Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia.

This study investigates the impact of combining psychophysical stress, induced by forced swim (FSS), with masseter inflammation on reactive oxygen species (ROS) production in trigeminal ganglia (TG), TRPA1 upregulation in TG, and mechanical hyperalgesia. In a rat model, we demonstrate that FSS potentiates and prolongs CFA-induced ROS upregulation within TG. The ROS levels in CFA combined with FSS group surpass those in the CFA-only group on days 4 and 28 post-treatment. FSS also enhances TRPA1 upregulation in TG, with prolonged expression compared to CFA alone. Furthermore, CFA-induced mechanical hyperalgesia is significantly prolonged by FSS, persisting up to day 28. PCR array analyses reveal distinct alterations in oxidative stress genes under CFA and CFA combined with FSS conditions, suggesting an intricate regulation of ROS within TG. Notably, genes like Nox4, Hba1, Gpx3, and Duox1 exhibit significant changes, providing potential targets for managing oxidative stress and inflammatory pain. Western blot and immunohistochemistry confirm DUOX1 protein upregulation and localization in TG neurons, indicating a role in ROS generation under inflammatory and stress conditions. This study underscores the complex interplay between psychophysical stress, inflammation, and oxidative stress in the trigeminal system, offering insights into novel therapeutic targets for pain management.

Age- and Sex-Dependent Effects of Moderate Exercise on Endogenous Pain Inhibition in Rats.

Diffuse noxious inhibitory controls (DNICs), or the pain inhibits pain phenomenon, refer to reduced pain-like behaviors that are displayed following a noxious conditioning stimulus located far from the test stimulus and have also been referred to as "descending control of nociception" when measured in awake-behaving animals. In this study, we sought to determine the impact of moderate long-term exercise on the DCN response and determine if this effect differed across age and sex. After a six-week exercise program consisting of 30 min of moderate treadmill running 5 days a week, the animals' forepaws were injected with capsaicin, and DCN responses were assessed using thermal withdrawal latencies of the hind paw. Young, exercised male and female rats displayed prolonged DCN responses relative to their sedentary counterparts, with the young exercised male group displaying longer-lasting DCN facilitation than the young exercised females. Exercise did not impact DCN responses in either male or female aged rats. Additionally, the serum testosterone levels did not change following exercise in any group. Importantly, the levels of corticosterone did not change following the exercise program, indicating that changes in the DCN response are not due to stress-induced analgesia. Our findings suggest that moderate exercise can facilitate the DCN response in young animals, even when this exercise does not change the levels of serum testosterone.

Technical, economic, and environmental feasibility of rice hull ash from electricity generation as a mineral additive to concrete.

A circular economy based on symbiotic relationships among sectors, where the waste from one is resource to another, holds promise for cost-effective and sustainable production. This research explores such a model for the agriculture, energy, and construction sectors in California. Here, we develop new an understanding for the synergistic utilization mechanisms for rice hull, a byproduct from rice production, as a feedstock for electricity generation and rice hull ash (RHA) used as a supplementary cementitious material in concrete. A suite of methods including experimental analysis, techno-economic analysis (TEA), and life-cycle assessment (LCA) were applied to estimate the cost and environmental performance of the system. TEA results showed that the electricity price required for break even on expenses without selling RHA is $0.07/kWh, lower than the market price. As such, RHA may be available at little to no cost to concrete producers. Our experimental results showed the viability of RHA to be used as a supplementary cementitious material, meaning it can replace a portion of the cement used in concrete. LCA results showed that replacing 15% of cement with RHA in concrete can reduce carbon dioxide equivalent (CO2e) emissions by 15% while still meeting material performance targets. While the substitution rate of RHA for cement may be modest, RHA generated from California alone could mitigate 0.2% of total CO2e from the entire cement production sector in the United States and 1% in California.

Economic and environmental performance of microalgal energy products - A case study exploring circular bioeconomy principles applied to recycled anaerobic digester waste flows.

