Identification of Stress-Responsive Metabolites in Plants Using an Untargeted Metabolomics Approach.

Stress can affect different groups of plant metabolites and multiple signaling pathways. Untargeted metabolomics enables the collection of whole-spectrum data for the entire metabolite content present in plant tissues at that point in time. We present a thorough approach for large-scale, untargeted metabolomics of plant tissues using reverse-phase liquid chromatography connected to high-resolution mass spectrometry (LC-MS) of dilute methanolic extract. MZmine is a specialized computer software that automates the alignment and baseline modification of all derived mass peaks across all samples, resulting in precise information on the relative abundance of hundreds of metabolites reflected by thousands of mass signals. Further processing with statistic and bioinformatic techniques will provide a comprehensive perspective of the variations and connections among groups of samples.

Metformin drugs under simulated gastric conditions can generate high nitrite-dependent levels of N-nitrosodimethylamine.

N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), group 2A carcinogens, were detected in finished drug products, including metformin, ranitidine, sartans and other drugs which caused multiple recalls in the USA and Europe. Important studies also reported the formation of NDMA when ranitidine and nitrite were added to simulated gastric fluid. Our objective was to screen finished drug products from Europe and USA for nitrosamine impurities and investigate the formation of NDMA in metformin finished drug products when added to simulated gastric fluid. One dosage unit of 30 different commercially available drugs, including metformin, sartans, and ranitidine were tested for NDMA, NDEA, and dimethylformamide (DMF) impurities, using a liquid chromatography-mass spectrometry (LC-MS) method. Then, 6 metformin finished drug products were tested in stomach conditions for 2 h at 37 °C in a 100 mL solution with a pH of 2.5 and different nitrite concentrations (40, 10, 1, 0.1 mM) and tested for NDMA, and DMF using LC-MS. We measured NDMA, NDEA, and DMF in 30 finished drug products. NDMA and DMF were quantified for metformin drug products in simulated gastric fluid with different nitrite concentrations. None of the 30 drugs showed concerning levels of NDMA, NDEA, or DMF when tested as single tablets. However, when metformin tablets are added to simulated gastric fluid solutions with high nitrite concentrations (40 mM and 10 mM), NDMA can reach amounts of thousands of nanograms per tablet. At the closest concentration to physiologic conditions we used, 1 mM, NDMA is still present in the hundreds of nanograms in some metformin products. In this in vitro study, nitrite concentration had a very important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs. 10 mM, 40 mM nitrite solutions generated NDMA amounts exceeding by more than a hundred times the acceptable daily intake set by the FDA of 96 nanograms. These findings suggest that metformin can react with nitrite in gastric-like conditions and generate NDMA. Thus, patients taking metformin could be exposed to NDMA when high nitrite levels are present in their stomach, and we recommend including a statement within the Patient Package Inserts/Instructions for use.

Investigation of variability in the matrix effect on stable isotope-labeled internal standards in liquid chromatography-tandem mass spectrometry analysis of 25 pesticides in vegetables.

The matrix effects (ME) in simultaneous analysis of pesticide residue using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were evaluated by comparing the slopes of matrix-matched and reagent-only calibrations of four types of vegetable samples. Both the sampling and measurement variances of the ME were also determined using one-way analysis of variance. Substantial ion suppression (ME<-20%) was observed in komatsuna, spinach, and tomato when a modified Japanese official method was implemented. The ME magnitude varied significantly due to sample variability for some pesticides, but it varied by no more than 4% as a result of analytical procedure variance. This study also showed that the addition of stable isotope-labeled internal standards at low concentrations improved the recovery of pesticides from samples at various residue levels. The findings of this study highlight the importance and practical application of internal standards and the matrix-matched calibration method in residue analysis using LC-MS/MS.

Qualitative and Quantitative Analysis of Three-Dimensional Fluorescence Spectra by Improved Parallel Factor Analysis with Internal Standard Sample Embedding.

