= Emerging. More than 5 years before clinical availability.
= Expected to be clinically available in 1 to 4 years.
= Clinically available now.

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MSACL 2019 EU : van den Ouweland

MSACL 2019 EU Abstract


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Topic: Troubleshooting

The Hurdles of Developing an LC-MS/MS Assay for Desmosine, a Biomarker for Elastin Degradation

Jody van den Ouweland (Presenter)
Canisius-Wilhelmina Hospital

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Authors: J.M.W. van den OUWELAND(1), M. SPANBROEK(2), R. JANSSEN(2), A. BEIJERS(1), H. van DAAL(1)
Department of Clinical Chemistry(1) and Pulmonary Disease(2), Canisius Wilhelmina Hospital, Nijmegen, The Netherlands

Short Abstract

Desmosine is a promising biomarker for estimating elastin degradation activity in diseases like chronic obstructive pulmonary disease and cystic fibrosis and for monitoring the effect of therapeutic interventions. We successfully developed an LC-MS/MS assay for the measurement of desmosine in urine and plasma, but encountered numerous obstacles during the developmental and validation process.

These varied from corrosion issues during sample preparation negatively impacting MS performance, to discontinuation of critical SPE material by the manufacturer and wrongful designation of the desmosine standard concentration by its supplier. As Molière stated “The greater the obstacle, the more glory in overcoming it.”

Introduction: Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. Its degradation gives rise to desmosine (DES) and isodesmosine (IDS) in body fluids. DES and IDS are promising biomarkers for estimating activity of elastin degradation, although their clinical validity and utility remain uncertain due to the lack of reliable and sensitive

assays. The objective of this study was to develop and validate a stable isotope dilution LC-MS/MS method for measurement of DES and IDS in urine and plasma. What was not anticipated were the many hurdles in the developmental process taking years for the assay could be introduced in daily practice.

Methods: The method is based on spiking with a isotope-labeled desmosine standard (D4-DES) to urine or plasma sample, acid hydrolysis (o/n at 110 °C), SPE using cellulose, C18 chromatography using 5mM NH4COOH in H2O/MeOH+ 0,1% HFBA and positive ESI MS/MS analysis (Xevo TQS, Waters) by SRM monitoring of transition ions 526-481 for DES, 526-397 for IDS and 530-486 for D4-DES. Lower limit of quantitation (LLOQ), imprecision profile, linearity and recovery were established.

Results: During assay development, we encountered numerous obstacles varying from corrosion issues during sample preparation negatively impacting MS performance, to discontinuation of critical SPE material by the manufacturer. We managed to obtain a sensitive (LLOQ 0.1 ng/ml) assay with base-line separation of DES and IDS. Intra- and inter-assay imprecision were <10%, assay linearity was up to 10 ng/ml, and DES and IDS recoveries were within 80-120%. We were surprised to observe a two-fold difference in measured concentrations when compared to data obtained from literature. It ultimately proved traced to an error in designation of the desmosine standard concentration by the supplier.

Conclusion: We have successfully developed a sensitive and specific assay for the measurement of DES and IDS in human urine and plasma. This method can be used to assess the potential of DES and IDS as biomarkers for estimating disease activity in COPD and the effect of therapeutic interventions.

Long Abstract

Problem

Method Information

Troubleshooting Steps

Outcome


References & Acknowledgements:


Financial Disclosure

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Grantsno
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Board MemberyesSci Com board MSACL EU
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

no