Ethyl Chloroformate Derivatization Research Articles

Rapid and Simultaneous Determination of Free Aromatic Carboxylic Acids and Phenols in Commercial Juices by GC-MS after Ethyl Chloroformate Derivatization

Natural phenol and phenolic acids are widely distributed in the plant kingdom and the major dietary sources include fruits and beverages derived therefrom. Over the past decades, these compounds have been widely investigated for their beneficial effects on human health and, at the same time, several analytical methods have been developed for their determination in these matrices. In the present paper, 19 different aromatic carboxylic acids and phenols were characterized by GC-MS using ethyl chloroformate as the derivatizing agent. This procedure occurs quickly at room temperature and takes place in aqueous media simultaneously with the extraction step in the presence of ethanol using pyridine as a catalyst. The analytical method herein developed and validated presents excellent linearity in a wide concentration range (25–3000 ng/mL), low LOQ (in the range 25–100 ng/mL) and LOD (in the range 12.5–50 ng/mL), and good accuracy and precision. As a proof of concept, ethyl chloroformate derivatization was successfully applied to the analysis of a selection of commercial fruit juices (berries, grape, apple, pomegranate) particularly rich in phenolic compounds. Some of these juices are made up of a single fruit, whereas others are blends of several fruits. Our results show that among the juices analyzed, those containing cranberry have a total concentration of the free aromatic carboxylic acids and phenols tested up to 15 times higher than other juices.

Ethylchloroformate Derivatization for GC-MS Analysis of Resveratrol Isomers in Red Wine.

Resveratrol (3,5,4′-trihydroxystilbene) is a natural compound that can be found in high concentrations in red wine and in many typical foods found in human diet. Over the past decades, resveratrol has been widely investigated for its potential beneficial effects on human health. At the same time, numerous analytical methods have been developed for the quantitative determination of resveratrol isomers in oenological and food matrices. In the present work, we developed a very fast and sensitive GC–MS method for the determination of resveratrol in red wine based on ethylchloroformate derivatization. Since this reaction occurs directly in the water phase during the extraction process itself, it has the advantage of significantly reducing the overall processing time for the sample. This method presents low limits of quantification (LOQ) (25 ng/mL and 50 ng/mL for cis- and trans-resveratrol, respectively) and excellent accuracy and precision. Ethylchloroformate derivatization was successfully applied to the analysis of resveratrol isomers in a selection of 15 commercial Italian red wines, providing concentration values comparable to those reported in other studies. As this method can be easily extended to other classes of molecules present in red wine, it allows further development of new GC–MS methods for the molecular profiling of oenological matrices.

Determination of atmospheric amines at Seoul, South Korea via gas chromatography/tandem mass spectrometry

Due to their important roles in salt-producing acid-base reactions, new particle formation (NPF), and as precursors in secondary organic aerosol (SOA) producing reactions, the atmospheric concentrations of particulate volatile amines (dimethylamine (DMA), ethylamine, diethylamine (DEA), propylamine, and butylamine) at Seoul were analyzed and evaluated. To quantify the presence of volatile amines in particulate matter with aerodynamic diameters less than or equal to a nominal 2.5 μm (PM2.5), an efficient and rapid analytical method based on in-matrix ethyl chloroformate (ECF) derivatization followed by headspace solid-phase microextraction (HS-SPME) was developed and validated using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) in the multiple reaction monitoring (MRM) mode. The annual mean concentration of the total 5 target amines was 5.56±2.76 ng/m3 and the seasonal difference was small. The concentrations of particulate amines measured in this study were lower than those observed in Zongludak, Turkey, Nanjing, China, and Jeju, Korea but slightly higher than that reported in Kobe, Japan. The concentrations of the nitrosamines (nitrosodimethylamine (NDMA) and nitrosodiethylamine (NDEA)), and of the nitramines (dimethylnitramine (DMN) and diethylnitramine (DEN)) measured along with those of the target amines were used in a simple linear regression analysis. It indicates the contribution of DMA to the formation of NDMA in all seasons (except the fall) and DEA to the formation of NDEA in the summer, while DMA and DEA did not significantly contribute to the formation of nitramines.

