Marine biotoxins occur naturally as a result of harmful algal blooms (HABs) in saltwater environments and bioaccumulate in filter-feeding bivalve molluscs. When mussels, oysters and some other shellfish are ingested by humans, these toxins & their metabolites can cause serious illness in humans. As such the level of presence of these in shellfish destined for human consumption is regulated in many countries in the world.
This presentation describes development of a rapid method to detect regulated and unregulated lipophilic marine biotoxins in various shellfish using ultra performance liquid chromatography and tandem quadrupole mass spectrometry.
Presentation during the Bureau of Agricultural Research (BAR) Seminar Series on June 22, 2017 at RDMIC Bldg., cor. Visayas Ave., Elliptical Rd., Diliman, Quezon City
Presentation during the Bureau of Agricultural Research (BAR) Seminar Series on June 22, 2017 at RDMIC Bldg., cor. Visayas Ave., Elliptical Rd., Diliman, Quezon City
Biosecurity measures in shrimp farming:-
- Biosecurity measures at the time of stocking
- Biosecurity measures at the initial time of culture period
- Biosecurity measures during mid culture period
- Biosecurity measures at the end of culture period
Seaweeds are taxonomically diverse group of marine plants from which the land plants diverged over fifty crore years ago, which are found in the coastal region between high tide to low tide and in the sub-tidal region up to a depth where 0.01 % photosynthetic light is available. Plant pigments, light, exposure, depth, temperature, tides and the shore characteristic combine to create a different environment that determines the distribution and variety among seaweeds. It contains photosynthetic pigments and with the help of sunlight and nutrient present in the seawater, they photosynthesize and produce food which have several health benefits and uses. The important to know about the ecology and distribution of seaweed and to distinguish the different algal groups based on their characteristics. In recent, the utilization of seaweed increased due to various available properties. The different usages are food, beauty enhancer, organic manure, fertilizer, feed complement, medicines, water treatments. This review is an attempt to highlights the seaweed with all the relevant application and uses.
The presentation on disease management in aquaculture is prepared for academic purpose. Farmers and entrepreneurs make sure that the proposed drug is permissible in their country. Make sure that you contact experts before you implement them.
Environmental analysis can be extremely challenging due to the low detection levels for toxic contaminants specified by legislation, particularly in drinking water, and the complexity of matrices encountered. Consequently highly selective and sensitive detection methods are required. This presentation provides an introduction to tandem quadrupole mass spectrometry and describes the use of high sensitivity tandem quadrupole mass spectrometry for the analysis of various environmental contaminants including pesticides, endocrine disruptors and polyfluorinated compounds such as PFOS.
Biosecurity measures in shrimp farming:-
- Biosecurity measures at the time of stocking
- Biosecurity measures at the initial time of culture period
- Biosecurity measures during mid culture period
- Biosecurity measures at the end of culture period
Seaweeds are taxonomically diverse group of marine plants from which the land plants diverged over fifty crore years ago, which are found in the coastal region between high tide to low tide and in the sub-tidal region up to a depth where 0.01 % photosynthetic light is available. Plant pigments, light, exposure, depth, temperature, tides and the shore characteristic combine to create a different environment that determines the distribution and variety among seaweeds. It contains photosynthetic pigments and with the help of sunlight and nutrient present in the seawater, they photosynthesize and produce food which have several health benefits and uses. The important to know about the ecology and distribution of seaweed and to distinguish the different algal groups based on their characteristics. In recent, the utilization of seaweed increased due to various available properties. The different usages are food, beauty enhancer, organic manure, fertilizer, feed complement, medicines, water treatments. This review is an attempt to highlights the seaweed with all the relevant application and uses.
The presentation on disease management in aquaculture is prepared for academic purpose. Farmers and entrepreneurs make sure that the proposed drug is permissible in their country. Make sure that you contact experts before you implement them.
