CACNA1S mutation-associated dental anomalies: A calcium channelopathy.

OBJECTIVES: To identify the molecular etiology of distinct dental anomalies found in eight Thai patients and explore the mutational effects on cellular functions. MATERIALS AND METHODS: Clinical and radiographic examinations were performed for eight patients. Whole exome sequencing, mutant protein modelling, qPCR, western blot analysis, scratch assays, immunofluorescence, confocal analysis, in situ hybridization, and scanning electron micrography of teeth were done. RESULTS: All patients had molars with multiple supernumerary cusps, single-cusped premolars, and a reduction in root number. Mutation analysis highlighted a heterozygous c.865A>G; p.Ile289Val mutation in CACNA1S in the patients. CACNA1S is a component of the slowly inactivating L-type voltage-dependent calcium channel. Mutant protein modeling suggested that the mutation might allow leakage of Ca2+ or other cations, or a tightening, to restrict calcium flow. Immunohistochemistry analysis showed expression of Cacna1s in the developing murine tooth epithelium during stages of crown and root morphogenesis. In cell culture, the mutation resulted in abnormal cell migration of transfected CHO cells compared to wildtype CACNA1S, with changes to the cytoskeleton and markers of focal adhesion. CONCLUSIONS: The malformations observed in our patients suggest a role for calcium signaling in organization of both cusps and roots, affecting cell dynamics within the dental epithelium.

Cannabinoid therapeutics in orofacial pain management: a systematic review.

The objective of this paper was to investigate the published evidence regarding effects of cannabinoids (natural and synthetic) on post-operative and/or out-of-office pain management in patients suffering from orofacial pain that presents in the dental setting. Three online databases (Ovid (MEDLINE), PubMed (MEDLINE), Scopus) were searched (July 2021). Additional studies were sought through grey literature searching (Cochrane Library Trials and ClinicalTrials.gov) and hand-searching the reference lists of included articles. All studies that analysed cannabinoid products and pain management of conditions that present in the general or specialist dental setting in the English language were included. Of the five articles included, one reported a significant effect on temporomandibular disorder pain relief using a topical cannabidiol formulation compared to a placebo. Four articles reported no significant effects of cannabinoids for pain management across various orofacial pain conditions. Although one study reported a positive effect, insufficient evidence exists to support a tangible clinical benefit of cannabinoids in managing orofacial pain, further research is recommended to investigate the benefits of cannabinoids' use. © 2022 Australian Dental Association.

SH3BP2-related fibro-osseous disorders of the maxilla and mandible: A systematic review.

Cherubism is a disorder of bony overgrowth of the jaws that manifests in childhood. SH3BP2 gene variants have been associated with cherubism; this gene plays a major role in bone homeostasis. Due to its rare occurrence, there is as yet no comprehensive understanding of the natural history and clinical course of the disease. The aim of this review was to compile and analyze all cases of SH3BP2-related cherubism and cherubism-like disorders. Thirty publications were identified, including 92 individuals from 34 families, who were diagnosed with SH3BP2-related fibro-osseous lesions of the jaw. Only 15% of cases included in this review had no known family history of the disease. The distribution of cherubism was equal with respect to biological sex. Missing teeth were reported in 38% of cases. Lesions were restricted to the mandible in 36% of cases and involved both the maxilla and mandible in 54% of cases. The clinical phenotypes reported in the articles analyzed varied greatly in detail, making comparisons between studies and conclusive analysis difficult. Further work is necessary to describe the connection between SH3BP2 gene variants and cherubism in order to advance its diagnosis and treatment.

Low rate of local recurrence detection by rectoscopy in follow-up of rectal cancer.

AIM: The main aim of this study was to examine the effectiveness of rectoscopy for detecting local recurrence of rectal cancer in patients following low anterior resection. METHOD: This was a retrospective study of 201 patients, who underwent low anterior resection for rectal or rectosigmoid cancer between 2007 and 2009 and who were followed up with rigid rectoscopy and imaging. A total of 91 patients were excluded from the analysis for various reasons, leaving 110 patients eligible for analysis. RESULTS: A total of 613 rectoscopies were performed, and 48 biopsies taken. Six local recurrences were detected in the 110 patients, three of which were first detected by rectoscopy and three by CT. Two of the local recurrences were detected outside the follow-up programme because of symptoms: one by rectoscopy and one by CT. Three of 613 (0.5%) rectoscopies led to detection of local recurrence. The sensitivity and specificity of rectoscopy to detect local recurrence was 0.50 and 0.93, respectively. Nineteen distant metastases were detected, and two patients had both local recurrence and distant metastasis. All local recurrences and distant metastases were detected within 48 months of surgery. CONCLUSION: Rigid rectoscopy is poor at detecting local recurrence. Only 3 out of 613 rectoscopies (0.5%) detected local recurrence. Due to extramural growth of some recurrences, the sensitivity is also very low. Based on our results, routine rectoscopy in the surveillance of asymptomatic patients cannot be recommended.

