Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite (III, H117/04) with the basic metabolites α-hydroxymetoprolol (II, H119/66), metoprolol (I) and guanoxan (IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 μl) using C₁₈ solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)–methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 μl) under vacuum, and aliquots (100 μl) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C₁₈ radial compression cartridge, 4 μm, 100×5 mm I.D.). Acetonitrile–methanol–TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear (r²≥0.996) within the concentration range of 0.25–40.0 mg/ml. Recoveries for all four analytes were greater than 76% (n=4). The assay method was validated with intra-day and inter-day variations less than 2.5%.     

Determination of naringin and naringenin in human plasma by high-performance liquid chromatography

An HPLC method for determining a flavonoid, naringin, and its metabolite, naringenin, in human plasma is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using genistin (for naringin) or daidzein (for naringenin) as an internal standard and solid-phase extraction using a Sep-Pak t C₁₈ cartridge. For the determination, HPLC was carried out using an Inertsil ODS-2 column (250x4.6 m I.D., 5 μm particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v; pH 7.1) for naringin and acetonitrile-0.1 M ammonium acetate solution-acetic acid (30:69:1, v/v; pH 4.9) for naringenin. The flow-rate was 1 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 280 nm for naringin and at 292 nm for naringenin. The detection limits on-column were about 0.2 ng for the two flavonoids.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Quantification of (+)-calanolide A, a novel and naturally occurring anti-HIV agent, by high-performance liquid chromatography in plasma from rat, dog and human

A HPLC method was validated for quantification of (+)-calanolide A (1), a novel anti-HIV agent, in rat, dog and human plasma. The synthetic intermediate (±)-12-oxocalanolide A (2) was found to be a suitable internal standard. Compounds were extracted from plasma using a solid-phase C₁₈ cartridge and quantified over the assay range of 12.5 to 800 ng/ml. The method was utilized to determine (+)-calanolide A pharmacokinetics in rats, dogs and humans. This is the first report of a validated HPLC assay for determination of (+)-calanolide A concentrations in rat and dog plasma as well as human plasma obtained from clinical trials. There was no evidence of in vivo epimerization of (+)-calanolide A to its inactive epimer (+)-calanolide B (3).     

Determination of rhoifolin and daidzin in human plasma by high-performance liquid chromatography

A method for determining flavonoids in human plasma is presented for application to pharmacokinetic studies of two flavonoids, rhoifolin and daidzin. Isocratic reversed-phase high-performance liquid chromatography (HPLC) was used with genistin as an internal standard and solid-phase extraction using a Sep-Pak C₁₈ cartridge. The mobile phases were acetonitrile—0.1 M ammonium acetate solution (20:80, v/v) for rhoifolin and methanol—0.1 M ammonium acetate solution (33:67, v/v) for daidzin. The detection limits on-column were 2 ng for rhoifolin and 0.5 ng for daidzin.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

Extraction and quantification of nicardipine in human plasma

A novel simple method of extraction, separation, identification and quantification of nicardipine in human plasma samples was completely studied. The human plasma samples were initially purified by solid-phase extraction (SPE) using a C₁₈ cartridge. The extracted samples were separated and nicardipine present in the samples was quantified by high-performance liquid chromatography (HPLC) on a reversed-phase C₁₈ column employing a mobile phase consisting of 60% (v/v) acetonitrile in 0.02 M NaH₂PO₄ with pH of 6.3 and a variable wavelength UV detector set at 254 nm. The recovery of nicardipine from plasma samples using selective SPE was 91±6.0% and had less interfering compounds in the HPLC analysis compared to the use of liquid–liquid (L/L) extraction. In the HPLC analysis, examining the effect of pH values of the mobile phase on the capacity factor (k′) of nicardipine revealed a method for selecting a critical k′ value of nicardipine to eliminate interfering peaks near the peak specific to the analyte. This method for quantification of nicardipine in human plasma samples was suitable for studying the pharmacokinetic profile of nicardipine administered as an intravenous bolus to cardiac surgical patients.     

Development of a high throughput 96-well plate sample preparation method for the determination of trileptal (oxcarbazepine) and its metabolites in human plasma

A high throughput preparation method for the determination of trileptal (oxcarbazepine, OXC) and its mono (MHD) and dihydroxy (DHD) metabolites in human plasma, using 96-well plate technology, has been developed and validated according to international regulatory requirements. Preparation of plasma samples (50 μl) containing the compounds to be analysed involved solid-phase extraction (SPE) on Empore C₁₈ 96-well SPE plates. Eluates from the plate were injected onto a reversed-phase column (Hypersil C₁₈, 3 μm) with UV detection at 210 nm. Detector response was linear over the ranges 0.2–10, 0.1–200 and 0.1–20 μmol/l, for OXC, MHD and DHD, respectively, with relative standard deviations from 1 to 10% and mean accuracies within 4% of the nominal values (number of standard curves=3 in duplicate). The limits of quantitation were 0.2, 0.1 and 0.1 μmol/l, respectively. The overall mean accuracies ranged from 96 to 106% and precision was in the range 4 to 11%. Cross validation indicated no significant difference between plasma concentrations obtained using the 96-well method and the previous method using a traditional SPE method with a 50 mg C₁₈ cartridge. About a threefold increase in sample throughput and a twofold decrease of plasma volume required for the assays, were the main advantages obtained from the previous method. The method was applied for the determination of 3000 plasma samples from clinical studies.     

