Quantitative analysis of colistin A and colistin B in plasma and culture medium using a simple precipitation step followed by LC/MS/MS

Abstract

An analytical method for quantitation of colistin A and colistin B in plasma and culture medium is described. After protein precipitation with acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), the supernatants were diluted with 0.03% TFA. The compounds were separated on an Ultrasphere C18 column, 4.6 mm x 250 mm, 5 microm particle size with a mobile phase consisting of 25% ACN in 0.03% TFA and detected with tandem mass spectrometry. The instrument was operating in ESI negative ion mode and the precursor-product ion pairs were m/z 1167.7-->1079.6 for colistin A and m/z 1153.7-->1065.6 for colistin B. The lower limit of quantification (LLOQ) for 100 microL plasma was 19.4 and 10.5 ng/mL for colistin A and B, respectively, with CV <6.2% and accuracy <+/-12.6%. For culture medium (50 microL+50 microL plasma), LLOQ was 24.2 and 13.2 ng/mL for colistin A and B, respectively, with CV <11.4% and accuracy <+/-8.1%. The quick sample work-up method allows for determination of colistin A and B in clinical samples without causing hydrolysis of the prodrug colistin methanesulfonate (CMS).