HPLC Column Research Articles

Planispine A Sensitized Cancer Cells to Cisplatin by Inhibiting the Fanconi Anemia Pathway.

The use of cisplatin as a chemotherapeutic drug is impeded by the development of drug resistance. Combination therapies of a chemosensitizer for cisplatin have been studied, but with little success, and the search for an effective combination therapy is continuing. Our earlier reports have shown that Zanthoxylum armatum DC. extract enhances the apoptotic effect of cisplatin in cancer cell lines. In this study, we purified and identified the bioactive phytocompound through bio-assay-guided purification, using column chromatography and HPLC. Chemical characterization using NMR and mass spectrometry revealed the compound as planispine A, with molecular structure C25H30O6 and molecular weight, 426.16 g/mol. Planispine A was found to inhibit cancer cell proliferation in a dose-dependent manner and to sensitize the cancer cells to cisplatin-augmented apoptotic cell death, in a caspase-dependent manner. A combination of planispine A and cisplatin induced S-phase cell cycle arrest, and reduced the expression of survival proteins such as cyclin D1. Interestingly, planispine A inhibits the Fanconi anemia pathway, as shown by reduced FANCD2 foci formation and FANCD2 monoubiquitination, which revealed the molecular mechanism of chemo-sensitization of cancer cells to cisplatin. Evaluation of this combination therapy in cisplatin-resistant tumors may lead to more efficient cisplatin treatment.

Total Synthesis of Loroxanthin

The first total synthesis of loroxanthin (1) was accomplished by Horner-Wadsworth-Emmons reaction of C25-apocarotenal 8 having a silyl-protected 19-hydroxy moiety with C15-phosphonate 25 bearing a silyl-protected 3-hydroxy-ε-end group. Preparation of apocarotenal 8 was achieved via Stille coupling reaction of alkenyl iodide 10 with alkenyl stananne 9, whereas phosphonate 25 was prepared through treatment of ally alcohol 23 with triethyl phosphite and ZnI2. The ally alcohol 23 was derived from the known (3R,6R)-3-hydroxy C15-aldehyde 20, which was obtained by direct optical resolution of racemate 20 using a semi-preparative chiral HPLC column.

Development and Validation of 96-Microwell-Based Spectrophotometric and High-Performance Liquid Chromatography with Fluorescence Detection Methods with High Throughput for Quantitation of Duvelisib and Seliciclib in Their Bulk Forms and Capsules

The Food and Drug Administration (FDA) has approved duvelisib (DUV) for managing follicular lymphoma, small lymphocytic lymphoma, and relapsed or refractory chronic lymphocytic leukemia. Seliciclib (SEL) is a candidate drug for these cancers, neurodegenerative disorders, renal diseases, several viral infections, and chronic inflammation disorders. This work describes the development and validation of a 96-microwell-based spectrophotometric method (MW-SPM) and a high-performance liquid chromatography with fluorescence detection method (HPLC-FD) for the quantitation of DUV and SEL in their bulk forms and capsules. The MW-SPM is based on the formation of colored charge transfer complexes (CTCs) as products for the reactions of DUV and SEL, as n-electron donors, with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), as a π-electron acceptor. The absorption intensity of the CTCs was measured by using an absorbance plate reader at 450 nm. The stoichiometric ratios of DUV:DDQ and SEL:DDQ were 1:1 and 1:2, respectively, and accordingly the reaction mechanisms were postulated. The HPLC-FD involved the chromatographic separation of DUV and SEL on a Hypersil™ Phenyl HPLC column (250 mm length × 4.6 mm i.d., 5 μm particle diameter) with a mobile phase composed of acetonitrile:acetate buffer, pH 4.5 (35:65, v/v) at a flow rate of 2.2 mL/min. DUV and SEL were detected at 370 nm after excitation at 280 nm. SEL was used as an internal standard (IS) for quantitation of DUV, and DUV was used as an IS for quantitation of SEL. Both MW-SPM and HPLC-FD were validated according to the guidelines of the International Council for Harmonization (ICH) for validation of analytical procedures. The linear ranges for both DUV and SEL were 14.52–200 µg/well (100 µL) and 0.12–3.2 µg/mL for MW-SPM and HPLC-FD, respectively. LOD values in MW-SPM for DUV and SEL were 4.4 and 3.17 µg/well, respectively; however, those for HPLC-FD were 0.03 and 0.05 µg/mL, respectively. The accuracy and precision of both methods were confirmed as the recovery values were ≥98.5% and the values of relative standard deviations (RSD) were ≤2.41%. Both methods were satisfactorily applied to the quantitation of DUV and SEL in their capsules; the mean recovery values were ≥99.2%. Both methods have simple procedures and high analytical throughput. Moreover, they consume a small volume of organic solvent; thus, they are economic and eco-friendly. Accordingly, the methods are valuable for routine use in quality control (QC) laboratories for quantitation of DUV and SEL in their bulk forms and capsules.

