Metabolomic Analysis Reveals Association between Decreased Ovarian Reserve and In Vitro Fertilization Outcomes.

In vitro fertilization (IVF) is a highly effective treatment for infertility; however, it poses challenges for women with decreased ovarian reserve (DOR). Despite the importance of understanding the impact of DOR on IVF outcomes, limited research has explored this relationship, particularly using omics approaches. Hence, we conducted a study to investigate the association between DOR and IVF outcomes, employing a metabolomic approach. We analyzed serum samples from 207 women undergoing IVF treatment, including 89 with DOR and 118 with normal ovarian reserve (NOR). Our findings revealed that DOR was significantly associated with unfavorable IVF outcomes, characterized by a reduced oocyte count, lower embryo quality, and decreased rates of pregnancy and live births. Furthermore, we identified 82 metabolites that displayed significant alterations in DOR patients, impacting diverse metabolic pathways. Notably, a distinct panel of metabolites, including palmitic acid, stearic acid, LysoPC(9:0(CHO)/0:0), PC(18:0/9:0(CHO)), and PC(16:0/9:0(CHO)), exhibited discriminatory power between the DOR and NOR groups, showcasing a strong correlation with IVF outcomes. These findings emphasize the crucial role of metabolomic disruptions in influencing IVF outcomes among women with DOR.

[Alignment method for metabolite chromatographic peaks using an N-acyl glycine retention index system].

Peak alignment is a crucial data-processing step in untargeted metabolomics analysis that aims to integrate metabolite data from multiple liquid chromatography-mass spectrometry (LC-MS) batches for enhanced comparability and reliability. However, slight variations in the chromatographic separation conditions can result in retention time (RT) shifts between consecutive analyses, adversely affecting peak alignment accuracy. In this study, we present a retention index (RI)-based chromatographic peak-shift correction (CPSC) strategy to address RT shifts and align chromatographic peaks for metabolomics studies. A series of N-acyl glycine homologues (C2-C23) was synthesized as calibrants, and an LC RI system was established. This system effectively corrected RT shifts arising from variations in flow rate, gradient elution, instrument systems, and chromatographic columns. Leveraging the RI system, we successfully adjusted the RT of raw data to mitigate RT shifts and then implemented the Joint Aligner algorithm for peak alignment. We assessed the accuracy of the RI-based CPSC strategy using pooled human fecal samples as a test model. Notably, the application of the RI-based CPSC strategy to a long-term dataset spanning 157 d as an illustration revealed a significant enhancement in peak alignment accuracy from 15.5% to 80.9%, indicating its ability to substantially improve peak-alignment precision in multibatch LC-MS analyses.

A dataset of branched fatty acid esters of hydroxy fatty acids diversity in foods.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a class of bioactive lipids that show therapeutic potential for diabetes, anti-cancer and inflammation. These FAHFAs can be obtained through dietary intake, potentially improving human health. However, there is currently inadequate knowledge regarding the presence and variety of FAHFAs in different foods. Herein, we profile FAHFAs from 12 typical food samples and 4 medicinal food samples with the aid of our previous established chemical isotope labeling-assisted liquid chromatography-mass spectrometry method and build a comprehensive dataset of FAHFA diversity. The dataset comprised a total of 1207 regioisomers belonging to 298 different families, with over 100 families being newly discovered for the first time. Therefore, our findings contribute valuable insights into the molecular diversity and presence of FAHFA in a range of foods. This dataset serves as a foundation for further exploration of the nutritional and medicinal functions of FAHFAs.

Determination of vitamin D metabolites in various biological samples through an improved chemical derivatization assisted liquid chromatography-tandem mass spectrometry approach.

