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A derivatizaton method combined with gas chromatography-mass spectrometry(GC-MS)was established for detection of isobutyl chloroformate(IBCF)residue in active pharmaceutical ingredient of agatroban.The extraction and derivatization reagents,derivatization time,qualitative and quantitative ions were selected and optimized,respectively.The possible mechanism of derivatization and characteristic fragment ions fragmentation were speculated.The agatroban samples were dissolved and extracted by methanol,and the residual IBCF was derived with methanol to generate methyl isobutyl carbonate(MIBCB).After 24 h static derivatization at room temperature,IBCF was completely transformed into MIBCB,which could be used to indirectly detect IBCF accurately.The results showed that the linearity of this method was good in the range of 25-500 ng/mL(R2=0.9999).The limit of detection(LOD,S/N=3)was 0.75 μg/g,and the limit of quantification(LOQ,S/N=10)was 2.50 μg/g.Good recoveries(95.2%-97.8%)and relative standard deviations(RSDs)less than 3.1%(n=6)were obtained from agatroban samples at three spiked levels of IBCF(2.50,25.00,50.00 μg/g),which showed good accuracy of this method.Good precision of detection results was obtained by different laboratory technicians at different times,the mean value of spiked sample solution(25.00 μg/g)was 24.28 μg/g,and the RSD was 2.1%(n=12).The durability was good,minor changes of detection conditions had little effect on the results.Under the original condition and conditions with initial column temperature±5℃,heating rate±2℃/min,column flow rate±0.1 mL/min,the IBCF content of spiked sample solution(25.00 μg/g)was detected,the mean value of detection results was 24.16 μg/g,and the RSD was 2.2%(n=7).Eight batches of agatroban samples from two manufacturers were detected using the established method,and the results showed that no IBCF residue was detected in any of these samples.The agatroban samples could be dissolved by methanol,and then the IBCF residue could be simultaneously extracted and derived with methanol as well.This detection method had the advantages of simple operation,high sensitivity,low matrix effect and accurate quantification,which provided a new effective method for detection of IBCF residue in agatroban.
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AIM To determine the contents of aspartic acid,glutamic acid,serine,glycine,threonine,citrulline,arginine,alanine,γ-amino-butyric acid,tyrosine,valine,phenlalanine,isoleucine,ornithine,leucine,lysine and proline in Gualoupi Injection and its intermediates,and to analyze their change laws.METHODS The OPA-FMOC online derivatization analysis was performed on a 45℃ thermostatic Waters XBridge C18 column(4.6 mm×100 mm,3.5 μm),with the mobile phase comprising of phosphate buffer solution-[methanol-acetonitrile-water(45 : 45 : 10)]flowing at 1 mL/min in a gradient elution manner,and the detection wavelengths were set at 262,338 nm.Principal component analysis and heatmap analysis were adopted in chemical pattern recognition for the corresponding intermediates in ten processes of six batches of samples.RESULTS Seventeen amino acids showed good linear relationships within their own ranges(R2>0.998 0),whose average recoveries were 83.4％-119.5％with the RSDs of 0.91％-7.94％.Different batches of samples in the same process were clustered,and the corresponding intermediates in different processed were clustered into three groups.Alcohol precipitation and cation exchange column demonstrated the biggest influences on amino acid composition.CONCLUSION This experiment can provide important references for the critical factors on quality control of Gualoupi Injection,thus ensure the stability and uniformity of final product.
