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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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@#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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Abstract The objective of this study was to evaluate the influence of vitamin C (VC) on the stability of stored liposomes under different climatic conditions. Liposomal formulations containing 1 mg/mL of VC (LIP-VC) and blank formulations (LIP-B) were prepared by the reverse-phase evaporation method. After preparation, they were characterized according to their refractive index, average vesicle diameter, polydispersity index (PDI), zeta potential, pH, content, encapsulation efficiency (EE%), morphology, stability and antioxidant activity. For stability, LIP-VC and LIP-B were stored in different climatic conditions (4 °C, 25 °C and 40 °C) for 30 days. The LIP-VC presented 1.3365 refractive index, 161 nm of mean diameter, 0.231 PDI, -7.3 mV zeta potential, 3.2 pH, 19.4% EE%, spherical morphology, 1 mg/mL of VC content, and antioxidant activity of 12 and 11.4 μmol of TE/mL for the radical DPPH and ABTS+, respectively. During stability, the LIP-B stored in 40 °C showed an instability in the parameters: PDI, vesicle size and zeta potential after 15 days, while the LIP-VC remained stable in its size and PDI for 30 days. After that, it is shown that VC can be used as an antioxidant and stabilizer in liposomes to increase the stability and shelf-life of vesicles.
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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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BACKGROUND: As carriers of enzymes, cells and drugs, magnetic polymer microspheres have been widely used in the fields of bioengineering, cytology, and biomedicine. OBJECTIVE: To prepare the magnetic polymer microspheres characterized by small particle size, good dispersion, strong magnetic response, safety, and non-toxicity. METHODS: Magnetic chitosan microspheres were prepared by reverse phase suspension process using Fe3O4 as core, paraffin as dispersed medium, Span-80 as emulsifier, and glutaraldehyde as cross-linking agent. The effects of factors including crosslinking time (0, 20, 40, 60, 80, 100, 120, 150 and 180 minutes), reaction temperature (20→50 °C, 30→60 °C, 40→70 °C, 50→80 °C), the concentration of chitosan (0. 01, 0. 02, 0. 03, 0. 04, 0. 05 g/mL), Fe3O4/chitosan mass ratio (1:1, 1:2, 1:3, 1:4), the amount of glutaraldehyde (8-10 mL), the amount of liquid paraffin (40, 60, 80, 100 mL), and stirring speed (0-2 000 r/min) on the properties of magnetic chitosan microspheres. The morphology, particle size, dispersion, and magnetic responsiveness of magnetic chitosan microspheres were characterized. RESULTS AND CONCLUSION: The optimum conditions for preparing magnetic chitosan microspheres were as follows: Starting with glutaraldehyde as crosslinking agent, the reaction was performed at 40 °C for 1 hour and then at 70 °C for 120 minutes. The concentration of chitosan was 0. 02 g/mL, the mass ratio of Fe3O4/chitosan was 1∶2, the dosage of liquid paraffin was 80 mL, the stirring speed was 1 200 r/min, and the dosage of glutaraldehyde was 8-10 mL. Magnetic chitosan microspheres had strong magnetic properties under the applied magnetic field and had good suspension stability in the natural state. The Fe3O4/chitosan composites were spherical, and the nanoparticles were encapsulated in the microspheres, which were core-shell structure. The surface of the microspheres was smooth and monodisperse. The magnetic chitosan microspheres prepared had a diameter of 1-15 μm, which is beneficial to the dispersion and magnetic separation of the microspheres in the reaction system.
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OBJECTIVE：To establish th e method for the determination of related substances in fidaxomicin raw material. METHODS：The detection ability of NP-HPLC-UV ，RP-HPLC-ELSD and RP-HPLC-UV systems for the related substances in fidamycin raw material was investigated and the best chromatographic system was selected . The HPLC detection method for the related substances was established. The detection was performed on Agilent Eclipse XDB C 18 column with mobile phase A consisted of 0.2％ triethylamine buffer solution （pH 3.8）-acetonitrile（55∶45，V/V），mobile phase B consisted of 0.2％ triethylamine buffer solution（pH 3.8）-acetonitrile（20∶80，V/V）at the flow rate of 1.0 mL/min（gradient elution ）；the detection wavelength was set at 230 nm，and column temperature was 35 ℃；the sample size was 10 µL. Calculation of the content of related substances was principal component self-control method without correction factor. RESULTS ：The impurities C and F could not be separated effectively in NP-HPLC-UV system. In RP-HPLC-ELSD system ，only impurities C ，D，E and F could be detected. In RP-HPLC-UV system ，11 impurities could be detected. In the study of methodology ，the linear ranges were 0.5-20.0 μg/mL for fidaxomicin（R2＝0.999 9）；the LOD was 0.05 ng，LOQ was 0.15 ng；RSDs of reproducibility and intermediate precision tests were less than 2.0％（n＝6）；average recovery was 98.4％（RSD＝3.6％，n＝9）. The sum of impurities in 3 batches of raw materials were 0.53％，0.51％，0.51％，respectively. CONCLUSIONS ：The effect of detecting impurities by RP-HPLC-UV are the best. Established method is specific and sensitive ，and can be used for the determination of related substance in fidaxomicin raw material.
