Paper Spray Ionization Mass Spectrometry Research Articles

Rapid discrimination of geographical origin of garlic (Allium sativum L.): A metabolomic approach applied to paper spray mass spectrometry data.

Distinguishing and categorizing the origin of garlic are highly significant, considering its widespread use as a flavoring agent. With billions of dollars annually in global trade, garlic is frequently susceptible to fraudulent practices. Paper spray ionization mass spectrometry (PS-MS) was employed to quickly analyze garlic samples from distinct geographic origins: China and Brazil. The so-generated PS-MS data were treated with metabolomic multivariate approaches, and the garlic samples from these different geographic regions were easily discriminated. Brazilian garlic was characterized to contain higher levels of amino acids, such as arginine, proline, and valine, and organosulfur compounds, such as allicin, alliin, and l-γ-glutamil-S-allyl-l-cysteine, compared to Chinese garlic. The PS-MS data were treated employing multivariate approaches, typically used in the metabolomics field, and this protocol was promptly able to discern among both types of samples. Hence, this combined strategy holds promise not only as an effective tool for the authentication of the geographical origin of garlic but also as a powerful means for biomarker discovery.

Synthesis, characterization, and application of a polymeric capillary fiber derived from methacrylic acid in the determination of cocaine in blood by FSI-MS

The development of probes/substrates that enable in-situ and in-natura biological analysis, capable of adsorbing analytes and enabling effective ionization, can be an alternative for detecting illicit drugs present in these matrices. Particularly, fiber spray ionization mass spectrometry (FSI-MS) shows promise because it enables straightforward and direct analyses without prior sample treatment or processing. In this study, a polymeric capillary fiber was developed and applied as a probe/substrate in FSI-MS experiments for the sampling and ionization of blood samples. The fiber was directly immersed in a solution of whole blood doped with cocaine (COC) and deuterated cocaine (COC-d3) to optimize the experimental conditions. The developed analytical approach was evaluated using whole blood samples taken from individuals suspected of drug abuse. Additionally, paper spray ionization mass spectrometry (PSI-MS) analyses were performed for comparison with the FSI-MS technique. The FSI analysis in positive mode, FSI(+), was effective in detecting COC and its metabolites with high sensitivity and accurate mass measurements. Evaluation of the whole blood sample collected from a suspected user revealed higher sensitivity for COC detection by FSI than by PSI. The quantification of COC in whole blood by FSI(+) showed detection and quantification limits of 32.0 and 106.7 ng mL−1, respectively. In addition, linearity was observed, with a coefficient of determination above 0.99 and a relative standard deviation ranging from 0.47 % to 8.58 %.

Simple Protein Analysis by Droplet Paper Spray Ionization Mass Spectrometry with Polyolefin Silica-Based Paper.

Paper spray ionization mass spectrometry (PSI MS) has emerged as a notable method for the rapid analysis of biological samples. However, the typical cellulose-based paper tip is incompatible with protein detection due to the strong interaction between cellulose hydroxyl groups and proteins. In this study, we utilized a commercially available polyolefin-based synthetic paper, Teslin®, as an alternative PSI substrate for simple protein analysis. We have named this method "droplet PSI" MS, as the aqueous protein solution droplet retains its shape on the Teslin® paper tip. For droplet PSI, no further chemical pretreatment was necessary for the Teslin® substrate; the only required preparation was shaping the Teslin® paper into a triangular tip. In droplet PSI MS, protein ion signals were instantly detected from a protein solution droplet upon applying a spray solvent in situ along with high voltage (HV). When compared with conventional PSI MS, our method demonstrated superior sensitivity. The droplet PSI MS utilizing Teslin® also showcased flexibility in real-time observation of protein alterations induced by an acid additive. Additionally, the effects of spray solvent composition and the application method were discussed.

Reactive Paper Spray Ionization Mass Spectrometry for Rapid Detection of Estrogens in Cosmetics.

