Comparison of HPLC and GLC methodologies for determination of glucosinolates using reference material

 The reference material rapeseed of known glucosinolate composition, was analysed by HPLC and GLC and two commercially available glucosinolates (glucotropaeolin and sinigrin) were used as internal standards. The HPLC method enabled determination of 11 different glucosinolates (as desulphoglucosinolates) that occur in the rapeseed. Only seven glucosinolates (as trimethylsilylated desulphoglucosinolates) were separated by GLC. The latter method did not allow the determination of methylsulphinylalkyl glucosinolates (glucoiberin, glucoraphanin and glucoallysin) and did not separate optical isomers of 2-hydroxy-3-butenyl glucosinolate (progoitrin and epi-progoitrin). Statistical evaluation of data (t-test, F-test) revealed no significant differences between the tested methods at the 95% confidence level. The advantages and disadvantages of both widely used chromatographic methods are discussed. Received: 26 May 1997 / Revised version: 11 July 1997     

HPLC determination of polycyclic aromatic hydrocarbons in olive oils

 In this paper, HPLC with spectrofluorimetric detection was applied to the determination of polycyclic aromatic hydrocarbons (PAHs) in olive oils. These compounds may sometimes contaminate vegetable oils because of their specific lipophilic characteristics, which are a significant problem for their extraction and purification from lipid matrices. Some improvements to previously published methods are introduced and satisfactory results for repeatability and recovery were obtained. Data on 51 authentic olive oil samples are reported and it was found that there is usually a limited presence of PAHs in extra virgin olive oils; furthermore, the analysis of some blends of refined and virgin oils shows that the distributions of light and heavy PAHs are different with the content of the former being lower in refined samples. As an example of this fact, two samples of lampante oil were followed throughout the refining step. Received: 23 September 1996 / Revised version: 17 December 1996     

Separation and characterization of cis-trans isomers of α-tocotrienol by HPLC using a permethylated β-cyclodextrin phase

 The behaviour of α-, β-, γ- and δ-tocotrienols (structurally related to tocopherols with naturally trans-configurated double bonds in the side-chain) during HPLC on a permethylated β-cyclodextrin phase was studied. A newly developed isocratic HPLC method is described with an acetonitrile/water (58:42 by vol.) eluent and fluorescence detection at an emission wavelength of 330 nm (excitation wavelength = 295 nm). Each of the separately injected tocotrienols exhibited four peaks. The four separated components of α-tocotrienol were identified by spectroscopy (MS, ¹H-NMR, FTIR) to be structural side-chain isomers. The order of retention times was determined to be all-cis-α-tocotrienol followed by cis-trans-/trans-cis-α-tocotrienol (differentiation between the two compounds was not possible) with all-trans-α-tocotrienol as the last eluting isomer. Received: 27 June 1997     

HPLC analysis of γ-irradiation-induced products of tryptophan in peptides and lysozyme

 The products of γ-irradiation of tripeptides (AWA, LWL, LWM) and lysozyme were determined by HPLC and UV/fluorescence detection. A fast and simple one-step hydrolysis with pronase E (30 – 60 min, 40°C) was developed to release the radiation products, without damage, from the peptide chain. N-Formylkynurenine (NFK), oxindolylalanine (OIA), 4-, 5-, 6- and 7-hydroxytryptophan were the main products of irradiation of peptides and lysozyme. It is possible that the nonphysiological hydroxytryptophan isomers 4-, 6- and 7-hydroxytryptophan could serve as marker substances for irradiated food with a high protein content. Received: 7 April 1997 / Revised version: 10 June 1997     

HPLC analysis of γ-irradiation-induced products of tryptophan in peptides and lysozyme

 The products of γ-irradiation of tripeptides (AWA, LWL, LWM) and lysozyme were determined by HPLC and UV/fluorescence detection. A fast and simple one-step hydrolysis with pronase E (30 – 60 min, 40°C) was developed to release the radiation products, without damage, from the peptide chain. N-Formylkynurenine (NFK), oxindolylalanine (OIA), 4-, 5-, 6- and 7-hydroxytryptophan were the main products of irradiation of peptides and lysozyme. It is possible that the nonphysiological hydroxytryptophan isomers 4-, 6- and 7-hydroxytryptophan could serve as marker substances for irradiated food with a high protein content. Received: 7 April 1997 / Revised version: 10 June 1997     

Development of an HPLC method for measurements of the stability of Irganox-type polymer antioxidants in fatty food simulants

