HPLC法测定大鼠血浆中EGCG的血药浓度

目的:建立快速、高效、灵敏的HPLC法测定大鼠血浆中的EGCG血药浓度。方法:以睾丸酮为内标物,血浆经盐酸和乙酸乙酯等去除蛋白。采用Agilent 20RBAX SB-C18色谱柱,乙腈-0.3%乙酸为流动相,流速1.0ml/min,检测波长为280nm,柱温40℃。结果:EGCG和内标的出峰时间分别为12.21min和15.4min。EGCG在0.05μg/ml-100μg/ml范围内线性关系良好(R2=0.9920),高、中、低3种浓度下的日内、日间精密度RSD均低于15%,相对回收率与绝对回收率均在100±15(%)范围内,稳定性好。结论:本方法灵敏、准确、高效,适用于大鼠血浆中EGCG的血药浓度检测。     

HPLC法测定槟榔中的多酚类物质

采用高效液相色谱法对槟榔中的多酚类物质进行分析.用甲醇提取槟榔中的多酚类物质,并依次用石油醚、乙酸乙酯、正丁醇萃取,萃取物经抽真空浓缩,流动相定容,高效液相色谱法测定.分析结果表明:槟榔幼果较槟榔成熟果中所含的多酚种类和数量少,槟榔成熟果中果仁的多酚种类和数量都远较皮中多;槟榔的甲醇提取物乙酸乙酯萃取部分中所含的多酚种...     

柱前衍生HPLC法测定大鼠血浆中的河蚌糖胺聚糖

目的:建立柱前衍生高效液相色谱法(HPLC)用于河蚌糖胺聚糖在大鼠血浆中的含量测定。方法:使用异硫氰酸荧光素(FITC)对河蚌糖胺聚糖进行荧光标记,色谱柱为Shodex SB-804HQ(8.0×300 mm),流动相为流速为0.5 m L·min-1的0.1 mol·L-1的Na H2PO4-Na2HPO4缓冲液(p H 7.8),柱温30℃,激发波长495 nm,发射波长520 nm。结果:FITC对河蚌糖胺聚糖的荧光标记率为70;线性范围为5~120μg·m L-1,批内和批间精密度均小于15.0%,提取回收率为51.5~58.6%,样品在血浆中稳定性良好。结论:血浆中内源性杂质不干扰样品的测定,建立的方法符合生物样本的分析要求。     

大鼠血浆中洛伐他汀的HPLC测定及药动学研究

目的:探索采用高效液相色谱法测定大鼠血浆中洛伐他汀浓度的方法。方法:血浆样品采用环已烷-二氯甲烷(3.5:1,V/V)提取,HPLC条件为色谱柱采用XTerra(?)MSC18柱(150×2.1 mm,5μm),流动相为乙腈-水(63L37,V/V),柱温35℃,检测波长为238nm。结果:大鼠血浆中洛伐他汀在0.01～5μg/mL线性范围内线性关系良好,最低检测浓度为0.01～g/mL,方法的提取回收率为81%～95%,日内、日间RSD均小于9.55%;药代动力学结果表明洛伐他汀在雌雄大鼠体内的Tp、Cmax和AUC均有显著性差异(p<0.05)。结论:建立的HPLC方法简便、灵敏度高,重现性好,药动学研究表明洛伐他汀在大鼠体内存在性别差异。     

HPLC法测定硝酸毛果芸香碱眼用凝胶中羟苯乙酯含量

目的:建立用HPLC法测定硝酸毛果芸香碱眼用凝胶中羟苯乙酯含量测定方法。方法:采用十八烷基硅烷键合硅胶为填充剂的色谱柱(250mm×4.6mm,5μm);以乙腈-1%的三氟乙酸(60:40)为流动相;流速1.2m L/min;检测波长为254nm,柱温为室温。结果:羟苯乙酯(1.41~14.10)?g/m L范围内呈良好线性关系,r=0.9999,平均回收率和RSD分别为99.51%和1.27%。结论:该方法简便、准确、重复性好,可作为硝酸毛果芸香碱眼用凝胶中羟苯乙酯含量的测定方法。     

