猪肌注盐酸多西环素的药物动力学和残留的研究
摘要
本研究建立了高效液相色谱(HPLC)法测定盐酸多西环素在猪血浆和组织中的方法，探讨了盐酸多西环素以2.5 mg／kg体重肌内注射后在猪(n=6)体内的药物动力学和残留消除规律。色谱条件是：色谱柱为：Agilent Zorbax TC-C18；流动相：乙腈：甲醇：0.01 mol／L草酸溶液(20：10：70)；流速：0.8 ml／min；检测波长：350 nm。样品处理：称取匀质样品，加Na_2EDTA-Mcllvaine缓冲溶液提取，然后用5％高氯酸溶液去除蛋白，上清液过SPE HLB oasis离子交换柱，取60μl进样分析。盐酸多西环素注射液在猪血浆、肌肉、肝脏、肾脏、皮和脂肪等组织中的线性范围均为0.05～12.8μg／g，线性方程和相关系数分别为Y=0.0078X-0.0682(R=0.9999)(血浆)；Y=0.0042X-0.0291(R=0.9998)(肌肉)；Y=0.0064X-0.0250(R=0.9997)(肝脏)；Y=0.0060X-0.0276(R=0.9997)(肾脏)；Y=0.0028X-0.0252(R=0.9999)(皮和脂肪)。测得该方法样品回收率大于69.4％，各样品日内变异系数小于5.90％，日间变异系数小于9.60％，血浆的最低检测限(0.025μg／ml)和最低定量限(0.05μg／ml)，组织的最低检测限(0.0125μg／g)和最低定量限(0.025μg／g)，均满足残留检测的要求。
结果表明，药物动力学模型符合有吸收一室模型，其药物动力学方程：C=3.372×(e~(-0.073t)-e~(-1.733t))。药物动力学参数：吸收半衰期(t_(1／2ka))、消除半衰期(t_(1／2 ke))分别为0.40±0.31 h、9.53±0.95 h，药时曲线下面积(AUC)为44.41±4.12μg·h／ml，最大血药浓度(C_(max))为2.81±0.13μg／ml，达峰时间(T_(max))为1.91±0.21 h。另外，以相同剂量肌内注射给猪(n=6)，每天一次，连续给药4 d后，在不同时间测定盐酸多西环素在猪的肌肉、肝脏、肾脏、皮和脂肪中的残留量。在给药16 d后，盐酸多西环素在各组织均能检测到且残留量均低于残留限量。盐酸多西环素注射液在猪体内消除缓慢，残留期较长，建议休药期不低于16 d。
High performance liquid chromatorgraphic method (HPLC) to detect Doxycyclinehydrochloride in pigs plasma and tissues were developed, pharmacokinetics and tissueresidues of Doxycycline hydrochloride were investigated, following intramuscularinjection at dose of 2.5 mg/kg body weight (b.w) in healthy pigs(n=6).chromatographyseparation was achieved on a Agilent Zorbax TC-C18 column using a mixature ofacetonitrile, methanol and 0.01M oxalic acid(v/v 20: 10: 70) as the mobile phase,thedetection wavelength is 350 nm and mobile speed is 0.8 ml/min. The Doxycyclinehydrochloride was extracted by by McIlvaine buffer from plasma and tissues. Thesupernatant was added 5% perchlorate to remove protein,and 60μl samples were injectedin high performance liquid chromatography to analyses after clean-up procedure bySPE HLB Oasis cartridge. Their linearity are all good over the concentration range(0.05～12.8μg/g) in plasma and tissues,The calibration of doxycycline hydrochloride andCorrelation coefficient(R) in plasma, muscle, liver, kidney, skin and fat were Y=0.0078X-0.0682(R=0.9999), Y=0.0042X-0.0291(R=0.9998), Y=0.0064X-0.0250(R=0.9997),Y=0.0060X-0.0276(R=0.9997), Y=0.0028X-0.0252(R=0.9999), respectively. Themean recoveries for all samples were all exceed 69.4%,Intra-day CV and inter-day CV areunder 5.90% and 9.60% respectively. The limits of detection (LOD) were 0.025μg/ml,0.0125μ/g in plasam, in tissues respectively. The limit of quatition (LOQ) were 0.05μg/ml, 0.025μg/g in plasam,in tissue respectively. Both can satisfy the need of theresidual detection.
