Conventional Liquid Chromatography Research Articles

Two-dimensional Mapping Using Different Chromatographic Separations Coupled with Mass Spectrometry for the Analysis of Ginsenosides in Panax Ginseng Root and Callus

Two-dimensional (2D) mapping using different chromatographic separations coupled with mass spectrometry is a rapid and simple method for the analysis of a mixture using conventional liquid chromatography mass spectrometry. The 2D map could be created from two different chromatograms obtained with the same detector and different columns or separation methods. In this study, 2D mapping was applied to the analysis of components contained in Panax ginseng, and was evaluated in terms of its effectiveness in the separation of these components. The several glycosides included in Panax ginseng could not be sufficiently separated by one-dimensional chromatography with a reverse phase or a hydrophilic interaction chromatography (HILIC) column, but the components of Panax ginseng could be separated and visualized as a component pattern by 2D mapping. We showed that the components contained in the calli and their quantities were altered by the culture conditions in which the calli were grown by 2D mapping. 2D mapping is expected to be a useful method for visualizing complex component patterns found in glycosides and unknown compounds in foods.

Possibilities of retention prediction in fast gradient liquid chromatography. Part 1: Comparison of separation on packed fully porous, nonporous and monolithic columns

Due to ever increasing importance of short analysis times in modern HPLC practice, fast gradient elution is becoming widely used both in one-dimensional and two-dimensional separations, especially in the second dimension where the speed of separation is of primary importance. For method development and optimization, prediction of retention in gradient elution from the isocratic data is very important. This is principally possible using the well-established theory of gradient elution, especially for reversed-phase separations. Feasibility of these approaches has been proven in conventional liquid chromatography and commercial prediction software is available for this purpose. However, fast gradient chromatography employing short columns and gradient times in between 1 and 2min or even less, impose additional stringent demands on the accuracy of prediction, due to rapid changes in mobile phase composition and increased role of extra-column contributions and instrumental dwell volume on the retention. In the present work, possibilities of prediction of the gradient elution times from the isocratic data measured only at two mobile phase concentrations were investigated in fast chromatography of alkylbenzenes, phenylurea and triazine pesticides on five columns differing in lengths: two monolithic ones and three packed with particles of different size. In the second dimension, gradient times are strongly restricted by the short cycle frequency. The effects of the flow-rate on the elution times, on the accuracy of their prediction and on peak capacity were studied under time constraint conditions. Gradient theory can be useful for the optimization of fast gradients.

Development of liquid microjunction extraction strategy for improving protein identification from tissue sections

MALDI Mass Spectrometry Imaging has shown important potential for molecular classification and pathology marker discovery. Protein markers identification is therefore of prime importance. Direct structural analysis from tissue sections has shown limitations for protein identification because of the high degree of complexity of tissues. Only proteins of major abundance are identified this way. On the contrary, conventional proteomics approaches clearly allow for reliable identification of complex protein extracts but do not provide fine correlation with protein location in their original context. Here is presented an approach to obtain identification of proteins of various abundances while keeping their localization within the section. On-tissue trypsin digestion followed by micro-extraction using a liquid micro-junction interface is an efficient strategy to extract tryptic peptides and further identify the associated proteins off tissues. It was shown that conventional Reverse Phase Liquid Chromatography separation on the extracted material followed by MS/MS analysis on a HR FTMS instrument enabled the identification of 1500 proteins on average with high confidence from an area of about 650μm in diameter, which corresponds to an estimated number of 1900 cells in average. The approach can be easily integrated in the MALDI MSI workflow and should provide interesting insights for clinical applications.

Lectin microarrays: concept, principle and applications

The lectin microarray is a novel platform for glycan analysis, having emerged only in recent years. Unlike other conventional methods, e.g., liquid chromatography and mass spectrometry, it enables rapid and high-sensitivity profiling of complex glycan features without the need for liberation of glycans. Target samples include an extensive range of glycoconjugates involved in cells, tissues, body fluids, as well as synthetic glycans and their mimics. Various procedures for rapid differential glycan profiling have been developed for glycan-related biomarkers. Such glycoproteomics targeting allows precise diagnosis of chronic diseases potentially related to cancer. Application of this method to evaluation of various types of stem cells resulted in the discovery of a new pluripotent cell-specific glycan marker. To explore this technology a more fundamental and extensive understanding of lectins is necessary in relation to the structural uniqueness of glycans. In this chapter, the essence of the lectin microarray is described with some focus on an evanescent-field-activated fluorescence detection principle as a system to achieve in situ (i.e., washing free) aqueous-phase observation under equilibrium conditions. The developed lectin microarray system allows even researchers with poor experience in glycan profiling to perform extensive high-throughput analysis targeting various forms of glycans and even cells.

