药学学报

Hydrolysis method	Hydrolysis reagent	Amino acid	Reference
Antioxidants and additives	Ethyl mercaptan; Mercaptoacetic acid; Phenol; Tryptamine	His; Met; Cys; Tyr; Trp	[5, 6]
Other acid hydrolysis	MSA; MESA; TSA	Trp; Met; Cys	[5, 7]
Alkaline hydrolysis	NaOH; LiOH; KOH; Ba (OH)₂	Trp (Not available for Ser; Thr; Arg; Cys)	[5, 8]
Enzymatic hydrolysis	Animal/plant/microorganism-derived proteases; compound enzyme	All AA	[8, 9]
Microwave hydrolysis	HCl; TFA	All AA	[6, 10]
Vapor-phase hydrolysis	HCl	All AA	[6, 10]

Hydrolysis method	Hydrolysis reagent	Amino acid	Reference
Antioxidants and additives	Ethyl mercaptan; Mercaptoacetic acid; Phenol; Tryptamine	His; Met; Cys; Tyr; Trp	[5, 6]
Other acid hydrolysis	MSA; MESA; TSA	Trp; Met; Cys	[5, 7]
Alkaline hydrolysis	NaOH; LiOH; KOH; Ba (OH)₂	Trp (Not available for Ser; Thr; Arg; Cys)	[5, 8]
Enzymatic hydrolysis	Animal/plant/microorganism-derived proteases; compound enzyme	All AA	[8, 9]
Microwave hydrolysis	HCl; TFA	All AA	[6, 10]
Vapor-phase hydrolysis	HCl	All AA	[6, 10]

Method	Classification	Analytical technique	Derivative reagent	Chiral additive	D/L Recognition	Reference
Direct method	Chiral stationary phase method	LC or CE	None	None	Difference in retention time of enantiomers in the chiral column	[12, 14, 15]
	Chiral mobile phase method	LC or CE	None	Cyclodextrin	Difference in retention time of non- enantiomers in the non-chiral column	[16, 17]
	Chiral spectroscopy method	MS/MS	None	Cu (Ⅱ)-L-Tyr; Zn (Ⅱ)-L-Trp; Phe/Pro/Ser-derivatives; Cyclodextrin	Difference of abundance ratio between daughter ion to parent ion of non-enantiomers	[18-21]
Indirect method	Chiral derivatization method	LC or CE	OPA; FLEC; FITC; FMOC-Cl; DABS-Cl; FDAA	None	Difference in retention time of non- enantiomers in the non-chiral column	[22-27]
	Non-chiral derivatization method	GC	Alkyl Methylsilane; Fluorinated alcohols; Chloroformate; Fluorinated anhydride	None	Difference in retention time of enantiomers in the chiral column	[28-37]

Method	Classification	Analytical technique	Derivative reagent	Chiral additive	D/L Recognition	Reference
Direct method	Chiral stationary phase method	LC or CE	None	None	Difference in retention time of enantiomers in the chiral column	[12, 14, 15]
	Chiral mobile phase method	LC or CE	None	Cyclodextrin	Difference in retention time of non- enantiomers in the non-chiral column	[16, 17]
	Chiral spectroscopy method	MS/MS	None	Cu (Ⅱ)-L-Tyr; Zn (Ⅱ)-L-Trp; Phe/Pro/Ser-derivatives; Cyclodextrin	Difference of abundance ratio between daughter ion to parent ion of non-enantiomers	[18-21]
Indirect method	Chiral derivatization method	LC or CE	OPA; FLEC; FITC; FMOC-Cl; DABS-Cl; FDAA	None	Difference in retention time of non- enantiomers in the non-chiral column	[22-27]
	Non-chiral derivatization method	GC	Alkyl Methylsilane; Fluorinated alcohols; Chloroformate; Fluorinated anhydride	None	Difference in retention time of enantiomers in the chiral column	[28-37]

