Electrospray Ionization Response Research Articles

Understanding mobile phase buffer composition and chemical structure effects on electrospray ionization mass spectrometry response

Mobile phase selection is of critical importance in liquid chromatography – mass spectrometry (LC-MS) based studies, since it affects retention, chromatographic selectivity, ionization, limits of detection and quantification, and linear dynamic range. Generalized LC-MS mobile phase selection criteria, suitable for a broad class of chemical compounds, do not exist thus far. Here we have performed a large-scale qualitative assessment of the effect of solvent composition used for reversed-phase LC separations on electrospray ionization (ESI) response for 240 small molecular weight drugs, representing various chemical compound classes. Of these 240 analytes 224 were detectable using ESI. The main chemical structural features affecting ESI response were found to all be surface area or surface charge-related. Mobile phase composition was found to be less differentiating, although for some compounds a pH effect was noted. Unsurprisingly, chemical structure was found to be the dominant factor for ESI response for the majority of the investigated analytes, representing about 85% of the replicating detectable complement of the sample data set. A weak correlation between ESI response and structure complexity was observed. Solvents based on isopropanol, and those containing phosphoric or di- and trifluoracetic acids, performed relatively poorly in terms of chromatographic or ESI response, whilst the best performing ‘generic’ LC solvents were based on methanol, acetonitrile using formic acid and ammonium acetate as buffer components, consistent with current practice in many laboratories.

Bisphenol A and its structural analogues in infant formulas available in the Brazilian market: Optimisation of a UPLC-MS/MS method, occurrence, and dietary exposure assessment

For the first time, structural analogues to bisphenol A were investigated in infant formulas marketed in Brazil. A fast and high throughput UPLC-MS/MS method was established for simultaneous analysis of bisphenol A, B, E, F, and S in complex infant formula matrices. The influence of mobile phase composition on electrospray ionization response in negative mode was studied to improve the detectability of the method. As also, the main sample preparation variables that could affect the extraction and cleanup were screened by the Plackett-Burman design. The method performance characteristics were adequate, including reliable limits of detection (5–10 µg kg−1) and quantification (10–20 µg kg−1) with suitable recoveries (84.2–108.9 %) and precision (≤18 %). Sixty-one infant formulas were analyzed, and 36 % of total samples contained at least one bisphenol analogue, whose levels ranged between 10.9 and 198.9 µg kg−1. Based on a deterministic approach, the estimated daily intakes for babies up to 6 months old, fed exclusively with infant formula, were below the temporary tolerable daily intake of 4 µg kg−1 body weight set for bisphenol A by the European Food Safety Authority.

Comprehensive assessment of the diminished statistical power caused by nonlinear electrospray ionization responses in mass spectrometry-based metabolomics

It seems to be well received that nonlinear electrospray ionization (ESI) distorts the signal distribution in mass spectrometry (MS) analysis, thus leading to diminished statistical power for t-test. However, the exact consequence and possible solutions to this quantitative issue have not been systematically explored. In this work, using a serial diluted urine metabolomics dataset, we demonstrated that over 80% of the metabolic features present nonlinear ESI response patterns, causing either left-skewed or right-skewed MS signal distributions. Among them, right-skewed MS distributions cannot be rescued using conventional data transformation (e.g., log transformation, power transformation). Furthermore, using a Monte Carlo simulation, we quantitatively assessed the reduced statistical power for t-test calculated using MS signal data in various sample sizes and effect sizes. In all these comparisons, t-test using MS signal data has consistently lower statistical power than t-test using metabolic concentration data. To address this statistical issue, we proposed a bioinformatic workflow, termed PowerU, to minimize the diminished statistical power caused by both the nonlinear ESI response and the intrinsic non-normal distribution of metabolic concentrations. The PowerU workflow is composed of two steps. The first step is to convert MS signals to quality control (QC) sample injection amounts to solve the skewed MS signal distributions. The second step is to perform a Shapiro-Wilk test to determine data normality and then use the normality results to guide the application of t-test and Mann-Whitney U test for the best statistical outcome. PowerU was tested using a metabolomics study of mouse cecum samples. Results demonstrate that the PowerU workflow can significantly boost statistical power for t-test and facilitate the discovery of significantly altered metabolites for downstream biological interpretation.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

