Heavy metals – know your limits

As analytical techniques get more sensitive, regulatory bodies are able to set lower drug impurity levels. Barbara Mason, European operations manager, Warwick Analytical Service, the analytical division of Exeter Analytical (UK), looks at the ever moving limits.

Heavy metals, toxic metals, trace metals, trace elements, minerals and micro-minerals are all terms that are used interchangeably to describe a group of elements, some of which are essential for biological activity, some of which are toxic at any level and all of which are toxic to some degree.

One definition of ‘heavy metals’ refers to the block of metals belonging to Groups 3 to 16 of the periodic table in periods 4 or greater. Another states that they are metallic elements with a high molecular weight and a density much greater (>5 times) than water. Other definitions rely on various chemical and toxicological properties. In short, there are many definitions for ‘heavy metals’, none of which is derived from an authoritative body.¹

The natural background levels of metals are generally controlled by the geological characteristics of their environment, with some being naturally of higher abundance, such as aluminium and iron, compared with mercury or silver. These rarer metals are known collectively as trace metals.²

Some 4% of the human body’s composition is mineral and there are a number of opinions as to how many are essential (cannot be synthesised by the body and must be obtained from food), although there are a number about which there is no debate. These are the macro elements where >100mg/day is required, and the trace elements of which much lower quantities are required.^3,4

Human activity over the millennia has redistributed many of these metals through the environment and many are commonly used in industrial processes, such as the manufacture of pharmaceutical, agrochemical and healthcare products, food preparation, processing and packaging and heavy industries, to name but a few. This has resulted in an increased likelihood of human exposure and environmental damage.

In 2002, the UK Drinking Water Inspectorate reported that tap water may contain contaminants, including toxic metals as well as pesticides, drugs and chemical waste from industry.⁵ According to the US Agency for Toxic Substances and Disease Registry,⁶ there are four heavy metals that are of particular concern: lead (Pb), cadmium (Cd), mercury (Hg) and inorganic arsenic (As); these are four of the top six hazards present in toxic waste sites. They are highly toxic even at very low concentrations, particularly as they are able to accumulate in the food chain. Being metals, these species are often present as cations, able to bind to negatively charged organic molecules and ultimately to be stored in human soft tissue such as the kidneys and hard tissue such as bone (see Table 1).⁷

Metal toxicity occurs in two overlapping categories:

Direct toxic effects causing tissue damage and disruption of metabolic processes

Heavy metals disrupt a vast array of metabolic processes – accumulation in coronary arteries causes a reduction in levels of nitric oxide, leading to impedance of blood flow and an increased risk of vascular blockage. Accumulation in the adrenal glands interferes with hormone production, resulting in early ageing, stress, decreased libido and aggravation of menopausal symptoms. Heavy metals can lead to an unresponsiveness to diabetic medication, an increase in the symptoms of neurological diseases, such as Alzheimer’s, Parkinson’s and multiple sclerosis, and in addition problems with skeletal development and maintenance, such as osteoporosis, as well as kidney and blood disorders.

Displacement or depletion of essential nutrients resulting in nutrient deficiency derived ill health

Heavy metals can bind preferentially and competitively to sites required by essential nutrients, reducing the capacity for their uptake and absorption and causing malnutrition of these elements.

Metal catalysts and reagents commonly used in the synthesis of food and pharmaceutical products can result in trace levels of metal residues or impurities in the final products. In an effort to limit the risks to health from exposure to these residues the European Medicines Agency produced a guideline on the specification limits for residues of metal catalysts or metal reagents, including guidelines on the permitted daily exposure (PDE) limits based on three classification categories depending on the extent of safety concerns of the metals and route of exposure (see Table 2).⁸

Stringent testing is clearly a pre-requisite for public health and historically this has been carried out using the Heavy Metals Limit Test, USP method – General Chapter <231>, a traditional wet chemistry colorimetric titration by comparison of the colour of a test solution of the metal sulphides against a lead standard solution.

While this method has been used successfully for more than a century it is limited by virtue of its qualitative, subjective endpoint and the limited range of metals that are sensitive to this method (lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper and molybdenum).

