Development Of Liquid Chromatographic Method Research Articles

Development and validation of high-performance liquid chromatography method for the simultaneous quantification of rivastigmine hydrogen tartrate and asiaticoside co-loaded in niosomes: A Box–Behnken design approach

Rivastigmine hydrogen tartrate (RHT), a reversible cholinesterase inhibitor, is considered as the first-line therapy for mild to moderate Alzheimer’s disease. Asiaticoside (AS), a pentacyclic triterpenoid saponin, is well known as cognitive enhancer due to its antioxidant effect. Based on the hypothesis of their synergistic therapeutic potential, RHT and AS were co-encapsulated in niosomal formulation. A simple, precise, and accurate high-performance liquid chromatography method was developed for simultaneous quantitative analysis. The chromatographic parameters were optimized by Box-Behnken experimental design. The separation was performed on a reversed-phase Phenomenex C18 (150 mm × 4.6 mm, 5 μm) column at 30 °C under the UV detection of 210 nm. The optimized mobile phase consisted of a mixture of 20 mM potassium dihydrogen phosphate buffer (pH 2.6) and acetonitrile (72:28 % v/v) under the isocratic mode at the flow rate of 0.9 mL/min. The developed method was fully validated under the ICH guidelines and could be successfully applied for simultaneous quantitative analysis of RHT and AS in niosomal formulation.

Quality by design‐engineered reversed‐phase high‐performance liquid chromatography method development and validation for simultaneous estimation of neomycin sulfate and beclomethasone dipropionate in bulk and pharmaceutical dosage form

AbstractThe aim of this study was to develop and validate a robust reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method for the simultaneous estimation of neomycin sulfate (NEO) and beclomethasone dipropionate (BECLO) in both bulk drug and pharmaceutical dosage forms. The analysis was conducted using the Box‐Behnken design. The separation of NEO and BECLO was conducted on a Phenomenex Luna C‐18 column (4.6 × 150 mm, 5 µm), employing a mobile phase comprising a mixture of methanol and trifluoroacetic acid in a ratio of 88:12% v/v. The separation was performed at a flow rate of 0.6 mL/min. NEO and BECLO were analyzed at a wavelength of 240 nm employing a photodiode array detector. The validation of the methodology followed the guidelines outlined in the International Council for Harmonization Q2 R (1). The validation process involved assessing critical parameters such as linearity, accuracy, system suitability, precision, and robustness. The results for each parameter were found to be within the acceptable range. The results indicate that the established RP‐HPLC method can effectively be employed for the routine analysis of NEO and BECLO in bulk drug and pharmaceutical dosage forms.

Enhancing LC×LC separations through multi-task Bayesian optimization

Method development in comprehensive two-dimensional liquid chromatography (LC×LC) is a challenging process. The interdependencies between the two dimensions and the possibility of incorporating complex gradient profiles, such as multi-segmented gradients or shifting gradients, make trial-and-error method development time-consuming and highly dependent on user experience. Retention modeling and Bayesian optimization (BO) have been proposed as solutions to mitigate these issues. However, both approaches have their strengths and weaknesses. On the one hand, retention modeling, which approximates true retention behavior, depends on effective peak tracking and accurate retention time and width predictions, which are increasingly challenging for complex samples and advanced gradient assemblies. On the other hand, Bayesian optimization may require many experiments when dealing with many adjustable parameters, as in LC×LC. Therefore, in this work, we investigate the use of multi-task Bayesian optimization (MTBO), a method that can combine information from both retention modeling and experimental measurements. The algorithm was first tested and compared with BO using a synthetic retention modeling test case, where it was shown that MTBO finds better optima with fewer method-development iterations than conventional BO. Next, the algorithm was tested on the optimization of a method for a pesticide sample and we found that the algorithm was able to improve upon the initial scanning experiments. Multi-task Bayesian optimization is a promising technique in situations where modeling retention is challenging, and the high number of adjustable parameters and/or limited optimization budget makes traditional Bayesian optimization impractical.