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@#Objective To develop an ultra performance liquid chromatography(UPLC)method for simultaneous determination of 19 amino acids in rat serum by employing the pre-column derivatization of phenyl isothiocyanate(PITC),verify and preliminarily apply the method.Methods The analysis was performed on an ACQUITY UPLC~?BEH C_(18) column(2.1mm×100 mm,1.7 μm) by using 0.1 mol/L sodium acetate buffer solution-acetonitrile as mobile phase at the flow rate of 0.3 mL/min in gradient elution.The column temperature was fixed at 52 ℃,and the detection wavelength was 254 nm.The specificity,linearity,precision,stability and accuracy of the method were verified based on the optimized chromatographic conditions and preparation method of sample solution,and the contents of 19 amino acids in rat serum were determined by using the developed method.Results All 19 amino acids in rat serum were separated well within 10 min,and the blank derivatization solvent and internal standard compounds had no interference with the analysis of amino acids.All the components exhibited good linear correlation in the given range(r ≥ 0.998 0),the limit of detection was 0.004 6-0.186 5 μg/mL,and the limit of quantitation was 0.013 9-0.559 6 μg/mL.The RSDs of precision and stability tests were all less than 4%.The average spike recoveries ranged from 90% to 110% and the RSDs were less than 5%.The contents of amino acids in rat serum ranged from 4.837 to 110.233 μg/mL.Conclusion The developed method is sensitive,simple and feasible,which is suitable for the analysis of amino acids in rat serum.
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The goal of the current study is to develop and validate a new chromatographic method for Fosfomycin Trometamol's determination in both pure form and pharmaceutical formulation. In the present study, a pre chromatographic derivatization of Fosfomycin Trometamol was done by forming an ion-pair complex of Heterocyclic nitrogen using the acidic dye methyl orange and phthalate buffer of pH-6. The yellow ion-pair complex was extracted with chloroform and it was further extracted with aqueous solution of 0.01M HCL. The ion-pair complex of Fosfomycin Trometamol and methyl orange obeyed Beer's law in the range of 30 -70 ?g/ml with a correlation coefficient (r2) of 0.9946. A Liquid chromatography system equipped with an Agilent ACE C18 column (250 X 4.6mm, 5?m) was used as a stationary phase in this work. The developed method was validated according to ICH guidelines. For determining the medication in bulk and formulation, the devised approach was proven to be accurate, sensitive, and repeatable
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This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 μL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.
Subject(s)
Amino Acids/metabolism , Eucommiaceae/chemistry , Chromatography, High Pressure Liquid/methods , Plant Leaves/chemistryABSTRACT
Background Diacetyl (DC) is widely used in the food flavoring industry and excessive occupational exposure to DC can cause serious respiratory diseases. However, there is no corresponding national standard method for the determination of DC in the air of workplace. Objective To establish a method for the determination of DC in workplace air by high performance liquid chromatography using 4-nitro-o-phenylenediamine (NPDA) as precolumn derivatization. Methods DC in the air of workplace was collected by solution absorption method. This experiment used NPDA as the derivatization reagent. By adjusting acidity of solution and optimizing concentration ration of DC/NPDA, derivatization temperature, and time, a method for the determination of DC in workplace air was proposed, and its performance indexes such as linearity, detection limit, and lower limit of quantification were obtained. Sampling efficiency was evaluated by relative comparison method, and sample stability was evaluated by sample preservation test. Accuracy and precision of the method were evaluated by standard addition recovery test with blank samples, and an interference test was carried out by adding standard samples. The established method was applied to actual samples to evaluate its adaptability. Results A combination of 60 °C for 2 h was selected for derivatization because a higher derivatization reaction temperature and a longer reaction time associated with a higher derivatization efficiency. The solution was separated by SB-C18 column (250 mm×4.6 mm, 5 μm) at 30 ℃, using a mixture of methanol and water (v/v, 65%/35%) as mobile phase with an elution flow rate of 1.0 mL·min−1, and was detected with a variable wavelength detector (λmax=257 nm) by qualitative analysis based on retention time and quantitative analysis based on external standard method. In terms of the proposed method, the linear range of detection was from 5 μg·L−1 to 2000 μg·L−1, with a correlation coefficient of 0.9999, and a detection limit of 1.3 μg·L−1, the quantitative detection of the lower limit was 4.3 μg·L−1, with a sampling volume V0 of 3.0 L, the minimum detection concentration was 4.3 μg·m−3, and the minimum quantitative concentration was 14.3 μg·m−3. The recovery rate was 99.1%-100.8%, the intra-batch precision was 0.5%-3.0%, and the inter-batch precision was 1.2%-2.0%. The average sampling efficiency of this method was 94.5%, and the sample could be stored at 4 °C for at least 14 d. The coexisting components in the air of the workplace did not interfere with the determination of DC. The DC content in the air of a flavor workplace was 5.86-8.85 mg·m−3. Conclusion A determination method for DC in workplace air by high performance liquid chromatography using NPDA as precolumn derivatization after being collected by 1.0% phosphoric acid absorbent is proposed and has the advantages of simple operation, high sensitivity, and good accuracy. With no DC loss and degradation, the method may satisfy the request for DC determination in the air of workplace.