Cytotoxic Properties of C17 Polyacetylenes from the Fresh Roots of Panax ginseng on Human Epithelial Ovarian Cancer Cells.

Although C17 polyacetylenes from Panax ginseng exhibit cytotoxic properties against various tumor cells, there have been few experiments on epithelial ovarian carcinoma cells. This study aimed to investigate the cytotoxic effects of C17 polyacetylenes from P. ginseng against ovarian cancer cell lines. Four unreported (1–4) and fifteen known (5–19) C17 polyacetylenes were obtained from the roots of P. ginseng using repeated chromatography (open column, MPLC, and preparative HPLC). The chemical structures of all the compounds were determined by analyzing their spectroscopic data (NMR, IR, and optical rotation) and HR-MS. The structures of new polyacetylenes were elucidated as (3S,8S,9R,10R)-(-)-heptadeca-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (1), (3S,8S,9R,10R)-(−)-heptadeca-1-en-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (2), (−)-haptadeca-9,10-epoxy-8-methoxy-4,6-diyne-3,11-diol (3), and (3R,9R,10R)-(+)-3-acetoxy-9,10-dihydroxyheptadeca-1-en-4,6-diyne (4), named ginsenoynes O, P, and Q, and 3-acetyl panaxytriol, respectively. Subsequently, in vitro experiments on A2780 and SKOV3 human epithelial ovarian carcinoma cells were performed to assess the cytotoxic properties of the isolates. Among the isolates, panaquinquecol 4 (15) exhibited the most remarkable cytotoxic effects on both human ovarian cancer cells A2780 (IC50 value of 7.60 μM) and SKOV3 (IC50 value of 27.53 μM). Therefore, C17 polyacetylenes derived from P. ginseng may warrant further investigation for their therapeutic potential in epithelial ovarian cancer.

Low level quantification of two potential genotoxic impurities in rilpivirine hydrochloride drug substance by HPLC technique

The goal of the research work was to develop new specific analytical method for the determination of potential genotoxic impurities 4-Iodo-2,6 Dimethyl aniline and 3-Iodo-2,6 Dimethyl aniline in Rilpivirine hydrochloride drug substances. The method was developed by using reverse phase high performance liquid chromatography technique. The comprehensive method development was done to accomplish right combination of chromatographic conditions and validated as per ICH guidelines. The method utilizes L1-octadecyl chemistry (250 millimeter (mm) x 4.6 mm ID, 5.0 micrometer (μm), HPLC column. Both impurities were detected by ultra violet detector at 210nm. The separation of both impurities was attained with 0.1% Ortho Phosphoric Acid in water and acetonitrile at 1.2 ml/min. The calibration curve exhibited good linearity over the concentration range of 0.12-0.45 μg/ml with coefficient of correlation value greater than 0.999. The accuracy in terms of % recovery of the added known amount was found in the range of 97-100 %. Based on experimental results, developed analytical method can be applied for the low level quantitation of both potential genotoxic impurities in Rilpivirine hydrochloride.