HPLC法测定甘薯叶片中的叶黄素

建立甘薯叶片中有效成分叶黄素含量的高效液相色谱测定方法,以寻求含量高的甘薯品种。采用Waters SunFire^TM C₁₈(150 mm×4.6 mm,5 μm )色谱柱;以甲醇—水为95:5(V/V)为流动相,流速为0.8 mL·min^-1;检测波长为445 nm;外标法定量。此色谱条件下,叶黄素含量在5~100 μg·mL^-1范围内时,与峰面积呈线性关系;样品平均回收率为99.5%;相对标准偏差 (RSD)为1.9%(n=5)。该方法灵敏、准确、专属性强,适用于甘薯叶片中叶黄素的测定;大部分供试品种间存在显著差异,其中以苏薯8号的含量最高。     

HPLC法测定葛花中鸢尾苷的含量

目的：测定8个产地葛花中鸢尾苷的含量,建立葛花中鸢尾苷含量测定的HPLC方法。方法：采用GRACEC18（250mm×4.6mm,5μm）色谱柱,流动相为乙腈-水梯度洗脱,流速为0.8mL/min,紫外检测波长为265nm;柱温为室温。结果：鸢尾苷的峰面积（Y）与浓度（X）在11.8～236.4μg/mL范围内具有良好线性关系,Y=34920X-1156.5,r=0.9995（n=7）;平均加样回收率为103.66%,RSD〈2%（n=9）;测得8批不同产地的葛花药材中鸢尾苷含量在37.00～113.1mg/g。结论：建立了高效液相色谱法测定葛花中鸢尾苷含量的方法,该法准确、可靠,可用于葛花中主要成分鸢尾苷的含量测定;不同产地葛花中均检测到鸢尾苷,但其含量有一定区别。     

HPLC法测定灵芝孢子粉中三萜含量

为准确测定灵芝孢子粉中三萜的含量,运用高效液相建立适合孢子粉的分析测定方法。通过对前处理条件的优化,确定40%乙醇为孢子粉中等极性三萜酸类的最佳提取溶剂,浓缩倍数是子实体提取条件的50倍。通过色谱柱和洗脱条件的优化,建立了包括灵芝酸I、灵芝烯酸C、灵芝酸C2等13种标准品测定方法,方法学考察显示该分析方法精密度、重复性、稳定性的RSD值均小于5%,可以用于灵芝孢子粉中三萜类成分的定量检测。通过5组样品的分析发现,灵芝酸C6、灵芝酸G、灵芝酸A、灵芝酸D、灵芝酸F是灵芝孢子粉中的主要三萜类成分,其中灵芝酸A含量最高,平均占样品三萜总量的比例达19.71%;三萜类成分的溶出量与是否破壁没有相关性。三萜类成分在灵芝孢子粉和灵芝孢子油产品中的含量非常低,孢子粉的三萜含量为14.24-99.70μg/g,仅为子实体的1/100,灵芝孢子油中三萜含量也均低于50μg/g,因此三萜类成分不适合作为灵芝孢子粉及其相关产品的定量检测指标。     

HPLC法测定小叶山葡萄叶中白藜芦醇含量

建立一种高效液相色谱法(HPLC)测定小叶山葡萄Vitis thunbergii var.taiwaniana叶中白藜芦醇含量的检测方法。Waters高效液相色谱仪:Hypersil C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇-水(72:28);流速:1 mL·min^-1;检测波长306 nm。结果表明,在上述色谱条件下,白藜芦醇含量在10~200μg·mL^-1范围内线性关系良好,相关系数r为0.9999。精密度、重现性、稳定性的RSD(n=5)分别为0.77%、0.41%、0.21%;平均加标回收率为99.92%,相对标准偏差为0.39%。该方法准确、灵敏、可靠,可用于白藜芦醇的定量和定性分析。     

