A Rapid and Simple High Pressure Liquid Chromatographic Method for Pharmacokinetic Study of Ciprofloxacin in Human Serum

Abstract

A rapid, accurate and sensitive method has been developed for the quantitative determination of ciprofloxacin, a new second-generation quinolone carboxylic acid antimicrobial agent, with high in vitro activity against a wide range of Gram- negative pathogens and Gram-positive cocci.

A Lichrosorb RP-18 250 x 4.0 mm, 5 μm analytical column was used with an eluting system consisting of a mixture of CH₃CN-CH₃OH-Citric acid 0.4 M (1:3:6 v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in a limit of detection of 0.2 ng per 20 μl injection. For the quantitative determination theophylline was used as chromatographic internal standard at a concentration of 1.56 ng/μl. A rectilinear relationship was observed up to 20 ng/μl. Analysis time was less than 6 min. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day calibration (n=8) and was found to be satisfactory with highly accurate and precise results. The method was applied to the direct determination of ciprofloxacin in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of ciprofloxacin in spiked samples was 98 ± 4% over the range of 0.5–5 mg/μl.     

Direct injection determination of theophylline and caffeine in blood serum by high-performance liquid chromatography using an ODS column coated with a zwitterionic bile acid derivative

An ODS column dynamically coated with zwitterionic bile acid derivative, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), was evaluated for direct injection determination of drugs in blood serum by HPLC. Polar functional groups such as sulfonate, ammonium and the three hydroxyl groups in CHAPS protruding towards an aqueous mobile phase formed a hydrophilic layer over the ODS reversed-phase surface, which resulted in high molecular mass compounds such as proteins being prevented from penetrating into the internal hydrophobic region. The bulk of the proteins were eluted as an unretained or nearly unretained band by using 0.2 mM sodium hydrogenphosphate solution (pH 7.4) as the mobile phase. In contrast, small molecules such as some inorganic anions and aromatic compounds were retained and thereby separated from one another. It was confirmed that the ODS column modified with CHAPS acts as a restricted access-type column with a hydrophobic interior and a hydrophilic exterior. Hence biological fluids could be directly injected into the CHAPS-coated ODS column. The present HPLC system using the CHAPS-coated ODS column was applied to the determination of theophylline and caffeine in human blood serum. The detection limits for the two drugs with UV absorption at 273 nm were 0.2 and 0.5 mg l-1 (injection volume 20 microliters) and the relative standard deviations of peak area measurements were < 1.4% and 2.2%, respectively, for 10 replicate measurements of serum spiked with 5 mg l-1 of each of the drugs.     

Determination of rare earth elements in human blood serum by inductively coupled plasma mass spectrometry after chelating resin preconcentration

The determination of all rare earth elements (REEs) in human blood serum by inductively coupled plasma mass spectrometry (ICP-MS) was performed with the aid of chelating resin (Chelex 100) preconcentration after acid digestion with HNO3 and HClO4. When chelating resin preconcentration was carried out at room temperature, the recoveries of heavy REEs were lower than those of light REEs because of their stable complex formation with residual organic compounds remaining in the digested serum solution. These problems were overcome by heating the solution at 80 degrees C during the chelating resin preconcentration process. As a result, the recoveries for all REEs were improved to 92-102% in the case of a concentration factor of 4, where the analytical detection limits for REEs were below 0.2 x 10(-12) g ml-1. Consequently, all REEs in individual human blood sera collected from five healthy volunteers could be determined by ICP-MS with good precision. The concentrations of REEs in human blood serum were extremely low, in the range from ca. 1 x 10(-12) g ml-1 of Eu to ca. 230 x 10(-12) g ml-1 of Ce.     

