bims-metlip 2020-06-07 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020–06–07
twenty papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

SWATH-MS for metabolomics and lipidomics: critical aspects of qualitative and quantitative analysis.
Identification and quantification of amino acids and related compounds based on Differential Mobility Spectrometry.
In matrix derivatization combined with LC-MS/MS results in ultra-sensitive quantification of plasma free metanephrines and catecholamines.
Quantitative profiling of eicosanoids derived from n-6 and n-3 polyunsaturated fatty acids by twin derivatization strategy combined with LC-MS/MS in patients with type 2 diabetes mellitus.
Applications of Chromatography-Ultra High-Resolution MS for Stable Isotope-Resolved Metabolomics (SIRM) Reconstruction of Metabolic Networks.
Determination of Polyphenols Using Liquid Chromatography-Tandem Mass Spectrometry Technique (LC-MS/MS): A Review.
SMfinder: Small Molecules Finder for Metabolomics and Lipidomics analysis.
High performance liquid chromatography - Mass spectrometric bioanalytical method for the determination of dapoxetine in human plasma: Application for bioequivalence study.
Derivatization assisted LC-p-MRM-MS with high CID voltage for rapid analysis of brassinosteroids.
MixONat, a software for the dereplication of mixtures based on 13C NMR spectroscopy.
Lipid Annotation by Combination of UHPLC-HRMS (MS), Molecular Networking, and Retention Time Prediction: Application to a Lipidomic Study of In Vitro Models of Dry Eye Disease.
Recommendations and Best Practices for Standardizing the Pre-Analytical Processing of Blood and Urine Samples in Metabolomics.
An Ultrasensitive Routine LC-MS/MS Method for Estradiol and Estrone in the Clinically Relevant Sub-Picomolar Range.
Exploring the Lipidome: Current Lipid Extraction Techniques for Mass Spectrometry Analysis.
High resolution mass spectrometry newborn screening applications for quantitative analysis of amino acids and acylcarnitines from dried blood spots.
LC-Swan probe: an integrated in-situ sampling interface for liquid chromatography separation and mass spectrometry analysis.
Hyphenation of packed column supercritical fluid chromatography with mass spectrometry: where are we and what are the remaining challenges?
Experimental and statistical protocol for the effective validation of chromatographic analytical methods.
[Tandem-mess spectrometry coupled with liquid chromatography (LCMS/MS) : a revolution in the clinical chemistry laboratory].
A Sensitive and Optimized HPLC-FLD Method for the Simultaneous Quantification of Hydroxychloroquine and Its Two Metabolites in Blood of Systemic Lupus Erythematosus Patients.

Metabolomics. 2020 Jun 05. 16(6): 71

SWATH-MS for metabolomics and lipidomics: critical aspects of qualitative and quantitative analysis.

Michel Raetz, Ron Bonner, Gérard Hopfgartner.

   INTRODUCTION: While liquid chromatography coupled to mass spectrometric detection in the selected reaction monitoring detection mode offers the best quantification sensitivity for omics, the number of target analytes is limited, must be predefined and specific methods developed. Data independent acquisition (DIA), including SWATH using quadrupole time of flight or orbitrap mass spectrometers and generic acquisition methods, has emerged as a powerful alternative technique for quantitative and qualitative analyses since it can cover a wide range of analytes without predefinition.
OBJECTIVES: Here we review the current state of DIA, SWATH-MS and highlight novel acquisition strategies for metabolomics and lipidomics and opportunities for data analysis tools.
METHOD: Different databases were searched for papers that report developments and applications of DIA and in particular SWATH-MS in metabolomics and lipidomics.
RESULTS: DIA methods generate digital sample records that can be mined retrospectively as further knowledge is gained and, with standardized acquisition schemes, used in multiple studies. The different chemical spaces of metabolites and lipids require different specificities, hence different acquisition and data processing approaches must be considered for their analysis.
CONCLUSIONS: Although the hardware and acquisition modes are well defined for SWATH-MS, a major challenge for routine use remains the lack of appropriate software tools capable of handling large datasets and large numbers of analytes.

Keywords:  Data independent acquisition; LC–MS; Lipids; Metabolites; Review; SWATH

DOI:  https://doi.org/10.1007/s11306-020-01692-0
Analyst. 2020 Jun 01.

