Lower Limit Of Quantitation Research Articles

Parallel artificial liquid membrane extraction coupled with UPLC-ESI-MS/MS method for high-throughput quantitation of repaglinide in diabetic patients

A high-throughput therapeutic monitoring method was developed for repaglinide (RPG) in diabetic patients, combining parallel artificial liquid membrane extraction (PALME) with ultraperformance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). PALME was performed using a 96-well donor plate comprising a donor solution containing a plasma sample, 50 mM phosphate buffer (pH = 8.0), and cetirizine (CTZ) as internal standard. A polypropylene (PP) porous membrane served as a selective support for the liquid membrane (SLM), preventing nonspecific binding produced by other membranes. The extraction was accomplished across SLM made of PP membrane with dodecyl acetate and 1 % trioctylamine (w/w), and the acceptor solution comprised DMSO and 200 mM formic acid (50:50, v/v). The simple workflow for PALME provided analyte enrichment, highly efficient sample cleanup, high throughput analysis, and excellent reproducibility. Method validation met FDA criteria, with a linear plasma calibration range (0.1–100 ng mL−1, r = 0.9995) and a lower limit of quantitation (LLOQ) of 0.1 ng mL−1. Recovery results at 98.9 % affirmed method reliability. The ability to analyze 198 samples per hour, coupled with a reduced amount of solvents, underscores the method's high throughput and eco-friendly profile. The PALME-UPLC-ESI-MS/MS method was successfully applied to therapeutic drug monitoring of RPG in diabetic patients following 2 mg RPG tablet administration, establishing its effectiveness.

A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

Bleeding Outcomes in Participants with Factor VIII Activity <5 IU/DL Post-Gene Transfer in GENEr8-1

Background: Valoctocogene roxaparvovec is expected to transfer a functional FVIII coding sequence that would eliminate the need for regular prophylaxis for people with severe hemophilia A (HA). At the same valoctocogene roxaparvovec dose, FVIII levels varied among participants in the phase 3 GENEr8-1 trial. The protective effect of low transgene-derived FVIII is unknown. Aim: To determine clinical outcomes for participants with low FVIII activity 3 years post-gene transfer in GENEr8-1. Methods: In the open-label, single-arm, phase 3 GENEr8-1 trial (NCT03370913), 134 adult males (intention-to-treat [ITT] population) with severe HA (FVIII activity <1 IU/dL) previously receiving regular FVIII prophylaxis and with no history of FVIII inhibitors received a single dose of 6x10 13 vg/kg valoctocogene roxaparvovec. Plasma FVIII activity was measured via chromogenic substrate assay (reported in text; lower limit of quantitation [LLOQ], 1.5 [previously 3.0] IU/dL) or one-stage assay (LLOQ, 1.0 IU/dL). For participants who resumed prophylaxis, the valid FVIII measurement closest to, but not after, return to prophylaxis (RTP) is reported. Presented FVIII activity at a visit week is the median value in the 4- or 6-week window around the target date. Week 156 FVIII activity was imputed as 0 for participants who discontinued; other missing FVIII measurements were imputed as the smaller of valid values immediately prior to or post week 156. Bleeds were self-reported during baseline and after cessation of regular FVIII prophylaxis (scheduled for week 4 post-infusion). RTP was defined per-protocol as usual FVIII prophylaxis administered ≥1 time/week for ≥4 consecutive weeks or ≥2 emicizumab injections/month. Outcomes are reported for up to 156 weeks. Results: Of 134 ITT participants, 131 completed the week 156 visit. Mean (standard deviation [SD]) FVIII activity at week 156 was 18.2 (30.6) IU/dL in the ITT population. At week 156, 46 of 134 (34.3%) ITT participants had median FVIII activity <5 IU/dL (range, 0 to 4.9 IU/dL). These 46 participants had annualized bleeding rate (ABR) for treated spontaneous and traumatic bleeds at baseline between 0 and 27.5 (mean, 4.8) bleeds/year and from post-prophylaxis to week 156 between 0 and 12.0 (mean, 1.7) bleeds/year. Of these 46 participants, 8 resumed prophylaxis before week 156 (range, 58-141 weeks; Figure 1) and 38 did not RTP before week 156 ( Figure 2). The 8 participants who resumed prophylaxis had FVIII activity between <1.5 and 4.5 IU/dL proximal to RTP; the 38 who did not RTP had median FVIII activity between 0 and 4.9 IU/dL at week 156. At week 156, 3 of 8 and 15 of 38 participants who did and did not RTP had FVIII of 0 IU/dL, respectively. For the 8 participants who resumed prophylaxis before week 156, baseline ABR for treated bleeds ranged from 0.0 to 22.0 (mean, 8.5) bleeds/year, compared with 0 to 27.5 (mean, 4.0) bleeds/year for the 38 participants who did not RTP before week 156. Treated ABR from post-prophylaxis to RTP ranged from 0.9 to 13.4 (mean, 4.8) bleeds/year for the 8 participants who resumed prophylaxis before week 156, compared with 0 to 12.0 (mean, 1.0) bleeds/year from post-prophylaxis to week 156 for the 38 participants who did not RTP before week 156. For 2 of 8 participants who resumed prophylaxis before week 156, treated ABR was higher during the post-prophylaxis period up to RTP than at baseline; treated ABR was higher post-prophylaxis to week 156 compared with baseline for 5 of 38 participants who did not RTP before week 156. Most of the 38 participants who did not RTP before week 156 had lower ABR for treated bleeds compared with baseline, low post-prophylaxis ABRs for treated bleeds, or no substantial treated spontaneous bleeds. Decisions to not RTP were multifactorial and based on participant-investigator shared decision making; narratives outlining determinants that may have influenced decisions around prophylaxis for participants who had week 156 FVIII activity <5 IU/dL will be presented. Conclusions: Most participants with low FVIII activity had low bleeding rates, suggesting that low endogenous FVIII expression may provide protective hemostatic benefits. Many participants who resumed prophylaxis had clinical presentation consistent with moderate hemophilia; the individual decision to RTP was multifactorial and influenced by FVIII activity, bleeding rates, desired physical activity levels, and personal preferences.

