**3. Results and discussion**

Table 1 shows the results of analysis and a regression line obtained from least-square analy‐ sis of Sample A, of which the slope and *y*-intercept were used for the calculation of LOQ. Twelve regression lines (six lines each for intraday and interday analyses) were performed in this study; slopes and *y*-intercepts of all 12 analyses are presented in Table 2. The calcula‐ tion for LOQ was based on the equation LOQ = 10 σ/S, where σ and S are the standard devi‐ ation of *y*-intercepts and the average slope of the 12 regression lines, respectively. In this study, the standard deviation of *y*-intercepts was 173.69 mV × L/µg and the average slope was 180,518 mV. The calculated LOQ was (10 \* 173.69)/180,518 = 0.01 µg/L. The accuracy of the method, expressed as % recovery, ranged from 88.8% to 94.1%, with an average value of 90.8%. The precision of the method, expressed as %RSD (percent relative standard devia‐ tion), ranged from 1.1% to 7.5%. Table 3 summarizes the accuracy and precision of determi‐ nation of AFM1 in goat milk samples fortified with AFM1 at four concentrations, with intraday and interday analyses. HPLC chromatograms of standard AFM1 (10 µg/L), a goat milk sample contaminated with AFM1 (0.05 µg/L), and an uncontaminated goat milk sample are presented in Figure 1. The retention time for AFM1 under the conditions in this study was approximately 6.8 min.

Table 4 shows the incidence and concentrations of AFM1 in raw and pasteurized goat milk samples. The incidence of AFM1 in raw goat milk collected in 2009, 2010 and 2011 was 46.7% (7/15), 66.7% (10/15) and 60.0% (9/15), respectively, while the incidence in pasteurized milk was 53.3% (8/15), 46.7% (7/15) and 53.3% (8/15), respectively. The total incidence of positive samples with respect to 90 samples analyzed in this study was 54.4% (49/90). Of the 49 posi‐ tive samples, only 7 samples (14.3%) were contaminated with AFM1 above the EU standard of 0.05 µg/L. The three-year average concentrations of AFM1 found in the raw and pasteur‐ ized milk samples were 0.043 and 0.040 µg/L, respectively. The maximum concentration found in this study was 0.086 µg/L, which was far below the U.S. regulatory limit of 0.5 µg/L. In this study, statistical analysis showed that there were no significant differences in AFM1 concentrations among the raw and pasteurized milk samples and across the two types of milk samples collected over a three-year period.

When compared to cow milk, goat milk has a lower percentage of positive samples and low‐ er AFM1 concentrations. Ghanem and Orfi [15] reported that the average concentration of AFM1 in raw goat milk (0.019 µg/L, n = 11), collected from markets in Syria between April 2005 and April 2006, was less than that in raw cow milk (0.143 µg/L, n = 74); the percentage of positive samples of goat milk (7 samples, 63.6%) was also less than that of cow milk (70 samples, 94.6%). Hussain et al. [16] found that 6 (20%) of 30 raw goat milk samples were contaminated with AFM1 at an average concentration of 0.002 µg/L, while 15 (37.5%) of 40 raw cow milk samples were contaminated with an average AFM1 level of 0.014 µg/L. Rahimi et al. [17] reported that the incidence of AFM1 in raw goat and cow milk samples collected from Ahvaz in Khuzestan province, Iran, between November 2007 and December 2008, was 31.7% (19/60) and 78.7% (59/75), respectively. Concentrations of AFM1 in raw milk samples of both species were 0.0301 and 0.0601 µg/L, respectively.


1 Average value of two determinations

**2.6. Statistical analysis**

210 Aflatoxins - Recent Advances and Future Prospects

**3. Results and discussion**

was approximately 6.8 min.

of milk samples collected over a three-year period.

