bims-metlip 2022-09-04 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–09–04
24 papers selected by
Sofia Costa, Matterworks

Coumarin-based derivatization reagent for LC-MS analysis of amino acids.
Benchmarking One-Phase Lipid Extractions for Plasma Lipidomics.
How Well Can We Predict Mass Spectra from Structures? Benchmarking Competitive Fragmentation Modeling for Metabolite Identification on Untrained Tandem Mass Spectra.
Simultaneous multiplexed quantification and C=C localization of fatty acids with LC-MS/MS using isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags and C=C epoxidation.
Clinical Usefulness of Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Low Serum Testosterone Measurement.
Determination of traditional and designer benzodiazepines in urine through LC-MS/MS.
Development of a Highly Sensitive and Rapid Liquid Chromatography-Tandem Mass Spectrometric Method Using a Basic Mobile Phase Additive to Determine the Characteristics of the Urinary Metabolites for Niemann-Pick Disease Type C.
Simultaneous quantitation of 17 endogenous adrenal corticosteroid hormones in human plasma by UHPLC-MS/MS and their application in congenital adrenal hyperplasia screening.
Mass Spectrometry Imaging technology in metabolomics: a systematic review.
On-tissue derivatization for isomer-specific mass spectrometry imaging and relative quantification of monosaccharides in biological tissues.
The Role of Mass Spectrometry Imaging in Pharmacokinetic Studies.
DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.
Analysis of illicit pills and drugs of abuse in urine samples using a 3D-printed open port probe hyphenated with differential mobility spectrometry-mass spectrometry.
Calibration Practices in Clinical Mass Spectrometry: Review and Recommendations.
Comparability of Steroid Collision Cross Sections Using Three Different IM-HRMS Technologies: An Interplatform Study.
MetaboAnnotator: An efficient toolbox to annotate metabolites in genome-scale metabolic reconstructions.
Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.
MIRTH: Metabolite Imputation via Rank-Transformation and Harmonization.
Comprehensive plasma metabolomics and lipidomics of benign and malignant solitary pulmonary nodules.
Validation of an LC-MS/MS assay for the simultaneous determination of bictegravir, doravirine, and raltegravir in human plasma.
Four-Dimensional Untargeted Profiling of N-Acylethanolamine Lipids in the Mouse Brain Using Ion Mobility-Mass Spectrometry.
Developing Transmission Mode for IR-MALDESI Mass Spectrometry Imaging.
A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass.
Resolving Enantiomers of 2-Hydroxy Acids by Nuclear Magnetic Resonance.

Talanta. 2022 Aug 05. pii: S0039-9140(22)00526-4. [Epub ahead of print]252 123730

Coumarin-based derivatization reagent for LC-MS analysis of amino acids.

Nguyễn Kim Ngân Bùi, Sigrid Selberg, Koit Herodes, Ivo Leito.

  In this paper, we report about the application of a sensitive fluorescent derivatization reagent Coumarin151-N-Hydroxysuccinimidyl Carbamate (Cou151DSC) for amino compounds using high-performance liquid chromatography (HPLC) compatible with ultraviolet (UV), fluorescence detector (FLD) and electrospray ionization - tandem mass spectrometry (ESI-MS/MS)-positive mode. We optimized derivatization procedure and validated an analytical method to determine 24 amino acids in Kvass drink using Norvaline as an internal standard. Compared to 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate (6-AQC), the derivatization with Cou151 DSC is faster and milder, for 5 min at 40°C instead of 15 min at 55°C. The limit of quantitation (LOQ, pmol on column) for 21 amino acids in this work is lower 1.1-30.0 times than values obtained with 6-AQC. The derivatives have excitation wavelength at 355 nm and emission wavelength at 486 nm. Their MS/MS fragmentation behaviors were examined together with 23 other amino compounds. We found three possibilities to lose a neutral group which can be Coumarin 151 isocyanate Cou151NCO (255 Da), amine Coumarin 151 (229 Da) or urea Cou151CONH2 (272 Da). The accuracy of the proposed method was within 83-107% with good relative standard deviations (RSDs) of equal or less than 6%. The recoveries were from 82 to 120% in four spiked concentrations, repeatability was between 0 and 14%. The intra- and inter-day precision are less than 13% and 18%, respectively.

Keywords:  Amino acids; Cou151DSC; Coumarin151-N-Hydroxysuccinimidyl Carbamate; Derivatization; ESI; LC-MS; MS/MS; Neutral loss

DOI:  https://doi.org/10.1016/j.talanta.2022.123730
Anal Chem. 2022 Sep 01.

Benchmarking One-Phase Lipid Extractions for Plasma Lipidomics.

Marcus Höring, Christoph Stieglmeier, Katja Schnabel, Tucker Hallmark, Kim Ekroos, Ralph Burkhardt, Gerhard Liebisch.

