Determination of Heavy metals

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Determination of heavy metal and pesticide residue and acceptance criteria as per different guidelines. (


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Increasing industrialization has been accompanied throughout the world by the extraction and distribution of mineral substances from their natural deposits. As certain heavy metals such as Lead, Cadmium, Mercury, Arsenic have been recognized to be potentially toxic within specific limiting values, a considerable potential hazard exists for human. Those metals are described as "heavy metals" which, in their standard state, have a specific gravity (density) of more than about 5 g/cm3. Some of them, such as copper, nickel, chromium and iron , for example, are essential in very low concentrations for the survival of all forms of life. These are described as essential trace elements. There are 60 heavy metals. These also include the precious metals platinum, silver and gold.

Commonly found Heavy Metals: 

Commonly found Heavy Metals Aluminum (Al) Antimony (Sb) Arsenic (As) Beryllium (Be) Boron (B) Cadmium (Cd) Chromium (Cr) Copper (Cu) Indium (In) Iron (Fe) Lead (Pb) Lithium (Li) Magnesium (Mg) Manganese (Mn) Molybdenum (Mo) Nickel (Ni) Osmium (Os) Palladium (Pd) Platinum (Pt) Rhodium (Rh) Rubidium (Rb) Selenium (Se) Strontium (Sr) Thallium (Tl) Tin (Sn) Tungsten (W) Zinc (Zn)

Sources of Heavy Metals in Drug Products: 

Sources of Heavy Metals in Drug Products API – Raw materials – Starting materials – Catalysts and metal reagents – Processing equipment Excipients – Starting materials from environmental/natural sources – Catalysts and metal reagents – Processing equipment Drug Product – Ingredients – Processing Equipment – Container closure

Heavy Metal Content of Ayurvedic Herbal Medicine Products: 

Heavy Metal Content of Ayurvedic Herbal Medicine Products 20 % of 70 Ayurvedic herbal medicinal products contained potentially harmful levels of toxic heavy metals (American Medical Association 2004) Ayurvedic medicines theory attributes an important therapeutic role to metals such as mercury, arsenic and lead 35 to 40 % of the medicines in the Ayurvedic formulary intentionally contain at least one metal Metal Range (mg/kg) Mean (mg/kg) USP Limit (mg/kg) ECP/EP Limit (mg/kg) Lead 5-37,000 40 1 3/5 Mercury 28 – 104,000 20,225 1.5 0.1/0.1 Arsenic 37 -8,130 430 1.5 -/-

Lead: Origin and Toxic Effects: 

Lead: Origin and Toxic Effects Lead has been mined since ancient times and has been processed in many ways, e.g. for water pipes, containers and, as acetate, even for sweetening wine ("lead sugar"). Lead can trigger both acute and chronic symptoms of poisoning. Lead is a typical cumulative poison. Such an intoxication leads to: Encephalopathies in the central nervous system (CNS); Disturbances in kidney and liver functions progressing as far as necrosis; Damage to the reproductive organs; Anemia's and many metabolic deficiency symptoms.

Cadmium: Origin and Toxic Effects: 

Cadmium: Origin and Toxic Effects It is spread by air and water (sewage sludge) far over sea and land, but especially in the vicinity of heavy industrial plants. It is absorbed by many plants and sea creatures and, because of its toxicity, presents a major problem for foodstuffs. Contamination through fertilizers becomes an increasing problem. Cadmium is concentrated particularly in the kidneys, the liver, the blood forming organs and the lungs. It most frequently results in kidney damage (necrotic protein precipitation) and metabolic anomalies caused by enzyme inhibitions. In humans, long-term exposure cause : renal dysfunction lung disease lung cancer bone defects (osteomalacia,osteoporosis) increased blood pressure

Mercury: Origin and Toxic Effects: 

