Pharmacokinetics and Milk Residues of Cefquinome in Lactating Chinese Dairy Cows After

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1 Pharmacokinetics and Milk Residues of Cefquinome in Lactating Chinese Dairy Cows After Intramammary Administration 1 LI Ya-fei, WANG Lin, GU Xiao-yan, ZENG Zhen-ling, HE Lim-in, YANG Fan, YUAN Bo, SHU Jian-hua and DING Huan-zhong Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou , P.R.China Abstract The purpose of the study was to investigate the pharmacokinetics of cefquinome in plasma and milk samples of lactating Chinese Holstein following a single intramammary administration into one quarter at the dose of 75 mg. Milk residue depletion of cefquinome administrated at one quarter following three consecutive infusions at the same dose were also carried out. Cefquinome concentrations in plasma and milk were determined by high-performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) method. A non-compartmental analysis was used to obtain the pharmacokinetic parameters of cefquinome. Following single treatment, cefquinome wasn t detected in any of the plasma samples. The concentration of cefquinome in milk reached peaked peak values (C max ) of (599.00±322.00) µg ml -1 at 2 h after administration (T max ), elimination half-life (t 1/2λz ) was (4.63±0.26) h, area under the concentration-time curve (AUC 0- ) was ( ± ) µg ml -1 h, and mean residence time (MRT) was (6.03±2.27) h. In residue depletion study, cefquinome concentrations in 5 out of 6 milk samples at 72 h were lower than maximum residue limit fixed by the European Regulatory agency (20 μg kg -1 for cefquinome) and cefquinome still could be detected in milk of treated quarters at 120 h post treatment. The maximum LI Ya-fei, Mobile: , yafei.lee@163.com; DING Huan-zhong, Tel: , Mobile: , hzding@scau.edu.cn

2 concentration (C max ) of cefquinome in milk from treated quarters was (486.50±262.92) µg ml -1 and arrived at 6 h after administration (T max ), elimination half-life (t 1/2λz ) was (6.30±0.76) h, area under the concentration-time curve (AUC 0- ) was ( ± ) µg ml -1 h, and mean residence time (MRT) was (10.09±1.40) h. This study showed that cefquinome has the feature of poor penetration into blood and was eliminated quickly from milk in lactating cows after intramammary administration. Key words: cefquinome, pharmacokinetics, residue, dairy cows INTRODUCTION It is known that cephalosporins play a prominent role in preventing and treating cow mastitis (Bradley and Green 2009). In view of cow milk as feeding source in many people s life, it is important to investigate the behavior of cephalosporins in milk of dairy cattle. In addition, from a food safety perspective, inappropriate usage of antibiotics may result in the drug residues in milk and lead to undesirable side effects such as resistance of pathogenic bacteria and finally threaten to people s health (Samanidou and Nisyriou 2008). Cefquinome (C 23 H 24 N 6 O 5 S 2, CEQ), an aminothiazolyl cephalosporin, is a fourth generation cephalosporin antibiotic, which has shown marked in vitro activity against both Gram-positive and Gram-negative bacteria such as Staphylococcus sp., Streptococcus sp., Pseudomonas sp., Escherichia coli, Salmonella sp., and Klebsiella pneumonia and is stable to the majority of chromosomally and plasmid-encoded β-lactamases (Chin et al. 1992; Guerin-Faublee et al. 2003; Limbert et al. 1991; Thomas et al. 2006). It was especially developed for veterinary use and has been approved to treat many diseases in cattle, pigs, sows and cows in European countries due to its

