Sample‐directed pseudotargeted method for the metabolic profiling analysis of rice seeds based on liquid chromatography with mass spectrometry

Rice is one of the most important food crops in the world. Metabolite composition in rice seeds varies significantly depending on genetic variety, climatic alternation and agricultural practice. Metabolomics is a powerful tool to reveal the metabolic response of rice to various conditions. In this work, a rice seed sample‐directed pseudotargeted metabolomics method was first established and validated based on ultra high performance liquid chromatography with triple quadrupole mass spectrometry in the multiple reaction monitoring mode. A total of 749 and 617 ion pairs in positive and negative modes were achieved, respectively. Among them, about 200 metabolites were identified or tentatively identified. The developed method showed better linearity and repeatability than those of non‐targeted metabolomics method. Good intra‐day and inter‐day precisions, recoveries and wide linear range were also obtained. Furthermore, the method was applied for the investigation of metabolic variation of rice seeds with two wild cultivars and their transgenic lines that were grown in two locations. Principal component analysis indicated that the effects of cultivar and location on metabolic variations were far more than those of gene modification. The nonparametric Mann–Whitney U test revealed that most metabolites were influenced by cultivar, location and gene modifications together.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Quantitative liquid chromatography/time-of-flight mass spectrometry

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.     

Metabolomics based on liquid chromatography with mass spectrometry reveals the chemical difference in the stems and roots derived from Ephedra sinica

To better understand different traditional uses of the stems (known as Mahuang) and roots (known as Mahuanggen) of Ephedra sinica, their chemical difference should be investigated. In this study, an ultra‐fast liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry untargeted metabolomics approach was established to reveal global chemical difference between Mahuang and Mahuanggen. Clear separation was observed in scores plots of principal component analysis and orthogonal partial least squares‐discriminant analysis. Twenty two chemical markers responsible for such separation were screened out and unambiguously/tentatively characterized. Then chemical markers of pharmacologically important ephedrine and pseudoephedrine were absolutely quantified using liquid chromatography coupled with tandem mass spectrometry under multiple reaction monitoring mode. The results showed that Mahuang was rich in ephedrine‐type alkaloids, while Mahuanggen was rich in macrocyclic spermine alkaloids. Additionally, different types of flavan‐3‐ols and flavones exist in Mahuang and Mahuanggen extracts. This research facilitates a better understanding of different traditional uses of Mahuang and Mahuanggen and provides references for chemical analysis of other medicinal plants.     

Determination of synthetic dyes in bean and meat products by liquid chromatography with tandem mass spectrometry

A sensitive and efficient method was developed for the simultaneous determination of eight synthetic dyes (Chrysoidin, Auramine O, Sudan(I–IV), Para Red, and Rhodamine B) in bean and meat products using high‐performance liquid chromatography with tandem mass spectrometry. A simple extraction procedure using acetonitrile has been applied for the extraction of these dyes from spiked bean and meat samples. Chromatographic separation was achieved on a Waters XTerra C₁₈ column (2.1 × 150 mm, 5 μm) with a multistep gradient elution. Detection and quantification were performed using mass spectrometry in multiple reaction monitoring mode. Linear calibrations were obtained with correlation coefficients R² > 0.99. The limits of detection and quantification for the eight dyes were in the ranges of 0.03–0.75 and 0.1–2.0 μg/kg depending on matrices, respectively. The recoveries of these dyes in different food matrices were between 71.2 and 116.9% with relative standard deviations <15.2%, suggesting that the developed method is promising for the accurate quantification of the eight dyes at trace levels in bean and meat products.     

Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high‐performance liquid chromatography with diode array detection and tandem mass spectrometry

The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry and high‐performance liquid chromatography with electrospray multistage tandem ion‐trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p‐coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high‐performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.     

Systematic approach to optimize a pretreatment method for ultrasensitive liquid chromatography with tandem mass spectrometry analysis of multiple target compounds in biological samples

In current approaches for new drug development, highly sensitive and robust analytical methods for the determination of test compounds in biological samples are essential. These analytical methods should be optimized for every target compound. However, for biological samples that contain multiple compounds as new drug candidates obtained by cassette dosing tests, it would be preferable to develop a single method that allows the determination of all compounds at once. This study aims to establish a systematic approach that enables a selection of the most appropriate pretreatment method for multiple target compounds without the use of their chemical information. We investigated the retention times of 27 known compounds under different mobile phase conditions and determined the required pretreatment of human plasma samples using several solid‐phase and liquid–liquid extractions. From the relationship between retention time and recovery in a principal component analysis, appropriate pretreatments were categorized into several types. Based on the category, we have optimized a pretreatment method for the identification of three calcium channel blockers in human plasma. Plasma concentrations of these drugs in a cassette‐dose clinical study at microdose level were successfully determined with a lower limit of quantitation of 0.2 pg/mL for diltiazem, 1 pg/mL for nicardipine, and 2 pg/mL for nifedipine.     

