Simultaneous Determination of Amlodipine with H₁-Receptor Antagonists by Reversed Phase High Performance Liquid Chromatography and Application to Interaction Studies

A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

A novel surface molecularly imprinted polymer as the solid-phase extraction adsorbent for the selective determination of ampicillin sodium in milk and blood samples

Surface molecularly imprinted polymers （SMIPs） for selective adsorption of ampicillin sodium were synthesized using surface molecular imprinting technique with silica gel as a support. The physical and morphological characteristics of the polymers were investigated by scanning electron microscope （SEM）, Fourier transform infrared spectroscopy （FT-IR）, thermogravimetric analysis （TGA）, elemental analysis and nitrogen adsorption-desorption test. The obtained results showed that the SMIPs displayed great adsorption capacity （13.5 lag/mg）, high recognition ability （the imprinted factor is 3.2） and good binding kinetics for ampicillin sodium. Finally, as solid phase extraction adsorbents, the SMIPs coupled with HPLC method were validated and applied for the enrichment, purification and determination of anapicillin sodium in real milk and blood samples. The averages of spiked accuracy ranged from 92.1% to 107.6%. The relative standard deviations of intra- and inter-day precisions were less than 4.6%. This study provides a new and promising method for enriching, extracting and determining ampicillin sodium in complex biological samples.

A highly sensitive SPE-liquid/liquid extraction-RPLC analytical method for the determination of 6β-hydroxycortisol and cortisol in cancer patients' urine

Objective: A highly sensitive SPE-liquid/liquid extraction RPLC method has been developed for the analysis of 6β-hydroxycortisol and cortisol in the urine of cancer patients. Methods: After SPE column purification and liquid-liquid extraction, the sample test solutions were analyzed with RPLC using a C18 analytical column. This improved analytical method has been validated for linearity, accuracy (recovery from urine), repeatability (within-day and between-day precision), specificity, sensitivity, and stability. This SPE-liquid/liquid extraction-RPLC is rapid, simple, accurate and reproducible. The technique is particularly useful for monitoring the CYP3A activity of cancer patients in clinical settings. The results are expressed as the ratio of 6β-hydroxycortisol to cortisol. Results: The CYP3A activity from a total of 153 samples was measured using this improved method. Considerable variation in the CYP3A activity of different cancer patients has been documented. Thus, personalized medical treatment based on the individual metabolic enzyme activity level is necessary. Conclusion: This new analytical method facilitates such individualized medical treatments.

PEAK IDENTIFICATION FROM INTERACTION INDEX IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The interaction index c which was derived from the fundamental retention equationlogk’ =a + cC_B in reversed--phase high--performancc liquid chromatography (RP-HPLC) quan-titatively describes the difference in the interaction between solute--strong. solvent andsolute--weak solvent; it has shown to be a constant for a specific solute even when columnsystems with different C18 packings are used. The theoretical basis for peak identificationby using interaction index has been proposed, which was based on the a,c values on stan-dard C18 column by utilizing linear a-a plots on column pairs and the linear relationship be-tween parameters a and c for the structural related compounds. Through the establishmentof parameters a,c data based on the standard C18 column for a certain type of compounds,the retention of thesc compounds on various C18 columns can be predicted. Typical exam-ples have been given to verify the correctness of this method.

高效液相色谱-质谱技术在蛋白质组学中的应用

蛋白质组学研究在生物医学领域发挥了重要作用,然而研究面临的主要难点在于其研究对象的复杂性和多样性。随着质谱技术的快速发展,高效液相色谱-质谱(HPLC-MS)分离分析复杂生物样品已经成为蛋白质组学研究的基础工具。蛋白质组学的研究从肽段分离,延伸到蛋白质和蛋白质复合物的分离,随着分析物的分子质量不断增大,其结构和组成复杂性也持续增加,分子特性也发生改变。面对不同的蛋白质组学研究对象,选择不同的分离模式、分离条件以及固定相参数是进行深度蛋白质组学研究的关键。本文综述了实验室常用的液相色谱分离模式,包括反相色谱(RPLC)、亲水相互作用色谱(HILIC)、疏水相互作用色谱(HIC)、离子交换色谱(IEC)和体积排阻色谱(SEC),以及其不同的组合模式与质谱联用在自下而上(bottom-up)分析、自上而下(top-down)分析、蛋白-蛋白相互作用分析中应用的研究。具体分析了色谱流动相与被分析对象之间的兼容性问题、色谱流动相与质谱兼容性问题,以及多维色谱中不同色谱模式之间流动相的兼容性问题。重点关注存在不兼容问题时研究者所提出的解决方案。此外,本文还评述了HPLC-MS结合样本前处理的方法在外泌体和单细胞蛋白质组学中的应用研究。总之,文章聚焦于近年来HPLC-MS技术在蛋白质组学中的研究进展,旨在为未来蛋白质组学领域的研究提供参考。