3 potent antimicrobial properties (Shpigel et al. 1997a; Thomas et al. 2004; Widmer et al. 2009). European Medicine Agency (EMA) for Veterinary Medicine has established the maximum residue limit (MRL) for CEQ in bovine milk as 20 μg kg -1 (CVMP 2003). Pharmacokinetic (PK) profiles on CEQ have been presented in several species (Al-Taher 2010; Hwang et al. 2011; Li et al. 2008; Liu et al. 2012; Tohamy 2011; Xie et al. 2013; Yuan et al. 2011); however, up to now, only one research has involved the behavior of CEQ in cows after intramammary (IMM) administration (Zonca et al. 2011a). Moreover, there seems to be no published paper about residue depletion of CEQ in dairy cows after IMM administration other than the EMA summary report about CEQ and MRL in milk (CVMP 1995). From the standpoint of ensuring public health, the problem arising from antibiotic residue in milk is still crucial. Chinese Holstein is different from American and European ones on some characteristics such as body size and milk production (Wen et al. 2010; Zhai et al. 2007). Due to the difference in physic-chemical properties of pharmaceutical preparations and animal species, the rate and extent of drug absorption and disposition may be altered. Therefore, it seems necessary to evaluate the pharmacokinetics and residue depletion of cefquinome in milk for a commercial formulation of cefquinome developed in China. An obvious characteristic of cow udder is that it is divided into front, rear, left, and right quarter, which has its own independent secretion systems and is mutual interlinked. Each nipple has an open channel for milk out. Considering the structure of the cow udder, the objectives of the present study, therefore, were to investigate the plasma and milk pharmacokinetics profiles CEQ following a single IMM administration into one quarter, and to understand disposition pattern of CEQ in milk at therapeutic dose to a treated quarter in lactating dairy according to Chinese guidelines.

4 RESULTS Method Validation The developed analytical method proved to be linear and reproducible for the detection of CEQ in milk samples (ranged from to 0.5 µg ml -1 ) and plasma samples (ranged from to 0.2 µg ml -1 ). The LOD and LOQ of the assay were and µg ml -1 for milk and and µg ml -1 for plasma, respectively, based on a signal to noise ratio 3 and 10. The LOQs were far below the MRL of 20 μg kg -1. In terms of the precision and accuracy of the assay, five replicates at three different concentrations (0.005, 0.05, 0.5 µg ml -1 ) were tested to evaluate coefficients of variation and recoveries. The recoveries of CEQ in milk ranged from % to 87.02%, with the intraday and interday variation coefficient less than 9% and 15%, respectively. In plasma, recoveries of CEQ varied from 89.41% to 98.25%, and the intraday and interday variation coefficients were less than 10% and 15% in all cases, respectively. These results indicated that this HPLC-MS/MS method was sensitive enough to provide the necessary detection of CEQ in milk and plasma samples. Pharmacokinetics No local and systemic adverse effects were observed in any cow after IMM administration. The semi-logarithmic graph of milk concentrations of CEQ from treated and untreated quarters following a single IMM administration at the dose of 75 mg per quarter was shown in Fig. 1. After IMM administration, CEQ attained to a maximum level of (598.00±323.00) µg ml -1 at 2 h after infusion and then declined in a mono-exponential manner to a level (0.01±0.005) µg ml -1 at 72 h post administration in treated quarters. At 72 h after single treatment, the concentration of CEQ in

5 milk from treated quarters reduced to below the MRL. Based on the milk concentration level of CEQ in treated quarters, main pharmacokinetic parameters of CEQ in milk were summarized in Table1. It was noted that the drug was eliminated quickly in milk samples from treated quarters (t 1/2λz 4.63±0.26 h). The area under the milk concentration time curve (AUC 0- ) was ( ± ) µg ml -1 h, and the mean residue time was (3.79±0.92) h. Concentrations of CEQ in milk from untreated quarters varied from µg ml -1 to µg ml -1, and CEQ could not be detected at 24 h post-treatment. The peak concentration of CEQ (0.08 µg ml -1 ) in untreated quarters was attained at 6 h after single administration and found only in one milk sample. Concentrations of CEQ in milk samples of 8 and 12 h from untreated quarters were all below the MRL (20 μg kg -1 ). It can be presumed that only a few of CEQ go into the untreated quarters because of diffusion or distribution between quarters. As a result, the milk drug concentration in untreated quarter is very low. Due to huge difference of CEQ concentrations from untreated quarters, pharmacokinetics parameters of milk samples collected from untreated quarters were not calculated. Besides, CEQ wasn t detected in plasma samples. Milk residue study After three consecutive infusions, CEQ could be detected in milk until 120 h from treated quarters (0.006 µg ml -1 ) and 48 h from untreated quarters (0.008±0.003 µg ml -1 ). The semi-logarithmic graphs of milk concentrations verse time from treated and untreated quarters were shown in Fig. 2. The observed concentrations of CEQ in milk samples after repeated treatments were slightly higher compared to those after single treatment collected at the same time point. For example, the concentrations of CEQ measured in milk from treated quarters in single infusion and

