Information-dependent Acquisition Research Articles

Identification of Potential Growth-Related Proteins in Chick Vitreous during Emmetropization Using SWATH-MS and Targeted-Based Proteomics (MRMHR).

The vitreous humor (VH) is a transparent gelatin-like substance that occupies two-thirds of the eyeball and undergoes the most significant changes during eye elongation. Quantitative proteomics on the normal growth period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia. In this study, a data-independent acquisition (SWATH-MS) was combined with targeted LC-ESI-MS/MS to identify and quantify the relative protein changes in the vitreous during the normal growth period (4, 7, 14, 21 and 28 days old) in the chick model. Chicks were raised under normal growing conditions (12/12 h Dark/light cycle) for 28 days, where ocular measurements, including refractive and biometric measurements, were performed on days 4 (baseline), 7, 14, 21 and 28 (n = 6 chicks at each time point). Extracted vitreous proteins from individual animals were digested and pooled into a left eye pool and a right pool at each time point for protein analysis. The vitreous proteome for chicks was generated using an information-dependent acquisition (IDA) method by combining injections from individual time points. Using individual pool samples, SWATH-MS was employed to quantify proteins between each time point. DEPs were subsequently confirmed in separate batches of animals individually on random eyes (n = 4) using MRMHR between day 7 and day 14. Refraction and vitreous chamber depth (VCD) were found to be significantly changed (p < 0.05, n = 6 at each time point) during the period. A comprehensive vitreous protein ion library was built with 1576 non-redundant proteins (22987 distinct peptides) identified at a 1% false discovery rate (FDR). A total of 12 up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, the cadherin family group, neurocan, and reelin, involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition. This study provided the first comprehensive spectral library of the vitreous for chicks during normal growth as well as a list of potential growth-related protein biomarker candidates using SWATH-MS and MRMHR during the emmetropization period.

Analysis of Intact In Vivo Peroxidized Phospholipids from Bovine Retina via LC-MS/MS and GC-MS/MS Using Autoxidized Retina Reference Standards.

Highly unsaturated fatty acids (HUFAs) are vulnerable to oxygen attack, thus making HUFA-rich, high metabolic rate/reactive oxygen species (ROS)-generating neurological tissue particularly susceptible to increased oxidative stress. Lipid oxidation is a putative early stage marker of neurodegenerative diseases, suggesting that reliable monitoring of oxidized neural lipids in vivo reveals early pathogenesis. Here, we present a novel methodology to detect and quantify intactin vivo ROS-driven peroxidized phospholipids (LPOx-PLs) in bovine retina extract. A protocol for preparing autoxidized pure phospholipids (PLs) and complex retinal extracts served as reference standards and was adapted to enable analytical parameter development. Fatty acid profiles of bovine retinas were first established with routine gas chromatography (GC) methods and used to customize mass spectrometry scanning for major HUFA-carrying PLs in the retinal extract. Targeted multiple reaction monitoring (MRM) scanning via triple quadrupole tandem mass spectrometry detected native (unoxidized) and oxidation-damaged PL regardless of the position of the O or O2 addition along the acyl chains and enabled quantification of relative signals from intact native and oxidized PL (5%-10% CV). MRM-triggered information-dependent acquisition (IDA) spectra confirmed the structure of peroxidized PLs, revealing that peroxidized species (+O-OH) dominated over single O-added species in vitro and in vivo. Positive identification and relative quantification are reported for 12 selected in vivo native and peroxidized phosphatidylcholines and phosphatidylethanolamines. These results enable future studies of the initial peroxidation due to toxins, genetics, or other initiating events influencing in vivo oxidation levels and potentially the effectiveness of strategies to mitigate this mechanism of action.

Simultaneous determination of 13 biogenic amines and their metabolites in liquid fermented food by HPLC-QTRAP-MS without derivatization

