Liquid‐Chromatography Quantitative Analysis of 20 Amino Acids after Derivatization with FMOC‐Cl and Its Application to Different Origin Radix isatidis

We developed a simple, rapid and reliable method for determination of 20 common amino acids based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl) and RP‐LC/UV, this method was first introduced into quantitative analysis of amino acids. The amino groups of amino acids were trapped with FMOC‐Cl to form amino acid‐FMOC‐Cl adducts which can be suitable for LC‐UV. Chromatographic separation was performed on a C₁₈ column with a mobile phase gradient consisting of acetonitrile and sodium acetate solution. This method was shown to be sensitive for 20 common amino acids. In the intra‐day precisions assay, the range of RSDs was 3.21‐7.67% with accuracies of 92.34‐102.51%; for the inter‐day precisions assay, the range of RSDs was 5.82‐9.19% with accuracies of 90.25‐100.63%. The results also indicated that solutions of amino acids‐FMOC‐Cl can be kept at room temperature for at least 24 h without showing significant losses in the quantified values. The validated method was successfully applied to the determination of major four kinds of amino acids in R. isatidis samples (Arg, Pro, Met and Val). The total content of amino acids in different origin R. isatidis was 13.32‐19.16 mg/g. The differences between R. isatidis samples were large using HCA.     

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‐COOC₂H₅]⁺ ions. The fragment ions due to loss of C₂H₄, CO₂, (CO₂ + C₂H₄) from [M‐COOC₂H₅]⁺ 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 ¹³C₂‐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.     

Simultaneous determination of homocysteine,methionine and cysteine in maternal plasma after delivery by HPLC‐fluorescence detection with DBD‐F as a label

An HPLC‐fluorescence (FL) method for determination of sulfur‐containing amino acids such as homocysteine (Hcy), methionine (Met) and cysteine (Cys) in human plasma was developed. The sulfur‐containing amino acids were labeled with 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F). Calibration curves in the range of 1–100 µm (Hcy and Met) and 5–500 µm (Cys) indicated good linearities (r ≥ 0.998). The limits of detection at a signal‐to‐noise ratio of 3 were 0.13 (Hcy), 0.02 (Met) and 0.11 µm (Cys), respectively. Acceptable results for accuracy and precision of intra‐ and inter‐day measurements were obtained. The results of Hcy and Cys obtained by the proposed method indicated good correlations with the conventional method (r > 0.911, n = 20). Furthermore, the method was applied to determination of the sulfur‐containing amino acids in maternal plasma (n = 200) after delivery. The concentrations of Hcy, Met and Cys as a median (inter quartile range, Q₁ and Q₃) were 5.37 (3.32–7.79) μm , 25.20 (20.10–31.06) μm and 147.25 (102.81–189.31) μm , respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of free amino acids in Amur sturgeon by ultra‐performance liquid chromatography using pre‐column derivatization with 6‐aminoquinolyl‐carbamyl

A rapid, sensitive, and reliable ultra‐performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6‐aminoquinolyl‐carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C₁₈ column with Acetonitrile–acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5–1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.     

Capillary electrophoresis–mass spectrometry as a tool for the noninvasive target metabolomic analysis of underivatized amino acids for evaluating embryo viability in assisted reproduction

Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 μL) of spent culture media collected after the cultivation of in vitro fertilized embryos. Special attention was focused on the sample preparation procedure. The sample was processed with acetonitrile, which facilitates online sample preconcentration via field-amplified sample stacking, and undesired sample evaporation was significantly reduced by the simultaneous addition of dimethyl sulfoxide. Key parameters that affected electrophoretic separation and mass spectrometric detection were investigated, including the type of buffers and organic solvent, optimization of their concentrations, and finally the settings for their ionization. The separation and quantification of 19 amino acids were achieved using 15% acetic acid as the background electrolyte with a sheath liquid consisting of an equimolar mixture of methanol and water. The applicability of the optimized system was demonstrated by determining the amino acid profile in 40 samples of spent cultivation medium in this pilot study. This developed method also has great potential for amino acid analyses in minute sample volumes of other biological matrices.     

