Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.     

Fast high-performance liquid chromatographic analysis of naphthodianthrones and phloroglucinols from Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) has been used in modern medicine for treatments of depression and neuralgic disorders. An HPLC method with photodiode array detection for the rapid determination of the major active compounds, naphthodianthrones and phloroglucinols, has been developed. The method permits the determination of hypericin, protohypericin, pseudohypericin, protopseudohypericin, hyperforin and adhyperforin in an extract in less than 5 min. Good linearity over the range 0.5-200 microg/mL for hyperforin and 0.02-100 microg/mL for hypericin was observed. Intra-assay accuracy and precision varied from 0.1 to 17% within these ranges. Lower levels of quantitative determination were 2 microg/mL for hyperforin and 0.5 microg/mL for hypericin, while detection limits were 0.1 and 0.02 microg/mL, respectively.     

Determination of ergometrine maleate by fluorescence detection.

A simple, accurate, sensitive and selective fluorescence analysis method for rapid determination of trace amounts of ergometrine maleate has been developed. The method is based on the fluorescence of ergometrine maleate. The calibration graph was linear in the range of 1 x 10(-4) to 0.2 microg/mL and the detection limit was 4 x 10(-5) microg/mL, with a relative standard deviation of 0.32% at 0.05 microg/mL (n = 11) ergometrine maleate. The influence of foreign compounds was tested. The proposed method was applied successfully to the determination of ergometrine maleate in pharmaceutical preparations and human plasma.     

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of bupropion in rat everted gut sacs

We have developed a rapid, sensitive and selective LC-MS method for the simultaneous assay of bupropion and its metabolite hydroxybupropion during its intestinal absorption, studied with the rat everted gut sac model. The method was validated in the concentration range of 1-15 microM (0.024-3.58 microg/mL) for bupropion and 0.005-1 microM (0.00127-0.25 microg/mL) for hydroxybupropion with 10 microL injected. Bupropion is used as a probe for the activity of the CYP2B6 isoenzyme of the P450 family of enzymes in man. Its major metabolite hydroxybupropion was found in the serosal media of the gut sac showing that the isoenzyme of the 2B group was active in the intestinal mucosa and metabolized bupropion during its passage across the mucosa. The metabolite was also quantified in the mucosal media indicating its ability to cross the apical membrane of the epithelial cells.     

In vitro metabolism of BYZX in human liver microsomes and the structural elucidation of metabolite by liquid chromatography-mass spectrometry method

In vitro phase I metabolism of BYZX, a novel central-acting cholinesterase inhibitor for the treatment of the symptoms of Alzheimer's disease, was studied in human liver microsomes (HLM) and the metabolite formation pathways were investigated by chemical inhibition experiments and correlation analysis. The residual concentration of substrate and the metabolite formed in incubate were determined by HPLC method. The calibration curves of BYZX were linear over the concentration range from 5.07 microM to 200.74 microM. The relative standard deviations of within day and between day were less than 5% (n=5). The limit of detection (LOD) was 0.18 microg/mL (S/N=3) and the limit of quantification (LOQ) was 0.55 microg/mL (R.S.D.=5.2%, n=5). The determination recoveries of BYZX were in the range of 98.2-104.8%. The apparent K(m) of BYZX in HLM was 53.25+/-17.2 microM, the V(max) was 0.94+/-0.77 microM/min/mg protein, and the intrinsic clearance value (Cl(int)) was 0.018+/-0.02 mL/min/mg protein. Ketoconazole and cyclosporin A were the most potent inhibitors on BYZX metabolism in HLM with IC(50) being 0.89 microM and 18.17 microM, respectively. And the inhibition constant (K(i)) of ketoconazole was 0.42 microM. The metabolite of BYZX was N-des-ethyl-BYZX elucidated by LC-MS-MS. The results demonstrated that the developed HPLC method was reliability, simple technique, and was applicable to be used for the researches of in vitro metabolism of BYZX. CYP3A4 was the major isozyme responsible for BYZX metabolism; N-dealkylation was the major metabolic pathway of BYZX. The predominant metabolite of BYZX was N-des-ethyl-BYZX detected in vitro phase I metabolism in HLM.     

A capillary zone electrophoresis method for the quantitative determination of hypoxoside in commercial formulations of African potato (Hypoxis hemerocallidea)

Hypoxoside is a norlignan diglucoside present in the corms of African potato, Hypoxis hemerocallidea, used as a popular African traditional medicine for its nutritional and immune boosting properties. A specific analytical method employing capillary zone electrophoresis has been developed and validated for the quantitative determination of this analyte. Sulfafurazole was used as internal standard, and electrophoretic separation of both analytes could be achieved within 12 min. Linearity of the method was established within the range 5-60 microg/mL and provided a high degree of accuracy (100 +/- 3%). The recovery of the method was found to be 100 +/- 5% and the RSDs of the intra- and inter-day precision were better than 5.19 and 2.52%, respectively. The limits of detection and quantification were calculated to be 0.5 and 2 microg/mL, respectively. The described method was used for the analysis and quality control of two commercially available products containing African potato. The method can also be used to determine product stability since it could separate the hypoxoside peak from its degraded products obtained from degradation studies.     

