Elaine Catherine Marley
email: elaine@r-biopharmrhone.com

Eric Mackay

G Young

for determination of vitamin B12 in a range of foods, juices and pharmaceutical 2 products using immunoaffinity clean-up and high performance liquid 3 chromatography

Keywords: Baby food, Fruit juices, Infant formulae, Nutritional supplements Vitamin B12, cyanocobalamin, immunoaffinity column, EASI-EXTRACT ® Vitamin B12, 32 HPLC-UV

Characterisation of vitamin B12 immunoaffinity columns and method development for determination of vitamin

B12 in a range of foods, juices and pharmaceutical products using immunoaffinity clean-up and high performance liquid chromatography with UV detection.

Introduction

3

Vitamin B12 is an important water-soluble vitamin; its primary functions are the formation of red 4 blood cells and the maintenance of a healthy nervous system. In addition it is needed to process 5 folic acid, another essential vitamin (Ball 1997). Deficiency can lead to pernicious anaemia due to 6 failure of red blood cell formation (Food Standards Agency 2003). Prolonged deficiency can also 7 lead to nerve degeneration and irreversible neurological damage due to failure to repair the myelin 8 sheath protecting nerve cells (Scott 1992). Vitamin B12 originates from bacteria, fungi and algae 9 (Tom 1994) and is found in virtually all types of meat, fish and dairy products. However, daily 10 intake is required because the amount absorbed is limited (approximately 0.002 mg/meal) by the 11 capacity of the (intrinsic factor-wall receptor) uptake system; consequently a range of foods are 12 fortified to reduce the incidence of deficiency related diseases. Groups at risk of deficiency 13 diseases include infants, the elderly and vegans. Hence all infant formulae are fortified with vitamin 14 B12 and foods that are produced for vegans, such as soya products are often fortified voluntarily, 15 as foods from plant sources do not contain vitamin B12.

17

Although the biologically active forms of vitamin B12 are methylcobalamin and adenosylcobalamin, 18 the synthetic form used in vitamin supplements, pharmaceuticals and in the fortification of food is 19 cyanocobalamin. Cyanocobalamin is converted to the biologically active methylcobalamin 20 following uptake by ileal enterocytes and prior to binding transcobalamin II (TCII); the majority of 21 cobalamin available for uptake from the circulatory system is associated with TCII (Food

22

Standards Agency 2003).

24

Vitamin supplements are widely used and manufacturers are required to test premixes and 25 finished product to confirm vitamin B12 levels. Also food and drink manufacturers are obliged by 26 law to determine the vitamin B12 content of a range of fortified commodities for process control 27 purposes and to enable accurate labelling.

29

Currently the determination of vitamin B12 in food products is routinely carried out by means of the 30 microbiological assay (MBA), which uses Lactobascillus leishmanii as the test organism 31 [START_REF] Anonymous | AOAC Official Methods of Analysis[END_REF]. Although the assay is extremely sensitive, the MBA lacks a high degree of 32 specificity as food commodities can contain inactive cobalamins that interfere with the growth of

7

A 10 µg/ml vitamin B12 stock solution was prepared by weighing 1 mg vitamin B12 into a 100 ml 8 amber volumetric flask. The vitamin B12 was then diluted to mark with water and placed in an 9 ultrasonic bath for 15 min to dissolve. The stock solution was stable for 3 months at 4 °C.

11

Serial dilutions of Vitamin B12 Stock solution were then used to prepare a five-point standard 12 curve; 2 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.25 µg/ml and 0.05 µg/ml. A correlation coefficient of >0.995 13 was obtained routinely. The limit of detection (LOD) was 1.5 ng of vitamin B12 injected and the 14 limit of quantification (LOQ) was 5 ng of vitamin B12 injected. The same protocol was used for the preparation of hydroxycobalamin stock and diluted solutions. The stability of the hydroxycobalamin 16 stock solution was 1 month at 4 °C.

2

A 5-15 ml aliquot (corresponding to 0.1-1.5 µg vitamin B12) of the sample extract was passed 3 through the column followed by 10 ml of water to wash the column. The vitamin B12 was eluted 4 with 3 ml methanol and subsequently evaporated to dryness by heating (50-60 °C) under a stream 5 of air and reconstituted in 300 µl of 0.025% TFA in water. When analysing infant formula the 6 sample volume applied to the column was restricted to ≤5ml as method development 7 demonstrated a decrease in vitamin B12 recoveries when applying volumes greater than 5ml. A 8 mean recovery of 99% (RSD r =0.71%, n=6) was obtained when 5ml of infant formula extract was 9 applied to the column, the recovery decreased to 88% (RSD r =3.22%, n=6) when 10ml was 10 applied and 78% (RSD r =3.57%, n=6) when 15ml was applied. The sample was ground to a fine powder with pestle and mortar. An amount of tablet containing 31 the equivalent of 2.5 µg vitamin B12 was dissolved in 100 ml water and placed in an ultrasonic 32 bath for 15 min. A 1 ml aliquot of the extract was made up to 1 litre with water in a volumetric flask.

33

The pH of the diluted extract was then confirmed to be between 4.5 and 7. If the pH was greater 34 than 7, it was adjusted to within range using citric acid. If the pH was less than 4.5 the sample was 35 discarded and re-extracted using phosphate buffered saline (PBS). up. In all cases the value of vitamin B12 given by NIST was established using the MBA and a 8 specified uncertainty was stated (Table 1). It should be noted that NIST 2383 and NIST 1546 were 9 complex matrices containing low levels of vitamin B12 and in all cases the results obtained using 10 immunoaffinity clean-up in conjunction with HPLC-UV were within the uncertainty limit obtained 11 using the MBA (Table 1). In particular, NIST 2383 Baby Food Composite was a very challenging 12 matrix to extract vitamin B12 from as the sample contained (in order of decreasing mass) orange 13 juice, infant formula, corn, rice flour, creamed spinach, carrots, papaya juice, tomato paste, beef, 14 macaroni, wheat flour, non-fat milk, Romano cheese, soya protein, onion powder, green pepper, 15 celery oil, oregano in conjunction with a low-level of vitamin B12 (0.44 µg/100 g). The sample 16 preparation and purification procedure clarified the sample filtrate (Figure 1) and concentrated the vitamin B12 sufficiently, to allow quantification by HPLC-UV analysis. In addition the sensitivity of

29

the exception of Brand 1 Plus Infant Formula all recoveries were greater than 100% of the 30 declared value with % RSD r < 7% (Table 1). EU legislative levels for vitamin B12 allow ± 15% of the declared value (European Commission Directive 2002). For Brand 3 Infant Formula and NIST 32 1846 Infant Formula the determined vitamin B12 levels were within the legislative range. For 33 Brand 1 and Brand 2 Infant Formula the determined values were 3% (RSD r = 1.6%) below the 34 lower limit and 2% (RSD r = 5.0%) above the upper limit respectively. Previous work by Casey et al.

35

(1982) demonstrated more efficient extraction of vitamin B12 when the sample was autoclaved for 36 10 min at 121 °C rather than heated on a boiling water bath for 10 min. However when autoclaved 37 Brand 1 Plus Infant Formula as described the recovery of vitamin B12 was found to decrease.

9

In addition two types of high protein powdered drinks and three types of high protein bars were 10 analysed. The protein powdered drinks contained high levels of vitamin B12; 103% and 94% of the 11 label claim were obtained (Table 2). For high-level vitamin B12 samples in complex matrices it is 12 critical the amount of vitamin B12 applied to the immunoaffinity column does not exceed 1.5 µg as 13 amounts greater than this are above the upper limit for optimum column performance. In contrast 14 protein bars are fortified with relatively low levels of vitamin B12. However, due to the 15 concentration effect of the immunoaffinity column the quantity of vitamin injected onto the HPLC 16 system was above the LOQ and good chromatographic separation was achieved. Recoveries relative to declared value of greater than 100% were obtained (Table 2).

3

Commercially available carbonated energy activation and rejuvenation drinks containing 0.1 4 µg/100 ml vitamin B12 according to the label claim were also tested. The drinks were found to 5 contain 0.11 µg/100 ml and 0.95 µg/100 ml respectively (Table 3). In order to obtain an indication 6 of column capacity, the activation and rejuvenation drinks were spiked with an additional 2 µg/100 7 ml and 15 µg/100 ml or 20 µg/100 ml vitamin B12. Recoveries of vitamin B12 from activation and 8 rejuvenation drinks spiked at 2 µg/100 ml were 95% and 99% respectively with 0.4-0.6µg vitamin 9 B12 being applied to the immunoaffinity column. In addition when the drinks were spiked at very 10 high levels (20 µg/100 ml or 15 µg/100 ml) the recoveries were 85% and 84% with 3-4.5 µg 11 vitamin being applied to the column. Hence the capacity of the column (defined as that level where 12 recovery is equal to 70%) exceeds 4.5 µg. It should be noted the performance of the column could 13 be affected by more complex matrices especially at levels of vitamin B12 close to column capacity.

14

Therefore for assessment of routine samples the recommended maximum amount of vitamin B12 applied to the immunoaffinity column should not exceed 1.5 µg. In addition, the dilution of 16 activation and rejuvenation drinks with PBS prior to passage through the column resulted in a 3-17 4% increase in recovery. This was not significant and therefore a change to protocol was not 18 recommended in this instance but dilution of sample with PBS may be used to greater effect when 19 analysing more complex matrices.

21

To challenge the immunoaffinity column with samples containing low levels of vitamin B12 (<0.14 22 µg/100 ml) four types of fruit drinks (apple, orange and peach, blackcurrant and apple, and 23 strawberry juice) were analysed and the vitamin B12 content quantified (Table 3). The innate level 24 of vitamin B12 in the fruit drinks was not declared. Therefore, to determine a percentage recovery 25 of the vitamin, the fruit drinks were spiked with 1 ug/100 ml vitamin B12 and recoveries in the 26 range of 78 -86% were obtained (Table 3).

33

Brand 2 with a declaration of 1000 µg/0.580 g, yielded 91% (RSD r = 3.7%) of the declared value.

34

Hence the described method can be successfully used to quantify the amount of vitamin B12 in

33 23 (

 23 the microorganism. Heudi et al. (2006) reviewed alternative methods such as an intrinsic factor 34 based assay using radioisotopic dilution, a protein binding assay with surface plasma 35 spectroscopy detection and solid phase extraction (SPE) techniques with subsequent 36 concentration procedures prior to analysis by HPLC-UV for the determination of vitamin B12 in 37 foods and biological samples. The majority of the methods previously identified lack sensitivity or Due to the need for accurate determination of vitamin B12 and the lack of sensitivity and specificity 5 with existing methods an immunoaffinity clean-up column was developed. The column consisted of 6 a monoclonal antibody with high affinity and specificity to vitamin B12 (cyanocobalamin) linked to 7 cyanogen bromide activated agarose beads. Immunoaffinity column clean-up with HPLC-UV could 8 be used for the routine analysis of vitamin B12 in food because it enables concentration of the 9 analyte and removal of interfering compounds; thus, allowing accurate quantification of vitamin 10 B12 at low levels. In addition, this work involved method development for the determination of 11 vitamin B12 in a range of food products and pharmaceuticals (NIST standard reference material, 12 infant formula, high protein powdered energy drinks and bars, breakfast cereal, carbonated soft 13 drinks, fruit juice drinks and vitamin B12 tablets) and consequently has expanded upon previous 14 work by Heudi et al. (2006). cyanocobalamin) (catalogue No. V2876), hydroxycobalamin (catalogue No. H7126), 20 sodium acetate (S8750), pepsin (EC 3.4.23.1, catalogue No. P7125), α-amylase (EC 21 3.2.1.1,catalogue No. A2771) and trifluoroacetic acid (TFA) were all purchased from Sigma-Aldrich 22 (Dorset, UK). Potassium cyanide (catalogue No. 424500250) was obtained from Acros Leicestershire, UK). HPLC grade acetonitrile and water were purchased from Lab-Scan Analytical 24 Sciences (Dublin, Ireland) and were used for mobile phase preparation and immunoaffinity column 25 elution solution. De-ionised water was obtained from a MilliQ water purification system (Millipore 26 UK Ltd) and was used for preparation of buffers and standards, sample dilution and immunoaffinity 27 column wash solution. NIST Standard Reference Materials were obtained from National Institute of 28 Standard and Technology (Gaithersburg, MD, USA). The immunoaffinity columns, EASI-29 EXTRACT ® Vitamin B12, (catalogue No. P80) are available from R-Biopharm Rhône Ltd 30 (Glasgow, UK).

  subject to light deterioration. Protect analytical work from bright light and use 34 actinic glassware for sample extracts and standard solutions.

