Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.     

Quantitative determination of the novel anticancer drug E7070 (indisulam) and its metabolite (1,4-benzenedisulphonamide) in human plasma, urine and faeces by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

E7070 (indisulam) is a novel anticancer drug currently undergoing clinical investigation. We present a sensitive and specific method for the quantitative determination of E7070 and its metabolite M1 (1,4-benzenedisulphonamide) in human plasma, urine and faeces. The analytes and their tetra-deuterated analogues, which were used as internal standards, were isolated from the biological matrix by solid-phase extraction with OASIS cartridges (0.5 mL plasma or 1 mL urine) and by liquid-liquid extraction with ethyl acetate at pH 5 (1 mL faecal homogenate). The analytes were separated on a C8 reversed-phase chromatographic column and analyzed using electrospray ionization and tandem mass spectrometric detection in the negative ion mode. The validated concentration ranges in plasma were 0.1-20 microg/mL for E7070 and 0.01-2 microg/mL for M1. In urine and faecal homogenate, a concentration range from 0.05-10 microg/mL or microg/g, respectively, was validated for both analytes. Validation of the plasma assay was performed according to the most recent FDA guidelines. The assay fulfilled all generally accepted requirements for linearity (r > 0.99, residuals between -8 and 10%), accuracy (-13.5 to 1.4%) and precision (all less than 11%) in the tested matrices. We investigated recovery, stability (working solutions at -20 degrees C and at room temperature, biological matrices at -20 degrees C, room temperature and after 3 freeze/thaw cycles; final extracts at room temperature) and robustness. All these parameters were found acceptable. This method is suited for mass balance studies or therapeutic drug monitoring, as demonstrated by a case example showing plasma concentrations and cumulative excretion of E7070 and M1 in urine and faeces. Furthermore, we show the presence of E7070 metabolites in patient urine.     

Determination of erythromycin A in rat plasma and urine by high-performance liquid chromatography with chemiluminescence detection using Tris(2,2'-bipyridine)ruthenium(II)

A simple and sensitive method was developed for the determination of erythromycin A (EA), decladinosyl erythromycin A (dClEA) and erythromycin B (EB) in rat plasma and urine by high-performance liquid chromatography with electrogenerated chemiluminescence detection using Tris(2,2'-bipyridine)ruthenium(II). The recovery rates of EA, dClEA and EB were 97, 94 and 85% from rat plasma and 89, 83 and 93% from rat urine, respectively. The calibration curves were linear over the concentration ranges 0.05-5 microg/mL for plasma and 0.5-50 microg/mL for urine. The precision and accuracy for all analytes in rat plasma were < or =9.0 and -6.3-7.2%, and those in urine were < or =9.4% and -6.1-7.6%, respectively. This method proved to be a powerful tool for determination of EA, dClEA and EB concentrations in samples from rats.     

Rapid quantification of HIV protease inhibitors in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

HIV protease inhibitors are important antiretroviral drugs which have substantially reduced the morbidity and mortality associated with HIV-1 infection. Recent data have shown relationships between plasma concentrations of the protease inhibitors and clinical response, which makes therapeutic drug monitoring valuable. We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of the six licensed protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) and the pharmacologically active nelfinavir metabolite M8 in plasma. The sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using only 100 microl of plasma. Chromatographic separation was performed on an Inertsil ODS3 column (50 x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml min(-1). The analytical run time was 5.5 min. The use of a 96-well plate autosampler allowed batch sizes up to 150 patient samples. The triple-quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 0.01-10 microg ml(-1) for indinavir and saquinavir, 0.1-10 microg ml(-1) for amprenavir, 0.05-10 microg ml(-1) for nelfinavir and ritonavir, 0.1-20 microg ml(-1) for lopinavir and 0.01-5 microg ml(-1) for M8. Saquinavir-d(5) and indinavir-d(6) were used as internal standards. The coefficients of variation were always <10% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/-15%). The validated concentration ranges proved to be adequate in daily practice. This robust and fast LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in our hospital.     

