1-4 certified reference material CRM. DSC HPLC ± 0. 4 % k = 2 P = GBW

2014 6 33 6 779 * 1 1 2 3 1 2 1. 100050 2. 277500 3. 100050 DSC HPLC 99. 5 ± 0. 4 % k 2 P 0. 95 GBW09587 R978. 7 R927. 1 A 1004-0781 2014 06-0779 - 06 Purity Determination and Uncertainty Evaluation of Ribavirin Purity Reference Material DU Hui 1 XING Cheng 1 SHI Yong-qiang 2 DU Guan-hua 3 LV Yang 1 2 1. Beijing City Key Laboratory of Pharmaceutical Polymorph Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100050 China 2. Shandong Province Key Laboratory of Pharmaceutical Polymorph Tengzhou 277500 China 3. Beijing City Key Laboratory of Drug Identification and Drug Screening Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100050 China ABSTRACT Objective To develop the ribavirin purity certified reference material CRM which has measurement traceability and high accuracy and establish an effective method of evaluation. Methods The homogeneity and the stability were checked by differential scanning calorimetry DSC. High performance liquid chromatography HPLC and DSC methods were used for purity determination of ribavirin. Results Ribavirin showed satisfactory homogeneity and stability. The certified value of ribavirin was 99. 5% with an uncertainty of 0. 4% k 2 P 0. 95. Conclusion Ribavirin purity CRM obtained in this paper has been proven to be a national primary CRM with high accuracy and traceability which can be used to validate analytical methods improve the accuracy of measurement data establish meteorological traceability of analytical results as well as control the quality of ribavirin in the pharmaceutical industry. KEY WORDS Ribavirin Purity certified reference material Calorimetry differential scanning Chromatography high performance liquid 1-4 certified reference material CRM 2013-11 - 01 2013-12 - 31 1959-9 010-63165212 E-mail luy @ imm. ac. cn * 5-7 CRM 2012ZX09301002-001-013 2013ZX09102110 200902008 1987-8 010-63030566 E-mail cong200767 @ 163. com 10-11 1956-010 - 63165184 E-mail dugh@ imm. ac. cn

780 Herald of Medicine Vol. 33 No. 6 June 2014 1 1. 1 1. 1. 1 differential scanning calorimetry DSC Mettler Toledo DSC-1 Al 2 O 3 DSC 40 μl Al 2 O 3 5. 0 K min - 1 130 ~ 180 chromatography HPLC Agilent 1260 2. 1. 2 HPLC DAD Agilent Eclipse XDB-C 18 250 mm 4. 6 mm 5 μm ph2. 53 100% 207 nm 30 25 ml 1. 0 ml min - 1 10 μl 1 000. 40 μg ml - 1 5 0. 1 Mettler Toledo V20 SX2. 5-10 0. 5 1. 0 2. 0 5. 0 ml 10 ml Agilent 10. 00 50. 02 7890A 100. 04 200. 08 500. 20 1 000. 40 μg ml - 1 J. T. Baker 1. 3 50 g 500 ml 24 h 60 2. 1. 2. 2 24 h 1 12. 50 mg HPLC 25 ml 98. 0% 500. 0 μg ml - 1 10 μl > 99. 0% 6 2 ml 13 008. 9 13 007. 4 13 019. 6 13 020. 8 12 998. 