Fragmentation Patterns of Phenolic C-Glycosides in Mass Spectrometry Analysis.

BACKGROUND: Many phenolic C-glycosides possess nutritional benefits and pharmacological efficacies. However, the MS/MS fragmentation pattern of phenolic C-glycosides analysis is understudied. This paper aims to determine the MS/MS fragmentation patterns of phenolic C-glycosides. METHOD: Ten compounds with different sugar moieties, aglycones, and substitutes were analyzed to determine the impact of these structural features on MS/MS fragmentation using UPLC-QTOF-MS analysis. RESULTS: The results showed that water loss followed by RDA reaction and alpha cleavage in the C-C bonded sugar moieties are the major fragmentation pathways. Additionally, the sugar cleavage was not affected by the skeleton and the substitute of the aglycones. These results suggested that the fragmentation patterns of phenolic C-glycosides differ from those in the O-glycosides, where the O-C glycosidic bond is the most cleavage-liable bond in MS/MS analysis. CONCLUSIONS: These MS/MS fragmentation patterns can be used for the identification of C-glycosides from dietary components and herbal medicine as well as developing robust methods using MRM methods to quantify C-glycosides.

Identifying the tissues-of-origin of circulating cell-free DNAs is a promising way in noninvasive diagnostics.

Advances in sequencing technologies facilitate personalized disease-risk profiling and clinical diagnosis. In recent years, some great progress has been made in noninvasive diagnoses based on cell-free DNAs (cfDNAs). It exploits the fact that dead cells release DNA fragments into the circulation, and some DNA fragments carry information that indicates their tissues-of-origin (TOOs). Based on the signals used for identifying the TOOs of cfDNAs, the existing methods can be classified into three categories: cfDNA mutation-based methods, methylation pattern-based methods and cfDNA fragmentation pattern-based methods. In cfDNA mutation-based methods, the SNP information or the detected mutations in driven genes of certain diseases are employed to identify the TOOs of cfDNAs. Methylation pattern-based methods are developed to identify the TOOs of cfDNAs based on the tissue-specific methylation patterns. In cfDNA fragmentation pattern-based methods, cfDNA fragmentation patterns, such as nucleosome positioning or preferred end coordinates of cfDNAs, are used to predict the TOOs of cfDNAs. In this paper, the strategies and challenges in each category are reviewed. Furthermore, the representative applications based on the TOOs of cfDNAs, including noninvasive prenatal testing, noninvasive cancer screening, transplantation rejection monitoring and parasitic infection detection, are also reviewed. Moreover, the challenges and future work in identifying the TOOs of cfDNAs are discussed. Our research provides a comprehensive picture of the development and challenges in identifying the TOOs of cfDNAs, which may benefit bioinformatics researchers to develop new methods to improve the identification of the TOOs of cfDNAs.

[Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n].

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Mass spectral analysis of secondary metabolites from Zingiber montanum rhizome extract using UHPLC-HR-ESI-QTOF-MS/MS.

INTRODUCTION: Zingiber montanum (J.Koenig) Link ex A.Dietr. is a popular medicinal plant in Thailand. Its rhizomes have been used as an ingredient in various Thai traditional medicine formulas. While many reports have focused on the chemical constituents and biological activities of this plant, a comprehensive study on secondary metabolite profiling using tandem mass spectrometry has, to this point, never been documented. OBJECTIVE: To analyze the chemical constituents in Z. montanum rhizomes using ultra-high performance liquid chromatography coupled with ultra-high-resolution electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-HR-ESI-QTOF-MS/MS) analyses and to utilize the characteristic fragmentation patterns of these compounds to facilitate their identification. METHODOLOGY: UHPLC-HR-ESI-QTOF-MS/MS in positive ion mode was used for chemical identification of secondary metabolites from the ethanolic extract of the plant material. MS/MS data of some known reference compounds, together with detailed fragmentation pattern information of several compounds obtained from the crude extract, were used to elucidate their chemical structures. RESULTS: In this work, one benzaldehyde, ten phenylbutenoid monomers, six curcuminoids, and nine phenylbutenoid dimers were assigned based on their characteristic fragment ions. Among these compounds, 2-(3,4-dimethoxystyryl)oxirane was tentatively suggested as a potential new compound. Several characteristic fragment ions from these compounds were assigned and the relative ion abundance of these was also used to differentiate the chemical structures of compounds having the same molecular mass. CONCLUSIONS: The results will benefit future high-throughput screening of bioactive compounds and method development for the quality control of raw materials and herbal drugs derived from Z. montanum rhizome extracts.