地表水中34种新兴污染物的超高效液相色谱-四极杆-飞行时间质谱快速筛查

目的 建立基于超高效液相色谱-四极杆-飞行时间质谱(ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry,UHPLC-Q-TOF-MS)技术快速筛查地表水中34种新兴污染物的方法。方法 通过正交试验设计优化固相萃取(solid phase extraction,SPE)前处理条件,样品经Oasis~? HLB和Oasis~? MCX固相萃取柱串联富集萃取。萃取物采用Kinetex~? EVO C18色谱柱分离,0.1%甲酸水溶液和0.1%甲酸甲醇溶液梯度洗脱,Q-TOF-MS “fullscan”和“targeted MS/MS”模式检测34种新兴污染物,建立34种新兴污染物母离子、子离子及保留时间的数据库。结果 地表水中34种新兴污染物在各自线性范围内呈良好的线性关系,相关系数(r)大于0.97。检出限为0.2~10 ng/L,回收率为81.2%~119.2%,日间精密度为0.78%~18.70%。将方法应用于多份地表水样品的检测,共检出6种新兴污染物,质量浓度为1.93~157.71 ng/L。结论 该方法能简单、快速地对地表水中的多种痕量级新兴污染物进行筛查分析。

初榨橄榄油中多酚类化合物含量随油橄榄果生长发育的累积变化

旨在为甘肃陇南橄榄油的质量评价和油橄榄鲜果采收时间提供指导,探究了不同品种初榨橄榄油(VOO)中多酚类化合物(PPs)随油橄榄果生长发育的累积变化。以甘肃陇南不同成熟度的8个品种油橄榄鲜果为原料,采用压榨法获得VOO,利用反相高效液相色谱法同时测定VOO中的9种PPs含量。结果表明:VOO中总多酚的含量主要由酪醇和橄榄苦苷决定,橄榄苦苷是含量最高的PPs,且其含量随油橄榄果成熟度指数(MI)变化最明显;VOO中木犀草苷含量均较低,阿魏酸和芹菜素仅在个别品种中检测到,芦丁在所测油品中均未检测到;‘阿斯’‘鄂植8号’‘中山24号’和‘佛奥’4个品种VOO的PPs评估数据表现良好,分别在MI为0~1.0、2.0~4.0、0~2.0和5.0~7.0表现最佳,建议这4个品种油橄榄鲜果的采收时间分别为9月下旬、11月上旬、9月下旬至10月上旬以及11月下旬,而‘切姆拉尔’VOO在PPs种类和总含量等评估数据中表现不佳。综上,可以依据不同MI的油橄榄鲜果制备的VOO中多酚含量,尤其是酪醇和橄榄苦苷含量,确定不同品种油橄榄鲜果的采收时间,并进行VOO的品质评价。

采收期、生长年限和加工方法对黄花乌头不同部位中关附甲素含量的影响

目的:为规范黄花乌头的人工栽培,通过优化高效液相法,对不同采收时期、生长年限黄花乌头不同部位中关附甲素的含量进行测定,比较不同加工方法对关附甲素含量的影响,为制定黄花乌头合理的采收时间和加工方式提供依据。方法:采用反相离子对高效液相色谱法,测定黄花乌头中不同部位中关附甲素的含量;色谱柱为安捷伦Zorbax SB-C18柱(250 mm×4.6 mm,5μm),流动相A为2 mg/mL庚烷磺酸钠水溶液(含0.2%三乙胺并以H_(3)PO_(4)调pH值至3.0),流动相B为乙腈,采用梯度洗脱,流速为1.0 mL/min,检测波长为205 nm,柱温20℃。结果:关附甲素的质量浓度在0.0251~0.2513 mg/mL 2范围内线性关系良好(R^(2)=1),精密度、重复性、稳定性试验结果的RSD均≤1.0%;平均加样回收率为97.83%,RSD为3.5%。不同采收期黄花乌头不同部位中9月份采集的样品子根含量最高,说明9月份为最佳采收期。不同生产年限样品中,三年生和三年以上生黄花乌头母根含量相差不大,但均大于两年生母根,地上部分中所含关附甲素都明显小于根部。不同加工方法所得关白附中关附甲素的含量大小依次为:阴干>晒干>炕干,说明不同条件对含量的影响较大。结论:建立的含量测定方法专属性强、精密度、重复性和稳定性较好,结果可靠,黄花乌头的最佳采收期为9月份,生长期限为3年,本研究为黄花乌头科学的人工栽培采收提供了实验依据。

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Rucaparib in Bulk and Pharmaceutical Dosage Form

The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min-1 at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.

