A metagenomics-based workflow for the detection and genomic characterization of GBS in raw freshwater fish.

The unexpected foodborne outbreak in Singapore in 2015 has accentuated Group B Streptococcus (GBS, Streptococcus agalactiae) sequence type 283 as an emerging foodborne pathogen transmitted via the consumption of contaminated raw freshwater fish. Isolation-based workflows utilizing conventional microbiological and whole-genome sequencing methods are commonly used to support biosurveillance efforts critical for the control management of this emerging foodborne pathogen. However, these isolation-based workflows tend to have relatively long turnaround times that hamper a timely response for implementing risk mitigation. To address this gap, we have developed a metagenomics-based workflow for the simultaneous detection and genomic characterization of GBS in raw freshwater fish. Notably, our validation results showed that this metagenomics-based workflow could achieve comparable accuracy and potentially better detection limits while halving the turnaround time (from 2 weeks to 5 days) relative to an isolation-based workflow. The metagenomics-based workflow was also successfully adapted for use on a portable long-read nanopore sequencer, demonstrating its potential applicability for real-time point-of-need testing. Using GBS in freshwater fish as an example, this work represents a proof-of-concept study that supports the feasibility and validity of metagenomics as a rapid and accurate test methodology for the detection and genomic characterization of foodborne pathogens in complex food matrices. IMPORTANCE: The need for a rapid and accurate food microbiological testing method is apparent for a timely and effective foodborne outbreak response. This is particularly relevant for emerging foodborne pathogens such as Group B Streptococcus (GBS) whose associated food safety risk might be undercharacterized. By using GBS in raw freshwater fish as a case example, this study describes the development of a metagenomics-based workflow for rapid food microbiological safety testing and surveillance. This study can inform as a working model for various foodborne pathogens in other complex food matrices, paving the way for future methodological development of metagenomics for food microbiological safety testing.

An optimized multi-technique based analytical platform for identification, characterization and quantification of nanoplastics in water.

Nanoplastics (NPs) have been identified as an emerging concern for the environment and our food chains in recent years. Monitoring the concentration and size of nanoplastics is essential to assess the potential risks that nanoplastic particles may pose. In this study, we presented a multi-technique based analytical platform to identify, characterize and quantify nanoplastics in water samples through a combination of sample pre-concentration, asymmetric flow field-flow fractionation coupled with multi-angle light scattering (AF4-MALS) and pyrolysis-GC/MS (Py-GC/MS). Models for predicting NPs concentration and particle number in unknown samples were established and validated using NPs standards of known size and AF4-MALS response. Py-GC/MS was applied for further identification of polymer type and quantification of mass concentration. Filtration conditions for pre-concentration were optimized to ensure a high recovery rate with minimal effect on original particle size. The addition of 0.05% SDS prior to filtration, using controlled filtration procedures, effectively improved the recovery. Furthermore, this study demonstrates the application of the analytical platform for the characterization and quantification of different nanoparticles (e.g. spiked PMMA and PS NPs) in the size range 60 nm-350 nm with detection limits down to 0.01 ppm in water samples. The established analytical platform can fill an analytical gap by offering a solution for quantifying size-resolved mass concentrations of nanoplastics and providing comprehensive data on size distribution, particle number and mass quantification with high sensitivity for detection.

Determination and Characterization of Gold Nanoparticles in Liquor Using Asymmetric Flow Field-Flow Fractionation Hyphenated with Inductively Coupled Plasma Mass Spectrometry.

