Comparison of highly-fluorinated chloroformates as direct aqueous sample derivatizing agents for hydrophilic analytes and drinking-water disinfection by-products

Four highly-fluorinated alkyl and aryl chloroformates, including 2,2,3,3,4,4,5,5-octafluoro-1-pentyl chloroformate (OFPCF), 2,3,4,5,6-pentafluorobenzyl chloroformate (PFBCF), 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octyl chloroformate (TDFOCF), and 2-(2,3,4,5,6-pentafluorophenoxy)-ethyl chloroformate (PFPECF), were synthesized and tested as reagents for the direct water derivatization of polar and hydrophilic analytes. The goal of this research was to develop an optimal derivatizing agent to aid in the identification of highly polar ozonation drinking water disinfection by-products (DBPs) that are believed to be missed with current analytical procedures. The chemical properties (reactivity, selectivity, derivatization products, and their chromatographic and spectral features) for the four chloroformates were investigated using a set of highly polar standard analytes, including malic and tartaric acids, hydroxylamine, valine, 2-aminoethanol, resorcinol, 1,3,5-trihydroxybenzene, and 2,4-dihydroxybenzoic acid. Upon derivatization, the analytes were extracted from the aqueous solvent and analyzed by gas chromatography (GC)-mass spectrometry (MS) in the electron capture negative ionization (ECNI) mode. Positive chemical ionization (PCI)-MS was used for confirmation of molecular ions that were weak or absent in ECNI mass spectra. Of the four derivatizing reagents tested, OFPCF showed the best performance, with good reaction efficiency, good chromatographic and spectroscopic properties, low detection limits (10-100 fmol), and a linear response more than two orders of magnitude. Further, the entire procedure from raw aqueous sample to ready-to-inject hexane solutions of the derivatives requires less than 10 min. PFBCF showed ideal applicability for derivatizing aminoalcohols and aminoacids. The two chloroformates with the highest intrinsic stability (TDFOCF and PFPECF) failed to derivatize some of the analytes. Finally, the OFPCF derivatizing agent was tested with simulated ozonated drinking water (aqueous fulvic acid treated with ozone), and three highly polar reaction by-products were determined.     

Ultratrace determination of highly hydrophilic compounds by 2,2,3,3,4,4,5,5-octafluoropentyl chloroformate-mediated derivatization directly in water

Highly hydrophilic compounds, with multiple carboxylic, hydroxylic, or aminic groups, have been determined at ultratrace level (3–30 fmol injected) in aqueous solution by direct derivatization with 2,2,3,3,4,4,5,5-octafluoropentyl chloroformate, followed by n-hexane extraction and analysis by gas chromatography-electron capture negative ion mass spectrometry (GC-ECNI-MS). The products have high molecular weights, but also high volatility, making their elution from the GC column efficient. The derivatizing agent was synthesized from the corresponding alcohol and phosgene. The derivatization reaction is catalyzed by a pyridine solution of N,N-dicyclohexylcarbodiimide. At 200 °C ion-source temperature, most negative ion mass spectra showed only a weak or no molecular ion, but a controlled and interpretable fragmentation, allowing the derivatives to be easily monitored by high-mass ion chromatograms. Typical distinctive fragments are located in the m/z 500–800 mass range. Optimization of the operating conditions for the derivatization was performed. This was done in order to minimize number and intensity of peaks due to the hydrolysis of chloroformate in the total ion chromatogram. Calibration curves proved linear over two orders of magnitude concentration.     

Pentafluorobenzyl chloroformate derivatization for enhancement of detection of amino acids or alcohols by electron capture negative ion chemical ionization mass spectrometry

Pentafluorobenzyl chloroformate (PFB-chloroformate) has been utilized as a derivatization reagent to impart electron affinity and provide structurally relevant fragmentation in electron capture negative ion chemical ionization mass spectrometry (ECNICI-MS). Phenylalanine (Phe) and decanol were used as model analytes. The conditions used for their derivatization and the chromatographic and mass spectrometric properties of the derivatives are reported. Phenylalanine in aqueous solution was derivatized in one step by using PFB-chloroformate and a mixture of water, ethanol, and pyridine. The phenylalanine N-pentafluorobenzyl-oxycarbonyl ethyl ester (N-PFBC-Phe-OEt) exhibited good gas chromatographic properties and in ECNICI-MS, a dominant [M — 181]^? fragment carries most of the ion current. Selected ion monitoring experiments on N-PFBC-Phe-OEt resulted in the facile detection of 400 fmol of material. Decanol was derivatized by using anhydrous conditions, and the resultant pentafluorobenzyl carbonate also exhibited a predominant [M — 181]^? ion in ECNICI-MS. Initial results indicate that the ECNICI-MS molar response of the decyl pentafluorobenzyl carbonate derivative is six-fold that of the decyl pentafluorobenzoate.     

