High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract

A sensitive and simple HPLC method has been developed and validated for the determination of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) in rat plasma. The plasma samples were extracted with ethyl acetate and analyzed using HPLC on an Aglient Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm, with a linear gradient of (A) acetonitrile and (B) 0.5% aqueous acetic acid (v/v), at a flow rate of 1.0 mL/min. The method was linear over the range of 0.1265-25.3 microg/mL for OXY and 0.117-23.4 microg/mL for RES. The extraction recovery for OXY, RES and internal standard ranged from 71.1 to 88.3%. The intra- and inter-day precisions were better than 10%, and the accuracy ranged from 89 to 108%. The validated method was used to study the pharmacokinetic profiles of OXY and RES in rat plasma after oral administration of Smilax china root extract.     

High-throughput determination of atrasentan in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Atrasentan (A-147627) is an endothelin antagonist receptor being developed at Abbott Laboratories for the treatment of prostate cancer. A quick and sensitive method for the determination of atrasentan in human plasma has been developed and validated using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A dual-column, single mass spectrometer system is used to provide a reliable and routine means to increase sample throughput. The analytical method involves liquid-liquid extraction and internal standard (A-166790). The plasma samples and internal standard are acidified with 0.3 M hydrochloric acid prior to being extracted into 1:1 (v/v) hexanes--methyl t-butyl ether. The organic extract was evaporated to dryness using heated nitrogen stream and reconstituted with mobile phase. Atrasentan and internal standard were separated with no interference in a Zorbax SB-C(18) analytical column with 2.1 x 50 mm, 5 microm, and a Zorbax C(8) guard column using a mobile phase consisting of 50:50 (v:v) acetonitrile--0.05 M ammonium acetate, pH 4.5, at a flow rate of 0.30 mL/min to provide 4 min chromatograms. For a 250 microL plasma sample volume, the limit of quantitation was approximately 0.3 ng/mL. The calibration was linear from 0.30 to 98.0 ng/mL (r(2) > 0.995). A significant advantage of the method is the ability to employ parallel HPLC separations with detection by a single MS/MS system to provide sensitivity and selectivity sufficient to achieve robust analytical results with a lower limit of quantitation of 0.30 ng/mL and high throughput.     

HPLC analysis and pharmacokinetics of icariin in rats

As a prerequisite to the determination of pharmacokinetic parameters of icariin in rats, an HPLC method using UV detection was developed and validated. Icariin and the internal standard, quercetin, were extracted from plasma samples using ethyl acetate after acidification with 0.05 mol/L NaH2PO4 solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB Cls column (250 x 4.6 mm id, 5 microm) equipped with a Shim-pack GVP-ODS C18 guard column (10 x 4.6 mm id, 5 microm) using a mobile phase of ACN/water/acetic acid (31:69:0.4 v/v/v) at a flow rate of 1.0 mL/ min. Detection was at 277 nm. The calibration curve was linear from 0.05 to 100.0 microg/mL with 0.05 microg/mL as the lower LOQ (LLOQ) in plasma. The intra- and interday precisions in terms of RSD were lower than 5.7 and 7.8% in rat plasma, respectively. The accuracy in terms of relative error (RE) ranged from -1.6 to 3.2%. The extraction recoveries of icariin and quercetin were 87.6 and 80.1%, respectively. The main pharmacokinetic parameters for rats were determined after a single intravenous administration of 10 mg/kg icariin: t1/2, 0.562 +/- 0.200 h; AUC0-infinity, 8.73 +/- 2.23 microg x h/mL; CLToT, 20.10 +/- 5.80 L/kg x h; Vz, 1.037 +/- 0.631 L/kg; MRT0-infinity, 0.134 +/- 0.040 h; and Vss, 0.170 +/- 0.097 L/kg.     

Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for lopinavir and 11-3,100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.     

Determination of optical impurity of pregabalin by HPLC with pre-column chiral derivatization

A new method for determining the optical impurity of Pregabalin is developed. The method is based on Pregabalin, and its isomers can be derivatized with Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide. These derivated compounds can be separated by an ordinary chromatography column (Inertsil ODS-2.5 microm, 250 mmx4.6 mm i.d.). Phosphoric acid buffer and acetonitrile (55:45, v/v) are used as mobile phase and 1.0 mL/min flow rate at room temperature. The detective wavelength is fixed at 340 nm. The results indicate that the limit of detection of R model optical impurity 1.1x10(-8) g/mL (signal-to-noise=3), accuracy, and repeatability is satisfied. Therefore, the method can be used for the quality control of Pregabalin.     