Vitamin D (VD) metabolites are involved in a variety of important metabolic processes and physiological effects in organisms. Profiling of VD metabolites favors a deep understanding of the physiological role of VD. However, VD metabolites are difficult to detect due to their high chemical structural rigidity, structural similarity, and low sensitivities under liquid chromatography-tandem mass spectrometry (LC-MS). Herein, we present a chemical derivatization assisted LC-MS/MS strategy for the detection of VDs, in which 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is employed to derivatize the conjugated diene of VD metabolites and provides sensitizing reporters for MS detection. After PTAD derivatization, the sensitivities of seven VD metabolites increased by 24-276 folds, with the limits of detection ranging from 3 to 20 pg mL-1. Using this method, we achieved a sensitive and accurate quantification of 7 VD metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 1,24,25-trihydroxyvitamin D3) of the VD metabolic pathway in different trace biological samples, including human serum, mouse tissues (namely liver, kidney, lung, and spleen), and cells. We believe that the present method can provide a promising tool for an in-depth analysis of VD metabolism.

Novel Peak Shift Correction Method Based on the Retention Index for Peak Alignment in Untargeted Metabolomics.

Peak alignment is a crucial step in liquid chromatography-mass spectrometry (LC-MS)-based large-scale untargeted metabolomics workflows, as it enables the integration of metabolite peaks across multiple samples, which is essential for accurate data interpretation. Slight differences or fluctuations in chromatographic separation conditions, however, can cause the chromatographic retention time (RT) shift between consecutive analyses, ultimately affecting the accuracy of peak alignment between samples. Here, we introduce a novel RT shift correction method based on the retention index (RI) and apply it to peak alignment. We synthesized a series of N-acyl glycine (C2-C23) homologues via the amidation reaction between glycine with normal saturated fatty acids (C2-C23) as calibrants able to respond proficiently in both mass spectrometric positive- and negative-ion modes. Using these calibrants, we established an N-acyl glycine RI system. This RI system is capable of covering a broad chromatographic space and addressing chromatographic RT shift caused by variations in flow rate, gradient elution, instrument systems, and LC separation columns. Moreover, based on the RI system, we developed a peak shift correction model to enhance peak alignment accuracy. Applying the model resulted in a significant improvement in the accuracy of peak alignment from 15.5 to 80.9% across long-term data spanning a period of 157 days. To facilitate practical application, we developed a Python-based program, which is freely available at https://github.com/WHU-Fenglab/RI-based-CPSC.

Uncovering the Carboxylated Metabolome in Gut Microbiota-Host Co-metabolism: A Chemical Derivatization-Molecular Networking Approach.

Gut microbiota-host co-metabolites serve as essential mediators of communication between the host and gut microbiota. They provide nutrient sources for host cells and regulate gut microenvironment, which are associated with a variety of diseases. Analysis of gut microbiota-host co-metabolites is of great significance to explore the host-gut microbiota interaction. In this study, we integrated chemical derivatization, liquid chromatography-mass spectrometry, and molecular networking (MN) to establish a novel CD-MN strategy for the analysis of carboxylated metabolites in gut microbial-host co-metabolism. Using this strategy, 261 carboxylated metabolites from mouse feces were detected, which grouped to various classes including fatty acids, bile acids, N-acyl amino acids, benzoheterocyclic acids, aromatic acids, and other unknown small-scale molecular clusters in MN. Based on the interpretation of the bile acid cluster, a novel type of phenylacetylated conjugates of host bile acids was identified, which were mediated by gut microbiota and exhibited a strong binding ability to Farnesoid X receptor and Takeda G protein-coupled receptor 5. Our proposed strategy offers a promising platform for uncovering carboxylated metabolites in gut microbial-host co-metabolism.

Chemical isotope labeling assisted liquid chromatography-mass spectrometry method for simultaneous analysis of central carbon metabolism intermediates.

Central carbon metabolism pathway (CCM) is one of the most important metabolic pathways in all living organisms and play crucial function in aspect of organism life. However, the simultaneous detection of CCM intermediates remains challenging. Here, we developed a chemical isotope labeling combined with LC-MS method for simultaneous determination of CCM intermediates with high coverage and accuracy. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, all CCM intermediates obtain better separation and accurate quantification at a single LC-MS run. The obtained limits of detection of CCM intermediates ranged from 5 to 36 pg/mL. Using this method, we achieved simultaneous and accurate quantification of 22 CCM intermediates in different biological samples. Take account of the high detection sensitivity of the developed method, this method was further applied to the quantification of CCM intermediates at single-cell level. Finally, 21 CCM intermediates were detected in 1000 HEK-293T cells and 9 CCM intermediates were detected in mouse kidney glomeruli optical slice samples (10∼100 cells).

