Anhydride In Ethyl Acetate Research Articles

Synthesis, SC XRD Based Structure Elucidation, Supramolecular Assembly Exploration Via Hirshfeld Surface Analysis, Computational and QTAIM Study of Functionalized Anilide

The anilide compound named (Z)-4-(2-methoxy-4-nitrophenyl)amino)-4-oxobut-2-enoic acid (MAOA) has been synthesized by the chemical reaction of 2-methoxy-4-nitroaniline and maleic anhydride in ethyl acetate. The synthesized compound was characterized by elemental analysis, FT-IR and UV-Vis spectroscopy, and TGA/DSC technique. Furthermore, the crystal structure was analyzed by the single crystal X-ray diffraction (SC XRD) technique. The supramolecular assembly of MAOA in terms of non-covalent interactions was explored by Hirshfeld surface analysis. Void analysis inferred that MAOA is expected to have good mechanical properties. The crystal packing environment was further investigated by interaction energy between molecular pairs and energy frameworks. Moreover, the result of the gas-phase DFT study showed that there is an intramolecular N–H···O and O–H···O hydrogen bond in MAOA due to the distance between D and A being smaller than the sum of their van der Waals radii. The result of the QTAIM study showed that there should also be an intramolecular CH···O hydrogen bond with a strength of 3.40 kcal/mol in MAOA.

GC-MS Studies on the Conversion and Derivatization of γ-Glutamyl Peptides to Pyroglutamate (5-Oxo-Proline) Methyl Ester Pentafluoropropione Amide Derivatives.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, γ-Glu-Cys-Gly) is the most abundant intra-cellular dicarboxylic tripeptide with multiple physiological roles. In biological samples, glutathione exists in its reduced form GSH and in two stable oxidized forms, i.e., in its symmetric disulfide form GSSG and as S-glutathionyl residue in proteins. S-Glutathionylation is a post-translational modification, which is involved in several pathophysiological processes, including oxidative stress. The GSH-to-GSSG molar ratio is widely used as a measure of oxidative stress. γ-Glutamyl is the most characteristic structural moiety of GSH. We performed gas chromatography-mass spectrometry (GC-MS) studies for the development of a highly specific qualitative and quantitative method for γ-glutamyl peptides. We discovered intra-molecular conversion of GSH, GSSG, γ-Glu-Cys and of ophthalmic acid (OPH; γ-glutamyl-α-amino-n-butyryl-glycine) to pyroglutamate (pGlu; 5-oxo-proline, also known as pidolic acid) during their derivatization with 2 M HCl/CH3OH (60 min, 80 °C). For GC-MS analysis, the methyl esters (Me) were further derivatized with pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v; 30 min, 65 °C) to their PFP derivatives. At longer reaction times, pGlu is hydrolyzed to Glu. Internal standards were prepared by derivatizing GSH, GSSG, γ-Glu-Cys and OPH in 2 M HCl/CD3OD. Quantification of the Me-PFP derivative of pGlu was performed in the electron-capture negative-ion chemical ionization (ECNICI) mode by selected-ion monitoring (SIM) of the mass-to-charge (m/z) ions 269 for unlabeled pGlu (d0Me-PFP-pGlu) and m/z 272 for the in situ prepared deuterium-labeled pGlu (d3Me-PFP-pGlu). Although not inherent to the analysis of small peptides, the present GC-MS method is useful to study several biochemical aspects of GSH. Using pentafluorobenzyl bromide (PFB-Br) as the derivatization reagent, we found that synthetic pGlu is converted in aqueous acetone (60 min, 50 °C) into its pentafluorobenzyl (PFB) ester (PFB-pGlu). This derivatization procedure is useful for the GC-MS analysis of free pGlu in the ECNICI mode. Quantitative analysis of PFB-pGlu by GC-MS requires the use of stable-isotope labeled analogs of pGlu as an internal standard.

