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Background: Persons with hemophilia experience joint bleeding that can lead to debilitating arthropathy, most commonly seen in ankles, knees, and elbows. Arthropathy can hinder participation in daily and athletic activities. We explored how hemophilic arthropathy impacts movement patterns in walking and bilateral squatting tasks in persons with hemophilia compared to healthy controls., Methods: Persons with hemophilia and healthy controls completed walking and squatting tasks while kinematic and kinetic motion capture data were collected. The Hemophilia Joint Health Score exam was performed to measure hemophiliac arthropathy. OpenSim was used to model muscle and joint reaction forces and calculate moments and angles. Peak values were compared using Cohen's d to estimate effect sizes of hemophilia on movement parameters., Findings: Nine persons with hemophilia and eight age-matched controls were analyzed. Temporal-spatial metrics were similar between hemophilia and control groups in both tasks. In walking, persons with hemophilia had higher peak ankle dorsiflexion angles, vertical ground reaction force weight acceptance peaks, and hip extension and flexion moments compared to controls. In squatting, persons with hemophilia had lower knee extension moments, ankle joint reaction force, and knee extensor forces, but had higher hip extension moments., Interpretation: Temporal-spatial metric similarity between hemophilia and controls suggests that kinetic and kinematic analyses are needed to identify movement pattern differences. These data identify potential compensatory strategies at the hip that may be used by persons with hemophilia to mitigate impact on the knee and ankle. Future work will confirm these data in a larger sample size and be used to develop physical therapy strategies., Competing Interests: Declaration of competing interest Beth Boulden Warren reports financial support was provided by National Institutes of Health. Beth Boulden Warren reports financial support was provided by Maternal and Child Health Bureau. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Catecholamine-induced cardiomyopathy is an uncommon complication of pheochromocytoma. Pheochromocytoma is a rare tumor that predominantly occurs in adults, making catecholamine-induced cardiomyopathy secondary to pheochromocytoma in children an exceedingly rare presentation. Treatment typically consists of medical management followed by surgical resection. Mechanical support, typically salvage therapy with extracorporeal membrane oxygenation, has been used in adult patients with cardiogenic shock and after cardiac arrest, but to our knowledge, the use of mechanical support has not been described in pediatric patients. Case Report: A 16-year-old female presented with cardiogenic shock resulting from catecholamine-induced cardiomyopathy secondary to pheochromocytoma. She was treated with a percutaneous left ventricular assist device to allow myocardial recovery while medical therapy was optimized. Given the early initiation, the patient's myocardial recovery was prompt, and only 3 days of device support were required. She was discharged in good condition and subsequently underwent uncomplicated laparoscopic resection of the tumor a few weeks later. Conclusion: In pediatric patients with catecholamine-induced cardiomyopathy secondary to pheochromocytoma, aggressive measures of support-including mechanical support and infrequently used options such as percutaneous left ventricular assist devices-should be considered early in treatment to maintain adequate cardiac output, avoid cardiac arrest, and allow for prompt myocardial recovery., (©2024 by the author(s); Creative Commons Attribution License (CC BY).)

Purpose: Since the introduction of vertebral body tethering (VBT) for adolescent idiopathic scoliosis (AIS), a variety of post-operative chest drainage systems have been utilized. Most surgeons use formal chest tubes with a Pleur-evac, while others use smaller bulb suction drains (e.g., Blake drain). In addition, some centers utilize pleural closure. This multicenter study evaluates whether drain type or pleural closure impact perioperative and 90 day complication rates., Methods: A retrospective review was conducted from three institutions with established VBT programs. All preoperative, perioperative and 90 day postoperative data were analyzed to determine differences in outcomes between three cohorts: standard chest tube (SCT), standard chest tube with pleural closure (SCTPC) and 10 French Bulb drain (BD)., Results: 104 patients were identified for the study. 57 SCT, 25 SCTPC and 22 BD. All data are listed in order: SCT, SCTPC, BD. Length of stay (3.7, 4.3, 3.0 days) was less in the BD group (p = 0.009); post-operative drainage (460, 761, 485 cc) was less in the SCT and BD groups (p < 0.001); intra-operative estimated blood loss (EBL) 146, 382, 64 cc was less in the BD group (p < 0.001). No significant difference in number of days (3.2, 3.2, and 2.8 days) drainage was in place, groups (p = 0.311). Complication profile was similar with 2 chest tube reinsertions in the SCT and one hemothorax that resolved spontaneously in BD group., Conclusions: In this series of 104 patients, SCT, SCTPC and BD all had a similar safety profile. All three methods were safe and effective in managing post-operative chest drainage after thoracic VBT. In the series, BD group had significantly shorter LOS than both groups that used chest tubes., Level of Evidence: Level III, Retrospective cohort study., (© 2022. The Author(s), under exclusive licence to Scoliosis Research Society.)

