Urinary Cortisol Quantitation Using Ultra High Pressure Liquid Chromatography/Compact Mass Spectrometry

Introduction

Cortisol is an important steroid hormone produced from cholesterol in the adrenal cortex. Its secretion is closely regulated by the Adrenocorticotropic hormone (ACTH). Most cortisol is protein-bound and only unbound cortisol is excreted in urine. The measurement of cortisol in urine is typically used to diagnosis of Cushing’s syndrome, a disorder of hypercortisolism.

Although immunoassay methods have extremely high sensitivity, they are subject to variable interferences from other steroids and their conjugates. Liquid chromatography with tandem mass spectrometry is used in clinical analysis because of its higher specificity and selectivity than immunoassay methods.

A simple and robust UHPLC/MS method using the Advion Interchim Scientific expression® Compact Mass Spectrometer (CMS) is introduced for urinary cortisol analysis and the dynamic range and sensitivity (LOD and LLOQ) of the UHPLC/CMS method will be evaluated.

Eberhard Karls University, Institute of Pharmaceutical Sciences, Tübingen, Germany

 

Q: WHAT IS THE FOCUS OF YOUR LAB’S RESEARCH?

A: We are a Medicinal Chemistry laboratory with a major focus on kinase inhibitors. Within the last decade, we have developed highly potent and selective chemical probes such as Skepinone L, a specific p38 MAP kinase inhibitor suitable for in vivo use. Our strategies involve the reversible targeting of kinases via ATP-competitive type I or less competitive type II inhibitors as well as intermediate type 11/2 inhibitors. More recently, we turned towards covalent kinase targeting by addressing non-catalytic cysteines. This strategy furnished excellent probes for JNK3 and JAK3. For example, we developed FM-381, an extremely isoform-selective JAK3 inhibitor, which is now available as a high-quality probe from the Chemical Probes Portal and the chemical probes program of the Structural Genomics Consortium.

Q: WHAT WAS YOUR PREVIOUS WORK EXPERIENCE?

A: Since our work mainly relies on organic synthesis, we have an urgent need for accurate and rapid characterization of novel compounds. While our group owns two NMR spectrometers and several HPLC systems, mass spectrometry was usually done at a shared service unit, which caused additional costs and delays. Although we were also equipped with several LC-MS devices, these were routinely used for biological samples or metabolism studies, thus adapting the workflows for chemical samples was always tedious. Consequently, a more practical solution was required.

Q: WHY DID YOU INCORPORATE THE EXPRESSION CMS INTO YOUR LABORATORY?

A: As mentioned, mass spectrometry was one of the bottlenecks in our synthesis endeavors. The purchase of an LC-MS system exclusively for reaction monitoring and compound analysis would have been an option. However, especially when dealing with crude mixtures, LC-MS devices are typically quite vulnerable and require a lot of maintenance. Moreover, if you do not have a UPLC system, LC-MS runs are time-consuming, limiting the number of samples to a maximum of a few dozens a day, which is a serious problem with respect to the size of our group.

Therefore, Advion’s expression CMS in combination with the Plate Express™ TLC plate reader was the perfect solution for us. It is easy to use, quite robust, offers a high throughput, and is suitable for almost the entire mass range of our compounds. Needless to say, the device is especially suited for reaction monitoring and the rapid assignment of product fractions from column chromatography.

Q: TO WHOM WOULD YOU RECOMMEND THE EXPRESSION CMS?

A: The expression CMS/Plate Express™ couple can be recommended to Organic or Medicinal Chemistry groups in general since it seamlessly integrates into organic synthesis workflows. Due to the affordable pricing, it is also a great solution for chemists in academia. Especially laboratories with high turnover of masters students and research interns will appreciate the robustness of the system.

Urinary Cortisol Quantitation Using Ultra High Pressure Liquid Chromatography/Compact Mass Spectrometry

Introduction

Cortisol is an important steroid hormone produced from cholesterol in the adrenal cortex. Its secretion is closely regulated by Adrenocorticotropic hormone (ACTH). Most cortisol is protein-bound, and only unbound cortisol is excreted in urine. Measurement of cortisol in urine is typically used in the diagnosis of Cushing’s syndrome, a disorder of hypercortisolism.

