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LABORATORY INFORMATION BULLETIN
DFS/ORA No 3966
THE DETERMINATION OF FREE UNDECYLENIC ACID
AND UNDECYLENIC ACID ZINC SALT
IN FOOT POWDER USING HPLC AND ATOMIC ABSORPTION
BY
DIANE T. OBRIEN,
BALTIMORE DISTRICT
The DOD drug section of the Baltimore District Laboratory received a product of foot powder for which free undecylenic acid and undecylenic acid zinc salt had to be determined. The USP XXIII monograph for Compound Undecylenic Acid Ointment determines the total amount of undecylenic acid and the amount of undecylenic acid zinc salt using two separate determinative steps: gas chromatography and atomic absorption. The total amount of undecylenic acid is determined by using several chloroform extractions, forming a derivative with the use of a silylating agent and finally injecting the derivative onto a GC column. Chloroform and the silylating agent are hazardous materials.1 Therefore, a method for free undecylenic acid was developed utilizing hexane as a diluent and HPLC as the determinative step.2 The retention time of the undecylenic acid was found to be about 6 minutes for this reproducible and linear method. The USP XXIII atomic absorption3 method for the determination of undecylenic acid zinc salt was modified to accomodate modern atomic absorption spectrometers. A linear range of 0.5 ppm to 2.0 ppm zinc was used. The product, which contains about 78% talc, 20% zinc undecylenate and 2% undecylenic acid can be analyzed for free undecylenic acid and zinc undecylenate using this efficient HPLC procedure and linear atomic absorption method.
NOTE: THE LABORATORY INFORMATION BULLETIN IS A TOOL FOR THE RAPID DISSEMINATION OF LABORATORY METHODS (OR INFORMATION) WHICH APPEAR TO WORK. IT MAY NOT REPORT COMPLETED SCIENTIFIC WORK. THE USER MUST ASSURE HIMSELF/HERSELF BY APPROPRIATE VALIDATION PROCEDURES THAT LIB METHODS AND TECHNIQUES ARE RELIABLE AND ACCURATE FOR THIS INTENDED USE. REFERENCE TO ANY COMMERCIAL MATERIALS, EQUIPMENT, OR PROCESS DOES NOT IN ANY WAY CONSTITUTE APPROVAL, ENDORSEMENT, OR RECOMMENDATION BY THE FOOD AND DRUGADMINISTRATION.
METHOD FOR THE DETERMINATION OF FREE UNDECYLENIC ACID IN FOOT POWDER
EQUIPMENT AND REAGENTS:
Instrumentation: ABI 783A variable wavelength detector set at 200 nm, Perkin Elmer Series 410 LC pump and Perkin Elmer ISS-100 autoinjector or an equivalent HPLC system.
Column: Waters Micro-Bondapak C-18, 10 micron, 300 mm x 3.9 mm i.d..
HPLC Mobile Phase: 40% water with 0.1% phosphoric acid : 60 % HPLC grade Acetonitrile. The water is prepared by adding 1 mL of 86.0% Baker Analyzed phosphoric acid to 1000 mL of deionized degassed water.
Reference standards: Undecylenic Acid, Sigma Chemical Co. catalogue number U-1754, 99.0% purity by gas chromatography.
Undecylenic Acid Zinc Salt, Sigma Chemical Co., catalogue number U-0625, 98.8% purity by gas chromatography. Talcum Powder, Allied Chemical lot # V193.
Phosphoric Acid: Baker Analyzed, assay 86.0%
Hexane and Acetonitrile: EM Science, OmniSolv, glass distilled, HPLC grade suitability.
PROCEDURE:
Preparation of working standard solution: Weigh accurately a portion of undecylenic acid equivalent to 25 mg into a 50.0 mL volumetric flask and dilute to volume with HPLC reagent grade hexane. The final concentration obtained should be 0.50 mg/mL undecylenic acid. Dilute this standard solution to obtain a working solution with a 0.15 mg/mL concentration. Keep standard solutions well capped and stored in refrigerator.
Preparation of standard for the linear curve: Weigh a 25 mg portion of undecylenic acid accurately into a 50 mL volumetric flask. Dilute to volume with hexane. This stock standard is equivalent to 0.5 mg/mL solution. All other working standards for the standard curve are prepared from dilutions of this stock solution. Prepare five standard working solutions having final concentrations of 0.06, 0.10, 0.12, 0.16, 0.20 mg/mL undecylenicacid and label them as STD A,B,C,D,E, respectively. Note: Although a standard curve does not need to be utilized for each analysis, it was prepared to ascertain that the products would be analyzed within a linear working range of the detector and HPLC system.
