A four-dimensional separation approach by offline 2D-LC/IM-TOF-MS in combination with database-driven computational peak annotation facilitating the in-depth characterization of the multicomponents from Atractylodis Macrocephalae Rhizoma (Atractylodes macrocephala)

Plant metabolites represent complex chemical system, which renders it difficult to clarify the chemical composition by conventional liquid chromatography/mass spectrometry (LC/MS) due to the limited selectivity and peak capacity. The rhizomes of Atractylodes macrocephala have been utilized as a traditional Chinese medicine Atractylodis Macrocephalae Rhizoma (Bai-Zhu), and have been reported containing multiple categories of plant metabolites. Targeting the multicomponents from A. macrocephala, an integral approach by offline two-dimensional liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) was established and validated. By configuring an XBridge Amide column of Hydrophilic Interaction Chromatography and an Atlantis Premier BEH C18AX column of mixed ion exchange and reversed-phase modes, the established 2D-LC/IM-QTOF-MS system showed high orthogonality up to 0.91. Dimension-enhanced, data-independent high-definition MS^E (HDMS^E) in the positive ESI mode was conducted on a Vion IM-QTOF mass spectrometer, and its hyphenation to offline 2D-LC could enable the four-dimensional separation (each dimension in 2D-LC, IM, and MS). Particularly, HDMS^E facilitated the acquisition of high-definition MS¹ and MS² spectra. In-house library-driven computational peak annotation by the bioinformatics platform UNIFI could efficiently process and annotate the HDMS^E data for the structural elucidation. By integrating reference compounds comparison, we could identify or tentatively characterize 251 components from A. macrocephala (including 115 sesquiterpenoids, 90 polyacetylenes, 11 flavonoids, 9 benzoquinones, 12 coumarins, and 14 others), which indicated large improvement in identifying those minor plant components, compared with the conventional LC/MS approach. Conclusively, offline 2D-LC/IM-QTOF-HDMS^E in combination with computational data interpretation proves to be powerful facilitating the in-depth multicomponent characterization of herbal medicine.     

Fast Characterization of Constituents in HuangKui Capsules Using UPLC�CQTOF-MS with Collision Energy and MassFragment Software

In this paper, ultra performance liquid chromatography (UPLC)?Cquadrupole-time-of-flight mass spectrometry (QTOF) with collision energy (MS ^E ) and MassFragment software were applied for fast separation and characterization of constituents in HuangKui Capsules (HKC). MS ^E was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of 12 constituents in HKC. When the reference compound was not available, MassFragment software was used to confirm the proposed structure, which increased our confidence in the MS fragment analysis of the proposed structure, facilitating the confirmation of the proposed structure and therefore easing the data processing procedure. In conclusion, UPLC?CQTOF-MS ^E and MassFragment software described in this paper proved to be useful for the identification of constituents in complex herbal medicines and could greatly decrease analytical time. In addition, this method may be applied to the analysis of more complex data found in Traditional Chinese Medicine.     

Resolving the microcosmos of complex samples: UPLC/travelling wave ion mobility separation high resolution mass spectrometry for the analysis of in vivo drug metabolism studies

In vivo drug metabolism studies with low concentrations of analytes and high matrix burden are challenging. Of special interest are ‘first-in-man’ studies in early stages of pharmaceutical development that do not use ¹⁴C labeled drug candidates. Beside conventional MS-fishing techniques which are biased towards known/expected metabolites and mass defect filtration procedures, this paper focuses on the untargeted/unbiased analysis of drug related compounds in complex matrices using two orthogonal separation techniques: UPLC and TWIMS. Standard sample material after oral administration of a drug compound to rats was investigated by UPLC/TWIMS in MS^E acquisition mode using interlaced collision energies for the parallel detection of [M+H]⁺ parent ions and fragments. Due to the fragmentation after ion mobility separation in the transfer region of the Synapt G2-triwave device, [M+H]⁺ ion species are aligned with their related fragments by virtue of possessing the same retention time and drift time profile. Four dimensional data analysis of the continuum raw data was performed by automated peak picking and alignment within the MS^E viewer software. As result, completely purified MS- and MS/MS-data of metabolites were extracted from raw mass data with high matrix burden and were used without compromise for structure elucidation. This analytical methodology is universally applicable for the unbiased/untargeted and robust analysis of any analyte of interest in complex matrices, including small molecules, peptides and proteins. The high quality data files can be used as data repositories for the purpose of retrospective analysis which is of particular interest for the long term process in drug development.     

