Simultaneous determination of amino-alpha-carbolines and amino-gamma-carbolines in cigarette smoke condensate by high-performance liquid chromatography

A method for the simultaneous detection of amino-alpha-carbolines (2-amino-alpha-carboline and 2-amino-3-methyl-alpha-carboline) and amino-gamma-carbolines (3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole and 3-amino-1-methyl-5H-pyrido [4,3-b]indole) by high-performance liquid chromatography has been developed. It consists of a three-step purification using three different columns with fluorometric detection. With this method, we have demonstrated that both amino-alpha-carbolines and amino-gamma-carbolines are present in cigarette smoke condensate. The method may be useful for detecting these carcinogens in various materials.     

Quantitation of mutagenic/carcinogenic heterocyclic aromatic amines in food products.

A method for screening genotoxic heterocyclic aromatic amines in cooked foods using solid-phase extraction and high-performance liquid chromatography with ultraviolet and fluorescence detection is described. Solid-phase extraction includes basic extraction on diatomaceous earth (Extrelut) and subsequent purification on propylsulphonic acid silica gel. This convenient procedure separates the analytes into a polar group and an apolar group. We have identified the following components in the two groups. The polar group contains aminoimidazoazaarenes i.e. 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine, and glutamic acid pyrolysates, i.e. 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-aminodipyrido[1,2-a:3',2'-d]-imidazole. The apolar group consists of five carbolines: 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-9H-pyrido[2,3-b]indole, 9H-pyrido[3,4-b]indole and 1-methyl-9H-pyrido[3,4-b]indole. The extraction efficiencies range from 45 to 90%, and the detection limits are in the low nanogram per gram range. The method was applied to the analysis of heterocyclic aromatic amines in pan-fried, oven-cooked and barbecued salmon.     

Thermospray liquid chromatographic-mass spectrometric analysis of mutagenic substances present in tryptophan pyrolysates

During protein pyrolysis, as can occur when broiling meat or fish, mutagenic substances are formed, as shown by in vitro mutagenicity assays. Some of the most active compounds have been shown to originate from tryptophan (Trp). Hundreds of grams of Trp had to be used previously to study the formation of these compounds by classical separation and detection methods. Studies have been made of the formation of two active heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by heating Trp at different temperatures and for different periods at time. Advantage was taken of the high selectivity and sensitivity of thermospray liquid chromatography-mass spectrometry coupling, which permitted the use of much smaller amounts (10 g) of starting material. These conditions permit a more accurate control of the pyrolysis temperature and the method of extraction can be shortened and simplified. The results show that Trp-P-1 and Trp-P-2 were already formed at 250 degrees C. These substances were detectable in the low ppb range, i.e., less than the threshold levels necessary to elicit a positive response in the Ames test under standard conditions.     

固相萃取-高效液相色谱法同时测定传统禽肉制品中的9种杂环胺类化合物

建立了固相萃取-高效液相色谱同时测定传统禽肉制品中9种杂环胺类化合物(HAAs)(包括2-氨基-3-甲基咪唑并［4,5-f］喹啉、2-氨基-3,4-二甲基咪唑并［4,5-f］喹啉、2-氨基-3,8-二甲基咪唑并［4,5-f］喹喔啉、2-氨基-3,4,8-三甲基咪唑并［4,5-f］喹喔啉、2-氨基-1-甲基-6-苯基-咪唑并［4,5-b］吡啶、3-氨基-1-甲基-5H-吡啶并［4,3-b］吲哚、3-氨基-1,4-二甲基-5H-吡啶并［4,3-b］吲哚、9H-吡啶并［4,3-b］吲哚、1-甲基-9H-吡啶并［4,3-b］吲哚)含量的分析方法。经过条件优化,肉样选用乙酸乙酯进行提取,提取液经丙基磺酸(PRS)和C18固相萃取小柱净化,采用TSK-gel ODS-80TM色谱柱,以乙腈和0.05 mol/L醋酸-醋酸铵缓冲液(pH 3.4)为流动相进行梯度洗脱分离,紫外-荧光检测器串联方式对目标化合物进行检测。通过波长扫描,确定紫外检测波长为263 nm,荧光激发波长/发射波长随时间切换程序为: 0～21 min, 300 nm/440 nm; 21～23.8 min, 315 nm/410 nm; 23.8～35 min, 265 nm/410 nm。在上述条件下,9种HAAs在35 min内实现基线分离。3个加标水平的平均回收率为60.47%～90.55%(n=6),相对标准偏差(RSD)为0.49%～9.74%(n=6),检出限(以信噪比为3计)为0.1～3.6 μg/kg。该方法简便快速、结果准确、灵敏度高,可作为测定传统禽肉制品中多种杂环胺类化合物的有效方法。     