Detection of ketamine and its metabolites in human hair using an integrated nanoflow liquid chromatography column and electrospray emitter fritted with a single porous 10 μm bead

Targeting metabolites incorporated into hair following drug administration is useful for evidential purposes as this approach can aid in differentiating between administration and passive exposure. Greater analytical sensitivity is required than for targeting the parent drug alone. A 20μm i.d. fused silica capillary column with an integrated electrospray emitter fritted with a single porous 10μm polymeric bead has been successfully fabricated to facilitate this purpose. The sensitivity gains through the use of these integrated single fritted columns coupled to a nanoelectrospray source (nanoflow–LC nanoESI) over conventional liquid chromatography–tandem mass spectrometry (LC–MS/MS) columns was explored by their application to the detection of ketamine and its phase I metabolites in human hair. Hair was collected from 4 volunteers following the administration of a small oral dose of ketamine (50mg) and subsequently analysed by the capillary–LC nanoESI approach. The drug and its metabolites were extracted from hair using solid phase extraction following a methanolic wash, pulverisation with a ball mill and acid digestion. From a 50μL extract, 1μL was injected and the method provided a limit of detection estimated to be 5fg on column for ketamine and norketamine and 10fg for dehydronorketamine. The single porous frit minimises extra column band broadening and offers an alternative fritting approach which reduces the blocking of the electrospray emitter, in comparison with other approaches such as sintering and polymerisation.

Determination of trans,trans‐muconic acid in workers' urine through ultra‐performance liquid chromatography coupled to tandem mass spectrometry

A novel method for the biological monitoring of benzene-exposed workers has been developed through ultra-performance liquid chromatography coupled to tandem mass spectrometry. The method uses trans,trans-muconic acid in urine as the benzene-exposure biomarker. The method was developed using a triple quadrupole mass spectrometer with enough sensitivity to facilitate diluting and injecting the urine samples directly, rather than performing a solid-phase extraction procedure as is common in the available protocols. Moreover, compared with a conventional high-pressure liquid chromatography system, the separation power provided by the ultra-performance liquid chromatography system allows a 10-fold reduction in run time. The method was adjusted to a dynamic range of between 198.9 and 4916.7 µg/L to cover the biological exposure index of trans,trans-muconic acid in urine. Also, the method demonstrated intra-day and inter-day precision at 98%, and accuracy within an acceptable range of 101 ± 8%. The method has been used to quantify various types of urine samples, such as workers' urine and inter-laboratory proficiency tests. Depending on the sample, the quantified levels ranged from less than the limit of quantitation to 3836.7 µg/L. No levels exceeding the calibration range were detected in the urine of workers, and the reported concentrations in urine for the proficiency tests were, as expected, based on known values. Moreover, the new method using sample dilution and faster chromatographic run was more effective, facilitating fast communication of results, as needed, to decision-makers.

High Speed Separation of PFCs in Human Serum by C18-Monolithic Column Liquid Chromatography-Tandem Mass Spectrometry

An analytical method has been developed for the rapid determination of perfluorinated compounds (PFCs) in human serum samples. The extraction and purification of PFCs from human serum were performed by the modified method of previous report. Ten PFCs were rapidly separated within 3.3 min by C18-monolithic column liquid chromatography (LC) and detected by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in negative ion mode. The runtime of PFCs on monolithic column LC was up to 4-fold faster than that on conventional column LC. The effect of triethylamine (TEA) to the mobile phase has investigated on the overall MS detection sensitivity of PFCs in ESI ionization. Quantification was performed by LC-MS/MS in multiple-ion reaction monitoring (MRM) mode, using 13 C-labeled internal standards. Method validation was performed to determine recovery, linearity, precision, and limits of quantification, followed by, the analysis of a standard reference material (SRM 1957 from NIST). The overall recoveries ranged between 81.5 and 106.3% with RSDs of 3.4 to 16.2% for the entire procedure. The calibration range extended from 0.33 to 50 ng mL �1 , with a correlation coefficient (R 2 ) greater than 0.995 and the limits of quantification with 0.08 to 0.46 ng mL �1 . This approach can be used for rapid and sensitive quantitative analysis of 10 PFCs in human serum with high performance and accuracy.

