homogeneity and stability - Springer Link

Anal Bioanal Chem (2011) 400:847–858 DOI 10.1007/s00216-011-4787-8

ORIGINAL PAPER

A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability Pearse McCarron & Håkan Emteborg & Sabrina D. Giddings & Elliott Wright & Michael A. Quilliam

Received: 23 November 2010 / Revised: 6 February 2011 / Accepted: 7 February 2011 / Published online: 6 March 2011 # Her Majesty the Queen in Right of Canada 2011

Abstract A candidate certified reference material (CRM) for multiple shellfish toxins (domoic acid, okadaic acid and dinophysistoxins, pectenotoxins, yessotoxin, azaspiracids and spirolides) has been prepared as a freeze-dried powder from mussel tissues (Mytilus edulis). Along with the certified values, the most important characteristics for a reference material to be fit-for-purpose are homogeneity and stability. Acceptable between-bottle homogeneity was found for this CRM. Within-bottle homogeneity was assessed using domoic acid, and it was shown that repeated subsampling of the CRM can be performed precisely down to 0.35 g. Both short- and long-term stability studies carried out under isochronous conditions demonstrated excellent stability of the various toxins present in the material. While degradation of some analytes was observed at +60°C in short-term studies, it was determined that shipping at ambient temperature is adequate. No instability was detected in long-term stability studies, and it was shown that the material can be held at +18°C safely for up to 1 year. To guarantee stability of the CRM over its lifetime the stock will be maintained at −20°C. The results of the homogeneity and stability testing show that CRM–FDMT1

P. McCarron (*) : S. D. Giddings : E. Wright : M. A. Quilliam National Research Council Canada, Institute for Marine Biosciences, 1411 Oxford St, B3H 3Z1 Halifax, NS, Canada e-mail: [email protected] H. Emteborg European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg 111, 2440 Geel, Belgium

is appropriate for its intended use in quality assurance and quality control of shellfish toxin analysis methods. Keywords Shellfish toxins . Phycotoxins . CRM–FDMT1 . Stability . Homogeneity . LC–MS . HPLC

Introduction In the field of shellfish toxin regulation and research, an increasing number of laboratories are moving away from use of bioassays towards chemical analytical methods. To facilitate the development and validation of these alternative methods, a project was undertaken to prepare a certified reference material (CRM) containing multiple groups of toxins. The preparation of this freeze-dried mussel tissue (FDMT) candidate CRM and the characterisation of its physical properties were described previously [1]. Briefly, a variety of mussel (Mytilus edulis) tissue materials contaminated with domoic acid (DA), azaspiracids (AZAs) okadaic acid and dinophysistoxins (OA and DTXs) were collected. Yessotoxins (YTXs), pectenotoxins (PTXs) and spirolides (SPXs) were obtained from algae or as purified toxins because insufficient quantities of naturally incurred mussels could be sourced. The various materials were blended to produce a bulk wet homogenate. The homogenate was then freeze-dried, milled and bottled in portions suitable for distribution and analysis. The most important parameters for consideration in the production of CRMs are that the material is homogenous and stable with respect to the analyte and the matrix [2]. To fulfil the various needs and uses of reference materials (RMs), and because of the time and expense that typically goes into their preparation, it is desirable that large quantities of materials are produced. It is necessary that

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the batch is homogenous so that all subsamples are equivalent at the minimum level of sample intake. During preparation of RMs, numerous factors can influence the homogeneity. Care must therefore be taken during every step of the mixing, homogenisation, dividing and filling to ensure that homogeneity is satisfactory. For dry powders such as CRM–FDMT1, the nature of the phase, the size of the solid particles, their distribution and the respective density is very important [3]. Following preparation of CRM–FDMT1, these parameters were assessed by means of laser diffraction particle size analysis and micrographs, and although this showed that the material was satisfactory with regard to these parameters it is of primary importance that there is good homogeneity of the analytes of interest within the matrix (i.e., shellfish toxins). The contribution from between-bottle heterogeneity to the expanded uncertainty of the certified value should ideally be as small as possible [4]. A CRM should be stable to ensure continued supply over long periods of time. Both the stability of the analytes and the matrix require consideration. While the stability of these components is obviously related, the latter is a particular concern for biological matrices. RMs are usually prepared in a quantity which is sufficient to last for a number of years, and for this reason they need to be produced in such a way that ensures stability throughout their expected lifetime. A range of factors can influence the stability of a material, including chemical and biological activity as well as physical effects. A freeze-dried matrix was prepared [1] based on a previous feasibility study that showed significant improvements in the stability of phycotoxins in a freeze-dried mussel material, when compared to a wet homogenate [5]. Stability testing of the candidate CRM is necessary to determine the most appropriate and economic storage and transport conditions. In addition to homogeneity and stability, the CRM should ideally present a matrix as similar as possible to “real-life” samples. It is recognized that a freeze-dried matrix is not the ideal representation of a CRM for a method dealing typically with wet tissue samples. However, the representativeness must often concede to the various processes and treatments that are carried out during preparation of a material to ensure that it is fit-for-purpose over the necessary periods of time. This paper describes extensive homogeneity and stability testing of CRM–FDMT1. Detailed information is available on various models for homogeneity testing [6, 7]. Betweenbottle homogeneity was evaluated to determine any variation between bottles of CRM–FDMT1. Within-bottle homogeneity testing was carried out to differentiate between method variability and any actual heterogeneity in material and also to establish a minimum recommended subsample intake. Additionally, as the preparation of this

