Ultra-performance Liquid Chromatography Method Research Articles

Method validation for the detection of formaldehydein fish using ultra performance liquid chromatography

Formaldehyde, the simplest aldehyde, tops the list of illegal and harmful food contaminants. Recently, formaldehyde has been used indiscriminately to preserve the freshness and quality of fish. Numerous incidents of formalin (a solution of formaldehyde) misuse during the storage and transportation of fish have been reported in several parts of India. In this context, it is pertinent to study formalin contamination in fish and shellfish. Protocol for detection of free and bound formaldehyde by ultra-performance liquid chromatography (UPLC) was attempted in the present study. Formaldehyde peak was observed in the expected retentiontime and standardisation of the protocol for formaldehyde determination was then carried out. UPLC method was developed and validated for specificity, repeatability, accuracy, and linearity. The average correlation coefficient (R2) was 0.99. The recovery rates of free and bound formaldehyde were 90.83 to 116.43% and 88.76 to 132.98%, respectively. The limit of detection (LOD) was found to be 0.5 ppm. The study investigated free and bound formaldehyde levels in commonly traded fish species across five major fish markets inTamil Nadu, India. Sphyraena barracuda had the highest recorded concentrations of free and bound formaldehyde at 9.8 and 6.45 mg kg-1, respectively, while Lethrinus lentjan exhibited the lowest concentrations at 0.8 mg kg-1 for free formaldehyde and 0.5 mg kg-1 for bound formaldehyde. Keywords: Formalin abuse, Limit of detection, Method validation, Retention time UPLC

Characterization, quantitation, and natural variability of phenolics in Rhododendron arboreum leaves of western Himalayan origin using UPLC-PDA and UHPLC-ESI-Q/TOF-MS/MS

Rhododendron arboreum Sm. is very popular in the Indian Himalayan region due to its significant ethnomedicinal importance and potential commercial applications. Apart from the ornamental value derived from its vibrant red flowers, fresh flowers are used to prepare medicinal juices, and dried are utilized as spices. Despite being a rich source of phenolics, these lack identified marker chemicals and validated methods. The present work encompasses the chemical investigation of R. arboreum leaves using UHPLC-ESI-Q/TOF-MS/MS. The chemotypic variability was assessed through a reverse phase ultra-performance liquid chromatography method coupled with a photodiode array detector (RP-UPLC-PDA) chemical signatures of R. arboreum leaves from different altitudes combined with chemometric analysis. The chromatographic method was developed and validated for the quantitative analysis of three vital secondary metabolites, i.e., Isoquercetin or Quercetin-3-O-glucoside (Qg), Reynoutrin or Quercetin-3-O-xyloside (Qx), and Quercitrin or Quercetin-3-O-rhamnoside (Qr)The hyphenation of the method with ESI-Q/TOF-MS/MS has confirmed the distribution of 11 chemicals (nine known and two unknown) in R. arboreum leaves belonging to the phenolic and hydrolyzable tannins class. Samples from higher altitudes >3000 m (above mean sea level) were exceptionally rich in Quercitrin. The chemometric investigation, comprising hierarchical cluster analysis (HCA), principal component analysis (PCA), and partial least square method-discriminant analysis (PLS-DA), successfully categorized thirty-three samples from diverse geographic regions into two distinct categories. This current study provides a validated method for the chemotypic discrimination and quality assessment of the R. arboreum leaves from Uttarakhand, Western Himalayan region of India, and laid the foundation for the comprehensive utilization of these renewable and sustainable resources.

A simple ultraperformance liquid chromatography mass spectrometry method for the determination of cortisol level in sea bass plasma

A simple, precise, and rapid ultra-performance liquid chromatography method for cortisol quantification in sea bass plasma, based on LC-ESI-UHR-QqTOF-MS, was optimised using tolperisone as internal standard. Sea bass plasma samples containing cortisol were spiked with tolperisone as internal standard and extracted with acetonitrile. Samples were vortexed and centrifuged at 15,000g for 10 min at 4 °C. The final extract, diluted in ammonium formate (1:1), was injected in the LC-ESI-UHR-QqTOF-MS system for analysis. Mass spectrometry acquisition parameters were set using positive ionisation mode over a m/z range from 150 to 2200. Cortisol and tolperisone were detected and quantified at m/z 363.21 and 246.18, respectively, in an Auto MS (MS/MS) mode. The detection and quantification limits of cortisol in sea bass plasma were 0.01 and 0.02 μg/mL, respectively. The mean extraction recovery of cortisol was of 99.1 ± 4.0 %. The within- and between-day precision presents a relative standard deviation (RSD) below 2.9 % and 5.3, respectively. Furthermore, the effect of four different diets (fish feed 1–4) in the basal and stress-induced plasma cortisol levels of sea bass was also assessed.

