Preventing false negatives with high-resolution mass spectrometry: the benzophenone case

Benzophenone (BP) is one of the many contaminants reported as present in foodstuffs due to its migration from food packaging materials. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is acknowledged in the literature as the method of choice for this analysis. However, cases have been reported where the use of this methodology was insufficient to unambiguously confirm the presence of a contaminant. In previous work performed by the authors, the unequivocal identification of BP in packaged foods was not possible even when monitoring two m/z transitions (precursor ion – product ion), since ion ratio errors higher than 20% were obtained. In order to overcome this analytical problem a fast, sensitive and selective liquid chromatography/high‐resolution mass spectrometry (LC/HRMS) methodology has been developed and applied to the analysis of BP in packaged foods. A direct comparison between LC/HRMS and LC/MS/MS data indicated better selectivity when working with LC/HRMS at a resolving power of 50 000 FWHM (full width at half maximum) than when monitoring two m/z transitions by LC/MS/MS. The resolving power used enabled the detection and identification of Harman as the compound impeding the confirmation of BP by LC‐MS/MS. Similar quantitative results were obtained by an Orbitrap mass analyser (Exactive?) and a triple quadrupole mass analyser (TSQ Quantum Ultra AM?). Copyright © 2011 John Wiley & Sons, Ltd.     

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.     

High-throughput logP measurement using parallel liquid chromatography/ultraviolet/mass spectrometry and sample-pooling

A novel approach to high-throughput logP measurement based on liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) is proposed. The logP value is determined by correlation with the logk value, where k is the capacity factor k = (t(r)-t(0))/t(0), with the logP value using a defined set of standards. Since the analyte retention time (t(r)) is determined from the appropriate extracted ion chromatogram (EIC), there are no interferences from impurities and this allows the pooling of multiple compounds into one injection. To ensure the accuracy and instrument robustness in a routine high-throughput environment, a simple and MS-friendly mobile phase consisting of 20 mM ammonium carbonate (pH 8.0) for basic compounds or 20 mM ammonium formate (pH 1.0) for acidic compounds, both in combination with methanol at a ratio of 45:55, is used. This approach has been successfully used on single as well as parallel multi-channel LC/UV/MS systems to screen small to large sets of lead compounds and their analogs. A high-throughput capability to analyze over 1000 compounds per day has been achieved.     

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.     

Use of high‐resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose‐dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high‐resolution mass spectrometry led to unequivocal identification of the interfering peak as an N‐desmethyl metabolite of the parent analyte. High‐resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the ¹³C‐isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry

In order to optimise the analysis of urinary nucleosides by high performance liquid chromatography/mass spectrometry (HPLC/MS), the HPLC separation of these compounds was performed at different 'flow rates' and 0.2mL/min was found to give both a better separation and ionisation. The ionisation conditions were optimised to give the best intensity of the molecules quasi-molecular ions. The ion distribution profile and ionisation in both positive and negative mode were examined and the detection of the protonated molecule in positive mode chosen for further analysis. The limits of detection of the method developed are reported and representative LC/MS and LC/MS/MS spectra shown. Typical urinary nucleoside chromatograms are presented.     

Qualitative–(semi)quantitative data acquisition of artemisinin and its metabolites in rat plasma using an LTQ/Orbitrap mass spectrometer

Artemisinin (QHS) is one of the first‐line antimalarials, and autoinduction of CYP‐mediated metabolism can result in its reduced exposure. To better understand the autoinduction of QHS, we evaluated the pharmacokinetics of QHS and its phase I metabolites in rats using an liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) method. The LC separation was improved, allowing the separation of QHS and its metabolites from their diastereomers, and seven metabolites of QHS with relatively high exposure were identified in rat plasma, including deoxyartemisinin (DQHS), three monoyhydroxylated plus deoxyl metabolites (M1–M3) and three monohydroxylated metabolites (M4–M6). For detection, a high‐resolution LTQ/Orbitrap mass spectrometer with an electrospray ionization (ESI) inlet in the positive ion mode was used. High‐resolution extracted ion chromatograms for each analyte were obtained by processing the full‐scan MS dataset with 10 ppm mass tolerance. The plasma samples were pretreated by protein precipitation with acetonitrile. The standard curve was linear (r² > 0.99) over the QHS and DQHS concentration range of 5.0–200.0 ng/ml in 50 µl of plasma, which offered sufficient sensitivity and accuracy for the determination of QHS and its metabolites. A 3‐day validation approach was used for absolute quantitation of QHS and DQHS. The other six metabolites of QHS were semiquantified based on the calibration curve of QHS. The present method was applied to the pharmacokinetic study of QHS in rats after a single oral administration. The data shown here also suggest that this type of mass analyzer will be capable of a quantitative–qualitative workflow. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma

