Chemical Quality Assurance

Module 4. Environmental contaminants in milk and milk products

Lesson 14
METHODS OF ESTIMATION OF PESTICIDES, ANTIBIOTICS AND HEAVY METALS

14.1 Introduction

Residues of antibiotics and pesticides are detrimental to health of the consumers as well as affect the quality of the dairy products. Presence of these contaminants in the milk and milk products is through the indiscriminate use of antibiotics in the health management of cattle and excessive use of pesticides in the fields. These contaminants not only affect the health and quality but also the export/ import potential of the dairy products. Similarly, heavy metals also have health hazards. In the present era of globalization the quality is the top most considered factor in running the business. Since, these contaminants are present in very small amounts, which are difficult to detect by the routine or old methods so sensitive methods are to be used. Thesis sensitive methods help in ensuring the presence or absence of these contaminants in the food stuffs. Some of the methods are as follows:

14.2 Methods for Pesticides

Isolation of organochloro pesticide residues (OCPR) in milk and milk products essentially is based upon adsorption clean-up (IDF standard 75 C: 27 method F, 1991). This involves mixing of test portion in presence of water with florisil until a homogenous powder is obtained, transfer of this mixture to florisil column, selective elution of pesticides and concentration of the eluate followed by HPLC analysis. The sample isolates are analysed by standardised HPLC conditions on octadecylsilyl (ODS) column, with solvent system methanol: water (80:20) at flow rate of 1.0 ml/min and at wavelength of 254 nm. Gas liquid chromatography (GLC) with electron capture detector (ECD) can also be used for the analysis of OCPR. Isolation of multiresidue of pesticides in milk is based upon solid phase extraction (SPE) over C18 cartridges. This involves blending of milk sample with acetonitrile followed by collection of supernatant over anhydrous sodium sulphate. The supernatant is concentrated to small volume, passed through SPE cartridges, eluant evaporated and again dissolved in known volume of acetonitrile followed by High pressure liquid chromatography (HPLC) analysis using binary gradient programming of acetonitrile and water solvent system. The sample isolates of multiresidue of pesticides are analysed by standardised HPLC conditions on ODS column, with solvent system acetonitrile: water (75:25) at flow rate of 0.5 ml/min and at wavelength of 200 nm. GLC with nitrogen phosphorus detector (NPD) can also be used for the analysis of organophosphates and carbamates.

Fig. 14.1 Florisil

Fig. 14.2 Florisil column filling

14.3 Methods for Antibiotics

14.3.1 Routine test methods for detection of antibiotic residues in milk

Rapid detection of antibiotic residues in milk is of immense importance to the dairy industry. Various rapid antibiotic detection methods have been commercialized in last two decades. Currently, seven types of detection methods are commonly used for detection of antibiotic residues in milk i.e. microbial growth inhibitor assay, microbial receptor assay, enzyme-colorimetric assay, receptor binding assay, spectrophotometric assay, chromatographic methods and immunoassay. These methods are qualitative, quantitative or semi-quantitative. However, they have one or more limitations in terms of precision, accuracy, sensitivity, cost and infrastructural requirement. Currently, microbial inhibitor & immuno-receptor tests have gained most popularity in the dairy industry at international level.

14.3.1.1 Reference method

The EU reference method for the determination of antibiotic residues in raw milk and in heat-treated milk is the International Dairy Federation microbial inhibition test. The IDF microbial inhibitor test uses B. stearothermophilus var. calidolactis, ATCC 10149 as the test organism due to its relatively high sensitivity to inhibitory substances. The IDF test procedure for detection of antibiotic residues in milk has been chosen as representative of similar procedures which in principle use B. stearothermophilus as the test organism. However, the IDF method is quite complex and lengthy to carry out as it involves the continual growth of large quantities of B. stearothermophilus spores. As the test involves a color change, which is dependent on the growth of B. stearothermophilus, if the organism fails to grow then a false negative result may occur. Therefore, due to the aforementioned technical difficulties in carrying out the reference method, microbial inhibitor test kit assays based on the IDF method, using B. stearothermophilus are the routine methods used for the determination of antibiotic residues in milk.

14.3.1.2 Microbial Inhibitor test

The 'traditional' tests for antibiotics in milk, known as 'microbial inhibitor' tests, involve incubating a susceptible organism in the presence of the milk sample. In the absence of an antibiotic, the organism grows and can be detected visually either by opacity of the agar growth medium or by a color change resulting from acid production. In the presence of an antibiotic, or any other inhibitor, the organism fails to grow and a zone of inhibition or lack of a color change is observed. Such tests are exceptionally sensitive to ß-lactam antibiotics. They are generally reliable and cost-effective but require incubation for several hours before the result can be visualized.

