Supramolecular solvents for making comprehensive liquid-liquid microextraction in multiclass screening methods for drugs of abuse in urine based on liquid chromatography-high resolution mass spectrometry.

Multiclass screening methods involving hundreds of structurally unrelated compounds are becoming essential in many control labs and research areas. Accurate mass screening of a theoretically unlimited number of chemicals can be undertaken using liquid chromatography coupled to high resolution mass spectrometry (LCHRMS), but the lack of comprehensive sample treatments hinders this unlimited potential. In this research, the capability of supramolecular solvents (SUPRAS) for making comprehensive liquid-liquid microextraction (LLME) in multiclass screening methods based on LCHRMS was firstly explored. For this purpose, a SUPRAS made up of 1,2-hexanediol, sodium sulphate and water was synthesized directly in the urine and applied to compound extraction and interference removal in the screening of eighty prohibited substances in sports by LC-electrospray ionization-time of flight mass spectrometry. Selected substances included a wide range of polarities (log P from -2.4 to 9.2) and functionalities (e.g. alcohol, amine, amide, carboxyl, ether, ester, ketone, sulfonyl, etc.). No interfering peaks were observed for any of the 80 substances investigated. Around 84-93% of drugs were efficiently extracted (recoveries 70-120%) and 83-94% of the analytes did not show matrix effects (±20%) in the ten tested urines. Method detection limits for the drugs were in the interval 0.002-12.9 ng mL-1, which are in accordance with the Minimum Required Performance Levels values established by the World Anti-Doping Agency. The applicability of the method was evaluated by the screening of thirty-six blinded and anonymized urine samples, previously analyzed by gas or liquid chromatography-triple quadrupole. Seven of the samples lead to an adverse analytical finding in line with the results obtained by the conventional methods. This research proves that LLME based on SUPRAS constitutes an efficient, economic, and simple sample treatment in multiclass screening methods, an application that is unaffordable for conventional organic solvents.

Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS.

A complete analytical protocol for the determination of 25 doping-related peptidic drugs and 3 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors, gonadotropin releasing factors and anti-diuretic hormones, with molecular weights ranging from 540 to 1320Da. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The methodology was validated in terms of selectivity, recovery, matrix effect, precision, sensitivity (limit of detection, LOD), cross contamination, carryover, robustness and stability. Recoveries ranged from 6 to 70% (microplates) and 17-95% (cartridges), with LODs from 0.1 to 1ng/mL. The suitability of the method was assessed by analyzing different spiked or excreted urines containing some of the target substances.

An automated sample preparation for detection of 72 doping-related substances.

Automation of sample preparation procedures in a doping control laboratory is of great interest due to the large number of samples that have to be analyzed, especially in large events where a high throughput protocol is required to process samples over 24 h. The automation of such protocols requires specific equipment capable of carrying out the diverse mechanical tasks required for accomplishing these analytical methodologies, which include pipetting, shaking, heating, or crimping. An automated sample preparation procedure for the determination of doping-related substances by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis, including enzymatic hydrolysis, liquid-phase extraction and derivatization steps, was developed by using an automated liquid handling system. This paper presents a description of the equipment, together with the validation data for 72 doping-related compounds including extraction efficiency, evaluation of carry-over, interferences, and robustness. Validation was approached as a comparison between the results obtained using the manual protocol and the transferred automated one. The described methodology can be applied for sample preparation in routine anti-doping analysis with high sample throughput and suitable performance.

Aplicación de eddopeptidasa alcalina en carnaza bovina para mejorar la calidad de la gelatina / Aplication of allkaline endopeptidase in bovine hide to improve the quality of the gelatine / Aplicaço da endoopeptidse alcalina na pele bovina para melhorar a quallidade da gelatina

El precio de la gelatina en el mercado internacional está determinado por sus propiedades físico-químicas que dependen a su vez de la materia prima, de la forma de extracción del colágeno y de su método de concentración. En este estudio, utilizando procesos de producción en planta piloto de gelatina tipo B, se evaluó el efecto de la aplicación de una endopeptidasa alcalina en el pretratamiento de tres tipos de carnaza bovina sobre la calidad final de la gelatina medida como bloom , viscosidad y claridad. Los resultados se compararon con procesos en los cuales se utilizaron las mismas materias primas sin pretratamiento enzimático. Los análisis estadísticos mostraron diferencias significativas entre los tratamientos. Los mejores resultados de bloom , viscosidad y claridad se presentaron utilizando la enzima en el pretratamiento de la carnaza entera; en trece horas de proceso se obtuvo el 61,5% de la gelatina con bloom alto superior a 300 g , el 51,3% con viscosidades altas mayores de 42 mp y el 82,1% con claridades altas menores de 39 unidades nefelométricas de turbidez. La carnaza entera sin pretratamiento enzimático presentó el 41% de valores altos de bloom , el 7,7% de viscosidades altas y el 76,9% de claridades altas .

The international market price of gelatine is determined by its physical-chemical properties, that depend on the raw material, the collagen extraction method and the concentration method. In this work, using class B gelatine production processes in pilot plant, the effect of application of alkaline endopeptidase was evaluated in the pre-treatment of three types of bovine hide on the gelatine final quality as measured by bloom, viscosity and clarity. The results were compared with processes in which the same raw materials were used without enzymatic pre-treatment. The statistical analysis showed significant differences among the treatments. The best bloom, viscosity and clarity results were presented using enzymatic pre- treatment in the whole hide, obtained in thirteen hours of process 61.5% of the gelatine with bloom higher than 300g, 51.3% with viscosities values higher than 42 mp and 82 ,1 % with high clarities value, with values lower than 39 nephelometric turbidity units. The whole hide without enzymatic pre-treatment showed 41% of high values of bloom, 7.7% of high viscosities and 76.9% of high clarities .

