In-house Standards Research Articles

Formulation Optimization and Preparation of Amlodipine and Telmisartan Double-layer Tablets (5/40Mg) using Wet Granulation Method

The two-layer tablet of amlodipine and telmisartan is an effective strategy to manage hypertension disease through a combination of various treatment mechanisms. However, the spray-drying method is currently used to prepare telmisartan tablets due to its extremely low solubility, which requires sophisticated equipment. In this study, wet granulation was applied to fabricate this two-layer tablet. Furthermore, design of experiments were deployed to design and optimize the formulation in order to obtain an in-vitro equivalence with the reference drug. As a result, a formulation of the two-layer table using the wet granulation method was successfully developed and optimized. The obtained tablet showed an in-vitro equivalence with the reference drug (with f2 superior to 50 for both amlodipine and telmisartan). Furthermore, the obtained tablet also met a set of in-house standards, including appearance, identification, assay, and dissolution rate. This study provided not a novel but effective and simple method of fabrication for amlodipine-telmisartan tablets using wet granulation. Furthermore, the obtained results also highlighted the significant contribution of a well-established design of experiments in the development of a complex tablet formulation.

Applying the German S2k-Guideline for Diagnosis and Treatment of Spondylodiscitis-A 5-Year Retrospective Evaluation of Patients without Neurological Symptoms.

Spondylodiscitis is a rather rare condition with an annual incidence of 1-7 per 100,000. Thus, empirical data on the treatment of this disease are limited. In 2020, the first German guideline for the diagnosis and treatment of spondylodiscitis was published. In a 5-year retrospective analysis, we examined the patient collective, the current diagnosis and treatment strategy, and the effect of Magnetic Resonance Imaging (MRI) diagnostics on therapeutic decisions of a consecutive monocentric cohort of 66 patients without neurological symptoms. The majority of the patients were male (55%) with a mean age of 74 years. Non-operative therapy was found to be associated with short-term treatment success in 54 (82%) of the patients. In 12 patients, who underwent surgical therapy, MRI diagnostics and clinical findings were equally important for the decision to perform a surgery. Patients treated operatively stayed for an average of 33.6 (±12.9) days in the hospital and thus significantly longer than non-operatively treated patients with 22.2 (±8.0) days. The in-house standard of care did not essentially deviate from the guideline's recommendations. Future research should address early detection of the need for surgical therapy, and immediate anti-infective treatment appropriate to the detected pathogen.

