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-performance liquid chromatographic method for the determination of a potential memory-enhancing compound (CL 275,838) and its desbenzyl metabolite in rat plasma or serum.

A rapid, selective, precise reversed-phase liquid chromatographic method has been developed for the determination of a potential memory-enhancing agent (CL 275,838) and its main metabolite (CL 286,527) in plasma and serum. The procedure includes isolation of compounds from proteins precipitated with acetonitrile, subsequent resolution by reversed-phase (Whatman Partisphere C8) high-performance liquid chromatography and ultraviolet detection. The assay was linear over the range 0.12-1.25 micrograms/ml of plasma or serum. The detection limit was 0.12 micrograms/ml, using 0.2 ml of plasma or serum. Intra- and inter-day validation studies indicated an acceptable precision and reproducibility of the method within the concentration range investigated, the overall coefficient of variation being less than 10%. The method is currently applied in support of pharmacological and toxicity studies of the compound in rodents.     

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.     

Analysis of plasmid DNA synthesis by double tracer method--differential effects of rifampicin and chloramphenicol on the replication of pSC101 and ColEl-amp in Escherichia coli K-12

An Escherichia coli strain, CR34 , harboring both pSC101 and ColEl -amp plasmids was exposed to media containing rifampicin (100 micrograms/ml) and/or chloramphenicol (180 micrograms/ml) and the cells were labeled for 20 min with 3H-thymine at 3, 25 and 50 min after exposure to drug(s). The plasmid DNA synthesis was assayed by DNA-DNA hybridization with 14C-labeled pSC 134 DNA as internal marker. In the presence of rifampicin, the replication of pSC 101 was from 57 to 104% that in its absence, and that of ColEl -amp was from 17 to 26%. The DNA replication of pSC 101 after addition of chloramphenicol was reduced to 35 to 75%, and that of ColEl -amp was reduced to 39% and then restored to 92%. This restoration was not observed in the presence of rifampicin.     

Determination of theophylline in serum and saliva in the presence of caffeine and its metabolites.

Because of marked variability in its metabolic clearance and its narrow therapeutic range (10-20 micrograms/ml) investigation of each patient's clearance of theophylline is desirable. The author reports here a rapid reversed-phase high-performance liquid chromatographic (HPLC) method to determine, within 3 min, the theophylline in serum and saliva in the 0.1-50 micrograms/ml range. A fast HPLC column, 10 x 4.6 mm, packed with 3-microns spherical ODS packing is used with acetonitrile-methanol-buffer pH 4.7 (4:7:89) to achieve separation of theophylline from paraxanthine and matrix components. Since theophylline is a major pediatric bronchodilator, the feasibility of assay in saliva was investigated as an alternative route for determining the clearance is stressed asthmatic children. Using this method it was found that the ratio of theophylline in simultaneous serum and saliva samples is very consistent over time in the same person (+/- 3.99%), but inter-individually this consistency is reduced ten-fold. Simultaneous serum and saliva samples need be taken only once to obtain the ratio and the kinetics followed further with salivary samples only.     

Fluorimetric determination of mexiletine in serum by high-performance liquid chromatography using pre-column derivatization with fluorescamine

A simple, specific and sensitive micro-scale method for the assay of the antiarrhythmic agent mexiletine in human serum is described. The method uses high-performance liquid chromatography, with pre-column fluorimetric derivatization by fluorescamine. Following extraction with diethyl ether, mexiletine and 4-methylmexiletine (an internal standard) were derivatized with fluorescamine under weakly alkaline condition (pH 9.0) and chromatographed on a reversed-phase column with aqueous methanol-2-propanol as the mobile phase. The two fluorescent derivatives of mexiletine and the internal standard were separated as clear single peaks, and no interfering peaks were observed on the chromatograms. The detection limit for mexiletine was 0.005 micrograms/ml from only 100 microliters of serum, and the calibration curves in the range 0.01-5 micrograms/ml were linear, with an overall coefficient of variation of less than 5%. The analytical recovery of a known amount of mexiletine added to serum was almost 100%. This method proved to be effective in the rapid monitoring of the serum concentrations in patients who received this potent antiarrhythmic drug.     

