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@#Objective To develop and verify a reversed phase high-performance liquid chromatography method for the determination of the purity of recombinant Mycobacterium tuberculosis(Mtb)Ag85b protein stock solution.Methods Fourfactor,three-level orthogonal test was designed,with the area,trailing factor,peak area and peak area RSD as the evaluation indexes to explore the optimal detection conditions. The methodology verification of specificity,linear range,precision and durability was conducted in accordance with the general principles of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition)9101.Results The results of all the evaluation indexes were good when the elution ratio of organic phase was30% ～ 95%,the detection temperature was 35 ℃,the sample volume was 3 μg,and the elution time of 95% organic phase was 15 min. The method had the linear correlation coefficient(R2)of 0. 998 5,the linear range of 1. 8 ～ 4. 2 μg,the reproducibility RSD of 0. 01%,and the intermediate precision RSD of 0. 16%,with good durability under slight changes of column temperature and flow rate.Conclusion The reversed phase high-performance liquid chromatography method for the purity determination of recombinant Mtb Ag85b protein stock solution was developed,which has good specificity,precision and durability,and can be used for the quality control of recombinant Mtb Ag85b protein stock solution.
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@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.
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Aim We want to increase the biological stability of short peptides by PEG modification. Methods Throughconnecting the maleimide group to one end of polyethylene glycol and adding a cysteine (Cys) to one end ofthe short peptide, the short peptide was finally modified by PEG through chemical bonds. We established areverse high-performance liquid chromatography (RP-HPLC) detection method to detect the change ofsubstances before and after the reaction; screened out the optimal detection method by orthogonal test;purified the modified short peptide by ultrafiltration; detected the reaction by infrared spectroscopy Changesin functional groups; tested the stability of RP-1 in rat plasma before and after modification. Result anddiscussion Through single factor test and orthogonal test, the pH in the reaction was 6, reaction temperaturewas 25 ˚C, reaction time was 1H, and reaction ratio was PEG: RP-1C 1:1.5. The solution does not contain RP-1Cafter ultrafiltration. Peripheral plasma stability testing found that the modified short peptides greatlyenhanced the stability. Conclusion Through experiments, we found the best conditions for the modification ofshort peptides, purification methods, and the stability of the modified short peptides was greatly improved.
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Objective: To establish a reversed phase high- performance liquid chromatography(RP- HPLC) method for determination of salvianolic acid B in rat plasma, and study the pharmacokinetic properties of salvianolic acid B in rats by the intragastric administration of Salvia miltiorrhiza extract and Yixingshu tablets. Methods: The SD rats were orally administered the S. miltiorrhiza extract and Yinxinshu tablets, respectively. The blood was sampled from the orbital venous plexus of rats and centrifuged to obtain plasma. Then, the content of salvianolic acid B in the rat plasma was determined after precipitation of proteins in plasma by adding methanol. The DAS2.0 software was used to analyze the pharmacokinetics parameters. Results: The established method for the determination of salvianolic acid B in rat plasma showed a good linearity in the concentration range of 0.253-10.120 μg/ml, and the limit of determination was 0.063 μg/ml. Meanwhile, the mean recovery of salvianolic acid B for the high, middle and low concentration test was 92.29%, 93.36%, and 96.50%, respectively, and the plasma concentration-time curves of salvianolic acid B for the S. miltiorrhiza extract and Yinxinshu tablets were consistent with the curves of two-compartment model. Compared with the S. miltiorrhiza extract, the Cmax and AUC0→∞ present were 2.50-fold and 2.71-fold, respectively. Conclusion: The present HPLC method was selective, accurate and sensitive for the determination of salvianolic acid B in rat plasma. The present result also indicated that the other medicinal herbs except S. miltiorrhiza in the Yixinshu tables could significantly accelerate the absorption of salvianolic acid B, so as to significantly improve the bioavailability of salvianolic acid B in the Yixinshu tables.
