Determination of a New Pleuromutilin Derivative in Broiler Chicken Plasma by RP-HPLC-UV and Its Application to a Pharmacokinetic Study

Determination of a New Pleuromutilin Derivative in Broiler Chicken Plasma by RP-HPLC-UV and Its... Abstract A simple, sensitive and reproducible high-performance liquid chromatography method was developed and validated for the determination of 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM), a new synthesized pleuromutilin derivative with potent antibacterial activity, in broiler chicken plasma after a single intravenous (i.v.), intramuscular (i.m.) or oral (p.o.) administration. Satisfactory separation was achieved on a ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) with UV detection at 279 nm, using a mobile phase comprising acetonitrile and ultrapure water (50:50, v/v). The elution was isocratic at ambient temperature with a flow rate of 1.0 mL/min. The method exhibited good linearity (R2 > 0.999) over the assayed concentration range (0.12–120.00 μg/mL) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied to the pharmacokinetic study of ATTM in chicken plasma after i.v. and p.o. administration. Introduction Pleuromutilin (Figure 1), constituted of a rather rigid 5–6–8 tricyclic carbon skeleton and a glycolic acid chain at C-14 (1, 2), was first discovered and isolated from Pleurotus mutilus and Pleurotus passeckerianus as a natural compound in 1951 (3). The modifications of the C-14 position have led to three drugs: tiamulin, valnemulin and retapamulin. Tiamulin and valnemulin are used in veterinary medicine for pigs and poultry (4, 5). Retapamulin was approved as a topical antimicrobial agent for the treatment of human skin infections in 2007 by Food and Drug Administration (6, 7). Extensive efforts were made to formulate BC-3781, BC-3205 and BC-7013 for human use (8, 9) after the success of retapamulin. Figure 1. View largeDownload slide Chemical structure of pleuromutilin and ATTM. Figure 1. View largeDownload slide Chemical structure of pleuromutilin and ATTM. Pleuromutilin derivatives selectively inhibit bacterial protein synthesis through interaction with prokaryotic ribosomes (10). Chemical footprinting studies showed that tiamulin and valnemulin bound to the bacterial ribosome at the peptidyl transferase center, thereby inhibiting the synthesis of peptide bond by hindering a correct location of the amino acid of tRNA (11). Crystallography data, utilizing a structure of 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin, demonstrated that the interactions of tricyclic core of the tiamulin are mediated through hydrophobic interactions and hydrogen bonds which are formed mainly by the nucleotides of domain V (12, 13). 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM, Figure 1) is a new derivative of pleuromutilin, with excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and Streptococcus agalactiae (14). The preliminary pharmacokinetic studies in rat showed that ATTM may serve as a possible lead compound for the development of antibacterial drug for veterinary use (14). The purpose of this study was to develop and validate a simple, sensitive and accurate high-performance liquid chromatography (HPLC) method for the quantification of ATTM in chickens plasma after administration. Moreover, the method has been applied to study the pharmacokinetics of ATTM in the chicken model. Experimental Materials and reagents ATTM was synthesized by our lab, and the structure was elucidated through infrared spectroscopy (IR), nuclear magnetic resonance (NMR) and high-resolution mass spectroscopy (HRMS) as well as comparison with the literature (14). Acetonitrile were HPLC grade and obtained from Fisher Scientific (Pittsburg, PA, USA). Ethyl acetate and methanol were purchased from the Tianjin Concord Chemical Reagent Co. Ltd (Tianjin, China). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other chemicals were of analytical grade or better. Equipment The analysis was carried out on a Waters 2695 HPLC (MA, USA) system equipped with a solvent degasser, a quaternary pump with controller, a manual injector and a diode array detector (DAD-2998). All instrument parts were automatically controlled by Empower software supplied from Waters Corporation. HPLC conditions The ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) was used for the separation with the column temperature maintained at 30°C. The injection volume was 10 μL and the total run time was 15 min. The isocratic elution with a mobile phase of acetonitrile-ultrapure water (50:50, v/v) pumped at a flow rate of 1.0 mL/min throughout the HPLC process. The mobile phase was filtered through a 0.22-μm filter and degassed ultrasonically for 15 min before use. ATTM was detected at the absorption wavelength 279 nm. Stock and working solutions Stock solutions of ATTM were prepared at a concentration of 120 μg/mL in acetonitrile and further diluted into 0.12, 1.20, 12.00, 60.00, 120.00 μg/mL for the preparation of working solutions. All solutions were stored at −70°C before use for no longer than 4 weeks. Calibration standards and quality control samples For the construction of calibration curves, a drug stock solution (120.00 μg/mL) was prepared by appropriate dissolution of ATTM in acetonitrile and further diluted to 0.12, 1.20, 12.00, 60.00, 120.00 μg/mL by spiking control chicken plasma. Three quality control (QC) samples were prepared to contain 2.00 μg/mL (QC low), 20 μg/mL (QC medium) and 200 μg/mL (QC high) by spiking drug-free plasma with ATTM for method validation studies. Sample preparation All the plasma samples were prepared by protein precipitation and extraction procedure from plasma. Ethyl acetate, methanol and acetonitrile were chosen to evaluated their protein precipitation and extraction efficiency according to the physicochemical property of ATTM. Three precise weighing ATTM samples (1.00 mg) were dissolved in 200-μL plasma, and 800 μL of three agents were added, respectively. The mixed samples were vortex-mixed for ~2 min and centrifuged in 15,000 rpm for 10 min. The supernatants were transferred to a new clean tube and following dryness with gentle nitrogen flow at 30°C. The dried samples were