Ultra high performance liquid chromatography tandem mass spectrometric detection of glucuronides resistant to enzymatic hydrolysis: Implications to doping control analysis

Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone) excreted after T administration. Due to their potential as biomarkers of T misuse, a UHPLC–MS/MS method for the direct quantification of these glucuronides was developed and validated. In addition, the main phase II metabolites of T that compose the steroid profile used for doping control purposes (glucuronides of T, epitestosterone, androsterone and etiocholanolone) were included. The method was found to be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were between 86% and 120%, the RSDs for the intra- and inter-day precision were below 15% and 25% respectively. The method showed low matrix effect. Samples obtained before and after the administration of T were analyzed by both the developed UHPLC–MS/MS method and the GC–MS/MS method currently used by anti-doping laboratories. Relevant disagreements between the results obtained for 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone quantitation were observed. These markers seemed to be more suitable for the screening of T misuse when detected by UHPLC–MS/MS. These discrepancies were further investigated in 50 urine samples from healthy volunteers. The two methods gave highly correlated results for all metabolites that are currently included in the athlete's steroid profile confirming the reliability of the UHPLC–MS/MS method. However, the quantification of 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone, was only possible by using the UHPLC–MS/MS method since three interfering compounds were observed when performing the GC–MS/MS analysis with the most intense ion transitions. These results confirm the potential of the resistant glucuronides as biomarkers of T misuse. Additionally, they suggest that previously reported reference ranges for these metabolites should be reevaluated.     

Development and validation of an HPLC method to quantify camptothecin in polymeric nanocapsule suspensions

A simple, rapid, and sensitive reversed-phase column high-performance liquid chromatographic method was developed and validated to quantify camptothecin (CPT) in polymeric nanocapsule suspensions. The chromatographic separation was performed on a Supelcosil LC-18 column (15 cm x 4.6 mm id, 5 microm) using a mobile phase consisting of methanol-10 mM KH2PO4 (60 + 40, v/v; pH 2.8) at a flow rate of 1.0 mL/min and ultraviolet detection at 254 nm. The calibration graph was linear from 0.5 to 3.0 microg/mL with a correlation coefficient of 0.9979, and the limit of quantitation was 0.35 microg/mL. The assay recovery ranged from 97.3 to 105.0%. The intraday and interday relative standard deviation values were < 5.0%. The validation results confirmed that the developed method is specific, linear, accurate, and precise for its intended use. The current method was successfully applied to the evaluation of CPT entrapment efficiency and drug content in polymeric nanocapsule suspensions during the early stage of formulation development.     

Faster and simpler determination of chlorophenols in water by fiber introduction mass spectrometry

Solid phase microextraction (SPME) of chlorophenols [2-chlorophenol (2CP), 2,4-dichlorophenol (24CP), 4-chloro-3-methylphenol (43CP), 2,4,6-tri-chlorophenol (246CP) and pentachlorophenol (PCP)] followed by direct mass spectrometric analysis has been performed by fiber introduction mass spectrometry (FIMS). Two SPME fibers (65 μm PDMS/DVB and 85 μm PA fibers) were tested, and FIMS was performed via selective ion monitoring (SIM). The extractions were evaluated at 10% ionic strength and pH 1. Best extraction times were determined for both fibers. Limits of detection (LOD) and limits of quantification (LOQ) for both fibers were in the low μg L⁻¹ range. Coefficients of correlation for the analytical curves showed linear responses and mineral water and river water samples spiked with 50 μg L⁻¹ presented high recoveries. FIMS, as compared to current EPA methods, is demonstrated to allow faster and simpler (elimination of pre-separation or derivatization steps) analysis of chlorophenols in water with the required sensitivity.     

