bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒05‒01
27 papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Methods Mol Biol. 2022 ;2504 157-173
      Lipidomics is an omics approach to comprehensively study lipid profiles in biological samples, such as plasma, serum, urine, and tissue specimens. Moreover, lipidomic analyses are useful for identifying novel lipid biomarkers, especially for various metabolic and malignant diseases in humans. Extracellular vesicles (EVs) are lipid bilayer-encapsulated nanoparticles secreted from various cells into the extracellular space. In particular, circulating EVs in the blood stream have attracted considerable research interest as they are considered the fingerprint of the cells from which they are secreted and are a promising source for less-invasive biomarker screening. Here, we describe the entire workflow for the lipidomic analysis of circulating EVs, including the methods for their purification from human plasma and serum, liquid chromatography coupled with high-resolution mass spectrometry-based lipid measurement, and data analyses for profiling EV lipids. Using this methodological workflow, over 260 lipid molecules belonging to the glycerophospholipid and sphingolipid groups can be detected.
    Keywords:  Extracellular vesicles; Lipid biomarkers; Lipidomics; Liquid biopsy; Liquid chromatography coupled with high-resolution mass spectrometry
  2. STAR Protoc. 2022 Jun 17. 3(2): 101302
      We describe here a user-friendly analysis protocol for semi-targeted polar metabolomics in human muscle biopsies using Zwitterionic Hydrophilic Interaction Liquid Chromatography and high-resolution full-scan mass spectrometry. Previously, this protocol has been used for Caenorhabditis elegans. Here we show that it can be successfully applied to human muscle biopsies with minor adjustments. Summarized instructions for other matrices are also provided. As peak integration in metabolomics can be challenging, we provide expected retention times and extensive peak descriptions to aid this process. For complete details on the use and execution of this protocol, please refer to Molenaars et al. (2021).
    Keywords:  Health Sciences; Mass Spectrometry; Metabolism; Metabolomics
  3. Talanta. 2022 Apr 16. pii: S0039-9140(22)00270-3. [Epub ahead of print]245 123474
      Retinoic acids (RAs), an important class of fatty acid derived from vitamin A, are closely associated with various human diseases including cancer. Hence, determination of endogenous RAs would help to uncover the mechanisms underlying RAs-related diseases. However, accurate quantification of RAs is still a challenge due to their high structure similarity, low abundance in biological samples and the lack of isotope internal standards (ISs). In this study, a liquid chromatography-mass spectrometry (LC-MS) method with high-sensitivity and high-throughput was developed to simultaneously determine 5 RAs (all-trans-retinoic acid, 13-cis-retinoic acid, 9,13-cis-retinoic acid, all-trans-4-oxoretinoic acid and 4-hydroxy-retinoic acid) in human serum. In the method, three derivatization reagents, N, N-dimethylethylenediamine (DMED), d4-N,N-dimethylethylenediamine (d4-DMED) and 13C2-N,N-dimethylethylenediamine (13C2-DMED), were used for triple chemical derivatization of RAs, thus the detection sensitivity and analysis throughput of endogenous RAs could be achieved. Benefiting from the developed strategy, the analysis throughput was enhanced and the detection sensitivity of RAs was increased by 14-398 folds. The limits of quantification (LOQs) of RAs were found to be between 8.4 and 130 pg/mL, which were better than previously reported methods. Good linearities of RAs were obtained with determination coefficient (R2) ranging from 0.9774 to 0.9999. The intra- and inter-day relative standard deviations (RSDs) were below 11.7% and 12.7%, respectively, indicating the acceptable reproducibility of the method. Using the developed method, we successfully quantified 4 RAs (all-trans-retinoic acid, 13-cis-retinoic acid, 9,13-cis-retinoic acid and all-trans-4-oxoretinoic acid) in health controls and cancer patient serum samples. Furthermore, t-test analysis showed that the concentration of three RAs (all-trans-retinoic acid, 13-cis-retinoic acid and all-trans-4-oxoretinoic acid) in cancer patient serum samples were significantly decreased compared with health controls, which indicated that RAs might play an important role in the formation and development of cancer.
    Keywords:  Accurate quantification; Derivatization; Liquid chromatography-mass spectrometry; Retinoic acid
  4. Clin Biochem. 2022 Apr 25. pii: S0009-9120(22)00114-X. [Epub ahead of print]
      OBJECTIVE: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly selective and sensitive method for the quantification of kinase inhibitors, yet not widely available in clinical routine for therapeutic drug monitoring (TDM). To provide a more accessible alternative, a high-performance liquid chromatography method with ultraviolet/diode array detection (HPLC-UV/DAD) to quantify cabozantinib, dabrafenib, nilotinib and osimertinib, was developed and validated. Results were compared to LC-MS/MS.METHOD: After liquid-liquid-extraction and reconstitution of the residue in 20mM potassium dihydrogen phosphate (KH2PO4) (pH4.6), acetonitrile and methanol (50:25:25,v/v/v), chromatographic separation was achieved in 20.0 min using a Luna® C18(2)-HST column (100×2mm, 2.5μm), protected by a C18 guard column (4×2mm) (column temperature: 30°C, autosampler: 10°C). Mobile phase A and B consisted of 20mM KH2PO4 (pH4.9) and acetonitrile (9:1,v/v) and acetonitrile:20mM KH2PO4 (pH4.9) (7:3,v/v), respectively. Gradient elution was performed at 200 µL/min. Analytes were quantified at 250, 280 and 330 nm, using sorafenib as internal standard.