reconstituted with 500 μL acetonitrile and injected in HPLC system for analysis. The recovery of three agents was measured by comparison chromatographic peak areas with that of same quantity of ATTM directly dissolved in 500 μL acetonitrile. This procedure was repeated in triplicate. Method validation Specificity The selectivity of the method was tested by analyzing six different batches of blank chicken plasma with or without ATTM by comparison of corresponding peaks to exclude potential endogenous interference. The interference was tested using the chromatographic/spectroscopic conditions. For the chromatographic peak of the six batches of blank chicken plasma spiked with ATTM, the purity angle and purity threshold were calculated using the chromatographic software. Linearity of calibration curves and range The linearity of the method was determined by analyzing the calibration standard samples ranged from 0.12 to 120.00 μg/mL, and the calibration curves were constructed by plotting peak area (y) of ATTM versus nominal concentration (x) in plasma. The lowest plasma level of ATTM on the calibration curves (0.12 μg/mL) was recognized as the lower limit of quantification (LLOQ) which can be quantified reliably, with an acceptable accuracy (80–120%) and precision (≤20%). The lower limit of detection and LLOQ were defined as a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively. Precision and accuracy The precision of this assay was determined by replicate analysis of LLOQ (0.12 μg/mL) and three concentration QC samples. An internal standard was not used due to the simplicity of the sample preparation procedure and use of a high-precision autosampler. The intra-day precision was determined by repeated analysis of the group of standards in a day (n = 6). The inter-day precision was determined by repeated analysis of the group of standards on three consecutive days (n = 6 series per day). The % recovery and % coefficient of variation (% CV) of QC samples were used to express the accuracy and precision, respectively. Recovery and matrix effect The assay recovery of plasma samples was determined by comparing the measured concentration with the added concentration to evaluate three concentration levels in blank plasma. The extraction recovery was determined by comparing peak areas of extracted spiked samples with those corresponding working solutions at the same concentrations. The matrix effect was evaluated by comparing the peak areas of the post-extracted spiked ATTM samples with those of corresponding working solutions. These procedures were repeated for five replicates at three concentration levels (0.12, 1.20 and 12.00 μg/mL) in blank plasma. Stability Spiked samples with ATTM at low, medium and high concentrations were used for stability evaluation under different storage and handling conditions, including three freeze–thaw cycles, ATTM was determined in six replicates at ambient temperature (25 ± 2°C) for 24 h and at 4°C in the autosampler tray for 24 h in processed samples. The stability was acceptable when 85–115% of the initial analytes was found. Pharmacokinetic study Animal About 35-week-old healthy white feather broilers (1.52–2.31 kg, 15 males and 15 females) were obtained from a commercial farm and housed under controlled conditions at 25 ± 2°C and humidity at 45–65% according to the requirements for this species. The birds had free access to water and non-medicated diets and were bred in a breeding room at 23°C, with 45 ± 5% humidity and a 12-h dark–light cycle. All animal procedures were conducted in accordance with the approved Institutional Animal Care and Use Committee (IACUC) protocols in Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS. Study design The animal was placed in metabolic cage, allowed to recover overnight, and fasted for 12 h before dosing. On the day of experiment, birds were weighed and assigned at random to each of the three groups, with the constraint that each group had to contain 10 chickens (5 males and 5 females). There was no significant difference between the average body weights of different groups. According to the results of subchronic oral toxicity of ATTM in rats (15), we selected 6-fold safe dose (30 mg/kg body weight) as a single intravenous (i.v.), intramuscular (i.m.) or oral (p.o.) administration, respectively. ATTM was formulated as a solution of 5% Tween in water and given in the right brachial vein, chest muscle and directly into the crop using a thin plastic tube attached to a syringe for i.v., i.m. and p.o. administration, respectively. Blood samples (~1 mL) were withdrawn from the heparinized catheter placed in the left brachial vein at 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after the administration. Blood samples were immediately centrifuged at 3,000 rpm for 10 min, and the plasma was separated and immediately frozen at −20°C until assayed. A total of 0.5 mL of the sample was mixed with 2 mL of acetonitrile in a 10-mL centrifuge tube. The sample was vortexed vigorously and then centrifuged at ~1,200 rpm for 10 min. The upper organic layer was transferred to clean centrifuge tube and the sample was re-extracted twice more with acetonitrile using the condition previously described. The whole organic extract was then evaporated to dryness under a nitrogen stream at 40°C and the residue was reconstituted in 200 μL of acetonitrile. Following this, an aliquot of the reconstituted extracts was transferred and filtrated through a 0.22-μm cellulose membrane filter and then 10 μL was injected into the HPLC system. Pharmacokinetic parameters A non-compartmental model was used to determine the pharmacokinetic parameters of ATTM. Pharmacokinetic parameters, including maximum plasma concentration (Cmax), time (Tmax), area under the plasma concentration versus time curve from zero to last sampling time (AUC0−t) and infinity (AUC0−∞) and elimination half-life (t1/2) were calculated using PKSolver Software (16). Data are reported as mean ± SD. Statistical analysis was conducted using SPSS 10.0 (IBM SPSS, USA). Results Synthesis and structural identification of ATTM The ATTM was synthesized with an overall yield of 81%. The purity was >98% by purifying with silica gel column chromatography. IR (KBr): 3,419, 3,330, 2,931, 1,731, 1,616, 1,507, 1,456, 1,417, 1,373, 1,282, 1,190, 1,152, 1,117, 1,019, 980, 953, 9,38,916 cm−1. 