scholarly journals Development of an UPLC/MS–MS method for quantification of intact IGF-I from human serum

Bioanalysis ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 53-65
Author(s):  
Nikunj N Tanna ◽  
Mary E Lame ◽  
Mark Wrona
Keyword(s):  
Protein Binding ◽  
Human Serum ◽  
Dynamic Range ◽  
Multiple Reaction Monitoring ◽  
Sodium Dodecyl ◽  
Elevated Serum ◽  
Analytical Sensitivity ◽  
Solid Core ◽  
Serum Samples ◽  
Igf I

Aim: Developing LC–MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC–MS/MS. Methods: IGF-I serum samples were denatured with sodium dodecyl sulfate, followed by organic protein precipitation to effectively disrupt protein binding and subsequent SPE of the resulting supernatant for sample cleanup and enrichment prior to LC–MS/MS analysis. Separation was performed on an analytical scale LC using a reversed-phase column containing <2 μm solid core particle followed by detection on a tandem quadrupole MS in multiple reaction monitoring mode. Results: Intact IGF-I was quantified from serum using the method described above at a LLOQ of 5 ng/ml with a dynamic range 5–1000 ng/ml (r2>0.99) and mean accuracy of 101.76%. Accuracies for quality control samples were between 93.9–107.7% with RSD <7%. Conclusion: The analytical sensitivity, linear dynamic range and excellent reproducibility of this method reliably measures endogenous and elevated serum IGF-I levels, demonstrating its utility in discovery, bioanalysis and clinical research.

scholarly journals Clinical Assay for AFP-L3 by Using Multiple Reaction Monitoring–Mass Spectrometry for Diagnosing Hepatocellular Carcinoma

2018 ◽  
Vol 64 (8) ◽  
pp. 1230-1238 ◽  
Author(s):  
Hyunsoo Kim ◽  
Areum Sohn ◽  
Injoon Yeo ◽  
Su Jong Yu ◽  
Jung-Hwan Yoon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatocellular Carcinoma ◽  
Multiple Reaction Monitoring ◽  
False Negative ◽  
Internal Standard ◽  
Analytical Sensitivity ◽  
Reaction Monitoring ◽  
Clinical Implementation ◽  
Clinical Value ◽  
Serum Samples

Abstract BACKGROUND Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA). However, LiBA does not always reflect AFP-L3 concentrations because of its low analytical sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring–mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum. METHODS The assay entailed the addition of a stable isotope-labeled internal standard protein analog, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using L. culinaris agglutinin lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation. RESULTS The lower limit of quantification of the MRM-MS assay (0.051 ng/mL) for AFP-L3 was less than that of LiBA (0.300 ng/mL). Thus, AFP-L3, which was not observed by LiBA in HCC samples (n = 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more analytical sensitive platform. The method was validated, meeting all the criteria in integrated multinational guidelines. CONCLUSIONS Because of the lower incidence of false-negative findings, the MRM-MS assay is more suitable than LiBA for early detection of HCC.

scholarly journals A Novel UPLC-MS/MS Assay for the Measurement of Linezolid and its Metabolite PNU-142300 in Human Serum and its Application to Patients With Renal Insufficiency

2021 ◽  
Vol 12 ◽  
Author(s):  
Yingying Wang ◽  
Er-min Gu ◽  
Xiaoxiang Du ◽  
Ren-ai Xu ◽  
Guanyang Lin
Keyword(s):  
Liquid Chromatography ◽  
Renal Insufficiency ◽  
Human Serum ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Gradient Elution ◽  
Ultra Performance Liquid Chromatography ◽  
Serum Samples ◽  
The Matrix ◽  
Liquid Chromatography Tandem Mass

