phase, resulting in distorted or split peaks; erroneous identification; unavailability, impurity and instability of carotenoid standards; quantification of highly overlapping peaks; low recovery from the HPLC column; errors in the preparation of standard solutions and in the calibration procedure; calculation errors. Illustrations of the possible errors in the quantification of carotenoids by HPLC are presented.
Article
Certified reference materials and materials distributed in proficiency testing need to be 'sufficiently homogeneous', that is, the variance in the mean composition of the distributed portions of the material must be negligibly small in relation to the variance of the analytical result produced when the material is in normal use. The requirement for sufficient homogeneity suggests the use of a formal test. Such tests as have been formulated rely on the duplicated analysis of the material from a number of portions, followed by analysis of variance. However, the outcome is not straightforward. If the analytical method used is very precise, then an undue proportion of the materials will be found to be significantly heterogeneous. If it is too imprecise, the test may be unable to detect heterogeneity. Moreover, the Harmonised Protocol Procedure (M. Thompson and R. Wood, Pure Appl. Chem., 1993, 65, 2123) seems to be unduly prone to the rejection of material that is in fact satisfactory. We present a simple new statistical approach that overcomes some of these problems.
Article
A variety of bonded phase parameters (endcapping, phase chemistry, ligand length, and substrate parameters) were studied for their effect on column retention and selectivity toward carotenoids. Decisions were made on how each of these variables should be optimized based on the separation of carotenoid and polycyclic aromatic hydrocarbon test probes. A column was designed with the following properties: high absolute retention, enhanced shape recognition of structured solutes, and moderate silanol activity. These qualities were achieved by triacontyl (C30) polymeric surface modification of a moderate pore size (approximately 20 nm), moderate surface area (approximately 200 m2/g) silica, without subsequent endcapping. The effectiveness of this "carotenoid phase" was demonstrated for the separation of a mixture of structurally similar carotenoid standards, an extract of a food matrix Standard Reference Material, and a beta-carotene dietary supplement under consideration as an agent for cancer intervention/prevention.
Article
The Horwitz ratio (HorRat) is a normalized performance parameter indicating the acceptability of methods of analysis with respect to among-laboratory precision (reproducibility). It is the ratio of the observed relative standard deviation among laboratories calculated from the actual performance data, RSDR (%), to the corresponding predicted relative standard deviation calculated from the Horwitz equation PRSDR (%) = 2C(-0.15), where C is the concentration found or added, expressed as a mass fraction. It is more or less independent of analyte, matrix, method, and time of publication (as a surrogate for the state of the art of analytical chemistry). It is now one of the acceptability criteria for many of the recently adopted chemical methods of analysis of AOAC INTERNATIONAL, the European Union, and other European organizations dealing with food analysis (e.g., European Committee for Standardization and Nordic Analytical Committee). The origin and applications of the formula are described. Consistent deviations from the ratio on the low side (values <0.5) may indicate unreported averaging or excellent training and experience; consistent deviations on the high side (values >2) may indicate inhomogeneity of the test samples, need for further method optimization or training, operating below the limit of determination, or an unsatisfactory method.
Things to know about the ring test [WWW Document
  • Ages Fao
FAO, AGES, 2015. Things to know about the ring test [WWW Document]. FAQ about FAO-IAG Ring Test. URL http://www.fao.org/ag/againfo/home/documents/2015_Announcement_Ring_test.pdf (accessed 7.16.20).
Vitamin A and Carotenoids: Chemistry, Analysis, Function and Effects (Food and Nutritional Components in Focus
  • P Crupi
  • V Preddy
  • R Antonacci
Crupi, P., Preddy, V., Antonacci, R., 2012. HPLC-DAD-MS (ESI+) determination of carotenoids in fruit, in: Preddy, V. (Ed.), Vitamin A and Carotenoids: Chemistry, Analysis, Function and Effects (Food and Nutritional Components in Focus, Volume 1).
Fruit and vegetables -your dietary essentials
  • Fao
FAO, 2020. Fruit and vegetables -your dietary essentials. FAO, Rome, Italy. https://doi.org/10.4060/CB2395EN
A comprehensive review on carotenoids in foods and feeds: status quo, applications, patents, and research needs
  • L Meulebroek
  • Van
  • N O'brien
Meulebroek, L. Van, O'Brien, N., 2021b. A comprehensive review on carotenoids in foods and feeds: status quo, applications, patents, and research needs. Crit. Rev. Food Sci. Food Nutr. https://doi.org/10.1080/10408398.2020.1867959