A key element of successful lipidomics analysis is a sufficient extraction of lipid molecules typically by two-phase systems such as chloroform-based Bligh and Dyer (B&D). However, numerous metabolomics and lipidomics studies today apply easy to use one-phase extractions. In this work, quantitative flow injection analysis high-resolution mass spectrometry was applied to benchmark the lipid recovery of popular one-phase extraction methods for human plasma samples. The following organic solvents were investigated: methanol (MeOH), ethanol (EtOH), 2-propanol (IPA), 1-butanol (BuOH), acetonitrile (ACN) and the solvent mixtures BuOH/MeOH (3:1) and MeOH/ACN (1:1). The recovery of polar lysophospholipids was sufficient for all tested solvents. However, nonpolar lipid classes such as triglycerides (TG) and cholesteryl esters (CE) revealed extraction efficiencies less than 5% due to precipitation in polar solvents EtOH, MeOH, MeOH/ACN, and ACN. Sample pellets also contained a substantial amount of phospholipids, for example, more than 75% of total phosphatidylcholine and sphingomyelin for ACN. The loss of lipids by precipitation was directly related to the polarity of solvents and lipid classes. Although, lipid recovery increased with the volume of organic solvent, recovery in polar MeOH remains incomplete also for less polar lipid classes such as ceramides. Addition of stable isotope-labeled internal standards prior to lipid extraction could compensate for insufficient lipid recovery for polar lipid classes including lysolipids and phospholipids but not for nonpolar CE and TG. In summary, application of one-phase extractions should be limited to polar lipid classes unless sufficient recovery/solubility of nonpolar lipids has been demonstrated. The presented data reveal that appropriate lipid extraction efficiency is fundamental to achieve accurate lipid quantification.

DOI: https://doi.org/10.1021/acs.analchem.2c02117
J Chem Inf Model. 2022 Aug 31.

How Well Can We Predict Mass Spectra from Structures? Benchmarking Competitive Fragmentation Modeling for Metabolite Identification on Untrained Tandem Mass Spectra.

Parker Ladd Bremer, Arpana Vaniya, Tobias Kind, Shunyang Wang, Oliver Fiehn.

Competitive Fragmentation Modeling for Metabolite Identification (CFM-ID) is a machine learning tool to predict in silico tandem mass spectra (MS/MS) for known or suspected metabolites for which chemical reference standards are not available. As a machine learning tool, it relies on both an underlying statistical model and an explicit training set that encompasses experimental mass spectra for specific compounds. Such mass spectra depend on specific parameters such as collision energies, instrument types, and adducts which are accumulated in libraries. Yet, ultimately prediction tools that are meant to cover wide expanses of entities must be validated on cases that were not included in the initial training and testing sets. Hence, we here benchmarked the performance of CFM-ID 4.0 to correctly predict MS/MS spectra for spectra that were not included in the CFM-ID training set and for different mass spectrometry conditions. We used 609,456 experimental tandem spectra from the NIST20 mass spectral library that were newly added to the previous NIST17 library version. We found that CFM-ID's highest energy prediction output would maximize the capacity for library generation. Matching the experimental collision energy with CFM-ID's prediction energy produced the best results, even for HCD-Orbitrap instruments. For benzenoids, better MS/MS predictions were achieved than for heterocyclic compounds. However, when exploring CFM-ID's performance on 8,305 compounds at 40 eV HCD-Orbitrap collision energy, >90% of the 20/80 split test compounds showed <700 MS/MS similarity score. Instead of a stand-alone tool, CFM-ID 4.0 might be useful to boost candidate structures in the greater context of identification workflows.

DOI: https://doi.org/10.1021/acs.jcim.2c00936
Anal Chim Acta. 2022 Sep 08. pii: S0003-2670(22)00786-3. [Epub ahead of print]1225 340215

Simultaneous multiplexed quantification and C=C localization of fatty acids with LC-MS/MS using isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags and C=C epoxidation.

Ting-Jia Gu, Yu Feng, Danqing Wang, Lingjun Li.

  Fatty acids (FAs) possess highly diverse structures and can be divided into saturated and unsaturated classes. For unsaturated FAs, both the numbers and positions of carbon-carbon double bond (C=C) determine their biological functions. Abnormal levels of FA isomers have been reported to be involved in various disease development, such as cancer. Despite numerous advances in lipidomics, simultaneous quantifying and pinpointing C=C bond positions in a high-throughput manner remains a challenge. Here we conducted epoxidation of C=C bonds of unsaturated FAs followed by the conjugation with isobaric SUGAR tags. With the assistance of LC-MS, FA isomers with the same masses were separated on the C18 column and individually subjected to MS/MS fragmentation. Upon higher-energy collisional dissociation, not only reporter ions for multiplexed quantification but also diagnostic ions for C=C localization were generated at the same time, allowing quantitative analyses of different unsaturated FA isomers in samples. The performance of this approach including epoxidation, labeling efficiencies, quantitation accuracy, and capability to pinpoint C=C bond position were evaluated. To evaluate our method, free FA extracts from healthy human serum were used to demonstrate the feasibility of this method for complex sample analysis. Finally, this method was also applied to investigate the changes of unsaturated FA isomers between heathy human and Alzheimer's disease (AD) patient serum.

