Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry

The glucuronide conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone were identified in human urine post-intravenous administration of Ketogan Novum. The human urine was extracted on a mixed-mode solid-phase micro-column before analysis with liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and tandem MS (MS/MS). Accurate mass and collision-induced dissociation product ion spectra were used for identification of the glucuronide conjugates. Two different TOF mass spectrometers were used and the accurate mass measurements were performed on three separate days with each instrument. The accuracy of the mass measurements was better than 2.1 ppm for two out of three conjugates and the inter-day relative standard deviation was within +/-0.00049%. The MS/MS fragmentation patterns of the conjugates were in accordance with those of the synthetic aglycones and included peaks originating from the [M + H](+) ion of the respective aglycone.     

Investigation into accurate mass capability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, with respect to radical ion species

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been shown to be an effective technique for the characterization of organometallic, coordination, and highly conjugated compounds. The preferred matrix is 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), with radical ions observed. However, MALDI-TOFMS is generally not favored for accurate mass measurement. A specific method had to be developed for such compounds to assure the quality of our accurate mass results. Therefore, in this preliminary study, two methods of data acquisition, and both even-electron (EE+) ion and odd-electron (OE+.) radical ion mass calibration standards, have been investigated to establish the basic measurement technique. The benefit of this technique is demonstrated for a copper compound for which ions were observed by MALDI, but not by electrospray (ESI) or liquid secondary ion mass spectrometry (LSIMS); a mean mass accuracy error of -1.2 ppm was obtained.     

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10^?9 torr. The increased pumping speed attainable with cryopumping (> 10⁵ L/s) allowed brief pressure excursions to above 10^?4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M_r 5734) and ubiquitin (M_r 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H₃PO₄ adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4⁺ charge state (m/z 1434) of insulin.     

Using accurate mass electrospray ionization-time-of-flight mass spectrometry with in-source collision-induced dissociation to sequence peptide mixtures

Although data-dependent LC-MS-MS with database searching has become au courant for identifying proteins, the technique is constrained by duty-cycle inefficiency and the inability of most tandem mass analyzers to accurately measure peptide product ion masses. In this work, a novel approach is presented for simultaneous peptide fragmentation and accurate mass measurement using in-source collision-induced dissociation (CID) on electrospray ionization (ESI)-time-of-flight (TOF) MS. By employing internal mass reference compounds, mass measurement accuracy within +/-5 ppm for tryptic peptide precursors and +/-10 ppm for most sequence-specific product ions was consistently achieved. Analysis of a complex solution containing several digested protein standards did not adversely affect instrument performance.     

Flow injection of the lock mass standard for accurate mass measurement in electrospray ionization time-of-flight mass spectrometry coupled with liquid chromatography

A method of flow injection of the lock mass for accurate mass measurement using electrospray ionization time-of-flight mass spectrometry is described. The reference compound is introduced in the chromatographic effluent via a six-port valve placed post-column, prior to the split connector. Flow injection is performed in such a way that the reference elution peak is superimposed in the total ion current and partially overlaps that of the investigated analyte, allowing independent ionization of the two compounds and thus accurate mass measurement with no ion suppression effects. Different lock mass molecules can be injected in a single analytical run to target various analytes. The performance of this methodology is demonstrated in both isocratic and gradient liquid chromatography modes. The molecular ion of the flow-injected lock mass could also be used as a reference for mass measurement of the in-source fragments of the analytes. Good mass accuracy, within 4 mDa of the theoretical values, was obtained.     

Investigation of space charge effects and ion trapping capacity on direct introduction ultra‐high‐resolution mass spectrometry workflows for metabolomics

Ultra‐high‐resolution mass spectrometry, in the absence of chromatography, is finding its place for direct analyses of highly complex mixtures, such as those encountered during untargeted metabolomics screening. Advances, however, have been tempered by difficulties such as uneven signal suppression experienced during electrospray ionization. Moreover, ultra‐high‐resolution mass spectrometers that use Orbitrap and ICR analyzers both suffer from limited ion trapping capacities, owing principally to space‐charge effects. This study has evaluated and contrasted the above two types of Fourier transform mass spectrometers for their abilities to detect and identify by accurate mass measurement, small molecule metabolites present in complex mixtures. For these direct introduction studies, the Orbitrap Fusion showed a major advantage in terms of speed of analysis, enabling detection of 218 of 440 molecules (<2 ppm error, 500 000 resolution at m/z 200) present in a complex mixture in 5 min. This approach is the most viable for high‐throughput workflows, such as those used in investigations involving very large cohorts of metabolomics samples. From the same mixture, 183 unique molecules were observed by FT‐ICR in the broadband mode, but this number was raised to 235 when “selected ion monitoring‐stitching” (SIM‐stitching) was employed (<0.1 ppm error, 7 T magnet with dynamic harmonization cell, 1.8 million resolution at m/z 200, both cases). SIM‐stitching FT‐ICR thus offered the most complete detection, which may be of paramount importance in situations where it is essential to obtain the most complete metabolic profile possible. This added completeness, however, came at the cost of a more lengthy analysis time (120 min including manual treatment). Compared to the data presented here, future automation of processing, plus the use of absorption mode detection, segmented ion detection (stepwise detection of smaller width m/z sections), and higher magnetic field strengths, can substantially reduce FT‐ICR acquisition times.     

