高效液相色谱法测定尿中美芬妥英及代谢产物4'-羟基美芬妥英

A simple, sensitive and reproducible HPLC assay is described for the determination of mephenytoin and 4'-hydroxymephenytoin in human urine. Phenobarbital was used as an internal standard.The compounds were separated on a U-Bondapack RP-C₁₈ column using a mobile phase of and the UV detectou was set at 210 nm. Calibration curves in the range 0.05～1.00ug/ml for mephenytoin and 0.5～100.0ug/ml for 4'-hydroxymephenytoin were linear (r=0.9998and r=0.9992,respectivesy). The average recovery was 95.10±2。95%,and the relative standard devia- tion within day and day to day was less than 10%。The detection limit for mephenytoin was 25mg/ml and 4‘-hydroxymephenytoin was 50mg。ml。The method was used to study the metabolism of S-mephenytoin 4'-hydroxylatoin in 10healthy volunteers.The 12 h urinary metabolic ratio (MR)and hydroxylation index(HI)were calculated to express interindividual variation in metabolism. Two of them exhibited defective 4'-hydroxylation of S-mephenytoin as poor metabllizers (HI:1349.18 and 409.57;MR:105.29 and 8.25).In the remaining 8 subjects, the ranged from 1.68 to 6.71 and the MR ranged from 0.002 to 0.014,as extensive metabolizers of S-mephenytoin.     

手性毛细管气相色谱法测定人尿中美芬妥英光学异构体含量的方法学研究

A gas chromatographic method equipped with nitrogen-phosphorus detector was developed for the determination of the S-and R-enantiomers of the anticonvulsant, mephenytoin (MP) in human urine. Dichioromethane (4 ml) was added to 1 ml urine, the mixture was shaken and centrifuged. The organic phase was transferred to another tube and blown to dryness under nitrogen on water bath (37℃). The residue was dissolved in 10 μl ethylacetate and 1～2 μl was injected into the GC. Our results showed that direct enantiomeric separation of mephenytoin was obtained by using a chiral capillary column, the retention times for S-and R-mephenytoin were 25. 5 and 26. 2 min respectively, with a detection limit less than 50 ng/ml of mephenytoin. Similar linear and reproducible standard curves were obtained over the concentration range of 53.2 to 2128. 0 ng/ml (for S-MP, r=0. 9914±0. 0070, n=6; and for R-MP, r=0. 9939±0. 0070, n=6), and the mean recoveries of S-and R-MP were 95. 4% and 95. 8% respectively. The within--day relative standard deviations were less than 8. 8% for both S-and R-MP, and that of between--days were less than 14.3%. There was a good reproducibility of the urine S/R mephenytoin determined in China and in Sweden by using similar method in 107 Chinese volunteers after a single oral dose of 100 mg racemic mephenytoin (r=0. 9091, P<0. 001).     

Extraction and separation of total flavonoids from Flos Puerariae and its antioxidant activity

Objective To study the optimum extraction and separation process of total flavonoids in the flowers of Flos Puerariae and its antioxidative activity.Methods The total flavonoids were extracted with assistance of ultrasonic wave and the content was determined at 265 nm wavelength by Spectrophotometric method.Orthogonal experiment L9(34)was carried out to investigate the effects of concentration of solvent,the ratio of material to liquid,time length of extraction,and frequency of extraction on extraction results of total flavonoids from Flos Puerariae.The extracted solution was purified by petroleum ether and ethanol sequently.Under these conditions,the total flavonoids was eluted gradiently with mixed mobile phase of methanol-chloroform solution in the silica gel column system,and then determined by UV scanning and HPLC.Fenton reaction was used to produce and detect hydroxyl radicals(·OH),and pyrogallol system was used to produce and detect the superoxide radical anion(O2-·).Results The optimum conditions were as follows;using 40%(V/V)methanol as extractor with the ratio of material to liquid at 1∶30,and extracting for 2.5 h a time for 3 times.The extraction yield of total flavonoids was 13.6%.Six isoflavone compounds were isolated from the flowers of Flos Puerariae by the method of silica gel column chromatography.Antioxidative test results showed good performance of flavonoid scavenging capacity in both hydroxyl radical system and superoxide radical system and its IC50 was 7.65 μg·mL-1 and 0.18 mg·mL-1,respectively.Conclusions This study provided scientific basis for further development and utilization of Flos Puerariae and make preparation for later pharmacological research.     

