LC Quantification of RT-B: The Active Metabolite of a New Resveratrol Derivative RT-A in Rat Plasma

RT-A, a new prodrug based on resveratrol, is currently under investigation. Preclinical studies in rats indicate that RT-A is readily absorbed and rapidly split into an active metabolite RT-B by lysase of the ester bond. An LC method was developed for the determination of RT-B in rat plasma. The assay was performed on a 5 μm Elite C₁₈ column (200 mm × 4.6 mm) with a mobile phase consisting of acetonitrile–0.1% phosphoric acid (28:72, v/v, pH 1.8) at a flow rate of 1.0 mL min^?1. Detection was at 318 nm, and baicalin was used as an internal standard. Calibration was linear over the range of 0.04–10 μg mL^?1 with a correlation coefficient of 0.9994. The mean extraction recoveries of RT-B determined over the concentrations of 0.1, 1.0, and 5.0 μg mL^?1 were (86.5 ± 6.8) %, (82.6 ± 2.0) %, and (92.7 ± 7.9) %. The RSD of intra- and inter-day precisions were all less than 10%. This method was successfully applied to evaluate the pharmacokinetics of RT-B after intravenous administration of RT-A.     

Determination of Phenolic Acids in Root Exudates of Allelopathic Rice by Solid Phase Extraction-Ion Chromatography with Conductivity Detection

A simple, effective, and green ion chromatography method with conductivity detection was developed for the determination of benzoic acid, cinnamic acid, syringic acid, and p-hydroxybenzoic acid in the root exudates of allelopathic rice. The analytes were well separated within 25 min in an anion exchange column (150 mm × 4.0 mm i.d., 5 µm particle size) with mixtures of 6.4 m mol L^?1 Na₂CO₃ and 2.0 m mol L^?1 NaHCO₃ as the mobile phase at a flow-rate of 0.7 mL min^?1. Detection limits of benzoic acid, cinnamic acid, syringic acid, and p-hydroxybenzoic acid were 0.05, 0.20, 0.50, and 0.05 µg mL^?1, respectively. Intra- and inter-day precision was ≤4.0% and 3.2%, respectively, and the intra- and inter-day accuracy, indicated by relative error, ranged from ?8.0% to 9.0%. The developed method was successfully used to determine phenolic acids in the root exudates of allelopathic rice. The average recoveries of the analytes were between 90.7% and 103.0%.     

Determination of Tectorigenin in Rat Plasma: Application to a Pharmacokinetic Study After Oral Administration of Tectorigenin or Its Prodrug Tectoridin

A simple, sensitive, and validated liquid chromatographic method has been developed for the determination of tectorigenin in rat plasma and application to a pharmacokinetic study after oral administration of tectorigenin or its prodrug tectoridin. The analysis was performed on a Kromasil C₁₈ analytical column using gradient elution with acetonitrile 0.1% phosphonic acid water at 0.8 mL min^?1. The detection wavelength for UV detection was set at 264 nm. The established method was fully validated with parameters as follows: the intra- and inter-day assay precisions (CV) of three analytes were in the range of 4.2–13.3% and accuracies were between 98.0 and 107.5%; the calibration curve was linear with r ² > 0.99 over a concentration range of 0.02–2 μg mL^?1; the lower limit of quantification was 0.02 μg mL^?1; tectorigenin showed stable in rat plasma after 12 h incubation at room temperature, 15 days storage at ?80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 24 h at 25 °C; and the mean recoveries of tectorigenin were 92.3 ± 3.2, 95.5 ± 2.9 and 94.5 ± 3.0% with quality control levels of 0.02, 0.2 and 2 μg mL^?1, respectively. In conclusion, this method is simple, economic, and sensitive enough for in vivo pharmacokinetic studies of tectorigenin.     

Determination of Tectorigenin in Rat Plasma: Application to a Pharmacokinetic Study After Oral Administration of Tectorigenin or Its Prodrug Tectoridin