HPLC法测定国产葡萄酒中白藜芦醇的含量

采用HPLC法测定国产葡萄酒中白藜芦醇的含量。色谱条件为 :Shim packCLC ODS柱 (15 0mm× 6 0mm) ,流动相 :0 2mol/LH3 PO4 CH3 CN(2 0∶80pH 3 0～ 3 5 ) ,在 30 6nm测定。测得 15种国产葡萄酒中通化干红葡萄酒的白藜芦醇含量最高为 5 6 0mg/L。该方法灵敏 ,重现性好     

Enantiomeric analysis of terfenadine in rat plasma by HPLC.

Enantiomeric pairs of the antihistaminic drug terfenadine and its carboxylic acid derivative were directly separated by HPLC using an ovomucoid protein column. Absolute configurations of terfenadine enantiomers were assigned by comparing their circular dichroism spectra with those of 1-phenyl-1-butanol enantiomers of known absolute stereochemistry. Terfenadine and its major carboxylic acid metabolite extracted from blood plasma following an oral administration of a racemic terfenadine to rats were found to be enriched in the (S)- and (R)-enantiomers, respectively. The results indicated that the (R)-enantiomer of an orally administered racemic terfenadine was preferentially oxidized in rats to form a carboxylic acid metabolite enriched in the (R)-enantiomer.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of sinefungin, a new antiprotozoal drug, in rat plasma has been developed and validated. Sample preparation was performed at 4°C by deproteinization with acetonitrile. Vidarabine was used as an internal standard. Both sinefungin and vidarabine were separated on a C₁₈ column with a mobile phase of ammmonium dihydrogenphosphate-acetonitrile (95:5, v/v) and detected by ultraviolet absorbance at 260 nm. Recoveries of sinefungin from plasma were 75 ± 3.2% and 81 ± 4.8% following dosage at concentrations of 10 μg/ml and 30 μ/ml, respectively. Using 25- μl of rat plasma the limit of quantitation was 1 μg/ml sinefungin, and the assay was linear from 1 to 30 μg/ml. This method appears sensitive enough to be used in further pharmacokinetic studies of sinefungin in animal models.     

Determination of dioscin in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for dioscin in rat plasma was developed. Ginsenoside Rh2 was employed as an internal standard. Dioscin is a naturally occurring saponin present in many traditional Chinese medicinal plants. Dioscin was determined after the acetonitrile-mediated plasma protein precipitation. The mobile phase consisted of acetonitrile:10 mmol/l aqueous ammonium acetate (95:5, v:v), which was pumped at 0.8 ml/min. The analytical column (100 mm x 4.6 mm i.d.) was packed with Hypersil ODS material (5 microm). The standard curve was linear from 1 to 100 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were <10%), precise and reproducible (within- and between-day coefficients of variation <10%). Dioscin in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 24 h. The utility of the assay was demonstrated by determining dioscin plasma concentrations in five rats for 120 h following a single oral gavage dose of 90 mg/kg.     

Determination of protodioscin in rat plasma by liquid chromatography-tandem mass spectrometry

Protodioscin (3-O-[alpha-L-rhamnopyranosyl-(1-->2)-{alpha-L-rhamnopyranosyl-(1-->4)}-beta-D-glucopyranosyl]-26-O-[beta-D-glucopyranosyl]-(25 R)-furost-5-ene-3 beta,26-diol) is a naturally occurring saponin present in many oriental vegetables and traditional medicinal plants, which has been associated with potent bioactivity. However, there is no specific and sensitive assay for quantitative determination of protodioscin in biological samples. We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples were prepared after plasma protein precipitation, and a aliquot of the supernatant was injected directly onto an analytical column with a mobile phase consisted of acetonitrile-water-formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive selected multiple reaction-monitoring mode. The lower limit of quantification (LLOQ) was 20.0 ng/mL and a linear range of 20-125,000 ng/mL. The intra- and inter-day relative standard deviation (R.S.D.) across three validation runs over the entire concentration range was <8.0%. Accuracy determined at three concentrations (50, 5000 and 50,000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma.     

