Advantages and limitations of HPLC in environmental analysis

Summary The application of HPLC to environmental analysis is often hindered by difficulty not experienced in other areas of analysis. Usually the components being determined are at parts per million levels or less and are usually in sample matrices that can yield many interferences. In order to develop successful methodology the prime requirements for an HPLC system are column efficiency and the sensitivity and selectivity of the detection system. In this presentation, examples are given to illustrate how HPLC can be used to advantage by comparing it to gas chromatographic (GC) methods and even TLC methods. For many classes of compounds, such as halogenated hydrocarbons (pesticides, PCB’s, dioxins), the GC methods may be preferred. However, for polycyclic aromatic hydrocarbons (PAH) HPLC with fluorescence detection has proven to be excellent for trace environmental analysis. Comparisons of HPLC with TLC for aflatoxins and with bioassays for paralytic shellfish toxins are made. Novel combinations such as headspace-HPLC analyses for SO₂ and HPLC-AA for organometallic compounds are discussed.     

Introduction of HPLC/orbitrap mass spectrometry as screening method for doping control

A new doping control screening method has been developed, for the analysis of doping agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and atmospheric pressure chemical ionization. The developed method allows the detection of 29 compounds, including agents with antiestrogenic activity, beta(2) agonists, exogenous anabolic steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm using an external reference. The detection limit for all compounds tested was better than 100 pg/ml. The recoveries of most analytes were above 70%. The measured median repeatability values for doping agents included in the method at concentrations of 1 and 10 ng/ml were 21 and 17%, respectively. The relative standard deviation (RSD) of the intraday precision (n = 6) ranged from RSD = 16-22%, whereas the interday precision (n = 18), ranged from RSD = 17-26%, depending on the solute concentration investigated.     

高效液相色谱法同时测定血浆中的10种蛋白同化激素

建立了一种用于10种蛋白同化激素的同时分离检测的高效液相色谱法。根据被分析物的性质,以C18反相色谱柱为分离柱,以乙腈和水为流动相,采用梯度洗脱方式,并在194～290 nm的范围内快速调节检测波长,使各物质均在最大吸收波长处被检出。在优化的条件下,10种被测组分在10 min内实现了快速的基线分离,检出限在0.01～0.10 μg/mL范围内。在兔血浆中进行加标回收率测定,10种被测组分的加标回收率为70.3%～120%。选取美雄醇为代表进行实际动物实验,成功检测到耳脉注射美雄醇后兔血浆内的美雄醇成分。实验结果表明该方法可行,快速简便,准确可靠。     

Application of HPLC to measure vanadium in environmental,biological and clinical matrices

Vanadate and vanadium compounds exist in many environmental, biological and clinical matrices, and despite the need only limited progress has been made on the analysis of vanadium compounds. The vanadium coordination chemistry of different oxidation states is known, and the result of the characterization and speciation analysis depends on the subsequent chemistry and the methods of analysis. Many studies have used a range of methods for the characterization and determination of metal ions in a variety of materials. One successful technique is high performance liquid chromatography (HPLC) that has been used mainly for measuring total vanadium level and metal speciation. Some cases have been reported where complexes of different oxidation states of vanadium have been separated by HPLC. Specifically reversed phase (RP) HPLC has frequently been used for the measurement of vanadium. Other HPLC methods such as normal phase, anion-exchange, cation-exchange, size exclusion and other RP-HPLC modes such as, ion-pair and micellar have been used to separate selected vanadium compounds. We will present a review that summarizes and critically analyzes the reported methods for analysis of vanadium salts and vanadium compounds in different sample matrices. We will compare various HPLC methods and modes including sample preparation, chelating reagents, mobile phase and detection methods. The comparison will allow us to identify the best analytical HPLC method and mode for measuring vanadium levels and what information such methods provide with regard to speciation and quantitation of the vanadium compounds.     

Analytical methods for anti-doping control in sport: anabolic steroids with 4,9,11-triene structure in urine

Doping control in sport is mandatory to detect and to control the use of prohibited substances. Due to the growing number of targets, the analysis of doping compounds and their metabolites is carried out using established screening methods. However, detection of anabolic steroids with 4,9,11-triene structure in urine is problematic, so it is necessary to improve the methods.We review the state of the art in doping-control analysis of 4,9,11-trien-3-one steroids, providing an overview of the screening and confirmatory methods developed for these analytes in human urine. First, we review chromatographic techniques. We discuss difficulties in the derivatization of those compounds prior to gas chromatography analyses. In recent years, liquid chromatography has been the preferred technique in drug testing in sport, due to the reduced sample pre-treatment, improved limits of detection and comprehensiveness. We also report on advances and limitations of immunochemical techniques for the analysis of this group of substances.     

