bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒06‒06
27 papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chim Acta. 2021 Jul 11. pii: S0003-2670(21)00436-0. [Epub ahead of print]1168 338610
      Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM).
    Keywords:  Boldenone; Doping control; Isotope ratio mass spectrometry; Multi-dimensional liquid chromatography; Steroids; Testosterone
  2. Front Chem. 2021 ;9 674265
      Hair is a unique biological matrix that adsorbs short-term exposures (e. g., environmental contaminants and personal care products) on its surface and also embeds endogenous metabolites and long-term exposures in its matrix. In this work, we developed an untargeted metabolomics workflow to profile both temporal exposure chemicals and endogenous metabolites in the same hair sample. This analytical workflow begins with the extraction of short-term exposures from hair surfaces through washing. Further development of mechanical homogenization extracts endogenous metabolites and long-term exposures from the cleaned hair. Both solutions of hair wash and hair extract were analyzed using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics for global-scale metabolic profiling. After analysis, raw data were processed using bioinformatic programs recently developed specifically for exposome research. Using optimized experimental conditions, we detected a total of 10,005 and 9,584 metabolic features from hair wash and extraction samples, respectively. Among them, 274 and 276 features can be definitively confirmed by MS2 spectral matching against spectral library, and an additional 3,356 and 3,079 features were tentatively confirmed as biotransformation metabolites. To demonstrate the performance of our hair metabolomics, we collected hair samples from three female volunteers and tested their hair metabolic changes before and after a 2-day exposure exercise. Our results show that 645 features from wash and 89 features from extract were significantly changed from the 2-day exposure. Altogether, this work provides a novel analytical approach to study the hair metabolome and exposome at a global scale, which can be implemented in a wide range of biological applications for a deeper understanding of the impact of environmental and genetic factors on human health.
    Keywords:  endogenous metabolites; exposome; hair metabolomics; liquid chromatography-mass spectrometry; long-term exposure; short-term exposures
  3. Metabolites. 2021 May 11. pii: 305. [Epub ahead of print]11(5):
      Lipids are essential cellular constituents that have many critical roles in physiological functions. They are notably involved in energy storage and cell signaling as second messengers, and they are major constituents of cell membranes, including lipid rafts. As a consequence, they are implicated in a large number of heterogeneous diseases, such as cancer, diabetes, neurological disorders, and inherited metabolic diseases. Due to the high structural diversity and complexity of lipid species, the presence of isomeric and isobaric lipid species, and their occurrence at a large concentration scale, a complete lipidomic profiling of biological matrices remains challenging, especially in clinical contexts. Using supercritical fluid chromatography coupled with high-resolution mass spectrometry, we have developed and validated an untargeted lipidomic approach to the profiling of plasma and blood. Moreover, we have tested the technique using the Dry Blood Spot (DBS) method and found that it allows for the easy collection of blood for analysis. To develop the method, we performed the optimization of the separation and detection of lipid species on pure standards, reference human plasma (SRM1950), whole blood, and DBS. These analyses allowed an in-house lipid data bank to be built. Using the MS-Dial software, we developed an automatic process for the relative quantification of around 500 lipids species belonging to the 6 main classes of lipids (including phospholipids, sphingolipids, free fatty acids, sterols, and fatty acyl-carnitines). Then, we compared the method using the published data for SRM 1950 and a mouse blood sample, along with another sample of the same blood collected using the DBS method. In this study, we provided a method for blood lipidomic profiling that can be used for the easy sampling of dry blood spots.
    Keywords:  Dry Blood Spot 4; Lipidomic 1; Plasma 3; Supercritical Fluids Chromatography 3
  4. Molecules. 2021 May 18. pii: 2989. [Epub ahead of print]26(10):
      The application of internal standards in quantitative and qualitative bioanalysis is a commonly used procedure. They are usually isotopically labeled analogs of the analyte, used in quantitative LC-MS analysis. Usually, 2H, 13C, 15N and 18O isotopes are used. The synthesis of deuterated isotopologues is relatively inexpensive, however, due to the isotopic effect of deuterium and the lack of isotopologue co-elution, usually they are not considered as good internal standards for LC-MS quantification. On the other hand, the preparation of 13C, 15N and 18O containing standards of drugs and their metabolites requires a complicated multistep de novo synthesis, starting from the isotopically labeled substrates, which are usually expensive. Therefore, there is a strong need for the development of low-cost methods for isotope-labeled standard preparations for quantitative analysis by LC-MS. The presented review concentrates on the preparation of deuterium-labeled standards by hydrogen-deuterium exchange reactions at the carbon centers. Recent advances in the development of the methods of isotopologues preparation and their application in quantitative analysis by LC-MS are evaluated.