全自动固相萃取-高效液相色谱-串联质谱法测定耕地周边地表水中6种嘧啶类杀菌剂的残留量

提出了全自动固相萃取-高效液相色谱-串联质谱法测定耕地周边地表水中乙嘧酚、嘧菌环胺、氟嘧菌胺、嘧菌腙、嘧霉胺、氯苯嘧啶醇等6种嘧啶类杀菌剂残留量的方法。分取预处理后的地表水样品500 mL,调节酸度至pH(7.00±0.05),以18 mL·min^(-1)流量过活化好的HLB固相萃取柱(500 mg/12 mL),用体积比1:1的甲醇-乙腈混合溶液洗脱,收集洗脱液,于45℃氮吹至近干,加入甲醇1 mL复溶,涡旋混匀后上机检测。结果表明:6种嘧啶类杀菌剂的质量浓度均在0.01~5.00μg·mL^(-1)内与对应的质谱响应值呈线性关系,检出限(3S/N)为0.007~0.033μg·L^(-1);按照标准加入法进行回收试验,回收率为84.8%~102%,测定值的相对标准偏差(RSD,n=5)为1.5%~3.6%,重复性RSD(n=6)为1.9%~2.9%。

在线固相萃取-超高效液相色谱-串联质谱法同时测定药厂周边地表水中6种利尿剂的残留量

提出了在线固相萃取-超高效液相色谱-串联质谱法(UHPLC-MS/MS)同时测定药厂周边地表水中布美他尼、呋塞米、氯噻嗪、氢氯噻嗪、安体舒通、氨苯喋啶等6种利尿剂残留量的方法。分取20 mL处理后的药厂周边地表水样品,置于在线固相萃取仪的自动进样盘中,以WATERS Oasis HLB固相萃取柱进行提取、净化和富集;所得溶液自动转入色谱系统中,以Shim-pack CLC-ODS C_(18)色谱柱为固定相,以不同体积比的0.1%(体积分数)甲酸溶液-甲醇的混合溶液为流动相进行梯度洗脱;分离后的6种目标物经电喷雾离子源正离子模式扫描,多反应监测模式检测,外标法定量。结果表明:6种目标物标准曲线的线性范围均为0.005~10.0 mg·L^(-1);检出限(3S/N)为0.0351~0.147μg·L^(-1);按照标准加入法对实际样品进行3个浓度水平的加标回收试验,回收率为83.3%~104%,测定值的相对标准偏差(n=6)均不大于5.0%。

全自动固相萃取-超高效液相色谱串联质谱法测定地表水中18种头孢菌素

建立了超高效液相色谱串联质谱(UPLC-MS/MS)法测定地表水中18种头孢菌素的方法。采用全自动固相萃取技术进行前处理,地表水样经HLB固相萃取小柱富集后,用1.0%(V/V)的甲酸溶液(溶剂为甲醇)洗脱。优选BEHShield RP18(1.7μm,2.1mm×100mm)色谱柱进行分离,在电喷雾正离子(ESI+)模式下电离,多反应模式(MRM)下检测,外标法定量。结果表明:18种头孢菌素在5.0~100μg/L范围内线性关系良好,相关系数(r)均>0.999,方法检出限为0.5~1.8ng/L,测定下限为2.0~7.2ng/L。对地表水样品进行加标回收实验,18种头孢菌素的相对标准偏差(RSD)为0.5%~14.5%,加标回收率为64.3%~92.8%。该方法自动化程度高,结果稳定可靠,适用于地表水中头孢菌素的测定。

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Bimatoprost 0.3% &Timolol 0.5% Pharmaceutical Ophthalmic Dosage Form

The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min-1 at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.

