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Untargeted Metabolomics Workshop Report: Quality Control

Considerations from Sample Preparation to Data Analysis
Prasad Phapale,* Vineeta Rai, Ashok Kumar Mohanty, and Sanjeeva Srivastava
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ABSTRACT: The Proteomics Society, India (PSI), hosted the Metabolomics workshop on experimental and data analysis training
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for untargeted metabolomics in December 2019. The workshop included six tutorial lectures and hands-on data analysis training
sessions presented by seven speakers from across the globe. The tutorials and hands-on data analysis sessions focused on workflows
for liquid chromatography−mass spectrometry (LC-MS) based on untargeted metabolomics. We review here three main topics from
the workshop, which were uniquely identified as bottlenecks for new researchers: (a) experimental design, (b) quality controls
during sample preparation and instrumental analysis, and (c) data quality evaluation using open source tools. Our objective here is to
present common challenges faced by novice researchers and present guidelines to address them. We provide resources and good
practices for researchers who are at the initial stage of setting up metabolomics workflows in their laboratories.
KEYWORDS: untargeted metabolomics, workshop report, LC-MS/MS analysis, quality control

(IIT) Bombay was the convener of all “Omics” workshops

M etabolomics research laboratories and facilities are
rapidly growing due to the advances in high-resolution
mass spectrometry and allied techniques.1 As these technologies
evolve, there is an urgency to provide adequate training to new
researchers joining the metabolomics community.2 Recently,
proteomics, biochemistry, and pharmaceutical research labo-
ratories, which are well equipped with high-resolution mass
spectrometers, are increasingly adopting the metabolomics
analysis workflows. This has created a unique need for several
mass spec researchers, students, and pharmaceutical profes-
sionals to opt for “face-to-face” and/or e-learning training
courses to fill the experimental and informatics knowledge gaps
about metabolomics analysis. Several e-learning portals and
video tutorials for the experimentation and data analysis
protocols are available on our Web site.3 Further, to make the
learning sessions more interactive, we initiated a live webcast at
the Indian Institute of Technology (IIT), Bombay (see
Supporting Information). Such initiatives further demanded
face-to-face metabolomics training (experimental and data
analysis) similar to ASMS short courses and fall workshops.
Such an opportunity came through the preconference work-
shops organized at 11th Annual meeting of Proteomics Society,
India (1−5 December 2019) at the National Dairy Research
Institute (NDRI), Karnal, India. The proceedings of the
conference included plenary talks over the 3 days, with
preconference workshops. The Metabolomics workshop con-
ducted on “Education Day” (1 December 2019) was the first of
its kind, which comprehensively covered the experimental as
well as data analysis part of the workshop. Over 70 participants
from both academia and the industry across India with the
beginner or intermediate metabolomics skillsets attended the
workshop. The workshop was coordinated by Prasad Phapale
(European Molecular Biology Laboratory, Germany) and
assisted by Vineeta Rai (North Carolina State University,
USA). Sanjeeva Srivastava (Indian Institute of Technology
© XXXX American Society for Mass
Spectrometry. Published by the
American Chemical Society. All rights
reserved.
conducted during this meeting.