Quality control of [177Lu]Lu-PSMA preparations using HPLC: effect of sample composition and ligand on recovery

Background[177Lu]Lu-PSMA is used for the treatment of metastatic castration-resistant prostate cancer. For in-house productions, quality control methods are essential for ensuring product quality, and thus patient safety. During HPLC method development for quality control of [177Lu]Lu-PSMA-I&T, we noticed an unpredictable variability in peak area and height with replicate measurements. After a run, irremovable radioactivity was measured over the whole the length of the HPLC column, with slightly higher activity at the beginning and end of the column. The uniform distribution suggests that [177Lu]Lu-PSMA-I&T interacts with the column. As a result of the interaction, incomplete and variable recovery of injected activity was observed leading to the variability in peak area and height. Therefore the aim of this study was to (1) investigate the effect of sample composition on the interaction of [177Lu]Lu-PSMA-I&T to the HPLC column (measured as recovery, peak area, and peak height), and (2) to compare this with same concentrations of the well-known [177Lu]Lu-PSMA-617.ResultsSample composition significantly affects recovery of [177Lu]Lu-PSMA-I&T, leading to a change in peak area and height. Recovery was 24% when diluted with 0.1 mM octreotide, 38% with water, and increased to 95% when diluted with 0.7 mM unlabeled PSMA-I&T. Peak area and height decreased to 26% and 17% when diluted in octreotide and to 41% and 29% when diluted in water, compared to a dilution in PSMA-I&T. Further experiments showed that recovery (and consequently peak area and peak height) reached a plateau of > 99% at concentrations of 0.27 mM and higher. [177Lu]Lu-PSMA-617 also interacts with the HPLC column, leading to lower, but less variable, recovery (9%). The low recovery of [177Lu]Lu-PSMA-617 could not be prevented with addition of unlabeled PSMA-617.Conclusion[177Lu]Lu-PSMA-I&T can undergo an irreversible binding with an HPLC column resulting in a decreased recovery. The recovery is can be highly dependent on sample composition. The addition of a surplus of unlabeled PSMA-I&T leads to an accurate analysis of [177Lu]Lu-PSMA-I&T.

Green Chromatographic Method for Determination of Active Pharmaceutical Ingredient, Preservative, and Antioxidant in an Injectable Formulation: Robustness by Design Expert.

We report an efficient HPLC method for simultaneous qualitative and quantitative analysis of lincosamide antibiotic injectable formulations containing Clindamycin phosphate (CMN), benzyl alcohol (BA), and ethylenediaminetetraacetic acid (EDTA) as major ingredients. The three components were separated by Phenomenex prodigy C8 (250 mm × 4.6 mm, 5 μm) HPLC column, flow rate 1.1 mL/min, injection volume 30 μL, and column temperature 35 °C, using 0.05 M sodium acetate buffer (pH 4.5) with acetonitrile (ACN) in the ratio of 80:20 (v/v). The detection wavelength was set as 240 nm. The method was validated as per International Conference on Harmonization (ICH) guidelines and was confirmed to be specific, precise, accurate, and linear. Method robustness was executed by utilizing quality in the design of the experiment. Accuracy results were found to be 99.3–100.5% for CMN, 99.3–100.8% for BA, and 99.1–100.3% for EDTA. Precision results were obtained as % relative standard deviation (RSD): 0.6% for CMN, 0.4% for BA, and 0.4% for EDTA. Correlation coefficient (r2) values were obtained as >0.999 for the three components. Analytical solutions are stable for 48 h at benchtop and refrigerator conditions. The greenness of the analytical method was evaluated by the Green Analytical Procedure Index (GAPI), National Environmental Method Index (NEMI), analytical eco-scale, and Analytical Greenness (AGREE) tools to confirm that the method is eco-friendly.

Secondary metabolites from the mangrove soil derived fungus Xylariaceae sp. SCSIO41212

ObjectiveTo obtain structurally novel and bioactive secondary metabolites from the marine fungus Xylariaceae sp. SCSIO41212. MethodsCompounds were purified by comprehensive chromatography methods of silica gel column, Sephadex LH-20, Ostade-cylsilane (ODS) and semi-preparative HPLC, and their structures were elucidated by means of physical and chemical properties, magnefic resonance imaging, spectroscopic analysis, and comparison with the reported literatures. ResultsChemical investigation of the fungus Xylariaceae sp. SCSIO41212 has led to the isolation of two new compounds, xylaolide B (1) and xylaolide C (2), together with nine known compounds (3–11). Their structures were determined on the basis of detailed analysis of the 1D and 2D NMR as well as MS data. Compounds 1–6 were evaluated for their antiviral, and cytotoxic activities, respectively. Compound 1 showed weak cytotoxicities against K562, MCF-7 and SGC7901 respectively. ConclusionThis study enriches the diversity of secondary metabolites of marine-derived fungi and provides an important reference for novel promising drug leads research.

