Determination and assay validation of pinosylvin in rat serum: application to drug metabolism and pharmacokinetics

A method of analysis of pinosylvin in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in natural products. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of pinosylvin and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL) were precipitated with acetonitrile after addition of the internal standard, 7-ethoxycoumarin. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 mm x 4.6 mm, ID, 5 microm) with UV detection at 308 nm. The calibration curves were linear ranging from 0.5 to 100 microg/mL. The mean extraction efficiency was >99%. Precision of the assay (coefficient of variation) was <10%, including the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 15%. The limit of detection was 100 ng/mL for a 0.1 mL sample. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of pinosylvin. Three metabolites of pinosylvin, two oxidative and one glucuronidated, have been identified. The two oxidative metabolites of pinosylvin have been identified as E- and Z-resveratrol.     

Stereospecific high-performance liquid chromatographic analysis of eriodictyol in urine

A stereospecific method of analysis of eriodictyol [5,7,3',4'-tetrahydroxyflavanone] in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism, and tissue distribution in fruits and humans. A simple high-performance liquid chromatographic method was developed for the stereospecific determination of eriodictyol in rat and human urine. Separation was achieved on a Chiralpak OJ-RH column with UV detection at 288 nm. The stereospecific calibration curves were linear ranging from 0.5 to 100 microg/ml. The mean extraction efficiency was >98.8%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 microg/ml). Bias of the assay was lower than 8%, and was within 6% at the limit of quantitation. The assay was applied successfully to the urinary excretion of eriodictyol in rats and humans, and to the stereospecific quantification of eriodictyol in raw lemon juice, conventional and organic lemonade.     

Stereospecific high-performance liquid chromatographic analysis of naringenin in urine

A method of analysis of naringenin [(+/-)-4',5,7-trihydroxyflavanone] in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism, tissue distribution in fruits and humans. A simple high-performance liquid chromatographic method was developed for simultaneous determination of naringenin enantiomers in rat and human urine. Urine (0.1 ml) was precipitated with cold acetonitrile after addition of the internal standard, daidzein. Separation was achieved on a Chiralcel OD-RH column with UV detection at 292 nm. The calibration curves were linear ranging from 0.5 to 100 microg/ml for each enantiomer. The mean extraction efficiency was >99%. Precision of the assay was <9.4% (CV), and was within 5.4% at the limit of quantitation (0.5 microg/ml). Bias of the assay was lower than 16%, and was within 15% at the limit of quantitation. The assay was applied successfully to the urinary excretion of naringenin in rats and humans.     

Stereospecific high-performance liquid chromatographic analysis of hesperetin in biological matrices

A method of analysis of hesperetin (+/--3,5,7-trihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and tissue distribution. A simple high-performance liquid chromatographic method was developed for simultaneous determination of hesperetin enantiomers in rat serum, and rat and human urine. Serum and urine (0.1 ml) were precipitated with cold acetonitrile after addition of the internal standard, 7-methoxycoumarin. Separation was achieved on a Chiralpak AD-RH column with UV detection at 298 nm. The calibration curve was linear ranging from 0.5 to 100 microg/ml for each enantiomer. The mean extraction efficiency was >98%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.5 microg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the urinary excretion of hesperetin in rats and humans.     

High-performance liquid chromatographic analysis of mometasone furoate and its degradation products: application to in vitro degradation studies

A method of analysis of mometasone furoate in pharmaceutical formulations and biological fluids is necessary to study the degradation kinetics and determine its stability. A simple high-performance liquid chromatographic method was developed for simultaneous determination of mometasone furoate and its degradation products in human plasma. Plasma (0.5 ml) was extracted with dichloromethane after addition of the internal standard, dexamethasone 21-acetate. Separation was achieved on a Beckman C(8) column with UV detection at 248 nm. The calibration curve was linear ranging from 0.2 to 100 microg/ml. The mean extraction efficiency was >86%. Precision of the assay was <10% (CV), and was within 10% at the limit of quantitation (0.2 microg/ml). Bias of the assay was lower than 7%. The limit of detection was 50 ng/ml for a 0.5-ml sample. The assay was applied successfully to the in vitro kinetic study of degradation of mometasone furoate in human plasma and simulated biological fluids.     