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material occurred in two stages, some testing of aliquots of the bulk wet homogenate sampled prior to freeze-drying will be described. To determine the risk of degradation during transport, short-term stability (STS) testing at elevated temperatures over a relatively short period is necessary. Examination of the long-term stability (LTS) of the CRM was carried out to determine its behaviour under controlled storage conditions.

Experimental Sample selection and shifting for homogeneity and stability studies At the Institute for Reference Materials and Measurements (IRMM), the samples required for homogeneity and stability testing were selected after bottling using a random stratified sampling procedure. This ensured analysis of samples representative of the entire CRM fill series. Twenty bottles of CRM–FDMT1 were selected to evaluate between-bottle homogeneity of the lipophilic toxins by liquid chromatography–mass spectrometry (LC–MS). An approach for homogeneity testing as outlined in ISO Guide 35 [7] was followed, where a single extraction was done for each bottle and the extracts were analysed in triplicate. Additionally, eight bottles of CRM–FDMT1 were used for a more thorough evaluation of within-bottle homogeneity, taking four subsamples from each bottle. In order to recommend the minimum portion size, four of the bottles used for within-bottle testing were subsampled, taking 0.7, 0.35, 0.14 and 0.07 g sized portions from each. Final extract volumes of these samples were scaled to facilitate equivalent volumetric control of the extractions for each subsample size (50, 25, 10 and 5 mL, respectively). Given that the high performance liquid chromatography-ultraviolet detection (LC-UVD) method of analysis for DA has better precision than the LC–MS methods for other analytes in CRM–FDMT1, within-bottle homogeneity testing was carried out only for DA. Isochronous designs of STS and LTS studies were applied [8]. Samples were placed at the selected storage conditions at the beginning of the study periods, and after pre-determined time-points samples were taken from the various storage conditions and placed in the reference condition (−70°C). For the STS study, time-points selected were 0, 2, 8, 16 and 30 days, with temperature conditions of −20, +4, +18 and +60°C, respectviely. For the LTS study, time-points selected were 0, 2, 3, 6, 9 and 12 months, with temperatures of −20, +4 and +18°C, respectively. Triplicate samples were taken for each timepoint/temperature condition. When all time-points were complete, the samples were sent to the National Research

Homogeneity and stability of a multi-toxin shellfish tissue CRM

849

Council (NRC) on dry ice for extraction and analysis. The main CRM–FDMT1 stock was kept at −20°C based on previous experience with similar materials [5] until the stability studies were complete.

was quantified in CRM–FDMT1 from the resulting calibration curves.

Standards and chemicals

A three-step extraction procedure was also applied for the lipophilic toxins in FDMT1 based on good overall recovery determined during method development [10]. After thorough mixing of the contents of the CRM–FDMT1 bottles, 0.35-g subsamples were weighed into 50-mL centrifuge tubes and re-constituted as above with 1.65 mL of deionised water. The samples were then extracted in triplicate with methanol (vortexing for the first step, then homogenization with an Omni-Prep for the second step, and vortexing again for the final step). The supernatants from each step were combined in 25-mL flasks and made up to volume with the extraction solvent. Studies on purified 13-desmethylspirolide C 13-desMe-SPX C showed instability at higher pHs (>5) [11] so a 1-mL subsample of the final methanol extract was taken and diluted to 5.0 mL with acidified methanol (0.1% formic acid). Aliquots of the various extracts were passed through 0.45-μm filters prior to analysis by LC–MS. Samples were analysed using an Agilent 1200 LC coupled to an Applied Biosystems API4000 QTrap equipped with a turbospray ionisation source using a multi-toxin LC–MS method developed for characterization of CRM-FDMT1 [10]. A binary mobile phase was used, with A=water and B=95% aqueous acetonitrile, each containing 5-mM ammonium acetate (pH 6.8). A Phenomenex Synergi MaxRP C12 column (50×2 mm i.d., 3 μm) was eluted at 300 μL/min with a linear gradient of 25– 100% B over 5 min, held for 3 min, decreased to 25% B over 0.1 min, and equilibrated at 500 μL/min for 2 min until the next run. For quantitation selective reaction monitoring (SRM) transitions in negative ionisation mode were; OA/DTX2 803.5>255.1m/z, DTX1 817.5>255.1, YTX 1,141.6>1,061.6. Positive ionisation mode transitions were; PTX2 876.5>823.5, AZA1 842.5>672.5, AZA2 856.5>672.5, AZA3 828.5>658.5, AZA6 842.5>658.5. Six-point calibrations were acquired using a mixed standard prepared from certified calibrants of OA, DTX1, DTX2, YTX, PTX2 and AZA1. AZA1 was used for calibration of AZA2, −3 and −6 assuming an equimolar response. The acidified extracts were measured separately for SPXs using the same LC separation. No negative effect of the higher pH mobile phase on SPX stability was noticed. In positive mode the SRM transition 692.5>164.1 was monitored for 13-desMe-SPX C, and 706.5>164.1 was monitored for SPX C and 20-Me-SPX G. For calibration of these SPXs, a five-point curve acquired using dilutions of a 13-desMe-SPX C calibrant was used, assuming equimolar responses for SPX C and 20-Me-SPX G.

