Accelerating Cross-Modality Reaction Optimization via Robotically Automated Vacuum Enabled Direct-inject Mass Spectrometry (RAVE MS)

Kampo is a form of traditional Japanese medicine, and its therapeutic strategy has been validated empirically over millennia, mainly in Asia. Kampo therapy aims to holistically prevent and treat disease based on the specific diagnosis Sho (in Japanese), in contrast with modern medical treatment which focuses on a patient's affected parts and local conditions. The medicines formulated using crude drugs derived from natural sources (Kampo formulas) are prescribed for patients according to their specific Sho, and thus the Kampo medication system is very complex. However, our previous study strongly suggested that Kampo medication theory could be explained by chemometrics and informatic approaches [Okada et al. in J Nat Med 70:107-114, 2016]. Here, we studied a group of seven formulas with Bupleurum Root and Scutellaria Root as the principal crude drugs. First, decoctions of the formulas were prepared and their supernatants were analyzed by non-targeted direct infusion mass spectrometry (MS) and principal component analysis, which is a type of unsupervised machine learning. Next, supervised machine learning was used to perform partial least squares modeling of the MS data matrix trained on the patients' constitution of Excess, Deficiency, or Medium between these two states (EDM) in Sho. The results showed that the correlation between the chemical fingerprints obtained by MS analysis and EDM could be modeled well using this approach. This cheminformatics modeling approach successfully interpreted part of the complex Kampo medication system studied using the fingerprints of formulas obtained by MS analysis and was consistent with the predicted Sho.

Metabolomics has demonstrated a great potential in numerous biomedical research fields in the last years, such as the study of the underlying pathology of diseases, discovery of diagnostic biomarkers or drug development. Nowadays, the main challenge in metabolomics is to obtain comprehensive and unbiased metabolomic profiles due to the huge complexity, heterogeneity and dynamism of metabolome. For this purpose, mass spectrometry represents a very interesting analytical platform, since complexity of metabolome may be overcome through the use of different orthogonal separation techniques, including liquid chromatography, gas chromatography and capillary electrophoresis. Alternatively, direct mass spectrometry analysis, either by direct infusion or flow injection, has been postulated as an alternative in metabolomics, complementing hyphenated approaches. These techniques exhibit several advantages such as the ability for high-throughput screening, fast analysis and wide metabolomic coverage, since there is not exclusion of compounds due to the separation device. The present work explores the potential of metabolomic platforms based on direct infusion mass spectrometry for metabolic fingerprinting of serum samples. The most important issues to be considered in this type of approaches were reviewed, including sample handling, comprehensive analysis, data processing, as well as further identification of metabolites and global characterization of metabolomic fingerprints.

The purpose of this investigation was to better understand lipid composition in human meibum. Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation. The absolute amount (μmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 α,ω Type II DEs, and 21 ω Type I-St DEs. Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive.

High-throughput analysis has become a critical component in chemical biology and analytical chemistry due to the large libraries of compounds that are screened every day for drug development. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein modifications, both chemically deposited and biological post-translational modifications (PTMs). With the advent of antibody drug conjugates (ADCs) and other novel protein-based conjugates, the demand for such an analysis has skyrocketed. Here, we present a new protocol that achieves quantitative data for modified peptides in approximately 30 s of MS acquisition time. This platform includes a direct injection MS approach coupled with new software named iFishMass to extract targeted signals from hundreds of runs. iFishMass automatically generates plots and statistics. This platform will enable a faster analysis of synthetic modifications installed on monoclonal antibodies to create ADCs, and it is potentially scalable to biological PTMs. Sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates, paving the way to an inexpensive but effective platform for high-throughput screening of conjugation sites on proteins.

Ziziphus joazeiro, a Saponin-Rich Brazilian Medicinal Plant: Pharmacognostic Characterization of Bark and Leaves

Rapid analysis of metabolites in biofluids is of great importance for disease diagnosis or new-born disease screening. Herein, we introduce an agarose hydrogel conditioning method to enhance the performance of paper spray ionization mass spectrometry. With facile and fast hydrogel conditioning, the signal intensity of therapeutic drugs spiked in urine was 5 to 15 fold higher than that in direct paper spray ionization mass spectrometry analysis. Consequently, the sensitivity of metabolites in urine was improved via hydrogel conditioning, resulting in 9 to 15 fold decrease in the possibility of detection (POD) levels. These results show that agarose hydrogel conditioning coupled with paper spray ionization mass spectrometry could serve as a facile ionization method for ambient mass spectrometry, which might be useful in fast screening of metabolites and therapeutic drugs in raw biofluids.

