High-Throughput and Sensitive Quantitation of Plasma Catecholamines by Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Using a Solid Phase Microwell Extraction Plate

Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC–MS/MS analysis run time is 2.0 min per sample. This UPLC–MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50–250 μL, e.g., from children and mice)

Additional file 2 of CUDC-907, a dual PI3K/histone deacetylase inhibitor, increases meta-iodobenzylguanidine uptake (123/131I-mIBG) in vitro and in vivo: a promising candidate for advancing theranostics in neuroendocrine tumors

Additional file 2. Additional data

Duloxetine blocked MDMA- and MDA-induced 5-HT and NE efflux.

<p>Duloxetine inhibited SERT-mediated 5-HT release by MDMA (<b>a</b>) and MDA (<b>b</b>). Duloxetine also inhibited NET-mediated NE release by MDMA (<b>c</b>) and MDA (<b>d</b>). Values are expressed as mean ± SEM (<i>n</i>  = 3–6) of retained radiolabeled substrate following incubation with various concentrations of MDMA and MDA.</p

Duloxetine inhibited the psychotropic effects of MDMA.

<p>MDMA produced stimulant-like (<b>b–d</b>) and “entactogenic" (<b>e, f</b>) effects compared with placebo (<i>p</i><0.001 for all scales). Duloxetine significantly inhibited MDMA-induced elevations in all of these subjective effects (<b>a–f</b>) (<i>p</i><0.001 for all scales). Values are expressed as mean+SEM (<i>n</i>  = 16).</p

Dexamethasone effects on primary tumor cells.

<p>(A) Whisker-box plot of PNMT mRNA relative ratio from PGL cells (tumors P16, P30, P35 and P42) incubated with <i>vs</i>. without dexamethasone 1μM for 24 hrs (reference). Reporter genes included the following: EEFIA1, GAPDH and TBP, ns; not statistically different. (B) Whisker-box plot of TH, DBH and PNMT mRNA relative ratio from PHEO cells (tumors P23, P36, P44, P45, P52, P53, P56, P61 and P62) incubated with <i>vs</i>. without dexamethasone 1μM for 24 hrs (reference). TH: FC 2.8, SE 1.28–7.55, CI 0.68–17.38, reporter genes included the following: EEFIA1, GAPDH and TBP. ** <i>P</i><0.01. (C) Western blot of PHEO cell incubated with (+) or without (-) dexamethasone (Dexa.) 1μM for 24 hrs.</p

Inhibition of MDMA-induced 5-HT or NE release by different inhibitors.

<p>95% CI, 95% confidence interval; na, not assessed.</p

Duloxetine increased MDMA exposure.

<p>Pharmacokinetics of MDMA, MDA, HMMA, and duloxetine (<b>a–d</b>). Duloxetine was administered 16 h and 4 h before MDMA, which was administered at the 0 h time point. Duloxetine increased the C_max and AUC_0–6 of MDMA (<b>a</b>), had no significant effect on MDA exposure (<b>b</b>), and decreased the C_max and AUC_0–6 of HMMA (<b>c</b>). Plasma duloxetine concentrations were similar in the duloxetine-placebo and duloxetine-MDMA groups before MDMA administration (at –4 h and 0 h). Duloxetine concentrations increased 1 h after MDMA administration in the duloxetine-MDMA <i>vs</i>. duloxetine-placebo group (<b>d</b>). Values are expressed as mean±SEM of 16 subjects. MDMA, 3,4-methylenedioxymethamphetamine; MDA, 3,4-methylenedioxyamphetamine; HMMA, 4-hydroxy-3-methoxymethamphetamine.</p

Duloxetine reduced the cardiostimulant response to MDMA.

<p>Duloxetine reduced the elevations in mean arterial blood pressure (<b>a</b>) and heart rate (<b>b</b>) in response to MDMA. Duloxetine also nonsignificantly lowered the MDMA-induced increase in body temperature (<b>c</b>). Values are expressed as mean+SEM of 16 subjects.</p

Duloxetine prevented the acute effects of MDMA in the Altered States of Consciousness (ASC) scale.

<p>MDMA significantly increased the ASC sum score, Oceanic Boundlessness (OB), Anxious Ego Dissolution (AED), and Visionary Restructuralization (VR) dimensions, and most of the subscales (*p<0.05, **p<0.01, ***p<0.001, placebo-placebo vs. placebo-MDMA). Duloxetine significantly reduced the effect of MDMA in all dimensions and subscales (^#p<0.05, ^##p<0.01, ^###p<0.001, placebo-MDMA vs. duloxetine-MDMA). Values are expressed as mean+SEM (n  = 16).</p

mRNA and protein quantification of TH, DBH and PNMT in PGL <i>vs</i>. mixed and NorAD PHEO.

<p>(A) Whisker-box plot of mRNA relative ratio from PGL (n = 11) <i>vs</i>. mixed PHEO (n = 28) (reference). TH: reaction efficiency (RE) 0.99, fold change to reference (FC) 0.29, Standard error (SE) 0.04–1.65, 95% confidence Interval (CI) 0.003–48.29; DBH: not statistically significant (ns); PNMT: RE 0.98, FC 0.024, SE 0.001–0.41, CI 0–4.82. Reporter genes included the following: EEFIA1, RE 0.99; GAPDH, RE 1.04, TBP, RE 0.97. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, (B) Ratio from PGL (n = 11) <i>vs</i>. NorAd PHEO (n = 22) (reference). (C) Ratio from NorAd PHEO (n = 22) <i>vs</i>. mixed PHEO (n = 28) (reference) PNMT: FC 0.07, SE 0.005–0.93, CI 0.001–10.99.</p