In Matrix Derivatization Combined with LC-MS/MS Results in Ultrasensitive Quantification of Plasma Free Metanephrines and Catecholamines

Abstract

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated L-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 μL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4−30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (β-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R 2 > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R 2 > 0.85). Reference intervals for L-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II

“…It was thus important to stabilize the analytes during sample preparation to prevent oxidation and also render them amenable for UPLC-MS analyses. We thus employed a derivatization method previously reported and validated by van Faassen et al, with minor modifications [ 32 ]. In addition, although many methods have been reported for DOPA analysis in plasma, few have evaluated brain levels.…”

“…We next assessed the linearity, precision, accuracy of the LC-MS method, and benchtop and autosampler stability of derivatized D-DOPA, as some modifications were made to the method reported and validated by van Faassen et al [ 32 ]. The results for these are presented in Table S1A–C .…”

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II

“…It was thus important to stabilize the analytes during sample preparation to prevent oxidation and also render them amenable for UPLC-MS analyses. We thus employed a derivatization method previously reported and validated by van Faassen et al, with minor modifications [ 32 ]. In addition, although many methods have been reported for DOPA analysis in plasma, few have evaluated brain levels.…”

“…We next assessed the linearity, precision, accuracy of the LC-MS method, and benchtop and autosampler stability of derivatized D-DOPA, as some modifications were made to the method reported and validated by van Faassen et al [ 32 ]. The results for these are presented in Table S1A–C .…”

Semantic Annotation of Experimental Methods in Analytical Chemistry

“… Graph representations of the linear experimental sections in van Faassen et al, 28 Lajin and Goessler, 29 and Kocurek et al 30 (left), ”parallel” experiments in McAvan et al, 31 Pérez-Diez et al, 32 and Yan et al 17 (middle), and more complex methods in Venzac et al, 33 Flint et al, 34 and Lebede et al 35 (right). The graphs were rendered and the nodes colored as in Figure 1 .…”

Plasma-Free Metanephrine and Normetanephrine Quantification for Clinical Applications Validated by Combining Solid-Phase Extraction and HPLC-MS/MS

“…The magnitude of matrix effects can vary depending on the sample preparation method, with SPE typically providing better matrix cleanup than simple protein precipitation [ 31 ]. However, completely eliminating matrix effects remains challenging, particularly at low analyte concentrations, where the signal-to-noise ratio is most susceptible to matrix interference [ 18 , 32 ].…”

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II

“…It was thus important to stabilize the analytes during sample preparation to prevent oxidation and also render them amenable for UPLC-MS analyses. We thus employed a derivatization method previously reported and validated by van Faassen et al, with minor modifications [ 32 ]. In addition, although many methods have been reported for DOPA analysis in plasma, few have evaluated brain levels.…”

“…We next assessed the linearity, precision, accuracy of the LC-MS method, and benchtop and autosampler stability of derivatized D-DOPA, as some modifications were made to the method reported and validated by van Faassen et al [ 32 ]. The results for these are presented in Table S1A–C .…”

Semantic Annotation of Experimental Methods in Analytical Chemistry

“… Graph representations of the linear experimental sections in van Faassen et al, 28 Lajin and Goessler, 29 and Kocurek et al 30 (left), ”parallel” experiments in McAvan et al, 31 Pérez-Diez et al, 32 and Yan et al 17 (middle), and more complex methods in Venzac et al, 33 Flint et al, 34 and Lebede et al 35 (right). The graphs were rendered and the nodes colored as in Figure 1 .…”

Plasma-Free Metanephrine and Normetanephrine Quantification for Clinical Applications Validated by Combining Solid-Phase Extraction and HPLC-MS/MS

“…The magnitude of matrix effects can vary depending on the sample preparation method, with SPE typically providing better matrix cleanup than simple protein precipitation [ 31 ]. However, completely eliminating matrix effects remains challenging, particularly at low analyte concentrations, where the signal-to-noise ratio is most susceptible to matrix interference [ 18 , 32 ].…”

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II

“…It was thus important to stabilize the analytes during sample preparation to prevent oxidation and also render them amenable for UPLC-MS analyses. We thus employed a derivatization method previously reported and validated by van Faassen et al, with minor modifications [ 32 ]. In addition, although many methods have been reported for DOPA analysis in plasma, few have evaluated brain levels.…”

“…We next assessed the linearity, precision, accuracy of the LC-MS method, and benchtop and autosampler stability of derivatized D-DOPA, as some modifications were made to the method reported and validated by van Faassen et al [ 32 ]. The results for these are presented in Table S1A–C .…”

Semantic Annotation of Experimental Methods in Analytical Chemistry

“… Graph representations of the linear experimental sections in van Faassen et al, 28 Lajin and Goessler, 29 and Kocurek et al 30 (left), ”parallel” experiments in McAvan et al, 31 Pérez-Diez et al, 32 and Yan et al 17 (middle), and more complex methods in Venzac et al, 33 Flint et al, 34 and Lebede et al 35 (right). The graphs were rendered and the nodes colored as in Figure 1 .…”

Plasma-Free Metanephrine and Normetanephrine Quantification for Clinical Applications Validated by Combining Solid-Phase Extraction and HPLC-MS/MS

“…The magnitude of matrix effects can vary depending on the sample preparation method, with SPE typically providing better matrix cleanup than simple protein precipitation [ 31 ]. However, completely eliminating matrix effects remains challenging, particularly at low analyte concentrations, where the signal-to-noise ratio is most susceptible to matrix interference [ 18 , 32 ].…”