 A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed to measure the stability of four Irganox-type polymer antioxidants (Irganox 245, Irganox 1035, Irganox 1098 and Irganox 3114) in an olive oil food simulant and isooctane, which has been proposed as an alternative fatty food simulant. The tests of stability in olive oil were carried out under three different conditions, i.e. 40°C for 10 days, 100°C for 1 h and 175°C for 1 h. The exposure conditions for isooctane were 60°C for 3 h. Results showed that for all additives tested no instability phenomena in olive oil or isooctane simulants were observed under the exposure conditions applied. The analytical methodology developed could eventually be used for stability testing and migration studies of other similarly structured antioxidants in fatty food simulants. Received: 11 September 1997     

Development of an HPLC method for measurements of the stability of Irganox-type polymer antioxidants in fatty food simulants

 A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed to measure the stability of four Irganox-type polymer antioxidants (Irganox 245, Irganox 1035, Irganox 1098 and Irganox 3114) in an olive oil food simulant and isooctane, which has been proposed as an alternative fatty food simulant. The tests of stability in olive oil were carried out under three different conditions, i.e. 40°C for 10 days, 100°C for 1 h and 175°C for 1 h. The exposure conditions for isooctane were 60°C for 3 h. Results showed that for all additives tested no instability phenomena in olive oil or isooctane simulants were observed under the exposure conditions applied. The analytical methodology developed could eventually be used for stability testing and migration studies of other similarly structured antioxidants in fatty food simulants. Received: 11 September 1997     

SPE and MSPD as pre-separation techniques for HPLC of tetracyclines in meat, milk and cheese

 Solid phase extraction (SPE) and Matrix solid phase dispersion (MSPD) have been tested as pre-separation procedures for high-performance liquid chromatography (HPLC) determination of tetracycline antibiotics in milk, meat and cheese. The extraction recoveries ranged from 48% to 86% for SPE and from 89% to 93% for MSPD at concentration levels of the maximal residual limits (MRL) recommended by the European Union to be 100 ng/g for the tetracyclines oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC). The detection limits were 15 – 22 ng/g for SPE and 30 ng/g for MSPD. Following the relatively simple SPE procedure we used a Lichrosorb RP-18 column and a diode array detector for HPLC. Results dealing with analysis of tetracyclines HPLC in cheese are published for the first time. Received: 7 March 1997     

HPLC determination of stevioside in plant material and food samples

 An HPLC method for the determination of sweet-tasting stevioside (STS) in the leaves of the plant Stevia rebaudiana and in some beverages (e.g. tea, orange juice) was developed. The pre-separation procedure consisted of extraction of STS from the plant material using boiling water and a solid-phase extraction (SPE). Recovery rates of the SPE for the analysed matrices ranged from 92.8% to 97.8% (for concentrations of STS of 105, 210 and 300 μg/ml; Relative Standard Deviation (RSD)≤3.3%). The chromatographic separations were realized using a C₁₈ column, the mobile phase consisting of methanol and water, and with UV detection at 210 nm. The limits of determination of STS were 5 μg/ml for the leaf extracts and the tea sample and 8 μg/ml for the juice sample. Received: 7 April 1998     

HPLC determination of stevioside in plant material and food samples

 An HPLC method for the determination of sweet-tasting stevioside (STS) in the leaves of the plant Stevia rebaudiana and in some beverages (e.g. tea, orange juice) was developed. The pre-separation procedure consisted of extraction of STS from the plant material using boiling water and a solid-phase extraction (SPE). Recovery rates of the SPE for the analysed matrices ranged from 92.8% to 97.8% (for concentrations of STS of 105, 210 and 300 μg/ml; Relative Standard Deviation (RSD)≤3.3%). The chromatographic separations were realized using a C₁₈ column, the mobile phase consisting of methanol and water, and with UV detection at 210 nm. The limits of determination of STS were 5 μg/ml for the leaf extracts and the tea sample and 8 μg/ml for the juice sample. Received: 7 April 1998     

HPLC determination of trimethoprim in meat and milk with an SPE preseparation procedure

 An HPLC method in combination with solid-phase extraction and dual wavelength detection has been developed in order to monitor trimethoprim residues in meat and milk. The extraction recoveries for the maximal residual limit concentration in food (50 ng/g) were 86±5% for meat and 73±6 % for milk samples. The limits of determination of trimethoprim were 5 ng/g at 229 nm and about 15 ng/g at 280 nm. The method has been developed in response to specific needs of food control laboratories. This publication introduces the assay for milk and meat samples. Received: 27 March 1996/Revised version: 23 July 1996     

On the reaction of L-ascorbic acid with propylamine under various conditions: quantification of the main products by HPLC/DAD

 The reaction of _L-ascorbic acid (AA) with proteins (protein ascorbylation) has a considerable impact on food processing and health. So far four products have been isolated and identified which can be formed from AA and lysine derivatives. Thus, 2-deoxy-2-(propylamino)ascorbic acid, 3-deoxy-3-(propylamino)ascorbic acid, oxalic acid monopropylamide and oxalic acid dipropylamide were synthesized and an HPLC system for their separation and quantification was developed. Then, mixtures of AA and propylamine, as a model compound for lysine derivatives, were reacted under various conditions and product yields were determined in relation to reaction time, temperature, amine concentration and pH value. The results were discussed in detail and can help to evaluate the contribution of the four products to protein and lysine ascorbylation under different conditions. Received: 2 September 1997 / Revised version: 27 November 1997     

