Mobile Phase Flow Rate Research Articles

Combined continuous nanoparticle synthesis with chromatographic size classification

In this paper, we report a combination of the continuous flow synthesis of gold nanoparticles (AuNPs) with subsequent purification and narrowing of the particle size distribution (PSD) by size-exclusion chromatography (SEC) by adapting the flow rates of synthesis and classification. First, we show scalability of chromatographic classification with respect to column dimension and the absence of irreversible nanoparticle adhesion on the column material. Two different syntheses lead to a large and widely distributed and a small and narrowly distributed AuNP dispersion, which are classified by a semipreparative column. The PSDs of individual fractions are characterized by analytical SEC. The broadly distributed AuNP dispersion was classified into three fractions with distinct PSDs. For the narrowly distributed AuNPs, the separation is almost independent of the mobile phase flow rate: coarse and fine fractions with almost identical PSDs and separation efficiency curves are observed irrespective of the flow rate. Even NP samples with narrow PSDs can be classified into multiple fractions with tailored PSDs while simultaneously removing dissolved impurities from the dispersion. With our study, we demonstrate the potential of a direct combination of continuous NP synthesis with chromatographic classification for the optimization of final PSDs and the simultaneous purification of nanoparticulate dispersions.

Green Alternatives in Pharmaceutical and Bioanalytical Analysis of TDM Required Drugs: Procainamide.

In drug analysis, using non-hazardous solvents instead of the ones harmful to humans and the environment is a green strategy to protect analysts and environmental health. Procainamide (PCA) is an antiarrhythmic drug requiring therapeutic drug monitoring (TDM) because of its narrow therapeutic window and serious side effects. The aim of this study is to develop validated green HPLC methods to be used in drug quality control and TDM analysis for PCA, thus indicating the further applicability in the analysis of TDM-required drugs, such as immunosuppressants, anti-cancer drugs, and psychiatric drugs. Human-friendly ethanol was selected as an organic solvent in the mobile phase. PCA was eluted from NUCLEODUR 100-5 C8 ec (5 μm, 150 x 4.6 mm) column by a mobile phase containing ethanol and 50 mM NaH2PO4 buffer (5:95, v/v). The mobile phase flow rate was 1.0 ml min-1, the column temperature was 35 °C, and the wavelength at the PDA detector was 278 nm. Retention time for PCA was 5.0 min and 7.7 min for paracetamol as an internal standard (IS). In the green HPLC method for pharmaceutical analysis, the highest relative standard deviation (RSD) and mean recovery values were 1.32% and 98.89%, respectively. In the analysis of plasma, the sample preparation step was only smooth protein precipitation by ethanol. Thus, the bioanalytical method was fully green having a limit of detection (LOD) of 0.3 μg ml-1 and a limit of quantification (LOQ) of 0.8 μg ml-1. The therapeutic plasma concentration for PCA was reported in the range of 4-12 μg ml-1. As a result, the green HPLC methods developed and validated in this study were selective, accurate, precise, reproducible, and trustable and have the quality for the application in pharmaceutical and TDM analysis of PCA, thus encouraging green HPLC analysis of other TDM required drugs.

Identification and Structural Characterization of Forced Degradation Impurities of Sacubitril by HRMS – Development and Validation of a Stability indicating RP-HPLC method

The current work identifies and characterises the stress degradation products of the anti-hypertensive drug sacubitril. The stability profile of sacubitril was evaluated under several stress settings including hydrolytic, oxidative, thermal and photolytic conditions in accordance with ICH recommendations. The degradation products of sacubitril were separated using reverse phase liquid chromatography and structural characterization was carried out using HRMS. The chromatographic separation was carried out by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) using a Fortis C18 column (150 mm X 4.6 mm; 5μm particle size) with mobile phase A (consisting of 0.1% TFA in Milli-Q water) and mobile phase B (100% Acetonitrile) in gradient mode. With a sample injection volume of 10μL, the mobile phase flow rate was 0.7 mL/min. A quadrupole time-of-flight (Q-TOF) mass spectrometer was used for the LC-MS analysis. Sacubitril is relatively stable in oxidative, thermal and photolytic conditions; however, considerable degradation was observed in acid, base and neutral hydrolysis. Three degradation products were identified and the structural characterization of these impurities was performed using the HRMS data and mass fragmentation. The proposed RP-HPLC method was robust, precise and specific in determining the impurity profile of sacubitril under different stress conditions.