Screening of non-polar heterocyclic amines in urine by microextraction in packed sorbent-fluorimetric detection and confirmation by capillary liquid chromatography

A rapid and simple procedure for the direct screening of urine samples is described. The method involves microextraction in a packed sorbent (MEPS) that is on-line coupled to a capillary liquid chromatograph with fluorimetric detection. The overall arrangement works as a screening/confirmatory system for monitoring non-polar heterocyclic aromatic amines (HAAs) in urine samples. This configuration allows the selective retention of HAAs from urine on a C₁₈ MEPS cartridge integrated in the needle of a micro-well plate autosampler. Retained HAAs were eluted with methanol/water (90:10, v/v) and directly injected into the fluorimetric detector. This screening method provides a yes/no binary response that may require confirmation. The samples for which the concentration of HAAs was close to or above the established threshold limit (30 ng mL⁻¹) were subjected to capillary liquid chromatography (CLC) for confirmation purposes. A mobile phase of acetonitrile and triethylamine (25 mM) at pH 2.5, through a gradient of composition at a flow rate of 20 μL min⁻¹, resulted in good separations between the analytes in less than 11 min. This confirmation method allowed the determination of the analytes in the 10-100 ng mL⁻¹ range for harmane and norharmane and from 20 to 200 ng mL⁻¹ for 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b] indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido-[4,3-b] indole (Trp-P-2), 2-amino-9H-pyrido-[2,3-b] indole (AαC) and 2-amino-3-methyl-9H-pyrido-[2,3-b] indole (MeAαC), with relative standard deviation (RSD) values between 2.12% and 3.73%, and limits of detection between 1.6 and 5.6 ng mL⁻¹ for all the HAAs.     

固相萃取-高效液相色谱法同时测定羊肉制品中9种杂环胺

建立了同时测定羊肉制品中9种杂环胺的固相萃取-高效液相色谱(SPE-HPLC)分析方法。样品经2 mol/L NaOH超声提取、80 mL二氯甲烷液-液萃取,利用阳离子交换柱(MCX柱)净化和富集后进行HPLC分析。采用反相C18色谱柱,以乙腈和0.01 mol/L磷酸溶液(用三乙胺调整pH 3.6)为流动相对杂环胺进行梯度洗脱,使用二极管阵列检测器(DAD)分别在228 nm(2-氨基-9H-吡啶并［2,3-b］吲哚(AaC)、2-氨基-3-甲基-9H-吡啶并［2,3-b］吲哚(MeAaC))、253 nm(2-氨基-3-甲基咪唑并［4,5-f］喹啉(IQ)、1-甲基-9H-吡啶并［3,4-b］吲哚(Harman)、2-9H-吡啶并［3,4-b］吲哚(Norharman))、263 nm(2-氨基-3,8-二甲基咪唑并［4,5-f］喹喔啉(MeIQx)、2-氨基-3,4,8-三甲基咪唑并［4,5-f］喹喔啉(4,8-DiMeIQx)、3-氨基-1-甲基-5H-吡啶并［4,3-b］吲哚(Trp-p-2))、321 nm(2-氨基-1-甲基-6-苯基咪唑并［4,5-b］吡啶(PhIP))处进行检测。实验结果表明,9种杂环胺分离效果良好,回收率为50.27%～94.77%(n=6),相对标准偏差为0.08%～4.42%;通过全波长扫描,确定检出限(以信噪比(S/N)=3计)为1.6～41.0 μg/L。该方法操作简单,结果准确,重现性好,可用于同时检测羊肉制品中9种杂环胺的含量。     

Optimization and validation of a gas chromatographic method for analysis of (RS,SR)-diastereoisomeric impurity in formoterol fumarate