Identification of anti‐inflammatory active ingredients from Tumuxiang by ultra‐performance liquid chromatography/quadrupole time‐of‐flight‐MSE

The dried roots of Inula helenium L. (IH) and Inula racemosa Hook f. (IR) are used commonly as folk medicine as ‘tumuxiang’ (TMX). The mixing and sharing of IH and IR in clinical use is a universal phenomenon. Modern pharmacological studies confirmed that IH and IR display anti‐inflammatory activities. However, the difference in anti‐inflammatory pharmacodynamic substances between these two herbs is still unknown. In the present study, the fingerprints of 18 IH and nine IR samples were established using UPLC/QTOF‐MS^E. A dimethylbenzene‐induced mouse ear vasodilation model was applied in evaluating the anti‐inflammatory properties of all 27 samples. Then, the spectrum–efficacy model between chemical characteristic peaks and anti‐inflammatory activities was investigated using principal component regression and partial least squares. Finally, the combination of UNIFI Scientific Information System with a library search of traditional Chinese medicines was employed to automatically characterize the peaks. UNIFI identified a total of 80 chemical components. Among the components, the 53 characteristic peaks showed correlation with anti‐inflammatory activities, pointing to phenolic and organic acids as primary anti‐inflammatory ingredients of TMX. This approach can efficiently and intelligently facilitate the identification of bioactive components from traditional Chinese medicine.     

Rapid characterization of chemical constituents of Platycodon grandiflorum and its adulterant Adenophora stricta by UPLC‐QTOF‐MS/MS

Platycodon grandiflorum (PG) is extensively used for treating cough, excessive phlegm, sore throat, bronchitis and asthma, whereas Adenophora stricta (AS) is commonly used to reduce phlegm, clear lung and tonify stomach. Due to similar appearances, PG is sometimes adulterated with cheap AS so as to gain profits. And this will inevitably result in different pharmacological property. In order to further clarify the differences in the chemical composition of these two Chinese herbs, the ultra‐high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry coupled with UNIFI platform was used to establish a reliable, simple, sensitive and rapid analytical method. Seventy‐five compounds, including triterpenoid saponins, organic acids, flavonoids, steroids, phenols, etc., were identified from PG based on MS^E data and retention time under the optimized conditions. Meanwhile, 57 compounds including triterpenoid saponins, organic acids, steroids, phenols, alkaloids, etc. were identified from AS. Among all the identified compounds, there were only 14 common components (mainly organic acids) existing in two herbs, and most of the other chemical compositions are totally different between the two herbs. Based on the results, AS cannot substitute for PG. In addition, PG adulterated with AS will lead a poor efficacy in clinical application. In addition, the systematic comparison of similarities and differences between two Chinese herbs will provide reliable characterization profiles to clarify the pharmacological fundamental substances.     

Holistic quality evaluation of Saposhnikoviae Radix (Saposhnikovia divaricata) by reversed-phase ultra-high performance liquid chromatography and hydrophilic interaction chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry-based untargeted metabolomics

