Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

Environmental analysis of polar herbicides in complex organic-rich matrices by high performance liquid chromatography atmospheric pressure ionization mass spectrometry (HPLC-API-MS)

A comprehensive forensic investigation of sensitive ecosystems in the Everglades Area is presented. Assessing the background levels of contamination in these ecosystems represents a vital resource to build up forensic evidence required to enforce future environmental crimes within the studied areas. This investigation presents the development and validation of a fractionation and isolation method for two families of herbicides commonly applied in the vicinity of the study area, including phenoxy acids like 2,4-D, MCPA, and silvex; as well as the most common triazine-based herbicides like atrazine, prometyne, simazine and related metabolites like DIA and DEA. Accelerated solvent extraction (ASE) and solid phase extraction (SPE) were used to isolate the analytes from abiotic matrices containing large amounts of organic material. Atmospheric-pressure ionization (API) with electrospray ionization in negative mode (ESP-), and Chemical Ionization in the positive mode (APCI+) were used to perform the characterization of the herbicides of interest.

Microphytobenthos composition in Heron Reef, Australia, by High Performance Liquid Chromatography (HPLC) (July 2012)

We quantified pigment biomarkers by high performance liquid chromatography (HPLC) to obtain a broad taxonomic classification of microphytobenthos (MPB) (i.e. identification of dominant taxa). Three replicate sediment cores were collected at 0, 50 and 100 m along transects 5-9 in Heron Reef lagoon (n=15) (Fig. 1). Transects 1-4 could not be processed because the means to have the samples analysed by HPLC were not available at the time of field data collection. Cores were stored frozen and scrapes taken from the top of each one and placed in cryovials immersed in dry ice. Samples were sent to the laboratory (CSIRO Marine and Atmospheric Research, Hobart, Australia) where pigments were extracted with 100% acetone during fifteen hours at 4°C after vortex mixing (30 seconds) and sonication (15 minutes). Samples were then centrifuged and filtered prior to the analysis of pigment composition with a Waters - Alliance HPLC system equipped with a photo-diode array detector. Pigments were separated using a Zorbax Eclipse XDB-C8 stainless steel 150 mm x 4.6 mm ID column with 3.5 µm particle size (Agilent Technologies) and a binary gradient system with an elevated column temperature following a modified version of the Van Heukelem and Thomas (2001) method. The separated pigments were detected at 436 nm and identified against standard spectra using Waters Empower software. Standards for HPLC system calibration were obtained from Sigma (USA) and DHI (Denmark).

Intact and core tetraether lipids in water column profiles of suspended particulate matter and of surface sediment samples off Cape Blanc, NW Africa

In the reconstruction of sea surface temperature (SST) from sedimentary archives, secondary sources, lateral transport and selective preservation are considered to be mainly negligible in terms of influencing the primary signal. This is also true for the archaeal glycerol dialkyl glycerol tetraethers (GDGTs) that form the basis for the TEX86 SST proxy. Our samples represent four years variability on a transect off Cape Blanc (NW Africa). We studied the subsurface production, vertical and lateral transport of intact polar lipids and core GDGTs in the water column at high vertical resolution on the basis of suspended particulate matter (SPM) samples from the photic zone, the subsurface oxygen minimum zone (OMZ), nepheloid layers (NL) and the water column between these. Furthermore we compared the water column SPM GDGT composition with that in underlying surface sediments. This is the first study that reports TEX86 values from the precursor intact polar lipids (IPLs) associated with specific head groups (IPL -specific TEX86). We show a clear deviation from the sea surface GDGT composition in the OMZ between 300 and 600 m. Since neither lateral transport nor selective degradation provides a satisfactory explanation for the observed TEX-derived temperature profiles with a bias towards higher temperatures for both core- and IPL -specific TEX86 values, we suggest that subsurface in situ production of archaea with a distinct relationship between lipid biosynthesis and temperature is the responsible mechanism. However, in the NW-African upwelling system the GDGT contribution of the OMZ to the surface sediments does not seem to affect the sedimentary TEX86 as it shows no bias and still reflects the signal of the surface waters between 0 and 60 m.

HPLC Determination of Angiotensin-Converting Enzyme Activity on Toyopearl HW-40S Column