Rapid detection of harmful estrogens in cosmetics is essential in protecting public health. To reduce time-consuming pretreatment and analytical procedures, a novel reactive paper spray ionization mass spectrometry (RPSI-MS) methodology was developed. RPSI-MS is suitable for quantitatively analyzing estrogens in cosmetics by utilizing an online derivatization reaction between estrogens and 2-fluoro-1-methyl-pyridinium-p-toluene-sulfonate (FluMP). Using estradiol valerate as the internal standard (I.S.), three estrogens, estradiol, estriol, and ethinyloestradiol, in cosmetics were quantitatively characterized within minutes. Multiple parameters were optimized including FluMP concentration and volume, triethylamine amount as well as the drying time. The three estrogens displayed good linearity ranging from 0.002 to 1 μg/mL, with R2 above 0.99. The recovery results of all the estrogens were within 80~111%. The limit of detection (LOD) was 0.001 μg/mL for the three estrogens. Compared to conventional paper spray ionization mass spectrometry (PSI-MS), extraction is not required and the detection sensitivity of RPSI-MS was improved by 34,000, 80,000, and 1400 times for estradiol, estriol, and ethinyloestradiol, respectively. The protocol established in this paper is sensitive, eco-friendly, and suitable for rapid testing of estrogens in cosmetics.

Authenticity study of commercial samples of St. John's wort by paper spray ionization mass spectrometry and chemometric tools

AbstractHypericum perforatum L. (St. John's wort) is one of the world's most consumed medicinal plants for treating depression and psychiatric disorders. Counterfeiting can occur in the medicinal plant trade, either due to the lack of active ingredients or the addition of substances not mentioned on the labels, often without therapeutic value or even harmful to health. Hence, 43 samples of St. John's wort commercially acquired in different Brazilian regions and other countries were analyzed by paper spray ionization mass spectrometry (PS‐MS) and modeled by principal component analysis. Hence, samples (plants, capsules, and tablets) were extracted with ethanol in a solid–liquid extraction. For the first time, PS‐MS analysis allowed the detection of counterfeit H. perforatum samples containing active principles typical of other plants, such as Ageratum conyzoides and Senna spectabilis. About 52.3% of the samples were considered adulterated for having at least one of these two species in their composition. Furthermore, out of 35 samples produced in Brazil, only 13 were deemed authentic, having only H. perforatum. Therefore, there is a clear need to improve these drugs' quality control in Brazil.

Spectrochemical approach combined with symptoms data to diagnose fibromyalgia through paper spray ionization mass spectrometry (PSI-MS) and multivariate classification

This study performs a chemical investigation of blood plasma samples from patients with and without fibromyalgia, combined with some of the symptoms and their levels of intensity used in the diagnosis of this disease. The symptoms evaluated were: visual analogue pain scale (VAS); fibromyalgia impact questionnaire (FIQ); Hamilton anxiety rating scale (HAM); Tampa Scale for Kinesiophobia (TAMPA); quality of life Questionnaire—physical and mental health (QL); and Pain Catastrophizing Scale (CAT). Plasma samples were analyzed by paper spray ionization mass spectrometry (PSI-MS). Spectral data were organized into datasets and related to each of the symptoms measured. The datasets were submitted to multivariate classification using supervised models such as principal component analysis with linear discriminant analysis (PCA-LDA), successive projections algorithm with linear discriminant analysis (SPA-LDA), genetic algorithm with linear discriminant analysis (GA-LDA) and their versions with quadratic discriminant analysis (PCA/SPA/GA-QDA) and support vector machines (PCA/SPA/GA-SVM). These algorithm combinations were performed aiming the best class separation. Good discrimination between the controls and fibromyalgia samples were observed using PCA-LDA, where the spectral data associated with the CAT symptom achieved 100% classification sensitivity, and associated with the VAS symptom achieved 100% classification specificity, with both symptoms at the moderate level of intensity. The spectral variable at 579 m/z was found to be substantially significant for classification according to the PCA loadings. According to the human metabolites database, this variable can be associated with a LysoPC compound, which comprises a class of metabolites already evidenced in other studies for fibromyalgia diagnosis. This study proposed an investigation of spectral data combined with clinical data to compare the classification ability of different datasets. The good classification results obtained confirm this technique is as a good analytical tool for the detection of fibromyalgia, and provides theoretical support for other studies about fibromyalgia diagnosis.

Rapid Detection of Estrogens in Cosmetics by Chemical Derivatization and Paper-Spray Ionization Mass-Spectrometry.

