Erythrocyte sequestration of metformin in horses: impact on matrix-specific pharmacokinetics and detection windows

Metformin is used for the treatment of type 2 diabetes and is one of the most prescribed medications in human medicine. It is less commonly prescribed in equine medicine, and its use is tightly regulated in several performance horse disciplines. In horseracing, it is considered a banned substance. A previous pharmacokinetic study of metformin in horses demonstrated a prolonged, unpredictable elimination phase. In the current study it was hypothesized that this was due to sequestration of metformin in a “deep” compartment, specifically red blood cells. This could result in pharmacokinetic differences between blood matrices (i.e. blood and serum). The objective of the current study was to assess red blood cell partitioning and the concentrations of metformin in different blood matrices following oral administration to horses. To that end, six horses received a single 15 g oral dose of metformin and plasma, serum, whole blood, red blood cells, and urine samples were collected starting at 5 min until 31 days post administration. Concentrations of metformin were determined using liquid chromatography-tandem mass spectrometry, and pharmacokinetic analysis performed. Red blood cells act as a reservoir for metformin in horses with the average blood to plasma ratio ranging from < 1 to > 10 at the later time points. The terminal half-life (mean ± SD) was 14.7 ± 7.25, 75.4 ± 32.2 and 49.1 ± 7.01 in plasma, serum, and red blood cells, respectively. The difference between serum and plasma concentrations was > 15% at several time points, especially at the later times. Concentrations in urine samples, fluctuated unpredictably over time. Red blood cells act as a reservoir for metformin leading to a prolonged detection time, necessitating an extended withdrawal time for oral administration prior to competition in performance horses to prevent inadvertent positive drug tests. Additionally, differences in metformin concentrations across various biological matrices may preclude the extrapolation of data from one sample type to another.