Ion-pair liquid chromatography-electrospray mass spectrometry for the analysis of cyclic nucleotides.
An LC-MS method has been developed combining ion-pair chromatography with an electrospray interface linking microbore and capillary HPLC to mass spectrometry. Separation of cyclic nucleotides on C18 reversed-phase columns, using tetrabutylammonium bromide as an ion pairing agent was evaluated with different mobile phase compositions. It was found that low ion-pairing agent concentration (50-500 microM) used in combination with low flow-rates (5-10 microl min(-1)) allowed the system to operate for up to several days without observing a reduced signal caused by source pollution. The loss of sensitivity expected in ion-pair chromatography could be remedied by using a 2-propanol coaxial sheath flow. Optimal conditions for negative ion electrospray resulted in a linear detection response in the femtomole to picomole range. Using biological samples this method was evaluated and compared with a classical ion-suppression RP-HPLC method using UV detection.
Witters
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Department of Biology, University of Antwerp, U.I.A., Belgium.
Van Dongen
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Esmans
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Van Onckelen
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HA
D000227:Adenine Nucleotides / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000032:analysis; D003563:Cyclic CMP / Q000032:analysis; D006152:Cyclic GMP / Q000032:analysis; D007289:Cyclic IMP / Q000032:analysis; D013058:Mass Spectrometry; D009712:Nucleotides, Cyclic / Q000032:analysis*; D000644:Quaternary Ammonium Compounds; D051381:Rats; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D000227:Adenine Nucleotides; D009712:Nucleotides, Cyclic; D000644:Quaternary Ammonium Compounds; D007289:Cyclic IMP; D003563:Cyclic CMP; C010565:2',3'-cyclic AMP; C009405:tetrabutylammonium; D000242:Cyclic AMP; D006152:Cyclic GMP