Environmental analysis can be extremely challenging due to the low detection levels for toxic contaminants specified by legislation, particularly in drinking water, and the complexity of matrices encountered. Consequently highly selective and sensitive detection methods are required. This presentation provides an introduction to tandem quadrupole mass spectrometry and describes the use of high sensitivity tandem quadrupole mass spectrometry for the analysis of various environmental contaminants including pesticides, endocrine disruptors and polyfluorinated compounds such as PFOS.
An overview of Pine Lake Laboratories capabilities involving oligonucleotides. Includes challenges, examples, method development, validation, and stability!
Mycotoxins are are secondary metabolites produced by fungi and are dangerous for feed and food chains as they can create contamination in pre- and post-harvest processes. Many are highly toxic and as such levels in food products are regulated in Europe, the US, Japan and other countries. This presentation is an overview of the application of ultra-performance liquid chromatography combined with tandem quadrupole mass spectrometry to analyse various food products for mycotoxins in line with regulatory requirements.
Accurate data are needed on threshold doses for allergenic foods. Data are also needed to demonstrate that available methods of analysis can detect and quantify allergens in foods at or around these threshold levels and that they are robust and fit for purpose. This is of major concern as no curative treatment is currently available for food allergy and accidental ingestion of the culprit food can lead to severe clinical symptoms. Elimination of the problem food ingredient from the diet reduces the risk of allergic reactions but this can lead to other issues such as deficiencies, eating disorders and growth retardation. Emergency medication is available including Antihistamines (H1 blockers), EpiPen (adrenaline-autoinjector) and Corticosteroids. This presentation describes investigations into peanut allergens and their quantitation in highly complex samples.
This presentation will provide an introduction to the technology and benefits of Waters ACQUITY Ultra Performance Convergence Chromatography system (UPC2) and illustrate how it can impact the analysis of chemicals and polymers.
Jane Cooper, Senior Applications Scientist
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
- Ability to run LC and GC amenable compounds in single analysis
-Fast 7 minute analysis of the 24 regulated allergens and 6 additional compounds containing:
– Different classes of compounds
– Different polarities
- UC2 with MS detection offers an orthogonal technique, which enables greater selectivity and specificity compared to either HPLC or GC analysis alon
- The developed 7 minute UPC2 method, is greater than 6 times faster than existing HPLC and GC
Analysis of Phenolic Antioxidants in Edible Oil/Shortening Using the PerkinEl...PerkinElmer, Inc.
Phenolic antioxidants are commonly used in food to prevent the oxidation of oils. Oxidized oil and fats cause foul odor and rancidity in food products, which is a major cause for concern to the food industry. Globally, regulations vary, but current maximum allowable levels are as low as 100 μg/g (100 ppm). This application note presents a UHPLC method for the analysis of the ten most common phenolic antioxidants that may be found in such products.
Feed ingredients and feed prices are increasing; it is becoming harder to maintain the nutritional balance of the feed without increasing too much the feed price. Now, the use of ingredients from less stringent quality is likely to increase. Though plant materials are usually more reasonable in price than animal products, they can present problems through the presence of naturally occurring contaminants. Indeed, contamination of feed commodities by microorganisms and mycotoxins is the first negative factor impacting animal feed quality. Numerous researches have studied the decrease of performances with contaminated feeds.
Measuring pKas, logP and Solubility by Automated titrationJon Mole
Presentation by Sirius Analytical covering measurement of pKa, LogP, LogD, Solubility, Supersaturation and precipitation kinetics.
For more details visit www.sirius-analytical.com
This presentation describes the investigation using Ion Mobility MS of previously reported issues with tandem quadrupole MS/MS methods for fluoroquinolone antibiotics in food of animal origin. The discovery of protomer species with different fragmentation pathways explains the variability of the tandem quadrupole MRM results.
A new reversed phase solid phase extraction device which simplifies sample preparation as no conditioning or equilibration steps are required. Matrix effects in MS can be reduced by removal of interferences, particularly fats and phospholipids.