Background: Undifferentiated pleomorphic sarcoma (UPS) is a high-grade neoplasm typically diagnosed in older adults and localized to the extremities or retroperitoneum. Because of poor response to therapy and high rates of recurrence, this neoplasm is associated with a poor prognosis. Case Report: A 12-year-old female presented with weight loss, abdominal pain, fatigue, and diarrhea. She was profoundly anemic with occult blood-positive stools. On endoscopy, a fungating cecal mass was biopsied and diagnosed as malignant sarcomatoid neoplasm. The neoplasm was resected with clear margins during subsequent surgery, and on final pathology was diagnosed as UPS. A suspicious lung nodule was also removed via video-assisted thoracoscopic surgery and found to be a granuloma positive for Histoplasma capsulatum for which the patient received antifungal therapy. The patient did not receive additional chemotherapy or radiotherapy and was doing well without signs of recurrence at 12 months postresection. Conclusion: This report of cecal UPS in a 12-year-old is rare because of the patient's age and tumor location. We have identified only 2 other case reports of pediatric gastrointestinal UPS. This case illustrates the need for a broad differential and prompt workup in pediatric patients presenting with weight loss and abdominal complaints. More information regarding the management and outcomes in cases of gastrointestinal UPS is needed to assist providers in determining the best treatment course and to allow for better prognostication., (©2023 by the author(s); Creative Commons Attribution License (CC BY).)

Background: Biliary dyskinesia (BD) is a well-established gallbladder pathology in adult patients and rates of cholecystectomy for BD continue to rise in the United States. Many pediatric patients with vague abdominal pain of variable duration are evaluated for biliary dyskinesia. It remains unknown which cohort of pediatric patients diagnosed with BD are most likely to have sustained improvement in symptoms following laparoscopic cholecystectomy. We aimed to determine whether cholecystectomy resulted in symptom relief and led to a reduction in the number of medical visits related to gastrointestinal (GI) symptoms after surgery., Methods: We performed a multi-institution retrospective review of all children < 18 years of age who underwent laparoscopic cholecystectomy for BD between January 2013 and April 2018 in our hospital system. GI symptoms and clinical visits related to a GI complaint were assessed preoperatively. Patients were followed for 2 years after surgery. At 6 months and 2 years postoperatively, symptoms and the rate of medical visits related to a GI complaint were quantified and compared to the preoperative values., Results: In total, 45 patients met our inclusion criteria. Of these, 82% of patients were female. The average age was 14 years old (± 2.6) and 56% of patients met the criteria for being overweight or obese. The mean gallbladder ejection fraction was 13% (± 10.8). All patients had abdominal pain, 82% (37/45) presented with nausea, and 51% (23/45) presented with post-prandial pain. Six months postoperatively, 58% of patients experienced resolution of their abdominal pain which decreased to 38% of patients after 2 years. Similarly, 59% had resolution of their nausea at 6 months compared to 43% at 2 years, and 100% had resolution of their post-prandial pain at 6 months compared to 91% at 2 years. The total number of clinical visits related to a GI complaint decreased from 2.6 (± 2.4) preoperatively to 1.0 (± 1.3) within 6 months postoperatively. When followed to 2 years postoperatively, the 6-month rate of clinical visits related to a GI complaint decreased from a mean of 2.6 preoperatively to 0.71 following surgery., Conclusions: Following cholecystectomy, we observed a high percentage of durable symptom resolution in those patients with BD who presented with post-prandial pain. Patients with non-food-related abdominal pain, with or without nausea and vomiting, had a lower rate of symptom resolution after surgery and the rate declined with time. For patients without post-prandial pain, evaluation and treatment of alternative sources of pain should be considered prior to surgery. Regardless of their presenting symptoms, patients who underwent surgery for BD had fewer clinical GI-related visits after surgery. However, no specific gallbladder ejection fraction or symptom alone was predictive of a lower rate of clinical visits postoperatively., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Surgical safety checklists (SSCs) aim to create a safe operating room environment for surgical patients. Provider attitudes toward checklists affect their ability to prevent harm. Pediatric surgeons' perceptions surrounding SSCs, and their role in improving patient safety, are unknown., Methods: American Pediatric Surgical Association members conducted an online survey to evaluate the use of and attitudes toward SSCs. The survey measured surgeons' perceptions of checklists, including the components that make them effective and barriers to participation. To better evaluate the available data on SSCs, the authors performed a systematic literature review on the use of SSCs with a focus on pediatric studies., Results: Of the 353 survey respondents, 93.6% use SSCs and 62.6% would want one used in their own child's operation, but only 54.7% felt that checklists improve patient safety. Reasons for checklist skepticism included the length of the checklist process, a distraction from thoughtful patient care, and lack of data supporting use. Literature review shows that checklists improve communication, promote teamwork, and identify errors, but do not necessarily decrease morbidity. Staff perception is a major barrier to implementation., Conclusions: Almost all pediatric surgeons participate in SSCs at their institutions, but many question their benefit. Better pediatric surgeon engagement in checklist use is needed to change the safety culture, improve operating room communication, and prevent harm.

