Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

Determination of abamectin in soil samples using high-performance liquid chromatography with tandem mass spectrometry

Abamectin, which is comprised of a mixture of avermectins B1a and B1b, is a natural pesticide used as an anti-parasitic agent in livestock, ornamental, and agricultural crops, which can potentially be transported to aquatic systems. These compounds are highly toxic to both aquatic vertebrates and invertebrates at low concentrations in water. This investigation developed high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) techniques to support automated extraction by an accelerated solvent extraction (ASE) system and chromatographic techniques to measure residues of avermectins in complex soil samples. HPLC along with atmospheric pressure chemical ionization (APCI) MS/MS was used for separation and determination of avermectin isomers in soil samples. Average method recovery for abamectin by UV was 91%, while detection by MS/MS resulted in a 68% recovery for abamectin. Individual method recoveries by MS/MS were 53.6% for avermectin B1a and 36.8% for avermectin B1b. The use of tandem technology eliminated matrix interferences and resulted in an approximately eight-fold increase in sensitivity.     

Application of Mn(III)-catalysed olefin hydration reaction to the selective functionalisation of avermectin B1

The Mn(dpm)₃-catalysed olefin hydration reaction of α,β-unsaturated esters and ketones discovered by Mukaiyama in 1990 and further developed by Magnus in 2000 was applied to the challenging environment of avermectin B₁. Different avermectin substrates such as 4″,7-OTMS-5-oxo-avermectin B₁3, avermectin B₁1 and Δ^2,3-avermectin B₁6 were thus treated with Mn(dpm)₃, PhSiH₃ in isopropanol under oxygen atmosphere to afford several novel analogues, including 3,4-dihydro-3-hydroxy-avermectin B₁8 with high level of regio- and stereoselectivity, 2-hydroxy-3,4-dihydro-avermectin B₁7, the first example of a 2-substituted avermectin and the novel 22,23-dihydro-22-hydroxy-avermectin B₁9a and 9b, epimeric at C(22). Biological activity of these new avermectin derivatives is also reported.     

A collaborative study: high-pressure liquid chromatographic determination of carbadox and pyrantel tartrate in animal feeds

Carbadox (CBX), an antibacterial agent, and pyrantel tartrate (PT), an anthelmintic, are formulated either separately or together in swine feeds. The official Association of Official Analytical Chemists (AOAC) spectrophotometric methods for both drugs are long, nonspecific, and require standard addition techniques. Results by this technique are positively biased. A simple, direct, specific, high-pressure liquid chromatographic (HPLC) method to determine either one or both drugs simultaneously with apparent accuracy and precision is developed. Drugs are released from feed matrices by water, extracted with dimethylformamide (DMF), cleaned up on alumina, and quantitated by direct comparison to standards using a Whatman Partisil 10 ODS-3 column and a mobile solvent containing 23.5 +/- 1.5% DMF in phosphate buffer (pH 2.0). Fourteen laboratories participated in a collaborative study of this method for determination of CBX and PT in animal feeds.     

改良的QuEChERS结合高效液相色谱-串联质谱同时测定水产品中7种阿维菌素类药物残留

建立了改良的QuEChERS结合高效液相色谱-串联质谱(HPLC-MS/MS)同时测定水产品中7种阿维菌素类药物(阿维菌素、伊维菌素、多拉菌素、塞拉菌素、乙酰氨基阿维菌素、莫西菌素和埃玛菌素)的分析方法。样品经0.2%(v/v)氨化乙腈提取,3 g无水硫酸镁和2 g无水硫酸钠除水剂和沉淀蛋白质,100 mg十八烷基硅烷(C18)和500 mg无水硫酸镁净化。以0.1%(v/v)甲酸乙腈(含5 mmol/L乙酸铵)-0.1%(v/v)甲酸水溶液(含5 mmol/L乙酸铵)为流动相,采用Varian Pursuit ULTRA C8色谱柱(100 mm×2.0 mm,2.8μm)进行分离。在加热电喷雾离子(HESI)源、正离子模式下采用多反应监测模式检测,基质匹配标准曲线外标法定量。阿维菌素、伊维菌素、多拉菌素、塞拉菌素、乙酰氨基阿维菌素和莫西菌素在2~200μg/L范围内、埃玛菌素在0.2~20μg/L范围内呈线性相关,相关系数(r)均≥0.997 2,水产品中阿维菌素类药物的加标回收率为71.6%~112.8%,相对标准偏差(RSD)为4.7%~13.1%,不同水产品的基质效应均小于15%。阿维菌素、伊维菌素、多拉菌素、塞拉菌素、乙酰氨基阿维菌素和莫西菌素的定量限均为5μg/kg,埃玛菌素的定量限为0.25μg/kg。该法操作简便,重复性好,适用于水产品中7种阿维菌素类药物残留量的同时测定。     