Stable-Isotope Dilution GC–MS Measurement of Metformin in Human Serum and Urine after Derivatization with Pentafluoropropionic Anhydride and Its Application in Becker Muscular Dystrophy Patients Administered with Metformin, l-Citrulline, or Their Combination

Metformin (N,N-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography–mass spectrometry (GC–MS) method for the quantitative analysis of metformin (metformin-d0) in 10-µL aliquots of human serum and urine using N,N-[dimethylo-2H6]guanylguanidine (metformin-d6) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 °C), and extraction into toluene. The negative-ion chemical ionization GC–MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge (m/z) ratios of m/z 383 for metformin-d0 and m/z 389 for metformin-d6. Our results suggest that all amine/imine groups of metformin-d0 and metformin-d6 are converted to their N,N,N-tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of m/z 383 and m/z 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 × 500 mg/d; metformin group; n = 10) or l-citrulline (3 × 1500 mg/d; citrulline group; n = 9) followed by a six-week supplementation with 3 × 500 mg/d of metformin plus 3 × 1500 mg/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC–MS is suitable to measure reliably circulating and excretory metformin in clinical settings.

Specific and sensitive GC–MS analysis of hypusine, Nε-(4-amino-2-hydroxybutyl)lysine, a biomarker of hypusinated eukaryotic initiation factor eIF5A, and its application to the bi-ethnic ASOS study

Hypusination is a unique two-step enzymatic post-translational modification of the Nε-amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography–mass spectrometry (GC–MS) method for the measurement of biological hypusine (Hyp), i.e., Nε-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH3OH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CD3OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d0Me) and the deuterium-labelled methyl esters (d3Me) derivatives: d0Me-Hyp-(PFP)5 and d3Me-Hyp-(PFP)5, respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for d0Me-Hyp-(PFP)5 and m/z 814 for the internal standard d3Me-Hyp-(PFP)5. Selected-ion monitoring of m/z 811 and m/z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1–5 µM Hyp was 91–94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black (n = 38, age 7.8 ± 0.7 years) and white (n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68–5.31] µM (range 0.54–9.84 µM) in the black boys and 3.87 [2.95–5.06] µM (range 1.0–11.7 µM) in the white boys (P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20–0.29] µmol/mmol (range 0.11–0.36 µmol/mmol) in the black boys and 0.26 [0.21–0.30] µmol/mmol (range 0.10–0.45 µmol/mmol) in the white boys (P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.

Development, validation of a GC–MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine

A gas chromatography-mass spectrometry (GC–MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of l-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC–MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50–1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC–MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC–MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Measurement of the tripeptides glutathione and ophthalmic acid by gas chromatography-mass spectrometry

Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is the most abundant intracellular dicarboxylic tripeptide with multiple physiological roles. GSH and its symmetric disulfide GSSG co-exist in biological samples. The GSH-to-GSSG molar ratio is high within cells and low in extracellular compartments. It is widely used as a biomarker of oxidative stress. Here, we report on a novel two-step derivatization procedure which allows for the specific quantitative measurement of GSH by gas chromatography-mass spectrometry (GC-MS) in the electron-capture negative-ion chemical ionization mode. The method is based on the derivatization of GSH first with pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C) and subsequently with 2 M HCl/CH3OH (30 min, 80 °C). Esterification in 2 M HCl/CD3OD is used for the in situ synthesis of deuterium-labelled GSH for use as internal standard. Quantification is performed by selected-ion monitoring of m/z 557 for GSH and m/z 560 for the internal standard (1 mM). Linear regression analysis between the peak area ratio of m/z 557 to m/z 560 (y) and the GSH concentration (x; range, 0–1 mM) resulted in the regression equations y = 0.02 + 0.62x (r2 = 0.976) in the absence and y = 0.02 + 0.63x (r2 = 0.997) in the presence of GSSG (range, 0–1 mM), underlying the linearity of the method for GSH and suggesting lack of interference by GSSG. γ-Glu-Cys and Cys-Gly also did not interfere. The GC-MS method demonstrated that in human hemolysate, nitrite at toxicological concentrations (range, 0–15 mM) depletes erythrocytic GSH. For the GC-MS analysis of the GSH tripeptide analogue ophthalmic acid, which lacks a Cys moiety, the derivatization order was HCl/CH3OH followed by pentafluoropropionic anhydride in ethyl acetate.

An Efficient Oxidation of Sulfides to Sulfones with Urea–Hydrogen Peroxide in the Presence of Phthalic Anhydride in Ethyl Acetate

A metal-free, environmentally benign oxidation of substituted sulfides directly to their corresponding sulfones is described. Using urea-hydrogen peroxide and phthalic anhydride in ethyl acetate clean conversion into the sulfone was achieved without observation of the possible sulfoxide oxidation product.

