Development of an Immunoaffinity Column for the Determination of T-2

Chapter 15

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Development of an Immunoaffinity Column for the Determination of T-2 and HT-2 Toxins in Cereals Using Liquid Chromatography with Fluorescence Detection C. Donnelly, A. Pollock, Y. Heidtmann, and E. Marley R-Biopharm Rhône Ltd., West of Scotland Science Park, Unit 3.06 Kelvin Campus, Maryhill Road, Glasgow, Scotland G20 OSP

With new European Union (EU) mycotoxin regulations under consideration for T-2 toxin (T-2) and HT-2 toxin (HT-2) in cereals, there is a need for new analytical methods to detect and quantify these toxins accurately. It is often difficult to quantify T-2 and HT-2 in cereals due to interfering components and pigments in the samples, which can often mask T-2 and HT-2 peaks in a liquid chromatography (LC) chromatogram leading to poor recoveries and sensitivity. An improved method has been developed using an immunoaffinity column in conjunction with LC-fluorescence. The protocol involved extraction with 90% methanol and filtration prior to dilution of the sample with water (for maize, wheat, and barley) or with 2% sodium chloride (for more complex oat samples) and application onto the immunoaffinity column. Use of the 2% sodium chloride for dilution of the oats extract was developed in conjunction with Nestle Research Centre in Switzerland and facilitated precipitation of proteins from the sample and removal of interfering components, resulting in improved chromatography for this matrix. This method was shown to be rapid and easy to use with a limit of detection of 1 ppb for HT-2 and 10 ppb for T-2 in cereals, which is significantly lower than proposed EU legislative limits (1). Furthermore the sensitivity of the method can be improved by passing a larger volume of sample extract through the immunoaffinity column prior to analysis by LC. Excellent recoveries of 70-105% for T-2 and between 100-119% for HT-2 were obtained with wheat, barley, maize, and oats and the method was found to be robust and sensitive. It can therefore be concluded that the method using immunoaffinity columns will meet forthcoming EU legislative limits and will satisfy recommended method performance criteria for these toxins. 276

© 2008 American Chemical Society

Siantar et al.; Food Contaminants ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


277 Currently no official method exists for the determination of T-2 toxin (T-2) and HT-2 toxin (HT-2); before legislation can be introduced it has been recommended that analytical methods for T-2 and HT-2 are required with acceptable recovery, accuracy, and analytical precision. There is also thought to be a need for reference materials, which can be used to check consistency, reliability, and traceability of analytical methods (2). These materials and methods can then be used in international collaborative studies for T-2 and HT2 to ensure that proposed legislative levels can be met. EASI-EXTRACT® T-2 and HT-2 immunoaffinity columns were developed to provide a suitable method for detection of T-2 and HT-2 in cereals. The columns were assessed in conjunction with a liquid chromatography (LC)-fluorescence system in order to improve the detection of T-2 and HT-2 in cereals by providing cleaner chromatography and better sensitivity than the more traditional solid phase extraction columns. T-2 and HT-2 occur in a wide range of cereal products including wheat, oats, and barley and often co-occur with other trichothecenes (J). These mycotoxins are often found in cereals and feed and can cause acute symptoms in livestock and poultry, including vomiting, feed refusal, and poor weight gain. They can also have chronic effects on animals such as immunosupression, oral lesions, and lethargy (4). T-2 and HT-2 are type A trichothecenes which means that LC with UV detection is not generally applicable since they lack a keto group at the C-8 position (5), although a variety of post-column derivatisation methods have been developed, including those involving detection of pnitrobenzoate or diphenylindenone sulfonyl esters of T-2 (6). For this reason, GC analysis is generally the chosen method for the determination of T-2 and HT-2. Most methods for type A trichothecenes are based on trimethylsilylation or fluoroacylation for derivatization to increase volatility and sensitivity. Detection is then carried out using mass spectrometry (MS) (7) or with an electron capture detector (ECD) for the fluoroacylated trichothecenes. The formation of fluoroacyl derivatives by trifluoroacetic anhydride (TFA), pentafluoropropionyl (PFP), or heptafluorobutyryl (HFB) derivatization increase the sensitivity of the LC-ECD procedure (6). Other newer methods for detection of trichothecenes by LC-MS/MS have also been developed using solid phase extraction columns or multifunctional cleanup columns (5, £). For extraction of T-2 and HT-2 from cereal samples different extraction solvents can be employed, including acetonitrile-water or methanol-water, and extraction can be facilitated by shaking or blending. Sample clean up is then often carried out using silica gel, florisil, cyano and C solid phase extraction columns. Typical detection limits for quantitative determination of T-2 and HT2 in cereals are 3 ng/g (T-2, LC-MS), 1 ng/g (HT-2, LC-MS), 10 ng/g (T-2 and HT-2, GC-MS) and 10 to 50 ng/g (T-2 and HT-2, GC-ECD). Typical recoveries can range from 70-120% (6). The problem with these techniques, however, is that the associated equipment is very expensive and is not widely available for use by smaller food companies and analytical laboratories. In addition highly experienced analysts must be employed to use and maintain these systems. 18