1

  Potassium cyanide is a highly toxic colourless crystalline compound. Consequently, solutions and 2 extracts should be handled with extreme care. Gloves and other protective clothing were worn as 3 a safety precaution during the handling of this compound. Potassium cyanide residues can be 4 decontaminated using 15% sodium hypochlorite.

  17

  samples were selected to demonstrate the clean-up capacity of the immunoaffinity 21 columns, these included; NIST Standard Reference Materials, whey-based Infant Formula, high 22 protein powdered drinks and bars, wheat breakfast cereal, carbonated soft drinks, fruit juices and 23 vitamin B12 tablets. Three test portions of each sample were analysed and the mean result it was critical to consider the saturation of the immunoaffinity column and the LOQ 31 of the HPLC-UV system; hence the optimum amount of vitamin B12 loaded onto the 32 immunoaffinity column should be within the range of 0.1-1.5µg. A 5-30g test portion of sample33(depending on label claim) was mixed with 100 ml of 50 mM sodium acetate buffer (pH 4.0), 1 ml 34 potassium cyanide (1%), 0.5 g α-amylase and 2 g pepsin for 10 minutes. The sample was 35 transferred to a shaking water bath set at 37 °C for 30 min followed by a further 30 min incubation 36 in a 100 °C shaking water bath. After cooling, the sample was filtered through a Schleicher &

11 12

 11 Analysis of vitaminB12 content in carbonated soft drinks and fruit juices 13 14 Sample preparation 15 Carbonated soft drinks were degassed by placing the sample in an ultrasonic bath for 10 min and 16 upon removal adjusting the pH to 7.0 with 1M sodium hydroxide solution. For fruit juices, the pH 17 was adjusted to 7.0 with 1M sodium hydroxide solution and centrifuged for 10 min at 4000 rpm.18Subsequently the supernatant was filtered through glass microfibre filter paper (GMF2, Satorius).

19 20

 19 Purification of vitamin B12 from carbonated soft drink and fruit juice extracts21A 20 ml-100 ml aliquot of sample (corresponding to 0.1-1.5 µg vitamin B12) was passed through 22 the immunoaffinity column. The column was washed with 10 ml of water and the vitamin B12 23 eluted with 3 ml methanol. The column eluate was evaporated to dryness by heating (50-60 °C) 24 under a stream of air and reconstituted in 500 µl of 0.025% TFA in water.

  Purification of vitamin B12 from tablet extract 1 Passed 20 ml of the sample extract through the immunoaffinity column. The column was washed 2 with 10 ml water and the vitamin B12 eluted with 3 ml methanol. The column eluate was 3 evaporated to dryness by heating (50-60 °C) under a stream of air and reconstituted in 1 ml of 4 0.025% TFA in water.Samples were analysed using a Dionex HPLC-UV system equipped with Chromeleon software, a 9 UVD 170U/340U detector, a P680 gradient pump, a TCC-100 thermostatted column compartment 10 and sample injections of 100 µl were made from an ASI-100 autosampler (Dionex, CA, USA). The 11 chromatographic separations were performed via an ACE 3 AQ, 150 mm x 3.0 mm column 12 (Hichrom, Reading, UK), at a flow rate of 0.250 ml/min. The mobile-phase consisted of 0.025% 13 TFA in HPLC grade water (solution A) and acetonitrile (solution B) and followed a series of 14 gradients (ratio of solution A: solution B): range 0-3.5 min 100:0, range 3.5-11 min 85:15, range 15 11-19 min 75:25, range 19-20 min 90:10 and range 20-35 min 100:0 (v/v). The column effluent 16 was monitored by UV at 361 nm.

  17

  the stock vitamin B12 (cyanocobalamin) and hydroxycobalamin solutions were

5

  22prepared in water to challenge the immunoaffinity column. When 200 ng vitamin B12 in water was 23 applied to the immunoaffinity column >90% mean recovery (RSD r =2.1%, n=10) was obtained and 24 when 200 ng hydroxycobalamin in water was applied to the column a mean recovery of 75%25(RSD r =1.0%, n=6) was achieved. Pure solutions of adenosylcobalamin and methylcobalamin 26 could not be obtained as hydroxycobalamin was always found to be present; adenosylcobalamin 27 and methylcobalamin are reduced cobalamin derivatives that are unstable and degrade to the 28 hydroxo compound (Hoffbrand 1979). The column was challenged with a mixture of 100 ng vitamin 29 B12 and 200 ng hydroxycobalamin in water; 91% (RSD r =1.7%, n=6) mean recovery of vitamin 30 B12 and 45% (RSD r =1.1%, n=6) mean recovery of hydroxycobalamin was obtained. Hence, both 31 cyanocobalamin and hydroxycobalamin were shown to interact with the antibody on the 32 immunoaffinity column. However, the results demonstrated the antibody had greatest affinity to 33 vitamin B12. This finding is particularly relevant because the synthetic form of cobalamin used in 34 vitamin B12 supplements, pharmaceuticals and in the fortification of food is cyanocobalamin.35 36At the preliminary stages of method development, the effectiveness of methanol and acetonitrile 37 as elution solvents for releasing bound vitamin B12 from the immunoaffinity column were 38 columns were challenged with 1500 ng vitamin B12 in water, washed with 10 ml 1 water and the vitamin B12 eluted with methanol or acetonitrile, the recoveries were >90% (RSD r 2 =6.2%,) and <5% (RSD r =8.3%) respectively. Hence methanol was used as elution solvent for all 3 further work.4Three NIST Standard Reference Materials (NIST 1846 Infant Formula, NIST 1546 Meat 6 Homogenate and NIST 2383 Baby Food Composite) were analysed using immunoaffinity clean

  7

18

  the method was further improved as immunoaffinity clean up removed interfering peaks from 19 HPLC chromatograms allowing accurate determination of vitamin B12 at low levels using λ (max.) 20 of 361 nm. In support of this Choi et al. (2003) showed without prior clean up monitoring at 550 nm 21 was necessary to avoid interfering peaks; monitoring vitamin B12 at 550nm resulted in a decrease 22 in sensitivity.

  NIST 1846, three other commercial brands of infant formula were purchased at a 28 local supermarket and the vitamin B12 content assessed and compared to the label claim. With

1

  Naturally occurring forms of vitamin B12 (methylcobalamin and adenosylcobalamin) are often 2 associated with proteins within food matrices. Pepsin was added to the extraction mixture prior to 3 incubation at 37 °C to aid protein digestion and release protein bound methylcobalamin and 4 adenosylcobalamin. Also α-amylase was added to promote digestion of the starch content of the 5 sample and aid filtration. The methylcobalamin and adenosylcobalamin were converted to 6 cyanocobalamin (vitamin B12) by the addition of potassium cyanide; the cyano group replaces the 7 methyl or adneosyl group to yield vitamin B12 (Heudi et al. 2006).

  flake sample 107% of the declared level (1.7 µg/100 g) was obtained with RSD r of 22 4.4% ; this is within ± 15% of the declared value and complies with EU legislation (European 23 Commission Directive 2002). Cereals are a challenging matrix because they absorb much of the 24 extraction buffer and generally contain low level vitamin B12. The use of immunoaffinity columns 25 to purify the vitamin from the sample filtrate coupled with the evaporation of the column eluate, 26 concentrates the vitamin B12 sufficiently to allow detection by HPLC-UV. 27 28 Vitamin B12 sample preparation procedures have the dual purpose of liberating protein-bound 29 cobalamins and converting the naturally occurring labile forms to cyanocobalamin. Consequently 30 total vitamin B12 content can be determined by the quantification of cyanocobalamin. However to 31 prevent the use of toxic cyanide solutions, required to form cyanocobalamin, it is possible to 32 convert all the vitamin B12 to sulphitocobalamin by reaction with metabisulphite (Na 2 S 2 O 5 )33(Eitenmiller 1999). To establish if this route could be a practical solution and so avoid the use of 34 cyanide solutions, optimisation of the HPLC-UV procedure would be required to assess the affinity 35 of the antibody present on the immunoaffinity column to sulphitocobalamin. An alternative solution 36 to this problem is to derive the α-ribozole, a fluorescent marker for the HPLC determination of foodstuffs(Pakin et al. 2005). The disadvantage associated with this method is the 1 overnight incubation required to form the α-ribozole.

  content of 2 different brands of vitamin B12 supplement tablets were analysed to 31 compare the percentage of vitamin B12 recovered relative to the declared value. Brand 1 with a 32 declaration of 2000 µg/0.849 g, yielded 100% (RSD r = 3.1%) of the declared vitamin value and

3the

  In conclusion the immunoaffinity column isolates and concentrates vitamin B12 from a wide range 4 of commodities prior to quantitative HPLC-UV determination at 361nm resulting in improved 5 sensitivity. Robust and reliable (RSD r = 0.8-10%) procedures for the accurate determination of 6 vitamin B12 in complex matrices (NIST 2383) containing low level vitamin B12 (0.44 µg/100 g) as 7 well as simpler matrices such as vitamin tablets containing high levels (2000 µg/0.849 g) of vitamin 8 B12 were developed. The analysis of NIST Standard Reference Materials demonstrates the 9 immunoaffinity column and the microbiological assay generate comparable results in complex 10 matrices. In addition the method is rapid, inexpensive, and easy to use compared to existing 11 methods for vitamin B12 analysis. Council of 10 June 2002. Official Journal of the European Communities. L183:51-57. 20 21 Food Standards Agency. 2003. Expert Group on Vitamins and Minerals Risk Assessment.

  Kilinç T, Fontannaz P, Marley E. 2006. Determination of Vitamin B12 in food products

  24 and in premixes by reversed-phase high performance liquid chromatography and immunoaffinity 25 extraction. J. Chromatogr. A. 1101:63-38. 26 27 Hoffbrand AV. 1979. Nutritional and biochemical aspects of Vitamin B12 in the importance of 28 vitamins to human health. TG Taylor. MTP Press Ltd Lancaster.. 41-46. 29 30 Pakin C, Bergaentzle M, Aoude-Werner D, Hasselmann C. 2005. α-Ribazole, a fluorescent marker 31 for liquid chromatographic determination of vitamin B12 in foodstuffs. J Chromatogr. A. 1081:182-M. 1992. Folate-Vitamin B12 interrelationships in the central nervous system. Proc. Nutr.

