Kentucky Derby winner Medina Spirit’s second blood test confirmed the presence of the banned steroid betamethasone, an attorney for the horse owner told CNBC on Wednesday.

The second positive test increases the likelihood that Medina Spirit’s victory on May 1 will be overturned by Kentucky race officials and Mandaloun, who finished second that day, will be declared the winner.

Hours after the test was released, the company that operates the Churchill Downs Racetrack – the site of the Kentucky Derby – said it immediately banned Baffert for two years.

Clark Brewster, attorney for Medina Spirit’s owner Amr Zedan, said officials are allowing another laboratory to analyze a third sample of the three-year-old stallion.

That test, Brewster said, could determine if there are any chemicals that would support coach Bob Baffert’s claim that the betamethasone came from an antifungal ointment applied to the horse rather than an injection.

If the third test gives that result, Brewster could argue that Medina Spirit will be disqualified from the Derby, the first jewel in the thoroughbred sport’s Triple Crown.

The attorney suggested that he could also question the accuracy and protocol of the first official test and the second blood analysis known as the split sample.

“I haven’t seen the papers showing that even the primary or split tests were properly approved,” Brewster said.

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

Brewster said that if a horse fails an initial drug test, a trainer usually has the option to send “the B sample” to a selected laboratory for analysis for a second, confirmatory test.

For the Medina Spirit B sample, Brewster said the horse’s team “requested that both the blood and urine be sent to such a laboratory.”

Medina Spirit’s coach Bob Baffert lifts the trophy after finishing 147th with Medina Spirit.

Andy Lyons | Getty Images

The lawyer said that if both substances were tested, the presence of chemical components could be detected that would indicate whether betamethasone was from an ointment.

“But she [racing officials] refused to send the urine, Brewster said.

The attorney said the Medina Spirit team was informed on Monday or Tuesday that the lab had found “betamethasone” in the split sample.

Brewster said the lab hadn’t released the steroid levels found in the blood “but they said it was there”.

“They estimated it was 25 picograms,” he said.

Baffert first announced at a press conference May 9 that Medina Spirit tested positive for the steroid, saying the first sample contained 21 picograms of betamethasone.

While this drug is legal for use as a therapeutic on a horse in Kentucky, any trace on race day is a reason for disqualification if a second test confirms it was in the blood that day.

A picogram is a trillionth of a gram, a point Brewster emphasized several times during a telephone interview on Wednesday.

The attorney said that in recent years testing laboratories have been able to detect tiny amounts of pharmaceutical substances, some of which can enter a horse’s or human’s system through accidental contact as opposed to deliberate administration.

The Kentucky Horse Racing Commission will have the final say on whether Medina Spirit’s Derby win will be invalidated.

“Hopefully they’ll make a reasonable judgment,” Brewster said.

“I think there will be consensus that this is a negligible amount that cannot have affected the race,” said the lawyer.

Baffert, who has failed five of his horses in drug tests so far this year, has been suspended indefinitely from the Churchill Downs Racetrack, where the derby is taking place, due to the first positive test from Medina Spirit.

Medina Spirit was later allowed to compete in the Preakness Stakes in Baltimore, the second leg of the Triple Crown, on May 15, under an agreement that she and another Baffert-trained horse, Concert Tour, would undergo “rigorous testing and surveillance,” the Maryland Jockey Club said.

Medina Spirit came third in the Preakness.

Medina Spirit will not be competing in the Belmont Stakes on Saturday in Long Island, New York, as Baffert was temporarily suspended from horse participation in that race, the third jewel of the Triple Crown or other major New York circuit, last month, the positive Drug test from the derby.

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

Baffert’s attorney W. Craig Robertson III later made a formal statement on Wednesday about the latest drug test.

“In response to inquiries, it confirms that the Medina Spirit split sample confirmed the betamethasone finding at 25 picograms,” said Robertson.

“There are other tests being done, including DNA testing,” Robertson said.

“We expect these additional tests to confirm that the presence of betamethasone is from the Otomax topical ointment and not from an injection,” said Robertson.

“Ultimately, we expect this case to be about treating Medina Spirit’s rash with Otomax. We won’t have anything to say until the additional tests are completed.”

Kristin Voskuhl, a spokeswoman for the Kentucky Horse Racing Commission, said in a statement that the commission “does not provide any comments or updates on the state of the investigation.”

“The KHRC values ​​fairness and transparency and will provide information to the media and the public upon completion of an investigation,” said Voskuhl.

Marty Irby, director of the Animal Wellness Action advocacy group, said in a statement: “The news of Medina Spirit’s second positive test is not a shock.”

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

“And we urge Churchill Downs to show no mercy and to permanently ban Bob Baffert and his horses from the Kentucky Derby and all Churchill Downs courses. It is time to end the cheating and drug abuse in the quickest two minutes in sports and an example “has to be done.”

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