X-Ray Fluorescence in Biological Sciences. Группа авторов

of As concentration in an exposed person. A stainless‐steel scissor was used to collect the hair sample obtained from different parts of the scalp at an interval of 1 cm. Collected hair samples, preserved in zipper polyethylene bags, were cut into small pieces, washed with a sufficient volume of acetone for a couple of minutes, and then thoroughly washed again several times with deionized water, and finally washed once more with acetone [5]. The sample was preliminarily air dried at room temperature for about three hours, and then again in an oven at 180 °C until the sample was completely dry.

      6.2.4 Sample Preparation

Initially the hair sample was prepared by grinding in a ball mill until powderized (Figure 6.1a), and then crushed to a fine homogeneous mass by a carbide mortar and pestle (Figure 6.1b). A pellet of 2.5 cm diameter was prepared from the powdered mass using a hydraulic press pellet maker (Specac) (Figure 6.1c), applying 10 tons pressure, for subsequent analysis by EDXRF. The pellets from the standards were also prepared similarly for construction of calibration curves. All pellets (Figure 6.1d) were preserved in desiccators until irradiation.

      6.2.5 Sample Analysis

The Epsilon 5 EDXRF spectrometer (Figure 6.2) equipped with a Gadolinium (Gd) tube at 100 kV is designed for fast, high precision, qualitative and quantitative elemental analysis. This was the machine used to determine Arsenic concentrations in hair samples [6]. It is microprocessor‐controlled and operated from an external PC connected to the spectrometer. The sample changer of the system can accommodate up to 133 samples. It has a programmable system that automates the loading of the sample into the measuring chamber. It is outfitted with stainless steel trays to hold samples in the cabinet and sample cups to hold samples in the measuring chamber during irradiation.

Figure 6.1 (a)–(d) Different steps of making sample pellet for EDXRF analysis. (a) Initially hair sample was grinding with ball mill. (b) Finally grinding with mortar and pestle to get fine powder for making pellets. (c) Making of pellets with Hydraulic press pellet maker. (d) Pellets is ready for irradiation.

Figure 6.2 Irradiation of sample pellets using compact energy dispersive X‐ray fluorescence (EDXRF) system.

      6.2.6 Accuracy and Precision of the Method

      6.2.6.1 Construction of Calibration Curve

In any analytical technique, validation of the method is the most important quality control issue. Accurate and precise results depend on the careful construction of the method or analytical technique which produces them. A calibration curve was constructed using three lab‐synthesized secondary standards of arsenic in varying concentrations (5.8, 9.5, and 14.8 ppm) prepared from commercially available 1000 ppm As standard (Mark, Germany). The secondary standards were prepared by doping 2 g of cellulose with different amount of As [7]. The accurate concentrations of the element in the standards was obtained by the source excited by EDXRF. Figure 6.3 shows the calibration plot of As reflecting a good regression value. A certified reference material (CRM) Orchard Leaf (NIST 1571) obtained from the US‐based National Institute of Standards & Technology, was prepared and analyzed under same condition as the real samples and used to check the accuracy and precision of the analytical method [8]. The calculated values of relative error (%) and variation (%) as shown in Table 6.1. was found in good agreement with the certified values and within an error margin of 10%, thus confirming the method is quite fit for purpose.

      6.2.6.2 Measured Condition

       Measuring time: 100 seconds

       Unit: ppm

       Arsenic (As) condition: Rb_Re‐Tl

       Secondary target: ZrFigure 6.3 Calibration curve for measurement of arsenic (As).Table 6.1 Analytical results of Orchard Leaf (NIST1571) obtained from Compact EDXRF system as the purpose to fulfill the requirement of method validation.ElementsElemental concentration (mg/kg)MeanSDCertified valueRE (%)CV (%)Exp‐1Exp‐2K14 90013 98714 44464614 7001.744.47Ca19 98119 62819 80525020 9005.241.26Mn90.8992.7291.811.2991−0.881.41Fe302311306.506.36300−2.172.08Cu12.2213.0112.620.5612−5.134.43Zn25.1925.5725.380.2725−1.521.06As9.489.659.570.12104.351.26Rb11.3711.4111.390.03125.080.25Sr39.2139.8839.550.4737−6.881.20Pb44.7145.0344.870.23450.290.50

6.3 Determination of Lead Concentrations in Human Whole Blood Using EDXRF Technique with Special Emphasis on Evaluating Association of Blood Lead Levels with Autism Spectrum Disorders (ASD)

      6.3.1 Background

Autism spectrum disorders is an emerging and increasingly relevant point of social concern. ASD is a range of potentially devastating childhood conditions associated with environmental, genetic, and epigenetic factors. Some of these effects appear to be transgenerational. ASD is a kind of neurodevelopment disorder that can produce features such as impaired psychosocial and adaptive skills functioning, as well as lack of social interaction, poor communication skills, and compulsive patterns of activity [9] are also reported regarding ASD. It is thought that autism is initiated by disruption of normal neurobiological mechanisms during the prenatal period, accompanied by strong genetic components, but nongenetic factors are likely involved as well with increased prevalence.

      A combination of genetic susceptibility and exposure to environmental toxins at critical periods during brain development has recently been hypothesized as the origin of autism [10] and several studies have observed elevated levels of heavy metals and essential minerals among children diagnosed with ASD [9]. Johnson and Myers [11] suggested that environmental exposure to heavy metals and essential minerals may act as a central nervous system teratogen in early gestational life. Among the toxic heavy metals, Pb is of particular concern as an undesirable contaminant originating from a variety of common sources such as motor vehicles, lead paint, contaminated soil etc. Pb can be ingested through air, drinking water, food etc. Developing fetuses and children are more sensitive to Pb exposure compared to adults because of the relative immaturity of the blood–brain barrier, increased gastrointestinal absorption, and the prevalence of hand‐to‐mouth behaviors. Hence the possibility of Pb exposure leading to the development of ASD in children is of greater concern to children than adults.

      Many studies from across the world support the association between exposure to Pb and ASD. But different, conflicting [12] opinions are also available and must be taken into consideration. Adams et al. [13] conducted a study where they compared 55 children with autism ages 5–16 years to 44 control subjects of similar age and gender and reported that the autism group had significantly higher levels of Pb in their red blood cells and higher urinary levels of lead,