基于质谱技术的手性氨基酸分析以控制消旋肽杂质的研究进展

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林洁虹 ¹^,² , 汪泓 ² , 邵泓 ² , 陈钢 ²^,^* , 林梅 ²^,^*

药学学报 | 综述 2019,54(11): 1958-1964

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药学学报 |综述 2019 , 54 (11) : 1958 -1964

基于质谱技术的手性氨基酸分析以控制消旋肽杂质的研究进展

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林洁虹¹^,², 汪泓², 邵泓², 陈钢²^,^*, 林梅²^,^*

作者信息

1.中国医药工业研究总院上海医药工业研究院, 上海 201203

2.上海市食品药品检验所, 上海 201203

通讯作者:

*陈钢, Tel: 86-21-50798175, E-mail: chengang@smda.gov.cn;

林梅, Tel: 86-21-50798202, E-mail: linmeish@163.com

Advances in research on mass spectrometry based chiral amino acid analysis for quality control of racemic peptide impurities

Jie-hong LIN¹^,², Hong WANG², Hong SHAO², Gang CHEN²^,^*, Mei LIN²^,^*

Affiliations

1. Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China

2. Shanghai Institute for Food and Drug Control, Shanghai 201203, China

出版时间: 2019-11-12 doi: 10.16438/j.0513-4870.2019-0335

文章导航

摘要

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手性氨基酸分析是一种灵敏、高效、经济的消旋肽杂质控制方法，尤其适用于氨基酸组成复杂的合成多肽药物。通过结合质谱检测的手性氨基酸分析可以获得消旋肽中非预期存在的氨基酸对映体组成，质谱技术还可以准确定位肽段中发生异构化的氨基酸手性中心，为消旋肽杂质的筛选，以及进一步实现微量消旋肽杂质的快速鉴定及定量奠定坚实基础，在控制合成多肽药物质量和以化学合成为基础的多肽药物设计研发中有重要作用。本文总结了多肽药物的水解方法；并对基于质谱技术的手性氨基酸分析的最新方法作简要综述，最后对合成多肽药物中消旋肽杂质的研究及控制方向进行了展望。

关键词

合成多肽药物 / 消旋肽杂质 / 多肽水解 / 手性氨基酸分析 / 质谱

Abstract

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Chiral amino acid analysis is a sensitive, efficient and economical method for controlling racemic peptide impurities, especially for synthetic polypeptide drugs with complex composition of amino acids. Unexpected amino acid enantiomers in racemic peptides can be measured by chiral amino acid analysis coupled with mass spectrometry. The position of amino acid isomerization in the peptide segment can be accurately mapped by mass spectrometry, which lays a solid foundation for screening of racemic peptide impurities and rapid identification or quantification of trace racemic peptide impurities. Combination of the two techniques is vital for quality control of the synthetic polypeptide drugs and for research of polypeptide drugs based on chemical synthesis. The strategies of peptide hydrolysis have been summarized in this review. The latest chiral amino acid analysis based on mass spectrometry is briefly reviewed. Based on our knowledge, we have pointed to the direction of research and control of racemic peptide impurities in synthetic polypeptide drugs.

Key words

synthetic polypeptide drug / racemic peptide impurity / peptides hydrolysis / chiral amino acids analysis / mass spectrometry

引用本文

林洁虹, 汪泓, 邵泓, 陈钢, 林梅. 基于质谱技术的手性氨基酸分析以控制消旋肽杂质的研究进展. 药学学报, 2019 , 54 (11) : 1958 -1964 . DOI: 10.16438/j.0513-4870.2019-0335

Jie-hong LIN, Hong WANG, Hong SHAO, Gang CHEN, Mei LIN. Advances in research on mass spectrometry based chiral amino acid analysis for quality control of racemic peptide impurities[J]. Acta Pharmaceutica Sinica, 2019 , 54 (11) : 1958 -1964 . DOI: 10.16438/j.0513-4870.2019-0335

