David Marshall (Presenter)
University Hospital South Manchester
Bio: I am currently a Trainee Clinical Scientist in the Department of Clinical Biochemistry at the University Hospital of South Manchester in England. My role involves LC-MS/MS method development, troubleshooting, and other tasks within the lab. I am currently completing an MSc in Blood Sciences as part of the course. I began in the NHS at the Royal Devon & Exeter hospital as a Medical Laboratory Assistant in 2011 where, after further studying & training I became a HCPC registered Biomedical Scientist. I then moved up to Manchester in 2014 and began training as a Clinical Scientist in September 2016. I have recently carried out placements in haematology, immunology and genetics which have broadened my knowledge and given me ideas for the future. I am currently working on method development for urine analysis (submitted) and androgen analysis using different sample types.
Authorship: David Marshall, Joanne Adaway, Brian Keevil
University Hospital South Manchester
Urine stone screens are important to identify patients at risk of nephrolithiasis. A new HPLC-MS/MS method was developed for oxalate and citrate to replace the current in house methods. Full method validation including method comparison was carried out prior to routine use. After introduction we noticed a positive bias for our EQA returns compared to WEQAS users using the oxalate oxidase enzymatic method. Recovery was good and any matrix effect was compensated for by the internal standard used therefore the differences seen may be due to variation in the sample prep, calibration and/or the enzymatic method itself.
Currently oxalate and citrate are analysed using different methods, and in order to streamline the process we decided to combine the two into a single method. Our previous mass spectrometry method for oxalate used a weak anion exchange (WAX) analytical column prior to injection into a mass spectrometer which took advantage of the compound’s anionic nature. In the new proposed method a WAX solid phase extraction (SPE) plate is used to suitably clean-up the sample for introduction into the HPLC-MS/MS.
Calibrator and acidified urine (50µL) was combined with 20µL of mixed internal standard (13C2 Oxalate/citrate-d4) and 500µL 5% v/v formic acid in water and passed through a Waters WAX SPE plate which was pre-conditioned with 100% MEOH then water. After 3 clean up steps (2% formic acid, water, then methanol) the plate was eluted with 5% NH3 in methanol, the eluate was dried down and re-constituted with 100µL distilled water, separation was then performed on an HSS T3 (2.1 x 50mm) column, flow rate of 0.5 ml/min using routine mobile phases. We used multiple reaction monitoring to detect citrate, oxalate and their respective internal standards.
After the new method was introduced we noticed a consistent proportional positive bias for our oxalate EQA results compared to the all laboratory trimmed mean (ALTM) which is calculated from 23 other WEQAS users using the oxalate oxidase enzymatic method, we were therefore concerned whether the difference was significant. In house QC and validation data show good inter/intra-assay precision and recovery respectively. Further investigation highlighted the differences in our calibration material and sample clean-up prior to analysis, consequently we decided to use the same calibrators to compare both methods and compare the sample clean-up procedure.
We compared oxalate results from our HPLC-MS/MS method with results from the enzymatic method with the charcoal clean-up step; this was carried out in a 96 well plate with optically inert wells for accurate spectrophotometric quantification. We also sent urine samples to an external laboratory using the enzymatic method for a direct comparison of oxalate. In addition we compared oxalate from our MS method with the enzymatic method using different clean-up procedures.
- Direct comparison between our MS method and an external lab using the enzymatic method showed a 15% positive bias for oxalate using mass spectrometry.
- When comparing our MS method and the enzymatic method done in house (with charcoal clean-up); preliminary data showed there to be a negative bias (10%) for oxalate using the mass spectrometry method thus conflicting with the above.
- When different sample clean-up techniques were used with the enzymatic method discrepant results were seen due to no internal standard compensating for loss of oxalate. The charcoal extraction showed better recovery than the different methods attempted.
Conclusions & Discussion
The discrepant EQA and comparison with the external lab points towards there being differences in the MS and enzymatic method, however, when the same calibrator set was used for MS vs enzymatic we found that charcoal recovered more oxalate; the opposite to what we saw before. Different calibrator sets were checked and compared well; therefore we postulate that the discrepancy in results may be due to variation from sample prep and the enzymatic reaction.
The variation from sample clean-up techniques could be explained by either loss in the plate or inhibitors of the reaction not being filtered out which have a marked effect on results from the enzymatic method. However, this problem doesn’t affect our mass spectrometry due to an internal standard being used which compensates for any loss or ion suppression.
Further work is being undertaken to elucidate why there are discrepancies in results between the different methodologies and clean up techniques, we intend to compare more patient samples to try and obtain firmer conclusions.
References & Acknowledgements:
IP Royalty: no
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