Similar to Routine Quantification of Lipophilic Marine Biotoxins in Shellfish by LC/MS/MS - Waters Corporation Food Safety (20)
This presentation describes the utilisation of microfluidic chromatography coupled with high resolution mass spectrometry incorporating ion mobility for separation, detection and identification of steviol glycoside isomers. Moving into the routine environment we then will show a simple, cost-effective solution for the determination of stevioside, Rebaudioside A and other non-nutritive sweeteners in a variety of food products.
Analysis of pesticides in food using both LC- and GC-MS/MS, with data and description of Atmospheric Pressure GC, available on the same system as UPLC-MS/MS with rapid changeover.
Overview of foodomics applications using high resolution mass spectrometry including profiling of natural products, dietary intake studies and an introduction of REIMS direct analysis.
Food fraud has become a major concern in recent years. Fruit juice is commonly adulterated with cheaper alternatives. High resolution MS in combination with 'omics data analysis approaches can identify markers of adulteration.
Bisphenol A is an additive used in the production of polycarbonate plastics and epoxy resins. These synthetic materials are widely used in food packaging to protect the safety and integrity of foods and beverages. BPA has been discovered to be an endocrine disruptor which can mimic the body's own hormones and may lead to negative health effects and this has generated concern over the leaching of the compound from packaging into food. This presentation describes the analysis of BPA and related compounds in baby food and infant formula using UPLC and tandem quadrupole mass spectrometry.
Food fraud is on the increase globally and checking for the authenticity of foodstuffs is a major challenge. Typical of food fraud is the substitution of a low value product for a higher value one to increase profit to the trader. For years, traders have been passing off a lesser quality rice, CSR 30, as the world's finest long-grained, aromatic rice, Basmati, in key markets like the US, Canada and the EU. In the process, the rice exports enjoy the duty exemption accorded to pure Basmati in the EU and thousands of consumers get duped. This presentation describes the use of Atmospheric Pressure Gas Chromatography coupled to high resolution MS to differentiate between types of rice and to identify marker compounds using statistical analysis software.
Allergens are a major food safety concern and incidences of food allergy in industrialised populations has increased in recent times. One of the most common food allergies is that of peanuts. Food regulations for allergens exist in many countries and are being modified regularly as more is understood about allergens and the reactions they cause. This presentation describes the use of time-of-flight mass spectrometry to locate, identify and quantify an allergenic protein in both raw and roasted peanuts. Typical food processing (e.g. food processing) can alter the markers peptides present and amount that they are present in the samples which adds complexity to the analysis.
This presentation compares wo methods for the detection of low-level pesticide residues in fruit juice. One involves the use of QuEChERS sample preparation and the other a 'dliute and shoot' approach. Sample preparation is utilised to remove the matrix effects associated with mass spectrometry (MS), using a 'dilute and shoot' approach requires the use of highly sensitive MS detection. It can be seen from the results shown that the 'dilute and shoot' approach can be used in many cases.
This presentation describes development of a routine tandem quadrupole LC/MS/MS method for milk and egg allergens based on proteomic studies to identify the allergenic peptide markers. Initial studies are done using a proteomic workflow and quadrupole time of flight mass spectrometry.
This presentation describes the operation and application of the Waters APGC (Atmospheric Pressure Gas Chromatography) ion source which provides a highly sensitive GC-MS, MS/MS capability for tandem quadrupole and time of flight MS systems. It is very easy to swap between APGC, Electrospray (for UPLC) and other ion sources without instrument venting in minutes.
APGC provides significant performance advantages over traditional GC/MS ionisation methods, giving high sensitivity and less fragmented spectra.
This presentation describes a new food testing solution that allows mass detection to be accessible for many of the routine analyses found within a food testing lab. The ACQUITY UPLC, QDa detector in combination with the Empower software solution is fit for purpose and is easy to use. You simply power on and you are ready to go
With the ACQUITY QDa detector many of the normal processes that are required for mass spectrometers (such as mass calibration and optimisation and manual adjustments that need to be made) have all been fully automated.