    Keywords:  N-methylated amino acids; hydrogen−deuterium exchange; liquid chromatography-mass spectrometry; quantitative LC-MS analysis
  5. Anal Chem. 2021 Jun 01.
      13C-isotope tracing is a frequently employed approach to study metabolic pathway activity. When combined with the subsequent quantification of absolute metabolite concentrations, this enables detailed characterization of the metabolome in biological specimens and facilitates computational time-resolved flux quantification. Classically, a 13C-isotopically labeled sample is required to quantify 13C-isotope enrichments and a second unlabeled sample for the quantification of metabolite concentrations. The rationale for a second unlabeled sample is that the current methods for metabolite quantification rely mostly on isotope dilution mass spectrometry (IDMS) and thus isotopically labeled internal standards are added to the unlabeled sample. This excludes the absolute quantification of metabolite concentrations in 13C-isotopically labeled samples. To address this issue, we have developed and validated a new strategy using an unlabeled internal standard to simultaneously quantify metabolite concentrations and 13C-isotope enrichments in a single 13C-labeled sample based on gas chromatography-mass spectrometry (GC/MS). The method was optimized for amino acids and citric acid cycle intermediates and was shown to have high analytical precision and accuracy. Metabolite concentrations could be quantified in small tissue samples (≥20 mg). Also, we applied the method on 13C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and 13C-isotope enrichments in a single 13C-isotopically labeled sample.
  6. J Chromatogr A. 2021 May 24. pii: S0021-9673(21)00394-0. [Epub ahead of print]1651 462270
      The development of a chiral separation strategy has always been a challenge of crucial importance, particularly in the pharmaceutical field. Chromatographic methods have become popular, particularly High Performance Liquid Chromatography and Supercritical Fluid Chromatography from a preparative scale point of view. A bioactive compound bearing three stereogenic centers was entrusted in our laboratory and the aim of this work was to obtain the complete resolution of the eight stereoisomers. Nine different polysaccharide-based columns were tested in SFC under various carbon dioxide-based mobile phases. The use of a single chiral column Lux Cellulose-2 under 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C, 120 bar outlet pressure allowed the obtention of eight peaks. To further improve the resolution of the two last isomers, two columns were serially coupled . The results obtained with the six different combinations are discussed. The tandem column supercritical fluid chromatography has demonstrated to be a useful technique to resolve the eight stereoisomers on Lux Cellulose-2//Cellulose-2 tandem of coupled columns with 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C and 120 bar outlet pressure, despite a long analysis time. In order to compare the two methods (i.e supercritical and liquid), chiral liquid chromatography under polar aqueous-organic mode, polar organic mode and normal-phase mode, was implemented. The last mode allowed the full baseline resolution of the eight isomers on Cellulose-5 CSP, with 20% 2-PrOH in n-heptane at a flow-rate of 0.8 mL/min, at 25°C, λ = 220 nm. The limits of detection and of quantification were determined for this method and the best values obtained for isomer 8 were equal to 2.84 and 9.37 nM respectively. Finally, a small-scale preparative separation of the multiple chiral centers compound was implemented on Cellulose-5 CSP within 10% 2-PrOH in n-heptane in order to study the stereoisomer elution order on Cellulose-2, Cellulose-5 and Chiralpak AD-H, under EtOH or 2-PrOH in n-heptane mobile phases, and partial reversal elution orders were observed.