Prodrugs of HIV protease inhibitors-saquinavir, indinavir and nelfinavir-derived from diglycerides or amino acids: synthesis, stability and anti-HIV activity

With the aim of improving the pharmacological properties of current protease inhibitors (PIs), the synthesis of various acyl and carbamate amino acid- or diglyceride-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis and their anti-HIV activity in CEM-SS and MT4 cells have been investigated. l-Leucine (Leu) and l-phenylalanine (Phe) were connected through their carboxyl to the PIs while l-tyrosine (Tyr) was conjugated through its aromatic hydroxyl via various spacer units. Hydrolysis of the prodrug with liberation of the active free drug was crucial for antiviral activity. The Leu- and Phe-PI prodrugs released the active free drug very rapidly (half-lives of hydrolysis in buffer at 37 degree C of 3-4 h). The Tyr-PI conjugates with a -C(O)(CH(2))(4)- linker exhibited half-lives in the 40-70 h range and antiviral activities in the 21-325 nM range (from 2 to 22 nM for the free PIs). The chemically very stable carbamate "peptidomimetic" Tyr-PI prodrugs (no hydrolysis detected after 7 days in buffer) displayed a very low anti-HIV activity or were even inactive (EC(50) from 2300 nM to >10 microM). A very low antiviral activity was measured for the diglyceride-substituted saquinavir and for all of the disubstituted indinavir and nelfinavir prodrugs. All these prodrugs probably released the active parent PI too slowly under the antiviral assay conditions. These results combined with those from transepithelial transport studies (Rouquayrol et al., Pharm. Res., 2002, 19, 1704-1712) indicate that conjugation of amino acids (through their carboxyl) to the PIs constitutes a most appealing alternative which could improve the intestinal absorption of the PIs and reduce their recognition by efflux carriers.     

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

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

Simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in plasma by high-performance liquid chromatography with UV detection

A specific, accurate, precise and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in human plasma. The method employed a simple liquid-liquid extraction of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib and internal standard (IS, DRF-4367) from human plasma (500 microL) into acetonitirile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid (pH 3)-acetonitrile-methanol-water at a flow rate of 1.0 mL/min. The eluate was monitored using an ultraviolet (UV) detector set at 235 nm. The ratio of peak area of each analyte to IS was used for quantification of plasma samples. Nominal retention times of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide, IS and celecoxib were 15.63, 17.20, 21.66, 24.95, 26.27, 30.24 and 32.22 min, respectively. The standard curve for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib was linear (r2 > 0.999) in the concentration range 0.1-50 microg/mL and for nimesulide (r2 > 0.999) in the concentration range 0.5-50 microg/mL. Absolute recovery was >83% from human plasma for all the analytes and IS. The lower limit of quantification (LLOQ) of nimesulide was 0.5 microg/mL and for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib the LLOQ was 0.1 microg/mL. The inter- and intra-day precisions in the measurement of QC samples, 0.1, 0.3, 15.0 and 40.0 microg/mL (for all analytes except nimesulide), were in the range 2.29-9.37% relative standard deviation (RSD) and 0.69-10.28% RSD, respectively. For nimesulide the inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.5, 1.5, 15.0 and 40.0 microg/mL, were in the range 3.21-7.37% RSD and 0.97-7.06% RSD, respectively. Accuracy in the measurement of QC samples for all analytes was in the range 91.03-106.38% of the nominal values. All analytes including IS were stable in the battery of stability studies, viz. bench top, autosampler and freeze-thaw cycles. Stability of all analytes was established for 21 days at -20 degrees C. The application of the assay in an oral pharmacokinetic study in rats co-administered with celecoxib and valdecoxib is described.     

Head-column field-amplified sample stacking in capillary electrophoresis for the determination of cimetidine, famotidine, nizatidine, and ranitidine-HCl in plasma.