Liquid biopsy with cell free DNA: new horizons for prostate cancer.

Although prostate cancer (PCa) is one of the most common tumors in European males, the only minimally invasive diagnostic tool in PCa setup is the determination of PSA in serum. Cell-free DNA (cfDNA) has been demonstrated to be helpful for PCa diagnosis but has not yet been integrated into the clinical setting. This review aims to provide a systematic update of cfDNA and its fragmentation patterns in PCa reported in literature published over the last twenty years. Due to the high variability of the scientific methods adopted and a lack of standardized median cfDNA levels, results fluctuate across different studies. These differences may be due to the cfDNA source, the quantification method, or the fragmentation pattern. Blood plasma is the most frequently analyzed biological fluid, but seminal plasma has been reported to contain higher cfDNA concentration due to its vicinity to the tumor origin. CfDNA has been shown to be composed of single-stranded (ssDNA) and double-stranded DNA (dsDNA), so the total cfDNA concentration should be preferred as it corresponds best to the tumor mass. Fluorometry and capillary electrophoresis (CE) may be quick and cost-effective tools for cfDNA assessment in a clinical setting. The greatest future challenge is the elaboration of common guidelines and standardized procedures for diagnostic laboratories performing cfDNA analysis. A multiparametric approach combining the analysis of total cfDNA (both ssDNA and dsDNA), cfDNA fragment length, and specific genetic mutations (ctDNA assessment) is required for optimal future applications of liquid biopsy.

Differentiation of bisphenol F diglycidyl ether isomers and their derivatives by HPLC-MS and GC-MS-comment on the published data.

Positional isomers of bisphenol F diglycidyl ether (BFDGE) have been analyzed by high-pressure liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry (HPLC-MS, GC-MS). Positional isomers of BFDGE derivatives (BFDGEx2H2O, BFDGExH2OxHCl) have been analyzed by HPLC-MS. On the basis of the obtained fragmentation patterns, the elution order of the isomers has been unequivocally determined, in standard solutions and in the sample of liquid obtained after rinsing an empty mackerel fish can with acetonitrile. Under HPLC condition, para,para isomers are eluted first, then ortho,para isomers' elution follows, and ortho,ortho isomers are eluted last. Under GC condition, the reverse elution order has been obtained. For the first time, two ortho,para isomers of BFDGExH2OxHCl have been detected and their elution order has been determined. The obtained results are of key importance for determination of the isomer distribution of BFDGE and its derivatives in food samples.

Comprehensive identification, fragmentation pattern, and metabolic pathways of gefitinib metabolites via UHPLC-Q-TOF-MS/MS: in vivo study of rat plasma, urine, bile, and faeces.

Gefitinib, the first approved inhibitor for oral epidermal growth factor receptor (EGFR), has been proved to be effective in non-small cell lung cancer with EGFR mutation. However, there are many metabolites of gefitinib that have not been identified in vivo. This study aims to identify the metabolites of gefitinib and its metabolic pathways in rats using ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) detector. Protein precipitation, solid-phase and ultrasonic extraction were used for the pre-treatment of plasma, urine, bile and faeces samples. In this study, a total of 28 compounds were identified in rat plasma, 29 in bile, 20 in urine and 16 in faeces. 20 new compounds were firstly reported as metabolites of gefitinib. Reduction, hydroxylation, dealkylation and dehalogenation were the major metabolic pathways in phase I. For phase II, the main pathways were sulphate and glucuronide conjugation. The fragment ions of gefitinib and its metabolites were usually generated via the fracture of C1-O bond of propoxy on the C6 position of aniline quinazoline ring. The results may be valuable and important for understanding the metabolic process of gefitinib in clinical application and drug safety.

