To develop the analytical method for the determination of Lead (Pb) content in hair using Graphite Furnace Atomic Absorption Spectrometry with Zeeman Correction.
To investigate the most suitable hair washing procedure that can effectively remove the exogenous contaminants without the loss of endogenous deposited Lead (Pb) in hair.
To compare the lead (Pb) concentration found in scalp hair and axillary hair and investigate the correlation of lead (Pb) concentration between them.
To study the relationship between the concentration of Lead (Pb) in scalp hair and sashimi daily intake of citizen in Hong Kong
2.1 Toxicological of lead to human
Lead is toxic heavy metals that can be found in irrespirable suspended particles. Lead particles which is present in the car exhausts with the size less than 10Î¼m will affect both adult and children. The consumer products improvement act (CPSIA) sets the permissible lead concentration limit in paint and similar surface coating, children metal and non-metal products in 2009. The concentration of lead in paint and similar surface coating should not be greater than 90 mg/kg while the lead concentration should not be greater than 300 mg/kg for children metal and non-metal products (1). This indicates that the lead exposure is a global concern in the world. Lead exposure will cause the damage of the nervous system, kidney, red blood cells and cause the increase of the blood pressure. Long term exposure of lead will cause the decrease of mental abilities and co-ordination. The effects of lead exposure can be treated by therapy. Otherwise, the brain will be damaged permanently. (2)
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2.2 Intake of toxic elements from sashimi
Sashimi is one of the popular foods in Hong Kong. They can be found in the conventional Japanese-style restaurants and self-service Sushi Restaurants. Different types of sushi and sashimi are displayed on the conveying belts and the customers can choose the sushi that they want. The pre-packed sashimi and sushi can be found in the supermarkets, or shops on the main streets and shopping centre. The plant sewage discharge or oil spill will cause serious water pollution. Heavy metals such as lead and mercury will be accumulated in its body. When larger fishes predate smaller fishes, the accumulated toxins in the small fish will be transferred to the large fish. As a result, the heavy metals will be accumulated up the food chain and the large fish which accumulate higher heavy metals content in its body than small fish. The Food Safety Report for July 2009 released from the Centre for Food Safety (CFS) found that a swordfish sashimi sample, a tuna sashimi sample and a fish fillet sample contain the mercury with the concentration from 0.68ppm to 1.3ppm, exceeding the legal limit of 0.5ppm (3). When human ingest the sashimi, the heavy metals in sashimi will be accumulated in the organ, bone and even scalp hair or axillary hair. As a result, the lead exposure of human can be examined by analyzing the lead concentration in scalp hair or axillary hair.
2.3 Structure and growth of scalp hair
Hair is located in the follicle below the skin surface in the dermis. The follicle has the glandular and connective tissue components. The dermal papilla which contains nerves, blood vessels and pigment forming cell is the generative zone of hair. The follicle cells generate the hair shaft which is composed of dead cells and form outermost layers of the epithelium. Hair is composed of hard keratin and the hair structure is shown as follows:
In the keratizination, the keratinized cell will be split and exposed the surface that is not exposed previously. The keratinized cells bound tightly to their neighbors in a complex array. The crannies between cells will be formed during the keratinization process. Scalp hair which contains a high proportion of the sulphydryl groups (-SH) containing amino acid. As a result, external contaminants deposit on the hair surface may become incorporated into the hair structure by the interaction with -SH groups in the hair (4, 5, 6). These allow the exogenous contaminants easy to be trapped in the outer surface of the hair. As a result, washing of hair is essential to remove the external contaminants. Scalp hair grows at an average rate of 1 centimeter per month (7), but the range of hair growth is from 0.6 to 3.36 cm/month (8). To investigate the human lead exposure within 2 - 3 months, 2 - 3 centimeters from the proximal of scalp hair should be taken for the analysis. Usually, most of the hair is in the anagen phase, which is the growing phase of hair and last for 2 to 6 years. The phase is longer for longer hair. In apoptosis, the hair will enter the catagen phase, the follicle will start to regress and move toward the surface. The next phase is telogen, the hair will fall out and enter the resting phase. The cycle will be completed and the anagen phase will be restarted again (9).
