Fast analysis using monolithic columns coupled with high-flow on-line extraction and electrospray mass spectrometric detection for the direct and simultaneous quantitation of multiple components in plasma

In this work, monolithic columns were used as a fast separation tool for multiple-component quantitative liquid chromatography-tandem mass spectrometry (LC-MS-MS) assays of drug candidates in biological fluids. A considerably reduced runtime was achieved while maintaining good chromatographic separations. This significantly improved separation speed demanded higher throughput on sample extraction. To this end, monolithic separations were coupled on-line with high-flow extraction, which allowed for the fast extraction and separation of samples containing multiple analytes. An evaluation of this system was performed using a mixture of fenfluramine, temazepam, oxazepam, and tamoxifen in plasma. A total cycle time of 1.2 min was achieved which included both sample extraction and subsequent monolithic column separation via column switching. A total of over 400 plasma samples were analyzed in less than 10 h. The sensitivity and responses were reproducible throughout the run. The system has been routinely used in the authors' laboratory for high-throughput quantitation of compounds in biological fluids in support of drug discovery programs. The assay for samples from a 9-in-1 dog pharmacokinetic study is shown as an example to demonstrate the capability of this system.     

Automated column-switching high-performance liquid chromatography method for the determination of 1-hydroxypyrene in human urine

An on-line sample treatment method to determine 1-hydroxypyrene (1-OHP), a metabolite of polycyclic aromatic hydrocarbons (PAHs), in human urine has been developed. The hydrolysed biological fluid was directly injected into the chromatographic system after only centrifugation. A miniature precolumn loop packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system was used for analyte enrichment. The analytes were non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure comprising two purification columns and an analytical column. Pre-treatment and analysis were performed within 2 and 20 min, respectively. Average 1-OHP recovery reached 99% in the 1–25 μg/l range of urine, and the quantitation limit was 20 ng/l for 100 μl of injected sample. A comparison with a more time-consuming off-line method was performed by analysing 120 urine samples of PAH-exposed and expected unexposed workers; the statistical treatment indicated that both methods are in agreement.     

Simultaneous determination of inosine, hypoxanthine, xanthine, and uric acid and the effect of metal chelators

We describe a sensitive, reproducible method for the simultaneous determination of the ATP catabolites inosine, hypoxanthine, xanthine, and uric acid in biological samples and organ perfusate using reverse-phase chromatography and multiwavelength detection at 254, 270, and 292 nm. Sample preparation includes precipitating proteins with perchloric acid, neutralizing the sample, passing the supernatant over a polyethyleneimine column, and analyzing the collected fractions by high-performance liquid chromatography. Addition of metal chelators to the perchloric acid resulted in increased values for xanthine, hypoxanthine, and uric acid. The method was sensitive (limit of detection, 0.08 nmol on column; S/N = 4) and linear over the range 0.5-30 microM. Precision and accuracy of the method were evaluated for lung tissue and lung perfusate. Coefficients of variation ranged from 2.8 to 6.1% for perfusate and from 1.7 to 12.6% for tissue. Recoveries for all compounds exceeded 90%. We applied this method to rat lung tissue, lung perfusate, and rat and human blood. Advantages of this method are simultaneous quantitation with excellent sensitivity of all compounds, simplified peak identification by using multiwavelength detection, and improved accuracy by preventing loss of compounds with metal chelators.     

LC-MS/MS quantitation of plasma progesterone in cattle

Quantitation of progesterone (P₄) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P₄ in plasma of cattle with no sample derivatization. The quantitation of plasma P₄ released from three nonbiodegradable, commercial, intravaginal P₄-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P₄ kinetics (P > 0.05) whereas results of P₄ quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P₄ limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P₄ quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies.     

A fast and simple assay for busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry using turbulent flow online extraction technology

Busulfan is used in myeloablative preparation regimens for hematopoietic bone marrow transplantation. Due to its narrow therapeutic range therapeutic drug monitoring of busulfan is recommended. In this study a fast and simple method for measuring busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed utilizing turbulent flow online extraction technology. Serum or plasma was mixed with acetonitrile containing d(8)-busulfan. After centrifugation the supernatant was injected onto a turbulent flow preparatory column then transferred to a C18 analytical column monitored by a tandem mass spectrometer set at positive electrospray ionization. The analytical cycle time was 4.0min. The method was linear from 0.15 to 41.90μmol/L with an accuracy of 87.9-103.0%. Inter- and intra-assay CVs across four concentration levels were 2.1-7.8%. No significant carryover or ion suppression was observed. No interference was observed from commercial control materials containing more than 100 compounds. Comparison with a well established LC-MS/MS method using patient specimens (n=45) showed a mean bias 1.3% with Deming regression of slope 1.02, intercept -0.02μmol/L, and a linear correlation coefficient 0.9883. The LC-MS/MS method coupled with turbulent flow online sample cleaning technology described here offers reliable busulfan quantitation in serum or plasma with minimum manual sample preparation and was fully validated for clinical use.     

