Radiopharmaceutical QC Review of Techniques

  1. Review the components of a 99mTc-chelate preparation
    1. Some radiopharmacies set their 99mTc-chelate to 95% or greater percent bound before allowing it for patient administration
    2. What are the other components found in solution after a 99mTc radiopharmaceutical is compounded?
      1. 99mTc7+ is reduced by Sn2+, usually to 99mTc4+
      2. There can still be some free 99mTc7+ in solution, 99mTcO4-
      3. Hydrolyzed 99mTc can occur in solution, to a by-product 99mTcO2. This usually occurs when there is an excessive amount o Sn2+. It can form 99mTc Sn(OH)2 stannous hydroxide. (This is a colloid)
      4. Once 99mTcO2 is formed it cannot chelate to the intended radiopharmaceutical
      5. Stannous ion can also become a colloid in the presence of moisture
      6. In the presences of air (O2) reduced technetium can re-oxidize and revert back to 99mTcO4-
      7. Some kits have ascorbic acid (antioxidant) to help retard the re-oxidation process
      8. More information about Sn can be found by clicking
    3. Radionuclide Purity – This is the fraction of the total desired radiopharmaceutical after compounding. Can you think of different examples?
    4. Radiochemical purity – Is the total amount or faction of the desired radioactive compound. Can you think of different examples?
      1. What about 99Mo in 99mTc, how would this effect our imaging results if too much Molly is in solution?
      2. Could radiolysis be an issue? What would happen if this occurred?
      3. Stability of the tag can be effected by time, light, temperature, or radiolysis
  2. Let us take a look at thin layer chromatography, ITLC, and how the impurities are separated for analysis
    1. Closer look at the ITLC strips

      2. TLC Strip

      1. A drop of the radiopharmaceutical is placed at the origin on the strip indicated by the blue dot
      2. The strip is then placed in a tube that contains a specific type of solvent
      3. The type of solvent varies depends on the type of radiopharmaceutical used
      4. The goal is to use a solvent that can separate the different compounds found in the radiopharmaceutical solution
      5. After the strip is placed in the solvent and via capillary action, the solution moves up the strip, indicated by the arrow
      6. As the solvent moves the different compounds in the radiopharmaceutical separate based on how the a solvent effects the agent
      7. Strip is then taken out after the solvent travels to the top
        1. The point of were the drop is placed is known as the origin
        2. The end point is known as the solvent front
      8. Reference front (Rf) value is then identified by finding the position on of a specific compound and determining its location on the strip
      9. There are two methods used to determine this the Rf value
      10. Usually you can cut the ITLC strip in half and determine the amount of activity
      11. Or if the department has a radiochromatographic scanner activity is counted along the strip and then the radioactive distribution can be graphically displayed
      12. Below is an image of a radiochromatographic scanner

        13. Types of TLC scanners

      13. From the strip above 99mTc-MAA was placed at the origin. The solvent used was acetone. HR 99mTc and 99mTc-MAA stay at the origin while 99mTcO4- moves to the top of the solvent front
      14. Rf values for each component is noted (assume the distance between the solvent front and the origin is 10 cm)
        1. 0 for HR 99mTc (stayed at the origin 0cm/10cm = 0) - this one is not a concern since MAA does not dissolve in aqueous solution
        2. 0 for 99mTcMAA (stayed at the origin 0cm /10cm = 0)
        3. 1.0 for 99mTcO4- (moved to the end of the solvent front 10cm/10cm = 1.0)
        4. Percent of bound can now be calculated by determining the amount of activity at Rf = 0 and at Rf = 1.0
        5. See diagram below


      15. Other points of interest
        1. Instant Thin-Layer Chromatography = ITCL are glass fiber impregnated with
          1. Silica gel ITLC (SG) or
          2. Polysilic acid ITLC (SA)
        2. Stationary phase are those components that do not move along the solvent front
        3. Mobile phase are those components that move along the solvent front
  3. Other types of tests used to determine radiochemical purity

    1. Gel chromatography
      1. A sample or drop is placed at the top of a column containing Sephadex gel which is soaked in a specific solvent (based on what needs to be separated)
      2. Separation of the radiopharmaceutical is dependent on molecular size, where the larger molecules move faster through the gelled solution
      3. Each fraction of the solution is then measured and % bound is determined
      4. An example of separation could be: 99mTc-MDP come out first(tube A), then 99mTc04- second (tube B), and 99mTc02 (R - remains)remains in the column. So how would you calculate %Bound? Assume A has 57,900 cpm, B has 1577 cpm, R has 995 cpm. Answer

    2. Paper or polyacrylamide gel electrophoresis
      1. Small sample of radiopharmaceutical is applied to the gel or paper
      2. Soaked in a buffer
      3. Voltage is applied across this material for a period of time
      4. Based on charge and ionic mobility the components within the radiopharmaceutical separate and percentages can be calculated

    3. Ion Exchange
      1. Sample of radiopharmaceutical is placed on in a column of ionic resin and the column is eluted with an appropriate solvent
      2. Different species are separated by the exchange of ions from the solution and the resin
      3. Percentages can then be calculated

