Derivatization Yield Research Articles

Developing a GC-EI-MS/MS method for quantifying warfarin and five hydroxylated metabolites generated by the Fenton reaction.

Since the 1950s, Warfarin has been used globally as both a prescription drug and a rodenticide. Research has shown that warfarin and other rodenticides are present in the environment and food chain. However, emerging contaminants are subject to degradation by biotic and abiotic processes and advanced oxidation processes. In some cases, detecting the parent compound may not be possible due to the formation of structurally changed species. This approach aims to identify hydroxylated transformation products of warfarin in a laboratory setting, even after the parent compound has undergone degradation. Therefore, the Fenton reaction is utilized to insert hydroxylation into the parent compound, warfarin, by hydroxyl and hydroperoxyl radicals generated by Fe2+/Fe3+ redox reaction with hydrogen peroxide. Using multiple reaction monitoring, a GC-MS/MS method, incorporating isotopically labeled reference compounds, is used to quantify the expected derivatized species. The analytes are derivatized using trimethyl-3-trifluoromethyl phenyl ammonium hydroxide, and the derivatization yield of warfarin is determined by using isotopically labeled reference compounds. The method has a linear working range of 30 to 1800ng/mL, with detection limits ranging from 18.7 to 67.0ng/mL. The analytes are enriched using a C18-SPE step, and the recovery for each compound is calculated. The Fenton reaction generates all preselected hydroxylated transformation products of warfarin. The method successfully identifies that 4'-Me-O-WAR forms preferentially under the specified experimental conditions. By further optimizing the SPE clean-up procedures, this GC-MS-based method will be suitable for detecting transformation products in more complex matrices, such as environmental water samples. Overall, this study provides a better understanding of warfarin's degradation and offers a robust analytical tool for investigating its transformation products.

Influence of pH and salts on DMF-DMA derivatization for future Space Applications

For gas chromatography - mass spectrometry (GC-MS) analyses performed in situ, pH and salts (e.g., chlorides, sulfates) may enhance or inhibit the detection of targeted molecules of interest for astrobiology (e.g. amino acids, fatty acids, nucleobases). Obviously, salts influence the ionic strength of the solutions, the pH value, and the salting effect. But the presence of salts may also produce complexes or mask ions in the sample (masking effect on hydroxide ion, ammonia, etc.). For future space missions, wet chemistry will be conducted before GC-MS analyses to detect the full organic content of a sample. The defined organic targets for space GC-MS instrument requirements are generally strongly polar or refractory organic compounds, such as amino acids playing a role in the protein production and metabolism regulations for life on Earth, nucleobases essential for DNA and RNA formation and mutation, and fatty acids that composed most of the eukaryote and prokaryote membranes on Earth and resist to environmental stress long enough to still be observed on Mars or ocean worlds in geological well-preserved records. The wet-chemistry chemical treatment consists of reacting an organic reagent with the sample to extract and volatilize polar or refractory organic molecules (i.e. dimethylformamide dimethyl acetal (DMF-DMA) in this study). DMF-DMA derivatizes functional groups with labile H in organics, without modifying their chiral conformation. The influence of pH and salt concentration of extraterrestrial materials on the DMF-DMA derivatization remains understudied. In this research, we studied the influence of different salts and pHs on the derivatization of organic molecules of astrobiological interest with DMF-DMA, such as amino acids, carboxylic acids, and nucleobases. Results show that salts and pH influence the derivatization yield, and that their effect depend on the nature of the organics and the salts studied. Second, monovalent salts lead to a higher or similar organic recovery compared to divalent salts regardless of pH below 8. However, a pH above 8 inhibits the DMF-DMA derivatization influencing the carboxylic acid function to become an anionic group without labile H. Overall, considering the negative effect of the salts on the detection of organic molecules, future space missions may have to consider a desalting step prior to derivatization and GC-MS analyses.

Rapidly identifying and quantifying of unsaturated lipids with carbon‐carbon double bond isomers by photoepoxidation

Unsaturated lipids play an essential role in life activities. Identifying and quantifying their carbon-carbon double bond (CC) isomers have become a hot topic in recent years. In lipidomics, the analysis of unsaturated lipids in complex biological samples usually requires high-throughput methods, which puts forward the requirements of rapid response and simple operation for identification. In this paper, we proposed a photoepoxidation strategy, which uses benzoin to open the double bonds of unsaturated lipids to form epoxides under ultraviolet light and aerobic conditions. Photoepoxidation is controlled by light and has a fast response. After 5 min, the derivatization yield can reach 80% with no side reaction products. Besides, the method has the advantages of high quantitation accuracy and a high yield of diagnostic ions. It was successfully applied to rapidly identify the double bond locations of various unsaturated lipids in both positive and negative ion modes, and to rapidly identify and quantitatively analyze the various isomers of unsaturated lipids in mouse tissue extract. So the method has the potential for large-scale analysis of unsaturated lipids in complex biological samples.

