Ultra‐high pressure LC for astaxanthin determination in shrimp by‐products and active food packaging

Nowadays, there is increasing interest in natural antioxidants from food by‐products. Astaxanthin is a potent antioxidant and one of the major carotenoids in crustaceans and salmonids. An ultra‐high pressure liquid chromatographic method was developed and validated for the determination of astaxanthin in shrimp by‐products, and its migration from new packaging materials to food simulants was also studied. The method uses an UPLC® BEH guard‐column (2.1 × 5 mm, 1.7 µm particle size) and an UPLC® BEH analytical column (2.1 × 50 mm, 1.7 µm particle size). Chromatographic separation was achieved using a programmed gradient mobile phase consisting of (A) acetonitrile–methanol (containing 0.05 m ammonium acetate)–dichloromethane (75:20:5, v/v/v) and (B) ultrapure water. This method was evaluated with respect to validation parameters such as linearity, precision, limit of detection, limit of quantification and recovery. Low‐density polyethylene films were prepared with different amounts of the lipid fraction of fermented shrimp waste by extrusion, and migration was evaluated into food simulants (isooctane and ethanol 95%, v/v). Migration was not detected under the tested conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic determination of neutraceuticals, glucosamine sulphate and chitosan, in raw materials and dosage forms

A simple, rapid, selective and specific high performance liquid chromatographic (HPLC) method was developed to quantitate glucosamine and its (β-1-4)-d-polymeric form chitosan. The chromatographic separation was achieved using an aminophase column and refractive index (RI) detection. The mobile phase consisted of ACN:H₂O:CH₃COOH (50:50:0.02) and pH was adjusted to 4.0. The standard curves for glucosamine sulphate showed linearity (r≥0.99) over the concentration range from 20 to 1000 μg ml⁻¹ for raw materials and dosage forms. The precision of the raw material assay expressed as the relative standard deviation (R.S.D.), was less than 3% at all concentrations.Chitosan, poly-(β-1-4)-d-glucosamine compounds, was hydrolysed with 6 M HCl at 100 °C for 10 h and the released glucosamine was determined by the same HPLC method. The proposed method showed linear relation in concentration ranges of 100-500 μg ml⁻¹.The suggested procedure was applied for the determination of glucosamine sulphate and chitosan in their dosage forms and the validity of the method was further checked by applying the standard addition technique. The method was found to be specific with good linearity, accuracy, precision and is well-suited for quantitation of glucosamine sulphate and chitosan in raw materials and pharmaceutical formulations.     

Sensitive and rapid analytical method for the quantification of glucosamine in human plasma by ultra high performance liquid chromatography with tandem mass spectrometry

A highly sensitive and rapid ultra high performance liquid chromatography with tandem mass spectrometry method has been developed and validated for the determination of glucosamine in human plasma using miglitol as the internal standard. Special attention was paid to achieve the high throughput and sensitivity of the established method, and the absence of a matrix effect on the analytes. The sample preparation procedure involved a simple deproteinization step. The chromatographic separation was achieved on a Waters ACQUITY HSS Cyano column using a mixture of acetonitrile/2 mM ammonium acetate solution containing 0.03% formic acid (80:20, v/v) as the mobile phase with a very short run time of 1.5 min. This method was validated over the concentration range of 10–3000 ng/mL for glucosamine. The intra‐ and inter‐batch precision was <13.9% for the low, medium, and high quality control samples. The established method is highly sensitive with a lower limit of quantification of 10 ng/mL, low enough to determine the circadian rhythm on endogenous glucosamine level in human plasma, which has not been reported in detail until now. The method was successfully applied to characterize the pharmacokinetic profile of glucosamine in healthy volunteers following a single oral administration of 750 or 1500 mg glucosamine hydrochloride.     