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n-Octanol/ water partition coefficients (Kow ) is an important parameter commonly used to explain toxicity, activity and transmembrane of drugs. However, it is difficult to be detected by direct experimental determination. In this work, a set of 29 neutral and acidic analogues of naphthalene and anthraquinone with reliable experimental Kow data was chosen as model compounds for establishing linear relationship between the logarithm of apparent n-octanol/ water partition coefficient (lgKow), and the logarithm of reversed phase-high performance liquid chromatography (RP-HPLC) retention factor of the solutes corresponding to neat aqueous fraction of mobile phase (lgkw ) as the quantitative structure-retention relationship (QSRR) model. Methanol-water mixture was used as mobile phase at various pH, and retention time (tR ) was rectified by a dual-point retention time correction (DP-RTC) in this method. The experiment results indicated that the proposed QSRR model had good correlation coefficient R2 = 0. 974 -0. 976 with satisfactory results of internal and external validation (the cross-validated correlation coefficient R2cv of 0. 970-0. 973, and 1. 4% ≤relative error (RE)≤7. 9% for all the 6 verification compounds). In addition, this QSRR model was compared with linear solvation energy relationship ( LSER) involved in different descriptors of molecular structure, showing no differences. The QSRR model was applied to measure Kow of 11 naphthalenes and anthraquinones, and the predicted data were compared with Shake-flask method (SFM) experimental ones, as well as calculated ones obtained by software. The results suggested that the proposed method for Kow determination in this work was more accurate, simple and fast. To the best of our knowledge, this is the first report on measuring Kow data for these compounds. The proposed strategy provides the possibility in determining Kow of lipophilic components in complex mixture more quickly and accurately by RP-HPLC.
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Objective To develop a simple method of determination of sorafenib in serum by reversed-phase high performance liquid chromatography (RP-HPLC) and to explore its application in sorafenib therapeutic drug monitoring (TDM).Methods Sorafenib extracted by ethyl ether-petroleum (9∶1) with internal standard of erlotinib from serum was wiped off in 60 ℃ water bath.Sorafenib was redissolved by mobile buffer and analyzed by 40 μl.Chromatographic column was Symmetry Rp18 (5 μm,4.6 mm×250 mm,waters) column in normal temperature.The mobile buffer was 28 mmol/L acetate buffer (pH 5.8)-acetonitrile (37∶63).Sorafenib and erlotinib were detected in 249 nm and 335 nm,respectively.Results The concentration range of sorafenib was 0.50-20.00 μg/ml (r =0.9999).The within-day and between-day accuracies of sorafenib were less than 4.77 ％ and 8.79 ％,respectively.The average recovery rate was 98.48 ％.Sorafenib was stable in serum or after extraction.The concentrations of sorafenib in two patients were detected.Conclusion Detection of sorafenib in serum by RP-HPLC is simple and accurate,which is available to determine sorafenib in serum.The TDM of sorafenib has clinical significance.
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Objective To develop a simple,sensitive,and accurate method for the determination of shionone in rat plasma after administration of shionone.Methods The separation was developed by HPLC on a Waters shieldTM RP18 column (150mm × 3.9mm,5 μm) with a mobile phase composed of acetonitrile-0.05％ phosphoric acid water (98:2) at a flow rate of 1.0 mL/min.UV Detector was set at 200nm.Results The linear range of the standard curves was 0.043-1.720 μg/ml with the correlation coefficient of 0.995.The intra-and inter-day precisions were all below 10％.Conclusion The developed method can be successfully applied to the pharmacokinetic study.
