Chemical derivatization strategies for enhancing the HPLC analytical performance of natural active triterpenoids

Triterpenoids widely exist in nature,displaying a variety of pharmacological activities.Determining triterpenoids in different matrices,especially in biological samples holds great significance.High-performance liquid chromatography(HPLC)has become the predominant method for triterpenoids analysis due to its exceptional analytical performance.However,due to the structural similarities among botanical samples,achieving effective separation of each triterpenoid proves challenging,necessitating significant improvements in analytical methods.Additionally,triterpenoids are characterized by a lack of ultraviolet(UV)absorption groups and chromophores,along with low ionization efficiency in mass spectrometry.Consequently,routine HPLC analysis suffers from poor sensitivity.Chemical derivatization emerges as an indispensable technique in HPLC analysis to enhance its performance.Considering the structural characteristics of triterpenoids,various derivatization reagents such as acid chlorides,rhodamines,isocyanates,sulfonic esters,and amines have been employed for the derivatization analysis of triterpenoids.This review comprehensively summarized the research progress made in derivatization strategies for HPLC detection of triterpenoids.Moreover,the limitations and challenges encountered in previous studies are discussed,and future research directions are proposed to develop more effective derivatization methods.

Analysis of Monosaccharide Composition of Pu-erh Tea Polysaccaride by Pre-column Derivatization HPLC

[Objective] This study aimed to establish a pre-column derivatization HPLC method for the identification and analysis of monosaccharide composition of Pu-erh tea polysaccharide. [Method] Pu-erh tea polysaccharide was extracted using the wa- ter extraction method, further isolated and purified by DEAE cellulose-52 columns. The obtained tea polysaccharide and four components TPS1, TPS2, TPS3 and TPS, were first derived by 1-phenyl-3-methyl-5-pyrazolone （PMP）, and then the PMP derivatives of monosaccharide were analyzed by high performance liquid chromatog- raphy （HPLC）. [Result] Pu-erh tea polysaccharide contained eight kinds of monosac- chaddes （mannose, rhamnose, glucuronic acid, galacturonic acid, grucose, galactose, arabinose, fucose）, without xylose; so it was the same with TPS1; each of TPS2, TPS3 and TPS4 contained seven monosaccharides, while no fucose. [Conclusion] This method is simplified and rapid, which can be used to determine the monosac- charide composition of Pu-erh tea polysaccharide and monosaccharide content.

A Pre-column Derivatization HPLC Method for the De-termination of Peimine and Peiminine in Bulbus Fritillar-iae

A pre-column derivatization HPLC method for the determination of peimine(P)and peiminine(PE)in Bulbus Fritillariae has been developed. Under the derivatization conditions optimized,the calibration curve is Y1=-3.83×103+1.33 ×105X1,r=0.998 for P and Y2=-7.86 × 102+6.33 × 104X2,r=0995 for PE,where Y is the peak area and X is the weight of the alkaloid; the average recovery is 98.0%(n=5, RSD=2.1%) for P and 101.0%(n=5,RSD=4.1%)for PE,the linear range is from 0.504 μg to 3.126μg for P and from 0.520μgto 3.328μgfor PE,respectively. Results of the determination of the two alkaloids in several samples of different Fritillaria species from various parts ofthe country are presented.The results suggest that P and PE are two major chemical constituents in bulbs of different Fritillaria species,and that the method developed is generally appli-cable to the determination of the hydroxy group on aliphatic fused ring systems without steric hin-drance.

Determination of 18 Kinds of Amino Acids in Fresh Tea Leaves by HPLC Coupled with Pre-column Derivatization

A rapid and accurate quantitative method of high performance liquid chromatography( HPLC) with fluorescence detector has been developed for the analysis of 18 kinds of amino acids in fresh tea leaves. The samples were minced and mixed,and extracted with ultra pure water at 90℃ for 20 min. The 6-aminoquinolyl N-hydroxy-succinimidyl carbamate( AQC) was used as pre-column derivatization reagent. Gradient HPLC separation was performed on a C_(18) column( Symmetry C_(18),3. 9 mm × 15 cm,4 μm). Good linearity between concentrations and peak areas was achieved in the concentration range of 5. 0-250 μmol/L for 18 kinds of amino acids. The method was validated by the analysis of five replicates. The 18 kinds of amino acid standards were spiked in fresh tea leaf samples and the average recovery rate was 86. 25%-109. 05% with relative standard deviations( n = 5) ranging from 6. 03% to 10. 56%. The limit of detection( LOD) for the analytes was0. 05-1. 27 μmol/L. The method was successfully applied to the analysis of the 18 kinds of amino acids in fresh tea leaves from east Dongting and west Dongting mountains in Suzhou. The results indicate that the method is simple,rapid,precise and reliable.

