MSACL 2016 EU Abstract

A Novel Approach to Urine Catecholamines LC-MS/MS Analysis

Marianne L. Bergmann (Presenter)
Lillebaelt Hospital, Vejle

Authorship: Marianne L. Bergmann (1), Anne Schmedes (1), Seyed Sadjadi (2)
(1) Lillebaelt Hospital, Vejle, Denmark, (2) Phenomenex, Torrance, CA

Short Abstract

The catecholamines, epinephrine (E) and norepinephrine (NE) are very small and polar molecules, causing additional challenges to LC-MS/MS method development, as they are poorly retained on reverse-phase C18 columns. This work presents the development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pair chromatography (IPC). Here, the ion-pair reagent 1-Heptane Sulfonic Acid (HSA) is added into the extracted samples instead of the mobile phases (MP) while using conventional LC/MS friendly MP for analysis. This overcomes some of the disadvantages of traditional IPC and the method shows very good performance characteristics.

Long Abstract

Introduction

Catecholamines are small, very polar molecules and are therefore poorly retained on reverse-phase C18 columns. This causes short retention times and difficulty in separating epinephrine (E), norepinephrine (NE) and other isobaric compounds from each other and from the matrix. Elution of E and NE too close to the solvent front will increase the risk of ion suppression caused by other compounds and hamper the sensitivity of the method. In addition, the small polar compounds elute in solvent with very low organic content (maximum 5%), which leads to poor ionization in the mass spectrometer.

In a recent development, a new approach to ion-pair chromatography (IPC) has been established that does not require the addition of an ion-pair reagent (IPR) into the mobile phases. Instead, the IPRs are added into the sample prior to injection on the LC column. This approach can overcome some of the disadvantages of traditional IPC, such as contamination of the mass spectrometer and severe ion suppression.

The aim of this work was to develop an LC-MS/MS method for determining catecholamines in urine samples, based on the new IPC approach, using 1-Heptane Sulfonic Acid (HSA) as IPR.

Methods

Sample extraction was performed on a Hamilton STARlet workstation, using solid phase extraction on Waters Sep-Pak Alumina B, 100 mg 96 well extraction plates or Phenomenex Strata Alumina-N, 100 mg 96 well extraction plates.

After elution the samples were diluted with 60mmol/L HSA in aqueous 0.1% formic acid and analyzed on a Waters Acquity UPLC with Xevo TQ-S tandem mass spectrometer operated in electrospray positive mode (ES+). The chromatographic separation was achieved with a Phenomenex Kinetex Biphenyl (100 x 2.1 mm, 2.6 µm) column and gradient elution with mobile phases consisting of 0.1% formic acid in water (mobile phase A) and methanol (mobile phase B).

Results

Chromatographic separation of E and NE was achieved with very stable retention time (RT) of E and NE. In 484 injections in routine use of the method the RT was steady with a CV% of less than 4 %. Furthermore, HSA was separated from E and NE, routing the HSA to waste instead of entering the mass spectrometer.

For routine use the method was calibrated with a single level ClinCal® calibrator (Recipe) with 0.686 µmol/L NE and 0.159 µmol/L E. During validation an eight point calibration curve was prepared and least squares linear regression showed linearity up to 2.0 µmol/L for both analytes. Mean recoveries were 103% (CV = 4.5%) for E and 106% (CV =3.7 %) for NE. Intermediate precision for control samples in the normal (n=31) and abnormal range (n=28) were below 7% for E and 7.5% for NE. Matrix effect experiments showed a 10% lower response for pre-extracted samples than for mobile phase samples containing HSA, indicating only slight ion suppression. No carryover was observed in the blank sample when injected after the highest calibrator.

Conclusion

We have developed and validated an LC-MS/MS method, using a novel approach to IPC, for simultaneous measurement of E and NE in urine. The assay has good performance characteristics and has been routinely used in clinical diagnostics for 6 months.


References & Acknowledgements:


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