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678

WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

FOOD CHEMICAL CONTAMINANTS

Liquid Chromatographic Analysis of Aflatoxin Using Post-Column Photochemical Derivatization: Collaborative Study 1

ARTHUR E. WALTKING

Waltking Associates, 482 Rock Rd, Glen Rock, NJ 07452

DAVID WILSON

University of Georgia, Coastal Plain Experiment Station, Tifton, GA 31793

Collaborators: D. Chan; E. Dunn; J. Humphries; H. Kandler; E. Sizoo; D. Wilson

Aflatoxin analysis, with post-column derivatization using a photochemical reactor for enhanced detection (PHRED) system for derivatization, has been compared to the officially recognized iodine and Kobra cell derivatization systems. This photochemical system has been extensively used for screening peanuts by some U.S. Department of

Agriculture laboratories for many years. From their periodic method checks, using standard spiked samples, an 80 sample series with each of the

3 derivatization methods was statistically analyzed. Paired comparisons, using the same sample extract, were also made between the PHRED and one of the other 2 methods, among laboratories in

4 different countries, on a variety of naturally

contaminated commodity products. The differences between the techniques were not significant for peanuts, but for corn the photochemical system consistently gave slightly higher values for aflatoxins B 1 and B 2 than the Kobra cell method. However, a comparison of all sample results showed no significant differences between methods. The Pearson correlation coefficients for aflatoxin B 1 in 102 test samples and aflatoxin B 2 in 94 test samples were 0.9994 and 0.9874, respectively. The probability factor was P < 0.0001, and the t-tests were not significantly different except for the corn. These indicated that the PHRED system is equivalent to the iodine and Kobra cell methods for peanuts relative to the current official procedures, but the PHRED system has a slightly high bias for corn compared to the iodine and Kobra cell systems.

Submitted for publication March 2006. The recommendation was approved by the Methods Committee on Natural Toxins and Allergens as First Action. See “Official Methods Program Actions,” (2006) Inside Laboratory Management, January/February issue.

Corresponding author’s e-mail: arthur.waltking@verizon.net
1

The study is a change of method proposal for AOAC Methods 991.31 and 999.07 and the modification (from TLC to LC) for AOAC Method

970.45.

W hen aflatoxins were found to adversely affect the

health of various animal species in the 1960s,

attempts were made to provide fast, inexpensive,

and comprehensive detection methods. Two methods developed by researchers of the U.S. Food and Drug Administration, requiring up to 5 h for 4 samples (1, 2), were the first significant improvements from the almost 24 h required with the initial method (3). The development of the Best Foods (BF) method permitted the analysis of 4 samples to be completed within 1 h (4, 5). These methods, in the 1960s, provided the basic tools for isolating aflatoxin contaminants from foods and commodities. The principal emphasis in the 1970s was on improving sampling techniques and statistical evaluations of the data (6–8). Studies also continued on many other aspects of the problem and improvements were made in the identification of the biological conversion to additional metabolites, the M 1 (milk toxins; 9) and in studies of the development and degradation of the aflatoxins during storage of the host commodity or during commercial food preparations (10–12). A major advance was the development of a confirmation of identity method by Przybylski (13), which readily proved the presence of the aflatoxins B 1 and G 1 by forming derivatives that have different transport positions on the thin-layer plates. Also, the analyses became more objective in form as the subjective evaluation of the thin-layer plates was automated by densitometers or supplanted by liquid chromatography (LC; 14–16).

The advances in the use of LC and various enhancement techniques were well documented in a review article in 1992 (17). Many of the currently used LC methods use derivative formation (18–23) and therefore result from the original chemical confirmation test (13). In 1993, aflatoxin analysis using a photochemical derivatization technique was introduced by Joshua (24). His paper critiques the merits of the 3 chemical approaches, using trifluoroacetic acid (TFA), iodine, or bromine, relative to the photochemical technique. Because the photochemical reactor for enhanced detection (PHRED) system provides the derivative formation using only UV light, the ability to obtain the same results with a

WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

679

simple sample preparation and less chemical waste is a distinct advantage in the present day laboratory. An important improvement in LC detection was reported recently by Joshua (25), who demonstrated near baseline separation of all 6 aflatoxins (B 1 ,B 2 ,G 1 ,G 2 ,M 1 , and M 2 ), as well as zearalenone and ochratoxin A on a single chromatogram when using post-column photochemical derivatization and fluorescence detection. The possibility of providing a single detector condition, which could offer a universal quantitative procedure for sample extracts for all 8 of these contaminants independent of the extraction method used, prompted this study.

