Raw materials of cell therapeutics are required to be examined for impurity causing safety issues. So, the purity test of advanced biopharmaceuticals is crucial for ensuring the safety and quality of ...final products. qRT-PCR is commonly used to detect impurities in final drug products, but it is needed more sensitive and accurate method for detecting undifferentiated cells. Recentily, a purity test using ddPR, which is characterized by more sensitivity and sesitivityy has been proposed as a new method for impurity. In this study, we conducted study on validation of assay for detecting undiffernetiated cells in cell based therapy products using ddPCR.
In this study, we prepared mixed cells using direct-reprogrammed iNSCs as main material and iPSCs as un differentiated cells. To detect undifferentiated cells, NANOG, POU5F1, and TDGF1, known as iPSC-specific genes, were set markers for validation. Validation parameters were set specificity, linearity, accuracy, precision (repeatability and intermediate precision), detection limit (DL), quantitation limit (QL), and robustness according to the guidelines of ICH (Q2) and MFDS (Korea). All results was analyzed QX Manager software 2.1 Standard Edtion. And Statical analyses were performed using GraphPad Prism software. According to the study, iPSCs were specifically expressed NANOG, POU5F1, and TDGF1 significantly higher than fibrolbast and iNSC. For linearity, five different concentirations of iPSCs were mixed, resulting in a coefficient R2 ≥ 0.999 of the standard curve. In the accuracy using low, medium, and high concentration samples, it was confirmed that the relative error between expected and mesured was within 20%. Repeatability intermediate precision between two analysts were confirmed no significant difference. And it was confirmed that QL was 0.1ng and DL was 0.01ng of iPSCs in iNSCs. In addition, robustness of this method was verified there was no significant difference despite different idle time after droplets generation in NANOG and TDGF1, except for the results of POU5F1 after 24h. In this study, we successfully assessed the impurities in cell-based therapy by ddPCR. From these results, we recommend ddPCR as a reliable and sensitive method to evaluate the impurity of cell-based therapeutics.
This research was supported by a grant (22201MFDS122) from the Ministry of Food and Drug Safety.
Organotin compounds (OTs) are well studied in various environmental compartments, with a critical focus on the water column as their primary entry point into aquatic ecosystems. In this context, a ...method for the analysis of organotin (OTs) in water using silicone rubber-based passive sampling was optimized, validated, and field-tested. Validation covered crucial parameters, including the limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, linearity, and matrix effect. The method was shown to be robust (R2 ≥ 0.99), with recoveries between 70.2 and 114.6%, and precise (CV < 12.8%) (N = 3). LODCw and LOQCw were ≤15 and ≤ 48 pg Sn L−1, respectively, for TBT and TPhT. The matrix effect showed to be low (>-20% ME < 20%) for all OTs but TPhT (69.4%). The silicone rubber–water partition coefficients (Log Ksr,w) were estimated at 3.37 for MBT, 3.77 for DBT, 4.17 for TBT, 3.49 for MPhT, 3.83 for DPhT, and 4.22 for TPhT. During the field study carried out between October 2021 and February 2022 at the entrance of the Port of Santos navigation channel (Southeastern Brazil), sampling rates ranged between 4.1 and 4.6 L d−1, and the equilibrium was achieved for MBT, DBT, MPhT, and DPhT after ∼45 days of deployment. The freely dissolved concentrations varied between 134 and 165 pg Sn L−1 for TBT, 388 and 610 pg Sn L−1 for DBT, and 1114 and 1509 pg Sn L−1 for MBT, while MPhT, DPhT, and TPhT were below the limit of detection. Results pointed out that J-FLEX® rubber-based passive sampling is a suitable and reliable alternative method for the continuous monitoring of OTs in the water column.
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•J-FLEX® silicone-based passive sampling device has been implemented for the analysis of OTs.•Sampler-water partition coefficients were determined for 6 OTs.•Matrix effect may be a matter of concern and must be appraised.•Freely dissolved OTs were detected at pg Sn L−1 levels in an estuarine water column, meeting guidelines requirements.•J-FLEX® rubber has proven to be a reliable tool for continuous monitoring of OTs in the water column.