The EU has approved the usage of gold as a food additive (E175) and it has been applied in numerous foods for coloring and decoration purposes. Different from the general assumption that edible gold is mainly present in the form of flakes or external coating in foods, this work demonstrated that gold nanoparticles (Au NPs) can be released from gold flakes and extracted under optimized conditions. To support future risk assessment associated with the exposure of Au NPs to human health, an effective approach was established in this study for both size characterization and mass determination of Au NPs released in a commercial gold-containing liquor using Asymmetric Flow Field-flow Fractionation (AF4) hyphenated with Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Our results showed that no Au NPs were detected in the original liquor product and only after ultrasonication for several minutes did Au NPs occur in the ultrasound-treated liquor. Particularly, Au NPs released in the liquor can be well extracted after 100-fold enrichment of gold flakes and the subsequent ultrasonication for 25 min. Size characterization of Au NPs was conducted by AF4-ICP-MS under calibration with Au NP standards. The gold particle sizes detected ranged from 8.3-398.0 nm and the dominant size of the released Au NPs was around 123.7 nm in the processed liquor. The mass concentration of gold particles determined in the liquor sample with gold flakes concentrated and subsequently sonicated was 48.1 µg L-1 by pre-channel calibration and the overall detection recoveries ranged over 82-95%. For the comparison control samples without ultrasonication, there was no detection of Au NPs. The established method was demonstrated to be useful for monitoring Au NPs in liquor and is possibly applied to other similar foodstuffs.

The detection, characterization, and quantification of dominant degradation products of nisin A and Z in selected dairy products by liquid chromatography-high-resolution mass spectrometry technique.

Nisin, a bacteriocin produced through fermentation using bacterium Lactococcus lactis, has several commercial variants such as nisin A and nisin Z. Nisin serves as a natural preservative with antimicrobial properties in various food products, including dairy and beverages, for extending product shelf life. The efficacy and safety of nisin A as a bacteriocin has been well characterized. However, there is limited evidence regarding the efficacy, stability, and safety of nisin Z as a food preservative, as it has not undergone comprehensive regulatory reviews. In this work, we studied the stability of nisin A and Z in a selection of yogurt drinks and found nisin to be unstable, particularly in fruit-flavored yogurt drinks. Both nisin A and Z could experience significant degradation leading to the nisin parent ion peaks dropping below detectable level before the product's expiry date. Compared with nisin A, the formation of oxidized metabolite nisin Z+O appeared to be the predominant reaction for nisin Z. These findings highlight the need for further scientific research to understand the behavior of nisin Z under different application conditions, which is crucial for assessing the efficacy and safety of nisin Z under these conditions. One potential application of this knowledge is to optimize the formulation of yogurt-based drinks to stabilize nisin Z and sustain its biopreservative function throughout the product's shelf life. Additionally, the current study shows that for the testing of the presence of nisin A or nisin Z, it is imperative to cover both the parent and the main degradant(s) of nisin. This is especially true for nisin Z, for which the regulatory approval status may vary in different markets. As such, the confirmative identification of nisin Z and its key metabolites in commercial products would be essential.

Surveillance of veterinary drug residues in food commonly consumed in Singapore and assessment of dietary exposure.

Non-judicious and indiscriminate use of veterinary drugs in animal husbandry may result in accumulation of residues in animal tissues, and consequently in food for human consumption. The abuse of veterinary drugs presents a potential risk to consumer health, especially if the residue level is higher than the health-based guidance value (HBGV) such as the acceptable daily intake (ADI). Contamination of drug residues in food also promotes the emergence of antimicrobial resistance (AMR) which poses a serious threat to public health globally. There has been limited information on the occurrence and dietary exposure to veterinary drug residues in Singapore to date. In this study, the occurrence of four classes of veterinary drugs, namely beta-agonists, coccidiostats, fluoroquinolones and macrolides, were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in food widely consumed by Singapore residents. The magnitude of dietary exposure was assessed based on the consumption profile of Singapore population. Out of 216 food samples, 9.72 % were detected positive with veterinary drug residues, where majority of the positive samples were poultry and its derived products, followed by eggs and egg products. 7 veterinary drugs, specifically ciprofloxacin, enrofloxacin, clopidol, diclazuril, lasalocid, nicarbazin and tilmicosin, were detected in the samples, with clopidol and enrofloxacin being the most frequently detected drugs. Dietary exposure was evaluated using the estimated daily intake (EDI) of the detected drugs and benchmarked against the corresponding acceptable daily intake (ADI). All the %ADI values were far less than 100 in both the average and high consumer scenarios, indicating that the health risk associated with dietary exposure to these drugs in Singapore is low.

