Test animals
Twelve clinically healthy cows of a mixed breed were housed under controlled experimental conditions at the animal facilities of Centre d’Economie Rurale (CER, Marloie, Belgium). These cows were 2 to 6 years of age and had a body weight between 370 and 600 kg. They were fed a commercial diet, with ad libitum access to water and hay. During the entire study, animals were kept in three separate groups (4 animals per group), all housed in a half-covered pen. Prior to the in vivo study, an initial acclimatization period of 10 days was foreseen, allowing adaptation to the environmental and feeding conditions. This study was approved by CER’s Ethical Committee (CE/Sante/ET/004).
Experimental protocol
After acclimatization (Fig. 1A), animals were subjected to a similar oral (per os, PO) and intramuscular (IM) prednisolone treatment sequence. First, a growth-promoting treatment (long-term, 40 mg per cow a day, PO and IM) was applied, followed by a therapeutic treatment (short-term, 0.5 mg per kg bodyweight a day, PO and IM) (Fig. 1A). Then, a washout period of 11 weeks was incorporated, after which tetracosactide hexaacetate (2 mg per day) was administered intramuscularly for 4 days to mimic stress (Fig. 1B). All types of treatment were executed at 8 h in the morning.
Phase I: Prednisolone treatments
The growth-promoting treatment started with 30 consecutive days of PO administration of 40 mg per day of prednisolone in capsules (prednisolone, Fagron, Belgium) (Day + 1 till Day + 30), followed by a washout period of 10 days. Next, IM injections of 40 mg per day of Solu-Delta-Cortef® (prednisolone sodium succinate, Zoetis, Belgium) were given for 30 consecutive days (Day + 41 till Day + 70). Before the start of the therapeutic prednisolone treatment, a washout period of 35 days was foreseen.
During the therapeutic treatment, a similar experimental set-up was implemented. First, the animals received 0.5 mg prednisolone per kg body weight PO for 5 days (Day + 106 till Day + 110), which was followed by a washout period of 25 days. Next, IM injections of 0.5 mg of Solu-Delta-Cortef® per kg body weight were given during 5 consecutive days (Day + 136 till Day + 140). Until 32 days (Day + 141 till Day + 172) after the last prednisolone administration, urine samples were collected in order to monitor the reconversion to the natural glucocorticoid body state.
Phase II: ACTH treatment
After phase I, a washout period of 11 weeks was considered. Subsequently, all animals received IM injections of 2 mg tetracosactide hexaacetate (Utrecht University, Faculty of Veterinary Medicine, Utrecht, The Netherlands), corresponding to 200 I.U. of ACTH, during 4 consecutive days.
Sample collection
During prednisolone treatments, urine samples were each time collected in the morning, prior to prednisolone administration. These samples were obtained by a veterinarian using a probe (to prevent faecal contamination) and were immediately portioned into 15-mL tubes, which were then stored in the dark at −80 °C until analysis. During the growth-promoting treatment, samples were collected every five days whereas during the therapeutic treatment, urine samples were collected the first, third and last day. In addition, samples were also collected every day during the acclimatization period and every five days during the washout periods. Also, urine samples were collected twice a day during the ACTH treatment period, whereby samples were collected prior to and at 4 h (Day + 1 and Day + 2) or at 6 h (Day + 3 and Day + 4) after tetracosactide hexaacetate administration.
Blood samples were collected in the morning, thereby applying a similar sampling strategy as for urine. In addition, blood samples for pharmacokinetic analysis were collected at the beginning and end of each treatment period at time 0 (just before administration), 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h (post-administration). With each collection, 5 mL of blood was sampled into heparin tubes. One hour after collection, blood was centrifuged at 600 x g during 15 min at 4 °C and divided into 2-mL plasma aliquots, which were then immediately stored at −20 °C until analysis.
Reagents and chemicals
Standards of prednisolone, prednisone, cortisone, cortisol, dihydrocortisone, aldosterone, allotetrahydrocortisol, urocortisol, tetrahydrocortisone, corticosterone, deoxycorticosterone, α-cortolone and 6β-hydroxycortisol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Internal standards were prednisolone-d8 (TRC, Canada), cortisol-d4 and prednisolonde-d4 (Sigma-Aldrich). Reagents were of analytical grade (Merck, Darmstadt, Germany) when used for extraction purposes and of liquid chromatography-mass spectrometry (LC-MS) Optima grade (Fisher Scientific, Loughborough, UK) for ultra-high performance LC (UHPLC) tandem MS (MS/MS) and high-resolution MS (HRMS) applications. Ultrapure water was obtained by usage of a purified-water system (Sartorius AG, Goettingen, Germany). Stock solutions were prepared in ethanol at a concentration of 200 μg mL−1 and stored in dark glass bottles at −20 °C.
Sample preparation
Urine
A detailed description of the analytical procedure, which was used for glucocorticoid extraction from urine, has been given in earlier work [21]. In brief, 5 mL of urine was enriched with the internal standards cortisol-d4 and prednisolone-d8 to reach final concentration of 10 μg L−1. Next, a twofold liquid-liquid extraction with tert-butyl methyl ether was applied, whereby the organic phases were collected, pooled and dried under a gentle stream of nitrogen at 50 °C. The residue was then dissolved in 100 μL of initial mobile phase conditions and transferred into an LC- vial.
Plasma
Two mL of vortexed plasma was spiked with the internal standards cortisol-d4 and prednisolone-d4 to obtain final concentrations of 10 μg L−1. Glucocorticoids were extracted by liquid-liquid extraction, thereby using 5 mL acetonitrile. After 30 min of extraction, samples were centrifuged at 3760 x g for 10 min at 10 °C. Then, the supernatants were collected and evaporated under a gentle stream of nitrogen at 40 °C. The residue was suspended in 200 μL of water-acetonitrile (80/20, v/v) and transferred into an LC-vial.
