柱前衍生-超高效液相色谱法测定鱼卵中的17种氨基酸

建立了一种快速、灵敏的柱前衍生-超高效液相色谱-光电二极管阵列检测器(UPLC-PDA)测定史氏鲟(Acipenser schrenckii)、达氏鳇(Huso dauricus)和小体鲟(Acipenser ruthenus)鱼卵中17种氨基酸含量的方法。采用6.0 mol/L的盐酸水解鱼卵,提取液经低压浓缩、碱性中和,然后以6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯(AQC)为衍生试剂在pH 8.8硼酸盐缓冲溶液中衍生化。采用的色谱分离柱为Waters BEH C18柱(100 mm×2.1 mm, 1.7 μm),流动相为30 mmol/L乙酸铵水溶液(pH 3.5)和乙腈(含0.15%(v/v)甲酸及30 mmol/L乙酸铵),梯度洗脱,流速为0.7 mL/min,在260 nm波长下检测。17种氨基酸在5.0～1000 μmol/L浓度范围内,峰面积与浓度之间的线性关系良好(r2≥0.9950)。以标准加入法测定回收率和相对标准偏差(RSD),在100、500、750 μmol/L的添加水平下,17种氨基酸的平均回收率为75.4%～107.3%, RSD为2.19%～12.3%。以3倍信噪比(S/N＞3)计方法的检出限,17种氨基酸的检出限为0.94～4.04 μmol/L。应用该方法检测了3种鲟鳇鱼鱼卵中的17种氨基酸含量。结果表明,该方法简便、准确、快速、可靠。     

柱前衍生-反相高效液相色谱法测定荔枝果蒂中的氨基酸

建立了AccQ· Tag柱前衍生-高效液相色谱法测定荔枝果蒂中氨基酸含量的方法.荔枝果蒂样品在120℃下真空水解22 h,再与AQC衍生剂进行衍生,并采用反相液相色谱法-荧光检测器进行分析,外标法计算样品中17种氨基酸的含量.17种氨基酸在35 min内可完全分离,荔枝果蒂中氨基酸的含量在2.5～25 μmol/L范围内与色谱峰面积呈良好的线性关系,相关系数不小于0.999.方法的加标回收率在71.2％～92.0％之间,测定结果的相对标准偏差为3.16％～9.94％(n=6),方法的检出限(S/N=3)在0.000 5～0.0012 mg/L之间.     

柱前衍生-反相高效液相色谱法测定不同方法煮制的猪肉及其汤汁中的游离氨基酸

建立了反相高效液相色谱(RP-HPLC)分离检测用不同方法煮制的猪肉及其汤汁中17种游离氨基酸的方法.样品经6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生后,采用Nova- PakTMC18色谱柱分离,以AccQ·Tag Eluent A稀释液、乙腈和超纯水为流动相,梯度洗脱,检测波长为248 nm,在4...     

柱前衍生化高效液相色谱-荧光检测法测定桑叶中的1-脱氧野尻霉素

采用柱前衍生化高效液相色谱-荧光检测法测定了桑叶中的1-脱氧野尻霉素(DNJ)。用0.05 mol/L HCl提取桑叶中的DNJ,采用6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)试剂在pH 8.5硼酸盐缓冲液下对DNJ进行衍生化,以0.02 mol/L磷酸二氢钾缓冲液(pH 5.0)-乙腈(体积比为85∶15)为流动相,利用C18色谱柱(5 μm,250 mm×4.6 mm)分离,在激发波长为250 nm、发射波长为395 nm条件下进行荧光检测,DNJ的AQC衍生物与衍生化试剂的水解产物分离良好。方法的线性范围为0.5～25 mg/L,检出限为0.02 mg/L（S/N=3）。实验测得桑叶中DNJ含量为0.12%;回收率为96.1%～98.6%。     

柱前衍生化-超高效液相色谱法快速测定酱油中的18种氨基酸

建立了一种6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生,超高效液相色谱(UPLC)对酱油中18种氨基酸进行快速分离检测的方法。采用BEH C18色谱柱分离,在260 nm波长下检测,以乙酸铵-乙酸-乙腈-水和乙腈-乙酸为流动相,将流动相梯度和流速梯度相结合,在12 min内实现了18种氨基酸衍生物的分离。方法的线性回归系数(r2)均大于0.999,检出限为0.032～0.12 mg/L,日间相对标准偏差(RSD)为0.72%～4.05%,在酱油中18种氨基酸的加标回收率为90.2%～103.7%。该方法前处理过程简单,分离时间短,是检测酱油中氨基酸的有效手段,可用于酱油的质量评定。     

