Johanna Konta let slip two match points as she was knocked out of the Rogers Cup by 20/1 shot Ekaterina Makarova.
The British number one served for a straight-sets win in the second-round contest but eventually fell 5-7 7-6 (7/4) 6-3 to the world number 42 in almost two-and-a-half hours.
Seventh seed Konta battled back from a break down in the first two sets but could not recover after dropping her serve in the eighth game of the decider in Toronto.
Sky Bet's joint 5/1 favourite Karolina Pliskova marked her first match as world number one with a straight-sets victory over Anastasia Pavlyuchenkova.
Pliskova secured a 6-3 6-3 success in Toronto to reach the last 16, where she will face Naomi Osaka.
The rest of the top six in the rankings also secured second-round wins, but six seeds were knocked out.
Second seed and defending champion Simona Halep beat Magdalena Rybarikova 6-3 6-4 as she looks to become the first player to win back-to-back titles since Martina Hingis in 2000.
Angelique Kerber, the third seed, battled past Donna Vekic 6-4 7-6 (7/5) while Wimbledon champion Garbine Muguruza beat Kirsten Flipkens 7-5 6-2.
Fifth seed Elina Svitolina beat Daria Kasatkina 7-6 (7/4) 6-4 to set up a clash with Venus Williams, a straight-sets winner against Katerina Siniakova, while 2010 champion Caroline Wozniacki raced past Ekaterina Alexandrova 6-3 6-0.
Svetlana Kuznetsova, seeded eighth, is also on her way home having gone down 6-4 7-5 to 18-year-old American Catherine Bellis while Sloane Stephens knocked out 2012 winner Petra Kvitova 7-6 (7/4) 3-6 6-2.
Agnieszka Radwanska, who lifted the title in 2014, took just 46 minutes to beat Timea Babos 6-0 6-1 but 11th seed Dominika Cibulkova lost 6-2 6-4 to Lucie Safarova.
Osaka beat 15th seed Anastasija Sevastova in straight sets, 16th seed Elena Vesnina was beaten in three by Ashleigh Barty and Caroline Garcia saw off Varvara Lepchenko 6-1 6-4.