Determination of thiazolidine-4-carboxylates in urine by chloroformate derivatization and gas chromatography-electron impact mass spectrometry

The derivatization method of thiazolidine-4-carboxylic acid (TZCA) and methyl-thiazolidine-4-carboxylic acid (Me-TZCA) in urine with alcohol/chloroformate was achieved. TZCA and Me-TZCA were derivatized in one step in urine with ethyl chloroformate in 1 min at room temperature. The derivatives of TZCA and Me-TZCA had very good chromatographic properties and offered very sensitive response for gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS). On the basis of derivatization, the method for simultaneous determination of TZCA and Me-TZCA in human urine was developed. Deuterated Me-TZCA (Me-TZCA-d(4)) was synthesized as the internal standard (IS) for the analysis of urine samples. TZCA and Me-TZCA were derivatized and extracted from urine at pH 9.5 with toluene, and then the dried extract was dissolved with 100 microl ethyl acetate and injected in GC/MS system. The recoveries of TZCA and Me-TZCA were about 102 and 103%, respectively, at the concentration of 0.05 mg/l. The method detection limits (MDL) were 1.0 and 0.5 microg/l, respectively, for TZCA and Me-TZCA in 1 ml human urine. The coefficients of variation of TZCA and Me-TZCA were less than 6% at the concentrations of 0.05 and 0.2 mg/l, respectively. To assess the formation of TZCA during inhalation with formaldehyde (FA) (about 3.1 and 38.1 ppm FA in air), urine samples from rats were taken during 3 days after initiation of treatment. The mean amount of TZCA determined was 0.07 mg/l in control group and 0.18 mg/l during treatment with 3.1 ppm. The TZCA levels increased up to about 1.01 mg/l during treatment with 38.1 ppm. It is planned to study whether urinary TZCA can be used as an indicator in the biological monitoring of exposure to FA.     

Determination of beryllium and selenium in human urine and of selenium in human serum by graphite-furnace atomic absorption spectrophotometry.

For human urine beryllium (Be), each sample (500 microl) was diluted (1+1) with Nash reagent (containing 0.2% (v/v) acetylacetone and 2.0 M ammonium acetate buffer at pH 6.0) and then a 20-microl volume of Triton X-100 (0.4%, v/v) aqueous solution was added. An aliquot (10 microl) of the diluted urine mixture was introduced into a graphite cuvette and was atomized according to a temperature program. The method detection limit (MDL, 3sigma) for Be was 0.37 microg/l in the undiluted urine sample and the calibration graph was linear up to 65.0 microg/l. Calibration graphs were prepared by the standard addition method. Accuracies of 98.6-102% were obtained when testing standard reference material (SRM 2670) freeze dried human urine samples. Precision (relative standard deviation, RSD) for urine Be was < or = 2.3% (withinrun, n = 5) and was < or = 3.0% (between-run, n = 3). For human urine and serum selenium (Se), samples (100 microl) were diluted with HNO3 (0.2%, v/v) to make a (1+1) dilution for urine analysis or a (1+4) dilution for serum analysis. An additional aliquot (10 microl) of Triton X-100 (0.1%, v/v) was added to each 200 microl of (1+1) diluted urine (or 20 microl of the Triton X-100 was added to each 500 microl of (1+4) diluted serum) sample. After the diluted sample mixture (10 microl) was introduced into a graphite cuvette, the corresponding chemical modifier (10 microl, containing Ni2+ + Pd + NH4NO3 in HNO3 (0.2%, v/v)) was added to it and the mixture was atomized. The MDL (3sigma) for Se in urine and in serum was 4.4 and 21.4 microg/l in undiluted sample, respectively, and the calibration graphs were linear up to 150 and 400 microg/l. Accuracies of urine Se were 98.9 - 99.4% by testing SRM 2670 (NIST) urine standards with RSD (between-run, n = 3) within 2.9%; and that of serum Se was 97.2% when testing a certified second-generation human serum (No. 29, #664) with RSD (between-run, n = 3) of 1.4%. The proposed method can be applied easily, directly, and accurately to the measurement of Be and Se in real samples (including six urine Se and four serum Se from patients of Blackfoot Disease in Taiwan).     

