Sample Preparation Conditions Research Articles

Micelle separation conditions based on particle size strongly affect carotenoid bioaccessibility assessment from juices after in vitro digestion

For assessing the carotenoid bioaccessibility during in vitro digestion, obtaining the micelle fraction that can diffuse through the mucin layer according to the particle size is an important step. However, the accuracy of the various approaches to obtaining the right fraction of micelles described in literature has not been investigated. In many studies the reported bioaccessible fraction might therefore be over- or underestimating the real bioaccessibility. The present study aimed to fill this gap and give approaches to get accurate data on the bioaccessible fraction from samples with different properties. Results illustrated that optimizing the centrifugal speed and duration of the digesta are essential in obtaining the micelle fraction. Different digesta have different optimum centrifugal parameters to obtain the bioaccessible fraction. 6,000 rpm and 40 min was selected as the optimum centrifugal parameter for combined juice (CJ). 6,000 rpm and 60 min was the optimum centrifugal parameter for CJ with oil, and CJ with emulsion using non-ionic emulsifier. 8,000 rpm and 20 min was the optimum centrifugal parameter for CJ with emulsion using ionic emulsifier. Polygalacturonanase (PG)-digested systems required higher centrifugal speed (10,000 rpm) compared with CJ-based systems (6,000 rpm or 8,000 rpm). A prediction model to determine the optimal centrifugation speed/time from the properties of the intestinal digesta was developed. Sample preparation conditions strongly affect carotenoid bioaccessibility assessment from juices during in vitro digestion. Based on these results, it is highly recommended to perform an optimized preparation procedure for bioaccessible fraction prior to carotenoid bioaccessibility analysis.

Automated high-throughput in vitro assays to identify metabolic hotspots and protease stability of structurally diverse, pharmacologically active peptides for inhalation

The inhalation of peptides comes with the advantage of directly targeting the lung as tissue of interest. However, peptides are often rapidly metabolized in lung tissue through proteolytic cleavage. We have developed an assay workflow to obtain half-life and metabolite ID data for peptides incubated with four proteases abundant in lungs of asthma and COPD patients. The assay system has been validated using 28 structurally diverse linear and cyclic peptides with a molecular weight between 708 and 5808 Da. Experimental conditions for incubation, sample preparation, chromatography, data acquisition and analysis are compatible with the required throughput in early stage peptide projects. Together with co-crystal structures and Ala scans, we are using the described assay workflow to guide the first chemical modifications of peptide hits in early respiratory drug discovery projects.

Determination of zinc by atomic absorption in automobile tire rubber as a source of ecotoxicants in atmospheric air

Annotation. A methodology for determining zinc in tires (as a source of ecotoxicants in the atmospheric air) by atomic absorption with microwave pre-treatment as a product of motor vehicle wear in St. Petersburg was developed. In the article, the author gives different ways of sampling for testing. One of the rubber samples was taken from one area no larger than 5x5 cm on one tyre (first sampling option), the second sample was taken cumulatively from all tyre sides around its circumference (second sampling option). Tests of selected samples were carried out allowing conclusions to be drawn about the optimal sampling method. A comparative analysis has been made of the test results of samples taken by different methods from the car tyre surface. Based on the comparative analysis conclusions were made about the homogeneity of the tire composition. Various physical and chemical methods of sample preparation for the heavy metals determination are presented. In the course of the work the author selected the optimal conditions for sample preparation for testing allowing to obtain the maximum possible extraction of the analyte at the stage of sample preparation. The atomic absorption method was used to determine the zinc content in automobile tire samples. On the basis of the results obtained conclusions about the optimal sampling methods and sample preparation were made and the test conditions of the samples were optimized.