6 113 three treatments were (266.00±80.00) µg ml -1, (486.00±263.00) µg ml -1 at 6 h post-administration, respectively. In the depletion study, the mean milk concentration of CEQ after dosing decreased progressively from peak value of ( ±65.73) µg ml -1 at 6 h to (7.70±3.79) µg ml -1 at 24 h (Fig. 2), and then declined to (1.25±0.37) µg ml -1 at 36 h. CEQ was still detectable at 120 h (0.002 µg ml -1 ), which was far below the MRL. Main pharmacokinetic parameters of CEQ in milk from treated quarters were shown in Table 2. The area under the milk concentration time curve (AUC 0- ) was ( ± ) µg ml -1 h, mean residue time was (10.09±1.40) h and the elimination half life (t 1/2λz ) was (6.30±0.76) h. CEQ could be detected in milk samples from untreated quarters of four cows at 6 h after treatment, but could not be detected in the other two cows. DISCUSSION Several previous reports have described the detection methods of CEQ in animal tissues, including high performance liquid chromatography (HPLC) (Uney et al. 2011), HPLC-MS/MS (Becker et al. 2004; Maes et al. 2007), screening methods and enzyme immunoassay (Suhren and Knappstein 2003; Thal et al. 2011). In this experiment, a high performance liquid chromatography-tandem mass spectrometry method with high sensitivity and excellent reproducibility was developed. The LOQ is µg ml -1 and this method can be applied in determining the residue concentration of CEQ in milk samples. Even though fourth generation cephalosporins have exhibited excellent antimicrobial activity, at present, they were not top-priority antimicrobial agents in treatment of animal infection. In view of the limitation of bacterial resistance, the fourth generation cephalosporins are considered to be in prudent use (Prescott 2006; FDA 2008). The use of CEQ should be reserved for infections where

7 susceptibility tests indicate that alternatives are not available. Until now, information on the pharmacokinetic characteristics of CEQ after IMM administration is still limited in China. Compared to the findings of previous study covered by Zonca et al. (2011), shorter t 1/2λz (4.63±0.26 h) and MRT (3.79±0.92 h) were observed in our study for cows with single intramammary infusion; however, when the cows were treated for three consecutive times, the t 1/2λz (6.30±0.76 h) was more or less equal to the report by Zonca et al. and the MRT (10.09±1.40 h) was much longer. CEQ was not detected in plasma samples in this study. The main reason for this may be that CEQ was administrated to only one quarter and infused once. Although report from CVMP in 1995 confirmed the systemic absorption of cefquinome; however, in the study of Cefquinome sulfate behavior reported by Zonca et al. in 2011, the C max of cefquinome in plasma serum was as low as 0.09 µg ml -1 when all quarters were treated for three consecutive times. The dissociation constant (pka) and lipid solubility of different chemotherapeutic agents after IMM administration can also influence the rate of their absorption (Bajwa et al. 2007a). Other factors such as the constituents of formulation, experimental animal species and so on perhaps also affect the pharmacokinetic process. In addition, milk fraction, to a certain degree, may have an effect on the process of cephalosporins in milk (Stockler et al. 2009). CEQ acts as time-dependent antimicrobial. One of the most important pharmacokinetics/pharmacodynamics parameters for this type drug is the time which is above MIC 90. MIC 90 of CEQ against various common species of bovine mastitis bacteria including E.coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and other pathogens lies in the range of >0.008-<4.0 μg ml -1 ( Ehinger et al. 2006; Schmid and Thomas 2002; Shpigel et al. 1997b; Tenhagen et al. 2006). According to the present findings, the time that an effective