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Axillary or pubic hair which grow slower than scalp hair and with much proportion in the resting phase. In addition, axillary or pubic hair is not normally exposed to the environment and as a result the chances of exogenous lead contamination by air should be reduced.
2.4 Correlation study of lead in scalp and axillary hair
In the research, the correlation of lead concentration between the scalp hair and axillary hair are investigated by determining the lead concentration in scalp and axillary hair from donors. At least 10 data should be obtained from different donor to get the correlation relationship. The concentration of lead in scalp hair versus concentration of lead in axillary hair is plotted and the correlation between scalp and axillary hair is determined by the slope of the curve and regression. The one-way ANVOA can be used to investigate whether the lead content in scalp and axillary hair has significantly different or not.
2.5 Advantages and limitations of hair analysis and correlation of lead in hair and blood
Hair analysis is an area of increasing interest in the fields of medical and biological science. Hair provides one of the most accurate records of the health and toxic heavy metals content in human body. Hair is an excellent indicator of past changes in metabolism and environmental exposure to heavy metals (6, 10). Compared with blood or urine analysis, hair has its own advantages oven them. The analysis of urine and blood can show the current status of body. However, hair shows a longer time frame. Heavy metals found in hair is higher levels than blood and urine so that to allow more sensitive and precise measurement. Hair is easier and safer to collect and store than blood or urine. This makes hair an excellent choice as a screening tool. If you do test for the toxicity of lead in the patient hair through the hair testing of heavy metals, then the patient should undergo the natural treatment of chelation therapy if they are suspected to have the possible lead toxicity. This is a safe and preferable procedure because it allows you to take an oral tablet that will bind the toxic heavy metals within the soft tissue of your body to be excreted through your urine. However, the applicability of hair analysis on an individual basis is strongly limited because it is difficult to distinguish exogenous and endogenous sources. Also, the information concerning the kinetics of uptake and binding of heavy metals during hair growth from the blood and the effect of hair washing, shampoo, perspiration on the concentration of heavy metals in hair are insufficient. Furthermore, the correction between heavy metals in hair and heavy metals in blood, urine or target organs is unknown. P.S.I. Barry published a paper titled "A comparison of concentrations of lead in human" in British Journal of Industrial Medicine at 1975 (11). From his study of concentrations of lead in tissues, the concentration of lead in hair is at least 30 times greater than lead concentration found in the blood. The results are tabulated as follows:
Unknown occupational exposure to lead
Children aged 16 years and under
Male adults aged 29 - 82 years with occupational exposure to lead
* British Journal of Industrial Medicine at 1975, 32, 119 - 139
In according to the Centre for Disease Control (CDC) , the blood lead levels of 10Î¼g /dL
(100Î¼g /L) or more suggests possible lead toxicity for children. In according to Occupational Safety and Heath Administration (OSHA), the blood lead levels of 50Î¼g /dL (500Î¼g /L) or more suggests possible lead toxicity. Due to the concentration of lead in hair is at least 30 times greater than that in blood. As a result, the concentration of hair should not be greater than 3 mg/kg for children and 15 mg/kg for adult. Otherwise, the lead blood test should be performed to confirm if lead exposure is over the tolerance limit. Attempts should be made at specifying hair lead levels to be considered high and dangerous. EI-Dakhakhny AA and EI-Sadik YM in "Lead in hair among exposed workers" stated that the lead concentration of hair greater than 30 mg/kg is indicating an excessive level and 110 mg/kg indicating as dangerous level (5, 6, 12).
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The US Agency for Toxic Substances and Disease Registry states that the effect of the concentration of heavy metals in hair to the human health is difficult to predict because of the insufficient correlation data. The US Centres for Disease Control and Prevention have compared hair and blood samples from 189 children to assess the accuracy of hair analysis in screening for lead poisoning. The method had 57% sensitivity and an 18% false-negative rate. The investigators concluded that measurement of lead content in hair is not an adequate method of screening for childhood lead poisoning. To obtain a reliable measure of individual lead exposure, the investigators concluded that it is necessary to assess the whole blood lead level (13). From an analytical point of view, hair analysis is not a robust diagnostic tool for heavy metal poisoning. The specimen is prone to exogenous contamination because hair is a perfect binding medium for dust. Personal habits such as the use of different kinds of shampoo or hair dyes contribute to the variability.
