Development Of Chromatographic Method Research Articles

Digitally Enabled Generic Analytical Framework Accelerating the Pace of Liquid Chromatography Method Development for Vaccine Adjuvant Formulations

Digitally Enabled Generic Analytical Framework Accelerating the Pace of Liquid Chromatography Method Development for Vaccine Adjuvant Formulations

Separation and identification of oligonucleotides impurities and degradation products by reversed phase ultra-high performance liquid chromatography using phenyl-bonded stationary phases without ion pairs - A step towards sustainability

This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3′N and 5′N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. The proposed separation methods without ion-pair reagents contribute to a more sustainable approach in oligonucleotide analysis.

Development of a Superheated Water Chromatography Method for the Sustainable Determination of Preservatives in Foods and Cosmetics

Development of a Superheated Water Chromatography Method for the Sustainable Determination of Preservatives in Foods and Cosmetics

Theoretical predictions to facilitate the method development in slalom chromatography for the separation of large DNA molecules

Slalom chromatography (SC) re-emerged in 2024 due to the availability of low adsorption ultra-high pressure liquid chromatography (UHPLC) packed columns/instruments and large modalities being investigated in the context of cell and gene therapies. The physico-chemical principles of SC retention combined with hydrodynamic chromatography (HDC) exclusion have been recently reported. In SC, DNA macromolecules are retarded because: (1) they can be stretched to lengths comparable to the particle diameter, and (2) their elastic relaxation time is long enough to maintain them in non-equilibrium extended conformations under regular UHPLC shear flow conditions. Here, a quantitative HDC-SC retention model is consolidated. A general plate height model accounting for the band broadening of long DNA biopolymers along packed beds is also derived for supporting method development and predicting speed-resolution performance in SC.For illustration, the chromatographic speed-resolution properties in SC are predicted for the separation of specific critical pairs (4.0/4.5, 10/11, and 25/27 kbp) of linear dsDNA polymers. The calculations are performed for two available custom-made particle sizes, dp= 1.7 and 2.5μm, at a constant pressure of 10,000 psi. The predictions are directly validated from experimental data acquired using low adsorption MaxPeakTM 4.6 mm i.d. Columns packed with 1.7μm BEHTM 45 Å (15 cm long column) and 2.5μm BEH 125 Å (30 cm long column) Particles, and by injecting six linear dsDNAs (λ DNA-Hind III Digest). The LC system is very low dispersion ACQUITYTM UPLCTM I-class PLUS System, and the mobile phase is a 100 mM phosphate buffer at pH 8. Maximum resolution is always achieved when the average extended lengths of linear dsDNAs are equal to a critical length, which is proportional to the particle diameter and to the square root of the applied shear rate. Most advantageously, the experimental results reveal that the relaxation times of linear dsDNAs observed under shear flow conditions are two orders of magnitude shorter than those expected in the absence of flow: this enables the detection of the longest linear dsDNAs up to 25 kbp without irremediable loss in column performance. Finally, the retention-efficiency model elaborated in this work can be used to rapidly anticipate and develop methods (selection of particle size, column length, and operating pressure) for any targeted DNA and time-resolution constraints.

Development and validation of a gas chromatography method for analysing polychlorinated biphenyls in fish roe

A method for determining polychlorinated biphenyls (PCBs) in fish roe by gas chromatography (GC) has been developed. The suitability (validation) of the process for the determination of 14 PCBs (PCB 18, PCB 31, PCB 28, PCB 52, PCB 44, PCB 101, PCB 149, PCB 118, PCB 153, PCB 138, PCB 180, PCB 170, PCB 194, PCB 209) in fish roe by GC using an electron capture detector (ECD) was evaluated according to the following criteria: selectivity, linearity, limits of detection (LOD), limits of quantification (LOQ), accuracy and precision. Automated Soxhlet extraction and sample clean-up by solid phase extraction (SPE) were proposed for extracting PCBs from fish roe. The results of the method selectivity study showed that the determination of PCBs in fish roe is not affected by other components in the sample. The correlation coefficients for fourteen PCBs ranged from 0.9962 to 0.9999 (R2 ≥0.995). The limits of detection (LOD) and limits of quantification (LOQ) of PCBs are below the maximum permissible levels set by the European Union (EU). The recovery percentage ranged from 81.5% to 107%, indicating the PCB extraction procedure's acceptability (R, 80 – 120%). The relative standard deviation (RSD, %) of the measurement results under convergence conditions ranged from 1.02% to 9.43% (RSD ≥15%). The obtained method suitability (validation) data meets the Commission Regulation (EU) No 589/2014 criteria.

