The method was validated according to ISO 5725-4 (2020), EURACHEM (2014) and DOQ-CGCRE-008 (2016). The limits of detection and quantification of the method for the nine organochlorines were α-HCH (1.2 and 12.6 µg kg−1), β-HCH (1.7 and 12.0 µg kg−1), γ-HCH (1.5 and 11.6 µg kg−1), δ-HCH (0.8 and 11.6 µg kg−1), heptachlor (1.0 and 10.8 µg kg−1), cis-heptachlor epoxide (0.9 and 11.5 µg kg−1), trans-heptachlor epoxide (0.9 and 11.5 µg kg−1), cis-chlordane (0.4 and 7.9 µg kg−1) and trans-chlordane (0.5 and 10.9 µg kg−1), respectively, and all of them were within the maximum limits recommended by the EPA for the compounds α-HCH (86.0 and 360.0 µg kg−1), β-HCH (300.0 and 1.3 × 103 µg kg−1), γ-HCH (570.0 and 2.5 × 103 µg kg−1), δ-HCH (not defined), heptachlor (130.0 and 630.0 µg kg−1), cis-/trans-heptachlor epoxide (7.0 and 330.0 µg kg−1), cis-/trans-chlordane (1.77 × 103 and 7.7 × 103 µg kg−1) in residential and industrial soil, respectively. Recovery results were between 65% and 105% for almost all compounds, which is an optimum result for multi-residue analytical methods, considering the complexity of the matrix used in the study. Caieiras presented contamination levels of α-HCH in the range of 2.0 to 66.0 µg g−1, which was higher than the limits established by EPA, corresponding to 0.077 µg g−1 for residential soil and 0.27 µg g−1 for industrial soil. According to the validation study, the analytical method proposed was reliable for organochlorine quantification, and the QuEChERS was considered efficient for organochlorine extraction from soil.Technological advances have promoted improvements in several science fields, especially related to environmental and analytical areas with the improvement of detection and development of environmentally friendly extraction techniques. This study applied Quick, Easy, Cheap, Effective, Rugged and Safe method (QuEChERS) for soil extraction and assessed its performance through a validation study using samples from the soil of a contaminated area in Caieiras, SP, Brazil. Nine organochlorine pesticides, including the isomers alpha, beta, gamma and delta- hexachlorocyclohexane;  The interest for green analysis methods using fewer solvents and chemical reagents has increased, and the environmental impact generated by human activities has been estimated through different techniques. A large number of studies over the world have contributed to advances in this field, and several matrices have been analyzed to quantify the presence of pesticides and industrial byproducts in air, water, soil, food, human blood and tissues ,4,5,6,7.Some efforts have been made for countries from all around the world to develop quantification methods for organochlorine contamination study as well as to estimate their impact on the environment and human health. Many studies related to pesticide quantification in the soil are available in the literature, particularly in the context of soil contamination ,9,10. Ho6Cl6), heptachlor (C10H5Cl7), cis- and trans-heptachlor epoxide (C10H5Cl7), and cis- and trans-chlordane (C10H6Cl8), which were considered in this study. In 2002, the Environmental Company of São Paulo state (CETESB) produced the first document of contaminated areas in Brazil in which 225 areas were registered accounting environmental passives and in 2003 initiated a partnership with the Health Surveillance Agency from Caieiras, SP, and studied soil and water samples from an illegal HCH landfill. The place is surrounded by a population that has been exposed to this contamination since the deposition. The study found the presence of HCH isomers in soil and water, although the study was not conclusive [Among these POPs are the isomers alpha (α), beta (β), gamma (γ) and delta (δ) hexachlorocyclohexane , initially developed by Anastassiades and colleagues, initially for food extraction, has led to excellent technical and economic results . The tecThe present study aimed to contribute to the accomplishment of the Stockholm treaty signed by Brazil, considering a large number of not studied landfills in the country and taking into account the soil diversity and the environmental passives available around the world which have never been studied nor monitored. In this sense, real samples were collected in Caieiras landfill, SP, Brazil for initial screening of the area and the determination of the contamination levels of the POPs mentioned previously. The determination and quantification were performed by gas chromatography coupled to an electron capture detector (GC-ECD) and the analytical method was validated following the ISO 5725-4 (2020), EURACHEM (2014) and DOQ-CGCRE-008: 2016 ,27,28. There are many analytical methods for pesticide determination in soil available in the literature, however, in terms of comparison of results, it is quite complex, because of the different soil properties found in the landfills reported. It is known that the analytical method response is impacted by the matrix effect. In the case of a soil matrix, some properties such as pH, which modulates the compound percolation, granulometry, which reveals the soil profile, and organic content, which modulates compound percolation, directly impact the outcome of the method. In this context, the present study is also relevant for providing soil characterization, which might help other studies. Concerning the contamination results, they were compared with the maximum limits for industrial and domestic soil recommended by the Environmental Protection Agency (EPA)  showed icis, trans-heptachlor epoxide, cis and trans-chlordane, and the reagents magnesium sulfate (MgSO4), sodium chloride (NaCl), sodium citrate (Na3C6H5O7), sodium hydrogen citrate sesquihydrate (C6H6Na2O7·1.5 H2O) and Bondesil-PSA were bought from Sigma Aldrich . Solvents such as acetonitrile (ACN) and hexane were purchased from J. T. Baker® . Plastic centrifugal tubes (50 and 30 mL) were purchased from Corning TM . Polypropylene syringe microfilters (diameter of 0.25 mm and pore size of 0.45 µm) were purchased from Advanced MFS Gebhardt .Individual standard solutions of α, β, γ, δ-HCH, heptachlor, The method was performed in a GC 17A with a nickel-63 electron capture detector model ECD-2010 Exceed . The gas chromatographic system was equipped with a 30 m DB-5 capillary column with stationary phase of trifluoropropyl methyl polysiloxane  with an internal diameter of 0.25 mm and 0.25 µm film thickness. Helium with a purity of 99.999%  was used as a gas carrier with a column head pressure of 12 p.s.i., split/splitless unit. Chromatographic data were collected and recorded using GC Analyst software .−1) was made by aliquoting the individual stock solutions of alpha-hexachlorocyclohexane (α-HCH), beta-hexachlorocyclohexane (β-HCH), gamma-hexachlorocyclohexane (γ-HCH), delta-hexachlorocyclohexane (δ-HCH), heptachlor, cis-heptachlor epoxide, trans-heptachlor epoxide, cis-chlordane and trans-chlordane (1.0 mg mL−1 in hexane). The analytical curve was prepared in two media, in acetonitrile (ACN) and soil matrix, and for that, the standard mixture was diluted in seven points, in the range of 100 to 1160 µg kg−1.The organochlorine pesticide stock standard mixture  and temperature gradient of 80 to 210 °C at 25 °C min−1 and 210 to 290 °C at 35 °C min−1. The representative scheme of the analytical method developed in this work is shown in The GC-ECD conditions applied were 250 °C for injection temperature, 280 °C for interface temperature, 2.5 kV for voltage detector, helium as the gas carrier (1.1 mL min−1) and in soil matrix (100 µg kg−1); accuracy, which was assessed by trueness, evaluated by the Z-score test of recovery, and precision, assessed through repeatability, through the analysis of the standard mixture prepared in soil matrix, in three different concentration levels, in seven replicates, in the same day, equipment and operator and calculating the limit of repeatability (r) from the relative standard deviation; reproducibility, considering three concentration levels and seven replicates analyzed in different days and analysts; limits of detection and quantification, determined by analytical curves, in solvent and in soil matrix; and recovery.The quality assurance was performed through validation tests based on the criteria established by the European Guide, EURACHEM: 2014 , Interna−1) for 24 hours, followed by extraction by the QuEChERS method. To compare the area values obtained from the chromatograms, the fortification was performed in the same concentrations as for the soil samples after the extraction procedure. The experiments were made in triplicate, and the extracts were evaluated by GC-ECD. Equation (1) shows the formula used to calculate the recovery, considering that R (%) means recovery expressed in percentage, A1 means fortification before extraction and A2 fortification after extraction. The blank soil was prepared and extracted by QuEChERS as well as the rest of the samples. All samples were assessed in triplicate.Recovery assays were performed by fortifying 10 g of soil sample collected at the same region of the samples analyzed, with a pesticide standard mixture prepared in two concentration levels  of the data were determined using Microsoft Office Excel 365 .cis and trans-heptachlor epoxide and cis and trans-chlordane organochlorines from soil samples. For that, some parameters such as the gradients of temperature were tested as follows: (1) Temperature (T) was set to 60 °C for 1 min, and then, T was increased to 180 °C at 23 °C min−1 for 6 min, and finally, T was increased to 330 °C at 35 °C min−1 for 1 min, and the speed of column gas carrier was 1.7 mL min−1; (2) T was set to 80 °C for 1 min, and then, T was increased to 210 °C at 25 °C min−1 for 6 min. After that, T was increased to 330 °C at 35 °C min−1 for 1 min, and the speed of column gas carrier was 1.5 mL min−1; (3) T was set to 90 °C for 1 min, then T was increased to 170 °C at 35 °C min−1 for 5 min. Posteriorly, T was increased to 250 °C at 40 °C min−1 for 1 min, and the speed of column gas carrier was 1.6 mL min−1; (4) T was set to 80 °C for 1 min, and then, T was increased to 210 °C at 25 °C min−1 for 7 min. T was increased to 290 °C at 35 °C min−1 for 1 min, and the speed of the column gas carrier was 1.7 mL min−1 (data not shown). The best performance was found for method 4, and its chromatogram is shown in The method was modulated to optimize chromatographic analysis time and to define the best parameters for quantification of α, β, γ, δ-HCH, heptachlor, The organic matter content of the soil was evaluated and resulted in 0.8%, showing that the soil presented low organic content and the matrix was not likely to retain organic contaminants, which allowed its leaching to deep levels. The granulometry assay showed a balance in the soil profile, in other words, the soil presented 37% of clay, 31% of silt and 32% of sand, indicating that the soil presented characteristic grain size of clay, silty and sandy soil in similar proportions.Linearity was expressed through the linear regression equation and determination coefficients, which were greater than 0.991 for all compounds in both solvent and matrix groups as observed in −1 was observed. By observing the chromatograms in which the standard solutions were prepared at the same concentration, it was possible to note the higher intensity in