2. DETERMINATION OF THE CHEMICAL STABILITY OF HEATED SAMPLES OF TETRACYCLINE HYDROCHLORIDE Presented at 9th COMMONWEALTH PHARMACEUTICAL ASSOCIATION + MALAYSIAN PHARMACEUTICAL SOCIETY PHARMACY SCIENTIFIC CONFERENCE 2007 PARKROYAL HOTEL, KUALA LUMPUR MALAYSIA 1st - 5th AUGUST, 2007 Presented at 9th COMMONWEALTH PHARMACEUTICAL ASSOCIATION + MALAYSIAN PHARMACEUTICAL SOCIETY PHARMACY SCIENTIFIC CONFERENCE 2007 PARKROYAL HOTEL, KUALA LUMPUR MALAYSIA 1st - 5th AUGUST, 2007

3. Presented by DR. NAEEM HASAN KHAN ASSOCIATE PROFESSOR HEAD DEPARTMENT OF PHARMACEUTICAL CHEMISTRY KULLIYYAH OF PHARMACY INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA KUANTAN CAMPUS, PAHANG DARUL MAKMUR MALAYSIA Presented by DR. NAEEM HASAN KHAN ASSOCIATE PROFESSOR HEAD DEPARTMENT OF PHARMACEUTICAL CHEMISTRY KULLIYYAH OF PHARMACY INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA KUANTAN CAMPUS, PAHANG DARUL MAKMUR MALAYSIA

4. STRUCTURE OF TETRACYCLINE

5. SUBSTITUTION

6. CHEMICAL TRANSFORMATION OF TETRACYCLINE

7.  Tetracycline (TC) can be prepared by the catalytic reduction of Chlortetracycline.  TC undergoes epimerisation at C-4, resulting in the formation of 4- epitetracycline (ETC).  Due to the presence of a hydroxyl group at C-6, acid degradation takes place resulting in the formation of anhydrotetracycline (ATC). This anhydro derivative also epimerizes at C-4, to give degradation product, 4- epianhydrotetracycline (EATC). This degradation product can also be formed by acid degradation of ETC.  Another related compound, 2- acetyl - 2 - decarboxamidotetracycline is also present as a fermented impurity.  Tetracycline (TC) can be prepared by the catalytic reduction of Chlortetracycline.  TC undergoes epimerisation at C-4, resulting in the formation of 4- epitetracycline (ETC).  Due to the presence of a hydroxyl group at C-6, acid degradation takes place resulting in the formation of anhydrotetracycline (ATC). This anhydro derivative also epimerizes at C-4, to give degradation product, 4- epianhydrotetracycline (EATC). This degradation product can also be formed by acid degradation of ETC.  Another related compound, 2- acetyl - 2 - decarboxamidotetracycline is also present as a fermented impurity.

8. CHEMICAL STABILITY OF TETRACYCLINE HYDROCHLORIDE WHEN HEATED FOR A LONG PERIOD OF TIME The stability studies of heated samples of TC hydrochloride was carried out following the references;  CONNORS K.A., AMIDON G.L. and KENNON L., CHEMICAL STABILITY OF PHARMACEUTICALS, WILEY (1978).  NAEEM HASAN KHAN, WERA P., ROETS E. and HOOGMARTENS J., JOURNAL OF LIQUID CHROMATOGRAPHY, 13(7),1351-1374 (1990).  NAEEM HASAN KHAN, ROETS E., HOOGMARTENS J. and VANDERHAEGHE H., JOURNAL OF CHROMATOGRAPHY,405,229 (1987).  UNITED STATES PHARMACOPEIA XXI, ROCKVILLE, MD (1985). The stability studies of heated samples of TC hydrochloride was carried out following the references;  CONNORS K.A., AMIDON G.L. and KENNON L., CHEMICAL STABILITY OF PHARMACEUTICALS, WILEY (1978).  NAEEM HASAN KHAN, WERA P., ROETS E. and HOOGMARTENS J., JOURNAL OF LIQUID CHROMATOGRAPHY, 13(7),1351-1374 (1990).  NAEEM HASAN KHAN, ROETS E., HOOGMARTENS J. and VANDERHAEGHE H., JOURNAL OF CHROMATOGRAPHY,405,229 (1987).  UNITED STATES PHARMACOPEIA XXI, ROCKVILLE, MD (1985).