Mercury: Origin and Toxic Effects Slurry is one of the main sources of mercury, which is not only toxic in compound form but also as a metal. The relative high vapour pressure of metallic mercury makes it hazardous. Organic mercury compounds are particularly toxic and include methylmercury, which is formed by the action of anaerobic organisms, and is biomagnified in aquatic food chains through fish and other aquatic species into the human organism. Inorganic mercury poisoning is associated with: Tremors Gingivitis minor psychological changes spontaneous abortion

Arsenic: Origin and Toxic Effects: 

Arsenic: Origin and Toxic Effects Plants absorb arsenic fairly easily, so that high-ranking concentrations may be present in food. The concentrations of the dangerous inorganic arsenics that are currently present in surface waters enhance the chances of alteration of genetic materials of fish. This is mainly caused by accumulation of arsenic in the bodies of plant-eating freshwater organisms. Birds eat the fish that already contain eminent amounts of arsenic and will die as a result of arsenic poisoning as the fish is decomposed in their bodies.

Chromium: Origin and Toxic Effects: 

Chromium: Origin and Toxic Effects Chromium is used in metal alloys and pigments for paints, cement, paper, rubber, and other materials. Low-level exposure effects : irritate the skin cause ulceration Long-term exposure effects : kidney and liver damage damage to circulatory and nerve tissue

Copper: Origin and Toxic Effects: 

Copper: Origin and Toxic Effects Copper is an essential substance to human life, but in high doses it can cause: Anemia liver and kidney damage stomach and intestinal irritation. People with Wilson's disease are at greater risk for health effects from overexposure to copper.

Techniques for Determination of Heavy Metals: 

Techniques for Determination of Heavy Metals In many pharmacopeias, color reactions with special reagents such as thioacetamide or diethyldithiocarbamate are given. Instrumental analyses have to be employed when the metals are present in trace quantities, in admixture, or when the analyses have to be quantitative. The main methods commonly used are atomic absorption spectrophotometry (AAS), inductively coupled plasma (ICP) and neutron activation analysis (NAA). Atomic absorption (flame, graphite furnace, cold vapor) ICP-MS XRF Ion Chromatography Flame Emission Spectroscopy

Limit Test for Arsenic: 

Limit Test for Arsenic A popular test method relies on the digestion of the plant matrix/food followed by subjection of the digestate to a comparative colorimetric test in a special apparatus. The method uses N-N-diethylmethyldithiocarbamate in pyridine and it reacts with hydrogen arsenide to afford a red–purple complex. The limit is expressed in terms of arsenic (III) trioxide (As 2 O 3 ). Figure 1: Apparatus for arsenic limit test A, generator bottle (capacity up to the shoulder: approximately 70 ml) B, exit tube C, glass tube (inside diameter: 5.6 mm, the tip of the part to be inserted in the absorber tube D is drawn out to 1 mm in diameter) D, absorber tube (inside diameter: 10 mm) E, small perforation F, glass wool (about 0.2 g) G, mark at 5 ml H and J, rubber stopper

Figure 1: 

Figure 1


…contd. Method Weigh the amount of the sample as directed in the monograph, and place it in a crucible of platinum, quartz or porcelain. Add 10 ml of a solution of magnesium nitrate hexahydrate in ethanol (95) (1 in 10), burn the ethanol, heat gradually, and ignite to incinerate. If carbonized material still remains after this procedure, moisten with a small quantity of nitric acid, and ignite again to incinerate in the same manner. After cooling, add 3 ml of hydrochloric acid, heat in a water bath to dissolve the residue, and designate it as the test solution. Place the test solution in the generator bottle A and, if necessary, wash down the solution in the bottle with a small quantity of water.