The strategy of parallel factor analysis, combined with the internal standard method, has been increasingly applied to the qualitative and quantitative analysis of three-dimensional fluorescence spectra of unknown mixed fluorophores. Nevertheless, the disparity in the number of fluorophores included in the internal standard sample set and the number included in test samples may impact the qualitative and quantitative outcomes of parallel factor analysis. In this work, we systematically established the framework of the parallel factor analysis with internal standard sample embedding (ISSE-PARAFAC) strategy. We applied this framework to six datasets representing two scenarios and a real dataset and conducted a detailed discussion on the effects of the disparity between the number of fluorophores in the internal standard sample set and the number in the test set on both qualitative and quantitative results. Additionally, we introduced an enhancement to PARAFAC by aggregating fluorophores with similar emission wavelengths, corresponding to the peaks of emission loadings (spectra) obtained from PARAFAC, as a single fluorophore. This aggregation aimed to mitigate the strong correlation between similar fluorophores. The results imply that the presence of irrelevant fluorophores in the internal standard sample set, whether increased or decreased, does not significantly affect the qualitative and quantitative analysis of target fluorophores in the test set. Moreover, we demonstrated that the improved parallel factor analysis with internal standard sample embedding not only fully decomposes the uncorrelated mixed fluorophores for qualitative analysis but also allows the established linear concentration model for fluorescent components to predict the corresponding fluorophore concentration of test samples, enabling quantitative analysis at the ppm level (mg/L).

Synergistic enhancement mediated sensitive SERS-based immunoassay of PSA using versatile PDMS@AgNPs@ZIF-67 biomimetic substrates.

Among various biomimetic polymer materials, polydimethylsiloxane (PDMS) stands out as an ideal matrix for surface-enhanced Raman scattering (SERS) due to its unique intrinsic Raman signal and tenacity. In order to realize the precise detection of prostate-specific antigen (PSA), we proposed a sandwich-type SERS-active immunostructure composed of PDMS@silver nanoparticles (Ag NPs)@ZIF-67 biomimetic film as the immunosubstrate and gold nanorods (Au NRs) as immunoprobes. Due to the synergistic effect of electromagnetic enhancement facilitated by biomimetic surfaces and chemical enhancement achieved by ZIF-67, this structure enabled an ultrasensitive and selective detection of PSA across a broad range from 10-3 to 10-9 mg/mL. The achieved limit of detection was as low as 3.0 × 10-10 mg/mL. Particularly, the intrinsic Raman signal of PDMS matrix at 2905 cm-1 was employed as a potential internal standard (IS) in the detection, achieving a high coefficient of determination (R2) value of 0.996. This multifunctional SERS substrate-mediated immunoassay holds vast potential for early diagnosis of prostate cancer, offering promising prospects for clinical applications.

Development of a rapid quantitative method of analysis of carbohydrate fatty acid ester reaction mixtures using 1H qNMR.

Two new analytical methods, applying absolute 1H qNMR, were developed to monitor product yield and quantify unreacted carbohydrate and fatty acid reactants, in the synthesis of carbohydrate fatty acid esters (CFAE). These methods provide a mass balance of the crude reaction mixtures and diversify the analytical screening and quantitation approaches available within the synthesis of these molecules. Both methods were validated for the model reaction of methyl α-d-glucopyranoside (MAG) and lauric acid (LA) to form the mono ester product, methyl 6-O-dodecanoyl-α-d-glucopyranoside. Analysis in CD3OD by 1H qNMR, with fumaric acid (FA) as an internal standard (IS), allowed monitoring of all reaction components. Alternatively, using CDCl3 and (E)-stilbene as IS enabled the analysis of CFAE and fatty acid. Parameters calculated for method validation included specificity and selectivity, linearity, accuracy, intermediate precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. Both methods provided excellent linearity with R2 > 0.997. The accuracy, precision, and robustness of the method in CD3OD was <2 % uncertainty making it suitable for complete reaction analysis. The method completed in CDCl3 resulted in accuracy, intermediate precision, and robustness of <5 %, except for accuracy in the lowest levels of concentration (>5 %). For all related analytes in the CD3OD and CDCl3 methods, the LOD and LOQ were determined to ensure applicability for the intended use in the assessment of reaction crude composition. Finally, the system suitability was assessed in a scaled lipase catalysed CFAE synthetic reaction. The determined qNMR product yields were verified against isolated purified product yields with <5 % uncertainty.