Understanding the electrospray ionization response factors of per- and poly-fluoroalkyl substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are used extensively in commercial products. Their unusual solubility properties make them an ideal class of compounds for various applications. However, these same properties have led to significant contamination and bioaccumulation given their persistence in the environment. Development of analytical techniques to detect and quantify these compounds must take into account the potential for these properties to perturb these measurements, specifically the potential to bias the electrospray ionization (ESI) process. Direct injection ESI analysis of 23 different PFAS species revealed that hydrophobicity and PFAS class can predict the ESI overall response factors. In this study, a method for predicting the behavior of individual PFAS compounds, including relative retention order in chromatography, is presented which is simply based on the number of fluorine atoms in the molecule as well as the class of the compound (e.g., perfluroalkylcarboxylic acids) vs. computational estimations (e.g., non-polar surface area and logP).

Overestimation of 3α- over 3β-25-Hydroxyvitamin D3 Levels in Serum: A Mechanistic Rationale for the Different Mass Spectral Properties of the Vitamin D Epimers.

The metabolism of vitamin D3 includes a parallel C-3 epimerization pathway-in addition to the standard metabolic processes for vitamin D3-reversing the stereochemical configuration of the -OH group at carbon-3 (β→α). While the biological function of the 3α epimer has not been elucidated yet, the additional species cannot be neglected in the analytical determination of vitamin D3, as it has the potential to introduce analytical errors if not properly accounted for. Recently, some inconsistent mass spectral behavior was seen for the 25-hydroxyvitamin D3 (25(OH)D3) epimers during quantification using electrospray LC-MS/MS. The present work extends that of Flynn et al. ( Ann. Clin. Biochem. 2014, 51, 352-559) and van den Ouweland et al. ( J. Chromatogr. B 2014, 967, 195-202), who reported larger electrospray ionization response factors for the 3α epimer of 25(OH)D3 in human serum samples as compared to the regular 3β variant. The present work was concerned with the mechanistic reasons for these differences. We used a combination of electrospray ionization, atmospheric pressure chemical ionization, and density functional theory calculations to uncover structural dissimilarities between the epimers. A plausible mechanism is described based on intramolecular hydrogen bonding in the gas phase, which creates a small difference of proton affinities between the epimers. More importantly, this mechanism allows the explanation of the different ionization efficiencies of the epimers based on kinetic control of the ionization process, where ionization initially takes place at the hydroxyl group with subsequent proton transfer to a basic carbon atom. The barrier for this transfer differs between the epimers and is in direct competition with H2O elimination from the protonated hydroxyl group. The "hidden" site of high gas phase basicity was revealed through computational calculations and appears to be inaccessible via direct protonation.

Isomer separation and analysis of amphiphilic polysialogangliosides using reversed-phase liquid chromatography-mass spectrometry.

Gangliosides are amphiphilic, acidic glycosphingolipids possessing one or more sialic acid residues and several isobaric structural isomers with different abundances and bioactivities. Therefore, the distinction between these isomers is crucial for their proper profiling. Although liquid chromatography-mass spectrometry has been successfully employed for this purpose, the distinction process can still be improved, particularly regarding liquid chromatography. Recently, a reversed-phase liquid chromatography method that could separate disialoganglioside isomers was reported; however, the distinction of trisialoganglioside isomers using reversed-phase liquid chromatography has not been demonstrated. Here, we investigated the practicality of a reversed-phase liquid chromatography with an octadecylsilane column for separating polysialoganglioside isomers and successfully achieved the isomer separation of disialogangliosides and trisialogangliosides for the first time. We also confirmed several crucial factors in the mobile-phase composition, which affect the differential retention and mass spectral response of the isomers. First, an organic modifier, acetonitrile, exhibited superior selectivity against polysialogangliosides over methanol. Second, ammonium bicarbonate was the best ammonium salt additive among those tested, in terms of the separation efficiency and mass spectral response. Third, as the ammonium salt concentration increased, the negative electrospray ionization response was extensively suppressed, and the retention of gangliosides increased.