…contd. Add 1 drop of methyl orange TS , and after neutralizing with ammonia TS , ammonia solution (NH4OH), or diluted hydrochloric acid, add 5 ml of diluted hydrochloric acid (1 in 2) add 5 ml of potassium iodide TS , and allow to stand for 2 to 3 minutes. Add 5 ml of acidic tin (II) chloride TS , and allow to stand for 10 minutes. Then add water to make 40 ml, add 2 g of zinc for arsenic analysis, and immediately connect the rubber stopper H fitted with B and C with the generator bottle A. Transfer 5 ml of the absorbing solution for hydrogen arsenide to the absorber tube D, insert the tip of C to the bottom of the absorber tube D, then immerse the generator bottle A up to the shoulder in water maintained at 25 °C, and allow to stand for 1 hour. Disconnect the absorber tube, add pyridine to make 5 ml, if necessary, and observe the color of the absorbing solution: the color produced is not more intense than the standard color.

Limit test for cadmium and lead: 

Limit test for cadmium and lead Preparation of the sample For the wet digestion method in an open system, place 200–250 mg of air-dried medicinal plant material, accurately weighed, finely cut and homogeneously mixed, into a cleaned silica crucible. Add 1.0 ml of the digestion mixture, cover the crucible without exerting pressure and place it in an oven with a controlled temperature and time regulator (computer-controlled, if available). Heat slowly to 100 °C and maintain at this temperature for up to 3 hours; then heat to 120 °C and maintain at this temperature for 2 hours. Raise the temperature very slowly to 240 °C, avoiding losses due to possible violent reactions, especially in the temperature range of 160–200 °C, and maintain at this temperature for 4 hours. Dissolve the remaining dry inorganic residue in 2.5 ml of nitric acid (~1000 g/l) TS and use for the determination of heavy metals. Every sample should be tested in parallel with a blank. Method The contents of lead and cadmium may be determined by inverse voltammetry or by atomic absorption spectrophotometry.

Limit test for total toxic metals as lead: 

Limit test for total toxic metals as lead Preparation of sample solution and blank solution Test solution : Place an amount of the sample, as directed in the monograph, in a quartz or porcelain crucible, cover loosely with a lid, and carbonize by gentle ignition. After cooling, add 2 ml of nitric acid and 5 drops of sulfuric acid, heat cautiously until white fumes are evolved, and incinerate by ignition between 500 °C and 600 °C. Cool, add 2 ml of hydrochloric acid, evaporate to dryness in a water-bath, moisten the residue with 3 drops of hydrochloric acid, add 10 ml of hot water, and warm for 2 minutes. Then add 1 drop of phenolphthalein TS , add ammonia TS drop by drop until the solution develops a pale red color, add 2 ml of dilute acetic acid, filter, if necessary, and wash with 10 ml of water. Transfer the filtrate and washings to a Nessler tube, and add water to make 50 ml. Designate this as the test solution.


…contd. Control solution Evaporate a mixture of 2 ml of nitric acid, 5 drops of sulfuric acid, and 2 ml of hydrochloric acid on a water-bath, further evaporate to dryness on a sand-bath, and moisten the residue with 3 drops of hydrochloric acid. Hereinafter, proceed as directed above for the test solution, and then add the volume of standard lead solution as directed in the monograph and sufficient water to make 50 ml. Procedure Add 1 drop of sodium sulfide TS both to the test solution and to the control solution, mix thoroughly, and allow to stand for 5 minutes. Then compare the colors of the two solutions by viewing the tubes downwards or transversely against a white background. The test solution has no more color than the control solution.

Detection of cadmium, copper, iron, lead, nickel and zinc: 

Detection of cadmium, copper, iron, lead, nickel and zinc Measure the content of (Cd), copper (Cu), iron (Fe), lead (Pb), nickel (Ni) and zinc (Zn) by the standard additions method using reference solutions of each heavy metal. Suitable instrumental parameters are listed in Table. The absorbance value of the compensation liquid ( blank solution ) is subtracted from the value obtained with the test solution.