N-(2-Chloro-5-cyanophenyl)-4,4,4-trifluorobutanamide

N-(2-Chloro-5-cyanophenyl)-4,4,4-trifluorobutanamide (3) was synthesized by reacting 4,4,4-trifluorobutanoic acid (1) with 2-amino-4-chlorobenzonitrile (2) in the presence of triethylamine and propylphosphonic anhydride in ethyl acetate. Character­ization of the compound was done by IR, 1H-NMR, 13C-NMR, LC-MS and CHN analysis.

Segmental hair analysis and estimation of methamphetamine use pattern

The aim of this study was to investigate whether the results of segmental hair analysis can be used to estimate patterns of methamphetamine (MA) use. Segmental hair analysis for MA and amphetamine (AP) was performed. Hair was cut into the hair root, consecutive 1cm length segments and 1-4cm length segments. Whole hair was also analyzed. The hair samples were incubated for 20h in 1mL methanol containing 1% hydrochloric acid after washing the hair samples. Hair extracts were evaporated and derivatization was performed using trifluoroacetic anhydride in ethylacetate at 65°C for 30min. Derivatized extract was analyzed by gas chromatography/mass spectrometry. The 15 subjects consisted of 13 males and two females and their ages ranged from 25 to 42 (mean, 32). MA and AP concentrations in the whole hair ranged from 3.00 to 105.10ng/mg (mean, 34.53) and from 0.05 to 4.76ng/mg (mean, 2.42), respectively. Based on the analysis of the 1cm length segmental hair, the results were interpreted in a way to distinguish between continuous use of MA (n = 10), no recent but previous use of MA (n = 3), and recent but no previous use of MA (n = 2). Furthermore, the individuals were interpreted as light, moderate, and heavy users based on concentration ranges previously published.

Solubility of 3-Chlorophthalic Anhydride and 4-Chlorophthalic Anhydride in Organic Solvents and Solubility of 3-Chlorophthalic Acid and 4-Chlorophthalic Acid in Water from (283.15 to 333.15) K

The solubility of 3-chlorophthalic anhydride and 4-chlorophthalic anhydride in ethyl acetate, acetone, and 1,4-dioxane and the solubility of 3-chlorophthalic acid and 4-chlorophthalic acid in water were measured using Schreinemaker’s wet residue method at temperatures ranging from (283.15 to 333.15) K at atmospheric pressure. Results of these measurements were correlated with the modified Apelblat equation.

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography–tandem mass spectrometry as methyl ester tri( N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; N G, N G-dimethyl- l-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic–tandem mass spectrometric (GC–tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [ 2 H 3 ]-methyl ester ADMA (d 3Me-ADMA) as the internal standard. Aliquots (100 μl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 μM for plasma and serum; 20 μM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 μl aliquot of 2 M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 μl aliquot of 2 M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 °C. After cooling to room temperature, the plasma or urine sample is combined with the d 3Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 °C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 μl aliquot of 0.4 M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 μl aliquot is injected into the GC–tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/ z 378 which is produced by collision-induced dissociation of the ions at m/ z 634 for endogenous ADMA and m/ z 637 for d 3Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39±0.06 μM ( n=12) and 3.4±1.1 μmol/mmol creatinine ( n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d 3Me-ADMA, respectively.

Demonstration of oxidation dyes on human hair

This paper describes a method of selected ion monitoring (SIM) analysis which can demonstrate the staining of human hair with oxidation hair dyes. Hair samples were decomposed with NaOHNa 2S 2O 4 solution by heating (100°C, 30 min) in a stream of nitrogen. Basic and neutral ether extracts from the reaction mixture were trifluoroacetylated with trifluoroacetic anhydride in ethyl acetate and were then analyzed by SIM. The minimum lengths of a single hair for the detection of the 5 components of oxidation hair dyes acting as indicators were 1 mm for toluene-2,5-diamine, 2 mm for p-phenylenediamine, 20 mm for p-aminophenol, 50 mm for m-aminophenol and 100 mm for o-aminophenol. This method was applied to practical cases and the results were good.