    RESULTS: Calibration curves were linear (35-2,000 ng/mL). Method validation assays met requirements by U.S. Food and Drug Administration and European Medicines Agency. Compared to the more sensitive and specific LC-MS/MS, HPLC-UV/DAD showed a good correlation and a strong positive association (Kendall's tau 0.811¬-0.963, p<0.05). Bland-Altman-plots revealed 100% (cabozantinib), 98.6% (dabrafenib), 98.6% (nilotinib) and 96.2% (osimertinib) of relative differences inside the limits of agreement. Regulatory agency criteria for sample reanalysis and cross validation were met (±20%-criterion:100% (cabozantinib), 94.3% (dabrafenib), 92% (nilotinib) and 84.6% (osimertinib).
    CONCLUSION: The developed HPLC-UV/DAD method is "fit-for-TDM" in clinical routine and serves as a genuine alternative to LC-MS/MS.
    Keywords:  HPLC-UV/DAD; TDM; cabozantinib; dabrafenib; nilotinib; osimertinib
  5. Methods Mol Biol. 2022 ;2484 3-12
      Metabolite profiling aiming at quantifying the metabolome of flowers is emerging as a suitable tool to understand the metabolic complexity of these reproductive organs and the associations between primary and secondary metabolites which characterize them. This chapter provides a general method for the combined analyses of primary and secondary metabolites via gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-mass spectrometry (LC-MS) of flower samples. We describe the preparatory steps, the procedure of metabolites' extraction and finally provide examples of data representation. The method described here can be applied to the analysis of metabolomes of entire flowers, as well as specific flower organs.
    Keywords:  Flower; GC-MS; LC-MS; Primary metabolites; Secondary metabolites
  6. Se Pu. 2022 May 08. 40(5): 433-442
      A high throughput screening method based on ultra performance liquid chromatography-quadrupole-time-of-flight high resolution mass spectrometry (UPLC-Q-TOF HRMS) was developed for the simultaneous and rapid confirmation of 73 prohibited compounds in cosmetics. The sample was dispersed in a saturated sodium chloride solution and ultrasonically extracted using acetonitrile containing 0.2% (v/v) formic acid. The resultant solution was centrifuged and then cleaned using dispersive solid phase extraction using a primary secondary amine (PSA) sorbent. The purified solution was centrifuged, and the supernatant was filtered through a 0.22 μm membrane before determination. The optimal pretreatment method was determined by comparing the recovery rates obtained using different extraction solvents and different amounts of purifying agents. The chromatographic separation conditions and mass spectrometry scanning mode were also optimized. Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) with gradient elution using 0.1% (v/v) formic acid aqueous solution and methanol as mobile phases. The eluent from the column was further detected using Q-TOF HRMS with the high resolution multiple reaction monitoring (MRM HR) scanning mode. Retention time, precise mass of parent ion, isotope abundance ratio, and precise mass of fragment ions were the parameters considered for rapid untargeted screening and confirmation. The matrix effects of water- and cream-based cosmetics were investigated. The matrix effects could be addressed using the matrix matched standard curve method. The correlation coefficients for the 73 prohibited compounds were all >0.99 in the corresponding linear concentration range. The limits of detection (LODs) were in the range of 5-150 μg/kg, and the limits of quantification (LOQs) were in the range of 15-450 μg/kg. Average recoveries were in the range of 60.3%-130.3% at three spiked levels, and the intra-day and inter-day precisions were 0.8%-10.0% (n=6) and 1.1%-15.0% (n=3), respectively. A total of 692 cosmetics samples were screened; 16 positive samples were detected, namely, sulfamethoxazole, meprednisone, lincomycin, 4-acetamidophenol, trimethoprim, alfacalcidol, betamethasone 17-valerate, brimonidine, chloramphenicol, chlorpheniramine, clobetasol propionate, crotamiton, econazole, ketoconazole, prednisone 21-acetate, and prednisone, with content in the range of 0.5-1136.1 mg/kg. The optimized method is accurate, fast, and simple, and it is suitable for the routine detection and rapid screening of common prohibited compounds in cosmetics. In addition, a screening and confirmation library was established for the 650 prohibited compounds using SCIEX OS and Library View software, using information-dependent acquisition (IDA)-MS/MS mode for MS data acquisition. The database contains multiple types of information, including formulas, theoretical exact mass, retention time, precise mass of parent ion, isotope abundance ratio, and fragment ion distribution. The library can be used for the simultaneous and rapid confirmation of prohibited compounds in cosmetics.
    Keywords:  cosmetics; prohibited compounds; quadrupole-time-of-flight high resolution mass spectrometry (Q-TOF HRMS); ultra performance liquid chromatography (UPLC)
  7. J Pharm Biomed Anal. 2022 Apr 16. pii: S0731-7085(22)00193-5. [Epub ahead of print]215 114772
      A highly sensitive method was developed for the quantification of vinblastine, vincristine, vinorelbine, and its active metabolite 4-O-deacetylvinorelbine in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Deuterated isotopes were used as internal standard and liquid-liquid extraction with tert-butyl methyl ether (TBME) was used for sample pre-treatment. The final extract was injected on a C18 column (50 × 2.1 mm ID, 5 µm). Gradient elution was used in combination with Reversed Phase chromatography to elute the analytes and internal standards from the column in 5 min and the API4000 triple quadrupole MS detector was operating in the positive ion mode. The calibration model, accuracy and precision, selectivity and specificity, dilution integrity, carryover, matrix factor and recovery, and stability were evaluated over a concentration range from 0.025 to 10 ng/mL for vinblastine, vinorelbine, and 4-O-deacetylvinorelbine and from 0.1 to 40 ng/mL for vincristine. The intra- and inter-assay bias and precisions were within ± 12.4% and ≤ 10.6%, respectively. This method was successfully applied to study the pharmacokinetics of vincristine in paediatrics and vinorelbine and 4-O-deacetylvinorelbine using in vivo mouse models.