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Trace amines(TAs)are metabolically related to catecholamine and associated with cancer and neuro-logical disorders.Comprehensive measurement of TAs is essential for understanding pathological pro-cesses and providing proper drug intervention.However,the trace amounts and chemical instability of TAs challenge quantification.Here,diisopropyl phosphite coupled with chip two-dimensional(2D)liquid chromatography tandem triple-quadrupole mass spectrometry(LC-QQQ/MS)was developed to simul-taneously determine TAs and associated metabolites.The results showed that the sensitivities of TAs increased up to 5520 times compared with those using nonderivatized LC-QQQ/MS.This sensitive method was utilized to investigate their alterations in hepatoma cells after treatment with sorafenib.The significantly altered TAs and associated metabolites suggested that phenylalanine and tyrosine metabolic pathways were related to sorafenib treatment in Hep3B cells.This sensitive method has great potential to elucidate the mechanism and diagnose diseases considering that an increasing number of physiological functions of TAs have been discovered in recent decades.
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Amino-containing compounds,including amino acids,aliphatic amines,aromatic amines,small peptides and catecholamines,are involved in various biological processes and play vital roles in multiple meta-bolic pathways.Previous studies indicated that some amino-containing metabolites are significant diagnostic and prognostic biomarkers of gastric cancer.However,the discovery of precise biomarkers for the preoperative diagnosis of gastric cancer is still in an urgent need.Herein,we established a polarity-regulated derivatization method coupled with liquid chromatography-mass spectrometry(LC-MS)for amino-containing metabolites profiling in the serum samples of patients with gastric cancer and healthy controls,based on our newly designed and synthesized derivatization reagent(S)-3-(1-(diisopropox-yphosphoryl)pyrrolidine-2-carboxamido)-N-hydroxysuccinimidyl ester(3-DP-NHS).Enhanced separa-tion efficiency and detection sensitivity for amino-containing metabolites were achieved after derivatization.This method exhibited good linearity,recovery,intra-and inter-day precision and accu-racy.Only 5 μL serum is needed for untargeted analysis,enabling 202 amino-containing metabolites to be detected.Statistical analysis revealed altered amino acid metabolisms in patients with gastric cancer.Furthermore,ultra high performance liquid chromatography coupled with mass spectrometry(UHPLC-MS/MS)analysis quantification revealed increased serum levels of tryptamine and decreased concen-trations of arginine and tryptophan in patients with gastric cancer.Receiver operating characteristic(ROC)curves indicated that an increased tryptamine/tryptophan ratio could serve as a potential biomarker for gastric cancer diagnosis.This study demostrated the possibility of using serum amino acid biomarkers for gastric cancer diagnosis,providing new avenues for the treatment of gastric cancer.
