Enhanced detectability of fluorinated derivatives of N,N-dialkylamino alcohols and precursors of nitrogen mustards by gas chromatography coupled to Fourier transform infrared spectroscopy analysis for verification of chemical weapons convention

N,N-Dialkylamino alcohols, N-methyldiethanolamine, N  -ethyldiethanolamine and triethanolamine are the precursors of VX type nerve agents and three different nitrogen mustards respectively. Their detection and identification is of paramount importance for verification analysis of chemical weapons convention. GC–FTIR is used as complimentary technique to GC–MS analysis for identification of these analytes. One constraint of GC–FTIR, its low sensitivity, was overcome by converting the analytes to their fluorinated derivatives. Owing to high absorptivity in IR region, these derivatives facilitated their detection by GC–FTIR analysis. Derivatizing reagents having trimethylsilyl, trifluoroacyl and heptafluorobutyryl groups on imidazole moiety were screened. Derivatives formed there were analyzed by GC–FTIR quantitatively. Of these reagents studied, heptafluorobutyrylimidazole (HFBI) produced the greatest increase in sensitivity by GC–FTIR detection. 60–125 folds of sensitivity enhancement were observed for the analytes by HFBI derivatization. Absorbance due to various functional groups responsible for enhanced sensitivity were compared by determining their corresponding relative molar extinction coefficients (ε^′_max${{\epsilon }^{\prime }}_{\text{max}}$) considering uniform optical path length. The RSDs for intraday repeatability and interday reproducibility for various derivatives were 0.2–1.1% and 0.3–1.8%. Limit of detection (LOD) was achieved up to 10–15 ng and applicability of the method was tested with unknown samples obtained in international proficiency tests.     

Optimization of simultaneous derivatization and extraction of aliphatic amines in water samples with dispersive liquid-liquid microextraction followed by HPLC

Dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) with simultaneous derivatization followed by high-performance liquid chromatography-diode array detection (HPLC-DAD) was applied for preconcentration and determination of primary and secondary aliphatic amines in environmental water samples. A ternary mixture consisting of a disperser, an extractant and a derivatization reagent was used for the simultaneous derivatization and extraction of aliphatic amines in different water samples. The effects of various experimental parameters on derivatization and extraction efficiency were studied simultaneously using experimental design. A Plackett-Burman design was performed for screening of variables in order to determine the significant variables affecting the extraction efficiency. Then, the significant factors were optimized by using a Box-Behnken design (BBD) and the response surface equations were derived. Under optimal conditions, the preconcentration factors were between 210 and 290. The limit of detections (LODs) ranged from 0.005 to 0.02 μg/L and dynamic linear ranges (DLRs) of 0.05-500 and 0.1-500 μg/L were obtained for most of analytes. The performance of the method was evaluated for extraction and determination of primary and secondary aliphatic amines in environmental water samples in micrograms per liter and satisfactory results were obtained (RSDs <12.5%).     

Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography-electrospray ionization mass spectrometry

Unusual amino acids such as beta-methoxytyrosine (beta-MeOTyr), allo-threonine (allo-Thr) and allo-isoleucine (allo-Ile) were derivatized with N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among beta-MeOTyr isomers was poor. The best separation of beta-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.     

Development and comparison of HPLC and MEKC methods for the analysis of cyclic sulfur mustard degradation products

In this study, novel, fast, and simple methods based on RP‐HPLC and MEKC with DAD are developed and validated for the qualitative and quantitative determination of five cyclic sulfur mustard (HD) degradation products (1,4‐thioxane, 1,3‐dithiolane, 1,4‐dithiane, 1,2,5‐trithiepane, and 1,4,5‐oxadithiepane) in water samples. The HPLC method employs a C18 column and an isocratic water‐ACN (55:45, v/v) mobile phase. This method enables separation of all five cyclic compounds within 8 min. With the CE method, the baseline separation of five compounds was achieved in less than 11 min by applying a simple BGE composed of a 10 mM borate buffer and 90 mM SDS (pH 9.15). Both methods showed good linear correlation (R ² > 0.9904). The detection limits were in the range of 0.08–0.1 μM for the HPLC method and 10–20 μM for MEKC. The precision tests resulted in RSDs for migration times and peak areas less than 0.9 and 5.5%, respectively, for the HPLC method, and less than 1.1 and 7.7% for the MEKC method, respectively. The developed methods were successfully applied to the analysis of five cyclic HD degradation products in water samples. With the HPLC method, the LODs were lowered using the SPE for sample purification and concentration.     

