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).     

Evaluation of the interrelationship between mass resolving power and mass error tolerances for targeted bioanalysis using liquid chromatography coupled to high‐resolution mass spectrometry

The determination of acceptable mass error tolerances for high‐resolution mass spectrometry based signals has been evaluated in a comprehensive way. This was achieved by using a technical approach which is based on the post‐column infusion of an analyte containing solution. This well‐known experimental setup was not used to spot signal suppression regions of a particular analyte, but to spot regions of the chromatogram where a systematic mass drift of the analyte ion can be observed (isobaric interference plot). Not the changing signal intensity but the stability of the measured analyte mass was observed. A wide range of different analytes in combinations with potentially interfering matrices has been evaluated. Furthermore, different mass resolving power settings were evaluated. Isobaric interferences between matrix compounds and analytes were common at mass resolving powers <50 000 full width at half maximum. The proposed post‐column infusion technique is a useful tool for the determination of the assay and matrix‐specific mass error tolerances. It aims to ensure the highest possible selectivity, at the same time preventing the encounter of detrimental mass error related peak deformations as well as false negative findings. Unlike conventional matrix spiking approaches, isobaric interference plots provide information of potential interferences across the whole chromatographic time range. This becomes relevant when there is a relative retention time shift between the analyte and potential interfering matrix compounds. Furthermore, the described setup can be used to study how the mass accuracy of any mass spectrometer is affected by a widely varying total ion current. Copyright © 2012 John Wiley & Sons, Ltd.     

Fourier transform laser microprobe mass spectrometry for the molecular identification of inorganic compounds

This study attempted to determine the molecular composition of inorganic analytes at the surface of solids by Fourier transform laser microprobe mass spectrometry (FT LMMS) with an external ion source. A database was established from the analysis of pure compounds. FT LMMS uses a similar ionization as the older LMMS instruments with time-of-flight (TOF) mass analyzer. However, apart from the mass resolution, the mass spectral patterns can be significantly different in FT LMMS compared to TOF LMMS. FT LMMS yields detailed information on the analyte by means of structural fragments, enabling us to specify the main building blocks, as well as adduct ions, consisting of the analyte molecule and a stable ion. Hence, deductive reasoning allows tentative characterization of the analogs without reference spectra, except for compounds with the same elements in different stoichiometries. In that case comparative data are needed.     

Accurate mass measurements of positive ions in the desorption chemical ionization mode

Desorption chemical ionization mass spectrometry employing ammonia as the reagent gas has been extensively used to obtain molecular mass and structural information on a wide variety of compounds. Mass-deficient reference standards normally used for calibration purposes in mass spectrometry do not provide adequate mass spectra under ammonia chemical ionization conditions. In order to overcome this problem a mixture of ammonia and methane as reagent gases was employed. In high-resolution accurate mass measurement experiments, this gas mixture allows the simultaneous detection of mass spectra of perfluorokerosene adequate for calibration purposes and spectra containing molecular mass information of the analyte. A needle valve system was used to control the composition of the gas mixture introduced into the ion source. For positive-ion accurate mass measurements of higher masses (up to m/z = 2300), Fomblin 18/8 oil was successfully used as a reference standard under ammonia, methane and isobutane desorption chemical ionization conditions.     

Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry

The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches. In these cases higher effective temperatures could be measured accompanied by increased analyte intensities, which can be explained by stronger volatilization processes caused by a hindered heat dissipation resulting in a raised analyte transfer into the gas phase. For all sublimed compounds, a strong increase of the initial ion velocity compared with DD preparations could be measured. Higher initial ion velocities correlate with a decrease in internal ion energy which might be attributed to the very uniform crystal morphology exhibited by sublimed compounds. For sublimed matrices without reduced grain size, at least slightly higher analyte intensities could be detected at raised laser fluences. Analyte accumulation in the uppermost matrix layers or the detected higher ion stability can be explanations for these results.     

