Open Science Repository Chemistry

doi: 10.7392/Chemistry.70081933


Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES


Awad A. Momen, Ali D. M. H., Mohammed A. Khalid, Malik A. Elsheikh

Department of Chemistry, Faculty of Applied Medical Sciences and Scientific Departments, Taif University, Saudi Arabia


Abstract

A simple and reliable analytical procedure for determination of selected trace elements such as Al, B, Ba, Cd, Cr, Cu, Mn, Pb and Zn in human blood samples was modified. Samples were collected from normal subjects, diabetic mellitus and hypertensive patients. Samples were taken from both genders of different ages from occupants of urban populations of Taif city, Saudi Arabia. Different sample preparation procedures with acids and oxidizing reagents were tested and well investigated. Analysis was made by inductively coupled plasma optical emission spectrometry after sample digestions. The overall recoveries of all determined elements were found in the range of (94.6–104.9%) of the expected values. The results of this study showed that the mean concentrations of the Al, B, Ba, Cd, Cu and Pb in human blood of diabetic mellitus and hypertensive patients were higher than the corresponding values of normal subjects. While the concentrations of Cr, Mn and Zn in human blood of diabetic mellitus and hypertensive patients were lower as compared to values of normal subjects, the differences found were non-significant (p=0.05).

Keywords: trace elements, digestion procedure, diabetes mellitus, hypertension, Inductively coupled plasma-optical emission spectrometry (ICP-OES).



Citation: Momen, A. A., Ali, D. M. H., Khalid, M. A., & Elsheikh, M. A. (2013). Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES. Open Science Repository Chemistry, Online(open-access), e70081933. doi:10.7392/Chemistry.70081933

Received: January 27, 2013.

Published: February 25, 2013.

Copyright: © 2013 Momen, A. A., Ali, D. M. H., Khalid, M. A., & Elsheikh, M. A. Creative Commons Attribution 3.0 Unported License.

Contact: research@open-science-repository.com



Introduction

Diabetes mellitus (DM) is a disease that prevails all over the world. It's prevalence rates differ from one country to another. It is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action associated with chronic hyperglycemia and disturbances of carbohydrate, lipid and protein metabolism. Long-term vascular complications represent a major cause of morbidity and mortality in patients with diabetes mellitus. In addition, various biochemical disorders associated with vascular complications, such as hyperlipidemia and oxidative stress, which frequently co-exist with diabetes mellitus, appear inadequate to explain the increased risk of vascular diseases. The observations suggest that additional factors may be involved in the acceleration of diabetic vascular disease [1, 2]. In the other hand, hypertension (HTN) is the force that exerted by the blood against the walls of the bleed vessels. It is characterized by the increase of blood pressure in vessels, arteries and veins. The prevalence of hypertension increases with advancing age. Moreover, nowadays the age criteria have been changed and even the younger have HTN problems due to lack of exercise, fast foods, coffee consumption, smoking and alcohol use. Genetic effect may also be a factor [3].

Current development of human health related studies requires a growing number of elements to be monitored in biological matrices. Few of the elements present in nature play a metabolic role in living organisms [4]. According to their abundance, these elements are classified as macro, micro or trace elements, representing 93%, 5% and around 1%, respectively, of the total body weight. The remaining percentage could be attributed to those elements with unknown biological functions, to others which are present only because of the exposure to polluted environment or to those intentionally introduced into the body for a special treatment [5, 6].

Trace elements (TEs) are defined as “any element having an average  concentration of less than about 100 ppm (100 μg ml-1)” [6, 7]. They (TEs) have recently been attracting the attention of scientists in various systems related to human health, such as in clinical and environmental analysis. Also, the measurement of TEs is increasingly attracting interest from physicians because deviations in TEs uptake and/or metabolism are known to be related to certain dysfunctions [8]. Analytical studies of TEs dealing with problems of microanalysis of biological samples also have been increasing due to the expanding health areas [9]. Moreover, a great effort has been expended on developing analytical procedures for TEs measurements and improving their sensitivity and specificity [8].