Patterned Signal Ratio Biases in Mass Spectrometry-Based Quantitative Metabolomics.

Despite the well-known nonlinear response of electrospray ionization (ESI) in mass spectrometry (MS)-based analysis, its complicated response patterns and negative impact on quantitative comparison are still understudied. We showcase in this work that the patterns of nonlinear ESI response are feature-dependent and can cause significant compression or inflation to signal ratios. In particular, our metabolomics study of serial diluted human urine samples showed that over 72% and 16% metabolic features suffered ratio compression and inflation, respectively, whereas only 12% of the signal ratios represent real metabolic concentration ratios. More importantly, these ratio compression and inflation largely exist in the linear response ranges, suggesting that it cannot be resolved by simply diluting the sample solutions to the linear ESI response ranges. Furthermore, we demonstrated that a polynomial regression model that converts MS signals to sample injection amounts can correct the biased ratios and, surprisingly, outperform the linear regression model in both data fitting and data prediction. Therefore, we proposed a metabolic ratio correction (MRC) strategy to minimize signal ratio bias in untargeted metabolomics for accurate quantitative comparison. In brief, by using the data of serial diluted quality control (QC) samples, we applied a cross-validation strategy to determine the best regression model, between linear and polynomial, for each metabolic feature and to convert the measured MS intensities to QC injection amounts for accurate metabolic ratio calculation. Both the studies of human urine samples and a metabolomics application supported that our MRC approach is very efficient in correcting the biased signal ratios. This novel insight of patterned ESI nonlinear response and MRC workflow can significantly benefit the downstream statistical comparison and biological interpretation for untargeted metabolomics.

Fold-Change Compression: An Unexplored But Correctable Quantitative Bias Caused by Nonlinear Electrospray Ionization Responses in Untargeted Metabolomics.

The nonlinear signal response of electrospray ionization (ESI) presents a critical limitation for mass spectrometry (MS)-based quantitative analysis. In the field of metabolomics research, this issue has largely remained unaddressed; MS signal intensities are usually directly used to calculate fold changes for quantitative comparison. In this work, we demonstrate that, due to the nonlinear ESI response, signal intensity ratios of a metabolic feature calculated between two samples may not reflect their real metabolic concentration ratios (i.e., fold-change compression), implying that conventional fold-change calculations directly using MS signal intensities can be misleading. In this regard, we developed a quality control (QC) sample-based signal calibration workflow to overcome the quantitative bias caused by the nonlinear ESI response. In this workflow, calibration curves for every metabolic feature are first established using a QC sample injected in serial injection volumes. The MS signals of each metabolic feature are then calibrated to their equivalent QC injection volumes for comparative analysis. We demonstrated this novel workflow in a targeted metabolite analysis, showing that the accuracy of fold-change calculations can be significantly improved. Furthermore, in a metabolomic comparison of the bone marrow interstitial fluid samples from leukemia patients before and after chemotherapy, an additional 59 significant metabolic features were found with fold changes larger than 1.5, and an additional 97 significant metabolic features had fold changes corrected by more than 0.1. This work enables high-quality quantitative analysis in untargeted metabolomics, thus providing more confident biological hypotheses generation.

A comparative study of the electrospray ionization response of β-O-4' lignin model compounds.

Electrospray ionization mass spectrometry has recently become the technique of choice for rapid characterization of lignin degradation products. However, the fundamental question of the relationship between lignin structure and ionization efficiency has not been explored. In this work, we studied the electrospray ionization response of five structurally similar β-O-4' model lignin compounds using lithium cationization in the positive electrospray ionization mode. The studied compounds have the same β-O-4' backbone structure but differ at the α-position by increasing nonpolar side chains. Our results show a correlation between the ionization response and the length of the nonpolar side chain, with analytes having the longest side chain recording the highest ESI response in the full scan mode. Factors affecting the formation of analyte ions and analyte cluster ions were also studied. We have shown for the first time in this work that the introduction of a nonpolar group onto a β-O-4' lignin compound can increase the lithium cationization ESI response in the positive ion mode.

