Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography

The determination of quinine, (3S)-3-hydroxyquinine, 2′-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 μl cold methanol. Urine samples were 10–100× diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024–0.081 μM.     

Highly sensitive assay for the measurement of serotonin in microdialysates using capillary high-performance liquid chromatography with electrochemical detection

A highly sensitive isocratic capillary high-performance liquid chromatographic (HPLC) method with electrochemical detection (ED) for the simultaneous measurement of serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in microdialysates has been developed using a 0.5 mm i.d. capillary column and a 11-nL detection cell. This method, validated on both pharmacological and analytical bases, can be performed using injection volumes as low as 1 microL. The limits of detection were 5.6 x 10(-11)mol/L and 3.0 x 10(-9)mol/L for 5-HT and 5-HIAA. Several applications of the present method are given on microdialysates from rodent brain and human spinal cord.     

Metabolism of benz[a]anthracene by the filamentous fungus Cunninghamella elegans.

The metabolism of the carcinogen benz[a]anthracene (BA), a tetracyclic aromatic hydrocarbon, by Cunninghamella elegans was investigated. C. elegans grown on Sabouraud dextrose broth transformed [14C]BA to labeled BA trans-8,9-dihydrodiol (90%), BA trans-10,11-dihydrodiol (6%), and BA trans-3,4-dihydrodiol (4%), but not to BA trans-5,6-dihydrodiol. These metabolites were separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography and were identified by UV and mass spectral techniques. A BA tetraol, 8 beta,9 alpha,10 alpha,11 beta-tetrahydroxy-8 alpha, 9 beta,10 beta,11 alpha-tetrahydro-BA, was also identified as a metabolite and may have arisen as an additional oxidation product of either BA 8,9- or 10,11-dihydrodiol. This is the first study in which a biologically produced BA tetraol has been identified. Our results suggest that the transformation of BA to trans-dihydrodiols by C. elegans is similar to the transformation of BA found in mammals, except that BA 5,6-dihydrodiol is not produced.     

Use of microbore LC-MS/MS for the quantification of oxcarbazepine and its active metabolite in rat brain microdialysis samples

A microbore LC-MS/MS method is developed and validated for the quantification of the anti-epileptic drug oxcarbazepine (OXC) and its active metabolite 10,11-dihydro-10-hydroxycarbamazepine (MHD) in rat brain microdialysates, together with the internal standard for microdialysis probe calibration, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide (m-CBZ). The benefits of gradient versus isocratic separation are shown, next to the improved sensitivity resulting from the addition of 0.1% formic acid to the mobile phase. The coupling of microdialysis with ESI-MS requires sample desalting for which column switching was applied. Using weighed regression to calculate the calibration curves (1-1000 ng/mL), the assay was validated in terms of linearity, accuracy and precision, yielding a sensitive (limit of quantification is 1 ng/mL) and selective method for quantification of OXC, MHD and m-CBZ. By applying this method, we were able to determine the extracellular concentrations of OXC and MHD during at least 4h after intraperitoneal (i.p.) administration of 10 mg/kg OXC.     

Capillary electrophoresis with laser-induced fluorescence detection, an adequate alternative to high-performance liquid chromatography, for the determination of ciprofloxacin and its metabolite desethyleneciprofloxacin in human plasma

A method to determine plasma concentrations of ciprofloxacin and its metabolite desethyleneciprofloxacin (M1) by CE with HeCd laser-induced fluorescence detection is described. Following precipitation of proteins and centrifugation supernatant is injected hydrodynamically (10 s, 0.5 p.s.i.) into the capillary. Overall analysis time for the quantification of both analytes was 7 min. The total amount of plasma needed for multiple injections (n>5) was 10–20 μl. Data on accuracy and precision are presented. The assay performance is compared to the specifications of a validated HPLC method, which is routinely used for the quantification of ciprofloxacin and M1 in body fluids. Both methods showed comparable accuracy and precision for both analytes throughout the whole working range (inter-day precision <9%; inter-day accuracy 96–110%). The limit of quantification (LOQ) of 20 μg/l (M1 10 μg/l) for the CE procedure was slightly higher than for the HPLC method, where 10 μg/l (M1 2.5 μg/l) was determined. However, application of the methods to human plasma samples derived from a clinical study proved that comparable results are obtained and that the sensivity of the HPCE method was sufficient to fully describe typical plasma concentration timie profiles of ciprofloxacin and its metabolite M1. Both the adequate sensitivity and the required smaller sample volume compared to HPLC indicate that the method is feasible for clinical studies where sample amounts are limited, e.g., studies to investigate pharmacokinetics in pediatric patients. Preclinical studies form another possible application of this technique.     