Analysis of amicarbazone and its two metabolites in grains and soybeans by liquid chromatography with tandem mass spectrometry

A sensitive, simple and reliable analytical method based on a modified quick, easy, cheap, effective, rugged, safe sample preparation and liquid chromatography with tandem mass spectrometry detection was developed for the simultaneous determination of amicarbazone and its two major metabolites desamino amicarbazone and isopropyl‐2‐hydroxy‐desamino amicarbazone residues in grains (rice, wheat, corn, buckwheat) and soybean. Several parameters, including liquid chromatography and tandem mass spectrometry conditions, extraction approaches and the adsorbents for clean‐up, which might influence the accuracy of the method, were extensively investigated. The established method was further validated by determining the linearity (R² > 0.99), fortified recovery (79–118%), precision (1–12%) and sensitivity (limit of quantification, 5 μg/kg for amicarbazone and desamino amicarbazone, and 10 μg/kg for isopropyl‐2‐hydroxy‐desamino amicarbazone). Finally, the established method was successfully applied to determine the residues of amicarbazone and its metabolites in 49 real samples of grain and soybean.     

Identification in residue analysis based on liquid chromatography with tandem mass spectrometry: Experimental evidence to update performance criteria

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the most widely used techniques for identification (and quantification) of residues and contaminants across a number of different chemical domains. Although the same analytical technique is used, the parameters and criteria for identification vary depending on where in the world the analysis is performed and for what purpose (e.g. determination of pesticides, veterinary drugs, forensic toxicology, sports doping). The rationale for these differences is not clear and in most cases the criteria are essentially based on expert opinions rather than underpinned by experimental data. In the current study, the variability of the two key identification parameters, retention time and ion ratio, was assessed and compared against requirements set out in different legal and guidance documents. The study involved the analysis of 120 pesticides, representing various chemical classes, polarities, molecular weights, and detector response factors, in 21 different fruit and vegetable matrices of varying degrees of complexity. The samples were analysed non-fortified, and fortified at 10, 50 and 200 μg kg⁻¹, in five laboratories using different LC-MS/MS instruments and conditions. In total, over 135,000 extracted-ion chromatograms were manually verified to provide an extensive data set for the assessment. The experimental data do not support relative tolerances for retention time, or different tolerances for ion ratios depending on relative abundance of the two product ions measured. Retention times in today’s chromatographic systems are sufficiently stable to justify an absolute tolerance of ±0.1 min. Ion ratios are stable as long as sufficient response is obtained for both product ions. Ion ratio deviations are typically within ±20% (relative), and within ±45% (relative) in case the response of product ions are close to the limit of detection. Ion ratio tolerances up to 50% did not result in false positives and reduced the false negative rate for pesticides with product ions in the low S/N range to <5%. Without ion ratio criterion, two false positives were obtained in 105 non-fortified samples. Although the study has been conducted for pesticides residues in fruits and vegetables, the impact of these findings is believed to extend towards other application areas and possibly support adjustment or consolidation of criteria across other analytical domains.     

Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: A review

This article reviews novel quantification concepts where elemental labelling is combined with flow injection inductively coupled plasma mass spectrometry (FI-ICP-MS) or liquid chromatography inductively coupled plasma mass spectrometry (LC–ICP-MS), and employed for quantification of biomolecules such as proteins, peptides and related molecules in challenging sample matrices. In the first sections an overview on general aspects of biomolecule quantification, as well as of labelling will be presented emphasizing the potential, which lies in such methodological approaches. In this context, ICP-MS as detector provides high sensitivity, selectivity and robustness in biological samples and offers the capability for multiplexing and isotope dilution mass spectrometry (IDMS). Fundamental methodology of elemental labelling will be highlighted and analytical, as well as biomedical applications will be presented. A special focus will lie on established applications underlining benefits and bottlenecks of such approaches for the implementation in real life analysis. Key research made in this field will be summarized and a perspective for future developments including sophisticated and innovative applications will given.     

