Direct Derivatization Research Articles

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.

Analysis of nonylphenol polyethoxycarboxylates and their related metabolites by on-line derivatization and ion-trap gas chromatography–mass spectrometry

This study presents a modified method to analyze nonylphenol polyethoxycarboxylates (NPEC) and their related metabolites (carboxyalkylphenol ethoxycarboxylates (CNPEC)) in water samples. The method involves extraction of samples by a graphitized carbon black (GCB) cartridge, and direct derivatization in the GC injection-port using a large-volume (10–20 μl) direct sample introduction (DSI) device with tetraalkylammonium (TAA) salts. The analytes are identified and quantitated by ion-trap GC–MS. The large-volume DSI injection-port derivatization technique provides sensitivity, fast and reproducible results for NPEC and their metabolites, to quantitation at 0.1 μg/l in 200 ml of water samples. The retention effect of TAA salts in the injection-port is not detected. In addition, the significant [M−29] + ions and molecular ions of butylated NPEC and CNPEC residues are observed. Recovery of NP1EC in spiked water samples ranges from 90 to 108%. Moreover, relative standard deviations of replicate analyses ranges from 1 to 9%. However, unsatisfactory on-line derivatization of CNPEC residues is observed. This finding maybe owing to their lesser dissociation with the ion-pair reagent in chloroform.

Analysis of linear alkylbenzenesulfonates in water samples by large-volume injection-port derivatization and gas chromatography–mass spectrometry

This work presents a modified method to analyze linear alkylbenzenesulfonates (LASs) in water samples. The method involves extraction of samples by a graphitized carbon black (GCB) cartridge, and direct derivatization in the GC injection port using a large-volume (10–20 μl) direct sample introduction (DSI) device with tetraalkylammonium (TAA) salts. The analytes were then identified and quantitated by ion-trap GC–MS. The large-volume DSI injection-port derivatization technique provides sensitivity, fast and reproducible results for LAS residues, to quantitation at 0.1 μg/l in 200 ml of water samples. The retention effect of TAA salts in the injection port was not detected. Enhanced selected mass chromatograms of [M−55] + ions of butylated C 10–C 13 LASs by electron impact ionization MS allows one to determine LAS residues at trace levels in environmental samples. Recovery of total LASs in spiked variety water samples ranged from 89 to 112% while RSDs ranged from 2 to 13%.

Simultaneous determination of sugars, sugar alcohols, acids and amino acids in apricots by gas chromatography–mass spectrometry

Our GC–MS method for the simultaneous quantitation of mono- di- and trisaccharides, sugar alcohols and acids, measured as their trimethylsilyl-oxime ether/ester derivatives, prepared in presence of the fruit matrix, from one solution by one injection has been extended. The reproducible determination of apricot constituents was performed both on the basis of total ion current (TIC) and selective fragment ion (SFI) values, ensuring identification and quantitation in a wide concentration range (∼1·10 −3 to ≥40%), calculated on dry matter basis of the samples). The GC–MS procedure was utilized to prove the advantage of the direct derivatization process, in the presence of the fruit matrix, and to determine the changes in the sugar/sugar alcohol/carboxylic acid/amino acid composition of two apricot cultivars, as a function of their harvesting dates and storage conditions. Reproducibility of quantitations, characterized by their RSD values, proved to be, 3.6% (TIC) and 4.3% (SFI).

Protein Sequencing by Matrix-Assisted Laser Desorption Ionization–Postsource Decay–Mass Spectrometry Analysis of theN-Tris(2,4,6-trimethoxyphenyl)phosphine-Acetylated Tryptic Digests