Two New Alkaloids from Fuzi and Their Metabolites Study.

Former study suggests alkaloids from herbs of Aconitum genus plants possess excellent bioactivities, which exert great value for related deeper chemical constituent investigation. Herein, chemical isolation was performed and four alkaloids were isolated from Fuzi, of which two were new ones, and the other two were reported NMR data for the first time. Their chemical structures were identified by NMR data, high resolution MS, UV and IR analysis. Additionally, the MS fragmentation patterns were explored, formerly, that of hetisane alkaloid was rarely reported, and fragmentation mechanism of the diagnostic ion was proposed. Based on these fragment pathway, metabolites and metabolic pathways of four compounds were investigated in rat liver microsomes using UPLC-Q/TOF-MS, and dehydrogenation product was firstly found from metabolites of hetisane alkaloid.

Development of a LC-HRMS based approach to boost structural annotation of isomeric citrus flavanones.

INTRODUCTION: The structural annotation of target relies on high-resolution mass spectrometry (HRMS) information resulting in dubious identities in most cases. The accurate annotation of isomeric structures is still challenging to be confirmed with significant bottleneck. OBJECTIVE: This study focused on the improvement of structural annotation of candidate structures via four pairs of isomeric flavanone-7-O-diglucosides and their basic flavanone aglycones commonly detected in citrus products. METHOD: An integrated liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) approach merging retention time, accurate mass, tandem mass spectrometry (MS/MS) information (diagnostic ions), ion ratio at selected collision energy was established successfully. RESULTS: Feasibility of this approach was validated confidently in biological samples with relative standard deviation (RSD) of ion ratio range from 3.91 to 12.28%. Differences of fragmentation patterns of citrus flavanones were illustrated reasonably. MS/MS fragments of (S)-hesperetin and (S)-isosakuranetin were complicated and showed typical radical ion [1,2 A - H]•- (m/z 164) in negative ESI mode due to the methoxyl group on B-ring, which showed huge difference with (R)-hesperetin and (R)-isosakuranetin. CONCLUSION: This study integrated multiple levels to boost the confidence of structural annotation relied on LC-HRMS, and provided important values in practice for precise identification of citrus flavanones in biological matrices.

Rapid identification and structural characterization of polyoxypregnane glycosides in Dregea sinensis by HPLC-MSn and HRMS.

By HPLC-MSn and HRMS analyses, the structures of 52 polyoxypregnane glycosides were rapidly inferred from Dregea sinensis Hemsl on the basis of their sodium-cationized molecules [M + Na]+ and predominant diagnostic ions resulting from the saccharic chain on C3 and the neutral loss of substituent on C11 and C12. Compounds 1 and 7 significantly inhibited LPS-stimulated splenocyte proliferation in vitro.[Formula: see text].

Identification of Antiprotozoal Compounds from Buxus sempervirens L. by PLS-Prediction.

Various nor-triterpene alkaloids of Buxus (B.) sempervirens L. have shown remarkable in vitro activity against the causative agents of tropical malaria and East African sleeping sickness. To identify further antiprotozoal compounds of this plant, 20 different fractions of B. sempervirens L., exhibiting a wide range of in vitro bioactivity, were analyzed by UHPLC/+ESI-QqTOF-MS/MS. The analytical profiles were investigated by partial least squares regression (PLS) for correlations between the intensity of LC/MS signals, bioactivity and cytotoxicity. The resulting models highlighted several compounds as mainly responsible for the antiprotozoal activity and thus, worthwhile for subsequent isolation. These compounds were dereplicated based on their mass spectra in comparison with isolated compounds recently reported by us and with literature data. Moreover, an estimation of the cytotoxicity of the highlighted compounds was derived from an additional PLS model in order to identify plant constituents with strong selectivity. In conclusion, high levels of antitrypanosomal and antiplasmodial activity were predicted for eight and four compounds, respectively. These include three hitherto unknown constituents of B. sempervirens L., presumably new natural products.