    Keywords:  Multiple chiral centers compound; Normal-phase mode; Polar aqueous-organic mode; Polar organic mode; Tandem-coupling columns
  7. Metabolites. 2021 May 05. pii: 295. [Epub ahead of print]11(5):
      In order to increase metabolite coverage in LC-MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume, or manipulation of the samples after the first analysis, potentially leading to loss of analytes. When sample material is precious, the number of analyses that can be performed is limited. To that end, an automated single-injection LC-MS method for sequential analysis of both the hydrophilic and lipophilic fractions of biological samples is described. Early eluting compounds in a HILIC separation are collected on a trap column and subsequently analyzed in the RPLC mode. The instrument configuration, composed of commercially available components, allows easy modulation of the dilution ratio of the collected effluent, with sufficient dilution to obtain peak compression in the RPLC column. Furthermore, the method is validated and shown to be fit for purpose for application in untargeted metabolomics. Repeatability in both retention times and peak areas was excellent across over 140 injections of protein-precipitated blood plasma. Finally, the method has been applied to the analysis of real perilymph samples collected in a guinea pig model. The QC sample injections clustered tightly in the PCA scores plot and showed a high repeatability in both retention times and peak areas for selected compounds.
    Keywords:  LC–MS; automated; chemical coverage; sequential columns; untargeted metabolomics
  8. Diagnostics (Basel). 2021 May 26. pii: 962. [Epub ahead of print]11(6):
      Metabolic alteration plays a functional role in kidney allograft complications. Metabolomics is a promising high-throughput approach in nephrology but is still limited by the lack of overlap in metabolite coverage. We performed an untargeted fecal metabolomic analysis of forty stable kidney allograft recipients and twenty non-transplant controls. First, we applied the ultra-high performance liquid chromatography (UHPLC) analysis coupled with the Diod Array detector. The potential biomarkers were then collected and identified by gas chromatography-mass spectrometry (GCMS). In order to allow for complete coverage of the fecal polar and non-polar metabolites, the performance of five organic solvents with increasing polarity was investigated successively. UHPLC analysis revealed that the fecal metabolite profiles following the five extractions were significantly different between controls and kidney allografts. GC-MS analysis showed that the best predictors' metabolites belonged mainly to long-chain fatty acids, phenolic compounds, and amino acids. Collectively, our results showed the efficiency of our pioneer method to successfully discriminate stable kidney-transplant recipients from controls. These findings suggest that distinct metabolic profiles mainly affect fatty acid biosynthesis and amino acid metabolism. In such a context, the novel insights into metabolomic investigation may be a valuable tool that could provide useful new relevant biomarkers for preventing kidney transplant complications.
    Keywords:  biomarkers; kidney transplantation; metabolites; metabolomics
  9. Metabolites. 2021 May 13. pii: 313. [Epub ahead of print]11(5):
      Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell's metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.
    Keywords:  HILIC chromatography; NADH; NADPH; glutathione; mass-spectrometry method; redox metabolite detection in mammalian cells; redox metabolites
  10. Life (Basel). 2021 May 31. pii: 512. [Epub ahead of print]11(6):
      Nicotinamide adenine dinucleotide (NAD+) and its metabolome (NADome) play important roles in preserving cellular homeostasis. Altered levels of the NADome may represent a likely indicator of poor metabolic function. Accurate measurement of the NADome is crucial for biochemical research and developing interventions for ageing and neurodegenerative diseases. In this mini review, traditional methods used to quantify various metabolites in the NADome are discussed. Owing to the auto-oxidation properties of most pyridine nucleotides and their differential chemical stability in various biological matrices, accurate assessment of the concentrations of the NADome is an analytical challenge. Recent liquid chromatography mass spectrometry (LC-MS) techniques which overcome some of these technical challenges for quantitative assessment of the NADome in the blood, CSF, and urine are described. Specialised HPLC-UV, NMR, capillary zone electrophoresis, or colorimetric enzymatic assays are inexpensive and readily available in most laboratories but lack the required specificity and sensitivity for quantification of human biological samples. LC-MS represents an alternative means of quantifying the concentrations of the NADome in clinically relevant biological specimens after careful consideration of analyte extraction procedures, selection of internal standards, analyte stability, and LC assays. LC-MS represents a rapid, robust, simple, and reliable assay for the measurement of the NADome between control and test samples, and for identifying biological correlations between the NADome and various biochemical processes and testing the efficacy of strategies aimed at raising NAD+ levels during physiological ageing and disease states.