506. Resistance Analyses from the Remdesivir Phase 3 REDPINE Study in Participants with Severely Reduced Kidney Function Who Were Hospitalized for COVID-19

Abstract Background Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in nonhospitalized and hospitalized adult and pediatric patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 REDPINE study, which demonstrated the safety of RDV in participants with severely reduced kidney function. Methods REDPINE (NCT04745351) was a double-blind, placebo-controlled trial in participants hospitalized for COVID-19 with severely reduced kidney function who were randomized 2:1 to receive RDV or placebo for 5 days. Full-genome deep sequencing of SARS-CoV-2 was performed on nasopharyngeal swab samples collected on Days 1 (baseline), 3, 5, 7, 14, 21, and/or 29. Emergent amino acid substitutions from RDV-treated participants were tested for RDV susceptibility in a SARS-CoV-2 replicon system. Results Of the 243 participants randomized and treated, 82 (RDV, 55; placebo, 27) met the resistance analysis criteria (all participants with all-cause death or invasive mechanical ventilation [IMV] through Day 29 or with SARS-CoV-2 RNA above the viral load assay lower limit of quantitation on Day 14). Sequencing data at both baseline and postbaseline timepoints were obtained from 60 participants (RDV, 41; placebo, 19). Among these, emergent amino acid substitutions in Nsp12 were observed in 8/41 (19.5%) in the RDV group and 1/19 (5.3%) in the placebo group. The proportions of participants with all-cause death or IMV through Day 29 were comparable between participants in the RDV group with emergent Nsp12 substitutions (5/8, 62.5%) or without emergent Nsp12 substitutions (30/33, 90.9%). In 4 participants in the RDV group, Nsp12 substitutions with low-level reduced susceptibility to RDV were identified in samples collected 9 days after cessation of RDV treatment (2.9- to 3.4-fold change in EC50): M794I (n = 2), C799F (n = 1), and E136V (n = 1). Three of these 4 participants had received solid organ transplants and were receiving concomitant immunosuppressive therapies during the study. Conclusion The results of resistance analyses in participants with severely reduced kidney function hospitalized for COVID-19 confirm a high barrier to clinically meaningful resistance to RDV in COVID-19 patients. Disclosures Kristen Andreatta, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jiani Li, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Lauren Rodriguez, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Dong Han, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Simin Xu, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jason K. Perry, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Yiannis Koullias, MD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Robert H. Hyland, DPhil, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Charlotte Hedskog, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds

Fast on-site simultaneous electroanalysis of fusidic acid and betamethasone in pharmaceuticals and water samples using novel stochastic platform

Fusidic acid and betamethasone are two pharmaceutically active compounds found in topical dosage forms. Sometimes, they can unintentionally wind up in surface water via skin washing. Therefore, a stochastic platform based on a type of calix[4]arene and polyaniline modified screen-printed electrode was developed for their on-site analysis. For both analytes, remarkably wide dynamic ranges (1.0 × 10−17–1.0 × 10−4 for fusidic acid, and 1.0 × 10−18–1.0 × 10−3 for betamethasone) and considerably low limits of quantitation (1.0 × 10−17 for fusidic acid, and 1.0 × 10−18 for betamethasone) were attained. Moreover, the proposed platform was successfully applied to real samples such as topical dosage form and surface water, obtaining recovery values higher than 90.0 % with relative standard deviation values below 0.1 %.