Investigation of the Variability of Alkaloids in Buxus sempervirens L. Using Multivariate Data Analysis of LC/MS Profiles.

Buxus sempervirens L. is a common ornamental plant in southern and central Europe, and has been used ethopharmacologically against a wide variety of diseases due to it containing nor-triterpene alkaloids of the nor-cycloartane type. Recently, we demonstrated the interesting antiprotozoal potential of some of these compounds. To characterize the temporal variability in the alkaloid profile of two different varieties and their leaves and twigs, 30 different extracts of B. sempervirens were evaluated by Ultra High Performance Liquid Chromatography/positive Mode-Electrospray Ionization Quadrupole Time-of-Flight-Tandem Mass Spectrometry (UHPLC/+ESI-QqTOF-MS/MS). The analytical profiles were thoroughly investigated by various methods of multivariate data analysis (MVDA). A principal component analysis (PCA) model elucidates the seasonal variation in the phytochemical composition of B. sempervirens var. arborescens and suffruticosa along with differences between the varieties. Analysis of a volcano plot illustrated the differences between the two organs, the leaf and twig. Eighteen compounds were highlighted by the models as constituents of the plant characteristic for a season, variety or organ. These compounds were dereplicated based on their chromatographic and +ESI-QqTOF-MS and -MS/MS data. In addition, mass spectral fragmentation pathways for already known alkaloids as well as new natural products could be postulated for the first time. In conclusion, the MVDA models give detailed information on the temporal variability in the alkaloid profile of two different varieties and their organs (leaf vs. twig) of B. sempervirens. Thus, the results of this study allow, e.g., the identification of characteristic compounds for the different varieties, plant organs, seasons, and the optimal harvesting time for the isolation of particular Buxus-alkaloids of interest for subsequent studies.

[Study on fragmentation patterns of coumarins in Notopterygium inchum with ultrahigh performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry].

To demonstrate the fragmentation patterns of simple coumarins furanocourmarin(C_7-C_8), furanocourmarin(C_6-C_7) and dihydrofuran coumarin by mass spectrometry, with fraxin, scopoletin, isopsoralen, pimpinellin, isoimperatorin, notopterol and noda-kenin as study subjects, so as to provide a basis for rapid identification of compounds in different subtypes of coumarins. Ultrahigh performance liquid chromatography combined with quardrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was implemented in both positive and negative ion modes. Masslynx software was employed to provide the elemental constituents of each detected ion based on its accurate molecular weight. Chemdraw 2014 was used to cultivate mass number of each inferred structure. The fragment pattern of each compound was determined based on the structures inferred from all the relevant ions. And the patterns were drawn by Chemdraw 2014. The deviation between the calculated molecular weight of the inferred structure and the detected value of the ions was used to assess the correctness of the inferred structures in the fragmentation patterns. The results showed that with UPLC-Q-TOF, neutral loss of CO_2 and CO was reflected in lactone and furan skeletons from the courmarin structure. An even mass was attributed to the loss of an odd number of methyl radicals from compounds with a methoxy substituent. Furanocourmarin(C_7-C_8) produced a protonated molecular ion([M+H]~+), while the other courmarin subtypes produced either a sodium adduct of the molecular ion([M+Na]~+) or a sodium adduct of the molecular ion([M+Na]~+) with a protonated molecular ion([M+H]~+). The m/z 203.03 was a diagnostic ion for furanocourmarin(C_6-C_7), and the m/z 147.04 was supplementary evidence for furanocourmarin(C_6-C_7) identification. The characteristic ion of furanocourmarin(C_7-C_8) was m/z 131.05, while m/z 187.04 was the characteristic ion of dihydrofuran coumarin. The m/z 203.03 ion for furanocourmarin(C_7-C_8) was pretty weak. In negative ion mode, furanocourmarin(C_7-C_8) did not have any signals that were different from the other subtypes of courmarins. The fragmentation patterns in negative ion mode for the other subtypes of courmarins were similar to those in positive ion mode. Four types of fragmentation patterns were identified as forcourmarins from Notopterygium inchum. This study provides the basis for the rapid identification of courmarin subtypes by mass spectrometry.

