Derivatisation
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Separation technologies play an important role in revealing biological processes at various omic levels, in pharmacological and clinical research. In this context, CE is a strong candidate for analyses of samples with rapidly increasing complexity. Even though CE is well known for its many advantages in this regard, the sensitivity of CE analyses is insufficient for many applications. Accordingly, there are generally three main options for enhancing the sensitivity of CE analyses - using special detection techniques, using sample pre-concentration and derivatisation. Derivatisation is often the method of choice for many laboratories, since it is simple and provides several advantages such as small sample volume demand and the possibility of automation. Although it can be performed in different ways depending on where the reaction takes place, this article reviews one of the simplest and at the same time most useful approaches on-capillary derivatisation. Even if in many cases the use of on-capillary derivatisation alone is enough to improve the detection sensitivity, on other occasions it needs to be employed in combination with the other above-mentioned strategies. After a simple discussion of derivatisation in general, special attention is focused on the on-capillary approach and methodologies available for on-capillary reactant mixing. Its applications in various fields are also described.
In 1996, the EU prohibited the use of substances with anabolic action for food-producing animals (EU Directive 96/22/EC). In cases of illegal use of steroid hormones, these substances are usually applied to the animals in the form of esters. The reliable determination of intact steroid esters in animal tissues or body fluids is an unequivocal proof of illegal treatment of animals with EU prohibited anabolic substances. Previously our laboratory developed a sensitive method for determination of oestradiol benzoate and other steroid esters in blood plasma using LC-MS/MS, validated according to Commission Decision 2002/657/EC. This study describes a GC-MS method which has been developed for five oestradiol esters in blood plasma. The sample preparation procedure consisted of protein precipitation, phospholipids removal and cleaning on an alumina column. Oestradiol esters were derivatised with 2, 3, 4, 5, 6-pentafluorobenzoyl chloride (PFBCl) and pyridine in dichloromethane. The measurement of oestradiol esters was carried out by GC-MS/NCI with Cool On-Column injection. Methane was used as a negative chemical ionisation reagent gas. The method for determination of oestradiol esters in blood plasma has been validated according to Commission Decision 2002/657/EC. Decision limits for all analytes were observed below 0.05 ng mL-1. The method is robust for bovine and porcine plasma analyses and can be applied both for screening and confirmatory determination in routine residue monitoring.
INTRODUCTION: Cannabinoids are organic compounds, natural or synthetic, that bind to the cannabinoid receptors and have similar pharmacological properties as produced by the cannabis plant, Cannabis sativa. Gas chromatography (GC), e.g. gas chromatography mass spectrometry (GC-MS), is a popular analytical tool that has been used extensively to analyse cannabinoids in various matrices. OBJECTIVE: To review published literature on the use of various GC-based analytical methods for the analysis of naturally occurring cannabinoids published during the past decade. METHODOLOGY: A comprehensive literature search was performed utilising several databases, like Web of Knowledge, PubMed and Google Scholar, and other relevant published materials including published books. The keywords used, in various combinations, with cannabinoids being present in all combinations, in the search were cannabinoids, Cannabis sativa, marijuana, analysis, GC, quantitative, qualitative and quality control. RESULTS: During the past decade, several GC-based methods for the analysis of cannabinoids have been reported. While simple one-dimensional (1D) GC-MS and GC-FID (flame ionisation detector) methods were found to be quite common in cannabinoids analysis, two-dimensional (2D) GC-MS as well as GC-MS/MS also were popular because of their ability to provide more useful data for identification and quantification of cannabinoids in various matrices. Some degree of automation in sample preparation, and applications of mathematical and computational models for optimisation of different protocols were observed, and pre-analyses included various derivatisation techniques, and environmentally friendly extraction protocols. CONCLUSIONS: GC-based analysis of naturally occurring cannabinoids, especially using GC-MS, has dominated the cannabinoids analysis in the last decade; new derivatisation methods, new ionisation methods, and mathematical models for method optimisation have been introduced.
