Simultaneous quantitative cassette analysis of drugs and detection of their metabolites by high performance liquid chromatography/ion trap mass spectrometry

A method using high performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (MS) for simultaneous quantification of multiple drugs and detection of their metabolites is described. The new approach offers a significant increase in analytical throughput and is illustrated with analysis of the in vitro metabolism of 19 alpha-1a receptor antagonists. The compounds were separated into four cassette groups by using a computer program as well as by manual examination. The samples from incubation with dog liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrospray (ESI) ion trap MS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 60 min, respectively. The quantitative results from the cassette analysis procedure agreed well with those obtained from conventional discrete analysis. In addition, the technique allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multi-stage tandem mass spectrometry (MS(n)) on the ion trap instrument allowed confirmation of the detected metabolites.     

Identification of Amino Acids with Modified Ninhydrin Reagents on Thin-Layer Chromatography Plates

JPC – Journal of Planar Chromatography – Modern TLC - The four new spray reagents (Benzoic acid and some of its parahaloderivatives) have been introduced. These reagents enable...     

Silanylidene and Germanylidene Anions: Valence‐Isoelectronic Species to the Well‐Studied Phosphinidene

The reduction of TipMCl₃ (Tip=2,4,6‐triisopropylphenyl) (M=Si, Ge) with KC₈ in the presence of cyclic alkyl(amino) carbene (cAAC) afforded the acyclic silanylidene and germanylidene anions in the form of potassium salt [K(cAAC)MTip]₂ (M=Si ( 1 ); Ge ( 2 )). The silanylidene and germanylidene anions are valence‐isoelectronic to the well‐studied phosphinidene and are a new class of acyclic anions of Group 14. Compounds 1 and 2 were isolated and well characterized by NMR and single‐crystal X‐ray structure analysis. Furthermore, the structure and bonding of compounds 1 and 2 was investigated by computational methods.     

Extraction and chemical investigation of Kulthi (Macrotylona uniflorus,Lam.) seed protein

Studies have been carried out on the protein solubility profile of Kulthi (Macrotylona uniflorus, Lam.) seed in aqueous solution over various pHs and at different concentrations of NaCl, Na₂SO₃, CaCl₂, and MgCl₂ at pH 8.0. Amino acid analysis of isolated protein identified 17 amino acids, 9 of which are essential. Gel-permeation chromatography on Sephadex G-200 revealed the presence of seven components in the protein fraction. Their molecular weights were determined by two comparable standard methods. Extractable Kulthi seed proteins in salt solutions were separated electrophoretically into eight fractions whose molecular weights were found to be 186,200, 131,800, 108,400, 91,200, 53,700, 44,700, 38,000, and 27,500.     

Aminotroponiminatogermaacid halides with a ge(e)x moiety (e = s, se; x = f, cl)

Fluorination of aminotroponiminate (ATI) ligand-stabilized germylene monochloride [(t-Bu)(2)ATI]GeCl (1) with CsF gave the aminotroponiminatogermylene monofluoride [(t-Bu)(2)ATI]GeF (2). Oxidative addition reaction of compound 2 with elemental sulfur and selenium led to isolation of the corresponding germathioacid fluoride [(t-Bu)(2)ATI]Ge(S)F (3) and germaselenoacid fluoride [(t-Bu)(2)ATI]Ge(Se)F (4), respectively. Similarly, reaction of aminotroponiminatogermylene monochloride [(i-Bu)(2)ATI]GeCl (9) with elemental sulfur and selenium gave the aminotroponiminatogermathioacid chloride [(i-Bu)(2)ATI]Ge(S)Cl (11) and aminotroponiminatogermaselenoacid chloride [(i-Bu)(2)ATI]Ge(Se)Cl (12), respectively. Compound 9 has been prepared through a multistep synthetic route starting from 2-(tosyloxy)tropone 5. All compounds (2-4 and 6-12) were characterized through the multinuclear NMR spectroscopy, and single-crystal X-ray diffraction studies were performed on compounds 2, 4, and 8-12. The germaselenoacid halide complexes 4 and 12 showed doublet (-142.37 ppm) and singlet (-213.13 ppm) resonances in their (77)Se NMR spectra, respectively. Germylene monohalide complexes 2 and 9 have a germanium center in distorted trigonal pyramidal geometry, whereas a distorted tetrahedral geometry is seen around the germanium center in germaacid halide complexes 4, 11, and 12. The length of the Ge═E bond in germathioacid chloride (11) and germaselenoacid halide (4 and 12) complexes is 2.065(1) and 2.194(av) ?, respectively. Theoretical studies (based on the DFT methods) on complexes 4, 11, and 12 reveal the nature of the Ge═E multiple bond in these germaacid halide complexes with computed Wiberg bond indices (WBI) being 1.480, 1.508, and 1.541, respectively.     

Silica gel assisted reductive cyclization of 2-nitro-β-piperidinostyrenes,derived from 2-nitrotoluenes,to indoles

An efficient modification of the Leimgruber-Batcho method of indole synthesis has been devised that facilitates the synthesis of a variety of 2,3-unsubstituted indoles containing halogen, methoxy and benzyloxy groups, from 2-nitrotoluenes, in high yields. The modified method involves the condensation of 2-nitrotoluenes with tripiperidinomethane followed by the reductive cyclization of the intermediate 2-nitro-β-piperidinostyrenes with iron and acetic acid in refluxing toluene in the presence of silica gel (column chromatography grade, 60–200 mesh).     