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OBJECTIVE: To study the preparation technology of baicalin glycosides liposomes. METHODS: Baicalin liposomes modified by vitamin E and Tween 80 were prepared by reverse phase evaporation method. The free drug was separated by ultracentrifugation and the encapsulation rate was determined by UV spectrophotometry. Based on the results from single factor tests, orthogonal experimental design was used to investigate the factors influencing the envelopment rate of liposomes. The baicalin liposomes prepared by the optimized process were characterized for particle size, Zeta potential, and morphology. RESULTS: The optimum conditions for the preparation of liposomes were as follows: the ratio of water phase to organic phase 1∶3, the concentration of baicalin 3 mg•mL-1, the ratio of cholesterol to soy lecithin 1∶6, dosage of vitamin E 2 mg,dosage of Tween 80 120 μL. The mean diameter was 52.2 nm, Zeta potential was -51.9 mV, the encapsulation rate was 70.22% and drug loading capacity was 3.18%. Transmission Electr Microscope(TEM) results showed that the shape of the liposomes was good, the particle size was relatively uniform and consistent with the results of laser granulometry. CONCLUSION: The stability of baicalin liposomes can be improved by the addition of vitamin E and Tween 80.
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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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Objective To establish a RP-HPLC method for simultaneously determining berberine hydrochloride, baicalin, emodin and chrysophanol in Sanhuang tablets. Methods The analysis was performed on an Thermo Hypersil GOLD aQ column (250 mm×4.6 mm, 5.0 μm) with gradient elution of methanol and 0.1％orthophosphoric acid at the flow rate of 1.0 ml/min. The column temperature was 30 , and the detection wave℃length was 254 nm. Results The peaks of berberine hydrochloride, baicalin, emodin and chrysophanol were successfully separated from each other. The liner ranges of calibration curves were 0-0.6348 μg (r=0.9999), 0-1.0172 μg (r=0.9999), 0-0.8024 μg (r=0.9999), 0-0.9672 μg (r=0.9999). The average recoveries (n=9) were 101.03％, 99.81％, 99.35％, 99.54％, respectively. The limits of quantification (LOQs) were 0.0363, 0.0210, 0.0497, 0.0793 mg/g, respectively. The sample solution was stable within 24 h, the RSD (n=5) were 0.48％, 0.56％, 0.68％, 0.49％, respectively. The robustness was investigated by varying the conditions of column temperature (± 1 ℃) and flow rate (± 0.1 ml/min), with the RSD s 0.57％ and 0.42％, 0.44％ and 0.65％, 0.74％ and 0.29％, 0.46％ and 0.56％, respectively. The contents of berberine hydrochloride, baicalin, emodin and chrysophanol in twelve samples were 4.6303-5.5866, 14.5514-18.8189, 0.1711-2.7984, 0.5299-2.9254 mg/tablet, respectively. Conclusion The establish method is validated to be suitable for quality control of Sanhuang tablets.
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Objective To screen and validate the prognostic biomarkers of patients with hepatocelluar carcinoma (HCC) based on reverse phase protein array (RPPA).Methods All public RPPA data (HCC patients) in TCGA database before December 2017 were enrolled in this study.Univariate and multivariate Cox analysis were used to screen the target proteins with prognostic value.From January 2013 to January 2014,121 HCC patients who were treated in the Affiliated Hospital of North Sichuan Medical College were enrolled in the study.Prognostic related proteins screened from RPPA data were detected in these patients' tumor specimen.Then,the correlations between the expression of the screened protein and prognosis was further analyzed.Results In the 218 protein,23 was related with HCC patient prognosis.Multivariate Cox regression analysis showed that high expression of NOTCH1 (HR=1.515,95％ CI:1.287～1.845,P＜0.05) and high expression of AKT1 (HR=1.119,95％ CI:1.033～ 1.203,P＜0.05) increased the risk of death,while high expression of NF2 (HR=0.865,95％ CI:0.783～0.956,P＜0.05) decreased the risk of death.The results of clinical data analysis indicated that there were significant differences of NOTCH1,AKT1 and NF2 level between HCC and adjacent tissue.Further survival analysis revealed that high expression of NOTCH1 and AKT1 were significantly associated with poor prognosis.The expression of NF2 was correlated with prognosis,but the difference was not statistically significant (P＞0.05).Conclusion RPPA,as a high throughput protein expression quantitative technique,can be used for screening prognostic markers,and the reliability of the screening results is higher in general.