The levels of 2-amino-9H-pyrido[2,3-b]indole (AalphaC or 2-amino-alpha-carboline), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC or 2-amino-3-methyl-alpha-carboline), 9H-pyrido[3,4-b]indole (norharman), and 1-methyl-pyrido[3,4-b]indole (harman) have been determined in the mainstream smoke condensate from three reference cigarettes, namely Kentucky reference 1R5F, Kentucky reference 2R4F, and CORESTA CM4. The amino-alpha-carbolines, and norharman and harman (beta-carbolines) can be classified as heterocyclic aromatic amines (HAAs) and are listed as biologically active agents in the mainstream smoke of cigarettes. For the analysis, the mainstream smoke condensate from cigarettes was collected on a filter pad, the analytes were isolated using solid-phase extraction (SPE), and quantified without derivatization on a GC-MS. Total amounts of carbolines found in the condensate increased from ultralight 1R5F to full-flavor CM4 cigarettes. The level of harman was about 250 ng/cigarette for the 1R5F cigarette and about 1025 ng/cigarette for the CM4 cigarette. Norharman was typically three times more abundant than harman. The AalphaC was found at about 10 times lower level compared to harman, and MeAalphaC was about 50 times lower than harman. The use of reference cigarettes can provide a common measure for laboratories to assess carboline amounts among cigarette brands.     

A reinvestigation of the synthesis of 3H-[1,2]diazepino[5,6-b]indoles. The synthesis of pyrido[4,3-b]indoles

Recently reported [1] syntheses of 6-methyl-1,2,4,5-tetrahydro-1,4-dioxo-3H[1,2]diazepino[5,6-b]indole ( 5 ) and 4-hydroxy-6-methyl-3H[1,2]diazepino[5,6-b]indole ( 12 ) were reinvestigated and shown to be in error. The correct assignments for these respective structures are 3-amino-1,9-dihydro-9-methyl-2H-pyrido[4,3-b]indol-2,4(3H)-dione ( 6 ) and 3-amino-3,9-dihydro-9-methyl-2H-pyrido[4,3-b]indol-2-one ( 13 ). Condensation of 6 and 13 with p-nitrobenzaldehyde produced benzylidene derivatives, which confirmed the presence of the amino groups.     

Synthesis of 3-methyl-6-r-6h-thiazolo-[4,3-<Emphasis Type="Italic">b</Emphasis>]-1,2,4-triazolo[4,3-<Emphasis Type="Italic">d</Emphasis>]-1,3,4-thiadiazoles

A one-reactor method has been developed for the synthesis of 3-methyl-6-R-6H-thiazolo[4,3-b]-1,2,4-triazolo[4,3-d]-1,3,4-thiadiazoles by the condensation of aromatic aldehydes, thioglycolic acid, and 4-amino-5-methyl-1,2,4-triazole-3(2H)-thione in a sulfuric acid medium.     

Suppressive effect of the SOS-inducing activity of chemical mutagen by citric acid esters from Prunus mume Sieb. Et Zucc. using the Salmonella typhimurium TA1535/pSK1002 umu test

A methanol extract from Prunus mume Sieb. Et Zucc. showed a suppressive effect of the SOS-inducing activity on the mutagen 3-amino-1,4-dimethyl-SH-pyrido[4,3-b]indole(Trp-P-1) in the Salmonella typhimurium TA1535/pSK1002 umu test. The methanol extract was re-extracted with hexane, dichloromethane, ethyl acetate, butanol and water. The dichloromethane and ethyl acetate fractions showed suppressive effect. Suppressive compounds were isolated by silica gel column chromatography and identified as trimethyl citrate (1) and dimethyl citrate (2) by GC-MS, IR and 1H and 13C-NMR spectroscopy. Compounds 1 and 2 suppressed 51 and 39% of the SOS-inducing activity at a concentration of 2.0 micromol/mL.     

Dynamic study of excited state hydrogen-bonded complexes of harmane in cyclohexane-toluene mixtures

Photoinduced proton transfer reactions of harmane or 1-methyl-9H-pyrido[3,4-b]indole (HN) in the presence of the proton donor hexafluoroisopropanol (HFIP) in cyclohexane-toluene mixtures (CY-TL; 10% vol/vol of TL) have been studied. Three excited state species have been identified: a 1:2 hydrogen-bonded proton transfer complex (PTC), between the pyridinic nitrogen of the substrate and the proton donor, a hydrogen-bonded cation-like exciplex (CL*) with a stoichiometry of at least 1:3 and a zwitterionic exciplex (Z*). Time-resolved fluorescence measurements evidence that upon excitation of ground state PTC, an excited state equilibrium is established between PTC* and the cationlike exciplex, CL*, lambdaem approximately/= 390 nm. This excited state reaction is assisted by another proton donor molecule. Further reaction of CL* with an additional HFIP molecule produces the zwitterionic species, Z*, lambda(em) approximately/= 500 nm. From the analysis of the multiexponential decays, measured at different emission wavelengths and as a function of HFIP concentration, the mechanism of these excited state reactions has been established. Thus, three rate constants and three reciprocal lifetimes have been determined. The simultaneous study of 1,9-dimethyl-9H-pyrido[3,4-b]indole (MHN) under the same experimental conditions has helped to understand the excited state kinetics of these processes.     

