On-column derivatization and analysis of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis

This work demonstrates the use of an in-capillary procedure for derivatization of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis (CE). Migrating in an uncoated fused-silica capillary, plugs of substrate and anhydride are injected separately and electrophoresed. Differential transport velocities permit the separate zones to penetrate each other under an applied field, thereby facilitating reaction. In initial experiments the extent of reaction between tryptophan and acetic anhydride was examined and product amounts quantitated by CE. In separate experiments a series of amino acids and peptides were injected into the capillary and reacted with phthalic anhydride on-column to yield the phthalic derivatized species. Finally, on-column derivatization of alkylamines with phthalic anhydride was investigated and electrophoretic mobility related to molecular weight of the derivatized amines. These procedures illustrate the use of the capillary as a microreactor in the facile synthesis of derivatized molecules and ease of quantitation of reaction products under conditions of electrophoresis.     

Control of liquid crystal pretilt angles by chemical derivatization reaction of polyvinyl alcohol films

A chemical derivatization technique was used to control the pretilt angle of a liquid crystal. A polyvinyl alcohol (PVA) alignment layer, which gives a very low pretilt angle when in contact with the liquid crystal (LC), was reacted with trifluoroacetic anhydride (TFAA) in the gas phase to change polar -OH groups to -OCOCF₃ groups. By introduction of the -OCOCF₃ groups in to the PVA, we obtained homeotropic alignment of the E7 LC molecules. The homeotropic alignment of E7 LC molecules in contact with the derivatized PVA alignment layer was confirmed by FTIR and microscopy with crossed polarizers. The change of liquid crystal molecules from homogeneous to homeotropic alignment may be caused by the decrease in surface tension of the PVA alignment layer, due to substitution of the polar -OH groups by -OCOCF₃ groups in the gas phase derivatization reaction.     

Single-drop microextraction with in-microvial derivatization for the determination of haloacetic acids in water sample by gas chromatography–mass spectrometry

A new approach using single-drop microextraction (SDME) and gas chromatography–mass spectrometry for the determination of six haloacetic acids (HAAs) in water samples was presented. n-Octanol was used as extractant and derivatization reagent. HAAs were derivatized both simultaneously during the extraction in the solvent microdrop, and after extraction, inside a glass microvial (1.1 mm I.D.). Trifluoroacetic anhydride (TFAA) was used as the reaction catalyst. The influence of catalyst amount, derivatization time and temperature on the yield of the in-microvial derivatization was investigated. Derivatization reaction was performed using 1.2 μL of TFAA at 100 °C for 20 min. Extraction was performed using 1.8 μL of n-octanol containing TFAA (10%, v/v). Experimental parameters, such as, exposure time, sample pH and extraction temperature were controlled and optimized. Analytical parameters such as linearity, precision and limit of detection were also evaluated. The proposed method was proved to be a suitable analytical procedure for HAAs in water with limits of detection 0.1–1.2 μg/L. The relative recoveries range from 82.5 to 97.6% for all the target analytes. Precision values were from 5.1 to 8.5% (as intra-day relative standard deviation, RSD) and 8.8–12.3% (as inter-day RSD).     

Detection of lysergic acid diethylamide, delta-9-tetrahydrocannabinol and related compounds by plasma chromatography.

Plasma chromatography as a method for ultratrace qualitative and quantitative detection of organic compounds is especially well suited for detection of gas chromatographic effluents. The optimum range of sample quantity is 10-6 to 10-12 g for detection and identification of a compound by use of its characteristic positive and negative mobility spectra. The type of reference mobility spectra produced by alkanes, aromatics, esters, halogenated compounds, nitrogenated compounds and organic acids have been previously reported. This study presents the reference mobility spectra produced for lysergic acid diethylamide (LSD), delta-9-tetrahydrocannabinol (delta-9-THC), digitoxigenin and several biochemical compounds of research significance. LSD and delat-9-THC in a mixture can be detected and identified by plasma chromatography positive mobility spectra in quantities of 10-7 g or less. All the compounds investigated in this study display strong MH-+ ions along with other ions primarily of the type (M)NO-+, (M)2H-+. None of these compounds exhibits negative mobility spectra.     