In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

A strategy for screening and identification of new amino acid-conjugated bile acids with high coverage by liquid chromatography-mass spectrometry.

Bile acids (BAs) are a class of vital gut microbiota-host cometabolites, and they play an important role in maintaining gut microbiota-host metabolic homeostasis. Very recently, a new mechanism of BA anabolic metabolism mediated by gut microbiota (BA-amino acid conjugation) has been revealed, which provides a perspective for the research on BA metabolism and gut metabolome. In this study, we established a polarity-switching multiple reaction monitoring mass spectrometry-based screening method to mine amino acid-conjugated bile acids (AA-BAs) derived from host-gut microbiota co-metabolism. In addition, a retention time-based annotation strategy was further proposed to identify the AA-BA isomers and epimers. Using the developed methods, we successfully screened 118 AA-BA conjugates from mouse and human feces, 28 of them were confirmed by standards, and 62 putatively identified based on their predicted retention times. Moreover, we observed that the levels of most AA-BAs were significantly downregulated in the feces of chronic sleep deprivation mice, suggesting that the AA-BA metabolism was closely related to the physiological state of the host.

Rapid and Economical Chemoselective Metabolomics Using Boronate Ester Formation on a Monolithic Substrate.

Although chemoselective labeling strategies show great potential in in-depth description of metabolomics, the associated time and expense limit applications in high-throughput and routine analysis. We report a fast and effective chemoselective labeling strategy based on multifunctionalized monolithic probes. A rapid pH-responsive boronate ester reaction was employed to immobilize and release probe molecules from substrate in 5 min. The mesoporous surface and hierarchically porous channels of the substrate allowed for accelerated labeling reactions. Moreover, the discernible boron beacons allowed for recognition of labeled metabolites with no need for expensive isotopic encoding. This new strategy has been successfully used for submetabolome analysis of yeast cells, serum, and faeces samples, with improved sensitivity for short chain fatty acids up to 1 600 times compared with non-labeled liquid chromatography-mass spectrometry (LC-MS) methods.

Profiling of Branched Fatty Acid Esters of Hydroxy Fatty Acids in Teas and Their Potential Sources in Fermented Tea.

Branched fatty acid ester of hydroxy fatty acid (FAHFA) is a class of natural lipid with important biological functions. In this study, we first profiled natural-origin FAHFAs in different teas using the chemical labeling-assisted liquid chromatography-mass spectrometry method. Consequently, we observed rich molecular diversity of FAHFAs with multiple regioisomers in teas. Additionally, the FAHFA contents had a positive relationship with the tea fermentation degree and a negative relationship with homologous fatty acids. Moreover, the highly accumulated FAHFAs (e.g., 3-MAHMA) in some postfermented teas (e.g., Fu brick tea) were also basically interpreted with regiospecificity of FAHFAs in both teas and fungus. This study revealed that tea is a rich natural source of FAHFAs, and some abundant FAHFAs might be the functional molecules accounting for the antidiabetic function of teas.

PTIP governs NAD+ metabolism by regulating CD38 expression to drive macrophage inflammation.

NAD+ metabolism is involved in many biological processes. However, the underlying mechanism of how NAD+ metabolism is regulated remains elusive. Here, we find that PTIP governs NAD+ metabolism in macrophages by regulating CD38 expression and is required for macrophage inflammation. Through integrating histone modifications with NAD+ metabolic gene expression profiling, we identify PTIP as a key factor in regulating CD38 expression, the primary NAD+-consuming enzyme in macrophages. Interestingly, we find that PTIP deletion impairs the proinflammatory response of primary murine and human macrophages, promotes their metabolic switch from glycolysis to oxidative phosphorylation, and alters NAD+ metabolism via downregulating CD38 expression. Mechanistically, an intronic enhancer of CD38 is identified. PTIP regulates CD38 expression by cooperating with acetyltransferase p300 in establishing the CD38 active enhancer with enriched H3K27ac. Overall, our findings reveal a critical role for PTIP in fine-tuning the inflammatory responses of macrophages via regulating NAD+ metabolism.