Three-dimensional spectrochromatographic determination of chlorogenic acid in Melampyrum stenophyllum Boiss. extracts by parallel factor analysis.

Co-elution is a common challenge in phytochemical chromatography. Full chromatographic separation often requires extensive optimization, long analysis times, and excessive solvent use. A viable alternative could be mathematical elution of analytes using three-dimensional decomposition. This study aimed to develop a method to determine chlorogenic acid in Melampyrum stenophyllum Boiss. extracts without complete chromatographic separation, to validate the method, and to cross-validate assay results against a classical ultra-performance liquid chromatography (UPLC) method. Ultra-performance liquid chromatography-photodiode array (UPLC-PDA) spectrochromatograms were arranged into a three-way data cube with dimensions of time, wavelength, and sample and then decomposed using parallel factor analysis to reveal chromatographic, spectral, and concentration profiles. The chromatographic and spectral profiles were used to identify chlorogenic acid in overlapping signals. The relative concentration profile was used to quantify it in the plant extract. The assay results were statistically compared with those from an in-house classical UPLC method. Chlorogenic acid was co-eluted at 1.45 min and quantified as 16.11 mg per gram dry weight of Melampyrum stenophyllum extracts (SD = 0.28), despite significant interference in a 4-min runtime. The analytical validity was confirmed by recovery calculations from standard solutions and standard addition samples (RSD < 2%), and the t-test resulted in a p-value of 0.09 (α = 0.05), indicating no significant difference between the results obtained from mathematical elution and chromatographic separation. Chlorogenic acid was quantified from plant material accurately despite the co-elution. Validation and cross-validation results support the method's applicability.

Green micellar UPLC and complementary eco-friendly spectroscopic techniques for simultaneous analysis of anti-COVID drugs: a comprehensive evaluation of greenness, blueness, and whiteness

The development of sustainable analytical methodologies that minimize hazards, waste generation, and energy consumption has become crucial. This study introduces pioneering green‒blue-white approaches for the simultaneous quantification of montelukast sodium (MLK) and fexofenadine hydrochloride (FEX) in combination formulations. The first approach employs an ultra-performance liquid chromatographic method (UPLC) with a green micellar mobile phase of 0.02 M sodium dodecyl sulfate and 10% 1-pentanol (65:35%). The method demonstrated excellent resolution, peak symmetry, and a short analysis time, with retention times of 3.53 min for MLK and 1.67 min for FEX. The MLK and FEX linearities were 1–260 and 1.2–312 μg/mL, respectively. The second approach involves complementary built-in spectroscopic techniques (second derivative, third derivative, and ratio difference methods) using water as a solvent, providing a green, simple, low-cost alternative in laboratories where expensive chromatographic devices may not be readily available. The MLK and FEX linearities were 3–50 and 3–60 μg/mL, respectively. All methods were comprehensively validated and showed satisfactory results. The proposed methods demonstrated excellent linearity (r2 ≥ 0.9990), accuracy (recovery 98.5–101.5%), and precision (RSD ≤ 2%) across wide concentration ranges. A multifaceted evaluation was conducted to assess the environmental sustainability, real-world applicability, and economic viability of the proposed methods in comparison with previously reported techniques. This comprehensive assessment leveraged several state-of-the-art tools, including NEMI, ComplexGAPI, AGREE, ESA, BAGI, and RGB12. The suggested approaches exhibited favorable quadrant profiles in the NEMI and ComplexGAPI assessments, coupled with higher AGREE scores (0.90, 0.86) than reported (0.62, 0.74, 0.75, 0.69, 0.74, 0.74, and 0.75), in addition to higher ESA score (88, 92) than reported (75, 84, 85, 79, 82, 82, and 83), collectively affirming their environmentally friendly credentials. Moreover, we embraced the innovative notions of 'blueness' and 'whiteness' assessment by harnessing the recently formulated BAGI and RGB12 algorithms. The higher BAGI score (90, 82.5) than reported (72.5, 70, 70, 67.5, 67.5, 67.5, and 72.5), confirmed the excellent real-world applicability of the proposed methods, while the notable RGB12 indices (89.8, 88.1) than reported (67.8, 72.8, 71.5, 67.1, 73.7, 70.3, and 73.2), validated their cost-effectiveness and overall sustainability, contributing to an eco-friendly future for quality control processes.