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In this work,a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry(LC-MS)method was developed for metabolite profiling of the glutathione anabolic pathway(GAP)in cancer tissues and cells.The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate(DMMIC)was used to label the amino group of metabolites,and a reductant of dithiothreitol(DTT)was employed to stabilize the thiol group.By combining DMMIC derivatization with LC-MS,it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples,which had good linearity(R2=0.9981-0.9999),precision(interday precision of 1.6％-19.0％and intraday precision of 1.4％-19.8％)and accuracy(83.4％-115.7％).Moreover,the recovery assessments in tissues(82.5％-107.3％)and in cells(98.1％-118.9％)with GSH-13C2,15N,and Cys-15N demonstrated the reliability of the method in detecting tissues and cells.Following a methodological evaluation,the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma(ESCC)and the effect of p-hydroxycinnamaldehyde(CMSP)on the GAP in KYSE-150 esophageal cancer cells.The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes,which can contribute to research on drugs and diseases.
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@#Objective To develop and verify a pre-column derivatization reverse phase-high performance liquid chromatography(RP-HPLC) method for determination of Glycine and Histidine content in recombinant proteins.Methods AccQ Tag-C 18(3.9 mm × 150 mm,4 μm) column was used as chromatographic column,6-aminoquinolyl-N-hydroxysccinimidyl carbamate(AQC) was used as pre-column derivatization reagent,while α-aminobutyric acid as internal standard.AccQTag Eluent A solution,acetonitrile solution and high-purity water were used as mobile phases.The UV detection wavelength was 248 nm,injection volume was 10 μL,flow rate was 1.0 mL/min,and column temperature was 37 ℃.The contents of Glycine and Histidine in samples were determined by the internal standard method,and the specificity,linearity,detection limit,quantitative limit,precision,accuracy and stability of the method were verified.Results The developed method effectively separated Glycine,Histidine and internal standard α-aminobutyric acid with high specificity.The standard curves of Glycine in the range of 2.25～11.25 μg/mL and Histidine in the range of 72.85～364.24 μg/mL showed good linearity,each correlation coefficient(R~2) 0.99.The detection limits were 2.25 μg/mL for Glycine and 18.21 μg/mL for Histidine.The quantitative limits were 4.69 μg/mL for Glycine and 32.86 μg/mL for Histidine.The relative standard deviation(RSD) of 6 replicates with the same concentration of Glycine and Histidine were 4.6% and 5.0%,and the RSD of recovery rate in intermediate precision test was 6.9% and 2.0%,respectively.The content of Glycine was close to the quantitative limit,and the average recoveries of high,medium and low concentrations of samples were within 75.9%～111.7%;The recoveries of Histidine ranged from 88.9% to 97.3%.The RSD of Glycine content and Histidine content was 7.7% and 3.3% respectively at 0,12,18,24,30 and 48 h in the same sample.Conclusion The pre-column derivatization RP-HPLC method has accurate and reliable results with high precision,which might be used for quality control of Glycine and Histidine content in recombinant proteins.
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@#In this paper, chemical derivatization-high performance liquid chromatography was used to determine the potential genotoxic impurities chloroacetyl chloride and chloroacetic acid, respectively, in the raw material of azintamide.Derivatization was carried out using 2-nitrophenylhydrazine followed by the determination.Separation was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5 μm), with mobile phase consisting of 0.1% phosphoric acid in water (A) and acetonitrile(B) by gradient elution, at a flow rate of 1 mL/min.The column temperature was 40 °C and the detection wavelength was 226 nm.The blank solvent, derivatization reagent, and azintamide did not interfere with the peak of the test substance, and the target component was well separated from the others.For impurities chloroacetyl chloride and chloroacetic acid, the limits of detection (LOD) were 7.5 ng/mL and 15 ng/mL respectively. There was a good linear relationship between the integral area and the concentration in the range of 30-300 ng/mL.The sample recovery rate was in the range of 87.37% ~ 109.75%.The two methods established in this study have good specificity, good precision, high sensitivity and simple operation, which can be used for the trace determination of potential genotoxic impurities chloroacetyl chloride and chloroacetic acid in the raw material of azintamide.
