HM Medical Clinic

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Microsoft word - harmonised_steroids.doc

IDENTIFICATION OF HYDROCORTISONE ACETATE, DEXAMETHASONE, BETAMETHASONE, 2/12/2005 ACM MAL 07 BETAMETHASONE 17-VALERATE AND TRIAMCINOLONE ACETONIDE IN COSMETIC PRODUCTS BY TLC AND HPLC THIN LAYER CHROMATOGRAPHIC TECHNIQUE (TLC) The method describes the identification of hydrocortisone acetate, dexamethasone, betamethasone, betamethasone 17-valerate and triamcinolone acetonide in cosmetic products. Liquid samples suspected of containing steroid compounds are neutralized and extracted with ethyl acetate. Cream samples suspected of containing steroid compounds are extracted with methanol. The extracted solutions are evaporated to dryness. Residues are dissolved in methanol for identification by thin layer chromatographic technique. All reagents must be of analytical grade. 3.1. Dichloromethane 3.2. Methyl 3.3. Water 3.4. Methanol 3.5. Anisaldehyde 3.6. Sulfuric acid (concentrated) 3.7. Hydrochloric acid (0.5 M) 3.8. Ammonium hydroxide (0.5 M) 3.9. Glacial acetic acid 3.10. Tetrazolium blue 3.11. Sodium hydroxide (pellets) 3.12. Ethyl acetate 3.13. Developing solvent: Dichloromethane/Methyl Acetate /Water, 100:50:50 Use the lower layer of the mixture. 3.14. Reference materials: 3.14.1. Hydrocortisone acetate 3.14.2. Dexamethasone 3.14.3. Betamethasone 3.14.4. Betamethasone 17-valerate 3.14.5. Triamcinolone acetonide 3.15. Standard solutions 3.15.1. Weigh 10 mg of every reference material into separate 10 mL volumetric flask. Add 5 mL of methanol. Sonicate for 5 min, make up to volume with methanol. 3.15.2. Mixture of standard solutions: weigh 10 mg of every reference material into one 10 mL volumetric flask. Add 5 mL methanol. Sonicate for 5 min and make up to volume with methanol. 3.16. Spray reagents 3.16.1. Into a 100 mLvolumetric flask, add 50 mL glacial acetic acid. Then, add 0.5 mL anisaldehyde, and finally 11 mL sulfuric acid. Shake gently. IDENTIFICATION OF HYDROCORTISONE ACETATE, DEXAMETHASONE, BETAMETHASONE, 2/12/2005 ACM MAL 07 BETAMETHASONE 17-VALERATE AND TRIAMCINOLONE ACETONIDE IN COSMETIC PRODUCTS BY TLC AND HPLC 3.16.2. Other spray reagent for information: Alkaline tetrazolium blue solution : mix 10 mL of a 0.2% w/v solution of tetrazolium blue in methanol with 30 mL of a 12% w/v solution of sodium hydroxide in methanol (this solution should be freshly prepared). Normal laboratory equipment, and: 4.1. TLC plates, ready for use: silica gel 60 F254 nm 4.2. TLC 4.4. UV lamp 254 nm 4.5. Oven 4.6. Filter paper, "1 PS" or equivalent 4.7. Water 4.8. Shaker 4.9. pH 4.10. 0.45 µm syringe membrane filter, PVDF or equivalent 5. PROCEDURE 5.1 Preparation of the sample Take about 15 mL of sample. Neutralize to pH 7 with 0.5 M HCl or 0.5 M NH4OH, extract 2 times with 20 mL ethyl acetate, discard aqueous layer. Filter – if necessary - the combined extract into evaporating dish and evaporate to dryness in a water bath (indication time: 30 min). Dissolve the residue in 5 mL of methanol and filter using the syringe membrane filter. Weigh about 5 g of sample in a centrifuge tube and dissolve by adding 20 mL of methanol. Warm on water bath for 10 minutes and shake vigorously for 5 minutes using a shaker. Centrifuge at 3000 – 4000 rpm for 10 minutes and leave it in the freezer for 10 minutes. Evaporate the clear supernatant solution to dryness in the water bath (indication time: 1h). Dissolve the residue in 5mL of methanol and filter the solution using the syringe membrane filter. 5.2.1 Saturate a chromatographic tank with the developing solvent. 5.2.2 On a plate, deposit: • 20 µL of every reference solution, • 20 µL of the mixture of reference solutions, • 20 µL of the sample solution. Develop until the solvent front has migrated 15 cm from the start. 5.2.3 Remove the plate and allow to dry at room temperature. IDENTIFICATION OF HYDROCORTISONE ACETATE, DEXAMETHASONE, BETAMETHASONE, 2/12/2005 ACM MAL 07 BETAMETHASONE 17-VALERATE AND TRIAMCINOLONE ACETONIDE IN COSMETIC PRODUCTS BY TLC AND HPLC 5.3.1 Observe the plate under UV light at 254 nm, and mark the position of the spots. 5.3.2 Spray the plate with reagent 3.16.1 . Let it dry and put the plate in the oven at 120°C for 10 min and observe the spots. 6. INTERPRETATION OF RESULT Colour of spot
Estimated
Steroids
Spray reagent 3.16.1 Spray reagent 3.16.2