Mass Spectrometry-Based Characterization of New Spirolides from Alexandrium ostenfeldii (Dinophyceae).

Spirolides belong to a group of marine phycotoxins produced by the marine planktonic dinophyte Alexandrium ostenfeldii. Composed of an imine moiety and a spiroketal ring system within a macrocylcle, spirolides are highly diverse with toxin types that vary among different strains. This study aims to characterize the spirolides from clonal A. ostenfeldii strains collected from the Netherlands, Greenland and Norway by mass spectral techniques. The structural characterization of unknown spirolides as inferred from high-resolution mass spectrometry (HR-MS) and collision induced dissociation (CID) spectra revealed the presence of nine novel spirolides that have the pseudo-molecular ions m/z 670 (1), m/z 666 (2), m/z 696 (3), m/z 678 (4), m/z 694 (5), m/z 708 (6), m/z 720 (7), m/z 722 (8) and m/z 738 (9). Of the nine new spirolides proposed in this study, compound 1 was suggested to have a truncated side chain in lieu of the commonly observed butenolide ring in spirolides. Moreover, there is indication that compound 5 might belong to new spirolide subclasses with a trispiroketal ring configuration having a 6:5:6 trispiroketal ring system. On the other hand, the other compounds were proposed as C- and G-type SPX, respectively. Compound 7 is proposed as the first G-type SPX with a 10-hydroxylation as usually observed in C-type SPX. This mass spectrometry-based study thus demonstrates that structural variability of spirolides is larger than previously known and does not only include the presence or absence of certain functional groups but also involves the triketal ring system.

Systematic characterization of the chemical constituents in vitro and prototypes in vivo of Dingkun Dan using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with the UNIFI™ software.

Dingkun Dan (DKD), a famous traditional Chinese medicine, has been widely used in the treatment of irregular menstruation, leucorrhea abnormality, and postpartum gynecological diseases since Qing dynasty (1739). It comprises 30 flavors of Chinese medicinal materials, which results in its complex chemical composition. In this study, an integrative method was developed to rapidly characterize the chemical components of DKD using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with the UNIFI™ software. A total of 234 compounds, including 47 triterpenoid saponins, 55 flavonoids, and 38 alkaloids, were identified. Of them, 170 compounds were characterized initially and 61 compounds were identified unambiguously using reference standards. Under the same analysis conditions, 43 prototypical components, which were tentatively assigned as triterpenoid saponins, flavonoids, alkaloids, terpenoids, phenylpropanoids, and others, were absorbed in rat by serum pharmacochemistry analysis. DKD exhibited diverse pharmacological activities through the combined effect of these components. This study was the first systematic study of chemical components in vitro originating from 30 medicinal materials and prototypes in vivo of DKD, which could provide scientific evidence for explaining its therapeutic effect.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

Electron Transfer Induced Decomposition in Potassium-Nitroimidazoles Collisions: An Experimental and Theoretical Work.

Electron transfer induced decomposition mechanism of nitroimidazole and a selection of analogue molecules in collisions with neutral potassium (K) atoms from 10 to 1000 eV have been thoroughly investigated. In this laboratory collision regime, the formation of negative ions was time-of-flight mass analyzed and the fragmentation patterns and branching ratios have been obtained. The most abundant anions have been assigned to the parent molecule and the nitrogen oxide anion (NO2-) and the electron transfer mechanisms are comprehensively discussed. This work focuses on the analysis of all fragment anions produced and it is complementary of our recent work on selective hydrogen loss from the transient negative ions produced in these collisions. Ab initio theoretical calculations were performed for 4-nitroimidazole (4NI), 2-nitroimidazole (2NI), 1-methyl-4- (Me4NI) and 1-methyl-5-nitroimidazole (Me5NI), and imidazole (IMI) in the presence of a potassium atom and provided a strong basis for the assignment of the lowest unoccupied molecular orbitals accessed in the collision process.