Stainless-steel substrates have grown in importance in the development of planar sorptive phases. However, the reduced wettability of polished sheets makes difficult their functionalization. This limitation can be solved by using amorphous silica gel microparticles as superficial guides. These particles increase both the wettability and the surface area of the final sorptive phases, which has a key influence on the extraction kinetics. In this article, double-sided thermal-resistant adhesive tape was used as the binder to pre-immobilize the silica particles over the stainless-steel surface, thus avoiding aggressive etching procedures. A linear polyester (synthesized by conventional free melt polycondensation from 1,4-butanediol and phenylsuccinic acid) was deposited onto the particulate area by the drop-casting technique. The resulting polyester-silica gel sheets (PSG-sheets) have been evaluated for the extraction of four selective serotonin reuptake inhibitors (SSRI) (fluoxetine, paroxetine, sertraline, and venlafaxine) in saliva samples, followed by direct infusion mass spectrometry analysis. The method allows the determination of the target compounds at the low µg·L−1 range, and it was successfully applied to analyze a saliva sample from a patient under paroxetine treatment. The method also fulfills the typical validation criteria in terms of precision (intrer-day precision better than 16.5% at the limit of quantification) and accuracy (relative recoveries in the interval 87–123% at the limit of quantification), both parameters having been evaluated at four concentration levels.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-025-07521-2.

In-situ sampling of lipids in tissues using a porous membrane microprobe for direct mass spectrometry analysis

Protein phosphorylation is one of the most critical post-translational modifications for regulating protein activity and function. Direct mass spectrometry (MS) analysis of phosphopeptides presents significant challenges due to their inherently low abundance and the inefficient ionization characteristics. In this study, we report the design of a novel pH-responsive magnetic adsorbent, denoted as Fe3O4@NH2@Rb, for highly selective capture of phosphopeptides. This innovative material leverages dynamic and reversible electrostatic interactions between bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane (Bis) and rhodamine B (Rb), which are responsive to pH changes. Under acidic conditions, Bis undergoes protonation while Rb transforms into a ring-opening zwitterionic form. The rapid and reversible transformation of Bis and Rb facilitates the efficient adsorption under acidic conditions and elution in alkaline environments. As anticipated, Fe3O4@NH2@Rb exhibits high enrichment performance for phosphopeptides from both standard protein digests and complex biological samples. This advancement holds great potential to significantly contribute to the phosphoproteomics research.

Chiral analysis in food science

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses.

Accelerating covalent binding studies: Direct mass shift measurement with acoustic ejection and TOF-MS

Liquid chromatography coupled to mass spectrometry (LC-MS) is an excellent technique to determine trace levels of polar and thermolabile pesticides and their degradation products in complex matrices. LC-MS can be equipped with several mass analyzers, each of which provides unique features capable to identify, quantify, and resolve ambiguities by selecting appropriate ionization and acquisition parameters. We discuss in this review the use of LC coupled to (quadrupole) time-of-flight mass spectrometry (LC-(Q)ToF-MS) to determine the presence of target and non-target pesticides in water and food. This technique is characterized by operating at a resolving power of 10,000 or more. Therefore, it gives accurate masses for both parent and fragment ions and enables the measurement of the elemental formula of a compound achieving compound identification. In addition, the combination of quadrupole-ToF permits tandem mass spectrometry, provides more structural information, and enhances selectivity. The purpose of this article is to provide an overview on the state of art and applicability of liquid chromatography time-of-flight mass spectrometry (LC-ToF-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) for the analysis of pesticides in environmental matrices and food. The performance of such techniques is depicted in terms of accurate mass measurement, fragmentation, and selectivity. The final section is devoted to describing the applicability of LC-(Q)ToF-MS to routine analysis of pesticides in food matrices, indicating those operational conditions and criteria used to screen, quantify, and identify target and "suspected" pesticides and their degradation products in water, fruits, and vegetables. The potential and future trends as well as limitations of LC-(Q)ToF-MS for pesticide monitoring are highlighted.