2.6 Sample collection of scalp hair
Concentration of lead in hair has been shown to increase from the proximal to distal end of hair (14, 15). As a result, homogenization of hair samples is very important, especially if long lengths of hair are collected. The scalp hair samples should be collected 2 - 3 cm closest to the scalp using a pair of stainless steel scissors washed with ethanol (16). To collect the axillary hair, the shaver washed with ethanol is used. The samples should be kept in sealed plastic bags prior to analysis. To ensure the hair sample is well homogenized, the sample should be cut into the dimension less than 2 mm and manually homogenized, the homogeneity is studied by performing duplicate analysis and relative standard deviation shows the homogeneity of sample (17). The growth rates of hair are different in different position of the scalp, the location that the sample is taken should be considered carefully to ensure the consistent measurement. The growth rate of hair at vertex (top), occipital (back) and temporal (side) regions is different. Centre for Disease Control (CDC) defined its standard protocol for the hair collection, 0.5 - 1 grams of occipital hair was collected (9). Clean hair that has not been dyed, permed, bleached, or straightened for three months and that only the newest hair growth should be sampled (18). This procedure is designed to prevent major sources of contamination, which include the environment (water, grease, dust, dirt, air) and hair treatments (dyes, perms and other chemical treatments). This protocol should be followed in the research to ensure the consistency in measurement. The hair sample should be collected in the zip bag. The information of sashimi daily intake should be collected from the donors of hair samples to investigate the relationship between the concentration of lead in hair and the daily intake of sashimi. The daily intake of sashimi is calculated by the following equation:
Daily intake of sashimi ( pieces /day) =
No. of pieces of sashimi per meal X Frequency of eating per week
The donor should fill in the questionnaire while the hair samples are collected. The questionnaire is shown as follows:
Sample No.: ________________ Date : __________________
Name : __________________ Sex : __________________
Age :___________________ Weight :_________________(kg)
Frequency of eating sashimi per week?
â-¡less than 1 â-¡1 - 2 â-¡more than 2
How many pieces of sashimi in each meal?
â-¡1 - 3 â-¡4 - 6 â-¡7 - 9 â-¡more than 9
What kind of seafood you prefer most?
â-¡ Tuna â-¡Salmon â-¡Clams, scallops and abalone
â-¡ Yellowtail â-¡Squid â-¡Others (Please specify: ___________________)
2.7 Investigation of the different washing protocol of scalp hair
After the hair is collected from the donor, the washing procedure should be employed to remove exogenous contaminants. The ideal washing procedure is to remove only external contaminants and leave endogenous lead intact. However, no washing method can distinguish between external and internal contaminations of lead (9). Studies by the International Atomic Energy Agency (IAEA) examined different washing procedures and found that even in cases where endogenous elements were removed it was not to the extent that would render the sample unusable. They concluded that while there are many different washing procedure, including incomplete removal of exogenous contamination or partial removal of endogenous elements, representative measurements could be made if standardized washing procedures were employed (19,20). As a result, lead content in hair provide the information of the potential lead exposure of the patients, but that does not tell us how many portion of lead is internally incorporated and how many portion of lead is externally incorporated. Smith (1964) showed that the concentrations of arsenic found in hair will vary depending on what washing methods are applied. No method shown to be able to remove all arsenic. The arsenic concentration in hair before the contamination is 0.14ppm. The hair is external contaminated with 12.08 ppm arsenic and the effect of different washing procedures are shown as follows :(21)
Washing time (mins)
5.03 - 6.21
4.20 - 4.93
4.92 - 6.26
0.4 - 0.7
* Smith H. 1964
If hair is not washed aggressively, exogenous lead will still remain. If hair is washed too aggressively, endogenous lead may be removed. The extent to which the hair should be washed depends on what kind of analytes is being studied. Washing is not necessary if the analyte which is being tested cannot be found in external source (e.g. methyl mercury). For the determination of lead content in hair, washing procedure is necessary because exogenous lead in the dust, dirt and air that will deposit on scalp hair. The major source of lead in air pollutant is from the exhaust of vehicles even if the Hong Kong government banned the lead petrol since April 1999. Different washing procedure can cause different analytical results so that to select the most appropriate washing protocol is the very important in the sample preparation.