Gas chromatography and mass spectrometry of methoxylated polybrominated diphenyl ethers (MeO-PBDEs)

Twenty-six methoxylated polybrominated diphenyl ethers (MeO-PBDEs) were characterized by gas chromatography (GC) on four different GC columns with different lengths and polarities, as well as by mass spectrometry using three ionization techniques, electron ionization (EI), electron capture negative ionization (ECNI) and positive ion chemical ionization (PICI). MeO-PBDE congeners with similar retention times on a nonpolar GC column were separated when analyzed on a polar GC column. EI can be used to determine the position of the methoxy substituent (ortho, meta or para) relative to the diphenyl ether oxygen in the MeO-PBDEs. The PICI ionization technique is shown to be valuable to generate structural information of the MeO-PBDEs, i.e. the degree of bromination on both the methoxy-substituted ring and the entirely brominated phenyl ring can be obtained. This structure information can also be achieved for certain MeO-PBDEs with the methoxy group in ortho position to the diphenyl ether bond in ECNI mode. Like other brominated compounds ECNI is preferable when analyzing environmental samples for quantification of MeO-PBDEs because of the formation of bromide ions, which enables low detection limits.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

GC separation of amino acid enantiomers via derivatization with heptafluorobutyl chloroformate and Chirasil‐L‐Val column

Heptafluorobutyl chloroformate (HFBCF), a recently introduced derivatization reagent, was examined in enantioseparation of amino acids (AAs) by GC. Twenty proteinogenic AAs, plus ornithine, cystine and 4‐fluorophenylalanine (internal standard) were treated with the reagent and separation properties of the derivatives were assessed on a Chirasil‐Val capillary column. Nineteen AA enantiomers were efficiently separated in 43 min except proline, arginine and cystine. The HFBCF derivatives of the studied DL ‐AAs show improved separation over other chloroformate‐based derivatives hitherto reported. A combination of the improved and faster separation with a simple derivatization protocol, involving an immediate one‐step reaction–extraction in two‐phase aqueous‐organic medium, and low elution temperatures extend application of HFBCF to chiral AA analysis.     

Enhancing the detection sensitivity of trace analysis of pharmaceutical genotoxic impurities by chemical derivatization and coordination ion spray-mass spectrometry

Many pharmaceutical genotoxic impurities are neutral molecules. Trace level analysis of these neutral analytes is hampered by their poor ionization efficiency in mass spectrometry (MS). Two analytical approaches including chemical derivatization and coordination ion spray-MS were developed to enhance neutral analyte detection sensitivity. The chemical derivatization approach converts analytes into highly ionizable or permanently charged derivatives, which become readily detectable by MS. The coordination ion spray-MS method, on the other hand, improves ionization by forming neutral-ion adducts with metal ions such as Na⁺, K⁺, or NH₄⁺ which are introduced into the electrospray ionization source. Both approaches have been proven to be able to enhance the detection sensitivity of neutral pharmaceuticals dramatically. This article demonstrates the successful applications of the two approaches in the analysis of four pharmaceutical genotoxic impurities identified in a single drug development program, of which two are non-volatile alkyl chlorides and the other two are epoxides.     

