قَالُوا سُبْحَانَكَ لا عِلْمَ لَنَا إلاَّ مَا عَلَّمْتَنَا إنَّكَ أَنتَ العَلِيمُ الحَكِيمُ

1. بسم الله الرحمن الرحيم قَالُوا سُبْحَانَكَ لا عِلْمَ لَنَا إلاَّ مَا عَلَّمْتَنَا إنَّكَ أَنتَ العَلِيمُ الحَكِيمُ

2. Stability Indicating Methods

3. Stability Indicating Methods Any method, that affords selective determination of the intact molecule in presence of its degradation products, is considered as stability indicating method. • The intact molecule is the active form of drug. • Although degradations are usually inactive yet sometimes they are harmful. • Choice of suitable stability indicating method to carry out stability study of certain drug. • Registration of new pharmaceutical formulations Importance:

4. Official methods: Most of official methods are not stability indicating. In the case of the official method the whole monograph must be applied, including: • Tests for identity • Limit tests for impurities and degradation products • Last step is the analysis.

5. Drug Decomposition Degradation Of Pharmaceutical Compounds Despite the large number of possible reactions leading to drug decomposition, perhaps most of them can be classified as either:- Hydrolysis or Oxidation

6. Hydrolysis O II R-C--- II) Other kind of hydrolysis I) Hydrolysis of carboxylic acid derivative Hydrolysis Of Carboxylic acid Derivatives We are concerned in this part with compounds possessing acyl group

9. Pharmaceutical Examples (I) Simple ester hydrolysis is exemplified by the hydrolysis of a) Procaine OH H2O + H2O H P-amino benzoic acid N-diethyl ethanolamine

10. b) Atropine + H2O H2O H OH c) Aspirin hydrolyses to salicylic acid & acetic acid + H2O H2O H OH

11. (2) Hydrolysis of lactones e.g Pilocarpine H OH + H2O H2O

12. (3) amide • Procainamide OH + H2O H2O H b) Nicotinamide OH H H2O + H2O NH3 b) Chloramphenicol OH H + H2O H2O

13. (4) Lactam ring Penicillin contains both a Lactam ring & an amide function, although both the Lactam & the amide can be hydrolyzed, it is observed that the Lactam is more labile than the amide, apparently because of strain induced by fusion of the four membered ring to 5- membered ring. Penicillin G + H2O H2O OH H

14. 5) Di-imide Barbituric acid derivative " phenobarbital" 1,2 + H2O H2O H2O 1,6

15. II) Other kinds of hydrolysis a) Alkyl halides hydrolysis to the corresponding alcohol e.g. hydrolysis of chloramphenicol to the corresponding aa dihydroxy derivative OH H + 2H2O HO HO H OH chloramphenicol + 2HCl aa dihydroxy derivative

16. O NH CH2 – C – CH3 + H2O CH3 – C– CH3 b) Hydrolysis as a reverse of condensation. Another kind of hydrolysis is the reverse of a “condensation” reaction, in which an amine e.g., adds to the carbonyl (compounds with elimination of water). hydrolysis Primary amine HO + NH3 condensation imine c) Hydration (addition of water) rather than hydrolysis, but there is no fundamental difference. + H2O Partial cleavage

17. Stabilization of Pharmaceuticals • When drug decomposition is the result of a hydrolysis reaction, an obvious & effective means of stabilization is • To limit access of the drug to water. • This is simply accomplished by using a solid dosage form, as with aspirin, which is normally available in tablets, capsules & suppositories because of its instability in liquid formulation. Antibiotic is supplied in dry form, water is added before use.

18. 2. Control of pH offers a mean for • So a compromise pH, at which the solubility and stability are both acceptable, must be selected. • Stability can always be increased by lowering temperature. • Changes in solvent composition of formulation can have significant effects.To decrease hydrolysis rate e.g. it may seem reasonable to replace water with an alcohol solvent; but one must be aware of possible complication (e.g. alcoholysis instead of hydrolysis as a decomposition reaction). Controlling stability & Affect solubility

19. Oxidation • Oxidation reaction is a complementary one, its partner is reduction. • One can not happen without the other. • oxidation /reduction (redox) reactions involve the transfer of electrons. (or one or more oxygen or hydrogen) • The oxidation state of carbon atom , & hence of a compound containing carbon atoms, is determined by the number of bonds from carbon to oxygen. Thus since oxygen is "added", we consider an aldehyde to be more oxidized than an alcohol, & So on.

20. Note that reduction can be thought of as the addition of hydrogen, accordingly we consider the addition of hydrogen to an olefin to be a reduction. Another example oxidation of hydroquinone (1,4 dihydroxybenzene ) to p. benzoquinone. In general oxidation is a loss of electrons & reduction is a gain of electrons. The rate of oxidation may be a function of the concentration of H + , i.e., of pH. Accordingly , many compounds are more resistant to oxidation at low pH values.