Synergistic effect of combining paeonol and cisplatin on apoptotic induction of human hepatoma cell lines

Aim： To investigate whether paeonol （Pae） has synergistic effects with cisplatin （CDDP） on the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and SMMC-7721. Methods： The cytotoxic effect of drugs was measured by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide assay. The coefficient of drug interaction was used to analyze the nature of drug interactions. Morphological changes were observed by acridine orange fluorescence staining. Cell cycle and the apoptosis rate were detected by flow cytometry. Bcl-2 and Bax expression were assayed by immunohistochemical staining. Results： Pae or CDDP had antiproliferative effect on the 2 cell lines in a dose-dependent manner, with different sensitivities to drugs. More interestingly, a synergistic inhibitory effect on the viability of the 2 cell lines was observed after treatment with a combination of Pae （15.63, 31.25, and 62.5 mg/L） with various concentrations of CDDP. Further study showed typical morphological changes of apoptosis if the cells were exposed to the two agents for 24 h. The apoptotic rate of the cells with combination treatment was significantly higher than that of cells treated with Pae or CDDP alone. The expression of Bcl-2 decreased and that of Bax increased in the treated groups, especially in the combination group, with the ratio of Bcl-2/Bax decreasing correspondingly. Additionally, a combination of Pae with CDDP resulted in a stronger S phase arrest, compared with Pae or CDDP alone. Conclusion： Pae, in combination with CDDP, had a significantly synergistic growth-inhibitory and apoptosis-inducing effect on the 2 human hepatoma cell lines, which may be useful in hepatoma treatment.     

高效液相色谱法同时测定人血清中普罗帕酮及其活性代谢物的浓度

A rapid, sensitive and simple high performance liquid chromatographicmethod for the simultaneous determination of propafenone (PPF) and its metabolites(5-hydroxypropafenone, 5-OHP; N-depropylpropafenone, NDP) in serum hasbeen developed. Separation of PPF, 5-OHP and NDP was achieved by reversed phasechromatography using a mobile phase consisting of 57% methanol and 43% 10 mmol/Lpotassium dibasic phosphate (pH 2.7)at a flow rate of 1.0 mi/rain on a 5μm ODS-C18column. The eluent was monitored at 254 nm. The method showed a good linearity. The recoveries of PPF, 5-OHP and NDP werefound to be 99.54±2. 13%, 100.02±3.66% and 100.48±3.10%, respectively. Precisionstudies for both within day and day-to-day at different concentrations provided RSDvalues of less than 5%. Some commonly used drugs can be determined in the same procedurewithout interference except phenytoin. This method is well adapted to the therapeuticmonitoring of PPF treated patients, as well as for pharmacokinetic studies.     

Regulating expressions of cyclin D1, pRb, and anti-cancer effects of deguelin on human Burkitt＇s lymphoma Daudi cells in vitro

Aim: To investigate anticancer effects and molecular mechanism of deguelin on human Burkitt‘ s lymphoma Daudi cells in vitro and compare the cytotoxicities of deguelin on Daudi ceils and human peripheral blood monocular ceils (PBMC). Methods: The effects of deguelin on the growth of Daudi cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptos iswere dectected through Hoechst 33258 staining and Annexin V/PI double-labeled cytometry. The effect of deguelin on the cell cycle of Daudi cells were studied by a propidium iodide method. The expressions of cyclin D1 and pRb were checked by Western blot. Results: The proliferation of Daudi cells were decreased in deguelin-treated group with a 24-h IC50 value of 51.55 nmol/L. Deguelin induced Daudi cells apoptosis was in a time- and dose-dependent manner. G0/G1 phase increased and S phase decreased in Daudi cells treated with deguelin. With deguelin 0, 5, 10, 20, and 40 nmol/L treatment for 24 h, G0/G1 phase increased from 37.34% to 56.56%, whereas S phase decreased from 37.72% to 21.36%. PBMC was less sensitive to the cytotoxic effect of deguelin than Daudi cells. The expression of cyclin D1 and pRb protein were decreased sharply in Daudi cells treated with deguelin. Conclusion: Deguelin is able to inhibit the proliferation of Daudi cells by regulating the cell cycle that arrested cells at Go/G, phase and inducing the cell apoptosis. Moreover, deguelin selectively induced apoptosis of Daudi cells with low toxicity in PBMC. The antitumor effects of deguelin were related to downregulating the expression of cyclin D 1 and pRb protein.     