Determination of streptomycin in serum by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for monitoring the serum concentration of streptomycin. The method includes clean-up using a Sep-Pak C18 cartridge and quantitation using dihydrostreptomycin as an internal standard. Streptomycin and dihydrostreptomycin were separated by reversed-phase ion-pair chromatography on LiChrosorb RP-18 and detected by UV absorption (195 nm). The calibration graph of serum streptomycin concentration was linear over the range 5-50 micrograms/ml. Streptomycin was added to serum at the level of 20.0 micrograms/ml and its concentration was determined to be 18.9 micrograms/ml with a coefficient of variation of 2.07% (n = 5). The clinical application of this method was confirmed by comparison with fluorescence polarization immunoassay.     

Enzyme immunoassay for the drug of anti-ulcer using avidin-biotin system.

We have developed a competitive enzyme immunoassay for a drug, which was a newly synthesized anti-ulcer agent, using an enzyme immunoassay. The polyclonal anti-drug antibody coupled to biotin, peroxidase labeled drug derivatives as a tracer, and a small column of Sepharose 4B covalently bound to avidin were used in the assay. This assay is simple and rapid, and the sensitivity and the measuring range can be controlled by the flow rate of the substrate solution. The correlation between serum drug concentrations (0.1-10 micrograms/ml) measured by gas chromatography and this assay was good (r = 0.991). This principle for the assay is very practical and applicable to the enzyme immunoassay for small and large molecules.     

Simultaneous quantitation of salivary carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbitone by high-performance liquid chromatography

A rapid, sensitive and accurate high-performance liquid chromatographic method for the simultaneous quantitation of phenobarbitone, phenytoin, carbamazepine and carbamazepine-10,11-epoxide in saliva is described. Only small volumes of saliva (100 microliters) are required. Separation of the drugs is achieved by reversed-phase chromatography on a Nova-Pak C18 column, with a mobile phase of acetonitrile-phosphate buffer at a flow-rate of 2.0 ml/min. Detection is effected by ultra-violet absorption at 215 nm. The total run time is under 12.5 min per assay. A precipitation but no extraction step is involved, simplifying the assay method. Salivary concentrations in the range 0.25-25 micrograms/ml for carbamazepine, 0.5-20 micrograms/ml for phenytoin and phenobarbitone and 0.4-20 micrograms/ml for carbamazepine-10,11-epoxide can be measured. Recovery varies from 94 to 108%. The method has been used for routine measurements of anticonvulsants in saliva collected daily from patients with intractable epilepsy.     

High-performance liquid chromatographic method for the determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma.

Determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma involved derivatization of the amino and hydroxyl groups with a ultraviolet-absorbing chromophore followed by extraction into an organic phase. Reversed-phase high-performance liquid chromatography with gradient elution was used for the separation of the analyte from the internal standard (2,3-butanediol). The assay was linear in the range 1.0-1000.0 micrograms/ml of plasma and the coefficient of variation varied between 9.6 and 16.3% whereas the accuracy varied between 90 and 108%. The limit of detection for the assay was 0.282 micrograms/ml. Stability of tris(hydroxymethyl)aminomethane in human plasma frozen at -20 degrees C was studied over a period of three month and the data indicated no significant change.     

Automated high-performance liquid chromatographic determination of chloramphenicol in milk and swine muscle tissue using on-line immunoaffinity sample clean-up.

