3-Methylhistidine determined in plasma by "high-performance" lipid chromatography

1990 ◽  
Vol 36 (3) ◽  
pp. 556-559 ◽  
Author(s):  
H M van Eijk ◽  
N E Deutz ◽  
A J Wagenmakers ◽  
P B Soeters
Keyword(s):  
High Performance ◽  
Venous Blood ◽  
Exchange Chromatography ◽  
Precolumn Derivatization ◽  
Automated Method ◽  
Human Volunteers ◽  
Peak Areas ◽  
Total Analysis ◽  
The Difference

Abstract In this fully automated method for determination of 3-methylhistidine (3MH) in plasma we use precolumn derivatization with o-phthaldialdehyde and subsequent separation by HPLC. Total analysis time is 36 min, and peak areas measured vary linearly with the amount of analyte injected, over the range of 0 to 20 pmol of 3MH (R2 = 0.995), with a coefficient of variation (CV) of 1.6%. The method is reliable, accurate, inexpensive, and at least 1000-fold more sensitive than conventional ion-exchange chromatography with ninhydrin. Because of its sensitivity, the method can be used to estimate venous-arterial differences. In four human volunteers the plasma 3MH concentration varied between 4.97 and 6.08 mumol/L, and the difference between "arterialized" and femoral venous blood for 3MH varied between 0.09 and 0.47 mumol/L.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

scholarly journals An HPLC-automated Derivatization for Glutathione and Related Thiols Analysis in Brassica rapa L.

Agronomy ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1157
Author(s):  
Francesco Nacca ◽  
Concetta Cozzolino ◽  
Petronia Carillo ◽  
Pasqualina Woodrow ◽  
Amodio Fuggi ◽  
...  
Keyword(s):  
Brassica Rapa ◽  
High Performance ◽  
Internal Standard ◽  
Plant Tissues ◽  
Cellular Functions ◽  
Sulphur Compounds ◽  
Stress Protection ◽  
Automated Method ◽  
Reduced Forms

The high content of glucosinolates and glutathione makes the Brassicaceae an important healthy food. Thiols and especially glutathione and γ-Glu-Cys-Gly tripeptide are involved in many fundamental cellular functions such as oxidative stress protection. Although several methods for sulphur compounds analysis in biological samples are actually used, the determination of glutathione and other sulphur derivatives in plant tissues is rather problematic due to their extreme susceptibility to oxidation, which can lead to their overestimation. The aim of this work was the improvement and validation of an automated method for determination of reduced and oxidised glutathione, cysteine and γ-glutamylcysteine in plant tissues. The method consists of a fully automated pre-column derivatization of thiols based on monobromobimane reagent, a high-performance liquid chromatography derivatives separation, and a fluorimetric detection and quantification. The method was successfully applied for determination of the oxidized and reduced forms of Cys, γ-GC and GSH content in leaves, petioles, inflorescences and roots of Brassica rapa L. subsp. Sylvestris. At harvest, in freshly cut plants, the average contents of GSH/2GSSG were 840/45, 345/70 and 150/70 nmol g−1 FW for the florets, leaf blades and stems, respectively; those of Cys/2Cys were 80/12, 29/12 and 24/6 nmol g-1 FW; while those of γ-GC/γ-GCCG-γ were 8.0/4.0, and 6.0/3.0, 3.0/2.0 nmol g−1 FW, respectively. Such amounts were lower in low-sulphur-grown plants at harvest. The very low coefficient of variation between repeated tests (maximum 1.6%), the high recovery of internal standard (>96%) and the linear correlation coefficient of the calibration (R2 > 0.99) support the efficiency of this method that allowed analysing about 50 samples/die in a totally automated manner with no operator intervention. Our results show that the reported method integrations can significantly improve thiols detection via HPLC.

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