Quantitative analysis of anabolics on thin-layer chromatographic plates using image analysis techniques

A new detection system is introduced for the quantitative analysis of thin-layer chromatographic plates, which is based on a relatively simple, cheap but advanced image analysis system. Both one- and two-dimensional plates can be analysed. Recording and analysis can also be performed from photographs or even slides. Applications are shown for a number of samples containing anabolic compounds.     

Determination of residual anabolic steroid in meat by gas chromatography-ion trap-mass spectrometer

The use of natural and synthetic anabolic steroids in animal fattening has been prohibited in Taiwan and many countries because of their potential toxic effect on public health. This paper describes a newly developed gas chromatography-ion trap-mass spectrometry (GC-IT-MS) method for the quantitative determination of various residual anabolic steroids in meat. Anabolic steroid was derivatized with N-methyl-N-trimethylsilytrifluoroacetamide prior to GC-IT-MS analysis. MS² was employed for quantitative measurement. In addition, 2d-estradiol was used as an internal standard. Quantitative determination was based on the ratio of peak area of steroid derivative to peak area of internal standard derivative. Good linearity of each compound, 0.03-1.0 μg/ml, was determined. Solvent extraction was used to extract residual anabolic compounds in meat samples and a solid phase extraction (SPE) procedure was utilized for sample cleanup and pre-concentration. The limits of detection of anabolic compounds approximately ranged from 0.1 to 0.4 μg/kg. The detection limit was comparable with or better than reported methods and was below the minimum required performance limits (MRPLs) established by the European Community (EC). The application of this newly developed method was demonstrated by analyzing various beef, pork, chicken and several animal internal organ samples from local markets.     

Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.     

Fast determination of tetracycline antibiotics in different media by high-performance liquid chromatography

Summary The use of high-performance liquid chromatography (HPLC) for the identification and determination of tetracycline antibiotics is reviewed. HPLC chromatograms provide fast identification by retention time, t_R, and precise quantitation by measurement of peak height or peak area. For separation of tetracycline compounds, most HPLC methods use reversed-phase C₁₈ or C₈ columns and UV detection. The HPLC solvent system should have a pH of about 6 to prevent steric changes in the tetracycline molecule. For accurate quantitation it is necessary to avoid tailing and this is accomplished by adding a zwitter ion to the solvent system. Methanol and acetonitrile are frequently used as organic modifiers in these solvent systems. In a single analysis, HPLC methods can be used to separate as many as nine or ten commercially used tetracycline compounds and to determine four to five tetracyclines in commercial tetracycline preparations or in biological fluids.     

Metabolism of some anabolic agents: toxicological and analytical aspects

The metabolism of the animal growth promotants diethylstilbestrol, zeranol and 17 beta-trenbolone and of a few anabolizing steroids used in humans is briefly reviewed. The possible role of reactive metabolic intermediates in the toxicity of some anabolic agents is discussed. Analytical implications of the metabolism of anabolizing agents are described and examples of the analysis of metabolites by means of recently developed techniques are given. It is proposed to utilize the covalent binding of reactive metabolites of anabolic compounds to blood proteins such as haemoglobin and serum albumin for retrospective doping analysis.     

Use of solid-phase microextraction for measuring oil-water partition coefficients and correlation with high-performance liquid chromatographic methods for lipophilicity

For flavour compounds, lipophilicity is often estimated by the partition coefficient between oil and water (log Koil-water), which is highly relevant to food. A modification of the shake-flask method is reported here where compounds are quantified in the two phases using solid-phase microextraction (SPME). SPME's highly sensitivity to non-polar compounds facilitates quantification in the water phase. Twelve flavour compounds representing a broad range of lipophilicities and functional groups were analysed by two methods. Their log Koil-water was determined using SPME quantitation and their log k(w) using a reversed-phase HPLC methodology. The isocratic capacity factor at 60% methanol and predicted log P value also showed high correlation factors with other methods. The octadecyl silylated surface of the HPLC column provides a matrix that interacts with lipophilic compounds where the retention time is the indication of lipophilicity. Both methods gave reproducible results (median 3% and 4% RSD) and similar but not identical values for lipophilicity. The relationship between the two methods is log k(w) =0.85 log Koil-water +0.48 with a correlation coefficient of 0.94. The new SPME detection method, with the ability to quantify limonene and 2-pentylfuran at 1 ppm in the water phase, is preferred for flavour compound analysis due to the applicability of oil-water partitioning in food.     