The contribution of the metabolites of linezolid to the associated myelosuppression is unknown in patients who are renal impairment. In this research, the purpose of our experiment was to explore and develop a quick and robust ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the determination of linezolid and its metabolite PNU-142300 in human serum simultaneously. The analytes were prepared using a simple and convenient approach with acetonitrile for protein crash, and then separated from the matrix on a Waters Acquity Ultra performance liquid chromatography (UPLC) BEH C18 (2.1 mm × 50 mm, 1.7 μm) column in a program of gradient elution, where the mobile phase was consisted of water with 0.1% formic acid and acetonitrile, and was placed at 0.40 ml/min flow rate. Multiple reaction monitoring (MRM) was employed and conducted for UPLC-MS/MS detection with ion transitions at m/z 338.01 → 296.03 for linezolid, m/z 369.96 → 327.98 for PNU-142300 and m/z 370.98 → 342.99 for tedizolid (Internal standard, IS), respectively. This method had good linearity respectively in the calibration range of 0.01–20 μg/ml for linezolid, and 0.05–100 μg/ml for PNU-142300. In the intra- and inter-day, the precision of linezolid and PNU-142300 was below 14.2%, and the accuracy in this method was determined to be from −9.7 to 12.8%. In addition, recovery and matrix effect of the analytes were all found to be acceptable, and the analytes during the assay and storage in serum samples were observed to be stable. The novel optimized UPLC-MS/MS assay was also successfully employed to determine the concentration levels of linezolid and PNU-142300 in human serum. The results showed that linezolid-associated myelosuppression occurs more frequently in patients with renal insufficiency, and the metabolite-to-parent concentration ratio of PNU-142300 is predicted to reduce this toxicity of myelosuppression.

scholarly journals Adalimumab and anti-adalimumab LISA-TRACKER immunoassays performance criteria for therapeutic drug monitoring of adalimumab-amgen biosimilar (ABP501)

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Fabien Francois ◽  
Loubna Naimi ◽  
Xavier Roblin ◽  
Anne-Emmanuelle Berger ◽  
Stephane Paul
Keyword(s):  
Human Serum ◽  
Dynamic Range ◽  
Polyclonal Antibodies ◽  
Performance Criteria ◽  
Clinical Samples ◽  
Therapeutic Drug ◽  
Serum Samples ◽  
Acceptance Criteria ◽  
Sample Stability ◽  
Validation Parameters

Abstract Background ABP501 is a biosimilar to Reference Adalimumab (HUMIRA®) produced by AMGEN. Adalimumab (ADA) has a marketing authorization for Crohn's disease, ulcerative colitis and other inflammatory or autoimmune diseases. The aim of this study was to evaluate the LISA-TRACKER assays developed by Theradiag (France), for the monitoring of ABP501 and anti-ABP501 antibodies in human serum. Results 68 ABP501 clinical samples were measured with the LISA TRACKER Duo Adalimumab assay. LISA TRACKER has been validated as suitable for quantification of ABP501 in human serum samples. Accuracy of the LISA-TRACKER was measured using 3 human serum matrices spiked with known levels of biosimilar, 3 levels spanning the dynamic range. Percentages of recovery were ranged from 90 to 120% for biosimilar batch1, and between 93 and 105% for biosimilar batch2. The acceptance criteria (CV < 20%) were met for intra-run (from 3.8 to 16.5%) and inter-run imprecision (from 4.4 to 13.9%) including the two batches. All results were comprised within ± 20% from results, obtained with the kit and sample unexposed in order to evaluate stability of the sample, stability of the kit and consistency of the results. In any case, but two, all percentages of inhibition were > 50% for specificity. Specificity was tested with Biosimilar spiked samples, Biosimilar with Humira® spiked samples, and clinical samples from patients treated with adalimumab biosimilar. All of these samples were spiked with polyclonal antibodies directed against Humira®. Specificity inhibition and specificity detection steps were also part of the validation parameters. Reagents made with ABP501 gave similar results than reagents made with Humira® meeting acceptance criteria. Conclusions LISA-TRACKER ADA and anti-ADA assays are reliable for the monitoring of patients treated with ABP501.

scholarly journals Reusable, Noninvasive, and Sensitive Fluorescence Enhanced ZnO-Nanorod-Based Microarrays for Quantitative Detection of AFP in Human Serum

