Delia B. Rodriguez-Amaya

A scheme for obtaining standards and HPLC quantification of leafy vegetable carotenoids

Abstract Reliability of data obtained by HPLC directly depends on the accuracy of the calibration. A major difficulty is obtaining and maintaining pure standards. This work presents a strategy for isolating standards by open column chromatography and quantification by HPLC, using leafy vegetables as examples. The purity of standards evaluated by HPLC was 91–97% for neoxanthin, 95–98% for violaxanthin, 97–100% for lactucaxanthin, 92–96% for lutein and 90–97% for β-carotene. Calculation procedures were also evaluated, demonstrating that results obtained with one-point recalibration, straight-line equation (until 30 days after construction of the full calibration curves) and response factors relative to β-carotene were similar (CVs of 1.6–4.0%), being well below between-sample lot natural variation (CVs of 6.1–42.5%). The scheme proposed is relatively low-cost, provides a constant supply of carotenoid standards, including those unavailable commercially, and high sample throughput.

Mycotoxin research in Brazil: the last decade in review

O numero de artigos de pesquisa (128 artigos) sobre micotoxinas publicados por pesquisadores brasileiros em 1991-2000 superou a soma de artigos (85 artigos) publicados nas tres decadas anteriores (1961-1990). Trinta por cento das publicacoes foi levantamento da ocorrencia de micotoxinas em alimentos e racoes. Aflatoxinas em amendoim e produtos de amendoim continua sendo um problema alarmante, e a alta incidencia e niveis elevados de fumonisinas em milho e produtos de milho tambem parecem ser um problema serio. A contaminacao com outras micotoxinas, como zearalenona, ocratoxina A e tricotecenos, foi baixo. A ocorrencia de aflatoxina M1 em leite e laticinios e de patulina em suco de maca precisa ser verificada, pois, ha uma certa divergencia nos resultados. Trabalhos sobre os metodos analiticos, estudos micologicos e efeitos toxicos constituiram 16, 13 e 13%, respectivamente, dos artigos publicados na decada avaliada. A busca de meios de prevencao/controle da contaminacao fungica e producao de micotoxinas aumentou notadamente, perfazendo 27% dos artigos, incluindo investigacoes sobre fatores influentes (por exemplo, resistencia de genotipos, conteudo/atividade de agua, umidade relativa, temperatura, presenca de metais, tipo de solo, infestacao com inseto) e o potencial antagonico de outros microrganismos contra os fungos produtores de micotoxinas. Os efeitos de extrato de planta, flavonoides, fungicidas e outros quimicos, sacos utilizados para estocagem, adsorventes e processamento de alimentos foram tambem estudados. Portanto, apesar das limitacoes de recursos, a pesquisa brasileira responde as necessidades do pais, reflete as preocupacoes internacionais e os desenvolvimentos recentes na area.

Assessment of the saponification step in the quantitative determination of carotenoids and provitamins A

Abstract The saponification step in the determination of carotenoids was reassessed. Synthetic carotenoids (β-apo-8′-carotenal, β-carotene) and food samples (tomato, kale, papaya) were used and different procedures were evaluated. Hot saponification resulted in greater losses and cis - and epoxycarotenoids were formed. The degradation was aggravated by a more direct contact between the carotenoids and the alkali. The AOAC procedure, performed in the presence of acetone, led to complete transformation of β-apo-8′-carotenal to citranaxanthin. Saponification was unnecessary for kale and tomato but was needed for good separation of papaya carotenoids which included carotenol esters. Saponification of the carotenoids dissolved overnight at room temperature in petroleum ether, with equal volume of 10% methanolic KOH, retained β-, γ-carotene, β-apo-8′-carotenal and lycopene and completely hydrolysed the carotenol esters. However, even with this mild saponification, lutein, zeaxanthin and violaxanthin degraded significantly. These losses could be reduced to insignificant levels by using an atmosphere of nitrogen or antioxidant.

Optimization of HPLC quantification of carotenoids in cooked green vegetables—Comparison of analytical and calculated data

Data on cooked vegetables is lacking in carotenoid databases. Analysis of cooked vegetables has problems somewhat different from those of the raw produce. Although cooking makes the extraction of carotenoids easier, incorporation of oil and formation of degradation products during cooking pose analytical difficulties. Thus, a method developed for raw vegetables was optimized for cooked vegetables, particularly in the pre-chromatographic steps. In the absence of data obtained by laboratory analyses, carotenoid concentrations have been calculated from raw sample results. Since carotenoids are prone to degradation, assessment of retention or loss during cooking/processing is also needed. Multiplying the carotenoid concentrations of the raw sample by the %CCs (concentration change factors) gave the same values obtained by analyses of the cooked samples. The same concentrations could be obtained with the %TRs (true retention factors) only if the products obtained by multiplication were divided by the yield factors. The %TR correctly reflected the degradation percentage during cooking. These results are understandable, considering that weight gain or loss due to uptake or release of water, oil or food solids, must be compensated for to appraise carotenoid degradation. On the other hand, concentration changes brought about by cooking include both degradation and weight changes.

