Development of hydrophilic fluorogenic derivatization reagents for thiols: 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

Sensitive, reactive, and hydrophilic fluorogenic reagents for thiols with the benzofurazan skeleton, 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (AcABD-F) and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (TCAcABD-F) have been developed. These reagents reacted with thiols within 10 min at 60 degrees C. AcABD-F and TCAcABD-F themselves do not fluoresce but are strongly fluorescent after the reaction with thiol compounds. The generated derivatives were highly water-soluble, since they dissociated a proton and ionized in the neutral pH region. The derivatives with four biologically important thiol compounds were separated on a reversed-phase HPLC column and detected fluorometrically at 504 nm with excitation at 388 nm. The detection limit attained for homocysteine with AcABD-F was 25 fmol on column (11 nM) (signal-to-noise ratio = 3), and that for glutathione with TCAcABD-F was 45 fmol on column (20 nM).     

Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C and pH 8.0 and the reaction rates were several times higher than those with ABD-F; it is suggested that the electron withdrawing effect of the dimethylsulphonamide group (SO2NMe2) is larger than that of the sulphonamide group (SO2NH2). No reaction occurred with alanine, proline, cystine or cysteic acid under the same conditions. The fluorescence intensities of the derivatives were found to be higher in neutral and acidic media than in alkaline solutions. The thiol derivatives of DBD-F were separated by high-performance liquid chromatography and detected fluorimetrically, the detection limits being 0.92, 0.16, 0.13, 0.16 and 0.32 pmol for cysteine, glutathione, homocysteine, N-acetylcysteine and alpha-mercaptopropionylglycine, respectively. The method was applied to the determination of thiols in rat tissues.     

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.     

Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.     

LC Determination of Thiols Derivatized with 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole after SPE

Solid-phase extraction (SPE) coupled with high-performance liquid chromatography?Cfluorescence detection (LC?CFL) was developed for the determination of three thiol compounds including glutathione, cysteine and acetylcysteine. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was used for derivatization of thiols. Factors affecting derivatization and extraction efficiency were optimized. Sample solution (2?mL) was extracted on a SPE column for 2?min and then eluted with 400???L methanol. The analytes were injected onto the LC system for separation on a C₁₈ column, and eluted with methanol?Cacetate buffer. The analytes were detected by fluorescence at an emission wavelength of 515?nm with excitation at 385?nm. The linearity of the method was in the range of 0.1?C60???M, with correlation coefficients ranging from 0.9979 to 0.9990. The detection limits of the method were in the range of 5?C20?nM. The proposed method was applied to the analysis of human plasma samples with recoveries of 86?C112.9%.     

4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as chemilumigenic reagent for high performance liquid chromatographic peroxyoxalate chemiluminescence detection

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), presented as a fluorogenic labelling reagent for amines and amino acids, is preferred for peroxyoxalate chemiluminescence (PO-CL) detection in high performance liquid chromatography. Amino acids and epinephrine derivatized with DBD-F were separated on a reversed phase column and detected at the femtomole level by the PO-CL detection system.     

Use of 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the beta-blocker metoprolol in serum by high-performance liquid chromatography.

Fluorogenic reagents having a benzofurazan moiety, viz., 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 7-fluoro-4-nitro-2,1,3-benzoxadiazole and 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole, were compared for the sensitive analysis of their derivatives by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection. Of the proline derivatives, DBD-proline was the most sensitive with a detection limit of 2 fmol. The optimum concentrations of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and H2O2 for the post-column reaction were 0.5 and 75 mmol dm-3 respectively and amino acids and beta-blockers derivatized with DBD-F were detected in the range 0.2-40 fmol (signal-to-noise ratio = 3) using the proposed method. The lower detection limit of metoprolol (a beta-blocker having an isopropylamino group) spiked in serum was 0.8 ng ml-1 using 20 microl of serum (signal-to-noise ratio = 5).     

High-performance liquid chromatography/chemiluminescence determination of biological thiols with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide

The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH group. The labelling reaction was carried out at 60 degrees C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODS-80 column (150 x 4.6 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23 +/- 0.15 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.     

Existence of low-molecular-weight thiols in Caenorhabditis elegans demonstrated by HPLC-fluorescene detection utilizing 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.     