Estrogens in personal care products are harmful to customers. Conventional methods such as HPLC and LC-MS require tedious sample pretreatment and long analytical time. Paper-spray ionization mass spectrometry (PSI-MS) is a powerful tool for the determination of compounds with little time and minimal pretreatment procedures. Since most estrogens show poor responses in PSI-MS, we developed a chemical derivatization and PSI-MS method to determinate three estrogens: estradiol, estriol and ethinyloestradiol with estradiol valerate as the internal standard (I.S.). After derivatization with 2-fluoro-1-methyl-pyridinium-p-toluene-sulfonate, the three estrogens could be quantified in seconds. This method showed good linearity in the range of 0.1~30 μg·mL-1, with R2 > 0.999. Their recovery results were all between 85%~115%. The limits of detection (LOD) were 0.04 μg·mL-1, 0.02 μg·mL-1 and 0.02 μg·mL-1 for estradiol, estriol and ethinyloestradiol respectively, which improved around 200, 2000, and 900 times compared to non-derivative PSI-MS. The method could quantitatively determine estrogens in cosmetics.

A simple, quick and non-destructive approach for sampling drugs of abuse in tablets and blotter for qualitative analysis by paper spray mass spectrometry.

This study presents the development of a simple, fast, and inexpensive approach for the direct analysis of new psychoactive substances (NPS) in seized tablets and blotter paper, with improved sample preservation and increased analytical frequency. Paper triangles were gently rubbed against the surface of the samples containing synthetic drugs and then subjected to analysis by paper spray ionization mass spectrometry (PS-MS). Seized samples containing lysergic acid diethylamide (LSD) and several other substances from the classes of amphetamines, N-benzyl-substituted phenethylamines, synthetic cathinones, and synthetic cannabinoids, were analysed. Three types of paper were tested (filter paper, blotter paper, and synthetic paper) and several combinations of spray solvents were studied for the optimization. All samples were weighed and photographed before and after sequences of analysis in order to attest to the sample preservation. The results revealed that the approach is excellent for sample preservation, with less than 5% of mass loss even after 27 consecutive analyses. Moreover, no significant signal decreases were observed in mass spectrometry (MS) even after the experiments. It was possible to unequivocally identify illicit substances from seized samples (pills and blotter paper). By overcoming the solubilization and wet extraction process used for sample preparation, the waste was restricted to a volume of only 10 μL of solvent for the PS-MS analysis. The main advantage of our approach over existing methods is the sample preparation, which is simple and quick since the samples are just rubbed against the PS paper. This brings enormous benefits in terms of analytical frequency, economy of time and low consumption of solvents. Another important point is that the sample can remain intact for further analysis, which is crucial in forensic analysis.

APLICAÇÃO DA ESPECTROMETRIA DE MASSAS COM IONIZAÇÃO POR PAPER SPRAY E TÉCNICAS RELACIONADAS NA ANÁLISE DIRETA DE MATRIZES BIOLÓGICAS PARA DETECÇÃO DE DROGAS - UMA REVISÃO

APPLICATION OF PAPER SPRAY IONIZATION MASS SPECTROMETRY AND RELATED TECHNIQUES IN THE DIRECT ANALYSIS OF BIOLOGICAL MATRICES FOR DETECTION OF DRUGS - A REVIEW. In recent years, technological development in analytical instrumentation has progressed vigorously, which has allowed the evolution of analytical techniques that play an indispensable role within society, highlighting mass spectrometry. Parallel to the technological advance of this technique, the development of new ionization methods that are simpler, faster, and allow direct analysis without the need for prior treatment or sample processing is underway. The methods of ambient ionization derived from paper spray have been developing rapidly in current days, mainly in the forensic field, due to the chemical modifications made in the paper substrate and the introduction of new types of organic materials that function as a probe/substrate to collect, store, and ionize the sample, then enabling more efficient and selective analyzes. This review highlights direct analyzes by applying paper spray ionization in the forensic field and some variations of this technique, including Thread Spray Ionization (TSI) when a fabric thread is used; Touch Spray Ionization (TSI), which uses materials such as metallic needles, medical swabs, and wooden sticks; Fiber Spray Ionization (FSI), which applies polymeric fibers as substrates.

Accelerated synthesis of phthalimide derivatives: Intrinsic reactivity of diamines towards phthalic anhydride evaluated by paper spray ionization mass spectrometry.

