Determination of amifloxacin and two of its principal metabolites in plasma and urine by high-performance liquid chromatography using automated column switching

The automated determination of amifloxacin and two of its principal metabolites in human plasma and urine by column-switching high-performance liquid chromatography is described. Plasma or urine samples, diluted 1:1 with 0.5 M sodium citrate buffer pH 2.5, were directly injected onto a cation-exchange pre-column. Following a 2.0-min wash of the pre-column with water at a flow-rate of 1.1 ml/min, the effluent from the pre-column was directed to the analytical column by a column-switching device. The precision of the plasma and urine methods ranged from a +/- 1.9 to +/- 3.6% for all compounds. The accuracies of the methods were within a range of -3.3% to 6.4% of the nominal values for all compounds. Linear responses were observed for all the standards in the range 0.10-5.0 micrograms/ml for plasma and 0.50-100 micrograms/ml for urine for all three compounds. The minimum quantifiable levels were 0.10 and 0.50 micrograms/ml for plasma and urine, respectively. The analytical methods may be used to quantify amifloxacin and the piperazinyl-N-desmethyl and piperazinyl-N-oxide metabolites in plasma and urine samples obtained from humans, monkeys, dogs and rats.     

Determination of a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.     

HPLC determination of cis-diamminedichloroplatinum(II) in plasma and urine with UV detection and column-switching

A method for determining cis-diamminedichloroplatinum(II) (CDDP), an anticancer drug, in plasma and urine by HPLC with UV detection and column-switching has been developed. Typical conditions were as follows. An apparatus was composed of two columns, two pumps, a UV detector, a sample injector with a 100 microL loop, a switching valve, a column oven and a recorder. A Rheodyne model 7125 sample injector was used as the switching valve. A precolumn (4.6 mm ID x 25 cm) was packed with MCI GEL CK10S (a strong cation exchanger), and an analytical column (4.6 mm ID x 5 cm) was packed with MCI GEL CDR10 (a strong anion exchanger). Both columns were connected in series via the switching valve. The CDDP-containing fraction of the effluent from the precolumn was loaded to the analytical column by column-switching and the effluent from the analytical column was monitored at 210 nm. An eluent of 0.3 M sodium dihydrogen phosphate was pumped at a flow rate of 1 mL/min and the columns were maintained at 40 degrees C. CDDP was eluted at about 11 min and the identity of the peak of CDDP on the chromatogram was confirmed by its 3-dimensional chromatogram and analysis of platinum in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and concentration of CDDP up to 100 microM. Correlation efficients were 0.998 for plasma and 0.999 for urine. The detection limit was 0.1 microM for CDDP in both plasma and urine (S/N = 3,0.005 AUFS). The reproducibility was within 3% for 10 determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

On-line solid-phase extraction of piroxicam prior to its determination by high-performance liquid chromatography

A direct method for the determination of piroxicam in plasma is described. Plasma is directly injected onto the extraction column (10 mm x 2 mm I.D., packed with 40-microns Bond Elut C2) where proxicam is separated from the plasma concomitants using a solid-phase extraction procedure. Using a laboratory-made on-line column-switching system, the drug is quantitatively transferred and separated on the analytical column (15 cm x 4.6 mm I.D., Supelcosil LC18 DB, 5 microns) followed by determination using ultraviolet absorption at 331 nm. Validation of the method demonstrated a good recovery (100%), sensitivity (limit of determination 0.2 microgram/ml, based on a 20-microliters sample volume), accuracy and precision (better than 5%). The developed method has been adopted for studying the steady-state pharmacokinetics of the drug.     

Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

A column-switching system for the direct injection of plasma or serum samples, followed by isocratic high-performance liquid chromatography and ultraviolet detection, is described for the simultaneous quantitation of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline and the E- and Z-isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline. The method included adsorption of amitriptyline and metabolites on a reversed-phase C8 clean-up column (10 microns; 20 mm x 4.6 mm I.D.), washing of unwanted material to waste and, after on-line column-switching, separation on a cyanopropyl analytical column (5 microns; 250 mm x 4.6 mm I.D.). The compounds of interest were separated and eluted using acetonitrile-methanol-0.01 M phosphate buffer (pH 6.8) (578:188:235, v/v) within less than 20 min. Various drugs frequently co-administered with amitriptyline or other antidepressants did not interfere with the determinations. In plasma samples spiked with 25-300 ng/ml, the recoveries were between 84 and 112% and the inter-assay coefficients of variation were 3-11%. After a minor modification, as little as 5 ng/ml could be quantitated. There were linear correlations (r greater than 0.99) between drug concentrations of 5-500 ng/ml and the detector signal. The method allows routine measurements of amitriptyline, nortriptyline and hydroxylated metabolites in blood plasma or serum of patients treated with amitriptyline or nortriptyline, and enables the results to be reported within 1 h.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. II. Simultaneous determination of etretinate, acitretin and 13-cis-acitretin in plasma

