André Liesener

Liquid chromatography with post-column electrochemical treatment and mass spectrometric detection of non-polar compounds

The first hyphenation of high performance liquid chromatography (HPLC), electrochemical online oxidation and mass spectrometry (MS) is described. Ferrocenecarboxylic acid esters of various alcohols and phenols have been synthesized, separated by reversed-phase HPLC and oxidized (ionized) coulometrically prior to single quadrupole MS analysis using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces. The dependence of the ionization on the electrochemical pretreatment is demonstrated. Limits of detection for selected derivatives range from 4 x 10(-9) to 4 x 10(-7) mol dm-3 depending on the individual compound and the selected interface.

Development of a fast liquid chromatography/mass spectrometry screening method for angiotensin-converting enzyme (ACE) inhibitors in complex natural mixtures like snake venom.

A new robust high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS)-based screening method for angiotensin-converting enzyme (ACE)-inhibiting substances in crude samples is described. The ACE assay is carried out in a typical offline setup by incubation of the samples with ACE and angiotensin I (AI), followed by stopping the reaction with acetonitrile containing val(5)-AI serving as internal standard (I.S.). AI and the product angiotensin II (AII) are extracted from the incubation mixture by turbulent-flow chromatography (TFC) applied in backflush mode as online solid-phase extraction and are directly quantified by ESI(+)-MS. The presence of ACE inhibitors (ACEi) is detected by an increase in AI signal intensity and a corresponding decrease of AII signal, as compared to the blank assay. The overall time of analysis of the TFC/ESI-MS method was 5 min, thus making the described setup suitable for a rapid screening method. The assay was validated using a known ACE inhibitor and the IC(50) values found were in good accordance with a common HPLC/UV method and literature data. The method was successfully applied for the screening of size-exclusion chromatography fractions of the venom of the pitviper Bothrops moojeni. Three of 18 analyzed fractions inhibited ACE, due to peptides present as components of this snake venom. These compounds were extracted from the two most-active fractions by means of TFC and isolated by means of HPLC. Three peptides with ACE inhibitory activity were characterized and their structures were elucidated with ESI-MS/MS-based de novo sequencing to be ZKWPPGKVPP, ZKWPRPGPEIPP and ZNWPRPGPEIPP, respectively (Z = pyroglutamic acid).

Assessing protease activity pattern by means of multiple substrate ESI-MS assays

The development of a simultaneous multiple substrate enzymatic assay based on electrospray ionization mass spectrometry (ESI-MS) detection is described. This multiplexing assay scheme was employed in a parallel proteolytic enzyme activity screening. As model systems, the respective activities of trypsin, thrombin, chymotrypsin, bromelain, ficin and elastase towards seven different substrates were assessed. The resulting activity patterns were evaluated semi-quantitatively ranking the enzymatic activities in five classes of activity (very high, high, medium, low and no activity) with respect to the individual substrates. The validity of the MS-based multiplexing assay scheme was proved by comparison with the results obtained from single substrate assays detected by means of UV/vis absorption at 405 nm, showing good agreement of the resulting activity patterns and classifications.

Pharmacokinetic Properties of Nintedanib in Healthy Volunteers and Patients With Advanced Cancer

Nintedanib, a triple angiokinase inhibitor, has undergone clinical investigation for the treatment of solid tumors and idiopathic pulmonary fibrosis. Nintedanib (Vargatef®) plus docetaxel is approved in the EU for the treatment of patients with adenocarcinoma non‐small cell lung cancer (NSCLC) after first‐line chemotherapy, and as monotherapy (Ofev®) in the United States and EU for the treatment of patients with idiopathic pulmonary fibrosis. Pharmacokinetics (PK) of nintedanib after oral single and multiple doses and intravenous (IV) administration were assessed using 3 data sets: (1) an absolute bioavailability trial that enrolled 30 healthy volunteers; (2) a pooled data analysis of 4 studies that enrolled a total of 107 healthy volunteers; and (3) a pooled data analysis of 4 studies that enrolled a total of 149 patients with advanced cancer. In the absolute bioavailability trial of healthy volunteers, nintedanib showed a high total clearance (geometric mean 1390 mL/min) and a high volume of distribution at steady state (Vss = 1050 L). Urinary excretion of IV nintedanib was about 1% of dose; renal clearance was about 20 mL/min and therefore negligible. There was no deviation from dose proportionality after IV administration in the dose range tested. Absolute bioavailability of oral nintedanib (100 mg capsule) relative to IV dosing (4‐hour infusion, 6 mg) was slightly below 5%. Nintedanib was quickly absorbed after oral administration. It underwent rapid and extensive first‐pass metabolism and followed at least biphasic disposition kinetics. In advanced cancer patients, steady state was reached at the latest at 7 days for twice‐daily dosing. Nintedanibs PK was time‐independent; accumulation after repeated administration was negligible.

Altered tryptophan catabolite concentrations in major depressive disorder and associated changes in hippocampal subfield volumes

BACKGROUND Tryptophan depletion is a well-replicated biological finding in Major Depressive Disorder (MDD). The kynurenine pathway (KP) and its rate-limiting tryptophan degrading enzyme, indolamine 2,3 dioxygenase (IDO), have been implicated in the pathogenesis of depression. IDO expression is driven by inflammatory cytokines, providing a putative link between inflammation and neuropathology. This study examined circulating concentrations of C-reactive protein (CRP), plasma tryptophan, kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) and whole blood mRNA expression of IDO in patients with major depressive disorder (MDD) compared with healthy controls (HC). METHODS A diagnosis of major depression was made according to DSM-IV. Depression severity was assessed using the Hamilton depression (HAM-D) rating scale. 74 MDD patients, 39 with a first presentation of MDD (fpMDD) and 35 with chronic or recurrent episodes (rMDD), and 37 HC were recruited to the study. Whole blood and plasma samples were collected. Expression of markers in whole blood were measured by PCR, circulating CRP by ELISA and KP metabolites by LC-MS/MS. Hippocampal cornu ammonis (CA) and subiculum volumes were determined by MRI and calculated using FreeSurfer. RESULTS Tryptophan concentrations were significantly reduced in MDD compared to HC. There was a positive correlation between QUIN and both CRP concentrations and whole blood IDO1 in MDD. KYNA concentrations were reduced in MDD patients presenting with a first episode (fpMDD) compared to those presenting with recurrent depression (rMDD) and HC. By contrast QUIN concentrations were elevated in rMDD compared to fpMDD and HC. KYNA/QUIN was reduced in MDD and rMDD but not fpMDD compared to HC. Hippocampal subfield volumes were smaller in MDD patients than HC for CA1 (left only), CA2/3 (left and right) and CA4 (right only). CRP and CA1 volumes were negatively correlated bilaterally in MDD patients. KYNA and subiculum volume were positively correlated bilaterally. DISCUSSION This study found evidence of KP metabolism imbalance in MDD patients in addition to tryptophan reduction and mild immune activation. Relationships between CRP and KYNA with some hippocampal subfield volumes in MDD patients suggest that this inflammatory signature may be associated with reduced hippocampal subfield volumes in depression.