A bioanalytical assay for estimation of thymoquinone in rats cerebrospinal fluid and brain tissues of nasally administrated thymoquinone loaded lipo-polymeric nanoshells and its pharmacokinetic profiling

Thymoquinone (TH) has been one of the major phytochemical used in the treatment of cancers since long time, especially in the management of glioblastoma multiforme (GBM). The formulation of lipo-polymeric nanoshells (LPNs) and their nasal delivery are fascinating approaches for overcoming the drawbacks of low solubility and poor bioavailability of TH. Hence targeting LPNs to the brain requires a validated bioanalytical method for the assessment of TH concentration in Cerebrospinal fluid (CSF) and brain tissue homogenates (BTH). Therefore, the current work focuses on the development and validation of high-performance liquid chromatography (HPLC) method in CSF by employing nasal simulated fluid (NSF) as one of the major components of the mobile phase. The developed method was checked for linearity in the range of 0.05 to 1.6 μg/mL, having an r2 value of 0.999 with mean % recovery >95% and % RSD values below <2.0%. The developed method gave a clear separation of TH at 6.021 ± 0.17 min with an internal standard at 4.102 ± 0.09 min and a CSF spike at 2.170 ± 0.12 min. The developed method assisted in determining the in-vitro and in-vivo drug release study of LPNs, pharmacokinetic profiling, qualitative in-vivo brain uptake study, in-vitro cellular uptake, and generating stability data of formulated LPNs proposed for intranasal administration in rats.

Improving the Accuracy of Predictive 2D-LC Optimization Strategies: Incorporation of Simulated Elution Profiles to Account for Injection Band Broadening in Online Comprehensive Two-Dimensional Liquid Chromatography.

Method development in online comprehensive two-dimensional liquid chromatography (LC × LC) requires the selection of a large number of experimental parameters. The complexity of this process has led to several computer-based LC × LC optimization algorithms being developed to facilitate LC × LC method development. One particularly relevant challenge for predictive optimization software is to accurately model the effect of second dimension (2D) injection band broadening under sample solvent mismatch and/or sample volume overload conditions. We report a novel methodology that combines a chromatographic numerical simulation model capable of predicting elution profiles of analytes under conditions where peak distortion occurs with a predictive multiparameter Pareto optimization approach for online LC × LC. Preliminary method optimization is performed using a theoretical model to predict 2D injection profiles, and optimal experimental configurations obtained from the Pareto fronts are then subjected to further optimization using the simulation model. This approach drastically reduces the number of simulations and therefore the computational demand. We show that the optimal experimental conditions obtained in this manner are similar to those obtained using a complete optimization using only the simulation model. Online HILIC × RP-LC separation of phenolic compounds was used to compare experimental data to simulated two- and three-dimensional contour plots. The main advantage of the proposed approach is the ability to predict the formation of split or deformed peaks in the 2D, a significant benefit in online LC × LC method optimization, especially for separation combinations with mismatched mobile phases. A further benefit is that simulated elution profiles can be used for the visualization of predicted two-dimensional chromatograms for method selection.

Development of an High Performance Liquid Chromatography Method for Simultaneous Determination of Hesperidin and Sodium Benzoate in Kien Ty Syrup

The Kien ty syrup is formulated from six medicinal materials, including Pericarpium Citri Reticulatae, Fructus Aurantii immaturus, Radix Codonopsis, Rhizoma Atractylodis macrocephalae, Fructus Crataegi, Fructus Hordei germinatus and sodium benzoate as a preservative. Hesperidin, a bioflavonoid found in Pericarpium Citri Reticulatae, Fructus Aurantii immaturus, is used to assess the syrup's quality in terms of assay. In this study, an HPLC method for simultaneous determination of hesperidin and sodium benzoate in Kien ty syrup was developed. The method was performed on an Agilent C18 column (250 × 4.6 mm; 5 μm) with the mobile phase being a mixture of 0.05 M phosphate buffer solution pH 5.5: acetonitrile (70:30, v/v), running in a isocratic elution mode. The flow rate was set at 1.0 mL/min, and the injection volume was 10 μL. The analytes were detected at 225 nm. The method was validated and met the requirements according to ICH, AOAC guidelines. Applying the developed quantification method, the analysis of three sirup lots revealed that the content of hesperidin and sodium benzoate ranged from 0.480-0.504 mg/mL and 2.869-2.306 mg/mL, respectively. The study results indicate that this quantification method can be applied for simultaneous determination of hesperidin and sodium benzoate in Kien ty syrup and similar products.&#x0D;

