Development and validation of a new HPLC-UV method for the simultaneous determination of triclabendazole and ivermectin B1a in a pharmaceutical formulation

A rapid, simple, and sensitive RP-HPLC analytical method was developed for the simultaneous determination of triclabendazole and ivermectin in combination using a C18 RP column. The mobile phase was acetonitrile-methanol-water-acetic acid (56 + 36 + 7.5 + 0.5, v/v/v/v) at a pH of 4.35 and flow rate of 1.0 mL/min. A 245 nm UV detection wavelength was used. Complete validation, including linearity, accuracy, recovery, LOD, LOQ, precision, robustness, stability, and peak purity, was performed. The calibration curve was linear over the range 50.09-150.26 microg/mL for triclabendazole with r = 0.9999 and 27.01-81.02 microg/mL for ivermectin with r = 0.9999. Calculated LOD and LOQ for triclabendazole were 0.03 and 0.08 microg/mL, respectively, and for ivermectin 0.07 and 0.20 microg/mL, respectively. The intraday precision obtained was 98.71% with RSD of 0.87% for triclabendazole and 100.79% with RSD 0.73% for ivermectin. The interday precision obtained was 99.51% with RSD of 0.35% for triclabendazole and 100.55% with RSD of 0.59% for ivermectin. Robustness was also studied, and there was no significant variation of the system suitability of the analytical method with small changes in experimental parameters.     

Determination and pharmacokinetic study of syringin and chlorogenic acid in rat plasma after administration of Aidi lyophilizer

A high-performance liquid chromatographic (HPLC) method was developed for the first time to simultaneously quantify syringin and chlorogenic acid in rat plasma using wavelength-transfer technology. The analysis was performed on a Diamonsil C(18) column (200 x 4.6 mm i.d., 5 microm particle size) with isocratic mobile phase consisting of acetonitrile-0.05% phosphoric acid (12:88, v/v). The linear ranges were 0.20-10 and 0.25-30 microg/mL, respectively. The lower limits of quantification were 0.20 and 0.25 microg/mL, respectively. The method was shown to be reproducible and reliable with intraday precision below 8.5 and 6.1%, interday precision below 7.1 and 5.5%, accuracy within +/-7.1 and +/-8.6%, and mean extraction recovery excess of 92.1 and 80.9%, respectively, which were all calculated from the blank plasma sample spiked with syringin and chlorogenic acid at three concentrations of 0.20, 1.0 and 6.0 microg/mL for syringin and 0.25, 2.0 and 20 microg/mL for chlorogenic acid. This method was validated for specificity, accuracy and precision and was successfully applied to the pharmacokinetic study of syringin and chlorogenic acid in rat plasma after intravenous administration of Aidi lyophilizer.     

Quantitation of transdermal tadalafil in human skin by reversed-phase high-performance liquid chromatography

A reliable and sensitive HPLC method was developed for the quantitation of tadalafil transdermal permeation through human skin. An RP column with UV detection at 290 nm was used for chromatographic separation at ambient temperature. The mobile phase was acetonitrile-water containing 20 mM pH 7 phosphate buffer (35/65, v/v) with a flow rate of 1.0 mL/min. The LOQ achieved was 1 ng/mL, and the calibration curve showed good linearity over the concentration range of 5-2000 ng/mL for tadalafil, with a determination coefficient (R2) of 0.998. The RSD values of intraday and interday analyses were all within 7%. Parameters of validation proved the precision of the method; this validated method was applied for the determination of tadalafil in transdermal permeation and drug deposition in human skin studies.     

Quantification of controlled release leuprolide and triptorelin in rabbit plasma using electromembrane extraction coupled with HPLC–UV

An electromembrane extraction followed by HPLC–UV technique was developed and validated for quantification of leuprolide and triptorelin in rabbit plasma. The influencing parameters on the extraction efficiency were optimized using experimental design methodology. The optimized conditions were found to be; supported liquid membrane: a mixture of 1‐octanol and 2‐ethyl hexanol (1:1) containing 10% v/v di(2‐ethylhexyl) phosphate, applied voltage: 5 V, extraction time: 5 min, pH of the donor phase: 4.5 and pH of the acceptor phase: 1.0. The optimized method was validated for linearity, intraday and interday precision, and accuracy in rabbit plasma. The range of quantification for both peptides was 0.5–1000 ng/mL with regression coefficients higher than 0.994. Relative recoveries of leuprolide and triptorelin were found to be 80.3 and 75.5%, respectively. Limits of quantification and detection for both peptides were found to be 0.5 and 0.15 ng/mL, respectively. The validated method was successfully applied to pharmacokinetic study of the 1‐month depot formulations of each peptide after subcutaneous administration to rabbits.     