    Keywords:  NAD+; ageing; biomarker; nicotinamide; plasma
  11. Molecules. 2021 May 06. pii: 2737. [Epub ahead of print]26(9):
      Mass spectrometry imaging is a powerful tool for analyzing the different kinds of molecules in tissue sections, but some substances cannot be measured easily, due to their physicochemical properties. In such cases, chemical derivatization could be applied to introduce the charge into the molecule and facilitate its detection. Here, we study cholesterol derivatization with betaine aldehyde from tissue slices and evaluate how different sample preparation methods influence the signal from the derivatization product. In this study, we have tested different solutions for betaine aldehyde, different approaches to betaine aldehyde deposition (number of layers, deposition nozzle height), and different MALDI matrices for its analysis. As a result, we proved that the proposed approach could be used for the analysis of cholesterol in different tissues.
    Keywords:  betaine aldehyde; cholesterol; mass spectrometry (MS); mass spectrometry imaging (MSI)
  12. J Proteome Res. 2021 May 31.
      Recently, the gut microbiota has been found to be associated with many diseases, such as inflammatory bowel disease, depression, Parkinson's disease, cancer, metabolic syndrome, and cardiovascular disease (CVD). Among various gut microbiota-derived metabolites (GMs), short-chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) metabolites are the most frequently discussed metabolites. LC-MS/MS shows advantages in quantifying the levels of metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time-consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with CVD. The results showed a significant reduction in plasma butyrate levels in CVD patients compared with healthy controls, suggesting its potentially protective role in CVD. In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.
    Keywords:  LC−MS; SCFAs; bile acids; cardiovascular disease; derivatization; gut metabolites
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jun 30. pii: S1570-0232(21)00249-X. [Epub ahead of print]1176 122768
      Favipiravir is a broad-spectrum inhibitor of viral RNA polymerase. It is currently used as a possible treatment for coronavirus disease 2019 (COVID-19). Pre-clinical or clinical trials of favipiravir require robust, sensitive, and accurate bioanalytical methods for quantitation of favipiravir levels. Recently, several studies have been reported about developing a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring favipiravir levels. However, these methods were validated predominantly for plasma samples, electrospray ionization was operated only in negative or positive mode, and clinical application of these methods has not been applied for patients with COVID-19. This study aimed was to develop a validated LC-MS/MS method for the measurement of favipiravir levels in positive and negative electrospray ionization mode and to perform a pilot study in patients with COVID-19 receiving favipiravir to demonstrate the applicability of this method in biological samples. Simple protein precipitation was used for the extraction of favipiravir from the desired matrix. Favipiravir levels were quantitated using MS / MS with an electrospray ionization source in positive and negative multiple reaction monitoring (MRM) mode. The chromatographic detection was performed on a reverse-phase Phenomenex C18 column (50 mm × 4.6 mm, 5 µm, 100 Å) with gradient elution using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase. The method was linear over the concentration ranges of 0.048-50 µg/mL (in negative ionization mode) and 0.062-50 µg/mL (in positive ionization mode) with a correlation coefficient (r2) better than 0.998. The total run time was 3.5 min. The intra-assay and inter-assay %CV values were less than 7.2% and 8.0%, respectively. A simple, rapid and robust LC-MS / MS method was developed for the measurement of favipiravir and validation studies were performed. The validated method was successfully applied for drug level measurement in COVID-19 patients receiving favipiravir.
    Keywords:  COVID-19; Favipiravir; Tandem mass spectrometry; Validation
  14. Metabolites. 2021 May 11. pii: 303. [Epub ahead of print]11(5):
      Metabolomics plays an important role in various fields from health to agriculture. However, the comprehensive quantitative metabolomic analysis of plants and plant metabolites has not been widely performed. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based plant metabolomics offers the sensitivity and breadth of coverage for both phenotyping and disease diagnosis of plants. Here, we report a high-coverage and quantitative MS-based assay for plant metabolite analysis. The assay detects and quantifies 206 primary and secondary plant metabolites, including many key plant hormones. In total, it measures 28 amino acids and derivatives, 27 organic acids, 20 biogenic amines and derivatives, 40 acylcarnitines, 90 phospholipids and C-6 sugars. All the analysis methods in this assay are based on LC-MS/MS techniques using both positive and negative-mode multiple reaction monitoring (MRM). The recovery rates of spiked plant samples at three different concentration levels (low, medium and high) ranged from 80% to 120%, with satisfactory precision values of less than 20%. This targeted plant metabolomic assay has been successfully applied to the analysis of large numbers of pine and spruce needle samples, canola root samples, as well as cannabis samples. Moreover, the assay was specifically developed in a 96-well plate format, which enables automated, high-throughput sample analysis. This assay has already been used to analyze over 1500 crop plant samples in less than two months.
