Liquid chromatography-tandem mass spectrometry - Application in the clinical laboratory

This review provides a concise survey of liquid chromatography tandem mass spectrometry (LCTMS) as an emerging technology in clinical chemistry. The combination of two mass spectrometers with an interposed collision cell characterizes LCTMS as an analytical technology on its own and not just as a more specific detector for HPLC compared with conventional techniques. In LCTMS, liquid chromatography is rather used for sample preparation but not for complete resolution of compounds of interest. The instrument technology of LCTMS is complex and comparatively expensive; however, in routine use, methods are far more rugged compared to conventional chromatographic techniques and enable highthroughput analyses with very limited manual handling steps. Moreover, compared to both gas chromatographymass spectrometry (GCMS) and conventional HPLC techniques, LCTMS is substantially more versatile with respect to the spectrum of analyzable compounds. For these reasons it is likely that LCTMS will gain far more widespread use in the clinical laboratory than HPLC and GCMS ever did. In this article, the key features of LCTMS are described, method development is explained, typical fields of application are discussed, and personal experiences are related

Tools to evaluate nanodiamond-mediated delivery of tiopronin for cataract prevention

”There is a growing demand for non-surgical means of cataract treatment. This dissertation presents three bodies of work that reflect the early-stage development of eye drop formulations aimed at delaying cataract progression. These formulations consist of the antioxidant 2-mercaptopropionylglycine (MPG) loaded onto nanodiamond particles. Cataractogenesis is linked to oxidative damage to lens proteins. To investigate the potential of MPG for protection against oxidative damage, A549 cells were incubated in 0.6 mM tert-butylhydroperoxide (tBHP). Cells exposed to tBHP without MPG exhibited elevated levels of reactive oxygen species, which led to the depletion of the vital antioxidant glutathione and, ultimately, apoptosis. Co-administration of 5 mM MPG protected cells from tBHP-induced damage, resulting in maintenance of cell viability. To monitor the uptake and fate of MPG in the eye, a rapid, high sensitivity HPLC method was developed for the analysis of MPG and its metabolite, 2-mercaptopropionic acid, in ocular tissues. Method validation experiments demonstrated the reliability of this method for quantifying MPG uptake and evaluating drug delivery strategies. Achieving effective drug concentrations in the lens poses a major challenge. Nanodiamond is biocompatible, and its surface chemistry can be tailored to specific applications. Thus, it is emerging as a candidate of interest for drug delivery. Nanodiamond surfaces were functionalized with carboxyl (ND-COOH), hydroxyl (ND- OH), and amino (ND-NH2) groups to investigate the effect of ND surface chemistry on adsorption and release of MPG. The ND-NH2 exhibited the highest adsorption capacity, but the ND-OH was the most effective for sustained release”--Abstract, page iv

Dendrimer-based Report-Eradication Antineoplasic Machines

Polyurea (PURE) dendrimers are a versatile platform for cancer nanotheranostics. The aim of this study was to improve the therapeutic efficacy of carboplatin by a buthionine sulfoximine (BSO) triggered inhibition of glutathione synthesis. BSO nanodelivery was achieved by controlled release from an encapsulated formulation using a folate target polyurea dendrimer of generation four (BSO@PUREG4-FA). Platinum-based anti-cancer drugs, such as cisplatin and carboplatin have been widely used in chemotherapy. In particular, carboplatins are used as standard chemotherapeutic in ovarian cancer, a silent killer, which is the most lethal gynecologic malignancy and the seventh most common cancer among women worldwide. However, carboplatin chemoresistance is a major problem and there is evidence that increased glutathione levels play an important role in the anticancer mechanism of action. Cell death assays using OVCAR3 (OSC) and ES2 (OCCC) ovarian cancer cell lines were used to determine the efficacy of BSO@PUREG4-FA nanoformulations. Cytotoxicity data showed that the encapsulated drug, if compared with the free drug, improve the efficacy of BSO, by reduction of the IC50, against both OVCAR3 (64-fold) and ES2 (146-fold) cell lines. The results showed that inhibition of glutathione synthesis improve the efficacy of carboplatin in both cell lines. In this study a new method for detection of BSO was also developed, based on UV detection upon BSO chemical derivatization

Mild, aqueous α-arylation of ketones : towards new diversification tools for halogenated metabolites and drug molecules

