Table of Contents Author Guidelines Submit a Manuscript
International Journal of Electrochemistry
Volume 2011, Article ID 427238, 4 pages
Research Article

Enantioselective Potentiometric Membrane Electrodes Based on Antibiotics for the Determination of L- and D-Glyceric Acids

1Laboratory of Electrochemistry and PATLAB Bucharest, National Institute of Research for Electrochemistry and Condensed Matter, 202 Splaiul Independentei Strada, 060021 Bucharest, Romania
2Department of Chemistry, Al-Aqsa University, Gaza, Palestine
3The Pharmaceutical and Drug Industries Research Division, Pharmaceutical and Medicinal Chemistry Department, National Research Centre, Dokki, Cairo 12311, Egypt

Received 9 February 2011; Accepted 16 March 2011

Academic Editor: Bengi Uslu

Copyright © 2011 Raluca-Ioana Stefan-van Staden et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Glyceric acid (GA) is a human metabolite existing in L- and D-configurations, which are considered the markers for the diseases L- and D-glyceric aciduria/academia, respectively. Enantioselective, potentiometric membrane electrodes based on carbon paste modified with antibiotics as chiral selectors, vancomycin, and teicoplanin were designed for the assay of L- and D-GA, respectively, in the concentration ranges of 10−9–10−7 and 10−4–10−2 moL/L with very low detection limits (1.5 × 10−10 moL/L for L-GA and 1.6 × 10−4  moL/L for D-GA, resp.). The surface of the electrodes can be regenerated simply by polishing in order to obtain a fresh surface ready to be used in a new assay. The proposed electrodes can be successfully applied for the enantioanalysis of L- and D-glyceric acids in serum samples.

1. Introduction

The enantiomers of the urinary organic acids are important markers for inborn errors of metabolism. Accordingly, there is a growing demand for determining the metabolic products in human blood (academia) and urine (aciduria). Different enantiomers may originate from separate metabolic pathway, due to enzyme deficiency.

Glyceric acid (2,3-dihydroxypropionic acid, GA) is a human metabolite existing in L- and D-configurations. These two enantiomers are vital biological markers for the diagnosis of two different metabolic diseases, primary hyperoxaluria type II (L-glyceric aciduria, PH2) and D-glyceric aciduria [16]. Therefore, enantioselective analysis of glyceric acid is necessary to differentiate between the two inherited metabolic diseases.

Up to date, the assay of GA was done using capillary gas chromatography [710], liquid chromatography [11], high-performance liquid chromatography [12], capillary electrophoresis [13], polarimetry [14], and colorimetric methods [15].

Enantioselective, potentiometric membrane electrodes (EPMEs) proved to be very reliable for the enantioanalysis of pharmaceutical compounds as well as of compounds of clinical importance [16]. Macrocyclic antibiotics represent a new class of chiral selectors used in the design of EPME, offering a high selectivity and enantioselectivity [17]. The macrocyclic antibiotics contain stereogenic centers and functional groups, which allow them to interact with chiral molecules by hydrophobic, dipole-dipole, π-π interactions, hydrogen bonding, steric repulsion [18, 19], and charge-to-charge repulsions [2022].

This paper describes the design, response characteristics, (enantio) selectivity, and applications of two EPMEs based on vancomycin and teicoplanin for the enantioanalysis of GA.

2. Experimental

2.1. Electrode Design

Paraffin oil and graphite powder were mixed in a ratio of 1 : 4 (w/w) to form the carbon paste. The modified carbon pastes were obtained by the addition of the aqueous solutions of vancomycin (pH = 4) or teicoplanin (pH = 6) (10−3 moL/L) (100 μL chiral selector solution to 100 mg carbon paste) to the carbon paste. The unmodified carbon paste was filled into a plastic pipette peak leaving a space of 3-4 mm into the top to be filled with the modified carbon paste.

The diameter of the proposed EPMEs was 3 mm. Electric contact was obtained by inserting an Ag/AgCl wire into the carbon paste. 0.1 moL/L KCl was used as internal solution. All the sensors tips were gently rubbed on fine abrasive paper to produce a flat surface. The surface of the sensors was wetted with deionized water and then polished with an alumina paper (polished strips 30144-011, Orion) before use. When not in use, the electrodes were immersed in 10−3 moL/L of L- or D-glyceric acid solution, respectively.

