bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒02‒19
thirty papers selected by
Sofia Costa

  1. bioRxiv. 2023 Feb 10. pii: 2023.02.09.527886. [Epub ahead of print]
      Poor chemical annotation of high-resolution mass spectrometry data limit applications of untargeted metabolomics datasets. Our new software, the Integrated Data Science Laboratory for Metabolomics and Exposomics â€" Composite Spectra Analysis (IDSL.CSA) R package, generates composite mass spectra libraries from MS1-only data, enabling the chemical annotation of LC/HRMS peaks regardless of the availability of MS2 fragmentation spectra. We demonstrate comparable annotation rates for commonly detected endogenous metabolites in human blood samples using IDSL.CSA libraries versus data dependent acquisition (DDA) MS2 libraries in validation tests. IDSL.CSA can create and search composite spectra libraries from any untargeted metabolomics dataset generated using high-resolution mass spectrometry coupled to liquid or gas chromatography. The cross-applicability of these libraries across independent studies can improve overall annotation rates in metabolomics and exposomics projects, providing access to new biological insights that may be missed due to the lack of MS2 fragmentation data. The IDSL.CSA package is available in the R CRAN repository ( . Detailed documentation and tutorials are provided at .
  2. Lab Invest. 2021 10;pii: S0023-6837(22)00403-2. [Epub ahead of print]101(10): 1403-1410
      Stable isotope labeling techniques have been widely applied in the field of metabolomics and proteomics. Before the measured mass spectral data can be used for quantitative analysis, it must be accurately corrected for isotope natural abundance and tracer isotopic impurity. Despite the increasing popularity of dual-isotope tracing strategy such as 13C-15N or 13C-2H, there are no accurate tools for correcting isotope natural abundance for such experiments in a resolution-dependent manner. Here, we present AccuCor2 as an R-based tool to perform the correction for 13C-15N or 13C-2H labeling experiments. Our method uses a newly designed algorithm to construct the correction matrices that link labeling pattern and measured mass fractions, then use non-negative least-squares to solve the labeling patterns. Our results show that the dual-isotope experiments often require a mass resolution that is high enough to resolve 13C and 15N or 13C and 2H. Otherwise, the labeling pattern is not solvable. However, this mass resolution may not be sufficiently high to resolve other non-tracer elements such as oxygen or sulfur from the tracer elements. Therefore, we design AccuCor2 to perform the correction based on the actual mass resolution of the measurements. Using both simulated and experimental data, we show that AccuCor2 performs accurate and resolution-dependent correction for dual-isotope tracer data. The authors developed AccuCor2 as the first resolution-dependent method for accurate isotope natural abundance correction of experimental data generated from dual-isotope tracers. They show that such correction requires a minimum resolution to resolve tracer isotopologues. AccuCor2 showed improved accuracy more than previously developed tools, which assume infinite resolution of the instrument.
  3. Clin Chem Lab Med. 2023 Feb 14.
      OBJECTIVES: We developed an isotope dilution (ID)-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for lamotrigine in human serum and plasma, using quantitative nuclear magnetic resonance-characterized reference standards to ensure traceability to the International System of Units.METHODS: A sample preparation protocol based on protein precipitation combined with LC-MS/MS analysis using a C18 column for chromatographic separation was established for the quantification of lamotrigine in human serum and plasma. Assay validation was performed according to current guidelines. Spiked serum and plasma samples were used to assess selectivity and specificity; a post-column infusion experiment and comparison of standard line slopes were performed to ascertain possible matrix effects. Precision and accuracy were determined in a 5 days validation experiment. Measurement uncertainty was determined per the Guide to the Expression of Uncertainty in Measurement.
    RESULTS: The method allowed the quantification of lamotrigine in serum and plasma in a range of 0.600-24.0 μg/mL without any observable matrix effects. The relative mean bias (n=6) ranged from 1.7 to 3.7%; intermediate precision, including variances in between-day, -calibration, and -injection, was ≤2.4%, independent of the level and matrix. Total measurement uncertainty for a single measurement was ≤2.6%; expanded uncertainty was ≤5.2% (coverage factor k=2).
    CONCLUSIONS: This candidate RMP based on ID-LC-MS/MS provides a traceable and reliable platform for the standardization of routine assays and the evaluation of clinical samples.
    Keywords:  SI units; isotope dilution-liquid chromatograph-tandem mass spectrometry; lamotrigine; qNMR; reference measurement procedure; traceability
  4. Clin Chem Lab Med. 2023 Feb 15.
      OBJECTIVES: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma.METHODS: Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines.
    RESULTS: The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200-5,700 ng/mL (0.01584-12.54 μmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5-2.1% for all analyte concentrations. Bias was -3.0 to 2.1% for samples within the measuring range and 0.8-4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%.
    CONCLUSIONS: This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.
