Trace Detection of Glycolic Acid by Electrophore Labeling Gas Chromatography -Electron Capture Mass Spectrometry Gang Shao and Roger W. Giese* Department of Pharmaceutical Sciences in the Bouve College of Pharmacy and Health Professions, Barnett Institute, and Chemistry Department, Northeastern University, Boston, Massachusetts 02115 As little as 1 0 pg of standard glycolic acid (glycolate) was detected in a method comprising the following sequence of steps: (1 ) add glycolate-2 ,2 -d 2 as an internal standard and exchange the carboxylate oxygens in hot HCl/ [ 18 O]- water; (2 ) form an amide derivative with a water-soluble carbodiimide and the electrophoric amine, AMACE1 ; (3 ) purify by bypass HPLC; (4 ) derivatize the residual hydroxy with butyric anhydride; (5 ) partition with acetonitrile/ 2 M NaCl; and (6) detect by GC-ECMS. At an intermediate stage in method development, 1 pg of glycolate-2,2,-d 2 could be detected by subjecting it to the above steps 2 -6, forming product in an overall, absolute yield of 7 6 %. Step 1 was added after an effort to fully overcome background contamination by glycolate was unsuccessful. For ex- ample, background contamination by glycolate could increase rather than decrease when the methanol reagent in the procedure was “carefully purified.” The work extends the sensitivity for glycolate detection by ∼100- fold and provides high-performance conditions for the analytical steps employed. We are interested in the trace detection of the polar metabolites that arise from oxidative damage to the sugars of DNA. 1 In part, the measurement of these metabolites is of interest to better understand how this type of damage is repaired enzymatically. 2 More broadly, oxidative damage to DNA is of interest because it may play a role in aging and some disease processes such as cancer and heart disease. We selected glycolic acid (glycolate) as an initial analyte to test. This compound can form as a secondary metabolite of phosphoglycolate, which in turn arises from oxidative damage at the 4′ position of the deoxyribose residues of DNA. 3 Due to its water solubility and ease of occurrence as a background contaminant, trace glycolate is a challenging analyte. Phosphoglycolate is a product of a reaction catalyzed by ribulose-1,5-biphosphate carboxylase-oxygenase (rubis- co), the most abundant protein on earth. 4 Glycolate is an important chemical industrially, environmentally, cosmetically, and clinically as has been summarized recently. 5 Prior assays for glycolate, whether as a standard, an industrial chemical, or a metabolite, have provided moderate sensitivity, when handling a detection limit 6 is considered. For example, a detection limit of 12 ng for standard glycolate was achieved recently using a plant tissue-based chemiluminescence flow biosensor. 7 An amperometric biosensor was used to detect as little as 90 ng (S/ N ) 6) of glycolate. 5 Yao and Porter reported the detection of spiked glycolate in serum down to 0.5 ng based on trimethylsilylation/ GC-FID, 8 while showing a chromatogram from a sample containing a spike of 25 ng. Soga and co-workers, using capillary electrophoresis-electrospray ionization-mass spectrom- etry, showed an electropherogram obtained by making an injection from a 20- μL sample containing 152 ng of glycolate and reported a corresponding detection limit of 9 ng considering S/ N )3. 9 In an HPLC procedure with UV detection, glycolate was measured in cosmetic products in the low-microgram range. 10 Although previously we reported the detection of a diluted standard of O 2 - pivalyl-3′,5′-bis(trifluoromethyl)benzylglycolate by GC-ECMS at the zeptomole level, anhydrous conditions were used for the derivatization, and the derivatization was performed at the mil- ligram level. 11 Methods also have been reported for measuring analogues of glycolate. For example, γ-hydroxybutyric acid has been detected at the low-nanogram level by gas chromatography-positive chemi- cal ionization mass spectrometry after conversion to the corre- sponding lactone, 12 and at the mid-nanogram level by HPLC with UV detection. 13 A low-nanogram level detection limit was reported for lactic acid when measured by trimethylsilylation-GC-FID. 14 Previously we reported the synthesis of an electrophoric reagent, “AMACE1”, which can be coupled onto carboxylic acids such as glycolate in an aqueous buffer, yielding, after further derivatization as necessary, products with excellent detection * To whom correspondence should be addressed. E-mail: r.giese@ neu.edu. (1) Von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor & Franis, London, 1987. (2) Demple, B.; Harrison, L. Annu. Rev. Biochem. 1994 , 63, 915-948. (3) Greenberg, M. M. Chem. Res. Toxicol. 1998 , 11, 1235-1248. (4) Conn, E. E.; Stumpf, P. K.; Bruening, G.; Dot, R. H. Outlines of Biochemistry; Wiley: New York, 1987; p 490. (5) Tsiafoulis, C. G.; Prodromidis, M. I.; Karayannis, M. I. Anal. Chem. 2002 , 74, 132-139. (6) Yang, C.; Shimelis, O.; Zhou, X.; Li, G.; Bayle, C.; Nertz, M.; Lee, H.; Strekowski, L.; Patonay, G.; Couderc, F.; Giese, R. W. J. Chromatogr., A 2002 , 979, 307-314. (7) Li, B.; Zhang, A.; Jin, Y. Anal. Chem. 2001 , 73, 1203-1206. (8) Yao, H. H.; Porter, W. H. Clin. Chem. 1996 , 42, 292-297. (9) Soga, T.; Ueno, Y.; Naraoka, H.; Ohashi, Y.; Tomita, M.; Nishioka, T. Anal. Chem. 2002 , 74, 2233-2239. (10) Scalia, S.; Callegari, R.; Villani, S. J. Chromatogr., A 1998 , 795A, 219-225. (11) Wang, P.; Murugaiah, V.; Yeung, B.; Vouros, P.; Giese, R. W. J. Chromatogr., A 1996 , 721, 289-296. (12) Frison, G.; Tedeschi, L.; Maietti, S.; Ferrara, S. D. Rapid Commun. Mass Spectrom. 2000 , 24, 2401-2407. Anal. Chem. 2004, 76, 3049-3054 10.1021/ac0304267 CCC: $27.50 © 2004 American Chemical Society Analytical Chemistry, Vol. 76, No. 11, June 1, 2004 3049 Published on Web 04/23/2004