    Keywords:  Human plasma; LC-MS/MS; Pharmacokinetics; Vinca-alkaloids
  8. J Mass Spectrom Adv Clin Lab. 2022 Apr;24 50-56
      Introduction: Ion mobility-mass spectrometry (IM-MS) is an emerging technique in the -omics fields that has broad potential applicability to the clinical lab. As a rapid, gas-phase structure-based separation technique, IM-MS offers promise in isomer separations and can be easily combined with existing LC-MS methods (i.e., LC-IM-MS). Several experimental conditions, including analyte cation adducts and drift composition further provide a means to tune separations for global and/or targeted applications.Objectives: The primary objective of this study was to demonstrate the utility of IM-MS under a range of experimental conditions for detection of glucocorticoids, and specifically for the separation of several isomeric pairs.
    Methods: LC-IM-MS was used to characterize 16 glucocorticoids including three isomer pairs: cortisone/prednisolone, betamethasone/dexamethasone, and flunisolide/triamcinolone acetonide. Collision cross section (CCS) values were measured for all common adducts (e.g., protonated and sodiated) using both step-field and single-field methods. Alternative alkali, alkaline earth, and transition metals were introduced, such that their adducts could also be measured. Finally, four different drift gases (helium, nitrogen, argon, and carbon dioxide) were compared for their relative separation capability.
    Results: LC-IM-MS offered a robust, multidimensional separation technique that allowed for the 16 glucocorticoids to be analyzed and separated in three-dimensions (retention time, CCS, and m/z). Despite the relatively modest resolution of isomer pairs under standard conditions (i.e., nitrogen drift gas, sodiated ions, etc.), improvements were observed for alkaline earth and transition metals (notable barium adducts) and in carbon dioxide drift gas.
    Conclusion: In summary, LC-IM-MS offers potential as a clinical method due to its ease of coupling with traditional LC-MS methods and its promise for tuning separations to better resolve targeted and/or global isomers in complex biological samples.
    Keywords:  CCS, collision cross section; CIMS, cyclic ion mobility spectrometry; DMS, differential mobility spectrometry; DTIMS, drift tube ion mobility spectrometry; FAIMS, field asymmetric ion mobility spectrometry; Glucocorticoids; IM-MS, ion mobility-mass spectrometry; IMS, ion mobility spectrometry; Ion mobility spectrometry; Ion mobility-mass spectrometry; SLIM, structures for lossless ion manipulations; TIMS, trapped ion mobility spectrometry; TWIMS, traveling wave ion mobility spectrometry
  9. Ann Lab Med. 2022 Sep 01. 42(5): 531-557
      Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized in clinical laboratories because it has advantages in terms of specificity and sensitivity over other analytical technologies. These advantages come with additional responsibilities and challenges given that many assays and platforms are not provided to laboratories as a single kit or device. The skills, staff, and assays used in LC-MS/MS are internally developed by the laboratory, with relatively few exceptions. Hence, a laboratory that deploys LC-MS/MS assays must be conscientious of the practices and procedures adopted to overcome the challenges associated with the technology. This review discusses the post-development landscape of LC-MS/MS assays, including validation, quality assurance, operations, and troubleshooting. The content knowledge of LC-MS/MS users is quite broad and deep and spans multiple scientific fields, including biology, clinical chemistry, chromatography, engineering, and MS. However, there are no formal academic programs or specific literature to train laboratory staff on the fundamentals of LC-MS/MS beyond the reports on method development. Therefore, depending on their experience level, some readers may be familiar with aspects of the laboratory practices described herein, while others may be not. This review endeavors to assemble aspects of LC-MS/MS operations in the clinical laboratory to provide a framework for the thoughtful development and execution of LC-MS/MS applications.
    Keywords:  Calibration; Liquid chromatography-tandem mass spectrometry; Operations; Quality control; Troubleshooting; Validation; Verification
  10. Biochem Res Int. 2022 ;2022 9186536
      Metabolomics is a comprehensive analysis of metabolites existing in biological systems. As one of the important "omics" tools, the approach has been widely employed in various fields in helping to better understand the complex cellular metabolic states and changes. Bacterial metabolomics has gained a significant interest as bacteria serve to provide a better subject or model at systems level. The approach in metabolomics is categorized into untargeted and targeted which serves different paradigms of interest. Nevertheless, the bottleneck in metabolomics has been the sample or metabolite preparation method. A custom-made method and design for a particular species or strain of bacteria might be necessary as most studies generally refer to other bacteria or even yeast and fungi that may lead to unreliable analysis. The paramount aspect of metabolomics design comprises sample harvesting, quenching, and metabolite extraction procedures. Depending on the type of samples and research objective, each step must be at optimal conditions which are significantly important in determining the final output. To date, there are no standardized nor single designated protocols that have been established for a specific bacteria strain for untargeted and targeted approaches. In this paper, the existing and current developments of sample preparation methods of bacterial metabolomics used in both approaches are reviewed. The review also highlights previous literature of optimized conditions used to propose the most ideal methods for metabolite preparation, particularly for bacterial cells. Advantages and limitations of methods are discussed for future improvement of bacterial metabolomics.
  11. RSC Adv. 2021 Sep 01. 11(47): 29797-29806
      A rapid and selective LC-MS/MS method is described for the simultaneous assay of Avanafil and Dapoxetine in human plasma via a protein precipitation (PP) sample preparation technique. Tadalafil was chosen as the internal standard reaching good recovery and reproducibility while diminishing the effects of the matrix. An Agilent Zorbax Eclipse XDB C18 column (4.6 × 50 mm, 1.8 μm) was used for the chromatographic separation and analysis, while 0.1% formic acid : acetonitrile (60 : 40, v/v) was utilized at a flow rate of 0.5 mL min-1. It was revealed that 6 min stop time accomplished the best separation. The assay was linear over the range of 10-6000 ng mL-1 for both drugs. The established bio-analytical method validation was demonstrated following US-FDA recommendations including sensitivity, selectivity, linearity, accuracy and precision. Furthermore, other validation parameters were assessed such as the dilution integrity, matrix effect, carryover, and analyte stability during both short- and long-term sample processing and storage. The adopted method was efficaciously applied to a clinical study for the concurrent determination of Avanafil and Dapoxetine in human plasma.
