Help on: Measurement of urinary citrate levels by HPLC

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Question:"We would like to measure 24 hrs urinary citrate levels in humans for our upcoming project. How can HPLC help us?"

Answer:
"To utilise HPLC for the measurement of urinary citrate you need to consider the nature of the analyte, in this case the citrate molecule. The relatively polar nature of this molecule makes it suitable for analysis by either ion exchange HPLC [1] or reversed phase partition HPLC. Reversed phase partition HPLC is usually preferred due to its availability in most laboratories.

Examples in the literature report the use of C18 columns for the analysis of citrate [2,3]. A low pH aqueous mobile phase was used combined with a column preconditioning step consisting of 100% methanol, this organic solvent rinse was repeated between injections to ensure that the chromatography was acceptable. The reason for using a 100% aqueous mobile phase is to obtain satisfactory retention of the relatively polar citrate molecule.

I suggest that you make use of an aqua column which can perform in 100% aqueous conditions, and should adequately retain the citrate molecule. The low pH of the mobile phase means that citrate is in its unionised form and maximises the retention on a RP column. It is important to control the pH adequately since citrate has 3 pKa values at 3.1, 4.7 and 5.4, changes in pH around these values may have an effect on the retention of the analyte and thus the reproducibility of the chromatography.

The citrate molecule contains a weak chromophore which will enable the use of UV detection. A low wavelength such as 210 nm will be suitable. LC-MS may also be used.

The other issue to consider when analysing samples of urine is the matrix and how much sample preparation is required. Khaskhali et al. [2] found that deproteinisation of the urine sample was adequate."

References:1. Kristina L. Penniston, Stephen Y. Nakada, Ross P. Holmes, Dean G. Assimos. Journal of Endourology. March 2008, 22(3): 567-570. ‘Quantitative Assessment of Citric Acid in Lemon Juice, Lime Juice, and Commercially-Available Fruit Juice Products’
2. M. Hassan Khaskhali, M. Iqbal Bhanger and F. D. Khand Journal of Chromatography B Volume 675, Issue 1, 12 January 1996, Pages 147-151. ‘Simultaneous determination of oxalic and citric acids in urine by high-performance liquid chromatography ‘
3. Keevil B. G. ; Owen L. ; Thorton S. ; Kavanagh J. ; Annals of clinical biochemistry 2005, vol. 42 (5), pp. 357-363 ‘Measurement of citrate in urine using liquid chromatography tandem mass spectrometry : comparison with an enzymatic method'

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