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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
@#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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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
@#To establish a method for the determination of formaldehyde and glyoxal simultaneously in varenicline tartrate active pharmaceutical ingredient (API) and its intermediate, formaldehyde and glyoxal were derivatized by 2, 4-dinitrophenylhydrazine (2,4-DNPH) to improve the HPLC retention and UV detection sensitivity. Separation was performed on a C8 (150 mm × 4.6 mm, 5 μm) column by linear gradient elution using acetonitrile and water as the mobile phase; the detective wavelength was set at 380 nm.Formaldehyde and glyoxal were quantitatively determined by an external reference method.Linear calibration was established for both formaldehyde and glyoxal in the range from 0.094 to 1.88 μg/mL.The detection and the quantification limits were 0.047 μg/mL (19 μg/g) and 0.094 μg/mL (38 μg/g), respectively.The recoveries were (95.0±1.1)% and (99.4 ± 2.6)% for formaldehyde and glyoxal, respectively.This method has been fully validated to be applicable to quantitative analysis of trace amount of formaldehyde and glyoxal in varenicline tartrate API and its intermediate.Test results demonstrated that the contents of both formaldehyde and glyoxal met the permitted daily exposure (PDE) limits for the finished products of varenicline tartrate API as well as its intermediate, though the glyoxal contents in the crude intermediates were likely to exceed the limit.The established method is valuable for the manufacturing process and quality control of varenicline tartrate.
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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 .
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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 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
A pre-Ultraperformance Liquid Chromatography (UPLC) column derivatization procedure was developed for thesimultaneous quantification of essential amino acids (EAAs) in the solid oral dosage pharmaceutical formulation.This analytical procedure has been validated with the help of the concept of total error. The total error approach (thecombination of systematic and random error) is a decision-making tool for ensuring the performance of the method.Fluorenylmethyloxycarbonyl chloride was used as a derivatization reagent. The amino acid derivatives were separatedon a C18 column (internal diameter 2.1 × 100 mm, 1.6 µm) by gradient elution with 0.1% trifluoroacetic acid andacetonitrile:water (90:10, v/v), respectively, as mobile phase A and B. About 10 EAAs could be detected at 265 nm in35 minutes with a flow rate of 0.25 ml/min. The linearity range of each amino acid was between 0.1 and 1.0 mg/ml.The accuracy and risk profiles were considered acceptable across the range. The precolumn derivatization procedureand the concept of the validation of total error could be used as an appropriate strategy to demonstrate the suitabilityof the analytical procedure for the separation and evaluation of EAA in solid oral dosage formulations.
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A pre-column derivatization and ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS) method was developed for qualitative and quantitative determination of medium- and short-chain fatty acids in mice feces, and was further applied to evaluate variations in the feces of mice before and after antibiotic treatment. This animal experiment had been approved by Animal Experimental Ethics Committee of Jiangsu Province Academy of Traditional Chinese Medicine. By optimizing the derivatization conditions and UHPLC-QTOF-MS/MS parameters a new UHPLC-QTOF-MS/MS method with 3-nitrophenylhydrazine as the derivatization reagent was developed for simultaneous determination of 16 medium- and short-chain fatty acids. Validation studies showed that the linearity of the calibration curves was good (R2>0.99), the RSD of intra-day and inter-day precision was less than 10%, the repeatability RSD was less than 6%, the recovery rate was between 80% - 120% at three spiked levels, and the stability RSD was less than 7% within 36 h. The types and amounts of the detected medium- and short-chain fatty acids in feces significantly changed after the mice were treated with antibiotics. The content of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and lactic acid decreased, whereas that of heptanoic acid and succinic acid increased significantly. All these results suggest that the newly established method is accurate and reliable, and can be used for determination of medium- and short-chain fatty acids in feces.
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OBJECTIVE：To establish pre -column derivatization-HPLC fingerprint of Polyporus polysaccharide ，and to determine the contents of main monosaccharide components ，so as to provide reference for quality evaluation of Polyporus umbellatus. METHODS ：Polyporus polysaccharide was extracted with boiling water and precipitated by ethanol and deproteinized by Sevage from 11 batches of P. umbellatus from different producing areas. The samples were firstly hydrolyzed with trifluoro-acetic acid （TFA）and then derivatized by 1-phenyl-3-methyl-5-pyrazolone（PMP）. HPLC analysis was then conducted. The determination was carried out on HypersiL BDS C 18 column with mobile phase composed of 0.1 mol/L phosphate buffer （pH 6.84）-acetonitrile（84∶16，V/V）by gradient elution at the flow rate of 1.0 mL/min. The detection wavelength was set at 254 nm， and column temperature was 30 ℃. The sample size was 20 µL. The similarity of 11 batches of Polyporus polysaccharide was evaluated by using TCM Chromatographic Fingerprint Similarity Evaluation System （2012A edition ），and the contents of main monosassharide components were detected. The peak was identified by comparing with the reference substance ，and cluster analysis was performed by using SPSS 23.0 software. RESULTS ：In HPLC fingerprints of the 11 batches of samples ，3 common peaks were identified ，namely mannose ，glucose and galactose. The similarity of all samples was above 0.94. Cluster analysis classified 11 batches of samples into three categories. S 1-S6，and S 8 were grouped into category 1；S7，S10 and S 11 were grouped into category 2；S9 was individually grouped into one category. Results of content determination showed that the contents of mannose ranged from 1.571 to 8.771 mg/g；those of glucose ranged from 26.072 to 132.194 mg/g，and those of galactose ranged from 3.420 to 36.593 mg/g. CONCLUSIONS ：Established pre-column derivatization HPLC fingerprints can provide reference for quality evaluation of P. umbellatus . The monosaccharide composition of different batches of Polyporus polysaccharide is the same ；there is no significant correlation between fingerprint characteristic peak and the origin of herbs ；there is significant difference in the content of monosaccharide of P. umbellatus .