In the research, different washing procedures are examined and the most suitable washing procedure is selected for the determination of lead content in hair. Different washing procedures including IAEA developed standard washing procedure (22.23). The hair is first cut into approximately 0.3 cm and mixed to homogeneous. Afterwards, the hair sample is washed with 1: 200 v/v of Triton X-100 by four times. The sample is rinsed with acetone and allowed to drain. This is followed by three rinses with deionized water and two rinses with acetone. The sample is dried in an oven at 75 Â± 5 oC. The IAEA washing procedure is compared with other washing procedures such as washing with deionized water, 5% hydrochloric acid or 5% sodium hydroxide for 30 minutes. To investigate the effectiveness of different washing procedure, the lead content of unwashed hair should be determined to get the background lead content in hair. Assume the hair sample is uncontaminated without the exogenous lead. All the lead content found in sample is endogenous lead. To fulfill this assumption, the axillary hair should be used instead of scalp hair because it will not expose in air and less chance to be contaminated by lead in air or dust. The hair samples with known mass in centrifuge tube is spiked with known amount of lead standard solution and the sample is dried in an oven at 75 Â± 5 oC to allow the interaction of lead with hair sample to give the contaminated hair sample. Afterwards, the effectiveness to remove the exogenous lead contaminant of different washing procedures is examined by determining the lead content of external contaminated hair sample after specific washing procedures. Also, the uncontaminated hair sample is washed with different washing procedure and the lead content found in different washing protocols are compared with the lead content found in unwashed hair sample to investigate the extent of removal of endogenous lead content. Three replicates should be performed in each washing procedure to determine the precision of measurement. The washing protocol that remove the exogenous lead effectively and retain the endogenous lead should be selected. Washing protocol that causes the loss of the endogenous lead should not be used to prevent the false negative result. Hair analysis is only the screening method to check the lead exposure of human body. In case, if the exogenous lead that causes the false positive result, the lead exposure should be confirmed by lead blood test. As a result, it is safer to select the washing protocol which retains the endogenous lead in hair.
2.8 Microwave digestion
After the hair is washed with appropriate washing procedure, it should be digested in concentrated acid to leach out the lead in hair. The microwave digestion will be used for the digestion of hair in the research. After the hair sample is washed, 0.1g of sample is taken for the analysis. To ensure that the homogenize sample is taken from the population, three replicates should be performed in each sample and precision is determined in term of relative standard deviation. The relative standard deviation should be less than 10%. In the microwave digestion, the sample is digested with concentrated nitric acid under the pressure in closed vessel. As a result, higher temperature, which is higher than the boiling point of the concentrated nitric acid at normal atmospheric pressure, can be used for the digestion. Microwave digestion has shorter digestion time (25 - 30 minutes per batch) when compare with traditional hotplate digestion and prevents the loss of volatile elements such as mercury, arsenic and selenium. Lesser concentrated acid is used for the digestion and prevent the cross-contamination. During the digestion, the temperature sensor will be inserted into the reference vessel to monitor the temperature of the internal environment of the vessel. The power output of the microwave will be controlled to ensure the temperature raise up and level off in accordance with the temperature profile of the digestion programme. In case, if the pressure builds up inside the vessel excess the maximum temperature that the vessel can sustain, the vessel cap will be moved up to release the excess pressure and closed again to prevent the loss of the analyte.
Lead is non-volatile element that will not volatile even if open beaker digestion method is used. However, lead will form the white precipitate in the presence of sulphate to form the insoluble lead sulphate. As a result, concentrated sulphuric acid should not be used for the digestion of hair sample. All the glassware should be immersed into the 1:1 nitric acid bath for overnight before using for the sample preparation to ensure that all the lead which adsorb on the glass surface is leached out.
