Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry

A family of small peptides has reached the focus of doping controls representing a comparably new strategy for cheating sportsmen. These growth hormone releasing peptides (GHRP) are orally active and induce an increased production of endogenous growth hormone (GH). While the established test for exogenous GH fails, the misuse of these prohibited substances remains unrecognized. The present study provides data for the efficient extraction of a variety of known drug candidates (GHRP-1, GHRP-2, GHRP-4, GHRP-5, GHRP-6, alexamorelin, ipamorelin, and hexarelin) from human urine with subsequent mass spectrometric detection after liquid chromatographic separation. The used method potentially enables the retrospective evaluation of the acquired data for unknown metabolites by means of a non-targeted approach with high-resolution/high-accuracy full-scan mass spectrometry with additional higher collision energy dissociation experiments. This is of great importance due to the currently unknown metabolism of most of the targets and, thus, the method is focused on the intact peptidic drugs. Only the already characterised major metabolite of GHRP-2 (d-Ala-d-2-naphthylAla-l-Ala, as well as its stable isotope-labelled analogue) was synthesised and implemented in the detection assay. Method validation for qualitative purpose was performed with respect to specificity, precision (<20%), intermediate precision (<20%), recovery (47–95%), limit of detection (0.2–1 ng/mL), linearity, ion suppression and stability. Two stable isotope-labelled internal standards were used (deuterium-labelled GHRP-4 and GHRP-2 metabolite). The proof-of-principle was obtained by the analysis of excretion study urine samples obtained from a single oral administration of 10 mg of GHRP-2. Here, the known metabolite was detectable over 20 h after administration while the intact drug was not observed.     

Drugs of abuse screening in urine as part of a metabolite-based LC-MS<Superscript>n</Superscript> screening concept

Today, immunoassays and several chromatographic methods are in use for drug screening in clinical and forensic toxicology and in doping control. For further proof of the authors’ new metabolite-based liquid chromatography-mass spectrometry (LC-MSⁿ) screening concept, the detectability of drugs of abuse and their metabolites using this screening approach was studied. As previously reported, the corresponding reference library was built up with MS² and MS³ wideband spectra using a LXQ linear ion trap with electrospray ionization in the positive mode and full scan information-dependent acquisition. In addition to the parent drug spectra recorded in methanolic solution, metabolite spectra were identified after protein precipitation of urine from rats after administration of the corresponding drugs and added to the library. This consists now of data of over 900 parent compounds, including 87 drugs of abuse, and of over 2,300 metabolites and artifacts, among them 436 of drugs of abuse. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs of abuse were determined using spiked human urine, and the resulting data have been acceptable. Using two automatic data evaluation tools (ToxID and SmileMS), the intake of 54 of the studied drugs of abuse could be confirmed in urine samples of drug users after protein precipitation and LC separation. The following drugs classes were covered: stimulants, designer drugs, hallucinogens, (synthetic) cannabinoids, opioids, and selected benzodiazepines. The presented LC-MSⁿ method complements the well-established gas chromatography-mass spectroscopy procedure in the authors’ laboratory.     

A Remarkably Simple Class of Imidazolium‐Based Lipids and Their Biological Properties

A series of imidazolium salts bearing two alkyl chains in the backbone of the imidazolium core were synthesized, resembling the structure of lipids. Their antibacterial activity and cytotoxicity were evaluated using Gram‐positive and Gram‐negative bacteria and eukaryotic cell lines including tumor cells. It is shown that the length of alkyl chains in the backbone is vital for the antibiofilm activities of these lipid‐mimicking components. In addition to their biological activity, their surface activity and their membrane interactions are shown by film balance and quartz crystal microbalance (QCM) measurements. The structure–activity relationship indicates that the distinctive chemical structure contributes considerably to the biological activities of this novel class of lipids.     

Capillary isoelectric focusing immunoassay as a new nanoscale approach for the detection of oligoclonal bands

The detection of oligoclonal bands (OCBs) in cerebrospinal fluid is an indicator of intrathecal synthesis of immunoglobulins which is a neurochemical sign of chronic inflammatory brain diseases. Intrathecally synthesized IgGs are typically observed in patients with multiple sclerosis. The current standard protocol for the detection of OCBs is IEF on agarose or polyacrylamide gels followed by immunoblotting or silver staining. These methods are time consuming, show substantial interlaboratory variation and cannot be used in a high throughput‐approach. We have developed a new nanoscale method for the detection of OCBs based on automated capillary IEF followed by immunological detection. Evidence for intrathecal IgG synthesis was found in all tested patients (n = 27) with multiple sclerosis, even in two subjects who did not have oligoclonal bands according to standard methods. The test specificity was at 97.5% (n = 19). Our findings indicate that the novel OCB‐CIEF‐immunoassay is suitable for the rapid and highly sensitive detection of OCBs in clinical samples. Furthermore, the method allows for a higher sample throughput than the current standard methods.     

Two 2,6‐Dioxabicyclo[3.3.1]nonan‐3‐ones from Phragmanthera capitata (Spreng.) Balle (Loranthaceae)

Phytochemical investigation of the leaves of Phragmanthera capitata collected on Cassia spectabilis tree led to the isolation of two natural lactones, rel‐(1R,5S,7S)‐7‐[2‐(4‐hydroxyphenyl)ethyl]‐2,6‐dioxabicyclo[3.3.1]nonan‐3‐one ( 1 ) and 4‐{2‐[rel‐(1R,3R,5S)‐7‐oxo‐2,6‐dioxabicyclo[3.3.1]non‐3‐yl]ethyl}phenyl 3,4,5‐trihydroxybenzoate ( 2 ) together with the known compounds betulinic acid ( 3 ), dodoneine ( 4 ), quercetin 3‐O‐α‐L ‐rhamnopyranoside ( 5 ), quercetin 3‐O‐α‐L ‐arabinofuranoside ( 6 ), quercetin ( 7 ), betulin ( 8 ), lupeol ( 9 ), and sitosterol ( 10 ). Their structures were established by means of modern spectroscopic techniques, and the relative configuration of compound 1 was confirmed by X‐ray analysis. Compounds 1 and 2 were tested in vitro for their antiplasmodial activity against the Plasmodium falciparum chloroquine sensitive‐strains NF54 and 3D7. Compound 2 exhibited good antiplasmodial activity against both strains with IC₅₀ of 2.4 and 4.9 μM , respectively, while compound 1 was inactive.     

Two CdII/CoII‐Imidazolate Coordination Polymers: Syntheses,Crystal Structures,Stabilities, and Luminescent/Magnetic Properties

Cadmium(II) based 2D coordination polymer [Cd(L1)₂(DMF)₂] ( 1 ) (L1 = 4,5‐dicyano‐2‐methylimidazolate, DMF = N,N′‐dimethylformamide) and 2D cobalt(II)‐imidazolate framework [Co(L3)₄] ( 2 ) (L3 = 4,5‐diamide‐2‐ethoxyimidazolate) were synthesized under solvothermal reaction conditions. The materials were characterized by elemental analysis, IR spectroscopy, thermogravimetric analysis, powder X‐ray diffraction measurement (PXRD) and single‐crystal X‐ray diffraction. Compound 1 has hexacoordinate Cd^II ions and forms a zigzag chain‐like coordination polymer structure, whereas compound 2 exhibits a 2D square grid type structure. The thermal stability analysis reveals that 2 showed an exceptional thermal stability up to 360 °C. Also, 2 maintained its fully crystalline integrity in boiling water as confirmed by PXRD. The solid state luminescent property of 1 was not observed at room temperature. Compound 2 showed an independent high spin central Co^II atom.