Analysis of L- and D-Amino Acids Using UPLC

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Journal of Bacteriology
Nov 2013



With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in crustaceans and mollusks. In this protocol, we describe a method for analysis of L- and D-amino acids using ultra-performance liquid chromatography (UPLC). To analyze D- and L-amino acids, the enantiomers are initially converted into diastereomers (diastereomers are stereoisomers that are not related as object and mirror image and are not enantiomers) using pre-column derivatization with o-phthaldialdehyde plus N-acylated cysteine (N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine). The resultant derivatives are fluorescent diastereomers. This is followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using UPLC, and the fluorescence is detected by a fluorescence detector included in UPLC system. Using this method, 16 kinds of D-amino acid can be analyzed.

Materials and Reagents

  1. N-Acetyl-L-cysteine (NAC) (Wako Pure Chemical Industries, catalog number: 017-05131 )
  2. N-tert-butyloxycarbonyl-L-cysteine (NBC) (Funakoshi, catalog number: 04-621 )
  3. o-Phthaldialdehyde (OPA) (Nacalai Tesque, catalog number: 27824-74 )
  4. Methanol (Wako Pure Chemical Industries, catalog number: 132-06471 )
  5. Acetonitrile (Wako Pure Chemical Industries, catalog number: 015-08633 )
  6. Trichloroacetic acid (Wako Pure Chemical Industries, catalog number: 203-04952 )
  7. Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 192-15985 )
  8. 0.4 M sodium borate buffer (see Recipes)
    1. Boric acid (Wako Pure Chemical Industries, catalog number: 021-02195 )
    2. Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 195-13775 )
  9. 50 mM sodium acetate buffer (see Recipes)
    1. Acetic acid (Wako Pure Chemical Industries, catalog number: 192-01075 )
    2. Sodium acetate (Wako Pure Chemical Industries, catalog number: 192-01075)
  10. 50% TCA (see Recipes)
  11. 1 M NaOH (see Recipes)


  1. ACQUITY UPLC-FLR system (Waters)
    1. AccQ-Tag Ultra 2.1 x 100 mm column (Waters, catalog number: 186003837 )
    2. ACQUITY binary solvent manager
    3. ACQUITY sample manager
    4. ACQUITY FLR detector
    5. Sample vial: 12 x 32 mm glass screw neck vial, Quick Thread, LectraBond cap, PTFE/silicone septa, Total Recovery (Waters, catalog number: 186000384c )
    6. Empower 2 software
  2. Vortex (Taitec, catalog number: 0061271-000 )
  3. Polytetrafluoroethylene membrane filters (4-mm diameter, 0.2-μm pore size) (Merck KGaA, catalog number: SLLGH04NL )
  4. Amicon Ulta 0.5 ml centrifugal filter 3 K device (Merck KGaA, catalog number: UFC500396 )