Methanol, acetonitrile and trifluoroacetic acid (TFA) were purchased from Caledon (Georgetown, ON, Canada). Ammonium acetate and formic acid were purchased from BDH laboratory reagents (Toronto, ON, Canada). Shellfish toxins calibrants were obtained from the NRC’s Certified Reference Material Program (CRMP; Halifax, NS, Canada). Distilled water was further purified using a Milli-Q purification system (Millipore Corp., Billerica, MA, USA). DA extraction and analysis After thorough mixing of the contents of the CRM– FDMT1 bottles, 0.7 g subsamples were weighed into 50-mL centrifuge tubes and re-constituted by adding 3.3 mL of de-ionised water, vortex mixing immediately and sonicating in an ultrasonic bath (Branson Ultrasonics Corp, CT, USA) for 1 min. The samples were then extracted with a three-step liquid/solid extraction technique using methanol/water (50:50 v/v; vortexing for the first step, then homogenization with an Omni-Prep (OMNI International, Kennesaw, GA, USA) for the second step and vortexing again for the final step). The supernatants from each step were combined in 50-mL volumetrics, made up to a volume with the extraction solvent and mixed. Aliquots of the extracts were filtered through 0.45μm filters prior to analysis. For testing a minimum subsample intake, smaller portions of the powder were sampled and extracted using the same procedure, maintaining a consistent sample-to-solvent ratio for all extractions (see above). For the subsamples of the bulk wet homogenate taken prior to freeze-drying, the described DA extraction procedure was applied without reconstitution. Analysis was carried out based on the procedure of Quilliam et al. [9], without the SAX clean-up step. An isocratic run at 900 μL/min was performed using 10% aqueous MeCN with 0.1% TFA on a Luna C18 (2) column (150 × 4.6 mm i.d., 3 µm) (Phenomenex, Torrance, CA, USA). The STS analyses were carried out using an Agilent 1100 LC system equipped with a model G1315B DAD. For the LTS analyses, an Agilent 1200 LC system equipped with a model G1315D DAD was used. For instrument calibration, a six-point dilution series was prepared from a certified DA calibration solution. The sum of DA and the 5′-epi-domoic acid isomer (epiDA)

Lipophilic toxin extraction and analysis

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Results and discussions Homogeneity testing The preparation of the CRM was performed in two main stages. Initially, a bulk wet material was prepared, which was subsequently freeze-dried to produce the final candidate CRM [1]. Previously, phycotoxin RMs and CRMs have been successfully prepared as wet homogenates [12– 14], however, these were generally on a much smaller scale than that reported in the current study (150 kg). Aliquots (4 g) of the bulk wet material taken prior to shipment to IRMM for freeze-drying were tested for homogeneity of DA and the lipophilic toxins at the NRC. The relative standard deviations were similar to the method repeatability for the major toxin analogues, which showed good homogeneity for the bulk wet material (data not shown). This demonstrated that the methodology used in the preparation of the bulk wet tissue homogenate was appropriate for preparation of fit-for-purpose RMs, and that it can be applied in future preparations where freeze-drying is not desirable. However, for the characterisation of CRM–FDMT1 and assigning associated with certified values, the homogeneity of the final product was a primary concern. Lipophilic toxins homogeneity Between-bottle homogeneity testing was carried out to determine possible differences between various bottles of the CRM–FDMT1 batch for the different toxins, and to determine whether detectable differences were within acceptable limits. Differences are foreseeable if segregation occurred during the preparation, homogenisation, or filling procedures, resulting in a heterogeneous bulk material or in the bottles filled last being different to those filled first. Bottles need to be selected across the entire fill series to determine the existence of any trend, and the analysis must be performed using a method with high precision under repeatability conditions [6, 15] where bottles are measured