2.2. Apparatus

A 663 VA Stand (Metrohm, Herisau, Switzerland) connected to a PGSTAT 100 and software (Eco Chemie version 4.9) was used for all potentiometric measurements. An Ag/AgCl (0.1 moL/L KCl) electrode was used as reference electrode in the cell.

2.3. Reagents and Materials

L- and D-glyceric acids, vancomycin, and teicoplanin were purchased from Sigma-Aldrich (USA). Graphite powder (1-2 μm) was purchased from Aldrich (Milwaukee, WI, USA); paraffin oil was purchased from Fluka (Buchs, Switzerland), and phosphate buffer (pH = 3.5) from Merck (Darmstadt, Germany).

Deionized water from a Modulab system (Continental Water Systems, San Antonio, Tex, USA) was used for all solutions preparation. L- and D-glyceric acid solutions were prepared from standard L- and D-GA solutions (1 × 10−1 moL/L) by serial dilutions. Serum and urine samples were buffered with phosphate buffer (pH = 3.5), sample : buffer = 1 : 1.

2.4. Recommended Procedure

Direct potentiometry was used for potential determination of each standard solution (10−10–10−2 moL/L). All measurements were performed at 25°C. The electrodes were placed in stirred standard solutions. Calibration graphs were obtained by plotting E(mV) versus pL-GA or pD-GA, respectively. The unknown concentrations were determined from the calibration graphs.

3. Results and Discussion

3.1. EPMEs Response Characteristics

The response characteristics of the EPMEs were determined at pH = 3.5 (phosphate buffer) using the potentiometric method. The response obtained for L-GA was linear and near-Nernstian only for the EPME based on vancomycin, while the response obtained for D-GA was linear and near-Nernstian only for the EPME based on teicoplanin. The following are the equations of calibration for the EPMEs based on vancomycin and teicoplanin: L-GA:E=574.658.6pL-GA,𝑟=0.9957,D-GA:E=206.050.0pD-GA,𝑟=0.9988,(1) where E (mV) is the potential of the electrochemical cell, pL-GA=log[L-GA],pD-GA=log[D-GA], and 𝑟 is the correlation coefficient. The response characteristics of the EPMEs are shown in Table 1. A very low detection limit was recorded for the assay of L-GA: 10−10 moL/L magnitude order. The electrodes responses displayed a good stability and reproducibility for the tests performed for 3 months, when daily used for measurements (RSD < 1.0%).

Table 1: Response characteristics of enantioselective, potentiometric membrane electrodes for L- and D-glyceric acidsa.

The response time recorded for the assay of the D-enantiomer was 2 min while the response time recorded for the assay of the L-enantiomer was 30 s.

3.2. The Influence of pH on the Responses of the Electrodes

The effect of pH on the response of the electrodes was determined by recording the emf of the cell containing solutions of L- or D-GA of different pH values. The pHs of the solutions of the enantiomers were adjusted using small volumes of HCl (0.1 moL/L) or NaOH (0.1 moL/L) solutions. E (mV) versus pH plots (Figure 1) show that the emf is not depending on the pH in the ranges of 4–9 and 3–8 for vancomycin- and teicoplanin-based EPME, respectively.

Figure 1: Effect of pH on the response of the EPMEs to L-glyceric acid (10−8 moL/L L-GA) and D-glyceric acid (10−3 moL/L) solutions. (I) Vancomycin-based EPME; (II) teicoplanin-based EPME.
3.3. Selectivity of the Electrode

The selectivity of both electrodes was checked using the mixed solutions method proposed by Ren [23], over L- or D-GA, creatine, and creatinine. The ratios between the concentrations of analyte and interferent were 1 : 10. The potentiometric selectivity coefficients (Table 2) obtained for EPMEs proved their enantioselectivity as well as their selectivity over creatine and creatinine. Inorganic cations such Na+, K+, and Ca2+ do not interfere in the analysis of L- and D-GA.

Table 2: Potentiometric selectivity coefficients for the electrodes proposed for the assay of L- and D-glyceric acidsa.
3.4. Analytical Applications

Solutions containing L- and D-GA in different ratios were prepared to test the recovery for each enantiomer in the presence of its antipode and the suitability of the EPMEs for the enantioanalysis of L- and D-GA in serum and urine samples. The recovery tests (Tables 3 and 4) obtained for each enantiomer proved the suitability of the electrodes for enantioanalysis. No significant differences in the recovery values were recorded for the ratios between L : D or D : L enantiomers varying from 1 : 9 to 1 : 99.99.