  1. Empower 2 software


  1. D, L-Amino acids are bound with OPA, and NAC (Aswad, 1984) or NBC (Hashimoto et al., 1992) using two methanolic solutions (A and B) containing the derivatizing reagents (see Notes 1-3). This derivatization results in fluorescent diastereomers. Methanolic solution A is prepared by dissolving 8 mg of OPA and 10 mg of NAC in 1 ml of methanol, while methanolic solution B is prepared by dissolving 10 mg of OPA and 10 mg of NBC in 1 ml of methanol. These methanolic solution A and B are not light sensitive.
  2. The reaction mixture (250 μl) for the derivatization contains 25 μl of amino acid sample, 50 μl of methanolic solution (A or B) and 175 μl of 0.4 M sodium borate buffer (pH 10.4) in a sample vial. Vortex the reaction mixture. To choose the correct methanolic solution for your sample, see Notes 2 and 3.
  3. After fluorescence derivatization for 2 min at room temperature in the dark, set the sample vial on vial holder in ACQUITY sample manager of UPLC system. Introduction of an aliquot (1 μl) of the reaction mixture into a UPLC system is performed automatically. Because the fluorescence derivatives are unstable, the introduction of an aliquot into a UPLC system should be performed immediately after the incubation.
  4. The system is operated at a flow rate of 0.25 ml/min at 30 °C. The UPLC gradient system for analysis of OPA-NAC derivatives [A = 50 mM sodium acetate (pH 5.9) and B = methanol] is 10-20% B for 3.2 min, 20% B for 1 min, 20-40% B for 3.6 min, 40% B for 1.2 min, 40-60% B for 3.8 min, 60% B for 1 min and 60-10% B for 0.01 min, and then the system is equilibrated with 90% A and 10% B for 4.19 min. About 4.0 ml of A and about 2.7 ml of B are needed for the analysis of one sample. The gradient system for analysis of OPA-NBC derivatives [A = 50 mM sodium acetate (pH 5.9) and B = acetonitrile] is 15-21% B for 7 min, 21-27.5% B for 1.5 min, 27.5% B for 2 min, 27.5-30% B for 1 min, 30-40% B for 2 min, 40% B for 0.5 min and 40-15% B for 0.01 min, and then the system is equilibrated with 85% A and 15% B for 3.99 min. About 3.8 ml of A and about 1.8 ml of B are needed for the analysis of one sample. The excitation and emission wavelengths for fluorescent detection of the diastereoisomeric amino acid derivatives are 350 nm and 450 nm, respectively. Before the first analysis, the system is pre-equilibrated with the equilibration condition described above for 16 min.
  5. The data is processed using Empower 2 that is control and analysis software for a UPLC system. The peak heights and retention times are used for amino acid quantification and identification, respectively (see Note 4). In the case of analysis for biological sample, identification of amino acid is performed based on retention times of amino acids provided from analysis of a control amino acid sample run alongside.
  6. The standard solution for calibration curves consists of 4 mM 32 kinds of amino acids: D- and L-forms of aspartate (Asp), glutamtamate (Glu), serine (Ser), alanine (Ala), leucine (Leu), phenylalanine (Phe), valine (Val), methionine (Met), tryptophan (Trp), arginine (Arg), histidine (His), threonine (Thr), asparagine (Asn), tyrosine (Tyr) and glutamine (Gln), plus L-isoleucine (L-Ile) and D-allo-isoleucine (D-allo-Ile). The standard solution was diluted to 7 concentrations (5, 25, 50, 100, 250, 500 and 1,000 μM) in 0.05 M HCl, after which the solutions were filtered through polytetrafluoroethylene membrane filters (4-mm diameter, 0.2-μm pore size). Based on chromatograms provided from UPLC analysis of each concentration of the standard solution, calibration curves of D or L-amino acid are calculated plotting peak height versus content of amino acid. The calculation of equation of a calibration curve and quantification of amino acid in your sample are performed using Empower 2 software.

Representative data

Instability of the column temperature negatively affects reproducibility of separation of two neighboring peaks (for example, peaks of D-histidine and L-histidine in the case of OPA-NAC derivatization). We recommend that you keep the column temperature at 30 °C accurately.


  1. In this method, amino groups in compounds are targets for diastereomeric and fluorescent derivatization, and not only amino acids but also proteins are derivatized with the derivatizing reagents. The derivatized fluorescent proteins cause contaminated peaks, and prevent identification and quantification of amino acids. Therefore, proteins in a sample should be eliminated by filtration using an Amicon Ulta 0.5 ml centrifugal filter 3 K device or trichloroacetic acid (TCA) treatment.
  2. TCA treatment is performed as described below. Add 125 μl of 50% TCA (see Recipes) to the 500 μl of a sample solution such as biological sample and food sample. After incubation for 5 min at room temperature, the mixture is centrifuged (10,000 x g for 15 min at 4 °C), and an aliquot (500 μl) of the supernatant is neutralized by addition of 300 μl of 1 M NaOH (see Recipes). When samples after TCA treatment are used for UPLC analysis, filter the sample solution through polytetrafluoroethylene membrane filters (4-mm diameter, 0.2-μm pore size).
  3. Using OPA-NAC derivatization, D- and L-forms of Asp, Ser, Gln, His, Arg, Ala, Trp, Val, Met, Tyr, Phe, Leu, D-Thr, D-allo-Ile and L-Ile can be analyzed at one time (Figure 1A).
  4. Using OPA-NBC derivatization, D- and L-forms of Glu, Asn, Asp, Ser, Gln, Ala, Tyr, Val, Met, Phe, D-Thr, D-Arg, D-Leu, L-His, L-Ile and L-Trp can be analyzed at one time (Figure 1B).
  5. In analyses using OPA-NAC and OPA-NBC, when calibration curves were calculated plotting peak height versus content of amino acid (from 0.5 to 100 pmol), the linearity was observed in all cases with regression coefficients above 0.998.