吡喃并[4,3-b]吡喃衍生物与牛血清白蛋白作用的荧光光谱

用荧光光谱法分析了在生理条件下一种吡喃并[4,3-b]吡喃衍生物—2-氨基-7-甲基-4-(4-硝基苯)-5-氧代-4H,5H-吡喃并[4,3-b]吡喃-3-腈与牛血清白蛋白的作用机制。求得不同温度下二者的结合常数和结合位点数,探讨了微量金属离子对实验体系结合常数的影响,并根据热力学参数确定了这种吡喃并[4,3-b]吡喃衍生物与牛血清白蛋白之间的作用力类型。根据Frster非辐射能量转移机理,测定了这种吡喃并[4,3-b]吡喃衍生物与牛血清白蛋白相互结合时,能量给体-受体间的作用距离和能量转移效率,并用同步和三维荧光技术讨论了其衍生物对牛血清白蛋白构象的影响。结果表明,吡喃并[4,3-b]吡喃衍生物主要以静态猝灭方式使牛血清白蛋白荧光强度显著降低,二者主要凭借氢键和范德华力结合。     

Studies on the syntheses of heterocyclic compounds—DLXIX : One-step synthesis of dihydropyridocarbazole derivatives

Heating indole and 4,5-dibromomethyl-3-hydroxy-2-methylpyridine hydrobromide (10), followed by acetylation, gave the desired dihydropyrido[4.3-b]carbazole (12) and its structural isomer, dihydropyrido[3.4-b]carbazole (13). The structures of (12 and 13) were determined by UV spectral investigations of the corresponding dehydrogenated products, 4-acetoxy-3-methyl-6H-pyrido[4.3-b]carbazole (11) and 4-acetoxy-3-methyl-10H-pyrido[3.4-b]carbazole (14).On the other hand, the same reaction in the presence of sodium iodide afforded 12 together with four other coupling products which were not cyclized, such as (16, 17, 18), and a dimer.     

Electron Ionization Mass Spectra of Organophosphorus Compounds Part I: The Mass Fragmentation Pathways of Cyclic α-Aminophosphonates Monoester Containing 1,2,4-Triazinone Moiety

Abstract

The electron impact induced fragmentation reactions of 3-(4-chlorophenyl)-3,4- dihydro-2-ethoxy-2-oxido-7-methyl-2H,6H-[1,2,4]triazino[4,3-e][1,4,5,2]thiadiazaphosphin in-6-one (1), 3,7-dimethyl-2-ethoxy-2-oxido-1,2,3,4-tetrahydro-6H-[1,2,4]triazino[4,3-b][1,2,4,5]triazaphosphinin-6-one (2), and 9-amino-3,7-dimethyl-4-ethoxy-4-oxido-2,3,4,9-tetrahydro-8H-[1,2,4]triazino[3,2-c][1,2,4,5]triazaphosphinin-8-one (3) are presented and compared. The 1,2,4-triazine rings have almost identical fragmentation routes. The 1,2,4-triazine rings are rather stable relative to the phosphorus rings. Therefore, fragmentation of the phosphorus rings is more favorable for the compounds than the stable 1,2,4-triazine rings.     

Molecular Diversity from the L-Proline-Catalyzed,Three-Component Reactions of 4-Hydroxycoumarin,Aldehyde, and 3-Aminopyrazole or 1,3-Dimethyl-6-aminouracil

The three-component reaction of 4-hydroxycoumarin, aldehyde, and aminopyrazole in the presence of a catalytic amount of L-proline in ethanol under reflux conditions provided the cyclized product (dihydrochromeno[4,3-b]pyrazolo[4,3-e]pyridine-6(7H)-one), whereas under similar reaction conditions, replacing aminopyrazole by 1,3-dimethyl-6-aminouracil provided the acyclic three-component product (6-amino-5-((4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione). This method is metal free and atom economical, and avoids column chromatographic purification as all the products can be purified by recrystallization.     

