Oxytocin chromatography

D. S. Brown and D. R. Jenke, Determination of trace levels oxytocin in pharmaceutical solutions by high-performance liquid chromatography, J. Chromatogr., 4/0 157 (1987). 

Oxytocin Standard chloramine T (without metabisulfite) 0.01-0.02 Adsorption chromatography on Sephadex G-25 column in acetic acid E. E. Thompson, P. Freychet, and J. Roth, Endocrinology, 91 1199 (1972)... 

When the unusual bromine cleavage of oxytocin and vasopressin, reported by du Vigneaud and his associates, was repeated with a simplified tripeptide model, N-carbobenzyloxy-(S-benzyl-L-cysteinyl-L-tyrosyl-L-iso-leucine (CXV) (Ressler and du Vigneaud, 1957), bromine or NBS liberated a ninhydrin-reactive material in 40% yield. Paper chromatography proved isoleucine to be the sole ninhydrin-reactive substance present. The ultraviolet spectrum of the reaction mixture showed the characteristic 260 mM absorption for a dienone (CXVI). 

Numerous immunoassays for measuring oxytocin in plasma or urme have been described. However, their routine clinical application has been limited because of a lack of physiological relevance to human reproductive disorders and because of a lack of easy-to-use immunoassays with acceptable sensitivity and specificity. With most plasma assays, a preliminary extraction procedure with column chromatography is required, not only to concentrate the minute amount of hormone that is present in the specimen, but also to remove nonspecific interfering substances. 

Figure 10.16 Separation of a peptide mixture by hydrophobic interaction chromatography (reproduced with permission from A.J. Alpert, J. Chromatogr., 444, 269 (1988)]. Conditions column, 20cm x 4.6mm i.d. stationary phase, polypropyl aspartamide 5pm mobile phase, 1mlmin of 2M ammonium sulfate with 0.025M sodium phosphate, pH 6.5-0.025M sodium phosphate pH 6.5, linear gradient as indicated detector, UV 220 nm. 1 = substance Ml—9) 2 = [Arg ]-vasopressin 3 = oxytocin 4 = substance P, free acid 5 —[Try ]-substance P, 6 = substance P,1 [Tyr ]-somatostatin 8 = somatostatin 9 = [Tyr ]-somatostatin.

Any chemist knows that the more steps there are in a chemical synthesis, the lower the final yield. For example, if each step in a 10-step synthesis furnishes a 90 percent yield of product, the yield of the final product will be only about 35 percent. That is why it is not possible to extend Du Vigneaud s masterful syntheses (see chapter 6) of the hormones oxytocin and vasopressin (9 amino acid residues each) to proteins, even small ones such as ribonuclease A (124 amino acid residues). In order to pursue this daunting challenge Robert Bruce Merrifield (1921-2006), at Rockefeller University, devised a new concept solid-phase synthesis. The idea is disarmingly simple covalently attach an amino acid to a macroscopic particle that can be exposed to the reaction, washed, and then separated by simple filtration. Each reaction step requires no chromatography and no crystallization, just washing and filtering. At the end, completed peptide chains are chemically released from the particles. 

The foremost advantage of the chemical assay is that the specificity of the analysis can be tested with the most rigid criteria. In the pituitary assay of oxytocin and vasopressin (Gruber et a/., 1976), specificity was demonstrated by the following four criteria (1) The oxytocin and vasopressin peaks were identified by their retention times. (2) Addition of synthetic oxytocin or vasopressin to the pituitary extract resulted in a quantitative increase in the height of the specific peak with no noticeable peak broadening. (3) After purification by reverse-phase chromatography, each of the nonapeptide fluorophors was collected, hydrolyzed in 6 N HCl, and then characterized... 

Extraction and concentration of oxytocin and 306 other neurohypophyseal hormones from dilute samples of biological fluids Affinity chromatography of L-arginine esterase 307 of human blood plasma... 

D. Yamashiro, Partition chromatography of oxytocin on Sephadex Nature (Lond.) 201 76-77 (1964) also D. Yamashiro, D. Gillessen, V. du Vigneaud, Oxytocein and deamino-oxytocein. Biochemistry 5 3711-3720 (1966)... 

Two fractions were obtained by chromatography on a column of Sephadex G-75 The first emerged with the void volxane and contained protein with a molecular weight greater than 50,000 this protein appeared to lack affinity for either oxytocin or vasopressin. The second fraction, accounting for over 80 per cent of the protein recovered had a molecular weight of less than 50,000 and in dialysis experiments bound both hormones. 

Inoue, T. Umemura, H. Yoshida, H. Todoroki, K. Nohta, H. Yamaguchi, M. Sensitive determination of oxytocin-related compounds by liquid chromatography with pre-column excimer fluorescence derivatization. Chromatography 2009, 30(Suppl. 1), 17-18. 

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