bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒04‒18
fifteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Talanta. 2021 Jul 01. pii: S0039-9140(21)00183-1. [Epub ahead of print]229 122262
      Measuring physiochemically diverse molecules (including lipids) which vary significantly in their concentrations poses a great analytical challenge. In untargeted lipidomics studies, reversed phase chromatography coupled with data-dependent MS/MS acquisition (DDA) is frequently applied. The optimal assay should deliver a high number of detected compounds with associated fragmentation data. In this work, we introduce novel 30 and 50 min UHPLC assays utilising lipid separation on a C30 stationary phase with a modified DDA strategy using smaller precursor m/z ranges scheduled for different lipid classes across the retention time range (defined as scheduled MS/MS). To evaluate the efficiency of the novel assays, mammalian tissue extracts (lamb liver, kidney and heart) were analysed and data were compared to a 15 min reversed phase C18 assay with multiple traditional DDA injections. The 30 min C30 assay detected double the number of detected compounds compared to the 15 min C18 assay. Applying the scheduled MS/MS DDA strategy with a single injection, a similar number of annotated lipids were reported compared to the traditional DDA strategy applied with five replicate injections on a C18 column. A longer 50 min C30 chromatographic assay did not result in an expected improvement in the chromatographic separation of overlapping isomer peaks compared to the 30 min method but did result in loss of accuracy of peak picking algorithms. We recommend the 30 min C30 assay with scheduled MS/MS acquisition as an efficient tool to analyse complex biological matrices and to annotate lipid species based on MS/MS data.
    Keywords:  C(18) reversed phase; C(30) reversed phase; Intelligent DDA; Lipidomics; Scheduled MS/MS
  2. Anal Chem. 2021 Apr 13. 93(14): 5805-5814
      Stereospecific recognition of metabolites plays a significant role in the detection of potential disease biomarkers thereby providing new insights in diagnosis and prognosis. D-Hdroxy/amino acids are recognized as potential biomarkers in several metabolic disorders. Despite continuous advances in metabolomics technologies, the simultaneous measurement of different classes of enantiomeric metabolites in a single analytical run remains challenging. Here, we develop a novel strategy for untargeted chiral metabolomics of hydroxy/amine groups (-OH/-NH2) containing metabolites, including all hydroxy acids (HAs) and amino acids (AAs), by chiral derivatization coupled with liquid chromatography-high resolution tandem mass spectrometry (LC-HR-MS/MS). Diacetyl-tartaric anhydride (DATAN) was used for the simultaneous derivatization of-OH/-NH2 containing metabolites as well as the resulting diastereomers, and all the derivatized metabolites were resolved in a single analytical run. Data independent MS/MS acquisition (DIA) was applied to positively identify DATAN-labeled metabolites based on reagent specific diagnostic fragment ions. We discriminated chiral from achiral metabolites based on the reversal of elution order of D and L isomers derivatized with the enantiomeric pair (±) of DATAN in an untargeted manner. Using the developed strategy, a library of 301 standards that consisted of 214 chiral and 87 achiral metabolites were separated and detected in a single analytical run. This approach was then applied to investigate the enantioselective metabolic profile of the bone marrow (BM) and peripheral blood (PB) plasma samples from patients with acute myeloid leukemia (AML) at diagnosis and following completion of the induction phase of chemotherapeutic treatment. The sensitivity and selectivity of the developed method enabled the detection of trace levels of the D-enantiomer of HAs and AAs in primary plasma patient samples. Several of these metabolites were significantly altered in response to chemotherapy. The developed LC-HR-MS method entails a valuable step forward in chiral metabolomics.