  12. J Am Soc Mass Spectrom. 2022 Apr 28.
      Postcolumn infusion has been widely used to study the matrix effect of analytical methods based on liquid chromatography coupled to mass spectrometry (LC-MS). Nevertheless, this methodology is usually only applied during a method development or validation. With this application note, we aim to demonstrate that the continuous use of postcolumn infusion can be also a very useful tool to monitor the quality of LC-MS analyses and easily detect flaws in the analytical method performance. Here we propose a protocol that can be transferred to other LC-MS platforms, and we show some real situations in bioanalysis in which postcolumn infusion proved to be extremely helpful in, for example, the evaluation of a sample treatment or the detection of unexpected sources of the matrix effect.
  13. Biomed Chromatogr. 2022 Apr 26. e5387
      In this, we report the development and validation of an LC-MS/MS method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid-liquid extraction method. The chromatographic separation of bendamustine, copanlisib, and the IS was achieved on a Atlantis C18 column using an isocratic mobile phase (5 mM ammonium acetate:methanol; 20:80, v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively with a total run time of 2.5 min. The calibration curve ranged from 3.99-2996 and 4.33-3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intraday precision and accuracy, stability in processed samples and up on storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01-5.05 and 2.74-6.13% and 1.98-7.64 and 8.62-9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at -80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.
    Keywords:  Bendamustine; LC-MS/MS; copanlisib; method validation; mouse plasma; pharmacokinetics
  14. Se Pu. 2022 May 08. 40(5): 477-487
      With the increasing use of chlorine-containing pesticides, hypochlorous acid disinfection water as well as aquatic product insecticides and fungicides are widely used in the cultivation of fish. This has led to the contamination of fish by chlorophenol compounds. However, currently, there is no standard method for the simultaneous determination of 19 chlorophenol compounds in fish. In this study, the optimum chromatography and mass spectrometry conditions were determined by investigating the instrument parameters. The 19 chlorophenol compounds were well separated using the DB-5MS capillary chromatographic column (30 m×0.25 mm×0.25 μm) with a carrier gas flow rate of 1 mL/min. Under this condition, the chromatographic peak was sharp and symmetric. An analytical method was developed for the simultaneous determination of the 19 chlorophenol compounds in fish using gas chromatography-mass spectrometry coupled with QuEChERS pretreatment. The improved QuEChERS method was used in sample preparation. The 19 chlorophenol compounds were extracted with organic solvents and purified with purifying agents. During the experiment, the effect of the kinds and volumes of the extraction solvent, as well as the types and dosages of the purifying agent, on the recoveries of the 19 chlorophenol compounds were investigated. Moreover, the temperature and time of derivatization, as well as the dosage of the derivatization agent, were optimized. All aforementioned analyses were conducted with the aim of determining the optimal pretreatment method. Finally, the optimized gas chromatography-mass spectrometry conditions were employed for the quantitative determination of 19 chlorophenol compounds in fish samples. Based on the experimental results, the best extraction method was determined to be the one where the extraction agent (10 mL ethyl acetate) was added to 3 g sodium chloride and 5 g anhydrous magnesium sulfate in the test tube, followed by ultrasonication for 15 min. The sample was centrifuged at 4500 r/min for 5 min, and 500 mg C18 was selected as the purifying agent to purify the supernatant. The purified supernatant was blown with nitrogen to less than 1 mL at 45 ℃, and then redissolved with ethyl acetate to 1 mL. Subsequently, the sample solution was passed through a 0.22 μm organic filter membrane, following which 50 μL bis(trimethylsilyl)trifluoroacetamide was added for derivatization at 45 ℃ for 30 min. Lastly, the 19 chlorophenol compounds were determined by gas chromatography-mass spectrometry with an electrospray ionization source and selecting ion monitoring mode. The 19 chlorophenol compounds were then quantitatively analyzed by the external standard method. The compounds showed good linearity in the concentration range of 0.4-10 μg/L, with correlation coefficients (R2) greater than 0.998. The limits of detection and limits of quantification were 0.01-0.05 μg/kg and 0.04-0.16 μg/kg, respectively. Moreover, the average recoveries of the 19 chlorophenol compounds were in the range of 70.6%-115.0% at three spiked levels, and the relative standard deviations were in the range of 2.6%-10.5%. The established method in this study was applied to detect and analyze chlorophenol compounds in actual samples. The experimental results showed that various levels of chlorophenol compounds could be detected in different fishes. Among them, the total amount of chlorophenol compounds detected in the Corvina was 8.74 μg/kg, followed by the Crucian carp at 7.59 μg/kg, and the minimum detected amount in rice fish (1.59 μg/kg). With its simple operation, high sensitivity, and good repeatability, the established method simplifies the pre-treatment of fish samples. It can also meet the requirements for the high-throughput detection of 19 chlorophenol compounds in fish, thereby significantly improving the detection efficiency of chlorophenols. Moreover, the method provides crucial technical support and a theoretical basis for the establishment of feasible detection standards for chlorophenols in China, as well as for the control of residue levels of chlorophenol compounds in fish. The findings have important practical significance to implement management measures during fish breeding and transportation.
