HPLC with programmed wavelength fluorescence detection for the simultaneous determination of marker compounds of integrity and P-gp functionality in the Caco-2 intestinal absorption model

A high sensitivity reversed-phase HPLC method is presented for the simultaneous determination of marker compounds of paracellular transport (atenolol), transcellular transport (propranolol) and P-gp functionality (talinolol) in the Caco-2 system. The Caco-2 system is presently commonly accepted as an in vitro cell culture model of the intestinal mucosa. A programmed wavelength fluorescence detection method was used to optimise the response of the marker compounds. This marker compound mixture and the corresponding HPLC assay can be used for in house validation of the Caco-2 system or to evaluate simultaneously the effect of test compounds or absorption enhancing strategies on monolayer integrity and P-gp functionality. The method can easily be adapted to determine the concentration of atenolol, propranolol and talinolol in blood, thus allowing to use the same compounds in the in situ rat perfusion system with blood sampling from the mesenteric vein.     

HPLC程序波长法同时测定黄连解毒汤中栀子苷和4种黄酮类成分的含量

目的:建立一种同时检测黄连解毒汤中栀子苷、黄芩苷、汉黄芩苷、黄芩素和汉黄芩素含量的 HPLC 方法。方法:采用Kromasil C_(18)色谱柱(150 mm×4.6 mm,5 μm),以乙腈(A)-5 mmol·L~(-1)磷酸二氢钠溶液(含0.05%磷酸,pH 3.0)(B)为流动相,线性梯度洗脱(0～8.5 min,86.5%B;8.5～17.0 min,86.5%B→80%B;17～30 min,80%B;30～40 min,80%B→60%B;40～51 min,60%B→52%B;51～55 min,52%B→42%B;55～60 min,42%B→86.5%B;60～65 min,86.5%B),流速1.0 mL·min~(-1),程序可变波长紫外检测(0～15 min,238 nm;15～50 min,276 nm)。结果:在58 min 内黄连解毒汤中栀子苷、黄芩苷、汉黄芩苷、黄芩素和汉黄芩素5种成分被完全分离;回归方程显示5种成分的峰面积与其浓度呈良好的线性;5种成分的加样叫收率(n=15)分别为102.2%,97.56%,98.61%,97.85%,98.41%;RSD 小于5.0%。结论:利用程序可变波长检测,建立了一种可靠的同时测定黄连解毒汤中5种标志性成分含量的 HPLC 方法。     

变波长高效液相色谱法测定布洛芬混悬液中6种防腐剂的含量

目的 建立布洛芬混悬液中6种常用防腐剂的分离测定方法。方法 Agilent C₁₈色谱柱(250×4.6 mm,5 μm),流动相：甲醇-0.02mol.L_-1乙酸铵溶液,梯度洗脱,流速：1.0 mL.min_-1,柱温：30℃,检测波长：变波长。用保留时间定性,以峰面积按标准曲线法定量。结果 6种防腐剂分离良好,在0.002～0.05mg. mL_-1的质量浓度范围内线性关系良好(r≥0.999),平均加样回收率在97.2%～100.6%, RSD%≤2.2%。结论 本法简单、准确,可用于布洛芬混悬液中防腐剂的定性和定量检测。     

多波长HPLC法同时测定青黛药材中靛蓝和靛玉红的含量

目的:利用多波长高效液相色谱法,建立同时测定青黛药材中2种有效成分(靛蓝和靛玉红)含量的方法,并对其质量特征进行分析.方法:采用HPLC法,Phenomenex C18色谱柱(250 mm×4.6 mm,5 μm),柱温30 ℃;以甲醇-水溶液(70∶30)为流动相洗脱,流速1.0 ml/min;检测波长286 nm(检测靛蓝)、292 nm(检测靛玉红),同时测定不同产地青黛药材中靛蓝和靛玉红含量.结果:在20 min内青黛中靛蓝和靛玉红2种有效成分被完全分离.靛蓝在5.19～83.04 μg/ml范围内线性关系良好(r=0.999 9),平均回收率为100.46％,RSD为2.18％;靛玉红在0.364～5.825 μg/ml范围内线性关系良好(r=0.999 9),平均回收率为101.57％,RSD为2.22％.结论:本方法合理、便捷、快速简便、准确、重复性好,能同时测定青黛中靛蓝和靛玉红含量,符合现行《中国药典》简便、快速、准确的要求.     