On the reaction of L-ascorbic acid with propylamine under various conditions: quantification of the main products by HPLC/DAD

 The reaction of _L-ascorbic acid (AA) with proteins (protein ascorbylation) has a considerable impact on food processing and health. So far four products have been isolated and identified which can be formed from AA and lysine derivatives. Thus, 2-deoxy-2-(propylamino)ascorbic acid, 3-deoxy-3-(propylamino)ascorbic acid, oxalic acid monopropylamide and oxalic acid dipropylamide were synthesized and an HPLC system for their separation and quantification was developed. Then, mixtures of AA and propylamine, as a model compound for lysine derivatives, were reacted under various conditions and product yields were determined in relation to reaction time, temperature, amine concentration and pH value. The results were discussed in detail and can help to evaluate the contribution of the four products to protein and lysine ascorbylation under different conditions. Received: 2 September 1997 / Revised version: 27 November 1997     

Comparison between two commercial ELISAs and a culture procedure for the detection of Listeria spp.

 The efficiency of two commercial enzyme-linked immunosorbent assays (ELISAs), the Oxoid Listeria rapid test and the Tecra Listeria visual immunoassay for the detection of Listeria spp. was compared with that of a culture method. Of the 60 samples examined by ELISA and the culture procedure, Listeria was detected and confirmed by culture in 20, 34 and 44 samples by the Tecra, Oxoid and microbiological methods, respectively. The overall sensitivity of the Tecra and Oxoid tests was 50% and 93%, respectively. Both ELISAs had a specificity of 100%. The efficiency of the Oxoid test was 95%, while that of the Tecra assay was 67%. Differences in the results of the Oxoid test and those of the culture method were not statistically significant; however, the differences between the results of the Tecra assay and the culture procedure were significant. The Oxoid test was less labour intensive, less time consuming and easier to perform than the Tecra assay. It was concluded that the Oxoid test is a good alternative method to the culture procedures when screening for the presence of Listeria spp. in foods, especially when a large number of samples have to be analysed. Received: 24 June 1997     

Comparison between two commercial ELISAs and a culture procedure for the detection of Listeria spp.

 The efficiency of two commercial enzyme-linked immunosorbent assays (ELISAs), the Oxoid Listeria rapid test and the Tecra Listeria visual immunoassay for the detection of Listeria spp. was compared with that of a culture method. Of the 60 samples examined by ELISA and the culture procedure, Listeria was detected and confirmed by culture in 20, 34 and 44 samples by the Tecra, Oxoid and microbiological methods, respectively. The overall sensitivity of the Tecra and Oxoid tests was 50% and 93%, respectively. Both ELISAs had a specificity of 100%. The efficiency of the Oxoid test was 95%, while that of the Tecra assay was 67%. Differences in the results of the Oxoid test and those of the culture method were not statistically significant; however, the differences between the results of the Tecra assay and the culture procedure were significant. The Oxoid test was less labour intensive, less time consuming and easier to perform than the Tecra assay. It was concluded that the Oxoid test is a good alternative method to the culture procedures when screening for the presence of Listeria spp. in foods, especially when a large number of samples have to be analysed. Received: 24 June 1997     

Determination of food contamination by mineral oil material from printed cardboard using on-line coupled LC-GC-FID

 Inks used for printing paper and cardboard are dispersions of synthetic organic pigments in a bonding agent system built up essentially of resins, vegetable oils and high-boiling-point mineral oil products (b.p. >250°C). The proportion of mineral oil material in the ink ranges between 20 and 30%. The more volatile mineral oil components slowly evaporate from the printed cardboard box and may migrate into the food product. They contaminated cereals and dry baby-food products at concentrations between 10 and 150 mg/kg. Received: 27 January 1997     

Evaluation of xanthine derivatives in chocolate – nutritional and chemical aspects

