等温微量热技术快速评价头孢曲松钠的体外抑菌效果

目的：开发一种基于等温微量热技术实时测定头孢曲松钠体外抑菌效果的快速检测方法。方法：摸索金黄色葡萄球菌株起始接种量的范围和头孢曲松钠溶液检测浓度的区间，确定可用于快速分析的热谱图参数，比较测试药品与原研药品（罗氏芬）在拉曼光谱、X射线衍射谱、抗生素效价测定、高效液相色谱和体外微量热图谱的差异。结果：以10⁵ CFU·mL^-1的金黄色葡萄球菌为起始浓度，头孢曲松钠溶液质量浓度（C_n）在0.25~0.80 mg·mL^-1时与含药物的微生物热谱图主峰相对出峰时间的归一化值（α）存在正相关线性关系（R²=0.99）；同时，头孢曲松钠溶液C_n和含药物的微生物热谱图曲线主峰斜率的归一化值（β）存在负相关线性关系（R²=0.89）。微量热法检测的测试药品体外抑菌活性比罗氏芬相比低约8%，其结果与传统管碟法测试结果相同。X射线衍射粉末衍射结果显示两者晶型组成不同。结论：微量热技术具有灵敏度高及精密度好的特点，可实时检测抗生素体外抑菌活性，在快速评价抗生素效价领域具备较好的应用前景。

Objective:To develop a rapid method of isothermal microcalorimetry detection technology for the real-time test of antibacterial activity of ceftriaxone sodium in vitro. Methods:The range of initial bacterial concentration, ceftriaxone sodium concentration and parameters from heat flow curves were tested and optimized using Staphylococcus aureus ATCC 6538, respectively. The generic ceftriaxone sodium and original drug (Rocephin) were compared by Raman spectra, X-ray powder diffraction spectra, cylinder-plate method and HPLC method. Results:When mass concentration (C_n) of ceftriaxone sodium was between 0.25 mg·mL^-1 and 0.80 mg·mL^-1, C_n had a positive linear relationship with the relative time difference (α) for the main heat-flow peak (R²=0.99), using 10⁵ CFU·mL^-1 of S. aureus initial concentration. Meanwhile, C_n had a negative linear relationship with the relative slope (β) for the main heat flow peak (R²=0.89). The antibacterial activity of generic ceftriaxone sodium in vitro by isothermal microcalorimetry was 8% lower than that of Rocephin, and the same result was confirmed by cylinder-plate method. However, X-ray powder diffraction spectra showed a significant difference in their crystal form. Conclusion:Isothermal microcalorimetry is a rapid, sensitive, precision and useful technology to test antibacterial activity in real-time with a good application prospect.