Abstract PO5-13-07: Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics

Abstract Background Early-stage triple-negative breast cancer (TNBC) is usually treated with neoadjuvant chemotherapy (NADC), but prognosis remains poor compared with other BC subtypes. Unfortunately, less than 30% of BC patients achieve pathological complete response (pCR) to NADC and the overall survival of TNBC when cancer spreads is estimated at 10-13 months. Complete response is associated with improved progression-free and overall survival rates, but more accurate prognostic markers are needed. In this study, we assessed the prognostic value of blood circulating metabolites by using targeted-based metabolomics. Methods Patients received standard neoadjuvant chemotherapy with epirubicin plus cyclophosphamide followed by paclitaxel. Blood samples were obtained upon completion of chemotherapy and before surgery. Pathological response to treatment was recorded according to the Miller & Payne system, and categorized as partial (MP 1, 2, & 3) or complete response (MP 4 & 5). Blood samples were collected in EDTA tubes, centrifuged and plasma was stored at -80°C. After QC standard addition and methanol extraction of proteins, four fractions of the resulting extract were analysed: two by reverse-phase/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one by reverse phase/UPLC-MS/MS with negative ion mode ESI, and one by hydrophilic interaction liquid chromatography/UPLC-MS/MS) by Metabolon using a Thermo Scientific Q-Exactive mass spectrometer with a heated electrospray ionization source and an Orbitrap mass analyser. Using Metabolon's hardware and software, raw data were extracted, identified, QC processed and quantified using the area under the curve (AUC). Identified compounds were compared with library entries of purified standards or recurrent unknown entities. The prognostic capacity of each metabolite was assessed using BRB ArrayTools. Selected metabolites were validated using a targeted approach using compound standards as follows. Plasma samples were re-analysed by UPLC/MS using a Vanquish LC coupled to an Orbitrap Exploris 240 MS (Thermo Fisher Scientific) with positive and negative ionization mode ESI under polarity switching. Identification of compounds were performed by comparison of retention times (against in-house authentic standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra. These analyses were carried out by MS-Omics (Denmark). Results Twenty patients treated in the Medical Oncology Unit of the University Hospital of Jaén were recruited. Median age was 50 (31-76). Twelve patients obtained a pathological complete response. Median follow-up after surgery was 25 months. Seven patients eventually had distant metastases (2 in the lungs, 1 in the brain, 1 in the bones and 3 multi-site). Metabolon untargeted analysis identified and quantified 985 metabolites, 789 after xenobiotics exclusion. Survival analysis identified 16 metabolites related with distant relapse (p< 0.05). The ten metabolites with lower p-value were selected for targeted validation. Ms-Omics pipeline allowed absolute quantification of 5 metabolites, 3 of them showing high correlation (R >0.7) between both measurements (targeted and untargeted). Validation of the prognostic value of the candidate metabolites is ongoing in a larger TNBC cohort. Conclusions Metabolomics is a useful tool for the detection of simply assessable and cost-effective prognostic biomarkers in TNBC. Easily monitoring the presence of minimal residual disease is worth to be settled up in the clinical practice for the most aggressive molecular subtype of breast cancer. In this work we have defined a set of metabolites that could predict distant relapse events in TNBC patients treated with neoadjuvant chemotherapy. Further analyses are being carried out to validate the prognostic value of the proposed candidate metabolites. Citation Format: Pedro Sánchez-Rovira, Rocio Urbano-Cubero, Angelo Gamez Pozo, Carmen González-Olmedo, Alicia Cano-Jimenez, Andrea Zapater- Moros, Leticia Díaz-Beltrán, Lucia Trilla-Fuertes, Mariana Díaz-Almirón, Enrique Espinosa, Pilar Zamora, Juan Angel Fresno Vara. Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-07.

Quantification and Potential Viability of Human Noroviruses in Final Effluent from Wastewater Treatment Works in Pretoria, South Africa

Growing global concerns over water scarcity, worsened by climate change, drive wastewater reclamation efforts. Inadequately treated wastewater presents significant public health risks. Previous studies in South Africa (SA) have reported high norovirus levels in final effluent and sewage-polluted surface water, indicating pathogen removal inefficiency. However, the viability of these virions was not explored. This study assessed human norovirus viability in final effluent from wastewater treatment works (WWTWs) in Pretoria, SA. Between June 2018 and August 2020, 200 samples were collected from two WWTWs, including raw sewage and final effluent. Norovirus concentrations were determined using in-house RNA standards. Viability of noroviruses in final effluent was assessed using viability RT-qPCR (vPCR) with PMAxx™-Triton X-100. There was no significant difference in GI concentrations between raw sewage (p = 0.5663) and final effluent (p = 0.4035) samples at WWTW1 and WWTW2. WWTW1 had significantly higher GII concentrations in raw sewage (p < 0.001) compared to WWTW2. No clear seasonal pattern was observed in norovirus concentrations. At WWTW1, 50% (7/14) of GI- and 64.9% (24/37) of GII-positive final effluent samples had no quantifiable RNA after vPCR. At WWTW2, the majority (92.6%, 25/27) of GII-positive final effluent samples showed a 100% RNA reduction post vPCR. PMAxx™-Triton X-100 vPCR provides a more accurate reflection of discharge of potentially viable noroviruses in the environment than standard RT-qPCR. Despite significant reductions in potentially viable noroviruses after wastewater treatment, the levels of potentially viable viruses in final effluent are still of concern due to the high initial load and low infectious dose of noroviruses.

Potential of external (in air) particle induced gamma-ray emission method for the preparation of isotopic composition of boron in-house reference standard in boron carbide matrix for quality control work

In-house reference standards for isotopic compositions of boron in natural and enriched boron carbide matrix were prepared using external PIGE methods.

Non-matrix-matched analysis of Fe isotopes in silicates by laser ablation MC-ICP-MS and potential silicate in-house standards for microbeam Fe isotopic analysis

Fe isotopic compositions of natural silicate minerals, including olivine, garnet, amphibole and biotite are directly calibrated against non-matrix-matched BCR-2G glass by LA-MC-ICP-MS under wet plasma conditions.

