An LC–MS/MS method for the determination of budesonide and 16α-hydroxyprednisolone in dog plasma

Graphical abstract


Method details
An LC-MS/MS method for the quantitative determination of budesonide (BUD) and one of its main metabolite, 16a-hydroxyprednisolone (16a-HP), in dog plasma was developed and validated. The simultaneous quantification of the two molecules, the rapidity and simplicity of the procedure, and the low volume of sample required make the proposed technique suitable for pharmacokinetic studies involving also small-sized dogs. In addition, the developed protocol was employed in a clinical trial on dogs of different breeds, orally treated with BUD once a day for 30 days, demonstrating to be suitable for the purpose [1].

Materials
BUD and betamethasone (BET) standards were purchased from Sigma-Aldrich (Milan, Italy); 16a-HP was purchased from Du-Hope International Group (Nanjing, China). The internal standard solution was prepared diluting BET in water:acetonitrile (1:1) at a concentration of 50 ng/mL.

Sample preparation
1. Add 20 mL of BET internal standard solution to 0.5 mL of plasma.
2. Activate the Strata-X cartridge (1 mL, 30 mg) (Phenomenex, Torrance, USA) with 1 mL of methanol and wash with 1 mL of water. 3. Load the sample on the cartridge, at a flow speed of 2 mL/min. 4. Wash the cartridge with 3 mL of a solution of 5% methanol in water. 5. Dry the cartridge under low vacuum conditions for 30 s. 6. Elute the analytes with 1.5 mL of methanol at a flow speed of 2 mL/min, and completely remove the remaining solvent through vacuum. 7. Evaporate the eluate to dryness under nitrogen stream and heating at 35 8C.
8. Dissolve the sample in 100 mL of a mixture of water: acetonitrile (1:1) containing 1% formic acid and vortex for 30 s. 9. Transfer the reconstituted extract to autosampler vial for LC-MS/MS analysis.

LC-MS/MS conditions
The liquid chromatograph was an Alliance 2695 system consisting of a quaternary pump, solvent degasser, auto sampler and column heater (Waters Corporation, Milford, USA). Chromatographic separation was achieved on a Waters XBridge MS C18 column (3.5 mm, 2.1 mm Â 150 mm) in combination with a protecting guard column (3.5 mm, 2.1 mm Â 5 mm) of the same type (Waters Corporation, Milford, USA). The mobile phase was water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B).
A small amount (0.5 mL) of plasma is required, making this approach suitable for pharmacokinetic studies also in small sized dogs.

Method validation
The proposed LC-MS/MS method was validated according to current European guidelines [2], taking into account specificity, linearity, accuracy, precision and limit of quantification (LOQ). The specificity was evaluated checking the ion chromatograms of blank samples collected from 21 dogs and analyzed for potential interfering compounds co-eluting at the specific retention times of the analytes.
The linearity of the method was assessed with five-points matrix-matched calibration curves, obtained by spiking pooled blank samples of plasma with standard solutions of BUD and 16a-HP at 0, 0.25, 0.5, 1, 5 and 10 ng/mL, and freshly prepared during different days. Peak area ratios between each analyte and BET (internal standard) were plotted against their concentration ratios, and a linear regression least square regression model was applied, showing a good linearity (R 2 > 0.99).
The precision and accuracy of the assay were tested by analyzing blank samples fortified at three concentrations (0.5, 1 and 5 ng/mL) for both analytes. In particular, 18 spiked samples for each level were processed on three different days (six samples per day). The precision was measured as relative standard deviation to the mean (CV%) of both repeatability (within-day) and reproducibility (between-days). The results of the accuracy and precision experiments ( Table 2) confirmed the good performances of the method.
The limit of quantification (LOQ) of the method, defined as the measured concentration with a signal to noise (S/N) ratio greater than 10, and acceptable precision and accuracy, was 0.25 ng/mL for both analytes.
The absence of matrix ion suppression was assessed by the post column infusion technique: during the injection of a blank matrix sample in the LC-MS system, BUD and 16a-HP standard solutions were directly and continuously infused into the MS interface [3].

Background
Corticosteroids are anti-inflammatory drugs frequently used for the treatment of a wide range of diseases both in human and veterinary medicine. BUD is a non-halogenated glucocorticoid recently introduced in veterinary medicine for the treatment of respiratory and inflammatory bowel diseases (IBD), particularly in dogs refractory to systemic steroids. However, BUD veterinary-labeled products are not available on the market to date. BUD is rapidly metabolized and one of the main products in human is 16a-HP [2]. Although many different works have been published on the detection of BUD in human plasma [4][5][6][7], only two studies are available on its determination in dog plasma, one of which written in Chinese [8,9]; in addition, none of them simultaneously investigated both BUD and 16a-HP.

Acknowledgement
MethodsX thanks the reviewers of this article for taking the time to provide valuable feedback.