Collaborative Study

Purpose

The purpose of the study is to determine the equivalency of the performance characteristics for an LC photochemical derivatization technique (PHRED) to that of other previously AOAC-approved post-column derivatization systems, iodine (20), and Kobra cell (26, 27), thereby recognizing the reduced cost and simplicity advantages. As a first step, a new technique must accurately measure aflatoxin with both corn and peanut crops, which are among the most highly regulated in the United States and Europe. However, this study was considered to be unnecessarily complex if it were to involve multiple commodities, using multiple aflatoxins with a number of official methods. Although for many collaborative studies, a precollaborative study would be performed to determine the efficacy or ruggedness of the method, in this case such studies have already been done in part by at least 2 laboratories (28, 29). In these cases, the data were reported to provide equivalent results for the photochemical method with the iodine and the Kobra cell methods. The data from the first of these references (28), which compared the PHRED to the iodine method (20), are included with the author’s permission in this data set. The iodine method was also used as one of the reference methods in the AOAC collaborative study, which compared 9 aflatoxin methods (30). The other reference (29) showed equivalency of the Kobra cell to the PHRED.

Performance

In ref. 29, where the relative standard deviation (RSD) values of the validation testing ranged from 0.3 to 1.8% for the PHRED and 0.9 to 2.0% for the Kobra cell, the equivalency of these systems is indicated.

Principle

The PHRED is a compact unit placed between the LC column and the detector in the same manner as the Kobra cell (31) to perform online post-column derivatization of aflatoxins to increase detectability and/or selectivity of response for the detector. Unlike the iodine and Kobra cell, it performs the derivatization photochemically without additional chemicals added to the mobile phase. The method for the PHRED is therefore an adjunct method. It is used only for the enhancement of aflatoxins B 1 and G 1 in the extract of

any of the official preparation methods using LC separation and post-column derivatization. Instead of a full collaborative study, it was proposed and accepted that a study of the PHRED consisting of a direct comparison of the same extracts with the Kobra cell or iodine systems would be appropriate for statistical analysis. This study used the procedure described in the Method section with the PHRED system.

Quality Assurance–Control

Three European laboratories actively involved with aflatoxin analysis were asked to use the PHRED equipment on a minimum of 4 sample extracts of a naturally contaminated material which was prepared and analyzed by their current method. They were provided a mixed aflatoxin standard prepared from a single supply, so that differences in the standard used would be negated. Their submission of the comparative results and photocopies of the chromatograms of the samples and standards were used to certify their capability prior to delivery of a collaborative sample extract series. Subsequently, with the change from a standard collaborative to an equivalency review, these data have been included with the extensive data available from the United States.

Participating Laboratories

Six laboratories participated in the study: RIVM (Bilthoven, The Netherlands); Kantonales Labor (Zurich, Switzerland); Central Science Laboratory (York, United Kingdom); University of Georgia (Tifton, GA); U.S. Department of Agriculture (USDA; Blakely, GA); USDA (Madill, OK).

Study Design

The USDA laboratory in Blakely, GA, has used the PHRED photochemical system as well as the iodine method for many years for screening peanuts while the laboratory in Madill, OK, used the Kobra cell. From their periodic method checks using peanut samples spiked with aflatoxin standards, the last 80 samples, with each of the 3 derivatization methods, were tabulated and analyzed as shown in Tables 1–4. All of their extracts were prepared using the extractions in AOAC Method 970.45. Segments of sample slurries which proved to be free of contamination were spiked with a mixed aflatoxin standard and carried through as confirmation of proper analysis. Earlier USDA results of 19 naturally contaminated peanuts from ref. 27, also using extracts from AOAC Method 970.45, are shown in Table 5. The extracts in this study compared the PHRED and the iodine system. An independent series of naturally contaminated corn and peanuts, 37 and 30 samples, respectively, were analyzed by PHRED and the Kobra cell system at the University of Georgia. These extracts, as shown in Table 6, were prepared using AOAC Method 991.31. Adding to these the 16 samples from the European checks of the PHRED equipment operation (Table 7), provided a total of 102 samples of naturally contaminated extracts which were statistically analyzed, independently and combined. The European laboratories reported using the following referenced methods for sample extract preparation: United

