An extended stable isotope-labeled signature peptide internal standard for tracking immunocapture of human plasma osteopontin for LC-MS/MS quantification

Abstract Background: A stable-isotope dilution method for the sensitive determination of d-galactose in human plasma was established. Methods: d-[13C]Galactose was added to plasma, and the concentration was measured after d-glucose was removed from the plasma by treatment with d-glucose oxidase and the sample was purified by ion-exchange chromatography. For gas chromatographic–mass spectrometric analysis, aldononitrile pentaacetate derivatives were prepared. Monitoring of the [MH-60]+ ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed the assessment of 1-12C-, 1-13C-, and U-13C6-labeled d-galactose, respectively. The d-galactose concentration was quantified on the basis of the 13C-labeled internal standard. Results: The method was linear (range examined, 0.1–5 μmol/L) and of good repeatability in the low and high concentration ranges (within- and between-run CVs <15%). The limit of quantification for plasma d-galactose was <0.02 μmol/L. Measurements in plasma of postabsorptive subjects yielded d-galactose concentrations (mean ± SD) of 0.12 ± 0.03 (n = 16), 0.11 ± 0.04 (n = 15), 1.44 ± 0.54 (n = 10), and 0.17 ± 0.07 (n = 5) μmol/L in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turnover study to evaluate endogenous d-galactose formation. Conclusions: The present findings establish that detection of d-galactose from endogenous sources is feasible in human plasma and show that erroneously high results may be obtained by enzymatic methods.

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Quantitation of 2-hydroxyglutarate in human plasma via LC–MS/MS using a surrogate analyte approach

Bioanalysis ◽

10.4155/bio-2020-0131 ◽

2020 ◽

Vol 12 (16) ◽

pp. 1149-1159

Author(s):

Feng Yin ◽

Yonghua Ling ◽

Jennifer Keller ◽

Dennis Kraus ◽

Rohini Narayanaswamy ◽

...

Keyword(s):

Clinical Study ◽

Stable Isotope ◽

Human Plasma ◽

Isocitrate Dehydrogenase ◽

Internal Standard ◽

Protein Precipitation ◽

Plasma Samples ◽

Target Engagement ◽

Regulatory Guidance ◽

Accuracy And Precision

Aim: 2-Hydroxyglutarate (2-HG) is a target engagement biomarker in patients after treatment with inhibitors of mutated isocitrate dehydrogenase (mIDH). Accurate measurement of 2-HG is critical for monitoring the inhibition effectiveness of the inhibitors. Materials & methods: Human plasma samples were spiked with stable isotope labelled internal standard, processed by protein precipitation, and analyzed using LC–MS/MS. This method was validated following regulatory guidance and has been successfully applied in a clinical study for mIDH inhibition. Results: An LC–MS/MS method with a surrogate analyte approach was developed and validated to measure 2-HG in human plasma with acceptable intra- and inter-assay accuracy and precision. Conclusion: A sensitive and robust LC–MS/MS method was developed and validated for measuring 2-HG in human plasma.

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Using stable isotope labeled internal standard for LC‐MS/MS quantitation of meloxicam in human plasma

Biomedical Chromatography ◽

10.1002/bmc.5217 ◽

2021 ◽

Author(s):

Cuijiao Zhan ◽

Changmao Wang ◽

Yaqin Wang ◽

Haitang Xie ◽

Jiru Chu ◽

...

Keyword(s):

Stable Isotope ◽

Human Plasma ◽

Internal Standard

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Selected Ion Monitoring Assay for the Antidepressant Mianserin in Human Plasma With Stable Isotope Labeled Analog as Internal Standard

Journal of Analytical Toxicology ◽

10.1093/jat/6.1.34 ◽

1982 ◽

Vol 6 (1) ◽

pp. 34-37 ◽

Cited By ~ 4

Author(s):

Satya P. Jindal ◽

Teresa Lutz ◽

Per Vestergaard

Keyword(s):

Stable Isotope ◽

Human Plasma ◽

Internal Standard ◽

Selected Ion Monitoring

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Development and Validation of a New Method for the Determination of Anti-hepatitis C Agent Simeprevir in Human Plasma using HPLC with Fluorescence Detection

Current Analytical Chemistry ◽

10.2174/1573411015666181217121619 ◽

2020 ◽

Vol 16 (4) ◽

pp. 428-435

Author(s):

Ahmed F.A. Youssef ◽

Yousry M. Issa ◽

Kareem M. Nabil

Keyword(s):

Hepatitis C ◽

Human Plasma ◽

Fluorescence Detection ◽

Internal Standard ◽

Protein Precipitation ◽

Assay Method ◽

Fluorescence Detector ◽

Limit Of Quantification ◽

Relative Standard

Background: Simeprevir is one of the recently discovered drugs for treating hepatitis C which is one of the major diseases across the globe. Objective: The present study involves the development of a new and unique High-Performance Liquid Chromatography (HPLC) method using fluorescence detection for the determination of simeprevir (SIM) in human plasma. Methods: Two methods of extractions were tested, protein precipitation using acetonitrile and liquidliquid extraction. A 25 mM dipotassium hydrogen orthophosphate (pH 7.0)/ACN (50/50; v/v), was used as mobile phase and C18 reversed phase column as the stationary phase. The chromatographic conditions were optimized and the concentration of simeprevir was determined by using the fluorescence detector. Cyclobenzaprine was used as an internal standard. Results: Recovery of the assay method based on protein precipitation was up to 100%. Intra-day and inter-day accuracies range from 92.30 to 107.80%, with Relative Standard Deviation (RSD) range 1.65-8.02%. The present method was successfully applied to a pharmacokinetic study where SIM was administered as a single dose of 150 mg SIM/capsule (Olysio®) to healthy individuals. Conclusion: This method exhibits high sensitivity with a low limit of quantification 10 ng mL-1, good selectivity using fluorescence detection, wide linear application range 10-3000 ng mL-1, good recovery and highly precise and validation results. The developed method can be applied in routine analysis for real samples.