Supercharging and multiple reaction monitoring of high-molecular-weight intact proteins using triple quadrupole mass spectrometry.

Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins. The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis. Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.5% formic acid to examine the electrospray ionization response for three model proteins: bovine serum albumin (66 kDa), holo-transferrin (78 kDa), and immunoglobulin G (150 kDa). The settings of ionization source temperature and mobile phase flow rate were also examined. MRM transitions were developed for a wide range of precursor ions for each protein, and limits of detection were determined for the proteins in the presence of favorable additive combinations. For most of the proteins, m-NBA (1%) and TFE (5%) worked most effectively, both to shift the charge state and increase intensity. This is the first report of the use of TFE as an effective agent for both increasing protein chargeability and ionization response. TFE increased ionization efficiency between 3- and 14-fold for the model proteins studied. Increases in both source temperature and flow rate reduced the magnitude of the average charge state observed. The MRM transitions of six to eight different precursor ions of the proteins were optimized and limits of detection in the nanogram quantity on column were determined. The feasibility for top-down quantitative analysis of high-molecular-weight proteins with a triple quadrupole mass spectrometer was demonstrated. Further, additives such as TFE can be highly beneficial for increased chargeability and response of the proteins.

Comparison of the electrospray ionization (ESI) responses of penicillins with ESI responses of their methanolysis products.

The electrospray ionization (ESI) responses, defined as the area of chromatographic peak of ion [M+H]+ obtained upon HPLC/ESI-MS analysis, of three β-lactam antibiotics, namely penicillin G, ampicillin and carbenicillin have been compared with the ESI responses of their methanolysis products. It has been found that methanolyzed penicillin G has much higher ESI response than the penicillin G. Methanolyzed ampicillin also has higher ESI response than ampicillin; however, the effect is less pronounced than for penicillin. Methanolyzed carbenicillin does not have pronouncedly higher ESI response than carbenicillin.

Ammonium hydroxide enhancing electrospray response and boosting sensitivity of bisphenol A and its analogs

As one of the most important analytical techniques for accurate quantification of bisphenol compounds, liquid chromatography coupled to online electrospray ionization (ESI) tandem mass spectrometry exhibits relative weak ESI response in negative mode, which greatly reduces their sensitivity and limits their detection at trace levels, especially in complex samples such as blood. Based on the facilitated deprotonation of bisphenol molecule under alkaline condition, post-column injection of ammonium hydroxide (NH3·H2O) to mass spectrometer was explored to enhance the ionization efficiency of BPA and its eight analogs and improve their analytical sensitivity. Parameters effecting response intensity and stability were investigated, including mass concentration and flow rate of NH3·H2O. Under the optimal conditions with the addition of 2% (w/w) of NH3·H2O at 4 μL min−1, the instrument detection limits for bisphenol compounds were greatly lowered to 0.001–0.04 ng mL−1, which were 2–28.6 times lower than the result obtained without injecting NH3·H2O, except TBBPA (0.03 ng mL−1 in either case). The relative standard deviations (RSDs) for instrument repeatability of BPA and its analogs at three different concentration levels were in a range of 1.2–20%. Furthermore, a decreased matrix effects (90–111%) for bisphenols (except TBBPA) analysis in serum extracts were found compared with the result obtained without NH3·H2O injection (43–111%). The results demonstrated that the improved instrumental method coupled with suitable pretreatment techniques is more feasible to analyze bisphenol compounds in complex bio-samples.

Physicochemical Parameters Affecting the Electrospray Ionization Efficiency of Amino Acids after Acylation.

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC–MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure–property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response.