A sensitive, rapid and specific quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of apomorphine (APO) in canine plasma. The analytes were prepared using one-step liquid-liquid extraction, and analyzed on a Waters Symmetry C(18) column interfaced with triple quadrupole tandem mass spectrometer. A mixture of methanol/0.1% formic acid in water (70: 30, v/v) was employed as the isocratic mobile phase. Positive electrospray ionization was utilized as the ionization source. The analyte and clenbuterol (internal standard) were both detected using multiple reaction monitoring (MRM) mode. The limit of detection (LOD) obtained was 0.03 ng/mL. The assay was linear over the concentration range of 0.1-100 ng/mL, and provided good precision (RSD) and good accuracy (RE). The analyte was stable by using antioxidants throughout the whole study. The experimental results show that LC/MS/MS is a rapid and sensitive method to analyze APO in plasma. Finally, the proposed method was successfully applied to a pharmacokinetic study of APO after intranasal administration of 0.5 mg apomorphine to 10 healthy beagle dogs.     

Bias reduction in the quantitative analysis of a target analyte present in a limited quantity in human plasma using dual-mode heart-cutting two-dimensional liquid chromatography coupled with isotope dilution mass spectrometry

Dual-mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS were performed with triple quadrupole mass spectrometry using folic acid standards and/or ¹³C₅-folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked ¹³C₅-folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma, eliminating matrix effects and saving analysis time.     

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.     

Determination of melanotan-II in rat plasma by liquid chromatography/tandem mass spectrometry: determination of pharmacokinetic parameters in rat following intravenous administration

A method is described for the determination of melanotan-II (MT-II), a synthetic cyclic heptapeptide analog of alpha-melanocyte-stimulating hormone (alpha-MSH), in rat plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analyte is recovered from plasma by solid-phase extraction (SPE) and subsequently analyzed by LC/MS/MS. A SPE procedure using OASIS 96-well plates is used for extraction of MT-II from rat plasma. Using the described approach a limit of quantitation of 5 ng/mL was achieved in rat plasma. This level of sensitivity allowed the determination of pharmacokinetic parameters, following intravenous administration of MT-II in rat.     

Assigning product ions from complex MS/MS spectra: The importance of mass uncertainty and resolving power

This study offers a unique insight into the mass accuracy and resolving power requirements in MS/MS analyses of complex product ion spectra. In the examples presented here, accurate mass assignments were often difficult because of multiple isobaric interferences and centroid mass shifts. The question then arose whether the resolving power of a medium-resolution quadrupole time-of flight (QqTOF) is sufficient or high-resolution Fourier-transform ion cyclotron resonance (FT-ICR) is required for unambiguous assignments of elemental compositions. For the comparison, two paralytic shellfish poisons (PSP), saxitoxin (STX) and neosaxitoxin (NEO), with molecular weights of 299 and 315 g x mol(-1), respectively, were chosen because of the high peak density in their MS/MS spectra. The assessment of QqTOF collision-induced dissociation spectra and FT-ICR infrared multiphoton dissociation spectra revealed that several intrinsic dissociation pathways leading to isobaric fragment ions could not be resolved with the QqTOF instrument and required FT-ICR to distinguish very close mass differences. The second major source of interferences was M + 1 species originating from coactivated 13C12Cc-1 ion contributions of the protonated molecules of the PSPs. The problem in QqTOF MS results from internal mass calibration when the MH+ ions of analyte and mass calibrant are activated at the same time in the collision or trapping cell. Although FT-ICR MS readily resolved these interfering species, the QqTOF did not provide resolving power >20,000 (full width at half maximum) required to separate most isobaric species. We were able to develop a semi-internal QqTOF calibration technique that activated only the isolated 12C isotope species of the protonated molecules, thus reducing the M + 1 interferences significantly. In terms of overall automated elemental formulas assignment, FT-ICR MS achieved the first formula hit for 100% of the product ions, whereas the QqTOF MS hit rate was only 56 and 65% for STX and NEO product ions, respectively. External mass calibration from commercial FT-ICR and QqTOF instruments gave similar results.     