The abnormal metabolism of TEs plays an important role in health and disease conditions, and studies about them have been attracting significant interest. It has been speculated that TEs may play a role in the pathogenesis of many diseases. Some of them form part of enzymes and others are involved in the synthesis of hormones [3, 10]. Others are known to be associated with certain diseases if they are present in the body in abnormally low concentrations. Several of them have been documented as being involved in blood pressure control and others may lead to intoxications in humans, if ingested in high concentrations. Many of them are excreted primarily in urine and some are transmitted to blood [11].

Blood usually is used for the diagnosis and treatment of chronic degenerative disease caused by some TEs. So, blood analysis can provide important information to the clinician that may not be readily available with urine analysis. Levels of TEs in the blood and the excreted urine are tightly controlled via metabolic, re–absorptive, and excretory mechanisms [12–14].

In view of the above facts, it is important to determine the TEs concentrations in human bloods (HB) having physiological disorders, such as DM and HTN. Various biopsy materials – such as bone and teeth, hair and nails, organs and blood and its components, urine, cerebrospinal, amniotic, synovial fluids and tears, saliva, perspiration, bile, milk –  are good indexes of exposure to elements, easily accessed and may be used as bioindicators for these purposes [15].

Biological samples (BSs) namely HB were chosen for this study as probability (representative) sampling. Sample collections were consisted of a number of healthy (normal subjects) and patients (DM and HTN) of different ages (30–75 years), selected from occupants of urban populations of Taif city (Saudi Arabia) on personal request. A questionnaire were employed in order to collect details concerning physical data, ethnic origin, health, dietary habits and consent of the donor. Some factors that affect analytical and biological variability of the concentrations to be determined, such as the route of absorption, the presence of sources of environmental pollution in certain areas of residence, physiological variables and life–styles, also were discussed.

There are several modern techniques for the determination of TEs in HB. The pretreatment procedures vary according to the nature of the samples, the available method of analysis, the elements to be determined and their concentration levels. Most techniques generally require the element to be in solution. In all cases, samples demand manipulation (sampling, subsampling, washing, etc.) prior to other processing and detection [6]. In most clinical inorganic determinations, the sample is digested or leached by oxidizing acidic mixtures aided by heat or ultrasound or microwave radiation for oxidizing the organic matter [16]. The main advantages of microwave-assisted procedures are that they require smaller amounts of sample and oxidizing materials, shorter digestion times and easiness of sample handling. These procedures have to be validated in order to ensure that no contamination and/or losses have occurred. The presence of these problems could affect the accuracy and the precession of final results. Thus, the validation of the whole procedure was made by using a certified reference materials and/or standard addition method and/or by comparing the results of two different certified analytical procedures [8, 17].

Biomonitoring of such elements present in a complex samples requires sensitive analytical methods with outstanding precision and high sample throughput. This is to cope with the low element concentrations and with the large number of samples that will have to be processed, eventually following an emergency [6]. The most common analytical techniques for measuring TEs concentrations in BSs like HB are flame and/or electrothermal atomic absorption spectroscopy [4, 8, 18, 19], inductively coupled plasma optical emission spectroscopy [17, 20], inductively coupled plasma mass spectrometry [11, 21–24] and high performance liquid chromatography [25].

It follows that analytical methods for determining minor and TEs in biological matrices such as HB should involve minimal sample handling and achieve detection limits relatively low, to permit easy and reliable determination of elements [11]. Considering these requirements,  inductively coupled plasma optical emission spectroscopy (ICP-OES) is a good solution, because it allows rapid and precise multi-element determination in a single solution, with sufficiently low detection limit and wide dynamic range and high accuracy [17, 26–28].

Although potentially harmful effects of trace elements are generally well known, limited studies are available regarding the investigation of relationship between these elements and diseases. This will be indicated by the determination of the concentrations of selected TEs like Cd and Pb in HB of DM and HTN patients. Then, by testing the increase or the decrease of these elements compared to control subjects. A total of 138 samples of HB were analyzed after 'wet digestion' for nine TEs using ICP-OES.


