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Determination of Norethindrone Stability on Red Delicious Apples
Determination of Norethindrone Stability on Red Delicious Apples DERRAL0.MAYBERRY*, MARIEKOW6b4NSKYtS, PHILIP A. LANE'',
AND PAUL
E. WRAY'
Received August 24, 1988, from the R. W. Johnson Pharmaceutical Research Institute, Orfho Pharmaceutical Corporation, Raritan, NJ 08869-0602. Accepted for publication November 8, 1989. 'Present address: National Starch, Bridgewater, NJ. _-._____
Abstract A stability-indicatingassay procedure for the determination of norethindrone on the surface of Red Delicious apple slices was developed. The apple slice is homogenized in pH 10 borate buffer solution and norethindrone is then partitioned into chloroform using a diffusionchamber that has a membranethat is permeableto hydrophobic compounds separating the buffer and chloroform. An aliquot of the chloroform extract is then evaporated to dryness and reconstituted in tetrahydrofuran. The reconstitutedsample is then chromatographed on a C,B reversed-phase column using a mobile phase consisting of water:tetrahydrofuran:methanol(63:26:11, v/v/v). Detection is in the UV range at 254 nm. The chromatographic system is specific for norethindrone in the presence of its autoxidationproducts. Stability data indicate that norethindroneis stable for up to 6 h on the surface of Red Delicious apples.
I
X
RiJxl!
JiUA
Norethindrone [( 17a)-17-hydroxy-19-norpregn-4-en-20-yn%one; 1; see structure] i s a progestational agent commonly used in oral contraceptive tablets in conjunction w i t h a n estrogen, such as ethinyl estradiol or mestranol. The clinical pharmacokinetics o f norethindrone have been reviewed by Orme e t al.1 Liquid chromatographic methods for the determination of norethindrone, alone o r in the presence o f estrogens, in a variety o f matrices have been published.2-12 To support a long-term norethindrone drug safety study in which norethindrone was fed to monkeys o n slices o f Red Delicious apples, it was necessary to develop a stability-indicating assay procedure for norethindrone, since n o methods for norethindrone o n apples have been published. This paper describes a stability-indicating H P L C method for the determination of norethindrone o n Red Delicious apples.
1
Y
Y
0
H
H
-OH
6
1
H
0
H
H
0
ti
OH
0
CH
Ii
-OH
dehydro
dehydro
=O
H
0
=O
Experimental Section Instruments-A liquid chromatograph consisting of a Waters model M6000 pump and model 440 fixed wavelength (254 nm) detector, and a Rheodyne model 7120 injector with a 10-pL sample loop was operated a t ambient room temperature. Samples were injected onto a Waters C,, flondapak reversed-phase column (30 cm x 3.9 mm i.d.). All data were collected using a Hewlett-Packard Laboratory Automation System. Materials-All chemicals were reagent grade unless noted otherwise and were purchased from commercial sources. Burdick and Jackson spectrograde methanol and tetrahydrofuran were used without additional purification. Norethindrone Reference Standard was obtained from the USP.The diffusion chamber was assembled using Bellco model 1972-00250 spinner flasks as shown in Figure 1. Cole-Palmer model 6370-00 3-blade stirrers equipped with tachometers were used to stir all solutions a t 500 rpm. Samples were homogenized using a Brinkman model pT10/35 homogenizer. The poly(dimethy1siloxane) membrane (Silastic; 0.005-inch thickness) used to separate the chloroform and aqueous compartments of the diffusion chamber was purchased from Dow-Corning (Midland, MI). Chromatographic Conditions-The mobile phase was water:tetrahydrofuran:methano1(63:26:11,v!v/v). The solution was sonicated for 15 min to deaerate the mixture, and then used to equilibrate the column at a rate of 1.7 mumin. 746 I Journal of Pharmaceutical Sciences Vol. 79, No. 8, August 1990
(+)
- Ring A is benzenoid
Preparation of pH 10 Buffer Solution-The pH 10 buffer solution was prepared by combining 30.9 g of boric acid, 37.3 g of KCI, and 462 mL of 0.05 M NaOH in a 1-L flask; this was then dissolved in and diluted to volume with distilled water. The pH was adjusted to 10 with either acid or base as necessary. Standard Solutions-Approximately 70 mg of norethindrone reference standard was accurately weighed and transferred into a 100-mL volumetric flask and diluted to volume with 95% ethanol. This solution is stable a t ambient room temperature for up to three months. A 10.0-mL portion of the stock norethindrone solution was pipetted into a 100-mL volumetric flask and diluted to volume with 95% ethanol. A 5.0-mL aliquot of the diluted stock solution was then pipetted into a stoppered 50-mL centrifuge tube and evaporated to dryness under a stream of nitrogen a t 4050°C. For apple slices containing 30-200 pg of norethindrone, the residue was reconstituted in 25.0 mL of tetrahydrofuran. For slices containing 200-3500 pg of norethindrone, 5.0 mL of tetrahydrofuran was added. A 10-pLaliquot of the corresponding standard and sample were then injected into the OO22-3549/90/08OO-O746$01 .OO/O 0 1990, American Pharmaceutical Association
7 S t l r r l n g Motors
-
Results and Discussion The chromatographic system described here separates intact norethindrone from its autoxidation products and from
1
L-Ff
A
+Chloroform
Aqueous
Dlsperslon of Homogenized hPP 1e L
Figure 1-Diagram
0.005" Pely( dlsethylsl loxane ) Uembrane
of the diffusion chamber.