Untargeted metabolomics more suits the quality evaluation of TCM because of its holistic property. To assess the holistic quality difference of Saposhnikoviae Radix (the roots of Saposhnikovia divaricata), we integrate ultra-high-performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay. A BEH C18 column in the reversed-phase mode and a BEH Amide column in Hydrophilic Interaction Chromatography (HILIC) mode were utilized for metabolites profiling, which enabled high coverage of the non-polar to polar components in Saposhnikoviae Radix. Moreover, the application of major components knockout strategy enlarged the exposure of those minor components. Integrated use of high-definition MS^E (HDMS^E) and data-dependent acquisition (DDA) could enhance the metabolites characterization by providing reliable fragmentation information and collision cross section values. Computational in-house library-driven automated peak annotation of the HDMS^E and DDA data assisted to characterize 104 components from Saposhnikoviae Radix. Chemometric analyses of the commercial Saposhnikoviae Radix samples (64 batches collected from 11 cultivars aging from 1 to 4 years), based on the positive MS^E data, in general could indicate large discrimination between Guan-Fang-Feng (from Heilongjiang) and the others, but negligible difference among Saposhnikoviae Radix from the other ten provinces of China and with different ages. Quantitative assays of prim-O-glucosylcimifugin and 4′-O-β-D-glucosyl-5-O-methylvisamminol, by a rapid and fully validated UHPLC-UV method, could primarily deduce that Guan-Fang-Feng aging 2 and 3 years exhibited better quality. The methods established can holistically assess the quality of TCM with wide spans of plant metabolites covered.     

Chemical composition database establishment and metabolite profiling analysis of Yangyin qingfei decoction

The chemical fingerprinting and metabolite profile in a rat plasma sample after intragastric administration of Yangyin qingfei decoction (YYQFD, 14 g/kg) were investigated. First, YYQFD was analyzed by UPLC/Q‐TOF MS to establish the chemical composition database by comparing their retention behavior, accurate molecular mass and MS² data with those of references or known compounds in the literature. In this database, 100 chemical constituents with information on retention time, molecular mass, molecular formula, MS² data and compound name were identified, which can provide compound information for further metabolite profiling studies. Furthermore, 64 compounds including 37 prototypes and 27 metabolites were detected in the dosed rat plasma sample, and the metabolic pathways of YYQFD were hydrolyzation, hydroxylation, dehydrogenation, glucuronidation, glucosylation, sulfation and mixed modes. Among the five component herbs in the YYQFD, Glycyrrhizae Radix et Rhizome and Fritillariae Thunbergii bulbs were actively metabolized, contributing 16 and 7 metabolites, respectively. It is suggested that chemical characterization and metabolite profiling studies are valuable to elucidate the material basis of herbal preparations.     

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS (in data‐independent acquisition mode, or MS^E), was improved by using a new MS/MS mode, ion mobility separation enhanced‐MS^E (HDMS^E), and applied to analyze the area of human plasma low‐density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid‐cut into 72 square gel pieces and subjected to quantitative LC‐MS/MS. Compared with MS^E, HDMS^E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC‐HDMS^E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B‐100 was the most abundant protein in the grid‐cut area, concentrated at pI ca. 5.4–6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39–42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B‐100. Twenty‐two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.     

Identification of the Major Constituents in Zhimu–Huangqi Herb-Pair Extract and Their Metabolites in Rats by LC–ESI-MSn

The Zhimu–Huangqi herb-pair is a famous Chinese herbal formula with a combination of Rhizoma Anemarrhenae (Zhimu in Chinese) and Radix Astragali (Huangqi in Chinese). This work describes a sensitive and specific LC–ES-MSⁿ methodology for identification of the major constituents in Zhimu–Huangqi herb-pair extract and their metabolites in rats after oral administration. A total of 30 compounds have been identified or tentatively characterized from the herb-pair extract, and 13 of them were unambiguously identified by comparing the retention times and mass spectra with those of reference standards, while the other 17 compounds were tentatively identified on the basis of their MSⁿ fragmentation behaviors and exact mass information from literature. Moreover, the metabolites in vivo were also identified. The Zhimu–Huangqi herb-pair extract was actively metabolized in rats, including four parent compounds and 8 metabolites in serum and seven parent compounds and 23 metabolites in urine. This study proposed a good example for the rapid identification of major constituents in complex systems such as herbal extract or traditional Chinese medicine formula, which facilitated the clarification of the metabolic pathway of the herbs in the body to better understand the action mechanism.     