1H NMR (400 MHz, DMSO) δ: 7.28 (s, 2 H), 6.08 (dd, J = 17.8, 11.2 Hz, 1 H), 5.52 (d, J = 8.1 Hz, 1 H), 5.03 (dd, J = 21.0, 14.7 Hz, 2 H), 4.51 (d, J = 5.9 Hz, 1 H), 4.02 (q, J = 7.1 Hz, 1 H), 3.90 (q, J = 16.0 Hz, 2 H), 3.45–3.32 (m, 2 H), 2.39 (s, 1 H), 2.19 (dd, J = 18.8, 10.8 Hz, 1 H), 2.11–1.97 (m, 4 H), 1.64 (dd, J = 18.4, 9.5 Hz, 2 H), 1.54–1.42 (m, 1 H), 1.36–1.21 (m, 6 H), 1.16 (d, J = 7.1 Hz, 1 H), 1.08–0.94 (m, 4 H), 0.82 (d, J = 6.7 Hz, 3 H), 0.59 (d, J = 6.7 Hz, 3 H). 13C NMR (100 MHz, DMSO) δ: 217.08, 169.73, 166.70, 148.89, 140.68, 115.39, 72.63, 70.27, 59.75, 57.23, 44.95, 44.14, 41.51, 36.33, 34.00, 30.10, 28.70, 26.60, 24.47, 20.75, 16.09, 14.51, 14.08, 11.54. HRMS (ESI): [M + H]+ calcd for C24H35N3O4S2, 494.2142; found, 494.2139. Sample preparation The protein precipitation and extraction from plasma and concentration of objective components should guarantee the preparation procedure with high recovery and avoiding their degradation. Three types of reagents (ethyl acetate, methanol and acetonitrile) for preparation were studied during the experiment. As shown in Figure 2, acetonitrile was the best agent in terms of perfect preparation and absence of interference. Figure 2. View largeDownload slide Effect of different kinds of extraction agents on the extraction efficiency. Figure 2. View largeDownload slide Effect of different kinds of extraction agents on the extraction efficiency. Chromatographic conditions A simple, accurate, fast and sensitive HPLC method was developed to routinely measure plasma levels and study pharmacokinetics of ATTM in our chicken model to correlate their pharmacological effects to plasma levels and pharmacokinetic behavior. This method is valid within a wide range of plasma concentrations (0.120–120.00 μg/mL) and may be proposed as a suitable method for pharmacokinetic studies. Good separation of ATTM peak from that of many interfering compounds with short run times was obtained using a mobile phase system of acetonitrile and water at a ratio of 50:50 v/v, at 1 mL/min flow rate (Figure 3). Figure 3. View largeDownload slide Chromatograms of ATTM in chicken plasma. (A) Blank chicken plasma. (B) Spiked with 12 μg/mL of ATTM (1). (C) Plasma sample collected from a chicken 30 min following a single i.v. administration of 30 mg/kg of ATTM (1). Figure 3. View largeDownload slide Chromatograms of ATTM in chicken plasma. (A) Blank chicken plasma. (B) Spiked with 12 μg/mL of ATTM (1). (C) Plasma sample collected from a chicken 30 min following a single i.v. administration of 30 mg/kg of ATTM (1). Assay validation Sensitivity and selectivity Plasma samples spiked with ATTM were assayed in decreasing concentrations. The LLOQ of ATTM in chicken plasma was 0.12 μg/mL. The specificity of this method was examined by analyzing six different blank chicken plasma samples and blank plasma spiked with ATTM. Typical chromatograms of blank plasma, blank plasma spiked with 12 μg/mL of ATTM, and chicken plasma collected at 30 min after i.v. administration of ATTM are shown in Figure 3. No interfering peak of endogenous substance that affects the determination of ATTM (~6.75 min) in chicken plasma was observed. Furthermore, the purity threshold and purity angle values of six batches of blank plasma spiked with ATTM are tabulated in Table I. It was observed that the purity angle values were always less than the purity threshold values indicating that the peak observed was pure. Table I. Purity Angel and Purity Threshold of Blank Plasma Spiked with ATTM No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 Table I. Purity Angel and Purity Threshold of Blank Plasma Spiked with ATTM No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 Linearity Calibration curves were constructed using five standards in plasma by plotting peak area versus the nominal concentration of ATTM. The established calibration curves were found to be linear over the entire concentration range of 0.12–120.00 μg/mL, and used to measure ATTM concentrations in pharmacokinetics analysis and validation assay. The coefficient of determination (R2) was 0.9994 in all calibration curves (n = 5). The mean regression equation for ATTM was y = 8401.41x + 400.75. Precision and accuracy The accuracy and precision data for the plasma samples are presented in Table II. The intra- and inter-day accuracy assessed by replicate analysis of QC samples in plasma on three consecutive days were 95.33–104.01% and 99.40–102.78%, respectively. The intra- and inter-day precision (% CV) ranged from 1.49% to 4.84% and 0.93% to 3.92%, respectively. Both intra- and inter-day accuracy and precision were within acceptable limits, which indicated that the established HPLC method was good at precision and accuracy. Table II. Intra- and Inter-Day Precision and Accuracy of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Table II. Intra- and Inter-Day Precision and Accuracy of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Recovery The assay recovery and extraction recovery of ATTM were determined at 0.12, 1.20 and 12.00 μg/mL (n = 6). The mean assay recoveries were 92.50%, 98.25% and 98.76% for ATTM of 0.12, 1.20 and 12.00 μg/mL in plasma, respectively. The data of extraction recovery were ranged from 90.83% to 96.02% with an RSD < 2%, which indicated that the extraction procedure was consistent and reproducible (Table III). The high recovery suggested that there was negligible loss during the drug extraction. Table III. Recovery of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Table III. Recovery of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Stability Stability tests of the ATTM were assessed using triplicates of spiked samples at three concentrations under different conditions. The results of the short-term (24 h) at room temperature, long-term (4 weeks) at −70°C and freeze–thaw are shown in Table IV. The ATTM did not degrade up to 24 h at room temperature and was found to be stable over a period of 4 weeks at the storage condition of −70°C. Stability of ATTM after three freeze–thaw