Five Significant Phenols from Phyllostachys glauca McClure Leaves Extracted Using Ultrasound-Assisted Deep Eutectic Solvent Extraction

Phyllostachys glauca McClure leaves (PML), as economical natural product material, contain abundant phenols, particularly flavonoids, with significant biological activities that contribute to their widespread applications in food, pharmaceutical, and cosmetic industries. To study the significant phenols in PML, an ultrasound-assisted deep eutectic solvent extraction method with high efficiency and low toxicity was established for extracting the phenols from PML and the bamboo leaves of 17 other bamboo species. Using the Ultra Performance Liquid Chromatography (UPLC) method, the content of phenols in the leaves of the 18 bamboo species was determined. PML were found to contain neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoorientin, and orientin at contents of 0.793 ± 00.17 mg/g, 0.491 ± 0.0081 mg/g, 0.485 ± 0.0043 mg/g, 0.465 ± 0.0021 mg/g, and 0.044 ± 0.0005 mg/g, respectively, with a total content of 2.278 mg/g. These contents were significantly higher than those found in the leaves of 17 other bamboo species. Additionally, the significant phenols, neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were found simultaneously in the leaves of 15 bamboo species, especially in PML. Therefore, PML can be viewed as the natural product material with considerable application values, owing to its abundant phenols, and can exhibit its importance in utilizing neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid as resources.

A validated stability‐indicating reversed‐phase‐UPLC method for simultaneous estimation of promethazine hydrochloride, methylparaben, propylparaben and sodium benzoate assay of cough suppressant and antihistamine liquid oral dosage forms

A quick, simple, sensitive, efficient and stability-indicating reverse-phase ultraperformance liquid chromatographic method for the estimation of propylparaben, methylparaben and sodium benzoate in a pharmaceutical liquid oral formulation was developed. A Waters Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 μm i.d. column was used to perform chromatographic separation with a 0.1% perchloric acid mobile phase used as solvent A and a mixture of 0.1 % perchloric acid and methanol in the ratio 20:80 (v/v), respectively, as solvent B. The experiments were carried out at a flow rate of 0.4 ml/min and the detection wavelength was 240 nm. The compartment temperature of the column was set at 40°C and the injection volume was set at 2 μl. The main aim of the research was to develop a single UPLC assay method for promethazine (active ingredient) and preservatives in the oral solution of promethazine HCl and dextromethorphan HBr that contains promethazine (active ingredient) and methylparaben, propylparaben and sodium benzoate (preservatives). An assay of dextromethorphan HBr was developed and validated by another HPLC method. The drug and preservatives were eluted at retention times of 19.3 min for promethazine HCl, 9.3 min for methylparaben, 18.9 min for propylparaben and 8.9 min for sodium benzoate. Validation of the developed method was carried out as stated by the International Conference on Harmonization guidelines ICH Q2B and under USP<1225>. The analytical parameters verified specificity/selectivity, linearity, accuracy, ruggedness and robustness. The linearity ranges of promethazine HCL, methylparaben, propylparaben and sodium benzoate were 10-100, 10-80, 1.0-8.0 and 10-80 μg/ml, respectively, with a correlation coefficient of active ingredients and preservatives of 1.00. Percentage recoveries of promethazine, propylparaben, methylparaben, and sodium benzoate were 100.0-100.2, 99.0-100.3, 99.5-98.0 and 99.0-100.0%. The validated analytical method proves that the method is specific, precise, linear, accurate, sensitive, rugged and stable, indicating the quantification of the active ingredient and all preservatives in liquid oral formulations.

