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Stability of 17α-methyltestosterone in fish feed
Aquaculture 271 (2007) 523 – 529 www.elsevier.com/locate/aqua-online
Stability of 17α-methyltestosterone in fish feed Terence P. Barry a,⁎, Ashok Marwah b , Padma Marwah b a
University of Wisconsin — Madison, Department of Animal Sciences, Laboratory of Fish Endocrinology and Aquaculture, 660 North Park Street, Madison, WI 53706, United States b Department of Biochemistry, Enzyme Institute, 1710 University Avenue Madison, WI 53726, United States Received 14 January 2007; received in revised form 27 April 2007; accepted 1 May 2007
Abstract The synthetic steroid 17α-methyltestosterone (MT) is used as a feed additive to produce predominately male populations of tilapia (Oreochromis sp.). The legal use of MT by the U.S. tilapia industry requires an approved New Animal Drug Application (NADA) from the U.S. Food and Drug Administration (FDA). The present study was conducted to satisfy the remaining FDA data requirements in the area of product chemistry. The results indicated that (1) MT-treated feed is homogenously mixed during the manufacturing and bagging process, (2) MT remains uniformly distributed throughout the feed during prolonged storage at room temperature, and (3) MT concentrations in fish feed are stable for at least several months when feed is stored at 4 °C or lower, but decline linearly with time at higher temperatures. The half-life of MT (i.e., the time required for the MT concentration to fall from 60 mg/kg to 30 mg/kg) was 1.1 and 4.8 months for feed stored at 40 °C and 22 °C, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: 17α-methyltestosterone; MT; Tilapia; Drug; FDA; NADA
1. Introduction The synthetic steroid 17α-methyltestosterone (MT, Fig. 1) is used as a feed additive to produce predominately male populations of tilapia (Oreochromis sp.). Mono-sex male production is superior to mixed-sex production because the absence of females prevents precocious spawning and eliminates behaviors that limit fish growth in mixed-sex populations (Beardmore et al., 2001). Allmale populations of tilapia are produced as follows. Tilapia in the early stages of sexual differentiation (i.e., 7 to 12 days post-hatch, 9 to 11 mm total length, 10 to 15 mg) are fed a diet containing 60 mg MT per kilogram ⁎ Corresponding author. Tel.: +1 608 262 6450; fax: +1 608 262 0454. E-mail address: [email protected] (T.P. Barry). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.05.001
of feed. The MT-treated feed is offered to the fish at an average of 15–20% body weight per day for 28 consecutive days. The efficacy of this treatment regime has been verified in numerous studies conducted under Investigational New Animal Drug (INAD) exemptions that allowed limited use of MT for sex-reversing fish (e.g., Gale et al., 1999; Wassermann and Afonso, 2003). The legal use of MT by the U.S. tilapia aquaculture industry will depend on the U.S. Food and Drug Administration (FDA) approving a New Animal Drug Application (NADA) for this product. The present study was conducted to satisfy the remaining FDA requirements in the area of product manufacturing. Specifically, the FDA required studies to: (1) document the stability of MT in feed under different storage conditions; (2) determine if MT is
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Fig. 1. Chemical structures of 17α-methyltestosterone (MT) and 3β-methoxy-17β-hydroxyandrost-5-en-7-one as internal standard (IS).
mixed homogeneously during the manufacturing process (i.e., verify that all bags from a given production run contain the same concentration of MT); and (3) address the concern raised by the FDA that MT might become associated with lipids in the fish feed and migrate to the bottom of the bag during prolonged storage. In all studies MT was measured using an FDAapproved high performance liquid chromatography (HPLC) method developed specifically for measuring MT in fish feed (Marwah et al., 2005).