■ AGENDA AND PARTICIPANT SURVEY
The workshop agenda was inspired and structured in the lines of
the ASMS fall workshops and preconference short courses,
which are considered as one of the best resources for beginners
as well as advanced researchers in the field. The goal of the
present workshop was to provide participants with tools and
guidelines to setup experimental workflows and data analysis
pipelines in their own laboratories. The particular focus was
given on quality controls for sample preparation, instrument
analysis, and data processing. The main objective was to
highlight the challenges and possible errors during experimental
design, sample preparation, and metabolite annotations. The
workshop was systematically divided into experimental and data
analysis tutorials. The presenting speakers were Prasad Phapale,
Vineeta Rai, Yashwant Kumar (Translational Health Science
and Technology Institute, India), Shubhra Agarwal (Elucidata,
Inc.), Saurabh Nagpal (Agilent Technologies), and Ashok
Mohanty (NDRI, India). Lecture topics included an introduc-
tion to metabolomics workflows, sample preparation, exper-
imental design, data analysis tutorials, functional metabolic
measurements, and possible ways of integration of metabolo-
mics data with proteomics and transcriptomics for multiomics
analysis (agenda in Supporting Information). The workshop
started with the orientation of the participants and an icebreaker
Received: June 13, 2020
Revised: July 17, 2020
Accepted: July 23, 2020
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Journal of the American Society for Mass Spectrometry
lecture on the introduction to metabolomics by Vineeta Rai to
bring all the participants on the same page. The participants
were provided with survey forms, demo data sets, and digital
copies of additional reference material days prior to the
workshop. The survey revealed that the majority of participants
were using LC-MS instruments, particularly Orbitrap-based
mass spec technology for untargeted metabolite profiling
(Supporting Information). Thus, the prime focus of the
experimental part of the workshop was on untargeted
metabolomics workflows based on a Q Exactive mass
spectrometer. The biological samples of interest ranged from
plants to mammalian cells and tissue to biofluids (plasma/serum
and urine). The sample preparation, experimental design, and
quality control(s) part were given equal importance due to its
newness to researchers from the proteomics background
(Supporting Information with survey results). Following the
experimental section, the hands-on data analysis training was
provided using open-source El-Maven software, which is an
adaptation of original Maven software developed at Joshua
Rabinowitz lab for comprehensive Metabolomics data anal-
ysis.4,5 Participants were introduced to the conversion of the raw
data obtained from different mass spectrometry platforms to
.mzXML using a freely available msConvert tool.6 The data
analysis demonstrations were conducted by Shubhra Agarwal.5
Further, interactive sessions were conducted based on the
interest of the participants, specific challenges from their
respective projects, and their data sets. The coursework
materials presented were of a practical nature with tools, tips,
and troubleshooting in which participants can practice in their
laboratories.
The most common concerns were related to run-to-run
variations within the lab, sample preparation issues, and
assessing overall data quality. The common themes of the
questions from participants were as follows:
• How to deal with sample processing variations and
normalization issues?
• How to track variability during sample processing and
instrumental analysis?
• Is the quality of acquired LC-MS data acceptable?
• Can I trust metabolite annotations given by my software
and spectral library match?
Considering the outlined common questions, the overall
workshop and tutorials were then focused on three main topics.
(i) Experimental design: the selection of metabolomics
approach. (ii) Quality controls during sample preparation and
instrumental analysis. (iii) Data quality evaluation of metabolite
features and their annotations. The metabolite features were
defined as unique m/z and retention time (RT) pairs.
Instead of summarizing individual lectures and tutorials, this
report summarizes topics, ideas, and resources shared with
participants and attempts to address the above-mentioned
common challenges faced during the initial setup of untargeted
metabolomics workflows. This report can be a brief guide and
resource for novice metabolomics researchers and students.
■ EXPERIMENTAL DESIGN AND SELECTION OF
UNTARGETED METABOLOMICS APPROACH
The major goal of the experimental design should be to
minimize as well as track the technical or analytical variability
and capture the biological variations among the sample groups.
To track the analytical variability, participants were strongly
advised to use process blanks, quality control samples (QC)
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with randomized samples analysis sequence as described in the
next section.4,5 Before planning the experiments, it is
recommended to discuss with biologists and statisticians to
decide on numbers of samples and replicates required to get a
statistical significance among the experimental groups. The
selection of untargeted metabolomics platforms should be given
proper thought due to challenges, pitfalls, and the amount of
time required for downstream data analysis. The simple question
researchers can ask is “do I have a hypothesis to test?” or “do I
have metabolites or biochemical pathways to target?”. If yes,
then it is always a good idea to use targeted metabolomics
methods due to their reliability, throughput, and quality of data.6
The untargeted approach is more suitable for hypothesis
generation.7 We also proposed a semitargeted approach where
one can focus on a broad set of metabolites for which good
quality MS/MS and RT libraries are already generated or
publicly available. Also, with semitargeted analysis, there is a
possibility of retrospectively mining the data for additional
metabolites and plan follow-up experiments.
■ QUALITY CONTROLS DURING SAMPLE
PREPARATION AND INSTRUMENTAL ANALYSIS
(a) Sample Processing. Among the participants, the sample
preparation and quality control were the most common
concerns for untargeted metabolomics analysis. In order to
derive meaningful biological conclusions, particularly for
longitudinal studies, the reproducibility of metabolomics data
is of great importance. The irreproducibility of untargeted
metabolomics data sets is often frustrating for new researchers.