8 therapeutic concentration of CEQ in milk is well above the MIC for the majority of pathogens causing mastitis could be maintained up to 48 h after single intramammary administration. Recent clinical trials have also shown beneficial efficacy of CEQ therapy in bovine mastitis (Bradley et al. 2011; Kasravi et al. 2011). The concentrations of drug in milk samples from untreated quarters were irregular, which was also observed in other study (Bajwa et al. 2007b). The structure of the mammary gland may affect the transport of drugs in milk (Gehring and Smith, 2006). Consequently, a big difference was found in milk concentration of CEQ between treated and untreated quarters. To the best of our knowledge, until in a recent study, behaviors of CEQ after IMM infusion to lactating dairy cows at the same dosage of this experiment have been reported (Zonca et al. 2011b). However, the drug maintenance time in milk is longer in our experimental work because of the lower LOQ. Furthermore, this study was of great significance in guiding the usage of CEQ in Chinese dairy cows because it was in consistent with the environments of Chinese field breeding and Chinese guidances CONCLUSION In this paper, a sensitive and convenient HPLC-MS/MS method was developed for the determination of CEQ in milk and plasma. The pharmacokinetic profile and residue depletion of CEQ in milk obtained in this study provide plenty of useful information on the intramammary administration of CEQ in clinical practice MATERIALS AND METHODS

9 Chemicals and reagents Cefquinome sulfate reference standard (cefquinome purity 80.1%) was purchased from the China Institute of Veterinary Drug Control (Beijing, China). CEQ sulfate intramammary infusion (75 mg CEQ of one syringe) was a commercial preparation provided by Eastern Along Pharmaceutical Co., Ltd (Guangzhou, Guangdong, China). This commercial formulation was given as an intramammary administration at the dose of 75 mg per udder for three consecutive times, as indicated by the manufacturer. HPLC-grade methanol (MeOH) and acetonitrile (ACN) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Distilled-deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). HPLC-grade formic acid (FA) was purchased from CNW Technologies GmbH (Düsseldorf Germany). All other reagents used in this study were of analytical grade and purchased in China. Animals This study was undertaken in a commercial dairy farm located in Guangzhou city (South of China) and lasted from late July to September in During this period, the climate was hot and humid. The mean milk yield was approximately 7200 kg/cow per year. All the cows were machine-milked twice daily at about 6 a.m. and 6 p.m. Twelve clinically healthy lactating cows based on the medical history, physical examination, food intake, and milk productions for each cow were involved in the study. The selected cows weighed (600±50) kg with the age of 3-5 years and yielded (20±2) kg milk per day. Most of the experimental cows were in their first or second lactation individually in a stall. They were fed with dry forage grass twice a day without any antibiotic and had free access to fresh water. During the experiment period, all of the cows were checked by a professional veterinarian daily for general health, behavior, and appearance. This

10 study was carried out in accordance with the approved Institutional Animal Care and Use Committee protocols in South China Agricultural University. Experimental design In single-dose treatment, 75 mg CEQ were administrated intramammarily into the left front quarters of six healthy Chinese Holstein cows after clear wash. Following administration with CEQ, after discarding the first two streams of milk, about 50 ml milk samples were collected by hand from treated quarters according to the following schedule: before and at 2, 4, 6, 8, 10, 12, 24, 36, 48, 60 and 72 h after administration. Milk samples were also collected before and at 2, 4, 6, 8, 12 and 24 h post-treatment from right front quarter. Moreover, plasma samples (about 10 ml) were drawn from jugular vein before and at 6, 12, and 24 h after drug administration. Blood samples were then centrifuged and plasma was frozen and stored at -20ºC until analysis. The other six Chinese Holstein cows were used to study the milk residue behavior of CEQ sulfate after IMM administration. 75 mg CEQ were infused into the left rear quarters of six cows for three consecutive times at a 12 h interval. After last drug treatment, milk samples of individual cow were collected from the treated quarters at 0 (before administration) and 6, 12 h and then every 12 h until 120 h and from the untreated quarters (right rear quarters) before administration and at 6, 12, 24, 36 and 48 h after administration. All the samples were stored at - 20ºC for further assay. Milk and plasma extraction procedure Briefly, milk sample (1 ml) was transferred into a 15 ml polypropylene centrifuge tube and 10% trichloroacetic acid (3 ml) was added. The mixture was briefly vortexed for 1 min and shaken for 10 min, centrifuged at about 9000 g for 10 min under 4ºC. Supernatant was transferred to a glass tube and the residue was then re-extracted once. The resulting supernatant was loaded onto a

