A novel, reliable and rapid high-performance liquid chromatography (HPLC) method with post-column derivatization was developed and validated. The HPLC method was used for the simultaneous determination of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) in various cereals and grains. Samples were extracted with 80:20 (v/v) methanol:water and purified using C18 (40–63 μm) solid-phase extraction cartridges. AFs were separated using a LiChroCART–RP-18 (5 μm, 250 × 4.0 mm²) column. The mobile phase consisted of methanol:acetonitrile:buffer (17.5:17.5:65 v/v) (pH 7.4) delivered at the flow rate of 1.0 mL min⁻¹. The fluorescence of each AF was detected at λ_ex = 365 nm and λ_em = 435 nm. All four AFs were properly resolved within the total run time of 20 min. The established method was extensively validated as a final verification of the method development by the evaluation of selectivity (AFB₁, AFB₂, AFG₁ and AFG₂), linearity ( R² ≥ 0.9994), precision (average SD ≤ 2.79), accuracy (relative mean error ≤ −5.51), robustness ( p < 0.0080), ruggedness ( p < 0.0100) and average recoveries (89.2–97.8%). The limits of quantification of AFB₁, AFB₂, AFG₁ and AFG₂ were 0.080, 0.073, 0.062 and 0.066 ng g⁻¹, respectively. Finally, the developed method was applied for the analysis of AFs in 45 samples comprising rice ( n = 20), wheat ( n = 15) and maize ( n = 10). The results showed that 65% of rice, 20% of wheat and 80% of maize samples were found contaminated with AFs. Thus, according to the achieved results, it is suggested that the newly developed HPLC method could be effectively applied for the routine analysis of the AFs in different cereals and grains.

Background: The adherence pattern of antiepileptic drugs (AEDs) among patients with epilepsy is relatively lower in the United States and different European countries. However, adherence and cost analysis of AEDs in Asian countries have not been thoroughly studied. Therefore, the present study aimed to analyze the cost and adherence of AEDs and its associated factors in patients followed in Pakistan. Methods: Data from prescriptions collected from patients with epilepsy who have visited the Outpatient Department (OPD) of different tertiary care hospitals at the cosmopolitan city of Karachi, Pakistan from December 2015 to November 2019. The mean follow-up period for each participant was about 22 months. Pairwise comparisons from Cox regression/hazard ratios were used to assess the predictors of adherence. Direct costs of AEDs were calculated and presented as the annual cost of drugs. Results: A total of 11,490 patients were included in this study, 51.2 % were male and 48.8 % were female with a mean age of 45.2 ± 15.8 y. Levetiracetam was found as the most prescribing AED in all study participants (32.9 %). Of them, 49.1 % of patients continued their initial recommended treatment. However, 31.3 % of patients have discontinued the therapy, while, 19.6 % were switched to other AED. Adherence with initial treatment was more profound in male (57.4 %) patients, compared to female with a mean age of 44.2 years. Lamotrigine users (60.6 %) showed a higher tendency to retain on initially prescribed drugs. The total cost of epilepsy treatment in the entire study cohort was 153280.5 PKR ($941.9). By applying the Cox regression analysis, it can be observed that the patients with increasing age (OR, 2.04), migraine (OR, 2.21), psychiatric disorders (OR, 4.28), other comorbidities (OR, 1.52) and users of other than top five prescribing AEDs (2.35) were at higher risk of treatment discontinuation. However, levetiracetam (OR, 0.69), valproic acid (OR, 0.52), carbamazepine (OR, 0.81), lamotrigine (OR, 0.80) or lacosamide (OR, 0.65) users have more chances to continue their initial therapy. Conclusions: Similar to western countries, the majority of patients with epilepsy exhibited low adherence with AEDs. Various associated factors for improving adherence were identified in this study.

Intracanal medicaments are an important adjunct to the effective disinfection of the root canal system. However, conventional intracanal medicaments do not provide adequate protection against Enterococcus faecalis, which is the organism of interest in many cases of root canal failures. This study aimed to evaluate the influence of biosynthesized calcium oxide nanoparticles (CaO NPs) on the antibacterial activity, pH, microleakage and cytotoxicity of intracanal medicaments. CaO NPs were biosynthesized by the direct thermal decomposition of eggshells (EGS) and the reduction of calcium nitrate with papaya leaf extract (PLE). These nanoparticles were mixed with a proprietary calcium hydroxide powder in 10% and 25% (w/w) concentrations and blended in analytical-grade coconut oil to formulate the experimental medicaments. These were then evaluated for antibacterial activity, pH, microleakage and cytotoxicity at 1 day, 7 days and 15 days. A proprietary calcium hydroxide paste formulation (MX) was used as the control. Means and standard deviations were calculated and analyzed using repeated-measures ANOVA for pH and three-way ANOVA for the antibacterial effect, microleakage and cytotoxicity, followed by LSD post hoc analysis. Significant antibacterial activity was noted against Enterococcus faecalis at all times, with zones of inhibition (ZOI) up to 19.60 ± 2.30 mm. pH levels up to 13.13 ± 0.35 were observed for the experimental groups. Microleakage remained comparable to the control, while cytotoxicity was not observed in any of the groups at any time. Intracanal medicaments formulated with 10% and 25% (w/w) of biosynthesized CaO NPs could be promising candidates for the disinfection of the root canal system compared to conventional counterparts.