  3. Anal Chem. 2021 Apr 15.
      Lipids and metabolites are of interest in many clinical and research settings because it is the metabolome that is increasingly recognized as a more dynamic and sensitive molecular measure of phenotype. The enormous diversity of lipid structures and the importance of biological structure-function relationships in a wide variety of applications makes accurate identification a challenging yet crucial area of research in the lipid community. Indeed, subtle differences in the chemical structures of lipids can have important implications in cellular metabolism and many disease pathologies. The speed, sensitivity, and molecular specificity afforded by modern mass spectrometry has led to its widespread adoption in the field of lipidomics on many different instrument platforms and experimental workflows. However, unambiguous and complete structural identification of lipids by mass spectrometry remains challenging. Increasingly sophisticated tandem mass spectrometry (MS/MS) approaches are now being developed and seamlessly integrated into lipidomics workflows to meet this challenge. These approaches generally either (i) alter the type of ion that is interrogated or (ii) alter the dissociation method in order to improve the structural information obtained from the MS/MS experiment. In this Perspective, we highlight recent advances in both ion type alteration and ion dissociation methods for lipid identification by mass spectrometry. This discussion is aimed to engage investigators involved in fundamental ion chemistry and technology developments as well as practitioners of lipidomics and its many applications. The rapid rate of technology development in recent years has accelerated and strengthened the ties between these two research communities. We identify the common characteristics and practical figures of merit of these emerging approaches and discuss ways these may catalyze future directions of lipid structural elucidation research.
  4. Anal Chim Acta. 2021 May 08. pii: S0003-2670(21)00207-5. [Epub ahead of print]1158 338381
      Prostate cancer, a leading cause of cancer-related deaths worldwide, principally occurs in over 50-year-old men. Nowadays there is urgency to discover biomarkers alternative to prostate-specific antigen, as it cannot discriminate patients with benign prostatic hyperplasia from clinically significant forms of prostatic cancer. In the present paper, 32 benign prostatic hyperplasia and 41 prostatic cancer urine samples were collected and analyzed. Polar and positively charged metabolites were therein investigated using an analytical platform comprising an up to 40-fold analyte enrichment step by graphitized carbon black solid-phase extraction, HILIC separation, and untargeted high-resolution mass spectrometry analysis. These classes of compounds are often neglected in common metabolomics experiments even though previous studies reported their significance in cancer biomarker discovery. The complex metabolomics big datasets, generated by the UHPLC-HRMS, were analyzed with the ROIMCR procedure, based on the selection of the MS regions of interest data and their analysis by the Multivariate Curve-Resolution Alternating Least Squares chemometrics method. This approach allowed the resolution and tentative identification of the metabolites differentially expressed by the two data sets. Among these, amino acids and carnitine derivatives were tentatively identified highlighting the importance of the proposed methodology for cancer biomarker research.
    Keywords:  Chemometrics; LC-HRMS; Polar compounds; Positively charged compounds; ROI-MCR-ALS; Untargeted metabolomics
  5. J Pharm Biomed Anal. 2021 Apr 05. pii: S0731-7085(21)00170-9. [Epub ahead of print]199 114059
      Endogenous steroid hormones with similar structure, poor content and high efficacy are difficult and vital to be quantitatively detected. In this study, a validated method was established for the simultaneous quantification of thirteen steroids in human serum, and applied to the study of type 2 diabetes mellitus (T2DM). An ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of thirteen steroid hormones in human serum, including androstenedione, corticosterone (B), cortisol (F), cortisone, 18-hydroxycortisol (18OHF), 11-deoxycorticosterone, 11-deoxycortisol, pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone, androstanolone and estradiol. Under the optimum conditions, method was achieved with a BEH Shield RP18 column within 18 min. The lower limits of quantitation for steroids were 0.08-7.81 ng/mL. The intra- and inter-day precision for all the analytes were less than 15 %, and the accuracy ranged from -14.19 % to 12.89 % at three quality control levels. The proposed method, indicating high steady and sensitivity, was successfully applied to the quantification of thirteen steroids levels in serum from patients with T2DM and healthy individuals. The serum concentrations of 18OHF and F were significantly increased in the patients compared with the healthy individuals, while B was significantly decreased. The fold change was 1.98, 1.25 and 0.79 respectively. The ratio of 18OHF to B (18OHF/B) exhibited a 2.51-fold increase in T2DM patients and presented a more significant change. 18OHF/B was identified as a prospective serum marker, which deserves further attention.