More from Waters Corporation - Food QC, Safety & Research (16)
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Picture shows the extent of an algal bloom (lighter blue) off the UK coastline
Brevetoxins – there are no good methods of analysis for these types of compounds available to date…
National Oceanic and Atmospheric Administration (NOAA) is a federal agency focused on the condition of the oceans and the atmosphere – this seems to be a very good programme for marine biotoxins
Useful link to all the NRLs within Europe: http://www.aesan.msps.es/en/CRLMB/web/enlaces/enlaces.shtml
In the European Union (EU) legislation various toxin groups are regulated and these toxins should be monitored in official control programs. The lipophilic biotoxins that should be monitored are okadaic acid (OA), dinophysistoxin-1, -2, -3 (DTX1, -2, -3), where DTX3 are the ester forms of respectively OA, DTX1 and -2, furthermore, pectenotoxin-1, -2 (PTX1, -2), yessotoxin (YTX), 45OH yessotoxin (45OH YTX), homo yessotoxin (homoYTX), 45OH homo yessotoxin (45OH homoYTX), azaspiracid-1, -2 and -3 (AZA1, -2, -3)
Reference: European Commission (2005) Laying down implementing measures for certain products under Regulation (EC) No 853/2004 of the European Parliament and of the Council and for the organisation of official controls under Regulation (EC) No 854/2004 of the European Parliament and of the Council and Regulation (EC) No 882/2004 of the European Parliament and of the Council, derogating from Regulation (EC) No 852/2004 of the European Parliament and of the Council and amending Regulations (EC) No 853/2004 and (EC) No 854/2004. Off J Eur Commun L338:40-41
Consumption of shellfish (mussels, oysters, clams, etc) contaminated with biotoxins can cause severe intoxications in humans such as Diarrhetic Shellfish Poisoning (DSP). Due to their lipophilic properties DSP toxins are often classified as lipophilic marine biotoxins. Marine biotoxins are naturally produced by different types of phytoplankton and are therefore also named phycotoxins. The complexity of the lipophilic marine biotoxins lies in the variety of physiochemical properties such as carboxylic acids, sulfonic acids, amino and imino functionalities.
Before July 2011, the official method was a bioassay based on the oral administration of shellfish meat to a rat or the interperitoneal injection of a shellfish extract in mice.
There were two main issues with this method
It was perceived as unethical
Not scientifically robust enough to determine the trace amounts of specific toxins. (The presence of cyclic imines would cause a physical reaction using the bioassay – the result often fatal for the animal. Cyclic imines (figure 1b) are also classified as lipophilic marine toxins - based on their physiochemical properties. Although these toxins are not currently regulated, the European Food Safety Authority (EFSA) indicated that more data on toxicity as well as their occurrence in shellfish should be collected)
Acidic Method
Get good chromatographic peaks
Perceived to be not as sensitive as the alkali method
(Fewer people have adopted this method in industry)
Alkali Method
Get poor peak shapes for some of the AZAs
Method separates by polarity:
ESI- compounds @ start
ESI+ compounds @ end
Improved LoDs compared to the acidic method
The journal puublication used an Alliance and Quattro Ultima under basic conditions and was recognised as an official method as part of the EU regulations
The aims are to produce a much faster routine analysis than the conventional LC method under alkaline conditions, and to include additional non-regulated compounds that are of interest to EFSA. In this application note, various classes of lipophilic marine biotoxins including some of the non-regulated cyclic imines were analysed in a five-minute analysis using a Waters ACQUITY UPLC system coupled to a Xevo TQ-S. The ACQUITY UPLC technology allows the analytical runtime to be reduced without compromising peak resolution and quality, whilst the Xevo TQ-S (tandem quadrupole mass spectrometer) will provide ultra-high sensitivity for the detection, and allows the simultaneous acquisition of multiple reaction monitoring (MRM) transitions in alternating electrospray + (ESI+) and - ionisation (ESI-) modes which is required for the analysis of these lipophilic marine biotoxins.