The simultaneous separation and determination of six flavonoids and troxerutin in rat urine and chicken plasma by reversed-phase high-performance liquid chromatography with ultraviolet-visible detection

The method of high-performance liquid chromatography (HPLC) with UV-vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm x 4.6 mm, 5.0 microm) maintained at 35.0 degrees C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10-70.00 microg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50-350.00 microg/mL (troxerutin). The detection limits were 0.010-0.050 microg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.     

A rapid and validated HPLC method to quantify racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction

A HPLC method with UV detection was developed and validated for the determination of thiorphan in human plasma. Nevirapine was used as the internal standard. Separation was performed by a Waters sunfire C18 reversed-phase column maintained at 35 degrees C. The mobile phase was a mixture of 0.05 M phosphate buffer with the pH adjusted to 2.6 and acetonitrile (74:26, v/v) at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with solid-phase extraction cartridges (Oasis HLB 3 mL, 60 mg). The extraction recovery for plasma samples of thiorphan at 0.1, 0.4 and 2.0 microg/mL was 93.5%, 98.2% and 97.8%, respectively. The calibration curve was linear with the correlation coefficient (r) above 0.9998. Linearity was verified over the range of 0.05-4 microg/mL thiorphan in plasma. The limit of quantification (LOQ) is 0.05 microg/mL. The mean accuracy was 92.7-99.6%. The coefficient of variation (precision) in the within- and between-batch was 2.2-8.4% and 4.1-8.1%, respectively. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of thiorphan.     

Simultaneous determination of abacavir and zidovudine from rat tissues using HPLC with ultraviolet detection

A simple high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of abacavir and zidovudine (AZT) in rat plasma, amniotic fluid, fetal, and placental tissues. Extraction of abacavir, AZT, and the internal standard, azidouridine (AZDU) in amniotic fluid was carried out by protein precipitation. Extraction from plasma, fetal and placental homogenates was achieved by using a salting out technique. Chromatographic separation was performed using a C8 column (150 mm x 4.6 mm, 5 microm). The mobile phase consisted of 12% acetonitrile in 25 mM sodium phosphate buffer (adjusted to pH 7 with sodium hydroxide) for the fetus, placenta, plasma and amniotic fluid samples at a flow rate of 0.8 mL/min. The method was validated over the range from 0.05 to 50 microg/mL for both abacavir and AZT in the four biological matrices. The absolute recovery of abacavir ranged from 79 to 94%, while AZT recoveries ranged from 79 to 90% in the different biological matrices. The internal standard recovery ranged from 90 to 92%. Acceptable intra- and inter-day assay precision (<10% R.S.D.) and accuracy (<10% error) were observed over 0.05-50 microg/mL for all four matrices.     

An HPLC method for determination of oridonin in rabbits using isopsoralen as an internal standard and its application to pharmacokinetic studies for oridonin-loaded nanoparticles

A simple and sensitive HPLC method has been developed and validated for the determination of oridonin (ORI) in rabbit plasma. A simple liquid-liquid extraction (LLE) method was applied to extract ORI and the internal standard (IS), isopsoralen, from rabbit plasma. Chromatographic separation of ORI and the IS was achieved with a Kromasil C18 5-mum column (250 mm x 4.6 mm) using methanol-water (50:50, v/v) as mobile phase at a flow rate of 1 mL/min. The ultraviolet (UV) detection wavelength was set at 241 nm. The lower limit of quantification (LLOQ) was 0.02 microg/mL. The calibration curves were linear over a concentration range of 0.02-10 microg/mL. The assay accuracy and precision were within the range of 95.1-113.5% and 5.4-8.6%, respectively. This HPLC method was applied successfully to the pharmacokinetic study of ORI-loaded poly(caprolactone)-poly(ethylene oxide)-poly(caprolactone) copolymer nanoparticles (ORI-PCL-PEO-PCL-NP) in rabbits, given as a single intravenous injection at the dose equivalent to 2mg of ORI/kg, and the pharmacokinetic parameters for ORI were compared with a single intravenous injection of a ORI solution at the same dose.     

High-throughput determination of fudosteine in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A 96-well protein precipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 microL) by the methanol (150 microL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 microL supernatant and 100 microL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC-MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol-water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178-->91 and m/z 284-->91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 microg mL(-1), with good linearity (r>0.999) over the linear range of 0.02-10 microg mL(-1). The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 microg mL(-1). The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.     

Simultaneous quantitation of five flavonoid glycosides in Herba Epimedii by high-performance liquid chromatography-tandem mass spectrometry

A rapid and accurate reversed-phase liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the quantitative determination of five flavonoid glycosides, icariin, epimedin A, epimedin B, epimedin C and hyperin in Herba Epimedii. Chromatographic separations were performed using a C(18) narrow-bore HPLC column; a mixture of an aqueous solution of ammonium formate (pH 4.0) and acetonitrile was used as the mobile phase, with compounds detected in the positive ion mode with multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with an electrospray ionisation interface. This method for the determination of the reported flavonoid glycosides was accurate and reproducible, with a lower limit of quantication of 0.5 microg/mL. The standard calibration curves for the above-mentioned compounds were linear (r(2) > 0.998) over the concentration range 0.5-10.0 microg/mL. The relative standard deviations for intra- and inter-day precision over the concentration range for the flavonoid glycosides were lower than 7.8% with accuracy between 90.1 and 111.0%. The established method was successfully applied to the quality assessment of samples of Herba Epimedii collected from Korea and China.     

Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid-liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow rate=1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25-40 microg/mL for plasma sample and 1.0-500 microM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7+/-7.86%, 96.8+/-3.20% and 102.7+/-9.72% recovery from 0.5, 10, and 40 microg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze-thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.     

Determination of indomethacin in plasma by micellar electrokinetic chromatography with UV detection for premature infants with patent ducts arteriosus

A simple and selective micellar electrokinetic chromatography (MEKC) is described for determination of indomethacin in plasma. Plasma proteins are precipitated by acetonitrile. An aliquot of supernatant was evaporated and reconstituted with Tris buffer for MEKC analysis. The separation of indomethacin was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (30 mM; pH 8.0) with 100 mM sodium octanesulfonate (SOS) as an anionic surfactant. Under this condition, a good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation of indomethacin were studied, including pH and concentrations of the Tris buffer and SOS. The linear range of the method for the determination of indomethacin was over 0.3-10.0 microg/mL; the detection limit (signal-to-noise ratio=3; injection 0.5 psi 5s) was 0.1 microg/mL. The proposed method for determination of indomethacin in premature infants with patent ducts arteriosus has been demonstrated.     

Development of a sensitive and rapid nucleic acid assay with tetraphenyl porphyrinatoiron chloride by a resonance light scattering technique.

Based on the interaction between nucleic acids and tetraphenyl porphyrinatoiron chloride (FeTPPCl), a novel method for the determination of nucleic acids at the nanogram level has been developed. Under the optimum conditions, the weak resonance light scattering (RLS) intensity of FeTPPCl was greatly enhanced by nucleic acids and the enhanced RLS intensity was proportional to the concentration of nucleic acids in the range 0.02-2.8 microg/mL for calf thymus DNA, 0.05-3.3 microg/mL for fish sperm DNA and 0.07-4.5 microg/mL for yeast RNA. The detection limits (3sigma) were 2.9 ng/mL for calf thymus DNA, 3.9 ng/mL for fish sperm DNA and 5.2 ng/mL for yeast RNA. Almost no interference could be observed from proteins, nucleosides and most of the metal ions. The proposed method showed good reliability, sensitivity, rapidity and reproducibility when applied to the determination of nucleic acids in synthetic and biochemical samples. The time savings make this method suitable for large routine analyses.     

Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.     

Determination and pharmacokinetics of orientin in rabbit plasma by liquid chromatography after intravenous administration of orientin and Trollius chinensis Bunge extract

A high-performance liquid chromatography (HPLC) method was developed and validated for the determination of orientin in rabbit plasma using ultraviolet (UV) absorbance detection. Orientin is the active constituent of purified herbal extract (TRO PE) from the flower of Trollius chinensis Bunge. Protein precipitation was used as the sample preparation technique. A Diamonsil C18 column (150 mmx4.6 mm, 5 microm) was equilibrated with a mobile phase composed of 0.1% acetic acid/methanol/acetonitrile (80/5/15, v/v/v). The calibration curve of orientin in rabbit plasma was linear in the concentration range of 0.530-53.0 microg/mL. This validated method was successfully applied to a pharmacokinetic study in rabbits after the intravenous administrations of orientin and TRO PE at three different doses.     

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1-20 microg mL(-1) for MTU and 0.1-10 microg mL(-1) for PTU, the detection limits were 0.03 microg mL(-1) for PTU, 0.03 microg mL(-1) for MTU and the quantification limits were 0.1 microg mL(-1) for PTU, 0.1 microg mL(-1) for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.     

Mechanism and inhibitory effect of galangin and its flavonoid mixture from Alpinia officinarum on mushroom tyrosinase and B16 murine melanoma cells

The whitening effects of the flavonoid constituents of Alpinia officinarum Hance were investigated on melanin biosynthesis in B 16 mouse melanoma cells, tyrosinase inhibition and UV absorption. The melanin content was reduced to 1.276 microg /10(5) cell for flavonoid mixture and 1.161 microg /10(5) cell for galangin while the melanin control was 1.632 microg/10(5) cell. Both flavonoid mixture and galangin reduced melanin production with an inhibition of 21.81% and 28.86% at a concentration of 26.5 microg/mL and 29 microg/mL (107.4 microM), respectively. Tyrosinase inhibition by the flavonoid mixture and galangin were higher at lower concentrations and galangin showed competitive inhibition at a concentration less than 21.23 microg/mL which was soluble. In addition, the flavonoid mixture and galangin showed a broad absorption band at 270 approximately 290 nm related to the UV-B area. These observations suggest that galangin may be a whitening agent and a promising candidate for prevention of skin cancer. This is the first full scale report on the evaluation of the whitening effect of galangin.