Direct determination of methionine sulfoxide in milk proteins by enzyme hydrolysis/high-performance liquid chromatography.

A direct and simultaneous HPLC/UV determination of methionine and methionine sulfoxide in enzyme-hydrolyzed milk proteins is described. Protein hydrolysis is accomplished by a three-enzyme (pronase, leucine aminopeptidase, prolidase) 20-h/37 degrees C digestion. A gradient elution reversed-phase HPLC system with UV detection at 214 nm and 280 nm is then used to determine the quantitative releases of methionine sulfoxide, methionine, tyrosine, and tryptophan. The ease of methionine oxidation by a wide variety of oxidants, coupled with the quantitative release of both methionine and its sulfoxide by the three-enzyme hydrolysis, renders the approach valuable for identifying oxidized milk proteins. The relatively simple method proved accurate and precise in its application to commercial milk products, finding methionine sulfoxide levels as high as 74% of the total methionine.

Cytoprotective agent in Lactobacillus bulgaricus extracts.

Adenosine 5'-diphosphoribose (ADP-ribose) has been identified as a significant contributor to the anti-cytotoxic activity of Lactobacillus bulgaricus extracts. Although the biological activities associated with the administration of probiotic bacteria and components thereof are sometimes attributed to the peptidoglycans that comprise a substantial portion of the Gram-positive bacterial cell wall, we found that the beta-nicotine adenine dinucleotide (NAD) hydrolysis product ADP-ribose was a significant contributor to the observed anti-cytotoxicity in our L. bulgaricus extracts. The ADP-ribose was isolated, identified, and quantitated by high performance liquid chromatography (HPLC) and by nuclear magnetic resonance (NMR) spectroscopy. ADP-ribose levels as low as 5 mg/L exhibited a measurable inhibition of tumor necrosis factor alpha (TNF-alpha) mediated cytotoxicity in an in vitro cell assay, whereas the ADP-ribose content of the L. bulgaricus extracts often exceeded 5 mg/g dry weight.

Glutamine in commercial liquid nutritional products.

Total glutamine concentrations in commercial nutritional products have been determined by enzymatic hydrolysis followed by HPLC quantification of free glutamine and free pyroglutamic acid. Hydrolysis was accomplished by a published three-enzyme (Pronase, leucine aminopeptidase, prolidase), 20-h/37 degrees C digestion. Glutamine was determined as its FMOC derivative by reverse phase HPLC-fluorescence, and pyroglutamic acid was determined directly by organic acid HPLC-UV. Approximately 4.11% of the released glutamine is converted to pyroglutamic acid during the 20-h digestion. Experimental ratios of enzyme hydrolysis glutamine to acid hydrolysis glutamic acid + glutamine + pyroglutamic acid (GLX) indicate that the method recovers >90% of the protein-bound glutamine. The nutritional products with casein dominant intact protein systems typically deliver >9 g of glutamine/100 g of protein, or approximately 40 g of glutamine/100 g of GLX.

Direct determination of free methionine in soy-based infant formula.

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.

Determination of isoflavones in ready-to-feed soy-based infant formula.

An alkaline hydrolysis/liquid chromatography (LC) method was developed for determination of isoflavones in ready-to-feed soy-based infant formula. The method consists of a 15 min methanol extraction, 10 min alkaline hydrolysis, HCl neutralization, gravity filtration, aqueous dilution, and 50 min LC analysis with UV detection at 262 and 250 nm to quantify 6 isoflavone analytes: daidzin, glycitin, genistin, daidzein, glycitein, and genistein. The concentration averages for 10 commercial batches (microg aglycone/g formula) were daidzein, 6.12 +/- 1.23; glycitein, 1.19 +/- 0.16; genistein, 12.8 +/- 2.35; and total, 20.1 +/- 3.61. Validation experiments demonstrated extraction completion and analyte stability to alkaline hydrolysis. Spike recoveries ranged from 97.6 to 104.1%, and a series of accuracy assessments showed that isoflavone concentration determined by the method was within 5% of the true value. The relative standard deviation values for repeatability ranged from 0.4 to 2.2% (n = 10), and from 0.3 to 2.7% (n = 4) for intermediate precision. Isoflavone peak purity was verified by comparing sample and standard peak area ratios (262/250 nm). The limits of detection and quantitation (microg/ formula) ranged from 0.02 to 0.05 and 0.08 to 0.18 microg/g, respectively. The difference between our concentrations and those reported by others in 1995-1998 is attributable to the well-established seasonal variation in soybean isoflavone levels. Although the method was applied exclusively to ready-to-feed formula in the present study, it is equally suitable for powder and concentrated liquid infant formulas.

Determination of acesulfame and sucralose in oral electrolyte maintenance solution by liquid chromatography.

A method was developed for the direct, simultaneous determination of acesulfame and sucralose in oral electrolyte maintenance solution (OEMS). Analyte separation and quantitation were achieved by gradient reversed-phase liquid chromatography (LC) and UV absorbance at 192 nm, respectively. Detection at a second wavelength, 214 nm, was used to check sucralose peak purity; 20 microL OEMS was injected without preparation or dilution. System linearity was demonstrated as 192 nm peak area versus analyte concentration at 80-120% OEMS sweetener fortification (r > 0.999, and all residuals < 0.5%, for both acesulfame and sucralose). Spike recoveries for OEMS samples prepared at 3 spiking levels (80, 100, and 120% sweetener fortification) ranged from 100.3 to 102.0% for acesulfame, and from 97.9 to 102.3% for sucralose. In a second assessment of method accuracy, the same spiked OEMS samples were tested by 2 alternative methods: acesulfame (LC/UV at 230 nm) and sucralose (anion exchange-pulsed amperometric detection). Results for the alternative acesulfame method were within 1.2%, and for the alternative sucralose method within 6.0%, of the corresponding results obtained by the 192 nm method. Repeatability and intermediate precision RSD values were < 1 % for acesulfame and < 3% for sucralose. The limits of quantitation were 1.6 and 32 mg/L for acesulfame potassium and sucralose, respectively. Despite the weak UV absorptivity of sucralose and the consequent small size of its LC peak, no evidence was found for sucralose interference in any of the commercial OEMS flavors.