Development and Validation of a High-Performance Liquid Chromatography Method to Quantify Marker Compounds in Lysimachia vulgaris var. davurica and Its Effects in Diarrhea-Predominant Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS), a common gastrointestinal disorder worldwide, is characterized by chronic abdominal pain, bloating, and disordered defecation. IBS is associated with several factors, including visceral hypersensitivity, gut motility, and gut-brain interaction disorders. Because currently available pharmacological treatments cannot adequately improve symptoms and may cause adverse effects, the use of herbal therapies for managing IBS is increasing. Lysimachia vulgaris var. davurica (LV) is a medicinal plant used in traditional medicine to treat diarrhea. However, information on whether LV can effectively improve diarrhea-predominant IBS (IBS-D) remains limited. In this study, using an experimental mouse model of IBS-D, we elucidated the effects of the LV extract. The methanol extract of LV decreased fecal pellet output in the restraint stress- or 5-hydroxytryptamine (5-HT)-induced IBS mouse model and inhibited 5-HT-mediated [Ca2+]i increase in a dose-dependent manner. Furthermore, we developed and validated a high-performance liquid chromatography method using two marker compounds, namely, chlorogenic acid and rutin, for quality control analysis. Our study results suggest the feasibility of the methanol extract of LV for developing therapeutic agents to treat IBS-D by acting as a 5-HT3 receptor antagonist.

Targeting Breast Cancer: Encapsulation and In-vitro Studies of α-Amyrin from Capparis zeylanica L.

Historically, Capparis zeylanica L. has garnered attention for its potential effectiveness in treating cancer. This has inspired the researchers to delve into the plant’s properties in search of new and innovative compounds with anticancer properties. Therefore, the current study aimed to encapsulate and characterize α-amyrin phytochemical from leaf extract of C. zeylanica L. and study it for the in-vitro cancer cell line. The high-performance liquid chromatography (HPLC) method development of C. zeylanica L. extracts furnished α-amyrin, whose characterization was established by high-performance liquid chromatography (HPLC), fourier-transform infrared spectroscopy (FTIR), liquid chromatography-mass spectrometry (LC-MS). Molecular docking was performed using Auto Dock Vina software to validate the mechanism of action. The α-amyrin nanosponges was synthesized through the emulsion solvent diffusion method, employing various drug-polymer ratios ranging from 1:1 to 1:5, with pullulan as the chosen polymer. Compatibility was revealed no interaction using FTIR. The cytotoxic properties were assessed on MCF-7 cell lines using MTT assay. The production yield and entrapment efficiency of F1-F5 batches in the range of 43 to 68.36% and 70 to 82.5%, respectively. Batch F5 showed the highest production yield, and entrapment efficiency. The average particle size ranges from 241.3 to 603.2 nm. The F5 formulation had shown the highest drug release (90.40 ± 0.88%). The IC50 value for optimized formulation was 34.54 μg/mL as observed in MCF-7 breast cancer, which was considerable as compared with 5-fluorouracil. In-silico studies revealed that α-amyrin showed good binding affinity to breast cancer PDBIDs and form a stable complex. In our study, α-amyrin has been successfully extracted and further validation through pharmaceutical formulation was done and warranted to solidify its potential as an effective anticancer agent.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

High-Performance Liquid Chromatography Method Development and Validation for Estimation of Mahanimbine in Curry Leaves

High-Performance Liquid Chromatography Method Development and Validation for Estimation of Mahanimbine in Curry Leaves

Analytical quality by design approach for the development of high-performance liquid chromatography method for simultaneous analysis of metformin and sitagliptin in the presence of major degradation products.

An analytical quality by design-based high-performance liquid chromatography method for determining metformin (MET) and sitagliptin (SIT) in stress-degraded samples was developed and validated. The analytical target profile and risk assessment-driven critical method variables, for example, pH, % aqueous, and buffer concentration, were studied for their effect on method responses of retention time and resolution using a central composite design. The correlation regression coefficient was more than 0.8, and variables interaction was significant on method responses with curvature effect. The method operable design region afforded an aqueous range of 55%-70% and an ortho-phosphoric acid buffer of 0.1% with a pH of 3.0-4.0 as a robust region for the suitable method performance characteristics. The separation of MET and SIT from their degradants (m/z 85.0509; m/z 193.0694) on the C8 column was achieved using a mobile phase consisting of 0.1% ortho-phosphoric acid and methanol (60:40% v/v; pH 3.0). The optimized method eluted MET and SIT at 4.3±0.2 and 7.1±0.2min, respectively, with acceptable specificity and resolution. The linearity ranges of 25-250μg/mL (r2 : 0.9982) and 5-50μg/mL (r2 : 0.9989) was established for MET and SIT, respectively. The % recovery (98.81%-102.17%), precision (0.55%-1.65%), and robustness study for method variables were acceptable.

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids.