正文

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多肽类药物因其生物活性高、特异性强、不良反应小等优势, 近年来被广泛用于肿瘤、代谢、心血管等疾病防治领域。多肽药物常用的制备方法有:化学合成、生物提取和基因重组技术。其中, 化学合成中的固相合成法因其操作简单、过程易控、制备周期短且设计性强等特点, 近年来备受关注, 也是目前最常用的多肽制备方法之一。但是该工艺易导致氨基酸的缺失、插入、氧化/还原、消旋和旋光异构等, 从而产生大量的有关物质, 增加了质量控制的难度。消旋肽是合成多肽中一类常见的杂质, 为多肽链中含一个或多个非预期手性构型的氨基酸残基所形成的肽链^[1]。对于由少则3个、多则几十个氨基酸组成的合成多肽药物来说, 用传统色谱方法可能需要合成2ⁿ-1个消旋肽来比对, 不仅工作量巨大且成本高昂。其次, 由于分子量和结构差异小, 消旋肽很难在保留时间上与主成分峰及其他杂质峰完全分离^[2]; 若其微量存在, 甚至容易被主成分峰掩蔽, 而无法检出。因此, 消旋肽杂质的控制相对于其他杂质更加困难。目前对消旋肽杂质的控制通常是将合成多肽完全水解成游离氨基酸, 再通过对不同构型的手性氨基酸混合物进行对映体分离并计算外消旋化的相对比例来间接进行测定。因此, 手性氨基酸的分离分析成为消旋肽杂质控制的关键技术。

基于质谱联用技术的手性氨基酸分析近年来发展迅速, 已成为合成和天然肽及蛋白质的基本分析方法, 还用于临床化学中的疾病诊断。1995年Goodlett等^[3]将手性氨基酸分析结合液相色谱-质谱法(liquid chromatography-mass spectrometry, LC-MS)方法成功用于人工合成胸腺五肽中小于0.1%消旋肽的鉴定, 使得人们看到了这一联用技术在合成多肽体系中鉴别微量消旋肽的可能性。对于序列更长的合成多肽来说, 该技术的优势更加明显。目前在药典中也将质谱技术应用于变构手性氨基酸的分析。以艾塞那肽为例, 其为胰高血糖素样肽-1的受体激动剂, 是一种人工合成的三十九肽。针对其消旋肽杂质控制, USP41规定使用氘代酸水解, 进行多步衍生化, 采用气相色谱-质谱法(gas chromatography-mass spectrometry, GC-MS)及对映异构体标记法对D-His和L-His的相对比例进行测定, 以控制含D-His消旋肽的含量。由于肽段仅含一个L-His位点, 因此通过限制D-His的相对比例不高于1.0%, 可以控制D-His¹消旋肽杂质含量。该方法快速、准确、灵敏, 且无需消旋肽对照品, 还能同时监测其他变构氨基酸存在的可能性。因此基于该联用技术的方法逐渐成为消旋肽杂质控制的分析模式之一。近年来, 随着氨基酸分析方法的成熟及新型质谱技术的出现, 可以从变构氨基酸的层面满足对合成多肽药物中消旋肽杂质控制的需求, 因此, 越来越多研究人员将该联用技术用于设计合成过程以及药品的质量控制阶段。

