Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel

Miniaturization embracing instrumentation, column particle size, and column dimensions is one of the major current trends in separation techniques. This leads to shortening of analysis time and great savings in solvent consumption. Ultra performance liquid chromatography (UPLC) is one of the new developments in liquid chromatography. An ultra-high pressure system allows using of small particle-packed columns with small diameter, which has a positive effect on both system efficiency and analysis time. An analytical method for determination of the active substance diclofenac, the degradation product 1-(2,6-dichlorphenyl)-2-indolinone, and the preservatives methylparaben and propylparaben was used for testing and comparing LC systems. Various octadecylsilica-based analytical columns were examined. Acquity UPLC BEH C18 (2.1 x 50 mm, 1.7 microm) and (2.1 x 100 mm, 1.7 microm) were tested for UPLC. The following analytical columns were used in a test for HPLC: Purospher RP 18e (125 x 4.0 mm, 5 microm), Zorbax Eclipse XDB C18 (75 x 4.6 mm, 3.5 microm), Zorbax Eclipse SB C18 (50 x 4.6 mm, 1.8 microm), as was a monolithic column (Chromolith Performance RP-18e (100 x 4.6 mm). Results of a System Suitability Test (SST) were calculated and compared for each chromatographic peak. System efficiency and analysis duration were compared with regard to solvent consumption and system maintenance     

反相高效液相色谱法同时测定咖啡豆中的6种酚酸类化合物

建立了同时测定咖啡豆中6种酚酸类化合物(咖啡酸、3-咖啡酰奎尼酸、4-咖啡酰奎尼酸、5-咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸、4,5-二咖啡酰奎尼酸)的反相高效液相色谱测定方法。采用Kromasil C18柱(200 mm×4.6 mm, 5 μm),以乙腈和0.1%甲酸水溶液为流动相进行梯度洗脱,二极管阵列检测器检测,45 min内可对6种目标物进行同时检测,且各化合物都能达到基线分离。经测定,样品中6种酚酸类化合物的加标回收率为90.76%～104.73%,相对标准偏差为0.7%～3.9%。该法简便、快速、灵敏度高,适用于咖啡豆中6种酚酸类化合物的同时分析以及咖啡豆原料与制品的质量控制和综合评价。     

Development of a method for fast analysis of phenolic molecular markers in biomass burning particles using high performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry

A high performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry (HPLC/APCI-MS) method for the fast analysis of 21 biomass burning tracers in particles samples has been developed. Separation was done with a Zorbax SB-C18 Rapid Resolution cartridge column (4.6 mm x 30 mm x 3.5 microm), using a CH3OH/H2O/CH3COOH gradient at a flow rate of 0.5 mL/min. The observed relative standard deviations (RSD) for the retention times and peak areas were <0.6 and <15%, respectively. With the short analytical column and the sensitive detector the total analysis time for the standard mixture was reduced to 15 min. Instrumental detection limits were <1 microM (S/N=3) for all standard compounds except homovanillic acid (4.3 microM). The suitability of the developed method for the analysis of biomass burning particles is demonstrated by the measurements of five different real biomass burning samples. The results of these measurements showed clear differences between the different kinds of biomass and they are in good agreement with results from earlier studies in the literature.     

在线柱浓缩-超高效液相色谱法测定水体中的痕量甲萘威和呋喃丹

采用在线柱浓缩-超高效液相色谱联用技术测定水体中痕量甲萘威和呋喃丹。水样过滤后直接进样,采用固相萃取小柱富集待测物,梯度洗脱后,利用阀切换技术将待测物反冲至分析柱Acclaim RSLC C18(100 mm×2.1 mm, 2.2 μm)上进行色谱分离,以10 mmol/L醋酸铵缓冲溶液(pH 5.0,用醋酸调节)和乙腈分别为流动相A和B,梯度洗脱,泵流速为0.8 mL/min,检测波长为280 nm,二极管阵列检测器检测。甲萘威和呋喃丹在1.0～100 μg/L范围内线性良好(相关系数r2 ＞ 0.9999),检出限(S/N=3)分别为0.5和0.25 μg/L,加标回收率为76.0%～120.0%。用所建立的方法测定了水中痕量的甲萘威与呋喃丹的含量,结果令人满意。     

Evaluation of an International Pharmacopoeia method for the analysis of nelfinavir mesilate by liquid chromatography

A gradient LC method for the determination of related substances in nelfinavir mesilate (NFVM) has been recently published in the International Pharmacopoeia. The method uses a base deactivated reversed phase C18 column (25 cm x 4.6 mm I.D.), 5 microm kept at a temperature of 35 degrees C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 225 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards NFVM components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm I.D.), 5 microm. A two level fractional factorial design was applied to examine the robustness of the method. The method shows good selectivity, precision, linearity and sensitivity. Seven commercial samples were examined using this method.     