Quantitative analysis of 17 amino acids in tobacco leaves using an amino acid analyzer and chemometric resolution

A method was developed for quantifying 17 amino acids in tobacco leaves by using an A300 amino acid analyzer and chemometric resolution. In the method, amino acids were eluted by the buffer solution on an ion‐exchange column. After reacting with ninhydrin, the derivatives of amino acids were detected by ultraviolet detection. Most amino acids are separated by the elution program. However, five peaks of the derivatives are still overlapping. A non‐negative immune algorithm was employed to extract the profiles of the derivatives from the overlapping signals, and then peak areas were adopted for quantitative analysis of the amino acids. The method was validated by the determination of amino acids in tobacco leaves. The relative standard deviations (n = 5) are all less than 2.54% and the recoveries of the spiked samples are in a range of 94.62–108.21%. The feasibility of the method was proved by analyzing the 17 amino acids in 30 tobacco leaf samples.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Microwave‐Assisted Synthesis of β‐Lactams and Cyclo‐β‐dipeptides

Different cyclo‐β‐dipeptides were prepared from corresponding N‐substituted β‐alanine derivatives under mild conditions using PhPOCl₂ as activating agent in benzene and Et₃N as base. To evaluate β³‐substituent influence, the amino acids 7 – 26 were synthesized, and a β‐lactam formation reaction was carried out instead of cyclo‐β‐dipeptide formation. The crystal structures of three derivatives of cyclo‐β‐peptides and one β‐lactam are presented.     

A UPLC–MS/MS approach for simultaneous determination of eight flavonoids in rat plasma,and its application to pharmacokinetic studies of Fu‐Zhu‐Jiang‐Tang tablet in rats

The purpose of this study is to establish and validate a UPLC–MS/MS approach to determine eight flavonoids in biological samples and apply the method to pharmacokinetic study of Fu‐Zhu‐Jiang‐Tang tablet. A Waters BEH C₁₈ UPLC column was employed with methanol/0.1% formic acid–water as mobile phases. The mass analysis was carried out in a triple quadrupole mass spectrometer using multiple reaction monitoring with negative scan mode. A one‐step protein precipitation by methanol was used to extract the analytes from blood. Eight major flavonoids were selected as markers. Our results showed that calibration curves for 3′‐hydroxypuerarin, mirificin, puerarin, 3′‐methoxypuerarin, daidzin, rutin, astragalin and daidzein displayed good linear regression (r ² > 0.9986). The intra‐day and inter‐day precisions (RSD) of the eight flavonoids at high, medium and low levels were <8.03% and the bias of the accuracies ranged from −5.20 to 6.75%.The extraction recoveries of the eight flavonoids were from 91.4 to 100.5% and the matrix effects ranged from 89.8 to 103.8%. The validated approach was successfully applied to a pharmacokinetic study in Sprague–Dawley rats after oral administration of FZJT tablet. Double peaks were emerged in curves of mean plasma concentration for 3′‐methoxypuerarin, which was reported for the first time.     

Lewis Acid‐Mediated Addition of Amino Acid‐Substituted α‐Allylsilanes to Aromatic Acetals

Unnatural amino acids extend the pharmacological formulator's toolkit. Strategies to prepare unnatural amino acid derivatives using Lewis acid‐activated allylsilane reactions are few. In this regard, we examined the utility of allylsilanes bearing an amino acid substituent in the reaction. Diastereoselective addition of methyl 2‐(N‐PG‐amino)‐3‐(trimethylsilyl)pent‐4‐enoate and methyl (E)‐2‐(N‐PG‐amino)‐3‐(trimethylsilyl)hex‐4‐enoate (PG=protecting group), 2 and 13 , respectively, to aromatic acetals in the presence of Lewis acids is described. Of those examined, TiCl₄ was found to be the most effective Lewis acid for promoting the addition. At least 1 equiv. of TiCl₄ was required to achieve high yields, whereas 2 equiv. of BF₃?OEt₂ were required for comparable outcomes. Excellent selectivity (>99% syn/anti) and high yield (up to 89%) were obtained with halo‐substituted aromatic acetals, while more electron‐rich electrophiles led to both lower yields and diastereoselectivities.     

Solid‐phase Total Synthesis of (−)‐Apratoxin A and Its Analogues and Their Biological Evaluation

Two approaches for the solid‐phase total synthesis of apratoxin A and its derivatives were accomplished. In synthetic route A, the peptide was prepared by the sequential coupling of the corresponding amino acids on trityl chloride SynPhase Lanterns. After cleavage from the polymer‐support, macrolactamization of 10 , followed by thiazoline formation, provided apratoxin A. This approach, however, resulted in low yield because the chemoselectivity was not sufficient for the formation of the thiazoline ring though its analogue 33 was obtained. However, in synthetic route B, a cyclization precursor was prepared by solid‐phase peptide synthesis by using amino acids 13 – 15 and 18 . The final macrolactamization was performed in solution to provide apratoxin A in high overall yield. This method was then successfully applied to the synthesis of apratoxin analogues. The cytotoxic activity of the synthetic derivatives was then evaluated. The epimer 34 was as potent as apratoxin A, and O‐methyl tyrosine can be replaced by 7‐azidoheptyl tyrosine without loss of activity. The 1,3‐dipolar cycloaddition of 38 with phenylacetylene was performed in the presence of a copper catalyst without affecting the thiazoline ring.     

Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages

5‐Dimethylamino‐1‐sulfonyl naphthalene (DNS, commonly referred as dansyl) is a functionality, bearing well‐established properties in directing the fragmentation, by mass spectrometry (MS), of the corresponding ionized sulfonylated derivatives. This property is shared also by its labeled analogs. The use of d₀/d₆ DNS derivatives is now exploited in the application of the well‐established isotope dilution mass spectrometric approach in the assay of complex mixtures. A new method for the quantitation of amino acids (AAs) in beverages is therefore presented, which relies on liquid chromatographic separation of their N‐dansylated derivatives followed by comparative electrospray tandem MS/MS of the d₀/d₆ isobaric mixtures. Labeled and unlabeled DNS derivatives of the selected AAs are readily available by microwave‐assisted synthetic protocols. The novelty of the method is represented by the use of heavy and light DNS‐isotopologue providing suitable reporter groups. Multiple‐reaction monitoring has been applied in the assay of AAs in wine, pineapple juice and bergamot juice with good‐to‐excellent results as proved by both relative standard deviation, lower than 15%, and by the accuracy values in the range 90–110%. Copyright © 2012 John Wiley & Sons, Ltd.     

Rational Topological Design for Fluorescence Enhancement upon Aggregation of Distyrylfuran Derivatives

A series of 2,5‐distyrylfuran derivatives bearing pentafluorophenyl‐ and cyanovinyl units have been synthesized for aggregation‐induced emission (AIE). The effect of the type and extent of the supramolecular connections on the AIE of the furan derivatives were examined and correlated with their X‐ray crystal structures. It was found that the simultaneous presence of cyano and perfluorophenyl units strongly enhances the fluorescence upon aggregation. Single‐crystal X‐ray diffraction analysis confirmed that C?H???F, F???F, C?H???nitrile, Ar???Ar_F (Ar=aryl, Ar_F=fluoroaryl), and nitrile???Ar_F intra‐ and intermolecular interactions drive the topology of the molecule and that solid‐state supramolecular contacts favor AIE of the furan derivatives.     

Application of 10‐ethyl‐acridine‐3‐sulfonyl chloride for HPLC determination of aliphatic amines in environmental water using fluorescence and APCI‐MS

A simple, sensitive method for the determination of aliphatic amines based on a sulfonylation reaction using 10‐ethyl‐acridine‐3‐sulfonyl chloride (EASC) as pre‐column labeling reagent with fluorescence detection and APCI‐MS identification has been developed. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by HPLC with an excitation maximum at λ_ex 270 nm and an emission maximum at λ_em 430 nm. Identification of derivatives was carried out by online post‐column MS in positive‐ion mode. Comparing with the widely used 5‐dimethylaminonaphthalene‐1‐sulfonylchloride (Dansyl‐Cl), EASC‐amine derivatives not only exhibited high fluorescence but also exhibited excellent MS ionizable potential. Detection limits obtained from 0.10 pmol injection, at a S/N of 3, were 4.0–12.7 fmol. The mean intra‐ and inter‐assay precision for all aliphatic amine levels were <3.84 and 3.21%, respectively. Excellent linear responses were observed with coefficients of >0.9995.     

Facile Synthesis of 7‐epi‐Taxane and Its Derivatives and Preliminary Evaluation of Anticancer Activity

7‐epi‐Taxane has been achieved efficiently in gram scale from natural taxane via inversion of the 7‐hydroxyl group simply using Ag₂O as catalyst and DMF as solvent. The catalyst could be quantitatively recovered by filtration without loss of catalytic activity. This condition is also applicable to the direct epimerization of taxane derivatives (e.g., docetaxel and paclitaxel) to 7‐epi‐taxane derivatives (e.g., 7‐epi‐docetaxel and 7‐epi‐paclitaxel). Furthermore, 33 ester derivatives of 7‐epi‐taxane with different amino acid moieties at the position of C‐13 were successfully synthesized via esterification without protecting C‐7‐OH. Bioassay results revealed that compounds 13 and 18 have good selectivity against prostatic cancer cell line DU145, with IC₅₀ value as low as 15.9 nmol/L for 18 .     

