Stepped collisional energy MSAll: an analytical approach for optimal MS/MS acquisition of complex mixture with diverse physicochemical properties

The analysis of complex mixtures is becoming increasingly important in various fields, such as nutrition, medicinal plants and metabolomics. The components contained in such complex mixtures are always characterized with diverse physiochemical properties that pose a major challenge during the optimization of various parameters using liquid chromatography‐mass spectrometer (LC‐MS). The parameter ‘CE energy’ that is normally set at a fixed value with a moderate range of CE spread during data‐dependent acquisition (DDA) analysis, a prevalent approach for untargeted identification, often fails to generate sufficient MS/MS fragment ions for untargeted identification of components from complex mixtures. Here we developed a simple and generally applicable acquisition method named stepped MS^All (sMS^All) in this study, aiming to obtain optimal MS/MS spectra for identification of chemically diverse compounds from complex mixtures. sMS^All collects serial MS^All scans acquired at low CE to gradually ramped‐up high CE values in a cycle that conventional DDA scans cannot afford. The resultant MS/MS spectra of each compound were compared and evaluated among serial MS^All scans, and the optimal spectra were used for identification. An untargeted data analysis strategy was then employed to analyze these optimal MS/MS spectra by searching common diagnostic ions and connecting the diagnostic ion families into a network via bridging components. This sMS^All‐based route enables identification of 71 natural products from a herbal preparation, whereas only 53 out of 71 compounds were identified using the classical DDA approach. Therefore, the sMS^All‐based approach is expected to find its wide applications for characterization of vastly diverse compounds with no priori knowledge from various complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.     

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3)

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM³ was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM³ modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM³ showed a similar range of linearity (R ²?>?0.99 for both methods) with protein concentrations above 1 μg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM³ methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS³ fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.     

Deciphering chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma by liquid chromatography with transformed multiple reaction monitoring mass spectrometry

In this study, we propose an integrated strategy for the efficient identification and quantification of herbal constituents using liquid chromatography with mass spectrometry. First, liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the chemical profiling of herbs, where a targeted following nontargeted approach was developed to detect trace constituents by using structural correlations and extracted ion chromatograms. Next, ion pairs and parameters of MS² of quadrupole time‐of‐flight mass spectrometry were selected to design multiple reaction monitoring transitions for the identified compounds on liquid chromatography with triple quadrupole mass spectrometry. The relative concentration of each constituent was then calculated using a semiquantitative calibration curve. The proposed strategy was applied in a study of chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma. A total of 140 compounds were identified or tentatively characterized from the herbs, 132 of which were relatively quantified. The visualized quantitative results clearly showed codecoction produced significant constituent concentration variations especially for those with a low polarity. The case study also indicated that the present methodology could provide a reliable, accurate, and labor‐saving solution for chemical studies of herbal medicines.     

Identification of triacylglycerol using automated annotation of high resolution multistage mass spectral trees

High complexity of identification for non-target triacylglycerols (TAGs) is a major challenge in lipidomics analysis. To identify non-target TAGs, a powerful tool named accurate MSⁿ spectrometry generating so-called ion trees is used. In this paper, we presented a technique for efficient structural elucidation of TAGs on MSⁿ spectral trees produced by LTQ Orbitrap MSⁿ, which was implemented as an open source software package, or TIT. The TIT software was used to support automatic annotation of non-target TAGs on MSⁿ ion trees from a self-built fragment ion database. This database includes 19108 simulate TAG molecules from a random combination of fatty acids and corresponding 500582 self-built multistage fragment ions (MS ≤ 3). Our software can identify TAGs using a “stage-by-stage elimination” strategy. By utilizing the MS¹ accurate mass and referenced RKMD, the TIT software can discriminate unique elemental composition candidates. The regiospecific isomers of fatty acyl chains will be distinguished using MS² and MS³ fragment spectra. We applied the algorithm to the selection of 45 TAG standards and demonstrated that the molecular ions could be 100% correctly assigned. Therefore, the TIT software could be applied to TAG identification in complex biological samples such as mouse plasma extracts.     