680 WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

Table 1.

extraction and iodine system for determinations performed by USDA (Blakely, GA) a

Peanuts and pistachio nuts spiked with 12.0 ng/g aflatoxin standard using AOAC Official Method 970.45 for

Aflatoxin, ng/g

Sample No.

B 1

B 2

G 1

G 2

Total

1

4.0

1.2

5.4

1.0

11.6

2

6.9

0.9

2.8

0.3

10.9

3

5.8

1.6

3.9

0.6

11.9

4

4.0

1.2

4.2

1.3

10.7

5

5.2

1.5

4.3

0.9

11.9

6

4.9

1.3

4.2

0.4

10.8

7

4.7

1.2

4.0

1.1

11.0

8

3.9

1.3

6.3

0.8

12.3

9

4.6

1.6

4.7

1.6

12.5

10

5.9

1.3

4.8

0.3

12.3

11

4.5

1.4

5.0

1.2

12.1

12

3.7

1.1

4.0

1.0

9.8

13

3.5

1.4

4.1

0.8

9.8

14

4.0

1.3

4.0

0.4

9.7

15

3.9

1.2

3.8

1.2

10.1

16

4.0

1.1

5.0

0.4

10.5

17

4.2

1.4

4.6

1.5

11.7

18

4.0

1.0

4.4

0.6

10.0

19

3.2

1.0

4.7

0.9

9.8

20

4.4

1.0

4.4

0.5

10.3

21

6.0

0.9

5.6

0.0

12.5

22

4.3

1.3

5.6

1.5

12.7

23

4.2

1.2

5.3

1.2

11.9

24

3.7

1.3

5.4

1.1

11.5

25

4.4

1.3

3.2

1.1

10.0

26

4.6

1.6

4.3

0.6

11.1

27

4.5

1.5

3.7

1.1

10.8

28

5.2

1.3

3.5

1.2

11.2

29

3.9

1.2

3.8

0.9

9.8

30

3.9

1.2

3.9

0.9

9.9

31

4.0

1.3

4.0

0.9

10.2

32

4.1

1.3

3.7

0.9

10.0

33

4.1

1.3

4.0

0.9

10.3

34

3.9

1.3

4.1

0.8

10.1

35

4.1

1.3

4.0

0.8

10.2

36

4.2

1.3

3.9

0.8

10.2

37

4.2

1.3

3.9

0.9

10.3

38

4.3

1.4

4.3

0.9

10.9

39

4.5

1.5

4.3

1.0

11.3

40

2.3

1.0

5.8

1.8

10.9

41

3.9

1.1

5.9

1.0

11.9

42

3.8

1.0

5.2

0.8

10.8

43

4.5

1.5

4.1

1.4

11.5

44

4.4

1.6

3.2

0.9

10.1

Table 1.

(continued)

WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

681

Aflatoxin, ng/g

Sample No.

