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@#Abstract： Objective To develop and validate a reverse phase⁃high performance liquid chromatography（RP⁃HPLC） method for determination of residual N⁃hydroxy succinimide（NHS）content in semaglutide. Methods A RP⁃HPLC method was developed based on the screening of chromatographic column and optimization of mobile phase（phosphate concentration and the ratio of acetonitrile），validated for specificity，suitability，accuracy，reproducibility and stability， and determined for linear range，limit of quantitation（LOQ）and limit of detection（LOD）. The NHS contents in three batches of semaglutide were determined by the developed method. Results The optimal condition for RP⁃HPLC was as follows：CAPCELL PAK ADME column（4. 6 mm × 150 mm，3 μm）was adopted，serving 0. 05 mol／L potassium dihy⁃ drogen phosphate solution⁃acetonitrile（98∶2）as mobile phase A，and 70% acetonitrile as mobile phase B with gradient elution（0 min，0% B；10 min，0% B；19 min，90% B；19. 1 min，0% B；25 min，0% B）at a flow rate of 0. 8 mL／min. The detection wave length was set at 260 nm，while the column temperature was 30 ℃. The developed method showed good specificity and systemic suitability，of which the linear range was 0. 2 ~ 3. 0 μg／mL（R2 = 1. 000 0），while the LOD and LOQ were 4. 8 and 9. 6 ng respectively. The RSD of recovery rates of NHS samples at three concentrations was 0. 58%， indicating a high accuracy. The RSD of NHS contents in six test samples was 0. 16%，indicating a high reproducibility. The RSD of peak areas of NHS after storage at room temperature for 0，4，8，12，16，20 and 24 h was 0. 34%，indicating a high stability. No NHS was detected in three batches of semaglutide by the developed method. Conclusion The developed RP⁃HPLC method is simple and sensitive，which may be used for the determination of NHS content in semaglutide.
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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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@#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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Objective: The present study was undertaken to develop and validate an RP-HPLC method for the combination of imiquimod and salicylic acid Methods: The method was carried out on Nucleodur C18 (250 mm × 4.6 mm I.D., 5 ????m) using low-pressure gradient elution mode. The mobile phase was used as 30M potassium dihydrogen phosphate and acetonitrile (45:55) pH 6.5 adjusted using ortho-phosphoric acid. The concentration of solvents was 1-20 µg/ml and the volume of injection was 20 mcl with the flow rate of 1.0 ml/min. The absorption maxima of salicylic acid and imiquimod were found 234 nm and 226 nm, respectively. Results: The method was validated and showed the linearity greater than 0.99% and with precision (RSD%<1). The limit of detection (LOD) and limit of quantification (LOQ) of salicylic acid was found to be 0.09756 µg/ml and 0.2956 µg/ml, respectively, and imiquimod was found to be 0.044031 µg/ml and 0.13334 µg/ml, respectively. Conclusion: The method developed in the present study was found to be sensitive, specific, and can be applied for the simultaneous estimation of imiquimod and salicylic acid.
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Objective: The objective of the present work was to establish a simple, precise, accurate and robust method for simultaneous estimation of gallic acid, curcumin and piperine from the marketed ayurvedic formulation by liquid chromatography. Methods: The separation was carried out on Hemochrom C18 Column (250 mm × 4.6 mm ID, 5 µm pore size) with a mobile phase methanol: acetonitrile: water (pH 3.2adjusted by using orthophosphate acid) in the ratio 70:20:10v/v by isocratic elution modeat 25 °C and the flow rate was setat0.8 ml/min. The analysis was carried out atisoabsorptive wavelength of 295 nm. Results: The retention time of gallic acid, curcumin and piperine was found to be 3.3(±0.2), 4.7 (±0.2) and 5.6 (±0.2) min, respectively. The linearity range for gallic acid, curcumin and piperine was found to be 10-70 μg/ml, 20-80 µg/ml and 2-14 µg/ml, respectively with the coefficient of linear regression greater than 0.99 for all markers. Mean percent recoveries for gallic acid, curcumin, and piperine were found within the limit of acceptance (99-100%). The percent relative standard deviation (%RSD) for precision and robustness was found less than 2%, which indicates the method is precise and robust. The developed method applied for quantification of these markers from the marketed ayurvedic formulation of Dekofcyn tablet. Conclusion: The developed method was found to be simple, rapid, precise and reproducible for standardization of Dekofcyn tablet and can be useful for other formulations containing these three markers.
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Objective To develop a method for the determination and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in reverse phase high performance liquid chromatography (RP-HPLC) system.Methods RP-HPLC system had Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm,5 ttm) with detection wavelength of 265 nm,column temperature of 25 ℃ and flow rate of 1 mL/min.Retention behaviors of vitamin D3 and its 3 isomers were studied by altering the mobile phase.Firstly,acetonitrile was mixed with different proportions of methanol,water and ethanol as the mobile phase to investigate the effects of these 4 mobile phase components on the retention behavior of vitamin D3 and its 3 main related substances (isomers) on a C18 column.Then,a suitable mobile phase was selected for content determination and impurity analysis according to the retention behavior study.Results The recovery was only 80.55％-84.37％ with 100％ acetonitrile as the mobile phase.The addition of ethanol in acetonitrile was found to make remarkably significant improvement.Recovery rate was achieved between 98.07 ％ and 103.23 ％ with V (acetonitrile) ∶V (ethanol) =90∶10 as the mobile phase,while improving pealk shape.The method showed good linearity [(0.52-5.2) x 10-4 t mol/L,R2＞0.999] and fine density (RSD＜2.32％) which can be used for determination.For impurities profile,it could be achieved using V (acetonitrile):V (water) =95:5 as the mobile phase which can obviate interference from soybean oil matrix.Conclusions The method established in this experiment can easily and accurately determine the content and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in a RP-HPLC system.