Antitumor drugs possessing topoisomerase I inhibition: applicable separation methods

Separation methods for antitumor drugs capable of topoisomerase I inhibition were reviewed in this study. Camptothecin (CPT) its related analogues seemed to be promising anticancer drugs that exhibit topoisomerase I inhibition. This group of compounds contain a closed α-hydroxy-δ-lactone ring (lactone form) that can undergo reversible hydrolysis to form the open-ring form (carboxylate form). In vitro pharmacological study showed that the antitumor activity of the lactone form was higher than that of the carboxylate form. Thus a quantitative method to separate these two forms is important to evaluate the pharmacokinetics and pharmacodynamics of these compounds. Nevertheless, current separation methods are complicated by the pH-dependent instability of the lactone moiety. High-performance liquid chromatography (HPLC) coupled with fluorometric detection has been widely used for the quantitation of the drug as the intact lactone form or as the total lactone carboxylate forms in biological matrices. In this report we reviewed current applicable chromatographic techniques for further bioanalytical studies of CPT derivatives including sample preparations, HPLC columns, mobile phases and additives.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

Direct determination of tamoxifen and its four major metabolites in plasma using coupled column high-performance liquid chromatography

A rapid, rugged and fully automated method has been developed for the determination of tamoxifen and its major metabolites in plasma. The system is based upon an in-line extraction process combined with column switching to a coupled analytical column. The plasma sample is deproteinated by the addition of acetonitrile before injection onto a semi-permeable surface (SPS) cyano guard column (1.0 × 0.46 cm I.D.). After washing the guard column briefly with water, the sample is eluted with a mobile phase composed of 35% acetonitrile in 20 mM potassium phosphate buffer (pH 3). The eluent is directed through a cyano analytical column (25 × 0.46 cm I.D.) and a photochemical reactor where the analytes are converted to highly fluorescent phenanthrene derivatives. Tamoxifen, 4-hydroxytamoxifen, N-desdimethyltamoxifen, N-desmethyltamoxifen and tamoxifen-ol are eluted in that order at a flow-rate of 1.0 ml/min. The method has been validated for use in a clinical study utilizing tamoxifen in the treatment of recurrent cerebral astrocytomas.     

Determination of lansoprazole and five metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of lansoprazole, a new proton-pump inhibitor, and five of its metabolites in human plasma is described. Lansoprazole, its metabolites, and internal standard (omeprazole) were extracted into diethyl ether-methylene chloride and separation was obtained using a reversed-phase column under isocratic conditions. The method features monochromatic ultraviolet detection at 285 nm, and single extraction, single evaporation sample handling. The lower limit of quantitation, based on standards with acceptable coefficients of variation, was 10 ng/ml for all compounds. No endogenous compounds were found to interfere. This method has been demonstrated to be suitable for pharmacokinetic studies in humans.     

Sensitive analysis of [ -Pen]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [ -Pen^2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C₁₈ solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm×50 μm I.D. capillary column with borate buffer (25 mM, pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Poster Sessions AP13: Novel Techniques and Technologies. A simple and sensitive HPLC‐method for simultaneous monitoring of oxidative stress indices and monoamine metabolites

Studies of the antioxidant defense system and the monoamine metabolic pathways are often complicated by cumbersome analytical methods, which require separate and multistep extraction and chemical reaction procedures. Thus, measurements of multiple parameters are limited in relatively small biological samples. High performance liquid chromatography (HPLC) coupled with a Coulometric Multi‐Electrode Array System (CMEAS) provides us a convenient and most sensitive tool to measure low molecular weight, redox‐active compounds in biological sample. The deproteinized sample was analyzed on a HPLC coupled with a 16‐channel CMEAS, which incremented from 60 to 960 mV in 60 mV steps. Each sample was run on a single column (Meta‐250, 4.6 × 250 mm) under a 150‐minute complex gradient that ranged from 0% B (A: 1.1% pentane sulfonic acid) to 20% B (B: 0.1 m lithium acetate in mixture of methanol, acetonenitrile and isopropanol), with a flow rate of 0.5 mL/min. We have developed an automated procedure to simultaneously measure various antioxidant, oxidative stress marker, and monoamine metabolites in a single column with binary gradient. No other chemical reactions are necessary. In order to reduce the running time and yet achieve a reproducible retention time by the autosampler injection, our gradient elution profile was modified to produce a shorter equilibration time and to compensate for the initial contamination of mobile phase B following the first injection. Without the use of two columns in series and peak suppresser/gradient mixer, we have simplified the previously published method to measure over 20 different antioxidants, oxidative stress markers and monoamine metabolites simultaneously in biological samples.     