    Keywords:  QuEChERS; chlorophenols; fish; gas chromatography-mass spectrometry (GC-MS)
  15. Analyst. 2022 Apr 26.
      Chain modifications on fatty acyls, such as methyl branching, are important to modulate the biochemical and biophysical properties of lipids. The current lipid analysis workflows which mainly rely on collisional-induced dissociation (CID) to obtain the structural information of lipids often fail in locating the chain modifications. Radical-directed dissociation (RDD) is a new type of tandem mass spectrometry (MS/MS) method capable of producing intrachain cleavages, thus allowing the detailed characterization of lipid structures. In this study, we have developed an RDD method induced by nitroxide radicals (NO˙) for the analysis of branched-chain fatty acids (BCFAs). Fatty acids (FAs) are first amidated by O-benzylhydroxylamine; MS2 CID of the lithium adduct ion of the derivatized FAs uncages the nitroxide radical, which subsequently initiates RDD along the chain. The location of methyl branching can be determined via characteristic 28 Da spacing due to cleavages on either side of the branching point, with enhanced fragmentation observed toward the carbonyl end. This nitroxide-RDD method has been integrated onto reversed-phase liquid chromatography and applied for the profiling of BCFAs from yak milk powder and pooled human plasma samples. Other than the more often encountered iso- and anteiso-BCFAs, we have identified FA n-5 17 : 0 as a minor component from human plasma, which has been rarely reported before.
  16. Front Mol Biosci. 2022 ;9 785232
      The status of metabolomics as a scientific branch has evolved from proof-of-concept to applications in science, particularly in medical research. To comprehensively evaluate disease metabolomics, multiplatform approaches of NMR combining with mass spectrometry (MS) have been investigated and reported. This mixed-methods approach allows for the exploitation of each individual technique's unique advantages to maximize results. In this article, we present our findings from combined NMR and MS imaging (MSI) analysis of human lung and prostate cancers. We further provide critical discussions of the current status of NMR and MS combined human prostate and lung cancer metabolomics studies to emphasize the enhanced metabolomics ability of the multiplatform approach.
    Keywords:  imaging; lung cancer; mass spectrometry; metabolomics; nuclear magnetic resonance spectroscopy; prostate cancer
  17. Se Pu. 2022 May 08. 40(5): 452-460
      In China, the detection methods for polychlorinated biphenyls (PCBs) in aquatic products are mainly effective for 6 indicative PCBs and 12 coplanar dioxin-like PCBs, which only account for a limited proportion of PCBs in organisms. In this study, to obtain the detailed concentration levels of PCBs in organisms, elucidate the metabolism and enrichment characteristics of PCBs in organisms, and accurately evaluate the exposure level and risks of PCBs to humans, an improved method for the simultaneous determination of 82 PCBs in fish and shellfish samples was developed using isotope dilution-high resolution gas chromatography-high resolution mass spectrometry (ID-HRGC-HRMS). The recovery and reproducibility of two extraction methods, i. e., oscillatory extraction and accelerated solvent extraction (ASE), were compared. Finally, ASE was chosen for subsequent experiments. Specifically, after adding 1 ng13C-labeled extraction internal standards, the samples were extracted under pressure by ASE using a mixture of n-hexane-dichloromethane (1∶1, v/v). The experimental conditions employed for this were a pressure of 10.3 MPa, heating temperature of 100 ℃, heating time of 5 min, static time of 8 min, flush volume of 60%, purging time of 120 s, and 34 mL cells. Subsequently, the extracts were loaded on an 8 g acid silica gel (44%) column (inner diameter: 15 mm) and eluted with 90 mL of n-hexane. After purification and concentration, the analytes were determined by HRGC-HRMS with a fused-silica capillary column (DB-5MS, 60 m×0.25 mm×0.25 μm). The temperature program was optimized to separate the most target compounds at the baseline. Specifically, the initial oven temperature was 120 ℃, which was held for 1 min, following by heating to 180 ℃ at 30 ℃/min, heating to 210 ℃ at 2 ℃/min and holding for 1 min, and further heating to 310 ℃ at 2.5 ℃/min and holding for 1 min. The injector and ion source temperatures were 270 ℃ and 280 ℃, respectively. With a static resolution of 10000, the HRMS instrument was operated in the selected-ion monitoring mode at an electron energy of 35 eV. The 82 PCBs were qualified by their retention time and two characteristic ions, and thereafter quantified using the mean relative corresponding factor (RRF). The results showed that the relative standard deviation (RSD) of the RRF obtained from six-point calibration standard solutions was less than 20%. The linearity ranges were from 0.1 to 200 μg/L, and the correlation coefficients (r2) were greater than 0.99. Under optimum conditions, the method detection limits (MDLs) for the PCBs of biological samples were in the range of 0.02-3 pg/g. To validate the method, the fish and shellfish samples were spiked with a low level (0.4 ng) and high level (3.6 ng) of native PCB standards. The spiked recoveries using low-concentration native PCBs were 71.3%-139% in fish and 76.9%-143% in shellfish, and the RSDs (n=7) were 2.1%-14% and 4.5%-14%, respectively. The spiked recoveries using high-concentration native PCBs were 77.6%-141% and 82.2%-131%, respectively, and the RSDs (n=7) were 1.4%-9.4% and 1.7%-11%, respectively. An analysis of fresh fish and shellfish samples showed that the contents of a single PCB ranged from "not detected" to 54.1 pg/g, where 12 coplanar dioxin-like PCBs were detected in the range of 12.6 pg/g to 74.5 pg/g, six indicative PCBs in the range of 30.9 pg/g to 62.1 pg/g, and 82 PCBs in the range of 174 pg/g to 672 pg/g. It was concluded that this method could be successfully applied for the determination of PCBs in biological samples with good accuracy and precision. This comprehensive analytical method of PCBs in aquatic products provides effective technical support for biological monitoring; it will also aid in ecological and environmental management and the implementation of the Stockholm Convention policies.