Keywords:  Carbon-carbon double bonds; Fatty acids; Human serum; LC-MS/MS; Multiplexed quantification; SUGAR tags

DOI:  https://doi.org/10.1016/j.aca.2022.340215
Ann Lab Med. 2023 Jan 01. 43(1): 19-28

Clinical Usefulness of Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Low Serum Testosterone Measurement.

Sung-Eun Cho, Jungsun Han, Ju-Hee Park, Euna Park, Geun Young Kim, Jun Hyung Lee, Ahram Yi, Sang Gon Lee, Eun Hee Lee, Yeo-Min Yun.

   Background: Mass spectrometry methods exhibit higher accuracy and lower variability than immunoassays at low testosterone concentrations. We developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantifying serum total testosterone.
Methods: We used an ExionLC UPLC (Sciex, Framingham, MA, USA) system and a Sciex Triple Quad 6500+ (Sciex) MS/MS system in electrospray ionization and positive ion modes with multiple reaction monitoring transitions to evaluate precision, accuracy, linearity, lower limit of quantitation (LLOQ), carryover, ion suppression, stability, and reference intervals. For method comparison, we measured serum testosterone concentrations using this method in 40 subjects whose testosterone concentrations ranged from 0.14 to 55.48 nmol/L as determined using the Architect i2000 immunoassay (Abbott Diagnostics, Abbott Park, IL, USA) and in an additional 160 sera with testosterone concentrations <1.67 nmol/L.
Results: The intra- and inter-run precision CVs were <2.81%, and the accuracy bias values were <3.85%, which were all acceptable. The verified linear interval was 0.03-180.84 nmol/L; the LLOQ was 0.03 nmol/L. No significant carryover and ion suppression were observed. The testosterone in serum was stable at 4°C, at -20°C, and after three freeze-thaw cycles. The reference intervals were successfully verified. The correlation was good at testosterone concentrations of 0.14-55.48 nmol/L; however, the Architect assay showed positive percent bias at concentrations <1.67 nmol/L.
Conclusions: The UPLC-MS/MS assay shows acceptable performance, with a lower LLOQ than the immunoassay. This method will enable the quantitation of low testosterone concentrations.

Keywords:  Performance; Quantitation; Tandem mass spectrometry; Testosterone

DOI:  https://doi.org/10.3343/alm.2023.43.1.19
Curr Pharm Des. 2022 Aug 31.

Determination of traditional and designer benzodiazepines in urine through LC-MS/MS.

Brian Rossi, Francesca Freni, Claudia Carelli, Matteo Moretti, Dario Galatone, Claudia Vignali, Luca Morini.

   BACKGROUND: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis.
OBJECTIVE: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases.
METHODS: 100 µL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode.
RESULTS: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine.
CONCLUSION: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

Keywords:  Benzodiazepines; LC-MS/MS; NPS; designer benzodiazepines; flubromazepam; forensic toxicology; urine

DOI:  https://doi.org/10.2174/1381612828666220831103224
Biol Pharm Bull. 2022 ;45(9): 1259-1268

Development of a Highly Sensitive and Rapid Liquid Chromatography-Tandem Mass Spectrometric Method Using a Basic Mobile Phase Additive to Determine the Characteristics of the Urinary Metabolites for Niemann-Pick Disease Type C.

Masamitsu Maekawa, Keitaro Miyoshi, Aya Narita, Toshihiro Sato, Yu Sato, Masaki Kumondai, Masafumi Kikuchi, Katsumi Higaki, Torayuki Okuyama, Yoshikatsu Eto, Hiroshi Sakamaki, Nariyasu Mano.

  As Niemann-Pick disease type C (NPC) is difficult to diagnose owing to its various clinical symptoms; biomarker tests have been developed. Previously, we revealed urinary sulfated cholesterol metabolites as noninvasive biomarkers for NPC. However, LC/tandem mass spectrometry (LC/MS/MS) requires long separation time and large urine volumes. Recently, a basic mobile phase was reported to increase the MS intensity. Thus, we developed a highly sensitive and rapid LC/MS/MS method for analyzing urinary cholesterol metabolites using a basic mobile phase additive. 3β-Sulfooxy-7β-N-acetylglucosaminyl-5-cholenic acid, its glycine and taurine conjugates, 3β-sulfooxy-7β-hydroxy-5-cholenic acid, and 7-oxo form were measured, with selected reaction monitoring in negative ion mode. Oasis HLB and L-column 3 were used for column-switching LC/MS/MS and urine diluted 10-fold was employed as the sample. After trapping, gradient separation was performed using solutions containing 1% (v/v) ammonium solution. On average, a 16-fold increase in peak areas was observed compared to that obtained at pH 5.5 with the mobile phases. Although the previous method needed 60 min for separation from interference peaks, we succeeded to separate them in 7 min with optimized LC condition. Further, all compounds showed good linearity from 0.3-1000 ng/mL, with satisfactory intra- and inter-day reproducibility. The developed method was applied to the urinalysis of healthy participants and NPC patients. Overall, the concentrations of metabolites correlated with those obtained using the previous method. Therefore, we succeeded to increasing MS intensity and shorten LC running time; and the method is useful for the noninvasive diagnostic screening of patients with NPC.

