Naringenin-Zinc Oxide Nanocomposites Amalgamated Polymeric Gel Augmented Drug Delivery and Attenuated Experimental Cutaneous Candidiasis in Balb/c Mice: In Vitro and In Vivo Studies.

Naringenin (NRG) inhibits the fungal 17ß-hydroxysteroid dehydrogenase accountable for ergosterol synthesis in Candida albicans (C. albicans), a causative agent for cutaneous candidiasis. In present research, NRG was complexed with ZnO nanomaterial (NRG-Zn2+) to synthesize NRG-Zn2+ nanocomposites. The particle size and ζ-potential of NRG-Zn2+ nanocomposites were respectively estimated to be 180.33 ± 1.22-nm and - 3.92 ± 0.35-mV. In silico data predicted the greater affinity of NRG-Zn2+ nanocomposite for 14α-demethylase and ceramide in comparison to NRG alone. Later, NRG-Zn2+ nanocomposites solution was transformed in to naringenin-zinc oxide nanocomposites loaded chitosan gel (NRG-Zn-CS-Gel) with viscosity and firmness of 854806.7 ± 52386.43 cP and 698.27 ± 10.35 g, respectively. The ex-vivo skin permeation demonstrated 70.49 ± 5.22% skin retention, significantly greater (P < 0.05) than 44.48 ± 3.06% of naringenin loaded chitosan gel (NRG-CS-Gel) and 31.24 ± 3.28% of naringenin solution (NRG Solution). NRG-Zn-CS-Gel demonstrated 6.71 ± 0.84% permeation of NRG with a flux value of 0.046 ± 0.01-µg/cm2/h. The MIC50 of NRG-Zn-CS-Gel against C. albicans was estimated to be 0.156-µg/mL with FICI (fractional inhibitory concentration index) of 0.018 that consequently exhibited synergistic efficacy. Further, NRG-Zn-CS-Gel demonstrated superior antifungal efficacy in C. albicans induced cutaneous candidiasis infection in Balb/c mice. The fungal burden in NRG-Zn-CS-Gel treated group was 109 ± 25 CFU/mL, significantly lower (P < 0.05) than positive control (2260 ± 446 CFU/mL), naringenin loaded chitosan gel (NRG-CS-Gel; 928 ± 127 CFU/mL) and chitosan gel (CS-Gel; 2116 ± 186 CFU/mL) treated mice. Further, histopathology examination and cytokine profiling of TNF-α, IL-1ß and IL-10 revealed the healing of skin and inflammation associated with cutaneous candidiasis infection. In conclusion, NRG-Zn-CS-Gel may be a potential candidate for translating in to a clinical viable topical nanotherapeutic.

Application of machine-learning model to optimize colonic adenoma detection in India.

AIMS: There is limited data on the prevalence and risk factors of colonic adenoma from the Indian sub-continent. We aimed at developing a machine-learning model to optimize colonic adenoma detection in a prospective cohort. METHODS: All consecutive adult patients undergoing diagnostic colonoscopy were enrolled between October 2020 and November 2022. Patients with a high risk of colonic adenoma were excluded. The predictive model was developed using the gradient-boosting machine (GBM)-learning method. The GBM model was optimized further by adjusting the learning rate and the number of trees and 10-fold cross-validation. RESULTS: Total 10,320 patients (mean age 45.18 ± 14.82 years; 69% men) were included in the study. In the overall population, 1152 (11.2%) patients had at least one adenoma. In patients with age > 50 years, hospital-based adenoma prevalence was 19.5% (808/4144). The area under the receiver operating curve (AUC) (SD) of the logistic regression model was 72.55% (4.91), while the AUCs for deep learning, decision tree, random forest and gradient-boosted tree model were 76.25% (4.22%), 65.95% (4.01%), 79.38% (4.91%) and 84.76% (2.86%), respectively. After model optimization and cross-validation, the AUC of the gradient-boosted tree model has increased to 92.2% (1.1%). CONCLUSIONS: Machine-learning models may predict colorectal adenoma more accurately than logistic regression. A machine-learning model may help optimize the use of colonoscopy to prevent colorectal cancers. TRIAL REGISTRATION: ClinicalTrials.gov (ID: NCT04512729).

Lesser prevalence of polyps/WNT-dysregulation and concomitant upregulation of gamma-catenin/MYC point to alternate pathways in colorectal cancer in India.