Assignation of inorganic mercury and methylmercury mass fractions in a soil matrix certified reference material by two analytical methodologies based on species-specific isotope dilution mass spectrometry and chromatographic separation.

Assignment of inorganic mercury and methyl mercury mass fractions at an ultratrace level in soil certified reference material EnvCRM 03 with a complex matrix composition was undertaken. Inorganic mercury and methyl mercury contents by species-specific isotope dilution inductively coupled plasma mass spectrometry with on-line high-performance liquid chromatography or with classical off-line chromatography were established. Different extraction protocols: sequential extraction ((1) H2 SO4 /KBr/CuSO4 ; (2) dichloromethane; (3) Na2 S2 O3 ) or one-step extraction (diluted HCl) in solid-liquid systems were verified. Sequential extraction allowed quantification and separation of inorganic mercury and methyl mercury on HPLC column in one chromatographic run and were found to be (316 ± 10) μg/kg (U = 3.2%, k = 2) and (0.53 ± 0.02) μg/kg (U = 3.8%, k = 2), respectively. Extraction by diluted HCl and application of classical off-line chromatography led to the separation of methyl mercury from predominant inorganic mercury form and was found to be (0.54 ± 0.03) μg/kg (U = 5.4%, k = 2). To the best-obtained literature knowledge, there was no available soil material aimed for speciation analysis of inorganic mercury and methyl mercury so far. Both developed analytical methodologies were found to be equally sensitive and could be successfully applied for mercury species determination in samples with the complex matrix.

Six pairs of phenylpropanoid enantiomers from Cinnamomum mollifolium

Six pairs of undescribed phenylglycerol benzoate enantiomers, (±)-mollifolines A−F, which can also be categorized into three two-pairs of epimers, were isolated from Cinnamomum mollifolium H. W. Li (Lauraceae). The relative configurations (threo or erythro) of the epimers were determined by conformational searching of the lowest energy conformers and analyses of the relationship between the dihedral angle of H-7'─C-7'─C-8'─H-8′ and the 3JH-7', H-8' coupling constant according to the Karplus equation. Furthermore, intramolecular hydrogen bonds were proved to play an important role in stabilizing the lowest conformations by using reduced density gradient (RDG) method for noncovalent interactions. Chiral resolutions of these enantiomer pairs were accomplished by immobilized polysaccharide derivative-based chiral HPLC columns. Absolute configurations of the 12 optically pure isomers were finally determined by quantum chemical time-dependent density functional theory (TDDFT) calculations of their electronic circular dichroism (ECD) spectra.

Simultaneous Estimation of Amoxycillin and Potassium Clavulanate in Injection Formulation by RP-HPLC

Amoxycillin (AMX) and Potassium clavulanate (PTC) are proven for their therapeutic benefits to treats various diseases. AMX and PTC are proven for their therapeutic benefits to treat various diseases. Due to their synergistic actions, a rapid and specific RP-HPLC method was processed and validated as per ICH guidelines. The chromatographic separation was performed with Inertsil 5μ, 250 mm X 4.6 mm, C-18 HPLC column by using phosphate buffer: methanol (80:20) as mobile phase, at 1mL/min of flow rate and 220nm m. The absorption maxima were found to be 246.6 nm for AMX and 257.6 nm for PTC in 0.1M NaOH used as the solvent. The linearity for these drugs at the selected wavelengths lies between 5-200 µg/ml for AMX and 5-200 µg/ml for PTC. Accuracy was determined by recovery study from injection dosages forms were found to be 99.352 for AMX and 99.533 for PTC. Precision of method was determined as repeatability, day to day and analyst to analyst variation and a show the value within acceptable limit i.e. R.S.D was found to be less than 2 percent).

Simultaneous quantification of volatile fatty acids and nonvolatile organic acids in Hevea brasiliensis latex.