cycles of plasma samples indicated it was stable when subjected to these conditions. Based on these results, there was no stability-related problem to influence the assay. Table IV. Stability of ATTM in Chicken Plasma (n = 6) Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Table IV. Stability of ATTM in Chicken Plasma (n = 6) Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Pharmacokinetics analysis The validated analytical method was successfully applied to investigate the pharmacokinetics of ATTM in broiler chicken plasma after a single i.v., i.m. or p.o. dose of 30 mg/kg, respectively. The main pharmacokinetic profiles are displayed in Table V and the mean plasma concentration versus time curve (as well as the log-plasma concentration versus time curve) of ATTM after i.v., i.m. and p.o. administration are shown in Figure 4. Table V. Pharmacokinetic Parameters of ATTM in Broiler Chickens after Administration Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 aMaximum concentration. bTime to reach Cmax. cHalf-life. dClearance. eVolume of distribution. fMean resident time. gMean resident time. gArea under the curve. hOral bioavailability. Table V. Pharmacokinetic Parameters of ATTM in Broiler Chickens after Administration Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 aMaximum concentration. bTime to reach Cmax. cHalf-life. dClearance. eVolume of distribution. fMean resident time. gMean resident time. gArea under the curve. hOral bioavailability. Figure 4. View largeDownload slide Plasma concentration–time curve (A) and log-concentration–time curve (B) after i.v., i.m. and p.o.administration of ATTM to broiler chickens. Figure 4. View largeDownload slide Plasma concentration–time curve (A) and log-concentration–time curve (B) after i.v., i.m. and p.o.administration of ATTM to broiler chickens. Discussion The elimination of plasma proteins or other interfering organic components is a prerequisite for a successful HPLC determination of analytes in the developing of an analytical method. In this study, a simple protein precipitation and liquid–liquid extraction procedure was optimized to suit the pharmacokinetic studies for ATTM. Protein precipitation and extraction with ethyl acetate and methanol gave poor recoveries, respectively. However, the preparation with acetonitrile was much better and showed no interfering chromatographic peaks due to plasma endogenous substances. A sensitive, specific, accurate and reproducible reversed phase HPLC method for the determination of ATTM in broiler chicken plasma was developed and validated in this study. This method was successfully applied to the study of pharmacokinetics of ATTM in broiler chickens for the first time and can be available for large number of biological samples very efficiently. The detection wavelength is very important for the analysis of biological samples. The scan of ATTM in acetonitrile using UV spectrophotometer revealed absorption maxima at 279 nm, and was therefore selected as the detection wavelength for HPLC studies. The establishment of chromatographic conditions started from selection of HPLC columns. ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) demonstrated symmetrical peak shapes and better separation for the analyte compared with the other chromatographic columns. It was found that base and acid in the mobile phase had a minimal effect on the retention time and peak shape of ATTM. Therefore, the various mixtures of pure water and acetonitrile were tested to enhance the peak resolution and to eliminate the peak tailing of the target compounds with an optimization procedure. After a single i.v. administration of ATTM (30 mg/kg) in broiler chickens, the mean value of t1/2 was 3.93 h which was close to that in SD rat with 3.40 h of t1/2 after a single i.v. administration (5 mg/kg) of ATTM (14) and reflected a high rate of elimination. Following p.o. administration, ATTM was absorbed rapidly, with Cmax values of 27.82 μg/mL at 4.00 h (Tmax), respectively. Then, the concentrations of the compound dropped with elimination half-life times (t1/2) of 6.08 h, respectively. Compared with SD rats after the same route at a dose of 5 mg/kg (Cmax, 3.07; Tmax, 0.75), ATTM was absorbed more slowly, but with a higher Cmax in chickens (14). The systemic bioavailability of ATTM was 72.03% following p.o. administration in chickens, which was higher than that in SD rats (F, 35.81). Following i.m. administration, ATTM was absorbed rapidly, with Cmax values of 29.03 μg/mL at 0.50 h (Tmax). Then, the concentrations of ATTM dropped with elimination half-life times (t1/2) of 3.92 h. The systemic bioavailability of ATTM was 75.29% following i.m. administrations in chickens. Conclusion An accurate and precise HPLC method for the determination of ATTM in chickens plasma has been developed and validated. Using this method, pharmacokinetic features of ATTM after i.v., i.m. and p.o. administration in chickens was investigated, which were characterized by high i.m. or p.o. bioavailability. The achieved pharmacokinetic results could be useful for further study on the active mechanism of ATTM. Acknowledgments This work was supported by the Basic Scientific Research Funds in Central Agricultural Scientific Research Institutions (No. 1610322016007) and the National Key Technology Support Program (No. 2015BAD11B02). 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All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Determination of a New Pleuromutilin Derivative in Broiler Chicken Plasma by RP-HPLC-UV and Its Application to a Pharmacokinetic Study

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Abstract