Although immunoassay methods have extremely high sensitivity, they are subject to variable interferences from other steroids and their conjugates. Liquid chromatography with tandem mass spectrometry is increasingly used in clinical analysis because of its higher specificity and selectivity than immunoassay methods. A simple and robust UHPLC/MS method will be introduced for urinary cortisol analysis and the dynamic range and sensitivity (LOD and LLOQ) of the UHPLC/CMS method will be evaluated.

Cannabis-related Bioanalysis (from Plant to Forensic) using LC-CMS (Single Quad)

Introduction

The legalization of marijuana and hemp provides commercial opportunities as well as analytical challenges. Accurate and precise quantitative analysis of plant and medicinal products is needed in this new industry to document composition and assure the safety of cannabis products. LC/MS techniques can provide unparalleled selective and sensitive measurements for these purposes. Here we describe the use of a relatively inexpensive compact mass spectrometer for SIM LC/MS analysis of cannabis-related applications.

This poster was presented at ASMS 2018 Annual Conference in San Diego, CA.

University of Leeds Webinar: Self-Optimizing Continuous Flow Reactors

Chemical processes can be performed in two ways; in batch or in flow, each method having set advantages and disadvantages associated with them.

Flow chemistry has gathered increasing attention over the past decade and is being readily adopted into academia to help improve synthetic routes, which maybe undesirable in batch, where outputs such as yield, productivity, E-factor or selectivity maybe improved via various methods of optimisation. On-line analytics within continuous flow chemistry allow reactions to be monitored in real-time ultimately allowing immediate characterisation and the ability to optimise in the easiest possible way.

Hosted by Jack Henion, Advion’s Chief Scientific Founder, Chris Horbaczewskyj of the University of Leeds outlines the work carried out within the Bourne group for process development applications:

  • Use of flow chemistry with online HPLC in the self-optimisation of reactions,
  • Use of online mass spectrometry for the optimisation of continuous flow reactions using an experimental design approach,
  • The development of new algorithms for multiple variable optimisation in chemical systems.

LC/CMS Determination of Polycyclic Aromatic Hydrocarbons (PAHS) in Water (EPA 610 Mix)

Polycyclic aromatic hydrocarbons (PAHs) are neutral compounds containing multiple aromatic fused rings that are present in fossil fuels and can be formed from the incomplete combustion of organic material. In recent years, it has been well documented that several PAHs can be potentially carcinogenic via exposure or ingestion.

In this application note, we analyze 16 PAHs by LC/CMS (EPA 610 Mix) on the EPA priority target list using the Advion expression Compact Mass Spectrometer (CMS) coupled with the Advion AVANT UHPLC system. 

Biscarbene gold(I) complexes: structure–activity-relationships regarding antibacterial effects, cytotoxicity, TrxR inhibition and cellular bioavailability

Claudia Schmidt, Bianka Karge, Rainer Misgeld, Aram Prokop, Mark Brönstrup, Ingo Ott

A series of gold(I) complexes with two N-heterocyclic carbene ligands (biscarbene gold complexes) were prepared and evaluated for their effects against cancer cells and pathogenic bacteria. Proliferation inhibition was observed in cancer cells and in Gram-positive bacteria, whereas Gram-negative bacteria were less sensitive towards the compounds. The protein binding and cellular uptake were quantified and the combined results indicated a strong correlation between cellular bioavailability and antiproliferative effects. The biscarbene gold complexes inhibited bacterial and mammalian TrxRs with low to moderate potency. However, based on the obtained structure–activity-relationships and the high cellular accumulation levels, TrxR inhibition can be considered as a relevant contributor to the cellular pharmacology of biscarbene gold(I) complexes.

The MS analysis was carried out using Advion Expression® CMS.