Analytical quality by design (AQbD) based optimization of RP-UPLC method for determination of nivolumab and relatlimab in bulk and pharmaceutical dosage forms

BackgroundThe Analytical Quality by Design (AQbD) methodology extends the application of Quality by Design (QbD) principles to the management of the analytical procedure life cycle, encompassing method creation, optimization, validation, and continuous improvement. AQbD assists in creating analytical procedures that are robust, reliable, precise, and cost-efficient. Opdualag™ is a combination of Nivolumab and Relatlimab, which are antibodies that block programmed death receptor-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) receptors, used to treat advanced melanoma. This work aims to develop and validate a reversed-phase ultra-performance liquid chromatography (RP-UPLC) method using AQbD principles to determine NLB and RTB in pharmaceutical products.ResultsA central composite design (CCD) comprising three factors arranged in five distinct levels was implemented via Design-expert® software to optimize the chromatographic conditions. A mathematical model was constructed and the effects of three independent factors namely flow rate (X1), percentage of methanol in the mobile phase (X2), and temperature (X3) on responses including retention time (Y1–2), resolution factor (Y3), theoretical plates (Y4–5), and tailing factor (Y6–7) were investigated. The software determined the optimal chromatographic conditions for the separation of NLB and RTB, which were as follows: 32.80% methanol in the mobile phase, 0.272 mL/min flow rate, 29.42 °C column temperature, and 260 nm UV detection. The retention time for NLB and RTB were 1.46 and 1.88 min, respectively. The method exhibited linearity across the concentration ranges of 4–24 µg/mL for RTB and 12–72 µg/mL for NLB. The limits of detection (LOD) and limit of quantification (LOQ) for NLB and RTB, respectively, were 0.89 µg/mL, 2.69 µg/mL and 0.15 µg/mL and 0.46 µg/mL. The percentage relative standard deviation (%RSD) of intraday and interday precision for NLB and RTB was below 2. The recovery percentages for NLB and RTB were determined to be 99.57–100.43% and 99.59–100.61%, respectively. Both drugs were found to be susceptible to oxidative and photolytic degradation in forced degradation studies.ConclusionsEmploying the AQbD-based methodology, a straightforward, fast, accurate, precise, specific, and stability-indicating RP-UPLC method has been established for the quantitative analysis of NLB and its RTB in pharmaceutical formulations.

A robust ultra performance liquid chromatography method for dacarbazine quantification in nanosponge formulation: A quality by design approach

A new, user-friendly Ultra Performance Liquid Chromatography (UPLC) method was developed using Analytical Quality by Design (QbD) for precise analysis of Dacarbazine in both bulk powder and Dacarbazine-loaded Nanosponges. This method follows ICH guidelines and uses a statistically designed approach to optimize the separation conditions for Dacarbazine. A central composite design was implemented to identify the ideal combination of organic solvent (mobile phase composition) and flow rate. The UPLC method was successfully optimized for rapid separation (1.3 minutes) for Dacarbazine using a mobile phase consisting of 70% Methanol: 30% Orthophosphoric acid (0.1%) at a flow rate of 1.0 mL/min, using a Hypersil C18 column. The method exhibited excellent linearity with a correlation coefficient value of 0.997 across a concentration range of 4-20 μg/mL, accuracy (drug recovery of 96-100%) and sensitivity (LOD 1.15 μg/mL, LOQ 3.50 μg/mL) and it successfully quantified the drug in the formulated Dacarbazine-loaded Nanosponges. This QbD-driven UPLC method offers a valuable tool for reliable Dacarbazine quality control in pharmaceuticals providing rapid analysis, high accuracy, and low detection limits.

In vivo determination of the bioavailability of folic acid through the utilization of the PBPK model in conjunction with UPLC

This paper employed a physiologically based pharmacokinetic model (PBPK) to investigate the transformations of folic acid and its metabolites in vivo. Additionally, an ultra-performance liquid chromatography (UPLC) method was developed to accurately measure the body's retention rate and conversion rate of folic acid, tetrahydrofolate, and 5-methyltetrahydrofolate. Furthermore, the bioavailability of folic acid in the body was assessed by combining this method with an evaluation technique for animal models. The study found that the gastric metabolism time was 2 h, while the small intestinal metabolism duration was 4 h. The maximum conversion rate was observed in plasma and liver after 6 h, and in the brain after 8 h. This serves as a framework for creating a model to assess the bioavailability of folic acid in living organisms, to enhance the safety and efficacy of folic acid intake.