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNβ in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8 + T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment. Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8 + T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR. See related commentary by Mittal et al., p. 1066 This article is highlighted in the In This Issue feature, p. 1047 ., (©2018 American Association for Cancer Research.)

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis., Competing Interests: The authors have declared that no conflict of interest exists.

Immunosuppression of tumour-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8(+) TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumour cells, leading to CD8(+) T-cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T-cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists.

MAP2K4 encodes a dual-specificity kinase (mitogen-activated protein kinase kinase 4, or MKK4) that is mutated in a variety of human malignancies, but the biochemical properties of the mutant kinases and their roles in tumorigenesis have not been fully elucidated. Here we showed that 8 out of 11 cancer-associated MAP2K4 mutations reduce MKK4 protein stability or impair its kinase activity. On the basis of findings from bioinformatic studies on human cancer cell lines with homozygous MAP2K4 loss, we posited that MKK4 functions as a tumor suppressor in lung adenocarcinomas that develop in mice owing to expression of mutant Kras and Tp53. Conditional Map2k4 inactivation in the bronchial epithelium of mice had no discernible effect alone but increased the multiplicity and accelerated the growth of incipient lung neoplasias induced by oncogenic Kras. MKK4 suppressed the invasion and metastasis of Kras-Tp53-mutant lung adenocarcinoma cells. MKK4 deficiency increased peroxisomal proliferator-activated receptor γ2 (PPARγ2) expression through noncanonical MKK4 substrates, and PPARγ2 enhanced tumor cell invasion. We conclude that Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing PPARγ2 levels.

Myelomeningocele (MMC) is a common and devastating malformation. As an alternative to fetal surgical repair, tissue engineering has the potential to provide a less invasive approach for tissue coverage applicable at an earlier stage of gestation. We have previously evaluated the use of gelatin hydrogel composites composed of gelatin sponges and sheets as a platform for tissue coverage of the MMC defect in the retinoic acid induced fetal rat model of MMC. In the current study, we compare our previous composite with gelatin microspheres as a scaffold for tissue ingrowth and cellular adhesion within the amniotic fluid environment. We also examine the relative efficacy of various bioactive protein coatings on the adhesion of amniotic fluid cells to the construct within the amniotic cavity. We conclude from this study that gelatin microspheres are as effective as gelatin sponges as a scaffold for cellular ingrowth and amniotic fluid cell adhesion and that collagen type I and fibronectin coatings enhance amniotic fluid cell adhesion to the gelatin-based scaffolds. These findings support the potential for the development of a tissue-engineered injectable scaffold that could be applied by ultrasound-guided injection, much earlier and less invasively than sponge or sheet-based composites.

Non-small cell lung cancer (NSCLC) cells with somatic mutations in K-ras recruit to the tumor a variety of cell types (hereafter collectively termed "stromal cells") that can promote or inhibit tumorigenesis by mechanisms that have not been fully elucidated. Here, we postulated that stromal cells in the tumor microenvironment alter the tumor cell secretome, including those proteins required for tumor growth and dissemination, and we developed an in vitro model to test this hypothesis. Coculturing a murine K-ras mutant lung adenocarcinoma cell line (LKR-13) with a murine lung stromal cell (macrophage, endothelial cell, or fibroblast) enhanced stromal cell migration, induced endothelial tube formation, increased LKR-13 cell proliferation, and regulated the secretion of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial-to-mesenchymal transition. Among these proteins, CXCL1 has been reported to promote NSCLC development, whereas interleukin-18 (IL-18) has an undefined role. Genetic and pharmacologic strategies to inhibit CXCL1 and IL-18 revealed that stromal cell migration, LKR-13 cell proliferation, and LKR-13 cell tumorigenicity required one or both of these proteins. We conclude that stromal cells enhanced LKR-13 cell tumorigenicity partly through their effects on the secretome of LKR-13 cells. Strategies to inhibit tumor/stromal cell interactions may be useful as therapeutic approaches in NSCLC patients.

A liquid chromatographic (LC) method with fluorescence detection was developed for concurrent determination of 4 fluoroquinolones: ciprofloxacin (CIPRO), enrofloxacin (ENRO), sarafloxacin (SARA), and difloxacin (DIFLX) in catfish, shrimp, and salmon. The procedure consists of extraction from fish tissue with acidified ethanol, isolation and retention on a cation exchange solid-phase extraction column, elution with basic methanol, and LC analysis with fluorescence detection. LC is performed by isocratic elution with acetonitrile-2% acetic acid (16 + 84) mobile phase, and a PLRP-S polymer column with fluorescence detection, excitation 278 nm and emission 450 nm. A target level of 20 ppb for each of the 4 fluoroquinolones has been established for this method. Fortified and incurred fish sample results are based on a 5-point standard curve calculation (10-160 ppb). Overall percent recoveries (% relative standard deviation) from fortified catfish were 78 (10), 80 (11), 70 (9.4), and 78 (10); from fortified shrimp, 69 (5.9), 85 (4.9), 79 (5.9), and 90 (4.5); and from fortified salmon, 56 (15), 93 (5.6), 61 (11), and 87 (5.0) for CIPRO, ENRO, SARA, and DIFLX, respectively. Data from the analysis of fluoroquinolone-incurred catfish, shrimp, and salmon are presented.