11 solid phase extraction (SPE) cartridge (Strata-X, 60 mg/3 ml), which had previously been activated with 3 ml of methanol and 3 ml of deionized water at a flow rate of approximately 1 ml/min. Sample solutions flowed through the SPE cartridge under gravity. SPE cartridge was then rinsed with 3mL water and dried under vacuum for 15 min. The retained drug was eluted from the cartridge with 1 ml 30% acetonitrile. Finally, the eluate was vortexed following by centrifugation at g for 10 min. The upper layer was subsequently collected into a small volume sample vial after filtering using a 0.22 μm nylon syringe filter and then analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Plasma sample (0.5 ml) was transferred into a 2 ml poly-propylene centrifuge tube together with acetonitrile (0.5 ml). The mixture was vortexed for 1 min followed by centrifugation at g under 4ºC for 10 min. The clear supernatant was collected into another 2 ml poly-propylene centrifuge tube and mixed with deionized water (0.8 ml) and filtered through a 0.22 μm syringe filter before LC-MS/MS analysis. LC-ESI-MS/MS conditions HPLC-MS/MS analyses were operated on a system consisting of an Agilent 1200 series HPLC system coupled to an API 4000 triple quadrupole mass spectrometer with electrospray ionization interface. HPLC separation was achieved using a Luna C 18 column ( mm, 5 μm). The mobile phases comprised solution A (water with 0.1% formic acid, v/v) and B (acetonitrile) at a flow rate of 0.25 ml min -1. Gradient conditions were as follows: 0-1 min, 5% B; 1-3 min, 5% B to 60% B; 3-6 min, 60% B; min, 60.00% B to 5% B; and min, 5% B. Injection volume of 5 μl was used for the HPLC-MS/MS analysis. The MS/MS system was performed using an electrospray source in positive ionization mode

12 (ESI+). Operational ESI parameters were manually optimized as follows: source temperature at 600ºC; ion spray voltage, 4.5 kv; curtain gas, 20 psi; ion source gas 1(GS1)and gas 2 (GS2) at 50 and 55 psi, respectively. Quantification was conducted using multiple reaction monitoring (MRM) mode for the transitions m/z 529.3>134.1 for CEQ with dwell time of 0.1s for transition. Validation procedure Stock solution at a concentration of 1000 µg ml -1 was prepared by dissolving CEQ sulfate in water, stored at -20ºC and shaded from light. Working solutions for milk (ranging from to 0.5 µg ml -1 ) and blood (ranging from to 0.2 µg ml -1 ) were obtained by further diluting the stock solution with water. All the working standard solutions were freshly prepared, stored at 4 ºC in the darkness. Calibration curve for measuring CEQ were prepared by matrix-matched standard calibration method. Blank matrix was obtained from blank plasma or milk by the procedures described above, then the serial working solutions were diluted with blank matrix to get calibration curve. Samples were diluted if their concentration exceeded the linear ranges. Parameters including recovery, repeatability, specificity, limit of detection (LOD), and limit of quantification (LOQ) in milk and plasma were determined. The intraday and interday coefficients of variation (CV %) were also calculated. Pharmacokinetic analysis Concentration-time data were analyzed using a WinNonlin software (version 6.1; Pharsight Corporation, USA) to obtain the pharmacokinetic parameters of CEQ in milk and plasma. A non-compartmental method was applied to calculated main parameters in this study. The linear trapezoidal rule was used to calculate the area under concentration-time curves (AUC) and area under the first moment curve (AUMC). The mean residence time (MRT) was determined as