We have recently reported a simple procedure by which low picomole quantities of peptides can be modified to the correspondingN-Tris(2,4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) derivatives (Z. H Huang, J. Wu, D. A. Gage, and J. T. Watson,Anal. Chem.69, 137–144, 1997). This modification significantly facilitates sequence interpretation by providing exclusively N-terminal product ions (mainly a-type ions) in the fast-atom bombardment–MS/MS and matrix-assisted laser desorption ionization–postsource decay(MALDI–PSD)–MS spectra. The TMPP-Ac derivatization approach has been extended now for the direct derivatization of tryptic digests originating from 1–5 μg of proteins with molecular weights from 10-120 kDa. Our new procedure involves tryptic digestion in aqueous solution buffered to pH 8–8.2 with phosphate or Tris–HCl, followed by reaction with TMPP-acetic acidN-hydroxysuccinimide ester (TMPP-AcOSu bromide, 2–4 nmol reagent/μg protein, rt, 20 min) to provide N-terminally derivatized products, while the ϵ-NH2groups in lysine remain unchanged. The resultant derivatized peptide mixture or its partially separated HPLC fractions are subsequently analyzed by MALDI–PSD–MS using 0.5- to 1-pmol aliquots, giving rise to product ion spectra that are easily interpretable. As there is no need for material transfer and change of buffer media, the tandem enzymatic–chemical reaction/MS analysis process is usually carried out with very high throughput (digestion, 1 h; reaction, 1/3 h; HPLC, 1 h; MALDI–PSD, 3–4 fragments/h). This procedure will be of potential use for obtaining sequence information directly from mixtures or as an adjunct of peptide mass mapping to provide protein identification with high confidence.

Improved analytical methods for determination of nitrogenous stress metabolites occurring in Limonium species

Efficient and reliable high-performance liquid chromatographic procedures have been developed for metabolic analyses of amino acids, polyamines and betaines in Limonium species. The adaptive significance of accumulated low-molecular-mass nitrogenous compounds in dry or salt environments is under study. HPLC profiles of dansylated water-soluble polyamines revealed 1,3-diaminopropane and tyramine as the most abundant amines in the species under study whereas common aliphatic di- and polyamines (i.e., putrescine, spermidine and spermine) were poorly represented as their free forms. Nevertheless acylated conjugates of putrescine, 1,3-diaminopropane and spermidine were also characterized, especially in L. vulgare, a halophytic salt marshes species. Direct derivatization of amino acids of the crude aqueous extracts with 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate allowed efficient determinations of most proteinic amino acids as well as non-proteinic ones such as ornithine, γ-aminobutyric acid and β-alanine also related to polyamine metabolism. Analysis of betaines was improved, especially for β-alanine betaine, which is quite uncommon in higher plants whose metabolic routes from β-alanine are poorly understood. β-Alanine betaine was first converted to acrylic acid through trimethylamine β-elimination under alkaline treatment of the crude extracts and then quantified by HPLC. Thus Limonium species ranged from high β-alanine betaine accumulators, to low accumulators and finally to a third group where it was not detected, since β-alanine betaine was replaced by glycine betaine as shown here by 1H-NMR investigations. Metabolic links between these nitrogenous solutes and the adaptive significance of such adjustments are discussed. Special emphasis is directed towards the possible involvement of aliphatic polyamine oxidative catabolism, which seems to be effective in these species, to precursor recycling for compatible solute biosynthesis.

Determination of valproic acid in human serum by high-performance liquid chromatography with fluorescence detection.

A simple and highly sensitive high-performance liquid chromatographic method is described for the determination of valproic acid in human serum. The method is based on the direct derivatization of serum sample with 6,7-methylenedioxy-1-methyl-2-oxo-1,2-dihydroquinoxaline-3-ylpr opionohydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37 degrees C. The resulting derivatives were separated on a reversed-phase column (YMC Pack ODS-A) with isocratic elution and were fluorimetrically detected at 440 nm with excitation at 365 nm. The detection limit (signal-to-noise ratio=3) for valproic acid added to serum sample was 0.1 microg (700 fmol)/ml serum sample (2.3 fmol on column). The method was applied to determine the unbound- and total-valproic acid levels in the serum obtained from three healthy volunteers after oral administration of the drug (600 mg).

High Arenophilicity and Water Tolerance in Direct Derivatization of Peptides and Proteins by Metal π-Coordination

High Arenophilicity and Water Tolerance in Direct Derivatization of Peptides and Proteins by Metal π-Coordination

Folate-mediated targeting of antisense oligodeoxynucleotides to ovarian cancer cells.

