Bace1 Deletion in the Adult Reverses Epileptiform Activity and Sleep-wake Disturbances in AD Mice.

Alzheimer's disease (AD) increases the risk for seizures and sleep disorders. We show here that germline deletion of ß-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) in neurons, but not in astrocytes, increased epileptiform activity. However, Bace1 deletion at adult ages did not alter the normal EEG waveform, indicating less concern for BACE1 inhibition in patients. Moreover, we showed that deletion of Bace1 in the adult was able to reverse epileptiform activity in 5xFAD mice. Intriguingly, treating 5xFAD and APPNL-G-F/NL-G-F (APP KI) mice of either sex with one BACE1 inhibitor Lanabecestat (AZD3293) dramatically increased epileptiform spiking, likely resulting from an off-target effect. We also monitored sleep-wake pathologies in these mice and showed increased wakefulness, decreased non-rapid eye movement sleep, and rapid eye movement sleep in both 5xFAD and APP KI mice; BACE1 inhibition in the adult 5xFAD mice reversed plaque load and sleep disturbances, but this was not seen in APP KI mice. Further studies with and without BACE1 inhibitor treatment showed different levels of plaque-associated microgliosis and activated microglial proteins in 5xFAD mice compared with APP KI mice. Together, BACE1 inhibition should be developed to avoid off-target effect for achieving benefits in reducing epileptic activity and sleep disturbance in Alzheimer's patients.SIGNIFICANCE STATEMENT BACE1 is widely recognized as a therapeutic target for treating Alzheimer's disease patients. However, BACE1 inhibitors failed in clinical trials because of inability to show cognitive improvement in patients. Here we show that BACE1 inhibition actually reduces sleep disturbances and epileptic seizures; both are seen in AD patients. We further showed that one of clinically tested BACE1 inhibitors does have off-target effects, and development of safer BACE1 inhibitors will be beneficial to AD patients. Results from this study will provide useful guidance for additional drug development.

Generating strategies for a national comeback in pancreas transplantation: A Delphi survey and US conference report.

In the United States, potential transplant candidates with insulin-dependent diabetes mellitus are inconsistently offered pancreas transplantation (PTx), contributing to a dramatic decline in pancreas allograft utilization over the past 2 decades. The American Society of Transplantation organized a workshop to identify barriers inhibiting PTx and to develop strategies for a national comeback. The 2-day workshop focused on 4 main topics: (1) referral/candidate selection, (2) organ recovery/utilization, (3) program performance/patient outcomes, and (4) enhanced education/research. Topics were explored through expert presentations, patient testimonials, breakout sessions, and strategic planning, including the identification of tasks for immediate focus. Additionally, a modified-Delphi survey was conducted among workshop members to develop and rate the importance of barriers, and the impact and feasibility of workgroup-identified improvement strategies. The panelists identified 16 barriers to progress and 44 strategies for consideration. The steps for a national comeback in PTx involve greater emphasis on efficient referral and candidate selection, better donor pancreas utilization practices, eliminating financial barriers to procurement and transplant, improving collaboration between transplant and diabetes societies and professionals, and increasing focus on PTx training, education, and research. Partnership between national societies, patient advocacy groups, and professionals will be essential to realizing this critical agenda.

Kidney Transplant Clinicians' Perceptions of Palliative Care for Patients With Failing Allografts in the US: A Mixed Methods Study.

RATIONALE & OBJECTIVE: Kidney transplant patients with failing allografts have a physical and psychological symptom burden as well as high morbidity and mortality. Palliative care is underutilized in this vulnerable population. We described kidney transplant clinicians' perceptions of palliative care to delineate their perceived barriers to and facilitators of providing palliative care to this population. STUDY DESIGN: National explanatory sequential mixed methods study including an online survey and semistructured interviews. SETTING & PARTICIPANTS: Kidney transplant clinicians in the United States surveyed and interviewed from October 2021 to March 2022. ANALYTICAL APPROACH: Descriptive summary of survey responses, thematic analysis of qualitative interviews, and mixed methods integration of data. RESULTS: A total of 149 clinicians completed the survey, and 19 completed the subsequent interviews. Over 90% of respondents agreed that palliative care can be helpful for patients with a failing kidney allograft. However, 46% of respondents disagreed that all patients with failing allografts benefit from palliative care, and two-thirds thought that patients would not want serious illness conversations. More than 90% of clinicians expressed concern that transplant patients and caregivers would feel scared or anxious if offered palliative care. The interviews identified three main themes: (1) transplant clinicians' unique sense of personal and professional responsibility was a barrier to palliative care engagement, (2) clinicians' uncertainty regarding the timing of palliative care collaboration would lead to delayed referral, and (3) clinicians felt challenged by factors related to patients' cultural backgrounds and identities, such as language differences. Many comments reflected an unfamiliarity with the broad scope of palliative care beyond end-of-life care. LIMITATIONS: Potential selection bias. CONCLUSIONS: Our study suggests that multiple barriers related to patients, clinicians, health systems, and health policies may pose challenges to the delivery of palliative care for patients with failing kidney transplants. This study illustrates the urgent need for ongoing efforts to optimize palliative care delivery models dedicated to kidney transplant patients, their families, and the clinicians who serve them. PLAIN-LANGUAGE SUMMARY: Kidney transplant patients experience physical and psychological suffering in the context of their illnesses that may be amenable to palliative care. However, palliative care is often underutilized in this population. In this mixed-methods study, we surveyed 149 clinicians across the United States, and 19 of them completed semistructured interviews. Our study results demonstrate that several patient, clinician, system, and policy factors need to be addressed to improve palliative care delivery to this vulnerable population.

