radiochemical methods of analysis,Gajanan naik

Slide 1:

RADIOCHEMICAL METHODS OF CHEMICAL ANALYSIS AND QUALITY CONTROL OF RADIO PHARMACEUTICALS 1 GAJANAN M.NAIK FIRST YEAR M.PHARM Q.A

Slide 2:

All atoms nuclei, except hydrogen, is made up of a collection of protons and neutrons. The chemical properties of an atom are determined by its atomic number, Z , the number of protons. The sum of neutrons and protons is the mass number, A . The nuclei of isotopes of an element contain the same number of protons, but have different numbers of neutrons. Radioactive isotopes (radionuclide's), undergo spontaneous disintegration, which ultimately leads to stable isotopes. Radioactive decay of isotopes occurs with the emission of electromagnetic radiation in the form of x-rays or gamma ray. Accompanying this emission is the formation of electrons, positrons and the helium nucleus. 2

Slide 3:

Some of the most chemically important types of radiation from radioactive decay are listed in the table. Four of these types - alpha particles, beta particles, gamma-ray photons and X-ray photons can be detected and counted. 3 Product Symbol Charge Mass Number Alpha particle  +2 4 Beta particles Negatron  - -1 1/1840 (~0) Positron  + +1 1/1840 (~0) Gamma ray  0 X-Ray  0 0 Neutron n 0 1 Neutrino v 0 0

Slide 4:

Alpha Decay: Alpha decay is a common radioactive process encountered with heavier isotopes. The alpha particle is a helium nucleus having a mass of 4 and a charge of +2. An example of alpha decay is shown. A Z  + A-4 (Z- 2) + 4 2 He2+ EXAMPLE:238 U( α ) 234 Th 4

Slide 5:

Beta Decay: Beta decay is a radioactive process in which, the atomic number changes but the mass number stays the same. Three types of  decay are encountered: negatron formation, positron formation and electron capture. Example of these three process are: negatron formation positron formation electron capture Negatrons (  - ) are electrons that form when one of the neutrons in the nucleus is converted to a proton. A positron (  + ), with the mass of the electrons, forms when the proton in the nucleus is converted to neutron. 5

Slide 6:

Gamma-Ray Emission: Gamma rays are produced by nuclear relaxations. Gamma-ray emission is the result of a nucleus in an excited state returning to the ground state in one or more quantized steps with the release of monoenergetic gamma rays. Generally, the lifetime of the excited states is very small, of the order of 10 -16 TO 10 -13 seconds. The γ radiation is emitted immediately after a preceding α or β decay. The gamma-ray emission spectrum is characteristic for each nucleus and is thus useful for identifying radioisotopes. Gamma radiation is highly penetrating 6

Slide 7:

RADIOACTIVITY DETECTORS Gas-filled Detectors Knowledge of the properties of nuclear radiation is needed for the measurement and identification of radionuclide's and in the field of radiation protection. The most important aspect is the interaction of radiation with matter. Charged high-energy particles or photons, such as protons, electrons, positrons, or X-ray photons, set off ionization processes in gases (positive ions and free electrons) . If an electric field is applied, the ions and the electrons move in opposite directions. The motion of the charged particles gives rise to a current that can be measured in an external circuit. 7

Slide 8:

a:Ionization Chambers Ionization chambers are constructed in various ways, for instance as grid ion chambers, guard ring chambers, current ion chambers or integrating ion chambers. Usually, the electrodes are parallel plates and enclosed in a gas-tight chamber, filled with air or a noble gas. Because of the low pulse heights to be measured,low voltage range applied;upto 200 volts, good electrical isolation of the electrodes is of great importance. Ionization chambers are used to measure α emitters . They are applied for calibration of radioactive sources, radiation monitoring and dosimetry in radiation protection. 8

Slide 9:

b:Proportional Counters Proportional counters may consist of a sealed cylinder serving as cathode, a thin wire as anode and a thin window, but they are often constructed as flow counters. In this type of counter ,a gas, preferably methane or a mixture of argon and methane, flows through the counter during operation and the sample is brought into the counter. The operational voltage depends on the nature and the pressure of the gas and varies between about 2 and 4 kV . Proportional counters are well suited to measure α and low-energy β radiation. As proportional counters have dead times of only several μs, high counting rates can be measured without losses. Because the internal counting efficiency of proportional counters for γ radiation is low (about 1%), they are not suited to measure γ radiation. However, proportional counters of special design and operating at high gas pressure are applied to X-ray and low energy γ ray spectrometry. 9

