Posted by: tvasailor | May 24, 2012

Overview “Known Unknowns”

New 9-29-20:
Use of Commercial and User EI Libraries with NIST Software for Unknown Characterization

New 11/1/2020:  Differences in EI GC Orbitrap and Standard Quadrupole Spectra

Systematic Process for the Identification of “Known Unknowns” in Commercial Products by GC-MS and LC-MS

Note: Details on other subjects are found in “My Topics” tab under sailboat picture above or on the sidebar links to the right..

Introduction:  In the last 34 years, we have developed a systematic process for the identification of “known unknowns” by GC-MS and LC-MS in commercial products.  We define “known unknowns” as non-targeted species which are known in the chemical literature or mass spectrometry reference databases, but unknown to the investigator.  The process is shown in the following simplified flowchart:


This process is described in detail in the February 2013 copy of LCGC, “MS-The Practical Art.”  The article is entitled:  “Identifying “Known Unknowns” in Commercial Products by Mass Spectrometry.”  A copy with associated ads is shown below:

LCGC PDF with Advertisements

The article originated from work presented at Pittcon in 2012.

NIST Search of EI and CID Spectra:  The initial step in our process utilizes computer searches of EI (GC-MS) or CID (LC-MS) spectra against reference databases using the NIST MS search.  The computer EI searches normally work better than CID ones, but the latter are still very useful.  We employ both purchased, in-house, and internet databases:

We use the NIST and Wiley commercial databases, but there are many other specialty databases that others might find useful.

The NIST search interfaces easily with a wide variety of manufacturers’ data processing and structural drawing programs:


“Spectra-Less” Database Searching:  If the NIST search is not successful, then accurate mass data is used to obtain a molecular formula (MF), a monoisotopic mass, or an average molecular weight.  This data is used to search very large databases such as the CAS Registry or ChemSpider  via web interfaces.  We define them as “spectra-less” databases because they contain no computer-searchable mass spectral data.  We had originally used this approach to search the TSCA database or our Eastman Chemical plant material listing.

The candidate structures from the CAS Registry or ChemSpider searches are prioritized by either the number of associated references or key words.  Other ancillary information such as mass spectral fragments in EI or CID spectra; isotopic abundances, UV spectra; types of ion adductsCI data; number of exchangeable protons;  etc. are used to narrow the list to one structure.  This website has many screenshots (SciFinder1, SciFinder2, ChemSpider) that illustrate these approaches with many examples.

Model EI and CID Spectra from NIST Structure Search:  The NIST MS Search program ranks model compounds employing structural searching of both our commercial and in-house databases.  This is particularly valuable for finding model compounds for a proposed structure found in searches of “spectra-less” databases such as the CAS Registry and ChemSpider.

As noted in the table above, there are ~800,000 structures associated with the EI spectra and 100,000 structures with CID mass spectra in our computer-searchable databases.  We use the NIST MS Interpreter  program to automatically correlate fragment ions in the EI and CID spectra with the component’s substructure.

“No Results” from Process:  There will be non-targeted species in the sample which are “unknown unknowns”, those not found in any reference libraries or “spectra-less” databases.  A few thoughts on their identification are discussed in another section.

Future Improvements Needed:  The approach works well for the majority of our samples which are fairly simple and contain components with molecular weights <500 daltons.  On the other hand, improvements are needed for complex samples and components with molecular weights >500 daltons.

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Above Image from Wikipedia

The Thermo GC Orbitrap is a very powerful tool for the identification of unknowns easily switching from EI to CI modes. However, one must be aware of the differences in the EI spectra obtained with the Orbitrap compared to standard quadrupole and magnetic spectra found in commercial and user libraries.

The following links shed some insight into those differences from a mechanistic point of view.

Study Comparing 480 Spectra

Literature Reference

Thermo and Personal User’s Libraries:

Thermo has and on-going effort to create a database of EI GC-Orbitrap spectra. Release Version 1.0 contains 766 spectra. They are spectra of contaminants related to PCB’s, environmental contaminants, and pesticides.