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Rapid and sensitive reversed phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (RP-UPLC) method with UV detection has been developed and validated for of parathyroid hormone (PTH)in presence of meta-cresol as a stabilizer in a pharmaceutical formulation.Chromatography was performed with phase containing 0.1％ Trifluoroacetic acid (TFA) in MilliQ water and 0.1％ TFA in acetonitrile with gradient and flow rate at 0.3 mL/min for HPLC and 0.4 mL/min for UPLC.Quantification was accomplished with internal reference standard (qualified against innovator product and National for for Biological Standards and Control (NIBSC) standard).The methods were validated for linearity (correlation coefficient=0.99),range,accuracy,precision and robustness.Robustness was confirmed by considering three factors; mobile phase composition,column temperature and flow rate/age of mobile phase.Intermediate precision was confirmed on different equipments,different columns and on different days.The relative standard deviation (RSD) (＜2％ for RP-HPLC and ＜1％ for UPLC n=30) indicated a good precision.Retention time was found about 17min and 2min by HPLC and UPLC methods,respectively.Both methods are simple,highly sensitive,precise and accurate and have the potential of being useful for routine quality control.
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An analytical method of reversed phase high performance liquid chromatography (RP-HPLC) was developed for simultaneous determination of 5-hydroxymethylfurfural and nine phenolic compounds (including (+)-catechin, (-)-epicatechin, chlorogenic acid, rutin, caffeic acid, protocatechuic acid, syringic acid, ferulic acid and p-coumaric acid) in 20 min.Ten components were detected and separated successfully by Diamonsil C_(18) column (150 mm x4.6 mm, 5 μm) at wavelength of 280 nm and column temperature of 42 ℃, with acetonitrile and 3% acetic acid solution as the mobile phase in gradient elution.The resultant correlation coefficients of the ten compounds were between 0.9911 and 0.9995 with detection limit from 0.2 to 0.5 mg/L, the RSD less than 2.4% and the average recoveries from 89.4% to 98.3%.These experimental results demonstrate that 5-hydroxymethylfurfural and the nine phenolic compounds in different rice wine samples can be determined with the new method for practical uses.
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A method was developed for the determination of sodium caprylate in human serum albumin by reversed phase high performance liquid chromatography(RP-HPLC) after pre-column derivatization. The caprylic acid, extracted from human serum albumin by hexane, was treated with ω-bromoacetophenone and 18-crown-6 for 30 min at 50 ℃, and analyzed on a Nova-Park C_(18)(150 mm×3.9 mm, 4 μm) column with methanol-water(75∶ 25, V/V) as the mobile phase. The internal standard was enanthic acid, the flow rate was 1.0 mL/min and the detection wavelength was 262 nm. The extraction yield of caprylic acid was 97.9% and that of enanthic acid was 98.2%. The linear range of sodium caprylate was 9.00×10~(-4)-1.44×10~(-2) mol/L(r=0.9995). The average recovery of caprylic acid was 99.7%, RSD was less than 0.9%. The present method is reliable and relatively simple and can be used for the determination of sodium caprylate in human serum albumin.
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Objective To determine diazepam, nitrazepam, oxazepam, estazolam, and al-prazolam simultaneously in human plasma by reversed phase high-performance liquid chromatography (RP-HPLC). Methods Ten microliter carbamazepine (50 mg/L)as the internal standard was added into 1 mL sample, which contained the 5 mixed sedative hypnotics as standard substance and human plasma as ground substance. They were extracted with acetoacetate from plasma samples, and then were dissolved by 100 μL mobile phase. The blood drug levels were analyzed by high perform-ance liquid chromatograph with 20 μL sample injection on a chromatographic column C 18 (4.6 mm×250 mm)at 30℃. The mobile phase consisted of methanol and water (65:35) , and the flow rate was 1.0 mL/min. The ultraviolet detection wavelength was 230 nm. Results The linearity range of the 5 drugs was 5～1 200 μg/L(r≥0.9966, P<0.05). The recovery rate was 95.5%～105.6%. The extraction recovery rate was more than 75%. The relative standard deviation (RSD) of intra-day and inter-day was less than 10% (n=5). Conclusion RP-HPLC method is convert-ient, accurate and sensitive for simultaneous determination of the concentration of diazepam, nitraze-pam, oxazepam, estazolam, and alprazolam in human plasma.
