Automated accurate mass data processing using a gas chromatograph/time-of-flight mass spectrometer in drug discovery

A gas chromatograph/time-of-flight (GCT) mass spectrometer, with high mass measurement accuracy to within 5 ppm, has been used for the automated accurate mass analysis of multicomponent mixtures and drug discovery compounds. A multicomponent mixture was analyzed several times over the course of a week to assess the reproducibility and ruggedness of the automated method while operating the GCT in electron ionization mode. For example, the data for 31 radical cations generated via electron ionization was processed using automated software (i.e. OpenLynx) to provide for mass accuracies less than 5 ppm for nearly 100% of the ions from multiple injection data. Mass accuracies of the radical anions of polyaromatic hydrocarbons generated via negative chemical ionization, and protonated pyridines and quinolines generated via methane chemical ionization, were mainly less than 5 ppm from multiple injection data. In addition, the automated method has been used for the accurate mass analysis of drug discovery compounds.     

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.     

Automated multiple-layer spotting for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of synthetic polymers utilizing ink-jet printing technology

Recently, a new multiple-layer matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) sample spotting technique for poly(ethylene glycol), offering improved analysis possibilities, was described. In this contribution the application of ink-jet printing to automated, multiple-layer MALDI-TOFMS sample preparation of synthetic polymers is presented, allowing accurate deposition of matrix, additive and analyte solutions. The new sample preparation technique was evaluated for poly(ethylene glycol) as well as poly(methyl methacrylate) standards, and optimized settings for both synthetic polymers have been obtained.     

Intercomparison study on accurate mass measurement of small molecules in mass spectrometry

The results from an intercomparison of accurate mass measurement of a small molecule (molecular weight 475 Da) across a broad range of mass spectrometers are reported. The intercomparison was designed to evaluate the relative capabilities and the optimum methodology of the diverse range of mass spectrometers currently used to record accurate mass measurements. The data will be used as a basis for developing guidance on accurate mass measurement. The need for guidance has resulted from the continued growth in the use of accurate mass measurements for assignment of elemental formula in the chemical and biochemical industries. This has been fuelled by a number of factors and includes the rapid pace of instrument development, which has enabled accurate mass measurements to be made in a less costly, yet robust fashion. The data from the intercomparison will allow us to compare those protocols that produced excellent accuracy and precision with those that produced poorer accuracy and/or precision for each type of mass spectrometer. The key points for best practice will then be established from this comparison for each type of mass spectrometer and accurate mass measurement technique. A compound was sent to the participating laboratories (in the UK, Europe, and USA), the identity of which was not revealed. Each laboratory was asked to record a minimum of five repeat mass measurements of the molecular species using their local protocols and their preferred ionization technique or techniques. To the best of our knowledge there were no interfering (unresolved) ions that originated from the sample. A questionnaire was also completed with the experimental work. The information from the questionnaires was used to evaluate the protocols used to record the measurements. Forty-five laboratories have reported their results. To summarize the performance of mass spectrometers in the intercomparison, magnetic sector field mass spectrometers used in peak matching mode and FTMS reported the highest mean mass measurement accuracy (88 and 83%, respectively, achieved < or =1 ppm). Magnetic sector field mass spectrometers used in voltage scanning produced 60% of the mean mass measurements with accuracy < or =1 ppm. Magnetic sector field mass spectrometers used in magnet scanning modes, quadrupole-TOF and TOF instruments generally achieved mean mass measurement accuracy between 5 and 10 ppm. The two low resolution triple quadrupoles used in the inter-comparison produced mean mass measurement accuracy of <2 ppm. The precision of the data from each instrument and experiment type is an important consideration when evaluating their relative capabilities. Using both the precision and accuracy, it will be possible to define the uncertainty associated with the elemental formulae derived from accurate mass measurements. Therefore, a thorough statistical evaluation of the data is underway and will be presented in a subsequent publication.     

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).     