Receptors for vitamin folic acid are frequently overexpressed on epithelial cancer cells, especially ovarian cancer cells. In this study, we examined whether this expression might be exploited to specifically deliver antisense oligodeoxynucleotides (ODN) to tumor cells. A conjugate was prepared by directly coupling folic acid to the 3' terminus of an anti-c-fos ODN and its cellular uptake and tumor inhibitory effect were evaluated using FD2008 cells that overexpress folate receptors. When a phosphorothioate (PS)/phosphodiester (PO) chimeric ODN was conjugated with folic acid, its uptake by FD2008 cells was increased by about 8-fold (P < 0.01). In contrast, conjugation of folate to the ODN did not increase its uptake by CHO cells that lack the expression of FBP (P > 0.05). Furthermore, the increase in the uptake of conjugated ODN by FD2008 cells could be blocked by adding an excess amount of folic acid. The PS/PO antisense ODN had some inhibitory effect on the growth of FD2008 cells. However, its activity was significantly increased following conjugation with folic acid (P < 0.01). ODN of scrambled sequences with and without conjugation with folic acid failed to inhibit the growth of FD2008 cells. Finally, the antisense effect of the conjugated ODN on FD2008 cells was inhibited by an excess amount of free folic acid, suggesting that the sequence-dependent effect of folate-antisense ODN conjugate was mediated by folate binding protein. Direct derivatization of ODN with folate significantly improves their targeting efficiency to tumor cells in vitro. The folate-conjugated ODN, due to their small size and possibly efficient extravasation at tumor site, has the potential for treating solid tumors that overexpress folate receptors.

A novel technique for direct derivatization of ionic organotin and alkyl-lead compounds in sediment: simultaneous determination of organotin and alkyl-lead compounds in sediment by gas chromatography-plasma atomic emission (GC-AED) dual-channel detection

A direct derivatization carried out on sediment slurries and simultaneous speciation of organotin and alkyl lead in sediments is described. Simultaneous determination was performed using dual-channel operation of the AED detector. Direct contact of phentylmagnesium bromide with the sediment improved drastically the recovery of the monobutyltin (MBT) species. The combined complexation/extraction/derivatization procedure and the dual-channel AED operation reduced the analysis time. Analysis of a Certified Reference sediment, PACS-1, demonstrated the accuracy of the method. The absolute detection limits (3×noise level) are 0.5 pg and 0.2 pg respectively for tin and lead. The relative detection limits for sediment (1 g sample) are 2.5 ng g−1 and 1 ng g−1 respectively for organotin and alkyl-lead, expressed as the metal. The results of the analysis of some environmental samples are given. © 1997 John Wiley & Sons, Ltd.

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method for the direct and simultaneous determination of estriol 3- and 16-glucuronides in pregnancy urine is described. The method is based on direct derivatization of the glucuronic acid moiety in estriol glucuronides in urine with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or urine sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 37°C. The resulting fluorescent derivatives were separated by column-switching chromatography using a first column (YMC-Pack C 4) for clean-up of the derivatives and a second column (YMC Pack Ph) for the complete separation of the derivatives. The derivatives were detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise ratio=3) for estriol 3- and 16-glucuronides were 150 and 180 fmol in a 5 μl of urine (14 and 17 ng ml −1 urine), respectively. The present method is highly sensitive and simple without any clean-up such as conventional solid-phase extraction.

Analysis of ifosforamide mustard, the active metabolite of ifosfamide, in plasma.

Ifosforamide mustard is the active metabolite of ifosfamide, a cytostatic drug. In this study a sensitive and selective method for the analysis of ifosforamide mustard in plasma is described. The method consists of direct derivatisation of ifosforamide mustard in plasma with diethyldithiocarbamate and subsequent solid-phase extraction of the resulting derivative. The analysis of the derivatisation product was performed by high-performance liquid chromatography with UV detection. The calibration graph was linear in the concentration range 0.45-45 microM and the minimum detectable concentration was 0.45 mumol. The samples were stabilised by addition of semicarbazide and sodium chloride. A patient's plasma sample was analysed by means of the described method. The ifosforamide mustard concentration was 2.3 microM.