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Objective: To establish the HPLC method for determination of oxysophocarpine (OSC) and optimize the extraction and purification technology of OSC from Sophora alopecuroides by inverse phase membrane. Methods: Based on single-factor test, the influence of aqueous phase and organic phase volume ratio, the concentration of sodium hydroxide and hydrochloric acid, and the extraction cycle time were investigated using orthogonal design method. Results: OSC was determined by Shim-pack VP-ODS chromatographic column (250 mm×4.6 mm, 5 μm), mobile phase was methanol-0.2% phosphoric acid aqueous solution (7:93), gradient elution, flow rate was 1 mL/min, column temperature was 30℃, and detection wavelength was 221 nm. The ratio of aqueous phase and organic phase volume was 1:1, hydrochloric acid concentration was 0.03 mol/L, sodium hydroxide concentration was 0.5 mol/L, water pump flow rate was 6 mL/min, and cycle time was 60 min. The extraction rate of OSC 98.21% in 60 min was under the best experimental conditions. OSC had good linearity relationship within the range of 0.01-0.7 mg/mL, r2=0.9978, and the respective average recovery rate was 97.47%, RSD=1.95%. Conclusion: This extraction technology is simple operation, with low organic solvent consumption, and can be used for alkaloids extraction.
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Objective To screen for the differential expression proteins in brain tissues of SD rat after diffuse axonal injury (DAI) by isobaric tag for relative and absolute quantification-liquid chromatographmass spectrometer/mass spectrometer (iTRAQ-LC-MS/MS),and to explore potential biomarkers available for the diagnosis of DAI.Methods Animal models of DAI were established with the Marmarou method as reference,and the subjects were divided into blank control group (n=4),sham strike group (n=4) and fatal strike group (n=4),respectively.The proteins in rat brain tissues were detected by iTRAQ-LC-MS/MS,and bioinformatics analysis and verification were performed on the results and screened for the differential expression proteins.Results A total of 2 016 proteins were identified and quantified.The bioinformatics analysis revealed that the proteins had wide distribution and function,and participated in different biological processes.There were 16 proteins showed differential expression in fatal strike group,including one up-regulated expression protein and 15 down-regulated expression proteins.The results of iTRAQLC-MS/MS were confirmed by Western blotting method.Conclusion Multiple differential expression proteins in rat brain tissues after DAI can be screened by iTRAQ-LC-MS/MS.This not only indicates a research direction for exploring the pathogenesis of DAI,but also provides potential biomarkers available for the diagnosis of DAI.
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Objective To find the efficient modification groups of anti-proteinase hydrolyzation in polypeptide by investigat-ing and comparing the relation between the functional groups and their ability to inhibit proteinase hydrolyzation. Methods Reverse phase-high performance liquid chromatography(RP-HPLC)method was developed to investigate in vitro metabolisms of new drug LXT101 and its structural modified analogs LZN series and LMP series in pancreatin system. All the separations of peptide drugs and their digested fragments were monitored at 225 nm. Results The good linear range was 4.0-400 μg/ml(r>0.9990)for new drug LXT101 and its structural modified analogs,i.e.,LZN series and LMP series. The recoveries of all peptide drugs ranged from 95.0%to 98.7%in pancreatin systems. The relative standard derivations(RSD)of intra-day and inter-day were less than 1.5%and 2.5%,re-spectively. The revealed order of digested half-life of the peptide drugs was LZN series>LMP series>LXT101. Conclusion The study of different sites and different functional groups on the lifetime indicates that the half-lives of peptides are prolonged by introducing the functional groups in the suitable sites of peptide,which feature as proteinase inhibitors,such as carbamoyl(Cbm),acetyl(Ac),para-amino-phenylalanine(Aph)or para-uramido-phenylalanine(Uph),which work as either proton donor or acceptor. Our results can pro-vide some useful and valuable information on structural design of peptide drug with long lifetime and high activity.