This study quantifies the financial and environmental impacts of a microalgal bioenergy system that attempts to maximize circular flows by recovering and reusing the carbon, nutrients, and water within the system. The system produces microalgal biomass using liquid digestate of an anaerobic digester that processes 45 metric tons of food waste and generates 28.6 m3 of permeate daily in California, and three energy production scenarios from the biomass are considered: producing biodiesel, electricity, and both. In all scenarios, the resulting energy products delivered only modest reductions in environmental impacts as measured by carbon dioxide equivalent emissions. The carbon intensities (CIs) of biodiesel from this study were 91.0 gCO2e/MJ and 93.3 gCO2e/MJ, which were lower than 94.71 gCO2e/MJ of conventional petroleum diesel, and the CI of electricity from this study was 70.6 gCO2e/MJ, lower than the average electricity grid CI in California (82.92 gCO2e/MJ). The economic analysis results show that generating electricity alone can be profitable, while biodiesel produced via this system is not cost competitive with conventional diesel due to high capital expenses. Thus, generating electricity in lieu of biodiesel appears to be a better option to maximize the use of waste flows and supply lower-carbon energy.

Forced mouth opening induces post-traumatic hyperalgesia and associated peripheral sensitization after temporomandibular joints injury in mice.

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. The pathophysiology of TMD is not fully understood, and it is necessary to understand pathophysiology underlying painful TMD conditions to develop more effective treatment methods. Recent studies suggested that external or intrinsic trauma to TMJ is associated with chronic TMD in patients. Here, we investigated the effects of the TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors. FMO increased mechanical hyperalgesia assessed by von Frey test, spontaneous pain-like behaviors assessed by mouse grimace scale, and anxiety-like behaviors assessed by open-field test. In vivo GCaMP Ca 2+ imaging of intact trigeminal ganglia (TG) showed increased spontaneous Ca 2+ activity and mechanical hypersensitivity of TG neurons in the FMO compared to the sham group. Ca 2+ responses evoked by cold, heat, and capsaicin stimuli were also increased. FMO-induced hyperalgesia and neuronal hyperactivities were not sex dependent. TG neurons sensitized following FMO were primarily small to medium-sized nociceptive afferents. Consistently, most TMJ afferents in the TG were small-sized peptidergic neurons expressing calcitonin gene-related peptides, whereas nonpeptidergic TMJ afferents were relatively low. FMO-induced intraneural inflammation in the surrounding tissues of the TMJ indicates potentially novel mechanisms of peripheral sensitization following TMJ injury. These results suggest that the TMJ injury leads to persistent post-traumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors.

Intraganglionic reactive oxygen species mediate inflammatory pain and hyperalgesia through TRPA1 in the rat.

Reactive oxygen species (ROS) are generated in nociceptive pathways in response to inflammation and injury. ROS are accumulated within the sensory ganglia following peripheral inflammation, but the functional role of intraganlionic ROS in inflammatory pain is not clearly understood. The aims of this study were to investigate whether peripheral inflammation leads to prolonged ROS accumulation within the trigeminal ganglia (TG), whether intraganglionic ROS mediate pain hypersensitivity via activation of TRPA1, and whether TRPA1 expression is upregulated in TG during inflammatory conditions by ROS. We demonstrated that peripheral inflammation causes excess ROS production within TG during the period when inflammatory mechanical hyperalgesia is most prominent. Additionally, scavenging intraganglionic ROS attenuated inflammatory mechanical hyperalgesia and a pharmacological blockade of TRPA1 localized within TG also mitigated inflammatory mechanical hyperalgesia. Interestingly, exogenous administration of ROS into TG elicited mechanical hyperalgesia and spontaneous pain-like responses via TRPA1, and intraganglionic ROS induced TRPA1 upregulation in TG. These results collectively suggest that ROS accumulation in TG during peripheral inflammation contributes to pain and hyperalgesia in a TRPA1 dependent manner, and that ROS further exacerbate pathological pain responses by upregulating TRPA1 expression. Therefore, any conditions that exacerbate ROS accumulation within somatic sensory ganglia can aggravate pain responses and treatments reducing ganglionic ROS may help alleviate inflammatory pain.

The degeneration-pain relationship in the temporomandibular joint: Current understandings and rodent models.

