Bioanalysis of erythromycin 2'-ethylsuccinate in plasma using phase-system switching continuous-flow fast atom bombardment liquid chromatography—mass spectrometry

A specific method for the determination of erythromycin 2'-ethylsuccinate (EM-ES) in plasma is described. The method involves a liquid—liquid extraction procedure followed by the analysis of extracts using phase-system switching (PSS) continuous-flow fast atom bombardment (CF-FAB) liquid chromatography—mass spectrometry (LC—MS). In PSS EM-ES is enriched after analytical separation on a short trapping column, from which it is desorbed to the LC—MS interface. In this way, favourable mobile phases can be used for the LC separation and for the MS detection. Using the PSS approach a flow-rate reduction from 1.0 ml/min in the LC system to 15 μl/min going into the mass spectrometer was achieved without splitting. The determination limit for EM-ES was 0.1 μg/ml.     

Determination of diclofenac in plasma using a fully automated analytical system combining liquid—solid extraction with liquid chromatography

A fully automated analytical system based on liquid—solid extraction combined with column liquid chromatography is described for the determination of diclofenac in plasma. After addition of pH 5 buffer and the internal standard solution to the plasma sample, both sample preparation via a C₁₈ disposable extraction column and injection were performed by a Gilson ASPEC system. Diclofenac and the internal standard were separated on a reversed-phase column, using methanol—pH 7.2 phosphate buffer (56:44, v/v) as mobile phase at a flow-rate of 0.4 ml/min. The reproducibility and accuracy of the method were acceptable over the concentration range 31–3140 nmol/l in plasma.     

Monitoring of met-enkephalin in vivo with 5-min temporal resolution using microdialysis sampling and capillary liquid chromatography with electrochemical detection

A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 μl/min. Separations were performed using 7 cm×25 μm I.D. fused-silica capillary columns packed with 5 μm Alltima C₁₈ particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 μl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K⁺-induced depolarization conditions.     

Suspension trapping (STrap) sample preparation method for bottom‐up proteomics analysis

Despite recent developments in bottom‐up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS‐based protein extraction with rapid detergent removal, reactor‐type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack—this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow‐through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.     

Use of guanidine hydrochloride and ammonium sulfate in comprehensive in-line sorption enrichment of xenobiotics in biological fluids by high-performance liquid chromatography

A novel approach has been developed for direct injection of physiological fluids on an in-line extraction pre-column followed by column switching in order to introduce the adsorbed xenobiotic onto the analytical column. The physiological fluid is pre-treated with guanidinium solution in water (200 μl of fluid plus 300 μl of a reagent containing 8.05 M guanidinium and 1.02 M ammonium sulfate) in order to denature protein binding sites and to serve as a universal solvent for a divergent range of polar to non-polar xenobiotics in a hydrophilic medium. A 0.5 M ammonium sulfate solution (500 μl) is used as a pre- and post-flush reagent for the extraction pre-column (30 mm × 2.1 mm I.D.). The pre-flush reagent prepares the sorbent environment of the C₁₈ pre-column for the hydrophobic retention of analytes. The post-flush reagent flushes non-retained sample proteins and salts to waste prior to switching the pre-column in-line with the analytical column. Universal chromatographic conditions for the analytical phase allows elution of a range of polar to non-polar xenobiotics within 20 min from an end-capped C₈ silica anaytical column (250 mm × 4.6 mm I.D.). This is effected by a linear gradient from a binary system consisting of solvent A (0.05 M KH₂PO₄) and solvent B (acetonitrile—isopropanol, 80:20, v/v).     