Alternating Dual-Collision Energy Scanning Mass Spectrometry Approach: Discovery of Novel Microbial Bile-Acid Conjugates.

Bile acids (BAs) are a type of gut microbiota-host cometabolites with abundant structural diversity, and they play critical roles in maintaining host-microbiota homeostasis. In this study, we developed a new N-(4-aminomethylphenyl) pyridinium (AMPP) derivatization-assisted alternating dual-collision energy scanning mass spectrometry (AMPP-dual-CE MS) method for the profiling of BAs derived from host-gut microbiota cometabolism in mice. Using the proposed method, we discovered two new types of amino acid conjugations (alanine conjugation and proline conjugation) and acetyl conjugation with host BAs, for the first time, from mouse intestine contents and feces. Additionally, we also determined and identified nine new leucine- and phenylalanine-conjugated BAs. These findings broaden our knowledge of the composition of the BA pool and provide insight into the mechanism of host-gut microbiota cometabolism of BAs.

Integration of Chemical Derivatization and in-Source Fragmentation Mass Spectrometry for High-Coverage Profiling of Submetabolomes.

In-source fragmentation-based high-resolution mass spectrometry (ISF-HRMS) is a potential analytical technique, which is usually used to profile some specific compounds that can generate diagnostic neutral loss (NL) or fragment ion (FI) in ion source inherently. However, the ISF-HRMS method does not work for those compounds that cannot inherently produce diagnostic NL or FI in ion source. In this study, a derivatization-based in-source fragmentation-information-dependent acquisition (DISF-IDA) strategy was proposed for profiling the metabolites with easily labeled functional groups (submetabolomes) by liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (LC-ESI-Q-TOF MS). As a proof-of-concept study, 36 carboxylated compounds labeled with N,N-dimethylethylenediamine (DMED) were selected as model compounds to examine performance of DISF-IDA strategy in screening the carboxylated metabolites and acquiring their MSn spectra. In ESI source, the DEMD-derived carboxylated compounds were fragmented to produce characteristic neutral losses of 45.0578, 63.0684, and/or 88.1000 Da that were further used as diagnostic features for screening the carboxylated metabolites by DISF-IDA-based LC-Q-TOF MS. Furthermore, high-resolution MSn spectra of the model compounds were also obtained within a single run of DISF-IDA-based LC-Q-TOF MS analysis, which contributed to the improvement of the annotation confidence. To further verify its applicability, DISF-IDA strategy was used for profiling carboxylated submetabolome in mice feces. Using this strategy, a total of 351 carboxylated metabolites were detected from mice feces, of which 178 metabolites (51% of the total) were positively or putatively identified. Moreover, DISF-IDA strategy was also demonstrated to be applicable for profiling other submetabolomes with easily labeled functional groups such as amino, carbonyl, and cis-diol groups. Overall, our proposed DISF-IDA strategy is a promising technique for high-coverage profiling of submetabolomes with easily labeled functional groups in biological samples.

Screening and Identification of Epoxy/Dihydroxy-Oxylipins by Chemical Labeling-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

