Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Chiral separation of 2,3-allenoic acid by capillary zone electrophoresis using cyclodextrin derivatives

In this paper, five of six samples of 2,3-allenoic acid enantiomers were separated by capillary zone electrophoresis (CZE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as chiral selectors. Using HP-beta-CD for chiral separation, three of the six enantiomers were separated. Five experimental conditions including HP-beta-CD concentration, pH, buffer concentration, temperature, and running voltage were investigated for their influence on separation and migration using enantiomers of 2-methyl-4-phenyl-2,3-butadienoic acid (A) and 2-(n-propyl)-4-phenyl-2,3-butadienoic acid (B) as samples. Good separation results were observed when [HP-beta-CD] = 3-12 mmol/L and pH = 7-9 for samples A and B. The temperature range of 15-25 degrees C can be selected for convenience. According to the chiral separation results, HP-beta-CD and HP-gamma-CD should be valuable selectors to separate 2,3-allenoic acids and HP-gamma-CD was suggested to separate the 2,3-allenoic acid samples with a group at 4-position bulkier than phenyl.     

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.     

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Validation of a rapid micellar electrokinetic capillary chromatographic method for the simultaneous determination of isoniazid and pyridoxine hydrochloride in pharmaceutical formulation

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 degrees C, at 50 mbar 4s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 microm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 microg mL(-1) for ISO and 0.40 microg mL(-1) for PYR and the method was linear at least in the range of 3.0-100 microg mL(-1) for ISO and 1.0-100 microg mL(-1) for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.     

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.     

Determination of josamycin in rat plasma by capillary electrophoresis coupled with post-column electrochemiluminescence detection

A novel determination method for josamycin (JOS) based on capillary electrophoresis-electrochemiluminescence detection has been described. In this study, platinum disk electrode (300 microm in diameter) was used as a working electrode and the conditions affecting separation and detection were investigated in detail. Under optimal condition: 40 cm separation capillary (75 microm i.d.); 1.25 V applied potential on the Pt disc of the ECL detector cell; 5 mM Ru(bpy)3(2+) and 50mM phosphate buffer (pH 7.5) in the detection cell; 12 kV separation voltage; 8s injection time; 10 kV injection voltage and 15 mM running buffer (pH 7.5), calibration curve was linear over the range from 10 ng/mL to 5.0 microg/mL with a detection limit of 3.1 ng/mL at a signal-to-noise ratio of 3. The method can be successfully applied for the determination of josamycin in rat plasma in 6 min and the extraction recoveries with spiked plasma samples were over 92%.     

Detection of d-Serine in rat brain by capillary electrophoresis with laser induced fluorescence detection

A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2-20.0 microM. The limit of detection for d-Serine was 3.0 x 10(-7)M.     

Solid phase synthesis and biological evaluation of enantiomerically pure wasp toxin analogues PhTX-343 and PhTX-12

PhTX-343 and PhTX-12, analogues of the natural polyamine wasp toxin PhTX-433, were synthesised in 40-60% yields as pure enantiomers using solid phase synthesis techniques. Capillary electrophoresis procedures were developed for chiral separation and determination of enantiomeric purity (ee) of the enantiomers of PhTX-343 and PhTX-12. The methods were optimised with respect to chiral selector, buffer pH, and temperature around the capillary. Thus, rac-PhTX-343 was resolved using a separation buffer containing 30 mM heptakis-(2, 6-di-O-methyl)-beta-cyclodextrin in 50 mM 6-aminocarproic acid (pH 4. 0) at 15 degrees C. rac-PhTX-12 was not resolvable in this system, but could be resolved using a separation buffer containing 10% w/v of dextrin 10, a linear maltodextrin, in 50 mM 6-aminocaproic acid (pH 4.0) at 15 degrees C. Using these methods, the optical purity of the synthetic enantiomers was determined to be ee > 99%. The enantiomers were also characterised by chiroptical methods. The antagonist potency of the enantiomers was tested on nicotinic acetylcholine receptors (human muscle-type nAChR) expressed in TE671 cells, ionotropic glutamate receptors in Xenopus laevis oocytes (expressing recombinant GluR1flop receptors), and locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The potencies of each pair of enantiomers were similar (eudismic ratio close to 1).     

Separation of cefoperazone enantiomers using beta-cyclodextrin as chiral additive by capillary zone electrophoresis

A capillary electrophoresis method was developed to separate the enantiomers of cefoperazone. Different cyclodextrins, including alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD), 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), and methyl-beta-cyclodextrin (Me-beta-CD), were tested as chiral additives in the running buffer. The effect of various parameters on enantioseparation such as concentration of NaH(2)PO(4), buffer pH, and CD concentration was also studied. The cefoperazone enantiomers were baseline separated under conditions of 0.04 mmol/L beta-CD, 75 mmol/L NaH(2)PO(4) buffer at pH 4.0. A fused silica capillary (40 cm effective length x 75 microm ID) was used. The applied voltage and capillary temperature were 20 kV and 25 degrees C, respectively. Under these conditions, linear calibration curves were obtained in the 5-500 microg/ml range using UV detection at 280 nm. The limit of detection for both isomers was 0.1 microg/ml. The method was used for the analysis of different pharmaceutical preparations (dose) and biological samples containing cefoperazone.     

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.     

