目的 结合氘代盐酸水解和柱前衍生化,建立超高效液相色谱-四极杆串联飞行时间质谱法(UPLC-Q-TOF-MS)同时测定胸腺法新原料药中9种痕量D-氨基酸含量的方法。方法 采用氘代盐酸水解,选择Nα-(2,4-二硝基-5-氟苯基)-L-丙氨酰胺[Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide,FDAA]进行柱前衍生化;选择ACCQ-TAGTM ULTRA C18色谱柱 (2.1 mm×100 mm,1.7 μm),以0.1%(V/V)甲酸/水溶液-乙腈为流动相,流速为0.3 mL·min-1,线性梯度洗脱;Xevo G2-S Q-TOF采用电喷雾离子(ESI)源和正离子监测模式,通过标准曲线法对胸腺法新原料药中9种D-氨基酸含量进行准确测定。结果 9种D-氨基酸衍生产物在一定范围内进样量与峰响应值呈良好的线性关系(r>0.991 2);定量限低至0.05~0.30 pmol;精密度和平均加标回收率等均符合2015年版《中国药典》方法学要求。5家企业的胸腺法新原料药中D-氨基酸含量测定结果为25.16~1 322.01 μg·g-1,其中D-Glu、D-Asp、D-Lys和D-Ser的含量较高,分别为1 105.45~1 322.01、614.15~740.50、358.06~388.76和138.78~291.60 μg·g-1。结论 所建立的方法灵敏、高效、可靠,可同时测定胸腺法新中9种痕量D-氨基酸的含量,为该合成多肽药物全面控制消旋肽杂质提供新的方法参考。
Abstract
OBJECTIVE To develop an ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry method for simultaneous determination of nine trace D-amino acids in thymalfasin, combined with deuterated acid hydrolysis and precolumn-derivatization. METHODS The sample was hydrolyzed by deuterated acid, followed by precolumn-derivatization with Nα-(2, 4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA). The analysis was then performed on an ACCQ-TAGTM ULTRA C18 column (2.1 mm×100 mm, 1.7 μm), with mobile phase comprising water containing 0.1% formic acid-acetonitrile gradiently eluted at a flow rate of 0.3 mL·min-1. Nine D-amino acids in thymalfasin were correctly quantified using standard curves by Xevo G2-S Q-TOF coupled with electrospray ion source in positive ion mode. RESULTS The standard curves of the nine D-amino acids derivatives had good linearity in the ranges of the tested concentrations (r>0.991 2). The limits of quantitation of all D-amino acids derivatives were as low as 0.05-0.30 pmol. The precision and recoveries met the requirements of Chinese Pharmacopoeia (2015). The contents of D-amino acids in the samples from five companies were determined to be 25.16-1 322.01 μg·g-1. Among them, D-glutamate, D-aspartate, D-lysine and D-serine had higher contents, which were 1 105.45-1 322.01, 614.15-740.50, 358.06-388.76 and 138.78-291.60 μg·g-1, respectively. CONCLUSION The method is sensitive, efficient and reliable, working well for simultaneous determination of nine trace D-amino acids in thymalfasin, which provides a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.
关键词
胸腺法新 /
消旋肽 /
氘代酸水解 /
柱前衍生化 /
超高效液相色谱-四极杆串联飞行时间质谱法
{{custom_keyword}} /
Key words
thymalfasin /
racemic peptide /
deuterated acid hydrolysis /
pre-column derivatization /
ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry
{{custom_keyword}} /
中图分类号：
R917
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] TIAN N N. Study on quality of thymalfasin [D]. Lanzhou:University of Lanzhou, 2015.
[2] GAO D M, WANG F S. Review on the characteristics and function of thymosin alpha 1 [J]. Chin J Biochem Pharm (中国生化药物杂志), 2007, 28(2): 136-139.
[3] D'HONDT M, BRACKE N, TAEVERNIER L, et al. Related impurities in peptide medicines [J]. J Pharm Biomed Anal, 2014, 101: 2-30.
[4] HU Y X, JIANG Y, HAN T J, et al. Quality control and related substances of synthetic peptide drugs [J]. Chin J New Drug (中国新药杂志), 2018(5): 502-508.
[5] MARIS N L, SHEA D, BLEEM A, et al. Chemical and physical variability in structural isomers of an L/D α-sheet peptide designed to inhibit amyloidogenesis [J]. Biochemistry, 2018, 57(5): 507-510.
[6] Ch.P (2015). VolⅡ(中国药典2015年版. 二部) [S]. 2015: 1162-1163.
[7] CHEN J X, WANG S L, ZHANG H Y, et al. Content determination of the related substances in thymalfasin for injection by RP-HPLC [J]. Mod Med Health (现代医药卫生), 2014, 30(22):3390-3392.
[8] CHENG L, TANG Y M, YIN G, et al. HPLC determination of impurities of thymalfasin and thymalfasin for injection [J]. Chin J Pharm Anal(药物分析杂志), 2013, 33(11): 1942-1947.
[9] GOODLETT D R, ABUAF P A, SAVAGE P A, et al. Peptide chiral purity determination: hydrolysis in deuterated acid, derivatization with Marfey's reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry [J]. J Chromatogr A, 1995, 707(2): 233-244.
[10] USP 41 [S]. 2018: 1667-1670.
[11] FRANK H, WOIWODE W, NICHOLSON G, et al. Determination of the rate of acidic catalyzed racemization of protein amino acids [J]. Eur J Org Chem, 1981, 3: 354-365.
[12] ISHIGO S, NEGISHI E, MIYOSHI Y, et al. Establishment of a two-dimensional HPLC-MS/MS method combined with DCl/D2O hydrolysis for the determination of trace amounts of D-amino acid residues in proteins [J]. Chromatography, 2015, 36: 45-50.
[13] WANG W H, YANG B. Ultra-high performance liquid chromatography combined with pre-column derivatization for quantification of free amino acids in Poria [J]. Chin Pharm J (中国药学杂志), 2017, 52(5): 372-376.
[14] TANG L, WEI W, ZHAO Y, et al. Determination of free amino acids in Hericium erinaceus mycelium by HPLC with precolumn derivatization [J]. Chin Pharm J (中国药学杂志), 2017, 52(21): 1899-1902.
[15] BRUCKNER H, ZIVNY S. High-performance liquid chromatographic resolution of (R,S)-α-alkyl-α-amino acids as diastereomeric derivatives [J]. Amino Acids, 1993, 4(1-2): 157-167.
[16] PETER A, TOROK G, TOTH G, et al. Enantiomeric separation of unusual secondary aromatic amino acids [J]. Chromatographia, 1998, 48: 53-58.
[17] MARFEY P. Determination of D-amino acids. Ⅱ. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene [J]. Carlsberg Re Commun, 1984, 49(6): 591-596.
[18] BRUCKNER H, KELLER-HOEHL C. HPLC separation of DL-amino acids derivatized with N, 2-(5-fluoro-2, 4-dinitrophenyl)-L-amino acid amides [J]. Chromatographia, 1990, 30(11): 621-629.
[19] HU P, ZHANG Y J, CAI J, et al. Simultaneous determination of amino acids, nucleosides and nucleobases in different parts of Metaplexis japonica (Thunb.) Makino processed by different methods by UFLC-QTRAP-MS/MS [J]. Chin Pharm J (中国药学杂志), 2017,52(13):1177-1182.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学青年基金项目资助(81803422)
{{custom_fund}}
PDF(4934 KB)