Determination of zinc by atomic absorption in automobile tire rubber as a source of ecotoxicants in atmospheric air

Annotation. A methodology for determining zinc in tires (as a source of ecotoxicants in the atmospheric air) by atomic absorption with microwave pre-treatment as a product of motor vehicle wear in St. Petersburg was developed. In the article, the author gives different ways of sampling for testing. One of the rubber samples was taken from one area no larger than 5x5 cm on one tyre (first sampling option), the second sample was taken cumulatively from all tyre sides around its circumference (second sampling option). Tests of selected samples were carried out allowing conclusions to be drawn about the optimal sampling method. A comparative analysis has been made of the test results of samples taken by different methods from the car tyre surface. Based on the comparative analysis conclusions were made about the homogeneity of the tire composition. Various physical and chemical methods of sample preparation for the heavy metals determination are presented. In the course of the work the author selected the optimal conditions for sample preparation for testing allowing to obtain the maximum possible extraction of the analyte at the stage of sample preparation. The atomic absorption method was used to determine the zinc content in automobile tire samples. On the basis of the results obtained conclusions about the optimal sampling methods and sample preparation were made and the test conditions of the samples were optimized.

Discovery and quantification of lipoamino acids in bacteria

Improving knowledge about metabolites produced by the microbiota is a key point to understand its role in human health and disease. Among them, lipoamino acid (LpAA) containing asparagine and their derivatives are bacterial metabolites which could have an impact on the host. In this study, our aim was to extend the characterization of this family. We developed a semi-targeted workflow to identify and quantify new candidates. First, the sample preparation and analytical conditions using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) were optimized. Using a theoretical homemade database, HRMS raw data were manually queried. This strategy allowed us to find 25 new LpAA conjugated to Asn, Gln, Asp, Glu, His, Leu, Ile, Lys, Phe, Trp and Val amino acids. These metabolites were then fully characterized by MS2, and compared to the pure synthesized standards to validate annotation. Finally, a quantitative method was developed by LC coupled to a triple quadrupole instrument, and linearity and limit of quantification were determined. 14 new LpAA were quantified in gram positive bacteria, Lactobacilus animalis, and 12 LpAA in Escherichia coli strain Nissle 1917.

Analytical performance of Compton/Rayleigh signal ratio by total reflection X‐ray fluorescence (TXRF): A potential methodological tool for sample differentiation

AbstractThe high sensitivity Compton and Rayleigh X‐ray scattering signals can be used to gain valuable information on the chemical composition of various matrices, by exploiting the ratio of those signals as a function of the effective atomic number (Zeff). Neither total reflection X‐ray fluorescence (TXRF) nor the effect of the experimental setup, including sample preparation, X‐ray excitation source selection, and band deconvolution procedure, has been assessed in this kind of approach. Here, a Compton/Rayleigh ratio and Zeff‐based TXRF method was set up and tested as an analytical tool for milk samples differentiation. The method was developed using a 90° scattering angle and assessed using different X‐ray excitation sources: a molybdenum tube (Mo Kα 17.5 KeV) and a tungsten tube (W Lα 8.5 KeV and W‐Brems 35 KeV). The evaluation of independent Compton and Rayleigh signals was performed by non‐Gaussian and Gaussian curve resolution methods, and both height and area‐based calculations were evaluated. Different sample preparation conditions were assessed. By using 11 standard materials, a calibration curve for Compton/Rayleigh ratio versus Zeff was established. The method was tested to determine the Zeff of milk samples, which enabled its use as a parameter to differentiate them. Good precisions were obtained with the Mo excitation source and the area‐based calculations, which allowed to differentiate undiluted milk samples by species, treatment, and fat content according to their Compton/Rayleigh ratio. This simple and rapid method has the potential to be used for the differentiation of various types of samples, including liquids, solids, and aerosols.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

N-Glycosylation Profiling of Human Blood in Type 2 Diabetes by Capillary Electrophoresis: A Preliminary Study.