    Keywords:  SI units; isotope dilution LC-MS/MS; methotrexate; qNMR; reference measurement procedure; traceability
  5. bioRxiv. 2023 Feb 03. pii: 2023.02.01.526566. [Epub ahead of print]
      Untargeted lipidomics allows analysis of a broader range of lipids than targeted methods and permits discovery of unknown compounds. Previous ring trials have evaluated the reproducibility of targeted lipidomics methods, but inter-laboratory comparison of compound identification and unknown feature detection in untargeted lipidomics has not been attempted. To address this gap, five laboratories analyzed a set of mammalian tissue and biofluid reference samples using both their own untargeted lipidomics procedures and a common chromatographic and data analysis method. While both methods yielded informative data, the common method improved chromatographic reproducibility and resulted in detection of more shared features between labs. Spectral search against the LipidBlast in silico library enabled identification of over 2,000 unique lipids. Further examination of LC-MS/MS and ion mobility data, aided by hybrid search and spectral networking analysis, revealed spectral and chromatographic patterns useful for classification of unknown features, a subset of which were highly reproducible between labs. Overall, our method offers enhanced compound identification performance compared to targeted lipidomics, demonstrates the potential of harmonized methods to improve inter-site reproducibility for quantitation and feature alignment, and can serve as a reference to aid future annotation of untargeted lipidomics data.
  6. J Lipid Res. 2023 Feb 09. pii: S0022-2275(23)00016-0. [Epub ahead of print] 100343
      Evaluating lipid profiles in human tissues and biofluids is critical in identifying lipid metabolites in dysregulated metabolic pathways. Due to various chemical characteristics, single-run lipid analysis has not yet been documented. Such approach is essential for analysing pathology-related lipid metabolites. Age-Related Macular Degeneration (AMD), the leading cause of vision loss in western countries, is emblematic of this limitation. Several studies have identified alterations in individual lipids, but the majority are based on targeted approaches. In this study, we analyzed and identified approximately 500 lipid species in human biofluids (plasma and erythrocytes) and ocular tissues (retina and retinal pigment epithelium) using the complementarity of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RPC), coupled to high-resolution mass spectrometry (HRMS). For that, lipids were extracted from human eyeglobes and blood from 10 subjects and lipidomic analysis was carried out through analysis in HILIC and RPC, alternately. Furthermore, we illustrate the advantages and disadvantages of both techniques for lipid characterization. RPC showed greater sensitivity in hydrophobicity-based lipid separation, detecting diglycerides (DAG), triglycerides (TAG), cholesterol (Chol), and cholesterol esters (CEs), whereas no signal of these molecules was obtained in HILIC. However, due to coelution, RPC was less effective in separating polar lipids like phospholipids, which were separated effectively in HILIC in both ionization modes. The complementary nature of these analytical approaches was essential for the detection and identification of lipid classes/subclasses, which can then provide distinct insights into lipid metabolism, a determinant of the pathophysiology of several diseases involving lipids, notably AMD.
    Keywords:  RPE/Choroid; age-related macular degeneration; erythrocytes; eye/retina; glycolipids; lipidomic analysis; mass spectrometry; phospholipids; plasma
  7. Methods Mol Biol. 2023 ;2622 227-239
      Liposomes are spherical, closed vesicles consisting of at least one lipid bilayer with a water chamber and are widely used to encapsulate bioactive molecules. Lipid membranes, composed of different types of lipids or lipophilic components, determine whether liposomes can achieve the desired purpose and determine the overall quality of liposomes. Thus, the quantification of lipid components and encapsulated molecules is essential to characterize and control the quality of liposomes. Moreover, multicomponent simultaneous determination is the preferred method for lipid component analysis in liposomes. Therefore, the present work describes an analytical methodology for the simultaneous determination of commonly used lipids in liposome formulations, using h igh-performance liquid chromatography coupled with a tandem mass spectrometry (MS) detector (HPLC-MS/MS). HPLC-MS/MS consists of a rapid and highly efficient chromatographic separation of the liposomal components with a C18 column and the subsequent detection of the ingredients through an MS detector, along with an accurate mass fragmentation pattern. The analytical process mainly includes lipid extraction, solution preparation, the optimization of chromatographic conditions, and method validation. We hope this analytical methodology is valuable and efficient and can be applied to the analysis of multiple types of lipids in liposomes, such as raw material quality analysis, formulation study, overall quality control, etc.
    Keywords:  HPLC-MS/MS; Lipid analysis; Liposome
  8. Bioinformatics. 2023 Feb 14. pii: btad088. [Epub ahead of print]
      MOTIVATION: Plasma ionization is rapidly gaining popularity for MS based studies of volatiles and aerosols. However, data from plasma ionization is delicate to interpret as competing ionization pathways in the plasma create numerous ion species. There is no tool for detection of adducts and in-source fragments from plasma ionization data yet, which makes data evaluation ambiguous.SUMMARY: We developed DBDIpy, a Python library for processing and formal analysis of untargeted, time-sensitive plasma ionization MS datasets. Its core functionality lies in the identification of in-source fragments and identification of rivaling ionization pathways of the same analytes in time-sensitive datasets. It further contains elementary functions for processing of untargeted metabolomics data and interfaces to an established ecosystem for analysis of MS data in Python.
