Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C₁₈ column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E₁ = +0.25 V and E₂ = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.     

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 ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 μm octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

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.     

Simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography–tandem mass spectrometry

Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC–MS–MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 μm). The mobile phase consisted of methanol–50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode.

The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95±2% and 95±4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5–100 ng/ml concentration range for the two analytes (r²=0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2–4.7% for the metabolite.The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.     

Simultaneous determination of malachite green and its metabolite leucomalachite green in eel plasma using post-column oxidation

A rapid HPLC method with solid-phase extraction (SPE) clean-up for malachite green (MG) and leucomalachite green (LMG) in eel plasma was developed. MG and LMG were extracted with a buffered methanolic solution. The extract was subjected to aromatic sulphonic acid SPE. MG and LMG were eluted from the SPE column with methanol after a treatment with ammonia gas. The reconstituted eluate was analyzed on a Chromspher B column with acetonitrile-ion-pair buffer (ph 4.0) (6:4, v/v) as the mobile phase and detection at 610 nm after post column oxidation with PbO₂. The average recoveries for MG and LMG over the linear range of applicability (20–2500 ng/ml) were 82±1% and 83±1%, respectively. The limits of quantification were 5.0 μg/1 for MG and 0.9 μ/1 for LMG.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C₁₈ column (250 × 4.6 mm I.D.) using 10 mM phosphoric acid—acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 μg/ml and the detection limits were 0.06 μg/ml for indomethacin and 0.08 μg/ml for mefenamic acid, for 50μl plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

High-performance liquid chromatographic determination of spectinomycin in swine, calf and chicken plasma

A rapid clean-up procedure based on ion-pair solid-phase extraction (SPE) for the high-performance liquid chromatographic (HPLC) determination of spectinomycin in swine, calf and chicken plasma at a limit of detection of 50 ng/ml is described. After dilution with water and adjustment of the pH to approximately 5.6, the plasma is applied to a high-hydrophobic C₁₈ SPE column treated with sodium dioctylsulphosuccinate. Spectinomycin is eluted with methanol and derivatized with 2-naphthalene sulphonyl chloride prior to chromatography. The HPLC set-up consists of a dual-column system using two Chromspher silica columns and dichloromethane—acetonitrile—ethyl acetate—acetic acid, in different ratios, as mobile phases. Detection is performed at 250 nm. Quantification is carried out using external standards prepared in blank cleaned plasma. Mean recoveries were 83 ± 3% (n = 5), 93 ± 6% (n = 5) and 92 ± 6% (n = 6) for swine, calf and chicken plasma, respectively, at the 0.1 μg/ml level.     

Synthesis of highly water-soluble fibrate derivatives via BGLation

Three water-soluble fibrates (fenofibrate, bezafibrate and chlofibrate) conjugated with a symmetrically branched glyceryl trimer (BGL003) were synthesized, and an evaluation of the fenofibrate–BGL003 conjugate as a candidate for anti-hyperlipemia drug was carried out using rats. The water-solubility of the fenofibrate–BGL003 conjugate was several thousand times greater than that of the original fenofibrate. The lipid-lowering effects of the fenofibrate–BGL003 conjugate were as strong as those of the same grams of fenofibrate. The actual active species of fenofibrate, fenofibric acid, was detected in rats’ blood, but neither the fenofibrate–BGL003 conjugate nor fenofibrate was detected, probably due to enzymatic hydrolysis of the ester bond. The plasma concentration of fenofibric acid derived from the fenofibrate–BGL003 conjugate was five times higher than that derived from fenofibrate 4 h after administration.     

High-performance liquid chromatography–electrospray ionization mass spectrometry method for the measurement of moclobemide and two metabolites in plasma

A rapid and sensitive liquid chromatography–electrospray ionisation mass spectrometry (HPLC–ESI-MS) assay has been developed for the measurement of moclobemide and metabolites, Ro12-5637 and Ro12-8095, in human plasma. Sample preparation (0.5 ml plasma) involves solid-phase extraction using C₁₈ cartridges. A Nova-Pak phenyl column (Waters, 4 μm, 150×2 mm I.D.) was employed for analyte separation with a mixture of 0.2 M ammonium formate buffer, pH 3.57 and acetonitrile as the mobile phase. The within- and between-day precisions of the assay were <18% and the limit of quantification for all analytes was 0.01 μg/ml. The total run-time was 6 min. The method described was used to measure moclobemide, Ro12-5637 and Ro12-8095 in human plasma following an oral 300 mg dose.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

High-performance liquid chromatographic determination of peroxisomicine A1 (T-514) in genus Karwinskia

A chromatographic method was developed for the T-514 determination in Karwinskia leaves, stems and roots. A C₁₈ analytical column and a mobile phase consisting of methanol and McIlvaine buffer (pH 3) were used. T-514 was detected using a diode array detector and the chromatograms were recorded at 269 and 410 nm. A linear dependence of a peak area on the T-514 concentration (r=0.9991) was obtained in the range of 0.126–12.6 μg/ml. Limits of T-514 quantification (signal-to-noise ratio 10) in plant samples were 126 ng/ml at 410 nm and 28 ng/ml at 269 nm. T-514 was extracted from the plant material with ethyl acetate. Optimal extraction conditions were studied: number of extraction steps, volume of extracting agent and extraction time. The extracts were cleaned up using solid-phase extraction (SPE). SPE recoveries of 99.9% and 98.4% were achieved for the T-514 concentrations of 1.4 μg/ml and 0.26 μg/ml, respectively.     

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 determination of methoxsalen in plasma after liquid—solid extraction

An improved method suitable for the determination of 8-methoxypsoralen in the range 50–1500 ng/ml in the plasma of psoriatic patients undergoing PUVA (psoralens and long-wave ultraviolet light) therapy is proposed. A 5-ml aliquot of plasma containing sodium citrate as anticoagulant was centrifuged, griseofulvin was added as internal standard and the sample was denatured with acetonitrile. The supernatant was applied to C₁₈ cartridges and 8-methoxypsoralen was eluted with methanol. The evaporated eluate was reconstituted in the mobile phase for high-performance liquid chromatography (HPLC) and applied to the HPLC column: mobile phase, acetonitrile—0.01 M phosphoric acid (34:66); flow-rate, 1 ml/min; temperature, 40°C; column, Spherisorb 5 ODS, 100 mm × 4.6 mm I.D., 5 μm particle size; UV detection at 248 nm; detection limit, 15 ng/ml of plasma.     

Determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma by high-performance liquid chromatography

A selective high-performance liquid chromatographic method to assess either bezafibrate, ciprofibrate or fenofibric acid plasma levels is described. Drugs are extracted with diethyl ether, after acidification with HCl. An isocratic acetonitrile-0.02 M H₃PO₄ (55:45) mobile phase, a C₁₈ (5 μm) column and UV detection are used. The LOQ found was 0.25 μg/ml for the three fibrates. Intra- and inter-assay accuracy ranges were 90–107% and 82–111%; 96–115% and 94–107%; 94–114% and 94–126% for bezafibrate, ciprofibrate and fenofibric acid, respectively. Intra- and inter-assay precision (C.V.% ranges) were 1.72–3.06% and 2.66–7.67%; 1.88–4.64% and 0.62–2.99%; 1.26–4.69% and 3.56–7.17% for the three fibrates studied. Its sensitivity, accuracy and precision make it a useful tool for monitoring plasma levels of these drugs in a clinical setting and for research purposes.     

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.