Figure 1 13

 1 Figure1

Figure 1

 1 Figure 1 Chromatograms obtained for the determination of vitamin B12 in (a) NIST Milkbased Infant Feed 1846 (3.9 µg/100 g), (b) NIST Meat Homogenate 1546 (0.6 µg/100 g) and (c) NIST Composite baby food (0.44 µg/100 g)

Table 1 . Percentage recoveries of vitamin B12 from a range of NIST SRM and Infant Formula

 1

	Product	Claim (µg/100 g)	Acceptable range (µg/100 g)	Amount determined (µg/100 g) (n=3)	Recovery relative to claim (%)	RSD r (%) 1
	NIST Meat Homogenate 1546	0.6	0.5-0.7 a	0.5	83	2.9
	NIST Baby Food Composite 2383 NIST Infant Formula 1846	0.44 3.9	0.25-0.63 a 3.6-4.2 a (3.23-4.49) b	0.55 4.1	125 106	2.1 0.8
	Brand 1 Plus infant Formula	1.37	1.16-1.58 b	1.13	82	1.6
	Brand 2 infant Formula	1.25	1.06-1.44 b	1.47	118	5.0
	Brand 3 Infant Formula	1.30	1.11-1.50 b	1.48	114	5.9
	a As defined by NIST
	b As defined by EU Legislation (± 15% of declaration)
	RSDr represents the repeatability when analysing 3 test portions of each sample.

Table 2 . Percentage recoveries of vitamin B12 from protein drinks and bars

 2 RSDr represents the repeatability when analysing 3 test portions of each sample.

	Product	Claim	Recovery relative to claim (%)	RSD r (%) 1
	Protein powdered drink 1	5.26 µg/0.580 g	103	2.7
	Protein powdered drink 2	5.26 µg/0.580 g	94	10.2
	Protein bar A	3.5 µg/100 g	124	7.8
	Protein bar B	0.90 µg/100 g	166	8.8
	Protein bar C	0.94 µg/100 g	183	5.4

Table 3 . Percentage recoveries of vitamin B12 fortified and spiked energy drinks and fruit drinks

 3 RSDr represents the repeatability data in analysing 2 different energy drinks at 3 levels of vitamin B12 using 3 test portions of juice at each level.RSDr represents the repeatability data in analysing 4 different types of fruit juice drinks spiked at 1 µg/100ml vitamin B12 using 3 test portions of juice. % Recovery values were corrected for natural vitamin B12 content.

	Product	Initial concentration (µg/100 ml)	Added concentration (µg/100 ml)	Concentration found (µg/100 ml)	Recovery (%)	RSD r (%) 1
	Activation energy drink	0.1	2	1.2	95	9.9
	Activation energy drink	0.1	20	17.1	85
	Rejuvenation fruit drink	0.1	2	2.1	99
	Rejuvenation fruit drink	0.1	15	12.7	84
						RSD r (%) 2
	Apple juice	Not declared	1	0.76	78	6.5
	Orange and peach juice	Not declared	1	0.96	86
	Blackcurrant
	and apple	Not declared	1	0.94	82
	juice
	Strawberry juice	Not declared	1	0.90	82
	1

2

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

European Commission Directive. 2002. Directive 2002/46/EC of the European Parliament and of


Chun-Kwan Wong

Patricia Hung

Kellie L H Lee

Kai-Man Kam

Solid-phase extraction clean-up of ciguatoxin-contaminated coral fish extracts for use in the mouse bioassay

Keywords: Clean-up, Extraction, Toxicology -in-vivo Additives/Contaminants: Ciguatoxins, Natural toxicants Food Types: Fish, Fish Ciguatera fish poisoning, Ciguatoxin recovery, Florisil, Mouse bioassay, Solid-phase extraction

Solid-phase extraction clean-up of ciguatoxin-contaminated coral fish extracts for use in the mouse bioassay

Chun-Kwan Wong

Introduction

Ciguatera fish poisoning (CFP) is the commonest marine biotoxin illness in the world [START_REF] Isbister | Neurotoxic marine poisoning[END_REF]. The disease is due to consumption of toxic species of coral reef fish captured in the circumtropical regions. These fish bio-accumulate lipid-soluble sodium channel toxins, ciguatoxins (CTXs), in their flesh and viscera via marine food chain until reaching humans to cause food intoxication. Toxic forms of epiphytic benthic marine dinoflagellates inhabiting in the coral reef areas such as Gambierdiscus toxicus are believed to be the toxin-causative microalgae [START_REF] Holmes | Strain dependent production of ciguatoxin precursors (gambiertoxins) by Gambierdiscus toxicus (Dinophyceae) in culture[END_REF][START_REF] Lewis | Origin and transfer of toxins involved in ciguatera[END_REF][START_REF] Lewis | The changing face of ciguatera[END_REF]. CFP is characterized by gastrointestinal, neurological and cardiovascular disorders [START_REF] Palafox | Ciguatera fish poisoning: review of clinical manifestations[END_REF][START_REF] Pearn | Neurology of ciguatera[END_REF][START_REF] Pottier | Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies[END_REF][START_REF] Keynan | Neurological symptoms in a traveler returning from Central America[END_REF]. The mechanism of toxicity is elicited by the binding ability of CTXs to site 5 on the voltage-dependent sodium channel of excitable tissues [START_REF] Lewis | Ciguatera toxins, pharmacology of toxins involved in ciguatera and related fish poisoning[END_REF][START_REF] Pearn | Neurology of ciguatera[END_REF]).

With the advance in global seafood business and international transportation, endemic disease of CFP has not been confined to certain tropical and subtropical areas (between 35 o north and 35 o south latitude) of the Pacific Ocean, western Indian Ocean and the Caribbean Sea [START_REF] Lehane | Ciguatera fish poisoning: a review in a risk-assessment framework[END_REF][START_REF] Goodman | Ciguatera poisoning in Vanuatu[END_REF][START_REF] Wong | Study of an outbreak of ciguatera fish poisoning in Hong Kong[END_REF]. The disease potentially poses large socio-economic and public health impacts in communities where fish is the major source of protein in the normal diet.

CTXs are amongst the most potent toxins (LD 50 = 0.25 µg/kg, intraperitoneal injection in mice for Pacific-CTX-1) known in the world that cause significant health impact on humans even at the level [START_REF] Lewis | Purification and characterization of ciguatoxins from moray eel (Lycodontis javanicus, Muraenidae)[END_REF][START_REF] Lehane | Ciguatera fish poisoning: a review in a risk-assessment framework[END_REF][START_REF] Lehane | Ciguatera: a recent advances but the risk remains[END_REF][START_REF] Halstead | The microbial biogenesis of aquatic biotoxins[END_REF]). The application of ordinary freezing or cooking does not destroy the toxins. In spite of the high toxicity and common morbidity of CTXs, much effort has been made to deal with toxicological and therapeutic mechanisms. Their research and development are however hindered by the lack of commercial CTX standards and surrogates, as well as the insufficient ciguateric fish for toxin extraction. There are also difficulties and complexities in laboratory synthesis of toxins by culturing CTX-producing dinoflagellates [START_REF] Legrand | Isolation and some properties of ciguatoxin[END_REF][START_REF] Holmes | Strain dependent production of ciguatoxin precursors (gambiertoxins) by Gambierdiscus toxicus (Dinophyceae) in culture[END_REF]. The present knowledge on CTXs relied mainly on field sampling of CTX-contaminated fish for studying toxin extraction methods, mechanisms, and structures of toxin congeners. Considerable efforts has gone into development of different diagnostic methods for CTXs detection and analysis such as immunoassays and chemical analysis by LC/MS (Manger et al. 1995;[START_REF] Se | Ciguatera toxins: chemistry and detection[END_REF][START_REF] Lewis | Ciguatera toxins, pharmacology of toxins involved in ciguatera and related fish poisoning[END_REF][START_REF] Lewis | Detection of toxins associated with ciguatera fish poisoning[END_REF]). The widely recommended method for CTXs testing in coral fish samples is still based on the mouse bioassay of fish ether extracts [START_REF] Hoffman | The mouse ciguatoxin bioassay: a dose-response curve and symptomatology analysis[END_REF][START_REF] Vernoux | The mouse ciguatoxin bioassay: directions for use to control fish for consumption[END_REF][START_REF] Ting | Ciguatera poisoning: a global issue with common management problems[END_REF]. Until establishment of a validated alternative analytical method for CTXs detection [START_REF] Hungerford | Committee on natural toxins and food allergens[END_REF], the mouse bioassay will still play an important role in ciguatera research and public health protection. Continuous development and improvement of the existing methodologies for this toxicity bioassay are needed for enhancing detection and accurate determination of CTXs in the fish tissues.

Florisil is a co-precipitate of silica and magnesia normal phase sorbent that has been commonly used to remove co-extracted interferences and contaminants in a variety of environmental and food samples [START_REF] Lopez-Avila | Cleanup of environmental sample extracts using Florisil solid-phase extraction cartridges[END_REF][START_REF] Schenck | Florisil solid-phase extraction cartridges for cleanup of organochlorine pesticide residues in foods[END_REF]. Its slightly basic surface may facilitate the adsorption of low to moderate polarity compounds from organic solutions. It has also been adopted to purify and isolate CTX-containing fraction from crude ether extract prior to analysis by analytical instruments such as HPLC/MS [START_REF] Lucas | Pacific ciguatoxin-1 associated with a large common-source outbreak of ciguatera in East Arnhem Land[END_REF][START_REF] Vernoux | Isolation and characterization of Caribbean ciguatoxins from the horse-eye jack (Caranx latus)[END_REF][START_REF] Hamilton | Isolation and characterisation of Indian Ocean ciguatoxin[END_REF].

In the present study we have investigated the efficacy of applying a commercially available Florisil solid-phase extraction cartridge to purify and isolate CTX-containing fraction from crude fish lipid extract, with the aim of studying the potential use of adding a clean-up step in routine laboratory test for improving the purity of coral fish ether extracts.

Materials and methods

Coral fish samples preparation

Coral fish species used for this study included Cheilinus undulates (humphead wrasse), Epinephelus areolatus (areolated grouper), Epinephelus lanceolatus (giant grouper), Epinephelus polyphekadion (camouflage grouper), Lutjanus stellatus (star snapper), Plectropomus areolatus (squaretail coralgrouper), Plectropomus leopardus (leopard coralgrouper) and Variola albimarginata (lyretail) (Table 1). All coral reef fish samples were collected during different periods from markets in Hong Kong, except a piece of giant grouper flesh, which was provided by a donor who had suffered from ciguatera symptoms after consumption of this fish flesh. Two retrospective coral fish samples, X and Y (CTX-positive samples), were used as positive controls for this study. All samples were kept

Sample extraction

The fish flesh (50/100 g) was cooked at 70 o C water bath for 15 min in a water bag. Sample was cooled in refrigerator, or put in -80 o C freezer for 5 min. The cooled fish sample was minced and extracted with acetone. A lipid-soluble extract was obtained by liquid-liquid partitioning procedures as previously described [START_REF] Wong | Study of an outbreak of ciguatera fish poisoning in Hong Kong[END_REF]. The final dried lipid ether extract was stored in -20 o C freezer prior to testing.

Mouse bioassay

In order to analyze the toxicity and potential presence of CTXs in the coral fish samples, all ether extracts were first screened for CTXs using a validated mouse bioassay as previously described [START_REF] Wong | Study of an outbreak of ciguatera fish poisoning in Hong Kong[END_REF]. The mice were closely monitored (1-h interval) for 6 h after injection. Raised bottom grids with white background were inserted into the bottom of the cages for monitoring the condition of diarrhea easily. Video camera was set to record the experimental setup for at least 24 h to determine the mice survival time and toxic symptoms beyond 6 h. The mice were then monitored for another 4 consecutive days. Rectal temperature and body weight were also recorded at days 1 and 4, respectively. All experimental mice used in the study were sacrificed in accordance with stipulated ethical standards and guidelines.

Spiked coral fish samples

Among 16 coral fish samples used in this study ( and giant grouper (Gg)) were detected to have trace amount of CTXs at <3.6 MU/100g flesh and ~4.7-9.4 MU/100g flesh, respectively. Others were determined to be CTX free (CTX-negative) after the examination. Five pieces of raw coral fish flesh (CTX-negative) including a humphead wrasse (Hw2), two squaretail coralgroupers (Sc2 and Sc2-a) and two leopard coralgroupers (Lc2 and Lc3) were spiked with pure Pacific-ciguatoxin-1 (P-CTX-1) purchased from Prof. Richard J.