Quality by Design (QbD): A Comprehensive Understanding and Implementation in Pharmaceuticals Development

Abstract: Quality by Design (QbD) is a systematic approach for improvement that stresses product and process and begins with a predetermined objective, as recommended by the USFDA and International Council Harmonization (ICH). Regulatory bodies frequently highlight the use of ICH quality criteria, which include Q8, Q9, Q10, and Q11. The differentiation between the traditional and QbD helps to study the risk assessment and technique for developing new products. There are a few steps involved in pharmaceutical and Analytical QbD. Various factors were used for the study of QbD, such as Analytical Target Product Profile (ATPP), Risk Assessment Quality Design Space, Control Strategy, etc. Critical Quality Attribute (CQA) may be understood and analyzed via a way of means of understanding the goods and technique and risk evaluation is useful for effective verbal exchange among FDA and industry, research/improvement and production, and amongst a couple of production sites inside the company. Life-cycle management of analytical procedure begins off evolving with the establishment of ATP and maintains until the approach is in use. The design of the experiment (DoE) involves the Q8 guidelines. DoE has been used in the rational development and optimization of analytical methods. Culture media composition, mobile phase composition, flow rate, and time of incubation are input factors (independent variables) that may be screened and optimized using DoE. Process analytical technology is implemented for the understanding and identification of developing a product and techniques. There are various benefits and applications of QbD in the pharmaceutical industry.

Novel green HPTLC and organic solvent-free micellar LC methods for the simultaneous determination of Ozenoxacin and benzoic acid; greenness assessment and applications

Ozenoxacin (OZ) is a novel synthetic quinolone antimicrobial agent that has been recently approved by US-FDA for the treatment of impetigo in combination with its preservative, benzoic acid (BA). Meanwhile, implementing greener chromatography has been a recent goal of a great ecological importance. In the proposed research, two novel green methods have been validated for the simultaneous determination of OZ and BA in skin creams for the first time. An organic-solvent free mixed-micellar HPLC technique was developed using RP-C8 core-shell column with a mobile phase consisting of 0.01 M Brij-35, 0.15 M sodium dodecyl sulfate, and 0.02 M ammonium acetate in water, adjusted to pH 4.5. The mobile phase flow rate was 1 mL min−1 and detecting analytes in short retention times (3.4 and 4.7 min for BA and OZ, respectively) at wavelength 235 nm. The working concentration ranges were 1–10 and 10–100 μg mL−1 for BA and OZ, respectively. Another eco-friendly high-performance thin-layer chromatography (HPTLC) method was designed using HPTLC silica-gel 60 F254 plates as a stationary phase and ethyl acetate–ethanol–ammonia (6:4:2, v/v/v) as mobile phase. The detection was also performed at λmax = 235 nm. The method was found linear across concentration range of 0.5–1.8 and 0.05–0.18 μg/band for OZ and BA, respectively which gave symmetric, compact band with Rf values of 0.21 and 0.69 for OZ and BA, respectively. The proposed methods were applied successfully in their combined marketed pharmaceutical cream. The proposed methods were typically based on safe biodegradable reagents, and their ecological superiority was proven using recent greenness metrics.