Instrumentation and conditions

A Varian 725–ES inductively coupled plasma-optical emission spectrometer, with radial viewing configuration, was used to analyze the standard and the sample solutions of Al, B, Ba, Cd, Cr, Cu, Mn, Pb and Zn. The ICP-OES operating conditions were well optimized and carefully selected in order to maximize the sensitivity for the desired elements and to obtain the best  precision and accuracy. Details of the operating conditions are summarized in Table 1. Each element was measured at two specific lines (nm) atomic (I) and/or ionic (II) lines characteristics of a particular element that gives maximum sensitivity [20]. Lead was measured at atomic (I) line only. The intensity of this emission is indicative of the concentration of the element within the sample. Selected emission lines (nm) for each element are summarized in Table 2.


Table 1: ICP–OES operating parameters for determination of selected TEs in HB samples

Table 1: ICP–OES operating parameters for determination of selected TEs in HB samples


Table 2: ICP–OES selected atomic (I) and/or ionic (II) emission lines (nm) for selected TEs

Table 2: ICP–OES selected atomic (I) and/or ionic (II) emission lines (nm) for selected TEs


Reagents and glassware

All reagents and chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany, www.merck.de). Mineral acids, chemical reagents and oxidizing agents 69–72 % (m/m) HNO3 (d = 1.41–1.51 kg l−1), 36.5–38 % (m/m) HCl (d = 1.18–1.19 kg l−1), 30% (m/m) H2O2 (d = 1.11–1.45 kg l−1), etc. were used. A multi-element stock standard solution (1000 mg l−1) was also used. Calibration standard solutions were obtained from the stock solution by suitable dilutions. De-ionized doubly distilled water (DDDW) was also used throughout the analyses for preparing reagent, standard and sample solutions. De-ionized doubly distilled water also was used for washing and rinsing of all apparatus and glassware. Acid-washed polypropylene bottles were used for preparing and storing solutions. All solutions were stored at −5°C until needed for analysis. Polypropylene storage bottles, glassware and the auto sampler cups were cleaned by soaking in 5 mole l-1 HNO3 for about 24 hrs, rinsing five times with DDDW, dried, and stored in a class-100 laminar-flow hood.

Sample collection and handling

For the present study, One hundred and thirty eight samples of venous HB were collected from healthy nonsmoking normal subjects (n = 45), DM (n = 52) and HTN patients (n = 41). Samples were taken from males and females of different ages ranged from 30 to 75 years from occupants of urban populations of Taif city, Saudi Arabia. Venous HB (~5 ml) was sampled by using metal-free safety vacutainer blood collecting tubes containing > 1.5 mg K2EDTA obtained from Becton Dickinson, Rutherford, USA. Samples were kept in the freezer (−5°C) till being analyzed.

Microwave–assisted acid digestion

Triplicate of 0.5 ml of HB samples, of each DM, HTN patients and normal subjects, were directly placed into porcelain crucible. Three milliliters of concentrated HNO3–H2O2 (2:1, v/v) was added to each crucible. The crucibles were covered and kept at room temperature (~35 oC) for about 5 min as a pre-digestion time, then placed in a microwave oven. Then, crucibles were heated following a one-stage digestion program at 30 % of total power (900 W). Complete digestions of all samples required 2-3 min. After the digestion was completed, the crucibles were left to cool at room temperature and the resulting solution (about 0.5 ml of semi-dried mass) was dissolved by 5 ml of 0.1 mol l-1 HNO3. Then, transferred quantitatively to 10 ml volumetric flasks, diluted with DDDW up to mark and transferred to a polyethylene storage bottle for further analysis. Blank and spike sample solutions were carried out simultaneously through the complete digestion procedures and similar acid matrices. The presence of ca. 0.1 mol l-1 HNO3 in the final solution was necessary to maintain acidic environment and avoid formation of insoluble hydroxides before measurement steps. This procedure is similar to that stated by Kazi et al. [2], Afridi et al. [10] and Memon et al. [29], with some modifications in digestion time and microwave oven program. The validity of the digestion procedure was checked by spiking of different HB samples of normal subjects with a known amounts of multi-element standard solution before (pre) and after (post) digestion procedures. All target elements were determined in the prepared solutions by ICP-OES.