Preparation of sample: Weigh accurately a 0.5 gram portion of the foot powder into a 100.0 mL volumetric flask. Add HPLC grade hexane to volume. Invert about five times, allow solids to settle, and then take a portion of the hexane supernatant for injection. Samples were not filtered. Solutions can be stored under refrigeration for short periods of time, although the extracts are not stable overnight.
Preparation of spiked placebo samples: Accurately weigh out a portion of Talc equivalent to 0.39 grams, a portion of zinc undecylenate equivalent to 0.10 grams and a portion of undecylenic acid equivalent to 0.01 grams into a 100 mL volumetric flask. Dilute to volume with HPLC grade hexane and treat as the sample.
Chromatographic Conditions:
Column: 300 mm x 3.9 mm, 10 micron, Micro-Bondapak C-18 or equivalent
Column Temperature: Maintained at 400C
Mobile Phase: 40% water 0.1% phosphoric acid : 60% Acetonitrile
Flow Rate: 1.0 mL/min
Variable Wavelength Detector-UV wavelength: 200 nm, 0.10 range at the 10mv setting
System Suitability: Five replicate injections of the working standard solution provide an RSD of NMT 2.0%. The tailing factor obtained for undecylenic acid is NMT 2.0. The theoretical plates can not be less than 1000.
Analytical procedure:
Inject 30 ul portions of the standards, sample and blank containing hexane onto the chromatograph system described above. Duplicate injections of samples should be made, as well asbracketing between standard solution injections. Calculate the % free undecylenic acid by using the following formula:
area of spl x conc. of std x dilution factor x 100= % free
area of std wt. of spl undecylenic
acid
Linearity summary: The system is linear (Corr. Coef. 0.9990) over the concentration range of 0.06 to 0.20 mg/mL using area counts. The values obtained for the samples were calculated using peak heights and areas with out much difference being noted in the final values. A copy of the linear regression curve is attached as Figure 1.
RESULTS AND DISCUSSION:
Recovery summary: Spiked Placebo samples were analyzed using the aforementioned method.
SPIKED PLACEBO CONTENTS RESULTS/RECOVERIES
78.9% talc, 19.9% zinc 2.05% undecylenic acid, 99.0% recovery
undecylenate and
2.07% undecylenic Acid
77.9% talc, 20.1% zinc 2.04% undecylenic acid, 98.0% recovery
undecylenate and
2.08% undecylenic acid
97.95% talc and 2.04% 2.08% undecylenic acid, 102.0% recovery
undecylenic acid
79.9% talc and 20.14% No undecylenic acid was found *
zinc undecylenate
*This proves that zinc undecylenate is not soluble in hexane and does not dissociate to the free acid.
Sample summary: Duplicate portions of two samples of foot powder were analyzed for free undecylenic acid using two different HPLC systems each fitted with a different HPLC C-18 column. They were found to contain:
Sample % undecylenic acid found
1a 1.53, 1.39 Avg = 1.46
1b 1.41, 1.47 Avg = 1.44
2a 1.50, 1.43 Avg = 1.46
2b 1.51, 1.36 Avg = 1.44
1a,2a Applied Biosystems Programmable Absorbance Detector
1b,2b Spectroflow 757 Variable Absorbance Detector
Chromatograms of the blank, sample and standard are shown in
Figure 2.
In conclusion, this HPLC method is reproducible, rapid and accurate for the determination of free undecylenic acid in foot powder.
METHOD FOR THE DETERMINATION OF ZINC
UNDECYLENATE IN FOOT POWDER
The following method was developed to replace the USP XXIII method for the determination of zinc undecylenate in Compound Undecylenic Acid Ointment. The modifications in the USP XXIII method are necessary to accomodate the newer atomic absorption spectrometers and specify a linear range for the analysis of zinc in undecylenic acid zinc salt contained in foot powder.
EQUIPMENT AND REAGENTS:
Instrumentation: Perkin Elmer 3100 set for Zinc EDL lamp,
214 nm wavelength, 0.7 nm slit width, air-acetylene flame. Maximize the signal by using the highest standard prepared.
Reference Standards: Zinc NIST Reference Standard Solution #3168 Concentration of 10.00 mg/mL Zinc in 10% HCl
Reference materials used for sample placebos: Undecylenic Acid, Sigma Chemical Co., catalogue number U-1754, 99.0% pure by gas chromatography. Undecylenic Acid Zinc Salt, Sigma Chemical Co., catalogue number U-0625, 98.8% purity by gas chromatography. Talcum Powder, Allied Chemical, lot# V193.
Phosphoric Acid: Baker Analyzed, assay 86.0%
Methanol: EM Science, OmniSolv, glass distilled, HPLC grade suitability.