The objectives of this study were to investigate the pH of common beverages and to evaluate the effects of common acidic beverages on the surface hardness and weight loss of human tooth specimens. A total of 106 beverages were conveniently purchased from supermarkets in Karachi, Pakistan. Prior to evaluation, beverages were refrigerated or stored at room temperature in accordance with the manufacturers’ recommendations. Beverages were categorized into six groups: ‘Sports and Energy drinks’, ‘Water’, ‘Fruit Juices and Drinks’, ‘Sodas’, ‘Milk and Flavored Milk’ and ‘Teas and Coffee’. Using a pH meter, the pH of each beverage was measured in triplicate at room temperature. In addition, the influence of five highly acidic beverages on the weight loss and surface hardness of human tooth specimens was evaluated using gravimetric analysis and the Vickers hardness tester, respectively. ‘Sports and Energy drinks’, ‘Fruits Juices and Drinks’ and ‘Sodas’ were the most acidic beverage categories, with a pH range of 3.00–5.00. A total of 33% of beverages tested in this study were highly acidic (pH less than 4.00), 29% of beverages were moderately acidic (pH 4.00–4.99) and 31% were mildly acidic (pH 5.00–6.99). Significant weight loss was observed in all immersed specimens compared to control counterparts (p < 0.05). Similarly, for surface hardness, five highly acidic beverages (Red Bull, Pepsi, Apple Cidra, Tang Mosambi and Tang Orange) significantly decreased the surface hardness of specimens (p < 0.05). The pH levels of commonly available beverages in Pakistan are highly acidic, which may encourage loss of minerals from teeth; hence, affecting their surface hardness.

This study aimed to isolate fungal pathogens and to subsequently quantify aflatoxin (AF; B1 + B2 + G1 + G2) contamination in wheat crops grown in Pakistan. Accordingly, a total of 185 wheat samples were collected from different areas of Pakistan and numerous potent fungal pathogens were isolated. AF contamination attributed to the presence of intoxicating fungal pathogens and resulting metabolic activities were quantified using a high performance liquid chromatography-fluorescence detector coupled with postcolumn derivatization. Additionally, the effect of fungal pathogens on seed germination was also examined. The results obtained showed that 50% of tested wheat samples were found to be contaminated with a diverse range of fungal species. The rate of recurrence of fungal pathogens were Aspergillus 31%, Penicillium 9%, Fusarium 8%, Rhizopus 3%, and Alternaria 2%. The presence of Tilletia indica and Claviceps purpurea species was found to be inevident in all tested wheat samples. AFB1 contamination was detected in 48 (26.0%) samples and AFB2 in 13 (7.0%) samples. AFG1 and AFG2 were not found in any of the tested samples. The contamination range of AFB1 and AFB2 was 0.05-4.78 μg/kg and 0.02-0.48 μg/kg, respectively. The total amount of AFs (B1 + B2) found in 48 (26.0%) samples had a mean level of 0.53 ± 0.40 μg/kg and a contamination range of 0.02-5.26 μg/kg. The overall results showed that in 137 (74.0%) samples, AFs were not found within detectable limits. Furthermore, in 180 (97.2%) samples, AF levels were found to be below the maximum tolerated levels (MTL) recommended by the European Union (4 μg/kg). In five (2.7%) samples, AF contamination was higher than the MTL of the European Union. However, these samples were fit for human consumption with reference to the MTL (20 μg/kg) assigned by the USA (Food and Drug Administration and Food and Agriculture Organization) and Pakistan (Pakistan Standards and Quality Control Authority). Germination rates in healthy and contaminated wheat kernels were 84.6% and 45.2%, respectively. Based on the obtained results, it was concluded that the levels of fungal pathogen and AF contamination in Pakistani-grown wheat are not a potential threat to consumer health. However, control procedures along with a strict monitoring policy are mandatory to further minimize the prevalence of fungal carriers and the potency of AFs in crops cultivated in Pakistan.

During 2012–2014, 170 samples of export quality raisins were collected from different vendors in Pakistan. The collected samples were analysed for the presence of aflatoxins (AFs) and Ochratoxin A (OTA) contamination using high-performance liquid chromatography technique. The limit of detection and limit of quantification of AFs/OTA were 0.12/0.10 and 0.36/0.30 µg kg⁻¹, respectively. Only 5% of the samples were contaminated with AFs, ranging 0.15–2.58 µg kg⁻¹ with a mean of 0.05 ± 0.26 µg kg⁻¹. None of the raisin samples exhibited AFs contamination above the maximum limit (ML = 4 µg kg⁻¹) as set by the European Union (EU). About 72% of the samples were contaminated with OTA, ranging 0.14–12.75 µg kg⁻¹ with a mean of 2.10 ± 1.9 µg kg⁻¹. However, in 95.3% of the tested samples, OTA level was lower than the ML of 10 µg kg⁻¹ as regulated by the EU. Apparently, a strict and continuous monitoring plan, including regulatory limits, improves food safety and quality for all types of commodities.