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.

Inconsistent internal standard response in LC–MS/MS bioanalysis: an evaluation of case studies

Bioanalysis ◽  
2019 ◽  
Vol 11 (18) ◽  
pp. 1657-1667 ◽  
Author(s):  
Daniela Fraier ◽  
Luca Ferrari ◽  
Katja Heinig ◽  
Elke Zwanziger
Keyword(s):  
Case Studies ◽  
Method Development ◽  
Internal Standard ◽  
Reliability Of Results ◽  
Root Cause ◽  
Standard Response ◽  
Response Variation

Aim: Monitoring the internal standard (IS) response is common practice in bioanalysis by LC–MS/MS. IS response variation may raise questions on assay quality and should trigger investigations into the root cause. Results: In two case studies with IS variability, re-analysis of diluted samples and spiking predose study samples revealed no effect of IS variability on results. The D17-labeled IS in a third case proved not to be suitable during method development and was replaced by a differently labeled IS. Conclusion: Determining the exact root cause for varying IS response is not always feasible; however, assay accuracy and reliability of results should be demonstrated. In some cases, assay re-development is needed to solve the problem.

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.

Learning how to interpret ‘dangerous’ internal standard behaviors

Bioanalysis ◽  
2019 ◽  
Vol 11 (18) ◽  
pp. 1679-1684 ◽  
Author(s):  
Eric J Woolf
Keyword(s):  
Case Studies ◽  
Matrix Effects ◽  
Internal Standard ◽  
Anomalous Behavior ◽  
Internal Standards ◽  
Root Cause ◽  
Potential Impact

Internal standard (IS) response has been an active topic of discussion within the bioanalytical community. Initial discussions focused on developing criteria for anomalous responses. Recently, understanding the cause and potential impact of variable IS response has been emphasized. Following a review of recommendations from industry discussions regarding variable IS responses, case studies where interferences with IS response resulted in quantitation inaccuracy are presented. The examples illustrate that variable IS response cannot always be attributed to compensation of matrix effects. Anomalous IS responses, even for stable label internal standards should be investigated and the root cause for the anomalous behavior should, if possible, be determined.

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.

Determination of alcohols in mixtures by 19F NMR spectroscopy using hexefluoroacetone reagent

1982 ◽  
Vol 47 (7) ◽  
pp. 1973-1978 ◽  
Author(s):  
Jiří Karhan ◽  
Zbyněk Ksandr ◽  
Jiřina Vlková ◽  
Věra Špatná
Keyword(s):  
Standard Deviation ◽  
Nmr Spectroscopy ◽  
Standard Method ◽  
Internal Standard ◽  
Internal Standard Method ◽  
Concentration Region ◽  
Experimental Conditions ◽  
Curve Method ◽  
Sample Preparation Procedure

The determination of alcohols by 19F NMR spectroscopy making use of their reaction with hexafluoroacetone giving rise to hemiacetals was studied on butanols. The calibration curve method and the internal standard method were used and the results were mutually compared. The effects of some experimental conditions, viz. the sample preparation procedure, concentration, spectrometer setting, and electronic integration, were investigated; the conditions, particularly the concentrations, proved to have a statistically significant effect on the results of determination. For the internal standard method, the standard deviation was 0.061 in the concentration region 0.032-0.74 mol l-1. The method was applied to a determination of alcohols in the distillation residue from an oxo synthesis.