Comparison of tetramethylammonium hydroxide (TMAH), trimethylsulfonium hydroxide (TMSH), and trimethylphenylammonium hydroxide (TMPAH) thermochemolysis for in situ space analysis of organic molecules in planetary environments

One of the main objectives of present and future space exploration missions dedicated to astrobiology is the detection of organic molecules of interest for life (e.g. amino and fatty acids). With this aim, a sample preparation and a gas chromatograph (connected to a mass spectrometer) are generally used. To date, tetramethylammonium hydroxide (TMAH) has been the first and only thermochemolysis reagent to be used for in situ sample preparation and chemical analysis of planetary environments. Although TMAH is widely used in terrestrial laboratories, numerous applications also leverage other thermochemolysis reagents that may be more relevant than TMAH to meet both scientific and technical objectives of space instrumentation. The present study compares the performance of tetramethylammonium hydroxide (TMAH), trimethylsulfonium hydroxide (TMSH), and trimethylphenylammonium hydroxide (TMPAH) reagents on molecules of interest to astrobiology. The study focuses on the analyses of 13 carboxylic acids (C7–C30), 17 proteinic amino acids, and the 5 nucleobases. Here we report the derivatization yield without stirring or adding solvents, the detection sensitivity with mass spectrometry, and the nature of the degradation products from the reagents produced during pyrolysis. We conclude that TMSH and TMAH are the best reagents for analyzing carboxylic acids and nucleobases. Amino acids are not relevant targets for a thermochemolysis over 300 °C as they are degraded and showed high limits of detection. As TMAH, and probably TMSH, meet the space instrumentation requirements, this study informs sample treatment approaches prior to GC-MS analysis in in situ space studies. The thermochemolysis reaction using TMAH or TMSH is also recommended for space return missions to extract organics from a macromolecular matrix, derivatize polar or refractory organic targets, and volatilize with the fewest organic degradations.

Separation and determination of 3-hydroxyaspartate by online concentration capillary electrophoresis/laser-induced fluorescence with microwave-assisted derivatization.

A chiral analytical method was proposed based on capillary electrophoresis with laser-induced fluorescence detection coupled with microwave-assisted derivatization for the simultaneous baseline separation and sensitive detection of four stereoisomers of 3-hydroxyaspartate. The derivatization reaction of 3-hydroxyaspartate with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was greatly accelerated by microwave irradiation. Under the optimized conditions, the derivatization yield was increased by 20% and the derivatization time was shortened by 20 min when compared with those from conventional water bath heating. In addition, the sensitivity was improved by online sample concentration methods. The detection limit of l-threo-3-hydroxyaspartate obtained by large-volume sample stacking with polarity switching was 5.3nmol/L, which was around 1000-fold lower than that of the capillary electrophoresis/laser-induced fluorescence without stacking. The excellent analytical performance in terms of linearity and precision was also achieved. Furthermore, the developed method was successfully applied to the determination of 3-hydroxyaspartate in the spiked urine, and satisfactory recoveries were obtained ranging from 90.5 to 107.0%.

Development, validation of a GC–MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine

A gas chromatography-mass spectrometry (GC–MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of l-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC–MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50–1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC–MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC–MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Gas chromatography/mass spectrometry analysis of organic acid profiles in human serum: A protocol of direct ultrasound-assisted derivatization.