in random order to enable separation of analytical drift from trends in the fill order. For the lipophilic toxins, a single extract from each of the 20 bottles was injected in triplicate and the average values with associated relative standard deviations are shown (Table 1). Good homogeneity for the major analytes was observed. The data has been determined to follow a normal distribution, and the experimental set-up allows direct evaluation of the data by one-way analysis of variance (ANOVA) to determine the contribution to the final uncertainty from the relative between-bottle heterogeneity. ANOVA allows for a differentiation of betweenbottle heterogeneity for the CRM (between mean squares (MS between)) and any variation in the analytical method (within mean squares (MS within)). Using mean square values, the uncertainty contributions from between-bottle homogeneity (Sbb) were calculated using the ISO Guide 35 approach [7]: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSbetween MSwithin Sbb ¼ ð1Þ n Since the instrumental variability was found in many cases to be the largest contributor to uncertainty in these analyses, an alternative between-bottle homogeneity (μbb*) evaluation was used to determine the maximum homogeneity that could be masked by analytical variability. In these instances, homogeneity uncertainties will be assigned using: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSwithin 4 2 » mbb ¼ ð2Þ dfMSwithin n Where dfMSwithin are the degrees of freedom from ANOVA for MSwithin and n is the number of replicate measurements. To reduce labour intensity a single extract was prepared from each bottle, and therefore only the variability from instrumental analysis (LC–MS) is separated from the uncertainty between bottles [7]. Since this alternative

Table 1 Between-bottle homogeneity data for the major lipophilic toxin analogues in CRM–FDMT1

Average (mg/kg) SD (n=20) % RSD μ*bb (%)

OA

DTX1

DTX2

YTX

AZA1

AZA2

AZA3

PTX2

13-desMe-SPX C

1.17 0.03 2.8 1.5

0.69 0.02 2.9 1.1

3.0 0.1 2.3 0.8

2.1 0.1 4.7 1.2

3.4 0.1 1.6 1.3

0.83 0.02 1.8 1.1

0.68 0.01 1.7 2.6

0.26 0.01 5.6 3.0

2.6 0.1 3.6 1.1

Bottles selected were representative of the entire production series. Note: concentrations stated are information values obtained from homogeneity testing and are not the final certified values for the CRM. Shown are the uncertainties for homogeneity (μ*bb) calculated using Eq. 2


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approach to homogeneity assessment was necessary, the combination of variance due to extraction and heterogeneity between bottles is less than the variability of the LC–MS analysis. Domoic acid homogeneity and subsampling study CRM–FDMT1 was filled in approximately 3 g portions providing sufficient material for replicate samplings of a single bottle, considering that 0.7 g of powder equates to approximately 4 g of wet tissue (at a water content of 83–84%). Homogeneity testing of eight bottles representing the fill series was done by taking four subsamples from each bottle, to determine both the between- and within-bottle homogeneity of DA. Within-bottle variance was not statistically different from between-group variance (p>0.05) which shows that replicate portions of CRM–FDMT1 could be successfully sampled from a single bottle (Table 2). When calculating the combined uncertainty for the final certified values, the contribution from between-bottle heterogeneity from this data set will be included following the Sbb approach (Eq. 1) due to the better precision associated with LC-UVD analysis. Using this approach, the final uncertainty from the relative betweenbottle heterogeneity is only 0.7% for DA showing that the CRM–FDMT1 is very homogeneous. Further evidence for the good between-bottle homogeneity was obtained from the samples in the stability study. In this study, a range of samples were stored at −20°C and at the reference temperature of −70°C. These were also selected from across the entire fill series using a random stratified sampling procedure at IRMM. Stability study samples were extracted on the same day and analysed in the same run ensuring repeatability of analysis, such that it was possible to obtain homogeneity information for the CRM as part of the stability work. The combined −70°C and −20°C data from the STS study were all comparable to the precision of the methods used for the analyses and demonstrated good homogeneity of the analytes between different bottles of the