    Keywords:  LC-MS/MS; cannabis buds; canola roots; conifer needles; crop plant; plant metabolomics
  15. J Chromatogr A. 2021 Apr 02. pii: S0021-9673(21)00257-0. [Epub ahead of print]1651 462133
      Aminoglycosides are mostly used as veterinary antibiotics. In France, their consumption accounts for about 10% of all prescribed animal medicine. Due to their high polarity nature (log Kow < -3), they require chromatographic separation by hydrophilic interaction liquid chromatography or ion-pairing chromatography. This study presents the development of an ion pairing liquid chromatography with alkanesulfonates coupled to tandem mass spectrometry for the analysis of 10 aminoglycosides (spectinomycin, streptomycin, dihydrostreptomycin, kanamycin, apramycin, gentamicin, neomycin and sisomicin) in wastewater samples. The novelty of this method lies in the addition of the ion paring salt directly and only into the sample vial and not in the mobile phase, lowering the amount of salt added and consequently reducing signal inhibition. The optimized method was validated and showed satisfactory resolution, performances suitable with the analysis of aminoglycosides in wastewater samples, with limits of quantifications less than 10 ng/mL for most of the compounds, low matrix effects, high accuracy (85%-115% recoveries) and reproducibility (2%-12%RSD). It was then applied successfully to raw and treated wastewater samples.
    Keywords:  Aminoglycosides; Ion-pairing chromatography; Wastewater
  16. Metabolites. 2021 May 04. pii: 294. [Epub ahead of print]11(5):
      Lipidomic approaches are widely used to investigate the relationship between lipids, human health, and disease. Conventional sample preparation techniques for the extraction of lipids from biological matrices like human plasma are based on liquid-liquid extraction (LLE). However, these methods are labor-intensive, time-consuming, and can show poor reproducibility and selectivity on lipid extraction. A novel, solid-phase extraction (SPE) approach was demonstrated to extract lipids from human plasma using a lipid extraction SPE in both cartridge and 96-well-plate formats, followed by analysis using a combination of targeted and untargeted liquid chromatography/mass spectrometry. The Lipid Extraction SPE method was compared to traditional LLE methods for lipid class recovery, lipidome coverage, and reproducibility. The novel SPE method used a simplified protocol with significant time and labor savings and provided equivalent or better qualitative and quantitative results than traditional LLE methods with respect to several critical performance metrics; recovery, reproducibility, and lipidome coverage.
    Keywords:  SPE; lipidomics; lipids; liquid chromatography; mass spectrometry; sample preparation
  17. Pharmaceuticals (Basel). 2021 May 13. pii: 460. [Epub ahead of print]14(5):
      Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration-effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines' requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.
    Keywords:  NRTIs; liquid chromatography; nucleoside analogues; tandem mass spectrometry; tenofovir alafenamide
  18. Int J Comput Assist Radiol Surg. 2021 May 29.
      PURPOSE: Intraoperative assessment of surgical margins is important for reducing the rate of revisions in breast conserving surgery for palpable malignant tumors. The hypothesis was that metabolomics methods, based on mass spectrometry, could find patterns of relative abundances of molecules that distinguish clusters of benign tissue and cancer in surgical resections.METHODS: Excisions from 8 patients were used to acquire 112,317 mass spectrometry signals by desorption electrospray ionization. A process of nonnegative matrix factorization and graph decomposition produced clusters that were approximated as affine spaces. Each signal's distance to the affine space of a cluster was used to visualize the clustering.
    RESULTS: The distance maps were superior to binary clustering in identifying cancer regions. They were particularly effective at finding cancer regions that were discontinuously distributed within benign tissue.
