Which of the following substrates cannot be used in gluconeogenesis?

Gluconeogenesis is the process of synthesizing glucose from non-carbohydrate precursors such as amino acids, lactate, and glycerol. However, not all substrates can be used in this process. Palmitic acid has a 16-carbon backbone and is a saturated long-chain fatty acid. Natural sources of palmitic acid include palm oil and palm kernel oil, as well as butter, cheese, milk, and meat.

Palmitic acid is a fatty acid that produces solely acetyl and hence it cannot be used as a substrate for gluconeogenesis. 

The other substrates listed in the question (aspartic acid, glutamic acid, and glycerol) can be used in gluconeogenesis because they can be converted to intermediates in the gluconeogenic pathway that can ultimately be used to synthesize glucose.