Core–shell column Tanaka characterization and additional tests using active pharmaceutical ingredients

In the last decade, core–shell particles have gained more and more attention in fast liquid chromatography separations due to their comparable performance with fully porous sub‐2 μm particles and their significantly lower back pressure. Core–shell particles are made of a solid core surrounded by a shell of classic fully porous material. To embrace the developed core–shell column market and use these columns in pharmaceutical analytical applications, 17 core–shell C₁₈ columns purchased from various vendors with various dimensions (50 mm × 2.1 mm to 100 mm × 3 mm) and particle sizes (1.6–2.7 μm) were characterized using Tanaka test protocols. Furthermore, four selected active pharmaceutical ingredients were chosen as test probes to investigate the batch to batch reproducibility for core–shell columns of particle size 2.6–2.7 μm, with dimension of 100 × 3 mm and columns of particle size 1.6 μm, with dimension 100 × 2.1 mm under isocratic elution. Columns of particle size 2.6–2.7 μm were also tested under gradient elution conditions. To confirm the claimed comparable efficiency of 2.6 μm core–shell particles as sub‐2 μm fully porous particles, column performances of the selected core–shell columns were compared with BEH C₁₈, 1.7 μm, a fully porous column material as well.     

Comparison of a new high‐resolution monolithic column with core‐shell and fully porous columns for the analysis of retinol and α‐tocopherol in human serum and breast milk by ultra‐high‐performance liquid chromatography†

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high‐throughput assay methods in bioanalysis. It can be achieved either by ultra‐high‐performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction—monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second‐generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core‐shell and fully porous sub‐2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.     

Separation of natural product using columns packed with Fused‐Core particles

Three HPLC columns packed with 3 μm, sub‐2 μm, and 2.7 μm Fused‐Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express^TM C18 column packed with Fused‐Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3^TM C18 column packed with 3 μm particles. Column lot‐to‐lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity^TM BEH column packed with sub‐2 μm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high‐efficiency and high‐speed separation to be performed using conventional HPLC instrumentation.     

New validated high-performance liquid chromatographic method for simultaneous analysis of ten flavonoid aglycones in plant extracts using a C18 fused-core column and acetonitrile-tetrahydrofuran gradient

An HPLC method of high resolution has been developed and validated for the simultaneous determination of ten prominent flavonoid aglycones in plant materials using a fused‐core C18‐silica column (Ascentis® Express, 4.6 mm × 150 mm, 2.7 μm). The separation was accomplished with an acetonitrile‐tetrahydrofuran gradient elution at a flow rate of 1 mL/min and temperature of 30°C. UV spectrophotometric detection was employed at 370 nm for flavonols (quercetin [QU], myricetin [MY], isorhamnetin [IS], kaempferol [KA], sexangularetin [SX], and limocitrin [LM]) and 340 nm for flavones (apigenin [AP], acacetin [AC], chrysoeriol [CH], and luteolin [LU]). The high resolution of critical pairs QU/LU (10.50), QU/CH (3.40), AP/CH (2.51), SX/LM (2.30), and IS/KA (2.70) was achieved within 30.3 min. The observed column back pressure was less than 4300 psi, thus acceptable for conventional HPLC equipment. The method was sensitive enough having LODs of 0.115–0.525 ng and good linearity (r > 0.9999) over the test range. The precision values, expressed as RSD values, were <7.5%, and the accuracy was in the range of 95.3–100.2% for all analytes except MY (73.8%). The method was successfully employed for the determination of flavonoids in several medicinal plants, such as Ginkgo biloba, Betula pendula, and a variety of Sorbus species.     