Inhibition of 17β-hydroxysteroid dehydrogenase (17β-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists

Various naturally occurring steroids, synthetic steroid derivatives and non-steroidal hormone agonists and antagonists were assayed as inhibitors of human placental 17β-HSD activities. Microsomal 17β-HSD was inhibited by C₁₈ -,C₁₉- and C₂₁-steroids. Soluble 17β-HSD was highly specific for C₁₈-steroids. In contrast to the soluble activity, the microsomal enzyme also had a strong affinity for ethinylestradiol (K_I=0.3 μM) and danazol (K_I=0.6 μM); anabolic steroids and norethisterone were weaker inhibitors. Of the non-steroids tested only diethylstilbestrol and o-demethyl CI-680 were inhibitors and they showed a greater affinity for soluble 17β-HSD.K_I-values for estradiol-17β, (0.8 μM), progesterone (27.0 μM) and 20α-dihydroprogesterone (1.5 μM) were comparable to reported tissue levels of these compounds, consistent with a possible competition in vivo among naturally occurring C₁₈-, C₁₉-, and C₂₁-steroids for the active site of microsomal 17β-HSD.     

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C₈ SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C₈/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP^® guard column (20×4.6 mm) connected to a Prism-RP^® analytical column (150×4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH=4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (λ_ex=260 nm, λ_em=375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5–500 ng/ml for human plasma and human urine yielded a linear response (R²>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.     

Simultaneous determination of a potassium channel opener and its metabolite in rat plasma with column-switching liquid chromatography using atmospheric pressure chemical ionisation

A specific LC–MS assay was developed for simultaneous determination of Ro 31-7837 (I) and its metabolite Ro 31-6930 (II) in rat plasma, using on-line SPE by column-switching reversed-phase HPLC combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry for detection in the selected reaction monitoring mode. The method involved precipitation of plasma proteins with ethanol and automatic injection of a 1-ml aliquot of the supernatant onto a standard bore trapping column (LC-ABZ, 20×4.6 mm) for compound retention. Using the backflush mode, the analytes were transferred onto the analytical column (Kromasil C₁₈, 125×4.0 mm) for chromatographic separation and mass spectrometric detection. The mean precision and accuracy for I and II in the concentration range 0.25–100 ng/ml were found to be 3.7% and 101%, and 3.5% and 106%, respectively. The data were assessed from QC samples during the validation phase of the assay. The lower limit of quantification for both I and II was 0.25 ng/ml, using a 0.5-ml plasma aliquot. This LC–MS method provided the requisite specificity, sensitivity, accuracy and precision to assess the pharmacokinetics of the compounds in the rat.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Ultrasensitive determination of NE-100, a novel sigma ligand,in human plasma by liquid chromatography and electrospray ionization tandem mass spectrometry combined with a column-switching technique

For the highly sensitive and selective determination of NE-100, a novel sigma ligand, at levels of low picogram per milliliter of human plasma, a method with excellent reliability employing liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS–MS) combined with a column-switching technique has been developed. The method involves the use of a stable isotope labeled compound as the internal standard (I.S.), liquid–solid extraction of a plasma specimen with a C₈ cartridge, automated on-line clean-up on a short trapping column, subsequent separation on a micro-bore C₁₈ column and detection with ESI-MS–MS using m/z 356 ([M+H]⁺) as a precursor ion and m/z 105 as a product ion in a selected reaction monitoring mode. The detection and the quantification limits of NE-100 in plasma were 0.5 pg/ml with a signal-to-noise ratio (S/N) of 3 and 2.3 pg/ml, respectively, with an S/N of 21. The good linearity of the calibration graph was obtained in the range of 2.3∼907.0 pg/ml with excellent reliability. The developed method was applied to the determination of NE-100 in plasma obtained from the clinical trail.     

High-performance liquid chromatographic assay for the determination of the novel etoposide derivative dimethylaminoetoposide (NK611) and its metabolites in urine of cancer patients

A simple, reproducible and specific urine assay for the novel epipodophyllotoxin derivative dimethylaminoetoposide (NK611, I) its picro form (III), the N-demethyl metabolite (II) and its picro form (IV) is reported. The method involves the addition of Pr-NK611 as internal standard, chloroform extraction and HPLC separation on a Nova-Pak C₁₈ column with a mobile phase of acetonitrile-0.05 M KH₂PO₄ (pH 6.4) (23:77, v/v). UV detection was used with absorbance monitored at 205 nm and the limit of quantification was 100 ng/ml. The intra- and inter-day precisions were within the ranges 1.1–3.4% and 1.9–2.4% for all analytes and the accuracy was 101–107%. The extraction recovery was more than 88% for I, II and IV and more than 83% for III. The assay is applicable to the urinary monitoring of I–IV in clinical pharmacokinetic investigations.     

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C₁₈ column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.     

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C₈ analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.     

Stereoselective determination of R(−)- and S(+)-MK-571, a leukotriene D4 antagonist,in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(−)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D₄ antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine—acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine—pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r²>0.999) for concentrations of enantiomers of 1 from 0.05 μg/ml, the lowest quantitation limit, up to 2.5 μg/ml. Intra-day coefficients of variation at 0.05 μg/ml were 2.4% for the R(−)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(−)- and S(+)-1 were 2.6 and 3.6%, respectively. The utilit of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.