    AVAILABILITY AND IMPLEMENTATION: DBDIpy is implemented in Python (Version ≥ 3.7) and can be downloaded from PyPI the Python package repository ( or from GitHub (
  9. Lab Invest. 2021 Apr;pii: S0023-6837(22)00647-X. [Epub ahead of print]101(4): 423-429
      Metabolic flux analysis (MFA) aims at revealing the metabolic reaction rates in a complex biochemical network. To do so, MFA uses the input of stable isotope labeling patterns of the intracellular metabolites. Elementary metabolic unit (EMU) is the computational framework to simulate the metabolite labeling patterns in a network, which was originally designed for simulating mass isotopomer distributions (MIDs) at the MS1 level. Recently, the EMU framework is expanded to simulate tandem mass spectrometry data. Tandem mass spectrometry has emerged as a new experimental approach to provide information on the positional isotope labeling of metabolites and therefore greatly improves the precision of MFA. In this review, we will discuss the new EMU framework that can accommodate the tandem mass isotopomer distributions (TMIDs) data. We will also analyze the improvement on the MFA precision by using TMID. Our analysis shows that combining the MIDs of the parent and daughter ions and the TMID for the MFA is more powerful than using TMID alone.
  10. Anal Chem. 2023 Feb 15.
      Metabolite identification represents a major bottleneck in contemporary metabolomics research and a step where critical errors may occur and pass unnoticed. This is especially the case for studies employing liquid chromatography-mass spectrometry technology, where there is increased concern on the validity of the proposed identities. In the present perspective article, we describe the issue and categorize the errors into two types: identities that show poor biological plausibility and identities that do not comply with chromatographic data and thus to physicochemical properties (usually hydrophobicity/hydrophilicity) of the proposed molecule. We discuss the problem, present characteristic examples, and propose measures to improve the situation.
  11. J Chromatogr A. 2023 Feb 04. pii: S0021-9673(23)00071-7. [Epub ahead of print]1692 463843
      The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) has proved effective in the LC × LC analysis of polyphenols due to the high degree of orthogonality associated with these separation modes for various classes of phenolic compounds. However, despite the growing number of such applications, HILIC is almost exclusively used as the first dimension (1D) separation mode, and RP-LC in the second dimension (2D). This is somewhat surprising in light of the potential advantages of swapping these separation modes. In this contribution, we present a detailed evaluation of the potential of online RP-LC × HILIC-MS for the analysis of phenolic compounds, comparing the performance of this system to the more established HILIC × RP-LC-MS configuration. Method development was performed using a predictive optimisation program, and fixed solvent modulation was employed to combat the solvent incompatibility between HILIC and RP-LC mobile phases. Red wine, rooibos tea, Protea and chestnut phenolic extracts containing a large diversity of phenolic compound classes were analysed by both HILIC × RP-LC- and RP-LC × HILIC-MS in order to compare the separation performance. Overall, the kinetic performance of HILIC × RP-LC was found to be clearly superior, with higher peak capacities and better resolution obtained for the majority of samples compared to RP-LC × HILIC analyses using similar column dimensions. Dilution of the 1D solvent combined with large volume injections proved insufficient to focus especially phenolic acids in the 2D HILIC separation, which resulted in severe 2D peak distortion for these compounds, and negatively impacted on method performance. On the other hand, a noteworthy improvement in the sensitivity of RP-LC × HILIC-MS analyses was observed due to higher ESI-MS response for the 2D HILIC mobile phase and greater sample loading capacity of the 1D RP-LC column, brought on by the high solubility of phenolic samples in aqueous solutions. As a result, a significantly higher number of compounds were detected in the RP-LC × HILIC-MS separations. These findings point to the potential advantage of RP-LC × HILIC as a complementary configuration to HILIC × RP-LC for phenolic analysis.
