Comparative study of three digestion methods for elemental analysis in traditional medicine products using atomic absorption spectrometry
© Uddin et al. 2016
Received: 2 October 2015
Accepted: 20 January 2016
Published: 30 January 2016
Traditional medicine mainly of herbal origin is widely used all around the world. Heavy metal contamination in such products is frequently reported. Accumulation of heavy metals in the human body leads to various health hazards. Thus, precise determination for such contaminants is required for safety assurance. Sample preparation is a significant step in spectroscopic analysis to achieve reliable and accurate results. Wet digestion methods are basically used for the dissolution of herbal product samples prior to elemental analysis.
This study has been designed to evaluate the efficiency of three acid digestion methods using different solvents. Five samples were digested with three different acid digestion methods namely method A (a combination of nitric-perchloric acids HNO3–HClO4 in a ratio 2:1), method B (only nitric acid HNO3), and method C (a mixture of nitric-hydrochloric acids HNO3–HCl in a ratio 1:3), to recommend the most efficient digestion method that gains the highest analyte recovery. The analysis of arsenic (As), cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn), and iron (Fe) was conducted using various techniques of atomic absorption spectrometry (AAS).
The statistical analysis revealed that method C which represented the combination of nitric-hydrochloric acids HNO3–HCl in a ratio 1:3 was the most efficient digestion method for herbal product samples as it had given a significant high recovery (p < 0.05) for all metals compared to method A and method B. Accuracy of the proposed method was evaluated by the analysis of standard reference material (SRM) 1515 Apple Leaves from the National Institute of Standards and Technology (NIST) which presented good recoveries for all metals ranging from 94.5 to 108 %.
Method C provides highest recovery for all the analytes under investigation using AAS in herbal medicine samples.
KeywordsTraditional medicine Acid digestion Heavy metals Nitric acid
Traditional medicine (TM) has a significant contribution to the global health care system (Chan 2003). A significant proportion of the world’s population relies on TM to support their basic health care needs (Jayaraj 2010). Therefore, safety and quality of such products become a major concern (Igweze et al. 2012). Inorganic contaminants such as heavy metals are often present in herbal medicine in various concentration levels (Saeed 2010; Hina et al. 2011; Qing-hua et al. 2001). The presence of heavy metals in such products is either referred to the ingredients itself or they might arise during the processing part (Sharma and Dubey 2005). Arsenic, cadmium, lead, and nickel are toxic heavy metals that might be present in TM (Uddin et al. 2012). Prolonged exposure to these metals may cause many adverse health effects including cancer (Ray and Ray 2009). Although zinc and iron are essential metals for the human body at trace concentrations yet, they are toxic if present in higher concentrations (Vaikosen and Alade 2011). Consequently, heavy metal content in TM products must be accurately determined. Highly sensitive spectroscopic techniques such as flame (FAAS), graphite furnace (GFAAS), and hydride generation atomic absorption spectrometries (HGAAS) are mainly applied for elemental analysis in various samples. Such techniques require aqueous samples. Thus, solid samples need to be regularly converted into solutions using an appropriate dissolution method (Charun and John 2006). Acid digestion methods are generally used for the dissolution of herbal product samples prior to elemental analysis (Duyusen and Görkem 2011). In a spectroscopic elemental analysis sample preparation, acid digestion is an important step of the entire analytical procedure. It has a substantial effect on the recovery of various analyte contents in highly complex matrices such as herb and plant materials. Therefore, it requires further improvement to provide a standard technique that is able to gain accurate results (Nabil 2010). It is essential to assess the digestion efficiency of various digestion methods to achieve the optimal sample preparation method with clearer background (low noise level). Majority of samples are dissolved by various acids prior to spectroscopic elemental analysis. Wet/acid digestion has the benefits of being effective on both organic and inorganic substances as it has the ability to destroy the sample matrix and consequently minimize the interference. However, at this preliminary stage of the analytical processes, there are still some sources of potential errors such as incomplete digestion. Rational selection of the acid combinations used for various sample digestions is very important to achieve the reliable analytical method. Nitric acid is often utilized for this purpose as an oxidant reagent either individually or mixed with other digestion reagents such as acids and/or hydrogen peroxide. The oxidizing capacity, accessibility, and the affordability of nitric acid make it prevalent in this respect (Sastre et al. 2002). This study was aimed to assess the digestion efficiency of three acid digestion methods namely A, B, and C which represented a combination of nitric-perchloric acids HNO3–HClO4 in a ratio of 2:1, only nitric acid HNO3, and a mixture of nitric-hydrochloric acids HNO3–HCl in a ratio of 1:3, respectively. Five TM samples of herbal origin were digested with the abovementioned methods. The digestion processes were conducted using the conventional open vessel heating system as it provided the advantage of low equipment cost (Güler and Arzu 2006). The analysis of heavy metals was conducted using various techniques of atomic absorption spectrometry (AAS).