Table 3: The results obtained for the determination of L-glyceric acid in the presence of D-glyceric acida.
Table 4: The results obtained for the determination of D-glyceric acid in the presence of L-glyceric acida.

The results obtained for the analysis of L-glyceric and D-glyceric acid in serum and urine samples are shown in Tables 5 and 6, respectively. Different serum samples and urine samples were collected from different patients suspected of L-glyceric academia (1–3) or aciduria (4–9) and D-glyceric academia (10–12) or aciduria (13–18) for the recovery of L- and D-glyceric acid. All the serum and urine samples were buffered with phosphate buffer pH = 3.5. The results obtained using the proposed EPMEs are in good concordance with those obtained using the standard method, which is an HPLC technique [24]. The advantage of the proposed method over the standard one was the high reliability measured through low values of RSD (%), short time of analysis, and low cost of the enantioanalysis.

Table 5: Recovery of L-glyceric acid in serum and urine samples, (%)a.
Table 6: Recovery of D-glyceric acid in serum and urine samples, (%)a.

4. Conclusions

The macrocyclic antibiotics vancomycin and teicoplanin proved to be viable chiral selectors for the design of EPMEs. The enantioselective, potentiometric membranes electrodes proposed can be reliably used for the enantioselective analyses of L- and D-glyceric acids in serum and urine samples. Accordingly, they can be used for the fast and reliable diagnosis of L- or D-glyceric academia/aciduria. The construction of the electrodes is simple, fast, and reproducible. The serum and urine samples need only to be buffered with phosphate buffer of pH of 3.5 before L- and D-glyceric acids were determined.