Identification and quantification of amino acids and related compounds based on Differential Mobility Spectrometry.

Francis Berthias, Yali Wang, Eskander Alhajji, Bernard Rieul, Fathi Moussa, Jean-François Benoist, Philippe Maître.

Amino acids and related compounds constitute a class of biomarkers which is analyzed for early diagnosis of metabolic diseases (MDs). Protocols based on liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS) are routinely used for MD diagnosis. Our ultimate objective is to evaluate the analytical performance of differential mobility spectrometry (DMS) hyphenated to MS/MS, in the perspective of using DMS-MS/MS as an alternative or complementary method for the topics of emergency in metabolic diagnosis and newborn rapid screening. The aim of the present study is to evaluate the robustness of a DMS-MS/MS protocol for the separation, identification, and quantification of amino acids and related compounds. Performance in terms of peak capacity and separation of isobaric and isomeric species is compared to those using drift tube type ion mobility spectrometry instruments. High reproducibility of the measurement of the DMS compensation voltage (CV) of metabolites shows that this CV parameter, or the corresponding electric field, could be used for application in metabolite identification. Multiple measurements show that the CV value of each AA or related compound is stable over a large period of time (6 months). Potential effects of matrix or concentration of the analytes on the DMS identifier are found to be negligible. Quantification of a selected set of metabolites in human plasmas has been carried out. The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis were assessed as adequate for both physiological and pathological conditions. Concentration levels of metabolites derived with our DMS-MS protocols were found to be in good agreement with those obtained with routine LC-MS/MS protocols used for the diagnosis of MDs at the Hospital Robert Debré (Paris).

DOI: https://doi.org/10.1039/d0an00377h
Anal Chem. 2020 Jun 02.

In matrix derivatization combined with LC-MS/MS results in ultra-sensitive quantification of plasma free metanephrines and catecholamines.

Martijn van Faassen, Rainer Bischoff, Karin Eijkelenkamp, Wilhelmina H A de Jong, Claude P van der Ley, Ido Peter Kema.

Plasma free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated L-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 µL plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (β-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R2 > 0.99) and a high-performance liquid chromatography with electrochemical detection method for measuring plasma catecholamines (R2 > 0.85). Reference intervals for L-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the sub-nanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods, and enables simultaneous quantification of several classes of amines.

DOI: https://doi.org/10.1021/acs.analchem.0c01263
Anal Chim Acta. 2020 Jul 11. pii: S0003-2670(20)30491-8. [Epub ahead of print]1120 24-35

Quantitative profiling of eicosanoids derived from n-6 and n-3 polyunsaturated fatty acids by twin derivatization strategy combined with LC-MS/MS in patients with type 2 diabetes mellitus.

Fangbo Xia, Chengwei He, Meng Ren, Feng-Guo Xu, Jian-Bo Wan.

  Eicosanoids derived from n-6 and n-3 polyunsaturated fatty acids (PUFAs), serving as important signaling molecules, are implicated in many physiological and pathological processes, including Type 2 diabetes mellitus (T2DM). However, the quantification of endogenous eicosanoids is challenged by high structural similarity, low abundance in biological sample and poor electrospray ionization efficiency. In the current study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify 65 eicosanoids derived from n-6 and n-3 PUFAs in plasma samples using twin derivatization strategy with a pair of reagents, 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) and (diethylamino) naphthalene-1-sulfonyl piperazine (Dens-PP). Dns-PP-derivatized plasma sample was mixed with the equal volume of Dens-PP-derivatized eicosanoid internal standards for LC-MS/MS analysis in multiple reaction monitoring (MRM) mode. After Dns-PP derivatization, the ionization efficiency and separation performance were substantially improved, resulting in the enhanced sensitivity by 446- to 1009-folds compared to intact eicosanoids. The quantitative accuracy determined by twin derivatization method was found to be comparable with stable isotope labeled internal standards (SIL-IS) method. The newly proposed method was successfully employed to quantify the target eicosanoids in plasma samples from healthy controls and the patients with T2DM. N-6 PUFA-derived eicosanoids, PGF2α, PGD2, PGE2, PGA2, PGB2, 20-HETE and LTC4, significantly increased in plasma sample of T2DM patients. Oppositely, n-3 PUFA-derived eicosanoids, RvE1, 12(S)-HEPE and RvD1, remarkably decreased. Spearman's correlation analysis indicated the strong correlations between these highlighted eicosanoids and clinical parameters of T2DM. Collectively, the sensitive and reliable eicosanoid quantification method may facilitate to elucidate the characteristics of eicosanoid metabolism and understand the role of eicosanoids in the pathogenesis of T2DM and other diseases.