Simultaneous monitoring of the bioconversion from lysine to glutaric acid by ethyl chloroformate derivatization and gas chromatography-mass spectrometry

Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (l-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.

Combination of ultrasound-assisted ethyl chloroformate derivatization and switchable solvent liquid-phase microextraction for the sensitive determination of l-methionine in human plasma by GC-MS.

A green and fast analytical method for the determination of l-methionine in human plasma is presented in this study. Preconcentration of the analyte was carried out by switchable solvent liquid phase microextraction after ethyl chloroformate derivatization reaction. Instrumental detection of the analyte was performed by means of gas chromatography-mass spectrometry. N,N-Dimethyl benzylamine was used in the synthesis of switchable solvent. Protonated N,N-dimethyl benzylamine volume, volume/concentration of sodium hydroxide, and vortex period were meticulously fixed to their optimum values. Besides, ethyl chloroformate, pyridine, and ethanol volumes were optimized in order to get high derivatization yield. After the optimization studies, limit of detection and quantitation values were attained as 3.30 and 11.0ng/g, respectively, by the developed switchable solvent liquid phase microextraction gas chromatography-mass spectrometry method that corresponding to 76.7-folds enhancement in detection power of the gas chromatography-mass spectrometry system. Applicability and accuracy of the switchable solvent liquid phase microextraction-gas chromatography-mass spectrometry method were also checked by spiking experiments. Percent recovery results were ranged from 97.8 to 100.5% showing that human plasma samples could be analyzed for its l-methionine level by the proposed method.

Chiral analysis of β-methylamino alanine (BMAA) enantiomers after (+)-1-(9-fluorenyl)-ethyl chloroformate (FLEC) derivatization and LC-MS/MS

A developed method for chiral analysis of neurotoxin β-methylamino alanine (BMAA) allowed detection of d-BMAA in cycad seed samples.

Quantification of Isotopologues of Amino Acids by Multiplexed Stable Isotope-Resolved Metabolomics Using Ultrahigh-Resolution Mass Spectrometry Coupled with Direct Infusion.

Stable isotope-resolved metabolomics (SIRM) is increasingly used among researchers for metabolic studies including amino acid metabolism. However, the classical GC- or HPLC-based methods for amino acid quantification do not meet the needs for multiplexed stable isotope-enriched analysis by ultrahigh-resolution Fourier transform mass spectrometry (UHR-FTMS). This is due to insufficient acquisition time during chromatographic separations and large dynamic range in concentrations of analytes, which compromises detection and quantification of the numerous metabolite isotopologues present in crude extracts. This chapter discusses a modified ethyl chloroformate derivatization method to enable rapid quantitative analysis of stable isotope-enriched amino acids using direct infusion ion introduction coupled with UHR-FTMS.

Enantioselective micellar electrokinetic chromatography of dl-amino acids using (+)-1-(9-fluorenyl)-ethyl chloroformate derivatization and UV-induced fluorescence detection.

Chiral analysis of dl‐amino acids was achieved by micellar electrokinetic chromatography coupled with UV‐excited fluorescence detection. The fluorescent reagent (+)‐1‐(9‐fluorenyl)ethyl chloroformate was employed as chiral amino acid derivatizing agent and sodium dodecyl sulfate served as pseudo‐stationary phase for separating the formed amino acid diastereomers. Sensitive analysis of (+)‐1‐(9‐fluorenyl)ethyl chloroformate‐amino acids was achieved applying a xenon‐mercury lamp for ultraviolet excitation, and a spectrograph and charge‐coupled device for wavelength‐resolved emission detection. Applying signal integration over a 30 nm emission wavelength interval, signal‐to‐noise ratios for derivatized amino acids were up to 23 times higher as obtained using a standard photomultiplier for detection. The background electrolyte composition (electrolyte, pH, sodium dodecyl sulfate concentration, and organic solvent) was studied in order to attain optimal chemo‐ and enantioseparation. Enantioseparation of 12 proteinogenic dl‐amino acids was achieved with chiral resolutions between 1.2 and 7.9, and detection limits for most derivatized amino acids in the 13–60 nM range (injected concentration). Linearity (coefficients of determination > 0.985) and peak‐area and migration‐time repeatabilities (relative standard deviations lower than 2.6 and 1.9%, respectively) were satisfactory. The employed fluorescence detection system provided up to 100‐times better signal‐to‐noise ratios for (+)‐1‐(9‐fluorenyl)ethyl chloroformate‐amino acids than ultraviolet absorbance detection, showing good potential for d‐amino acid analysis.