In this study, low concentrations of histamine2-receptor (H2-)antagonists were effected across a water plug, with separation taking place in a binary buffer comprising ethylene glycol and NaH2PO4 (pH 5.0), and detection at 214 nm. Liquid-liquid extraction with ethyl acetate- isopropanol is shown to provide extracts that are sufficiently clean. The calibration curves were linear over a concentration range of 0.1-2.00 microg/mL cimetidine, 0.2-5.0 microg/mL ranitidine-HCl, 0.3-5.0 microg/mL nizatidine, and 0.1-3.0 microg/mL famotidine. Mean recoveries were > 82%, while the intra- and interday relative standard deviations (RSDs) and relative errors (REs) were all < 13%. The method is sensitive with a detection limit of 3 ng/mL cimetidine, 30 ng/mL ranitidine HCl, 50 ng/mL nizatidine and 10 ng/mL famotidine (S/N = 3, electric-driven injection 90 s). This newly developed capillary electrophoresis (CE) method was applied for the determination of analytes extracted from plasma taken from a volunteer dosing a cimetidine, ranitidine, and nizatidine tablet simultaneously. These three H2-antagonists can be detected in real samples by this method, excluding the low dosing of famotidine tablet.     

A sensitive and selective RP-HPLC method for simultaneous determination of picroside-I and picroside-II in rat plasma and its application in pharmacokinetics studies

A sensitive and selective HPLC method with UV detection for the simultaneous determination of picroside-I and picroside-II (active components of total glycoside of Picrorhiza scrophulariiflora Pennell) was developed and validated in rat plasma. After simple deproteinization using acetonitrile, analysis was performed on an RP-C18 column (250 mm x 4.6 mm id, 5 microm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min used in a gradient elution program. The UV detection wavelength was set at 262 and 277 nm. Linear calibration curves were obtained in the concentration range of 0.10-50 microg/mL for picroside-I and 0.25-200 microg/mL for picroside-II. The lower limits of quantification were 0.1 and 0.25 microg/mL for picroside-I and picroside-II, respectively. The recoveries from spiked control samples were up to 80% for both picroside-I and picroside-II. Accuracy and precision of the validated method were both within the acceptable limits of <15% at three quality control concentrations. The analytes were stable after three freeze-thaw cycles. The method was successfully used to determine concentrations of picroside-I and picroside-II after intravenous administration of total glycoside of Picrorhiza scrophulariiflora Pennell to rats.     

Quantification of the plant-derived hallucinogen Salvinorin A in conventional and non-conventional biological fluids by gas chromatography/mass spectrometry after Salvia divinorum smoking

A gas chromatography method with mass spectrometric detection is described for the determination of Salvinorin A, the main active ingredient of the hallucinogenic mint Salvia divinorum. The method was validated in plasma, urine, saliva and sweat using 17-alpha-methyltestosterone as internal standard. The analytes were extracted from biological matrices with chloroform/isopropanol (9:1, v/v). Chromatography was performed on a 5% phenyl methyl silicone capillary column and analytes were determined in the selected ion monitoring mode. The method was validated over the concentration range 0.015-5 microg/mL plasma, urine and saliva and 0.01-5 microg/patch in the case of sweat. Mean recoveries ranged between 77.1 and 92.7% for Salvinorin A in different biological matrices, with precision and accuracy always better than 15%. The method was applied to the analysis of urine, saliva and sweat from two consumers after smoking 75 mg plant leaves to verify the presence of the active ingredient of S. divinorum in human biological fluids as a biomarker of plant consumption. Salvinorin A was detected in urine (2.4 and 10.9 ng/mL) and saliva (11.1 and 25.0 ng/mL), but not in sweat patches from consumers.     

Simultaneous quantification of chlorogenic acid and caffeic acid in rat plasma after an intravenous administration of mailuoning injection using liquid chromatography/mass spectrometry

A simple, rapid and sensitive method was developed for the simultaneous quantification of chlorogenic acid (CGA) and caffeic acid (CA) in rat plasma using a high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile followed by centrifugation. The analytes and internal standard ferulic acid were separated on an Intersil C8-3 column (5 mm; 250 x 2.1 mm) with acetonitrile/0.05% triethylamine solution (70:30, v/v) as mobile phase at a flow rate of 0.2 mL/min with an operating temperature of 30 degrees C. Detection was performed on a quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operated in selected ion monitoring (SIM) mode. Negative ion ESI was used to form deprotonated molecules at m/z 353 for chlorogenic acid, m/z 179 for caffeic acid, and m/z 193 for the internal standard ferulic acid. Linear detection responses were obtained for CGA concentrations ranging from 0.005 to 2.0 microg/mL and for CA concentrations ranging from 0.010 to 2.0 microg/mL and the lower limits of quantitation (LLOQs) for CGA and CA were 0.005 and 0.01 microg/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.0% for both analytes. Deviation of the assay accuracies was within +/-10.0% for both analytes. Their average recoveries were greater than 88.0%. Both analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of CGA and CA following an intravenous dose of 5 mL/kg mailuoning injection to rats.     