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Steady improvement in mass spectrometers technology has transformed the targeted proteome analysis into a new stage. Parallel reaction monitoring (PRM) technology has evolved from the basic multiple reaction monitoring (MRM) targeted proteomics methods in recent years. PRM performs with a higher sensitivity, throughput and reproducibility in targeted quantification, however its limitations in effectiveness and accurate quantification of samples with higher complexity still remain unsolved. In this study through improving the chromatographic conditions of PRM we established a simple and robust platform for targeted proteomic quantification. The newly established PRM system is equipped with columns with increased inner diameter (150 μm) and decreased total length (8 cm); faster liquid phase elution rate (800 nL/min) and shortened elution gradient (35 min). These modifications enable PRM platform to combine with dual reverse phase chromatography, to quantify up to 400 low abundance peptides in human 293T cells whole cell extract. Our findings would benefit the promotion of PRM technology, especially providing a technical option for accurate quantification of low abundance proteins.
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A simple, highly sensitive stability indicating reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of sumatriptan succinate in bulk and tablet dosage form. The analysis was performed on reverse phase C18 ODS Inertsil (250×4.6mm, 5μm) column, with a mobile phase containing buffer: aetonitrile: methanol (80:10:10 v/v/v), pH was adjusted to 2.5 with orthophosphoric acid (OPA) at 221nm, by an isocratic elution mode with 1ml/min flow rate using photo diode array (PDA) detector at ambient temperature. The injection volume and retention time was found 20 μl and 4.4 minutes respectively. The method produced linear responses in the concentration range of 5-150 μg/ml, with a correlation coefficient of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) values for HPLC method were found to be 1.967 and 5.961 μg/ml respectively. The recovery of the method was 98% of the labelled value. This method was validated for accuracy, precision, linearity and robustness. Sumatriptan subjected to different ICH prescribed stress conditions of acid, alkali, peroxide, reduction, thermal, photolytic and humidity degradation. This method can easily and conveniently take up for routine quantitative analysis of sumatriptan in bulk and pharmaceutical dosage form by easily available materials with low cost.
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Cetuximab (CTX) is a potent chimeric mouse/human monoclonal antibody (mAb) approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses per-formed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-perfor-mance liquid chromatography with diode array detection ((RP)HPLC/DAD) is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmo-nization guidelines (ICH) for biotechnological drugs; therefore, we evaluated linearity, accuracy, preci-sion, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by compre-hensive chromatographic analysis by subjecting CTX to several informative stress conditions. As de-monstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also suc-cessfully used to quantify CTX in a long-term stability study performed under hospital conditions.
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abstract A simple stability-indicating RP-HPLC/UV method was validated for determination of beclomethasone dipropionate (BD) in nanocapsule suspensions. Chromatographic conditions consisted of a RP C18column (250 mm x 4.60 mm, 5 µm, 110 Å), using methanol and water (85:15 v/v) as mobile phase at 1.0 mL/min with UV detection at 254 nm. The calibration curve was found to be linear in the concentration range of 5.0-25.0 µg/mL with a correlation coefficient > 0.999. Precision was demonstrated by a relative standard deviation lower than 2.0%. Accuracy was assessed by the recovery test of BD from nanocapsules (98.03% to 100.35%). Specificity showed no interference from the components of nanocapsules or from the degradation products derived from acid, basic and photolytic conditions. In conclusion, the method is suitable to be applied to assay BD in bulk drug and in nanocapsules, and it can be employed to study stability and degradation kinetics.
resumo Um método simples de CLAE-FR/UV indicativo de estabilidade foi validado para a determinação do dipropionato de beclometasona (BD) em suspensões de nanocápsulas. As condições cromatográficas foram: coluna C18 fase reversa (250 mm x 4,60 mm, 5 µm, 110 Å), usando como fase móvel metanol e água (85:15 v/v) a 1,0 mL/min, com detecção UV a 254 nm. A curva de calibração foi linear no intervalo de 5,0-25,0 µg/mL com coeficiente de correlação >0,999. A precisão foi demonstrada por um desvio padrão relativo menor que 2,0%. A exatidão foi avaliada pelo teste de recuperação do BD a partir das nanocápsulas (98,03% a 100,35%). O teste de especificidade não mostrou interferência dos componentes das nanocápsulas e nem dos produtos de degradação derivados de condições ácidas, básicas e fotolíticas. Em conclusão, o método é adequado para ser aplicado na avaliação do BD puro e em nanocápsulas e pode ser empregado para o estudo de estabilidade e degradação cinética.