278 Furthermore some complex food matrices such as animal feed and oats can cause quenching of the T-2 and HT-2 signals making accurate quantification difficult. Analysis by LC is more widely available to smaller companies and many laboratories already use immunoaffinity columns in conjunction with LC for analysis of other mycotoxins. Moreover, 1-anthroylnitrile (1-AN) has been shown to be an efficient labelling reagent for the determination of T-2 by LCfluorescence (P). The use of a highly selective immunoaffinity column for T-2 and HT-2 in conjunction with LC-fluorescence will make this type of analysis accessible to a greater number of laboratories. The EASI-EXTRACT® T-2 and HT-2 immunoaffinity columns use a monoclonal antibody bound to a gel support, which is highly specific for both T2 and HT-2. The column not only cleans up sample extracts prior to quantification by LC-fluorescence but also concentrates the toxin, therefore improving detection and sensitivity. As with previous methods, T-2 and HT-2 were extracted from cereals using 90% aqueous methanol (v/v) before filtering and dilution with deionised water followed by immunoaffinity column cleanup. The toxin was released from the immunoaffinity column with methanol and the eluate evaporated to dryness before derivatization with 1-anthroylnitrile in the presence of 4-dimethylaminopyridine for fluorescence detection (P).

Materials and Methods Reagents and Consumables 4-Dimethylaminopyridine (DMAP) (Sigma, product code: D5640-25G); 1anthroylnitrile (1-AN) (WAKO, product code: 017-12101); toluene (high grade, Fluka, product code: 89676); filter paper (Whatman N° 113 or N° 4); glass microfiber filter paper (Sartorius MGB); T-2 standard (Sigma, product code: T4887); HT-2 standard (Sigma, product code: T4138); and T-2 and HT-2 immunoaffinity columns (EASI-EXTRACT®, R-Biopharm Rhône Ltd., product code: P43 and P43B).

Sample Preparation Test portions of 50 g cereal samples were extracted with 250 mL of methanol- water (9:1, v/v) plus 5 g of sodium chloride by blending at high speed for 2 min then filtering through a Whatman No. 113 filter paper.

Immunoaffinity Column Clean up and Derivatization Procedure A 7 mL aliquot of filtrate was diluted with 28 mL of deionised water for wheat, barley, and maize samples. For more pigmented samples like oats, 7 ml