在线衍生HPLC快速测定聚酯纤维中TPA与IPA的含量

聚酯纤维在氢氧化钾-乙醇溶液中解聚,解聚液用水定容后采用在线衍生高效液相色谱法(HPLC)分析苯二甲酸,建立了快速测定聚酯纤维中对苯二甲酸(TPA)与间苯二甲酸(IPA)含量的方法。结果表明:聚酯纤维在65℃氢氧化钾-乙醇溶液中振荡处理35min后完全解聚;在190~360nm检测波长范围内,TPA和IPA的最大吸收波长分别为196、212nm,次吸收波长分别为241、220nm,流动相为乙腈-水(0.1%甲酸,V_(乙腈)∶V_(水)=15∶85)时的保留时间分别为6.038、7.776min;TPA和IPA的检出限分别为0.50、1.58mg/L,加标回收率高,相对标准偏差小。该方法样品前处理简单,分析快捷,结果可靠。

Enantioresolution of a Series of Chiral Benzyl Alcohols by HPLC on a Dinitrobenzoylphenylglycine Stationary Phase after Achiral Pre-Column Derivatization

High performance liquid chromatography method for the separation of a series of chiral benzyl alcohols on N-(3,5-dinitrobenzoyl)-D-phenylglycine stationary phase (Macherey Nagel, Chiral-2) after pre-column achiral derivatization was developed. Cheap and easy available aromatic acid chlorides were used as derivatization agents. Good to excellent separations of the enantiomers were achieved in all cases in relatively short analytical runs. It was shown that the enantiorecognition depends on the substituents both in the starting alcohol and in the acid chloride. The method presents an efficient alternative to the direct analyses on polysaccharide and cyclodextrine-derived stationary phases.

HPLC-柱后衍生荧光法测定水产品中河豚毒素含量结果的不确定度评定

通过建立HPLC-柱后衍生荧光法测定水产品中河豚毒素含量的不确定度评定方法,量化分析测定结果的不确定度分量,并对各分量相对贡献进行比较。结果表明:测定结果的不确定度主要来源于标准品的校准过程、样品称量、实验过程移取及定容、样品重复实验误差、前处理过程中的回收率不同等。其中校准不确定度中标准溶液配制的不确定度贡献最多,称样量不确定度最小;当水产品中河豚毒素的含量为90.6μg/kg时,其扩展不确定度为0.69μg/kg(包含因子k=2)。

HPLC of Amino Acids and Oligopeptides by Pre-Column Fluorescence Derivatization with 9-Acridine Formyl Chloride

A highly sensitive HPLC method for the detection of amino acids and oligopeptides with 9-acridine formyl chloride by pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, histidine, triglycine and glutathione were separated on a reversed-phase C-18 column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The five derivatives were eluted in 28 min with a good reproducibility. Linear range of the calibration graph was 0.08-260 nmol/ml(-1). The relative standard deviations(n=6) are < 5%. Detection limits (signal-to-noise ratio=3) for the five derivatives are 20-40 fmol.

