Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

Dual parallel mass spectrometry for lipid and vitamin D analysis

There are numerous options for mass spectrometric analysis of lipids, including different types of ionization, and a wide variety of experiments using different scan modes that can be conducted. Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) provide complementary types of information that are both desirable. However, the duty cycle of the mass spectrometer places limits on the number of experiments that can be performed, and instruments usually employ only one type of ionization at a time. This work describes the approaches we have used that employ two mass spectrometers in parallel or in a column-switching configuration that allows multiple ionization modes and types of experiments to be conducted simultaneously during a single chromatographic run. These data demonstrate how use of two systems can reduce or eliminate the need for repeat injections and repetitive experiments. Approaches are described that employ two mass spectrometers connected in parallel as detectors for a single chromatographic system (LC1/MS2) or that employ two liquid chromatographs and two mass spectrometers in a column-switching arrangement (LC2/MS2). Examples of LC1/MS2 analyses of triacylglycerols (TAGs), sphingolipids, and vitamin D are given, as well as an example of an LC2/MS2 experiment that is used to perform analysis of both polar and non-polar lipids in a total lipid extract.     

Tandem LC columns for the simultaneous retention of polar and nonpolar molecules in comprehensive metabolomics analysis

The tandem use of hydrophilic interaction LC columns with RP columns in series configuration has resulted in the retention of both polar and nonpolar components in complex biological samples (mouse serum) in a single analysis. This approach successfully coupled various columns with orthogonal separation characteristics, employed a single solvent gradient program compatible with the two columns and used ESI coupled to a TOF mass spectrometer for detection. Ion suppression, a common problem in ESI, was virtually eliminated for components eluting with apparent capacity factors >0.7. Retention time reproducibility with the tandem columns performed over three days with over 100 injections was comparable to that observed for single columns alone. This method was applied to the analysis of a pooled mouse serum sample and afforded highly reproducible data for up to 3000 mass spectral features. This approach was implemented with a conventional LC–MS system and should find broad applicability in the comprehensive analysis of complex mixtures containing a wide range of compound polarities.     

Direct simultaneous analysis of plasma samples for a drug discovery compound and its hydroxyl metabolite using mixed-function column liquid chromatography-tandem mass spectrometry

A polymer-coated mixed-function (PCMF) column was evaluated for direct plasma injection for the simultaneous determination of a drug candidate and its hydroxyl metabolite by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS-MS) in support of pharmacokinetic studies. Each diluted monkey plasma sample containing internal standard was directly injected on to the PCMF column for sample clean-up, enrichment and chromatographic separation. The proteins and macromolecules were first eluted from the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column were then eluted with a strong mobile phase using a gradient separation technique at a constant flow rate of 1.0 ml min(-1). When not diverted, the column effluent was connected either to the atmospheric pressure chemical ionization (APCI) source or the electrospray ionization (ESI) source as part of the mass spectrometer system used for quantification. The calibration curve was linear over the range 5-2500 ng ml(-1) for both analytes. The retention times for the analytes and the internal standard were both consistent and no column deterioration was observed for at least 500 injections. The recovery through the column and reproducibility of the dosed compound and its hydroxyl metabolite in monkey plasma samples were > 90% (RSD < 6%). The total analysis time was < 8 min per sample. The analytical results obtained by the proposed direct plasma injection method were in good agreement with those obtained by the conventional LC-MS-MS method.     