Anticancer Constituents and Cytotoxic Activity of Methanol-Water Extract of <em>Polygonum Bistorta</em> L.

This study was specifically designed to identify anticancer constituents in methanol-water extract of Polygonum bistorta L. and evaluate its cytotoxicity. For this purpose methanol-water (40:60 v/v) extract was subjected to conventional preparative high pressure liquid chromatography and 13 fractions were obtained. Constituents of obtained fractions were separated and identified with the help of GC-MS and LC-DAD-ESI-MS. Anticancer phenolic compounds such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, catechol, 4-methyl catechol, syringol and pyrogallol and fatty acids such as linoleic acid, myristic acid and palmitic acid were separated from different fractions. Fractions were evaluated for their cytotoxic activity on a rarely studied human hepatocellular carcinoma cell line (HCCLM3). 11 fractions showed good to strong cytotoxicity in a range of 200 µg/mL-800 µg/mL, whereas 2 fractions did not show any activity even at 800 µg/mL and no anticancer constituent was detected from them. 50 percent growth inhibition (GI50) values for five most active fractions were calculated and results were in a range of 86.5 (±3) µg/mL-126.8 (±3) µg/mL. 3 out of these 5 most active fractions were found to contain phenolic content in them whereas all other fractions containing phenolic content did possess cytotoxic activity that may suggest the importance of phenolic constituents in anticancer activity. Moreover, the results also showed a definite dose dependent relationship between amount of fractions and cytotoxic activity.

A novel method for hydrophobin extraction using CO2 foam fractionation system

Due to the exceptional properties and many potential applications of hydrophobins, special fungal proteins, it becomes necessary to develop a real scale procedure for their production and purification. In our previous study (Deckers et al., 2010) [CO2–hydrophobin structures acting as nanobombs in beer, Brew. Sci. 63:54–61], the strong interaction of CO2–hydrophobin was demonstrated. For the first time, in an approach to isolate hydrophobin HFBII from the growth media of Trichoderma reesei, a foam fractionation system using CO2 as the sparging gas was investigated in this study. Using CO2 foam fractionation, the concentration of HFBII was increased from 0.10±0.02mg/mL up to 0.57±0.04mg/mL. This was shown after a purification step by conventional liquid chromatography and identification of the goal protein using MALDI-TOF. The obtained molecular weight of the protein was 7.042kDa which corresponds to the complete molecule of HFBII, minus the last aminoacid. Micro-spectrophotometry was used for quantification of purified HFBII. Moreover, different parameters of the foam fractionation system were optimized. The concentration of the protein after treatment by CO2 followed by liquid chromatography was increased from 0.32±0.02 to 0.44±0.06mg/mL when the flow rate of gas injection was changed in the range of 1–3L/min. The highest amount of HFBII equal to 0.57±0.04mg/mL was obtained by the highest ratio of liquid height over the column height. Using the larger pore size frits causes increased protein absorption as well. The gushing potential of samples revealed that in contrast to the samples before CO2 treatment, interestingly, no gushing was observed for the samples after treatment. The possibility that stable aggregates of HFBII molecules are formed as a consequence of their high concentration is discussed in this paper. By using DLS analysis of the overfoam, 100nm particle size of CO2 nanobubbles coated by HFBII was obtained. The final concentration of the protein was carried out using Amicon® ultracentrifuge device with the average recovery of 63.8±8.2%.

Modeling Nonspecific Toxicity of Organic Compounds to the Fathead Minnow Fish by Means of Chromatographic Systems

The performance of chromatographic systems to mimic aquatic toxicity to the fathead minnow fish is evaluated taking into account the factors that contribute to the variance of biological-chromatographic correlations. These factors are the precision to measure the fathead minnow toxicity, the precision of the surrogate chromatographic system, and the error from the dissimilarity between the fathead minnow and chromatographic systems. The precisions are estimated through the characterization of the systems by the solvation parameter model. Several chromatographic systems as well as the common reference octanol-water partition system have been selected to test their ability to model the nonspecific toxicity to the fathead minnow by means of the proposed approach. Predictions and experimental tests show that the micellar electrokinetic chromatography system of sodium taurocholate and chromatographic measurements using an immobilized artificial membrane column provide the most precise estimations of this biopartitioning property. The octanol-water partition system, the conventional C18 high-performance liquid chromatography systems, and the micellar electrokinetic chromatography system of sodium dodecylsulfate show worse performances.

