Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Evaluation of online extraction/mass spectrometry for in vivo cassette analysis

An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC-MS software (Masslynx 3.5, Waters Corporation, Beverly, MA). Various analytical conditions such as extraction column, mobile phases, run time and wash solvent were optimized to establish an analytical method that was simple, easy to set up and generic. Sample preparation effort was minimal, which included dilution of plasma with water and centrifugation conducted in 96-well plate format. The system was used to analyze in vivo plasma samples from rat n-in-one cassette dosing studies. Concentration and pharmacokinetic (PK) data obtained from the online extraction method were comparable with data obtained from the protein precipitation extraction method. Overall, the simple, robust online extraction system provides cost savings by minimizing sample preparation and method development time. The system was used to analyze compounds from different structural classes. These studies suggest that calculated lipophilicity of a compound can be used as a tool for pre-selection of extraction column, which would save method development time for early discovery studies.     

Determination of remifentanil in human plasma by liquid chromatography-tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation

Remifentanil is a synthetic short-acting opioid with a short half-life that is being used during anaesthesia of small children. In this work an LC-MS/MS method for remifentanil quantification in 20 μL volume of human plasma was developed and validated in connection with a clinical study on neonatal children. Sample preparation was performed with micro extraction in packed syringe (MEPS), which is a miniaturization of solid phase extraction. For this method a mixed phase sorbent M1 (C8, cation exchange), and a protocol for basic compound extraction was followed. Remifentanil-(13)C(6) was used as internal standard. For chromatographic separation, a C18 analytical column with gradient elution was used with mobile phase consisting of aqueous 0.1% formic acid and methanol. The total analysis time was 5.0 min and the measuring range was between 0.05 and 50 ng/mL. Precision and accuracy were with acceptance criteria of ±15%. Plasma samples were stable for 5 weeks at -20°C and for 4h at room temperature while 50% was lost after 24h. This method was successfully applied for remifentanil determination in clinical samples and results agreed with a reference method. With this method using MEPS, a low limit of quantification and much reduced sample volume was obtained as compared with previous methods.     

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.     

High-throughput solid-phase extraction for the determination of cimetidine in human plasma

For the implementation and validation of an automated `high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C₁₈ sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.     

Solid-phase extraction on Styrosorb cartridges as a sample pretreatment method in the stereoselective analysis of propranolol in human serum

Sample pretreatment using solid-phase extraction (SPE) on cartridges filled with small-particle Styrosorb porous polystyrene-based sorbent has been used in the analysis of propranolol enantiomers in human serum by high-performance liquid chromatography (HPLC) with fluorescent detection. SPE on Sep-Pak C₁₈ cartridges was used as a reference pretreatment method. The propranolol content of the samples was determined by achiral normal-phase HPLC and the enantiomeric ratio of propranolol (S/R) was then determined by chiral HPLC on a column with silica-bonded cellulose-tris(3,5-dimethylphenyl carbamate). Recoveries of propranolol from serum using SPE on Styrosorb and C₁₈ phases were 97±5% and 96±5%, respectively. Detection and quantification limits for propranolol enantiomers were 4 and 7 ng/ml, respectively.     

Liquid chromatographic determination of oxcarbazepine and its metabolites in plasma of epileptic patients after solid-phase extraction

A method based on high-performance liquid chromatography with UV detection in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of the antiepileptic drug oxcarbazepine and its main metabolites in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective solid-phase extraction procedure using hydrophilic-lipophilic balance cartridges. The separation was obtained on a reversed-phase column (C(18), 150x4.6 mm I.D., 5 micrometer) using a phosphate buffer-acetonitrile-methanol-triethylamine mixture (final apparent pH* 3.5) as the mobile phase. Under these chromatographic conditions, oxcarbazepine and its metabolites 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and the internal standard are baseline separated in less than 9 min. The extraction yield values were >94% for all analytes and the precision, expressed by the RSD%, was in the low percentage range. For the entire method, including sample pre-treatment and HPLC determination, the linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.995; the limit of quantitation was 15 ng ml(-1). The method was applied to plasma samples from patients undergoing chronic treatment with oxcarbazepine, both in monotherapy and in polytherapy. Based on the analytical parameters precision, accuracy, limit of quantitation and analysis time the method is suitable for routine application in therapeutic drug monitoring.     

High-performance liquid chromatographic determination of lamivudine in human serum using liquid-liquid extraction; application to pharmacokinetic studies

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.     

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using high-performance liquid chromatography

We have developed an assay for the simultaneous determination of methotrexate (MTX) and its main metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N¹⁰-methylpteroic acid (DAMPA) in plasma, urine and saliva meeting the requirement of rapidity for routine use in high-dose MTX therapy and the requirement of sensitivity for its potential use in therapeutic drug monitoring in low-dose MTX therapy. Sample preparation is based on solid-phase extraction using C₈ Isolute cartridges. Chromatographic separation was achieved with a reversed-phase column (C₁₈), and quantitation by subsequent exposure to UV light of 254 nm, which converted MTX and its two metabolites by photolytic oxidation to fluorescent products. The recoveries of MTX, 7-OHMTX and DAMPA from plasma at 100 nmol/1 were 85.8, 91.1 and 102.3%, respectively. The limits of detection for MTX, 7-OHMTX and DAMPA in plasma and saliva were 0.1 nmol/1. In urine the limit of detection was 10 nmol/1 for all compounds. The limits of quantitation in plasma and saliva were 0.5 nmol/1 for all compounds.     