"One-pot" ethyl chloroformate derivatization and liquid-liquid extraction of reduced glutathione in erythrocyte and its quantitative GC-MS analysis.

A simple "one-pot" derivatization and liquid-liquid extraction (LLE) procedure was developed for GC-MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol-pyridine media, the derivative separated and detected by gas chromatography-mass spectrometry using a short non-polar capillary GC column at a high column-head pressure. Total analysis time was 11min. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4-813.4μM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparison of this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, thus proving that the described GC-MS method could be employed for fast and simple GSH analysis in clinical studies.

N ε-(carboxymethyl)-l-lysine content in cheese, meat and fish products is affected by the presence of copper during elaboration process

Formation of dietary advanced glycation end products has been extensively studied, principally with the aim to decrease their intake. In this work, the relationship between copper potentially present during food elaboration and N e-(carboxymethyl)-l-lysine (CML) concentrations, has been examined for the first time. For CML determination, a reversed phase liquid chromatography-electrospray ionization-ion trap tandem mass spectrometry procedure, based on acid hydrolysis, ethyl chloroformate derivatization and quantification in MRM mode was set-up, yielding method quantification limit of 98 µg kg−1; copper was determined by ICP-MS. For eleven commercial cheeses, CML and Cu were found in the ranges 3.70–8.58 µg g−1 and 0.08–15.5 µg g−1, respectively, suggesting an inverse relation between these two parameters. For beef, chicken, Mexican pork “carnitas” and salmon, the CML concentration was lower in the item cooked in Cu casserole while element concentration was increased, as compared to this same raw material prepared in Teflon™ (except for “carnitas”). Concentration-dependent effect of Cu, manifest by decreased CML formation, was confirmed evaluating conversion percentage of chemically protected lysine (ZLys) to ZCML in the absence and in the presence of different Cu concentrations (50.0% and 20.4% conversion for Cu:ZLys molar ratio 0:1 and 0.04:1, respectively). Consistent results obtained in the analysis of three different sample types point to the inhibitory effect of copper during CML formation; however, it should be stressed that Cu is only one parameter within a complex set of factors/conditions involved in glycation process. Although better understanding of the observed effect at molecular level is needed, the results obtained in this work strongly suggest beneficial effect of copper, inhibiting glycation process during food elaboration.

Chloroformate derivatization for tracing the fate of Amino acids in cells and tissues by multiple stable isotope resolved metabolomics (mSIRM)