A UPLC Method for Simultaneous Estimation of Trastuzumab and Hyaluronidase in Bulk and Pharmaceutical Dosage Form

Objective: The proposed research aimed at establishing a quick, accurate, and exact ultra-performance liquid chromatography (UPLC) methodology for contemporaneously analyzing trastuzumab and hyaluronidas in bulk and pharmaceutical formulation, with a focus on the stability-indicating properties of the assay. Method: Using a Water X-Bridge C18 column (50 mm x 4.6 mm x 2.5 μ) and a mobile phase of ACN: Buffer (pH 2.5) [50% v/v], pushed at 0.5 mL/min, compounds were separated chromatographically. An ultraviolet (UV) detector fixed at 260 nm was employed to identify the isolated compounds. Results: The findings showed that 1.3529 and 2.404 minutes were optimal for separating Trastuzumab and Hyaluronidase, correspondingly. The current procedure has been verified in accordance with ICH standards Q2 R1, and stability-indicating tests have been performed in accordance with ICH standards Q1A R2. Both the intra- and inter-day precisions were determined to be satisfactory. The suggested approach showed linearity between 30 to 180 μg/mL of trastuzumab and between 12.50-75μg/ mL of hyaluronidase. It was determined that the limit of detection (LoD) and limit of quantitation (LoQ) for trastuzumab were 0.36 and 1.2 μg/mL, correspondingly, whereas those for hyaluronidase were 0.15 and 0.5 μg/mL. The approach was shown to have a recovery rate around 98.6 to 99.7%. Conclusion: The suggested approach successfully separated the chemical compounds from their by-products. Therefore, trastuzumab and hyaluronidase routine evaluation and stability-indicating tests were both effectively implemented using the approach.

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.

In vitro evaluation of the percutaneous absorption of progesterone in anhydrous permeation-enhancing base using the Franz skin finite dose model and mass spectrometry

Progesterone is used for hormone replacement therapy through various routes of administration. This study was conducted to (a) evaluate the stability of progesterone in a proprietary anhydrous permeation-enhancing base (APEB) and the efficiency of its skin permeation, and (b) determine the appropriateness of mass spectrometry as a method of analysis for permeated progesterone. Using a proven stability-indicating ultra-performance liquid chromatographic method, the compounded hormone (100 mg progesterone/g APEB gel) was determined to be physically and chemically stable at room temperature for six months. Skin permeation analysis using the Franz skin finite dose model and mass spectrometry imaging showed an optical density of 1699 for the permeated progesterone compounded in APEB and 550 for the permeated progesterone in a water containing VBC, which is a statistically significant different (P = 0.029). The study suggests that APEB can be used as a compounding base for effective skin permeation of progesterone, and mass spectrometry is a reliable method for visualization and quantitative analysis of permeated progesterone.

Quality assessment of oral antimalarial and antiretroviral medicines used by public health systems in Sahel countries

Malaria and Human Immunodeficiency Virus infections are among the top 10 causes of death in low income countries. Furthermore, many medicines used in these treatment areas are substandard, which contributes to the high death rate. Using a monitoring system to identify substandard and falsified medicines, the study aims to evaluate the quality of antimalarial and antiretroviral medicines in Sahel countries, assessing site conditions, compliance of medicines with pharmacopoeia tests, formulation equivalence with a reference medicine, and the influence of climate on quality attributes. Ultra Performance Liquid Chromatography methods for eight active pharmaceutical ingredients were validated following the International Conference for Harmonization guideline for its detection and quantification. Quality control consists of visual inspections to detect any misinformation or imperfections and pharmacopeial testing to determine the quality of pharmaceutical products. Medicines which complied with uniformity dosage units and dissolution tests were stored under accelerated conditions for 6 months. Artemether/Lumefantrine and Lopinavir/Ritonavir formulations failed uniformity dosage units and disintegration tests respectively, detecting a total of 28.6% substandard medicines. After 6 months stored under accelerated conditions (40 °C // 75% relative humidity) simulating climatic conditions in Sahel countries, some medicines failed pharmacopeia tests. It demonstrated the influence of these two factors in their quality attributes. This study emphasizes the need of certified quality control laboratories as well as the need for regulatory systems to maintain standards in pharmaceutical manufacturing and distribution in these countries, especially when medicines are transported to rural areas where these climatic conditions are harsher.