    Keywords:  Human serum; Liquid chromatography-tandem mass spectrometry; Steroids; Type 2 diabetes mellitus
  6. J Chromatogr A. 2021 Mar 30. pii: S0021-9673(21)00248-X. [Epub ahead of print]1644 462124
      Supercritical fluid chromatography (SFC), combined with mass spectrometry (MS), was employed for the determination of five chiral fungicides, from two different chemical families (acylalanine and triazol) in wine and vineyard soils. The effect of different SFC parameters (stationary phase, chiral selector, mobile phase modifier and additive) in the resolution between enantiomers and in the efficiency of compounds ionization at the electrospray source (ESI) was thorougly described. Under final working conditions, chiral separations of selected fungicides were achieved using two different SFC-MS methods, with an analysis time of 10 min and resolution factors from 1.05 to 2.45 between enantiomers. In combination with solid-phase extraction and pressurized liquid extraction, they permitted the enantiomeric determination of target compounds in wine and vineyard soils with limits of quantification in the low ppb range (between 0.5 and 2.5 ng mL-1, and from 1.3 to 6.5 ng g-1, for wine and soil, respectively), and overall recoveries above 80%, calculated using solvent-based standards. For azolic fungicides (tebuconazole, myclobutanil and penconazole) soil dissipation and transfer from vines to wines were non-enantioselective processes. Data obtained for acylalanine compounds confirmed the application of metalaxyl (MET) to vines as racemate and as the R-enantiomer. The enantiomeric fractions (MET-S/(MET-S+MET-R)) of this fungicide in vineyard soils varied from 0.01 to 0.96; moreover, laboratory degradation experiments showed that the relative dissipation rates of MET enantiomers varied depending on the type of soil.
    Keywords:  Enantiomeric fraction; Fungicides; Soil; Supercritical fluid chromatography; Wine
  7. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Apr 02. pii: S1570-0232(21)00164-1. [Epub ahead of print]1173 122684
      Sphingolipids (SLs) are endogenously bioactive molecules with diverse structures, and its metabolic disorders are involved in the progression of many diseases. In this study, an ultra-performance liquid chromatography quadrupole exactive mass spectrometry (UPLC-Q-Exactive-MS) method was established to comprehensively profile SLs in plasma. First, the fragment patterns of SL standards of each subclass were investigated. Then, the SL species in plasma were characterized based on the fragmentation rules. Finally, a total of 144 endogenous SL species consisting of 216 regioisomers were identified in plasma of human, golden hamster and C57BL/6 mice, which was the most comprehensive identification for SLs in plasma. In addition to the known species, 19 SL species that have never been reported were also identified. The profile of SLs in plasma of human and two rodent species was compared subsequently. It was worth noting that a total of 9 SL molecular species consisting of 11 regioisomers with low abundance were successfully identified in human plasma through comparison among species. Those findings contribute to a deeper understanding of SLs in human plasma and provide scientific basis for the selection of animal model. The established profile of SLs in plasma could be used for screening of lipid biomarkers of various diseases.