基于HPLC研究柏子仁中黄曲霉毒素的污染状况与风险分析

目的基于HPLC研究柏子仁中4种黄曲霉毒素的污染情况,并进行风险分析,评估柏子仁的用药安全。方法采用Agilent Eclipse Plus C 18(4.6×250 mm 5μm)为色谱柱,柱后光化学衍生法检测,以甲醇∶乙腈∶水(35∶10∶55)为流动相,流速1.0 mL·min^(-1);柱温:40℃;荧光检测器检测。结果黄曲霉毒素B 1、B 2、G 1、G 2分别在0.35~20.8μg·L^(-1)、0.13~7.6μg·L^(-1)、0.36~21.6μg·L^(-1)、0.13~7.6μg·L^(-1)范围内呈良好的线性关系,黄曲霉毒素B 1、B 2、G 1、G 2平均回收率分别为90.6%、83.46%、87.84%、86.58%,相对偏差分别为2.9%、3.6%、3.6%、4.7%。23批柏子仁中有14批检出黄曲霉毒素B 1和总量,残留量分别在1~4μg·kg^(-1)和1~5μg·kg^(-1)之间,均符合规定,但检出率高达61%,存在安全隐患。结论该方法简单、准确、方便,能有效评价柏子仁中黄曲霉毒素的污染情况。警示柏子仁易受黄曲霉毒素的污染,需加强柏子仁监管力度和不定期的专项抽检,降低安全风险,以确保临床用药安全。

PMP柱前衍生HPLC法测定八月瓜果皮多糖中单糖组成

为建立同时测定八月瓜果皮中6种单糖组分的色谱分离方法,采用1-苯基-3-甲基-5-吡唑啉酮(1-phenyl-3-methyl-5-pyrazolone,PMP)柱前衍生和高效液相色谱法测定八月瓜果皮中6种单糖组分。色谱柱为SHIMADZU-C18(4.6 mm×250 mm,5μm),流动相为乙腈(A)-0.05 mol/L甲酸铵溶液(B)梯度洗脱(0~10 min:18%A;10~20 min:18%~20%A;20~40 min:20%~23%A;40~60 min:23%A),柱温30℃,流速1 m L/min,检测波长245 nm。结果表明,所建立的PMP-HPLC柱前衍生化法可准确地测定八月瓜果皮中的D-甘露糖、L-鼠李糖、D-半乳糖醛酸、D-葡萄糖、D-半乳糖和D-阿拉伯糖含量,6种单糖成分在各自质量浓度范围内线性关系良好(R2≥0.998 3),加样回收率为92.58%~100.02%。10批八月瓜果皮样品中均检出6种单糖,D-甘露糖、L-鼠李糖、D-半乳糖醛酸、D-葡萄糖、D-半乳糖和D-阿拉伯糖平均物质的量比为1.18∶1.00∶23.25∶11.73∶3.51∶5.88,其中D-半乳糖醛酸含量为51.84~212.84 mg/g,D-葡萄糖含量为22.44~73.23 mg/g,D-阿拉伯糖含量为6.14~60.01 mg/g,是八月瓜果皮中的单糖主要成分。该试验所建立的方法操作简便、重复性好和准确度高,适用于八月瓜果皮多糖中单糖成分的分析。

Determination of Content of Amino Acid in Abalone by Precolumn Derivatization and its Nutritional Value Evaluation

[Objectives] This study was conducted to optimize the determination conditions of amino acids from abalone. [Methods] The sample was treated by acid hydrolysis method and subjected to 2,4-2 nitro fluorobenzene column derivatization. The amino acid content in abalone was determined by HLPC,and the nutritional value of the amino acids was evaluated with egg protein model put forward by Institute of Nutrition and Food Hygiene,Chinese Academy of Preventive Medicine. [Results] Abalone contains full amino acids. According to the FAO/WHO ideal,it is a high-quality protein source and suitable for supplement of protein source for human body. [Conclusions]The experimental method has simple operation and could achieve a good effect with wide linear range and correlation coefficient over 0. 999 8,and the obtained results are satisfactory.

Determination of Sparfloxacin in Human Urine by Reversed-Phase High Performance Liquid Chromatography With Nitrous Acid and Hydroiodic Pre-Column Derivatization

Sparfloxacin can be oxidized by nitrous acid, then react with hydroiodic acid to form a fluorescent derivative. Based on this, a reversed-phase high performance liquid chromatographic pre-column derivatization new method is described for the determination of sparfloxacin in human urine. The linear range is 0.05 mg/L to 4.0 mg/L, the recoveries are 91.5%similar to 95.7% and the RSD is 1.2%similar to4.2%. The results showed that this method is suitable for the determination of sparfloxacin in human urine.