    Keywords:  Comprehensive two-dimensional liquid chromatography (LC × LC); High resolution mass spectrometry (HR-MS); Hydrophilic interaction chromatography (HILIC); Phenolics; Reversed phase liquid chromatography (RP-LC)
  12. Anal Chem. 2023 Feb 15.
      To meet the ever-increasing need for high-throughput screening in metabolic engineering, information-rich, fast screening methods are needed. Mass spectrometry (MS) provides an efficient and general approach for metabolite screening and offers the capability of characterizing a broad range of analytes in a label-free manner, but often requires a range of sample clean-up and extraction steps. Liquid extraction surface analysis (LESA) coupled MS is an image-guided MS surface analysis approach that directly samples and introduces metabolites from a surface to MS. Here, we combined the advantages of LESA-MS and an acoustic liquid handler with stable isotope-labeled internal standards. This approach provides absolute quantitation of target chemicals from liquid culture-dried droplets and enables high-throughput quantitative screening for microbial metabolites. In this study, LESA-MS was successfully applied to quantify several different metabolites (itaconic acid, triacetic acid lactone, and palmitic acid) from different yeast strains in different mediums, demonstrating its versatility, accuracy, and efficiency across a range of microbial engineering applications.
  13. Methods Mol Biol. 2023 ;2628 489-504
      Mass spectrometry remains one of the gold standard approaches in examining the lipidome in biological samples. Recently, advancements in chromatography and mass spectrometry approaches have enabled broad coverage of the lipidome. However, many limitations still exist, and lipidomic analysis often requires a fine balance between coverage of the lipidome, structural detail, and sample throughput. For biomedical and clinical research using human samples, the diversity and natural variation between different individuals necessitate larger sample numbers to identify significant associations with clinical outcomes and account for potential confounding factors. Here we describe a targeted lipidomics workflow that enables reproducible profiling of thousands of plasma samples in a systematic manner, while maintaining good structural detail and high coverage of the lipidome.
    Keywords:  Cohort studies; Lipidomics; Mass spectrometry; Plasma; Targeted lipidomics
  14. Biochim Biophys Acta Gen Subj. 2023 Feb 13. pii: S0304-4165(23)00027-2. [Epub ahead of print] 130329
      BACKGROUND: Metals are pervasive throughout biological processes, where they play essential structural and catalytic roles. Metals can also exhibit deleterious effects on human health. Powerful analytical techniques, such as mass spectrometry imaging (MSI), are required to map metals due to their low concentrations within biological tissue.SCOPE OF REVIEW: This Mini Review focuses on key MSI technology that can image metal distributions in situ, describing considerations for each technique (e.g., resolution, sensitivity, etc.). We highlight recent work using MSI for mapping trace metals in tissues, detecting metal-based drugs, and simultaneously imaging metals and biomolecules.
    MAJOR CONCLUSIONS: MSI has enabled significant advances in locating bioactive metals at high spatial resolution and correlating their distributions with that of biomolecules. The use of metal-based immunochemistry has enabled simultaneous high-throughput protein and biomolecule imaging.
    GENERAL SIGNIFICANCE: The techniques and examples described herein can be applied to many biological questions concerning the important biological roles of metals, metal toxicity, and localization of metal-based drugs.
    Keywords:  DESI; LA-ICP-MS; MALDI; Metallomics; SIMS; Spatial metabolomics; cyTOF
  15. Sci Rep. 2023 Feb 13. 13(1): 2534
      Andrographis paniculata, a medicinal plant in Thailand national list of essential medicines, has been proposed for treatment of patients with mild to moderate coronavirus disease 2019. This study aims to develop a highly selective and sensitive liquid chromatography triple quadrupole tandem mass spectrometry method for quantitative determination of major diterpenoids in plasma and urine with application in pharmacokinetics. Chromatographic separation was performed on C18 column using a gradient mobile phase of water and acetonitrile. Mass spectrometry was analyzed using multiple reaction monitoring with negative ionization mode. This validated analytical method was very sensitive, less time consuming in analysis, and allowed the reliability and reproducibility on its application. The clinical pharmacokinetics was evaluated after single oral administration of A. paniculata extract (calculated as 60 mg of andrographolide). The disposition kinetics demonstrated that major diterpenoids could enter into systemic circulation, but they are mostly biotransformed (phase II) into conjugated glucuronide and sulfate metabolites. These metabolites are predominantly found in plasma and then extremely eliminated, in part through urinary excretion. The successful application of this analytical method supports its suitable uses in further clinical benefits after oral administration of A. paniculata.
  16. Clin Chem Lab Med. 2023 Feb 13.
      OBJECTIVES: Sex hormone binding globulin (SHBG) is a hormone binding protein which plays an important role in regulating the transport and availability of biologically active androgens and estradiol to target cells and used to calculate free testosterone concentrations.METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, featuring an albumin removal step followed by a tryptic digestion. After a reduction step with dithiothreitol and alkylation with iodoacetamide three signature peptides were used for the quantification of SHBG.
    RESULTS: The method enables the quantification of serum and plasma SHBG over the clinically relevant range of 200-20,000 ng/mL and was validated according to the most recent guidelines. The LC-MS/MS method correlates well with the Abbott Alinity immunoassay (R2>0.95), but the LC-MS/MS results are on average 16-17% lower than the immunoassay results, which is consistent for all three signature peptides.
