In-house Validation Research Articles

In-House Validation of an SPE-GC-FID Method for the Detection of Free and Esterified Hydroxylated Minor Compounds in Virgin Olive Oils.

Minor compounds in vegetable oils are distributed between free and esterified forms, and the ratio of these two fractions could represent an important parameter for assessment of oil authenticity. A simple method based on offline SPE-GC-FID for the analysis of free and esterified hydroxylated minor compounds in olive and sunflower oils has been developed and in-house validated. A satisfactory repeatability relative standard deviation (<7.5%) was obtained in all cases. The method, which requires simple instrumentation, allows for reliable quantification in a single chromatographic run with the advantages of minimizing sample manipulation, use of toxic solvents and reagents, and time consumption. The analytical procedure was applied to pure oil samples, including 15 authentic extra virgin olive oils collected from different European countries (Spain, Italy, Greece, and Portugal). Finally, the proposed SPE-GC-FID methodology could detect changes in the ratio between the free and esterified forms in pure extra virgin olive oil when mixed with refined sunflower oil at different percentages of 2, 5, 10, 15, and 20% (w/w) to simulate adulteration.

In-house and on-field validation of the multiplex PCR assay developed for authentication of three commercially important shrimp species

In this study, a multiplex PCR assay was developed for the authentication of three commercially important shrimp species viz., Penaeus monodon, Penaeus vannamei, and Penaeus indicus. Three new species-specific primers designed from the mitochondrial 16S rRNA gene yielded distinct amplicon size of 360, 277, and 203 bp for P. monodon, P.vannamei, and P.indicus, respectively. The sensitivity, accuracy, specificity, and suitability of the multiplex PCR assay was determined as per standard protocol. The assay exhibited high specificity to the target shrimp species. The sensitivity ranged from 1 to 0.1 ng/μl of DNA concentration. In-house validation with eleven types of mixed-species gave 100% correlation in the authentication of the target shrimp species. Field validation with 22 processed commercial shrimp products revealed 22% mislabeling. The developed multiplex PCR assay authenticated the three commercially important Penaeids in processed shrimp products from other related shrimp, fish as well as crab species in a single run.

In-house validation of AF4-MALS-UV for polystyrene nanoplastic analysis.

The suitability of asymmetric flow field-flow fractionation (AF4) coupled on-line to multi-angle light scattering (MALS) and UV diode array (UV-DAD) detectors was tested to simultaneously detect polystyrene nanoplastics (PS-NPs) and collect information about their size. A mixture of four sizes of PS-NPs at 20nm, 60nm, 100nm and 200nm was prepared by dilution with ultrapure deionized water and gentle mixing and was used as test sample for a polydisperse nanoplastic system. The AF4 method separated each single size of PS-NP mixture in a total time of 48min by using 0.2%SDS as carrier solution. Then, the PS-NPs were sized and detected by following their MALS (90° scattering angle) and UV (215 nm) signals. Quality control (QC) performances as linearity, between-day repeatability, resolution factor, trueness/recovery, limit of detection (LoD) and selectivity were calculated, according to the ISO/TS 21362:2018. Method uncertainty was calculated following the ISO/TS 21748:2002 by summing between-day repeatability and trueness or recovery uncertainties. In-house validation results demonstrated good peak resolution and selectivity, R2 linearity of 0.998-0.999 in the range 50-1000μg/mL, between-day repeatability of ca. 10%, trueness/recovery above 90% and LoD between 15μg/mL (20nm) and 33μg/mL (200nm). Expanded uncertainty was 16.1-17.9% on PS-NP size between 60 and 200nm and 10.4-14.7% on PS-NP concentration between 100 and 1000μg/mL. Compared to traditional single-technique analysis, this hyphenated method offers great promise for separating and analysing diverse populations of PS-NPs present in real matrices, which is critical for health and risk assessment studies and any regulatory action.

Development and Validation of Optical Methods for Zeta Potential Determination of Silica and Polystyrene Particles in Aqueous Suspensions.