Amino acids have crucial roles in central metabolism, both anabolic and catabolic. To elucidate these roles, steady-state concentrations of amino acids alone are insufficient, as each amino acid participates in multiple pathways and functions in a complex network, which can also be compartmentalized. Stable Isotope-Resolved Metabolomics (SIRM) is an approach that uses atom-resolved tracking of metabolites through biochemical transformations in cells, tissues, or whole organisms. Using different elemental stable isotopes to label multiple metabolite precursors makes it possible to resolve simultaneously the utilization of these precursors in a single experiment. Conversely, a single precursor labeled with two (or more) different elemental isotopes can trace the allocation of e.g. C and N atoms through the network.Such dual-label experiments however challenge the resolution of conventional mass spectrometers, which must distinguish the neutron mass differences among different elemental isotopes. This requires ultrahigh resolution Fourier transform mass spectrometry (UHR-FTMS). When combined with direct infusion nano-electrospray ion source (nano-ESI), UHR-FTMS can provide rapid, global, and quantitative analysis of all possible mass isotopologues of metabolites. Unfortunately, very low mass polar metabolites such as amino acids can be difficult to analyze by current models of UHR-FTMS, plus the high salt content present in typical cell or tissue polar extracts may cause unacceptable ion suppression for sources such as nano-ESI.Here we describe a modified method of ethyl chloroformate (ECF) derivatization of amino acids to enable rapid quantitative analysis of stable isotope labeled amino acids using nano-ESI UHR-FTMS. This method showed excellent linearity with quantifiable limits in the low nanomolar range represented in microgram quantities of biological specimens, which results in extracts with total analyte abundances in the low to sub-femtomole range. We have applied this method to profile amino acids and their labeling patterns in 13C and 2H doubly labeled PC9 cell extracts, cancerous and non-cancerous tissue extracts from a lung cancer patient and their protein hydrolysates as well as plasma extracts from mice fed with a liquid diet containing 13C6-glucose (Glc).The multi-element isotopologue distributions provided key insights into amino acid metabolism and intracellular pools in human lung cancer tissues in high detail. The 13C labeling of Asp and Glu revealed de novo synthesis of these amino acids from 13C6-Glc via the Krebs cycle, specifically the elevated level of 13C3-labeled Asp and Glu in cancerous versus non-cancerous lung tissues was consistent with enhanced pyruvate carboxylation. In addition, tracking the fate of double tracers, (13C6-Glc + 2H2-Gly or 13C6-Glc + 2H3-Ser) in PC9 cells clearly resolved pools of Ser and Gly synthesized de novo from 13C6-Glc (13C3-Ser and 13C2-Gly) versus Ser and Gly derived from external sources (2H3-Ser, 2H2-Gly). Moreover the complex 2H labeling patterns of the latter were results of Ser and Gly exchange through active Ser-Gly one-carbon metabolic pathway in PC9 cells.

Characterization of N-methylated amino acids by GC-MS after ethyl chloroformate derivatization.

Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α-amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N-methyl and N,N-dimethyl amino acids were synthesized by the methylation of α-amino acids and characterized by a GC-MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC-MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N-methyl (1-18) and N,N-dimethyl amino acids (19-35) showed abundant [M-COOC2 H5 ]+ ions. The fragment ions due to loss of C2 H4 , CO2 , (CO2 + C2 H4 ) from [M-COOC2 H5 ]+ were of structure indicative for 1-18. The EI spectra of 19-35 showed less number of fragment ions when compared with those of 1-18. The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H]+ ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds (1-35) were confirmed by high-resolution mass spectra data and further substantiated by the data obtained from 13 C2 -labeled glycines and N-ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd.

Identifying inhibitory compounds in lignocellulosic biomass hydrolysates using an exometabolomics approach

BackgroundInhibitors are formed that reduce the fermentation performance of fermenting yeast during the pretreatment process of lignocellulosic biomass. An exometabolomics approach was applied to systematically identify inhibitors in lignocellulosic biomass hydrolysates.ResultsWe studied the composition and fermentability of 24 different biomass hydrolysates. To create diversity, the 24 hydrolysates were prepared from six different biomass types, namely sugar cane bagasse, corn stover, wheat straw, barley straw, willow wood chips and oak sawdust, and with four different pretreatment methods, i.e. dilute acid, mild alkaline, alkaline/peracetic acid and concentrated acid. Their composition and that of fermentation samples generated with these hydrolysates were analyzed with two GC-MS methods. Either ethyl acetate extraction or ethyl chloroformate derivatization was used before conducting GC-MS to prevent sugars are overloaded in the chromatograms, which obscure the detection of less abundant compounds. Using multivariate PLS-2CV and nPLS-2CV data analysis models, potential inhibitors were identified through establishing relationship between fermentability and composition of the hydrolysates. These identified compounds were tested for their effects on the growth of the model yeast, Saccharomyces. cerevisiae CEN.PK 113-7D, confirming that the majority of the identified compounds were indeed inhibitors.ConclusionInhibitory compounds in lignocellulosic biomass hydrolysates were successfully identified using a non-targeted systematic approach: metabolomics. The identified inhibitors include both known ones, such as furfural, HMF and vanillin, and novel inhibitors, namely sorbic acid and phenylacetaldehyde.