Determination of a new antibacterial agent (Ro 23-9424) by multidimensional high-performance liquid chromatography with ultraviolet detection and direct plasma injection

Analysis of a new antibacterial agent, Ro 23-9424 (I), in plasma has been complicated by the fact that its metabolite, fleroxacin (II), is formed not only in vivo, but also nonenzymatically by the hydrolysis of the ester bond of I. In order to minimize sample preparation time and possible hydrolysis during sample preparation, a high-performance liquid chromatographic procedure was developed which features direct injection of plasma and multidimensional chromatography. The first dimension size-exclusion separation allows plasma proteins to elute with the column void volume. The second dimension reversed-phase column provides a high-resolution separation dependent upon the hydrophobicity of the sample species. With a 5-μl injection, the limit of quantitation of the method is 0.35 μg/ml for I and 0.27 μg/ml for II. The method was used to determine steady state plasma vs. time profiles for I and II from 750 mg i.v. doses of I administered twice daily.     

High-performance liquid chromatography coupled with tandem mass spectrometry for the detection of amyloid Beta peptide related with Alzheimer's disease.

Recent studies show that quantitative and qualitative differences in amyloid beta (Abeta ) peptides may be implicated in the development of Alzheimer's disease. New evidence seems to support the existence of a dynamic equilibrium between Abeta peptide in the brain and peripheral blood circulation. The quantitation of Abeta in the blood may allow the development of the potential value of Abeta peptides as a biomarker in the development of Alzheimer's disease. In this communication, quantitation of Abeta peptides using high-performance liquid chromatography coupled with tandem mass spectrometry in a linear ion trap mode is presented. RP-HPLC was performed using a Waters Xterra MS C8 column (3.0 mm x 150 mm). Abeta(1-40) peptide was eluted using a gradient elution program. Eluate from the RP-HPLC column was split to both the UV detector and electrospray ionization MS source. The product ion scan was performed in a linear ion trap mode utilizing the transition of a multiply charged molecular ion of Abeta(1-40) to a singly charged product ion. The detection limit of 31.25 ng in column load using a 3.0-mm-diameter conventional C8 column was achieved. The Abeta(1-40) standard calibration curves show excellent linearity from 34 ng to 2500 ng Abeta(1-40) of column sample load. The product ion scan enhances sensitivity 10 times compared with the best previously achieved by a single-quadrupole instrument in the selective ion monitoring mode. Moreover, the product ion scan of Abeta(1-40) provides superior selectivity and specificity, which is very important in the quantitation of Abeta(1-40) in a complex biological matrix.     

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10 000 pg/ml. Sample extraction (efficiencies >86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantited using post-column UV-photochemical cyclization coupled with fluoremetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Sensitive quantification of chosen drugs by reversed-phase chromatography with electrochemical detection at a glassy carbon electrode

Reversed-phase-high-performance liquid chromatographic method with electrochemical detection has proven to be a highly sensitive and selective method for determination of trace components in complex biological samples, and the electrochemical detector becomes an important alternative tool to ultraviolet and fluorescence detectors. A rapid and sensitive method for the accurate determination of metoclopramide, hydrochlorothiazide, imipramine and diclofenac in serum or plasma samples is described. The method is based on liquid-liquid extraction. The compounds were separated on C-18 column as stationary phase with a different binary mixture as mobile phase. Proposed method was validated with respect to specificity, linearity range, limit of detection and quantitation, precision, accuracy and successfully applied in a pharmacokinetic studies.     

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.     

Determination of lorazepam in plasma and urine as trimethylsilyl derivative using gas chromatography–tandem mass spectrometry

A procedure based on gas chromatography–tandem mass spectrometry for identification and quantitation of lorazepam in plasma and urine is presented. The analyte was extracted from biological fluids under alkaline conditions using solid-phase extraction with an Extrelut-1 column in the presence of oxazepam-d₅ as the internal standard. Both compounds were then converted to their trimethylsilyl derivatives and the reaction products were identified and quantitated by gas chromatography–tandem mass spectrometry using the product ions of the two compounds (m/z 341, 306 and 267 for lorazepam derivative and m/z 346, 309 and 271 for oxazepam-d₅ derivative) formed from the parent ions by collision-induced dissociation in the ion trap spectrometer. Limit of quantitation was 0.1 ng/ml. This method was validated for urine and plasma samples of individuals in treatment with the drug.     

A modified method for the determination of tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine by solid phase extraction using a molecularly imprinted polymer and liquid chromatography tandem mass spectrometry

The work described in this paper represents an improvement over a previously published method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine by solid phase extraction on a molecularly imprinted polymer column coupled with HPLC and -MS/MS detection. The influence of ion suppression due to sample matrix effect was evaluated, and found to influence the response of NNAL. By changing the liquid chromatography conditions, the response for this method was enhanced approximately 25-fold through avoidance of ionization suppression that was found with a previously published method and sample throughput has been improved. The dynamic range of the assay extends from 20 to 2500 pg/mL with a mean r² > 0.998. The lower limit of quantitation for the assay was 20 pg/mL despite the use of an inherently lower sensitivity instrument. The method was validated according to current FDA guidelines for bioanalytical method validations.