本文总结了多肽药物不同的水解方法, 且对基于质谱技术的手性氨基酸分析的最新方法作简要综述, 旨在为消旋肽杂质的深入研究与质量控制提供一些有益的参考。

1 多肽的水解方法

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为了获得多肽中手性氨基酸的组成信息, 需要先将多肽进行水解, 既要求多肽完全水解成氨基酸, 还要保证氨基酸的结构不受水解条件的干扰。常用的水解条件是110 ℃盐酸水解反应24 h, 可以使大部分多肽完全水解。然而个别氨基酸, 例如Trp和Cys^[4], 在高温酸性条件下十分不稳定。为了减少氨基酸被氧化破坏, 需加入保护剂^{[5, 6]}或采用其他的水解方法, 见表 1。甲磺酸(methanesulfonic acid, MSA)是一种不具氧化性的有机强酸, 已被用于含有Trp、Met或Cys的多肽水解, 但其主要缺点是不易挥发, 因此水解后MSA不能挥发除去。此外, MSA不能用于气相水解^[6], 类似的还有巯基乙磺酸和对甲苯磺酸等^{[5, 7]}。碱水解^{[5, 8]}虽然可用于回收在基本条件下稳定的Trp, 但也会破坏Ser、Thr、Arg和Cys, 还容易导致其他氨基酸发生外消旋。酶解法^{[8, 9]}的反应条件温和且无消旋作用, 但由于某些残基的蛋白酶具有较高的特异性, 为获得所有氨基酸残基, 样品应与几种蛋白酶一起培养, 反应时间冗长且商品酶的价格高昂。在保证完全水解的前提下, 通过缩短反应时间或降低温度等条件优化可以减少对氨基酸的破坏, 或者选择其他高效的加热方式, 如微波辅助水解或HCl蒸汽水解^{[6, 10]}, 加速水解过程从而减少氨基酸暴露于恶劣条件下的时间。这将减少敏感氨基酸的降解, 提高回收率, 缩短分析周期。针对不稳定氨基酸水解方法的研究已较为成熟, 美国药典和欧洲药典均有相关通则。除此之外, 手性氨基酸分析还要求考虑水解条件对原始手性纯度的影响。

自然界存在的氨基酸大多为L型。组成多肽的氨基酸是羧酸α位碳原子上的氢原子被氨基取代后的化合物, 作为氨基酸手性中心之一, α碳上的氢原子活性高, 容易在酸水解条件下发生氢原子交换, 使原始的L构型扭转成D型, 即氨基酸发生外消旋化。Frank等^[11]提出, 多肽中的氨基酸消旋化通常发生在水解初始阶段的肽链上和长时间暴露(通常24 h)于盐酸的游离氨基酸中, 且二者发生外消旋化的速率不同。通过将D-氨基酸百分比与水解时间的线性曲线外推到零的方法可以排除后者干扰。为了进一步区分多肽中氨基酸的原始手性和整个水解过程发生的外消旋化, 近年来的方法均采用氢同位素交换-质谱联用技术^{[2, 12]}。Miyamoto等^[13]采用DCl/D₂O对合成模型肽进行水解, 使用液相色谱/串联质谱分析氨基酸对映体, 并比较了原始D-Ala和水解生成D-Ala的质荷比, 由于后者的分子量增加了1, 通过高分辨质谱可以将二者区分开, 随后将其成功应用于黄体生成素释放激素(D-Phe^{2, 6}-LHRH)衍生物十肽的分析, 还观察到对于设计含有D-氨基酸的合成肽, 非天然氨基酸D-Phe也可能在水解中发生外消旋化生成L-Phe。因此, 氘代盐酸水解成为合成多肽中手性氨基酸分析的重要手段之一。

2 手性氨基酸的分析方法

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多肽经水解成氨基酸后, 需要采用合适的分析手段将其分离、灵敏检测及准确定量。手性氨基酸的分析方法有直接分析法和衍生化间接分析法, 其中, 衍生化法又分为手性衍生化和非手性衍生化。常用的手性氨基酸分析方法已有许多相关报道, 见表 2^{[12, 14-37]}。在此只介绍近几年应用较为广泛的GC-MS、LC-MS和毛细管电泳-质谱法(capillary electrophoresis-mass spectrometry, CE-MS), 包括较新的色谱柱、衍生试剂和仪器技术等。