Uncertainty in analyte mass for samples containing small numbers of particles.

A sampling equation was derived that relates the standard deviation in analyte mass to the number of particles in the sample, the fractions of the different types of particles in the mixture and the masses and analyte concentrations of the individual particles. The equation, which is applicable to samples containing any number of particles, was verified by sampling and analysis of two cereal grain mixtures for manganese, potassium, chlorine and magnesium, and by Monte Carlo computer simulation. Comparison of the sampling precision of analyte mass with the analytical measurement precision was also studied, and it was shown that use of the equation allows the calculation of the minimum number of particles required to hold the sampling relative standard deviation to that of the analytical measurement.     

Cavitation of liquid flow in electrospray mass spectrometry especially for addition of internal calibrant in 'accurate mass' measurements

'Accurate mass' measurements in electrospray mass spectrometry are becoming more prevalent with the increasing availability of mass spectrometers with sufficient resolution. A reference compound is ideally admitted separately but almost simultaneously with the analyte and this is achieved by use of a 'dual sprayer' or voltage switching between reference and sample sprayers. This paper describes a novel third method, relying on cavitation (segmentation) of the liquid stream containing the reference compound, allowing the sample to ionise independently from the sample, thus preventing interference. The technique may also find application in kinetic experiments, such as protein folding studies. A high-performance liquid chromatography (HPLC) injector was also used to provide a reference compound, producing Gaussian-shaped profiles of varying ion intensity, thus allowing easier selection of a desirable measurement point where the intensities of the reference and analyte were similar.     

Structural analysis of diols by electrospray mass spectrometry on boric acid complexes

A method is presented to characterize diols using negative ion electrospray (ES) mass spectrometry in combination with collision-induced dissociation tandem mass spectrometry (MS/MS). The analyte diol is added to a solution containing an ethylene glycol/boric acid [2:1] complex and then subjected to infusion ES. The following boric acid complexes are formed: (i) a complex with two ethylene glycol molecules, (ii) a mixed ethylene glycol/analyte complex, and (iii) a complex with two analyte molecules. The first complex serves as a reference for the assessment of the extent of complex formation with the analyte. The ES mass spectra of acyclic vicinal diols all feature intense mixed complex signals, indicative of efficient complex formation. Chemical fine tuning is achieved by MS/MS experiments. Thus, although the (2R,3R)-(-)-2,3-butanediol and meso-2,3-butanediol stereo-isomers show the same complexation efficiency, MS/MS experiments reveal pronounced structure characteristic differences. By contrast, 1,3- and 1,4-diols are less prone to complex formation as they give only weak signals relative to the reference. For cyclic vicinal diols only the cis isomer produces an intense mixed complex, whose MS/MS spectrum is characteristically different from that of the trans form. The above procedure does not permit an unambiguous differentiation of acyclic polyhydroxy compounds like mannitol and sorbitol. However, structurally related methyl glycosides show characteristic MS/MS spectra. Our findings indicate that the above simple procedure may be useful to probe the presence and structure of diols and other polyols in aqueous solutions. Copyright 1999 John Wiley & Sons, Ltd.     

Thin-layer chromatography combined with diode laser desorption/atmospheric pressure chemical ionization mass spectrometry

The desorption of an analyte by a continuous wave diode laser from a porous surface of a thin-layer plate covered with a graphite suspension is presented. The thermally desorbed analyte molecules are ionized in the gas phase by a corona discharge at atmospheric pressure. Therefore, both essential processes--the desorption and the ionization of analyte molecules, which are often performed in one step--are separated. The target preparation is easy and fast since no additional extraction process is required. The mass spectrometric background signal was mostly limited to the low mass range showing no interference with typical compounds of interest. In this study, the calmative and antihypertensive drug reserpine was chosen as model analyte, which is often used for specification of mass spectrometers. No fragmentation was observed because of efficient collisional cooling under atmospheric pressure. The influence of diode laser power and the composition of the graphite suspension were investigated, and a primary optimization was performed.     