The current method used in latex industries to determine the volatile fatty acids contents of Hevea brasiliensis latex is steam distillation. However, the accuracy of the method has been debated for some time. We assessed the accuracy of the method and developed a new, more reliable high-performance liquid chromatographic method of determining acids in latex. The volatile fatty acids (formic, acetic, propionic, butyric, and valeric acids) and nonvolatile organic acids (oxalic, malic, lactic, citric, and succinic acids) in latex are directly determined simultaneously for the first time with high sensitivity and without losses during sample preparation. To avoid errors from derivatization, an acid-resistant Prevail HPLC column and a gradient mobile phase of 25mM potassium dihydrogen phosphate (pH 2.5) and acetonitrile were employed. Under optimum conditions, the calibrations of both types of acids demonstrated satisfactory correlation coefficients of ≥0.990, with limits of detection ranging from 0.02 to 395mM. The developed method demonstrated the profiles of acids in field and concentrated latex of the same batch. Moreover, the evolution of the profiles of all studied acids in both types of latex during a 3-month period was also revealed.

Estimation of Dapagliflozin in Pure and Marketed Formulation by Validated Reverse Phase-High Performance Liquid Chromatographic Method

Dapagliflozin is a highly selective, orally active and reversible inhibitor of the human sodium glucose co- transporter 2 (SGLT2). The dapagliflozin is a diabetic drug which is used with proper diet and exercise in adults with type 2 diabetes to improve the glycemic control. The aim of this study is to develop a rapid, precise and accurate RP- HPLC method for estimation of dapagliflozin in bulk and formulation as per ICH guidelines. The chromatography was carried on Sunsil C18 (150 X4.5mm, 5µ) column HPLC (Water’s) using a mixture of methanol and water at different ratios and finally optimized with 85:15 v/v as mobile phase with flow rate 1.0ml/min at 225nm. The correlation regression is found to be 0.999. The retention time was found to be 2.74min. The accuracy of the present method was evaluated at 50%, 100% and 150%. The recoveries of dapagliflozin API and tablet were found to be in the range of 98.5% - 100.6%. Precision studies were carried out and the relative standard deviation values were less than two. The method was found to be robust. The limit of detection and limit of quantification were determined as 0.011µg/ml and 0.034µg/ml. The proposed method was found to be rapid, precise, accurate and robust and can be used for the estimation of dapagliflozin in API and Pharmaceutical dosage form.

Analytical method development and validation of rivaroxaban in bulk and pharmaceutical dosage form by using RP-HPLC

A simple, specific, accurate, and precise reverse phase high performance liquid chromatography (RP-HPLC0 method was developed and validated to analyse Rivaroxaban bulk dosage form. Using Sun Q C18 HPLC column separation was carried out. This was maintained at ambient temperature. During separation mobile phase consist of Acetonitrile : Buffer(sodium acetate buffer) 80:20 was delivered at a flow rate of 1mL/min. Using UV detector analysis was carried out at the wavelength 249 nm. RP-HPLC method was validated by using various parameter like system suitability, linearity, specificity, precision, limit of detection, limit of quantification and robustness. The RP-HPLC method was found to be linear over the concentration range from 5-30 µg/mL (r2 =0.994). Retention time for bulk rivaroxaban was found to be 3.69 min. LOQ of method was 1.331 µg/mL and LOD 0.439 µg/mL. Thus the developed RP-HPLC method was found to be robust which can be applied for the regular analysis of Rivaroxaban in the bulk as well as pharmaceutical dosage form

Analytical method development and validation for estimation of dolutegravir in tablet dosage form

A simple, specific, accurate, and precise reverse phase high performance liquid chromatography method was developed and validated to analyse Dolutegravir Sodium bulk dosage form. Using Princeton C18 HPLC column separation was carried out. This was maintained at 25ºC temperature. Shimadzu LC -20A prominence with LC solution softwarering separation mobile phase consist of Methanol: Water (0.1% OPA) in ratio of 80:20 v/v was delivered at a flow rate of 1mL/min. Using UV detector analysis was carried out at the wavelength 254 nm. RP-HPLC method was validated by using various parameter like system suitability, linearity, specificity, precision, limit of detection, limit of quantification and robustness. The RP-HPLC method was found to be linear over the concentration range from 10-60 µg/mL (r2 =0.9998). Retention time for bulk Dolutegravir Sodium was found to be 4.383 min. LOQ of method was 1.3274 µg/mL and LOD 0.4380 µg/mL. Thus the developed RP-HPLC method was found to be robust which can be applied for the regular analysis of Dolutegravir Sodium in the bulk as well as pharmaceutical dosage form.