Simultaneous determination of preservatives in beverages,vinegar, aqueous sauces,and quasi-drug drinks by stir-bar sorptive extraction (SBSE) and thermal desorption GC-MS

A method for the simultaneous determination of seven preservatives - sorbic acid, benzoic acid, and p-hydroxybenzoic acid ethyl, isopropyl, propyl, isobutyl, and butyl esters - in beverages, vinegar, aqueous sauces, and quasi-drug drinks has been developed using the stir-bar sorptive extraction technique then thermal desorption GC-MS analysis. The extraction conditions - pH, sample volume, extraction temperature, salt addition, and extraction time - were examined. d5-Benzoic acid and p-hydroxybenzoic acid sec-butyl ester were added as surrogate internal standards to compensate for the effect of sample matrix and coexisting analytes on the sorptive extraction. The linearity of the method was good over the concentration range from 1 to 1000 microg mL(-1) for sorbic acid, 10-1000 microg mL(-1) for benzoic acid, and 0.1-100 microg mL(-1) for p-hydroxybenzoic acid ethyl, isopropyl, propyl, isobutyl, and butyl esters, and the correlation coefficients were higher than 0.9984. The limit of detection ranged from 0.015 to 3.3 microg mL(-1). The recoveries (95-105%) and precision (RSD: 0.86-6.0%) of the method were examined by analyzing a sparkling soft drink, white wine, red wine, balsamic vinegar, soy sauce, and quasi-drug drink samples fortified at the 5 to 50 microg mL(-1) level.     

Simultaneous determination of pantoprazole and its two metabolites in dog plasma by HPLC

A simple and sensitive high-performance liquid chromatography (HPLC) method is developed and validated for simultaneous determination of pantoprazole and its two metabolites (pantoprazole sulfone and pantoprazole thioether) in dog plasma and applied to a pharmacokinetic study in Beagle dogs. Following a protein precipitation procedure, the samples are separated using reversed-phase HPLC (C18) by a gradient of acetonitrile and ammonium acetate (pH 6.0) at a flow rate of 1.0 mL/min and quantitated using UV detection at 290 nm. Omeprazole is selected as the internal standard. The method has a lower limit of quantitation of 0.025 microg/mL for pantoprazole and its two metabolites, using 0.1-mL aliquots of plasma. The linear calibration curves are obtained in the concentration range of 0.025-10.0 microg/mL for three analytes. The intra- and interrun precision (relative standard deviation), calculated from quality control (QC) samples, is less than 13% for three analytes. The accuracy determined from QC samples is between -6.4% and 12%.     

Screening for and validated quantification of phenethylamine-type designer drugs and mescaline in human blood plasma by gas chromatography/mass spectrometry

In recent years, several newer designer drugs of the so-called 2C series such as 2C-D, 2C-E, 2C-P, 2C-B, 2C-I, 2C-T-2, and 2C-T-7 have entered the illicit drug market as recreational drugs. Some fatal intoxications involving 2C-T-7 have been reported. Only scarce data have been published about analyses of these substances in human blood and/or plasma. This paper describes a method for screening and simultaneous quantification of the above-mentioned compounds and their analog mescaline in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode, after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. Validation data for 2C-T-2 and 2C-T-7 were unacceptable. For all other analytes, the method was linear from 5 to 500 microg/L and the data for accuracy (bias) and precision (coefficient of variation) were within the acceptance limits of +/-15% and <15%, respectively (within +/-20% and <20% near the limit of quantification of 5 microg/L).     

