Upper Limit Of Quantification Research Articles

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a recombinant humanized anti-IL-4Rα monoclonal antibody CM310 in serum and its application to pharmacokinetic study in Sprague-Dawley Rats

CM310 is a recombinant humanized monoclonal antibody targeting Interleukin (IL)-4 receptor alpha (IL-4Rα). IL-4Rα blockade prevents IL-4 and IL-13 from binding to their receptor, thereby inhibiting downstream signaling pathways that drive Type 2 helper T-cell (Th2) inflammation. CM310 holds potential for treating Th2-related inflammatory diseases, such as asthma, atopic dermatitis and chronic sinusitis with nasal polyposis. In this study, a direct enzyme-linked immunosorbent assay (ELISA) was developed to measure the concentrations of CM310 in rat serum. Seven calibration standards (ranging from 25 to 1600 ng/mL) and three quality controls (70, 500 and 1250 ng/mL) were defined. The limit of detection (LOD), lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 13, 25 and 1600 ng/mL, respectively. The method exhibited excellent precision and accuracy and successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring CM310 in Sprague-Dawley rats. The development and validation ELISA method met the acceptable criteria, which suggested that these can be applied to quantify CM310, as well as in PK studies.

LC-MS-MS quantification of Δ8-THC, Δ9-THC, THCV isomers and their main metabolites in human plasma.

In recent years, potential therapeutic applications of several different cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), its isomer Δ8-THC and Δ9-tetrahydrocannabivarin (Δ9-THCV), have been investigated. Nevertheless, to establish dose-effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid/liquid chromatography-tandem mass spectrometry (LC/LC-MS-MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ8 and Δ9 isomers of THC, THCV and those of their major metabolites in human plasma. Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 µL was extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC-atmospheric pressure chemical ionization-MS-MS running in the positive multiple reaction monitoring mode. The lower limit of quantification ranged from 0.5 to 2.5 ng/mL, and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9% to 109% and 4.3% to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ8-THCV, 236 were positive for Δ8-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8-11.9 ng/mL), 383 for the major metabolite (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabivarin (Δ8-THCV-COOH) (95.4 ng/mL; 20.7-328 ng/mL), 260 for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (Δ9-THCV-COOH) (5.8 ng/mL; 2.5-16.1 ng/mL), 157 for (-)-11-hydroxy-Δ8-tetrahydrocannabivarin (11-OH-Δ8-THCV) (1.7 ng/mL; 1.0-3.7 ng/mL), 49 for Δ8-THC-COOH (1.7 ng/mL; 1.4-2.3 ng/mL) and 42 for Δ9-THCV (1.3 ng/mL; 0.8-1.6 ng/mL). We developed and validated the first LC/LC-MS-MS assay for the specific quantification of Δ8-THC, Δ9-THC and THCV isomers and their respective metabolites in human plasma. Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH were the major Δ8-THCV metabolites in human plasma after oral administration of 98.6% pure Δ8-THCV.

Profiling of Oxylipins as Markers of Oxidative Stress in Biological Samples.

Oxylipins are oxidized metabolites of polyunsaturated fatty acids (PUFAs). They represent a class of risk markers and/or therapeutic targets for diseases associated with inflammation, including cardiovascular disease and brain disorders. Because the biological activities of free PUFAs and oxylipins depend on their chemical structures and concentrations, monitoring PUFAs and oxylipin levels in biological systems is critical for understanding their roles in health and disease. Traditionally, accurate quantification of free PUFAs and oxylipins in biological samples was performed separately, as PUFAs are often 1000-fold more abundant than the derived oxidized fatty acids (oxylipins). This article describes a liquid chromatography multiple reaction monitoring tandem mass spectrometry method for the quantitative analysis of five free PUFAs and 88 oxylipins in various biological fluids, including plasma, platelet supernatants, and tissues. The same approach can also be used in conjunction with an alkaline hydrolysis step to quantify total oxylipins in fish oils. We observed that in some samples, linoleic acid levels in plasma and eicosapentaenoic acid and arachidonic acid levels in brain tissue were above the upper limit of quantification. To address this issue, we developed a data analysis method to obtain PUFA and oxylipin concentrations in these samples without additional sample preparation, thus significantly saving time and labor. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantification of polyunsaturated fatty acids (PUFAs) and oxylipins using liquid chromatography multiple reaction monitoring tandem mass spectrometry Support Protocol 1: Preparation of internal standard mixed working solution Support Protocol 2: Preparation of standard mixed stock solution Support Protocol 3: Preparation of standard mixed working solution Alternate Protocol 1: Extraction and quantitation of free PUFAs and oxylipins from mouse brain tissue Alternate Protocol 2: Extraction and quantitation of total PUFAs and oxylipins from fish oil.