Exact mass measurement on an electrospray ionization time-of-flight mass spectrometer: error distribution and selective averaging

An automated, accurate and reliable way of acquiring and processing flow injection data for exact mass measurement using a bench-top electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Using Visual Basic programs, individual scans were selected objectively with restrictions on ion counts per second for both the compound of interest and the mass reference peaks. The selected "good scans" were then subjected to two different data-processing schemes ("combine-then-center" and "center-then-average"), and the results were compared at various ion count limit settings. It was found that, in general, the average of mass values from individual scans is more accurate than the centroid mass value of the combined (same) scans. In order to acquire a large number of good scans in one injection (to increase the sampling size for statistically valid averaging), an on-line dilution chamber was added to slow down the typically rapid mass chromatographic peak decay in flow-injection analysis. This simple addition worked well in automation without the need for manual sample dilution. In addition, by dissolving the reference compound directly into the mobile phase, manual syringe filling can be eliminated. Twenty-seven samples were analyzed with the new acquisition and process routines in positive electrospray ionization mode. For the best method found, the percentage of samples with RMS error less than 5 ppm was 100% with repetitive injection data (6 injections per sample), and 95% with single injection data. Afterwards, 31 other test samples were run (with MW ranging from 310 to 3493 Da, 21 samples in ESI+ and 10 in ESI- mode) and processed with similar parameters and 100% of them were mass-calculated to RMS error less than 5 ppm also.     

Parallel column liquid chromatography with a single multi-wavelength absorbance detector for enhanced selectivity using chemometric analysis

The selectivity of high performance liquid chromatography (HPLC) separations is increased using a parallel column configuration. In this system, an injected sample is first split between two HPLC columns that provide complementary separations. The effluent from the two columns is recombined prior to detection with a single multiwavelength absorbance detector. Complementary stationary phases are used so that each chemical component produces a detected concentration profile consisting of two peaks. A parallel column configuration, when coupled with multivariate detection, provides increased chemical selectivity relative to a single column configuration with the same multivariate detection. This enhanced selectivity is achieved by doubling the number of peaks in the chromatographic dimension while keeping the run time constant. Unlike traditional single column separation methodology, the parallel column system sacrifices chromatographic resolution while actually increasing the chemical selectivity, thus allowing chemometric data analysis methods to mathematically resolve the multivariate chromatographic data. The parallel column system can be used to reduce analysis times for partially resolved peaks and simplify initial method development as well as provide a more robust methodology if and when subsequent changes in the sample matrix occur (such as when new interferences show up in subsequent samples). Here, a mixture of common aromatic compounds were separated with this system and analyzed using the generalized rank annihilation method (GRAM). Analytes that were significantly overlapped on both stationary phases applied, ZirChrom PBD and CARB phases, when used in traditional single column format, were successfully quantified with a R.S.D.% of typically 2% when the same stationary phases were used in the parallel column format. These results indicate that a parallel column system should substantially improve the chemical selectivity and quantitative precision of the analysis relative to a single-column instrument.     

AutoScan: an automated workstation for rapid determination of mass and tandem mass spectrometry conditions for quantitative bioanalytical mass spectrometry

An automated flow injection analysis (FIA) mass spectrometry system (AutoScan) was developed to allow rapid unattended determination of optimal conditions during mass (ms) and tandem mass spectrometry (ms/ms) on new chemical entities (NCEs) arranged in 96-well plates. The 96-well plate is placed on the deck of a modified Gilson Multiprobe autosampler for injection into a PE Sciex API 2000 triple quadrupole mass spectrometer. A customized software interface is used to create the necessary scan experiments by associating each 96-well plate of NCEs to be scanned with an index file containing data on the identity of each analyte and its expected molecular weight. Analytes are injected four at a time into a custom injection manifold and conventional mass spectra are acquired in both polarities (+/-) using an alternating positive/negative Q1 scan function. The software determines the optimal polarity and definitive precursor ion for all analytes and uses the results to build the injection sequence for product ion scanning. The samples are automatically re-injected under MS/MS conditions, and product ion scans that loop among different collision energies are collected for each analyte. The resulting data are processed automatically and the optimal MS/MS transitions for each analyte are selected. A color-coded graphical interface facilitates data review. Any unusual ion transitions or transposition errors made during plate preparation are noted and corrected. Complete MS and MS/MS conditions are obtained for 96 compounds in about one hour and the resulting data are available for download as sample control injection sequence files.     