Basic Principles of MLC

Micellar liquid chromatography (MLC) is an efficient alternative to conventional reversed-phase liquid chromatography with hydro-organic mobile phases. Almost three decades of experience have resulted in an increasing production of analytical applications. Current concern about the environment also reveals MLC as an interesting technique for “green” chemistry because it uses mobile phases containing 90% or more water. These micellar mobile phases have a low toxicity and are not producing hazardous wastes. The stationary phase is modified with an approximately constant amount of surfactant monomers, and the solubilising capability of the mobile phase is altered by the presence of micelles, giving rise to a great variety of interactions (hydrophobic, ionic, and steric) with major implications in retention and selectivity. From its beginnings in 1980, the technique has evolved up to becoming in a real alternative in some instances (and a complement in others) to classical RPLC with aqueous-organic mixtures, owing to its peculiar features and unique advantages. The addition of an organic solvent to the mobile phase was, however, soon suggested in order to enhance the low efficiencies and weak elution strength associated with the mobile phases that contained only micelles.

Comparison of liquid chromatography-microchip/mass spectrometry to conventional liquid chromatography–mass spectrometry for the analysis of steroids

The feasibility of a microfluidic-based liquid chromatography-electrospray ionization/mass spectrometric system (HPLC-Chip/ESI/MS) was studied and compared to a conventional narrow-bore liquid chromatography-electrospray ionization/mass spectrometric (LC-ESI/MS) system for the analysis of steroids. The limits of detection (LODs) for oxime derivatized steroids, expressed as concentrations, were slightly higher with the HPLC-Chip/MS system (50–300pM) using an injection volume of 0.5μL than with the conventional LC-ESI/MS (10–150pM) using an injection volume of 40μL. However, when the LODs are expressed as injected amounts, the sensitivity of the HPLC-Chip/MS system was about 50 times higher than with the conventional LC-ESI/MS system. The results indicate that the use of HPLC-Chip/MS system is clearly advantageous only in the analysis of low-volume samples. Both methods showed good linearity and good quantitative and chromatographic repeatability. In addition to the instrument comparisons with oxime derivatized steroids, the feasibility of the HPLC-Chip/MS system in the analysis of non-derivatized and oxime derivatized steroids was compared. The HPLC-Chip/MS method developed for non-derivatized steroids was also applied to the quantitative analysis of 15 mouse plasma samples.

Application of a Tandem Mass Spectrometer and Core–Shell Particle Column for the Determination of 151 Pesticides in Grains

A comparison of ultrahigh performance liquid chromatography (UHPLC) with a 2.6 μm core-shell particle column (Kinetex C(18)) and conventional liquid chromatography (LC) with a 3 μm porous particle column (Atlantis dC(18)), coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS), for the determination of 151 pesticides in grains is presented in this study. Pesticides were extracted from grain samples using a procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Quantification, with an analytical range from 5 to 500 μg/kg, was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analogue as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated using a designed experiment, that is, the nested design. The UHPLC (Kinetex C(18)) was superior to conventional LC (Atlantis dC(18)) as it yielded a shorter analytical run time, increased method sensitivity, and improved method performance. For UHPLC/ESI-MS/MS (Kinetex C(18)), 90% of the pesticides studied had recoveries between 81 and 110%, 88% of the pesticides had intermediate precision ≤20%, and 84% of the pesticides showed measurement uncertainty ≤40%. As compared to UHPLC/ESI-MS/MS (Kinetex dC(18)), the LC/ESI-MS/MS (Atlantis dC(18)) showed a relatively lower sensitivity, less repeatability, and larger measurement uncertainty. UHPLC/ESI-MS/MS with 2.6 μm core-shell particle column and scheduled MRM proved to be a good choice for quantification or determination of pesticides in grains.