Implications for Metabolite Quantification by Mass Spectrometry in the Absence of Authentic Standards.

Quantification of metabolites by mass spectrometry in the absence of authentic reference standards or without a radiolabel is often called "semiquantitative," which acknowledges that mass spectrometric responses are not truly quantitative. For many researchers, it is tempting to pursue this practice of semiquantification in early drug discovery and even preclinical development, when radiolabeled absorption, distribution, metabolism, and excretion studies are being deferred to later stages of drug development. The caveats of quantifying metabolites based on parent drug response are explored in this investigation. A set of 71 clinically relevant drugs/metabolites encompassing common biotransformation pathways was subjected to flow injection analysis coupled with electrospray ionization (ESI) mass spectrometry. The results revealed a large variation in ESI response even for structurally similar parent drug/metabolite pairs. The ESI response of each metabolite was normalized to that of the parent drug to generate an ESI relative response factor. Overall, relative response factors ranged from 0.014 (>70-fold lower response than parent) to 8.6 (8.6-fold higher response than parent). Various two-dimensional molecular descriptors were calculated that describe physicochemical, topological, and structural properties for each drug/metabolite. The molecular descriptors, along with the ESI response factors, were used in univariate analyses as well as a principal components analysis to ascertain which molecular descriptors best account for the observed discrepancies in drug/metabolite ESI response. This investigation has shown that the practice of using parent drug response to quantify metabolites should be used with caution.

Using the partial least squares method to model the electrospray ionization response produced by small pharmaceutical molecules in positive mode.

The signal intensity in electrospray ionization mass spectrometry (ESI-MS) positive mode is affected by parameters that are related to the physicochemical properties and structural features of a molecule. Accordingly, the combined interactions of an analyte and the mobile phase used is still an area that demands further clarifications since there is no general pattern regarding the nature of a molecule and the mechanism by which vapor-phase ions are produced. A multivariate analysis method, such as Partial Least Squares (PLS), provides the opportunity to correlate the effect of a large number of parameters interpreting this complex procedure with the use of appropriate mathematical algorithms. This work involves the development of models containing up to 84 X variables which characterize the analytes studied (99) focusing on their positive or negative effect on the vapor-phase ion formation process. These descriptors are correlated with the signal response of the positively charged analyte ions which corresponds to the Y variable. The results showed that parameters referring or directly related to the ionization percentage of basic or acidic groups of an analyte can be used to determine the signal response on positive ESI-MS mode. Structural characteristics, polar surface area, lipophilicity, the ability of analytes to acts as hydrogen bond donors or acceptors, water solubility, density of a solid, surface tension of the substance and the number of free rotatable bonds are descriptors which are of secondary importance, still they cannot be considered negligible. The models derived are proved to be reliable for the investigation of such mechanisms, with a small number of components and good linearity (R(2) >83%, Q(2) >70%).

Spatial effects on electrospray ionization response

The position of the spray-head, the solvent, and a variety of additional instrumental parameters were independently adjusted during electrospray ionization mass spectrometric (ESI-MS) analysis of an equimolar mixture of two different ions. These parameters were found to have drastic effects on the distribution of signal intensity from one ion to another, and therefore the resulting usefulness of acquired spectra. The analytes studied were bis(triphenylphosphine)iminium (PPN) chloride and tetramethylammonium (TMA) chloride, two chemically distinct ions. The use of these two ions in a test solution yielded information regarding ESI probe spatial effects for two very different analytes, while probing the issue of sampling efficiency. Each experimental parameter was individually adjusted prior to rastering the spray head across the operational plane in order to observe how adjustment to a particular parameter affects analyte signal in relation to the distance from the MS aperture. Following acquisition, the intensities of both ions were plotted as ion contour maps demonstrating the intensity change with respect to capillary position in relation to the mass spectrometer aperture. The sharp contrast in ion intensity, and even differential ion activity, with relatively minor instrument changes (such as temperature programming, gas flow rates and solvent choice) clearly demonstrates the importance of finding the “sweet spot” for the ESI spray head, especially when signal intensity and a quality analysis are key.