High-performance liquid chromatography assay of gnetol in rat serum and application to pre-clinical pharmacokinetic studies

A method of analysis of gnetol (2,3′,5′,6-tetrahydroxy-trans-stilbene) in biological fluids is necessary for the study of its kinetics and disposition in plants. A simple high-performance liquid chromatography (HPLC) method was developed for the determination of gnetol in rat serum using a reverse-phase, isocratic system. Separation was achieved using a Phenomenex^® Luna C₁₈ (2) column with ultraviolet detection at 305 nm. The standard curves were linear ranging from 0.5 to 100 μg/ml. The mean extraction efficiency was >90.5%. Precision of the assay was <14% (R.S.D.), and was within 14% at the limit of quantitation (0.5 μg/ml). Bias of the assay was lower than 15%, and was within 15% at the limit of quantitation. The assay was applied successfully to the study of serum disposition of gnetol in rats.     

Stereospecific high-performance liquid chromatographic validation of homoeriodictyol in serum and Yerba Santa (Eriodictyon glutinosum)

A stereospecific method of analysis of racemic homoeriodictyol (eriodictyol 3′-methyl ether) in biological fluids is necessary to study pharmacokinetics and disposition in fruits and herbs. A simple high-performance liquid chromatographic method was developed for the determination of homoeriodictyol enantiomers. Separation was achieved in a Chiralcel^® OJ-RH column with UV-detection at 288 nm. The standard curves in serum were linear ranging from 0.5 to 100.0 μg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 μg/ml). Bias of the assay was <15%, and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of homoeriodictyol enantiomers in rats and to the quantification of homoeriodictyol enantiomers in Yerba Santa (Eriodictyon glutinosum).     

Direct high-performance liquid chromatographic analysis of D-tocopheryl acid succinate and derivatives

A method of analysis of a Vitamin E derivative D-tocopheryl acid succinate (TS) in biological fluids and commercially available products is necessary to study the kinetics of in vitro and in vivo metabolism, tissue distribution, and content uniformity. A simple and inexpensive high-performance liquid chromatographic method was developed for the direct determination of D-tocopheryl acid succinate in commercially available products, rat serum, and rat tissues. This method can also be applied to the determination of 15 Vitamin E derivatives. Rat serum (0.1 ml) was extracted with sodium dodecyl sulfate, ethanol, hexane, and then dried under nitrogen gas after addition of the internal standard, DL-alpha-tocopherol acetate. Separation was achieved on a C18 column with UV detection at 205 nm. The calibration curve for D-tocopheryl acid succinate was linear ranging from 0.025 to 100 microg/ml. The mean extraction efficiency was >92%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.025 microg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the serum and tissue distribution of D-tocopheryl acid succinate in rats, various Vitamin E derivatives, and content uniformity in commercially available products containing D-tocopheryl acid succinate.     

Simultaneous determination of 6,7-dimethylesculetin and geniposide in rat plasma and its application to pharmacokinetic studies of Yin Chen Hao Tang preparation

A method for the rapid and simultaneous determination of 6,7-dimethylesculetin (CAS 120-08-1) and geniposide (CAS 24512-63-8) in rat plasma has been developed, using validated high performance liquid chromatography (HPLC) with solid phase extraction (SPE). The HPLC analysis was performed on a commercially available column (200 mm x 4.6 mm, 5 microm) with acetonitrile-methanol-0.1% aqueous formic acid as mobile phase and the UV detection at 343 nm and 238 nm for 6,7-dimethylesculetin and geniposide, respectively. The calibration curves for 6,7-dimethylesculetin and geniposide were linear over the range 0.4-25.6 microg/mL and 1.12-71.68 microg/mL, respectively. The lower limits of quantitation were 0.40 microg/ mL and 1.12 microg/mL, and the lower limits of detection were 0.06 microg/mL and 0.09 microg/ mL, respectively. The intra-day and inter-day precision for 6,7-dimethylesculetin and geniposide were < 5%, whereas the absolute recovery percentages were > 74%. A successful application of the developed HPLC analysis was demonstrated for the pharmacokinetic study of a Traditional Chinese Medicine formula of Yin Chen Hao Tang preparation.     