Lewis (University of Queensland, Australia). The P-CTX-1 was dissolved in chloroform-methanol (1:1, v:v) and evenly spiked into the raw coral fish flesh (500 ng P-CTX-1/100g flesh) carefully.

The spiked flesh samples were kept in room temperature for ~15 min before storage in -80 o C freezer. The samples were defrosted in 4 o C refrigerator a day prior to sample extraction.

Purification of fish lipid extract (ether extract) with Florisil

Fish ether extracts were purified using commercially available Florisil cartridges (Waters, USA).

The elution procedures were based on the method previously described by [START_REF] Vernoux | The mouse ciguatoxin bioassay: directions for use to control fish for consumption[END_REF]Lewis (1997), and[START_REF] Hamilton | Isolation and characterisation of Indian Ocean ciguatoxin[END_REF], with slightly modifications. The CTX-containing fraction was purified by eluting the cartridge with hexane-acetone-methanol solvent mixtures of increasing polarity. Briefly, ether extract (20 or 40 mg) was dissolved in 0.8 ml of hexane-acetone (4:1, v:v) and loaded onto a 125 µm 60Å Florisil cartridge pack (Waters, 3 ml, 500 mg, 0.8 ml bed volume) which had been pre-washed with three bed volumes (V b ) of acetone-methanol (7:3, v:v) and then five V b of hexane-acetone (4:1, v:v). Fraction hexane-acetone (fraction HA) was eluted into a sample vial with four additional V b of hexane-acetone (4:1, v:v) solvent mixture which was applied to remove hydrophobic contaminants in the lipid extract. The target acetone-methanol (fraction AM) fraction, was subsequently collected by applying eight V b of acetone-methanol (7:3, v:v) solvent mixture onto the cartridge. Three additional methanol (M) fractions (M1, M2 and M3) were collected separately by eluting two V b of 100% methanol for fraction M1 and five V b of 100% methanol twice, respectively for fractions M2 and M3. Therefore, each fraction collected was equivalent to the corresponding quantity contained in 20 or 40 mg of crude ether extract. Residues of ether extract (fraction ERC) retained inside the cartridge were discarded. Extraction of fish ether extracts through Florisil cartridge was performed by using Vacuum Manifold Processing Station (Agilent) under atmospheric gravity. All eluted fractions (fractions HA, AM, M1, M2 and M3) were collected into V-Vials (Wheaton), dried under nitrogen gas, weighed by electronic balance and then stored in -20 o C freezer until testing for CTX toxicity in each fraction with validated mouse bioassay. Each dried fraction was suspended in 0.5 ml of 1% Tween 60/0.9% saline, sonicated for 5-10 min to suspend the dried fraction, heated at 37 o C and injected carefully into mice by i.p. route.

In order to study extraction efficiency of the Florisil cartridge and potential toxicity of CTX in each fraction, different conditions of acetone-methanol solvent mixtures (9:1 and 7:3, v:v) and lipid extract quantities (20 and 40 mg) were tested for establishing optimal condition for CTX recovery and isolation.

P-CTX-1 for calibration and toxicity study

The calibration equation log (MU) = 2.3 log (1+1/T) was used to establish an in-house CTX [START_REF] Lewis | Recovery of ciguatera from fish flesh[END_REF][START_REF] Lewis | Detection of ciguatoxins and related benthic dinoflagellate toxins: in vivo and in vitro methods[END_REF][START_REF] Lewis | Detection of toxins associated with ciguatera fish poisoning[END_REF]. MU and T represent the number of mouse unit and survival time (hours), respectively for each mouse. The quantity of CTXs in the sample was evaluated by determining dose (log MU) versus time-to-death (log (1+1/T)) relationship from local mice species (female ICR mice) using pure P-CTX-1. Dosages between 0.01 and 12.5 MU (62.5 pg-70 ng) of P-CTX-1 were applied to study their toxic effects on mice and to differentiate the range of lethal and sub-lethal doses. A lethal dose calibration was established by injecting diluted standard toxins (in the range of 10-70 ng/0.5 ml of 1% Tween 60/0.9% saline) into mice through i.p. route. LD 50 was determined by analyzing relationship between injected dose (d) and survival time (T) using the derived equation log d = a log (1+1/T) + b, where d represents dosage in ng/kg, T is survival time in hours, a and b are minimum survival time and extrapolated smallest dose that kills mice at infinite time, respectively [START_REF] Molinengo | The curve doses vs survival time in the evaluation of acute toxicity[END_REF][START_REF] Meier | Approximate LD 50 determinations of snake venoms using eight to ten experimental animals[END_REF][START_REF] Legrand | Isolation and some properties of ciguatoxin[END_REF]. A linear relationship between log d and log (1+1/T) was produced with intercept on y-axis for evaluating the value of LD 50 . Overall, 47±4.6% (mean±S.E.M.) of 20 mg ether extract was eluted out while 53±4.6% of residues (ERC) were retained inside the cartridge after purification. Among the eluates, it composed of 3.0±0.7% HA, 15±2.9% AM, 29±2.7% M (M1, M2 and M3). The percentage of target fraction (fraction AM) isolated from the crude extracts ranged from 2.5 to 33% (0.6 to 7.8 mg). The average weight of target fraction AM isolated from the 20 mg ether extracts was 3.7±0.5 mg under the acetone-methanol (7:3, v:v) solvent mixture condition. Over 80% of components from the 20 mg crude ether extracts were removed after Florisil purification. This represents the clean-up efficiency of the cartridge on the target fraction AM. 52±7.8% of residues (ERC) were retained inside the cartridge after the purification. Among the eluates, it composed of 4.4±1.8% HA, 21±5.1% AM, 31±3.7% M (M1, M2 and M3). The percentage of target fraction (fraction AM) isolated from the crude extracts ranged from 7.1 to 42%

Results

220±10

mg of ether extracts from P-CTX-1-spiked and CTX-positive samples

(1.8 to 11 mg). The average weight of target fraction AM isolated from the 20 mg ether extract was 4.8±0.6 mg under the acetone-methanol (7:3, v:v) solvent mixture. By using the condition of acetone-methanol (9:1, v:v) solvent mixture instead of the (7:3, v:v) condition for performing Florisil purification on Sc2 extracts (n=4), the amount of target fraction AM (1.1±0.3%) collected was only 5.6% of those collected under the acetone-methanol (7:3, v:v) condition. The percentages of residues retained inside the column after purification were 43±3.7% and 75±3.1% under the elution of acetone-methanol (7:3, v:v) and (9:1, v:v), respectively. With regard to the clean-up efficiency of the cartridge on the target fraction AM, over 70% of components from the 20 mg crude ether extracts were removed after Florisil purification under the condition of acetone-methanol (7:3, v:v) solvent mixture. v:v) and (7:3, v:v) solvent mixtures, were used to elute target fraction AM from the 40 mg crude ether extracts. Under the condition of acetone-methanol (9:1, v:v) solvent mixture, 4.2±0.2% of target fraction AM was collected from both ether extracts of spiked samples Sc2 and Lc3. By contrast, 28±6.8% of such fraction could be obtained by using the condition of acetone-methanol (7:3, v:v) solvent mixture. According to the two acetone-methanol solvent mixture conditions applied, the latter one eluted more amount of fraction AM and the residues remained inside the cartridge (39±3.2%) was also less than those of the acetone-methanol (9:1, v:v) elution (59±8.2%).

Overall, 55±6.7% of 40 mg ether extract was eluted out while 45±6.7% of residues (ERC) were retained inside the cartridge after the purification under the condition of acetone-methanol (7:3, v:v) solvent mixture. Among the eluates, it composed of 12±3.6% HA, 15±5.9% AM, 27±5.1% M (M1, M2 and M3). The percentages of target fraction AM isolated from the 40 mg crude extracts ranged from 1.9 to 38% (0.9 to 20 mg). The average weight of target fraction AM isolated was 8.4±3.1 mg.

With regard to the clean-up efficiency of the cartridge for extracting the target fraction AM, over 70% of components from the 40 mg crude ether extracts were removed after Florisil purification under the elution of acetone-methanol (7:3, v:v) solvent mixture.

In addition to fractions HA and M3 collected from 40 mg ether extracts, there was no significant difference (P > 0.

Mouse bioassay analysis

In this study, 5.6 ng of pure P-CTX-1 was determined as LD 50 for 20 g mice according to the dose versus time to death relationship curve of standard P-CTX-1. Intraperitoneal injection of 20 mg ether extract of CTX-negative samples to each mouse did not elicit any typical toxic symptoms of CTXs. Rectal temperatures of the treated mice were at 37.3±0.0 o C (mean±S.E.M.) (n=33) and their body weight showed average 0.0±0.1 g (mean±S.E.M.) and 2.1±0.2 g increase at days 1 and 4, respectively. Mice injected with Tween 60/0.9% saline showed rectal temperature of mice maintained at 37.3±0.0 o C (n=32) and their body weight increased 0.2±0.1 g and 2.2±0.1 g at days 1 and 4, respectively, during the experimental period. Body weight gain at days 1 and 4 were Recovery of spiked P-CTX-1 ranged from 32.6 to 69.4% (29.1 to 62.0 MU/100g flesh) for six spiked P-CTX-1 coral fish samples including a humphead wrasse (Hw2), three squaretail coralgroupers (Sc2 Pt1, Sc2 Pt2 and Sc2-a), and two leopard coralgroupers (Lc2 and Lc3) (Table 2).

Analysis of the results revealed that recovery of spiked P-CTX-1 for the six fish samples averaged 44.8±5.2%. Overall, the degree of P-CTX-1 recovery also varied between the tested samples. By comparing P-CTX-1 recovery between three samples of squaretail coralgroupers (Sc2 Pt1, Sc2 Pt2 and Sc2-a), no significant difference (P > 0.05; Student's t-test) could be found between the samples from the same species of fish and between the samples taken from the same piece of fish flesh.

Significant difference in P-CTX-1 recovery (P < 0.05; Student's t-test), however, was found between the two leopard coralgrouper samples (Lc2 and Lc3) of same species. All mice injected with 20 mg ether extracts of the spiked P-CTX-1 samples (Hw2, Sc2 Pt1, Sc2 Pt2, Sc2-a, Lc2 and Lc3) experienced significant hypothermia within an hour without recovery and died within 6 h.

Recovery of spiked P-CTX-1 from coral fish flesh after Florisil extraction

For 20 mg ether extracts that passed through Florisil cartridge, P-CTX-1 was retrieved in fraction AM by applying the acetone-methanol (7:3, v:v) solvent condition. P-CTX-1 recovery for all tested samples was improved (overall recovery increased by more than 90%) as compared with those without Florisil purification (Table 2). The recovery percentage of spiked P-CTX-1 in samples Hw2, Sc2 Pt2, Sc2-a and Lc2 increased from 32.6 to 76.3%, 41.3 to 84.8%, 43.1 to 72.5% and 38.9 to 69.7%, respectively. The average recovery percentage of the tested samples after Florisil extraction was 75.8±3.3%. On the other hand, the average recovery percentage of P-CTX-1 for two squaretail coralgroupers (Sc2 Pt2 and Sc2-a) under the elution of acetone-methanol (9:1, v:v) solvent mixture maintained at 43.4%, which was comparable to that without performing purification through Florisil cartridge. In addition, there was no significant difference (P > 0.05; Student's t-test-Paired) in recovery of spiked P-CTX-1 between samples Sc2 Pt2 and Sc2-a of same species. Further application of 40 mg ether extract (twofold dosage) for Florisil purification showed that no significant improvement could be found even under the acetone-methanol (7:3, v:v) condition.