2种HPLC-柱后衍生化-荧光检测法测定远志中黄曲霉毒素的比较

对远志中测定黄曲霉毒素的2种柱后衍生化方法(碘衍生化和光化学衍生化)进行分析比较。样品经过免疫亲和柱净化,HPLC-柱后衍生化-荧光检测器检测;采用C_(18)(250 mm×4.6 mm,5μm)色谱柱,荧光检测。柱后衍生化系统:(1)碘衍生化法,以甲醇-乙腈-水(25∶20∶55)为流动相;衍生溶液为0.05%碘溶液,流速为0.3 m L·min^(-1),衍生反应温度为70℃;(2)光化学衍生化法,以甲醇-乙腈-水(35∶15∶50)为流动相。碘衍生化方法中,黄曲霉毒素B_(2)、G_(2)在3.8~19 pg范围内线性关系良好,B_(1)在10.4~52 pg范围内线性关系良好,G_(1)在10.8~54 pg范围内线性关系良好;在光化学衍生化方法中,黄曲霉毒素B_(2)、G_(2)在1.9~45.6 pg范围内线性关系良好,B_(1)在5.2~124.8 pg范围内线性关系良好,G_(1)在5.4~129.6 pg范围内线性关系良好,r>0.9999;回收率在85%~105%之间。2种衍生化方法的测定结果比较接近,但是光化学衍生化方法更加灵敏,且操作简单、分析速度快。

柱前衍生HPLC-UV法检测蜂蜜中二羟基丙酮的方法研究

建立一种柱前衍生HPLC-UV法检测蜂蜜中二羟基丙酮的方法。采用2mL3.0g/L的二硝基苯肼溶液进行二羟基丙酮的柱前衍生,在364nm处进行检测。该方法在2~40mg/kg范围内线性关系良好,提取回收率在102%~110%之间,方法的检测限为0.5mg/kg。该方法检测限低,准确度高,可用于蜂蜜中二羟基丙酮的检测。

AQC柱前衍生高效液相色谱方法测定康复新液中游离氨基酸和总肽的含量

目的应用6-氨基喹啉基-N-羟基琥珀酰亚氨基氨基甲酸酯(AQC)柱前衍生化技术,建立康复新液中游离氨基酸和总肽的高效液相色谱(HPLC)含量测定方法,并以总肽含量为指标对市售样品进行检测。方法以十八烷基硅烷键合硅胶为填充剂(Kromasil 100-5 C18色谱柱);60%乙腈(A)-0.14 mol·L^(-1)三水合乙酸钠溶液,用磷酸调节pH值至5.0(B)为流动相,梯度洗脱;柱温:39℃;流速:1.0 mL·min^(-1),检测波长:248 nm。结果14种氨基酸均能达到良好的分离效果;门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、精氨酸、苏氨酸、丙氨酸、脯氨酸、缬氨酸、赖氨酸、异亮氨酸、亮氨酸和苯丙氨酸分别在2.0~99.7、3.4~168.5、2.8~139.6、4.0~201.4、4.8~238.1、6.7~336.9、3.3~167.5、9.7~487.3、4.1~202.5、4.4~221.6、5.4~270.5、3.3~166.8、4.8~240.8、4.7~236.6μg·mL^(-1)范围内呈现良好的线性关系(r=0.9995~1.0000);游离氨基酸的平均加样回收率(n=6)为86.8%~108.1%,RSD为2.8%~4.4%,总肽酸水解氨基酸的平均加样回收率(n=6)为83.2%~102.7%,RSD为0.1%~3.1%;仪器精密度、重复性、稳定性实验的RSD均小于5.0%。结论该方法与现行质量标准的紫外分光光度法比较,二者测定的总氨基酸含量结果基本一致,但前者的专属性、重现性更优;以具有生物活性的总肽为质控指标,针对性更强,可为康复新液的质量评价提供更科学、合理的方法。