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis bioanalytical technique, for a high-resolution determination of the N-glycan profile in human blood samples of patients with type 2 diabetes (T2D). To achieve the profile information of these complex oligosaccharides, linked by asparagine to hIgG in the blood, the glycoproteins of the samples needed to be cleaved, labelled, and purified with sufficient yield and selectivity. The resulting samples were analyzed by capillary electrophoresis, with laser-induced fluorescence detection. After separation parameter optimization, the capillary electrophoresis technique was implemented for efficient N-glycan profiling of whole blood samples from the diabetic patients. Our results revealed that there were subtle differences between the N-glycan profiles of the diabetic and control samples; in particular, two N-glycan structures were identified as potential glycobiomarkers that could reveal significant changes between the untreated/treated type 2 diabetic and control samples. By analyzing the resulting oligosaccharide profiles, clinically relevant information was obtained, revealing the differences between the untreated and HMG-CoA reductase-inhibitor-treated diabetic patients on changes in the N-glycan profile in the blood. In addition, the information from specific IgG N-glycosylation profiles in T2D could shed light on underlying inflammatory pathophysiological processes and lead to drug targets.

Sample preparation and culture condition effects on MALDI-TOF MS identification of bacteria: A review.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI-TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI-TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.

Low-cost natural carbon dioxide sorbents available in the Czech Republic

The article deals with the issue of carbon dioxide adsorption on mineral samples, two of which are rich in montmorillonite and one in kaolinite. The last comparative sample is clinoptilolite, which is widely used as a sorbent in agriculture, water treatment, etc. The theoretical part summarizes several current researches on the use of bentonites as adsorbents, both in their raw form and after various chemical treatments. The study presented here does not suggest any modification procedure, but tests untreated samples and samples subjected to calcinations at temperatures of 250-750 ° C. The calcination of units of grams was carried out by means of a carousel TGA, which made it possible to record curves of mass changes and to obtain a sufficient amount of calcinates for further analyses at the same time. From the point of view of achieving the highest specific surface area and the total pore volume, the optimal calcination temperature for the phyllosilicate samples ranged from 250 to 450 °C. Natural zeolite, on the other hand, showed a deterioration of both of these parameters at any temperature exceeding 150 °C. The same temperature dependence was found in the case of adsorption capacities determined by an automatic analyser Autosorb IQ using pure CO2. Measurements on this instrument also confirmed that selected inexpensive natural materials provide comparable adsorption capacities as the commercially available 13X molecular sieve used as a reference sample. Based on the performed analyses, the initial conditions of sample preparation for the upcoming measurement of adsorption properties on a larger apparatus operating in the PSA/TSA mode were determined. The primary aim of the tests using the selfdesigned high-pressure adsorption unit will be to determine the adsorption capacities that will take into account the temperature and pressure conditions in a real postcombustion carbon dioxide capture system. Unlike the automatic analyser described above, it will be possible to quantify the influence of important factors such as: flue gas humidity, the presence of other permanent gases (especially SO2) and last but not least various CO2 partial pressures and absolute pressure during adsorption and desorption. The experiments will verify the extent to which the presence of noncondensing moisture in the gaseous mixture is problematic. In the case of phyllosilicates, it is not just the parallel adsorption of H2O that affects the adsorption capacity available for CO2 capture. It will be empirically determined to what extent the swelling of the sorbent occurs in the wet gas, changing the gas flow through the layer and especially the pressure loss. The results of measurements on high-pressure apparatus will be the basis for the design and construction of a larger pilot scale unit.

Application of Homogeneous Liquid-Liquid Microextraction With Switchable Hydrophilicity Solvents to the Determination of MDMA, MDA and NBOMes in Postmortem Blood Samples.