Slide 10:

c:Geiger-Muller Counters Geiger-Muller counters are operated at relatively high voltages of several kV. The avalanche-like spreading of the ionization processes leads to the production of a great number of positive ions and electrons The neutralization of the electrons at the anode wire gives rise to the emission of photons, which react with the gas by emission of photoelectrons. These trigger further avalanches and the processes continue until the build-up of the positive ion sheath in the vicinity of the anode wire ,reduces the electrical field strength so far that no more events can be counted. The advantages of Geiger-Muller counters are their simplicity and the fact that further amplification is not needed. 10

Slide 11:

Most Geiger-Muller counters are equipped with windows and are therefore inexpedient for measuring α and low-energy β radiation. Energy discrimination is not possible. Gamma radiation can be counted with a low internal counting efficiency of about 1%. 11 counting efficiency is equal to CPM/DPM, the ratio of Counts per Minute (CPM) to Disintegrations per Minute (DPM) expressed as a percentage.

Slide 12:

SCINTILLATION COUNTERS SOLID SCINTILLATION COUNTERS In the transparent crystal or liquid the radiation is absorbed and photons are emitted . At the photocathode of the photomultiplier tube the photons release electrons which are multiplied by the dynodes of the multiplier to give pulses of several mV. Scintillation counters are applied primarily for measuring gamma radiation and low energy beta radiation. If gamma radiation is to be measured, thick scintillating crystals of high density are used in order to absorb as much gamma radiation as possible. NaI or CsI crystals doped (activated) by addition of small quantities of TI are well suited and at a size of 1 to 2 inches (2.5 to 5cm) they give an internal counting efficiency of 15to 30%. 12

Slide 13:

Inorganic crystals NaI ( Tl ) crystals CsI (T1) crystals ZnS (Ag) Inorganic crystals have the advantage of high density, so that energetic particles can be brought to rest in a fairly small crystal. 13 Organic crystals Anthracene trans- Stilbene p- Terphenyl

Slide 14:

LIQUID SCINTILLATION COUNTERS For counting low-energy beta radiation, the crystal is substituted by a scintillating liquid , and the sample is dissolved in the liquid (internal-source liquid scintillation counting). The method is also used to measure weak X-ray and γ ray emitters. The scintillating liquid is prepared by dissolving a primary scintillator (e.g.2,5diphenyloxazole) and, if necessary, a secondary scintillator (e.g. p- bis [2-(5-phenyloxazolyl)] in a suitable solvent mainly organic compounds are used, e.g. toluene, benzene, p- xylene or dioxane . and adding a solution containing the radioactive sample. Under these conditions, self-absorption of the radiation in the sample, absorption of the radiation in the air and the window of the detector, and back scattering of beta particles are avoided. The main advantage of liquid scintillation counting is the relatively high counting efficiency, which can amount to about 90 to 100% 14

Slide 15:

15

Slide 16:

RADIOCHEMICAL ANALYSIS METHODS 16

Slide 17:

NEUTRON ACTIVATION ANALYSIS In typical NAA, stable nuclides ( A Z, the target nucleus) sample undergo neutron capture reactions in a flux of (incident) neutrons . The radioactive nuclides ( A+1 Z, the compound nucleus) produced in this activation process usually decay by emission of a beta particle (ß - ) and gamma ray(s) with a unique half-life. A high-resolution gamma-ray spectrometer is used to detect these ‘delayed’ gamma rays in the presence of the artificially induced radioactivity in the sample for both qualitative and quantitative analysis . 17

Slide 18:

The incident neutron hits the target nucleus, which captures the neutron and is converted into a new compound nucleus. The latter immediately emits radiation called prompt gamma radiation and forms the radionuclide, which then kicks out a beta particle and emits the delayed gamma radiation (since it is emitted after some time delay), forming the product nucleus. The energies of the delayed gamma rays are used to identify component sample elements. The count of gamma rays of a specific energy indicates the amount of an element in the sample. For example, when a silver sample is irradiated, A fraction of the 109 Ag atoms in the sample will capture a neutron and become 110 Ag. The radioactive 110 Ag atoms have a half-life of 24.6 seconds. Beta decay of 110 Ag atoms to 110 Cd occurs with emission of a 658 keV gamma ray. The amount of silver in the original sample can be determined by measuring the count of 658 keV gamma-rays emitted from the sample in a given time interval after irradiating the sample. 18