material (data not shown). Additionally, no significant effects from the fill order (i.e. trends) were visible in the STS and LTS study results from the bottles selected over the entire fill series (Fig. 1). During this work the contents of the bottles were mixed by a combination of rolling and inversion between subsampling and it is recommended that this is practiced by end-users when taking replicate samples from a bottle of CRM–FDMT1. Different aliquot sizes (0.7, 0.35, 0.14 and 0.07 g) were analysed to determine the minimum quantity of sample required to ensure representative subsampling. There was no statistically significant increase in variability with smaller sample sizes (p>0.05). However, there is a suggested trend of decreasing DA concentration when going to smaller sample sizes (Fig. 2). Therefore, based on experience a minimum sample size of 0.35 g is recommended due to potential issues with handling and accurate weighing of very small quantities of the dry powder. Stability testing Short-term stability In the STS study, the sum of DA+epiDA displayed excellent stability with no significant concentration reductions occurring at any of the storage conditions investigated up to +60°C (Table 3). In previous testing of freeze-dried RMs for phycotoxins, similar stability was also achieved, whereas significant degradation was observed in an equivalent wet tissue homogenate [5]. In that work, the highest temperature investigated was +40°C. For CRM– FDMT1, +60°C was investigated and the consistency observed virtually guarantees the stability during shipping and transport. The concentration values discussed for CRM– FMDT1 are the sum of DA and epiDA in the material (Fig. 3a), which are typically combined for regulatory purposes. DA has been shown to isomerise under extreme conditions of heat [16] and this conversion to its 5′-epimer

Table 2 Within- and between-bottle homogeneity for CRM–FDMT1 assessed by replicate sampling of individual bottles and determination of DA+epiDA levels by LC-UVD Bottle number Replicate Replicate Replicate Replicate

#1 #2 #3 #4

Average (mg/kg) SD %RSD

284

1392

2217

3109

4085

4943

5583

6349

128.4 135.5 136.5 134.8

135.1 135 132.9 131.8

136 135.5 136.2 135.8

135.4 134.8 138.1 135.5

134.7 132.3 132.8 133.4

131.5 134.1 137.5 135.5

136.9 124.8 130.5 131.9

133.7 134.6 137.3 135.4

133.8 3.7 2.7

133.7 1.6 1.2

135.9 0.31 0.2

136 1.5 1.1

133.3 1.1 0.8

134.6 2.5 1.9

131 5 3.8

135.2 1.5 1.1

Overall average 134.2 1.6 1.2

0.90 0.05

0.00 0.07 g

0.14 g

0.35 g

0.7 g

Sub-sampling size

Fig. 2 Evaluation of sample intake size on recovery of DA and epiDA from CRM–FDMT1. Error bars represent ±1 standard deviation (SD, n=4). Different aliquot sizes were extracted with proportionate volumes of solvent resulting in equivalent sample-tosolvent ratios for each size

(2.7) (2.4) (11) (2.4) 98.1 95.6 82.0 79.4 (7.2) (3.7) (5.9) (9.3) 93.7 94.3 76.7 68.9 (3.5) (11) (3.5) (7.6) 93.6 84 63.8 57.6 (3.3) (2.5) (0.9) (1.9) 95.4 72.9 60.0 45.7 (4.7) (4.9) (3.3) (2.8) 89.0 71.2 57.1 46.9 (8.2) (2.1) (5.2) (6.4) 99.3 95.5 93.5 87.6 (2.8) (3.0) (3.6) (4.7) 95.9 96.3 94.0 88.0 (2.9) (3.8) (3.3) (4.7) 98.8 97.9 93.0 78.2 (7.3) (4.1) (4.1) (3.9) 104.6 106.0 101.2 101.3 (9.3) (2.2) (1.0) (4.6) 102.5 101.5 103.2 100.2 Numbers in parentheses are standard deviations (SD, n=3)

0.95

(10) (5.7) (4.5) (7.7)

1.00

98.0 98.4 97.3 96.9

1.05

(7.4) (2.6) (2.6) (2.6)

DA + epiDA values normalised to max (0.7 g)

(epiDA) was observed in CRM–FDMT1 at the 60°C temperature condition (Fig. 4a and e). At −20°C epiDA comprised approximately 1% of the total DA+epiDA peak area; however, following 30 days at +60°C epiDA contributed approximately 8% of the total. This isomerisation was only observed at the +60°C condition, with the DA level remaining consistent at the other temperatures investigated (Fig. 3b and c). Based on these results, shipping at ambient temperature would be appropriate for this CRM with respect to DA, but to avoid any possible issues of isomerisation, chilled packaging could be applied as a precaution. OA, DTX1 and DTX2 displayed excellent stability at all conditions examined in the STS study (Fig. 5). The results are consistent with a feasibility study carried out for OA

102.9 95.8 101.8 95.1

Fig. 1 Combined DA+ epiDA concentrations in stability study samples (−70 and −20°C) plotted against CRM–FDMT1 bottle number. This shows absence of trend in values due to bottle fill series. The %RSD was approximately 2% the for combined data

(0.9) (0.8) (0.3) (0.7)

6000

98.5 100.5 100.2 98.8

5000

2 8 16 30

4000

13-desMe-SPX C SPX C

3000

Fill series (bottle #)

AZA6

2000

AZA3

1000

AZA2

0

AZA1

0.7

PTX2

Average ± 1 x SD STS LTS

0.8

YTX

0.9

DTX2

1.0

DTX1

1.1

Timepoint (days) DA+epiDA OA

Normalised DA + epiDA values

1.2

20-Me-SPX G

P. McCarron et al. Table 3 Concentrations of toxins monitored in short-term stability studies on CRM–FDMT1 over 30 days at +60°C expressed as a percentage of the respective day 0 samples that had been stored at the reference condition (−70°C)