Statistical analysis

All results were statistically evaluated by Student ttest and ANOVA test (p=0.05). In addition, Microsoft Excel and Origin software's were also used to assess the significance of the differences between the variables investigated in patients and normal subjects. The concentration values obtained were expressed as mean value ± standard deviation (p=0.05). All statistical analyses were based upon triplicate measurements of all standards and sample solutions.

Analytical figures of merit

The validity and efficiency of the microwave digestion method was checked by analyzing spike solutions with a multi-element standard solution. The spike solutions were added to a known amounts of the HB samples of normal subjects before and after digestion, which had also been through the digestion steps. The detection limit (LOD) was defined as 3s m-1, where s is the standard deviation corresponding to ten blank injections and m is the slope of the calibration graph. The LOD was 1 μg l-1 for Cu, 0.9 μg l-1 for Al and Cr, 0.6 μg l-1 for B, and Cd, 0.15 μg l-1 for Ba, 0.08 μg l-1 for Mn, 5 μg l-1 for Pb and 0.5 μg l-1 for Zn.

Results and discussion

All results were expressed as x ± s, where x mean values and s is standard deviation. To ensure that no contamination and/or loss of elements occurs during sample preparations and measurement methodology, a recovery test was demonstrated by standard addition methodology. It was performed using a bulk sample which had also been through all digestion procedures. A multi-element standard solution spike was added to a known amount of the HB samples of normal subjects both before and after digestion, to assess the validity of the digestion procedure. The recoveries of the pre-digestion (pre-spiked) sample were ranged between 94.6 and 103.7 %. While the recoveries of the post-digestion (post-spiked) sample were between 95.7 and 104.9 %. This indicates that there was no contamination and/or loss of elements during sample preparations and measurement steps. Thus, no significant differences were observed (p=0.05). The accuracy observed was found to be quite satisfactory. The recovery test results are given in Tables 3. The percentage recoveries of selected elements in HB samples of normal subjects were calculated according to equation [30, 32]:
 
% Recovery = [Xs / (Xu + K)] x 100

Where: Xs = measured mean value for spiked sample; Xu = measured mean value for unspiked sample; K = known value for the spike in the sample.


Table 3: Percent recoveries results of TEs in HB of normal subjects at the selected conditions

Table 3: Percent recoveries results of TEs in HB of normal subjects at the selected conditions

a: a mean value (n=3)


Table 4 shows the results obtained for the determined selected TEs in HB samples of normal subjects, DM and HTN patients. From the results it was found that the Al levels in HB of DM patients (0.87±0.06 mg l-1) and HTN patients (0.89±0.08 mg l-1) were higher than the corresponding value of normal subjects (0.76±0.05 mg l-1). Likewise, Ba values in HB (0.29±0.04 mg l-1) of DM and HTN patients (0.35±0.04 mg l-1) were high vs. normal subjects (0.04±0.01 mg l-1), but the differences found were not significant (p=0.05). Moreover, very close ranges of B were detected in both HB (0.65±0.05 mg l-1) of DM and (0.72±0.05 mg l-1) HTN patients as compared to normal subjects (0.60±0.04 mg l-1), but the differences found were non-significant (p=0.05). Furthermore, high Cu values were found in both HB of DM (0.83±0.07 mg l-1) and HTN patients (0.89±0.08 mg l-1) as compared to normal subjects (0.51±0.05 mg l-1), with no significant differences (p=0.05).

In contrast, low Cr, Mn and Zn values were found in both HB (0.07±0.03 mg l-1), (0.09±0.02 mg l-1), (0.43±0.04 mg l-1) of DM and (0.06±0.01 mg l-1), (0.05±0.01 mg l-1), (0.06±0.02 mg l-1) HTN patients as compared to normal subjects (0.09±0.02 mg l-1), (0.25±0.04 mg l-1), (0.68±0.05 mg l-1), respectively, but the differences found were not significant (p=0.05). Moreover, it is bad worth to mentioned that high levels of both Cd and Pb were detected in HB of both DM (0.05±0.02 mg l-1),(0.34±0.04 mg l-1) and HTN patients (0.09±0.03 mg l-1), (0.62±0.06 mg l-1), as compared to normal subjects (0.03±0.01 mg l-1), (0.09±0.03 mg l-1), respectively, but the differences found were insignificant (p=0.05).