Aflatoxin M1 and ochratoxin A in a human milk bank in the city of São Paulo, Brazil

Because infants are more susceptible to the adverse effects of mycotoxins, this work was carried out to determine aflatoxin M1 (AFM1) and ochratoxin A (OA) in milk from the Human Milk Bank of the Southern Regional Hospital, São Paulo, Brazil. Analytical methods were first established and evaluated. The methods involved the extraction of AFM1 with methanol and OA with 1% aqueous sodium bicarbonate solution and methanol, clean-up with immunoaffinity columns having antibodies specific for each mycotoxin and quantification by high performance liquid chromatography (HPLC) with fluorescence detection. The method established for AFM1 had mean recovery percentages of 94, 77 and 82% and coefficients of variation of 17.5, 3.4 and 4.2% at 0.01, 0.03 and 0.05 ng ml−1, respectively. For the OA method, the corresponding values were 84, 84 and 75% for recovery and 14.1, 3.7 and 4.0% for the coefficient of variation. The limit of quantification for both methods was 0.01 ng ml−1. Of a total of 50 samples analysed, only one was contaminated with AFM1, at 0.024 ng ml−1, and two with OA, at 0.011 and 0.024 ng ml−1. Although the incidence observed was low, it is recommended that the study be extended to other milk banks of the city of São Paulo.

Simultaneous determination of total lipid, cholesterol and fatty acids in meat and backfat of suckling and adult pigs

Abstract Total lipid, cholesterol and fatty acids were determined in the meat, backfat and skin of suckling pigs and in meat cuts and backfat of adult pigs. Mean total lipid was similar (3.2–3.8%) in the meat of the suckling pigs and the adult pork loin, but lower (2.4%) in fresh ham. In the backfat, the values were 50, 68 and 92% for suckling (15 and 21 days) and adult pigs, respectively. Mean cholesterol levels were similar (98 and 95 mg/100 g) in the meat of the suckling pigs, and higher than those of pork loin and fresh ham (49 and 44 mg/100 g). The values obtained in the backfat were 102, 79 and 33 mg/100 g for the 15 and 21 day old and adult pigs, respectively. Of 47 fatty acids quantified, the principal components were 18:1n9, 16:0, 18:2n6, 16:1n7, 18:0, 18:1n7 and 14:0, although there was some inversion of the order.

Comparison of the cholesterol content of Brazilian chicken and quail eggs

There was no significant difference in the cholesterol levels of large, white- and dark-shelled eggs. Although slightly lower, the cholesterol concentration of small, white-shelled eggs was also not significantly different in terms of mg/g yolk. The cholesterol content of the quail egg in mg/g yolk was very similar to that of the chicken egg. No significant difference was seen also when the cholesterol levels of raw and cooked chicken eggs were compared. The overall average of the cholesterol content of the chicken egg was 12.0 mg/g yolk and that of the quail egg was 12.1 mg/g yolk.

Carotenoid composition of cooked green vegetables from restaurants

It is generally recognised that databases should be in terms of the food as eaten, but data on cooked foods are lacking. In this work, β-carotene, lutein, violaxanthin and neoxanthin in cooked green vegetables from restaurants were determined by HPLC. In general, there was no statistical difference in the carotenoid concentrations of vegetables taken from different restaurants. This result is surprising at first glance because of the many factors that can cause variation in the raw materials, aside from those brought about by the cooking conditions. However, the interplay of many factors might have masked individual effects. Moreover, taking the analytical sample from a large restaurant batch might have compensated the individual variations, giving more representative average concentrations. Between-lot variations of samples from the same restaurant were sometimes appreciable. Violaxanthin was the carotenoid most affected by cooking.

Occurrence of aflatoxin M1 in milk and dairy products commercialized in Campinas, Brazil

One hundred and fifty-two samples of pasteurized milk, powdered milk, cheese and yoghurt, marketed in Campinas, Brazil in 1989-1990, were analysed for aflatoxin M1 by the AOAC TLC method (visual quantitation) 980.21. Fifty-two pasteurized milk samples were also analysed in 1992 by the AOAC HPLC method 986.16. Aflatoxin M1 was not detected in the 1989-1990 samples. Four milk samples of the 1992 batch were contaminated at 73-370 ng/1. Except for the sample with 370 ng/l, which would have been also found positive by the TLC method, the detection of aflatoxin M1 in 1992 reflects the higher sensitivity of the HPLC method, not a greater occurrence of the toxin. Contamination of milk and milk products with aflatoxin M1 does not appear to be a serious public health problem in the city of Campinas at the moment.

Caracterização físico-química e capacidade antioxidante de pitanga (Eugenia uniflora L.)

Este estudo foi realizado para obter mais informacoes sobre as propriedades fisico-quimicas, composicao e atividade antioxidante de frutos de pitanga (Eugenia uniflora L.), especialmente os do Rio Grande do Sul (Brasil). Foram comparadas pitangas com diferentes coloracoes de polpa (roxa, vermelha e laranja) de selecoes cultivadas na Embrapa Clima Temperado (RS-Brasil). Foram observadas pequenas diferencas nos parâmetros de qualidade e na composicao centesimal e de acidos graxos entre as frutas com diferentes coloracoes de polpa. Os extratos de pitanga roxa apresentaram maiores conteudos totais de fenolicos e de antocianinas, bem como, a maior capacidade antioxidante. A capacidade antioxidante (valores de DPPH e FRAP) dos extratos metanolicos de pitanga apresentou alta correlacao com o conteudo de fenolicos totais, mas nos extratos etanolicos, o conteudo de antocianinas correlacionou-se apenas com a capacidade antioxidante avaliada pelo metodo de FRAP. A pitanga de cor laranja apresentou maiores teores de β-criptoxantina e β-caroteno, enquanto que a de cor vermelha continha alto teor de licopeno. Os resultados indicam que a pitanga de cor roxa, cultivada no Rio Grande do Sul, e uma fonte rica de compostos fenolicos e possui alta capacidade antioxidante. As de cor vermelha e laranja, por outro lado, sao fontes ricas de carotenoides.