Development and validation of a high-performance liquid chromatography method with post-column derivatization for the detection of aflatoxins in cereals and grains.

A novel, reliable and rapid high-performance liquid chromatography (HPLC) method with post-column derivatization was developed and validated. The HPLC method was used for the simultaneous determination of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in various cereals and grains. Samples were extracted with 80:20 (v/v) methanol:water and purified using C18 (40-63 µm) solid-phase extraction cartridges. AFs were separated using a LiChroCART-RP-18 (5 µm, 250 × 4.0 mm(2)) column. The mobile phase consisted of methanol:acetonitrile:buffer (17.5:17.5:65 v/v) (pH 7.4) delivered at the flow rate of 1.0 mL min(-1) The fluorescence of each AF was detected at λex = 365 nm and λem = 435 nm. All four AFs were properly resolved within the total run time of 20 min. The established method was extensively validated as a final verification of the method development by the evaluation of selectivity (AFB1, AFB2, AFG1 and AFG2), linearity (R(2) ≥ 0.9994), precision (average SD ≤ 2.79), accuracy (relative mean error ≤ -5.51), robustness (p < 0.0080), ruggedness (p < 0.0100) and average recoveries (89.2-97.8%). The limits of quantification of AFB1, AFB2, AFG1 and AFG2 were 0.080, 0.073, 0.062 and 0.066 ng g(-1), respectively. Finally, the developed method was applied for the analysis of AFs in 45 samples comprising rice (n = 20), wheat (n = 15) and maize (n = 10). The results showed that 65% of rice, 20% of wheat and 80% of maize samples were found contaminated with AFs. Thus, according to the achieved results, it is suggested that the newly developed HPLC method could be effectively applied for the routine analysis of the AFs in different cereals and grains.

Aflatoxins contamination in Pakistani brown rice: a comparison of TLC, HPLC, LC-MS/MS and ELISA techniques.

Advancement in the field of analytical food-chemistry has explored various experimental techniques for aflatoxins (AFs) quantification. The present study was aimed to compare four different techniques; thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) for the analysis of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in brown rice (n = 120) being collected from Karachi, Pakistan. All the four assays provide precised, accurate and comparable results. However, some differences were observed. For instance, TLC, HPLC and LC-MS/MS methodologies offered the advantage of the quantification of individual toxins in contrast to ELISA technique. The contamination ranges of AFB1/AFB2 as determined by TLC, HPLC and LC-MS/MS were 1.18-9.97/0.59-1.52, 0.16-10.54/0.26-1.35 and 0.11-10.88/0.38-1.48 µg/kg, respectively. However, AFG1 and AFG2 were not detected in any tested samples. Furthermore, owing to low-detection limit and sensitivity, HPLC and LC-MS/MS methodologies have identified greater number of contaminated samples in comparison to TLC and ELISA techniques. The overall average results of total AFs as provided by HPLC (3.79 µg/kg) and LC-MS/MS (3.89 µg/kg) were found higher in comparison to TLC (3.68 µg/kg) and ELISA (3.70 µg/kg). On the basis of achieved results, it was concluded that TLC, HPLC, LC-MS/MS and ELISA techniques are valuable tool for the quantification of AFs in cereals and grains. Furthermore, HPLC and LC-MS/MS techniques offer an added advantage for the detection of AFs in diminutive levels.

Aflatoxin B1 in betel nuts (Areca catechu L.) imported to Pakistan from different regions of South Asia.

Aflatoxin B1 (AFB1) levels were evaluated in betel nuts (Areca catechu L.) being imported to Pakistan during 2010-2011. In total, 278 betel nut samples (India = 21, Indonesia = 51, Sri-Lanka = 34 and Thailand = 172) were received from the Department of Customs and were analysed by thin layer chromatography (TLC). All Indian origin betel nuts showed AFB1 contamination ranging from 11.7-262.0 µg kg(-1) with a mean of 92.5 µg kg(-1). Among Indonesian and Sri Lankan shipments, 80.4% and 73.5% betel nuts were contaminated with AFB1 ranging between 3.3-39.2 and 6.5-103.4 µg kg(-1) with a mean of 11.6 and 35.0 µg kg(-1), respectively. However, only 30.2% of Thailand origin samples showed AFB1 contamination ranging 3.3-77.0 µg kg(-1) with a mean of 6.6 µg kg(-1). The widespread occurrence of AFB1 increases the hazard associated with betel nuts. Thus, strict control is a pre-requisite for the production and import/export of psychoactive substances as betel nuts.

Aflatoxins contamination and prevention in red chillies (Capsicum annuum L.) in Pakistan.

During 2006-2011, 331 red chilli samples (226 whole, 69 powdered and 36 crushed) were collected from all over Pakistan for the estimation of total aflatoxins (AFs = AFB1 + AFB2 + AFG1 + AFG2) contamination by thin layer chromatography (TLC). Mean AFs levels in whole, powdered and crushed chillies were 11.7, 27.8 and 31.2 µg kg(-1), respectively. AFs levels in 62.4% of whole, 26.1% of powdered and 19.4% of crushed chillies were found lower than the maximum limit (ML = 10 µg kg(-1)) as assigned by the European Union. Furthermore, whole (27.9%), powdered (28%) and crushed (27.8%) chillies showed AFs contamination which ranged between 10 and 20 µg kg(-1). However, 9.7% of whole, 46% of powdered and 52.8% of crushed chillies showed AFs levels beyond the ML of 20 µg kg(-1) as assigned by the USDA. It was concluded that AFs contamination in chillies requires further investigation, monitoring and routine analysis. Furthermore, proper harvesting, drying, handling, storage and transport conditions need to be employed.

Occurrence of aflatoxins contamination in brown rice from pakistan.

BACKGROUND: The objective of this study was to determine the distribution of an economically-important class of mycotoxins, the aflatoxins (AFs) in Pakistani Brown Rice. METHODS: A total of 262 of brown rice samples were collected from different vendors during July 2006 to June 2011. Samples were analyzed for the occurrence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) by thin layer chromatography (TLC) technique. RESULTS: AFB1 was detected in 250 (95.4%) samples, whereas AFB2 was detected in 20 (7.6%) samples. Furthermore, AFG1 and AFG2 were not found in any sample. The contamination range of AFB1 and AFB2 was found 1.07-24.65 µg/kg and 0.52-2.62 µg/kg, respectively. Total AFs were quantified in 250 (95.4%) samples with an average of 3.89 µg/kg and contamination range was noted to be between 1.07-27.27 µg/kg. The overall results indicated that in 12 (4.6%) samples, AFs were not found within detectable limits. Furthermore, in 188 (71.7%) samples, AFs level was found below than maximum tolerated levels (MTL) as recommended by the European Union (4 µg/kg). Moreover, in 61 (23.3%) samples, AFs range was found between 4-20 µg/kg, which were fit for human consumption as per MTL (20 µg/kg) assigned by USA (FDA and FAO) and Pakistan (PSQCA). While only one sample (27.27 µg/kg) exceeded the above mention regulation limits. CONCLUSION: Low level of AFs occurs frequently in brown rice, and can be improved using proper harvesting practices, storage and transportation conditions. The small quantities of AFs warrant performing further investigation, monitoring and routine analysis on regular basis.