chromatograph for quantitation. Solutions of norethindrone in tetrahydrofuran were prepared daily since norethindrone was found to be stable for only a few days in tetrahydrofuran. Norethindrone Autoxidation Decomposition ProductsNorethindrone drug substance was subjected to autoxidative degradation in an oxygen atmosphere (82 psi) at 170 "C for 4.5 h. The following compounds were isolated and characterized (see structure: ethinyl estradiol(Z),6,7-dehydronorethindrone(3),6&hydroxynorethindrone (4), 10P-hydroxynorethindrone( 5 ) ,6-keto-ethinylestradiol (6), and 6-keto-norethindrone(7). Recovery Studies-One half of an apple was homogenized in 200 mL of pH 10 buffer solution and was transferred into one of the compartments of the diffusion chamber. The homogenizer and homogenizing vessel were each rinsed with 50 mL of pH 10 buffer solution which was then added to the homogenized apple solution. A 500-pL aliquot of ethanol containing either 35, 350, or 3500 pg of norethindronewas added directly to the homogenized apple solution. Approximately 350 mL of chloroform was placed into the other compartment of the chamber. After stirring both solutionsat 500 rpm for at least 18 h, the chloroform solution was then carefully poured into a 500-mLgraduated cylinder and the volume was recorded. After filtering the chloroform solution through a Millipore filter (#FHLP02500),a 50-mL portion of the filtrate was pipetted into two 50-mL centrifuge tubes and evaporated to dryness under a stream of nitrogen at 40-50 "C. At this point in the analysis, the samples can be stored at ambient room temperature for several days before continuing,The residue was then dissolved in tetrahydrofuran using 0.5, 1.0, or 10.0 mL for the 35-, 350-, and 3500-pg samples, respectively. A 1 0 - 4 aliquot of the appropriate standard and sample was then injected into the HPLC for analysis. Apple Slices Containing Norethindrone-Apple slices containing norethindrone were cut into several pieces and were placed into a homogenizing vessel containing 200 mL of pH 10 buffer solution.The same procedure described above in the recovery study was employed with the exception of the addition of the ethanolic norethindrone aliquots. The volume of tetrahydrofuran used to reconstitute the residue for HPLC analysis depended on the amount of norethindrone present in the sample. The amount of norethindrone in the apple slice is calculated using the following equation:
any potential interfering substances from the apple slice. The selectivity of the chromatographic system to separate norethindrone from its autoxidation products is shown in Figure 2. The chromatographic characteristics of norethindrone and its autoxidation products are shown in Table I. The chromatogram in Figure 2 shows that norethindrone (1) is baseline separated from all of its autoxidation products, except 6,7dehydronorethindrone (3) and 6-keto-ethinyl estradiol (6). Although 3 and 6 are not baseline separated from norethindrone, their presence i n the apple samples will be observed. Preliminary attempts to extract norethindrone from the apple matrix using conventional liquid-liquid partitioning techniques indicated that substantial amounts of interfering substances were being extracted from the apple. Varying the pH, buffer composition, and extraction solvents did not satisfactorily remove the interfering substances. Since the conventional approach to isolating norethindrone was unsuccessful, the use of a diffusion chamber with a nonporous poly(dimethylsiloxane) membrane separating the compartments was investigated. The poly(dimethylsi1oxane) membrane was chosen because it is permeable to hydrophobic compounds and the apple constituents (primarily polar compounds such as carbohydrates and polysaccharides) would be excluded by the membrane and would not diffuse into the chloroform. Nonpolar apple compounds present i n the aqueous phase, however, are transported into the chloroform along with norethindrone. Exploratory experiments over a pH range of 3 to 10 indicated that fewer components from the apple matrix were extracted into chloroform at pH 10 than at the lower pHs. Based on these experiments, pH 10 was chosen as the
2
pg of norethindronelapple slice =
0 where RtSAMand &Dl are the peak areas of norethindrone in the samples and standards, respectively, C i s the concentration of norethindrone in CLglmL in the working standard; V,,, is the volume of tetrahydrofuranused to reconstitute the sample, V,,, is the volume of chloroform measured after extraction and 1/50 is the dilution factor.
