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1.
Recently direct plasma injection LC/MS/MS technique has been increasingly used in pharmaceutical research and development due to the demand for higher throughput of sample analyses. In this work, two on-line extraction methods including high flow LC/MS/MS and high flow column switching LC/MS/MS were investigated. The evaluations were conducted and focused on their performances with respect to peak responses, separation efficiency, and signal to-noise ratio in a multiple-component LC/MS/MS assay. Two HPLC pumps were used-with one for high flow delivery and one for gradient elution. A CTC autosampler was used to inject plasma samples. High flow LC was achieved by the use of 4 ml/min flow rate on a 1×50 mm Waters Oasis column. A 2×100 mm YMC column was coupled via a column-switching valve. The extracted analytes were analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole MS/MS. As a rapid and simple procedure, vortex-mixing plasma and internal standard directly in sample vials completed sample preparation. The high flow column switching method (two-column system) provided sharper peak shape than the conventional high flow method. This effect increased analyte signal-to-noise ratio and sensitivity. Narrower peak width resulted in much better separation efficiency, which was required for multiple compound (N-in-1) analysis. A 2 mm I.D. column resulted in better peak shape and resolution than using a smaller I.D. column. The selected method achieved acceptable recoveries for most of the compounds tested, and it was successfully applied to a 10-in-1 pharmacokinetic (PK) study. The results showed that the dynamic range, lower limit of quantitation, assay accuracy and precision were acceptable for all compounds. Rapid sample preparation eliminated labor intensive and time consuming processes and improved productivity. This high throughput on-line extraction high flow column switching method has been proven particularly useful for multiple component analysis in PK studies.  相似文献   

2.
LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology™ Robot and BenchMate™ Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid—liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate™ performed buffer and organic solvent addition, liquid—liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2–100 ng ml−1 in plasma and 0. 1–20 μg ml−1 in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.  相似文献   

3.
We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement. On-line solid phase extraction, on the other hand, compared well with off-line solid phase extraction, using our LC extraction column design, as contamination of the extraction column was minimized by back flushing using the Gilson syringe pump. Finally, on-line solid phase extraction for support of Caco-2 permeability studies worked very well for both single components and mixtures as the matrix was much simpler, presenting fewer contamination problems.  相似文献   

4.
The aim of this work is to present a new concept, called on-line desorption of dried blood spots (on-line DBS), allowing the direct analysis of a dried blood spot coupled to liquid chromatography mass spectrometry device (LC/MS). The system is based on an inox cell which can receive a blood sample (10 μL) previously spotted on a filter paper. The cell is then integrated into LC/MS system where the analytes are desorbed out of the paper towards a column switching system ensuring the purification and separation of the compounds before their detection on a single quadrupole MS coupled to atmospheric pressure chemical ionisation (APCI) source. The described procedure implies that no pretreatment is necessary in spite the analysis is based on whole blood sample.  相似文献   

5.
LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.  相似文献   

6.
CGS 26214 is a racemic compound having cholesterol-lowering activity in rats, dogs, and monkeys. This compound has two equipotent chiral components CGS 28934(-) and CGS 28935(+). An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of the chiral components in human plasma following clinical doses of 1 mg or less. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compounds. First, the compounds were esters and susceptible to hydrolysis under experimental conditions. Second, a lower limit of quantitation (LLOQ) of 0.4 ng/ml was needed. Third, positive electrospray ionization of CGS 26214 did not yield sufficient sensitivity needed for the studies in humans. Consequently, LC/MS/MS conditions were optimized for negative ion mode of detection. Fourth, sample preparation steps proved to be critical in order to reduce the possibility of microbore chiral-HPLC column (100 x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix mediated electrospray ion suppression. Although the present method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, the method was successfully applied to generate plasma concentration-time profiles for human subjects after oral doses (0.9 mg) of the racemate as well as the optically pure isomers.  相似文献   

7.
To enhance to compatibility of the on-line coupling of liquid chromatography (LC) with mass spectrometry (MS) for the analysis of basic pharmaceuticals, the use of volatile mobile phase systems in combination with miniaturised LC was investigated. Multifactor analysis of variance (MANOVA) was used to evaluate the data obtained for the various variables (modifier, stationary phase, buffer, buffer pH and buffer concentration) on the resolution, peak symmetry and retention of four basic compounds analysed using LC columns with internal diameters (I.D.) of 0.3, 1.0 and 4.6 mm (conventional). Preliminary results obtained with the investigated micro and conventional columns showed similar behaviour with respect to ruggedness. The various investigated variables showed that miniaturisation by simply downscaling dimensions can result in varying selectivity and peak shapes for basic compounds. When comparing volatile mobile phases (containing ammonium acetate or ammonium citrate) and a conventional non-volatile mobile phase (containing sodium phosphate) under pH 3 conditions, similar separation performances were observed. In the present study, ammonium citrate as the buffering salt, a high buffer concentration and methanol as the modifier showed the best peak symmetry.  相似文献   

