Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

High-Pressure Liquid Chrohatographic Assay of Cefotaxime and Desacetylcefotaxime in Human Myometrium

Abstract

An analytic high-pressure liquid chromatographic (HPLC) procedure for the assay of desacetylcefotaxime and cefotaxime in gynecologic tissue was developed. Normal individuals undergoing elective hysterectomy were subjects in this study. Blood and myometrium were removed up to four hours after a 1-g intramuscular dose of cefotaxime. Since cefotaxime is unstable in homogenized tissue at room temperature, the specimens must be maintained at 4°C during homogenization and extraction. Mean serum desacetylcefotaxime and cefotaxime levels were 3.2 ± 2.0 μg/ml and 6.8 ± 4.4 μ/ml, respectively. The mean myometrium concentrations of desacetylcefotaxime and cefotaxime were 8.4 ± 10.0 μg/g and 6.3 ± 8.9 μg/g, respectively. The cefotaxime to desacetylcefotaxime ratios in serum and tissue were 2.12 and 0.75, respectively. Our results suggest that in antimicrobial synergistic studies evaluating serum and tissue levels, the optimal ratio of one part cefotaxime to at least one part desacetylcefotaxime.     

High-performance liquid chromatographic assay of cefotaxime, desacetylcefotaxime and ceftriaxone in rat plasma

A simple and selective high-performance liquid chromatographic method is described for the analysis of the cephalosporins cefotaxime (CXM), desacetylcefotaxime (DACXM) and ceftriaxone (CFX) in rat plasma. Plasma was deproteinized with methanol, and the supernatant was directly injected into the chromatograph and monitored at 254 nm. For determination of the unbound drugs, a centrifugal ultrafiltration method was employed. The calibration curves were linear (r = 0.999) from 2.5 to 500 micrograms/ml; the detection limits were 100 ng/ml for DACXM and 250 ng/ml for CXM and CFX. The method was not interfered with by other plasma components, nor by barbital sodium or caffeine, and has been applied to study the pharmacokinetics of the cephalosporins in rats.     

Analytical procedure for the determination of rufloxacin, a new pyridobenzothiazine, in human serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantification of rufloxacin in human serum and urine has been developed and validated. The compounds, rufloxacin and internal standard, are extracted from buffered serum and urine using dichloromethane. They are then separated on an anion-exchange column using 0.05 M phosphate buffer-acetonitrile (80:20, v/v). The eluate is quantified by measuring the ultraviolet absorbance at 296 nm. The lower limit of detection for the analyte is 0.1 microgram/ml in serum and 0.05 micrograms/ml in urine. The method is linear from 0.3 to 10 micrograms/ml for serum and 0.1 to 10 micrograms/ml for urine. The method has been applied in a pharmacokinetic study in volunteers.     

高效液相色谱-串联质谱法同时测定鸡蛋中头孢噻肟及其主要代谢物残留

建立了高效液相色谱-串联质谱(HPLC-MS/MS)法同时测定鸡蛋中头孢噻肟及其代谢物去乙酰头孢噻肟残留量的检测方法.样品经乙腈-水(9∶1, V/V)提取,正己烷除脂,C18固相分散萃取除杂,Agilent Eclipse Plus C18色谱柱(100 mm×2.1 mm, 3.5 μm)分离,以0.2%(V/V)甲酸-乙腈为流动相,进行梯度洗脱,目标物采用电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式检测,基质匹配标准溶液外标法定量.结果表明,头孢噻肟和去乙酰头孢噻肟分别在1.0~143.0 μg/L和1.0~120.0 μg/L浓度范围内线性关系良好(R.2>0.999).方法检出限(LOD, S/N=3)分别为0.07和0.14 μg/kg,定量限(LOQ, S/N=10)分别为0.23和0.99 μg/kg. 在5.0、50.0和100.0 μg/kg 3个添加水平下,头孢噻肟和去乙酰头孢噻肟的回收率分别为83.1%~103.0%和88.2%~101.0%,相对标准偏差(RSD, n=6)均介于2.0%~6.2%.实际样品测定结果表明,本方法简便、快速、灵敏、准确, 可用于鸡蛋中头孢噻肟及去乙酰头孢噻肟的残留分析检测.     

Determination of neomycin in plasma and urine by high-performance liquid chromatography. Application to a preliminary pharmacokinetic study.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in plasma and urine. The plasma was deproteinated with trichloroacetic acid and centrifuged. The supernatant was mixed with ion-pair concentrate and centrifuged again. The resultant supernatant was analyzed by HPLC. Urine was centrifuged to remove debris, if any, mixed with ion-pair concentrate and analyzed directly by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall average recovery of neomycin was 97 and 113% from plasma spiked at 0.25-1.0 micrograms/ml, using standard curves prepared in plasma extract and in water, respectively, and 94% for urine spiked at 1-10 micrograms/ml using a standard curve prepared in water. The method was used to detect neomycin in plasma and urine obtained from animals injected intramuscularly with neomycin. Various pharmacokinetic parameters of neomycin were also determined from its profile of plasma concentration versus time.     

Improved High-Performance Liquid Chromatographic (HPLC) Assay Method for Ceftizoxime

Abstract

We improved a high-performance liquid chromatographic method for the quantitative determination of ceftizoxime in human serum and urine using cefotaxime as internal standard. It employs a μ Bondapak Alkyl Phenyl column, elution with acetonitrile-phosphate buffer and measurement of UV absorption at 254 nm. Results obtained using the HPLC assay were compared to those obtained using a microbiological assay. The correlation coefficient was 0.987 (n:25). The method is rapid, accurate and reproducible with a sensitivity of 2.5 μg/ml of ceftizoxime. Cefotaxime and its major metabolite, the desacetylcefotaxime, can also be quantitated by this procedure.     