Identification and Characterization of Mercury Contamination in Vegetables and Herbs Cultivated on a Commercial Vertical Indoor Farming System with Light-Emitting Diode Lighting: Unveiling an Unusual Food Safety Risk of Some Improperly Manufactured High-Density Agricultural Production Systems.

Artificial grow lights, such as light-emitting diodes (LEDs) and fluorescent grow lights, are commonly used in modern day indoor farming, citing advantages in energy efficiency and a higher controlled environment. However, the use of LEDs poses a risk in mercury contaminations as a result of its production process, specifically LEDs with polyurethane encapsulates that were traditionally produced using mercury resins as a catalyst. A total of 10.0 ppm of mercury was detected in a curly kale sample harvested from an indoor hydroponic vegetable farm, exceeding Singapore Food Regulation's limit of 0.05 ppm. Vegetables, farming inputs, and surface swabs from the affected farm were analyzed using wet acid digestion followed by cold vapor atomic absorption spectroscopy analysis. The investigation found high concentrations of mercury in the LED encapsulant, and the encapsulant material was identified to be polyurethane by Fourier transform infrared spectroscopy and pyrolysis-gas chromatography-mass spectrometry analysis, indicating the source of mercury contamination to be the LED polyurethane encapsulant.

Occurrence and Dietary Exposure to Acrylamide from Foods Consumed within and outside Main Meals in Singapore.

This study investigated the influence of 'snackification' in Singaporean diets, leading to increased dietary acrylamide exposure. Acrylamide concentrations in commonly consumed foods within and outside the main meals were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). High acrylamide concentrations were detected in vegetables cooked at high temperatures (ranging from 0.5 to 478.4 µg/kg) and potato-based crackers and chips (ranging from 81.8 to 2095.8 µg/kg). The estimated total dietary exposure for the Singapore population was 0.165 µg/kg bw/day for general consumers and 0.392 µg/kg bw/day for high consumers (95th percentile). The acrylamide exposure from outside main meals was nearly equivalent to that from within the main meals. The calculated margins of exposure (MOE) were below 10,000, indicating potential human health concern. These findings highlight the need for industry practices and consumer advisories to reduce acrylamide exposure from foods consumed both within and outside main meals.

Occurrence and Dietary Exposure of 3-MCPD Esters and Glycidyl Esters in Domestically and Commercially Prepared Food in Singapore.

This study investigated the prevalence and occurrence of 3-monochloropropanediol esters (3-MCPDEs) and glycidyl esters (GEs) in domestically and commercially prepared food in Singapore and assessed the total dietary exposure for the Singaporean population. Minimal impact on the formation of 3-MCPDEs and GEs was observed from the domestic cooking methods commonly practiced in Singapore such as deep frying and stir frying. The estimated total dietary exposure to 3-MCPDEs for the Singaporean population (aged 15 to 92) was 0.982 µg/kg bw/day for general consumers and 2.212 µg/kg bw/day for high consumers (95th percentile), which accounted for 49.1% and 110.6% of the tolerable dietary intake (TDI) at 2 µg/kg bw/day by the European Food Safety Authority (EFSA). The calculated margins of exposure (MOE) for GEs based on the dietary exposure for general consumers at 0.882 µg/kg bw/day and 2.209 µg/kg bw/day for high consumers were below 10,000, indicating a potential health concern. Our study showed that the occurrence of 3-MCPDEs and GEs varied among vegetable oils, and domestic cooking methods did not significantly impact the levels of 3-MCPDEs and GEs in prepared food. The critical factor influencing the prevalence and occurrence of 3-MCPDEs and GEs was the choice of oil used for cooking, which absorbed into the cooked food. It is essential to encourage the food industry to continue its innovation on mitigation measures to control and reduce 3-MCPDEs and GEs in vegetable oil production. Consumers are advised to make informed choices on food consumption and cooking oil for food preparation to reduce their exposure to 3-MCPDEs and GEs.