A randomized block experiment was used to evaluate the differences in AFM1 concentra‐ tions in the two types of milk samples and among the three collection years. Duncan's multi‐ ple comparison test was applied to obtain significance levels between the raw milk and pasteurized milk, and among each year of individual milk products (*P* < 0.05). SPSS Statis‐

Table 1 shows the results of analysis and a regression line obtained from least-square analy‐ sis of Sample A, of which the slope and *y*-intercept were used for the calculation of LOQ. Twelve regression lines (six lines each for intraday and interday analyses) were performed in this study; slopes and *y*-intercepts of all 12 analyses are presented in Table 2. The calcula‐ tion for LOQ was based on the equation LOQ = 10 σ/S, where σ and S are the standard devi‐ ation of *y*-intercepts and the average slope of the 12 regression lines, respectively. In this study, the standard deviation of *y*-intercepts was 173.69 mV × L/µg and the average slope was 180,518 mV. The calculated LOQ was (10 \* 173.69)/180,518 = 0.01 µg/L. The accuracy of the method, expressed as % recovery, ranged from 88.8% to 94.1%, with an average value of 90.8%. The precision of the method, expressed as %RSD (percent relative standard devia‐ tion), ranged from 1.1% to 7.5%. Table 3 summarizes the accuracy and precision of determi‐ nation of AFM1 in goat milk samples fortified with AFM1 at four concentrations, with intraday and interday analyses. HPLC chromatograms of standard AFM1 (10 µg/L), a goat milk sample contaminated with AFM1 (0.05 µg/L), and an uncontaminated goat milk sample are presented in Figure 1. The retention time for AFM1 under the conditions in this study

Table 4 shows the incidence and concentrations of AFM1 in raw and pasteurized goat milk samples. The incidence of AFM1 in raw goat milk collected in 2009, 2010 and 2011 was 46.7% (7/15), 66.7% (10/15) and 60.0% (9/15), respectively, while the incidence in pasteurized milk was 53.3% (8/15), 46.7% (7/15) and 53.3% (8/15), respectively. The total incidence of positive samples with respect to 90 samples analyzed in this study was 54.4% (49/90). Of the 49 posi‐ tive samples, only 7 samples (14.3%) were contaminated with AFM1 above the EU standard of 0.05 µg/L. The three-year average concentrations of AFM1 found in the raw and pasteur‐ ized milk samples were 0.043 and 0.040 µg/L, respectively. The maximum concentration found in this study was 0.086 µg/L, which was far below the U.S. regulatory limit of 0.5 µg/L. In this study, statistical analysis showed that there were no significant differences in AFM1 concentrations among the raw and pasteurized milk samples and across the two types

When compared to cow milk, goat milk has a lower percentage of positive samples and low‐ er AFM1 concentrations. Ghanem and Orfi [15] reported that the average concentration of AFM1 in raw goat milk (0.019 µg/L, n = 11), collected from markets in Syria between April 2005 and April 2006, was less than that in raw cow milk (0.143 µg/L, n = 74); the percentage

tics version 17.0 for Windows was used for statistical analysis.

2 Residual peak area = peak area of AFM1-fortified sample – peak area of blank sample

**Table 1.** Linear regression analysis of AFM1-fortified sample A for the determination of LOQ


**Year Samples Positive1 AFM1 concentration (ng/ml)2 AFM1 incidence3**

2009 15 7 (46.7) 0.042 ± 0.012 0.022–0.061 6 (85.7) 1 (12.5) 2010 15 10 (66.7) 0.049 ± 0.018 0.025–0.086 8 (80.0) 2 (20.0) 2011 15 9 (60.0) 0.036 ± 0.015 0.018–0.066 8 (88.9) 1 (11.1) Total 45 26 (57.8) 0.043 ± 0.017 0.018–0.086 22 (84.6) 4 (15.4)

2009 15 8 (53.3) 0.039 ± 0.017 0.015–0.075 7 (87.5) 1 (12.5) 2010 15 7 (46.7) 0.045 ± 0.015 0.022–0.061 6 (85.7) 1 (14.3) 2011 15 8 (53.3) 0.035 ± 0.019 0.014–0.073 7 (87.5) 1 (12.5) Total 45 23 (51.1) 0.040 ± 0.016 0.014–0.073 20 (87.0) 3 (13.0) **Overall 90 49 (54.4) 0.041 ± 0.016 0.014–0.086 42 (85.7) 7 (14.3)**