A fully automated method for accurate mass determination using high-performance liquid chromatography with a quadrupole/orthogonal acceleration time-of-flight mass spectrometer

A generic LC/ESI(+)-oaTOFMS method has been developed for routine automated high accuracy mass determinations of different classes of substances. The system makes use of micro-high-performance liquid chromatography and a hybrid quadrupole/orthogonal acceleration time-of-flight (Q-oaTOF) mass spectrometer. Reproducible and accurate mass measurements were obtained using an electrospray dual sprayer with reserpine as reference compound, introduced into the mass spectrometer alternating with the samples. Experiments were performed to optimize analyte/reference response ratio, statistical algorithm correction setting, and analyte concentration. In these experiments, a clear dependence of the mass measurement error on the analyte/reference response ratio was observed. The dependence of average mass error versus different dead time correction algorithm settings (Np factors) was also explored. In the final automated procedure, verified for a statistically significant set of compounds ( approximately 550) obtained from a medicinal chemistry department, about 70% of the analyzed samples satisfied the acceptance criteria fixed at a maximum error of +/-5 ppm (mass range 150-800 Da).     

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 automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities

Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r(2) = 0.9998) over two orders of magnitude of concentration of rac-1-phenylethylamine (PEA), which is converted to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (< or =5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes.     

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.     

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.     

Practical quantitative biomedical applications of MALDI-TOF mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) is used to obtain fast and accurate determinations of molecular mass, but quantitative determinations are generally made by other techniques. In this study we illustrate the practical utility of automated MALDI-TOFMS as a tool for quantifying a diverse array of biomolecules covering an extensive molecular weight range, and present in biological extracts and fluids. Growth hormone was measured in rat pituitary tissue; insulin in human pancreatic tissue; homovanillic acid in human urine; and LVV-hemorphin-7, epinephrine and norepinephrine in human adrenal and pheochromocytoma tissues. Internal standards including compounds of similar molecular weight, structural analogs or isotopomers were incorporated into each analysis. We report on the current practical limitations of quantitative MALDI-TOFMS and highlight some of the potential benefits of this technique as a quantitative tool.     

The use of nonpolar matrices for matrix-assisted laser desorption/ionization mass spectrometric analysis of high boiling crude oil fractions

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with nonpolar matrices has been investigated for its applicability to the characterization of atmospheric resid crude oil fractions. The data obtained by use of nonpolar matrices was compared with that from polar matrices as well as from direct LDI-MS and field ionization mass spectrometry. Nonpolar matrices, such as anthracene or 9-cyanoanthracene, yield only a single radical molecular cation upon LDI. Thus, no interfering matrix-related ions are present during the MALDI-TOFMS analysis of the crude oil sample. Nonpolar matrices yield molecular mass distributions from linear mode MALDI-TOFMS that are comparable to distributions found with LDI-MS. An advantage of nonpolar matrices is the increased production of analyte ions, which allows reflectron mode MALDI-TOFMS to be performed. Nonpolar matrices are also shown to be less sensitive to solvent and sample preparation conditions than conventional polar matrices. These results suggest that nonpolar matrices may be favorable alternatives to more traditional polar or acidic matrices commonly used in the MALDI mass spectral characterization of crude oil related samples.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A2 and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase A2 and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained. In addition to the dominant protein [M + H]+ ions, multimeric and multiply charged ions were also observed in the mass spectra. The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected. Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes. Mass accuracies of 0.1 and 0.3% were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants.     

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.     

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.     

