Liquid chromatography tandem mass spectrometry: challenges in introducing published methods into the clinical laboratory

We read with interest the recent article by Marshall et al. ¹ on combined quantification of urinary citrate and oxalate using liquid chromatography tandem mass spectrometry (LC-MS/MS). We have attempted to develop a method for the simultaneous measurement of urinary citrate and oxalate using a ‘dilute and shoot’ approach on an Acquity liquid chromatography module coupled to a Xevo TQS tandem mass spectrometer (Waters, UK) with limited success.

Oxalate tuning experiments were performed using an oxalate solution (9 mg/mL, Sigma Aldrich), which was diluted in deionised water to produce a solution of 1 mg/mL (pH 2). Equipment was operated in electrospray negative ionisation mode (capillary voltage 2.7 kV, collision energy 9.0 eV, source temperature 150°C, desolvation temperature 200°C and desolvation gas flow 500 L/hr). Mobile phase A was deionised water and mobile phase B methanol. Both mobiles phases contained 2 mmol/L ammonium acetate (Sigma-Aldrich, Dorset, UK) and 0.1% formic acid (Biosolve BV, Netherlands). The composition of the mobile phase was manipulated to pH 4.2 (oxalic acids highest pKa of 4.2). An oxalate precursor ion was generated (m/z 88.9), but it proved challenging to generate an optimal product ion (m/z 60.8) even at micromolar concentrations (collision energies of 5–50 eV evaluated). Consequently, further assay development for oxalate was not performed. The exact reason for the poor product ion signal is unknown, but one may postulate that it results from oxalate being a small molecule that has poor ionisation efficiency ² and is fragile in the stepwave and/or collision cell of the mass spectrometer used in our study.

In contrast, citrate ionised more efficiently to generate both precursor and product ions (191.1 > 111.0). An Atlantis C18 column (3 × 100 mm, 3.0 µm, Waters, UK) was used for chromatographic isolation of citrate (run time 5 min). Citrate’s analytical measuring range was linear up to 10,000 μmol/L, the lower limit of the measuring interval was 50 μmol/L. Interassay precision was less than 15%, between 200 and 10,000 μmol/L. Method comparison with a citrate lyase enzymatic assay (ILab Aries, Cheshire, UK) showed a mean bias of 31.4% (95% CI 24.0–38.8). Postcolumn infusion of citrate (1 mg/mL) did not show ion suppression at the area of citrate elution (retention time 2.29 min). However, matrix factor experiments revealed ion enhancement at 1 mmol/L and ion suppression at 9 mmol/L. The percentage coefficient of variation for the internal standard normalised matrix factor was 19.4 and 13% at 1 and 9 mmol/L, respectively. This was likely due to the use of phosphate-buffered saline (PBS)-based calibrators. In contrast, Marshall et al. ¹ reported a matrix factor of <10% for citrate using PBS calibrators. This is likely due to the choice of column (0.8 μm HSS T3 Vanguard column coupled to an HSS T3 2.1 × 50mm 1.8 μm, Waters, UK) as well as the solid phase extraction step. ³ Our findings make an excellent case for undertaking both qualitative (i.e. postcolumn infusion) and quantitative assessments of the matrix effects when developing LC-MS/MS methods, as per recommendations of the CLSI C62A guidance for LC-MS/MS based methods in the clinical laboratory. ³

LC-MS/MS has increasingly become a popular technique in the clinical laboratory. Nonetheless, it is still not main stream for measurement of citrate and oxalate. A review of the urinary citrate and oxalate methods used by the WEQAS scheme participants shows that they are dominated by enzymatic methods (personal communication, WEQAS 2017). This is not surprising as they are robust and avoid the need for technical expertise.

Our experience highlights some of the difficulties in introducing published LC-MS/MS methods in the clinical laboratory. With harmonisation gaining momentum in the clinical laboratory accreditation process and with the introduction of LC-MS/MS specific guidance, ³ it is hoped that the assay development process for newly published methods should become more robust and less daunting.