Epoxy/dihydroxy-oxylipins are important biologically active compounds that are mainly formed from polyunsaturated fatty acids (PUFAs) in the reactions catalyzed by the cytochrome P450 (CYP 450) enzyme. The analysis of epoxy/dihydroxy-oxylipins would be helpful to gain insights into their landscape in living organisms and provide a reference for the biological studies of these compounds. In this work, we employed chemical labeling-assisted liquid chromatography (LC) coupled with high-resolution mass spectrometry (CL-LC-HRMS) to establish a highly sensitive and specific method for screening and annotating epoxy/dihydroxy-oxylipins in biological samples. The isotope reagents 2-dimethylaminoethylamine (DMED) and DMED-d4 were employed to label epoxy/dihydroxy-oxylipins containing carboxyl groups so as to improve the analysis selectivity and MS detection sensitivity of epoxy/dihydroxy-oxylipins. Based on a pair of diagnostic ions with a mass-to-charge ratio (m/z) difference of 15.995 originating from the fragmentation of derivatives via high-energy collision dissociation (HCD), the potential epoxy/dihydroxy-oxylipins were rapidly screened from the complex matrix. Furthermore, the epoxy/dihydroxy groups could be readily localized by the diagnostic ion pairs, which enabled us to accurately annotate the epoxy/dihydroxy-oxylipins detected in biological samples. The applicability of our method was demonstrated by profiling epoxy/dihydroxy-oxylipins in human serum and heart samples from mice with high-fat diet (HFD). By the proposed method, a total of 32 and 62 potential epoxy/dihydroxy-oxylipins including 42 unreported ones were detected from human serum and the mice heart sample, respectively. Moreover, the relative quantitative results showed that most of the potential epoxy/dihydroxy-oxylipins, especially the oxidation products of linoleic acid (LA) or α-linolenic acid (ALA), were significantly decreased in the heart of mice with HFD. Our developed method is of high specificity and sensitivity and thus is a promising tool for the identification of novel epoxy/dihydroxy-oxylipins in biological samples.

A mathematical method for calibrating the signal drift in liquid chromatography - mass spectrometry analysis.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) has become the most versatile analytical tool for profiling small-molecule compounds and increasingly been applied in many fields. Nevertheless, LC-MS based quantification still face some challenges, such as signal drift in LC-MS, which may affect the validity of the obtained data and lead to misinterpretation of biological results. Here, we established a calibration method known as "RIM" to compensate the signal drift of LC-MS. To this end, a mixture of d4-2-dimethylaminoethylamine (d4-DMED)-coded normal fatty acids (C5-C23) was used as calibrants to construct RIM calibration. With the addition of calibrants, not only the MS signal drift, but also the mass accuracy and LC retention time can be calibrated, thereby improving the reliability of quantitative data. The effectiveness of RIM was carefully validated using a human serum extract spiked with 34 standards and then RIM was applied for rat brain untargeted metabolome research. In addition, to expand the functionality and flexibility of RIM for data handling, we generated a MATLAB-based RIM program, which implements the above concepts and allows automatic data process. Taken together, the proposed RIM method has potential application in large-scale quantitative study of complex samples.

Boron Isotope Tag-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry for Discovery and Annotation of cis-Diol-Containing Metabolites.

cis-Diol-containing metabolites are widely distributed in living organisms, and they participate in the regulation of various important biological activities. The profiling of cis-diol-containing metabolites could help us in fully understanding their functions. In this work, based on the characteristic isotope pattern of boron, we employed a boronic acid reagent as the isotope tag to establish a sensitive and selective liquid chromatography-high-resolution mass spectrometry method for the screening and annotation of cis-diol-containing metabolites in biological samples. Boronic acid reagent 2-methyl-4-phenylaminomethylphenylboronic acid was used to label the cis-diol-containing metabolites in biological samples to improve the selectivity and MS sensitivity of cis-diol-containing metabolites. Based on the characteristic 0.996 Da mass difference of precursor ions and the peak intensity ratio of 1:4 originating from 10B and 11B natural isotopes, the potential cis-diol-containing metabolites were rapidly screened from biological samples. Potential cis-diol-containing metabolites were annotated by database searching and analysis of fragmentation patterns obtained by multistage MS (MSn) via collision-induced dissociation. Importantly, the cis-diol position could be readily resolved by the MS3 spectrum. With this method, a total of 45 cis-diol-containing metabolites were discovered in rice, including monoglycerides, polyhydroxy fatty acids, fatty alcohols, and so forth. Furthermore, the established method showed superiority in avoiding false-positive results in profiling cis-diol-containing metabolites.

In-Depth Annotation Strategy of Saturated Hydroxy Fatty Acids Based on Their Chromatographic Retention Behaviors and MS Fragmentation Patterns.