    CONCLUSIONS: The LC-MS/MS method which includes an albumin depletion step allows quantification of SHBG in serum and plasma without an immunocapture step at clinically relevant SHBG levels, thus contributing to better lab-to-lab consistency of results.
    Keywords:  albumin depletion; biomarker; human sex hormone binding globulin (SHBG); liquid chromatography-tandem mass spectrometry (LC-MS/MS)
  17. Biomed Chromatogr. 2023 Feb 16. e5606
      Zika still poses a threat to global health due to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma, cerebrospinal (CSF), and seminal fluid (SF) and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C18 columns. Analytical detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. The validated ranges for sofosbuvir were 0.5 - 2000 ng/mL (plasma) and 0.5 - 100 ng/mL (CSF and SF), while for the metabolite they were 2.0 - 2000 ng/mL (plasma), 5.0 - 200 ng/mL (CSF), and 10 - 1500 ng/mL (SF). The intra-day and inter-day accuracy (90.8 to 113.8 %) and precision (1.4 to 14.8%) were within the acceptable values. The developed methods fulfilled all validation parameters concerning selectivity, matrix effect, carryover, linearity, dilution integrity, precision, accuracy, and stability, confirming their suitability for the analysis of clinical samples.
    Keywords:  GS-331007; LC-MS/MS; Sofosbuvir; Zika; human matrices
  18. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Feb 02. pii: S1570-0232(23)00029-6. [Epub ahead of print]1217 123619
      Assessing human exposure to commonly used, highly toxic, but non-persistent organophosphates (OPs) is challenging because these toxicants are readily biotransformed into dialkyl phosphates (DAPs) and other metabolites. Growing hair accumulates toxicants and their metabolites, which makes hair a valuable non-invasively sampled matrix that can be used to retrospectively examine chemical exposure. However, the efficient quantification of hydrophilic DAP compounds in hair is challenging due to complex hair matrix effects. To improve upon existing methods, we first examined the acid dissociation constants (pKa) of DAPs and amino acids (major components in hair) and identified the best pH conditions for minimizing matrix effects. We hypothesized that under basic pH conditions DAPs and amino acids would be negatively charged and have weak interactions favorable to DAP dissociation from the matrix. To test this, we compared the efficiency of various pH conditions of suitable solvents to extract six DAPs from hair samples, and we quantified these DAPs using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). As expected, a basic extraction (methanol with 2% NH4OH) approach had the highest extraction efficiency and yielded satisfactory recoveries for all six DAPs (72%-152%) without matrix effects. Additionally, the alkaline extract can be directly injected into the LC-MS/MS. This relatively rapid and simple procedure allowed us to process up to 90 samples per week with reproducible results. To our knowledge, this is the first method to quantify all six DAPs simultaneously in hair using LC-MS/MS with electrospray ionization (ESI) in negative ion mode. Finally, we demonstrated the feasibility of measuring DAP levels in hair samples from patients affected with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease potentially linked to OP exposure. Due to our optimized solvent extraction process, the method we have developed is compatible with the rapidity and sensitivity needed for hair analysis applied to population biomonitoring.
    Keywords:  Dialkyl phosphates; ElectroSpray Ionization; Hair matrix; LC-MS/MS; Organophosphate pesticides; Solvent pH
  19. Front Chem. 2023 ;11 1100150
      A simple and efficient vortex-assisted matrix solid phase dispersion with a ultra-high-performance liquid chromatography-triple quadrupole mass spectrometer (VA-MSPD-UHPLC-MS/MS) was applied for simultaneous extraction and determination of seven alkaloids and three organic acids from Uncariae Ramulas Cum Unicis. The optimal extraction conditions of the target components were obtained by Box-Behnken design (BBD) combined with response surface methodology (RSM). The results of the method validation showed that this analytical method displayed good linearity with a correlation coefficient (r) no lower than 0.9990. The recoveries of ten active ingredients from Uncariae Ramulas Cum Unicis ranged from 95.9% to 103% (RSD ≤ 2.77%). The RSDs of intra-day and inter-day precisions were all below 2.97%. The present method exhibited not only lower solvent and sample usage, but also shorter sample processing and analysis time. Consequently, the developed VA-MSPD-UHPLC-MS/MS method could be successfully and effectively used for the extraction and analysis of ten active components from Uncariae Ramulas Cum Unicis.