A novel method for simultaneous measurement of concentration and enrichment of NO synthesis-specific amino acids in human plasma using stable isotopes and LC/MS ion trap analysis

Stable isotope studies offer the opportunity to study the in-depth metabolic pathway of glutamine, citrulline, and arginine amino acids involved in NO synthesis. The use of multiple stable isotopes can be used to elucidate the exact transformation of glutamine to citrulline and arginine de novo synthesis. This novel method provides a purification step using cation exchange resin in combination with a rapid and easy derivatization procedure for a precise and robust measurement of the concentration and isotopic enrichments of NO synthesis-specific amino acids using a liquid chromatography mass spectrometry (LC/MS) ion trap system with high sensitivity and selectivity. The ethyl chloroformate derivatization procedure is beneficial in terms of robustness, velocity, simplicity, and derivative stability. In addition, the ethyl chloroformate derivatization can be performed at room temperature in an aqueous environment without incubation and the isolation of the derivatives from the reaction mixture also serves as a purification step. The concentration and enrichment of NO synthesis-specific amino acids as well as phenylalanine and tyrosine to determine protein turnover, were measured with good inter-day precision for the concentration (<7.4%) and enrichment (<12.7%) in plasma samples at low and high levels. The low limit of quantification was 0.2μmol/L for most of the amino acids and the purification method showed to have good recoveries between 78% and 98%. No ion-suppression was observed by post-column infusion experiments. In order to develop new nutritional strategies, this novel method can be used to support the elucidation of the effect of administration of specific supplements on the glutamine-citrulline-arginine pathway by using stable isotope studies.

Determination of t,t-muconic acid in urine samples using a molecular imprinted polymer combined with simultaneous ethyl chloroformate derivatization and pre-concentration by dispersive liquid–liquid microextraction

The present communication describes the preparation and evaluation of a molecularly imprinted polymer (MIP) as a solid-phase extraction (SPE) sorbent and simultaneous ethyl chloroformate (ECF) derivatization and pre-concentration by dispersive liquid-liquid microextraction (DLLME) for the analysis of t,t-muconic acid (t,t-MA) in urine samples using gas chromatography-mass spectrometry. The imprinting polymer was prepared using methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, 2,2-azobisisobutyronitrile as the initiator and t,t-MA as a template molecule. The imprinted polymer was evaluated for its use as a SPE sorbent by comparing both imprinted and non-imprinted polymers in terms of the recovery of t,t-MA from urine samples. Molecular modelling studies were performed in order to estimate the binding energy and efficiency of the MIP complex formed between the monomer and the t,t-MA. Various factors that can affect the extraction efficiency of MIP, such as the loading, washing and eluting conditions, were optimized; other factors that can affect the derivatization and DLLME pre-concentration were also optimized. MIP in combination with ECF derivatization and DLLME pre-concentration for t,t-MA exhibits good linearity, ranging from 0.125 to 2 μg mL(-1) (R(2) = 0.9971), with limit of detection of 0.037 μg mL(-1) and limit of quantification of 0.109 μg mL(-1). Intra- and inter-day precision was found to be <6%. The proposed method has been proven to be effective and sensitive for the selective pre-concentration and determination of t,t-MA in urine samples of cigarette smokers.

Rapid and simultaneous determination of twenty amino acids in complex biological and food samples by solid-phase microextraction and gas chromatography–mass spectrometry with the aid of experimental design after ethyl chloroformate derivatization

Amino acids play a vital role as intermediates in many important metabolic pathways such as the biosynthesis of nucleotides, vitamins and secondary metabolites. A sensitive and rapid analytical method has been proposed for the first time for the simultaneous determination of twenty amino acids using solid-phase microextraction (SPME). The protein samples were hydrolyzed by 6M HCl under microwave radiation for 120min. Then the amino acids were derivatized by ethyl chloroformate (ECF) and the ethoxy carbonyl ethyl esters of amino acids formed were extracted using SPME by direct immersion. Finally the extracted analytes on the SPME fiber were desorbed at 260°C and analyzed by gas chromatography–mass spectrometer (GC–MS) in electron ionization mode. Factors which affect the SPME efficiency were screened by Plackett–Burmann design; most significant factors were optimized with response surface methodology. The optimum conditions for SPME are as follows: pH of 1.7, ionic strength of 733mg, extraction time of 30min and fiber of divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The recovery of all the amino acids was found to be in the range of 89.17–100.98%. The limit of detection (LOD) of all derivatized amino acids in urine, hair and soybean was found to be in the range of 0.20–7.52μgL−1, 0.21–8.40μgL−1 and 0.18–5.62μgL−1, respectively. Finally, the proposed technique was successfully applied for the determination of amino acids in complex biological (hair, urine) and food samples (soybean). The method can find wide applications in the routine analysis of amino acids in any biological as well as food samples.