Development and validation of a fast and sensitive chromatographic assay for all-trans-retinol and tocopherols in human serum and plasma using liquid-liquid extraction

A sensitive HPLC assay for all-trans-retinol, alpha-tocopherol, and gamma-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol-chloroform mixture (3:1, v/v) without I.S. addition. After removal of the precipitated protein, 20 microl aliquots of the supernatant (equivalent to 6.7 microl of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C(18) S3 ODS2 column with a methanol-water step gradient (97:3 to 100) at 1.0 ml/min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for alpha-tocopherol and 250 ng for gamma- and delta-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C₁₈ column using a mobile phase consisting of water–acetonitrile–1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 μl of plasma, the standard curve was linear from 0.05 to 10.0 μg/ml.     

Liquid—liquid extraction and high-performance liquid chromatography for the determination of a novel antidysrhythmic agent (UK-68,798) in human urine

A routine method for the determination of a novel class III antidysrhythmic agent, 1-(4-methane-sulphonamidophenoxy)-2-[N-(4-methanesulponamidophenethyl)-N-methylamino]ethane, in human urine has been developed. The method involves solvent extraction followed by high-performance liquid chromatography on an unmodified silica column with ultraviolet detection. Despite a low recovery of drug through the three-stage extraction procedure a reliable assay with high precision (coefficient of variation less than 6%) and a limit of determination of 2.5 ng/ml was achieved. The method has been applied to the analysis of samples following single oral and intravenous doses of 1–12.5 μg/kg of the drug to human volunteers.     

A comparison of heavy liquid floatation and microwave digestion techniques for the extraction of fossil phytoliths from sediments

This paper reports the results of a comparative analysis of two techniques used for the extraction of fossil phytoliths from sediments. The first is a standard heavy liquid floatation extraction technique that has been successfully used and proven to provide reliable phytolith assemblage data. A second technique using a Perkin-Elmer Multiwave Microwave Sample Preparation System is introduced, and the results are compared to those of the conventional technique. Results for both techniques, as well as the advantages and disadvantages of their use are discussed. This study provides an introduction to an alternative technique for the extraction of fossil phytoliths and possibly starch grains from sediments. The microwave technique is fast, inexpensive, omits the need for heavy liquid floatation and most importantly results in the appropriation of interpretable and replicable fossil phytolith assemblage data.     

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensivity of 0.005 μg/ml for naproxen, and 0.05 μg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefanamic acid as the quantitation limit in human urine using indomethacin as an internal standard.     

High-performance liquid chromatographic-tandem mass spectrometric method for the determination of clemastine in human plasma

A highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method (HPLC-MS-MS) has been developed to quantitate clemastine in human plasma for the purpose of pharmacokinetic studies. Sample preparation was carried out by liquid-liquid extraction using deuterated clemastine as an internal standard. Chromatographic separation used a C18 reversed phase polymer column giving an extremely fast total run time of 2 min. The method was validated and used for the bioequivalence study of clemastine tablets in healthy male volunteers (n=28). The lower limit of detection proved to be 0.01 ng/ml for clemastine.     

Rapid quantitation of fluoxetine and norfluoxetine in serum by micro-disc solid-phase extraction with high-performance liquid chromatography-ultraviolet absorbance detection

A rapid, robust and sensitive method for the extraction and quantitative analysis of serum fluoxetine (FLX) and norfluoxetine (N-FLX) using a solid-phase extraction (SPE) column and high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed and validated. The sample clean-up step was performed by simple micro-disc mixed-mode (non-polar and strong cation exchange (SCX)) SPE cartridges. Separation of analytes and internal standard (IS) clomipramine (CLO) from endogenous matrix interference was achieved using a Waters Symmetry C(8) (150 mm x 2.1 mm i.d., 5 microm) reversed-phase narrow bore column. The relative retention times were 8.5, 9.6 and 10.5 min for FLX, N-FLX and CLO, respectively with a low isocratic flow rate of 0.3 ml/min. Chromatographic run time was completed in 15 min and peak area ratios of analytes to IS were used for regression analysis of the calibration curve. The latter was linear from 10 to 4000 nmol/l using 0.5 ml sample volume of serum. The average recovery was 95.5% for FLX and 96.9% for N-FLX. The lowest limit of quantitation (LLOQ) for serum FLX and N-FLX was 10 nmol/l (on-column amount of 200 fmol). The method described was used to analyse serum samples obtained from rats given chronic FLX treatment and to examine the relationship between steady state serum drug concentrations and neurochemical changes in several brain regions.     

Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2'-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC-MS/MS

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2'-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. (15)N(5)-8-OHdG ((15)N(5)-8-hydroxy-2'-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284→168 for 8-OHdG and m/z 289→173 for (15)N(5)-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.     

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 μm) using water–acetonitrile–methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 μg/ml. The limit of quantitation was 0.08 μg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.