Continuous chromatographic separation of a baclofen precursor (N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone) in a simulated moving bed using a polysaccharide carbamate as chiral stationary phase

Liquid chromatography is known as one of the most flexible, efficient and cost-effective methods to resolve racemic mixture in order to attend the growing demand of the pharmaceutical industry for pure enantiomeric compounds. Cellulose tris(3,5-dimethylphenylcarbamate) is frequently used as a stationary phase for enantiomeric separations because of its attractive properties, including high enantioselectivity, high loading capacity and good mechanical stability. In this study, we investigated the usefulness of cellulose tris(3,5-dimethylphenylcarbamate) as the stationary phase and of ethanol and hexane mixtures as the mobile phases for the chromatographic separation of potential pharmaceutical intermediates. Using adsorption equilibrium data, we determined the optimal operational conditions for the separation of the N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone enantiomers - a baclofen precursor - in a semi-preparative scale simulated moving bed unit. This unit was used to obtain high purity enantiomers on a scale of 1g/day. The outlet streams were analyzed by an on-line system that consisted of a UV-vis spectrophotometric unit, a polarimeter, and HPLC. Enantiomeric purities of up to 97% were obtained for the raffinate stream and up to 90% for the extract stream.     

Reactivity of cyclometallated platinum complexes with chiral ligands

The reaction of [Pt₂Me₄(μ-SMe₂)₂] with 3-substituted iminic thiophenes and 2-phenylpyridine gives platinum (II) [C,N] cyclometallated complexes which contain a labile ligand (SMe₂ or CH₃CN). Several platinum (II) complexes have been synthesized by substitution reactions with phosphine or sulfoxide ligands to introduce, in most cases, a second chiral center. The new complexes’ reactions with methyl iodide were subsequently studied and showed results that are dependent on the steric and electronic effects of both the cyclometallated ligand and the ancillary phosphine or sulfoxide ligand. The structure of [PtMe((R)-C₁₀H₇CHMeNCHC₄H₂S)(CH₃CN)], a synthetic precursor, is also reported.     

Synthesis, reactivity and crystal structures of platinum (II) and platinum (IV) cyclometallated compounds derived from 2- and 4-biphenylimines

The reaction of [Pt₂Me₄(μ-SMe₂)₂] with ligands 4-C₆H₅C₆H₄CHNCH₂CH₂NMe₂ (1a) and 2-C₆H₅C₆H₄CHNCH₂CH₂NMe₂ (1b) carried out in acetone at room temperature produced compounds [PtMe₂{4-C₆H₅C₆H₄CHNCH₂CH₂NMe₂}] (2a) and [PtMe₂{2-C₆H₅C₆H₄CHNCH₂CH₂NMe₂}] (2b), respectively, in which the imines act as bidentate [N,N′] ligands. Cyclometallated [C,N,N′] compounds [PtMe{4-C₆H₅C₆H₃CHNCH₂CH₂NMe₂}] (3a) and [PtMe{2-C₆H₅C₆H₃CHNCH₂CH₂NMe₂}] (3b), were obtained by refluxing toluene solutions of compounds 2a or 2b. Reaction of [Pt₂Me₄(μ-SMe₂)₂] with ligands 4-C₆H₅C₆H₄CHNCH₂Ph (1c) and 2-C₆H₅C₆H₄CHNCH₂Ph (1d) produced compounds [PtMe{4-C₆H₅C₆H₃CHNCH₂Ph}SMe₂] (5c) and [PtMe{2-C₆H₅C₆H₃CHNCH₂Ph}SMe₂] (5d) containing a [C,N] ligand, from which triphenylphosphine derivatives 6c and 6d were also prepared. In all cases, metallation took place to yield five-membered endo-metallacycles and formation of seven-membered or of exo-metallacycles was not observed. The reactions of 3a, 3b, 6c and 6d with methyl iodide were studied in acetone and gave the corresponding cyclometallated platinum (IV) compounds. All compounds were characterised by NMR spectroscopy and compounds 3b, 4a, 6c and 6d were also characterised crystallographically.     