Similar results were also found by application of 40 mg ether extract of sample Lc3 to pass through Florisil cartridge under both conditions (7:3 and 9:1, v:v) of acetone-methanol solvent mixture separately. Figure 1 shows the rectal temperature patterns in mice injected with both crude ether (non-Florisil purified) extracts and fraction AM (Florisil purified) of the P-CTX-1-spiked samples (Sc2 Pt2, Sc2-a, Lc2 and Hw2). There was a similar pattern and trend of hypothermia in both sets of mice. In general, mice survival times showed characteristic difference between the toxicity of crude ether (non-Florisil purified) extracts and AM fractions (Florisil purified). Mice receiving AM fractions experienced a much more rapid death than those receiving non-Florisil purified ether extracts.

Other fractions of eluates including HA, M1, M2 and M3 were also collected by applying 20 or 40 mg ether extracts of CTX-negative, CTX-positive and P-CTX-1-spiked samples to pass through Florisil cartridges under acetone-methanol (7:3, v:v) solvent mixture condition. Mouse bioassay analysis showed no significant toxic effect in mice injected with those fractions of eluates. The average rectal temperature of mice was 37.5±0.0 o C during the course of experiment while the body weight increased 0.0±0.0 g and 2.2±0.1 g, respectively at days 1 and 4, respectively.

Toxicity of CTXs from CTX-positive samples before and after Florisil purification

Toxicity of CTXs from CTX-positive samples before Florisil purification

Mouse bioassay results from two CTX-positive and two retrospective field samples (CTX-positive) are summarized in Table 3. For mice injected with 20 mg ether extracts of samples Hw3 (Humphead wrasse) and Gg (Giant grouper), sub-lethal doses of CTXs, <3.6 MU/100g flesh and 4.7-9.4 MU/100g flesh respectively, were determined by using validated mouse bioassay. Signs of It should be noted, however, that significant body weight changes (2.2±0.3 g decrease) and growth depression (0.0±0.9 g increase) were observed at days 1 and 4, respectively. Sublethal dose of CTXs present in sample Hw3 was then estimated to be less than 3.6 MU/100g flesh.

Intraperitoneal injection of 20 mg ether extracts of Gg into mice (n=7), during 6-hour observation period at day 0, caused a decrease in rectal temperature (Figure 2), which ranged between 30.7 and 35.6 o C (averaged at 33.4±0.2 o C). Hypothermia (rectal temperature below 33 o C) of treated mice was more conspicuous after 3 h observation. Lower rectal temperature (34.2±0.9 o C) in mice was also detected at day 1, which returned to normal at day 4 (37.1±0.1 o C). It should be noted that significant body weight change (2.5±0.4 g decrease) and growth depression (0.5±0.7 g increase)

were also observed at days 1 and 4, respectively. Sublethal dose of CTXs in sample Gg was estimated to be 4.7-9.4 MU/100g flesh. Hypothermia in mice during the course of experiment was more prominent than those obtained after injection with 20 mg ether extract of Hw3 (non-Florisil purified). In addition, hypothermia persisted (32.1±3.5 o C) together with significant body weight decrease (1.5±0.7 g decrease) at day 1. One of the mice died at day 4 while the one that survived still experienced persistent lower rectal temperature (35.7 o C) and a significant (3.0 g) decrease in body weight. On the other hand, injection of fraction AM of sample Gg into mice (n=2) induced significant hypothermia within an hour (Figure 2). The rectal temperature of the mice during 6-hour period ranged between 24.2 and 32.8 o C (averaged at 29.7±0.6 o C). Hypothermia in mice during the course of experiment was also more prominent than those injected with 20 mg ether extract of Gg (non-Florisil purified). The treated mice died at 6.6±0.2 h with terminal convulsion. The amount of CTXs was estimated to be at sublethal dose ranging from 6.5 to 12.9 MU/100 g flesh for Hw3 and lethal dose of 25.4 MU/100g flesh for Gg, respectively.

For mice (n=2 for each set) injected with each fraction of other eluates (HA, M1, M2 and M3) from depression was detected in mice at days 1 (0.0 ±0.1 g increase and 0.3. ±0.2 g decrease for Hw3 and Gg, respectively) and 4 (2.0 ±0.2 g increase and 2.1 ±0.2 g increase for Hw3 and Gg, respectively) for both samples.

For the two retrospective samples X and Y (positive control samples), there was significant enhancement of CTXs toxicity (22.2 to 33.6 MU/100g flesh) (P < 0.05; Student's t-test-Paired) on mice after injecting Florisil-purified fraction AM of sample X (Table 3). No prominent toxicity change, however, was found in mice injected with Florisil-purified fraction AM of sample Y.

Nevertheless, the agreement in rectal temperature patterns between mice injected with fraction AM and crude ether extract of sample Y showed that their toxic responses were comparable to each other (data not shown). These results confirmed that CTXs could mostly be isolated in fraction AM from 20 mg ether extract of both samples X and Y by applying a Florisil purification step under the condition of acetone-methanol (7:3, v:v) solvent mixture. On the other hand, no conspicuous sign of toxic effect or CTX poisoning symptom was found in mice injected with each fraction of the other eluates (HA, M1, M2 and M3) from both retrospective samples.

Discussion

Since reliable rapid kits are lacking [START_REF] Pearn | Neurology of ciguatera[END_REF] for only ciguatoxins (CTXs) detection, nor are there validated extraction and clean-up procedures for chemical analysis, information on the mouse bioassay is still central to the toxicological study and detection of CTXs in coral fish. In order to prevent micro-epidemic outbreak of ciguatera, surveillance and detection of CTXs in coral fish samples is one of the better means to prevent people from suffering ciguatera intoxication [START_REF] Johnson | Miscellaneous natural intoxicants[END_REF]. Most of ciguateric fish that had been implicated in clinical ciguatera or sporadic outbreak cases, have very often been eaten or not easily available for further laboratory analysis.

Therefore, fish samples collected from these cases are vital for checking the presence and toxicity of CTXs using traditional mouse bioassay. However, results of the bioassay often depended on toxicity responses of mice injected with crude ether extracts, which are partially purified from standard extraction of coral fish flesh. Both quantity and quality of lipid residues or components in the ether extracts may vary between different fish species and sizes. The previously described standard extraction method has a CTX recovery at ~63%, ranging between 49 and 71%, from studies using CTX-spiked samples [START_REF] Lewis | Recovery of ciguatera from fish flesh[END_REF][START_REF] Lewis | Detection of ciguatoxins and related benthic dinoflagellate toxins: in vivo and in vitro methods[END_REF]. This method has also been documented to be useful in extracting CTXs from ciguateric fishes [START_REF] Lewis | Detection of ciguatoxins and related benthic dinoflagellate toxins: in vivo and in vitro methods[END_REF][START_REF] Lewis | Detection of toxins associated with ciguatera fish poisoning[END_REF]. [START_REF] Lewis | Recovery of ciguatera from fish flesh[END_REF] revealed that there was no significant interference of lipids on mice if standard toxins were added to 20 mg ether extracts of unspiked coral fish. The present study showed that such matrix interference might exist by spiking the standard Pacific-CTX-1 (P-CTX-1) into raw flesh of certain coral fish. From the results of recovery test on P-CTX-1-spiked samples, the average The extraction efficiency of Florisil cartridges varies with the specified gradient of solvent mixtures. Increasing polarity (hexane-acetone (4:1, v:v) < acetone-methanol (7:3, v:v) < 100% methanol) optimized the overall recovery of CTXs in the target fraction AM. The application of 20 mg fish ether extract facilitated the separation of ~17% (~4.2 mg) of target fraction AM with good retention of standard toxin. Although lipid extraction efficiency of Florisil cartridges (such as percentages of the target fraction AM collected and residues remained inside the cartridge) on both 20 mg and 40 mg ether extracts were comparable to each other, the recovery efficiency for CTXs showed a better result by using 20 mg ether extract for performing this Florisil extraction. In addition, there was significant difference in toxin recovery by using different conditions of acetone-methanol solvent mixture (9:1, v:v) and (7:3, v:v). The latter condition optimized the overall recovery of CTXs from the ether extracts. The recovery of standard toxin from spiked samples under the condition of acetone-methanol (9:1, v:v), the eluting solvent condition previously used with Florisil by [START_REF] Lucas | Pacific ciguatoxin-1 associated with a large common-source outbreak of ciguatera in East Arnhem Land[END_REF] and [START_REF] Hamilton | Isolation and characterisation of Indian Ocean ciguatoxin[END_REF], was less efficient than that under the condition of acetone-methanol (7:3, v:v) applying 40 mg (twofold dose) ether extracts for such purification, however, showed that no significant improvement on CTX recovery could be demonstrated in mouse bioassay by injecting with the target fractions. This may be due to overloading of the cartridge by the 40 mg ether extract, or some CTXs were still trapped or retained on the adsorbent surfaces or lipid matrix without eluting out together with the target fractions. Poor elution efficiency and toxin recovery found by using acetone-methanol (9:1, v:v) condition could also be explained by the fact that most of CTXs were still retained on Florisil adsorbent surface before complete elution of the target analytes from the cartridge.

In order to eliminate the possibility of interferences, the maximum amount of extract used for CTX test in mouse bioassay is set at 20 mg for a 20 g mouse by intraperitoneal route [START_REF] Lewis | Recovery of ciguatera from fish flesh[END_REF]. Extraction through Florisil cartridge using the condition of acetone-methanol (7:3, v:v) solvent mixture facilitated the separation of 4.2±0.4 mg target fraction AM from 20 mg ether extracts (CTX-positive, CTX-negative and CTX-spiked samples). The target fraction collected was acceptably below the maximum level of extract required for the bioassay. Therefore, if necessary, an amount of pooled target fractions below the maximum level requirement may be administered into mice for eliciting more conspicuous toxic responses of CTXs, especially for borderline cases.

In summary, a Florisil solid-phase extraction step using acetone-methanol (7:3, v:v) condition demonstrated an efficient way to purify and isolate ciguatoxins containing fraction from ciguateric

  80 o C in Biotoxin Laboratory upon collection and were then put into 4 o C refrigerator one day before extraction.

  .7 mg (mean±S.E.M.) of fish ether extracts, ranged between 135 and 285 mg, were obtained from each 100 g of fish samples after extraction.Extraction and isolation efficiency of Florisil solid-phase extraction cartridge 20 mg of ether extracts from CTX-negative samples 20 mg ether extracts were obtained from nine CTX-negative samples, including two humphead wrasses (Hw1 and Hw2), two camouflage groupers (Cg1 and Cg2), two areolated groupers (Ag1 , a squaretail coralgrouper (Sc1), a leopard coralgrouper (Lc1) and a lyretail (Lt). These were passed through Florisil cartridges for purification and fractionization under the acetone-methanol (7:3, v:v) solvent mixture condition. Different eluates (fractions HA, AM, M1, M2 and M3) were obtained after Florisil purification of 20 mg of CTX-negative extracts (n=18).