Moving from fast to ballistic gradient in liquid chromatography/tandem mass spectrometry pharmaceutical bioanalysis: Matrix effect and chromatographic evaluations

The paper describes the steps taken by the authors to move from a fast to a ballistic gradient in routine liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of plasma samples from pharmacokinetic (PK) profiling of new chemical entities. The reduction of column dimensions from 50 x 4.6 mm to 30 x 2.1 mm followed by optimization of chromatographic separation led to a decrease in the typical runtime from 5 (fast) to 2 min (ballistic) using an API4000 tandem mass spectrometer in Turbo Ionspray mode for detection. Three analytical standards representing typical molecular structures from our sample repository were used to spike plasma from four different species (rat, dog, human and mouse). Two different approaches were used to evaluate matrix effect: post-column infusion and comparison of the peak areas of neat standards and standards spiked after extraction into different pools of plasma; the influence of PEG400 as a typical dosing vehicle was also considered. Two different protein precipitation procedures were taken into account for sample extraction prior to injection. Peak shape, width and height, selectivity and sensitivity of the method were taken into account for chromatographic evaluation. The ballistic method was successfully cross-validated with the conventional fast gradient chromatographic assay.     

Full utilization of a mass spectrometer using on‐demand sharing with multiple LC units

The applicability of liquid chromatography–mass spectrometry (LC/MS) is often limited by throughput. The sharing of a mass spectrometer with multiple LCs significantly improves throughput; however, the reported systems have not been designed to fully utilize the MS duty cycle, and as a result to achieve maximum throughput. To fully utilize the mass spectrometer, the number of LC units that a MS will need to recruit is application dependent and could be significantly larger than the current commercial or published implementations. For the example of a single analyte, the number may approach the peak capacity to a first degree approximation. Here, the construction of a MS system that flexibly recruits any number of LC units demanded by the application is discussed, followed by the method to port a previously developed LC/MS method to the system to fully utilize a mass spectrometer. To demonstrate the performance and operation, a prototypical MS system of eight LC units was constructed. When 1‐min chromatographic separations were performed in parallel on the eight LCs of the system, the average LC/MS analysis time per sample was 10.5 s when applied to the analysis of samples in 384‐well plate format. This system has been successfully used to conduct large‐volume biochemical assays with the analysis of a variety of molecular entities in support of drug discovery efforts. Allowing the recruitment of the number of LC units appropriate for a given application, this system has the potential to be a plug‐and‐play system to fully utilize a mass spectrometer. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

Capillary supercritical fluid chromatography-mass spectrometry

Capillary supercritical fluid chromatography and its combination with mass spectrometry (SFC-MS) is an important analytical methodology for the analysis of thermally labile and high molecular weight compounds. The mass spectrometer provides sensitive and highly selective detection for the chromatographic effluent. The same physical-chemical properties of supercritical fluids that provide important chromatographic advantages are also important for the transport and gas phase introduction of analyte molecules into the mass spectrometer. The low mobile phase flow rates of small diameter (< 100 μm i.d.) capillary columns allow the total chromatographic effluent to be introduced with conventional, but often slightly modified, ion source configurations for quadrupole mass spectrometers with either electron impact (EI) or chemical ionization (CI) detection. The full range of CI reagents can also be used to obtain additional selectivity and complementary structural data. The instrumentation and operating parameters for capillary SFC-MS methods are described. The applicability and usefulness of various SFC-MS analysis methods are demonstrated using a variety of samples.     

Detecting and Identifying in vivo Metabolites of Brodimoprim via LC/ESI-MS with Data-dependent Scanning

The present article covers a simple approach to detect and subsequently identify in vivo metabolites of brodimoprim, using high performance liquid chromatography coupled to ion trap mass spectrometer（LC/ESI-MS）, which is based on a data-dependent acquisition of isotope ions and result verified by full scan mass spectrum. The distinguished advantage of data-dependent scan is rapidness because it requires minimum sample preparation, and all the necessary data can be obtained in one chromatographic run. In addition, it is highly sensitive and selective, allowing detection of trace metabolites even in the presence of complex biomatrix. As a result, four phase-Ⅰ（M1--M4） and four Phase-Ⅱ（M5--M8） metabolites of brodimoprim were identified in urine after the oral administration of hrodimoprim to Wistar rats. Their chemical structures were proposed based on the interpretation of their CID fragmentation characterizations and the metabolic pathway was exhibited in this article.     