A method for the determination of theophylline in serum by isotope dilution mass spectrometry

Summary A method for the determination of theophylline in human serum by the isotope dilution/mass spectrometric technique is described. As an internal standard labelled (1,3-¹⁵N₂-2-¹³C)theophylline is added to the serum sample. The analyte and internal standard are extracted with chloroform/2-propanol (90:10) and converted to the trimethylsilyl derivatives. The extraction and silylation procedures are checked by adding theophylline and internal standard in various concentrations to blank serum and determining the recovery. The trimethylsilyl derivatives of labelled and non-labelled theophylline are separated and detected by GC-MS with the mass spectrometer set to m/z 252 and 255. The amounts of theophylline in the serum are calculated from the isotope ratios measured by selected ion monitoring. The accuracy, precision and recovery of this GC-MS method are presented and discussed. The coefficient of variation determined from duplicate samples was less than 2.5%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1.Part of the work was presented at the 32th Kongreß der Deutschen Gesellschaft für Laboratoriumsmedizin, Frankfurt 1991     

Pharmacokinetics of theophylline and phenobarbital during peritoneal dialysis compared with intestinal dialysis in rats

The transfer rates of theophylline and phenobarbital from blood into the peritoneal cavity were investigated after their intravenous administrations at a dose of 10 mg/kg each in rats. Appreciable amounts of both drugs were transported into the dialysate with their transfer rates decreasing as the serum concentrations declined. The average amount of theophylline transferred into the dialysate was 17% and 24% of dose in 2 and 4 h, respectively, while that of phenobarbital was 13% and 45% of dose in 2 and 8 h, respectively. The amounts appeared to be greater than those transported into the intestinal lumen which was previously reported by the authors. Peritoneal dialysis decreased the serum half-life and the area under the serum concentration-time curve (AUC) value of theophylline from 2.1 to 1.5 h and from 38 to 26 micrograms.h/ml, respectively and increased the total body clearance by 47% as compared with the control. The dialysis also decreased the serum half-life and the AUC value of phenobarbital from 8.3 to 5.3 h and from 116 to 83 micrograms.h/ml, respectively and increased the total body clearance by 112% as compared with the control. Overall, the effectiveness of peritoneal dialysis in removing theophylline and phenobarbital appears to be not very different from that of intestinal dialysis with oral activated charcoal which has been previously reported by the authors in spite of the difference in transport into the intestinal lumen and peritoneum.     

Increased transport of theophylline into gastrointestinal lumen and gastrointestinal dialysis by activated charcoal in rats with hepatic cirrhosis

The characteristics of exsorption and/or excretion of theophylline into the small intestinal lumen in rats with hepatic cirrhosis (HC rats) induced by carbon tetrachloride were investigated by an in situ single-pass perfusion technique. The serum concentrations of theophylline after i.v. administration of aminophylline (10 mg/kg) in the HC rats were significantly higher than those in normal rats during the experimental period. Moreover, the exsorption of theophylline from blood into the intestinal lumen was significantly increased in the HC rats compared with the normal rats. Treatments with oral activated charcoal reduced the serum theophylline levels in the HC rats. Consequently, gastrointestinal dialysis by oral administration of activated charcoal may be a useful method to remove poisonous drugs from the blood in patients with hepatic failure (including cirrhosis), which decreases the systemic clearance.     

Evaluation of a radioimmunoassay of alpha 1-microglobulin (alpha 1-m) with simplified procedures

A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25 microliters, and thus 200 mg/l of alpha 1-m standard was prepared using 50 microliters of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intra-assay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about alpha 1-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of alpha 1-m were less than 25 mg/l in serum and less than 10 mg/l in urine. The present results indicate that the determination of alpha 1-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function.     

New simplified microassay for the quantitation of theophylline and its major metabolites in serum by high-performance liquid chromatography

A high-performance liquid chromatographic technique is presented for simultaneous determination of theophylline, 3-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid and caffeine in serum using beta-hydroxyethyltheophylline (BHET) as an internal standard. An aliquot of a serum sample (100 microliters) was added to 40 microliters of an internal standard solution (BHET; 100 micrograms/ml) and vortexed. A 20% trichloroacetic acid solution (60 microliters) was then added; the mixture was vortexed, centrifuged and 100 microliters were injected onto a C8 column maintained at 45 degrees C. Inter- and intra-day variability of the assay for all compounds was less than 4.3%. Sensitivity ranged from 10 ng/ml for 1-methyluric acid to 25 ng/ml for theophylline. Drugs commonly coadministered with theophylline did not interfere with the assay.     

