Plasma Catecholamines by LC/MS/MS
Applications | 2016 | Agilent TechnologiesInstrumentation
Measuring plasma catecholamines such as norepinephrine, epinephrine, and dopamine is essential for diagnosing autonomic disorders, monitoring stress responses, and supporting clinical research into neuroendocrine function.
This study aimed to establish a highly sensitive and specific LC/MS/MS protocol for simultaneous quantitation of the three major catecholamines in human plasma, achieving a broad dynamic range, excellent linearity, and robust precision.
Sample preparation involved two key steps:
Chromatographic separation was performed on an Agilent 1290 Infinity LC system equipped with a Pursuit PFP column (2×150 mm, 3 µm) using a methanol/water gradient containing 1 mM ammonium fluoride. Detection employed an Agilent 6460 Triple Quadrupole MS with Jet Stream ESI+ in MRM mode. Deuterated analogs served as internal standards to correct for matrix effects and variability.
The method resolved all analytes within a 10-minute run and achieved linear calibration from 5 to 2,500 pg/mL (R² > 0.9997). Matrix effects ranged from 42 to 119 %, while recoveries fell between 56 and 59 %, fully compensated by internal standards. Intra- and inter-day precision remained below 5 % CV across the dynamic range. Quality control samples showed consistent accuracy and reproducibility (CV < 4 %).
This LC/MS/MS approach offers high sensitivity and reproducibility with minimal sample volume, making it suitable for routine clinical assays for pheochromocytoma diagnosis, therapeutic monitoring, and research on stress physiology.
Future developments may include automated SPE workflows, integration with high-throughput platforms, exploration of alternative column chemistries, and coupling to high-resolution MS to expand the panel of neurochemical biomarkers.
A robust, precise, and sensitive LC/MS/MS method was demonstrated for the simultaneous quantitation of plasma catecholamines, meeting clinical research requirements for dynamic range and reproducibility.
Sample Preparation, Consumables, LC/MS, LC/MS/MS, LC/QQQ
IndustriesClinical Research
ManufacturerAgilent Technologies
Summary
Significance of the topic
Measuring plasma catecholamines such as norepinephrine, epinephrine, and dopamine is essential for diagnosing autonomic disorders, monitoring stress responses, and supporting clinical research into neuroendocrine function.
Objectives and overview
This study aimed to establish a highly sensitive and specific LC/MS/MS protocol for simultaneous quantitation of the three major catecholamines in human plasma, achieving a broad dynamic range, excellent linearity, and robust precision.
Methods and instrumentation
Sample preparation involved two key steps:
- Protein precipitation and lipid removal via Agilent Captiva NDLipids filtration.
- Selective cleanup using Agilent BondElut PBA SPE cartridges.
Chromatographic separation was performed on an Agilent 1290 Infinity LC system equipped with a Pursuit PFP column (2×150 mm, 3 µm) using a methanol/water gradient containing 1 mM ammonium fluoride. Detection employed an Agilent 6460 Triple Quadrupole MS with Jet Stream ESI+ in MRM mode. Deuterated analogs served as internal standards to correct for matrix effects and variability.
Main results and discussion
The method resolved all analytes within a 10-minute run and achieved linear calibration from 5 to 2,500 pg/mL (R² > 0.9997). Matrix effects ranged from 42 to 119 %, while recoveries fell between 56 and 59 %, fully compensated by internal standards. Intra- and inter-day precision remained below 5 % CV across the dynamic range. Quality control samples showed consistent accuracy and reproducibility (CV < 4 %).
Benefits and practical applications
This LC/MS/MS approach offers high sensitivity and reproducibility with minimal sample volume, making it suitable for routine clinical assays for pheochromocytoma diagnosis, therapeutic monitoring, and research on stress physiology.
Future trends and opportunities
Future developments may include automated SPE workflows, integration with high-throughput platforms, exploration of alternative column chemistries, and coupling to high-resolution MS to expand the panel of neurochemical biomarkers.
Conclusion
A robust, precise, and sensitive LC/MS/MS method was demonstrated for the simultaneous quantitation of plasma catecholamines, meeting clinical research requirements for dynamic range and reproducibility.
References
- Whiting MJ. Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Ann Clin Biochem. 2009;46:129–136.
- Agilent Technologies. Phenylboronic Acid (PBA) Solid Phase Extraction Mechanisms and Applications. Technical Overview SI-02442, 2010.
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