Synchronous high-performance liquid chromatography with a photodiode array detector and mass spectrometry for the determination of citrinin, monascin, ankaflavin, and the lactone and acid forms of monacolin K in red mold rice

The Monascus fermentation product red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK) in both its lactone (MKL) and acid (MKA) forms and the mycotoxin citrinin (CT). The yellow pigments in RMR, namely, monascin (MS) and ankaflavin (AK), have been reported to exhibit antimetastatic and antiangiogenic activities. Currently, MK and these yellow pigments are usually detected in RMR by different analytical methods that are inconvenient, expensive, and time-consuming. The goal of this study was to establish a rapid, synchronous analytical method for determination of the MKA, MKL, MS, AK, and CT levels in RMR. MKA, MKL, MS, AK, and CT were extracted by the same extraction method, then separated by RP-HPLC with a C18 column. The effluent from the column was passed through a photodiode array detector and then introduced directly into a fluorescence detector. The results showed that high recovery rates of MKA, MKL, MS, AK, and CT are possible if RMR powder is extracted with 75% ethanol (10 mL) at 80 degrees C for 30 min. With regard to the optimal conditions of the HPLC, the peaks of MKA, MKL, MS, AK, and CT can be clearly separated from any noise peaks by isocratic elution with a mobile phase comprising 0.05% trifluoroacetic acid in acetonitrile-water (62.5 + 37.5, v/v).     

HPLC analysis of antioxidants

Qualitative and quantitative HPLC methods are described for the analysis of mixtures of twelve antioxidants. For identification of the components present, gradient elution with a convex profile from 35:65 v/v water-methanol to pure methanol is used, on a Waters 5 mum C(18) Resolve column, with an ultraviolet detector. Propyl gallate and methyl p-hydroxybenzoate cannot be separated, however. For quantitative analysis, with ultraviolet and electrochemical detectors in series, the 35:65 water-methanol mixture or pure methanol is used as the eluent, under isocratic conditions, with lithium perchlorate as supporting electrolyte. An applied potential ranging from +0.8 to + 1.7 V allows detection of all the antioxidants tested. Both modes of detection are very sensitive, with limits of detection as low as 61 pg (UV, methyl p-hydroxybenzoate) and 360 pg (electrochemistry, butylhydroxyanisole).     

An HPLC method for determination of azadirachtin residues in bovine muscle

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.     

High-performance liquid chromatographic stability indicating assay method of tianeptine sodium with simultaneous fluorescence and UV detection

The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay of tianeptine (TIA) in bulk drug and tablet form. TIA is subjected to different stress conditions, including UV-light, oxidation, acid base-base hydrolysis, and temperature. TIA and its possible degradation products are analyzed on Agilent-Zorbax-XDB-C18 column using gradient elution with acetonitrile and 0.02M sodium acetate (pH 4.2). The samples are monitored simultaneously with photo-diode array at 254 nm and fluoroscence detector set to 350 nm (ex) and 425 nm (em). TIA is integrated from its UV-chromatogram, and the photodecomposition products are integrated from the fluoroscence-chromatogram. TIA and its photodecomposition products are separated by TLC using ethyl acetate-n-hexane-glacial acetic acid-methanol (10.0:14.0:0.2:1.0, v/v) as developing system. One potential photodegradation product is detected by fluoroscence in TIA-tablet form and separated by TLC. The linear range of TIA is between 0.5 to 50 microg/injection with limits of quantitation and detection values of 30 and 8 ng/injection, respectively. The inter-assay percentage of deviation is not more than 0.03%, and the day-to-day variation is not more than 0.1%.     

乳及乳制品中多种防腐剂和甜味剂的同时测定

建立了高效液相色谱法同时测定乳及乳制品中安赛蜜、苯甲酸、糖精钠、山梨酸和阿斯巴甜的方法。通过加入适量沉淀剂除去样品中绝大部分蛋白质后,采用C18色谱柱分离,以甲醇-0.05 mol/L磷酸二氢钾溶液为流动相梯度洗脱,用二极管阵列检测器于230 nm波长处检测安赛蜜、苯甲酸和山梨酸,于210 nm波长处检测糖精钠和阿斯巴甜。被测物的回收率为96.0%～103.5%,精密度(以相对标准偏差(RSD)计)为1.93%～2.76%,安赛蜜、苯甲酸、糖精钠、山梨酸和阿斯巴甜的检出限分别为1.0, 1.0, 0.5, 1.0, 1.5 μg/g。该方法可用于乳及乳制品中这5种添加剂的同时测定。     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.     