Development and validation of an HPLC/MS/MS method for determining the thiazolidinone PG15 in rat plasma

A rapid, sensitive, and simple HPLC/MS/MS method was developed and validated for the determination of (5Z,E)-3-[2-(4-chlorophenyl)-2-oxoethyl]-5-(1H-indol-3-ylmethylene)-thiazolidine-2, 4-dione (PG15) in rat plasma using chlortalidone as an internal standard (IS). Analyses were performed using a C18 column and isocratic elution with acetonitrile-water (90 + 10, v/v) containing 10 mM ammonium hydroxide (pH 8.0) as the mobile phase pumped at 0.3 mL/min. Detection was performed by MS with negative ion mode electrospray ionization. Rat plasma samples were prepared by deproteinizing with acetonitrile. Detected fragments were 395.1 > 171.9 for PG15 and 337.3 > 189.9 for the IS. Calibration curves were linear from 10 to 1000 ng/mL, with the determination coefficient > 0.99. The intraday and interday precisions were less than 12.2 and 11.3%, respectively. The applicability of the HPLC/MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after oral administration of PG15 to rats, and it provided the necessary sensitivity, linearity, precision, accuracy, and specificity.     

Determination of apomorphine in canine plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to a pharmacokinetic study

An LC-ESI-MS method was developed and validated for the assay of apomorphine in canine plasma using one-step liquid-liquid extraction. The analytes were separated on a Phenomenex Gemini C18 (150 mm x 2.0 mm id 3 microm) column and determined by MS in the positive ion mode. The linear range was 0.4-40 ng/mL with an LOD of 0.2 ng/mL for apomorphine in plasma. The intraday and interday precision and accuracy of quality control samples were < 5.9% RSD and < 7.5% bias for apomorphine. Extraction recoveries were > 80%. The validated method was successfully applied to analyze canine plasma samples in a pharmacokinetic study of apomorphine in dogs and detailed pharmacokinetic parameters were calculated.     

Determination of streptomycin residue in cucumber and Chinese cabbage by high-performance liquid chromatography with postcolumn derivatization and fluorometric detection

A sensitive and accurate method was developed for the determination of streptomycin using HPLC followed by postcolumn derivatization and fluorometric detection. The analyte was extracted, using aqueous solution from cucumber and Chinese cabbage, by a two-step SPE procedure. The extraction, cleanup, and chromatography conditions were optimized, and the performance of the analysis method was evaluated. The conditions of chromatography were as follows: the separation was performed on a C18 column; the isocratic mobile phase consisted of acetonitrile and a mixed solution containing 10 mM sodium 1,2-naphthoquinone-4-sulfonate and 0.4 mM sodium 1-heptanesulfonate (25+75, v/v); and the flow rate was 1 mL/min. The fluorescence detector was set at an excitation wavelength of 263 nm and an emission wavelength of 435 nm. The calibration curve was linear over the range of 50-2000 ng/mL, with a correlation coefficient of 0.9995. The LOD and LOQ were 10 and 30 ng/g, respectively, in both cucumber and Chinese cabbage. The method was validated for selectivity, linearity, precision, and accuracy. The intraday and interday precision and accuracy were within 10%. The mean recoveries from spiked samples were more than 75%, with RSD lower than 10%.     

HPLC determination and pharmacokinetics of chlorogenic acid in rabbit plasma after an oral dose of Flos Lonicerae extract