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Objective::To establish a pre-column derivatization reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of 17 amino acids in Cynomorii Herba from different producing areas and conduct a multivariate statistical analysis. Method::RP-HPLC with pre-column derivatization was employed, with phenyl isothiocyanate (PITC) as derivatization reagent. Separation was performed on a WondaSil C18-WR column (4.6 mm×150 mm, 5 μm), with 0.05 mol·L-1 sodium acetate solution (pH 6.5) as mobile phase A, and acetonitrile-methanol-water (3∶1∶1) as mobile phase B for gradient elution at a flow rate of 0.8 mL·min-1. The detective wave length was set at 254 nm, and the column temperature was maintained at 35 ℃. Principal component analysis (PCA) and systematic cluster analysis (HCA) models were established for multivariate statistical analysis and quality evaluation. Result::17 Kinds of amino acid were detected in Cynomorii Herba, 7 of which were essential amino acids. The 17 amino acids showed good linearity in respective concentration range, r = 0.999 0-0.999 9.The average recoveries were between 98.03%-103.89%with RSD<3.5%. The results of PCA and HCA were basically the same, and both methods can be used to clearly distinguish Cynomorii Herba from 12 municipal producing areas into 6 regions. PCA can be used to classify Cynomorii Herba according to different municipal or provincial production areas, and HCA can be used to classify it according to provincial production areas. It showed that the amino acid contents in Cynomorii Herba from different municipal and provincial producing areas had differences, and the content distribution showed obvious geographical clustering characteristics. PCA showed that Cynomorii Herba from Gansu province and Inner Mongolia had higher amino acid contents and better quality as compared with other producing areas. Conclusion::The established method can be used for content determination of 17 amino acids in Cynomorii Herba from different producing areas, and provide a reference for its comprehensive quality evaluation.
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OBJECTIVE: To develop an ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry method for simultaneous determination of nine trace D-amino acids in thymalfasin, combined with deuterated acid hydrolysis and precolumn-derivatization. METHODS: The sample was hydrolyzed by deuterated acid, followed by precolumn-derivatization with Nα-(2, 4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA). The analysis was then performed on an ACCQ-TAGTM ULTRA C18 column (2.1 mm×100 mm, 1.7 μm), with mobile phase comprising water containing 0.1% formic acid-acetonitrile gradiently eluted at a flow rate of 0.3 mL•min-1. Nine D-amino acids in thymalfasin were correctly quantified using standard curves by Xevo G2-S Q-TOF coupled with electrospray ion source in positive ion mode. RESULTS: The standard curves of the nine D-amino acids derivatives had good linearity in the ranges of the tested concentrations (r>0.991 2). The limits of quantitation of all D-amino acids derivatives were as low as 0.05-0.30 pmol. The precision and recoveries met the requirements of Chinese Pharmacopoeia (2015). The contents of D-amino acids in the samples from five companies were determined to be 25.16-1 322.01 μg•g-1. Among them, D-glutamate, D-aspartate, D-lysine and D-serine had higher contents, which were 1 105.45-1 322.01, 614.15-740.50, 358.06-388.76 and 138.78-291.60 μg•g-1, respectively. CONCLUSION: The method is sensitive, efficient and reliable, working well for simultaneous determination of nine trace D-amino acids in thymalfasin, which provides a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.
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Agmatine is an endogenous amine synthesized from the decarboxylation of arginine.It has a rich biological effects and presents in plants, bacteria and mammalian tissues.Agmatine is highly polar and has a low molecular weight.There are no UV and fluorescent absorption groups in agmatine structure, so it is difficult to gasify.In addition, the content of endogenous agmatine is low, and there are many interference components in biological samples, and the general method is difficult to detect.The pre-column derivatization of agmatine with fluorescence reagents not only increase the molecular weight of agmatine, but also make them with fluorescence, which greatly improve the detection sensitivity and enable the endogenous agmatine to be well separated.It is an important method to determine the content of agmatine.In this paper, several kinds of precolumn derivatives are summarized, and the advantages and disadvantages of various derivatives, the best derivation conditions and detection methods were also analyzed comprehensively.The aim is to determine the content of agmatine and to provide methods and ideas for its biological research.