Colorectal cancer (CRC) is known to follow adenoma carcinoma sequence (ACS) in majority of the tumors and the driver variants and associated pathways are well delineated. However, most of the published data are from the west and information in other ethnicities is sparse. We therefore comprehensively evaluated the CRC tumors from Indian ethnicity for the prevalence of ACS. In this cohort study, clinical data of 100,497 patients who attended hospital between 2013 and 2018 were accessed. Tumors from patients (n = 130) with CRC who were treated primarily by surgery were included. DNA and RNA were isolated to assess variants (direct sequencing) and WNT-pathway dysregulation in genes related to ACS. Global gene expression was generated and analyzed on microarrays (Affymetrix; N = 10) and next generation sequencing platforms (Illumina; N = 25). Gene expression at mRNA (qRT-PCR) and protein level (IHC) of JUP/CTNNB1/MYC were assessed. Correlation between expression of JUP and MYC was evaluated by Karl Pearson's correlation coefficient. The prevalence of polyps was 16.75%, while 18.26% variants in APC/CTNNB1, 20.00% in KRAS, and 18.33% WNT dysregulation were noted. Interestingly, 29/60 (48.33%) tumors showed only MYC upregulation with normal APC/CTNNB1 expression. Global gene expression and validation in an independent tumor cohort confirmed concomitant upregulation of JUP (gamma-catenin) & MYC (r = 0.71; p = 0.001) at mRNA and protein in sizeable number of tumors (45/96; 46.88%). Our study provides evidence for limited prevalence of ACS in the Indian ethnicity. Preventive colonoscopies for early identification and management of CRC may not be an effective strategy in this ethnicity.

Technical performance and diagnostic yield of motorised spiral enteroscopy compared with single-balloon enteroscopy in suspected Crohn's disease: a randomised controlled, open-label study (the MOTOR-CD trial).

OBJECTIVE: Recent studies have shown that motorised spiral enteroscopy (MSE) enables deeper and total small bowel evaluation compared with single-balloon enteroscopy (SBE) in suspected Crohn's disease (CD) when analysed per procedure. However, no randomised controlled study has compared bidirectional MSE with bidirectional SBE in suspected CD. DESIGN: Patients with suspected CD requiring small bowel enteroscopy were randomly assigned to either SBE or MSE between May 2022 and September 2022 in a high volume tertiary centre. Bidirectional enteroscopy was done if intended lesion could not be reached on unidirectional study. Comparison was made with regard to technical success (ability to reach lesion), diagnostic yield, depth of maximal insertion (DMI), procedure time and total enteroscopy rates. Depth:time ratio was calculated to avoid confounding for the location of lesion. RESULTS: Among 125 suspected patients with CD (28% female, 18-65 years, median 41 years), 62 and 63 underwent MSE and SBE, respectively. The overall technical success (98.4 %: MSE, 90.5 %: SBE; p=0.11), diagnostic yield (95.2%: MSE; 87.3%: SBE, p=0.2) and procedure time were not significantly different. However, MSE appeared to have higher technical success (96.8% vs 80.7%, p=0.08) in deeper small bowel (distal jejunum/proximal ileum) with higher DMI, higher depth:time ratio and total enteroscopy rates when attempted (77.8% vs 11.1%, p=0.0007). Both the modalities were safe although minor adverse events were more common with MSE. CONCLUSION: MSE and SBE have comparable technical success and diagnostic yield for small bowel evaluation in suspected CD. MSE scores over SBE with regard to deeper small bowel evaluation with complete small bowel coverage and higher depth of insertion in a shorter time. TRIAL REGISTRATION NUMBER: NCT05363930.

Surgical management of cystic duct stump calculi causing post-cholecystectomy syndrome: A prospective study.

Aim: Remnant cystic duct stump calculi are an uncommon but important cause of 'post-cholecystectomy syndrome'. High index of suspicion is needed to diagnose this condition in a symptomatic post-cholecystectomy patient. We present our experience with the surgical management of this condition. Patients and Methods: This prospective study included 19 patients with residual gallstone disease who underwent completion cholecystectomy between August 2016 and October 2021. Investigations included abdominal ultrasound and magnetic resonance cholangiopancreatography. The demographic, clinical, surgical and early post-operative variables of these patients were prospectively maintained and analysed. Results: The study included 14 women and 5 men. The mean age was 42.1 years (range, 14-80 years). The median duration between index surgery and completion cholecystectomy was 36 months (range, 2-178 months) (interquartile range, 105 months). The follow-up duration was 2 months. The initial surgery was open cholecystectomy in 17 and laparoscopic cholecystectomy in 2 patients. All patients with residual stump stone presented with pain, while 10 out of 19 patients complained of dyspepsia. Completion cholecystectomy could be performed laparoscopically in 16 cases, whereas 3 patients underwent open surgery. The mean operative time was 80 min (range, 55-140 min), and the mean blood loss was 100 ml (range, 50-160 ml). The mean hospital stay was 3 days (range, 2-10 days). No post-operative mortality or major morbidity was recorded in any of our patients. Conclusion: Laparoscopic excision of the cystic duct stump is feasible and safe even after previous open cholecystectomy. It is increasingly becoming the treatment of choice where expertise is available.