The participants were using a variety of sample types in their
laboratories (see Supporting Information for survey results).
They were advised to consider the sample collection, quenching,
storage, and processing based on specific sample types. Some
researchers work with mass spec facilities for the LC-MS
analysis, and they were advised on shipment, freeze−thaw
stability, and storage requirements of metabolomic samples.
Participants were encouraged to read sample-specific storage,
shipments, and preprocessing guidelines prior to planning the
experiments.8
(b) Sample Amount Normalization. There was a concern
about tracking the variability of biological amounts in samples
such as cell numbers and dry weight. Although there is no
standard method available for sample amount normalization,
various approaches were suggested based on sample type, which
gives an estimation of sample amount (Figure 1). The cell
number counting and dry sample weights should always be
preferred as a measurement of the sample amount. However,
such measurements are often not possible under given
Figure 1. Considerations and measures to track variability during
sample preparation and instrumental analysis for untargeted metab-
olomics workflow.
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Journal of the American Society for Mass Spectrometry
experimental designs and alternative protein or DNA measure-
ments, and a UV assay can be used as an estimate of sample
amounts. These orthogonal measurements can be used for
preacquisition corrections of the sample amounts to minimize
their effect on individual metabolite levels.9
(c) Sample Preparation. During the preparation of the
samples, participants were advised to use two-stage internal
standard (ISD) systems to track variations in sample preparation
and analytical instrumentation. The first set of internal standards
should be spiked before the sample processing and another set of
internal standards can be included during sample reconstitution
before LC-MS injections to track sample preparation and
instrumental variations, respectively. It is also emphasized to
process a blank in the same way as other samples (process blank)
in order to account for background peaks appearing from sample
processing. The equal amounts of samples from the same cohort
can be mixed to prepare pooled quality controls (QC) and
process them along with the sample batch. Further guidelines
were given for optimization of extraction protocols for metabolic
quenching, recovery, and stability based on sample types and
metabolites of interest (Figure 1 and Supporting Information).
(d) Instrumental Analysis. Although most of the
participants were familiar with mass spec instruments, they
were unfamiliar with the concepts such as pooled QC, two-stage
ISD, LC-MS system conditioning, and data quality evaluation.
We demonstrated the use of pooled QC as a powerful approach
to tracking the intrabatch analytical variability using PCA plots
visualization and setting standard deviation limits for selected
features. The QC samples can also be used for the conditioning
of the LC-MS system to ensure its stability before starting the
analysis sequence.5 Although some recommendations about
ISDs, system suitability standards, technical replicates, number
of QCs, and acceptance criteria (Figure 1) were provided,
participants were encouraged to test the suitability for these
methods for their platforms.
■ DATA QUALITY EVALUATION
The data analysis, metabolite identification, and evaluation of
data quality are significant challenges in metabolomics research.
The hands-on data analysis section on El-Maven was conducted
by Shubhra Agarwal.10,11 The data sets for the training were
generated at the EMBL Metabolomics Core facility and available
on MetaboLights Repository study number MTBLS1301.1,10
The focus was on the conceptual understanding of the software
parameters, manual evaluation of software output, peak
curation, and validation of metabolite annotations. A database
search for metabolite identification with different available mass
spectral libraries was performed, including open EMBL-MCF
LC-MS/MS spectral library and databases from MassBank of
North America (MoNA).12 The impact of different spectral
libraries, spectral matching scores, and possibilities of mis-
annotations was demonstrated. The use of quality MS/MS
databases, along with RTs from standards, was recommended to
avoid false-positive metabolite identification. It was striking for
the proteomics researchers to know that, unlike automated
peptide identification pipelines, metabolite identifications due
to their inherent chemical complexity still require expert
evaluations as there are no established false discovery rate
criteria.
