A novel method for the determination of urinary citrate levels: Ion-pair and high performance liquid chromatography with liquid membrane extraction technique

Abstract

In the clinical evaluation, and treatment of the patients with urinary calcium oxalate stones, determination of urinary citric acid levels is as important as oxalic acid levels. Presence of specific matrix proteins, and other anions in urine complicates its measurement. Therefore we aimed to develop a routine high performance liquid chromatography (HPLC) method which could determine citric acid levels in urine, and other body fluids. One hundred patients referred from various clinical sections were included in the study, and their urinary citrate levels in spot or 24-hour urine samples were measured as required. Measurement of oxalic acid was performed with a liquid membrane technique using a 314 nmUV detector at a stationary phase of C18 column, under 25oC ambient temperature, and at a flow rate of 1.7 mL/min Internal standard method was used for the determination of citrate concentration. For citrate standards at two distinct concentrations (1.25 mmol/L, and 5 mmol/L) respective intraday, and interday coeffıcient of variation (CV) values were found to be 4.29 vs 5.37%, and 2.78 vs 3.13% with a linearity interval of 0.03-20 mmol/L. In three different concentrations mean recovery rates of 101.2, 100, and 99.7% were obtained, respectively, and functional sensitivity of the assay was determined as 0.009 mmol/L. In conclusion, thanks to this method we developed, problems encountered during analysis of biological samples with complex matrix appears to be solved at greater extent. Besides compared with other available methods, this HPLC assay yields results in a shorter time frame with higher cost-effectiveness, simplicity, reproducibility, and reliability Therefore, we think that it can be used routinely in the determination of citrate levels in urine, and other body fluids.

Keywords

• Citrate
• High performance liquid chromatography
• Liquid membrane extraction technique
• Urine

References

1. Akinci, M., Esen, T., Tellaloğlu, S., 1991. Urinary stone disease in Turkey: An updated epidemiological study. Eur. Urol. 20, 200-203. PMID: 1823043.
2. Amaro, C.R., Goldberg, J., Amaro, J.L., Padovani, C. R., 2005. Metabolic assessment in patients with urinary lithiasis. Int. Braz. J. Urol. 31, 29-33. doi: 10.1590/S1677-55382009000600004.
3. Ansari, M.S., Gupta, N.P., 2003. Impact of socioeconomic status in etiology and management of urinary stone disease. Urol. Int. 70, 255-261. doi: 10.1159/000070130.
4. Balaji, K.C., Menon, M., 1997. Mechanism of stone formation. Urol. Clin. North Am. 24, 1-11. doi:10.1016/S0094-0143(05)70350-5.
5. Beutlere, M., Yeh, M.K.Y., 1959: A simplified method for the determination of citric acid. J. Lab. Clin. Med. 54, 125-131. PMID:13665158.
6. Dempsey, E.F., Forbes, A.P., Melick. P.A., Henneman. P.H., 1960. Urinary oxalate excretion. Metabolism. 9, 52-58. PMID:13815869.
7. Fredric, L.C., Andrew E., Elaine, W., 2005. Kidney stone disease. J. Clin. Invest. 115, 2598-2608. doi:10.1172/JCI26662.
8. Holmes, R.P., 1995. Measurement of urinary oxalate and citrate by capillary electrophoresis and indirect ultraviolet absorbance. Clin. Chem. 4179, 1297-1301. PMID:7656440.

9. Holmes, R.P., Martha., K., 1999. Urinary oxalate and citrate. Clinical applications of capillary electrophoresis. Methods Mol. Med. 27, 199-202. doi:10.1385/1-59259-689-4:199.
10. Keevil, B.G., Owen, L., Thornton, S., Kavanagh, J.,2005. Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method. Ann. Clin. Biochem. 42, 357-363. doi: 10.1258/0004563054889963.
11. Keevil, B.G., Thornton, S., 2006. Quantification of urinary oxalate by liquid chromatography-tandem mass spectrometry with online weak anion exchange chromatography. Clin. Chem. 52, 2296-2299. doi: 10.1373/clinchem.2006.075275.
12. Khaskhali, M.H., Iqbal, B., Khand, F.D., 1996. Simultaneous determination of oxalic and citric acids in urine by high-performance liquid chromatography. J. Chromatogr. B. Biomed. Appl. 675, 147-151. doi:10.1016/0378-4347(95)00338-X.
13. Kok, D.H., Socrates E., Papaouls, S.E., Bijvoet, O.L.M., 1990. Kidney Int. 37, 51-56. doi:10.1038/ki.1990.7.
14. Lewis, B.D., 1990. Determination of citrate in urine by simple direct photometry. Clin. Chem. 36, 578.
15. Li, M.G., Madappally, M.M., 1989. Rapid enzymatic determination of urinary oxalate. Clin. Chem. 35, 2330-2333.
16. Moe, O.W., 2006. Kidney stones. Lancet. 367, 333-344. doi:10.1016/S0140-6736(06)68071-9.
17. Ogawa, Y., Yamaguchi, K., Tanaka, T., 1987. Evaluation of the enzymatic method using oxalate oxidase for urinary oxalate assay. Acta Urol. Jpn. 33, 1951-1954.
18. Öner, A., 2009. Üriner sistem taş hastalığında metabolik değerlendirme. Turkiye Klinikleri J. Urol. Special Topics. 2, 6-8.
19. Pearle, M.S., Calhoun, E.A., Curhan, G.C., 2005. Urologic disease in America Project: Urolithiasis. J. Urol. 173, 848-857. doi:10.1097/01. ju.0000152082.14384.d7.
20. Petrarulo, M., Facchini, P., Cerelli, E., 1995. Citrate in urine determined with a new citrate lyase method. Clin. Chem. 41, 1518-1521.
21. Smith, L.H., 1989. The medical aspect of urolithiasis: An overview. J. Urol. 141, 707-710. PMID:2645427.
22. Wahl, C., Hess, A., 2000. Kidney calculi-nutrition a trigger or treatment? Ther. Umsch. 57, 138-145. PMID:2645427.