Sam A. Margolis

Analysis of anthocyanins in foods by liquid chromatography, liquid chromatography-mass spectrometry and capillary electrophoresis.

This article reviews recent developments in the methodology for the measurement of anthocyanins that offer several advantages over classical methods of analysis. The use of UV-diode array and mass spectrometric (MS) detectors, with improved methods of liquid chromatography analysis has facilitated identification of these analytes. The use of capillary electrophoresis (CE) analysis of the anthocyanins under acid conditions has significantly increased peak resolution and improved the detection limits by several orders of magnitude. CE offers the advantage of economies of very small sample size, very small solvent consumption, and short analysis times along with the future possibility of being combined with MS detection.

Separation of blackcurrant anthocyanins by capillary zone electrophoresis

The four major anthocyanins present in juice of the blackcurrant (Ribes nigrum) may be completely separated by capillary zone electrophoresis under strongly acidic conditions. The separation, resolution and peak shapes of the anthocyanins are critically influenced by the pH of the running buffer and the presence of an organic solvent. Fused-silica and polyacrylamide-coated capillary columns were evaluated for their ability to resolve the closely migrating analytes. Optimum qualitative separation was achieved on a fused-silica capillary with a phosphate running buffer containing 30% (v/v) acetonitrile at an apparent pH of 1.5.

Nutritional status and growth in juvenile rheumatoid arthritis

The specific cause of short stature in juvenile rheumatoid arthritis (JRA) is unknown. One hypothesis links altered growth to inadequate dietary intake. In this study, nutritional status was assessed in 34 children with JRA (8 with systemic JRA, 14 with polyarticular JRA, and 12 with pauciarticular JRA) and 9 healthy controls using 3-day diet records, anthropometrics, and biochemical analyses. Differences in growth were found among the three types of JRA. One third of all subjects were at or below the 10th percentile in height for age (these being predominantly among the systemic and polyarticular groups). With few exceptions, the mean dietary intake for calories and essential nutrients was found to be adequate for each of the three groups. However, more than half of those with systemic JRA reportedly consumed less than the recommended caloric intake for their age and weight. No significant correlations were found linking dietary intake to growth percentiles in any of the groups studied. Biochemical abnormalities were found among the systemic and polyarticular groups. These abnormalities included low plasma levels of vitamins A and C, proteins (albumin, prealbumin, and retinol binding protein) and zinc; and increased levels of copper and glutathione peroxidase activity. Plasma selenium and vitamin E levels were unchanged. The discrepancy between intake and certain circulating nutrient levels may reflect alterations in the requirements, absorption, or use of these nutrients in the presence of chronic inflammation.

Liquid chromatographic measurement of l-ascorbic acid and d-ascorbic acid in biological samples

D- and L-Ascorbic acids have been separated using liquid chromatography (LC) on a polymer-coated silica-based NH2 column and the L-isomer has been quantified in human serum, rat serum, rat lung, rat lung perfusate, infant formula (SRM 1846) and mixed food sample (SRM 2383). The D-isomer was observed only in trace amounts in the mixed food sample. The results demonstrate that ascorbic acid was stable on the column and completely recovered from supplemented samples of human serum and that this method of analysis is accurate, precise and has broad application exhibiting no dependence on the nature of the matrices evaluated herein.

Comparison of methods for extraction of flavanones and xanthones from the root bark of the osage orange tree using liquid chromatography

Abstract This study compares conventional solid–liquid extraction, supercritical fluid extraction (SFE), and pressurized fluid extraction (PFE) for their efficiency in extracting xanthones and flavanones from the root bark of the osage orange tree (Maclura pomifera). Seven compounds were extracted from the plant material by solvent extraction at room temperature for 48 h. The same compounds were removed from the root bark by 45- and 35-min extractions using SFE and PFE, respectively, and under optimized conditions, in same or higher yields than those obtained by the conventional 48-h solvent extraction. Although all seven compounds were present in the SFE extracts when only CO2 was used as the fluid, the addition of 20 vol.% methanol (MeOH) to the CO2 proved essential for achieving high yields. Use of SFE with CO2–MeOH also led to the recovery of an additional flavanone from a wet sample of root bark. This flavanone is absent from the conventional solvent extracts and appears in small amounts in the PFE extracts. An optimized LC separation for the analysis of the different extracts is presented, and it is demonstrated that the separation of xanthones and flavanones is considerably improved by the use of deactivated C18 columns in conjunction with a mobile phase containing acetonitrile and a weak organic acid.

Preliminary application of liquid chromatography-electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma.

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C18-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC-MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/mnl.

Characterization of prenylated xanthones and flavanones by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

Reversed-phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) in the positive-ion mode was utilized to analyze crude ether extracts from the root bark of Maclura pomifera, a tree known to have a high content of prenylated xanthones and flavanones. Identification of three xanthones and two flavanones was based on their unique mass spectra. Under optimum conditions peaks corresponding to the [MH](+) ion and characteristic fragments for each compound were observed. (1)H NMR data were used to confirm the identities of two xanthones that had the same molecular mass and similar fragmentation patterns. Fragmentation of the analytes was achieved by application of an electrostatic potential at the entrance of the single quadrupole mass spectrometer. The optimum voltage for fragmentation was found to be related to the class of compounds analyzed and, within each class, to be dependent on the structure of the prenyl moiety. Collision-induced pathways consistent with precedent literature describing the MS characterization of similar compounds and with the observed fragmentation patterns are tentatively proposed.

Chromatographic separations of serum proteins on immobilized metal ion stationary phases.

The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.

Synthesis of nitrogen-15-labeled 2-amino(glycofurano)oxazolines via glycosylamine intermediates☆

Abstract A new, efficient synthesis of doubly 15N-labeled 2-amino-oxazoline derivatives of pentoses and hexoses has been delineated that involves treatment either of unprotected or O-isopropylidenated glycosylamines with cyanamide-15N2 in methanol to give 2-amino(glycofurano)oxazolines-15N2. A probable mechanism for these reactions is presented. These techniques provide a practical means by which a variety of stable or radioactive isotopes can be introduced into any of several known, clinically significant pyrimidine anhydronucleosides, such as 2,2′-anhydro-(1-β- d -arabinofuranosylcytosine) (cyclo-C).

The high-performance liquid chromatographic analysis of diastereomers and structural analogs of angiotensins I and II

Diastereomers, alpha- and beta-aspartic acid forms, and partial sequences of angiotensin I and II were resolved by reversed-phase high-performance liquid chromatography (HPLC). Nearly all of the peptides which were examined contained significant amounts of peptides whose amino acid composition differed from the designated peptide. This chromatographic procedure combined with amino acid analysis clearly offers the investigator a rapid method for analyzing and quantifying the purity of angiotensins which are intended for use as reference substances for radioimmunoassay and biological assay.