Simultaneous analysis of three avermectins in soils by high-performance liquid chromatography with fluorescence detection

A simple and sensitive method has been developed and validated for the determination of abamectin B_1a (ABA B_1a), emamectin B_1a (EMA B_1a) benzoate and ivermectin H₂B_1a (IVM H₂B_1a) in soils. The avermectins (AVMs) residues were extracted from soils with acetonitrile/water (9?:?1, v/v) and then were purified on C₁₈ solid-phase extraction (SPE) cartridge. After being derivatised by N-methylimidazole (N-MIM) and trifluoroacetic anhydride (TFAA), the residues of three AVMs were analysed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The method was validated in terms of system suitability, linearity, selectivity, precision, recovery, specificity and stability. There was a good linear relationship (R ^2?>?0.99) for three AVMs ranged from 0.01 to 5?µg?mL^?1. The LOD and LOQs of ABA B_1a, EMA B_1a benzoate and IVM H₂B_1a for standard solutions were 1.1–1.7 and 3.6–5.7?µg?L^?1 respectively. The accuracy of AVMs in soils was from 83.7 to 115.5% with precision less than or equal to 12.4%. Using the developed method, 9 soil samples with 9.3–12806.3?µg?kg^?1 of AVMs residues had been detected.     

Characterization and determination of genipin-1-β-gentiobioside in gardenia fruit by high-performance liquid chromatography with ultraviolet detection following electrospray ionization mass spectrometry

A practical method for the analysis of genipin-1-β-gentiobioside in gardenia fruit was developed by high-performance liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry characterization (HPLC/UV/ESI-MS). Efficiency of ultrasonic extraction (UE), accelerated solvent extraction (ASE) and high-speed homogenizer extraction (HHE) on genipin-1-β-gentiobioside in gardenia fruit was compared. UE gave higher extraction efficiency and lower matrix interference, and it was employed. Analysis of genipin-1-β-gentiobioside was performed using a Supelco C₁₈ column with methanol–20 mM NH₄Ac (30:70, v/v) as the eluent. Flow rate was at 0.8 ml/min, and the detection was at 240 nm. The limit of detection (LOD), using HPLC/UV, was about 0.25 μg/ml, whereas the overall recovery was better than 96.0%. Both full scan MS and MS² of genipin-1-β-gentiobioside with positive polarity were obtained and elucidated. The specific ions were chosen to characterize genipin-1-β-gentiobioside in gardenia fruit sample. The proposed method has been successfully applied to measure the real samples and the content profiles of genipin-1-β-gentiobioside in gardenia fruit samples were obtained and evaluated.     

Mechanisms of a Cyclobutane-Fused Lactone Hydrolysis in Alkaline and Acidic Conditions

Searching for functional polyesters with stability and degradability is important due to their potential applications in biomedical supplies, biomass fuel, and environmental protection. Recently, a cyclobutane-fused lactone (CBL) polymer was experimentally found to have superior stability and controllable degradability through hydrolysis reactions after activation by mechanical force. In order to provide a theoretical basis for developing new functional degradable polyesters, in this work, we performed a detailed quantum chemical study of the alkaline and acidic hydrolysis of CBL using dispersion-corrected density functional theory (DFT-D3) and mixed implicit/explicit solvent models. Various possible hydrolysis mechanisms were found: B_AC2 and B_AL2 in the alkaline condition and A_AC2, A_AL2, and A_AL1 in the acidic condition. Our calculations indicated that CBL favors the B_AC2 and A_AC2 mechanisms in alkaline and acidic conditions, respectively. In addition, we found that incorporating explicit water solvent molecules is highly necessary because of their strong hydrogen-bonding with reactant/intermediate/product molecules.     

Istamycin aminoglycosides profiling and their characterization in Streptomyces tenjimariensis ATCC 31603 culture using high‐performance liquid chromatography with tandem mass spectrometry

A high‐performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry method was developed and validated for the robust profiling and characterization of biosynthetic congeners in the 2‐deoxy‐aminocyclitol istamycin pathway, from the fermentation broth of Streptomyces tenjimariensis ATCC 31603. Gradient elution on an Acquity CSH C₁₈ column was performed with a gradient of 5 mM aqueous pentafluoropropionic acid and 50% acetonitrile. Sixteen natural istamycin congeners were profiled and quantified in descending order; istamycin A, istamycin B, istamycin A₀, istamycin B₀, istamycin B₁, istamycin A₁, istamycin C, istamycin A₂, istamycin C₁, istamycin C₀, istamycin X₀, istamycin A₃, istamycin Y₀, istamycin B₃, and istamycin FU‐10 plus istamycin AP. In addition, a total of five sets of 1‐ or 3‐epimeric pairs were chromatographically separated using a macrocyclic glycopeptide‐bonded chiral column. The lower limit of quantification of istamycin‐A present in S. tenjimariensis fermentation was estimated to be 2.2 ng/mL. The simultaneous identification of a wide range of 2‐deoxy‐aminocyclitol‐type istamycin profiles from bacterial fermentation was determined for the first time by employing high‐performance liquid chromatography with tandem mass spectrometry analysis and the separation of istamycin epimers.     