Calibration process for rechargeable cell and battery test systems.

Coulombic efficiency is a powerful metric for evaluating the performance of materials in rechargeable cells and batteries. The ideal Coulombic efficiency, the ratio of charge removed to charge inserted, is unity. Some specialized systems can accurately measure cell capacity and Coulombic efficiency within 0.001%, which requires precise control and measurement of current, voltage, time, and temperature. Most battery electrode and electrolyte research is not performed with such precise but complex systems. The purpose of this paper is to demonstrate a simple, robust procedure to measure and possibly improve the accuracy of capacity and Coulombic efficiency measurements on standard systems in their as-used state. This approach is built on a commercially available thin film rechargeable cell for micro or milliampere currents and can be extended to, e.g., 18 650, cells for higher currents. An improved method to display Coulombic efficiency data is also presented. Regular, consistent calibration of testing systems and reporting of system resolution at specified test conditions is encouraged.

Rate of Anastomotic Leakage After Rectal Anastomosis Depends on the Definition: Pelvic Abscesses are Significant.

BACKGROUND AND AIMS: The International Study Group of Rectal Cancer has proposed that a pelvic abscess in the proximity of the anastomosis is considered an anastomotic leak, whether or not its point of origin is detectable. This study describes how the inclusion of pelvic abscesses alters the leakage rate. MATERIAL AND METHODS: Risk factors and postoperative complications in patients with visible anastomotic leakage ("direct leakage"), pelvic abscesses alone in the vicinity of a visibly intact anastomosis ("abscess leakage"), and no leakage were retrospectively evaluated. RESULTS: In total, 341 patients operated with anterior resections and who received an anastomosis within 15 cm as measured from the anal verge were included. A total of 37 patients (10.9%) had direct leakage, 13 (3.8%) had abscess leakage, and 291 (85.3%) had no leakage. The overall anastomotic leakage rate was 14.7% (50 patients). In accordance with the grading system outlined by International Study Group of Rectal Cancer, 7 patients (2.1%) experienced Grade A leakage, 19 (5.6%) Grade B, and 24 (7.0%) Grade C. Direct leak patients had more often a reoperation due to anastomotic complications (odds ratio = 19.7, p = 0.001), a permanent stoma (odds ratio = 28.5, p = 0.001), and a longer hospital stay than abscess leak patients (29.0 vs 15.5 days, p = 0.030). CONCLUSION: Abscess leakage accounted for over one-fourth of the overall leakage rate, raising the leakage rate. Direct leak patients were at a higher risk of requiring a reoperation, permanent stoma, and longer hospital stay than abscess leak patients. Abscess leak patients were at a greater risk for a urinary tract infection, wound infection, and postoperative intestinal obstruction than non-leak patients.

Gas chromatography-isotope dilution mass spectrometry method validation for target pesticides in soybeans.

The production of certified reference materials requires the application of highly accurate methods for characterisation. A gas chromatography-isotope dilution mass spectrometry method, setting ambitious performance criteria, was developed for eight selected pesticides in soybeans. Pressurised liquid extraction was followed by automated gel-permeation chromatography and solid-phase extraction clean-up. Pesticides identification respected a Commission Decision and guidelines of the Directorate General for Health and Food Safety (DG SANTE). Reliable quantification involved stable isotopically labelled analogues as internal standards. Validation, according to ISO/IEC 17,025 and DG SANTE guidelines, assessed linearity, LOD/LOQ, trueness, selectivity, precision, stability and robustness. Mean recoveries ranges (83-109%, relative standard deviations < 3%), repeatability (2.2-4.8%), day-to-day variation (0.6-4.2%) and combined uncertainty (1.2-4.2%) were fit for purpose. The method is highly accurate and suitable for certification of the selected pesticides in soybean matrix reference material. Chemical compounds studied in this article: Diazinon (PubChem CID: 3017); malathion (PubChem CID: 4004); chlorpyrifos (PubChem CID: 2730); captan (PubChem CID: 8606); endosulfan (PubChem CID: 3224); tebuconazole (PubChem CID: 86,102); iprodione (PubChem CID: 37,517); cypermethrin (PubChem CID: 2912).