Determination of zearalenone content in cereals and feedstuffs by immunoaffinity column coupled with liquid chromatography

The zearalenone content of maize, wheat, barley, swine feed, and poultry feed samples was determined by immunoaffinity column cleanup followed by liquid chromatography (IAC-LC). Samples were extracted in methanol-water (8 + 2, v/v) solution. The filtered extract was diluted with distilled water and applied to immunoaffinity columns. Zearalenone was eluted with methanol, dried by evaporation, and dissolved in acetonitrile-water (3 + 7, v/v). Zearalenone was separated by isocratic elution of acetonitrile-water (50 + 50, v/v) on reversed-phase C18 column. The quantitative analysis was performed by fluorescence detector and confirmation was based on the UV spectrum obtained by a diode array detector. The mean recovery rate of zearalenone was 82-97% (RSD, 1.4-4.1%) on the original (single-use) immunoaffinity columns. The limit of detection of zearalenone by fluorescence was 10 ng/g at a signal-to-noise ratio of 10:1 and 30 ng/g by spectral confirmation in UV. A good correlation was found (R2 = 0.89) between the results obtained by IAC-LC and by the official AOAC-LC method. The specificity of the method was increased by using fluorescence detection in parallel with UV detection. This method was applicable to the determination of zearalenone content in cereals and other kinds of feedstuffs. Reusability of immunoaffinity columns was examined by washing with water after sample elution and allowing columns to stand for 24 h at room temperature. The zearalenone recovery rate of the regenerated columns varied between 79 and 95% (RSD, 3.2-6.3%). Columns can be regenerated at least 3 times without altering their performance and without affecting the results of repeated determinations.     

高效液相色谱法测定水产品中7种兽药残留量

提出了高效液相色谱法同时测定水产品中呋喃唑酮、噁喹酸、萘啶酸及4种磺胺类药物等7种兽药残留量的方法。样品经乙腈提取,正己烷脱脂,用中性氧化铝柱和C18固相萃取柱进行分离及净化。以Symmetry C18色谱柱为分离柱,以不同体积比混合的乙腈与0.08mol.L-1乙酸溶液为流动相进行梯度洗脱,紫外检测器于波长265nm处检测。7种兽药的质量浓度与其峰面积均在0.05～1.0mg.L-1范围内呈线性关系,检出限(3S/N)为0.01～0.04mg.kg-1。方法的回收率在68.2%～97.3%之间,相对标准偏差(n=6)在1.6%～14%之间。     

Column reversed-phase high-performance liquid chromatographic method for simultaneous determination of rabeprazole sodium and domperidone in combined tablet dosage form

A new, simple column reversed-phase high-performance liquid chromatographic (HPLC) method for simultaneous determination of rabeprazole sodium (RAB) and domperidone (DOM) in a combined tablet dosage form has been developed and validated. Determination was performed using a Jasco HPLC system with a HiQ SiL octadecylsilane (C18) column (250 x 4.6 mm id), acetonitrile-0.1 M ammonium acetate (50 + 50, v/v) mobile phase, and paracetamol as an internal standard. The detection was performed using a UV detector set at 280 nm. The method was validated with respect to linearity, accuracy, precision, and robustness. Beer's law was obeyed in the concentration range of 1.0-10.0 and 0.5-5.0 microg/mL for RAB and DOM, respectively. The method has been successfully applied for the analysis of drugs in a pharmaceutical formulation.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

Amino acid, fatty acid, and carbohydrate content of Artocarpus altilis (breadfruit)

