Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC‐MS/MS

Collection of oral fluid (OF) is easy and non‐invasive compared to the collection of urine and blood, and interest in OF for drug screening and diagnostic purposes is increasing. A high‐throughput ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method for determination of 21 drugs in OF using fully automated 96‐well plate supported liquid extraction for sample preparation is presented. The method contains a selection of classic drugs of abuse, including amphetamines, cocaine, cannabis, opioids, and benzodiazepines. The method was fully validated for 200 μL OF/buffer mix using an Intercept OF sampling kit; validation included linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, stability, and carry‐over. Inter‐assay precision (RSD) and accuracy (relative error) were <15% and 13 to 5%, respectively, for all compounds at concentrations equal to or higher than the lower limit of quantification. Extraction recoveries were between 58 and 76% (RSD < 8%), except for tetrahydrocannabinol and three 7‐amino benzodiazepine metabolites with recoveries between 23 and 33% (RSD between 51 and 52 % and 11 and 25%, respectively). Ion enhancement or ion suppression effects were observed for a few compounds; however, to a large degree they were compensated for by the internal standards used. Deuterium‐labelled and ¹³C‐labelled internal standards were used for 8 and 11 of the compounds, respectively. In a comparison between Intercept and Quantisal OF kits, better recoveries and fewer matrix effects were observed for some compounds using Quantisal. The method is sensitive and robust for its purposes and has been used successfully since February 2015 for analysis of Intercept OF samples from 2600 cases in a 12‐month period. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization and determination of antioxidant components in the leaves of Camellia chrysantha (Hu) Tuyama based on composition–activity relationship approach

Camellia chrysantha (Hu) Tuyama (CCT), an ornamental plant possessing antioxidant activity, has been infused as tea and drank for its health benefits. The antioxidant components in CCT, however, had not been clearly characterized. To quickly identify the antioxidant constituents of CCT, a composition–activity relationship strategy based on ultra high-pressure liquid chromatography coupled with linear ion trap hybrid orbitrap mass spectrometry and orthogonal partial least-squares method has been applied. As a result, 16 variables were found to make significant contributions to the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Six of them were identified as catechin (1), epicatechin (5), vitexin (8), isovitexin (10), quercetin-7-O-β-D-glucopyranoside (12) and kaempferol (16). The strength of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity was found to be 12 > 1 > 5 > 16 > 8 > 10 by validation test. Meanwhile, a liquid chromatography-electrospray ionization-mass spectrometry method was established for quantitative determination of six marker compounds in CCT samples from different preparations. The validation of the method, including linearity, sensitivity (limitation of detection and limitation of quantification), repeatability, precision, stability, and recoveries, was carried out and demonstrated to meet the requirements of quantitative analysis. This is the first report on the comprehensive characterization and determination of chemical constituents in CCT by ultra high-pressure liquid chromatography coupled with linear ion trap hybrid orbitrap mass spectrometry. The results indicate that the composition–activity relationship approach may be a useful method for the discovery of active constituents in natural plants and the quality control of medicinal herbs.     

An improved validated ultra high pressure liquid chromatography method for separation of tacrolimus impurities and its tautomers

A selective, specific and sensitive ultra high pressure liquid chromatography (UHPLC) method was developed for determination of tacrolimus degradation products and tautomers in the preparation of pharmaceuticals. The chromatographic separation was performed on Waters ACQUITY UPLC system and BEH C₈ column using gradient elution of mobile phase A (90:10 v/v of 0.1% v/v triflouroacetic acid solution and Acetonitrile) and mobile phase B (90:10 v/v acetonitrile and water) at a flow rate of 0.6 mL min⁻¹. Ultraviolet detection was performed at 210 nm. Tacrolimus, tautomers and impurities were chromatographed with a total run time of 25 min. Calibration showed that the response of impurity was a linear function of concentration over the range 0.3–6 µg mL⁻¹ (r² ≥ 0.999) and the method was validated over this range for precision, intermediate precision, accuracy, linearity and specificity. For precision study, percentage relative standard deviation of each impurity was < 15% (n = 6). The method was found to be precise, accurate, linear and specific. The proposed method was successfully employed for estimation of tacrolimus impurities in pharmaceutical preparations. Copyright © 2010 John Wiley & Sons, Ltd.     