•An LC-MS/MS method for VAMS was developed for quantifying Lurasidone using a liquid–liquid extraction technique.•The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and ...achieved acceptable precision and accuracy.•The recovery for analyte from VAMS was approximately 40% at four different concentrations.•A comparative study between both conventional (plasma) and microsampling (DBS and VAMS) techniques.•The study supports the advantages of microsampling while maintaining scientific judgement.
The ecological impact of biological, chemical, and analytical research practices, including toxic reagents and biohazardous waste, has led to the development of alternative sampling and extraction techniques for bioanalysis. Microsampling (sample volume < 50 µL) aligns with the 3Rs principle, allowing multiple sampling points from the same animal at different time points and improving animal welfare. A bioanalytical method was developed to investigate factors related to bioanalytical challenges and the implementation of microsampling techniques. An LC-MS/MS method for Volumetric Absorptive Microsampling (VAMS), 20 µL, was developed for quantifying Lurasidone using a liquid–liquid extraction technique. The method uses a C18, Phenomenex column for chromatographic separation and a mobile phase composition of Methanol, Acetonitrile, and Water with 0.1 % HFBA. The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. The recovery for analyte from VAMS was approximately 40% at four different concentrations and is consistent (%CV < 15), with no significant differences among HCT levels. The matrix factor ranged between 85.00 and 115.00 %, showing no substantial issues with reduced or enhanced signal. The stability data showed no significant degradation of LUR in VAMS samples when stored at room temperature for 15 days. The newly established method for Lurasidone confirmed the use of VAMS sampling method and its analysis on LC-MS/MS. Further, the data obtained from microsampling techniques was compared with conventional (plasma) technique, as proof-of-concept, and it confirms the agreement between the two methods. The study supports the advantages of microsampling in protecting the environment and animals while maintaining scientific judgement.
The main objective of method validation process is to prove that an analytical method is acceptable for its intended purpose. The necessity for laboratories to use fully validated methods is now ...universally accepted as a way to obtain reliable results. There are diverse documents for method validation including information about different performance parameters. The classical performance characteristics are accuracy, limit of detection, precision, recovery, robustness, ruggedness, selectivity, specificity and trueness. Unfortunately, contradictory information is normally present among the method validation documents used by laboratories. The inconsistency about the performance parameters can generate some degree of confusion in the complete method validation process. This manuscript addresses controversial and discrepant information, focusing specifically on several national and international method validation guidelines published by prominent organizations and institutions which serve as guidance to validate new analytical methods by practitioners working in different fields.
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•Analytical method validity depends on selected validation guideline.•In-depth assessment of performance parameters for method validation.•Evaluation of controversial and discrepant information.•Lack of common terminology for method validation.
Matrix components of a sample can affect the quantitative measurement of the target analytes. This singularity is named “matrix effects” and should be considered for method validation procedure. Two ...main strategies to overcome matrix effects were suggested in the scientific literature: reducing the presence of components responsible for the effects and compensating the influence of matrix effects in the calibration methodology. It has been recommended that the best option to surpass matrix effects at research level is to establish a systematic optimization by the combination of approaches. On the other hand, from the method validation regulatory viewpoint, the information contained in the different method validation guidelines about matrix effects was uncommon. In fact, only in 54% of the evaluated regulatory documents the term matrix effects was mentioned explicitly in their contents. In addition, in the rest of the guidance documents addressing matrix effects, the subject was typically considered only superficially.
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•Matrix effects are critical for validation of chromatography-mass spectrometry methods.•Detailed evaluation of matrix effects for method validation.•Discrepant information for matrix effects from research versus regulatory points of views.•Contradictory information about matrix effects among different method validation guidelines.
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•A novel, comprehensive analytical method validation to quantify 53 phytochemicals.•Increased credibility by using isotopically labeled internal standards.•Potential natural sources ...for rosmarinic acid, protocatechuic aldehyde and cosmosiin.•Several compounds were detected for the first time in many plant species.