A liquid chromatographic/mass spectrometric (LC/MS) electrospray confirmation method has been developed to confirm 4 ionophores (monensin, lasalocid, salinomycin, and narasin) in a variety of animal feeds using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane-ethyl acetate and isolated using a silica solid-phase extraction cartridge. These ionophores were confirmed in both medicated feeds and nonmedicated feeds fortified with these drugs at the 1-50 ppm level. In addition, this method was used to confirm residues of monensin in a nonmedicated feed that was collected from a feed mill immediately after the production of a similar feed that was medicated with high levels of monensin.

A gas chromatographic (GC) method is presented for determining residues of chloramphenicol (CAP), florfenicol (FF), florfenicol amine (FFa), and thiamphenicol (TAP) in shrimp tissues, with meta-nitrochloramphenicol (mCAP) as the internal standard. The composited shrimp is extracted with basic ethyl acetate, followed by an acetonitrile-basic ethyl acetate mixture. This extract is centrifuged, filtered, evaporated, and reconstituted in water; the reconstituted extract is acidified, defatted with hexane, and passed through a propylsulfonic acid (PRS) and C18 solid-phase extraction (SPE) system. The C18 SPE column is eluted with methanol, and the PRS SPE column is eluted with basic MeOH plus counter ion. The combined eluates are evaporated, reconstituted in acetonitrile, and derivatized with Sylon BFT. After derivatization, the addition of toluene directly to the sample, followed by the addition of basic water, quenches the derivatization process. After centrifugation, the organic layer is carefully removed, and the analytes are determined by GC with electron capture detection. Shrimp tissues were fortified with fenicols (i.e., CAP, FF, FFa, and TAP) at 5, 10, 20, 40, and 80 ng/mL. Overall recoveries were 88, 101, 91, and 84% with overall interassay (between-day) variabilities (i.e., relative standard deviations) of 5.3, 9.4, 12.8, and 7.4% for CAP, FF, FFa, and TAP, respectively. The method detection limits were calculated as 0.7, 1.4, 2.4, and 1.3 ng/g (ppb) for CAP, FF, FFa, and TAP, respectively, based on a 10 g sample. The quantitation limit as determined empirically by this method is the lower limit of the standard curve, which is about 5 ng/g (ppb) for each analyte.

A liquid chromatographic (LC) method with fluorescence detection (FLD) is described for determining residues of the pesticide azamethiphos (AZA) in salmon tissue. The sample is extracted with ethyl acetate, centrifuged, dehydrated with anhydrous sodium sulfate, evaporated, reconstituted in water, and defatted with hexane. The aqueous phase is passed through a C18 solid-phase extraction (SPE) column. The SPE column is eluted with methanol, and the eluate is evaporated to dryness and then taken up in 10% acetonitrile (ACN) in water. The analyte is determined by LC using a C18 column, ACN-H2O (32 + 68) mobile phase, and FLD with excitation at 230 nm and emission at 345 nm. Composited salmon tissues were fortified with AZA at 5, 10, 21, 42, and 83 ng/g or ppb (target level, X = 10 ng/g). Overall recoveries were 86%, with between-day variability of 5.3%. The method detection limit was calculated as 1.2 ppb AZA based on a 5 g sample. The limit of quantitation as determined empirically by this method is the lower limit of the standard curve, approximately 5 ppb.

A multi-residue LC/MS method has been developed to confirm avermectin drug residues in several food matrices. Ivermectin (IVR), doramectin (DOR), eprinomectin (EPR) and moxidectin (MOX) are confirmed using atmospheric pressure chemical ionization (APCI) with negative ion detection and selected ion monitoring of three to four ions for each compound. The drug residues are extracted from tissue or milk using previously published procedures. IVR and DOR are confirmed at 20 ppb levels in fortified salmon muscle; IVR is also confirmed in tissue from salmon dosed with the drug. Residues of DOR, IVR, and EPR are confirmed in fortified milk at the 20 ppb level and in fortified beef liver at 40 ppb. Residues of MOX can also be confirmed in these matrices, but at slightly higher levels (40-80 ppb).

