Quantitative Analysis of Orotic Acid by LC-ESI-MS/MS James Cooper - PowerPoint PPT Presentation

Quantitative Analysis of Orotic Acid by LC-ESI-MS/MS James Cooper Willink Biochemical Genetics Genetic Medicine St Mary’s Hospital Manchester

Overview — Increased orotate excretion a biomarker for several IEMs — Quantitation of orotate useful since: — Can help with differential diagnosis ÷ QAA not conclusive ÷ Qualitative organic acid analysis misleading — Stable isotope dilution LC-ESI-MS/MS method [1] — Sample preparation rapid and simple — Good accuracy — Good precision — Capable of detecting elevated concentrations of clinical significance Adapted from [1] Marca GI, Casetta B and Zammarchi E. Rapid determination of orotic acid in urine by a fast liquid chromatography/tandem mass spectrometric method. Rapid Commun Mass Spectrom 2003; 17:788-793.

Why Quantitate? [Orotate] uM / mmol creatinine Hereditary orotic aciduria 1000 750 Argininaemia 500 Citrullinaemia OCT 250 ASA LPI HHH syndrome 0 CPS Deficiency & Unaffected [2] Salerno C, Crifco C. Diagnostic value of urinary orotic acid levels: Applicable Separation Methods. J. Chrom B 781 (2002) 57–71

Choice of Instrumentation HPLC GC-MS • Long procedure • Long procedure – Clean up – Clean-up/extraction – Derivatisation • Drug interference LC-ESI-MS/MS — Simple, rapid sample preparation: 20ul sample + 150ul 15 N 2 -orotic acid (IS) (200µM ) — ÷ Calibration standards 0-200µM ÷ EQA samples used as IQC — C18 reversed phase LC separation — 5µ x 4.6mm x 15omm Phenomenex Gemini — 60% acetonitrile with 2.5mM ammonium acetate

Multiple Reaction Monitoring (MRM) LC-ESI(-ve)-MS/ MS in MRM mode m/ z 155.1 à 111.1 Orotic acid [M-H] - [M-H – CO 2 ] - m/ z 157.1 à 113.1 15 N2-Orotic acid [M-H] - [M-H – CO 2 ] - Q 1 Q 3 q2 m/z 111.1 CID m/z 155.1 m/z 113.1 m/z 157.1

Linearity — Linear to 200uM — r 2 = 0.9977

Accuracy — 21 EQA samples analysed — Direct comparison of results and target values — r 2 = 0.9898

Precision Inter-Assay Intra-Assay • EQA samples • 22 replicates of EQA material analysed – C 1 = 2.6 µM C = 36.5 µM – C 2 = 39.9 µM • – C 3 = 89.5 µM • CV = 6% • CV 1 = 29% (n=7) • CV 2 = 8 % (n=11) • CV 3 = 7 % (n=11)

Limit of Detection • LOD = 3.125 µM • S/N = 42 • Fit for purpose – clinically significant increase detectable OA110608005 MRM of 2 Channels ES- S/N:RMS=100.39 155 > 111 100 2.56e3 2.35 2.43 % 2.45 2.53 2.73 2.26 0.45 0.69 3.09 2.85 4.50 0.16 0.94 1.77 4.28 4.80 4.98 0.60 1.97 3.43 3.65 3.82 4.11 1.03 1.33 1.36 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 OA110608004 MRM of 2 Channels ES- 155 > 111 100 2.56e3 S/N:RMS=58.60 2.49 2.54 2.55 % 2.56 2.62 2.85 3.43 3.97 0.04 0.44 0.66 1.66 1.94 3.62 0.36 2.19 3.51 4.08 4.32 4.94 1.63 2.97 4.37 4.74 1.06 1.37 0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00