International standards for monoclonal antibodies for assessing the biological activity of medicines: A status update

Scientific relevance. The clinical effects and the expiration of patents for original (reference) biotechnological medicines based on monoclonal antibodies (mAbs) stimulated the development of biosimilar mAbs. The quality profile of a biosimilar mAb should correspond to the quality of the reference medicinal product. When demonstrating biosimilarity and determining the activity of medicines as part of batch quality control, analysts should study the biological properties of mAbs using suitable reference standards. The lack of international standards (ISs) makes mAb manufacturers use in-house reference standards. There is a risk of obtaining non-uniform quality and efficacy data because of the use of in-house reference standards, the heterogeneity and structural complexity of mAbs, and the relationship between the biological activity and efficacy of mAbs.Aim. This study aimed to analyse the relevance of and need for ISs for the biological activity of biotherapeutic mAbs and to define the role of reference medicinal products and ISs in assessing biosimilarity and testing medicines throughout their lifecycle.Discussion. This review covers the issues arising from the lack of ISs for assessing the biological activity of mAbs and the role and significance of reference products and ISs for biosimilars. The authors describe the specifics of studying the biological properties of mAbs and summarise the data on the need to develop and use ISs for the standardisation of biological tests. This review presents the results of studies on the first ISs established by the World Health Organisation to assess the biological activity of mAbs; these results suggest the need to standardise mAbs using ISs to ensure the quality, safety, and efficacy of mAb therapy.Conclusions. The use of ISs for mAbs plays a key role in harmonising biological activity assessments. Publicly available ISs serve as primary standards for the calibration of secondary reference materials. Moreover, ISs are required for the harmonisation of activity evaluation (in IU) between laboratories and for the consistency of the activity of various medicinal products from different manufacturers that share the same INN. The use of ISs by mAb manufacturers will contribute to ensuring the quality of mAbs and clinical monitoring of the effectiveness of their use.

Nanogram-scale boron isotope analysis through micro-distillation and Nu Plasma 3 MC-ICP-MS

The determination of boron isotopes (δ11B) represents a powerful tool for a variety of applications such as the reconstruction of past ocean pH and atmospheric pCO2 from the analysis of marine biogenic carbonates. In recent years, MC-ICP-MS has gained popularity over other techniques thanks to its superior sample throughput and high ionization efficiency. This study evaluates, for the first time, the performance of the Nu Instruments Plasma 3 MC-ICP-MS for measuring δ11B using different sample introduction systems and detector configurations. The main goal is to provide a detailed methodology for nanogram-scale boron isotope analysis through a straightforward approach that can be easily adopted. Boron (B) purification from the carbonate matrix was performed through micro-distillation, using a temperature of 95 °C and a minimum heating duration of 15 h, allowing the full recovery of B from up to 3 mg of carbonate mass. We attained blank values (on average 14 ± 6 pg, 1 SD, n = 27) comparable to the lowest micro-distillation blanks reported in the literature. Three sample introduction systems were tested, and the 30 μL min−1 nebuliser system outperformed the 50 and 170 μL min−1 systems in terms of signal intensity per mass of B. Two detector configurations were used based on the total boron signal intensity achieved: (1) FC11/FC12, with two Faraday cups fitted to 1011 Ω and 1012 Ω amplifier resistors to detect 11B and 10B ion beams, respectively, and (2) FC12/IC, with which we investigated, for the first time, the feasibility of combining an ion counter for detecting 10B, and a Faraday cup fitted to a 1012 Ω amplifier for 11B. The FC12/IC configuration provided accurate results compared to the use of two Faraday cups for total boron signals lower than 0.35 V (∼12 ng of B in the analysed solution). The proposed analytical procedure was validated through the analysis of several reference materials with varying boron amounts, including clam JCt-1, coral JCp-1, NIST RM 8301 Foram and Coral solutions, and boric acid ERM-AE121. Furthermore, the long-term reproducibility was assessed with two in-house standards (coral CLD-1 and foraminifera GINF-1), providing values of 25.68 ± 0.23 ‰ (2SD, n = 53; with 14–36 ng of B) and 14.90 ± 0.16 ‰ (2SD, n = 12; with 11–16 ng of B), respectively.

In vitro activity of cefiderocol against comparators (ceftazidime-avibactam, ceftazidime-avibactam/ aztreonam combination, and colistin) against clinical isolates of meropenem-resistant Klebsiella pneumoniae from India.