Introduction to qualification and validation of an immunoassay.

Immunoassays play an important role in drug development of products targeting the immune system. Consistent quality of the results from an immunoassay is essential to make unbiased and accurate claims about the drug product during preclinical and clinical development stages. Assay qualification and validation shed light on the performance of the assay. It is the first evaluation and the verification, respectively, of the assay's performance. This tutorial explains and illustrates the calculation methodology for important assay qualification parameters including precision, relative accuracy, linearity, the lower limit of quantification (LLOQ), the upper limit of quantification (ULOQ), the assay range and dilutability. This tutorial focuses on assays used for (pre-) clinical purposes, characterized by a lognormal distribution of the measurements on its original untransformed scale and by the lack of well characterized reference material. Statistical calculations are illustrated with qualification data from an enzyme-linked immunosorbent assay (ELISA) vaccine immunoassay.

Preliminary Results of a Phase 2a Clinical Trial to Evaluate Safety, Tolerability and Antiviral Activity of Intravenous Brincidofovir (BCV IV) in Immunocompromised Patients with Adenovirus Infection

Background: Adenovirus (AdV) may cause life threatening or fatal infections in immunosuppressed patients including recipients of allogeneic hematopoietic cell transplant (allo-HCT). Brincidofovir (BCV) is a lipid conjugate of cidofovir, that crosses target cell membranes by means of facilitated and passive diffusion and has a long intracellular half-life. The safety, tolerability and antiviral activity of intravenous BCV (BCV IV) were evaluated in a dose ascending Phase 2a study (BCV-PA01: ATHENA study, NCT04706923) in sequential cohorts. Methods: Eligible patients aged 2 months or older who were immunocompromised due to allo-HCT, organ transplant, immunosuppressive medications, or congenital immunodeficiency, and had AdV viremia or disseminated AdV disease were included. AdV viremia was defined as 1) AdV viral load in the blood > 10,000 copies/mL OR 2) two samples greater than 48 hours apart with the second result higher than the first and both greater than 1000 copies/mL, within 7 days prior to initiation of BCV IV treatment. BCV IV was administered intravenously twice weekly for 4 weeks or until resolution of AdV viremia up to 14 weeks, whichever occurred later. The dose escalation was as follows: 0.2 mg/kg (Cohort 1), 0.3 mg/kg (Cohort 2), or 0.4 mg/kg (Cohort 3) for patients weighing < 50 kg and 10 mg/dose, 15 mg/dose, or 20 mg/dose, respectively for patients weighing ≥ 50 kg. The patients were subsequently followed up for 30 days after the last BCV IV dosing. AdV viral loads in blood were monitored weekly by quantitative real-time polymerase chain reaction tests (lower limit of detection (LOD) 25 copies/mL, lower limit of quantification 190 copies/mL, upper limit of quantification 1 x 10 9 copies/mL). Viral clearance was defined as two consecutive plasma viral load test results below the LOD. Results: Of 27 patients treated with BCV IV, 8 were in Cohort 1, 9 in Cohort 2 and 10 in Cohort 3. Baseline characteristics of patients are shown in Table 1. Treatment-related adverse events (TRAE) were observed in 7 patients, as shown in Table 2. No serious TRAEs including gastrointestinal and hepatic toxicities were observed. All TRAEs were reversible and resolved after the completion of the treatment. AE-related discontinuations of treatment were observed in 3 of 8 patients in Cohort 1; 2 of 9 patients in Cohort 2; and 1 of 10 patients in Cohort 3 (Table 2). The reason for discontinuation of treatment beyond 4 weeks of treatment in Cohort 3 was successful clearance of AdV viremia. Antiviral activity was dose-dependent, and viral clearance was achieved in 25% (2/8) in Cohort 1; 56 % (5/9) in Cohort 2; and 100% (10/10) in Cohort 3. The duration of treatment with BCV IV and viral responses are summarized in Table 2. In Cohort 3, it is notable that 90 % of patients achieved AdV viremia clearance in ≤ 4 weeks of treatment with BCV IV. In Cohorts 2 and 3, all patients who achieved AdV viremia clearance maintained undetectable AdV in the blood through the follow-up period. Conclusions: BCV IV was found to be safe and well tolerated in immunocompromised patients. Notably, the potentially serious gastrointestinal and hepatic toxicities described with the oral BCV formulation were not observed. BCV IV was highly effective in a dose-dependent manner, led to rapid clearance of AdV viremia within a short treatment period and provided sustained virologic response. In view of promising results and in the absence of any other approved treatments for AdV infection, our results support the need for further exploration and additional clinical trials of BCV IV as a treatment for AdV infection.

