Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters
Rowan Hellier1, Luke Griffiths1, Chandra Sundas1
1Department of Medical Biochemistry, Immunology and Toxicology, University Hospital of Wales, Cardiff, Wales, UK.
Objectives:
Quantitation of plasma amino acids (AA) is critical for the diagnosis and monitoring of inherited disorders of AA metabolism. AA analysis using ion-exchange chromatography (IEC) with post-column ninhydrin derivatization is time consuming, with run times of ∼2 h, limiting sample throughput. Liquid chromatography mass-spectrometry can potentially address some of the current challenges.
Methods:
Performance of components of the Waters Kairos Amino Acid Kit using liquid chromatography single quadrupole mass-spectrometry (LC-MS) following derivatization of samples with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ•Tag™ Ultra Derivatization Reagent) was evaluated. Results were compared with the Biochrom-IEC method using patient specimens (n=115), ClinChek® control and external quality assessment (EQA) material.
Results:
The kit reagents and our developed method had a 19-min analysis time, demonstrated acceptable inter-assay imprecision (CV<10 %) and bias vs. IEC-method (overall mean bias <2 %). Excellent correlation (concordance correlation coefficient (CCC) >0.99) with IEC was demonstrated for 10/23 analytes, good correlation (CCC >0.95) for 10/23, with the remaining three amino acids (aspartate, histidine and tryptophan) demonstrating moderate concordance (CCC ≥0.90 but <0.95). 1/23 AAs had a mean bias >10 % using EQA material. The method demonstrated a lower limit of quantitation of ≤2.5 μmol/L for all AA, making this assay suitable for CSF analysis. Calibration stability bias was <5 % over 12-weeks. Derivatized AAs were stable for ≤17 days. The analytical column supplied demonstrated good retention time stability (<0.4 %) and was capable of >2000 injections.
Conclusions:
The tested methodology demonstrated good analytical performance and correlation with IEC. This approach confers practical advantages over IEC, including analytical selectivity and workflow time efficiency.