Fast liquid chromatography coupled to electrospray tandem mass spectrometry peptide sequencing for cross-species protein identification

Using a parallel microcolumn switching liquid chromatography set-up coupled to a quadrupole time-of-flight mass spectrometer, a rapid liquid chromatography/mass spectrometric (LC/MS) protein identification method is presented. Without prior sample clean-up up to 300 protein digest samples a day can be processed. Using data-directed acquisition, up to 10 fragmentation analyses for each protein sample can be acquired in the same chromatographic run that can be used for database searching. Using internal peptide sequence information, protein databases and the various nucleic acid databases can both be queried for cross-species identification of the protein sample. The method was evaluated and put into force to generate data for a tobacco cell culture protein database.     

Zirconia-based column high-performance liquid chromatography/atmospheric pressure photoionization tandem mass spectrometric analyses of drug molecules in rat plasma

A method using zirconia-based column high-performance liquid chromatography (HPLC) interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer (MS/MS) was developed for the quantitative determination of new chemical entities in rat plasma in support of pharmacokinetics studies. The ionization suppression resulting from endogenous components of the biological matrices on the quantitative zirconia-based column HPLC/APPI-MS/MS method was investigated using the post-column infusion technique. The analytical results for 'rapid rat pharmacokinetics' for 12 drug discovery compounds, obtained by both silica-based phase (S-phase) and zirconia-based phase (Z-phase) chromatographic separation, are in good agreement in terms of accuracy. The application of a Z-phase column for high-temperature fast HPLC/MS/MS methods was explored to reduce the analysis time from 3 min to 30 s for column temperatures of 25-110 degrees C, respectively. The chromatographic retention times and peak responses of all analytes were found to be reproducible under high-temperature conditions following 100 continuous injections, with %CV less than 0.4 and 5, respectively.     

Determination of polycyclic aromatic hydrocarbon metabolites in milk by a quick,easy, cheap,effective, rugged and safe extraction and capillary electrophoresis

We describe the first application of a quick, easy, cheap, effective, rugged and safe extraction technique to the CZE analysis of monohydroxylated metabolites of polycyclic aromatic hydrocarbons in milk. Complete resolution of 2‐hydroxyfluorenene, 1‐hydroxynaphthalene, 2‐hydroxynaphthalene, 3‐hydroxyphenanthrene, and 9‐hydroxyphenanthrene was accomplished in 4 min of electrophoretic run. Limits of detection at the parts‐per‐billion were obtained with a single solvent (acetonitrile) for metabolite extraction and sample stacking. The small sample volume (1.2 mL) and the conservative usage of chemicals provided a simple and rapid procedure for the simultaneous extraction of numerous samples. Adding 4 min of electrophoretic run per sample, it should be possible to screen ten samples in approximately 1 h of analysis time. The nanoliter extract volume required for sample injection allows for further chromatographic usage and confirmation of positive samples. The unique electrophoretic pattern of the studied metabolites demonstrates the potential for the unambiguous determination of positional isomers with very similar chromatographic behaviors and undistinguishable mass fragmentation patterns.     