Stability-indicating liquid chromatographic method development and validation for quantification of trifarotene in pure and topical drug product

BackgroundTrifarotene is effective for treating acne and other skin issues. To ensure its quality and meet regulatory standards, a reverse phase liquid chromatography (RP-LC) stability-indicating method was developed and validated for its quantification in pure and topical dosage forms. An isocratic elution chromatographic method was employed, using an octadecylsilyl silica gel-packed column (150 mm × 4.6 mm, 3 µm particle size). The mobile phase was a mixture of phosphate buffer and acetonitrile (40:60 v/v). Chromatographic conditions included a flow rate of 0.5 mL/min, column temperature of 40 °C, detection at 265 nm, and injection volume of 20 µL.ResultsThe developed analytical method reports the retention time of trifarotene 11.2 min, higher theoretical plate count, asymmetric peak, and good resolution between the peaks of trifarotene, phenoxyethanol, and environmentally generated impurities.ConclusionThe analytical method has been found to be linear, accurate, robust, specific, and selective for impurities produced during forced degradation studies. The proposed analytical method can be utilized for routine pharmaceutical analysis of trifarotene to judge its quality and safety.

Development of a high-performance liquid chromatography method for simultaneous quantification of sixteen polyphenols and application to tomato

The main purpose of the current work was to develop a new method to evaluate and quantify sixteen polyphenol compounds from tomato fruit using high-performance liquid chromatography (HPLC). The separation of 16 polyphenols from tomato fruit was achieved in < 60 min by using a Waters Symmetry C18 column (250 × 4.6 mm i. d, 5 µm particle sizes) with a gradient system of ultrapure water (1 % acetic acid) and 100 % methanol, a temperature of 30 °C, an injection volume of 10 μL and a flow rate of 1.1 mL/min, respectively. The analytical characteristics of evaluation method provide sufficient sensitivity for all tomato polyphenols compounds within normal range 0.1–20 μg·mL−1 (R2≥0.999) with 0.069–0.365 μg·mL−1 LOD, and 0.171–1.106 μg·mL−1 LOQ, with good system suitability (<2 % RSD of retention time, peak area, and tailing factor, 6,000–1,336,000 N, and >1.5 peak resolution), <10 % RSD of precision, stability, repeatability, and robustness, and 99.2 - 105.0 % of recovery. The applicability of this method was demonstrated by the determination of polyphenols in nine cultivars of tomatoes. The results showed that ‘184′ possessed the highest content of total polyphenols (1249.53 μg·g−1 DW) followed by ‘Disease resistance 184′ (1064.93 μg·g−1 DW). The main polyphenol components were rutin, quercetin, gallic acid, chlorogenic acid, 2,5-dihydroxy benzoic acid, caffeic acid and benzoic acid in tomato fruits. In conclusion, this novel HPLC method is useful and acceptable to analyze polyphenols in tomato fruit.

Development and Validation of a High-Performance Thin-Layer Chromatography Method for Simultaneous Qualitative and Quantitative Analysis of Scutellarin and Scutellarein in S. barbata D. Don

Development and Validation of a High-Performance Thin-Layer Chromatography Method for Simultaneous Qualitative and Quantitative Analysis of Scutellarin and Scutellarein in S. barbata D. Don

Box-Behnken design in optimization of the green liquid chromatographic method for the quantification of Afatinib in drug product: AQbD approach

The aim of this research work was to quantitatively determine the tyrosine kinase inhibitor, Afatinib, in tablets using an eco-friendly chromatographic method. The development of a green liquid chromatographic method using analytical quality-by-design (AQbD) approach principles to estimate Afatinib via Box-Behnken design. A simple and effective green liquid chromatographic method was developed and validated. The analysis was done on the Zodiac C18 column by isocratic elution mode. Mobile phase composed of pH 3.0 potassium dihydrogen phosphate buffer and ethanol in the ratio of 65:35%V/V. The flow rate was set to 0.6 mL/min. with an injection volume of 10 µL and at detection wavelength of 254 nm. The optimized method was found to be specific, linear, precise, accurate, and robust. Established linearity in the range of 24.6 to 73.9 µg/mL. The developed method exhibited excellent greenness. Based on the greenness assessment tools, the analytical eco-score value of 94 and the pictograms of NEMI, Modified NEMI, GAPI, Complex GAPI, AGREE, and AGREE prep expressed the method’s greenness. Hence, the AQbD approach based green method and the validation data indicate that the method can be employed in routine analysis for the determination of Afatinib in pharmaceutical dosage form in quality control laboratory use.