2 50 mg 13 003. 1 RSD 0. 07% HPLC 500 2 2. 1 2. 1. 1 DSC 2. 1. 1. 1 6 1. 5 ~ 3. 5 mg 1. 50 1. 72 2. 16 2. 51 3. 01 3. 52 mg 40 μl Al 2 O 3 DSC 1 040. 1 1 043. 5 1 042. 8 1 051. 9 RSD 0. 42% HPLC 1. 5 ~ 3. 5 mg 2. 2 Y 192. 672 7X - 5. 993 3 R 2 2. 1. 1. 2 6 2. 50 mg 40 μl 6 192. 42 194. 87 187. 67 192. 48 187. 57 1. 5 ~ 3. 5 mg 191. 88 RSD 1. 53% DSC 1. 1. 2 high performance liquid 2. 1. 2. 1 1 25. 01 mg 1. 2 10 μl 090225 3 GBW13202 429. 75 ± 0. 01 K Millipore 10. 00 ~ 1 000. 40 μg ml - 1 0. 2 μm Y 25. 43X + 41. 387 R 2 0. 999 8 n 6 HPLC 2. 1. 2. 3 6 12. 50 mg 25 ml 500. 0 μg ml - 1 10 μl 3 6 1 040. 3 1 041. 2 2. 2. 1 500 0. 999 3 n 6 DSC 15 3 DSC

2014 6 33 6 781 3 15 15 6 1 1 2 3 F F 1. 44F 0 1 2 4 6 12 F 14 30 2. 04F < F 14 30 t t t 4 2. 776 2. 2. 2 DSC t4 0 7 0. 802 t 4 0 14 0. 000 t 4 0 7 0. 728 t 4 0 14 60 90% ± 5% 25 0. 000 t 4 0 7 0. 000 t 4 0 14 4 500 ± 500 lx 0 7 14 d 0. 567 3 3 1 Tab. 1 1 Results of homogeneity test on purity certified reference material of ribavirin Y ij /% 1 2 3 n i N Y i i j j Y j N Y 2i j i j 1 99. 47 99. 47 99. 48 3 2. 984 2 0. 994 7 2. 968 5 0. 000 1 2 99. 48 99. 51 99. 51 3 2. 985 0 0. 995 0 2. 970 1 0. 000 2 3 99. 51 99. 50 99. 52 3 2. 985 3 0. 995 1 2. 970 7 0. 000 1 4 99. 49 99. 48 99. 49 3 2. 984 6 0. 994 9 2. 969 3 0. 000 1 5 99. 48 99. 50 99. 51 3 2. 984 9 0. 995 0 2. 969 9 0. 000 2 6 99. 49 99. 49 99. 47 3 2. 984 5 0. 994 8 2. 969 1 0. 000 1 7 99. 47 99. 49 99. 50 3 2. 984 6 0. 994 9 2. 969 3 0. 000 2 8 99. 47 99. 50 99. 51 3 2. 984 8 0. 994 9 2. 969 7 0. 000 2 9 99. 50 99. 48 99. 50 3 2. 984 8 0. 994 9 2. 969 7 0. 000 1 10 99. 48 99. 47 99. 49 3 2. 984 4 0. 994 8 2. 968 9 0. 000 1 11 99. 49 99. 49 99. 47 3 2. 984 5 0. 994 8 2. 969 1 0. 000 1 12 99. 50 99. 48 99. 49 3 2. 984 7 0. 994 9 2. 969 5 0. 000 1 13 99. 49 99. 47 99. 50 3 2. 984 6 0. 994 9 2. 969 3 0. 000 2 14 99. 48 99. 49 99. 50 3 2. 984 7 0. 994 9 2. 969 5 0. 000 1 15 99. 48 99. 51 99. 51 3 2. 990 7 0. 996 9 2. 981 4 0. 000 2 0. 994 9 0. 000 1 s i Tab. 2 / 2 Results of short-term stability test on purity certified reference material of ribavirin /% / d 1 2 3 % 0 99. 47 99. 49 99. 50 99. 49 0. 000 152 8 7 99. 50 99. 47 99. 48 99. 48 0. 000 152 8 14 99. 49 99. 48 99. 49 99. 49 0. 000 057 7 0 99. 51 99. 51 99. 49 99. 50 0. 000 115 5 7 99. 51 99. 47 99. 48 99. 49 0. 000 208 2 14 99. 48 99. 51 99. 50 99. 50 0. 000 152 8 0 99. 46 99. 50 99. 50 99. 49 0. 000 230 9 7 99. 52 99. 48 99. 48 99. 49 0. 000 230 9 14 99. 50 99. 48 99. 52 99. 50 0. 000 200 0