21. Oxidative Pathways of Pharmaceutical Interest Autoxidation:- This term is used to refer to oxidations that takes placespontaneously under mild conditions. The majority of the reactions involved are free radical in type withorganic peroxides often being intermediate or final products. Free radicals are atoms or molecules that have one or more unshared valence electrons ; e.g. Free radicals are formed by: Thermal or Photolytic hemolytic cleavage of covalent bond. Redox processes involving one electron transfer steps e.g. 2CH3 * CH3 CH3

22. The first 1st step is called initiation & takes a period of time called theinduction period. The length of the induction period depends on The initiation step in a hydrocarbon reaction is written as follows. The second step is propagation hydro peroxide formation occurs The final step is termination Reactions occur that break the chain Termination may take place by coupling (two radicals combine to form a non radical), R H R*+ *H R* + O2 ROO* ROO* + RH ROOH + R*

23. A few selected oxidative reactions of pharmaceutical interest are illustrated here Many drugs have been reported to be subjected to autoxidation including. - apomorphine - Cyanocobalamin - ascorbic acid - epinephrine - chlorpromazine - ergometrine - phenothiazine derivative - heparin - hydrocortisone - kanamycin - morphine - neomycin - p. amino bezoic acid - penicillin - Phenylephrine - prednisolone - prednisone - procaine - riboflavin - streptomycin - sulfadiazine - terpenes - tetracyclines - thiamine - vitamins A, D & E - rancidity of oils & fats.

24. Morphine Morphine dimerizes when oxidized + HOO* H2O2 H OH the dimer (bimorphine or pseudomorphine) + H* Then the morphine free radical couples with a morphine molecule ( at the free position ortho to phenolic oxygen) to give the dimer (bimorphine or pseudomorphine) & H. Hydrogen peroxide is then produced. hydrogen peroxide can cause additional, oxidation to the N-oxide.

25. Chlorpromazine It forms yellow green color initially

26. The marked effect of UV light on an oxidation reaction 5% solutions of 2 therapeutically useful phenothiazine salts (A) Chlorpromazine hydrochloride & (B) Prochlorperazine ethane disulfonate were placed in Warburg respirometer to permit measurement of oxygen up take & were then exposed to a sun light. The solutions became colored shortly after the light was turned on, & they continue to darken. As shown in the following figure. Plot of oxygen – uptake data for chlorpromazine hydrochloride (A) & Prochlorperazine ethane disulfonate (B) illustrate induction period, the linearity of uptake with time, the extreme light dependence of the oxidative degradation. Induction period

27. Inhibition of Oxidation • Protection from light: • The choice of suitable container is very important . • Note also that if a product is light sensitive, it is important that this fact should be stated on the label. • To exclude light, four main techniques are available • Wrap – around label • Container coatings (some may incorporate UV absorbing material). • Various cartooning procedure. • The use of so –called light resistant containers.

28. 2) Exclusion of oxygen: • Removing O2 from a formulation is an obvious way to prevent oxidation. • This can be done in several ways: • Oxygen may be expelled from aqueous preparations by boiling the water beforehand, although some oxygen will redissolve during the cooling process. • A better way is bubbling nitrogen through the solvent to flush the O2 out of solution. • Another way is to flush the head space of the container with N2 just prior sealing or capping.

29. 3) addition of antioxidants: • antioxidants are materials that act by being more readily oxidized than the agents they are to protect. • Thus in a closed system (e.g. an ampoule) they may consume essentially all the oxygen present & there by protect the drug. • Open system may require higher antioxidant concentration than closed system. antioxidants may also function by being inhibitors of free radicals, i.e., they provide an H. to break the chain reaction. • To enhance the effectiveness of the antioxidant approach it is sometimes useful to use more than one antioxidant. • It has been found that a combination of two antioxidants along with a chelating agent to complex metals (thus inhibiting their catalytic effect of the antioxidant). • Emulsions may need two antioxidant systems water soluble one & an oil soluble one. • The concentration used generally vary between 0.01% 1%, • 0.1% is a good place to start when formulating.

30. Chelating agents are: • citric acid • phenylalanine • EDTA • sorbitol • tartaric acid • Oil soluble antioxidants are: • Butylate dihydroxyanisole (BHA) • ascorbyl palmitate • hydroquinone • lecithin, propyl gallate • Water soluble antioxidants are: • Na bisulphite • cysteine hydrochloride • thioglycolic acid • ascorbic acid • Na sulphite

31. 4- Control of pH: - The principle of lowering pH was discussed before. - For purpose of pharmaceutical products, the pH range of 3-4 is generally found most useful. - This technique is not for use for drugs that precipitate or decompose at lower pH values.