A simple, sensitive, and validated liquid chromatographic method has been developed for the determination of tectorigenin in rat plasma and application to a pharmacokinetic study after oral administration of tectorigenin or its prodrug tectoridin. The analysis was performed on a Kromasil C₁₈ analytical column using gradient elution with acetonitrile 0.1% phosphonic acid water at 0.8 mL min⁻¹. The detection wavelength for UV detection was set at 264 nm. The established method was fully validated with parameters as follows: the intra- and inter-day assay precisions (CV) of three analytes were in the range of 4.2–13.3% and accuracies were between 98.0 and 107.5%; the calibration curve was linear with r ² > 0.99 over a concentration range of 0.02–2 μg mL⁻¹; the lower limit of quantification was 0.02 μg mL⁻¹; tectorigenin showed stable in rat plasma after 12 h incubation at room temperature, 15 days storage at −80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 24 h at 25 °C; and the mean recoveries of tectorigenin were 92.3 ± 3.2, 95.5 ± 2.9 and 94.5 ± 3.0% with quality control levels of 0.02, 0.2 and 2 μg mL⁻¹, respectively. In conclusion, this method is simple, economic, and sensitive enough for in vivo pharmacokinetic studies of tectorigenin.

     

Determination of Free and Total Piperacillin–Tazobactam in Plasma by HPLC–MS–MS: An Adapted Method for Neonates

A sensitive and specific liquid chromatography electrospray ionization-tandem mass spectrometry method for determination of total and free piperacillin–tazobactam in human plasma has been developed and validated. Plasma deproteinization was achieved with Amicon^® Ultra-0.5 mL centrifugal filter device (Millipore, Bedford, USA). Chromatography was performed on a Capcell Pak C₁₈ MG column (ID 2 mm × 100 mm, 5 μm, Shiseido, Kyoto, Japan) with isocratic elution using a mobile phase containing water and acetonitrile with an addition of 0.02% of formic acid. Detection was achieved by an Applied Biosystems API 3000 triple quadrupole mass spectrometer (ABI-SCIEX, Toronto, Canada). Electrospray ionization (ESI) was used for ion production. The limits of quantification were 100 ng mL⁻¹ for piperacillin and 30 ng mL⁻¹ for tazobactam. The precision and accuracy for both intra- and inter-day determination of piperacillin ranged from 2.8 to 9.1% and from 94.9 to 104.4%. The precision and accuracy for intra- and inter-day determination of tazobactam ranged from 2.9 to 9.3% and from 88.9 to 99.8%. The precision and accuracy for intra- and inter-day determination of free piperacillin ranged from 4.4 to 14.7% and from 89.0 to 109.6%. The precision and accuracy for intra- and inter-day determination of free tazobactam ranged from 2.8 to 14.4% and from 93.9 to 108.0%. Fifty and 150 μL plasma were used for total and free piperacillin–tazobactam analysis, respectively. The validation results of this analytical method made it feasible for being used in a further pilot study of population pharmacokinetics of piperacillin–tazobactam in neonates.

     

LC Determination of a Novel Synthetic Thiazolidinedione (BP-1107) in Rat Plasma and Its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatographic method for the quantification of BP-1107 in rat plasma has been established. Plasma samples were prepared by extraction with tert-butyl methyl ether, and troglitazone was used as an internal standard. The analytical separation was performed on a C₁₈ column using acetonitrile–0.3% phosphoric acid in water (pH 4.00 adjusted with triethylamine) (75:25, v/v) as a mobile phase. A detailed validation of the method was performed as per USFDA guidelines. For BP-1107 at the concentrations of 2.42, 16.11 and 32.22 μg mL^?1 in rat plasma, the extraction recoveries were 114.14 ± 9.75, 95.37 ± 12.06 and 90.00 ± 6.46%, respectively. The mean recovery for internal standard was 91.96 ± 2.51%. The lower limit of quantitation of BP-1107 was 16 ng. The linear quantification range of the method was 0.81–53.70 μg mL^?1 in rat plasma with a correlation coefficient greater than 0.999. The intra-day and inter-day accuracy for BP-1107 at 2.42, 16.11 and 32.22 μg mL^?1 levels in rat plasma fell between 97.10–110.02 and 97.52–108.04%. The intra-day and inter-day precision were in the ranges of 1.91–5.63 and 4.43–6.28%, respectively. The method was successfully applied to a pharmacokinetic study of BP-1107 in rats after an intravenous administration.     