A comparative study of metallating agents in the synthesis of [C,N,N′]-cycloplatinated compounds derived from biphenylimines

The reactions of ligands 4-C₆H₅C₆H₄CHNCH₂CH₂NMe₂ (1a) and 2-C₆H₅C₆H₄CHNCH₂CH₂NMe₂ (1b) in front of cis-[PtCl₂(dmso)₂] or cis-[PtPh₂(SMe₂)₂] produced compounds [PtCl₂{4-C₆H₅C₆H₄CHNCH₂CH₂NMe₂}] (2aCl) and [PtCl₂{2-C₆H₅C₆H₄CHNCH₂CH₂NMe₂}] (2bCl) or [PtPh₂{4-C₆H₅C₆H₄CHNCH₂CH₂NMe₂}] (2aPh) and [PtPh₂{2-C₆H₅C₆H₄CHNCH₂CH₂NMe₂}] (2bPh). From all these compounds, the corresponding cyclometallated [C,N,N′] platinum(II) compounds 3aCl, 3bCl, 3aPh and 3bPh were obtained although under milder conditions and with higher yields for the phenyl derivatives. The reaction of compounds 3aPh and 3bPh with methyl iodide gave cyclometallated [C,N,N′] platinum(IV) compounds 4aPh and 4bPh of formula [PtMePhI{C₆H₅C₆H₃CHNCH₂CH₂NMe₂}]. Compounds 3aCl and 3bCl containing a chloro ligand, although unreactive towards methyl iodide, undergo oxidative addition of chlorine to produce the corresponding platinum(IV) compounds [PtCl₃{4-C₆H₅C₆H₃CHNCH₂CH₂NMe₂}] (6aCl and 6bCl). All compounds were characterised by NMR spectroscopy and crystal structures of compounds 3bCl and 6bCl are also reported.     

A practical one-pot procedure for the synthesis of pyrazino[2′,3′:4,5]thieno[3,2-d]pyrimidinones by a tandem aza-Wittig/heterocumulene-mediated annulation strategy

A simple one-pot and efficient method is described for the synthesis of pyrazino[2′,3′:4,5]thieno[3,2-d]pyrimidinone derivatives 6 via a tandem aza-Wittig/heterocumulene-mediated annulation process. The iminophosphorane 3 reacted with aryl isocyanates, followed by heterocyclization on addition of secondary amines to give the corresponding guanidine intermediates 5, which were cyclized in the presence of a catalytic amount of potassium carbonate to tricyclic compounds 6. Similarly, iminophosphorane 3 reacts with phenols, thiophenol, or ROH to give 2-aryl(alkyl)oxy(thio)pyrazino[2′,3′:4,5]thieno[3,2-d]pyrimidinone derivatives 7 in good yields. The corresponding carbodiimide 4c and guanidine-type intermediate compounds 5 could be isolated and characterized, thus confirming the suggested reaction pathway. However, two isomeric pyrazinothienopyrimidinones 8 and 9 may be produced in the reaction of iminophosphorane 3 with aromatic isocyanates and subsequent reaction with primary amines in the presence of a catalytic amount of potassium carbonate. The effects of the nucleophiles and isocyanates on the regioselectivity of the cyclization have been investigated.     

Development of a new sequential injection in-line cloud point extraction system for flame atomic absorption spectrometric determination of manganese in food samples

A preconcentration method for manganese determination by sequential injection cloud point extraction with subsequent detection by flame atomic absorption spectrometry (FAAS) has been developed. The enrichment of Mn was performed after a preliminary on-line cloud point extraction and entrapment of manganese-containing surfactant aggregated within a minicolumn packed with cotton. The laboratory-made reagent 4-(5′-bromo-2′-thiazolylazo)orcinol (Br-TAO) and the surfactant Triton X-114 were used for cloud point extraction. The manganese ions were eluted with sulphuric acid solution and directly introduced into the FAAS. Chemical and flow variables affecting the preconcentration were studied. Using a sample volume of 2.80 mL the limit of detection and enrichment factor were calculated to be 0.5 μg L⁻¹ and 14, respectively. The sample frequency is 48 h⁻¹, considering a total run cycle of 75 s. The accuracy of the proposed method has been demonstrated by the analysis of the certified reference biological materials rice flour and tomato leaves. The method has been applied to determination of manganese in food samples.