Biogenic amines and their metabolites (BAs) are among the most frequently detected compounds in liquid fermented foods, raising significant concerns about the potential risks they pose. To rapidly and selectively determine BAs, this study proposed and validated a method for the simultaneous determination of 13 BAs in liquid fermented foods using high performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry (HPLC-QTRAP-MS) without derivatization. The simultaneous separation and determination of histamine (HIS), tyramine (TYR), putrescine (PUT), octopamine (OCT), 2-phenylethylamine (2-PHE), dopamine (DOP), spermidine (SPD), epinephrine (EPI), agmatine (AGM), 5-hydroxytryptamine (5-HYD), tryptamine (TRY), L-noradrenaline (L-NOR), cadaverine (CAD) were achieved using a Waters Atlantis Premier BEH Z-HILIC (1.7 μm particle size, 100 mm × 2.1 mm id) column with a mobile phase consisting of water (containing 0.025 % formic acid) and acetonitrile (containing 0.1 % formic acid). The analysis of target compounds was performed with electrospray ionization (ESI) in positive ion mode, multiple reaction monitoring (MRM), information dependent acquisition (IDA), enhanced product ion (EPI), and multi-step mode analysis combined with EPI library searching. To enhance the extraction efficiency of BAs, factors such as extraction solvent, filter membranes, extraction methods, pH, oscillation speed, time and temperature were systematically investigated. The results demonstrated that the method exhibited low limits of detection (LOD) and quantification (LOQ), ranging from 0.05 to 2.00 µg/L and 0.16 to 6.60 µg/L, respectively. The method also showed satisfactory reproducibility, with intra-day and inter-day relative standard deviations ranging from 0.90 % to 3.80 % and 2.65 % to 8.27 %, respectively. Additionally, it had a broad linear range from 0.05 to 200.00 µg/L with excellent linearity (R>0.99) and an average recovery rate between 65.68 % and 115.14 %. Overall, the results demonstrated that the established method was convenient to operate, offered a short analysis time, and achieved high accuracy and sensitivity. It met the qualitative and quantitative requirements for detecting 13 BAs in liquid fermented food, thus providing strong technical support for BAs detection.

Sample-Specific MS/MS Methods in High-Throughput Mass Spectrometry.

The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.

Determination of bioactive substances in 27 edible flowers based on LC-MS/MS

Edible flowers, as a food ingredient, are rich in bioactive substance and have received a lot of attention. In this study, we have established a pseudo-targeting method based on Multi Reaction Monitoring (MRM) - Enhanced Product Ions (EPI) in the Information Dependent Acquisition (IDA) mode. The results showed that a total of 43 metabolites, including flavonoids, phenolic acids, and terpenes were detected in 27 edible flowers using integrated liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy. Rutin was measured in all edible flowers, but the levels varied significantly among the different varieties of flowers, with the highest being about 3500 times the lowest. Albiflorin and paeoniflorin are polyphenolic substance unique to Paeonia suffruticosa. The cyanidin 3-O-glucoside chloride content in reddish-purple flowers is higher than that of other colors. Dicoumarin and 4-hydroxycoumarin were discovered in edible flowers for the first time. This study established a pseudo-targeted detection method to reveal the distribution of polyphenolic metabolites and enriched the research on polyphenolic metabolites in edible flowers. It can also serve as a general strategy to detect metabolites in other agricultural products, providing valuable information about potential metabolites for consumers.

Determination of nine bisphenol analogues in human urine by high-throughput solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis

Bisphenol analogues (BPs) are a class of typical environmental endocrine-disrupting chemicals (EDCs). This study aimed to establish a highly sensitive and high-throughput method utilizing 96-well solid-phase extraction (96-well SPE) in conjunction with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) employing multiple reaction monitoring (MRM), information-dependent acquisition (IDA), and enhanced product ion (EPI) scan modes for the identification and quantitative analysis of nine BPs in human urine. Urine samples were initially thawed to room temperature, followed by digestion using β-glucuronidase in an ammonium acetate buffer solution at 37 °C overnight. Subsequently, they were purified using 96-well SPE and finally analyzed by UHPLC-MS/MS. The limits of detection (LOD) for the nine BPs ranged from 0.05 μg∙kg−1 to 0.3 μg kg−1. Average recoveries fell within the range of 92.8 % to 111.7 %. Moreover, both the intra-day and inter-day precisions were satisfactory, with relative standard deviations (RSDs) ranging from 2.2 % to 6.7 % and 3.5 % to 6.3 %, respectively. The targets in the samples exhibited a perfect match, with a purity fit value exceeding 70 % from the self-built library. The analytical method developed in this study demonstrates high accuracy and sensitivity. In addition, the MRM-IDA-EPI mode can effectively identifies the target BPs and prevents false positive detection of analytes in the urine.

Metabolite profiling of liquiritin in acute myocardial infarction model rat after intragastric administration using an information-dependent acquisition-mediated ultra-high-performance liquid chromatography-tandem mass spectrometry method.

