Comparison of a high‐performance liquid chromatography method for quantification of carbamazepine with chemiluminescent microparticle immunoassay

Carbamazepine is an antiepileptic drug widely used for the treatment of epilepsy. In the National Institute of Neurology, monitoring has been performed using the technique chemiluminescent microparticle immunoassay (CMIA) in an automated way during the last five years. The aim of this study was to develop a simple and rapid HPLC analytical method coupled to DAD‐UV detection for the determination of plasma concentrations of carbamazepine and compare its feasibility with those used in routine analysis. The developed HPLC method was fully validated and the applicability of the proposed method was verified through the analysis of plasma samples of patients and later compared with the quantification of the same plasma samples with the CMIA method. The limit of quantification obtained was 0.5 μg/mL. The mean value for recovery was 99.05% and the coefficient of variation (CV) was 5.6%. The precision and accuracy of this method were within the acceptable limits; inter‐ and intraday CV values were <10%. The correlation between the CMIA method and the developed HPLC method was very good (r ≈ 0.999). A Bland–Altman plot showed no significant bias between the results. The HPLC‐DAD method may be an alternative to determine and monitoring the carbamazepine levels in human plasma or serum. Copyright © 2015 John Wiley & Sons, Ltd.     

Validation of a high‐performance liquid chromatography method for the determination of (−)‐α‐bisabolol from particulate systems

A reversed‐phase high performance liquid chromatography method has been developed and validated for determination and quantitation of the natural sesquiterpene (−)‐α‐bisabolol. Furthermore the application of the method was done by characterization of chitosan milispheres and liposomes entrapping Zanthoxylum tingoassuiba essential oil, which contains appreciable amount of (−)‐α‐bisabolol. A reversed‐phase C₁₈ column and gradient elution was used with the mobile phase composed of (A) acetonitrile–water–phosphoric acid (19:80:1) and (B) acetonitrile. The eluent was pumped at a flow rate of 0.8 mL/min with UV detection at 200 nm. In the range 0.02–0.64 mg/mL the assay showed good linearity (R² = 0.9999) and specificity for successful identification and quantitation of (−)‐α‐bisabolol in the essential oil without interfering peaks. The method also showed good reproducibility, demonstrating inter‐day and intra‐day precision based on relative standard deviation values (up to 3.03%), accuracy (mean recovery of 100.69% ± 1.05%) and low values of detection and quantitation limits (0.0005 and 0.0016 mg/mL, respectively). The method was also robust for showing a recovery of 98.81% under a change of solvent in standard solutions. The suitability of the method was demonstrated by the successful determination of association efficiency of the (−)‐α‐bisabolol in chitosan milispheres and liposomes. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for quantitation of indomethacin in rat plasma and its application to a pharmacokinetic study in rats

A highly sensitive and rapid bioanalytical method has been developed and validated for the estimation of indomethacin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of indomethacin and phenacetin (internal standard, IS) from rat plasma with acetonitrile. Chromatographic separation was achieved with 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 3.0 min. The MS/MS ion transitions monitored were 357.7 → 139.1 for indomethacin and 180.20 → 110.10 for IS. Method validation and pharmacokinetic study plasma analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.51 ng/mL and the linearity was observed from 0.51 to 25.5 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.00–10.2 and 5.88–9.80%, respectively. This novel method has been applied to an oral pharmacokinetic study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the quantification of methyl protodioscin in rat plasma: application to a pharmacokinetic study

A high performance liquid chromatography/tandem mass spectrometry assay was first developed and validated for the quantification of methyl protodioscin (MPD), a natural furostanol saponin with distinct antitumor activity, in rat plasma with 17alpha-ethinylestradiol as internal standard (IS). Methanol-mediated protein precipitation was employed for plasma sample pretreatment. The separation was achieved on a C(18) column (150 x 4.6 mm, i.d., 5 microm) by isocratic elution with methanol-water (72:28, v/v) as mobile phase at a flow rate of 1.0 mL/min. Ion acquisition was performed in selective reaction monitoring positive mode by monitoring the transition of m/z 1085.7 --> 1053.7 for MPD, and in selective ion monitoring negative mode by monitoring the deprotonated ion m/z 295.5 for IS. The assay was linear over the concentration range of 2.024-270.0 microg/mL with 2.024 microg/mL as the lower limit of quantification. It was specific, accurate, precise and reproducible with intra- and inter-run RSD <8.3% and RE between -11.5 and 12.8%. The assay was successfully applied to a preclinical pharmacokinetic study after an intravenous dose of 40 mg/kg MPD to rats.     