Development, validation and comparison of two microextraction techniques for the rapid and sensitive determination of pregabalin in urine and pharmaceutical formulations after ethyl chloroformate derivatization followed by gas chromatography–mass spectrometric analysis

The present article reports first time the use of solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME) to extract pregabalin (PRG) from urine and pharmaceutical formulations followed by GC-MS analysis after ethyl chloroformate (ECF) derivatization. PRG is an antiepileptic and analgesic drug, which is a structural analogue of γ-amino-butyric acid (GABA). It is approved by Food and Drug Administration (FDA) for the treatment of central nervous system (CNS) disorders and neuropathic pain. Initially PRG was derivatized with ECF in the presence of pyridine at room temperature for 30s. Experimental parameters were investigated for derivatization, SPME and DLLME conditions. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.019 μg/ml and 0.063 μg/ml for SPME and 0.022 μg/ml and 0.075 μg/ml for DLLME respectively. The percentage recovery, in case of SPME was in the range of 83-98% while for DLLME it is in the range of 84-98%. The intra and inter-day precisions were found to be less than 6%. The developed methods after ECF derivatization were found to be simple, fast, efficient and inexpensive. DLLME has several advantages like lesser extraction time and cost effectiveness as compared to SPME. The developed methods may find wide application for the routine determination of PRG in biological as well as in quality control samples of pharmaceutical formulations.

Response to Letter to the Editor regarding “GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia”

Dear Sir, Dr Husek Petr argued that “Ourmethod should not be pursued as a general tool suitable for serum-based metabolic profiling study due to several possible defects for amino acid measurement although he believed that our method might be applied to identify the metabolites associated with human uremia” [1]. We are pleased to know scientists from areas of conventional analytical chemistry are also interested in our work in addition to those from the emerging metabolomics field [1–4]. We acknowledge the contribution made by many pioneering researchers, including Husek’s group. However, the purpose of our study [5] was to identify differential metabolites between subjects diagnosed with uremia and healthy controls by use of a global profiling approach—“metabolomics” and/or “metabonomics”. To ensure the efficiency, reproducibility, and reliability of sample-preparation procedures that are capable of handling hundreds of clinical samples, we selected the ECF method developed by Husek [6] in preference to many other methods and optimized it for global metabolite analysis of human serum samples. Before addressing technical concerns raised by Husek [1], we would like to provide a brief introduction to metabolomics because this field is rather different from routine analytical chemistry. Metabolomics/metabonomics has evolved quickly in recent years and can be defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification” [7]. The strength of metabolomics lies in its capability to identify a metabolic fingerprint, which provides global metabolic information associated with a pathophysiological state. The ability to acquire the complete set of smallmolecule metabolites (the whole metabolome) from biospecimens by use of appropriate analytical instrumentation will ensure the discovery of abnormally regulated key metabolites and metabolic pathways most relevant to the specific disease and useful for mechanistic interpretation [7, 8]. In reality, however, it is not possible to recover all metabolites of a great variety of chemical classes from biological samples. Additionally, the concentration ranges of these metabolites span more than a dozen orders of magnitude with compounds such as glucose in the millimolar range in blood and compounds such as some lipids in the femtomolar range [9]. Therefore, optimum sample-preparation procedures in metabolic profiling studies are usually a compromise among sensitivity, number of samples analyzed, number of metabolites detected, and specific aims of the study design. In this scenario, it is very This article is the response to the “Letter to the Editor” to be found at http://dx.doi.org/10.1007/s00216-011-5615-x

Letter to the Editor regarding “GC–MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia”