Green Liquid Chromatography Method for the Determination of Related Substances Present in Olopatadine HCl Nasal Spray Formulation, Robustness by Design Expert.

Dual therapeutic nature drug mast cell stabilizer and histamine receptor antagonist olopatadine hydrochloride (OPT) nasal spray does not have an official monograph, and no literature is available. Eye drops formulation had the official monograph for impurities, but the determination was done in two methods. A simple and effective green liquid chromatography method to develop and validate for the related substances of OPT nasal spray formulation. A 25 min gradient method was employed to separate impurities and OPT with a 1.0 mL/min flow rate using a Boston green C8 (150 mm × 4.6 mm, 5 µm) HPLC column. The set wavelength and column oven temperatures were 299 nm and 30°C, respectively. pH 3.5 phosphate buffer-acetonitrile in the ratio of (70:30, v/v) as mobile phase A and (50:50, v/v) ratio as mobile phase B. A Quality by Design (QbD) based Design of Experiments (DoE) was employed to evaluate the robustness characteristics of the analytical method validation. The obtained RSD from the precision and intermediate precision was 0.4 to 4.1%. The % recovery of the impurities from LOQ to 150% of specification level was 87.5 to 110.3%. The linear regression curves for the impurities with a correlation coefficient of >0.999 indicate that all peak responses are linear with the concentration. The sample and standard solutions were stable for 24 h at benchtop and refrigerator conditions. All the critical peaks were well separated from the forced degradation studies' diluent, placebo, and generated degradation peaks. The method validation data and QbD based robustness study results indicate that the developed impurities method fits the routine quality control laboratory use. National Environmental Index (NMEI), Green Analytical Procedure Index (GAPI), Analytical Eco-scale and Analytical Greenness (AGREE) tools expressed the method's greenness. The proposed method is QbD utilized and green chemistry assessed impurities determination method for OPT in nasal spray formulation.

Synthesis of C8F13-SiO2 stationary phase for chromatographic separation of highly polar compounds

Up to now, the chromatographic separation and analysis of highly polar compounds are still challenging because of low retention ability and selectivity of conventional stationary phases (SPs). Developing new SPs to achieve high-resolution and high-efficiency separation of polar compounds is of great importance for health and medicine, food safety, environmental protection, etc.. Herein, an influential multi-step modification strategy (modification with 1H,1H,2H,2H-perfluorooctyltriethoxysilane on the silica twice and end-capped with trimethylchlorosilane once) is introduced to prepare C8F13-SiO2 SP. The physical properties of C8F13-SiO2 SP, including morphologies, pore structures and carbon content were fully characterized by various instrumental techniques. The packed HPLC column with C8F13-SiO2 SP exhibited high column efficiency, good peak shape (tailing factors for most analytes are between 0.9 and 1.3), and resolution for simultaneous separation of ginsenosides, sulfonamides, nucleosides, and benzoylurea insecticides (BUs); meanwhile, the retention mechanism of typical reversed-phase liquid chromatography (RPLC) mode was observed. A column efficiency of 30,220 plates/m for separation of ginsenoside Re was obtained. Furthermore, the resulting columns benefit from the advantages of supreme reproducibility, repeatability and stability. Importantly, the chromatographic properties (e.g. column efficiencies, chromatographic peak shapes, retention ability and resolution) of the C8F13-SiO2 SP for the polar analytes were superior to those of the commercial SPs (including commercial amino-modified silica SP, diol-modified silica SP and commercial PFP-SiO2 SP). Thus, the C8F13-SiO2 SP is able to satisfy the strict requirements of highly polar compounds separation.