6 mL of 65 - 69% concentrated nitric acid and 2 mL of hydrogen peroxide are added into 0.1g of washed hair sample in the digestion vessel. Wait for 10 minutes for the complete of initial reaction before the vessel is covered with cap. The sample is digested with the following temperature profile.
After the digestion, the vessel should be cooled down until the temperature of the reference vessel drops below 50oC. The vessel can be opened and deliver to 25 mL volumetric flask. The phosphoric acid should be added into the sample solution so that the final concentration of phosphoric acid in sample solution is 1%. Phosphoric acid is the matrix modifier in graphite furnace atomic absorption spectrometry to stabilize the lead at higher temperature by decreasing the volatility of lead element. Addition of matrix modifier greatly reduces the matrix interferences. Finally, make up to its volume with deionized water. Check all the sample solution to ensure no incomplete digestion. The sample solution should be clear without precipitate. In case, if some white precipitates are present in the sample solution, it should be filtered off with 0.45 Î¼m cellulose acetate membrane filter. The concentration of lead in the sample solution is analyzed with graphite furnace atomic absorption spectrometry. Duplicate samples should be performed to calculate the precision and it is one of the major budgets in estimating the uncertainty of the test method.
2.9 Optimization of graphite furnace atomic absorption spectrometry (GFAAS)
It is very important to optimize the analytical instrument and determine the limit of detection and quantification limit. In our research, the graphite furnace atomic absorption spectrometry will be used for the analysis. As a result, all parameters that affect the sensitivity and precision of measurement should be optimized and adjusted before using for the analysis.
Graphite Furnace Atomic Absorption Spectrometry (GFAAS) is based on the absorption by an atom in its ground state of radiation at specific wavelength produced by atoms of the same element contained in a hollow cathode lamp. Light absorbed by the atomized sample is measured and can be related to the concentration of the element in the sample. The important components are the light source, which emits the characteristic narrow-line spectrum of the element of interest; the electrically heated atomizer which atomize the analyte in the graphite tube; a monochromator for dispersion of the light into its discrete spectral wavelengths that can be selected for analysis with slits of variable width; a photomultiplier that converts photons into an electrical signal.
The sensitivity of graphite furnace atomic absorption spectrometry is affected by light path, temperature programme, lamp current and bandpass. The light path can be optimized by adjusting the height and lateral adjustments to obtain the maximum signal strength. The lamp current of the hollow cathode lamp will changes the lamp energy and power incidence to the atom cell. The intensity of light source from the hollow cathode lamp is proportional to the square of lamp current. The increase in the cathode lamp current results in an increase in the kinetic energy of the ionized argon gas causing more atoms to be sputtered. However, too high lamp current will cause the self-absorbance and lower the intensity of light. It is because when the lamp current is too high, a cloud of neutral residual unexcited atoms will be formed in front of the cathode and results in self absorbance. Bandpass must be adjusted to suite the operation needs. A large width will generate a good signal-to-noise ratio. However the resonance line cannot be isolated from other lines and cause the problem in measurement due to the low resolution of the spectral line. At the same time, too narrow spectral bandwidth will cause the low signal-to-noise ratio because of the reduction of light passing through the sample and reach the detection. The lamp current and bandpass should be adjusted to achieve high signal to noise ratio and sensitivity. The temperature programme (ashing and atomization steps) should be optimized to achieve the maximum sensitivity and reduce the background absorption. Non-specific absorption can affect the accuracy of the analysis. Non-specific absorption is a false signal due to either molecular absorption or light scattering. It can be corrected for by accurate simultaneous Zeeman background correction. As a result, the slit width, lamp current and the temperature programme should be optimized by the injection of 0.1 mg/L lead standard solution. The absorption wavelength for lead should be 283.3 nm. The testing parameters for the optimization of graphite furnace atomic absorption spectrometry are shown as follows:
Slit width (nm)
0.2, 0.5 and 1.0 nm
Lamp current (mA)
2, 3, 4, 6, 8, 10 mA
Ashing temperature: 300, 450, 600, 650, 700oC
Atomization temperature: 1800, 1900, 2000, 2100oC
Graphite furnace atomic absorption spectrometry (GFAAS) have lower detection limit than flame atomic absorption spectrometry (FAAS) because the residence time of the analyte in the graphite tube is longer than in flame. However, the matrix effect that causes the non-atomic absorption will affect the accuracy and precision of GFAAS. It is important to use the Zeeman correction to minimize the non-atomic absorption. When compare with ICP-MS, GFAAS cannot perform the multi-element analysis. In our research, only lead is the target of interest. If other heavy metals need to be analyzed, then ICP-MS should be used instead of GFAAS.