Cefiderocol (FDC), a novel siderophore drug, is active against Gram-negative bacteria producing carbapenemases, including metallo-beta-lactamases. The objective of this study is to compare the in vitro activity of FDC with ceftazidime-avibactam (CZA), CZA/aztreonam (AT) combination, and colistin (CST), in clinical isolates of meropenem-resistant (MER-R) Klebsiella pneumoniae. From the 2,052 clinical specimens submitted for culture testing, 245 K. pneumoniae isolates were recovered within a 6-month period in 2021. One hundred three non-duplicate, non-outbreak, MER-R (minimum inhibitory concentration, MIC >4 µg/mL) strains were included in the study. Identification and susceptibility were performed using VITEK-2 (bioMérieux). Meropenem-susceptible isolates (n = 10) served as controls. For FDC, broth microdilution (BMD) was performed after in-house standardization. Disk diffusion (Liofilchem, Italy) and broth microdilution (ComASP, STC, Liofilchem, Italy) were used for susceptibility testing of CZA and CST, respectively. Synergy testing for CZA and AT was performed using disk approximation method. CLSI breakpoints were used for the interpretation of the results. For FDC, MIC50 and MIC90 were 2 and 8 µg/mL, respectively. A total of 80% of isolates were susceptible to FDC, 26.2% of isolates were susceptible to CZA, synergy testing with CZA/AT was positive for 74 (72%) of the isolates, and 89.3% were intermediate to CST. Nine (8.7%) were susceptible only to FDC. FDC is active in vitro against MER-R K. pneumoniae >CZA/AT > CZA > CST, as observed in this study, applying CLSI criteria. Clinico-microbiological studies should be performed to assess the clinical efficacy of this novel drug in this region with a high prevalence of carbapenem resistance among Gram-negative organisms. IMPORTANCE Management of infections with multi-drug resistant Klebsiella pneumoniae is a major challenge in hospital settings, with few treatment options. In this study, the authors aim to assess the in vitro susceptibility of these clinical isolates to cefiderocol, a novel siderophore. Comparators are colistin, ceftazidime-avibactam, and ceftazidime-avibactam/aztreonam synergy, which are currently available options for treatment in this region. Baseline-resistance rates against cefiderocol are higher than those in the previously published studies, with MIC50 and MIC90 at 2 and 8 µg/mL, respectively.

Pollen allergen products: current standardisation issues

Scientific relevance. Pollen allergen medicines are in high demand, and their therapeutic benefits directly correlate with their standardisation. Better diagnosis and treatment of allergic diseases require state-of-the-art procedures for assessing the allergenic activity of pollen allergen products using reference standards and physicochemical testing methods.Aim. The study aimed at developing methodological approaches to the standardisation of pollen allergen products in order to shift to measuring their potency in allergenic activity units (AAU) and bring their quality in line with the requirements of the State Pharmacopoeia of the Russian Federation.Materials and methods. The study used pollen allergen reference standards by Microgen, the WHO International Standard for timothy grass (Phleum pratense) pollen extract, a gel filtration standard kit of molecular weight markers ranging from 1.35 to 670 kDa, bovine serum albumin, serum samples with specific IgE obtained from donors sensitised to the study pollen allergens, labelled anti-human IgE antibodies, and reference standards for determining residual volatile solvents by gas chromatography. The identification of in-house reference standards for the potency of pollen allergens involved Western blotting (for allergenic components). The total protein content was determined by Bradford’s assay. In addition, the authors used high-performance liquid chromatography to study protein fractions and gas–liquid chromatography to determine the content of residual organic solvents.Results. To substitute the existing method of non-specific characterisation of allergenic activity in protein nitrogen units (PNU), the authors developed and tested a new method to control allergenic activity in allergenic activity units (AAU) based on an in vitro competitive enzyme-linked immunosorbent assay (ELISA) procedure developed and validated in this study. Furthermore, the authors developed and certified 15 primary in-house reference standards with allergenic activity established in AAU/mL using skin tests in vivo. The experimental data were analysed to standardise the allergenic activity of the pollen allergens manufactured by Microgen. The authors developed physicochemical methods for the certification of in-house reference standards and validated these methods in accordance with the State Pharmacopoeia of the Russian Federation. The study involved selecting chromatographic separation conditions for residual organic solvents (acetone and diethyl ether) and establishing system suitability criteria for the chromatographic system. The allergenic activity of secondary in-house reference standards was certified against that of primary in-house reference standards using competitive ELISA. Thus, the authors managed to shift to the standardisation of pollen allergen products in vitro.Conclusions. The authors developed their competitive ELISA-based method to standardise pollen allergen products by comparing the inhibition of immune responses to a product and a standard. The study demonstrated the feasibility of substituting allergenic activity quantification (in AAU) for protein nitrogen content determination (in PNU) and showed the first example of using AAU for the certification of in-house reference standards. Additionally, the authors developed and validated an analytical procedure for determining the content of residual organic solvents in pollen allergen products by gas–liquid chromatography.