Arylethyl (= Phenylethanoid) Glycosides and Oligosaccharide from the Stem of Cistanche tubulosa

Three new analogues of the arylethyl (= phenylethanoid) glycoside echinacoside ( 5 ), namely cistantubulosides A ( 1 ), B₁/B₂ ( 2a / 2b ), and C₁/C₂ ( 3a / 3b ), and one new oligosaccharide, cistantubulose A₁/A₂ ( 4a / 4b ), were isolated from the stems of Cistanche tubulosa (Schenk ) R. Wight , together with five known compounds, i.e., campneoside I, campneoside II ( 6 ), echinacoside ( 5 ), tubuloside, and cistanoside F. Among the compounds isolated, 2a / 2b, 3a / 3b, 4a / 4b , campneoside I, campneoside II ( 6 ), and cistanoside F each consisted of a pair of epimers. The structures of the new compounds were elucidated on the basis of spectral data.     

Effects of CuCl2��6H2O and ZnCl2��6H2O on the viscosity of aqueous ethanol mixtures

The effects of CuCl₂ and ZnCl₂ on the viscosity in aqueous ethanol mixtures (10%–50% v/v) were studied in the concentration range 1.0×10⁻²–8.0×10⁻² mol·dm⁻³ at different temperatures. It was found that the viscosities increased with an increase in the concentration of the salts and percent composition of ethanol content, whereas it decreased with an increase in temperature. Ion-ion and ion-solvent interactions are determined with the help of A- and B-coefficients of Jones-Dole equation. The values of A- and B-coefficients are irregular and increase with a rise in temperature and also with an increase in ethanol contents for both salts. Negative values of B-coefficients show that ion solvent interactions is comparatively small and suggest that CuCl₂ and ZnCl₂ behave as structure breakers in aqueous ethanol mixtures. Thermodynamic parameters like the energy of activation (E _η ) and change in entropy of activation (ΔS*) were also evaluated which confirm the structure breaker behavior of salts in aqueous ethanol mixtures.     

Preparative HPLC for the Facile Isolation of Drug Glucuronide Conjugates from Crude Extracts of Urine

Abstract

Reverse-phase preparative HPLC has been used to advantage for the isolation of crystalline quantities of drug glucuronide conjugates from crude urinary extracts. Following chronic administration of large doses of diazepam, levorphanol and hydroxyethylflurazepam to dogs, urine was collected and the water soluble drug conjugates adsorbed on a column of Amberlite XAD-2 resin. In each instance elution with methanol and solvent evaporation yielded a crude oil (approx. 3 g) which was chromatographed in one portion on either PrepPAK 500 C₁₈ (Waters) or Magnum 40 ODS-3 (Whatman) columns using aqueous methanol solvent systems. A greater than 90% purification was achieved in this single initial chromatographic step. Employing a combination of subsequent semi-preparative HPLC steps on either C₁₈ or silica gel columns, milligram quantities of the glucuronides of oxazepam, levorphanol and hydroxyethylflurazepam were isolated. The isolation procedures provide a general approach for obtaining milligram quantities of intact drug conjugates which may otherwise be difficult to obtain by chemical synthesis. Such conjugates can be used as authentic standards in the quantitation of certain drug metabolites in biological media during pharmacokinetic/biopharmaceutic studies.     

Preparative separation of six antimycin A components from antimycin fermentation broth by high-speed counter-current chromatography

A method of using high-speed counter-current chromatography (HSCCC) was established for preparative isolation and purification of antimycin A components from antimycin fermentation broth. Six antimycin A components were successfully purified for the first time by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (5:2:4:1, by volume). Total of 20 mg antimycin A₄(a or b), 25 mg antimycin A₃(a or b), 21 mg antimycin A₈(a or b), 34 mg antimycin A₂(a or b), 26 mg antimycin A₁(a or b) and 34 mg antimycin A₁(a or b) with the purities of 93.2, 98.6, 96.2, 94.1, 94.9 and 96.7%, respectively, determined by high-performance liquid chromatography (HPLC), were yielded from 200 mg crude sample only in one HSCCC run.     