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A RP-HPLC method for the quantification of the six major bovine milk proteins (κ-casein (CN),α_(s2)-CN,α_(s1)-CN,β-CN,Whey,immunoglobulins (Igg) ) is described. Separation and quantification were achieved by a reversed phase analytical column (Agilent Zorbax 300SB-C_8,250 mm×4.6 mm ,5μm) and the gradient elution solvents of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile at a flow rate of 0. 8 mL/min. Column temperature was set at 45℃ and the sample was monitored with photodiode array detector at 214 nm. A linear relationship( r >0. 999) between the concentrations of proteins and peak areas was observed over the concentration range. Recoveries of six target proteins spiked in milk were form 74. 8% to 132.5%. Nine kinds of milks of different brands were analyzed,and the difference of the concentration and relative ration of κ-CN,α_(s2)-CN,α_(s1)-CN,β-CN and Whey were found.
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A reversed phase high-performance liquid chromatography method for the simultaneous analysis of 23 kinds of phenolic components in water extract of porpolis (WEP) was developed. The separation was performed on ZORBAX Eclipse XDB C_(18) column (150 mm×4.6 mm, 5 μm) by gradient elution. The mobile phase consisted of ethanol with 0.1% formic acid at the flow rate of 1.0 mL/min. The detection wavelengths were 256 and 280 nm. The injection volume was 20 μL, and the column temperature was maintained at 35 ℃. The method showed good linear relationship, precision and repeatability. The recoveries were between 93.3% and 106.6%. Eighteen Reference compounds were detected in WEP of Hehei by this method. The content of Catechin was the highest(30.50 mg/g), the next was 3,4-dimethoxycinnamic acid (15.41 mg/g). Nine Reference compounds were detected in WEP of Yunnan. The content of catechin was also the highest (11.23 mg/g), the next was chrysin (15.41 mg/g). Similarity of WEPs between the products of Hebei and Yunnan were 0.099 (256 nm) and 0.194 (280 nm). The chemical compositions of two WEPs were remarkably different.
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An analytical method of fluorescent dye-labeled oligonucleotides was established by ion pair reversed phase high performance liquid chromatography(IP-RP-HPLC) which was improved by optimizing the effects of triethylamine-acetic acid(TEAA)(0-0.15 mol/L), pH(4.5-7.0) and gradient. Comparing the retention of 5, 10 and 15-mer unlabeled oligonucleotides with that of 5'-carboxyfluorescein(5'FAM) labeled oligonucleotides, the mechanism of fluorescent dye-labeled oligonucleotides retention was studied. In addition, TaqMan~(TM) probes as wellas other common fluorescent dye-labeled oligonucleotides were concerned. The results showed that the best resolution of different length fluorescent dye-labeled oligonucleotides was observed under the condition of 0.01 mol/L TEAA and pH 7.0. The retention behavior of fluorescent dye-labeled oligonucleotides was different from that of unlabeled oligonucleotides significantly, and therefore they can be separated completely. The results indicated that the retention of unlabeled oligonucleotides enhanced with the increase of the length of molecule. In contrast, the retention of fluorescent dye-labeled oligonucleotides was reduced with the increase of the length of molecule. For the hydrophobicity of fluorescent dyes made a great impact on the retention, a longer retention time the labeled oligonucleotides would take while the hydrophobicity of fluorescent dyes was higher. However, the effect of the hydrophobicity was limited as the length was increased to a certain level.
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To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatog-raphy (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejec-tion (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differ-entially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were ex-cised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor,apolipoprotein A-Ⅳ precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor,etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into un-derstanding the mechanisms and potential treatment strategy of acute rejection.
