A HILIC-ESI-MS method for profiling biomarkers of Maple syrup urine disease and methyl malonic acidemia in urineElizabeth Mary Mathew1

, Leslie Lewis2, Pragna Rao3, Asha Kamath4, Sudheer Moorkoth1

1Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education-576104, Karnataka,India.2Department of Paediatrics, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India3Department of Biochemistry, Kasturba Medical College, Manipal Academy of Higher Education: Manipal, 576104, Karnataka, India4Department of Statistics, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India

3-methyl oxovaleric acid,2-hydroxy -3-methyl butyric acid

BCKDH

Acetoacetate, acetyl CoA, propionyl CoA

Methyl malonic acid

Succinic acid

Methyl malonyl CoA mutase

Introduction

To develop and validate a novel derivatisation free and sensitive bioanalytical LC-MS/MS method for the simultaneous quantification of methyl malonic acid, 3-methyl-2-oxovaleric acid, 4-methyl-2-oxovaleric acid and 2-hydroxy-3-methyl butyric acid from urine. validate a novel derivatisation

Ethical clearance: IEC 767/2015Instrument: LC-MSn (Thermo Scientific, US)

Objectives

Methodology

Results

Leucine, isoleucine, valine

Optimised chromatographic conditions

Column: A SeQuant ZIC-cHILIC column (250 x 4.6 mm, 3 μm)Temperature of column oven and autosampler: 25⁰C and 4⁰CMobile phase: 25μM formic acid in acetonitrile: 25μM formic acid in water(55:45) Flow rate:0.8mL/minSample injection volume :2µLSample preparation: dilute and shoot approach i.e. by 100 times dilution of urine sampleMass spectrometry conditions: Spray voltage: 8V, vaporizer temperature: 300°C, nitrogen sheath gas flow : 55 arbitrary units, auxiliary gas flow: 12 arbitrary units, sweep gas flow: 2 arbitrary units, ion transfer capillary temperature: 35°C with a voltage of -31.00V and tube lens :-78.39, multipole00 offset :3.50V, lens0: 7.50V, multipole0 offset: 9.25V, lens voltage: 16V, gate lens: 56V, multipole1 offset :14V ,multipole RF amplitude :400 V.p.p. and front lent lens : 9.25 V. The optimised SRM transition for each analyte were as follows; MMA (m/z 117 → 73, CE:40),3MOVA (m/z 129 → 84, CE:50), 2H3MB (m/z 117 → 71, CE:35) and tropic acid (m/z 166 → 135,CE:32) in SRM mode

Optimised chromatogram

Summary of validation results

References

Poster no:8b

Analyte Calibration

range(µg/mL)

LOQ(µg/mL

)

L0D(µg/mL

)

QC Levels

(µg/mL)

Accuracy Precision

(%CV)

Percent

Recovery

3MOVA 0.05-10 0.05 0.01

LLOQ(0.05) 92.6- 93.08% 4.33% -LQC(0.5)

98.68- 104.61 % 3.00% 97.88%MQC(2)HQC(8)

MMA 0.05-90 0.05 0.01

LLOQ(0.05) 89.12 - 98.86% 1.25% -LQC(1)

93.62- 110.76% 7.38% 91.53%MQC(18)HQC(72)

2H3MB 0.05-25 0.05 0.01

LLOQ(0.05) 98.86- 104.04% 7.50% -LQC(1)

98.86-110.76 % 1.51% 103.43%MQC(18)HQC(72)

4MOVA 0.05-10 0.05 0.01

LLOQ(0.05) 95.7- 98.08% 3.33% -LQC(1)

95.61-101.62% 1.00% 95.98%MQC(18)HQC(72)

Summary of stability results

Summary of method comparison studies

The authors acknowledge Manipal University for providing the infrastructure facility to conduct this study. The authors acknowledge MSACL for the young investigator travel grant and Indian council of medical education and research for the International travel grant for Non ICMR scientists ( 3/2/TG-94/HRD-2018 (22)). The authors also acknowledge Vision group of science and technology, Government of Karnataka (GRD-308) and Grand Challenges Canada (R-ST-POC-1707-07275) for providing the financial support to undertake the work.

Baumgartner, M. R., Hörster, F., Dionisi-Vici, C., Haliloglu, G., Karall, D., Chapman, K. A., … Chakrapani, A.; Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia.; Orphanet Journal of Rare Diseases, (2014);9:130

Acknowledgement

A derivatization free HILIC-MS method have been developed for the simultaneous estimation of 3MOVA,2H3MB, MMA and 4MOVA from urine samples

Conclusion