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.

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 The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques.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.

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.

Development of a Rapid and Efficient Liquid Chromatography Method for Determination of Gibberellin A4 in Plant Tissue, with Solid Phase Extraction for Purification and Quantification

A new, rapid and efficient reverse phase Liquid Chromatography (RP-LC) method was developed for determination of Gibberellin A4 (GA4) in samples of flower stalk of Dasylirion cedrosanum and vegetative tissue of Epithelantha micromeris. Purification of GA4 was carried out by solid phase extraction (SPE), in Epithelantha micromeris. In the chromatography method was obtaining a retention time of 2.1 min, using Hypersil GOLD C-18 column (100 × 4.6 mm dim and size particle 5 μ), mobile phase 50/50 acetonitrile/water and a flow 1.0 ml/min. Detection was carried out by a UV detector set at 205 nm, and a quantization limit of 0.4 mg/L. The obtained correlation coefficient was 0.995.

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

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.

A liquid chromatography with tandem mass spectrometry method for quantitating total and unbound ceritinib in patient plasma and brain tumor

A rapid, sensitive, and robust reversed-phase liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of total and unbound ceritinib, a secondgeneration ALK inhibitor, in patient plasma and brain tumor tissue samples. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C_(18) column using a 4-min gradient elution consisting of mobile phase A(0.1% formic acid in water) and mobile phase B(0.1% formic acid in acetonitrile), at a flow rate of 0.4 m L/min. Ceritinib and the internal standard([^(13)C_6]ceritinib) were monitored using multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantitation(LLOQ) was 1 n M of ceritinib in plasma. The calibration curve was linear over ceritinib concentration range of 1–2000 n M in plasma. The intra-and interday precision and accuracy were within the generally accepted criteria for bioanalytical method( o15%).The method was successfully applied to assess ceritinib brain tumor penetration, as assessed by the unbound drug brain concentration to unbound drug plasma concentration ratio, in patients with brain tumors.

Purification of RhIFN- in Bacteria Bodies with Acetic Acid-Water as Mobile Phase in RPLC

Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu I-ICI and most impure proteins can be separated by this way. Compared with the usual dilution method, the bioactivity recovery of the purified rhIFN- was found to be over 500%. In addition, compared to common organic solvents employed ill RPLC, acetic acid has higher freezing point, and therefore, it is easy to concentrate the aim-protein by freeze-drying when acetic acid-water is used as mobile phase ill RPLC.

烟草中甲霜灵的手性分离方法差异研究

对比反相液相色谱-串联质谱(RPLC-MS/MS)和超临界流体色谱-串联质谱(SFC-MS/MS)手性分离烟草中甲霜灵的差异。烟草样品经乙腈提取、盐析分层、快速滤过型净化(multi-Plug Filtration Cleanup,m-PFC)柱净化后,分别采用RPLC-MS/MS和SFC-MS/MS进行手性分离。从多个性能参数(分离效率、线性、选择性、回收率、重复性、灵敏度、基质效应等)对两种方法进行了全面比较。采用不同的分离方法,在10~500 ng/mL范围内,甲霜灵的不同异构体均可呈现良好的线性关系(R^(2)≥0.9993)。在各异构体加标浓度为0.1、0.5、2.0 mg/kg水平下,采用不同的分离方法均可获得满意的回收率(88.7%~96.2%)和良好的重复性(RSD<7.0%)。结果表明:RPLC-MS/MS和SFC-MS/MS具有互补性,均适用于手性分离和测定烟草基质中的甲霜灵。

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。

二维液相色谱分离鉴定桑葚中多酚类化合物

目的建立一套亲水作用色谱×反相色谱二维液相色谱系统对桑葚中多酚类化合物进行鉴定。方法首先采用单因素实验对色谱系统中一维色谱的流动相A的组成、二维色谱的色谱运行条件以及两维系统接口处的样品环的体积进行优化,然后利用优化系统对桑葚中多酚类化合物进行分离鉴定。结果当一维色谱流动相A中乙酸浓度为2.0%、二维色谱流动相流速为3.3 mL/min、二维色谱分析时间为1.3 min、接口处的样品环体积为150μL时,桑葚提取物中多酚类化合物分离效果最好;使用优化后的系统对桑葚提取物中多酚类化合物进行分离鉴定,成功分离鉴定了桑葚提取物中5种酚类化合物。结论本研究的优化方法可行,优化后的系统可以高效、准确分离桑葚提取物中多酚类化合物,为分离鉴定桑葚中多酚类化合物提供了一定的实践基础。