Expanding the access to kidney transplantation: Strategies for kidney transplant programs.

Kidney transplantation is the most successful kidney replacement therapy available, resulting in improved recipient survival and societal cost savings. Yet, nearly 70 years after the first successful kidney transplant, there are still numerous barriers and untapped opportunities that constrain the access to transplant. The literature describing these barriers is extensive, but the practices and processes to solve them are less clear. Solutions must be multidisciplinary and be the product of strong partnerships among patients, their networks, health care providers, and transplant programs. Transparency in the referral, evaluation, and listing process as well as organ selection are paramount to build such partnerships. Providing early culturally congruent and patient-centered education as well as maximizing the use of local resources to facilitate the transplant work up should be prioritized. Every opportunity to facilitate pre-emptive kidney transplantation and living donation must be taken. Promoting the use of telemedicine and kidney paired donation as standards of care can positively impact the work up completion and maximize the chances of a living donor kidney transplant.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

OBJECTIVES: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 µg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

OBJECTIVES: Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. METHODS: A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 µg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. CONCLUSIONS: A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma.

OBJECTIVES: A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. METHODS: To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. RESULTS: The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 µg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. CONCLUSIONS: In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making it suitable for standardizing routine assays and assessing clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of primidone in human serum and plasma.

OBJECTIVES: Primidone is an anticonvulsive drug used in the treatment of epilepsy and essential tremor. It offers beneficial effects in controlling seizures, but its usage is also associated with possible side effects. To ensure optimal therapy, it is crucial to measure its concentration through accurate quantification methods. Therefore, our main goal was to develop and validate a new reference measurement procedure (RMP) for accurately measuring primidone levels in human serum and plasma. METHODS: In our study, we focused on the separation of primidone from both known and unknown interferences using a C18 column. To achieve accurate sample preparation, we developed a protocol involving protein precipitation followed by a high dilution step. The validation of the assay and determination of measurement uncertainty were carried out following guidelines from organizations such as the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. These rigorous validation processes ensure the reliability and accuracy of our method for quantifying primidone levels in human serum and plasma samples. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interference. It can be used to quantify primidone in the range of 0.150-30.0 µg/mL. Intermediate precision was less than 4.0 %, and repeatability CV ranged from 1.0 to 3.3 % across all concentration levels. The relative mean bias ranged from 0.1 to 3.9 % for native serum levels, and from -2.6 to 2.8 % for lithium-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were 1.5-4.1 % and 0.9-1.0 %, respectively. CONCLUSIONS: In this study, we introduce an innovative LC-MS/MS-based candidate RMP specifically designed for primidone in human serum and plasma. Our RMP offers a traceable platform, facilitating the standardization of routine assays and enabling the evaluation of clinically relevant samples. With this novel approach, we aim to enhance the accuracy and reliability of primidone measurements, ultimately benefiting the field of clinical research and patient care.

Gastrointestinal bleeding in a kidney transplant recipient.

Opportunistic infections are common in transplant recipients, but gastrointestinal bleed is rarely reported to be due to opportunistic fungal infections, and hence could present as a diagnostic challenge. We report a case of disseminated histoplasmosis in a kidney transplant recipient whose initial presentation was acute lower gastrointestinal bleeding with no other symptoms. The colonoscopy showed scattered punchout circular colonic ulcers with biopsy revealing budding yeasts consistent with a diagnosis of histoplasmosis. The patient was successfully treated with a prolonged course of intravenous amphotericin B followed by oral itraconazole.

Poor utilization of deceased donor pancreata in the United States: Time for action.

