In-line Fluorescence Research Articles

Analysis of β-Carotene in Medical Food by Liquid Chromatography with Matrix Solid-Phase Dispersion

A liquid chromatographic method is described for analysis of beta-carotene in medical food. The nutrient is extracted from medical food without saponification by matrix solid-phase dispersion and quantitated by isocratic normal-phase chromatography with a Si 60 column and a mobile phase of hexane containing 0.125% (v/v) isopropyl alcohol. The limit of quantitation is 0.02 microgram/mL at 436 nm. Standard response was linear over the concentration range of 0.02-1.0 microgram/mL (r2 = 0.99998). Recoveries were determined on a zero control reference material containing added beta-carotene at various levels. Recoveries averaged 91.2% (n = 25) with coefficients of variation from 0.50 to 3.10%. The method provides a rapid, specific, sensitive, and easily controlled assay for analysis of beta-carotene in fortified medical food. In addition, retinyl palmitate can be assayed simultaneously with an in-line fluorescence detector.

High-Performance Liquid Chromatographic Assay for Tryptase Based on the Hydrolysis of Dansyl-Vasoactive Intestinal Peptide

A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While theKmvalue was unaffected, thekcatvalue decreased, yielding akcat/Kmratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in thekcat/Kmratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.

High-performance liquid chromatographic determination of an arginine-containing octapeptide antagonist of vasopressin in human plasma by means of a selective post-column reaction with fluorescence detection

A novel high-performance liquid chromatographic method was developded for the determination in plasma samples of the synthetic octapeptide SK&F 105494 { O-ethyl- d-tyrosyl- l-phenylalanyl- l-asparaginyl-5-[1-(carboxymethyl)cyclohexyl]- l-norvalyl- l-arginyl- d-arginamide, cyclic (5–1) peptide}, and active aquaretic agent which significantly increases water excretion in experimental animal models through competitive antagonism of renal epithelial vasopressin receptors. The method involves isolation of Sk&F 105494 from plasma samples by solid phase extraction prior to chromatogrpahic analysis. Following chromatographic separation, in-line fluorescence detection was accomplished via a selective-post-column reaction of the guanidino group of the arginine moiety of the peptide withalkaline ninhydrin to generate a highly fluorescent product. Optimization of the post-column reaction conditions and the use of high-performance liquid chromatography columns of reduced internal diameter (2.1 mm), resulted in an on-column detection limit of 50pg (45 fmol). The limit of sensitivity in plasma was 0.5 ng/ml. The assay was linear over the range 0.5–100 ng/ml. Precision and accuracy were within 11% across the calibration range. The assay was shown to be suitable to study the pharmacokinetics of SK&F 105494 in human subjects and animals. In addition, the methodology developed has general applicability in the detection of arginine-containing peptides in biololgical matrices.

Distribution of Endogenous Indole-3-Acetic Acid and Compression Wood Formation in Reoriented Branches of Douglas-Fir

Five-year-old segments of intact 7-year-old branches of Douglas-fir (Pseudotsuga meziesii [Mirb.] Franco) were reoriented to determine the relation between indole-3-acetic acid (IAA) and the formation of compression wood. Eight branches per treatment were either left at their original angle (mean of 69 degrees , the control), or bent proximal to the segment to reorient it up or down 30 degrees . Differentiating xylem tissue from the upper and lower sides of each segment was collected and extracted separately for IAA analysis by in-line fluorescence detection of free IAA and IAA methyl ester after sequential C(16) reversed-phase high performance liquid chromatography. The IAA methyl ester was confirmed by gas chromatography-mass spectroscopy. Compression wood formed on the upper side of branches reoriented up and on the lower side of controls or branches reoriented down. IAA was present in all samples. The difference in IAA concentration between upper and lower sides was either not correlated, or negatively correlated in segments reoriented down, with both the occurrence of compression wood and the rate of new tracheid production. Mean concentrations for whole branch segments were not affected by the treatments, regardless of whether IAA concentrations were expressed on a surface area, weight, or cell basis.