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of chlorogenic acid (3-O-caffeoyl-D-quinic acid) in plasma and applied to its pharmacokinetic study in rabbits after administration of Flos Lonicerae extract. Plasma samples are extracted with methanol. HPLC analysis of the extracts is performed on a C(18) reversed-phase column using acetonitrile-0.2% phosphate buffer (11:89, v/v) as the mobile phase. The UV detector is set at 327 nm. The standard curves are linear in the range 0.0500-1.00 microg/mL (r = 0.9987). The mean extraction recovery of 85.1% is obtained for chlorogenic acid. The interday precision (relative standard deviation) ranges from 5.0% to 7.5%, and the intraday precision is better than 9.0%. The limit of quantitation is 0.0500 microg/mL. The plasma concentration of chlorogenic acid shows a C(max) of 0.839 +/- 0.35 microg/mL at 34.7 +/- 1.1 min and a second one of 0.367 +/- 0.16 microg/mL at 273.4 +/- 39.6 min.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM02734, a novel antineoplastic agent, in dog plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM02734, in dog plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM02734 was established using PM02734 standards from 0.05 to 100 ng/mL in blank plasma. The dominating ions were doubly charged molecular ions [M+2H]2+ at m/z 740.0 instead of singly charged ones at m/z 1478.4. The selected reaction monitoring (SRM), based on the m/z 740.0 --> 212.2 transition, was specific for PM02734, and that based on the m/z 743.8 --> 212.2 transition was specific for deuterated PM02734 (the internal standard, IS); no endogenous materials interfered with the analysis of PM02734 and IS from blank plasma. The assay was linear over the concentration range 0.05-100 ng/mL. In terms of sensitivity of assay 0.05 ng/mL is a very low LLOQ, especially considering PM02734 is a peptide. The correlation coefficients for the calibration curves ranged from 0.9990 to 0.9999. The mean intraday and interday accuracies for all calibration standards (n = 9) ranged from 93 to 111% (< or =11% bias) in dog plasma, and the mean interday precision for all calibration standards was less than 6.4%. The mean intra- and interday assay accuracy for all quality control replicates in dog plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 85-111% (< or =15% bias) and from 99-109% (< or =9% bias), respectively. The mean intra- and interday assay precision was less than 12.1 and 13.3% for all QC levels, respectively. The assay has been used to support preclinical pharmacokinetic (PK) and toxicokinetic studies. The results showed that preclinical samples could be monitored for PM02734 up to 168 h after dosing, which allowed us to identify multiple elimination phases and accurately estimate PK information.     

Determination of antiviral nucleoside analogues AM365 and AM188 in perfusate and bile of the isolated perfused rat liver using HPLC

Development, validation and application of an HPLC assay for new antiviral nucleoside analogues AM365 and AM188 in isolated perfused rat liver perfusate and bile were performed. An analytical column (Phenosphere-NEXT, 250 x 4.6 mm, C(18), 4 microm, Phenomenex) was used in tandem with a guard column (4 x 3 mm, C(18), Phenomenex) and operated at 25 degrees C. The mobile phase [methanol:10 mmol/L sodium orthophosphate buffer (pH 7.0), 15:85, v/v] was pumped at 1 mL/min. The signal from a diode array detector was collected from 190 to 300 nm. The chromatogram was processed at 220 and 252 nm for AM365 and AM188, respectively. The HPLC method was validated by six intraday and seven interday runs. Standard curves were linear in the range 0.125-8.00 microg/mL for AM365 and AM188, and the lower limit of quantification for AM365 and AM188 was 0.125 microg/mL. Mean interday precision and accuracy of IPL perfusate quality control samples were within 8.8%, and mean intraday precision and accuracy were within 13.1%. The assay has been successfully used in the study of metabolism and disposition of AM365 in the isolated perfused rat liver.     

Liquid chromatographic determination of cetirizine in oral formulations

The development and validation of a reversed-phase liquid chromatographic (LC) method for the determination of cetirizine dihydrochloride in oral formulations are described. An isocratic LC analysis was performed on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size). The mobile phase was 1% orthophosphoric acid solution, pH 3.0-acetonitrile (60 + 40, v/v), pumped at a constant flow rate of 1.0 mL/min. Measurements were made at a wavelength of 232 nm. The calibration curves were linear over the range of 10-30 microg/mL (r2 = 0.9999). The relative standard deviation (RSD) values for intraday precision were 0.94 and 1.43% for tablets and compounded capsules, respectively. The RSD values for interday precision were 0.13 and 0.82% for tablets and compounded capsules, respectively. Recoveries ranged from 97.7 to 101.8% for tablets and from 98.4 to 102% for compounded capsules. No interferences from the excipients were observed. Because of its simplicity and accuracy, the method is suitable for routine quality-control analysis for cetirizine in tablets and compounded capsules.     

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.     

High-performance liquid chromatographic determination of doxazosin in human plasma for bioequivalence study of controlled release doxazosin tablets

A highly sensitive high-performance liquid chromatographic quantification method with fluorescence detection was developed and validated for the determination of doxazosin in human plasma. The developed method employed one-step extraction of doxazosin from plasma matrix with ethyl acetate using propranolol as an internal standard. Chromatographic separation was obtained within 8.0 min using a reverse-phase Capcell-Pak C(18) column (150 x 4.6 mm i.d., 5 microm) and the mobile phase consisted of methanol-water containing 10 mM perchloric acid and 1.8 mM sodium heptane sulfonic acid (50:50, v/v) and was set at a flow rate of 1.5 mL/min. The calibration curve constructed was linear in the range of 0.3-50.0 ng/mL. The proposed method achieved a lower limit of quantification of 0.3 ng/mL, better than the reported HPLC methods. Average recoveries of doxazosin and the internal standard from human plasma matrix were 87.0 and 85.9%, respectively. The present method was validated by evaluating the precision and accuracy for inter- and intraday variation in the concentration range 0.3-50 ng/mL. The precision values expressed as relative standard deviations in the inter- and intraday validation were 1.17-6.29 and 0.84-5.94%, respectively. This method was successfully applied to the bioequivalence study of two doxazosin controlled release tablets in healthy, male human subjects.     