Assisted Active Learning for Model-Based Method Development in Liquid Chromatography.

In recent decades, there has been a growing interest in fully automated methods for tackling complex optimization problems across various fields. Active learning (AL) and its variant, assisted active learning (AAL), incorporating guidance or assistance from external sources into the learning process, play key roles in this automation by enabling the autonomous selection of optimal experimental conditions to efficiently explore the problem space. These approaches are particularly valuable in situations wherein experimentation is costly or time-consuming. This study explores the application of AAL in model-based method development (MD) for liquid chromatography (LC) by using Bayesian statistics to incorporate historical data and analyte information for the generation of initial retention models. The process involves updating the model parameters based on new experiments, coupled with an active data selection method to choose the most informative experiment to run in a subsequent step. This iterative process balances model exploitation and experimental exploration until a satisfactory separation is achieved. The effectiveness of this approach is demonstrated via two practical examples, resulting in optimized separations in a limited number of experiments by optimizing the gradient slope. It is shown that the ability of AAL to leverage past knowledge and compound information to improve accuracy and reduce experimental runs offers a flexible alternative approach to fixed design methods.

A Quality by Design (QbD) driven gradient high performance liquid chromatography method development for the simultaneous estimation of dasatinib and nilotinib in lipid nanocarriers

Tyrosine kinase inhibitors (TKIs) are effective as a targeted treatment for chronic myeloid leukemia (CML), which can selectively suppress BCR-ABL1 kinase activity. CML therapy with TKIs combination has been supported by in-vitro, in-vivo, and patient-based data where the nilotinib-dasatinib co-administration has exerted superior anticancer efficacy with greater cellular uptake, less resistance to chemotherapy, and no additive adverse events encountered. Therefore, it is essential to develop a suitable analytical method for the simultaneous estimation of these drugs in the developed novel lipid nanocarriers like liposomes. Design of Experiment (DoE) has been implemented as a tool of QbD to systematically investigate the relation between the HPLC method attributes and analytical responses, i.e., chromatographic detection, quantification, and peak properties for dasatinib and nilotinib. An Ishikawa diagram is constructed to delineate possible influencing variables to the analytical performances. Afterward, 4 factors 2 level full factorial design (FFD) was employed to model and identify the main effects and interaction effects between the factors selected after the initial risk assessment. The suggested design space for optimized chromatographic conditions by QbD analysis is linear within the selected range of drug concentrations, accurate and precise, sensitive, and robust according to the ICH guidelines. The optimal method is comprised of a 1 mL/min flow rate of mobile phase (ACN and 20 mM KH2PO4 of pH 7.00) in gradient mode at 25 °C column temperature for 20 μL sample injection volume and detection wavelength fixed at 297 nm. Most importantly, this novel HPLC method is simple and selective enough to evaluate dasatinib and nilotinib content in the lipid nanocarriers.

Advanced stability-indicating gradient elution reverse phase-high performance liquid chromatography method development and validation for simultaneous estimation of rutin and curcumin using an analytical quality by design approach

This research delves into a systematic Analytical Quality by Design (AQbD) approach to develop and validate a stability-indicating robust RP-HPLC method for the simultaneous determination of Rutin and Curcumin, compounds known for their anti-inflammatory, antioxidant, and wound-healing properties. Utilizing QbD principles, critical method parameters (CMPs) were identified and screened using design of experiments (DoE) and optimized via response surface methodology (RSM) with Central Composite Design (CCD). The optimized chromatographic separation used a Phenomenex Luna C18 column with gradient elution, using methanol and 0.1% formic acid, a flow rate of 1 ml/min, and detection at 345 nm (isosbestic point). The retention times were 5.63 mins for Rutin and 17.01 mins for Curcumin. The stability indication of the developed HPLC method was performed, and validation parameters were meticulously conducted following the ICH Q2(R1) guidelines, demonstrating linearity over 2-10 μg/ml for Rutin and 5-25 μg/ml for Curcumin, with correlation coefficients of 0.9999 and 0.9998, respectively. Precision studies showed %RSD within acceptable limits, and accuracies ranged between 97-100%. This validated HPLC method offers a reliable, sensitive approach for simultaneous quantifying Rutin and Curcumin, suitable for routine analysis in pharmaceutical laboratories and quality control.