High-performance anion-exchange chromatography of ultraviolet-absorbing constituents of human urine

A 100-μl urine sample was chromatographed on a column packed with a strongly basic macroreticular anion-exchange resin (Diaion CDR-10, 5– μm diameter with a nominal 35% cross linkage). The elution was performed with a linear acetate gradient from 0 to 6.0 M at an average flow-rate of 0.72 ml/min and at an average pressure of 104 kg/cm². The relative standard deviation of retention times and peak height was ± 4% or less. The properties of the macroreticular anion-exchange resin, the effect of the particle size, the pH of acetate buffers, and the effect of the flow-rate of the eluent on the separation were investigated. Thirty three components of urine were then resolved and named.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1–250 μg/ml using 12.5 μl of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C₁₈ column with isocratic elution with acetate buffer (25 mM, pH 4.5)—methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35°C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 μg/ml for BA and HA and 5.0 μg/ml for PAA and PA with a 20-μl injection.     

Gas chromatographic determination of zopiclone in plasma after solid-phase extraction

A gas chromatographic technique for determining zopiclone based on a solid-phase extraction procedure with C₁₈ cartridge for sample clean-up is presented. Quantification can be achieved with 1 ml of plasma. The method uses prazepam as internal standard. Zopiclone is separated on a 5% phenyl methyl silicone analytical column and detected with an electron-capture detector, which consequently allows a limit of quantitation of 2 μg/l. It is thus simple, rapid, sensitive and linear over the range 5–2000 μg/l.     

Determination of α-tocopherol in plasma and erythrocytes by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of α-tocopherol in plasma or erythrocytes with photodiode-array detection is described. Using this detector, information about the spectrum, absorption maxima and purity of the peak is obtained. Tocopherol was separated on a 5-μm Spherisorb ODS-2 column with methanol as element at a flow-rate of 1.0 ml/min. As little as 100 μl of plasma or 150 μl of erythrocytes can be used for accurate analysis with direct extraction without saponification. The speed, specificity, sensitivity and reproducibility of this technique make it particularly suitable for the routine determination of α-tocopherol in plasma or erythrocytes.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Determination of diazepam and its pharmacologically active metabolites in blood by bond elut™ column extraction and reversed-phase high-performance liquid chromatography

A rapid and quantitative analytical micro method for the determination of diazepam and its major pharmacologically active metabolites utilizing high-performance liquid chromatography (HPLC) is reported. The drug and its metabolites were extracted from 50–100 μl samples of whole blood, serum or plasma using Bond Elut™ C_15f column and quantitated by high-performance liquid chromatography, using Technicon Fast-LC-C-8 (RP 5 μm) bonded column and a mobile phase consisting of 53% methanol, 1% acetonitrile in KH₂PO₄ buffer and 10 μl/l triethylamine. Methyl nitrazepam and medazepam were used as internal and external standards, respectively. The extraction and recovery of diazepam and its major pharmacologically active metabolites, i.e., 3-hydroxydiazepam, desmethyldiazepam and oxazepam from blood were higher than 88% for all compounds. The minimum detection range of each compound was approximately 2.5 ng per 100-μl sample. This micro method of simultaneous quantitation of diazepam and its major pharmacologically active metabolites provides a valuable technique for the study of diazepam pharmacokinetics in a small animal model without disturbance of normal hemodynamics from excess blood loss, as well as in clinical evaluation of pediatric patients.     

Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay for the determination of sanfetrinem in human plasma

A rapid, selective and accurate high-performance liquid chromatography–tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 μl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C₁₈(2), 50×2.0 (5 μm) column with a mobile phase consisting of water–acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 μg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.     

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.     

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.     

Simultaneous on-line extraction and analysis of sirolimus (rapamycin) and ciclosporin in blood by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific semi-automated liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous quantification of sirolimus and ciclosporin in blood. Following a simple protein precipitation step, the supernatants were injected into the HPLC system and extracted on-line. After column switching, the analytes were backflushed from the extraction column onto the analytical narrow-bore column and eluted into the ESI-MS system. The assay was linear from 0.4 to 100 μg/l sirolimus and from 2 to 1500 μg/l ciclosporin. The mean recoveries of sirolimus and ciclosporin were 98 and 96%, respectively. The mean interday precision/accuracy was 8.6%/−4.8% for sirolimus and 9.3%/−2.9% for ciclosporin.     

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.