    CONCLUSIONS: Desorption electrospray ionization mass spectrometry, which has been shown to be useful intraoperatively, can acquire signals that distinguish malignant from benign breast tissue in surgically excised tumors. The method may be suitable for real-time surgical decisions based on cancer margins.
    Keywords:  Breast cancer; Mass spectrometry imaging; Metabolomics
  19. Molecules. 2021 May 05. pii: 2715. [Epub ahead of print]26(9):
      Untargeted metabolomics based on liquid chromatography coupled with mass spectrometry (LC-MS) can detect thousands of features in samples and produce highly complex datasets. The accurate extraction of meaningful features and the building of discriminant models are two crucial steps in the data analysis pipeline of untargeted metabolomics. In this study, pure ion chromatograms were extracted from a liquor dataset and left-sided colon cancer (LCC) dataset by K-means-clustering-based Pure Ion Chromatogram extraction method version 2.0 (KPIC2). Then, the nonlinear low-dimensional embedding by uniform manifold approximation and projection (UMAP) showed the separation of samples from different groups in reduced dimensions. The discriminant models were established by extreme gradient boosting (XGBoost) based on the features extracted by KPIC2. Results showed that features extracted by KPIC2 achieved 100% classification accuracy on the test sets of the liquor dataset and the LCC dataset, which demonstrated the rationality of the XGBoost model based on KPIC2 compared with the results of XCMS (92% and 96% for liquor and LCC datasets respectively). Finally, XGBoost can achieve better performance than the linear method and traditional nonlinear modeling methods on these datasets. UMAP and XGBoost are integrated into KPIC2 package to extend its performance in complex situations, which are not only able to effectively process nonlinear dataset but also can greatly improve the accuracy of data analysis in non-target metabolomics.
    Keywords:  KPIC2; LC–MS; Pure Ion Chromatogram; UMAP; XGBoost
  20. Pharmaceuticals (Basel). 2021 May 15. pii: 466. [Epub ahead of print]14(5):
      Paracetamol (acetaminophen) (PAR), caffeine (CAF) and tramadol hydrochloride (TRA) are important drugs widely used for many clinical purposes. Determination of their contents is of the paramount interest. In this respect, a quick, simple and sensitive isocratic RP-HPLC method with photodiode array detection was developed for the determination of paracetamol, caffeine and tramadol in pharmaceutical formulations. An improved sensitive procedure was also evolved for tramadol using a fluorescence detector system. A C18 column and a mobile phase constituted by methanol/phosphate were used. LODs were found to be 0.2 μg/mL, 0.1 μg/mL and 0.3 μg/mL for paracetamol, caffeine and tramadol hydrochloride, respectively, using photodiode-array detection. Alternatively, LOD for tramadol decreased to 0.1 μg/mL with the fluorescence detector. Other notable analytical figures of merit include the linear concentration ranges, 0.8-270 μg/mL, 0.4-250 μg/mL and 1.0-300 (0.2-40) μg/mL, for the same ordered analytes (including the fluorescence detector). The proposed method was successfully applied for the quantitative determination of the three drugs in tablet dosage forms.
    Keywords:  HPLC; caffeine; paracetamol; photodiode-array; tramadol
  21. Bioanalysis. 2021 Jun 03.
      Aim: Quantification of stereoisomers in biological matrices is of pivotal importance for drug development. Supercritical fluid chromatography paired with chiral stationary phases is the gold standard for resolution of enantiomers. However, this technique often proves inadequate for resolution of polar stereoisomers. Materials & methods: A combination of achiral chemical derivatization with supercritical fluid chromatography using chiral stationary columns to improve enantiomeric resolution is described. Results: Separation of four possible stereoisomers of linerixibat was achieved after derivatization with 3N HCl in n-butanol within 12 min (case1). Derivatization with acetic, propionic, butyric, isobutyric, valeric and isovaleric anhydrides significantly improved the separation of stereoisomers (case 2 and 3) within 10 min. The best stereoisomeric resolution was achieved using valeric and isovaleric anhydrides.