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Aim To study the effect of tetramethylpyrazine(TMP) on binding of 125I-VEGF to VEGF receptor. Methods The mice sera were collected after peritoneal injection with big-dose TMP,low-dose TMP,protamine and NS. A reversed-phase high performance liquid chromatography(RP-HPLC) method was used to determine the TMP in mice serum. The culture medium of ECV304 was treated with the mice sera in different groups. Radioligand binding assay(RBA) of receptor and Scatchard pot were performed to observe the changes of the maximum binding capacity(B_ max) and dissociation constant(K_d).Results The sera of big-dose TMP inhibited 125I-VEGF binding to its receptor, K_d=343.30?36.64 pmol?L-1,B_ max=46.26?5.85 fmol/2?10~5 cells(P0.05),but B_ max decreased(P
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AIM: To study the effects of polysaccharides of ferment cultured cryptoporus volvatus (CVPS) on release of leukotrienes from guinea pig lung and Schultz - Dale reaction. METHODS: The bioassay method and reversed phase high-performance liquid chromatography (RP-HPLC) were used to analyze SRS-A or LTD4 from sensitized or normal guinea pig, after challenged with antigen or A23187 respectively. The antiallergic effects of CVPS were evaluated with Schultz-Dale reaction. RESULTS: The release of SRS-A or LTD4 from sensitized or normal guinea pigs were greatly reduced by CVPS after antigen or A23187 challenge. The CVPS could also inhibit the Schultz-Dale reaction of sensitized guinea pig (IC50 = 0.49 g·L-1). CONCLUSION: The effects of CVPS involved in inhibiting release of leukotrienes from lung and antiallergy.
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The pharmacokinetics and absolute bioavailability of 1-hexyl-carbamoyl-5-fluorouracil ( HCFU ) (10 mg/kg ) after oral and intravenous administration were studied in 5 dogs with cross-over design. The concentration of HCFU in serum was determined by reversed-phase high performanee liquid chromatography. After intravenous administration, the curve of serum HCFU concentration vs time was fit to a two-compartment opened model and the phar-macokinetic parameters were. T1/2?=1 .67 min, T1/2? = 34.55 min, Vc= 0.2525L/kg,C1 = 0.3205 L/kg?h~-1 & AUCiv =1.9375 mmol/min?L~-1. When HCFU tablets were tiken orally, the curve of concentration vs time was fit to an one-compartment opened model and its pharmacokinetie parameters were: T1/2ke=12.13 min, T1/2ke=38.51 min, Tmax=23.46 min,Cmax=8.140?10~-3mmol/L & AUCpo=1.5856 mmol/min?L~-1 . The absolute bioavailability calculated from AUCpo and AUCiv was 0.8214.
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The influence of cimetidine on the pharmacokinetics of ftorafur ( FT-207 ) in 9 rats after intragastric administration were investigated with cross-over design. The concentration of ftorafur in serum was determined by reversed-phase HPLC. Its curve of concentration vs time was fit to one compartment opened model. The serum concentrations and areas under the curve ( AUC ) vs time were increased when cimetidine orally administered daily for 5d or single dose. The peak of serum and AUC of ftorafur were increased by 21 .0%(P
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For controlling the quality of sea buckthornoil, a reversed phase-high performance liquid chro-matography (RP-HPLC) method was establishedto determine the a-Tocopherol contents. Aftersaponated, the sea buckthorn oil samples were ex-tracted by ethyl ether. The extraction liquid wascondensed and tben dissolved in anhydreousethanol for liquid chromatographic analysis. Chro-matographic conditions were as follows: ZORBAXODS column (4. 6 ? 150 mm); methanol as mobilephase (1. 0 ml/min); the wavelength of UV detec-tor: 280 nm. Three branches of sea buckthornseed oil and three branches of fruit oil which wereproduced by Yongshou County, Shaanxi Provincewere determined. The average contents of a-Toco-pherol were 989 mg/kg, and 1745 mg/kg, respec-tively. The recoveries of this method were 98. 6%(for seed oil), 102. 2% (for fruit oil) with CV lessthan 5. 44%.