Temporomandibular disorders (TMD) represent a group of musculoskeletal conditions involving the temporomandibular joints (TMJ), the masticatory muscles and associated structures. Painful TMD are highly prevalent and conditions afflict 4% of US adults annually. TMD include heterogenous musculoskeletal pain conditions, such as myalgia, arthralgia, and myofascial pain. A subpopulations of TMD patients show structural changes in TMJ, including disc displacement or degenerative joint diseases (DJD). DJD is a slowly progressing, degenerative disease of the TMJ characterized by cartilage degradation and subchondral bone remodeling. Patients with DJD often develop pain (TMJ osteoarthritis; TMJ OA), but do not always have pain (TMJ osteoarthrosis). Therefore, pain symptoms are not always associated with altered TMJ structures, which suggests that a causal relationship between TMJ degeneration and pain is unclear. Multiple animal models have been developed for determining altered joint structure and pain phenotypes in response to various TMJ injuries. Rodent models of TMJOA and pain include injections to induce inflammation or cartilage destruction, sustained opening of the oral cavity, surgical resection of the articular disc, transgenic approaches to knockout or overexpress key genes, and an integrative approach with superimposed emotional stress or comorbidities. In rodents, TMJ pain and degeneration occur during partially overlapping time periods in these models, which suggests that common biological factors may mediate TMJ pain and degeneration over different time courses. While substances such as intra-articular pro-inflammatory cytokines commonly cause pain and joint degeneration, it remains unclear whether pain or nociceptive activities are causally associated with structural degeneration of TMJ and whether structural degeneration of TMJ is necessary for producing persistent pain. A thorough understanding of the determining factors of pain-structure relationships of TMJ during the onset, progression, and chronification by adopting novel approaches and models should improve the ability to simultaneously treat TMJ pain and TMJ degeneration.

Comparison of sensory outcomes in patients with successful motor outcome versus recurrent exotropia after surgery for intermittent exotropia.

Here, we compared sensory outcomes between patients with successful motor outcomes and recurrent exotropia after intermittent exotropia surgery. We retrospectively analyzed 303 patients who underwent surgery for intermittent exotropia, divided into two groups: successful motor outcome defined as an alignment between 10 prism diopters (PD) exodeviation and 5PD esodeviation at the final follow-up (Group A, n = 177) and residual or recurrent exotropia defined as exodeviation > 10 PD (Group B, n = 126). Preoperative and postoperative (at final visit) sensory outcomes were compared using the Titmus stereotest and distance Worth 4-dot test. Stereoacuity significantly improved postoperatively in both successful motor outcome group (Group A) and residual or recurrent exotropia group (Group B). However, stereoacuity did not differ between groups preoperatively and postoperatively. On the other hand, fusion rates for the Worth 4-dot test were significantly higher in group A than in group B, preoperatively and postoperatively, and significantly increased postoperatively only in group A. Therefore, the distance Worth 4-dot test may be useful for evaluating postoperative prognosis and preoperative sensory status.

Optimization and validation of a method for the determination of acidic pesticides in cabbage and spinach by modifying QuEChERS procedure and liquid chromatography-tandem mass spectrometry.

A quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was developed and combined with liquid chromatography-tandem mass spectrometry to analyze 12 acidic pesticides in cabbage and spinach. The extraction solvents, phase partition salts and sorbents effect was studied to optimize the method followed by dilution before sample injection. The extraction involved 5% formic acid in acetonitrile, and the liquid-liquid partition was salt-induced. Carbopack Z, a high surface area graphitized carbon black, was a new sorbent used in the clean-up. The results show that Carbopack Z effectively removes interferences with little loss of acidic pesticides. All tested pesticide recoveries were satisfactory when Carbopack Z was combined with C18 in the clean-up at optimized condition. After clean-up, the extract was subjected to 10-fold dilution to sufficiently reduce the matrix effect (<20%). The limit of quantification (LOQ) was 1-5 ng/g, and the mean recovery was between 95 and 110% with a relative standard deviation <20% (between 2% and 10%) for the spiking of three concentrations: 5, 50, and 500 ng/g. The extract was less pigmented in the modified QuEChERS method than its original version. Thus, the modified method is a useful alternative for investigating the acidic pesticide residues in cabbage and spinach.

Pain modulatory network is influenced by sex and age in a healthy state and during osteoarthritis progression in rats.

Old age and female sex are risk factors for the development of osteoarthritis (OA) and chronic pain. We investigated the effects of sex and age on pain modulatory networks in a healthy state and during OA progression. We used functional MRI to determine the effects of sex and age on periaqueductal gray functional connectivity (PAG FC) in a healthy state (pre-OA) and during the early and late phases of monosodium iodoacetate-induced OA in rats. We then examined how sex and age affect longitudinal changes in PAG FC in OA. In a healthy state, females exhibited more widespread PAG FC than males, and this effect was exaggerated with aging. Young males had moderate PAG FC changes during the early phase but recruited additional brain regions, including the rostral anterior cingulate cortex (ACC), during the late phase. Young females exhibited widespread PAG FC in the early phase, which includes connections to insula, caudal ACC, and nucleus accumbens (NAc). Older groups had strong PAG FC with fewer regions in the early phase, but they recruited additional brain regions, including NAc, in the late phase. Overall, our findings show that PAG FC is modulated by sex and age in a healthy state. A widespread PAG network in the early phase of OA pain may contribute to the transition from acute to chronic OA pain and the increased risk of developing chronic pain for females. Enhanced PAG FC with the reward system may represent a potential mechanism underlying chronic OA pain in elderly patients.