    Keywords:  GSK2330672; GSK3326595; chemical derivatization; chiral inversion; chromatographic separation; polar compounds; supercritical fluid chromatography
  22. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jun 30. pii: S1570-0232(21)00227-0. [Epub ahead of print]1176 122747
      Pharmaceutical compounds ingested by humans are metabolized and excreted in urine and feces. These metabolites can be quantified in wastewater networks using wastewater-based epidemiology (WBE) methods. Standard WBE methods focus on samples collected at wastewater treatment plants (WWTPs). However, these methods do not capture more labile classes of metabolites such as glucuronide conjugates, products of the major phase II metabolic pathway for drug elimination. By shifting sample collection more upstream, these unambiguous markers of human exposure are captured before hydrolysis in the wastewater network. In this paper, we present an HPLC-MS/MS method that quantifies 8 glucuronide conjugates in addition to 31 parent and other metabolites of prescription and synthetic opioids, overdose treatment drugs, illicit drugs, and population markers. Calibration curves for all analytes are linear (r2 > 0.98), except THC (r2 = 0.97), and in the targeted range (0.1-1,000 ng mL-1) with lower limits of quantification (S/N = 9) ranging from 0.098 to 48.75 ng mL-1. This method is fast with an injection-to-injection time of 7.5 min. We demonstrate the application of the method to five wastewater samples collected from a manhole in a city in eastern Massachusetts. Collected wastewater samples were filtered and extracted via solid-phase extraction (SPE). The SPE cartridges are eluted and concentrated in the laboratory via nitrogen-drying. The method and case study presented here demonstrate the potential and application of expanding WBE to monitoring labile metabolites in upstream wastewater networks.
    Keywords:  Epidemiology; Glucuronide; HPLC-MS/MS; Metabolite; Opioid; Sewage; Wastewater-based
  23. J Chromatogr A. 2021 May 24. pii: S0021-9673(21)00397-6. [Epub ahead of print]1651 462273
      This study presents an accurate and precise analytical strategy for the determination of chloroquine phosphate at trace levels in human body fluids (urine, serum, and saliva). Simultaneous derivatization-spraying based fine droplet formation-liquid phase microextraction (SD-SFDF-LPME) method was used to derivatize and preconcentrate the analyte prior to gas chromatography-mass spectrometry (GC-MS) measurements. Acetic anhydride was employed as derivatizing agent in this study. After optimizing the SD-SFDF-LPME method, the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.16 and 0.53 mg/kg, respectively. Quadruple isotope dilution (ID4) was coupled to the SD-SFDF-LPME method in order to alleviate matrix effects and promote accuracy/precision of the method. Chloroquine acetamide-d3 was firstly synthesized in our research laboratory and used as the isotopic analogue of the analyte in the ID4 experiments. Superior percent recovery results (99.4% - 101.0%) with low standard deviation values were obtained for the spiked samples. This validated the developed SD-SFDF-LPME-ID4-GC-MS method as highly accurate and precise for the determination of chloroquine phosphate at trace levels. In addition, the isotopic analogue of the analyte was obtained via the acetamide derivative of the analyte, which is an alternative to obtain isotopic analogues of organic compounds that are not accessible or commercially available.
    Keywords:  Chloroquine phosphate; Gas chromatography mass spectrometry; Liquid phase microextraction; Quadruple isotope dilution
  24. Talanta. 2021 Sep 01. pii: S0039-9140(21)00359-3. [Epub ahead of print]232 122438
      UPLC-MS/MS methods are the gold standard for routine, high-throughput measurements of biogenic monoamines for the diagnosis of catecholamine-producing tumors. However, this cannot be achieved without employing efficient sample pretreatment methods. Therefore, two pretreatment methods, thin-film solid phase microextraction (TF-SPME) and packed fibers solid phase extraction (PFSPE), were developed and evaluated for the analysis of biogenic monoamines and their metabolites in urine. A hydrophilic-lipophilic balance (HLB) coating was chosen for the thin-film blade format SPME method and compared with a Polycrown ether (PCE) composite nanofiber used as an adsorbent for the PFSPE method. Under optimal conditions, the absolute extraction recovery and relative matrix effect of the newly developed TF-SPME method were determined to be 35.7-74.8% and 0.47-3.63%, respectively. The linearity was 0.25-500 ng mL-1 for norepinephrine, epinephrine, dopamine, normetanephrine 3-methoxytyramine, serotonin, histamine, and 0.1-500 ng mL-1 for metanephrine. The intra-and inter-assay coefficients of variation were 0.7-8.7%, and the respective accuracies were calculated to be 90.8-104.7% and 89.5-104.5% for TF-SPME. Compared with the PFSPE method, the TF-SPME method had a higher extraction efficiency, lower matrix effects and a wider linear range for eight target substances, which ensured higher accuracy of simultaneous detection of all compounds of interest. Therefore, the proposed TF-SPME method can be employed for the high throughput screening for neuroendocrine tumors in a routine clinical setting and other relative research by simultaneous quantitation of urine eight biological monoamines in a single run.