Ultra-high performance liquid chromatographic method for the determination of polycyclic aromatic hydrocarbons in a passive environmental sampler

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds released in the environment by different sources. The aim of the present work was to validate a solid‐phase extraction (SPE) and a rapid ultra‐high performance liquid chromatographic (UHPLC) method for the analysis of PAHs in a passive environmental sampler, namely a Dacron® (the commercial name of a synthetic fiber based on polyethylene terephthalate) textile. The elution temperature was optimized to improve the resolution of early‐eluted compounds, namely acenaphthene (Ac) and fluorene (F). The UHPLC method lasts about 10 min and showed good linearity for all the 16 PAHs considered, with regression coefficients over 0.99. Recoveries, limits of detection (LODs), and limits of quantification (LOQs) of the SPE method were well within the performance criteria fixed by the Regulation n. 836/2011, namely 0.3 and 0.9 μg/kg, respectively.     

High performance liquid chromatography for the determination of glucosamine sulfate in human plasma after derivatization with 9-fluorenylmethyl chloroformate

In this study, we developed a simple, rapid, sensitive, and reliable method for the determination of glucosamine sulfate in human plasma, which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC-FLD. For the first time, FMOC-Cl was introduced into derivatization of glucosamine sulfate in human plasma. The amino groups of glucosamine sulfate and vertilmicin sulfate (the internal standard) were trapped with FMOC-Cl to form glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts, which can be very suitable for HPLC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C18 column (DIAMONSIL 150 x 4 mm id, 5 microm) with a mobile phase gradient consisting of acetonitrile and water at a flow-rate of 1 mL/min. The retention times of glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts were 8.9 and 21.2 min, respectively. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 15 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-10 mg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 5.2-8.1% and 6.1- 8.5%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 90%. The validated method was successfully applied to the determination of glucosamine sulfate in human plasma samples.     

Adsorption and recovery issues of recombinant monoclonal antibodies in reversed‐phase liquid chromatography†

The poor recovery of large biomolecules is a well‐known issue in reversed‐phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state‐of‐the‐art reversed‐phase wide‐pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150 000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene‐bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42 000 and 443 000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions.     

Simultaneous and rapid determination of deoxynivalenol and its acetylate derivatives in wheat flour and rice by ultra high performance liquid chromatography with photo diode array detection

A simple and reliable method of ultra high performance liquid chromatography coupled with photo‐diode array detection has been proposed for the simultaneous determination of deoxynivalenol and its acetylated derivatives in wheat flour and rice, especially focusing on the optimization of sample extraction, cleanup, and chromatographic separation conditions. Sample pretreatment consisted of a first step using a quick, easy, cheap, effective, rugged, and safe based extraction procedure and a subsequent cleanup step based on solid‐phase extraction. The method was extensively validated in wheat flour and rice, obtaining satisfactory analytical performance with good linearity (R² ≥ 0.999), acceptable recoveries (80.0–104.4%), and repeatability (RSDs 1.3–10.7%). The limits of detection (21.7–57.4 μg/kg) and quantitation (72.3–191.4 μg/kg) for deoxynivalenols were lower than those usually permitted by various countries’ legislation in these food matrices. The method was applied to 34 wheat and rice samples. The results were further compared with results of ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry.     

Hydrostatic countercurrent chromatography and ultra high pressure LC: Two fast complementary separation methods for the preparative isolation and the analysis of the fragrant massoia lactones

Using a one‐step preparative hydrostatic countercurrent chromatography method, the fragrant massoia lactones were purified from the crude massoia bark oil, in less than 3 h. The fractionation was performed with the biphasic solvent system c‐hexane–methanol–water (10:9:1, v/v/v), leading to target compounds with purity over 96%, as determined by GC‐MS and ultra high pressure LC‐MS analyses. Together with C‐10, C‐12 and C‐14 massoia lactones, two other aromatic compounds used in perfumes, benzyl benzoate and benzyl salicylate, were also obtained as pure compounds. In parallel, an easy and efficient ultra high pressure LC method was developed for the ultra‐fast analysis of massoia lactones, as an alternative to long GC‐MS methods.     

HPLC assay for determination of amphotericin B in biological samples

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse-phase C(18) column using an acetonitrile-acetic acid-water (52:4.3:43.7, v/v/v) mixture as mobile phase. The flow rate was 1 mL/min and the effluent was monitored at 406 nm. A linear response over the concentration range 0.1-10.0 microg/mL was obtained. Intra-day and inter-day RSDs were below 5% for all the sample types. This new HPLC method was applied to assay amphotericin B in plasma and several tissue samples such as kidney, liver, spleen and bone marrow. Application of this method to pharmacokinetic studies in mice and dog is provided.     