ISNT rule satisfaction in Korean non-glaucomatous subjects.

PURPOSE: To evaluate the efficacy of the ISNT rule in normal eyes of Koreans. METHODS: We retrospectively reviewed medical records of 890 subjects with fundus photographs and evaluated the presence of the cup and ISNT rule satisfaction. If the ISNT rule was violated, the quadrants in which the neuroretinal rim was thinnest and thickest, respectively, were evaluated. RESULTS: Among 890 eyes, 84.7% showed the cup. The subjects without the cup were significantly younger and more hyperopic. Among 754 eyes with the cup, 53.5% showed ISNT rule satisfaction. In 351 eyes violating the ISNT rule, the most common quadrant showing the thickest neuroretinal rim was the inferior (65.5%), and the thinnest, the temporal (98.3%). CONCLUSION: The cup was absent in 15% of the assessed eyes. Only about half of eyes with the cup showed ISNT rule satisfaction. Even in eyes violating ISNT rule, the inferior was the most common quadrant with the thickest neuroretinal rim and the temporal, the thinnest.

Compositional cyber-physical epidemiology of COVID-19.

The COVID-19 pandemic has posed significant challenges globally. Countries have adopted different strategies with varying degrees of success. Epidemiologists are studying the impact of government actions using scenario analysis. However, the interactions between the government policy and the disease dynamics are not formally captured. We, for the first time, formally study the interaction between the disease dynamics, which is modelled as a physical process, and the government policy, which is modelled as the adjoining controller. Our approach enables compositionality, where either the plant or the controller could be replaced by an alternative model. Our work is inspired by the engineering approach for the design of Cyber-Physical Systems. Consequently, we term the new framework Compositional Cyber-Physical Epidemiology. We created different classes of controllers and applied these to control the disease in New Zealand and Italy. Our controllers closely follow government decisions based on their published data. We not only reproduce the pandemic progression faithfully in New Zealand and Italy but also show the tradeoffs produced by differing control actions.

The Role of DNA Methylation in Transcriptional Regulation of Pro-Nociceptive Genes in Rat Trigeminal Ganglia.

Epigenetic modulation by DNA methylation is associated with aberrant gene expression in sensory neurons, which consequently leads to pathological pain responses. In this study, we sought to investigate whether peripheral inflammation alters global DNA methylation in trigeminal ganglia (TG) and results in abnormal expression of pro-nociceptive genes. Our results show that peripheral inflammation remotely reduced the level of global DNA methylation in rat TG with a concurrent reduction in DNMT1 and DNMT3a expression. Using unbiased steps, we selected the following pro-nociceptive candidate genes that are potentially regulated by DNA methylation: TRPV1, TRPA1, P2X3, and PIEZO2. Inhibition of DNMT with 5-Aza-dC in dissociated TG cells produced dose-dependent upregulation of TRPV1, TRPA1, and P2X3. Systemic treatment of animals with 5-Aza-dC significantly increased the expression of TRPV1, TRPA1, and PIEZO2 in TG. Furthermore, the overexpression of DNMT3a, as delivered by a lentiviral vector, significantly downregulated TRPV1 and PIEZO2 expression and also reliably decreased TRPA1 and P2X3 transcripts. MeDIP revealed that this overexpression also significantly enhanced methylation of CGIs associated with TRPV1 and TRPA1. In addition, bisulfite sequencing data indicated that the CGI associated with TRPA1 was methylated in a pattern catalyzed by DNMT3a. Taken together, our results show that all 4 pro-nociceptive genes are subject to epigenetic modulation via DNA methylation, likely via DNMT3a under inflammatory conditions. These findings provide the first evidence for the functional importance of DNA methylation as an epigenetic factor in the transcription of pro-nociceptive genes in TG that are implicated in pathological orofacial pain responses.

