Therapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatography system

A direct in-line pre-column extraction technique in which guanidinium and ammonium sulfate are used, followed by column switching, was employed to analyze serum, plasma and cerebrospinal fluid samples of patients treated for tuberculous meningitis. Resolution of a wide range of polar to non-polar xenobiotics was obtained on a C₈ silica column by using a linear gradient from a binary system consisting of solvent A (0.05 M KH₂PO₄) and solvent B (acetonitrile—isopropanol, 4:1, v/v). Apart from the anti-tuberculosis drugs (isoniazid, pyrazinamide, ethionamide and rifampicin) the patients received up to sixteen different medicines for prevention of complications and the treatment of symptoms. Qualitative resolution of all the drugs was obtained by the chromatographic system. Quantitation of pyrazinamide and ethionamide was achieved with high precision and low inter-sample variation.     

Microbore high-performance liquid chromatographic determination of cisapride in rat serum samples using column switching

For the determination of cisapride from serum samples, an automated microbore high-performance liquid chromatographic method with column switching has been developed. After serum samples (100 μl) were directly injected onto a Capcell Pak MF Ph-1 pre-column (10×4 mm I.D.), the deproteinization and concentration were carried out by acetonitrile–phosphate buffer (20 mM, pH 7.0) (2:8, v/v) at valve position A. At 2.6 min, the valve was switched to position B and the concentrated analytes were transferred from MF Ph-1 pre-column to a C₁₈ intermediate column (35×2 mm I.D.) using washing solvent. By valve switching to position A at 4.3 min, the analytes were separated on a Capcell Pak C₁₈ UG 120 column (250×1.5 mm I.D.) with acetonitrile–phosphate buffer (20 mM, pH 7.0) (5:5, v/v) at a flow-rate of 0.1 ml/min. Total analysis time per sample was 18 min. The linearity of response was good (r=0.999) over the concentration range of 5–200 ng/ml. The within-day and day-to-day precision (CV) and inaccuracy were less than 3.7% and 3.8%, respectively. The mean recovery was 96.5±2.4% with the detection limit of 2 ng/ml.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

Column liquid chromatographic determination of paroxetine in human serum using solid-phase extraction

A 0.5-ml aliquot of a serum sample, after the addition of a 100-μl aliquot of a 5 μg/ml solution of dibucaine as the internal standard, is vortex-mixed with 0.5 ml of acetonitrile and centrifuged. The supernatant is applied to a 1-ml BondElut C₁₈ silica extraction column conditioned with subsequent washings with 1 M HCl, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid—methanol (1:40, v/v). A 7-μl aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₈ silica particles and eluted at ambient temperature with a mobile phase of 10 mM phosphate buffer-acetonitrile (2:1, v/v) (pH 3.2). The peaks are detected with a fluorescence detector (excitation at 295 nm, emission at 365 nm). The resulting chromatogram is clean with no extraneous peaks. Paroxetine and dibucaine give sharp peaks which are well separated from each other and from the solvent peaks. The extraction recovery of the drug and the internal standard is in the range of 90% which allows a highly sensitive determination of paroxetine.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C₁₈ column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E₁ = +0.25 V and E₂ = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.     

Colorimetric determination of hydroxyurea in human serum using high-performance liquid chromatography

A high-performance liquid chromatography assay for hydroxyurea in human serum was developed based on a commercial colorimetric assay kit for urea (Sigma Diagnostics). Serum (0.5 ml), spiked with methylurea as an internal standard, was treated with 70% perchloric acid. Supernatant (0.2 ml) was combined with 0.7 ml of BUN acid reagent and 0.6 ml of BUN color reagent. The resulting colored reactant (100 μl) was analyzed on a 300×3.9 mm Bondclone 10 C₁₈ column coupled with a UV–Vis detector, at 449 nm. The mobile phase was 13% acetonitrile in water. Retention times of colored derivatives of hydroxyurea and methylurea were 6.5 and 12.2 min, respectively. The log–log calibration curve was linear from 0.0065 to 1.31 mM. Average accuracy was 99.9±4.0% and the intra- and inter-day error of assay did not exceed 11%.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Determination of ceftazidime in plasma using high-performance liquid chromatography and electrochemical detection: Application for individualizing dosage regimens in elderly patients