Adrenaline and cortisol levels are lower during nighttime than daytime hypoglycaemia in children with type 1 diabetes.

AIM: We investigated children's counter regulatory hormone profiles during a hyperinsulinaemic hypoglycaemic clamp procedure at day and night. METHODS: In 2013, we assessed the counter regulatory response to hypoglycaemia in eight outpatients with type 1 diabetes, recruited from the Herlev Hospital, Denmark, at a mean age of 9.6 ± 2.3 years. Hyperinsulinaemic 80 mU/m2 /min clamps were performed with a stepwise reduction in plasma glucose from euglycaemia (7-9 mmol/L) to hypoglycaemia (<3.5 mmol/L) and the glucose nadir (≤2.2 mmol/L) during the day and night. Adrenaline, cortisol, glucagon and growth hormone levels were assessed. RESULTS: Adrenaline and growth hormone levels were higher during the day versus the night (p = 0.04 and p = 0.01, respectively). However, at the glucose nadir, the level of adrenaline was lower during the night than the day (0.6 ± 0.2 versus 1.9 ± 0.5 nmol/L, p = 0.016) and cortisol was lower during the day than the night (42 ± 15 versus 319 ± 81 nmol/L, p = 0.016). No differences were demonstrated for glucagon and growth hormone levels based on the same criteria. CONCLUSION: The adrenaline response was blunted during nocturnal iatrogenic hypoglycaemia in our study cohort, and no increase in cortisol levels was demonstrated.

No difference in time-dependent improvement in functional outcome following closing wedge versus opening wedge high tibial osteotomy: a randomised controlled trial with two-year follow-up.

AIMS: The aim of this prospective randomised study was to compare the time course of clinical improvement during the first two years following a closing or opening wedge high tibial osteotomy (HTO). It was hypothesised that there would be no differences in clinical outcome between the two techniques. PATIENTS AND METHODS: Between 2007 and 2013, 70 consecutive patients were randomly allocated to undergo either a closing or opening wedge HTO. All patients had medial compartment osteoarthritis (OA), and were aged between 30 years and 60 years. They were evaluated by independent investigators pre-operatively and at three and six months, and one and two years post-operatively using the Knee Injury and Osteoarthritis Outcome Score (KOOS), the Oxford Knee Score (OKS), the Lysholm score, the Tegner activity score, the University of California, Los Angeles (UCLA) activity scale and range of movement (ROM). RESULTS: There were no significant differences at any time between the two techniques for any clinical outcome score (p > 0.05). The mean scores for all the systems, except UCLA and Tegner, significantly improved until six months post-operatively (p < 0.001). For some scores, the improvement continued until one and two years. CONCLUSION: This prospective randomised study suggests that there are no differences in the time course of the clinical improvement between the closing and opening wedge techniques for HTO during the first two post-operative years. Patients can expect continued improvement in physical function for between six months and one year after HTO regardless of the technique used. Cite this article: Bone Joint J 2017;99-B:1157-66.

Observation of Quantum-Limited Spin Transport in Strongly Interacting Two-Dimensional Fermi Gases.

We measure the transport properties of two-dimensional ultracold Fermi gases during transverse demagnetization in a magnetic field gradient. Using a phase-coherent spin-echo sequence, we are able to distinguish bare spin diffusion from the Leggett-Rice effect, in which demagnetization is slowed by the precession of a spin current around the local magnetization. When the two-dimensional scattering length is tuned to be comparable to the inverse Fermi wave vector k_{F}^{-1}, we find that the bare transverse spin diffusivity reaches a minimum of 1.7(6)ℏ/m, where m is the bare particle mass. The rate of demagnetization is also reflected in the growth rate of the s-wave contact, observed using time-resolved spectroscopy. The contact rises to 0.28(3)k_{F}^{2} per particle, which quantifies how scaling symmetry is broken by near-resonant interactions, unlike in unitary three-dimensional systems. Our observations support the conjecture that, in systems with strong scattering, the local relaxation rate is bounded from above by k_{B}T/ℏ.