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Beclometasona/farmacocinética , Cromatografía Líquida de Alta Presión/métodos , Nanocápsulas , Nanopartículas , Cromatografía de Fase InversaRESUMEN
Objective To evaluate whether the reverse phase protein array (RPPA) method can be used for detecting TORCH infection in pregnant women .Methods The RPPA method was established for detecting TORCH infection .The positive coinci‐dence rates of TORCH infection detected by the RPPA method and ELISA method in 2000 fresh serum samples from pregnant women were compared for evaluating the feasibility of RPPA in TORCH detection .Results The positive coincidence rates of estab‐lished RPPA and ELISA for detecting TORCH infection was 100 .0% ,91 .1% ,97 .2% ,91 .3% and 93 .0% respectively ,indicating that the detection results of various indexes by RPPA and ELISA had better consistency (P>0 .05) ,but the positive detection rates of RPPA for Rubellavirus ,CMV and HSV‐1 ,2 were higher than those of correspondent ELISA method .Conclusion RPPA method for detecting TORCH infection has the advantages of simpleness ,rapidness ,high sensitivity and strong specificity ,is an effective method of auxiliary diagnosis for bearing and rearing better children in clinical ,and is worthy of being promoted and used in the fu‐ture .
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Hydroxymethylnitrofurazone (NFOH) is a new compound with potential leishmanicidal and trypanocidal activity. Despite its effectiveness, the formulators have to overcome its poor aqueous solubility. Recently, polymeric nano-scale drug delivery systems have proposed for the treatment of neglected diseases. As several studies have confirmed the advantages of such formulations, and this approach provides new analytical challenges, including the need to detect trace amounts of the drug. A suitable method was developed and validated for NFOH determination bound to poly (n-butylcyanoacrylate) (PBCA) nanoparticles. The chromatographic separation was achieved using a C18 column maintained at 25 ºC and an isocratic mobile phase consisting of water and acetonitrile: 80:20 (v/v) at a flow rate of 1.2 mL min-1 and UV-detection at 265 nm. Investigated validation parameters included selectivity, linearity, accuracy, precision and robustness (changes in column temperature, mobile phase composition and flow). The method was specific, the peak of NFOH had no interference with any nanoparticle excipients and no co-elution with main degradation product (nitrofurazone). Linearity was over the range of 0.94 13.11 μg mL-1 (r2=0.999). The method was accurate and precise, recovery of 100.7%, RSD of 0.4%; intra-day and inter-day RSD range 9.98-9.99 μg mL-1 and 0.3% to 0.5%, respectively. Robustness confirmed that method could resist the applied changes. Application of the optimized method revealed an encapsulation efficiency of 64.4% (n=3). Therefore, the method was successfully developed and validated for the determination of the encapsulation efficiency of NFOH-PBCA nanoparticles.
Hidroximetilnitrofural (NFOH) é um novo composto que possui atividade leishmanicida e tripanomicida potencial. Um método apropriado foi desenvolvido e validado para a determinação de NFOH em nanopartículas de poli(n-butil cianoacrilato) (PBCA). A separação cromatográfica foi obtida usando uma coluna C18 (5 µm de tamanho de partícula, 4,6 mm de diâmetro e 150 mm de comprimento), mantida a 25 °C, fase móvel composta de água e acetonitrila 80:20 (v/v), fluxo de 1,2 mL min- 1 e detecção por UV a 265 nm. Investigaram-se os seguintes parâmetros de validação: seletividade, linearidade, exatidão, precisão e robustez (mudanças na temperatura de coluna, proporção da fase móvel e fluxo). O método mostrou-se específico, o pico de NFOH não apresentou interferência dos picos provenientes dos excipientes das nanopartículas e separado do principal produto de degradação (nitrofural). A linearidade foi obtida na faixa de 0,94-13,11 μg mL- 1 (r2=0,999). O método mostrou exatidão (recuperação de 100,7%, DPR de 0,4 %) e precisão (intra-dia e inter-dia, 9,98-9,99 μg mL- 1 e DPR 0,3% a 0,5%, respectivamente). A robustez provou que o método pode resistir às mudanças propostas. Aplicação do método otimizado revelou eficiência de encapsulação de 64,4% (n=3). Portanto, o método foi desenvolvido e validado com sucesso para a determinação da eficiência de encapsulação de nanopartículas de NFOH-PBCA.