The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 5 - Rapid analysis of catecholamines and metanephrines in biological fluids by automated online solid-phase extraction LC/MS/MS
Sylvie Beaudet1; Martin Sibum2; Luce Boulanger3
1MDS Analytical Technologies, Concord , Canada ; 2Spark Holland Inc., Emmen, NETHERLANDS; 3CHUM St-Luc Hospital, Montreal, Canada
Novel Aspect: This high-throughput SPE/LC/MS/MS method enables automatic extraction, concentration and separation of plasma catecholamines in a very short analysis time.
Introduction: Quantitation of plasma free catecholamines has been used for clinical diagnosis of phenochromocytoma, paraganglioma and neuroblastoma, which are tumors that produce excessive amounts of catecholamines. A rapid, sensitive and fully automated high-throughput online solid-phase extraction/Liquid Chromatography/Tandem mass spectrometry (SPE/LC/MS/MS) method is proposed. This online method enables extraction, concentration and separation of plasma catecholamines in a very short analysis time. It also reduces potential for human error associated with labor intensive steps usually required in offline solid-phase extraction while maintaining sample quality and throughput.
Methods: We used an automated online solid-phase extraction system coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer. The following catecholamines were studied: norepinephrine, epinephrine, normetanephrine, metanephrine, dopamine, 3,4-dihydroxyphenylalanine, vanillomandelic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Plasma samples and deuterated analogs were picked up separately by the autosampler and injected onto the SPE cartridge in one injection operation. The eluted sample was then transferred onto an analytical column for chromatographic separation and subsequent MS/MS detection. The acquisition was performed in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI) and structural confirmation obtained with MRM triggered Enhanced Product Ion (EPI).
Results: This method is specific and allows rapid testing for disorders associated with increased catecholamine concentrations. The use of SPE led to significant cleaner samples and was highly effective in decreasing interferences present in the matrix that can cause problems with many HPLC-based methods. Several SPE materials were tested and C18 sorbent was selected based on the recovery, reproducibility and peak shape. HILIC chromatography was used because metanephrines are polar analytes that are not well-retained on most reverse-phase columns. By using HILIC chromatography, excellent separation was achieved and the presence of higher organic composition as the analytes elute provides increased response, resulting in better sensitivity.
Integrating Information Dependant Acquisition (IDA) into the automated LC/MS/MS workflow provides increased information versus the MRM-only catecholamine quantitation. The ability of the system to automatically trigger full scan MS/MS and MS3 spectra from the MRM transitions can be used to distinguish the target compounds from their metabolites. This additional degree of confirmation helps eliminate erroneous metabolite identifications and confirm the presence and structure of the respective products.
The combination of the online solid-phase extraction with the hybrid-triple quadrupole/Linear Ion Trap mass spectral detection provide an automated, sensitive and selective method to identify and quantitate metanephrines in these complex samples in a single run. The automated sample preparation, combined with fast LC/MS/MS runtimes, is compatible with the high-throughput demands in bioanalytical and clinical research labs.