In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Analytical Ultracentrifugation-Calibrated Anion-Exchange Chromatography for Sensitive and Intact Determination of Osteopontin in Infant Formula and Dairy Products.

Osteopontin is a crucial protein ingredient that has been applied in fortified dairy products and infant formula. It has great significance to infant gut health and brain development. However, current techniques including enzyme-linked immunosorbent assay and liquid chromatography coupled with mass spectrometry are still facing the bottleneck of low sensitivity and indirect quantification. Moreover, the unavailable certified commercial OPN standard hinders its accurate quantification. Herein, a novel method of anion-exchange chromatography was established to determine OPN concentration in several dairy matrices. The polarity-reversed capillary isoelectric focusing was utilized to measure the exact isoelectric point (pI) to support method development for OPN separation. Analytical ultracentrifugation was used to calibrate the purity of intact OPN to develop an in-house reference standard. The method showed the merits of limits of detection to 0.04 mg/100 g, relative standard deviation of reproducibility <5% for 13 out of 14 tested matrices, and an average recovery rate of 101.3%. This method has shown the potential to be adopted as an international standard method for the quantification of intact OPN in infant formula and dairy products.

Safety and Efficiency of Low-Dose Spinal Analgesia Compared to Epidural Analgesia in Treatment of Pain during Labour: A Case Control Study.

The epidural catheter for analgesia has been used for decades and has become the gold standard in pain therapy for pregnant women in labour. However, procedural parameters such as time to pain relief and duration to implementation pose hurdles for patients shortly before delivery. Low-dose spinal analgesia (LDSA) is an alternative procedure that was investigated in the study with regard to patient satisfaction and complication rates compared to epidural catheter. In a retrospective monocentric study, a total of 242 patients receiving low-dose spinal analgesia or epidural catheters were evaluated using propensity score matching. Subjective patient satisfaction as well as complication rates were primarily analysed. We hypothesise that LDSA is a safe procedure and provides a similar level of satisfaction compared with the epidural catheter. For this purpose, both procedures were performed according to in-house standards and the patients were interviewed afterwards. Patients who required surgical delivery were excluded to prevent bias. The LDSA was rated on average as very good [1.09 ± 0.311 vs. 1.07 ± 0.431] in terms of satisfaction by the patients compared to the epidural catheter without showing a significant difference (p = 0.653). Complications were in the low single-digit non-significant range for both procedures [6 (5%) vs. 7 (6%); p = 0.776]. The evaluation showed more perineal tears I° and II° in the low-dose spinal analgesia group [I°: 28 (23%) vs. 3 (2%); p < 0.001-II°: 30 (25%) vs. 2 (2%); p < 0.001]. Neonatal parameters differed significantly only in umbilical cord base excess and umbilical cord venous pH [-5.40 vs. -6.40; p = 0.005]. LDSA represents a low complication procedure for patients at the end of labour with a high satisfaction level. With the LDSA in the repertoire of pain relief during childbirth, it is possible to also achieve pain reduction for women with deliveries of high velocity without compromising patient satisfaction or perinatal morbidity.

Untargeted metabolomics on first trimester serum implicates metabolic perturbations associated with BMI in development of hypertensive disorders: a discovery study.