Ethylene glycol monobutyl ether (EGBE), an industrial solvent, is absorbed by the body not only by inhalation but also by dermal absorption (liquid or vapour). EGBE is metabolized to butoxyacetic acid (BAA). Pooled freeze-dried urine candidate reference material (RM) was prepared from urine obtained from persons occupationally exposed to EGBE. This material has the advantage of containing butoxyacetic acid in both the free and conjugated (glutamine and glycine) forms, as found in native urine. In all GC method modifications used, acid hydrolysis was used to release BAA from its conjugated form. The amount of butoxyacetic acid in homogeneity and stability testing was measured by GC after derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Detection was by MS in EI mode, in the authors' laboratory. For interlaboratory comparison of the reference material GC methods with MS, FID, and ECD were used. Different extraction solvents (dichloromethane-isopropanol 2:1, ethyl acetate, or dichloromethane) and derivatisation reagents (trimethylsilyldiazomethane, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide) were used. Using ANOVA (at the statistical level alpha = 0.05) no changes were found in the concentration of butoxyacetic acid during fifteen month isochronous stability testing, or in homogeneity testing. The uncertainty contributions were u (h) = 8.8 mg L(-1) and u (s) = 6.5 mg L(-1). The concentration of butoxyacetic acid in freeze-dried urine RM was evaluated from the results of eight laboratory data sets within an interlaboratory comparison by use of the interactive statistical software IPECA. The contribution to total uncertainty derived from interlaboratory comparison was u (i) = 12.7 mg L(-1). The reference value (c = 273 +/- 33 mg L(-1)) is an unweighted arithmetic average of accepted results. The value is traceable to the pure butoxyacetic acid (98% w/w; Acros Organic #257760010) used as calibrant. The uncertainty given is combined expanded uncertainty derived from the results from interlaboratory comparison, and from homogeneity and stability tests (k = 2). The reference material will be used to verify method performance in the biological monitoring of occupational exposure to EGBE.
- MeSH
- ethylenglykoly metabolismus MeSH
- glykoláty normy moč MeSH
- lidé MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- pracovní expozice MeSH
- referenční standardy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A method for determination of low concentrations of chloramphenicol in urine, feed water, milk and honey was developed. A comparison was carried out between a routinely used analytical method based on solid phase extraction (SPE-C18) for cleaning the extract and the new procedure for the sample preparation using columns based on the molecular imprinted polymers (MIP) principle. The extracts obtained from the MIP clean-up procedure were clean enough for chromatografic analyses. Confirmatory analyses were conducted using GC/MS-NCI after derivatisation (silylation). The described method was fully validated according to CD 2002/657/EC. This method is considerably robust and allows very dirty samples to be processed. The described MIP procedure is very simple and low-time-consuming, and provides high throughput of the samples examined. This could be used for routine screening and confirmatory analyses as well.
- MeSH
- antibakteriální látky moč MeSH
- chloramfenikol moč MeSH
- chromatografie metody přístrojové vybavení MeSH
- krmivo pro zvířata analýza MeSH
- med analýza MeSH
- mléko chemie MeSH
- molekulový imprinting MeSH
- polymery chemie MeSH
- prasata MeSH
- skot MeSH
- voda analýza MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
An enzyme-linked immunosorbent assay (ELISA) method is described for the semi-quantitative determination of semicarbazide (SEM), the marker residue for the banned nitrofuran drug, nitrofurazone, in chicken eggs. The sample homogenate is subjected to acid hydrolysis and derivatisation with o-nitrobenzaldehyde, followed by ethyl acetate/hexane extraction and detection by ELISA. The ELISA procedure has been validated using 0.3, 1.0 and 3 microg kg(-1) of SEM in fortified samples. Detection capability (CC(ss)) was based on the acceptance of 5% false compliant results for a given concentration level according to Commission Decision 2002/657/EC and was determined to be 0.3 microg kg(-1) with a respective limit of detection of 0.13 microg kg(-1). A validated LC-MS/MS method was used for the analysis of incurred egg samples and the results compared with ELISA. A good correlation between the results obtained from ELISA and LC-MS/MS within the concentration range 0.12-20.3 microg kg(-1) was observed in samples collected from chickens fed with a medicated ration of nitrofurazone (r = 0.992, n = 14). Validated ELISA enabled reliable monitoring of SEM levels in eggs collected from incurred chickens over a 90-day period.