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

Perxanthate determination in flotation solutions by ultraviolet spectrophotometry

Alkyl perxanthates (ROCSSO(-)) can be determined in solutions from flotation plants or other systems by direct ultraviolet spectrophotometry or after extraction into a solvent. Direct determination is preferred for visually clear solutions. In alkaline solution (pH > 8) at 348 nm perxanthates have a molar absorptivity of (1.042 +/- 0.013) x 10(4) l.mole(-1).cm(-1) and Beer's law holds up to 25 mg/l. The detection limit with a 1-cm cell is 0.2 mg/l. Interferences are few; nickelocyanide (>30 mg/l.) interferes slightly. After acidification to pH < 2, with an aqueous:organic phase-volume ratio of 1:1, more than 99% of the perxanthate is extracted into chloroform. In chloroform perxanthic acids have an apparent molar absorptivity of (5.47 +/- 0.09) x 10(3)l.mole(-1).cm(-1) at 302 nm. The absorbance of the chloroform extract at 302 nm is proportional to perxanthate concentration in the original aqueous phase up to 25 mg/l. The detection limit with a 1:1 phase-volume ratio and a 1-cm cell is 0.2 mg/l. Interferences include mercaptobenzothiazole (>1 mg/l.), sodium sulphite (>25 mg/l.) and cuprocyanide; xanthate interferes if it is not decomposed before extraction.     

Development of a fluoroimmunosensor for theophylline using immobilised antibody

A flowthrough theophylline fluoroimmunosensor with an antibody covalently immobilised on a solid support has been developed. The immobilisation technique proposed in this paper used Protein-A on control pore glass (Protein A-CPG) in an immunoreactor and dimethylsuberimidate as a cross-linking agent. Several supports and cross-linking reagents were tested in order to obtain oriented immobilisation and thus efficiency of the immunological reaction and reusability of the immunosensor. The immunosensor performance characteristics were established. The precision expressed as RSD, was 1.6%; the detection limit was 3 mug l(-1); the immunoreactor lifetime was established in 80 assays and there were no interferences with structurally similar compounds such as aminophylline, dihydroxypropyltheophylline and caffeine in the determination of the analyte. This fluoroimmunosensor was applied to determine theophylline in human serum samples from patients of the Puerta de Hierro Hospital in Madrid. The results obtained show that there are no significant differences between the proposed immunosensor and the high-pressure liquid chromatographic method with UV detection used by the Hospital, thus demonstrating the validity of the method.     

High-performance liquid chromatographic separation of caffeine, theophylline, theobromine and paraxanthine in rat brain and serum.

Caffeine and its metabolites theophylline, theobromine and paraxanthine have been determined in rat brain and serum samples by high-performance liquid chromatography with ultraviolet detection. The recovery, 85-103%, allowed quantification by external standard methods. The variability was found to be less than 3 and 7% for intra-day and inter-day assays, respectively. The detection limit, 1.57 ng of methylxanthines on column, allowed the determination of 62.5 ng/g or ml in biological material. Rats treated with 30 mg/kg caffeine (subcutaneously) were sacrificed at different times (1, 6, 12 and 24 h). Higher concentrations of methylxanthines (specially paraxanthine) were observed in the striatum than in the rest of the brain, and it was also observed that the clearance of methylxanthines was faster in serum than in brain structures.     

Evaluation of the Acculevel Theophylline Assay

Abstract

As a quality assurance study, we evaluated the rapid on-site AccuLevel theophylline assay and compared the results to the reference method of High Performance Liquid Chromatography (HPLC). The rapid result was needed in order to provide more timely treatment for patients presented to the Emergency Medical Department (EMD) who required a quantitative serum theophylline determination early in the course of treatment.

A good correlation between the theophylline concentrations simultaneously determined by AccuLevel and HPLC was observed; r² =0.916, n=96. A slight positive bias by AccuLevel was noted with the mean of concentrations being 11.7 mg/L and 13.2 mg/L for HPLC and AccuLevel, respectively. Precision of the assay was found to be 4.6%. Because of the delay in transporting specimens from the EMD to the Clinical Laboratories at our Medical Center, the EMD was able to obtain quantitative theophylline serum values from 30 minutes to 1.5 hours sooner by using the AccuLevel, on-site test.     