Proposal of novel staging system CNM (Crohn’s primary site, nodes, mesentery) to predict postoperative recurrence of Crohn’s disease

After oncologic resection, histological grading and staging of the tumor give important prognostic information about the future risk of recurrence and hence influence the subsequent management plan. Several studies and their meta-analysis have shown that various histological features (e.g., microscopic positive resection margins, plexitis, granuloma, mesenteric inflammatory activity) can predict postoperative clinical/endoscopic/surgical recurrence after resection in Crohn’s disease (CD). Inclusion of mesentery in surgical resection specimens has been shown to reduce surgical recurrence after ileocolonic resection in CD. However, there is no uniform histopathological staging system for risk stratification in postoperative CD to systematically predict postoperative recurrence. This is because the prediction to date is based on clinical characteristics (smoking status, disease phenotype, surgical history). Histopathological predictors are still not adopted in routine clinical practice due to the lack of a uniform staging system, heterogeneity of published studies and lack of standardized definition of histological features. In this article, we attempted to incorporate all such histological features in a single histological staging system CNM (Crohn’s primary site [resection margin positivity, plexitis, granuloma, depth of infiltration], nodes [presence of granuloma], mesentery [involved or not]) in surgical resection specimen in CD. The proposed CNM classification would help to enable systematic reporting, design future clinical trials, stratify postoperative recurrence risk and choose appropriate postoperative prophylaxis.

A novel LC-MS/MS method for simultaneous estimation of acalabrutinib and its active metabolite acalabrutinib M27 in human plasma and application to a human pharmacokinetic study.

A simple, specific, selective and accurate bioanalytical method was developed and validated for simultaneous estimation of acalabrutinib and its active metabolite in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Deuterated analogs of both the analytes were used as internal standards. The extraction of analytes and internal standards were evaluated from the human plasma by liquid-liquid extraction technique using methyl tertiary butyl ether (TBME). The separation of the analytes was carried out on Zorbax Eclipse XDB-C18 (150 × 4.6 mm, 5 µm) column with a mixture of acetonitrile and 10 mM ammonium formate in 0.1% formic acid buffer (65 : 35, v/v) as mobile phase at a flow rate of 1 mL min-1. The method linearity was determined in the widen concentration range from 5.000 ng mL-1 to 1600 ng mL-1 with r 2 > 0.99. The entire method validation was carried out as per the USFDA guidelines on bioanalytical method validation and all validation experiment results were found within acceptable limits. Clinical pharmacokinetic study of both the parent drug and its active metabolite was successfully performed on six healthy volunteers under fasting conditions by applying the present method.

EUS versus MRCP to perform ERCP in patients with intermediate likelihood of choledocholithiasis: a randomised controlled trial.

OBJECTIVE: In patients with an intermediate likelihood of choledocholithiasis, European Society of Gastrointestinal Endoscopy (ESGE) guidelines recommend endoscopic ultrasound (EUS) or magnetic resonance cholangiopancreatography (MRCP) to diagnose choledocholithiasis to make the indication for endoscopic retrograde cholangiopancreatography (ERCP) treatment; there is no randomised control trial to compare both in this setting. DESIGN: Patients with suspected choledocholithiasis satisfying ESGE guideline's intermediate likelihood were screened for this single-centre randomised controlled trial between November 2019 and May 2020. The enrolled patients were randomised to either EUS or MRCP. ERCP was performed in stone positive cases or if clinical suspicion persisted during follow-up. Negative cases underwent a further 6-month clinical follow-up. Main outcome was accuracy (sensitivity/specificity) of both tests to diagnose choledocholithiasis, with ERCP or follow-up as a gold standard. RESULTS: Of 266 patients, 224 patients (mean age: 46.77±14.57 years; 50.9 % female) were enrolled; overall prevalence of choledocholithiasis was 49.6%, with a higher frequency in the MRCP group (63/112 vs 46/112 for EUS). Both sensitivity of EUS and MRCP were similarly high (92%-98%), without significant differences between the two groups. The negative predictive value and likelihood ratio + were significantly higher in EUS arm (p<0.05). The percentage of ERCPs either incorrectly halted back (false negatives: EUS: 2 vs MRCP: 5) or performed unnecessarily (false positives: EUS: 1 vs MRCP: 2) was low in both groups. CONCLUSION: The performance parameters of both EUS and MRCP are comparable for detecting choledocholithiasis in the intermediate-risk group of choledocholithiasis and the choice of a test should be based on local expertise, availability of resources and patient preference. TRIAL REGISTRATION NUMBER: NCT04173624.