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Endogenous ribonucleotides(RNs)and deoxyribonucleotides(dRNs)are important metabolites related to the pathogenesis of many diseases.In light of their physiological and pathological significances,a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltri-fluoroacetamide(MTBSTFA)was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).A one-step extraction of cells with 85％methanol followed by a simple derivatization reaction within 5 min at room temper-ature contributed to shortened analysis time.The derivatives of 22 nucleoside mono-,di-and tri-phosphates were retained on the typical Cig column and eluted by ammonium acetate and acetonitrile in 9 min.Under these optimal conditions,good linearity was achieved in the tested calibration ranges.The lower limit of quantitation(LLOQ)was determined to be 0.1-0.4 μM for the tested RNs and 0.001-0.1 μM for dRNs.In addition,the precision(CV)was＜15％and the RSD of stability was lower than 10.4％.Furthermore,this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT116 exposed to 10-hydroxycamptothecin.In conclusion,our method has proven to be simple,rapid,sensitive,and reliable.It may be used for specific expanded studies on intracellular pharmacology in vitro.
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@#Abstract: Objective - - To establish a pre column derivatization high performance liquid chromatography method for detecting Methods dimethyl sulfate (DMS) in workplace air. DMS in workplace air was collected with mercaptopyridine impregnated （ silicone tube. The derivative of DMS and mercaptopyridine was eluted by mobile phase phase A: water, phase B: acetonitrile, ∶ the volume ratio was 40 60) , and separated with a C18 column, then detected with diode array detector and quantitated by a Results - standard curve. The linear range of DMS was 0.17 40.00 mg/L, with the correlation coefficient of 0.999 95. The detection limit and the lower limit of quantitation were 0.05 and 0.17 mg/L respectively. The minimum detection concentration and minimum quantitation concentration were 0.02 and 0.04 mg/m³, respectively (air sample volume of 4.5 L, 1.0 mL sample - - - solution). The average desorption efficiency was 98.40% 102.00%. The within run and between run relative standard deviations - - were 0.61% 3.92% and 1.71% 6.00%, respectively. The samples could be stored at room temperature for at least 14 days. Conclusion This method can be used to detect DMS in workplace air.
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Abstract L-Malic acid is the Active Pharmaceutical Ingredient of the latest generation of compound electrolyte injection (STEROFUNDIN ISO, Germany) and plays a very important role in the rescue of critically ill patients. The optical purity of L-malic acid is a Critical Quality Attributes. A new reversed-phase high performance liquid chromatography (RP-HPLC) method for pre-column derivatization of D-malic acid enantiomer impurity in L-malic acid bulk drug was established. The derivatization reaction was carried out using (R)-1-(1-naphthyl)ethylamine ((R)-NEA) as a chiral derivatization reagent. The Kromasil C18 column was used with a detection wavelength of 225 nm, a flow rate of 1.0 mL·min-1, and a column temperature of 30 °C. The mobile phase was acetonitrile-0.01 mol·L-1 potassium dihydrogen phosphate solution (containing 20 mmol·L-1 sodium heptanesulfonate, adjusted to pH 2.80 with phosphoric acid) (at a ratio of 45:55) and the resolution of D-malic acid and L-malic acid derivatization products reached 1.7. The proposed method possesses the advantages of simple operation, mild conditions, stable derivatization products and low cost. Also it gave better separation and was more accurate than previous methods
Subject(s)
Chromatography, High Pressure Liquid/methods , Malicum Acidum/analysis , Chromatography, Reverse-Phase/methods , Patients/classification , Total Quality Management/classificationABSTRACT
Low ionizability and poor chemical instability make some target analytes not suitable for direct LC-MS/MS analysis. To improve the detection performance, chemical derivation is frequently imported. However, such technology involves multiple disciplines such as laboratory medicine, organic chemistry and separation science. At present, it′s still a challenge for general application of chemical derivatization in routine clinical laboratories in our country. This article summarizes the key scientific connotation of chemical derivatization in the detection of vitamins, steroids, neurotransmitters and therapeutic drugs, and describes the derivatization approaches for development of chemical mass spectrometry in the future.
