Improved high-performance liquid chromatographic method for the determination of polycyclic aromatic hydrocarbon metabolites in human urine

A reversed-phase HPLC method with fluorescence detection was evaluated for utility in determination of urinary metabolites of polycyclic aromatic hydrocarbons as biomarkers of environmental exposure. The method, which was developed for use in studies of high-level occupational exposure, was found to be unreliable for relatively low-level environmental exposures. The method was modified to include quantitation by standard addition in order to compensate for matrix effects at levels as low as 0.1 ng/ml. The standard addition modification increased both qualitative and quantitative performance, with recovery of 1-hydroxypyrene spikes improved from 164% to 114% at 0.36 ng/ml. The modified method was successfully applied in an environmental exposure study.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid—liquid extraction. Separation was achieved on a C₁₈ reversed-phase column. The limit of detection of ciprofloxacin is 0.05 μg/ml and that of its three metabolites is 0.25 μg/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

On-line solid-phase extraction and high-performance liquid chromatographic determination of chlorthalidone in urine

A simple and rapid on-line method for the determination of chlorthalidone in urine is proposed. The sample containing the internal standard is injected in a CN precolumn. After a 2-ml water rinsing, the precolumn is coupled for 30 s to the HPLC column via a switching valve, allowing the on-line elution of the compounds of interest. Analysis is carried out by reversed-phase chromatography with an acetonitrile-0.01 M phosphate buffer pH 7 (20:80, v/v) eluent, using UV detection at 214 nm. While the LC separation is performed, the precolumn is regenerated and conditioned, and is ready to receive the next sample at the end of the run. Accurate (>95%) and precise (<10%) analyses, in the range of 0.1–20 μg/ml of chlorthalidone in urine, have been achieved using this method.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

High-performance liquid chromatographic determination of drugs and metabolites in human serum and urine using direct injection and a unique molecular sieve

Silicalite is a molecular sieve that contains an intricate system of channels approximately 6 Å in diameter. These channels are hydrophobic and have been shown to retain relatively small hydrophobic and hydrophilic molecules from aqueous and biological samples. Silicalite is shown to be a restricted-access medium that permits the injection of biological fluids directly onto a HPLC column packed with Silicalite, eliminating the need for sample preparation. The sample macromolecules elute with high recovery mostly at the extraparticulate void. Simultaneously, Silicalite allows various drugs and metabolites to enter the channels and be retained. Recoveries >90% were generally obtained for a wide variety of drugs and their metabolites from human serum and urine.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25–100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25–100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

Improved high-performance liquid chromatographic analysis of teniposide in human plasma

A simple and practical high-performance liquid chromatographic analysis has been developed for measuring teniposide (VM26) in human plasma. The present analytical method has improved extraction efficiency from human plasma, therefore allowing determination of VM26 in a clinical setting using ultraviolet detection alone. Furthermore, sample preparation was simplified and shortened through use of a one-step extraction procedure. VM26 and internal standard (ibuprofen) were extracted from human plasma (0.5 ml) with ethyl acetate. A phenyl μBondapak column eluted with a mobile phase, consisting of acetonitrile–distilled water–acetic acid (30:68:2, v/v/v) was used for separation, and quantitation was achieved with a UV monitor set at 240 nm. Average extraction efficiency was 96.8±6.6% for VM26 between 1 and 25 μg/ml, and 91.4±4.3% for internal standard, with both intra- and inter-day coefficients of variation being less than 10%. The detection limit with a 100-μl injection was estimated at 0.2 μg/ml with a signal-to-noise ratio of 3 for VM26 in human plasma. The stability data of VM26 in plasma, standard and stock solutions were also obtained. The present method was found to be an alternative to the previously reported method with an electrochemical detection, and can be easily applied to routine clinical pharmacokinetic studies of VM26.     

Simple direct injection high-performance liquid chromatographic method to determine quinidine in plasma

High-performance liquid chromatographic methods that use direct injection of plasma include column-switching procedures, modified mobile phases and small-pore modified stationary phases. By using a large-pore (300 Å) Selectosil C₁₈ column, developed for the analysis of macromolecules, we have shown that quinidine in plasma and protein solutions can be assayed accurately and rapidly by directly injecting 2 μl plasma or protein solution onto the column. Column life is not reduced, and the limit of quantitation is 0.01 μM.     