    4. Solvent extraction
      1. Radiopharmaceutical is placed into an liquid and shaken
      2. Separation of the radiopharmaceutical within the solution depends on preferential solubility of the compounds or elements that are in solution
      3. The different components within the radiopharmaceutical separate into different immiscilbe liquids
      4. These components can assessed for the amount of radiochemical purity

    5. High-performance liquid chromatography (HPLC)
      1. Radiopharmaceutical is placed in a column containing packing material and solution
      2. The sample is then forced through a column via electrical pumps
      3. Different solutions are passed though this system at high pressure where separation of the radioactive components occur
      4. This results is a high resolution speed separation as the different components within each solution are assayed


      http://www.ncerthelp.com/text.php?contype=Concept&class_id=9&sub_id=S&chapter_id=CH2&q_no=13

    6. Distillation
      1. Via high vapor pressure radiopharmaceutical is distilled leaving other compounds
      2. Best for determining free iodine or noble gases
  4. Chemical purity
    1. This is defined as the fraction of material desired
    2. Al+3 in a 99mTcO4- is an example
    3. Additive in solution such as reducing and anti-oxidizing agents are not considered impurities
  5. Biological testing – the issues here are sterility, apyrogenicity, and toxicity that are issues when evaluating radio-purity
    1. Sterility
      1. There is/are no viable bacteria or microorganisms present
      2. Several methods are employed to assure sterility

    2. Autoclaving
      1. Water is heated to 121oC creating steam that is pressurized at 18 psi for 15 minutes
      2. Destroys microorganisms
      3. Certain radiopharmaceuticals cannot tolerate this method since it will break down the compound (ex. Protein). Hence a radiopharmaceutical must be thermostable
      4. This technique is not useful with short lived radionuclides (ex. PET)

    3. Membrane filtration
      1. Filtering a radiopharmaceutical to remove microorganisms
      2. Milli-pore filter most commonly used is 0.45 μm, however, 0.22 μm may also be used to filter out smaller components (ex. Blood)
      3. Method is best used for short-lived radiopharmaceuticals
      4. Method is also used to generate filtered colloid - Do you recall the filter's size?
    4. Sterility testing


      5. https://www.quora.com/What-is-turbidity-in-regards-to-microbiology

      1. Method I - Take a radiopharmaceutical sample and place into a thioglycollate media. Incubate at 30 – 35oC for 14 days
      2. Method II - Use a soybean-casein digest media and incubate at 20 – 25oC for 14 days
      3. These methods are usually done by the manufacturer after the pharmaceutical has been made
      4. For shorter lived radionuclides 14C-glucose in a trypticase soy broth culture. Generation of 14C-glucose in a gas that ionization chamber indicates that presence of microorganisms. This process only takes 3 -24 hours
    5. Pyrogens or endotoxins
      1. While a radiopharmaceutical may be sterile after or before compounding there can be residual components left from bacteria:
        1. Pyrogens cause fever or reaction that may be associated with the endotoxin and are fragments of bacterial cell wall. Reactions may occur: fever, chills, malaise, leukopenia, pain in the joints, flushing, sweating, headache, and/or pupil dilation. Usually they are composed of polypeptides
        2. Endotoxins is a subclass of Pyrogens and are reminiscence of gram-negative bacteria cell wall, specifically lipopolysaccharides, may be considered poisonous, and will cause fever
      2. Symptoms occur between 30 minutes and 2 hours post dose and usually dissipate 10 to 12 hours later
    6. Pyrogenicity testing

      1. USP Rabbit Test
        1. Three rabbits are kept in a controlled environment and injected with a radiopharmaceutical
        2. Dose is calculated based on weight, 3 – 10 times the human equivalent
        3. Rectal temperatures are taken at 1, 2, and 3 hours post injection
          • Less than 0.6oC change per rabbit or nothing greater than 1.4oC summed means there are no endotoxins present
          • If positive, then the test is repeated with 5 rabbits where less than 0.6oC change per rabbit or a sum of less than 3.7oC indicates the lack of endotoxins

      2. LAL test (Limulus amebocyte lysate)
        1. Limulus polyphemus is extracted from the blood of a Horseshoe Crab
        2. Usually 0.l mL of LAL is mixed with the radiopharmaceutical and incubated at 37oC for 15 -60 minutes at a pH between 6 to 8
        3. If the compound turns opaque gel it is considered positive. The thicker the gel the greater the amount of pathogens
    7. Toxicity
      1. Does the radiopharmaceutical have a toxin effect on the human system?
      2. Example of this would be thallium is toxic to the human body, so how can we use it?
      3. Animals are injected (mice, rat, rabbit, or dog) with the radiopharmaceutical. After 2 to 6 weeks the animals are scarified and an autopsy is done to evaluate the animal’s systems.
      4. LD 50/30 - NRC defines the median death rate in humans
        1. Two types of animals are given radioactive doses at increasing levels
        2. This establishes when 50% die at 30 days
        3. However, this test has, for the most part, been replaced with cell cultures and computer modeling
        4. Here are some other facts from WIKI

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