Validated HPLC method for the determination of ranitidine in plasma

A validated HPLC method for the determination of ranitidine in human plasma is presented. Sulfanilamide as internal standard (IS) was used. Plasma samples were purified by solid phase extraction (SPE) using a copolymeric [poly(divinyl-benzene-co-N-vinylpyrrolidone)] column ("Oasis Waters"). Mobile phase consisting of dibasic potasium phosphate 0.08 M/acetonitrile/methanol/triethylamine 0.05% (89.5:3:7:0.05) pH5 was used at a flow rate of 0.9 ml/min on a C18 column (Nova-Pack, 3,9 x 300 mm, Waters). The eluate was monitored using an UVNis detector set at 300 nm. Ratio of peak area of ranitidine to sulfanilamide was used for the quantitation of plasma samples. FDA criteria for bioanalytical validation was used to validate the method. Linearity was assessed between 100-1600 ng/ml, the limit of quantitation was 100 ng/ml and recovery was greater than 94%. Accuracy, precision and selectivity met the current recommendations for bioanalytical method validation. The method was successfully used in a bioavailability study of a ranitidine tablet in healthy volunteers.     

Rapid and sensitive HPLC method for the simultaneous determination of dorzolamide hydrochloride and timolol maleate in eye drops with diode-array and UV detection

A rapid and sensitive HPLC method has been developed for the simultaneous determination of dorzolamide hydrochloride and timolol maleate. The drugs were monitored with a diode-array detector at two fixed wavelengths (lambda = 250.0 nm for dorzolamide hydrochloride and 300.0 nm for timolol maleate). Liquid chromatography was performed on a RP-YMC pack ODS A-132 C18 (5 microm, 15 cm x 6.0 mm) column and the mobile phase consisted of an acetonitrile: phosphate buffer (pH 2.5): methanol (5:85:10 v/v/v) mix and a flow rate of 1.2 ml x min(-1). The linearity of the method ranged between 4.0-45.0 microg x ml(-1) for dorzolamide hydrochloride and and 2.0-20.6 microg x ml(-1) for timolol maleate in binary mixture. The procedure was successfully applied to the determination of these compounds in pharmaceutical preparations and gave a high recovery, good accuracy and precision without any interference by the excipients.     

波长切换HPLC法同时测定甘草及其炮制品中7个物质的含量

目的:建立高效液相色谱法测定甘草及炮制品中芹糖基甘草苷、甘草苷、芹糖基异甘草苷、异甘草苷、甘草素、甘草酸、异甘草素的含量。方法:采用Dikma Technologies-C18(200 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-0.05%磷酸水溶液,梯度洗脱,流速:0.8mL.min-1,检测波长为276 nm(0～16 min)、360 nm(16～24 min)、276 nm(24～28 min)、248 nm(28～38min)、370 nm(38～42 min),柱温30℃。结果:在本文色谱条件下,芹糖基甘草苷、甘草苷、芹糖基异甘草苷、异甘草苷、甘草素、甘草酸、异甘草素的进样量分别在0.056～0.56,0.095～0.95,0.026～0.26,0.015～0.15,0.013～0.13,0.348～3.48,0.003～0.03μg范围内与色谱峰面积呈良好的线性关系;加样回收率(n=6)均在96.1%～98.1%,RSD均小于2.6%。16个来源甘草药材及其4种不同炮制品中7个物质的含量有一定差异。结论:本法快速、准确,重复性好,可更好地控制甘草药材及其炮制品的质量。     