HPLC/MS Analysis of Historical Pharmaceutical Preparations of Heroin and Cocaine

Pharmaceutical preparations of heroin and cocaine more than seventy years old were analyzed using RP-HPLC. The composition of mobile phase was optimized. The components were identified by MS² or MS³, and the APPI fragmentation mechanisms of compounds found were proposed. The sample of heroin hydrochloride injection solution consists of 96.1% morphine and 3.9% of codeine. The sample of cocaine hydrochloride injection solution consists of 26.9% cocaine, 31.5% benzoylecgonine, 17.4% ecgonine, and 24.2% ecgonine methyl ester.     

LC–MS3 Strategy for Quantification of Carbamazepine in Human Plasma and Its Application in Therapeutic Drug Monitoring

This study developed a detection method based on the strategy of HPLC/MS³ and verified its suitability by quantifying carbamazepine in human plasma. The high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS³) system was performed using a Shimadzu UFLC XR liquid chromatography and a SCIEX QTRAP^® 5500 linear ion trap triple quadrupole mass spectrometer. The specific operation was as follows: the sample protein was firstly precipitated using methanol, then carbamazepine and carbamazepine-D₂N¹⁵ were separated on an ACQUITY UPLC HSS T3 column using the gradient elution with solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.25 mL/min. Each sample was run for 7 min. This method was validated for various parameters including accuracy, precision, selectivity, linearity, LLOQ, etc. Only 5 μL of sample plasma could obtain the result of LLOD 0.5 µg/mL. The intra-day and inter-day precision was <8.23%, and accuracy was between −1.74% and 2.92%. This method was successfully used for monitoring the blood concentration of epilepsy patients after carbamazepine treatment.     

A comprehensive library‐based,automated screening procedure for 46 synthetic cannabinoids in serum employing liquid chromatography‐quadrupole ion trap mass spectrometry with high‐temperature electrospray ionization

Considering the vast variety of synthetic cannabinoids and herbal mixtures – commonly known as ‘Spice’ or ‘K2’ – on the market and the resulting increase of severe intoxications related to their consumption, there is a need in clinical and forensic toxicology for comprehensive up‐to‐date screening methods. The focus of this project aimed at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography‐ion trap‐MS (LC‐MSⁿ) system and a spectra library‐based approach, currently including 46 synthetic cannabinoids and 8 isotope labelled analogues. In the process of method development, a high‐temperature ESI source (IonBooster^TM, Bruker Daltonik) and its effects on the ionization efficiency of the investigated synthetic cannabinoids were evaluated and compared to a conventional ESI source. Despite their structural diversity, all investigated synthetic cannabinoids benefitted from high‐temperature ionization by showing remarkably higher MS intensities compared to conventional ESI. The employed search algorithm matches retention time, MS and MS²/MS³ spectra. With the utilization of the ionBooster source, limits for the automated detection comparable to cut‐off values of routine MRM methods were achieved for the majority of analytes. Even compounds not identified when using a conventional ESI source were detected using the ionBooster‐source. LODs in serum range from 0.1 ng/ml to 0.5 ng/ml. The use of parent compounds as analytical targets offers the possibility of instantly adding new emerging compounds to the library and immediately applying the updated method to serum samples, allowing the rapid adaptation of the screening method to ongoing forensic or clinical requirements. The presented approach can also be applied to other specimens, such as oral fluid or hair, and herbal mixtures and was successfully applied to authentic serum samples. Quantitative MRM results of samples with analyte concentrations above the determined LOD were confirmed as positive findings by the presented method. Copyright © 2014 John Wiley & Sons, Ltd.     

An UPLC‐MS3 Method for Rapid Separation and Determination of Four Tobacco‐specific Nitrosamines in Mainstream Cigarette Smoke

An ultra‐high‐pressure liquid chromatography/MS³ (UPLC‐MS³) method has been developed and validated for the quantitative determination of four major TSNAs in mainstream cigarette smoke using MS³ scan mode on a hybrid triple quadrupole‐linear ion trap mass spectrometer. The new method combining the UPLC with MS³ scan mode offers decreased sample analysis time and good selectivity. After mainstream cigarette smoke was collected on a Cambridge filter pad, the particulate matter was extracted with ammonium acetate solution and analyzed by UPLC‐MS³ using isotopically labeled analogues as internal standards. Four TSNAs were completely separated on an Agilent XDB‐C₁₈ UPLC column using isocratic elution during a 6 min LC run time. Excellent linearity was obtained over the concentration range of 1.0‐150.0 ng/mL for all TSNAs with values for correlation coefficient (r) between 0.9985‐0.9994. Limits of detection (LOD) of each TSNA varied from 0.023 to 0.067 ng/mL, and lower limits of quantification (LLOQ) varied from 0.077 to 0.223 ng/mL, respctively. The recovery of each TSNA was from 89.2 to 106.8%. The method achieved excellent reproducibility with RSD 2.1‐6.8% for intra‐assay and 3.4‐9.1% for inter‐assay. This method can be used as an effective approach to significantly improve the detection capability for TSNAs in complex matrices.     