Qualitative and quantitative determination of the major coumarins in Zushima by high performance liquid chromatography with diode array detector and mass spectrometry

A method, high-performance liquid chromatography coupled with diode array and electrospray tandem mass spectrometry (HPLC-DAD-ESI-MS), was developed to qualitatively identify and quantitatively determine the 10 major active coumarins of Zushima. The analysis was performed by using a ZORBAX SB-C18 analytical column (250 mm x 4.6 mm ID, 5 microm) at gradient elution of 0.5% aqueous formic acid and acetonitrile with diode array detection (325 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. The proposed method was successfully applied for the qualitative and quantitative analysis of 10 coumarins in five different species of Zushima which had great variation on the contents of investigated coumarins.     

Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine,clozapine and N-desmethylclozapine in human plasma

A specific and sensitive direct-injection high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the rapid identification and quantitative determination of olanzapine, clozapine, and N-desmethylclozapine in human plasma. After the addition of the internal standard dibenzepin and dilution with 0.1% formic acid, plasma samples were injected into the LC/MS/MS system. Proteins and other large biomolecules were removed during an online sample cleanup using an extraction column (1 x 50 mm i.d., 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought inline with the analytical column by automatic valve switching. Analytes were separated on a 5 microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm) with a mobile phase of acetonitrile/0.1% formic acid (20:80, v/v) at a flow rate of 0.5 mL/min. The total analysis time was 6 min per sample. The inter- and intra-assay coefficients of variation for all compounds were <11%. By eliminating the need for extensive sample preparation, the proposed method offers very large savings in total analysis time.     

A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography

A sensitive and reliable RP-HPLC method was developed using a C18 CLC-ODS (M) - 4.6x250 mm(2)column and gradient elution for the analysis of phenolic compounds in propolis raw material and its products. A procedure for the extraction of phenolic compounds using aqueous ethanol (90%) with the addition of veratraldehyde as the internal standard (IS) was developed allowing to quantify ten compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, drupanin, artepellin C, baccharin, and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid (DCBEN). The developed method gave good detection response and linearity in the range of 20.83-533.33 microg/mL.     

Quality evaluation of Rhizoma Belamcandae (Belamcanda chinensis (L.) DC.) by using high-performance liquid chromatography coupled with diode array detector and mass spectrometry

A high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) method was developed to evaluate the quality of Rhizoma Belamcandae (Belamcanda chinensis (L.) DC.) through establishing chromatographic fingerprint and simultaneous determination of seven phenolic compounds. The analysis was achieved on an Alltima C(18) analytical column (250 mm x 4.6 mm i.d. 5 microm) using linear gradient elution of acetonitrile-0.1% trifluoroacetic acid. The correlation coefficients of similarity were determined from the HPLC fingerprints, and they shared a close similarity. By using an online APCI-MS/MS, twenty phenols were identified. In addition, seven of these phenols including mangiferin, 7-O-methylmangiferin, tectoridin, resveratrol, tectorigenin, irigenin and irisflorentin were quantified by the validated HPLC-DAD method. These phenols are considered to be major constituents in Rhizoma Belamcandae, and are generally regarded as the index for quality assessment of this herb. This developed method by having a combination of chromatographic fingerprint and quantification analysis could be applied to the quality control of Rhizoma Belamcandae.     

