Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

Isotopic effects on retention times of caffeine and its metabolites 1,3,7-trimethyluric acid, theophylline, theobromine and paraxanthine

Physicochemical parameters that influence gas chromatographic separation are numerous. Consequently, isotope labelling, because it modifies physicochemical properties, can induce isotopic effects on retention time. Caffeine has been chosen to study this influence because as itself and its metabolites, it allows the preparation of different methylxanthine isotopomers and thus is one of the best models to study isotopic effects induced by stable isotope labelling. Using a caffeine molecule labelled with deuterium at different positions and rat hepatocytes to obtain metabolites, it was possible to study the influence of labelling on retention time [(14% cyanopropylphenyl)methylpolysiloxane] and to point out the role of each labelled site. It appears that isotopic effects induced by the labelling depend not only on the number of labelling atoms but also on whether this labelling is at position 1, 3 or 7 and, consequently, on the role of the labelled site on the function of the molecule.     

Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C(18) reversed-phase column (250 mm x 4.6 mm I.D., particle size 5 microm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6-99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2-5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.     

Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in biological fluids

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.     

Simple and rapid high-performance liquid chromatography method for the determination of ofloxacin concentrations in plasma and urine

A high-performance liquid chromatographic method for the determination of ofloxacin in human plasma and urine was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was linear from 0.5 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 5%. The average recovery of ofloxacin from plasma was 93%. The method was evaluated in samples from healthy subjects whose drug levels were already measured by microbiological assay.     

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml⁻¹ using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile–aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 μg ml⁻¹. Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 μg ml⁻¹ and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

Simultaneous high-performance liquid chromatographic determination of caffeine and theophylline for routine drug monitoring in human plasma

An HPLC method for the simultaneous determination of both caffeine and theophyllie in human plasma is described, using a reversed-phase chromatography column, heated by a thermostatic oven at 35°C, with UV detection and isocratic elution. The linearity and reproducibility of the method are verified. For the two drugs, the limit of detection is 0.1 μg ml⁻¹. This analytical method is rapid and reliable and allows routine controls of therapeutic levels of theophylline and caffeine, especially in premature infants where the volume of plasma samples is very small.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in saliva

A high performance liquid chromatographic method for determination of moxifloxacin in human saliva was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.25 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from saliva was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in plasma

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C₁₈ column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).     

Simple method for determination of paraquat in plasma and serum of human patients by high-performance liquid chromatography

A simple and fast HPLC system is presented for quantifying paraquat in human plasma and serum using 1,1'-diethyl-4,4'-bipyridyldiylium (diethyl paraquat) as an internal standard. An octadecyl-silica column is used with an eluent of 10% acetonitrile (v/v) containing sodium 1-octanesulphonic acid (3.0 mM) and a diethylamine-orthophosphoric acid buffer (pH 3). Unlike with other techniques, sample treatment requires only the precipitation of protein contents by 6% perchloric acid (v/v) in methanol. The method has a limit of detection of 0.1 microg/ml and is linear up to 10 microg/ml. The serum of four patients and the plasma of one patient with paraquat intoxication's were analysed and positive identification and quantification was readily achieved. One of those patients survived, partially given the rapid disclosure of his levels of paraquat. Therefore, this method is suitable for quantification of paraquat in toxicological samples. It may be used as a prognostic tool in critical case detoxification and to quickly identify potentially salvageable patients for enrollment in new hemofiltration studies.     

Simple and rapid liquid chromatography method for determination of efavirenz in plasma

A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C(18) column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 microg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at -20 degrees C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.     

Simple and sensitive method for the determination of celecoxib in human serum by high-performance liquid chromatography with fluorescence detection

A simple method is described for the determination of the cyclooxygenase-2 specific inhibitor celecoxib in human serum by HPLC using the demethylated analogue as internal standard. After protein precipitation with acetonitrile, samples were extracted with chloroform. Separation was achieved on a Prontosil C18 AQ column (150x3 mm I.D., 3-microm particle size) at a flow-rate of 0.35 ml/min using water-acetonitrile (40:60, v/v) as the mobile phase. Using fluorescence detection with excitation at 240 nm and emission at 380 nm, the limit of quantification was 12.5 ng/ml for a sample size of 0.5 ml of serum. The assay was linear in the concentration range of 12.5-1500 ng/ml and showed good accuracy and reproducibility. At all concentrations intra- and inter-assay variabilities were below 11% with less than 9% error. The method was applied to the determination of celecoxib for pharmacokinetic studies in man.     

