Limit Of Assay Quantification Research Articles

Supercharging reagents in LC-MS/MS hormone analyses: Enhancing ionization, not limit of quantification

One of the critical steps during LC-MS/MS hormone analyses that affects the sensitivity of the assay is the ionization process. Enhancing ionization efficiencies by the addition of supercharging reagents might be one way to improve sensitivity and reduce the limit of quantification (LOQ). Therefore, we investigated whether the addition of the supercharging reagents m-nitrobenzyl alcohol (m-NBA), sulfolane, propylene carbonate, and o-nitroanisole (o-NA) increased ionization efficiency and improved assay LOQ of insulin, oxytocin, sex steroids, and corticosteroids in test solutions. Additionally, the influence of the supercharging reagents was tested in serum samples after sample pretreatment to determine whether ionization would be enhanced similarly in routine analyses and, subsequently, lead to improved sensitivity. The screening experiments showed that the impact of the supercharging reagents varied for each hormone; although the addition of m-NBA increased the signal of all hormones, the other reagents only enhanced ionization efficiencies for some hormones. While the addition of 0.05 v/v% m-NBA and 0.05 v/v% o-NA did result in an increase in peak area in both test solutions and serum samples, it did not significantly improve the signal-to-noise ratio, as a simultaneous increase in noise was observed. In conclusion, even though supercharging reagents can enhance ionization efficiencies of hormones significantly, the addition of these reagents does not result in an improved LOQ for hormone measurements with LC-MS/MS.

Comparison of empirical and dynamic models for HIV viral load rebound after treatment interruption.

To compare empirical and mechanistic modeling approaches for describing HIV-1 RNA viral load trajectories after antiretroviral treatment interruption and for identifying factors that predict features of viral rebound process. We apply and compare two modeling approaches in analysis of data from 346 participants in six AIDS Clinical Trial Group studies. From each separate analysis, we identify predictors for viral set points and delay in rebound. Our empirical model postulates a parametric functional form whose parameters represent different features of the viral rebound process, such as rate of rise and viral load set point. The viral dynamics model augments standard HIV dynamics models-a class of mathematical models based on differential equations describing biological mechanisms-by including reactivation of latently infected cells and adaptive immune response. We use Monolix, which makes use of a Stochastic Approximation of the Expectation-Maximization algorithm, to fit non-linear mixed effects models incorporating observations that were below the assay limit of quantification. Among the 346 participants, the median age at treatment interruption was 42. Ninety-three percent of participants were male and sixty-five percent, white non-Hispanic. Both models provided a reasonable fit to the data and can accommodate atypical viral load trajectories. The median set points obtained from two approaches were similar: 4.44 log10 copies/mL from the empirical model and 4.59 log10 copies/mL from the viral dynamics model. Both models revealed that higher nadir CD4 cell counts and ART initiation during acute/recent phase were associated with lower viral set points and identified receiving a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based pre-ATI regimen as a predictor for a delay in rebound. Although based on different sets of assumptions, both models lead to similar conclusions regarding features of viral rebound process.

Detection of residues in urine and tissues of sheep treated with trace levels of dietary ractopamine HCl.