Rapid enantiomeric analysis of zopiclone in serum by supercritical fluid chromatography-tandem mass spectrometry.

Zopiclone (ZOP) is a hypnotic drug prescribed to treat insomnia. Due to the chiral nature of ZOP, the psychologically active S-form and inactive R-form need to be determined enantiomerically in a forensic drug analysis. In the present study, a supercritical fluid chromatography (SFC) method was designed with a faster analysis ability than that of previously reported techniques. The SFC-tandem mass spectrometry (SFC-MS/MS) method was optimized using a column with a chiral polysaccharide stationary phase (Trefoil CEL2). ZOP was extracted from pooled human serum using solid-phase extraction (Oasis HLB) and analyzed. The developed SFC-MS/MS method achieved the baseline separation of S-ZOP and R-ZOP within 2 min. The fit-for-purpose method validation indicated that the optimized solid-phase extraction achieved near complete recovery and approximately 70% of the matrix effect. Both the retention time and peak area showed sufficient precision. The lower and upper limits of quantification (LOQ) were 5.7 × 10-2 ng/mL and 25 ng/mL for R-ZOP, and 5.2 × 10-2 ng/mL and 25 ng/mL for S-ZOP. The calibration line was linear in the range from lower LOQ to upper LOQ. The stability test indicated that ZOP in serum stored in a refrigerator (4°C) degraded and about 55% remained in 31 days. The quick analysis of the SFC-MS/MS method makes it a valid option for the enantiomeric analysis of ZOP.

Quantification of cefaclor in human plasma using SIL-IS LC-ESI-MS/MS for pharmacokinetics study in healthy Chinese volunteers.

A steady, high-efficiency, and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method was established and validated using cefaclor-d5 as the stable isotope-labeled internal standard for quantification of cefaclor in human plasma. One-step protein precipitation was applied to extract human plasma samples using methanol as precipitant. An Ultimate XB C18 column (2.1 × 50.0mm, 5.0μm) was used to achieve chromatographic separation. Mobile phases of gradient elution were an aqueous solution containing 0.1% formic acid (mobile phase A) and an acetonitrile solution containing 0.1% formic acid (mobile phase B). Electrospray ionization in positive-ion mode was applied to detect under multiple reaction monitoring mode. Target fragment ion pairs of cefaclor and stable isotope-labeled internal standard, respectively, were m/z 368.2 → 191.1 and m/z 373.2 → 196.1. Linear range of this method was between 20.0 and 10,000.0ng/ml, with coefficient of determination (R2 ) >0.9900. Seven concentrations of quality control samples were used: 20.0ng/ml (lower limit of quantitation), 60.0ng/ml (low quality control), 650 ng/ml (middle quality control), 5000 ng/ml (arithmetic average middle quality control [AMQC]), 7500 ng/ml (high quality control), 10,000 ng/ml (upper limit of quantification), and 40,000 ng/ml (dilution quality control [DQC]). The method was validated for selectivity, lower limit of quantitation, linearity, accuracy, precision, recovery, matrix effect, dilution reliability, stability, carryover, and incurred sample reanalysis. This stable isotope-labeled internal standard liquid chromatography-electrospray ionization-tandem mass spectrometry approach has been successfully applied to study the pharmacokinetics of cefaclor dry suspension among healthy Chinese volunteers.