Determination of hydroxy-PCBs in urine by gas chromatography/mass spectrometry with solid-phase extraction and derivatization

A sensitive derivatization and extraction method is proposed for the determination of hydroxy-PCBs in urine. Phenolic hydroxyl groups of PCBs were allowed to react with five different reagents such as iodomethane, iodoethane, iodopropane, BSTFA and MTBSTFA. Propylated products at 100 °C for 30 min showed the best sensitivity with mass selective detector. Extraction recoveries and relative standard deviations of hydroxy-PCBs by SPE using C2 column were in the range of 78.0-112.3% and 2.5-9.6%, respectively. Instrumental detection limits for derivatized hydroxy-PCBs were in the range of 1-2 pg and were 10-1000 times more sensitive than those of non-derivatized hydroxy-PCBs. The correlation coefficients of the linear regression curves exceed 0.99, and the intra- and inter-day precisions were evaluated by RSDs within 10% at the concentrations of 0.4 and 4.0 ng/mL.     

In situ aqueous derivatization and determination of non-steroidal anti-inflammatory drugs by salting-out-assisted liquid-liquid extraction and gas chromatography-mass spectrometry

A new analytical method for the determination of trace levels of five non-steroidal anti-inflammatory drugs (NSAIDs: clofibric acid, ibuprofen, naproxen, diclofenac and ketoprofen) in water samples is described. The analytical procedure involves in situ aqueous derivatization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 2,2,2-trifluoroethylamine hydrochloride (TFEA) and salting-out liquid-liquid extraction (SALLE), followed by gas chromatography-programmed temperature vaporizer-mass spectrometry (GC-PTV-MS). The influence of several parameters on the efficiency of the derivatization (stirring time, reaction time, reagent concentration and pH), and the extraction (solvent, volume, salts and stirring time) and injection steps (liner, injection volume, liner temperature, injection time, venting time and venting flow) was investigated. The detection limits of the method in water varied from 0.042 μg/L for ibuprofen to 1.2 μg/L for ketoprofen. The relative standard deviations (RSD) values were found to be relatively low (<10% for all compounds). The methodology developed was applied to the determination of NSAIDs in several environmental matrices including tap, river, sea and influent and effluent waste water samples. The results obtained show the presence of ibuprofen and naproxen in the influent waste water sample.     

Modifications and insights into a method for the analysis of the nitrogen mustard mechlorethamine by high-performance liquid chromatography

Previously, a method was presented for the analysis of mechlorethamine by derivatization of this unstable nitrogen mustard to bis(2-phenylthioethyl)methylamine (PTEMA), a stable compound suitable for analysis by HPLC with UV detection [J.C. Reepmeyer, J. Chromatogr. A, 1085 (2005) 262]. Mechlorethamine HCl served as a reference standard and it was derivatized in situ simultaneously with samples of mechlorethamine HCl in ointment preparations. This paper presents the synthesis of PTEMA on a gram scale, synthesis of its picrate salt, bis(2-phenylthioethyl)methylamine picrate (PTEMAP), and isolation of the picrate as a crystalline solid. PTEMAP may serve as a reference standard replacing the toxic mechlorethamine HCl. Insights into the handling, storage, drying, and hygroscopic properties of mechlorethamine HCl and PTEMAP are discussed. In addition, one step following the derivatization procedure in the original method is recognized as a potential for error, and a procedure relating to the order of addition of reagents is presented to avoid this error. The method has been extended to the analysis of mechlorethamine in aqueous solutions.     

Electrophoresis PDMS/glass chips with continuous on-chip derivatization and analysis of amino acids using naphthalene-2,3-dicarboxaldehyde as fluorogenic agent

In this work, we developed a PDMS electrophoresis device able to carry out on-chip derivatization and quantification of amino acids (AAs) using naphthalene-2,3-dicarboxaldehyde (NDA) as a fluorogenic agent. A chemical modification of the PDMS surface was found compulsory to achieve the derivatization of AAs with NDA and a limit of detection (LOD) of 40 nM was reached for glycine. Finally, we suggested the applicability of this microdevice for the analysis of real biological samples such as a rat hippocampus microdialysate.     

In situ derivatization reaction and determination of ibuprofen in water samples using headspace generation-programmed temperature vaporization-gas chromatography-mass spectrometry

The aim of the present work is to propose a method for the determination of ibuprofen, as a typical representative of pharmaceutical compounds, in aqueous samples. To do so, an in situ derivatization reaction in aqueous medium was employed in the vial of a headspace sampler (HS), after which instrumental measurements were made with gas chromatography-mass spectrometry (GC-MS). As the injection system we propose a programmed temperature vaporizer (PTV) where, in solvent vent mode, better results can be obtained than with the conventional split and splitless injection modes. Since the derivatization reaction takes place in the HS vial, after the mixing of reagents and the sealing of the vial, the whole process takes place on-line, with no need for intermediate steps. The simplicity and speed of the method – analysis throughput: 10.5 min – together with the limit of detection obtained (0.23 μg/L), bearing in mind that no preconcentration step or later clean-up step are required, make this a good method for the analysis of ibuprofen in aqueous samples of urban waste water.     