Dependence of negative-mode electrospray ionization response factors on mobile phase composition and molecular structure for newly-authenticated neutral acylsucrose metabolites.

Authentic standards of known concentrations serve as references for accurate absolute quantification of plant metabolites using liquid chromatography/mass spectrometry (LC/MS). However, often such standards are not commercially available or not amenable for custom syntheses. Despite the widespread use of electrospray ionization for metabolite analyses, the fundamentals needed for reliable prediction of molecular response factors have yet to be explored in detail for analytes that lack ionized functional groups. In order to lay a foundation for quantifying unknown neutral plant metabolites in absence of authentic standards, sub-milligram quantities of purified homologous acylsucrose metabolites were authenticated by subjecting each to basic hydrolysis and quantifying the sucrose product using stable-isotope dilution ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with multiple reaction monitoring (MRM). Once authenticated, molar response factors of [M + formate](-) ions for the acylsucrose metabolites were determined at different mobile phase compositions ranging from 40%-80% acetonitrile, and demonstrated relationships of response factors with mobile phase composition and metabolite structural features including nonpolar surface areas, the length of the longest acyl chain, and the number of hydroxyl groups. This approach was employed to calculate predicted response factors for three authenticated acylsucroses based on mean values for all isomers with a common number of total acyl carbon atoms. Absolute UHPLC-MS quantification was performed on these three metabolites in an extract from leaves of the wild tomato Solanum habrochaites LA1777, yielding deviations of 26%, 6.7%, and 7.3% from values established using compound-specific response factors.

Counter-ion and solvent effects in electrospray ionization of solutions of alkali metal and quaternary ammonium salts

Despite the wide use of electrospray ionization (ESI) mass spectrometry, the factors determining the relative signal intensities of different analytes are not yet fully understood. We have performed a systematic study to assess the influence of selected counter-ions (Br−, ClO4−, BF4−, BPh4−) on the ESI response of alkali-metal (Li+, Cs+) and quaternary ammonium ions (NEt4+, NBu4+) over a concentration range of 0.025–2.5mmolL−1. Specifically, we have subjected solutions containing equimolar mixtures of these analytes in H2O, CH3OH, CH3CN, and tetrahydrofuran, respectively, to ESI and determined the relative signal intensities of the ions thus produced. Especially in the aprotic solvents, variation of the counter-ion can significantly change the outcome of the ESI process, which we ascribe to ion pairing. The smaller counter-ions Br−, ClO4−, and BF4− undergo more selective ion pairing in that their tendencies to pair up with the cations strongly decrease in the order Li+>Cs+>NEt4+>NBu4+. In contrast, ion pairing of the larger BPh4− counter-ion with the different cations is much less selective. As a result, the smaller counter-ions Br−, ClO4−, and BF4− suppress the ESI response of Li+ and Cs+ ions significantly more strongly than does BPh4−. Additional evidence for selective ion pairing comes from the observation of ionic aggregates enriched in the alkali-metal ions relative to the quaternary ammonium ions.

Compounds having thiourea moiety as derivatization reagents in liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS): synthesis of derivatization reagents for carboxylic acids

The derivatization reagents for carboxylic acids, N-(Pyridin-3-yl)hydrazinecarbothioamide, N-[4-(dimethylamino)phenyl]hydrazinecarbothioamide, 1-(2-aminoethyl)-3-(pyridin-3-yl)thiourea, 1-(2-aminoethyl)-3-[4-(dimethylamino)phenyl]thiourea and 4-(2-aminoethyl)-N-phenylpiperazine-1-carbothioamide were synthesized. These reagents reacted with carboxylic acids at 60°C for 45 min in the presence of the condensation reagents. The generated derivatives were favorably separated on the reversed-phase column and sensitively detected by electrospray ionization tandem mass spectrometry. These reagents enhanced the electrospray ionization response of the analyte and generated a particular product ion efficiently by collision-induced dissociation, and thus they were suitable for MS/MS detection.