A magnetic molecularly imprinted polymer based on an eco-friendly deep eutectic solvent for the selective recognition of dichlorodiphenyl trichloroethane and its degradation products in fruits and vegetables

A new magnetic molecularly imprinted polymer was successfully synthesized using a ternary deep eutectic solvent derived from caffeic acid-choline chloride-formic acid as a functional monomer, thymol-menthol deep eutectic solvent as a template, ethylene glycol dimethacrylate as a cross-linker, potassium peroxodisulfate as an initiator, and aqueous ethanol solution (90% (v/v)) as a porogen. The synthesized material was characterized and applied for magnetic solid-phase extraction of dichlorodiphenyl trichloroethane and its degradation products. Optimization of the extraction condition was carried out using the central composite design and response surface methodology. The good analytical performance of magnetic solid phase extraction/gas chromatography‒mass spectrometry using the proposed adsorbent shows a wide linear range of 0.07–500 ng g−1 with R2 greater than 0.992. Low detection limits and quantitation limits were observed in the ranges of 0.01–2.00 ng g−1 and 0.07–2.50 ng g−1, respectively. The precisions shown in terms of relative standard deviations were lower than 7.0% for intraday (n=5) and 8.6% for interday (n=5 × 3) experiments. The proposed method was applied for preconcentration and determination of dichlorodiphenyl trichloroethane and its degradation products in fruit and vegetable samples. The satisfactory recoveries of the real samples at three spiked concentrations were obtained in the range of 79.1%–110.9% with RSDs lower than 7.5%. The findings highlight the importance of developing efficient sorbents for the enrichment of persistent organic pollutants in food matrices.

Analysis of 4-Hydroxyphenyllactic Acid and Other Diagnostically Important Metabolites of α-Amino Acids in Human Blood Serum Using a Validated and Sensitive Ultra-High-Pressure Liquid Chromatography-Tandem Mass Spectrometry Method.

The profile of and dynamic concentration changes in tyrosine, phenylalanine, and tryptophan metabolites in blood are of great interest since they could be considered potential biomarkers of different disorders. Some aromatic metabolites, such as 4-hydroxyphenyllactic, 4-hydroxyphenylacetic, phenyllactic, and 4-hydroxybenzoic acids have previously demonstrated their diagnostic significance in critically ill patients and patients with post-COVID-19 syndrome. In this study, a sensitive method, including serum protein precipitation with methanol and ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection, was developed and validated for six phenyl- and five indole-containing acids in human serum. The liquid-liquid extraction was also examined, but it demonstrated unsatisfactory results based on analyte recoveries and the matrix effect. However, the recoveries for all analytes reached 100% and matrix effects were not observed using protein precipitation. This made it possible to use deionized water as a blank matrix. The lower limits of quantitation (LLOQs) were from 0.02 to 0.25 μmol/L. The validated method was used for the analysis of serum samples of healthy volunteers (n = 48) to reveal the reference values of the target analytes. The concentrations of the most clinically significant metabolite 4-hydroxyphenyllactic acid, which were revealed using UPLC-MS/MS and a previously developed gas chromatography-mass spectrometry method, were completely comparable. The proposed UPLC-MS/MS protocol can be used in the routine clinical practice of medical centers.

One Tube 23 Color Full Spectral Flow Cytometry Panel in Detecting Minimal Residual Disease in Pediatric B-Cell Acute Lymphoblastic Leukemia