Keywords:  LC–electrospray ionization–tandem mass spectrometry; Niemann–Pick disease type C; ammonium solution; basic condition LC; cholesterol metabolite; urinary biomarker

DOI:  https://doi.org/10.1248/bpb.b22-00185
Front Chem. 2022 ;10 961660

Simultaneous quantitation of 17 endogenous adrenal corticosteroid hormones in human plasma by UHPLC-MS/MS and their application in congenital adrenal hyperplasia screening.

Qiaoxuan Zhang, Min Zhan, Huihui Wu, Pinning Feng, Xing Jin, Zemin Wan, Jun Yan, Pengwei Zhang, Peifeng Ke, Junhua Zhuang, Jiuyao Zhou, Liqiao Han, Xianzhang Huang.

  Accurate investigation of adrenal hormone levels plays a vital role in pediatric endocrinology for the detection of steroid-related disorders. This study aims to develop a straightforward, sensitive UHPLC-MS/MS method to quantify 17 endogenous adrenal corticosteroid hormones in human plasma. These hormones are the main ingredients in the synthetic and metabolic pathways of adrenal corticosteroid hormones. Chromatographic separation was achieved on a C18 column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode with a run time of 7 min. The samples were extracted by liquid-liquid extraction and required no derivatization. Analytical performance was evaluated, including linearity, analytical sensitivity, accuracy, precision, and specificity. Plasma specimens from 32 congenital adrenal hyperplasia (CAH) patients and 30 healthy volunteers were analyzed to further reveal the diagnostic value of multiple steroid hormones in the synthetic and metabolic pathways of adrenal corticosteroid in CAH diagnosis. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference of isomers or structural analogues. The imprecisions were <10%. The lower limits of quantification varied from 0.05 to 15.0 ng/ml. Good linearity coefficients (r 2 > 0.998) were also obtained for most hormones in the required concentration range, except for 21-deoxycortisol (r 2 = 0.9967) and androstenediol (r 2 = 0.9952). The recoveries for the steroid hormones ranged from 91.7 to 109.8%. We developed the UHPLC-MS/MS method for the simultaneous measurement of steroid hormones. The results showed that measurement of steroid hormones simultaneously could improve the diagnostic efficiency of CAH.

Keywords:  UHPLC-MS/MS; adrenal hormones; congenital adrenal hyperplasia; diagnostic efficiency; simultaneous quantitation

DOI:  https://doi.org/10.3389/fchem.2022.961660
Biomed Chromatogr. 2022 Aug 31. e5494

Mass Spectrometry Imaging technology in metabolomics: a systematic review.

Si-Qi Gao, Jin-Hui Zhao, Yue Guan, Ying-Shu Tang, Ying Li, Li-Yan Liu.

  Mass spectrometry imaging (MSI) is a powerful label-free analysis technique that can provide simultaneous spatial distribution of multiple compounds in a single experiment. By combining the sensitive and rapid screening of high-throughput mass spectrometry with spatial chemical information, metabolite analysis and morphological characteristics are presented in a single image. MSI can be used for qualitative and quantitative analysis of metabolic profiles and it can provide visual analysis of spatial distribution information of complex biological and microbial systems. Matrix assisted laser desorption ionization, laser ablation electrospray ionization and desorption electrospray ionization are commonly used in MSI. Here, we summarize and compare these three technologies, as well as the applications and prospects of MSI in metabolomics.

Keywords:  Mass spectrometry; biological sample; imaging; metabolomics

DOI:  https://doi.org/10.1002/bmc.5494
Anal Chim Acta. 2022 Sep 08. pii: S0003-2670(22)00812-1. [Epub ahead of print]1225 340241

On-tissue derivatization for isomer-specific mass spectrometry imaging and relative quantification of monosaccharides in biological tissues.

Yuhao Han, Yisheng Zhao, Panpan Chen, Lei Wang, Qiongzheng Hu, Xiao Wang, Chenglong Sun.

  Spatially-resolved profiling of tissue monosaccharides not only gives an insight into the spatial heterogeneity of monosaccharides, but also helps to decipher the possible roles of monosaccharides in biological processes. Here, we develop an on-tissue derivatization method, coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to image and quantify the aldose and ketose isomers of monosaccharide in biological tissues. A new derivatization reagent, 1-naphthaleneacethydrazide (NAH) was synthesized for the on-tissue derivatization of monosaccharides, and it significantly enhanced the imaging sensitivity of monosaccharides. Moreover, the NAH-derivatized aldose and ketose can generate isomer-specific diagnostic ions during MALDI-MS/MS analysis, and thus paves way for the isomer-specific MS imaging of aldose and ketose monosaccharides. On this basis, we further constructed a quantitative MALDI-MS imaging model based on isomer-specific diagnostic ions, and calculated the expression contents of aldose and ketose monosaccharide isomers in different tissue regions of carrot section. We expect that the development of this method should provide more precise view on the spatial distributions and contents of different monosaccharides in heterogeneous biological tissues.