A peer-verified, liquid chromatographic (LC) method for simultaneous determination of residues of flumequine (FLU), nalidixic acid (NAL), oxolinic acid (OXO), and piromidic acid (PIR) in catfish muscle is presented. Sample workup involves homogenizing tissue with acetone, defatting with hexane, and extracting quinolones into chloroform. Sample is purified further by partitioning into base and then subsequently back-extracting into chloroform after acidifying the aqueous phase. After solvent is evaporated, the residue is diluted with mobile phase, and analytes are introduced into an LC system where separations are made with a 5 microns, reversed-phase polymer column and an isocratic, buffered acetonitrile-tetrahydrofuran mobile phase. Determinations are made by UV detection at 280 nm for PIR and by fluorescence detection (excitation at 325 excitation and emission at 365 nm) for the other 3 analytes. Each quinolone was used to fortify catfish muscle at 5, 10, 20, 40, and 80 ng/g. The following recoveries and relative standard deviation (RSD) values represent an average of the 5 levels for each analyte: FLU, 79.7% (RSD = 5.7%); OXO, 80.8% (RSD = 6.3%); PIR, 75.0% (RSD = 5.9%); and NAL, 87.1% (RSD = 10%). Assay of 5 levels (base incurred catfish, plus 4 dilutions with control catfish) of catfish muscle incurred with the 4 quinolones gave the following averages: FLU: base, 198 ng/g (RSD = 2.3%); dilutions, 98.0 ng/g (RSD = 4.2%), 61.6 ng/g (RSD = 4.4%), 21.6 ng/g (RSD = 2.8%), 9.24 ng/g (RSD = 8.7%); OXO, base, 257 ng/g (RSD = 6.9%); dilutions, 146 ng/g (RSD = 5.5%), 95.0 ng/g (RSD = 4.1%), 30.7 ng/g (RSD = 3.8%), 13.7 ng/g (RSD = 4.6%); PIR, base, 22.1 ng/g (RSD = 4.2%); dilutions, 13.7% ng/g (RSD = 6.7%), 6.49 ng/g (RSD = 15%), 2.65 ng/g (RSD = 15%); and NAL, base, 75.1 ng/g (RSD = 3.8%); dilutions, 42.3 ng/g (RSD = 5.1%), 24.1 ng/g (RSD = 6.3%), 8.59 ng/g (RSD = 4.8%). A second multiresidue analysis of the 4 quinolones was performed by an outside analyst. Average recoveries from catfish fortified at 5, 10, 20, and 40 ng/g were FLU, 75.9% (RSD = 4.0%); OXO, 84.0% (RSD = 5.5%); NAL, 85.6% (RSD = 8.9%); and PIR, 66.2% (RSD = 8.7%).

A gas chromatographic (GC) method is described for determining residues of chloramphenicol (CAP), florfenicol (FF), and thiamphenicol (TAP) in raw milk, with meta-nitrochloramphenicol (mCAP) as internal standard. Milk is extracted with acetonitrile, centrifuged, evaporated, reconstituted in water, and passed through a C18 solid-phase extraction (SPE) column. The SPE column is eluted with 60% methanol, and then the eluate is evaporated and derivatized with Sylon BFT ¿N,O-bis(trimethylsilyl)trifluoroacetamide [BSTFA]-trimethylchlorosilane [TMCS], 99 + 1¿. After derivatization, toluene is added directly to the sample, followed by water, to quench the derivatization process. After centrifugation, the organic layer is carefully removed. Analytes are determined by GC with electron capture detection (ECD). Milk was fortified with fenicols (the collective name for CAP, FF, and TAP) at 5, 10, 20, 40 and 80 ng/mL (target level = 10 ng/mL). Overall recoveries were 92, 100, and 104% for CAP, FF, and TAP, respectively. Overall interassay (between-day) variabilities were 6.1, 6.7, and 6.0% for CAP, FF, and TAP, respectively. Raw milk samples containing incurred residues of FF were also analyzed.

A multiresidue liquid chromatography/mass spectrometry (LC/MS) confirmation method for fluoroquinolones in catfish muscle was developed by using an electrospray interface. Residues of ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin were positively identified in catfish muscle fortified at 10-80 ppb as well as in incurred tissue. The extraction procedure is based on an LC method with fluorescence detection for determination of these compounds in catfish. Residues were extracted from catfish muscle with an acidic ethanol solution, and the extracts were cleaned up on a propyl sulfonic acid solid-phase extraction column. Chromatographic conditions were optimized to be compatible with the electrospray interface. Internal electrospray voltages were optimized so that 3 fragment ions, in addition to the protonated molecular ion, could be monitored for each residue. To obtain maximum sensitivity, separate MS acquisition programs were developed for ciprofloxacin/enrofloxacin and sarafloxacin/difloxacin pairs.

A liquid chromatographic method was developed for determination of ivermectin B1a (IVR) extracted from raw fortified and incurred Atlantic salmon muscle tissues. The method was also used to determine fortified doramectin (DOR) in Atlantic salmon. Tissue extract was applied to C8 solid-phase extraction (SPE) column, followed by a silica SPE column. Residues in the eluate were treated with trifluoroacetic anhydride and methylimidazole to dehydrate the IVR molecule and form an aromatic fluorescent moiety with a trifluoroacetic ester. This product was subsequently treated with ammonium acetate in methanol to cleave the ester and convert the functional group back to a stable alcohol form. The analytes were determined by fluorescence with excitation at 272 nm and emission at 465 nm. A C18 Hypersil column was used for analysis with a mobile phase of acetonitrile-water (90 + 10, v/v) and an oven temperature of 65 degrees C. IVR and DOR were determined at 5 fortification levels (1, 5, 10, 20, and 40 ppb). Intra-assay absolute recoveries ranged from 75 to 89% for IVR and from 73 to 85% for DOR. Relative standard deviations (RSDs) were < 7% in all cases. The limit of detection (3 x baseline noise) was 0.25 ppb extracted from tissue. Incurred tissues had an average concentration of 32 ppb, with an RSD of 3%.