Zeta potential is frequently used to examine the colloidal stability of particles and macromolecules in liquids. Recently, it has been suggested that zeta potential can also play an important role for grouping and read-across of nanoforms in a regulatory context. Although the measurement of zeta potential is well established, only little information is reported on key metrological principles such as validation and measurement uncertainties. This contribution presents the results of an in-house validation of the commonly used electrophoretic light scattering (ELS) and the relatively new particle tracking analysis (PTA) methods. The performance characteristics were assessed by analyzing silica and polystyrene reference materials. The ELS and PTA methods are robust and have particle mass working ranges of 0.003 mg/kg to 30 g/kg and 0.03 mg/kg to 1.5 mg/kg, respectively. Despite different measurement principles, both methods exhibit similar uncertainties for repeatability (2%), intermediate precision (3%) and trueness (4%). These results confirm that the developed methods can accurately measure the zeta potential of silica and polystyrene particles and can be transferred to other laboratories that analyze similar types of samples. If direct implementation is impossible, the elaborated methodologies may serve as a guide to help laboratories validating their own methods.

Determination of Pharmaceutical Residues by UPLC-MS/MS Method: Validation and Application on Surface Water and Hospital Wastewater.

In this study, an analytical method for the simultaneous determination of 7 major pharmaceutical residues in Vietnam, namely, carbamazepine, ciprofloxacin, ofloxacin, ketoprofen, paracetamol, sulfamethoxazole, and trimethoprim, in surface water and hospital wastewater has been developed. The method includes enrichment and clean-up steps by solid phase extraction using mix-mode cation exchange, followed by identification and quantification using an ultrahigh-performance liquid chromatography and tandem mass spectrometry and employing electrospray ionization (UPLC-ESI-MS/MS). Seven target compounds were separated on the reversed phase column and detected in multiple reaction monitoring (MRM) mode within 6 minutes. The present study also optimized the operating parameters of the mass spectrometer to achieve the highest analytical signals for all target compounds. All characteristic parameters of the analytical method were investigated, including linearity range, limit of detection, limit of quantification, precision, and accuracy. The important parameter in UPLC-ESI-MS/MS, matrix effect, was assessed and implemented via preextraction and postextraction spiking experiments. The overall recoveries of all target compounds were in the ranges from 55% to 109% and 56 % to 115% for surface water and hospital wastewater, respectively. Detection limits for surface water and hospital wastewater were 0.005–0.015 µg L−1 and 0.014–0.123 µg L−1, respectively. The sensitivity of the developed method was allowed for determination of target compounds at trace level in environmental water samples. The in-house validation of the developed method was performed by spiking experiment in both the surface water and hospital wastewater matrix. The method was then applied to analyze several surface water and hospital wastewater samples taken from West Lake and some hospitals in Vietnam, where the level of these pharmaceutical product residues was still missed. Sulfamethoxazole was present at a high detection frequency in both surface water (33% of analyzed samples) and hospital wastewater (81% of analyzed samples) samples.

In-house validation of accelerated solvent extraction-gas chromatography-mass spectrometry for the determination of bound 3- and 2-monochloropropanediols (MCPD) and glycidol in food products

ABSTRACT The study aimed to establish the detection method for bound 3-, 2-MCPD, and glycidol using accelerated solvent extraction (ASE) and gas chromatography mass spectrometry (GC-MS). The ASE was modified for reduced solvent volume and process time to extract lipid from the chocolate spread, infant formula, potato chips, and sweetened creamer. The solvent selected for ASE was a mixture of iso-hexane and acetone at 100°C with the lipid and analyte recovery ranging from 96.9% to 98.6% and 84.1% to 107.5%, respectively. The derivatisation of analytes was adopted from the AOCS method Cd29a-13 for GC-MS analysis. The results showed that the coefficient of determination (R2) of all analytes was >0.99. The limit of detection (LOD) was 0.1 mg kg−1 expressed in lipid basis for both bound 3- and 2-MCPD and 0.2 mg kg−1 expressed in lipid basis for bound glycidol. The limit of quantitation (LOQ) was 0.3 mg kg−1 expressed in lipid basis for both bound 3- and 2-MCPD and 0.6 mg kg−1 expressed in lipid basis for bound glycidol. A blank spiked with 3-monochloropropanediols fatty acid esters (MCPDE) and 2-MCPDE (0.3, 2.1, and 7.2 mg kg−1) and glycidol esters (0.6, 4.7, and 16.6 mg kg−1) were chosen for accuracy and precision tests. The recoveries were 91.7% to 105.9%. Both repeatability and within-laboratory reproducibility of the analysis were within the acceptable level of precision ranging from 1.7% to 16%. This is the first time that a full validation procedure extending to both accuracy and precision tests has been carried out for sweetened creamer and chocolate spread. Overall, the combined protocol of ASE and AOCS Cd29a-13 was successfully validated for both solid and liquid food samples with lipid content from 10% to 30%.