Keywords:  Biological tissues; Isomer; Mass spectrometry imaging; Monosaccharides; On-tissue derivatization; Quantification

DOI:  https://doi.org/10.1016/j.aca.2022.340241
Xenobiotica. 2022 Sep 01. 1-48

The Role of Mass Spectrometry Imaging in Pharmacokinetic Studies.

Chukwunonso K Nwabufo, Omozojie P Aigbogun.

  Although liquid chromatography-tandem mass spectrometry is the gold standard analytical platform for the quantification of drugs, metabolites, and biomarkers in biological samples, it cannot localize them in target tissues.The localization and quantification of drugs and/or their associated metabolites in target tissues is a more direct measure of bioavailability, biodistribution, efficacy, and regional toxicity compared to the traditional substitute studies using plasma.Therefore, combining high spatial resolution imaging functionality with the superior selectivity and sensitivity of mass spectrometry into one analytical technique will be a valuable tool for targeted localization and quantification of drugs, metabolites, and biomarkers.Mass spectrometry imaging (MSI) is a tagless analytical technique that allows for the direct localization and quantification of drugs, metabolites, and biomarkers in biological tissues, and has been used extensively in pharmaceutical research.The overall goal of this current review is to provide a detailed description of the working principle of MSI and its application in pharmacokinetic studies encompassing absorption, distribution, metabolism, excretion, and toxicity processes, followed by a discussion of the strategies for addressing the challenges associated with the functional utility of MSI in pharmacokinetic studies that support drug development.

Keywords:  ADMET; COVID-19; LC-MS/MS; SARS-CoV-2; desorption electrospray ionization; mass spectrometry imaging; matrix-assisted laser desorption ionization; pharmacokinetics

DOI:  https://doi.org/10.1080/00498254.2022.2119900
J Mass Spectrom. 2022 Sep;57(9): e4882

DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.

Saleh M Khalil, Richard R Sprenger, Martin Hermansson, Christer S Ejsing.

  Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA-imaging technology where high-resolution MSI is combined with parallel acquisition of lipid tandem MS2 spectra on a hybrid ion trap-Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA-imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA-imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA-imaging on the Orbitrap Elite. Altogether, our work shows that DDA-imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.

Keywords:  MALDI-imaging mass spectrometry; lipid identification; lipidomics; mouse brain

DOI:  https://doi.org/10.1002/jms.4882
Analyst. 2022 Aug 30.

Analysis of illicit pills and drugs of abuse in urine samples using a 3D-printed open port probe hyphenated with differential mobility spectrometry-mass spectrometry.

Piotr Sosnowski, Victor Marin, Xiaobo Tian, Gérard Hopfgartner.

The present work describes the application of an in-house developed 3D-printed open port probe (3DP-OPP) with differential ion mobility spectrometry (DMS) mass spectrometry. Targeted quantitative analysis in urine was performed with a triple quadrupole mass spectrometer in the selected reaction monitoring mode (OPP-DMS-SRM/MS) and illicit pill screening using data independent acquisition (OPP-DMS-SWATH/MS). The combination of compensation voltage (CoV) scanning in DMS using modifiers with SWATH/MS acquisition for MS/MS spectrum generation enabled the differentiation of isobaric signals with a large dynamic range and enhance the information contained in the screening of illicit ecstasy pills. As for any direct MS introduction technique where no chromatographic separation is applied DMS with acetonitrile as a modifier allows the separation of cocaine and tramadol, and their isomeric metabolites in urine samples. Quantitative application using OPP-DMS-SRM/MS is presented without the need for sample preparation with a lower limit of quantification at 10-25 ng mL-1 for the analytes and less than 40 seconds for sample to sample analysis.

DOI: https://doi.org/10.1039/d2an00925k
Ann Lab Med. 2023 Jan 01. 43(1): 5-18

Calibration Practices in Clinical Mass Spectrometry: Review and Recommendations.

IFCC Working Group on Method Evaluation Protocols

   Background: Calibration is a critical component for the reliability, accuracy, and precision of mass spectrometry measurements. Optimal practice in the construction, evaluation, and implementation of a new calibration curve is often underappreciated. This systematic review examined how calibration practices are applied to liquid chromatography-tandem mass spectrometry measurement procedures.
Methods: The electronic database PubMed was searched from the date of database inception to April 1, 2022. The search terms used were "calibration," "mass spectrometry," and "regression." Twenty-one articles were identified and included in this review, following evaluation of the titles, abstracts, full text, and reference lists of the search results.
Results: The use of matrix-matched calibrators and stable isotope-labeled internal standards helps to mitigate the impact of matrix effects. A higher number of calibration standards or replicate measurements improves the mapping of the detector response and hence the accuracy and precision of the regression model. Constructing a calibration curve with each analytical batch recharacterizes the instrument detector but does not reduce the actual variability. The analytical response and measurand concentrations should be considered when constructing a calibration curve, along with subsequent use of quality controls to confirm assay performance. It is important to assess the linearity of the calibration curve by using actual experimental data and appropriate statistics. The heteroscedasticity of the calibration data should be investigated, and appropriate weighting should be applied during regression modeling.
Conclusions: This review provides an outline and guidance for optimal calibration practices in clinical mass spectrometry laboratories.

