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.     

Simultaneous determination of dopamine, dopac and homovanillic acid : Direct injection of supernatants from brain tissue homogenates in a liquid chromatography—electrochemical detection system

A simple method based on high-performance liquid column chromatography with electrochemical detection is described for the simultaneous determination of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in discrete brain regions of rats. The supernatant of a tissue homogenate is injected directly onto a liquid chromatograph, thus omitting the commonly adopted adsorption step. Of the four different supports tested Nucleosil C₁₉ (5 μm) was found superior with respect to chromatographic performance. The effects of pH, methanol and the ion-pairing agent hexyl sulfate on the retention were studied. The mobile phase used in the final studies consisted of citrate buffer pH 4.25—methanol (92:8, v/v) containing hexyl sulfate (1.7 · 10⁻³M). Standard curves of dopamine, DOPAC and HVA were found linear up to about 600 pmol per injection for each compound. The precisions of the chromatographic step were (s_rel. %): 0.72% (dopamine), 1.26% (DOPAC) and 2.69% (HVA).     

Graphitized macroporous carbon microarray with hierarchical mesopores as host for the fabrication of electrochemical biosensor

A novel graphitized ordered macroporous carbon (GMC, pore size 380 nm) with hierarchical mesopores (2–30 nm) and high graphitization degree was prepared by nickel-catalyzed graphitization of polystyrene arrays. The obtained GMC possessed high specific surface area, large pore volume, and good electrical conductivity, which was explored for the enzyme entrapment and biosensor fabrication by a facile method. With advantages of novel nanostructure and good electrical conductivity, direct electrochemistry of hemoglobin (a model protein) was observed on the GMC-based biocomposite with a formal potential of −0.36 V (vs. Ag/AgCl) and an apparent heterogeneous electron transfer rate constant (k_s) of 1.2 s⁻¹ in pH 7.0 buffer. Comparative studies revealed that GMC offered significant advantages over carbon nanotubes (CNTs) in facilitating direct electron transfer of entrapped Hb. The fabricated biosensor exhibited good sensitivity (101.6 mA cm⁻² M⁻¹) and reproducibility, wide linear range (1–267 μM), low detection limit (0.1 μM), and good long-term stability for H₂O₂ detection. GMC proved to be a promising matrix for enzyme entrapment and biosensor fabrication, and may find wide potential applications in biomedical detection and environmental analyses.     

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.     

Application of capillary gas chromatography to the study of hydrolysis of the nerve agent VX in rat plasma

We present here a gas chromatography technique allowing the detection and quantification of VX [O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate] a as well as its P---S bond hydrolysis product diisopropylaminoethanethiol directly from spiked rat plasma. This technique was applied to study VX hydrolysis in rat plasma. We observed that 53±4% of 374 μM VX disappeared from spiked plasma after 2 h. VX disappearance was mainly related to enzymatic cleavage of the P---S bond (K_m=2.5 mM and V_max=13.3 nmol min⁻¹ of rat plasma). The activity was totally inhibited by 1 mM Hg²⁺ and was also inhibited by metal chelators.     

Biological monitoring of hexamethylene diisocyanate by determination of 1,6-hexamethylene diamine as the trifluoroethyl chloroformate derivative using capillary gas chromatography with thermoionic and selective-ion monitoring

A GC method using a novel derivatization reagent, 2′,2′,2-trifluoroethyl chloroformate (TFECF), for the derivatization of primary and secondary aliphatic amines with the formation of carbamate esters is presented. The method is based on a derivatization procedure in a two-phase system, where the carbamate ester is formed. The method is applied to the determination of 1,6-hexamethylene diamine (HDA) in aqueous solutions and human urine, using capillary GC. Detection was performed using thermionic specific detection (TSD) and mass spectrometry (MS)—selective-ion monitoring (SIM) using electron-impact (EI) and chemical ionization (CI) with ammonia monitoring both positive (CI)⁺ and negative ions (CI)⁻. Quantitative measurements were made in the chemical ionization mode monitoring both positive and negative ions. Tetra-deuterium-labelled HDA (TDHDA; H₂NC²H₂(CH₂)₄C²H₂NH₂) was used as the internal standard for the GC—MS analysis. In CI⁺ the m/z 386 and the m/z 390 ions corresponding to the [M + 18]⁺ ions (M = molecular ion) of HDA—TFECF and TDHDA—TFECF were measured; in CI⁻ the m/z 267 and the m/z 271 ions corresponding to the [M — 101]⁻ ions. The overall recovery was found to be 97 ± 5% for a HDA concentration of 1000 μg/l in urine. The minimal detectable concentration in urine was found to be less than 20 μg/l using GC—TSD and 0.5 μg/l using GC—SIM. The overall precision for the work-up procedure and GC analysis was ca. 3% (n = 5) for 1000 μg/l HDA-spiked urine, and ca. 4% (n = 5) for 100 μg/l. The precision using GC—SIM for urine samples spiked to a concentration of 5 μg/l was found to be 6.3% (n = 10).     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Determination of retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection

A method for separating and detecting retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection is described. Packed columns with an inner diameter of 180 μm were employed for the separation using a C₁₈ stationary phase and a mobile phase containing acetonitrile-water-methanol (65:32.5:2.5, v/v/v) with 1% tetrabutylammonium perchlorate and 0.174 M acetate buffered at pH 5. The detection cell consisted of a carbon fiber barrel electrode held at 0.9 V versus an Ag/AgCl reference. Injection volumes of 2 μl produced detection limits of 2.73, 0.472, 0.428, and 0.267 fmol (or 410, 64.1, 60.9, and 38.2 pg ml⁻¹) for 13-cis-retinoic acid, all-trans-retinoic acid, retinaldehyde, and retinol, respectively. This represents an improvement in detection limits of at least three orders of magnitude for similar analyses using liquid chromatography and UV absorbance detection. The detector signal was linear over two orders of magnitude of analyte concentration. Retinoid concentrations in bovine serum were determined and found to be in good agreement with previously reported values.     