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.     

Dispersive liquid-liquid microextraction based on the solidification of floating organic droplet for the determination of polychlorinated biphenyls in aqueous samples

In the proposed method, an extraction solvent with a lower toxicity and density than the solvents typically used in dispersive liquid-liquid microextraction was used to extract seven polychlorinated biphenyls (PCBs) from aqueous samples. Due to the density and melting point of the extraction solvent, the extract which forms a layer on top of aqueous sample can be collected by solidifying it at low temperatures, which form a layer on top of the aqueous sample. Furthermore, the solidified phase can be easily removed from the aqueous phase. Based on preliminary studies, 1-undecanol was selected as the extraction solvent, and a series of parameters that affect the extraction efficiency were systematically investigated. Under the optimized conditions, enrichment factors for PCBs ranged between 494 and 606. Based on a signal-to-noise ratio of 3, the limit of detection for the method ranged between 3.3 and 5.4 ng L⁻¹. Good linearity, reproducibility and recovery were also obtained.     

Homogeneous liquid-liquid extraction of trace amounts of mononitrotoluenes from waste water samples

Homogeneous liquid-liquid extraction method was studied based on a phase separation phenomenon in a ternary solvent system. According to this procedure, mononitrotoloenes were extracted by single-phase extraction in a water/methanol/chloroform, homogeneous ternary solvent system. Methanol and chloroform were used as consolute and extraction solvents, respectively. The homogeneous solution was broken by the addition of salt and a cloudy solution was formed. After centrifugation, the fine droplets of the extraction solvent were sedimented in the bottom of the conical test tube. Analysis of the extracts was carried out by gas chromatography. The optimization procedure was performed using Box-Behnken design. The variables involved were: sample and extraction solvent volumes, consolute solvent volume and phase separator reagent concentration. Optimum results were obtained under the following conditions: sample volume of 5 mL, extraction solvent volume of 55 μL, consolute solvent volume of 1 mL and phase separator reagent concentration; 5% (w/v). Under these conditions, the enrichment factors of 354, 311 and 300, dynamic linear ranges of 0.5-500, 1-500 and 1-500 μg L⁻¹, and limit of detections (LODs) of 0.09, 0.09 and 0.1 μg L⁻¹ were obtained for o-nitrotoluene, m-nitrotoluene and p-nitrotoluene, respectively. Finally, the method was successfully applied to the extraction and determination of MNTs in the waste water samples in the range of micrograms per liter with R.S.Ds. < 13.2%.     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.     

Dispersive liquid-liquid microextraction based on solidification of floating organic droplet method for the determination of diethofencarb and pyrimethanil in aqueous samples

A dispersive liquid-liquid microextraction method was developed for the determination of fungicides (diethofencarb and pyrimethanil) in aqueous samples. It is based on the use of solidified floating organic drops combined with high-performance liquid chromatography. Extraction solvent and dispersive solvent, extraction time and salt effect were optimized. Under optimized conditions, the enrichment factors for a 5?mL water sample are between 145 and 161. The limits of detection for diethofencarb and pyrimethanil are 0.24 and 0.09???g ? L^?1, respectively. The method offers good repeatability and high recovery. Compared with dispersive liquid-liquid microextraction, it has a higher enrichment factor, high precision due to the ease with which the solidified floating phase is transferred, thus avoiding the loss of analyte. Toxic solvents were replaced by 1-dodecanol with its much lower toxicity. The method has been successfully applied to the determination of the two fungicides in tap water, lake water, and river water.     

Trace analysis of surfactants derived from fatty alcohols I. Optimization of derivatization reaction

Summary Pre-column derivatization of the primary hydroxyl group in fatty alcohols and fatty alcohol ethoxylates using carbazole-9-carbonyl chloride (CC−Cl) and FMOC-Cl is described and compared with derivatization with 1-naphthoyl chloride (N−Cl). As the excess of derivatization reagent leads to a broad and strongly tailing reagent peak, it hinders trace determination of fatty alcohols and fatty alcohol ethoxylates. Therefore, an off-line as well as an on-line solid-phase extraction (SPE) method for removal of excess reagent are described. The on-line method which is based on column switching, shows better reproducibility higher pre-concentration, lower risk of contamination and can be easily automated, while the off-line method is better suited for the analysis of derivatized, fatty alcohol ethoxylates. An example of the trace analysis of fatty alcohols with a concentration of 2 ppb is given.     