Liquiritin (LQ), a kind of flavonoid isolated from licorice, was proven to have great potential in treating heart failure. Pharmacokinetic evaluation is important for demonstrating clinical efficacy and mechanisms, and the prototype drug and its metabolite profiling are important for drug discovery and development. However, the metabolism of LQ in acute myocardial infarction (AMI) model rats still needs to be studied in depth. An information-dependent acquisition (IDA)-ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was applied to profile LQ metabolites in AMI model rat plasma. Protein precipitation and extraction were used for sample preparation. Chromatographic separation was achieved using an XSelect BEH C18 column (2.1 × 150 mm, 2.5μm) using gradient elution method combining 0.1% formic acid and acetonitrile with a flow rate of 0.3mL/min. Twelve metabolites were identified in IDA mode, sulfation, glucuronidation, methylation, methyl esterification, glutamine conjugation, and valine conjugation, and their composite reactions were presumed as the primary pathways of LQ metabolism. The variation in the peak areas showed that the time to reach the peak drug concentration of LQ and 12 metabolites was within 5h. In summary, IDA-bridged UHPLC-MS/MS from characteristic fragment ions toward confidence-enhanced identification could effectively screen and profile metabolites.

Targeted and untargeted screening for impurities in losartan tablets marketed in Germany by means of liquid chromatography/high resolution mass spectrometry

Technical advances in the field of quality analysis allow an increasingly deeper look into the impurity profile of drugs. The ability to detect unexpected impurities in addition to known impurities ensures the supply of high-quality drugs and can prevent recalls due to the detection of harmful unexpected impurities, as has happened recently with the N-nitrosamine and azido impurities in losartan (LOS) drug products. In the present study, the LC-MS/HRMS approach described by Backer et al. was applied to an even more complex system, being the investigation of 35 LOS drug products and combination preparations purchased in 2018 and 2022 in German pharmacies. The film-coated tablets were analysed by means of four LC-MS/HRMS method variants. For the separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and for mass spectrometric detection a qTOF mass spectrometer with electrospray ionization in positive and negative mode was used. An information-dependent acquisition method was applied for the acquisition of high-resolution mass spectrometry data. The combination of an untargeted and a targeted screening approach revealed the finding of eight impurities in total. Beside the five LOS related compounds, LOS impurity F, J, K, L, M, and related compound D from amlodipine besilate, LOS azide and an unknown derivative thereof were detected. Identification and structure elucidation, respectively, were successfully performed using in silico fragmentation. Differences in the impurity profiles of drug products from 2018 and 2022 could be observed. This study shows that broad screening approaches like this are applicable to the analysis of drug products and can be an important enhancement of the quality assurance of medicinal products.

Target and suspect screening of psychoactive substances in seizures and oral fluid exploiting retention time prediction and LC-MS/MS analysis

BackgroundNovel psychoactive substances (NPS) are a group of substances, mainly of synthetic origin, characterized by toxicological properties extremely dangerous. The main difficulty in recognizing NPS in seizures and biological samples lies in their dynamic nature, related to the continuous synthesis and introduction on the market of new drugs, often with very similar structures to existing ones. The aim of this study was the creation of a robust and versatile method for the analysis of traditional drugs and NPS in different matrices. ResultsBoth target analysis and suspect screening methodologies were developed. The strategy used for suspect screening allowed to collect data through a scheduled multi reaction monitoring (sMRM) survey which triggered the collection of enhanced product ion (EPI) spectra when a compound met information dependent acquisition (IDA) criteria. The retention time of the different drugs, which was crucial to define the sMRM survey scan parameters, was predicted with a Quantitative Structure Retention (Chromatographic) Relationship (QSRR) model by Multiple Linear Regression. The model was validated through the evaluation of training set predictions, an external validation set and a leave-one out strategy; the results showed that the method fit for its purpose. The target method was validated in oral fluid as a testing matrix, with excellent results in term of recovery, accuracy, precision and matrix effect. Finally, the performances of the suspect method were evaluated by analysing a mixture containing 8 reference standards not included in the initial dataset, as well as seizures and real oral fluid samples. Four NPS were putatively identified in the analysed samples. SignificanceThe advantage of the proposed approach is the possibility of quantifying 65 classical drugs of abuse and NPS and, at the same time, detect and putatively identify 146 additional drugs in one single LC-MS/MS run. This is an innovative strategy for multi analyte detection and enables detection of low concentrations of drugs in complex biological matrices such as oral fluid. Considering the highly dynamic drug market, a strength of this strategy is that the analytical method can be kept up to date through the addition of new compounds based on the last drug monitoring bodies alerts without the need of authentic standards.

