Tranmere's Johnstone's Paint Trophy campaign came to an end&nbsp;with a 4-3 defeat at Chesterfield.
Rovers led twice in the contest, first through a superb strike from Adam McGurk
only for the home side to score twice either side of half time through Alex Mendy and Jordan Bowery.
Tranmere responded with two quick goals of their own from substitute Enoch Showunmi and Mustafa Tiryaki but Bowery headed in his second of the game to level.
As penalties approached, Chesterfield grabbed the winner in injury time and a place in the northern quarter finals when Craig Westcarr headed in from a free kick.
There were three changes to the side that started against Colchester United as Ian Goodison and Andy Robinson were rested and Dave Raven dropped to the bench. Mark McChrystal came in for his first game since picking up an injury in the first round against Port Vale with Danny Holmes making his first appearance since rejoining the Club in the summer. Also coming into the starting line up was Zoumana Bakayogo who took up the left midfield role as Adam McGurk
occupied a more central role in support of Mustafa Tiryaki.
An early Robbie Weir cross had to be punched away by Chesterfield keeper Ole Soderberg and the midfielder tested the Newcastle loanee on six minutes with a low effort from the edge of the area.
McChrystal cleared from just in front of his line after Holmes slipped while winning the ball off Tope Obadeyi on the right side of the Rovers area and Jordan Bowery headed wide moments later for the hosts.
Tranmere took the lead in style on nine minutes. A long ball forward was chested into the path of McGurk by Tiryaki and the striker took one touch before firing into the bottom right corner from just outside the area. It was a fine strike from the 22-year old for his second goal of the season.
McChrystal cleared a header from Alex Mendy on 11 minutes with Owain Fon Williams saving from the corner that followed, though the referee's whistle blew for a foul in the build-up and Craig Westcarr's effort wouldn't have counted had it gone in.
Ash Taylor did well to clear from inside his area as Westcarr shaped to shoot on 20 minutes and a free kick taken by Lee Johnson struck the Rovers wall as the home side pressed for an equaliser.
A quickly taken throw in from Lucas Akins saw McGurk pulled back by Dwayne Mattis, with Rovers winning a free kick in dangerous position just outside the area. However, McGurk's curled effort went straight into the arms of Soderberg.
Akins almost doubled the lead on 34 minutes after running on to a superb pass from Weir, taking on a defender and seeing his low shot well saved by Soderberg at his right hand post, with Rovers unable to take advantage of the rebound.
Bakayogo headed over the bar from an Akins cross on 39 minutes, moments after he'd gained possession for Tranmere by putting Simon Ford under pressure in the Chesterfield area.
Fon Williams cut out Westcarr's square ball across the six yard box on 42 minutes while McChrystal produced a block to deny the striker moments later, however, the home side drew level a minute from half time. Mendy picked up the ball midway inside the Rovers half, ran towards the area and sent a low shot into the bottom right corner beyond the dive of Fon Williams. Like McGurk's earlier strike, it was an effort that gave the keeper no chance of a save.
Fon Williams comfortably claimed an early cross as Chesterfield attacked at the start of the second half while a McGurk shot went well wide of the target soon after.
However, the home side took the lead on 54 minutes. After McGurk lost possession the ball reached Westcarr out wide and his cross was headed in by Jordan Bowery from close range.
Buchanan did well to end an attack from Westcarr on 57 minutes, while Showunmi replaced Bakayogo soon after, a move that saw McGurk switch to left midfield.
Two goals in quick succession earned Rovers the lead. First, a free kick was floated into the area by John Welsh on 71 minutes, headed back across goal by Taylor and turned in by Showunmi for his second goal of the campaign.
Then, with Tranmere attacking down the right, the ball dropped to Tiryaki on the right side of the area. The striker aimed a shot goalwards which was easily caught by Soderberg at his near post but as the keeper landed he dropped it backwards over his line and into the net.
The goals drew a fantastic reaction from the travelling 253 travelling fans but they were disappointed to see the hosts equalise for a second time on 79 minutes. A cross from the right was cleared out of the area but it was played back in by Mendy and Bowery headed in his second of the night from six yards out.&nbsp;&nbsp;
Showunmi sent a header over from eight yards from a Welsh corner on 81 minutes while Max Power came on in place of Tiryaki with five minutes of normal time remaining.
As the game went into five minutes of added time, the Spireites scored their fourth of the night, as Westcarr headed in from close range from a free kick.
It proved to be the final chance of the match as Rovers exited the competition.
Chesterfield: Soderberg, Talbot (Ford 20), Downes, Mattis (Whitaker 76), Smith, Mendy, Johnson (Allott 76), Grounds, Obadeyi, Westcarr, Bowery. Substitutes: Fleming, Allott, Clay.
Tranmere: Fon Williams, Holmes, Taylor, McChrystal, Buchanan, Akins, Weir, Welsh, Bakayogo (Showunmi 58), McGurk (Labadie 70), Tiryaki (Power 86). Substitutes: Coughlin, Raven.
Referee: Geoff Eltringham
Yellow cards: Mattis (32)
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