Determination of polypeptide antibiotics in animal tissues using liquid chromatography tandem mass spectrometry based on in-line molecularly imprinted solid-phase extraction

Effective purification and enrichment of polypeptide antibiotics in animal tissues is always a challenge, due to the co-extraction of other endogenous peptides which usually interfere their final determination. In this study, a molecularly imprinted column was prepared by packing polymyxin E-imprinted particles into a 100 mm × 4.6 mm i.d. HPLC column. The as-prepared imprinted columns were able to tolerate 100% aqueous phase and exhibited good stability and high column efficiency. Polypeptides antibiotics with similar molecular size or spatial structure to polymyxin E were well retained by the imprinted column, suggesting class selectivity. After optimization of mobile phase conditions of imprinted column, polypeptide antibiotics in animal tissue extracts were enriched and cleaned up by in-line molecularly imprinted solid-phase extraction, allowing the screening of target analytes in complex samples at low concentration levels by UV detection. Eluate fraction from the imprinted column was collected, and further dried and re-dissolved with methanol-0.5% formic acid aqueous solution (80:20, v/v) for final LC-MS/MS analysis. Analysis was accomplished using multiple reaction monitoring (MRM) in positive electrospray ionization mode and analytes quantified using the matrix-matched external calibration curves. The results showed high correlation coefficients for target analytes in the linear range of 2 ∼ 200 μg kg−1. At four different concentration levels (limit of quantification, 50, 100 and 200 μg kg−1), recoveries of four polypeptide antibiotics in swine, cattle and chicken muscles ranged from 66.7 to 94.5% with relative standard deviations lower than 16.0%. The limits of detection (LOD) were 2.0 ∼ 4.0 μg/kg, depending upon the analyte and sample. Compared with a conventional pretreatment method, the imprinted column was able to remove more impurities and to significantly reduce matrix effects, allowing the accurate analysis of polypeptide antibiotics.

Quantification of prefusion conformation for HIV vaccine using size-exclusion chromatography

A closed prefusion conformation or an open (non-prefusion) conformational state of a protein vaccine candidate molecule can determine if it effectively elucidates a desired immunity. A quick and reliable method to monitor conformational state is important during vaccine development. In addition to our existing immunoassays, we have developed a unique physicochemical approach using size-exclusion chromatography to assess binding between antibody and the structurally desired antigen protein. Through the bound monoclonal antibody protein vaccine peak shift in the size-exclusion chromatography profile, this method determines the percent closed (prefusion) conformation present in a sample. Since only the closed prefusion conformation binds to the specific antibody, the population of the closed versus the open conformation of the vaccine molecule can be monitored without the need for a reference calibrator. This new method can be applied broadly to vaccine development, as well as for antibody selection during antibody drug discovery. The mAb CAP256V2LS (250 µg/mL) specific to prefusion conformation was mixed with HIV trimer (250 µg/mL) at 2:1 volume ratio, incubated at 37 °C for 30 mins and injected onto HPLC column. The percent of non-prefusion conformation was calculated based on ratio of peak area of unbound trimer and total area of control trimer sample (without mAb).

Development and validation of green chemistry HPLC-RI method for the simultaneous estimation of glucosamine and chondroitin sulfate from drug products

Design and development of analytical methods with environment friendly reagents and solvents is the need of the hour for labs engaged in analytical services. Every year thousands of chemical and pharmaceutical laboratories worldwide are generating tons of toxic chemical wastage causing environmental pollution thereby endangering the human existence. The objective of this study was to develop and validate a sensitive, stability-indicating, accurate and precise green chemistry RP-HPLC method with RI detector for the simultaneous quantitation of glucosamine and chondroitin sulfate from various pharmaceutical dosage forms. Complete separation of both the actives was achieved in isocratic mode by using Hypersil BDS Phenyl (250 x 4.6 mm, 5µm) HPLC column. Purified water as a diluent and phosphoric acid buffer pH 2.5 was used as a mobile phase at a flow rate of 0.2 mL/min. The column temperature was maintained at 40°C. Both the molecules being non-chromophoric in nature, refractive index (RI) detector was used for detection. The proposed method being environment friendly does not require organic solvents, gradient elution and complex derivatization unlike the reported methods. The developed method is successfully validated as per ICH guidelines. The method is stability indicating, sensitive and economical. Hence, it can be successfully used for the routine analysis of commercial batches of these combination products.