Development and validation of a RP‐HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C₁₈ column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182–5035 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the range of 1.41–11.2 and 3.66–8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

A simple and sensitive HPLC‐UV determination of 7‐O‐succinyl macrolactin A in rat plasma and urine and its application to a pharmacokinetic study

A simple, sensitive and reproducible isocratic reversed‐phase (C₁₈) high‐performance liquid chromatography (HPLC) method was developed to determine 7‐O‐succinyl macrolactin A (SMA) in rat plasma and urine samples using UV detector set at 230 nm. Lamotrigine was used as internal standards (IS) to ensure the precision and accuracy of the method. The retention times of SMA and IS for the plasma sample were 9.2 and 4.4 min, respectively, and those for the urine samples were 7.9 and 4.3 min, respectively. The intra‐ and inter‐day variations of the analytical responses, expressed in terms of relative standard deviation, were less than 14.9%. The accuracy, in terms of average analytical recovery, ranged from 90.4 to 119%. The lower limits of quantification of SMA in rat plasma and urine samples were 0.02 and 0.1 µg/mL, respectively. This method is applicable for the pharmacokinetic studies of SMA in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

高效液相色谱法同时测定化妆品中的9种水溶性着色剂

建立了用高效液相色谱-二极管阵列检测器(HPLC-DAD)同时检测化妆品中9种水溶性着色剂（溶剂绿7、食品黄3、食品红17、酸性黄1、酸性红33、食品红4、食品红1、橙黄I、酸性橙7）的检测方法。不同种类的化妆品采用不同的样品前处理方法提取后,用Diamonsil C18色谱柱分离,以乙腈-磷酸二氢钾缓冲溶液(pH 6)为流动相进行梯度洗脱,检测波长为240 nm,15 min内可对9种目标物同时进行检测,且各化合物都达到基线分离。经测定,该方法的平均回收率(n = 9)为85.33%～100.2%,相对标准偏差(RSD)为3.68%～8.20%,检出限为0.01～0.1 mg/L。方法简单、快速,能有效地提取、分离和测定化妆品中9种水溶性着色剂。将该方法用于实际化妆品的检测,结果令人满意。     

Development and application of methods for determination of residual monomer in dental acrylic resins using high performance liquid chromatography

Two high-performance liquid chromatographic methods for determination of residual monomer in dental acrylic resins are described. Monomers were detected by their UV absorbance at 230 nm, on a Nucleosil C18 (5 microm particle size, 100 A pore size, 15 x 0.46 cm i.d.) column. The separation was performed using acetonitrile-water (55:45 v/v) containing 0.01% triethylamine (TEA) for methyl methacrylate and butyl methacrylate, and acetonitrile-water (60:40 v/v) containing 0.01% TEA for isobutyl methacrylate and 1,6-hexanediol dimethacrylate as mobile phases, at a flow rate of 0.8 mL/min. Good linear relationships were obtained in the concentration range 5.0-80.0 microg/mL for methyl methacrylate, 10.0-160.0 microg/mL for butyl methacrylate, 50.0-500.0 microg/mL for isobutyl methacrylate and 2.5-180.0 microg/mL for 1,6-hexanediol dimethacrylate. Adequate assay for intra- and inter-day precision and accuracy was observed during the validation process. An extraction procedure to remove residual monomer from the acrylic resins was also established. Residual monomer was obtained from broken specimens of acrylic disks using methanol as extraction solvent for 2 h in an ice-bath. The developed methods and the extraction procedure were applied to dental acrylic resins, tested with or without post-polymerization treatments, and proved to be accurate and precise for the determination of residual monomer content of the materials evaluated.     