    Keywords:  Plasma; Profile; Sphingolipid; UPLC-Q-Exactive-MS
  8. J Vet Diagn Invest. 2021 Apr 13. 10406387211005433
      Vitamin E deficiencies can impact normal growth and development in humans and animals, and assessment of circulating levels of vitamin E and its metabolites may be an important endpoint for evaluation. Development of a sensitive method to detect and quantify low concentrations of vitamin E and metabolites in biological specimens allows for a proper diagnosis for patients and animals that are deficient. We developed a method to simultaneously extract, detect, and quantify the vitamin E compounds alpha-tocopherol (α-TP), gamma-tocopherol (γ-TP), alpha-tocotrienol (α-TT), and gamma-tocotrienol (γ-TT), and the corresponding metabolites formed after β-oxidation of α-TP and γ-TP, alpha-carboxymethylbutyl hydroxychroman (α-CMBHC) and alpha- or gamma-carboxyethyl hydroxychroman (α- or γ-CEHC), respectively, from equine plasma and serum. Quantification was achieved through liquid chromatography-tandem mass spectrometry. We applied a 96-well high-throughput format using a Phenomenex Phree plate to analyze plasma and serum. Compounds were separated by using a Waters ACQUITY UPLC BEH C18 column with a reverse-phase gradient. The limits of detection for the metabolites and vitamin E compounds were 8-330 pg/mL. To validate the method, intra-day and inter-day accuracy and precision were evaluated along with limits of detection and quantification. The method was then applied to determine concentrations of these analytes in plasma and serum of horses. Alpha-TP levels were 3-6 µg/mL of matrix; the metabolites were found at much lower levels, 0.2-1.0 ng/mL of matrix.
    Keywords:  alpha-carboxymethylbutyl hydroxychroman; gamma-carboxyethyl hydroxychroman; liquid chromatography–mass spectrometry; vitamin E; α/γ-tocopherol
  9. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Mar 29. pii: S1570-0232(21)00157-4. [Epub ahead of print]1173 122677
      Climbazole is a preservative and an anti-dandruff ingredient with applications in various cosmetic products. The general population is therefore exposed to this chemical, and exposure monitoring is desirable. We have postulated a pathway for the human metabolism of climbazole, leading to two specific metabolites which can be excreted via urine. An analytical method for the determination of these metabolites in human urine was developed and validated. The sample preparation includes an enzymatic hydrolysis protocol. The measurement as such is based on online solid phase extraction (SPE), coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Intra- and inter-series coefficients of variation (CV) were determined in the concentration range from 1 µg/l to 100 µg/l with spiked pooled urine samples, and they were consistently below 15%, mostly below 10%. The corresponding accuracies (mean relative recovery rates) in spiked pooled urine varied from 97% to 103%. The robustness of the method was estimated by spiking individual urine samples. At 1 µg/l, the robustness was rather limited due to interfering matrix peaks in several samples, but excellent results were obtained at 10 µg/l and 100 µg/l, with CVs between 7% and 14% and accuracies from 101% to 110%. Matrix interferences often seemed to be associated with higher creatinine contents (≥2.0 g/l) of the samples. We subsequently applied the method to urine specimens from a human metabolism study involving documented climbazole exposures. We were able to identify and quantify the postulated metabolites in those real samples, thus validating our metabolism hypothesis. We also investigated the precision and accuracy of the enzymatic deconjugation with the real samples. The deconjugation step was found to be highly repeatable and largely quantitative. Both metabolites formed glucuronides, though varying fractions were also excreted in unconjugated (free) forms. Phase II conjugates other than glucuronides did not seem to be produced in significant amounts. With our method, both climbazole metabolites can be reliably quantified in the range between about 1.5 µg/l (depending on matrix interferences in individual samples) and at least 500 µg/l.
    Keywords:  Anti-dandruff agents; Cosmetic ingredients; Human biomonitoring; Online SPE; Preservatives; UHPLC-MS/MS
  10. Sci Rep. 2021 04 13. 11(1): 8017
      Hydroxychloroquine (2-[[4-[(7-Chloroquinolin-4-yl) amino]pentyl](ethyl) amino]-ethanol, HCQ), an effective anti-malarial drug, has been tested in the clinics for potential treatment of severe coronavirus disease 2019 (COVID-19). Despite the controversy around the clinical benefits of HCQ, the existence of a chiral center in the molecule to possess two optical isomers suggests that there might be an enantiomeric difference on the treatment of COVID-19. Due to their poor resolution and the inability of quantification by previously reported methods for the analysis of HCQ enantiomers, it is necessary to develop an analytical method to achieve baseline separation for quantitative and accurate determination of the enantiomeric purity in order to compare the efficacy and toxicity profiles of different enantiomers. In this study, we developed and validated an accurate and reproducible normal phase chiral high-performance liquid chromatography (HPLC) method for the analysis of two enantiomers of HCQ, and the method was further evaluated with biological samples. With this newly developed method, the relative standard deviations of all analytes were lower than 5%, and the limits of quantification were 0.27 μg/ml, 0.34 μg/ml and 0.20 μg/ml for racemate, R- and S-enantiomer, respectively. The present method provides an essential analytical tool for preclinical and clinical evaluation of HCQ enantiomers for potential treatment of COVID-19.
