Variations in internal standard response: some thoughts and real-life cases

Bioanalysis ◽  
2019 ◽  
Vol 11 (18) ◽  
pp. 1715-1725 ◽  
Author(s):  
Olivier Le Blaye
Keyword(s):  
Bioanalytical Methods ◽  
Real Life ◽  
Internal Standard ◽  
Internal Standards ◽  
Standard Response ◽  
Scientific Judgment

Internal standards are essential to ensure the reliability of chromatographic bioanalytical methods. However, this only holds true if the response of the internal standard is adequately monitored. A difference needs to be made between isolated variations in a limited number of samples, which can be easily handled using objective criteria triggering the re-analysis of the affected samples and more complex situations such as trends or systematic differences, which may require the use of scientific judgment, trigger further investigations and ultimately result in the rejection of analytical runs.

Very complex internal standard response variation in LC–MS/MS bioanalysis: root cause analysis and impact assessment

Bioanalysis ◽  
2019 ◽  
Vol 11 (18) ◽  
pp. 1693-1700 ◽  
Author(s):  
Nico C van de Merbel ◽  
Remco A Koster ◽  
Corey Ohnmacht
Keyword(s):  
Internal Standard ◽  
Experimental Conditions ◽  
Internal Standards ◽  
Large Variability ◽  
Sample Handling ◽  
Root Cause ◽  
Analytical Response ◽  
Standard Response ◽  
Complex Variation ◽  
Response Variation

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC–MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability.

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

2016 ◽  
Vol 5 (03) ◽  
pp. 4862 ◽  
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.

scholarly journals Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 26 ◽  
Author(s):  
Kayla Borland ◽  
Jan Diesend ◽  
Taku Ito-Kureha ◽  
Vigo Heissmeyer ◽  
Christian Hammann ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Isotope Labeling ◽  
Rna Stability ◽  
Internal Standard ◽  
Modified Nucleosides ◽  
Rna Modification ◽  
Mouse Tissue ◽  
Rna Modifications ◽  
Internal Standards ◽  
Translation Fidelity

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

Systematic internal standard variability and issue resolution: two case studies

Bioanalysis ◽  
2019 ◽  
Vol 11 (18) ◽  
pp. 1685-1692 ◽  
Author(s):  
Tom Verhaeghe
Keyword(s):  
Stable Isotope ◽  
Case Studies ◽  
Internal Standard ◽  
Structural Analog ◽  
High Variability ◽  
Quality Controls ◽  
First Case ◽  
Standard Response

Two case studies are presented of validated assays where the internal standard showed high variability, and there was a clear response difference between study samples and standards and quality controls. In the first case a co-eluting peak boosted the stable isotope labeled internal standard response in samples from hepatically impaired subjects. In the second case the blank plasma matrix suppressed the structural analog internal standard response. For both assays the issue could be resolved by adapting the chromatographic conditions and re-validating the assay (case 1) or by diluting the study samples with blank plasma (case 2).

A Survey of Mathematical Correction Procedures in X-Ray Spectrometry

1975 ◽  
Vol 19 ◽  
pp. 1-17 ◽  
Author(s):  
Ronald Jenkins
Keyword(s):  
First Principles ◽  
Quantitative Data ◽  
Internal Standard ◽  
Characteristic Line ◽  
X Rays ◽  
Matrix Correction ◽  
Internal Standards ◽  
X Ray ◽  
Correction Equations ◽  
Secondary Fluorescence

X-ray spectrometry is an old technique dating back some sixty-odd years and although most of the early interest revolved around the qualitative aspects of the method it wasn't long before attempts were made to obtain quantitative data. One of the first recorded attempts was that by Coster and von Hevesey who in 1923 accurately determined the amount of hafnium in zirconium using tantalum as an internal standard. Glocker and Schreiber were the first to attempt calculation of X-ray characteristic line intensity from first principles although no attempt was made at that time to correct for secondary fluorescence. In von Hevesey's book, “Chemical Analysis by X-Rays,” published in 1932 , a whole chapter is devoted to what is called “Disturbing effects and their avoidance.” Among the effects discussed were primary and secondary absorbtion and third element effects. Matrix correction equations were developed although most of the quantitative work at that time was done using internal standards.

A sensitive liquid-chromatographic method for determination of 3'-azido-3'-deoxythymidine (AZT) in plasma and urine.

1988 ◽  
Vol 34 (8) ◽  
pp. 1565-1568 ◽  
Author(s):  
M A Hedaya ◽  
R J Sawchuk
Keyword(s):  
Human Plasma ◽  
Prescription Drugs ◽  
Chromatographic Method ◽  
Internal Standard ◽  
Infectious Complications ◽  
Over The Counter ◽  
Internal Standards ◽  
Liquid Chromatographic Method ◽  
Liquid Chromatographic

Abstract We describe a liquid-chromatographic assay for AZT in human plasma and urine. This assay involves the use of two internal standards, allowing reference of AZT peaks to the appropriate internal standard, the choice depending on the range of concentrations encountered. This method is isocratic, specific, sensitive enough to allow quantification of AZT in concentrations observed clinically, and requires only 13 min of chromatographic time. We saw no interference from various over-the-counter and prescription drugs often used in treating the infectious complications of AIDS.

scholarly journals Internal Standards for Quantitative Analysis of Chemical Warfare Agents by the GC/MS Method: Nerve Agents