Determination of a novel phosphodiesterase4 inhibitor, 3‐[1‐(3cyclopropylmethoxy‐4‐difluoromethoxybenzyl)‐1H‐pyrazol‐3‐yl]‐benzoic acid (PDE‐423) in rat plasma using liquid chromatography–tandem mass spectrometry

A method for determining a novel phosphodiesterase‐4 inhibitor, 3‐[1‐(3cyclopropylmethoxy‐4‐difluoromethoxybenzyl)‐1H‐pyrazol‐3‐yl]‐benzoic acid (PDE‐423), in rat plasma was developed and validated using liquid chromatography–tandem mass spectrometry for further pharmacokinetic study for development as a novel anti‐asthmatic drug. PDE‐423 in the concentration range of 0.02–10 µg/mL was linear with a correlation coefficient of >0.99, and the mean intra‐ and inter‐assay precisions of the assay were 7.50 and 3.86%, respectively. The validated method was used successfully for a pharmacokinetic study of PDE‐423 in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

A robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of 5‐azacytidine

The DNA methyltransferase inhibitor 5‐azacytidine is being evaluated clinically as an oral formulation to treat various solid tumors. A sensitive, reliable method was developed to quantitate 5‐azacytidine using LC‐MS/MS to perform detailed pharmacokinetic studies. The drug of interest was extracted from plasma using Oasis MCX ion exchange solid‐phase extraction 96‐well plates. Chromatographic separation was achieved with a YMC J'sphere M80 C₁₈ column and isocratic elution with a methanol–water–formic acid (15:85:0.1, v/v/v) mobile phase over a 7 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5‐azacytidine. The assay range was 5–500 ng/mL and proved to be accurate (97.8–109.1%) and precise (CV ≤ 9.8%). Tetrahydrouridine was used to stabilize 5‐azacytidine in blood/plasma samples. With the addition of tetrahydrouridine, long‐term frozen plasma stability for 5‐azacytidine at ?70°C has been determined for at least 323 days. The method was applied for the measurement of total plasma concentrations of 5‐azacytidine in a cancer patient receiving a 300 mg oral daily dose. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an RP‐HPLC method for the quantitation of Orteronel (TAK‐700), a CYP17A1 enzyme inhibitor,in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography assay method has been developed and validated for the estimation of Orteronel in rat plasma. The bioanalytical procedure involves extraction of Orteronel and phenacetin (internal standard) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1 mL/min and a C₁₈ column maintained at ambient room temperature. The eluate was monitored using a photodiode array detector set at 242. Orteronel and internal standard eluted at 4.8 and 6.2 min, respectively and the total run time was 9 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 100–3149 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the ranges of 0.31–7.87 and 3.97–6.35, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of Orteronel in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative determination of rupestonic acid in rat plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A sensitive and specific high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC‐ESI‐MS/MS) method was developed and validated for determination of rupestonic acid in rat plasma. Protein precipitation method was used to extract rupestonic acid and the internal standard (IS) warfarin sodium from rats plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol–0.1% formic acid in water (40:60, v/v), pumped at 0.4 mL/min. Rupestonic acid and the internal standard (IS) warfarin sodium were detected at m/z 247.2 → 203.1 and 307.1 → 161.3 in positive ion and multiple reaction monitoring mode respectively. The standard curves were linear over the concentration range of 2.5–5000 ng/mL (r² > 0.99). The within‐day and between‐day precision values for rupestonic acid at four concentrations were 4.7–5.7 and 4.4–8.7%, respectively. The method described herein was fully validated and successfully applied to the pharmacokinetic study after an intravenous administration of rupestonic acid in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, 5‐hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λ_ex/λ_em = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005–1.0 μg/mL for tryptamine, 2‐phenethylamine and spermidine, 0.025–1.0 μg/mL for putrescine, 0.001–1.0 μg/mL for cadaverine, 0.25–20 μg/mL for histamine, 0.25–10 μg/mL for 5–hydroxytryptamine and dopamine, and 0.01–1.0 μg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3–75.0 ng/mL and 1.0–250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra‐day and ≤6.58% for inter‐day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.     