Paper spray (PS) is a simple and innovative ambient ionization technique for mass spectrometry (MS) analysis. Under PS-MS conditions, chemical reactions, which usually occur slowly on a bulk scale, are accelerated. Moreover, the formation of products and transient species can be easily monitored. In this manuscript, reactions between phthalic anhydride and diamines were conducted and monitored using a PS-MS platform. The reaction products (phthalimides) have many pharmaceutical applications, but their traditional syntheses can take hours under reflux, requiring laborious purification steps. In situ reactions were performed by dropping methanolic solutions of phthalic anhydride and diamines on a triangular paper. The analyses were achieved by positioning the triangle tip in front of the mass spectrometer entrance, whereas a metal clip was attached to the triangle base. After adding methanol to the paper, a high voltage was applied across the metal clip, and the mass spectra were acquired. The intrinsic reactivity of alkyl and aromatic diamines was evaluated. The carbon chain remarkably influenced the reactivity of aliphatic diamines. For aryl diamines, the ortho isomer was the most reactive. Moreover, for aryl amines with electron-withdrawing substituents, no reaction was noticed. Taking advantage of the unique characteristics of PS-MS, it was possible to investigate the intrinsic reactivity of model alkyl (ethylene versus propylene) and aryl (o-phenylene versus m-phenylene and p-phenylene) diamines towards phthalic anhydride. Some crucial parameters that affect the intrinsic reactivity of organic molecules, such as isomerism, intramolecular interaction, and conformation, were easily explored.

Carbon fiber paper spray ionization mass spectrometry

Carbon fiber paper (CFP) is commonly used as a proton exchange membrane in fuel cells due to its prominent areal electrosorption capacity, exceptional conductivity and excellent chemical stability. In this paper, we first explored the feasibility of carbon fiber paper as a specific paper substrate in paper spray ionization mass spectrometry (PSI-MS). The results demonstrated that CFPSI-MS combines the merits of PSI and carbon fiber ionization (CFI) and exhibits better performance of various compound analyses than either of these techniques alone. The application of CFP can highly enhance the signal stability and detection sensitivity of a diverse array of analytes, especially in negative ionization mode. The ion intensity of target analytes such as saccharides and flavonoids was improved 2-90-fold. Many nonpolar/low-polarity analytes, such as polycyclic aromatic hydrocarbons, which are difficult to ionize by traditional PSI-MS, were successfully detected by CFPSI-MS with a 2.5 kV high voltage. Moreover, CFPSI-MS presents high sensitivity in semiquantitative analysis, and the limits of detection (LODs) of cyclic adenosine monophosphate (CAMP), naringin and tivantinib in whole blood were improved 2-100-fold compared to those in traditional PSI-MS. In real sample analysis, CFPSI-MS also exhibits excellent capability in human breath analysis and blood metabolomic profiling.

Bisphenol determination in UHT milk and packaging by paper spray ionization mass spectrometry

This study evaluates the use of paper spray ionization mass spectrometry (PSI-MS) for rapid determination of bisphenol A (BPA) and bisphenol S (BPS) in UHT milk and milk packaging. The packages were analyzed by cutting the cartons into triangular shapes and submitting them to PSI-MS analysis. The milk samples were subjected to a simple liquid–liquid extraction and the supernatant was deposited onto a triangular paper that was subsequently used for PSI-MS analysis. In milk, BPS and BPA levels ranged from 60.0 to 150.8 ng mL−1. The LOD and LOQ values were 1.5 and 4.8 ng mL−1 for BPA, and 4.8 and 16.0 ng mL−1 for BPS, respectively. Linearity was R2 > 0.98 for both compounds. Precision values were below 20%, and recoveries close to 100%. The PSI-MS can be used as a simple, rapid, and accurate methodology to determine bisphenols in milk and milk packaging.

Rapid determination of aldehydes in food by high-throughput reactive paper spray ionization mass spectrometry

Due to endogenous existence and hazardous properties, the quantification of fatty and aromatic aldehydes in food matrices is indispensable to food safety. Current analytical methodologies for aldehydes in food suffer from several drawbacks (typically complicated pretreatment, long analysis time and low analytical throughput, low sensitivity, etc.). In this work, a highly sensitive, high-throughput and sample pretreatment-free method for aldehyde analysis was developed to address the above problems, where in situ extraction and derivatization of aldehydes with hydrazines to hydrazones were integrated into reactive paper spray ionization mass spectrometry (reactive PSI-MS) considering weakly ionizable properties of aldehydes. Four aldehydes, formaldehyde, acetaldehyde, propionaldehyde and benzaldehyde were reacted with dansyl hydrazine in 1.5 % v/v HAc acidified methanol on the qualitative fast-speed filter paper for 5 min, and then a voltage (+4 kV) was applied to initiate paper spray for mass spectrometry detection with internal calibration by dansyl amide. Once the completion of MS analysis, the glass plate was moved to introduce the second triangular tip towards the MS inlet (5 mm apart) for next trial, achieving high-throughput reactive PSI-MS analysis (60 h−1). The four aldehydes showed good linearity in the concentration range from 2 to 150 μM with low detection limits of 0.03–0.15 μM. The method was applied to direct quantification of four aldehydes in 15 kinds of foods such as vegetables, fruits and meat products. The proposed method has the advantages of simple operation, high efficiency and high sensitivity, making it attractive to routine analysis of aldehydes in food, environmental and biological samples.