Chromatographic method development for simultaneous determination of serotonin, melatonin, and L‐tryptophan: Mass transfer modeling, chromatographic separation factors, and method prediction by artificial neural network

AbstractThis work endeavored to develop a high performance liquid chromatography (HPLC) method for simultaneous quantification of three important biotherapeutic molecules namely, L‐tryptophan, serotonin, and melatonin, present in low amounts in agro‐commodities. In the first approach, using pure chemical standards of the same in a mixture, chromatogram separation parameters such as peak sharpness, peak shouldering, and peak separation were judged by a human panel and biasness‐cum‐decision uncertainties were averted using fuzzy logic analysis. In the second approach, the separation parameters such as peak resolution and separation factors were evaluated to obtain a well‐resolved chromatogram. The parameters of the said separation process were successfully modeled by dimensionless numbers of mass transfer of the analytes in the column; and post‐fitting of mass transfer equations, high R2 was obtained suggesting successful development of the chromatographic process. Euclidean distances between the values of each chromatographic separation parameter and their respective ideal values; the defuzzified scores; findings of mass transfer study; and separation factors concomitantly established that the mobile phase of composition 1% acetic acid in HPLC water (A)–HPLC grade methanol (B) in gradient elution conditions (90% A–10% B) at a flow rate of 1 mL/min could simultaneously quantify the three molecules (μg) in button mushrooms with good resolution. The presence of the said biomolecules in the extract of button mushrooms was also confirmed by electrospray ionization (ESI)‐mass spectrometer (MS). An artificial neural network model (high R2) was also developed for chromatographic users, which would allow accurate prediction of the chromatographic parameters by varying mobile phase composition and flow rates.

Evaluation of ethanol-based aqueous two-phase systems performance for application in centrifugal partition chromatography (CPC)

Aqueous two-phase systems (ATPS) have recently been proposed for Centrifugal Partition Chromatography (CPC). This type of biphasic system has several advantages over conventional aqueous-organic liquid–liquid systems, namely, high biocompatibility owing to its large water content. The most commonly used ATPS are polymer-based systems. However, owing to their poor phase separation hydrodynamics compared to aqueous-organic systems, the use of short-length water-miscible alcohols has been evaluated as an alternative to polymers. In this study, an ATPS composed of ethanol and dipotassium hydrogen phosphate (K2HPO4) is proposed to overcome the common hydrodynamic drawbacks of polymer-based ATPS. The Ethanol/K2HPO4 system exhibited a very short settling time (ts = 30.85 ± 0.80 s), high interfacial tension (IFT = 6.38 mN/m), significant density differences between the phases (Δρ = 353.40 kg/m3), and low viscosity of the coexisting phases at 25 °C (maximum µ = 3.79 ± 0.11 mPa.s), making it an ideal choice for implementation in CPC apparatus. A comprehensive study of this biphasic system was conducted using CPC equipment by carefully adjusting the operating parameters, specifically, the mobile phase flow rate (F), rotational speed (ω), and mode of operation, that is, ascending (upper phase is mobile) or descending (lower phase is mobile). In this particular CPC equipment, the use of polymers as phase-forming compounds in ATPS resulted in higher CPC pump pressures compared to ethanol or phosphate salt aqueous solutions, thus confirming the superior performance of the ethanol/salt ATPS. The results obtained using this ATPS demonstrated that high F and ω values lead to lower stationary phase retention (SF) and higher backpressures, respectively. SF remained relatively constant and above 50% for F ≤ 6 mL/min but significantly decreased for F > 6 mL/min. This trend confirms that hydrostatically stable conditions can be achieved by employing the Ethanol/K2HPO4 system in the CPC mode by controlling F (i.e., the hydrodynamics of the mobile phase). The partition of the model solutes gallic acid and L-tyrosine was also evaluated using the ethanol/K2HPO4 system. The critical operating CPC parameters were compared, confirming the significant potential of the ethanol/salt ATPS for successful commercial application of this chromatographic technique in the separation of small value-added biomolecules.

Development of Analytical Quality by Design Compliant Chaotropic Chromatography Method for Ziprasidone and Its Five Impurities Determination.

In this study, an AQbD-compliant chaotropic chromatography method for ziprasidone and the determination of its five impurities was developed. The influence of critical method parameters (initial and final methanol fraction in the mobile phase, gradient duration) on the set of selected critical method attributes (t_imp. V, t_imp. V - t_imp. I, S and <WUSP>) was studied by Box-Behnken design. The errors resulting from the calculation of the model coefficients were propagated to the selected responses by Monte Carlo simulations, and their predictive distribution was obtained. The design space was computed (π ≥ 80%), and a working point was selected: initial methanol fraction 38.5%, final methanol fraction 77.5%, and gradient duration 16.25 min. Furthermore, the quantitative robustness of the developed method was tested using the Plackett-Burman design. P_imp II and P_imp V were found to be significantly affected, the first by mobile phase flow rate and the second by gradient duration. Finally, the method was validated, and its reliability for routine quality control in capsules was confirmed.

