Quantitative analysis of clindamycin in human plasma by liquid chromatography/electrospray ionisation tandem mass spectrometry using d1-N-ethylclindamycin as internal standard

A new method for the quantitative analysis of clindamycin in human plasma by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) is presented. Recently published methods possess a disadvantage because of their use of internal standards with extraction and ionisation properties different from those of clindamycin. To avoid these problems, d(1)-N-ethylclindamycin was synthesised for use as internal standard by N-demethylation and subsequent d(1)-N-ethylation. Plasma sample preparation was done by an easy and rapid liquid-liquid extraction using ethyl acetate. The method was validated in the expected concentration range for a pharmacokinetic study. Calibration graphs were linear within the range 0.05-3.2 microg/mL plasma. Intra-day precision was between 0.90% (2.8 microg/mL) and 3.25% (0.05 microg/mL), inter-day variability was found to be between 1.33% (0.7 microg/mL) and 2.60% (0.05 microg/mL). Inter-day accuracy showed deviations between 0.4% (0.05 microg/mL) and -4.8% (0.2 microg/mL). The method is simple and robust, and has been applied to the batch analysis of clindamycin during a pharmacokinetic study.     

Determination of cefuroxime in human serum or plasma by liquid chromatography with electrospray tandem mass spectrometry

Cefuroxime is a second-generation cephalosporin used against different kinds of bacterial infections. To be able to optimize the dosing it is necessary to characterize the pharmacokinetics of cefuroxime which requires a selective and sensitive analytical method for cefuroxime in plasma or serum. A new rapid liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using cefotaxime as internal standard, was developed for analysis of cefuroxime in human serum. The work-up procedure consisted of protein precipitation with acetonitrile/cefotaxime, and after centrifugation the supernatant was dissolved in mobile phase. The sample was injected on a SB-CN column and the detection was performed using tandem mass spectrometry (MS/MS). The limit of quantification was determined to 0.025 microg/mL. The method was linear in the range 0.025-50 microg/mL with a coefficient of correlation >0.999. The limit of quantification and intra-day variability were found to be the same for plasma samples, which indicates that the method is valid for serum as well as plasma samples.     

Simple, sensitive and rapid liquid chromatography/atmospheric pressure chemical ionization mass spectrometric method for the quantitation of Ranolazine in rat plasma

A sensitive and specific method using liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) has been developed and validated for the identification and quantification of Ranolazine in rat plasma. A simple liquid-liquid extraction procedure was followed by injection of the extracts onto a C18 column with isocratic elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different batches of blank plasma. Linearity was established for the concentration range 0.02-10.0 microg/mL, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (relative standard deviation (RSD) %) was lower than 10%, and accuracy ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.01 microg/mL with 20 microL plasma. The proposed method enables the unambiguous identification and quantification of Ranolazine for pre-clinical and clinical studies.     

Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry

A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the limit of detection was 0.125 ng/mL. The excretion profile was determined after administration of 500 microg salmeterol (Serevent) to four standard-bred mares via a metered dose inhaler (MDI) with an Equinehaler adapter. Salmeterol was detected from 1 h until 12 h post-administration. Maximum urinary concentrations varied between 2.3 and 14.9 ng/mL.     

Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms

Plant diseases caused by plant pathogens substantially reduce crop production every year, resulting in massive economic losses throughout the world. Accurate detection and identification of plant pathogens is fundamental to plant pathogen diagnostics and, thus, plant disease management. Diagnostics and disease-management strategies require techniques to enable simultaneous detection and quantification of a wide range of pathogenic and non-pathogenic microorganisms. Over the past decade, rapid development of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for characterization of microorganisms has enabled substantially improved detection and identification of microorganisms. In the biological sciences, MALDI-TOF MS is used to analyze specific peptides or proteins directly desorbed from intact bacteria, fungal spores, nematodes, and other microorganisms. The ability to record biomarker ions, in a broad m/z range, which are unique to and representative of individual microorganisms, forms the basis of taxonomic identification of microorganisms by MALDI-TOF MS. Recent advances in mass spectrometry have initiated new research, i.e. analysis of more complex microbial communities. Such studies are just beginning but have great potential for elucidation not only of the interactions between microorganisms and their host plants but also those among different microbial taxa living in association with plants. There has been a recent effort by the mass spectrometry community to make data from large scale mass spectrometry experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MALDI-TOF MS as a universal technique for detection of plant pathogens and non-pathogens. The effects of experimental conditions are sufficiently understood, reproducible spectra can be obtained from computational database search, and microorganisms can be rapidly characterized by genus, species, or strain.     