Use of pulp, peel, and seed of Annona crassiflora Mart. in elaborating extracts for fingerprint analysis using paper spray mass spectrometry

The Brazilian cerrado is considered one of the most critical biomes in the world. Araticum (Annona crassiflora Mart.) is a native plant from the Brazilian cerrado, abundant in nutrients and highly energetic. This study aimed to obtain chemical fingerprints of different parts of the araticum fruit, i.e. pulp, peel, and seed. Extracts from these three parts were prepared using different solvents (ethanol, water, and mixtures of ethanol and water) and later analyzed by paper spray ionization mass spectrometry. In general, ethanol extracted more metabolites than the other solvents. The chemical profiles varied according to the fruit part, geographic location, and extractor solvent. Among the metabolites, acetogenins (62.3%) and alkaloids (20.7%) predominated. Principal component analyses revealed that the samples were grouped according to the fruit part, regardless of the extractor solvent used. Araticum shows remarkable potential due to the beneficial properties of the metabolites for human health. The insertion of araticum in the human diet is still underexplored but is a promising alternative.

Paper spray ionization mass spectrometry applied for quantification of pesticides and discrimination from tomato varieties (Solanum lycopersicum)

The determination of pesticide residues in tomatoes is usually performed by GC–MS and LC–MS, which provide high analytical performance but require several steps of sample preparation. Thus, this work employed a simpler and faster technique, the Paper Spray Ionization Mass Spectrometry (PSI-MS), combined with Principal Component Analysis (PCA) to differentiate the tomatoes varieties and for quantification of pesticides residues. It was determined a metabolic profiling by PSI-MS and the samples were grouped according to the variety using PCA. A quantitative method was developed for sensitive and accurate quantitative analysis of the fungicide cyazofamid by PSI(+)-MS using fruit peels of two varieties of tomatoes. The method showed a calibration curve with R2> 0.99, and low limits of detection (LOD) and quantification (LOQ) of 10 ng.mL−1 and 25 ng.mL−1, respectively. The intra-day and inter-day recoveries percentage were in a range of 93.87–109.32 % and 99.54–100.81 % for variety Sweet Heaven, respectively. Similar results were obtained for variety BS II0012 with a satisfactory average recovery ranged from 89 % to 108 %, within the standards established by the regulatory agency. The results obtained reinforce the potential of PSI-MS as a rapid and simple analytical tool for determination of pesticide residue in agricultural products.

Data-Driven Soft Independent Modeling of Class Analogy in Paper Spray Ionization Mass Spectrometry-Based Metabolomics for Rapid Detection of Prostate Cancer.

Sensitive, rapid, and meaningful diagnostic tools for prostate cancer (PC) screening are urgently needed. Paper spray ionization mass spectrometry (PSI-MS) is an emerging rapid technology for detecting biomarker and disease diagnoses. Due to lack of chromatography and difficulties in employing tandem MS, PSI-MS-based untargeted metabolomics often suffers from increased ion suppression and subsequent feature detection, affecting chemometric methods for disease classification. This study first evaluated the data-driven soft independent modeling of class analogy (DD-SIMCA) model to analyze PSI-MS-based global metabolomics of a urine data matrix to classify PC. The efficiency of DD-SIMCA was analyzed based on the sensitivity and specificity parameters that showed 100% correct classification of the training set, based on only PC and test set samples, based on normal and PC. This analytical methodology is easy to interpret and efficient and does not require any prior information from the healthy individual. This new application of DD-SIMCA in PSI-MS-based metabolomics for PC disease classification could also be extended to other diseases and opens a rapid strategy to discriminate against health problems.