固相萃取-反相高效液相色谱法同时测定饲料中的角黄素和虾青素

建立了一种同时测定饲料中角黄素和虾青素的固相萃取-高效液相色谱法（HPLC）。样品由乙腈提取,经LC-NH2固相萃取小柱净 化,洗脱剂为乙腈-甲苯（体积比为3∶1）,洗脱液被浓缩后进行HPLC分析,色谱柱为ZORBAX Eclipse XDB-C18柱(150 mm×4.6 mm,5 μ m),流动相为乙腈-甲醇（体积比为95∶5）,流速1.0 mL/min,采用二极管阵列检测器检测,检测波长为474 nm;外标法定量。角黄素和 虾青素的线性范围分别为1.0～30.0 mg/L和1.0～20.0 mg/L,相关系数分别为0.9990和0.9991,回收率为90%～101%,相对标准偏差为 0.62%～3.68%,检出限分别为0.84和0.60 mg/L。该方法简便、快速、准确,可用于饲料中角黄素和虾青素的同时测定。     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

A validated method for analysis of chromium picolinate in nutraceuticals by reversed phase high performance liquid chromatography

A validated high performance liquid chromatographic method was developed for the determination of chromium picolinate in pharmaceutical dosage forms. The analysis was performed at room temperature using a reversed-phase Supelcosil LC-18 (250 x 4.6 mm, 5 microm) column. The mobile phase consisted of acetonitrile:water (40:60 v/v) at a fl ow rate of 0.8 mL/min. The UV-detector was set at 264 nm. The developed method showed a good linear relationship in the concentration range from 0.125 to 12.5 microg/mL with a correlation coefficient from 0.999. The limit of detection and limit of quanti fi cation were 0.091 and 0.181 microg/mL, respectively.     

Determination of picroside II in dog plasma by HPLC and its application in a pharmacokinetics study

A sensitive and simple high-performance liquid chromatography method with UV detection was developed and validated for determining picroside II in dog plasma. Paeoniflorin was employed as internal standard and the sample pre-treatment procedure consists of deproteinization by addition of acetonitrile. Chromatographic separations were performed on a Shimadzu VP-ODS column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-0.1% acetic acid aqueous (v/v), 23:77, v/v, at a rate of 1 mL/min. Detection was carried out at a wavelength of 266 nm. Calibration standards ranged from 0.25 to 500 microg/mL in dog plasma and the mean correlation coefficient of 0.9981 was found for the linear calibration curves (n = 6). The limit of quantification (LOQ) was 0.25 microg/mL. Intra- and inter-assay RSD ranged from 0.70 to 7.5%. Accuracy (%bias) ranged from -6.3 to 6.0%. This method was applied to the pharmacokinetic study of picroside II in dogs. The study demonstrated the plasma picroside II concentration-time curves were fitted to the two-compartment open model and showed linear pharmacokinetics.     

Quantification of myrislignan in rat plasma by solid-phase extraction and reversed-phase high-performance liquid chromatography

A rapid and simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of myrislignan in rat plasma after intravenous administration. The analytes extracted from plasma samples by solid-phase extraction were successfully carried out on a Diamonsiltrade mark ODS C(18) column (250 x 4.6 mm i.d., 5 microm) with an RP(18) guard column (8 x 4.6 mm i.d., 5 microm) and a mobile phase of MeOH-H(2)O (4:1, v/v). The UV detector was set at a single wavelength of 270 nm. The linear ranges of the standard curves were 0.5-30.0 microg/mL with the correlation coefficients greater than 0.9992. The lower limits of detection and quantification were 0.1 and 0.3 microg/mL for myrislignan. Intra- and inter-day precisions were 2.4-7.5 and 1.3-5.7%, respectively. The extraction recovery from plasma was more than 90%. This assay method has been successfully used to study the pharmacokinetics of myrislignan in rats.     