    Keywords:  UHPLC-MS/MS; Uncariae Ramulas Cum Unicis; response surface methodology; silica; vortex-assisted matrix solid phase dispersion
  20. Food Sci Nutr. 2023 Feb;11(2): 688-695
      Dispersive liquid-liquid microextraction was used in conjunction with liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry to quantitate vitamins K1 and K2 in vitamin-fortified emulsions, and vital microextraction parameters were optimized using response surface methodology coupled with Box-Behnken design. Under optimal microextraction conditions, highly linear (R 2 > .999) calibration curves were obtained for both vitamins in a broad concentration range (1-1000 μg/L), and vitamin recoveries exceeded 90%. The detection and quantitation limits equaled 1.89 and 5.72 μg/L for vitamin K1, respectively, and 5.00 and 15.15 μg/L for vitamin K2, respectively. When applied to vitamin-K-loaded nanoemulsions and solid lipid nanoparticles, the developed method achieved excellent results, outperforming the currently employed Korean Food Code method, and therefore holding great promise for the quantitation of vitamin K in vitamin-fortified food products.
    Keywords:  dispersive liquid–liquid microextraction; liquid chromatography–tandem mass spectrometry; response surface model; vitamin K
  21. J Chromatogr A. 2023 Feb 03. pii: S0021-9673(23)00048-1. [Epub ahead of print]1692 463820
      Typical chromatographic analysis of chiral compounds requires the use of achiral methods to evaluate impurities or related substances along with separate methods to evaluate chiral purity. The use of two-dimensional liquid chromatography (2D-LC) to support simultaneous achiral-chiral analysis has become increasingly advantageous in the field of high-throughput experimentation where low reaction yields or side reactions can lead to challenging direct chiral analysis. Advancements in multi-dimensional chromatography have led to the development of robust 2D-LC instrumentation with reversed phase solvent systems (RPLC-RPLC) enabling this simultaneous analysis, eliminating the need to purify crude reaction mixtures to determine stereoselectivity. However, when chiral RPLC cannot separate a chiral impurity from the desired product, there are few viable commercial options. The coupling of NPLC to RPLC (RPLC-NPLC) continues to remain elusive due to solvent immiscibility between the two solvent systems. This solvent incompatibility leads to lack of retention, band broadening, poor resolution, poor peak shapes, and baseline issues in the second dimension. A study was conducted to understand the effect of various water-containing injections on NPLC and applied to the development of robust RPLC-NPLC methods. Following thoughtful consideration and modifications to the design of a 2D-LC system in regards to mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility, proof of concept has been demonstrated with the development of reproducible RPLC-NPLC 2D-LC methods to perform simultaneous achiral-chiral analysis. Second dimension NPLC method performance proved comparable to corresponding 1D-NPLC methods with excellent percent difference in enantiomeric excess results ≤ 1.09% and adequate limits of quantitation down to 0.0025 mg/mL for injection volumes of 2 µL, or 5 ng on-column.
  22. Biotechnol J. 2023 Feb 16. e2200444
      Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for Hela carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol: chloroform: water (1:1:1, v/v/v), 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent Hela carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids, adenosine nucleotides and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33 nmol/million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile was found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect amino acid metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to 2D cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of Hela carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment. This article is protected by copyright. All rights reserved.
    Keywords:  3D tumor spheroids; Hela; extraction; isotope dilution mass spectrometry; metabolomics; quenching; sample preparation
  23. Methods Mol Biol. 2023 ;2622 289-302
      Nanomedicine offers the possibility of modifying the distribution of encapsulated drugs and biomolecules. Nanomedicine could limit the transplacental passage and/or enhance the concentration of drugs in placental tissue; this approach could be exploited for the treatment of pregnancy disorders. In the context of pregnancy, tackling the biological fate of both the nanocarrier and the drug has high importance in ensuring both the mother's and the fetus' safety.In this study, we propose a method for quantifying the uptake of liposomes inside placental tissue using covalently labeled liposomes and adapting a high-performance liquid chromatography (HPLC) method using a fluorescent detector. An optimized protocol for liquid-liquid extraction of fluorescent lipids from placental tissue extracts, followed by HPLC analysis, is detailed in this chapter. The HPLC method allows the quantification of fluorescent lipids using a calibration curve, including the biological matrix and extraction procedures. The internalization rate of fluorescent liposomes within human villous placental explants was quantitatively assessed, thanks to the HPLC developed method and suitable analytical tools.
    Keywords:  Ex vivo model; Fluorescence; HPLC; Liposomes; Placenta; Placental explants
  24. Sci Rep. 2023 Feb 16. 13(1): 2776
      Glycans play an important role in biology with multiple cellular functions ranging from cell signaling, mobility and growth to protein folding and localization. The N-glycosylation state within a tissue has been found to vary greatly between healthy and diseased patients and has proven to have an important clinical diagnostic value. Matrix assisted laser-desorption ionization (MALDI) mass spectrometry imaging (MSI) allows for untargeted analysis of biomolecules, including N-glycans, on a tissue section and provides a spatial context of the analyte. Until now, N-glycans have been predominantly analyzed using MALDI MSI on formalin-fixed paraffin embedded (FFPE) tissue sections, however this greatly reduces the clinical applicability, as the FFPE embedding process alters the biological environment of the tissue. Here we developed a protocol that allows for MALDI MSI of N-glycans from fresh frozen tissue that matches the current standard of FFPE analysis. By optimizing several steps in the sample preparation, we see orders of magnitude increase in signal intensity. Furthermore, this method limits delocalization of released N-glycans, thus improving the effective spatial resolution of the label-free molecular images. This protocol provides a novel perspective towards clinical application of MALDI MSI and capitalizes on the diagnostic value of N-glycan analysis.
