method validation
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Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 457
Elżbieta Gniazdowska ◽  
Wojciech Goch ◽  
Joanna Giebułtowicz ◽  
Piotr J. Rudzki

Background: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. Methods: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes—tramadol and its major metabolite (O-desmethyl-tramadol)—in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). Results: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85–115% acceptance criteria. Conclusions: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

Molecules ◽  
2022 ◽  
Vol 27 (1) ◽  
pp. 297
Essam Ezzeldin ◽  
Muzaffar Iqbal ◽  
Yousif A. Asiri ◽  
Gamal A. E. Mostafa ◽  
Ahmed Y. A. Sayed

Pexidartinib is the first drug approved by the U.S. Food and Drug Administration specifically to treat the rare joint tumor tenosynovial giant cell tumor. In the current study, a validated, selective, and sensitive UPLC-MS/MS assay was developed for the quantitative determination of pexidartinib in plasma samples using gifitinib as an internal standard (IS). Pexidartinib and IS were extracted by liquid-liquid extraction using methyl tert-butyl ether and separated on an acquity BEH C18 column kept at 40 °C using a mobile phase of 0.1% formic acid in acetonitrile: 0.1% formic acid in de-ionized water (70:30). The flow rate was 0.25 mL/min. Multiple reaction monitoring (MRM) was operated in electrospray (ESI)-positive mode at the ion transition of 418.06 > 165.0 for the analyte and 447.09 > 128.0 for the IS. FDA guidance for bioanalytical method validation was followed in method validation. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.999 with a limit of quantitation of 0.5 ng/mL. Moreover, the metabolic stability of pexidartinib in liver microsomes was estimated.

2021 ◽  
Vol 22 (2) ◽  
pp. 243-251
Muhammad Abdurrahman Munir ◽  
Khairiah Haji Badri ◽  
Lee Yook Heng ◽  
Muhammad Mukram Mohamed Mackeen ◽  
Edison Eukun Sage

Histamine is commonly present in food containing proteins, like in mackerel. Consuming fish is imperative for the improvement of human muscles. Nevertheless, some studies reported ingesting fish containing histamine more than 50 mg·kg-1 can cause toxicity. This study analyzed and determined the composition of histamine in mackerel and its products commonly consumed in Malaysia, especially on the East Coast of Malaysia. These included processed mackerel such as canned products, satay (skewed fish) and keropok lekor (fish cake/ cracker). Histamine analysis was performed using High Performance Liquid Chromatography (HPLC) equipped with a fluorescence detector. A derivatizing reaction was applied to increase the sensitivity of HPLC to histamine using 9-flourenilmethylchloroformate (FMOC-Cl). The chromatographic separation was achieved in 15 min. Method validation was in accordance to Commission Decision 657/2002/CE. The linear range was at 0.16 – 5.00 µg·mL-1 (histamine) with the LOD at 0.10 µg·mL-1 and LOQ at 0.30 µg·mL-1 . Method applicability was checked on seven real samples involving raw, cooked, and dry products, yielding acceptable recovery.

Food Research ◽  
2021 ◽  
Vol 5 (6) ◽  
pp. 211-220
I.S. Maulidyah ◽  
D.N. Faridah ◽  
H.N. Lioe

The presence of heavy metals in infant formula has become a global concern. The most common method to determine heavy metals is AAS. However, as this technique is lacking in several aspects, including the instrument’s low sensitivity, a more sensitive instrument such as ICP-MS is necessary. The Inductively Coupled Plasma Mass Spectrometry (ICPMS) was used in accordance with the standard method AOAC 2015.01 with modifications on the microwave condition and the addition of hydrochloric acid (HCl) during the sample digestion process. The modified standard method requires a validation process. This research aimed to validate the method of analysis for the determination of Pb, Cd, Hg, As in infant formula using ICP-MS and its application in formula milk. This research consists of five stages: 1) instrumental performance; 2) homogeneity test; 3) method orientation; 4) method validation; and 5) the application of the validated method to other products. The findings in the research were: the method linearity was confirmed at working concentration 5-30 µg/kg for all the heavy metals with R2 value of nearly 1,000; the method limits of detection (LOD) were 0.74 µg/kg (Pb), 0.41 µg/kg (Cd), 0.08 µg/kg (Hg), 0.50 µg/kg (As), while the method’s limits of quantification (LOQ) were 2.48 µg/kg (Pb), 1.36 µg/kg (Cd), 0.27 µg/kg (Hg), 1.67 µg/kg (As); the method was found precise with Relative Standard Deviation (RSD) below 2/3 RSD Horwitz and all the recovery values were found to fall within the acceptable range (60–115%); the % RSD intra-lab reproducibility was below RSD Horwitz; and the method was robust, indicating that it was unaffected by small changes in its variables. The validated method can be applied routinely to determine heavy metals in infant formula and formula milk.

2021 ◽  
Vol 18 ◽  
Diaa Shakleya ◽  
Sonal Mazumder ◽  
Naresh Pavurala ◽  
Sara Mattson ◽  
Patrick J. Faustino

Background: Transdermal drug delivery systems (TDS) are widely used to deliver a number of different drug therapeutics. The design delivery can be impacted by excipients and, more broadly, organic solvents. Organic or residual solvents are routinely monitored due to safety concerns. However, there is little information on the mechanical properties and delivery performance of TDS. Objective: The objective of this study was to develop and validate an efficient GC-Headspace method to determine the residual solvents (n-heptane, o-xylene, and ethyl acetate) in transdermal patches. The analytical method was applied to monitor residual solvents in TDS and evaluate the potential effect of the residual solvent levels on the TDS adhesion properties. Methods: An Agilent GC 7890A was integrated with an Agilent headspace analyzer 7697A system and was used for method development, analytical method validation, and the testing phases of the study. For the analysis of residual solvents in TDS, 2cm x 3cm, a TDS sample was placed in a 20 mL Headspace vial containing 2 mL of a DMSO/water (1:1, v/v) solvent mixture, and an external standard (cyclohexane) was extracted by the headspace analyzer. The system suitability test was conducted according to USP <621>, and analytical method validation was conducted according to USP <1225> over 3 days for validation and was also performed during in-study sample analysis. Results: The resolution between the solvents was acceptable (2.5, %RSD = 8.0). Intra- and inter-day accuracy and precision of all quality control standards as well as the spiked standards in the transdermal patches were found to be acceptable with RSD% ≤ 10% and accuracy ≥ 85%, respectively. Linearity was > 0.99 for all analytes. Conclusion: The validated GC-Headspace method was successfully applied to a pilot study for in-house manufactured TDS patches to study the impact of residual solvent concentration on adhesion performance.

2021 ◽  
Vol 18 ◽  
Behrouz Seyfinejad ◽  
Abolghasem Jouyban

AAPS Open ◽  
2021 ◽  
Vol 7 (1) ◽  
Deanna J. Nelson ◽  
G. Dean Marbury

AbstractIon chromatography (IC) has evolved into one of the most widely used separation techniques of analytical chemistry. Consequently, the number of users of this method is continuously growing. Analysts often assume that widely used guidelines for HPLC method validation in regulated environments routinely apply to IC. This manuscript provides an analysis of the potential shortcomings of traditional approaches to development and validation of IC methods using suppressed conductivity detection and a risk-based alternative approach to these activities. The goal of the alternative approach is a reduction in the risk of erroneous determinations of analytes when IC methods using suppressed conductivity detection are employed.

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