Accurate mass measurement using Fourier transform ion cyclotron resonance mass spectrometry for structure elucidation of designer drug analogs of tadalafil, vardenafil and sildenafil in herbal and pharmaceutical matrices

Phosphodiesterase type 5 (PDE-5) inhibitors are a class of drugs used primarily in the treatment of erectile dysfunction. The Food and Drug Administration (FDA) approved PDE-5 inhibitors include sildenafil citrate, vardenafil hydrochloride and tadalafil. In this study, accurate mass measurements were made by electrospray ionization (ESI) using Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) to elucidate the structures of sildenafil, tadalafil and vardenafil analogs that were found in products marketed as dietary supplements. Initial detection of these analogs was accomplished through routine screening of suspect samples by liquid chromatography/electrospray ionization multi-stage mass spectrometry (LC/ESI-MS(n)) on a low-resolution ion trap instrument. The chromatographic behavior and mass spectrometric fragmentation patterns observed were often similar to those observed for FDA approved PDE-5 inhibitors. The mass accuracy and resolving power associated with FTICRMS allows for the determination of elemental compositions. Elucidation of the product ion structures for the analogs was accomplished through the use of accurate mass measurements with the aid of Mass Frontier software (version 4.0). Using FTICRMS, accurate masses with measurement errors averaging <0.4 ppm were achieved, allowing assignment of one possible elemental formula to each fragment ion. The mass measurement errors associated with [M + H](+) for the analogs aminotadalafil, piperidino vardenafil, hydroxyacetildenafil and piperidino acetildenafil were 0.1, 0.0, 0.1 and 0.5 ppm, respectively. Based on the accuracy of the measurements, structural assignments could be made with a high degree of confidence.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Exact mass measurements using a 7 tesla fourier transform ion cyclotron resonance mass spectrometer in a good laboratory practices-regulated environment

Fourier transform ion cyclotron resonance mass spectrometry has been found to produce reliable exact mass measurements using two different internal calibration methods. For these measurements, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) were utilized both individually and in tandem. For internal calibration with a co-dissolved polyethylene glycol standard, measurements of 41 compounds resulted in an average absolute mass determination error of 0.7 ppm, with a standard deviation of 0.9 ppm. For comparison, internal calibration was effected through the simultaneous use of ESI and MALDI, with the former being used for the introduction of analyte ions and the latter for formation of polymethylmethacrylate calibrant ions. This technique led to mass measurements with an average absolute error of 0.8 ppm and a standard deviation of 1.0 ppm. In addition, exact mass measurements of tandem mass spectrometry fragment ions were made for 35 compounds using external calibration with a single internal mass standard. The observed average absolute error was 0.7 ppm with a standard deviation of 1.0 ppm.     

Parallel high-throughput accurate mass measurement using a nine-channel multiplexed electrospray liquid chromatography ultraviolet time-of-flight mass spectrometry system

A nine-channel multiplexed electrospray (MUX) liquid chromatography ultraviolet time-of-flight mass spectrometry (LC/UV/TOFMS) system has been used to simultaneously measure accurate masses of eluting components from eight parallel gradient LC columns. Accuracies better than 5 and 10 ppm were achieved for 50 and 80% of samples, respectively, from a single batch analysis of ten plates (960 samples) of a Fmoc-Asp(OtBu)-OH and reserpine mixture. Combinatorial library compounds were analyzed using this parallel high-throughput system in both positive and negative modes to rigorously verify expected products and identify side products. A mass accuracy of 10 ppm root mean square (RMS) is routinely obtained for combinatorial library samples from this high-throughput accurate mass LC/MS system followed by automated data processing. This mass accuracy is critical in revealing combinatorial synthesis problems that would be missed by unit mass measurement.     