Fumonisin B1 analysis in maize by Molecularly Imprinted Polymer Paper Spray Ionization Mass Spectrometry (MIP-PSI-MS)

Fumonisin B1 (FB1) is the most abundant and important mycotoxin for agriculture, food, and health among all fumonisins due to its widespread presence and toxicity. This compound is a concern for industries involving maize and maize-based products. This study proposes a new approach using molecularly imprinted polymer (MIP) combined with paper spray ionization mass spectrometry (PSI-MS) to enhance the sensitivity of analysis of FB1 in maize samples. MIP was synthesized directly on a cellulose membrane using 1,2,3-propanetricarboxylic acid as a template molecule. The analytical performance of MIP-PSI-MS provided a linear calibration curve with R2>0.99. Precision and accuracy were less than 5.5 % for all analyses. Finally, the MIP-PSI-MS method was applied to quantify the contents of FB1 in commercial maize samples, and 0.439 ± 0.060 and 0.486 ± 0.061 μg mL−1 were found in two samples.

Assessing the environmental ruggedness of paper spray ionization (PSI) coupled to a portable mass spectrometer operated under field conditions

Significant efforts have went towards coupling portable mass spectrometric systems and ambient ionization methods, with a culminating goal of field-based, point-of-need screening of harmful chemicals. While fieldable, ambient MS methods are an intriguing combination, ruggedness of these ionization methods in regard to naturally-occurring environmental conditions while operating in-situ, such as ambient temperature, relative humidity (RH) and wind, bears further investigation, as such reports are limited. In this work, a systematic characterization of paper spray ionization-mass spectrometry (PSI-MS) performance under variable environmental conditions was undertaken on a field-deployable MS system. Of all conditions, the presence of wind speeds greater than 3 m/s (∼7 MPH) was shown to have the most substantial effect on the spectral attributes of signal intensity and duration of the PSI-MS spraying event. Generally, moderate ambient temperature and %RH produced the best spectral performance for PSI-MS. Heat maps were generated from field-collected data across variable temperature and %RH in an effort to inform general PSI-MS performance trends and guide on-site operation by potential users.

Rapid determination of lutein in fresh and commercial food samples using paper spray ionization mass spectrometry

The current strategy reports a simple, rapid and precise paper spray ionization mass spectrometry method for trace determination of lutein in matrices of different complexities. The established method was based upon optimization of the applied, cone and RF voltages and mobile phase. Under the optimized parameters, a linear concentration range (1.0–50 μg/g) of lutein was successfully achieved with low limits of detection (3σ/b) and quantification (10σ/b) of 0.5 and 1.665 μg/mL, respectively. The proposed method was successfully applied for trace determination of lutein residues in fresh and commercial foods (green and black tea, and tomato paste) purchased from local markets in Canada. The measured concentration of lutein in green bean was 2.76 ± 0.11 μg/g while the highest level (51.5 ± 0.05 μg/g) of lutein was found in the mint stems.

On-surface multicomponent Povarov reaction examined by paper spray mass spectrometry

Povarov multicomponent reaction (MCR) is an acid-catalyzed process that uses three distinct classes of reactants. i. e. an aldehyde, aniline, and activated alkene. We employ paper spray ionization mass spectrometry (PS-MS) as a convenient platform for investigating the on-surface Povarov reaction using the following suitable prototype starting reactants: the tag-charged aldehyde 1, 4-bromoaniline, 2,3-dihydrofuran, and formic acid. The PS(+)-MS revealed, besides the formation of the Povarov product (a tetrahydroquinoline compound), the transitional imine. Moreover, the oxonium intermediate was also detected as the water and formic acid adducts. Other alkenes (2,3-dihydropyran and styrene), acid catalysts (acetic acid and p-sulfonic acid calix[4]arene), and aldehydes (4-nitrobenzaldehyde, 4-anisaldehyde, 4-methoxycinnamaldehyde, citronellal, and valeraldehyde) were employed to verify the reaction generality. We observed that styrene (an unactivated alkene), p-sulfonic acid calix [4] arene (an acid with a non-nucleophilic conjugated base), and aromatic aldehydes (4-nitrobenzaldehyde and 4-anisaldehyde) showed lower reactivity than the other reactants. In conclusion, an exciting possibility emerges as PS-MS can be widely employed to investigate similar on-surface organic reactions to verify the connections between the on-surface and condensed phase processes.