Effective cleanup for the determination of six quinolone residues in shrimp before HPLC with diode array detection in compliance with the European Union Decision 2002/657/EC

A high‐performance liquid chromatographic method was developed for the determination of six quinolone residues (ciprofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine) in shrimp tissue samples. Separation was carried out by a LiChrospher® 100 RP‐8e column, running at a 22 min gradient elution program, and the mobile phase consisted of citric acid (0.4 mol/L), acetonitrile and methanol. Detection was achieved by a diode array detector, monitoring at 255 and 275 nm. Sample preparation included initial extraction with citric acid solution and further clean‐up by solid‐phase extraction, employing Lichrolut RP‐18 cartridges. Validation was performed according to the European Union Decision 2002/657/EC. The detection capability was 127.2 μg/kg for ciprofloxacin, 115.2 μg/kg for enrofloxacin, 126.2 μg/kg for sarafloxacin, 113.1 μg/kg for oxolinic acid, 125.2 μg/kg for nalidixic acid, and 239.0 μg/kg for flumequine. Recoveries ranged between 83.0 and 121.6%. The Youden test was applied to study the method ruggedness.     

LC method for the direct and simultaneous determination of four major furan derivatives in coffee grounds and brews

The simultaneous quantification of two potential genotoxic hydroxymethyl furan derivatives in coffee (furfuryl alcohol and 5‐hydroxymethylfurfural) alongside their carboxylic acid derivatives (2‐furoic acid and 5‐hydroxymethyl furoic acid, respectively) was carried out. Their extraction from ground roasted coffee using sonication, simple shaking or heat‐assisted shaking lead to similar results. A minimum of 97.3% of the four furan derivatives were extracted during the first extraction cycle using water, whereas methanol showed considerably lower extraction efficiency. A simple high‐performance liquid chromatography method coupled with diode array detection was developed for the simultaneous determination and was applied to roasted coffee extracts or brews. No sample pre‐treatment except for centrifugation was needed. The diode array detector was used to assess the purity of the peaks of interest in analyzed samples against authentic standards. The linearity according to Mandel, accuracy (recovery ≥ 89.9%) and precision (inter‐ and intraday relative standard deviation ≤ 4.5%) were checked. The values for the limit of detection and quantification ranged within 0.11–0.76 and 0.35–2.55 μg/mL, respectively. Filtered and espresso brews were analyzed for the four furan derivatives where furfuryl alcohol showed double the concentration of 5‐hydroxymethylfurfural and about ten times the concentrations of 2‐furoic acid or 5‐hydroxymethyl furoic acid.     

Simultaneous determination of saflufenacil and its two metabolites in soil samples by ultra‐performance liquid chromatography with tandem mass spectrometry

Saflufenacil is a new protoporphyrinogen‐IX‐oxidase inhibitor herbicide. When used, it can enter the soil and has a high risk to reach and contaminate groundwater and aquatic systems. A rapid and sensitive method of ultra‐performance LC with MS/MS was developed for the simultaneous determination of saflufenacil and its two metabolites in soil samples. A modified quick, easy, cheap, effective, rugged, and safe method was applied as the pretreatment procedure. The method was validated by five types of soil samples collected from several regions of China, which all showed good linearity (R² ≥ 0.9914) and precision (RSD ≤ 26.2%). The average recoveries of the three analytes ranged between 74.1 and 118.9% at spiking levels of 3–300 μg/kg. The method limits of detection (S/N 3:1) and method limits of quantification (S/N 10:1) achieved are in the ranges of 0.25–2.75 and 0.83–9.16 μg/kg, respectively. This indicated that the developed ultra‐performance LC with MS/MS method is a promising analytical tool for monitoring the environmental risks posed by saflufenacil.     