    Figure 1. Chromatograms of NAC-OPA A and NBC-OPA B derivatives of free amino acids. A. Each peak corresponds to 100 pmol of amino acid. B. Each peak corresponds to 20 pmol of amino acid.


  1. 0.4 M sodium borate buffer (pH 10.4, 100 ml)
    Mix 2.47 g of boric acid with 80 ml of ultra pure water
    pH to 10.4 with sodium hydroxide
    Add ultra pure water to 100 ml
    Filtration (0.22 μm)
    Stored at room temperature
  2. 50 mM sodium acetate buffer (pH 5.9, 1 L)
    Mix 4.1 g of sodium acetate with 900 ml of ultra pure water
    pH to 5.9 with acetic acid.
    Add ultra pure water to 1 L
    Filtration (0.22 μm)
    Stored at room temperature
  3. 50% TCA
    Mix 25 g of TCA with 30 ml of ultra pure water
    Add ultra pure water to 50 ml
    Stored at 4 °C
  4. 1 M NaOH
    Mix 4.0 g of sodium hydroxide with 80 ml of ultra pure water
    Add ultra pure water to 100 ml
    Stored at room temperature


In this method, we have modified Aswad’s and Hashimoto’s methods for the derivatization of amino acids using OPA and chiral thiols (NAC and NBC). In addition, this work was supported by a grant for Promotion of Basic Research Activities for Innovate Bioscience from the Bio-oriented Technology Research Advancement Institution (BRAIN) and JSPS KAKENHI Grant Number 2402734.


  1.  Aswad, D. W. (1984). Determination of D- and L-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde. Anal Biochem 137(2): 405-409.
  2.  Hashimoto, A., Nishikawa, T., Oka, T., Takahashi, K. and Hayashi, T. (1992). Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatogr 582(1-2): 41-48.


除了甘氨酸,α-氨基酸具有光学活性,并且可以形成每个氨基酸的两种光学异构体(L-和D-)。分析技术的最新发展表明,几种游离D-氨基酸如D-天冬氨酸,D-丝氨酸和D-丙氨酸存在于包括人在内的许多种生物中,并且具有生物学上的重要作用。 D-天冬氨酸调节动物和人的生殖活性。 D-丝氨酸作为Nε-甲基-D-天冬氨酸受体的共激动剂,其介导谷氨酸能神经传递。 D-丙氨酸在甲壳类动物和软体动物中起到类似渗透物的作用。在该协议中,我们描述了使用超高效液相色谱(UPLC)分析L-和D-氨基酸的方法。为了分析D-和L-氨基酸,首先将对映异构体转化为非对映异构体(非对映异构体是与物镜和镜像无关的立体异构体,并且不是对映异构体),使用前柱衍生化,邻苯二甲醛加上Nε - 酰化的半胱氨酸(Nε - 乙酰基-L-半胱氨酸或Nε - 叔丁基氧羰基-L 。所得衍生物是荧光非对映异构体。然后使用UPLC在十八烷基硅烷基固定相上分离所得荧光异吲哚衍生物,并通过包括在UPLC系统中的荧光检测器检测荧光。使用该方法,可以分析16种D-氨基酸。


  1. N-乙酰基-L-半胱氨酸(NAC)(Wako Pure Chemical Industries,目录号:017-05131)
  2. (Nak)(Funakoshi,目录号:04-621)。
  3. o - 酞醛(OPA)(Nacalai Tesque,目录号:27824-74)
  4. 甲醇(Wako Pure Chemical Industries,目录号:132-06471)
  5. 乙腈(Wako Pure Chemical Industries,目录号:015-08633)
  6. 三氯乙酸(Wako Pure Chemical Industries,目录号:203-04952)
  7. 氢氧化钠(Wako Pure Chemical Industries,目录号:192-15985)
  8. 0.4 M硼酸钠缓冲液(见配方)
    1. 硼酸(Wako Pure Chemical Industries,目录号:021-02195)
    2. 氢氧化钠(Wako Pure Chemical Industries,目录号:195-13775)
  9. 50 mM乙酸钠缓冲液(见配方)
    1. 乙酸(Wako Pure Chemical Industries,目录号:192-01075)
    2. 醋酸钠(Wako Pure Chemical Industries,目录号:192-01075)
  10. 50%TCA(参见配方)
  11. 1M NaOH(见配方)