the peaks of compounds 1–4 in the solvent, which might mean that the matrix presented a suppression effect over the signals of such analytes. It was possible to conclude that the signal intensities of compounds 5–9 were the same when assessed in the matrix, revealing a very small matrix effect, and thus evidencing that the matrix affects differently the evaluated analytes. Selectivity was also evaluated by F and t-tests. The results are presented in Selectivity and sensitivity were observed in the chromatograms of the standard solution in solvent and soil matrix, as observed in Precision was evaluated in terms of variation coefficient, repeatability and reproducibility. The variation coefficient was expressed in percentage, and three concentration levels were considered, and all variation coefficients were lower than 20% as observed in The limit of repeatability (r) and reproducibility (R) values, as well as the concentration levels and the relative standard deviations are shown in the −1. The previously mentioned results are shown in Trueness was evaluated through the Z-score test. Limits of detection and quantification were determined by the analytical curve parameters with 95–99% of assurance, analyte concentration higher than zero, and recovery, determined by two soil fortification assays, using concentrations of 1 and 2 µg g6 ± 0.17 µg kg−1 of α-HCH and 9.7 × 105 ± 0.05 µg kg−1 of β-HCH. The HCH isomers were quantified in other samples in the range of 1.75 × 104 to 2.04 × 106 µg kg−1, mainly by α-HCH, showing high contamination levels considering the maximum acceptable limits for residential and industrial soil according to the EPA, established as 86 and 360 µg kg−1 for α-HCH, 300 and 1.3 × 103 µg kg−1 for β-HCH, 570 and 2.5 × 103 µg kg−1 for γ-HCH. There is no established limit for δ-HCH [Sample 1 (n1) presented contamination for all HCH isomers, and the chromatogram is shown in or δ-HCH .The method proposed yielded optimum separation and signal of the nine organochlorines including all isomers within 18 min. Concerning the soil characterization, Caldas and colleagues evaluated the soil samples from a rice field and found a soil with low organic matter content of 0.6%, clay content of 16% and pH of 5.2 .Quinete and collaborators developed an analytical method to quantify some organochlorine pesticides in water and soil to monitor the impact of the agriculture activities expansion in the near border of Atlantic Rain Forest situated in southeastern Brazil. One of the soil samples was classified as Udorthent Eutrophic, predominantly sandy, and the other as Chernosol Argiluvic Orthic and Ultisol Dystrophic soil, mostly clayey. They found soil samples with pH in the range of 3.87 to 4.50, which is considered high acidity for soil .According to the granulometric assay, the soil analyzed in this study presented contents of 31% silt, 37% clay and 32% sand, a balanced and different profile in comparison with the above-mentioned studies. A very low organic matter content of 0.8% was identified and a pH of 3.5, which is considered extremely acid for soil and tends to facilitate leaching, similarly as reported by Quinete and collaborators. Thus, considering the viability of applying the method for the evaluation of soil matrices from different places, the soil characterization parameters should be considered due to the impact of the matrix characteristics on the analytical response. Therefore, soil characterization is essential for understanding the ordinary interactions between the matrix and the contaminant, and ultimately, to determine the effects of the matrix over the quantification.2) obtained for the solvent and matrix assays also revealed that the analytical curves of this study held optimum linearity with r2 higher than 0.991 for all compounds from both groups. The determination coefficient is frequently used to indicate linear fitting among the assayed points in which values higher than 0.81 are required to assure adequate linearity of the system. Due to the complexity of soil analyzed, the effects of the matrix could potentially influence the chromatographic response and, consequently, the analyte quantification. In this context, such effect was studied to ensure reliability in the analytical results by analyzing the organochlorines in ACN and in the soil matrix extract. Concerning selectivity and sensitivity, the nine compounds were perfectly-identified and separated, including the impurities originated from the soil matrix. The −1, and all of them presented VC lower than 5% for concentrations of 500 and 1000 µg kg−1, which is considered an optimum performance for multi-compound analytical methods, mainly concerning those which present a complex matrix. With regards to repeatability and reproducibility results  of three different concentration levels for all compounds at each concentration assayed . The VC  results , the ave results  and the −1 and 11.41 to 79.23 µg kg−1, respectfully. For the soil of low organic matter content, the results were 6.11 to 14.78 µg kg−1 and 20.37 to 49.27 µg kg−1. In this study, the ranges obtained for limits of quantification and detection were 7.9 to 14.3 µg kg−1 and 0.3 to 3.8 µg kg−1, respectively [Concerning the matrix effect, Fernandes and collaborators (2013), as an example, observed the matrix effect over 13 pesticides such as α, β-HCH, HCB, endrin, o, p’-DDT, among others . In termectively . Concerning the matrix effect in the present study, normally the matrix presents a signal increment, but in some cases, there is a reduction, as observed for compounds α, β, γ and δ-HCH in −1 of soil ranged from 70% to 120% with RSD < 15% [As for the recovery assay, Caldas and colleagues found results between 70.3% and 120% and relative standard deviation (RSD) lower than 18.2% . FernandSD < 15% . CorreiaSD < 15% . RissatoSD < 15% .With respect to the present study, almost all compounds presented results between 65% and 105% , which a−1 and of 44.2% at soil fortification of 2.0 µg g−1. These variations might take place due to the nature of the soil matrix and the high matrix impact on the analyte at lower concentrations. On the other hand, the isomer γ-HCH presented recovery of 211.7% at soil fortification of 2.0 µg g−1, which might be attributed to the matrix increment, while it presented 88.6% recovery at soil fortification of 1.0 µg g−1. Additionally, many other validation criteria were assessed in the present study, and the results pointed out that the method is reliable for organochlorine determination under the specified conditions. Concerning lower concentrations for the recovery test, initially, the concentrations of 0.25, 0.5 and 0.75 µg g−1 were evaluated, but the results were not reliable, presenting a high variation coefficient due to the contamination promoted by the spike being lower than the contamination already present in the samples, as the blank soil adopted was collected in the region and showed some contamination level that was previously determined and subtracted in the calculation. Additionally, in a multiscreen method, some compounds usually present more or less detectability by the method at a given concentration range. As an example, α-HCH presented recovery of 92.1% at soil fortification of 1.0 µg g−1, 0.15 µg kg−1 of β and γ-HCH, 0.07 µg kg−1 of δ-HCH and 0.05 µg kg−1 of heptachlor epoxide in soil samples from three different regions of northeast São Paulo, Brazil [−1 of γ-HCH in soil samples in Rio de Janeiro, Brazil [Comprising the contamination study, in the last decades, there has been an increasing number of studies regarding the determination of contaminated areas as well as an increase in research related to the development and optimization of analytical methodologies to assess different kinds of matrixes. Rissato and collaborators determined α-HCH in the range of 0.06 to 0.26 µg kg, Brazil . Nearby,, Brazil . −1 of γ-HCH in integrated pest management soil and 15 µg kg−1 of the same compound in organic farming soil, both from Portugal [−1) than in a family farm village (0.01–0.35 µg kg−1) [−1, with the highest level found at the urban site—Riverdale (0.98 µg kg−1), followed by the rural site—Borden (0.38 µg kg−1), the suburban sites—Aurora (0.11 µg kg−1) and North York (0.19 µg kg−1) in Canada [Finally, tracing an international comparative for pesticides in soil studies, Fernandes and colleagues determined 19 µg kge 0.01–0. µg kg−1 n Canada . By comparing results from the present study  and otheBased on the presented data from different places around the world, the contamination quantified in the present study was found in higher levels if compared to samples analyzed by other studies in different regions, indicating a strong need for remediation, especially considering the exposure of the surrounding population. Concerning the distribution of soil contamination, n4 and n5 presented the highest levels of HCH isomers, and such sampling points were collected inside the borders of the landfill, followed by n8, which was situated outside the landfill limits. HCH isomers were found in twelve of the total fifteen samples at higher levels than the limits established by EPA for residential and industrial soil as shown in cis and trans-heptachlor epoxide and cis and trans-chlordane. The linearity and sensitivity were evaluated through the analytical curve assay whereby the method was considered adequate based on the regression coefficients above than 0.99127 and the working range within the limits established by EPA, thus validating the method for the application proposed. The method presented adequate selectivity, highlighting its capacity of separating the compounds in the study even in the presence of isomers, which are compounds of hard separation. The precision was evaluated through the variation coefficient data, and all results were lower than 20%, indicating adequate precision. Trueness was assessed by Z-score test in which all results were lower than 2 and thus being in accordance with the established criteria. The limits of detection and quantification were lower than the maximum acceptable limits for organochlorine contaminants residues in soil according to EPA evidencing adequate sensitivity and determination capacity. The recovery was measured in the range of 65% to 105% for almost every compound and was considered acceptable for the evaluation of complex matrixes. The technique was particularly interesting considering its easy and quick performance, as well as its low cost and low reagent amounts, which makes its use environmentally justifiable.The multi-residue method developed presented adequate performance according to the national and international guides for validation of analytical methods. GC-ECD technique showed great performance to quantify the compounds α, β, γ, δ-HCH, heptachlor, The study enabled the application of the method to organochlorine extraction in soil matrix with suitable accuracy and contributed to Brazilian input to the Stockholm treaty, in terms of the international commitment to identify and quantify contamination and soil remediation when applicable. Moreover, the evaluation of an unstudied landfill area where considerable amounts of pesticides were illegally discarded and which is currently surrounded by a community is of interest to public health, and its economic aspects are of relevance to society.