Cloud-point extraction combined with HPLC for determination of larotaxel in rat plasma: a pharmacokinetic study of liposome formulation

A simple and efficient method based on cloud-point extraction combined with high-performance liquid chromatography was developed and validated for the determination of larotaxel in rat plasma. Nonionic surfactant Triton X-114 was chosen as the extraction solvent. Variable parameters affecting the cloud-point extraction efficiency, for example the nature and concentration of surfactant, NaOH concentration, incubation temperature, and time were evaluated and optimized. Chromatographic separation was accomplished on a Diamonsil C(18) column (150 mm × 4.6 mm, 5 μm) using a mobile phase consisting of acetonitrile and 0.1% phosphoric acid with pH 4.0 (60:40, v/v). The calibration curve showed good linearity over the range of 0.05-10 μg/mL. Under the optimum conditions, the method was shown to be reproducible and reliable with intraday precision below 5.7%, interday precision below 7.2%, accuracy within ±3.5%, and mean extraction recovery of 91.8-94.2%. The validated method was successfully applied to the pharmacokinetic study of larotaxel in rat plasma after a single intravenous administration of larotaxel injection and larotaxel-loaded liposome, respectively. The results indicated that the larotaxel-loaded liposome led to significant differences in pharmacokinetic profile.     

Pharmacokinetic application of a bio‐analytical LC‐MS method developed for 5‐fluorouracil and methotrexate in mouse plasma,brain and urine

In the past we have reported significant cognitive deficits in mice receiving 5‐fluorouracil in combination with low‐dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio‐analytical method was therefore developed and validated for 5‐fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer. Chromatographic separation was accomplished by Agilent® Zorbax® SB‐C₁₈ column, with isocratic elution (5 mM ammonium acetate and methanol, 70:30, %v/v) at a flow rate of 300 μL/min. The limit of quantitation for both drugs was 15.6 ng/mL (plasma and brain) and 78.1 ng/mL (urine), with interday and intraday precision and accuracy ≤15% and a total run time of 6 min. This bio‐analytical method was used for the pharmacokinetic characterization of 5‐fluorouracil and methotrexate in mouse plasma, brain and urine over a period of 24 h. This method allowed characterization of the brain concentrations of 5‐fluorouracil over a period of 24 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of naproxen in human plasma by GC–MS

This paper describes a GC–MS method for the determination of naproxen in human plasma. Naproxen and internal standard ibuprofen were extracted from plasma using a liquid–liquid extraction method. Derivatization was carried out using N‐methyl‐N‐(trimethylsilyl)trifluoroacetamide. The calibration curve was linear between the concentration range of 0.10–5.0 μg/mL. Intra‐ and interday precision values for naproxen in plasma were <5.14, and accuracy (relative error) was better than 4.67%. The extraction recoveries of naproxen from human plasma were between 93.0 and 98.9%. The LOD and LOQ of naproxen were 0.03 and 0.10 μg/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of naproxen in six healthy Turkish volunteers who had been given 220 mg naproxen.     

高效液相色谱法同时测定发酵液中赤藓糖醇和L-赤藓酮糖的含量

建立了采用高效液相色谱(HPLC)同时测定发酵液中底物赤藓糖醇和产物L-赤藓酮糖含量的方法。采用Lichrospher 5-NH2色谱柱(250 mm×4.6 mm),柱温30 ℃,以乙腈-水(体积比为9:1)为流动相,流速1.0 mL/min。用示差折光检测器检测赤藓糖醇,检测器温度为35 ℃。用紫外检测器在室温下检测L-赤藓酮糖,检测波长为277 nm。所得赤藓糖醇的线性范围为1.00～100.00 g/L,相关系数为0.9985,检出限为0.10 g/L,定量限为0.45 g/L;所得L-赤藓酮糖的线性范围为1.00～100.00 g/L,相关系数为0.9958,检出限为0.50 g/L,定量限为0.87 g/L;赤藓糖醇的日内和日间相对标准偏差(RSD)分别小于3.28%和5.30%, L-赤藓酮糖的日内和日间RSD分别小于2.16%和2.25%;回收率均大于99%。取不同时间的发酵液样品分别用上述方法测定,结果表明所建立的HPLC法不受发酵液中其他组分的影响,可同时测定底物赤藓糖醇和产物L-赤藓酮糖的含量。     