Body mass index (BMI) in early pregnancy is a critical risk factor for hypertensive disorders of pregnancy (HDP). The pathobiology of the interplay between BMI and HDP is not fully understood and represents the focus of this investigation. BMI and 1st-trimester serum samples were obtained from the Global Alliance to Prevent Prematurity and Stillbirth repository for 154 women (105 without HDP and 49 with HDP). Metabotyping was conducted using ultra-high-performance liquid-chromatography high-resolution mass spectrometry (UHPLC HR-MS). Multivariable linear regression and logistic models were used to determine metabolites and pathway perturbations associated with BMI in women with and without HDP, and to determine metabolites and pathway perturbations associated with HDP for women in categories of obese, overweight, and normal weight based on the 1st trimester BMI. These outcome-associated signals were identified or annotated by matching against an in-house physical standards library and public database. Pathway analysis was conducted by the Mummichog algorithm in MetaboAnalyst. Vitamin D3 and lysine metabolism were enriched to associate with BMI for women with and without HDP. Tryptophan metabolism enrichment was associated with HDP in all the BMI categories. Pregnant women who developed HDP showed more metabolic perturbations with BMI (continuous) than those without HDP in their 1st-trimester serum. The HDP-associated pathways for women with normal weight indicated inflammation and immune responses. In contrast, the HDP-associated pathways for women of overweight and obese BMI indicated metabolic syndromes with disorders in glucose, protein, and amino acid, lipid and bile acid metabolism, and oxidative and inflammatory stress. High first-trimester BMI indicates underlying metabolic syndromes, which play critical roles in HDP development. Vitamin D3 and tryptophan metabolism may be the targets to guide nutritional interventions to mitigate metabolic and inflammatory stress in pregnancy and reduce the onset of HDP.

Perioperative pain management in dermatosurgery - current practice in Germany: Data analysis of a Germany-wide online survey among dermatosurgeons.

Adequate pain management should be part of the standard of care in surgical procedures. However, there is a paucity of data in the field of dermatosurgery. In a standardized online survey among dermatosurgeons working in Germany, the current practice of perioperative pain management was investigated. Members of the German Society for Dermatosurgery (DGDC) and heads of dermatosurgical departments were asked to participate. Questions were related to practical implementation of perioperative pain management, pain documentation and personal sources of information on the topic. 116 questionnaires were analyzed. While prophylactic analgesia is rarely used, the vast majority (86%) reported the use of postoperative on-demand medication. The majority of surgeons do not have a fixed regimen. Mostly NSAIDs and occasionally low potency opioids are used. Pain is documented by the majority (59.1%) as free text. Personal experience (69%) and in-house standards (51%) are the most important factors in pain management. The use of guidelines (25%) plays a minor role. Perioperative pain management in dermatosurgery is strongly influenced by personal experience and may vary depending on the surgery performed. Consensus-based standardized recommendations are lacking. For adequate perioperative analgesia, the development of demand-oriented pain concepts is desirable. This requires prospective studies that address the specific patient population and surgical procedures in dermatology.

Botulinum toxin preparations: areas for improvement and issues of standardisation

Scientific relevance. Botulinum toxin preparations are a good example of using a deadly toxin as a unique therapeutic agent. However, there are many unresolved issues related to biotechnology, biological activity, interchangeability, and standardisation of botulinum toxin preparations. Aim. To review current opportunities for improving therapeutic botulinum toxin preparations.Discussion. This review covers botulinum toxin type A preparations and unresolved issues related to them. In the absence of international non-proprietary names recommended by the World Health Organisation or by the Board of the Eurasian Economic Commission, domestic and imported botulinum toxin type A preparations approved in Russia have only similarity-based grouping names. In addition, manufacturers name botulinum toxin preparations at their discretion. Therefore, classifying these preparations under a common nomenclature is essential for clear identification, adequate selection, and correct prescription. Several studies have shown significant variability across botulinum toxin type A preparations. Due to the identified differences in qualitative and quantitative characteristics, botulinum toxin type A preparations cannot be considered similar, which raises the issue of their interchangeability and bioequivalence. To resolve this issue, a unified classification and naming system for botulinum toxin preparations should be established and documented in regulatory standards. According to the literature, manufacturers of botulinum toxin preparations use in-house reference standards. Hence, the same activity unit resulting from toxicity and efficacy studies may express a different protein load for each botulinum toxin preparation. Keeping that in mind, the authors discuss the development of a single international potency standard for existing and pipeline botulinum toxin type A preparations. Conclusions. The article describes novel pharmaceutical compositions containing botulinum toxin, including those in late development. Summarised data from clinical studies on the safety, efficacy, and cost-effectiveness of botulinum toxin type A preparations can guide prescribing decisions.

