Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH₂ bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 μg/ml for plasma, 3 μg/ml for urine and 0.03 μg/ml for saliva using UV detection.     

Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis determination method with UV detection of grepafloxacin and clinafloxacin has been developed. The separation was performed in 35 mM borate-35 mM phosphate buffer solution (pH 8.6), containing 6% (v/v) of acetonitrile. Analyses were realised using fused-silica capillaries (57 cm length x 75 microm I.D.) and the operating conditions were: 15 kV applied voltage, 30 degrees C and detection at 279 nm. Piromidic acid was used as an internal standard. The linear concentration range of application was 1.0-120.0 microg ml(-1) for both compounds, with a detection limit of 0.2 microg ml(-1) for grepafloxacin and 0.3 microg ml(-1) for clinafloxacin. The analysis yielded good reproducibility (RSD between 3.37 and 1.74%). It was applied to the determination of grepafloxacin and clinafloxacin in human and rat urine samples. The method was validated using HPLC as a reference method. Recovery levels were between 94.5 and 103%.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

Multiresidue determination of quinolone antibacterials in eggs of laying hens by liquid chromatography with fluorescence detection

An analytical method for the simultaneous determination of seven quinolones (ciprofloxacin, enrofloxacin, danofloxacin, difloxacin, flumequine, oxolinic acid and sarafloxacin) in egg samples of laying hens was developed. Their use is totally prohibited in animals from which eggs are produced for human consumption. Protein precipitation was achieved by addition of acetonitrile and ammonia, removal of acetonitrile with dichloromethane, the quinolones remaining in the basic aqueous extract. The aqueous extract was analysed by liquid chromatography with fluorescence detection (LC-FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Norfloxacin was used as an internal standard. The limits of detection found were 4-12 ng g(-1). These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in different tissues of eggs-producing animals.     

RP-HPLC method with fluorescence detection for determination of small quantities of triamcinolone in plasma in presence of endogenous steroids after derivatization with 9-anthroyl nitrile; pharmacokinetic studies

A new indirect RP-HPLC method was developed for determination of small, ng/ml, concentrations of triamcinolone (TMC) in human plasma, in presence of endogenous corticosteroids: cortisol (hydrocortisone, F), cortisone (E) and their metabolites, after administration of TMC in a free alcohol form. After solid phase extraction (SPE) in cartridges with octadecyl phase, TMC and prednisolone (I.S.) were derivatized by treatment with 9-anthroyl nitrile (9-AN) in a basic mixture, consisting of triethanolamine and quinuclidine, to receive fluorescent esters at 21-hydroxyl group of the steroid chain. Optimal conditions were also established to purify fluorescent TMC and I.S. derivatives before injection into HPLC column. The fluorescent esters were determined using an isocratic RP-HPLC technique in a C18 column. The mobile phase consisted of acetonitrile and 0.3 mM ortho-phoshoric acid. The method was validated before using to pharmacokinetic studies. Calibration curve of TMC was linear in the range of 2.5-100.0 ng/ml. Intra- and inter-day measurement precision and accuracy were equal to or lower than 15%. Percent recovery, and limits of detection (LOD) and quantification (LOQ) of TMC were also determined. The method was applied for in vivo conditions after administration of tablets containing TMC to healthy volunteers. Moreover, the method provided potential to determine TMC and, simultaneously, other glucocorticoids: E, F and their metabolites in one analytical run. Column interactions were observed between endogenous metabolites of E. Usefulness of the elaborated method was confirmed in pharmacokinetic studies following administration of a small (4 mg) dose of TMC to human volunteers. The method can provide an alternative to HPLC coupled with RIA in determination of small quantities of TMC.     

Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry

Acyl-CoAs have important role in fat and glucose metabolism of the cells. In this study we have developed an on-line HPLC-ESI-MS/MS method for determination of long-chain acyl-CoA compounds in rat liver samples. Six long-chain acyl-CoAs (C16:0, C16:1, C18:0, C18:1, C20:0 and C20:4) were separated with a C4 reversed-phase column using triethylamine acetate and acetonitrile gradient. Negative electrospray ionization is very suitable for acyl-CoA compounds and excellent MS/MS spectra for long-chain acyl-CoAs can be obtained. MS/MS method with an ion trap mass spectrometer makes it possible to identify and quantitate individual acyl-CoAs simultaneously. The method proved to be sensitive enough for determination of all compounds of interest using 0.4-0.7 g of tissue and was validated in the range of 0.1-15.0 pmol/microl.     

Sensitive quantification of diphemanil methyl sulphate in human plasma by liquid chromatography-tandem mass spectrometry

A simple detection system with a high-performance liquid chromatography (HPLC) with positive ionisation-tandem mass spectrometry (ESI-MS/MS) for determining diphemanil methylsulphate (DMS) levels in human plasma using 4-diphemanylmethylene,1-methylpiperidine as an internal standard (I.S.), is proposed. The acquisition was performed with the multiple reactional monitoring (MRM) mode, by monitoring the transitions: m/z 278>262 for DMS and m/z 263>247 for the I.S. The method involved a simple single-step deproteinisation with acetonitrile. The analyte was chromatographed on a Zorbax C18 reversed-phase chromatographic column by isocratic elution with 10(-3)M ammonium acetate and 10(-3)M hexafluorobutyric acid, adjusted to pH 7.0 with ammoniac/acetonitrile (40/60, v/v). The results were linear over the studied range (0.5-50.0 ng mL(-1)) and the total analysis time for each run was 10 min. The mean extraction apparent recoveries expressed at the 95% intervals of confidence were 94-104% for DMS and 92-106% for the I.S. The intra- and inter-assay precisions were 4.6-8.4% and 2.9-10.6%, respectively. The limit of quantification was 0.15 ng mL(-1). The devised assay was successfully applied to the residual concentrations monitoring in infant.     

Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase V radioassay

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-d-mannopyranosyl]-β-D-glucopyranoside (IPGMG, 1) and its radiolabeled form, [(125)I]IPGMG ([(125)I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-2,3,4-tri-O-acetyl-β-D-glucopyranoside (21), was synthesized as a precursor for the preparation of [(125)I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [(125)I]1. When [(125)I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of β1-6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a K(m) of 23.7 μM and a V(max) of 159 pmol/h. μg protein. [(125)I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry

A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.     

Validation and application of a liquid chromatography-tandem mass spectrometric method for the determination of SCH 211803 in rat and monkey plasma using automated 96-well protein precipitation

A rapid, sensitive, specific, accurate, and reproducible automated liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the quantitative determination of 1'-(2-amino-3-methylbenzoyl)-4-[[[(3-chlorophenyl)sulfonyl]phenyl]methyl]-1,4'-bipiperidine hydrochloride (SCH 211803) in plasma has been developed. The method was validated in rat and monkey plasma over the concentration range of 0.5-250 ng/ml using 2H(4)-SCH 211803 as the internal standard (IS). Automated 96-well plate protein precipitation (PP) with acetonitrile (ACN) was used for sample processing. The method employed a Betasil C18 column with a fast gradient for the separation of analyte and internal standard from the plasma matrix and a triple quadrupole mass spectrometer operated in positive ion multiple reaction monitoring (MRM) mode for detection. The method was used for the determination of SCH 211803 plasma concentrations to support pre-clinical studies.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.     

Analysis of the second generation antidepressant venlafaxine and its main active metabolite O-desmethylvenlafaxine in human plasma by HPLC with spectrofluorimetric detection

A high-performance liquid chromatographic method has been developed for the determination of the recent serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine and its main active metabolite, O-desmethylvenlafaxine, in human plasma. Separation was obtained by using a reversed-phase column (C8, 150 x 4.6 mm I.D., 5 microm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 6.8 and 25% acetonitrile. Fluorescence detection was used, exciting at lambda=238 nm and monitoring the emission at lambda=300 nm. Citalopram was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C1 cartridges (100 mg, 1 mL). The limit of quantification (LOQ) was 1.0 ng mL(-1) and the limit of detection (LOD) was 0.3 ng mL(-1) for both analytes. The method was applied with success to plasma samples taken from patients undergoing treatment with venlafaxine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method is suitable for therapeutic drug monitoring of venlafaxine and its main metabolite in depressed patients' plasma.     

Determination of spinosin in rat plasma by reversed-phase high-performance chromatography after oral administration of Suanzaoren decoction

A sensitive, simple, and accurate method for determination of spinosin in rat plasma with sulfamethoxazole (SMZ) as internal standard was developed using RP-HPLC with UV detection. Sample preparations were carried out by protein precipitation with acetonitrile, followed by the evaporation of the acetonitrile to dryness. The resultant residue was then reconstituted in mobile phase and injected onto a Hypersil C(18) (200 x 4.6 mm I.D., 5 microm) analytical column. The mobile phase consisted of acetonitrile-water (15:85, v/v) with 1% glacial acetic acid. The assay was shown to be linear over the range of 18.07-903.5 ng/ml (R(2)=0.995). Mean recovery was determined as 93.6%. Within- and between-day precisions were 相似文献   

A HPLC method for determination of nicousamide in dog plasma and its application to pharmacokinetic studies

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for determination of nicousamide, an inhibitor of rennin and transforming growth factor-beta1 (TGF-beta1) type II receptors, has been developed and validated. Following acetonitrile deproteiniation, samples were separated by isocratic reversed-phase HPLC on an Aichrom Bond-AQ C(18) column and quantified using UV detection at 320 nm. The mobile phase was acetonitrile/water (ratio 62:38 containing 0.1% H(3)PO(4)), with a flow-rate of 1.0 ml/min. A linear curve over the concentration range 5-200 ng/ml (r(2)=0.9978) was obtained. The coefficients of the variation for the intra- and inter-day precisions ranged from 1.4-10.7% and 1.8-7.1%, respectively. The percentage of relative recovery was 91.56-105.45%. The method was used to determine the plasma concentration-time profiles for nicousamide after oral doses of 30, 100 and 300 mg/kg in dogs. A nonlinear pharmacokinetics was found in dogs at doses from 30 to 300 mg/kg. Following 30 mg/kg oral dose, the C(max) and AUC in females were lower than that in male. There is a potential for accumulation in dogs following multiple doses.     

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid-liquid extraction following the derivatization. The conjugate was analyzed by ion-pair HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching : I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17x4.6 mm I.D.), filled with C₁₈ Corasil 37–53 μm. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-1000 ng/ml was between 1.0 and 4.7% for all compounds.     

Determination of ZD9583, a thromboxane receptor antagonist, in human plasma and urine by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

A liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS–MS) method is described for the determination of a thromboxane receptor antagonist (4Z)-6-((2S,4S,5R)-2-(1-(2-cyano-4-methylphenoxy)-1-methylethyl)-4-(3-pyridyl)-3-dioxan-5-yl)hex-4-enoic acid (ZD9583, I) in human plasma and urine. Proteins in plasma and urine samples are precipitated using acidified acetonitrile. The resulting supernatant is chromatographed on a C₈ reversed-phase chromatography column. Following the diversion of the solvent front from the mass spectrometer by a switching valve, the column eluate is passed on to the mass spectrometer via a heated nebulizer interface where the analyte is detected by multiple reaction monitoring (MRM). The method has a chromatographic run time of less than 2 min, a linear calibration curve with a range of 1–500 ng ml⁻¹ and intra- and inter-day precision estimates of less than 10% over the calibration range.