Cmax) of HCQ was 292.33 ng/mL in blood and 36.90 ng/mL in plasma after single dose of 3 mg/kg. The value of area under curve (AUC0–∞) was determined as 5,978.94 and 363.31 h* ng/mL for the blood and plasma, respectively. The descending order of the tissue-to-plasma concentration ratio was liver > spleen > kidney > lung > heart > subcutaneous fat > brain. The tissue-to-plasma concentration ratio and the tissue-to-blood concentration ratio for lung were found to be time-dependent with 267.38 and 5.55 at 120 h postdose, respectively. A five-compartment model with first-order oral absorption and elimination best described the plasma, blood, and lung tissue pharmacokinetics. The estimated elimination rate constant (ke) for a typical monkey was 0.236 h−1. The volume of distribution in central (Vc/F) and other two peripheral compartments (Vb/F and Vl/F) were 114, 2.68, and 5.55 L, respectively. Model-based simulation with PK parameters from cynomolgus macaques showed that the ratio of the blood or plasma to lung tissue was a dynamic change course, which suggested that the rate of HCQ concentration decrease in the blood or plasma was faster than that in the lung tissue. HCQ was found to be accumulated in tissues, especially in the liver, kidney, lung, and spleen. Also, the tissue-to-plasma concentration ratio increased over time. The population pharmacokinetic model developed could allow for the assessment of pharmacokinetics–pharmacodynamics relationships, especially relevant tissue concentration-response for HCQ. Determining appropriate treatment regimens in animals allows translation of these to clinical studies.To evaluate the biodistribution of hydroxychloroquine (HCQ) in cynomolgus macaques and receive dynamic quantitative relationship between plasma, blood, and lung tissue concentration using the population pharmacokinetic modeling method, seventeen cynomolgus macaques were divided into six groups according to different HCQ dosing regimens over 5 days. The monkeys were euthanized, and blood, plasma, urine, feces and ten tissues were collected. All the samples were prepared by protein precipitation and analyzed by HPLC-MS/MS detection. The population pharmacokinetics of HCQ in the plasma, red blood cells, and lung tissue was conducted and simulated via ADAPT program. Results demonstrated that the maximum concentration ( As a 4-aminoquinoline compound with the hydroxyl group based on structure of chloroquine, hydroxychloroquine (HCQ) has a similar therapeutic effect but less side effects compared with other antimalarial drugs. Several clinical practices confirmed that HCQ has good safety and reliable efficacy in various rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, it has been used as basic treatment for rheumatic immune diseases.50 values with different cell lines or different sampling times. Thus, it is necessary to conduct additional pharmacokinetic studies to understand how the concentration in the lung tissue relates to its efficacy.Since the outbreak of the novel coronavirus disease (COVID-19) in December 2019, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are more than 21.2 million confirmed cases of those infected and nearly 770,000 deaths . It has in vitro experimental data, and human physiological parameters to predict the pharmacokinetic characteristics of the drug in vivo in a mechanistic manner.However, there are still some difficulties in clinical studies, such as the ethical issues and operability of obtaining tissue samples. Physiologically based pharmacokinetic (PBPK) modeling, a powerful means of optimizing the dosing regimen, can integrate the drug physicochemical properties, In this study, we first established a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for determining the concentration of HCQ and N-desethyl hydroxychloroquine (DHCQ) in cynomolgus monkeys. Then, we performed a biodistribution study following different HCQ dosing regimens in monkeys. We also conducted a population pharmacokinetics study to characterize the time-dependent pharmacokinetic characteristics, and then HCQ concentration in the lung, blood, and plasma were predicted so as to support the verification of HCQ physiologically based pharmacokinetic (PBPK) model to assist clinical dosing regimen optimization.The reference standard of hydroxychloroquine sulfate (100% purity), hydroxychloroquine sulfate-D4 , and N-desethyl hydroxychloroquine (DHCQ) bromide were purchased from TLC Pharmaceutical Standards Ltd. . Hydroxychloroquine sulfate (99.90% purity), the experimental drug, was supplied by Jiangsu Shenhua Pharmaceutical Co., Ltd. Ammonium formate of analytical grade was purchased from Sinopharm Chemical Reagent Co., Ltd. . Methanol, acetonitrile, and formic acid of HPLC grade were purchased from Fisher Scientific . Purified water obtained from a PURELAB Option-Q7  system was used throughout the experiment.Cynomolgus macaques (4.13 ± 0.43 kg) were supplied by Shanghai Beijing Institute of Xieerxin Biology Resource (SCXK: 2015-0011). The animal experiments were carried out in accordance with the guidance from the Animal Care and Use Committee of the Pharmaron Beijing Co. Ltd. (IACUC No.16-120).Biological samples were analyzed using an HPLC–MS/MS system. The liquid chromatography system  was equipped with an LC-20ADXR binary liquid pump. Chromatographic separation was performed on a Venusil C18 Plus chromatographic column , and the column temperature was maintained at 35 °C. The mobile phase consisted of 0.1% formic acid and 20 mM ammonium formate in water (mobile phase A) and acetonitrile (mobile phase B). The chromatographic separation was achieved using 3-min gradient elution. The gradient was 5% B at 0.0–0.01 min, 5%–30% B at 0.01–1.40 min, 30%–95% B at 1.40–1.50 min, 95% B at 1.50–2.30 min, 95%–5% B at 2.30–2.40 min, and 5% B at 2.40-3.00 min. The flow rate was set to 0.8 mL/min. The injection volume was 2.0 µL for all samples.2) and ion source gas 2 (N2) was both set to 50 psi. The air curtain gas (N2) pressure was 30 psi. Optimal multiple reaction monitoring (MRM) was selected, and the dwell time for each analyte was 150 ms. The ion pairs used for quantitative analysis were m/z 336.1→247.0 (hydroxychloroquine), m/z 308.2→247.0 (N-desethylhydroxychloroquine), and m/z 340.1→247.0 . The collision energy (CE) was set to 30 eV for each analyte.Mass spectrometric detection was performed on an AB Triple Quad 4500 mass spectrometer, equipped with an electrospray ionization (ESI) source (Turbo Spray ESI source) and Analyst 1.6.3 quantitative analysis software (American Applied Biosystems company). The ESI source was set in the positive mode, with a spray voltage of 5500 V. The ion source temperature was set to 600 °C. The pressure of ion source gas 1 , and the diluent was collected for HPLC-MS/MS analysis after thorough vortex mixing for 1 min. While for the plasma samples, the preparation method was the same as the blood samples, but without zinc sulfate solution.For blood samples, an aliquot of 50 µL whole blood or plasma was mixed with 25 µL IS (500 ng/mL) solution and 50 µL zinc sulfate solution (0.2 mol/L). After thorough vortex mixing for 1 min, 400 µL acetonitrile containing 2% formic acid was added for protein precipitation. After thorough vortex mixing for 1 min, the samples were centrifuged at 1700g for 15 min, an aliquot containing 100 µL of the supernatant was diluted with 200 µL 90% acetonitrile  was added. The mixture was homogenized with a high-speed homogenizer, and the produced homogenate was stored at -70 °C until analysis. Similar to the preparation of the plasma, the tissue samples (50 µL) were mixed with 25 µL of IS (500 ng/mL) and 400 µL of acetonitrile containing 2% formic acid, then vortex mixed for 1 min, and centrifuged at 14,000 rpm for 5 min. An aliquot of 100 µL of the supernatant was diluted with 200 µL 90% acetonitrile , and the diluent was then analyzed by LC-MS/MS.Cynomolgus macaque  tissue samples were removed from the -70 °C refrigerator and placed on the ice tray to be cut into pieces. Tissues were placed in the homogenate tubes, and 10 times equivalent volume of 50% methanol . The matrix effect was evaluated by comparing the peak areas of the QC samples postspiked in the blank blood, plasma, and tissue homogenate with those in the mobile phase (n = 6). The extraction recovery and matrix effect of the IS were determined in the same way. The stability of the tested analytes was evaluated using the QC samples spiked in blank monkey biological samples (n = 3) under the following conditions: (1) store at room temperature for 24 h; (2) three freeze-thaw cycles from −80 °C to room temperature; and (3) store at−80 °C for 30 days.Cynomolgus macaques  were randomly divided into six groups from A to F. In group A, monkeys were given with 3 mg/kg hydroxychloroquine. In group B, monkeys were treated with 3 mg/kg hydroxychloroquine twice at D1 and D2, with an interval of 4 h. In group C, 2 mg/kg hydroxychloroquine was administered twice at D1, with an interval of 4 h, and then 1 mg/kg hydroxychloroquine twice daily was treated at D2 and D3, with an interval of 4 h. In group D, 6 mg/kg hydroxychloroquine was administered twice at D1, with an interval of 4 h, and then 2 mg/kg hydroxychloroquine twice daily was treated at D2 and D3, with an interval of 4 h. In group E, 21 mg/kg hydroxychloroquine was administered twice at D1, with an interval of 4 h. In group F, 21 mg/kg hydroxychloroquine was administered twice at D1, with an interval of 4 h, and then 7 mg/kg hydroxychloroquine twice daily was treated at D2∼D5, with an interval of 4 h. Hydroxychloroquine was administered intragastrically in all groups.2-EDTA as anticoagulant in groups A ∼ D. About 2 mL blood samples were collected at predose and 1, 2, 4, 8, 24, 48, 72, 144, 192, 240, and 264 h postdose with the same anticoagulant in group E. And about 2 mL blood samples were collected at predose and 1, 2, 4, 8, 24, 48, 72, 96, 97, 98, 100, 104, and 120 h postdose with the same anticoagulant in group F. Then, 2 mL blood sample was divided equally into two parts, one of which was stored at -70 °C until analysis without any treatment, while the other one was centrifuged at 2000 g for 10 min at 4 °C, and then, the harvested plasma samples were stored at -70 °C until analysis. The pharmacokinetic parameters t1/2 , CLz , and AUC0–t (area under curve)were calculated from the plasma concentration–time data according to noncompartmental methods by WinNonlin  in a noncompartmental manner.Blood samples (approximately 2 mL) were collected before and after dosing from the hind limb vein in polyethylene tubes at 1, 2, 4, 8, 24, 48, and 72 h with KCynomolgus macaques  were randomly divided into six groups ). All individuals in each group were euthanized at 504, 336, 192, 240, 264, and 120 h following administration of hydroxychloroquine. Tissues  were harvested and homogenized by the methods described in “Total 17 cynomolgus macaques were included in this analysis. The dose regimen administered including a) 11.82–11.97 mg , b) the first two loading doses in the range of 8.12–9.4 mg, following the 4 dose in the range of 4.06–4,72 mg , c) first two loading doses in range of 24.72–27.78 mg, following the 4 dose in the range of 8.24–9.26 mg . Intensive plasma (141) and blood sampling (149) were performed. Fourteen of 17 cynomolgus macaques with one sample from the lung were collected at 120, 198, 240, 264, and 480 h.Initially, integrated models were established by