Quantitation of 17alpha-ethinylestradiol in aquatic samples using liquid-liquid phase extraction, dansyl derivatization, and liquid chromatography/positive electrospray tandem mass spectrometry

A rapid and sensitive method for the analysis of 17alpha-ethinylestradiol (EE2) in environmental aqueous samples has been developed. Aquatic samples were extracted using liquid-liquid extraction, and organic phase extracts were concentrated and derivatized with dansyl chloride. Analysis was performed using high-performance liquid chromatography with positive electrospray ionization and tandem mass spectrometry (HPLC/ESI-MS/MS). Deuterated 17alpha-ethinylestradiol was used as internal standard and was added to samples before extraction. A limit of quantitation of 1 ng/L was obtained using a 25 mL aqueous sample. The average recovery of EE2 spiked into a 25 mL tapwater sample was 100%. This highly sensitive quantitation method is useful for measuring low levels of EE2 in aqueous environmental samples.     

Quantification of cyclamate and cyclohexylamine in urine samples using high-performance liquid chromatography with trinitrobenzenesulfonic acid pre-column derivatization

An HPLC isocratic method with pre-column derivatization and UV detection for the quantification of cyclamate and cyclohexylamine in urine samples is described. The method requires very little sample preparation. Free cyclohexylamine is analysed in a first run and subsequently cyclamate is analysed as cyclohexylamine, after the simple process of oxidation of the sample by means of hydrogen peroxide. Cycloheptylamine is used as internal standard. Trinitrobenzenesulfonic acid (TNBS) appears to be a good reagent for the pre-column derivatization. The time per run is 15 min; the coefficients of variation of the assays range from 1.1 to 5.5%; the limits of detection are 0.09 and 0.11 ppm for cyclohexylamine and cyclamate anion, respectively. The system described has always performed efficiently, with a high degree of stability, in daily routine work.     

Improved solid-phase extraction/micellar procedure for the derivatization/preconcentration of benzaldehyde and methyl derivatives from water samples

A simple, rapid and sensitive method has been developed for the determination of aromatic low-molecular mass aldehydes (LMMAs) such as benzaldehyde (BA) and methyl derivatives in water samples through the use of liquid chromatography-diode array detection (LC-DAD). The method is based on the continuous in situ derivatization of the aldehydes with 2,4-dinitrophenylhydrazine (DNPH) on a LiChrolut EN solid-phase extraction (SPE) column in the presence of sodium dodecyl sulfate (SDS) micelles. After elution, hydrazones were successfully separated on a RP-C₁₈ column using a linear gradient mobile phase of acetonitrile (ACN)-water at 75-95% ACN for 10 min. Linearity was established over the concentration range 0.4-200 μg L^-1 and limits of detection (LODs) from 120 to 200 ng L^-1; the inter-day precision expressed as the relative standard deviation (RSD) of the aldehydes ranged from 3.0% to 3.5%. The method was successfully applied to the analysis of aromatic and aliphatic LMMAs in water samples with average recoveries ranging between 93.6% and 99.5%. The proposed method surpasses other chromatographic alternatives in terms of LODs, sample requirements for analysis and cost.     

High-performance liquid chromatographic analysis of hexosamines, hexosaminitols, N-acetylhexosamines and Nacetylhexosaminitols by ultraviolet and fluorescence detection at picomole levels

A simple and rapid method for the preparation of 9-fluorenylmethoxycarbonyl (Fmoc) derivatives of hexosamines (glucosamine, galactosamine), hexosaminitols (glucosaminitol, galactosaminitol), N-acetylhexosamines (N-acetylglucosamine, N-acetylgalactosamine) and N-acetylhexosaminitols (N-acetylglucosaminitol, N-acetylgalactosaminitol), is described. The derivatization was performed at room temperature for 5–10 min under neutral and slightly alkaline conditions. The separation of Fmoc-amino sugar and amino sugar alcohol derivatives on various reversed-phase columns (Cosmosil C₁₈, Sinopak-S C₈ and Develosil C₁₈) with isocratic elution was examined. The determination limits of hexosaminitol, N-acetylhexosamine, and N-acetylhexosaminitol derivatives were 0.4, 1.4 and 1.6 pmol with fluorescence detection and 4.6, 16 and 20 pmol with ultraviolet detection, respectively.