A modified HPLC method for the determination of aspartic acid racemization in collagen from human dentin and its comparison with GC.
The D/L ratio of aspartic acid enantiomers in proteins of low turnover is generally accepted as a reliable procedure for age determination. In our study, twelve samples of eyetooth dentin were analyzed for age determination. The pure insoluble collagen isolated from eyetooth dentin was obtained by an EDTA demineralization process. Free amino acids obtained after collagen hydrolysis were converted into o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) derivatives for HPLC analysis under modified conditions and into trifluoroacetic acid isopropyl esters for GC analysis, respectively. The modified HPLC procedure used phosphate buffer and acidified sample matrix prior to injection which resulted in suppression of peak tailing of both diastereomers, thus allowing achievement of both good selectivity and good resolution. To ensure the high accuracy of the developed method the other parameters, i.e. specificity, precision, linearity, LOD, and LOQ, were also determined. Nine collagen samples covering the age range of 18 to 84 years were used for the determination of coefficient of racemization (KR) and calculation of parameters for age estimation. The regression equations for the data set analyzed were as follows: KR= 0.0005 x age + 0.0262 (R2 = 0.9639) for HPLC, and KR= 0.0006 x age + 0.0319 (R2 = 0.9374) for GC, respectively.
Benesová
Terezie
T
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20 Prague 4, Czechia.
Ales
Honzátko
H
Pilin
Alexandr
A
Votruba
Jaroslav
J
Flieger
Miroslav
M
D001224:Aspartic Acid / Q000737:chemistry* / Q000302:isolation & purification; D002849:Chromatography, Gas / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003804:Dentin / Q000737:chemistry*; D006801:Humans; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D001224:Aspartic Acid