considering four and five compartment models with and without an absorption lag phase or with and without peripheral compartment (model selection based on the likelihood ratio test) using measurements from the plasma, blood, and lung simultaneously. A model of the plasma, blood, and lung tissue was final developed by fitting a five-compartment model to represent the relationship between the plasma, blood concentrations, and lung tissue. The five-compartment model with first-order oral absorption  and elimination  was evaluation simultaneously to describe the time course of HCQ in the plasma . The parcbmax and Kclmax denote the maximum rate of transfer from the central compartment to the blood and lung, respectively, while Acb50 (mg) and Acl50 (mg) denote the amount of plasma compartments where the rate to blood is half-maximal of Kcbmax and Kclmax, respectively. Kpc (1/h) and Kcp (1/h) denote the rate of transfer between the peripheral compartment and central compartment.Following Model evaluation was performed using goodness-of-fit (GOF) plots with several diagnostic scatter plots and the comparison of observation and individual concentration time profiles, including (1) observed vs. population-predicted concentration (DV vs. PRED), (2) observed vs. individual-predicted concentration (DV vs. IPRED), (3) conditional standardized residuals vs. time (CSRES vs. TIME), and (4) conditional standardized residuals vs. population-predicted concentration (CSRES vs. PRED).The simulation of the pharmacokinetic profile following multiple HCQ doses was conducted using the typical parameters of the final model. The simulated HCQ concentration in the plasma, blood, and lung tissue was plotted over time.Population analysis was used to develop an overall pharmacokinetic model for describing the HCQ concentration sampled from each of the four components. Maximum likelihood estimates for model parameters were obtained through the application of the expectation maximization algorithm to the parametric, nonlinear mixed-effects maximum likelihood model, as proposed an developed by Schumitzky  and WalkHCQ and DHCQ were spiked into blank plasma and various tissue samples, respectively, to determine the calibration curve. The linear responses for HCQ were observed over the corresponding concentration ranges of 5.00-1,000.00 ng/mL, 2.00-200.00 ng/mL, and 0.40-200.00 ng/mL in the blood, plasma, tissue, respectively. While the linear responses for DHCQ were observed over the corresponding concentration ranges of 2.50-500.00 ng/mL, 0.20-200.00 ng/mL, 0.40-200.00 ng/mL in the blood, plasma, tissue, respectively.All the correlation coefficients were greater than 0.99, indicating a good linearity of the calibration curves. The lowest limit of quantification (LLOQ) of HCQ in blood, plasma, and tissue samples were 5.00 ng/mL, 2.00 ng/mL and 0.40 ng/mL, respectively. While the lowest limit of quantification (LLOQ) of DHCQ in blood, plasma, and tissue samples were 2.50 ng/mL, 0.20 ng/mL, and 0.40 ng/mL, respectively.max) of HCQ was 292.33 ± 114.66 ng/mL in blood and 36.90 ± 22.52 ng/mL in plasma. The value of area under curve (AUC0–∞) were determined as 5,978.94 ± 1981.30 and 363.31 ± 195.38 h*ng/mL for blood and plasma, respectively. HCQ were detected in the blood and plasma up to 72 h, which was the last time point monitored. The percentage extrapolation of the AUC from the last measured time point to infinity was less than 10% and 20% in the blood and plasma, respectively.The developed and validated LC–MS/MS method was successfully applied to the determination of HCQ pharmacokinetics and tissue distribution after intragastric administration of HCQ in monkeys following the dose regimen described in “tissue/blood) were ≥1, indicating accumulation of HCQ in tissues. The HCQ Kp_tissue/blood ratio for the various tissues was observed in the descending order of thymus > kidney > spleen > liver > heart > subcutaneous fat > brain > lung in group E, whereas the Kp_tissue/blood ratio was different in group F with the descending order of liver > thymus > spleen > kidney > lung > heart > subcutaneous fat > brain. But the last sampling time of group E was 264 h, and the corresponding plasma concentration was below the limit of quantification, resulting the Kp_tissue/plasma was only available in group F. Similar to the Kp_tissue/blood ratio, the descending order of the Kp_tissue/plasma ratio in group F was liver > spleen > kidney > lung > heart > subcutaneous fat > brain. The Kp_tissue/plasma ratio and Kp_tissue/blood ratio for the lung was 267.38 ± 42.93 and 5.55 ± 0.46, respectively.Concentrations of HCQ and DHCQ were detected in all studied tissues –3. In ad−1, and Ke was estimated to be 0.236 h−1. The volume of distribution in the central (Vc/F) and other two peripheral compartments (Vb/F and Vl/F) were 114, 2.68 and 5.55 L, respectively. Estimates of Acb50, Acl50, Kbc, Klc, Kcbmax, Kclmax, Kcp, and Kpc between the central compartment and different peripheral compartments were 7.05 mg, 0.498 mg, 0.718 h−1, 0.159 h−1, 2.48 h−1, 1.92 h−1, 0.600 h−1, and 0.514 h−1, respectively. The interindividual variability (IIV) was estimated for Ka, Ke, Vc/F, Vb/F, Vl/F, Acb50, Acl50, Kbc, Klc, Kcbmax, Kclmax, Kcp, and Kpc. The IIV for PK parameters were expressed as CV% and ranged from 29 to 88.7%, except for IIV of Kcl (160%). The final model parameter estimates are summarized in A five-compartment model with first-order oral absorption and elimination was selected as the base model for HCQ concentration in the plasma, blood, and lung tissue . The absGOF plots for the final model were generated, as depicted in One dosing regimen of HCQ was simulated using the final population PK model. The simulated HCQ mean concentration in plasma, blood, and lung tissue is shown in in vitro to identify candidates to repurpose for SARS-CoV-2. The antimalarial HCQ has demonstrated antiviral activity against SARS–CoV-2 in vitro and in several clinical studies with small sample size. However, the efficacy of HCQ in the treatment of COVID-19 was quite controversial in clinical studies. It appeared that the free plasma concentration of HCQ was lower than extracellular EC50in vitro (submitted data). However, it is quite complicated to extrapolate efficacy from in vitro to humans just based on this simple comparison, as pointed out by 50, which could be compared with lung tissue concentration, and there are still many challenges, such as possible differences in mechanism, active metabolites (unpublished data). Although we knew more about its cellular kinetics and its distribution in monkeys, the recommendation of appropriate dosage to treat COVID-19 still needs to consider safety and efficacy margin to allow possible difference between in vivo and in vitro.SARS-CoV-2 can attack cells expressing angiotensin I-converting enzyme 2 (ACE2), leading to serious infections of the lungs, the gastrointestinal tract, and other tissues . MoreoveConsidering that it is impossible to carry out biodistribution research in clinical studies, the additional pharmacokinetics experiments in animals are necessary to understand its exposure in tissues. It is especially helpful when the PBPK modeling strategy was recognized to be supportive but need more validated data, such as lung tissue or lung lining fluid concentrations . It has The pharmacokinetics data obtained from different dosing regimens could be used simultaneously to perform population pharmacokinetics model and evaluate the pharmacokinetics characteristic of HCQ. Since the HCQ concentration in the lung tissue is higher than that in the plasma or blood, we could confirm that it might be effective in the lung. In addition, there has been simulated that lung pH reduction from 6.7 to 6 in COVID-19 patients could cause 4.0-fold increases in lung exposure of HCQ, indicating that higher concentrations of HCQ in patients’ lung tissue with COVID-19 were sustained for a longer period after administration . Many otFinally, as stereoisomeric drug, HCQ is administered as racemates, whereas enantiomeric differences may be existing in pharmacokinetics . TherefoIn summary, a population pharmacokinetics model for HCQ in cynomolgus macaques was developed and constructed using data from monkeys. The derived population PK model should be able to evaluate the dynamic process of HCQ in the plasma, blood, and lung tissue concentration to support the clinical applications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee(IACUC) Pharmaron Beijing Co., Ltd, Beijing, China.DL conceived and designed the study. HL and CS directed this whole study. KW developed and optimized the hydroxychloroquine population PK model. QL and GB designed the dose regimen of hydroxychloroquine and analyzed the experiment data. XG and ST performed population PK model analysis. XT, MX, and JW carried out the animal experiments. GC, ML, BW, and HJ conducted the bioanalysis of hydroxychloroquine. QL, GB, and DL prepared this manuscript.This work was supported by the Bill & Melinda Gates Foundation (INV-015694), the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2020YFC0844500), and the National Natural Science Foundation of China (Project Nos. 81672133 and 81874010).Authors GC, ML, BW, and HJ were employed by the company HR-Biopharm Technology Co., Ltd. Authors XT, MX, and JW were employed by the company Pharmaron Beijing Co., Ltd. Author KW was employed by the company Shanghai Qiangshi Information Technology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Duhaldea nervosa  A. Anderberg (Asteraceae), is a folk medicine used to treat a variety of diseases including fracture and rheumatoid arthritis. Despite its widespread use, the metabolism of isochlorogenic acid A in vivo has not been fully studied. Isochlorogenic acid A, one of the main components of  An analytical strategy based on UHPLC-Q-Exactive Orbitrap MS is proposed for the detection and identification of the metabolites of isochlorogenic acid A in rats. Six male Sprague-Dawley rats (180 ± 20 g) were randomly divided into two groups. Then, blood and tissue samples were obtained after oral administration of isochlorogenic acid A (200 mg/kg). All the samples were pre-treated by the Solid Phase Extraction (SPE) method. Next, the samples were analysed by UHPLC-Q-Exactive Orbitrap MS. Finally, the metabolites were identified based on the metabolomic workflow template. A total of 33 metabolites were identified in rat plasma, with 30 of them being reported for the first time. The distribution of all metabolites in tissues was first investigated, three of them were widely distributed in liver, lungs, and kidneys. The corresponding reactions including methylation, hydrolysis, sulphate conjugation, glucuronide conjugation, as well as their composite reactions, are reported in this study.in vivo. This method has wide-scale application prospects in the identification of metabolites. Considering that limited research has been conducted in this area, this study proposes metabolic pathways to further understand mechanisms of isochlorogenic acid A and the forms that are truly effective  Duhaldea nervosa  A. Anderberg (Asteraceae)  is a major component of n fragment information . Both acetonitrile  and formic acid  were of LC-MS grade, while methanol of LC grade was provided by Fisher Scientific . Additional analytical reagents were obtained from Aladdin Industrial Corporation.The standard compounds were identified by NMR spectrum, and the purity of no less than 