Stereospecific high-performance liquid chromatographic analysis of flurbiprofen: application to pharmacokinetic studies

A method of analysis of flurbiprofen (+/- 2-(2-fluoro-4-biphenyl)-propionic acid) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and tissue distribution. A simple high-performance liquid chromatographic method was developed for simultaneous determination of flurbiprofen enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane-isopropanol (95:5, v/v) after addition of the internal standard (IS), S-naproxen and acidification with H(2)SO(4). Separation was achieved on a Chiralpak AD-RH column with UV detection at 247 nm. The calibration curve was linear ranging from 0.05 to 50 microg/ml for each enantiomer. The mean extraction efficiency was >95%. Precision of the assay was <11% (CV), and was within 12.6% at the limit of quantitation (LOQ) (0.05 microg/ml). Bias of the assay was lower than 13.1%, and was within 12.8% at the LOQ. The assay was applied successfully to the in vivo kinetic study of flurbiprofen in rats.     

Stereospecific high-performance liquid chromatographic validation of homoeriodictyol in serum and Yerba Santa (Eriodictyon glutinosum)

A stereospecific method of analysis of racemic homoeriodictyol (eriodictyol 3′-methyl ether) in biological fluids is necessary to study pharmacokinetics and disposition in fruits and herbs. A simple high-performance liquid chromatographic method was developed for the determination of homoeriodictyol enantiomers. Separation was achieved in a Chiralcel^® OJ-RH column with UV-detection at 288 nm. The standard curves in serum were linear ranging from 0.5 to 100.0 μg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 μg/ml). Bias of the assay was <15%, and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of homoeriodictyol enantiomers in rats and to the quantification of homoeriodictyol enantiomers in Yerba Santa (Eriodictyon glutinosum).     

GC-MS-MS determination of gamma-hydroxybutyrate in blood and urine

A gas chromatographic-tandem mass spectrometric (GC-MS-MS) method for determining trace concentrations of gamma-hydroxybutyrate (GHB) in blood and urine has been developed. Multiple reaction monitoring was used to detect parent and daughter ions of GHB, 233 and 147, respectively, following liquid-liquid extraction with acetonitrile and derivatisation with N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA). Deuterated GHB was used as an internal standard. The assay produced excellent linearity and sensitivity without conversion to gamma butyrolactone. The lower limit of quantitation (LLOQ) in 50 microL of sample was 2.5 microg/mL. The expanded uncertainty values for intra- and interassay results were +/- 0.097 and +/- 0.123 ng/mL at a confidence level of 95% for blood and urine, respectively. Endogenous concentrations of GHB were found to be in the range of 0.3 to 6 microg/mL in urine and 0.5 to 2.3 microg/mL in blood, confirming previously suggested cut-off values for forensic analysis.     

Development and Validation of a Rapid RP-HPLC-DAD Analysis Method for the Simultaneous Quantitation of Paclitaxel and Lapatinib in a Polymeric Micelle Formulation

A robust and rapid analysis method was developed and validated for the simultaneous assay of paclitaxel (PTX) and lapatinib (LPT) in a polymeric micelle formulation as a novel drug delivery system using high-performance liquid chromatography (HPLC). The assay was performed using the C18 MZ-Analytical Column (5 μm, 150 × 4.6 mm, OSD-3) which was protected with the C18 pre-column (5 μm, 4.0 × 4.6 mm, OSD-3). The mobile phase was composed of acetonitrile and water (70/30; V/V) with a flow rate of 0.5 mL/min and detection wavelength of 227 nm. Accuracy was reported as the relative error and was found to be less than 6.8%. The interday assay was evaluated to be 3.22% and 5.76% RSD for PTX and LPT, respectively. The intraday precision was found to be at its maximum value of 5.83% RSD. The limit of detection for both PTX and LPT was found to be 1 µg/mL by means of the newly developed method. The limit of quantitation for PTX and LPT was found to be 5 µg/mL. The calibration curves for both drugs were linear in the concentration range of 5 to 80 μg/mL. In vitro release for both drugs from the polymeric micelle was evaluated using the newly developed analysis method.     