B 1

B 2

G 1

G 2

Total

45

4.7

1.5

4.2

0.6

11.0

46

4.7

1.7

3.9

0.6

10.9

47

5.0

1.6

5.2

1.4

13.2

48

3.7

1.5

4.0

1.2

10.4

49

4.9

1.4

3.7

0.6

10.6

50

4.4

1.3

4.6

0.5

10.8

51

4.2

1.3

4.6

0.9

11.0

52

4.0

1.2

4.6

0.8

10.6

53

3.7

1.0

4.4

1.0

10.1

54

4.0

1.1

4.8

0.9

10.8

55

4.4

1.2

5.4

1.3

12.3

56

4.0

1.1

4.8

0.8

10.7

57

3.8

1.1

4.6

1.2

10.7

58

4.5

1.2

5.4

1.2

12.3

59

4.6

1.3

5.5

1.2

12.6

60

3.9

1.1

4.9

1.0

10.9

61

3.9

1.2

5.2

1.0

11.3

62

3.9

1.0

4.7

1.1

10.7

63

3.9

1.2

5.0

1.0

11.1

64

3.7

0.9

4.3

1.1

10.0

65

3.8

1.0

4.6

1.1

10.5

66

4.4

1.3

5.5

1.3

12.5

67

3.6

0.9

4.4

1.0

9.9

68

5.1

1.3

5.5

1.2

13.1

69

4.1

1.0

5.5

0.9

11.5

70

3.6

1.1

5.0

1.0

10.7

71

3.4

1.0

4.5

1.2

10.1

72

3.8

0.9

4.6

0.8

10.1

73

4.1

1.1

5.2

0.9

11.3

74

4.2

1.1

5.4

0.9

11.6

75

4.4

1.1

5.5

1.1

12.1

76

3.6

0.9

4.7

0.8

10.0

77

3.9

1.1

5.5

1.1

11.6

78

3.9

1.0

5.2

0.9

11.0

79

4.4

1.1

5.9

0.9

12.3

80

4.0

1.0

5.4

0.9

11.3

a All samples are peanuts except Nos. 20, 32, 44, 48, and 69 which were pistachio. Standard concentrations (ng/g) used for spiking all samples: B 1 at 4.6; B 2 at 1.4; G 1 at 4.6; G 2 at 1.4.

682 WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

Table 2.

PHRED system for determinations at USDA (Blakely, GA) a

Peanuts spiked with 12.0 ng/g aflatoxin standard using AOAC Official Method 970.45 for extraction and

Aflatoxin, ng/g

Sample No.

B 1

B 2

G 1

G 2

Total

1

4.5

1.4

5.0

1.3

12.2

2

4.1

1.6

3.8

0.8

10.3

3

3.2

2.0

5.0

0.6

10.8

4

4.4

1.1

5.5

0.9

11.9

5

4.1

1.1

4.7

0.8

10.7

6

3.2

1.2

5.0

0.6

10.0

7

4.3

1.6

5.0

0.9

11.8

8

4.5

1.4

4.7

0.7

11.3

9

5.2

1.2

4.6

0.9

11.9

10

4.7

1.2

4.4

0.4

10.7

11

5.3

1.0

6.1

0.5

12.9

12

3.6

1.0

4.4

1.2

10.2

13

7.8

1.2

4.0

0.2

13.2

14

5.1

0.9

5.5

0.9

12.4

15

6.1

1.1

4.7

0.9

12.8

16

3.4

1.2

4.4

0.8

9.8

17

5.3

1.2

5.6

1.1

13.2

18

4.3

1.2

5.3

1.0

11.8

19

4.1

1.3

6.0

0.9

12.3

20

4.1

1.3

5.2

1.6

12.2

21

4.1

1.0

4.4

0.8

10.3

22

5.2

1.4

5.6

0.8

13.0

23

4.0

1.2

5.4

0.8

11.4

24

5.2

1.0

5.3

0.8

12.3

25

4.9

1.3

5.8

1.2

13.2

26

3.6

1.1

4.7

0.9

10.3

27

4.0

1.2

4.4

0.7

10.3

28

4.9

1.3

5.0

1.6

12.8

29

4.2

1.2

3.8

0.7

9.9

30

3.7

0.8

4.6

1.0

10.1

31

4.2

1.1

4.7

1.0

11.0

32

4.2

1.0

5.3

0.9

11.4

33

4.1

1.2

5.2

0.8

11.3

34

3.7

1.2

4.4

0.9

10.2

35

4.1

1.1

3.9

0.7

9.8

36

4.7

1.2

4.3

0.9

11.1

37

4.9

1.9

4.4

0.9

12.1

38

4.4

1.2

4.5

1.2

11.3

39

4.1

1.2

4.2

0.8

10.3

40

4.5

1.3

4.4

1.0

11.2

41

4.5

1.3

4.5

1.4

11.7

42

4.4

1.3

4.6

1.2

11.5

43

4.8

1.5

5.3

1.4

13.0

44

3.7

1.1

4.2

0.8

9.8

Table 2.

(continued)

WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

683

Aflatoxin, ng/g

Sample No.