Traditional medicine samples
Finished products of traditional medicine samples were collected from three different states in the East Coast region of Peninsular Malaysia, namely Pahang, Terengganu, and Kelantan, from various commercial places of the sampling area. Finished herbal products used for medical purposes are herbal preparations that underwent all stages of production including packaging. They might consist of different herbs/plants, various parts of the same plants, and plant extracts. Five samples of herbal origin in capsule and tablet dosage forms were used to perform the optimization of acid digestion method.
Chemicals and sample preparation
All chemicals and reagents used in this study were of analytical and trace metal grades. Trace metal grades 65 % HNO3, 37 % HCl, and 70 % HClO4 were obtained from Fisher Malaysia. Stock standard solutions for each metal arsenic (As), cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn), and iron (Fe) with a concentration of 1000 ppm were supplied by Perkin Elmer USA. Deionized water was used throughout the study. Sodium borohydride (NaBH4), sodium hydroxide (NaOH), l-ascorbic acid (C6H8O6), and potassium iodide (KI) were from Merck (Germany). A standard reference material (SRM) 1515 Apple Leaves was obtained from the National Institute of Standards and Technology (NIST, USA). All glassware were soaked in 5 % (v/v) HNO3 overnight then rinsed with deionized water and dried using lab dryer FDD-720 prior to use.
Methods of digestion
Samples were accurately weighed (0.5 g each) and placed in a 100-mL PTFE beaker. The samples were subjected to three different acid digestion methods, as will be explained, to identify the most appropriate digestion method to determine the contents of As, Pb, Cd, Ni, Zn, and Fe in TM samples by AAS.
Method A (nitric-perchloric acid digestion 2:1)
To the sample, 5 mL of 65 % HNO3 was added, and then the mixture was boiled gently for 30–45 min. After cooling, 2.5 mL of 70 % HClO4 was added, and the mixture was gently boiled until dense white fumes appeared. Later, the mixture was allowed to cool, and 10 mL of deionized water was added followed by further boiling until the fumes were totally released (Hseu 2004).
Method B (nitric acid digestion)
To the sample, 5 mL of 65 % HNO3 was added, and then the mixture was boiled gently over a water bath (90 °C) for 1–2 h or until a clear solution was obtained. Later, 2.5 mL of 65 % HNO3 was added, followed by further heating until total digestion (Zheljazkov and Nielson 1996).
Method C (nitric-hydrochloric acid digestion 1:3)
To the sample, 9 mL of freshly prepared acid mixture of 65 % HNO3 was added, and 37 % HCl was added. Then, the mixture was boiled gently over a water bath (95 °C) for 4–5 h (or until the sample had completely dissolved) (Ang and Lee 2005).
During the digestion procedures, the inner walls of the beakers were washed with 2 mL of deionized water to prevent the loss of the sample, and at the last part of the digestion processes, the samples were filtered with Whatman 42 (2.5-μm particle retention) filter paper. Then, a sufficient amount of deionized water was added to make the final volume up to 50 mL.
Instrumental parameters for FAAS of Zn and Fe analysis; GFAAS for Cd, Pb, and Ni analysis; and HGAAS for As analysis
Atomization temp. (°C)
Analysis of the standard reference material
The accuracy of the optimize method was verified by the analysis of SRM 1515 Apple Leaves.
Results were expressed as the mean of triplicates ± standard deviation (SD). The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons using SPSS.