Increased mortality in a Danish cohort of young people with Type 1 diabetes mellitus followed for 24 years.

AIM: To determine the mortality rate in a Danish cohort of children and adolescents diagnosed with Type 1 diabetes mellitus compared with the general population. METHODS: In 1987 and 1989 we included 884 children and 1020 adolescents aged 20 years and under, corresponding to 75% of all Danish children and adolescents with Type 1 diabetes, in two nationwide studies in Denmark. Those who had participated in both investigations (n = 720) were followed until 1 January 2014, using the Danish Civil Registration System on death certificates and emigration. We derived the expected number of deaths in the cohort, using population data values from Statistics Denmark to calculate the standardized mortality ratio. Survival analysis was performed using Cox proportional hazards model. RESULTS: During the 24 years of follow-up, 49 (6.8%) patients died, resulting in a standardized mortality ratio of 4.8 (95% confidence interval 3.5, 6.2) compared with the age-standardized general population. A 1% increase in baseline HbA1c (1989), available in 718 of 720 patients, was associated with all-cause mortality (hazard ratio = 1.38; 95% confidence interval 1.2, 1.6; P < 0.0001). Type 1 diabetes with multiple complications was the most common reported cause of death (36.7%). CONCLUSION: We found an increased mortality rate in this cohort of children and adolescents with Type 1 diabetes compared with the general population. The only predictor for increased risk of death up to 24 years after inclusion was the HbA1c level in 1989. This emphasizes the importance of achieving optimal metabolic control in young people with Type 1 diabetes.

Retinal vascular geometry and its association to microvascular complications in patients with type 1 diabetes: the Danish Cohort of Pediatric Diabetes 1987 (DCPD1987).

PURPOSE: To examine associations between retinal vascular geometry (tortuosity, branching coefficient [BC] and length-diameter ratio [LDR]) and diabetic proliferative retinopathy (PDR), nephropathy, and peripheral neuropathy in patients with type 1 diabetes mellitus (T1DM). METHODS: A cohort of patients with T1DM participated in a clinical examination in 2011. Blood and urine analyses were done and retinal images taken. PDR was defined as Early Treatment Diabetic Retinopathy Study level 61 or above, nephropathy as albumin-creatinin ratio ≥300 mg/g, and neuropathy as vibration perception threshold >25 Volt. Retinal vessel parameters were measured using semi-automated software. Multiple logistic regressions were performed to investigate correlations between retinal vascular parameters and outcomes. Models were adjusted for other variables (sex, age, duration of diabetes, systolic and diastolic blood pressure, HbA1c, and presence of microvascular complications). Odds ratios were given per standard deviation in retinal vascular parameter. RESULTS: Retinal vascular analyses were performed in 181 patients. Mean age and duration of diabetes were 37.0 years and 29.4 years respectively, and 50.8% were male. Prevalence of PDR, nephropathy, and neuropathy were 26.5%, 6.8%, and 10.1% , respectively. Patients with increased arteriolar BC had a higher risk of nephropathy (OR: 3.10, 95% CI: [1.01-9.54]). Patients with increased venular BC had a higher risk of neuropathy (OR: 2.11, 95% CI: [1.11-4.03]). No associations were found in patients with PDR. CONCLUSIONS: By analyzing the retinal vascular tree in patients with T1DM, we found a higher risk of complications in kidneys and nerves when BC was increased. This might indicate a suboptimal construction of the vascular tree in these patients.

Influenza A Virus in Backyard Pigs and Poultry in Rural Cambodia.

Surveillance of influenza virus in humans and livestock is critical, given the worldwide public health threats and livestock production losses. Livestock farming involving close proximity between humans, pigs and poultry is often practised by smallholders in low-income countries and is considered an important driver of influenza virus evolution. This study determined the prevalence and genetic characteristics of influenza A virus (IAV) in backyard pigs and poultry in Cambodia. A total of 751 animals were tested by matrix gene-based rRT-PCR, and influenza virus was detected in 1.5% of sampled pigs, 1.4% of chickens and 1.0% of ducks, but not in pigeons. Full-length genome sequencing confirmed triple reassortant H3N2 in all IAV-positive pigs and various low pathogenic avian influenza subtypes in poultry. Phylogenetic analysis of the swine influenza viruses revealed that these had haemagglutinin and neuraminidase genes originating from human H3N2 viruses previously isolated in South-East Asia. Phylogenetic analysis also revealed that several of the avian influenza subtypes detected were closely related to internal viral genes from highly pathogenic H5N1 and H9N2 formerly sequenced in the region. High sequence homology was likewise found with influenza A viruses circulating in pigs, poultry and wild birds in China and Vietnam, suggesting transboundary introduction and cocirculation of the various influenza subtypes. In conclusion, highly pathogenic subtypes of influenza virus seem rare in backyard poultry, but virus reassortment, involving potentially zoonotic and pandemic subtypes, appears to occur frequently in smallholder pigs and poultry. Increased targeted surveillance and monitoring of influenza circulation on smallholdings would further improve understanding of the transmission dynamics and evolution of influenza viruses in humans, pigs and poultry in the Mekong subregion and could contribute to limit the influenza burden.