Screening of biologically active and industrially important phytochemicals by reliable and reproducible analytical techniques is crucial as the chemical diversity and complexity of the plant extracts may cause troubles for identification methods. The goal of this study was to develop, optimize and validate a comprehensive and robust LC–MS/MS (liquid chromatography tandem mass spectrometry) method to qualify and quantify 53 phytochemicals in 33 medicinal and aromatic plant species (10 endemic species) from different families which are important in food, cosmetic, and medicinal industry. A detailed analytical method validation procedure was conducted comprising linearity, inter-day and intra-day precision (repeatability), accuracy (recovery), detection and quantification limits (LOD/LOQ), and relative standard uncertainty (U% at 95 % confidence level (k = 2)). Three isotopically labelled internal standards (quercetin D3, rutin D3, and ferulic acid D3 used for flavonoids, flavonoid glycosides, and the rest, respectively) were used to increase the reliability of the developed method and credibility of the results by compensating for the matrix effects and analyte losses during sample preparation and analyses. The validated analytical method was used to screen acetone, methanol, and water extracts of the selected medicinal and aromatic plant species. In conclusion, it can be seen that water extracts of Carduus pycnocephalus and Alcea hohenackeri, methanol extract of Melissa officinalis, acetone extract of Verbascum pinetorum, and acetone, methanol, and water extracts of Capsella bursa-pastoris have food preservation potentials and could be used as natural source for fumaric acid (144.7, 85.4 mg analyte/g extract), rosmarinic acid (88.3 mg analyte/g extract), protocatechuic aldehyde (44.2 mg analyte/g extract), and cosmosiin (21.9, 20.5, and 22.3 mg analyte/g extract), respectively.
N-Nitrosamines (NAs) pose a threat to food safety due to their carcinogenic and mutagenic properties. In this study, we developed and validated a QuEChERS-based LC-MS/MS method for the simultaneous ...analysis of 11 NAs in 74 processed fish meat, processed meat, and salted fish products. Sample preparation was optimized by screening two versions of QuEChERS buffer, four extraction methods, and eight purification methods. The optimal analytical approach was validated for three product categories in terms of linearity, matrix effects, accuracy, and precision. Satisfactory precision and accuracy were demonstrated, with relative recoveries of 70–120% for the 11 NAs. The limits of detection for fish meat, processed meat, and salted fish products were 0.12–7.50, 0.12–4.14, and 0.10–7.81 ng·g−1, respectively. Among the 11 NAs, nine were detected in all 74 samples. This methodology could be applied to monitor NA levels to ensure the safety and quality of food products.
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•Simultaneous analysis method for 11 volatile and non-volatile N-nitrosamines (NAs).•Levels of 11 NAs in 74 samples ranged from 0.41 to 5.92 ng·g−1.•NDEA, NDBA, and NPIP commonly found in fish, meat, and salted fish products.•Sorbent combination for clean-up should be tailored to the specific product type.
•New FDA Guidance on bioanalytical method validation is similar to the EMA Guideline.•Differences include suggested validation parameters and terminology.•The EMA describes the practical conduct of ...experiments more precisely.•The FDA presents reporting more comprehensively.•Advantages of both documents should be combined in a global ICH guideline.
Bioanalysis concerns the identification and quantification of analytes in various biological matrices. Validation of any analytical method helps to achieve reliable results that are necessary for proper decisions on drug dosing and patient safety. In the case of bioanalytical methods, validation additionally covers steps of pharmacokinetic and toxicological studies – such as sample collection, handling, shipment, storage, and preparation.
We drew our attention to the difference of both the newest FDA Guidance and the EMA Guideline on bioanalytical method validation. We aimed to point out advantages of both documents from the laboratory perspective.
The FDA and the EMA documents are similar, but not identical. The EMA describes the practical conduct of experiments more precisely, while the FDA presents reporting recommendations more comprehensively. There are also differences in recommended validation parameters. We hope that the International Council for Harmonisation will combine advantages of both documents to avoid confusing differences in terminology as well as the unnecessary effort of being compliant with two or more guidelines.
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