TFAA chemical derivatization and XPS. Analysis of OH and NHx polymers

The determination of functional groups on complex polymer surfaces by X‐ray photoelectron spectroscopy (XPS) can be improved considerably by derivatization reactions. Simple polymers containing hydroxyl groups or amino groups were investigated as reference materials for the derivatization with trifluoroacetic anhydride (TFAA). ‐1 Poly(vinyl alcohol) (PVA), poly(hydroxyethyl methacrylate) (PHEMA), poly(vinyl butyral) (PVB), poly(allylamine) (PAAm), and poly(diallyl amine) (PDAAm) were derivatized using TFAA and analyzed with XPS. Polyethylene (PE) was used as an independent external reference for the binding energy (BE). Applying this procedure, the BE scales of all measurements were referenced to the carbon atoms of PE. It was found that the BE of the CF₃ component in the C1s region is different when bonded as an acetate or as an amide. The CF₃ BE is also influenced by the density of these groups in the polymer molecule. In TFAA‐PVA, where every second main chain carbon atom carries a trifluoroacetate (TFAc) group, the BE is 294.3 eV while in TFAA‐PVB with only isolated groups, the BE is 293.6 eV. The BE of the CF₃ component in the trifluoroacetamides (TFAAms) prepared from PAAm and PDAAm was found to be 292.5 and 292.3 eV, respectively. Compared with the analog fluorine free compounds, the BE is shifted toward higher values also for the ester carbon atom, the amide carbon atom, and the carbon atom to which the ester or amide is bonded. The data suggest that the gas phase reaction of TFAA with a polymer surface is diffusion limited. The actual ester or amide formation is a fast reaction and runs as a wave into the surface. Copyright © 2009 John Wiley & Sons, Ltd.     

The synthesis of P-chiral amino acid-derived phosphonamidic anhydrides

The synthesis of an array of P-chiral amino acid-derived phosphonamidic anhydrides is described. These anhydrides are prepared by condensation of allylated amino acids 19-22 with methyl- or vinylphosphonic dichlorides 23 or 24 to produce three diastereomeric anhydrides 4-11a-c in good to excellent yields. The mechanistic issues concerning anhydride formation are discussed and supported by experimental results. Vinylphosphonamidic anhydrides 8-11 are further derivatized via the ring-closing metathesis (RCM) reaction to yield amino acid-derived bicyclic phosphonamidic anhydrides.     

Control of liquid crystal pretilt angles by chemical derivatization reaction of polyvinyl alcohol films

A chemical derivatization technique was used to control the pretilt angle of a liquid crystal. A polyvinyl alcohol (PVA) alignment layer, which gives a very low pretilt angle when in contact with the liquid crystal (LC), was reacted with trifluoroacetic anhydride (TFAA) in the gas phase to change polar –OH groups to –OCOCF₃ groups. By introduction of the –OCOCF₃ groups in to the PVA, we obtained homeotropic alignment of the E7 LC molecules. The homeotropic alignment of E7 LC molecules in contact with the derivatized PVA alignment layer was confirmed by FTIR and microscopy with crossed polarizers. The change of liquid crystal molecules from homogeneous to homeotropic alignment may be caused by the decrease in surface tension of the PVA alignment layer, due to substitution of the polar –OH groups by –OCOCF₃ groups in the gas phase derivatization reaction.     

A Sensitive Gas Chromatographic Method for the Determination of Metoprolol in Human Plasma

Abstract

A sensitive method for the determination of metoprolol in plasma has been developed. The procedure is based on gas chromatographic measurements of derivatized metoprolol, using 9-bromophenanthrene as internal standard. Metoprolol is derivatized with pentafluoropropionic anhydride. The resulting derivative gives a four-fold increase in sensitivity compared to the published methods where trifluoroacetic anhydride was used for derivatization.     

Microwave-accelerated derivatization prior to GC-MS determination of sex hormones

A new microwave-accelerated derivatization method was developed for rapid determination of 13 natural sex hormones in feeds. Sex hormones were isolated from the sample matrix by ultrasonic extraction, followed by solid-phase extraction, derivatized under microwave irradiation, and then analyzed directly by gas chromatography-mass spectrometry (GC-MS) in selective ion monitoring (SIM) mode. The key parameters affecting derivatization efficiency, including microwave irradiation time, microwave power, and reaction solvent were studied. Under microwave power of 360 W and microwave irradiation for 3 min, 13 natural sex hormones were simultaneously derivatized using heptafluorobutyric acid anhydride (HFBA) as derivatization reagent. This method was applied to the determination of 13 natural sex hormones in different feed samples, and the obtained results were compared with those obtained by the traditional thermal derivatization. The recoveries from 58.1 to 111% were obtained at sex hormone concentrations of 10-300 μg/kg with RSDs ≤12.0%. The results showed that the proposed method was fast, simple, efficient and can be applied to the determination of 13 natural sex hormones in different feed samples.     