Development of a novel UPLC approach to co-prediction of four active compounds in a multi-component pharmaceutical preparation

In this work, a new ultra-performance liquid chromatography method based on photodiode array detection (UPLC-PDA) was first developed for the quantitative analysis of the quaternary mixture of ascorbic acid (AA), paracetamol (PAR), caffeine (CAF) and chlorpheniramine maleate (CPA) in a commercial dosage form. The developed UPLC-PDA method offered a new possibility for the co-determination of four active ingredients in a drug combination with short run time and simple sample preparation. The successful chromatographic separation of the four drugs was performed using a Waters Acquity UPLC BEH C18 column (1.7 µm 2.1 × 100 mm) (Mildford, USA) and a mobile phase consisting of water (12 %), acetonitrile (13 %) and 0.1 M H3PO4 (75 %) at a flow rate of 0.25 mL/min. The validation of the proposed UPLC-PDA approach was verified by analyzing synthetic mixtures, inter- and intra-day experiments, and commercial powder samples and provided satisfactory results.

Stability-Indicating UPLC-PDA-QDa Methodology for Carvedilol and Felodipine in Fixed-Dose Combinations Using AQbD Principles

The development of analytical procedures, in line with the recent regulatory requirements ICH Q2 (R2) and ICH Q14, is progressing, and it must be able to manage the entire life cycle of the methodology. This is also applicable to and especially challenging for combinations of drug substances and dosage form. A reliable and efficient, stability-indicating, MS-compatible, reverse-phase ultra-performance liquid chromatographic (UPLC®) method was developed for the determination of carvedilol and felodipine in a combination oral dosage form. The development of the method, performed using analytical quality by design (AQbD) principles, was in line with the future regulatory requirements. Furthermore, the fixed-dose combination dosage forms are a clear solution to the polypharmacy phenomenon in the elderly population. The main factors evaluated were the mobile phase buffer, organic modifier, column, flow, and column temperature. The optimum conditions were achieved with a Waters Acquity HSS T3 (100 × 2.1 mm i.d., 1.8 µm) column at 38 °C, using ammonium acetate buffer (5 mM, pH 4.5) (Solution A) and MeOH (Solution B) as mobile phases in gradient elution (t = 0 min, 10% B; t = 1.5 min, 10% B; t = 12.0 min, 90% B; t = 13.0 min, 10% B; t = 15.5 min, 10% B) at a flow rate of 0.2 mL/min and UV Detection of 240 and 362 nm for carvedilol (CAV) and felodipine (FLP), respectively. The linearity was demonstrated over concentration ranges of 30–650 µg/mL (R2 = 0.9984) (CAV) and 32–260 µg/mL (R2 = 0.9996) (FLP). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability, and fixed-dose combination dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with improved knowledge, leading to high-quality data, lower operation costs, and minimum regulatory risk. Furthermore, this work paves the way for providing a platform of robust analytical methods for the simultaneous quantification of innovative on-demand new dose combinations.

Evaluation of chemical components and quality in Xinhui Chenpi (Citrus reticulata 'Chachi') with two different storage times by GC-MS and UPLC.

Xinhui Chenpi (XHCP) is a well-known type of Chenpi (CP) widely used as both a Chinese herb and a food ingredient. While previous studies have explored how the quality of CP changes over time, there has been limited research specifically on XHCP. This study aims to assess the chemical components and quality of XHCP based on total flavonoid content (TF), antioxidant activity (AA), and color value (CV) at two stages: freshly harvested (XHCP-0Y) and after 3 years of storage (XHCP-3Y). Thirty-eight common volatile compounds were identified, and the content of 17 compounds among them, nine nonvolatile compounds, which included one alkaloid (synephrine), three phenolic acids (PA, protocatechuic acid, vanillic acid, and ferulic acid), and five flavonoids (narirutin, hesperidin, sinensetin, nobiletin, and tangeretin), were firstly detected by the newly developed gas chromatograph-mass spectrometer (GC-MS) and ultra-performance liquid chromatography (UPLC) methods. Compared to XHCP-0Y, the content of 17 volatile compounds and synephrine decreased in XHCP-3Y to varying degrees, while the content of PA, five flavonoids, TF, AA, and CV increased. The reduction of dryness caused by volatile compounds and the enhancement of efficacy related to PA, flavonoids, and AA suggested improved quality of XHCP after 3 years of storage. The methods developed in this study show promise for evaluating the quality of XHCP during the aging process.