  11. J Mass Spectrom. 2021 Apr 02. e4727
      Design of experiments (DoE) is a valuable tool for optimization of a quantitative bioanalytical method utilizing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Liquid chromatography mass spectrometry (LC-MS) is composed of several processes, including, liquid introduction and analyte ionization. The goal is to transfer analytes from atmospheric pressure to vacuum and maintain conditions that are compatible for both the LC and the MS. These processes involve many experimental factors which need to be simultaneously optimized to obtain maximum sensitivity and resolution at minimum retention time. In this tutorial the basic concepts of DoE will be explained with focus on practical use of DoE, and then three case studies about utilization of DoE for optimization of liquid chromatography tandem mass spectrometry (LC-MS/MS) quantitative assays will be presented.
    Keywords:  D-optimal design; bioanalysis; clinical mass spectrometry; design of experiments (DoE); response surface methodology; tutorial
  12. Anal Methods. 2021 Apr 14. 13(14): 1731-1739
      The accurate identification of unknown illegal additive compounds in complex health foods continues to be a challenging task in routine analysis, because massive false positive results can be screened with ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry-based untargeted techniques and must be manually filtered out. To address this problem, we developed a chemometric-based strategy, in which data analysis was first performed by using XCMS, MS-DIAL, Mzmine2, and AntDAS2, to select those that provided acceptable results to extract common features (CFs), which can be detected by all of the selected methods. Then, CFs whose contents were significantly higher in the suspected illegal additive group were screened. Isotopic, adduct, and neutral loss ions were marked based on the CFs by using a new adaptive ion annotation algorithm. Fragment ions originating from the same compound were identified by using a novel fragment ion identification algorithm. Finally, a respective mass spectrum was constructed for each screened compound to benefit compound identification. The developed strategy was confirmed by using a complex Chinese health food, Goujiya tea. The features of all illegal additive compounds were precisely screened by the developed strategy, and massive false positive features from the current data analysis method were greatly reduced. The constructed respective mass spectra can benefit compound identification and avoid the risk of identifying ions from the same illegal compound as different compounds. Moreover, unknown compounds that are contained in an illegal compound library can be screened.
  13. Biomed Chromatogr. 2021 Apr 15. e5143
      A UPLC-MS/MS method was developed to determine the levels of five traditional antipsychotics (chlorprothixene, perphenazine, fluphenazine, thioridazine, and promethazine) in human plasma with carbamazepine as the internal standard (IS). Samples were extracted using simple liquid-liquid extraction (ethyl acetate/methyl tert-butyl ether, 2:3 v/v); then, the analytes were subjected to gradient elution chromatography with a mobile phase composed of 0.1% formic acid in water and acetonitrile. The analytes were separated using a Waters XBridge BEH C18 column (100 mm × 2.1 mm, 2.5 μm). The linear ranges of chlorprothixene, perphenazine, fluphenazine, thioridazine, and promethazine are 2-250 ng/mL, r > 0.995. The limit of quantitation is 2 ng/mL, and the limit of detection is in the range of 0.1-0.5 ng/mL. The relative standard deviations (RSDs) of interday and intraday are less than 10%, and the relative errors (REs) are in the range of -5.70% to 7.20%. The recoveries of five drugs are in the range of 70-109%. The results of methodology verification indicate that this method is simple, economical, and sensitive, and suitable for the simultaneous quantification of five traditional antipsychotics in human plasma.