hydrocortisone acetate triamcinolone acetonide betamethasone 17- 7.1 This method is also applicable to identify the following steroids; cortisone acetate, prednisolone, prednisone, flucinolone acetonide, betamethasone 21-valerate, hydrocortisone. 7.2 Further screening by HPLC shall be carried out. 7.3 The limit of detection (LOD) shall be reported. This method specifies a procedure for further identification of hydrocortisone acetate, dexamethasone, betamethasone, betamethasone 17-valerate and triamcinolone acetonide in cosmetic products. Liquid samples suspected of containing steroid compounds are neutralized and extracted with ethyl acetate. Cream samples suspected of containing steroid compounds are extracted with methanol. The extracted solutions are evaporated to dryness. Residues are dissolved in methanol for identification by reversed phase liquid chromatography with UV detection. All reagents must be of analytical quality. Water used must be distilled water, or water of at least equivalent purity 3.1 Acetonitrile, HPLC grade 3.2 Ammonium hydroxide (0.5 M) 3.3 Methanol, HPLC grade 3.4 Ethyl 3.5 Hydrochloric acid (0.5 M) 3.6 Mobile phase : Acetonitrile : Water IDENTIFICATION OF HYDROCORTISONE ACETATE, DEXAMETHASONE, BETAMETHASONE, 2/12/2005 ACM MAL 07 BETAMETHASONE 17-VALERATE AND TRIAMCINOLONE ACETONIDE IN COSMETIC PRODUCTS BY TLC AND HPLC Time (min)
Ratio (%)
Acetonitrile
Note: the above gradient conditions should be adjusted when necessary to the type of column in use. 3.7 Reference 3.7.1 Hydrocortisone 3.7.2 Dexamethasone 3.7.3 Betamethasone 3.7.4 Betamethasone 3.7.5 Triamcinolone acetonide 3.8.1 Weigh 10 mg of every reference material into separate 10 mL volumetric flask. Add 5 mL of methanol. Sonicate for 5 minutes, make up to volume with methanol. Filter using a syringe membrane filter. 3.8.2 Mixture of standard solutions: weigh 10 mg of every reference material into one 10 mL volumetric flask. Add 5 mL methanol. Sonicate for 5 minutes and make up to volume with methanol. Filter using a syringe membrane filter. Normal laboratory equipment and: 4.1 Water bath, capable of maintaining a temperature of 60 °C 4.2 High-performance liquid chromatograph with a variable wavelength UV detector and 20 µL Stainless steel chromatographic column, length 250 mm, internal diameter 4,6 mm, (packed with ODS), particle size 5 µm, or equivalent. 4.4 Filter paper, 1 PS or equivalent 4.5 Ultrasonic 4.6 Shaker 4.7 pH 4.8 Syringe membrane filter (0.45 µm pore size)
5. PROCEDURE
Take about 15 mL of sample. Neutralize to pH 7 with 0.5 M HCl or 0.5 M NH4OH, extract 2 times with 20 mL ethyl acetate, discard aqueous layer. Filter – if necessary - the combined extract into evaporating dish and evaporate to dryness in a water bath (indication time: 30 min). Dissolve the residue in 5 mL of methanol and filter using the syringe membrane filter. IDENTIFICATION OF HYDROCORTISONE ACETATE, DEXAMETHASONE, BETAMETHASONE, 2/12/2005 ACM MAL 07 BETAMETHASONE 17-VALERATE AND TRIAMCINOLONE ACETONIDE IN COSMETIC PRODUCTS BY TLC AND HPLC Weigh about 5 g of sample in a centrifuge tube and dissolve by adding 20 mL of methanol. Warm on water bath for 10 minutes and shake vigorously for 5 min using a shaker. Centrifuge at 3000 – 4000 rpm for 10 min and leave it in the freezer for 10 min. Evaporate the clear supernatant solution to dryness in the water bath (indication time: 1h). Dissolve the residue in 5mL of methanol and filter the solution using the syringe membrane filter. 5.2 High performance liquid chromatography 5.2.1 Adjust the flow rate of the mobile phase to 1.2 mL/min and set the detector wavelength to • 20 µL of every reference solution, • 20 µL of the mixture of reference solutions, • 20 µl of the sample solution. The relative standard deviation (RSD) of the retention time should be less than 1%, and the RSD of 6 replicate injections should be less than 2%. 5.2.4 Compare the retention time of the chromatograms obtained for sample and reference 6. INTERPRETATION 6.1 The retention time of reference and sample solution should not differ by more than ± 1 %. 6.2 The estimated retention (RT) for the steroids are as follows: Estimated retention time (RT) (min) Betamethasone 15 Dexamethasone 16 Triamcinolone Acetonide Hydrocortisone Acetate Betamethasone 17-valerate 7. Limit of detection (LOD) and limit of quantitation (LOQ): IDENTIFICATION OF HYDROCORTISONE ACETATE, DEXAMETHASONE, BETAMETHASONE, 2/12/2005 ACM MAL 07 BETAMETHASONE 17-VALERATE AND TRIAMCINOLONE ACETONIDE IN COSMETIC PRODUCTS BY TLC AND HPLC Hydrocortisone Acetate Triamcinolone Acetonide Betamethasone 17-valerate This method is also applicable to identify the following steroids; cortisone acetate, prednisolone, prednisone, flucinolone acetonide, betamethasone 21-valerate and hydrocortisone Combine the results from TLC and HPLC to identify the presence of the steroids within the scope of the method. Harmonised method:

Issued by the chemical analysis group at the harmonization workshop in Kuala-
Lumpur, on September 13th to 17th, 2004
Approved by the harmonization workshop delegates workshop in Kuala-Lumpur, on
September 13th to 17th, 2004,
Modified after the Kuala-Lumpur training, Dec 6 th to Dec 10 th, 2004
Modified and approved after the Brunei workshop, Aug 30th to 31st, 2005
Modified and approved after the final review in Singapore, Nov 30th to Dec 2nd, 2005

Source: http://www.asean.org/storage/images/archive/MRA-Cosmetic/Doc-8.pdf

Letter

KPMG IFRG Limited Tel +44 (0) 20 7694 8089 Fax +44 (0) 20 7694 8429 8 Salisbury Square DX 38050 Blackfriars London EC4Y 8BB sylvia.smith@kpmgifrg.com Technical Director International Auditing and Assurance International Federation of Accountants 545 Fifth Avenue, 14th Floor New York, New York 10017 Contact Sylvia Smith IAASB Exposure Draft, Proposed International Standard on Review Engagements: ISRE 2400 (Revised), Engagements to Review Historical Financial Statements

Microsoft powerpoint - eating disorders2006

Chisoo Choi, M.D. Brookhaven Hospital "There were times I felt fat. I had a distorted image of myself" Ana Carolina Reston Learning Objectives  Diagnostic criteria  Differential diagnosis  Medical consequences  Treatment approach  Psychotropic medications Learning Objectives  Identify 3 major types of eating disorders  List the diagnostic criteria for each  Determine the healthy weight & BMI