Identification and quantification of common microplastics in table salts by a multi-technique-based analytical method.

Microplastics (MPs) are considered as contaminants of emerging concern to the environment and our food chains in recent years. In this study, we presented a multi-technique-based analytical method for detection of MPs through a combination of microscope-FTIR (µ-FTIR) with pyrolysis-GC/MS (Py-GC/MS) to achieve 3-dimensional (3D) information for the identification of polymer type, characterization of particle size and morphology, and quantification of MPs based on both particle number and mass of plastics. Plastics that are commonly used and disposed of, including polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), poly vinyl chloride (PVC), polyamide (PA), and poly(methyl methacrylate) (PMMA), were covered in this study. Sample extraction and separation procedures were optimized for these microplastics in table salts where good recoveries (> 75%) were achieved. To further enhance the detection sensitivity in simultaneous quantification of multiple polymers in a sample, a serial dissolution approach with different solvents was developed for the detection of all 7 types of plastics. The established sample preparation process and multi-technique-based analytical method were validated with polymer standards in table salts, resulting in satisfactory qualification and quantification for all samples tested. A retail survey of MPs in table salts was conducted with the developed analytical method, revealing that MPs were present in all commercially available table salts. The total number of MP particles varied from 20 to 125 particles/kg and the total mass contents of seven types of plastics ranged from 30 to 530 µg/kg in table salts.

Detection, Identification and Size Distribution of Silver Nanoparticles (AgNPs) in Milk and Migration Study for Breast Milk Storage Bags.

The engineered silver nanoparticles (AgNPs) have been widely used in various food contact materials (FCMs) based on their antibacterial properties. This widespread use of nanosilver has, however, increased the risk of exposure of AgNPs to human due to their migration from FCMs causing a potential hazard present in foods. Therefore, it is important to establish a reliable and practical method for the detection of AgNPs in food matrices to support risk assessment on AgNPs exposure. Taking the examples of milk and AgNPs-containing breast milk storage bags, this study established an approach for size characterization and quantification of AgNPs in milk and evaluated the relevant silver migration, based on enzymatic digestion and the analysis by asymmetric flow field-flow fractionation (AF4) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) and single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). No migration of AgNPs was found from breast milk storage bags under various simulated storage conditions as well as extreme scenarios. The suitability and reliability of this method were also validated by the determination of multiple parameters, including accuracy, repeatability, limit of detection (LOD), limit of quantification (LOQ), and recovery, for AF4-ICP-MS and SP-ICP-MS, respectively, with good and overall acceptable evaluation results obtained for all. The established and validated approach was demonstrated to be suitable for the characterization and quantitation of AgNPs in milk as well as the analysis of their migration from breast milk storage bags.

Separation and size characterization of highly polydisperse titanium dioxide nanoparticles (E171) in powdered beverages by using Asymmetric Flow Field-Flow Fractionation hyphenated with Multi-Angle Light Scattering and Inductively Coupled Plasma Mass Spectrometry.