Abstract A simple, sensitive and reproducible high-performance liquid chromatography method was developed and validated for the determination of 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM), a new synthesized pleuromutilin derivative with potent antibacterial activity, in broiler chicken plasma after a single intravenous (i.v.), intramuscular (i.m.) or oral (p.o.) administration. Satisfactory separation was achieved on a ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) with UV detection at 279 nm, using a mobile phase comprising acetonitrile and ultrapure water (50:50, v/v). The elution was isocratic at ambient temperature with a flow rate of 1.0 mL/min. The method exhibited good linearity (R2 > 0.999) over the assayed concentration range (0.12–120.00 μg/mL) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied to the pharmacokinetic study of ATTM in chicken plasma after i.v. and p.o. administration. Introduction Pleuromutilin (Figure 1), constituted of a rather rigid 5–6–8 tricyclic carbon skeleton and a glycolic acid chain at C-14 (1, 2), was first discovered and isolated from Pleurotus mutilus and Pleurotus passeckerianus as a natural compound in 1951 (3). The modifications of the C-14 position have led to three drugs: tiamulin, valnemulin and retapamulin. Tiamulin and valnemulin are used in veterinary medicine for pigs and poultry (4, 5). Retapamulin was approved as a topical antimicrobial agent for the treatment of human skin infections in 2007 by Food and Drug Administration (6, 7). Extensive efforts were made to formulate BC-3781, BC-3205 and BC-7013 for human use (8, 9) after the success of retapamulin. Figure 1. View largeDownload slide Chemical structure of pleuromutilin and ATTM. Figure 1. View largeDownload slide Chemical structure of pleuromutilin and ATTM. Pleuromutilin derivatives selectively inhibit bacterial protein synthesis through interaction with prokaryotic ribosomes (10). Chemical footprinting studies showed that tiamulin and valnemulin bound to the bacterial ribosome at the peptidyl transferase center, thereby inhibiting the synthesis of peptide bond by hindering a correct location of the amino acid of tRNA (11). Crystallography data, utilizing a structure of 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin, demonstrated that the interactions of tricyclic core of the tiamulin are mediated through hydrophobic interactions and hydrogen bonds which are formed mainly by the nucleotides of domain V (12, 13). 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM, Figure 1) is a new derivative of pleuromutilin, with excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and Streptococcus agalactiae (14). The preliminary pharmacokinetic studies in rat showed that ATTM may serve as a possible lead compound for the development of antibacterial drug for veterinary use (14). The purpose of this study was to develop and validate a simple, sensitive and accurate high-performance liquid chromatography (HPLC) method for the quantification of ATTM in chickens plasma after administration. Moreover, the method has been applied to study the pharmacokinetics of ATTM in the chicken model. Experimental Materials and reagents ATTM was synthesized by our lab, and the structure was elucidated through infrared spectroscopy (IR), nuclear magnetic resonance (NMR) and high-resolution mass spectroscopy (HRMS) as well as comparison with the literature (14). Acetonitrile were HPLC grade and obtained from Fisher Scientific (Pittsburg, PA, USA). Ethyl acetate and methanol were purchased from the Tianjin Concord Chemical Reagent Co. Ltd (Tianjin, China). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other chemicals were of analytical grade or better. Equipment The analysis was carried out on a Waters 2695 HPLC (MA, USA) system equipped with a solvent degasser, a quaternary pump with controller, a manual injector and a diode array detector (DAD-2998). All instrument parts were automatically controlled by Empower software supplied from Waters Corporation. HPLC conditions The ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) was used for the separation with the column temperature maintained at 30°C. The injection volume was 10 μL and the total run time was 15 min. The isocratic elution with a mobile phase of acetonitrile-ultrapure water (50:50, v/v) pumped at a flow rate of 1.0 mL/min throughout the HPLC process. The mobile phase was filtered through a 0.22-μm filter and degassed ultrasonically for 15 min before use. ATTM was detected at the absorption wavelength 279 nm. Stock and working solutions Stock solutions of ATTM were prepared at a concentration of 120 μg/mL in acetonitrile and further diluted into 0.12, 1.20, 12.00, 60.00, 120.00 μg/mL for the preparation of working solutions. All solutions were stored at −70°C before use for no longer than 4 weeks. Calibration standards and quality control samples For the construction of calibration curves, a drug stock solution (120.00 μg/mL) was prepared by appropriate dissolution of ATTM in acetonitrile and further diluted to 0.12, 1.20, 12.00, 60.00, 120.00 μg/mL by spiking control chicken plasma. Three quality control (QC) samples were prepared to contain 2.00 μg/mL (QC low), 20 μg/mL (QC medium) and 200 μg/mL (QC high) by spiking drug-free plasma with ATTM for method validation studies. Sample preparation All the plasma samples were prepared by protein precipitation and extraction procedure from plasma. Ethyl acetate, methanol and acetonitrile were chosen to evaluated their protein precipitation and extraction efficiency according to the physicochemical property of ATTM. Three precise weighing ATTM samples (1.00 mg) were dissolved in 200-μL plasma, and 800 μL of three agents were added, respectively. The mixed samples were vortex-mixed for ~2 min and centrifuged in 15,000 rpm for 10 min. The supernatants were transferred to a new clean tube and following dryness with gentle nitrogen flow at 30°C. The dried samples were reconstituted with 500 μL acetonitrile and injected in HPLC system for analysis. The recovery of three agents was measured by comparison chromatographic peak areas with that of same quantity of ATTM directly dissolved in 500 μL acetonitrile. This procedure was repeated in triplicate. Method validation Specificity The selectivity of the method was tested by analyzing six different batches of blank chicken plasma with or without ATTM by comparison of corresponding peaks to exclude potential endogenous interference. The interference was tested using the chromatographic/spectroscopic conditions. For the chromatographic peak of the six batches of blank chicken plasma spiked with ATTM, the purity angle and purity threshold were calculated using the chromatographic software. Linearity of