Keywords:  Chemical isotope labeling; Eicosanoids; LC-MS/MS; Quantification; Twin derivatization; Type 2 diabetes mellitus

DOI:  https://doi.org/10.1016/j.aca.2020.04.064
Trends Analyt Chem. 2020 Feb;pii: 115676. [Epub ahead of print]123

Applications of Chromatography-Ultra High-Resolution MS for Stable Isotope-Resolved Metabolomics (SIRM) Reconstruction of Metabolic Networks.

Qiushi Sun, Teresa W-M Fan, Andrew N Lane, Richard M Higashi.

  Metabolism is a complex network of compartmentalized and coupled chemical reactions, which often involve transfers of substructures of biomolecules, thus requiring metabolite substructures to be tracked. Stable isotope resolved metabolomics (SIRM) enables pathways reconstruction, even among chemically identical metabolites, by tracking the provenance of stable isotope-labeled substructures using NMR and ultrahigh resolution (UHR) MS. The latter can resolve and count isotopic labels in metabolites and can identify isotopic enrichment in substructures when operated in tandem MS mode. However, MS2 is difficult to implement with chromatography-based UHR-MS due to lengthy MS1 acquisition time that is required to obtain the molecular isotopologue count, which is further exacerbated by the numerous isotopologue source ions to fragment. We review here recent developments in tandem MS applications of SIRM to obtain more detailed information about isotopologue distributions in metabolites and their substructures.

Keywords:  13C/15N positional isotopologues; Ion chromatography; data independent MS2; multiplexed stable isotope resolved metabolomics (mSIRM); nucleotides; pathway reconstruction; ultra high-resolution FT-MS

DOI:  https://doi.org/10.1016/j.trac.2019.115676
Antioxidants (Basel). 2020 Jun 02. pii: E479. [Epub ahead of print]9(6):

Determination of Polyphenols Using Liquid Chromatography-Tandem Mass Spectrometry Technique (LC-MS/MS): A Review.

Olalla López-Fernández, Rubén Domínguez, Mirian Pateiro, Paulo E S Munekata, Gabriele Rocchetti, José M Lorenzo.

  In recent years, the consumption of polyphenols has been increasing, largely due to its beneficial effects on health. They are present in a wide variety of foods, but their extraction and characterization are complicated since they are mostly in complex matrices. For this reason, the use of selective, sensitive, and versatile analytical techniques such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is necessary. In this review, the most relevant studies of the last years regarding the analysis of polyphenols in different matrices by comprehensive LC-MS/MS are discussed. Relevant steps such as extraction, sample purification, and chromatographic analysis methods are emphasized. In particular, the following methodological aspects are discussed: (a) the proper selection of the extraction technique, (b) the extraction and elution solvents, (c) the purification step, (d) the selection of both stationary and mobile phases for the chromatographic separation of compounds, and (e) the different conditions for mass spectrometry. Overall, this review presents the data from the most recent studies, in a comprehensive way, thus providing and simplifying the information of the great variety of works that exist in the literature on this wide topic.

Keywords:  LC–MS/MS; analytical methods; anthocyanins; electrospray ionization; flavonols; phenolic compounds

DOI:  https://doi.org/10.3390/antiox9060479
Anal Chem. 2020 Jun 05.

SMfinder: Small Molecules Finder for Metabolomics and Lipidomics analysis.

Giuseppe Martano, Michele Leone, Pierluca D'oro, Vittoria Matafora, Angela Cattaneo, Marco Masseroli, Angela Bachi.

Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user friendly open-source software, named SMfinder, for the robust identi-fication and quantification of small molecules. The software introduces a MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and tar-geted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted and flux analysis.