[1] 李娅萍, 薛晶, 杨智慧, 等.采用新型混合模式离子交换柱HPLC-ELSD法测定头孢曲松钠中钠离子含量以及成盐率[J].中国抗生素杂志, 2013, 38(7):524 LI YP, XUE J, YANG ZH, et al.Determination the content of sodium and salt-forming rate in ceftriaxone sodium for injecion by an HPLC-ELSD method using a new type of mixed-mode ion exchange column[J].Chin J Antibiot, 2013, 38(7):524
[2] 李丹, 张永信. 头孢曲松钠的特性及临床应用[J].上海医药, 2013, 34(13):12 LI D, ZHANG YX.Characteristics of ceftriaxone sodium and its clinical application[J].Shanghai Med Pharm J, 2013, 34(13):12
[3] 胡昌勤,尹立辉,朗雅宁.头孢唑林钠水合物新晶体及其理化特性的研究[J].药学学报, 2008, 43(8):868 HU CQ, YIN LH, LIANG YN.Physical and chemical characteristics of a new cefazolin sodium hydrate crystal[J].Acta Pharm Sin, 2008, 43(8):868
[4] 涂林, 余方键, 胡昌勤.国产注射用头孢曲松钠的质量现状[J].中国药事, 2002, 16(1):45 TU L, YU FJ, HU CQ.The quality of ceftriaxone sodium in China[J].Chin Pharm Aff, 2002, 16(1):45
[5] HICKEY MB, PETERSON ML, MANAS ES, et al.Hydrates and solid-state reactivity:a survey of β-lactam antibiotics[J].J Pharm Sci, 2007, 96(5):109
[6] 谢景文, 王荣, 贾正平, 等, 毛细管电泳测定头孢曲松钠对映体含量[J].中国药房, 2002, 13(1):43 XIE JW, WANG R, JIA ZP, et al.Determination of the enantiomers of ceftriaxone sodium by capillary electrophoresis[J].China Pharm, 2002, 13(1):43
[7] ARNET I, ALTERMATT M, ROGGO Y, et al.Pharmaceutical quality of eight generics of ceftriaxone preparation for injection in Eastern Asia[J].J Chemother, 2015, 27(6):337
[8] BAUER J, SPANTON S, HENRY R, et al.Ritonavir:an extraordinary example of conformational polymorphism[J].Pharm Res, 2001, 18(6):859
[9] LAMBERT PA, CONWAY BR. Pharmaceutical quality of ceftriaxone generic drug products compared with Rocephin[J].J Chemother, 2003, 15(4):357
[10] 中华人民共和国药典2015年版.四部[S].2015:160 ChP 2015.Vol Ⅳ[S].2015:160
[11] ZULUAGA AF, AGUDELO M, RODRIGUEZ CA, et al.Application of microbiological assay to determine pharmaceutical equivalence of generic intravenous antibiotics[J].BMC Clin Pharmacol, 2009, 9:1
[12] 黄英.管碟法测定抗生素效价的影响因素分析[J].中国药师, 2005, 8(8):689 HUANG Y.Influence factors and analysis of cylinder-plate method for antibiotics[J].China Pharm, 2005, 8(8):689
[13] BRAISSANT O, WIRZ D, GOPFERT B, et al.Use of isothermal microcalorimetry to monitor microbial activities[J].FEMS Microbiol Lett, 2010, 303(1):1
[14] 胡艳军, 蒋风雷, 欧阳宇, 等.微量热法在药物活性评价中的应用[J].中国科学:化学, 2010, 40(9):1276 HU YJ, JIANG FL, OUYANG Y, et al.Microcalorimetry in drug activity evaluation[J].Sci China Ser B, 2010, 40(9):1276
[15] MA J, QI WT, YANG LN, et al.Microcalorimetric study on the growth and metabolism of microencapsulated microbial cell culture[J].J Microbiolog Methods, 2007, 68(1):172
[16] BRAISSANT O, WIRZ D, GÖPFERT B, et al.Biomedical use of isothermal microcalorimeters[J].Sensors (Basel), 2010, 10(10):9369
[17] von AH U, WIRZ D, DANIELS AU.Rapid differentiation of methicillin-susceptible Staphylococcus aureus from methicillin-resistant S.aureus and MIC determinations by isothermal microcalorimetry[J].J Clin Microbiol, 2008, 46(6):2083
[18] WENZLER T, STEINHUBER A, WITTLIN S, et al.Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum[J].PLoS Negl Trop Dis, 2012, 6(6):e1668
[19] GAREDEW A, SCHMOLZ E, LAMPRECHT I.Microbiological and calorimetric investigations on the antimicrobial actions of different propolis extracts:an in vitro approach[J].Thermochim Acta, 2004, 422(1):115
[20] von AH U, WIRZ D, DANIELS AU.Isothermal micro calorimetry-a new method for MIC determinations:results for 12 antibiotics and reference strains of E.coli and S.aureus[J].BMC Microbiol, 2009, 9:106
[21] BALDONI D, HERMANN H, FREI R, et al.Performance of microcalorimetry for early detection of methicillin resistance in clinical isolates of Staphylococcus aureus[J].J Clin Microbiol, 2009, 47(3):774
[22] BONKAT G, BRAISSANT O, WIDMER AF, et al.Rapid detection of urinary tract pathogens using microcalorimetry:principle, technique and first results[J].BJU Int, 2012, 110(6):892
[23] CHEN XY, HU XL, XIA CF, et al.Antibacterial evaluation of novel organoarsenic compounds by the microcalorimetric method[J].Biol Trace Elem Res, 2013, 153(1-3):382
[24] KONG W, JIN C, XIAO X, et al.Comparative study of effects of two bile acid derivatives on Staphylococcus aureus by multiple analytical methods[J].J Hazard Mater, 2010, 179(1-3):742
[25] FURUSTRAND TAFIN U, CLAUSS M, HAUSER PM, et al.Isothermal microcalorimetry:a novel method for real-time determination of antifungal susceptibility of Aspergillus species[J].Clin Microbiol Infect, 2012, 18(7):E241