Results and discussion
Concentration of different metals (μg/g) (±SD) in traditional medicine samples (TM 1–TM 5) using different acid digestion methods
Recovery percentages and concentrations of As, Cd, Pb, Ni, Zn, and Fe in SRM (1515)
SRM conc. μg/g (±SD)
Measured conc. μg/g (±SD)
All the TM samples were contained heavy metals at different concentrations. Highest analyte recoveries for all TM samples were gained using method C that ranged 0.3–1.9; 0.14–0.33; 2.5–4.66; 4.7–7.02; 14.3–56.8; and 127–688 μg/g for As, Cd, Pb, Ni, Zn, and Fe, respectively. Based on the fact that TM are highly consumed worldwide, a significant concern from many health institutes in different countries had imposed permissible limits of heavy metals in raw/finished herbal product. In Canada, the maximum limits for As, Pb, and Cd are 5, 10, and 0.3 ppm, respectively; in India, 10, 10, and 0.3 ppm for As, Pb, and Cd, respectively (Gupta et al. 2010). It is equitable to assume that heavy metal intake through such products has significant influence on human’s health. Therefore, an adequate method for their determination is of importance.
Previous studies suggested various methods for digesting different samples for metal analysis (Nabil 2010). Aqua regia has been proposed as the best digestion method for samples with low carbonate or organic matter contents such as sediments and agricultural soils (Sastre et al. 2002). Another study reported that there was no significant differences between the digesting capacity of HNO3 acid and HNO3–HClO4 acid mixture in the measurement of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) contents of barley (Hordeum vulgare L. cv. Minorimugi) and rice (Oryza sativa L. cv. Akihikari) seedlings (Shaibur, et al. 2010). Nitric acid digestion was proposed as the most efficient method for recovering Cd, Mn, and Ni in the majority of composts samples (Hseu 2004). Another study recommended the combination of HNO3–HCl in a ratio of 1:2 as the most efficient digestion method which yielded the highest recovery of Pb, Zn, and Fe in canned sardines samples (Fong et al. 2006). However, TM samples have complex matrices as they are made from either one herb or a mixture of herbs from any part of a plant such as leaves, roots, seeds, and flowers that might have different chemical properties. The digestion capacity of hydrochloric-nitric acids HNO3–HCl in a ratio of 1:3 mixture had proven to be the best acid combination suitable for the decomposition of TM samples due to the ability of such mixture to release the metal ions from such complex matrices of herbal materials and subsequently to minimize the noise level during the detection procedure.
Sample preparation is a crucial step in spectroscopic elemental analyses as it can considerably affect the accuracy of results. Significant differences between the digesting capacities of different methods were identified. The digestion capacity using a mixture of hydrochloric-nitric acids HNO3–HCl in a ratio of 1:3 was the most efficient method in terms of the recovery of As, Cd, Pb, Ni, Zn, and Fe in herbal medicine samples.