- MeSH
- chromatografie kapalinová metody MeSH
- ELISA metody MeSH
- financování organizované MeSH
- hmotnostní spektrometrie metody MeSH
- karcinogeny analýza MeSH
- kontaminace potravin analýza MeSH
- kontrola léčiv a omamných látek MeSH
- kur domácí MeSH
- nitrofurazon MeSH
- rezidua léčiv analýza MeSH
- semikarbazidy analýza MeSH
- vejce analýza MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- validační studie MeSH
Na stanovení koncentrace isepamicinu v séru byla zavedena HPLC metoda s derivatizací. Isepamicin a tobramicin (vnitřní standard) se extrahovaly ze séra na kolonkách plněných Amberlitem XAD-2. Derivatizace se prováděla 1-fluor-2,4-dinitrobenzenem 1 hod na vodní lázni při teplotě 80 °C. Na chromatografickou analýzu se použila microbore kolona 1x150 mm s reversní octadecylovou fází, mobilní fáze byla složena následovně: acetonitril/methanol/voda/triethylamin 20:80:140:0,05 v/v/v/v, pH 3,8. Detekce se prováděla při 365 nm. Linearita metody byla testována v rozsahu 0-100 mg/ml, recovery bylo 96-102 % a variační koeficient okolo 5 % v koncentracích 10, 25 a 100 mg/l. U 17 pediatrických pacientů (osm bylo mladších než šest let a devět starších) byla stanovována minimální a maximální koncentrace isepamicinu. Průměrný věk dětí byl 8,4±5 let a hmotnost 26,7±13,4 kg. Dávka 13,3±2,9 mg/kg isepamicinu se podávala ve 30 minutové infuzi jednou denně. Průměrné údolní koncentrace isepamicinu byly 1,05±1,5 mg/l a v píku 35,4±19,5 mg/l. Farmakokinetické parametry se získaly pomocí farmakokinetického programu MW- PHARM 3.30. Jako model se použila populační farmakokinetika gentamicinu pro různé věkové skupiny. U dětí pod šest let jsme zjistili následující farmakokinetické parametry: CL 2,41±1,71 ml/min/kg, Vd 0,49±0,12 l/kg, t1/2 3,03±2,12 hod., Cmax 28,77±19,46 mg/l, Cmin 0,58±0,75mg/l a tmax 0,51±0,24 hodin. U dětí nad 6 let: CL 1,50±1,45 ml/min/ kg, Vd 0,32±0,087 l/kg, t1/2 3,83±1,97 hod.,Cmax 41,23±23,81 mg/l, Cmin 1,47±1,92 mg/l a tmax 0,40±0,17 hod. Zavedení HPLC metody a farmakokinetické modelování může snížit riziko toxicity isepamicinu.
HPLC method with derivatisation for measuring isepamicin concentration in serum has been developed. The assay utilised extraction of isepamicin and tobramicin (IS) from serum on Amberlite XAD-2 columns. Derivatisation was performed with 1-fluor-2,4-dinitrobenzene for 1 hour in water bath at 80 °C. Chromatography was carried out using microbore column (1 × 150 mm) with reverse octadecyl phase and acetonitril/methanol/water/triethylamine 20:80:140:0,05 v/v/v/v, pH 3,8 as mobile phase. Separation was monitored at 365 nm. Linearity was 0-100 mg/ml, recovery 96-102 % and coefficient of variations about 5 % in tested concentrations (10, 25 and 100 mg/ml). Trough and peak serum levels of isepamicin were measured during the treatment of 17 paediatric patients (8 were under 6 and 9 over 6 years). Mean age was 8,4±5 years and weight 26,7±13.4 kg. The dose of isepamicin 13,3±2,9 mg/kg was given in 30 minutes infusion once daily. Trough level was found 1,05±1,51 mg/l and peak level 35,4±19,5 mg/l, respectively. Pharmacokinetic data were obtained by the aid of pharmacokinetic program MW-PHARM 3.30. Gentamicin population pharmacokinetic model for different age groups was used. In the group of children under 6 year the pharmacokinetic data were CL 2,41±1,71 ml/min/kg, Vd 0,49±0,12 l/kg, t1/2 3,03±2,12 hours, Cmax 28,77±19,46 mg/l, Cmin 0,58±0,75mg/l and tmax 0,51±0,24 hours. While in the group of children over 6 year were CL 1,50±1,45 ml/min/kg, Vd 0,32±0,08 l/kg, t1/2 3,83±1,97 hours, Cmax 41,23±23,81 mg/l, Cmin 1,47±1,92 mg/l and tmax 0,40±0,17 hours. Implementation of HPLC method and pharmacokinetic modelling may reduce risk of isepamicin toxicity.
