Development of an Assay for Citrate and Oxalate in Urine by Hydrophilic Interaction LC-MS/MS After Weak Anion Exchange SPE Albert Dijkhuizen (Presenter) Leiden University Medical Center

Authors: A.Dijkhuizen, M.M.Pieterse, P.A.Spigt, P.Schippers, J.A.Bakker, C.M.Cobbaert
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center

Traditionally urinary oxalate and citrate were analyzed with enzymatic and colorimetric methods. Today chromatographic-mass spectrometric methods become more common. We developed a HILIC LC-MS/MS method including SPE for the determination of oxalate and citrate.
After sample clean-up with a Waters Oasis WAX SPE-column, extracts are injected on a Waters BEH-amide column. Elution is performed with a linear acetonitrile to water (pH=10.0) gradient. Retention-times are 2.1 and 2.8 min. for oxalate and citrate respectively. Calibration-curves are linear from 10-1000 µM for oxalate and 50-5000 µM for citrate. The method is free of interfering peaks and ion suppression.

Introduction

Traditionally oxalate and citrate in urine are analyzed with enzymatic and colorimetric methods (1,2). Recently, methods based on gas or liquid chromatography combined with mass spectrometry, were introduced . The high specificity of these methods makes the assays more preferred. At the moment oxalate and citrate are mostly measured by gas chromatography/mass spectrometry(3). The drawback of this method is the time-consuming sample preparation with derivatization. Marshall et al. measured oxalate and citrate by liquid chromatography/tandem mass spectrometry using a reversed phase column (4). Due to the polar nature of the analytes retention times are very short and oxalate quantitation is affected by considerable ion suppression (-23 to + 65%). Although this suppression is compensated for by an internal standard this is not an ideal situation. The polar nature of oxalate and citrate makes them perfect candidates for Hydrophilic Interaction Liquid Chromatography (HILIC). Oxalate and citrate have several pKa-values between 1 and 7 so for good chromatography the pH must be lower than -1 or higher than 9. Yet, most HILIC- columns cannot operate in caustic conditions. This problem was overcome by using a Waters BEH amide column that has a maximum pH-limit of 11. The method presented here is,- to our best knowledge-, the first LC-Tandem-MS method that analyses citrate and oxalate after a simple sample-preparation with no ion suppression and with sufficient retention.

Method development

Our first selection of analytical column and mobile phase gave satisfying results and needed only little tweaking. First, direct injection of urine was tested. Ion suppression was monitored for this and revealed that massive ion suppression was present.

SPE with weak anion exchange (WAX) was compared to SPE with strong ion exchange (SAX) . In this comparison SAX gave better extraction-recovery but needed a higher volume to elute the analytes. Further investigation of the results revealed that the biggest losses with WAX take place when the column is washed with methanol. Investigation of the ion suppression when load-, wash-, and elution-steps are injected revealed that the load-step and first wash step ( 0,1 M HCL) suffer the most from ion suppression. The second wash step (methanol) shows no ion suppression so this wash step in the protocol does not add to the elimination of ion suppression in the final method. Because this wash step also lowers recovery this wash step is eliminated from the final protocol.

When tested with patient samples this method shows that samples contain a compound that interferes with citrate when certain MRM’s are chosen. Careful selection of the two MRM’s used for quantification and conformation eliminated this interference.

Methods

Calibrators were prepared by adding certified stock-solutions (Cerilliant) to certified steroid-free serum( Golden West Biologicals).

Standardized aliquots of urine are acidified with 25 µl HCL 4 M and internal standards (citrate-D4 and oxalate- 13C2) are added. After centrifugation the sample is applied to a Waters Oasis WAX-column. After washing with 0,1 M HCl the analytes are eluted with 0.5 ml 5% ammonia in methanol. 0.5 µl of this extract was directly injected on the LC-MS-system.

The LC-system consist of an Agilent Infinity II autosampler and UHPLC-pump. The column is a Waters BEH-amide 100x2.1 mm, 1.7 µm pd. Eluens A is 50 mM ammoniumbicarbonaat pH=10.0 in water and eluens B is 50 mM ammoniumbicarbonaat pH=10.0 in acetonitrile/water 70/30. The linear gradient starts with 0 % A at t=0 min, and ends with 25% A at t= 5 min. Equilibration time is 3 min.

The Agilent 6495 triple quadrupole mass spectrometer was operating in the negative ion-mode.

Results

MRM’s selected are m/z 89>45 and 89>61 for oxalate, 91>46 and 91>62 for oxalate-13C2 , 191>87 and 191>111 for citrate and 195>88 and 191>113 for citrate-D4.

Retention time of oxalate is 2.1 min. and of citrate 2.8 min. Calibration curves are linear from 10-1000 µM for oxalate and 50-5000 µM for citrate ( r2>0,999). The method has showed to be free of interfering peaks and ion suppression. A full validation of this method is currently underway.

Conclusions

Our in house developed LC-MS method enables specific and accurate quantitation of citrate and oxalate in urine in the clinical relevant concentration range. The method is free from matrix interferences due to specific MRM’s, sufficient retention and the use of stable isotope labeled internal standards. It is anticipated that the ongoing analytical validation will demonstrate that the analytical performance requirements for clinical utility are fulfilled, this LC-MS test will be applied for clinical use.

1 Mingen G., Mathew M.(1989). Rapid enzymatic determination of urinary oxalate. Clin.Chem, 35(12), 2330-2333.

2 Jan K. Kirejczyk, Tadeusz Porowski,corresponding author Jerzy Konstantynowicz, Agata Kozerska, Andrzej Nazarkiewicz, Bernd Hoppe, and Anna Wasilewska (2014). Urinary citrate excretion in healthy children depends on age and gender Pediatr Nephrol, 29(9), 1575–1582.

3 Michelle Lopez, Mendel Tuchman, Jon I. Scheinman (1985). Capillary gas chromatography measurement of oxalate in plasma and urine. Kidney International, 28(1), 82-84

4 Marshall DJ, Adaway JE, Keevil BG (2017). A combined liquid chromatography tandem mass spectrometry assay for the quantification of urinary oxalate and citrate in patients with nephrolithiasis. Ann Clin Biochem. doi: 10.1177/0004563217739035 [Epub ahead of print]