Endoscopic full-thickness plication for the treatment of PPI-dependent GERD: results from a randomised, sham controlled trial.

BACKGROUND: The majority of endoscopic antireflux procedures for GERD are cumbersome to use and randomised long-term data are sparse. We conducted such a trial to determine the efficacy and safety of a novel, easy to use endoscopic full-thickness fundoplication (EFTP) device in patients with GERD. DESIGN: Patients with proton pump inhibitor (PPI)-dependent GERD were randomised to either EFTP or a sham procedure in 1:1 ratio. The primary endpoint was ≥50% improvement in the health-related quality of life (GERD-HRQL) score at 3 months. Secondary end points included improvement in GERD-HRQL, reflux symptom scores, PPI usage, oesophageal acid exposure and reflux episodes and endoscopic findings at 3, 6 and 12 months. RESULTS: Seventy patients were randomised; 35 in each group with a median (IQR) age of 36 (29-42) years, 71.4% males. 70% had non-erosive reflux disease on endoscopy with a mean DeMeester score of 18.9 (±19.93). The mean (±SD) duration of EFTP procedure was 17.4 (±4) min. The primary end point was more frequently achieved in the EFTP group (65.7% vs 2.9%; p<0.001). Median (IQR) % improvement in GERD-HRQL was significantly higher in the EFTP group at 6 (81.4 (60.9-100.0) versus 8.0 (2.2-21.6); p<0.001) and 12 (92.3 (84.4-100.0) versus 9.1 (4.8-36.0); p<0.001) months. In the EFTP group, 62.8% patients were off-PPI at 12 months compared with 11.4% in the sham group (p<0.001). pH-metry parameters partially improved at 3 months, (n=70; total reflux episodes in EFTP arm and non-acid reflux episodes for EFTP vs sham) but not at 12 months (n=27); endoscopic oesophagitis was seen in 0% in the treatment (n=18) and 5 (29.4%) in the control group (n=17) at 12 months. No major procedure-related adverse events were encountered in either group. CONCLUSION: EFTP using a novel device is safe and effective in improving quality of life in patients with PPI dependent mostly non-erosive reflux disease at short and long terms; objective parameters showed a limited response rate. TRIAL REGISTRATION NUMBER: NCT03322553.

Comparison of Oncostatin M in Patients with Chronic Periodontitis with and without Diabetes

Abstract Objective: To compare the Oncostatin M (OSM) concentrations in tissues of patients with chronic periodontitis with and without diabetes. Material and Methods: Sixty-four subjects visiting the dental outpatient department were categorized as "healthy" (Group 1), "periodontitis" (Group 2), and "diabetes with periodontitis" (Group 3) groups. The clinical oral examination included assessment of plaque, gingivitis, probing depth, clinical attachment level. Blood glucose was assessed for group 3 patients. OSM concentration in the tissues was assessed using ELISA in all groups. Results: The mean OSM was 0.02 ± 0.04 pg/mg in the healthy group, 0.12 ± 0.09 pg/mg in the chronic periodontitis group and 0.13 ± 0.10 pg/mg in the diabetes-periodontitis group. A significantly higher mean OSM was seen in Group 2 and Group 3 than Group 1. The amount of OSM positively correlated with probing depth and clinical attachment level. Conclusion: Periodontal disease causes a rise in Oncostatin M, independent of the diabetic status. Expression of OSM in the gingival tissues can serve as an inflammatory marker.

Oversized donor heart transplantation-clinical experience with an underestimated problem.