    Keywords:  Traditional antipsychotics; UPLC-MS/MS; liquid-liquid extraction; plasma
  14. Rapid Commun Mass Spectrom. 2021 Apr 16. e9104
      RATIONALE: Gabapentin has shown initial promise as an opioid-sparing medication in pain patients as well as a treatment for opioid withdrawal and LC-MS/MS is often used for clinical monitoring. Despite reports of validated tandem masspectormetric methods for the determination of gabapentin and buprenorphine, mechanisms for the collision-induced fragmentation have not been adequetly described.METHODS: A rapid analytical method has been developed to determine the gabapentinoid, gabapentin, and partial opioid agonist, buprenorphine in 20 microliters of human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a chromatographic run time of 2 minutes. A simplified sample cleanup procedure using methanol precipitation of serum proteins/lipids followed by evaporation and reconstitution in mobile phase was demonstrated. Gabapentin and buprenorphine were detected following positive ion electrospray ionization using multiple-reaction-monitoring. The internal standard approach was used for quantitation with labeled gabapentin-D10 and buprenorphine-D4 serving as internal standards. Using organic reaction principals and stable isotope labels, collision-induced fragmentation mechanisms for both gabapentin and buprenorphine are proposed. The method was validated according to the FDA Guidance for Industry - Bioanalytical Method Validation.
    RESULTS: Accuracy was demonstrated by error values ≤15% for buprenorphine and ≤6% for gabapentin. The inter-day precision was ≤4.88% and 15.59% for gabapentin and buprenorphine and the intra-day precision was ≤5.20% and 11.65% for gabapentin and buprenorphine. The lower limit of quantitation corresponded to 10 ng/mL for gabapentin and 1 ng/mL for buprenorphine in serum. Recoveries were 104% ± 2.55 and 85% ± 2.03 for gabapentin and buprenorphine, respectively.
    CONCLUSIONS: Concentrations of gabapentin and buprenorphine were determined for 5 authentic human serum samples to further validate the utility of the method and applicable to therapeutic drug monitoring beyond its use as a drug screening assay. Furthermore, new mechanisms for the collision-induced dissociation of gabapentin and buprenorphine have been proposed.
  15. Zhonghua Yu Fang Yi Xue Za Zhi. 2021 Apr 06. 55(4): 528-534
      Objective: Explore the establishment of a fast, stable and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detecting the level of m6A modification in RNA and its application. Methods: The degree of m6A in RNA can be expressed as the ratio of m6A and adenosine (A) in concentration, which can be determined by ESI source positive ion multiple reaction monitoring (MRM) mode. The established method was verified by analyzing three quality control samples (m6A: 4, 40, 400 nmol/L; A: 40, 400, 4 000 nmol/L) with three different concentrations of low, medium, and high. The method was used to detect the degree of m6A in RNA from mouse spleen T cells treated in different ways. The t test was used to compare the differences between the two groups of data. Results: The established method had a good Linearity (R2>0.99) in a range of 1-500 nmol/L for m6A and 10-5 000 nmol/L for A. The limit of detection (LOD) was 1 nmol/L for m6A and 10 nmol/L for A. The recoveries were between 98.9% and 116.5%. The intra-day (n=5) RSDs and the inter-day (n=15, 5 days) RSDs were 2.4%-9.5% and 4.4%-9.6%, respectively. And this method was used to detect the degree of m6A in the RNA from mouse spleen T cells cultured in different conditions. The results showed that the m6A modification level in the RNA of primary CD8+T cell was 0.271 5±0.017 9, and the m6A modification level in the RNA of primary CD8+T cell with IL-27 was 0.251 7±0.015 0, indicating that primary CD8+T cells have a higher level of RNA methylation. Conclusion: This research has established a fast, simplemethylation degree in RNA with HPLC-MS/MS. This method is easy to be popularized and is suitable for the detection of large quantity of samples, and of great significance in analyzing the relationship between methylation and diseases.