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

Use of micellar mobile phases and microbore column switching for the assay of drugs in physiological fluids

The feasibility of directly assaying drugs in physiological fluids using on-line preconcentration and microbore high-performance liquid chromatography has been demonstrated. The untreated sample is injected onto a hydrophobic pre-column, using micellar sodium dodecyl sulfate (SDS) in the case of serum or phosphate buffer in the case of urine, as the load mobile phase. This traps the components of interest which are then backflushed onto a microbore analytical column using a stronger mobile phase. This procedure was then applied to diazepam in serum and phenobarbital in urine. Recovery was linear and quantitative over the range 30-3000 ng/ml for diazepam in serum and 2-200 micrograms/ml for phenobarbital in urine. The diazepam method was specific against caffeine and the three major metabolites of diazepam: oxazepam, temazepam, and nordiazepam. The effects of varying pre-column dimensions, pre-column loading time, and SDS concentration volume were evaluated.     

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 micrograms/ml for plasma, 3 micrograms/ml for urine and 0.03 micrograms/ml for saliva using UV detection.     

High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Simultaneous determination of cefotaxime and desacetylcefotaxime in real urine sample using voltammetric and high-performance liquid chromatographic methods

Two rapid, accurate and sensitive methods are developed and validated for the quantitative simultaneous determination of cefotaxime (CFX) and its active metabolite desacetylcefotaxime (DCFX) in urine.Based on the previous results which showed the four electron reduction of CFX at ≈ −0.5 V, and the new findings that DCFX reduction occurred at more positive potential (−0.23 V), the new adsorptive stripping differential pulse voltammetric (AdSDPV) method was developed for determination of CFX in the presence of DCFX. Linear responses were observed over a wide concentration range (0.07-0.52 μg/ml for CFX and 0.22-1.3 μg/ml for DCFX) in urine.The second assay involves subsequent separation on a reversed-phase HPLC column, with ultraviolet detection at 262 nm. Retention times were 4.057 and 1.960 min for CFX and DCFX, respectively. Linear responses were observed over a wide range, 0.55-6.60 μg/ml for CFX and 1.10-11.00 μg/ml for DCFX, in urine.The statistical evaluation for both methods was examined by means of within-day repeatability (n = 5) and day-to-day precision (n = 3) and was found to be satisfactory with high accuracy and precision.     

Simultaneous determination of ampicillin and sulbactam by liquid chromatography: post-column reaction with sodium hydroxide and sodium hypochlorite using an active hollow-fibre membrane reactor

A high-performance liquid chromatographic method has been developed for the simultaneous determination of ampicillin (ABPC) and sulbactam (SBT) in serum and urine. The method involves separation of ABPC and SBT from the background components of serum and urine on a C18 column, post-column reaction with sodium hydroxide and sodium hypochlorite using an active hollow-fibre membrane reactor, and detection at 270 nm. At ABPC and SBT concentrations of 10 and 5 micrograms/ml in urine and serum samples, the precisions (relative standard deviations) were 0.9-2.5% (n = 8). The detection limits were 20 and 5 ng for ABPC and SBT, respectively, at a signal-to-noise ratio of 3.     

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.     

High-performance liquid chromatographic method for the routine determination of sulphadimidine, its hydroxy metabolites and N4-acetylsulphadimidine in body fluids and cell culture media.

A simple high-performance liquid chromatographic method is presented for the determination of trace amounts of sulphadimidine (SDD), its hydroxylated metabolites and N4-acetyl-SDD in blood plasma, urine, hepatocyte culture media and microsomal incubations. The synthesis of 5-hydroxy-SDD and an improved method for the isolation of 4-methylhydroxy-SDD from urine are described and their respective specific absorption coefficients at 265 nm are calculated by on-line radiochemical and ultraviolet detection. The limit of detection of the analytical method is 0.05 micrograms/ml for SDD and its hydroxy metabolites and 0.2 micrograms/ml for N4-acetyl-SDD. Linear calibration graphs for SDD and its metabolites were constructed from 0.2 to 50 micrograms/ml. The method has been applied to biotransformation studies in vivo and in vitro.     

Determination of the monocyclic beta-lactam antibiotic carumonam in plasma and urine by ion-pair and ion-suppression reversed-phase high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatography method has been developed for the determination of the new monocyclic beta-lactam antibiotic carumonam in plasma and urine. The method for plasma involves protein precipitation with acetonitrile and removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantification are achieved using a mobile phase based on either ion-suppression or ion-pair chromatography on a reversed-phase column with UV detection. The limit of determination is 0.5 micrograms/ml plasma, using a 0.5-ml specimen, and 25 micrograms/ml urine, using a 50-microliter specimen. The inter-assay reproducibility is generally better than 4% when an internal standard is used. Since beta-lactam antibiotics may degrade on storage, close attention must be paid to the stability of these drugs in biological fluids; novel measures to prevent degradation on storage are described. The assay has been successfully applied to the analysis of several thousand samples from pharmacokinetic studies, including a study involving patients with impaired renal function.     

Simultaneous determination of tranilast and metabolites in plasma and urine using high-performance liquid chromatography

A method has been developed for the simultaneous determination of Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid (N-5'), and metabolites in plasma and urine from humans, dogs and rodents administered N-5'. Total N-5' and metabolite N-3 conjugates were determined in human urine. Detection limits in plasma were 0.2 micrograms/ml for metabolite N-3-S and N-5' and 0.1 micrograms/ml for metabolites N-3 and N-4. In urine, detection limits were 2 micrograms/ml for metabolite N-3-S and N-5' and 1 micrograms/ml for metabolites N-3 and N-4. Metabolite N-4 was not identified in any sample assayed.     

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.