(a) Feature Quality Evaluation. The software mostly
generates a list of thousands of features from an LC-MS run;
however, many of these features correspond to artifacts from
chemical noise, contaminants, instrument background, and
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redundant metabolite signals (isotopes, in-source fragmenta-
tion, adducts).13 Such artifacts and feature redundancy were
demonstrated to the participant from process blanks and known
standard mixture data sets. The parameters such as chromato-
graphic peak shapes (peak width and symmetry), signal
intensities, variation in QC injections, and presence in process
blank can be considered for feature quality evaluation (Figure
2). El-Maven software assists feature quality evaluation though
Figure 2. Data quality evaluation guidelines.
peak quality measures, chromatogram overlays, and visualization
which allows users to flag off bad peaks before further data
analysis. It was strongly advised during initial method develop-
ment to spend time on such evaluations and get trained in data
quality inspections as these factors are greatly influenced by
sample types, protocols, and LC-MS methods used.
(b) Validation of Metabolite Annotation. The critical
aspects of reporting metabolite identification levels proposed by
the Metabolite Standard Initiative (MSI) were explained to the
participants.7,14 The automated metabolite identification from a
software often performs miss annotations of LC-MS peaks or
features. The earlier described feature quality filtration process
can significantly avoid noisy features and false annotations. Also,
the in-house-generated RT and MS/MS data from standards
(LC-MS/MS libraries) can be a gold standard for confident
annotations of features from untargeted metabolomics data sets.
In the absence of such customized libraries, researchers are
advised to use public MS/MS databases.15 However, caution
must be taken to ensure the quality of MS/MS matches and
verify those identifications with authentic standards whenever
possible. Another common concern was the lack of MS/MS data
for many features in an LC-MS run due to MS/MS acquisition
speed and coeluting peaks. It was suggested to perform solid-
phase extraction or fractionation to reduce the sample
complexity and conduct targeted MS/MS acquisition (e.g., use
of inclusion and exclusion lists) for the features of interest
(Figure 2). The participants were also suggested the settings of
DIA and DDA scans to generate comprehensive MS/MS
databases from their representative samples.
■
ATTENDEE FEEDBACK
Attendees were provided anonymous feedback after the
workshop via an online survey form. Overall, more than 80%
of participants found the workshop useful and were willing to
attend and recommend such workshops in the future.
Participants were suggested to have extended duration (4−5
days) of the training and include lipidomics workflows with
more extensive data analysis sessions.
■
CONCLUDING REMARKS
Metabolomics is a fast-growing field with a wider implication in
major scientific research areas. When the metabolomics data is
combined with other “omics’’ data sets, especially proteomics, it
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Journal of the American Society for Mass Spectrometry
provides better insights into the biological processes. During the
past decade, due to government support, many new mass
spectrometry laboratories and facilities have been setup in India.
Thus, many laboratories with mass spec facilities have gained
momentum to perform metabolomics analysis. This has led to
the organization of this metabolomics workshop in the
Proteomics Society, India (PSI), conference. Also, there is a
growing interest among pharmaceutical researchers, bioanalyt-
ical scientists, and drug discovery professionals to setup LC-MS
metabolomics workflows within their bioanalytical labs.
However, due to the chemical diversity of the metabolome,
various experimental considerations are needed before embark-
ing on a metabolomics study. The particular consideration
should be given to metabolomic experimental designs with
quality controls and data evaluation criteria. The biological
sample-specific preparation and normalization protocols should
be optimized to reduce variability while capturing the dynamics
of cellular metabolism. This workshop report thus highlights
some guidelines addressing practical challenges for mass
spectrometrists who are a novice to the metabolomics
workflows. We emphasize that, although the LC-MS-based
“untargeted metabolomics” is appealing, whenever possible, the
experiments should be hypothesis-driven “targeted metabolo-
mics” and not resource-driven. When setting up untargeted
metabolomics workflows, each lab should have its own sample or
project-specific standard practices such as use of two-stage ISD,
pooled QCs, and data quality evaluation criteria. Such practices
can generate quality data sets that should be validated for
features and their annotations using suitable data analysis
platforms. We strongly encourage submitting such standardized
workflows and data sets to the public repositories, which will
guide new laboratories to establish untargeted metabolomics.1
These data sets along with standard mass spectral libraries can be
used as a benchmark in optimizing and developing computa-
tional tools to automate the metabolite annotation and data
quality evaluation processes. All of these community efforts
should lead to the establishment of quality control guidelines for
metabolomics analysis similar to “FDA Bioanalytical Method
Validation Guidance for Industry”.16 Such guidelines and open-
source computational resources can ease the transition of new
mass spec laboratories to metabolomics research and train
existing laboratories in generating and reporting quality-
controlled results. The workshops similar to this one can
provide training to proteomics, pharmaceutical, and metab-
olomics researchers within the mass spec community and are
extremely important in the future to exchange ideas, challenges,
resources, and to provide guidelines.