Keywords:  Calibration; Linearity; Mass spectrometry; Regression; Statistics

DOI:  https://doi.org/10.3343/alm.2023.43.1.5
J Am Soc Mass Spectrom. 2022 Sep 01.

Comparability of Steroid Collision Cross Sections Using Three Different IM-HRMS Technologies: An Interplatform Study.

Max L Feuerstein, Maykel Hernández-Mesa, Andrea Kiehne, Bruno Le Bizec, Stephan Hann, Gaud Dervilly, Tim Causon.

Steroids play key roles in various biological processes and are characterized by many isomeric variants, which makes their unambiguous identification challenging. Ion mobility-mass spectrometry (IM-MS) has been proposed as a suitable platform for this application, particularly using collision cross section (CCS) databases obtained from different commercial IM-MS instruments. CCS is seen as an ideal additional identification parameter for steroids as long-term repeatability and interlaboratory reproducibility of this measurand are excellent and matrix effects are negligible. While excellent results were demonstrated for individual IM-MS technologies, a systematic comparison of CCS derived from all major commercial IM-MS technologies has not been performed. To address this gap, a comprehensive interlaboratory comparison of 142 CCS values derived from drift tube (DTIM-MS), traveling wave (TWIM-MS), and trapped ion mobility (TIM-MS) platforms using a set of 87 steroids was undertaken. Besides delivering three instrument-specific CCS databases, systematic comparisons revealed excellent interlaboratory performance for 95% of the ions with CCS biases within ±1% for TIM-MS and within ±2% for TWIM-MS with respect to DTIM-MS values. However, a small fraction of ions (<1.5%) showed larger biases of up to 7% indicating that differences in the ion conformation sampled on different instrument types need to be further investigated. Systematic differences between CCS derived from different IM-MS analyzers and implications on the applicability for nontargeted analysis are critically discussed. To the best of our knowledge, this is the most comprehensive interlaboratory study comparing CCS from three different IM-MS technologies for analysis of steroids and small molecules in general.

DOI: https://doi.org/10.1021/jasms.2c00196
Bioinformatics. 2022 Sep 01. pii: btac596. [Epub ahead of print]

MetaboAnnotator: An efficient toolbox to annotate metabolites in genome-scale metabolic reconstructions.

Ines Thiele, German Preciat, Ronan M T Fleming.

MOTIVATION: Genome-scale metabolic reconstructions have been assembled for thousands of organisms using a wide-range of tools. However, metabolite annotations, required to compare and link metabolites between reconstructions remain incomplete. Here, we aim to further extend metabolite annotation coverage using various databases and chemoinformatic approaches.
RESULTS: We developed a COBRA toolbox extension, deemed MetaboAnnotator, which facilitates the comprehensive annotation of metabolites with database independent and dependent identifiers, obtains molecular structure files, and calculates metabolite formula and charge at pH 7.2. The resulting metabolite annotations allow for subsequent cross-mapping between reconstructions and mapping of, e.g., metabolomic data.
AVAILABILITY: MetaboAnnotator and tutorials are freely available at https://github.com/opencobra.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btac596
Clin Chem. 2022 Sep 03. pii: hvac135. [Epub ahead of print]

Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.

Jordi Farré-Segura, Caroline Le Goff, Pierre Lukas, Gaël Cobraiville, Marianne Fillet, Anne Catherine Servais, Pierre Delanaye, Etienne Cavalier.

   BACKGROUND: Parathyroid hormone (PTH) measurement is important for patients with disorders of calcium metabolism, including those needing bone-turnover monitoring due to chronic kidney disease-mineral bone disorder. There are currently 2 generations of PTH immunoassays on the market, both having cross-reactivity issues and lacking standardization. Therefore, we developed an LC-MS/MS higher-order method for PTH analysis.
METHODS: The method was calibrated against the international standard for 1-84 PTH (WHO 95/646). Antibody-free sample preparation with the addition of an isotope-labeled internal standard was performed by solid-phase extraction. Extracts were analyzed by LC-MS/MS. EDTA-K2 plasma was used throughout the development and validation. Bias and uncertainty sources were tested according to ISO 15193. Clinical Laboratory Standards Institute guidelines and reference measurement procedures were consulted for the design of the validation. Patient samples and external quality controls were compared between LC-MS/MS and 2 third-generation immunoassays.
RESULTS: The method was validated for 1-84 PTH from 5.7 to 872.6 pg/mL. The interassay imprecision was between 1.2% and 3.9%, and the accuracy ranged from 96.2% to 103.2%. The measurement uncertainty was <5.6%. The comparison between LC-MS/MS and the immunoassays showed a proportional bias but moderate to substantial correlation between methods.
CONCLUSIONS: This LC-MS/MS method, which is independent of antibodies, is suitable for a wide range of PTH concentrations. The obtained analytical performance specifications demonstrate that development of a reference measurement procedure will be possible once a higher order reference standard is available.

