Posted by: tvasailor | May 24, 2012

Overview “Known Unknowns”




New 1/2/2021:  Unknown Identification Using NIST Search and EI and MS/MS (Tandem) Libraries

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:

flowchart3

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.

I have developed a two “webinar” series courses that detail the use of the NIST search with EI and tandem libraries:

Link to free webinar courses

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:

types_of_databases_searches
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:

nist_search

“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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Posted by: tvasailor | January 11, 2021

Bread Recipes from Betty Ottenfeld

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In the early 80’s, my Wife Sandra took a bread baking course at the Presbyterian Church. Our family was highly appreciative. Sandra scanned the cookbook handout from the course.

Link to Cookbook Handout in PDF

Picture of Betty, a bird-lover, below:

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Posted by: tvasailor | January 1, 2021

Buying NIST “20” EI and Tandem (MSMS) Libraries

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The new libraries for 2020 include the NIST MS Search Software at no additional cost.

There were major increases in the number of compounds and major software enhancements.

Link to 2020 NIST Updates

The NIST 2020 Tandem library is ridiculously inexpensive, very high quality, and contains 1.3 M spectra!

Be sure to shop around. Here is the total list of distributors:

Total List of Distributors

Here are some good websites that I have noted for distributors. Let me know if you have found others.

Diablo Analytical (convenient same-day ftp download)
Wiley
Adaptas Solutions (formerly SIS)
Cerno Biosciences
GC Image
MS Wil
SpectralWorks
Stanton Scientific

The EI library package contains Lib2NIST utility, but not the Tandem one.

Link for Lib2NIST Utility

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MS/MS (Tandem) spectra can be used to identify unknowns employing library searches. This is accomplished in much the same approach as that employed for EI GC-MS with the NIST search software. The much improved NIST Search Version 2.4 is included with the 2020 library release. I will attempt to introduce the user to the use of the NIST Version 2.4 search software employing NIST, Wiley, “crowd-sourced (MoNA)”, and user libraries for the identification of unknowns.

NIST has an ambitious program to extend their already comprehensive, high-quality MS/MS databases. See the following link:

NIST Pipeline for Extending MS/MS Libraries

This involves a very comprehensive process for selecting pertinent compounds for purchase and subsequent analyses. The problem with variability of the spectra is reduced by analyzing the samples on a variety of instruments at a variety of energy levels (20 steps). In addition, many different precursor ions and fragment ions are characterized (MSn). This has already led to a very large high quality library of accurate mass MS/MS spectra in the 2020 release version (31K compounds/1.3 M spectra) and the effort is ongoing.

YouTube Videos

Part I: Overview (17 min)
Part II: NIST MS/MS Search (18 min)
Part III: Detailed Discussion Hybrid MS/MS Search (18 min)
Part IV: Importing MSMS Spectra (11 min)
Part V: NIST Structure Searches (12 min)
Part VI: MS Interpreter (12 min)
Part VII: Creating-Using MSMS Libraries (12 min)
Part VIII: “Spectraless Libraries” (12 min)

Higher Quality Videos Zipped Pt. I-VIII

Detailed Handouts

Part I: Overview 12/27/20
Part II: NIST MSMS Search 12/27/20
Part III: Detailed Discussion Hybrid MS/MS Search 12/27/20
Part IV: Importing MSMS Spectra 12-27-20
Part V: NIST Structure Searches 12-27-20
Part VI: MS Interpreter 12-27-20
Part VII: Using-Creating MSMS Libraries 12-27-20
Part VIII: “Spectraless” Libraries 12-27-20

Handouts Pt. I-VIII_Zipped-12-27-20

Resources

Buy a copy! Distributors of NIST Libraries Shop Around for Best Price!
NIST Tandem Quick-Start Guide
DeltaMass Table in Nominal Mass (hopefully accurate mass in future?)
Beyond the Top Hit: Information from Hybrid Similarity Search Hit Lists
Lib2NIST (not included with NIST tandem library)
Importing Spectra to NIST Search (all internet links)
Components in MoNA Libraries
MoNA MSMS Library in NIST Format
MoNA EI Library in NIST Format
MoNA Download Site in MSP and SDF Formats
Lib2NIST_documentation
Resources for NIST EI Searches
PowerShell Script for Adding Precursor_m/z Field to User Library
Users’ Manual for NIST 2.4
NIST Downloads Libraries-Tools-Software
NIST MS 2020 at ASMS

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Posted by: tvasailor | December 11, 2020

Identifying Unknowns Using LCMS and GCMS with Library Searching

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This is a free instructional series including YouTubes, detailed handouts, and miscellaneous resources.

EI GC-MS Identifications: The first part is for EI GC-MS identifications and is accessed at the following link:

Link to EI GC-MS ID of Unknowns with NIST Search

LC-MSMS Identifications: The second part is for LC-MS-MS identifications and is accessed at the following link:

Link to LC-MS-MS ID of Unknowns with NIST Search

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Thanks to Wikipedia for Graphics in Header

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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 are also finding to be useful in accurate mass Tandem (MS/MS) hybrid searches in LC-MS analyses.

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 (Jan 2021, 502 entries)

YouTube and Associated Handout for NIST Hybrid EI Search

YouTube Advanced NIST Hybrid Search of EI Spectra
YouTube Advance NIST Hybrid Search for MS/MS (Tandem) Spectra
Handout NIST Hybrid Search of EI Spectra
Handout NIST Hybrid Search of Tandem (MS/MS) Spectra

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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:
Downloads

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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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