An automated gradient high-performance liquid chromatographic method for the determination of etretinate, acitretin and 13-cis-acitretin in plasma was developed, using a column-switching technique. After protein precipitation with ethanol, 0.5 ml of the supernatant was injected onto a precolumn (17 mm x 4.6 mm I.D.), filled with 37-53 microns C18 Corasil. Polar plasma components were washed out using 1% ammonium acetate and 1% acetic acid-acetonitrile (8:2, v/v); the retained retinoids were then transferred to the analytical column (125 mm x 4 mm I.D., filled with 5-microns ODS material) in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. The limit of quantification was 2 ng/ml and the inter-assay precision in the concentration range 20-1000 ng/ml was between 0.9 and 4.0% for all three compounds. To optimize the recovery for etretinate (greater than 60%), protein was precipitated from plasma with ethanol before injection, instead of direct injection of plasma samples, and a mobile phase containing 20% acetonitrile, instead of pure water or buffer, was used.     

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.     

Simultaneous determination of cefamandole and cefamandole nafate in human plasma and urine by high-performance liquid chromatography with column switching

A high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of cefamandole and cefamandole nafate in plasma and urine. The plasma and urine samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard solution in 0.05 M phosphoric acid. Polar plasma and urine components were washed out using 0.05 M phosphoric acid. After valve switching, the concentrated drugs were desorbed in back-flush mode and separated by a reversed-phase C8 column with methanol-5 mM tetrabutylammonium bromide (45:55, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.5 microgram/ml. The total analysis time per sample was less than 30 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9%. The method has been successfully applied to plasma and urine samples for human volunteers after intravenous injection of cefamandole nafate.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.     

Routine determination of hydroxyphenytoin in urine by high-performance liquid chromatography using an automatic column-switching technique

A high-performance liquid chromatographic method for the determination of the main phenytoin metabolite, hydroxyphenytoin, in the urine of epileptic patients is described. The use of an automated column-switching technique greatly simplifies the pretreatment steps. Thereby, both time and chemicals are saved. The possibility of error arising during the several pretreatment steps is considerably reduced. Following acid hydrolysis of the hydroxyphenytoin glucuronic acid conjugate the sample is diluted with water and after centrifugation is injected onto the pre-column. After washing for a short time with water, the substances which were absorbed on the head of the pre-column were backflushed with water--acetonitrile as eluent onto the analytical column. Separation is achieved by gradient elution using an ODS reversed-phase column with a particle size of 5 microns.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with UV-Vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV-Vis detection has been developed and validated for the determination of vigabatrin (VG) in human plasma and urine. The samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A good chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) gradient elution. Tranexamic acid was used as an internal standard (I.S.). The method was linear over the concentration range of 0.8-30.0 microg/ml for both samples. The method is precise (relative standard deviation (R.S.D.) <9.13%) and accurate (relative mean error (RME) <-8.75%); analytical recoveries were 81.07% for plasma and 83.05% for urine. The assay was applied to pharmacokinetic study in a healthy volunteer after a single oral administration of 1 g of vigabatrin.     

硅胶基硝化苯硼酸亲和色谱材料的合成及应用

合成了硅胶基硝化苯硼酸亲和色谱材料,首先对间氨基苯硼酸进行硝基化,制得3-氨基-4-硝基苯硼酸功能基团,通过γ-缩水甘油醚氧丙基三甲氧基硅烷把功能基团键合到硅胶基体上,在20.7MPa压力下装成亲和色谱预柱(35mm×4.6mmi.d.)。用该预柱通过六通阀后接ODS分析柱(250mm×4.6mmi.d.),构成中心切割二维HPLC。该系统能对含有顺二羟基结构的化合物进行在线富集,实现生物复杂样品直接进样分离分析。使用该系统对尿中多种修饰核苷进行了分离分析,以pH值7.95的0.25mol/LNH4Ac碱性缓冲液把实际尿样(100μL)中核苷保留在预柱上,生物大分子无保留通过,再切换六通阀,以pH4.50的25mmol/LKH2PO4酸性洗脱液把保留在预柱上的核苷洗脱,进样到下一级ODS分析柱柱头上聚焦,然后用梯度洗脱法(pH4.50的25mmol/LKH2PO4缓冲液与体积比60∶40的甲醇-水梯度混合构成洗脱液)完成核苷在ODS分析柱上的分离(紫外检测260nm),11种目标核苷的分离分析取得了良好的定性定量结果。     

Trace-level determination of 3'-azido-3'-deoxythymidine in human plasma by preconcentration on a silver (I)-thiol stationary phase with on-line reversed-phase high-performance liquid chromatography

A method was developed for the determination of 3'-azido-3'-deoxythymidine (AZT) in plasma. The method is based on the trace enrichment of AZT on a pre-column packed with a silver-loaded thiol stationary phase at pH 11.6. On-line desorption to the reversed-phase liquid chromatographic system is performed by injecting a plug of 50 microliters of 1 M perchloric acid on the silver (I)-thiol pre-column. Two different sample pretreatment methods - protein precipitation with perchloric acid and on-line clean-up via a polymeric PRP-1 pre-column - were applied for the determination of AZT in human plasma. The latter method allows the direct injection of plasma samples into the analytical system and can therefore easily be automated. With both methods detection limits in the order of 10(-8) M AZT were obtained after preconcentration of 1.0 ml of plasma, using UV detection at 267 nm.     