The application of titanium dioxide as E171 food additive has become an issue of debate due to numerous reports that titanium dioxide nanoparticles (TiO2 NPs) inside the products may pose risks to human health. However, there is still a lack of an official standardized methodology for the detection and size characterization of TiO2 particles in foods containing E171. In this study, a method was presented for size characterization of TiO2 particles with various independent verifications in coffee creamer and instant drink powders, using Asymmetric Flow Field-Flow Fractionation hyphenated with Multi-Angle Light Scattering and Inductively Coupled Plasma Mass Spectrometry (AF4-MALS-ICP-MS). TiO2 particles from these products were well extracted, followed by their optimized AF4 separation using anionic surfactant Sodium Dodecyl Sulfate (SDS) (0.05%, pH 9) and mixed surfactant NovaChem (0.2%), respectively. Size determination of TiO2 NPs was conducted based on AF4 calibration with polystyrene nanospheres and verification with TiO2 NPs standard suspension of 100 nm under two different AF4 conditions. The TiO2 particle sizes detected ranged from 24.4 - 544.3 nm for coffee creamer and 27.7 - 574.3 nm for instant drink powders, with the TiO2 NPs detection recoveries of 75% and 92%, respectively. Hydrodynamic diameters from AF4 size calibration could be independently validated by the gyration diameters from online MALS measurement. The established approach was demonstrated to be reliable and pragmatic for size profiling of highly polydisperse TiO2 particles and thus useful for monitoring E171 in similar foodstuffs.

Isotopic dilution assay development of nisin A in cream cheese, mascarpone, processed cheese and ripened cheese by LC-MS/MS method.

The current food safety concern for food integrity demands the availability of an accurate, easy and reliable analytical tool for assay development of nisin A in cheese. To address this, we report the application of isotopically labelled peptide sequence MSTKDFNLDLVSVSKKDSGASP(R) (without thioether bridges) as internal standard for determination of nisin A in cream cheese, mascarpone, processed cheese and ripened cheese without the need for matrix-matched calibration by triple-quadrupole mass spectrometry. Full method validation was performed according to the modified Commission Decision 2002/657/EC criteria and method robustness was checked on 10 random cheese samples. Internal standard provided significant improvement (p < 0.05) in method precision for determination of nisin A in all four types of cheese. Significant losses (p < 0.05) for Nisin A in cheese was observed one week later. A fit-for-purpose method using internal standard procedure for accurate quantitation of Nisin A in cheese becomes available.

Analytical Methods for Mycotoxin Detection in Southeast Asian Nations (ASEAN).

Aflatoxins B1 (AFB1) and B2 (AFB2) and G1 and G2 remain the top mycotoxins routinely analyzed and monitored by Association of Southeast Asian Nations (ASEAN) national laboratories primarily for food safety regulation in the major food commodities, nuts and spices. LC tandem fluorescence detection (LC-fluorescence) represents a current mainstream analytical method, with a progressive migration to a primary method by LC tandem MS (MS/MS) for the next half decade. Annual proficiency testing (PT) is conducted by ASEAN Food Reference Laboratories (AFRLs) for mycotoxin testing as part of capability building in national laboratories, with the scope of PT materials spanning from naturally mycotoxin-contaminated spices and nuts in the early 2010s to the recent contamination of corn flour in 2017 for total aflatoxin assay development. The merits of the mainstream LC-fluorescence method are witnessed by a significant improvement (P < 0.05) in PT z-score passing rates (≤2) from 11.8 to 79.2% for AFB1, 23.5 to 83.3% for AFB2, and 23.5 to 79.2% for total aflatoxins in the last 5 years. This paper discusses the journey of ASEAN national laboratories in analytical testing through AFRLs, and the progressive collective adoption of a multimycotoxin LC-MS/MS method aided by an isotopic dilution assay as a future primary method for safer food commodities.

Stable nitrogen and carbon isotopes in sediments and biota from three tropical marine food webs: Application to chemical bioaccumulation assessment.