New analytical method development and validation for estimation of molnupiravir in bulk and tablet dosage form by RP-HPLC method

A new simple, selective, rapid, precise reversed-phase high-performance liquid chromatography method has been developed and validated for the estimation of Molnupiravir in bulk and its pharmaceutical dosage form. The separation was made using Symmetry ODS C18 (4.6×150mm, 5µm) column. The mobile phase used contained Methanol. Phosphate Buffer pH-4.2 adjusted with Orthophosphoric acid solution in the ratio of 35:65% v/v in an isocratic mode at a wavelength of 236nm. The mobile-phase flow rate and the sample volume injected were 1 ml/min and 10 μL, respectively. The retention time of Molnupiravir was found to be 2.8 ±0.2mins. A good linear relationship of Molnupiravir r =0.999) was observed over a concentration range of 20 to 100µg/ml of Molnupiravir. The limit of detection (LOD) and limit of quantification (LOQ) for Molnupiravir was found to be 2.6µg/ml and 6.35µg/ml. The recovery percentage was observed in the range of 98-102%. The relative standard deviation for the precision study was found <2%. The developed method is simple, precise, specific, accurate and rapid, making it suitable for the estimation of Molnupiravir in bulk and marketed pharmaceutical dosage form. It was concluded that in the present developed RP- HPLC method is simple, rapid, and accurate, hence can be used for routine quality control analysis in the Pharmaceutical industry.

Optimization of a HPLC-UV bioanalytical method using Box-Behnken design to determine the oral pharmacokinetics of neratinib maleate in Wistar rats.

The current research work reports the development and validation of a sensitive, robust and reproducible bioanalytical method for quantifying neratinib maleate in rat plasma. More than 85% of the drug was extracted from the plasma samples by protein precipitation. The method was optimized using Box-Behnken design, a response surface method. The effect of three critical factors, viz., the pH of the buffer (X1 ), the aqueous phase proportion in the mobile phase (X2 ) and the mobile phase flow rate (X3 ), was studied on two response variables, retention time (Y1 ) and United States Pharmacopoeia (USP)width (Y2 ). With the highest overall desirability function value of 0.943, the obtained optimized method conditions were: X1 = 2.4 ± 0.1; X2 = 66.7 ml, and X3 = 0.85 ml/min. Under the optimized conditions, the values of Y1 and Y2 for a sample containing 1 ppm of the drug were found to be 14.1 min and 0.50 ± 0.003, respectively. Single-dose intravenous bolus (7.5 mg/kg) and oral (15 mg/kg) pharmacokinetic studies were performed to determine the absolute bioavailability of the drug. The optimized bioanalytical method was sensitive enough to capture 95% of the drug eliminated from the body. The absolute oral bioavailability of the drug was 49.30%.

A novel process for developing a quantitative chromatographic fingerprint analysis method based on analytical quality by design: A case study of Xiaochaihu capsules

In this study, an improved Analytical Quality by Design (AQbD) process was proposed to establish quantitative fingerprints of Chinese medicines. A quantitative fingerprint method for the strong polar components in Xiaochaihu capsules was developed as an example. Different method evaluation indicators were used to characterize this method, and these indicators were measured at different stages of quantitative fingerprint development. Through this method, the difficulty in determining common peaks in the early stage of quantitative fingerprint development is eliminated. The method parameters that needed to be optimized were the mobile phase gradient conditions, column temperature and flow rate through the Ishikawa diagram. Single-factor experiments were used to determine some parameters. Definitive screening design (DSD) experiments were used to optimize critical method parameters (CMPs). The common peaks were determined, and indices were optimized. The critical method attributes (CMAs) were the resolution of guanosine, the percentage of common peak to the total peak area, retention time of the last peak and the common peak number. A stepwise regression method was used to build quantitative models for critical method attributes and critical method parameters. The method operable design region (MODR) was calculated by the experimental error simulation method and successfully verified. After the analysis conditions were optimized, four components, uridine, adenine, 5-hydroxymethylfurfural, and guanosine, were quantitatively determined. There were 7 common peaks in the fingerprints. The common peak area accounted for 90.64%. Both fingerprint and quantitative detection analysis methods were methodologically validated. The new AQbD process proposed in this study can be potentially applied to develop other Chinese medicines fingerprint methods.