One sample multi-point calibration curve as a novel approach for quantitative LC-MS analysis: The quantitation of six aflatoxins in milk and oat-based milk as an example

Preparing of calibration curves are critical steps for accurate quantitative LC-MS bioanalysis. Traditional multi-sample external calibration curve (MSCC) is labor-intensive and prone to error. In this study, a novel strategy of one sample multi-point calibration curve (OSCC) using multiple isotopologue reaction monitoring (MIRM) was proposed and validated using LC-MS for the quantitation of six aflatoxins in milk and oat-based milk samples. The developed MIRM-OSCC methodology is comprehensively validated and the results indicated that the established method exhibits good performance in selectivity, sensitivity, accuracy and precision. Furthermore, the OSCC could realize sample dilution by monitoring the MIRM channel with less intensity for samples beyond the upper limit of quantification, without the need of sample dilution, which improves the assay throughput. Considering the advantages of excluding the MSCC preparation and sample dilution in OSCC, this strategy can be widely applied in various fields such as drugs, food safety and environmental analysis.

Validated Bio-Analytical Method for Cinacalcet Concentrations in Rat Plasma by Reversed Phase-High Performance Liquid Chromatography- Photodiode Array Detector Method

The goal of this work was to create and verify a simple reversed phase-high performance liquid chromatography-photodiode array detector-based method for quantifying cinacalcet in rat plasma. Six albino Wistar rats were given cinacalcet orally by oral gavage. Blood was obtained at regular intervals using retro-orbital vein puncture and processed to get plasma. Cinacalcet was extracted from plasma using a 70:30 v/v ethanol:dichloromethane combination, with an 82.80 %-104.08 % recovery rate. The reversed phase-high performance liquid chromatography-photodiode array detector technique was used to determine cinacalcet plasma concentrations. The mobile phase was acetonitrile:0.1 M dipotassium phosphate buffer (pH 6.8) (50:50 v/v) at a flow rate of 1.0 ml/min. 241.0 nm was used as the detecting wavelength. The method's linearity for cinacalcet was proven by a correlation coefficient (R2) of more than 0.9996 and a linear range of 1 μg/ml to 24 μg/ml for cinacalcet. The lower and upper limits of quantification were found to be 1 μg/ml and 24 μg/ml respectively. At the cinacalcets retention time, no interfering peaks were seen. This reversed phase-high performance liquid chromatography-photodiode array detector approach is a straightforward and effective way to determine pharmacokinetic parameters by measuring plasma cinacalcet levels.

The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics.

In this article, we have presented the development and validation of a rapid and sensitive reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of vincristine (VCR) in patient serum samples. Chromatographic separation was achieved on a Kinetex® (Singapore) column using a mobile phase consisting of 25 mM acetic acid and 0.3% formic acid (A) and methanol (B) in a gradient elution mode at a flow rate of 0.3 mL/min. The VCR and internal standard (vinblastine) were monitored using the multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantification (LLOQ) was 0.67 ng/mL, and the upper limit of quantification (ULOQ) was 250 ng/mL for VCR. The calculated values of LOD and LOQ for VCR were 0.075 and 0.228 ng/mL, respectively. The calibration curve was linear over the VCR concentration range of 1.0-250 ng/mL in serum. The intra- and inter-day precision and precision were within the generally accepted criteria for the bioanalytical method (<15%). The method was successfully applied to the analysis of serum samples in clinical practice.

Analytical Validation of Surface-Enhanced Raman Scattering Paper Lateral Flow Assays for Detection of Traumatic Brain Injury Biomarker

Traumatic brain injury (TBI) can cause brain function loss injury-related deaths. Early TBI management is significant, but requires a timely, accurate, and sensitive assessment solution. However, current radiation- or imaging-based TBI diagnostic technologies are either less effective or time-consuming and expensive. Here, a surface-enhanced Raman scattering paper-based lateral flow strip (SERS-PLFS) is developed for rapid detection of neuron-specific enolase (NSE), a TBI biomarker in clinical blood plasma samples. The SERS-PLFS device is validated analytically with clinical human blood plasma samples. The performance metrics including accuracy, precision, reportable range, lower limit of quantification and upper limit of quantification, recovery, uncertainty, selectivity, robustness, are assessed using statistical methods. The validation results show that the SERS-PLFS is reliable in measuring the NSE levels in TBI patients.