Strategies to avoid false negative findings in residue analysis using liquid chromatography coupled to time-of-flight mass spectrometry

Liquid chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (LC/TOF) provides an attractive alternative to liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS/MS) in the field of multiresidue analysis. The sensitivity and selectivity of LC/TOF approach those of LC/MS/MS. TOF provides accurate mass information and a significantly higher mass resolution than quadrupole analyzers. The available mass resolution of commercial TOF instruments ranging from 10 000 to 18 000 full width at half maximum (FWHM) is not, however, sufficient to completely exclude the problem of isobaric interferences (co-elution of analyte ions with matrix compounds of very similar mass). Due to the required data storage capacity, TOF raw data is commonly centroided before being electronically stored. However, centroiding can lead to a loss of data quality. The co-elution of a low intensity analyte peak with an isobaric, high intensity matrix compound can cause problems. Some centroiding algorithms might not be capable of deconvoluting such partially merged signals, leading to incorrect centroids.Co-elution of isobaric compounds has been deliberately simulated by injecting diluted binary mixtures of isobaric model substances at various relative intensities. Depending on the mass differences between the two isobaric compounds and the resolution provided by the TOF instrument, significant deviations in exact mass measurements and signal intensities were observed. The extraction of a reconstructed ion chromatogram based on very narrow mass windows can even result in the complete loss of the analyte signal. Guidelines have been proposed to avoid such problems. The use of sub-2 microm HPLC packing materials is recommended to improve chromatographic resolution and to reduce the risk of co-elution. The width of the extraction mass windows for reconstructed ion chromatograms should be defined according to the resolution of the TOF instrument. Alternative approaches include the spiking of the sample with appropriate analyte concentrations. Furthermore, enhanced software, capable of deconvoluting partially merged mass peaks, may become available.     

Determination of a correction to improve mass measurement accuracy of isotopically unresolved polymerase chain reaction amplicons by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The experimental determination of average mass by mass spectrometry is limited for large molecules due to the negative bias introduced by the natural distribution of isotopic abundances. This results in the measurement of the top-of-centroid (ToC) as opposed to the true centroid. We have developed a practical correction factor that is applied to the ToC measurement to largely remove the systematic bias introduced by nature. The correction factor is calculated easily using the average molecular mass (<100 kDa) of the analyte molecule and the full-width half maximum resolving power (<3,500) of the measurement. In addition, an approach to calculating resolving power is described that accurately predicts resolving power achievable for Fourier transform ion cyclotron resonance (FT-ICR) mass analysis of large molecules. A combination of internal calibration with a dual-electrospray source and application of the correction factor to average mass measurements improved the mass error from 192.5 to -35.0 ppm for a 44 kDa PCR amplicon.     

Immuno-mass spectrometry assay of EPI-HNE4, a recombinant protein inhibitor of human elastase

In order to increase the sensitivity of liquid chromatography/mass spectrometry (LC/MS) assays of recombinant proteins for pharmacokinetics studies, we have developed an immuno-mass spectrometry assay for EPI-hNE4, a 6237 Da protein currently developed for respiratory distress syndromes. After immunocapture of the analyte in human plasma with magnetic beads coated with anti-EPI-hNE4 antibodies, the intact protein was eluted and separated in reversed-phase LC and then analysed by tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) mode. The problem of analytical interference due to endogenous binding antibodies was addressed by successive steps of acidification and neutralisation before immunocapture. Furthermore, potential variations in the recovery of analyte during sample extraction were compensated for by addition of an internal standard recognised by the antibodies. The precision of the assay remained therefore below 15%. A significant increase in assay sensitivity was achieved since the extraction step allowed sample concentration and removal of matrix components interfering with the electrospray ionisation process. Using 0.4 mL of plasma, a limit of quantification at 0.5 ng/mL (80 pM) was reached, which represents a 10-fold improvement in sensitivity over our previous work using sample precipitation. This technique was able to monitor EPI-hNE4 kinetics in the plasma of human subjects for 36 h after an intravenous administration of 0.125 mg/kg.     