Biphasic recognition chiral extraction: A novel method for separation of mandelic acid enantiomers

This article reports a new chiral separation method-biphasic recognition chiral extraction for the separation of mandelic acid enantiomers. Distribution behavior of mandelic acid enantiomers was studied in the extraction system with O,O'-di-benzoyl-(2S,3S)-4-toluoyl-tartaric acid (D-(+)-DTTA) in organic phase and beta-CD derivatives in aqueous phase, and the influence of the types and concentrations of extractants and pH on extraction efficiency was investigated. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD), hydroxyethyl-beta-cyclodextrin (HE-beta-CD), and methyl-beta-cyclodextrin (Me-beta-CD) have stronger recognition abilities for S-mandelic acid than those for R-mandelic acid, among which HP-beta-CD has the strongest ability. D-(+)-DTTA preferentially recognizes R-mandelic acid. pH and the concentrations of extractants have great effects on chiral separation ability. A high enantioseparation efficiency with a maximum enantioselectivity of 1.527 is obtained at pH of 2.7 and the ratio of 2:1 of [D-(+)-DTTA] to [HP-beta-CD]. The obtained results indicate that the biphasic recognition chiral extraction is of stronger chiral separation ability than the monophasic recognition chiral extraction. It may be very helpful to optimize the extraction systems and realize the large-scale production of pure enantiomers.     

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.     

Determination of enantiomeric excess for 2,3-dihydroxy-3-phenylpropionate compounds by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as chiral selector

A new capillary zone electrophoresis (CZE) method was developed to separate three chiral 2,3-dihydroxy-3-phenylpropionate enantiomers using neutral hydroxypropyl-beta-CD (HP-beta-CD) as chiral selector and borate as background electrolyte. The results showed that HP-beta-CD exhibited good enantioselectivity and high resolution was achieved under the optimum condition of pH 10.3, 200 mM borate buffer containing 6% methanol and 50 mM HP-beta-CD at 15 kV and 20 degrees C within 16 min. The precision of the method was <0.9% for migration time and 4.5% for corrected peak area. In addition, the developed method was successfully applied to the determination of enantiomeric excess (ee) of synthetic 2,3-dihydroxy-3-phenylpropionate samples. With this method, low as 0.2% impurity of the undesirable enantiomer in the presence of high amount of target enantiomer was determined. The results demonstrated that the proposed CZE method is a simple and useful technique and is applicable to ee assay of 2,3-dihydroxy-3-phenylpropionate enantiomers.     

Investigation of inclusion complexation of paclitaxel by cyclohenicosakis-(1-->2)-(beta-D-glucopyranosyl), by cyclic-(1-->2)-beta-D-glucans (cyclosophoraoses), and by cyclomaltoheptaoses (beta-cyclodextrins)

Inclusion complexation of the poorly soluble drug, paclitaxel, was investigated with various host cyclooligosaccharides such as a family of isolated neutral cyclohenicosakis-(1-->2)-(beta-D-glucopyranosyl) (cyclic-(1-->2)-beta-D-glucans, cyclosophoraoses), dimethyl cyclomaltoheptaose (cyclodextrins, DM-beta-CD) and hydroxypropyl cyclomaltoheptaose (cyclodextrins, HP-beta-CD). Quantitative analysis with high-performance liquid chromatography (HPLC) indicated that all three cyclic oligosaccharides could increase the solubility of paclitaxel, where DM-beta-CD gave the best results and a family of cyclosophoraoses and HP-beta-CD, both gave similar results. Complexation of host molecules with paclitaxel was studied by NMR and fluorescence spectroscopic analyses. NMR spectroscopic analysis showed that the aromatic regions of paclitaxel experienced noticeable changes of the chemical shifts or peak shapes upon interaction with host molecules. The relatively bulky cyclosophoraoses allowed favorable accessibility to either the B-ring or A-ring of paclitaxel, while DM-beta-CD and HP-beta-CD allowed accessibility to all the aromatic rings including the C ring. The interaction of DM-beta-CD with paclitaxel greatly increased the fluorescence intensity compared with other host molecules, suggesting the more effective partitioning of a moderate fluorophore into a hydrophobic cluster adjacent to the C-ring of paclitaxel.     

Chiral resolutions of (9-anthryl)methoxyacetic acid and (9-anthryl)hydroxyacetic acid by capillary electrophoresis

The resolutions of (9-anthryl)methoxyacetic acid (9AMAA) and (9-anthryl)hydroxyacetic acid (9AHAA) were performed by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as a chiral selector. Various factors affecting migration time and resolutions of these compounds were investigated with a run voltage of 20 kV, column temperature 20 degrees C and 20 mM Tris-H(3)PO(4) buffer (pH 6.5) containing 5 mM HP-beta-CD for 9AMAA, or 10 mM HP-beta-CD for 9AHAA, (+/-)-9AMAA and (+/-)-9AHAA were successfully separated at Rs 3.27 and 1.92, respectively.     

Optimization of the separation lactic acid enantiomers in body fluids by capillary electrophoresis

The optimization of the separation conditions of the two optical isomers of lactic acid by a factorial design is reported. Initially, different chiral selectors were systematically investigated and then a experimental design with three quantitative factors (cyclodextrin concentration and background buffer pH and concentration) were evaluated. Optimal conditions for obtaining a resolution higher than 1.5 were: phosphate buffer 200 mM at pH=6.0 with 413 mM 2-hydroxypropyl-beta-cyclodextrin added (HP-beta-CD), 20 degrees C, -20 kV of applied potential and polyacrylamide-coated capillary. The method was validated for the measurement in plasma and it was applied to the identification of both isomers in body fluids such as urine, amniotic fluid and cerebrospinal fluid. Samples were centrifuged and diluted (1:4) prior to the analysis.     

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

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.