Improved analytical validation and pharmacokinetics of valsartan using HPLC with UV detection

The primary objective of the study was to validate a simple and sensitive method of determining valsartan concentration in human plasma samples using high performance liquid chromatography (HPLC) combined with ultraviolet (UV) detection. Methanol appeared to be the best with a high recovery efficiency compared to other solvents such as acetonitrile, ethylacetate and methyl-tert-butyl ether. After a simple protein precipitation using methanol, the analytes were separated on a Phenomenex Luna C(18) column using 42% acetonitrile with 15 mM potassium dihydrogenphosphate in water (pH 2.0; adjusted with phosphoric acid) as the mobile phase at a flow rate of 1.2 mL/min. The standard calibration curve constructed in the concentration range of 50-2000 ng/mL showed good linearity (r(2)>0.9997). Spironolactone was used as an internal standard (IS). Valsartan and IS eluted at 10.25 and 12.17 min, respectively. The intra-day and inter-day precision and accuracy were satisfactory with relative standard deviations of less than 15%. No interference peaks or matrix effects were observed in human plasma. Valsartan concentration in human plasma was well established following a single 80 mg oral dose (Diovan capsule) to eight healthy volunteers. The current determination of valsartan concentration by protein precipitation using methanol followed by analysis using HPLC with UV detection was rapid and sensitive, and provide an alternative to the analysis of valsartan by studying its clinical applications.     

Validated HPLC method for determination of sennosides A and B in senna tablets

This study developed an efficient and reliable ion-pair liquid chromatographic method for quantitation of sennosides A and B in commercial senna tablets. Separation was conducted on a Hypersil C 18 column (250 x 4.6 mm, 5 microm) at a temperature of 40 degrees C, using a mixture of 0.1 M acetate buffer (pH 6.0) and acetonitrile (70:30, v/v) containing 5 mM tetrahexylammonium bromide as mobile phase. Sennosides A and B were completely separated from other constituents within 14 min. The developed method was validated. Both run-to-run repeatability (n=10) and day-to-day reproducibility (n=3) of peak area were below 0.4% RSD. Linearity of peak area was tested in the range 30-70 microg/ml (r>0.9997). Accuracy was assessed with recovery and the recoveries for sennosides A and B were 101.73+/-1.30% and 101.81+/-2.18% (n=3 x 6), respectively. Robustness of the analytical method was tested using a three-leveled Plackett-Burman design in which 11 factors were assessed with 23 experiments. Eight factors (column, concentration of ion pair reagent, % of organic modifier (acetonitrile), buffer pH, column temperature, flow rate, time constant and detection wavelength) were investigated in a specified range above and below the nominal method conditions. It was found that: (1) column and % acetonitrile affected significantly resolution and retention time, (2) column, % acetonitrile, column temperature, flow rate and time constant affected significantly the plate number of sennoside A, and (3) column and time constant affected significantly the tailing factor.     

高效液相色谱法同时测定大鼠肠灌流液中L-肌肽和酚红的浓度

目的建立HPLC方法同时测定大鼠肠灌流液中L-肌肽和酚红的质量浓度。方法采用KromasilNH2色谱柱（200mm×4．6mm,5μm）,流动相为乙腈-磷酸盐缓冲液（1mmol．L^-1磷酸二氢钾,0．2mmol．L^-1磷酸氢二钾）（体积比50：50）,流速1．0mL·min^-1,柱温35℃,检测波长210nm,进样量20μL。结果L-肌肽和酚红分别在质量浓度22．6～158mg·L^-1（r=0．9998）、14．2～99．4mg·L^-1（r=0．9991）与峰面积线性关系良好,2种成分回收率分别为99．1％、98．8％,RSD均〈3％。结论所用方法准确、简便、专属性好,可准确快速测定大鼠肠灌流液中L-肌肽和酚红的含量;L-肌肽的摄取过程依赖于H^＋电位梯度。     

