Method For Accurate Quantification Research Articles

A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment.

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

An accurate spinal cord partition and quantification method to monitor atrophy in multiple sclerosis

An accurate spinal cord partition and quantification method to monitor atrophy in multiple sclerosis

International co-validation on absolute quantification of single nucleotide variants of KRAS by digital PCR.

The precise quantification of KRAS single nucleotide variant (SNV) is critical for the treatment and prognosis of lung and colorectal cancer. Validation of digital PCR (dPCR) as a method for accurate quantification of KRAS SNV has great clinical importance. An international co-validation on absolute quantification of KRAS SNV by dPCR was conducted among three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ). A candidate reference material (RM) was provided by NIM and three measurands were reported: copy number concentration (Tc) of KRAS G12A mutation and wild type and KRAS G12A fractional abundance (FA). Homogeneity and stability assessment showed that the study materials provided by NIM were sufficiently homogeneous and stable during the study period. En number performance statistics was used to evaluate equivalence of the study among the three NMIs. All En values for both Tc and KRAS G12A FA≤1 showed good agreement and consistency with the reference value within the expanded uncertainty. This indicates that dPCR with full uncertainty evaluation can serve as a candidate primary reference measurement procedure (PRMP) for the KRAS SNV measurement and value assignment of reference materials.

An analytical quality by design approach towards a simple and novel HPLC-UV method for quantification of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline

N-acetyl-seryl-aspartyl-lysyl proline (Ac-SDKP) is a tetrapeptide possessing anti-fibrotic, angiogenic, anti-inflammatory, anti-apoptotic, and immunomodulatory properties. Currently, the main method to quantify the peptide is liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), both of which are labour intensive and require expensive equipment and consumables. Furthermore, these techniques are generally utilised to detect very low or trace concentrations, such as in biological samples. The use of high concentrations of analyte might overload the extraction column or the separation column in LC-MS/MS or the ELISA plates, so the response could be a non-linear relationship at high analyte concentrations. Thus, they are not ideal for formulation development where detection of dose-equivalent concentrations is typically required. Therefore, a cost-effective, simple, and accurate quantification method for the peptide at a higher concentration needs to be developed. In this study, a simple and novel HPLC-UV method is proposed and validated using an Analytical Quality by Design (AQbD) approach. The method is first screened and optimised using chromatographic responses including capacity factor, resolution, tailing factor, and theoretical plate counts, fulfilling the International Council for Harmonisation (ICH) Q2 (R1) guidelines. The resultant optimised chromatography conditions utilised 10 mM phosphate buffer at pH 2.5 and acetonitrile as mobile phases, starting at 3% (v/v) acetonitrile and 97% (v/v) buffer and increasing to 9.7% (v/v) acetonitrile and 90.3% (v/v) buffer over 15 min at a flow rate of 1 mL/min at the column temperature of 25 °C. The injection volume is set at 10 μL and the VWD detector wavelength is 220 nm. The method established is suitable for detecting the peptide at a relatively high concentration, with a quantifiable range from 7.8 μg/mL to 2.0 mg/mL. In addition, the use of a relatively simple HPLC-UV approach could significantly reduce costs and allow easier access to quantify the peptide concentration. A limitation of this method is lower sensitivity compared with using LC-MS/MS and ELISA methods but running costs are lower and the methodology is simpler. The method is capable to quantify the peptide in various tested matrix solutions, with successful quantitation of the peptide in samples obtained from in vitro drug release study in PBS and from a chitosan-TPP nanogels formulation. Therefore, the method developed here offers a complementary approach to the existing quantification methods, quantifying this peptide at increased concentrations in simple to intermediately complex matrix solutions, such as HBSS, DMEM and FluoroBrite cell culture media.