Stability‐indicating HPLC assay for lysine–proline–valine (KPV) in aqueous solutions and skin homogenates

A simple, sensitive and stability‐indicating high‐performance liquid chromatographic (HPLC) assay method was developed and validated for a bioactive peptide, lysine–proline–valine (KPV) in aqueous solutions and skin homogenates. Chromatographic separation was achieved on a reversed phase Phenomenex C₁₈ column (4.6 × 250 mm, packed with 5 µm silica particles) with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). The proposed HPLC method was validated with respect to accuracy, precision, linearity, repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The calibration curve was linear with a correlation coefficient (r) of 0.9999. Relative standard deviation values of accuracy and precision experiments were <2. The LOD and LOQ of KPV were 0.01 and 0.25 µg/mL, respectively. Under stress conditions (acid, alkali and hydrogen peroxide) KPV yielded lys–pro–diketopiperazine as major degradation product, which was identified by flow injection MS analysis. The developed HPLC method was found to be efficient in separating the active peptide from its degradation products generated under various stress conditions. Also, the validated method was able to separate KPV from other peaks arising from endogenous components of the skin homogenate. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioselective HPLC-UV method for determination of eslicarbazepine acetate (BIA 2-093) and its metabolites in human plasma

Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug undergoing clinical phase III trials for epilepsy and phase II trials for bipolar disorder. A simple and reliable chiral reversed-phase HPLC-UV method was developed and validated for the simultaneous determination of eslicarbazepine acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma. The analytes and internal standard were extracted from plasma by a solid-phase extraction using Waters Oasis HLB cartridges. Chromatographic separation was achieved by isocratic elution with water-methanol (88:12, v/v), at a flow rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (beta-cyclodextrin, 5 microm) column at 30 degrees C. All compounds were detected at 225 nm. Calibration curves were linear over the range 0.4-8 microg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4-80 microg/mL for each licarbazepine enantiomer. The overall intra- and interday precision and accuracy did not exceed 15%. Mean relative recoveries varied from 94.00 to 102.23% and the limit of quantification of the assay was 0.4 microg/mL for all compounds. This method seems to be a useful tool for clinical research and therapeutic drug monitoring of eslicarbazepine acetate and its metabolites S-licarbazepine, R-licarbazepine and oxcarbazepine.     

Development and validation of a highly sensitive LC-MS/MS-ESI method for the determination of nobiletin in rat plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of nobiletin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of nobiletin and citalopram (internal standard, IS) from rat plasma with liquid–liquid extraction. Chromatographic separation wa s achieved using an isocratic mobile phase (0.2% formic acid–acetonitrile, 20:80, v/v) at a flow rate of 0.6 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1 °C) with a total run time of 2.0 min. The MS/MS ion transitions monitored were 403.2 → 373.0 for nobiletin and 325.2 → 109.0 for IS. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range extended from 0.05 to 51.98 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.96–14.3 and 6.21–12.1, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous analysis of catechol‐O‐methyl transferase activity,S‐adenosylhomocysteine and adenosine

Novel HPLC method utilizing UV‐detection was developed to analyse catechol‐O‐methyltransferase (COMT) products, vanillic acid and isovanillic acid, S‐adenosylhomocysteine (SAH) and adenosine formed from dihydroxybenzoic acid and S‐adenosyl‐L‐methionine (SAM) by incubation of the rat tissues. Entacapone, a COMT inhibitor, prevented the formation of SAH only partially in the striatal homogenate whereas in the kidney homogenate the increase of SAH was prevented by entacapone. In conclusion, this method was reliable, rapid and simple. COMT seemed to be partially responsible on the SAM utilizing methylations in the striatal homogenates while in the high COMT activity tissue, COMT was the main SAH producing methyltransferase. Copyright © 2009 John Wiley & Sons, Ltd.     

Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84+/-4.6 micromol/mg protein (n=3).