Stereospecific high-performance liquid chromatographic assay of ketoprofen in human plasma and urine

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of ketoprofen (KT), a 2-arylpropionic acid (2-APA) non-steroidal antiinflammatory drug (NSAID), in plasma and urine was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), plasma containing the KT enantiomers and I.S. was extracted by liquid-liquid extraction at an acidic pH. After evaporation of the organic layer, the drug and I.S. were reconstituted in mobile phase and injeted into the HPLC system. The enantiomers were separated at ambient temperature on a commercially available 250 × 4.3 mm amylose carbamate-packed chiral column (Chiralpak AD) column with hexane-isopropyl alcohol-trifluoroacetic acid (80:19.9:0.1, v/v/v) as the mobile phase pumped at 1.0 ml/min. The enantiomers of KT were quantified by ultraviolet detection with the wavelength set at 254 nm. The assay described allows for the direct quantification of KT enantiomers without pre-column derivatization, and is suitable for clinical studies of KT enantiomers in human plasma and urine after administration of therapeutic doses.     

Rapid high-performance liquid chromatographic determination of vancomycin in human plasma

A rapid simple and robust reversed-phase HPLC method was developed for rapid screening in bioavailability studies or comparative bioequivalence studies. The method is specific for vancomycin as no interference from acetylsalicylic acid, paracetamol and caffeine was observed. The mean intra-day precision was from 11.7% (low concentration) to 0.3% (high concentration) and the within-day precision from 15.0 to 0.3%, determined on spiked samples. The accuracy of the method was 106.4–99.8% (intra-day) and 103.5–100.2% (inter-day).     

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.     

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.     

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

Novel liquid chromatographic determination of cystatin C in human urine

A rapid and sensitive method for quantitative determination of cystatin C (CC) protein in human urine via HPLC was developed and validated. Acetone has been used as a precipitating agent of CC protein from the urine biological matrix. Separation and detection were accomplished by ion pair liquid chromatography and UV detection. Gradient elution mode was utilized to elute CC with a UV detection of 224 nm. The analysis time was 14 min per sample using Ace C8 (150 × 4.6 mm i.d., 5 μm) as a chromatographic column with a flow rate of 1.0 mL/min. Calibration curve with good linearity (r² = 0.99) within the range from 0.390 to 0.001 mg/mL was obtained. Limits of detection and quantification were 0.001 and 0.002 mg/mL, respectively. Inter-assay and intra-assay variabilities were <15% for all levels and <20% at the limit of quantification level. Major advantages of the method: specific where no false positive results might be obtained and fast where sample pretreatment needs only 1 h.     

High-performance liquid chromatographic determination of PEG 600 in human urine

Polyethylene glycols (PEGs) are non-ionic, water-soluble synthetic polymers which have been widely used for many applications. Since they are of very low toxicity and are readily excreted in urine, PEGs in the molecular weight range 400–6000 have been used extensively in the study of intestinal physiology in man. A high-performance liquid chromatographic (HPLC) method has been developed for the determination of PEG 600 in human urine, which includes a pre-column derivatisation step. The dibenzoate derivatives of PEG 600 can be quantitatively prepared, and this, coupled with ultraviolet detection at 230 nm, has greatly improved the limit of detection for the determination of PEGs by HPLC. A suitable extraction procedure has also been developed which enabled PEG levels in urine to be monitored with much greater sensitivity than any previously reported method.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) are chromatographed on a column packed with Spherosil XOA 600 (5 μm) using a 7:3 (v/v) mixture of diisopropyl ether—isooctane (1:1, v/v) + 0.2% triethylamine and diisopropyl ether—methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. < 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

High-performance liquid chromatographic determination of finasteride in human plasma using direct injection with column switching

A fully automated column-switching high-performance liquid chromatographic (HPLC) method was developed for the quantification of finasteride [N-(1,1-dimethylethyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide] in human plasma. Plasma samples were diluted with an equal volume of ethylene glycol-water (40:60, v/v), then the diluted sample (150 μl) was injected into the HPLC system without clean-up. The analyte was retained on a pretreatment column, whereas plasma proteins and other endogenous components were washed out to waste. The analyte was transferred to the analytical column in the heart-cut mode and then detected at 210 nm. A quantification limit of 1 ng/ml was attained. There was a linear relationship between peak height and drug concentration in plasma in the range 1–50 ng/ml. This method was validated and applied to the assay of plasma samples to characterize pharmacokinetic parameters in clinical studies.