title: Simultaneous determination of rifampicin, clarithromycin and their metabolites in dried blood spots using LC-MS/MS.
abstract: BACKGROUND
  Rifampicin (RIF) and clarithromycin (CLR) are common drugs for the treatment of infections like Mycobacterium tuberculosis and Mycobacterium ulcerans. Treatment for these diseases are long-term and the individual pharmacokinetic variation, drug-drug interactions or non-adherence may introduce sub-therapeutic exposure or toxicity. The application of therapeutic drug monitoring (TDM) can be used to ensure efficacy and avoid toxicity. With the use of dried blood spot (DBS), TDM may be feasible in rural areas. During DBS method development, unexpected interactions or matrix effects may be encountered due to endogenous components in the blood. Another complication compared to plasma analysis is that RIF can form chelate complexes with ferric ions or can bind with hemes, which are potentially present in the extracts of dried blood spots.
  
  
  METHODS
  The investigation focused on the interaction between RIF and the endogenous components of the DBS. The use of ethylenediaminetetraacetic acid (EDTA) and deferoxamine (DFX) as chelator agents to improve recoveries and matrix effects were investigated. A rapid analytical method was developed and validated to quantify RIF and CLR and their active metabolites desacetyl rifampicin (DAc-RIF) and 14-hydroxyclarythromcin (14OH-CLR) in DBS samples. A clinical application study was performed in tuberculosis patients by comparing DBS concentrations with plasma concentrations.
  
  
  RESULTS
  The interaction between RIF and the DBS matrix was avoided using the complexing agents EDTA and DFX, which improved recoveries and matrix effects. The developed sample procedure resulted in a simple and fast method for the simultaneous quantification of RIF, CLR and their metabolites in DBS samples. High stability was observed as all four substances were stable at ambient temperature for 2 months. Deming regression analysis of the clinical application study showed no significant differences for RIF, DAc-RIF, CLR and 14OH-CLR between patient plasma and DBS analysis. The slopes of the correlation lines between DBS and plasma concentrations of RIF, DAc-RIF, CLR and 14OH-CLR were 0.90, 0.99, 0.80 and 1.09 respectively. High correlations between plasma and DBS concentrations were observed for RIF (R(2)=0.9076), CLR (R(2)=0.9752) and 14OH-CLR (R(2)=0.9421). Lower correlation was found for DAc-RIF (R(2) of 0.6856).
  
  
  CONCLUSIONS
  The validated method is applicable for TDM of RIF, CLR and their active metabolites. The stability of the DBS at high temperatures can facilitate the TDM and pharmacokinetic studies of RIF and CLR even in resource limited areas. The role of EDTA and DFX as complexing agents in the extraction was well investigated and may provide a solution for potential applications to other DBS analytical methods.
authors.affiliation: University of Groningen, Department of Pharmacotherapy and Pharmaceutical Care, Groningen, The Netherlands; University of Groningen, University Medical Center Groningen, Department of Hospital and Clinical Pharmacy, PO Box 30.001, 9700 RB Groningen, The Netherlands; Hanoi University of Pharmacy, Hanoi, Vietnam.
authors.forename: D H
authors.initials: DH
authors.lastname: Vu
authors.affiliation: University of Groningen, University Medical Center Groningen, Department of Hospital and Clinical Pharmacy, PO Box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: R A
authors.initials: RA
authors.lastname: Koster
authors.affiliation: University of Groningen, University Medical Center Groningen, Department of Hospital and Clinical Pharmacy, PO Box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: M S
authors.initials: MS
authors.lastname: Bolhuis
authors.affiliation: University of Groningen, University Medical Center Groningen, Department of Hospital and Clinical Pharmacy, PO Box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: B
authors.initials: B
authors.lastname: Greijdanus
authors.affiliation: University of Groningen, University Medical Center Groningen, Tuberculosis Center Beatrixoord, Haren, The Netherlands.
authors.forename: R V
authors.initials: RV
authors.lastname: Altena
authors.affiliation: Hanoi University of Pharmacy, Hanoi, Vietnam.
authors.forename: D H
authors.initials: DH
authors.lastname: Nguyen
authors.affiliation: University of Groningen, Department of Pharmacotherapy and Pharmaceutical Care, Groningen, The Netherlands.
authors.forename: J R B J
authors.initials: JR
authors.lastname: Brouwers
authors.affiliation: University of Groningen, University Medical Center Groningen, Department of Hospital and Clinical Pharmacy, PO Box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: D R A
authors.initials: DR
authors.lastname: Uges
authors.affiliation: University of Groningen, University Medical Center Groningen, Department of Hospital and Clinical Pharmacy, PO Box 30.001, 9700 RB Groningen, The Netherlands. Electronic address: j.w.c.alffenaar@umcg.nl.
authors.forename: J W C
authors.initials: JW
authors.lastname: Alffenaar
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D017291:Clarithromycin / Q000097:blood*; D015203:Reproducibility of Results; D012293:Rifampin / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D017291:Clarithromycin; D012293:Rifampin
keywords: Clarithromycin; Deferoxamine; Dried blood spot (DBS); Ethylenediaminetetraacetic acid (EDTA); Liquid chromatography tandem mass spectrometry (LC–MS/MS); Rifampicin