DOI: https://doi.org/10.1021/acs.analchem.0c00585
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 15. pii: S1570-0232(20)30020-9. [Epub ahead of print]1149 122154

High performance liquid chromatography - Mass spectrometric bioanalytical method for the determination of dapoxetine in human plasma: Application for bioequivalence study.

Rana Said, Basel Arafat, Tawfiq Arafat.

  Dapoxetine is an oral medication used for treatment of premature ejaculation (PE) in men aged (18-64 years). In this study, we present a validated, precise and sensitive method for determination of dapoxetine in human plasma by liquid chromatography/ electrospray ionization-tandem mass spectrometry. Dapoxetine and the internal standard (Dapoxetine- d6) were extracted from plasma via liquid-liquid extraction (LLE). The LC separation was performed utilizing ACE C8 (4.6 X50) mm, 5 µm column. The mobile phase was composed of acetonitrile and buffer (0.01 M Ammonium acetate +0.02% Formic acid solution) (85:15, v/v). The method was linear within the concentration range of 5.0-600 ng/mL for Dapoxetine in human plasma. Short analytical run was achieved with 1.6 min run time. Intra-day and inter-day accuracy was between 97 and 106% with precision (CV, %) of ≤ 5% achieved across all the quality control samples. Dapoxetine was stable in several conditions with recovery rates > 90%. This method was utilized successfully in clinical pharmacokinetic study following oral administration of 60 mg Dapoxetine tablets in 36 healthy male subjects. The result for all 90% confidence intervals were within the preset ranges. The method proved to be highly reproducible and sensitive and thus can be employed in bioequivalence studies and large scale sample analysis of Dapoxetine.

Keywords:  Bioequivalence; Dapoxetine; LC-MS/MS; LLE

DOI:  https://doi.org/10.1016/j.jchromb.2020.122154
Talanta. 2020 Sep 01. pii: S0039-9140(20)30349-0. [Epub ahead of print]217 121058

Derivatization assisted LC-p-MRM-MS with high CID voltage for rapid analysis of brassinosteroids.

Na An, Quan-Fei Zhu, Lei Yu, Yong-Ting Chen, Sheng-Li Chen, Yu-Qi Feng.

  In this study, a derivatization-assisted pseudo-multiple reaction monitoring with high CID voltage (HV-p-MRM) strategy was proposed for the analysis of brassinosteroids (BRs) by liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ MS). The concept of the HV-p-MRM strategy was proposed on the basis of an assumption that the precursor ion of analyte is stable in collision cell and less prone to fragmentation at high CID voltage, while co-existing ions (impurity) of easy fragmentation can break down into smaller fragment ions. In such case, by increasing the CID voltage, the co-existing ions that are introduced due to the low resolution of the quadrupole 1 (Q1) can be filtered out by quadrupole 3 (Q3), while the stable precursor ion of analyte will pass through Q3, thereby that the signal-to-noise ratio (S/N) of the analysis can be improved. As a proof-of-concept study, BRs were derivatized with rhodamine B-boronic acid (RhB-BA) and then the derivatives were used to investigate their variations in MS signal, background noise, and S/N upon the CID voltage and MS scanning resolution. The results showed that S/N of these derivatives can be improved in HV-p-MRM mode. To further demonstrate the feasibility of HV-p-MRM strategy, a RhB-BA derivatization assisted LC-HV-p-MRM-MS method was developed for the analysis of BRs. Using this method, rapid and sensitive determination of BRs in different organs of rape flower was achieved. It can be expected that HV-p-MRM may be suitable for the analytes that are stable or can be converted into compounds of high stability in collision cell at high CID voltage.

Keywords:  Brassinosteroids; CID voltage; Derivatization; Multiple reaction monitoring; Triple quadrupole mass spectrometry

DOI:  https://doi.org/10.1016/j.talanta.2020.121058
Anal Chem. 2020 Jun 01.

MixONat, a software for the dereplication of mixtures based on 13C NMR spectroscopy.

Antoine Bruguière, Séverine Derbré, Joël Dietsch, Jules Leguy, Valentine Rahier, Quentin Pottier, Dimitri Bréard, Sorphon Suor Cherer, Guillaume Viault, Anne-Marie Le Ray, Frédéric Saubion, Pascal Richomme.

