HPLC法测定发酵液中非达霉素的含量

目的：建立高效液相色谱法定量检测微生物发酵液中的非达霉素。方法：采用Agilent 5 TC-C₁₈色谱柱（250 mm×4.6 mm，5 μm），以甲醇-0.1%甲酸水为流动相，流速1.0 mL·min^-1，检测波长228 nm，柱温40℃。结果：非达霉素质量浓度在6.59~79.08 μg·mL^-1范围内与峰面积呈现良好的线性关系（r=0.999 9，n=7）；平均回收率为98.6%（n=9，RSD=0.91%）；精密度、重复性和稳定性良好，RSD均小于2%；3批发酵液中非达霉素的测定结果分别为59.4、52.5、53.6 μg·mL^-1。结论：本方法可用于非达霉素的菌株筛选以及发酵过程产物的控制。

Objective: To establish an HPLC method for quantitative determination of fidaxomicin in fermentation broth. Methods: The separation was carried out on the Agilent 5 TC-C₁₈ (250 mm×4.6 mm, 5 μm)column at the detection wavelength of 228 nm and the column temperature was set at 40℃. The mobile phase consisted of methanol-0.1% formic acid solution at a flow rate of 1.0 mL·min^-1. Results: The mass concentration of fidaxomicin and peak area showed excellent linearity within the range of 6.59-79.08 μg·mL^-1, and the correlation coefficient was 0.999 9 (n=7). The average recovery (n=9) was 98.6% (RSD=0.91%). The precision, reproducibility and stability were satisfactory with RSD of less than 2%. The contents of fidaxomicin in three batches of fermentation broths were 59.4, 52.5 and 53.6 μg·mL^-1, respectively. Conclusion: The developed method is suitable for strain screening and fermentation control of fidaxomicin.

[1] ZHANEL GG,WALKTY AJ,KARLOWSKY JA.Fidaxomicin:a novel agent for the treatment of Clostridium difficile infection[J].Can J Infect Dis Med Microbiol,2015,26(6):305
[2] MICHAEL G,GRIT A.OPT-80,a macrocyclic antimicrobial agent for the treatment of clostridium difficile infection:a review[J].Expert Opin Investig Drugs,2008,17(4):547
[3] 姜春梅,刘洋,王京晶,等.抗感染新药非达霉素的药理作用与临床评价[J].中国新药杂志,2011,20(23):2283 JIANG CM,LIU Y,WANG JJ,et al.Pharmacology and clinical evaluation of the new anti-infective drug fidaxomicin[J].Chin New Drugs J,2011,20(23):2283
[4] CRUZ MP.Fidaxomicin(Dificid),a novel oral macrocyclic antibacterial agent for the treatment of Clostridium difficile-associated diarrhea in adults[J].Pharm Ther,2012,37(5):278
[5] MANTHEY CF,ECKMANN L,FUHRMANN V.Therapy for Clostridium difficile infection:any news beyond metronidazole and vancomycin[J].Expert Rev Clin Pharmacol,2017,10(11):1239
[6] SHUE YK,FRANK CJ,CHIOU MH,et al.Tiacumicin Production:US,2006/0257981 A1[P].2006-11-16
[7] THERIAULT RJ,KARWOWSKI JP,JACKSON M,et al.Tiacumicins,a novel complex of 18-membered macrolide antibiotics.Ⅰ.Taxonomy,fermentation and antibacterial activity[J].J Antibiot,1987,40(5):567
[8] SANGHVI S,ROACH M,ZHOU JF,et al.Compositions of Stable Tiacumicins:US,2008/0176927 A1[P].2008-07-24
[9] 肖毅,李苏梅,马亮,等.台勾霉素产生菌指孢囊菌NRRL 18085遗传操作体系的建立[J].微生物学报,2010,50(8):1014 XIAO Y,LI SM,MA L,et al.Genetic manipulation system for tiacumicin producer Dactylosporangium aurantiacum NRRL 18085[J].Acta Microbiol Sin,2010,50(8):1014
[10] 江宏磊,方志锴,陈名洪,等.大环内酯类抗生素产生菌的基因筛选及其代谢产物研究[J].天然产物研究与开发,2017,29(11):1895 JIANG HL,FANG ZK,CHEN MH,et al.Gene screening of macrolide-producing strain and its metabolites[J].Nat Prod Res Dev,2017,29(11):1895
[11] SHUE YK,HWANG CK,CHIU YH,et al.18-Membered Macrocycles and Analogs Thereof:US,2010/0009925 A1[P].2010-02-14
[12] KURABACHEW M,LU SH,KRASTEL P,et al.Lipiarmycin targets RNA polymerase and has good activity against multidrug-resistant strains of Mycobacterium tuberculosis[J].J Antimicrob Chemoth,2008,62(4):713
[13] HOCHLOWSKI JE,SWANSON SJ,RANFRANZ LM,et al.Tiacumicins,a novel complex of 18-membered macrolide antibiotics.Ⅱ.Isolation and structure determination[J].J Antibiot,1987,40(5):575
[14] CORONELLI G,WHITE RJ,LANCINI GC,et al.Lipiarmycin,a new antibiotic from Actinoplanes.Ⅱ.Isolation,chemical,biological and biochemical characterization[J].J Antibiot,1975,28(4):253
[15] XIAO Y,LI SM,NIU SW,et al.Characterization of tiacumicin B biosynthetic gene cluster affording diversified tiacumicin analogues and revealing a tailoring dihalogenase[J].J Am Chem Soc,2011,133:1092