Determination of Mercury by Cold Vapor Atomic Absorption Spectrophotometer in Tongkat Ali Preparations Obtained in Malaysia

Abstract

The DCA (Drug Control Authority), Malaysia, has implemented the phase 3 registration of traditional medicines on 1 January 1992, with special emphasis on the quality, efficacy, and safety (including the presence of heavy metals) in all pharmaceutical dosage forms of traditional medicine preparations. As such, a total of 100 products in various pharmaceutical dosage forms of a herbal preparation, containing Tongkat Ali, were analyzed for mercury content using cold vapor atomic absorption spectrophotometer. Results showed that 36% of the above products possessed 0.52 to 5.30 ppm of mercury and, therefore, do not comply with the quality requirement for traditional medicines in Malaysia. Out of these 36 products, 5 products that possessed 1.05 to 4.41 ppm of mercury were in fact have already registered with the DCA, Malaysia. However, the rest of the products that contain 0.52 to 5.30 ppm of mercury still have not registered with the DCA, Malaysia. Although this study showed that only 64% of the products complied with the quality requirement for traditional medicines in Malaysia pertaining to mercury, they cannot be assumed safe from mercury contamination because of batch-to-batch inconsistency.

Over the years, herbal medicinal products have become more and more popular in countries such as the United States, the United Kingdom, Australia, and Malaysia (MacLennan, Wilson, and Taylor 1996; Eisenberg et al. 1998; Ernst 2000a; Ernst and White 2000; Zaidi 2002; Saras 2003). Therefore, a critical evaluation of their safety is extremely important (Ernst 2000b). As such, the DCA (Drug Control Authority), Malaysia, has implemented the phase 3 registration of traditional medicines on 1 January 1992, with special emphasis on the quality, efficacy, and safety (including the presence of heavy metals) in all pharmaceutical dosage forms of traditional medicine preparations ( Guidelines for application for registration of pharmaceutical products (containing scheduled poisons and non-scheduled poisons) 1993; Guidelines on good manufacturing practice (GMP) for traditional medicines 1999; Ramli 2001).

So far, until 2000, since the implementation of this phase, the DCA, Malaysia managed to receive a total of 20,291 applications for registration, out of which only 8550 products successfully registered, with 4400 products obtained from local manufacturers whilst 4150 products were imported (Newsletter of the Drug Control Authority Malaysia 2001). Currently, there are around 140 Malaysian manufacturers registered with the Ministry of Health Malaysia that are producing herbal preparations (Shakri 2000).

Mercury is the most toxic element after polonium (Ramli 2001). It can be presented in the form of different inorganic species, mercury (0), mercury (I) ion, and mercury (II) ion (Mahan and Meyers 1987). Among these three species, elemental Hg in the gaseous state is the most dangerous because it is rapidly absorbed by the lungs and then passes into the blood and brain or in the form of organomercury compounds, of which the short-chain-length alkylmercuries are responsible for causing severe damages to the central nervous system and the gastrointestinal tract, kidney damages, and other illnesses (Antonovich and Bezlutskaya 1996).

In addition, human consumption and disposal of mercury (together with environmental factors, such as rain and altered flow of rivers) have increased the risk of harmful exposure to mercury. Mass mercury poisonings of humans in Minamata Bay and Iraq enhanced the research of mercury transport, fate, bioaccumulation, and toxicity (Goldwater 1971; D’ltri and D’ltri 1977; Hamada and Osame 1996).

A large number of studies in mercury determination have been published. The four most common and reliable techniques for mercury determination are spectrophotometry after chelation with dithizone, atomic absorption and emission spectroscopy, neutron activation analysis, and gas chromatography. Of these, CVAAS (cold vapor atomic absorption spectrophotometer) has become the most popular technique because the mercury compounds can be reduced to elemental mercury that occurs as vapor (Lam Leung, Cheng, and Lam 1991; Landi, Fagioli, and Locatelli 1992; Navarro, Lopez, and Lopez 1992; Guo and Baasner 1993; Adeloju, Dhindsa, and Tandon 1994). Regardless of the digestion method used, the organic matter has to be destroyed and if CVAAS technique is applied, all mercury forms present must be oxidized to mercury (II) prior to reduction to elemental mercury (Ping and Dasgupta 1989; Baxter and Frech 1990; Welz, Tsalev, and Sperling 1991).