Keywords:  LC-MS/MS; intact parathormone; liquid chromatography; mass spectrometry; reference measurement procedure

DOI:  https://doi.org/10.1093/clinchem/hvac135
Genome Biol. 2022 Sep 01. 23(1): 184

MIRTH: Metabolite Imputation via Rank-Transformation and Harmonization.

Benjamin A Freeman, Sophie Jaro, Tricia Park, Sam Keene, Wesley Tansey, Ed Reznik.

  Out of the thousands of metabolites in a given specimen, most metabolomics experiments measure only hundreds, with poor overlap across experimental platforms. Here, we describe Metabolite Imputation via Rank-Transformation and Harmonization (MIRTH), a method to impute unmeasured metabolite abundances by jointly modeling metabolite covariation across datasets which have heterogeneous coverage of metabolite features. MIRTH successfully recovers masked metabolite abundances both within single datasets and across multiple, independently-profiled datasets. MIRTH demonstrates that latent information about otherwise unmeasured metabolites is embedded within existing metabolomics data, and can be used to generate novel hypotheses and simplify existing metabolomic workflows.

Keywords:  Imputation; Matrix factorization; Metabolomics; Missing data; Unmeasured metabolites

DOI:  https://doi.org/10.1186/s13059-022-02738-3
Metabolomics. 2022 Aug 29. 18(9): 71

Comprehensive plasma metabolomics and lipidomics of benign and malignant solitary pulmonary nodules.

Wei Zhou, Lili Lin, Lian-Yong Jiang, Jin-Long Wu, Wei-Chen Xu, Yang Zhou, Ma-Jie Wang, Xiang-Ming Cao, Hui-Qing Lin, Jian Yang, Li-Chun Deng, Zhi-Hao Zhang, Jin-Jun Shan.

   INTRODUCTION: Solitary pulmonary nodules (SPNs) are commonly found in imaging technologies, but are plagued by high false-positive rates.
OBJECTIVE: We aimed to identify metabolic alterations in SPN etiology and diagnosis using less invasive plasma metabolomics and lipidomics.
METHODS: In total, 1160 plasma samples were obtained from healthy volunteers (n = 280), benign SPNs (n = 157) and malignant SPNs (stage I, n = 723) patients enrolled from 5 independent centers. Gas chromatography-triple quadrupole mass spectrometry (GC‒MS) and liquid chromatography-Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometry (LC‒MS) were used to analyze the samples for untargeted metabolomics and lipidomics.
RESULTS AND CONCLUSION: GC‒MS-based metabolomics revealed 1336 metabolic features, while LC‒MS-based lipidomics revealed 6088 and 2542 lipid features in the positive and negative ion modes, respectively. The metabolic and lipidic characteristics of healthy vs. benign or malignant SPNs exhibited substantial pattern differences. Of note, benign and malignant SPNs had no significant variations in circulating metabolic and lipidic markers and were validated in four other centers. This study demonstrates evidence of early metabolic alterations that can possibly distinguish SPNs from healthy controls, but not between benign and malignant SPNs.

Keywords:  Less invasive diagnosis; Lipidomics; Metabolomics; Solitary pulmonary nodules

DOI:  https://doi.org/10.1007/s11306-022-01929-0
J Pharm Biomed Anal. 2022 Aug 20. pii: S0731-7085(22)00431-9. [Epub ahead of print]220 115010

Validation of an LC-MS/MS assay for the simultaneous determination of bictegravir, doravirine, and raltegravir in human plasma.

Amanda P Schauer, Craig Sykes, Mackenzie L Cottrell, Arkaitz Imaz, Daniel Podzamczer, Angela Dm Kashuba.

  Bictegravir (BIC), an integrase inhibitor, and doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, were recently approved by the US FDA for HIV treatment and are recommended first line treatment options. Because certain clinical scenarios warrant using them in combination, we developed a fully validated LC-MS/MS method for simultaneous measurement of BIC and DOR, along with a legacy integrase inhibitor, raltegravir (RAL), in human plasma over a clinically relevant 1000-fold range for each analyte. These analytes were extracted from the plasma by protein precipitation with their stable, isotopically labeled internal standards (BIC-d5, 13C6-DOR, and RAL-d6). Following extraction, samples were analyzed by reverse phase chromatography on a Waters Atlantis T3 C18 (50 ×2.1 mm, 3 µm particle size) column with subsequent detection by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The assay was linear (R2 >0.994) over the selected calibration ranges (20.0-20,000 ng/mL (BIC), 3.00-3000 ng/mL (DOR), and 10.0-10,000 (RAL)). The assay was accurate (inter-assay %Bias ≤ ± 8.5) and precise (inter-assay %CV ≤11.4). This method was validated according to FDA guidance for industry and can be used to assess the pharmacokinetics of two newly approved antiretrovirals, or to support therapeutic drug monitoring for modern antiretroviral therapy.