Low-molecular-weight organic acids that generally contain one to three carboxyl groups are involved in many important biological processes; therefore, it is important to develop a quantitative method for analyzing organic acids in serum in order to allow an evaluation of metabolic changes. In this study, we evaluated a protocol for detecting 26 organic acids in serum based on ultrasound-assisted derivatization by gas chromatography/mass spectrometry (GC/MS). Serum samples were prepared using ultrasound-assisted silane derivatization before GC/MS analysis to quantify concentrations of organic acids. Additionally, we investigated the variables affecting derivatization yields, including the extraction solvent, derivatization reagents, and derivatization conditions (reaction temperature, duration, and sonication parameters). The protocol was ultimately applied to detect organic acid profiles related to obesity. We used acetone as the extraction solvent and determined suitable derivatization conditions, as follows: BSTFA + 1% TMCS, 50°C, 10min, and 100% ultrasound power. The protocol showed satisfactory linearity (r = 0.9958-0.9996), a low limit of detection (0.04-0.42 μmol/L), good reproducibility (coefficient of variation (CV) %: 0.32-13.76%), acceptable accuracy (recovery: 82.97-114.96%), and good stability within 5days (CV%: 1.35-12.01% at room temperature, 1.24-14.09% at 4°C, and 1.01-11.67% at -20°C). Moreover, the protocol was successfully applied to obtain the organic acid profiles from obese and healthy control subjects. We identified and validated a protocol for ultrasound-assisted derivatization prior to GC/MS analysis for detecting 26 kinds of organic acids in serum. The results suggest the efficacy of this protocol for clinical applications to determine metabolic changes related to fluctuations in organic acid profiles.

Dual derivatization strategy for the comprehensive quantification and double bond location characterization of fatty acids by ultra-high performance liquid chromatography-tandem mass spectrometry

Comprehensive analysis of fatty acids (FAs) has long been challenging due to their poor ionization efficiency, lack of characteristic fragment ions and difficulty of identifying C=C bond locations. In this study, a high coverage ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for the quantification and C=C bond location characterization of FAs using two structural analogues, 2-hydrazinyl-4,6-dimethylpyrimidine (DMP) and 2-hydrazinylpyrimidine (DP), as dual derivatization reagents. DP-labeled FA standards were used as internal standards to reduced matrix effects, which guaranteed the accurate quantification of FAs. The derivatization yields of FAs were larger than 99% and the sensitivities were increased by 400-fold compared with non-derivatized FAs. Pretreatment and instrumental analysis of FAs can be completed in 20 minutes. Only 5 μL rat plasma can satisfy the quantification of 36 FAs with good linearity (r>0.99). Both intra-day and inter-day accuracies were in the range of 85-105%, and the precisions were less than 15%. The extraction recoveries were investigated to be in the range of 88-112%. No obvious matrix effects were observed for the derivatized FAs. In addition, the locations of C=C bonds in DMP-derivatized FAs could be identified by diagnostic fragment ions generated from 1,4-hydrogen elimination and allylic cleavage under low energy collision induced dissociation (CID). The new method was finally employed for FA profiling in plasma from rats with moxifloxacin-induced liver injury. Significant downregulation of butyric acid was observed in moxifloxacin treated model rats, which was believed to be related to the liver injury.

GC-MS analysis of organic acids in rat urine: A protocol of direct ultrasound-assisted derivatization.

The aim of the present study was to develop a novel ultrasound-assisted derivatization method for analysis of urine that can be used for preliminary screening and monitoring of metabolic disorders. Here we describe an ultrasound-assisted derivatization method followed by GC-MS analysis to quantify 26 organic acids in urine. The optimum levels of the variables affecting the yield of derivatization were investigated, including urease doses, derivatization reagents and derivatization conditions (duration time, reaction temperature and sonic power). The method exhibited the best results with 80 μl urease. The optimal reaction conditions were 100 μl BSTFA, 80% ultrasound power, 70°C and 40 min. This method showed satisfactory linearity, good reproducibility and an acceptable limit of detection and accuracy. Therefore, it could potentially be used to as a standard method to enable comparisons between laboratories. Finally, we applied our method to urine samples from pregnant rats administered 2 or 10 mg/kg folic acid supplementation.

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.

Selective Gas Chromatography Mass Spectrometry Method for Ultratrace Detection of Selenocyanate

The recent interest in the determination of selenocyanate (SeCN-) in wastewater systems has spurred the development of analytical methods for its determination at the ultratrace level. Since most of the current procedures require complex and costly instrumental configurations, we have developed a simple and rapid gas chromatography tandem mass spectrometry (GC/MS/MS) method able to detect SeCN- in water samples with a LOD of 0.1 ng/g Se. A 1 mL volume of aqueous sample was buffered with sodium bicarbonate and treated with triethyloxonium tetrafluoroborate for conversion of the analyte into volatile EtSeCN. The derivatization yield was higher than 90%, and it could tolerate concentrations of chloride or sulfate up to 2%. The EtSeCN was extracted in chloroform and could be detected in electron ionization and also in negative chemical ionization mode with a further gain in signal-to-noise ratio by a factor of 2. The method was applied for the analysis of natural waters with quantitation of SeCN- in the low ng/g region. The Se13C15N- internal standard could be used for isotope dilution. Quantitative spike recoveries of 1 ng/g Se were obtained from seawater and river water, and 1 ng/g Se could be quantified within a standard uncertainty of 15%.