Zero‐valent Iron Nanoparticles Modified Screen‐printed Electrode for FIA or HPLC Amperometric Detection of Metronidazole in Pharmaceutical Formulations

A zero‐valent iron nanoparticles modified electrode was employed as an amperometric detector in flow conditions to determine metronidazole and 2‐methyl‐5‐nitroimidazole in pharmaceutical formulations. Flow injection analysis at ?0.6 V (vs. Ag) achieved limits of detection of 2 and 6 μM for metronidazole and 2‐methyl‐5‐nitroimidazole, respectively, but the analysis was not discriminative between the two compounds. When reverse phase high performance liquid chromatography was applied previous to the electrochemical detection both analytes could be analysed simultaneously. However, the limits of detection slightly increased (10 μM) as a consequence of the use of an organic solvent and a lower sample volume. The relative standard deviation of analytical repeatability was <4.0 % in both techniques. The methods were validated by comparing the results obtained from the analysis of commercial samples with those provided by HPLC‐DAD and no significant differences were detected. Results probed that the modified electrode was a successful tool in the FIA and HPLC analysis of nitro compounds.     

On-Line HPLC with Biochemical Detection for Screening Bioactive Compounds in Complex Matrixes

On-line high-performance liquid chromatography (HPLC) coupled with biochemical detection (BCD) has been developed to screen compounds showing antioxidant action, enzyme inhibition and receptor affinity in complex matrixes. This review summarizes HPLC methods combining different post-column detection methods, such as diode-array detection (DAD), mass spectrometry (MS), chemiluminescence (CL) and nuclear magnetic resonance, for antioxidant screening. The methods based on a single relatively stable reagent such as DPPH^• and ABTS^•+ were the most popular. Oxygen free radical scavengers mainly depended on post-column CL detection. The on-line hyphenated HPLC–BCD systems based on post-column UV/DAD fluorescence and MS detection were also widely applied to screen enzyme- and receptor-active compounds. These strategies provide a convenient tool for quick identification and quantification of active compounds in complex matrixes.

     

Targeted three-dimensional liquid chromatography: a versatile tool for quantitative trace analysis in complex matrices

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.     

Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most abundant ion. Results of validation experiments are presented. The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.     

Determination of organic alkyl- and 1-hydroxy hydroperoxides with chemiluminescence, fluorescence and GC/MS

Summary In this study three methods for the determination of peroxy compounds with HPLC are presented. The applicability of these methods with respect to atmospheric chemistry is discussed. In rain samples 1-hydroxy hydroperoxides are determined with a detection limit of 0.058 mol/l. Alkyl hydroperoxides are determined in a mixture of hydrocarbon, hydrogen peroxide and air which has been irradiated with light before analysis. A new method for the detection of peroxides in such photolysis mixtures by GC/MS is presented.     

类固醇兴奋剂分析方法的研究进展

蛋白同化雄性类固醇是一类滥用最为普遍的兴奋剂物质,对其进行有效的控制和检测关系到运动员的身心健康和体育比赛的公平公正。对类固醇兴奋剂分析方法的改进和发展是目前兴奋剂检测的重要任务。本文主要是对自2002年以来类固醇兴奋剂样品的预处理和检测手段的研究进展做一概述,包括气相色谱-质谱法、液相色谱-质谱法、免疫法、电化学方法以及质谱法等。     

Determination of a new anti-allergenic agent, 1-[4-[3-[4-[bis-(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone, and its active acidic metabolite in plasma by high-performance liquid chromatography

High-performance liquid chromatographic (HPLC) methods were developed for the analysis of two compounds in a series of new antiallergenic agents, 1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone and its active acidic metabolite in plasma. The methods utilize ultraviolet or fluorescence detection, liquid-liquid extraction or solid-phase extraction and reversed-phase HPLC. The drugs were quantitated in samples from bioavailability studies performed in dogs. Calibrations were in the ng/ml concentration range for both compounds in plasma.     

Radiochemical analysis of the polar agrochemicals validamycin and the metabolite melamine by ion-exchange chromatography

Suitable analytical methods to determine the radiochemical purity of validamycin and melamine are not described in the literature. High performance ion-exchange chromatography for the detection of underivatized validamycin and melamine has been proposed. The compounds were separated on HPLC using a styrene-divinylbenzene copolymer based cation-exchanger. For the separation of validamycin a resin in the hydrogen-form has given a good separation and for melamine in the Na⁺-form. For the first time an ion-exchange separation of validamycin is described allowing the separation of at least four components of commercial validamycin: validamycin A, validamycin B or G, validoxylamine A and validoxylamine B or G without prior derivatization. The methods were applied to determine the radiochemical purity of ¹⁴C- or ³H labeled compounds.This revised version was published online in November 2005 with corrections to the Cover Date.