UV-spectrophotometric determination of imatinib mesylate and its application in solubility studies

A new, simple and sensitive UV-spectrophotometric method was developed for the determination of imatinib mesylate in bulk and pharmaceutical formulations (tablets and nanoparticles). The developed spectroscopic method was validated for selectivity, linearity and range, precision, accuracy and sensitivity. The method has demonstrated excellent linearity over the range of 2.5-25 microg/mL with regression equation: absorbance (AU) = 0.047 x concentration (microg/mL) + 0.008 and r2 = 0.9998. The developed method demonstrated consistent high recoveries (99-102%) and low relative standard deviation (< 5%) at 285 nm. Moreover, the method was found to be highly sensitive with low limit of detection (0.57 microg/mL) and limit of quantitation (1.71 microg/mL). The apparent molar absorptivity and Sandell's sensitivity was found to be 2.75 x 10(3) L/M cm and 2.15 microg/cm2 respectively. The validated method was successfully employed for the drug content analysis from tablets and nanoparticles preparations. Additionally, the method was successfully employed for pH metric solubility analysis of the drug.     

HPLC assay and pharmacokinetic study of atorvastatin in beagle dogs after oral administration of atorvastatin self-microemulsifying drug delivery system

A specific and accurate reversed-phase HPLC with UV detection was developed for the assay of atorvastatin in beagle dog plasma. Indomethacin was used as the internal standard. Atorvastatin was extracted by protein precipitation, the extracts were injected into a Kromasil C8 column (150 mm x 4.6 mm, 5 microm) with UV wavelength set at 270 nm. The mobile phase consisted of acetonitrile:0.1 mol/L ammonium acetate buffer (pH 4.0) (65:35% v/v) at a flow rate of 1.0 ml/min. The column was at ambient temperature (25 degrees C). The injection volume was 25 microl. The blank plasma did not interfere with the determination of atorvastatin and indomethacin. A good linear relationship was obtained between the peak area ratio of atorvastatin to indomethacin and the concentration of atorvastatin over the range of 0.05 to 2.5 microg/mL. The limit of quantification was 25 ng/mL, the limit of detection was 8 ng/ml. The total chromatographic analysis time was within 9 min. The method is accurate, precise and fast for the assay of atorvastatin in plasma following oral administration of an atorvastatin SMEDDS to healthy beagle dogs.     

Quantitative determination and sampling of azathioprine residues for cleaning validation in production area

Cleaning validation is an integral part of current good manufacturing practices in any pharmaceutical industry. Nowadays, azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9 mm x 150 mm, 4 microm) and methanol-water-acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0 mL min(-1). UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 microg mL(-1) with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit (QL) were 0.09 and 0.29 microg mL(-1), respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. Nevertheless, the simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.     

Fast RPLC-UV method on short sub-two microns particles packed column for the assay of tenoxicam in plasma samples

An extraction-less sample preparation technique followed by a RPLC-UV method on sub-two microns particles packed short column were used for the assay of tenoxicam in plasma samples. Protein precipitation was made by means of trichloroacetic acid addition. Supernatant was injected to the chromatographic column without any further pH adjustment. The mobile phase consisted in a mixture of acetonitrile and aqueous 0.1% phosphoric acid, at 2 mL/min flow rate and gradient elution. The Zorbax SB-C18 column (50 mm length, 4.6 mm internal diameter and 1.8 microm particle size) was thermostated at 60 degrees C. The mobile phase gradient composition program allowed separation of tenoxicam and piroxicam (internal standard), column clean-up and re-equilibration within 4 min. UV detection was achieved at 368+/-10 nm. The method is characterized by a low limit of quantitation of 25 ng/mL for tenoxicam, with a linearity interval up to 5500 ng/mL. The use of a low volume detection cell and detector high frequency data acquisition rate produced high precision and accuracy through a whole bioequivalence study of tenoxicam in two commercially available tablet formulations, after a single oral administration dose. Full method validation is presented. The high throughput characteristic of the proposed method allowed full validation and bioanalytical study completion within a 96 h period.     