虫草花与冬虫夏草腺苷成份的比较研究

Objective To compare the content difference of adenosine in Cordyceps sineasis and Cordyceps Flower.Methods HPLC method was applied to determine the contents of adenosine,with a column of Shim-pack VP-ODS 4.6 nun×200mm,5μm) and a mobile phase of phosphate buffer solution(pH=6.5)-methanol(17:3),flow rate was 1ml/min,detection wavelength was 260nm.Results The linear range of adenosine was 0.07835-0.39988μg,the result showed good linearity.The average recovery was 96.22%,RSD 0.21%.Conclusion The content of adenosine in Cordyceps Flower was higher.It is suggested that Cordyceps Flower has the same tonic efficacy as Cordyceps sinensis.     

Influence of SLYWG on malignant tumor ascites

Objective To search the effect of Chinese traditional medicine "Shang Lu Yu Wang Gao"(SLYWG)on the ascites induced by tumor,and its mechanism.Methods The tumor ascites model and diuretic experiments were introduced to evaluate the effect of SLYWG.Physical characteristics,the tumor cell counting,volume of the ascites,protein content in ascites,the characters of ascites,the life duration of S180 tumor bearing animals as the indexes of evaluation.the diurtic experiments were performed on rats nad rabbits,the osmotic pressure,K+,Na+,Cl-,Ca2+ and pH in urine were determined.Results The inhibitions to ascites of SLYWG were displayed in three dosage(30 g·kg-1,15 g·kg-1 and 7.5 g·kg-1).Ascites caused by tumor was significantly inhibited by the local administration of SLYWG.The increase of mice ascites was slow down,the content of ALB and the TP in ascites were decreased,the surviving time of mice was extended.SLYWG had remarkable diuresis effect on the rats and rabbits,it could reduce the osmotic pressure of urine,decrease the exclude of K+ but had no effect on the Na+,Cl-,Ca2+ and pH in urine.Conclusions Tumor ascites was significantly inhibited by the ventral administration of SLYWG.SLYWG had diuretic effect in rats and rabbits,it reduced the osmotic pressure of urine,decrease the exclude of K+ but had no effect on Na+,Cl-,Ca2+ and pH of urine.     

Determination of JBP485 by LC/MS and its pharmacokinetics in rats

Objective Cyclo-trans-4-L-hydroxyprolyl-L-serine(JBP485)is a dipeptide isolated from Laennec,and Laennec is a hydrolyzate of human placenta.Evidence has indicated that JBP485 exhibited potent anti-hepatitis activity.In this study,we developed a method for rapid and sensitive determination of JBP485 in rat biologic samples by LC/MS,and then studied its pharmacokinetics.We investigated the main excretion pathway of JBP485.Methods Following protein-precipitation with methanol,the analyte and internal standard(Paracetamol)were separated from rat plasma using an isocratic mobile phase on an Cap cell pack C18 UG120 column with a mobile phase consisting of methanol-water-formic acid(30∶70∶0.2,V∶V∶V)at a flow rate of 0.5 mL·min-1.An API 3200 tandem mass spectrometer equipped with Turbo Ion Spray ionization source was used as detector and was operated in the positive ion mode.Multiple reaction monitoring transporter precursor to product ion combinations of m/z 201.1→86.1 and m/z 152.1→110.1 were performed to quantify JBP485 and internal standard,respectively.After JBP485 was injected intravenously at a dose of 25 mg·kg-1 to rats,blood,bile and urine was collected at different time up to 8 h.The plasma concentration-time curve was plotted.The main pharmacokinetic parameters of JBP485 were obtained by 3P97 software.Results Linear calibration was generated over a concentration range of 0.1-200 μg·mL-1 for biologic samples by using LC/MS,with the lower limit of quantification of 0.1 μg·mL-1.Intra-and inter-days precision and accuracy were acceptable for all quality control samples.The mean recovery of JBP485 was above 90%,respectively.Pharmacokinetic parameters were estimated as follows:AUC(9103.96±513.85)μg·min·mL-1,t1/2β(77.98±4.06)min and CL(0.002±0.0001)mL·kg-1·min-1.The cumulative urinary excretion for 8 hours after administration was 30% of the dose.The cumulative biliary excretion for 8 hours after administration was 2%.Conclusions The LC/MS method was suitable for the pharamacokinetic study of JBP485 in rat with the advantages of specificity,sensitivity,accuracy and high speed.Renal excretion was the main excretive route of JBP485.     

