Determinations of geniposide using LC/MS/MS methods via forming ammonium and acetate adducts

In this paper, we report method development work to determine geniposide using LC/MS/MS via the formation of positive and negative ion adducts. Geniposide, which has been recognized to have choleretic effects, is the major iridoid glycoside component of Gardenia herbs. To enhance the sensitivity of LC/MS detection of geniposide, a small amount of volatile additives such as ammonium acetate and acetic acid are added into mobile phase solvents to form positive and negative adducts, which can then ionize via electrospray processes. The formation of positive adducts is due to the complexation between geniposide and ammonium ions ([M + NH₄]⁺). The formation of anionic adducts [M + CH₃COO]⁻ is believed to occur via hydrogen bonds bridging acetate ions and glucose groups on the geniposide molecule. Mobile phase solvents containing acetonitrile and aqueous solution (0.2 mM ammonium acetate or 0.1% acetic acid) at the ratio 15: 85 are employed to elute geniposide using C8 reverse phase liquid chromatography columns with electrospray tandem mass spectrometry determinations. Using geniposide standards, the methods are validated at the concentration ranges of 5 to 1000 ng/mL and 20 to 5000 ng/mL using ammonium and acetate adducts respectively. The correlation coefficients of the standard curves are 0.9999 using both ammonium and acetate adducts. The detection limits of using ammonium and acetate adducts are 1 and 5 ng/mL respectively. The measurement accuracy and precision of using ammonium adducts are within 12% and 3% respectively, whereas the accuracy and precision are within 6 and 11% respectively using acetate adducts. When the validated calibration curves of the ammonium adduct of geniposide are used to determine spiked control samples in rat blood dialysates, the determination errors of accuracy and precision are within 12% and 10% respectively.     

哈茨木霉CGMCC 2979生物转化栀子中的京尼平苷制备京尼平

采用微生物直接转化药材的方法,将栀子中的京尼平苷转化为京尼平,无需糖苷酶和京尼平苷的制备. 在培养温度为30 ℃,pH 6.1以及栀子载量为80 g/L的条件下,48 h京尼平苷的转化率为97.8%. 转化后的京尼平通过XAD-16N大孔树脂偶联硅胶层析的方法,制备得到纯度大于95%的京尼平,收率为62.3%. 在催化、转化机制研究中,从哈茨木霉CGMCC2979的发酵液中分离得到了分子量为74.4 kDa的京尼平苷β-葡萄糖苷酶,该酶最优催化条件为50 ℃和pH 4.0-5.0. K_m和V_max分别为3.6 mmol/L和775 μmol/h/mg蛋白. 本文提供了一种简便、高效制备京尼平的新方法.     

HPLC测定蒙药沏其日甘-5中栀子苷的含量

建立了沏其日甘-5（化痰五味散）中栀子苷的含量测定方法。采用BDS（150×4.6mm,5μm）色谱柱,以乙腈-水（15∶85）为流动相,检测波长为238nm,流速为0.8mL/min。栀子苷在1.3—6.5μg范围内具有良好的线性关系,r=0.9998。     

大孔吸附树脂分离纯化杜仲中活性成分

比较了AB-8．NKA-9．NKA-11．D-140．D-101．HPD-600、S-8及聚酰胺等8种吸附树脂对杜仲中的活性成分京尼平甙和松脂醇二葡萄糖甙的吸附及脱附性能．在静态吸附研究的基础上，筛选出效果较好的树脂进行动态实验研究．实验表明：最佳的同时分离纯化松脂醇二葡萄糖甙和京尼平甙的树脂为S-8．并进行了该树脂对杜仲中两种活性成分的吸附和脱附研究，确定了最佳的工艺参数。     

高效液相色谱法同时测定栀子中三种活性成分

采用高效液相色谱法(HPLC)，在C18反相柱上，以甲醇：乙酸溶液(3 397)=18：82为流动相进行洗脱，同时对栀子中的金尼泊甙、金尼泊甙酸及绿原酸进行了分析测定。试验结果表明，该法可使三种有效成分达到基线分离，峰形对称，测定快速，结果准确．     

HPLC同时测定清胃黄连丸中的4种有效成分

建立同时测定清胃黄连丸中栀子苷、黄芩苷、小檗碱和巴马汀含量的HPLC方法。结果表明,栀子苷、黄芩苷、小檗碱和巴马汀分别在2.07—66.2μg/mL（r=0.9997）、2.72—86.9μg/mL（r=0.9998）、2.23—71.3μg/mL（r=0.9998）、2.13—68.1μg/mL（r=0.9998）范围内与峰面积呈良好的线性关系;平均回收率分别为103.2%（RSD=3.27%）、106.7%（RSD=2.46%）、103.5%（RSD=2.13%）、95.7%（RSD=2.68%）。该方法简便可行、重现性好、能更好的控制清胃黄连丸的质量。     