Studies of trophodynamics and contaminant bioaccumulation in tropical marine ecosystems are limited. The present study employed stable isotope and trace contaminant analysis to assess sources of primary productivity, trophic interactions, and chemical bioaccumulation behavior in 2 mangrove food webs and 1 offshore coastal marine food web in Singapore. Samples of sediment, phytoplankton, mangrove leaves, clams, snails, crabs, worms, prawns, and fishes were analyzed for stable carbon and nitrogen isotope values, as well as concentrations of persistent organic pollutants. In the mangrove food webs, consumers exhibited similar δ13 C values, probably because of the well-mixed nature of these systems. However, the 2 primary consumers (common nerite and rodong snail) exhibited distinct δ13 C values (-21.6‰ vs -17.7‰), indicating different carbon sources. Fish from Singapore Strait exhibited similar δ13 C values, indicating common carbon sources in this offshore marine food web. The highest trophic level was found in glass perchlet (trophic level = 3.3) and tilapia (trophic level = 3.4) in the 2 mangrove food webs and grunter (trophic level = 3.7) in the Singapore Strait food web. Concentrations of polychlorinated biphenyl (PCB 153) and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) concentrations ranged from 0.9 to 84.6 ng/g lipid weight and from <0.2 to 267.4 ng/g lipid weight, respectively. The trophic magnification factors of PCB 153 and p,p'-DDE ranged between 1.63 and 4.62, indicating biomagnification in these tropical marine food webs. The findings provide important information that will aid future chemical bioaccumulation assessment initiatives. Environ Toxicol Chem 2017;36:2521-2532. © 2017 SETAC.

Multi-mycotoxin screening reveals separate occurrence of aflatoxins and ochratoxin a in Asian rice.

The determination of important regulated mycotoxins in rice has been reported previously but not in the individual matrix of white, brown, red, and basmati rice with respect to the matrix effect, recovery, and stability. A total of 190 Asian rices were examined for regulated mycotoxin contamination by the LC-ESI-MS/MS method. Significant variation (p < 0.05) in the matrix effect was observed for fumonisins. Methanol improved the limits of detection (LOD) for HT-2 from 50 µg/kg to 2.3 µg/kg by promoting ionization efficiency of the ammonium-adduct. LOD and limits of quantitation ranged from 0.1 to 18 µg/kg and 0.2-31 µg/kg, respectively. All analytes degraded by more than 50% on storage, except fumonisins. Acetic acid (1%) provided significant improvement (p < 0.05) in recovery for all analytes in selected white rice from Thailand and China. Mean recovery ranged from 70 to 120%. RSD values were lower than 15% for all analytes. Five AFB1 and single OTA positive samples were detected. No correlation between mycotoxin contamination and rice species (r = 0) exists.

Identification of edible Bird's nest with amino acid and monosaccharide analysis.

This study describes the approach of amino acid and monosaccharide combined with Hotelling T2 range plot to identify edible bird nests (EBN) and non-EBN. Prior to the approach, an analytical method was developed and validated to quantify monosaccharides in EBN. Hotelling T2 range plots of both compounds were successful in predicting the different types of EBN and differentiating EBN and non-EBN. This outcome suggests EBN contains a group of glycoproteins which is not affected by the EBN's coloration, country of origin, and/or the processing method of the food item. In addition, the glycoproteins were shown to be unique to EBN. EBN were revealed to be rich in protein and essential amino acids as well as contain a wider variety of monosaccharides than most food items. The overall findings suggest that amino acid and monosaccharide provide information not only on the detected compounds and also insights into the glycoproteins of EBN.

Quantitative assessment of moniliformin in cereals via alternative precipitation pathways, aided by LC-LIT-MS and LC-Q-TOF-MS.

The availability of a simple chemical precipitation workflow aided by targeted and untargeted mass spectrometry would provide an accurate diagnostic platform for the direct determination of moniliformin in cereals for food safety control. In-house method validation was performed at six concentration levels of 8, 40, 80, 200, 400, and 600 ng g(-1) in cereal flours of wheat, corn, rye, oats and barley. Spiking experiments were made at three concentration levels of 20, 40 and 100 ng g(-1). Protein precipitation and "PHREE" column cleanup strategy provided recoveries of 81-108% for all cereals matrices using external calibrants. "PHREE" purification provided significant (p < 0.05) ion signal enhancement reduction advantage for all matrices except corn flour. Moniliformin underwent significant (p < 0.05) degradation over 2 weeks when prepared in acidified water. A simple, low-cost and fit-for-purpose procedure for the identification and quantitation of moniliformin in cereals becomes available to support prospective regulatory function.