Table 4: Selected TEs concentrations in HB samples of normal subjects, DM and HTN patients

Table 4: Selected TEs concentrations in HB samples of normal subjects, DM and HTN patients

a: a mean value ± standard deviation (n=3)


Figure 1 show the distribution of the concentrations of nine TEs in HB of normal subjects, DM and HTN patients under study. It shows that lower concentrations were observed for Cr, Mn and Zn. The opposite was true for Al, B, Ba, Cd, Cu and Pb.


Figure 1: Comparison of selected TEs concentrations in HB samples of normal subjects, DM and HTN patients 

Figure 1: Comparison of selected TEs concentrations in HB samples of normal subjects, DM and HTN patients

There is wide variation in the published data for the TEs concentrations in BSs such as HB of DM and HTN patients of different countries [2–4, 6, 11, 27, 31–34]. These variations in values are due to the variation in food habits and probably to the exposure of various substances causing high variation of trace elements levels in HB. To compare the reference ranges determined in the present study with those found by other authors is difficult, because there is a lack of coherence in the levels of TEs found by various laboratories. One possible explanation for the different ranges of TEs may come from the fact that with higher analytical sensitivity, the presence of contaminants becomes increasingly important. This is especially the case with elements that are physiologically present at very low concentrations, such as Mn, Cd, Ba and Cr.


Conclusions

The goal of the work described here is to provide a fast, cheap, sensitive, and reliable procedure for TEs analyses in a range of clinical matrices, i.e., HB (DM and HTN patients) using high resolution ICP-OES. To assess this, a suite of clinically important TEs such as Al, B, Ba, Cd, Cr, Cu, Mn, Pb and Zn was quantified. However, after all conditions had been established, measurements became very efficient. Since the ICP-OES has excellent sensitivity, multi-element data can be obtained with very short acquisition times. Three replicates for nine TEs were performed in only about 30 seconds, 138 samples of each of the nine TEs can be measured in few hours (~ 2 h). We can conclude that there is evidence that the metabolism of several TEs like Cr, Cd, Pb, Zn and Cu might have specific roles in the pathogenesis and progress of diseases such as DM and HTN [1–3, 35]. Also, these results indicate that additional studies are necessary for investigation of possible roles of trace elements in DM, HTN and other diseases.


Acknowledgments  

The authors gratefully thanks the dean of the Deanship of Scientific Research, Taif University, Saudi Arabia, for sponsoring this project. Also, we acknowledge all the individuals who kindly participated in the study including the staff of CPL, Ministry of Petroleum, Khartoum, Sudan, for samples processing.


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Cite this paper

APA

Momen, A. A., Ali, D. M. H., Khalid, M. A., & Elsheikh, M. A. (2013). Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES. Open Science Repository Chemistry, Online(open-access), e70081933. doi:10.7392/Chemistry.70081933

MLA

Momen, Awad A. et al. “Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES.” Open Science Repository Chemistry Online.open-access (2013): e70081933. Web. 25 Feb. 2013.

Chicago

Momen, Awad A., D. M. H. Ali, Mohammed A. Khalid, and Malik A. Elsheikh. “Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES.” Open Science Repository Chemistry Online, no. open-access (February 25, 2013): e70081933. http://www.open-science-repository.com/assessment-of-digestion-procedure-for-determination-of-trace-elements-by-icp-oes.html.

Harvard

Momen, A.A. et al., 2013. Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES. Open Science Repository Chemistry, Online(open-access), p.e70081933. Available at: http://www.open-science-repository.com/assessment-of-digestion-procedure-for-determination-of-trace-elements-by-icp-oes.html.

Science

1. A. A. Momen, D. M. H. Ali, M. A. Khalid, M. A. Elsheikh, Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES, Open Science Repository Chemistry Online, e70081933 (2013).

Nature

1. Momen, A. A., Ali, D. M. H., Khalid, M. A. & Elsheikh, M. A. Assessment of Digestion Procedure for Determination of Trace Elements by ICP-OES. Open Science Repository Chemistry Online, e70081933 (2013).


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