Simple determination of azasetron in rat plasma by column-switching high-performance liquid chromatography

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17?mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17?mM potassium phosphate buffer (pH 3.0)) and detected at 250?nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800?ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0?mg/kg to rats.     

高效液相色谱法测定盐酸芬氟拉明及其在片剂中含量

应用高效液相色谱法测定了盐酸芬氟拉明及其片剂的含量,使用２５ｃｍ×０．４６ｃｍｉ．ｄ．分析柱,填充ＨｙｐｅｒｓｉｌＢＤＳＣ１８（５μｍ）,检测波长２６４ｎｍ,流动相为Ｖ（乙腈）∶Ｖ（０．４ｍｏｌ／Ｌ乙酸铵）∶Ｖ（三乙胺）＝３０∶７０∶２。选用咖啡因作内标物,用内标法进行定量测定,在０．１～０．５ｇ／Ｌ范围内呈良好的线性关系,相关系数为０．９９９９。片剂的平均回收率为９９．９８％。方法简单、快速、灵敏度高、重现性好。     

On-line preconcentration of niobium(V) and tantalum(V) as 4-(2-pyridylazo) resorcinol-citrate ternary complexes in geological samples by ion interaction high-performance liquid chromatography

4-(2-Pyridylazo) resorcinol (PAR) and citrate were used as pre-column complexing agents for the determination of Nb(V) and Ta(V) as ternary complexes in geological samples. Aliquots of 2 ml of the standard and sample solutions containing the Nb(V) and Ta(V) complexes were loaded onto a concentrator column (C18, 0.4 cm x 4.6 mm) with a carrier mobile phase comprising 20% (v/v) methanol and containing 5 mM acetic acid, 5 mM citric acid and 10 mM tetrabutylammonium bromide (TBABr), pH 6.5 at 2 ml/min for 2 min, with the effluent being directed to waste. An automatic switching valve was then switched to flush both complexes from the concentrator column onto a C18 analytical column using a mobile phase comprising 32% (v/v) methanol and containing 5 mM acetic acid, 5 mM citric acid and 3 mM TBABr, pH 6.5 for 2.5 min. The switching valve was then switched back to the original position, and cleaned with methanol for 7 min to eliminate unwanted species still adsorbed to the concentrator column. This procedure prevented later eluting compounds from reaching the analytical column, which reduced the overall run time. The detection limits of Nb(V) and Ta(V) (determined at a signal-to-noise ratio of 3, detection wavelength of 540 nm and a 2-ml sample volume) were 0.012 and 0.039 ppb for Nb(V) and Ta(V), respectively. Recoveries of Nb(V) and Ta(V) were 99.4 and 96.2%, respectively. The HPLC results obtained from the reference granite and basalt samples agreed well with inductively coupled plasma MS and certified values, but the HPLC method yielded slightly low values of the Nb/Ta ratio.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography.

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 x 4.0 mm I.D., 5 microns particle size) protected by a guard column (Perisorb RP-18, 30 x 4.0 mm I.D., 30-40 microns particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5-100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) - 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0-97.8%; method comparison c(bioassay) = 1.092c(HPLC) - 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.     

Determination of rotenone in river water utilizing packed capillary column switching liquid chromatography with UV and time-of-flight mass spectrometric detection

Fast and sensitive packed capillary column switching liquid chromatography methodology has been developed for the determination of the pesticide rotenone in river water. Sample volumes of up to 1 ml are loaded onto a 23 x 0.25 mm, 5 microm Kromasil C18 packed capillary precolumn using a noneluting solvent composition of water-acetonitrile (99:1, v/v) at flow-rates up to 100 microl/min prior to solute backflushing onto a 200 x 0.32 mm, 3.5 microm Kromasil C18 packed capillary analytical column using a mobile phase of water-acetonitrile (30:70, v/v) at a flow-rate of 5 microl/min. The method was evaluated using river water samples spiked with rotenone in the concentration range 0.5-50 ng/ml using UV detection. The within-assay precision was between 5.0 and 7.7% relative standard deviation (RSD, n = 6) and the between assay precision was between 7.5 and 8.9% RSD (n = 6). The method was linear within the investigated mass range displaying a calibration curve correlation factor of 0.997. The mass limit of detection was 10 pg corresponding to a concentration limit of detection of 10 pg/ml, using time-of-flight mass spectrometry.     

Determination of oxytetracycline in blood serum by high-performance liquid chromatography with direct injection.

A direct injection analysis by high-performance liquid chromatography has been developed for oxytetracycline in serum of animals and fish. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of methanol-0.2 M oxalic acid (10:90, v/v, pH 7.0) with ultraviolet detection at 360 nm were used. The standard calibration curves in serum of chicken, hog, cattle and rainbow trout were linear over the range 0.1-20 micrograms/ml. The recoveries of oxytetracycline from all serum samples determined at two different concentrations (0.5 and 2.0 micrograms/ml) were 88-103%. The detection limit was 0.05 micrograms/ml for every serum sample.