NIRS法对栀子不同炮制品栀子苷含量的快速检测

以96批栀子不同炮制品为研究对象,高效液相色谱测定栀子苷含量为参考值,利用近红外光谱仪积分球漫反射测定其光谱图,建模波段取8 660~7 500,6 650~5 600和4 900~4 000 cm-1,以标准正态变换(SNV)和二阶导数法(2nd derivative)为预处理方法,主成分数为8,采用偏最小二乘法(PLS)对83批栀子样品建立栀子苷的定量校正模型,最终以13批栀子不同炮制品对模型进行验证。结果,定量模型的内部交叉验证决定系数(R2)为0.992 85,校正均方差(RMSEC)为0.240,预测均方差(RMSEP)为0.254,内部交叉验证均方差(RMSECV)为0.386 91,RMSEP/RMSEC=1.06。模型验证得到的相对分析误差(RPD)为8.81,绝对偏差范围-0.39%~0.23%,说明模型预测性较好。通过相关系数法,优选样品装样量、扫描次数、重复次数、分辨率实验条件;并由近红外一阶导和二阶导图,除去温湿度和样品水分影响波段,结合栀子苷对照品近红外光谱图,确定建模波段。首次利用NIRS法建立栀子不同炮制品栀子苷定量校正模型,方法简单快速,模型稳定可靠、准确性高,可同时应用于不同炮制品栀子中栀子苷含量的预测。     

两种大孔吸附树脂结合分离纯化京尼平甙

比较了H103、NKA-II、HPD100A、HPD400A及D141等5种大孔吸附树脂对栀子提取液中栀子黄色素和京尼平甙的吸附性能。在通过静态吸附实验研究其吸附量、吸附动力学特征的基础上,确定了用H103和HPD100A两种非极性大孔树脂进行京尼平甙的分离纯化,并确定了工艺参数。首先用H103树脂吸附京尼平甙,用蒸馏水洗脱杂质,再用一定浓度的乙醇洗脱;所得的京尼平甙洗脱液再用HPD100A树脂吸附,进一步除去栀子黄色素等杂质,得到的京尼平甙纯度达到81.3%,回收率为88.5%。     

Quality control of medicinal herbs Fructus gardeniae, Common Andrographis Herb and their preparations for their active constituents by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

Dehydroandrographolide, andrographolide and geniposide are the main active constituents of many herbal medicines, e.g., Fructus gardeniae, Common Andrographis Herb. They are used as the markers to control the quality of such herbal medicines and their herbal preparations. In this paper, a simple and sensitive high-performance liquid chromatographic (HPLC) method coupled with photodiode array detection (DAD) and electrospray mass spectrometry (ESI/MS) were developed to determine the three compounds simultaneously in extracts of medicinal herbs and herbal preparations produced by different companies. The extracts were separated on a C₁₈ reversed phase HPLC column, with a gradient solvent system, the time for the separation of the three target analytes was 10 min. The abundance ions were recorded using selected ion monitoring (SIM) mode with m/z 297.3, 297.3 and 411.1 for dehydroandrographolide, andrographolide and geniposide, respectively. The limit of detection for dehydroandrographolide, andrographolide and geniposide were 20, 30 and 150 ng mL⁻¹, respectively. The proposed method was successfully applied to the determination of the contents of the compounds in related to medicinal herbs and preparations.     

Colorimetric determination of amino acids using genipin from Gardenia jasminoides

Genipin, a hydrolysate of geniposide from gardenia fruits, produces blue pigments on reaction with amino acids. The colorimetric detection of amino acids using this genipin reaction was evaluated and compared with the well-known ninhydrin reaction. The molar absorptivities of the blue pigments, the reaction products of genipin with various amino acids, were greater than those of the respective ninhydrin reaction products. When asparagine was reacted with genipin, the molar absorptivity was about 14 times higher than with ninhydrin. The absorbance of the genipin–amino acids increased linearly with increase of amino acid concentration, indicating that genipin could be a useful reagent for quantitation of amino acids. Thin layer chromatographic analysis showed that the genipin reaction produces clear and stable colored spots. The blue ninhydrin reaction spots were usually bleached in 24 h at room temperature, while the genipin reaction spots remained unchanged for several months. The addition of 0.1 mM Cu²⁺ and Fe³⁺ decreased the absorbance of Gly–ninhydrin pigment by 50% and 98%, respectively, but those metal ions did not affect the absorbance of the Gly–genipin pigment.