Sensitive chiral high-performance liquid chromatographic assay for labetalol in biological fluids

The four stereoisomers of the combined α- and β-adrenoceptor antagonist labetalol were separated and quantified at therapeutic concentrations by normal-phase high-pressure liquid chromatography using a chiral stationary phase and fluorescence detection. Drug in plasma or urine was recovered by solid-phase extraction with 83±5% efficiency. Limits of detection from biological samples (3 ml) were between 1.5–1.8 ng ml⁻¹. Intra-day and inter-day variation at 25 ng ml⁻¹ were ≤2.7% and ≤5.80% respectively for all stereoisomers. The assay was applied to an examination of the disposition of labetalol stereoisomers after a single oral dose of racemate to a human volunteer. Labetalol appears to undergo enantioselective metabolism leading to relatively low plasma concentrations of the pharmacologically active enantiomers.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml⁻¹ for gastric juice aspirate and plasma and 0.010 μg g⁻¹ for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml⁻¹ for plasma and gastric juice aspirate and up to 40 μg g⁻¹ for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2′-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2′-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN^®. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml⁻¹ (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml⁻¹ urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg⁻¹ creatinine (range 0.17–5.9 ng mg⁻¹ creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.     

Determination of the GABAB receptor agonist CGP 44532 (3-amino-2-hydroxypropylmethylphosphinic acid) in rat plasma after pre-column derivatization by micro-high-performance liquid chromatography combined with negative electrospray tandem mass spectrometry

An assay, based on pre-column derivatization and micro-high-performance liquid chromatography–tandem mass spectrometry, was developed for the determination of the GABA_B agonist CGP 44532 in rat plasma. CGP 44532, a highly polar 3-amino-2(S)-hydroxypropylmethylphosphinic acid, presented difficulties in developing a chromatographic method for the analysis of the compound in rat plasma. Instead of analyzing the target compound directly, it was derivatized prior to separation to a 4-nitrobenzylcarbamate isopropyliden derivative. In order to reach the required quantitation limit, on-line solid-phase extraction was utilized for sample clean-up and reversed-phase micro-column high-performance liquid chromatography, for separation of the plasma samples. The separated compounds were detected by negative electrospray tandem mass spectrometry in selected reaction monitoring mode. The derivatives show good chromatographic and mass spectrometric properties and both the target compound and the internal standard, could be eluted as symmetrical peaks with good signal/noise ratio. The MS–MS detection was selective and sensitive due to the straight fragmentation pattern. After injection of 200-μl sample aliquots, the limit of quantification was 10 ng ml⁻¹. The analytical assay is useable in the range of 10–500 ng ml⁻¹.     

Determination of idarubicin and idarubicinol in rat plasma using reversed-phase high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of idarubicin and idarubicinol in rat plasma. Blood samples were analyzed from 16 rats which had received an intravascular dose of 2.25 mg kg⁻¹ idarubicin. After deproteinization with acetonitrile, the separation was performed with a LiChrospher 100 RP-18 column (5 μm), using fluorescence detection (excitation: 485 nm/emission: 542 nm). The mean recovery was 95.6% for idarubicin and 90.7% for idarubicinol, respectively. The detection limit was 0.25 ng ml⁻¹ using an injection volume of 50 μl. Daily relative standard deviation (RSD) was 3.2% (10 ng idarubicin/ml, n=10) and 4.4% (10 ng idarubicinol/ml, n=10).     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Sensitive method for the determination of bisphenol-A in serum using two systems of high-performance liquid chromatography

The aim of this study was to establish an easy and accurate method for the determination of bisphenol-A (BPA) in the body liquid such as serum and urine. Two high-performance liquid chromatography (HPLC) systems, HPLC with electrochemical detector (ED), and HPLC with mass spectrometry (MS) using electrospray ionization (ESI) interface were used for the assay in the serum samples prepared with solid-phase extraction method. Water or EtOH at a concentration below 50% was suitable for the extraction of BPA from serum. The limit of detection of BPA was 0.2 ng ml⁻¹ for the HPLC-ED method and 0.1 ng ml⁻¹ for HPLC–MS. There was a good correlation between the data obtained by the two HPLC systems. BPA concentrations in healthy human serum were low (0–1.6 ng ml⁻¹). From various commercial fetal bovine serum and sheep plasma, however, significant amounts of BPA were detected. Since no BPA was detected from sheep plasma immediately after collection, the high amounts of BPA were considered to be caused by the handling of blood during the preparation of the products after blood collection. In vitro study showed that the amount of BPA leached from polycarbonate tube into sheep plasma were 40 times larger than those into water and the leached amount of BPA depended on the temperature (37°C>20°C>5°C).     

Determination of trace lead in biological and water samples with dispersive liquid-liquid microextraction preconcentration

A new method for the determination of trace lead was developed by dispersive liquid–liquid microextraction preconcentration and graphite furnace atomic absorption spectrometry. In the proposed approach, 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) was used as a chelating agent, and carbon tetrachloride and ethanol were selected as extraction and dispersive solvents. Some factors influencing the extraction efficiency of lead and its subsequent determination, including extraction and dispersive solvent type and volume, pH of sample solution, concentration of the chelating agent, and extraction time, were studied and optimized. Under the optimum conditions, the enrichment factor of this method for lead was reached at 78. The detection limit for lead was 39 ng L⁻¹ (3σ), and the relative standard deviation (RSD) was 3.2% (n = 7, c = 10 ng mL⁻¹). The method was successfully applied to the determination of trace amounts of lead in human urine and water samples.     

Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma

A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (−)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C₁ stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (−)-nicardipine was possible in a concentration range of 5–100 ng ml⁻¹ and the limit of detection in plasma was 2.5 ng ml⁻¹. The recoveries of (+)- and (−)-nicardipine added to plasma were 91.4–98.4% and 93.3–96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (−)-nicardipine in plasma from healthy volunteers.     

Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay

A novel assay method using nuclease protection assay integrated with sandwich hybridization (NPA-SH) for qualitative and quantitative detection of microalgae has been developed. Two species-specific nuclease-protection-assay (NPA) probes targeted 28S ribosomal RNA of Prorocentrum minimum and Prorocentrum micans, respectively, were designed in this study. The assay consists of S1 nuclease protection, sandwich hybridization and signal detection. The specificity of the probes was verified with cultured algae in the laboratory and field sample from Jiaozhou Bay, and the quantity by NPA-SH analysis showed good agreement with that of cell-counting with a light microscope. The optical absorbance of probe binding on the target showed good linear fit with cell amount. A standard curve for P. minimum was established to correlate the optical absorbance to cell density on a basis in the linear range between 15 and 475 cells ml⁻¹ seawater, and the equation deducted was ‘y = 0.0053 × x + 0.0658’ (R² = 0.992, n = 4). The assay was sensitive to detect 15 cells ml⁻¹ seawater. And for P. micans, with linear range between 0.6 and 20 cells ml⁻¹ seawater, the equation deducted was ‘y = 0.1174 × x + 0.1106’ (R² = 0.996, n = 4); the assay was sensitive to detect less than 1 cell ml⁻¹ seawater. The inter-assay coefficients of variation (CVs) were 12.4 and 10.9%, respectively. The good specificity, sensitivity and reproducibility of the NPA-SH implied that this new technique could be extremely useful for qualitative and quantitative assay of P. minimum and P. micans at low abundance.     

Determination of 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo[1,5-a]quinoxalin-4(5H)-one in serum by high-performance liquid chromatography

A high-performance liquid chromatographic assay with solid-phase extraction (SPE) for the rapid and sensitive quantitation of 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo[1,5-a]quinoxalin-4(5H)-one (I, U-78875) in serum is described. The validation results indicated that the present method had excellent intra- and inter-assay precision (≤ 9.5%, mean ± S.D. = 3.9±3.0%, n = 25) and accuracy (≤ 10.0%, mean ± S.D. = 3.0 ± 2.9%, n = 25), as well as improved sensitivity (2 ng/ml, using a 100-μl injection). Each chromatographic run is only 10 min and the organic solvent for the extraction of I and internal standard (U-82217) from serum was only 300 μl. The application results obtained from the SPE method were in good agreement with the advanced automated sample preparation method.     

Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography–mass spectrometry

A method for the determination of clenbuterol (4-amino-3,5-dichloro-α[(tert.-butylamino)methyl]-benzyl alcohol hydrochloride) in hair of living cows has been developed. Hair samples were digested in an alkaline medium. The diphasic dialysis technique is a semi-permeable membrane technology developed for the direct extraction of relatively low-molecular-mass analytes such as clenbuterol. In this case, we used sodium citrate buffer to homogenize the digested hair, dichloromethane was used as the extraction solvent at 37°C, and stirring was applied at 150 rpm for 4 h. The analysis was carried out using gas chromatography–mass spectrometry. The calibration curve for clenbuterol in hair was linear in the range from 12.5 to 400 ng g⁻¹. The detection limit of clenbuterol was 5 ng g⁻¹ and the quantification limit was 12.5 ng g⁻¹, in hair. A good inter-day reproducibility was obtained (R.S.D.=7.08%). The repeatability and intra-day reproducibility (50 ng g⁻¹ of hair, n=10) show R.S.D.s of 7.1 and 9.5%, respectively.     

Determination of clenbuterol in beef liver and muscle tissue using immunoaffinity chromatographic cleanup and liquid chromatography with ultraviolet absorbance detection

Clenbuterol, a beta-agonist, was determined in samples of beef liver and muscle. The method employed an acidic aqueous extraction followed by protein precipitation. The supernatant liquid was passed through a weak cation-exchange cartridge and then through a commercially available immunoaffinity cartridge. Clenbuterol was eluted from the immunoaffinity cartridge with 80% ethanol in water. The eluate was concentrated and analysed directly by reversed-phase liquid chromatography using gradient elution and UV detection at 245 nm. Detection limits were estimated to be 0.3 ng g⁻¹ clenbuterol. A single immunoaffinity cartridge was used for ten sample extracts with no significant loss in capacity. No organic solvents other than ethanol and methanol were employed in the procedure. Recoveries of clenbuterol from samples of beef liver and muscle spiked at 2 and 5 ng g⁻¹ carried through the entire procedure were 63±11% (range, 53–74%) compared to pure standards. Absolute recoveries of pure standards (30 ng clenbuterol) carried through the same analytical steps were 70±5% (n = 6), the losses being primarily due to the ion-exchange step.     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO₄), allows a quantification limit of 2.52 μg ml⁻¹ and a detection limit of 0.83 μg ml⁻¹. Recoveries and absolute recoveries of cefepime from plasma were 96.13–99.44% and 94–102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 μg ml⁻¹ (n=10).The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.