高效液相色谱法测定北柴胡地下部分黄酮类化合物的含量

建立了高效液相色谱法测定北柴胡地下部分黄酮类化合物含量的方法。采用SepaxGP-C18色谱柱(250×4.6 mm,5μm),流动相为乙腈-0.4%磷酸(体积比35∶65),检测波长360 nm,柱温30℃,流速1.0 mL/min。结果表明,北柴胡地下部分含有槲皮素。芦丁、木犀草素、槲皮素、山奈酚、芹菜素分别在0.0050～0.0248、0.0050～0.0248、0.0051～0.0256、0.0046～0.0232、0.0054～0.0272 mg/mL范围内线性关系良好,相关系数分别为0.9957、0.9995、0.9998、0.9998、0.9998,槲皮素的平均回收率为98.34%,相对标准偏差(RSD)为0.76%。该法简便,快速,准确,重复性好,可作为北柴胡药材质量控制的方法。     

Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 μm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 μm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 μm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 μm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 μm average particle size materials for clinical sample analysis.     

Determination of the polyphenolic content of a Capsicum annuum L. extract by liquid chromatography coupled to photodiode array and mass spectrometry detection and evaluation of its biological activity

The present study was aimed to investigate the polyphenolic profile of a pepper (Capsicum annuum L.) extract from Algeria and evaluate its biological activity. The total polyphenol content of the extract was determined as 1.373 mg of gallic acid equivalents (±0.0046), whereas the flavonoids were determined as 0.098 mg of quercetin (±0.0015). The determination of the complete polyphenolic profile of the extract was achieved by liquid chromatography with an RP‐amide column in combination with photodiode array and mass spectrometry detection through an electrospray ionization interface. A total of 18 compounds were identified, of which five were reported for the first time in the sample tested. Quercetin rhamnoside was the most abundant compound (82.6 μg/g of fresh pepper) followed by quercetin glucoside (19.86 μg/g). The antioxidant activity and antimicrobial effects were also determined. For the antimicrobial tests assessed against Gram‐positive and Gram‐negative bacteria, kaempferol showed the strongest inhibitory effect followed by quercetin and caffeic acids. In the study of the cytotoxicity of the extract, the cancer cells (U937) were more affected than the normal cells (peripheral blood mononucleated cells), with more than 62% inhibition at the highest concentration.     

High‐resolution peptide separations using nano‐LC at ultra‐high pressure

We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability (RSD values below 0.18%) was demonstrated applying ultra‐high pressure conditions.     

Nano and rapid resolution liquid chromatography–electrospray ionization–time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil

The applicability of nanoLC‐ESI‐TOF MS for the analysis of phenolic compounds in olive oil was studied and compared with a HPLC method. After the injection, the compounds were focused on a short capillary trapping column (100 μm id, effective length 20 mm, 5 μm particle size) and then nanoLC analysis was carried out in a fused silica capillary column (75 μm id, effective length 10 μm, 3 μm particle size) packed with C18 stationary phase. The mobile phase was a mixture of water + 0.5% acetic acid and ACN eluting at 300 nL/min in a gradient mode. Phenolic compounds from different families were identified and quantified. The quality parameters of the nanoLC method (linearity, LODs and LOQs, repeatability) were evaluated and compared with those obtained with HPLC. The new methodology presents better sensitivity (reaching LOD values below 1 ppb) with less consumption of mobile phases, but worse repeatability, especially inter‐day repeatability, resulting in more difficulties to get highly accurate quantification. The results described in this article open up the application fields of this technique to cover a larger variety of compounds and its advantages will make it especially useful for the analysis of samples containing low concentration of phenolic compounds, as for instance, in biological samples.     

Capillary electrochromatography‐mass spectrometry for the determination of 5‐nitroimidazole antibiotics in urine samples

The separation of eight antibiotics belonging to 5‐nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 μm particle size; Cogent^TM Bidentate C18, 4.2 μm; Pinnacle II? Phenyl, 3 μm; Pinnacle II? Cyano, 3 μm), Cogent? Bidentate C18 (4.2 μm) gave the best performance. For CEC‐MS coupling, a laboratory assembled liquid‐junction‐nano‐spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC‐ESI‐MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica‐60 (5 μm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 μg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5‐nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67–103% range were obtained. Furthermore, for the selected antibiotics, CEC‐MS² spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Integrated microfluidic devices with enhanced separation performance: application to phosphoproteome analyses of differentiated cell model systems

