Pesticides are synthetic chemical compounds that are toxic and may cause toxicity when exposed in... more Pesticides are synthetic chemical compounds that are toxic and may cause toxicity when exposed in large quantities. The leading site of action of these compounds is the inhibition of acetylcholinesterase. Quantifying the pesticide levels in the human body can help understand and study pesticide exposure in humans. The study aimed to develop and validate a rapid and accurate liquid chromatography–tandem mass spectrometry method for the quantitative determination of pesticide concentration in human blood samples. The analytes were separated using an Agilent Stable Bond C18 column and a mobile phase containing 10 mM ammonium formate in water and acetonitrile. The limits of detection and quantitation from the developed method were 0.023–0.161 and 0.072–0.487 ng/mL, respectively, while the recoveries from the spiked blood samples were 78.01–104.36%. The intraday and interday accuracies and precision were 40–105% and 0.5–13 RSD% and 40–113% and 3–14 RSD%, respectively. Linear regression was obtained at a value of > 0.9921, indicating the method to be accurate and precise. Validation was further performed on pesticide exposure and control blood samples effectively. The developed method can be employed to estimate the levels of exposure in blood samples since the method has been found to be accurate and reliable under the European standard guidelines.
Rationale: Occupational or environmental exposure to organophosphate pesticides widely used world... more Rationale: Occupational or environmental exposure to organophosphate pesticides widely used worldwide can lead to significant health problems. Monitoring the levels of dialkyl phosphates (DAP) metabolites in human urine is a common biomarker for assessing such exposure. In this study developing a new screening method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (UFLC-MS/MS) to detect six DAP metabolites in human urine. Methods: Three different compound extraction methods, namely QuEChERS, lyophilization, and liquid extraction, were compared in this study. The performance of each method was evaluated based on the recovery rate. The liquid extraction method provided better results and required only 20 minutes for analysis. The recovery rate, repeatability and reproducibility were determined to assess the method reliability. Additionally, the limits of detection and limits of quantitation were established to determine the lowest concentrations at which t...
Several methods have been reported to estimate Acetylcholinesterase (AChE) enzyme activity in blo... more Several methods have been reported to estimate Acetylcholinesterase (AChE) enzyme activity in blood samples. The Ellman assay is the most important among all but with several shortcomings, and there is a need to develop a method which is accurate, sensitive and quick for analyzing AChE. Therefore, we have developed an assay utilizing RP-HPLC with UV detection for the determination of AChE activity. This method measured the conversion of 1-naphthol acetate to 1-naphthol to estimate AChE activity in blood samples. Performance was judged on the basis of reproducibility, sensitivity, accuracy, and the ability to screen enzyme activity within 20minutes. A series of experiments were performed, varying the concentration of blood and substrate, with optimal sensitivity using 50 μM substrate and 10μL blood. The validation parameters such as linearity (R2 of ≥ 0.9842 for 1-naphthol and ≥ 0.9897 for 1-naphthol acetate), precision (94.21–96.41%), accuracy (85.2%–99.6% and 82.6%–99.3% for 1-naph...
OBJECTIVE In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and val... more OBJECTIVE In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and validation of oxidized product by a validated ultra high-performance liquid chromatography (UHPLC) method. METHODS Two hundred milligrams of EGCG was oxidized in 5 mL of hydrogen peroxide (H2O2) and was identified by a validated UHPLC method with precision and robustness. Confirmation of parameters like C-H stretching and mass was carried out using infrared spectroscopy and mass spectroscopy, respectively. Identification of oxidized EGCG (O-EGCG) was done by UHPLC. RESULTS The infrared spectroscopy chromatograms observed less intensity C-H stretching as compared to O-EGCG. The mass of EGCG and O-EGCG were 459.09 and 915.16, respectively. Structure elucidation revealed a loss of one proton in O-EGCG as compared to EGCG. Validation of the developed method was specific, with linear correlation coefficient 0.9981 and 0.9917, respectively for EGCG and O-EGCG, the accuracy rate of 95.2%-99.6% for EGCG, and 99.18%-101.5% for O-EGCG. CONCLUSION Together, the results of this study demonstrate the formation of a dimer also the UHPLC method developed for identification of both EGCG and O-EGCG is validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.
Guazuma ulmifolia Lam. belonging to the family Sterculiaceae and commonly called Pigeon wood or B... more Guazuma ulmifolia Lam. belonging to the family Sterculiaceae and commonly called Pigeon wood or Bastard cedar, in Telugu known as Bhadraksh or Rudraksha. It is Native to Tropical America and widely distributed in tropical America from Mexico to northern part of Argentina & the middle part of Brazil. The bark and leaves have been traditionally used antibacterial, antidysenteric, antifungal, anti-inflammatory, antimicrobial, astringent, depurative, diaphoretic, emollient, febrifuge, hepatoprotective, pectoral, refrigerant, stomachic, styptic and sudorific. Phytochemically plant contains fatty acids, steroids, flavanoids, tannins, glycosides, carbohydrates, mucilage. Preclinical study on plant performed till date Anti-diabetic action, Anti-Hypertensive, Anti-microbial action, Anti-Oxidant, Antiulcer Action, Hair growth promoter, Weight loss agent.