  1. ACQUITY UPLC-FLR系统(Waters)
    1. AccQ-Tag Ultra 2.1×100mm柱(Waters,目录号:186003837)
    2. ACQUITY二进制溶剂管理器
    3. ACQUITY示例管理器
    4. ACQUITY FLR检测器
    5. 样品瓶:12 x 32 mm玻璃螺丝颈瓶,Quick Thread,LectraBond盖,PTFE /硅胶隔片,Total Recovery(Waters,目录号:186000384c)
    6. Empower 2软件
  2. Vortex(Taitec,目录号:0061271-000)
  3. 聚四氟乙烯膜过滤器(4mm直径,0.2μm孔径)(Merck KGaA,目录号:SLLGH04NL)
  4. Amicon Ulta 0.5ml离心过滤器3K装置(Merck KGaA,目录号:UFC500396)


  1. Empower 2软件


  1. 使用含有衍生化试剂的两种甲醇溶液(A和B)使D,L-氨基酸与OPA和NAC(Aswad,1984)或NBC(Hashimoto等人,1992)注1-3)。这种衍生化导致荧光非对映异构体。甲醇溶液A通过将8mg的OPA和10mg的NAC溶解在1ml的甲醇中制备,而甲醇溶液B通过将10mg的OPA和10mg的NBC溶解在1ml的甲醇中制备。这些甲醇溶液A和B对光不敏感
  2. 用于衍生化的反应混合物(250μl)在样品瓶中含有25μl氨基酸样品,50μl甲醇溶液(A或B)和175μl0.4M硼酸钠缓冲液(pH 10.4)。涡旋反应混合物。要为样品选择正确的甲醇溶液,请参见注释2和3
  3. 在室温下在黑暗中荧光衍化2分钟后,将样品瓶放在UPLC系统的ACQUITY样品管理器的小瓶支架上。将反应混合物的等分试样(1μl)引入a UPLC系统自动执行。因为荧光衍生物不稳定,所以将等分试样引入UPLC系统应当在孵育后立即进行。
  4. 该系统在30℃下以0.25ml/min的流速操作。用于分析OPA-NAC衍生物[A = 50mM乙酸钠(pH5.9)和B =甲醇]的UPLC梯度系统为10-20%B,3.2分钟,20%B,1分钟,20-40% 3.6分钟,40%B持续1.2分钟,40-60%B持续3.8分钟,60%B持续1分钟,60-10%B持续0.01分钟,然后系统用90%A和10%B平衡4.19分钟。需要约4.0ml的A和约2.7ml的B用于一个样品的分析。用于分析OPA-NBC衍生物[A = 50mM乙酸钠(pH5.9)和B =乙腈]的梯度系统为15-21%B 7分钟,21-27.5%B 1.5分钟,27.5%B 2分钟min,27.5-30%B持续1分钟,30-40%B持续2分钟,40%B持续0.5分钟,40-15%B持续0.01分钟,然后系统用85%A和15%B 3.99分钟。需要约3.8ml的A和约1.8ml的B用于一个样品的分析。用于非对映异构体氨基酸衍生物的荧光检测的激发和发射波长分别为350nm和450nm。在第一次分析之前,系统用上述平衡条件预平衡16分钟。
  5. 使用Empower 2处理数据,所述Empower 2是用于UPLC系统的控制和分析软件。峰高和保留时间分别用于氨基酸的定量和鉴定(见注4)。在分析生物样品的情况下,基于从对照氨基酸样品的分析提供的氨基酸的保留时间来进行氨基酸的鉴定。
  6. 标准曲线的标准溶液由4mM 32种氨基酸组成:天冬氨酸(Asp),谷氨酸(Glu),丝氨酸(Ser),丙氨酸(Ala),亮氨酸(Leu),苯丙氨酸Phe,缬氨酸,Val,蛋氨酸Met,色氨酸Trp,精氨酸Arg,组氨酸His,苏氨酸Thr,天冬酰胺Asn,酪氨酸Tyr和谷氨酰胺Gln。 - 异亮氨酸(L-Ile)和D-异亮氨酸 - 异亮氨酸(D-allo-Ile)。将标准溶液在0.05M HCl中稀释至7个浓度(5,25,50,100,250,500和1,000μM),之后将溶液通过聚四氟乙烯膜过滤器(直径4mm,孔径0.2μm)过滤, )。基于从每种浓度的标准溶液的UPLC分析提供的色谱图,计算D或L-氨基酸的校准曲线,绘制峰高对氨基酸含量的曲线。 a的方程的计算 校准曲线和样品中氨基酸的定量使用Empower 2软件进行