LC-MS/MS Method Development and Validation for Determination of Favipiravir Pure and Tablet Dosage Forms.

Analytical method development and validation for determination of favipiravir (FVPR) in pure and tablet dosage forms by liquid chromatography with tandem mass spectrometry/mass spectrometry (LC-MS/MS) technique. A simple LC-MS/MS method was developed for determination of a new antiviral drug, FVPR in pharmaceutical formulations. The stationary phase employed was a Shim pack GISS, C18 (100 mm × 2.1 mm, 1.9 μm) column and mobile phase used in pump A was 10.0 mM ammonium acetate and in pump B methanol was used. The gradient program was used with fixed mobile phase flow rate at 0.4 mL min-1. Total run time was 5.0 min. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines. The established method found better outcomes. The linearity graph was found in the range of 50-200 μg/mL and the correlation coefficient value (R2) obtained was found to be 1.0. The limit of detection (LOD) and limit of quantification (LOQ) were 4.044 μg/mL and 12.253 μg/mL, respectively. Tremendous recovery outcomes were observed and found to be 101%, 99.0%, and 99.5% for FVPR at 150% upper, 100% middle, and 50% lower concentrations, respectively. All outcomes obtained comply with ICH guidelines. The developed method was simple, unique, accurate, robust, precise, and reproducible for determination of FVPR in tablet formulation. The method is novel and could be adopted in formulation industry.

Open tubular liquid chromatography: Recent advances and future trends.

Nano-liquid chromatography (nanoLC) is gaining significant attention as a primary analytical technique across various scientific domains. Unlike conventional high-performance LC, nanoLC utilizes columns with inner diameters (i.ds.) usually ranging from 10 to 150μm and operates at mobile phase flow rates between 10 and 1000 nl/min, offering improved chromatographic performance and detectability. Currently, most exploration of nanoLC has focused on particle-packed columns. Although open tubular LC (OTLC) can provide superior performance, optimized OTLC columns require very narrow i.ds.(<10μm) and demand challenging instrumentation. At the moment, these challenges have limited the success of OTLC. Nevertheless, remarkable progress has been made in developing and utilizing OTLC systems featuring narrow columns(<2μm). Additionally, significant efforts have been made to explore larger columns (10-75μm i.d), demonstrating practical applicability in many situations. Due to their perceived advantages, interest in OTLC has resurged in the last two decades. This review provides an updated outlook on the latest developments in OTLC, focusing on instrumental challenges, achievements, and advancements in column technology. Moreover, it outlines selected applications that illustrate the potential of OTLC for performing targeted and untargeted studies.

Simultaneous Determination of Posaconazole and Hemp Seed Oil in Nanomicelles through RP-HPLC via a Quality-by-Design Approach.

The present study involves the development of a reverse-phase HPLC method employing the quality-by-design methodology for the estimation of posaconazole and hemp seed oil simultaneously in nanomicelles formulation. The successful separation of posaconazole and hemp seed oil was achieved together, and this is the first study to develop and quantify posaconazole and hemp seed oil nanomicelles with linoleic acid as the internal standard and developed a dual drug analytical method employing a quality-by-design approach. The study was performed on a Shimadzu Prominence-I LC-2030C 3D Plus HPLC system with a PDA detector and the Shim-pack Solar C8 column (250 mm × 4.6 mm × 5 μm) for analysis with a mobile phase ratio of methanol:water (80:20% v/v) maintaining the flow rate of 1.0 mL/min. The final wavelength was selected as 240 nm and the elution of hemp seed oil and posaconazole was obtained at 2.7 and 4.6 min, respectively, with a maximum run time of 8.0 min. Box Behnken design was employed to optimize the method, keeping the retention time, peak area, and theoretical plates as dependent variables, while the mobile phase composition, flow rate, and wavelengths were chosen as independent variables. Parameters such as specificity, accuracy, robustness, linearity, sensitivity, precision, ruggedness, and forced degradation study were performed to validate the method. The calibration curves of posaconazole and hemp seed oil were determined to be linear throughout the range for concentration. The suggested approach can be effectively utilized for estimating the content of drugs from their nanoformulation and proved suitable for both in vivo and in vitro research.