 Organic substances containing nitrogen are widespread throughout the whole of the natural world. Also included amongst these are the methylxanthine derivatives caffeine, theobromine and theophylline. These are very closely related and are to be found in extremely varying contents in different plants. Because of their pharmacological effect, which is fundamentally of a stimulative nature, methylxanthines have been significant since time immemorial as luxury, non-essential foodstuffs and as medication. These substances are often referred to as alkaloids. Theobromine is the major alkaloid in cacao (Theobroma cacao). Caffeine, on the other hand, is only to be found in cacao in very small quantities and theophylline only in trace amounts. Methylxanthines are said to contribute towards the typically bitter taste of cacao. In addition they represent important analytical parameters with regard to the evaluation of the quality of cacao-containing and chocolate products. The amount of methylxanthines in cacao depends on various influencing factors, the most essential ones being processing procedures, genotype, geographical origin and cacao bean weight. By determining the amount of theobromine and caffeine in cacao and/or cacao products, the amount of fat-free dry cacao can also be estimated. There are various methods available for the detection and determination of methylxanthines. UV-spectrophotometric determination and analysis using HPLC have attained major significance here. With regard to human nutrition, cacao and cacao products are the main natural sources of theobromine, but significantly less so with regard to caffeine. The presence of methylxanthines in cacao products leads to that they are sometimes the subject of consumer criticism, particularly because they are considered as luxury foodstuffs. The concluding evaluation of methylxanthines by dieticians and toxicologists reveals that plants that contain methylxanthines have been consumed for as long as can be remembered without having led to damage to health. Scientific research has shown that the consumption of these substances has no consequence whatsoever, as long as consumption is not excessive, which is the case for all foodstuffs. Methylxanthines in cacao products have no negative effects on the health of humans because their amounts in chocolates and other processed foodstuffs containing cacao are so low that they account for only a very small proportion of the whole of the human diet. Received: 15 November 1996 / Revised version: 22 January 1997     

Evaluation of xanthine derivatives in chocolate – nutritional and chemical aspects

 Organic substances containing nitrogen are widespread throughout the whole of the natural world. Also included amongst these are the methylxanthine derivatives caffeine, theobromine and theophylline. These are very closely related and are to be found in extremely varying contents in different plants. Because of their pharmacological effect, which is fundamentally of a stimulative nature, methylxanthines have been significant since time immemorial as luxury, non-essential foodstuffs and as medication. These substances are often referred to as alkaloids. Theobromine is the major alkaloid in cacao (Theobroma cacao). Caffeine, on the other hand, is only to be found in cacao in very small quantities and theophylline only in trace amounts. Methylxanthines are said to contribute towards the typically bitter taste of cacao. In addition they represent important analytical parameters with regard to the evaluation of the quality of cacao-containing and chocolate products. The amount of methylxanthines in cacao depends on various influencing factors, the most essential ones being processing procedures, genotype, geographical origin and cacao bean weight. By determining the amount of theobromine and caffeine in cacao and/or cacao products, the amount of fat-free dry cacao can also be estimated. There are various methods available for the detection and determination of methylxanthines. UV-spectrophotometric determination and analysis using HPLC have attained major significance here. With regard to human nutrition, cacao and cacao products are the main natural sources of theobromine, but significantly less so with regard to caffeine. The presence of methylxanthines in cacao products leads to that they are sometimes the subject of consumer criticism, particularly because they are considered as luxury foodstuffs. The concluding evaluation of methylxanthines by dieticians and toxicologists reveals that plants that contain methylxanthines have been consumed for as long as can be remembered without having led to damage to health. Scientific research has shown that the consumption of these substances has no consequence whatsoever, as long as consumption is not excessive, which is the case for all foodstuffs. Methylxanthines in cacao products have no negative effects on the health of humans because their amounts in chocolates and other processed foodstuffs containing cacao are so low that they account for only a very small proportion of the whole of the human diet. Received: 15 November 1996 / Revised version: 22 January 1997     

Quantitative estimation of the action of α-amylase from Bacillus subtilis on native corn starch by HPLC and HPTLC

 The optimal conditions of corn starch liquefaction and dextrinization using α-amylase from Bacillus subtilis to obtain maltodextrins with a dextrose equivalent of 12.9–23.5 were determined. The pattern of action of B. subtilis α-amylase on native corn starch was monitored using high-performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC). It was possible to separate malto-oligosaccharides of up to 9 glucose units by HPLC and those of up to 12 glucose units by HPTLC, respectively. The results suggest that α-amylase from B. subtilis is an endo-specific enzyme which mainly produces two oligosaccharides, DP3 and DP6. It was shown that both methods can be successfully applied for the characterization of maltodextrins. Received: 22 August 1997     

Quantitative determination of carbohydrates in orange juice by gas chromatography

 A gas chromatographic method using a capillary column is described for the quantitative analysis of fructose, glucose, sucrose and myo-inositol in orange juice. The method is evaluated for precision and recovery using phenyl-β-glucoside as an internal standard. The results support the suitability of the method. Carbohydrates (fructose, glucose, sucrose and myo-inositol) were determined in different kinds of orange juice. The determination of carbohydrate composition and ratios of the carbohydrate constituents provide a method to assess orange juice quality, especially the myo-inositol content and myo-inositol/fructose ratio. These new indices, which were found to be lower in samples made from concentrates, provide information on the quality and genuineness of orange juice. Received: 10 June 1997