    Keywords:  accelerated solvent extraction (ASE); environmental monitoring; fish; isotope dilution-high resolution gas chromatography-high resolution mass spectrometry (ID-HRGC-HRMS); polychlorinated biphenyls (PCBs); shellfish
  18. Se Pu. 2022 May 08. 40(5): 409-422
      To ensure the success of large-scale sporting events, prevent the contamination of food by prohibited substances, and evaluate the risk of foodborne stimulants and other hormones in food, it is necessary to establish a high-throughput, rapid, and accurate detection method for foodborne stimulants and other hormones. In this study, a QuEChERS method is proposed for the simultaneous determination of 44 foodborne stimulants and 6 progestogens using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analyzed foodborne stimulants include 19 β2-agonists, 3 β-blockers, 11 anabolic agents, 8 glucocorticoids, and 3 diuretics. A meat sample was crushed and homogenized, following which the internal standard was added. Subsequently, the sample was shaken and extracted with water and an acetonitrile solution containing 0.5% acetic acid, then dehydrated and centrifuged with sodium chloride and anhydrous magnesium sulfate. The supernatant was purified by PSA, C18, neutral alumina, and anhydrous magnesium sulfate. It was then dried with nitrogen and concentrated. The concentrated extracts were separated using an ACQUITY BEH C18 column (100 mm×2.1 mm, 1.7 μm) with gradient elution using 0.1% formic acid-5 mmol/L ammonium acetate solution and methanol as mobile phases. The target compounds were detected by ultra-performance liquid chromatography-tandem mass spectrometry with electrospray ionization and positive ion scanning (ESI+) in the multiple reaction monitoring (MRM) mode, and quantified by the internal standard method. The linear ranges of β2-agonists and β-blockers were 0.1-20 μg/L, the linear ranges of glucocorticoids were 0.5-200 μg/L, and those of the others were approximately 0.2-50 μg/L. The linear relationships of 50 compounds were good, with correlation coefficients >0.99 in the linear ranges, and limits of quantification (LOQs) in the range of 0.1-0.4 μg/kg. The recoveries of the 50 target compounds spiked in chicken, pork, beef, lamb samples at three levels ranged from 50.3% to 119.9%, while the relative standard deviations (RSDs, n=6) ranged from 0.42% to 15.1%. Nine meat samples (including 3 beef, 3 pork, 2 chicken, and duck samples) were tested by this method and the national standard method (GB/T 21981-2008). The t test was used for statistical analysis of the hydrocortisone and cortisone contents, and no significant difference was found between the results obtained by the two methods. The developed method was used to analyze 12 beef samples from a farm. In all, 4 compounds were detected, while the other 46 were not detected. The content ranges and detection rates of the compounds were as follows: hydrocortisone: 3.3-22.6 μg/kg, 100%; cortisone: 1.5-2.1 μg/kg, 67%; androstenedione: 0.7-1.2 μg/kg, 17%; and testosterone: 0.6-1.5 μg/kg, 42%. In conclusion, the proposed method is simple, accurate, and sensitive, and hence, is suitable for the detection of foodborne stimulants and progestogens in different kinds of raw meat.
    Keywords:  QuEChERS; foodborne stimulants; meat; progestogens; ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
  19. Se Pu. 2022 May 08. 40(5): 443-451
      Cases of toxic mushroom poisoning occur frequently in China every year. In particular, mushrooms containing amanitins can cause acute liver damage, with high mortality rates. The symptoms of acute liver damage are experienced 9-72 h after consumption of the mushrooms. At this time, the concentration of amanitins in blood and urine is too low to be detected even by the highly sensitive ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS), thus rendering clinical diagnosis and treatment difficult. To this end, a method was developed for the determination of α-amanitin, β-amanitin and γ-amanitin in urine and plasma by UPLC-MS/MS. Urine and plasma samples were extracted and cleaned up by using an immunoaffinity column. A sample of 2.00 mL urine or 1.00 mL of plasma was diluted with 8.00 mL of phosphate buffer solution (PBS) and then loaded onto the immunoaffinity column at a flow rate of 0.5-1.0 mL/min. After washing the column with 10 mL of PBS and 13 mL of water successively, the bound amanitins were eluted with 3.00 mL of methanol-acetone (1∶1, v/v). The eluent was dried under nitrogen at 55 ℃. The residue was dissolved in 100 μL of 10% (v/v) methanol aqueous solution. The amanitins in urine were concentrated 20 times, while those in plasma were concentrated 10 times. Chromatographic separation was performed on a Kinetex Biphenyl column (100 mm × 2.1 mm, 1.7 μm) with gradient elution using methanol and 0.005% (v/v) formic acid aqueous solution as mobile phases. The three amanitins were detected by negative electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode and quantified by the solvent standard curve external standard method. Method validation was performed as recommended by the European Drug Administration (EMEA). Four levels of quality control (QC) samples were prepared, which covered the calibration curve range, viz., the limit of quantification (LOQ), within three times the LOQ (low QC), medium QC, and at 85% of the upper calibration curve range (high QC), and used to test the accuracy, precision, matrix effect, extraction recovery, and stability. The calibration curves for the three amanitins showed good linear relationships in the range of 0.1-200 ng/mL, and the correlation coefficients (r) were greater than 0.999. The matrix effects and extraction efficiencies of the three amanitins in urine and plasma were 92%-108% and 90%-103%, respectively, and the coefficients of variation were less than 13%. The accuracies of the three amanitins in urine were within -9.4%-8.0%. The repeatability and intermediate accuracies were 3.0%-14% and 3.5%-18%, respectively. When the sampling volume was 2.00 mL, the limits of detection of the three amanitins in urine were 0.002 ng/mL. The accuracies of the three amanitins in plasma were within -13%-8.0%. The repeatability and intermediate accuracies were 3.9%-9.7% and 5.5%-12%, respectively. When the sampling volume was 1.00 mL, the limits of detection of the three amanitins in plasma were 0.004 ng/mL. The developed method is simple, sensitive, and accurate. During toxic mushroom poisoning detection, 0.0067 ng/mL of α-amanitin and 0.0059 ng/mL of β-amanitin were detected in the urine of poisoned patients 138 h after ingesting poisonous mushrooms. This method has successfully solved the problem of detecting ultra-trace levels of amanitins in the urine and plasma of poisoned patients. It has important practical significance for the early diagnosis, early treatment, and mortality reduction of suspected poisoning patients. This method can also provide reliable technical support for future research on the toxicological effects and in vivo metabolism of these toxins.