Whether chemists or biologists, researchers dealing with metabolomics require tools to decipher complex mixtures. As a part of metabolomics and initially dedicated to identifying bioactive natural products, dereplication aims at reducing the usually time-consuming process of known compounds isolation. Mass spectrometry and nuclear magnetic resonance are the most commonly reported analytical tools during dereplication analysis. Though low sensitive, 13C-NMR has many advantages for such a study. Noteworthy, it is nonspecific allowing simultaneous high-resolution analysis of any organic compounds including stereoisomers. Since NMR spectrometers nowadays provide useful dataset in a reasonable time frame, we have embarked upon writing a software dedicated to 13C-NMR dereplica-tion. The present study describes the development of a freely distributed algorithm, namely MixONat and its ability to help researchers decipher complex mixtures. Based on Python 3.5, MixONat analyses a {1H}-13C NMR spectrum optionally combined with DEPT-135 and 90 data - to distinguish carbon types (i.e. CH3, CH2, CH and C) - as well as a MW filtering. The software requires predicted or experimental carbon chemical shifts (δc) databases and displays results that can be refined based on user interactions. As a proof of concept, this 13C-NMR dereplication strategy was evaluated on mixtures of increasing complexity and exhibiting pharmaceuti-cal (poppy alkaloids), nutritional (rosemary extracts) or cosmetics (mangosteen peel extract) applications. Associated results were com-pared with other methods commonly used for dereplication. MixONat gave coherent results that rapidly oriented the user towards the correct structural types of secondary metabolites, allowing the user to distinguish between structurally close natural products, including stereoisomers.

DOI: https://doi.org/10.1021/acs.analchem.0c00193
Metabolites. 2020 May 29. pii: E225. [Epub ahead of print]10(6):

Lipid Annotation by Combination of UHPLC-HRMS (MS), Molecular Networking, and Retention Time Prediction: Application to a Lipidomic Study of In Vitro Models of Dry Eye Disease.

Romain Magny, Anne Regazzetti, Karima Kessal, Gregory Genta-Jouve, Christophe Baudouin, Stéphane Mélik-Parsadaniantz, Françoise Brignole-Baudouin, Olivier Laprévote, Nicolas Auzeil.

  Annotation of lipids in untargeted lipidomic analysis remains challenging and a systematic approach needs to be developed to organize important datasets with the help of bioinformatic tools. For this purpose, we combined tandem mass spectrometry-based molecular networking with retention time (tR) prediction to annotate phospholipid and sphingolipid species. Sixty-five standard compounds were used to establish the fragmentation rules of each lipid class studied and to define the parameters governing their chromatographic behavior. Molecular networks (MNs) were generated through the GNPS platform using a lipid standards mixture and applied to lipidomic study of an in vitro model of dry eye disease, i.e., human corneal epithelial (HCE) cells exposed to hyperosmolarity (HO). These MNs led to the annotation of more than 150 unique phospholipid and sphingolipid species in the HCE cells. This annotation was reinforced by comparing theoretical to experimental tR values. This lipidomic study highlighted changes in 54 lipids following HO exposure of corneal cells, some of them being involved in inflammatory responses. The MN approach coupled to tR prediction thus appears as a suitable and robust tool for the discovery of lipids involved in relevant biological processes.

Keywords:  dry eye disease; hyperosmolarity; lipidomic; liquid chromatography; molecular network; tandem mass spectrometry

DOI:  https://doi.org/10.3390/metabo10060225
Metabolites. 2020 Jun 03. pii: E229. [Epub ahead of print]10(6):

Recommendations and Best Practices for Standardizing the Pre-Analytical Processing of Blood and Urine Samples in Metabolomics.

Raúl González-Domínguez, Álvaro González-Domínguez, Ana Sayago, Ángeles Fernández-Recamales.

  Metabolomics can be significantly influenced by a range of pre-analytical factors, such as sample collection, pre-processing, aliquoting, transport, storage and thawing. This therefore shows the crucial need for standardizing the pre-analytical phase with the aim of minimizing the inter-sample variability driven by these technical issues, as well as for maintaining the metabolic integrity of biological samples to ensure that metabolomic profiles are a direct expression of the in vivo biochemical status. This review article provides an updated literature revision of the most important factors related to sample handling and pre-processing that may affect metabolomics results, particularly focusing on the most commonly investigated biofluids in metabolomics, namely blood plasma/serum and urine. Finally, we also provide some general recommendations and best practices aimed to standardize and accurately report all these pre-analytical aspects in metabolomics research.