GC‐MS Characterization of Cyclic Fatty Acid Monomers and Isomers of Unsaturated Fatty Acids Formed During the Soybean Oil Heating Process

In this work, the possibility of formation and isolation of cyclic fatty acid monomers (CFAM) from polyunsaturated fatty acids via prolonged heat treatment of soybean oil are studied. The structures of CFAM are determined using Gas chromatography‐Mass spectrometry (GC‐MS) of their 4, 4‐dimethyloxazoline (DMOX) derivatives, the yield of derivatization is 98%. Among CFAM, the cyclohexyl fatty acids with ring from C12 to C17 and the cyclohexneyl with ring from C10 to C15 formed acid are detected by GC‐MS. The new cyclopentyl with ring at C14 position or between C13 to C17 are detected from linoleic acid. Also, the variation of major fatty acids composition of soybean oil during heated treatment at 150, 180, 210, and 240 °C for 60 h are analyzed. The results indicate that after heating, soybean oil contains four isomers of conjugated linoleic acid (CLA), four isomers of C18:1 and four isomers of C18:3. The heating process, the enrichment steps, and CFAM concentration can modify the profile of CFAM and permit the emergence of new compounds, which can be useful to know the edible oil structural changes during the phases of extraction and analysis.Practical Applications: When heated at high temperature for a long time, soybean oil undergoes isomerization and cyclization reactions. In fact, CFAM structures with five and six membered carbon rings are identified from heated oil. A new mass spectrum of the CFAM derivative from the intramolecular C18:2 cyclization is also described. Although CFAM are present at very low concentrations in heated oils, these compounds are the most toxic metabolites produced in soybean oil heated at high temperature. In addition to the four isomers of conjugated linoleic acid, four isomers of C18:1 and four of C18:3 are detected during the heating process. The data clearly show that it is necessary to avoid heating the soybean oil to a temperature above 210 °C for a period of more than 60 h which can minimize the production of CFAM.The gap of 68 amu between the molecular ion (333) and m/z 265 indicate the location of cyclopentyl ring at C14, which is first determined in heated soybean oil.

Optimization of reactions between reducing sugars and 1-phenyl-3-methyl-5-pyrazolone (PMP) by response surface methodology

Reducing sugars have strong reactivity with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form the sugar-PMP derivatives, which can be more accurately analyzed by HPLC-UV at 248 nm. Glucose and glucosamine reacted with the PMP based on the response surface methodology within a range of temperature between 60 °C and 80 °C, and time between 60 and 180 min. The optimal conditions for the glucose-PMP and glucosamine-PMP reactions were obtained at 71 °C for 129 min, and 73 °C for 96 min, respectively. Subsequently, other sugars and their derivatives, including xylose, ribose, fructose, galactose, mannose, lactose, maltose, sucrose, glucuronic acid, sorbitol, mannitol, xylitol, and cyclodextrins were investigated and compared under the optimized condition for glucose. All of the above compounds, except the fructose, sugar alcohols and non-reducing sugars, could form the sugar-PMP derivatives. This study demonstrated that different chemical structures of sugars and their derivatives could significantly influence the rate and yield of the PMP derivatization.

An Enhanced GC/MS Procedure for the Identification of Proteins in Paint Microsamples.

The chemical characterization of materials used in works of art is extremely useful for gaining a better knowledge of the artistic heritage and to guarantee its preservation. A derivatization GC/MS procedure for the identification of proteins in a microsample from painted works of art has been optimized. The amino acid fraction is derivatized using anhydrous dimethylformamide (DMF) as solvent instead of pyridine (Py), commonly used to facilitate the reaction. Although pyridine is often considered a silylation catalyst, there are many instances in which silylation reactions actually are slower in pyridine than other solvents. In addition, pyridine also may have other undesirable effects such as the promotion of secondary products and other chromatographic anomalies. Using DMF, the formation of artifacts is limited and the derivatization yield of hydrophilic amino acids such as proline and hydroxyproline has improved, thus making the identification of organic paint media more straightforward. The method has been validated and successfully applied to identify the binder of the sample taken from the pictorial cycle of the 12th century monastery of Santa Maria delle Cerrate (Lecce, Italy), thus highlighting the use of eggs as a binding medium.