混合型离子交换反相吸附固相萃取-超高效液相色谱-三重四极杆质谱法测定水中23种非甾体抗炎药的残留

非甾体抗炎药(NSAIDs)作为一种新型有机污染物,在环境水体中普遍检出,对生态系统及人体健康造成潜在的威胁,开发准确、可靠的测定水中痕量非甾体抗炎药的检测方法至为重要.本文采用Oasis MCX固相萃取柱对水样进行富集,建立测定水中23种非甾体抗炎药的超高效液相色谱-三重四极杆质谱分析方法.采用酸性条件(pH=4)萃取水样,上样的流速为2 mL·min^(−1),用4 mL甲醇-4 mL5%氨水甲醇进行洗脱,浓缩定容后用ACQUITY UPLCTM BEH C18柱(2.1 mm×100 mm,1.7μm),以2 mmol·L^(−1)乙酸铵+0.05%(V/V)甲酸水溶液-乙腈作为流动相进行梯度洗脱,多反应选择离子监测(MRM)模式进行检测,内标法定量.23种NSAIDs在相关线性范围内线性良好(r=0.9951—0.9992),回收率为80.2%—120%,相对标准偏差为0.4%—12.5%,方法检出限为0.20—4.84 ng·L^(−1).将该方法应用于10份地表水的检测,结果显示有10种NSAIDs检出,质量浓度在ND—83.5 ng·L^(−1)之间.该方法简单、灵敏、高效,可应用于环境水体中NSAIDs的检测.

固相萃取-C_(30)-RPLC法测定南极磷虾油中的虾青素

建立用固相萃取预分离、测定南极磷虾油样品中虾青素3种同分异构体的高效液相色谱法。样品经固相萃取小柱预分离净化,加NaOH-甲醇溶液皂化反应后,经YMC-Carotenoid C30色谱柱分离得到虾青素3种同分异构体,采用外标法定量。南极磷虾油中虾青素的最佳净化条件为:以LC-NH2为固相萃取小柱,正己烷-丙酮-甲醇(1:2:2,V/V)为洗脱溶剂,洗脱体积为8mL,洗脱流速控制在2～4mL/min。样品经LC-NH2固相萃取小柱净化后平均添加回收率为86.1%～94.3%,相对标准偏差为0.79%～1.91%。结果表明,方法重现性好,回收率高,适用于南极磷虾油中虾青素含量分析。

混合离子对试剂体系下寡核苷酸的IP-RPLC保留行为

利用离子对反相液相色谱(IP-RPLC)对寡核苷酸在混合离子对试剂三乙胺/丙胺-乙酸盐(TEA/PAAA)体系下的保留行为进行了研究,并与经典离子对试剂三乙胺乙酸盐(TEAA)体系下的保留行为进行了对比.实验结果表明,相同离子对试剂浓度下,寡核苷酸在TEA/PA-AA体系下的保留均弱于TEAA体系下的保留,且寡核苷酸的保留均随着离子对试剂浓度(20~120 mmol/L)的增加而增强.同型寡核苷酸(dC)_n作为特例,当n>10时,保留基本趋于稳定,这是由于(dC)_n随着离子对试剂浓度的增加保留增长较快,在较低的离子对试剂浓度下即可达到最大保留.同时发现,短链同型寡核苷酸(dT)_n和异型寡核苷酸在TEA/PA-AA体系下的分离均优于TEAA体系,而同型寡核苷酸(dA)_n和(dC)_n的分离则在TEAA体系下更优.通过研究流动相中离子对试剂总浓度c_p与寡核苷酸保留因子k之间的关系,推断出2种体系下寡核苷酸的保留机理均以离子对模型占主导地位.总体而言,分离相同长度的异型寡核苷酸时,TEA/PA-AA混合离子对体系尤其在中等离子对浓度下比TEAA体系具有明显优势,低的离子对试剂浓度可增加与后续电喷雾质谱(ESIMS)的兼容性,有利于寡核苷酸的定性分析.