Synthetic drugs for recreational purposes are in constant evolution, and their consumption promotes a significant increase in intoxication cases, resulting in damaging public health. The development of analytical methodologies to confirm the consumption of illicit drugs in biological matrices is required for the control of these substances. This work exploited the development of an extraction method based on homogenous liquid-liquid microextraction with switchable hydrophilicity solvent (SHS) as extraction phase for the determination of the synthetic drugs 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and N-methoxybenzyl-methoxyphenylethylamine derivates (25B, 25C and 25I) in postmortem blood, followed by liquid chromatography coupled to mass spectrometry in tandem. The optimized sample preparation conditions consisted of using 250 µL of ZnSO4 10% and 50 µL of NaOH 1 mol/L in the protein precipitation step; N,N-dimethylcyclohexylamine was used as SHS, 650 μL of a mixture of SHS:HCl 6 mol/L (1:1 v/v), 500 μL of whole blood, 500 μL of NaOH 10 mol/L and 1 min of extraction time. The proposed method was validated, providing determination coefficients higher than 0.99 for all analytes; limit of detection and limit of quantitation ranged from 0.1 to 10 ng/mL; intra-run precision from 2.16% to 9.19%; inter-run precision from 2.39% to 9.59%; bias from 93.57% to 115.71% and matrix effects from 28.94% to 51.54%. The developed method was successfully applied to four authentic postmortem blood samples from synthetic drugs users, and it was found to be reliable with good selectivity.

Optimization of conditions for esterification of thiodiacetic acid in urine using mathematical planning for biological monitoring

Introduction. To assess the impact of the toxicants vinyl chloride (VC) and 1,2-dichloroethane (DCE) on humans, personalized biomonitoring of thiodiacetic acid (TDAA) is of most significant importance. Determination of TDAA in urine was carried out using the method of gas chromatography-mass spectrometry GC-MS. Materials and methods. Sample preparation consists of the analyte’s esterification in a biological matrix with methyl alcohol (with 10% boron trifluoride), extraction of the derivative by liquid extraction with ethyl acetate. We used an Agilent 7890A gas chromatograph with an HP-5MS capillary column and a mass-selective detector. TDAA was identified in the form of a dimethyl ester on a mass chromatogram according to the retention time and the ratio of the intensities of the registered ions. Results. To determine the rational parameters of the sample preparation process for the determination of TDAA in urine studies were carried out under the method of planning the experiment, which makes it possible to obtain the most accurate mathematical description of the processes. Optimization of the conditions for esterification of TDAA was carried out using mathematical planning, varying the temperature, process time, and the nature of the catalyst (BF3 or H2SO4). The scheduling matrix included eight experiments; the degree of conversion of TDAA served as an optimization parameter. Interpretation of the model showed that temperature makes more contribution to the formation of the degree of conversion than the processing time. The nature of the catalyst does not affect the degree of conversion. Conclusion. A mathematical model developed for optimizing the conditions for sample preparation of the biomarker of exposure to VC (TDAA) in urine, showed the contribution of three factors (reaction temperature, reaction time, catalyst nature) to the conversion rate, of which the reaction temperature makes the greatest contribution to the choice of optimal esterification conditions. The nature of the catalyst (BF3 or H2SO4) does not affect the conversion rate.

Evaluation of Effects on Tritium Measurement According to HTO Type Sample Preparation Conditions

Evaluation of Effects on Tritium Measurement According to HTO Type Sample Preparation Conditions

A quality optimization approach to image Achilles tendon microstructure by phase-contrast enhanced synchrotron micro-tomography

Achilles tendons are mechanosensitive, and their complex hierarchical structure is in part the result of the mechanical stimulation conveyed by the muscles. To fully understand how their microstructure responds to mechanical loading a non-invasive approach for 3D high resolution imaging suitable for soft tissue is required. Here we propose a protocol that can capture the complex 3D organization of the Achilles tendon microstructure, using phase-contrast enhanced synchrotron micro-tomography (SR-PhC-μCT). We investigate the effects that sample preparation and imaging conditions have on the resulting image quality, by considering four types of sample preparations and two imaging setups (sub-micrometric and micrometric final pixel sizes). The image quality is assessed using four quantitative parameters. The results show that for studying tendon collagen fibers, conventional invasive sample preparations such as fixation and embedding are not necessary or advantageous. Instead, fresh frozen samples result in high-quality images that capture the complex 3D organization of tendon fibers in conditions as close as possible to natural. The comprehensive nature of this innovative study by SR-PhC-μCT breaks ground for future studies of soft complex biological tissue in 3D with high resolution in close to natural conditions, which could be further used for in situ characterization of how soft tissue responds to mechanical stimuli on a microscopic level.