A validated HPLC method for the determination of donepezil in human plasma after derivatization with 9-fluorenylmethyl chloroformate

A new HPLC method has been developed for determining donepezil in human plasma. To find the optimum conditions, a derivatization reaction was performed in different media, and the reaction product was identified by NMR and GC-MS after a semi-preparative HPLC separation. Under optimized conditions, donepezil was derivatized by 9-fluorenylmethyl chloroformate in chloroform and carbonate buffer at pH 9.5 in the presence of NaI after solid-phase extraction from a plasma sample. The reaction product was quantified on a reversed-phase TRACER EXCEL ODS-A, 5 μm column using a mixture of acetonitrile–10 mM acetate buffer（pH 6.0）–THF（60：35：5, v/v/v） as the mobile phase with fluorescence detection at 264 nm（ex） and 313 nm（em）. Fluoxetine was used as the internal standard. The total run-time of the analysis was about 10 min, and a clean chromatogram was obtained. The developed method was linear over the range of 1–100 ng/mL in 500 μL of plasma samples（r20.998）. The intra-day and inter-day precision values were in the range of 2.6%–11.6%. The limit of quantification was 1 ng/mL.     

高效液相色谱法测定血浆及尿中氯霉素的含量

采用高效液相色谱法,以非那西汀为内标物在λ_(max)=278nm下,研究了血浆及尿中氯霉素的含量。本法测定结果:保留时间:氯霉素7 min,非那西汀11min;最低检测浓度:血样为52.3%μg/ml,尿样为37μg/ml;平均回收率:血样为100.5%,尿样为99.7%;线性范围:血样及尿样均为5～50μg/ml;日内变异系数:血样在1.10%以下,尿样在1.19%以下;日间变异系数:血样在5.3%以下,尿样在5.9%以下;新诺明等药物对测定无干扰。     

Determination of a new phosphodiesterase V inhibitor,DA-8159, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.     

Determination of rufloxacin, a new tricyclic fluoroquinolone in biological fluids using high-performance liquid chromatography with ultraviolet detection.

A simple, specific, and sensitive high-performance liquid chromatography method has been developed for routine monitoring of the antimicrobial agent rufloxacin in human serum and urine. Serum or urine-spiked with internal standard pipemidic acid, were vortex-mixed for 2 min with dichloromethane at pH 7.4. The evaporated extract was dissolved in 0.05 M NaOH. The mobile phase consisted of orthophosphoric acid, tetrabutylammonium iodide, and methanol. Drugs were resolved at ambient temperature on a 10 micron Viosfer LC-RP-18 column (250 x 4.6 mm I.D.) equipped with a guard column. Flow rate was 1.8 ml/min, and monitoring was performed at 295 nm. The calibration curve was linear from 0.1 to 10 micrograms/ml for human serum and from 0.05 to 10 micrograms/ml for urine. Retention times were 3.1 and 6.3 min for internal standard and rufloxacin. The detection limit of rufloxacin was 0.05 microgram/ml for human serum and 0.03 microgram/ml for urine. No interference from other commonly administered drugs or endogenous substances was observed. The assay demonstrated sufficient sensitivity and specificity for the study of the pharmacokinetics of rufloxacin in humans.     