High-Throughput Screening of Ten Microcystins and Nodularins, Cyanobacterial Peptide Hepatotoxins, by Reversed-Phase Liquid Chromatography-Electrospray Ionisation Mass Spectrometry

Liquid chromatography-electrospray ionisation mass spectrometry was evaluated in the high-throughput analysis of microcystins and nodularins, cyanobacterial peptide hepatotoxins. Extracts originating from cyanobacterial strains and field material were separated on a 30 mm × 4 mm I.D. Merck Purospher STAR RP-18e column using a rapid gradient of aqueous formic acid and acetonitrile, ionised by electrospray technique and analysed on a Micromass Quattro II triple-quadrupole mass spectrometer operated in the selected ion recording (SIR) mode. The total analysis time per sample was 2.8 min corresponding to 514 samples a day. The system showed good robustness during a series of 320 repetitive injections of a field sample containing three major microcystins.     

Automation of a Fourier transform ion cyclotron resonance mass spectrometer for acquisition, analysis, and e-mailing of high-resolution exact-mass electrospray ionization mass spectral data

A system has been designed to automatically acquire high-resolution (>50,000 FWHM), exact-mass (mass measurement error ≤3 mmu) electrospray ionization mass spectra with a commercial Fourier transform ion cyclotron resonance mass spectrometer equipped with a high-field (9.4 tesla) superconducting magnet and a commercial autosampler. Upon the injection of each individual sample, the autosampler transmits a contact closure signal to the previously tuned and calibrated mass spectrometer to initiate data acquisition. A software package was designed to run off-line and to accept a sample list with input information for each of the samples. Then for each of the samples, the software automatically processes the acquired data, interprets the exact-mass data by correlating the observed masses with predicted masses computed from proposed elemental formulas, and then finally prints the spectra, peak lists, and exact-mass reports, and e-mails the exact-mass reports to the submitting chemists. With this automation package, large numbers of samples can be run unattended while obtaining exact masses for all the abundant ions in the spectra. Sample turnaround times are reduced with a corresponding increase in sample throughput. The performance of the system was evaluated with nearly 700 samples with a precalibrated instrument, without the presence of an internal standard. The system was found to be reliable and robust with a fitted standard deviation of 0.32 mmu and a small average systematic mass error of ?0.28 mmu. Typical data acquired with the system have resolving powers >50,000 (FWHM) and mass errors <1.0 mmu.     

A high-throughput multicomponent screening method for diuretics, masking agents, central nervous system (CNS) stimulants and opiates in human urine by UPLC-MS/MS

A simple and rapid multicomponent screening method of 130 substances for direct injections of urine samples has been developed. The fully automated method based on ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) is used for three different classes of doping agents: diuretics, central nervous system stimulants (CNS stimulants) and opiates. The samples are diluted with buffer containing internal standards (IS) by a pipetting robot system into 96-well plates. Samples are injected on a reversed phase sub 2-microm particle column connected to a fast polarity switching and rapid scanning tandem mass spectrometer with an electrospray interface. The software used to evaluate the results produced reports containing a small-sized window for each component and a data table list with flags to indicate any adverse analytical findings in the sample. The report can also be processed automatically using an application software, which interpret the data and indicate if there is a suspicious sample. One 96-well plate can be analyzed within 16 h.     

Direct screening and confirmation of priority volatile organic pollutants in drinking water

A screening tool was proposed for the rapid detection of eight priority volatile organic pollutants according to European standards in drinking water. The method is based on the direct coupling of a headspace sampler with a mass spectrometer, using a chromatographic column heated to 175 degrees C as an interface. The water sample was subjected to the headspace extraction process and the volatile fraction was introduced directly into the mass spectrometer, without prior chromatographic separation, achieving low detection limits (0.6-1.2 ng/ml) for all compounds. The mass spectrum resulting from the simultaneous ionization and fragmentation of the mixture of molecules constitutes the volatile profile of each sample. An appropriate chemometric treatment of these signals permitted them to be classified, on the basis of their volatile composition, as contaminated or uncontaminated with respect to the legally established concentration levels for these compounds in drinking water, and providing no false negatives. A conventional confirmation method was carried out to analyze positive water samples by using the same instrumental setup as in the screening method, but using an appropriate temperature program in the chromatographic column to separate, identify and quantify each analyte.     

A novel mass spectrometry cluster for high-throughput quantitative proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.