Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene

A coupled column liquid chromatographic (LC–LC) method for high-speed analysis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t-MA from urine samples was obtained using a 3 μm C₁₈ column (50×4.6 mm I.D.) as the first column (C-1) and a 5 μm C₁₈ semi-permeable surface (SPS) column (150×4.6 mm I.D.) as the second column (C-2). The mobile phases applied consisted, respectively, of methanol–0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol–0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol–0.05% TFA in water (25:75, v/v) was used for cleaning C-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC–LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50–4000 ng/ml) showed excellent linearity (r>0.999). The method was validated using urine samples collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%.     

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.     

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.     

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.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

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.     

Determination of zolpidem in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 μl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C₁₈ column, 5 μm particle size with an isocratic mobile phase consisting of methanol–acetonitrile–26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.     

Measurement of cefuroxime in human bronchoalveolar lavage fluid by high-performance liquid chromatography after solid-phase extraction

A sensitive and selective method for the determination of cefuroxime in bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography (HPLC) with UV detection at 280 nm after solid-phase extraction with C₁₈ cartridges was developed. A Waters symmetry C₁₈ column was used and the mobile phase was acetonitrile-0.05 M ammonium phosphate buffer (pH 3.2) (15:85, v/v). The method enabled the determination of cefuroxime at concentrations below 100 ng/ml, with a linear calibration curve at concentrations of 5–100 ng/ml for 400 μl of BAL. The intra- and inter-assay coefficient of variations for 10, 40 and 80 ng/ml were between 5.3 and 8.9%. Analytical recoveries were between 92.7 and 106.2%. The detection limit was 1 ng/ml at a signal-to-noise ratio of 3:1 using 400 μl of BAL. The method was successfully used for the analysis of BAL fluid from patients after oral administration of 500 mg cefuroxime axetil twice daily.     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

Determination of concentrations of flecainide in human serum by high-performance liquid chromatography on a fluorocarbon-bonded silica gel column

An optimized method for the determination of flecainide in serum is presented. Extraction using a solid-phase C₁₈ column and chromatography on a stabilized fluorocarbon-bonded silica gel column effectively separate flecainide from an internal standard (a positional isomer of flecainide). The HPLC apparatus and conditions were as follows: analytical column, Fluofix 120N; sample solvent, 20 μl; column temperature, 40°C; detector, Shimadzu RF-5000 fluorescence spectrophotometer (excitation wavelength=300 nm, emission wavelength=370 nm); mobile phase, 0.06% phosphoric acid containing 0.1% tetra-n-butyl ammonium bromide–acetonitrile (75:25, v/v); flow-rate, 1.0 ml/min. The standard curves for flecainide were linear in the concentration range examined (10–2000 ng/ml). The regression equation was y=0.08+0.0078x (r=0.9998). The minimum detectable amount of flecainide was approximately 5 ng/ml. In the within-day study, the precision coefficients of variation were 2.66, 2.18, 2.54, 2.72, 2.88, 2.24, and 3.29% for the 10, 50, 100, 200, 500, 1000, and 1500 ng/ml standards, respectively. The absolute recovery rates of flecainide at each concentrations were 94–100%. The method described provides analytical sensitivity, specificity and reproducibility suitable for both biomedical research and therapeutic drug monitoring.     

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 nortriptyline in human serum by fully automated solid-phase extraction and on-line high-performance liquid chromatography in the presence of antipsychotic drugs

A fully automated on-line method for determination of nortriptyline in human serum was developed using an ASPEC XL (Gilson) solid-phase extraction apparatus in combination with high-performance liquid chromatography. Solid phase extraction was performed on cyanopropyl cartridges. HPLC was carried out using a C₁₈ column with a mobile phase of acetonitrile–0.01 M triethylamine (34:66 v/v) buffer, pH 3.0. UV detection was at 242 nm. The Inter-day CV% was <5%. Comparison with liquid–liquid extraction of serum from patients treated with nortriptyline showed good agreement. Studies of analytical interference from coadministered psychoactive drugs revealed that only imipramine and a methotrimeprazine metabolite interfered.     

Direct analysis of urinary trans,trans-muconic acid by coupled column liquid chromatography and spectrophotometric ultraviolet detection: method applicability to human urine

A coupled column liquid chromatographic (LC–LC) method for the direct analysis in human urine of the ring opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. The method was tested using urine samples collected from five refinery workers exposed to low concentrations of airborne benzene (0.2–0.5 ppm), and from non-exposed volunteers. The analytical columns used were of 50×4.6 mm I.D. packed with 3 μm p.s. Microspher C₁₈ material as the first column (C-1), and a 100×4.6 mm I.D. column packed with 3 μm p.s. Hypersil ODS material as the second one (C-2). The mobile phases applied consisted, respectively, of methanol–0.074% trifluoroacetic acid (TFA) in water (4:96, v/v) on C-1, and of methanol–0.074% TFA in water (10:90, v/v) on C-2. Under these conditions t,t-MA eluted 15 min after injection. The present method, coupling the LC–LC technique with UV detection at 264 nm, permits the quantitation of t,t-MA directly in urine at levels as low as 0.05 mg/l. The determination is performed with a sample throughput of 2 h⁻¹ requiring only pH adjustment and centrifugation of the sample. Calibration plots of standard additions of t,t-MA to pooled urine taken from five non-exposed subjects were linear (r>0.999) over a wide concentration range (0.05, 0.1, 0.5, 1.0, 2.0 mg/l). The precision of the method (RSD) was in the range of 0.5 to 3.8%, and the within-session repeatability on workers urine samples (levels 0.06, 0.1, 0.2, 1.0 mg/l) was in the range of 3 to 8%. The present method improves the applicability of routine t,t-MA analysis, where it is most desirable that a large number of biological samples can be processed automatically or with minimal human labour, at low cost, and with a convenient turn-around time.     

Quantitative analysis of human serum corticosterone by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

An original method based upon high-performance liquid chromatography coupled to electrospray ionization mass spectrometry has been developed for corticosterone (B) quantification in human serum. After extraction by diethyl ether using triamcinolone (T) as an internal standard, solutes are separated on a C₁₈ microbore column (250×1.0 mm, I.D.), using acetonitrile–water–formic acid (40:59.9:0.1, v/v/v) as the mobile phase (flow-rate 40 μl/min). Detection is performed on an API 1 single quadrupole mass spectrometer equipped with a ESI interface and operated in positive ionization mode. Corticosterone quantifications were realized by computing peak area ratios (B/T) of the serum extracts analyzed in SIM mode (m/z 347 and m/z 395 for B and T, respectively), and comparing them with the calibration curve (r=0.998).     

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C₁₈ Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C₁₈, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r≥0.992) and 200 ng/ml for the active metabolites (r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.     

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.     

Determination of clozapine and its metabolite, N-desmethylclozapine, in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single solvent extraction step high-performance liquid chromatographic method is described for quantitating clozapine and its metabolite, N-desmethylclozapine, in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–28.6 mM sodium acetate buffer, pH 2.6 (10:20:70, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of clozapine after an intravenous bolus dose (2.5 mg/kg).