The number of solid organ pancreas transplants performed in the United States has declined over the past two decades despite improving outcomes and the known benefits associated with this procedure. Although the reasons are multifactorial, high rates of deceased donor pancreata nonrecovery and nonuse have at least in part contributed to the reduction in pancreas transplant activity. The pancreas has higher nonrecovery and nonuse rates compared to the kidney and liver because of more stringent donor selection criteria, particularly with respect to donor age and body mass index, although even marginally inferior donor pancreata likely still benefit some patients compared to alternative therapies. In this editorial, we present several donor-, candidate-, and center-specific factors that are either confirmed or suspected of being associated with inferior outcomes, which contribute to high pancreas nonrecovery and nonuse rates. In addition, we have discussed several measures to increase pancreas recovery and reduce pancreas nonutilization.

Comprehensive review: Frailty in pancreas transplant candidates and recipients.

Well-selected patients with kidney disease and diabetes mellitus who undergo simultaneous kidney-pancreas transplantation often experience dramatic improvements in quality of life and long-term survival compared to those who remain on medical therapy. Over the past several years the importance of frailty in the pancreas transplant candidate and recipient populations has grown. More patients with advanced age have entered the waitlist, and complications from prolonged diabetes, even in younger patients, have created increased evidence of risk for frailty. Given these concerns, and the broad challenges facing pancreas transplantation volumes overall, we generated this review to help establish the impact and implications. We summarize the interplay of immunological factors, aging, environmental factors, diabetes mellitus, and chronic kidney disease that put these patients at risk for frailty. We discuss its measurement and recommend a combination of two instruments (both well-validated and one entirely objective). We describe the outcomes for patients before and after pancreas transplantation who may have frailty, and what interventions can be taken to mitigate its effects. Broader investigation into frailty in the pancreas transplant population is needed to better understand how to select patients for pancreas transplantation and to how manage its consequences thereafter.

The Perspectives of General Nephrologists Toward Transitions of Care and Management of Failing Kidney Transplants.

The management of failing kidney allograft and transition of care to general nephrologists (GN) remain a complex process. The Kidney Pancreas Community of Practice (KPCOP) Failing Allograft Workgroup designed and distributed a survey to GN between May and September 2021. Participants were invited via mail and email invitations. There were 103 respondents with primarily adult nephrology practices, of whom 41% had an academic affiliation. More than 60% reported listing for a second kidney as the most important concern in caring for patients with a failing allograft, followed by immunosuppression management (46%) and risk of mortality (38%), while resistant anemia was considered less of a concern. For the initial approach to immunosuppression reduction, 60% stop antimetabolites first, and 26% defer to the transplant nephrologist. Communicating with transplant centers about immunosuppression cessation was reported to occur always by 60%, and sometimes by 29%, while 12% reported making the decision independently. Nephrologists with academic appointments communicate with transplant providers more than private nephrologists (74% vs. 49%, p = 0.015). There are heterogeneous approaches to the care of patients with a failing allograft. Efforts to strengthen transitions of care and to develop practical practice guidelines are needed to improve the outcomes of this vulnerable population.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of lamotrigine in human serum and plasma.

OBJECTIVES: We developed an isotope dilution (ID)-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for lamotrigine in human serum and plasma, using quantitative nuclear magnetic resonance-characterized reference standards to ensure traceability to the International System of Units. METHODS: A sample preparation protocol based on protein precipitation combined with LC-MS/MS analysis using a C18 column for chromatographic separation was established for the quantification of lamotrigine in human serum and plasma. Assay validation was performed according to current guidelines. Spiked serum and plasma samples were used to assess selectivity and specificity; a post-column infusion experiment and comparison of standard line slopes were performed to ascertain possible matrix effects. Precision and accuracy were determined in a 5 days validation experiment. Measurement uncertainty was determined per the Guide to the Expression of Uncertainty in Measurement. RESULTS: The method allowed the quantification of lamotrigine in serum and plasma in a range of 0.600-24.0 µg/mL without any observable matrix effects. The relative mean bias (n=6) ranged from 1.7 to 3.7%; intermediate precision, including variances in between-day, -calibration, and -injection, was ≤2.4%, independent of the level and matrix. Total measurement uncertainty for a single measurement was ≤2.6%; expanded uncertainty was ≤5.2% (coverage factor k=2). CONCLUSIONS: This candidate RMP based on ID-LC-MS/MS provides a traceable and reliable platform for the standardization of routine assays and the evaluation of clinical samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of topiramate in human serum and plasma.

