The 2016 Olympic champion's blistering display sliced more than one-tenth of a second off the previous record of 52.34 sec, set by Russia's Yuliya Pechonkina.
Olympic champion Dalilah Muhammad smashed one of athletics' oldest world records when she stormed to the women's 400 metres hurdles title in 52.20 seconds at the United States championships in Iowa on Sunday.
The blistering run shaved more than a tenth of a second from the previous mark of 52.34 seconds set by Russian Yuliya Pechonkina in 2003.
"I'm just shocked," said the 29-year-old Muhammad, who suffered a concussion after a fall during training two weeks ago.
"I've been kind of hitting that time in practice consistently and my coach was like, 'There's no way you can't do it'."
Teenager Sydney McLaughlin was second in 52.88, just 0.13 shy of her personal best, with Spencer third in 53.11, as all three runners made the US team for September's world championships in Doha.
Noah Lyles also qualified after running 19.78 seconds to win his 200 metres showdown with 100m champion Christian Coleman.
The two were side-by-side before Lyles pulled away in the last 60 metres.
Coleman claimed second (20.02) with Ameer Webb third (20.45).
The women's race went to Dezerea Bryant in 22.47 seconds.
Craig Engels edged Olympic champion Matthew Centrowitz in the men's 1,500m by four-hundredths of a second, winning in 3:44.93.
The men's 110m hurdles was the reverse of the US collegiate final with Daniel Roberts winning over Grant Holloway in 13.23. Holloway clocked 13.36.
World indoor champion Sandi Morris cleared 4.85 metres to win the women's pole vault, while world champion Emma Coburn won her sixth consecutive US 3,000m steeplechase title in 9:25.63.
Lopez Lomong used a late surge to defeat Olympic 5,000m silver medallist Paul Chelimo in the men's event and claim his second title of the meet after winning the 10,000.
Lomong will not be doubling up in the 5,000 and 10,000 at Doha, however, because he lacks the standard in the longer distance.