The urea cycle has been found to be closely associated with certain types of cancers and other diseases such as cardiovascular disease and chronic kidney disease. An analytical method for the precise quantification of urea cycle amino acids (arginine, ornithine, citrulline, and argininosuccinate) by off-line two-dimensional liquid chromatography (2D-LC) combined with fluorescence-based detection was developed. Before analysis, the amino acids were derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to obtain NBD-amino acids. The first dimension involved the reversed-phase separation, in which NBD derivatives of urea cycle amino acids were completely separated from each other and mostly separated from the 18 NBD-proteinogenic amino acids. The samples were eluted with stepwise gradient using 0.02% trifluoroacetic acid in water-acetonitrile as the mobile phase. In the second dimension, an amino column was used for the separation of NBD-ornithine, -citrulline, and -argininosuccinate, while a sulfonic acid column was used to separate NBD-arginine. The developed 2D-LC system was used to analyse human plasma samples. The fractions of NBD-urea cycle amino acids obtained in the first dimension were collected manually and introduced into the second dimension. By choosing appropriate mobile phases for the second dimension, each NBD-urea cycle amino acid eluted in the first dimension was well separated from the other proteinogenic amino acids and interference from endogenous substance. This could not be achieved in the first dimension. The urea cycle amino acids in human plasma sample were quantified, and the method was well validated. The calibration curves for each NBD-urea cycle amino acid showed good linearity from 3 (ASA) or 15 (Orn, Cit, and Arg) to 600 nM, with correlation coefficients higher than 0.9969. The intraday and interday precisions were less than 7.9% and 15%, respectively. The 2D-LC system is expected to be useful for understanding the involvement of the urea cycle in disease progression.

Ultra‐high‐performance liquid chromatography method development for the quantification of Molnupiravir and its process‐related impurities using Box‐Behnken experimental design

AbstractMolnupiravir, a promising antiviral agent, has gained significant attention for its potential in treating viral infections, particularly in the context of the coronavirus disease 2019 pandemic. This study aimed to develop and validate an ultra‐high‐performance liquid chromatography (UHPLC) method for the quantification of Molnupiravir and its associated impurities (Imp‐I, Imp‐II, Imp‐III, and Imp‐IV). The method involved a systematic investigation of critical method parameters and their optimization using Quality by Design principles. The percentage of organic modifiers, column temperature, and flow rate were systematically optimized using the Box‐Behnken design. The developed method was assessed for specificity, system suitability, accuracy, precision, linearity, and detection limits. The results demonstrate strong specificity, with the analysis remaining accurate even in the presence of impurities. The developed method exhibits excellent precision, with repeatability and intermediate precision showing relative standard deviation values ranging from 0.78% to 1.14% for impurities and 0.82% to 1.91% for Molnupiravir. Furthermore, the method displays exceptional linearity, covering a wide range of concentrations. The linear regression analysis yields high coefficients of determination (r2, 9993–0.9997), confirming the linearity. The developed UHPLC method is well‐suited for the accurate and reliable analysis of Molnupiravir and its impurities, making it a valuable tool for quality control and pharmaceutical research applications.

Impurity Profiling and a Stability Indicating Advanced Liquid Chromatographic Method Development and Validation for the Estimation of Related Impurities of Anagrelide

A novel, streamlined and highly selective isocratic liquid chromatographic method has been developed for the quantification of impurities present in anagrelide. Exceptional resolution between anagrelide and its impurities was accomplished utilizing a Waters Nova Pack C18 column (250 mm × 4.6 mm, 4 µm), employing a phosphate buffer with a pH of 4.4 as mobile phase A and a blend of acetonitrile and methanol with buffer in a 30:40:30 v/v/v ratio as mobile phase B. The column oven was maintained at 35 ºC and the mobile phase flowed at a constant rate of 1.0 mL/min, while detection was set at 254 nm. Rigorous validation was conducted to assess accuracy, precision, specificity, linearity and sensitivity. Validation studies have unequivocally established the HPLC method’s precision, specificity, rapidity, reliability and reproducibility. Linearity was ascertained for both anagrelide and its impurities, exceeding an R2 value of 0.95. The limits of detection (LOD) and limit of quantitation (LOQ) were determined to be more than sufficient for precise estimation. This method was validated in accordance with the stringent ICH guidelines. The relative standard deviation (RSD) for intra- day and inter-day precision remained consistently below 5%. Percentage recovery demonstrated excellent agreement, affirming the simplicity, specificity, precision and enhanced accuracy of method for determining related substances in pharmaceutical substances and various anagrelide-containing dosage forms.