9.  Using the approximate formula of Connors et.al. ; ( ∆ T/10) t 90 (T2) = t 90 ( t1) / Q 10 with T2 = T1+ ∆ T where t 90 = time needed to reach 10% decomposition, T = temperature ºC, Q 10 = factor by which the rate constant increases for 10º C Temperature increase, T2 = T1 + ∆ T  Using the approximate formula of Connors et.al. ; ( ∆ T/10) t 90 (T2) = t 90 ( t1) / Q 10 with T2 = T1+ ∆ T where t 90 = time needed to reach 10% decomposition, T = temperature ºC, Q 10 = factor by which the rate constant increases for 10º C Temperature increase, T2 = T1 + ∆ T

10. Shelf life Calculated t 90 values (years) of TC hydrochloride Q 10 screw capped vials rubber stoppered vials 40º C 70º C 40º C 2 80 10 24 3 600 23 180 Q 10 / 2 = the estimate for the lower limit Q 10 / 3 = gives most likely estimate Shelf life Calculated t 90 values (years) of TC hydrochloride Q 10 screw capped vials rubber stoppered vials 40º C 70º C 40º C 2 80 10 24 3 600 23 180 Q 10 / 2 = the estimate for the lower limit Q 10 / 3 = gives most likely estimate

11. The values obtained from the, screw capped samples heated for 6 years, show that the stability is good as ; t 90 (40º C) = 80 years for the Q 10/2. t 90 (70º C) = 10 years for the Q 10/3 The values obtained from the, screw capped samples heated for 6 years, show that the stability is good as ; t 90 (40º C) = 80 years for the Q 10/2. t 90 (70º C) = 10 years for the Q 10/3

12. H.PL.C. CONDITIONS Column : PLRP-S (polystyrene-divinylbenzene copolymer) Mobile phase : Tert.-butanol 6.9 gm ; 0.2 M phosphate buffer pH 9.0 (10.0 ml) ; 0.02 M tetrabutylammonium hydrogen sulphate solution pH 9.0 (15.0 ml) ; 0.01 M sodium edetate solution pH 9.0 (10.0 ml) ; double distilled water upto 100.0 ml. Flow rate : 1 ml / min. Detection : UV at 254 nm Temperature : 60º C Elution time : 35 minutes Column : PLRP-S (polystyrene-divinylbenzene copolymer) Mobile phase : Tert.-butanol 6.9 gm ; 0.2 M phosphate buffer pH 9.0 (10.0 ml) ; 0.02 M tetrabutylammonium hydrogen sulphate solution pH 9.0 (15.0 ml) ; 0.01 M sodium edetate solution pH 9.0 (10.0 ml) ; double distilled water upto 100.0 ml. Flow rate : 1 ml / min. Detection : UV at 254 nm Temperature : 60º C Elution time : 35 minutes

13. Influence of heating on the composition of laboratory made Tetracycline Base sample TC Base (Heated) TC % ETC % ADTC % EATC % ATC % Water % Total % at 40 º C (open bottle) for 48 hours 76.7 1.6 0.3 0.2 19.6 98.6 at 40 º C (closed bottle ) for 48 hours 76.2 1.7 0.3 0.2 20.6 99.2 at 80 º C (open bottle) for 48 hours 88.9 3.2 0.3 0.2 1.4 2.2 95.8

14. Discussion  In the rubber stoppered vials, the decomposition was faster as compared to the screw capped vials. This can probably be explained by the fact that the samples in screw capped vials remain completely dry since small amount of water, liberated upon formation of ATC, is evaporated by evaporation. In the rubber stoppered vials, this water is probably trapped and thus facilitates further acid catalysed decomposition of TC hydrochloride. This confirms the influence of water content on the stability of TC which was also confirmed by the H.P.L.C. analysis of these samples.  In the rubber stoppered vials, the decomposition was faster as compared to the screw capped vials. This can probably be explained by the fact that the samples in screw capped vials remain completely dry since small amount of water, liberated upon formation of ATC, is evaporated by evaporation. In the rubber stoppered vials, this water is probably trapped and thus facilitates further acid catalysed decomposition of TC hydrochloride. This confirms the influence of water content on the stability of TC which was also confirmed by the H.P.L.C. analysis of these samples.

15. SUMMERY After stability studies of TC hydrochloride by; H.P.L.C., For content identification,separation and quantity determination CARL FISCHER, for water content It may be concluded that TC hydrochloride when kept for a longer period at relatively low temperature and under conditions of low humidity, is a stable product physico - chemically. After stability studies of TC hydrochloride by; H.P.L.C., For content identification,separation and quantity determination CARL FISCHER, for water content It may be concluded that TC hydrochloride when kept for a longer period at relatively low temperature and under conditions of low humidity, is a stable product physico - chemically.

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