Determination of a new reversible proton pump inhibitor,DBM-819, in human plasma and urine,and rat tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new proton pump inhibitor, DBM-819, in human plasma and urine and rat tissue homogenates using KR-60461 as an internal standard. A 100-microl aliquot of acetonitrile (containing 0.5 microg/ml of the internal standard) and a 200-microl aliquot of 0.1 M Na(2)HPO(4) (adjusted pH 11 with 1 N NaOH) were added to a 100-microl aliquot of biological sample. After vortex-mixing, the mixture was extracted with 1 ml of ethylacetate. After centrifugation at 12000 x g for 3 min, the organic layer was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-microl aliquot of mobile phase, and a 40-microl aliquot was injected onto the HPLC column. The mobile phase, 0.02 M phosphate buffer (pH 5): acetonitrile: methanol (46:44:10, v/v/v), was run at a flow rate of 0.5 ml/min and the column effluent was monitored by the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 470 nm. The retention times for DBM-819 and the internal standard were approximately 10.5 and 12 min, respectively. The detection limits of DBM-819 in human plasma and urine, and rat tissue homogenates were 0.01, 0.02 and 0.02 (or 0.05) microg/ml. respectively. The coefficients of variation (CV) of the assay were below 11% for human plasma and urine, and rat tissue homogenates. No interferences from endogenous substances were found.     

HPLC柱切换法测定血浆和尿样中头孢克肟浓度

用双泵HPLC柱切换系统直接进样测定血浆和尿样中头孢克肟(cefixime,CFIX)的浓度。血浆和尿样的回收率分别为99.1%和98.6%;最低检测浓度分别为0.05和0.2μg/ml;日内和日间的RSD小于5%;血浆和尿样浓度分别在0.1～3.2和1.0～32μg/ml范围内呈线性相关。此方法集样品净化、富集成分和色谱分析一次连续进行,操作简便、快速,可以进较大的样品量,灵敏度相对明显提高。     

Simultaneous analysis of thiamphenicol and its prodrug thiamphenicol glycinate in human plasma and urine by high performance liquid chromatography: application to pharmacokinetic study

A simple and sensitive method for simultaneous determination of the active compound, thiamphenicol (TAP) and its prodrug, thiamphenicol glycinate (TG) in human plasma and urine is described. The procedure involved extraction of TG and TAP with ethyl acetate (plasma) or 100-fold dilution with the mobile phase (urine) followed by determination by reversed-phase high performance liquid chromatography (HPLC) with UV detection at 224 nm. Separation of the compounds was achieved on a column packed with Hypersil ODS2. The mobile phase consisted of acetonitrile-water containing 0.003 M tetrabutyl ammonium bromide and 0.056 M ammonium acetate (87:13, v/v) with a flow rate of 1.0 ml/min. The chromatograms did not contain interfering peaks due to the suitable extraction procedure and chromatographic conditions. The calibration curves of TG and TAP were linear ranging from 0.78 to 100 microg/ml in plasma and in urine. The intra-day and inter-day relative standard deviations (S.D.) were less than 10%. The recoveries of TG and TAP in plasma and urine were above 80%. TG was not stable in plasma samples and after extraction at ambient temperature or in freeze-thaw cycles, and hence the samples for injection on HPLC column should be stored in refrigerator or under ice cooling prior to analysis, and the plasma samples should not experience the freeze-thaw cycle more than one time. Unlike TAP, TG could not be detected in most urine samples. Application of this method demonstrated that it was feasible for the clinical pharmacokinetic study.     

Determination of methotrexate and indomethacin in urine using SPE-LC-DAD after derivatization

A validated HPLC-DAD assay is presented for the simultaneous quantification of methotrexate and indomethacin in a drug combination which is used synergistically to intervene with tumoral or inflammatory tissue microenvironments. The analytes were isolated from urine via solid phase extraction. The method is based on derivatizing both analytes with a soluble carbodiimide coupler and 2-nitrophenylhydrazine directed to their commonly occurring carboxylate functions. The chromatographic separation was accomplished on an octylsilica column in less than 15 min using acetate buffer (pH 4; 10 mM)-methanol (60:40, v/v) as eluent at 1.5 ml/min and monitored at 400 nm. The selectivity of the method was demonstrated in a pre-dosed rheumatoid arthritis patient. Quality control samples were prepared and analyzed to reveal the validity of the method. Life samples collected from a healthy volunteer were monitored for both drugs and their urinary levels were determined.     