HPLC Analysis and Pharmacokinetic Study of Paeoniflorin after Intravenous Administration of a New Frozen Dry Powder Formulation in Rats

A simple and specific high performance liquid chromatographic (HPLC) method with UV detection using picroside II as the internal standard was developed and validated to determine the concentration of paeoniflorin in rat plasma and study its pharmacokinetics after an single intravenous administration of 40 mg kg^?1 paeoniflorin to Wistar rats. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 0.05 mol L^?1 NaH₂PO₄ solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB C₁₈ column (250 × 4.6 mm I.D., 5 μm) with a Shim-pack GVP-ODS C₁₈ guard column (10 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–water–acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 mL min^?1. The UV detection was performed at a wavelength of 230 nm. The linear calibration curves were obtained in the concentration range of 0.05–200.0 μg mL^?1 in rat plasma with the lower limit of quantification (LLOQ) of 0.05 μg mL^?1. The intra- and inter-day precisions in terms of % relative standard deviation (RSD) were lower than 5.7 and 8.2% in rat plasma, respectively. The accuracy in terms of % relative error (RE) ranged from ?1.9 to 2.6% in rat plasma. The extraction recoveries of paeoniflorin and picroside II were calculated to be 69.7 and 56.9%, respectively. This validated method was successfully applied to the pharmacokinetic study of a new paeoniflorin frozen dry power formulation. After single intravenous administration, the main pharmacokinetic parameters t _1/2, AUC_0-∞, CL_TOT, V _Z, MRT_0-∞ and V _ss were 0.739 ± 0.232 h, 43.75 ± 6.90 μg h mL^?1, 15.50 ± 2.46 L kg^?1 h^?1, 1.003 ± 0.401 L kg^?1, 0.480 ± 0.055 h and 0.444 ± 0.060 L kg^?1, respectively.     

LC Assay of Eletriptan in Tablets and In Vitro Dissolution Studies

Eletriptan (ELT) is a new selective serotonin agonist approved for the treatment of acute migraine headaches. A simple and rapid liquid chromatographic method was developed and validated for the assay of ELT in tablets. Chromatography was carried out on a 250 mm × 4.6 mm C₁₈ column at 30 °C. Acetonitrile–15 mM triethylamine solution (adjusted to pH 7.0 using concentrated o-phosphoric acid) (60:40, v/v) mixture was used as mobile phase at 1.0 mL min^?1 flow rate and UV detector was set at 225 nm. A linear response (r ²  = 0.9999) was observed in the range of 0.1–1.6 μg mL^?1. The method showed good recoveries (100.08 %) and the RSD values for intra- and inter-day precision were 0.78–1.93 and 1.10–2.15%, respectively. The method can be used for quality control assays and in vitro dissolution studies of ELT in tablets.     

Determination of Pyrethroid Pesticides in Environmental Waters Based on Magnetic Titanium Dioxide Nanoparticles Extraction Followed by HPLC Analysis

A simple and fast method based on magnetic separation for extraction of pyrethroid pesticides including beta-cyfluthrin, cyhalothrin and cyphenothrin from environmental water samples has been established. Magnetic titanium dioxide was used as sorbent, which was synthesized by coating TiO₂ on Fe₃O₄ in liquid-state co-precipitation method. The sorbent has been characterized by scanning electron microscopy and Fourier-transform infrared spectrometry, and the magnetic properties were investigated with physical property measurement system. Various parameters affecting the extraction efficiency were evaluated to achieve optimal condition and decrease ambiguous interactions. The analytes desorbed from the sorbent were detected by high performance liquid chromatography. Under the optimal condition, the linearity of the method is in the range of 25–2,500 ng L^?1. The detection limits and quantification limits of pyrethroid pesticides are in the range of 2.8–6.1 ng L^?1 and 9.3–20.3 ng L^?1, respectively. The relative standard deviations of intra- and inter-day tests ranging from 2.5 to 7.2 % and from 3.6 to 9.1 % were obtained. In all three spiked levels (25, 250 and 2,500 ng L^?1), the recoveries of pyrethroid pesticides were in the range of 84.5–94.1 %. The proposed method was successfully applied to determine pyrethroids in three water samples. Cyphenothrin was found in one river water near farmlands, and its concentration was 52 ng L^?1.     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL⁻¹. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL⁻¹) and limit of quantitation (10.16 ng mL⁻¹). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.

     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL^?1. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL^?1) and limit of quantitation (10.16 ng mL^?1). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.     