Development and Validation of a Novel Stability-Indicating Reverse Phase High-Performance Liquid Chromatography Method for the Quantification of Capivasertib in Bulk Drug and Pharmaceutical Dosage Form

The quantification of capivasertib has been achieved using a Waters Symmetry column with UV detection at 260 nm through a novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method that has been established and verified. The analysis can be completed in just 5 minutes. The separation and collection of capivasertib occurred at a retention time of 3.3475 minutes. The concentration range of capivasertib was shown to be linear, ranging from 50 to 300 μg/mL. The regression equations for capivasertib were determined as y = 13485.85x + 1723.04. The detection limit for capivasertib was determined to be 0.6 μg/mL, while the quantification limits were established at 2.00 and 0.5000 μg/mL, respectively.

Development and optimization of high-performance extraction chromatography method for separation of rare earth elements

The effectiveness of commonly used extractants for chromatographic separation of rare earth elements (REEs) was compared. Columns loaded with similar molar concentrations of tributyl phosphate (TBP), di-(2-ethylhexyl) phosphoric acid (HDEHP), and N-Methyl-N, N, N-tri-octyl-ammonium chloride (Aliquat-336), with mineral acid as eluent were evaluated. Retention factors were determined, and separation efficiency was assessed based on the resolution data of the REEs acquired under the same elution conditions for each column. HDEHP demonstrated the best separation efficiency for the entire REE series (mean Rs = 2.76), followed by TBP (mean Rs = 1.52), while Aliquat-336 exhibited the lowest performance (mean Rs = 1.42). The HDEHP-coated column was then used to optimize the extraction chromatographic separation of the REEs. The primary challenge was to completely elute the heavy REEs (Tb – Lu) while maintaining adequate separation of the light REEs (La – Gd) within a reasonably short time. The stepwise gradient elution procedure improved the resolution between adjacent REEs, allowing the complete separation of the entire REE series within 25 minutes. Better separation efficiency for light REEs was achieved at higher column temperatures and a mobile phase flow rate of 1.5 mL/min in the tested domain of 20–60 °C, and 0.5–2.0 mL/min, respectively, resulting in plate heights (H) ranging from 0.011 to 0.027 mm.

Analytical QbD for the optimization of a multimode HPLC method for the investigation of hydrochlorothiazide, diltiazem and propranolol release from 3D printed formulation

Since 3D printing technology is an emerging field in pharmaceutical technology, the present study aimed at the development of a mixed-mode liquid chromatographic method for the separation and determination of hydrochlorothiazide, diltiazem, and propranolol to investigate their in-vitro release performance from 3D printed tablets. Due to the unique properties of the mixed-mode stationary phase, the three drugs were separated in less than 8 min under isocratic elution. Method development was accomplished following the Analytical Quality by Design principles and was evaluated using risk assessment and multivariate analysis. The influences of critical method parameters on critical method attributes (were screened using a 2-level fractional factorial design and subsequently optimized through a central composite design. The method operable design region was approved by the establishment of a robust zone using Monte Carlo simulation and capability analysis. The validation of the HPLC method was performed based on the total error concept. The relative bias was varied between ─ 11.6 % and 10.5 % and the RSD values for repeatability and intermediate precision were below 4.4 % in all cases. The limits of detection (LOD) ranged between 0.17 – 0.90 μg/mL and were adequate for the specific application. The developed method was successfully applied to the analysis of the studied drugs in in-vitro drug release samples obtained from 3D-printed tablets combining the above-mentioned active pharmaceutical ingredients (APIs).

Development and Validation of Ultra-Performance Liquid Chromatography (UPLC) Method for Simultaneous Quantification of Hydrochlorothiazide, Amlodipine Besylate, and Valsartan in Marketed Fixed-Dose Combination Tablet

Fixed-dose combination therapy is considered a practical approach in the treatment of various diseases, as it can simultaneously target different mechanisms of action that achieve the required therapeutic efficacy through a synergistic effect. A combination of hydrochlorothiazide (HTZ), amlodipine (AMD), and valsartan (VLS) has been created for the treatment of hypertension. Therefore, the aim of this study was to develop an optimized UPLC method for the simultaneous quantification of this combination. A DoE at a level of 32 was used to investigate the effects of column temperature (20, 30, and 40 °C) and formic acid concentration (0.05, 0.15, and 0.25%) on the retention time of each active pharmaceutical ingredient (API), the peak area, and the peak symmetry, as well as the resolution between HTZ-AMD and AMD-VLS peaks. The optimized analytical method was validated and used to extract the three APIs from the marketed product. The optimized analytical condition with a column temperature of 27.86 °C and a formic acid concentration of 0.172% showed good separation of the three APIs in 1.62 ± 0.006, 3.59 ± 0.002, and 3.94 ± 0.002 min for HTZ, AMD, and VST, respectively. The developed method was linear with the LOQ for a HTC, AMD, and VST of 0.028, 0.038, and 0.101 ppm, respectively. Moreover, the developed assay was sustainable and robust, with an RSD % of less than 2%. The application of this method in the extraction of HTZ, AMD, and VST from the Exforge® marketed product showed good separation with a measurable drug content of 23.5 ± 0.7, 9.68 ± 0.1, and 165.2 ± 5.2 mg compared to the label claims of 25/10/160 for HTZ, AMD, and VST, respectively.