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Conc. normalised to day zero mean (-70°C)


1.2

A: domoic + epi-domoic acid

1.0

0.8 Average T0 (-70°C) ± 2 × SD -20°C +4°C +18°C +60°C

0.6

0.4 1.2

B: domoic acid

1.0

0.8

0.6

0.4 8

C: epi-domoic acid

7 6 5 4 3 2 1 0 0

5

10

15

20

25

30

Time point (days)

Fig. 3 Individual STS graphs for a DA+epiDA (sum), b DA, and c epiDA in CRM–FDMT1. Isomerisation of DA to epiDA is observed at +60°C over the study duration. Error bars represent ± SD (n=3). Note: stability study results are illustrated as values normalized to reference temperature (time zero (T0), −70°C)

and DTX2 in a freeze-dried mussel tissue matrix [5]. In the initial stages of CRM-FDMT1 preparation, characterization of stock mussel tissues showed that some contained quantities of the acyl esters of the dinophysistoxins [1]. A study by Rodrigues et al. showed that while free OA and DTX2 were stable in fresh mussel tissues stored at −30°C over a period of 1 month, the concentration of acyl esters of these compounds decreased over the same study period [17]. However, separate studies on the development of shellfish tissue RMs for phycotoxins determined that the presence of acyl esters in tissues used for preparation of a RM can have a very important bearing on the stability of

853

parent analogue levels [18]. It was shown that levels of free OA and DTX2 in raw tissue RMs (i.e., not cooked or thermally processed prior to bottling) can undergo increases at temperatures above freezing, which is probably due to an enzymatic hydrolysis of the acyl esters. This was observed in tissues from a number of shellfish species including mussels. It was also shown that heat treatment of the tissues prior to RM preparation resulted in much more stable levels of free OA, DTX2 and their acyl esters. In CRM–FDMT1, no significant changes in concentration were observed at any condition, which can probably be attributed to the freeze-drying of the matrix as well as inactivation of enzyme activity by thermal processing of all tissues prior to bulk homogenisation [1]. PTX2 showed good stability in the STS study with the exception of some significant degradation at +60°C after the 30-day time-point (Fig. 6). In samples at this treatment condition, a small isomer of PTX2 not present samples stored at the reference temperature eluted after PTX2 (Fig. 4). Although no stability studies have been reported for PTX2 in a freeze-dried shellfish tissue matrix to date, an assessment of PTX2 in a wet mussel homogenate RM showed that PTX2 was quite unstable and began to degrade significantly after 1 week at +20°C [18]. Therefore, it can be concluded that freeze-drying is also highly effective for stabilisation of mussel tissue RMs for PTX2. Enzymatic transformation of PTX2 to PTX2 seco acid (PTX2sa) and a C-7 epimer of PTX2sa has been reported in raw shellfish [19]. Low levels of these components present in the CRM were monitored as part of the stability studies and they displayed similar stability to PTX2 with significant degradation occurring at +60°C in the STS study. Based on these results, CRM–FDMT1 could be shipped appropriately at ambient temperature with respect to PTXs; however, chilling could be applied as a precautionary measure due to unforeseen delay in transit. Stability of YTX was excellent in the STS (Table 3), with no significant concentration changes being detected at any of the conditions; 45-OH YTX is also present in FDMT1 at a lower concentration, and this displayed similar stability to YTX (data not shown). AZA1 and AZA2 displayed equivalent stabilities during the STS study (Table 3). Concentrations were stable up until 18 days at +60°C; however, degradation was significant at the 30-day time point (P164.1 of 13-desMe–SPX C showing degradation and formation of isomer (asterisk) at the stress condition (b, f). LC–MS of m/z 876.5>823.5 showing degradation of PTX2 and formation of isomer (asterisk) at the stress condition (c, g). LC–MS at m/z 828.5> 658.5 showing degradation of AZA3 (d, h). Peaks (1 and 2) are present in reference sample due to extensive thermal sterilization of stock tissue (d) and increase of re-arrangement isomer at stress condition in stability study (H, 2*)

P. McCarron et al.

4

5

6

3

4

6

G

C

2.0e+3 PTX2

PTX2

1.0e+3

*

0.0 4.5 1.2e+4

Intensity (cps)