Application of liquid chromatography-electrospray ionization time-of-flight mass spectrometry for analysis and quality control of compound Danshen preparations

A liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) method has been developed to evaluate the quality of three formulas of compound Danshen preparations (CDPs), through a simultaneous determination of 22 marker constituents (nine major phenolic acids, eight major saponins and five major diterpenoids). Optimum separations were obtained with a Zorbax C(18) column, using a gradient elution with 0.1% aqueous formic acid and acetonitrile. Limits of detection and quantification were in ranges of 1.58-10.10 and 4.85-28.56 ng/mL. All calibration curves showed good linear regression (r(2 ) > 0.9900) within the test range, and the recoveries were between 78.4 and 103.1% for all analytes. The assay was successfully utilized to analyze the 22 marker components in 26 samples. The overall results demonstrated that this method is sensitive, accurate and reliable for the quality control of CDPs.     

Development of a simple and reliable accurate mass liquid chromatography/electrospray ionization mass spectrometry method for high-resolution accurate mass determinations of new drug entities on a triple quadrupole mass spectrometer

Accurate mass measurements are used to determine the elemental composition and formulae of molecules to confirm their identity or to assist in their characterization. Currently, the most widely used techniques for measuring exact masses employ magnetic sector instruments, Fourier transform ion cyclotron resonance mass spectrometers and lower resolution instruments such as time-of-flight (TOF) and quadrupole-TOF. This paper reports the accurate mass measurement using a triple quadrupole mass spectrometer. Indeed, the recently introduced triple quadrupole mass spectrometer, with unique enhanced mass-resolution capability, has demonstrated simple data acquisition methods and requires few experiments to measure exact masses with accuracy and determines elemental compositions of both protonated and deprotonated molecules. All the accurate mass measurements were performed using both positive and negative electrospray ionization in enhanced mass-resolution mode (peak width of 0.1 Th FWMH). Several new drug entities were investigated as simulated unknowns and analyzed by means of an accurate mass liquid chromatography/electrospray ionization mass spectrometry (AM-LC/ESI-MS) method. The accurate mass measurements resulted in only one proposed elemental composition for all tested compounds, using reasonable elemental limits and mass tolerance for the calculation. Moreover, all the experimentally determined accurate mass measurements gave satisfactory results in terms of accuracy (lower than 5 ppm).     

Automated high resolution mass spectrometry for the synthetic chemist

Exact mass measurement at high resolution is an important tool alongside other spectroscopic methods to help confirm the structure of a novel compound prepared by the synthetic chemist. Exact mass measurement is used in the pharmaceutical industry to confirm the expected empirical formula of a product when problems have been experienced using elemental analysis. Because of the amount of manual intervention necessary when acquiring exact mass measurements, especially when using probe ionization techniques such as fast atom bombardment ionization or electron ionization, this method has been seen to be time consuming and labor intensive for the mass spectrometrist. An automated high resolution mass spectrometric method has been developed at Pfizer Central Research which has streamlined exact mass measurement. The method, which uses electrospray ionization on a double focusing mass spectrometer, is described. The samples are analyzed using a flow injection technique, with sodiated polyethylene glycol present in the mobile phase to provide mass reference peaks. The data are acquired and processed using a macro developed “in house.” This automated technique can process 15–20 samples an hour including data processing and report generation, using very small amounts of compound (∼25 μg), but more importantly it can be left to run unattended overnight. This allows the instrument to be used for more complex experiments during the day when it is important to have a mass spectrometrist present. The results presented here demonstrate that this method gives exact mass measurements within an acceptable limit of 5 ppm, and the variation on one sample, injected 10 times, is not excessively high (−1.8 to +1.6 mDa).     

The automation of a commercial Fourier transform mass spectrometer to provide a quick and robust method for determining exact mass for the synthetic chemist

Automation of a commercially available Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer for the routine analysis of the synthetic products from high-speed chemistry is described. The automation includes software written by the instrument manufacturer and in-house developed software; allowing electronic submission of samples from the chemist and e-mailing of results back to the chemist. The use of samples of relatively high concentration (ca 1 mg x mL(-1)) is possible due to the protocol that has been developed, which includes dilution by the autosampler during sample injection. Though high concentrations are used for speed and convenience the amount of sample consumed is still small ca 15 microg per injection. The results from this method have been shown to be both accurate (average error +/- 0.91 ppm) and precise (-0.70 ppm to 2.26 ppm). The system is capable of analysing up to 800 samples per 24 hours. As high speed chemistry becomes more highly utilised within discovery the number of samples requiring accurate mass analysis will rise, and the method we have described will prevent high resolution mass spectrometry becoming the bottleneck in new chemical entity production. The accuracy and precision demonstrated by this method allows high confidence levels in assigned molecular formulae for expected compounds and reduces the number of possible formulae to consider when working with a compound that is not the desired product of a given reaction.     