Assessing the quality ecology of endemic tree species in China based on machine learning models and UPLC methods: The example of Eucommiaulmoides Oliv.

Climate change significantly affects the suitability distributions of natural populations, especially relict species such as Eucommia ulmoides Oliv. (E. ulmoides). However, the driving eco-factors of the distribution of E. ulmoides and the relationship between geo-distribution patterns and quality are still unclear. Here, a new approach was established by combining machine learning models (MaxEnt, Random Forest, and Biomod2 model), ultra-performance liquid chromatography (UPLC) and spatial analysis to address this question. The results reveal that temperature is the most critical variable influencing the distribution of E. ulmoides including Bio1, Bio10, and Bio11, followed by precipitation. Multi-model predictions of suitable habitats for E. ulmoides cover an area ranging from 113.33 × 104 km2 to 285.98 × 104 km2, mainly distributed in the mountainous areas of southwestern China, including the eastern regions of the Dalou, Wuling, and Qinling-Daba Mountains. Under different future scenarios, suitable habitat shifted northward by 184.90–424.88 km, and the contraction area is greater than the expansion area, with the difference ranging from 49.28 × 104 km2 to 98.39 × 104 km2. Eight common components (e.g., chlorogenic acid and pinoresinol doglucoside) and two endemic components (caffeic acid and geniposidic acid) were found in the leaves of the northern and southern medicinal districts (SDM and NMD). The contents of eight key secondary metabolites of E. ulmoides leaves were better distributed in SDM than in NDM. Among these, Xinyang in Henan, Lu'an and Fuyang in Anhui, and the southeastern region of Zhejiang are suitable growing areas for the high-quality cultivation of E. ulmoides, which deserve special attention. The seasonal precipitation (Bio15) was negatively correlated with chlorogenic acid and pinoresinol diglucoside while longitude significantly positively correlated with pinoresinol diglucoside. Overall, this study provides a novel assessment pattern for quality assessment and effective protection of E. ulmoides.

Feasibility of excitation-emission fluorescence spectroscopy in tandem with chemometrics for quantitation of trans-resveratrol in vine-shoot ethanolic extracts.

trans-Resveratrol (TR) is a well-known phytochemical compound with important biological properties. It can be recovered from agri-food by-products or wastes, such as vine shoots. Once recovered, its concentration should be measured, possibly in a green, non-destructive, and efficient manner. With these premises, this work aimed to explore the feasibility of excitation-emission fluorescence spectroscopy combined with chemometrics for the analysis of TR in raw extracts obtained from vine shoots. A total of 75 extracts were produced and analyzed by ultra-performance liquid chromatography method with diode array detection (UPLC-DAD) and spectrofluorimetry. Then, the feasibility of two calibration strategies for TR quantitation was assessed - a parallel factor analysis (PARAFAC)-based calibration and the N-way partial least squares (NPLS) regression. The extracts showed variable TR content, the excitation/emission maxima of which were at around 305/390 nm, respectively. The best PARAFAC-based calibration allowed a root mean square error of prediction (RMSEP) of 22.57 mg L-1 , and a relative prediction deviation (RPD) of 2.91 to be obtained but a large number of PARAFAC components should be considered to improve the predictions. The results of the NPLS regression were slightly better, with a RMSEP of 19.47 mg L-1 , and an RPD of 3.33 in the best case. Fluorescence could be an alternative analytical technique to measure TR in complex samples. Chemometric tools allowed the identification of the TR signal in the fluorescence landscapes, which could be further used for its non-destructive quantitation. The need for a more accurate criterion for optimal PARAFAC complexity emerged. © 2024 Society of Chemical Industry.

Eco-friendly micellar ultra-performance liquid chromatography (UPLC) method for simultaneous determination of non-communicable diseases drugs in bulk powder, spiked human plasma and in individual dosage forms

Eco-friendly micellar ultra-performance liquid chromatography (UPLC) method for simultaneous determination of non-communicable diseases drugs in bulk powder, spiked human plasma and in individual dosage forms