Pt–NiCo Nanostructures with Facilitated Electrocatalytic Activities for Sensitive Determination of Intracellular Thiols with Long‐Term Stability

A Pt–NiCo nanomaterial has been synthesized for developing the sensitive electrochemical determination of biological thiols that include L ‐cysteine (CySH), homocysteine (HCySH), and gluthione (GSH) with high sensitivity and long‐term stability, in which the Pt nanoparticles are well supported on amorphous NiCo nanofilms. The electrochemical oxidation of thiols has been successfully facilitated on the optimized Pt–NiCo nanostructures, that is, two oxidation peaks of CySH have been clearly observed at potentials of +0.06 and +0.45 V. The experimental results demonstrate that the first peak for CySH oxidation may be attributed to a direct oxidation from CySH to L ‐cystine (CySSCy), whereas the second peak possibly results from a sequential oxidation from CySSCy to cysteic acid (CySO₃H), together with a direct oxidation of CySH into CySO₃H. The enhanced electrocatalytic activities at the Pt₂₃–NiCo nanostructures have provided a methodology to determine thiols at a very low potential of 0.0 V with relatively high sensitivity (637 nA μM cm^?2), a low detection limit (20 nM ), and a broad linear range. The striking analytical performance, together with the characteristic properties of the Pt–NiCo nanomaterial itself, including long‐term stability and strong antipoisoning ability, has established a reliable and durable approach for the detection of thiols in liver cancer cells, Hep G2.     

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.     

Synthesis of 4(6)-aryloxy- and 4-arylthio-2,1,3-benzoxadiazoles

Reactions of 4,6-dibromo-2,1,3-benzoxadiazole with phenols or arenelthiols lead to 4-aryloxy-6-bromo-2,1,3-benzoxadiazoles or 4-arylthio-6-bromo-2,1,3-benzoxadiazoles, respectively. Isomeric 6-aryloxy-4-bromo-2,1,3-benzoxadiazoles were synthesized by replacement of the fluorine atom in 2,6-dibromo-4-fluoro-1-nitrosobenzene with phenols and treatment of the resulting 4-aryloxy-2,6-dibromo-1-nitrosobenzene with sodium azide.     

High performance liquid chromatography with chemiluminescence detection of methamphetamine and its related compounds using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole.

A high performance liquid chromatographic assay of methamphetamine (MP) and its related compounds, i.e. ephedrine (EP), norephedrine (NE), p-hydroxymethamphetamine (p-HMP), p-hydroxyamphetamine (p-HAP) and amphetamine (AP), with peroxyoxalate chemiluminescence detection has been developed. 4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was used as a fluorescent labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate in acetonitrile was used as a postcolumn chemiluminogenic reagent. DBD derivatives of MP and its related compounds were separated by a gradient elution with acetonitrile and 0.01 M imidazole buffer (pH 7.0) within 65 min. The detection limits (S/N = 3) for the proposed method for MP, AP, EP, NE, p-HMP and p-HAP were 27, 100, 40, 133, 25 and 133 fmol on column, respectively. The recoveries of these compounds with normal urine samples were 87.4-106.4%. The method was successfully applied to the assay of MP and its metabolites in urine samples from MP addicts. A good linear correlation for the resulted amounts of MP or AP between the proposed method and gas chromatography was obtained (r = 0.993 for MP or 0.991 for AP).     

Sensitivity of Positive Ion Mode Electrospray Ionization Mass Spectrometry in the Analysis of Thiol Metabolites

High signal intensities of glutathione (GSH), [GSH+H]⁺ (m/z 308), cysteine (CySH), [CySH+H]⁺ (m/z 122), and homocysteine (hCySH), [hCySH+H]⁺ (m/z 136), are observed in ESI MS with on‐line electrochemistry (EC). Dimers formed by H‐bonding, which are not electrochemical products, are detected as [2GSH+H]⁺ (m/z 615), [2CySH+H]⁺ (m/z 243) and [2hCySH+H]⁺ (m/z 271) together with disulfide dimers GSSG, CySSCy and hCySSCyh, [GSSG+H]⁺ (m/z 613), [CySSCy+H]⁺ (m/z 241) and [hCySSCyh+H]⁺ (m/z 269). When dopamine is present a thiol/dopamine quinone (DAQ) adduct is observed. Formation of this adduct is proposed to occur by an electrochemical mechanism during ESI. Catalysis of thiol oxidation and analysis of thiol mixtures is addressed.     

Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.     