11.25
22.50
T IME, MIN showing separation of norethindrone from its autoxidation products. Key: (1) norethindrone; (2)ethinyl estradiol; (3) 6,7-dehydronorethindrone; (4) Gphydroxynorethindrone; (5) lophydroxynorethindrone; (6) 6-keto-ethinyl estradiol; (7) 6-ketonorethindrone; (8) impurity in 4; (9) impurity in 7. Figure 2-Chromatogram
Journal of Pharmaceutical Sciences I 747 Vol. 79, No. 8, August 1990
Table Whromatographlc Characteristics of Norethlndrone and Its Autoxldatlon Products a R8 K No. Compound 1 2
Norethindrone Ethinyl estradiol
3
6,7-Dehydronorethindrone
1 .Ob 2.6 0.90
6pHydroxynorethindrone 1Op-Hydroxynorethindrone 6-keteEthinyl estradiol 6-keteNorethindrone
4 5
6 7
0.31 0.40 1.1
0.71
-
4.3 11.1 3.9
10.0 0.9 8.9 7.0 0.5 2.8
1.3 1.a
4.8 3.1
Q
'Resolution as defined in USP XXII, p. 1567.
cn
Retention time of norethindrone was 11.6 min.
w
Iy
optimum pH of the aqueous phase for use with chloroform in subsequent method development. Reconstitution of dried aliquots of the chloroform extract in either methanol, acetonitrile, or tetrahydrofuran showed that tetrahydrofuran dissolved less of the apple residue and afforded additional sample cleanup prior to HPLC analysis. A chromatogram obtained from a Red Delicious apple without added norethindrone after extraction and reconstitution in tetrahydrofuran is shown in Figure 3. A stability study of norethindrone dissolved in tetrahydrofuran showed a 3 and 7% loss after 3 and 7 days of storage at 25 "C, respectively, indicating the need to prepare norethindrone standard solution in tetrahydrofuran on a daily basis. A linear regression analysis of the detector response (area) over the range of potential norethindrone concentration (15-350 pglmL) yielded a straight line with a coefficient of determination (R2) of 0.9999. Experiments carried out to determine the length of time required for complete extraction of norethindrone from pH 10 buffer solution into chloroform showed that a minimum of 9 h was required for complete transport of norethindrone into chloroform. Due to the length of time required for complete extraction, a minimum overnight extraction time of a t least 18 h was employed.