The single lab single day validation study was performed in order to assess the performance of the developed UPLC-MS/MS method. The following characteristics criteria were assessed; linearity, recovery, repeatability, within-laboratory reproducibility (empirically calculated from the repeatability), selectivity and decision criteria (CCα). Blank shellfish from the Dutch national monitoring program were used for validation, the shellfish species that were included in the validation were mussels (Mytilus edulis), oysters (Crassosrea gigas), cockles (Cerastoderma edule) and ensis (Ensis directus). In order ensure that the shellfish species used for the validation were blank, they were subject to analysis for the toxins shown in Table (2?) prior to the beginning of the study. Seven different shellfish samples (4 mussel, 1 oyster, 1 cockle and 1 ensis) were extracted and spiked at 0, 0.5, 1 and 1.5 times the validation level.
Homogenized whole flesh shellfish tissue (1 g) was extracted in triplicate with 3 mL methanol. After each addition of methanol, the extract was vortex-mixed. After vortex-mixing the extract was centrifuged for 5 min at 2000 g and the supernatant was transferred to a 10 mL volumetric flask. After the third extraction (9mL in total), the volume was made up to 10 mL with methanol. The crude shellfish extract was then filtered through a 0.2 µm membrane filter prior to spiking or analysis.
In order to determine the amount of DTX3 (ester forms of OA, DTX1 and -2) extracts were also subjected to alkaline hydrolysis using 2.5 M sodium hydroxide. After heating the alkaline mixture for 40 min at 76 °C, the mixture was cooled to room temperature and subsequently neutralized using 2.5M hydrochloric acid.
Certified standards OA, DTX1, -2, PTX2, YTX, AZA1, -2, -3, gymnodimine (GYM) and 13-desmethyl spirolide C (SPX1): National Research Council, Institute for Marine Biosciences (NRC-CNRC), Halifax, Canada.
Semi-purified standards for pinnatoxin-E (PnTX-E), -F (PnTX-F) and –G (PnTX-G): Cawthron Institute, Nelson, New Zealand
For each toxin a standard stock solution was prepared in methanol.
From the stock solutions, the matrix matched standards (MMS) calibration curves were prepared in blank mussel extract.
Blank extract was prepared according to the sample extraction procedure.
The levels of the matrix matched standard were 0, 0.125, 0.25, 0.5, 1.0 and 1.5 times the validation level
The individual stock solutions of the cyclic imines were diluted in methanol to obtain tuning parameters with IntelliStart Technology.
IntelliStart greatly simplifies the use of LC-MS systems by automating instrument setup, compound tuning, and performing system suitability checks.
Currently for some of the regulated toxins, standards are not yet available (PTX1, 45OH YTX, homoYTX and 45OH homoYTX). In order to determine these toxins, MRM transitions are included in the method based on the structural relation with the toxin from which a standard is available, respectively PTX2 and YTX.
Furthermore, MRM transitions of other non-regulated lipophilic toxins are included which could be screened for using this confirmatory method
The method parameters that that have been determined in these experiments are now integrated into the Quanpedia database: this allows any user of this system to have access to this method protocol.
MRM chromatograms of matrix matched standard in mussel matrix at 1.0 x validation level.
Ticks = toxins currently regulated
Cross = non-regulated – (as per the EU regulations).