2.1 GC-MS

GC-MS法分析手性氨基酸具有较高的分辨率和灵敏度, 常与手性毛细管柱联用。氨基酸为极性物质, 需要先对其进行非手性衍生化以提高挥发性。主要的衍生化方法有:酯化、酰胺化和硅烷化等。常见的衍生试剂有叔丁基二甲基甲硅烷^[28]、氯甲酸酯^[29]、脂肪醇、氟化醇^{[29, 30]}和氟化酸酐^[29-31]。GC-MS可以用较少的花费实现手性氨基酸高通量分析, 目前已有许多研究报道。Waldhier等^[31]分别在结合了GC-MS的两种手性毛细管柱(Chirasil-L-Val柱和RT-γDEXsa柱)上分析20种蛋白性氨基酸的3种氯甲酸酯和6种酸酐衍生产物, 得到D-氨基酸衍生物的检测限和定量限最低分别可达到3.2 nmol·L^-1和0.031 μmol·L^-1, 并将该方法用于直接测定人血清和尿中游离的L/D-氨基酸, 结果表明GC-MS方法受基质影响较小且精密度好。用GC-MS分析手性氨基酸的分离效果和灵敏度受到多因素影响, 包括衍生产物的稳定性、衍生试剂组合^[29-32]、色谱柱及质谱仪器类型等。Lorenzo等^[33]在采用异丙醇/五氟丙酸酐两步反应法对氨基酸进行衍生化时, 为提高Cys和Met的稳定性, 添加了丁基化羟基甲苯作为抗氧化剂, 随后通过GC-MS技术在Chirasil-L-Val柱上成功分离出14对D/L氨基酸衍生物。手性柱能够分离异构体氨基酸衍生物, 但价格昂贵。Luan等^[34]近期推出了一种不对称标记法, 可以选择性在氨基酸终端标记上不同的烷基, 有效提高结构识别的准确性, 结合正化学电离-GC-MS/MS技术就能用非手性毛细管柱(HP-5MS)将同分异构体(Ile/Leu、l-Ala/β-Ala)分开。与传统GC-MS相比, 多维气相色谱(multidimensional gas chromatography, MDGC)^{[35, 36]}在分辨率和灵敏度方面具有更大优势。Waldhier等^[37]通过实验发现, 利用γ-CD手性柱-GC-MS无法将D-Ile/L-Leu、L-Thr/L-Asp和L-Ser/D-Met分离, 随后结合手性柱(Rt-DEXsa)与极性柱(ZB-AAA), 建立二维气相色谱飞行时间质谱方法解决了该问题, 并在66 min内成功分离20个氨基酸的氯甲酸甲酯衍生物。由于第二根柱子有效提高了分离度, 将该方法应用于L-氨基酸中痕量存在的D-氨基酸测定, 可以实现基线分离且得到较低的定量限。