Quantitative analysis of clavulanic acid in porcine tissues by liquid chromatography combined with electrospray ionization tandem mass spectrometry

The purpose of this study was to develop and validate a method for the determination of clavulanic acid (CLAV) residues in edible tissues of swine by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). After a simple extraction of CLAV using an aqueous phosphate buffer solution of pH 6.0, an ultrafiltration step was performed for protein removal. Chromatography of CLAV and the internal standard tazobactam (TAZO) was achieved on a reversed-phase PLRP-S polymeric column (150 mm × 2.1 mm i.d., 100 Å) using a mixture of 0.05 (v/v)% formic acid in water and acetonitrile. The mass spectrometer was operated in the MS/MS selected reaction monitoring (SRM) mode. The method was validated for the analysis of porcine muscle, skin plus fat, liver and kidney, according to the requirements defined by the European Community. Calibration curves were prepared for all tissues and good linearity was achieved over the concentration ranges tested (correlation coefficient ≥0.99 and goodness-of-fit coefficient ≤10%). Limits of quantification of 50 ng g⁻¹ were obtained for the analysis of CLAV in the various tissues which corresponds in all cases to at least half the maximum residue limits (MRLs). Limits of detection ranged between 8.0 and 15.14 ng g⁻¹. The within-day, between-day precisions and trueness fell within the ranges specified in the EMEA/CVMP/573-00/FINAL document. Biological samples from pigs that received an oral or intravenous bolus of a commercial amoxicillin/clavulanic acid formulation were analyzed using the described method.     

Rapid identification of anti‐inflammatory compounds from Tongmai Yangxin Pills by liquid chromatography with high‐resolution mass spectrometry and chemometric analysis

We present an integrated approach to rapidly identify anti‐inflammatory compounds of TongmaiYangxin Pills (TMYXP), a botanical drug for the treatment of cardiovascular disease. Liquid chromatography coupled with high‐resolution mass spectrometry was used to analyze the chemical composition of TMYXP. Eighty compounds of TMYXP including flavonoids, coumarins, iridoid glycosides, saponins, and lignans, were identified unambiguously or tentatively. After the rapid isolation and bioassay, 18 fractions of TMYXP were obtained and their anti‐inflammatory activities were evaluated in lipopolysaccharide‐stimulated RAW 264.7 macrophages. We performed chemometric analysis to reveal the correlation between the chemical and pharmacological information of the fractions to facilitate the identification of active compounds. To verify the reliability of the proposed method in discovering active components from a complex mixture, activities of seven compounds, which were positively or negatively related to bioactivity according to calculation, were validated in vitro. Results indicated that six active compounds with high R values exerted certain anti‐inflammatory effects in a dose‐dependent manner with IC₅₀ values of 53.6–204.1 μM. Our findings suggest that the integrated use of identification based on high‐resolution mass spectrometry and chemometric methods could rapidly identify active compounds from complex mixture of natural products.     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry

Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography(RPLC)/ hybrid quadrupole ion trap/time-of-flight mass spectrometry(QIT/TOFMS).Tautomers gave different MS/MS spectra in negative mode.Different mass spectra were also obtained by hydrogen/deuterium exchange LC/MS/MS in positive mode.Our results suggest that enol form is the major form in the solution(water/acetonitrile).     

Separation and identification of oligomeric ethyl silicates by liquid chromatography with electrospray ionization mass spectrometry

Reversed‐phase liquid chromatography coupled with electrospray ionization mass spectrometry was used to study the molecular structures of components and molar mass distributions in ethyl silicate‐40, a versatile liquid precursor for silicon‐based materials. Identity testing by standard spectroscopic techniques showed that a commercial sample of ethyl silicate‐40 was composed of linear/branched ethoxysiloxane oligomers with the silicon atoms ranging from 2 to 12 together with minor monocyclic species. Analysis of the sample by liquid chromatography coupled with evaporative light scattering detection resulted in an elution profile consisting of a series of peak clusters. Peak identification showed that the linear/branched homologous series of oligomers were eluted in the order of increasing number of silicon atoms in the molecules and the time duration (width) of the resulting peak clusters increased in the same fashion corresponding to increasing number of geometric isomers. In addition, small amounts of monocyclic oligomers present in the sample were found to be less retained than each linear/branched counterpart. Finally, the molar mass distribution parameters for ethyl silicate‐40 determined by the developed method were in good agreement with the literature values. Overall, this work demonstrates that reversed‐phase liquid chromatography coupled with electrospray ionization mass spectrometry is an indispensable tool for the comprehensive characterization of complex mixtures of this type.     