Spectrophotometric stability indicating procedures are described for determination of glafenine i... more Spectrophotometric stability indicating procedures are described for determination of glafenine in pure and dosage forms. First derivative of ratio spectra ( 1 DD) and multivariate calibration chemometric methods are proposed for determination of glafenine in presence of its ...
A simple, sensitive, accurate, precise, reproducible and inexpensive UV Spectrophotometric method... more A simple, sensitive, accurate, precise, reproducible and inexpensive UV Spectrophotometric method has been developed and subsequently validated for the determination of Naftopidil in bulk and its pharmaceutical formulation. Methanol was used as a solvent in the present investigation. The UV spectrum was scanned between 200 to 400 nm and 232 nm was selected as maximum wavelength for absorption. Beer’s law was obeyed in the concentration range of 2-10 µg/ml with correlation coefficient r = 0.9994.The percent recoveries of naftopidil were found to be 99.75 to 100.293.The intra and inter day precision percent relative standard deviation values in the range of 0.41 to 0.86 and 0.33 to 0.74.The LOD and LOQ were found to be 0.089 µg/ml and 0.27197 µg/ml. The method has been successfully utilized to determine the naftopidil in tablets and can be extended for routine analysis in bulk drugs. Results of the analysis were validated as per ICH guidelines.
Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and fo... more Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and form oxidised products or dimers. In the present study, we aimed to evaluate the therapeutic effects, including antioxidation and immunomodulatory action, of the Oxidised Epigallocatechin Gallate (O-EGCG) as compared to native EGCG and the action of these compounds on main protease (Mpro) docking against SARS-CoV-2. HCT-116 (Human Colon Cancer) cell lines were used to estimate the total antioxidant capacity and lipid peroxidation levels and pro-inflammatory markers (human IL-6, IL-1β, TNF-α). Further, molecular docking analysis was performed by AutoDock and visualised in Discovery studio. Improved antioxidant capacity of O-EGCG was observed, and there was a significant decrease in the inflammatory markers (IL-1β, IL-6, and TNF-α) when O-EGCG was applied as compared to EGCG. The O-EGCG was shown to be strongly associated with the highest docking score and active site residues of IL-1, IL-6,...
Pesticides are synthetic chemical compounds that are toxic and may cause toxicity when exposed in... more Pesticides are synthetic chemical compounds that are toxic and may cause toxicity when exposed in large quantities. The leading site of action of these compounds is the inhibition of acetylcholinesterase. Quantifying the pesticide levels in the human body can help understand and study pesticide exposure in humans. The study aimed to develop and validate a rapid and accurate liquid chromatography–tandem mass spectrometry method for the quantitative determination of pesticide concentration in human blood samples. The analytes were separated using an Agilent Stable Bond C18 column and a mobile phase containing 10 mM ammonium formate in water and acetonitrile. The limits of detection and quantitation from the developed method were 0.023–0.161 and 0.072–0.487 ng/mL, respectively, while the recoveries from the spiked blood samples were 78.01–104.36%. The intraday and interday accuracies and precision were 40–105% and 0.5–13 RSD% and 40–113% and 3–14 RSD%, respectively. Linear regression was obtained at a value of > 0.9921, indicating the method to be accurate and precise. Validation was further performed on pesticide exposure and control blood samples effectively. The developed method can be employed to estimate the levels of exposure in blood samples since the method has been found to be accurate and reliable under the European standard guidelines.
Rationale: Occupational or environmental exposure to organophosphate pesticides widely used world... more Rationale: Occupational or environmental exposure to organophosphate pesticides widely used worldwide can lead to significant health problems. Monitoring the levels of dialkyl phosphates (DAP) metabolites in human urine is a common biomarker for assessing such exposure. In this study developing a new screening method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (UFLC-MS/MS) to detect six DAP metabolites in human urine. Methods: Three different compound extraction methods, namely QuEChERS, lyophilization, and liquid extraction, were compared in this study. The performance of each method was evaluated based on the recovery rate. The liquid extraction method provided better results and required only 20 minutes for analysis. The recovery rate, repeatability and reproducibility were determined to assess the method reliability. Additionally, the limits of detection and limits of quantitation were established to determine the lowest concentrations at which t...