  1. H. E. Williams and L. H. Smith Jr., “L-glyceric aciduria. A new genetic variant of primary hyperoxaluria,” The New England Journal of Medicine, vol. 278, no. 5, pp. 233–238, 1968. View at Google Scholar
  2. E. Van Schaftingen, “D-Glycerate kinase deficiency as a cause of D-glyceric aciduria,” The FEBS Letters, vol. 243, no. 2, pp. 127–131, 1989. View at Publisher · View at Google Scholar
  3. S. K. Wadman, M. Duran, and D. Ketting, “D glyceric acidemia in a patient with chronic metabolic acidosis,” Clinica Chimica Acta, vol. 71, no. 3, pp. 477–484, 1976. View at Google Scholar
  4. N. J. Brandt, S. Brandt, K. Rasmussen, and F. Schnoheyder, “Letter: hyperglycericacidaemia with hyperglycinaemia: a new inborn error of metabolism,” British Medical Journal, vol. 4, no. 5940, pp. 344–347, 1974. View at Google Scholar · View at Scopus
  5. E. Van Schaftingen, J. P. Draye, and F. Van Hoof, “Coenzyme specificity of mammalian liver D-glycerate dehydrogenase,” European Journal of Biochemistry, vol. 186, no. 1-2, pp. 355–359, 1989. View at Publisher · View at Google Scholar · View at Scopus
  6. P. D. Dawkins and F. Dickens, “The oxidation of d- and l-glycerate by rat liver,” The Biochemical Journal, vol. 94, pp. 353–367, 1965. View at Google Scholar
  7. A. Kaunzinger, A. Rechner, T. Beck, A. Mosandl, A. C. Sewell, and H. Böhles, “Chiral compounds as indicators of inherited metabolic disease: simultaneous stereodifferentiation of lactic-, 2-hydroxyglutaric- and glyceric acid by enantioselective cGC,” Enantiomer, vol. 1, no. 3, pp. 177–182, 1996. View at Google Scholar · View at Scopus
  8. J. P. Kamerling, G. J. Gerwig, J. F. G. Vliegenthart, M. Duran, D. Ketting, and S. K. Wadman, “Determination of the configurations of lactic and glyceric acids from human serum and urine by capillary gas-liquid chromatography,” Journal of Chromatography B, vol. 143, no. 2, pp. 117–123, 1977. View at Google Scholar · View at Scopus
  9. D. J. Dietzen, T. R. Wilhite, D. N. Kenagy, D. S. Milliner, C. H. Smith, and M. Landt, “Extraction of glyceric and glycolic acids from urine with tetrahydrofuran: utility in detection of primary hyperoxaluria,” Clinical Chemistry, vol. 43, no. 8, pp. 1315–1320, 1997. View at Google Scholar · View at Scopus
  10. M. Petrarulo, C. Vitale, P. Facchini, and M. Marangella, “Biochemical approach to diagnosis and differentiation of primary hyperoxalurias: an update,” Journal of Nephrology, vol. 11, supplement 1, pp. 23–28, 1998. View at Google Scholar
  11. M. S. Rashed, H. Y. Aboul-Enein, M. Alamoudi et al., “Chiral liquid chromatography tandem mass spectrometry in the determination of the configuration of glyceric acid in urine of patients with D-glyceric and L-glyceric acidurias,” Biomedical Chromatography, vol. 16, no. 3, pp. 191–198, 2002. View at Publisher · View at Google Scholar · View at Scopus
  12. M. Petrarulo, M. Marangella, D. Cosseddu, and F. Linari, “High-performance liquid chromatographic assay for L-glyceric acid in body fluids. Application in primary hyperoxaluria type 2,” Clinica Chimica Acta, vol. 211, no. 3, pp. 143–153, 1992. View at Publisher · View at Google Scholar · View at Scopus
  13. A. García, M. Muros, and C. Barbas, “Measurement of nephrolithiasis urinary markers by capillary electrophoresis,” Journal of Chromatography B, vol. 755, no. 1-2, pp. 287–295, 2001. View at Publisher · View at Google Scholar · View at Scopus
  14. M. Fontaine, N. Porchet, C. Largilliere et al., “Biochemical contribution to diagnosis and study of a new case of D-glyceric acidemia/aciduria,” Clinical Chemistry, vol. 35, no. 10, pp. 2148–2151, 1989. View at Google Scholar · View at Scopus
  15. G. R. Bartlett, “Human red cell glycolytic intermediates,” The Journal of Biological Chemistry, vol. 234, no. 3, pp. 449–458, 1959. View at Google Scholar · View at Scopus
  16. R. I. Stefan, J. F. van Staden, and H. Y. Aboul-Enein, Electrochemical Sensors in Bioanalysis, Marcel Dekker, New York, NY, USA, 2001.
  17. Armstrong D. W., “A new class of chiral selectors for enantiomeric separations by LC, TLC, GC, CE and SFC,” in Proceedings of the Pittsburg Conference Abstracts, p. 572, Pittconn, 1994.
  18. D. W. Armstrong and U. B. Nair, “Capillary electrophoretic enantioseparations using macrocyclic antibiotics as chiral selectors,” Electrophoresis, vol. 18, no. 12-13, pp. 2331–2342, 1997. View at Publisher · View at Google Scholar · View at Scopus
  19. T. J. Ward and T. M. Oswald, “Enantioselectivity in capillary electrophoresis using the macrocyclic antibiotics,” Journal of Chromatography A, vol. 792, no. 1-2, pp. 309–325, 1997. View at Publisher · View at Google Scholar · View at Scopus
  20. M. P. Gasper, A. Berthod, U. B. Nair, and D. W. Armstrong, “Comparison and modeling study of vancomycin, ristocetin A, and teicoplanin for CE enantioseparations,” Analytical Chemistry, vol. 68, no. 15, pp. 2501–2514, 1996. View at Publisher · View at Google Scholar · View at Scopus
  21. T. J. Ward, C. Dann, and A. Blaylock, “Enantiomeric resolution using the macrocyclic antibiotics rifamycin B and rifamycin SV as chiral selectors for capillary electrophoresis,” Journal of Chromatography A, vol. 715, no. 2, pp. 337–344, 1995. View at Publisher · View at Google Scholar · View at Scopus
  22. T. J. Ward, “Macrocyclic antibiotics—the newest class of chiral selectors,” LC GC, vol. 14, no. 10, pp. 886–894, 1996. View at Google Scholar
  23. K. Ren, “Selectivity problems of membrane ion-selective electrodes: a method alternative to the IUPAC recommendation and its application to the selectivity mechanism investigation,” Fresenius' Journal of Analytical Chemistry, vol. 365, no. 5, pp. 389–397, 1999. View at Google Scholar
  24. I. D. P. Wootton, Micro-Analysis in Medical Biochemistry, J. A. Chuchill Ltd., London, UK, 4th edition, 1964.