This study describes a sensitive HPLC–electrochemical detection method for the analysis of ceftazidime, a third-generation cephalosporin, in human plasma. The extraction procedure involved protein precipitation with 30% trichloroacetic acid. The separation was achieved on a reversed-phase column (250×4.6 mm I.D., 5 μm) packed with C₁₈ Kromasil with isocratic elution and a mobile phase consisting of acetonitrile–25 mM KH₂PO₄–Na₂HPO₄ buffer, pH 7.4 (10:90, v/v). The proposed analytical method is selective, reproducible and reliable. The assay has a precision of 0.2–15.1% (C.V.) in the range of 5–200 μg ml⁻¹. (corresponding to 0.5 to 20 ng of ceftazidime injected onto the column), and is optimised for assaying 50 μl of plasma. The extraction recovery from plasma was approximately 100%. The method was highly specific for ceftazidime and there was no interference from either commonly administered drugs or endogenous compounds. This assay was used to measure ceftazidime in elderly patients for therapeutic drug monitoring.     

Capillary reversed-phase high-performance liquid chromatographic determination of acetyl-methylprednisolone in feline spinal cord

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) was used to determine acetylmethylprednisolone (A-MP) that had been administered to feline spinal cord tissue. The method used a 300 mm × 0.32 mm I.D. packed capillary octadecylsilyl (ODS) column and an isocratic mobile phase of 40 mM triethylamine formate (TEAF, pH 3.2)-acetonitrile (50:50, v:v). The chromatographic behavior of A-MP was evaluated with respect to peak-area and peak-height by varying the A-MP concentration (12–190 μM) with a fixed injection volume (1 μl), and by varying the injection volume (1–10 μl) with a fixed concentration (12 μM) of A-MP. The limit of detection (signal-to-noise ratio, 3:1) was 250 pg (600 fmol) of synthetic A-MP. Various amounts of A-MP directly spiked into feline spinal cord segments were solvent extracted, separated, and plotted against peak-area (r² = 1.00). Background tissue without A-MP gives minimal (<1%) interference at 243 nm. The method also detects exogenous A-MP that was administered into feline spinal cord via an intrathecal injection. Furthermore, the presence of A-MP was confirmed via its molecular ion and corresponding product ions that were obtained by fast-atom bombardment tandem mass spectrometry (FAB-MS-MS).     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Post-column enzyme reactors for chemiluminometric detection of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in an anion-exchange chromatographic system

A liquid chromatographic system consisting of a co-immobilized 3-hydroxybutyrate dehydrogenase-NADH oxidase reactor and an immobilized pyranose oxidase reactor in series and a chemiluminometer was developed for the simultaneous determination of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in plasma. The enzymes were immobilized on toresylated poly(vinyl alcohol) beads. Separation was achieved on a TSK gel SAX column (40×4 mm I.D.) with an eluent of 50 mM NaOH containing 30 mM sodium butyrate. The hydrogen peroxide produced was detected by measuring the chemiluminescence emitted on admixing with luminol and potassium hexacyanoferrate(III). The calibration curves were linear from 0.8 to 500 μM (7 ng−4 μg) for glucose, from 0.8 to 400 μM (7 ng−3 μg) for 1,5-anhydroglucitol and from 1 to 700 μM (5 ng−4 μg in a 50-μl injection) for 3-hydroxybutyrate. The sample throughput was four per hour. The reactors were stable for at least ten days.     