Chiral separation of homocysteine by derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole followed by capillary electrophoresis using gamma-cyclodextrin

Homocysteine was derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to form an inclusion complex with cyclodextrin and to facilitate UV detection. ABD-homocysteine showed interaction with beta- and gamma-cyclodextrin in capillary electrophoresis at pH 2.25 as indicated by the decreased migration time. However, chiral separation of D,L-ABD-homocysteine was observed using gamma-CD only. Optimal separation was obtained at pH 2.25, 50 mM gamma-CD concentration, and 20 kV applied voltage. L-ABD-Homocysteine migrated faster than the D-isomer as demonstrated by a spiking experiment using dithiothreitol-reduced L-homocystine.     

Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection

The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H₃PO₄/CH₃CN (96:4) to 0.15 M H₃PO₄/CH₃CN (85:15) over 30 min followed by isocratic elution with 0.15 M H₃PO₄/CH₃CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g^-1) and cystine (0.62 μmol g^-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g^-1) was observed in liver, spleen, heart and testicle.     

Synthesis and application of 4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxadiazole (MDBDH) as a new derivatizing agent for aldehydes

In a five step synthesis, 4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxadiazole (MDBDH) was prepared in high yields as a stable new derivatizing agent for carbonyl compounds. Reagent and derivatives have not been described in literature before. Major advantage of this substance compared with similar reagents is its improved solubility in polar solvents, e.g. methanol and ethanol. MDBDH reacts with aldehydes in the presence of an acidic catalyst under formation of the corresponding hydrazones. These are separated by means of reversed-phase liquid chromatography and detected UV/vis spectroscopically at wavelengths around 450 nm, depending on the individual hydrazone. MDBDH reacts with oxidizers as nitrogen dioxide and nitrite to only one product, 4-(N,N-dimethylaminosulfonyl)-7-methylamino-2,1,3-benzoxadiazole (MDBDA), which can easily be separated from the hydrazones of lower aldehydes. Due to large molar absorptivities and absorption maxima at wavelengths > 430 nm, limits of detection range from 4 x 10(-8) to 6 x 10(-8) mol.L-1, and limits of quantification range from 1 x 10(-7) to 2 x 10(-7) mol.L-1 for the individual hydrazones. The method was applied to the determination of aldehydes in automobile exhaust.     

S-nitrosocysteine and cystine from reaction of cysteine with nitrous acid. A kinetic investigation

The formation of the S-nitrosocysteine (CySNO) in aqueous solution starting from cysteine (CySH) and sodium nitrite is shown to strongly depend on the pH. Experiments conducted within the pH range 0.5-7.0 show that at pH below 3.5 the NO+ (or H2NO 2 +) is the main nitrosating species, while at higher pH (>3.5) the nitrosating species is most likely the N2O3. A kinetic study provided a general kinetic equation, V(CySNO) = k1[HNO2][CySH]eq [H+] + k2[HNO2]2. The first term of this equation is predominant at pH lower than 3.5, in agreement with the literature for the direct nitrosation of thiols with nitrous acid; the value for the third-order rate constant, k(1) = 7.9 x 10(2) L(2) mol(-2) min(-1), was calculated. For experiments at pH higher than 3.5, the second term becomes prevalent and the second-order rate constant k(2) = (3.3 +/- 0.1) x 10(3) L mol(-1) min(-1) was calculated. A competitive oxidation process leading to the direct formation of cystine (CySSCy) has been also found. Most likely also for this process two different mechanisms are involved, depending on the pH, and a general kinetic equation, V(CySSCy) = k3[CySH](eq)[HNO2][H+] + k3'[CySH]eq[HNO2], is proposed.     

Investigations in the field of 2,1,3-thiadiazole and 2,1,3-selenadiazole

The action of sodiomalonic ester on 4-bromomethylbenzo-2,1,3-thiadiazole forms a malonate which is converted by acid hydrolysis into 4-(-carboxyethyl)benzo-2,1,3-thiadiazole. When this reaction is carried out with 5-bromomethylbenzo-2,1,3-thiadiazole, mono- and disubstituted malonic esters are formed the acid hydrolysis of which gives the corresponding acids. The nitration of 4-and 5-(-carboxyethyl)benzo-2,1,3-thiadiazoles forms, respectively, 4-(-carboxyethyl)-5,7-dinitrobenzo-2,1,3-thiadiazole and 5-(-carboxyethyl)4-nitrobenzo-2,1, 3-thiadiazole. The reaction of 4-bromomethylbenzo-2,1, 3-thiadiazole with potassium cyanide forms two products: 4-cyanomethylbenzo-2,1, 3-thiadiazole and 1,2-di(benzo-2,1, 3-thiadiazole-4-yl)-2-cyanoethane.For part LVIII, see [1].