- Klíčová slova
- ISEPAMICIN,
- MeSH
- dítě MeSH
- monitorování léčiv MeSH
- nežádoucí účinky léčiv MeSH
- tobramycin farmakokinetika krev MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- dítě MeSH
The aim of this study was to evaluate the combined effect of temperature (10, 20 and 37°C), pH (4, 5, 6, 7 and 8), and NaCl content (0, 1, 3, 4, 5 and 6% w/v) on the growth and putrescine and cadaverine production of Serratia marcescens CCM 303 under model conditions. The decarboxylase activity of S. marcescens was monitored in broth after cultivation. The cultivation medium was enriched with selected amino acids (ornithine, arginine and lysine; 0.2% w/v each) serving as precursors of biogenic amines. Levels of putrescine and cadaverine in broth were analysed by high-performance liquid chromatography after pre-column derivatisation with o-phthalaldehyde reagent. S. marcescens produced higher amounts of putrescine (up to 2096.8 mg L(-1)) compared to cadaverine content (up to 343.3 mg L(-1)) in all cultivation media. The highest putrescine and cadaverine concentrations were reached during cultivation at 10-20°C, pH 5-7 and NaCl content 1-3% w/v. On the other hand, the highest BAs production of individual cell (recalculated based on a cell; so called "yield factor") was observed at 10°C, pH 4 and salt concentration 3-5% w/v as a response to environmental stress.
- MeSH
- biogenní aminy metabolismus MeSH
- chlorid sodný farmakologie MeSH
- kadaverin analýza biosyntéza MeSH
- koncentrace vodíkových iontů MeSH
- kultivační média chemie farmakologie MeSH
- putrescin analýza biosyntéza MeSH
- Serratia marcescens účinky léků růst a vývoj metabolismus MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this paper two fast and highly sensitive ultra-high performance liquid chromatography (UHPLC) methods for the determination of tetracycline antibiotics (oxytetracycline, tetracycline, doxycycline, demeclocycline, chlortetracycline, minocycline and degradation product epitetracycline) in surface waters have been developed using fluorescence (FL) and mass spectrometry (MS) detection. ACQUITY UPLC BEH C8 and ACQUITY CSH C18 columns were employed for FL and MS detection, respectively, both packed with 1.7μm particles. Mixed-mode separation mechanism of CSH (charged surface technology) sorbent was found particularly useful in analysis of TCs, which possess problematic amphoteric structures. The FL methodology was based on chelation of tetracyclines with calcium ions to perform on-column derivatisation. The developed methods were compared in the terms of validation parameters including linearity, sensitivity, precision and accuracy. The linearity range for FL detection was within 7ngmL(-1) to 50μgmL(-1) with method limit of detection (MLOD) as low as 0.2ngmL(-1) for most of the analytes. MS detection showed even higher sensitivity reaching MLOD of 0.003ngmL(-1), which is the highest sensitivity reported so far in analysis of TCs. Matrix matched calibration curves in the range of 0.01-50ngmL(-1) were used for quantification to compensate for matrix effects with the correlation coefficients demonstrating good linearity (0.9940-0.9999). The extraction of the antibiotics from surface waters was performed using solid phase extraction with Oasis HLB cartridges. Accuracy was expressed as recovery with values ranging from 96.52% to 127.30% and from 91.66% to 123.70% for FL and MS detection, respectively.
- MeSH
- antibakteriální látky analýza MeSH
- chemické látky znečišťující vodu analýza MeSH
- extrakce na pevné fázi MeSH
- fluorescenční spektrometrie MeSH
- lineární modely MeSH
- řeky chemie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- tetracykliny analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