Slide 19:

The measured count rate ( R) of the gamma rays from the decay of a specific isotope ( 110 Ag) in the irradiated sample can be related to the amount (n) of the original, stable isotope ( 109 Ag) in the sample through the following equation R = ε I γ R = measured gamma-ray count rate (cps) ε = absolute detector efficiency I γ = absolute gamma-ray abundance 19

Slide 20:

In most cases, however, a standard is irradiated and counted under similar conditions as the sample, and the mass of the element in the sample ( W sam ) is found by comparing the measured count rates (R) for the sample and standard through the following equation W sam = mass of element in sample (g) W std = mass of element in standard (g) R sam = sample gamma-ray count rate (cps) R std = standard gamma-ray count rate (cps) 20

Slide 21:

ISOTOPE DILUTION ANALYSIS There are two general types of isotope dilution methods. These are (a) direct, (b) reverse, These methods are based on the same fundamental principles but differ in technique and procedure Direct Isotope Dilution – Determination of an Inactive Compound by Dilution with an radioactive Compound. used to determine the quantity of a nonradioactive or untagged constituent in a mixture of closely related compounds which are difficult to separate quantitatively by conventional methods . 21

Slide 22:

The technique consists of: . Step 1 Addition of a "spike" consisting of a known amount of isotopically labeled compound with a known specific activity S' to the unknown mixture containing the same compound made up of stable isotopes. The two are thoroughly mixed.to obtain a uniform distribution Step 2. Suitable treatment of the mixture to isolate the same compound in pure form. It is essential that the isolated compound be pure, but it is not necessary that all of the compound be recovered from the mixture. Thus, it is possible to avoid tedious processes required for quantitative separations and frequently to carry out analysis otherwise impractical. Step 3.Determination of the isotope content of the isolated portion by measurement of its specific activity S. The ratio of active and inactive molecules depends on the relative masses of (active) substance added m' and (inactive) substance originally present m . 22

Slide 23:

If R' = activity(cps) in spike used for assay m' = mass of spike used for assay m = mass of inactive substance in unknown used in assay then the specific activity of the spike S' is given by S' = R'/m' (1) and the specific activity of the substance mixture isolated in pure form from the mixture is S = R'/(m' + m) (2) It is desired to know the mass m of substance in the unknown in terms of easily measurable quantities. This may be accomplished by dividing equation(1) by equation (2) and solving for m. Thus, 23

Slide 24:

Reverse isotopic dilution analysis Reverse isotopic dilution analysis involves estimation of radionuclide by dilution with an inactive nuclide (inactive compound),so called reverse isotope dilution. This application is very valuable if the radionuclide is present in carrier-free form. Again, quantitative separation is avoided; 24

Slide 25:

Radiometric analysis Radiometric analysis is also based on the use of radiotracers. However, in contrast to isotope dilution analysis, stoichiometric relations are applied in radiometric methods. The substance to be determined is brought into contact with another substance labelled with a radionuclide or containing a radionuclide. Reaction between these two substances yields a radioactive product that either can be separated and measured or can be measured continuously in the course of the reaction. The activity is proportional to the amount of substance to be determined 25

Slide 26:

The following applications are distinguished - radio reagent methods; - radio release methods; - isotope exchange methods; - radiometric titration 26

Slide 27:

Radio reagent methods; the substance to be determined is reacted with a radioactive reagent the radioactive product of the reaction between the substance to be determined and a radioactive reagent is separated by various methods, such as precipitation or liquid-liquid extraction. Cl -, Br- or I- in concentrations down to 0.5 pg/l can be determined by addition of an excess of phenyl mercury nitrate labelled with 203 Hg. The complexes formed with the halide ions are extracted into benzene, whereas the phenyl mercury nitrate stays in the aqueous phase. From the difference between the activities in the aqueous phase before and after the reaction, the amount of halide ions is calculated. 27

Slide 28:

Radio release methods The substance to be determined is brought into contact with another substance containing a radionuclide reagent . by their interaction a certain amount of the radionuclide is released and measured. By reaction with oxygen 85 Kr is released and can be measured continuously. Oxygen dissolved in water can be measured by reaction with 204 Tl deposited on Cu; 204 Tl is oxidized and released into the water where it can be measured. 28