An enzyme-linked immunosorbent assay for therapeutic drug monitoring of infliximab

An enzyme-linked immunosorbent assay (ELISA) measuring serum infliximab concentrations in treated patients was developed. Microtiter plates were sensitized with tumor necrosis factor alpha (TNF-alpha) and saturated with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Samples diluted 1:100 in PBS-1% BSA were added and bound infliximab was detected using peroxidase-conjugated goat anti-human immunoglobulin G specific for Fc fragment (HRP-anti hIgG). Reading was performed using an ELISA plate reader. The limit of detection, calculated by assaying 10 replicates of a drug-free serum sample or blank sample and defined as the lowest concentration distinguishable from zero at 2 standard deviations, was 0.014 microg/mL. Each quality control sample was tested on 7 occasions on 1 day and on 5 separate days. The intraday precision indices of the method were (percent coefficients of variation, CV%) 11.7%, 6.2%, and 6.9% for 0.04 microg/mL, 2 microg/mL, and 4.5 microg/mL, respectively. The corresponding bias measures (percent deviation) were -5.5%, -1.9%, and -7.9%, respectively. The between-days precision was 9.8%, 5.3%, and 5.3% for 0.04 microg/mL, 2 microg/mL, and 4.5 microg/mL, respectively. The corresponding bias were +0.3%, -0.3%, and -7.8%, respectively. Lower limit of quantitation and upper limit of quantitation were 0.04 microg/mL and 4.5 microg/mL, respectively. Trough serum concentrations of infliximab were measured in 6 adult patients with various diseases and in 5 pediatric patients with Crohn's disease. For the latter group, samples drawn 1 hour after the end of the infusion and repeated measurements also were available. Data were described using a 1-compartment population pharmacokinetic model. Terminal elimination half-life was 10.9 days. This method is rapid, accurate, and reproducible, and may be useful in therapeutic drug monitoring of infliximab.     

Development and validation of a solid-phase extraction-liquid chromatographic method for determination of amoxicillin in plasma

A bioanalytic method for the determination of amoxicillin in plasma by hydrophilic interaction solid-phase extraction and liquid chromatography has been developed and validated. Plasma was precipitated with acetonitrile before samples were loaded onto a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) solid-phase extraction column. Amoxicillin was analyzed by liquid chromatography on an Aquasil (150 x 4.6 mm) LC column with mobile-phase acetonitrile: phosphate buffer (pH 2.5; 0.1 mol/L) (7:93, v/v) and UV detection at 230 nm. A regression model using 1/concentration weighting was found the most appropriate for quantification. The intraassay precision for plasma was 3.3% at 15.0 microg/mL and 10.9% at 0.200 microg/mL. The interassay precision for plasma was 1.8% at 15.0 microg/mL and 7.5% at 0.200 microg/mL. The total-assay precision for plasma over 4 days using a total of 20 replicates was 13.2%, 5.5%, and 3.8% at 0.200 microg/mL, 3.00 microg/mL, and 15.0 microg/mL, respectively. The lower limit of quantification and the limit of detection were 0.050 microg/mL and 0.025 microg/mL, respectively, for 100 microL plasma. Long-term storage stability studies of amoxicillin in plasma indicate that a temperature of -80 degrees C is necessary to prevent degradation of amoxicillin.     

Comparison of UV spectrophotometric method and high performance liquid chromatography for the analysis of flunarizine and its application for the dissolution test

This study aimed to develop a simple UV spectrophotometric method for the analysis and the dissolution test of flunarizine in capsules. The UV absorbance was both measured directly and by the first derivative measurements at 254 and 268 nm, respectively. The developed methods were validated for their linearity, accuracy, precision, limit of detection (LOD) and limit of quantitation (LOQ) in comparison with the reported HPLC method. The UV spectrophotometric method illustrated excellent linearity (r2 > 0.9999) in the concentration range of 6-24 microg/mL. Precision (%R.S.D. < 1.50) and recoveries were good (%R > 99.62). The LOD of direct UV and first derivative measurements were 0.09 and 0.84 microg/mL, respectively, and the LOQ were 0.26 and 2.55 microg/mL, respectively. Results from the assay of flunarizine in capsules by the UV spectrophotometric methods, both direct and first derivative measurements were not significantly different from those of the HPLC method (P > 0.05). Additionally, the method was successfully used for the dissolution test of flunarizine capsule and was found to be reliable, simple, fast, and inexpensive.