Reaction of 5-bromo-6-amino-3-(4-methylaminobutyl)pyridine with potassamide

It has been found that potassamide reacts with 5-bromo-6-amino-3-(4-methylamino-butyl)pyridine to give a mixture of the starting material, 6-aminonicotine, and 1-methyl-8-amino-2,3,4,5-tetrahydro[4,3-b]azepine. The latter has been obtained independently from 1-methyl-9-cyano-2,3,4,5,7,8-hexahydropyrido[4,3-b]azepin-8-one.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 3, pp. 364–367, March, 1986.     

Reaction of diketene with cyanothioacetamide: A convenient and regioselective method for the preparation of new 4(1H)-pyridone derivatives

The reaction of diketene with cyanothioacetamide in dry dioxane in the presence of triethylamine gives triethylammonium 3-cyano-6-methyl-4-oxo-1,4-dihydro-2-pyridinethiolate. The regioselective S-alkylation of this thiolate is a convenient method for the preparation of substituted 4(1H)-pyridones and also derivatives of thiazolo[3,2-a]pyridine and thieno[2,3-b]pyridine. The action of 2-amino-1,1,3-tricyanopropene on this thiolate leads to its transformation into a new heterocyclic system, namely, 5H-pyrido[2′,3′:4,5]thiopyrano[2,3-b]pyridine; treatment with iodine yields the oxidation product, namely, the corresponding bis(2-pyridyl) disulfide. The structure of isopropyl (3-cyano-6-methyl-4-oxo-1,4-dihydro-2-pyridinyl)thioacetate was confirmed by X-ray diffraction structural analysis. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 716–725, May, 2007.     

New sesquiterpenes from Ferula ferulaeoides (Steud.) Korovin. VI. Isolation and identification of three new dihydrofuro[2,3-b]chromones

Three novel 2-prenyl-dihydrofurochromone-type sesquiterpenoid derivatives, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-2-[4,8-dimethyl-3(E),7-nonadienyl]-furo[2,3-b]chromone, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E),7-pentenyl]-furo[2,3-b]chromone, and 2,3-dihydro-7-hydroxy-2R*,3R*-dimethyl-2-[4-methyl-5-(4-methyl-2-furyl)-3(E),7-pentenyl]-furo[2,3-b]chromone, were isolated from the roots of Ferula ferulaeoides. The structures were established by comprehensive spectral analysis. The biosynthetic pathway leading to these 2-prenyl-dihydrofurochromone-type sesquiterpenoids is proposed based on their structures.     

2-Dicyanomethylidene-3-ethoxymethylidene-2,3-dihydroindole in the synthesis of fused tri- and tetracyclic systems

The reactions of 2-dicyanomethylidene-3-ethoxymethylidene-2,3-dihydroindole with hydrazine hydrate and phenylhydrazine afforded 2,3-diamino-4-cyanopyrido[4,3-b]indole and 3-amino-2-anilino-4-cyanopyrido[4,3-b]indole, respectively, and the reactions of the latter compounds with dimethylformamide diethyl acetal were studied. The reactions of 2,3-diamino-4-cyanopyrido[4,3-b]indole with benzaldehyde, ethyl acetoacetate, and acetylacetone were investigated. First representatives of new heterocyclic systems, viz., [1,2,4]triazolo[1’,5’:1,6]-pyrido[4,3-b]indole and pyrazolo[1’,5’:1,2]pyrido[4,3-b]indole, were synthesized. The structure of ethyl 6-cyano-5-[(E)-(dimethylamino)methylideneamino]-2-methyl-7H-pyrazolo-[1’,5’:1,2]pyrido[4,3-b]indole-1-carboxylate was established by X-ray diffraction.     

Synthesis of some condensed benzofurans

The condensation of O-phenylhydroxylamine (Ia) with tetrahydro-4-thiopyrone, 1-methyl-4-piperidone (IIb), 1,2,5-trimethyl-4-piperidone, and 4-thiochromanone and of O-(4-tolyl)-hydroxylamine with IIb was used to synthesize the corresponding benzofuran derivatives (IIIa-e), from which IIIa and IIIb were obtained by deamination of 8-amino-3,4-dihydro-1H-thiopyrano-[4,3-b]benzofuran and 2-methyl-8-amino-1,2,3,4-tetrahydrobenzofuro[3,2-c]pyridine.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 740–742, June, 1972.