**2.3. Extraction and determination of aflatoxin M1**

The extraction procedure was performed using the manufacturer's recommendations, as previously described by Ruangwises et al. [13]. Briefly, 50 ml of raw milk or pasteurized milk sample was pipetted into a 50-ml plastic centrifuge tube. Milk samples were defatted by centrifugation at 3,500 *g* for 20 min at 4o C. Fat was separated; the resulting skimmed milk was then transferred into a 50-ml plastic syringe with a Luer tip which was attached to an immunoaffinity column. The skimmed milk was allowed to flow into the column by gravity at a flow rate of approximately 1 ml/min. After the skimmed milk had run through, 20 ml of HPLC water was used to wash the column. AFM1 was eluted from the column with 1.25 ml of acetonitrile:methanol (3:2) and 1.25 ml of HPLC water. The eluate (a total volume of 2.5 ml) was filtered through a nylon syringe filter for HPLC with pore size 0.45 µm (Whatman, UK). AFM1 in the final solution was determined using HPLC. Each milk sample was extract‐ ed and analyzed for AFM1 in duplicate.

#### **2.4. Instrument**

The law that regulates the quality of milk products in Thailand is the Notification of the Ministry of Public Health No. 265, which regulates only cow milk products. However, the law does not specify the regulatory standards for AFM1 but states that "…*milk products may be contaminated with aflatoxins at a level that is not harmful to human health*" [6]. The only guide‐ line that regulates the quality of raw goat milk is the Thai Agricultural Standard TAS 6006-2008 of the National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives [7]. Like Notification No. 265 for cow milk products, the TAS 6006-2008 guideline does not specify the recommended limit for AFM1 in goat milk.

In Thailand, the number of dairy goats is approximately 5% that of dairy cows [8–10]. Goat milk is consumed by only a small percentage of the country's population, particularly Thai people who have an allergy to cow milk. Goat milk has been shown to form finer and softer curds than cow milk following acidification under conditions similar to those in the stom‐ ach, thus making it more readily digested [11]. It has been reported that micellar caseins of human and goat milk were 96% hydrolyzed by pepsin and trypsin in *in vitro* studies, while the hydrolytic rate of cow milk was 76–90% [12]. With the knowledge that goat milk is more easily digested, some Thai adults prefer goat milk products. As a result, the number of dairy goats in Thailand has been gradually increasing in recent years. In 2009, the number of dairy goats in Thailand was 20,830; the numbers increased to 22,630 and 33,363 in 2010 and 2011,

Thailand is administratively divided into four regions: central, north, northeast and south. The central region was selected for this study, since this region has the highest number of dairy goats and the highest rate of goat milk production, accounting for approximately 60% of the national total [8–10]. There are no internationally published reports regarding the

The purpose of this study was to investigate whether the concentrations of AFM1 in raw and pasteurized goat milk produced in Thailand are within the acceptable level for consumption.

AFM1 reference standard (from *Aspergillus flavus*) was purchased from Sigma-Aldrich (St.

USA). Solvents (HPLC grade) – acetonitrile, methanol, and water – were purchased from

Raw goat milk samples were collected from private farms, while pasteurized goat milk sam‐ ples were purchased from supermarkets in the central region of Thailand. In Thailand, com‐

for at least 15 s [6]. All milk samples were collected over three years: January–February of

TM immunoaffinity columns were obtained from Vicam (Nixa MO,

C for 30 min or at 72 o

C

quality and levels of AFM1 in goat milk produced in Thailand.

respectively [8–10].

208 Aflatoxins - Recent Advances and Future Prospects

**2. Materials and methods**

Louis MO, USA). AflaM1

Merck (Darmstadt, Germany).

**2.2. Milk sample collection and sample preparation**

mercial pasteurized milk is produced by heat treatment, either at 63 o

**2.1. Chemicals**

A complete liquid chromatographic system (ProStar; Varian, Palo Alto CA, USA) consisted of a HPLC pump (model 240), an auto injector (model 410), a column oven (model 510), and a fluorescence detector (model 363). The HPLC conditions for analysis of AFM1 were as fol‐ lows: column, Spherisorb ODS-2 (Waters, Milford MA, USA); column temperature, 40 °C; mobile phase, water:methanol:acetonitrile (57:23:20); flow rate, 1 ml/min; and detector, fluo‐ rescence spectrophotometer (excitation 360 nm; emission 440 nm).

#### **2.5. Determination of limit of quantification**

The Q2B procedure of the U.S. Food and Drug Administration [14] was used for determina‐ tion of the limit of quantification (LOQ) for AFM1. Milk samples (50 ml) were fortified with standard AFM1 at four concentrations of 0.025, 0.050, 0.125 and 0.250 µg/L, while blank sam‐ ples were not fortified with standard AFM1. Concentrations of AFM1 in AFM1-fortified milk samples and blank samples were quantified as described above in Section 2.3 using AflaM1 TM immunoaffinity columns. All samples were analyzed for AFM1 in duplicate.

Individual linear regression lines were obtained from least-square regression analyses of the residual peak areas versus the four concentrations of fortified AFM1 (0.025, 0.050, 0.125 and 0.250 µg/ml). The residual peak areas were peak areas of AFM1-fortified samples minus the peak area of blank sample. A total of 12 regression lines (six regression lines each for intra‐ day and interday analyses) were obtained by least-square linear regression. The LOQ of the method was calculated using the equation LOQ = 10 σ/S, where σ is the standard deviation of *y*-intercepts and S is the average slope of the 12 linear regression analyses [14].

#### **2.6. Statistical analysis**

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‐ tics version 17.0 for Windows was used for statistical analysis.

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

**Residual peak area2**

**(mV)**

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

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

211

of both species were 0.0301 and 0.0601 µg/L, respectively.

**Peak area1 (mV)**

0 6,410 -

0.025 11,126.5 4,716.5

0.050 16,144.5 9,734.5

0.125 29,251 22,841

0.250 52,773 46,363

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

slope = 184,141; *y*-intercept = 197.86

1 Average value of two determinations

**AFM1 added (µg/L)**