Mass spectrometric detection
UHPLC-Orbitrap MS for urine
Glucocorticoid analysis of urine was performed by UHPLC-Orbitrap MS, according to the method of De Clercq et al. (2013) [21]. Chromatographic separation of the target analytes was thereby achieved using an Accela UHPLC system (Thermo Fisher Scientific, San José, USA), equipped with a Nucleodur Isis C18 column (1.8 μm, 100 mm × 2 mm, Macherey-Nagel, Düren, Germany). High-resolution mass spectrometric analysis was performed with an Exactive™ single-stage Orbitrap mass spectrometer (Thermo Fisher Scientific), equipped with a heated electrospray ionization probe (HESI II), operating in polarity switching mode. Instrument control and data processing were carried out by Xcalibur 2.1 software (Thermo Fisher Scientific, San José, USA).
UHPLC-MS/MS analysis for plasma
The chromatographic analysis of glucocorticoids in plasma was performed by a Waters Acquity system (Waters, Manchester, UK) according to Delahaut et al. (2014) [16]. Chromatographic separation of the target analytes was thereby achieved using an Acquity C18 column (1.7 μm, 125 × 3 mm). Mass spectrometric analysis was performed with a Xevo TSQ tandem mass spectrometer (Waters Corporation, Manchester, UK), operating in the positive ion electrospray mode and applying multiple reaction monitoring (MRM). For each target compound, two transitions were monitored (Additional file 2), the first being the quantifier and the second being the qualifier. For quantification, two internal standards were used: prednisolone-d4 and cortisol-d4. Instrument control and data processing were carried out by MassLynx and QUANLYNX software (Waters Corporation, Manchester, UK) respectively.
A brief validation of the newly developed method for glucocorticoid analysis of plasma was performed based on Commission Decision 2002/657/EC guidelines [22]. The method performance in terms of repeatability, within-laboratory reproducibility, recovery, CCα and specificity was thereby assessed. Plasma samples that were used for validation were obtained from non-medicated cows (n = 3), which were housed at the animal facilities of CER. Linearity was evaluated based on eight-point matrix-matched calibration curves with concentration levels ranging from 0.25 to 20 μg L−1 for prednisolone, prednisone, 20α-dihydroprednisolone, and 20β-dihydroprednisolone and from 0.5 to 40 μg L−1 for cortisol, cortisone, and dihydrocortisone. Repeatability was determined by analysis of samples that were spiked with the target compounds, thereby considering seven replicates for two different concentration levels, i.e. 0.50 and 5 μg L−1 for prednisolone, prednisone, 20α-dihydroprednisolone, and 20β-dihydroprednisolone and 1 and 10 μg L−1 for cortisol, cortisone, and dihydrocortisone. For evaluation of the within-laboratory reproducibility, the specified analyses were performed on two different occasions, with a different operator on the second occasion. The CCα was derived from chromatograms and corresponded to a concentration giving a peak with a signal-to-noise ratio of 3. Specificity was evaluated by analyzing a potential interfering substance (methylprednisolone) to verify potential cross-talk during analysis.
Quantitation and normalization
Due to the broad concentration range expected in the urine samples during the different prednisolone treatments, quantitation of the various urinary glucocorticoid compounds was based on two eight-point calibration curves (using area ratios), which were prepared in urine matrix. Samples were thereby fortified with all glucocorticoid standards to reach concentrations from 0.50 to 75 ng mL−1 and from 100 to 200 ng mL−1 for cortisol, cortisone, dihydrocortisone, prednisolone, prednisone, methylprednisolone, 20α-dihydroprednisolone, and 20β-dihydroprednisolone. The employed urine matrix was previously verified to contain no residues of prednisolone, prednisone, 20α-dihydroprednisolone and 20β-dihydroprednisolone, but the other glucocorticoids were found to be endogenously present. Therefore, the endogenous concentration levels of cortisol, cortisone, and dihydrocortisone were determined as the average of five non-fortified urine samples and taken into account during quantitation. In addition, since urine is a matrix prone to dilution, normalization by means of the specific gravity (Pocket Refractometer™, Atago, Tokyo) was applied using the Levine-Fahy eq. [23].
Pharmacokinetic analysis
Pharmacokinetic analysis was performed with WinNonlin 6.3 (Pharsight Corporation, St-Louis, USA). Plasma concentration-time profiles were modeled using a one-compartmental model for PO and a two-compartmental model for IM prednisolone administration. The most important pharmacokinetic parameters were the peak plasma concentration (Cmax), time to reach the peak plasma concentration (Tmax), area under the plasma concentration-time curve from time 0 to time inf (AUC0-inf), absorption rate constant (ka), absorption half-life (T1/2a), apparent clearance (Cl/F) and apparent volume of distribution (Vd/F). Additionally, for two-compartmental methods, the distribution rate constant (kelα), the elimination rate constant (kelβ) and elimination half-life (T1/2elβ) were also determined. The coefficient of determination (R2) was hereby used as an indicator for the goodness-of-fit. For the main metabolites, only Cmax, Tmax, AUC0-inf, kel en T1/2el were calculated.
Statistical analysis
All pharmacokinetic parameters were compared between administration routes according to dose (therapeutic and growth-promoting), thereby using one-way analysis of variance (ANOVA) (p-value ≤ 0.05) (SPSS 21, IBM, USA). Urinary concentrations were statistically evaluated using Student’s t-test and one-way ANOVA with post-hoc Tukey’s multiple comparisons test.