98% was determined by HPLC-UV. Isochlorogenic acid A , isochlorogenic acid B , isochlorogenic acid C , neochlorogenic acid , cryptochlorogenic acid , and chlorogenic acid  were provided by ChengDu ManSiTe Bio-Technology .Six male Sprague-Dawley rats (180 ± 20 g) were purchased from Hunan SJA Laboratory Animal Company in China and housed at a constant temperature of 24 ± 2 °C for a week of acclimatisation. The rats were randomly divided into two groups after fasting for 12 h with free access to water: group A, drug group for plasma and tissue; and group B, blank group for blank plasma and tissue. In accordance with the national law on the use of experimental animals, the experiment was approved by the Medicine Ethics Review Committee for Animal Experiments at the Hunan University of Medicine.3,5-DiCQA was dissolved in deionised water and orally administered at a dose of 200 mg/kg body weight to group A, followed by blood samples collected from the posterior orbital venous plexus at 0, 0.5, 1, 2, and 4 h. Each blood sample was then centrifuged at 4000 rpm for 15 min to obtain the plasma. The tissues from the heart, spleen, lung, kidneys, liver, and brain were immediately dissected and flushed with cold biological saline. All plasma and tissue samples were stored at −80 °C until further pre-treatment.The standard solution including isochlorogenic acid A, B, C, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid was dissolved in methanol and stored at 4 °C before analysis.All samples were pre-treated using the Solid Phase Extraction (SPE) method. Initially, the SPE column  was activated and equilibrated by flushing with 3 mL methanol and 3 mL water containing 0.5% formic acid. Then, the sample including 0.1 mL plasma or 1 mL supernatant of tissue homogenate was loaded on the preactivated column, followed by eluting with 3 mL water and 3 mL methanol containing 0.5% formic acid successively. Afterward, the methanol eluate was dried with a stream of nitrogen at room temperature to obtain the residue, which was again re-dissolved in 100 μL 5% acetonitrile-water and centrifuged at 13,000 rpm at 4 °C for 30 min. Finally, 2 μL of supernatant was injected into the UHPLC-Q-Excative Orbitrap MS for analysis.The tissue was homogenised in 1:5 g/mL biological saline using a homogeniser . The homogenates were then centrifuged at 4000 rpm for 15 min to obtain the supernatant, which was further pre-treated by the SPE method as mentioned above.A total of 25 μL β-glucuronidase  was added to 100 μL of plasma, followed by the addition of 75 μL Tris HCL . Afterward, the sample was sealed and incubated at 37 °C for an hour. Finally, the sample was pre-treated according to the SPE method as described above.UHPLC analysis was performed using the Ultimate 3000 system  equipped with an online vacuum degasser, a quaternary pump, and an automatic sampler. The samples were separated by an HYPERSIL GOLD C18 column . Water with 0.1% formic acid (A) and acetonitrile (B) was used as the mobile phase. The flowing gradient at the rate of 0.3 mL/min was applied at 35 °C: 0–2 min, 2–5% B; 2–5 min, 5–8% B; 5–10 min, 8–15% B; 10–15 min, 15–18% B; 15–20 min, 18–40% B; 20–24 min, 40–80% B; 24–26 min, 80% B; 26–27 min, 80–2% B; and 27–30 min, 2% B.n analyses were performed on Q-Exactive Focus Orbitrap MS  and the mass spectrometry was analysed using a negative heating electrospray ionisation source . The optimised parameters were set as follows: the flow rate of sheath gas (nitrogen) was 30 arb and the flow rate of auxiliary gas was 10 arb; Spray voltage 3.0 kV; Capillary temperature 320 °C; and auxiliary gas heater temperature 350 °C; High- resolution MS scanning was conducted at full MS scan in the mass range of m/z 100–1200 with a resolution of 35,000 and MS2 at a resolution of 17,500 triggered by parallel reaction monitoring mode; nitrogen (99.999% purity) was used as a collision gas; and the energy was set to 30% normalised collision energy.ESI-MS30 H60 O20 S4 N10 CL4; MS and MS2 with mass tolerances of 5 and 10 ppm, respectively; the tolerance of retention time was 12 s; and a different analysis was selected for post-processing.The Xcalibur version 4 workstation  was used to acquire all the raw data. The data was then analysed by Compound Discover version 3 with the metabolomics workflow template to detect the differential components between the drug and blank samples. The optimised parameters of metabolomics workflow template were set as follows: the minimum peak intensity was set at 10000; the maximum element count was set at CIn order to profile and identify the metabolites of 3,5-DiCQA comprehensively, an analytical strategy was based on UHPLC-Q-Exactive Orbitrap MS. Initially, the comparable samples including plasma and tissue were prepared using the SPE method. The samples were then analysed by UHPLC-Q-Exactive Orbitrap MS acquired by full mass scanning mode to obtain the full mass raw data. The data was processed on the Compound Discover version 3 workstation using the metabolomics workflow template to detect the differential components between the drug and blank samples. Afterward, the sample was injected into the UHPLC-Q-Exactive Orbitrap MS again to obtain the fragmentation ions acquired by parallel reaction monitoring mode triggered by differential components detected above. Finally, the metabolites of 3,5-DiCQA were characterised based on accurate MS, fragmentation ions, retention time and bibliography.Supplementary Table S1, respectively. The high-resolution extraction ion chromatography of these compounds is illustrated in A total of 33 metabolites as well as the parent drug itself  were detected and identified based on UHPLC-Q-Exactive Orbitrap MS, while 30 of these metabolites were reported for the first time. The retention time and mass spectrometry data of these metabolites are shown in Peaks 2, 9, 12, 21, 22, and 28 were accurately identified as 3-CQA, 5-CQA, 4-CQA, 3,4-DiCQA, 3,5-DiCQA, and 4,5-DiCQA, respectively by comparing the retention time, accurate MS, and fragmentation ions.− at m/z 353.08936 , and 515.11938 , respectively as peaks 2, 9, 12, and 21, 22, 28. By comparing the fragment ion at m/z 191.0547 and the retention time as reported in another study , and 529.13397 , 14 Da (CH2) more than CQA  and DiCQA , suggesting that they were methylation products of CQA and DiCQA, respectively. Peaks 11 and 31 yielded the fragmentation ion at m/z 193.0502 and 191.0554, respectively; therefore, peaks 11 and 31 were tentatively identified as 3-FQA, and 3 C, 5FQA, respectively , 28 Da (2CH2) more than DiCQA, suggesting that they were dimethylation products of DiCQA , which yielded the base peak ion at m/z 179.034 by the loss of the SO3 moiety (80 Da). Their MS2 spectrum is consistent with the spectrum of caffeic acid (CA), therefore, they were tentatively characterised as sulphation products of CA. In the same way, peaks 1 and 4 were tentatively confirmed to be sulphation products of dihydrocaffeic acid (DHCA); peaks 8 and 10 were sulphation products of dihydroferulic acid (DHFA); and peak 13 was sulphation products of ferulic acid (FA).Peaks 5 and 6 were eluted at 6.44 and 7.14 min, respectively. Both displayed the same pseudomolecular ions [M–H]− at m/z 355.0669 , 355.0670  and 355.0670 , respectively, and yielded the base peak ion at m/z 179.034 by the loss of 176 Da glucuronide. This data suggested that these metabolites were glucuronide conjugation. In the MS2 spectrum, ions at m/z 179.034 and 135.044 were observed, indicating that it could be attributed to CA. Therefore, they were tentatively inferred as glucuronide conjugation of CA. Peak 16 was eluted at 9.27 min with the pseudomolecular ions [M–H]− at m/z 369.08221 , 14 Da (CH2) more than peaks 3, 7, and 14, suggesting that it might be the methylation and glucuronide conjugation of CA. This finding was confirmed by the natural loss at m/z 176.033  in the MS2 spectrum. Therefore, peak 16 was tentatively identified as glucuronide conjugation of FA.Peaks 3, 7, and 14 produced their [M-H]− at m/z 691.1510  691.15063 , 691.14923  and 691.15143 , 176 Da (C6H8O6) more than DiCQA, indicating that they might be glucuronide conjugation of DiCQA. Their fragmentation ions were similar to those of peaks 21, 22, 24, and 28, respectively; therefore, they were tentatively inferred as glucuronide conjugation of 3, 4-DiCQA, 3, 5-DiCQA, 1, 5-DiCQA, and 4, 5-DiCQA, respectively. In addition, peaks 26 and 27 were tentatively characterised as methylation and glucuronide conjugation of DiCQA, respectively. Lastly, peaks 23, 25, 29, and 30 were plausibly identified as dimethylation and glucuronide conjugation of DiCQA.Peaks 17–20 were eluted at 13.41, 13.59, 13.94, and 14.08 min, respectively yielding the pseudomolecular ions [M–H]In this study, the glucuronide conjugation metabolites proposed by mass spectrometry were further confirmed using β-glucuronidase hydrolysis. The peak area of glucuronide conjugation metabolites including peaks 3, 7, 14, 16–20, 23, 25–27, 29, and 30 was decreased, while the area of peaks 2, 9, 12, 21, 22, and 28 was increased after glucuronidase treatment. These results were consistent with the assignation of glucuronide conjugation metabolites based on mass spectrometry.The relative abundances of those metabolites of 3,5-DiCQA were compared by the peak area of those precursor ions in MS, which was displayed in In this study, a total of 33 metabolites were unambiguously or tentatively identified based on UHPLC-Q-Exactive Orbitrap MS along with metabolomics and parallel reaction monitoring mode. The major metabolic pathways of 3,5-DiCQA in rat plasma are proposed in Supplementary Table S1). These results showed that a total of 4 metabolites which include peaks 7, 22, 32, and 33, were found in lung and liver; two metabolites that include peaks 32 and 33 were observed in the spleen; peak 22 was found in the heart; no metabolites were detected in the brain; 11 metabolites including peaks 7, 11, 14, 16, 22, 23, 25, 29, 30, 32, and 33 were detected in the kidney, indicating that the kidney might be the main metabolic organ. Peaks 22, 32, and 33 were distributed more widely than the other metabolites, suggesting that these metabolites might be the truly effective forms for exerting pharmacological effects.The distribution of all the metabolites in rats tissues was studied for the first time (shown in in vivo. Therefore, further research is warranted to confirm these findings. In conclusion, this study proposes the metabolic pathways of 3,5-DiCQA for the first time, which will be helpful to further understand its mechanism and truly effective forms in vivo.An analytical strategy based on UHPLCQ-Exactive Orbitrap MS technology with metabolomics and parallel reaction monitoring mode was established to detect and identify the metabolites of 3,5-DiCQA. A total of 33 metabolites were characterised in rat plasma, while 30 of these were reported for the first time. The corresponding reactions including methylation, hydrolysis, sulphate conjugation, glucuronide conjugation, and their composite reactions were all observed in this study. The results demonstrated that this practical strategy has the potential to be widely applicable for the detection and identification of metabolites. A total of 11 metabolites were detected and identified in organs. Among these, it is suspected that the parent drug and dimethylation products of DiCQA play an important pharmacological role Click here for additional data file.