Determination of estradiol and its degradation products by liquid chromatography

A novel HPLC method for simultaneous determination of estradiol and its seven degradation products in topical gel was developed. Zorbax SB-CN (150 mm x 4.6 mm, 5 microm) analytical column and mobile phase composed of acetonitrile, phosphoric acid 0.085%, and tetrahydrofurane (27:63:10, v/v/v) at flow-rate 1.0 ml min(-1) were used for the chromatographic separation using UV detection at 225 nm. The active substance estradiol was separated from all its known degradation products successfully. Two degradation products estrone and Delta(9(11))-estrone were not separated sufficiently, their peaks were evaluated as a sum of two components. The method was validated according to ICH guideline recommendations and thereafter it was successfully applied for stability tests of topical cream Estrogel HBF in the quality control laboratory. Limits of detection for degradation products ranged from 1.03 x 10(-5) to 1.14 x 10(-4) mg ml(-1), limits of quantitation for degradation products were in the range 3.43 x 10(-5) to 3.81 x 10(-4) mg ml(-1). The developed method is selective, precise, accurate and sensitive enough for determination of estradiol and its known degradation products.     

反相高效液相色谱法分离测定墨旱莲中的鳢肠醛

对墨旱莲中的有效成分鳢肠醛进行了分离和结构鉴定,并对其含量进行了分析。色谱条件:Kromasil C18柱(200 mm×4.6 mm,5 μm),流动相为乙腈-0.1%磷酸水溶液(体积比为65∶35),流速为1.0 mL/min,柱温为30 ℃,检测波长为365 nm,进样量为10 μL。结果表明,鳢肠醛的峰面积与其质量浓度有良好的线性关系(r=0.9993)。方法的加样回收率为96.7%～100.0%。该方法简便、快速、准确,可作为墨旱莲质量控制的一个有效方法。     

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC

In the present study, a simple and rapid reversed-phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (lambda(detection) = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy.     

高效液相色谱-质谱法测定发酵液中的喷司他丁

建立了测定发酵液中喷司他丁含量的反相高效液相色谱-质谱分析方法。采用的色谱条件: 色谱柱为Hypersil ODS2柱(250 mm×4.6 mm, 5 μm);流动相为甲醇/乙腈/10 mmol/L乙酸铵(pH 7.6)(2.5/2.5/95, v/v/v),流速为1.0 mL/min;检测波长为280 nm;柱温为40 ℃;进样量为10 μL。喷司他丁在1.0～100 mg/L范围内有良好的线性关系,相关系数为0.9999。该方法精密度好,稳定性高,能简便、快速、准确地测定发酵液中喷司他丁的含量。     

AQC柱前衍生化RP-HPLC法测定蒜氨酸及其有关物质的含量

采用6-氨基喹啉-N-(羟基琥珀酰亚胺基)氨基甲酸酯(6-aminoquinolyl -N- Hydroxysuccinimide Carbamate ,AQC)为柱前衍生化试剂,建立了AQC柱前衍生化RP-HPLC法测定蒜氨酸及其有关物质的含量。该衍生化方法反应瞬间完成,衍生化产物稳定。色谱条件为：Kromasil C18柱（250mm×4.6mm,5mm）,流动相A为0.1%乙酸铵(含0.03%乙酸),流动相B为水-乙腈（40∶60）,线性梯度洗脱,流速1.0ml/min,检测波长248nm。蒜氨酸在1.1719~1500μg /ml浓度范围内线性关系良好（r=0.9998）, 日内、日间精密度良好(RSD <1.8%,n=5), 加样回收率为99.1%（RSD1.9%,n=5）,检测限为3ng,该方法准确、方便、快速。     

Quantitative analysis of D-24851, a novel anticancer agent, in human plasma and urine by liquid chromatography coupled with tandem mass spectrometry