Several methods have been reported to estimate Acetylcholinesterase (AChE) enzyme activity in blo... more Several methods have been reported to estimate Acetylcholinesterase (AChE) enzyme activity in blood samples. The Ellman assay is the most important among all but with several shortcomings, and there is a need to develop a method which is accurate, sensitive and quick for analyzing AChE. Therefore, we have developed an assay utilizing RP-HPLC with UV detection for the determination of AChE activity. This method measured the conversion of 1-naphthol acetate to 1-naphthol to estimate AChE activity in blood samples. Performance was judged on the basis of reproducibility, sensitivity, accuracy, and the ability to screen enzyme activity within 20minutes. A series of experiments were performed, varying the concentration of blood and substrate, with optimal sensitivity using 50 μM substrate and 10μL blood. The validation parameters such as linearity (R2 of ≥ 0.9842 for 1-naphthol and ≥ 0.9897 for 1-naphthol acetate), precision (94.21–96.41%), accuracy (85.2%–99.6% and 82.6%–99.3% for 1-naph...
OBJECTIVE In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and val... more OBJECTIVE In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and validation of oxidized product by a validated ultra high-performance liquid chromatography (UHPLC) method. METHODS Two hundred milligrams of EGCG was oxidized in 5 mL of hydrogen peroxide (H2O2) and was identified by a validated UHPLC method with precision and robustness. Confirmation of parameters like C-H stretching and mass was carried out using infrared spectroscopy and mass spectroscopy, respectively. Identification of oxidized EGCG (O-EGCG) was done by UHPLC. RESULTS The infrared spectroscopy chromatograms observed less intensity C-H stretching as compared to O-EGCG. The mass of EGCG and O-EGCG were 459.09 and 915.16, respectively. Structure elucidation revealed a loss of one proton in O-EGCG as compared to EGCG. Validation of the developed method was specific, with linear correlation coefficient 0.9981 and 0.9917, respectively for EGCG and O-EGCG, the accuracy rate of 95.2%-99.6% for EGCG, and 99.18%-101.5% for O-EGCG. CONCLUSION Together, the results of this study demonstrate the formation of a dimer also the UHPLC method developed for identification of both EGCG and O-EGCG is validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.
Guazuma ulmifolia Lam. belonging to the family Sterculiaceae and commonly called Pigeon wood or B... more Guazuma ulmifolia Lam. belonging to the family Sterculiaceae and commonly called Pigeon wood or Bastard cedar, in Telugu known as Bhadraksh or Rudraksha. It is Native to Tropical America and widely distributed in tropical America from Mexico to northern part of Argentina & the middle part of Brazil. The bark and leaves have been traditionally used antibacterial, antidysenteric, antifungal, anti-inflammatory, antimicrobial, astringent, depurative, diaphoretic, emollient, febrifuge, hepatoprotective, pectoral, refrigerant, stomachic, styptic and sudorific. Phytochemically plant contains fatty acids, steroids, flavanoids, tannins, glycosides, carbohydrates, mucilage. Preclinical study on plant performed till date Anti-diabetic action, Anti-Hypertensive, Anti-microbial action, Anti-Oxidant, Antiulcer Action, Hair growth promoter, Weight loss agent.
Spectrophotometric stability indicating procedures are described for determination of glafenine i... more Spectrophotometric stability indicating procedures are described for determination of glafenine in pure and dosage forms. First derivative of ratio spectra ( 1 DD) and multivariate calibration chemometric methods are proposed for determination of glafenine in presence of its ...
A simple, sensitive, accurate, precise, reproducible and inexpensive UV Spectrophotometric method... more A simple, sensitive, accurate, precise, reproducible and inexpensive UV Spectrophotometric method has been developed and subsequently validated for the determination of Naftopidil in bulk and its pharmaceutical formulation. Methanol was used as a solvent in the present investigation. The UV spectrum was scanned between 200 to 400 nm and 232 nm was selected as maximum wavelength for absorption. Beer’s law was obeyed in the concentration range of 2-10 µg/ml with correlation coefficient r = 0.9994.The percent recoveries of naftopidil were found to be 99.75 to 100.293.The intra and inter day precision percent relative standard deviation values in the range of 0.41 to 0.86 and 0.33 to 0.74.The LOD and LOQ were found to be 0.089 µg/ml and 0.27197 µg/ml. The method has been successfully utilized to determine the naftopidil in tablets and can be extended for routine analysis in bulk drugs. Results of the analysis were validated as per ICH guidelines.
Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and fo... more Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and form oxidised products or dimers. In the present study, we aimed to evaluate the therapeutic effects, including antioxidation and immunomodulatory action, of the Oxidised Epigallocatechin Gallate (O-EGCG) as compared to native EGCG and the action of these compounds on main protease (Mpro) docking against SARS-CoV-2. HCT-116 (Human Colon Cancer) cell lines were used to estimate the total antioxidant capacity and lipid peroxidation levels and pro-inflammatory markers (human IL-6, IL-1β, TNF-α). Further, molecular docking analysis was performed by AutoDock and visualised in Discovery studio. Improved antioxidant capacity of O-EGCG was observed, and there was a significant decrease in the inflammatory markers (IL-1β, IL-6, and TNF-α) when O-EGCG was applied as compared to EGCG. The O-EGCG was shown to be strongly associated with the highest docking score and active site residues of IL-1, IL-6,...
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Papers by Ramakrishna Ungarala