Sensitive determination of nefopam and its metabolite desmethyl-nefopam in human biological fluids by HPLC

Nefopam (NEF) and desmethyl-nefopam (DMN) were assayed simultaneously in plasma, globule and urine samples using imipramine as internal standard. A liquid-liquid extraction procedure was coupled with a reverse phase high-performance liquid chromatography system. This system requires a mobile phase containing buffer (15 mM KH(2)PO(4) with 5 mM octane sulfonic acid: pH 3.7) and acetonitrile (77:33, v/v) through (flow rate=1.5 ml/min) a C(18) Symmetry column (150x4.6 I.D., 5 micrometer particle size: Waters) and a UV detector set at 210 nm. Internal standard was added to 1 ml of plasma or globule sample or 0.5 ml of urine sample, prior to the extraction under alkaline ambiance with n-hexane. The limits of quantification were 1 and 2 ng/ml for both molecules in plasma and globule, respectively; 5 and 10 ng/ml for NEF and DMN in urine, respectively. The method proved to be accurate and precise: the relative error at three concentrations ranged from -13.0 to +12.3% of the nominal concentration for all molecule and biological fluid; the within-day and between-day precision (relative standard deviation %) ranged from 1.0 to 10.1% for all the molecules and biological fluids. The method was linear between 1 and 60 ng/ml for both molecules in the plasma; 2 and 25 ng/ml for both molecules in the globule; 25 and 250 ng/ml for NEF and 50 and 500 ng/ml for DMN in the urine: correlation coefficients of calibration curves (determined by least-squares regression) of each molecule were higher than 0.992 whatever the biological fluid and during the pre-study and in-study validations. This method was successfully applied to a bio-availability study of NEF in healthy subjects.     

High performance liquid chromatographic analysis of a novel aminoalkylpyridine anticonvulsant 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane

A simple, rapid and specific high performance liquid chromatographic (HPLC) method for the quantitation of 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane (AAP-Cl) and identification of its putative metabolite, 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethanol (beta-AA) in rat blood and urine has been developed. AAP-Cl, beta-AA and an appropriate internal standard were extracted from rat biofluids by a solid phase extraction technique using C18 cartridges prior to the HPLC analysis. The extractibility was 92% for AAP-Cl and 98% for beta-AA. The HPLC analysis employed a symmetrical or standard reversed-phase HPLC column (Apex ODS, 5 microm, 25 cm x 0.46 cm) for blood or urine analysis, a mobile phase of water methanol acetonitrile (40:30:30) containing 20 microl 100 ml(-1) diethylamine at a flow rate of 1 ml min(-1), and UV detection at 254 nm. The limit of detection was 100 ng ml(-1) for both analytes in both blood and urine. The calibration curves for AAP-Cl in rat biofluids were shown to be linear in both low and high concentration ranges (blood: 0-1 and 1-10 microg ml(-1); urine: 0-10 and 10-100 microg ml(-1)) with intra- and inter-day coefficients of variation of no more than 18% for blood and 14% for urine. The method developed was successfully applied to a preliminary analysis of intact AAP-Cl in both blood and urine obtained from rats dosed with AAP-Cl.     

Determination of depleted uranium, pyridostigmine bromide and its metabolite in plasma and urine following combined administration in rats

A simple and reliable method was developed for the quantification of depleted uranium, the anti nerve agent drug pyridostigmine bromide (PB;3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The method involved using solid phase extraction and spectrophotometric determination of uranium, and high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 280 nm for PB and its metabolite. Uranium was derivatized using dibenzoylmethane (DBM) then the absorbance was measured at 405 nm. PB and its metabolite were separated using a gradient of 1--40% acetonitrile in 0.1% triflouroacetic acid water solution (pH 3.2) at a flow rate of 0.8 ml/min in a period of 14 min. Limits of detection were 2 ng/ml for uranium and 50 ng/ml for PB and its metabolite. Limits of quantitation were between 10 and 100 ng/ml for uranium and the other two analytes, respectively. Average percentage recovery of five spiked plasma samples were 83.7+/-8.6, 76.8+/-6.7, 79.1+/-7.1, and from urine 82.7+/-8.6, 79.3+/-9.5 and 78.0+/-6.2, for depleted uranium, PB and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear for standards between 100 and 1000 ng/ml for all three analytes. This method was applied to analyze the above chemicals and metabolites following combined administration in rats.