Development of acompetence catalogue for physicians in training for curriculum creation with respect to delivery room training

Anesthesiologic expertise is used at various points in the delivery room. The natural turnover of professionals requires continuous education and training for patient care. In afirst survey among consultants and trainees, the desire for adelivery room-specific anesthesiologic curriculum has emerged. In order to enable acurriculum with decreasing supervision, acompetence-oriented catalogue is used in many medical fields. The gain in competence develops gradually. The participation of practitioners should be obligatory to avoid adifferentiation between theory and practice. The structural framework of curriculum development by Kern etal. provides the learning objective analysis after further evaluation. In the sense of specific learning objective definition, the present study aims to describe the competences for anesthetists in the delivery room. An expert group (active in the anesthesiology delivery room environment) developed aset of items via atwo-step online Delphi survey. The experts were recruited from the German Society for Anesthesiology and Intensive Care Medicine (DGAI). We evaluated the resulting parameters for relevance and validity in alarger collective. Lastly, we used factorial analyses to identify factors that could be used to group items into relevant scales. In total, 201participants took part in the final validation survey. During the prioritization process of Delphi analyses, competencies such as neonatal care were not followed up. Not all items developed are exclusively delivery room-related, such as managing a difficult airway. Other items are specific to the environment of obstetrics. One example is integration of spinal anesthesia into the obstetric context. Some items are exclusively related to the delivery room, such as in-house standards of care in obstetrics as abasic skill. After validation, acompetence catalogue with 8scales with atotal of 44 competence items resulted (Kayser-Meyer-Olkin criterion 0.88). Acatalogue of relevant learning objectives for anesthetists in training could be developed. It specifies the generally required content of anesthesiologic training in Germany. Specific patient groups, such as patients with congenital heart defects, are not mapped. Competencies that could also be learned outside the delivery room, should be learned before the rotation. This enables the focus on the delivery room items, especially for those to be trained who do not work in ahospital with obstetrics. The catalogue needs to be revised for completeness for its own working environment. Particularly in hospitals that do not have apediatrician available, neonatal care becomes significant. Didactic methods, such as entrustable professional activities, have to be tested and evaluated. These enable competence-based learning with decreasing supervision and reflect the reality in hospitals. As not every clinic can provide the necessary resources for this anationwide provision of documents would be helpful.

Dual UHPLC-HRMS Metabolomics and Lipidomics and Automated Data Processing Workflow for Comprehensive High-Throughput Gut Phenotyping.

In recent years, feces has surfaced as the matrix of choice for investigating the gut microbiome-health axis because of its non-invasive sampling and the unique reflection it offers of an individual's lifestyle. In cohort studies where the number of samples required is large, but availability is scarce, a clear need exists for high-throughput analyses. Such analyses should combine a wide physicochemical range of molecules with a minimal amount of sample and resources and downstream data processing workflows that are as automated and time efficient as possible. We present a dual fecal extraction and ultra high performance liquid chromatography-high resolution-quadrupole-orbitrap-mass spectrometry (UHPLC-HR-Q-Orbitrap-MS)-based workflow that enables widely targeted and untargeted metabolome and lipidome analysis. A total of 836 in-house standards were analyzed, of which 360 metabolites and 132 lipids were consequently detected in feces. Their targeted profiling was validated successfully with respect to repeatability (78% CV < 20%), reproducibility (82% CV < 20%), and linearity (81% R2 > 0.9), while also enabling holistic untargeted fingerprinting (15,319 features, CV < 30%). To automate targeted processing, we optimized an R-based targeted peak extraction (TaPEx) algorithm relying on a database comprising retention time and mass-to-charge ratio (360 metabolites and 132 lipids), with batch-specific quality control curation. The latter was benchmarked toward vendor-specific targeted and untargeted software and our isotopologue parameter optimization/XCMS-based untargeted pipeline in LifeLines Deep cohort samples (n = 97). TaPEx clearly outperformed the untargeted approaches (81.3 vs 56.7-66.0% compounds detected). Finally, our novel dual fecal metabolomics-lipidomics-TaPEx method was successfully applied to Flemish Gut Flora Project cohort (n = 292) samples, leading to a sample-to-result time reduction of 60%.

Profiling flavourings in strawberry-flavoured e-liquid using headspace-gas chromatography-mass spectrometry.