Identification of the Major Constituents in Zhimu–Huangqi Herb-Pair Extract and Their Metabolites in Rats by LC–ESI-MSn

The Zhimu–Huangqi herb-pair is a famous Chinese herbal formula with a combination of Rhizoma Anemarrhenae (Zhimu in Chinese) and Radix Astragali (Huangqi in Chinese). This work describes a sensitive and specific LC–ES-MSⁿ methodology for identification of the major constituents in Zhimu–Huangqi herb-pair extract and their metabolites in rats after oral administration. A total of 30 compounds have been identified or tentatively characterized from the herb-pair extract, and 13 of them were unambiguously identified by comparing the retention times and mass spectra with those of reference standards, while the other 17 compounds were tentatively identified on the basis of their MSⁿ fragmentation behaviors and exact mass information from literature. Moreover, the metabolites in vivo were also identified. The Zhimu–Huangqi herb-pair extract was actively metabolized in rats, including four parent compounds and 8 metabolites in serum and seven parent compounds and 23 metabolites in urine. This study proposed a good example for the rapid identification of major constituents in complex systems such as herbal extract or traditional Chinese medicine formula, which facilitated the clarification of the metabolic pathway of the herbs in the body to better understand the action mechanism.

     

MassBank: a public repository for sharing mass spectral data for life sciences

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron‐ionization mass spectrometry(EI‐MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)‐MSⁿ data of 2337 authentic compounds of metabolites, 11 545 EI‐MS and 834 other‐MS data of 10 286 volatile natural and synthetic compounds, and 3045 ESI‐MS² data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI‐MS² data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass‐to‐charge ratio are optimized to the ESI‐MS² data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI‐MS² data on an identical compound under different collision‐induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21–23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid detection and structural characterization of verapamil metabolites in rats by UPLC–MSE and UNIFI platform

High-resolution mass spectrometry (HRMS) is an important technology for studying biotransformations of drugs in biological systems. In order to process complex HRMS data, bioinformatics, including data-mining techniques for identifying drug metabolites from liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or multistage mass spectrometry (MSⁿ) datasets as well as elucidating the detected metabolites’ structure by spectral interpretation software, are important tools. Data-mining technologies have widely been used in drug metabolite identification, including mass defect filters, product ion filters, neutral-loss filters, control sample comparisons and extracted ion chromatographic analysis. However, the metabolites identified by current different technologies are not the same, indicating the importance of technique integration for efficient and complete identification of metabolic products. In this study, a universal, high-throughput workflow for identifying and verifying metabolites by applying the drug metabolite identification software UNIFI is reported, to study the biotransformation of verapamil in rats. A total of 71 verapamil metabolites were found in rat plasma, urine and faeces, including two metabolites that have not been reported in the literature. Phase I metabolites of verapamil were identified as N-demethylation, O-demethylation, N-dealkylation and oxidation and dehydrogenation metabolites; phase II metabolites were mainly glucuronidation and sulfate conjugates, indicating that UNIFI software could be effective and valuable in identifying drug metabolites.     

Medium-Pressure Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry for Separation and On-Line Characterization of Flavonoids from Asparagus officinalis

A novel method for separation and on-line characterization of flavonoids from Asparagus officinalis by medium-pressure liquid chromatography coupled to electrospray ionization multi-stage mass spectrometry (MPLC-ESI-MSⁿ) was successfully established. The hyphenation between MPLC and ESI-MSⁿ was designed to keep the split ratio exactly in the range from 1:100 to 1:300. The separation procedure was guided by the chromatogram of ion current of MSⁿ and the structures of compounds were characterized by fragments information at the same time. Consequently, it was proved that MPLC coupled with ESI-MSⁿ was an effective method for separation of compounds from multi-component mixtures with high purity and desired amounts and simultaneous elucidation of chemical structures.