Quantitative determination and validation of avermectin B1a in commercial products using quantitative nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance is defined as a quantitative spectroscopic tool that enables a precise determination of the number of substances in liquids as well as in solids. There is few report demonstrating the application of NMR in the quantification of avermectin B_1a (AVB_1a); here, a proton nuclear magnetic resonance spectroscopy (¹H NMR) using benzene [1‐methoxy‐4‐(2‐nitroethyl) (PMN)] as an internal standard and deuterochloroform as an NMR solvent was tested for the quantitative determination of AVB_1a. The integrated signal of AVB_1a at 5.56 ppm and the signal of PMN at 8.14 ppm in the ¹H NMR spectrum were used for quantification purposes. Parameters of specificity, linearity, accuracy, precision, intermediate precision, range, limit of detection (LOD), limit of quantification (LOQ), stability and robustness were validated. The established method was accurate and precise with good recovery (98.86%) and relative standard deviation (RSD) of assay (0.34%) within the linearity of the calibration curve ranging from 5.08 to 13.58 mg/ml (R² = 0.9999). The LOD and LOQ were 0.009 and 0.029 mg/ml, which indicated the excellent sensitivity of the method. The stability of the method was testified by a calculated RSD of 0.11%. The robustness was testified by modification of four different parameters, and the differences among each parameter were all less than 0.1%. Comparing with the assay described by the manufacturer of avermectin tablets, there was no significant difference between the assay obtained by HPLC and quantitative NMR (qNMR), which indicated qNMR was a simple and efficient method for the determination of AVB_1a in commercial formulation products. Copyright © 2014 John Wiley & Sons, Ltd.     

The Use of High Pressure Liquid Chromatography for the Identification and Preparation of Pigments Concerned in Photosynthesis

Abstract

The chlorophylls and carotenoids present in preparations from chloroplasts of marine algae can be extracted and separated by high pressure liquid chromatography (H.P.L.C.). The reverse-phase columns; Partisil 10 ODS (Whatman), μ Bondapak C18 and μ Bondapak CN (Waters Associates) gave good separation of the different pigments. Addition of the ion-pairing agent tetrabutylamnonium phosphate to the methanol/water solvents gave improved separations with complex mixtures and identification was facilitated by examination of the column eluant at 440nm where a ll the pigments absorbed and at 650nm where only chlorophylls absorbed. The method allowed the isolation of individual pigments for further study.     

Conformational transitions of end-adsorbed triblock copolymers in a nonselective solvent

Conformational dynamics of triblock copolymers end-adsorbed onto a surface from a nonselective solvent have been studied by a Monte Carlo lattice simulation technique. The triblock copolymers are A B A with N_A = 10 and N_B = 5, 10, 20 and 40, at surface interaction parameters ε = −0.5 and −1.0. A is the adsorbing block and B is the nonadsorbing block. The number of chains in the periodic box is varied over the range 100 ≤ n ≤ 500. The triblock copolymer can exist in three states: loop (L), tail (T) and free (F) chain. Fractions of those conformations at different system parameters (n, N_B and ε) and the associated lifetimes are calculated from the simulations. A kinetic scheme is constructed and the corresponding transition rate matrix is used in the master formalism equation, the solution of which yields the dynamic modes of the system. A correlation function is defined to produce a single overall view of the rate of transitions between different conformational states. The degree of surface interaction has strong influence on the rate of transitions between the states, in particular, longer average lifetimes and a wider distribution of lifetimes of the loop conformation contribute to the slower decay of the correlation curves. At high surface adsorption, larger N_B decrease the rate of all transitions at all values of n. A weak dependence on n is observed for all sizes of N_B at both surface energies.     

固相合成甲胺基阿维菌素苯甲酸盐

To implement the solid phase synthesis of 4““-epi-methylamino-4““-deoxyavermecfin B1 benzoate, tert-butyldimethylsilylchloride was chosen for the first solution synthesis. Then a novel silyl chloride resin 1, achieved from hydroxymethyl polystyrene resin and dimethyldichlorosilane, was used successfully for the attachment of avermectin B1 2. Through oxidation, amination formation, cleavage, and benzoate formation, resin bounded avermectin B1 9 gave 4““-epi-methylamino-4““-deoxyavermectin B1 benzoate 3.     

MRSDCI studies of low-lying electronic states of the CF 2 + ion

Summary The equilibrium geometries, excitation energies, force constants and vibrational frequencies for four low-lying electronic statesX ² A ₁,² B ₁,² B ₂ and² A ₂ of the CF ₂ ⁺ ion have been calculated at the MRSDCI level with a double zeta plus polarization basis set. Our calculated excitation energies for these states and vibrational frequencies for the ground state are in good agreement with experimental data via photoelectron spectroscopy of the CF₂ radical (carbene). The electronic transition dipole moments, oscillator strengths for the² B ₁ X ² A ₁ and² B ₂ X ² A ₁ transitions, radiative lifetimes for the² B ₁ and² B ₂ states and the spin properties for theX ² A ₁ state are calculated based on the MRSDCI wavefunctions.