2.2 LC-MS

LC-MS可获得保留时间、分子量(质荷比)及含量的三维信息, 在多组分分析中具有独特的优势。LC-MS用于手性氨基酸分析主要分为直接分析法和基于手性衍生化的间接分析法。直接法指氨基酸无需衍生化, 直接使用手性柱进行分离, 避免了衍生化反应不可控的问题。Konya等^[14]建立了手性柱Crownpak CR-I和飞行时间质谱结合的直接分析方法, 在15 min内对18种蛋白性氨基酸(除Pro)实现了基线分离, 随后将该方法应用于食醋里痕量D-氨基酸的测定。直接法大大减少前处理步骤, 但手性柱成本高且易老化。另外, 手性柱的流动相多含不挥发性盐, 与质谱兼容性低, 这也限制了手性柱的条件优化。基于化学衍生化的方法能够一定程度改善化合物的分离度和灵敏度, 例如在色氨酸类似物的分离中, 使用Marfey试剂衍生物的间接法比采用替考拉宁手性柱分离的结果更佳^[22]。目前利用间接法分析手性氨基酸的应用广泛, 常用的衍生试剂有邻苯二甲醛(orthophthalaldehyde, OPA)、Nα-(2, 4-二硝基-5-氟苯基)-L-丙氨酰胺[Nα-(2, 4-dinitro-5-fluorophenyl)-L-alaninamide, FDAA]、乙酰葡萄糖异硫氰酸酯(glucopyranosyl isothiocyanate, GITC)^[23]、4-氟-7-硝基-2, 1, 3-苯并恶二唑(4-fluoro-7-nitro-2, 1, 3-benzoxadiazole, NBD-F)^[2]、4-二甲胺基苯基偶氮苯磺酰氯(4-N, N-dimethylaminoazobenzene-4'-sulfonyl chloride, DABS-Cl)^[4]、(S)-N-(4-硝基苯氧羰基)苯丙氨酸甲氧基乙酯[(S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester, S-NIFE]^[24]等。Mochizuki等^[25]利用一种新型三嗪型手性衍生化试剂DMT-(S)-Pro-OSu, 结合三重四级杆质谱(triple quadrupole-mass spectrometer, TQ-MS), 在非手性反相柱上对人唾液中的多种手性氨基酸进行高灵敏度和选择性检测。步骤繁琐且衍生产物不可控是间接法的主要缺陷, 因此开发自动化方法具有重大意义。OPA与手性硫醇的组合相对便宜、安全、省时且衍生化完全, 当与超高效液相色谱-四级杆-飞行时间质谱(ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, UPLC-Q-TOF-MS)联用时可以得到优异的分离效率、灵敏度和准确性^{[15, 26]}, 但其衍生产物稳定性差。Kühnreich等^[27]最近开发了一种自动衍生化的分析方法, 通过在自动取样针头内发生衍生化反应, 快速、准确地实现氨基酸定量。作为常用的氨基酸分析方法, 常有报道对GC-MS和LC-MS方法进行比较^[38], 前者制备时间较长(约6~8 h), 而后者仅花费60 min。另外, GC衍生化过程可能导致外消旋化。因此, 虽然二者都准确可靠, 但LC-MS具备更多优势, 比如无需手性柱、省时、灵敏, 更适用于痕量的氨基酸外消旋化分析。除了TQ-MS与Q-TOF-MS, UPLC/HPLC还能与轨道阱高分辨质谱(orbitrap MS)联用, 由于质谱的真空度更高, 且增加了具有离子聚集作用的弯曲线性离子肼部分, 从而可以得到更高的精密度和灵敏度。

2.3 CE-MS

CE-MS具有CE分离效率高、样品和试剂消耗量少等优点, MS高灵敏度和强大的结构解析功能正好可以弥补前者因上样量低引起的部分成分难以检测的缺陷, 现已成为氨基酸、多肽、蛋白质和多糖等分析的重要手段之一。Sánchez-Hernández等^[16]开发了两种分别以环糊精β-CD和γ-CD作为手性选择剂的CE-ESI-MS²方法, 实现了13种蛋白质氨基酸和鸟氨酸的手性分离和鉴定, 经过方法优化获得LOD在0.02~0.8 μmol·L^-1内, 有望用于检测小于1%的对映体杂质。为了更好地用于微量手性氨基酸分析, 需要解决CE-MS方法兼容性的问题。CE-MS联用要求前者选择的背景缓冲液、添加剂^[17]以及表面活性剂等易离子化, 且要求毛细管壁的涂层足够稳定^[39], 避免因其脱落而污染离子源。与LC-MS相似, 衍生化处理不仅可以改善CE-MS对手性氨基酸的分离效率, 还可以提高电离效率和降低MS背景噪声。常用的CE衍生化试剂有异硫氰酸荧光素(fluorescein isothiocyanate, FITC)^{[17, 39]}、丹磺酰氯和芴甲氧羰酰氯(9-fluorenylmethyl chloroformate, FMOC-Cl)^[40]。FITC和丹磺酰氯的主要缺点是衍生化反应通常需要数小时, 相反FMOC衍生化反应一般只需10 min。另外, 微芯片CE-MS (microchip electrophoresis-mass spectrometry, MCE-MS)是目前的研究热点之一, 具有分析耗时短^[41], 灵敏度高等优势。Li研究小组^[42]利用MCE-nano ESI-MS, 通过向辅助液添加1.5 mmol·L^-1的NBD-F实现了氨基酸混合物中Ser、Ala的在线柱后衍生化, 使其灵敏度提高了约50倍, 随后将优化过的方法应用于孵育后的细胞内外Ser对映体定量, 从而发现PC-12神经细胞对L/D-Ser的摄取规律。