**Table 4.** Incidence and concentrations of AFM1 in raw and pasteurized goat milk samples collected within the central

High incidence and concentrations of AFM1 in cow milk have also been found in Thailand. Ruangwises and Ruangwises [18] reported that all of 240 raw cow milk samples collected from 80 milk tanks at a milk collecting center in the central region of Thailand were found to be con‐ taminated with AFM1 at an average concentration of 0.070 µg/L. For pasteurized milk sam‐ ples, our previous studies showed that AFM1 was found in 349 (83.1%) of 420 pasteurized milk samples, collected from 40 provinces in all four regions of Thailand from May 2006 to January

Table 5 shows the incidence and concentrations of AFM1 in raw and pasteurized goat milk from various countries. For raw goat milk, Assem et al. [20] found that all of the three raw milk samples collected from markets in Lebanon between March–July 2010 contained AFM1 less than the LOQ of 0.005 ng/ml. Ozdemir [21] found that the mean concentration of AFM1 in 93 positive samples out of 110 raw milk samples collected from the city of Kilis, Turkey, from March–April 2006 was 0.019 µg/L. For pasteurized milk, Oliveira and Ferraz [22] deter‐ mined the concentrations of AFM1 in 12 pasteurized goat milk samples collected from the state of Sao Paulo, Brazil, and found that 7 samples (58.3%) were contaminated with an

2008, with AFM1 concentrations ranging between 0.012 and 0.114 µg/L [13,19].

*Raw milk*

*Pasteurized milk*

region of Thailand

1Numbers in parentheses are percentages for each year

3AFM1 incidence of the positive samples

average concentration of 0.034 µg/L.

2Means and ranges of AFM1 concentrations in the positive samples

Numbers in parentheses are percentages with respect to the positive samples

**analyzed (%) Mean Range 0.010–0.050 > 0.05**

Occurrence of Aflatoxin M1 in Raw and Pasteurized Goat Milk in Thailand

**µg/L µg/L**

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213

**Table 2.** Slopes and *y*-intercepts of 12 regression lines used for determination of LOQ for AFM1


a Values are mean ± SD

b % RSD = percent relative standard deviation.

**Table 3.** Accuracy and precision of determination of AFM1 in goat milk


1Numbers in parentheses are percentages for each year

2Means and ranges of AFM1 concentrations in the positive samples

3AFM1 incidence of the positive samples

**Sample Slope** *y***-intercept**

A 184,141 197.86 B 180,733 293.38 C 183,706 141.26 D 179,857 549.02 E 180,039 207.84 F 181,224 109.74

G 181,454 127.39 H 175,861 432.76 I 185,285 223.45 J 179,462 442.02 K 175,904 339.74 L 178,545 639.60

**Mean 180,518 (S) 308.67 SD 2,955.5 173.69 (σ)**

**(µg/L) Founda %RSDb Recovery Founda %RSDb Recovery**

0.025 0.023 ± 0.001 4.3 92.1 0.024 ± 0.002 7.5 94.1 0.050 0.046 ± 0.001 2.2 91.9 0.046 ± 0.002 3.5 91.4 0.125 0.112 ± 0.003 2.7 89.3 0.111 ± 0.004 3.9 88.8 0.250 0.225 ± 0.002 1.1 89.8 0.222 ± 0.005 2.1 89.0

**(µg/L) (%) (µg/L) (%)**

**Table 2.** Slopes and *y*-intercepts of 12 regression lines used for determination of LOQ for AFM1

**added Intraday (***n* **= 6) Interday (***n* **= 6)**

Intraday (*n* = 6)

212 Aflatoxins - Recent Advances and Future Prospects

Interday (*n* = 6)

**Overall (n = 12)**

**AFM1**

a

Values are mean ± SD

b % RSD = percent relative standard deviation.

