1. Biological Evaluation (Lecture – 9) Reference standards Joseph O. Oweta B. Pharm (MUST)

2. Objectives Overview & basic principles Sources & Preparation of reference standards Assay of Digitaloid drugs

3. Overview and Basic Principles “Reference standards….are highly characterised specimens reflective of specified drugs and foods (drug substances, biologics, excipients, dietary supplements, food ingredients, impurities, degradation products, reagents and performance verification standards.” USP

4. Overview and Basic Principles “A REFERENCE STANDARD is a highly purified compound, substance or product (or a standardized procedure) that is well characterized.” Hence may serve as guides to ensure the quality of subsequent compounds, procedures or products are in conformity with set (lay down) specifications of the standard. Reference Standards are divided into four main categories: – Primary, – Secondary, – Reagent grades – and Pharmacopeia

5. Primary Standard Is an analytical reagent of generally high purity and documentation confirming the results four or five of the following tests: HPLC, NMR, GC, TLC, LCMS, Karl Fischer and a Residual Solvents analysis. The parameters of the method of testing will also be provided along with these results of testing.

6. Secondary Standard Is generally of a lesser purity than a Primary standard. The defining difference between the two is that secondary standards have only one or two test results, usually HPLC and one other. These standards are also provided with a method of testing.

7. Reagent / Standard Grade Are separated from primary and secondary reference standards due to a lack of characterization and documentation of the batch compound. There is a Certificate of Analysis provided with the compound however no guaranteed method of analysis will be provided.

8. Pharmacopeias Reference Standard Pharmacopeia is predominantly a collection of specifications to primarily serve as a guide to manufacturers, pharmacists and regulatory agencies in carrying out their respective drug related activities with respect to the quality of drugs and pharmaceutical products.

9. Pharmacopeias Reference Standard Specifications 1.Purity 2.Potency 3.Test methodology 4.Standards for independent evaluation of drug quality 5.Identification properties and physical characteristics 6.Ultimate composition of substances as well as medicinal formulae 7.Presence or/and absence of traces of foreign substances

10. Pharmacopeias Reference Standard Importance 1.Promotes improvement of pharmaceutical products. 2.Ensures the maintaining of high quality products within and across countries. 3.Serves as a quick reference for drugs, medicinal drugs product, raw material and (small scale) manufacturing procedure. 4.Provides readily available reference to verify drugs and pharmaceutical products in the international market. 5.Easy reference for drug storage, labelling, use, dose regimen, strength etc.

11. Pharmacopeias Reference Standard Specifications British Pharmacopeia (BP) United States Pharmacopeias (USP) British Pharmaceutical Codex (BPC) European Pharmacopeia International Pharmacopeia (IP) African Pharmacopeia (AP) Pharmacopeia of India

12. Overview and Basic Principles (Recap – of principles of BA) The reference standard and test sample should have the same pharmacological effects and should have the same mode of action….Parallelism The test solution and reference standard should be compared for their established pharmacological effect using a specified pharmacological technique

13. Reference Standards Used to minimise error resulting from animal variation in certain bioassay procedures. Principle consists of testing unknown and standard on: – Two gps of similar animals – Same animal and organ (Some cases)

14. Reference Standards Amount of unknown rqd. to produce and effect in ref. to the std. is inv. Proportional to the potency. Potency of unknown can be expressed as a percentage of that of the standard. Precise methods of determining potency may be based upon observation on relative and not equal effects.

15. Assay of Digitaloid Drugs Group of drugs including digitalis (Dried leaves of D. Purpurea). Contains cardioactive glycosides – Digitoxin – Gitoxin Digitonin - saponin like glycoside largely devoid of cardiac effects of digitalis Complex mixture – Digitoflavin – Digitophyllin – Lipids – Carbohydrates

16. Assay of Digitaloid Drugs Cardioactive structures are similar: – Chemical structure – Pharmacodynamics (+ inotropic effect) Differ markedly in: – milligram potency. – Onset of activity – Duration of action Crude drugs also vary in: – Total glycoside content – Relative concentration of each active principle

17. Assay of Digitaloid Drugs Chemical Assays?? May determine total glycosides or total aglycones. Cannot measure the total p’dynamic activity of: – Crude drug – Galenical preparations Such drugs must be subjected to bioassays.

18. Bioassay of Digitalis Based on amount of a test material required to cause death due to cardiac arrest in the anaesthetised pigeon…. …relative to the amount of standard required to produce the same effect.

19. Bioassay of Digitalis Challenges/ limitations Fractional intermittent intravenous administration is used to model oral administration. – Variations in clinical effectives of different constituents not accounted for. Toxic end point considered instead of a therapeutic end point However, its argued that the toxic effects on the heart constitute an extension of the beneficial. No alternative presents a comparative advantage over the official method.

20. Bioassay of Digitalis Seperation and x-terisation has led to the availability of pure relatively pure cardiotonic glycosides. – Digoxin – Digitoxin – Acetyldigitoxin – Ouabain The above preparations are assayed quantitatively using spectroscopic methods.

21. Bioassay of Digitalis Chemical assays enable precise determination. However patient response varies for digitaloid drugs. A clinical assay must be performed with each patient…regardless of: – Chemical assay (single drug formulations) – Bioassay (homeopathy)

22. Digitalis Assay Digitalis Powdered digitalis Digitalis capsules Digitalis

23. Next week….Insulin & Glucagon assays