A user should also consider adding spectra to their personal NIST library noting the origin of the spectrum. I believe that in 70-80% of the cases the hit will be lower (700-800 fit). If no other spectrum from a standard quadrupole is present, the user will still find the EI orbitrap spectrum at the top of the list. Smaller MW compounds and TMS derivatives might be more confusing yielding much lower hits.

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Posted by: tvasailor | October 11, 2020

Excel DeltaMass Table for Hybrid Searches

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The Excel spreadsheet of DeltaMass values is very useful for determining the identity of a compound in a NIST hybrid EI or tandem library search. The values listed in the spreadsheet are common ones that I have found in my evaluation of EI library spectra. They would be even more useful when employing in conjunction with accurate mass measurements.

An odd DeltaMass value is indication that the number of nitrogens has changed by an odd number. Similar to nitrogen rule for molecular ions and their fragments in EI interpretation.

Excel DeltaMass Table

YouTube and Associated Handout for NIST Hybrid EI Search

YouTube Advanced NIST Hybrid Search of EI and MS/MS Spectra
Handout NIST Hybrid Search of EI and MS/MS Spectra-9-27-20

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Posted by: tvasailor | October 6, 2020

Agilent Deconvolution Reporting Software (DRS)

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Agilent DRS combines Chemstation-like software with AMDIS and NIST search to generate customer reports of target compound analyses.

Link to Software Products

Links to YouTube Videos

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Posted by: tvasailor | September 30, 2020

Downloads of NIST Software/Libraries/Tools

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All these resources can be found at:
NIST Libraries/Tools/Service Web Site

NIST Search Software 2.3 Version (2017) and Demo Library:
Download Installation Program

MS Interpreter Version 3.4:
Download Version 3.4

AMDIS Version 2.73:
Download AMDIS

Free Libraries and Tools:

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Posted by: tvasailor | September 21, 2020

NIST’s Tutorials and Suggested Settings for AMDIS Filters

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Gary Mallard wrote a series of articles including suggested filter settings in AMDIS. In my work, I have found these filter settings to be very useful for limiting the number of peaks deconvoluted.

The links are noted at the bottom of this post for the series of articles. I have captured the suggested filter settings and his comments below which were found in Figure 4 for Part 3 of his series.

AMDIS: Setting Up and Running a Deconvolution and Target Analysis-Part 1
AMDIS: Setting Up and Running a Deconvolution and Target Analysis-Part 2
AMDIS: Setting Up and Running a Deconvolution and Target Analysis-Part 3

Other AMDIS tips from NIST:

Developing Libraries
Extracting HIgh-Quality Spectra from Complex GC/MS Data
Determining Retention Indices Powerpoint
Determining Retention Indices Text Explanation

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Posted by: tvasailor | September 20, 2020

Wiley/NIST Webinar: Example MS Data Files and Spectra

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I used several test files and spectra in the Wiley Webinars to demonstrate the software. I have placed them here if you would like to reproduce my work.

The spectra are stored in a NIST library format. So you must download them and then transfer them to SpecList for searching using the following instructions:

Downloading/Using Example Spectra

Files and Spectra in Zip Files:

Example MS Data Files for Part III AMDIS Webinar
Example Spectra/Structures in Library Format Parts I-V

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Posted by: tvasailor | September 19, 2020

Mass Spec Training from David Sparkman

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David Sparkman has trained many in the “art” of mass spectrometry. He is an Adjunct Professor at the University of the Pacific in Stockton, California, and an independent contractor for NIST and Wiley.