Qualitative assays are sometimes used as the sole basis for detecting drug residues in live animals or in animal products. Such assays have become increasingly sensitive as detection technologies have improved, yet the limitations of such assays to discriminate purposeful and accidental drug exposures remain poorly defined. A study was conducted to determine the ability of a ractopamine lateral flow assay to accurately detect incurred ractopamine residues in contaminated feeds and in sheep fed trace quantities of ractopamine HCl. False positive and negative samples were determined using a quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Ractopamine HCl was fed to sheep at 0 (Zero), 1 (Low), 10 (Med), or 100 (High) µg/kg of diet ( = 4 per level, 0.5 kg of feed/d) for 7 consecutive d and urine was collected daily about ∼16 h post exposure. On-site lateral flow assays were able to reliably (0% false negatives) detect 20 μg of ractopamine HCl per kg of feed. Urine from treated sheep tested positive for ractopamine residues by lateral flow assay in 7.4 (Zero), 0 (Low), 82 (Med), and 86% (High) of the urine samples from each group. Parent ractopamine was below the assay limit of quantification (LOQ, 0.7 ng/mL) in all urine samples using LC-MS/MS. After hydrolysis of ractopamine conjugates, total ractopamine (parent + hydrolyzed metabolites) in urine of Low animals was always less than the LOQ, but in 7 of 28 samples were above the limit of detection (LOD, 0.22 ng/mL). In contrast, urine in Med animals contained 1.08 to 9.13 ng/mL of total ractopamine, while urine of High animals contained 4.85-32.82 ng/mL of total ractopamine. Ractopamine is rapidly eliminated; nevertheless, > 80% of urine samples from sheep exposed to 5 µg/d (M) of ractopamine HCl had detectable residues by the screening assay and a 100% of samples had measurable ractopamine using LC-MS/MS methods. Tissue residues of ractopamine were not detected in any of the sheep. The sensitivity with which the rapid, qualitative assay detected ractopamine was sufficient to reveal trace ractopamine exposures; these data suggest that the use of qualitative tests to indicate purposeful treatment of animals (i.e., for doping or growth enhancement), in the absence of collaborating quantitative data, is inappropriate.

Modeling the pharmacokinetic-pharmacodynamic relationship of the monoclonal anti-macaque-IL-15 antibody Hu714MuXHu in cynomolgus monkeys.

Hu714MuXHu is a recombinant chimeric murine‐human monoclonal antibody directed against interleukin‐15 (IL‐15), a proinflammatory cytokine associated with memory CD8+ and natural killer (NK) T‐cell activation and implicated in the pathogenesis of inflammatory diseases. A pharmacokinetic‐pharmacodynamic (PK/PD) model was developed to describe the NK cell count reduction in cynomolgus monkeys after treatment with Hu714MuXHu. Cynomolgus monkeys were dosed with Hu714MuXHu in three studies: as a single dose at 0.1 or 1 mg·kg−1 i.v.; weekly for 5 weeks at 0, 30, 60, or 150 mg·kg−1 i.v. or 150 mg·kg−1 s.c.; weekly for 13 weeks at 0, 5, 30, or 150 mg·kg−1 s.c. Serum Hu714MuXHu concentration‐time data were analyzed using noncompartmental analysis and the PK/NK cell count relationship was assessed via simultaneous PK/PD modeling. Hu714MuXHu PK was approximately dose‐proportional between 0.1–150 mg·kg−1 for i.v. and 5–150 mg·kg−1 for s.c. administration with an elimination half‐life of 12.7–18 days. Hu714MuXHu administration resulted in rapid and marked reductions in NK cell counts after the first dose which recovered fully after the serum Hu714MuXHu concentrations approached 0.1 μg·mL−1 (assay limit of quantification). PK/PD modeled Hu714MuXHu effects on NK cells had an EC 50 of 0.09 μg·mL−1. In summary, weekly i.v. or s.c. doses with Hu714MuXHu for up to 3 months in cynomolgus monkeys demonstrated linear PK and significant NK cell count reduction, which was described using PK/PD modeling. This approach may be used to guide investigative product dose selections for inflammatory diseases where NK cell count alterations are quantifiable.

Ethylone-Related Deaths: Toxicological Findings.

Synthetic cathinones are an emerging class of designer drugs, frequently with deceptive labels and a multitude of analogs to circumvent drug control regulations. Research regarding the pharmacological effects and toxicity of these amphetamine derivatives is scarce, heightening the risk to the public health and safety. The composition of synthetic cathinone products continually changes and laboratories began to notice ethylone-positive products in late 2011. This report presents nine postmortem cases in whom ethylone was identified. Ethylone was isolated using solid-phase extraction and detected by gas chromatography-mass spectrometry. Seven of the cases had measurable concentrations of ethylone in blood, ranging from 38 to 2,572 ng/mL; ethylone was detected in the blood sample of one case with a concentration below the assay limit of quantification (25 ng/mL), and one case did not have detectable ethylone in blood. Besides ethylone, all but one case were also positive for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; seven cases had other drugs quantified in blood, including ethanol, alprazolam, benzoylecgonine, diphenhydramine, morphine and tramadol. In five cases where ethylone was present at blood concentrations >400 ng/mL, no other drugs excluding ethanol, cannabis metabolite and doxylamine (one case) were found. The assay also tested for mephedrone, methylone and three dimethoxyamphetamine analogs; no case was positive for these analytes. The present report documents postmortem blood concentrations of ethylone, a novel synthetic cathinone, along with other concurrently identified substances. The findings provide valuable information for developing analytical assays and evaluating a toxic concentration range of ethylone.