Optimization of a Simplified and Effective Analytical Method of Pesticide Residues in Mealworms (Tenebrio molitor Larvae) Combined with GC-MS/MS and LC-MS/MS.

An effective analytical method was optimized for residues including chlorpyrifos-methyl, deltamethrin, fenoxanil, thiobencarb and fludioxonil in mealworms, the larval form of Tenebrio molitor. They are listed for pest control during wheat cultivation and can be found in wheat-bran feed for growing mealworms in South Korea. Analytes were extracted using acetonitrile and salt packet. Four clean-up methods ((1) MgSO4 + 25 mg PSA + 25 mg C18; (2) MgSO4 + 50 mg PSA + 50 mg C18; (3) EMR-lipidTM tube; and (4) 10 mL n-hexane) were investigated and the method (1) was selected due to its robustness. Low-temperature precipitation of fat and proteins improved the recoveries. Recoveries from the Method (1) were satisfying with 70-120% with <20% relative SD at a spiking level of 0.01 mg/kg. With the simultaneous sample preparation, fenoxanil, thiobencarb and fludioxonil were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and chlorpyrifos-methyl and deltamethrin by gas chromatography tandem mass spectrometry (GC-MS/MS). Quantification limits for LC-MS/MS and GC-MS/MS were 0.5 and 2.5 µg/L, respectively. No pesticides of interest were detected in 30 real samples collected across the nation. However, the data can be provided for establishing maximum residue limits for the pesticides in mealworms in response to the positive list system.

Monitoring and risk assessment of tepraloxydim in banana (Musa paradisiaca) and sweet orange (Citrus sinensis).

This study was conducted to analyze the residue levels of tepraloxydim in banana and sweet orange. Successive liquid-liquid extraction and cartridge clean-up method for tepraloxydim determination in banana and sweet orange were developed and validated by HPLC. The developed method was validated, and the recovery and LOQ of tepraloxydim were 79.3-99.5% and 0.02 mg kg-1, respectively. Among the 48 banana and 34 sweet orange samples, tepraloxydim was detected in two (0.03 mg kg-1) and four samples (0.03-0.05 mg kg-1), respectively. A risk assessment of tepraloxydim in banana and sweet orange was conducted by calculating the percent ratio of estimated daily intake (EDI) and acceptable daily intake (ADI). The ADI of tepraloxydim was 0.05 mg kg-1 day-1, and the EDIs of it from banana and sweet orange were 6.3 × 10-6 and 5.1-8.5 × 10-6, respectively. The percent of EDI to ADI of tepraloxydim was 0.013 and 0.010-0.017%, respectively. These results showed that the tepraloxydim levels in this study might not be harmful to human beings.

Brain networks and endogenous pain inhibition are modulated by age and sex in healthy rats.

Endogenous pain inhibition is less efficient in chronic pain patients. Diffuse noxious inhibitory control (DNIC), a form of endogenous pain inhibition, is compromised in women and older people, making them more vulnerable to chronic pain. However, the underlying mechanisms remain unclear. Here, we used a capsaicin-induced DNIC test and resting-state functional MRI to investigate the impact of aging and sex on endogenous pain inhibition and associated brain circuitries in healthy rats. We found that DNIC was less efficient in young females compared with young males. Diffuse noxious inhibitory control response was lost in old rats of both sexes, but the brain networks engaged during DNIC differed in a sex-dependent manner. Young males had the most efficient analgesia with the strongest connectivity between anterior cingulate cortex (ACC) and periaqueductal gray (PAG). The reduced efficiency of DNIC in young females seemed to be driven by widespread brain connectivity. Old males showed increased connectivity between PAG, raphe nuclei, pontine reticular nucleus, and hippocampus, which may not be dependent on connections to ACC, whereas old females showed increased connectivity between ACC, PAG, and more limbic regions. These findings suggest that distinct brain circuitries including the limbic system may contribute to higher susceptibility to pain modulatory deficits in the elderly population, and sex may be a risk factor for developing age-related chronic pain.

Age and Sex Differences in Acute and Osteoarthritis-Like Pain Responses in Rats.