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Objective To establish a method for determination of the azide ions in blood by gas chromatography-mass spectrometry （GC-MS） following pentafluorobenzyl derivatization. Methods A blood sample of 0.2 mL was placed into a 10 mL glass test tube, and the internal standard sodium cyanide, derivatization reagent pentafluorobenzyl bromide and catalyst tetradecyl benzyl dimethyl ammonium chloride were added in turn. After vortex mixing, the mixture was heated with low-power microwave for 3 min. After centrifugation, the organic phase was taken for GC-MS analysis. Results The azide ions in blood had a good linear relationship in the mass concentration range of 0.5 to 20 μg/mL. The lowest detection limit was 0.25 μg/mL and the relative recovery was 91.36%-94.58%. The method was successfully applied to a case of death from sodium azide poisoning. The mass concentration of azide ions in the blood of the dead was 11.11 μg/mL. Conclusion The method developed in this paper has strong specificity and is easy to operate, which is suitable for the rapid detection of azide ions in blood.
Subject(s)
Humans , Azides , Gas Chromatography-Mass Spectrometry , IonsABSTRACT
OBJECTIVE： To establish pre- column derivatization-HPLC fingerprint of polysaccharide from Achyranthes bidentata，and to determine the contents of monosaccharide components ，so as to provide reference for quality evaluation of A. bidentata decoction pieces. METHODS ：Taking 10 batches of A. bidentata decoction pieces from different manufacturers as samples，the polysaccharides were extracted by water extraction and alcohol precipitation ，hydrolyzed by trifluoroacetic acid ，and then derivatized by 1-phenyl-3-methyl-5-pyrazolone for HPLC analysis. The determination was performed on Hanbon Hedera C 18 column with column temperature of 30 ℃ at the flow rate of 1.2 mL/min. The mobile phase consisted of acetonitrile- 0.02 mol/L ammonium acetate solution （gradient elution ）. The detection wavelength was set at 250 nm，and sample size was 20 μL. HPLC fingerprint was established and similarity evaluation was performed for 10 batches of A. bidentata polysaccharide by using TCM Chromatogramic Fingerprint Similarity Evaluation System （2012A edition ）. The common peak was identified by comparing with the reference substance ，and cluster analysis was performed by using SPSS 25.0 software. The contents of identified monosaccharides were determined by HPLC. RESULTS ：The similarity of 10 batches of A. bidentata polysaccharide were all higher than 0.95. Nine common peaks were fixed and a total of 5 common peaks were identified ，namely anhydrous glucose （peak 1）， mannose（peak 2），rhamnose（peak 4），galacturonic acid （peak 5）and arabinose （peak 7）. Results of cluster analysis showed that S1 sample was classified into one category ；S2，S5，S8 and S 9 samples were clustered into one category ；S3，S4，S6，S7 and S10 samples were clustered into one category. Results of content determination showed that the contents of anhydrous glucose in 10 batches of samples ranged from 6.17 to 17.55 mg/g；those of mannose ranged from 3.31 to 7.66 mg/g；those of rhamnose ranged from 38.80 to 73.97 mg/g；those of galacturonic acid ranged from 2.49 to 8.95 mg/g；those of arabinose ranged from 11.30 to 28.58 mg/g. CONCLUSIONS ：Established pre-column derivatization HPLC fingerprints and content determination method can provide reference for quality evaluation of A. bidentata decoction pieces.