PROCEDURE:
Preparation of glassware: Collect all necessary volumetric glassware and rinse with (3 + 1) nitric acid : water solution.
Preferably fill volummetric to top, stopper and remain soaking overnight. Rinse well with distilled water prior to use.
Preparation of working standard solution: Pipette 2.0 mL of the NIST Zinc standard reference solution into a 200.0 mL volumetric. (final concentration = 100 ppM zinc) Pipette 10.0 mL of this solution into a 100.0 mL volumetric flask (final concentration = 10.0 ppM zinc)
Preparation of standard for the linear curve: Prepare astandard curve using the 10.0 ppM zinc standard solution having final concentrations of 0.5 ppM, 1.0 ppM and 2.0 ppM zinc. All dilutions are made using distilled water. A standard blank of distilled water is also prepared for analysis.
Preparation of sample: Weigh accurately into a 100.0 mL volumetric flask 0.5 grams of product. Add enough methanol (about 2-3 mLs) to wet the powder, add 1.0 mL of phosphoric acid, swirl to mix, then add about 50 mLs of methanol. Place in sonicator for 15 minutes. Remove from sonicator, allow to come to room temperature, bring to volume with methanol. Pipette 2.0 mL of the solution to a 200.0 mL volumetric and dilute to volume with water. These solutions are ready for the atomic absorption determinative step.
Preparation of spiked placebo samples: Accurately weigh out a portion of talcum powder equivalent to 0.39 grams, a portion of zinc undecylenate equivalent to 0.10 grams and a portion of undecylenic acid equivalent to 0.01 grams into a 100 mL volumetric flask. As with the samples, add enough methanol (about 2-3 mLs) to wet the powder, add 1.0 mL of phosphoric acid, swirl to mix, then add about 50 mLs of methanol. Place in sonicator for 15 minutes. Remove from sonicator, allow to come to room temperature, bring to volume with methanol. Pipette 2.0 mL of the solution to a 200.0 mL volumetric and dilute to volume with water. These solutions are ready for the atomic absorption determinative step.
Preparation of a placebo blank: Accurately weigh out a portion of talcum powder equivalent to 0.39 grams, add enough methanol to wet the powder, add 1.0 mL of phosphoric acid, swirl to mix and dilute with methanol to 100.0 mL. Pipette 2.0 mLs into a 200.0 mL volumetric and dilute to volume with water.
Analytical procedure: the determinative step: Set your atomicabsorption spectrophotometer to obtain the maximum signal using the 2.0 ppm zinc standard solution. Aspirate the standards, standard blank, sample blank, samples and a standard curve. Determine the ppm of zinc in each sample and calculate the amount of zinc undecylenate using the following formula:
ppm zinc x 431.92 x 10,000mL x 1 gram x 100 = gram zinc
65.38 1,000,000 ug undecylenate
where:
ppm zinc comes from the linear regression curve and is expressed in ppm (ug/mL)
431.92 = molecular weight of zinc undecylenate
65.38 = molecular weight of undecylenic acid
10,000 mL = dilution factor
grams of zinc undecylenate found x 100 = % zinc undecylenate grams of sample weighed found
% zinc undecylenate found x 100 = % of declared % declared on label
RESULTS AND DISCUSSION:
Recovery summary: Spiked placebo samples were analyzed using the aforementioned method.
Spiked placebo contents Results/Recoveries 77.71% talc, 20.23% zinc 21.10% zinc undecylenate, 104.3% undecylenate and 2.07% recovery undecylenic acid 77.41% talc, 19.89% zinc 21.01% zinc undecylenate, 105.6% undecylenate and recovery 2.697% undecylenic acid Linearity summary: The system is linear over the range of 0.5 ppm to 2.0 ppm zinc. A correlation coefficient of 0.999 was found. A copy of the linear regression curve is attached as Figure 3.
Sample results: Duplicate portions of two samples were analyzed and found to contain:
Sample % zinc % declared
undecylenate
found
1a 19.97 99.85
1b 20.67 103.3
Avg 20.32 101.6
2a 20.41 102.1
2b 20.46 102.3
Avg 20.435 102.2
In conclusion, this is a linear method which should be utilized for the determination of zinc undecylenate in foot powder.
REFERENCES
(1) The Merck Index, Tenth Edition, 1983, #9650.
(2) Undecylenic acid by Low Wavelength HPLC, Laboratory Information Bulletin No. 3299.
(3) Compound Undecylenic Acid Ointment, USP XXIII, p. 1617.
Graphic Attachments
Page 10 - Linear Regression Curve for undecylenic acid standards.