B 1

B 2

G 1

G 2

Total

45

3.7

1.1

4.2

0.8

9.8

46

4.6

1.1

4.5

0.8

11.0

47

3.7

1.3

4.4

0.7

10.1

48

3.8

1.4

4.5

1.3

11.0

49

5.1

1.2

5.2

1.2

12.7

50

5.1

0.7

4.8

1.2

11.8

51

5.4

0.6

4.4

0.5

10.9

52

3.9

1.3

4.5

1.0

10.7

53

4.0

1.2

3.8

1.1

10.1

54

3.8

1.2

4.4

0.8

10.2

55

4.1

1.3

4.9

0.7

11.0

56

4.3

1.5

5.4

1.3

12.5

57

3.7

1.1

4.7

1.0

10.5

58

4.3

1.5

5.4

1.3

12.5

59

4.6

1.5

4.5

0.9

11.5

60

4.6

1.1

5.1

0.9

11.7

61

4.4

1.2

5.1

1.4

12.1

62

4.1

1.1

4.3

0.9

10.4

63

4.1

1.1

4.5

1.4

11.1

64

4.0

1.1

5.3

1.5

11.9

65

5.1

1.3

5.7

0.9

13.0

66

3.6

0.9

4.5

0.6

9.6

67

3.7

1.0

4.3

0.7

9.7

68

4.5

1.2

5.3

1.0

12

69

4.5

1.4

5.2

1.5

12.6

70

4.4

1.0

4.5

0.6

10.5

71

3.7

1.0

5.8

1.6

12.1

72

3.6

1.0

4.6

1.0

10.2

73

3.7

1.1

4.9

0.6

10.3

74

3.5

1.0

4.5

0.8

9.8

75

3.8

1.0

4.5

0.9

10.2

76

3.3

0.9

4.7

0.9

9.8

77

4.5

1.3

6.0

0.4

12.2

78

4.3

0.9

5.0

0.7

10.9

79

5.0

1.1

5.3

0.4

11.8

80

5.0

1.2

4.6

0.9

11.7

a Standard concentrations (ng/g) used for spiking all samples: B 1 at 4.6; B 2 at 1.4; G 1 at 4.6; G 2 at 1.4.

684 WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

Table 3.

Kobra cell system for determinations at USDA (Madill, OK) a

Peanuts spiked with 12.0 ng/g aflatoxin standard using AOAC Official Method 970.45 for extraction and

Aflatoxin, ng/g

Sample No.