BSE control: detection of gelatine-derived peptides in animal feed by mass spectrometry

The epidemic of bovine spongiform encephalopathy (BSE) is thought to have resulted from feeding scrapie-infected sheep to cattle. This has led to a ban of feeding animals with "processed animal protein"(PAP). We report a novel approach for the mass spectrometric detection of PAP contamination in animal feedstuffs by detecting gelatine, a derivative of the major animal protein collagen. A method was developed to hydrolyse gelatine standards with hydrochloric acid, followed by detection of the derived hydrolytic peptides at m/z 828, 915, 957 and 1044 by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS-MS). The marker peptides can be detected at concentrations of 100 ng ml(-1) gelatine in water with MALDI. The procedure was adapted to obtain a suitable peptide map of gelatine extracted from spiked feed. The ratio of signal area of the gelatine-derived peptide at m/z 1044 to the internal standard at m/z 556 is shown to relate to the total amount of gelatine present in the sample.     

A rapid and sensitive liquid chromatography/tandem mass spectrometry method for determination of docetaxel in human plasma

A novel, rapid and sensitive isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for quantification of docetaxel in human plasma with paclitaxel as internal standard. The high sensitivity and specificity of MS/MS detection enabled the use of a small volume of plasma (0.05 mL) and a simple liquid-liquid extraction procedure. Furthermore, a very short run-time (3 min) fulfilled the need for monitoring plasma levels of docetaxel from large-scale clinical studies. The calibration curve for docetaxel was linear over the range 5-1000 ng/mL with coefficients of correlation >0.999 using only 0.05 mL plasma. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 96 to 110%. The applicability of the method was demonstrated in a pharmacokinetic study of a 1-h infusion of docetaxel with dosages of 75 mg/m(2). Possible conjugated metabolites of docetaxel were not detected in patients' samples.     

Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies

A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4-hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), debrisoquine/5-hydroxydebrisoquine (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid-phase extraction. The chromatography was performed using a C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05-5 microg/mL for caffeine and paraxanthine, 0.02-2 microg/mL for tolbutamide, 0.1-20 microg/mL for 4-hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5-hydroxydebrisoquine, 5-2500 ng/mL for omeprazole and 5-hydroxyomeprazole, and 1-100 ng/mL for midazolam and 1'-hydroxymidazolam. The intra- and inter-day precision were 0.3-13.7% and 1.9-14.3%, respectively, and the accuracy ranged from 93.5-107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five-probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects.     

Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).     

Simultaneous determination of paracetamol and dextropropoxyphene in human plasma by liquid chromatography/tandem mass spectrometry: application to clinical bioequivalence studies

A liquid chromatography/mass spectrometry method for simultaneous determination of paracetamol and dextropropoxyphene in human plasma is described. Paracetamol and dextropropoxyphene, together with their internal standards (tolbutamide and pyrroliphene), were extracted from 0.5 mL of plasma using solid-phase extraction. The chromatography was performed using a Thermo Hypersil APS-2 Amino column (250 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile and 0.4% glacial acetic acid in water (20:80). The total run time was 6 min for each sample. The triple-quadrupole mass spectrometer was operated in both positive (for detection of dextropropoxyphene and its IS pyrroliphene) and negative (for detection of paracetamol and its IS tolbutamide) modes using a polarity-switching technique. Multiple reaction monitoring was used for quantification. The method was linear over the concentration range of 0.1-20 microg/mL for paracetamol and 0.5-80 ng/mL for dextropropoxyphene. The intra- and inter-day precision were less than 10%, and the accuracy ranged from 92.2-110.9%. The lower limits of quantification were 0.1 microg/mL for paracetamol and 0.5 ng/mL for dextropropoxyphene. The present method provides a robust, fast and sensitive analytical tool for both paracetamol and dextropropoxyphene, and has been successfully applied to a clinical bioequivalence study in 14 subjects.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented.     

Using high-concentration trypsin-immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature

We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 microg/microL) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly.     

Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis‐stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO‐mimetic peptides is under development from which peginesatide (also referred to as Hematide?), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase‐III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO‐mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL). Copyright © 2011 John Wiley & Sons, Ltd.     

Progress and possible applications of miniaturised separation techniques and elemental mass spectrometry for quantitative, heteroatom-tagged proteomics

The application of miniaturised separation techniques such as capillary LC, nano LC or capillary electrophoresis offers a number of advantages in terms of analytical performance, solvent consumption and the ability to analyse very small sample amounts. These features make them attractive for various bioanalytical tasks, in particular those related to the analysis of proteins and peptides. The skillful combination of such techniques with inductively coupled plasma mass spectrometry (ICP-MS) has recently permitted the design of combined analytical approaches utilising either elemental or molecule-specific detection techniques such as electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry in a highly complementary manner for, as an example, proteomics-orientated research (heteroatom-tagged proteomics). Such hybrid approaches are, in particular, providing promising new options for the fast screening of complex samples for specific metal-containing or—more generally speaking—heteroatom-containing biomolecules, as well as the accurate absolute quantification of biomolecules, which is still an unsolved problem in bioanalysis. Here, progress in as well as the potential and the special requirements of hyphenating miniaturised separation techniques with ICP-MS are reviewed and critically discussed. In addition, selected applications are highlighted to indicate current and possible future trends within this emerging area of research.     

