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.

Development of an LC-MS/MS Determination Method for T-2 Toxin and Its Glucoside and Acetyl Derivatives for Estimating the Contamination of Total T-2 Toxins in Staple Flours.

A determination method previously validated for trichothecenes and zearalenone by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was adapted for the quantification of T-2 toxin (T-2) as well as its glucoside and acetyl derivatives, T-2-3-glucoside (T-2-3G) and 3-acetyl-T-2 (3A-T-2). HT-2 toxin (HT-2) and its acetyl derivative 3-acetyl-HT-2 (3A-HT-2) were also included as the target chemicals. Staple flours (56 samples collected from the Singapore market) were examined for contamination from T-2 and/or HT-2 and their derivatives. Among them, 16 flours were found to be contaminated with T-2 and/or HT-2, whereas none was contaminated with T-2-3G and 3A-HT-2, except for trace 3A-T-2 detected in 2 rye samples. Rye flour samples were frequently contaminated with both T-2 and HT-2. Some of the reference materials (RMs) were further analyzed, and T-2-3G and 3A-T-2 were quantitatively detected in corn and wheat RMs. The ratio of T-2-3G to T-2 in the RMs seemed to be much lower than the ratio of deoxynivalenol-3-glucoside to deoxynivalenol usually reported in former studies. To the best of our knowledge, the natural contamination of 3A-T-2 in staple flour is reported here for the first time.

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.

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.

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.

Feed-forward neural network assisted by discriminant analysis for the spectroscopic discriminantion of cracked spores Ganoderma lucidum: A prospective biotechnology production tool.

A major problem for manufacturers of cracked spores Ganoderma lucidum, a traditional functional food/Chinese medicine (TCM), is to ensure that raw materials are consistent as received from the producer. To address this, a feed-forward artificial neural network (ANN) method assisted by linear discriminant analysis (LDA) and principal component analysis (PCA) was developed for the spectroscopic discrimination of cracked spores of Ganoderma lucidum from uncracked spores. 120 samples comprising cracked spores, uncracked spores and concentrate of Ganoderma lucidum were analyzed. Differences in the absorption spectra located at ν1 (1143 - 1037 cm-1), ν2 (1660 - 1560 cm-1), ν3 (1745 - 1716 cm-1) and ν4 (2845 - 2798 cm-1) were identified by applying fourier transform infra-red (FTIR) spectroscopy and used as variables for discriminant analysis. The utilization of spectra frequencies offered maximum chemical information provided by the absorption spectra. Uncracked spores gave rise to characteristic spectrum that permitted discrimination from its cracked physical state. Parallel application of variables derived from unsupervised LDA/PCA provided useful (feed-forward) information to achieve 100% classification integrity objective in ANN. 100% model validation was obtained by utilizing 30 independent samples. ν1 was used to construct the matrix-matched calibration curve (n = 10) based on 4 levels of concentration (20%, 40%, 60% and 80% uncracked spores in cracked spores). A coefficient of correlation (r) of 0.97 was obtained. Relative standard deviation (RSD) of 11% was achieved using 100% uncracked spores (n = 30). These results demonstrate the feasibility of utilizing a combination of spectroscopy and prospective statistical tools to perform non destructive food quality assessment in a high throughput environment.

Discriminating authentic Nostoc flagelliforme from its counterfeits by applying alternative ED-XRF and FTIR techniques.

Nostoc flagelliforme is an edible blue-green algae belonging to the Nostocaceae family. It is recognised as a Chinese delicacy in south-eastern Asia and is widely consumed. Due to its high economic value and diminishing supply, as a result of overharvesting, counterfeits have often been found in the retail markets. Methods involving microscopy and histochemistry were conventionally applied to differentiate the authentic N. flagelliforme from its counterfeits. In this paper, we report an alternative analytical approach, using a combination of non-destructive energy dispersive X-ray fluorescence (ED-XRF) and Fourier-transform infrared (FTIR) spectroscopy, to achieve the objective of authentic N. flagelliforme verification. In view of the scarcity of this Chinese delicacy, such a non-destructive methodology would be ideal to preserve the integrity of the sample and yet provide a means to discriminate between authentic and counterfeit samples.