  25. Metabolomics. 2023 Feb 13. 19(2): 13
      INTRODUCTION: This study sought to compare between metabolomic changes of human urine and plasma to investigate which one can be used as best tool to identify metabolomic profiling and novel biomarkers associated to the potential effects of ultraviolet (UV) radiation.METHOD: A pilot study of metabolomic patterns of human plasma and urine samples from four adult healthy individuals at before (S1) and after (S2) exposure (UV) and non-exposure (UC) were carried out by using liquid chromatography-mass spectrometry (LC-MS).
    RESULTS: The best results which were obtained by normalizing the metabolites to their mean output underwent to principal components analysis (PCA) and Orthogonal Partial least squares-discriminant analysis (OPLS-DA) to separate pre-from post-of exposure and non-exposure of UV. This separation by data modeling was clear in urine samples unlike plasma samples. In addition to overview of the scores plots, the variance predicted-Q2 (Cum), variance explained-R2X (Cum) and p-value of the cross-validated ANOVA score of PCA and OPLS-DA models indicated to this clear separation. Q2 (Cum) and R2X (Cum) values of PCA model for urine samples were 0.908 and 0.982, respectively, and OPLS-DA model values were 1.0 and 0.914, respectively. While these values in plasma samples were Q2 = 0.429 and R2X = 0.660 for PCA model and Q2 = 0.983 and R2X = 0.944 for OPLS-DA model. LC-MS metabolomic analysis showed the changes in numerous metabolic pathways including: amino acid, lipids, peptides, xenobiotics biodegradation, carbohydrates, nucleotides, Co-factors and vitamins which may contribute to the evaluation of the effects associated with UV sunlight exposure.
    CONCLUSIONS: The results of pilot study indicate that pre and post-exposure UV metabolomics screening of urine samples may be the best tool than plasma samples and a potential approach to predict the metabolomic changes due to UV exposure. Additional future work may shed light on the application of available metabolomic approaches to explore potential predictive markers to determine the impacts of UV sunlight.
    Keywords:  LC–MS; Metabolomic profiling; Orthogonal partial least squares- discriminant analysis (OPLS-DA); Plasma; Principal components analysis (PCA); Ultraviolet radiation (UV); Urine
  26. Talanta. 2023 Feb 03. pii: S0039-9140(23)00075-9. [Epub ahead of print]257 124324
      This review provides an overview of the online hyphenation of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) with separation methods to date. The online coupling between separation techniques (gas and liquid chromatography, capillary electrophoresis) and FT-ICR MS essentially raises questions of compromise and is not look as straightforward as hyphenation with other analyzers (QTOF-MS for instance). FT-ICR MS requires time to reach its highest resolving power and accuracy in mass measurement capabilities whereas chromatographic and electrophoretic peaks are transient. In many applications, the strengths and the weaknesses of each technique are balanced by their hyphenation. Untargeted "Omics" (e.g. proteomics, metabolomics, petroleomics, …) is one of the main areas of application for FT-ICR MS hyphenated to online separation techniques because of the complexity of the sample. FT-ICR MS achieves the required high mass measurement accuracy to determine accurate molecular formulae and resolution for isobar distinction. Meanwhile separation techniques highlight isomers and reduce the ion suppression effects extending the dynamic range. Even if the implementation of FT-ICR MS hyphenated with online separation methods is a little trickier (the art of compromise), this review shows that it provides unparalleled results to the scientific community (the art of the possible), along with raising the issue of its future in the field with the relentless technological progress.
    Keywords:  Capillary electrophoresis (CE); Environment; FT-ICR MS hyphenation; Gas chromatography (GC); Liquid chromatography (LC); Omics
  27. Anal Chem. 2023 Feb 17.
      Electrospray may exhibit inadequate ionization efficiency in some applications. In such cases, atmospheric-pressure chemical ionization (APCI) and photoionization (APPI) can be used. Despite a wide application potential, no APCI and APPI sources dedicated to very low sample flow rates exist on the market. Since the ion source performance depends on the transfer of analytes from the liquid to the gas phase, a nebulizer is a critical component of an ion source. Here, we report on the nebulizer with a gas dynamic virtual nozzle (GDVN) and its applicability in APCI at microliter-per-minute flow rates. Nebulizers differing by geometrical parameters were fabricated and characterized regarding the jet breakup regime, droplet size, droplet velocity, and spray angle for liquid flow rates of 0.75-15.0 μL/min. A micro-APCI source with the GDVN nebulizer behaved as a mass-flow-sensitive detector and provided stable and intense analyte signals. Compared to a classical APCI source, an order of magnitude lower detection limit for verapamil was achieved. Mass spectra recorded with the nebulizer in dripping and jetting modes were almost identical and did not differ from normal APCI spectra. Clogging never occurred during the experiments, indicating the high robustness of the nebulizer. Low-flow-rate APCI and APPI sources with a GDVN sprayer promise new applications for low- and medium-polar analytes.