The crux of C1-INH testing in everyday lab work

BackgroundThe determination of functional C1-INH is complex and depends on methodology, sample transport, and storage conditions. In clinical practice, we encounter individuals with pathological values which then cannot be proved true, and HAE patients in whom the values were wrongly found to be normal under non-optimum conditions. We aimed to test realistic real-life sample processing conditions for accurate C1-INH determination. MethodsWe conducted two national inter-laboratory comparisons with optimal sample preparation but different dispatch conditions. We also investigated variations of temperature and time, and their influence on C1-INH. ResultsC1-INH levels showed a significantly wider dispersion under suboptimal transport conditions than under optimal conditions (p < 0.00001). Two putatively healthy patient samples turned out to be pathological. Contrary to our expectations, we found no significant trend in a specific direction when the variables of temperature, time and sample material were combined and varied under realistic conditions. However, the range of variation in [%] functionality was markedly greater in supposedly healthy volunteers. Thus, under experimental conditions we obtained false pathological results that were not far from reality. ConclusionC1- INH determination is crucial for the diagnosis of HAE. Time, temperature, and sample handling have a significant impact on this laboratory value, sometimes leading to incorrect values, inaccurate diagnoses, and inappropriate therapies. This underlines the importance of proper handling of samples. If a patient has ambiguous C1-INH values despite optimized conditions, thus hindering a conclusive diagnosis of HAE, we recommend genetic testing.

Utilizing natural grids structure of thin wood slices as alternative products to commercial TEM grids

Wood as renewable and earth-abundant biomass material, has attracted much research interest revolving around the utilization of its natural pore structure recently. The highly ordered microtexture of wood has been used to design multitudinous functional products which offer good potential for high value utilization of wood resource. Here, a novel application has been developed by using thin wood slice with transparent grid structure to fabricate TEM grids, which could entirely replace commercially products in TEM observation and even widen their application condition. Wood slice with simple inclusions removal process was used as holey grids directly, and a facile method was employed to uniformly cover graphene oxide/polyvinyl formal films on wood slice skeleton as holeless TEM grids. It has been proved that both the fabricated holey and holeless TEM grids are successfully applied to clearly observing the morphology and structure of nano-materials from zero dimension, one dimension to two dimensions. Meanwhile, it demonstrates wooden TEM grids are feasible in strong corrosive sample preparation condition, while commonly used TEM grids are impracticable. The designed TEM grids substitute material has the obvious superiorities in preparation process, low cost, corrosion resistance, hydrophilicity, environment and biocompatibility. The utilization of thin wood slice for TEM grids products also expanded the application field of wood and is very promising to be commercial products.

Determination of cortisol in hair using liquid chromatography-tandem mass spectrometry: a short review.

Cortisol is considered a particularly relevant biomarker in the context of stress evaluation. This study aims to review of the available literature on the determination of cortisol in hair using LC-MS/MS. Currently, there is no standardized procedure for the measurement of cortisol concentrations in hair, and different sample preparation, chromatographic separation and mass spectrometric detection conditions were described. Simple methanolic extraction, reversed-phase separation and MRM detection in negative ion mode are the most common employed analytical approaches. Reported assays presented acceptable sensitivity for clinical purposes. The increasing use of mass spectrometry in clinical laboratories may contribute to the establishment of LC-MS/MS as the method of choice for the determination of cortisol concentrations in hair.