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A RP HPLC method for estimation of sildenafil citrate (SC) in tablet dosage form and seminal fluid was developed and validated. Best resolution was obtained with column Waters Spherisorb® C18 bonded silica, (5 μm, 4.6 x 250 mm) at 230 nm with retention time of 5.01 min. The mobile phase used was TEA (0.2%) pH adjusted at 3 with OPA and ACN (60:40) with flow rate of 1.0 mL/min. The method for estimation of sildenafil citrate in tablet dosage form was found to be linear, accurate, precise, sensitive and selective. Whereas the bioanalytical estimation of SC in seminal fluid was found to be in the range of 100 ng/mL to 10μg/mL. Method was found to be highly sensitive as LOD and LOQ were found to 0.3 μg/ml and 0.9μg/ml. The repeatability and reproducibility were within the range i.e. less than 2%.The accuracy of the method was 99.3%. The percentage purity was calculated for market formulation was 102.8%. The internal standard used for bioanalytical methods was Diclofenac sodium. The drug was extracted from seminal fluid by protein precipitation using ACN as precipitating agent. The linearity range was from 100.0ng/mL to 2.0μg/mL. The LOD and LOQ were found to 0.03μg/mL and 0.1μg/mL. The repeatability and reproducibility were within the range i.e. % RSD less than 15%. The accuracy of the method was 90.36%.and the extraction efficiency was found to be 98.25%. The stability of drug was found to be within the range i.e. less the 15%.
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DMAE glycolate (DG) and sunscreens have been used associated in anti-aging dermocosmetic formulations. Despite extensive use of these substances, methods for quantification of DG as raw material and in cosmetic formulations, especially when associated, are not described in the literature. RP-HPLC and non-aqueous titration methods, with determination potentiometric end-point (PT), were developed and validated for rapid assay of DG as raw material and in a topic emulsion in association with sunscreens. Both methods are simple, selective, linear, accurate and precise. The PT method was chosen for stability study of DG in the formulation developed. The proposed formulation presented good stability performance as regards aspect, pH, apparent viscosity, and SPF, with less than 5% of DG degradation compared to initial conditions.
Glicolato de DMAE (DG) e protetores solares têm sido utilizados associados em formulações dermocosméticas antiidade. Apesar da ampla utilização dessas substâncias, métodos de quantificação para DG matéria-prima e em formulações cosméticas, especialmente quando associados, não estão descritos na literatura. Neste trabalho foram desenvolvidas e validadas metodologias por CLAE-FR e titulação em meio não-aquoso, com determinação do ponto final por potenciométrica (TP), para a rápida análise de DG matéria-prima e em emulsão tópica em associação com fotoprotetores. Ambos os métodos são simples, seletivos, lineares, exatos e precisos. O método TP foi escolhido para o estudo da estabilidade do DG na formulação desenvolvida. A formulação proposta apresentou um bom desempenho no que se refere a estabilidade, aspecto, pH, viscosidade aparente e SPF, com menos de 5% degradação do DG comparado as condições iniciais.
Subject(s)
Deanol/administration & dosage , Deanol/analysis , Deanol/pharmacology , Sunscreening Agents/pharmacology , Cosmetic Technology , Chromatography, High Pressure Liquid/statistics & numerical dataABSTRACT
Objective To explore the optimal method of extraction and determination of rhynchophylline and isorhynchophylline in Ramulus Uncariae cum Uncis. Methods A RP-HPLC was performed on a Phenomenex C18 column (4. 6 mm× 150 mm, 5m) at 30℃. The mobile phase consisted of methanol-0. 01 mmol/L triethylamine solution (70: 30, pH 7.0 adjusted by glacial acetic acid) at the flow rate of 0. 5 mL/min. The automatic sample injector was set at 5℃ and the ultraviolet detector was operated at 245 nm. And then, the effect of different extraction condition on the contents of rhynchophylline and isorhyn-ehophylline in Ramulus Uncariae cum Uncis was investigated. ResultsA good linearity of rhynchophylline was in the range of 2. 5μg/mL to 80. 0μg/mL (Y=76. 7170X-0. 0727,r=0. 999 8),the average recoveries were from 99. 84 ％ to 116. 91％, and the RSD (n=6) were less than 8.8 ％. A good linearity of isorhynchophylline was in the range of 2. 0 μg/mL to 80. 0 μg/mL (Y=87. 4729X-0. 3666, r=0.999 7), the average recoveries were from 87.08 ％ to 104. 97 ％, and the RSD (n=6) were less than 7.3 ％. The contents of rhynchophylline and isorhynchophylline in the methanol-extracted solu- tion were approximately ten times as much as those in water-decocted solution. The main factors which had effect on the ex-traction of rhynchophyUine and isorhynchophylline in methanol solution were supersonic time,methanol amount and cold-dousing time, in a decreasing sequence. The best condition selected by orthogonal experiment wasb as follows: extracting the medicinal material with 20-fold volumes of methanol, cold maceration for 24h and supersonic extraction for 1 h. Conclusion The extraction percentage of rhynchophylline and isorhynchophylline in the extraction condition of cold maceration with methanol and supersonic extraction is superior to that in the water-decocting condition. The method is simple, fast and accurate, and it can be used for the quality control of Ramulus Uncariae cum Uncis.
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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.