Slide 29:

Isotope exchange method The isotope exchange method is based on the exchange between two different forms or compounds of the element M to be determined : The labelled species M*Y is added and, after equilibration, the specific activity is the same in (1) and (2): After separation of compounds (1) and (2), their activity is measured and the mass can be calculated using equation An advantage of isotope exchange methods is that in special cases individual chemical forms (species) can be determined with high sensitivity. 29

Slide 30:

Radiometric titration In radiometric titration, the radioactivity of one component or in one phase is recorded as a function of addition of titrant. The compound formed is separated by precipitation, extraction or ion exchange in the course of the titration, and the end point is determined from the change in the activity in the residual solution. Radiometric titration may be applied in different ways: Inactive test solution and active titrant (activity in the solution is low at the beginning and begins to rise at the endpoint); active test solution and inactive titrant (activity in the solution decreases continuously, until the endpoint is reached); Both the test solution and the titrant active (activity in the solution decreases until the endpoint is reached and then increases again). 30

Slide 31:

RADIOIMMUNOASSAY Radioimmunoassay ( RIA ) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity. The Technique A mixture is prepared of radioactive antigen(tracer) radioactive isotopes 125 I or 131 I or3H (beta)are often used. antibodies against that antigen. Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies At increasing concentrations of unlabeled antigen added , an increasing amount of radioactive antigen is displaced from the antibody molecules. 31

Slide 32:

The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured. From these data, a standard binding curve, can be drawn. After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve. 32

Slide 33:

Separating Bound from Free Antigen Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first. For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by adding an antirabbit-IgG antiserum. The antigen-specific antibodies can be coupled to the inner walls of a test tube . After incubation, the contents ("free") are removed; the tube is washed ("bound"), and the radioactivity of both is measured The antigen-specific antibodies can be coupled to particles, like Sephadex. Centrifugation of the reaction mixture separates the bound counts (in the pellet) from the free counts in the supernatant fluid. Radioimmunoassay is widely-used because of its great sensitivity Using antibodies of high affinity (K 0 = 10 8 –10 11 M −1 ), it is possible to detect a few picograms (10 −12 g) of antigen in the tube. 33

Slide 34:

34

Slide 35:

QUALITY CONTROL OF RADIOPHARMACEUTICALS Since radiopharmaceuticals are intended for human administration, it is imperative that they undergo strict quality control measures. The quality control of radiopharmaceutical include the purity , potency , product identity, biologic safety, efficacy of the radiopharmaceuticals. QC INCLUDES PHYSICOCHEMICAL TESTS BIOLOGICAL TESTS 35

Slide 36:

Physicochemical Tests Physical Characteristics Recognize the colour and state of a radiopharmaceutical. Colloidal or aggregated preparations size identify Tc-sulfur colloid : 80 to 500 nm Tc-labeled albumin microshperes : (checked with haemocytometer under a light microscope) larger than 150µm - pulmonary arterial blockade embolism smaller than 10µm - localize in the reticuloendothelial system 36

Slide 37:

pH and Ionic strength 1.ideal pH of radiopharmaceutical should be 7.4 ( pH of the blood ) 2.However, radiopharmaceutical can vary between 2 and 9 because of high buffer capacity of blood. 3.Correct ionic strength can be achieved by adding a proper acid, alkali, or electrolyte and can be calculated from the concentrations of added electrolytes. 37

Slide 38:

Radionuclidic Purity RADIONUCLIDIC PURITY : It is defined as the fraction of total radioactivity in the form of the desired radionuclide present in a radiopharmaceutical. impurity sources 1. from extraneous nuclear reactions 2.fission heavy elements in reactor Radiopharmaceutical purity is determined by measuring the half-lives and characteristic radiations emitted by individual radionuclides. γ-ray emission radionuclides : checked with multi-channel analyzer pure β emission radionuclides checked with β-spectrometer or a liquid scintillation counter . 38

Slide 39:

Radiochemical Purity ; radioactivity in the desired chemical 1. RCP % = -------------------------------------------------------- x 100% Radioactivity of total radiopharmaceutical 2.Radiochemical impurities arise from decomposition due to (1)action of solvent (2)change in temperature or pH (3)light (4)presence of oxidizing or reducing agents (5)radiolysis Decomposition of labelled compound by radiolysis depends on the specific activity of the radioactive material the energy of the emitted radiation half-life of the radionuclide H 2 O 2 and H 2 O(decomposition of water) 39