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OBJECTIVE: To investigate the differences and correlation between the methods of on-line column extraction high performance liquid chromatography(HPLC) and pre-column derivatization HPLC for the determination of valproic acid(VPA) in plasma and provide reference for the clinical monitoring of VPA concentration. METHODS: One hundred and twenty samples were detected by the two methods respectively, and the differences of the test results were evaluated by statistical methods. RESULTS: The linear regression equation between the results determined by the pre-column derivatization HPLC method(X) and the on-line column HPLC method(Y) was as follows:Y=0.804 2X-2.611 3(r2=0.973 9). There was significant difference between the results determined by the two methods(P<0.05). CONCLUSION: Both methods can be used to monitor the concentration of VPA in clinical practice. However, it is necessary to pay attention to the difference between the two methods. And the on-line column HPLC method is more suitable for monitoring the concentration of VPA in clinical practice because it is simple to operate and has less interference from the metabolites.
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@#To develop a rapid and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of endogenous glutathione in rat plasma. Glycyltyrosine was used as the internal standard(IS)and 4-(N-maleimido)phenyl trimethylammonium iodide(MPTA)was used as the derivation reagent. Chromatographic separation was achieved on a Zorbax HILIC PLUS column(4. 6 mm×100 mm, 3. 5 μm)and the mobile phase consisted of acetonitrile and 0. 1% formic acid(75 ∶25)pumped at a flow rate of 1. 0 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by selected reaction monitoring(SRM)in the can meet positive ion mode. The linearity ranged from 3. 000 to 2 000 ng/mL(r=0. 997 1); and the limit of detection of glutathione in rat plasma was 10 pmol/L. Matrix effect, stability, precision and accuracy of the method met the requirements. The proposed method was proved to be selective and sensitive, which is suitable for the quantification of endogenous glutathione in rat plasma.
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Objective To develop a HPLC method for determining 14 hydrolyzed amino acids in Pfaf fia .Methods The sample was derivatized with phenyl isothiocyanate (PITC) .Amino acids were separated on Waters XBridge Shield RP18 (4.6 mm × 250 mm ,5 μm) column at the flow rate of 0.8 ml/min ,detected at 254 nm .The column temperature was 25 ℃ . Results The response was linear for 14 amino acids with a correlation coefficient r>0 .9990 .The average recoveries (n=6) were 90 .2%-105 .1% .Amino acids derivative solution remained stable in 24 hours .Conclusion This well-established method is very reliable .It can be used as a quantitative determination method for 14 amino acids in Pfaf fia .
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OBJECTIVE: To establish the quantification method for free amino acids including threonine, alanine, lysine and tyrosine in the inner part of Poria cocos. METHODS: AQC (6-quinolyl-N-hydroxy-succinimidyl-carboxylate) was used as deriving reagent, and pre-column derivatization combined with ultra-high performance chromatography was applied to quantify threonine, alanine, lysine and tyrosine in 24 batches of the inner part of Poria cocos from Yunnan, Hubei and Anhui provinces. The experimental data were analyzed by partial least squares discriminant analysis (PLS-DA) using Simca-p+11 software. RESULTS: Alanine existed in every batch of sample, and its content ranged from 16.97-69.92 μg·g-1 in samples collected from different origins. Threonine, lysine and tyrosine were not found in some samples. The PLS-DA result showed that the samples from Yunnan and Anhui provinces clustered well. While the amino acid contents in the samples from Hubei province varied greatly. CONCLUSION: AQC combined with pre-column derivatization is a convenient, rapid and sensitive method to quantify the free amino acids in Poria.
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Free amino acids play a great role in traditional Chinese medicine in injections of animal products, as they may take part in peptide synthesis and exhibit a bioactivity in vivo. However, most of the national standards for drugs and peer-reviewed papers only focus on the total amount of amino acids after peptide hydrolysis. We compare the advantage and disadvantage among high performance thin layer chromatography (HPTLC), pre-column derivatization ultra performance liquid chromatography (UPLC) and ion chromatography. As a result, the HPTLC and pre-column derivatization UPLC are suitable for quality analysis, while there is high matrix influence for ion chromatography analysis. The verified pre-column derivatization UPLC method is utilized in quantitative analysis. 24 kinds of amino acid were detected by this method, and 8 of them were reported for the first time from the injection. The system has high repeatability and accuracy with LOD on the level of pmol·mL-1.
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Objective:To compare the content differences of 18 amino acids in plancenta histolysate determined by pre-column de-rivatization HPLC and post-column derivatization cation-exchange chromatography ( AARO) . Methods: The HPLC method was per-formed on a C18 column and 2, 4-dinitro chlorobenzene ( DNCB) was used for pre-column derivatization, and then the determination was carried out after adding 0. 1 mol·L-1 borax buffer (pH=9. 1), and the AARO method was used for the direct determination with a strong acid cation-exchange chromatographic column and post-column derivatization. Results: The RSD for reproducibility of the AARO method was 1. 84%-0. 91%, while that of the HPLC method was 1. 87%-1. 04%. Conclusion:Both AARO method and HPLC method can be used for the quantitative determination of 18 amino acids in plancenta histolysate with the similar results. However, pre-column derivatization HPLC method may cause incomplete derivatization and instable derivatives.