279 of filtrate was diluted with 28 mL of 2% sodium chloride solution; the solution was gently shaken and left to settle for 5 min for precipitation of proteins from the extract to occur. The diluted filtrates were then further filtered through a Sartorius MGB glass microfibre filter paper. Twenty five mL of solution was passed through the EASI-EXTRACT® T-2 and HT-2 immunoaffmity columns at a slow flow rate of 3 mL per min. The immunoaffinity column was then washed with 20 mL of deionised water to remove any unbound residues from the column. Toxin was released from the immunoaffinity column using 1.5 mL methanol and the eluate was evaporated to dryness at 50 °C under a gentle stream of air. Fifty \iL of 1-anthroylnitrile and 50 \iL of 4-dimethylaminopyridine were added and mixed gently by vortex for 1 min; the mixture was placed on a heating block at 50 °C for 15 min. cooled in an iced water bath for 15 min, and evaporated to dryness at 50 °C under a gentle stream of air. The residue was reconstituted in 1 mL of mobile phase and mixed on a vortex mixer for 30 sec, before injecting 100 \iL into the LC system.

In-House Validation The method using EASI-EXTRACT® immunoaffinity columns with L C fluorescence detection was assessed using a range of spiked cereal extracts including extracts of maize, barley, wheat, and oats spiked at 100 ppb (total T-2 and HT-2, 1:1) to check the performance of the immunoaffinity columns and the quality of the LC traces following column clean-up (Table II). The performance of the complete method, including extraction with methanol-water (9:1, v/v) and

Table I. L C Conditions Guard cartridge: Analytical column: Mobile phase: a binary gradient was applied

LC pump Fluorescence detector: Column heater: Integrator Injector Injection volume:

Supelco guard filter (0.5 urn). Analytical column, Luna 5ju Phenyl-Hexyl LC column (150 mm x 4.6mm, 5^m). Initial composition = acetonitrile-water (70:30, v/v) 5 min = acetonitrile- water (70:30, v/v) 15 min = acetonitrile- water (85:15, v/v) 25 min = acetonitrile- water (85:15, v/v) 27 min = 100% acetonitrile 32 min = 100% acetonitrile 35 min = acetonitrile- water (70:30, v/v) To deliver mobile phase at flow rate: 1.0 mL/min Excitation: 381nm; emission: 470 nm Maintain guard and analytical columns at 40 °C From preferred supplier Autosampler /Rheodyne valve 100 nL


280


immunoaffinity column cleanup, was assessed on a range of spiked cereal samples. Samples of wheat, maize, and barley were spiked directly with 100 ppb total T-2 and HT-2 (1:1) (Table III). A naturally contaminated barley reference material was also analysed using the new method (Table IV) and results compared to GC-MS combined with trimethylsilyl derivatization (7); the same sample was then analysed over several days using LC-fluorescence.

Table II. Extracts Spiked with 100 ppb total toxin (T-2 and HT-2,1:1) Sample Pearl barley Maize flour Maize fine grits Wheat Maize Barley Barley Shredded wheat Shredded wheat Oats

No. of Average ppb Analyses T-2 HT-2

Average Recovery, % T-2 HT-2

%CV T-2

HT-2

(n=3)

51

51

102

102

10.0

10.8

(n=3)

38

46

76.3

91.8

10.0

4.9

(n=3)

38

56

76.3

113

5.6

4.7

(n=3) (n=l) (n=3) (n=3)

42 50 36 36

53 51 39 36

83.9 100.1 71.6 71.2

106 101 77.0 72.2

5.9

13.8

4.0 8.4

4.8 14.8

(n=3)

47

58

94.3

117

5.5

3.3

(n=D

47

49

93.5

98.8

-

-

(n=6)

51

51

103

102

5.0

5.6

-

-

Table III. Matrix Spiked with T-2 and HT-2 (1:1)