Rapid HPLC analysis of the antiepileptic lamotrigine and its metabolites in human plasma

A liquid chromatographic method with diode array detection (DAD) has been developed for the analysis of the antiepileptic agent lamotrigine (LTG) and its metabolites, lamotrigine 2-N-glucuronide and 2-N-methylated in plasma samples. The analytes were separated on a C8 RP column, using a mobile phase composed of methanol and a 0.45 mM, pH 3.5 phosphate buffer containing 0.17% triethylamine (24:76 v/v). Melatonin was used as the internal standard (IS). The DAD detector was set at 220 nm for the detection of all the analytes. A simple protein precipitation with methanol guaranteed high extraction yield values (>90%) and good purification from matrix interference. Good linearity was obtained in the 0.1-15.0 microg/mL range for LTG and lamotrigine 2-N-glucuronide and in the 0.1-2.0 microg/mL range for lamotrigine 2-N-methylated. The analytical method was validated in terms of precision, extraction yield, and accuracy. These assays gave RSD% values for precision always lower than 4.3% and mean accuracy higher than 80%. The method seems to be suitable for the analysis of plasma samples from patients treated with Lamictal.     

Identification of allantoin, uric acid, and indoxyl sulfate as biochemical indicators of filth in food packaging by LC.

A liquid chromatographic (LC) method was developed for the determination of allantoin, uric acid, and indoxyl sulfate in mammalian urine contaminated packaging material including paper bagging, corrugated cardboard, grayboard, and burlap bagging. The procedure involves solvent extraction and isolation of the 3 analytes by reversed-phase LC with ultraviolet detection at 225 nm for allantoin and 286 nm for uric acid and indoxyl sulfate. The composition of authentic mammalian urine such as mouse, rat, cat, dog, and human were also determined with regard to the 3 compounds of interest. A linear concentration range of 0.11-20.4, 0.02-10.0, and 0.04-30.0 microg/mL was obtained for allantoin, uric acid, and indoxyl sulfate, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.0104 and 0.0345 microg/mL for allantoin; 0.0018 and 0.0060 microg/mL for uric acid; and 0.0049 and 0.0165 microg/mL for indoxyl sulfate, respectively. Interday relative standard deviation values for a mixture of standard allantoin, uric acid, and indoxyl sulfate (n = 5) were 0.97, 0.80, and 0.94%, respectively. Analyte composition for 5 types of authentic mammalian urine varied from 0.19-6.88 mg/mL allantoin; 0.08-0.57 mg/mL uric acid; and 0.03-0.78 mg/mL indoxyl sulfate. Analyte content for 8 samples including 2 samples each for paper, cardboard, grayboard, and burlap bagging each contaminated with mouse or rat urine ranged from 相似文献   

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.     

Determination of 3',4',5',5,7-pentamethoxyflavone in the plasma and intestinal mucosa of mice by HPLC with UV detection

In a preliminary experiment 3',4',5',5,7-pentamethoxyflavone (PMF) inhibited adenoma development in Apc(Min) mice, a model of the human heritable condition familial adenomatous polyposis. An HPLC method for tricin was modified and validated to permit measurement of PMF in mouse plasma and intestinal mucosa. HPLC analysis was carried out on a Hypersil-BDS C(18) column with detection at 324 nm and tricin as internal standard. The assay was linear in the range of 100-2000 ng/mL plasma and 1.0-40 microg/mL mucosa. PMF in plasma was efficiently extracted using solid-phase columns. In the case of mucosa organic solvent protein precipitation displayed satisfactory accuracy and precision. The assay recovery at low, medium and high concentrations was between 85 and 103% for both biomatrices, with a relative standard deviation of <15%. The lower limits of quantitation for plasma and mucosa were 100 ng/mL and 1.0 microg/mL, respectively. This method allowed measurement of PMF steady-state median concentrations in plasma (1.08 nmol/mL, n = 11; 10th and 90th percentiles: 0.633 and 2.385 nmol/mL) and mucosa (108.5 nmol/g, n = 9; 10th and 90th percentiles: 38.9 and 164.4 nmol/g) in mice which had received PMF (0.2%, w/w) with their diet.