Evaluation of triple stage mass spectrometry as a robust and accurate diagnostic tool for determination of free cordycepin in designer egg.

Direct determination of free cordycepin in designer egg using a highly selective mass spectrometric (MS) technique aided by a rapid and efficient dilute-and-shoot workflow would enhance their application as diagnostic tools in food fraud control. Here, triple stage mass spectrometry (MS(3)) demonstrated excellent analyte selectivity capability even when incomplete chromatographic separation was performed. Method validation was performed at six concentration levels of 100, 200, 400, 800, 1200 and 1600ngg(-1). Spiking experiments were examined at three concentration levels of 200, 400, and 1200ngg(-1) in individual egg white and egg yolk, measured over 2days. MS(3) enabled ion chromatograms with zero-background interference to be made in egg extracts. MS(3) eliminated severe over recovery (p<0.05) observed in all fortified samples, a challenge that MRM-transition could not address in a single step. Matrix-matched calibrants were needed to compensate for over recovery observed under MRM-transition mode.

A flow-injection mass spectrometry fingerprinting scaffold for feature selection and quantitation of Cordyceps and Ganoderma extracts in beverage: a predictive artificial neural network modelling strategy.

Flow-injection mass spectrometry (FI/MS) represents a powerful analytical tool for the quality assessment of herbal formula in dietary supplements. In this study, we described a scaffold (proof-of-concept) adapted from spectroscopy to quantify Cordyceps sinensis and Ganoderma lucidum in a popular Cordyceps sinensis /Ganoderma lucidum -enriched health beverage by utilizing flow-injection/mass spectrometry/artificial neural network (FI/MS/ANN) model fingerprinting method with feature selection capability. Equal proportion of 0.1% formic acid and methanol (v/v) were used to convert extracts of Cordyceps sinensis and Ganoderma lucidum into their respective ions under positive MS polarity condition. No chromatographic separation was performed. The principal m/z values of Cordyceps sinensis and Ganoderma lucidum were identified as: 104.2, 116.2, 120.2, 175.2, 236.3, 248.3, 266.3, 366.6 and 498.6; 439.7, 469.7, 511.7, 551.6, 623.6, 637.7 and 653.6, respectively. ANN models representing Cordyceps sinensis and Ganoderma lucidum were individually trained and validated using three independent sets of matrix-free and matrix-matched calibration curves at concentration levels of 2, 20, 50, 100, 200 and 400 µg mL-1. Five repeat analyses provided a total of 180 spectra for herbal extracts of Cordyceps sinensis and Ganoderma lucidum. Root-mean-square-deviation (RMSE) were highly satisfactory at <4% for both training and validation models. Correlation coefficient (r2) values of between 0.9994 and 0.9997 were reported. Matrix blanks comprised of complex mixture of Lingzhi fermentation solution and collagen. Recovery assessment was performed over two days using six sets of matrix blank (n = 6) spiked at three concentration levels of approximately 83, 166 and 333 mg kg-1. Extraction using acetonitrile provided good overall recovery range of 92-118%. A quantitation limit of 0.2 mg L-1 was reported for both Cordyceps sinensis and Ganoderma lucidum. Intra-day and inter-day RMSE values of 7% or better were achieved. Application of the scaffold in a high-throughput routine environment would imply a significant reduction in effort and time, since the option of having a model driven analytical solution is now available.

Analytical method for the accurate determination of tricothecenes in grains using LC-MS/MS: a comparison between MRM transition and MS3 quantitation.

The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS(3) quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS(3) (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 µg kg(-1) (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90-108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 µg kg(-1). A quantitation limit of 2-6 µg kg(-1) was achieved by applying an MRM transition quantitation strategy. Under MS(3) mode, a quantitation limit of 4-10 µg kg(-1) was achieved. Relative standard deviations of 2-10% and 2-11% were reported for MRM transition and MS(3) quantitation, respectively. The successful utilization of MS(3) enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are unavailable under MRM transition.