Application of advanced high resolution mass spectrometric techniques for the analysis of losartan potassium regarding known and unknown impurities

Recalls of medicinal products can cause supply bottlenecks. This is often due to the findings of unexpected impurities that pose a health risk to patients. A recent example is losartan potassium which was contaminated with azido-impurities. The choice of the analytical method determines which substances can be detected and thus controlled. In this study a combination of an untargeted screening approach for impurities and a targeted evaluation of high-resolution mass spectrometry data was applied to search for impurities not described so far, leaving out a precise quantification. Six losartan potassium samples were studied regarding known and unknown impurities and hence highlight the applicability and capability of the approach. For separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and an electrospray ionization in positive and negative mode for mass spectrometric detection was used. An information-dependent acquisition method was applied for the measurement of losartan potassium samples. The untargeted data evaluation using general unknown comparative screening revealed the presence of N-methyl-2-pyrrolidone (NMP) and another impurity from synthesis. The identity of NMP was corroborated by a spiking experiment and the amount was estimated by means of standard addition. A targeted data evaluation by generating extracted ion chromatograms resulted in finding of four additional impurities. Combined approaches like this are needed to detect and respond to changes in the quality of drugs precociously.

SpecLipIDA: a pseudotargeted lipidomics approach for polyunsaturated fatty acids in milk.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), play an important role in the nutritional value of milk lipids. However, a comprehensive analysis of PUFAs and their esters in milk is still scarce. In this study, we developed a novel pseudotargeted lipidomics approach, named SpecLipIDA, for determining PUFA lipids in milk. Triglycerides (TGs) and phospholipids (PLs) were separated using NH2 cartridges, and mass spectrometry data in the information-dependent acquisition (IDA) mode were preprocessed by MS-DIAL, leading to improved identification in subsequent targeted analysis. The target matching algorithm, based on specific lipid cleavage patterns, demonstrated enhanced identification of PUFA lipids compared to the lipid annotations provided by MS-DIAL and GNPS. The approach was applied to identify PUFA lipids in various milk samples, resulting in the detection of a total of 115 PUFA lipids. The results revealed distinct differences in PUFA lipids among different samples, with 44 PUFA lipids significantly contributing to these differences. Our study indicated that SpecLipIDA is an efficient method for rapidly and specifically screening PUFA lipids.

Development of a Robust Method for Screening PDE-5 Inhibitor Additives in Dietary Supplements Using UPLC-MS/MS

A novel ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed to detect the illegal additive of phosphodiesterase type 5 (PDE-5) inhibitors in dietary supplements. With the optimized chromatographic program, vardenafil, sildenafil, and tadalafil were separated within 5 minutes. The MS1, MS2, and retention time of PDE-5 inhibitors were acquired simultaneously within the information dependent acquisition mass spectrometry mode. Quantification was achieved via the quantity ion current chromatogram that was extracted from the total ion current. The linear range of vardenafil and sildenafil was 0.5-48 mg/L while the range of tadalafil was 0.3-36 mg/L. The correlation coefficients of the calibration curves of three PDE-5 inhibitors were all greater than 99.9%. At three concentration levels, the RSD values of the five repeat tests were all better than 1.30%. Sildenafil was detected in one dietary supplement. The comprehensive method of this study is reliable and may be a powerful tool for routine PDE-5 inhibitor screening and determination.

SWATH-F: A Novel Nontarget Strategy Based on the SWATH-MS Deconvolution Method Assisting in Annotating PFAS Homologues in Multisample Studies.