In this study, we investigated age and sex differences in acute and chronic pain in rats. Groups of young (3-6 months) and aged (20-24 months) male and female Fischer 344 rats were used to assess basal thermal and mechanical thresholds, capsaicin-induced acute nocifensive responses and c-Fos expression in the spinal cord, and monoiodoacetate (MIA)-induced knee osteoarthritis (OA)-like pain responses. There was a significant sex, but not age, effect on thermal threshold on the hindpaw and mechanical threshold on the knee joint. No significant age and sex differences in capsaicin-induced nocifensive and c-Fos responses were observed. MIA induced a greater peak reduction of weight-bearing responses in aged males than young rats. Aged females developed the most profound weight-bearing deficit. With knee joint sensitivity as a primary outcome measure, MIA induced more pronounced and longer-lasting hyperalgesia in older rats, with aged female rats showing the worst effect. These data suggest that age may not have significant effect on acute nociceptive processing, but it significantly impacts OA-like pain, making aged rats, especially females, more vulnerable to chronic pain conditions. These preclinical models should provide important tools to investigate basic mechanisms underlying the impact of age and sex in chronic pain conditions.

Phosphorylation of TRPV1 S801 Contributes to Modality-Specific Hyperalgesia in Mice.

Transient receptor potential vanilloid subtype 1 (TRPV1) is a nonselective cationic channel activated by painful stimuli such as capsaicin and noxious heat, and enriched in sensory neurons of the pain pathway. During inflammation, chemical mediators activate protein kinases (such as PKC) that phosphorylate TRPV1 and thereby enhance its function, with consequent increases in nociceptor sensitization. However, the causal relationships between TRPV1 phosphorylation and pathological pain remain unexplored. To directly investigate the roles of one specific TRPV1 phosphorylation event in vivo, we genetically altered a major PKC phosphorylation site, mouse TRPV1 S801, to alanine. The TRPV1 expression pattern in sensory neurons of S801A knock-in (KI) mice was comparable to that in WT controls. However, sensitization of capsaicin-mediated currents after the activation of PKC was substantially impaired in sensory neurons from KI mice. Thermal hyperalgesia induced by PMA or burn injury in KI was identical to WT. Inflammatory thermal hyperalgesia was only marginally attenuated in KI mice. In contrast, PMA-evoked nocifensive responses and sensitization of capsaicin responses were significantly attenuated in the hindpaws of KI mice. Ongoing pain from inflamed masseter muscle was also reduced in KI mice, and was further inhibited by the TRPV1 antagonist AMG9810. These results suggest that PKC-mediated phosphorylation of TRPV1 S801 contributes to inflammation-mediated sensitization of TRPV1 to ligand, but not heat, in vivo Further, this suggests that interference with TRPV1 S801 phosphorylation might represent one potential way to attenuate inflammatory pain, yet spare basal sensitivity and produce fewer side effects than more general TRPV1 inhibition.SIGNIFICANCE STATEMENT Transient receptor potential vanilloid subtype 1 (TRPV1) has been considered a potential target for pain intervention. Global inhibitors of TRPV1 function, however, produce side effects which could compromise their clinical utility. By precisely removing a unique PKC phosphorylation site (TRPV1 S801) in mice through CRISPR/Cas9 editing, we provide in vivo evidence for a highly specific inhibition that leaves basal TRPV1 function intact, yet alleviates some forms of hyperalgesia. These findings support inhibition of TRPV1 S801 phosphorylation as a potential intervention for pain management.

Application of high-surface-area graphitized carbon black with primary secondary amine as an alternative quick, easy, cheap, effective, rugged, and safe cleanup material for pesticide multi-residue analysis in spinach.

A multi-residue method has been developed and validated to determine 46 pesticides in spinach using liquid chromatography tandem mass spectrometry. The method is based on modified quick, easy, cheap, effective, rugged, and safe sample preparation, where high-surface-area graphitized carbon black was used first as sorbent material in the dispersive solid-phase extraction. The method was compared with the quick, easy, cheap, effective, rugged, and safe method. The morphology, surface area, pore size, and pore volume of the sorbent was determined. The results obtained show that the sorbent consists of high surface area (233 m2 /g) and large pore volume (1.5 cm3 /g). The calibration curve correlation coefficient (R2 ) of the method was at least 0.99. The average recoveries ranged from 74 to 116%, and limits of detection and quantification from 0.0001 to 0.002 mg/kg and from 0.0002 to 0.005 mg/kg, respectively. Using the method, the pesticides exhibited low matrix effect (< 20%), except for nicosulfuron (29.86%), methomyl (26.77%), and flufenoxuron (24.65%). The method showed better potential to remove pigments than the quick, easy, cheap, effective, rugged, and safe method. It is demonstrated that the proposed method could be useful alternative for sample preparation of spinach and other matrices in future.