Introduction Minimal residual disease (MRD) is a normalized tool for risk stratification in the treatment of pediatric acute lymphoblastic leukemia (ALL) and has been introduced into many standardized chemotherapy regimens. Flow cytometry (FC) plays an important role in the monitoring of MRD. However, FC-MRD can't reliably detect residual leukemic cells below level of 0.01% in real clinical scenarios and some patients with negative MRD relapsed, suggesting the current used set of markers are not sensitive enough or specific enough. Published studies has shown that more markers with more reliable combination of antibodies may help improve the sensitivity and specificity of FC-MRD. Spectral multicolor flow cytometry (SMFC) has shown an advantage over traditional FC that more fluorescent markers could be applied simultaneously and more complex marker combinations could be exploited to distinguish leukemic blasts from normal B cell precursors with high specificity. Here we designed a SMFC-based panel of 26 markers with 23 fluorescent colors in one tube to monitor B-ALL MRD. Method The one-tube 23-color panels was designed according to previously published studies and our clinical experience based on standardized EuroFlow 8-color MRD panel. All the leukemia-associated immunophenotypes (LAIPs) were defined based on its deviation from hematogones. For CD10, CD19, CD22, CD34, CD58, CD73, CD86, CD123, CD200and CD304, the overexpression was considered as LAIP, while the under-expression was considered as LAIP for CD24, CD38, CD45 and CD81. Asynchronous co-expression of CD34 and CD20, CD34 and CD21 were also included. The aberrant cross-lineage expression was taken as LAIP for CD2, CD56, CD65, CD15, CD13, CD33, CD66c, NG2 and CD133. For CD44, under-expression or overexpression can both be considered as LAIP. To verify the effectiveness of the panel, 317 bone marrow samples from 317 patients with B-ALL diagnosed between 2009 to 2014 at the Shanghai Children's Medical Center (SCMC) were included. These samples were collected at day 19 to 283 (median: day 35), with 213 (67.19%) collected at around day 35, which was a regular time point to monitor MRD and to readjust the risk stratification. The results were compared with those of conventional 8-color FC-MRD. Results The limit of blank (LOB), limit of detection (LOD), and lower limit of quantitation (LLOQ) were firstly determined, the results showed reproducible sensitivity up to 0.001% (1-in-10 5) after acquisition of 4.8 million cells for 23-color panel. The 8-color and 23-color MRD panels showed high correlation of the quantitation of MRD (r 2=0.80, p<0.0001). Among cases of discordant between 8-color MRD negative (with cutoff <0.01%) and 23-color MRD negative (with cutoff <0.001%), relapse only occurred in patients with 23-color MRD positive and 8-color MRD negative, rather than patients with 23-color MRD negative and 8-color MRD positive, suggesting a higher sensitivity and specificity for 23-color MRD assay (See Figure). Patients with detectable MRD by either 8-color or 23-color had a significantly higher incidence of relapse (CIR, p<0.05) and shorter event free survival (EFS, p<0.05). Positive 23-color MRD seemed to be an even stronger factor impacting CIR and EFS. Compared to patients with 8-color MRD negative and 23-color MRD positive, patients with 8-color MRD positive and 23-color MRD negative had a significantly lower CIR (p<0.05) and a better EFS (p=0.068). Patients with positive 23-color MRD lower than 0.01% showed a trend towards poorer EFS and OS (p=0.083% and p=0.061%, respectively) compared to patients with 23-color MRD negative. Conclusion Based on these evidences, SMFC-based one-tube 23-color MRD panel for B-ALL was convenient and maneuverable with higher sensitivity and specificity compared to conventional 8-color FC-MRD panel. It would be a promising method to improve the risk stratification.

Site-specific imprinting of dengue virus non-structural 1 antigen on a polydopamine-based sensing film for early detection and prognosis of dengue

Serum levels of dengue virus (DENV) non-structural 1 (NS1) antigen can serve as a valuable prognostic indicator of severe dengue infections. A quartz crystal microbalance (QCM)-based biosensor with a biomimetic recognition element was designed to quantitatively detect DENV NS1 as an early disease biomarker. To mitigate the reliance on costly viral antigens during the molecular imprinting process, a synthetic peptide mimicking a DENV NS1 epitope was used as a surrogate template for the synthesis of an epitope-imprinted polydopamine (EMIPDA) sensing film on the biosensor surface. The maximal frequency shift for DENV NS1 was obtained with an EMIPDA film synthesised using 5 mg mL−1 of dopamine monomer and 0.5 mg mL−1 of peptide template. The EMIPDA-QCM biosensor achieved low detection and quantitation limits of 0.091 μg mL−1 and 0.436 μg mL−1, respectively, allowing acute-phase detection of dengue and prognosis of the disease progression. The EMIPDA-QCM biosensor exhibited remarkable selectivity with up to 68-fold larger frequency responses towards DENV NS1 compared to a major serum protein. The site-specific imprinting approach not only enhanced the biosensing performance but also enabled a 26-fold cost reduction for biosensor functionalisation, providing a cost-effective strategy for label-free biosensing of the dengue biomarker via the biopolymer film.

Validation of the Randox colorimetric assays for serum copper and zinc.

Serum copper and zinc are measured to assess deficiency and toxicity. Atomic absorption spectrophotometry and mass spectrometry methods are expensive and require highly trained staff. Colorimetric assays are available from Randox which are inexpensive and can be automated. We validated serum copper and zinc colorimetric assays on the Binding Site Optilite analyser including comparison with flame atomic absorption spectrophotometry (FAAS) and inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy, imprecision, lower limit of quantitation, and linearity were ascertained. The impact of triglycerides, bilirubin, nickel, and iron on assay performance was also investigated. Comparison of results from colorimetric analysis of patient and external quality assurance samples with those obtained by FAAS and ICP-MS was undertaken. Intra-, and inter-assay imprecision was <9%. Serum copper and zinc assays were linear between 1.8-35.6 and 2.3-45.7 µmol/L, respectively. Agreement was good between colorimetry and FAAS (intercept = -0.7, slope = 1.04) and ICP-MS (intercept = 0.6, slope = 0.99) for serum copper in patients' samples. For serum zinc, agreement was poor between colorimetry and FAAS (intercept = 2.2, slope = 0.87) and ICP-MS (intercept = 1.9, slope = 0.98) in patients' samples. There was a poor concordance in assessment of hypozincaemia between colorimetry and FAAS/ICP-MS. The Randox colorimetric assay for serum copper on the Optilite is simple to perform, has a short analysis time, and measured concentrations compare well with FAAS and ICP-MS. Due to poor agreement with FAAS and ICP-MS, colorimetry is not suitable for the measurement of serum zinc.