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@#The changes of metabolic profile are closely related to external stimulus, and the concentration of the metabolite can directly reflect the physiological or pathological states of organisms. Therefore, the quantitative detection of metabolites is necessary. However, traditional targeted metabolomic methods have such drawbacks as narrow coverage and low sensitivity. In recent years, derivatization techniques have developed rapidly in the field of metabolomics. Derivatization reagents for amine, hydroxyl, carboxyl, carbonyl, hydrosulphonyl and other groups have been used in metabolomics research. This paper introduces various derivatization reactions and their applications according to group classification and reviewes the characteristics of multi-group derivatization techniques, with a propect of their research directions and challenges.
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The present study compared the appearance and chemical composition of fruits of Perilla frutescens var. arguta(PFA) and P. frutescens var. frutescens(PFF). VHX-6000 3 D depth of field synthesis technology was applied for the appearance observation. The metabolites were qualitatively and quantitatively analyzed by pre-column derivatization combined with gas chromatography-mass spectrometry(GC-MS). Finally, cluster analysis(CA), principal component analysis(PCA), and orthogonal partial least-squares discriminant analysis(OPLS-DA) were applied for exploring the differences in their chemical compositions. The results indicated that the size and color of PFA and PFF fruits were different. PFF fruits were significantly larger than PFA fruits. The surface color of PFA fruits was brown, while PFF fruits were in multiple colors, such as white, grayish-white, and brown. Amino acids, saccharides, organic acids, fatty acids, and phenolic acids were identified in PFA and PFF fruits. The results of CA, PCA, and OPLS-DA indicated significant differences in the content of components between PFA and PFF fruits. Three metabolites, including D-glucose, rosmarinic acid, and D-fructose, which were significantly higher in PFA fruits than in PFF fruits, were screened out as differential metabolites. Considering the regulation on the content of rosmarinic acid in Perillae Fructus in the Chinese Pharmacopoeia(2020 edition), the medicinal value of PFA fruits is higher than that of PFF. In conclusion, there are differences in appearance and chemical composition between PFA fruits and PFF fruits. These results are expected to provide fundamental data for specifying plant source and quality control of Perillae Fructus.
Subject(s)
Fatty Acids , Fruit , Gas Chromatography-Mass Spectrometry , Perilla frutescens , Plant ExtractsABSTRACT
OBJECTIVE：To analyze and compare th e contents of 6 kinds of monosaccharide in Astragalus membranaceus from different growth years . METHODS ：2-4 years old A. membranaceus from three areas were extracted with water extraction and alcohol precipitation ，Sevage deproteinization to obtain A. membranaceus polysaccharide. The samples were firstly hydrolyzed with trifluoroacetic acid （TFA）and then derivatized by 1-phenyl-3-methyl-5-pyrazolone（PMP）. HPLC analysis was adopted to determine the contents of 6 kinds of monosaccharide as mannose ，rhamnose，galacturonic acid ，glucose，galactose，arabinose. The determination was performed on Symmetry C 18 column with phosphate buffer solution （pH 6.8）-acetonitrile（84∶16，V/V）as mobile phase at the flow rate of 1.0 mL/min. The detection wavelength was 245 nm，and column temperature was 35 ℃. The sample size was 20 µL. RESULTS ：The contents of mannose ，rhamnose，galacturonic acid ，glucose，galactose and arabinose were 0.50-0.94， 0.76-1.60，3.35-7.86，87.33-275.77，1.95-8.96，2.35-14.04 mg/g，respectively. Total contents of monosaccharide from 2，3，4 years old A. membranaceus were 98.26-139.92，173.81-295.71，122.37-182.41 mg/g，respectively. There was significant difference in the contents of glucose between 3 old years A. membranaceus and 2，4 old years A. membranaceus （162.71-275.77 mg/g vs. 87.33-107.70，111.54-167.26 mg/g，P＜0.05）. CONCLUSIONS ：Above 6 monosaccharides are detected in 2，3，4 years old A. membranaceus，among which the content of glucose is the highest. The content of glucose in 3 years old A. membranaceus is higher than that in 2 and 4 years old A. membranaceus .