Simultaneous determination of three herbicides in wheat,wheat straw,and soil using a quick,easy, cheap,effective, rugged,and safe method with ultra high performance liquid chromatography and tandem mass spectrometry

In this study, a sensitive and effective analytical method for the extraction and detection of three herbicide residues (florasulam, fluroxypyr, and halauxifen‐methyl) in wheat and soil was developed. Samples were extracted with acetonitrile/water followed by salting out, dispersive solid‐phase extraction cleanup, and detection using ultra high performance liquid chromatography coupled with tandem mass spectrometry. The target analytes were detected within a 5 min runtime using an ultra high performance liquid chromatography high‐strength silica trifunctional column connected to an electrospray ionization source in positive mode. The method was validated in five replicates at three fortification concentrations in each matrix. Adequate pesticide quantification and identity confirmation were attained, even at the lowest concentration levels. The method showed very good accuracy and precision. Good recoveries were observed for the three herbicides and mostly ranged between 75.8 and 114.6%, with intraday relative standard deviations <6.01% and interday relative standard deviations <4.02%. The limits of quantification ranged between 0.14 and 7.68 μg/kg for each herbicide. The method was successfully applied for the simultaneous analysis of the three herbicides in actual trial samples, and the results proved that the proposed method was effective in detecting these three herbicides.     

Fast determination of five components of coumarin,alkaloids and bibenzyls in Dendrobium spp. using pressurized liquid extraction and ultra‐performance liquid chromatography

A fast method for simultaneous determination of five compounds (one coumarin, two alkaloids and two bibenzyls) in Dendrobium spp. using pressurized liquid extraction and ultra‐performance liquid chromatography coupled with photo diode array detection was developed. The separation was performed on an Acquity UPLC BEH C₁₈ column (50 mm×2.1 mm id, 1.7 μm) with gradient elution of phosphate buffer (pH=7.1, 20 mM) and acetonitrile within 6 min. The method was validated for linearity, LOD and LOQ, precision and accuracy. All calibration curves showed good linearity (R²≥0.9999) within test ranges. The overall intra‐ and inter‐day variations (RSDs) of five analytes were less than 2.5%, and the detection recoveries were between 95.6 and 102.3%. The developed method was successfully applied to quantitative analysis of five investigated compounds in different species of Dendrobium. The results showed that there were great variations of their contents, though all these materials were officially used as Chinese herb, Shihu. In addition, the chromatographic fingerprints of Dendrobium spp. were also investigated.     

Quantitative determination of bovine caseinoglycomacropeptide in infant formulas by ultra-high-performance liquid chromatography-electrospray-ionization mass spectrometry

An ultra-high-performance liquid chromatography-electrospray ionization coupled to mass spectrometry method has been developed for determining caseinoglycomacropeptide (CGMP) in infant formulas by selected ion reaction and area monitoring modes. The present study focused on the optimization of sample pretreatment, chromatographic resolution and mass spectrometry parameters. After a simple sample pretreatment, the two genetic variants of caseinoglycomacropeptide, CGMP(A) and CGMP(B), were separated using a BEH300 C(18) column by gradient elution. The established method was extensively validated by determining the linearity (R(2)>0.999), average recovery (95.8-118.4%), inter-day precision (relative standard deviation ≤7.81%) and intra-day precision (relative standard deviation ≤6.99%) based on two scan modes. To further verify the applicability of the method, 21 brands of commercial available infant formulas were analyzed. The results showed that the present method is selective, sensitive and reliable for separating and quantifying two genetic variants (CGMP(A) and CGMP(B)) of caseinoglycomacropeptide in infant formulas with complex matrix.     

Recent developments in the HPLC separation of phenolic compounds

Phenolic compounds represent a class of highly complex naturally occurring molecules that possess a range of beneficial health properties. As a result, considerable attention has been devoted to the analysis of phenolics in a variety of samples. HPLC is the workhorse method for phenolic separation. However, conventional HPLC methods provide insufficient resolving power when faced with the complexity of real-world phenolic fractions. This limitation has been traditionally circumvented by extensive sample fractionation, multiple analysis methods and/or selective detection strategies. On the other hand, there is an increasing demand for improved throughput and resolving power from the chromatographic methods used for phenolic analyses. Fortunately, during the last decade, a number of important technological advances in LC have demonstrated significant gains in terms of both speed and resolution. These include ultra high-pressure liquid chromatography (UHPLC), high-temperature liquid chromatography (HTLC), multi-dimensional separations as well as various new stationary phase chemistries and morphologies. In recent years, these technologies have also found increasing application for phenolic analysis. This review seeks to provide an updated overview of the application of recent advances in HPLC to phenolic separation, with the emphasis on how these methodologies can contribute to improve performance in HPLC analysis of phenolics.     

