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.

[Orotate] uM / mmol creatinine

1000 500 250 750 OCT Citrullinaemia ASA Argininaemia LPI

CPS Deficiency & Unaffected

HHH syndrome Hereditary orotic aciduria

[2] Salerno C, Crifco C. Diagnostic value of urinary orotic acid levels: Applicable Separation

• Methods. J. Chrom B 781 (2002) 57–71

Why Quantitate?

Choice of Instrumentation

HPLC

• Long procedure

– Clean up

• Drug interference

GC-MS

• Long procedure

– Clean-up/extraction – Derivatisation

— Simple, rapid sample preparation:

—

20ul sample + 150ul 15N2-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

LC-ESI-MS/MS

Multiple Reaction Monitoring (MRM)

Q1

m/z 155.1 m/z 157.1

q2

CID

Q3

m/z 111.1 m/z 113.1

LC-ESI(-ve)-MS/ MS in MRM mode

Orotic acid [M-H] - m/ z 155.1 à 111.1 [M-H – CO2] -

15N2-Orotic acid [M-H] -

m/ z 157.1 à 113.1 [M-H – CO2] -

Linearity

— Linear to 200uM — r2 = 0.9977

Accuracy

— 21 EQA samples analysed — Direct comparison of results and target values — r2 = 0.9898

Precision

Inter-Assay

• EQA samples

– C1 = 2.6 µM – C2 = 39.9 µM – C3 = 89.5 µM

• CV1= 29% (n=7)
• CV2 = 8 % (n=11)
• CV3 = 7 % (n=11)

Intra-Assay

• 22 replicates of EQA material

analysed

• C = 36.5 µM
• CV = 6%

Limit of Detection

• LOD = 3.125 µM
• S/N = 42
• Fit for purpose – clinically

significant increase detectable

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