Development and validation of a simple HPLC method for simultaneous in vitro determination of amoxicillin and metronidazole at single wavelength

A simple, rapid, sensitive and robust reversed phase-HPLC method was developed and validated to measure simultaneously the amount of amoxicillin and metronidazole at single wavelength (254 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new floating-sustained release tablet formulation and its subsequent stability studies. An isocratic elution of filtered sample was performed on C18 column with buffered mobile phase (pH 4.0) and UV detection at 254 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.15 and 600 microg/ml for amoxicillin and 0.13 and 300 microg/ml for metronidazole were established. Intra and inter-day precision were less than 2.5%. The limits of detection (LOD) and quantification were 0.05 and 0.15 microg/ml for amoxicillin and 0.10 and 0.13 microg/ml for metronidazole. The determination of the two active ingredients was not interfered by the excipients of the products. Samples were stable in the release media (37 degrees C) and the HPLC injector at least for 12 h.     

Validated HPLC method for determination of chlorzoxazone in human serum and its application in a clinical pharmacokinetic study

A high performance liquid chromatographic (HPLC) method for the determination of chloroxazone in human serum using phenacetin as internal standard (IS) is described. Protein precipitation is used for preparation of the sample. A mobile phase consisting of acetonitrile and 0.5% acetic acid in water mixture (40:60 v/v) was used at a flow rate of 1 ml/min on a C18 column. The eluate was monitored using an UV/VIS detector set at 287 nm. Ratio of peak area of analyte to IS was used for quantification of serum samples. The absolute recovery was greater than 96% over a concentration range of 1 to 100 micrograms/ml and the limit of quantitation was 0.05 microgram/ml. The intra-day relative standard deviation (RSD) measured at 1, 10, 50, and 100 micrograms/ml ranged from 0.9 to 5.1%. The inter-day RSD ranged from 0.6 to 3.0%. The method is simple, sensitive and has been successfully used in pharmacokinetic study conducted in healthy human volunteers.     

高效液相色谱切换波长法同时测定大鼠血浆中绿原酸和栀子苷的浓度

目的：建立同时测定大鼠血浆中绿原酸和栀子苷浓度的方法。方法：以甲醇为溶剂,采用蛋白沉淀法处理血浆,采用高效液相色谱（HPLC）法测定药物浓度。色谱柱为大连依利特C₁₈柱（250 mm×4.6 mm,5 μm）,流动相为乙腈-0.1%甲酸（14：86）,内标为紫丁香苷。内标、绿原酸、栀子苷的检测波长分别为266 nm（0~5.9 min）、328 nm（6.0~7.9 min）和234 nm（8.0~12 min）。结果：绿原酸和栀子苷分别在1.625~104.0,1.925~123.2 μg·mL^-1内线性关系良好,回收率分别在92.25%~99.36%和91.96%~98.57%范围内,RSD均低于6%。结论：该方法简便、准确,可用于同时测定绿原酸和栀子苷的血药浓度。     