Rapid approach to the quantitative determination of topiramate (2, 3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate) in human plasma by liquid-liquid extraction and flow-injection negative-ion electrospray mass spectrometry.

Topiramate, a sulfamate-substituted monosaccharide (2,3:4, 5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate), is a new antiepileptic drug, which has been approved for adjunctive therapy in adult patients with partial-onset seizures. Liquid-liquid extraction followed by flow-injection negative-ion electrospray mass spectrometry was evaluated as a means for the quantitative analysis of Topiramate in human plasma. Prednisone (1,4-pregnadiene-17-alpha, 21-diol-3,11,20-trione [15 microg/mL]) was used as the internal standard because its solubility and molecular weight are similar to those of Topiramate. Calibration curves for Topiramate were linear over a range of 1 to 30 microg/mL plasma (signal-to-noise ratio >4) and were highly reliable (r(2) = 0.994). This approach offers several advantages: (i) the extraction of Topiramate from human plasma using chloroform is simple and reproducible; (ii) the quantitative determination of Topiramate, in the presence of an internal standard, by flow-injection negative-ion electrospray mass spectrometry with selected-ion recording, is rapid and accurate and does not require chromatographic separation; (iii) the assay possesses adequate sensitivity (2-25 microg/mL) for the quantitative analysis of Topiramate in plasma from patients.     

Determination of alpha-tocopherol in infant foods by liquid chromatography combined with atmospheric pressure chemical ionisation mass spectrometry

A novel, sensitive and specific method for the quantification of alpha-tocopherol in two infant foods (milk and cereals) using liquid chromatography on-line with positive atmospheric pressure chemical ionisation mass spectrometry detection (LC/APCI-MS) has been developed. The samples were first saponified in order to eliminate fats and to transform tocopherol esters into free tocopherol, followed up by a liquid-liquid extraction of the analyte in petroleum benzine/diisopropyl ether (75:25, v/v) prior to injection onto the LC system. For the quantification, deuterium-labelled tocopherol was used as internal standard and the samples were monitored in selected ion monitoring (SIM) mode. Calibration curves between 1-40 microg/mL of alpha-tocopherol showed a good linear correlation (r(2) = 0.99994), and the detection limit was determined to be 2.5 ng/mL. The within-day and between-day precision were determined for several dietetic infant formulae and certified reference samples, and found to be below 3.5%. The accuracy determined on a Nestlé reference sample (milk powder) was calculated to be 115.2 +/- 1.2%, which confirms the robustness of the proposed method. This study shows that single quadrupole LC/MS can be applied for the quantification of vitamins in food and the method offers better sensitivity and selectivity than traditional method such as LC-UV. This would simplify the preparation of the food samples and consequently enhance the vitamin analysis throughput in the food area.     

Development and validation of a reversed-phase liquid chromatographic method for separation and simultaneous determination of COX-2 inhibitors in pharmaceuticals and its application to biological fluids

An isocratic reversed-phase high-performance liquid chromatographic method has been developed for separation and simultaneous determination of COX-2 inhibitors, viz., celecoxib, rofecoxib, valdecoxib, nimesulide and nabumetone, using 4-chloro-2-nitroaniline as internal standard. Good chromatographic separation was achieved using a reversed-phase Inertsil C(18) column with mobile phase consisting of methanol and 0.05% aqueous glacial acetic acid (68:32 v/v) using photodiode array (PDA) detector at 230 nm. It was validated with respect to accuracy, precision, linearity, limit of detection and quantification. The linearity range was found to be 1.0--20 microg/mL and the percentage recoveries were between 97.55 and 100.14. The method is suitable not only for the estimation of active ingredients in pharmaceutical dosage forms but also in vitro estimations in human plasma. It is simple, rapid, selective and capable of detecting and determining COX-2 inhibitors with a detection limit of 0.127--1.040 microg/mL simultaneously.     

Rapid screening of polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography-tandem mass spectrometry

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.     

Simultaneous determination of new thioamphetamine designer drugs in plasma by capillary electrophoresis coupled with mass spectrometry

A simple method for the simultaneous identification and quantification of four 2,5-methylenedioxy derivatives of 4-thioamphetamine (ALEPH series) in plasma samples was developed. The method consists of solid-phase extraction (SPE) using a Bond Elut C(18) cartridge and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE/ESI-MS). The SPE method used required only simple steps and provided a clean extract from which identification of each drug was feasible, even at low concentrations. The method was validated according to international guidelines. The calibration curves were linear over the concentration range of 50 to 1000 ng/mL for all drugs with correlation coefficients that exceeded 0.998. The lower limits of detection of the drugs were 23-43 ng/mL. The absolute recoveries for the drugs were 64-92% and 75-96% at concentrations of 100 and 500 ng/mL, respectively. The validation data (precision, accuracy, and recovery) show the reproducibility and selectivity of the method. This clean and simple method allows the routine detection of designer drugs such as thioamphetamines which may become a serious problem in the control of illegal drugs.