• The second test of blood from Kentucky Derby winner Medina Spirit confirmed the presence of the banned steroid betamethasone, a lawyer for the horse's owner said.
• The second positive test increases the risk that Medina Spirit's victory will be overturned by Kentucky racing officials.
• Medina Spirit trainer Bob Baffert has said the betamethasone may have come from an ointment used on the thoroughbred.
• Baffert-trained horses have been banned from the Belmont Stakes, the third jewel in racing's Triple Crown.

The second test of blood from Kentucky Derby winner Medina Spirit confirmed the presence of the banned steroid betamethasone, a lawyer for the horse's owner told CNBC on Wednesday.

The second positive test sharply increases the chance that Medina Spirit's victory on May 1 will be overturned by Kentucky racing officials and that Mandaloun, which ran second that day, will be declared the winner.

Hours after disclosure of the test, the company that operates Churchill Downs Racetrack — the site of the Kentucky Derby — said it had immediately suspended Baffert for two years.

Clark Brewster, the attorney for Medina Spirit's owner, Amr Zedan, said officials are allowing another lab to analyze a third sample from the 3-year-old colt.

That test, Brewster said, could determine whether there are chemicals that would support the claim by trainer Bob Baffert that the betamethasone may have come from an antifungal ointment applied to the horse, and not an injection.

If the third test gives that result, Brewster could use it to argue against Medina Spirit being disqualified from the Derby, which is the first jewel in thoroughbred racing's Triple Crown.

The attorney suggested he also might challenge the accuracy and protocol of the first official test, and the second analysis of blood, known as a split sample.

"I have not seen the paperwork to conclude that even the primary or split tests were properly admitted," Brewster said.

The second failed test was first reported Wednesday by The New York Times.

Brewster said that if a horse fails a first drug test, a trainer normally has the option of "sending the B sample" for analysis at a selected lab for a second, confirmatory test.

For Medina Spirit's B sample, Brewster said, the horse's team "requested both the blood and urine to be sent to" such a lab.

The attorney said that if both substances were tested, it could detect the presence of chemical components that would indicate whether betamethasone came from ointment.

"But they [racing officials] refused to send" the urine, Brewster said. "They only sent the blood."

The lawyer said that on Monday or Tuesday, Medina Spirit's team was informed the lab "found betamethasone" in the split sample.

Brewster said the lab did not release the level of that steroid found in the blood, "but they said it's there."

"They estimated it was 25 picograms," he said.

Baffert at a May 9 news conference first revealed that Medina Spirit had tested positive for the steroid, saying the first sample was found to have 21 picograms of betamethasone.

While that drug can be legally used as a therapeutic in Kentucky on a horse, any trace of it on race day is grounds for disqualification if a second test confirms it was in the blood on that day.

A picogram is a trillionth of a gram, a point Brewster made several times during a phone interview Wednesday.

The lawyer said testing labs in recent years have become able to detect minute levels of pharmaceutical substances, some of which can enter a horse's or human's system by incidental contact, as opposed to intentional administration.

The Kentucky Horse Racing Commission will have the final say over whether to void Medina Spirit's Derby win.

"Hopefully they will make a reasonable judgment," Brewster said.

"I think there will be unanimity on the subject that this is an infinitesimal amount that could not have affected the race," the lawyer said.

Baffert, who so far this year has seen five of his horses fail drug tests, was suspended indefinitely from Churchill Downs Racetrack, where the Derby is held, as a result of Medina Spirit's first positive test.

Medina Spirit was later allowed to race May 15 in the Preakness Stakes in Baltimore, the second leg of the Triple Crown, under an agreement that it and another Baffert-trained horse, Concert Tour, submit to "rigorous testing and monitoring," the Maryland Jockey Club said.

Medina Spirit finished third in the Preakness.

Medina Spirit will not run in the Belmont Stakes on Saturday on Long Island, New York, because Baffert was temporarily suspended last month from entering horses in that race, the third jewel of the Triple Crown, or other major New York tracks as a result of the positive drug test from the Derby.

Baffert has trained two Triple Crown winners. He has trained seven Kentucky Derby winners, counting Medina Spirit.

Baffert's lawyer W. Craig Robertson III on Wednesday later issued a formal statement on the most recent drug test.

"In response to the inquiries, this will acknowledge that the Medina Spirit split sample confirmed the finding of betamethasone at 25 picograms," Robertson said.

"There is other testing that is being conducted, including DNA testing," Robertson said.

"We expect this additional testing to confirm that the presence of the betamethasone was from the topical ointment, Otomax, and not an injection," Robertson said.

"At the end of the day, we anticipate this case to be about the treatment of Medina Spirit's skin rash with Otomax. We will have nothing further to say until the additional testing is complete."

Kristin Voskuhl, a spokeswoman for the Kentucky Horse Racing Commission, said in a statement that the commission "does not provide comment or updates on the status of ongoing investigations."

"The KHRC values fairness and transparency, and will provide information to the media and public at the close of an investigation," Voskuhl said.

Marty Irby, executive director of the advocacy group Animal Wellness Action, in a statement, said, "The news of Medina Spirit's second test confirmed positive is no shock."

"Churchill Downs, the Kentucky Derby, and the Kentucky Horse Racing Commission must stand firmly together in agreement to take the 2021 Derby title away from Bob Baffert and the horse," Irby said.

"And we call on Churchill Downs to show no mercy and permanently ban Bob Baffert and his horses from the Kentucky Derby and all of Churchill Downs' tracks. It's time to end the cheating and medication abuse in the fastest two minutes in sports and an example must be made."

Disclosure: CNBC parent NBCUniversal owns NBC and NBC Sports, which broadcast the Triple Crown races.