8.
A sensitive and specific direct-injection high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass-spectrometry (HPLC-APCI-MS-MS) method has been developed for the rapid identification and quantitation of seven tricyclic antidepressants-amitriptyline, nortriptyline, doxepin, dosulepin, dibenzepin, opipramol, and melitracen-in human plasma. After the addition of the internal standard lofepramine and dilution with 0.1% formic acid, plasma samples were injected into the LC-MS-MS system. Proteins and other large biomolecules were removed during an on-line sample cleanup using an Oasis extraction column (1 x 50 mm, ID, 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought in-line with the analytical column by automatic valve switching. Analytes were separated on a 5-microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm, ID) using a step gradient of acetonitrile-0.1% formic acid at a flow rate of 0.6 mL/min. The total analysis time was only 12 minutes per sample. The interday and intraday coefficients of variation for all compounds were 相似文献   

9.
For the quantitation of angiotensin II receptor antagonists (ARA-II) in human plasma, a method using liquid-chromatography (LC)-electrospray ionization tandem mass spectrometry (MS/MS) has been developed with respect to simple sample clean-up and investigation of ion suppression effects. For sample preparation, protein precipitation using zinc sulphate and methanol showed advantages in speed, recovery, and reproducibility over solid-phase extraction. A triple quadrupole mass spectrometer (Sciex API 365) with turbo ionspray source was used for detection of compounds with multireaction monitoring (MRM) of two transitions per compound. Suppression effects caused by endogenous matrix compounds were investigated by post-column infusion of analytes and LC analysis of precipitates of blank plasma samples and could be excluded. A validation was performed for the ARA-II drugs (valsartan, irbesartan, losartan and its active metabolite EXP 3174, eprosartan, candesartan, and telmisartan). The developed method showed good intra- and interday precision (<12% relative standard deviation) and accuracy (<11.5% bias) at different concentrations for all the studied compounds. The calculated lower limits of quantitation were between 7 and 13 ng/mL, and the compounds were stable during the analytical process. These rather expensive drugs against hypertension are prescribed with increasing numbers in Europe and the industrialized nations. Complications might arise from overdosage or metabolic disorders. However, drug monitoring is not usually performed. Because the therapeutic concentrations range from a few nanograms to hundreds of nanograms per milliliter for the different drugs, and they are not amenable to gas chromatography/MS analysis because of their high molecular weight and polarity, the LC-MS/MS method is the golden standard for therapeutic drug monitoring and for clinical and forensic toxicology of ARA-II drugs.  相似文献   

10.
A variety of analytical procedures have been described for the determination of cisplatin and its analogues in biological fluids (plasma, plasma ultrafiltrate and urine), as well as in solid tissues. This paper attempts to review those methods which have been most commonly used in practice. These analytical methods may be conveniently divided into non-selective methods which detect only the platinum metal and selective methods which are capable of detecting the intact compounds. The non-selective methods include X-ray fluorescence, proton induced X-ray emission, flameless atomic absorption (FAA) and high-performance liquid chromatography (HPLC). The latter method requires pre-column derivatization with diethyldithiocarbamate. The selective methods generally employ a fractionation step using HPLC followed by either on-line or off-line detection. Off-line detection by FAA requires the collection of fractions from the HPLC column and is somewhat tedious. On the other hand, sample preparation is minimal and biological fluids may be injected directly onto the column. The most sensitive HPLC methods for the determination of cisplatin and its analogues in biological fluids employ on-line electrochemical detection or post column derivatization with bisulphite.  相似文献   

11.
Wong P  Hsieh F  Pham R  James CA 《Bioanalysis》2012,4(1):89-93
p38 MAP kinase is a key enzyme in the proinflammatory response and a large number of compounds have been studied as potential therapeutic drugs. This review summarizes the bioanalytical methods used for the analysis of p38 MAP kinase inhibitors, with a special focus on sample preparation and chromatographic analysis. Biological sample extraction techniques utilized included protein precipitation, liquid-liquid extraction and SPE. Applications include determinations of compounds in a variety of biological fluids and tissues. Extracted samples are typically separated by reverse-phase LC and quantitated either by UV or MS/MS detection. The benefits and limitations of each sample preparation strategy are discussed. The importance of chromatographic separation to avoid matrix effect and interference from endogenous compounds or drug-related biotransformation products are also discussed herein.  相似文献   