In order to identify emerging per- and polyfluoroalkyl substances (PFASs) and their alternatives in the environment or population, we need to perform extensive profiling of PFASs to determine their distribution in samples. The sequential window acquisition of all theoretical fragment-ion spectra (SWATH mode) is capable of obtaining a wide range of MS2 spectra but is difficult for direct identification of PFASs due to its complex MS2 spectra, and the nontarget screening method is difficult to identify due to its lack of a priori information. In this study, we demonstrated the great potential of SWATH-F, a nontarget fragment-based homologue screening method in combination with the SWATH-MS deconvolution, for detecting PFASs. We evaluated the application of SWATH-F to gradient spiked samples and real population serum samples, compared it with nontarget homologue screening in the information-dependent acquisition mode (IDA mode), and obtained better results for SWATH-F with 276% improvement (IDA:17 PFASs, SWATH-F: 64 PFASs) in identification. In addition, we automated the screening and identification process of SWATH-F to facilitate its use by researchers. SWATH-F is freely available on GitHub (https://github.com/njuIrene/SWATH-F) under an MIT license.

Comparison of IDA, SWATH, and MSALL techniques in the analysis of compounds of Huangqi Guizhi Wuwu decoction by UHPLC-Q-TOF-MS.

Huangqi Guizhi Wuwu decoction (HGWD) is an effective traditional Chinese medicine prescription, which is used for treating blood arthralgia in the clinic. However, its material basis has not been studied yet. Herein, a new and highly sensitive ultra-high-performance liquid chromatography-quadrupole-time of flight-MS (UHPLC-Q-TOF-MS) technique is proposed and used for the high-resolution and accurate identification of the material basis of HGWD. Seventy-eight compounds have been identified in HGWD. The advantages of information-dependent acquisition (IDA), sequential window acquisition of all theoretical fragment-ion spectra (SWATH), and MSALL in the quantitative and qualitative analyses of compounds were compared. For the identification of compounds, the best mode with the highest accuracy is the IDA. For the quantification of compounds, MSALL shows the best repeatability and linearity. This research provides a theoretical basis for the study of quality control of traditional Chinese medicine preparations.

Identification of low-level impurities in drug prototypes of carbocisteine by means of liquid chromatography-high-resolution mass spectrometry and general unknown comparative screening

High-resolution tandem quadrupole time-of-flight mass analysers enable new automated workflows for untargeted data evaluation of complex samples like drug products. An example of such procedure is the so-called general unknown comparative screening (GUCS), which is used for software-assisted, automated identification of components that are only present in a sample and not in a reference. The GUCS approach has been employed for the first time to detect both degradation products and reaction products in drug products. Two different carbocisteine containing syrup prototypes – one with sucrose and the other with artificial sweeteners – were selected as examples after nine months of storage at 40 °C and 75% relative humidity. The samples were analysed chromatographically using a Coresep SB mixed-mode column and high-resolution MS and MS/MS data were recorded in information dependant acquisition mode on a Sciex X500R quadrupole time-of-flight mass spectrometer. Data analysis was considerably facilitated using the corresponding placebo formulation as reference samples. With the GUCS approach two hitherto unknown degradation products of carbocisteine, i.e. the carbocisteine lactam of the sulfoxides and the disulfide between l-cysteine and thioglycolic acid, were detected at low concentrations in both of the syrup formulations. The presumed structures were confirmed by in silico analysis of the fragment spectra and high-resolution LC-MS experiments with reference substances. Two additional impurities were found in the sucrose-containing sample and identified as the N-glycosides of carbocisteine and its lactam, respectively, using binary mixtures with a 13C-labelled monosaccharide.

Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

Rapid Analysis of 18 Flavonoids in Tea by Ultrahigh-Performance Liquid Chromatography Coupled with Quadrupole-Time of Flight Mass Spectrometry

A method was established for the determination of 18 flavonoids in tea by ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The tea samples were extracted by 70% (V/V) methanol aqueous solution, and separation was achieved on a Kinetex F5 column (2.1 mm × 100 mm, 2.6 μm) with methanol and 0.1% formic acid in water as the mobile phase with a gradient elution. The samples were detected in TOF/MS and information-dependent acquisition (IDA)-MS/MS modes. The results showed that the relative standard deviations of 18 flavonoids were less than 5.0 ppm. The correlation coefficients (R2) of the linear equation were greater than 0.998 in the range of 0.10–200 ng/mL. The limits of detection were 0.0010–0.040 ppm, and the limits of quantification were 0.0020–0.10 ppm. The recoveries ranged from 73.8% to 107% at spiked levels of 0.0020–1.0 ppm, with relative standard deviations (RSDs) being less than 10%. The method was simple, specific, and reliable. It could be used for the rapid screening and quantitative analysis of flavonoids in tea.

Analysis of Phospholipids in Digestion Using Hybrid IDA and SWATH Acquisition: An Example for Krill Oil.