Development of a liquid chromatographic method for enantioseparation of Eflornithine using (S)-α-ethyl benzylamine as a chiral derivatizing agent

ABSTRACT Eflornithine (2-fluoromethyl-DL-ornithine) is a potent and irreversible inhibitor that selectively targets ornithine decarboxylase, a key enzyme in the polyamine biosynthesis pathway. Due to the lack of chromophoric moiety in the eflornithine structure, its detection via UV detector is difficult. Thus, direct high-performance liquid chromatographic (HPLC) separation of enantiomers of Eflornithine is not feasible, and pre-column derivatization is required for its determination through chiral HPLC columns. We developed an indirect HPLC method using (S)-α-ethyl benzylamine as a chiral derivatizing agent for the enantioseparation of Eflornithine. The diastereomers thus produced were then separated via LiChrospher C18 column (5 μm particle size, L × I. D. 25 cm × 4.6 mm). The mobile phase used was a mixture of acetonitrile and 0.1% aq. TFA and varied in linear gradients of 30–70% of acetonitrile for 30 min run at a flow rate of 1.0 mL min−1 and UV detection at 320 nm. The separation parameters were optimized by altering the mobile phase composition and flow rate. The findings revealed that the chromatographic separation was accomplished within 15 min, with resolution values greater than 4.5 for Eflornithine enantiomers. The detection and quantitation limits were 9.26 ng mL− 1 and 18.52 ng mL− 1 for Eflornithine enantiomers.

RP-HPLC method for estimation of miglitol and metformin hydrochloride in pharmaceutical formulation

A simple, rapid and sensitive RP-HPLC method was developed for the quantitative determination of metformin hydrochloride and miglitol in combined tablet dosage form. The chromatographic analysis was carried out on Phenomenex Gemini C18 (250 x 4.6 mm, 5 μm) with mobile phase containing methanol: TEA buffer (50:50 v/v). The flow rate of mobile phase was 1.0 mL/min and effluents were monitored at 272 nm. The retention times of miglitol and metformin hydrochloride were 2.121 min and 3.643 min, respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity and robustness.The method was found to simple, rapid and sensitive and was successfully applied to the estimation of metformin hydrochloride and miglitol in combined dosage form.

Reverse Phase High Performance Liquid Chromatography for Validation of Epinastine Hydrochloride in Bulk and Pharmaceutical Dosage Form

The validation method of epinastine hydrochloride from combined dosage form i.e. eye drops was described by high performance liquid chromatography method with separation of drugs on Water symmetry RP18 (150 x 4.6 mm i.d.) and 5 µ particle size. A mixture of buffer and acetonitrile (73:27 % (v/v)) was constituted as mobile phase. The chromatograms were studied at 220 nm as wavelength. The mobile phase was also used as a diluent. A validated of method was studied for linear regression, accuracy, method as well as system precision. The robustness study was done for change in wavelength, mobile phase composition and flow rate as per ICH guidelines. The method has been successfully used to analyze epinastine hydrochloride from dosage form i.e. eye drops.