The kinetics of Doxycycline Hydrochloride had been fit to one compartment modelwith first order absorption after intramuscular injection at dose of 2.5 mg/kg body weight(b.w) in pigs. The main parameters were as follows: the half-lives of absorption (t_(1/2 ka))and elimination (t_(1/2 ke)) were0.400±0.312 h, and 9.530±0.956 h respectively, area underthe plasma drug concentration-time curve from 0 to∞(AUC) was 44.414±4.123μg.h/ml, the peak plasma concentration (C_(max)) was 2.811±0.136μg/ml, peaking at 1.910±0.213 h (T_(pn))after dosing. Moreover, each healthy pig was administered DoxycyclineHydrochloride i.m. once a day at dose of 2.5 mg/kg body weight (b.w) for four days (n=6).The tissue residues of Doxycycline Hydrochloride were determined in muscle, liver,kidney, skin and fat. Tissue residues of Doxycycline Hydrochloride was to be lower to themaximum residue limit (MRL) after 16 days. The results indicated that the elimination ofDoxycycline Hydrochloride in tissue was slow. It is proposed that withdrawal periodshould be not less than 16 days after an intramuscular injection at a dose of 2.5 mg/kgb.w.
结果表明，药物动力学模型符合有吸收一室模型，其药物动力学方程：C=3.372×(e~(-0.073t)-e~(-1.733t))。药物动力学参数：吸收半衰期(t_(1／2ka))、消除半衰期(t_(1／2 ke))分别为0.40±0.31 h、9.53±0.95 h，药时曲线下面积(AUC)为44.41±4.12μg·h／ml，最大血药浓度(C_(max))为2.81±0.13μg／ml，达峰时间(T_(max))为1.91±0.21 h。另外，以相同剂量肌内注射给猪(n=6)，每天一次，连续给药4 d后，在不同时间测定盐酸多西环素在猪的肌肉、肝脏、肾脏、皮和脂肪中的残留量。在给药16 d后，盐酸多西环素在各组织均能检测到且残留量均低于残留限量。盐酸多西环素注射液在猪体内消除缓慢，残留期较长，建议休药期不低于16 d。
High performance liquid chromatorgraphic method (HPLC) to detect Doxycyclinehydrochloride in pigs plasma and tissues were developed, pharmacokinetics and tissueresidues of Doxycycline hydrochloride were investigated, following intramuscularinjection at dose of 2.5 mg/kg body weight (b.w) in healthy pigs(n=6).chromatographyseparation was achieved on a Agilent Zorbax TC-C18 column using a mixature ofacetonitrile, methanol and 0.01M oxalic acid(v/v 20: 10: 70) as the mobile phase,thedetection wavelength is 350 nm and mobile speed is 0.8 ml/min. The Doxycyclinehydrochloride was extracted by by McIlvaine buffer from plasma and tissues. Thesupernatant was added 5% perchlorate to remove protein,and 60μl samples were injectedin high performance liquid chromatography to analyses after clean-up procedure bySPE HLB Oasis cartridge. Their linearity are all good over the concentration range(0.05～12.8μg/g) in plasma and tissues,The calibration of doxycycline hydrochloride andCorrelation coefficient(R) in plasma, muscle, liver, kidney, skin and fat were Y=0.0078X-0.0682(R=0.9999), Y=0.0042X-0.0291(R=0.9998), Y=0.0064X-0.0250(R=0.9997),Y=0.0060X-0.0276(R=0.9997), Y=0.0028X-0.0252(R=0.9999), respectively. Themean recoveries for all samples were all exceed 69.4%,Intra-day CV and inter-day CV areunder 5.90% and 9.60% respectively. The limits of detection (LOD) were 0.025μg/ml,0.0125μ/g in plasam, in tissues respectively. The limit of quatition (LOQ) were 0.05μg/ml, 0.025μg/g in plasam,in tissue respectively. Both can satisfy the need of theresidual detection.