We measured cell mass and metabolic viability, mitochondrial transmembrane potential, superoxide anion content, and activity-like of caspase-3 and caspase-9 following treatment with the extracts and/or Dox. Also, selenium and vitamin C contents were measured in the plant extracts.  Bark extracts of these plants (1 and 25  The results confirmed that Dox treatment decreased cell mass, mitochondrial membrane potential and metabolic viability, increased mitochondrial superoxide anion, and stimulated caspase-3 and caspase-9-like activities. Pretreatment of the cells with the plant extracts significantly inhibited Dox cytotoxicity, with more significant results at the higher concentration. Measurements of selenium and vitamin C in the extracts revealed higher concentration of both when compared with other Cameroonian spices. A. lepidophyllus and M. myristica were effective against Dox-induced cytotoxicity, most likely due to their content in antioxidants. Both extracts of  Doxorubicin (Dox) is an anthracycline antibiotic commonly used alone or in combination with other therapeutics to treat several solid and hematologic tumors in both adult and pediatric patients , 2. Dox β) inhibition is also involved in the cardiomyopathy caused by anthracyclines [β cause DNA breaks and inhibit mitochondrial biogenesis in cardiomyocytes [Despite being a potent anticancer drug, the clinical use of Dox is impaired by its significant cumulative dose-dependent cardiotoxicity resulting in acute or long-term ventricular dysfunction and heart failure , 5. The cyclines . Animal myocytes , 12. We myocytes .in vivo and in vitro to counteract Dox cardiotoxicity, although with mixed results [Antioxidants have been used  results –16. Plan results . Phenoli results , 19.in vitro and in vivo [Extracts of plants used in traditional medicine, often validated over thousands of years, are often ignored in terms of their potential therapeutic effect and not considered for drug development purposes , 21. Alt in vivo , there i in vivo . It is n in vivo .Monodora myristica (Gaertn.) Dunal (M. myristica), commonly known as “African nutmeg,” belongs to the family Annonaceae, and it is widely distributed in Central and West African countries. Its fruits are used as spices in various Cameroonian dishes [M. myristica tree is important economically [n dishes . Studiesomically . Specifiomically . Previouomically .Afrostyrax lepidophyllus Mildbr. (A. lepidophyllus), commonly known as “country onion,” belongs to the family Huaceae, and has a more restricted distribution, occurring in the tropical forest of some Central and West African countries [A. lepidophyllus are used in the treatment of urinary infections, indigestions, enema, female frigidity, and snake bites [A. lepidophyllus against human glioblastoma (T98 G), human breast adenocarcinoma (MDA-MB 231), human malignant melanoma (A375), and human colon carcinoma (HCT116) cell lines have also been demonstrated [M. myristica and A. lepidophyllus against Dox cytotoxicity in cardiac cells were never investigated.ountries . Its fruountries . The barke bites . Althougke bites . The ant MDA-MB 2, human mnstrated . Still, M. myristica and A. lepidophyllus decrease the cytotoxicity of Dox on H9c2 cardiomyoblasts, a cell line often used as a model for cardiac cells and previously used to investigate potential interventions against Dox cytotoxicity [In this work, our objective was to test the hypothesis that hydroethanolic bark extracts from both toxicity –36.Penicillin, streptomycin, fetal bovine serum (FBS), and trypsin were purchased from Gibco-Invitrogen . High-glucose Dulbecco's modified Eagle's medium (DMEM), doxorubicin, dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), protease inhibitor cocktail , sulforhodamine B (SRB), and Bradford reagent were obtained from Sigma-Aldrich . Tetramethylrhodamine-ethyl ester (TMRE) was purchased from Invitrogen . Nitric acid, perchloric acid, ethanol, hydrochloric acid, sodium borohydride, 2,4-dinitrophenylhydrazine, and sulfuric acid were purchased from Sigma-Aldrich . Dox was dissolved in Milli-Q water at a stock solution of 25 mM.A. lepidophyllus and M. myristica were harvested in 2012 at Kala, a small village in the Center region of Cameroon. The plants were identified at the National Herbarium of Cameroon (YA) and voucher specimens were deposited under the numbers 39020 HNC and 27690/SFR/CAM, respectively. Both names were checked with Plant List (http://www.theplantslist.org). The samples were dried at room temperature, grounded into powder, and kept in dry conditions.The barks of 2O pH = 3) to obtain the extract. The mixtures were then filtered using Buchner funnel and Whatman No.1 filter paper. The process was repeated after 24 hours. The supernatant was concentrated using a rotavapor and dried in a freeze-drier  for 24 hours. The dried powders were conditioned and kept at 4°C for further experiments. Before the experiments, both lyophilized powder extracts were solubilized in 30/70 water/ethanol in a 50 mg/mL stock solution.Powders were macerated at the ratio of 1:10 (w/v) for 48 hours in a mixture of water/ethanol . After hydride generation using a NaBH4 solution, the absorbance measurement was completed in a quartz cuvette, heated over an air-acetylene flame at 196.0 nm.Determination of selenium (Se) content was performed as previously described by Diaz-Alarcon et al. . This me2SO4, produces a red-colored complex [µg/mL). Then, 0.13 mL of thiourea (10%) was added to remove the excess of bromine. A volume of 1 mL of 2,4-DNPH was added to the mixture, and the preparation was incubated for 3 hours at 37°C in a thermostated bath. The sample was then allowed to cool in an ice bath for 30 minutes and treated with 5 mL of H2SO4 (85%), with constant stirring. The solution was allowed to stand for 30 minutes, and the absorbance was read at 521 nm. The results were expressed using a standard curve obtained with several dilutions of pure ascorbic acid.This assay is based on the oxidation of ascorbic acid to dehydroascorbic acid in the presence of a bromine solution. The L-dehydroascorbic acid formed then reacts with 2,4-DNPH to produce an osazone, which, when treated with 85% H complex . A volumµg/mL of antimycotic (Fungizone® Gibco-Invitrogen), and 100 µg/ml of streptomycin in 75 cm2 tissue culture flasks at 37°C in a humidified atmosphere of 5% CO2. The culture media were changed every two-three days, and cells were passaged once they attained approximately 80% of confluence to avoid cell fusion and formation of multinucleated myotubules [µg/mL or 25 µg/mL in water/ethanol (30/70 v/v). As control, water/ethanol (30/70 v/v) (maximum of 0.05%) was used. The cells were then preincubated for 3 hours with the extracts before being coincubated for 24 hours with Dox at the concentrations of 0.5µM and 1µM.  .The H9c2 cell line , initially isolated from embryogenic rat heart tissue after several passages , shares µg/mL for 6, 24, or 48 hours. After treatment, the culture medium was removed, and the cells were rinsed with phosphate-buffered saline (PBS). Then, cells were then fixed in ice-cold methanol, supplemented with 1% acetic acid, and stored at −20°C for 24 hours. Following this process, the methanol/acetic acid was removed, and the plates were dried at 37°C. Later, 250 µL of 0.05% of SRB in 1% acetic acid was added. The fixed cells were incubated with the SRB solution for 1 hour at 37°C. The SRB solution was then discarded, and the plates were rinsed with 1% acetic acid solution, to remove the excess of unbound SRB, dried at room temperature. A volume of 500 µL of 10 mM Tris buffer (pH10) was added to dissolve the protein-bound SRB. The optical density was measured at 540 nm using a Biotek Cytation 3 reader . In order to assess the cytoprotection of the plant extracts against Dox-induced H9c2 cell death, H9c2 cells were pretreated with the plant extracts at the concentrations of 1 or 25 µg/mL for 3 hours before Dox treatment. H9c2 cells were then treated with 0.5 or 1 µM of Dox for 24 hours, as previously described [H9c2 cell mass was measured using sulforhodamine B (SRB) assay . This teescribed . After DµL of culture medium supplemented with 10 µg/mL of resazurin. Reduction to resorufin was measured by fluorimetry at 570 nm excitation and 600 nm emission using a Biotek Cytation 3 reader .The metabolic viability of H9c2 cells was assessed by the resazurin reduction assay . This asψm was indirectly assessed by staining H9c2 cells with the TMRE dye and its fluorescence determined in a microplate reader [µL of basal medium. A volume of 100 µL of TMRE (300 nM in HBSS medium) was added to the cells, which were incubated with the dye for 1 hour. The basal fluorescence was then read at 555 nm excitation wavelength and 580 nm emission wavelength using a Cytation 3 multiplate reader. Thereafter, 1 µL of a solution of FCCP plus oligomycin (100 µM/0.1 mg/mL) was added to the wells and the fluorescence was read at 555 nm excitation wavelength and 580 nm emission wavelength to obtain the maximum fluorescence. The cells were then fixed with ice-cold methanol in acetic acid, and the SRB assay was performed to normalize the fluorescence results to cell mass content.The Δe reader . The asse reader . This ase reader . The expe reader  with somµL of MitoSOX working solution  was added to the cells, followed by an incubation with the dye solution for 20 minutes at 37°C. The fluorescence was read for 90 min, at 37°C, with 510 nm excitation wavelength and 580 nm emission wavelength using a Cytation 3 multiplate reader. The cells were then fixed with ice-cold methanol in acetic acid, and the SRB assay was performed to normalize the results.The effect of the plant extracts on mitochondrial superoxide anion was determined using the fluorescence dye MitoSOX µL of lysis buffer  supplemented with 2 mM dithiothreitol (DTT) and protease inhibitor cocktail  was added [After treating H9c2 cells as previously described , in 150-as added . The celas added .μg and 25 μg of protein, respectively, were incubated in reaction buffer containing 25 mM HEPES (pH 7.5), 10% sucrose, 10 mM DTT, 0.1% CHAPS, and 100 µM caspase substrates Ac-LEHD-pNA (caspase-9) or Ac-DEDV-pNA (caspase-3), for 2 hours at 37°C. Caspase-like activities were determined by following the colorimetric detection of the chromophore p-nitroanilide at 405 nm after cleavage from the labeled substrate. The method was calibrated with known concentrations of p-nitroanilide (pNA), and the results were expressed as % pNA released.To measure caspase-9 and caspase-3-like activities, aliquots of cell extracts containing 50 t-test. Significance was accepted when p value < 0.05.Data statistical analyses were performed by using GraphPad Prism 6.0 program . Data is expressed as means ± SD, of six independent experiments, with 6 biological replicates for each condition. Multiple comparisons were performed using two-way analysis of variance (2-way ANOVA) followed by Dunnett's multiple comparisons test. Comparisons between two treatment groups were performed using a Student's A. lepidophyllus and 1.05 ± 0.01 mg/Kg for M. myristica (Echinops giganteus (<0.5 ± 0.00 mg/Kg) and Aframomum daniellii (0.7 ± 0.03 mg/Kg) [A. lepidophyllus (Afro) and M. myristica (Mono) extracts. Our results showed that their respective content in vitamin C (2.57 ± 0.87 mg/Kg for Afro and 2.89 ± 0.55 mg/Kg for Mono) was higher than that of several plants with similar properties  . The con3 mg/Kg) , was detoperties .µg/mL) previously tested in a pilot dose-response study. The data shows that plant extracts, per se, did not interfere with cell mass after 24-hour treatment. At the 48-hour time point, a small, but significant decrease of cell mass was observed for both extracts  partly prevented the loss of cellular metabolic viability by around 6% (Afro) and 9% (Mono), but only in the highest concentration of Dox tested (1 µM)  is a critical mediator for ATP generation and for several mitochondrial roles in the cell . A decre Figures .A. lepidophyllus and M. myristica plant extracts, respectively, at 25 µg/mL prior to Dox incubation (1 µM)  when cells were treated with 1 µM Dox, as shown in Figures Caspase-dependent cell death is implicated in the toxicity of Dox on H9c2 cells . Our nexreatment . After treatment , caspaseA. lepidophyllus and M. myristica extracts against Dox-induced cardiotoxicity in H9c2 cells. These two plants were chosen because of their uses in traditional Cameroonian medicine for the treatment of a variety of pathological conditions and because of previous publications reporting their high content in phenolic compounds [Doxorubicin is a highly active therapeutic agent used against a vast number of malignant diseases, including hematological and solid tumors ; howeverompounds , 31, 59 ompounds , 61.