14.3.1.3 Commercially available microbial inhibitor test

Based on the microbial inhibitor test principle several commercially available kits are popularly used. The Delvotest (Gist-brocades BV, The Netherlands) is the best known microbial inhibitor test. The first version developed, in the 1970s, was the Delvotest P, designed to detect ß-lactams. The target organism, B. stearothermophilus, is encapsulated in an agar medium containing a pH indicator, a nutrient tablet and the substantial excretion of these residues into milk sample both being dispensed onto the agar surface. The 'ampoule version' is designed for individual tests or small-scale testing whilst a micro-tire plate version is designed for mass testing where 96 tests can be undertaken simultaneously. A negative result is indicated by a color change from purple to yellow, due to acid development during incubation at 64°C for 2½ hours. The Delvotest P has been used throughout the world and has sensitivity to penicillin G of 0.005 IU/ml. A more recent development, the Delvotest SP, is capable of detecting a wider spectrum of substances, notably sulphonamides, but also has increased sensitivity to tylosin, erythromycin, neomycin, gentamicin, trimethoprim and other antimicrobials. The Delvotest SP appears identical to the Delvotest P, the only difference being the need to incubate the Delvotest SP for 2¾ hours. The Delvotest SP is sold throughout the world and, universally, has sensitivity to penicillin G of 0.003-0.004 IU/ml.

The Delvotest was introduced into the UK in 1994 for testing individual animals as well as bulk tank milk and is identical to the ampoule version of the Delvotest P, differing only in its packaging. Although the Delvotest is by far the most widely used microbial inhibitor test, Charm Sciences Inc. (USA) has manufactured three similar tests. The Charm AIM-96 test is a micro-tire plate test, similar to the Delvo test and capable of detecting ß-lactams, sulphonamides, tetracycline, macrolides and amino glycosides in 96 samples simultaneously. Unlike the Delvotest, however, it employs a liquid medium instead of agar. The inoculated micro-tire plate is incubated on a heating block, programmed to provide a time-temperature profile suited to the batch of B. stearothermophilus spores being used; the incubation period is typically 3-4 hours, at the end of which a blue-yellow color change indicates that a sample is negative. The Charm Farm test is a 'test-tube' version of the AIM-96 test, designed for on-farm use and employs the same microbial inhibitor principle with a color change.

Fig. 14.3 Microbial inhibitor test

14.3.2 Rapid test kits for the detection of antibiotics in milk

Microbial inhibition tests are lengthy and test for a broad spectrum of antibiotics, whereas rapid test kits generally detect a specific family of antibiotics. To avoid delays at milk intake points, rapid antibiotic screening tests are often used on raw milk prior to completion of the Delvo^® SP test.

14.3.2.1 Immuno-receptor test

The desire for a more rapid and reliable result has promoted the development of tests that employ the 'immune receptor' test principle, which is a variation of the well-established enzyme-linked immunosorbent assay (ELISA). Essentially, a specific target antibiotic group is captured by immobilized antibodies, or by a broader-spectrum receptor such as a bacterial cell. Most tests involve a competitive principle in which antibiotic in the sample competes with an internal antibiotic standard for the immune receptor. The antibody-antibiotic complex is then usually linked to an enzyme that catalyses a color or fluorescence reaction and a comparison of the intensity of the 'test' reaction with that of a 'control' determines whether the sample is positive or negative. Because of their competitive principle, a low intensity usually means 'positive' whilst a high intensity is regarded as 'negative'. Immune receptor tests can be made quantitative but are generally used to provide a 'pass/fail' result. They are generally more expensive than microbial inhibitor tests but only detect substances that react immunologic ally with the immobilized receptor and they provide a result in less than 10 minutes.