0
20
10 T I ME (MINI
Figure 3-Chromatogram of placebo Red Delicious apple. Table Il-Norethlndrone Recovery from Red Dellclous Apples Three, Four, and SIX Hours after Preparatlon Norethindrone Norethindrone Recovered, 9'0 Added, pg
35 350 3500
3h
4h
6h
94.3 4 6.8 (n = 6) 93.6 2 4.6 (n = 4) 96.1 4 5.4 (n = 6)
101.3 ? 7.0
(n = 5)
106.6 t 3.8 (n = 4)
-
-
99.3 2 4.5 (n = 6)
748 I Journal of Pharmaceutical Sciences Vol. 79,No. 8, August 1990
99.8
(n
2
=
3.4 4)
c W
n
0
I0 TIME (MINI
Flgure 4-Chromatogramof norethindrone on a R e d Delicious apple. Having established the optimum pH, the minimum extraction time, and best solvent for reconstitution of the apple residue, norethindrone recovery studies from the apple matrix were designed using the following guidelines: (I) the quantity of norethindrone that could be administered to the monkey would range from 35 to 3500 pg of norethindrone per apple slice; (2)10 to 300 pL of an ethanolic solution of norethindrone would be volumetrically added by syringe onto the surface of the apple slice; and (3) the apples would be consumed by the monkeys within 2 h of preparation. When portions of an ethanolic solution containing either 35,350, or 3500 pg of norethindrone were added directly to a homogenized half slice of apple in pH 10 buffer solution in the diffusion chamber, aliquots of the chloroform phase withdrawn after 18 h of extraction afforded a n average recovery of the added norethindrone of 112.8 5 9.7% (n = 6),103.5 2 3.8% (n = 41, and 97.3 +- 4.0% (n = 6),respectively. A 24-h aliquot of the 35-pg sample was also assayed and afforded an average recovery of 112.4 5 3.8% (n = 4). The average recovery for the 18- and 24-h samples was not statistically different a t the 95% confidence level, but their standard deviations were. The recovery results for the 35-pg samples were considerably higher than those obtained for the 350- and 3500-pg levels. Since different volumes of ethanol would be used to add varying amounts of norethindrone to apple slices, the effect of ethanol in the buffer solution was investigated. When 35 pg of norethindrone was added directly to a homogenized apple buffer solution with 500 p L of ethanol, the average recovery was 112.8 2 9.7% (n = 6). When the ethanol was removed by evaporation prior to adding the homogenized apple solution, the average norethindrone recovery was 101.6 +- 4.1% (n = 6). Based on these data, it was concluded that when ethanol is present in the aqueous homogenized apple buffer solution some of the water-insoluble apple constituents dissolve and diffuse into the chloroform, causing a positive bias in the determination of norethindrone. When this conclusion was tested by substituting a stronger organic solvent, tetrahydrofuran, for ethanol in the buffer solution, a larger positive
bias (114 2 28%,n = 3) was obtained. Since the norethindrone-containing apple slices would be consumed by the monkeys within 2 h of preparation, the stability of norethindrone on the apple slice was studied by preparing apple slices and allowing them to age for up to 12 h. The data in Table I1 show that no degradation of norethindrone occurs for up to 6 h on the apple surface and that complete recovery is obtained. When norethindrone was in contact with the apple for 8 and 12 h, considerable loss of norethindrone was obtained. A typical chromatogram showing norethindrone on a Red Deliciousapple is shown in Figure
4. Sw nnerton, N. F.; Fischer, J. B. J. Liq. Chromatogr. 1980,3, ld5 5. Hirai, S.;Hussain, A.; Babhair, S. J.Pharm. Sci.1980,69,857. 6. Johnston, M.A. J. Chromatogr. 1981,216,269. 7. Sundaresan, G.M.; Goehl, T. J.; Prasad, V. K. J. Pharm. Sci. 1981,70,702. 8. Loo, J. C. K.; Brien, R. J. Liq. Chromatogr. 1981,4,871. 9. Carignan, G.;Lodge, B. A,; Skakum, W. J. Chromatogr. 1984, 315,470. 10. Papas, A.; Marchese, S. M.; Delaney, M. F. Liq. Chromatogr. Mag. 1985,3,354. 11. Fasanmade, A. A.; Fell, A. F.; Scott, H. P.Anal. Chim.Acta 1986,
4.
12. Lee, G.;Oyans, M.; Bautista, J.; Kushinsky, S. J. Liq. Chromatogr. 1987,10, 2305.
187., 233.
References and Notes 1. O F e , M. L'E.; Back, D. J.; Breckenridge, A. M. Clin. Pharmacokm. 1983;8, 95-136 2. Schroff, A,; Mo er, E. S. Analytical Pro les ofDrug Substances, Vol. 4;Flory, $.,Ed.; Academic: New ork, 1975;p 269. 3. Gluck, J. A.; Shek, E. J. Chromatogr. Sci. 1980,18,631.
#
Acknowledgments The authors wish to thank Drs. M. L. Cotter and A. Oylerofthe PRI Molecular S ectroscopy Department for providing authentic samples of the noretiindrone autoxidation products.