Sol-gel immobilized cyano-polydimethylsiloxane coating for capillary microextraction of aqueous trace analytes ranging from polycyclic aromatic hydrocarbons to free fatty acids

Sol-gel coating containing highly polar cyanopropyl and nonpolar poly(dimethylsiloxane) components (sol-gel CN-PDMS coating) was developed for capillary microextraction (CME). The sol-gel chemistry provided an efficient means to immobilize the CN-PDMS coating by establishing chemical anchorage between the coating and the fused silica capillary inner surface. This chemical bond provided excellent thermal and solvent stability to the created sol-gel coating. For the extraction of polar and nonpolar analytes, the upper allowable conditioning temperatures were 330 degrees C and 350 degrees C, respectively. To our knowledge, this is the first time when a CN-PDMS thick coating survived such a high operation temperature. The prepared sol-gel CN-PDMS coating provided effective extraction of polar and nonpolar analytes simultaneously from aqueous samples. The cyanopropyl moiety in sol-gel CN-PDMS coatings provided effective extraction of highly polar analytes such as free fatty acids, alcohols, and phenols without requiring derivatization, pH adjustment or salting out procedures. The PDMS moiety, on the other hand, provided efficient extraction of nonpolar analytes. The extraction properties of the sol-gel CN-PDMS coatings can be fine tuned via manipulation of relative proportions of 3-cyanopropyltriethoxysilane and hydroxy-terminated PDMS in the sol solution used to create the coatings. Detection limits of nanogram/liter (ng/L) were achieved for both highly polar and nonpolar analytes directly extracted from aqueous media using sol-gel CN-PDMS coated microextraction capillaries followed by GC analysis.     

Sensitive determination of methylenedioxylated amphetamines by liquid chromatography

Different strategies for the liquid chromatographic determination of methylenedioxylated amphetamines were evaluated: separation and detection of underivatized analytes by (i) UV or (ii) fluorescence, (iii) derivatization with 3,5-dinitrobenzoyl chloride followed by separation and UV detection of the derivatives formed and (iv) derivatization with 9-fluorenylmethyl chloroformate (FMOC) and subsequent separation and fluorimetric detection of the derivatives. The compounds tested were 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDE). On the basis of these studies, a new procedure for the chromatographic determination of MDA, MDMA and MDE is proposed, based on derivatization with FMOC. The described procedure allows the quantification of the tested compounds with adequate linearity, reproducibility and accuracy in the concentration interval 0.5-20.0 micrograms mL-1. The limits of detection were 0.01 microgram mL-1 for MDA and 0.025 microgram mL-1 for MDMA and MDE. The utility of the described assay was tested by determining methylenedioxylated amphetamines in plasma and urine.     

Evaluation of various derivatization approaches for gas chromatography-mass spectrometry analysis of buprenorphine and norbuprenorphine

Various chemical derivatization approaches have been adapted for the analysis of buprenorphine and its major metabolite (norbuprenorphine) by GC-MS based methodologies. These approaches included alkylation, acylation, and silylation resulting in the formation of methyl, acetyl, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, and trimethylsilyl derivatives. This study conducted a comprehensive evaluation on the merits of these approaches based on the following criteria: reaction yields and ionization efficiency of the derivatization products; chromatographic characteristics; and cross-contributions to the intensities of ions designating the analytes and the internal standards. Under acidic derivatization conditions, the analytes could form three artifact products. Overall, derivatization by acetyl anhydride resulted in best performance characteristics.     

Sol-gel immobilized short-chain poly(ethylene glycol) coating for capillary microextraction of underivatized polar analytes

Sol-gel coating with covalently bonded low-molecular-weight (MW<300 Da) poly(ethylene glycol) (PEG) chains was developed for capillary microextraction (CME). The sol-gel chemistry proved effective in the immobilization of low-molecular-weight PEGs thanks to the formation of chemical bonds between the organic-inorganic hybrid sol-gel PEG coating and the fused silica capillary inner surface. This chemical anchorage provided excellent thermal and solvent stability to the created sol-gel PEG coating as is evidenced by its high upper limit of allowable conditioning temperature (340 degrees C) and its practically identical performance before and after rinsing with various solvents. The prepared sol-gel PEG coating provided simultaneous extraction of moderately polar and highly polar analytes from aqueous samples without requiring derivatization, pH adjustment or salting-out procedures. Detection limits on the order of nanogram per liter (ng/L) were achieved in CME-GC-flame ionization detection experiments designed for the preconcentration and trace analysis of both highly polar and moderately polar compounds extracted directly from aqueous media using sol-gel short-chain PEG coated microextraction capillaries.     