Electrochemical-based quantitative fingerprint evaluation strategy combined with multi-markers assay by monolinear method for quality control of herbal medicine

BackgroundImproving the quality control (QC) criterion of herbal medicine (HM) is an ongoing challenge. A rapid and convenient electrochemical analysis technique is now emerging as a promising application for HM QC. So far, extraction and analysis of the overall electroactive components is a key issue need to be solved to improve its application in integral HM QC. PurposeIn this work, using compound liquorice tablets (CLQTs) as an example, we like to put forward a more reliable and accurate quantification method of multi-components for the precise QC of HM. Furthermore, we propose an electrochemical fingerprint-based data mining, extract and synthesis strategy for in-depth and comprehensive QC of HM, qualitatively and quantitatively. Methods and designFirstly, the electrochemical quantitative fingerprint of 54 batches of CLQTs from nine manufacturers were developed using B-Z oscillatory system. Secondly, eight characteristic parameters were recorded and compared among samples using intuitive information and PCA. Then, tund was used to establish the correlation with sample dosage for determination of the relative content of overall electroactive components (Rc). The quantitative determination of five quality markers (Q-markers) were also performed using the novel method, called multi-markers assay by monolinear method (MAML). Finally, after using area integral calculus for electrochemical fingerprint, average linear quantitative fingerprint method (ALQFM) was successfully proposed to extract all latent characteristics for integral quality evaluation of samples. ResultsThe tund and dosage showed a good correlation, by which the obtained Rc displayed different fluctuation among nine manufacturers. Moreover, the contents of five Q-markers obtained by MAML displayed no significant difference with the traditional quantification method. Samples evaluated by ALQFM manifested the integral information and were divided into eight quality grades. The deduced results of correlation between Pl with P5C, Rc and PA were more persuasive for demonstrating the reliability and integrity of ALQFM in quality evaluation of HM electrochemical fingerprint. ConclusionThe study confirmed the idea that quantification of Q-markers combined electrochemical-based quality evaluation strategy could be used as a reliable method for HM QC qualitatively and quantitatively from point (precise) to face (integral).

An Improved Method to Quantify Short-Chain Fatty Acids in Biological Samples Using Gas Chromatography-Mass Spectrometry.

Gut microbial metabolites, short-chain fatty acids (SCFAs), are found at multiple locations in the host body and are identified as important metabolites in gut microbiome-associated diseases. Quantifying SCFAs in diverse biological samples is important to understand their roles in host health. This study developed an accurate SCFA quantification method by performing gas chromatography–mass spectrometry (GC/MS) in human plasma, serum, feces, and mouse cecum tissue. The samples were acidified with hydrochloric acid, and the SCFAs were extracted using methyl tert-butyl ether. In this method, distilled water was selected as a surrogate matrix for the quantification of SCFAs in target biological samples. The method was validated in terms of linearity, parallelism, precision, recovery, and matrix effect. The developed method was further applied in target biological samples. In conclusion, this optimized method can be used as a simultaneous SCFA quantification method in diverse biological samples.

Development and Validation of a New Stereoselective RP-HPLC Method for Simultaneous Quantification of Tadalafil, its One Enantiomer, and Two Diastereomers in API and Tablet Form

A new, simple, and stereoselective RP-HPLC method was developed and validated for the simultaneous quantification of tadalafil, its one enantiomer, and two diastereomers in API and tablets. Using varied compositions of water, acetonitrile, and acetic acid as mobile phases in gradient mode at a 0.40 mL/min flow rate and detection at 285 nm, all this separation was achieved on the Lux Cellulose-3 (150 mm x 4.6 mm, 3 µm) column at a 30.0°C oven temperature. All isomers were eluted within 24 minutes, with a resolution of more than 2.3 between any two isomers. With 10.0 µL injection volume, the LOD and LOQ were determined to be 0.06 µg/mL and 0.10 µg/mL, respectively. The linearity of tadalafil (0.10-400 µg/mL), one enantiomer, and two diastereomers (0.10-4.0 µg/mL) was confirmed with a correlation coefficient of 0.999. The forced degradation study revealed the specificity for all the peaks as well as the conversion of tadalafil into diastereomers (6S, 12aR) in acidic conditions and into diastereomers (6R, 12aS) in alkaline conditions. At lower concentrations, the recoveries for all isomers ranged from 100.0 ± 15.0%, while the assay values for tadalafil were within 100.0 ± 2.0%. According to the validation outcome as per ICH guidelines, the proposed method is an accurate, precise, linear, and robust stereoselective method for simultaneous quantification.