Angiotensin-converting enzyme (EC3.4.15. I; ACE), isa membrane-bounddipeptidyl carboxypeptidase that mediates the cleavage of the C-terminal dipeptide His-Leu of the decapeptide angiotensin, generating the most powerful endogenous vaso-constricting angiotensin.
Some ACE inhibitors, such as Captopril, have been used as anti-hypertensive drugs. Moreover in recent years, large quantities of ACE inhibitors have been identijied and isolated from peptides derivedfrom food material such as casein, soy protein, jish protein and so on. Functional food with hypotensive effect has been developed on the basis of these works.
Typicalprocedures for screening hypotensive peptides offood origins are separationof products of peptic and tryptic digestion of proteins followed by inhibitory activitydetermination of each fraction. A method developed by Cushman has been the mostwidely used, in which ACE activity is determined by the amount of hippuric acid
generated as a product of enzymatic reaction of ACE with tripeptide of hippuryl-Lhistidyl-L-leucine. Hippuric acid is determined spectrophotometrically at 228 nm after its isolation from the reaction system by ethylacetate extraction, which not only requires alarge quantity of reagent but also results in large error.
An improved method based on Cushman ’s method is proposed in this paper. In this method, an enzymatic reaction system is based on Cushman’s method, while isolation and determination of hippuric acid is performed by medium perjormance gel chromatography on a Toyopearl HW-40s column. Due to the size exclusion nature of the column with somewhat hydrophobic properties, complete separation of four existing fractions in the reaction system is obtained within a smallfraction of the time necessary in Cushman’s method, with ideal reproducibility.

YHTEISTYÖTÄ VAI YLIMÄÄRÄISTÄ TYÖTÄ? Opettajien kokemuksia kolmiportaisen tuen asiakirjojen käytöstä esi- ja alkuopetuksen nivelvaiheessa

Suomessa esi- ja perusopetuksessa otettiin 1.1.2011 käyttöön uusi oppimisen kolmiportainen tukimalli, joka korostaa kaikkien lasten oikeutta oppimiseen ja koulunkäyntiin aivan tavallisilla luokilla. Tuen malli muotoutui vuoden 2010 perusopetuslakimuutoksessa. Tukimallin jokaiseen eri vaiheeseen kuuluu kiinteästi tuen toteuttamiseen liittyviä asiakirjoja. Niihin kirjataan lapsen tukeen liittyvät asiat. Asiakirjoja ovat: pedagoginen arvio, oppimissuunnitelma, pedagoginen selvitys sekä henkilökohtainen oppimisen järjestämistä koskeva suunnitelma (HOJKS). Tämän tutkimuksen tarkoitus oli tutkia asiakirjojen käytettävyyttä esi- ja alkuopetuksen nivelvaiheessa. Tutkimuksessa selvitettiin, millaisia kokemuksia esi- ja alkuopettajilla on kolmiportaisen tuen asiakirjoista sekä niiden vaikutuksista heidän yhteistyöhönsä. Tutkimukseen osallistui viisi esiopettajaa ja viisi alkuopettajaa. Tutkimus suoritettiin teemahaastatteluna fenomenologista lähestymistapaa noudattaen. Analyysimenetelmänä käytettiin aineistolähtöistä sisällönanalyysiä. Tutkimustulosten mukaan esi- ja alkuopettajat kokivat, että kolmiportaisen tuen asiakirjoihin kirjattu tieto vaikuttaa positiivisesti lapsen saamaan tukeen. Asiakirjat ohjaavat tukea tarvitsevan lapsen oppimisen polkua inkluusioperiaatteen mukaisesti kohti omaa lähikoulua. Opettajat näkivät kuitenkin asiakirjoihin liittyviä epäkohtia. Asiakirjat olivat epäselviä ja eri kunnissa hyvin erilaisia. Esiopettajat kokivat niiden kirjaamisen työläänä ja osittain turhana, sillä heillä ei ollut tietoa, lukevatko alkuopettajat heidän esiopetuksesta lähettämiään asiakirjoja. Tuen eri asteiden ”rajapinnat” olivat opettajille epäselviä. Ei ollut selvää, milloin esimerkiksi oppimissuunnitelma oli aiheellista kirjoittaa. Alkuopettajien mielestä lasten pulmia oli vaikea ennakoida, koska eri esiopettajat kirjasivat samoja asioita eri tavalla. Asiakirjoihin liittyvät yhteistyö- ja siirtokäytänteet eivät myöskään olleet selviä. Tutkimus osoitti, että kolmiportaisen tuen asiakirjoja pitäisi esiopetuksessa selkeyttää ja yhdenmukaistaa. Esimiesten tulisi antaa yhtenevät ohjeet asiakirjojen kirjaamis- ja siirtokäytänteisiin: kuka tekee mitä ja millä ajalla. Erityisopettajien työpanosta asiakirjojen suhteen tulisi myös tarkentaa. Opettajien lisäkoulutukseen oli selkeästi tarvetta.