Simple,rapid and micro high-pressure liquid chromatographic method for the simultaneous determination of tolbutamide and carboxy tolbutamide in plasma

A rapid high-pressure liquid chromatographic (HPLC) assay is described for the quantitative analysis of tolbutamide and its major metabolite, carboxy tolbutamide, in plasma. An aliquot (25–100 μl) of plasma was prepared for chromatography by deproteinization as follows. One volume of plasma and 2.5 volumes of acetonitrile were vortex mixed for a few seconds and then centrifuged for approx. 1 min. A 50-μl sample of the clear supernatant was injected into the chromatograph. A μBondapak C₁₂ reversed-phase column was used with a mobile phase of acetonitrile-0.05% phosphoric acid (45:55) at a flow-rate of 1.5 ml/min. The column effluent was monitored by a variable-wavelength UV detector set at 200 nm. Tolbutamide and its metabolite had retention times of 5.75 and 3.25 min, respectively. The procedure yields reproducible results with sensitivity adequate for routine clinical monitoring of plasma levels or for single-dose pharmacokinetic studies. A number of commonly used drugs do not interfere with the method. A single plasma sample can be analyzed in approx. 9 or 10 min.     

Prevalidation statistical design to assess analytical methods example of a quick liquid chromatographic assay of itraconazole in serum

(A) An analysis-of-varience spreadsheet program is presented which allows to readily test and/or quantitate in a single run analytical linearity, matrix effect on recovery, repeatability of measurement and of extraction and the ruggedness of these features for up to three sessions. Owing to napierian logarithmic transformation, ANOVA mean squares directly read as relative standard deviations and checking linearity is straightforward. (B) A quick assay for therapeutic drug monitoring of itraconazole and its main metabolite was devised with the help of the program, and subsequently validated according to current quality control recommendations. The assay involves acetonitrile demixing extraction, reversed-phase HPLC and UV detection and shows acceptable performance from 0.06 to 5.0 mg/l (limit of detection about 0.03 mg/l). The prevalidation design fairly predicted precision and accuracy, was more informative about matrix effect and was even more demanding about analytical linearity.     

On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis

Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) does not provide detection limits sufficient to quantify glutathione (GSH) and glutathione disulfide (GSSG) in biological samples such as liver microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. This stacking technique allowed large volumes of high ionic strength sample injection without deterioration of the separation efficiency and resolution. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The limit of detection for GSH and GSSG was found to be 0.75 microM (S/N=6) and 0.25 microM (S/N=6), respectively. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73+/-2.08 microM (n=7) and 5.52+/-3.66 microM (n=7), respectively.     

High-performance liquid chromatographic determination of carbamazepine and metabolites in human hair

To study the use of hair analysis in monitoring drug compliance and historical changes in pharmacokinetics we developed a method for the quantitative determination of the anti-epileptic drug carbamazepine (CBZ) and trans-10,11-dihydro-10,11-dihydroxy-carbamazepine (CBZ-diol) in hair from carbamazepine users. Digestion by 1 M NaOH was found to be the best method for isolating CBZ and CBZ-diol from hair, followed by solid-phase extraction and reversed-phase HPLC with UV detection. Recoveries from spiked hair samples were 76–86%. Within-day precision (C.V.; n=10) for CBZ and CBZ-diol in hair of a CBZ user containing 10.9 μg/g CBZ and 3.2 μg/g CBZ-diol were 1.7 and 5.0%, respectively. Sectional hair analysis of a patient on a constant dosage of CBZ demonstrates an exponential decrease in hair concentrations of CBZ and CBZ-diol with increasing distance from the root, probably caused by shampooing. No CBZ-10,11-epoxide (CBZ-epox) could be detected. However, one component in the chromatogram is probably CBZ-β-hydroxythioether, an adduct of CBZ-epox with cysteine, or acridinethioacetal, its rearrangement product. The concentration of this component does not decrease with increasing distance from the root.     