Hydroxy fatty acids are a class of bioactive compounds in a variety of organisms. The identification of hydroxy fatty acids in biological samples has still been a challenge because of their low abundance, high structural similarity, and limited availability of authentic hydroxy fatty acid standards. Here, we present a strategy for the annotation of saturated monohydroxyl fatty acids (OH-FAs) based on the integration of chromatographic retention rules and MS2 fragmentation patterns. Thirty-nine authentic OH-FA standards were used to investigate their retention behavior on a reversed-phase stationary phase (C18) of liquid chromatography, and we found that their retention simultaneously follows two kinds of "carbon number rules". Using the "carbon number rules", the retention index (RI) of all OH-FAs that contain carbon numbers from 8 to 18 (C8-18) can be predicted. Additionally, by studying the MS2 fragmentation of OH-FAs under collision-induced dissociation, we found that the intensity ratio (IR) of the characteristic fragment ions ([M + H]+-63 and [M + H]+-45) is closely related to the position of the hydroxyl group on the OH-FA structure, which is helpful to further identify and confirm the OH-FA isomers. As a result, 97 of 107 potential OH-FAs detected in honey, human serum, and rice seedling by chemical isotope labeling-assisted liquid chromatography-mass spectrometry were annotated upon the RI matching and IR confirming. Furthermore, in order to simplify the annotation process of OH-FAs, we constructed an OH-FA library to facilitate the annotation of OH-FAs. Overall, this study provides a new and promising tool for the in-depth annotation of OH-FA isomers.

The mechanism of chlorogenic acid inhibits lipid oxidation: An investigation using multi-spectroscopic methods and molecular docking.

Endogenous lipase and lipoxygenase play important roles in accelerating lipid oxidation. Polyphenols are a series of commonly used chemicals for preserving fish and seafood products, due to their positive inhibitory effects on lipid oxidation. However, the mechanism involved is still unknown. The inhibitory effects of chlorogenic acid (CGA) on lipase and lipoxygenase were investigated and explored with multi- spectroscopic and molecular docking approaches. Results showed that CGA could inhibit the activities of lipase and lipoxygenase with concentration increased in a highly dose-dependent manner. CGA quenched intrinsic fluorescence intensities of enzymes by static quenching and binding with CGA which led to changes in 3D structures of enzymes. Results of the molecular docking confirmed binding modes, binding sites and major interaction forces between CGA and enzymes, which reduced the corresponding activity. Thus, this study could provide basic mechanisms of the inhibitory effects of polyphenols on lipid oxidation during food preservation.

Derivatization assisted LC-p-MRM-MS with high CID voltage for rapid analysis of brassinosteroids.

In this study, a derivatization-assisted pseudo-multiple reaction monitoring with high CID voltage (HV-p-MRM) strategy was proposed for the analysis of brassinosteroids (BRs) by liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ MS). The concept of the HV-p-MRM strategy was proposed on the basis of an assumption that the precursor ion of analyte is stable in collision cell and less prone to fragmentation at high CID voltage, while co-existing ions (impurity) of easy fragmentation can break down into smaller fragment ions. In such case, by increasing the CID voltage, the co-existing ions that are introduced due to the low resolution of the quadrupole 1 (Q1) can be filtered out by quadrupole 3 (Q3), while the stable precursor ion of analyte will pass through Q3, thereby that the signal-to-noise ratio (S/N) of the analysis can be improved. As a proof-of-concept study, BRs were derivatized with rhodamine B-boronic acid (RhB-BA) and then the derivatives were used to investigate their variations in MS signal, background noise, and S/N upon the CID voltage and MS scanning resolution. The results showed that S/N of these derivatives can be improved in HV-p-MRM mode. To further demonstrate the feasibility of HV-p-MRM strategy, a RhB-BA derivatization assisted LC-HV-p-MRM-MS method was developed for the analysis of BRs. Using this method, rapid and sensitive determination of BRs in different organs of rape flower was achieved. It can be expected that HV-p-MRM may be suitable for the analytes that are stable or can be converted into compounds of high stability in collision cell at high CID voltage.