OBJECTIVES: Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma. METHODS: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM). RESULTS: The method enabled topiramate quantification within the range of 1.20-36.0 µg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was ≤3.2 % and repeatability was 1.4-2.5 % across all concentration levels. The relative mean bias was -0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be ≤2.9 % (k=2) independent of the concentration level and the nature of the sample. CONCLUSIONS: In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. METHODS: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. RESULTS: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels. CONCLUSIONS: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of gabapentin in human serum and plasma.

OBJECTIVES: To describe and validate a reference measurement procedure (RMP) for gabapentin, employing quantitative nuclear magnetic resonance (qNMR) spectroscopy to determine the absolute content of the standard materials in combination with isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) to accurately measure serum and plasma concentrations. METHODS: A sample preparation protocol based on protein precipitation in combination with LC-MS/MS analysis using a C8 column for chromatographic separation was established for the quantification of gabapentin. Assay validation and determination of measurement uncertainty were performed according to guidance from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. ID-LC-MS/MS parameters evaluated included selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement. RESULTS: The use of qNMR provided traceability to International System (SI) units. The chromatographic assay was highly selective, allowing baseline separation of gabapentin and the gabapentin-lactam impurity, without observable matrix effects. Variability between injections, preparations, calibrations, and days (intermediate precision) was <2.3%, independent of the matrix, while the coefficient of variation for repeatability was 0.9-2.0% across all concentration levels. The relative mean bias ranged from -0.8-1.0% for serum and plasma samples. Passing-Bablok regression analysis indicated very good inter-laboratory agreement; the slope was 1.00 (95% confidence interval [CI] 0.98 to 1.03) and the intercept was -0.05 (95% CI -0.14 to 0.03). Pearson's correlation coefficient was ≥0.996. Expanded measurement uncertainties for single measurements were found to be ≤5.0% (k=2). CONCLUSIONS: This analytical protocol for gabapentin, utilizing traceable and selective qNMR and ID-LC-MS/MS techniques, allows for the standardization of routine tests and the reliable evaluation of clinical samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma.

OBJECTIVES: An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of carbamazepine. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800-18.0 µg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from -0.1 to 0.6 % for native serum and from -0.3 to -0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing-Bablok regression equation with a slope of 1.01 and an intercept of -0.01. The bias in the patient cohort was found to be 0.9 %. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of levetiracetam in human serum and plasma.

OBJECTIVES: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for levetiracetam quantification in human serum and plasma. METHODS: Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to characterize the RMP material to ensure traceability to SI units. To quantify levetiracetam, an LC-MS/MS method was optimized using a C8 column for chromatographic separation following protein-precipitation-based sample preparation. Spiked matrix samples of serum and plasma were used to test selectivity and specificity. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Precision and accuracy were evaluated over 5 days. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of levetiracetam within the range of 1.53-90.0 µg/mL. Intermediate precision was <2.2% and repeatability was 1.1-1.7% across all concentrations. The relative mean bias ranged from -2.5% to -0.3% across all levels and matrices within the measuring range. Diluted samples were found with a mean bias ranging from -0.1 to 2.9%. The predefined acceptance criterion for measurement uncertainty was met and determined for individual measurements independently of the concentration level and sample type to be ≤4.0% (k=2). CONCLUSIONS: We present a novel LC-MS/MS)-based candidate RMP for levetiracetam in human serum and plasma. Its expanded measurement uncertainty of ≤4.0% meets the clinical needs in levetiracetam monitoring. Utilizing qNMR to characterize levetiracetam reference materials allowed metrological traceability to SI units.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of methotrexate in human serum and plasma.

OBJECTIVES: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma. METHODS: Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines. RESULTS: The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200-5,700 ng/mL (0.01584-12.54 µmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5-2.1% for all analyte concentrations. Bias was -3.0 to 2.1% for samples within the measuring range and 0.8-4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%. CONCLUSIONS: This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.

Stroke and kidney transplantation.

PURPOSE OF REVIEW: This review will focus on the epidemiological data, risk factors, and management of stroke before and after kidney transplant. Stroke is highly prevalent in waitlisted patients as well as kidney transplant recipients and is associated with impaired transplant outcomes. Multiple traditional, nontraditional, and transplanted risk factors increase the risk of stroke. RECENT FINDINGS: Although the risk of stroke is reduced after kidney transplantation compared with remaining on dialysis, the morbidity and mortality from stroke after transplantation remain significant. SUMMARY: Early screening for risk factors before and after a kidney transplant and following the Kidney Disease Improving Global Outcomes (KDIGO) management guidelines could minimize the incidence of stroke and transplant outcomes.