O preço da gelatina no mercado internacional é determinado pelas suas propriedades físico-químicas as quais dependem da matéria-prima, da forma de extração do colágeno bovino e do método de concentração. Neste estudo, utilizando processos de produção em planta piloto de gelatina tipo B, foi avaliado o efeito da aplicação da protease alcalina no pré-tratamento de três tipos de pele bovina sobre a qualidade final da gelatina determinada como bloom , viscosidade e transparência. Os resultados foram comparados com processos nos quais se utilizaram as mesmas matérias-primas sem pré-tratamento enzimático. As análises estatísticas mostraram diferenças significativas entre os tratamentos. Os melhores resultados de bloom , viscosidade e transparência foram obtidos utilizando protease alcalina no pré-tratamento da pele inteira, se obtendo em treze horas do processo o 61,5% de gelatina com bloom alto superior a 300 g , o 51,3% com viscosidades altas maiores a 42 mp e o 82,1% com transparências altas menores a 39 unidades nefelométricas de turbidez . A pele inteira sem pré-tratamento enzimático apresentou o 41% dos valores altos de bloom , o 7,7% de viscosidades altas e o 76,9% de transparências altas.

New chiral ruthenium(II) catalysts containing 2,6-bis(4'-(R)-phenyloxazolin-2'-yl)pyridine (Ph-pybox) ligands for highly enantioselective transfer hydrogenation of ketones.

Treatment of complex trans-[RuCl(2)(eta(2)-C(2)H(4))[kappa(3)-N,N,N-(R,R)-Ph-pybox]] [(R,R)-Ph-pybox = 2,6-bis[4'-(R)-phenyloxazolin-2'-yl]pyridine] with phosphines or phosphites in dichloromethane at 50 degrees C leads to the formation of novel ruthenium(II)-pybox complexes trans-[RuCl(2)(L)[kappa(3)-N,N,N-(R,R)-Ph-pybox]] [L = PPh(3) (1 a), PPh(2)Me (2 a), PPh(2)(C(3)H(5)) (3 a), PPh(2)(C(4)H(7)) (4 a), PMe(3) (5 a), PiPr(3) (6 a), P(OMe)(3) (7 a) and P(OPh)(3) (8 a)]. Likewise, reaction of trans-[RuCl(2)(eta(2)-C(2)H(4))[kappa(3)-N,N,N-(R,R)-Ph-pybox]] with PPh(3) or PiPr(3) in refluxing methanol leads to the complexes cis-[RuCl(2)(L)(kappa(3)-N,N,N-(R,R)-Ph-pybox] [L = PPh(3) (1 b), PiPr(3) (6 b)]. No trans-cis isomerisation of complexes 1 a-8 a has been observed. Complexes 1 a-8 a, 1 b, 6 b together with the analogous trans-[RuCl(2)[P(OMe)(3)][kappa(3)-N,N,N-(S,S)-iPr-pybox]] (10 a) and the previously reported trans- and cis-[RuCl(2)(PPh(3))[kappa(3)-N,N,N-(S,S)-iPr-pybox]] (9 a and 9 b, respectively) are active catalysts for the transfer hydrogenation of acetophenone in 2-propanol in the presence of NaOH (ketone/cat/NaOH 500:1:6). cis-Ph-pybox derivatives are the most active catalysts. In particular, cis complexes 1 b and 6 b led to almost quantitative conversions in less than 5 min with a high enantioselectivity (up to 95 %). A variety of aromatic ketones have also been reduced to the corresponding secondary alcohols with very high TOF and ee up to 94 %. The overall catalytic performance seems to be a subtle combination of the steric and/or electronic properties both the phosphines and the ketones. A high TOF (27 300 h(-1)) and excellent ee (94 %) have been found for the reduction of 3-bromoacetophenone with catalyst 6 b. Reductions of alkyl ketones also proceed with high and rapid conversions but low enantioselectivities are achieved.

Rhodium(I) and Rhodium(III) complexes containing the chiral ligand 2,6-bis[4'(S)-isopropyloxazolin-2'-yl]pyridine (pybox): an unprecedented monohapto coordination of pybox.

The complex [Rh(kappa(3)-N,N,N-pybox)(CO)][PF(6)] (1) has been prepared by reaction of the precursor [Rh(mu-Cl)(eta(2)-C(2)H(4))(2)](2), 2,6-bis[4'(S)-isopropyloxazolin-2'-yl]pyridine (pybox), CO, and NaPF(6). Complex 1 reacts with monodentate phosphines to give the complexes [Rh(kappa(1)-N-pybox)(CO)(PR(3))(2)][PF(6)] (R(3) = MePh(2) (2), Me(2)Ph (3), (C(3)H(5))Ph(2) (4)), which show a previously unseen monodentate coordination of pybox. Complex 1 undergoes oxidative addition reactions with iodine and CH(3)I leading to the complexes [RhI(R)(kappa(3)-N,N,N-pybox)(CO)][PF(6)] (R = I (5); R = CH(3) (6)). Furthermore, a new allenyl Rh(III)-pybox complex of formula [Rh(CH=C=CH(2))Cl(2)(kappa(3)-N,N,N-pybox)] (7) has been synthesized by a one-pot reaction from [Rh(mu-Cl)(eta(2)-C(2)H(4))(2)](2), pybox, and an equimolar amount of propargyl chloride.