Simultaneous determination of 20 components in red wine by LC‐MS: Application to variations of red wine components in decanting

The decanting of red wines has a long tradition in red wine service from the perspective of modifying the aroma or taste of a wine. A simple and sensitive liquid chromatography‐mass spectrometry method was developed for the simultaneous determination of 20 organic acids and polyphenols in decanting red wine. The separation was performed on a Diamonsil C₁₈ column (250 mm × 4.6 mm, 5 μm) using a mobile phase composed of methanol‐0.1% acetic acid under gradient elution. Analysis was performed in selected ion monitoring mode with negative electrospray ionization interface. All the linear regressions showed good linear relationships (r² > 0.9973) between the peak area and concentration of each marker. The assay was reproducible with overall intra and interday variation of less than 5.0%. The recoveries for the quantified compounds were observed over the range of 92.1–108.3% with RSD values less than 5.7%. The method developed was successfully applied to determine the variations of the 20 components in red wine after decanting in different conditions. Concentrations of most organic acids and polyphenols investigated in the red wine were decreased in decanting. In addition, increment of duration, temperature, and light intensity would intensify the changes.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

A high-sensitivity UPLC-MS/MS method for simultaneous determination and confirmation of triptolide in zebrafish embryos

A high‐sensitivity ultra‐performance liquid‐chromatography (UPLC) coupled with tandem mass spectrometric method was developed for simultaneous quantification and confirmation of triptolide in both zebrafish embryos and the aqueous‐exposure solution on a tandem quadrupole mass spectrometer (TQ‐MS). This was achieved by performing quantification using the multiple reaction monitoring (MRM) acquisition with simultaneous characterization of the MRM peak using product ion confirmation (PIC) acquisition as it elutes from the chromatographic system. Separation was achieved on a 1.7 µm C₁₈ UPLC column using 0.1% formic acid water–acetonitrile mobile phase with a cycle time of 6 min. The linear range of 0.115–360 ng/mL, and lower limits of detection of 0.02 ng/mL and quantification of 0.064 ng/mL were established. This method was successfully applied to determine the time course of triptolide absorption by zebrafish embryos and the amount of triptolide remaining in the culture medium after administration of two triptolide dosages at three time points. This coupled MRM with PIC approach could provide both qualitative and quantitative results without the need for repetitive analyses. This resulted in the reduction of further confirmative experiments and analytical time, and ultimately increased laboratory productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Michael-Addition Reaction of Malononitrile with α,β-Unsaturated Cycloketones Catalyzed by KF／Al2O3

A series of KF/Al2O3 catalyzed Michael-addition reactions between malononitrile and α,β-unsaturated cycloketones in DMF solution were studied. At room temperature, 2-cyano-3-aryl-3-(1,2,3,4-tetrahydronaphthalen-1-one-2-yl) propionitrile derivatives were synthesized by the reaction between 2-arylmethylidene-1,2,3,4-tetra-hydronaphthalen-1-one and malononitrile. However, if the temperature was increased to 80℃, 2-amino-3-cyano-4-aryl-4H-benzo[h]chromene derivatives were obtained in high yields. When the α,β-unsaturated ketones were replaced by 2,6-biarylmethylidenecyclohexanone or 2,5-biarylmethylidenecyclopentanone, another series of 2-amino-3-cyano-4H-pyran derivatives was isolated successfully. The structures of the products were confirmed by X-ray diffraction analysis.     

Phase‐Transfer‐Catalyzed Asymmetric SNAr Reaction of α‐Amino Acid Derivatives with Arene Chromium Complexes

Although phase‐transfer‐catalyzed asymmetric S_NAr reactions provide unique contribution to the catalytic asymmetric α‐arylations of carbonyl compounds to produce biologically active α‐aryl carbonyl compounds, the electrophiles were limited to arenes bearing strong electron‐withdrawing groups, such as a nitro group. To overcome this limitation, we examined the asymmetric S_NAr reactions of α‐amino acid derivatives with arene chromium complexes derived from fluoroarenes, including those containing electron‐donating substituents. The arylation was efficiently promoted by binaphthyl‐modified chiral phase‐transfer catalysts to give the corresponding α,α‐disubstituted α‐amino acids containing various aromatic substituents with high enantioselectivities.