**Table 3.** Accuracy and precision of determination of AFM1 in goat milk

**(mV × L/µg) (mV)**

Numbers in parentheses are percentages with respect to the positive samples

**Table 4.** Incidence and concentrations of AFM1 in raw and pasteurized goat milk samples collected within the central region of Thailand

High incidence and concentrations of AFM1 in cow milk have also been found in Thailand. Ruangwises and Ruangwises [18] reported that all of 240 raw cow milk samples collected from 80 milk tanks at a milk collecting center in the central region of Thailand were found to be con‐ taminated with AFM1 at an average concentration of 0.070 µg/L. For pasteurized milk sam‐ ples, our previous studies showed that AFM1 was found in 349 (83.1%) of 420 pasteurized milk samples, collected from 40 provinces in all four regions of Thailand from May 2006 to January 2008, with AFM1 concentrations ranging between 0.012 and 0.114 µg/L [13,19].

Table 5 shows the incidence and concentrations of AFM1 in raw and pasteurized goat milk from various countries. For raw goat milk, Assem et al. [20] found that all of the three raw milk samples collected from markets in Lebanon between March–July 2010 contained AFM1 less than the LOQ of 0.005 ng/ml. Ozdemir [21] found that the mean concentration of AFM1 in 93 positive samples out of 110 raw milk samples collected from the city of Kilis, Turkey, from March–April 2006 was 0.019 µg/L. For pasteurized milk, Oliveira and Ferraz [22] deter‐ mined the concentrations of AFM1 in 12 pasteurized goat milk samples collected from the state of Sao Paulo, Brazil, and found that 7 samples (58.3%) were contaminated with an average concentration of 0.034 µg/L.

The levels of AFM1 in goat milk are influenced by both feeding practices and the types of feedstuffs. Virdis et al. [23] determined the concentrations of AFM1 in goat milk collected from two groups of farms with different feeding practices – extensive and intensive farms – in Sardinia, Italy, between the years 2003 and 2004. In extensive farms, goats were principal‐ ly fed on grass and naturally growing bushes which were often present in marginal areas, supplemented with low levels of concentrates consisting of broad bean (*Vicia faba*) and gar‐ den pea (*Pisum sativum*). In intensive farms, goats were mainly fed silo maize, maize grains, and alfalfa (*Medicago sativa*). The incidence of AFM1 in goat milk samples from extensive and intensive farms was 11.2% (9/80) and 71.4% (20/28), respectively. Concentrations of AFM1 found in positive samples from both farms were 0.009 and 0.0177 ng/ml, respectively.

**Country Year**

*Raw milk*

*Pasteurized milk*

1 Concentrations of AFM1 in positive samples

2 Values in parentheses are ranges

**Samples analyzed** **Positive (%)**

2010 (2011)

Dec 2008 (2010)

2007 (2010)

Apr 2006 (0.008–0.054) (2009)

Lebanon Mar–July 3 0 (0) < 0.005 Assem et al.

Iran Nov 2007 – 60 19 (31.7) 0.0301 ± 0.0183 Rahimi et al.

Pakistan Jan–Dec 30 6 (20) 0.002 ± 0.005 Hussain et al.

Turkey Mar–Apr 110 93 (84.5) 0.019 Ozdemir (2007) 2006 (0.005–0.117)2 Syria Apr 2005 – 11 7 (63.6) 0.019 ± 0.0138 Ghanem and Orfi

Thailand Jan 2008 – 45 26 (57.8) 0.036 ± 0.015 Present study Feb 2011 (0.011–0.064)

Brazil Oct 2004 – 12 7 (58.3) 0.072 ± 0.048 Oliveira and Ferraz

Thailand Jan 2008 – 45 23 (51.1) 0.034 ± 0.014 Present study Feb 2011 (0.010–0.058)

**Table 5.** Incidence and concentrations of AFM1 in raw and pasteurized goat milk in various countries

cows and goats are similar to those in Egypt and Pakistan [25].