CLL-461 Humoral Response to COVID-19 Vaccine: A Challenge in CLL

Chronic lymphocytic leukaemia (CLL) is associated with some degree of immune dysfunction as a result of the disease itself and/or treatment. COVID-19 has a major impact on patients with CLL who are at increased risk for severe disease and death. In this study, we aimed to understand the efficacy of anti-SARS-CoV-2 vaccines in patients with CLL. From January 2021, we collected data on 166 vaccinated patients with CLL followed at our site. Median age was 68 years (range 41-92); 43 (26%) were treatment-naïve (TN), 25 (15%) were previously treated, 95 (57%) were on active therapy, and 3 (2%) were experiencing relapse. Most patients received BNT162b2 (87%), followed by mRNA-1273 (4%) and ChAdOx1-S (3%); data is missing in 6%. Serology testing was performed with the SARS-CoV-2 S1/S2 IgG assay (Elecsys® Anti-SARS-CoV-2) 2 to 3 weeks after second and third vaccine doses and considered negative for antibody titers below 0.4 U/ml. Vaccine response was evaluated post-dose 2 in 119 patients and post-dose 3 in 74 patients. Post second dose, a higher seroconversion rate was observed in TN patients and those with sustained clinical response after therapy discontinuation (42% and 46% respectively) compared with actively treated patients (20.5%; [p=0.024; p=0.048]). Antibody response rate in patients receiving BTKi was considerably lower 19.7% (12/61). Three (42.9%) out of 7 patients who received venetoclax monotherapy seroconverted. None of the patients exposed to anti-CD20 antibodies (3/8 with targeted therapy, 2/8 with chemotherapy, 3/8 as single agent) <12 months before vaccination responded. Among patients actively treated who failed to achieve a humoral response after two-dose, 25.6% responded to the third dose of vaccine, although with a weak antibody level (median 8.64 U/ml, range 0.55-175). Overall, post third dose a higher median (IQR) antibody titer (127.9 U/mL; 0.55-2500) was observed compared to one post second dose (19.2 U/ml; 0.86-2500) in patients on therapy. Notably, all patients in clinical remission after treatment present titers above the upper limit of quantification (>2500 U/mL) post third dose. Conclusions: Humoral immune response to the COVID-19 vaccine is impaired in most patients with CLL and correlates with treatment status.

LC-MS-MS Determination of Cytostatic Drugs on Surfaces and in Urine to Assess Occupational Exposure.

The ever-increased usage of cytostatic drugs leads to high risk of exposure among healthcare workers. Moreover, workers are exposed to multiple compounds throughout their lives, leading to cumulative and chronic exposure. Therefore, multianalyte methods are the most suitable for exposure assessment, which minimizes the risks from handling cytostatic drugs and ensures adequate contamination containment. This study describes the development and full validation of two liquid chromatography-tandem mass spectrometry methods for the detection of gemcitabine, dacarbazine, methotrexate, irinotecan, cyclophosphamide, doxorubicinol, doxorubicin, epirubicin, etoposide, vinorelbine, docetaxel and paclitaxel in working surfaces and urine samples. The urine method is the first to measure vinorelbine and doxorubicinol. For surfaces, limits of detection (LOD) and limits of quantification (LOQ) were 5-100 pg/cm2, and linearity was achieved up to 500 pg/cm2. Inaccuracy was between -11.0 and 8.4%. Intra-day, inter-day and total imprecision were <20%, except for etoposide and irinotecan (<22.1%). In urine, LOD and LOQ were 5-250 pg/mL, with a linear range up to 1,000-5,000 pg/mL. Inaccuracy was between -3.8 and 14.9%. Imprecision was <12.4%. Matrix effect was from -58.3 to 1,268.9% and from -66.7 to 1,636% in surface and urine samples, respectively, and extraction efficiency from 10.8 to 75% and 47.1 to 130.4%, respectively. All the analytes showed autosampler (6°C/72 h), freezer (-22°C/2 months) and freeze/thaw (three cycles) stability. The feasibility of the methods was demonstrated by analyzing real working surfaces and patients' urine samples. Contamination with gemcitabine, irinotecan, cyclophosphamide, epirubicin and paclitaxel (5-4,641.9 pg/cm2) was found on biological safety cabinets and outpatients' bathrooms. Analysis of urine from patients under chemotherapy identified the infused drugs at concentrations higher than the upper LOQ. These validated methods will allow a comprehensive evaluation of both environmental and biological contamination in hospital settings and healthcare workers.

Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium.

Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.

Application of Ion-Mobility Spectrometry to Chemical Analysis at High Concentrations

Ion-mobility spectrometry (IMS) can perform qualitative and quantitative analysis of multicomponent chemical mixtures in real time, which is difficult for commonly used instruments such as gas chromatography–mass spectrometry and photo-ionization detectors. IMS is commonly applied in microanalytical (ppb) sensing of toxic gases. Thus, its application to quantitative analyses of chemical substances with a high proton affinity is generally not possible at high concentrations (~1000 ppm) because multimeric complexes are generated. In a previous study, we found that calibration curves derived from shifts in nominal arrival-time spectra of chemical substances overlapping with water clusters enable quantitative analysis at high concentrations. This study investigated the applicability of high-concentration quantitative analysis using IMS, including the lower and upper limits of quantification and their chemical dependence on methyl ethyl ketone and ethanol. We found that the magnitude of the shift in the nominal arrival-time spectrum at low concentrations is different from that at high concentrations, and that the concentration of a chemical substance can be determined with high precision from the shift in the arrival-time spectrum, even at low concentrations. Proton affinity has a significant effect on spectral shift and quantification limits. Our results indicate that shifts in nominal arrival-time spectra allow accurate quantitative analysis at both low and high concentrations. Our calibration technique is derived from the shift in nominal peaks including multimeric complexes at high concentrations, which resultantly recognized the highest dynamic range ever. We could measure the dynamic range of chemical substances over three orders using this method.

A newly developed and validated LC–MS/MS method for measuring 7-dehydrocholesterol (7DHC) concentration in human skin: a tool for vitamin D photobiology research

BackgroundUVB absorption by 7-dehydrocholesterol (7DHC) in the skin triggers the production of vitamin D and its metabolites, which maintain calcium homeostasis. Detection and measurement of 7DHC in skin using modern liquid chromatography–tandem mass spectrometry (LC–MS/MS) techniques have been lacking, yet there is need for such a technique to provide more information on 7DHC concentration and its UVB responses in human skin.ObjectivesTo develop and validate a reliable method to measure 7DHC concentration in skin.MethodsHuman skin punch biopsies of 5 mm diameter obtained through the Manchester Skin Health Biobank were utilised. 7DHC was extracted with ethyl acetate:methanol 1:1 (v/v) and derivatised using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), to allow for improved ionisation of 7DHC through Electrospray Ionisation Mass Spectrometry (ESI–MS). Solid supported liquid extraction (SLE) was also employed to allow the removal of larger lipids from 7DHC and minimise potential matrix effects.ResultsThe LC–MS/MS assay satisfied International Council for Harmonisation research standards for method validation. Calibration curve was linear with a typical r2 of 0.997, coefficient of variation was 11.1% and 4.32% for inter-assay and intra-assay imprecision, respectively. Lower limit of quantification was 1.6 µg/g and upper limit of quantification was 100 µg/g, SLE recovery of 7DHC was on average 91.4%.ConclusionsWe have developed a robust, precise and accurate assay for the detection and quantification of 7DHC in small samples of human skin (0.2 cm2 surface area). This novel method of extraction and quantification will be valuable to future vitamin D photobiology research.Graphical

Magnetic restricted-access carbon nanotubes for SPME to determine cannabinoids in plasma samples by UHPLC-MS/MS