An on-line high-performance liquid immunoaffinity chromatographic (HPLIAC) system for the direct determination of chloramphenicol in milk and swine muscle tissue is described. The system consisted of a dual-column system in which an HPLIAC column was directly coupled to an RP-8 high-performance liquid chromatographic column. Skimmed and deproteinated milk or aqueous muscle tissue extract was directly injected into the HPLIAC column. After a washing step with phosphate-buffered saline, chloramphenicol was desorbed by a glycine-NaCl buffer (pH 2.8) and directly concentrated on the RP-8 column. Next, chromatography was carried out using acetonitrile-sodium acetate buffer as the mobile phase. Chloramphenicol was detected at 280 nm. Mean recoveries from spiked raw milk were 70 +/- 2% (1-50 micrograms/kg) and from spiked swine muscle tissue 64 +/- 2% (10-50 micrograms/kg). The calibration curves were linear in the range 1-200 micrograms/kg spiking levels. Limits of determination were 1 microgram/kg for milk and 10 micrograms/kg for muscle tissue.     

Gas chromatographic assay for the new antitumor agent sulfamic acid diester (NSC 329680) and its stability in buffer, blood and plasma

A sensitive gas chromatographic assay with electron-capture detection has been developed for sulfamic acid diester (sulfamic acid 1,7-heptanediyl ester, NSC 329680) based on its conversion to 1,7-diiodoheptane in the presence of excess sodium iodide. The assay is linear up to 1 microgram/ml sulfamic acid diester and has a lower limit of detection of 25 ng/ml from 0.5 ml plasma. The coefficient of variation of the assay is 6.4% at 1 microgram/ml and 8.0% at 100 ng/ml. Sulfamic acid diester is relatively stable in 0.9% sodium chloride and 0.1 M sodium phosphate buffers, pH 7.0-9.0, with half-lives greater than 38 h. The major breakdown product of sulfamic acid diester is sulfamic acid 1,7-heptane-monoyl ester. When added to whole blood sulfamic acid diester shows concentration-dependent breakdown. At 50 and 100 micrograms/ml sulfamic acid diester, the half-time in whole blood is 6.9 h and 65% of the drug is sequestered by the blood cells. At 10 micrograms/ml sulfamic acid diester in blood, there is no detectable breakdown of the drug over 24 h and all of the drug is sequestered by the blood cells. Protein binding of sulfamic acid diester in human plasma is 82% at 10 micrograms/ml and 68% at 100 micrograms/ml.     

Direct injection HPLC analysis of some non-steroidal anti-inflammatory drugs on restricted access media columns

Direct serum injection methods are described for the HPLC determination of selected non-steroidal anti-inflammatory drugs (NSAIDS) on restricted access media (RAM) columns, using either a Pinkerton (ISRP) or semi-permeable surface (SPS) column. Twenty microlitres of a filtered serum sample was injected directly onto each column. Calibration curves were prepared for ketoprofen (1-20 micrograms/mL), fenbufen (0.8-20 micrograms/mL), sulindac (0.8-20 micrograms/mL) and probenecid (1.5-40 micrograms/mL) on the SPS column, and nabumetone (1.5-40 micrograms/mL) and indomethacin (1.5-40 micrograms/mL) on the Pinkerton column. The percentage error and imprecision of the methods were found to be less than 10%. The inter- and intra-day variability for analytes were in the range of 0.15-10%. The limits of quantitation and detection were in the range of 800-1500 and 300-800 ng/mL, respectively, for the analytes studied. A hydrophobic shielded phase (Hisep) column was also investigated and was found to be generally too retentive (> 29 min) for assay of these analytes.     

Antidepressants in serum determined by isolation with two on-line sorbent cartridges and liquid chromatography

A selective method for measuring tricyclic antidepressants in serum is reported. A single assay can be done within ca. 30 min and eight samples can be assayed in less than 150 min. A 1-ml serum sample was diluted and the drugs were extracted from it by passage through a graphitized carbon black (Carbopack B) cartridge. After one washing, this cartridge was connected on line to another one containing a silica-based strong acid exchanger. The tricyclics were removed from the Carbopack surface and selectively readsorbed onto the cation-exchange surface by passing 4 ml of methylene chloride-methanol (60:40, v/v) through the two cartridges. After another wash, the drugs were desorbed from the cation-exchange surface with 0.8 ml of acetonitrile-methanol-water (72:18:10, v/v) saturated with potassium chloride. An aliquot of this solution was chromatographed on a cyano column, and the absorbance of the effluent was measured at 215 nm. The mean analytical recoveries of tricyclic antidepressants added to serum within the range 10-200 micrograms/l exceeded 90%, except for 8-hydroxyamoxapine (mean recovery 85.3%) and amoxapine (mean recovery 83.8%) at the lowest serum concentration considered.     