FRI057 Drug Concentrations And Dosing Considerations For Mibavademab, A Novel Leptin Receptor Agonist, In Individuals With Generalized Lipodystrophy

Abstract Disclosure: J. Mendell: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. Y. Wang: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. B.A. Olenchock: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. S. Podgrabinska: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. O.E. Pagovich: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. E.A. Oral: Advisory Board Member; Self; Regeneron Pharmaceuticals. Consulting Fee; Self; Third Rock Ventures, Rejuvenate Inc. Grant Recipient; Self; Amryt Pharamceuticals, Ionis Pharmaceuticals Inc., Gemphire Theraputics. Research Investigator; Self; Regeneron Pharmaceuticals, Novo Nordisk, Rhythm pharmaceuticals, Fractyl Laboratories, GI Dynamics. Other; Self; Has IP rights to Metreleptin manufactured by Amryt Pharmaceuticals and receives royalty payments. R.J. Brown: Research Investigator; Self; Regeneron Pharmaceuticals. J. Davis: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. Background: Generalized lipodystrophy (GLD) is characterized by deficiency of adipose tissue and, consequently, low levels of the adipose-derived hormone leptin. Leptin deficiency in GLD causes metabolic abnormalities and complications that are often refractory to treatment with usual glucose/lipid-lowering agents. Mibavademab is a first-in-class human leptin receptor (LEPR) agonist monoclonal antibody that activates LEPR. We investigated baseline serum leptin in patients with GLD, pharmacokinetics (PK) of mibavademab, and soluble LEPR (sLEPR) levels from a phase 2 study. Methods: In this phase 2 trial (NCT04159415), patients with GLD (≥12 yrs old) underwent a single-blind placebo run-in, followed by 1:1 randomization to one of two fixed sequence arms: Treatment A (placebo for 8 weeks, then mibavademab low dose for 8 weeks, then mibavademab high dose for 20 weeks) or Treatment B (mibavademab low dose for 8 weeks, then mibavademab high dose for 28 weeks). A loading dose of intravenous mibavademab was administered before low-dose treatment. Mibavademab and sLEPR concentrations in serum were serially measured during mibavademab treatment using validated bioanalytical methods. Results: Sixteen patients were enrolled with median body weight (BW) of 51 kg, corresponding to the approximate BW cut point for mibavademab. Baseline serum leptin was below the lower limit of quantitation (0.078 mg/L) in 5 patients with a median concentration of 0.345 mg/L (n=15). Baseline sLEPR concentrations with Treatments A and B were 30.6 and 33.9 μg/L, respectively. At Week 8, mean (SD) mibavademab trough concentrations (Ctrough) with Treatments A and B were 0 (0) and 1.61 (1.71) mg/L, respectively. Low-dose PK were less than predicted from a first in human study in healthy volunteers with higher leptin levels, reflecting higher target mediated clearance in patients with GLD. At Week 16, Ctrough for Treatments A and B increased to 2.31 and 43.0 mg/L, respectively, but were still below predicted. At Week 36, Ctrough for Treatments A and B were 60 (35.3) and 67.7 (44.2) mg/L, respectively. Clinically meaningful metabolic benefit (i.e reflected by reductions in fasting triglycerides and glycated hemoglobin) was observed at Week 36 with both Treatments A and B. Conclusions: Mibavademab concentrations by Week 36 were associated with clinically meaningful improvement in metabolic parameters. Presentation: Friday, June 16, 2023

High-performance liquid chromatography with fluorescence detection method for measuring colistin in human serum and its application in critically ill patients treated with colistin methanesulfonate sodium

Colistin, in the form of colistin methanesulfonate sodium (CMS), has a narrow therapeutic window, and colistin levels must be monitored during treatment. The aim of this study was to develop and validate a high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for measuring colistin in human serum. Colistin was extracted from human serum using trichloroacetic acid and methanol for protein precipitation, followed by in-solid phase extraction derivatization with 9-fluorenylmethyl chloroformate. HPLC-FLD system using a reverse-phase HPLC C18 column and a mobile phase consisting of acetonitrile, tetrahydrofuran, and water (80%:4%:16%) were used for the quantification of colistin A, colistin B, and polymyxin B (internal standard) in human serum. The extraction recoveries were between 71% and 103%. Linear calibration curves were obtained for colistin in concentrations from 0.3 to 8.0 μg/mL, with good fit (r2 = 0.9993). The lower limit of quantitation was 0.3 μg/mL. The intra- and inter-day precision of the assay was 0.5–5% and 3.5–9.4%, respectively. The accuracy ranged from 98% to 100%. This validated method was successfully applied to the analysis of serum-receiving CMS in critically ill patients. This method will be useful to assist colistin dosage adjustment based on individual blood colistin levels for optimization of colistin therapy.