  For P-CTX-1-spiked and CTX-positive samples, 20 mg ether extracts from six samples including three P-CTX-1-spiked samples (humphead wrasses: Hw1, squaretail coralgrouper: Sc2 and leopard coralgrouper: Lc2) and three CTX-positive samples (humphead wrasses: Hw3, giant grouper: Gg and sample X) were passed through Florisil cartridges for purification and fractionization under acetone-methanol (7:3, v:v) solvent mixture condition. Different eluates (fractions HA, AM, M1, M2 and M3) were obtained after Florisil purification of 20 mg of P-CTX-1-spiked (n=13) and CTX-positive (n=6) extracts. Overall, 48±7.8% of 20 mg ether extract was eluted out while

  40 mg of ether extracts from P-CTX-1-spiked and CTX-negative samples (Pilot study) 40 mg ether extracts from two P-CTX-1-spiked samples (squaretail coralgrouper: Sc2 and leopard coralgrouper: Lc3) and three CTX-negative samples (camouflage grouper: Cg2, areolated grouper: Ag2 and star snapper: Ss) were passed through Florisil cartridges for purification and fractionization under the acetone-methanol (7:3, v:v) solvent mixture condition. Different eluates (fractions HA, AM, M1, M2 and M3) were obtained after Florisil purification of 40 mg of P-CTX-1-spiked (n=6) and CTX-negative (n=3) extracts. The two eluants, acetone-methanol (9:1,

  05; Student's t-test) in percentage of fractions AM, M1, M2 and ERC isolated between the application of 20 mg and 40 mg of ether extracts under the acetone-methanol (7:3, v:v) solvent mixture condition. Application of 40 mg ether extracts for Florisil purification yielded more AM as compared with those yielded from 20 mg ether extracts. In general, the average weight of fractions AM collected after Florisil purification (under acetone-methanol (7:3, v:v) solvent mixture condition) of 20 mg ether extracts of CTX-negative, P-CTX-1-spiked and CTX-positive fish samples was 4.2±0.4 mg (17±1.6% of 20 mg ether extracts). Percentage of other non-target components that were excluded after the purification was 83±1.6%. For the application of 40 mg ether extract under the same condition of solvent mixture, the average percentage of target fraction AM collected was 15±5.9% and those non-target components excluded after the purification was 83±6.0%. As a result, no significant difference (P > 0.05; Student's t-test) in extraction efficiency of the cartridge could be found between the use of 20 mg and 40 mg ether extracts for performing such purification.

  with CTX-negative samples (P > 0.05; Student's t-test).Recovery of spiked P-CTX-1 from coral fish flesh before and after Florisil extractionRecovery of spiked P-CTX-1 from coral fish flesh before Florisil extractionFor mice injected with ether extracts containing spiked P-CTX-1 (500 ng/100g flesh), they all exhibited typical signs of CTX poisoning including hypothermia (rectal temperature <33 o C within 2 h), severe diarrhoea, significant reduced locomotor activity and breathing difficulty during the course of experiment. No recovery from hypothermia was noted after injection and all mice experienced terminal convulsions before death.

  hypothermia, mild diarrhoea, reduced locomotor activity were observed in all mice after i.p. injection of the ether extracts.Hypothermia was detected within an hour in mice (n=4) injected with 20 mg ether extracts of sample Hw3 (Figure2), but there was subsequent recovery within 5 hours at day 0. Rectal temperatures of treated mice during the 6 hours ranged between 32.1-37.3 o C (averaged at 35.1±0.3 o C). Rectal temperature of mice returned to normal at both days 1 (37.5±0.4 o C) and 4 (37.4±0.2 o C).

  Toxicity of CTXs from CTX-positive samples after Florisil purificationFive fractions of eluates including HA, AM, M1, M2 and M3 were collected by applying 20 mg ether extracts of Hw3 and Gg to pass through Florisil under the condition of acetone-methanol (7:3, v:v) solvent mixture. Injection of Florisil purified fraction AM (target analyte) of sample Hw3 into mice (n=2) induced significant decrease in rectal temperature during 6-hour observation period at day 0 (Figure2). The rectal temperatures ranged between 27.1 and 35.7 o C, averaged at 31.5±0.6 o C.

  Gg, there was no conspicuous sign of toxic effect of CTX poisoning after injection of each fraction from both samples. Mice rectal temperature during the whole course of experiment, ranged between 36.7 and 38.3 o C (averaged at 37.7±0.0 o C) for sample Hw3, and between 36.6 and 38.4 o C (averaged at 37.5±0.0 o C) for sample Gg. No prominent body weight

  after injecting the Florisil-purified target fractions (fraction AM) of 20 mg ether extracts. The P-CTX-1 recovery was significantly enhanced by 97% as compared with that (~45%) from only ether extracts without performing such purification. It is of interest to note that mice injected with the target fractions induced a more rapid death than those injected with non-Florisil purified ether extracts. It is most likely that the Florisil clean-up step simply removed crucial lipid component(s) or residue(s) in the ether extracts which hampered the toxic action of P-CTX-1 on the target site(s) (e.g. sodium channels of excitable membranes) of mice. The degree of such interference effects by the lipid matrix might vary widely depending on fish species as well as the extraction procedures. In the CTX-positive field samples Hw3 (Humphead wrasse) and Gg (Giant grouper), prominent difference in pattern and trend of hypothermia was noted between mice injected with Florisil-purified fraction AM extracts and non Florisil-purified ether extracts. All mice exhibited a more serious hypothermia shortly after intraperitoneal injection of the Florisil-purified extracts. Based on the degree of toxic responses of mice in hypothermia during the course of experiment, it can be seen that the presence of unnecessary lipid matrix has a substantial masking effect on the toxic response of mice to CTXs. Although it is still speculative to confirm the possible existence of any chemical interaction or biochemical mechanism between CTXs and other lipid component(s) of the field coral fishes, the results from this study showed that these unnecessary lipid component(s) might suppress or retard the toxicological actions of CTXs on mice. Eliminating such component(s) in the extracts might facilitate the mechanistic action of the toxins on the target sites, and thus enhance the toxic responses of mice to the toxins. From the results of CTX-positive Y, there was no significant enhancement of toxic responses of mice to CTXs even injecting the Florisil-purified target fraction isolated from its ether extract. It is suggested that the bioassay may not sufficiently sensitive to differentiate such low levels of CTXs by observing the patterns of hypothermia in mice alone.

  because most of elements and CTXs in the ether extracts were still trapped inside the cartridge. Preliminary examination of the Florisil cartridges by

  extract. Though small sample sizes were used in this study, the results showed that the modified condition in the elution would optimize overall recovery of CTXs while minimizing the possible interferences of lipid matrix from co-extractants on mice. Further work would be necessary to evaluate the application of this extraction step in suspect cases and borderline case samples, and to examine its use in routine tests for various species of fish samples.

Figure 1

 1 Figure legends:

Figure 2

 2 Figure 2 Changes of rectal temperature in mice after i.p. injection of (A) 20 mg ether extracts of

  Figure1

Figure 1 .

 1 Figure 1. Change of rectal temperature in mice after i.p. injection of (A) 20 mg ether extracts of P-CTX-1 spiked samples Sc2 Pt2 and Sc2-a, and their target fractions AM; (B) 20 mg ether extract of samples Hw2 and Lc2, and their target fractions AM. Data are represented by mean±SEM.

Figure 1 Page

 1 Figure1

Figure 2 .

 2 Figure 2. Change of rectal temperature in mice after i.p. injection of (A) 20 mg ether extracts of sample Hw3, and their target fractions AM; (B) 20 mg ether extracts of sample Gg, and their target fractions AM. Data are represented by mean±SEM.

Figure 2 Page

 2 Figure2

Table 1 )

 1 , two samples (humphead wrasse (Hw3)

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

The authors are indebted to the Director of Health, Dr. P. Y. Lam, for the permission to publish this report and particularly grateful to the anonymous donor for supplying the ciguatoxin-contaminated coral fish samples.

Declaration

All commercial products and trade names mentioned in this article do not represent any suggestion or endorsement for use by our department.


Keywords: Chromatography -HPLC, Traceability Additives/Contaminants: Mycotoxins deoxynivalenol (DON), durum wheat, Fusarium spp., milling, mycotoxin, Triticum

The milling behavior of two naturally infected samples of durum wheat grain with contrasting levels of 3 mycotoxins was studied. Although the two samples showed a similar milling behavior, an increase of about 4 twenty percent in deoxynivalenol (DON) levels was found in semolina from the sample containing the higher 5 level of mycotoxin. However, even if the highest concentration of DON was found in fractions originating 6 from the grain outer layers, the mycotoxin contamination in semolina and flours were not related to the 7 amount of two compounds (ash or phytic acid) that could be used as markers to monitor these external 8 tissues. The presence of the trichothecene-producing fungi in the most internal semolina fraction was also 9 shown using specific DNA primers and PCR amplification. Comparison of DON concentrations in the feed 10 stock and corresponding output at each milling step or grinding of semolina fractions followed by sizing 11 showed that concentration of the mycotoxin occurs in the finest particles at the first processing steps.

12 Therefore, DON contamination of the milling fractions is not simply due to the presence of peripheral grain 13 tissues.

Fusarium head blight (FHB) or scab is a worldwide disease of wheat (Triticum aestivum and 2 durum) and other small grain cereals which is caused by several Fusarium species (Parry et al. 3 1995;[START_REF] Bai | Resistance to Fusarium head blight and 4 deoxynivalenol accumulation in wheat[END_REF]). In Europe, Fusarium graminearum is one of the most frequently encountered 4 species on wheat [START_REF] Goswami | Heading for disaster: Fusarium graminearum on cereal crops[END_REF]) mainly when wet and warm weather coincided with 5 the wheat anthesis and the beginning of grain filling [START_REF] Bottalico | Toxigenic Fusarium species and mycotoxins associated with head 7 blight in small-grain cereals in Europe[END_REF]Champeil et al. 6 2004). This fungal disease leads to the formation of shrivelled, light test-weight kernels that reduce 7 grain yield and quality and affect milling yield [START_REF] Edwards | Influence of agricultural practices on Fusarium infection of cereals and 16 subsequent contamination of grain by trichothecene mycotoxins[END_REF]). 8

In addition to grain yield loss due to FHB, production by Fusarium of toxic secondary metabolites 9 known as mycotoxins (Bennett and Klich 2003) may also impair the end-use of the harvested 10 cereals. One of these mycotoxins, deoxynivalenol (DON), which belongs to the trichothecene 11 (TCT) family could be considered as the most important wheat contaminant, as demonstrated by 12 surveys on its occurrence in grain [START_REF] Bottalico | Toxigenic Fusarium species and mycotoxins associated with head 7 blight in small-grain cereals in Europe[END_REF]. Its ingestion by animals was found 13 to lead to different symptoms depending on the sensitivity of species such as a decrease in weight 14 gain and nutritional efficiency, anorexic syndromes and altered immune function [START_REF] Pestka | Deoxynivalenol: Toxicity, mechanisms and animal health risks[END_REF]. [START_REF] Edwards | Influence of agricultural practices on Fusarium infection of cereals and 16 subsequent contamination of grain by trichothecene mycotoxins[END_REF] Due to its proved toxicity and its potential effect on the immune system, the European Community 16 have strictly fixed (EC regulation number 1881, 2006) the maximum DON authorized level in 17 unprocessed durum or common wheat grains at 1750 and 1250 µg.kg -1 respectively and in resulting 18 flours or semolina at 750 µg.kg -1 . 19 Some control of DON contamination in the field could probably be obtained by the selection of 20 resistant cultivars [START_REF] Gervais | 18 Mapping of quantitative trait loci for field resistance to Fusarium head blight in an European 19 winter wheat[END_REF]Jiang et al. 2007) and a better knowledge of the 21 environmental factors which are involved [START_REF] Edwards | Influence of agricultural practices on Fusarium infection of cereals and 16 subsequent contamination of grain by trichothecene mycotoxins[END_REF][START_REF] Koch | Evaluation of environmental and management effects on 6 Fusarium head blight infection and deoxynivalenol concentration in the grain of winter wheat. 7[END_REF]). However, the 22 fractionation process, as one of the final steps leading to the cereal end-products, remains critical to 23 control the mycotoxin content in consumed fractions. If the first stages of Fusarium contamination 24 appear to be limited to the grain outer layers [START_REF] Mckeehen | Evaluation of wheat (Triticum aestivum L.) phenolic 14 acids during grain development and their contribution to Fusarium resistance[END_REF]), contamination may be 25 reduced by the milling process that separates the endosperm from the bran containing these outer 1 layers, the fungi and the toxins could also be found in the endosperm [START_REF] Kang | Immunocytochemical localization of fusarium toxins in infected 4 wheat spikes by Fusarium culmorum[END_REF]2 Jackowiak et al. 2005) that leads to flours or semolina. Previous studies on durum wheat showed 3 that a significant reduction of the average DON level could be achieved at each step of the grain 4 processing [START_REF] Nowicki | Retention of the Fusarium 19 mycotoxin deoxynivalenol in wheat during processing and cooking of spaghetti and noodles[END_REF][START_REF] Dexter | Effect of Fusarium head blight on semolina 14 milling and pasta-making quality of durum wheat[END_REF]Visconti et al. 2004;[START_REF] Manthey | Microbial loads, 11 mycotoxins, and quality of durum wheat from the 2001 harvest of the Northern Plains region of 12 the United States[END_REF]). 5