High-throughput analysis in drug discovery: application of liquid chromatography/ion-trap mass spectrometry for simultaneous cassette analysis of alpha-1a antagonists and their metabolites in mouse plasma

The application of liquid chromatography/ion-trap mass spectrometry for simultaneous quantification of multiple drugs and detection of their metabolites for in vitro experiments was reported recently. In the current study, the use of these techniques was extended to in vivo pharmacokinetic (PK) studies of alpha-1a antagonists. In combination with limited time-point PK, greatly increased throughput was demonstrated for the in vivo screening and investigation of in vivo-in vitro correlation. In addition to quantitative analyses, the technique allowed simultaneous detection of major in vivo metabolites without having to reanalyze the plasma samples. The drugs were individually dosed in mice intravenously via tail vein injection and the blood samples were collected 5 min and 2 h after dosing. After the plasma samples for the different drugs had been prepared separately, they were pooled for cassette analysis. The concentrations of five test compounds in the plasma samples at 2 h ranged from 36-1062 ng/mL, whereas their 5-min plasma levels were similar. From the same cassette analysis, major metabolites in the samples were also detected simultaneously through the interpretation of full-scan mass spectra. The metabolite identification confirmed the results from a previous report that the major sites of metabolism are hydroxylation of the phenyl ring not bearing the alkylsulfonamide substitutent, piperidine N-dealkylation, and N-demethylation of the alkylsulfonamide group.     

Mechanism of autoxidation of 5,7-dihydroxytryptamine: effect of fluorine substitution at positions 4 and/or 6

Analogs of 5,7-dihydroxytryptamine (5,7-DHT), namely, 4-fluoro-, 6-fluoro-, and 4,6-difluoro-5,7-DHT's (30a-c) were synthesized starting from 4-fluorophenol (7a), 4-fluorobenzyl alcohol (12) and 2,4-difluorophenol (7b), respectively. Regiospecific hydroxylation and formylation ortho to fluoro groups, both via aryllithium intermediates, were made possible by the blocking effect of tert-butyldimethylsilyloxy functions and allowed the conversion of the starting materials to the key intermediates, namely, 3,5-bis(tert-butyldimethylsilyloxy)-2-fluoro-, 4-fluoro- and 2,4-difluorobenzaldehydes (11a, b and 19, respectively). The latter were converted in one step to the corresponding benzyloxybenzaldehydes, from which indole-2-carboxylates 22a-c were synthesized via azidostyrenes 21a-c, respectively. Decarbonylation of the indole-2-carboxaldehydes (24a-c) produced from 22a-c in two steps gave 2,3-unsubstituted indoles 25a-c, respectively. Introduction of the aminoethyl side chains on C-3 of 25a-c via the corresponding indole-3-acetonitriles, and subsequent debenzylation generated the hydroxytryptamines, which were isolated as their creatinine sulfate salts 30a-c, respectively. Cyclic voltammetric studies indicated that like 5,7-DHT, 30a-c undergo electrochemical oxidation in 1 M H2SO4 via the corresponding p-quinoneimine derivatives 31a-c by an electrochemical-chemical-electrochemical (ECE) process. The voltammetrically detectable products of the ECE process appear to be the corresponding 5-hydroxytryptamine-4,7-dione (6) derivatives 33a-c. The nature of the interaction of dissolved O2 with 30a-c at pH 7.4 appears to be strikingly different from that of 5,7-DHT, which undergoes autoxidation at pH 7.4 via the 4-hydroperoxy derivative 4 to the quinone 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pseudohalogenogermylenes versus Halogenogermylenes: Difference in their Complexation Behavior towards Group 6 Metal Carbonyls

Pseudohalogenogermylenes [(iBu)₂ATI]GeY (Y=NCO 4 , NCS 5 ) show different coordination behavior towards group 6 metal carbonyls in comparison to the corresponding halogenogermylenes [(iBu)₂ATI]GeX (X=F 1 , Cl 2 , Br 3 ) (ATI=aminotroponiminate). The reactions of compounds 4 – 5 and 1 – 3 with cis‐[M(CO)₄(COD)] (M=Mo, W, COD=cyclooctadiene) gave trans‐germylene metal complexes {[(iBu)₂ATI]GeY}₂M(CO)₄ (Y=NCO, M=Mo 6 , W 11 ; Y=NCS, M=Mo 7 ) and cis‐germylene metal complexes {[(iBu)₂ATI]GeX}₂M(CO)₄ (M=Mo, X=F 8 , Cl 9 , Br 10 ; M=W, X=Cl 12 ), respectively. Theoretical studies on compounds 7 and 9 reveal that donor–acceptor interactions from Mo to Ge atoms are better stabilized in the observed trans and cis geometries than in the hypothetical cis and trans structures, respectively.     

Independent synthesis of 5-hydroxytryptamine-4,7-dione - the neurocytotoxic product of autoxidation of 5,7-dihydroxytryptamine

Synthesis of 5-hydroxytryptamine-4,7-dione hydrochloride ( 20 ) is described starting from 3-bromo-4,5-dimethoxybenzaldehyde ( 6 ). Compound 6 was converted to 2,3,5-tris(benzyloxy)benzaldehyde ( 10 ) in 4 steps. Nitromethylenation of 10 followed by nitration and subsequent reductive cyclization gave 4,5,7-tris(benzyloxy)indole ( 13 ). Introduction of the aminoethyl (hydrochloride) side chain on C-3 of 13 , via the corresponding indole-3-acetonitrile, and subsequent debenzylation generated in situ, 4,5,7-trihydroxytryptamine hydrochloride ( 19 ) which underwent rapid autoxidation in ethanol to give 20 . 4,5,7-Trihydroxyindole ( 21 ) and 3-[2-(ethoxycarbonylamino)ethyl]-4,5,7-trihydroxyindole ( 24 ), both generated in situ, were also found to undergo rapid autoxidation to the corresponding 5-hydroxyindole-4,7-diones.