Dear Sir, In connection with the paper published by Tao et al. [1] I have been asked by some researchers, as the inventor of the chloroformate methodology [2, 3], to comment on the “optimized protocol” as claimed in the abstract. The procedure, presented as a technique for “global metabolic profiling” is a lot else than that. It might work for the purpose to which it was applied, i.e. identification of themetabolites associatedwith human uremia, but it should not be pursued as a general tool “suitable for serum-based metabolic profiling studies” (Abstract). The method is substantially inferior in both ECF-mediated derivatization of amino acids, compared with the original detailed study [4], and the method used for serum sample workup, as described, e.g., in 1995 [5]. Let me briefly draw attention to the basic faults that reduce the effectiveness of the assay. First, replacing chloroform by hexane in the extraction step hampers transfer of polar products (serine, asparagine, glutamine) into the organic phase, and strongly reduces extraction of some others (glycine), as is apparent from the chromatographic record. The comment that “extraction efficiencies of chloroform and n-hexane are comparable” is thus completely incorrect, and to justify use of the latter by “facilitated removal of the hexane upper layer from the vial” is correct regarding sample handling but incorrect analytically. Second, the reason for the proposed two-step ECF derivatization, with intermediate basification of the aqueous layer, is declared as “exhibiting better derivatization efficiency and providing more useful information”. In which way? The opposite seems to be true. One would expect at least augmented yields of the dibasic amino acids but lysine, ornithine, and histidine are absent from the record. Comparing the figure with the GC record of the original study [4] one can see this lapse, especially in yields of the later eluted analytes. The repeated treatment of the aqueous layer after the first extraction of the products did not result in any improvement, as already shown in 1997; the efficiency of extraction of the derivatized amino acids with chloroform was close to 100% [6]. Third, treating serum with ethanol directly leads to disruption of lipoproteins, so massive amounts of neutral lipids, cholesterol esters, acylglycerols, and phospholipids are liberated and co-extracted with the derivatized amino acids. Their injection into the system leads to progressive poisoning of the inlet and the stationary phase, because the low-volatility species cannot be eluted from common stationary phases within the temperature range used. This phenomenon was, surprisingly, not discussed, although clear evidence of worsening GC–MS conditions was mentioned (p. 2887): “all of the samples from each batch were analyzed within 48 h, and maintenance of the equipment was carried out after every 48-h analysis”, without giving the reason for this. Simple removal of the neutral lipids by hexane extraction, while retaining amino, fatty, and other carboxylic acids as salts in the aqueous phase for the subsequent derivatization step, was published by me among the first pioneering studies [5]; this paper was not cited, although the assay afforded much better analytical performance and a better chance of successful metabolic profiling of that kind. Finally, the large peak on the chromatographic record (Nr. 1) was falsely assigned to propionic acid instead of to lactic acid, i.e. 3-hydroxypropionic acid. P. Husek (*) Biology Centre, Academy of Sciences of the Czech Republic, Institute of Entomology, 370 05 Ceske Budějovice, Czech Republic e-mail: husek@bclab.eu

Application of ethyl chloroformate derivatization for solid-phase microextraction–gas chromatography–mass spectrometric determination of bisphenol-A in water and milk samples

A simple and rapid analytical method based on in-matrix ethyl chloroformate (ECF) derivatization has been developed for the quantitative determination of bisphenol-A (BPA) in milk and water samples. The samples containing BPA were derivatised with ECF in the presence of pyridine for 20 s at room temperature, and the non-polar derivative thus formed was extracted using polydimethylsiloxane solid-phase microextraction (SPME) fibres with thicknesses of 100 μm followed by analysis using gas chromatography-mass spectrometry. Three alkyl chloroformates (methyl, ethyl and isobutyl chloroformate) were tested for optimum derivatisation yields, and ECF has been found to be optimum for the derivatisation of BPA. Several parameters such as amount of ECF, pyridine and reaction time as well as SPME parameters were studied and optimised in the present work. The limit of detection for BPA in milk and water samples was found to be 0.1 and 0.01 μg L(-1), respectively, with a signal-to-noise ratio of 3:1. The limit of quantitation for BPA in milk and water was found to be 0.38 and 0.052 μg L(-1), respectively, with a signal-to-noise ratio of 10:1. In conclusion, the method developed was found to be rapid, reliable and cost-effective in comparison to silylation and highly suitable for the routine analysis of BPA by various food and environmental laboratories.