32. DETERMINATION

33. 1) Functional group approach: • The intact molecule • of the drug • via certain • functional group • Which does not persist • in any of the degradation • products • e.g. hydroxamic acid Method for the determination of penicillins Via-Lactam ring Is determined

34. e.g. hydroxamic acid Method for the determination of penicillins Via-Lactam ring Fe+++ reddish violetComplex of ferric hydroxamate 1 : 1

35. Separation and measurement 2) Combination of Operations Solvent extraction or Chromatographic methods Separation may be Carried out either by

36. Cu Cl A) Solvent extraction: Liquid – Liquid extraction It is the transfer of a solute From to one liquid phase another partition coefficient K = where Cu = concentration of the solute in the upper Phase & Cl = " " " " " " lower “

37. the intact molecule from Its degradation Products The feasibility of resolving depends on Ki where Separability factor a = Kd K's are the partition coefficient of the two substances. If  ~ 1 → no separation  much deviated from unity → good separation This can be accomplished by suitable choice of solvent, pHand ionic strength

38. I) Choice of Solvents: • Sometimes the analyst may select both solvents but often the sample is in aqueous solution. • The difference in partition coefficient of both intact & degradation products • The chemical nature of the 2nd solvent. • The difference in the chemical structure of the intact molecule & its degradation product. • -The choice of the suitable solvent is very important for the success of separation process. Ki Kd by will be markedly influenced

39. II) Control of Ionic strength: the salt concentration of an aqueous solution Is made very high ↑ → will be decreased  the solubility of non - electrolyte - This reduction of solubility by an increase in ionic strength is " the Salting out effect" - The ions of salt will reduce the availability of water molecules which act as a solvent for non electrolyte by tying up much water as a hydration shell around the ions. - The salt also helps to break the emulsions that may be formed when shaking the two phases together. - Salting out effect alters the apparent partition coefficient of many substances.

40. III) Control of pH: acids or bases Most of the drugs encountered by the pharmaceutical analyst are weak The solubility of these substances depends upon their ionic forms The ionic species are usually soluble in polar (specially aqueous) solvents A base & an acid They are readily separated by solvent extraction If we have a mixture of In acid medium complete extraction of the acid could be achieved while the base will be retained as its salt in aqueous layer.

41. H N O - C e.g. OH H - COOH + - NH3+ H+ - COOH + - NH2 OH- amide linkage - COO- + - NH2 H+ (HCl) ether Acetylsalicylic acid + antihistaminic in aqueous medium acetylsalicylic acid in ether Antihistaminic as hydrochloride salt in aqueous layer

42. Suppose, it is desired to retain weak acid, with pKa = 5 in aqueous layer while extracting a 2nd basic substance. • [A-] • pH = PKa + log________ • [HA] • at pH = 5 → [A-] = [HA] • at pH = 6 → about 10% remains as [HA] • at pH = 7 → “ 1% remains as [HA] • at pH = 8 → “0.1% remains as [HA] • So the pH should be at least 3 units more basic than pKa to ensure complete conversion to the anion form.

43. A F- b) Chromatographic Separation : a large number of stability indicating methods entail several chromatographic techniques. Zr4+ + eriochrome cyanine R  colored product Mineralization of F-  bleaching of the color  ZrF62- - TLC (densitometry) . - Column, - GLC - HPLC etc … at 525nm

44. If it is possible to determine the degradation in presence of intact. III- Determination via degration by quantitative conversion of intact molecule of drug into its degradates e.g. : penicillin chlorazepate dipotassium . In this case we determine the degradation products before and after degradation. By simple subtraction, the concentration of the intact drug can be obtained e.g.

45. degradation D (10) + Intact (90) 10 (D) + 90 (D) = 100 I = 100 - 10 = 90 100 degradation D1 (10) + D2 (10) + I (90) D1 (10) + D2 (10) + D1 (90) + D2 (90) I = 100 (D1) - 10 (D1) = 90 via (D1) or I = 100 (D2) - 10 (D2) = 90 via (D2) 100 • On the condition that • Method should be suitable for the determination of degradation without any contribution from the intact or D2 • Degradation is not affected during the process of degradation. • degradation should be quantitative.

46. 240nm A 280nm  Stability Indicating Method For The Determination of Chlorazepate dipotassium. Via its degrading products 2N HCl At room Temp ½hr 6N HCl at 100=Co for 1hr • H2N – CH2 - COOH • Glycine in aqueous layer ninhydin + pyridine bluish violet measured at 560nm * Extracted from neutralized hydrolyzate with diethylether the extract is evaporated & the residue is dissolved in CHCl3 & its absorbance is measured at either 240nm or 380nm

47. IV- Determination of the intact molecule of the drug in presence of its degradation products, without separation, by the use of suitable selective method : a) Organic polarography : It offers the desired specificity if the difference in E ½ the intact molecule and Its degradation products is big enough to prevent overlapping. (0.4 )

48. Applied voltage polarogram. current Intact + Deg inactive active (via degradation) active inactive (no problem) active active (the difference in E ½ should be at least 0.4 v )

49. b) Spectroscopic method : • 1) IR: • It has very few quantitative application in stability evaluation because of its limited sensitivity . • 2) NMR: It offers specificityalongwith simplicity Sensitivity & precision but it lacks as well It depends on suitable choice of one resonance band of the intact molecule, which is free from any overlap by the others, specially the resonance bands of the degradation products.

50. H OH H2O Pathway of Degradation of Clonazepam