UHPLC-Quadrupole/Orbitrap MS同步检测十字花科植物中的游离氨基酸

采用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱联用技术(UHPLC-Quadrupole/Orbitrap MS)结合柱前衍生法建立了可同时测定28种游离氨基酸的分析方法,并对十字花科植物中的游离氨基酸进行检测和分析。样品用超纯水提取后,经6-氨基喹啉基-N-羟基琥珀酰亚胺基甲酸酯(AQC)衍生,采用Waters BEH C18柱作为色谱柱,以pH 5.0乙酸铵缓冲溶液和80%乙腈水溶液作为流动相进行梯度洗脱。质谱检测器采用电喷雾离子源,在正离子模式下进行检测。实验结果表明,十字花科植物中含有25种以上游离氨基酸,其中包括人体必需的8种氨基酸。25种氨基酸在线性范围内相关性良好,平均加标回收率为80.5%~104.4%,相对标准偏差为0.6%~4.4%。不同氨基酸检测灵敏度不同,定量下限为0.01~1.45μmol/L。该方法杂质干扰小,分析速度快,灵敏度高,适用于植物样品中游离氨基酸的同步检测。     

微波水解衍生高效液相色谱法测定饲料中的氨基酸

建立了一种微波水解衍生高效液相色谱同时测定饲料中17种氨基酸含量的方法。采用2,4-二硝基氯苯作为柱前衍生试剂,利用C_(18)色谱柱分离。二极管阵列检测器进行检测,检测波长为360 nm,并对微波水解时间及温度进行优化。17种氨基酸在2.5~50 mg/L范围内呈良好线性关系,相关系数为0.990 7~0.999 9;相对标准偏差为0.88%~4.2%;加标回收率为90.6%~107.2%;检出限为0.15~2.37 mg/L。研究结果表明,150℃下微波水解16 min的结果与传统加热水解(110℃,24 h)的效果基本相同。该方法分析时间较短,灵敏度较高,可用于饲料中氨基酸含量的检测。     

脑蛋白水解物中氨基酸的柱前衍生高效液相色谱测定

通过升高色谱柱温度,结合线性梯度洗脱的分离模式调整色谱分离的选择性,提出了一种测定脑蛋白水解物注射液中氨基酸含量的2,4-二硝基氟苯(DNFB)柱前衍生高效液相色谱方法。本法采用Kromasil C18(250×4.6 mm i.d.,5μm)色谱柱,柱温44℃,在30 min内以3个线性梯度能完全分离17种氨基酸,定量线性范围为0.2~1.0mg/L;加标回收率为89%~108%。该法具有适用性好、稳定性高等特点,也可以应用于其它种类样品中氨基酸的测定。     

反相高效液相色谱-蒸发光散射检测法同时测定阿胶中的17种未衍生氨基酸

建立了反相高效液相色谱-蒸发光散射检测法(HPLC-ELSD)同时测定中药阿胶中17种未衍生氨基酸含量的方法。采 用PrevailTMC18色谱柱 (250 mm×4.6 mm i.d., 5 μm)，以乙腈-0.7%三氟醋酸溶液(含5.0 mmol/L七氟丁酸）为流 动相进行线性梯度洗脱，流速为0.8 mL/min，在漂移管温度115 ℃、氮气流量2.5 L/min条件下，在25 min内即可完成 对阿胶中17种氨基酸的分离测定。氨基酸质量浓度为0.073～2.327 g/L时,其峰面积的对数值与质量浓度的对数值线性 关系良好；17种氨基酸的加样回收率为93.5%～104.8%；信噪比为3时,测得氨基酸的最低检测限介于18.2 mg/L与54.6 mg/L之间。该法快速、简便、准确,可作为阿胶中氨基酸的直接测定方法,亦为其他药物中氨基酸的分析提供了参考。     

土壤中氨基酸分析方法的研究进展

本文对土壤中氨基酸的分析方法做了较详细的评述,并针对土壤氨基酸的特点,就土壤样品的前处理(主要包括水解和纯化)及目前常用的色谱分离分析方法进行了总结(主要包括柱后衍生阳离子交换色谱、气相色谱、柱前衍生反相高效液相色谱、阴离子交换色谱-积分脉冲安培检测等方法)。     

Enantiomer separation by CEC——Enantiomer separation by packed-capillary electrochromatography

Three chiral compounds were successfully separated in a short time with two enantiomer separation models on packed-capillary electrochromatography (CEC). (i) 75 μm I.D. capillaries were packed with 5 μm β-cyclodextrin (β-CD) chiral stationary phase (CSP). Effects of voltage, pH and concentration of organic modifier on electroosmotic flow (EOF) and chiral separations were investigated systematically. Enantiomers of a neutral compound (benzoin) and a neutral drug (mephenytoin) were separated within a short time with high efficiency. Efficiency of 32 000 theoretical plates per meter and resolution (R_s) of 1.42 were achieved for enantiomers of benzoin using a βCD packed column with 6.2 cm packed length. Efficiency of 45 000 theoretical plates per meter and R_s of 3.40 were obtained for enantiomers of mephenytoin. Especially, the enantiomer separation of mephenytion was performed in just 3.4 min with R_s of 2.60. (ⅱ) 75 μm I.D. capillary was packed with octadecylsilica particles (ODS). Chiral separat     