Development of a high-performance liquid chromatography (HPLC) method for analysis of charantin in bitter gourd (Momordica charantia) fruits

Development of a high-performance liquid chromatography (HPLC) method for analysis of charantin in bitter gourd (Momordica charantia) fruits

Development of a liquid chromatographic method with a different selectivity for the quantification of eighteen phytocannabinoids in hemp

To quantify phytocannabinoids in hemp, liquid chromatography diode array detector (LC-DAD) methods are favored, but their selectivity depends on baseline separation of all phytocannabinoids and unknown compounds in an extract. Therefore, development of a LC-DAD method with a different selectivity has become highly desirable. Currently, most LC-DAD methods use the water/acetonitrile eluting system, while this study aimed to use the water/methanol eluting system. A systematic investigation of various chromatographic parameters on LC separation of eighteen phytocannabinoids, the maximum number that has been quantified in hemp so far, plus two potential internal standards, led to a four-step isocratic mobile phase that was able to baseline separate the twenty compounds with a significantly different eluting order from published methods. Although changes in the mobile phase composition caused baseline drifts, consequent difficulty in quantification was avoided through detection at wavelengths longer than 230 nm. Subsequently, the method was validated according to the ISO 17025 guidelines, calibrated between 0.04 and 50 µg/mL, and used to analyze phytocannabinoids in nine strains of hemp flowers that were extracted using methanol between 0.04 and 50 % (w/w). Extraction recovery was tracked in real-time by spiking one of the two potential internal standards, i.e., abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp. Method selectivity was further assessed by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS), indicating minimum interferences. In addition, five untargeted/unknown phytocannabinoids were identified by ESI/TOFMS, including two structural isomers of Δ9-tetrahydrocannabinol (Δ9-THC), two structural isomers of Δ9-tetrahydrocannabinolic acid (Δ9-THCA), and one structural isomer of Δ9-THC acetate.

Evaluating the effectiveness of different household washing techniques for removal of insecticides from spinach and chickpea leaves by micellar liquid chromatography

In the past few decades, the employment of green analytical approaches in chromatographic method development has attracted the analytical separation community. The greenness of the developed method depends upon the toxicity of solvents and the amount of generated post-analysis waste generated. In this concern, micellar liquid chromatography (MLC) is a simple and rapid technique that generates very low toxic waste compared to traditional chromatographic pesticide detection methods. Here, MLC method has been validated and applied for the determination of monocrotofos (MCF), imidacloprid (ICP), dimethoate (DM) and profenofos (PFF) in spinach and chickpea leaves. The optimized mobile phase was 0.065 M SDS-2 % 1-propanol, 0.01 M NaH2PO4 buffered to pH 7. A C18 column was used for separation with a flow rate of 1 mL/min. The developed method has been validated following the guidelines of SANTE/11,312/2021 and ICH guidelines for; limit of quantification (0.05–0.20 mg/kg), linearity (r2> 0.997–0.999), precision (<6.3 %), accuracy (96.3 %–99.8 %) and robustness (<6) in real samples. ICP and MCF, apart from DM and PFF, were detected in the present work. After detecting insecticides in spinach and chickpea leaves both were washed with different household chemicals i.e. normal, lukewarm, common salt, lemon juice water and commercial ozonizer. Based on five washing techniques with insecticide concentration time intervals reduction rates were calculated for each washing treatment. The results show that lemon juice, common salt water, and ozonizer can be used as washing techniques for the reduction of superficial and systematic residues of ICP and MCF. Common salt and lemon juice water were better for washing over vinegar and potassium permanganate (KMnO4) as they enhance the colour of the green leafy vegetables and are available in every Indian kitchen. They can be easily used by lower socioeconomic classes who cannot afford KMnO4 and vinegar.

Development and Validation of High-Performance Thin Layer Chromatographic Method for Estimation of Acyclovir

Development and Validation of High-Performance Thin Layer Chromatographic Method for Estimation of Acyclovir