Revised method for the quantitative determination of 5-hydroxytryptamine in human plasma by gas chromatography--mass spectrometry--selected ion monitoring

To estimate the 5-hydroxytryptamine level in human plasma by gas chromatography--mass spectrometry--selected ion monitoring (GC--MS--SIM), a recovery method from plasma and derivatization conditions with pentafluoropropionic anhydride were investigated. By heating 5-hydroxytryptamine at 140 degrees C for 4 h in the presence of pentafluoropropionic anhydride the peak intensity of derivatized 5-hydroxytryptamine increased more than three times in comparison with the reported method. There was a marked difference in the plasma levels of 5-hydroxytryptamine obtained using the GC--MS--SIM method compared to those obtained using fluorometric assay.     

Determination of fatty alcohol ethoxylates with diphenic anhydride derivatization and liquid chromatography with spectrophotometric detection: A comparative study with other anhydrides

A method for the determination of fatty alcohol ethoxylates (FAEs) using diphenic anhydride as derivatization reagent and RP-HPLC separation with UV–vis detection is presented and compared to derivatization with maleic, phthalic, and other cyclic anhydrides. With these anhydrides, the reaction rates increased when urea was added to the reaction medium, and the yields did not decrease when the samples contained moderate amounts of water. Gradient elution on a C8 column was performed with water/acetonitrile in the presence of 0.1% acetic acid. The use of diphenic anhydride was advantageous for both the chromatographic separation and the detection. Specifically, sensitivity at 200 and 220 nm was significantly better for the FAE diphenates, resulting in lower limits of detection at both wavelengths for the diphenates than for the maleates and phthalates (up to 30 and 4.3 times lower at 220 nm, respectively). Response factors for the diphenates decreased less than those of the phthalates when the number of ethylene oxide units, m, increased, reaching a constant value of ca. 0.62 when m > 3. Peak symmetries and efficiencies were also better than those found for the other anhydrides. The optimized procedure was applied to the characterization and determination of FAEs in the effluent of a wastewater treatment plant and in sea water.     

A comparison of 2-D chromatography separations using UV and (18)O quantification of proteins in similar proteomes

Proteins present in two mitochondria preparations were separated by 2-D chromatography using the ProteomeLab PF-2D Protein Fractional System, protein fractionation in two dimensions (PF-2D). The proteins in each first-dimension fraction were determined by trypsinization and LC-MS/MS. Chromatography peaks were quantified by UV detection using the "Mapping Tools" software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF-2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for (18)O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using (18)O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF-2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.     

Extractionless GC/MS analysis of gamma-hydroxybutyrate and gamma-butyrolactone with trifluoroacetic anhydride and heptafluoro-1-butanol from aqueous samples

gamma-Hydroxybutyrate (GHB) is a DEA Schedule I drug of abuse commonly spiked into beverages to incapacitate victims of sexual assault. GHB is a challenging drug for analysis by GC/MS because of its small size, charged nature, low volatility, and intramolecular esterification leading to gamma-butyrolactone (GBL). In this work an extractionless technique has been developed that allows for the use of an aqueous sample for direct derivatization. The technique uses a solution of trifluoroacetic anhydride (TFAA) and 2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB) to derivatize the active hydrogens of GHB. The conversion of GBL into GHB can be forced under alkaline conditions by diluting the sample in 10 mM borate buffer, pH 12. GBL found in beverages intended for human consumption is treated as a Schedule I control substance analogue. Spikes of the two compounds into several beverage matrices gave quantitative recovery of GHB by GC/MS. The derivatization produces higher molecular mass products whose fragmentation pattern provides multiple peaks for confirmation and quantification. The concentration of GBL can also be indirectly determined by the method developed. Therefore, this extractionless technique is rapid, sensitive, and selective for the confirmation of the presence of GHB and GBL in commercial beverages.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Derivatizing Reagents Based on Ferrocene for HPLC-Ecd Determination of Peptides and Proteins