Keywords:  Antiretroviral; Bictegravir; Doravirine; Human Plasma; LC-MS/MS; Raltegravir

DOI:  https://doi.org/10.1016/j.jpba.2022.115010
Anal Chem. 2022 Aug 31.

Four-Dimensional Untargeted Profiling of N-Acylethanolamine Lipids in the Mouse Brain Using Ion Mobility-Mass Spectrometry.

Wenbin Liu, Wei-Dong Zhang, Tongzhou Li, Zhiwei Zhou, Mingdu Luo, Xi Chen, Yuping Cai, Zheng-Jiang Zhu.

N-Acylethanolamines (NAE) are a class of essential signaling lipids that are involved in a variety of physiological processes, such as energy homeostasis, anti-inflammatory responses, and neurological functions. NAE lipids are functionally different yet structurally similar and often have low concentrations in biological systems. Therefore, the comprehensive analysis of NAE lipids in complex biological matrices is very challenging. In this work, we developed an ion mobility-mass spectrometry (IM-MS) based four-dimensional (4D) untargeted technology for comprehensive analysis of NAE lipids. First, we employed the picolinyl derivatization to significantly improve ionization sensitivity of NAE lipids by 2-9-fold. Next, we developed a two-step quantitative structure-retention relationship (QSRR) strategy and used the AllCCS software to curate a 4D library for 170 NAE lipids with information on m/z, retention time, collision cross-section, and MS/MS spectra. Then, we developed a 4D untargeted technology empowered by the 4D library to support unambiguous identifications of NAE lipids. Using this technology, we readily identified a total of 68 NAE lipids across different biological samples. Finally, we used the 4D untargeted technology to comprehensively quantify 47 NAE lipids in 10 functional regions in the mouse brain and revealed a broad spectrum of the age-associated changes in NAE lipids across brain regions. We envision that the comprehensive analysis of NAE lipids will strengthen our understanding of their functions in regulating distinct physiological activities.

DOI: https://doi.org/10.1021/acs.analchem.2c02650
Rapid Commun Mass Spectrom. 2022 Sep 02. e9386

Developing Transmission Mode for IR-MALDESI Mass Spectrometry Imaging.

Alena N Joignant, Hongxia Bai, Jacob P Guymon, Kenneth P Garrard, Mark Pankow, David C Muddiman.

RATIONALE: The development and characterization of the novel NextGen infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) source catalyzed new advancements in IR-MALDESI instrumentation, including the development of a new analysis geometry.
METHODS: A vertically-oriented transmission mode (tm) IR-MALDESI setup was developed and optimized on thawed mouse tissue. Additionally, glycerol was introduced as an alternative energy-absorbing matrix for tm-IR-MALDESI since the new geometry does not currently allow for the formation of an ice matrix. The transmission mode was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm).
RESULTS: It was found that tm-IR-MALDESI produces comparable results to rm-IR-MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm-IR-MALDESI ion abundances.
CONCLUSIONS: This work has successfully demonstrated the adaptation of the NextGen IR-MALDESI source through the feasibility of tm-IR-MALDESI MSI on mammalian tissue, expanding future biological applications of the method.

DOI: https://doi.org/10.1002/rcm.9386
J Microbiol Methods. 2022 Aug 27. pii: S0167-7012(22)00156-7. [Epub ahead of print] 106561

A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass.

Rebecca Rocchi, Kees van Kekem, Walter H Heijnis, Eddy J Smid.

  Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B1 in fresh yeast biomass.

Keywords:  Extraction; Liquid chromatography; Saccharomyces cerevisiae; Thiamine; Thiochrome; Yeast

DOI:  https://doi.org/10.1016/j.mimet.2022.106561
Anal Chem. 2022 Aug 30.

Resolving Enantiomers of 2-Hydroxy Acids by Nuclear Magnetic Resonance.

Penghui Lin, Daniel R Crooks, W Marston Linehan, Teresa W-M Fan, Andrew N Lane.

Biologically important 2-hydroxy carboxylates such as lactate, malate, and 2-hydroxyglutarate exist in two enantiomeric forms that cannot be distinguished under achiral conditions. The D and L (or R, S) enantiomers have different biological origins and functions, and therefore, there is a need for a simple method for resolving, identifying, and quantifying these enantiomers. We have adapted and improved a chiral derivatization technique for nuclear magnetic resonance (NMR), which needs no chromatography for enantiomer resolution, with greater than 90% overall recovery. This method was developed for 2-hydroxyglutarate (2HG) to produce diastereomers resolvable by column chromatography. We have applied the method to lactate, malate, and 2HG. The limit of quantification was determined to be about 1 nmol for 2HG with coefficients of variation of less than 5%. We also demonstrated the method on an extract of a renal carcinoma bearing an isocitrate dehydrogenase-2 (IDH2) variant that produces copious quantities of 2HG and showed that it is the D enantiomer that was exclusively produced. We also demonstrated in the same experiment that the lactate produced in the same sample was the L enantiomer.

DOI: https://doi.org/10.1021/acs.analchem.2c00490