柱前衍生高效液相荧光检测法测定复方氨基酸注射液中甲硫氨酸亚砜的含量

目的建立测定复方氨基酸注射液中甲硫氨酸亚砜质量分数的高效液相色谱法。方法采用柱前衍生HPLCFLD法,色谱柱为Agilent Poroshell 120 EC-C18柱(3.0 mm×100 mm,2.7μm)或效能相当的色谱柱,以乙酸钠四氢呋喃溶液为流动相A,乙酸钠溶液-乙腈-甲醇(体积比20∶40∶40)为流动相B,流速为1.0 mL/min,采用荧光检测器,激发波长为233 nm,发射波长为441 nm。结果甲硫氨酸亚砜质量浓度在0.02605~2.605μg/mL范围内线性关系良好(r=0.9996),检测限为1.3×10^(-3)ng,平均回收率为100.1%,RSD为0.3%。结论建立的方法操作简单、准确、灵敏度高,适用于复方氨基酸注射液中甲硫氨酸亚砜的测定。

柱前衍生/高效液相色谱-串联质谱法测定氨基酸工业品中26种游离手性氨基酸含量

采用柱前衍生/高效液相色谱-串联质谱法(HPLC-MS/MS)测定氨基酸工业品中26种游离手性氨基酸含量。样品经0.1 mol/L HCl溶液溶解提取后,在碱性条件下与Marfey试剂进行衍生化反应,使用Phe⁃nomenex Kinetex F5(250 mm×5 mm,4.6μm)色谱柱,以10 mmol/L乙酸铵溶液-乙腈为流动相进行梯度洗脱,质谱分析采用电喷雾离子源负离子(ESI-)模式和多反应监测(MRM)模式。结果表明,各氨基酸衍生物之间的分离度良好,26种手性氨基酸的质量浓度在一定范围内与对应的峰面积呈线性关系,相关系数(r^(2))不小于0.9940,定量下限为0.5~12.5 ng/mL,加标回收率为80.3%~118%,相对标准偏差为0.47%~9.8%。应用该方法对180个工业样品进行检测,在L-型氨基酸纯品试剂、L-型稳定同位素标记氨基酸内标试剂、单品L-型氨基酸原料及复合氨基酸原料粉中均检出D-氨基酸。该研究首次评估了国内相关工业产品中手性氨基酸的质量状况,发现复合氨基酸原料需加强手性构型的监测管理,并深入分析了利用相关氨基酸纯品试剂作为标准物质或同位素内标时,对食品、环境、化工、医药等领域样品检测结果准确性可能造成的影响。该方法可为科研试剂、生物制药、化工食品和营养健康等领域提供质量管理和健康评测的有效技术手段。

光化学衍生法结合HPLC测定食品和饲料中的黄曲霉毒素

黄曲霉毒素的急性毒性很强,是重要的致癌物质。本文采用光化学衍生法结合HPLC测定了食品和饲料中的黄曲霉毒素,研究了光化学衍生的效果。结果表明,经光化学柱后衍生,四种黄曲霉毒素的检测灵敏度都比较高,检出限可达到3. 25pg。

柱前荧光衍生化RP-HPLC测定环维黄杨星D含量

目的 建立环维黄杨星D含量测定方法。方法 环维黄杨星D同异氰酸萘酯反应后 ,RP HPLC荧光法测定。色谱柱为C1 8柱 ;流动相为甲醇 水 (85∶15 ) ;流速 :1mL·min- 1 ;荧光检测激发波长 30 5nm ,发射波长 385nm ;柱温35℃。结果 衍生化反应重复性好 ,RSD为 1 2 1% ,主峰与相关物质分离良好。结论 本法简单、准确 ,可用于环维黄杨星D及其相关物质含量测定。

柱前衍生化RP-HPLC法分析龟板中氨基酸

目的采用柱前衍生RP-HPLC法对龟板中13种氨基酸进行分析.方法用6 mol/L HCl水解提取龟板中总氨基酸,以异硫氰酸苯酯(PITC)为柱前衍生化试剂,采用外标法,梯度洗脱的方式分析.结果13种氨基酸在40 min内均可得到很好的分离,回收率(除甘氨酸外)为91.68%～106.26%,RSD为0.718%～4.386%.结论所建立的方法分离效果好、灵敏、准确、简便,且采用普通的C18色谱柱,可广泛用于含有多种氨基酸样品的分析.