The kinetics of Doxycycline Hydrochloride had been fit to one compartment modelwith first order absorption after intramuscular injection at dose of 2.5 mg/kg body weight(b.w) in pigs. The main parameters were as follows: the half-lives of absorption (t_(1/2 ka))and elimination (t_(1/2 ke)) were0.400±0.312 h, and 9.530±0.956 h respectively, area underthe plasma drug concentration-time curve from 0 to∞(AUC) was 44.414±4.123μg.h/ml, the peak plasma concentration (C_(max)) was 2.811±0.136μg/ml, peaking at 1.910±0.213 h (T_(pn))after dosing. Moreover, each healthy pig was administered DoxycyclineHydrochloride i.m. once a day at dose of 2.5 mg/kg body weight (b.w) for four days (n=6).The tissue residues of Doxycycline Hydrochloride were determined in muscle, liver,kidney, skin and fat. Tissue residues of Doxycycline Hydrochloride was to be lower to themaximum residue limit (MRL) after 16 days. The results indicated that the elimination ofDoxycycline Hydrochloride in tissue was slow. It is proposed that withdrawal periodshould be not less than 16 days after an intramuscular injection at a dose of 2.5 mg/kgb.w.
引文
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69. Concordet D, Toutain P L.The withdrawal time estimation of veterinary drugs:a non-parametric approach,J Vet Pharmacology Therap 1997b,20:374-379
70. George Rigos, Maria A, Argyro A. Pharmacokinetics and tissue distribution of oxytetracycline in sea bass, Dicentrarchus labrax,at two water temperatures. Aquaculture, 2002, 210: 59-67
71. Goodman L S, Gilman AG (eds). The pharmacological basis of therapeutics, 9th edn. McGraw-Hill, New York, 1996
72. Ibrahim A. Alsarra I A, Niazy E M, Al-Sayed Y M, Al-Khamis K I, Ibrahim M E -A. High performance liquid chromatographic method for the determination.of doxycycline in human plasma and its application in pharmacokinetic studys. Saudi Pharmaceutical Journal, 2005, 13(1): 42-47
73. Jha V K, Jayachandran C, Singh M K, Singh S D. Pharmacokinetic data on doxycycline and its distribution in different biological fluids in female goats. Veterinary Research Commumications, 1989, 13: 11-16.
74. Kazuaki U , Takahiko A , Walai K. Pharmacokinetics of oxytetracycline in black tiger shrimp, Penaeus monodon, and the effect of cooking on the residues. Aquaculture, 2006, 11: 24-31.
75. Kuhne M, Wegmann S, Kobe A, Fries R, M. Tetracycline residues in bones of slaughtered animals. Food Control, 2000, 11: 175-180.
76. Michel G. Pharmacokinetics and tissue localization of doxycycline polyphosphate and doxycycline hydrochloride in the rat. Eur J Drug Metab Pharmacokinet, 1984, 9(2): 149-153
77. Martinez M, Friedlander P, Condon R, Meneses J, Orangers J, Weber N, Miller M.Response to criticisms of the US FDA parametric approach for withdrawal time estimation:rebuttal and comparison to the nonparametric method proposed by Concordet and Toutain. J Vet Pharmacol Therap, 2000, 23: 21-35
78. Ole-Mapenay I M, Mitema E S, Maitho T E. Aspects of the pharmacokinetics of doxycycline given to healthy and pneumonic East African dwarf goats by intramuscular injection. Veterinary Research Communications, 1997, 21(6): 453 -462.
79. Pascale D, Gordon J, Lamster I, Mann P, Seiger M, Arndt W. Concentration of doxycycline in human gingival fluid. J Clin Periodontol, 1986, 13(9): 841-844
80. pijper A, Van K B, Schoevers E J,Verheijden J H M, Van M. In vitro activity of five tetracyclines and some other antimicrobial agents against four porcine respiratory tract pathogens. Journal of veterinary pharmacology and therapeutics, 1989, 12: 267 -276.
81. Riond.J.L & Riviere,J.E.Pharmacokintics and metabolic inertness of doxycycline in young pigs. American Journal of Veterinary Research , 1998, 51: 1271-1275.
82. Ruth R, Dina F, Arie L. Bioequivalence study of two formulations of doxycycline.Current therapeutic research, 1998, 59(5): 315-324.