A study is conducted to determine the amino acid, fatty acid, and carbohydrate content of breadfruit using high-performance liquid chromatography (HPLC) and gas chromatography (GC). An HPLC method is used for the determination of amino acids and fatty acids in breadfruit. Representative amino acid samples are derivatized with phenylisothiocianate and the resulting phenylthiocarbamyl derivatives are separated on a reversed-phase column by gradient elution with a 0.05M ammonium acetate buffer and 0.01M ammonium acetate in acetonitrile-methanol-water (44:10:46, v/v). Representative fatty acid samples are derivatized with phenacyl bromide and the resulting fatty acid phenacyl esters are separated on a reversed-phase column by gradient elution with acetonitrile and water. Amino acid and fatty acid derivatives are detected by ultraviolet detection at 254 nm. The analysis of the carbohydrates in breadfruit employs a GC method. Carbohydrates are derivatized using trimethylchlorosilane and hexamethyldisilazane to form trimethylsilyl ethers. Compounds in the samples are separated by the temperature programming of a GC using nitrogen as the carrier gas. Percent recoveries of amino acids, fatty acids, and carbohydrates are 72.5%, 68.2%, and 81.4%, respectively. The starch content of the breadfruit is 15.52 g/100 g fresh weight.     

高效液相色谱法测定中药润燥止痒胶囊中大黄素和大黄素甲醚

提出了高效液相色谱法测定润燥止痒胶囊中大黄素和大黄素甲醚含量的方法。样品经甲醇-盐酸(10+0.3)混合溶液加热提取水解30min,采用Hypersil ODS 2C18色谱柱为分离柱,以不同体积比混合的甲醇和磷酸(0.1+99.9)溶液为流动相进行梯度洗脱,检测波长为254nm。大黄素和大黄素甲醚的平均回收率分别为100.2%和99.7%。     

五加生化胶囊的高效液相色谱指纹图谱研究

应用高效液相色谱(HPLC)法建立五加生化胶囊的指纹图谱。采用ANGLAVenusil XBP-C18色谱柱(250×4.6mm,5μm),以甲醇-0.1%磷酸溶液为流动相,梯度洗脱,检测波长为327nm,五加生化胶囊多数峰达到基线分离。采用2004A中药色谱指纹图谱相似度评价系统对11批样品的指纹图谱进行峰匹配,确定12个共有峰,11批五加生化胶囊指纹图谱的相似度均在0.90以上。结果表明:五加生化胶囊的HPLC指纹图谱特征性和专属性强,可较系统地用于五加生化胶囊的质量控制。     

基于荷移反应的高效液相色谱法测定氨基丁醇

建立了一种基于荷移反应的高效液相色谱测定氨基丁醇含量的分析方法。在pH 8.4的硼砂-硼酸缓冲溶液中,氨基丁醇与四氯苯醌于60℃反应60 min,利用高效液相色谱法-紫外检测器进行分析。荷移络合物采用Agilent Extend C18色谱柱（250 mm×4.6 mm,5 μm）分离,以0.001%（体积分数）三乙胺甲醇溶液为流动相进行梯度洗脱,流速为1 mL/min,检测波长为350 nm。该方法对氨基丁醇的定量限为0.01 g/L,线性范围为0.1~0.6 g/L,相关系数（R²）为0.9994;方法的加标回收率为98.3%~103.6%,相对标准偏差（RSD）为0.9%~1.6%。该法简便快捷,适用于氨基丁醇含量的快速检测。     

高效液相色谱法测定奶粉和液态奶中的三聚氰胺

建立了奶粉和液态奶中三聚氰胺的高效液相色谱（HPLC）检测方法。经阳离子交换固相萃取柱净化后的样品采用HPLC测定。优化的色谱条件：C18柱（4.6 mm ×200 mm,5 μm）,流动相为乙腈-0.01 mol/L庚烷磺酸钠（pH 3.3）（体积比为10∶90）,流速为1.0 mL/min,检测波长为236 nm,柱温为40 ℃,进样量为20 μL。方法的线性范围为1~500 mg/L,检出限为0.2 mg/kg （S/N=3）,定量限为1 mg/kg （S/N=15）,回收率为81.4%~83.7%,相对标准偏差为3.3%~8.5%（n=6）。     

