UKRG Handbook IDENTIFICATION TESTS
The issue of a product licence to a medicinal product gives assurance to the user that the product has been extensively evaluated in terms of quality, efficacy and safety. Products which do not have licences may be equally safe and efficacious but lack the documentary proof to this effect. The onus is therefore on the user to adopt a suitable programme of quality assurance and to take whatever steps are considered necessary to allow the product to be used with confidence. At the very least this should involve obtaining from the supplier a copy of a certificate of analysis for the particular batch of product being used, or a certificate of compliance with a previously agreed set of parameters for the particular batch. It is also necessary to demonstrate that the product received is in fact what it is claimed to be, and this involves the adoption of appropriate tests of identity. In general, a suitable test for identity should conform to the following parameters in that the procedure should be:
a) simple
b) rapid
c) non-destructive
d) undemanding in terms of complex equipment such as HPLC
Radiopharmaceutical kits for reconstitution with technetium-99m generally comprise a ligand, a reducing agent, and various excipients including buffering salts, antioxidants and fillers. Chemical identification of these components is possible, but generally demanding in terms of equipment and time, and also of material since the tests are usually destructive. It is less demanding, and much more appropriate, to consider identification of the radiolabelled components after reconstitution. Chemical analysis is usually inappropriate in this context due to the minute amounts of material present. Rapid test methods for radiochemical purity, including instant thin-layer chromatography (ITLC) are an established part of the quality control programme in many radiopharmacy units. However, such tests alone are not considered appropriate as tests of identity as they isolate and quantify impurities rather than the main radiopharmaceutical component. Rf values tend to be either 0 or 1 and are generally not unique to a particular product.
Radioactive pharmaceutical products and radiochemicals generally contain minute amounts of the radioactive component, so again, chemical analysis is inappropriate. Identification depends on comparing the behaviour of the radioactive species with that of a chemically authenticated reference species under identical conditions. There are often additional constraints in terms of the time available for completion of the test. Generally this has to be performed on the actual batch delivered for clinical use, and must be performed within the restrictions imposed by delivery schedules, patient administration times, and the rate of decay of the product.
Simple, rapid procedures or combinations of procedures are required which allow the isolation of the major radioactive component of a product in a uniquely identifiable way. Suitable techniques include chromatography, electrophoresis and filtration.
Chromatography
Chromatographic systems in which the radiochemical species migrate with unique Rf values are helpful in establishing product identity. This must be confirmed by comparing migration with that of a chemically identified reference substance, which can be located colorimetrically or fluorimetrically. The technique can be applied, for example, to iodinated or 14C- labelled compounds where it is possible to obtain reference substances. This is not the case with 99mTc radiopharmaceuticals where the chelate is chemically different to the non-radioactive ligand.
Electrophoresis.
Electrophoresis is based on the principle of separation of molecules on the basis of their electrical charge. Depending on the nature of the support medium used, secondary features including molecular size and shape also have influence on the extent of separation. Electrical charge is determined by the extent of ionisation, and important factors to be controlled in any system are therefore the pH and ionic strength of the electrolyte. As with chromatography, migration of an unknown compound should be compared with that of a reference substance It is often helpful to include as a standard a visible marker with known migration characteristics. Thus the migration of a radioactive component can be compared with the migration of the visible marker under identical conditions of separation. Results are generally available sufficiently quickly to allow identification, release and use of the product without having to set aside potentially expensive kit vials purely for purposes of analysis.
Filtration
Filtration may result from a simple sieving action. Membrane filters are pierced by pores of similar size and there is a single cut-off point at which all particles greater in size than the pores are retained. In depth filtration, filters are generally thicker, and retention of particles occurs by progressive impedance to flow through a tortuous path. There is therefore no precise cut-off point, but rather an average minimum value for the size of particle retained. Precise particle sizing may be performed using thin, non-retentive filter membranes of controlled pore size, which act essentially as sieves. For identification purposes, however, complete particle size distribution analysis is unnecessary and the requisite information can be generated in the form of a limit test using readily available depth filters of the type used for filtration sterilisation.
Combined tests for identity
In some circumstances, the required information may not be obtainable using a single system, and results from more than one type of assessment may be required.
Identification of specific radiopharmaceuticals |
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| 99mTc- Human Albumin; 125I Human Albumin by Electrophoresis | |||
| System: | |||
| Support medium | Cellulose Acetate (Sepraphore III< Gelman Sciences) | ||
| Paper dimensions | 120 mm x 25 mm | ||
| Electrolyte | Tris/glycine buffer pH 8 | ||
| Power supply | 300 volts constant current | ||
| Duration | 30 minutes | ||
| The sample is mixed with an authenticated sample of human albumin
prior to application to the support medium. After separation, divide the
strip lengthwise and stain one half using Ponceau S in 7.5% trichloroacetic
acid, followed by destaining in 5% acetic acid and washing in water. Albumin
is stained bright pink. Using the stained half of the strip as a guide,
divide the other half into zones and count for the presence of activity.
Most of the activity (> 95%) should have migrated the same distance as the pink stained zone. |
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| 14C Urea by chromatography | |||
| System: | |||
| Support medium | Cellulose | ||
| Developing solvent | n-butanol:water:acetic acid [120:50:30] | ||
| Reference marker | Urea | ||
| Urea stain | 10% p-dimethylaminobenzaldehyde in HCl : Acetone [1:4] | ||
| The sample is either mixed with an authenticated sample of urea, prior to application to the chromatographic plate or is applied to the plate adjacent to an authentic sample. After development, the radioactive zone is visualised autoradiographically by exposing the developed chromatogram to a sheet of X-ray film. Urea is identified chemically by spraying with a stain comprising 10% p-dimethylaminobenzaldehyde in HCl:Acetone (1:4). Most of the activity (> 95%) should be associated with a zone having an identical Rf (approximately 0.6) to that of the stained authenticated urea sample. | |||
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| Conclusions. | |||
| Simple, non destructive tests can be devised which adequately identify non-licensed radiopharmaceuticals and radiochemicals. In conjunction with an appropriate certificate of analysis or certificate of compliance, and compliance with other relevant standards, these tests give sufficient assurance of quality to allow safe use of the products in nuclear medicine. | |||
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