Flow behavior of protein solutions in a lab-scale chromatographic system

A fluid dynamics model has been developed to describe flow behavior in a lab-scale chromatographic system dedicated for protein processing. The case study included a detailed analysis of elution pattern of a protein, which was a monoclonal antibody, glycerol, and their mixtures in aqueous solutions. Glycerol solutions mimicked viscous environment of the concentrated protein solutions. The model accounted for concentration dependences of solution viscosity and density, and dispersion anisotropy in the packed bed. It was implemented into a commercial computational fluid dynamics software using user-defined functions. The prediction efficiency was successfully verified by comparing the model simulations in the form of the concentration profiles and their variances with the corresponding experimental data. The contribution of the individual elements of the chromatographic system to protein band broadening was evaluated for different configurations: for the extra-column volumes in the absence of the chromatographic column, for the zero-length column without the packed bed and for the column containing the packed bed. The influence of the operating variables, including: the mobile phase flowrate, the type of the injection system, i.e., the injection loop capillary or the superloop, the injection volume and the length of the packed bed, on band broadening of the protein was determined under nonadsorbing conditions. For protein solutions having viscosity comparable with the mobile phase, the flow behavior either in the column hardware or in the injection system made major contributions to band broadening, which depended on the type of the injection system. For highly viscous protein solution, the flow behavior in the packed bed exerted a dominant influence on band broadening.

Isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus using centrifugal partition chromatography

Replacement of synthetic colorants with natural ones is a current marketing trend. Nevertheless, the naturally occurring blue color is rare compared to other colours. In this work, centrifugal partition chromatography (CPC) process was developed as a more efficient and sustainable alternative to reversed phase column chromatography (RP-CC) for the preparative-scale purification of portisins. The strategy began with the extraction of anthocyanins from blueberry surplus and hemi-synthesis of respective portisins. Then, the CPC method development started with the biphasic solvent system selection followed by the optimization of the operating parameters and ended up with a comparison with RP-CC. Aiming at maximizing the portisin content, process throughput, efficiency, and minimizing the environmental risk factor, the effect of sample load (100–500 mg/100 mL of column volume), mobile phase flow rate (10–20 mL/min), and rotation speed (1000–1600 rpm) was evaluated. The two-phase solvent system consisted of tert‑butyl‑methyl ether, n-butanol, acetonitrile, and water (volume ratio 2:2:1:5) acidified with 0.1 vol.% of HCl was selected. The best conditions were 464 mg of sample/100 mL of column volume, 20 mL/min of mobile phase flow rate, and 1600 rpm of rotation speed at reversed phase mode, allowing the purification of portisins by 5-fold. Compared to the RP-CC, the CPC process efficiency was 2.4 times higher, while the CPC process environmental risk factor was 5.5 times lower. Overall, this study suggests that CPC can be considered an effective, and sustainable alternative process for the preparative isolation of portisins. With this purification approach, the blueberry surplus has been valorized and a naturally derived product has been prepared, allowing its subsequent use as a natural blue colorant.

RP-UFLC based Bioanalytical Method Development, Optimization, and Validation for the Estimation of Isradipine in Rabbit Serum

Introduction: The objective of this study is to provide a rapid, sensitive, consistent, and costeffective method for quantifying isradipine using ultra-fast liquid chromatography. Methods: Quality by Design principles will form the basis of this approach, grounded on response surface analysis. Shimadzu liquid chromatographic system equipped with a photodiode array detector and LC solution software was used to conduct the RP-UFLC method development and validation. An ODS C18 (250 x 4.6 mm; 5 μm) UFLC column was used to complete the analysis. The RSM methodology utilized a central composite design to perform the optimization studies. Results: The mobile phase ratio and flow rate were considered crucial method parameters, as well as the peak area, retention time, and USP plate were considered critical analytical attributes. The optimal conditions for chromatographic separation were followed using 80% acetonitrile and water (20% v/v) as mobile phase, a 1 mL/min flow rate, an injection volume of 20 μL, 40°C of column oven temperature, and maximum absorption at λmax 254 nm using graphical optimization technique. When examining concentrations between 5 and 150 ng/mL, linearity was observed with an R2 of 0.999. The method created was validated by employing stability testing per the recommendations provided by ICH Q2 (R1). The analysis of blood serum was modified so that it could be used to examine the pharmacokinetic parameters. Conclusion: As a result, high accuracy, precision, sensitivity, linearity, and robustness were established for predicting the amount of isradipine present in its freeze-dried nano-formulations.