As the “omics” research is moving toward the integrated-
omics approach to get a “systems-level” understanding of
biological processes, such cross-disciplinary training is of
immense value. Specific modules on lipidomics workflows due
to the significance of lipid research could be an important
addition to such workshops. Moreover, considerable efforts are
required in the training and development of data analysis tools
for reliable and efficient extraction of biological information
from mass spec data. The multi-omics data integration challenge
is still huge and will require effort across research communities.
The report summarizes our perspective from the workshop
and brief guidelines for the novice researchers to optimize
metabolomics workflows from sample preparation to data
analysis.
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ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jasms.0c00224.
Resources from the PSI metabolomics workshop (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Prasad Phapale − Metabolomics Core Facility, European
Molecular Biology Laboratory, Heidelberg 69117, Germany;
orcid.org/0000-0002-9487-597X; Email: prasad.phapale@
embl.de
Authors
Vineeta Rai − Department of Entomology and Plant Pathology,
North Carolina State University, Raleigh, North Carolina 27695,
United States
Ashok Kumar Mohanty − Animal Biotechnology Centre,
National Dairy Research Institute, Karnal, Haryana 132001,
India
Sanjeeva Srivastava − Proteomics Laboratory, Department of
Biosciences and Bioengineering, Indian Institute of Technology
Bombay, Mumbai 400076, India; orcid.org/0000-0002-
0651-4438
Complete contact information is available at:
https://pubs.acs.org/10.1021/jasms.0c00224
Notes
All training material is available on Github (https://github.
com/prasadpb/Workshop). The demo data files used during
data analysis sessions are available on MetaboLights Repository
study number MTBLS1301.
■ ACKNOWLEDGMENTS
We are thankful to the Proteomic Society, India (PSI);
organizing committee ICAR-NDRI; students of Protein Science
Lab, Animal Biotechnology Centre, ICAR-NDRI. We are
thankful for the valuable feedback and guidance from Rose
Gathungu and Theodore Alexandrov of the EMBL Metab-
olomics Core Facility staff.
■
REFERENCES
(1) Kale, N. S.; Haug, K.; Conesa, P.; Jayseelan, K.; Moreno, P.;
Rocca-Serra, P.; Nainala, V. C.; Spicer, R. A.; Williams, M.; Li, X.; Salek,
R. M.; Griffin, J. L.; Steinbeck, C. MetaboLights: An Open-Access
Database Repository for Metabolomics Data. Curr. Protoc. Bioinfor-
matics 2016, 53, 14.13.1−14.13.18.
(2) Weber, R. J. M.; Winder, C. L.; Larcombe, L. D.; Dunn, W. B.;
Viant, M. R. Training Needs in Metabolomics. Metabolomics 2015, 11
(4), 784−786.
(3) EMBL. Metabolomics Core Facility video tutorials https://www.
embl.de/mcf/metabolomics-core-facility/video-tutorials/index.html
(accessed 2020-03-29).
(4) Begou, O.; Gika, H. G.; Theodoridis, G. A.; Wilson, I. D. Quality
Control and Validation Issues in LC-MS Metabolomics. In Metabolic
Profiling: Methods and Protocols; Theodoridis, G. A., Gika, H. G.,
Wilson, I. D., Eds.; Springer: New York, 2018; pp 15−26.
(5) Broadhurst, D.; Goodacre, R.; Reinke, S. N.; Kuligowski, J.;
Wilson, I. D.; Lewis, M. R.; Dunn, W. B. Guidelines and Considerations
for the Use of System Suitability and Quality Control Samples in Mass
Spectrometry Assays Applied in Untargeted Clinical Metabolomic
Studies. Metabolomics 2018, 14 (6), 72.