Journal of Pharmaceutical Sciences I 749 Vol. 79, No. 8, August 1990
AND PAUL
E. WRAY'
Received August 24, 1988, from the R. W. Johnson Pharmaceutical Research Institute, Orfho Pharmaceutical Corporation, Raritan, NJ 08869-0602. Accepted for publication November 8, 1989. 'Present address: National Starch, Bridgewater, NJ. _-._____
Abstract A stability-indicatingassay procedure for the determination of norethindrone on the surface of Red Delicious apple slices was developed. The apple slice is homogenized in pH 10 borate buffer solution and norethindrone is then partitioned into chloroform using a diffusionchamber that has a membranethat is permeableto hydrophobic compounds separating the buffer and chloroform. An aliquot of the chloroform extract is then evaporated to dryness and reconstituted in tetrahydrofuran. The reconstitutedsample is then chromatographed on a C,B reversed-phase column using a mobile phase consisting of water:tetrahydrofuran:methanol(63:26:11, v/v/v). Detection is in the UV range at 254 nm. The chromatographic system is specific for norethindrone in the presence of its autoxidationproducts. Stability data indicate that norethindroneis stable for up to 6 h on the surface of Red Delicious apples.
I
X
RiJxl!
JiUA
Norethindrone [( 17a)-17-hydroxy-19-norpregn-4-en-20-yn%one; 1; see structure] i s a progestational agent commonly used in oral contraceptive tablets in conjunction w i t h a n estrogen, such as ethinyl estradiol or mestranol. The clinical pharmacokinetics o f norethindrone have been reviewed by Orme e t al.1 Liquid chromatographic methods for the determination of norethindrone, alone o r in the presence o f estrogens, in a variety o f matrices have been published.2-12 To support a long-term norethindrone drug safety study in which norethindrone was fed to monkeys o n slices o f Red Delicious apples, it was necessary to develop a stability-indicating assay procedure for norethindrone, since n o methods for norethindrone o n apples have been published. This paper describes a stability-indicating H P L C method for the determination of norethindrone o n Red Delicious apples.
1
Y
Y
0
H
H
-OH
6
1
H
0
H
H
0
ti
OH
0
CH
Ii
-OH
dehydro
dehydro
=O
H
0
=O
Experimental Section Instruments-A liquid chromatograph consisting of a Waters model M6000 pump and model 440 fixed wavelength (254 nm) detector, and a Rheodyne model 7120 injector with a 10-pL sample loop was operated a t ambient room temperature. Samples were injected onto a Waters C,, flondapak reversed-phase column (30 cm x 3.9 mm i.d.). All data were collected using a Hewlett-Packard Laboratory Automation System. Materials-All chemicals were reagent grade unless noted otherwise and were purchased from commercial sources. Burdick and Jackson spectrograde methanol and tetrahydrofuran were used without additional purification. Norethindrone Reference Standard was obtained from the USP.The diffusion chamber was assembled using Bellco model 1972-00250 spinner flasks as shown in Figure 1. Cole-Palmer model 6370-00 3-blade stirrers equipped with tachometers were used to stir all solutions a t 500 rpm. Samples were homogenized using a Brinkman model pT10/35 homogenizer. The poly(dimethy1siloxane) membrane (Silastic; 0.005-inch thickness) used to separate the chloroform and aqueous compartments of the diffusion chamber was purchased from Dow-Corning (Midland, MI). Chromatographic Conditions-The mobile phase was water:tetrahydrofuran:methano1(63:26:11,v!v/v). The solution was sonicated for 15 min to deaerate the mixture, and then used to equilibrate the column at a rate of 1.7 mumin. 746 I Journal of Pharmaceutical Sciences Vol. 79, No. 8, August 1990
(+)
- Ring A is benzenoid
Preparation of pH 10 Buffer Solution-The pH 10 buffer solution was prepared by combining 30.9 g of boric acid, 37.3 g of KCI, and 462 mL of 0.05 M NaOH in a 1-L flask; this was then dissolved in and diluted to volume with distilled water. The pH was adjusted to 10 with either acid or base as necessary. Standard Solutions-Approximately 70 mg of norethindrone reference standard was accurately weighed and transferred into a 100-mL volumetric flask and diluted to volume with 95% ethanol. This solution is stable a t ambient room temperature for up to three months. A 10.0-mL portion of the stock norethindrone solution was pipetted into a 100-mL volumetric flask and diluted to volume with 95% ethanol. A 5.0-mL aliquot of the diluted stock solution was then pipetted into a stoppered 50-mL centrifuge tube and evaporated to dryness under a stream of nitrogen a t 4050°C. For apple slices containing 30-200 pg of norethindrone, the residue was reconstituted in 25.0 mL of tetrahydrofuran. For slices containing 200-3500 pg of norethindrone, 5.0 mL of tetrahydrofuran was added. A 10-pLaliquot of the corresponding standard and sample were then injected into the OO22-3549/90/08OO-O746$01 .OO/O 0 1990, American Pharmaceutical Association
7 S t l r r l n g Motors
-
Results and Discussion The chromatographic system described here separates intact norethindrone from its autoxidation products and from
1
L-Ff
A
+Chloroform
Aqueous
Dlsperslon of Homogenized hPP 1e L
Figure 1-Diagram
0.005" Pely( dlsethylsl loxane ) Uembrane
of the diffusion chamber.