    Keywords:  amanitins; plasma; quadrupole-time-of-flight high resolution mass spectrometry (Q-TOF HRMS); ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS); urine
  20. Anal Bioanal Chem. 2022 Apr 23.
      Herein, we describe a modular solid-phase extraction (SPE) setup, combining three sorbents, for the effective extraction of neutrals, acidic, and basic micropollutants from wastewater, followed by their further elution in three independent extracts. The performance of this approach was demonstrated for a suite of 64 compounds, corresponding to different chemical families, using liquid chromatography tandem-mass spectrometry (LC-MS/MS). Target compounds were effectively extracted from wastewater samples; moreover, 62 out of 64 species were isolated in just one of the three fractions (neutrals, acids, and bases) obtained from the combination of sorbents. Globally, the efficiency and the selectivity of the SPE methodology improved the features obtained using generic SPE polymers, displaying just reversed-phase interactions. The overall recoveries of the analytical method, calculated against solvent-based calibration standards, stayed between 80 and 120% for 57 and 60 compounds, in raw and treated wastewater, respectively. Procedural limits of quantification (LOQs) varied from 1 to 20 ng L-1. Analysis of urban wastewater samples identified a group of 19 pollutants showing either negligible median removal efficiencies (± 20%) during wastewater treatment, or even a noticeable enhancement (case of the biodegradation product of the drug valsartan), which might be useful as markers of wastewater discharges in the aquatic environment.
    Keywords:  Fractionation; Liquid chromatography tandem-mass spectrometry; Modular solid-phase extraction; Wastewater
  21. Curr Res Transl Med. 2022 Apr 26. pii: S2452-3186(22)00014-9. [Epub ahead of print]70(4): 103346
      It has been known as almost all the cells consists a lipid molecule which has a considerable impact in various biological processes. Lipids have been investigated with a potential role for the formation of cellular membrane and thereby maintaining the structural integrity. Omics has placed as a combined technologies utilized for an exploaration of mechanistic actions in several kinds of molecules that make up the cells of an organism. Lipidomics has been recognized as a newly emerged branch of omics technology. This technology has the captivating factors to classify and characterize almost all the cellular lipids with the help of various analytical techniques and computational biological plateform. In lipidomics studies, structural display of several lipid biomarkers could also be analyzed and considered for actual disease diagnosis procedures. This could also replace certain traditional diagnostics method at all over the globe. Our review focuses how important this lipidomics particularly in disease diagnosis and also covers various analytical techniques and computational methods or bioinformatics tools in for the diagnosis of disease. In addtion, we also pinponted the possible role of lipids in several kinds of cellular disorders including cancer, neurodegenerative diseases, cardiovascular diseases, diabetes and obesity in human population. .
    Keywords:  Cellular disorders; Computational biology; Disease diagnosis; Lipidomics; Omics technology
  22. Anal Chem. 2022 Apr 28.
      Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N-glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N-glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N-glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N-glycans as an additional or orthogonal method to current analytical approaches.
  23. J Pharm Biomed Anal. 2022 Apr 16. pii: S0731-7085(22)00195-9. [Epub ahead of print]215 114774
      A robust analytical method based on liquid chromatography coupled to tandem mass spectrometry was developed and validated to quantify rifapentine and 25-O-desacetyl rifapentine in human breast milk to aid in determining the breastfed infant risk to the excreted drug in human milk. Samples were extracted by a combination of protein precipitation and solid phase extraction using rifampicin-d3 as an internal standard. An Agilent® Poroshell 120 EC-C18 (4.6 mm × 50 mm, 2.7 µm) column was used for chromatographic separation employing an isocratic mobile phase consisting of acetonitrile: methanol: 0.1% formic acid (55/5/40, v/v/v) at a flow rate of 450 µL/min, and with a total run time of four minutes. Mass detection was on an AB Sciex API 4000 mass spectrometer using electrospray ionization in the positive mode and based on multiple reaction monitoring data acquisition. Rifapentine was accurately quantified across a concentration range of 2.00-2000 ng/mL and 25-O-desacetyl rifapentine from 4.00 to 2000 ng/mL. During validation, the inter- and intra-day accuracy and precision at the tested QC concentrations (N = 18) for rifapentine were between 97.4% and 100.6%, and 3.1% and 8.3%, respectively. The inter- and intra-day accuracy and precision for 25-O-desacetyl rifapentine were between 96.4% and 106.3%, and 6.7% and 11.8%, respectively. No significant matrix effects were observed, and the method was shown to be specific for rifapentine and 25-O-desacetyl rifapentine. Human milk samples (N = 22) generated during a phase I/II clinical trial were successfully analysed for rifapentine and 25-O-desacetyl rifapentine using this validated method. Concentrations for rifapentine and 25-O-desacetyl rifapentine in human milk samples (N = 22) ranged from 11.2-1180 ng/mL and 7.11-573 ng/mL, respectively.