Keywords:  blood; collection; metabolomics; pre-analytics; processing; storage; urine

DOI:  https://doi.org/10.3390/metabo10060229
J Endocr Soc. 2020 Jun 01. 4(6): bvaa047

An Ultrasensitive Routine LC-MS/MS Method for Estradiol and Estrone in the Clinically Relevant Sub-Picomolar Range.

Bjørn-Erik Bertelsen, Ralf Kellmann, Kristin Viste, Anne Turid Bjørnevik, Hans Petter Eikesdal, Per Eystein Lønning, Jørn V Sagen, Bjørg Almås.

   Background: Current analytical routine methods lack the sensitivity to monitor plasma estrogen levels in breast cancer patients treated with aromatase inhibitors. Such monitoring is warranted for premenopausal patients treated with an aromatase inhibitor and an LH-releasing hormone analogue in particular. Therefore, we aimed to develop a routine tandem mass spectroscopy combined with liquid chromatography (LC-MS/MS) method for estradiol (E2) and estrone (E1) for use in the sub-picomolar range.
Method: Calibrators, quality controls (QC), or serum samples were spiked with isotope-labeled internal standard and purified by liquid-liquid extraction. The reconstituted extracts were analyzed by LC-MS/MS in negative electrospray ionization mode. QCs at 6 levels made from pooled patient sera were used to validate the accuracy, sensitivity, and precision of the method.
Results: We achieved limits of quantification of 0.6 pmol/L (0.16 pg/mL) for E2 and 0.3 pmol/L (0.07 pg/mL) for E1. The coefficient of variation was below 9.0% at all QC levels for E2 (range, 1.7-153 pmol/L), and below 7.8% for E1 (range, 1.7-143 pmol/L). The method is traceable to the E2 reference standard BCR576. Reference ranges for E2 and E1 in healthy, postmenopausal women were obtained, for E2: 3.8 to 36 pmol/L, for E1: 22 to 122 pmol/L. We measured and confirmed ultra-low E2 and E1 concentrations in sera from patients on the aromatase inhibitors letrozole or exemestane.
Conclusion: This ultrasensitive LC-MS/MS method is suitable for routine assessment of serum E1 and E2 levels in breast cancer patients during estrogen suppression therapy. The method satisfies all requirements for measurement of E2 in the clinical setting as stated by the Endocrine Society in 2013.
Precis: We report an ultrasensitive LCMS/MS routine assay that measures pretreatment and suppressed levels of estradiol/estrone during aromatase inhibitor treatment of postmenopausal breast cancer patients.

Keywords:  LC-MS/MS; aromatase inhibitor; breast cancer; estradiol; estrogen suppression therapy; estrone

DOI:  https://doi.org/10.1210/jendso/bvaa047
Metabolites. 2020 Jun 03. pii: E231. [Epub ahead of print]10(6):

Exploring the Lipidome: Current Lipid Extraction Techniques for Mass Spectrometry Analysis.

Julian Aldana, Adriana Romero-Otero, Mónica P Cala.

  In recent years, high-throughput lipid profiling has contributed to understand the biological, physiological and pathological roles of lipids in living organisms. Across all kingdoms of life, important cell and systemic processes are mediated by lipids including compartmentalization, signaling and energy homeostasis. Despite important advances in liquid chromatography and mass spectrometry, sample extraction procedures remain a bottleneck in lipidomic studies, since the wide structural diversity of lipids imposes a constrain in the type and amount of lipids extracted. Differences in extraction yield across lipid classes can induce a bias on down-stream analysis and outcomes. This review aims to summarize current lipid extraction techniques used for untargeted and targeted studies based on mass spectrometry. Considerations, applications, and limitations of these techniques are discussed when used to extract lipids in complex biological matrices, such as tissues, biofluids, foods, and microorganisms.

Keywords:  extraction; lipids; targeted lipidomics; untargeted lipidomics

DOI:  https://doi.org/10.3390/metabo10060231
Anal Chim Acta. 2020 Jul 11. pii: S0003-2670(20)30494-3. [Epub ahead of print]1120 85-96

High resolution mass spectrometry newborn screening applications for quantitative analysis of amino acids and acylcarnitines from dried blood spots.