Elimination of matrix effects in urine for determination of ethyl glucuronide by liquid chromatography–tandem mass spectrometry

RationaleAlcohol use disorder affects 4% to 5% of the world's population. Analysis methods are available for various biological fluids to detect this disorder. Determination of ethyl glucuronide in urine by the liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is frequently used in forensic toxicology. These analyses are known to cause matrix effects.MethodsThe presented study describes the elimination of matrix effects for ethyl glucuronide. This study used two different LC/MS/MS systems containing orthogonal and z‐spray ion sources. Ethyl glucuronide was analyzed in negative polarity in electrospray ionization. A different dilution method was chosen for each study. The methods were developed and validated according to the European Medicines Agency bioanalytical method validation parameters.ResultsThe lower limit of quantitation of the developed methods was 0.025 μg/mL for ethyl glucuronide. The calibration curve of ethyl glucuronide was between 0.025 and 100 μg/mL with a correlation coefficient of &gt;0.99 for the two methods.ConclusionsIt was determined that the analyses using the z‐spray ion source were more affected by the matrix effect. The two validated methods involve rapid analysis time and simple sample preparation. Also, the methods were applied to real patients' urine.

B-187 Evaluation of Clinical Utility of LC-MS/MS Method for Thyroglobulin Measurement in Comparison with Immunoradiometric Assay and Chemiluminescence Microparticle Immunoassay

Abstract Background Thyroglobulin (Tg) is the follow-up index of choice for patients with differentiated thyroid cancer (DTC), especially who were undergone total thyroidectomy. However, due to the commonly used immunoassay’s inherent susceptibility to interference combined with the high prevalence of thyroglobulin antibody (TgAb) in DTC patients, the need for more reliable measurement for Tg independent from TgAb has been brought up. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for Tg measurement and compared its clinical usefulness and analytical performance with immunoradiometric assay (IRMA) and chemiluminescence microparticle immunoassay (CMIA). Methods We developed trypsin digestion-peptide immunoaffinity LC-MS/MS methods for Tg measurement. Analysis was performed using an LC-MS/MS system consisting of Agilent 1290 LC (Agilent Technologies, Santa Clara, CA) and Qtrap 5500+ MS/MS system(ABSciex, Framingham, MA). Tryptic Tg specific peptide (FSPDDSAGASALLR) and cleavable internal standard peptide (LSFYWTTTGDPFFDSHSDSLL*RSGPYMPQ, L*: 13C6, 15N) were quantitated against 5-point linear calibration curve with Access Thyroglobulin Calibrators (Beckman Coulter, Pasadena, CA). Three levels of serum control were prepared (3.95, 12.5, and 39.4 ng/mL). Lower limit of quantitation (LLoQ), linearity, precision, and carryover were validated for the assay. For method comparison, serum from patients with DTC or Hashimoto thyroiditis was collected. Tg was also measured with IRMA using BRAHMS TG Plus (Thermo Fisher Scientific, Waltham, MA), and CMIA using Architect Thyroglobulin (Abbott laboratories, Illinois, CA). TgAb was measured with IRMA using BRAMS Anti-Tg (Thermo Fisher Scientific) and the cut-off for TgAb was 60 IU/mL. Also, interference of TgAb was evaluated by comparing the Tg recovery of five sets of TgAb negative samples spiked with diluents and high TgAb samples. Results Within-run and between-run imprecisions were CV 7.8%–13.0% and CV 8.9%–12.7%, respectively. LLoQ was 0.5 ng/mL. The assay was linear through 0.5–650 ng/mL. No significant carryover was observed within the analytical measurement range. Out of the total 193 samples, the number of samples above LLoQ in both LC-MS/MS, and IRMA or CMIA were 73 samples (45 TgAb-positive and 28 TgAb-negative samples) and 69 samples (42 TgAb-positive and 27 TgAb-negative samples), respectively. The result well correlated between LC-MS/MS and IRMA (R2 = 0.99), and LC-MS/MS and CMIA (R2 = 0.98) in overall samples. When analyzed according to TgAb status, LC-MS/MS and IRMA (R2 = 0.84), and LC-MS/MS and CMIA (R2 = 0.93) correlated rather poorly in TgAb-positive samples, while TgAb-negative samples were consistent (R2 = 0.99 for IRMA, R2 = 0.97 for CMIA). We found one case with systemic metastatic thyroid cancer that showed significantly high Tg levels in IRMA and LC-MS/MS (above the upper limit of quantitation), whereas below LLoQ in CMIA. The interference of TgAb was negligible in LC-MS/MS, while significant interference was observed in CMIA, with a mean recovery rate of 57.6% (4.3%–82.5%). In one sample, when measured with CMIA, thyroglobulin level was 3.29 ng/mL when mixed with diluent, but below LLoQ (0.14 ng/mL) when spiked with TgAb-positive samples (TgAb 1700 IU/mL). Conclusion The LC-MS/MS method for Tg measurement was less prone to antibody interference than immunoassays. Measurement of Tg using LC-MS/MS would be useful for reliable diagnosis and proper management in TgAb-positive patients.