calibration curves and range The linearity of the method was determined by analyzing the calibration standard samples ranged from 0.12 to 120.00 μg/mL, and the calibration curves were constructed by plotting peak area (y) of ATTM versus nominal concentration (x) in plasma. The lowest plasma level of ATTM on the calibration curves (0.12 μg/mL) was recognized as the lower limit of quantification (LLOQ) which can be quantified reliably, with an acceptable accuracy (80–120%) and precision (≤20%). The lower limit of detection and LLOQ were defined as a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively. Precision and accuracy The precision of this assay was determined by replicate analysis of LLOQ (0.12 μg/mL) and three concentration QC samples. An internal standard was not used due to the simplicity of the sample preparation procedure and use of a high-precision autosampler. The intra-day precision was determined by repeated analysis of the group of standards in a day (n = 6). The inter-day precision was determined by repeated analysis of the group of standards on three consecutive days (n = 6 series per day). The % recovery and % coefficient of variation (% CV) of QC samples were used to express the accuracy and precision, respectively. Recovery and matrix effect The assay recovery of plasma samples was determined by comparing the measured concentration with the added concentration to evaluate three concentration levels in blank plasma. The extraction recovery was determined by comparing peak areas of extracted spiked samples with those corresponding working solutions at the same concentrations. The matrix effect was evaluated by comparing the peak areas of the post-extracted spiked ATTM samples with those of corresponding working solutions. These procedures were repeated for five replicates at three concentration levels (0.12, 1.20 and 12.00 μg/mL) in blank plasma. Stability Spiked samples with ATTM at low, medium and high concentrations were used for stability evaluation under different storage and handling conditions, including three freeze–thaw cycles, ATTM was determined in six replicates at ambient temperature (25 ± 2°C) for 24 h and at 4°C in the autosampler tray for 24 h in processed samples. The stability was acceptable when 85–115% of the initial analytes was found. Pharmacokinetic study Animal About 35-week-old healthy white feather broilers (1.52–2.31 kg, 15 males and 15 females) were obtained from a commercial farm and housed under controlled conditions at 25 ± 2°C and humidity at 45–65% according to the requirements for this species. The birds had free access to water and non-medicated diets and were bred in a breeding room at 23°C, with 45 ± 5% humidity and a 12-h dark–light cycle. All animal procedures were conducted in accordance with the approved Institutional Animal Care and Use Committee (IACUC) protocols in Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS. Study design The animal was placed in metabolic cage, allowed to recover overnight, and fasted for 12 h before dosing. On the day of experiment, birds were weighed and assigned at random to each of the three groups, with the constraint that each group had to contain 10 chickens (5 males and 5 females). There was no significant difference between the average body weights of different groups. According to the results of subchronic oral toxicity of ATTM in rats (15), we selected 6-fold safe dose (30 mg/kg body weight) as a single intravenous (i.v.), intramuscular (i.m.) or oral (p.o.) administration, respectively. ATTM was formulated as a solution of 5% Tween in water and given in the right brachial vein, chest muscle and directly into the crop using a thin plastic tube attached to a syringe for i.v., i.m. and p.o. administration, respectively. Blood samples (~1 mL) were withdrawn from the heparinized catheter placed in the left brachial vein at 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after the administration. Blood samples were immediately centrifuged at 3,000 rpm for 10 min, and the plasma was separated and immediately frozen at −20°C until assayed. A total of 0.5 mL of the sample was mixed with 2 mL of acetonitrile in a 10-mL centrifuge tube. The sample was vortexed vigorously and then centrifuged at ~1,200 rpm for 10 min. The upper organic layer was transferred to clean centrifuge tube and the sample was re-extracted twice more with acetonitrile using the condition previously described. The whole organic extract was then evaporated to dryness under a nitrogen stream at 40°C and the residue was reconstituted in 200 μL of acetonitrile. Following this, an aliquot of the reconstituted extracts was transferred and filtrated through a 0.22-μm cellulose membrane filter and then 10 μL was injected into the HPLC system. Pharmacokinetic parameters A non-compartmental model was used to determine the pharmacokinetic parameters of ATTM. Pharmacokinetic parameters, including maximum plasma concentration (Cmax), time (Tmax), area under the plasma concentration versus time curve from zero to last sampling time (AUC0−t) and infinity (AUC0−∞) and elimination half-life (t1/2) were calculated using PKSolver Software (16). Data are reported as mean ± SD. Statistical analysis was conducted using SPSS 10.0 (IBM SPSS, USA). Results Synthesis and structural identification of ATTM The ATTM was synthesized with an overall yield of 81%. The purity was >98% by purifying with silica gel column chromatography. IR (KBr): 3,419, 3,330, 2,931, 1,731, 1,616, 1,507, 1,456, 1,417, 1,373, 1,282, 1,190, 1,152, 1,117, 1,019, 980, 953, 9,38,916 cm−1. 1H NMR (400 MHz, DMSO) δ: 7.28 (s, 2 H), 6.08 (dd, J = 17.8, 11.2 Hz, 1 H), 5.52 (d, J = 8.1 Hz, 1 H), 5.03 (dd, J = 21.0, 14.7 Hz, 2 H), 4.51 (d, J = 5.9 Hz, 1 H), 4.02 (q, J = 7.1 Hz, 1 H), 3.90 (q, J = 16.0 Hz, 2 H), 3.45–3.32 (m, 2 H), 2.39 (s, 1 H), 2.19 (dd, J = 18.8, 10.8 Hz, 1 H), 2.11–1.97 (m, 4 H), 1.64 (dd, J = 18.4, 9.5 Hz, 2 H), 1.54–1.42 (m, 1 H), 1.36–1.21 (m, 6 H), 1.16 (d, J = 7.1 Hz, 1 H), 1.08–0.94 (m, 4 H), 0.82 (d, J = 6.7 Hz, 3 H), 0.59 (d, J = 6.7 Hz, 3 H). 