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

Three steps towards comparability and standardization among molecular methods for characterizing insect communities.

Molecular methods are currently some of the best-suited technologies for implementation in insect monitoring. However, the field is developing rapidly and lacks agreement on methodology or community standards. To apply DNA-based methods in large-scale monitoring, and to gain insight across commensurate data, we need easy-to-implement standards that improve data comparability. Here, we provide three recommendations for how to improve and harmonize efforts in biodiversity assessment and monitoring via metabarcoding: (i) we should adopt the use of synthetic spike-ins, which will act as positive controls and internal standards; (ii) we should consider using several markers through a multiplex polymerase chain reaction (PCR) approach; and (iii) we should commit to the publication and transparency of all protocol-associated metadata in a standardized fashion. For (i), we provide a ready-to-use recipe for synthetic cytochrome c oxidase spike-ins, which enable between-sample comparisons. For (ii), we propose two gene regions for the implementation of multiplex PCR approaches, thereby achieving a more comprehensive community description. For (iii), we offer guidelines for transparent and unified reporting of field, wet-laboratory and dry-laboratory procedures, as a key to making comparisons between studies. Together, we feel that these three advances will result in joint quality and calibration standards rather than the current laboratory-specific proof of concepts. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

First Synthesis of Ergotamine-13CD3 and Ergotaminine-13CD3 from Unlabeled Ergotamine.

Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.

[Determination of perchlorate and chlorate in drinks by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a method for determination of perchlorate and chlorate in drinks by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) based on isotopic internal standard method. METHODS: The perchlorate and chlorate residue in liquid drinks were extracted with methanol, in solid drinks with acetic acid solution, then centrifuged. The supernatant was cleaned-up with PSA/C18 cleanup tube. The separation of perchlorate and chlorate was carried out on a Acquity CSH fluorophenyl column(100 mm×2.1mm, 1.7 µm) and the detection was performed with tandem mass spectrometry with internal standard method for quantification. RESULTS: The peak area ratio of perchlorate and chlorate had a good linear relationship with their mass concentration within their respective linear ranges, with correlation coefficients(r) greater than 0.999. The limits of detection of perchlorate and chlorate were 0.2and 1 µg/L respectively and the limits of quantification were 0.5 and 3 µg/L respectively. The mean recoveries of two compounds were from 84.0% to 105.5% with relative standard deviations from 4.2% to 17.0% and 82.7% to 112.1% with relative standard deviations from 5.5% to 18.4%(n=6), respectively. The perchlorates in 11 kinds of beverage samples were 0.53-4.12 µg/L, chlorates were 3.27-61.86 µg/L. CONCLUSION: This method is simple, sensitive, accurate and reliable, which is suitable for the determination of perchlorate and chlorate in drinks.

Quantitative Analysis of Trace Analytes with Highly Sensitive SERS Tags on Hydrophobic Interface.

Surface-enhanced Raman scattering (SERS) presents a promising avenue for trace matter detection by using plasmonic nanostructures. To tackle the challenges of quantitatively analyzing trace substances in SERS, such as poor enrichment efficiency and signal reproducibility, this study proposes a novel approach using Au@internal standard@Au nanospheres (Au@IS@Au NSs) for realizing the high sensitivity and stability in SERS substrates. To verify the feasibility and stability of the SERS performances, the SERS substrates have exhibited exceptional sensitivity for detecting methyl blue molecules in aqueous solutions within the concentration range from 10-4 M to 10-13 M. Additionally, this strategy also provides a feasible way of quantitative detection of antibiotic in the range of 10-4 M to 10-10 M. Trace antibiotic residue on the surface of shrimp in aquaculture waters was successfully conducted, achieving a remarkably low detection limit of 10-9 M. The innovative approach has great potential for the rapid and quantitative detection of trace substances, which marks a noteworthy step forward in environmental detection and analytical methods by SERS.

Dispersive Solid-Phase Extraction and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry-A Rapid and Accurate Method for Detecting 10 Macrolide Residues in Aquatic Products.