An HPLC/UV method for the determination of RGH-1756 in dog and rat plasma

RGH-1756 (1-(2-methoxy-phenyl)-4-{4-[4-(6-imidazo[2,1-b]-thiazolyl)-phenoxy]-butyl}-piperazine dimethansulphonate) is a novel atypical antipsychotic candidate of Gedeon Richter Ltd. A new HPLC method has been developed and validated for the quantitative determination of RGH-1756 in dog and rat plasma. The compound and the internal standard are extracted from the biological samples by a simple and fast liquid–liquid extraction method, using 1-chlorobutane. The recovery for RGH-1756 is about 90%. The extracts are analyzed by reversed phase HPLC (column: Supelcosil-LC-18-DB 250*4.6 mm, 5 μm, eluent:acetonitrile:methanol:0.2 molar ammonium-acetate 40:25:35, λ=254 nm). The assay is specific for RGH-1756. The standard curves are linear in the range between 10 and 2000 ng ml⁻¹. The overall precision (expressed as CV%) and accuracy (expressed as bias%) of quality controls and calibration standards are within 15%. The validated lower limit of quantification is 10 ng/ml. No indications have been found for possible instabilities of RGH-1756 in plasma, in the extraction solvent, or after repeated thawing-freezing cycles. The method has been succesfully applied for the bioavailability studies of RGH-1756 in the two animal species. In these studies results of the inprocess method validation have shown the reliability of the method, too. CV% of quality controls in the rat study has been found between 7.4 and 10.0%, in the dog study between 4.1 and 12.5%. The bias has ranged from 0.4 to 3.8% and from −4.5 to 1.2% in the rat and dog study, respectively.     

Validated HPLC method for determination of PAT-5A, an insulin sensitizing agent, in rat plasma

A high performance liquid chromatographic method for the determination of PAT-5A (a potent insulin sensitizer) using DRF-2095 (a thiazolidinedione) as internal standard (I.S.) is described. A 1:1 v/v ethylacetate and dichloromethane solvent mixture was used for extraction of PAT-5A from plasma. A Kromasil KR100-5C18-250A, 5 microm, 4.6 x 250 mm SS column was used for the analysis. Mobile phase consisting of sodium dihydrogen phosphate (pH 4.0, 0.05 M) and methanol mixture (25:75, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector set at 345 nm. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. Using this method the absolute recovery of PAT-5A from rat plasma was > 90% and the limit of quantification was 0.05 microg/ml. The intra-day relative standard deviation (RSD) ranged from 2.19 to 4.98% at 1.0 microg/ml, 1.05 to 3.68% at 10.0 microg/ml and 3.14 to 5.08% at 50 microg/ml. The inter-day RSD were 1.6, 2.24 and 1.54% at 1, 10 and 50 microg/ml, respectively. The method was applied to measure the plasma concentrations of PAT-5A in pharmacokinetic and bioavailability studies in male Wistar rats.     

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C₁₈ column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25–8.0 μg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 μg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 μg/ml, 7.44 to 13.81% at 1.5 μg/ml and 6.10 to 13.94% at 6.0 μg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 μg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.     

HPLC method with fluorescence detection for the determination of ligustilide in rat plasma and its pharmacokinetics

Context: Few methods have been reported for the quantification of ligustilide (LIG) in biosamples: the pretreatment of the biological samples were laborious and time-consuming.

Objective: A high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) for the determination of LIG in rat plasma was developed and validated. Pharmacokinetics and bioavailability of LIG were determined by systematic investigation in Sprague-Dawley rats.

Materials and methods: LIG was isolated from the volatile oil of Radix Angelica sinensis and further purified by silica gel column chromatography. Podophyllotoxin was used as an internal standard. The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 290 and 395?nm during 0–4?min, and 336 and 453?nm during 4–14?min, respectively. LIG pharmacokinetics was studied in rats after oral and intravenous administration of 12.5, 25 and 50?mg/kg doses.

Results: Two calibration curves (Y?=?133.49 X???14.27 (r?=?0.9995), Y?=?145.61 X?+?13.76 (r?=?0.9996)) were constructed in the range of 2.44–10?000?ng/mL for LIG with a lower limit of quantitation of 2.44?ng/mL. Both intra-day and inter-day precision were less than 6%. Accuracy ranged from 88.93 to 99.52%. The recovery ranged from 89.07 to 99.71%. The absolute bioavailability values were 71.36, 68.26 and 75.44% for oral doses of 12.5, 25 and 50?mg/kg, respectively.

Conclusion: The present HPLC-FLD method was rapid, sensitive and reliable. LIG was absorbed and eliminated rapidly in rat.