Courses and Books

Mass Spec Training Courses

Introduction to Mass Spectrometry: Instrumentation, Applications, and Strategies for Data Interpretation

Gas Chromatography and Mass Spectrometry: A Practical Guide

Mass Spectrometry Desk Reference

Posted by: tvasailor | September 11, 2020

Wiley/NIST Webinar YouTube Channel

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My YouTubes:

I have recorded my own personal versions of the 5-part Wiley webinar series for using NIST software with Wiley and NIST libraries.

Part I: Spectral Searches with NIST MS Search
Part II: Structure Searches with MS Search and Using MS Interpre
Part III: AMDIS (NIST) for Processing EI Mass Spectral Data Files

Part IV: Advanced NIST Hybrid Search of EI and MS/MS Spectra
Part V: Creating and Sharing User EI and MS/MS Libraries

This allowed me to more easily change between different applications while demonstrating the software. In addition, the NIST windows are of higher quality created with my Zoom application versus Wiley’s WorkCast approach.

YouTube Videos from Others:

Agilent DRS -AMDIS/NIST Search/Customer Reports
Importing Shimadzu GC/MS Data into AMDIS and NIST
Accessing NIST Library Search in Agilent GC-MSD

Original Wiley Videos:

Webinars recorded by Wiley

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Posted by: tvasailor | August 20, 2020

Most Current User Manuals for NIST Search and AMDIS

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nist-amdis picI try to keep the most current manuals for AMDIS and NIST MS Search as they are updated.  Sometimes they could appear here before actually updated on NIST site.  Makes it convenient for keeping my links in presentations current.  See the links below:



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I presented a 5-part Wiley Webinar Series in August-September, 2020.  The series demonstrates the effective use of NIST software such as MS Search, MS Interpreter, and AMDIS with the Wiley, NIST, and user-created libraries.  The following links are for either my personal YouTube or Wiley recordings:

YouTube Recordings of Parts I-V

Wiley Website and Recorded Videos

Detailed Handouts including tables of content are shown below:

Part I: Spectral Searches with NIST MS Search-9-27-20
Part II: Structure Searches with MS Search and Using MS Interpreter
Part III:  AMDIS (NIST) for Processing EI Mass Spectral Data Files 9-22-20

Part IV: Advanced NIST Hybrid Search of EI and MS/MS Spectra-9-27-20
Part V: Creating and Sharing User EI and MS/MS Libraries

Other Webinar Resources:

DeltaMass Table for Hybrid Search in EI-9-21-20-(252 entries)
NIST Tutorials and Settings for AMDIS-10-15-20
Importance of Library Searches in the Chemical Industry
Example Files for Part III AMDIS Webinar
Example Spectra/Structures in Library Format Parts I-V
Wiley Library User Manual with Spectral Source Codes
NIST Free Downloads of Software/Libraries/Tools-9-29-20
AMDIS and NIST Search User Manuals-9-7-20
Description of NIST2020 Software and Library Release
by David Sparkman
MS Interpretation Training by David Sparkman

Wiley, NIST, and user-created mass spectral libraries are very powerful tools for the identification of organic compounds in complex mixtures by gas chromatography-mass spectrometry (GC-MS).  This series of seminars will demonstrate the use of the NIST (National Institute of Standards and Technology) software suite for searching these EI libraries.  The software includes MS Search, MS Interpreter, and AMDIS plus ChemSketch for structural drawing. The series should be useful to a wide variety of research and developmental scientists involved in medical, environmental, industrial, forensic, and biological disciplines.

Hybrid Search Resources from Others:

Gary Mallard from NIST a “real-world” example of a fentanyl-related compound from his work.

Hybrid EI Fentanyl Example

Arun Moorthy and Gary Mallard also presented an excellent workshop showing applications of the hybrid search in forensic applications.  The PDF file used in the seminar are shown below.  One will have to register on the website to see the video.

Hybrid vs. Similar Search Slides in PDF:  Identifying Seized Drugs Using Mass Spectral Library Searching
Hybrid vs. Similar Search Webinar:  Identifying Seized Drugs Using Mass Spectral Library Searching

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