Solid‐phase microextraction with fast GC combined with a high‐speed triple quadrupole mass spectrometer for targeted and untargeted food analysis

The present contribution is focused on the evaluation of a high‐speed triple quadrupole mass spectrometer, carried out under moderately fast GC conditions (analysis time: 16.6 min). The mass spectrometric instrument can be operated under high‐speed GC conditions, in both full‐scan (maximum scan speed: 20 000 amu/s) and multiple reaction monitoring (MRM) modes (minimum dwell time: 0.01 s). Additionally, the mass spectrometric system can generate full scan and MRM information, simultaneously and rapidly. A headspace solid‐phase microextraction with fast GC coupled to triple quadrupole MS approach was developed for the: (i) qualitative untargeted analysis of brewed tea volatiles, and (ii) MRM qualitative and quantitative analysis of targeted volatiles (also in brewed tea), namely 30 phytosanitary contaminants. The performance of the triple quadrupole instrument was satisfactory both for identification and quantification purposes. Furthermore, the method sensitivity was more than sufficient for the requirements of current legislation. Method validation, related to the MRM analysis, was performed considering: precision of quantification data (maximum coefficient of variation value: 12.0%) and quantification/qualification ion ratios (maximum coefficient of variation value: 14.4%), along with limits of detection (4 parts per trillion–5 parts per billion range) and quantification (14 parts per trillion–16 parts per billion range).     

Chromatographic Tandem Mass Spectrometric Detection of Physostigmine and its Major Metabolites in Rat Urine

Abstract

A rapid, sensitive, and specific liquid chromatographic‐electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of physostigmine and its metabolites in rat urine. 300 µg kg^–1 of physostigmine were used as a safe oral gavage dose for studies on its metabolites. 0–24 h urine was purified using a C18 solid‐phase extraction cartridge, and then detected by an on‐line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their MSⁿ spectra with physostigmine. Six metabolites and unchanged physostigmine existed in rat urine. All of the metabolites were reported for the first time.     

Ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MSE analysis of RNA oligonucleotides

Fast and efficient ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence‐related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS^E (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS^E technique is as efficient as the traditional MS/MS method, yet MS^E is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2′‐ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS^E approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS^E. Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time‐of‐flight (QTOF) MS/MS and MS^E methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

超高效液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于血浆中苦杏仁苷及其代谢产物野黑樱苷的定性和定量分析

建立了大鼠灌胃麻杏石甘汤后血浆中苦杏仁苷、野黑樱苷的定性及定量方法。样品经液液萃取净化处理,定性采用超高效液相色谱-串联四极杆飞行时间质谱仪（UPLC-QTOF-MS/MS）,经Shim-pack XR-ODS Ⅲ色谱柱（75 mm×2.0 mm,1.6 μm）分离,定量采用超高效液相色谱-串联三重四极杆质谱仪（UPLC-Q-TRAP-MS）,经Agilent C₁₈色谱柱（50 mm×2.1 mm,1.7 μm）分离,电喷雾负离子化（ESI）及MRM模式测定,流动相均为乙腈-0.1%（v/v）甲酸水溶液。结果显示苦杏仁苷、野黑樱苷在相应浓度范围内线性关系良好（相关系数分别为0.9990、0.9970）,精密度（RSD）小于9.20%,回收率为82.33%~95.25%,检出限（LOD）约为0.50 ng/mL。本方法快速简便,为血浆样品中苦杏仁苷、野黑樱苷的定性和定量分析提供良好参考。     

