title: Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry.
abstract: Selective and sensitive methods for the determination of the cationic dye and anti-malarial methylene blue in human liquid whole blood, dried whole blood (paper spot), and plasma depending on protein precipitation and cation exchange chromatography coupled to electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) have been developed, validated according to FDA standards, and applied to samples of healthy individuals and malaria patients within clinical studies. Acidic protein precipitation with acetonitrile and trifluoroacetic acid was used for liquid whole blood and plasma. For the extraction of methylene blue from paper spots aqueous acetonitrile was used. Sample extracts were chromatographed on a mixed mode column (cation exchange/reversed phase, Uptisphere MM1) using an aqueous ammonium acetate/acetonitrile gradient. Methylene blue was quantified with MS/MS in the selected reaction monitoring mode using ESI and methylene violet 3RAX as internal standard. Depending on the sample volume (whole blood and plasma 250 microL, and 100 microL on paper spots) the method was linear at least within 75 and 10,000 ng/mL and the limit of quantification in all matrices was 75 ng/mL. Batch-to-batch accuracies of the whole blood, plasma, and paper spot methods varied between -4.5 and +6.6%, -3.7 and +7.5%, and -5.8 and +11.1%, respectively, with corresponding precision ranging from 3.8 to 11.8% CV. After a single oral dose (500 mg) methylene blue concentrations were detectable for 72 h in plasma. The methods were applied within clinical studies to samples from healthy individuals and malaria patients from Burkina Faso.
authors.affiliation: Department of Internal Medicine VI, Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. juergen.burhenne@med.uni-heidelberg.de <juergen.burhenne@med.uni-heidelberg.de>
authors.forename: Jürgen
authors.initials: J
authors.lastname: Burhenne
authors.forename: Klaus-Dieter
authors.initials: KD
authors.lastname: Riedel
authors.forename: Jens
authors.initials: J
authors.lastname: Rengelshausen
authors.forename: Peter
authors.initials: P
authors.lastname: Meissner
authors.forename: Olaf
authors.initials: O
authors.lastname: Müller
authors.forename: Gerd
authors.initials: G
authors.lastname: Mikus
authors.forename: Walter E
authors.initials: WE
authors.lastname: Haefeli
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Walter-Sack
mesh_terms: D000284:Administration, Oral; D000962:Antimalarials / Q000097:blood*; D002138:Calibration / Q000592:standards; D002411:Cation Exchange Resins / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004791:Enzyme Inhibitors / Q000097:blood* / Q000493:pharmacokinetics; D006801:Humans; D008288:Malaria / Q000097:blood* / Q000188:drug therapy*; D008751:Methylene Blue / Q000032:analysis*; D015394:Molecular Structure; D010640:Phenothiazines / Q000097:blood / Q000592:standards; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000962:Antimalarials; D002411:Cation Exchange Resins; D004791:Enzyme Inhibitors; D010640:Phenothiazines; D008751:Methylene Blue; C060547:methylene violet