Aging Aging

212 Requena et al.Fig. 3. RP-HPLC analysis of N,N'-diacetyl-pentosidine, the precursor of thepentosidine standard. Hydrolysis of this compound yields pure pentosidine, or aslightly contaminated product that can be further purified by analytical RP-HPLC.3. After 48 h, dilute 300 µL of the resulting light brown solution to 3 mL with 1%TFA and apply to a 3-mL C-18 solid-phase extraction minicolumn previouslyequilibrated with 1% TFA.4. Wash the column with 1% TFA. Collect fractions (2 mL) and monitor absorbanceat 226 and 326 nm. After the initially high absorbance at 226 nm decreases to~0.2 and stabilizes (~25 fractions), elute the column with 5% CH 3 CN containing1% TFA. Collect and pool fractions, with peak absorbance at 326 nm, correspondingto elution of N,N'-diacetyl-pentosidine, and dry in vacuo (see Note 1).5. Redissolve sample in 500 µL of deionized water and purify N,N'-diacetylpentosidineby RP-HPLC (Fig. 3). Apply to 15 × 0.46 cm C-18 column, equilibratedwith CH 3 CN:0.1% HFBA in H 2 O (1:6), and elute isocratically at a flowrate of 1 mL/min. Monitor absorbance at 326 nm. A single major peak correspondingto N,N'-diacetyl-pentosidine should appear. Collect and pool peak fractions;dry in vacuo.6. Prepare pentosidine standard by hydrolysis of N,N'-diacetyl-pentosidine in 2 MHCl for 4 h at 110°C. Dry the hydrolysate and redissolve in 1 mL of deionized