Abstract
Vanillylmandelic acid (VMA) is one of the most important catecholamine metabolites, and it is usually used to aid in diagnosis of pheochromocytoma and paraganglioma. A new digital control microextraction by packed sorbent (MEPS) procedure coupled to liquid chromatography-mass spectrometry (LC-MS/MS) method has been developed to determine VMA in human urine. We evaluated important parameters influencing MEPS efficiency, including stationary phase, extracting cycles, and sample dilution. In optimized MEPS conditions, Only 10 μL of sample volume and 3 min preparation time for one sample were needed. Chromatographic separation was achieved with a hydrophilic interaction liquid chromatography (HILIC) column using gradient elution. VMA was detected using multiple reaction monitoring (MRM) with an electrospray source operating in negative ion mode. The method was validated for linearity, limit of quantification, accuracy, imprecision, matrix effect, and interference. Linearity was 0.5–100 μg/mL for VMA. Intra-assay, inter-assay, and total imprecision were less than 9.6%. Interferences precluding quantitation of VMA in dilute-and-shoot approach were reduced significantly using a MEPS approach. Method comparison of LC-MS/MS and homogeneous enzyme immunoassay was performed, and the reference interval was established. The developed MEPS-LC-MS/MS method certainly contributes to method robustness and makes it suitable for measurement of urinary VMA in routine clinical biochemistry laboratories.
Highlights
Pheochromocytoma and paraganglioma (PPGL) are rare tumors originating from adrenal and extra-adrenal chromaffin cells that are characterized by excessive secretion of large amount of catecholamines (Monteleone et al 2013)
Solid phase extraction (SPE) (Magera et al 2003) and dispersive liquid-liquid microextraction (DLLME) (Konieczna et al 2016) that were reported for sample cleanup, but dilute-and-shoot method was opted for sample preparation as it was easy and fast (Shen et al 2015; Clark et al 2017; Shen et al 2019; Grouzmann et al 2018)
The aim of the study was to develop and validate a fast, simple, and reliable eVol®—microextraction by packed sorbent (MEPS) procedure combined with the LC-MS/MS method to increase throughput, improve analytical specificity for Vanillylmandelic acid (VMA) in urine, and further establish reference interval of VMA to assist in diagnosis of PPGL
Summary
Pheochromocytoma and paraganglioma (PPGL) are rare tumors originating from adrenal and extra-adrenal chromaffin cells that are characterized by excessive secretion of large amount of catecholamines (Monteleone et al 2013). Solid phase extraction (SPE) (Magera et al 2003) and dispersive liquid-liquid microextraction (DLLME) (Konieczna et al 2016) that were reported for sample cleanup, but dilute-and-shoot method was opted for sample preparation as it was easy and fast (Shen et al 2015; Clark et al 2017; Shen et al 2019; Grouzmann et al 2018). The semi-automation of sample processing is compatible with various analytical instruments, including LC-MS equipment This technique, eVol®-MEPS, can meet the requirements of clinical laboratory for the simplicity, rapidity, miniaturization, and small sample volumes of sample preparation and maintain sufficient selectivity, precision, and accuracy
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