Anthocyanin Extraction Method and Sample Preparation Affect Anthocyanin Yield of Strawberries

Anthocyanins are a group of pigments with antioxidant activities that are present naturally in plants. The role of the pigment in human health and its quantitative analysis has attracted a lot of attention globally. A well-known and accurate method of anthocyanin quantification is based on spectrophotometric methods. However, these methods are subject to interference from impurities and need to be optimized for different plant matrixes and extraction conditions. Two experiments were designed to study (1) the effect of plant preparation methods (eg, fresh, frozen, and freeze-dried puree) on anthocyanin yield and (2) the effect of five anthocyanin extraction methods on anthocyanin yield of freeze-dried strawberry puree. Sample preparation methods did not have any effect on anthocyanin yield. Freeze-dried samples were used for their stability (ease of use and flexibility) to compare extraction methods. The anthocyanin yield was affected by the extraction method. Two methods containing chloroform gave the highest anthocyanin yield. One method with methanol:water:HCl gave intermediate results, and the pH differential and the other method with methanol:water:HCl (80:20:1) gave the lowest anthocyanin yields. Processing time (incubation time) was lowest in the pH differential method; however, the haze produced in this method may interfere with the spectrophotometric assessment of anthocyanins.

Method Development and Validation of Gas Chromatography Methods using Nitrogen Phosphorus Detector (GC-NPD) and Isotope Dilution Triple Quadrupole Mass Spectrometry (GC-IDMS/MS) for the Determination of Chlorpyrifos in Mango

Pesticides residue determination in fruits like chlorpyrifos is important due to its health effects on humans. Thus, there is a need for sensitive and accurate methods for better quantification. In this study, the performance of two gas chromatographic detection methods, nitrogen phosphorus detector (GC-NPD) and isotope dilution triple quadrupole mass spectrometer (GC-IDMS/MS), for the determination of chlorpyrifos in mango was evaluated to be used for the development of candidate reference material. Other than liquid-liquid extraction (LLE), Quick, Easy, Cheap, Efficient, Rugged and Safe (QuEChERS) sample extraction and dispersive solid phase extraction (dSPE) clean-up procedures were optimized to extract chlorpyrifos from the sample matrix. Comprehensive method validations were performed in GC-NPD and GC-IDMS/MS with HP-5 capillary column to establish linearity, the limit of detection (LOD), the limit of quantification (LOQ), accuracy, repeatability, and intermediate precision using freeze-dried mango samples. The LOD and LOQ were 2.35 and 3.47 µg kg-1 for GC-NPD, and 1.23 and 1.85 µg kg-1 for GC-IDMS/MS, respectively. Matrix-matched calibration curves showed excellent linearity of r2=0.999 for both GC-NPD and GC-IDMS/MS protocols. Acceptable repeatability was obtained for three spike concentrations of 10, 30, and 60 µg kg-1 (n=10) and intermediate precision for 0.5-, 1- and 3-months in the same three-spike concentrations (n=3) expressed as relative standard deviation (RSD) ranging from 0.34-5.61 % for GC-NPD and 0.46-4.22 % for GC-IDMS/MS. Satisfactory mean recoveries (n=10) were achieved: 88.48-109.81% in GC-NPD and 97.61-108.40% in GC-IDMS/MS. GC-IDMS/MS and GC-NPD methods were fit for the purpose of quantifying chlorpyrifos in Philippine mango accurately with the use of isotope dilution technique and traceability to international standards by the application of gravimetric sample preparation.