”Ei kaikille samalla kauhalla syötetä” Koulutulokkaiden valmiudet esi- ja alkuopettajien näkökulmasta

Esiopetuksella pyritään luomaan tasa-arvoiset lähtökohdat kouluun siirtymiselle. Esiopetuksen muuttuminen velvoittavaksi parantaa näitä lähtökohtia, koska tällöin kaikki koulutulokkaat ovat osallistuneet esiopetukseen. Esiopetuksella on suuri merkitys lapsen elämässä. Siellä lapsi saa oppia asioita leikin kautta sekä harjoitella koulussa tarvittavia taitoja, joista tärkeimpiä ovat sosiaaliset taidot ja itsesäätelyn taidot. Siirtyminen esiopetuksesta alkuopetukseen on iso askel sekä lapselle että hänen perheelleen. Helpottaakseen tämän ison askeleen ottamista, lapset käyvät tutustumassa kouluun etukäteen. Onnistuneeseen kouluun siirtymään päästään yhteistyöllä, jota tulisi tehdä niin esi- ja alkuopetuksen välillä kuin lapsen vanhempienkin kanssa. Tutkimuksen tavoitteena oli selvittää esi- ja alkuopettajien näkemyksiä koulutulokkaiden valmiuksista ja sitä, millainen esiopetus tukee näitä valmiuksia. Tutkimusta varten haastateltiin neljää lastentarhanopettajaa, jotka työskentelevät koulun yhteydessä sijaitsevissa esiopetusyksikössä ja neljää luokanopettajaa, jotka työskentelevät alkuopetuksessa. Koulutulokkaiden valmiuksia käsiteltäessä, tutkimuksessa esiin tuli erityisesti lasten sosiaaliset taidot ja itsesäätelyn taidot. Lasten tulisi kouluun tullessa pystyä tekemään tehtäviä sekä yksin että ryhmässä ja keskittymään pieniin tehtäviin. Erot lastentarhanopettajien ja alkuopetuksessa työskentelevien luokanopettajien näkemyksissä olivat melko vähäisiä. Tuloksissa esille tuli myös se, että kouluvalmiuksia tukeva esiopetus on lähtöisin lapsen omista kiinnostuksen kohteista, huomioi lapsen yksilöllisesti sekä tukee lapsen myönteisen oppimiskäsityksen muodostumista.

Otimização e validação de metodologia analítica empregando SPE e LC-ESI-MS/MS para determinação de multiclasses de agrotóxicos e metabólitos em água de superfície e de abastecimento público

A aplicação de agrotóxicos nas práticas agrícolas aumentou muito nos últimos anos. Isto vem ocorrendo devido ao crescimento populacional, demandando maior produção de alimentos. O uso de agrotóxicos e seus resíduos tornaram-se um problema devido a possível contaminação das águas de superfície e subterrânea, podendo impactar o meio ambiente e causar danos à saúde pública. Na cidade de Rio Grande, RS, Brasil, o suprimento de água potável é realizado pela CORSAN (Companhia Riograndense de Saneamento), que capta a água do Canal São Gonçalo, o qual estabelece uma ligação entre as duas lagoas: Lagoa dos Patos e Lagoa Mirim. Em suas margens há também a captação de água para irrigação das culturas agrícolas. Esta interação entre o uso da água das lagoas e a agricultura, pode resultar na contaminação das águas que são captadas para abastecimento dos municípios situados na região. Uma metodologia analítica empregando Extração em Fase Sólida (SPE) e Cromatografia Líquida acoplada a uma fonte de ionização por Electrospray tandem Espectrometria de Massas (LCESI-MS/MS) foi desenvolvida e validada para a determinação de dezoito agrotóxicos multiclasses (herbicidas, inseticidas e fungicidas) e dois metabólitos em amostras de água superficial e de abastecimento público. Esta metodologia foi aplicada para monitoramento durante dez meses na água superficial do Canal São Gonçalo e na água de consumo da cidade de Rio Grande, após o tratamento pela CORSAN. Os agrotóxicos selecionados foram: clomazona, bispiribaque-sódio, diurom, atrazina, simazina, imazetapir, imazapique, metsulfuron-metílico, quincloraque, penoxsulam, 2,4-D, pirazosulfuron-etílico, bentazona, propanil, irgarol, tebuconazol, fipronil e carbofurano. Os metabólitos foram: 3,4-DCA e 3-hidroxicarbofurano. Os limites de detecção do método variaram entre 0,4 – 40,0 ng L -1 , enquanto para os limites de quantificação a variação foi de 4,0 – 100,0 ng L -1 . Todos os compostos apresentaram excelente linearidade, com coeficiente de determinação maior do que 0,99. As recuperações empregando SPE com cartuchos contendo 500 mg de C18ec, variaram entre 70 a 120%, para 95% dos compostos, apresentando %RSD 20%. Através do monitoramento de múltiplas reações (MRM), duas transições diferentes (íon precursor – íon produto) foram selecionadas para cada composto, uma para quantificação e outra para confirmação, o que aumentou a seletividade do método. Para as amostras analisadas, foram detectados agrotóxicos nível de ng L -1 . O método desenvolvido é sensível, rápido e apresenta elevada seletividade, permitindo a identificação e a quantificação dos agrotóxicos em águas superficiais e de abastecimento público, atendendo os níveis requeridos pelos órgãos reguladores como da União Européia (98/83/EC) e do Brasil segundo a Portaria Nº. 518 (25/03/2004).