Determination of arbidol in rat plasma by HPLC-UV using cloud-point extraction

A method based on cloud-point extraction (CPE) was developed to determine arbidol in rat plasma by high performance liquid chromatography separation and ultraviolet detection (HPLC-UV). The non-ionic surfactant Triton X-114 was chosen as the extract solvent. Variable parameters affecting the CPE efficiency were evaluated and optimized. A Zorbax SB-C(18) column (4.6 mm i.d. x 150 mm, 5 microm particle size) was used for isocratic elution separation at 40 degrees C with detection wavelength at 316 nm. Under the optimum conditions, the method was shown to be reproducible and reliable with intraday precision below 6.6%, interday precision below 8.8%, accuracy within +/-5.0% and mean extraction recovery more than 89.7%, which were all calculated using a range of spiked samples at three concentrations of 0.2, 2 and 16 microg/ml for arbidol in plasma. The linear range was from 0.08 to 20 microg/ml. After strict validation, the method was successfully applied to the pharmacokinetic study of arbidol in rats after oral and intravenous administration, respectively.     

Determination of polymyxin E1 in rat plasma by high-performance liquid chromatography

A precise and accurate HPLC assay for polymyxin E(1) in rat and dog plasma has been validated. Samples and standards are extracted from plasma with a 96-well C(8) extraction disk plate. Sample extracts are derivatized with dansyl chloride, and polymyxin E(1) derivative is quantitated on a C(8) column by HPLC with fluorescence detection. The assay is linear in the range of 0.050-5.00 micro g/ml for polymyxin E(1). The precision and accuracy of polymyxin E(1) plasma assay was well within the recommended limits set in the FDA Guidance for Bioanalytical Method Validation. Polymyxin E(1) stability in rat and dog plasma for 24 h at room temperature and through three freeze-thaw cycles was demonstrated.     

Determination of p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C₁₈ column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2 → 120.2 and 152.1 → 110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1–500 μg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.     

Determination and pharmacokinetics of gastrodin in human plasma by HPLC coupled with photodiode array detector

In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X²) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: C_max, 1484.55 ± 285.05 ng/ml; T_max, 0.81 ± 0.16 h; t_1/2α, 3.78 ± 2.33 h; t_1/2β, 6.06 ± 3.20 h; t_1/2Ka, 0.18 ± 0.53 h; K₁₂, 0.18 ± 0.41/h; K₂₁, 0.20 ± 0.16/h; K₁₀, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC_0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC_0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS.     

Determination of bencycloquidium bromide in rat plasma by liquid chromatography-electrospray ionization-mass spectrometry

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) assay for the determination of bencycloquidium bromide (BCQB) in rat plasma was firstly developed and validated. After addition of 1-ethyl-bencycloquidium bromide as an internal standard (I.S.), the plasma samples were deproteinized with methanol and the supernatant was assayed by LC-ESI-MS. Chromatographic separation was achieved with a Hanbon Lichrospher 5-C18 column. The mobile phase consisted of methanol-40 mM ammonium acetate buffer-formic acid (75:25:0.25, v/v/v) and delivered at the flow rate of 1.0 ml/min. LC-ESI-MS was carried out on a single quadrupole mass spectrometer using electrospray ionization (ESI) and positive selected-ion monitoring (SIM). Target ions were monitored at [M](+)m/z 330.2 for BCQB and [M] (+)m/z 344.2 for I.S. Calibration curve was linear over the range of 3-1500 ng/ml. The lower limit of quantification (LLOQ) was 3.0 ng/ml. The intra- and inter-run relative standard deviations (R.S.D.%) of the assay were less than 7.1 and 12.3%, respectively. The accuracy determined at the concentrations of 3.0, 100.0, 500.0 and 1500 ng/ml for BCQB were within +/-15.0%. The established method has been applied successfully to study the pharmacokinetics of BCQB in rats after intranasal administration.