3 消旋肽的序列测定

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即使获得了微量存在的D-氨基酸含量, 仍无法确定其在多肽上发生外消旋化的位点, 需要多肽组学^[43]方法提供复杂多肽及其杂质信息。消旋肽的序列测定方法特异性强且准确度高, 对质谱仪器的要求较高。消旋肽序列测定的质谱方法主要分为三类联用技术, 分别是合成肽段对照-LC/MS技术^{[44, 45]}、自由基导向解离(radical directed dissociation, RDD)/碰撞诱导(collision induced dissociation, CID)技术^{[46, 47]}和离子淌度质谱(ion mobility mass spectrometry, IMMS)技术^{[48, 49]}。其中, IMMS是一种新型的二维分离质谱技术。与LC-Q-TOF仪器相比, LC-Q-IM-TOF的三个分析维度(保留时间、质荷比、漂移时间)相结合可以更好地、特异性地鉴定目标化合物, 同时排除共洗脱异构体的干扰或具有相同质荷比的基质离子, 并提高信噪比以提高定量的灵敏度。

4 结语

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基于该联用技术的方法逐渐成为消旋肽杂质控制的分析模式之一。通过结合质谱检测的手性氨基酸分析可以获得消旋肽中非预期存在的氨基酸对映体组成, 利用质谱技术定位肽段中发生异构化的氨基酸手性中心, 筛选出必要的消旋肽杂质对照品, 实现微量消旋肽杂质的快速鉴定及定量。本文对手性氨基酸的分析策略进行探讨, 结合多肽不同的水解方法, 综述了目前研究中所采用质谱技术进行手性氨基酸分析的特点。GC-MS具有高通量分析手性氨基酸的特点, 灵敏度较高, MDGC在分辨率上具有更大优势, 但均需结合价格昂贵的手性毛细管柱, 且非手性衍生化操作繁琐, 可能发生外消旋化; LC-MS手性衍生化方法在手性氨基酸分析中应用广泛, 无需手性柱、省时、灵敏, 更适用于痕量的氨基酸外消旋化分析; CE-MS和MCE-MS作为相对较新的技术, 也开始被应用于氨基酸及多肽的分析, 具有高分辨率、高灵敏度、快速高效、绿色环保等优点, 但也面临系统不兼容的问题, 限制了分离度与灵敏度进一步提升。

随着合成多肽药物在疾病防治领域日益瞩目, 其质量控制远不止目前药典中列出的质量控制项目, 还要求对有关物质开发精准的、有专属性的分析方法, 并结合杂质谱对具体有关物质进行合理的限度设置。结合现代质谱技术的手性氨基酸分析为杂质谱的完善奠定了基础。因此, 减少水解过程中氨基酸破坏和外消旋化的干扰、提高分离效率和实现灵敏准确的测定将成为多肽药物中氨基酸对映体分析方法的发展方向。另外, 开发并优化分析方法, 不仅服务于药品商品阶段的质量控制, 还有利于指导药物处方及合成工艺的设计, 帮助药物开发者充分理解和实施“质量源于设计”的理念, 对治疗用多肽药物的安全性与有效性具有重大意义。

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国家自然科学青年基金资助项目(81803422)

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2019年第54卷第11期

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doi: 10.16438/j.0513-4870.2019-0335

接收时间：2019-04-28
首发时间：2026-01-26
出版时间：2019-11-12

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收稿日期：2019-04-28
修回日期：2019-07-01

基金

国家自然科学青年基金资助项目(81803422)

作者信息

1.中国医药工业研究总院上海医药工业研究院, 上海 201203

2.上海市食品药品检验所, 上海 201203

通讯作者:

*陈钢, Tel: 86-21-50798175, E-mail: chengang@smda.gov.cn;