Determination of cefepime and cefpirome in human serum by high-performance liquid chromatography using an ultrafiltration for antibiotics serum extraction

The aim of this study was to describe a high-performance liquid chromatography (HPLC) assay for the determination of cefepime and cefpirome in human serum without changing chromatographic conditions. The assay consisted to measure cefepime and cefpirome which were unbound to proteins having a molecular mass of 10 000 or more by ultrafiltration followed by HPLC with a Supelcosil ABZ+ column and UV detection at a wavelength of 263 nm. The assay was been found to be linear and has been validated over the concentration range 200 to 0.50 μg/ml for both cefepime and cefpirome, from 200 μl serum, extracted. In future, the assay will support therapeutic drug monitoring for cefepime and cefpirome in neutropenic patients in correlation with microbiological parameters such as MIC₉₀ (minimal inhibitory concentration of antibiotic which kills 90% of the initial bacterial inoculum) and clinical efficacy.     

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.     

Simultaneous high-performance liquid chromatography determination of carbamazepine and five of its metabolites in plasma of epileptic patients

A high-performance liquid chromatographic method with UV detection for the simultaneous analysis of the antiepileptic drug carbamazepine and five of its metabolites in human plasma has been developed. The analysis was carried out on a reversed-phase column (C₈, 150×4.6 mm I.D., 5 μm) using acetonitrile, methanol and a pH 1.9 phosphate buffer as the mobile phase. Under these chromatographic conditions, carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine are baseline separated in less than 18 min. The extraction of the analytes from plasma samples was performed by means of an original solid-phase extraction procedure using Oasis HLB cartridges. The method requires only 250 μl of plasma for one complete analysis. The repeatability (RSD%<2.4), intermediate precision (RSD%<3.5) and extraction yield (84.8–103.0%) were very good for all analytes. The method is suitable for reliable therapeutic drug monitoring of patients undergoing chronic treatment with carbamazepine and for kinetic–metabolic studies of this drug.     

Modified high-performance liquid chromatographic method to measure both dextromethorphan and proguanil for oxidative phenotyping

The activities of the polymorphic enzymes cytochromes P450 2D6 and 2C19 can be assessed by administering the probe drugs, dextromethorphan and proguanil, respectively. An existing high-performance liquid chromatographic technique, which measures dextromethorphan and its metabolites, has been modified to also measure proguanil and its polymorphic metabolite, cycloguanil in urine. Proguanil and cycloguanil are assayed in separate aliquots of urine to that used for dextromethorphan/dextrorphan as pretreatment with β-glucuronidase is required for the analysis of dextrorphan. To assay all four compounds a common extraction procedure is used and a single reversed-phase column and isocratic mobile phase with UV and fluorescence detectors connected in series are required. This technique is specific and sensitive for each analyte (limits of detection, dextrorphan/dextromethorphan/proguanil: 0.1 μg/ml, cycloguanil: 0.2 μg/ml). All assays are linear over the concentration ranges investigated (dextromethorphan/dextrorphan: 0.5–10 μg/ml, proguanil/cycloguanil: 1–20 μg/ml). The method described therefore uses laboratory resources very efficiently for all the assays required for hydroxylation phenotyping using proguanil and dextromethorphan.     

Determination of plasma levels of spirorenone,a new aldosterone antagonist,and one of its metabolites by high-performance liquid chromatography

A method for the determination of plasma concentrations of spirorenone, a new aldosterone antagonist, and one of its metabolites, chromatographically characterized as 1,2-dihydro-spirorenone, is described. The assay utilizes high-performance liquid chromatography with UV detection. Reproducible results can be obtained with standard deviations of about 5% and the limit of detection is less than 5 ng/ml. Plasma levels of drug and metabolite have been measured after oral doses of 10 and 40 mg, respectively, administered to two male volunteers.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Extremely sensitive and selective antibodies against the explosive 2,4,6-trinitrotoluene by rational design of a structurally optimized hapten

Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)-activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 10(9) l/mol (rabbit R1) and (2.3 ± 0.2) × 10(9) l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC(50) value) of 0.1 ± 0.01 μg/l (R1) and 0.2 ± 0.02 μg/l (R2) and a working range of 0.005 to 150 μg/l (R1) and 0.007 to 200 μg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved.     