Automated sub-ppm mass accuracy on an ESI-TOF for use with drug discovery compound libraries

An automated, routine method to obtain sub-ppm accurate mass data on a benchtop electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Standards in the mass range 114 to 734 Da were analyzed over a 5-day period to demonstrate intra- and interday precision and mean mass accuracy less than 1 ppm. One hundred drug discovery pharmaceutical compounds were used to demonstrate an absolute average mass accuracy of 0.47 +/- 0.31 ppm. This is in contrast to previous reports of accurate mass analysis using time-of-flight mass spectrometry (TOFMS) technology that operates within 3 to 5 ppm. The same 100 samples were also analyzed using Fourier transform mass spectrometry (FTMS) technology and yielded comparable results to the TOFMS analysis.     

Accurate mass measurements for the confirmation of Sudan azo-dyes in hot chilli products by capillary liquid chromatography-electrospray tandem quadrupole orthogonal-acceleration time of flight mass spectrometry

The potential of capillary liquid chromatography (microLC)-quadrupole/time-of-flight mass spectrometry (Q-TOF MS) for the confirmation of Sudan I, II, III and IV azo-dyes as contaminants in hot-chilli food products was demonstrated. Using the microLC-electrospray ionization (ESI)-Q-TOF MS technique, accurate mass measurements of Sudan dyes were performed both on standard solutions and on matrices. Precision of exact mass measurements was calculated taking into account the ion statistics according to the number of ion sampled in the measurement. Accurate mass measurements by MS/MS experiments were performed to elucidate azo-dye fragmentation patterns. Selectivity of the microLC-Q-TOF MS method was assessed by evaluating matrix suppression effects by pre-column injection of blank hot chilli tomato sauce matrices. The results were compared with those obtained on a LC-triple quadrupole-MS system. Confirmation of Sudan I present in hot chill tomato sauce samples was obtained by accurate mass measurements. In real samples trueness of exact mass measurements was estimated to be 1.6 and 4.4 ppm when calculated for hot chilli tomato sauce and hot chilli tomato with cheese sauce samples, respectively; precision was calculated around 9.5 ppm.     

Identification of phospholipid structures in human blood by direct-injection quadrupole-linear ion-trap mass spectrometry

Direct-injection electrospray ionization mass spectrometry in combination with information-dependent data acquisition (IDA), using a triple-quadrupole/linear ion trap combination, allows high-throughput qualitative analysis of complex phospholipid species from child whole blood. In the IDA experiments, scans to detect specific head groups (precursor ion or neutral loss scans) were used as survey scans to detect phospholipid classes. An enhanced resolution scan was then used to confirm the mass assignments, and the enhanced product ion scan was implemented as a dependent scan to determine the composition of each phospholipid class. These survey and dependent scans were performed sequentially and repeated for the entire duration of analysis, thus providing the maximum information from a single injection. In this way, 50 different phospholipids belonging to the phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin classes were identified in child whole blood.     

An introduction to quadrupole-time-of-flight mass spectrometry

A brief introduction is presented to the basic principles and application of a quadrupole-time-of-flight (TOF) tandem mass spectrometer. The main features of reflecting TOF instruments with orthogonal injection of ions are discussed. Their operation and performance are compared with those of triple quadrupoles with electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometers. Examples and recommendations are provided for all major operational modes: mass spectrometry (MS) and tandem MS (MS/MS), precursor ion scans and studies of non-covalent complexes. Basic algorithms for liquid chromatography/MS/MS automation are discussed and illustrated by two applications.     

Reduced matrix effects for anionic compounds with paired ion electrospray ionization mass spectrometry

It is well-known that matrix effects in high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) can seriously compromise quantitative analysis and affect method reproducibility. Paired ion electrospray ionization (PIESI) mass spectrometry is an approach for analyzing ultra-low levels of anions in the positive ion mode. This approach uses a structurally optimized ion pairing reagent to post-column associate with the anionic analyte, subsequently forming positively charged complexes. These newly formed complex ions are often more surface-active as compared to either the native anion or the ion pairing reagent. No studies have examined whether or not the PIESI approach mitigates matrix effects. Consequently, a controlled study was done using five analytes in highly controlled and reproducible synthetic groundwater and urine matrices. In addition, two different mass spectrometers (linear ion trap and triple quadrupole) were used. Compared to the negative ion mode, the PIESI-MS approach was less susceptible to matrix effects when performed on two different MS platforms. Using PIESI-MS, less dilution of the sample is needed to eliminate ionization suppression which, in turn, permits lower limits of detection and quantitation.