The development of a liquid chromatography/tandem mass spectrometric assay for the quantitative analysis of the novel tubulin inhibitor D-24851 in human plasma and urine is described. D-24851 and the deuterated internal standard were extracted from 250 microL of plasma or urine using hexane/ether (1:1, v/v). Subsequently, 10-microL aliquots of reconstituted extracts were injected onto an Inertsil ODS analytical column (50 x 2.0 mm i.d., 5 microm particle size). An eluent consisting of methanol/5 mM ammonium acetate, 0.004% formic acid in water (80:20, v/v) was pumped at a flow rate of 0.2 mL/min. An API 365 triple quadrupole mass spectrometer was used in the multiple reaction monitoring mode for sensitive detection. For human plasma a dynamic range of 1-1000 ng/mL was validated, and for human urine a range of 0.25-50 ng/mL. Validation was performed according to the most recent FDA guidelines and all results were within requirements. The assay has been successfully applied to support a phase I clinical trial with orally administered D-24851.     

高效液相色谱-程序波长紫外检测法同时测定血浆中的色氨酸及其代谢物

建立了一种高效液相色谱-程序波长紫外检测法同时测定血浆中色氨酸(Trp)及其主要代谢产物犬尿氨酸(Kyn)和5-羟色胺(5-HT)。以茶碱为内标(IS),采用BDS-Hypersil-C8柱(150 mm×4.6 mm, 5 μm)分离。流动相为10 mmol/L醋酸钠缓冲液(pH 4.5)-乙腈(94:6, v/v),流速为0.6 mL/min;柱温为25 ℃;紫外检测波长设定: Kyn和IS为360 nm, 5-HT为220 nm, Trp为302 nm。3种物质的平均回收率为87%～113%;线性范围分别为3.97～400 μmol/L(Trp), 0.421～20.2 μmol/L(Kyn), 4.36～980 nmol/L(5-HT);检出限分别为0.134 μmol/L(Trp), 0.0160 μmol/L(Kyn), 2.03 nmol/L(5-HT)。利用该方法对15例抑郁症患者和15例健康志愿者的血浆进行测定,结果表明两组间Trp的代谢存在显著的差异。     

Quantitative determination of DRF-1042 in human plasma by HPLC: validation and application in clinical pharmacokinetics

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DRF-1042, a novel orally active camptothecin (CPT) analog, in human plasma. The sample preparation was a simple deproteinization with acidified methanol yielding almost 100% recovery of DRF-1042. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 x 4.6 mm, 5 microm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a fl ow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. The standard curves were linear (r(2) > 0.999) in the concentration ranges 5.0-2004 ng/mL. The lower limit of quantification (LLQ) of the assay was 5 ng/mL. The mean measured quality control (QC) concentrations (range 5 ng/mL to 40 microg/mL) deviated from the nominal concentrations in the range of -10.5-0.08 and -14.5-7.97%, inter- and intra-day, respectively. The inter- and intra-day precisions in the measurement of QC samples at four tested concentrations, were in the range 0.64-5.89% relative standard deviation (RSD) and 0.33-14.7% RSD, respectively. The method was found to be suitable for measurement of plasma concentrations above the calibration curve after serial dilutions. Stability of DRF-1042 was confirmed in a battery of studies, viz., on bench-top, in the auto-sampler, in the stock solutions, after four quick freeze-thaw cycles, up to one month at -20 degree C in human plasma and up to 2 months in the ex vivo samples. The method is simple, sensitive and reliable and has been successfully implemented to investigate the clinical pharmacokinetics of DRF-1042 in cancer patients in a phase I clinical trial.     

Establishment of a HPLC method for preclinical pharmacokinetic study of the novel anti-Parkinson's disease candidate drug FLZ in rats

An HPLC method was established and validated for the determination of compound FLZ, a synthetic novel anti-Parkinson's disease candidate drug, in rat plasma. FLZ and the internal standard bicyclol were extracted from plasma by solid-phase extraction method and analyzed on a Restek C18 column (4.6 x 250 mm, 5 microm) with a mobile phase consisting of methanol and water (60:40, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 320 nm. The calibration curve was linear within the concentration range from 25 to 500 ng/mL (r2 > 0.999), the limit of quantitation was 25 ng/mL and the average recovery was 92.0% with the RSD less than 5.9%. The relative standard deviation for intra- and inter-day precision was less than 3.8 and 6.9%, respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and applied to study the preclinical pharmacokinetics of FLZ in rat plasma, and it was the first time that the pharmacokinetics of FLZ had been investigated.