Corona discharge ion mobility spectrometry for the determination of theophylline and guaifenesin in human serum

This paper describes the determination of theophylline and guaifenesin in human serum using ion mobility spectrometry with positive corona discharge as source ionization. The optimization of parameters that could influence ion mobility spectrometry was investigated. Under optimum conditions (Temperature; injection: 220 and oven: 175 °C, Flow rate; carrier: 300 and drift: 600 mL min^?1, Voltage; corona: 2300 and drift: 7000 V, pulse width: 100 μs), calibration curves were linear in the ranges of 2 to120 and 6 to 120 ng for theophylline and guaifenesin, respectively. The relative standard deviation (n?=?10) was below 12 %. The detection limits were found to be 0.1 ng for theophylline and 0.5 ng for guaifenesin. The recovery results for the theophylline and guaifenesin determination in human serum was about 80 % that indicate the proposed method can be applied for the two drugs analysis in real sample. Furthermore, the proposed method has been evaluated in the simultaneous determination of the two drugs.     

Determination of methamphetamine in urine samples with sodium 1,2-naphthoquinone-4-sulphonate

Optimal conditions have been studied for the determination of methamphetamine in urine samples by an extractive-spectrophotometric method with sodium 1,2-naphthoquinone-4-sulphonate (NQS) as reagent. These conditions are: NaHCO₃ pH 10, NQS 6.3 × 10^–3 mol/l and heating for 5 min at 45°C. The accuracy and precision of the method were tested. The detection limits were 0.2 mg/l in the standard and 0.9 mg/l when 5 ml of urine sample were taken. The standard deviation of blank urine was evaluated from 12 different samples. The relative errors found in the determination of methamphetamine in urine were lower than 10% if the methamphetamine-amphetamine ratio was higher than 4.     

An RNA aptamer-based electrochemical biosensor for detection of theophylline in serum

An electrochemical RNA aptamer-based biosensor for rapid and label-free detection of the bronchodilator theophylline was developed. The 5'-disulfide-functionalized end of the RNA aptamer sequence was immobilized on a gold electrode, and the 3'-amino-functionalized end was conjugated with a ferrocene (Fc) redox probe. Upon binding of theophylline the aptamer switches conformation from an open unfolded state to a closed hairpin-type conformation, resulting in the increased electron-transfer efficiency between Fc and the electrode. The electrochemical response, which was measured by differential pulse voltammetry, reaches saturation within a few minutes after addition of theophylline, and the dynamic range for detecting theophylline is 0.2-10 muM. The electrode displays an inhibited response when applied directly in serum samples treated with RNase inhibitors; however a full response to the theophylline serum concentration was obtained by transferring the electrode to blank serum-free buffer solutions. It was demonstrated that theophylline is detected with high selectivity in the presence of caffeine and theobromine.     

Determination of 1-vinyl-2-pyrrolidone in human serum by high-performance liquid chromatography

Summary A simple method is described which allows the quantification of 1-vinyl-2-pyrrolidone (NVP) in human serum. NVP is extracted from serum with diethylether and determined with HPLC/UV-detection. 1-Cyclohexyl-2-pyrrolidone serves as an internal standard. The detection limit is 0.1 mg/l. The method has shown that NVP can enter the organisms of workers occupationally exposed to this substance.     

Stopped-flow injection liquid-liquid extraction spectrophotometric determination of palladium in airborne particulate matter and automobile catalysts

A stopped-flow injection liquid-liquid extraction (SF-EX-FIA) spectrophotometric method is reported for the determination of palladium(II), using the 2,2'-dipyridyl-2-pyridylhydrazone (DPPH) as a color forming reagent. The colored complex Pd(II)-DPPH was extracted in CHCl(3) and the absorbance was monitored at 560 nm. An injection valve was used as a commutator in order to combine the stopped-flow technique with liquid-liquid extraction FI system. The calibration graph was linear up to 12 mg l(-1) (s(r)=0.27%; r=0.9999) with a detection limit of c(L)=0.007 mg l(-1). The sampling rate was 20 injections per hour. The proposed method has been successfully applied to the determination of palladium in airborne particulate matter (APM) and in automobile exhaust gas converter catalysts.