Pediatric anesthesia during the coronavirus disease epidemic - One pediatric surgical hospital's rapid transition back to care.

Shriners Hospitals for Children - Northern California provides highly specialized pediatric surgical care. The rapid spread of coronavirus disease 2019 (COVID-19) required timely suspension of our elective surgeries. Mitigating disease spread and protecting patients and staff became paramount. Because pediatric patients in particular carry and may spread SARS-CoV-2 while asymptomatic, and due to the large amount of respiratory aerosols produced during airway manipulation, an unprecedented risk of provider infection came to exist. Surgical cases were tiered based on urgency and only emergent or urgent cases proceeded using airborne precaution personal protective equipment (PPE)1 . Furthermore, asymptomatic patients infected with SARS-CoV-2 undergoing surgery have higher postoperative morbidity and mortality2 . However, surgery in children is rarely completely elective and could not be postponed indefinitely. The ability to resume pediatric surgical care during this pandemic would only be possible if a completely new path was forged.

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.

Cannabinoids Determination in Brain: A Supplemental Helpful in Postmortem Evaluation

The scientific interest in cannabis has been documented by a wide literature, but postmortem studies and interpretations of autopsy findings are lacking or limited to few cases, few matrices analyzed or a small number of analytes. The present study describes the development and full in-house validation of a sensitive and simple method based on an optimized rapid clean-up procedure combined with a robust and highly sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) technique, designed to simultaneous determination of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid (THC-COOH) and 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid glucuronated (THC-COOH gluc.) in postmortem samples: central blood (CB), femoral blood (FB) and brain tissue (BR). The developed method was validated and applied to 24 postmortem cases involving cannabinoids. In this study, we presented a full optimization and validation of target analyses for each matrix. The procedure had proven to be reliable and accurate. This study adds new data, particularly about the cannabinoids concentrations in BR samples. Combined pattern (CB, FB, BR) can be used in the interpretation of postmortem cases, proving and strengthening the assessments made on blood data. BR matrix is a helpful supplement in the investigation of the role of cannabinoids as crucial or contributory factor in leading to death.

Determination of Cholesterol Content in Butter by HPLC: Up-to-Date Optimization, and In-House Validation Using Reference Materials.

This work deals with up-to-date optimization of cholesterol content determination when saponification and extraction procedures as well as HPLC conditions were studied. As found, optimal conditions for saponification process were identified by 15 min heating in the presence of 0.015 L of methanolic KOH solution with a concentration 1 mol/L with subsequent 0.015 L n-hexane–chloroform binary mixture (1:1, v/v) double extraction. HPLC separation consisted of isocratic elution with flow rate of 0.5 mL/min mobile phase composed of acetonitrile/methanol 60:40 (v/v) and stationary phase Zorbax Eclipse Plus C18 column 2.1 × 100 mm, 3.5 μm particle size diameters with detector wavelength 205 nm. The method passed through in-house validation criteria and its suitability was verified by analysis of butter reference materials. In final, the average content of cholesterol content in butter was determined at 2271.0 mg/kg. Thus, the method is suitable for the determination of cholesterol content in butter and probably also in other dairy products.

First Detection of Tetrodotoxin in Bivalves and Gastropods from the French Mainland Coasts.