This manuscript describes the development of magnetic restricted-access carbon nanotubes (M-RACNTs) for use as SPME sorbent to determine cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) in human plasma samples by UHPLC-MS/MS. The adsorptive phase was immobilized on an SPME device by electromagnetic interactions between the M-RACNTs and a cylindrical neodymium magnet (3-mm diameter x 8-mm height) attached to a stainless-steel rod (3-mm diameter x 40-mm height). The M-RACNTs were synthesized by incorporating Fe3O4 magnetic nanoparticles (MNPs) into commercial carbon nanotubes (CNTs); then the surface of the resulting sorbent was further coated with a layer of bovine serum albumin (BSA). Characterization techniques (SEM, FTIR, and Zeta potential) confirmed the presence of both MNPs and BSA layer dispersed through the structure of the CNTs. The M-RACNTs presented adequate sorption capacity, stable physical/chemical characteristics, and appropriate magnetic properties. Protein exclusion capacity (about 98.5%) was attributed to the chemical diffusion barrier created by the BSA network at the outer surface of the sorbent. The SPME parameters (sample pH, equilibrium time, and desorption conditions) were optimized by design of experiments (fraction factorial planning). The method (validated according to the FDA guidelines) presented adequate selectivity and linearity (coefficient of determination higher than 0.99) at concentrations ranging from the lower limit of quantification (LLOQ) (10 ng mL−1) to the upper limit of quantification (ULOQ) (300 ng mL−1) for both CBD and THC. Precision and accuracy varied from 4.47 to 19.84% (LLOQ) and −6.90 to 17.78% (LLOQ), respectively. Carry-over and matrix effect were not significant. The method was successfully applied to determine plasmatic CBD levels in healthy volunteers attending a single session of oral drug administration and THC levels in frequent cannabis smokers.

Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification.

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at ~3 × 103 bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 107 bacteria/mL, anda linearity R2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.

Development and Validation of an Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry method with Liquid-Liquid Extraction for The Quantification of Sitagliptin in Human plasma using Nebivolol as Internal Standard

We aimed to investigate the performance of the development method for sitagliptin quantification analysis in human plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS) using liquid-liquid extraction (LLE) and Nebivolol as internal standard (IS). The parameters of this presented validation method are selectivity, the lower limit of quantification (LLOQ), linearity, accuracy, precision with five different concentration (LLOQ, Low QC, Medium QC, High QC, Upper Limit Of Quantification (ULOQ)), Integrity of dilution, matrix effect, and test for stability. Based on this study, the multiple reaction monitoring (MRM) transitions were m/z 408,23 → 127,02 for Sitagliptin and m/z 406,25 → 151,06 for IS Nebivolol. The selectivity test obtained % interference of sitagliptin and IS nebivolol by (0.00 - 0.38) % and (0.04 - 0.24) %, respectively. LLOQ test obtained a concentration value of 10.32 ng/mL and % RSD (n = 5) of 5.27 %. When the Sitagliptin concentration ranged from 1 to 1000 ng/mL, the method showed strong linearity with a coefficient of correlation of 0.9991. Accuracy test obtained % differentiation of (-12.58 - 7.77) % and precision test obtained % RSD of (1.62 - 5.32) %. The dilution integrity test obtained % differentiation at 4 and 2 times dilution was -9.20 % and -9.16 %. Matrix effect and stability data were in line with the stipulated European Medicines Agency (EMA) guidelines for validating the bioanalytical method.

An UPLC-PDA assay for simultaneous determination of seven antibiotics in human plasma

Appropriate antibiotic dosing in critically ill patients requires concentration monitoring due to the occurrence of pathophysiological changes and frequent extracorporeal therapy that could significantly alter the normal pharmacokinetics of drugs. Herein, we describe an ultra-performance liquid chromatography with photodiode array (UPLC-PDA) for the simultaneous concentration determination of seven frequently used antibiotics (meropenem, cefotaxime, cefoperazone, piperacillin, linezolid, moxifloxacin, and tigecycline) in plasma from critically ill patients. The analytes were extracted from 200 μL human plasma by the addition of methanol for protein precipitation. The chromatographic separation was achieved using an ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a water (containing 0.1% trifluoroacetic acid)/acetonitrile linear gradient at a flow rate of 0.5 mL/min in a 4.5 min turn-around time. PDA detection wavelength was set individually for the analytes. The method was fully validated according to the European Medicines Agency (EMA) guideline. The lower limits of quantification for the analytes were between 0.05 and 0.8 μg/mL. The method is accurate (intra/inter-assay bias −8.4 to +12.4%) and precise (intra/inter-assay coefficient of variations 0.9–10.1%) over the clinically relevant plasma concentration ranges (upper limits of quantification 5–400 µg/mL). The applicability of the method has been successfully demonstrated by analyzing plasma samples collected from critically ill patients undergoing continuous renal replacement therapy.