μM for 24 hours) caused cytotoxicity on H9c2 cells, as indicated by cell mass loss, decreased metabolic viability, loss of ΔΨm, increased levels of mitochondrial superoxide anion, and increased caspase-3 and caspase-9-like activity, consistent with our previous studies in the same model [µg/mL) prevented Dox-induced cytotoxicity in H9c2 cells, especially at the highest concentration tested. It is noteworthy to observe that the extracts, per se, showed no relevant toxicity, when cell mass was measured after 24 hours of incubation. As for the 48-hour treatment time point, a small decrease in cell mass was observed.Under our experimental conditions, Dox  [Diverse mechanisms are involved in Dox-induced apoptosis, including binding to mitochondrial membranes and pro-oxidant redox cycles . Our rese (AMPK) .One explanation for the results is that the plant extracts' antioxidant activity may be responsible for the observed protection against Dox cytotoxicity. We previously demonstrated the high phenolic content of these same extracts , 29. PolThese extracts are also endowed with a significant content of selenium and vitamin C, which can also contribute to reducing Dox-induced damage in the cells. In fact, selenium and vitamin C concentrations in the two extracts were higher than those found in other commonly used Cameroonian spices .Selenium is a critical component of the antioxidant enzyme glutathione peroxidase (GPx) active site, playing an essential role in its spatial conformation and its catalytic activity . The conµM) decreased ROS generation and increased H9c2 viability after Dox treatment [A. lepidophyllus and M. myristica is higher than those found in Mondia whitei and Aframomum daniellii [M. myristica and A. lepidophyllus apart from the previously studied plants in this field.Recent findings reported that vitamin C (25 reatment . Vitaminaniellii . Therefoaniellii . AlthougM. myristica showed a higher protective effect against Dox than A. lepidophyllus. One possibility is that the higher content in vitamin C present in M. myristica could account for the difference, although both plants' phenolic composition and their intrinsic antioxidant activity may also explain at least some of the differences [When comparing the two extracts, ferences , 67.A. lepidophyllus and M. myristica exerted protection against in vitro Dox cytotoxicity on H9c2 cells by decreasing mitochondrial superoxide anion, preserving mitochondrial membrane potential, and reducing caspase-like activity. Therefore, this initial study indicates that both extracts should be further investigated in animal models of Dox-induced cardiotoxicity to potentially validate a novel antioxidant therapeutic strategy to counteract Dox cardiotoxicity.In conclusion, the current study showed that extracts from


The present investigate was intended for adsorption of heavy metals i.e. Pb, Cu, Cr, Zn, Ni and Cd onto activated charcoal prepared from neem leaf powder (AC-NLP) using batch and column studies. Batch adsorption was performed using different variables like adsorbent dose, temperature and contact duration. Thermodynamic analysis of batch treatment concluded that adsorption is thermodynamically feasible and endothermic. This adsorption followed the Pseudo second-order kinetic model derived from correlation coefficient values of chemical kinetic studies. For column study, interpretation of breakthrough curves and parameters were conducted by varying flow rate, initial concentration and bed height; and reveal that optimum conditions were lower flow rate (5 mL/min) and lower initial concentration (5 mg/L) and higher bed height (20 cm). Comparisons of batch and column study through isotherm models were evaluated and column study is more preferred than batch treatment. Maximum Thomas adsorption capacity was achieved upto 205.6, 185.8, 154.5, 133.3, 120.6, 110.9 mg/g for Pb, Cu, Cd, Zn, Ni and Cr respectively. This removal pattern is elucidated by metal ionic properties. Various adsorbing agents such as acids and bases were utilized for adsorption–desorption of AC-NLP. These metals are available from earth’s crust in very low concentration and found in their metallic, elemental form or chemical bounded with other inorganic materials like carbonate, sulphate, oxide or rock. Metal is considered as a one of the potential pollutant after dye, as it is lethal even at very low concentrations, last long effect, and persistent nature non‐biodegradable i.e. do not undergo microbial or chemical degradation2. Due to their highly solubility in aquatic environments, heavy metals are spontaneously immersed by fishes and vegetables. When these metals are come in contact with human, they are penetrating into our body by water, air, food or absorption through skin. Air , soil  and water  are main sources of heavy metal by which they are accessible in environment4. These metals are came into environment via various natural processes  as well as anthropogenic activities . Out of these, some industries, agriculture and technological expansion are aggressively enhanced the potential exposure onto human significantly. Heavy metal may be affect on nervous system, damage various body organs such as liver, kidney, lungs, brain, and blood, causes Alzheimer disease, etc.5. Toxic effects of various metals were reviewed by various scientists8.Water pollution is one of serious problem facing all over the World today, in which wastewater containing heavy metals are continuously released into environment. In few years, there is drastically increasing the ecological and global public health fear connected with atmospheric contamination using these metals. Out of 91 metals, ten metals such as Ni, Co, Mn, Zn, Cr, Cu, Se, Ti and Sb are prime public concern as per World Health organization (WHO)9. Activated charcoal is used as an adsorbent due to its remarkably high porosity, enhanced pore size and higher adsorption capacities. However due to high cost, it operation is someway inadequate. Currently, investigations are ongoing for preparation of charcoal using low cost materials, natural materials, agricultural waste, etc.10. Regarding adsorption process, it is performed by batch, up-flow or down-flow continuous fixed bed, continuous fluidized bed, continuous moving bed and oscillated (pulsed) bed, in which batch and up-flow continuous fixed bed adsorption investigations are more feasible due to easy and cheap technique and can removed most of contaminations11. The Neem (scientific name: Azadirachta indica and Family Meliaceae—Mahogany) is a large evergreen plant that found most part of Asia and other countries. Its leave, fruits, branches and roots are being used as range of therapeutic treatment such as from controlling digestive disorders to diabetes and high cholesterol to cancer. It is also used as a household pesticide and in toiletries, fertilizer, lubrication, cosmetic, etc.12. Previously neem leaf powder was utilized as an adsorbent and corrosion inhibitor16. The sorption–desorption process is required to reduce solid waste pollution load and cost effective process9.Though several methods such as adsorption, coagulation, membrane, oxidation, biological, chemical precipitation, flotation, ion exchange, and electrochemical deposition with their merits and demerits, but adsorption process is widely utilized for removal of contaminations including metals from raw waterIn this paper, the feasibility for adsorption of heavy metals onto AC-NLP was analyzed using batch and fixed bed column study. Several process parameters like temperature, adsorbent dose and contact duration; thermodynamic studies and chemical kinetic analyses were conducted for batch treatment. For column study, breakthrough curves and parameters were accomplished for different initial concentration, flow rate and bed height. Different isotherms were utilized for evaluation the performance by their capacities. Also, experiments of regeneration of AC-NLP were conducted.17. This AC-NLP was characterized by particle size porosity, pore diameter and pore volume using Mercury Porosimeter and Thermo Quest (Model No.: PASCAL—140). Also, surface area of AC-NLP was analyzed by multipoint Brunauer–Emmett–Teller (BET) model. Degassing was conducted at temperature of 240–250 °C at time duration 10–12 h under vacuum before BET measurement. Experiments were conducted by preparing stock solutions of Cadmium(II), Copper(II), Chromium(II), Zinc(II), Nickel(II) and Lead(II) using CdCl2·H2O, CuSO4·5H2O, CrCl2·5H2O, Zn(NO3)2·6H2O, NiSO4·6H2O and Pb(NO3)2 respectively. Thereafter, stock solutions were further diluted as per required of initial concentrations. All the chemicals utilized in these experiments were purchased from Sigma Aldrich and used as it is.For batch and column experiments, mature leaves of Neem were collected from near-by area of Pacific School of Engineering, Gujarat. These leaves were thoroughly rinsed with double distilled water to eliminate dust and water soluble impurities, followed by oven dried. The oven dried leaves were powdered and sieved with a < 200 µm to get dried neem leaf powder (NLP). Thereafter, 50 g NLP was impregnated with 200 mL of 1 M phosphoric acid solution with stirred at temperature of 85 °C for 4 h. For preparation of carbonization, impregnated NLP was heated at 330–350 °C in a muffle furnace for 8 h and allowed to cool in desiccators; and used as an adsorbent. The selection of activation agent i.e. phosphoric acid was based on previous experiments conducted by Alau and co-scientists18. The batch experiments were performed with constant metal concentration (100 mg/L) and stirring speed of 200 rpm. A water bath shaker , Double beam UV–Vis Spectroscopy  and Flame Atomic Absorption Spectroscopy (AAS)  was used for analysis of metals in all the adsorption experiments. In batch experiments, thermodynamic parameters such as change in Gibbs-free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) were determined by differentiating the temperatures (300 to 325 K) and using Langmuir isotherm constant as follows.Batch and column operations were carried out as per Table L vs. T having slope of ΔH° and the intercept yields the value of ΔS°/T was plotted. The values of these parameters are suggested the important properties such as thermodynamic nature, spontaneity system, type of adsorption 19. Chemical kinetic studies using Pseudo first order, Pseudo second order, Elovich, Intra-particle diffusion model for batch process were also conducted and their correlation coefficients were calculated. The details of kinetic models, importance, their linear equations and plots were previously represented20. Further, column study was performed using up-flow continues fixed bed glass column  as per Fig. Straight line graph of ln K22. To analysis reusability, the sorption–desorption study was carried out onto previously exhausted column of 20 cm with flow rate of 10 mL/min and initial metal concentration of 50 mg/L. Different acids  and bases  having different concentrations  were passed though exhausted column at up-flow direction at flow rate of 2 mL/min. The amounts of metal from column outlet were determined. After elution of metal i.e. regeneration of column, it was washed with distilled water at flow rate of 2 mL/min, until pH became 6.5–7.5. Thereafter, adsorption of metal using regenerated column was conducted at flow rate, initial metal concentration and bed height of 10 mL/min, 50 mg/L and 20 cm respectively. This sorption–desorption cycles were conducted for seven times and regeneration efficiency was determined for each cycle using following equation.reg and qorg (mg/g) is adsorptive capacity of regenerated column and original capacity of the adsorbent respectively.For column study, various breakthrough parameters are analyzed for establish the efficiency and efficaciously of column study. Table 25. In batch and column isotherm, graph having straight line was plotted and slop and intercept value of this line was calculated and; thereafter, parameters are calculated.Adsorption isotherm plays vital role as it provide certain useful information such as understanding mechanism of adsorption, surface characteristics and affinity of adsorbate–adsorbent. It also describes the capacity of adsorbent. Various adsorption isotherms are available. The details of most of isotherm models are given previously3/g and 612 m2/g respectively. Previously, we had utilized NLP as adsorbent and; their particle size and surface area