基于多级质谱的蛋白质组定量新方法新技术进展

定量蛋白质组学已经成为蛋白质组学的一个重要分支,以生物质谱为核心的定量蛋白质组方法日益发展。按照定量所依据的质谱信号来源于一级质谱谱图还是多级质谱谱图可以将定量蛋白质组方法分为一级质谱定量和多级质谱定量。本文主要综述基于多级质谱的定量方法和技术进展,分析比较了这些方法的优缺点,并对基于多级质谱的定量方法发展进行了展望。     

Structure elucidation of nanogram quantities of unknown designer drugs based on phenylalkylamine derivates by ion trap multiple mass spectrometry

Multiple mass spectra (MS¹ to MS⁶) of 55 phenylalkylamine derivatives were recorded with ion-trap mass spectrometry employing electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Fragmentation patterns were studied in detail and a generally applicable scheme was established for elucidation of the structures of phenylalkylamine derivatives. HPLC combined with ion-trap multiple mass spectrometry was used to identify the structure of reaction by-products in ecstasy samples from the black market. Low nanogram amounts were sufficient for on-line HPLC–MS ⁿ structure elucidation of unknowns.     

Assignment of the Stereochemistry and Anomeric Configuration of Sugars within Oligosaccharides Via Overlapping Disaccharide Ladders Using MSn

A systematic approach is described that can pinpoint the stereo-structures (sugar identity, anomeric configuration, and location) of individual sugar units within linear oligosaccharides. Using a highly modified mass spectrometer, dissociation of linear oligosaccharides in the gas phase was optimized along multiple-stage tandem dissociation pathways (MSⁿ, n = 4 or 5). The instrument was a hybrid triple quadrupole/linear ion trap mass spectrometer capable of high-efficiency bidirectional ion transfer between quadrupole arrays. Different types of collision-induced dissociation (CID), either on-resonance ion trap or beam-type CID could be utilized at any given stage of dissociation, enabling either glycosidic bond cleavages or cross-ring cleavages to be maximized when wanted. The approach first involves optimizing the isolation of disaccharide units as an ordered set of overlapping substructures via glycosidic bond cleavages during early stages of MSⁿ, with explicit intent to minimize cross-ring cleavages. Subsequently, cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic product ions (m/z 221). Finally, fingerprint patterns that establish stereochemistry and anomeric configuration were obtained from the diagnostic ions via CID. Model linear oligosaccharides were derivatized at the reducing end, allowing overlapping ladders of disaccharides to be isolated from MSⁿ. High confidence stereo-structural determination was achieved by matching MSⁿ CID of the diagnostic ions to synthetic standards via a spectral matching algorithm. Using this MSⁿ (n = 4 or 5) approach, the stereo-structures, anomeric configurations, and locations of three individual sugar units within two pentasaccharides were successfully determined.

Phenolic Profile,Antioxidant Capacity and Antimicrobial Activity of Nettle Leaves Extracts Obtained by Advanced Extraction Techniques

Nettle is a widely known plant whose high biological activity and beneficial medicinal effects are attributed to various bioactive compounds, among which polyphenols play an important role. In order to isolate polyphenols and preserve their properties, advanced extraction techniques have been applied to overcome the drawbacks of conventional ones. Therefore, microwave-assisted extraction (MAE) has been optimized for the isolation of nettle leaves polyphenols and it was compared to pressurized liquid extraction (PLE) and conventional heat-reflux extraction (CE). The obtained extracts were analyzed for their individual phenolic profile by UPLC MS² and for their antioxidant capacity by ORAC assay. MAE proved to be the more specific technique for the isolation of individual phenolic compounds, while PLE produced extracts with higher amount of total phenols and higher antioxidant capacity. Both techniques were more effective compared to CE. PLE nettle extract showed antimicrobial activity against bacteria, especially against Gram-negative Pseudomonas fragi ATCC 4973 and Campylobacter jejuni NCTC 11168 strains. This suggests that PLE is suitable for obtaining a nettle extract with antioxidant and antimicrobial potential, which as such has great potential for use as a value-added ingredient in the food and pharmaceutical industry.     