Quantitative performance of liquid chromatography coupled to Q-Exactive high resolution mass spectrometry (HRMS) for the analysis of tetracyclines in a complex matrix

The presence of antibiotics in the environment is of increased interest and, as modern mass spectrometers become more efficient, we are increasingly aware of traces of pharmaceuticals appearing in a wide range of environmental and biological matrices. The Q-Exactive mass spectrometer is part of these innovative hybrid high-resolution mass spectrometers (HRMS) which is often associated with peptide sequencing or metabolomics but with a limited number of studies focusing on its application to the quantification of small molecules in environmental and biological matrices. It combines the high resolving power (RP) performance of the Orbitrap with the high performance selectivity of the quadrupole. Tetracyclines (TCs) are a family comprising some of the most widely used antibiotics in veterinary medicine. This study presents the quantitative performances of the Q-Exactive by illustrating a new approach to quantify TCs using liquid chromatography coupled to a HRMS in a complex matrix, i.e., swine manure. The Q-Exactive was used at high-resolution in both full scan (FS) and targeted ion fragmentation (tMS²) modes. These two modes were optimized and compared to determine the most reliable and efficient approach to quantify TCs with good accuracy. The proposed method was optimized to obtain the best selectivity and sensitivity, thus eliminating false positive and allowing the detection of trace levels of analyte. The TCs were extracted from the matrix by sonication using McIlvaine buffer followed by an off-line solid phase extraction method to concentrate and clean the extracts. Both FS and tMS² modes presented good linearity (R² > 0.991) and repeatability (RSD < 15%). Mass accuracy was acceptable with values below 2 ppm. The method detection limits (MLD) calculated from the calibration curves ranged from 2.0 to 12 ng g⁻¹ for FS mode and from 1.5 to 3.6 ng g⁻¹ for tMS² mode. Accuracy and interday/intraday relative standard deviations were below 21% for both modes studied. TCs were quantified in real samples of swine manure with concentrations ranging from 29 to 75 ng g⁻¹. This study showed the possibility of using hybrid HRMS for trace detection and quantification of TCs in a complex matrix, thus avoiding false positive while achieving good selectivity and sensitivity.     

Multiresidue analysis of 36 pesticides in soil using a modified quick,easy, cheap,effective, rugged,and safe method by liquid chromatography with tandem quadruple linear ion trap mass spectrometry

A new method for simultaneous determination of 36 pesticides, including 15 organophosphorus, six carbamate, and some other pesticides in soil was developed by liquid chromatography with tandem quadruple linear ion trap mass spectrometry. The extraction and clean‐up steps were optimized based on the quick, easy, cheap, effective, rugged, and safe method. The data were acquired in multiple reaction monitoring mode combined with enhanced product ion to increase confidence of the analytical results. Validation experiments were performed in soil samples. The average recoveries of pesticides at four spiking levels (1, 5, 50, and 100 μg/kg) ranged from 63 to 126% with relative standard deviation below 20%. The limits of detection of pesticides were 0.04–0.8 μg/kg, and the limits of quantification were 0.1–2.6 μg/kg. The correlation coefficients (r²) were higher than 0.990 in the linearity range of 0.5–200 μg/L for most of the pesticides. The method allowed for the analysis of the target pesticides in the lower μg/kg concentration range. The optimized method was then applied to the test of real soil samples obtained from several areas in China, confirming the feasibility of the method.     

Enantioselective determination of acaricide etoxazole in orange pulp,peel, and whole orange by chiral liquid chromatography with tandem mass spectrometry

An efficient enantioselective method for the determination of etoxazole in orange pulp, peel, and whole orange was developed using liquid chromatography with tandem mass spectrometry. The enantioseparation was performed on a Chiralpak AD‐3R column at 30ºC using acetonitrile with 0.1% formic acid solution (80:20, v/v) as the mobile phase in less than 5 min. Quantification was achieved using matrix‐matched standard calibration curves. The overall mean recoveries for two enantiomers from orange pulp and whole orange were 91.0–99.6% and the orange peel was 92.6–103.1%, with relative standard deviations of 0.8–5.4% intraday and 2.0–4.8% interday at 1, 10, and 100 μg/kg levels, and 1.3–5.2% intraday and 3.5–4.3% interday at 5, 50, and 500 μg/kg levels, respectively. The limits of quantification for all enantiomers in three matrices did not exceed 5 μg/kg. Moreover, the absolute configuration of etoxazole enantiomers had been determined by the combination of experimental and predicted electronic circular dichroism spectra, and the first eluted enantiomer was confirmed as (S)‐etoxazole on a Chiralpak AD‐3R column while (R)‐etoxazole was first on three cellulose chiral columns. The application of the proposed method to real sample analysis suggests its potential use in enantioselective determination of etoxazole enantiomers in citrus.