Slide 40:

Methods used to detect the radiochemical impurities (1)precipitation (2)paper, thin-layer, and gel chromatography (3)gel electrophoresis (4)ion exchange (5)solvent extraction (6)high performance liquid chromatography (7)Distillation 40

Slide 41:

Biological Tests To examine the sterility, pyrogenicity , toxicity of radiopharmaceuticals. It is possible for a particular radiopharmaceutical solution to be sterile but still be highly pyrogenic when injected into patients. Sterility Sterility indicates the absence of any viable bacteria or microorganisms in a radiopharmaceutical. 41

Slide 42:

Methods of sterilization Autoclaving 1. heating in steam at 121 o C under 18 pounds per square inch (psi) for 15 to 20 min. 2. suitable for thermostable aqueous solution. 3. not suitable for short-lived radionuclides . Membrane Filtration 1.sieving mechanism 2.Commercially available Millipore filters are membrane filters made of cellulose esters Pore size : 0.45 and 0.22 μ m. 3. The most common method of sterilization in nuclear pharmacy. Used for : (1) short-lived radionuclides (2) heat-labile radiopharmaceuticals 42

Slide 43:

Sterility Testing 1. To prove that radiopharmaceuticals are free of viable microorganisms. 2. Incubate the radiopharmaceutical sample in (1) fluid thioglycollate medium at 30 to 35 o C for 7 to 14 days; or in (2) soyabean -casein digest medium for incubation at 20 to 25 o C for 7 to 14 days. 43

Slide 44:

APYROGENICITY It indicates the absence of pyrogens in a  radiopharmaceutical. 1. Pyrogens are either polysaccarides or proteins produced by the metabolism of microorganisms. 2. Bacterial products, endotoxins are the prime example of pyrogens. 3. various chemicals also can add pyrogens to a radiopharmaceutical solution. 44

Slide 45:

Pyrogenicity Testing USP Rabbit Test 3 normal rabbits ( wt > 1.5 kg ) lived in uniform temperature inject the test volume as equivalent to human dose via the ear vain measured rectal temperatures at 1, 2, 3 hr after injection the rise in temperature in individual animals is less than 0.6 0 C or the sum of the temperature rises in all three animals does not exceed4 0 CThe sample is considered apyrogenic. LAL Test A more rapid method, called the limulus amebocyte lysate (LAL) test. is empolyed for the detection of endotoxin-type pyrogens. lysate of amebocytes from the blood of the horsehoe crab, Limulus polyphemus . principle of the test :pyrogenic sample + 0.1 ml LAL [ at 37 0 C for 15 to 60 min (pH 6-8)] formation of an opaque gel 45

Slide 46:

TOXICITY TESTING 1. Toxic effects due to radiopharmaceutical administration include, alterations in the histology or physiologic functions of different organs in the body or even death. 2. The LD 50/30 , describes the toxic effect of a radiopharmaceuticals it is the dose required to produce 50% mortality in 30 days in any species after administration of the radiopharmaceutical. The test must be carried out in at least two species of animals. 3. LD 50/60 4. In most radiopharmaceuticals, toxicity arises from the pharmaceutical part of the radiopharmaceutical, not from the radionuclide part. 46

Slide 47:

BIBLIOGRAPHY Nuclear and Radiochemistry :Fundamentalsand Applications byKarl Heinrich Lieser. G. F. Knoll, Radiation Detection and Measurement ,John Wiley & Sons. Advanced Research Techniques In Basic Medical Sciences Associate Professor Dr. Özhan EyigörUludag University College of MedicineDepartment of Histology & Embryology Instrumental methods of chemical analysis,Galen wood Ewing. Horst Wahl, Quarknet lecture, June 2002 j o u r n a l o f p r o t e o m i c s 7 2 ( 2 0 0 9 ) 7 4 0 – 7 4 www.elsevier.com/locate/ j p r o t Quality Controlof Radiopharmaceuticals by 0. Wallen and Dr. E. Komarov (WHO) The Isotopes Project http://ie.lbl.gov/education/isotopes.htm The ABCs of Radioactivity http://www.lbl.gov/abc Radiochemistry by Gopal Saha. 47