However, the relationship between these steps and the tissue origin of the different fractions have 6 not been investigated neither the effect of the level of contamination on the DON distribution. 7

In this paper, the grain characteristics and milling behavior at a semi-industrial scale of two 8 naturally infected samples of the same durum wheat cultivar but displaying distinctly different 9 levels of DON were analyzed. Furthermore, the impact of the milling process on the DON 10 distribution in the outcoming fractions was compared and discussed. 11 12

Material and methods 13

Wheat samples and grain characterization 14

Two samples of Triticum durum (Acalou cultivar) cultivated in France in 2003 were stored at 4°C 15 before milling. [START_REF] Niessen | Group specific PCR-detection of potential trichothecene producing 17 Fusarium species in pure cultures and cereal samples[END_REF] Test weight and determination of Thousand Kernel Weight (TKW) determination were performed 17 respectively with standard AACC method 55-10 (AACC 2000) and ISO method (N°520, 1977). [START_REF] Nowicki | Retention of the Fusarium 19 mycotoxin deoxynivalenol in wheat during processing and cooking of spaghetti and noodles[END_REF] Grain vitreousness was assessed by analysis of kernel cross-sections (obtained with a Pohl kernel 19 cutter, Versuchs und Lehranstalt für Brauerei, Berlin, Germany) and expressed by the percentage of 20 vitreous versus mealy grains. 21

Particle Size Index (PSI) was determined according to the adapted AACC method 55-30 ( AACC 22 2000). Twenty grams of each wheat sample were ground with a laboratory mill 3303 (Perten 23

Instruments AB, Huddinge, Sweden) and the product obtained was sieved for 10 min using 75µm 24 sieve (LS-PRO, Hosokawa Alpine AG, Germany). The PSI corresponds to the percentage weight of 25 particles able to pass through the sieve. 26

Milling conditions 2

Cleaned grain was tempered to 17% water content before milling. Durum wheat milling was 3 performed using a semi-industrial semolina mill (150 kg.h -1 ) as described before (Chaurand et al. 4 1999). It was equipped with four break rolls or four reduction rolls followed by sifters for sizing and 5 six sizing purifiers (Figure 1). This led to the production of four break flours (BF2 to BF5) and four 6 reduction flours (RF1 to RF4); six purified semolina (SE1 and SE3 which corresponds to the inner 7 endosperm, SE2 and SE4 originating from more external part of the endosperm compared with SE1 8 and SE3, SE5 and SE6 coming from the most peripheral part of the endosperm) and four bran or 9 middlings: coarse bran (CB), purified fine bran (PFB), sized fine bran (SFB), and shorts (SH). Each 10 fraction was collected and its yield expressed as weight percentage of the total milled grain amount. 11

Furthermore, an aliquot sample of the 14 feeding fractions from each roll or purifier were also 12 recovered. The collected fractions were stored at 4°C. 13

Biochemical analyses 17

Water and ash content of the grain were determined in triplicate according to standard AACC 18 methods 44-19 and 08-12 respectively (AACC 2000). The phytic acid content was measured at 500 19 nm from acidic extract of ground grains or milling fractions using a colorimetric method described 20 by [START_REF] Latta | A simple and rapid colorimetric method for phytate determination[END_REF]

DON measurement 1

All the fractions were homogenized before their division to obtain a representative sample and 2 eventually ground in order to reduce the particle size below 500 µm. DON was then extracted at 3 room temperature with 100 mL of 84 % (v/v) acetonitrile in water using 25 g of each fractions 4 except for bran and middling fractions which allowed the amount to be reduced to 15 g. The 5 mixture was shaken 30 min and homogenized with an Ultra Turrax for 3 min. After filtering 6 through Whatman No. 1 filter paper, 8 mL of the filtrate were passed through a Mycosep 225 7 column (Romer Labs., Union, MO, USA). The recovered purified solution (4 mL) was transferred 8

to a new vial and the solvent evaporated to dryness under a stream of nitrogen at 50°C. The dry 9 residue was then stored in a tightly closed glass bottle at -20°C until use or suspended in 500 µL of 10 methanol 20 % (v/v) and centrifuged (20000g, 6 min) before RP-HPLC analysis. 11

The DON extract (50 µL) was injected on a NovaPak C 18 column (60Å, 4 µm, 150 × 3.6 mm, 12

Waters SAS, Guyancourt, Fr.) and equilibrated with 7% (v/v) acetonitrile at a flow rate of 0.7 13 mL.min -1 . Elution was obtained by increasing the acetonitrile concentration in water to 80 % after 14 15 min. DON was detected at 218 nm with a 2487 dual λ UV detector (Waters SAS, Guyancourt, 15

Fr.). Commercial DON (Sigma-Aldrich, St Quentin Fallavier, Fr) was used to prepare calibration 16 standards ranging from 0.1 to 1 µg.mL -1 . To assess a calibration curve with the standards, a linear 17 regression was fitted on the data. DON concentration in the extracts were computed from the 18 regression curve. Mean DON recovery was estimated at 98.5 % with a 5.9 % coefficient of 19 variation from a wheat standard flour sample (FAPAS, T2210, with an average concentration of 463 20 µg.kg -1 ) and based on triplicate measurement on four distinct extractions. contained 25 pmoles of each primers (Tox5-1: 5'-GCTGCTCATCACTTTGCTCAG; Tox5-2: 5'-1 CTGATCTGGTCACGCTCCATC) defined according to previous studies (Niessen and Vogel 2 1998), 2.5 U of DNA polymerase (Invitrogen Co., Cergy-Pontoise, Fr.), 400 µM of each 3 deoxynucleotide triphosphate (Promega, Charbonnières, Fr.). Reactions were performed using the 4 following PCR conditions: denaturation at 95°C for 1min 30, 40 cycles of denaturation at 95°C for 5 30 s, annealing at 55°C for 40 s and extension at 72°C for 30s, final extension at 72°C for 5 min, 6

followed by cooling at 4°C. Amplification products were analysed on a 1% agarose gel stained with 7 ethidium bromide. Genomic DNA extracted from the fungi F. graminearum was kindly given by L. 8

Pinson-Gadais (INRA, Bordeaux). 9 10

Results and discussion 11 12

Wheat characterization and milling behavior 13

Two durum wheat samples from the cultivar Acalou were selected as their levels of natural 14 contamination with DON were found to be different. The first sample (A1) was found to contain 15 382 µg.kg -1 (d. m.) of DON and thus could be considered acceptable in accordance to the European 16 Community legislation whereas the other (A2) displayed a DON level around 4203 µg.kg -1 (d. m.). 17

As Fusarium infection could lead to important grain damage that might severely affect the milling 18 behavior, the physical characteristics (test weight, TKW, vitreousness, PSI, water content) of wheat 19 grains from both samples were measured and compared. [START_REF] Goswami | Heading for disaster: Fusarium graminearum on cereal crops[END_REF] 21

Table 1 22 23

Table 1 clearly illustrates that even if the DON level in A2 is more than ten fold higher than that in 24 A1, no apparent differences between grain characteristics of the two wheat grain samples were 25 observed. As these samples shared common grain characteristics, it was interesting to compare their 26 milling behavior, deoxynivalenol concentration and distribution in the generated fractions. The 27 yield of each fraction obtained after grain milling at a semi-industrial scale (150 kg.h -1 , milling 1 scheme in Figure 1) are summarized in Table 2. Even if the samples displayed a different level of 2 toxin, which could either be due to a different growth of the fungi or a distinct toxin production 3 level, no drastic changes were observed between the milling yields of the different fractions from 4 both of the samples, taking into account the coefficient of variation. 5 6 Table 2 7 8

DON concentration and distribution in the milling fractions 9

The DON concentration in all of the milling fractions were then determined and compared (Table 10 2). According to previous studies [START_REF] Nowicki | Retention of the Fusarium 19 mycotoxin deoxynivalenol in wheat during processing and cooking of spaghetti and noodles[END_REF]Visconti et al. 2004), the most contaminated 11 fractions were the coarse bran (CB) and the purified fine bran (PFB). Indeed, these fractions contain 12 the major part of the grain outer layers that are the first to be infected by Fusarium (Jackowiak et al. 13 2005). These fractions showed a two to four fold concentration of DON compared to the grains as 14 demonstrated by the concentration ratio reported in Table 2. [START_REF] Edwards | Influence of agricultural practices on Fusarium infection of cereals and 16 subsequent contamination of grain by trichothecene mycotoxins[END_REF] Whatever the wheat sample, the other most contaminated fractions were the first two break flours 16 with DON concentration close to twice the toxin concentration in the grain. High amounts of DON 17 in these flours could be due to contamination with the outer layers at the grain breaking step or to 18 higher amounts of DON in the tissues from which these flours are originated. In contrast, the DON 19 concentration in the reduction flours however were found similar to those in the raw material. As 20 expected, the lowest concentration of DON was found in total semolina. However, the 21 concentration ratio between the toxin concentration in semolina and grains pointed out that 22 reduction of the DON concentration was more efficient for the less contaminated wheat sample (40 23 % in A1 compared to 30 % in A2). [START_REF] Jackowiak | Scanning electron microscopy of Fusarium 25 damaged kernels of spring wheat[END_REF] Comparison between distribution of the dry mass and DON amount in each type of the milling 25 fractions were calculated and summarized in Table 3. 26

Table 3 2

A distinct distribution of the total amount of DON in the milling fractions was obtained depending 3 on the wheat sample. Indeed, in the most contaminated grain sample, the highest toxin proportion 4 was found in semolina whereas, in the less contaminated sample, this highest toxin proportion was 5 found in bran and shorts. Two hypotheses could explain this difference in DON distribution: a 6 higher friability of the outer layers in the most contaminated grain sample or a higher amount of 7 toxin in the endosperm of these grains. In order to test a potential link between the increase of DON 8 proportion in semolina and contamination of this milling fraction with the outer layers, ash and 9 DON concentrations in semolina and flours were analyzed and compared (Figure 2). 10 11

Figure 2 12 13

Results clearly demonstrated that fractions displaying the highest DON concentration were not the 14 same as those showing the highest ash level. This lack of correlation between ash and DON 15 concentration was observed in both of the durum wheat samples. Therefore, it suggests that the 16 highest amount of DON in semolina from the A2 sample could not be simply due to the presence of 17 grain outer layers in these fractions. These results are in accordance with a previously published 18 study dealing with common wheat milling fractions (Seitz et al. 1985). Furthermore, the phytic acid 19 concentration, that could be used to track the aleurone cell content [START_REF] Greffeuille | Differences in the aleurone 23 layer fate between hard and soft common wheats at grain miling[END_REF], was 20 also determined in semolina and flours from the most infected grains. Results confirmed the 21 absence of relationships between DON concentration in these milling fractions and presence of the 22 grain peripheral tissues. Indeed, the phytic acid concentration was found to increase from the inner 23 to the outer endosperm semolina (5.0 mg.g -1 d.m. in SE1 to 15.5 mg.g -1 d.m. in SE6) as well as 24 from the first break and reduction flours compared to the last one (respectively between 12.6 and 25 27.0 mg.g -1 d.m. from BF2 to BF5 and between 15.2 to 34.6 mg.g -1 d.m. from RF1 to RF4). 26 compared to the last ones was not only due to a contamination with the grain outer layers but rather 2 resulted from highly infected particles obtained at the first steps of grain breaking. Production of 3 these fine particles from infected parts of the endosperm could be generated by the fungus 4 penetration and by degradation of this tissue as already suggested for common wheat (Seitz et al. 5