Busting Myths in Compound Handling Practices for Assay Developers

2021 ◽  
pp. 247263032110233
Author(s):  
Rosalia Gonzales ◽  
Travis Mathewson ◽  
Jefferson Chin ◽  
Holly McKeith ◽  
Lane Milde ◽  
...  
Keyword(s):  
The Other ◽  
Common Goal ◽  
Collaborative Relationship ◽  
Sample Handling ◽  
Root Cause ◽  
The Common ◽  
Recommended Practices ◽  
Handling Practices ◽  
The One

Since the advent of modern-day screening collections in the early 2000s, various aspects of our knowledge of good handling practices have continued to evolve. Some early practices, however, continue to prevail due to the absence of defining data that would bust the myths of tradition. The lack of defining data leads to a gap between plate-based screeners, on the one hand, and compound sample handling groups, on the other, with the latter being the default party to blame when an assay goes awry. In this paper, we highlight recommended practices that ensure sample integrity and present myth busting data that can help determine the root cause of an assay gone bad. We show how a strong and collaborative relationship between screening and sample handling groups is the better state that leads to the accomplishment of the common goal of finding breakthrough medicines.

SIMULTANEOUS ESTIMATION OF CURCUMIN AND GEFITINIB IN BULK AND TISSUE SAMPLES (PLASMA AND BRAIN HOMOGENATE) BY RP-HPLC: APPLICATION TO A DISTRIBUTION STUDY

INDIAN DRUGS ◽  
2019 ◽  
Vol 56 (07) ◽  
pp. 59-68
Author(s):  
H Mahajan ◽  
S Savale ◽  
P Nerkar ◽  
Keyword(s):  
Flow Rate ◽  
Mobile Phase ◽  
Internal Standard ◽  
Brain Homogenate ◽  
Reversed Phase ◽  
Hplc Method ◽  
Simultaneous Estimation ◽  
Bulk Analysis ◽  
Experimental Conditions ◽  
Rp Hplc

The present study was aimed at developing a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of curcumin (CRM) and gefitinib (GFT) in bulk, plasma and brain homogenate. hydrochlorothiazide was used as an internal standard (IS). A new simple, rapid, selective, precise and accurate RP-HPLC method has been developed. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.) coupled with a guard column of silica, mobile phase consisted of acetonitrile: water with 0.1% formic acid (30:70 v/v). The flow rate was 0.2 ml/min and the drug was detected using PDA detector at the wavelength of 242 nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation and flow rate, were optimised to provide high-resolution and reproducible peaks. The method was developed and tested for linearity range of 10-60 μg/mL for bulk analysis and 200-800 ng/mL for plasma and brain homogenate. The developed method was validated as per ICH guidelines, in terms of linearity, application of the proposed method to bulk sample, recovery, precision, repeatability, ruggedness, sensitivity (LOD and LOQ) and robustness and stability study (short and long-term stabilities, freeze/thaw stability, post-preparative). The low value of % RSD showed that the method was precise within the acceptance limit of 2%. The developed method was successfully applied for the analysis of the drug in bulk as well as various marketed formulation and drug in plasma and brain distribution studies.

Determination of Tryptophan Using Nanocomposite-Based Sensor

2013 ◽  
Vol 829 ◽  
pp. 675-680 ◽  
Author(s):  
Akbar Islamnezhad ◽  
Esmaeil Farzaneh Jobaneh
Keyword(s):  
Catalytic Effect ◽  
Basic Solution ◽  
Carbon Paste ◽  
Experimental Conditions ◽  
Chemically Modified ◽  
Solvent Free Condition ◽  
Analytical Response ◽  
Paste Electrode ◽  
Effect Of The Composition

In this study, the nanoComposite Silica Perchloric Acid Poly (2-Methyl Aniline) (NSPPMA) was synthesized under the solvent-free condition and used as modifier in preparation of chemically modified electrode (CME). The effect of the composition of carbon paste electrode on its voltammograms were evaluated in basic solution with 5.0 × 105 M Trp. It was found that addition of NSPPMA to the carbon paste would generate the peak current of Trp because of its catalytic effect on redox process. The best analytical response was obtained at pH 12.0. The anodic peak currents were proportional to Trp concentrations in the range of 2.5 × 109 5.0 × 10-4 M under the optimized experimental conditions. The detection limit was 1.7 × 10-10 M. The correlation of the peak currents against v1/2 (v is the scan rate) is linear. The proposed method was applied to the determination of Trp in pharmaceuticals formulations successfully.

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).

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