Measurement of hydroxyl radical in rat blood vessel by microbore liquid chromatography and electrochemical detection: an on-line microdialysis study

Salicylic acid (0.5 mM) is used as a trapping reagent of hydroxyl radical, and the formed 2,3- and 2,5-dihydroxybenzoic acids were collected via an on-line microdialysis device from the blood vessels. This study revealed the use of a sensitive liquid chromatographic system with electrochemical detection for the determination of 2,3- and 2,5-dihydroxybenzoic acids. Mobile phase consisted of 0.1 M monochloroacetic acid, 10 mM EDTA, 0.5 mM sodium octylsulfate, 20% acetonitrile and 5% tetrahydrofuran in 1 l (pH 3.0 adjusted with 1 M NaOH), and the flow-rate of 0.05 ml/min were found to be optimum. Isocratic separation of these adducts on a microbore column (reversed-phase C₁₈, 150×1 mm I.D., 5 μm) was achieved within 10 min. The optimal applied potential of dihydroxybenzoic acids was set at 750 mV based on a hydrodynamic study. This method has the detection limits of 1.3 pmol/ml (or 0.2 ng/ml) for 2,3- and 2,5-dihydroxybenzoic acids in Ringer solution (at signal-to-noise ratio=3).     

Convenient method for the determination of arginine and its related compounds in rumen fluid by reversed-phase high-performance liquid chromatography

In order to clarify arginine (Arg) metabolism by rumen microorganisms and by the tissues of ruminant animals, a convenient method for the simultaneous determination of Arg, citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV), and 4-aminobutyric acid (4AB) and lysine (Lys), incidentally, in goat rumen fluid was established by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out by stepwise isocratic elution with two mobile phases (solvent A and solvent B) on a LiChrospher 100 RP-18 column (150×4.6 mm I.D., 5 μm particle size) equipped with a guard column (4.0×4 mm, 5 μm particle size). Solvent A is composed of acetonitrile–sodium citrate buffer (pH 7.2) (15:85, v/v) containing tetrahydrofuran (5 ml/100 ml), with solvent B comprising acetonitrile–sodium citrate buffer (pH 5.4) (40:60, v/v). Five compounds (Cit, Arg, Pro, 4AB and 5AV) were separated within 33 min in solvent A and the other two (Orn and Lys) in solvent B. Solvent A was automatically switched to solvent B with the help of a valve controller. Complete separation needs 62 min after sample injection in a single chromatogram. Samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and detected on a fluorescence detector at excitation and emission wavelengths of 263 and 611 nm, respectively. The minimum detectable concentrations (μM) (signal-to-noise ratio, S/N 3:1) of these compounds were: 0.65 for Cit, 0.65 for Arg, 1.9 for Pro, 1.3 for 4AB, 1.9 for 5AV, 0.12 for Orn and 0.48 for Lys. When applied to rumen fluid from goats, recoveries of all compounds added to the rumen fluid were 96.6–100.6% for an intra-day study and 93.9–99.4% for inter-day (5 days) studies. The average contents of Orn, 5AV and Lys in the rumen fluid of three goats before morning feeding were 7.3, 13.5 and 3.6 μM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 μM) and 5AV (497.6 μM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 mM) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125×4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 μl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 μg/ml and was capable of detecting a minimum concentration of 0.029 μg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 × 4.0 mm I.D., 5 μm particle size) protected by a guard column (Perisorb RP-18, 30 × 4.0 mm I.D., 30–40 μm particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5–100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) − 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0–97.8%; method comparison c(bioassay) = 1.092c(HPLC) − 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.     

Determination of sodium artesunate in plasma using ion-pairing high-performance liquid chromatography

A chromatographic method is described for the determination of sodium artesunate in plasma. This includes cetyltrimethylammonium bromide as a cationic pairing ion in a reversed-phase system using an octadecylsilica 100×4.6 mm I.D. 3 μm analytical column with a mobile phase of acetonitrile/acetate buffer at pH7. Column switching incorporating a 5 μm octadecylsilica 100×4.6 mm I.D. precolumn is used in addition to off-line solid-phase extraction for pretreatment of plasma samples in order to eliminate interference from endogenous components. Detection is by post-column derivatisation with 1.0 M methanolic KOH followed by UV detection at 289 nm. Calibration is linear over the range 100–1600 ng ml⁻¹ and the limit of detection is estimated as 20 ng ml⁻¹. Illustrative results are shown of the artesunate plasma levels determined by the proposed method following the administration of artesunate as tablets and as suppositories to healthy volunteers.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.