Automated Solid-Phase Extraction for the Analysis of Metanephrine and Normetanephrine from Plasma using ACQUITY UPLC-MS/MS for Clinical Research

Significance of the Topic

Measuring metanephrine and normetanephrine in plasma is essential for diagnosing and monitoring disorders related to catecholamine excess. Traditional techniques such as HPLC with electrochemical detection or immunoassays often lack analytical specificity and require lengthy sample preparation and chromatography. Integrating automation and mass spectrometry enhances throughput, sensitivity, and robustness for clinical research applications.

Objectives and Overview of the Study

This study aimed to develop and validate an automated offline solid phase extraction workflow for quantifying plasma metanephrine and normetanephrine. The method combines Oasis WCX microelution plates with a Tecan liquid handling system, ACQUITY UPLC separation on a BEH Amide column, and Xevo TQ MS detection using multiple reaction monitoring.

Methodology and Used Instrumentation

Sample preparation involved spiking stripped serum with authentic metanephrine and normetanephrine salts to generate calibrators and blank correction. A deuterated internal standard mixture was prepared daily. Plasma samples were centrifuged and loaded onto the Tecan platform for automated SPE using Oasis WCX microelution plates. Automated steps included sample mixing with internal standard, conditioning, washing and elution with formic acid in acetonitrile.

• Liquid handling system Tecan Freedom EVO 100
• Oasis WCX microelution plates
• ACQUITY UPLC system with BEH Amide column (1.7 micron, 2.1 x 50 mm)
• Xevo TQ mass spectrometer operating in positive ESI and MRM mode

Chromatography used a gradient of 100 mM ammonium formate buffer at pH 3.0 and acetonitrile at 200 µl/min. The run time was 5 minutes per injection. MS detection monitored the [M+H-H2O]+ transitions 180>148 for metanephrine and 166>134 for normetanephrine at unit resolution.

Main Results and Discussion

The method achieved lower limit of quantification of 45 pmol/L for metanephrine and 127 pmol/L for normetanephrine, with LOD at half those values. Within-day and between-day imprecision were below 7.6% and 12.8% coefficient of variation, respectively. Detector response was linear up to 24.6 nmol/L (r2>0.995). SPE recovery exceeded 95% with acceptable matrix effects compensated by internal standard. Comparison with an online SPE method showed no significant bias for metanephrine and negligible bias for normetanephrine within clinical ranges. Investigation of potential isobaric interferences highlighted the need for unit resolution at MS2.

Benefits and Practical Applications

• Reduced operator involvement and risk of error through automation
• High sample throughput with short chromatography and microelution format
• Elimination of evaporation and reconstitution steps increases robustness
• Enhanced analytical specificity and sensitivity supports clinical research into catecholamine disorders

Future Trends and Potential Applications

The approach can be extended to multiplexed assays for other polar metabolites and adapted to high-resolution mass spectrometry platforms. Further automation integration may enable real-time monitoring and point-of-care applications. Advances in microfluidics and plate formats may improve throughput and reduce sample volumes.

Conclusion

An automated offline SPE-UPLC-MS/MS workflow provides a fast, sensitive, and reliable method for quantifying plasma metanephrine and normetanephrine. The combination of Oasis WCX microelution, Tecan automation, and Xevo TQ MS detection streamlines sample preparation and enhances analytical performance for clinical research.

References

1. Lenders JW et al. Determination of metanephrines in plasma by liquid chromatography with electrochemical detection. Clin Chem 1993;39(1):97–103.
2. De Jong WHA et al. Plasma free metanephrine measurement using automated online solid-phase extraction HPLC-tandem mass spectrometry. Clin Chem 2007;53(9):1684–1693.
3. Clarke MW et al. Improved plasma free metadrenaline analysis requires mixed mode cation exchange solid-phase extraction prior to liquid chromatography tandem mass spectrometry. Ann Clin Biochem 2011;48:352–357.