Accurate mass determination by multiple sprayers nano-electrospray mass spectrometry on a magnetic sector instrument

A new technique for accurate mass determination by using multiple sprayers nano-electrospray ionization mass spectrometry (nano-ESI-MS) on a magnetic sector instrument is described. Metal coated glass capillaries were used as nano-ESI sprayers. One of the sprayers was used for the reference compound solution, and others were used for the introduction of sample solutions. The spectra of the different compounds were obtained by shifting each sprayer's position relative to the sampling orifice. The accurate masses of several standard compounds were obtained with good accuracy, without problems arising from differences in ionization efficiency between the sample compounds and reference compound.     

Matrix effects on accurate mass measurements of low-molecular weight compounds using liquid chromatography-electrospray-quadrupole time-of-flight mass spectrometry

Liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) represents a powerful technique for the identification and/or confirmation of small molecules, i.e. drugs, metabolites or contaminants, in different matrices. However, reliability of analyte identification by HRMS is being challenged by the uncertainty that affects the exact mass measurement. This parameter, characterized by accuracy and precision, is influenced by sample matrix and interferent compounds so that questions about how to develop and validate reliable LC-HRMS-based methods are being raised. Experimental approaches for studying the effects of various key factors influencing mass accuracy on low-molecular weight compounds (MW < 150 Da) when using a quadrupole-time-of-flight (QTOF) mass analyzer were described. Biogenic amines in human plasma were considered for the purpose and the effects of peak shape, ion abundance, resolution and data processing on accurate mass measurements of the analytes were evaluated. In addition, the influence of the matrix on the uncertainty associated with their identification and quantitation is discussed. A critical evaluation on the calculation of the limits of detection was carried out, considering the uncertainty associated with exact mass measurement of HRMS-based methods. The minimum concentration level of the analytes that was able to provide a statistical error lower than 5 ppm in terms of precision was 10 times higher than those calculated with S/N = 3, thus suggesting the importance of considering both components of exact mass measurement uncertainty in the evaluation of the limit of detection.     

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.     

Selective isotopic exchange of polyfimctional ions in tandem mass spectrometry: Methodology,applications and mechanism

Isotopic exchange of mass-selected odd- and even-electron molecular ions of aromatic compounds upon collision with deuterated gases was investigated as a function of reagent gas, interaction time and collision energy. Use of ND₃ as reagent allows exchange of all active hydrogens for the compound types studied, providing a count of the total number of active hydrogens present in the analyte. CH₃OD exchanges specific types of active hydrogens, such as phenolic and carboxylic hydrogens, without exchanging amino hydrogens. This selectivity assists in the identification and enumeration of different types of active hydrogens present in polyfunctional compounds. The H–D exchange patterns serve to differentiate isomeric aromatic compounds containing methoxy, amino, hydroxy and carboxylic acid substituents. Trapping of mass-selected ions in the collision region of a triple quadrupole mass spectrometer greatly enhances the degree of H–D exchange, thereby facilitating determination of the number of active hydrogens in the analyte. Triple stage mass spectrometric experiments, performed in a pentaquadrupole mass spectrometer, help elucidate the exchange process. Isotopic exchange in the collision region of a tandem mass spectrometer also provides insights into the site of protonation in molecules containing several functional groups. The proximity of the functional groups and the proton affinity difference between the analyte and the reagent gas are important factors in site-specific H–D exchange in polyfunctional compounds. An investigation of the effects of collision energy reveals that cluster ion formation plays a major role in the exchange mechanism operating in the triple quadrupole and that H–D exchange, ion-molecule adduct formation and endothermic fragmentation are competitive reaction channels.     

