BCFAs are known to dominate many bacterial lipid profiles. For example, BCFAs have been suggested to constitute more than 75% of the total fatty acid profile of Bacillus subtilis. While BCFAs are minor lipid components of internal mammalian tissues, they have been detected as major components of meibomian gland secretions (within the eyelid) and vernix caseosa, the sebaceous wax coating human newborns. Specific short BCFAs, such as valproic acid are used as a mood-stabilizing and anti-epileptic drug but also cause neurotoxic effects and anti-convulsant effects related to neural tube defects, congenital defects and fetal growth. Among the most common BCFAs are saturated FAs with a methyl group at either the antepenultimate or penultimate carbon, known as the anteiso-or iso-position, respectively. Traditionally, gas chromatography-mass spectrometry (GC-MS) is deployed to resolve BCFAs from their straight-chain isomers and to facilitate identification and quantitation in complex biological samples. In general, GC-MS analysis first requires the conversion of FA structures to fatty acid methyl esters (FAMEs) to increase analyte volatility. Unfortunately, branched-chain fatty acid methyl esters (BCFAMEs) often co-elute with other FAMEs, even with the use of long, highly-polar columns. The problem of resolution and unambiguous identification is further compounded by high degree of similarity in the conventional (70 eV) electron ionization (EI) mass spectra of isomeric FAMEs. Thus, the presence of structurally distinct FAs can be masked in both chromatographic and mass spectrometric dimensions that further hinders the detection, identification, and quantitation of BCFAs. Moreover, BCFAME identification is often accomplished using spectral matching or via the use of retention time alignment, both of which require the utilization of reference standards that are often costly and rarely available. To circumvent these issues, Murphy and co-workers combined electron-ionization with collision-induced dissociation demonstrating that activation of the molecular ions of FAMEs gives rise to mass spectra that were much more sensitive to molecular structure; including the position(s) of methyl-chain branching. Brenna and co-workers have utilized this approach to provide detailed structure elucidation of branched-chain fatty acids in lipid extracts from human cell lines and food. Notably, this approach enables the mass spectrometric resolution of co-eluting GC peaks and is thus capable of both quantitative and qualitative analyses of BCFAMEs without reference standards. One challenge with this approach is that the molecular ion abundance resulting from EI of FAMEs is low and can impact sensitivity. Alternative GC-MS approaches that can increase the sensitivity for detection and identification of BCFAs rely upon wetchemical derivatization strategies prior to analysis that are designed to promote sensitive detection and structurally diagnostic fragmentation upon EI. Two of the most successful and widely adopted examples include 4,4-dimethyloxazoline (DMOX) and 3-pyridylcarbinol (historically -and incorrectly-referenced as 'picolinyl') ester derivatives of FAs. Both derivatives produce EI mass spectra with fragmentation patterns that can identify structural motifs including branching points in the acyl chain. Both methods require offline wet-chemical preparation prior to GCMS. However, complex lipid hydrolysis of fatty acyl chains from complex lipid precursors results in the loss of valuable information regarding origin and function of BCFAs.