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。

反相液相色谱-二极管阵列法测定消毒产品中苯扎氯铵3种同系物的含量

目前标准方法检测苯扎氯铵时仅用了一个平均相对分子质量洁尔灭来表示两种或几种混合物,无法区别苯扎氯铵的几种同系物,为了测定苯扎氯铵同系物的含量,进行了题示研究。取消毒产品1.0000 g,用水稀释并定容至10 mL,超声提取5 min,静置,过0.45μm聚砜醚水相滤膜,滤液中苯扎氯铵3种同系物十二烷基二甲基苄基氯化铵、十四烷基二甲基苄基氯化铵、十六烷基二甲基苄基氯化铵在Agilent XDB-C_(18)色谱柱(250 mm×4.6 mm,5μm)上分离,以体积比70:30的乙腈-含0.1%(体积分数)三乙胺的0.5 g·L^(-1)乙酸铵缓冲溶液(pH 5.0)为流动相进行等度洗脱,采用二极管阵列检测器在262 nm处检测。结果表明,扫尾剂三乙胺的体积分数从1%降低到0.1%,避免了扫尾剂三乙胺对色谱柱的可能损害,解决了出现鬼峰的问题。苯扎氯铵3种同系物的质量浓度在10~500 mg·L^(-1)内与对应的峰面积呈线性关系,检出限(3S/N)分别为1.1,1.2,1.5 mg·L^(-1)。按照标准加入法进行回收试验,回收率为98.8%~105%,测定值的相对标准偏差(n=8)均小于2.0%。方法用于实际消毒产品的分析,检出量为0.027%~44.60%,从微量到常量均有涉及。

RPLC法测定饲料添加剂中的脂溶性维生素

利用反相高效液相色谱法,简单、快速、准确地同时定量测定饲料添加剂中脂溶性维生素——V_K,V_A,V_D和V_E的含量.相邻各维生素色谱峰之间的分离度R_S在1.0～2.5之间,各个维生素的回收率在93～89％范围内.变异系数在5.9～1.2％之间.色谱条件为:μ-Bondapak C_(18)径向加压柱,流动相为甲醇,流速为0.8ml/min.紫外可变波长检测器采用MAXPL操作方式。使V_A在325nm检测;V_E在292nm检测;V_D和V_K在265nm检测.最低检出量V_A和V_D为5ng,V_K为2ng,V_E为40ng.

RPLC法测定饲料添加剂中的B族维生素

本文提出了利用反相离子对色谱法,简单,快速,准确同时定量测定饲料添加剂中的烟酸,烟酰胺,盐酸吡哆辛,氰钴胺素,核黄素和硫胺素。样品前处理简单,六个维生素能在12min内获得满意的分离,各组份之间的分离度Rs大于1.60。各个维生素的回收率在87.0～101.0％,相对偏差为3.1～2.0％。色谱柱为LichrosorbRP-18 250×φ5mm,流动相为甲醇/水,0.25％TEA,1.25mmol/l PICB-5,3.75mmol/l PICB-7,pH3.00,紫外可变波长检测器采用MAXPL操作方式λ 1=254nm,λ_2=280nm,λ_3=354nm,流速1.1 nl/min。

RP-HPLC-ELSD法测定逍遥颗粒中柴胡皂苷c、a、d的含量

目的:采用反相高效液相色谱-蒸发光散射检测(RP-HPLC-ELSD)法同时测定逍遥颗粒中柴胡皂苷c、a、d三种成分的含量。方法:采用安捷伦(Agilent)SB-C18色谱柱(4.6mm×250mm,5μm),流动相为乙腈(A)-水(B)。梯度洗脱程序为0~18min,30%A;18~26min,35%~45%A;26~35min,45%~55%A。流速为1.0ml/min,柱温为30℃,气体流速为2.5L/min,蒸发温度为60℃,进样量为10μl。结果:柴胡皂苷c、a、d分别在0.950~19.000μg、1.088~21.760μg、1.520~30.400μg的范围内线性关系良好。柴胡皂苷c平均回收率为99.49%,RSD为0.35%(n=9);柴胡皂苷a平均回收率为99.99%,RSD为0.66%(n=9);柴胡皂苷d平均回收率为99.12%,RSD为0.47%(n=9)。结论:该方法准确、灵敏、重复性好,可作为逍遥颗粒中柴胡皂苷c、a、d的含量测定方法。

Characters of the Plateau of Methanol Increment in Frontal Analysis in Reversed Phase Liquid Chromatography

With insulin methanol water, and the ion pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chromatography (RPLC) was investigated by on line UV spectrometry and identified with nuclear magnetic resonance (NMR) spectrometry and mass spectrometry. The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis (FA). When the insulin concentration was limited to a certain range, the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten. The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components. The displaced methanol was quantitatively determined by NMR and on line UV spectrometries. TFA with high UV absorbance can not be used as an ion pairing agent for the investigation of the FP in RPLC, but it can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC. A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC. From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC, while solute can penetrate into it in FA of RPLC. The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.