Preanalytical and analytical challenges in gas chromatographic determination of cholesterol synthesis and absorption markers

IntroductionCholesterol homeostasis disruption contributes to the development of different pathologies. Non-cholesterol sterols (NCSs) serve as cholesterol synthesis markers (desmosterol and lathosterol), and cholesterol absorption surrogate markers (campesterol, stigmasterol and β-sitosterol). The study aimed to resolve certain new pre-analytical and analytical problems and ensure a reliable and validated method. Materials and methodsMethod optimization, validation and stability studies were executed in human serum and plasma. Freeze-thaw cycles were done with and without antioxidant. Gas chromatography-mass spectrometer (GC-MS) was used for NCSs confirmation and plasticizer identification, while GC-flame ionization detector (GC-FID) was used for NCSs quantitation. ResultsIntra- and inter-assay variabilities for all NCSs were 2.75–9.55% and 5.80–7.75% for plasma and 3.10–5.72% and 3.05–10.92% for serum, respectively. Recovery studies showed satisfactory percentage errors for all NCSs: 93.4–105.7% in plasma and 87.5–106.9 in serum. Derivatized samples were stable up to 7days at −20°C and derivatization yield was affected by presence of plasticizers. Fatty acid amids were identified as interfering plastic leachates. Statistically different NCSs concentrations were observed after the 1st freeze-thaw cycle, in antioxidant-free samples, and after the 4th cycle in antioxidant-enriched samples. ConclusionsAll of the in-house procedures proved to be useful for minimizing the preanalytical and analytical variations, as proven by the validation results.

Highly sensitive method for aldehydes detection: Application to furfurals analysis in raisin and bovine milk powder

A pre-column fluorescent derivatization method based on nitrone formation has been applied to determine furfurals (e.g. furfural (F), 5-methyfurfural (5-MF) and 5-hydroxymethylfurfural (5-HMF)) in food samples for the first time. An N-substituted hydroxylamine reagent 4-((hydroxyamino)butyl)-7-hydroxycoumarin (HAHC) was used to react with the aldehyde group of furfurals to form stable nitrone derivatives with high fluorescence intensities. The reactions proceeded under mild conditions in 30 min with high derivatization yields (>93%). A baseline-separation of three furfurals derivatives was subsequently achieved within 25 min on a reversed-phase column. The detection limits were at the low femtomol level (S/N = 3, 20 μL per injection). The linear range of the calibration curve was 0.4–4000 nM with good correlation coefficients (R2 ≥ 0.9991). The proposed method was further applied for food sample analysis, such as bovine milk powder and raisin. Satisfactory recoveries were obtained in the range of 94.7%–103.5%. Above all, this pre-column derivatization method is simple, fast and highly sensitive, providing an effective and promising way for future studies of aldehydes in different matrices.

2-(4-Amino)-Phenyl-1-Hydrogen-Phenanthrene [9,10-d] Imidazole as a Novel Fluorescent Labeling Reagent for Determination of Fatty Acids in Raspberry

A novel method has been established for the rapid separation and determination of free fatty acids from 37 different varieties of raspberry. In this study, a new fluorescent labeling reagent for fatty acids, 2-(4-amino)-phenyl-1-hydrogen-phenanthrene [9, 10-d] imidazole (PIA), has been synthesized and successfully applied to the high-performance liquid chromatography (HPLC) determination of fatty acids in raspberry. The novel method has been optimized by HPLC with fluorescence detection and online mass spectrometry identification (HPLC–FLD–MS/MS). The 22 main fatty acids (FAs) present in raspberry were derivatized by PIA and separated on a reversed-phase Hypersil GOLD column with gradient elution. The main experimental parameters affecting extraction efficiency and derivatization yield were investigated and optimized by response surface methodology (RSM) combined with Box–Behnken design (BBD). Under the optimum conditions, the method was successfully applied for the analysis of 22 fatty acids in 37 different varieties of raspberry. Good linear correlations were observed for all fatty acids with correlation coefficients of > 0.9978. Limits of detection and quantification (LOD and LOQ) were in the range of 0.12 to 0.49 ng/mL and 1.07 to 2.81 ng/mL, respectively. Furthermore, the results indicated that the raspberries were rich in fatty acids, but the contents of the fatty acids varied among the different varieties.