Sample

Organic wheat grain Maize fine grits Pearl barley Maize flour Oats

Spiking Level Total Toxin

Average ppb

Average Recovery,%

%CV

T-2

HT-2

T-2

HT-2

T-2

HT-2

100 ppb

35

50

70.6

101

4.8

5.7

100 ppb

45

53

89.9

106

8.1

9.7

100 ppb

53

57

106

114

13.5

13.5

500 ppb 100 ppb

249 49

298 47

99.6 97.8

119 94.2

5.5 3.7

5.7 12.1


281 Table IV. Naturally Contaminated Barley Average GC-MS Result (ppb) (n=3)


Reference Material

T-2 26

HT-2 71

Average EASI-EXTRACT® T-2 and HT-2 Result (ppb) (n=3) T-2 HT-2 19 60

Results and Discussion When the EASI-EXTRACT® T-2 and HT-2 columns were challenged with equal amounts of T-2 and HT-2 toxins the recovery was 100% for both toxins demonstrating the antibody has equal affinity for T-2 and HT-2. The capacity of the column was also shown to be at least 1.5 \ig total toxin (T-2/ HT-2 - 1:1), while the limit of detection (LOD), at a signal to noise ratio of 3:1, was 1 ppb for HT-2 and 10 ppb for T-2 using the recommended LC conditions. The limit of quantification (LOQ), defined as 3 x LOD, was 3 ppb for HT-2 and 30 ppb for T-2. Both the LOD and the LOQ could be further improved by passing a greater volume of sample extract through the immunoaffinity column. * Maize, barley, wheat, and oats extracts were spiked at 100 ppb (50 ng T-2 /g and 50 ng HT-2/g). Recoveries ranged from 71% to 117% for both toxins in all cereal extracts tested with a % CV of 3.3% to 14.8% (Table II). The results confirmed that the EASI-EXTRACT® T-2 and HT-2 columns were suitable for analysis of T-2 and HT-2 in a variety of cereal extracts. Following the prescribed protocol, cereal samples were extracted and spiked following filtration with 100 ppb toxins (50 ng/g T-2 and 50 ng/g HT-2) or 500 ppb toxins (250 ng T-2 Ig and 250 ng HT-2/g). Recoveries ranged from 71% to 119% for both toxins with a % CV of 3.7% to 13.5% (Table III). Figures 1 and 2 show the effect of including a 2% sodium chloride dilution step for the analysis of oats. This step clearly facilitates protein precipitation prior to using the immunoaffinity column, resulting in much cleaner chromatography. Finally a barley reference material was analysed over several days using the immunoaffinity columns with LC-fluorescence (Figure 3) and results compared to GC-MS following trimethylsilyl derivatization (6). The LC results compared well to those obtained using GC-MS following trimethylsilyl derivatization (Table IV) and were reproducible over several days (Table V).

Conclusions Recoveries of T-2 and HT-2 using EASI-EXTRACT® T-2 and HT-2 columns on cereal samples spiked with total T-2 and HT-2 toxins at 100 ppb and


282 Fluoresce

,200

,000-


800

600

400-

200-

-200 15.0

20.0

25.0

30.0

35.0

Figure 1. Oats spiked with 100 ppb total T-2 and HT-2 toxin (1:1) analysed without a 2% sodium chloride dilution. 1,200

Fluoresce mV

1,000

800

600 2 -HT-2 -19.098

400 1-T-2-10.073

200-

-2000.0

5.0

10.0

15.0

20.0

25.0

30.0

min 35.0

Figure 2. Oats spiked with 100 ppb total T-2 and HT-2 toxin (1:1) analysed with the inclusion of2% sodium chloride dilution step to aid precipitation ofproteins.


283 CROMATOGRAFO X


UBI A, Ex=381, bm=4/0 (IZAIH)M)/U1.U)

Figure 3. LC Trace ofNaturally Contaminated Barley Reference Material using EASI-EXTRACT® T-2 & HT-2 Columns

Table V. Naturally Contaminated Barley: Analysed over 2 Days using EASI-EXTRACT® T-2 & HT-2 IAC Columns Sample Type Sample A Day 1 Sample A Day 2 Sample B Day 1 Sample B Day 2

T-2 22 24 13 15

HT-2 80 90 33 41

500 ppb were within the European Union (EU) specification of 60% to 120%. Analysis of naturally contaminated reference samples for T-2 and HT-2 also showed consistent results over two days giving a percentage relative standard deviation (% RSD) of

Development of an Immunoaffinity Column for the Determination of T-2

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