Determination of low plasma concentrations of 3-methoxy-4-hydroxyphenylethylene glycol using gas chromatography—negative-ion chemical ionization mass spectrometry

Gas chromatography—negative-ion chemical ionization mass spectrometry (GC—NICI-MS) allowed the detection of extremely low plasma concentrations of 3-methoxy-4-hydroxy-phenylethylene glycol (MHPG). Glucuronide and sulphate conjugates of MHPG were determined after enzymatic hydrolysis of plasma with β-glucuronidase—arylsulphatase. A 1-ml plasma sample was extracted at the pH of the hydrolysis (pH 4.8) with ethyl acetate, and the dry extract was derivatized with pentafluoropropionic anhydride in ethyl acetate. After evaporation of the solvent, the residue was dissolved in benzene and an aliquot was analysed by GC—NICI-MS. A trideuterated analogue of MHPG was used as an internal standard. Negative-ion chemical ionization of the pentafluoropropionyl derivatives was carried out using ammonia. The ion—molecule adducts at m/e 766 and 785 (MHPG) and m/e 769 and 788 (internal standard) were formed from the pentafluoropropionyl derivatives with the ions of m/e 163 (CF 3,CF 2,COO −) and m/e 144 (loss of fluorine from m/e 163). The concentrations of the ions of m/e 163 and 144 play a major role in the sensitivity and precision of this technique, which allows the detection of free MHPG plasma concentrations as low as 100 pg/ml in routine analysis.

Determination of isoxsuprine in human plasma by gas chromatography with electron capture detection.

A rapid and sensitive gas chromatographic method for the determination of the beta-adrenergic agent isoxsuprine in human plasma has been developed. The procedure involves the extraction of the drug with ether and an internal standard (propranolol) from plasma at alkaline pH, solvent evaporation, and the formation of a tri-trifluoroacetyl derivative by reaction with trifluoroacetic anhydride in ethyl acetate. Analyses were carried out by gas-liquid chromatography on a 3% OV-17 column using an electron capture detector. The minimum detectable amount of isoxsuprine was 0.5 ng/ml of plasma and the electron capture detector response was tested to be linear (r2 greater than 0.999) between 0.5 and 20 ng/ml. No interferences from endogenous substances were found. Precision of the method was found to be 9.9, and 6.1% coefficient of variation at 1 ng/ml and 10 ng/ml of plasma, respectively. Determination of isoxsuprine at the nanogram level in cord plasma samples from newborns at the time of the delivery was possible using the described procedure.

Hydrogenation of substituted phthalic anhydrides over palladium

Catalytic hydrogenation of phthalic anhydride in ethyl acetate over 10% palladium–carbon gives o-toluic acid. Under similar conditions 3- and 4-substituted phthalic anhydrides give phthalides (8), and/or 2-(hydroxy-methyl)benzoic acids (7) in addition to o-toluic acids (9). Mechanisms of formation of these products are discussed.

Determination of neutral and amino sugars in glycoproteins by gas chromatography.

A gas chromatographic method for the determination of neutral and amino sugars in glycoproteins was developed by using trifluoroacetyl derivatives of sugar alcohols and by selecting trifluoroacetic acid as acid catalyst for hydrolysis. The method is as follows : As the hydrolytic condition, 2.5 N TFA, 100°, 7 hr was common to obtain excellent recovery of neutral and amino sugars in many glycoproteins. In the case of having enough sample, it was recommendable to select 4 and 7 hr for hydrolysis of neutral sugars and 7 and 10 hr for amino sugars. In sample preparation for gas chromatography, sugars were reduced with sodium borohydride to corresponding sugar alcohols and trifluoroacetylated with trifluoroacetic anhydride in ethyl acetate. This method was further improved for micro-analysis of sugars in glycoproteins, taking full advantage of the sensitivity of an electron capture detector (ECD) to trifluoroacetyl derivatives. Consequently, it was suggested to be able to analyze constituent neutral and amino sugars in microgram amounts of glycoproteins.

Gas Chromatographic Determination of 3, 4-Dihydroxyphenylalanine (DOPA) in Urine

Urinary 3, 4-dihydroxyphenylalanine (DOPA) was extracted with alumina after removal of the fatty substances by shaking with ether-benzene (5 : 2), converted to n-butyl ester with n-butanol-HCl (5N) at 100°for 20 min, trifluoroacetylated with trifluoroacetic anhydride in ethyl acetate and determined by gas chromatography using electron capture detector. The overall recoveries of the added DOPA (50, 75, 100, and 150 ng) from urine (2 ml) were 66.0% ±3.55% (n=8). The pressence of DOPA in normal human urine was confirmed by gas chromatographymass spectroscopy.