RP-HPLC法同时测定紫苏叶提取物没食子酸、迷迭香酸、木犀草素含量研究

目的建立安徽地区紫苏叶中没食子酸、迷迭香酸和木犀草素成分的含量同时检测方法。方法采用反相高效液相色谱法,Symmetry C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇(A)-0.1%磷酸溶液(B)为流动相梯度洗脱,流速1.0 mL/min,柱温30℃,检测波长272 nm同时检测没食子酸、迷迭香酸和木犀草素含量。采用SPSS 17.0对测定结果进行分析,比较本地主产区紫苏叶中各成分的含量差异。结果没食子酸、迷迭香酸和木犀草素分别在33.80~101.2μg/mL,33.60~100.8μg/mL,34.00~102.0μg/mL呈线性关系,平均加样回收率分别为98.04%(RSD=1.50%,n=6),96.40%(RSD=0.53%,n=6)和96.49%(RSD=1.89%,n=6)。结论此方法简单方便,稳定可重复,可作为安徽地区主产地紫苏叶没食子酸、迷迭香酸和木犀草素成分含量的检测依据。

在线固相萃取-高效液相色谱-串联质谱法测定地表水中8种全氟化合物

建立了在线固相萃取-高效液相色谱-串联质谱法(On-Line SPE-HPLC-MS/MS)检测地表水中8种全氟化合物(perfluorinated compounds,PFCs)的分析方法.该方法用电喷雾离子源(ESI)在选定的多反应监测(MRM)负模式下确定目标化合物并使用同位素内标法定量,测定了地表水中的PFCs.8种PFCs的样品加标回收率在82.1%—103%之间,线性范围在0.66—500 ng·L^(-1)之间,相关系数(R^(2))在0.991—0.998之间,精密度(RSD)在3.95%—6.29%之间,检出限(LOD)在0.20—2.43 ng·L^(-1)之间,定量限(LOQ)在0.66—8.09 ng·L^(-1)之间.方法应用于大凌河及小清河地表水的分析,分析结果与该领域其他研究的结果一致,表明该方法具有实际应用意义.

RP-HPLC法同时检测石榴皮3种多酚类提取物含量研究

采用RP-HPLC法,Symmetry C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇(A)-0.2%磷酸溶液(B)为流动相梯度洗脱,流速1.0 mL/min,柱温30℃,检测波长272 nm同时检测安石榴苷、鞣花酸和没食子酸含量。采用SPSS 17.0对测定结果进行分析,比较不同品种石榴皮中各成分的含量差异。结果表明,安石榴苷、鞣花酸、没食子酸分别在13.120~209.92μg/mL,13.325~213.20μg/mL和13.275~212.40μg/mL与峰面积呈线性关系,平均加样回收率为97.58%(RSD:1.02%,n=6),99.31%(RSD:0.94%,n=6)和98.63%(RSD:0.70%,n=6)。白玉石榴皮中安石榴苷、鞣花酸的含量分别为7.13%和1.00%,比红酸石榴皮中的含量高。安徽地区石榴皮中安石榴苷、鞣花酸、没食子酸3种成分含量同时检测方法的灵敏度高、准确度好、重现性一致,可用于安徽本地石榴皮部位中安石榴苷、鞣花酸、没食子酸成分含量同时检测。

反相高效液相色谱法测定大豆磷脂中磷脂酰胆碱含量

建立一种通过反相高效液相色谱-紫外测定大豆磷脂中磷脂酰胆碱含量的方法。结果表明:方法的流动相为异丙醇-甲醇-水,且大豆磷脂酰胆碱在0.4615~2.3075 mg/mL范围内线性关系良好(R^(2)=0.9977);方法的检出限为1.4 mg/g、平均回收率为99.8%、相对标准偏差(RSD)为2.41%。方法的精确度高、重复性好,适用于大豆磷脂中磷脂酰胆碱含量的测定。

大豆低聚肽的分离纯化及呈鲜序列鉴定

为了分析大豆低聚肽中鲜味肽成分,本文通过纳滤分离结合感官评价对大豆低聚肽进行了初步分离得到鲜味最佳组分Pb-3,通过凝胶过滤层析从Pb-3中分离出鲜味和增鲜效果最强的Fb-2,进一步采用反相高效液相色谱分离出鲜味效果最佳的Kb-2,采用超高效液相色谱-串联质谱法从Kb-2中鉴定出鲜味肽.结果显示:与对照组相比,相对分子质量为300~500的大豆低聚肽Kb-2具有较强的鲜味和增鲜效果;鉴定出3种鲜味肽,分别为2个四肽Val-Thr-Val-Glu(VTVE)、Leu-Glu-Lys-Asp(LEKD)和1个三肽Trp-Glu-Arg(WER).本研究为大豆低聚肽生产复合调味品提供了理论支持.