This work reports on the application of a microfluidic device integrating nanoscale LC to nanoelectrospray MS (nano-LC-chip-MS) for the analysis of complex protein digests. Peak profile analyses of more than 700 peptide ions, reproducibly detected across replicate nano-LC-chip-MS runs (n = 5), indicated that the system provided RSD values of 0.24% on retention time, +/- 30 ppm on m/z measurement and +/- 30% variation on intensity over three orders of magnitude. RP adsorbant media with different alkyl chains and particle size packed in both trapping and separation channels were investigated to improve the chromatographic performance of this system. A two-fold improvement in chromatographic peak capacity was achieved using microfluidic devices comprising a 5 mircrom C3 trap with 2.5 microm C18 trap separation channel compared to the traditional 5 microm C18 stationary phase. Enhanced sample selectivity for the identification of phosphopeptides was obtained by combining immobilized metal affinity media prior to peptide separation on the RP microfluidic device. This system was evaluated in the context of differential phosphoproteome analyses to identify changes in signaling events and protein expression of human monocytes following the administration of phorbol ester.     

A particle size distribution analysis of stressed HPLC column packing material

Summary A particle size distribution analysis has been completed on three different HPLC column packing materials including silica gel (Si60) and two bonded phases (RP8 and RP18). The stationary phases were subjected to 18 hours stress with 1 N or 3 N KOH and found to have quantitatively different distribution patterns initially, at 13 hours and finally at 18 hours although the average particle diameters for the Si60 and RP8 were the same or higher at 18 hours as initially. Thirteen hoursstress with sodium octanesulfonate, tetrabutylammonium phosphate and ammonium acetate at exaggerated conditions also resulted in distributional changes with the Si60 and RP8 decreasing in average particle diameter when exposed to ammonium acetate and tetrabutylammonium stressing respectively.     

Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: Comparison of sub‐2 μm particles and conventional C18 columns

A simple and effective multi‐residue analysis method is presented for the extraction and determination of eleven quinolones (pipemidic acid, enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in fish tissues. In this study, multi‐residue separations on four columns packed with 5 μm or sub‐2 μm particles were simultaneously developed for the purpose of comparison. Various gradients were optimized and best resolutions were achieved on each column. A short and sub‐2 μm particle‐sized HPLC column was chosen for its advantages in analysis time and column performance. Additionally, considering the matrix effect of the complex crude fish tissue, an effective extraction protocol was also established for sample pre‐treatment procedure. Good recoveries (71–98%) were obtained from samples fortified with a mix of eleven quinolones at three levels, with satisfactory relative standard deviations and limits of detection. As a result, the sub‐2 μm HPLC column and proposed analytical procedures have been evaluated and applied to the analysis of different fish tissues. Detectable residues were observed in 8 of 30 samples, at concentrations ranging from 4.74 to 23.27 μg/kg.     

Determination of Steroid Hormones in Pharmaceutical Preparations by High Performance Liquid Chromatography

Abstract

The aim of this research was to standardize an high performance liquid chromatographic method for the determination of steroid hormones contained in commercially available pharmaceutical preparations. A Merck LiChrospher^® 100 RP–18 (5 μm) in LiChroCART^® (125-4) column, a rotative valve injector (20 μL loop), ambient temperature, a mobile phase consisting of water-methanol and UV detection at 254nm and 212nm make possible the quantitative determination of dexamethasone acetate, prednisone, ethynylestradiol and norgestrel contained in pharmaceutical preparations.     