83. Sader H S, Pfaller M A, Tenover F C. Evalution and characterization of multiresistant Enterococcus faecium from 12 U. S.medical centers. J Clin Microbiol, 1994, 32(11): 2840-2842
84. Sanders P, Cicquel.M, Hurtaud.D, Chapel A M,Gaudiche.C & Bousquet.E.Absolute bioavailabilitu of doxycycline after oral aministration in medicated feed to pigs. Proceedings of the 14th IPVS Congress. 1996, 663
85. Siska M C , Kristina E I, Vanoosthuyze C H, Van P. Use of metal chelate affinity chromatography and membranebased ion-exchange as clan-up procedure for trace residue analysis of tetracyclines in animal tissues and egg. Journal of chromatography, 1997, 690: 173-179
86. Sun Y, Peng Y, Aksornkoae N, Johnson J R, Boring J G., Scruggs D, Cooper R C, Laizure S C, Shukla A J. Controlled release of oxytetracycline in sheep. Journal of Controlled Release, 2002, 85(1): 125-134
87. Terlaak E A, Pijper A, Noordergraaf H, Schoevers E J, Verheijden J H M. A comparison of methods for in vitro susceptibility testing of porcine mycoplasma species. Thesis (A.Pijper) PhD University of Utrecht, 1991,35(2): 228-233
88. Vranic M L,Marangunich L,Fernandez Courel H,Fernandez Suarez A.Estimation the withdrawal period for Veterinary drugs used in food producing animals.Analytica Chimica Acta,2003,483:251-257
89. Wang W F, Lin H, Xue C H. Elimination of chloramphenicol, sulphamethoxazole and oxytetracycline in shrimp, Penaeus Chinensis following medicated-feed treatment. Environment International. 2004, 30: 367-373
90. Yoshimura H, Osawa N, Rasa F S. Hermawati D, Werdiningsih S. Isriyanthi N M, Sugimori T. Food Addit. animal Contam, 1991, 8: 65
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59. Abd El-Aty A M, Goudah A, Zhou H H. Pharmacokinetics of doxycycline after administration as a single intravenous bolus and intramuscular doses to non-lactating Egyptian goats. Pharmacol Res, 2004,49: 487-491
60. Anadon A, Martinez-Larranaga M R, Diaz M J, Bringas P, Fernandez M C, Fernandez-Cruz M L, Iturbe J, Martinez M A. Pharmacokinetics of doxycycline in broiler chickens. Avian Pathology, 1994,23: 79-90
61. Anadon A, Martinez-Larranaga M R, Fernandez-Cruz M L, Diaz M J, Fernandez R, Sevil B, Anton R. Pharmacokinetics of doxycycline in pigs.Proceedings of the 14th IPVS Congress, 1996:670
62. Axisa B, Naylor A R, Bell P R F, Thompson M M. Simple and reliable method of doxycycline determination in human plasma and biological tissues. Journal of Chromatography B, 2000, 744:3 59-365
63. Baret K. Pharmacokinetics and oral bioavailability of a doxycycline formulation (doxycycline 75%)in nonfasted young pigs.J.vet.pharmacol.therap. 2000,23:45-48
64. Bousquet E, Morvan H, Aitken I, Morgan J H. Comparative in vitro activity of doxycycline and oxytetracycline against porcine respiratory pathogens. veterinary Record, 1997, 141:37-40
65. Choi D H, Moon I S, Choi B K. Effects of subantimicrobial dosedoxycycline therapy on crevicular fluid MMP-8, and gingival tissue MMP-9, TIMP-1 and 1L-6 levels in chronic periodntitis. J Periodontol Res, 2004, 39(1): 20-26
66. Cinquina A L, Longo F, Anastasi G, Giannetti L. Validation of a high-performance liquid chromatography method for the determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline in bovine milk and muscle. Journal of Chromatography A, 2003,987: 227-233
67. Croubels S. Liquid chromatographic separation of doxycycline and 4-epidoxycycline in a tissue depletion study of doxycycline in turkeys. Journal of Chromatography B, 1998, 708: 145-152
68. Concordet D, Toutain P L.The withdrawal time estimation of Veterinary drugs revised, 1997a,20:280-386
69. Concordet D, Toutain P L.The withdrawal time estimation of veterinary drugs:a non-parametric approach,J Vet Pharmacology Therap 1997b,20:374-379
70. George Rigos, Maria A, Argyro A. Pharmacokinetics and tissue distribution of oxytetracycline in sea bass, Dicentrarchus labrax,at two water temperatures. Aquaculture, 2002, 210: 59-67
71. Goodman L S, Gilman AG (eds). The pharmacological basis of therapeutics, 9th edn. McGraw-Hill, New York, 1996
72. Ibrahim A. Alsarra I A, Niazy E M, Al-Sayed Y M, Al-Khamis K I, Ibrahim M E -A. High performance liquid chromatographic method for the determination.of doxycycline in human plasma and its application in pharmacokinetic studys. Saudi Pharmaceutical Journal, 2005, 13(1): 42-47
73. Jha V K, Jayachandran C, Singh M K, Singh S D. Pharmacokinetic data on doxycycline and its distribution in different biological fluids in female goats. Veterinary Research Commumications, 1989, 13: 11-16.