Background: The effect of direct oral anticoagulants (DOAC) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa, is a well-known problem and can cause both false positive and negative results. In particular, the situation in patients who develop lupus anticoagulant (LA) antibodies is highly complex. To evaluate the effectiveness of DOAC therapy in lupus-positive patients, 31 samples were enrolled in this retrospective study. All patient samples were spiked with three types of DOAC (dabigatran, DABI; rivaroxaban, RIVA; and apixaban, API) in a concentration that significantly influenced the screening test for LA and thus can mask the presence of LA. Subsequently, the DOAC was always unbound by the DOAC-Stop procedure. DOAC levels before and after binding were determined by functional assays, followed by liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Methods: The determination of DOAC levels was performed by direct thrombin assay and determination of anti-Xa activity with specific calibration as functional tests for DABI and xabans (API and RIVA). To determine concentration levels of API, DABI, and RIVA, our in-house LC-MS method was used. Results: The results of LA-positive samples show significant differences between functional tests and the LC-MS method both before and after DOAC binding. Conclusions: The acute findings of the presence of LA-type antibodies fundamentally affects the determination of DOAC by functional tests, and in this case, it is necessary to use LC-MS analysis to determine the true value. If patients treated with DOAC develop LA of medium and higher titers, we do not recommend checking DOAC levels with functional tests.

Perfluoroalkyl and polyfluoroalkyl substances （PFAS） possess desirable properties， including hydrophobicity， oleophobicity， surface activity， and thermal and chemical stability. Their extensive production and widespread application have resulted in the pervasive presence of PFAS in diverse environmental media. However， accumulating evidence indicates that PFAS are persistent， capable of long-range transport， bioaccumulative， and toxic； consequently， their adverse effects on ecosystems and humans are of widespread concern. Aquatic environments serve as a major transport pathway and contamination route for PFAS， making accurate measurement of PFAS levels in water crucial for assessing associated environmental and health risks. However， accurate quantification requires multi-step procedures， including sample filtration， enrichment， nitrogen blow-down concentration， and reconstitution， such as solid-phase extraction （SPE） and accelerated solvent extraction （ASE）. These methods are often labor-intensive and time-consuming. Although research on fully automated SPE technology is increasing， it necessitates installation of online SPE systems， which entail high costs and may present limitations in sample throughput per run. With continuous advancements in mass spectrometry， instrumental sensitivity has improved considerably， making direct injection of water samples for multi-analyte analysis technically feasible. However， reports on the use of direct injection methods for detecting PFAS in water remain limited， and the number of target analytes covered in such studies is relatively small. In this study， a direct injection-ultra performance liquid chromatography-triple quadrupole mass spectrometry （UPLC-MS/MS） method was developed for the determination of 31 PFAS in water. To optimize the chromatographic separation， enhance the detection sensitivity of target analytes， and minimize undesirable adsorption losses， the method was meticulously optimized with respect to solvent selection， injection volume， and syringe filter type. Our method involves the following procedure： 0.5 mL of water is aliquoted， mixed with 0.5 mL of methanol spiked with 2 ng of internal standard， and filtered through a 0.22 μm polypropylene membrane. The PFAS were analyzed by UPLC-MS/MS with an injection volume of 35 µL. The analytes were ionized in electrospray ionization negative mode （ESI-） with scheduled multiple-reaction monitoring （sMRM）. The MS parameters， including precursor and product ions， collision energy， and declustering voltage were optimized. Through optimization of the analytical column and mobile phases， the analytes were separated on an RSLC 120 C18 column with a gradient of methanol and 5 mmol/L ammonium acetate aqueous solution as the mobile phase in a gradient elution program. The results were quantified by the internal standard method. The method demonstrated excellent linearity （R²>0.994） across a defined concentration range. The limits of detection （LODs） and quantification （LOQs） were 0.007 1-3.0 ng/L and 0.024-10 ng/L， respectively. Recoveries at spiked levels of 2， 10， and 500 ng/L ranged from 67.2% to 130.2%， with relative standard deviations （RSDs） of 0.30% to 18%. To quantify the effective equivalence between the enrichment efficiency of SPE and the sensitivity of direct injection methods， a comparative analysis of analyte recovery rates was performed for both approaches. Furthermore， for long-chain PFAS， direct injection demonstrated consistent and favorable recovery performance. The method was applied to analyze PFAS in groundwater samples. The results showed that 24 PFAS were detectable with the total PFAS content （∑PFAS） ranging from 20.6 to 521 ng/L， with perfluorooctanoic acid （PFOA） and perfluorobutanoic acid （PFBA） being the primary pollutants. This approach is simple， rapid， highly sensitive， and provides broad coverage of target analytes， making it suitable for the quantitative analysis of PFAS in urban groundwater. It offers an efficient and reliable technical solution for determining trace-level PFAS in environmental water samples.