chromatograph for quantitation. Solutions of norethindrone in tetrahydrofuran were prepared daily since norethindrone was found to be stable for only a few days in tetrahydrofuran. Norethindrone Autoxidation Decomposition ProductsNorethindrone drug substance was subjected to autoxidative degradation in an oxygen atmosphere (82 psi) at 170 "C for 4.5 h. The following compounds were isolated and characterized (see structure: ethinyl estradiol(Z),6,7-dehydronorethindrone(3),6&hydroxynorethindrone (4), 10P-hydroxynorethindrone( 5 ) ,6-keto-ethinylestradiol (6), and 6-keto-norethindrone(7). Recovery Studies-One half of an apple was homogenized in 200 mL of pH 10 buffer solution and was transferred into one of the compartments of the diffusion chamber. The homogenizer and homogenizing vessel were each rinsed with 50 mL of pH 10 buffer solution which was then added to the homogenized apple solution. A 500-pL aliquot of ethanol containing either 35, 350, or 3500 pg of norethindronewas added directly to the homogenized apple solution. Approximately 350 mL of chloroform was placed into the other compartment of the chamber. After stirring both solutionsat 500 rpm for at least 18 h, the chloroform solution was then carefully poured into a 500-mLgraduated cylinder and the volume was recorded. After filtering the chloroform solution through a Millipore filter (#FHLP02500),a 50-mL portion of the filtrate was pipetted into two 50-mL centrifuge tubes and evaporated to dryness under a stream of nitrogen at 40-50 "C. At this point in the analysis, the samples can be stored at ambient room temperature for several days before continuing,The residue was then dissolved in tetrahydrofuran using 0.5, 1.0, or 10.0 mL for the 35-, 350-, and 3500-pg samples, respectively. A 1 0 - 4 aliquot of the appropriate standard and sample was then injected into the HPLC for analysis. Apple Slices Containing Norethindrone-Apple slices containing norethindrone were cut into several pieces and were placed into a homogenizing vessel containing 200 mL of pH 10 buffer solution.The same procedure described above in the recovery study was employed with the exception of the addition of the ethanolic norethindrone aliquots. The volume of tetrahydrofuran used to reconstitute the residue for HPLC analysis depended on the amount of norethindrone present in the sample. The amount of norethindrone in the apple slice is calculated using the following equation:
any potential interfering substances from the apple slice. The selectivity of the chromatographic system to separate norethindrone from its autoxidation products is shown in Figure 2. The chromatographic characteristics of norethindrone and its autoxidation products are shown in Table I. The chromatogram in Figure 2 shows that norethindrone (1) is baseline separated from all of its autoxidation products, except 6,7dehydronorethindrone (3) and 6-keto-ethinyl estradiol (6). Although 3 and 6 are not baseline separated from norethindrone, their presence i n the apple samples will be observed. Preliminary attempts to extract norethindrone from the apple matrix using conventional liquid-liquid partitioning techniques indicated that substantial amounts of interfering substances were being extracted from the apple. Varying the pH, buffer composition, and extraction solvents did not satisfactorily remove the interfering substances. Since the conventional approach to isolating norethindrone was unsuccessful, the use of a diffusion chamber with a nonporous poly(dimethylsiloxane) membrane separating the compartments was investigated. The poly(dimethylsi1oxane) membrane was chosen because it is permeable to hydrophobic compounds and the apple constituents (primarily polar compounds such as carbohydrates and polysaccharides) would be excluded by the membrane and would not diffuse into the chloroform. Nonpolar apple compounds present i n the aqueous phase, however, are transported into the chloroform along with norethindrone. Exploratory experiments over a pH range of 3 to 10 indicated that fewer components from the apple matrix were extracted into chloroform at pH 10 than at the lower pHs. Based on these experiments, pH 10 was chosen as the
2
pg of norethindronelapple slice =
0 where RtSAMand &Dl are the peak areas of norethindrone in the samples and standards, respectively, C i s the concentration of norethindrone in CLglmL in the working standard; V,,, is the volume of tetrahydrofuranused to reconstitute the sample, V,,, is the volume of chloroform measured after extraction and 1/50 is the dilution factor.