The authors thank the European Research Council (FP7/2007-2013/ERC grant agreement no 614779 to RJMG) and (FP7 2009-2014/ERC agreement no 227817 to SPN) for generous funding.The palladium-catalyzed aqueous α-arylation of ketones was developed and tested for a large variety of reaction partners. These mild conditions enabled the coupling of aryl/ alkyl-ketones with N-protected halotryptophans, heterocyclic haloarenes, and challenging base-sensitive compounds. The synthetic potential of this new methodology for the diversification of complex bioactive molecules was exemplified by derivatising prochlorperazine. The methodology is mild, aqueous and flexible, representing a means of functionalizing a wide range of halo-aromatics and therefore has the potential to be extended to complex molecule diversification.PostprintPeer reviewe

Quantification of 3α-hydroxytibolone in human plasma by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS): Application in a bioequivalence study in healthy postmenopausal volunteers

Abstract A sensitive, specific and fast method to quantify 3α-hydroxytibolone in human plasma using deuterated 3α-hydroxytibolone (d5) as internal standard is described. The analyte and the internal standard were extracted from plasma (900 μL) by liquid-liquid extraction using ethyl ether/hexane (50/50, v/v) and ammonium hydroxide (50%). The extracts were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry without derivatization. Chromatography was performed isocratically on a Gemini-NX™ C18 5 μm (150 × 4.6 mm i. d.) column. The method had a chromatographic run time of 3.75 min and a linear calibration curve over the range 1–100 ng/mL. The limit of quantification validated was 1 ng/mL. This method was used to assess the bioequivalence between two different tibolone oral formulations: Livolon (1.25 mg tablet) provided by Biolab Sanus Farmaceutica (Brazil), as the test formulation, and Libiam™ (1.25 mg tablet) produced by Libbs Farmaceutica (Brazil), as the reference formulation. A single 3.75 mg dose of each formulation was administered to 46 postmenopausal female healthy volunteers. The study was conducted in an open, randomized, two-period crossover balanced design with a 2 week washout interval between the doses. The 90% confidence interval for Cmax, AUC(0-last) and AUC(0-inf) individual test/reference ratios were 97.48–111.51, 95.35–103.20 and 96.42–103.86, respectively. It is concluded that Livolon (1.25 mg tablet) is bioequivalent to Libiam™ (1.25 mg tablet), with regards to both rate and extent of absorption

Synthesis and late-stage functionalization of complex molecules through C-H fluorination and nucleophilic aromatic substitution.

We report the late-stage functionalization of multisubstituted pyridines and diazines at the position α to nitrogen. By this process, a series of functional groups and substituents bound to the ring through nitrogen, oxygen, sulfur, or carbon are installed. This functionalization is accomplished by a combination of fluorination and nucleophilic aromatic substitution of the installed fluoride. A diverse array of functionalities can be installed because of the mild reaction conditions revealed for nucleophilic aromatic substitutions (S(N)Ar) of the 2-fluoroheteroarenes. An evaluation of the rates for substitution versus the rates for competitive processes provides a framework for planning this functionalization sequence. This process is illustrated by the modification of a series of medicinally important compounds, as well as the increase in efficiency of synthesis of several existing pharmaceuticals

Residue Determination in Honey

The use of antibiotics to fight bacterial and fungal honeybee diseases is documented since 1940s. Although at present in some countries certain antibiotics are authorized in apiculture, only few law systems provide maximum residue limits in honey. In addition, residues of worldwide banned antibiotics such as chloramphenicol, nitrofurans and nitroimidazoles have been frequently found. Therefore, the availability of reliable analytical methods able to detect concentrations at few parts per billions is fundamental. After a general overview of the available sample treatment strategies and analytical techniques, the most significantly published methods are discussed. Aminoglycosides and, to a lesser extent, tetracyclines are the more difficult classes to analyse. The current trends are the development of multiclass procedures and of micro-extraction techniques to improve the cost-effectiveness of residues control in the globalization era

Accessing Nature’s diversity through metabolic engineering and synthetic biology

In this perspective, we highlight recent examples and trends in metabolic engineering and synthetic biology that demonstrate the synthetic potential of enzyme and pathway engineering for natural product discovery. In doing so, we introduce natural paradigms of secondary metabolism whereby simple carbon substrates are combined into complex molecules through “scaffold diversification”, and subsequent “derivatization” of these scaffolds is used to synthesize distinct complex natural products. We provide examples in which modern pathway engineering efforts including combinatorial biosynthesis and biological retrosynthesis can be coupled to directed enzyme evolution and rational enzyme engineering to allow access to the “privileged” chemical space of natural products in industry-proven microbes. Finally, we forecast the potential to produce natural product-like discovery platforms in biological systems that are amenable to single-step discovery, validation, and synthesis for streamlined discovery and production of biologically active agents