…contd. Table: Instrumental parameters for heavy metals Cd Cu Fe Ni Pb Zn Wavelength nm 228.8 324.8 248.3 232 283.5 213.9 Slit width nm 0.5 0.5 0.2 0.2 0.5 0.5 Hollow-cathode lamp current mA 6 7 5 10 5 7 Ignition temperature °C 800 800 800 800 800 800 Atomization temperature °C 1800 2300 2300 2500 2200 2000 Background corrector on on on on on on Nitrogen flow Litre/min 3 3 3 3 3 3

Detection of arsenic and mercury: 

Detection of arsenic and mercury Measure the content of arsenic (As) and mercury (Hg) in comparison with reference solutions containing these elements at a known concentration by direct calibration using an automated continuous-flow hydride vapour generation system. The absorbance value of the compensation liquid (blank solution) is automatically subtracted from the value obtained with the test solution. Arsenic Sample solution: To 19 ml of the test solution or of the blank solution as described above, add 1 ml of a 200 g/l solution of potassium iodide R. Allow the test solution to stand at room temperature for about 50 min or at 70 °C for about 4 min. Acid reagent: Heavy metal-free hydrochloric acid R. Reducing reagent: A 6 g/l solution of sodium tetrahydroborate R in a 5 g/l solution of sodium hydroxide R. The instrumental parameters in Table A3.2 may be used. Mercury Sample solution: Test solution or blank solution, as described above. Acid reagent: A 515 g/l solution of heavy metal-free hydrochloric acid R. Reducing reagent: A 10 g/l solution of stannous chloride R or sodium tetrahydroborate in dilute hydrochloric acid R. The instrumental parameters in Table A3.2 may be used.


…contd . Table A3.2. Instrumental parameters for determination of arsenic and mercury As Hg Wavelength nm 193.7 253.7 Slit width nm 0.2 0.5 Hollow-cathode lamp current mA 10 4 Acid reagent flow rate ml/min 1.0 1.0 Reducing reagent flow rate ml/min 1.0 1.0 Sample solution flow rate ml/min 7.0 7.0 Absorption cell Quartz (heated) Quartz (unheated) Background corrector on on Nitrogen flow rate litre/min 0.1 0.1 Heating 800 °C 100 °C

Guidelines for Heavy Metals: 

Guidelines for Heavy Metals (European Committee) Regulation 466/2001 = Establishes Maximum levels for certain contaminants in foodstuff: Heavy metals (Pb, Hg, Cd) , other contaminants

Contaminants in medicinal herbs: Heavy metals: 

Contaminants in medicinal herbs: Heavy metals Limits currently applied in Germany Lead max. 5.0 ppm Cadmium max. 0.2 ppm Mercury max. 0.1 ppm Special regulations: –Flax seeds, hawthorn and yarrow (max. 0.3 ppm Cd) –Birch leaves, St. John’s wort, willow bark and holly leaves (max. 0.5 ppm Cd)

Element limits for oral materials (USP) : 

Element limits for oral materials (USP) Element Oral Permitted Daily Exposure for Dosage Forms, μg/day USP Oral Limit, μg/g Arsenic (As) 15 1.5 Cadmium (Cd) 25 2.5 Copper (Cu) 500 50 Lead (Pb) 10 1 Mercury (Hg) 15 1.5 Zinc (Zn) 15,000 1500

EMEA Guidelines: 

EMEA Guidelines For the purpose of this guideline, permitted daily exposure (PDE) has been employed as the key indicator of the maximum safe intake limit for individual elements. PDE is defined as the maximum patient exposure (expressed in mcg/kg/day) to an element, possibly on a chronic basis, that is unlikely to produce any adverse health effects. Oral PDEs are derived as recommended in the monograph. Parenteral PDEs (apart from that of Pt) are obtained by multiplying the oral PDE by the estimated oral bioavailability of the particular element ranging from 5 to 50%.

New Zealand Limits: 

New Zealand Limits Element New Zealand Food Standard (mg/kg) Cadmium, in any food other than shellfish 1.0 Copper, all beverages 2.0 Lead, all beverages 0.2


References and+pesticide+residue+in+herbal+products&hl=en&ct=clnk&cd=9&gl=in

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