Analysis of Asiaticoside in Beagle Dog Plasma by Liquid Chromatography/ Electrospray Ionization Tandem Mass Spectrometry and its Application to Pharmacokinetic Studies

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC – MS/MS) method was developed and validated for the determination of asiaticoside in beagle dog plasma. Plasma samples were extracted by protein precipitation with methanol. The chromatographic separation was performed on a C18 column with the isocratic mobile phase comprising of 78% methanol in water and 10 mM ammonium acetate buffer. Asiaticoside and the IS were detected with a triple-quadrupole tandem mass spectrometer in the negative ion mode for drug quantification. The total analytical run time was relatively short (4 min), and the limit of assay quantification (LLOQ) was 2.4 ng/mL using 300 μL of dog plasma. Asiaticoside and the internal standard (nimodipine) were monitored in the multi-reaction-monitoring (MRM) mode as follows: m/z 957.4→469.4 and m/z 417.2→122.0, respectively. The standard curve was linear over a concentration range from 2 to 190 ng/mL, and the correlation coefficients were greater than 0.998. The recoveries of asiaticoside from plasma were larger than 81.7%, and RSD of inter-day and intra-day assay were below 8.2%. The validated method was firstly applied to investigate the pharmacokinetics of asiaticoside in dogs. Keywords: Asiaticoside, LC-MS/MS, Electrospray ionization, Dog plasma, Pharmacokinetics, Limit of assay quantification (LLOQ), Multi-reaction-monitoring (MRM), Centella asiatica, Matrix Effect (ME), Accuracy, Precision, Stability

Determination of rimantadine in rat plasma by liquid chromatography/electrospray mass spectrometry and its application in a pharmacokinetic study

A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of rimantadine in rat plasma. Rimantadine was extracted by protein precipitation with methanol, and the chromatographic separation was performed on a C 18 column. The total analytical run time was relatively short (4.6 min), and the limit of assay quantification (LLOQ) was 2 ng/mL using 50 μL of rat plasma. Rimantadine and the internal standard (amantadine) were monitored in selected ion monitoring (SIM) mode at m/ z 180.2 and 152.1, respectively. The standard curve was linear over a concentration range from 2 to 750 ng/mL, and the correlation coefficients were greater than 0.999. The mean intra- and inter-day assay accuracy ranged from 100.1–105.0% to 100.3–104.0%, respectively, and the mean intra- and inter-day precision was between 1.3–2.3% and 1.8–3.0%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in rats after oral administration of rimantadine hydrochloride at the dose of 20 mg/kg.

Determination of roxatidine in human plasma by liquid chromatography/electrospray mass spectrometry and application to a clinical pharmacokinetic study

A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of roxatidine in human plasma. Roxatidine was extracted by single liquid-liquid extraction with tert-butyl methyl ether, and the chromatographic separation was performed on a C8 column. The total analytical run time was relatively short (5 min), and the limit of assay quantification was 2 ng/mL using 0.1 mL of human plasma. Roxatidine and the internal standard, propranolol, were monitored in selected ion monitoring (SIM) mode at m/z 307.3 and 260.3, respectively. The standard curve was linear over a concentration range from 2-500 ng/mL, and the correlation coefficients were >0.999. The mean intra- and inter-day assay accuracy ranged from 103.4-108.8% and 102.3-110.0%, respectively, and the mean intra- and inter-day precision was between 3.3-8.8% and 5.3-6.2%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers after oral administration of roxatidine acetate hydrochloride at a dose of 75 mg.