13C NMR (100 MHz, DMSO) δ: 217.08, 169.73, 166.70, 148.89, 140.68, 115.39, 72.63, 70.27, 59.75, 57.23, 44.95, 44.14, 41.51, 36.33, 34.00, 30.10, 28.70, 26.60, 24.47, 20.75, 16.09, 14.51, 14.08, 11.54. HRMS (ESI): [M + H]+ calcd for C24H35N3O4S2, 494.2142; found, 494.2139. Sample preparation The protein precipitation and extraction from plasma and concentration of objective components should guarantee the preparation procedure with high recovery and avoiding their degradation. Three types of reagents (ethyl acetate, methanol and acetonitrile) for preparation were studied during the experiment. As shown in Figure 2, acetonitrile was the best agent in terms of perfect preparation and absence of interference. Figure 2. View largeDownload slide Effect of different kinds of extraction agents on the extraction efficiency. Figure 2. View largeDownload slide Effect of different kinds of extraction agents on the extraction efficiency. Chromatographic conditions A simple, accurate, fast and sensitive HPLC method was developed to routinely measure plasma levels and study pharmacokinetics of ATTM in our chicken model to correlate their pharmacological effects to plasma levels and pharmacokinetic behavior. This method is valid within a wide range of plasma concentrations (0.120–120.00 μg/mL) and may be proposed as a suitable method for pharmacokinetic studies. Good separation of ATTM peak from that of many interfering compounds with short run times was obtained using a mobile phase system of acetonitrile and water at a ratio of 50:50 v/v, at 1 mL/min flow rate (Figure 3). Figure 3. View largeDownload slide Chromatograms of ATTM in chicken plasma. (A) Blank chicken plasma. (B) Spiked with 12 μg/mL of ATTM (1). (C) Plasma sample collected from a chicken 30 min following a single i.v. administration of 30 mg/kg of ATTM (1). Figure 3. View largeDownload slide Chromatograms of ATTM in chicken plasma. (A) Blank chicken plasma. (B) Spiked with 12 μg/mL of ATTM (1). (C) Plasma sample collected from a chicken 30 min following a single i.v. administration of 30 mg/kg of ATTM (1). Assay validation Sensitivity and selectivity Plasma samples spiked with ATTM were assayed in decreasing concentrations. The LLOQ of ATTM in chicken plasma was 0.12 μg/mL. The specificity of this method was examined by analyzing six different blank chicken plasma samples and blank plasma spiked with ATTM. Typical chromatograms of blank plasma, blank plasma spiked with 12 μg/mL of ATTM, and chicken plasma collected at 30 min after i.v. administration of ATTM are shown in Figure 3. No interfering peak of endogenous substance that affects the determination of ATTM (~6.75 min) in chicken plasma was observed. Furthermore, the purity threshold and purity angle values of six batches of blank plasma spiked with ATTM are tabulated in Table I. It was observed that the purity angle values were always less than the purity threshold values indicating that the peak observed was pure. Table I. Purity Angel and Purity Threshold of Blank Plasma Spiked with ATTM No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 Table I. Purity Angel and Purity Threshold of Blank Plasma Spiked with ATTM No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 No. Purity angel Purity threshold 1 0.182 2.135 2 0.175 2.106 3 0.179 2.068 4 0.208 2.264 5 0.167 2.087 6 0.190 2.153 Mean 0.184 2.136 Linearity Calibration curves were constructed using five standards in plasma by plotting peak area versus the nominal concentration of ATTM. The established calibration curves were found to be linear over the entire concentration range of 0.12–120.00 μg/mL, and used to measure ATTM concentrations in pharmacokinetics analysis and validation assay. The coefficient of determination (R2) was 0.9994 in all calibration curves (n = 5). The mean regression equation for ATTM was y = 8401.41x + 400.75. Precision and accuracy The accuracy and precision data for the plasma samples are presented in Table II. The intra- and inter-day accuracy assessed by replicate analysis of QC samples in plasma on three consecutive days were 95.33–104.01% and 99.40–102.78%, respectively. The intra- and inter-day precision (% CV) ranged from 1.49% to 4.84% and 0.93% to 3.92%, respectively. Both intra- and inter-day accuracy and precision were within acceptable limits, which indicated that the established HPLC method was good at precision and accuracy. Table II. Intra- and Inter-Day Precision and Accuracy of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Table II. Intra- and Inter-Day Precision and Accuracy of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Nominal concentration (μg/mL) Intra-day Inter-day Accuracy (%) Precision (% CV) Accuracy (%) Precision (% CV) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0.12 100.72 95.40 95.33 1.99 4.69 2.67 99.40 2.91 2.00 104.01 100.46 98.85 4.68 3.47 4.82 102.78 3.92 20.00 102.29 102.79 99.80 4.84 3.30 4.31 101.06 2.81 100.00 100.28 99.70 100.21 2.97 1.49 1.77 100.20 0.93 Recovery The assay recovery and extraction recovery of ATTM were determined at 0.12, 1.20 and 12.00 μg/mL (n = 6). The mean assay recoveries were 92.50%, 98.25% and 98.76% for ATTM of 0.12, 1.20 and 12.00 μg/mL in plasma, respectively. The data of extraction recovery were ranged from 90.83% to 96.02% with an RSD < 2%, which indicated that the extraction procedure was consistent and reproducible (Table III). The high recovery suggested that there was negligible loss during the drug extraction. Table III. Recovery of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Table III. Recovery of ATTM in Chicken Plasma (n = 6) Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Nominal concentration (μg/mL) Observed concentration (μg/mL) Recovery (%) RSD (%) Assay recovery  0.12 0.111 92.50 2.36  1.20 1.179 98.25 0.54  12.00 11.851 98.76 0.91 Extraction recovery  0.12 0.109 90.83 1.64  1.20 1.115 92.92 1.81  12.00 11.522 96.02 1.49 Stability Stability tests of the ATTM were assessed using triplicates of spiked samples at three concentrations under different conditions. The results of the short-term (24 h) at room temperature, long-term (4 weeks) at −70°C and freeze–thaw are shown in Table IV. The ATTM did not degrade up to 24 h at room temperature and was found to be stable over a period of 4 weeks at the storage condition of −70°C. Stability of ATTM after three freeze–thaw