C Austin Pickens, Konstantinos Petritis.

  Amino acid and acylcarnitine first-tier newborn screening typically employs derivatized or non-derivatized sample preparation methods followed by FIA coupled to triple quadrupole (TQ) MS/MS. The low resolving power of TQ instruments results in difficulties distinguishing nominal isobaric metabolites, especially those with identical quantifying product ions such as malonylcarnitine (C3DC) and 4-hydroxybutylcarnitine (C4OH). Twenty-eight amino acids and acylcarnitines extracted from dried blood spots (DBS) were analyzed by direct injection (DI)-HRMS on a Q-Exactive Plus across available mass resolving powers in SIM, in PRM at 17,000 full width at half maximum (FWHM), and a developed SIM/PRM hybrid MS method. Most notably, quantitation of C3DC and C4OH was successful by HRMS in non-derivatized samples, thus, potentially eliminating sample derivatization requirements. Quantitation differed between SIM and PRM acquired data for several metabolites, and it was determined these quantitative differences were due to collision energy differences or kinetic isotope effects between the unlabeled metabolites and the corresponding labeled isotopologue internal standards. Overall quantitative data acquired by HRMS were similar to data acquired on TQ MS/MS platform. A proof-of-concept hybrid DI-HRMS and SIM/PRM/FullScan method was developed demonstrating the ability to hybridize targeted newborn screening with metabolomic screening.

Keywords:  Amino acids and acylcarnitines; Dried blood spot; High resolution mass spectrometry; Metabolic disorders; Metabolomics; Newborn screening

DOI:  https://doi.org/10.1016/j.aca.2020.04.067
Anal Chem. 2020 Jun 04.

LC-Swan probe: an integrated in-situ sampling interface for liquid chromatography separation and mass spectrometry analysis.

Di-Qiong Jin, Shao-Wen Shi, Yan Ma, Qun Fang.

In-situ sampling mass spectrometry (MS) systems can achieve rapid analysis of samples while most of them do not have the pretreatment capability of chromatographic separation. This paper describes the design, fabrication and application of a swan-shaped in-situ sampling MS probe with liquid chromatography (LC) separation capacity. The LC-Swan probe was fabricated based on a single capillary with a micrometer-sized hole at its U-shaped bottom for sampling, a monolithic column for separation, and a tapered tip for electrospray. Four functions including in-situ sampling, sample injection, chromatographic separation, and MS electrospray were integrated in the LC-Swan probe. A direct sampling and a contacting-dissolution-injection sampling modes were developed to perform in-situ sampling and injection of liquid samples and dry spot samples, respectively, in the high flow-resistance LC system. A pressing-sealing method was also developed using a polydimethylsiloxane (PDMS) sealer to achieve the sealing of the probe sampling hole during the high-pressure chromatographic separation process. The LC-Swan probe-based system exhibited effective desalting capacity in the analysis of angiotensin II with similar relative standard deviations (RSDs) of retention time and peak area below 3% and 19% (n = 3) for both salt-containing and salt-free samples. The present system was applied for analyzing cytochrome C digest to test its separation capability for samples with complex compositions, and 19 peptides were detected in 13 min with an amino acid coverage of 85%. We also applied the system in metabolite analysis of mouse organ sections of brain, liver and kidney, to preliminarily demonstrate its application potential in MS imaging analysis.

DOI: https://doi.org/10.1021/acs.analchem.0c01555
Anal Bioanal Chem. 2020 May 30.

Hyphenation of packed column supercritical fluid chromatography with mass spectrometry: where are we and what are the remaining challenges?

Laura Akbal, Gérard Hopfgartner.

  Over the last decades, packed column supercritical fluid chromatography (pSFC) using carbon dioxide (CO2) as supercritical fluid has gained interest as a complementary separation technique to liquid chromatography (LC). Various commercial solutions for the hyphenation to atmospheric pressure ionization (API) including electrospray (ESI) and atmospheric pressure chemical ionization (APCI) have been described using split-flow or full-flow introduction approaches. This review discusses various aspects of the hyphenation using these two approaches. It also illustrates the difference between LC-MS and SFC-MS. The benefits and challenges of the decoupling of the separation mobile phase from the ionization conditions are also pointed out. The effect of CO2 on ESI performance and the adduct reduction are also discussed. Finally, limitation of current hardware and the limited use of smaller column internal diameters (i.d.) are discussed. Graphical abstract.