Strategies for the microextraction of polar organic contaminants in water samples

In this paper the most recent developments in the microextraction of polar analytes from aqueous environmental samples are critically reviewed. The particularities of different microextraction approaches, mainly solid-phase microextraction (SPME), stir-bar-sorptive extraction (SBSE), and liquid-phase microextraction (LPME), and their suitability for use in combination with chromatographic or electrically driven separation techniques for determination of polar species are discussed. The compatibility of microextraction techniques, especially SPME, with different derivatisation strategies enabling GC determination of polar analytes and improving their extractability is revised. In addition to the use of derivatisation reactions, the possibility of enhancing the yield of solid-phase microextraction methods for polar analytes by using new coatings and/or larger amounts of sorbent is also considered. Finally, attention is also focussed on describing the versatility of LPME in its different possible formats and its ability to improve selectivity in the extraction of polar analytes with acid-base properties by using separation membranes and buffer solutions, instead of organic solvents, as the acceptor solution.     

Reactions of Alkyl 1H,1H-Perfluoroalkyl Sulfones with Ammonia, Amines, and Hydrazines

2,2,3,3-Tetrafluoropropyl and 2,2,3,3,4,4,5,5-octafluoropentyl alkyl sulfones react with ammonia, primary and secondary amines, hydrazine hydrate, phenylhydrazine, and N,N-dimethylhydrazine to afford, depending on the length of the polyfluoroalkyl chain and reaction conditions, the corresponding enamines, imines, or mono- and bis-hydrazones. The bis-hydrazone obtained from phenylhydrazine and 2,2,3,3,4,4,5,5-octafluoropentyl benzyl sulfone is capable of undergoing further dehydrofluorination to give 5-difluoromethyl- or 5-unsubstituted 1-phenylpyrazole.     

Development of chromatographic methods to monitor the synthesis of (tributylstannyl)methanol through unstable lithiated intermediates

Simple and robust derivatization methods for monitoring the formation of (tributylstannyl)methanol from tributyltin hydride via unstable lithiated intermediates have been developed and validated. These analytes present both chromatographic and detection difficulties in their native states due to low volatility, poor aqueous solubility/stability, and lack of a chromophore. Derivatization of these analytes to trimethylsilyl analogues for gas chromatographic analysis or to benzenesulfonyl urethane analogues for reversed-phase liquid chromatographic analysis was evaluated. The derivatization/gas chromatographic methods developed were demonstrated to be more specific and sensitive for impurity monitoring during (tributylstannyl)methanol preparation.     

Synthesis of Highly Fluorinated Chloroformates and Their Use as Derivatizing Agents for Hydrophilic Compounds and Drinking‐Water‐Disinfection By‐Products

A rapid, safe, and efficient procedure was developed to synthesize, on a small scale, fluorinated chloroformates often required to perform analytical derivatizations. This new family of agents allows straightforward derivatization of highly polar compounds (with multiple hydroxy, carboxy, and amino substituents) in the aqueous phase, compatible with GC and GC/MS analysis. A goal of this work was to develop a derivatization procedure that would enable the detection and identification of highly polar disinfection by‐products in drinking water.     

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters.

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.     

Determination of <Emphasis Type="SmallCaps">D</Emphasis>- and <Emphasis Type="SmallCaps">L</Emphasis>-amino acids produced by cyanobacteria using gas chromatography on Chirasil-Val after derivatization with pentafluoropropyl chloroformate

A rapid and simple method was developed for the determination of free amino acids (AAs) released from cyanobacteria. The procedure involves trapping of AAs from the centrifuged cyanobacterial culture fluid on a cation-exchange resin, their release together with the resin by direct treatment with the reaction medium, followed by immediate derivatization with a corresponding chloroformate. The extractive alkylation transfers the analytes into an organic phase, an aliquot of which is subjected to GC analysis. Identification and quantification of AAs was performed by GC/MS and GC/FID, respectively, using propyl chloroformate (PCF) as the derivatization reagent. For chiral analysis, the cyanobacteria extracts were treated with 2,2,3,3,3-pentafluoropropyl chloroformate (PFPCF) to create more volatile analytes. Separation of the AA enantiomers was accomplished on a Chirasil-Val capillary column and the D/L enantiomeric ratios were determined. AAs of cyanobacteria are considered to be important for the assessment of energy flow in an aquatic food web, nutrition value of cyanobacteria in a food web and for cell–cell communication within cyanobacteria. The highest levels of AAs were found in the summer period at the beginning of the season (July). In the September and October samples, the amount of AAs was lower, the number of D-AAs decreased and the D/L ratio was higher than in the July sample. Based on the obtained results it can be assumed that young populations excrete AAs in higher concentrations and a different composition compared to actively growing populations. Figure PFPCF derivatization scheme     

