Material and methodology

Sample Collection

Peanuts sample were obtained from the local markets (Akbari Mandi) and retailers store in Lahore.
Standard samples are obtained from the Peshawar. The samples were brought to UVAS (Institute of
Microbiology) and were stored at 4 degrees until analysis

Chemicals

The chemicals for this experiment were sourced from reputable suppliers. Acetonitrile and methanol
solutions were obtained from Scientific Traders in Lahore, Pakistan. Working solutions were prepared
by diluting the mixed solutions with a 60:20:20 (v/v) mixture of methanol, water, and acetonitrile.
HPLC-grade methanol was procured from Lab Sonic, while deionized water was used throughout the
experiment. Analytical-grade NaCl was also purchased from Sonic Lab, ensuring quality and accuracy
in the analysis. Mereck C18 Columns and glass microfiber filters with a particle size of 1.5 µm were
provided by Scientific Traders, further enhancing the experiment's reliability and precision.

Standard Sample preparation

The standards of Aflatoxins were used which were prepared concentration of 1 µg/ml by diluting in
methanol/acetonitrile (98:2; v/v), wrapped with aluminium foil, and stored at 4 °C until the analysis.

Solvents:

In the comprehensive process of aflatoxin analysis, different solvents are used. An appropriate
organic solvent, such as acetonitrile or methanol, serves as the medium for extracting aflatoxins from
the peanut sample, effectively releasing them from the matrix for subsequent analysis. Aflatoxin
standards, consisting of pure compounds with known concentrations, are essential for calibration in
analytical methods, enabling the accurate quantification of aflatoxin levels in the sample.

Depending on the analytical method employed, buffer solutions are used to adjust PH. In certain
cases, reagents like for trifluoroacetic acid are needed for derivatization. Sodium sulfate is utilized to
eliminate residual moisture from the sample, as dry conditions are crucial for accurate aflatoxin
analysis. The choice of a solvent or mobile phase in HPLC analysis plays a vital role in separating and
detecting aflatoxins, with common solvents including water, acetonitrile, or methanol in various
proportions.

HPLC Conditions

In this chromatography setup, the mobile phase is composed of a mixture of water, methanol, and
acetonitrile in a specific ratio of 60% water, 20% methanol, and 20% acetonitrile, all measured by
volume. The flow rate is precisely controlled at 1 milliliter per minute, ensuring a consistent flow of
the mobile phase through the system. The mobile phase in this chromatography setup is made up of
ratio of water, methanol, and acetonitrile, measured by volume: 60% water, 20% methanol, and 20%
acetonitrile.

A Merck C18 chromatographic column, measuring 250 millimeters in length and 4.6 millimeters in
internal diameter, is the one used in this procedure. The column is packed with particles that are 5
micrometers in size, which is suitable for separating smaller molecules. The temperature of the
column is kept at 40 degrees Celsius to maintain the accuracy of the separation process.

Detection wavelength
Fluorescent detectors are used for compound detection, with excitation and emission wavelengths
set at 360 nm and 440 nm, respectively, enhances sensitivity and specificity in the detection of
aflatoxins.

Apparatus

A variety of glassware is needed for the analysis of aflatoxin in peanuts. Beakers and volumetric
flasks help in reagent handling and accurate solution preparation. Glass bottles are vital for storing
standards and sample extracts, preserving their integrity. Glass pipettes ensure controlled liquid
transfer for accurate dilutions. Glass vials are used to contain sample extracts for injection into
analytical instruments like HPLC. Glass syringes facilitate the precise injection of sample solutions.

Procedure

Sample preparation

In order to properly prepare peanuts for aflatoxin analysis, it is necessary to collect the peanuts and
subsequently grind them into a fine powder using a mortar and pestle, or alternatively, a grinder. The
ultimate objective of this process is to ensure that a finely ground sample is obtained. After this step,
a precisely weighed portion of the peanut sample, typically weighing approximately 25 grams, is
accurately measured using an analytical balance. Once the sample has been accurately weighed, it is
then transferred to a clean container that is suitably labeled to ensure the preservation of sample
integrity.

Extraction

In this procedure,25 grams of the sample are ground and weighed. Subsequently, a solvent mixture
of 80 ml acetonitrile and 20 ml water, which includes 5 grams of NaCl, is added to the sample. The
choice of solvent depends on the specific nature of the sample and can be selected. The sample is
then vigorously shaken for a duration of 1 hour to facilitate the extraction and dissolution of target
compounds. This step ensures that the sample is effectively prepared for further analysis, depending
on the intended analytical method and the specific characteristics of the sample under investigation.

Centrifugation and Filtration

To prepare the sample for aflatoxin analysis, it undergoes a centrifugation process at a high speed,
usually between 3000-4000 rpm, for a brief period. This process efficiently separates the solid phase
from the liquid phase, ensuring that any particles or debris are removed. The liquid portion or
supernatant is then carefully transferred to a clean container using either a syringe or a pipette. To
ensure that the sample is completely clean and free from impurities, an additional filtration step is
performed using a syringe filter or similar apparatus. A filter with a specific pore size, such as 0.45
µm or 0.22 µm, is used to ensure thorough purification and to obtain a clean and clarified sample
ready for subsequent analysis. These centrifugation and filtration procedures are crucial in
maintaining the quality and integrity of the sample, enabling accurate aflatoxin analysis.

Then, 9 ml of the sample is transferred to a Mycosep glass tube, and then 70 µl of acetic acid is
added. The mixture is vortexed for 30 seconds before being pushed through a Mycosep column.
From the eluate, 2 ml is removed and evaporated to dryness. To redissolve the aflatoxins, 200 µl of
hexane is added, and the sample is vortexed for 30 seconds.

Then, 50 µl of trifluoroacetic acid (TFA) is added, and the mixture is vortexed again for 30 seconds
before being left in the dark for 5 minutes. Next, 1.95 ml of acetonitrile and water in a 1:9 (v/v) ratio
is added to the mixture, and the layers are separated by vortexing. The lower layer is isolated and
filtered, and finally, 20 µl of the resulting solution is injected into the HPLC for aflatoxin analysis.
These precise steps ensure that the sample is thoroughly prepared for accurate and reliable results.

In this study, we found AFB1, AFB2, AFD1, and AFD2 Aflatoxins in Marketed in Pakistan in the peanut
samples of Lahore region with rates of 70, 51.7, 3.3, and 2.5%, respectively. Although the majority of
samples of peanut in Lahore (Pakistan) were safe for consumption, monitoring of AFs must be carried
out on a regular basis in the case of peanut oil consumed in this region. Continuous surveillance
measures must be done for reduction of AFs risks in the local consumers. This study suggested that
farmers, food processors, and local processors should be aware of acceptable hygiene practices for
the cultivation, protection, transportation, processing, and handling of peanut.