林梅, Tel: 86-21-50798202, E-mail: linmeish@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Hydrolysis method	Hydrolysis reagent	Amino acid	Reference
Antioxidants and additives	Ethyl mercaptan; Mercaptoacetic acid; Phenol; Tryptamine	His; Met; Cys; Tyr; Trp	[5, 6]
Other acid hydrolysis	MSA; MESA; TSA	Trp; Met; Cys	[5, 7]
Alkaline hydrolysis	NaOH; LiOH; KOH; Ba (OH)₂	Trp (Not available for Ser; Thr; Arg; Cys)	[5, 8]
Enzymatic hydrolysis	Animal/plant/microorganism-derived proteases; compound enzyme	All AA	[8, 9]
Microwave hydrolysis	HCl; TFA	All AA	[6, 10]
Vapor-phase hydrolysis	HCl	All AA	[6, 10]

Hydrolysis method

Hydrolysis reagent

Amino acid

Reference

Antioxidants and additives

Ethyl mercaptan; Mercaptoacetic acid; Phenol; Tryptamine

His; Met; Cys; Tyr; Trp

[5, 6]

Other acid hydrolysis

MSA; MESA; TSA

Trp; Met; Cys

[5, 7]

Alkaline hydrolysis

NaOH; LiOH; KOH; Ba (OH)₂

Trp (Not available for Ser; Thr; Arg; Cys)

[5, 8]

Enzymatic hydrolysis

Animal/plant/microorganism-derived proteases; compound enzyme

All AA

[8, 9]

Microwave hydrolysis

HCl; TFA

All AA

[6, 10]

Vapor-phase hydrolysis

HCl

All AA

[6, 10]

Method	Classification	Analytical technique	Derivative reagent	Chiral additive	D/L Recognition	Reference
Direct method	Chiral stationary phase method	LC or CE	None	None	Difference in retention time of enantiomers in the chiral column	[12, 14, 15]
	Chiral mobile phase method	LC or CE	None	Cyclodextrin	Difference in retention time of non- enantiomers in the non-chiral column	[16, 17]
	Chiral spectroscopy method	MS/MS	None	Cu (Ⅱ)-L-Tyr; Zn (Ⅱ)-L-Trp; Phe/Pro/Ser-derivatives; Cyclodextrin	Difference of abundance ratio between daughter ion to parent ion of non-enantiomers	[18-21]
Indirect method	Chiral derivatization method	LC or CE	OPA; FLEC; FITC; FMOC-Cl; DABS-Cl; FDAA	None	Difference in retention time of non- enantiomers in the non-chiral column	[22-27]
	Non-chiral derivatization method	GC	Alkyl Methylsilane; Fluorinated alcohols; Chloroformate; Fluorinated anhydride	None	Difference in retention time of enantiomers in the chiral column	[28-37]

Method

Classification

Analytical technique

Derivative reagent

Chiral additive

D/L Recognition

Reference

Direct method

Chiral stationary phase method

LC or CE

None

Difference in retention time of enantiomers in the chiral column

[12, 14, 15]

Chiral mobile phase method

LC or CE

None

Cyclodextrin

Difference in retention time of non- enantiomers in the non-chiral column

[16, 17]

Chiral spectroscopy method

MS/MS

None

Cu (Ⅱ)-L-Tyr; Zn (Ⅱ)-L-Trp; Phe/Pro/Ser-derivatives; Cyclodextrin

Difference of abundance ratio between daughter ion to parent ion of non-enantiomers

[18-21]

Indirect method

Chiral derivatization method

LC or CE

OPA; FLEC; FITC; FMOC-Cl; DABS-Cl; FDAA

None

Difference in retention time of non- enantiomers in the non-chiral column

[22-27]

Non-chiral derivatization method

Alkyl Methylsilane; Fluorinated alcohols; Chloroformate; Fluorinated anhydride

None

Difference in retention time of enantiomers in the chiral column

[28-37]