NIST20,23,26 Advanced Course LC-MS/MS Identification of Unknowns

This course teaches the practical application of the NIST MS Search software for identifying unknown compounds by LC-MS/MS. The concepts presented—including MS/MS library searching, hybrid searching, structure searching, MS Interpreter, and the effective use of commercial and user-created libraries—apply to all recent versions of the NIST software. The material is intended for a broad range of scientists working in medical, environmental, industrial, forensic, and biological applications.

If you are using NIST26, this is the recommended software because it includes expanded MS/MS libraries, improved workflows, and many new capabilities.

If your laboratory is still using NIST20 or NIST23, these tutorials remain highly applicable. Although some menus and screenshots differ, the fundamental approach to identifying unknown compounds is essentially unchanged. Once the underlying concepts are understood, transitioning between software versions is straightforward.

This is an advanced course in the NIST search software. With the V4.0 (2026) release, NIST has integrated chromatogram-level analysis directly into its MS/MS library search program. This is a significant advance that could position MS/MS libraries as central to LC-MS workflows as EI libraries have long been for GC-MS.

Together, the new LC-MS/MS analysis capabilities and the major expansion of the NIST MS/MS library create a powerful combination for compound identification. In addition, multiple libraries can be searched including free/crowd-sourced and user libraries when available in NIST MSP format.

In Versions 3.0 and 2.4, MS/MS data could only be processed within vendor-specific software before being imported into NIST Search. I’ve included guidance for processing Agilent (MassHunter) and Thermo (Freestyle) data, and similar workflows may be available from other vendors (see link)(opens in new tab)

NIST26

Recommended for all new users. Current software and largest MS/MS libraries.

NIST23

Fully supported by these tutorials. Interface differences. Start with V23 V3.0 Part 0

NIST20

Excellent fundamentals and still widely used in many laboratories, start NIST2020 V2.4 Part 1

• V4.0 (2026): start with NIST26
• V3.0 (2023): start with Part 0
• V2.4 (2020): start with Part I

Topics in V3.0 and 2.4 will be useful after completing the content for V4.0.

Video Resources:

Parts 0-VIII Zipped(opens in new tab)

Detailed Handouts:

Resources:

Large Group of MSMS Libraries in NIST Format:

>500K MS/MS Spectra with >150K structures from various crowd-sourced collections

FREE NIST Libraries(opens in new tab)

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