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Bioanalytical method development and validation of simultaneous analysis of telmisartan and hydrochlorthiazide in human plasma using LC-MS/MS

International Journal of Bioassays ◽

10.21746/ijbio.2016.03.003 ◽

2016 ◽

Vol 5 (03) ◽

pp. 4862 ◽

Cited By ~ 1

Author(s):

Mathew George* ◽

Lincy Joseph ◽

Arpit Kumar Jain ◽

Anju V.

Keyword(s):

Human Plasma ◽

Retention Time ◽

High Performance ◽

Method Development ◽

Matrix Effects ◽

Solid Phase ◽

Internal Standard ◽

Assay Method ◽

Buffer System ◽

Internal Standards

A simple, sensitive, rapid and economic high performance thin layer chromatographic method and a mass spectroscopic assay method has been developed for the quantification of telmisartan and hydrochlorthiazide combination in human plasma. The internal standards and analytes were extracted from human plasma by solid-phase extraction with HLB Oasis1cc (30mg) catridges. The scanning and optimization for the samples are done using methanol: water (50:50). The samples were chromatographed using reverse phase chromatography with C-18 column of different manufacturers like Ascentis C18 (150×4. 6, 5µ) using the buffer system Acetonitrile: Buffer (80:20%v/v) which consist of 2±0. 1Mm ammonium format at a flow rate of 0. 7ml/min at a column oven temperature 35±10c. The internal standard used was hydrochlorthiazide13c1, d2 and telmisartand3. The extraction techniques include conditioning, loading, washing and elution, drying followed by reconstitution of the dried samples. The volume injected was 10µl with the retention time of 3-4 min for telmisartan, 1-2 min for hydrochlorthiazide and for the internal standards the retention time was 3-4 min for telmisartand3 and 1-2 min for hydrochlorthiazide c13d2. The rinsing solution was Acetonitrile: HPLC grade water in the ratio (50:50). The above developed method was validated using various parameters like selectivity and sensitivity, accuracy and precision, matrix effects, % recovery and various stability studies. The method was proved to be sensitive, accurate, precise and reproducible. The preparation showed high recovery for the quantitative determination of telmisartan and hydrochlorthiazide in human plasma.

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Development and Validation of a Method for Quantification of Favipiravir as COVID-19 Management in Spiked Human Plasma

Molecules ◽

10.3390/molecules26133789 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3789

Author(s):

Mohammad Hailat ◽

Israa Al-Ani ◽

Mohammed Hamad ◽

Zainab Zakareia ◽

Wael Abu Dayyih

Keyword(s):

Human Plasma ◽

Internal Standard ◽

Weighting Factor ◽

Hplc Method ◽

Bioanalytical Method Validation ◽

Spiked Human Plasma ◽

Fda Guidance ◽

Significant Difference ◽

Us Fda ◽

Carry Over

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1–60.0 µg/mL with regression coefficient (r2) = 0.9976. However, with acceptable r2, the calibration data’s heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday’s % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at −20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.

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Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Biomedicines ◽

10.3390/biomedicines9060621 ◽

2021 ◽

Vol 9 (6) ◽

pp. 621

Author(s):

Aurélien Millet ◽

Nihel Khoudour ◽

Jérôme Guitton ◽

Dorothée Lebert ◽

François Goldwasser ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Plasma ◽

High Resolution Mass Spectrometry ◽

Internal Standard ◽

Therapeutic Drug ◽

Limit Of Quantification ◽

Immunoglobulin G4 ◽

Positive Mode ◽

Surrogate Peptide ◽

First Time

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

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Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of lenalidomide in human plasma and its application on bioequivalence studies

Journal of Analytical Science & Technology ◽

10.1186/s40543-019-0195-z ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

R. Gopinath ◽

S. T. Narenderan ◽

M. Kumar ◽

B. Babu

Keyword(s):

Liquid Chromatography ◽

Human Plasma ◽

Internal Standard ◽

Spectrometric Method ◽

Tandem Mass ◽

Mass Spectrometric Method ◽

Mass Detection ◽

Triple Quadrupole Mass Spectrometer ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectrometric

AbstractA simple, sensitive, and specific liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) method was developed and validated for the quantification of lenalidomide in human plasma. The separation was carried out on a symmetry, C18, 5-μm (50 × 4.6 mm) column as stationary phase and with an isocratic mobile phase of 0.1% formic acid in water-methanol in the ratio of (15:85, v/v) at a flow rate of 0.5 mL/min. Protonated ions formed by electrospray ionization in the positive mode were used to detect analyte and fluconazole (internal standard). The mass detection was made by monitoring the fragmentation of m/z 260.1/148.8 for lenalidomide and m/z 307.1/238.0 for internal standard on a triple quadrupole mass spectrometer. The developed method was validated over the concentration range of 10–1000 ng/mL for lenalidomide in human plasma with a correlation coefficient (r2) was 0.9930. The accuracy and precision values obtained from six different sets of quality control samples analyzed on separate occasions ranged from 99.41 to 106.97% and 2.88 to 4.22%, respectively. Mean extraction recoveries were 98.06% and 88.78% for the analyte and IS, respectively. The developed method was successfully applied for analyzing lenalidomide in human plasma samples.

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