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Tomas Capoun ◽  
Jana Krykorkova
Keyword(s):  
Internal Standard ◽  
Chemical Warfare Agents ◽  
Chemical Warfare ◽  
Linear Functions ◽  
Mass Detection ◽  
Response Factors ◽  
Internal Standards ◽  
Rescue Service ◽  
Warfare Agents ◽  
Peak Areas

General conditions and requirements for an internal standard useful in the determination of chemical warfare agents (CWAs) by the method of gas chromatography coupled with mass detection (GC/MS) were defined. The determination is based on a GC/MS analysis of a mixture of a CWA with an internal standard, conversion of the TIC chromatogram to a chromatogram extracted at a particular m/z ratio, and calculation of the CWA concentration from the internal standard concentration, response factor, and chromatographic peak areas. Available internal standards were identified, and they were verified for seven organophosphorus nerve-paralysing agents. Corresponding response factors were determined as a ratio of gradients of the linear functions of the peak area and compound concentration. Linearity, repeatability, and accuracy of the measurements were evaluated. The determination can be performed on all GC/MS systems of the Fire Rescue Service of the Czech Republic (FRS), where no CWA standards are available.

Liquid Chromatography of Liquid Formulations Containing 2,4-Dichlorophenoxyacetic Acid, Dicamba, and 2-(2-Methyl-4-chlorophenoxy)propionic Acid as Their Salts

1983 ◽  
Vol 66 (5) ◽  
pp. 1220-1225 ◽  
Author(s):  
Robert B Grorud ◽  
John E Forrette
Keyword(s):  
Propionic Acid ◽  
Internal Standard ◽  
Solvent System ◽  
Dichlorophenoxyacetic Acid ◽  
Binary Solvent ◽  
Internal Standards ◽  
Liquid Chromatographic Method ◽  
Standard Solution ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Liquid Chromatographic

Abstract A high pressure liquid chromatographic method has been developed for liquid herbicide combinations that contain different combinations of 3 active ingredients including 2,4-dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy)propionic acid (MCPP), and dicamba. A reverse phase column in the ion suppression mode and a binary solvent system separate all 3 herbicides quantitatively on a single chromatogram. The internal standard solution may contain 2 internal standards, salicylic acid and butyrophenone, for use with certain combinations of the herbicides. The solvent system resolves the compounds of interest from all significant impurities.

Quantitative Determination of Chlorpromazine. HC1 in Tablets, Spansules, Injectables, and Bulk Chemical by Nuclear Magnetic Resonance Spectroscopy

1978 ◽  
Vol 61 (1) ◽  
pp. 52-54
Author(s):  
John E Zarembo ◽  
Richard J Warren ◽  
David B Staiger
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Internal Standard ◽  
Resonance Spectroscopy ◽  
Internal Standards ◽  
Tetramethylammonium Bromide ◽  
Bulk Chemical ◽  
Characteristic Signal ◽  
Nuclear Magnetic

Abstract A nuclear magnetic resonance (NMR) procedure is described for the quantitative analysis of chlorpromazine. HC1 in bulk chemical as well as in final dosage forms—tablets, spansules, and injectables. The method is based on measurement of a characteristic signal of chlorpromazine relative to an internal standard. Three different internal standards are specified: Cyclohexane was selected because of the convenience and rapidity with which samples could be prepared for assay. Piperonal was used to verify the method and to show that precision and accuracy were not affected by the volatility of the cyclohexane. Tetramethylammonium bromide was used as an internal standard for Thorazine injectable. No interferences were found from stearates and other tablet excipients. The NMR procedure provides a simple, direct, and specific assay with a precision of ± 1–2%.

Comparing Internal and External Standards in Voice Quality Judgments

1993 ◽  
Vol 36 (1) ◽  
pp. 14-20 ◽  
Author(s):  
Bruce R. Gerratt ◽  
Jody Kreiman ◽  
Norma Antonanzas-Barroso ◽  
Gerald S. Berke
Keyword(s):  
Quality Evaluation ◽  
Context Effects ◽  
Voice Quality ◽  
Internal Standard ◽  
Internal Standards ◽  
Quality Dimension ◽  
Vocal Qualities ◽  
Scale Point ◽  
Descriptive Framework ◽  
Physical Signal

A new descriptive framework for voice quality perception (Kreiman, Gerratt, Kempster, Erman, & Berke, 1993) states that when listeners rate a voice on some quality dimension (e.g., roughness), they compare the stimulus presented to an internal standard or scale. Hypothetically, substituting explicit, external standards for these unstable internal standards should improve listener reliability. Further, the framework suggests that internal standards for vocal qualities are inherently unstable, and may be influenced by factors other than the physical signal being judged. Among these factors, context effects may cause drift in listeners’ voice ratings by influencing the internal standard against which judgments are made. To test these hypotheses, we asked 12 clinicians to judge the roughness of 22 synthetic stimuli using two scales: a traditional 5-point equal-appearing interval (EAI) scale and a scale with explicit anchor stimuli for each scale point. The stimulus set included a relatively large number of normal and mildly rough voices. We predicted that this would produce an increase in the perceived roughness of moderately rough stimuli over time for the EAI ratings, but not for the explicitly anchored ratings. Ratings made using the anchored scale were significantly more reliable than those gathered using the unanchored paradigm. Further, as predicted, ratings on the unanchored EAI scale drifted significantly within a listening session in the direction expected, but ratings on the anchored scale did not. These results are consistent with our framework and suggest that explicitly anchored paradigms for voice quality evaluation might improve both research and clinical practice.

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