Simple,rapid and sensitive method for the determination of indomethacin in plasma by high-performance liquid chromatography with ultraviolet detection

A micro method for determination of indomethacin in plasma was developed. Following deproteinization of plasma with acetonitrile containing internal standard (mefenamic acid), the separation of indomethacin and internal standard was achieved by high-performance liquid chromatography using a 7 μm LiChrosorb-RP18 column (250×4 mm I.D.) at 50°C. The mobile phase was 6 mM phosphoric acid–acetonitrile (50:50). The flow-rate was kept at 2.0 ml/min and the column effluent was monitored at 205 nm. The coefficients of variation of the method estimated at 0.2 and 1.0 μg/ml were 4.2 and 2.3%, and the detection limit of the drug was about 0.05 μg/ml (S/N=5). The method requires minimum pretreatment of the plasma with a small sample volume (25 μl), and is very suitable for therapeutic drug monitoring of indomethacin in premature infants with symptomatic patent ductus arteriosus.     

Simple and rapid method for determining nicotinamide adenine dinucleotide synthetase activity by high-performance liquid chromatography

A method is described for the determination of nicotinamide adenine dinucleotide synthetase (NADS) activity in human blood. Using high-performance liquid chromatography (HPLC), the formed NAD is separated from the substrates and the other blood components in less than 13 min. The activity of NADS determined by HPLC is closely correlated with that determined by the conventional spectrophotometric method, which requires two steps of enzyme reaction. The present method is simple and reliable and facilitates the routine analysis of NADS activity.     

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).     

Extractionless method for the simultaneous high-performance liquid chromatographic determination of urinary caffeine metabolites for N-acetyltransferase 2, cytochrome P450 1A2 and xanthine oxidase activity assessment

Urinary metabolic ratios of caffeine are used in humans to assess the enzymatic activities of cytochrome P450 isoenzyme 1A2 (CYP1A2), xanthine oxidase (XO) and for phenotyping individuals for the bimodal N-acetyltransferase 2 (NAT2), all of them involved in the activation or detoxification of various xenobiotic compounds. Most reported analytical procedures for the measurement of the urinary metabolites of caffeine include a liquid–liquid extraction of urine samples prior to their analysis by reversed-phase HPLC. At neutral to basic pH however, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), a metabolite of caffeine, spontaneously decomposes to 5-acetylamino-6-amino-3-methyluracil (AAMU). Since AAMU is not extracted in most organic solvents, the extent of AFMU decomposition cannot be precisely assessed. Although the decomposition reaction can be minimized by immediate acidification of the urine, accurate results can only be obtained when both AAMU and AFMU are monitored, or alternatively, if AAMU is measured after complete transformation of AFMU into AAMU in basic conditions. We report a liquid chromatographic method for the simultaneous quantitative analysis of the five urinary metabolites of caffeine used for the CYP1A2, XO and NAT2 phenotyping studies: AAMU, AFMU, 1-methylxanthine, 1-methyluric acid and 1,7-dimethyluric acid. These metabolites are satisfactory separated from all other known caffeine metabolites as well as endogenous urinary constituents. Sample treatment does not require any liquid–liquid extraction procedure. Urine samples are diluted and centrifuged before being injected (10 μl) onto a YMC-Pack Polyamine II (250×4.6 mm) column. A step-wise gradient elution program is applied using acetonitrile–0.75% (v/v) formic acid: (91:9) at 0 min→(75:25) at 25 min→(65:35) at 35 min→(65:35) at 45 min, followed by a re-equilibration step to the initial solvent composition. The flow-rate is 1.0 ml/min and the separations are monitored by UV absorbance at 260 and 280 nm. The procedure described here represents a substantial improvement over previous methods: a single analysis and a minimal urine sample treatment enables the simultaneous quantitation of five caffeine metabolites, notably AFMU and AAMU, used for the determination of CYP450 1A2, XO and NAT2 enzyme activity. Importantly enough, phenotyping individuals for the bimodal NAT2 is made possible without the uncertainty associated with the deformylation of AFMU, which is likely to happen at all steps prior to the analysis, during sample storage and even in the bladder of the subjects.