In 2015, tetrodotoxins (TTXs) were considered a potential threat in Europe since several studies had shown the presence of these toxins in European bivalve molluscs. In this study, we investigated the occurrence of TTXs in 127 bivalve samples (mussels and oysters) and in 66 gastropod samples (whelks) collected all along the French mainland coasts in 2017 and 2018. Analyses were carried out after optimization and in-house validation of a performing hydrophilic interaction liquid chromatography associated with tandem mass spectrometry (HILIC-MS/MS) method. The concentration set by European Food Safety Authority (EFSA) not expected to result in adverse effects (44 µg TTX equivalent/kg) was never exceeded, but TTX was detected in three mussel samples and one whelk sample (1.7–11.2 µg/kg). The tissue distribution of TTX in this whelk sample showed higher concentrations in the digestive gland, stomach and gonads (7.4 µg TTX/kg) than in the rest of the whelk tissues (below the limit of detection of 1.7 µg TTX/kg). This is the first study to report the detection of TTX in French molluscs.

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension.

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most "active" component of a nanoparticle dose. Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1-1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration). We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20-100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

In-house validation method for quantification of formaldehyde in silage and feedingstuffs with the use HPLC-DAD technique.

An HPLC-DAD method was developed for the determination of formaldehyde in animal feed and silage. The method is based on the determination of the product of chemical reaction between formaldehyde and 2,4-dinitrophenylhydrazine. A 3 g of feed or silage were extracted with Milli-Q water with phosphoric acid and next formaldehyde was derivative with the use 2,4-dinitrophenyl- hydrazine in acetronitrile solution. The extract was purified with 0.45 µm syringe filters and separeted on Zorbax Eclipse XDB C18 column and detection was carried out at 360 nm. Formal- dehyde was eluted with a mobile phase consisting of acetonitrile/water in isocratic elution. This method provided average recoveries of 90.6% to 102.2%, with CVs of 2.6% to 6.4% for feed and from 91.3% to 108.7% with CVs of 1.1% to 4.1% for silage in the ranged of 50 to 1000 mg/kg feeds and silage. The LOD and LOQ for formaldehyde in feed and silage ranged from 1.6 to 2.6 and 2.7 to 5.7 mg/kg, respectively. The methodology was applied for the analysis of feed and silage samples collected from poultry, pigs and cows farms.

Implementing NGS-based BRCA tumour tissue testing in FFPE ovarian carcinoma specimens: hints from a real-life experience within the framework of expert recommendations

AimsNext Generation Sequencing (NGS)-based BRCA tumour tissue testing poses several challenges. As a first step of its implementation within a regional health service network, an in-house validation study was compared...

Papanicolaou Staining and Processing Troubleshooting Exercise Using Digital Pathology: Peer Comparison in an External Quality Assurance Setting

Background: The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) offers a comprehensive range of External Quality Assurance programs for all disciplines of pathology and is committed to delivering programs to advance outcomes in patient care and promote excellence in laboratory diagnostic and technical proficiency. Proficiency testing in cytopreparatory techniques became available for formal enrolment in 2018. The 2019 technical program focused on the demonstration of a laboratory to troubleshoot problems encountered during staining and processing. The exercise provided a peer comparison of a laboratory’s ability to (1) identify the problem viewed from a digital image and (2) describe the troubleshooting steps to rectify the problem using assessment guidelines. Methods: Ten troubleshooting exercises including whole-slide z-stacked virtual images, a reference slide, and an online Result Entry questionnaire were provided. Common staining and processing problems were demonstrated in these exercises. Multiple-choice responses were made available to participants for each case with 1 response requiring submission. Participants were then allowed free text to describe how they would rectify the problem. Results: Of the 72 participants who participated, 62 participants (86%) achieved a satisfactory assessment, 6 participants (8%) received a borderline assessment, and 4 participants (6%) received an unsatisfactory assessment. The average mark obtained was 32.7 out of a total of 45 marks, which equates to a satisfactory assessment. The highest mark obtained for this survey was 42.5. Conclusions: Quality cytopathology preparatory techniques are integral to mitigating the risk of diagnostic error. The digital pathology platform allowed homogeneity of samples for all participants and appeared to be a satisfactory mode for demonstrating the technical problems to participants. Laboratories participating in this survey showed good performance in identifying processing and staining problems on virtual images and working through the troubleshooting steps. The exercise also identified a method for troubleshooting formalin vapour-affected slides using citrate buffer which may be useful for laboratories following in-house validation.

