A modified HPLC method for the determination of aspartic acid racemization in collagen from human dentin and its comparison with GC

Abstract

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.

Aspartic Acid Racemization Method for Age Estimation in Human Tissues: A Review

“…HPLC method is usually applied in racemization method in recent times (Fu et al 1995;Yekkala et al 2006). There are several advantages of HPLC method such as high sensitivity, shorter analysis time, simple procedure, high reproducibility, lower cost (Benešová et al 2004;Ogino & Ogino 1988;Qudsia et al 2014;Yekkala et al 2006). The data obtained from detector system was the chromatogram.…”

Enantiomeric Ratio of Amino Acids as a Tool for Determination of Aging and Disease Diagnostics by Chromatographic Measurement

“…The effects of sample preparation and buffer conditions were studied. The usual approach to estimating the age from teeth is by D/L-Asp ratio from hydrolyzed dentin [23][24][25][26][27]. A general problem that must be taken into account is possible AA racemization during acidic hydrolysis of proteins or peptides [28,29].…”

“…The authors described some additional racemization at higher temperatures [27]. Benešová et al performed an interesting study [26] in which two well-established, routinely used methods were compared. (i) AAs from hydrolyzed collagen from dentin derivatized with o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) were separated by RP HPLC on C8 column; (ii) AAs for GC analysis were converted into trifluoroacetic acid isopropyl esters.…”

“…Chiral XE-60-S-VAL-SA-PEA fused silica column was used for GC measurement. The following equation was used for the calculation of the age [26,34]:…”

An improved sample preparation for an LC method used in the age estimation based on aspartic acid racemization from human dentin

“…The aim of this study was to evaluate the efficiency of the LC method coupled with fluorescence detection used in the age estimation based on the determination of the D/L ratio of Asp in living human beings' dentin samples [24]. In the present paper, a modified sample preparation is proposed for better stability of o-phthaldialdehyde-Nacetyl-L-cysteine (OPA-NAC) derivatives of D/L-Asp (due to the instability below pH 7).…”