  28. ACS Meas Sci Au. 2022 Aug 17. 2(4): 361-369
      Ion mobility spectrometry coupled to mass spectrometry (IMS-MS) is slowly becoming a more integral part in omics-based workflows. With the recent technological advancements in IMS-MS instrumentation, particularly those involving traveling wave-based separations, ultralong pathlengths have become readily available in commercial platforms (e.g., Select Series Cyclic IMS from Waters Corporation and MOBIE from MOBILion). However, a tradeoff exists in such ultralong pathlength separations: increasing peak-to-peak resolution at the cost of lower signal intensities and thus poorer sensitivity of measurements. Herein, we explore the utility of temporal compression, where ions are compressed in the time domain, following high-resolution cyclic ion mobility spectrometry-mass spectrometry-based separations on a commercially available, unmodified platform. We assessed temporal compression in the context of various separations including those of reverse sequence peptide isomers, chiral noncovalent complexes, and isotopologues. From our results, we demonstrated that temporal compression improves IMS peak intensities by up to a factor of 4 while only losing ∼5 to 10% of peak-to-peak resolution. Additionally, the improvement in peak quality and signal-to-noise ratio was evident when comparing IMS-MS separations with and without a temporal compression step performed. Temporal compression can readily be implemented in existing traveling wave-based IMS-MS platforms, and our initial proof-of-concept demonstration shows its promise as a tool for improving peak shapes and peak intensities without sacrificing losses in resolution.
  29. Methods Mol Biol. 2023 ;2628 301-320
      Extracellular vesicles (EVs) are naturally occurring membranous particles that can be isolated from blood and other biofluids. EVs have drawn considerable attention for their potential as a minimally invasive biomarker source for a range of conditions, based on tissue-specific expression of proteins and other molecular information. To promote robust characterization of EV isolates, the International Society for Extracellular Vesicles (ISEV) has established consensus minimal requirements for the study of extracellular vesicles (MISEV) reporting guidelines. A core element of MISEV guidance is the recommendation for the analysis of protein markers in samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the sample matrix. Furthermore, there is growing interest in circulating EVs enriched for tissue-specific origin, and in this context, the degree of nontarget EV "contamination" (e.g., EVs derived from blood cells) may inform assessment of sample purity. The increasing application of EVs as a liquid biopsy for clinical applications requires a high-throughput multiplexed approach that enables analysis of protein markers from small volumes of starting material, ideally utilizing the same platform for measuring biomarkers of interest. To this end, targeted liquid chromatography mass spectrometry using multiple reaction monitoring (LC-MRM-MS) is a key platform for the quantitative assessment of target proteins within EV samples. Here we describe a protocol for the isolation of EVs from blood and parallel analytical methods targeting general EV markers and blood cell-derived EV markers, along with guidance of best practice for sample collection and processing.
    Keywords:  EV characterization; Extracellular vesicles; Liquid chromatography tandem mass spectrometry; Multiple reaction monitoring; Plasma; Protein markers; Serum
  30. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Feb 04. pii: S1570-0232(23)00034-X. [Epub ahead of print]1217 123624
      Retention time (RT) can provide orthogonal information different from that of mass spectrometry and contribute to identifying compounds. Many machine learning methods have been developed and applied to RT prediction. In application, the training data size is usually small in most chromatography systems. To enhance the performance of RT prediction, this study proposes a RT prediction method based on multi-data combinations and adaptive neural network (MDC-ANN). MDC-ANN establishes the RT prediction model for the target chromatographic system through transfer learning and a base deep learning model trained on a big dataset. It selects the optimal molecular representation combination from the multiple input candidates and automatically determines the neural network structure according to the determined input combination. MDC-ANN was compared with two new efficient deep learning methods, three transferring methods and four popular machine learning methods on 14 small datasets and showed advantages in MAE, MedAE, MRE and R2 in most cases. The experiment results illustrated that integrating multiple molecular representations can provide more information, improve the performance of RT prediction and contribute to compound annotation, different chromatographic systems may use different molecular representation combinations to obtain good RT prediction performance. Hence, MDC-ANN which automatically determines the best combination of molecular representations for a specific system is promising for predicting RTs accurately in real applications.
    Keywords:  Compound Annotation; Multi-Data Combinations; Neural Network; Retention Time Prediction; Transfer Learning