Regioselectivity of olefin oxidation by iodosobenzene catalyzed by metalloporphyrins : control by the catalyst

The regioselectivity of the oxidation of three monosubstituted olefins, 6-phenoxyhex-1-ene, hex-1-ene and styrene, by iodosobenzene in the presence of various Fe-, Mn- or Cr-tetraaryl-porphyrins, was studied. It was found that, besides epoxides, known products from such systems, allylic alcohols and aldehydes were formed, the latter not being derived from the corresponding epoxides. The relative importance of these reactions greatly depends upon both the metal and porphyrin constituents of the catalyst. More particularly, the competition between epoxidation and allylic hydroxylation can be efficiently controlled by non-bonded interactions between the olefin and porphyrin substituents. No hydroxylation of the aromatic rings and no oxidative dealkylation of the ether function was detected.     

Glycosidase inhibition by macrolide antibiotics elucidated by STD-NMR spectroscopy

A glycosynthase approach was attempted to glycodiversify macrolide antibiotics, using DesR, a family-3 retaining beta-glucosidase involved in the self-resistance mechanism of methymycin production. STD-NMR was used to probe enzyme-substrate interactions. Analysis of competitive STD-NMR experiments between erythromycin A and a chromogenic substrate (pNP-beta-d-glucose) with the hydrolytically inactive nucleophile mutants led us to discover a family of unprecedented glycosidase inhibitors. Analysis of kinetic data with wild-type DesR determined that erythromycin is a competitive inhibitor of the glucosidase (IC50 = 2.8 +/- 0.3 microM and Ki = 2 +/- 0.2 microM) with respect to the hydrolysis of pNP-beta-d-glucose. Comparable inhibitory data was obtained for clarithromycin; however, the inhibitory effect of azithromycin was weak and no significant inhibition was observed with methymycin or d-desosamine. This report documents significant inhibition of glycosidases by macrolide antibiotics and provides insight into the design of novel glycosidase inhibitors based on the macrolactone ring of macrolide antibiotics.     

Antibiotic recognition by binuclear metallo-beta-lactamases revealed by X-ray crystallography

Metallo-beta-lactamases are zinc-dependent enzymes responsible for resistance to beta-lactam antibiotics in a variety of host bacteria, usually Gram-negative species that act as opportunist pathogens. They hydrolyze all classes of beta-lactam antibiotics, including carbapenems, and escape the action of available beta-lactamase inhibitors. Efforts to develop effective inhibitors have been hampered by the lack of structural information regarding how these enzymes recognize and turn over beta-lactam substrates. We report here the crystal structure of the Stenotrophomonas maltophilia L1 enzyme in complex with the hydrolysis product of the 7alpha-methoxyoxacephem, moxalactam. The on-enzyme complex is a 3'-exo-methylene species generated by elimination of the 1-methyltetrazolyl-5-thiolate anion from the 3'-methyl group. Moxalactam binding to L1 involves direct interaction of the two active site zinc ions with the beta-lactam amide and C4 carboxylate, groups that are common to all beta-lactam substrates. The 7beta-[(4-hydroxyphenyl)malonyl]-amino substituent makes limited hydrophobic and hydrogen bonding contacts with the active site groove. The mode of binding provides strong evidence that a water molecule situated between the two metal ions is the most likely nucleophile in the hydrolytic reaction. These data suggest a reaction mechanism for metallo-beta-lactamases in which both metal ions contribute to catalysis by activating the bridging water/hydroxide nucleophile, polarizing the substrate amide bond for attack and stabilizing anionic nitrogen intermediates. The structure illustrates how a binuclear zinc site confers upon metallo-beta-lactamases the ability both to recognize and efficiently hydrolyze a wide variety of beta-lactam substrates.     

DNA damage by sulfite autoxidation catalyzed by cobalt complexes

DNA damage was investigated in the presence of sulfite, dissolved oxygen and cobalt(II) complexes with glycylglycylhistidine, glycylhistidyllysine, glycylglycyltyrosylarginine and tetraglycine. These studies indicated that only Co(II) complexed with glycylglycylhistidine (GGH) induced DNA strand breaks at low sulfite concentrations (1-80 microM) via strong oxidants formed in the reaction. In the presence of the other complexes, some damage occurred only in the presence of high sulfite concentrations (0.1-2.0 mM) after incubation for 4 h. In the presence of GGH, Co(II) and dissolved O2, DNA damage must involve a reactive high-valent cobalt complex. The damaging effect was increased by adding S(IV), due to the oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by the complex. SO3 -, HO and H radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline N-oxide). The results indicate that Co(II) binds O2 in the presence of GGH, and leads to the formation of a DMPO-HO adduct without first forming free superoxide or hydroxyl radical, supporting the participation of a reactive high-valent cobalt complex.