The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin (KIT), josamycin (JOS), spiramycin (SPI), tilmicosin (TIL), tylosin (TYL), and roxithromycin (ROX), was determined using solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The residues were extracted with 1% ammonia acetonitrile solution and purified by neutral alumina adsorption. Chromatographic separation was completed on an ACQUITY UPLC BEH C18 column with acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and mass spectrometry detection was performed by multiple reaction monitoring scanning with the positive mode in an electrospray ion source (ESI+). Five isotopically labeled compounds were used as internal standards for quality control purposes. The findings indicated that across the mass concentration span of 1.0-100 µg/L, there was a strong linear correlation (R2 > 0.99) between the concentration and instrumental response for the 10 MALs. The limit of detection of UPLC-MS/MS was 0.25-0.50 µg/kg, and the limit of quantitation was 0.5-1.0 µg/kg. The added recovery of blank matrix samples at standard gradient levels (1.0, 5.0, and 50.0 µg/kg) was 83.1-116.6%, and the intra-day precision and inter-day precisions were 3.7 and 13.8%, respectively. The method is simple and fast, with high accuracy and good repeatability, in line with the requirements for accurate qualitative and quantitative analysis of the residues for 10 MALs in aquatic products.

Simultaneous determination of 28 illegal drugs in sewage by high throughput online SPE-ISTD-UHPLC-MS/MS.

This study developed an online solid-phase extraction ultra-high performance liquid chromatography-tandem mass spectrometry (Online-SPE-UHPLC-MS/MS) method for the analysis of 28 illegal drugs in sewage. To achieve this, 28 isotope internal standards (ISTDs) were added to 3 mL sewage samples, the pH was adjusted to 7-8 using hydrochloric acid or 20% ammonia water, followed by centrifugation, filtration, and analysis using UHPLC-MS/MS. The results indicated an excellent linearity of 1-300 ng L-1, and cotinine in the concentration range of 20-6000 ng L-1, linear correlation coefficient R2 > 0.995, with the limit of detection (LOD) of 0.01-6 ng L-1, and a limit of quantification (LOQ) of 0.1-20 ng L-1. The addition of three concentrates of low (2 ng L-1/40 ng L-1), medium (20 ng L-1/400 ng L-1), and high concentration (200 ng L-1/4000 ng L-1) demonstrated the matrix effect of the target compound between ± 22.0%. The extraction recovery was 70.0-119.4%, and a percent accuracy of 75.7-118.1%. Similarly, the intra- and inter-day precisions were 1.8-20.0% and 1.5-18.9%, respectively. The results cemented the sensitivity, accuracy, reliability, strong specificity, and reproducibility, which can be used to screen 28 illegal drugs in sewage for trace analysis.

Reliable quantitative detection of uric acid in urine by surface-enhanced Raman spectroscopy with endogenous internal standard.

Abnormal levels of uric acid (UA) in urine serve as warning signs for gout and metabolic cardiovascular diseases, necessitating the monitoring of UA levels for early prevention. However, the current analytical methods employed suffer from limitations in terms of inadequate suitability for home-based applications and the requirement of non-invasive procedures. In this approach, creatinine, a metabolite with a constant excretion rate, was incorporated as an endogenous internal standard (e-IS) for calibration, presenting a rapid, pretreatment-free, and accurate strategy for quantitative determination of UA concentrations. By utilizing urine creatinine as an internal reference value to calibrate the signal fluctuation of surface-enhanced Raman spectroscopy (SERS) of UA, the quantitative accuracy can be significantly improved without the need for an external internal standard. Due to the influence of the medium, UA, which carries a negative charge, is selectively adsorbed by Au@Ag nanoparticles functionalized with hexadecyltrimethylammonium chloride (CTAC) in this system. Furthermore, a highly convenient detection method was developed, which eliminates the need for pre-processing and minimizes matrix interference by simple dilution. The method was applied to the urine detection of different volunteers, and the results were highly consistent with those obtained using the UA colorimetric kit (UACK). The detection time of SERS was only 30 s, which is 50 times faster than UACK. This quantitative strategy of using urinary creatinine as an internal standard to correct the SERS intensity of uric acid is also expected to be extended to the quantitative detection needs of other biomarkers in urine.