B-011 Analytical Validation of a New Vasoactive Intestinal Peptide Radioimmunoassay

Abstract Background Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases. Objective Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method. Method Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay. Results Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was &amp;lt;86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively. Conclusion We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.

B-366 Thyroglobulin Reflex to Manage Thyroglobulin Autoantibody Interference in an Academic Medical Center: A Comparison of Two Immunoassays and an LC-MS/MS Assay in the Presence of Thyroglobulin Autoantibodies

Abstract Background Thyroglobulin (Tg) measurement is clinically important during the management of patients with differentiated thyroid carcinoma (DTC). Enzyme immunoassays (EIA) offer an affordable and highly automated method for measuring Tg. However, in the presence of thyroglobulin autoantibodies (TgAb), Tg levels may be falsely low in EIA assays due to interference. Tg testing using liquid chromatography tandem mass spectrometry (LC-MS/MS) is recommended when TgAb are detected. We implemented a reflex test to Tg by LC-MS/MS when TgAb are present and may cause interference in EIA tests. Reflex cutoffs need to be refined to minimize interferences while preventing unnecessary reflex testing. Methods Our objective was to compare two Tg EIAs and an LC-MS/MS assay in the presence of TgAb, and the prevalence and clinical performance of two TgAb EIAs. TgAb was measured on the Abbott Architect and Beckman Coulter DxI assays in 602 samples from patients with DTC and other suspected thyroid conditions between 11/29/2022 and 2/1/2023. In all samples, Tg was measured by two EIAs, the Siemens Immulite and Beckman Coulter DxI. When TgAb by the Abbott assay was &amp;gt;14.4 IU/mL, Tg was tested by LC-MS/MS in a reference lab. Results Fifty-seven specimens reflexed to LC-MS/MS testing based on the cutoff &amp;gt;14.4 IU/mL for the Abbott assay. Of these 57 samples, 42 samples would have reflexed by the Abbott and Beckman Coulter assay (cutoff ≥ 4.0 IU/mL) and 15 would not have reflexed using the Beckman Coulter assay. Fifteen specimens would have reflexed to LC-MS/MS using the Beckman Coulter cutoff, but not according to the Abbott assay. The TgAb positivity rate was 9.5% for both assays, and the overall reflexing agreement was 95.0% in the entire cohort of 602 results. For the 15 specimens that the Beckman Coulter assay would not have reflexed, LC-MS/MS and the EIAs demonstrated clinically matching results in 13 specimens. Twenty-nine of 57 specimens had undetectable Tg levels (&amp;lt;0.5 ng/mL) by LC-MS/MS, also had Tg &amp;lt;0.5 ng/mL by the Beckman Coulter assay, while 27 measured at below the limit of quantitation (&amp;lt;0.9 ng/mL) for the Siemens Immulite assay. Five results were discrepant near detectable ranges (detectable by LC-MS/MS and falsely low by EIA), with one result more closely agreeing to the Siemens EIA (a patient with no DTC diagnosis) and the other four more closely agreeing to the Beckman Coulter EIA. Linear regression comparing the Siemens Immulite (LC-MS/MS = 0.7 * Immulite + 8.7, r = 0.99) and Beckman Coulter DxI (LC-MS/MS = 1.2 * DXI – 0.6, r = 1.00) to the LC-MS/MS assay, demonstrated biased results but strong correlations. Conclusion The Beckman Coulter EIA demonstrated better agreement with LC-MS/MS results at or near lower limits of quantitation in patients with TgAb. While linear regression demonstrated strong correlation between assays, the five discrepant results indicate that TgAb interference impacts Tg results by EIA. The TgAb threshold for reflex testing should continue to be investigated.