11.25
22.50
T IME, MIN showing separation of norethindrone from its autoxidation products. Key: (1) norethindrone; (2)ethinyl estradiol; (3) 6,7-dehydronorethindrone; (4) Gphydroxynorethindrone; (5) lophydroxynorethindrone; (6) 6-keto-ethinyl estradiol; (7) 6-ketonorethindrone; (8) impurity in 4; (9) impurity in 7. Figure 2-Chromatogram
Journal of Pharmaceutical Sciences I 747 Vol. 79, No. 8, August 1990
Table Whromatographlc Characteristics of Norethlndrone and Its Autoxldatlon Products a R8 K No. Compound 1 2
Norethindrone Ethinyl estradiol
3
6,7-Dehydronorethindrone
1 .Ob 2.6 0.90
6pHydroxynorethindrone 1Op-Hydroxynorethindrone 6-keteEthinyl estradiol 6-keteNorethindrone
4 5
6 7
0.31 0.40 1.1
0.71
-
4.3 11.1 3.9
10.0 0.9 8.9 7.0 0.5 2.8
1.3 1.a
4.8 3.1
Q
'Resolution as defined in USP XXII, p. 1567.
cn
Retention time of norethindrone was 11.6 min.
w
Iy
optimum pH of the aqueous phase for use with chloroform in subsequent method development. Reconstitution of dried aliquots of the chloroform extract in either methanol, acetonitrile, or tetrahydrofuran showed that tetrahydrofuran dissolved less of the apple residue and afforded additional sample cleanup prior to HPLC analysis. A chromatogram obtained from a Red Delicious apple without added norethindrone after extraction and reconstitution in tetrahydrofuran is shown in Figure 3. A stability study of norethindrone dissolved in tetrahydrofuran showed a 3 and 7% loss after 3 and 7 days of storage at 25 "C, respectively, indicating the need to prepare norethindrone standard solution in tetrahydrofuran on a daily basis. A linear regression analysis of the detector response (area) over the range of potential norethindrone concentration (15-350 pglmL) yielded a straight line with a coefficient of determination (R2) of 0.9999. Experiments carried out to determine the length of time required for complete extraction of norethindrone from pH 10 buffer solution into chloroform showed that a minimum of 9 h was required for complete transport of norethindrone into chloroform. Due to the length of time required for complete extraction, a minimum overnight extraction time of a t least 18 h was employed.