cycles of plasma samples indicated it was stable when subjected to these conditions. Based on these results, there was no stability-related problem to influence the assay. Table IV. Stability of ATTM in Chicken Plasma (n = 6) Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Table IV. Stability of ATTM in Chicken Plasma (n = 6) Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Time and condition of storage Nominal concentration (μg/mL) Percent of nominal RSD (%) Short-term (25°C, 24 h) 0.12 93.54 3.58 1.20 95.58 4.62 12.00 92.74 5.28 Long-term (−70°C, 4 weeks) 0.12 96.75 4.23 1.20 93.56 2.95 12.00 94.64 3.54 Freeze–thaw cycles (n = 3) 0.12 95.86 2.67 1.20 92.58 3.45 12.00 91.35 2.68 Pharmacokinetics analysis The validated analytical method was successfully applied to investigate the pharmacokinetics of ATTM in broiler chicken plasma after a single i.v., i.m. or p.o. dose of 30 mg/kg, respectively. The main pharmacokinetic profiles are displayed in Table V and the mean plasma concentration versus time curve (as well as the log-plasma concentration versus time curve) of ATTM after i.v., i.m. and p.o. administration are shown in Figure 4. Table V. Pharmacokinetic Parameters of ATTM in Broiler Chickens after Administration Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 aMaximum concentration. bTime to reach Cmax. cHalf-life. dClearance. eVolume of distribution. fMean resident time. gMean resident time. gArea under the curve. hOral bioavailability. Table V. Pharmacokinetic Parameters of ATTM in Broiler Chickens after Administration Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 Parameters i.v. i.m. p.o. Cmaxa (μg/mL) 92.14 ± 4.23 29.03 ± 1.16 27.82 ± 1.81 Tmaxb (h) 0.08 ± 0.02 0.50 ± 0.16 4.00 ± 0.52 T1/2c (h) 3.93 ± 0.72 3.92 ± 0.61 6.08 ± 1.06 Cld (L/h·kg) 0.16 ± 0.05 Vze (L/kg) 0.90 ± 1.58 MRTf (d) 4.09 ± 0.72 5.20 ± 0.93 10.17 ± 1.50 AUC0→tg (μg·h/mL) 174.87 ± 33.47 138.26 ± 30.55 121.28 ± 38.50 Fh (%) 75.29 ± 8.52 72.03 ± 9.63 aMaximum concentration. bTime to reach Cmax. cHalf-life. dClearance. eVolume of distribution. fMean resident time. gMean resident time. gArea under the curve. hOral bioavailability. Figure 4. View largeDownload slide Plasma concentration–time curve (A) and log-concentration–time curve (B) after i.v., i.m. and p.o.administration of ATTM to broiler chickens. Figure 4. View largeDownload slide Plasma concentration–time curve (A) and log-concentration–time curve (B) after i.v., i.m. and p.o.administration of ATTM to broiler chickens. Discussion The elimination of plasma proteins or other interfering organic components is a prerequisite for a successful HPLC determination of analytes in the developing of an analytical method. In this study, a simple protein precipitation and liquid–liquid extraction procedure was optimized to suit the pharmacokinetic studies for ATTM. Protein precipitation and extraction with ethyl acetate and methanol gave poor recoveries, respectively. However, the preparation with acetonitrile was much better and showed no interfering chromatographic peaks due to plasma endogenous substances. A sensitive, specific, accurate and reproducible reversed phase HPLC method for the determination of ATTM in broiler chicken plasma was developed and validated in this study. This method was successfully applied to the study of pharmacokinetics of ATTM in broiler chickens for the first time and can be available for large number of biological samples very efficiently. The detection wavelength is very important for the analysis of biological samples. The scan of ATTM in acetonitrile using UV spectrophotometer revealed absorption maxima at 279 nm, and was therefore selected as the detection wavelength for HPLC studies. The establishment of chromatographic conditions started from selection of HPLC columns. ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) demonstrated symmetrical peak shapes and better separation for the analyte compared with the other chromatographic columns. It was found that base and acid in the mobile phase had a minimal effect on the retention time and peak shape of ATTM. Therefore, the various mixtures of pure water and acetonitrile were tested to enhance the peak resolution and to eliminate the peak tailing of the target compounds with an optimization procedure. After a single i.v. administration of ATTM (30 mg/kg) in broiler chickens, the mean value of t1/2 was 3.93 h which was close to that in SD rat with 3.40 h of t1/2 after a single i.v. administration (5 mg/kg) of ATTM (14) and reflected a high rate of elimination. Following p.o. administration, ATTM was absorbed rapidly, with Cmax values of 27.82 μg/mL at 4.00 h (Tmax), respectively. Then, the concentrations of the compound dropped with elimination half-life times (t1/2) of 6.08 h, respectively. Compared with SD rats after the same route at a dose of 5 mg/kg (Cmax, 3.07; Tmax, 0.75), ATTM was absorbed more slowly, but with a higher Cmax in chickens (14). The systemic bioavailability of ATTM was 72.03% following p.o. administration in chickens, which was higher than that in SD rats (F, 35.81). Following i.m. administration, ATTM was absorbed rapidly, with Cmax values of 29.03 μg/mL at 0.50 h (Tmax). Then, the concentrations of ATTM dropped with elimination half-life times (t1/2) of 3.92 h. The systemic bioavailability of ATTM was 75.29% following i.m. administrations in chickens. Conclusion An accurate and precise HPLC method for the determination of ATTM in chickens plasma has been developed and validated. Using this method, pharmacokinetic features of ATTM after i.v., i.m. and p.o. administration in chickens was investigated, which were characterized by high i.m. or p.o. bioavailability. The achieved pharmacokinetic results could be useful for further study on the active mechanism of ATTM. Acknowledgments This work was supported by the Basic Scientific Research Funds in Central Agricultural Scientific Research Institutions (No. 1610322016007) and the National Key Technology Support Program (No. 2015BAD11B02). 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Journal of Chromatographic ScienceOxford University Press

Published: Apr 13, 2018

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