12.
An on-line purification method for cationic compounds and their metabolites in rat bile was investigated using a column-switching technique. 8-Hydroxyquinoline and its glucuronide were used as test compounds. Bile samples were injected directly into the system and successful on-line extraction with high purification efficiency for analytes was achieved using two-dimensional extraction LC; that is, reversed-phase chromatography followed by cation-exchange chromatography. After removal of the endogenous component by extraction LC, chromatographic separation of the target analyte was performed on an analytical ODS column, followed by identification using UV detection. The quantitative ability of the method was evaluated on the basis of injection repeatability, linearity and accuracy. This novel method was also applied to LC/MS analysis in order to characterise the pharmacokinetics of propranolol in rats, and the metabolites were successfully identified.  相似文献   

13.
In previously reported applications of salting-out assisted liquid/liquid extraction (SALLE) with acetonitrile, only inorganic salts were evaluated and implemented as the salting-out reagents. A potential concern of the method for the subsequent LC-MS analysis of biological samples was that a portion of the added salt (typically of high concentration) might be extracted and affect the chromatography separation and ionization of chromatography effluents in a mass spectrometer. Here we report, for the first time, the use of a mass spectrometry friendly organic salt, ammonium acetate, as a salting-out reagent in SALLE with acetonitrile for the simultaneous quantitation of an Abbott investigational new drug ABT-869 and its hydrophilic metabolite in human plasma. The performance of SALLE with ammonium acetate was compared with that of a previously reported method with a conventional liquid/liquid extraction technique using a set of pooled incurred samples. The % differences of the measured concentrations for 24 samples from these two methods were found to be within acceptance criteria, demonstrating SALLE with ammonium acetate as a reliable sample preparation technique. The SALLE method is simple, fast (25 min/plate), easy for automation, free of drying down step, and environmentally friendly. SALLE with mass spectrometry friendly salts has been applied to regulated sample analysis of both hydrophilic and hydrophobic compounds. It is envisioned that SALLE with acetonitrile and ammonium acetate be a universal method for high throughput automated sample preparation for bioanalytical chemistry.  相似文献   

14.
The hydroalcoholic extract of Tinnevelli senna is widely used as a laxative phytomedicine. In order to improve the knowledge of the chemical composition of this extract, LC/MS and LC/MS(n) studies were performed, allowing the on-line identification of most of the known constituents, i. e., flavonoids, anthraquinones and the typical dianthronic sennosides. However, the identity of four compounds could not be ascertained on-line under the given LC/MS conditions. These substances were isolated and their structures elucidated as kaempferol, the naphthalene derivative tinnevellin 8-glucoside and two new carboxylated benzophenone glucosides.  相似文献   

15.
Health Canada reported recently that medical devices containing di(2-ethylhexyl) phthalate (DEHP) should not be used in the clinical treatment of infants, young boys, pregnant women, and nursing mothers. The risk assessment of DEHP released from PVC medical devices is an important issue for hospitalized patients. In this study, a simple, accurate, low-contamination and high-throughput analytical technique for the determination of DEHP in intravenous (IV) solution was developed using column-switching liquid chromatography/mass spectrometry (LC/MS) with an extraction mini-column. The sample preparation for on-line extraction involved simply mixing IV solution with internal standard as DEHP-d4 in LC glass vials. The IV fat emulsion drug sample cannot be analyzed directly, hence this sample spiked with DEHP-d4 solution was extracted by hexane and measured by column-switching LC/MS yielding an average recovery of 92.2% (C.V.=7.8%, n=5). A linear response was found for a variety of drugs tested within the validated range of 0.1 or 0.5–10 μg/ml with correlation coefficients (r) greater than 0.99. These results suggest that this method can assay background exposure to DEHP released from PVC medical devices in the patients. The method was applied to various IV solution samples to establish the first screening method for DEHP released from medical devices with respect to their safety.  相似文献   