Negative ion electrospray ionization mass spectral study of dicarboxylic acids in the presence of halide ions

The negative ion electrospray ionization (ESI) mass spectra of a series of dicarboxylic acids, a pair of isomeric (cis/trans) dicarboxylic acids and two pairs of isomeric (positional) substituted benzoic acids, including a pair of hydroxybenzoic acids, were recorded in the presence of halide ions (F(-), Cl(-), Br(-) and I(-)). The ESI mass spectra contained [M--H](-) and [M+X](-) ions, and formation of these ions is found to be characteristic of both the analyte and the halide ion used. The analytes showed a greater tendency to form adduct ions with Cl(-) under ESI conditions compared with the other halide ions used. The isomeric compounds yielded distinct spectra by which the isomers could be easily distinguished. The collision-induced dissociation mass spectra of [M+X](-) ions reflected the gas-phase basicities of both the halide ion and [M--H](-) ion of the analyte. However, the relative ordering of gas-phase basicities of all analyte [M--H](-) and halide ions could not account for the dominance of chloride ion adducts in ESI mass spectra of the analytes mixed with equimolar quantities of the four halides.     

Estimation of the minimum uncertainty of DNA concentration in a genetically modified maize sample candidate certified reference material

Homogeneity testing and the determination of minimum sample mass are an important part of the certification of reference materials. The smallest theoretically achievable uncertainty of certified concentration values is limited by the concentration distribution of analyte in the different particle size fractions of powdered biological samples. This might be of special importance if the reference material is prepared by dry mixing, a dilution technique which is used for the production of the new and third generation of genetically modified (GMO) plant certified reference materials. For the production of dry mixed PMON 810 maize reference material a computer program was developed to calculate the theoretically smallest uncertainty for a selected sample intake. This model was used to compare three differently milled maize samples, and the effect of dilution on the uncertainty of the DNA content of GMO maize was estimated as well. In the case of a 50-mg sample mass the lowest achievable standard deviation was 2% for the sample containing 0.1% GMO and the minimum deviation was less than 0.5% for the sample containing 5% GMO.     

Characterization of humic substances by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and laser desorption/ionization (LDI-)TOFMS have been used to characterize Suwannee River humic substances, obtained from the International Humic Substances Society (IHSS), and Armadale soil fulvic acid (ASFA). An array of MALDI matrices were tested for use with humic substances, including alpha-cyano-4-hydroxycinammic acid (CHCA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,5-dihydroxybenzoic acid (DHBA), sinapinic acid, dithranol and norharmane. DHBA yielded the best results, exhibiting superior ionization efficiency, low noise, broad applicability to the analytes of interest, and most importantly producing an abundance of high mass ions, the highest observed being m/z 1848. A number of sample preparation modes were investigated; the overlayer method improved sample/matrix homogeneity and hence shot-to-shot reproducibility. The choice of the matrix, mass ratio of analyte to matrix, and the sample preparation protocol, were found to be the most critical factors governing the quality of the mass spectra. Matrix suppression was greatly enhanced by ensuring good mixing of matrix and analyte in the solid phase, proper optimization of the matrix/analyte ratio, and optimizing delayed extraction to ensure complete matrix-analyte reaction in the plume before ions are moved to the flight tube. A number of common features, in particular specific ions which could not be attributed to the matrices or to contaminants, were present in the spectra of all the humic substances, regardless of origin or operational definition. Additionally, a prominent repeating pattern of peaks separated by 55, 114 and 169 Da was clearly observed in both LDI and MALDI, suggesting that the humic compounds studied here may have quasi-polymeric or oligomeric features.     

Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry

An experimental investigation and theoretical analysis are reported on charge competition in electrospray ionization (ESI) and its effects on the linear dynamic range of ESI mass spectrometric (MS) measurements. The experiments confirmed the expected increase of MS sensitivities as the ESI flow rate decreases. However, different compounds show somewhat different mass spectral peak intensities even at the lowest flow rates, at the same concentration and electrospray operating conditions. MS response for each compound solution shows good linearity at lower concentrations and levels off at high concentration, consistent with analyte "saturation" in the ESI process. The extent of charge competition leading to saturation in the ESI process is consistent with the relative magnitude of excess charge in the electrospray compared to the total number of analyte molecules in the solution. This ESI capacity model allows one to predict the sample concentration limits for charge competition and the on-set of ionization suppression effects, as well as the linear dynamic range for ESI-MS. The implications for quantitative MS analysis and possibilities for effectively extending the dynamic range of ESI measurements are discussed.     

Functional group selective ion/molecule reactions: mass spectrometric identification of the amido functionality in protonated monofunctional compounds

A mass spectrometric method was developed for the screening of the amido functionality in monofunctional protonated analytes. This method is based on selective gas-phase derivatization of protonated analytes by (N,N-diethylamino)dimethylborane in a Fourier transform ion cyclotron resonance (FT-ICR) and triple quadrupole mass spectrometer. Examination of a series of protonated analytes demonstrated that only the compounds containing the amido functionality react with the aminoborane by the derivatization reaction. The mechanism involves proton transfer from the protonated analyte to the borane, followed by addition of the amide to the boron center, which leads to the elimination of neutral diethylamine. The derivatized analytes are readily identified on the basis of a shift of 40 m/z units relative to the m/z value of the protonated analyte and characteristic boron isotope patterns. Collision-activated dissociation was used to provide support for the structures assigned to the derivatized analytes. The structural information gained from this gas-phase derivatization method will aid in the functional group identification of unknown compounds and their mixtures.     

Solid state chemical ionization for characterization of organic compounds by laser mass spectrometry

A new technique involving the addition of a compound to the analyte to serve as a source of "reagent" ions has been developed for negative-ion laser mass spectrometry. This "solid state chemical ionization" leads to ions characteristic of the analyte, owing to ion-molecule reactions between the "reagent" ion and the neutral analyte in the laser-generated plume. Polycyclic aromatic hydrocarbons show formation of an ion corresponding to (M + O - H)(-) in their negative-ion laser mass spectra when mixed with compounds such as sym-trinitrobenzene, sodium nitrate and sodium peroxide. NO(-)(2), O(-), and O(-)(2) serve as "reagent" ions in these compounds. Formation of (M + Cl)(-) is seen in the laser mass spectra of glycosides mixed with hexachlorobenzene. Chloride serves as the "reagent" ion in this case.     

Thin layer chromatography/mass spectrometry

Thin layer chromatography (TLC)--a simple, cost-effective, and easy-to-operate planar chromatographic technique--has been used in general chemistry laboratories for several decades to routinely separate chemical and biochemical compounds. Traditionally, chemical and optical methods are employed to visualize the analyte spots on the TLC plate. Because direct identification and structural characterization of the analytes on the TLC plate through these methods are not possible, there has been long-held interest in the development of interfaces that allow TLC to be combined with mass spectrometry (MS)--one of the most efficient analytical tools for structural elucidation. So far, many different TLC-MS techniques have been reported in the literature; some are commercially available. According to differences in their operational processes, the existing TLC-MS systems can be classified into two categories: (i) indirect mass spectrometric analyses, performed by scraping, extracting, purifying, and concentrating the analyte from the TLC plate and then directing it into the mass spectrometer's ion source for further analysis; (ii) direct mass spectrometric analyses, where the analyte on the TLC plate is characterized directly through mass spectrometry without the need for scraping, extraction, or concentration processes. Conventionally, direct TLC-MS analysis is performed under vacuum, but the development of ambient mass spectrometry has allowed analytes on TLC plates to be characterized under atmospheric pressure. Thus, TLC-MS techniques can also be classified into two other categories according to the working environment of the ion source: vacuum-based TLC-MS or ambient TLC-MS. This review article describes the state of the art of TLC-MS techniques used for indirect and direct characterization of analytes on the surfaces of TLC plates.