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15 Shpigel N Y, Levin D, Winkler M, Saran A, Ziv G, Böttner A. 1997b. Efficacy of cefquinome for treatment of cows with mastitis experimentally induced using Escherichia coli. Journal of dairy science, 80, Stockler R, Morin D, Lantz R, Hurley W, Constable P Effect of milk fraction on concentrations of cephapirin and desacetylcephapirin in bovine milk after intramammary infusion of cephapirin sodium. Journal of veterinary pharmacology and therapeutics, 32, Suhren G, Knappstein K Detection of cefquinome in milk by liquid chromatography and screening methods. Analytica Chimica Acta,483, Tenhagen B A, Köster G, Wallmann J, Heuwieser W Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany. Journal of dairy science,89, Thal J, Steffen M, Meier B, Schneider E, Adriany A, Usleber E Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment. Analytical and Bioanalytical Chemistry,399, Thomas E, Roy O, Skowronski V, Zschiesche E, Martin G, Bottner A Comparative field efficacy study between cefquinome and gentamicin in neonatal calves with clinical signs of septicaemia. Revue de médecine vétérinaire,155, Thomas E, Thomas V, Wilhelm C Antibacterial activity of cefquinome against equine bacterial pathogens. Veterinary Microbiology, 115, Tohamy M A Age-related intramuscular pharmacokinetics of cefquinome in sheep. Small Ruminant Research, 99, Uney K, Altan F, Elmas M Development and Validation of a High-Performance Liquid Chromatography Method for Determination of Cefquinome Concentrations in Sheep Plasma and Its Application to Pharmacokinetic Studies. Antimicrobial Agents and Chemotherapy,55, Wen H, Pan B, Wang Y, Wang F, Yang Z, Wang M Plasma and milk kinetics of eprinomectin following topical or oral administration to lactating Chinese Holstein cows. Veterinary parasitology,174, Widmer A, Kummer M, Eser M W, Fürst A Comparison of the clinical efficacy of cefquinome with the combination of penicillin G and gentamicin in equine patients. Equine Veterinary Education,21, Xie W, Zhang X, Wang T, Du S Pharmacokinetic analysis of cefquinome in healthy chickens. British Poultry Science, 54,

16 Concentration(μg/mL) Journal of Integrative Agriculture Yuan L, Sun J, Wang R, Sun L, Zhu L, Luo X, Fang B, Liu Y Pharmacokinetics and bioavailability of cefquinome in healthy ducks. American Journal of Veterinary Research, 72, Zhai S, Liu J, Wu Y, Ye J Predicting urinary nitrogen excretion by milk urea nitrogen in lactating Chinese Holstein cows. Animal Science Journal,78, Zonca A, Gallo M, Locatelli C, Carli S, Moroni P, Villa R, Cagnardi P. 2011a. Cefquinome sulfate behavior after intramammary administration in healthy and infected cows. Journal of Dairy Science,94, Zonca A, Gallo M, Locatelli C, Carli S, Moroni P, Villa R, Cagnardi P. 2011b. Cefquinome sulfate behavior after intramammary administration in healthy and infected cows. Journal of Dairy Science,94, treated quarters untreated quarters Time(h) Fig. 1 Mean milk concentration (± SD) in the treated and untreated quarters during intramammary administration of cefquinome at a dose of 75 mg to one quarter (n=6).

17 Concentration(μg/mL) treated quarter untreated quar Fig. 2 Mean milk concentrations (±SD ) in the treated and untreated quarters during intramammary administration of cefquinome at a dose of 75 mg to one quarter for three consecutive times at a 12 h interval (n=6) Time (h) Table 1 Pharmacokinetic parameters (mean±sd) of cefquinome in milk following single intramammary infusion of 75 mg cefquinome in each treated quarter of six cows Parameter unit Cow number x ±S.D. Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ λz /h ±0.009 t 1/2λz h ±0.26 T max h ±0.82 C max µg /ml ± MRT h ±0.92 AUC 0- µg h/ml ± λz=the terminal rate constant; t 1/2λz = elimination half-life; T max = time to C max ; C max = maximum drug concentration;

18 MRT=mean residence time; AUC=the area under the milk concentration time curve from 0 to. Table 2 Pharmacokinetic parameters (mean±sd) of cefquinome in milk after three consecutive intramammary infusions of 75 mg cefquinome in each treated quarter of six cows Parameter unit Cow number x ±S.D. Ⅶ Ⅷ Ⅸ Ⅹ Ⅺ Ⅻ λz /h ±0.01 t 1/2λz h ±0.76 T max h ±0.00 C max µg /ml ± MRT h ±1.40 AUC 0- µg h/m ± L λz=the terminal rate constant; t 1/2λz = elimination half-life; T max = time to C max ; C max = maximum drug concentration; 382 MRT=mean residence time; AUC=the area under the milk concentration time curve from 0 to.