High-performance liquid chromatographic evaluation of biogenic amines in foods an analysis of different methods of sample preparation in relation to food characteristics

Biogenic amines are compounds formed by amino acid decarboxylation in fermented foods. Most of the methods for amine determination involve acid extraction followed by a liquid-liquid purification step. The different parameters which can influence amine recoveries are considered; experience with different foods such as cheese, fish and meat preserves are reported and for each of them the optimized analytical procedure is described. Data concerning recovery and repeatability of the method are also reported and the various factors that influence amine extraction are discussed. The possibility of applying direct derivatization without any other purification step is also considered.

Determination of carboxylic acid salts in pharmaceuticals by high-performance liquid chromatography after pre-column fluorogenic labelling.

2-Bromoacetyl-6-methoxynaphthalene (Br-AMN) was used as a fluorogenic labelling reagent for high-performance liquid chromatographic (HPLC) analyses of pyroglutamic acid, 4-hydroxybutyric acid and thioctic acid in pharmaceutical formulations. The reaction was carried out in aqueous medium in the presence of a cationic surfactant to obtain the direct derivatization of the salts of 4-hydroxybutyric acid and thioctic acid or in acetonitrile for pyroglutamic acid. The resulting naphthacylesters were then chromatographed under reversed-phase (C-18) conditions and detected fluorimetrically (lambda exc, 300 nm; lambda em, 460 nm). The method proved to be suitable for the sensitive and selective analysis of commercial formulations of the cited acidic drugs.

Determination of mono-, poly- and hydroxy-carboxylic acid profiles of beverages as their 2-nitrophenylhydrazides by reversed-phase ion-pair chromatography

A simple, rapid and accurate method for the derivatization and quantitative reversed-phase ion-pair chromatographic analysis of mono-, poly- and hydroxy-carboxylic acids in wines, fruit juices, beer and Japanese “sake” is presented. Nine hydrazine derivatives were prepared in high yield by direct derivatization of the carboxylic acids with 2-nitrophenyl-hydrazine hydrochloride in sample matrix. Isocratic separation of the carboxylic acid hydrazides was performed within 18 min by a suitable combination of pH, the polarity of eluent and the size of the counter ion. The analytical results showed good recovery and reproducibility using 3-methylglutaric acid as an internal standard. Due to its superior selectivity and sensitivity, the present method can serve as a useful tool for routine analysis of carboxylic acids in foods and beverages.

Molecular heterogeneity of PAF in normal human mixed saliva: quantitative mass spectral analysis after direct derivatization of PAF with pentafluorobenzoic anhydride

Platelet-activating factor (PAF), a family of phospholipid autacoids with potent pro-inflammatory activities, is present in saliva. The current study has quantitated various species of PAF isolated from normal human mixed saliva. Choline-containing, sn-2 acetylated phospholipids with sn-1 ether- or ester-linked fatty alcohol/acid moieties (alkyl-PAF or acyl-PAF, respectively) were evaluated after direct defvatization with pentafluorobenzoic (PFB) anhydride. Individual species of PFB-derivatized PAF were separated by gas chromatography prior to mass spectral analysis; quantitative estimates of six different species of PAF in saliva were made by comparison to corresponding authentic, synthetic PAF standards. In each saliva sample, all six species of PAF were readily detected by this facile procedure. The predominant PAF was 1- O-hexadecyl-2-acetyl- sn-glycero-3-phosphocholine or 16:0-alkyl-PAF (0.75 ± 0.09 pmol/ml saliva; mean ± S.E.; n = 5) which represented only 30.4 ± 1.5% of the total PAR Substantial amounts of 18:1- and 18:0-alkyl-PAF and 16:0-acyl-PAF were also identified (0.52 ± 0.07, 0.35 ± 0.06, and 0.35 ± 0.02 pmol/ml saliva, respectively). In summary, mass spectrometric analysis of PAF after direct derivatization with PFB anhydride has revealed that at least six different species of PAF are present in normal human mixed saliva. This structural diversity may represent an important aspect of homeostasis in the healthy oral cavity.