PURPOSE: Heart transplantation is the definitive treatment for end-stage heart failure. With respect to donor-recipient size matching, the problems with undersized heart transplantation have been widely discussed, but there is a paucity of information on oversized transplants due to the presumed advantage of large hearts. We intend to share our center's experience with oversized heart transplantation and its associated problems which would help to expand the knowledge on oversized cardiac allografts. METHODS: Patients who underwent isolated heart transplantation at our hospital between March 1, 2008, and March 1, 2020, were included. For adults, a donor-recipient predicted heart mass percentage difference exceeding 30% and for children, a donor-recipient weight ratio < 0.8 and > 2.0 was considered a mismatch. We collected data from the in-patient medical records and analyzed the in-hospital outcomes and survival post-transplant among various other parameters. RESULTS: Out of the 43 patients included in this study, 32 (74.4%) patients received a matched heart and 11 (25.6%) patients received oversized hearts. None of the patients received an undersized heart. The in-hospital mortality rate of oversized transplants was 18.2% whereas that of matched transplants was 9.4% (p = 0.432). The post-operative characteristics and 1-year survival were comparable between the groups. We encountered problems specific to oversizing in 5 of the 11 patients (45.4%) which are discussed. CONCLUSION: With the liberalization of donor criteria to overcome organ shortage, oversized heart transplantation poses certain unique challenges, which when efficiently managed offers acceptable outcomes.

Isolated renal hydatid disease: laparoscopic approach to an uncommon entity (case report).

Hydatidosis is a parasitic disease, endemic in various parts of the World. It frequently involves liver and lungs and, rarely, other organs as well. Isolated renal hydatidosis is a rare entity that accounts for less than 3% of all hydatid cases. Surgery remains the mainstay of treatment. We hereby report a case of isolated renal hydatid cyst involving left kidney that was managed by laparoscopic approach.

A novel LC-MS/MS method for simultaneous estimation of obeticholic acid, glyco-obeticholic acid, tauro-obeticholic acid in human plasma and its application to a pharmacokinetic study.

A rapid, robust, simple, selective, and sensitive liquid chromatography-tandem mass spectrometry method was developed for the simultaneous estimation of obeticholic acid and its two pharmacologically active metabolites, glyco-obeticholic acid, and tauro-obeticholic acid in human plasma. The analytes and their heavy stable isotope-labeled internal standards were extracted from 250 µL human plasma by a solid-phase extraction technique. The method linearity was established over a concentration range of 0.410 to 120.466 ng/mL for obeticholic acid, 0.414 to 121.708 ng/mL for glyco-obeticholic acid, and 0.255 to 75.101 ng/mL for tauro-obeticholic acid. The method was fully validated as per current guidelines on bioanalytical method validation of "United States of Food and Drug Administration" and "European Medicines Agency." The method was successfully applied to study the pharmacokinetics of obeticholic acid, glyco-obeticholic acid, and tauro-obeticholic acid following oral administration of obeticholic acid tablets to healthy male volunteers. All the measured concentrations were within calibration curve ranges.

Safety and immunogenicity trial of an inactivated SARS-CoV-2 vaccine-BBV152: a phase 1, double-blind, randomised control trial

BackgroundBBV152 is a whole-virion inactivated SARS-CoV-2 vaccine formulated with a TLR 7/8 agonist molecule adsorbed to alum (Algel-IMDG). MethodsWe conducted a double-blind randomized controlled phase 1 clinical trial to evaluate the safety and immunogenicity of BBV152. A total of 375 participants were randomized equally to receive three vaccine formulations (n=100 each) prepared with 3 g with Algel-IMDG, 6 g with Algel-IMDG, and 6 g with Algel, and an Algel only control arm (n=75). Vaccines were administered on a two-dose intramuscular accelerated schedule on day 0 (baseline) and day 14. The primary outcomes were reactogenicity and safety. The secondary outcomes were immunogenicity based on the anti-IgG S1 response (detected with an enzyme-linked immunosorbent assay [ELISA] and wild-type virus neutralization [microneutralization and plaque reduction neutralization assays]). Cell-mediated responses were also evaluated. ResultsReactogenicity was absent in the majority of participants, with mild events. The majority of adverse events were mild and were resolved. One serious adverse event was reported, which was found to be unrelated to vaccination. All three vaccine formulations resulted in robust immune responses comparable to a panel of convalescent serum. No significant differences were observed between the 3-g and 6-g Algel-IMDG groups. Neutralizing responses to homologous and heterologous SARS-CoV-2 strains were detected in all vaccinated individuals. Cell-mediated responses were biased to a Th-1 phenotype. ConclusionsBBV152 induced binding and neutralising antibody responses and with the inclusion of the Algel-IMDG adjuvant, this is the first inactivated SARS-CoV-2 vaccine that has been reported to induce a Th1-biased response. Vaccine induced neutralizing antibody titers were reported with two divergent SARS-CoV-2 strains. BBV152 is stored between 2{degrees}C and 8{degrees}C, which is compatible with all national immunization program cold chain requirements. Both Algel-IMDG formulations were selected for the phase 2 immunogenicity trials. Further efficacy trials are underway. Clinicaltrials.gov: NCT04471519