(6) Adusumilli, R.; Mallick, P. Data Conversion with ProteoWizard
msConvert. Methods Mol. Biol. 2017, 1550, 339−368.
J. Am. Soc. Mass Spectrom. XXXX, XXX, XXX−XXX
Journal of the American Society for Mass Spectrometry pubs.acs.org/jasms Conference Review

(7) Schrimpe-Rutledge, A. C.; Codreanu, S. G.; Sherrod, S. D.;

McLean, J. A. Untargeted Metabolomics Strategies-Challenges and
Emerging Directions. J. Am. Soc. Mass Spectrom. 2016, 27 (12), 1897−
1905.
(8) Kirwan, J. A.; Brennan, L.; Broadhurst, D.; Fiehn, O.; Cascante,
M.; Dunn, W. B.; Schmidt, M. A.; Velagapudi, V. Preanalytical
Processing and Biobanking Procedures of Biological Samples for
Metabolomics Research: A White Paper, Community Perspective (for
“Precision Medicine and Pharmacometabolomics Task Group”-The
Metabolomics Society Initiative). Clin. Chem. 2018, 64 (8), 1158−
1182.
(9) Wu, Y.; Li, L. Sample Normalization Methods in Quantitative
Metabolomics. J. Chromatogr. A 2016, 1430, 80−95.
(10) Rusu, P.; Shao, C.; Neuerburg, A.; Acikgöz, A. A.; Wu, Y.; Zou,
P.; Phapale, P.; Shankar, T. S.; Döring, K.; Dettling, S.; Körkel-Qu, H.;
Bekki, G.; Costa, B.; Guo, T.; Friesen, O.; Schlotter, M.; Heikenwalder,
M.; Tschaharganeh, D. F.; Bukau, B.; Kramer, G.; Angel, P.; Herold-
Mende, C.; Radlwimmer, B.; Liu, H.-K. GPD1 Specifically Marks
Dormant Glioma Stem Cells with a Distinct Metabolic Profile. Cell
Stem Cell 2019, 25, 241.
(11) Agrawal, S.; Kumar, S.; Sehgal, R.; George, S.; Gupta, R.; Poddar,
S.; Jha, A.; Pathak, S. El-MAVEN: A Fast, Robust, and User-Friendly
Mass Spectrometry Data Processing Engine for Metabolomics. In High-
Throughput Metabolomics: Methods and Protocols; D’Alessandro, A., Ed.;
Springer: New York, 2019; pp 301−321.
(12) Palmer, A.; Phapale, P.; Fay, D.; Alexandrov, T. Curatr: A Web
Application for Creating, Curating, and Sharing a Mass Spectral
Library. Bioinformatics 2018, 34, 1436.
(13) Baker, E. S.; Patti, G. J. Perspectives on Data Analysis in
Metabolomics: Points of Agreement and Disagreement from the 2018
ASMS Fall Workshop. J. Am. Soc. Mass Spectrom. 2019, 30 (10), 2031−
2036.
(14) Sumner, L. W.; Amberg, A.; Barrett, D.; Beale, M. H.; Beger, R.;
Daykin, C. A.; Fan, T. W.-M.; Fiehn, O.; Goodacre, R.; Griffin, J. L.;
Hankemeier, T.; Hardy, N.; Harnly, J.; Higashi, R.; Kopka, J.; Lane, A.
N.; Lindon, J. C.; Marriott, P.; Nicholls, A. W.; Reily, M. D.; Thaden, J.
J.; Viant, M. R. Proposed Minimum Reporting Standards for Chemical
Analysis Chemical Analysis Working Group (CAWG) Metabolomics
Standards Initiative (MSI). Metabolomics 2007, 3 (3), 211−221.
(15) Vinaixa, M.; Schymanski, E. L.; Neumann, S.; Navarro, M.; Salek,
R. M.; Yanes, O. Mass Spectral Databases for LC/MS- and GC/MS-
Based Metabolomics: State of the Field and Future Prospects. TrAC,
Trends Anal. Chem. 2016, 78, 23−35.
(16) Meesters, R. J. W.; Voswinkel, S. Bioanalytical Method
Development and Validation: From the USFDA 2001 to the USFDA
2018 Guidance for Industry. Journal of Applied Bioanalysis. 2018, 4, 67−
73.

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