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine

A dual Hg–Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl⁻ and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl⁻ were not separated at the optimum detection pH of 5.2. The signal from Cl⁻ was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl⁻ from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl⁻ in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 μl injected).     

Fosfomycin determination in serum by capillary zone electrophoresis with indirect ultraviolet detection

Capillary zone electrophoresis with indirect ultraviolet detection was used for the determination of fosfomycin in serum. Running buffer consisted of a mixture of 200 mM sodium borate with 10 mM phenylphosphonic acid used as ultraviolet absorbing background electrolyte. Relationships between the pH of the buffer and the efficiency of the separation (migration times and selectivities) or the sensitivity of detection were investigated. The method was then validated over a 10–100 μg ml⁻¹ concentration range to be applied to further therapeutic drug monitoring. The choice of ethylphosphonic acid as internal standard is discussed. The specificity and the linearity of the technique are demonstrated. The inter-day precision was satisfactory with a relative standard deviation of less than 2%. Accuracy was calculated with a standard error near 0.5 and 18% for 100 and 10 μg ml⁻¹, respectively.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E_M) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2 mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%.The E_M values of cortical cells of Lotus roots varied between −115 and −144 mV. Treatment with 250 μM of AlCl₃ (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51 mV (cv. UFGRS) and 16 mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K⁺ from Al-treated roots (2 mM Al) and resulted in a decrease of the diffusion potential (E_D). The total amount of K⁺ in Al-treated roots dropped from 31.4 to 16.8 μmol g⁻¹ FW in sensitive cv. INIA Draco, or from 26.1 to 22.7 μmol g⁻¹ FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21–34% of the control.     

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.     

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.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

New sensitive assay of vancomycin in human plasma using high-performance liquid chromatography and electrochemical detection

A method using reversed-phase high-performance liquid chromatography with electrochemical detection for the analysis of vancomycin in human plasma was developed. Chromatographic conditions included an octadecyl column, a mobile phase of acetonitrile–sodium phosphate buffer (pH 7) (12:88), a total run time of 12 min, and coulometric electrochemical detection at +700 mV. Linear detector response was found in the range 5–100 μg ml⁻¹ after a 1:80 dilution or from 0.5 to 50 μg ml⁻¹ after a 1:20 dilution of the samples. In both cases the correlation coefficient (r) of the calibration curve standard was better than 0.995. Vancomycin determination was based on a denaturation of plasma proteins with methanol, then a dilution with mobile phase was performed. Recovery of vancomycin from plasma was 103.1±3.9%, and no interference from commonly used drugs or endogenous compounds was observed. A significant correlation was shown with the EMIT assay (r=0.92, P<0.001) using clinical samples from children. This HPLC technique is simple, sensitive, rapid, precise, selective and requires only 100 μl of plasma for completion.     

Direct oxidation of boronates by peroxynitrite: Mechanism and implications in fluorescence imaging of peroxynitrite

In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO⁻) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO⁻, hypochlorous acid (HOCl), and hydrogen peroxide (H₂O₂) and found that ONOO⁻ reacts with 4-acetylphenylboronic acid nearly a million times (k = 1.6 × 10⁶ M^− 1 s^− 1) faster than does H₂O₂ (k = 2.2 M^− 1 s^− 1) and over 200 times faster than does HOCl (k = 6.2 × 10³ M^− 1 s^− 1). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO⁻ formed in cellular and cell-free systems.     

High-performance liquid chromatographic assays for protoporphyrinogen oxidase and ferrochelatase in human leucocytes

Rapid, sensitive and specific high-performance liquid chromatographic assays are described for protoporphyrinogen oxidase and ferrochelatase in human leucocytes. The enzyme reaction products were separated and quantitated by reversed-phase high-performance liquid chromatography with fluorescence detection. The optimal pH for the protoporphyrinogen oxidase assay was 8.6 and the Michaelis constant for protoporphyrinogen IX was 9.78 ± 0.96 μM (mean ± S.D.). The mean (± S.D.) activity of protoporphyrinogen oxidase in fourteen apparently healthy subjects was 0.146 ± 0.023 nmol protoporphyrin IX per min per mg protein. In one patient with variegate porphyria, the activity was 0.028 nmol protoporphyrin IX per min per mg protein. The optimal pH for ferrochelatase was 7.4 and with protoporphyrin and Zn²⁺ as substrates, the Michaelis constants were 1.49 and 8.33 μM, respectively. The mean activity of ferrochelatase in ten control subjects was 0.24 nM Zn—protoporphyrin or 2.05 nM Zn—mesoporphyrin formed per h per mg protein.     

High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). Chromatographic separation is achieved on a Hypersil BDS C₁₈ column (100×2.1 mm, 5 μm) with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.6, containing 1.0·10⁻⁴ M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.