Determination of DNA with cetyltrimethylammonium bromide by the measurement of Resonance Light Scattering.

A simple assay of DNA was developed based on the measurements of enhanced signals of Resonance Light Scattering (RLS) of cetyltrimethylammonium bromide (CTMAB) by DNA. The enhanced RLS signals, measured by simultaneously scanning the excitation and emission monochromators of a common spectrofluorometer with lambda ex = lambda em, was optimized for the DNA assay with CTMAB. On the conditions of pH 2.21 and ionic strength 0.002, the enhanced RLS intensity at 470.0 nm, delta I, was found to be proportional to the concentration of DNA in the range 0-2.5 micrograms/ml if 1.5 x 10(-5) M CTMAB was used. Limits of determination for calf thymus DNA and fish sperm DNA were 4.9 ng/ml and 9.2 ng/ml, respectively. Synthetic samples were determined with the recovery ratio ranging from 93.2% to 105.1%, and the RSD is lower than 2.7%.     

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.     

Photometric and fluorimetric assay of alkaline phosphatase with new coumarin-derived substrates

Summary Two new coumarin-derived synthetic substrates for use in the direct and continuous kinetic assay of alkaline phosphatase are presented. They have been studied with respect to optimum pH (9.5) and rate of enzymatic hydrolysis (1.5–1.8 nmol/min at pH 9.5) by alkaline phosphatase from calf intestine. Detection limits were 0.0005 units/ml for the photometric assay, and 0.00001 units/ml for the fluorimetric one. The relatively longwave shifted absorption and emission maxima of the new substrates in addition to the large Stoke's shifts allow the determination of enzyme activities in a spectral range distinctly outside the intrinsic fluorescence of biological matter such as serum.     

High-performance liquid chromatographic determination of (S)-(-)-ofloxacin and its metabolites in serum and urine using a solid-phase clean-up

A sensitive and selective method for the simultaneous determination of (S)-(-)-ofloxacin [(S)-(-)-OFLX] and its metabolites in serum and urine was developed using isocratic high-performance liquid chromatography with a specific solid-phase extraction procedure. (S)-(-)-OFLX and its metabolites, desmethyl-(S)-(-)-OFLX and (S)-(-)-OFLX N-oxide, were eluted from a C8 solid-phase column with recoveries of more than 98%. These compounds were separated and determined by means of a reversed-phase column with fluorimetric detection. Validation studies showed that the results were linear for (S)-(-)-OFLX in serum over the range 10-1200 ng/ml and in urine over the range 1-200 micrograms/ml. Analysis for (S)-(-)-OFLX and its metabolites showed good precision and accuracy with a relative standard deviation of less than 6%.     

High-performance liquid chromatographic method for simultaneous determination of enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2-dihydrobenzofuran-2, carboxylic acid and its N-monodemethyl metabolite in monkey plasma and urine after chiral derivatization

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.     

Determination of pioglitazone in dog serum using solid-phase extraction and high-performance liquid chromatography with ultraviolet (229 nm) detection

An analytical method is described for the determination of the free base of pioglitazone hydrochloride (U72, 107A, AD-4833) in dog serum. The method used solid-phase extraction of pioglitazone from serum followed by high-performance liquid chromatographic analysis on an octadecylsilane column with an eluent of acetonitrile-water (41:59, v/v) containing 1.2 ml/l acetic acid (pH 6.0 +/- 0.05). The column effluent was monitored at 229 nm. The analytical procedure has a linear range of 25 ng/ml to 20 micrograms/ml, a minimum quantifiable level of 25 ng/ml, absolute recovery of greater than 90% (n = 15), and precision of less than or equal to 8.8% (n = 45). The method was used in a preliminary dose proportionality study in the dog.