高效液相色谱法测定头孢他啶的含量及杂质

采用高效液相色谱法测定了头孢他啶的含量及杂质。以Alltima C18色谱柱(250 mm×4.6 mm,5 μm)为分离柱,以乙腈和磷酸盐缓冲溶液(pH 3.9)分别为流动相A和流动相B进行梯度洗脱,流速1.3 mL/min,柱温35 ℃,紫外检测波长255 nm。从头孢他啶药物中共分出14个杂质,且14个杂质间具有良好的分离度。头孢他啶在0.267~1069 μg/mL范围内与峰面积具有良好的线性关系(r=1.0000);其定量限(S/N=10)和最低检出限(S/N=3)分别为3.1 ng和0.93 ng。3个浓度的日内测定值的相对标准偏差(RSD）为0.72%(n=3),日间测定值的RSD为0.91%(n=3)。头孢他啶溶液在4 ℃避光条件下放置24 h保持稳定。本方法与欧洲/英国药典和日本药局方的方法比较,具有分离杂质数量多、分离度好的优点。     

Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2'-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.     

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Direct injection versus liquid-liquid extraction for plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry.

Direct injection versus liquid-liquid extraction for post-dose human plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) have been studied using a drug candidate compound. For the direct-injection method, an Oasis(R) HLB column (1 x 50 mm, 30 micrometer) was used as the on-line extraction column and a conventional Waters symmetry C18 column (3.9 x 50 mm, 5 micrometer) was used as the analytical column. Each plasma sample (100 microL) was mixed with 100 microL of a working solution of the internal standard in aqueous 0.05 M ammonium acetate (pH 6.9), and portions (10 microL) of these samples were then injected into the LC/MS/MS system. For the liquid-liquid extraction method, a YMC Basic C18 column (2.0 x 50 mm, 5 micrometer) was used as the analytical column. Each sample (0.5 mL) was extracted with methyl tert-butyl ether and the extract was reconstituted and injected into the LC/MS/MS system. The total analysis time for both methods was 2.0 min per sample. The accuracy, inter-day precision and intra-day precision obtained from the quality control samples were within 8% for both methods. The analysis results of post-dose human plasma samples showed that the deviations of 91% of the concentrations obtained using the direct-injection method were within +/-20% from the concentrations obtained using the liquid-liquid extraction method, and the overall average percentage deviation was -1.5%. The results showed that the two methods were equivalent in terms of total chromatographic run time, accuracy and precision. However, for a batch of 100 samples, the sample preparation time for the direct-injection method was only about 25% of the time required for liquid-liquid extraction. This decrease in sample preparation time resulted in the doubling of the overall sample analysis throughput.     

Comprehensive two-dimensional liquid chromatography with ultraviolet, evaporative light scattering and mass spectrometric detection of triacylglycerols in corn oil

An improved comprehensive two-dimensional (LC x LC) HPLC system for the analysis of triacylglycerols was developed. In the first-dimension, a Ag(I)-coated cation exchanger (250 mm x 2.1 mm, 5 microm) was employed with a gradient from 100% MeOH to 6% MeCN in MeOH at 20 microL/min. Using a 10-way valve with two switching loops, 1 min sections of the first-dimension were introduced in the second-dimension consisting of a 30 mm x 4.6 mm C18 (1.8 microm) column with an isocratic mobile phase of methanol-methyl tert-butyl ether (70:30) at 3.0 mL/min. As the second-dimension solvent was stronger than the first-dimension solvent, focusing in the second-dimension took place, leading to better separations than in previously reported analyses in which hexane was the main constituent of the first-dimension eluent. Compounds differing by 2 in their partition number were baseline separated in the second-dimension. Detection took place by UV at 210 nm, evaporative light scattering and (+)-atmospheric pressure chemical ionisation-MS with the latter giving the best results. Corn oil was investigated and 44 compounds could be detected: 34 triacylglycerols (TAGs), 8 oxygenated TAGs, and 2 TAGs containing a trans double bond. Data manipulation allowed the construction of contour plots and the automated calculation of the first- and second-dimension retention times and peak areas. Quantitative results are compared with a fatty acid methyl ester analysis, and with literature data.     

Quality evaluation of Ganoderma through simultaneous determination of nine triterpenes and sterols using pressurized liquid extraction and high performance liquid chromatography

A method combining HPLC and pressurized liquid extraction was developed for simultaneous quantification of nine components, including eight triterpenes (ganoderic acid A, ganoderic acid Y, ganoderic acid DM, ganoderol A, ganoderol B, ganoderal A, methyl ganoderate D and ganoderate G) and a sterol (ergosterol), in Ganoderma. The determination was achieved by using a Zorbax ODS C18 analytical column (4.6 x 250 mm id, 5 microm) and gradient elution with diode-array detection. All calibration curves showed good linearity (r2 > 0.9997) within the test ranges. The developed method showed good repeatability for the quantification of the nine investigated components in Ganoderma with intra- and inter-day variations of less than 2.4% and 4.1%, respectively. The validated method was successfully applied to quantify the nine components in two species of Ganoderma, i.e. G. lucidum and G. sinense, used as Lingzhi in China. Furthermore, hierarchical clustering analysis based on the nine components in HPLC profiles from the tested 11 samples showed that chemical characteristics were significantly different between G. lucidum and G. sinense, which suggested that clinical investigation should be performed so as to ensure the safety and efficacy of medication.     