色谱指纹图谱在苹果酒质量评价中的应用

采用反相高效液相色谱-电化学检测法研究了14种苹果酒样品的指纹图谱。以标准品绿原酸进行定位,通过对图谱分析和相对保留时间计算,确定了8个共有峰。根据共有峰的峰面积用相关系数法和向量夹角余弦法计算相似度,两种方法的计算结果一致。实验结果表明同一厂家生产的苹果酒相似度较好。该法为苹果酒的产品分析提供了有效的微观信息,为苹果酒的质量控制、新产品的研发以及苹果酒行业标准的制定提供一种可行思路。     

Optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related compounds

A validated stability-indicating HPLC method was developed for the analysis of azithromycin (AZ) and its related compounds in raw materials, capsule, and suspension using an Xterra RP C18 column at 50 degrees C with UV detection at 215 nm. Isocratic elution was employed using the mobile phase 14 mM disodium hydrogen phosphate (pH 10.5, adjusted by 1 M NaOH)-methanol-acetonitrile-tetrahydrofuran (40.0 + 30.0 + 30.0 + 0.1, v/v/v/v). AZ and 14 of its related compounds were separated and quantified. The described method was linear over the range of 2-1800 microg/mL AZ with (r = 0.9999). The stability of AZ was studied under accelerated acidic, alkaline, and oxidative conditions. The proposed method was used to investigate the kinetics of acidic and alkaline hydrolysis process of AZ at different temperatures, and the apparent pseudo first-order rate constant, half-life, and activation energy were calculated. The major peak detected from the degradation of AZ in alkaline and acidic conditions was decladinosylazithromycine, while azithromycin N-oxide was detected from the oxidative degradation. Long-term stability studies for capsule and oral suspension were carried out. The proposed stability-indicating method was completely validated according to the U.S. Food and Drug Administration requirements.     

反相高效液相色谱法测定乳品及乳制品中的黄曲霉毒素M_1

采用反相高效液相色谱测定乳品及乳制品中黄曲霉毒素M_1(AFM_1)的含量。样品经氯仿提取,过硅胶固相萃取柱净化,用氯仿-丙酮(1+1)混合溶液将黄曲霉毒素M_1从固相萃取柱上洗脱下来。以ZORBAX SB C_(18)色谱柱为分离柱,水和乙腈为流动相梯度淋洗,用荧光检测器检测,外标法定量。黄曲霉毒素M_1在1.0～25μg·L~(-1)质量浓度范围内与其峰面积呈线性关系,检出限(3S/N)为0.05μg·kg~(-1)。应用此法测定了牛奶和乳粉中AFM_1的含量,并测得其平均回收率分别在76.0%～80.0%和76.7%～90.8%之间,相对标准偏差(n=6)均小于7.0%。     

Separation and identification of 20 chemical constituents in the traditional Chinese medicinal preparation Shenbao tablet by LC-ESI-MS3

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization multiple-stage mass spectrometry for the simultaneous separation and identification of 20 chemical constituents in the traditional Chinese medicinal preparation of the Shenbao tablet is established. The samples are separated with an Alltima C(18) column (250 x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.5, v/v) and acetonitrile (B; 0 min, 76:24; 15 min, 70:30; 40 min, 53:47; 50 min, 30:70; and maintain 10 min) as the mobile phase at a flow rate of 1.0 mL/min. The ion trap mass spectrometer is coupled to the HPLC system. Satisfactory results are obtained within 60 min for the simultaneous separation and identification of the 20 constituents. This is the first report on the analysis of main chemical constituents in the Shenbao tablet.     

虎纹捕鸟蛛毒素-Ⅴ去折叠过程的色谱分析

 将纯化的天然虎纹捕鸟蛛毒素-Ⅴ经盐酸胍变性30 min后,在pH 3.0、反应温度为37 ℃的条件下与三羧甲基磷酸(TCEP)反应12 min,用反相高效液相色谱分离得到其全部去折叠中间体,通过基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行鉴定,并利用烷基化反应对这些去折叠中间体予以进一步确证。 根据其保留时间,分析虎纹毒素-Ⅴ各去折叠中间体的色谱行为,初步探讨了多肽或蛋白质构象异构体反相色谱行为的多样性。