Ratiometric SERS quantification of SO2 vapor based on Au@Ag-Au with Raman reporter as internal standard.

Practical gas sensing application requires sensors to quantify target analytes with high sensitivity and reproducibility. However, conventional surface enhanced Raman scattering (SERS) sensor lacks reproducibility and quantification arising from variations of "hot spot" distribution and measurement conditions. Here, a ratio-dependent SERS sensor was developed for quantitative label-free gas sensing. Au@Ag-Au nanoparticles (NPs) were filtered onto anodic aluminum oxide (AAO) forming Au@Ag-Au@AAO SERS substrate. 4-MBA was encapsulated in the gap of Au@Ag-Au and served as the internal standard (IS) to calibrate SERS signal fluctuation for improved quantification ability. Combined with headspace sampling method, SO2 residue in traditional Chinese medicine (TCM) can be extracted and captured on the immediate vicinity of Au@Ag-Au surface. The intensity ratio I613 cm-1/I1078 cm-1 showed excellent linearity within the range of 0.5 mg/kg-500 mg/kg, demonstrating superior quantification performance for SO2 detection. Signals for concentration as low as 0.05 mg/kg of SO2 could be effectively collected, much lower than the strictest limit 10 mg/kg in Chinese Pharmacopoeia. Combined with a handheld Raman spectrometer, handy and quantitative TCM quality evaluation in aspect of SO2 residue was realized. This ratiometric SERS sensor functioned well in rapid on-site SO2 quantification, exhibiting excellent sensitivity and simple operability.

3D hierarchic interfacial assembly of Au nanocage@Au along with IS-AgMNPs for simultaneous, ultrasensitive, reliable, and quantitative SERS detection of colorectal cancer related miRNAs.

Simultaneous, reliable, and ultra-sensitive analysis of promising miRNA biomarkers of colorectal cancer (CRC) in serum is critical for early diagnosis and prognosis of CRC. In this work, we proposed a novel 3D hierarchic assembly clusters-based SERS strategy with dual enrichment and enhancement designed for the ultrasensitive and quantitative analysis of two upregulated CRC-related miRNAs (miR-21 and miR-31). The biosensor contains the following: (1) SERS probe, Au nanocage@Au nanoparticles (AuNC@Au NPs) labeled with Raman reporters (RaRs). (2) magnetic capture unit, Ag-coated Fe3O4 magnetic nanoparticles (AgMNPs) modified with internal standard (IS). (3) signal amplify probes (SA probes) for the formation of hierarchic assembly clusters. Based on this sensing strategy, the intensity ratio IRaRs/IIS with Lg miRNAs presents a wide linear range (10 aM-100 pM) with a limit of detection of 3.46 aM for miR-21, 6.49 aM for miR-31, respectively. Moreover, the biosensor shows good specificity and anti-interference ability, and the reliability and repeatability of the strategy were then verified by practical detection of clinical serum. Finally, the biosensor can distinguish CRC cancer subjects from normal ones and guide the distinct tumor, lymph node, and metastasis (TNM) stages. Overall, benefiting from the face-to-face coupling of hierarchic assembly clusters, rapid magnetic enrichment and IS signal calibration of AgMNPs, the established biosensor achieves ultra-sensitive and simultaneous detection of dual miRNAs and opens potential avenues for prediction and staging of CRC.

Moxidectin is a candidate for use as an in vivo internal standard in pharmacokinetic studies, as demonstrated with use in simultaneous tissue cage and ultrafiltration fluid collection.