Chiral separation of 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, a metabolite of carbamazepine with two asymmetric carbons, in human serum

Chiral separation of 10,11-dihydro-10,11-trans-dihydroxycarbamazepine (CBZ-diol), a metabolite of carbamazepine (CBZ) with two asymmetric carbons, in serum taken from epileptic patients receiving CBZ alone for a long period, was performed by high-performance liquid chromatography using a polysaccharide stationary phase with n-hexane-ethanol (75:25, v/v) as the mobile phase. The enantiomeric ratio (S,S-/R,R-CBZ-diol) was 10.74 ± 1.13 (mean ± S.D.), which could demonstrate the presence of the stereospecificity in the hydrolysis of 10,11-dihydro-10,11-epoxycarbamazepine (CBZ-epoxide) to CBZ-diol and/or in the conversion of CBZ-diol to some metabolite such as 9-hydroxymethyl-10-carbamoylacridan. This is the first paper to report the determination of each enantiomer and the enantiomeric ratio of CBZ-diol in serum of epileptic patients who received CBZ.     

The formation of dihydrodiols by the chemical or enzymic oxidation of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene.

When benz[a] anthracene was oxidised in a reaction mixture containing ascorbic acid, ferrous sulphate and EDTA, the non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxybenz[a] anthracene and trans-3,4-dihydro-3,4-dihydroxybenz[a] anthracene together with small amounts of the 8,9- and 10,11-dihydrodiols were formed. When oxidised in a similar system, 7,12-dimethylbenz[a] anthracene yielded the K-region dihydrodiol, trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and the non-K-region dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-10,11-dihydro-10,11-dihydroxy-7,12-dimethylbenz[a] anthracene and a trace of the 1,2-dihydrodiol. The structures and sterochemistry of the dihydrodiols were established by comparisons of their UV spectra and chromatographic characteristics using HPLC with those of authentic compounds or, when no authentic compounds were available, by UV, NMR and mass spectral analysis. An examination by HPLC of the dihydrodiols formed in the metabolism, by rat-liver microsomal fractions, of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene was carried out. The metabolic dihydriols were identified by comparisons of their chromatographic and UV or fluorescence spectral characteristics with compounds of known structures. The principle metabolic dihydriols formed from both benz[a] anthracene and 7,12-dimethylbenz[a] anthracene were the trans-5,6- and trans-8,9-dihydrodiols. The 1,2- and 10,11-dihydrodiols were identified as minor products of the metabolism of benz [a] anthracene and the tentative identification of the trans-3,4-dihydriol as a metabolite was made from fluorescence and chromatographic data. The minor metabolic dihydriols formed from 7,12-dimethylbenz[a] anthracene were the trans-3,4-dihydrodiol and the trans-10,11-dihydriol but the trans-1,2-dihydrodiol was not detected in the present study.     

Extractionless high-performance liquid chromatographic method for the simultaneous determination of piroxicam and 5′-hydroxypiroxicam in human plasma and urine

A simple and rapid (extractionless) high-performance liquid chromatographic method with UV detection, at 330 nm, was developed for the simultaneous determination of piroxicam and its major metabolite, 5′-hydroxypiroxicam, in human plasma and urine. Acidified plasma and alkali-treated urine samples are used and naproxen is added as internal standard. The separation is performed at 40°C on a C₁₈ Spherisorb column with acetonitrile-0.1 M sodium acetate (33:67, v/v, pH 3.3) as mobile phase. The retention time is 2.2 min for 5′-hydroxypiroxicam, 2.6 min for piroxicam and 3.2 min for naproxen. The detection limit is 0.05 μg/ml using a 100-μl loop.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.