B 1

B 2

G 1

G 2

Total

1

4.2

1.3

4.2

0.9

10.6

2

3.8

1.2

3.9

0.9

9.8

3

4.1

1.3

4.4

1.0

10.8

4

3.8

1.1

4.3

0.8

10

5

3.7

1.1

4.4

1.0

10.2

6

4.0

1.2

4.6

0.8

10.6

7

4.1

1.3

4.8

0.7

10.9

8

5.1

1.5

5.5

0.8

12.9

9

4.4

1.4

5.0

1.1

11.9

10

3.8

1.2

4.3

0.9

10.2

11

4.3

1.3

4.8

0.9

11.3

12

4.6

1.5

5.2

1.1

12.4

13

4.2

1.3

4.7

0.9

11.1

14

3.9

1.2

4.3

1.0

10.4

15

4.6

1.4

5.0

1.3

12.3

16

4.1

1.3

4.6

1.2

11.2

17

4.1

1.3

4.5

1.0

10.9

18

4.4

1.4

4.9

0.9

11.6

19

4.7

1.3

4.6

0.9

11.5

20

4.0

1.2

4.4

0.8

10.4

21

4.2

1.2

4.7

0.7

10.8

22

4.3

1.3

4.7

0.8

11.1

23

4.8

1.5

5.3

1.0

12.6

24

4.2

1.3

4.6

0.8

10.9

25

3.8

1.2

4.1

0.8

9.9

26

3.9

1.2

4.2

0.8

10.1

27

4.7

1.4

4.8

0.6

11.5

28

5.5

1.2

4.1

0.5

11.3

29

4.5

1.4

4.7

0.7

11.3

30

4.0

1.3

4.4

1.0

10.7

31

3.9

1.2

4.3

0.9

10.3

32

4.2

1.3

4.5

0.9

10.9

33

4.5

1.4

4.9

0.9

11.7

34

4.9

1.4

4.3

0.9

11.5

35

4.1

1.3

4.4

0.8

10.6

36

4.3

1.4

4.8

0.8

11.3

37

4.5

2.2

4.5

0.7

11.9

38

3.9

1.2

4.1

0.6

9.8

39

4.7

1.5

5.1

0.8

12.1

40

4.1

1.3

4.6

0.8

10.8

41

4.7

1.5

5.2

0.7

12.1

42

4.3

1.4

5.0

1.0

11.7

43

3.8

1.2

4.5

0.9

10.4

44

4.2

1.4

4.8

0.9

11.3

Table 3.

(continued)

WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

685

Aflatoxin, ng/g

Sample No.

B 1

B 2

G 1

G 2

Total

45

4.1

1.3

4.5

0.8

10.7

46

4.3

1.4

4.9

0.9

11.5

47

4.6

1.5

5.1

1.0

12.2

48

5.4

1.6

5.3

0.6

12.9

49

5.1

1.6

5.3

0.7

12.7

50

5.3

1.5

5.0

1.1

12.9

51

4.7

1.5

5.0

1.0

12.2

52

4.6

1.4

4.8

0.9

11.7

53

4.5

1.4

4.7

0.8

11.4

54

4.4

1.3

4.7

0.8

11.2

55

4.2

1.4

4.6

0.7

10.9

56

4.5

1.4

4.9

0.6

11.4

57

4.6

1.4

5.0

0.8

11.8

58

4.2

1.4

4.7

1.1

11.4

59

4.3

1.3

4.5

0.8

10.9

60

4.3

1.3

4.5

0.7

10.8

61

5.1

1.4

4.6

0.7

11.8

62

4.2

1.3

4.6

0.7

10.8

63

4.6

1.5

5.1

1.3

12.5

64

4.2

1.3

4.6

1.0

11.1

65

4.9

1.4

4.5

0.9

11.7

66

3.7

1.2

4.4

0.8

10.1

67

3.9

1.2

4.6

0.8

10.5

68

3.9

1.2

4.5

0.7

10.3

69

4.4

1.4

4.9

0.6

11.3

70

5.0

1.5

5.0

0.7

12.2

71

4.5

1.5

5.2

0.8

12.0

72

4.7

1.1

4.1

0.4

10.3

73

3.6

1.3

5.2

0.8

10.9

74

4.7

1.5

5.3

0.7

12.2

75

4.5

1.2

4.4

0.6

10.7

76

4.1

1.2

4.4

0.8

10.5

77

4.1

1.2

4.4

0.7

10.4

78

4.4

1.3

4.7

0.9

11.3

79

4.2

1.3

4.7

0.8

11.0

80

4.2

1.3

4.8

0.8

11.1

a Standard concentrations (ng/g) used for spiking all samples: B 1 at 4.6; B 2 at 1.4; G 1 at 4.6; G 2 at 1.4.

686 WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

Table 4.

extraction a

Summary comparison of the 3 systems (iodine, PHRED, and Kobra cell) using AOAC Method 970.45

Technique

Mean, ng/g

SD, ng/g

RSD r , %

PRSD R , %

HorRat

 

Aflatoxin B 1

Iodine

0.64

15.13

36.06

0.42

4.23

Kobra

4.35

0.41

9.43

35.91

0.26

PHRED

4.34

0.70

16.13

35.93

0.45

 

Aflatoxin B 2

Iodine

1.22

0.22

16.39

43.46

0.38

Kobra

1.34

0.15

11.19

42.85

0.26

PHRED

1.19

0.22

18.49

43.62

0.42

 

Aflatoxin G 1

Iodine

4.62

0.72

15.58

35.59

0.44

Kobra

4.68

0.34

7.26

35.52

0.20

PHRED

4.81

0.54

11.23

35.38

0.32

 

Aflatoxin G 2

Iodine

0.95

0.31

32.63

45.12

0.72

Kobra

0.84

0.16

19.05

45.96

0.41

PHRED

0.93

0.30

32.26

45.26

0.71

 

Total aflatoxin

Iodine

11.02

0.89

8.08

31.24

0.26

Kobra

11.21

0.78

6.96

31.16

0.22

PHRED

11.27

1.05

9.32

31.13

0.30

a Tables 1–3 samples spiked with 12.0 ng/g aflatoxin standard (n = 80). For each set in Tables 1–3, the following assessments of the data were obtained: mean; standard deviation (SD); percent relative standard deviation for repeatability (RSD r , %); predicted relative reproducibility as calculated from the Horwitz equation (PRSD R , %) and the HorRat based on intralaboratory repeatability (RSD r ).