14.3.2.2 Commercially available immuno-receptor test

The commercially available immune receptor tests employ several variations of capture mechanism and color reaction but most possess the common features of an immunological reaction coupled with a change in color (or fluorescence). There are, however, two exceptions. The Penzym test (UCB Byproducts, Belgium) employs the inhibition of an enzyme reaction (DD-carboxypeptidase’s activity), instead of an immune reaction, to detect the presence of a ß-lactam and it visualizes this by a color change. The test produces pink color when a sample contains no antibiotics while a yellow color is interpreted as positive. Conversely, the Charm II assay (Charm Sciences Inc., USA) employs an immune reaction to bind the antibiotic to a microbial receptor but detects this complex using a low-level 3H or 14C radio-label, instead of an enzyme reaction. The Charm II assay (Charm Sciences Inc., USA) is not a single test but a family of separate tests for specific groups of antibiotics, notably ß-lactams, sulphonamides, tetracycline, novobiocin, amino glycosides and macrolides, as well as various other substances such as chloramphenicol. The Charm II assay is an immune receptor test but is suitable for large laboratories only, requiring a range of laboratory equipment, including a centrifuge and sample mixers to prepare samples as well as a scintillation counter to detect the radio-label. Calibration curves need to be prepared for each group of antibiotics and a 'negative control' sample must be tested each day. The charm ΙΙ β-lactam test uses bacteria with specific receptor sites that bind all β-lactam drugs. The bacteria are added to a milk sample along with an exact amount of [14C] labeled penicillin G. Any β-lactam already in milk competes for the binding sites with the labeled penicillin G. The amount of [14C]-penicillin G that binds to the receptor sites is measured compared to a previously determined control point or to a standard curve. The greater the amount of [14C]-penicillin G measured, the lower the β-lactam concentration in the sample. The Charm MRL test (Charm Sciences, USA) is very similar to the Beta STAR test and detects penicillin and cephalosporin in 8 minutes. The test strip is placed in a heating block, the milk sample is added to an absorbent pad at one end and the test is incubated. Two lines appear on the dipstick, a sample being considered positive if the 'test' line is lighter than the 'control' line. The results can be read visually or using an image reader.

14.4 Methods for Heavy Metals

14.4.1 Atomic absorption spectrometery

For routine analysis of heavy metals the method of choice is the atomic absorption spectrometry (AAS) which makes use of aqueous digest of the sample. The main approaches in this technique are either the wet acidic digestion of milk and/or milk products or the dry ashing to yield the final inorganic extract, suitable for flameless determination in the AAS apparatus. Most preparation procedures lead to undesirable high backgrounds levels from chemicals and/or the digestive apparatus and may therefore lead to false results. For the determination of arsenic, selenium, and mercury, special AAS techniques with the thermal decomposition of arsenic gas, selenium hydride or the cold absorption of mercury vapors are frequently used and yield reliable results. Neutron activation analysis (NAA) is based on element specific γ radiation of irradiated elements, but is not suitable for routine as not all elements of interest can be analysed due to nuclear safety regulation. Other procedures are spectrophotometric methods, voltametric and isotop dilution mass spectrometry.

14.4.2 Inductively coupled plasma atomic emission spectroscopy (ICP-AES)

ICP-AES, also referred to as inductively coupled plasma optical emission spectrometry (ICP-OES), is an analytical technique used for the detection of trace metals. It is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element.

14.4.3 Inductively coupled plasma-mass spectrometry (ICP-MS)

ICPMS is a relatively new technique for the determination of trace elements in solution. It offers better sensitivity than graphite furnace AA with the multi-element speed. It is a type of mass spectrometry that is highly sensitive and capable of the determination of a range of metals and several non-metals at concentrations below one part in 1012. It is based on coupling together a high-temperature ICP (inductively coupled plasma) source with a mass spectrometer. The ICP source converts the atoms of the elements in the sample to ions. These ions are then separated and detected by the mass spectrometer. In a typical application, metals are placed in solution by acid digestion. The solution is sprayed into flowing argon and passed into a torch which is inductively heated to approximately 10,000^oC. At this temperature, the gas and almost everything in it is atomized and ionized, forming a plasma which provides a rich source of both excited and ionized atoms. In ICPMS, positive ions in the plasma are focused down a quadrapole mass spectrometer. By acquiring the mass spectrum of the plasma, data can be obtained for almost the entire periodic table in just minutes with detection limits vary from metal to metal and ranges from 0.1 – 100 ppb depending upon the type of elements. This method requires a very small amount of sample about 10 mg. This technique has the following advantages:

i. Detection limits for most elements equal to or better than those obtained by Graphite Furnace Atomic Absorption Spectroscopy (GFAAS)
ii. Higher throughput than GFAAS
iii. The ability to handle both simple and complex matrices with a minimum of matrix interferences due to the high-temperature of the ICP source
iv. Superior detection capability to ICP-AES with the same sample throughput
v. The ability to obtain isotopic information.