In the present study, we analyzed the mercury content using CVAAS in a total of 100 products (both registered and unregistered with the DCA, Malaysia) in various pharmaceutical dosage forms of traditional preparations, containing one of the most popular herbal remedies, Eurycoma longifolia Jack, known as Tongkat Ali in Malaysia (Nooteboom 1972; Gimlette and Thomson 1977). These products, either single or combined preparations, were bought in the Malaysian market, after performing a simple random sampling on them, to enable any sample of an equal chance of selection (Newman 1995) and unbiased (Schefler 1984).

MATERIALS AND METHODS

Instrumentation

GBC 906 AA model atomic absorption spectrophotometer complete with inbuilt window-based 906 program software was used in this study. Conventional hollow cathode lamp was used for mercury.

Reagents

All reagents were analytical grade. Hydrochloride acid 37% (specific gravity 1.33; Merck); nitric acid 65% (specific gravity 1.40; Merck), and stannous chloride dihydrate (BDH, product no. 10270) were used in this study. Mercury stock solution, 1000 ppm (BDH, product no. 141454 K), was diluted to the approximate concentration and were prepared immediately before use. All chemicals were high quality technical grade. Deionized water (Deionizer Elga B113) was used throughout the study.

Treatment of Glasswares

All glasswares were soaked with aqua regia (HCl:HNO₃ = 3:2) for 2 h and then washed with deionized water prior to use.

Wet Digestion of Samples

A total of 100 samples were digested using aqua regia wet digestion. Approximately 1.5 g of the sample were weighed and placed in a 100-ml Quickfit round bottom flask. It was then added with 25.0 ml of freshly prepared aqua regia and then the mixture was refluxed over water bath for 6 to 8 h or until all the samples had dissolved in the aqua regia. The solution was allowed to cool and then filtered whilst the residue was washed with deionized water. The combined aqueous extract was then made up to 50.0 ml with deionized water. This extraction procedure was repeated for the same sample (replicate) and the blank (containing aqua regia only). Coarse particles, such as tablets, pills or powders, capsules, and the contents were grounded to fine powder or particles prior to wet digestion.

CVAAS

One-milliliter extract or blank or standard solution was added to 70 ml deionized water in a 150-ml Quickfit conical flask. The water was then added up to 2 ml of 10% stannous chloride and then was aspirated by gas stream into the flameless pathway of the monochromatic light. The absorbance was recorded when stable and the data were analyzed. The operating procedures used were slit width: 0.5 nm, current: 3.0 mA, and wavelength: 253.7 nm.

Preparation of Stannous Chloride (10%)

Ten grams of stannous chloride dihydrate (SnCl₂·2H₂O) was dissolved in 20 ml of hot concentrated HCl and diluted to 100 ml with deionized water. The resulting solution was heated with a metallic tin until the precipitate disappeared. The solution was stored in a glass bottle containing tin powder.

Mercury Standard Solution

Mercury stock solution (1000 ppm) was diluted serially to produce a standard solution of 0.1 μg/ml. Seven 150-ml Quickfit conical flasks were added to 70 ml of deionized water each. They were then added to 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml of 0.1 μg/ml of mercury stock solution to produce 0, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.30 μg of mercury, respectively. Two milliliters of 10% stannous chloride were added to each flask and were aspirated by a gas stream into the flameless pathway of the monochromatic light. The absorbance was recorded when stable and the data were analyzed.

RESULTS

Mercury Content Evaluation

Results showed that 36 out of 100, viz. 36%, of these products possessed 0.52 to 5.30 ppm of mercury and, therefore, do not comply with the quality requirement for traditional medicines in Malaysia (Tables 1 and 2). The quality requirement for traditional medicines in Malaysia is not exceeding 0.5 ppm for mercury (Jaafar 1995; http://www.bpfk.gov.my/default.asp; Regulating supplements 2003). Out of these 36 products, 5 products with 1.05 to 4.41 ppm of mercury in fact have already registered with the DCA Malaysia (Table 1). However, the rest of the products with 0.52 to 5.30 ppm of mercury still have not registered with the DCA Malaysia (Table 2).

