Russian Samsonova, who has won 20 of her last 22 matches, smacked 21 winners and broke the powerful Belarussian four times en route to a 6-4 2-6 6-2 upset win over the world number four.
The WTA Finals featuring the world's top eight players start on October 31st.
Iga Swiatek, Ons Jabeur and Jessica Pegula had already locked up the first three spots before Guadalajara so with the addition of American Gauff and France's Garcia, three openings remain.
Despite the loss, Sabalenka has not been eliminated from making the elite eight, according to the WTA.
She currently holds a qualifying spot and will now wait to see if her points total holds up against the field.
Fifth seed Gauff was not at her best but was still good enough to see off Italy's Elisabetta Cocciaretto 7-6(1) 6-3.
The American teenager double faulted five times and converted just four of 13 break points but was the better player on the biggest points, rolling through the first set tiebreak and carrying that momentum into the second set.
Danielle Collins and Jelena Ostapenko, who both have an outside shot of making the WTA Finals, won their second round matches on Wednesday, while other hopefuls including Madison Keys, Belinda Bencic and top seed Paula Badosa play later.