782 Herald of Medicine Vol. 33 No. 6 June 2014 Tab. 3 3 Results of long-term stability test on purity certified reference material of ribavirin /% / / 1 2 3 4 5 6 % 0 99. 53 99. 49 99. 49 99. 51 99. 50 99. 46 99. 50 0. 000 234 1 99. 48 99. 50 99. 53 99. 51 99. 47 99. 47 99. 49 0. 000 242 2 99. 47 99. 46 99. 48 99. 48 99. 52 99. 50 99. 49 0. 000 217 4 99. 48 99. 51 99. 50 99. 47 99. 52 99. 46 99. 49 0. 000 237 6 99. 50 99. 49 99. 47 99. 51 99. 48 99. 45 99. 48 0. 000 216 12 99. 49 99. 50 99. 49 99. 48 99. 51 99. 48 99. 49 0. 000 117 x y 100% y - 0. 000 006x + 0. 994 926 a - 0. 000 006 S 2 n Y i - b - ax i 2 /n - 2 4. i 1 07 10-9 n - 2 6-2 4 P 0. 95 95% t 2. 776 a < t4 S a 12 2. 3 f i A i 10 G 1. 461 G 0. 95 10 G 1. 631 G 0. 95 10 2. 176 G < G 0. 95 10 10 2. 176 G < G 0. 95 10 10 10 10 HPLC DSC 99. 72% 99. 49% A i C i 100% f i A i C i A i f i 2. 3. 1 4 DSC DSC DSC Tab. 4 Determination results of purity certified reference material of ribavirin by DSC methods < 2% Van t Hoff / / / x 1 - T mg % mg % 0 - T m ΔH 2 100% 1 2. 16 99. 49 6 2. 71 99. 47 RT 0 2 2. 72 99. 47 7 2. 72 99. 46 T 0 3 K T m 4 2. 67 2. 51 99. 53 99. 50 8 9 3. 17 2. 72 99. 45 99. 51 K R 8. 314 J K - 1 mol - 1 x 5 1. 62 99. 50 10 2. 30 99. 49 H J mol - 1 T 0 - T m 10 12. 50 mg 10 25 ml 500 μg ml - 1 1. 5 ~ 3. 5 mg 40 μl 10 μl 3 Al 2 O 3 5 4 10 HPLC 2. 3. 2 HPLC HPLC 0. 188 1%

2014 6 33 6 783 Tab. 5 A 1 5 HPLC Determination results of purity certified reference material of ribavirin by HPLC methods A A 2 A A 3 A A A S S S 1 13 308. 0 13 337. 4 13 298. 1 13 340. 3 13 304. 5 13 330. 4 13 303. 5 13 336. 0 5. 02 5. 09 99. 76 2 13 291. 9 13 345. 2 13 292. 2 13 328. 6 13 312. 6 13 325. 1 13 298. 9 13 333. 0 11. 87 10. 74 99. 72 3 13 300. 2 13 358. 4 13 308. 7 13 332. 1 13 361. 5 13 343. 5 13 323. 5 13 344. 7 33. 21 13. 19 99. 72 4 13 307. 2 13 396. 5 13 362. 1 13 398. 3 13 359. 9 13 393. 3 13 361. 0 13 396. 0 1. 56 2. 53 99. 74 5 13 345. 8 13 381. 2 13 346. 4 13 383. 5 13 360. 0 13 405. 7 13 353. 2 13 390. 1 9. 62 13. 53 99. 71 6 13 344. 1 13 376. 4 13 345. 4 13 386. 0 13 351. 5 13 393. 3 13 348. 5 13 385. 2 4. 31 8. 48 99. 71 7 13 187. 1 13 233. 8 13 203. 2 13 246. 4 13 174. 6 13 206. 0 13 188. 9 13 228. 7 20. 22 20. 67 99. 69 8 13 181. 4 13 212. 8 13 190. 8 13 233. 7 13 180. 8 13 215. 1 13 184. 3 13 220. 5 5. 61 11. 46 99. 73 9 13 168. 9 13 205. 1 13 178. 5 13 214. 8 13 182. 1 13 214. 5 13 176. 5 13 211. 5 6. 82 5. 52 99. 74 10 13 188. 1 13 226. 5 13 185. 0 13 218. 6 13 195. 8 13 239. 5 13 189. 6 13 228. 2 5. 56 10. 55 99. 71 13 272. 8 13 307. 4 99. 72 / % u 2 B1 DSC + u 2 A1 DSC + u 2 B2 DSC + u 2 B3 DSC 0. 000 334 6 0. 037 6% 2. 4. 4 HPLC HPLC 0. 024 5% 0. 250 2% 99. 72% 100% - 0. 250 2% 99. 47% 2. 3. 3 DSC HPLC 6 DSC t t 2. 02 18. 89 Tab. 6 Combined standand uncertainty results of DSC t 19 2. 093 method 99. 47% + 99. 49% / 2 99. 48 % u B1 DSC 2. 77 10-3 9. 