Advantages of monolithic over particulate columns for multiresidue analysis of organic pollutants by in-tube solid-phase microextraction coupled to capillary liquid chromatography

The performance of a monolithic C 18 column (150 mm × 0.2 mm i.d.) for multiresidue organic pollutants analysis by in-tube solid-phase microextraction (IT-SPME)-capillary liquid chromatography has been studied, and the results have been compared with those obtained using a particulate C 18 column (150 mm × 0.5 mm i.d., 5 μm). Chromatographic separation has been carried out under isocratic elution conditions, and for detection and identification of the analytes a UV-diode array detector has been employed. Several compounds of different chemical structure and hydrophobicity have been used as model compounds: simazine, atrazine and terbutylazine (triazines), chlorfenvinphos and chlorpyrifos (organophosphorous), diuron and isoproturon (phenylureas), trifluralin (dinitroaniline) and di(2-ethylhexyl)phthalate. The results obtained revealed that the monolithic column was clearly advantageous in the context of multiresidue organic pollutants analysis for a number of reasons: (i) the selectivity was considerably improved, which is of particular interest for the most polar compounds triazines and phenyl ureas that could not be resolved in the particulate column, (ii) the sensitivity was enhanced, and (iii) the time required for the chromatographic separation was substantially shortened. In this study it is also proved that the mobile-phase flow rates used for separation in the capillary monolithic column are compatible with the in-valve IT-SPME methodology using extractive capillaries of dimensions similar to those used in conventional scale liquid chromatography (LC). On the basis of these results a new method is presented for the assessment of pollutants in waters, which permits the characterization of whole samples (4 mL) in less than 30 min, with limits of detection in the range of 5–50 ng/L.

On-line analysis of carbonyl compounds with derivatization in aqueous extracts of atmospheric particulate PM 10 by in-tube solid-phase microextraction coupled to capillary liquid chromatography

A new device for carbonyl compounds based on coupling on-line and miniaturizing both, sample pretreatment and chromatographic separation, is reported. Two capillary columns, a GC capillary column (95% methyl–5% phenyl substituted backbone, 70 cm × 0.32 mm i.d., 3 μm film thickness) in the injection valve for in-tube solid-phase microextraction (IT-SPME) and a Zorbax SB C18 (150 mm × 0.5 mm i.d., 5 μm particle diameter) LC capillary column were employed. Different combinations of IT-SPME and derivatization using 2,4-dinitrophenylhydrazine (DNPH) were examined for mixtures containing 15 carbonyl compounds (aliphatic, aromatic and unsaturated aldehydes and ketones). A screening analysis of aqueous extracts of atmospheric particulate PM 10 was carried out. Moreover, the possibility of coupling IT-SPME and conventional liquid chromatography is also tested. Derivatization solution and IT-SPME coupled to capillary liquid chromatography provided the best results for achieving the highest sensitivity for carbonyl compounds in atmospheric particulate analysis. Detection limits (LODs) using a photodiode array detector (DAD) were ranged from 30 to 198 ng L −1, improving markedly those LODs reported by conventional SPME–LC–DAD.

Highly sensitive and accurate screening of 40 dyes in soft drinks by liquid chromatography–electrospray tandem mass spectrometry

A method combining solid phase extraction with high performance liquid chromatography–electrospray ionization tandem mass spectrometry was developed for the highly sensitive and accurate screening of 40 dyes, most of which are banned in foods. Electrospray ionization tandem mass spectrometry was used to identify and quantify a large number of dyes for the first time, and demonstrated greater accuracy and sensitivity than the conventional liquid chromatography–ultraviolet/visible methods. The limits of detection at a signal-to-noise ratio of 3 for the dyes are 0.0001–0.01 mg/L except for Tartrazine, Amaranth, New Red and Ponceau 4R, with detection limits of 0.5, 0.25, 0.125 and 0.125 mg/L, respectively. When this method was applied to screening of dyes in soft drinks, the recoveries ranged from 91.1 to 105%. This method has been successfully applied to screening of illegal dyes in commercial soft drink samples, and it is valuable to ensure the safety of food.