液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于检测牛肉中的刚果红

建立了牛肉中刚果红的检测方法。定性方法采用液相色谱-串联四极杆飞行时间质谱对未知物进行质谱谱图库匹配,定量分析采用超高效液相色谱-串联三重四极杆质谱。牛肉样品中的刚果红经液液萃取净化后,采用Agilent ZORBAX Eclipse Plus C₁₈ Rapid Resolution HD色谱柱(50 mm×2.1 mm, 1.8 μm)进行分离,流动相为95%(体积分数)甲醇,流速为0.2 mL/min。AB 4000⁺三重四极杆质谱仪在电喷雾负离子化(ESI)及MRM模式下定量。结果显示,刚果红在0.03~1 mg/L浓度范围内,线性关系良好(相关系数为0.9998),精密度良好(RSD小于5%),回收率为88%~91%,检出限约为0.01 mg/L。本方法快速简便,重现性好,可以为牛肉及其他肉制品中刚果红的定量提供良好的解决方案。     

Neuropeptide imaging on an LTQ with vMALDI source: The complete ‘all-in-one’ peptidome analysis

Direct tissue imaging was performed on dissected insect tissue using a MALDI ion trap to visualize endogenous neuropeptides. Coupling tissue imaging to tandem MSⁿ allows for the identification of previously known species and the ability to identify new ones by de novo sequencing, as searchable databases for insects are sparse. Direct tissue imaging is an attractive technique for the study of neuropeptides as minimal sample preparation is required prior to mass spectrometry. We successfully identified neuropeptides present in the corpora cardiaca and allata of Acheta domesticus (the house cricket). Diagnostic fragments at low m/z were used to distinguish between lipids and neuropeptides. The distribution of peptides appears to be more differentially localized than that of phospholipids, which seem to be more evenly distributed within the tissue.     

Studies on the aconitine-type alkaloids in the roots of Aconitum Carmichaeli Debx. by HPLC/ESIMS/MS

Studies of aconitine-type alkaloids in the Chinese herb Aconitum Carmichaeli were performed by HPLC/ESIMS/MSⁿ and FTICR/ESIMS in positive ion mode. The characteristic fragmentation pathways in the MSⁿ spectra were summarized based on previously published research literature and further study. According to the fragmentation pathways of mass spectrometry, results from the analysis of standard compounds and reports from literature, 111 compounds were identified or deduced in a total of 117 found compounds in A. Carmichaeli. In the 11 monoester-diterpenoid alkaloids (MDA), 10 diesterditerpenoid alkaloids (DDA) and 81 lipo-alkaloids, the novel alkaloids including 1 MDA, 2 DDA and 48 lipo-alkaloids were detected. In addition, 1 DDA, 7 lipo-alkaloids and 2 alkaloids with small molecular weights that possess C19-norditerpenoid skeleton were reported in A. Carmichaeli for the first time.     

Mass spectrometric profiling of flavonoid glycoconjugates possessing isomeric aglycones

In fields such as food and nutrition science or plant physiology, interest in untargeted profiling of flavonoids continues to expand. The group of flavonoids encompasses several thousands of chemically distinguishable compounds, among which are a number of isobaric compounds with the same elemental composition. Thus, the mass spectrometric identification of these compounds is challenging, especially when reference standards are not available to support their identification. Many different types of isomers of flavonoid glycoconjugates are known, i.e. compounds that differ in their glycosylation position, glycan sequence or type of interglycosidic linkage. This work focuses on the mass spectrometric identification of flavonoid glycoconjugate isomers possessing the same glycan mass and differing only in their aglycone core. A non‐targeted HPLC‐ESI‐MS/MS profiling method using a triple quadrupole MS is presented herein, which utilizes in‐source fragmentation and a pseudo‐MS³ approach for the selective analysis of flavonoid glycoconjugates with isomeric/isobaric aglycones. A selective MRM‐based identification of the in‐source formed isobaric aglycone fragments was established. Additionally, utilizing the precursor scanning capability of the employed triple quadrupole instrument, the developed method enabled the determination of the molecular weight of the studied intact flavonoid glycoconjugate. The versatility of the method was proven with various types of flavonoid aglycones, i.e. anthocyanins, flavonols, flavones, flavanones and isoflavones, along with their representative glycoconjugates. The developed method was also successfully applied to a commercially available sour cherry sample, in which 16 different glycoconjugates of pelargonidin, genistein, cyanidin, kaempferol and quercetin could be tentatively identified, including a number of compounds containing isomeric/isobaric aglycones. Copyright © 2015 John Wiley & Sons, Ltd.