液相色谱法同时测定维药蜀葵花中芦丁、槲皮素和山柰酚

维药是祖国医药学不可分割的组成部分。维药现代化,即利用现代技术研究维药的有效成分,是维药科学化、标准化、规范化、商品化和产业化的必经之路。本文建立了维药蜀葵花中有效成分芦丁、槲皮素和山柰酚的选择性提取方法,优化了高效液相色谱法(HPLC)同时测定这3种有效成分的分析条件。采用HC-C₁₈色谱柱(250 mm×4.6 mm, 5 μm)和甲醇-0.4%磷酸(50:50, v/v)流动相,在柱温30 ℃和流速1.00 mL/min的条件下实现了3种物质之间以及和干扰物之间的基线分离。维药蜀葵花中芦丁、槲皮素及山柰酚的线性范围分别为12.5~150 μg/mL (r=0.9998), 12.5~125 μg/mL (r=0.9999)及12.5~125 μg/mL (r=0.9988),加标回收率(n=5)分别为100.3%(RSD=1.1%)、97.60%(RSD=0.47%)、97.75%(RSD=0.71%)。该方法实现了同时测定维药蜀葵花中芦丁、槲皮素及山柰酚,为其他黄酮类物质的开发应用提供了科学依据,同时也可为其他维药分析提供借鉴。     

Sequential injection-bead injection-lab-on-valve coupled to high-performance liquid chromatography for online renewable micro-solid-phase extraction of carbamate residues in food and environmental samples

A sequential injection‐bead injection‐lab‐on‐valve system was hyphenated to HPLC for online renewable micro‐solid‐phase extraction of carbamate insecticides. The carbamates studied were isoprocarb, methomyl, carbaryl, carbofuran, methiocarb, promecarb, and propoxur. LiChroprep^® RP‐18 beads (25–40 μm) were employed as renewable sorbent packing in a microcolumn situated inside the LOV platform mounted above the multiposition valve of the sequential injection system. The analytes sorbed by the microcolumn were eluted using 80% acetonitrile in 0.1% acetic acid before online introduction to the HPLC system. Separation was performed on an Atlantis C‐18 column (4.6×150 mm, 5 μm) utilizing gradient elution with a flow rate of 1.0 mL/min and a detection wavelength at 270 nm. The sequential injection system offers the means of performing automated handling of sample preconcentration and matrix removal. The enrichment factors ranged between 20 and 125, leading to limits of detection (LODs) in the range of 1–20 μg/L. Good reproducibility was obtained with relative standard deviations of <0.7 and 5.4% for retention time and peak area, respectively. The developed method has been successfully applied to the determination of carbamate residues in fruit, vegetable, and water samples.     

Comparison of commercial organic polymer‐based and silica‐based monolithic columns using mixtures of analytes differing in size and chemistry

Commercially available silica‐based monolithic columns Chromolith RP‐8e, Chromolith RP‐18, and Chromolith HR RP‐18, and polymer‐based monolithic columns ProSwift RP‐1S, ProSwift RP‐2H, and ProSwift RP‐3U varying in pore size and bonded phase have been tested for the fast separation of selected sets of analytes. These mixtures of analytes included small molecules (uracil, caffeine, 1‐phenylethanol, butyl paraben, and anthracene), acylated insulins, and intact proteins (ribonuclease A, cytochrome C, transferrin, apomyoglobin, and thyroglobulin), and covered wide range of chemistries and sizes. Small molecules were well separated with a height equivalent to theoretical plate of 11–26 μm using silica‐based monolithic columns, while organic polymer‐based monoliths excelled in the fast sub 1 min baseline separations of large molecules. A peak capacity of 37 was found for separation of acylated insulins on Chromolith columns using a 3 min gradient at a flow rate of 3 ml/min. Poor recovery of proteins from Chromolith columns and significant peak tailing of small molecules using ProSwift columns were the major obstacles in using monolithic columns in those applications.     

HPLC‐DAD methodology for the quantification of organic acids,furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo‐diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250×4.6 mm, 5 μm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan‐3‐ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans‐resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n=5) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rosé and fortified wines.     

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro‐liquid chromatography system. Fully porous, core–shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub‐2 μm fully porous as well as the 2.7 μm core–shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56–2.69) before correction for extra‐column contribution compared to normal‐bore columns. Moreover, the influence of extra‐column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub‐2 μm fully porous particle packed column for ultra‐fast liquid chromatography.