Preclinical Safety and Biodistribution of Adenovirus-Based Cancer Vaccines After Intradermal Delivery

The recombinant adenoviral (Ad) vector is being considered as a cancer vaccine platform because it efficiently induces immune responses to tumor antigens by intradermal immunization. The aims of this study were to evaluate the potential toxicities and biodistribution after a single dose or six weekly intradermal doses of Ad2/gp100v2 and Ad2/MART-1v2, which encode tumor-associated antigens gp100 and MelanA/MART-1, respectively. The only dose-related toxicities associated with intradermal administration of these Ad vectors were inflammatory cell infiltrates in the draining lymph nodes and injection sites that persisted 83 days after administration. The biodistribution of Ad DNA as detected by real-time polymerase chain reaction was largely confined to the injection sites and draining lymph nodes of mice treated with either a single dose or multiple doses of Ad vector and in the spleens of mice treated with multiple doses of Ad vector. Adenoviral DNA was transiently detected in the bone marrow, lung, or blood of only one animal for each site and was below the limit of assay quantification (<10 copies/microg DNA). The vector persisted in the skin and lymph nodes as long as 92 days after the last dose. We conclude that Ad vectors delivered by intradermal administration provide a safe, genetic vaccine delivery platform that induces desirable immune responses at the immunization sites and the lymph nodes that, ultimately, result in immune responses specific to the tumor antigens.

Plasma, urinary and biliary residues in cattle following intramuscular injection of nortestosterone laurate.

The synthetic androgen 19-nortestosterone (beta-NT) has been used illegally as a growth promoter in cattle production in the European Union. Elimination of beta-NT and its metabolites in plasma, urine and bile was studied in three cattle with cannulated gallbladders following intramuscular injection at a single site of 500 mg of the laurate ester (NTL) containing 300.5 mg beta-NT. Using enzyme immunoassay quantification, plasma Cmax of free beta-NT was 0.5 +/- 0.15 microgram/L (mean +/- SEM). Concentrations of free beta-NT in plasma were consistently greater than the assay limit of quantification (0.12 microgram/L) for 32.7 +/- 13.42 days. Mean residence time for the beta-NT in plasma was 68.5 +/- 20.75 days. Following sample preparation by immunoaffinity chromatography, high-resolution GC-MS was used to quantify beta-NT and alpha-NT in urine and bile. beta-NT was detected irregularly in urine from two of the three animals post injection. The principal metabolite present in the urine, alpha-NT, was detected for 160.3 +/- 22.67 days post injection. Cmax for alpha-NT in urine was 13.7 +/- 5.14 micrograms/L. Mean urinary AUC0-183 days for alpha-NT was 845.7 +/- 400.90 (microgram h)/L. In bile, alpha-NT was the only metabolite detected for 174.3 +/- 8.67 days post treatment. Cmax for alpha-NT in bile was 40.8 +/- 12.70 micrograms/L and mean biliary AUC0-183 days for alpha-NT was 1982.6 +/- 373.81 (microgram h)/L. Concentrations of alpha-NT in bile samples were greater than those in urine samples taken at the same time. The mean ratio of biliary:urinary AUC0-183 days was 3.0 +/- 0.72. It is concluded that bile is a superior fluid for detection of alpha-NT following injection of NTL, owing to the longer period during which residues may be detected after administration.

Assay of Erythromycin from Human Serum by High Performance Liquid Chromatography with Electrochemical Detection

Abstract A sensitive and selective method has been developed for the determination of serum concentrations of erythromycin A by high performance liquid chromatography with electrochemical detection. Erythromycin was extracted from alkalinized serum samples with methyl t-butyl ether. After evaporation of the ether, the samples were reconstituted in acetonitrile/ammonium acetate and washed with hexane. Aliquots were injected onto a Sepralyte diphenyl column. The mobile phase consists of acetonitrile/sodium perchlorate/ammonium acetate/methanol under isocratic conditions. Eluted peaks were detected by dual coulometric electrodes operated in the oxidative screen mode. The recovery of erythromycin from serum was 84%. Assay limit of quantification was 0.05 μg/ml serum, and dynamic linear range was 0.05–1.5 μg/ml. This method was used to quantitate both erythromycin and its gastric degradation products from human serum. Additionally, other macrolide antibiotics could be quantified by electrochemical detection. A...