Suitability of an orbitrap mass spectrometer for the screening of pesticide residues in extracts of fruits and vegetables

For about 500 pesticides, the sensitivity of a benchtop high-resolution mass spectrometer using the Orbitrap for mass separation was compared to that of a widely used (low-resolution) tandem mass spectrometer. Both instruments were coupled to LC and used electrospray ionization. The selectivity of the Orbitrap in the full-scan acquisition mode without fragmentation was evaluated at a resolution of 100 000 full width at half maximum for all pesticides detectable with sufficient sensitivity. For this purpose, quasimolecular ions were extracted within 5 ppm windows from total ion chromatograms of two types of extracts of cucumber, lemon, wheat flour, raisin, and tea. In each of the obtained reconstructed ion chromatograms (individual chromatograms for 500 pesticides, each pesticide in 10 different extracts) the sum of signals not arising from the analyte was used to get a measure on selectivity. In addition, the target analyte list was checked for ions of similar mass. The influence of matrix on the ability to detect low concentrations of fortified pesticides was also studied, with the help of spiked extracts. This part of the survey tested whether analyte peaks were lost because of insufficient mass resolution or an early closing C-Trap (used to control the ion current into the Orbitrap). Finally, the stability of the ion ratio [M+H]+/[M+Na]+ was tested, which may be helpful to confirm the identity of an analyte.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

Determination of caudatin-2,6-dideoxy-3-O-methy-beta-d-cymaropyranoside in rat plasma using liquid chromatography-tandem mass spectrometry

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method has been developed for the determination of caudatin-2,6-dideoxy-3-O-methy-beta-d-cymaropyranoside (CDMC) in rat plasma. This method involves a plasma clean-up step using liquid-liquid extraction, followed by LC separation and positive electrospray ionization mass spectrometry detection (LC/ESI-MS/MS). Chromatographic separation of the analytes was achieved using a C(18) column with a mobile phase of acetonitrile and water (70:30, v/v) at a flow rate of 1.0 mL/min. Low energy collision tandem mass spectrometric analysis (CID-MS/MS) using the multiple reaction monitoring (MRM) mode was used for analyte quantification. For the MRM analysis of CDMC, the following transition at m/z 658.4 --> 529.6 derived from the protonated molecule [M + Na](+). A calibration curve was linear in the 5-500 ng/mL range for CDMC, and the limit of detection was 5 ng/mL. The inter- and intra-day precisions (RSD) were 相似文献   

Comparison between a high‐resolution single‐stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs

The capabilities of a high‐resolution (HR), accurate mass spectrometer (Exactive‐MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50 000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive‐MS was compared with that of a triple quadrupole mass spectrometer (TQ‐MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole‐N‐oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR‐MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR‐MS offers several benefits over TQ‐MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR‐MS delivers comparable results to TQ‐MS in routine quantitative drug analyses. Considering the advantages of HR‐MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery

A novel liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based depletion method for measuring compound partitioning between human plasma and red blood cells (RBC) in a drug discovery environment is presented. Conventionally, RBC partitioning is determined by separate measurements of drug concentrations in equilibrating plasma and whole blood or RBC using separate standards prepared in their respective matrices, i.e., in plasma and whole blood or RBC lysates. The process is very tedious, labor-intensive, and difficult to automate. In addition, interferences from the heme and other highly abundant cellular composites make the measurement of the drug concentration in whole blood or RBC inevitably variable even with a highly specific LC/MS/MS method. Therefore, there is an imminent need to develop a straightforward and fast method to assess the partitioning of drug-like compounds in RBC.This work describes an LC/MS/MS-based depletion assay that measures the compound concentration in plasma that has been equilibrating with RBC. Compounds were spiked into fresh human whole blood and plasma respectively to a final concentration of 500 nM. Both the spiked whole blood and plasma control were incubated at 37 degrees C for up to 60 min. During the time course, aliquots of plasma and whole blood from both incubation mixtures were sampled at 10 and 60 min. The whole blood samples were centrifuged to yield the plasma. The plasma samples from both incubations were extracted using a protein precipitation method, and analyzed using LC/MS/MS under the multiple-reaction monitoring (MRM) mode. The RBC partitioning ratio was calculated using the analyte peak area responses of the plasma samples through an equation deduced in this work.The method was first tested using two commercial compounds, phenoprobamate and acetazolamide, to determine the optimal incubation conditions and the concentration dependency of the assay. The assay reproducibility was also assessed by three inter-day assays for phenoprobamate. This method was further evaluated using 20 commercial compounds of different classes with a wide range of RBC partitioning coefficients and the results were compared with those reported in the literature. Excellent correlation (R2=0.9396) was found between the measured and literature values. In addition, several proprietary compounds were assayed using both the new and traditional methods and the measured partitioning ratios from the two methods are equivalent. The experiments in this work demonstrate that the LC/MS/MS-based depletion method can provide direct and accurate measurement of RBC partitioning for compounds in drug discovery.