Advances in the sensitivity of analytical instrumentation, toxicological measurements and an improvement in prediction algorithms as well as a greater understanding of the modes of action and in vivo consequences of exposure to these metals led to a lowering of the maximum permissible exposure limits and consequently provoked much discussion in recent years in the search for a more robust, quantitative method of determining the concentrations of these metals.⁹

The USP has since revised the standards for metal impurities in pharmaceutical and food products and a new method has been introduced, replacing the General Chapter <231> with three new chapters:

• Elemental Impurities – Limits <232>
• Elemental Impurities – Procedures <2232>
• Elemental Contaminants in Dietary Supplements – (SGS Bulletin)

The methods use closed vessel microwave digestion of the samples and analysis by Inductively Coupled Plasma – Optical Emission Spectroscopy (ICP-OES) or Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) to deliver a quantitative endpoint for an additional 21 heavy metal impurities with part per billion (ppb) detection levels.¹⁰

With its capacity for higher temperatures and pressures, closed vessel microwave digestion methods are widely acknowledged as the sample preparation method of choice for a wider range of samples for metals analysis than would be possible using a traditional hot plate.

These methods allow real-time feedback of reaction parameters and customised programs to be developed for specific sample matrices requiring multi-stage programs and power ramping. Enclosure of the samples eliminates the risk of cross contamination or the loss of volatiles and high quality digestion vessels result in minimal blank contamination.

ICP-OES instrument with dual view capability

With judicious choice of digestion matrix, this method delivers a stable, acidified sample digestate. These high pressure systems also allow the sample matrix to decompose with a minimal quantity of acid and, with the associated increase in boiling points, digestion may be possible using a single acid rather than mixtures, ultimately reducing the dilution of analytes in the final solution as well as the risk of contamination or matrix interference particularly for axial ICP-OES.

Until relatively recently, the normal analytical zone (NAZ), or region of the plasma from which emission radiation is sampled, has been side-on with the plasma operating in a vertical direction, referred to as ‘radial viewing’.

In the 1990s an alternative method was introduced where the plasma was rotated into the horizontal position and viewed end-on, referred to as ‘axial viewing’. This method produced a longer path length leading to higher analyte emission, enabling an increase in sensitivity and an improvement in detection limits of 5- to 10-fold. However, axial viewing causes an increase in spectral and matrix-induced interferences.

Self absorption effects are also a potential problem resulting in a reduced linear dynamic range of the source although with the introduction of a shear gas self absorption problems have largely been overcome and spectral interferences can be reduced by selecting an alternate spectral line with less interference or applying an interference correction factor.¹¹

In the case of complex samples, as are typically analysed for heavy metals contamination, it may not be possible to overcome all the limitations associated with axial viewing and this method may be inappropriate. Modern ICP-OES spectrometers are typically ‘dual view’ instruments where the analyst is able to select the most appropriate orientation for viewing the plasma based on the behaviours of the sample.

Such instruments, in combination with the closed vessel microwave digestion procedures previously discussed, have served to improve significantly the range of metal impurities, their detection limits and analysis times.

Although for historical reasons some institutions prefer to continue to use the original USP General Chapter <231> methodology, contract analytical organisations, such as Exeter Analytical (UK)’s analytical division Warwick Analytical Service, are now routinely running the new USP method as described here for the determination of heavy metal contamination in a wide variety of sample media with much success.

references:

1. J.H. Duffus, Pure Appl. Chem., 2002 74(5) 793-807

2. Z.L. He, X. E. Yang, P.J. Stoffella; J Trace Elem Med Biol 2005 19(2-3) 125-40

3. www.healthtree.com/articles/supplements/trace-elements

4. Medline Plus (2007) Trace Elements

5. http://dwi.defra.gov.uk/about/annual-report/2002/index.htm

6. www.atsdr.cdc.gov/cercla/

7. www.lef.org/protocols/prtcl-156.shtml

8. European Medicines Agency (EMEA) Doc ref: EMEA/CHMP/SWP/4446/2000, Guideline on the specification limits for residual metal catalysts or metal reagents

9. D.R. Abernethy; Heavy Metals – USP Perspective IPC-USP 7th Annual Scientific Meeting Hyderabad, 2008

10. USP Elemental Impurities, Heavy Metals, Life Science Technical Bulletin, SGS, Issue No 28 September 2010

11. C.B. Boss, K.J. Fredeen, (2004) Concepts, Instrumentation and Techniques in Inductively Coupled Plasma Optical Emission Spectrometry

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