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Saima Rafique ◽  
Farukh Kiyani ◽  
Sumbal Jawaid ◽  
Rubina Nasir ◽  
Mahmoosh Ahmad ◽  
...  
Keyword(s):  
Human Serum ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Zno Nanorods ◽  
Protein Microarrays ◽  
Great Promise ◽  
Quantitative Detection ◽  
Zno Nanorod ◽  
Serum Samples ◽  
Protein Assay

The fabrication of sensitive protein microarrays such as PCR used in DNA microarray is challenging due to lack of signal amplification. The development of microarrays is utilized to improve the sensitivity and limitations of detection towards primal cancer detection. The sensitivity is enhanced by the use of ZnO-nanorods and is investigated as a substrate which enhance the florescent signal to diagnose the hepatocellular carcinoma (HCC) at early stages. The substrate for deposition of ZnO-nanorods is prepared by the conventional chemical bath deposition method. The resultant highly dense ZnO-nanorods enhance the fluorescent signal 7.2 times as compared to the substrate without ZnO-nanorods. The microarray showed sensitivity of 1504.7 ng ml-1 and limit of detection of 0.1 pg ml-1 in wide dynamic range of 0.05 pg-10 μg ml-1 for alpha fetoprotein (AFP) detection in 10% human serum. This immunoassay was successfully applied for human serum samples to detect tumor marker with good recoveries. The ZnO-nanorod substrate is a simple protein microarray which showed a great promise for developing a low-cost, sensitive, and high-throughput protein assay platform for several applications in both fundamental research and clinical diagnosis.

scholarly journals Adalimumab and Anti-adalimumab Lisa-tracker Immunoassays Performance Criteria for Therapeutic Drug Monitoring of Adalimumab-amgen Biosimilar (Abp501)

2020 ◽  
Author(s):  
Fabien FRANCOIS ◽  
Loubna NAIMI ◽  
Xavier ROBLIN ◽  
Anne-Emmanuelle Berger ◽  
Stephane Paul
Keyword(s):  
Human Serum ◽  
Dynamic Range ◽  
Polyclonal Antibodies ◽  
Performance Criteria ◽  
Clinical Samples ◽  
Therapeutic Drug ◽  
Serum Samples ◽  
Acceptance Criteria ◽  
Sample Stability ◽  
Validation Parameters

Abstract Background. ABP501 is a biosimilar to Reference Adalimumab (HUMIRA®) produced by AMGEN. Adalimumab (ADA) has marketing authorization for Crohn's disease, ulcerative colitis and other inflammatory or autoimmune diseases. The aim of this study was to evaluate the LISA-TRACKER assays developed by Theradiag (France), for the monitoring of ABP501 and anti-ABP501 antibodies in human serum. Methods. Accuracy of the LISA-TRACKER was measured using 3 human serum matrices spiked with known levels of biosimilar, 3 levels spanning the dynamic range. Specificity was tested with Biosimilar spiked samples, Biosimilar with Humira® spiked samples, and clinical samples from patients treated with adalimumab biosimilar. All of these samples were spiked with polyclonal antibodies directed against Humira®. Intra-run, inter-run imprecision, inhibition, kit’s stability, specificity inhibition and specificity detection steps were also part of the LISA-TRACKER Duo Adalimumab assay validation parameters. Results. 68 ABP501 clinical samples were measured with the LISA TRACKER Duo Adalimumab assay. LISA TRACKER has been validated as suitable for quantification of ABP501 in human serum samples. Concerning accuracy, percentages of recovery were ranged from 90–120% for biosimilar batch1, and between 93% and 105% for biosimilar batch2. The acceptance criteria (CV < 20%) were met for intra-run (from 3.8–16.5%) and inter-run imprecision (from 4.4–13.9%) including the two batches. All results were comprised within +/-20% from results, obtained with the kit and sample unexposed in order to evaluate stability of the sample, stability of the kit and consistency of the results. In any case but two, all percentages of inhibition were > 50% for specificity. Reagents made with ABP501 gave similar results than reagents made with Humira® meeting acceptance criteria. Conclusions. LISA-TRACKER ADA and anti-ADA assays are reliable for the monitoring of patients treated with ABP501.

