Development, validation and application of a novel HPLC-MS/MS method for the measurement of minocycline in human plasma and urine

Mark A.J. Bayliss*, Katarina Rigdova, Mathew Kyriakides, Sally Grier, Andew M. Lovering, Hannah Williams, David C. Griffith, Alasdair MacGowan

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

13 Citations (Scopus)
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New treatments are urgently required to treat infections caused by multi-drug resistant Acinetobacter baumanni,. To address this need, a new formulation of Minocin® (minocycline for injection) has been developed that allows for higher doses of minocycline to be administered. Phase 1 clinical trials were conducted in healthy volunteers to assess the safety and pharmacokinetics (PK) of this new formulation at higher doses. In order to generate PK data, novel, selective and simple HPLC-MS/MS based assays were developed and validated for the determination of minocycline (MC) in human plasma and urine. The respective working ranges were 0.05 to 30 mg/L and 0.1 to 30 mg/L. Removal of endogenous proteins with trichloroacetic acid was used as a simple means of extracting MC from the samples. An analogue, tetracycline was used as the internal standard (IS). Chromatographic separation, including that of MC from its 4-epimer (4-EMC), was achieved on a Waters XBridge BEH C18 column (50 x 4.6 mm ID, 5 μm) with gradient elution. The mobile phases comprised water containing 5 mM ammonium formate at a pH of 2.5, and methanol containing 5 mM ammonium formate. The internal standard (IS) was tetracycline, a structural analogue of minocycline. The methods were fully validated and met regulatory acceptance criteria for intra-run and inter-run accuracy and precision, carryover, dilution integrity and matrix effects. Mean extraction recoveries ranged between 64.3% and 84.6% for MC and 64.3% for the IS. There was no significant ion suppression or enhancement for MC or the IS. The validated assays were successfully applied to 1423 plasma and 689 urine samples from a Phase 1 clinical study. There was no evidence of instability, or significant interconversion between MC and 4-EMC, in stored clinical samples, spiked plasma and urine samples, or their extracts, under various test conditions.

Original languageEnglish
Pages (from-to)90-98
Number of pages9
JournalJournal of Pharmaceutical and Biomedical Analysis
Early online date26 Feb 2019
Publication statusPublished - 30 May 2019


  • Assay validation
  • Epimer
  • Liquid chromatography–mass spectrometry
  • Minocycline
  • Plasma
  • Urine


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