Determination of hydroxycarbamates in aqueous matrices by direct derivatization and GC‐MS analysis in chemical ionization mode

AbstractA rapid and effective procedure, developed for the determination of polar compounds in water, has been applied to hydroxycarbamates. In few minutes, it is possible to perform a catalyzed derivatization of such compounds directly in the aqueous medium, using n‐hexyl chloroformate. Extraction with n‐hexane and injection into a benchtop GC‐MS system are the only two further steps to complete the analysis. Chemical ionization proved to be more effective than electron impact to produce valuable mass spectra in terms of selectivity and sensitivity. Linear quantitative responses were demonstrated over a two‐order‐of‐magnitude range. The minimum detectable concentration was 3 ppb for acetohydroxamic acid and 10 ppb for hydroxyurethane, obtained from Po river water samples spiked with the two hydroxycarbamate standards.

Assay of the concentration and stable isotope enrichment of short‐chain fatty acids by gas chromatography/mass spectrometry

AbstractIsotope dilution gas chromatographic mass spectrometric assays were developed for the concentration of shortchain fatty acids and of lactate in plasma. The assays involve spiking the samples with 2H‐ and/or 13‐C‐labeled internal standards and direct derivatization with 2,4‐difluoroaniline, using 1,3‐dicyclohexylcarbodiimide as a coupling agent. The assay can be used to determine the isotopic enrichment or mass isotopomer distribution of the fatty acids and of lactate. Blanks by ubiquitous formate, acetate and lactate are minimized by the one‐step derivatization procedure without deproteinization, using reagents dissolved in hydrocarbons. The assays were applied to experiments conducted in live dogs, pigs and monkeys, and in perfused rat livers. These techniques, in conjunction with appropriate stable isotope models, can be used to study carbohydrate fermentation in humans, ruminants and non‐ruminant mammals.

Simple and highly sensitive high-performance liquid chromatographic method for separating enantiomeric diacylglycerols by direct derivatization with a fluorescent chiral agent, ( S)-(+)-2- tert.-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid

A simple and highly sensitive HPLC method was developed for the determination of the absolute configuration and the optical purity of diacylglycerols. The method involves direct fluorescent labelling of diacylglycerols with ( S)-TBMB-carbonyl chloride in pyridine and HPLC separation of the derived diastereomeric ( S)-TBMB-carboxylated diacylglycerol derivatives [( S)-TBMB = ( S)-(+)-2- tert.-butyl-2-ethyl-1,3-benzodioxole]. Complete separation of the diastereomeric ( S)-TBMB-carbonyl-diacylglycerol derivatives and thus indirectly of the enantiomers of the parent diacylglycerols and of the sn-1,3-regioisomer was achieved using normal-phase silica gel HPLC within 30 min for every saturated single acid (R 1 = R 2) diacylglycerol (C 12:0–C 22:0) examined.

Fluorescein derivatization of fibrinogen for flow cytometric analysis of fibrinogen binding to platelets.

Dog and human fibrinogen were derivatized with N-hydroxysuccinimido-fluorescein and utilized for flow cytometric estimation of fibrinogen binding to activated platelets. Fluorescein-fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was saturable, dependent on agonist activation, and inhibited by unlabeled fibrinogen. In addition, EDTA and barbourin, a KGD-containing peptide, were found to inhibit the binding of fluorescein-fibrinogen. Fluorescein-fibrinogen bound to dog platelets with an apparent affinity of 0.31 microM after stimulation with either adenosine-5'-diphosphate (ADP) or plateletactivating factor. The labeled fibrinogen was also used to study the fibrinogen binding capacity of aged, biotinylated platelets. Aged platelets were indistinguishable from young platelets with regard to fibrinogen binding in response to ADP. These studies document that direct derivatization of fibrinogen with fluorescein generates a useful probe for analyzing fibrinogen binding to platelets with flow cytometry.