67 10-5 B u 2. 4 A1 DSC 7. 75 10-5 7. 79 10-5 A u B2 DSC 4. 39 10-4 2. 71 10-4 B 3 u B3 DSC 1. 62 10-6 1. 52 10-4 B 2. 4. 1 u H MS 2 - MS2 /n u 2. 50 10-8 2-1. 73 10-8 2 A S 2 / 10 13. 785 4 / 3 1. 04 10-8 u A 2. 4. 2 S 2 / 10 11. 300 0 S a S 6. 38 10-5 u c x peak 99. 47% n X i - X - 2 u ( A ) + u 2 ( A ) i 1 u t t. S a 12 6. 38 10-5 0. 00 133 7. 66 10-4 u c x H2 O s / 10 0. 000 006 6 2. 4. 3 DSC DSC u c x ash s / 10 6 0. 000 009 5 JJFl059-1999 u c x solvent s / 10 u c x DSC /99. 49% 0. 000 013

784 Herald of Medicine Vol. 33 No. 6 June 2014 HPLC u c x HPLC u c x peak 2 + u c x H2 O 2 + u c x ash 2 2 + u c x solvent 0. 001 330 2 REICHARD O ANDERSON J SCHVARCZ R et al. Ribavirin treatment for chronic hepatitis C J. Lancet 1991 2. 4. 5 337 u c ( ) x x - ( u c ( x ) ) 2 + ( u c ( x ) ) /x DSC DSC 99.48% 0.000 334 6 2 2 + 0.001 323 /x 2 HPLC HPLC 0.001 488 2. 4. 6 u c u 2 c x + u 2 H + u 2 t 0. 001 56 4 FONTINE H VALLET A SELVES J et al. Histopathologic 2. 4. 7 efficacy of ribavirin monotherapy in kidney allograft 0. 95 k 2 recipients with chronic hepatitis C J. Transplantation U ku c 0. 003 12 2004 78 6 853-857. 2. 5 5. ISO Guide 34 S. 2009. 6. ISO Guide 35 S. 2006. 99. 5% ± 0. 4% k 2 P 7 National Institute of Standards and Technology OL. http / / 0. 95 www. nist. gov / srm / index. cfm. 1 VINCENT M ELISABETH N PHIIPPE S. Brief communication case reports of ribavirin treatment for chronic hepatitis E J. Ann Int Med 2010 153 2 85-89. 8749 1058-1061. 3 GARNIER J L CHEVALLIER P DUBERNARD J M et al. Treatment of hepatitis C virus infection with ribavirin in kidney transplant patients J. Transplant Proc 1997 29 1 783. 3 8 DUEWER D L LIPPA K A LONG S E et al. Demonstrating the comparability of certified reference materials J. Anal Bioanal Chem 2009 395 1 155-169. 9 COMAR DB. http / /www. comar. bam. de /en /. 10. M. GBW09587 1985. 11. HPLC S. JJF 1006-1994. 12-14 15 16 12 ARIANNA L MARIA C S SILVANA P. Measurement of ribavirin and evaluation of its stability in human plasma by DSC HPLC high performance liquid chromatography with UV detection J. J Chromatogr B 2007 856 1-2 358-364. HPLC 13 ELISA Z TERESA G BARBARA L et al. Development of a rapid LC-MS / MS method ro ribavirin determination in rat DSC brain J. J Pharm Biomed Anal 2011 54 4 889-892. 14 LIU Y XU C YAN R. Sensitive and specific LC-MS /MS DSC method for the simultaneous measurements of viramidine and ribavirin in human plasma J. J Chromatogr B 2006 832 1 17-23. DSC HPLC 15 MICHAEL C B REGULA T. Determiantion of ribavirin in human serum and plasma by capillary electrophoresis J. Electrophoresis 2004 25 10-11 1615-1622. 16. J. 2010 23 8 195-196. DOI 10. 3870 /yydb. 2014. 06. 026