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Identification and quantitation of N ‐(carboxymethyl)valine adduct in hemoglobin by gas chromatography/mass spectrometry
Author(s) -
Cai Jian,
Hurst Harrell E.
Publication year - 1999
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199905)34:5<537::aid-jms806>3.0.co;2-h
Subject(s) - chemistry , chromatography , mass spectrometry , valine , adduct , hemoglobin , gas chromatography–mass spectrometry , gas chromatography , organic chemistry , amino acid , biochemistry
A sensitive, specific and reproducible method was developed for thequantitation of the hemoglobin (Hb) adduct N ‐(carboxymethyl)valine (CMV). Thisadduct is one of various products from the Maillard reaction,involving reducing sugars and amino acids, proteins or othermolecules with a free amino group. Such adducts, including N ε ‐(carboxymethyl)lysine(CML), are called advanced glycation end products(AGE) and have been correlated with aging and severity ofdiabetes in human tissues. This method was developed to examine theCMV–Hb adduct as a possible AGE formed by reaction of Hb withglucose or other oxidation products. CMV was cleaved selectively fromisolated globin using pentafluorophenyl isothiocyanate(PFPITC) in a modified Edman degradation at pH 9.5. Thecarboxyl group of the adduct was derivatized to its methyl ester withdiazomethane. The resulting derivative,5‐isopropyl‐1‐(methylacetate)‐3‐pentafluorophenyl‐2‐thiohydantoin, was detected by gas chromatography/mass spectrometry with selected ion monitoring (GC/SIM/MS). Quantitation was based on the response factor of the derivative molecular ion ( m / z 396) from synthesized CMV and N ‐(2‐carboxyethyl)valine (molecular ion m / z 410) as internal standard. This method exhibits reproducibility and linearity in the range 0.2–100 ng CMV. The limit of quantitation (0.2 ng CMV) gave a signal‐to‐noise ratio greater than 5: 1 using a 1: 30 sample aliquot. The GC/SIM/MS method can detect CMV adduct in 5 mg globin samples with relative standard deviations less than 5%. This approach avoids tedious acid hydrolysis and interference from other amino acids. The molecular ion and other CMV derivative ion assignments from samples were confirmed by accurate mass determinations using GC/high resolution SIM/MS. Measurements from random mouse, rat and human globin samples gave mean CMV levels of about 6, 5 and 14 nmol g −1 Hb in these species, respectively. Copyright © 1999 John Wiley & Sons, Ltd.