  1. 在该方法中,化合物中的氨基是非对映异构体和荧光衍生化的目标,并且不仅氨基酸而且蛋白质用衍生化试剂衍生化。衍生的荧光蛋白引起污染的峰,并防止氨基酸的鉴定和定量。因此,应使用Amicon Ulta 0.5ml离心过滤器3K装置或三氯乙酸(TCA)处理通过过滤来消除样品中的蛋白质。
  2. 如下所述进行TCA处理。向500μl的样品溶液如生物样品和食品样品中加入125μl的50%TCA(参见Recipes)。在室温下温育5分钟后,将混合物离心(10,000×g,在4℃下15分钟),并且通过加入300μl的上清液中和等分试样(500μl)的上清液1M NaOH(参见配方)。当使用TCA处理后的样品进行UPLC分析时,通过聚四氟乙烯膜过滤器(直径4mm,孔径0.2μm)过滤样品溶液。
  3. 使用OPA-NAC衍生化,将Asp,Ser,Gln,His,Arg,Ala,Trp,Val,Met,Tyr,Phe,Leu,D-Thr,D-allo - 可以一次分析Ile和L-Ile(图1A)
  4. 使用OPA-NBC衍生化,Glu,Asn,Asp,Ser,Gln,Ala,Tyr,Val,Met,Phe,D-Thr,D-Arg,D-Leu,L-His,L - 可以一次分析Ile和L-Trp(图1B)
  5. 在使用OPA-NAC和OPA-NBC的分析中,当计算峰高相对于氨基酸含量(0.5至100pmol)的标准曲线时,在所有情况下观察到具有回归系数的线性0.998。

    图1.游离氨基酸的NAC-OPA A和NBC-OPA B衍生物的色谱图。A.每个峰对应于100pmol的氨基酸。 B.每个峰对应于20pmol的氨基酸


  1. 0.4M硼酸钠缓冲液(pH 10.4,100ml) 将2.47g硼酸与80ml超纯水混合 用氢氧化钠将pH调至10.4 将超纯水加至100 ml
  2. 50mM乙酸钠缓冲液(pH5.9,1L) 将4.1g乙酸钠与900ml超纯水混合 用乙酸将pH调至5.9 将超纯水加入1 L
  3. 50%TCA
    将25g TCA与30ml超纯水混合
    将超纯水加入到50ml ml / 储存在4°C
  4. 1 M NaOH
    将4.0g氢氧化钠与80ml超纯水混合 将超纯水加至100 ml


在这种方法中,我们已经修改了Aswad和Hashimoto的使用OPA和手性硫醇(NAC和NBC)衍生化氨基酸的方法。 此外,这项工作是由一个基础研究促进基金支持 生物技术研究推进机构(BRAIN)和JSPS KAKENHI编号2402734的创新生物科学活动。


  1.   Aswad,D. W.(1984)。 在衍生化后,通过高效液相色谱法测定氨基酸混合物中的D-和L-天冬氨酸 一种手性加合物 - 邻苯二醛。 Anal Biochem 137(2):405-409。
  2. & Hashimoto,A.,Nishikawa,T.,Oka,T.,Takahashi,K.and Hayashi,T。(1992)。 通过高效液相色谱法测定大鼠脑和血清中的游离氨基酸对映异构体, H] - 叔丁氧基羰基-L-半胱氨酸和β-巯基乙醛。 58 Chromatogr 582(1-2):41- 48.
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引用:Mutaguchi, Y. and Ohshima, T. (2014). Analysis of L- and D-Amino Acids Using UPLC. Bio-protocol 4(17): e1231. DOI: 10.21769/BioProtoc.1231.