Fatty acid ethyl ester (FAEE) associated acute pancreatitis: An ex-vivo study using human pancreatic acini.

BACKGROUND & AIM: Fatty acid ethyl esters (FAEEs), are produced by non-oxidative alcohol metabolism and can cause acinar cell damage and subsequent acute pancreatitis in rodent models. Even though experimental studies have elucidated the FAEE mediated early intra-acinar events, these mechanisms have not been well studied in humans. In the present study, we evaluate the early intra-acinar events and inflammatory response in human pancreatic acinar tissues and cells in an ex-vivo model. METHODS: Experiments were conducted using normal human pancreatic tissues exposed to FAEE. Subcellular fractionation was performed on tissue homogenates and trypsin and cathepsin B activities were estimated in these fractions. Acinar cell injury was evaluated by histology and immunohistochemistry. Cytokine release from exposed acinar cells was evaluated by performing Immuno-fluorescence. Serum was collected from patients with AP within the first 72 h of symptom onset for cytokine estimation using FACS. RESULTS: We observed significant trypsin activation and acinar cell injury in FAEE treated tissue. Cathepsin B was redistributed from lysosomal to zymogen compartment at 30 min of FAEE exposure. IHC results indicated the presence of apoptosis in pancreatic tissue at 1 & 2hrs of FAEE exposure. We also observed a time dependent increase in secretion of cytokines IL-6, IL-8, TNF-α from FAEE treated acinar tissue. There was also a significant elevation in plasma cytokines in patents with alcohol associated AP within 72 h of symptom onset. CONCLUSION: Our data suggest that alcohol metabolites can cause acute acinar cell damage and subsequent cytokine release which could eventually culminant in SIRS.

Secure Closure of Right Ventricular Perforation Reinforced With Coronary Artery Wall.

Accidental entry into the right ventricular cavity is a common occurrence during exposure of the intra-myocardial left anterior descending artery. Several techniques have been described for repair of the perforation. Although these methods can be used, there is still a danger of persistent bleeding or distal ischemia. We describe a method that is safe and reproducible.

Identification, isolation and characterization of dolutegravir forced degradation products and their cytotoxicity potential.

Dolutegravir was approved by USFDA, Canada and European regulatory authorities as antiretroviral medication. In this article, DLG forced degradation studies as per the International Council for Harmonization (ICH) prescribed stress conditions was conducted and the resulting degradants were fully characterized. DLG was stable in basic, thermal and photolytic stress conditions, whereas DLG was found to unstable in acidic and oxidative conditions. One degradant each from acid and peroxide treated solutions was resolved on LC-MS and labelled as DP-1 and DP-2 with RT 1.80 min and 1.41 min, respectively. DP-1 and DP-2 were isolated by preparative HPLC with C18 column using gradient elution method. Subsequently DP-1 and DP-2 peaks were subjected to HRMS for accurate mass. Molecular mass of DP-1 and DP-2 were m/z 420.1379 (positive mode) and m/z 214.0319 (negative mode), respectively. Further, DP-1 & DP-2 were subjected to NMR spectroscopic analysis (including 2D) for structural confirmation. DP-1 was identified as N-(2,4-difluorobenzyl)-9-hydroxy-2-(4-hydroxybutan-2-yl)-1,8-dioxo-2,8-dihydro-1H-pyrido[1,2-a]pyrazine-7-carboxamide and it is earlier reported by Gudisela et al. [19] as DLG process impurity. DP-2 was identified as 2-(2,4difluorobenzylamino)-2-oxoacetic acid which is novel DLG degradant and not reported earlier to the best of our knowledge. DLG along its forced degradation products were found to be non-cytotoxic in in vitro assay conditions using HepG2 cells.