     

Untargeted metabolomics analysis to unveil the chemical markers for the differentiation among three Gleditsia sinensis-derived herbal medicines by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Precise identification and differentiation among those congeneric Traditional Chinese Medicines (TCMs) or derived from the same plant trend to be more challenging, particularly in the absence of appearance characteristics. Three TCMs, involving Gleditsiae Sinensis Fructus (GSF), Gleditsiae Fructus Abnormalis (GFA), and Gleditsiae Spina (GS), recorded in Chinese Pharmacopoeia (2020 edition) are derived from Gleditsia sinensis, but prescribed for different clinical uses. The documents aimed to compare their chemical differences are rare, to date. An untargeted metabolomics approach, based on ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS), was elaborated to unveil the potential chemical markers to differentiate among GSF, GFA, and GS. Good chromatographic separation of all the GSF/GFA/GS components was achieved within 33 min by utilizing a BEH C18 column, while data-independent MS^E in the positive mode was selected for profiling the metabolic features. Notably, the high-mass saponins (1300–2500 Da) gave unique protonated precursors ([M + H]⁺) in the positive ESI mode, compared with those complicated ion species occurring in the negative mode. Pattern recognition chemometrics analysis of 45 batches of G. sinensis samples could unveil 70 significantly altered ions assigned as 46 potentially differential components. The positive/negative high-accuracy MS² data analysis, phytochemical isolation/NMR analysis, and searching of an in-house library of G. sinensis, were utilized for structural elucidation. Three compounds (saikachinoside A, locustoside A, and locustoside B) rich in GSF could be the markers to differentiate from GFA/GS, while four components were characteristic for GS. These results obtained can greatly benefit the quality control of TCMs derived from G. sinensis.     

Characterization and identification of iridoid glucosides,flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MSⁿ). The optimized separation condition was obtained using an Agilent ZorBax SB‐C₁₈ column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC‐ESI‐MSⁿ method.     

Comprehensive characterization of multiple components and metabolites of Xiaojin Capsule based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Xiaojin Capsule, a classic traditional Chinese medicine formula, has been used to treat mammary cancer, thyroid nodules, and hyperplasia of the mammary glands. However, its systematic chemical information remained unclear, which hindered the interpretation of the pharmacology and the mechanism of action of this drug. In this research, an ultra high performance liquid chromatography coupled with a quadrupole time‐of‐flight mass spectrometry method was developed to identify the complicated components and metabolites of Xiaojin Capsule. Two acquisition modes, including the MS^Energy mode and fast data directed acquisition mode, were utilized for chemical profiling. As a result, 156 compounds were unambiguously or tentatively identified by comparing their retention times and mass spectrometry data with those of reference standards or literature. After the oral administration of Xiaojin Capsule, 53 constituents, including 24 prototype compounds and 29 metabolites, were detected in rat plasma. The obtained results were beneficial for a better understanding of the therapeutic basis of Xiaojin Capsule. A high‐resolution and efficient separation method was firstly established for systematically characterizing the compounds of Xiaojin Capsule and the associated metabolites in vivo, which could be helpful for quality control and pharmacokinetic studies of this medicine.     

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.     

Using In Silico Fragmentation to Improve Routine Residue Screening in Complex Matrices

Targeted residue screening requires the use of reference substances in order to identify potential residues. This becomes a difficult issue when using multi-residue methods capable of analyzing several hundreds of analytes. Therefore, the capability of in silico fragmentation based on a structure database (“suspect screening”) instead of physical reference substances for routine targeted residue screening was investigated. The detection of fragment ions that can be predicted or explained by in silico software was utilized to reduce the number of false positives. These “proof of principle” experiments were done with a tool that is integrated into a commercial MS vendor instrument operating software (UNIFI) as well as with a platform-independent MS tool (Mass Frontier). A total of 97 analytes belonging to different chemical families were separated by reversed phase liquid chromatography and detected in a data-independent acquisition (DIA) mode using ion mobility hyphenated with quadrupole time of flight mass spectrometry. The instrument was operated in the MS^E mode with alternating low and high energy traces. The fragments observed from product ion spectra were investigated using a “chopping” bond disconnection algorithm and a rule-based algorithm. The bond disconnection algorithm clearly explained more analyte product ions and a greater percentage of the spectral abundance than the rule-based software (92 out of the 97 compounds produced ≥1 explainable fragment ions). On the other hand, tests with a complex blank matrix (bovine liver extract) indicated that the chopping algorithm reports significantly more false positive fragments than the rule based software.

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