Development and Validation of the Ultra-Performance Liquid Chromatography Method for Quantification of Belzutifan in Pharmaceutical Dosage Form

Development and Validation of the Ultra-Performance Liquid Chromatography Method for Quantification of Belzutifan in Pharmaceutical Dosage Form

Advances in High-Performance Liquid Chromatography (HPLC) Method Development and Validation: A Comprehensive Review

High-Performance Liquid Chromatography (HPLC) stands as the cornerstone technique for the detection, separation, and quantification of drugs in various matrices. Method optimization necessitates a thorough investigation of multiple chromatographic parameters, encompassing sample pretreatment, mobile phase composition, column selection, and detector configuration. This article aims to comprehensively address the processes of method development, optimization, and validation within the realm of HPLC. The inherent advantages of HPLC, including its rapidity, specificity, accuracy, precision, and automation feasibility, render it an indispensable tool for analyzing a wide spectrum of drugs in complex multi-component dosage forms. Furthermore, HPLC method development and validation hold paramount importance across the spectrum of drug discovery, development, and manufacturing, as well as in diverse human and animal studies. The validation of analytical methods during drug development and manufacturing is imperative to ensure their fitness for the intended purpose. Adhering to Good Manufacturing Practice (GMP) requirements, pharmaceutical industries must establish comprehensive validation policies outlining the validation procedures. This article primarily delves into the optimization of HPLC conditions, emphasizing the critical role of method refinement in ensuring robust and reliable analytical methods.

Quality by Design Based Stability Indicating Quantitative Reverse Phase High Performance Liquid Chromatography Method Development and Validation for Bilastine in Tablet Dosage Form

Quality by Design Based Stability Indicating Quantitative Reverse Phase High Performance Liquid Chromatography Method Development and Validation for Bilastine in Tablet Dosage Form

Response Surface Methodology Assisted RP-HPLC Method for the Determination of Meropenem in Human Plasma: Application to a Pharmacokinetic Study

A simple, accurate and precise development of an reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of meropenem in human plasma samples for therapeutic drug monitoring (TDM) was developed and validated after optimization of various chromatographic parameters. The best recovery of meropenem was achieved through sample preparation based on a simple extraction method that included deproteinization and extraction with acetonitrile. The developed method reported acceptable values for selectivity, linearity, accuracy and precision, carry-over, dilution integrity and stability. The developed method was efficiently shown to be applicable to a real-time monitoring and pharmacokinetic study of meropenem in critically ill patients.

Isolation, In-silico Studies, and Biological Evaluation of Higenamine from Annona squamosa L. against Breast Cancer

The efficacy of Annona squamosa L. in cancer treatment has been documented, inspiring the authors to investigate the plant further for potential novel anticancer compounds. Therefore, the current study aimed to isolate and characterize Higenamine from leaves extract of A. squamosa L. and studied for the in-vitro cancer cell line and molecular docking. The high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) method development of A. squamosa L. leaves extract furnished Higenamine of which characterization was established by HPLC, infrared radiation (IR), liquid chromatography-mass spectrometry (LC-MS) and its acute toxicity study and cytotoxic properties using MCF-7 cell lines by MTT assay were assessed. Molecular docking was done using Auto Dock Vina software to validate the anticancer activity. The extraction batches by ultrasonication and maceration methods showed good results using 80% methanol. Acute toxicity studies, after a single dose (2000 mg/kg) administration of methanolic extract don’t show clinical symptoms of toxicity and mortalities. From the extract, Higenamine was eluted and the dried fraction showed 66% of enrichment of Higenamine. The developed HPLC and TLC methods showed Higenamine (HGN) peak at 6.21 minutes band at Rf 0.61, respectively. The isolated compound was identified as Higenamine with FTIR spectroscopic analysis which revealed various characteristic band values with functional groups. HPLC-MS showed m/z at 272.1 (M+H+) corresponding to the molecular formula C16H17NO3. The IC50 value for Higenamine was 42.39 μg/mL were observed in MCF-7 breast cancer which was better than 5-Fluorouracil having 39.22 μg/mL. In-silico studies explored that higenamine showed good binding affinity to breast cancer PDBIDs and forms a stable complex. Furthermore, absorption, distribution, metabolism, and excretion (ADME) properties of isolated compound was calculated using the Swiss ADME online tool in which higenamine was found to have a good pharmacokinetic profile as compared with 5-fluorouracil and doxorubicin. Higenamine has been isolated and identified as a potential anticancer agent. Further research can explore its effectiveness through pharmaceutical formulation and in vitro cell line studies.

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of &gt;75%.