A liquid chromatographic (LC) method with fluorescence detection is presented for the analysis of 4 fluoroquinolones; enrofloxacin (ENRO), ciprofloxacin (CIPRO), sarafloxacin (SARA), and difloxacin (DIFLX) in milk. The procedure consists of extraction of milk with acidified ethanol, isolation and retention on a cation exchange solid-phase extraction column, elution with basic methanol, and LC analysis with fluorescence detection. LC analysis is performed by isocratic elution using an acetonitrile-2% acetic acid (15 + 85) mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 278 and 450 nm, respectively. A target level of 10 ppb for each of the 4 fluoroquinolones has been established for this method. Average recovery from fortified raw milk samples (5-100 ppb each) based on a 5-point standard curve calculation was 70-90%, with relative standard deviations of < 15%.

Methylene blue (MB) is a thiazine dye that, although not regulated for use with edible fish, may sometimes be used as a chemotherapeutic agent in the aquaculture industry. A liquid chromatographic (LC) method was developed for the determination of MB in fish muscle. MB was extracted from catfish tissue with an acetonitrile-acetate buffer solution containing hydroxylamine and toluenesulfonic acid, partitioned into methylene chloride, and cleaned up on alumina and carboxylic acid solid-phase extraction cartridges. MB concentrations were determined by LC with visible detection at 660-665 nm. Recoveries of MB from catfish fortified at 10-50 ng/g were 75-90% (RSDs, 5-12%). Leucomethylene blue also can be recovered from catfish tissue as the MB colored form at low levels with similar recoveries. Analysis of catfish exposed to MB in a bath treatment at 5 ppm MB for 1 h recovered 10-20 ppb of the drug in the muscle tissue. The low tissue concentration suggests poor absorption of this drug compared with other antifungal dyes that tend to concentrate and remain in fish tissue at high levels.

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5, 10, 20, 40, and 80 ng/g. Average recoveries and relative standard deviations (RSDs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.

A liquid chromatographic (LC) method was adapted and optimized for the determination of malachite green and its metabolites in fish plasma and muscle. Residues in plasma were extracted with acetonitrile, the extract was evaporated to dryness, and residues were resolubilized for LC analysis. Residues in muscle were extracted with an acetonitrile-acetate buffer mixture, reextracted with acetonitrile, and partitioned into methylene chloride with final cleanup on alumni and propylsulfonic acid solid-phase extraction columns. Residue levels were determined by using an LC cyano column with a PbO2 postcolumn and visible detection (618 nm). Overall mean recoveries of parent malachite green (MG-C) and its major metabolite, leuco-malachite green (MG-L), from plasma were 93 and 87%,respectively, at fortification levels ranging from 25 to 250 ppb. Overall mean recoveries of MG-C and MG-L from muscle were 85 and 95%, respectively, at fortification levels ranging from 5 to 100 ppb. Relative standard deviations (RSDs) of recoveries at all fortification levels ranged from 3.9 to 7.0% for plasma and from 2.1 to 5.2% for muscle. The method was applied to incurred residues in tissues sampled from catfish after waterborne exposure to [14C]MG-C. Mean recoveries of total radioactive residues in plasma and muscle throughout the extraction and cleanup process were 88 and 87%, respectively, and corresponding RSDs for MG-C and MG-L were in the same range as those for fortified tissues. MG-L was confirmed as the major metabolite of MG-C in catfish.

A gas chromatographic/mass spectrometric (GC/MS) method was developed to confirm the presence of leucomalachite green (LMG), a metabolite of the triphenylmethane dye malachite green (MG), in catfish tissue. Residues were isolated according to a previously described liquid chromatographic (LC)/VIS spectrometric analysis of MG and LMG in fish. In our isolation procedure, analytes are extracted from tissue with acetonitrile-buffer, partitioned into CH2Cl2, and applied to neutral alumina and propylsulfonic acid solid-phase extraction cartridges. Before GC/MS analysis, extracts prepared for the LC determinative method are eluted from a cyano solid-phase extraction cartridge, extracted into organic solvent, and concentrated for GC/MS analysis. Selected ion monitoring was performed by using 5 diagnostic ions (m/z 330, 329, 253, 210, and 165) of LMG. The method was validated by confirming LMG in tissue fortified with mixtures of MG and LMG (5 and 10 ng/g each) and in tissue from fish that had been exposed to low levels of MG.