Keywords:  Electrospray; Hyphenation; Ionization; Mass spectrometry; Review; Supercritical fluid chromatography

DOI:  https://doi.org/10.1007/s00216-020-02715-4
MethodsX. 2020 ;7 100919

Experimental and statistical protocol for the effective validation of chromatographic analytical methods.

Eugenio Alladio, Eleonora Amante, Cristina Bozzolino, Fabrizio Seganti, Alberto Salomone, Marco Vincenti, Brigitte Desharnais.

  The validation of analytical methods is of crucial importance in several fields of application. A new protocol for the validation of chromatographic methods has been proposed. The overall protocol is described in a parallel paper, where the case of a multi-targeted gas chromatography - mass spectrometry (GC-MS) method for the determination of androgens in human urine is in-depth discussed. The purpose of this paper is to report the details about the GC-MS separation and detection of the target analytes, and to provide the mathematical formulas needed to perform the validation of the principal parameters. Briefly, the validation protocol foresees the repetition of three calibration curves in three different days, providing a total amount of nine replicates. Such a structured design allows to use the same experiments to•perform a rigorous calibration study, by the evaluation of heteroscedasticity, comparison of several weights and linear/quadratic calibration curves.•determine several parameters which are traditionally computed from dedicated experiments, namely intra- and inter-day accuracy and precision, limit of detection, specificity, selectivity, ion abundance repeatability, and carry over.•Finally, few further experiments are necessary to evaluate the retention time repeatability, matrix effect and extraction recovery.

Keywords:  Chromatographic method; GC-MS; Multiresidual analysis; Validation protocol

DOI:  https://doi.org/10.1016/j.mex.2020.100919
Rev Med Liege. 2020 May;75(5-6): 457-459

[Tandem-mess spectrometry coupled with liquid chromatography (LCMS/MS) : a revolution in the clinical chemistry laboratory].

E Cavalier, C Le Goff.

Manual or automated immunoassays are largely used in clinical chemistry laboratories for measuring various compounds like steroid or peptide hormones. However, these methods can lack sensibility and specificity. Hence, during this last decade, tandem-mess spectrometry coupled with liquid chromatography (LC-MS/MS) have emerged as a technique of choice to precisely quantify those molecules. However, these instruments remain quite expensive and need highly trained people.

Keywords: Immunoassay; LC; MS/MS
J Chromatogr Sci. 2020 Jun 01. pii: bmaa023. [Epub ahead of print]

A Sensitive and Optimized HPLC-FLD Method for the Simultaneous Quantification of Hydroxychloroquine and Its Two Metabolites in Blood of Systemic Lupus Erythematosus Patients.

Xuemei Luo, Ying Peng, Weihong Ge.

Quantification of hydroxychloroquine (HCQ) and its two metabolites desethylchloroquine and desethylhydroxychloroquine in human blood can provide insight into the pharmacokinetic/pharmacodynamic characteristics of HCQ for the treatment of systemic lupus erythematosus (SLE), which is crucial for the optimization of the therapy. A simple, sensitive and optimized high performance liquid chromatography with fluorescence detection method has been developed and validated for the simultaneous determination of HCQ and its two metabolites in human blood. After addition of internal standard chloroquine, the blood sample was deproteinized with 2-fold acetonitrile and separated on an YMC-Triart C18 column (250 × 4.6 mm, 5 μm) with a mobile phase of 20 mM sodium phosphate buffer solution containing 0.25% triethylamine (pH 8.0)-acetonitrile (60:40, v/v). The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 337 and 405 nm, respectively. The method was linear over the range of 3-3000 ng/mL for all three analytes and the chromatographic run time was 9 min. The values for intra- and inter-day precisions were ranged from 1.3 to 7.3. This method was successfully applied to quantify the concentrations of HCQ and its two metabolites in blood of 92 SLE patients.

DOI: https://doi.org/10.1093/chromsci/bmaa023