(Pectenotoxin 1, homo-yessotoxin, 45OH yessotoxin and 45OH homo yessotoxin are not shown in this example)
Can see that the MRM mode of the tandem quad is a very selective mode and there are only the peaks of interest that can be seen
UPLC separation provided good results for all different toxins classes; only peak shapes for azaspiracids were somewhat negatively affected by the alkaline conditions. Due to the high selectivity and sensitivity of the Xevo TQ-S this is not problematic for the routine application, as the peaks can be still easily detected at the sensitivity levels required by regulation. The resulting MRM chromatograms from the five-minute UPLC separation of various toxins are shown. For each compound two optimised MRMs are shown: one for confirmation and the other for quantification. The ion ratio between the results of two MRMs is one of the analytical criteria that will determine whether or not a biotoxin has been positively identified. Another regulatory criterion is the maximum residue limit (MRL) of the compound. Within the processing software, TargetLynx, it is possible to automatically flag parameters that are set within the regulatory framework so that the analyst can very easily see when these criteria have not met the permitted levels.
Sensitivity for the various toxins is good, even at levels of 0.125 times the validation level (regulatory level for regulated toxins) a signal-to-noise above 3 could be obtained. Currently for some of the regulated toxins, standards are not yet available (PTX1, 45OH YTX, homoYTX and 45OH homoYTX). In order to determine these toxins, MRM transitions are included in the method based on the structural relation with the toxin from which a standard is available, respectively PTX2 and YTX. Furthermore, MRM transitions of other non-regulated lipophilic toxins are included which could be screened for using this confirmatory method (Table 2). The method parameters that that have been determined in these experiments are now integrated into the Quanpedia database: this allows any user of this system to have access to this method protocol.
With respect to single-day validation, different method performance parameters were assessed such as linearity, recovery, repeatability, within-laboratory reproducibility, selectivity and CCα. For all toxins the linearity of the matrix matches standard calibration curve was excellent (R2 > 0.990) for the concentration range used (0.125 - 1.5 × PL). The recoveries obtained for the various toxins were good and ranged from 91 to 104%.
The only exception to this was PnTx-E (non-regulated) where a recovery of 122% was obtained. This early eluting (tr 1.2 min) compound tends to form methyl esters when extracts are prepared in methanol and this behaviour can be used to explain the higher than expected recovery as well as the poor repeatability (RSDr 23.1%) when compared to the other analytes.
For all regulated compounds the repeatability observed was good. For the non-regulated toxins, only PnTx-E and SPX1 were somewhat higher than expected. For SPX1 this was caused by the ensis matrix by removing the results of the ensis and re-calculating the repeatability results were acceptable, respectively 14.6% and 12.8% with and without ensis matrix. In order to decide if a sample is non-compliant the toxin level should be at or above the decision limit (CCα). The decision limit is a concentration at which we can conclude with a probability of 1-α or 95% (α = 5%) that the sample is above the permitted level and thus non-compliant.
Validation results of the single day validation.
1 including the ensis matrix
2 without the ensis matrix,
* poor reproducibility.
UPLC separation provided good results for all different toxins classes; only peak shapes for azaspiracids were somewhat negatively affected by the alkaline conditions. Due to the high selectivity and sensitivity of the Xevo TQ-S this is not problematic for the routine application, as the peaks can be still easily detected at the sensitivity levels required by regulation.
Sensitivity for the various toxins is good, even at levels of 0.125 times the validation level (regulatory level for regulated toxins) a signal-to-noise above 3 could be obtained. Currently for some of the regulated toxins, standards are not yet available (PTX1, 45OH YTX, homoYTX and 45OH homoYTX). In order to determine these toxins, MRM transitions are included in the method based on the structural relation with the toxin from which a standard is available, respectively PTX2 and YTX. Furthermore, MRM transitions of other non-regulated lipophilic toxins are included which could be screened for using this confirmatory method (Table 2). The method parameters that that have been determined in these experiments are now integrated into the Quanpedia database: this allows any user of this system to have access to this method protocol.
Single-day validation, using the different method performance parameters were assessed such as linearity, recovery, repeatability, within-laboratory reproducibility, selectivity and CCα. For all toxins the linearity of the matrix matches standard calibration curve was excellent (R2 > 0.990) for the concentration range used (0.125 - 1.5 × PL).