scholarly journals Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

2001 ◽  
Vol 8 (4) ◽  
pp. 776-784 ◽  
Author(s):  
Christophe Camilla ◽  
Laurent Mély ◽  
Antoine Magnan ◽  
Brice Casano ◽  
Sabine Prato ◽  
...  
Keyword(s):  
Simultaneous Determination ◽  
Whole Blood ◽  
Dynamic Range ◽  
Internal Standard ◽  
Multiplex Assay ◽  
Interleukin 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Flow Cytometric ◽  
Ifn Γ

ABSTRACT The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-γ (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-γ than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

scholarly journals Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 425
Author(s):  
Thi-Thao-Linh Nguyen ◽  
Van-An Duong ◽  
Dang-Khoa Vo ◽  
Jeongae Jo ◽  
Han-Joo Maeng
Keyword(s):  
Simultaneous Determination ◽  
Lung Tissue ◽  
Whole Blood ◽  
Internal Standard ◽  
Reversed Phase ◽  
Selected Reaction Monitoring ◽  
Liquid Chromatography Tandem Mass ◽  
Tissue Homogenates ◽  
Distribution Studies

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5–50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

A Validated LC–MS/MS Method for Simultaneous Determination of 3-O-Acetyl-11-Keto-β-Boswellic Acid (AKBA) and its Active Metabolite Acetyl-11-Hydroxy-β-Boswellic Acid (Ac-11-OH-BA) in Rat Plasma: Application to a Pharmacokinetic Study

2020 ◽  
Vol 58 (6) ◽  
pp. 485-493
Author(s):  
Tarun Sharma ◽  
Snehasis Jana
Keyword(s):  
Simultaneous Determination ◽  
Pharmacokinetic Study ◽  
Active Metabolite ◽  
Dynamic Range ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Rat Plasma ◽  
Boswellic Acid ◽  
Mass Detection

Abstract The aim of this study was to develop and validate a new, rapid, sensitive, selective and reliable liquid chromatography–tandem mass spectrometry method for simultaneous determination of 3-O-Acetyl-11-keto-β-boswellic acid (AKBA) and its active metabolite 3-O-Acetyl-11-hydroxy-β-boswellic acid (Ac-11-hydroxy-BA) in rat plasma. Both analytes (AKBA and Ac-11-hydroxy-BA) and the internal standard (IS, ursolic acid) were extracted from 100 μL of rat plasma by protein precipitation. Chromatographic separation was achieved on PRP-H1 RP-C18 column (75 mm × 2 mm, 1.6 μm) using acetonitrile–water (95.5 v/v) as the mobile phase. Mass detection was conducted by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. A linear dynamic range of 1–1,000 ng/mL for both AKBA and Ac-11-hydroxy-BA was established with mean correlation coefficient (r (1)) of 0.999. Intra- and inter-day precision (% CV) of analysis were found in the range of 1.9–7.4%. The accuracy determined for these analytes ranged from 92.4 to 107.2%. The extraction recoveries for both analytes ranged from 92.6 to 97.3% for spiked plasma samples and were consistent. The % change in stability samples compared to nominal concentration ranged from 0.4 to 4.2%. This method was successfully tested to a pharmacokinetic (PK) study for estimation of AKBA and acetyl-11-hydroxy-BA in rat plasma following oral administration of AKBA. This method has been validated with the advantage of shorter run time that can be used for high-throughput analysis and has been successfully applied to the pharmacokinetic study of AKBA in rats.

Simultaneous Determination of Chloroquine and Pyrimethamine with Cetirizine in an Active Form and Human Serum by RP-HPLC

2021 ◽  
Author(s):  
Hina Shamshad ◽  
Ali Sayqal ◽  
Jahan Zeb ◽  
Agha Zeeshan Mirza
Keyword(s):  
Human Serum ◽  
Simultaneous Determination ◽  
Active Form ◽  
Internal Standard ◽  
Hplc Method ◽  
Serum Samples ◽  
Rp Hplc ◽  
Cetirizine Hydrochloride ◽  
Bulk Drug

Abstract A simple, accurate and precise RP-HPLC method was developed for the simultaneous determination of chloroquine, pyrimethamine and cetirizine hydrochloride concentrations in bulk drug and human serum. The assay was performed using a mobile phase of methanol: water (70:30) at pH of 2.8 ± 0.05 on the Purospher C-18 column with UV detection at 230 nm and rosuvastatin used as an internal standard. The retention times observed for chloroquine, pyrimethamine and cetirizine hydrochloride were 3.5, 2.5 and 5.5 minutes, respectively. The method was found to be specific for the assayed drugs showing a linear response in the concentration range of 1–100 μg mL−1 with coefficients of determination values of (r = 0.999). The method was developed and validated according to ICH guidelines. The method was used to monitor the serum samples and was found to be sensitive for therapeutic purposes, showing the potential to be a useful tool for routine analysis in laboratories.