Evidence to support the hypothesis of tuberculosis as a cause of extreme osteonecrosis and osteomyelitis of the mandible in a West African population.

Maxillofacial tuberculosis (TB) is rare. The cases of 19 patients showing extreme bony destruction in the mandible, collected over a 3-month period in West Africa, are presented. Clinical, radiographic, and histological evidence indicated Mycobacterium tuberculosis as a possible cause. Further studies are in progress.

Optimum bolus wizard settings in insulin pumps in children with Type 1 diabetes.

AIM: To evaluate current insulin pump settings in an optimally regulated paediatric population using bolus wizard. METHODS: We used a retrospective study design to analyse data from 124 children on insulin pump therapy who had optimum HbA1c levels [< 59 mmol/mol (< 7.5%)] and no history of severe hypoglycaemic events. Bolus wizard settings were used to calculate the insulin to carbohydrate factors and insulin sensitivity factors. Multiple regression analysis was used to analyse the variables associated with the calculation factors. RESULTS: Insulin to carbohydrate factor varied from 276 in the youngest group to 424 in the oldest group, and increased according to age. Insulin sensitivity factor was highest in the group aged 6 to < 12 years, with a value of 125. Age, amount of carbohydrates, number of boluses per day and insulin per kg were all significantly associated with both calculation factors. Furthermore, duration of insulin pump treatment was significantly associated with insulin sensitivity factor and percentage bolus/basal was significantly associated with insulin to carbohydrate factor. Gender, diabetes duration and BMI were not associated with any of the calculation factors. CONCLUSION: Optimum insulin pump settings at pump initiation depend on both insulin requirements and use of the pump. Settings need to be individualized because the standardized calculation factors are not constant for children. There is a need to develop specific age- and insulin dose-dependent calculation factors.

Col2-Cre and tamoxifen-inducible Col2-CreER target different cell populations in the knee joint.

OBJECTIVE: Collagen type 2 (Col2)-Cre or tamoxifen-inducible Col2-CreER transgenic mouse lines have been used for studies to explore the cellular and molecular pathogenesis of osteoarthritis (OA). The purpose of this study is to investigate whether the targeted cells are the same or different in the two mouse lines. METHODS: We crossed tamoxifen inducible Col2-CreER and Col2-Cre mice with Rosa tdTomato reporter mice and analyzed the labeling patterns at different time points. RESULTS: In the Col2-CreER mice, 90.8 [95% confidence interval (CI) (88.3, 93.2)] and 82.8 (77.4, 88.3) % of the articular surface cells are Tomato positive when tamoxifen was administered at 2 and 2.5 weeks of age and strong activity was observed even 4.5 months after injection. However, 46.0 (32.8, 59.1) and 22.2 (11.7, 32.6) % of the surface cells were Tomato positive when tamoxifen was administered at 3 and 4 weeks of age, respectively. Little to no Tomato activity in the articular surface cells was observed when tamoxifen was administered at 8 weeks of age. At any stage of tamoxifen injection, the Tomato activity was detected in growth plate and epiphyseal bone in addition to articular chondrocytes, but little in endosteum and not in the synovium and ligament. In contrast, the targeted tissues in the Col2-Cre mouse line were articular cartilage, growth plate, meniscus, endosteum, ligament, bone and synovium. CONCLUSIONS: This study demonstrates that the pattern of targeted cells in the inducible Col2-CreER mice are partially overlapping with but different from that of targeted cells in Col2-Cre mice and the pattern varies dependent on when tamoxifen is administered.

Characterization of metabolic responders on CSII treatment amongst children and adolescents in Denmark from 2007 to 2013.