Photolysis and photocatalysis of ibuprofen in aqueous medium: characterization of by‐products via liquid chromatography coupled to high‐resolution mass spectrometry and assessment of their toxicities against Artemia Salina

The degradation of the pharmaceutical compound ibuprofen (IBP) in aqueous solution induced by direct photolysis (UV‐A and UV‐C radiation) and photocatalysis (TiO₂/UV‐A and TiO₂/UV‐C systems) was evaluated. Initially, we observed that whereas photocatalysis (both systems) and direct photolysis with UV‐C radiation were able to cause an almost complete removal of IBP, the mineralization rates achieved for all the photodegradation processes were much smaller (the highest value being obtained for the TiO₂/UV‐C system: 37.7%), even after an exposure time as long as 120 min. Chemical structures for the by‐products formed under these oxidative conditions (11 of them were detected) were proposed based on the data from liquid chromatography coupled to high‐resolution mass spectrometry (LC‐HRMS) analyses. Taking into account these results, an unprecedented route for the photodegradation of IBP could thus be proposed. Moreover, a fortunate result was achieved herein: tests against Artemia salina showed that the degradation products had no higher ecotoxicities than IBP, which possibly indicates that the photocatalytic (TiO₂/UV‐A and TiO₂/UV‐C systems) and photolytic (UV‐C radiation) processes can be conveniently employed to deplete IBP in aqueous media. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination and pharmacokinetic study of four phenol compounds in rat plasma by ultra‐high performance liquid chromatography with tandem mass spectrometry after oral administration of Echinacea purpurea extract

A rapid and sensitive assay based on ultra‐high performance liquid chromatography with electrospray ionization tandem mass spectrometry was established and validated for the simultaneous determination of cichoric acid, chlorogenic acid, quinic acid, and caffeic acid in rat plasma after oral administration of Echinacea purpurea extract using butylparaben as the internal standard. Samples were pretreated by liquid–liquid extraction with ethyl acetate. The separations for analytes were performed on an ACQUITY UPLC HSS C₁₈ column (1.8 μm 2.1 × 100 mm) using a gradient elution program with acetonitrile/10 mM ammonium acetate (pH 5.6) at a flow rate of 0.3 mL/min. The analytes were detected in multiple reaction monitoring mode with negative electrospray ionization. The lower limit of quantification of each analyte was not higher than 10.85 ng/mL. The relative standard deviation of the intraday and interday precisions was less than 14.69%. The relative errors of accuracies were in the range of –13.80 to 14.91%. The mean recoveries for extraction recovery and matrix effect were higher than 80.79 and 89.98%, respectively. The method validation results demonstrated that the proposed method was sensitive, specific, and reliable, which was successfully applied to the pharmacokinetic study of four components after oral administration of Echinacea purpurea extract.     

超高效液相色谱法同时测定护肤品中的川芎嗪、甘草苷与荷叶碱

建立了一种同时测定水剂、乳液、膏霜、啫喱类护肤品中川芎嗪、甘草苷和荷叶碱的超高效液相色谱(UPLC)分析方法。不同基质样品经甲醇超声提取后,采用Waters BEH C18色谱柱(2.1 mm×50 mm,1.7μm),以0.05%磷酸水溶液-乙腈为流动相进行梯度洗脱分离,二极管阵列检测器检测,结合保留时间和光谱图定性,以标准曲线定量。实验结果表明,3种待测物在0.05~50.0 mg/L浓度范围内线性关系良好,相关系数为0.999 4~0.999 9;川芎嗪、甘草苷和荷叶碱的方法检出限(S/N=3)分别为0.6、0.6、0.3 mg/kg,方法定量下限(S/N=10)分别为2.0、2.0、1.0 mg/kg;加标水平为1~200 mg/kg时,3种待测物的平均回收率为85.6%~98.2%,相对标准偏差(RSD,n=6)为1.1%~6.1%。该法前处理简单、回收率高、精密度好,适用于不同护肤品中川芎嗪、甘草苷和荷叶碱的测定。