A liquid chromatographic (LC) method is described for the simultaneous determination of the triazine herbicides, simazine (SIM), atrazine (ATZ), and propazine (PRO) in the 12.5-100 ppb range in catfish. The herbicides are extracted from catfish homogenates with ethyl acetate, followed by solvent partitioning between acetonitrile and petroleum ether and additional cleanup on a C18 cartridge. A Supelcosil LC-18-DB column is used for LC separation, and UV determination is at 220 nm. The isocratic mobile phase is a mixture of methanol, acetonitrile, and water. Mean recoveries from catfish were 88.7, 96.9, and 91.7%; standard deviations were 6.84, 7.78, and 6.26%; and coefficients of variation were 7.72, 8.03, and 6.82% for SIM, ATZ, and PRO, respectively.

To determine residues of malachite green (MG) and its metabolite, leucomalachite green (LMG), in catfish tissue, analytes are extracted with acetonitrile-buffer and the extract is partitioned into methylene chloride. Final cleanup and isolation are performed on neutral alumina solid-phase extraction (SPE) and propylsulfonic acid cation-exchange SPE columns before analysis by liquid chromatography with visible detection. PbO2 postcolumn oxidation is performed by isocratic elution with a buffered mobile phase from a cyano column. Recoveries and relative standard deviations (RSDs) from fortified catfish tissues were 72.9% (RSD, 1.92%; 23 ppb), 75.5% (RSD, 6.85%; 11 ppb), and 69.6% (RSD, 6.93%; 5.7 ppb) for MG and 87.4% (RSD, 2.92%; 21 ppb), 88.1% (RSD, 5.94%; 10 ppb), and 82.6% (RSD, 11.5%; 5.3 ppb) for LMG. The method was applied to MG-incurred catfish at depuration times of 0, 2, 4, 8, and 24 h. Average levels of residual MG and LMG in the 24 h depuration catfish tissue were 73.4 and 289 ppb, respectively.

A liquid chromatographic (LC) method is described for the determination of 5-100 ppb halofuginone hydrobromide (HFG) in eggs. HFG as the free base is extracted from eggs with ethyl acetate. The extract is cleaned up on an acidic Celite 545 column. A Waters C18 column is used for LC separation with UV determination at 243 nm. The isocratic mobile phase is a mixture of water-acetonitrile-ammonium acetate buffer (12 + 5 + 3) and acetic acid. The interassay average recovery from eggs was 90.4%, with a standard deviation of 5.11 and a relative standard deviation of 5.65%.

An interlaboratory study of a gas chromatographic method for determining chloramphenicol (CAP) residues in shrimp was conducted. An internal standard (Istd), the meta isomer of CAP, was added to the shrimp, and the treated shrimp were homogenized with ethyl acetate. The ethyl acetate extract was defatted with hexane, and the CAP was partitioned into ethyl acetate from an aqueous salt solution. The ethyl acetate was evaporated, and the dried residue was treated with Sylon, a trimethylsilyl derivatizing agent, to yield the trimethylsilyl derivative of CAP. A portion of the solution containing the derivative was injected into a gas chromatograph equipped with an electron capture detector. Levels of fortified and incurred CAP were calculated from the peak area ratio of standard CAP to Istd. Recoveries of CAP from tissue directly fortified at 5 ppb were 102% (within-laboratory relative standard deviation [RSDr] = 5.6%), 104% (RSDr = 5.5%), and 108% (RSDr = 6.3%) from Laboratories 1, 2, and 3, respectively. Incurred-CAP residues at 5 and 10 ppb levels were also determined, with the following results: Laboratory 1: composite A, 4.56 ppb (RSDr = 14.0%); composite B, 8.38 ppb (RSDr = 11.6%); Laboratory 2: composite A, 4.17 ppb (RSDr = 12.5%); composite B, 8.90 ppb (RSDr = 5.60%); Laboratory 3: composite A, 4.66 ppb (RSDr = 14.9%); composite B, 11.0 ppb (RSDr = 11.8%).

A liquid chromatographic (LC) method is described for the simultaneous determination of xylazine (XY) and its major metabolite, 2,6-dimethylaniline (2,6-DMA), in bovine and swine kidney in the 25-100 ppb range. XY and 2,6-DMA are extracted from kidney with chloroform, followed by cleanup on an acidic Celite 545 column. A mu Bondapak phenyl column is used for LC separation with UV determination at 225 nm. The mobile phase is a mixture of acetonitrile, water, sodium acetate, and acetic acid. Mean recoveries from bovine kidney were 78.3% for XY, with a standard deviation (SD) of 7.45 and a coefficient of variation (CV) of 9.51%, and 87.2% for 2,6-DMA, with an SD of 8.38 and a CV of 9.61%. Mean recoveries from swine kidney were 80.8% for XY, with an SD of 5.92 and a CV of 7.33%, and 86.7% for 2,6-DMA, with an SD of 6.16 and a CV of 7.10%.

A liquid chromatographic (LC) method is described for the determination of chlortetracycline hydrochloride (CTC) in poultry/swine and ruminant feeds in the 10-100 ppm range and in premix. CTC is extracted from ground feed/premix with acidified acetone, and the extract is filtered through a Millex-HV filter or disposable C18 column. The filtrate is partitioned with methylene chloride when additional cleanup is necessary. A Nova-Pak C18 column is used for LC separation with determination at 370 nm. The average recovery of CTC from premix was 95% with a standard deviation (SD) of 1.70 and a coefficient of variation (CV) of 1.79%. The overall average recovery from feeds was 77% with an SD of 3.18 and a CV of 4.10%.