1985). 6 7

Fusarium detection in the milling fractions 8

In order to monitor the TCT-producing Fusarium species, DNA extraction was performed from 9 each type of milling fractions and amplified by PCR. The primers used for amplification were 10 derived from the gene Tri5 encoding the first enzyme (trichodiene synthase) involved in the TCT 11 biosynthetic pathway, that was already been demonstrated to be a pertinent probe to track TCT-12 producing fungi [START_REF] Niessen | Group specific PCR-detection of potential trichothecene producing 17 Fusarium species in pure cultures and cereal samples[END_REF]. Results of the PCR amplification, reported in Figure 3, 13 revealed the expected specific fragment (658 bp). 14 11

Relationship between roll and purifier feedings and corresponding outputs 1

In order to clarify the DON distribution along milling, DON concentration in the feedings from all 2 rolls and purifiers were determined and summarized in Table 4. BR2 corresponds to meal fraction 3 obtained after the two first break rolls, BR3 to BR5 are the feedings corresponding to the following 4 break rolls, SP1 to SP6 are the feedings from the first to the sixth purifiers and RR1 to RR4 are the 5 feedings from the first to the fourth reduction rolls (Figure 1). 6 7

Table 4 8 9

The DON concentration in the feedings from each roll and purifier and the corresponding 10 obtained fraction (semolina or flours) could thus be compared: SP1 to SP6, BR2 to BR5 and RR1 to 11 RR4 with respectively SE1 to SE6, BF2 to BF5 and RF1 to RF4 as reported in Table 2. If DON 12 concentration in semolina was always found lower than those of the corresponding feedings 13 (ranging from identical to two or three-fold less concentrated), the ratio between break and 14 reduction flours and the corresponding feedings decreased from the first to the last step of the 15 milling process. Indeed, the DON concentrations were found to be 1.5 to 3-fold higher in the first 16 break and reduction flours compared to the corresponding feedings. Thus, at the first breaking and 17 reduction steps of the milling process, DON appeared to be concentrated in the flours, i.e. particles 18 with the smallest size. 19 20 Table 5 21 22 In order to test the hypothesis that grain breaking or endosperm reduction at the first milling steps 23 could lead to a preferential DON concentration in the finest fractions, the semolina SE3 and SE1 24 (originating from the most internal part of the grain) from sample A2 were sifted. Comparison between dry mass of the sifted semolina and their respective DON concentration showed that the 1 highest DON concentration occurred in the finest fractions (Table 5, a1, b1). 2 Each semolina was then ground, after removal of the finest particles (i.e. particles under 315 µm 3 for SE3 which accounted for 4 % of the semolina dry mass and displayed a DON concentration of 4 3358 µg.kg -1 ; particles under 450 µm for SE1 which accounted for 26 % of the semolina dry mass 5 and displayed a DON concentration of 4151 µg.kg -1 ). The resulting grinded material was classified 6

by size in order to analyze the DON concentration and distribution in each of the obtained fractions. 7 Table 5 (a2, b2) clearly shows that DON concentration was again higher (3 to 5 fold) in the fraction 8 characterized by the finest particle size. 9

Moreover, analysis of the DON amount distribution after semolina grinding and sizing compared 10 to those of the dry mass showed that DON amount in the largest size fractions was less than a half 11 the amount expected if DON was equally distributed in the material. In contrast, fractions 12 corresponding to the smallest particles concentrated the toxin with an about two fold factor 13 compared to the expected level. 14 Therefore, absence of relationships between DON contamination of flours and semolina and the 15 ash and phytic acid contents, as well as evidence of DON concentration in the finest particles from 16 the endosperm (Table 5) could explain the unexpected distribution of DON in the milling fractions. 17

It thus revealed that contamination of semolina or flours could not only be originated from the grain 18 outer layers but also from other contaminated parts of the grain that were shown to be friable. It 19 appeared particularly significant for grains containing a high level of mycotoxin and for fractions 20 coming out from the first steps of the process. Thus, higher DON level in the finest particles from 21 the semolina fraction of the most infected wheat sample could result from a deeper fungi 22 penetration in grains or a higher damage of the grain internal structure due to an increased synthesis 23 of hydrolytic enzymes, that are known to be produced by Fusarium [START_REF] Jackowiak | Scanning electron microscopy of Fusarium 25 damaged kernels of spring wheat[END_REF]. in semolina. This could be potentially achieved by improving the sizing efficiency of the sifters 2 after the two first break rolls or by increasing the efficiency of the first two sizing purifiers in order 3 to better eliminate the finest particles at the first milling steps even if this will results in a decrease 4 of the semolina yield. Indeed as semolina are the fractions used for pasta making, this control could 5 constitute a key factor in order to reduce the DON level in corresponding consumed wheat products.

6 a

 6 a , N. ZAKHIA-ROZIS b , M. CHAURAND a , F. RICHARD-FORGET c , M.F. SAMSON a , J. 4 ABECASSIS a & V. LULLIEN-PELLERIN a* . 5 INRA, UMR 1208, Ingénierie des Agropolymères et Technologies Emergentes, 2 Place Pierre 7 Viala, F-34060 Montpellier cedex1, France ; b CIRAD, UMR 1208, Ingénierie des Agropolymères et 8 Technologies Emergentes, TA 40/16 73 Rue JF Breton 34398 Montpellier cedex 5, France ; c INRA, 9 U.R. 1264, Mycologie et Sécurité des Aliments, Domaine de la Grande Ferrade, 71 Av. Edouard 10 Bourleaux, BP 81, 33883 Villenave-d'Ornon cedex, France.

Figure 1 15 16

 16 Figure 1 15

  observed higher DON concentration in flours from the first break and reduction rolls 1

Figure 3 16 17

 16 Figure 3 16

  of the finest particles during milling, in order to lower the DON amount 1

6 7 8 Figure 1 : 8 Figure 2 : 11 Figure 3 :

 68182113 Figure captions 1 2 Figure 1: Milling diagram of the semi industrial pilot mill (INRA, Montpellier). BR1 to BR5 are 3 feedings of break rolls, BF2 to BF5 are corresponding break flours, SP1 to SP6 are feedings of 4 purifiers, SE1 to SE6 are semolina; RR1 to RR4 are feedings of reduction rolls, RF1 to RF4 are 5 reduction flours, CB, PFB and SFB are respectively coarse bran, purified fine bran and sized fine 6 bran, SH are shorts. 7 8 Figure 2: Comparison of DON (mg.kg -1 d. m.) and ash (% d. m.) concentration in the milling 9 fractions (except brans and shorts) from the two durum wheat samples (means of two replicates). 10 11 Figure 3 : Detection of TCT-producing fungi in grains and milling fractions from durum wheat 12Acalou samples A1 and A2 after PCR with primers derived from the gene encoding trichodiene 13 synthase. F. graminearum corresponds to DNA extracted from the fungus. ST are total semolina, 14 SE1 and SE3 are semolina from the most inner part of the endosperm, BF2 are the first break flours, 15 RF1 are the first reduction flours, CB, SH, SFB and PFB are respectively coarse bran, shorts, sized 16 fine bran and purified fine bran. 17

Table 1 :

 1 Main physical characteristics and total DON concentration of the analyzed Acalou wheat samples naturally infected with Fusarium a . Values are means and standard deviation of three replicates.

	A1
	DON (mg kg ) Ash% -1

Table 2 :

 2 Yield and DON concentration in the different milling fractions from the two durum wheat samples. means of two replicates, C. V. < 5% for semolina and flours and < 10% for brans and shorts. c Ratio between DON concentrations in the analyzed fraction versus those in cleaned grains.

				A1			A2
	Milling Fractions	Yield a (% d. m.)	Concentrations DON b (µg.kg -1 d.m . ) Ratio c	Yield a (% d. m.)	Concentrations DON b (µg.kg -1 d.m . ) Ratio c
	Semolina SE1	6.2	266	0.70	6.0	3497	0.83
		SE2	26.0	260	0.68	25.3	3359	0.80
		SE3	26.8	180	0.47	25.9	2600	0.62
		SE4	10.5	139	0.36	10.5	2324	0.55
		SE5	0.1	121	0.32	0.7	2440	0.58
		SE6	6.8	230	0.60	7.5	3850	0.92
	Total semolina	76.4	220	0.58	75.9	2940	0.70
	Break flours	BF2	0.7	707	1.85	0.4	7737	1.84
		BF3	0.5	808	2.12	0.8	6206	1.48
		BF4	1.0	618	1.62	1.0	5341	1.27
		BF5	1.0	416	1.09	1.0	3208	0.76
	Reduction flours	RF1	1.1	431	1.13	1.1	5679	1.35
		RF2	1.1	368	0.96	1.1	5239	1.25
		RF3	0.5	300	0.79	0.6	3349	0.80
		RF4	1.0	400	1.05	1.4	3601	0.86
	Bran and Shorts	SFB	0.7	575	1.51	0.7	5196	1.24
		PFB	6.9	1146	3.00	7.2	10645	2.53
		CB	7.7	1463	3.83	7.6	14416	3.43
		SH	1.6	450	1.18	1.2	6091	1.45
	a C. V. < 5 %.

b

Table 3 :

 3 Comparison of yield and DON distribution in percent of the total grain mass or DON total content. calculated taking into account the fraction yield and DON concentration and expressed as a percentage of the total amount of DON in the sample.

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		A1	A2
	Milling Fractions	Yield a (% d.m.)	DON b (% d.m.)	Yield a (% d.m.)	DON b (% d.m.)
	Total Semolina	76.4	40.8	75.9	49.6
	Total Break Flours	3.2	4.8	3.3	3.7
	Total Reduction Flours	3.7	3.5	4.2	4.1
	Total Brans and Shorts	16.8	50.8	16.6	42.6

a Total yield was obtained from Table

2

. b http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 4 :

 4 DON concentration (µg.kg -1 d.m.) in the feedings of rolls and purifiers and ratio between DON concentrations in flours or semolina (reported in Table2) versus those in the feedings.

			A1		A2
	Fractions		DON a	Ratio	DON a	Ratio
	Break roll feedings	BR2	250	2.83	4891	1.58
		BR3	369	2.19	5865	1.06
		BR4	300	2.06	5946	0.90
		BR5	594	0.70	8571	0.37
	Sizing purifier feedings	SP1	262	1.01	4476	0.78
		SP2	352	0.74	4343	0.77
		SP3	200	0.90	3563	0.73
		SP4	201	0.69	3477	0.67
		SP5	320	0.38	5747	0.42
		SP6	349	0.66	4143	0.93
	Reduction roll feedings	RR1	242	1.78	2719	2.09
		RR2	248	1.48	3450	1.52
		RR3	401	0.75	3561	0.94
		RR4	312	1.28	4640	0.78

a means of two replicates, C. V. < 7%.

Table 5 :

 5 Distribution in dry mass (% d.m.), DON concentration (µg.kg -1 d.m.) and DON distribution (%) in sieved fractions from semolina SE3 or SE1 of the wheat sample A2 before (a1, b1) and after (a2, b2) grinding. DON concentration of each analyzed fraction was specified in brackets at the top of each table. (3496 µg.kg -1 d.m.) SE1 A2 (3189 µg.kg -1 d.m.)

	a1

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Mini Kit from Qiagen (Qiagen S.A., Les Ulis, Fr.) according to the manufacturer instructions. All

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yield loss. Theor. Appl Genet. 115: 1043-1052.