16.
A semi-automated liquid-liquid extraction (LLE) technique for biological fluid sample preparation was introduced for the quantitation of four drugs in rat plasma. All liquid transferring during the sample preparation was automated using a Tomtec Quadra 96 Model 320 liquid handling robot, which processed up to 96 samples in parallel. The samples were either in 96-deep-well plate or tube-rack format. One plate of samples can be prepared in approximately 1.5 h, and the 96-well plate is directly compatible with the autosampler of an LC/MS system. Selection of organic solvents and recoveries are discussed. Also, precision, relative error, linearity and quantitation of the semi automated LLE method are estimated for four example drugs using LC/MS/MS with a multiple reaction monitoring (MRM) approach. The applicability of this method and future directions are evaluated.  相似文献   

17.
Fast and comprehensive qualitative and quantitative methods preferably by gas chromatography–mass spectrometry (GC–MS) and/or liquid chromatography–mass spectrometry (LC–MS) are needed to support the (differential) diagnosis of acute poisonings in emergency toxicology. One option is a commercially available qualitative screening solution based on LC–MSn (Bruker Daltonik Toxtyper?, TT). Identified and toxicologically relevant compounds should be quantified to assess severity of poisonings. The aim of the present study was to test the TT system for quantification simultaneous with the screening process in blood plasma exemplified for 22 relevant drugs and two active metabolites. A standard liquid–liquid extraction was used for sample work‐up followed by 1:5 dilution of the final extracts. They were analyzed using the TT system consisting of a Bruker amaZon speed ion trap and a Thermo Fisher Dionex Ultimate 3000 LC system. Plasma levels were assessed using full‐scan data and an electronically stored five‐point calibration. The calibration model was linear for the studied ranges and could be used for at least two months. The method was validated according to international guidelines. The acceptance criteria recommended for emergency toxicology for accuracy and precision were fulfilled for all tested compounds, but bromazepam, lorazepam, oxycodone, and prothipendyl could reliably be determined only above the therapeutic range. In conclusion, the presented procedure allowed the combination of a comprehensive LC–MSn screening with fast automated assessment of plasma levels for emergency toxicology of tested compounds.  相似文献   

18.
An analytical method based on a green approach is proposed for clinical analysis. The proposed procedure involves the reduction of the sample preparation steps, the amounts of reagents and organic solvents. This simple and sensitive method for the analysis of clinical drug and biomarkers in human plasma was developed using LC connected to tandem mass spectrometry (LC–MS/MS) with a nanospray ion source. In this study, the desired drug and proteins were separated on a 5 and 10 cm RP C18 nano-flow column. Undesired polar substances in human plasma were washed out by using ACN:1% FA = 20:80 (v/v) as the loading mobile phase for drug analysis and good linearity was attainable. Only a small volume of human plasma (10 μL) was utilized for the monitoring of drug plasma concentration and significant proteins under clinical studies. All the sample preparation procedures and the analytical scheme were at microliter level. This strategy would lower the consumption of reagents and organic solvents and make a contribution toward the goal of reduction of pollution from analytical methods in general.  相似文献   

19.
An on-line SPE-CE system is described for the determination of insulin derivatives in urine, serum and plasma. By combining techniques based on different separation mechanisms, in this case reversed-phase SPE and CE, a more selective sample clean-up is obtained. The described on-line SPE-CE procedure is able to desalt and clean biological samples, resulting in more repeatable electrophoretic results as well as a good linearity for urine, serum and plasma samples spiked with insulin derivatives, thus proving the elimination of detrimental effects caused by the sample matrix. The on-line SPE-CE system was linear for urine, serum and plasma samples spiked with insulin derivatives between 5 and 80 mg/l. The repeatability in migration time was below 1% relative standard deviation (R.S.D.). The repeatability of the peak was better (<2.4% R.S.D.) when no off-line precipitation reaction (<6.2% R.S.D.) was used, proving the beneficial characteristics of on-line sample pretreatment procedures over off-line sample pretreatment procedures which are prone to sample losses and contamination.  相似文献   

20.
The present paper describes the on-line extraction of drugs in plasma using a restricted-access media (RAM) column in a column-switching high performance liquid chromatography (HPLC) apparatus that was equipped with an on-line dilution system. The use of a six- to eightfold on-line dilution ratio for plasma samples resulted in almost 100% recovery of both acidic and basic drugs from plasma. It was found that the relationship between the on-line dilution times and drug recovery efficiencies from plasma was explained in terms of the binding constant between the drug and albumin. The applicability of column-switching HPLC with an on-line dilution system and the effectiveness of the extraction procedure were confirmed by a simultaneous determination of the basic compound, ER-118585, and its metabolites in canine plasma.  相似文献   

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