An approach to analysis of primary amines by a combination of thin‐layer chromatography and matrix‐assisted laser desorption ionization mass spectrometry in conjunction with post‐chromatographic derivatization

On‐spot derivatization has been suggested for the modification of primary amine containing compounds for their analysis by thin‐layer chromatography hyphenated with matrix‐assisted laser desorption ionization mass spectrometry. The proposed approach was based on post‐chromatographic treatment of separated analytes inside the chromatographic zones on the thin‐layer chromatography plates by tris(2,6‐dimethoxyphenyl)methilium reagent. The derivatives, containing permanent positive charge, reveal exceptionally intense peaks of their cationic moieties and high signal/noise ratio in mass spectra recorded directly from the plates. The method was tested on a series of aliphatic, aromatic, and amine‐containing pharmaceuticals.     

p-Tolyl isocyanate derivatization for analysis of CWC-related polar degradation products by mass spectrometry

Most of the precursors and/or degradation products related to the Chemical Weapons Convention (CWC) are polar. Identification of these molecules in environmental samples provides clues regarding the alleged usage and/or synthesis of the parent toxic chemicals. Such polar compounds need to be derivatized in order to analyze them by gas chromatography–mass spectrometry (GC–MS). In this study, we developed a new derivatizing reagent, para-tolyl isocyanate (PTI), for derivatization of polar CWC-related compounds. The PTI reagent selectively derivatizes the –OH and/or?SH functional groups with high efficiency, but does not react with carboxylic acid (?COOH) or phosphonic acid (?(O)P(OH)₂) groups. The PTI derivatives of dialkyl aminoethanols, dialkyl aminoethanol-N-oxides, and 3-quinuclidinol were successfully eluted through GC, and their electron ionization (EI) mass spectra were distinct and provided the structure information by which the isomeric compounds can be easily distinguished. We also calculated the GC-retention index values that can be used for further confirmation of the target compounds. All the studied PTI derivatives can be analyzed by EI-MS with direct insertion probe and/or by direct electrospray ionization mass spectrometry (ESI-MS) together with the MS–MS data; both sets of data provide full structure information. The PTI reagent was found to be better in some respects than the conventional bistrimethylsilyl trifluoroacetamide (BSTFA), a trimethyl silylating reagent. The PTI reagent is commercially available, and the PTI derivatives are highly stable for months and are not sensitive to moisture. The applicability of the PTI derivatization for trace-level determination of the target CWC-related polar compounds in environmental matrices and in human plasma samples is also evaluated.

Dispersive derivatization liquid-liquid extraction of degradation products/precursors of mustards and V-agents from aqueous samples

A new derivatization and extraction technique termed as dispersive derivatization liquid-liquid extraction (DDLLE) speeds up the analysis process by removing the requirement for drying of the sample. The derivatization process takes place at the interface between the analyte containing aqueous phase and derivatization agent laden organic phase. The organic phase is highly dispersed using disperser solvent so that the total surface area is large. The derivatizing agent used is 1-(heptafluorobutyryl)imidazole and the resulting heptafluorobutyryl (HFB) derivatized analytes are partitioned into the organic phase. In addition to reduced sample preparation time, for some of the analytes, the HFB derivatives provide better spectral differentiation between isomers than conventional trimethylsilyl (TMS) derivatives. Method parameters for the DDLLE, such as extraction, and disperser solvent and their volume, type and amount of base, amount of heptafluorobutyrylimidazole and extraction time were optimized on diisopropylaminoethanol (DiPAE), ethyldiethanolamine (EDEA), triethanolamine (TEA) and thiodiglycol (TDG). The DDLLE was also used on various real world samples, which also includes few OPCW organized proficiency test and a spiked urine sample. The observed limit of detection (LOD) with 1mL of sample for DDLLE in full scan with AMDIS was 10ng/mL and with methane chemical ionization, multiple reaction monitoring (MRM) was 100pg/mL, i.e., 100fg on-column.