In-house validation of a lamp kit for diagnosis of Plasmodium, Plasmodium falciparum and Plasmodium vivax in Vietnam

Vietnam announced the elimination of malaria in 25 provinces in 2019 and and advance to eliminate malaria nationwide by 2030. Quick, accurate diagnosis and prompt treatment play a very important role in malaria eradication strategies. In this study, we developed the LAMP technique to detect Plasmodium spp., P. falciparum, P. vivax from different kinds of sample such as whole blood, dried blood spot, etc. The advantages of this method included high specificity, fast detection time, and simple equipment use. Primer set was designed for founding on 18S rRNA gene. A positive reaction was visualised with the naked eye, using the color indicator, Malachite Green.

Compact and tunable stretch bioreactor advancing tissue engineering implementation. Application to engineered cardiac constructs

Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro. In tissue engineering applications, bioreactors have become fundamental and effective tools for providing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on decellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation.

Validation of a radiochemical method for the determination of 55Fe and 63Ni in water and steel samples from decommissioning activities

We present the validation of a radiochemical procedure for the determination of 55Fe and 63Ni in water and steel samples from nuclear decommissioning activities. The in-house validation was an essential requirement of the accreditation to ISO/IEC 17025. A combination of co-precipitation, anion chromatography and extraction chromatography steps was used for separating and purifying both radionuclides. The activities were measured by liquid scintillation counting. Due to the lack of reference materials, the method was validated by analysing simulated samples and by the standard addition technique. The parameters precision/accuracy, repeatability/linearity, selectivity/specificity, decision threshold, detection limit and uncertainty of the method were evaluated.

Fatty Acid Ethyl Esters in Virgin Olive Oils: In-House Validation of a Revised Method.

The content of fatty acid ethyl esters (FAEEs) is one of the quality parameters to define if an olive oil can be classified as extra virgin as these compounds are considered markers for virgin olive oils obtained from poor-quality olives. In addition, FAEEs can also be indirect markers to detect soft deodorization treatment. In this study, an off-line HPLC-GC-FID method for determination of FAEEs is presented, revising the preparative step and the GC injector required by the official method (EU Reg. 61/2011). After optimization, the method was validated in-house by analyzing several parameters (linearity, limit of detection LOD, limit of quantification LOQ, robustness, recovery, precision, and accuracy) to determine its effectiveness. Linearity was measured in the 2.5–50 mg/L range; furthermore, intra-day and inter-day precision values were lower than 15%, while the LOD and LOQ were lower than 1 and 1.5 mg/kg, respectively, for all compounds considered. The main advantages of this revised protocol are: (i) significant reduction in time and solvents needed for each analytical determination; (ii) application of HPLC as an alternative to traditional LC, carried with manually packed glass columns, thus simplifying the separation step.

Multi-Toxin Quantitative Analysis of Paralytic Shellfish Toxins and Tetrodotoxins in Bivalve Mollusks with Ultra-Performance Hydrophilic Interaction LC-MS/MS-An In-House Validation Study.

Ultra-performance hydrophilic interaction liquid chromatography tandem mass spectrometry system (UP-HILIC–MS/MS) was used in multi-toxin analysis of paralytic shellfish toxins (PSTs) and tetrodotoxins (TTXs) in sample matrices from bivalve molluscan species commercially produced for human consumption in Sweden. The method validation includes 17 toxins of which GTX6 and two TTX analogues, TTX and 4,9-anhydroTTX, were previously not analyzed together with hydrophilic PSTs. 11-deoxyTTX was monitored qualitatively with a non-certified reference standard. The performance of the method was evaluated for selectivity, repeatability, and linearity by analyzing spiked samples which generated linear calibration curves across the concentration ranges used (R2 > 0.99). The in-house reproducibility (RSD) was satisfactory including the LOD and LOQ for both PST and TTX toxins being far below their regulatory action limits. The major advantage of the method is that it allows direct confirmation of the toxin identity and specific toxin quantification using a derivatization-free approach. Unlike the PST-chemical methods used in routine regulatory monitoring until now for food control, the UP-HILIC-MS/MS approach enables the calibration set-up for each of the toxin analogs separately, thereby providing the essential flexibility and specificity in analysis of this challenging group of toxins. The method is suitable to implement in food monitoring for PSTs and TTXs in bivalves, and can serve as a fast and cost-efficient screening method. However, positive samples would, for regulatory reasons still need to be confirmed using the AOAC official method (2005.06).