atomic absorption spectrometer
American Organization of Analytical Chemistry
analysis of variance
electrodeless discharge lamp
flame atomic absorption spectrometer
graphite furnace atomic absorption spectrometer
- HClO4 :
- HNO3 :
- NaBH4 :
hydride generation atomic absorption spectrometer
- Mg(NO)3 :
National Institute of Standards and Technology
Statistical Package for Social Sciences
standard reference material
The authors would like to express their gratitude to all the academic and technical staff of the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, IIUM, for their support. They are also thankful to the Research Management Centre (RMC), IIUM, and Ministry of Higher Education (MOHE), Malaysia, for funding this study.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Ang H, Lee K. Analysis of mercury in Malaysian herbal preparations: a peer-review. Biomed Sci. 2005;4:31–6.Google Scholar
- Chan K. Some aspects of toxic contaminants in herbal medicines. Chemosphere. 2003;52:1361–71.View ArticleGoogle Scholar
- Charun Y, John GF. Comparative study of acid-extractable and total digestion methods for the determination of inorganic elements in peat material by inductively coupled plasma-optical emission spectrometry. Anal Chim Acta. 2006;557:296–303.View ArticleGoogle Scholar
- Duyusen EG, Görkem A. Comparison of acid digestion techniques to determine heavy metals in sediment and soil samples. GU J Sci. 2011;24:29–34.Google Scholar
- Fong SS, Kanakaraaju D, Ling SC. Evaluation of acid digestion with different solvent combination for the determination of iron, zinc and lead in canned sardines. M J Chem. 2006;8:10–5.Google Scholar
- Güler S, Arzu NU. The effect of acid digestion on the recoveries of trace elements: recommended policies for the elimination of losses. Turk J Chem. 2006;30:745–53.Google Scholar
- Gupta S, Pandotra P, Gupta AP, Dhar JK, Sharma G, Ram G, et al. Volatile (As and Hg) and non-volatile (Pb and Cd) toxic heavy metals analysis in rhizome of Zingiber officinale collected from different locations of North Western Himalayas by atomic absorption spectroscopy. Food Chem Toxicol. 2010;48(10):2966–71.View ArticleGoogle Scholar
- Hina B, Rizwani GH, Naseem S. Determination of toxic metals in some herbal drugs through atomic absorption spectroscopy. Pak J Pharm Sci. 2011;24:353–8.Google Scholar
- Hseu ZY. Evaluating heavy metal contents in nine composts using four digestion methods. Bioresource Technol. 2004;5:53–9.View ArticleGoogle Scholar
- Igweze ZN, Orisakwe OE, Obianime A. Nigerian herbal remedies and heavy metals: violation of standard recommended guidelines. Asian Pac J Trop. 2012;2:1423–30.View ArticleGoogle Scholar
- Jayaraj P. Regulation of traditional and complementary medicinal products in Malaysia. Int J Green Pharm. 2010;4:10–4.View ArticleGoogle Scholar
- Nabil RB. Sample preparation for flame atomic absorption spectroscopy: an overview. Rasayan J Chem. 2010;4:49–55.Google Scholar
- Qing-hua Y, Qing W, Xiao-qin M. Determination of major and trace elements in six herbal drugs for relieving heat and toxicity by ICP-AES with microwave digestion. J Saudi Chem Soc. 2001;16:287–90.View ArticleGoogle Scholar
- Ray SA, Ray MK. Bioremediation of heavy metal toxicity-with special reference to chromium. Ameen J Med Sci. 2009;2:57–63.Google Scholar
- Saeed M. Analysis of toxic heavy metals in branded Pakistani herbal products. J Chem Soc Pak. 2010;32(471):475.Google Scholar
- Sastre J, Sahuquillo A, Vidal M, Rauret G. Determination of Cd, Cu, Pb and Zn in environmental samples microwave-assisted total digestion versus aqua regia and nitric acid extraction. Anal Chim Acta. 2002;462:59–72.View ArticleGoogle Scholar
- Shaibur MR, Shamim AHM, Imamul Huq SM, Kawai S. Comparison of digesting capacity of nitric acid and nitric acid-perchloric acid mixture and the effect of lanthanum chloride on potassium measurement. Nat Sci. 2010;8:157–62.Google Scholar
- Shah AQ, Kazi TG, Arain MB, Jamali MK, Afridi HI, Jalbani N, et al. Comparison of electrothermal and hydride generation atomic absorption spectrometry for the determination of total arsenic in broiler chicken. Food Chem. 2009;113:1351–5.View ArticleGoogle Scholar
- Sharma P, Dubey RS. Lead toxicity in plants. Braz J Plant Physiol. 2005;17:35–52.View ArticleGoogle Scholar
- Uddin ABMH, Khalid RS, Abba S. Determination of heavy metal concentration of different traditional medicine formulations available at the East Coast Region of Malaysia. Afr J Pharm Pharacol. 2012;6:1487–91.Google Scholar
- Vaikosen EN, Alade GO. Evaluation of pharmacognostical parameters and heavy metals in some locally manufactured herbal drugs. J Chem Pharm Res. 2011;3:88–97.Google Scholar
- Zheljazkov VD, Nielson NE. Effect of heavy metals on peppermint and cornmint. Plant Soil. 1996;178:59–66.View ArticleGoogle Scholar