Characterization of the molecular mass distribution of pullulans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using 2,5-dihydroxybenzoic acid butylamine (DHBB) as liquid matrix

The performances of several matrices were investigated for the accurate determination of the molecular mass distributions of pullulans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The ionic liquid matrix (ILM) 2,5-dihydroxybenzoic acid butylamine (DHBB) gave better and more reliable results than the crystalline matrices 2,5-dihydroxybenzoic acid (DHB) and 2,4,6-trihydroxyacetophenone (THAP). With the ILM it was possible to obtain spectra of pullulans up to more than 100 kDa, the highest molar mass reported thus far. Owing to the known advantages of liquid matrices providing better spot-to-spot reproducibility, an almost noise-free spectrum and constant baselines were obtained when working under optimized conditions. In particular, the extent of in-source fragmentation occurring with this group of fragile polymers was considerably and decisively reduced. Thus, a more reliable representation of the true oligomer and polymer distributions is experimentally attainable, especially for distributions with small polydispersity values. The maximum error in the measured distribution associated with fragmentation was estimated by model calculations describing the changes in the polymer distribution upon different probabilities of fragmentation events. These simulation results indicated that the data obtained by MALDI-TOFMS using the liquid DHBB matrix were of high reliability. In particular, the average value of the distributions, M(w), and the polydispersity were obtained with predicted uncertainties of between 3 and 15% depending on the width of the distribution and the mass of the polymers. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometry

An efficient method was developed for toxicological drug screening in urine by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The method relies on a large target database of exact monoisotopic masses representing the elemental formulae of reference drugs and their metabolites. Mass spectral identification is based on matching measured accurate mass and isotopic pattern (SigmaFit) of a sample component with those in the database. Data post-processing software was developed for automated reporting of findings in an easily interpretable form. The mean and median of SigmaFit for true-positive findings were 0.0066 and 0.0051, respectively. The mean and median of mass error absolute values for true-positive findings were 2.51 and 2.17 ppm, respectively, corresponding to 0.65 and 0.60 mTh. For routine screening practice, a SigmaFit tolerance of 0.03 and a mass tolerance of 10 ppm were chosen. Ion abundance differences from urine extracts did not affect the accuracy of the automatically acquired SigmaFit or mass values. The results show that isotopic pattern matching by SigmaFit is a powerful means of identification in addition to accurate mass measurement.     

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass measurement accuracy by using delayed extraction

Mass measurement accuracy of a reflectron time-of-flight mass spectrometer equipped with delayed extraction is evaluated. Mass resolution of 10,000–15,000 is achieved routinely for peptides in the mass range of 1–6 ku ionized by matrix-assisted laser desorption ionization. The mass measurement accuracy of peptides with molecular weights of 1–4 ku is 10–15 ppm by using external calibration and better than 5 ppm by using internal calibration. The mass calibration remains accurate over a broad mass-to-charge ratio range (1–4 ku), even when the calibration curve is extrapolated several thousand mass units.     

Improved mass accuracy in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides

One problem of matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry is the moderate mass accuracy that typically can be obtained in routine applications, Here we report improved mass accuracy for peptides, even when low amounts and complex peptide mixtures are used. A new procedure for preparing matrix surfaces is used, and there is no need to mix the matrix with the sample or to add internal standards. Examples are shown with a mass accuracy better than 50 ppm in a peptide mixture. Peptide mapping as well as sequencing by creating “ragged ends” or “ladder sequencing” should benefit especially from the improved mass accuracy.     

Improved method for peak picking in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for peak picking for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. The method is based on the assumption that two sets of ions are formed during the ionization stage, which have Gaussian distributions but different velocity profiles. This gives rise to a certain degree of peak skewness. Our algorithm deconvolutes the peak and utilizes the fast velocity, bulk ion distribution for peak picking. Evaluation of the performance of the new method was conducted using peptide peaks from a bovine serum albumin (BSA) digest, and compared with the commercial peak-picking algorithms Centroid and SNAP. When using the new two-Gaussian algorithm, for strong signals the mass accuracy was equal to or marginally better than the results obtained from the commercial algorithms. However, for weak, distorted peaks, considerable improvement in both mass accuracy and precision was obtained. This improvement should be particularly useful in proteomics, where a lack of signal strength is often encountered when dealing with weakly expressed proteins. Finally, since the new peak-picking method uses information from the entire signal, no adjustments of parameters related to peak height have to be made, which simplifies its practical use.