Quantification of sedative-hypnotics in human urine and plasma via polystyrene-based solid phase extraction-LC-MS/MS analysis

Owing to the adverse effects of the overuse of common sedative-hypnotics on human health, the development of an efficient analytical method for the detection of drugs in clinical emergencies and forensic science is significant. Although conventional analytical methods, such as immunoassay, liquid chromatography (LC), gas chromatography, and mass spectrometry (MS) are reliable, they exhibit drawbacks such low-throughput screening and high costs. Thus, in this study, we developed a novel high-throughput method consisting of a polystyrene-based solid phase extraction (SPE) and an LC with tandem MS analysis for the detection of drugs in biological samples and investigated its precision and reliability via the detection of twelve sedative-hypnotics in human urine and plasma samples. Good linear relationship (r ≥ 0.99) were achieved within the concentration range of 0.1–20 ng/mL for the 12 analytes in urine samples. Whereas, in the plasma samples, the correlation coefficient was greater than 0.99 in the concentration range 1–100 ng/mL for lorazepam and clonazepam and in the range 0.5–100 ng/mL for the remaining analytes. The intra- and inter-day precision, autosampler and freeze–thaw stabilities, and lower limit of quantitation (LLOQ) for all twelve analytes in the urine and plasma samples were favorable. Furthermore, sedative-hypnotics were detected in clinical samples obtained from the Hebei General Hospital using this method. These results indicated that the analytical method proposed in this study can be effectively applied in toxicology screening and drug abuse monitoring.The method developed in this study could be applied in clinical and forensic toxicology laboratories for sedative-hypnotic drug screening, providing support for drug abuse monitoring and clinical diagnosis.

Determination of cefuroxime in breast milk by LC-MS/MS using SALLME technique.

The World Health Organization recommends that infants be exclusively breastfed for the first 6months. Antibiotics are among the most commonly prescribed drugs for pregnant and lactating women. The vast majority of drugs pass into breast milk, which may create a risk for the infant. In cases where drug exposure may pose a risk, breastfeeding should be discontinued. Therefore, the mother's drug use should be decided by considering the most accurate and recent data. Cefuroxime is a second-generation cephalosporin antibiotic with a broad spectrum of activity against Gram-negative and -positive microorganisms. In this study, we aimed to develop the LC-MS/MS method using salt-assisted liquid-liquid micro-extraction (SALLME) for the determination of cefuroxime in breast milk. The method was validated according to the European Medicines Agency (EMA) guidelines. Cefuroxime and the internal standard cefixime were extracted from plasma by a SALLME technique. The results obtained from the entire validation study are at an acceptable level according to the EMA criteria. The calibration curve of cefuroxime was between 25 and 1000 ng/ml, with correlation coefficients of >0.99. The lower limit of quantitation was 25 ng/ml for cefuroxime. Furthermore, the developed method was applied for the determination of cefuroxime in real patient breast milk.

Quantification of human serum albumin by combining chymotrypsin/trypsin digestion coupled with LC-MS/MS technique

The quantification of albumin is important in clinical medicine because the concentration of albumin in biological fluids is closely related to human health. In this study, we developed a highly selective and robust assay to determine human serum albumin (HSA) in human plasma by combining chymotrypsin/trypsin digestion coupled with targeted LC-MS/MS technique. Human plasma samples were denatured, reduced, alkylated, and digested with both chymotrypsin and trypsin to generate surrogate peptides. A unique chymotryptic peptide (NAETF) arising from human serum albumin was finally selected for targeted LC-MS/MS detection and quantification. Numerous parameters related to the targeted LC-MS/MS assay were evaluated, including lower limit of quantitation (LLOQ), linearity range, enzyme digestion efficiency, accuracy and precision. The LC-MS/MS assay was linear in the concentration range 0.05–1 mg/mL with intra-day and inter-day precision <10.2% and accuracy ranging from −3.94% to 4.89%. The assay was successfully applied to determine HSA in 148 human plasma samples.

Establishment of a sensitive UPLC-MS/MS method to quantify safinamide in rat plasma.

A fast, simple, and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for the quantification of safinamide in rat plasma. Plasma samples were treated with acetonitrile for protein precipitation, and diazepam was used as an internal standard (IS). The analytes were separated on an Acquity UPLC C18 (2.1mm × 50mm, 1.7μm) chromatographic column with gradient elution using a mobile phase (0.1% formic acid-acetonitrile). Then, the eluates were detected by electrospray ionization (ESI) in positive ion mode. The analytes were quantified by multiple reaction monitoring (MRM) using the transition m/z 303.3→215.0 of safinamide and m/z 285.0→154.0 of IS. Safinamide had good linearity in the concentration range of 1.0-2000ng/mL, and the lower limit of quantitation (LLOQ) was 1.0ng/mL. The intra- and inter-day precision and accuracy of safinamide were less than 7.63%, while the average recovery rate was 92.98%-100.29%. The method was validated to be stable and had low noise, short chromatographic run time, wide linear range, small sample volumes, low sample injection volumes, and high sensitivity. Therefore, it can be used in pharmacokinetics and preclinical and clinical studies.