AIM: This prospective study aimed to identify and estimate the frequency of responders offered Continuous subcutaneous insulin infusion (CSII) from baseline data and during follow-up, and secondly to characterize CSII users with good adherence to pump therapy among 463 children and adolescents with Type 1 diabetes mellitus. METHODS: A response was defined as lowering HbA1c with 1% or achieving an HbA1c<7.5% (58 mmol/mol). Good adherence was defined as measuring ≥7 self monitored blood glucoses (SMBGs) and taking ≥7 boluses daily. Logistic regression was used to estimate the effect of demographic and clinical variables prior to and during pump treatment. RESULTS: At 24 months follow-up 32% qualified as responders. Stratifying for age at onset, 45% of the children aged <6 yrs qualified as responders vs. 32% and 28% of the youngsters and adolescents aged 6-12 yrs. and 12-19 yrs., respectively (p=0.02). Responders were characterized by their HbA1c-level at pump onset (p=0.001), taking more daily boluses (7.64 ± 3.33 vs. 6.40 ± 3.18 p=0.003) and measuring more SMBGs per day at follow-up (6.88 ± 2.35 vs. 6.31 ± 2.54 p=0.03). The incidence of severe hypoglycemia decreased from 14.3 to 3.3 events per 100 person years (p<0.0001). Twenty percent did not respond despite a good adherence toward CSII therapy. CONCLUSION: Age <6 years, high or low HbA1c at pump initiation and number of daily boluses were associated with improved or sustained near-normal metabolic outcome. The incidence of severe hypoglycemia was significantly reduced. Twenty percent of the population had good adherence without any metabolic improvement.

Dissemination of the multidrug-resistant extended-spectrum ß-lactamase-producing Escherichia coli O25b-ST131 clone and the role of house crow (Corvus splendens) foraging on hospital waste in Bangladesh.

Two hundred and thirty-eight faecal samples from crows foraging on hospital wastes were analysed for extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. ESBL-producing crow isolates were characterized and compared with 31 patient isolates. Among the crows, 59% carried ESBL producers. These included Escherichia coli, Klebsiella pneumoniae, Raoultella terrigena and Enterobacter cloacae harbouring the genes for CTX-M-1, CTX-M-15, CTX-M-55, CTX-M-79, and CTX-M-14. Human isolates carried only the CTX-M-15 gene. Two-thirds of crow E. coli isolates and all human E. coli isolates were multidrug resistant. Crows and patients shared E. coli sequence types, including the epidemic E. coli O25b-ST131 clone. The scavenging behaviour of crows at poorly managed hospital waste dumps made them potential reservoirs of antibiotic resistance, including ESBLs.

Accurate determination of selected pesticides in soya beans by liquid chromatography coupled to isotope dilution mass spectrometry.

A sensitive, accurate and simple liquid chromatography coupled with mass spectrometry method for the determination of 10 selected pesticides in soya beans has been developed and validated. The method is intended for use during the characterization of selected pesticides in a reference material. In this process, high accuracy and appropriate uncertainty levels associated to the analytical measurements are of utmost importance. The analytical procedure is based on sample extraction by the use of a modified QuEChERS (quick, easy, cheap, effective, rugged, safe) extraction and subsequent clean-up of the extract with C18, PSA and Florisil. Analytes were separated on a C18 column using gradient elution with water-methanol/2.5 mM ammonium acetate mobile phase, and finally identified and quantified by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Reliable and accurate quantification of the analytes was achieved by means of stable isotope-labelled analogues employed as internal standards (IS) and calibration with pure substance solutions containing both, the isotopically labelled and native compounds. Exceptions were made for thiodicarb and malaoxon where the isotopically labelled congeners were not commercially available at the time of analysis. For the quantification of those compounds methomyl-(13)C2(15)N and malathion-D10 were used respectively. The method was validated according to the general principles covered by DG SANCO guidelines. However, validation criteria were set more stringently. Mean recoveries were in the range of 86-103% with RSDs lower than 8.1%. Repeatability and intermediate precision were in the range of 3.9-7.6% and 1.9-8.7% respectively. LODs were theoretically estimated and experimentally confirmed to be in the range 0.001-0.005 mg kg(-1) in the matrix, while LOQs established as the lowest spiking mass fractionation level were in the range 0.01-0.05 mg kg(-1). The method reliably identifies and quantifies the selected pesticides in soya beans at appropriate uncertainty levels, making it suitable for the characterization of candidate reference materials.