HPLC-ESI-MSn Identification and NMR Characterization of Glucosyloxybenzyl 2R-Benzylmalate Deriva-Tives from Arundina Graminifolia and Their Anti-Liver Fibrotic Effects In Vitro.

Four new glucosyloxybenzyl 2R-benzylmalate derivatives, named Arundinoside H (2), I (5), J (6), K (8) as well as four known compounds Arundinoside D (1), G (3), F (4), E (7) were isolated and characterized by a combination of chemical and spectroscopic methods, including HR-ESI-MS, 1D and 2D NMR experiments. Besides, 24 unreported compounds were inferred from ESI-MSn data. The anti-liver fibrotic activities of the isolates were determined as proliferation inhibition of lipopolysaccharide (LPS)-induced activation of rat hepatic stellate cells (HSC-T6). The result suggested Arundinosides D, H, F, I and K showed moderate inhibitory effects in vitro.

Phytochemical Profiling of Coryphantha macromeris (Cactaceae) Growing in Greenhouse Conditions Using Ultra-High-Performance Liquid Chromatography⁻Tandem Mass Spectrometry.

Chromatographic separation combined with mass spectrometry is a powerful tool for the characterization of plant metabolites because of its high sensitivity and selectivity. In this work, the phytochemical profile of aerial and radicular parts of Coryphantha macromeris (Engelm.) Britton & Rose growing under greenhouse conditions was qualitatively investigated for the first time by means of modern ultra-high-performance liquid chromatography⁻tandem mass spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS). The UHPLC-PDA-HESI-Orbitrap-MS/MS analysis indicated a high complexity in phenolic metabolites. In our investigation, 69 compounds were detected and 60 of them were identified. Among detected compounds, several phenolic acids, phenolic glycosides, and organic acids were found. Within this diversity, 26 metabolites were exclusively detected in the aerial part, and 19 in the roots. Twenty-four metabolites occurred in both plant parts. According to the relative abundance of peaks in the chromatogram, ferulic and piscidic acids and their derivatives may correspond to one of the main phenolic compounds of C. macromeris. Our results contribute to the phytochemical knowledge regarding C. macromeris and its potential applications in the pharmaceutical and cosmetic industries. Besides, some metabolites and their fragmentation patterns are reported here for the first time for cacti species.

Multi-Steps Fragmentation-Ion Trap Mass Spectrometry Coupled to Liquid Chromatography Diode Array System for Investigation of Olaparib Related Substances.

A high-performance liquid chromatography-diode array-mass spectrometric (LC-DAD-MS) method was developed and validated to investigate the related substances of olaparib (OLA) in bulk form. OLA was exposed to acid⁻base hydrolysis, boiling, oxidation with hydrogen peroxide, and UV light followed by LC-DAD-MS analysis. OLA and OLA-related substances were simultaneously and quantitatively monitored by DAD at 278 nm and triple quadrupole mass spectrometry (QQQ-MS). The investigated compounds were auto-scanned by an ion trap MS which applied positive and negative modes separately. The fragmentation pathway was confirmed by applying multi-steps fragmentation to identify the resulted cleaved ions and their parent ion. OLA was found to be sensitive to the alkaline hydrolysis and less sensitive to UV light. Two major hydrolytic degradation products, including the protonated molar ions m/z 299 and m/z 367, were identified. Three potential impurities were also characterized. The LC-MS limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 ng/µL, respectively. The quantitative results obtained by LC-DAD was comparable with that of LC-QQQ-MS. The proposed method shows good intra-day and inter-day precision with relative standard deviation (RSD) <2%.