5

5.0

5.5

6.0

6.5

7.0

4.5

5.0

5.5

6.0

6.5

7.0

6.5

7.0

H

D

9.0e+3

6.0e+3

AZA3

AZA3

3.0e+3

2* 1 2

1

0.0

4.5

5.0

5.5

6.0

Time (min)

tissues [5]. Oxidised metabolites of AZA1 and AZA2, namely AZA17 and AZA19, respectively [21], were recently shown to decarboxylate at elevated temperatures in shellfish to form AZA3 and AZA6, respectively [22]. AZA3 and AZA6 demonstrated similar stabilities in CRM– FDMT1 (Table 3). In a previous study, AZA3 concentrations increased in a freeze-dried material prepared using uncooked mussel tissues [5]. However, the AZA3 increase in the freeze-dried matrix was much less than was observed for an equivalent wet tissue material in the same study, and subsequent degradation was not as significant either. Thorough heat treatment of the stock tissues during the initial preparation stages of CRM–FDMT1 [1] ensured that

6.5

7.0

4.5

5.0

5.5

6.0

Time (min)

increased levels of AZA3 could not occur due to complete decarboxylation of AZA17, leading to an overall improvement in the stability of AZA3 in CRM–FDMT1. For shipment of CRM–FDMT1, precautionary chilled transport could be used to prevent the decomposition of AZA analogues that was observed at the +60°C temperature condition. For inclusion of SPXs in the candidate multi-toxin CRM, an algal paste of an Alexandrium ostenfeldii culture was used [23]. Preliminary screening of an acidified methanol extract of CRM–FDMT1 showed that the three primary SPXs present were 13-desMe-SPX C, SPX C and 20-Me-SPX G. Each of these analogues displayed excellent stability in the

1.4 1.2 1.0 0.8 0.6 Average T0 (-70°C) ± 2 × SD -20°C +4°C +18°C +60°C

0.4 0.2 0.0 0

5

10

15

20

25

30

855 Conc. normalised to day zero mean (-70°C)




0.4 0.2 0.0 0

5

10

15

20

25

30

Time point (days)

Fig. 5 Short-term stability results for DTX1 in CRM–FDMT1. This is also indicative of the good stability also observed for OA and DTX2 present in the material. Error bars represent ± standard deviation (SD, n=3)

Fig. 7 Short-term stability results of AZA3 in CRM–FDMT1. Error bars represent ± standard deviation (SD, n=3)

STS study at −20°C, +4°C and +18°C. Degradation was significant for 13-desMe-SPX C at 8 days and +60°C (Fig. 8), while 20-Me-SPX G and SPX C displayed slightly better stability as degradation was not significant until 16 days at +60°C (Table 3). Some isomerisation of 13desMe-SPX C began to occur at +60°C with an earlier eluting unresolved peak being observed (Fig. 4). No isomer peaks were seen with this method for the SPX C and 20-Me-SPX G analogues, but it is possible that they were not resoled from the original compounds. In the STS studies, the only condition where degradation became significant was +60°C, and this was only for some of the toxin analytes. AZA3 and AZA6 displayed the most significant degradation, with concentrations dropping after 2 days. Degradation of AZA1, AZA2, PTX2 and the SPXs

was observed at the latter stages of the STS study, with isomerisation of DA to epiDA also occurring at this condition. In spite of these susceptibilities at higher temperatures, the toxins displayed excellent stability in the freeze-dried matrix, equivalent to or better than stabilities previously reported for other shellfish toxin RMs [5, 18]. In order to achieve the stability required for confident shipping and handling of such quality control materials, the “real-life wet sample” representativeness of the CRM conceded to the preparation of a freeze-dried matrix. Conveniently, it is a relatively simple step to rehydrate the freeze-dried powder prior to extraction. However, differences in the matrices of RMs and real-life samples should not be a major issue to analysts performing environmental monitoring, as it has to be accepted that natural samples



Time point (days)


0.4 0.2 0.0 0

5

10

15

20

25

30

Time point (days)

Fig. 6 Short-term stability results of PTX2 in CRM–FDMT1. Error bars represent ± standard deviation (SD, n=3)

1.4 1.2 1.0 0.8 0.6

Average T0 (-70°C) ± 2 × SD -20°C +4°C +18°C +60°C

0.4 0.2 0.0 0

5

10

15

20

25

30

Time point (days)

Fig. 8 Short-term stability results of 13-desMe-SPX C in CRM– FDMT1. Error bars represent ± standard deviation (SD, n=3)

(1) (6) (1) (3) (4) 95 98 98 102 105 (1) (4) (12) (9) (9) 101 100 99 104 102 (4) (5) (1) (2) (4) 101 100 103 104 106 (11) (11) (13) (11) (14) 97 94 99 104 101 (5) (8) (11) (13) (15) 99 95 97 102 105 (4) (11) (9) (8) (9) 102 96 95 105 106 (7) (11) (10) (6) (13) 99 96 95 104 106 (3) (14) (5) (8) (7) 98 99 97 106 102 (2) (9) (2) (5) (9) 102 99 91 100 102 (10) (3) (6) (4) (6) 100 95 94 100 102 (4) (7) (15) (6) (8) 100 100 100 102 100