Electrosonic spray ionization—An ideal interface for high-flow liquid chromatography applications

Electrosonic spray ionization (ESSI) has been studied as an interface between high-performance liquid chromatography (HPLC) and mass spectrometry (MS), using sample flow rates up to 3.0 ml min −1. This ionization interface was compared with pneumatically assisted electrospray ionization (ESI) using mass spectrometry for detection. For experiments that did not involve direct comparison of different flow rates, the ESI experiments were performed using post column splitting to work at optimal conditions. ESSI allows the interfacing of conventional or high-resolution liquid chromatography (LC) methods to mass spectrometry without post column splitting. High sample flow rates could be handled without a significant loss of signal intensity using a nebulization gas flow rate of 5.5 L min −1. Since ESI needs to be operated with lower sample flow rates, it is limited to micro/nano LC systems, or post column splitting must be used. In particular, nano LC systems have to be treated with great care and require constant maintenance. When using post-column splitting, the increased diffusion can become a problem especially when using systems with very small void volumes. In all experiments ESSI showed better signal intensities than a commercially available, pneumatically assisted ESI source. ESSI does not require heating of the nebulizer gas, which should help to preserve the original structure of thermally unstable molecules. Therefore, ESSI is presented as an alternative to the commercially available heated ESI sources of AB SCIEX, Thermo Fischer, Agilent and Waters. The observed LC-ESSI-MS ion chromatograms are shown to be very stable even when using flow rates higher than 1.0 ml min −1, which could be very suitable for ultra high performance LC, where sample flow rates up to 2.0 mL min −1 with backpressures up to 1200 bar are used. Also, a difference in the relative intensities of singly and doubly protonated peptide monomers and dimers was observed between the two ionization methods. The coefficients of determination for the calibration of instrument response for Val–Tyr–Val and Met-Enkephalin showed excellent linearity over a wide concentration range (0.1–100 μM), while ESI results were only linear over a much smaller range (0.1–20 μM). The observed behavior is thought to be caused by insufficient ionization efficiency of solutions above ∼20 μM by ESI, exemplifying the robustness of ESSI as an interface between LC and MS.

Nanotechnology and chip level systems for pressure driven liquid chromatography and emerging analytical separation techniques: A review

Pressure driven liquid chromatography (LC) is a powerful and versatile separation technique particularly suitable for differentiating species present in extremely small quantities. This paper briefly reviews main historical trends and focuses on more recently developed technological approaches in miniaturization and on-chip integration of LC columns. The review emphasizes enabling technologies as well as main technological challenges specific to pressure driven separations and highlights emerging concepts that could ultimately overcome fundamental limitations of conventional LC columns.

Rapid quantification of major reaction products formed during thermochemical pretreatment of lignocellulosic biomass using GC–MS

Accurate quantification of reaction products formed during thermochemical pretreatment of lignocellulosic biomass would lead to a better understanding of plant cell wall deconstruction for production of cellulosic biofuels and biochemicals. However, quantification of some process byproducts, most notably acetamide, acetic acid and furfural, present several analytical challenges using conventional liquid chromatography methods. Therefore, we have developed a high-throughput gas chromatography based mass spectrometric (GC–MS) method in order to quantify relevant compounds without requiring time-consuming sample derivatization prior to analysis. Solvent extracts of untreated, ammonia fiber expansion (AFEX) treated and dilute-acid treated corn stover were analyzed by this method. Biomass samples were extracted with acetone using an automated solvent extractor, serially diluted and directly analyzed using the proposed GC–MS method. Acetone was the only solvent amongst water, methanol and acetonitrile that did not contain detectable background levels of the target compounds or facilitate a buildup of plant-derived residues in the GC injector, which decreased analytical reproducibility. Quantitative results were based on the method of standard addition and external standard calibration curves.

Thermal Stability of Thiazide and Related Diuretics During Superheated Water Chromatography

The separations of eleven thiazide and related sulfonamide diuretics using superheated water chromatography at up to 200 °C on an XBridge C18 column were compared with conventional liquid chromatography at ambient temperature. Most of the sulfonamide diuretics were thermally stable, but many of the thiazides were degraded. The most sensitive were the dihydrothiazides, including cyclopenthiazide and hydrochlorothiazide, which were converted to the corresponding aminobenzenedisulfonamides in the pre-column heater. The N-methyl thiazides, including methyclothiazide, appeared to be more stable but degraded on the column and of the thiazides, chlorothiazide eluted largely unchanged although some on-column degradation was observed for benzthiazide. The results confirmed that in superheated water chromatography problems may be encountered with analytes with low storage stability at ambient temperature.