scholarly journals Simultaneous Determination of Cyclosporine A, Tacrolimus, Sirolimus, and Everolimus in Whole-Blood Samples by LC-MS/MS

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Mustafa Karapirli ◽  
Murat Kizilgun ◽  
Ozgur Yesilyurt ◽  
Husamettin Gul ◽  
Zeki Ilker Kunak ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Simultaneous Determination ◽  
Cyclosporine A ◽  
Whole Blood ◽  
Immunosuppressive Drugs ◽  
Tandem Mass ◽  
Blood Samples ◽  
Whole Blood Samples

Objectives. Cyclosporine A (CyA), tacrolimus (TRL), sirolimus (SIR), and everolimus (RAD) are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of CyA, TRL, SIR, and RAD in whole-blood samples.Materials and Methods. We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS) in the multiple reaction monitoring (MRM) detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients.Results. System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes.Conclusion. This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD) in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

Simultaneous Determination of Aflatoxin B1 and Aflatoxin M1 in Food Matrices by Enzyme-Linked Immunosorbent Assay

2012 ◽  
Vol 6 (3) ◽  
pp. 767-774 ◽  
Author(s):  
Wenxiao Jiang ◽  
Zhanhui Wang ◽  
Greta Nölke ◽  
Jing Zhang ◽  
Lanlan Niu ◽  
...  
Keyword(s):  
Simultaneous Determination ◽  
Immunosorbent Assay ◽  
Aflatoxin B1 ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Food Matrices

scholarly journals GASTROESOPHAGEAL REFLUX DISEASE AND BRONCHIAL ASTHMA: MECHANISMS OF COMORBID FORMATION AND COURSE.

2020 ◽  
Vol 2 ◽  
pp. 3-7
Author(s):  
Aleksey Oparin ◽  
Givi Akhvlediani ◽  
Anatoliy Oparin ◽  
Sergei Pavlov
Keyword(s):  
Bronchial Asthma ◽  
Interleukin 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clear Correlation ◽  
The Third ◽  
Gender And Age ◽  
Galectin 3 ◽  
Degree Of Certainty ◽  
Anti Inflammatory

The aim of the study: To estimate the level of Galectin-3 with the parallel tracing of the content of pro- and anti-inflammatory cytokines Interleukin-6 (IL-6) and Interleukin-4 (IL-4) for patients with GERD and BA both in case of separate nosologies, and in case of their combined course during the period of exacerbation of the diseases. Methods. The study was conducted in three groups of patients, homogeneous by gender and age. The first group included 18 patients with GERD. The second group included 19 patients with intermittent or persistent-mild bronchial asthma. The third group included 22 patients suffering from GERD with concomitant BA intermittent or persistent-mild severity. Determination of the level of galectin-3 and interleukins (IL-4 and IL-6) in the blood serum was carried out by enzyme-linked immunosorbent assay. Results. Analyzing the results of the study, we found that the level of galectin - 3 was increased on average in both groups of patients with isolated GERD (and in patients with BA). In patients of the third group with comorbid pathology, the level of galectin-3 was statistically significantly higher than not only the norm, but also the average of patients with isolated BA and GERD. At the same time, we found the rise in the level of pro-inflammatory (IL-6) and anti-inflammatory (IL-4) cytokines. Moreover, in patients with GERD, the level of IL-6 was increased with a higher degree of reliability, and the level of IL-4 was increased with a lower degree of reliability. In patients with BA, on the contrary, the level of IL-4 was determined more often and higher, and the level of IL-6 was lower. Conclusions. Analyzing result of the study, a clear correlation and features of changes in the level of galectin-3, IL-4, IL-6 in patients with isolated GERD, BA, as well as with the comorbidity of these diseases, were revealed. In patients with BA, the level of galectin-3 increases with the same degree of certainty as in the group of patients with GERD. In the cytokine system, on the contrary, the level of anti-inflammatory (IL-4) cytokines increases with a greater degree of certainty than the level of pro-inflammatory (IL-6) cytokines. In patients with GERD with concomitant BA, the level of galectin-3 increases with a greater degree of certainty. It is observed also a higher rising of pro-inflammatory (IL-6) cytokines and a slightly pronounced increasing of the level of anti-inflammatory (IL-4) cytokines in comparison with the group of patients with isolated GERD.

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