May 2005 (2007)

The observation that the incidence and concentrations of AFM1 in goat milk are relatively low‐ er than those in cow milk can be explained in terms of the feeding procedure and the carryover rate of AFB1 in feedstuffs to AFM1 in the milk. Cows are generally fed with several major AFB1-contaminated feedstuffs: corn, cotton seed, and concentrated feed. Unlike cows, goats are fed with fresh grass but not corn or cotton seed; the main AFB1-contaminated feedstuffs fed to goats are concentrate feedstuffs. Motawee et al. [24] explained the different feeding patterns of cows and goats in Egypt. Cows are generally kept in enclosed areas and fed with a large pro‐ portion of AFB1-contaminated feedstuffs, with a short period of time for grazing on pasture; while goats are allowed to graze on pasture in the morning and are brought back into the en‐ closed areas for concentrate feedstuffs in the evening. Hussain et al. [16] explained that goats in Pakistan are mainly fed by grazing on pasture. AFB1-contaminated feedstuffs – corn, cotton seed, and concentrate feed – are not used to feed goats. In Thailand, the feeding procedures for

**Concentration**

Occurrence of Aflatoxin M1 in Raw and Pasteurized Goat Milk in Thailand

**(µg/L)1 Reference**

http://dx.doi.org/10.5772/52723

215

**Figure 1.** HPLC chromatograms of AFM1 with a retention time of approximately 6.8 min: (A) standard 10 µg/L AFM1, (B) goat sample contaminated with 0.05 µg/L AFM1, and (C) uncontaminated goat milk sample


1 Concentrations of AFM1 in positive samples

2 Values in parentheses are ranges

The levels of AFM1 in goat milk are influenced by both feeding practices and the types of feedstuffs. Virdis et al. [23] determined the concentrations of AFM1 in goat milk collected from two groups of farms with different feeding practices – extensive and intensive farms – in Sardinia, Italy, between the years 2003 and 2004. In extensive farms, goats were principal‐ ly fed on grass and naturally growing bushes which were often present in marginal areas, supplemented with low levels of concentrates consisting of broad bean (*Vicia faba*) and gar‐ den pea (*Pisum sativum*). In intensive farms, goats were mainly fed silo maize, maize grains, and alfalfa (*Medicago sativa*). The incidence of AFM1 in goat milk samples from extensive and intensive farms was 11.2% (9/80) and 71.4% (20/28), respectively. Concentrations of AFM1

214 Aflatoxins - Recent Advances and Future Prospects

found in positive samples from both farms were 0.009 and 0.0177 ng/ml, respectively.

(A)

(B)

(C)

**Figure 1.** HPLC chromatograms of AFM1 with a retention time of approximately 6.8 min: (A) standard 10 µg/L AFM1,

(B) goat sample contaminated with 0.05 µg/L AFM1, and (C) uncontaminated goat milk sample

**Table 5.** Incidence and concentrations of AFM1 in raw and pasteurized goat milk in various countries

The observation that the incidence and concentrations of AFM1 in goat milk are relatively low‐ er than those in cow milk can be explained in terms of the feeding procedure and the carryover rate of AFB1 in feedstuffs to AFM1 in the milk. Cows are generally fed with several major AFB1-contaminated feedstuffs: corn, cotton seed, and concentrated feed. Unlike cows, goats are fed with fresh grass but not corn or cotton seed; the main AFB1-contaminated feedstuffs fed to goats are concentrate feedstuffs. Motawee et al. [24] explained the different feeding patterns of cows and goats in Egypt. Cows are generally kept in enclosed areas and fed with a large pro‐ portion of AFB1-contaminated feedstuffs, with a short period of time for grazing on pasture; while goats are allowed to graze on pasture in the morning and are brought back into the en‐ closed areas for concentrate feedstuffs in the evening. Hussain et al. [16] explained that goats in Pakistan are mainly fed by grazing on pasture. AFB1-contaminated feedstuffs – corn, cotton seed, and concentrate feed – are not used to feed goats. In Thailand, the feeding procedures for cows and goats are similar to those in Egypt and Pakistan [25].

The carry-over rate of AFB1 in feedstuffs to AFM1 in milk is relatively lower in goats than in cows. The carry-over rates in cows have been reported to vary from 0.3% to 6.2%, with a mean value of 1.81% (n = 42) [26]. In Thailand, Ruangwises and Mhosatanun [27] deter‐ mined the carry-over rates during the early lactation period (the first 4 weeks of lactation) in nine cows fed with feedstuffs naturally contaminated with AFB1. The carry-over rates ranged between 1.96% and 3.12%, with an average value of 2.02%. For goats, Smith et al. [28] reported an average carry-over rate of 0.55% in three goats which were fed with feedstuffs containing 100 ppb AFB1. Mazzette et al. [29] found an average carry-over rate of 0.26% in three goats within 72 h after receiving a single oral dose of 0.8 mg of AFB1.