(1) (1) (2) (1) (1)

102 92 94 102 98

(2) (9) (9) (12) (12)

104 94 102 99 106

SPX C 13-desMe-SPX C AZA6 AZA3 AZA2 AZA1 PTX2 YTX DTX2 DTX1

Numbers in parentheses are standard deviations (SD, n=3)

ð3Þ

2 3 6 9 12

m1ts ¼ mb t1ts :

OA

In the LTS studies no degradation was observed for any of the toxins at any of the conditions tested (Table 4). This shows that temperatures up to +18°C are appropriate for storage of the CRM for periods of up to 1 year. In comparison with the STS, no isomerisation of DA was observed in the LTS study (data not shown). The excellent stability is in good agreement with a prior study of a freeze-dried mussel matrix, which showed that it was stable for a period of more than 8 months at +40°C [5]. A previous LTS study of YTX in a wet mussel tissue RM showed that degradation was significant at +40°C after 6 months [18]. Although a higher temperature was investigated in the previous study, these results for CRM–FDMT1 indicate that freeze-drying is beneficial for the stability of YTX when compared to wet tissue homogenates. The good LTS observed for AZA3 in CRM– FDMT1 is highly satisfactory considering the known stability issues for this toxin [5, 22]. When considering long-term storage of a CRM, the least stable analyte dictates the storage condition [7]. Degradation of some toxins was observed at temperatures greater than +18°C in the short-term studies, and although concentrations were stable for 12 months at +18°C in the long-term studies, it is conceivable that additional years of storage at this condition could result in some degradation. Therefore, frozen storage (−20°C) will be employed for long-term management of CRM–FDMT1. No significant trends or degradation could be discerned from the LTS study for any of the toxins. An uncertainty contribution from the LTS study results will be calculated for all certified toxins for a 12-month shelf life. Regression analysis was used to determine the slope of stability plots at the specified storage condition (−20°C). Slopes were not significantly different from zero. The standard uncertainty about the slope (μb) along with the chosen shelf life (tlts) of the material will be used to determine the uncertainty component from stability (μlts) according to the ISO Guide 35 [7]:

DA+epiDA

Long-term stability

Timepoint (month)

routinely vary significantly in composition and analytical methods must be robust enough to deal with these variations in samples [3]. CRM–FDMT1 is suitable for shipping at +18°C; however, shipments may be chilled as a precaution in certain situations. Once STS stability testing had been completed the remaining stock of CRM– FDMT1 at the IRMM (>6,000 units) was shipped overnight to the NRC by air under chilled conditions for long-term storage.

20-Me-SPX G

P. McCarron et al. Table 4 Concentrations of toxins monitored in long-term stability studies on CRM–FDMT1 over 12 months at +18°C expressed as a percentage of the respective day 0 samples that had been stored at the reference condition (−70°C)

856


Conclusions Comprehensive studies were performed evaluating the homogeneity and stability of the various toxins present in CRM–FDMT1. The material showed good homogeneity for all toxins present, both between bottles selected over the entire fill series, and also within bottles, which was assessed by taking replicate samples from the individual bottles. For CRM–FDMT1, a minimum sample size of 0.35 g is recommended (approximately 2 g wet tissue equivalent). The excellent stability of all toxins in the STS and in the LTS studies highlights the effectiveness of freezedrying for producing an extremely stable RM, bearing in mind the instability of some of these toxins in wet mussel tissue homogenates stored at similar conditions. This outcome alone clearly justifies the extensive effort put into the freeze-drying and associated processes for the production of CRM–FDMT1. Because of the scarcity of some of the tissue materials used in the preparation and the value of the purified and semi-purified toxins, it is highly satisfactory that such good stability was obtained. To assess the stability of the various toxins over the life of CRM–FDMT1, stability checks on bottles of the material maintained at the selected storage condition (−20°C) will be performed on a periodic basis in real-time. The satisfactory results of the homogeneity and stability studies show that CRM–FDMT1 is fit for its intended use as a quality control and quality assurance tool in chemical analysis of multiple groups of shellfish toxins. Work on this project is on-going and is focused on final optimization of accurate and precise extraction and analysis methods, followed by application of these procedures for assignment of certified values and uncertainties. Acknowledgements The work of the RM-processing team at the IRMM on the preparation of CRM–FDMT1 is appreciated. Conchi Contreras and Thomas Linsinger are acknowledged for the help in setting up and executing the isochronous shifting of samples for stability studies. Dr. Philipp Hess (formerly of the Marine Institute in Ireland) is acknowledged for his contribution to the organisation of this project. Dr. Jeremy Melanson at the NRC is acknowledged for reviewing this manuscript. This is NRCC publication number 51793.

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