Evaluation of the HemoCue® WBC System as a Point of Care Diagnostic Tool for White Blood Cell Quantification in Pinnipeds

Point of care (POC) hematology testing can be valuable in triage and field settings. We assessed the accuracy between the HemoCue® WBC system and two comparative analyzers, as well as the precision of the HemoCue® WBC system in five different pinniped species: Zalophus californianus, Arctocephalus townsendi, Callorhinus urcinus, Phoca vitulina, and Mirounga angustirostris for white blood cell (WBC) quantification. In Zalophus (n = 164; 106 from U.S. Navy Marine Mammal Program (Navy); 58 from The Marine Mammal Center (TMMC)), the HemoCue® was compared to two hematology analyzers, Sysmex Xe-5000 and Vet ABC Plus. In Phoca (n = 50; TMMC), Callorhinus (n = 29; TMMC), Arctocephalus (n = 17; TMMC), and Mirounga (n = 67; TMMC), the HemoCue® was compared to Vet ABC Plus only. Bland–Altman and Passing–Bablok agreement of HemoCue® with Sysmex Xe-5000 and Vet ABC Plus analyzers were good for Zalophus, Arctocephalus, Phoca, and Mirounga but marginal with Callorhinus; bias = 0.56 × 109/L (Zalophus; Navy), −2.13 × 109/L (Zalophus; TMMC), −1.59 × 109/L (Arctocephalus), −2.48 × 109/L 0.917 (Phoca), −0.01 × 109/L (Mirounga), and −6.05 × 109/L (Callorhinus). The coefficient of variation from triplicate runs of samples were within acceptable limits for all species (2.50% ± 1.63 (Zalophus; TMMC), 3.09% ± 2.14 (Arctocephalus), 2.47% ± 1.35 (Callorhinus), 2.88% ± 1.75 (Phoca), and 3.44% ± 2.53 (Mirounga)), respectively. The presence of nucleated red blood cells (nRBC; 1–37 nRBC/100 WBC) did not significantly interfere with WBC counts in Zalophus, Callorhinus, and Phoca at the population level, but their presence should be evaluated at the individual level. The HemoCue® provides an accurate method for WBC quantification with WBC counts up to 30 × 109/L (upper limit of linearity of the analyzer) in Zalophus, Arctocephalus, Phoca, and Mirounga, but is less accurate in Callorhinus, and showed good precision in all species.

Development and Validation of RP-HPLC Method for Estimation of Lacosamide in Bulk and Its Pharmaceutical Formulation

Objective - The present paper describes a simple, accurate, and precise reversed-phase high-performance liquid chromatography (HPLC) method for rapid and simultaneous quantification of Lacosamide in bulk and pharmaceutical dosage form. Methods: The chromatographic separation was achieved on C18 (250mmx4.6mm, 5µ id). Mobile phase contained a mixture of methanol: phosphate buffer in the ratio of 35:65(PH 3) v/v, flow rate 1.0 ml/min and ultraviolet detection at 210 nm. Results: The retention time Locosamide was 0.21 min. The proposed method shows a good linearity in the concentration range of 02 to 12μg/ml for locosamide under optimized conditions. Precision and recovery study results are in between 95.37 to 97.95 % w/w.. In the entire robustness conditions, percentage relative standard deviation is. Conclusion: This method is validated for different analytical performance parameters like linearity. Precision, accuracy, limit of detection, limit of quantification, robustness, and pharmacokinetic study were determined according to the International Conference of Harmonization (ICH) Q2B guidelines. All the parameters of validation were found in the acceptance range of ICH guidelines.