UHPLC测定芎菊上清丸中9种成分含量及化学模式分析

目的 建立UHPLC波长切换法同时测定芎菊上清丸中9种成分的含量方法。方法 采用Agilent Ecilipse C₁₈（2.1 mm×100 mm,1.6 μm）色谱柱,流动相：甲醇-0.05%磷酸水溶液,梯度洗脱;流速为0.3 mL·min^-1;检测波长：327,237,320,345,278,254 nm;柱温30℃;进样量2 μL;并采用SPSS 22.0统计软件对含量测定结果进行主成分分析与聚类分析。结果 绿原酸、3,5-二咖啡酰奎宁酸、栀子苷、甘草苷、阿魏酸、盐酸小檗碱、黄芩苷、升麻素苷、5-O-甲基维斯阿米醇苷线性范围分别为4.30~68.80 μg·mL^-1（r=0.999 0）、6.66~106.56 μg·mL^-1（r=0.999 2）、7.67~122.72 μg·mL^-1（r=0.999 4）、4.88~78.08 μg·mL^-1（r=0.999 1）、2.37~37.92 μg·mL^-1（r=0.999 1）、6.50~103.92 μg·mL^-1（r=0.999 2）、8.85~141.60 μg·mL^-1（r=0.999 4）、0.88~14.08 μg·mL^-1（r=0.999 7）、0.74~11.92 μg·mL^-1（r=0.999 3）;平均加样回收率（n=9）均在99.42%~103.10%,RSD均<2.0%。主成分分析与聚类分析均可将不同生产厂家的芎菊上清丸很好地分类,且分类结果一致。结论 所建立的多成分方法快捷、准确、重复性好,可用于芎菊上清丸的质量控制。     

Detection of minor constituents in TongMai Granules using NP×RP off-line two-dimensional liquid chromatography coupled with orbitrap mass spectrometry

TongMai Granules is a Chinese medicine patent drug to treat cardiovascular diseases. It is prepared from the water extract of Dan-shen, Chuan-xiong and Ge-gen. The main compounds of TongMai Granules are isoflavone glycosides, isoflavones, and phenolic acids. However, it is difficult to detect low-polar compounds such as tanshinones and phthalides though they may also contribute to the therapeutic effect. In this work, an off-line two-dimensional chromatography method was developed to enrich the minor constituents in TongMai Granules. The extract was subjected to an SEP-PAK silica SPE column and eluted with petroleum ether, ethyl acetate–methanol (6:1, v/v), and methanol, successively. The three fractions were then analyzed using ultra-high performance liquid chromatography coupled with orbitrap mass spectrometry (UHPLC/orbitrap-MS). The isoflavones from Ge-gen, phenolic acids from Dan-shen and phthalides from Chuan-xiong were distributed in different fractions. A total of 131 compounds, including 59 from Ge-gen, 26 from Dan-shen, 43 from Chuan-xiong, and 3 common ones from Dan-shen and Chuan-xiong were tentatively identified. Among them, 26 compounds were identified by comparing with reference standards. This study set a good example for comprehensive analysis of the constituents in traditional Chinese medicine formula.     

UHPLC/MS~n质谱树技术与Q/TOF联用在利舒康胶囊成分鉴定中的应用

目的建立UHPLC/MS~n质谱树技术与Q/TOF联用的方法,对中药复方制剂利舒康胶囊成分进行快速识别和鉴定。方法以含有0.5%乙酸的甲醇-水(3∶1)混合溶液作为提取液制备供试液,采用Thermo Syncronis C_(18)色谱柱(100 mm×2.1 mm,1.7μm)分离,以乙腈-甲醇(1∶1)-0.1%甲酸水溶液为流动相进行梯度洗脱,线性离子阱质谱采用正、负离子同时扫描模式并进行多级质谱碎裂,Q/TOF高分辨质谱分别采用正、负离子扫描模式并进行二级质谱碎裂。结果在线性离子阱正、负离子多级质谱树的验证下,高分辨质谱在中药复方制剂利舒康胶囊中同时确认出生物碱类、黄酮类、三萜类、有机酸类、苯丙素类等各种电离模式下响应的小分子化合物,22个成分经与对照品比对而准确鉴定。结论 UHPLC-Q/TOF高分辨质谱与UHPLC/MS~n质谱树推导技术的联用可以快速鉴定复杂体系中的化学成分,同时也为该复方制剂的质量评价指标选择和药效物质研究奠定了基础。     