0
20
10 T I ME (MINI
Figure 3-Chromatogram of placebo Red Delicious apple. Table Il-Norethlndrone Recovery from Red Dellclous Apples Three, Four, and SIX Hours after Preparatlon Norethindrone Norethindrone Recovered, 9'0 Added, pg
35 350 3500
3h
4h
6h
94.3 4 6.8 (n = 6) 93.6 2 4.6 (n = 4) 96.1 4 5.4 (n = 6)
101.3 ? 7.0
(n = 5)
106.6 t 3.8 (n = 4)
-
-
99.3 2 4.5 (n = 6)
748 I Journal of Pharmaceutical Sciences Vol. 79,No. 8, August 1990
99.8
(n
2
=
3.4 4)
c W
n
0
I0 TIME (MINI
Flgure 4-Chromatogramof norethindrone on a R e d Delicious apple. Having established the optimum pH, the minimum extraction time, and best solvent for reconstitution of the apple residue, norethindrone recovery studies from the apple matrix were designed using the following guidelines: (I) the quantity of norethindrone that could be administered to the monkey would range from 35 to 3500 pg of norethindrone per apple slice; (2)10 to 300 pL of an ethanolic solution of norethindrone would be volumetrically added by syringe onto the surface of the apple slice; and (3) the apples would be consumed by the monkeys within 2 h of preparation. When portions of an ethanolic solution containing either 35,350, or 3500 pg of norethindrone were added directly to a homogenized half slice of apple in pH 10 buffer solution in the diffusion chamber, aliquots of the chloroform phase withdrawn after 18 h of extraction afforded a n average recovery of the added norethindrone of 112.8 5 9.7% (n = 6),103.5 2 3.8% (n = 41, and 97.3 +- 4.0% (n = 6),respectively. A 24-h aliquot of the 35-pg sample was also assayed and afforded an average recovery of 112.4 5 3.8% (n = 4). The average recovery for the 18- and 24-h samples was not statistically different a t the 95% confidence level, but their standard deviations were. The recovery results for the 35-pg samples were considerably higher than those obtained for the 350- and 3500-pg levels. Since different volumes of ethanol would be used to add varying amounts of norethindrone to apple slices, the effect of ethanol in the buffer solution was investigated. When 35 pg of norethindrone was added directly to a homogenized apple buffer solution with 500 p L of ethanol, the average recovery was 112.8 2 9.7% (n = 6). When the ethanol was removed by evaporation prior to adding the homogenized apple solution, the average norethindrone recovery was 101.6 +- 4.1% (n = 6). Based on these data, it was concluded that when ethanol is present in the aqueous homogenized apple buffer solution some of the water-insoluble apple constituents dissolve and diffuse into the chloroform, causing a positive bias in the determination of norethindrone. When this conclusion was tested by substituting a stronger organic solvent, tetrahydrofuran, for ethanol in the buffer solution, a larger positive
bias (114 2 28%,n = 3) was obtained. Since the norethindrone-containing apple slices would be consumed by the monkeys within 2 h of preparation, the stability of norethindrone on the apple slice was studied by preparing apple slices and allowing them to age for up to 12 h. The data in Table I1 show that no degradation of norethindrone occurs for up to 6 h on the apple surface and that complete recovery is obtained. When norethindrone was in contact with the apple for 8 and 12 h, considerable loss of norethindrone was obtained. A typical chromatogram showing norethindrone on a Red Deliciousapple is shown in Figure
4. Sw nnerton, N. F.; Fischer, J. B. J. Liq. Chromatogr. 1980,3, ld5 5. Hirai, S.;Hussain, A.; Babhair, S. J.Pharm. Sci.1980,69,857. 6. Johnston, M.A. J. Chromatogr. 1981,216,269. 7. Sundaresan, G.M.; Goehl, T. J.; Prasad, V. K. J. Pharm. Sci. 1981,70,702. 8. Loo, J. C. K.; Brien, R. J. Liq. Chromatogr. 1981,4,871. 9. Carignan, G.;Lodge, B. A,; Skakum, W. J. Chromatogr. 1984, 315,470. 10. Papas, A.; Marchese, S. M.; Delaney, M. F. Liq. Chromatogr. Mag. 1985,3,354. 11. Fasanmade, A. A.; Fell, A. F.; Scott, H. P.Anal. Chim.Acta 1986,
4.
12. Lee, G.;Oyans, M.; Bautista, J.; Kushinsky, S. J. Liq. Chromatogr. 1987,10, 2305.
187., 233.
References and Notes 1. O F e , M. L'E.; Back, D. J.; Breckenridge, A. M. Clin. Pharmacokm. 1983;8, 95-136 2. Schroff, A,; Mo er, E. S. Analytical Pro les ofDrug Substances, Vol. 4;Flory, $.,Ed.; Academic: New ork, 1975;p 269. 3. Gluck, J. A.; Shek, E. J. Chromatogr. Sci. 1980,18,631.
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Acknowledgments The authors wish to thank Drs. M. L. Cotter and A. Oylerofthe PRI Molecular S ectroscopy Department for providing authentic samples of the noretiindrone autoxidation products.
Journal of Pharmaceutical Sciences I 749 Vol. 79, No. 8, August 1990