This study showed that only 64% of the products complied with the quality requirement for traditional medicines in Malaysia pertaining to mercury.

DISCUSSION

The use of aqua regia in the acid digestion could ensure total extraction of metals from both inorganic and organic samples (Lee and Laufmann 1971). Some metals, such as gold, are in- soluble in HCl and also HNO₃, but they are soluble in a mixture of these two acids because the enhanced dissolving power in aqua regia is due to the oxidizing ability of nitrate ion in strong acid plus complexing ability of chloride ion (Sienko and Plane 1984). In addition, aqua regia digestion of biological materials virtually eliminates loss of mercury compared to conventional wet digestion (Lee and Laufmann 1971). Wet digestion using either H₂SO₄–HClO₄ or HNO₃–H₂SO₄ with dithizone extraction is susceptible to mercury loss through equipment absorption or volatilization.

AAS was used in this study because it was found that this method is the most suitable method for the mercury analysis, especially for the determination of low concentration of mercury present. It is also relatively easy, cheap, and also more specific compared to other analytical methods such as dithizone and colorimetry methods (Lindstedt 1970).

CVAAS is the predominant technique for mercury analysis (Environmental Protection Agency [EPA] 1986; Beaty and Kerber 1993; Adeloju, Dhindsa, and Tandon 1994; Bruhn et al. 1994; Landi and Fagioli 1994) due to its high selectivity and sensitivity. This approach does not suffer from any important interference. Therefore, it is limited to mercury (Beaty and Kerber 1993) because all metals are solid at normal room temperature. Hence, the elemental mercury, which is liquid at normal room temperature, has a high vapor pressure to be generated into the vapor state.

The sensitivity of the CV technique is far greater than can be achieved by the conventional AAS. This improved sensitivity is achieved through 100% sampling efficiency. All of the mercury in the sample solution placed in the reaction flask is chemically atomized and transported to the sample cell for measurement (Beaty and Kerber 1993).

Stannous chloride was chosen as the reducing agent in the analysis to increase precision and accuracy as compared to sodium tetrahydroborate because in the latter, it was found that the pH dependence of sodium tetrahydroborate for mercury (II) ion reduction introduced variance to mercury recoveries. In addition, variable amounts of unreacted HNO₃ could react vigorously with sodium tetrahydroborate, thus reduce nitrate ion instead of mercury (II) ion (Adair and Cobb 1999).

Although this study showed that only 64% of the tested products complied with the quality requirement for traditional medicines in Malaysia pertaining to mercury, these products cannot be assumed safe from mercury contamination because of batch-to-batch inconsistency (Ko 1998). As such, the DCA, Malaysia, has specified that the validity of pharmaceutical products (nonpoisons) to be limited to a maximum of 5 years. Applicants will have to submit an application for reregistration for the reassessment of the quality, efficacy, and safety of their products. In addition, the DCA may reject, cancel, or suspend the registration of any product if deemed necessary ( Newsletter of the Drug Control Authority Malaysia 2001).

The traditional belief that the traditional medicines, particularly the natural herbal remedies, are of natural origin and hence, inherently safe (Friedman 1996; Ernst 1999) and without any side effects is sometimes unfounded (Ramli 2001), because mercury poisoning through traditional Chinese medicines has been reported (Kang-Yum and Oransky 1992; Li et al. 2000).

In conclusion, heavy metal poisoning such as mercury has been repeatedly associated with traditional medicines. Given the increasing popularity of such treatments (MacLennan, Wilson, and Taylor 1996; Eisenberg et al. 1998; Ernst 2000a; Ernst and White 2000; Zaidi 2002; Saras 2003), it is important that doctors are aware of the risks associated with these products and finding ways to minimize them (Ernst 2000b), besides including questions pertaining to the use of these remedies during the routine taking of a patient’s history (Ernst 1999). This will definitely go far beyond the possibility of heavy metal contaminations, also covering areas like herbal toxicity and herbal-drug interactions (De Smet 1995; De Smet and D’ Arcy 1996; Ernst 1999; Ernst et al. 2001).