D-最优设计响应面法结合UHPLC优选补骨脂药材炮制工艺

目的采用D-最优设计响应面法结合UHPLC技术优选补骨脂药材炮制工艺。方法以补骨脂药材炮制工艺的关键参数闷润时间、炒制温度及加盐量为自变量,进行D-最优设计;以UHPLC检测获得的补骨脂药材中9种主要指标成分补骨脂素、异补骨脂素、新补骨脂异黄酮、补骨脂甲素、补骨脂定、补骨脂乙素、补骨脂宁、补骨脂查耳酮和补骨脂酚炮制前后的质量分数变化为因变量,进行响应面分析;并结合单因素试验优化炒制时间,优选出补骨脂药材炮制工艺。结果补骨脂药材的最佳炮制工艺为在100 g补骨脂药材中加入2.10 g盐,闷润12 h,在80℃炒制30 min。结论利用D-最优设计响应面法结合UHPLC能够优选补骨脂药材炮制工艺参数,提高炮制工艺的稳定性和重复性,为提高补骨脂盐炙品的质量均一性和临床用药安全性提供技术支持。     

基于熵权TOPSIS模型和灰色关联度分析的土茯苓质量评价

目的 用熵权TOPSIS模型和灰色关联度法,探究2种颜色规格土茯苓的多元指标质量差异。方法 采用UHPLC测定32批土茯苓落新妇苷的含量,并测定水分、醇浸出物和水浸出物的含量,借助熵权法、灰色关联度法以及熵权结合灰色关联度法构建质量指标的综合评价模型。测定饮片的色度值,并计算与指标含量的相关性。结果 3种评价方法的排序结果基本一致,红棕色土茯苓的质量高于类白色土茯苓。色度相关性研究表明,样品颜色越深,质量较好。结论 所建立的方法结合2种评价模型的特点,可为土茯苓品质研究提供一定的研究基础。     

Characterization of forced degradation products of toloxatone by LC-ESI-MS/MS

Forced degradation of toloxatone in solutions under basic, acidic, neutral, photo UV–VIS, photo UVC and oxidative stress conditions was investigated and structural elucidation of its degradation products was performed with the use of UHPLC system coupled ESI-Q-TOF mass spectrometer. Eight degradation products were found and their masses and formulas were obtained with high accuracy (0.09–3.79?ppm). The structure of unknown degradation products were elucidated from MS/MS fragmentation spectra of all analyzed compounds. Additionally, whole signals of decomposed substances were compared chemometrically. It was found that toloxatone is fragile towards basic hydrolysis, oxidative conditions and UVC irradiation. Finally, the toxicity of transformation products was computationally evaluated and compared in multivariate manner.     

UHPLC同时测定进口西洋参4个化学成分的含量

目的 建立UHPLC同时测定进口西洋参中4个活性成分（人参皂苷Rg₁、人参皂苷Re、人参皂苷Rb₁和人参皂苷Rd）含量的方法。方法 采用Zorbax SB C₁₈（2.1 mm×100 mm,1.8 μm）色谱柱,以乙腈为流动相A,水为流动相B,梯度洗脱,流速0.5 mL·min^-1,柱温30℃,检测波长为203 nm。结果 4种成分的分离度良好,且在各自浓度范围内呈现良好的线性关系（r≥0.999 9）,平均回收率为95.7%~100.3%（RSD1.4%~1.8%）。结论 该方法操作简单,缩短了分析时间,重复性好,为评价进口西洋参的质量提供了可靠的方法。