Highly sensitive and specific derivatization strategy to profile and quantitate eicosanoids by UPLC-MS/MS

Eicosanoids are signaling molecules mainly oxidized from arachidonic acid (ARA) and eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). They have attracted increasing attention from the scientists attributing to their essential physiological functions. However, their quantification have long been challenged by the low abundance, high structure similarity, poor stability and limited ionization efficiency. In this paper, an ultra-high performance liquid chromatograph coupled with tandem mass spectrometry (UPLC-MS/MS) strategy was developed for the comprehensive profiling of more than 60 eicosanoids based on an efficient derivatization reagent 2,4-bis(diethylamino)-6-hydrazino-1,3,5-triazine (T3) and general multiple reaction monitoring (MRM) parameters. Carboxylic acid of eicosanoid was converted to amide in 30 min at 4 °C with derivatization yield larger than 99%. Limits of quantitation (LOQs) for derivatized eicosanoids varied from 0.05 to 50 pg depending on their structures. The sensitivities of derivatized eicosanoids were enhanced by 10- to 5000-folds compared to free eicosanoids. Stabilities of T3 modified eicosanoids were also highly improved compared to free eicosanoids. This new method can also be used to quantify eicosanoids in bio-samples using isotopic internal standards with high efficiency and reliability within 19 min. 46 and 50 eicosanoids in rat plasma and heart tissue from control and acute myocardial ischemia (AMI) model rats were respectively profiled and quantitated using this new method. And 24 of 46 and 25 of 50 eicosanoids were found to be significantly changed between control and model groups. The changed eicosanoids related to AMI modeling were further statistically analyzed and interpreted based on eicosanoid metabolism pathway.

Determination of Triterpene Acids from 37 Different Varieties of Raspberry using Pre-column Derivatization and HPLC Fluorescence Detection

In this work, we have developed an efficient method for the rapid extraction and separation of triterpene acids from 37 different varieties of raspberry via ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME). The triterpene acids were then determined by high-performance liquid chromatography (HPLC) with fluorescence detection using benzimidazo-[2,1-b]quinazolin-12(6H)-one-5-ethyl-p-toluenesulfonate (BQETS) as the labeling agent. Five triterpene acids, including asiatic acid (AA), maslinic acid (MA), corosolic acid (CA), oleanolic acid (OA) and betulinic acid (BA), were extracted by UA-DLLME using chloroform and acetone as the extracting and dispersing solvents, respectively. After extraction and nitrogen flushing, the extracts were simultaneously characterized by HPLC based on pre-column derivatization using BQETS, a new labeling agent synthesized in our laboratory. Several key parameters affecting the extraction efficiency and derivatization yields were investigated and optimized by response surface methodology (RSM) combined with Box–Behnken design (BBD). The method was further validated for linearity (correlation coefficient R 2 > 0.9979), precision (RSD = 0.23–2.45 %), and recovery (RSD = 90–106.5 %). The limits of detection (LODs) and the limits of quantification (LOQs) were determined to be within the range of 1.83–7.69 µg/L and 6.06–25.47 µg/L, respectively. This is the first report of the use of BQETS as a pre-column derivatization agent for the determination of triterpene acids in real samples. The proposed method has been applied to the determination of five triterpene acids in 37 different raspberry varieties with significantly increased sensitivity compared to other methods. The results obtained indicate that the contents of triterpene acids vary significantly across different raspberry varieties.

Large‐scale Enrichment of Sulfur‐containing Acid in Acetonitrile Using Triazine Analog as the Derivatizing Reagent for Desorption from Humic‐fraction‐modified Silica Gel

AbstractThe analog of triazine is used as the derivatizing reagent for enriching large‐scale acid (e.g. amino acid) containing a sulfur atom on the humic‐fraction‐modified silica gel in acetonitrile and desorbing from the adsorbent in hexane, respectively. The percent yield of the chemical derivatization under alkaline conditions, ranging from about 8 to almost 100 %, is pH dependent, and varies significantly among these examined analytes, believed to be due to the structure of the analyte, not the derivatizing reagent. The percentage of enrichment, not reproducible under an aqueous environment and independent of the type of triazine analogs, reaches almost 100 % in all cases. The force leading to the adsorption is the complexation between carboxyl groups on analyte and a humic‐fraction‐modified adsorbent based on the adsorption equilibrium results. Consequently, these results are not reproducible under ethyl ether or methanol environments due to the competition for binding sites from solvent molecules.