The composition and digestion of phospholipid-rich foods have important effects on the health of the body. Herein, a model-assisted liquid chromatography coupling mass spectrometry (LC-MS) method was established to analyze the phosphatidylcholine (PC) and lyso-phosphatidylcholine (LPC) species in krill oil before and after digestion. According to the confirmed PC and LPC species in the IDA (information dependent acquisition) results, three categories of mathematical models were set up, involving the retention time (RT), carbon number and unsaturation degree of the fatty acyl chain. All of the regression coefficient values (R2) were greater than 0.90, showing satisfactory fitting results. On this basis, using the computationally created precursor ion mass of PC and LPC species, 12 extra PC species and 4 LPC species were found in the SWATH (sequential windowed acquisition of all theoretical fragment ions) results. The PC and LPC compositions in the final digestive products had obvious differences among the different krill oils with different phospholipid content. Furthermore, more than half of the LPC species in the final digestive products were newly generated, indicating that LPC was one of basic constituents in the digestive products of krill oil. In conclusion, model-assisted hybrid IDA and SWATH acquisition has excellent detection performance, contributing to deep studies of the formations and functions of phospholipids.

Rapid determination of five common toxic alkaloids in blood by UPLC–MRM–IDA–EPI: Application to poisoning case

Toxic alkaloids are typically found in herbal medicines and have strong pharmacological effects and a broad therapeutic spectrum. On the other hand, toxic alkaloids exert toxicological activities in vivo; as such they have a narrow therapeutic window and can induce poisoning due to incorrect dose or misuse. In this view, there is an urgent need to develop a rapid and sensitive assay to detect these toxic alkaloids. This study developed a method for determining five common toxic alkaloids in blood, including brucine, strychnine, aconitine, mesaconitine, and hypaconitine using ultra-high liquid chromatography-tandem quadrupole/linear ion trap mass spectrometry (QTRAP UPLC–MS/MS). The analytes in this investigation were extracted with ether and detected using multiple reaction monitoring (MRM)-information-dependent acquisition (IDA)-enhanced product ion (EPI) scanning modes. SKF525A served as the internal standard (IS). The approach demonstrated excellent linearity, with a correlation coefficient (R) > 0.9964, and satisfactory sensitivity, with the limit of detection (LOD) of 0.31 ∼ 3.26 ng/mL and a limit of quantification (LOQ) of 1.13 ∼ 11.52 ng/mL. The extraction recovery (ER) was 78.8 ∼ 116.2%, the matrix effect (ME) was −12.3 ∼ 21.2%, and the method accuracy was 0.8 ∼ 12.8%. In addition, the intra-day precision and the inter-day precision (RSD) were 0.7% ∼ 7.4% and 0.4% ∼ 13.5%, respectively. The developed approach is sensitive and efficient, and offer significant application prospect in clinical monitoring and forensic detection of poisoning.

Impact of Matrix Species and Mass Spectrometry on Matrix Effects in Multi-Residue Pesticide Analysis Based on QuEChERS-LC-MS.

With the popularity of multi-residue pesticide analysis based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) cleanup and liquid chromatography-mass spectrometry (LC-MS), matching optimal matrix-matched calibration protocols and LC-MS conditions to reduce matrix effects (MEs) has become a crucial task for analysts in their routines. However, dozens to hundreds of pesticide analytes in a single run generate increasingly multi-dimensional ME data, requiring appropriate tools to handle these data sets. Therefore, we established an ME analysis strategy by drawing on analytical thinking and tools from metabolomics analysis. Using this, matrix species-induced and mass spectrometry-induced systematic ME variations were distinguished, and pesticides contributed to the variations were scanned out. A simultaneous weakening of MEs on 24 pesticides in 32 different matrices was achieved using the time-of-flight-mass spectrometry (TOF-MS) scan under the information-dependent acquisition (IDA) mode of high-resolution mass spectrometry (HR-MS), compared to multiple reaction monitoring (MRM) scanning by tandem mass spectrometry (MS/MS). Bay leaf, ginger, rosemary, Amomum tsao-ko, Sichuan pepper, cilantro, Houttuynia cordata, and garlic sprout showed enhanced signal suppression in the MRM scan for 105 differential MRM transitions for 42 pesticides and in IDA mode for 33 pesticides, respectively. This study revealed the interference of matrix species and mass spectrometry on MEs and provided a novel strategy for ME analysis.