Simultaneous Estimation of Vinpocetine and Its Metabolite, Apovincaminic Acid, in Beagle Plasma by LC-MS-MS

A method was developed to determine vinpocetine and its metabolite, apovincaminic acid, in beagle plasma by LC-MS-MS. After protein precipitation with methanol, the supernatant of the sample was concentrated and injected into an Agilent Zorbax XDB-C₁₈ column. The sample was separated by a mobile phase consisting of acetonitrile and 0.2% formic acid solution, and the reading was determined on an Agilent 6410 Triple Quad Tandem mass spectrometer in multiple reaction monitoring mode with the following transitions: m/z 351.5 ?? 280.2/266.3 for vinpocetine, 323.2 ?? 236.1/280.2 for apovincaminic acid, and 411.2 ?? 191.1 for the internal standard. The intra- and inter-day variances were less than 15% (RSD%), and average recoveries were higher than 80%. The linearity ranges (LR) between 0.1 and 20.0 ng mL^?1 for vinpocetine (r ² = 0.9980) and between 1.0 and 200.0 ng mL^?1 for apovincaminic acid (r ² = 0.9995) were established. In summary, this method is sensitive, specific, and appropriate for in vivo study of various dosage forms of vinpocetine.     

Simultaneous Determination of Ramipril and Its Active Metabolite Ramiprilat in Human Plasma by LC–MS–MS

A selective, rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed for the simultaneous determination of ramipril and ramiprilat in human plasma using enalapril as the internal standard via one-step extraction with ethyl acetate under acidic condition. The analysis was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of 1% formic acid-acetonitrile (25:75, v/v) at a constant flow rate of 0.5 mL min^?1. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring mode via electrospray ionization. Linear calibration curves of ramipril and ramiprilat were obtained in the concentration range of 0.107–107.0 and 0.262–105.0 ng mL^?1, respectively. The intra- and inter-day precision (RSD) values were below 8.2 and 4.8% for ramipril, 10.4 and 12.3% for ramiprilat, and accuracy (RE) were within ±5.5 and ±3.2%, respectively at all QC levels. The method was utilized to support clinical pharmacokinetic studies in healthy volunteers following oral administration of ramipril tablets.     

LC–ESI–MS Determination of Imperatorin in Rat Plasma After Oral Administration and Total Furocoumarins of Radix Angelica dahuricae and its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatography–electrospray ionization mass spectrometry method for the quantification of imperatorin in rat plasma after oral administration and total furocoumarins of Radix Angelica dahuricae has been established. The plasma samples were deproteinized by adding internal standard (IS) osthole solution, which was prepared by acetonitrile. The analysis was performed on a Shim-pack C₁₈ column (150 × 2.0 mm i.d., 5 μm) using acetonitrile and 0.5% formic acid solution (70:30, v/v) as a mobile phase. The detection was performed on a quadrupole mass spectrometer detector with an ESI interface operated in the selected ion monitoring mode. The linear quantification range of the method was 2–4000 ng mL^?1 in rat plasma with a correlation coefficient greater than 0.99, the limit of detection (LOD) was 0.5 ng mL^?1 and the lower limit of quantification (LLOQ) 2 ng mL^?1. The intra- and inter-day relative standard deviations (RSD) were less than 2.5 and 3.5%, respectively. The recoveries were above 90%. The validated method was successfully applied to a pharmacokinetic study of imperatorin in rats after oral administration and total furocoumarins of Radix Angelica dahuricae.     

Determination of Jasminoidin in Rabbit Plasma for the Pharmacokinetic Investigation after Single Dose Oral Administration of Gardenia jasminoides Ellis and Gardenia jasminoides Ellis Coupling Coptis chinensis Franch Extracts

A simple, sensitive and rapid method for the analysis of jasminoidin in rabbit plasma by liquid chromatography coupled to tandem mass spectrometry was developed. Detection was by positive ion electrospray ionization in multiple reactions monitoring mode. The method included a chromatographic run of 5.0 min using a C₁₈ analytical column and the calibration curve was linear over the concentration range of 0.5–2,000 ng mL^?1 with a correlation coefficient R of 0.998 or better. The intra- and inter-day precision ranged from 3.4 to 5.6% and 4.3 to 8.2%. The intra- and inter-day assay accuracy was between ?7.4 and 8.6%. The method was successfully applied for the pharmacokinetic study on jasminoidin in rabbit after a single dose oral administration of Gardenia jasminoides Ellis (Gardenia) and Gardenia coupling Coptis chinensis Franch (Coptidis) extracts.     

