title: Development and validation of a liquid chromatography tandem mass spectrometry assay for the measurement of faecal metronidazole.
abstract: BACKGROUND
  Metronidazole is an oral antibiotic which is widely used in the treatment of patients with Clostridium difficile associated disease.
  
  
  METHODS
  This article describes the validation of a LC-MS/MS assay for the measurement of metronidazole in human faecal samples.
  
  
  RESULTS
  Matrix matched and aqueous standards showed no significant difference in performance for the routine calibration of the assay. D4deuterated metronidazole internal standard eluted with a different retention time to the undeuterated metronidazole on chromatography, hence zidovudine was used as an internal standard. Ion suppression was noted for both metronidazole and zidovudine due to unidentified compounds present in the faecal matrix and this was improved by extracting a smaller quantity of faeces and diluting the extract prior to analysis. Measurement uncertainty was 13% at 28,400ng/ml, 7.2% at 3300ng/ml, 3.9% at 320ng/ml, 13.6% at 109ng/ml and 30.9% at 20ng/ml. The assay was shown to be linear on dilution and the sensitivity of the assay was superior to HPLC assays using UV detection. The limit of detection was 5ng/ml, the limit of quantitation was 66ng/ml and the upper limit of the working range was 30,000ng/ml. Patient samples were stable at -20°C for 12months and extracted faecal samples were stable on storage for 1week at 4°C. There were no specific requirements for patient preparation or time of sample collection relative to taking metronidazole.
  
  
  CONCLUSIONS
  Metronidazole can be quantified in faecal samples using LC-MS/MS which opens up opportunities for further research in this area.
authors.affiliation: Derriford Combined Laboratory, Derriford Hospital, Plymouth PL6 8DH, UK.
authors.forename: Jinny
authors.initials: J
authors.lastname: Jeffery
authors.affiliation: Derriford Combined Laboratory, Derriford Hospital, Plymouth PL6 8DH, UK.
authors.forename: Zoe J
authors.initials: ZJ
authors.lastname: Vincent
authors.affiliation: Derriford Combined Laboratory, Derriford Hospital, Plymouth PL6 8DH, UK.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Ayling
authors.affiliation: Department of Gastroenterology, Derriford Hospital, Plymouth PL6 8DH, UK. Electronic address: sjl@doctors.org.uk.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lewis
mesh_terms: D000890:Anti-Infective Agents / Q000032:analysis / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D016360:Clostridioides difficile / Q000187:drug effects*; D003015:Clostridium Infections / Q000188:drug therapy / Q000378:metabolism* / Q000382:microbiology; D005243:Feces / Q000737:chemistry* / Q000382:microbiology; D006801:Humans; D057230:Limit of Detection; D008795:Metronidazole / Q000032:analysis* / Q000494:pharmacology; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000890:Anti-Infective Agents; D008795:Metronidazole
keywords: Assay validation; Faecal sample; Faeces; Mass spectrometry; Metronidazole