E-liquids typically contain nicotine and flavourings in a matrix of propylene glycol (PG) and vegetable glycerine (VG). Some nicotine-free e-liquids are flavouring only in the aerosol carrier with the option for users to add their own nicotine. It is only the nicotine that is monitored in terms of level, as specified by the manufacturers. Little is known of the toxicological effect for some of the flavourings in the context of vaping as these are only regulated for ingestion and not inhalation. A method was developed to analyse volatile organic compounds (VOCs) evolved when e-liquids are vaporised based on headspace-gas chromatography-mass spectrometry (HS-GC-MS) for e-liquids. An in-house standard was prepared with sample matrix and purchased strawberry flavouring to simulate a simple e-liquid but with known levels. This standard was then used to optimise the analysis for use with e-liquid samples but not for full quantification purposes. These were purchased from a range of retailers and with different batches but mainly focussed on strawberry flavour. The results identified three key components indicative of strawberry flavour (ethyl-3-methyl butanoate, ethyl 2-methyl butanoate and ethyl butanoate) and showed considerable variation between both manufacturers and batches. Flavouring VOCs areregulated for ingestion but are not regulated for e-liquid inhalation, so these could have toxicological implications. In addition, the inconsistency between samples suggests further issues when users add their own nicotine to the e-liquids as the viscous sample matrix makes homogeneous mixing difficult.

Abstract 5116: Analytical performance of ELSA-seq, a blood-based test for early detection of multiple cancers

Abstract Introduction: Detection of cancer is its early stages can potentially improve therapeutic effectiveness. Previously, we demonstrated that ELSA-seq, a machine-learning-aided methylation profiling test, can detect and locate multiple cancers with high accuracy in plasma samples. Here, we evaluated the analytical performance of a refined test version of ELSA-seq, including analytical sensitivity, specificity, repeatability/reproducibility, and robustness. Methods: The classification algorithm and cut-offs for the ELSA-seq test were established in the THUNDER study (NCT04820868). Here, we describe the analytical performance using both plasma samples from the THUNDER study and DNA blends from cell lines. Analytical sensitivity was established by defining the limit of detection (LoD) using in-house cfDNA Methylation Reference Standards (MRS) with lowest DNA input for the assay. In brief, fragmented genomic control DNA (NA24385, Coriell Institute) was used as a diluent to contrive human cancer cell lines with defined mixing ratios (tumor fractions), which was further verified by digital droplet PCR (ddPCR). The LoD was determined by the lowest tumor fractions at which accurate DOC and TOO was reported in at least 95% of replicates. As variant allele frequency (VAF) is widely used as a surrogate measurement for tumour fractions, we also used ultra-deep mutation sequencing to attain VAF as an independent piece of evidence. Analytical specificity was assessed by the true negative rate in 120 plasma samples from age-matched healthy donors. A batch-to-batch repeatability/reproducibility study was carried out using 168 clinical samples processed across multiple reagent lots, instruments, and operators. Robustness was evaluated using common interfering substances that potentially could be present in plasma samples such as hemoglobin, bilirubin, triglycerides, and genomic DNA. Testing was performed using 24 cancer and 24 non-cancer samples, with or without interfering substances. Results: At 5ng input mass (approximating from 1ml plasma), the LOD95 was estimated down to 0.05% (tumor fractions) or 0.02% (VAF) among 6 cancer cell lines. 2 false positives were detected in 120 age-matched healthy donor samples, yielding a specificity of 98.3% (95%CI: 93.5-99.7%). All test results were concordant across multiple reagent lots, instruments, and operators (100% repeatability and reproducibility), and high Pearson correlation coefficients (&amp;gt;99%) were observed in the pair-wise comparison. In addition, none of the substances tested interfered with the assay. Conclusions: These results suggest that ELSA-seq is a highly sensitive test for detection of the trace amount of tumor-derived methylation signals in plasma samples. The performance is highly reproducible and robust, which is critical for clinical implementations. Citation Format: Bingsi Li, Jing Su, Guangliang Zhang, Jiayue Xu, Jianlong Peng, Ya Zhou, Fujun Qiu, Shuai Fang, Xiaofang Wen, Guoqiang Wang, Jing Zhao, Hao Wang, Shangli Cai, Zhihong Zhang. Analytical performance of ELSA-seq, a blood-based test for early detection of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5116.