Determination of patulin and penicillic acid in unroasted cocoa beans.
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Penicillic acid (PA) is a carcinogenic food contaminant produced by several food-borne fungi. PA was resolved as a sharp peak by reversed-phase high-performance liquid chromatography on a small-particle (10 micrometer) column in 3-4 min by an elution system composed of acetonitrile (AN), water and glacial acetic acid (AC) with detection by ultraviolet (UV) absorbance at 254 nm. Peak height and peak area were related linearly to the amount of PA injected over a range of 5-500 ng. Reproducibility of retention time, peak height and peak area was demonstrated. The lower detection limit was 5 ng in two elution systems [AN-H2O-AC(45:55:2), flow-rate (F) 1 ml/min; AN-H2O-AC(40:60:2), F = 1.2 ml/min] and 10 ng in a third system [AN-H2O-AC(25:75:2), F = 1.6 ml/min]. Based on the sensitivity and separation of PA from (40:60:2), F = 12 ml/min; urine, AN-H2O-AC (25:75:2), F = 1.6 ml/min. Good recovery (89-98%) over a range of 1-50 microgram/ml of PA was obtained from PA-spiked plasma samples treated first with 25% HPO3 followed by extraction with chloroform. A single peak detected either by UV absorbance or by radioactivity was obtained when plasma samples spiked with [14C]PA were extracted. Good recovery (92-105%) of PA also was obtained from spiked urine and bile samples.
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The application of C7 aliphatic carboxylic acids (heptanoic, 2-methylhexanoic, 5-methylhexanoic and 2,2-dimethyl-n-valeric acids) as eluents in ion-exclusion chromatography with conductimetric detection for C1-C6 aliphatic carboxylic acids (formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic and caproic acids) was carried out using a highly sulfonated styrene-divinylbenzene co-polymer resin (TSKgel SCX) in the H+ form as a stationary phase. When using 0.05 mM sulfuric acid at pH 4.0 as the eluent, peak shapes of hydrophobic carboxylic acids (isovaleric, valeric, isocaproic and caproic acids) were tailed strongly. In contrast, when using 1 mM these C7 carboxylic acids at pH ca. 4 as the eluents, although system peaks (vacant peaks) corresponding to these C7 carboxylic acids appeared, peak shapes of these hydrophobic acids were improved drastically. Excellent simultaneous separation and relatively high sensitive conductimetric detection for these C1-C6 aliphatic carboxylic acids were achieved in 25 min on the TSKgel SCX column (150 x 6 mm I.D.) using 1 mM 5-methylhexanoic acid at pH 4.0 as the eluent.
Screening for medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency by urinary 3-phenylpropionylglycine may not be reliable in early infancy because young infants are not colonized with adult-type colonic flora. In this study we delineated the microbes that produce 3-phenylpropionic acid, the precursor of 3-phenylpropionylglycine. We found that the use of some antibiotics may alter gut flora, thereby confounding this method of screening for MCAD deficiency.
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