    Keywords:  Human milk; Liquid chromatography with tandem mass spectrometry; Rifapentine; Tuberculosis
  24. Talanta. 2022 Apr 22. pii: S0039-9140(22)00293-4. [Epub ahead of print]246 123497
      It is established that vitamin D deficiency is correlated with the disease severity in COVID-19 patients. However, the reliable and sensitive quantitation of vitamin D3 (D3) and its metabolites remains a difficult challenge. Herein, a novel ultrasensitive and reliable UHPLC-ESI-MS/MS method was developed and validated for the quantitation of D3 and its major metabolites in COVID-19 patients. The mass spectral sensitivity was augmented via controlled microwave-assisted derivatization reaction (CMDR) with 2-nitrosopyridine (Pyr-NO) at 65 °C for 2 min. CMDR hyphenation with UHPLC-MS/MS improves detection sensitivity while shortening separation and derivatization reaction times. The precursor to product ion transitions for D3, 25-hydroxy D3 (25(OH)D3), 1,25-dihydroxy D3 (1,25-(OH)2D3) and calcipotriol (CPT) as an internal standard were m/z 493.4 → 231.3, m/z 509.4 → 231.3, m/z 525.4 → 247.3, and m/z 521.4 → 247.3; respectively. The separation of the formed derivatives was conducted using a gradient elution mode with mobile phase A: formic acid (0.1%) in water and mobile phase B: formic acid (0.1%) in acetonitrile. The elution started with 40% (v/v) of B for 0.3 min then increased linearly to 90% (v/v) at 2 min on an Agilent EclipsePlus C18 (50 × 2.1 mm, 1.8 μm) column at a flow rate of 0.3 mL min-1. The method was validated using FDA standards for bioanalytical method validation over a concentration range of 0.02-50 ng mL-1 with correlation coefficient ≥0.9987 and the lower limit of quantitation (LLOQ) were 0.02-0.05 ng mL-1 in human plasma. The developed method has demonstrated excellent comparability to a well-established chemiluminescent immunoassay (CLIA) method for the analysis of D3 metabolites in human samples. The developed UHPLC-ESI-MS/MS method was implemented for routine and reliable quantitation of D3 and its major metabolites in COVID-19 patients.
    Keywords:  1,25-dihydroxyvitamin D3; 2-nitrosopyridine; 25-hydroxyvitamin D3; CMDR; COVID-19; UHPLC-ESI-MS/MS; Vitamin D3
  25. RSC Adv. 2021 May 27. 11(32): 19874-19884
      Magnetic nanoparticles modified with porous titanium dioxide were used as clean-up nanospheres for the detection of eight drug poisons in human blood by high-performance liquid chromatography-mass spectrometry. The magnetic clean-up nanospheres (Fe3O4@mTiO2) with a mesoporous structure were successfully synthesized and characterized by scanning electron microscopy/energy dispersive spectroscopy, transmission electron microscopy, X-ray diffractometry, vibrating sample magnetometry, infrared spectroscopy, and Brunauer-Emmett-Teller techniques. Lipid co-extractives, such as phosphatidic acid and fatty acids, which are major interferences in HPLC-MS analysis causing ion suppression in the MS spectra of blood, could be efficiently removed by Fe3O4@mTiO2 based on the Lewis acid-Lewis base interactions. Following the optimization of the quantities of Fe3O4@mTiO2, the method was applied to the determination of eight drugs in spiked blood. The analytical ranges typically extended from 2 to 500 ng mL-1, and the recoveries ranged from 79.5-99.9% at different concentrations of blood. The limits of quantitation for drug poisons were 0.14-1.03 ng mL-1, which makes the method a viable tool for drug poison monitoring in blood.
  26. Environ Int. 2022 Apr 18. pii: S0160-4120(22)00166-0. [Epub ahead of print]164 107240
      Inter-chemical correlations in metabolomics and exposomics datasets provide valuable information for studying relationships among chemicals reported for human specimens. With an increase in the number of compounds for these datasets, a network graph analysis and visualization of the correlation structure is difficult to interpret. We have developed the Chemical Correlation Database (CCDB), as a systematic catalogue of inter-chemical correlation in publicly available metabolomics and exposomics studies. The database has been provided via an online interface to create single compound-centric views. We have demonstrated various applications of the database to explore: 1) the chemicals from a chemical class such as Per- and Polyfluoroalkyl Substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalates and tobacco smoke related metabolites; 2) xenobiotic metabolites such as caffeine and acetaminophen; 3) endogenous metabolites (acyl-carnitines); and 4) unannotated peaks for PFAS. The database has a rich collection of 35 human studies, including the National Health and Nutrition Examination Survey (NHANES) and high-quality untargeted metabolomics datasets. CCDB is supported by a simple, interactive and user-friendly web-interface to retrieve and visualize the inter-chemical correlation data. The CCDB has the potential to be a key computational resource in metabolomics and exposomics facilitating the expansion of our understanding about biological and chemical relationships among metabolites and chemical exposures in the human body. The database is available at site.
    Keywords:  Biomonitoring; Database; Exposomics; Inter-chemical correlation; Metabolic pathways; Metabolomics; NHANES; Software
  27. Biomed Chromatogr. 2022 Apr 26.
      Penindolone (PND) is a novel Influenza A virus dual inhibitor that blocks hemagglutinin-mediated adsorption and membrane fusion. A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine PND in rat plasma. Plasma samples preparation was a simple deproteinization with acetonitrile followed by centrifugation. Chromatographic separation was performed on a C18 column with a gradient mobile phase of acetonitrile-water containing 0.1% formic acid. Detection was carried out by electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. Linear detection responses were obtained for PND ranging from 1 to 1000 ng/mL. The intra-day and inter-day precision (relative standard deviations, RSD) were within 6.5%, and accuracy (relative error, RE) was within ±11.0%. The extraction recovery data of PND and IS were more than 96.0%. PND were proved to be stable during the sample storage, preparation and analytic procedures. The validated method was successfully applied to the pharmacokinetic and bioavailability studies of PND in rats. The results showed the existence of twin peaks, gender difference and nonlinear pharmacokinetics of PND. In addition, two oxidation metabolites and three glucuronidation metabolites of PND were detected by ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS).
    Keywords:  metabolism; method validation; penindolone; pharmacokinetics