2.10 Method validation, estimation of uncertainty and quality control
After the optimization of the instrument, the limit of detection and quantification limit should be determined. The limit of detection is determined by aspirating the matrix blank solution by ten times and it is calculated by YLOD = Yblank + 3 x SB at 95% confidence limit, where SB is the standard deviation of matrix blank. The limit of quantification limit is determined by aspirating the standard solution at 3 - 5 times of limit of detection for ten times. The lead concentration of hair greater than 30 mg/kg is indicating an excessive level. The minimum quantification limit to fulfill the testing requirement is (30 x 0.1 / 25 = 0.12 mg/L). It is better to achieve the quantification limit which is ten times less than the compliance limit. As a result, 0.01 mg/L, 0.015 mg/L and 0.02 mg/L lead standard solutions are aspirated for ten times to obtain the quantification limit. The quantification limit should be the concentration of lead that gives the recovery within 90 - 110% and the calculated quantification limit (10 x SD) should be less than the concentration of lead solution aspirated. The calibration curve should be prepared with matrix solution. The calibration points of 0.01, 0.05, 0.1, 0.2 mg/L should be used and the linearity should be performed across three days and the percentage error of each calibration point and regression are calculated. The percentage error should be within 10% and the regression should be at least 0.995. To verify and validate the test method, the sample spike at the lowest quantification limit and the compliance limit (30 mg/kg) should be performed across a certain period of time for ten replicates. The average recovery should be within 90 - 110% and the precision should be less than 5% RSD. For each batch of sample preparation and analysis, the blank and duplicate sample spike should be performed. Blank is prepared by following the whole testing procedure without the sample. It is used to check for the contamination. The sample spike is used to study the matrix effect and to ensure each batch of sample preparation fulfill the defined acceptable criteria. The sample spike of different batch of sample analysis will be used to estimate the measurement uncertainty of the test method. The bottom up approach will be used to estimate the uncertainty. Precision and trueness studies are two major budgets that contribute to the uncertainty of measurement. The one-way ANOVA is used to verify the precision within day and between day are statistically different or not. Trueness of sample spike across a period of time can be calculated by the following equation (24):
Where u() = uncertainty of recovery,= average recovery, n = number of replicate,
= standard deviation, = average concentration, = uncertainty of standard solution, = concentration of standard spike in sample. The uncertainty of precision and trueness is combined to calculate the combined uncertainty. The expanded uncertainty is calculated by the multiply the combined uncertainty with the coverage factor k which is equal to 2 for 95% confidence limit. The t-test should be performed using the following equation to check for any bias in the test method (24).
In case if the analytical method have any bias, it should be solved and corrected. If it cannot be corrected, the uncertainty should be extended to include the bias measurement.
Schedule of the research work
October 2010 - November 2010
Optimization of GFAAS including slit width, lamp current, ashing and atomization temperature
Determination of instrument detection limit, quantification limit
December 2010 - January 2011
Investigate and select the best washing protocol (IAEA washing protocol, water, 5% HCl, 5% NaOH)
Perform the method validation (linearity, method of detection limit and quantification limit)
February 2011 - July 2011
Collection of scalp and axillary hair from the donor
Correlation study of lead content in scalp and axillary hair.
Study of relationship between lead in hair and daily intake of sashimi