Abstract

Several ferrocenic compounds for derivatization of peptides and proteins were synthesized and then tested by reaction with bovine serum albumin (BSA). The reactivity of the reagents and the electroactivity of the derivatized BSA were estimated by the height of the ECD-signal after an HPLC analysis run. The most suitable reagent was 3-ferrocenylpropionic anhydride which reacts with BSA within 15 minutes at room temperature. The anhydride presented itself as a stable compound which can be synthesized in high yields. Up to pH 9–10 it is only slowly hydrolyzed, and its derivatization products are highly electroactive. Another reagent is especially suited to derivatize those proteins whose isoelectric points are higher than 10: Ferrocenylmethyl-succinimidyl-glycine-hydrochloride. This compound develops its derivatization activity only with pH values higher than 9, but it is rather difficult to prepare.     

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded ¹H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The ¹H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same ¹H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The ¹H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in ¹H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by ¹H qNMR was not feasible.     

Rapid determination of bisphenol A in drinking water using dispersive liquid‐phase microextraction with in situ derivatization prior to GC‐MS

This paper described a novel approach for the determination of bisphenol A by dispersive liquid‐phase microextraction with in situ acetylation prior to GC‐MS. In this derivatization/extraction method, 500 μL acetone (disperser solvent) containing 30.0 μL chlorobenzene (extraction solvent) and 30.0 μL acetic anhydride (derivatization reagent) was rapidly injected into 5.00 mL aqueous sample containing bisphenol A and K₂CO₃ (0.5% w/v). Within a few seconds the analyte was derivatized and extracted at the same time. After centrifugation, 1.0 μL of sedimented phase containing enriched analyte was determined by GC‐MS. Some important parameters, such as type and volume of extraction and disperser solvent, volume of acetic anhydride, derivatization and extraction time, amount of K₂CO₃, and salt addition were studied and optimized. Under the optimum conditions, the LOD and the LOQ were 0.01, 0.1 μg/L, respectively. The experimental results indicated that there was linearity over the range 0.1–50 μg/L with coefficient of correlation 0.9997, and good reproducibility with RSD 3.8% (n = 5). The proposed method has been applied for the analysis of drinking water samples, and satisfactory results were achieved.     

Nitric oxide-releasing/generating polymers for the development of implantable chemical sensors with enhanced biocompatibility

This paper reports a derivatization, mass fragmentation study relating to the most common, non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, ketoprofen and diclofenac, identified and quantified in the aquatic environment. Derivatizations have been performed with four silylation reagents in order to select the most proper one, taking into account analytical and financial points of view, equally. The tested reagents were N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) and for this purpose at the first time, hexamethyldisilazan (HMDS)+trifluoroacetic acid (TFAA). Varying derivatization time and temperature, taking into consideration chemical and financial advantages, HMDS+TFAA proved to be the optimum selection. Responses of derivatives have been compared, as a function of the ionization technique (external/internal ionization), as well as on the treatment of compounds' selective fragment ions (SFIs): (i) extracting the corresponding, characteristic m/z masses from TIC elutions and (ii) from SIM elutions, in parallel. Reproducibilities of measurements, expressed in relative standard deviation percentages (R.S.D.%), including the nanogram and the low picogram levels of injected derivatives, provided an average between 0.93 R.S.D.% and 4.11 R.S.D.%. NSAIDs' enrichment was performed with solid-phase extraction (SPE), applying the Oasis HLB (Waters) cartridges: recoveries in the 1-6 microg L(-1) range varied between 84% and 111%, with an average reproducibility of 6.4 R.S.D.%. The utility of the optimized derivatization method is presented, on monthly basis, by the identification and quantitation of the ibuprofen, naproxen, ketoprofen and diclofenac content of the influent and effluent waste-water samples obtained from a Hungarian waste-water treatment plant.     

蛋白质荧光标记中协同效应的高效液相色谱

以牛血清白蛋白和胰岛素为研究对象, 经1,5-I-AEDANS衍生后, 采用高效液相色谱(HPLC)进行分离分析, 发现两者的衍生存在明显的协同作用.     

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.