In vivo ultrafiltration has been used in veterinary pharmacokinetics since the early 2000's as an improvement on the tissue cage model which enables sampling of fluids from extra-circulatory compartments. Variability in analyte recovery from ultrafiltration samples, due to membrane fouling or tissue inflammation, has been a concern for this technique. Internal standards may be used to scale or verify the unknown result, such as is common in analytical extractions and in vivo microdialysis. Eight merino sheep were implanted with subcutaneous tissue cages and 2 weeks prior to the initiation of the study the sheep were injected with 0.2 mg/kg moxidectin subcutaneously. On the day of the study ultrafiltration probes were inserted subcutaneously. At time zero 4 mg/kg of carprofen was injected intravenously. Plasma, tissue cage, and ultrafiltration samples were taken 30 min before and 0.5, 1, 2, 3, 4, 5, 7, 24, 36, 48, 72 h after dosing. Carprofen and moxidectin concentrations were measured by LC-MS/MS. Pharmacokinetic parameters were estimated using Monolix for both the carprofen concentrations and the moxidectin corrected carprofen concentrations. The ultrafiltration probes failed to consistently produce enough sample volume to analyse. Moxidectin concentrations in the plasma and tissue cage fluid were stable throughout the 72 h sampling window. Moxidectin proved to be suitable as an in vivo internal standard for pharmacokinetic research using, tissue cages, plasma sampling and ultrafiltration probes, but the application of ultrafiltration techniques requires refinement.

Realization of qualitative to semi-quantitative trace detection via SERS-ICA based on internal standard method.

Surface-enhanced Raman spectroscopy (SERS) can quickly identify molecular fingerprints and has been widely used in the field of rapid detection. However, the non-uniformity inherent in SERS substrate signals, coupled with the finite nature of the detection object, significantly hampers the advancement of SERS. Nowadays, the existing mature immunochromatographic assay (ICA) method is usually combined with SERS technology to address the defects of SERS detection. Nevertheless, the porous structure of the strip will also affect the signal uniformity during detection. Obviously, a method using SERS-ICA is needed to effectively solve signal fluctuations, improve detection accuracy, and has certain versatility. This paper introduces an internal standard method combining deep learning to predict and process Raman data. Based on the signal fluctuation of single-antigen SERS-ICA test strip, the double-antigen SERS-ICA test strip was constructed. The full spectrum Raman data of double-antigen SERS-ICA test strip was normalized by the sum of two characteristic peaks of internal standard molecules, and then processed by deep learning algorithm. The Relative Standard Deviation (RSD) of Raman data of bisphenol A was compared before and after internal standard normalization of double-antigen SERS-ICA test strip. The RSD processed by this method was increased by 3.8 times. After normalization, the prediction accuracy of Root Mean Square Error (RMSE) is improved by 2.66 times, and the prediction accuracy of R-square (R2) is increased from 0.961 to 0.994. The results showed that RMSE and R2 were used to comprehensively predict the collected data of double-antigen SERS-ICA test strip, which could effectively improve the prediction accuracy. The internal standard algorithm can effectively solve the challenges of uneven hot spots and poor signal reproducibility on the test strip to a certain extent, so as to improve the semi-quantitative accuracy.

Comparison of calibration strategies for accurate quantitation by isotope dilution mass spectrometry: a case study of ochratoxin A in flour.

Analysis of low-level organic contaminants in complex matrices is essential for monitoring global food safety. However, balancing sample throughput with complex experimental designs and/or sample clean-up to best reduce matrix effects is a constant challenge. Multiple strategies exist to mitigate these effects, with internal standard-based methods such as isotope dilution mass spectrometry (IDMS) being the most advantageous. Here, multiple internal calibration strategies were investigated for the quantification of ochratoxin A (OTA) in wheat samples by liquid chromatography-mass spectrometry (LC-MS). Internal standard-based quantitation methods such as single (ID1MS), double (ID2MS), and quintuple (ID5MS) isotope dilution mass spectrometry, as well as external standard calibration, were explored and compared. A certified reference material (CRM) of OTA in flour, MYCO-1, was used to evaluate the accuracy of each method. External calibration generated results 18-38% lower than the certified value for MYCO-1, largely due to matrix suppression effects. Concurrently, consistently lower OTA mass fractions were obtained for the wheat samples upon quantitation by external calibration as opposed to ID1MS, ID2MS, and ID5MS. All isotope dilution methods produced results that fell within the expected range for MYCO-1 (3.17-4.93 µg/kg), validating their accuracy. However, an average 6% decrease in the OTA mass fraction was observed from results obtained by ID1MS compared to those by ID2MS and ID5MS. Upon scrutiny, these differences were attributed to an isotopic enrichment bias in the isotopically labelled internal standard [13C6]-OTA that was used for ID1MS, the OTAL-1 CRM. The advantages and limitations of each isotopic method are illustrated.