Determination of epinephrine in human serum using a Pt electrode modified by polyaniline–poly(3-methylthiophene)–poly(3,3′-diaminobenzidine)

2015 ◽  
Vol 93 (11) ◽  
pp. 1239-1244 ◽  
Author(s):  
Emine Ülker ◽  
Muammer Kavanoz
Keyword(s):  
Human Serum ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Oxidation Potential ◽  
Serum Samples ◽  
Pt Electrode ◽  
Limit Of Quantification ◽  
Amperometric Determination ◽  
Best Response

A Pt electrode modified by polyaniline–poly(3-methylthiophene)–poly(3,3′-diaminobenzidine) was used for amperometric determination of epinephrine in a solution of NaHSO4/Na2SO4 (pH 2.0). For these studies, potentials between 0.40 and 0.50 V were applied, and the best response was obtained at 0.45 V. The limit of detection, limit of quantification, and the linear dynamic range were 1.23 × 10−4, 4.10 × 10−4, and 4.10 × 10−4 to 100.0 mmol L−1, respectively. These results were compared with determinations using Pt electrodes that were coated and uncoated with homopolymers. To check the accuracy of the developed method and the matrices interference, determination of epinephrine was performed in human serum samples using this modified electrode. The epinephrine concentrations were adjusted to 1.0 and 5.0 mmol L−1, and recovery values were calculated as 100.4% and 96.8%, respectively. It is significant that the determination of epinephrine was carried out at a lower potential (0.45 V) than the oxidation potential of epinephrine (0.65 V) without any matrix effect.

scholarly journals The 95000-Mr gelatin-binding protein in human serum and plasma

1985 ◽  
Vol 228 (3) ◽  
pp. 605-608 ◽  
Author(s):  
T Vartio
Keyword(s):  
Human Serum ◽  
Binding Protein ◽  
Blood Clot ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Human Monocyte ◽  
Secretory Component ◽  
Serum Samples ◽  
Lower Quantity

A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.

Simultaneous Monitoring of Febuxostat and Uric Acid in Human Serum Samples Using the Direct Square-Wave Voltammetric Method

2019 ◽  
Vol 15 (6) ◽  
pp. 678-684
Author(s):  
Biljana Nigović ◽  
Jakov Vlak
Keyword(s):  
Uric Acid ◽  
Human Serum ◽  
Oxidase Inhibitor ◽  
Serum Samples ◽  
Boron Doped Diamond ◽  
Fast Analysis ◽  
Xanthine Oxidase Inhibitor ◽  
Voltammetric Method ◽  
Square Wave ◽  
Human Serum Samples

Background: High uric acid serum level, hyperuricemia, is now associated with many diseases such as gout, chronic kidney disease, hypertension, coronary artery disease and diabetes. Febuxostat is a novel selective xanthine oxidase inhibitor approved for the treatment of hyperuricemia. Objective: The aim of this study was to develop a first analytical method for the simultaneous determination of febuxostat and uric acid. Methods: An unmodified boron-doped diamond electrode provided concurrent quantitation of drug at low levels and uric acid, which has clinical significance in the diagnosis and therapy of hyperuricemia, at relatively high concentrations. The direct square-wave voltammetric method was applied to the analysis of both analytes in human serum samples. Results: Under the optimized conditions, the linear response of peak current on febuxostat concentration was achieved in the range from 7.5 × 10-7 to 3 × 10-5 M, while uric acid showed two linear ranges of 5 × 10-6 - 5 × 10-5 M and 5 × 10-5 - 2 × 10-4 M. The method was successfully utilised for quantification of both analytes in human serum samples. Good recoveries were obtained without interference from common inorganic cations and anions as well as glucose, dopamine, ascorbic and folic acids at concentrations expected in physiological conditions. Conclusion: The great benefits of developed method are fast analysis (only 7.5 s for run), low cost and simplicity of performance.

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.

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