    Keywords:  Biogenic monoamines; Hydrophilic-lipophilic balanced polymeric particles; Liquid chromatography-tandem mass spectrometry; Solid phase microextraction; Urine
  25. J Proteome Res. 2021 Jun 04. 20(6): 3114-3123
      Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography-mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides.
    Keywords:  ion chromatography; phosphoinositides; targeted lipidomics
  26. J Pharm Biomed Anal. 2021 May 26. pii: S0731-7085(21)00285-5. [Epub ahead of print]203 114174
      The reported method aims to be a powerful aid for the simultaneous determination of tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and tetrahydrocannabivarin (THCV) in oily based preparations. The chromatographic separation was carried out using an Hypersil Gold PFP (50 × 2.1 mm, 1.9 μm) column, using H2O + 2 mM ammonium formate + 0.2 % formic acid (M1) and Methanol + 2 mM ammonium formate + 0.2 % formic acid (M2) as mobile phases. The flow rate was set 0.4 mL/min. Specifically, this method was validated in terms of linearity, limit of detections and quantifications (LODs and LOQs), accuracy (precision and trueness, both intra and interday), selectivity, and matrix effects. This procedure allowed quantifying seven phytocannabinoids in less than 10 min. The validated method shows a good linearity within the range 0.25-1000 ng/mL, while precision and trueness (intra- and inter-day) were below <13.25 % and 7.59 %, respectively. Regarding the matrix effect, the method satisfies all the requirements, except for the THC and THCV, where it reaches about 120 %. This element does not affect the method performances as it has been observed that this value is constant and reproducible and therefore does not involve errors in the quantitative analysis. The method was tested and applied on more 70 different oily based preparations. Furthermore, starting from four different cannabis cultivar (FM2, Bedrolite, Bedrocan, and Bediol), it allowed to evaluate the reproducibility of the magistrali preparations. The real samples, in fact, derive from different local pharmacies, and were analyzed by the accredited UNI CEI EN ISO/IEC 17025:2018, Pharmatoxicology Laboratory (ACCREDIA, lab n. 2274 ASLPE, accreditation number 1822 L), accordingly to the current regulations.
    Keywords:  Dilute and shoot procedure; LC–MS/MS; Oily based preparations; Pharmaceutical formulation; Pharmacotoxicological application; Phytocannabinoids
  27. Methods Mol Biol. 2021 ;2276 357-382
      Untargeted metabolomics has rapidly become a profiling method of choice in many areas of research, including mitochondrial biology. Most commonly, untargeted metabolomics is performed with liquid chromatography/mass spectrometry because it enables measurement of a relatively wide range of physiochemically diverse molecules. Specifically, to assess energy pathways that are associated with mitochondrial metabolism, hydrophilic interaction liquid chromatography (HILIC) is often applied before analysis with a high-resolution accurate mass instrument. The workflow produces large, complex data files that are impractical to analyze manually. Here, we present a protocol to perform untargeted metabolomics on biofluids such as plasma, urine, and cerebral spinal fluid with a HILIC separation and an Orbitrap mass spectrometer. Our protocol describes each step of the analysis in detail, from preparation of solvents for chromatography to selecting parameters during data processing.
    Keywords:  Accurate mass; Data-dependent acquisition; HILIC; High-resolution; Liquid chromatography; Mass spectrometry; Metabolites; Metabolomics; Profiling; Quality assurance; Quality control