Determination of 3-hydroxy pterocarpan, a novel osteogenic compound in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetics study

A rapid, sensitive and selective LC‐MS/MS method for the quantitative analysis of 3‐hydroxy pterocarpan (S006‐1709) in female rat plasma has been developed and validated. A Discovery RP₁₈ column was used for the chromatographic elution using acetonitrile and 0.1% acetic acid in water as mobile phase (80:20 v/v) at the flow rate of 0.5 mL/min. MS/MS analysis was performed using a triple quadrupole mass spectrometer with electrospray ionization in negative ion mode using biochanin as an internal standard (IS). Extraction of S006‐1709 and IS from rat plasma was done by liquid–liquid extraction method using diethyl ether. The LC‐MS/MS method was sensitive with 1.95 ng/mL as the limit of detection and 3.9 ng/mL as the lower limit of quantification. The method was linear in the concentration range of 3.9–1000 ng/mL. The percentage bias for intraday and interday accuracy was not greater than 4.2 and the %RSD for intraday and interday precision was not greater than 13.2. The recoveries of S006‐1709 and IS were 73.9–79.3 and 85.7%, respectively. S006‐1709 was found to be stable in various stability studies. The validated LC‐MS/MS method was successfully applied for the oral pharmacokinetics study of S006‐1709 at 10 mg/kg in female Sprague–Dawley rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Enantiomeric separation of mirtazapine and its metabolites by nano-liquid chromatography with UV-absorption and mass spectrometric detection

Mirtazapine (MIR) and two of its main metabolites, namely, 8-hydroxymirtazapine and N-desmethylmirtazapine, were separated in totheir enantiomers by nanoLC in a laboratory-made fused-silica capillary column (75 microm ID) packed with a vancomycin-modified silica stationary phase. The simultaneous separation of the three couples of the studied enantiomers was achieved in less than 33 min, using an experimentally optimized mobile phase delivered in the isocratic mode. Optimization of the mobile-phase composition was achieved by testing the influence of the buffer pH and concentration, the water concentration, the organic modifier type and concentration, and on the retention and resolution of the analytes. The optimum mobile-phase composition contained 500 mM ammonium acetate pH 4.5/water/MeOH/MeCN, 1:14:40:45 v/v/v/v. Using a UV detector at 205 nm, the method was validated studying several experimental parameters such as LOD and LOQ, intraday and interday repeatability, and linearity. Good results were achieved: LOD and LOQ were in the range 5-15 and 10-40 microg/mL, respectively (the highest value was obtained for the DEMIR enantiomers); correlation coefficients, 0.9993-0.9999; the intraday and interday precision was acceptable (RSD < 2%) using an internal standard. The method was tested for the separation of the studied enantiomers in an extracted (solid-phase) serum sample spiked with standard racemic mixture of MIR and its two metabolites. Finally, the nanoLC system was connected to a mass spectrometer through a nanoelectrospray interface and the MS, MS2, and MS3 spectra were acquired showing the potential of the system used for characterization and identification of the separated analytes.     

Development and validation of a high-performance liquid chromatographic method for determination of eprosartan in bulk drug and tablets

A simple, precise, and accurate isocratic RP-HPLC method was developed and validated for determination of eprosartan in bulk drug and tablets. Isocratic RP-HPLC separation was achieved on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase 0.5% formic acid-methanol-acetonitrile (80 + 25 + 20, v/v/v, pH 2.80) at a flow rate of 1.0 mL/min. The retention time of eprosartan was 7.64 +/- 0.05 min. The detection was performed at 232 nm. The method was validated for linearity, precision, accuracy, robustness, solution stability, and specificity. The method was linear in the concentration range of 10-400 microg/mL with a correlation coefficient of 0.9999. The repeatability for six samples was 0.253% RSD; the intraday and interday precision were 0.21-0.57 and 0.33-0.71% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.86-100.92%. The drug was subjected to the stress conditions hydrolysis, oxidation, photolysis, and heat. Degradation products produced as a result of the stress conditions did not interfere with detection of eprosartan; therefore, the proposed method can be considered stability-indicating.