74. Kazuaki U , Takahiko A , Walai K. Pharmacokinetics of oxytetracycline in black tiger shrimp, Penaeus monodon, and the effect of cooking on the residues. Aquaculture, 2006, 11: 24-31.
75. Kuhne M, Wegmann S, Kobe A, Fries R, M. Tetracycline residues in bones of slaughtered animals. Food Control, 2000, 11: 175-180.
76. Michel G. Pharmacokinetics and tissue localization of doxycycline polyphosphate and doxycycline hydrochloride in the rat. Eur J Drug Metab Pharmacokinet, 1984, 9(2): 149-153
77. Martinez M, Friedlander P, Condon R, Meneses J, Orangers J, Weber N, Miller M.Response to criticisms of the US FDA parametric approach for withdrawal time estimation:rebuttal and comparison to the nonparametric method proposed by Concordet and Toutain. J Vet Pharmacol Therap, 2000, 23: 21-35
78. Ole-Mapenay I M, Mitema E S, Maitho T E. Aspects of the pharmacokinetics of doxycycline given to healthy and pneumonic East African dwarf goats by intramuscular injection. Veterinary Research Communications, 1997, 21(6): 453 -462.
79. Pascale D, Gordon J, Lamster I, Mann P, Seiger M, Arndt W. Concentration of doxycycline in human gingival fluid. J Clin Periodontol, 1986, 13(9): 841-844
80. pijper A, Van K B, Schoevers E J,Verheijden J H M, Van M. In vitro activity of five tetracyclines and some other antimicrobial agents against four porcine respiratory tract pathogens. Journal of veterinary pharmacology and therapeutics, 1989, 12: 267 -276.
81. Riond.J.L & Riviere,J.E.Pharmacokintics and metabolic inertness of doxycycline in young pigs. American Journal of Veterinary Research , 1998, 51: 1271-1275.
82. Ruth R, Dina F, Arie L. Bioequivalence study of two formulations of doxycycline.Current therapeutic research, 1998, 59(5): 315-324.
83. Sader H S, Pfaller M A, Tenover F C. Evalution and characterization of multiresistant Enterococcus faecium from 12 U. S.medical centers. J Clin Microbiol, 1994, 32(11): 2840-2842
84. Sanders P, Cicquel.M, Hurtaud.D, Chapel A M,Gaudiche.C & Bousquet.E.Absolute bioavailabilitu of doxycycline after oral aministration in medicated feed to pigs. Proceedings of the 14th IPVS Congress. 1996, 663
85. Siska M C , Kristina E I, Vanoosthuyze C H, Van P. Use of metal chelate affinity chromatography and membranebased ion-exchange as clan-up procedure for trace residue analysis of tetracyclines in animal tissues and egg. Journal of chromatography, 1997, 690: 173-179
86. Sun Y, Peng Y, Aksornkoae N, Johnson J R, Boring J G., Scruggs D, Cooper R C, Laizure S C, Shukla A J. Controlled release of oxytetracycline in sheep. Journal of Controlled Release, 2002, 85(1): 125-134
87. Terlaak E A, Pijper A, Noordergraaf H, Schoevers E J, Verheijden J H M. A comparison of methods for in vitro susceptibility testing of porcine mycoplasma species. Thesis (A.Pijper) PhD University of Utrecht, 1991,35(2): 228-233
88. Vranic M L,Marangunich L,Fernandez Courel H,Fernandez Suarez A.Estimation the withdrawal period for Veterinary drugs used in food producing animals.Analytica Chimica Acta,2003,483:251-257
89. Wang W F, Lin H, Xue C H. Elimination of chloramphenicol, sulphamethoxazole and oxytetracycline in shrimp, Penaeus Chinensis following medicated-feed treatment. Environment International. 2004, 30: 367-373
90. Yoshimura H, Osawa N, Rasa F S. Hermawati D, Werdiningsih S. Isriyanthi N M, Sugimori T. Food Addit. animal Contam, 1991, 8: 65