高效液相色谱法测定化妆品中的12种紫外吸收剂

建立了高效液相色谱同时测定化妆品中12种紫外吸收剂含量的方法。样品用甲醇提取,高速离心,过滤,以SB-C8柱(250 mm×4.6 mm, 5 μm)为分离色谱柱,甲醇和0.1%(v/v)甲酸水溶液为流动相,梯度洗脱,以311 nm为检测波长进行定性、定量分析。该方法前处理简单、易操作,12种紫外吸收剂分离效果良好;在1.0～500 mg/L范围内呈线性关系,相关系数大于0.9995;方法检出限为0.002～0.1 mg/L;实际样品中的加标回收率为97.4%～107.5%,相对标准偏差为1.54%～4.98%。该方法简便、准确,能够满足化妆品中紫外吸收剂的检测要求。     

Rapid qualitative and quantitative ultra high performance liquid chromatography method for simultaneous analysis of twenty nine common phenolic compounds of various structures

Twenty nine phenolic compounds comprising nine phenolic acids, sixteen flavonoids (including eight tea catechins, glycosides and aglycones), four coumarins plus caffeine were analysed within 20 min using ultra high performance liquid chromatography (UHPLC) with PDA detection. UHPLC system was equipped with C18 analytical column (100 mm × 2.1 mm, 1.7 μm), utilising 0.1% formic acid and methanol mobile phase in the gradient elution mode. The developed method was tested for the system suitability: resolution, asymmetry factor, peak capacity, retention time repeatability and peak area repeatability. The method was fully validated in the terms of linearity (r² > 0.9990 for all 30 compounds), range (typically 1-100 mg L⁻¹), LOD, LOQ, inter/intra-day precision (<3% and <9% respectively) and inter/intra-day accuracy (typically 100 ± 10%). Subsequently the method was applied to the identification (spectral information and peak purity calculations were profited) and quantification of phenolic compounds and caffeine present in tea infusions and extracts.     

Comparison of phenyl-type columns in the development of a fast liquid chromatographic system for eighteen opiates commonly found in forensic toxicology

We report a precise and reliable method for the detection of 18 of the most commonly found opiates on the Belgian legal and illicit market, by ion-exchange, reversed-phase high-performance liquid chromatography, using a conventional phenyl-type analytical column (150x4.6 mm I.D., particle size 5 microm) and diode-array detection. We also describe a performance (efficiency and sensitivity) comparison of this column to a recently developed "high-speed" column (53x7.0 mm I.D., particle size 3 microm) packed with the same stationary phase, and used under slightly adjusted flow and gradient conditions. The final method, using the "high-speed" column, showed a significant reduction (55%) in analysis time without loss of resolution and sensitivity.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

固相萃取-反相高效液相色谱法同时测定饲料中的角黄素和虾青素

建立了一种同时测定饲料中角黄素和虾青素的固相萃取-高效液相色谱法（HPLC）。样品由乙腈提取,经LC-NH2固相萃取小柱净 化,洗脱剂为乙腈-甲苯（体积比为3∶1）,洗脱液被浓缩后进行HPLC分析,色谱柱为ZORBAX Eclipse XDB-C18柱(150 mm×4.6 mm,5 μ m),流动相为乙腈-甲醇（体积比为95∶5）,流速1.0 mL/min,采用二极管阵列检测器检测,检测波长为474 nm;外标法定量。角黄素和 虾青素的线性范围分别为1.0～30.0 mg/L和1.0～20.0 mg/L,相关系数分别为0.9990和0.9991,回收率为90%～101%,相对标准偏差为 0.62%～3.68%,检出限分别为0.84和0.60 mg/L。该方法简便、快速、准确,可用于饲料中角黄素和虾青素的同时测定。