Kingdom (32); Switzerland (26); The Netherlands: Set 1 (26), Set 2 (33). Statistical analyses of all the natural contamination series were performed only for the B 1 and B 2 aflatoxins because of absence of G aflatoxins in so many of the products. A univariate statistical analysis of the spiked samples showed similarity of performance of the aflatoxin G 1 series to the aflatoxin B 1 results.

AOAC Official Method 2005.08 Analysis of Aflatoxin

Liquid Chromatography with Post-Column Photochemical Derivatization

First Action 2005

[The photochemical reactor for enhanced detection (PHRED) is applicable to determination of aflatoxins in test extracts of corn and peanuts when using AOAC Methods 991.31, 999.07, or 970.45 with LC. Although no significant difference exists for the PHRED in comparison to other post-column methods with peanuts, a slightly high bias is obtained with corn when compared with the iodine or Kobra cell possibly due to higher recovery.]

Caution: Mycotoxins are toxic substances. Perform manipulations in a hood wherever possible, taking particular precautions, such as using a glove box, when toxins are in dry form because of their electrostatic nature and tendency to disperse. Swab any accidental spills and all glassware and waste materials with 5% NaOCl bleach. Use UV glasses if there is exposure to any direct or reflected UV light from the light source. Aflatoxins must be handled with extreme caution because they are known to be carcinogens. Use hypochlorite bleach for cleaning glassware and when disposing of waste materials.

A. Principle

Post-column derivatization of aflatoxins can increase detectability and/or selectivity of responses for the HPLC detector. By performing the derivatization photochemically, the derivative structures B 2a and G 2a are apparently obtained, providing the enhanced signals for the B 1 and G 1 aflatoxins without effect on the B 2 and G 2 aflatoxins.

B. Apparatus

Note: Evaluate any leakage of UV light from source equipment and if detected, provide shielding or protective

WALTKING & WILSON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 3, 2006

Table 5.

Method 970.45 extraction

Comparison of PHRED and iodine systems with naturally contaminated peanuts using AOAC Official

687

 

PHRED

Iodine

 

Aflatoxin, ng/g

 

B 1

B 2

G 1

G 2

Total

B 1

B 2

G 1

G 2

Total

2.44

0.57

ND a

ND

3.01

2.36

0.60

ND

ND

2.96

31.53

4.91

0.42

0.21

37.07

30.62

5.06

ND

ND

35.68

42.22

6.52

ND

0.10

48.84

38.90

6.97

ND

ND

45.87

20.68

3.12

ND

0.15

23.96

19.36

3.32

ND

ND

22.68

37.22

5.79

ND

0.10

43.12

35.63

6.33

ND

ND

41.96

42.53

6.14

ND

0.13

48.81

50.69

ND

ND

ND

50.69

42.69

6.57

ND

0.12

49.38

45.62

6.79

ND

ND

52.41

33.13

4.96

ND

ND

38.09

32.88

5.05

ND

ND

37.93

25.40

2.99

ND

0.13

25.52

22.74

3.33

ND

ND

26.07

16.38

2.47

ND

ND

18.86

15.03

2.58

ND

ND

17.61

2.74

0.42

ND

ND

3.16

2.60

0.43

ND

ND

3.03

33.53

4.91

ND

ND

38.44

35.77

5.86

ND

ND

41.63

0.67

0.04

ND

ND

0.72

1.03

0.21

ND

ND

1.24

2.44

0.51

ND

ND

2.95

2.69

0.66

ND

ND

3.35

2.80

1.85

0.06

0.11

4.82

2.49

2.11

ND

ND

4.60

2.33

1.27

0.52

0.19

4.31