Quantification of lithium dendrite and solid electrolyte interphase (SEI) in lithium-ion batteries

The unexpected plating of lithium on the anode is a common issue for lithium-ion batteries (LIBs), which shortens the cycle life by consuming active Li+ and causes the severe safety hazard due to the formation of Li dendrites. However, the quantitive detection of deposited metallic Li is hindered by the lack of feasible and precise method. Herein, we utilized the differential scanning calorimetry (DSC) technique to quantify the deposited metallic Li by measuring the heat of metallic Li melting. This technique provides an accurate quantification method for the “dead Li” and solid electrolyte interphase (SEI) evolution of the graphite electrode. The measured minimum amount of metallic Li reaches 0.19 μg (0.72 μAh). We found that SEI growth consumes more capacity in the period of the nucleation of lithium dendrites than that in the period of the growth of lithium dendrites. The formation of SEI is the main cause for the capacity loss during the Li plating/stripping on both graphite and copper electrodes. Only 1.4–1.6% of capacity loss of the commercial pouch cell aged at low temperature is consumed to form “dead Li”.

Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification.

Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis.

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4°C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30months demonstrating they are suitable for method development.

Novel long non-coding RNAs of relevance for ulcerative colitis pathogenesis.

Background and aimsThe study aimed to identify yet unknown and uncharacterized long non-coding RNAs (lncRNAs) in treatment-naïve ulcerative colitis (UC), and to define their possible roles in UC pathogenesis. For that purpose, accurate quantification methods for lncRNA transcript detection, multiple and “stringent” strategies were applied. New insights in the regulation of functional genes and pathways of relevance for UC through expression of lncRNAs are expected.MethodsThe study was based on sequencing data derived from a data set consisting of treatment-naïve UC patients (n = 14) and control subjects (n = 16). Two complementary aligners were used to identify lncRNAs. Several different steps were used to validate differential expression including plotting the reads over the annotation for manual inspection. To help determine potential lncRNA involvement in biological processes, KEGG pathway enrichment was done on protein-coding genes which co-expressed with the lncRNAs.ResultsA total of 99 lncRNAs were identified in UC. The lncRNAs which were not previously characterized (n = 15) in UC or other autoimmune diseases were selected for down-stream analysis. In total, 602 protein-coding genes correlated with the uncharacterized lncRNAs. KEGG pathway enrichment analysis revealed involvement of lncRNAs in two significantly enriched pathways, lipid and atherosclerosis, and T-cell receptor signaling.ConclusionThis study identified a set of 15 yet uncharacterized lncRNAs which may be of importance for UC pathogenesis. These lncRNAs may serve as potential diagnostic biomarkers and might be of use for the development of UC treatment strategies in the future.

Quantifying the electrochemical active site density of precious metal-free catalysts in situ in fuel cells

Advances in the development of precious-group metal-free (PGM-free) catalysts for the oxygen reduction reaction (ORR) in fuel cell cathodes have produced active catalysts that reduce the performance gap to the incumbent Pt-based materials. However, utilization of state-of-the-art PGM-free catalysts for commercial applications is currently impeded by their relatively low durability. Methods designed to study catalyst degradation in the operation of fuel cells are therefore critical for understanding durability issues and, ultimately, their solutions. Here we report the use of Fourier-transform alternating current voltammetry as an electrochemical method for accurate quantification of the electrochemically active site density of PGM-free cathode catalysts, and to follow their degradation in situ during the operation of polymer electrolyte fuel cells. Using this method, we were able to quantify the electrochemical active site density, which will enable the elucidation of degradation mechanisms of PGM-free ORR catalysts in situ in fuel cells.

Design-based stereology in alpaca skin (Vicugna pacos): Impacts on fiber production

Alpacas are reared mainly for fiber extraction, which is a highly valued product in the textile industry. The quality of fiber has close relation to the skin. For this reason, this work aims to evaluate the skin of different body regions; scapular, costal and lateral femoral in Huacaya and Suri alpacas by means of stereology, an accurate quantification method. The total volume of the skin, derma, follicular groups and sebaceous and sweat glands was estimated. Total volume of skin (Vref) and total volume of follicular groups (VGF) were different for body regions. Femoral region showed higher values for VGF. Colour and breed were also different for Vref and total volume of derma (VD). Comparing the two breeds the femoral region presented higher fiber production. Dark animals had more derma and it was reported close relationship between total skin volume and their fractions volumes: derma, follicular groups and sweat glands.

Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets

Apple scar skin viroid (ASSVd), of the genus Apscaviroid, causes serious pome fruit diseases, such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. This study aimed at establishing a sensitive and accurate method for quantification of ASSVd in apple leaves and plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity was analyzed using other apple viruses, and the negative amplification of the cross-reaction assay demonstrated the high specificity of RT-ddPCR. The detection limit of ASSVd by RT-ddPCR was 1.75 × 102 copies/μL (0.14 concentration), and the sensitivity was ten-fold higher than that of RT-qPCR. Similarly, positive detection in apple plantlet samples by RT-ddPCR was higher than that by RT-qPCR. The RT-ddPCR assay represents a promising alternative for accurate quantitative detection and diagnosis of ASSVd infection in ASSVd-free certification programs.

Application of individual response factors for accurate quantitation of alkylated PAH homologs in complex environmental samples using gas chromatography/triple quadrupole mass spectrometry (GC–MS/MS)

Alkylated polycyclic aromatic hydrocarbons (alkyl PAHs) are abundant in the environment. They can be more toxic and persistent than their non-alkylated parent PAH compounds, and thus alkyl PAHs can pose an increased threat to environmental quality. However, the US EPA has not listed alkyl PAHs as priority pollutants, which is primarily due to a lack of reliable and accurate analytical method for routine identification and quantification of alkyl PAHs in complex environmental samples. A multiple reaction monitoring (MRM) method in GC–MS/MS has been proposed for more accurate quantification of target alkyl PAHs in complex sample matrices. However, the lack of certified analytical standards for alkyl PAH isomers and the historic use of relative response factors (RFs) of parent PAH to quantify its alkylated PAH isomers can compromise the quantitative results obtained from MRM analyses. In this study, we used commercially available parent and alkyl PAH standards to develop and optimize both SIM and MRM analytical methods. The optimized methods were used to determine and compare RFs of alkyl PAHs to their respective parent PAHs in both SIM and MRM modes. The results showed that the conventional approach of quantifying alkyl PAHs by using the RFs of corresponding parent PAHs underestimates alkyl PAHs by 1.2–6.4 (2.2 ± 1.2) and 1.2–6.7 (3.4 ± 2.0) times, respectively, for SIM and MRM analyses. The results further demonstrated that the use of RFs of parent PAHs to quantify their alkylated homologs affects MRM-based analysis more than SIM-based analysis, underestimating the alkyl PAHs in MRM mode to as low as only 15% of the actual concentrations obtained by using individual RFs for each alkylated PAH homologs. The results showed that a GC–MS/MS in MRM mode, with baseline separation and higher selectivity, is a powerful analytical tool for the analysis of alkyl PAHs in complex matrices, however, the use of at least one of the individual RFs for target PAH analyte clusters is critical for accurate quantitative MRM analysis. An accurate quantification of alkyl PAHs is important for determining their transport, fate, and environmental health impacts for next generation of oil spill studies as well as for updating existing EPA priority pollutant list by including alkyl PAHs in the list.

Rapid and concise quantification of mycelial growth by microscopic image intensity model and application to mass cultivation of fungi

The microbial food fermentation industry requires real-time monitoring and accurate quantification of cells. However, filamentous fungi are difficult to quantify as they have complex cell types such as pellet, spores, and dispersed hyphae. In this study, numerous data of microscopic image intensity (MII) were used to develop a simple and accurate quantification method of Cordyceps mycelium. The dry cell weight (DCW) of the sample collected during the fermentation was measured. In addition, the intensity values were obtained through the ImageJ program after converting the microscopic images. The prediction model obtained by analyzing the correlation between MII and DCW was evaluated through a simple linear regression method and found to be statistically significant (R2 = 0.941, p < 0.001). In addition, validation with randomly selected samples showed significant accuracy, thus, this model is expected to be used as a valuable tool for predicting and quantifying fungal growth in various industries.