Ducatina umbilicata gen. et sp. nov., a remarkable Trapeliaceae from the subantarctic islands in the Indian Ocean

Damien Ertz, Ulrick Søchting, Alice Gadea, Maryvonne Charrier, Roar S. Poulsen

The new genus and species Ducatina umbilicata is described from Îles Crozet and Îles Kerguelen. This lichen is characterized by an umbilicate thallus with a black verrucose lower surface and a greyish to dark olivaceous smooth upper surface having large verrucae, large semiimmersed cephalodia, semi-immersed apothecia with a prominent thalline margin, simple, mainly ellipsoid ascospores of 23–42Å~12–25 μm and the presence of unknown chemical compounds. Phylogenetic analyses using nuLSU and mtSSU sequences place Ducatina in the Trapeliaceae (Baeomycetales). The new taxon is closely related to Orceolina antarctica and O. kerguelensis, two other lichens endemic to these subantarctic islands, differing by its morphology and the lack of chemical compounds. Ducatina is the only genus in the Trapeliaceae to develop a large umbilicate thallus.

The HPLC/MS analysis was carried out using Advion Expression® CMS ESI.

Synthesis of a Bioreversibly Masked Lipophilic Adenosine Diphosphate Ribose Derivative

Katharina Pahnke, Chris Meier

The design of a bioreversibly protected lipophilic sugar nucleotide as a potential membrane‐permeable precursor of adenosine diphosphate ribose (ADPR) is described. ADPR is the most potent activator of the transient receptor potential melastatin 2 (TRPM2) ion channel. Membrane‐permeable, lipophilic derivatives of ADPR are of great interest as tools for study of the mechanism of TRPM2. The approach described here was based on our recently disclosed “DiPPro” and “TriPPPro” prodrug approaches developed for the intracellular delivery of nucleotides. A lipophilic, bioreversibly masked ADPR analogue containing an enzymatically cleavable 4‐pentanoyloxybenzyl (PB) mask at the phosphate moiety next to the 5′‐position of adenosine, together with O‐acetyl groups, was prepared in high yields. Chemical and enzymatic hydrolysis studies in phosphate buffer (pH 7.3) were performed to assess chemical stability and possible (selective) enzymatic demasking of the ADPR analogue. HPLC‐MS revealed that the PB group was readily cleaved enzymatically. In addition, the formation of partially deacetylated ADPR compounds and also of fully unprotected ADPR was observed.

The MS analysis was carried out using Advion Expression® CMS.

New chemical and radiochemical routes to [18F]Rho6G-DEG-F, a delocalized lipophilic cation for myocardial perfusion imaging with PET

J. A. H. Inkster, S. Zhang, V. Akurathi, A. Belanger, S. Dubey, S. T. Treves, A. B. Packard

New chemical and radiochemical syntheses are described for the preparation of [18F]Rho6G-DEG-F, an 18F-labeled analogue of the fluorescent dye rhodamine 6G, which has shown promise as myocardidal perfusion imaging agent. Tosylated precursors of [18F]Rho6G-DEG-F amenable to 18F-labeling were obtained either through a two-step synthesis from rhodamine 6G lactone (33% yield), or in one step from rhodamine 575 (64% yield), then purified by preparative C18 chromatography. Manual synthesis of [18F]Rho6G-DEG-F was achieved in a single radiochemical step from either the tosylate salt or the tosylate/formate double salt in DMSO under standard nucleophillic aliphatic 18F-fluorination conditions (K[18F]F/K2CO3/Kryptofix 2.2.2.). Incorporation of the [18F]F− was found to be satisfactory (≥34% by TLC), despite the protic character of the precursor molecules. [18F]Rho6G-DEG-F was manually synthesized in final decay-corrected radiochemical yields of 11–26% (tosylate salt) and 9–21% (tosylate/formate double salt). The protocol was transferred to an automated synthesis unit, where the product was obtained in 3–9% radiochemical yield (n = 3) decay corrected to start-of-synthesis, >99% radiochemical purity, and a molar activity of 122–267 GBq μmol−1 (3.3–7.2 Ci μmol−1).

The MS analysis was carried out using Advion Expression® CMS.