Simultaneous Determination of Sildenafil,Tadalafil, and Vardenafil in Pharmaceutical Preparations by High-Temperature Gas Chromatography/Mass Spectrometry

Sildenafil (SD), tadalafil (TD), and vardenafil (VD) are drugs used to treat erectile dysfunction (ED). Pharmaceutical counterfeiting-related SD, TD, and VD compounds are becoming a critical problem due to their harmful side-effects. Especially, SD and TD are two major counterfeit ED drugs in South Korea. In this study, a new high-temperature gas chromatography/mass spectrometry (HTGC/MS) method was developed for the rapid determination of SD, TD, and VD in pharmaceutical preparations. A HT GC column was introduced for the rapid analysis of high molecular weight and high boiling point compounds. High-speed centrifugation led to shortened time for results. Calibration curves were linear (R ² ≥ 0.9972) over the concentration ranges of 12.5–100 µg mg⁻¹ for TD and VD, and 25–175 µg mg⁻¹ for SD. The intra- and inter-day precisions were within 7.5 %, while the intra- and inter-day accuracies ranged from –3.0 to 8.0 %. The limits of quantification were 0.7 µg mg⁻¹ (SD and TD) and 0.13 µg mg⁻¹ (VD). The recoveries were in the range of 87.3–102.4 %. The developed method was rapid and accurate both for routine quantitative measurement of SD, TD, and VD in pharmaceutical preparations as well as screening their suspected counterfeit compounds.

     

An Optimized Ion-Pair HPLC Method for Simultaneous Analysis of Nucleoside Triphosphate Levels in Hepatoma Cell Line

An improved ion-pair HPLC method was developed for the simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in HepG2 cell extracts. HPLC conditions, flow rate and column temperature were optimized and good linearity (r ²  > 0.9993) was obtained over the investigated concentration ranges. Reproducibility was evaluated by intra- and inter-day assays and RSD values were below 5.39%. Recoveries ranged from 98.2 ± 3.49% to 103.1 ± 1.75%, respectively. Finally, the method was successfully applied to the analysis of eight compounds present in HepG2 cell extracts.     

Determination of Tiopronin in Human Plasma and Pharmacokinetics by LC-ESI-MS

A rapid, sensitive and specific method based on high performance liquid chromatography-electrospray ionization-mass spectrometry was developed for the determination of tiopronin in human plasma. In this study, vitamin C and dithiothreitol were used as the reducer and to release and stabilize tiopronin from dimeric mixed forms with endogenous thiols encountered during pretreatment of plasma samples. The separation was successfully achieved on an Agilent SB-Aq column packed with 5 μm C₁₈ silica, using an aqueous formic acid solution (pH 4.5–4.7), 0.5 mM tris(hydroxymethyl)aminomethane and methanol (95:5, v/v) as the mobile phase. Mass spectra were acquired in selective ion monitoring mode, using the [M ? H] ^? ions of tiopronin at m/z 162.0 and the [M ? H]^? of the internal standard sodium cyclamate at m/z 178.0, respectively. This quantitative assay was fully validated with respect to precision, repeatability and accuracy. The correlation coefficients were >0.9995 in the range 0.025–8.15 μg mL^?1 in human plasma. The mean recoveries were above 85%. The limit of quantitation was 0.012 μg mL^?1 with a relative standard deviation of inter-day and intra-day accuracy of less than 15%. This LC-ESI-MS method was also successfully applied to a pharmacokinetic study after oral administration of formulated tiopronin to healthy volunteers. The elimination half-life (T _1/2) was 21.5 ± 11.1 h.     

Pharmacokinetics and Tissue Distribution Study of Tryptanthrin in Rats by RP-HPLC with DAD Detector

A simple RP-LC-UV method was established for the determination of tryptanthrin in plasma and different tissues of rats. The separation was achieved by HPLC on a C₁₈ column with a mobile phases composed of acetonitrile–water (47:53, v/v), UV detection was used at 251 nm. Good linearity was found between 0.0183–1.1712 μg mL⁻¹ (r ² = 0.999) for plasma and 0.0937–1.7568 μg mL⁻¹ for the tissue samples, respectively (r ² ≥ 0.9932). The intra- and inter-day precisions expressed as the relative standard deviation for the method were 0.92–6.01 and 1.06–9.11 %, respectively. The relative recoveries of tryptanthrin ranged from 95.26 to 97.89 % for plasma and 82.55 to 114.99 % for tissue homogenates (except heart). The developed method was successfully applied to the pharmacokinetics and tissue distribution research after orally administration of a 56-mg kg⁻¹ dose of tryptanthrin to healthy SD rats. The main pharmacokinetics distribution results showed that liver, lung, small intestine, and large intestine were the major distribution tissues of tryptanthrin in rats, and that tryptanthrin had difficulty in crossing the blood–brain barrier.