were 122 and 612 m2/g respectively. It shows that AC-NLP was increased the surface active sites during charcoal preparation and activation and may be more potential than raw NLP as an adsorbent.The percentage porosity, particle size, pore diameter, pore volume and surface area of AC-NLP was found to be 41%, 201 mesh, 9.5–9.9 nm, 0.098 cm26. Maximum percentage removal of Pb, Cu, Cr, Zn, Ni and Cd was found to be 100, 91.5, 83.5, 73.0, 64.4 and 50.9 respectively using this variable of adsorbent dose. So, order of adsorption capacity for metals are Pb > Cu > Cr > Zn > Ni > Cd. Experiments for evaluate the influence of temperature (300 to 325 K) were conducted at constant adsorbent dose and contact duration of 6 g/L and 120 min respectively for adsorption of heavy metals onto AC-NLP. Percentage removal and other thermodynamic parameters are presented in Table Figure 28.Influence of temperature can be explained using following four assumptions: (1) Increasing the temperature may be increase the diffusion rate of adsorbate molecules covering the external boundary layer and in the interior pores of the adsorbent particle, thus decrease in the viscosity of the solution. And therefore, it will alter the equilibrium capacity of the adsorbent and change the equilibrium towards the endothermic path, (2) The mobility of chemicals contributing pollutions like metals may be increasing with increasing the temperature and thus adsorption process is increased, (3) Increasing temperature may be boosts the energy of adsorbate, which lead to more interaction with active sites at the surface, and (4) At higher temperature, swelling effect of inner composition of adsorbent may be occur, which may be increase the penetration ability of adsorbate onto adsorbent29. Thereafter, straight line indicated that equilibrium after 90 min contact duration. It indicated that all vacant sites of AC-NLP are exhausted. The plots of chemical kinetics i.e. Pseudo first order, Pseudo second order, Elovich, Intra-particle diffusion model were drawn and mentioned in Fig. 2) were calculated. R2 values obtained were 0.970–0.933, 0.994–0.974, 0.938–0.844 and 0.974–0.926 for Pseudo first order [ln(Qe – Qt) vs t], Pseudo second order (t/Qe vs t), Elovich (Qt vs t), Intra-particle diffusion model (Qt vs t1/2) respectively. It shows that the adsorption of heavy metals onto AC-NLP approaches the Pseudo second order kinetic models. It was expected that metal adsorption of AC-NLP is chemisorptions and rate-limited step. Also, chemical bond is formed between metal species and AC-NLP and contributes to find site that maximize their coordination number with the surface. This phenomenon was previously study adsorption of Indigo Carmine30.The effect of contact duration on metal adsorption at different contact duration namely 30, 60, 90, 120, 150 and 180 min was studied at AC-NLP dose of 6 g/L and temperature of 315 K and results are mentioned in Fig. b and te); and breakthrough and exhaust volume (vb and ve) are decreased with increasing the flow rate and initial concentration. Also, it was observed that height of MTZ was decreasing with increasing flow rate and initial metal concentration. The adsorption capacity at 50% breakthrough capacity was decreased from 62,500.0 to 12,993.42 mg/g with increasing flow rate of metals from 5 to 20 mL/min respectively for Pb. The adsorption capacity at 50% breakthrough capacity was decreased from 21,381.58 to 6907.89 mg/g with increasing flow rate of metals from 5 to 20 mL/min respectively for Cr. Also, The adsorption capacities at 50% breakthrough capacity was decreased from 25,000.0 to 13,651.3 mg/g with increasing initial Pb metals from 25 to 100 mg/L respectively. Also, The adsorption capacities at 50% breakthrough capacity was decreased from 8717.11 to 7236.84 mg/g with increasing initial Cr metals from 25 to 100 mg/L respectively. These statements stated the adsorption capacity was decreasing with increasing flow rate and initial metal concentration.Figure 32. The breakthrough: exhaustion time for Pb and Cr achieved at 128:177 and 58:107 min respectively and these times for other metals i.e. Cu, Cd, Zn and Ni were in-between at flow rate, initial metal concentration and bed height of 5 mL/min, 50 mg/L and 10 cm respectively.About flow rate, volume required for exhaustion was extensively increasing with decreasing flow rate. For smaller flow rates, removal efficiency of heavy metal is increases and column was difficult to exhaust. This is due to decrement of contact time between adsorbate and adsorbent, which diminish in adsorption capacity and service time of the bed. Also, effect of flow rate is explanted by mass transfer prerequisites. The rate of mass transfer is increased at higher flow rate and thus, reduces the residence time for metal adsorption onto the surface of AC-NLP. With enhancing the initial concentration of metal, the column is saturate rapidly and thus, decline the breakthrough timeb and te); and breakthrough and exhaust volume (vb and ve) are increased with increasing the bed depth. The adsorption capacities at 50% breakthrough are calculated from breakthrough parameters and shown that it was increasing from 3714.29 to 11,967.21 mg/g with increasing bed depth from 5 into 10 cm respectively for lead. And same was increasing from 7540.98 to 15,714.29 mg/g with increasing bed depth from 5 into 10 cm respectively for chromium. This is due increases number of sorption site i.e. total surface area of AC-NLP with in bed height. Moreover, enlargement of mass transfer zone and height of MTZ is due to fact that decreasing slope of breakthrough curve with increasing bed height. The circumstance of axial dispersion in the mass transfer was more potential with decreasing bed height. This reduces the diffusion of the solute which has not enough time to contact and hence, diffuse into entire adsorbent mass33. Also, heights of MTZ (HMTZ) were below 3 cm, so, fixed bed adsorption of heavy metals onto newly prepared adsorbent is feasible. It is due the fact that column can be functioned over a long time before becoming completely exhausted. From column parameters and breakthrough parameters, it was concluded that lead required the highest time and chromium required least time period to reach the breakthrough concentration that metals, which revealed that adsorption of lead is highest and Cadmium is lowest onto AC-NLP among investigated metals.Breakthrough behaviour of the metal column adsorption process onto AC-NLP at different bed height  with constant inlet metal concentration and of flow rate 10 mg/L and 10 mL/min respectively was investigated and shown as breakthrough curves in Fig. 2) i.e. nearer to one for all models indicates the applicability of all models. For batch isotherm, values of Langmuir affinity constant (KL) and Freundlich sorption intensity (n) are between 0 and 1 for all metals, which confirmed the adsorption is favourable. Maximum monolayer uniform adsorption capacity of Langmuir isotherms are 174.7, 154.5, 144.2, 133.4, 122.5 and 111.5 mg/g for Pb, Cu, Cd, Zn, Ni and Cr respectively. Temkin model indicates the nature of adsorption is endothermic and heat required for enhancement of adsorption as Temkin parameter related to the heat adsorption b (J/mol) > O. From correlation coefficient (R2) mentioned in Table 2 ≤ 0.9948) was closely fits to experimental data than other investigated model i.e. Freundlich model (0.9810 ≤ R2 ≤ 0.9884) and Temkin model (0.9721 ≤ R2 ≤ 0.9762), which confirmed that Langmuir model was significantly expressed metal adsorption onto AC-NLP. However, Langmuir model is depends on presumption of identical adsorption heat and monolayer adsorption. So, it suggests the monolayer coverage of metals on the flat surface of the AC-NLP having homogeneous distribution of active sites available for sorption on the adsorbent surface. For column study, optimum parameters were selected i.e. lower flow rate (5 mL/min) and initial concentration (5 mg/L) and higher bed height (20 cm) derived from column parameters and breakthrough variables for adsorption of heavy metals i.e. Pb, Cu, Cd, Zn, Ni and Cr onto AC-NLP. So, these parameters were selected for column isotherm study. Maximum Thomas adsorption capacity were achieved up to 205.6, 185.8, 154.5, 133.3, 120.6 and 110.9 mg/g for heavy metals, Pb, Cu, Cd, Zn, Ni and Cr respectively. Derived from correlation coefficient (R2), Thomas model is more preferred than other investigated isotherms such as BDST and; Adam & Bohart model, which indicates that follow second-order reversible reaction kinetics and also no axial dispersion. Table Table +2 is lower; hence adsorption of lead is highest. Further, order of ionic radius: Pb+2 (120 pm) > Cd+2 (95 pm) > Zn+2 (74 pm) > Cu+2 (73 pm) > Ni+2 (69 pm) > Cr+2 (63 pm). Due to steric overcrowding on the surface, large ionic radius encourages a quick saturation of adsorption sites. As the lead ion (Pb+2) has higher ionic radii, so, lead has maximum adsorption capacity compared to other metal ions. While increasing ionic strength, the distribution coefficients (KD) are decreases and initial metal concentrations of metals are increases, which trends to reduction in adsorption process. The value of KD for lead is lowest than other investigated metals, so, adsorption of lead is higher.From batch as well as column study, order of removal for heavy metals was lead > copper > Cadmium > Zinc > Nickel > Chromium. This trend of removals is observed due to mobility of heavy metal and thereafter, their ionic properties in the aqueous phase, because adsorption is also depends upon various ionic properties such electronegativity, ionic radius, hydration radius, degree of hydration, ionic strength, etc. The electronegativies of Pb, Cu, Cd, Zn, Ni and Cr are 1.90, 1.87, 1.65, 1.91, 1.69 and 1.66 respectively. More electronegativity tents higher cation ion exchange at the surface and thus, enhances the adsorption. Also, ion exchange process also depends upon their degree of hydration and hydration radius. The degree of hydration and hydration radius of Pb2SO4) may be destroyed the structure of exhausted AC-NLP, and lower concentration (0.1 M) and weak acid  required more time and volume to regenerate the AC-NLP due to may be strong bond formation between heavy metal and AC-NLP. From sorption–desorption data, it was indicated that regeneration efficiencies was slow decreasing from 98.2 ± 3.5 to 65.9 ± 4.9% for all metals in consecutive seven cycles. Also, weight loss of 2% was found in each cycle. It implied that adsorption of heavy metals onto AC-NLP was reversible process, as per discussed in chemical kinetic study. Further, total regeneration was not achieved due to adsorption was chemisorption and strong bond was formed between heavy metal and AC-NLP during adsorption; and not disintegrated even strong acid, hydrochloric acid40.Among acids and bases having different concentrations, all acids (i.e. low pH) particularly, 0.5 M HCl was found more effective for seven successive sorption–desorption cycles for all instigated metals  using AC-NLP. Suitability of acid was explained by proton exchange principle, which stated that higher number of protons were required for desorption of cationic heavy metals, thus, release the deposited metal from AC-NLP  and; regenerate of AC-NLP. So, regeneration capacity of acid was very high and that of alkali was least. Further, higher concentration of HCl (1.0–2.0 M) and strong acid , endothermic and favourable.2), order of chemical kinetin model for pertinence using adsorption data was Pseudo second order > Intra-particle diffusion model > Pseudo first order > Elovich. It suggested that adsorption follows the Pseudo second order kinetic models, considering that adsorption was chemisorption and rate-limited step.From correlation coefficient values  and initial concentration (5 mg/L) and higher bed height (20 cm).2 values.Parameters including maximum adsorption capacity of batch  and column  isotherm were calculated. Langmuir and Thomas isotherm was more convenient model that other investigated models for batch and column model respectively, derived from RThe order of metal removals was Pb > Cu > Cd > Zn > Ni > Cr, which was explained using various ionic parameters of metals.Regeneration efficiencies of AC-NLP was achieved from 98.2 ± 3.5 to 65.9 ± 4.9% in consecutive seven cycles using 0.1 M HCl, which concluded that adsorption was reversible and chemisorption.Batch and column adsorption study is conducted for removal of heavy metals such as Pb, Cu, Cd, Zn, Ni and Cr using activated charcoal prepared from neem leaf powder; and following conclusions were made.