A method is presented for determination of residues of gentian violet (GV), its demethylated metabolites (pentamethyl and tetramethyl), and leucogentian violet (LGV) in chicken tissue. The analytes are extracted from tissue with acetonitrile/buffer and partitioned into methylene chloride. Polar lipids are removed on an alumina column followed by partitioning into methylene chloride from a citrate buffer. The compounds of interest are isolated on a disposable carboxylic acid cation exchange column and then eluted with 0.02% HCl in methanol. GV, its metabolites, and LGV are determined by liquid chromatography using isocratic elution with a buffered mobile phase from a cyano column and amperometric electrochemical detection at +1.000 V. Average recoveries of GV and LGV from commercially purchased chicken liver fortified with 20 ppb of each compound were 92% [standard deviation (SD) = 7, coefficient of variation (CV) = 7.6%] and 86% (SD = 7, CV = 8.1%), respectively. Average recoveries of GV, LGV, the pentamethyl metabolite, and 1 of the tetramethyl metabolites from control chicken liver (provided by the Center for Veterinary Medicine) fortified with 20 ppb of each compound were 80% (SD = 7, CV = 8.8%), 76% (SD = 3, CV = 3.9%), 83% (SD = 6, CV = 7.2%), and 76% (SD = 8, CV = 10.5%), respectively. Mean results from 10 analyses of residue-incurred chicken liver were 31 ppb GV (SD = 3, CV = 9.7%), 34 ppb pentamethyl metabolite (SD = 3, CV = 8.8%), and 40 ppb tetramethyl metabolite(s) (SD = 2, CV = 5.0%), for an average value of 105 ppb total residues (SD = 6, CV = 5.7%); no LGV was found. Data are also presented to show applicability of the method to muscle tissue.

The metabolite leucogentian violet (LGV) was found in chicken fat obtained from chickens dosed with gentian violet (GV); however, no residues of the parent compound, GV, and its oxidized metabolites were found. Therefore, a rapid method was developed for the specific determination of LGV in chicken fat. Chicken fat containing LGV is separated from the cellular protein with methylene chloride. LGV is then separated from the fat by partition extraction with an aqueous acid phase in which LGV is protonated, and the fat is discarded with the methylene chloride layer. The aqueous solution is neutralized, LGV is re-extracted into methylene chloride, and the methylene chloride is evaporated. An acetonitrile-water solution containing LGV is filtered before liquid chromatography using a cyano column, an acetate buffer-acetonitrile mobile phase, and an electrochemical detector set at a potential of +1.000 V. Average recoveries of LGV from chicken fat were 83.9% with a coefficient of variation (CV) of 12.9% for the 5 ppb level; 82.8% with a CV of 13.5% for the 10 ppb level; and 77.7% with a CV of 2.56% for the 20 ppb level. Levels of incurred LGV in chicken fat averaged 49.3 ppb with a CV of 2.43%.

A method is presented for the derivatization and determination of carboxylic acids by high-performance liquid chromatography with electrochemical and ultraviolet detection. The derivatizing reagents used in this study were synthesized, and their suitability was investigated for determination of drugs and metabolites with carboxylic acid groups. Quinoxaline-2-carboxylic, benzoic and salicylic acids each labeled with 1-(4-hydroxyphenyl)-, 1-(2,4-dihydroxyphenyl)- and 1-(2,5-dihydroxyphenyl)-2-bromoethanone were the principal esters studied; in addition, some antibiotics and their salts were also esterified. Conditions of derivatization are relatively mild at 60 degrees C for 60 min or less, and the reaction is 76% complete. The detection limits are as low as 1 pmol for some acids. Clean-up steps are not required to remove excess derivatizing reagent.

A method of determining total penicillins begins with an enzymatic hydrolysis of the beta-lactam ring to form their respective penicilloate product. Acetonitrile precipitates much of the casein and protein, which are then separated from the liquid by centrifugation. The lipids are removed from the aqueous fraction with methylene chloride. Mercuric chloride is added, which reacts with the penicilloate to liberate the side chain that has a terminal aldehyde. These penilloaldehyde products are extracted with methylene chloride and are subsequently reacted with dansyl hydrazine. The resulting fluorolabeled side chains are separated by liquid chromatography on a C18 column with acetonitrile-water as mobile phase. The fluorescence is measured by the mercury line at 254 nm excitation wavelength and a 500 nm filter on the emission side. The overall average recoveries from milk spiked at 25, 50, and 100 ppb are benzyl penicillin 79.4%; phenoxymethyl penicillin 59.7%; phenethicillin 75.9%; nafcillin 87.7%; methacillin 47.5%; oxacillin 57.6%; cloxicillin 37.3%; and dicloxicillin 26.4%.