**References**

[1] JECFA (Joint FAO/WHO Expert Committee on Food Additives). (2001). Safety Evalu‐ ation of Certain Mycotoxins in Food: Aflatoxin M1. WHO Food Additives Series. (47),

Occurrence of Aflatoxin M1 in Raw and Pasteurized Goat Milk in Thailand

http://dx.doi.org/10.5772/52723

217

[2] Jay, J. M., & Loessner, Golden. D. A. (2005). Modern Food Microbiology (7th edition).

[3] IARC (International Agency for Research on Cancer). (2002). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans,. 82, Some Traditional Herbal Medi‐ cines, Some Mycotoxins, Naphthalene and Styrene. Lyon, France: World Health Or‐

[4] FAO (Food and Agriculture Organization of the United Nations). (2004). Worldwide Regulations for Mycotoxins in Food and Feed in 2003. *Food and Nutrition Paper*, 81.

[5] European Commission. Commission Regulation (EC) No. (2006). Setting Maximum Levels for Certain Contaminants in Foodstuffs. *Official Journal of the European Union*,

[6] Ministry of Public Health. (2003). Notification of the Ministry of Public Health. B.E. 2545): Cow's Milk., *The Royal Thai Government Gazette* [265], , 120 Special Issue 4D

[7] Ministry of Agriculture and Cooperatives. (2008). Thai Agricultural Standard (TAS) 606-2008 Raw Goat Milk. The Royal Thai Government Gazette, 139(139D), August

[8] DLD (Department of Livestock Development). (2009). Livestock Infrastructure Infor‐ mation in Thailand:. Bangkok: Department of Livestock Development, Ministry of

[9] DLD. (2010). Livestock Infrastructure Information in Thailand:. Bangkok: Depart‐ ment of Livestock Development, Ministry of Agriculture and Cooperatives; 2010 (in

[10] DLD. (2011). Livestock Infrastructure Information in Thailand:. Bangkok: Depart‐ ment of Livestock Development, Ministry of Agriculture and Cooperatives; 2011 (in

[11] Park, Y. W. (2007). Rheological Characteristics of Goat and Sheep Milk. *Small Rumi‐*

[12] Jasinska, B. (1995). The Comparison of Pepsin and Trypsin Action on Goat, Cow, Mare and Human Caseins. *Roczniki Akademii Medycznej w Białymstoku*, 40, 486-493. [13] Ruangwises, N., Saipan, P., & Ruangwises, S. (2011). Estimated Daily Intake of Afla‐ toxin M1 in Thailand. In: Guevara-González RG (ed.). Aflatoxins- Biochemistry and

Geneva, Switzerland: World Health Organization.

New York: Springer Science and Business Media.

ganization (WHO), IARC Press.

Rome: FAO.

364-5, L 364/24.

2008) (in Thai).

Thai).

Thai).

*nant Research*, 68, 73-87.

(January 2003) (in Thai).

Agriculture and Cooperatives; 2009 (in Thai).

This study showed that 49 samples (54.4%) of the 90 goat milk samples collected within the central region of Thailand in January–February of the years 2009–2011 were contaminated with AFM1 equal to or more than the LOQ of 0.01 µg/L. Concentrations of AFM1 were not significantly different among the raw and pasteurized milk samples and across the two types of milk samples collected over three years. Of the 49 positive samples, 7 samples (14.3%) had AFM1 greater than the EU regulatory limit of 0.05 µg/L. All 90 goat milk sam‐ ples contained AFM1 below the U.S. regulatory limit of 0.5 µg/L. This study presents the first internationally published report on the contamination of AFM1 in raw and pasteurized goat milk produced in Thailand. The present study and our three previous reports on the occur‐ rence of AFM1 in cow milk products [13,18,19] suggest that regulatory standards be adopted for AFM1 to ensure the quality of raw milk and milk products in Thailand.
