MSACL 2019 EU Abstract

Towards a LC-MS/MS Method for the Quantification of Serum Symmetric Dimethylarginine and Asymmetric Dimethylarginine for Use in a Routine Clinical Laboratory

David Marshall, Brian Keevil
Manchester University NHS Foundation Trust

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David Marshall (Presenter) Manchester University NHS Foundation Trust

Presenter Bio: I am currently a trainee Clinical Scientist in the Department of Clinical Biochemistry at Wythenshawe Hospital 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 which will be completed this summer.
I began my career in the NHS 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.
Previous projects I have worked on include development of a HPLC-MS/MS method for analysis of T, A4 and 17OHP collected using the Mitra microsampling devices. I have also validated methods for analysis of tacrolimus and creatinine on on blood samples collected onto Mitra devices.

Relevant Financial Disclosures (within past 24 months) Salary Waters Conflict mitigation NOT REQUIRED.

Background
Symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) are metabolites of the amino acid L-arginine. Recently there has been increasing interest in the utility of SDMA and have shown potential as an alternative to estimated glomerular filtration rate (eGFR), however, the use of GC-MS or long analytical run times has hindered the availability of analysis in routine clinical laboratories. Here, we describe the development of a rapid and simple LC-MS/MS method to help better-understand the utility of SDMA and ADMA in a routine clinical biochemistry lab.

Objectives
To develop and validate an LC-MS/MS method for measurement of serum SDMA and ADMA. The method has then been applied to undertake an in-house comparison with a published HILIC method. The methods were used to look at the correlation between SMDA and ADMA in patients with normal (eGFR >90) and impaired (eGFR <90) renal function.

Methods
The protocol from the published HILIC method was adapted and performed on a Waters Xevo TQS mass spectrometer; samples were introduced and subjected to a protein crash and subsequent dilution with acetonitrile prior to introduction into the LC-MS/MS system. The new method utilised a cation exchange column (CEX) and was undertaken on a Waters Xevo TQS Micro mass spectrometer, the sample preparation was the same as above as it was fast and simple. Partial validation was carried out for both methods, sample comparisons were undertaken to both investigate the suitability of the CEX method and to see how well SDMA, ADMA and ratios of both compared with creatinine concentration which was measured using an automated platform.

Results
The method was validated against an industry standard with recovery, ion suppression and imprecision all within acceptable limits for both methods. However, sample comparisons showed there to be slight differences in the CEX method from the HILIC method even though the same calibrators and preparation procedure were used. SDMA concentrations showed good correlation with creatinine in normal patients.

Conclusion
Extraction and analysis of SDMA/ADMA is very pH sensitive, therefore we postulate that pH differences between the HILIC and CEX methods may account for the small positive bias. However, due to there being no reference materials available it is difficult to identify which assay is accurate.