IMPROVEMENT OF THE ANALYTICAL CONTROL FOR APPLICATION OF PESTICIDES IN THE SYSTEM OF CHEMICAL PROTECTION OF CHICKPEA

Relevance. To date, there are no approved methods in Ukraine for the determination of azoxystrobin, thiabendazole, fludioxonil and pyridate in grain chickpeas. Therefore, there is a need for scientific substantiation of the method selection, the development of conditions for sample preparation of chickpea grain, qualitative identification and quantification of pesticides in this matrix used in protection system of chickpea was given in the article, which will allow to control the established hygienic standards of pesticides and minimize pesticide negative impact on population health and the environment.&#x0D; Objective of the research is to develop methods for the determination of azoxystrobin, thiabendazole, fludioxonil and pyridate in grain chickpea.&#x0D; Materials and methods. Chromatographic analysis was performed by Shimazu (Japan) liquid chromatographs using ultraviolet and fluorescent detection.&#x0D; The package of IBM SPSS StatisticsBase v.22 and MS Exel statistical programs was used for statistical processing of results.&#x0D; Results. Optimal conditions of sample preparation of chickpea grain and chromatographic determination of azoxystrobin, fludioxonil, thiabendazole and pyridate (as the sum of pyridate and its metabolite – pyridafol) with the limits of quantitative determination of 0.1; 0.1; 0.01 and 0.05 mg/kg, respectively, wеre developed to provide the determination of the analyzed compounds at the required level (the measure of correctness is (72–75)%) and the control of the established hygienic standards of these compounds in chickpea grain.&#x0D; Conclusions. Developed methods for determination of azoxystrobin, fludioxonil, thiabendazole and pyridate in chickpea grain by high-performance liquid chromatography allow to control the established hygienic standards, to obtain representative information on the content of pesticide residues, which is a prerequisite for risk assessment of plant protection products.

Metabolic Profiling and Quantitative Analysis of Cerebrospinal Fluid Using Gas Chromatography-Mass Spectrometry: Current Methods and Future Perspectives.

Cerebrospinal fluid is a key biological fluid for the investigation of new potential biomarkers of central nervous system diseases. Gas chromatography coupled to mass-selective detectors can be used for this investigation at the stages of metabolic profiling and method development. Different sample preparation conditions, including extraction and derivatization, can be applied for the analysis of the most of low-molecular-weight compounds of the cerebrospinal fluid, including metabolites of tryptophan, arachidonic acid, glucose; amino, polyunsaturated fatty and other organic acids; neuroactive steroids; drugs; and toxic metabolites. The literature data analysis revealed the absence of fully validated methods for cerebrospinal fluid analysis, and it presents opportunities for scientists to develop and validate analytical protocols using modern sample preparation techniques, such as microextraction by packed sorbent, dispersive liquid–liquid microextraction, and other potentially applicable techniques.

Optimized method for analysis of ethanolamines, hydrolysis products of nitrogen mustards, from urine samples using LC-MS/MS

Highly polar ethanolamines (EAs), excreted in urine, are hydrolysis products of nitrogen mustards (NMs), which are prohibited by the Chemical Weapons Convention (CWC). The methods established for biological matrices are essential for verification analysis of the CWC related chemicals. This paper describes a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed for qualitative and quantitative analysis of EAs, N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA) and triethanolamine (TEAOH) from urine samples. After optimization of sample preparation and chromatographic conditions, the method was fully validated. Silica solid-phase extraction (SPE) cartridges and a porous graphite carbon (PGC) column were selected for validation studies. The method is linear from 5 to 500, 0.5 to 250, and 0.5 to 500 ng/mL for TEAOH, EDEA, and MDEA, respectively. It is also precise and accurate. A minimum sample amount of 0.5 mL urine was used. The limit of quantification using this approach was 0.4, 5.5, and 6.3 ng/mL for MDEA, EDEA and TEAOH, respectively. The combination of the PGC column and high pH eluents in analysis retained and separated the studied EAs. Retention times were 2.11, 2.56 and 2.98 min for MDEA, EDEA and TEAOH, respectively. The method is applicable for verification analysis of the CWC.