Chromatographic method for determination of glycated proteinaceous species in blood

  • US 5,843,788 A
  • Filed: 09/21/1994
  • Issued: 12/01/1998
  • Est. Priority Date: 12/05/1988
  • Status: Expired due to Term
First Claim
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1. A method of analyzing a liquid sample containing both glycated and nonglycated blood protein fractions using high pressure liquid chromatographic separation techniques, said method comprising the steps of:

  • providing a high pressure liquid chromatographic column including therein synthetic resin polymer particles having a diameter of from about 3-20 microns with a phenylboronic acid covalently coupled to said polymer particles;

    contacting said column with said liquid sample having a volume of from 0.01-0.125 μ

    L of red blood cells or from about 0.2-2.5 μ

    L of serum or plasma, and with a transport solution for said nonglycated protein fraction, said transport solution containing a buffering agent and a salt, causing said glycated protein fraction to complex with said phenylboronic acid, and continuously separating said transport solution and nonglycated protein fraction from the column as a first analyte;

    thereafter contacting said column and complexed glycated proteins with an eluant for separating the glycated proteins from the column, said eluant containing mannitol and a salt, and continuously separating said eluant and said separated glycated proteins from the column as a second analytesaid contacting steps being carried out at a superatmospheric column pressure; and

    continuously spectrophotometrically analyzing each of said first and second analytes in serial order using light of a desired wavelength, said spectrophotometric analysis being carried out without blanking of the spectrophotometer with said eluant,the spectrophotometric analysis of said first and second analytes yielding data, which if graphically plotted, would present a pair of separate, single, sharp integratable peaks respectively corresponding to the quantities of said first and second analytes present in said sample,said transport solution and eluant being spectrophotometrically balanced such that, in the quantitative high pressure liquid chromatographic determination of glycated protein in a 0.5 ul. liquid test sample containing 6 to 8 g./dl. of total protein and a glycated protein content of the test sample, said determination being carried out at said desired wavelength, the calculated signal to noise ratio is equal to or greater than about 20, where the signal is the height of the absorbance peak produced by the glycated fraction of the test sample, and the noise is the greatest distance of deviation observed when a blank sample of the transport solution is measured at said desired wavelength and at otherwise identical test conditions, both said height and distance being measured relative to an idealized baseline established by the absorbance at said desired wavelength of the transport solution.

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