to analysis by an online single quadruple mass detector to ensure the homogeneity of peaks (MT and IS) and integrity of the data. In brief, the samples were analyzed as follows. Feed samples were extracted with methanol and MT and the IS were partitioned between 80% methanol and hexane. The methanol layer was evaporated, purified by solid phase extraction, and subjected to chromatography on a C18 column using a water: acetonitrile gradient (Marwah et al., 2005). 2.2. Freezer storage validation experiment
2. Methods 17α-methyltestosterone (USP, cGMP, N99% pure) was purchased from Spectrum Chemical (Gardena, CA). The internal standard (IS) was supplied by Steraloids, Inc. (Newport, USA). All other solvents and reagents were HPLC grade and purchased from Sigma Chemical Company (St. Louis, MO) or Fischer Scientific (Hampton, NH). Control and MT-treated feeds were obtained from Rangen, Inc. (Buhl, ID). The MT-treated feed has a MT concentration of approximately 60 mg MT per kg of feed, and goes by the brand name, Masculinizing Feed for Tilapia®. The control diet was identical to the experimental diet but contained no MT. Both the control and MT-treated feeds are 40 to 50% crude protein with a particle size of 400 to 1000-μm in diameter. The feed was initially packaged in commercial-sized plastic bags, 46 cm wide by 86 cm high, and containing 20 kg of feed. 2.1. Analytical procedures Concentrations of MT in feed samples were measured by high performance liquid chromatography-mass spectrometric analysis (LC-MS) using 3β-methoxy17β-hydroxyandrost-5-en-7-one as an internal standard (IS). The analytical method was developed specifically for measuring MT in fish feed, and is highly accurate, reproducible, and robust (Marwah et al., 2005). Representative HPLC chromatograms were subjected
All feed samples for the homogeneity, segregation, and storage studies were temporarily stored in a − 40 °C freezer before analysis. Thus, it was necessary to determine if frozen storage had an effect on the concentration of MT in the samples. Feed samples containing known concentrations of MT (both feed obtained from Rangen, and “fortified” feed samples prepared by adding 60 mg/kg MT to control feed in the laboratory) were prepared and stored at −20 °C or −40 °C for various times up to 18 months. At the end of the storage periods the MT levels in the samples were measured. Fresh “fortified” feed samples were also prepared and analyzed for MT at the end of the storage period for comparison with MT levels in the stored samples. 2.3. Homogeneity study The homogeneity study was conducted using two separate batches of feed produced at Rangen on a commercial scale (i.e., minimum 180 kg). Duplicate 50 g samples were collected from bags of feed at the (1) beginning, (2) middle and (3) end of the batch process as per FDA-Center for Veterinary Medicine (CVM) Guideline No. 5. Specifically, feed was sampled from the first, fifth and tenth bags of a normal 180–225 kg production run. The samples were collected in 50 ml polypropylene centrifuge tubes from the top of each bag by personnel at Rangen, and shipped by overnight mail on dry ice to researchers at UW-
T.P. Barry et al. / Aquaculture 271 (2007) 523–529 Table 1 Effect of freezing on the concentration of MT in fish feed Storage temperature
Storage time (months)
Initial MT concentration before storage (mg/kg feed)
MT concentration after storage (mg/kg feed)
− 20 °C − 20 °C − 40 °C − 40 °C − 40 °C
6 18 3 5 11
59.9 + 0.6 56.0 + 0.1 a 59.9 + 0.6 59.9 + 0.6 59.9 + 0.6
59.6 + 0.7 53.6 + 0.1⁎⁎ 60.5 + 0.3 58.8 + 1.5 58.3 + 0.7
⁎⁎Significantly different than initial MT levels at p b 0.01. a The batch of MT-treated feed used in the 18-month sample was different than the batch used in the other samples.
Madison for analysis of MT concentrations. All samples from both batches were analyzed at the same time. The samples were stored at −40 °C until analysis. 2.4. Segregation study The segregation study was designed to evaluate the potential of MT to segregate in different parts of the feed or storage bag during transport and storage. It is possible, for example, that MT may associate with oils in the feed, and the oils may migrate to the bottom of the bag during transportation and prolonged storage. The experiment was conducted using one commercialsized batch of feed as follows. Three full 20 kg bags of feed from the middle of a production run were shipped by semi-truck from Buhl, ID to Madison, WI where they were stored upright at room temperature. Each bag was sampled and tested after 1 and 3 months of storage. The samples were collected using a 20-cm metal feed
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probe. Duplicate ∼ 50 g samples were collected from the top, middle, and bottom of each bag. The holes from which the samples are taken on month 1 were sealed with tape, and the bags were stored for an additional 2 months at which time the month-3 samples were collected from the top, middle, and bottom of each bag using the feed probe. All samples were stored at − 40 °C until analysis. 2.5. Storage studies Long-term storage experiments were conducted at three different temperatures: “Freezer” (−20 ± 2°C), “Refrigerator” (4± 2 °C), and “Room Temperature” (22± 2 °C). The experiment was conducted using a standard commercial refrigerator/freezer (− 20 and 4 °C). Samples were collected at times 0, 1, 2, 3 months. The experiment was conducted as follows. A 20 kg bag of feed was shipped from Rangen in Buhl, ID to Madison, WI. The feed was thoroughly mixed by pouring the feed back and forth 25 times between two large plastic containers. Duplicate 50 g time-zero samples were collected and stored frozen at −40 °C. The remaining feed was then placed into 1 kg plastic bags and sealed with tape. The 1 kg bags were made by cutting and heat-sealing 20 kg feed storage bags obtained from Rangen. For each of the three environmental conditions, there were three replicate bags per sampling period. After 1, 2 and 3 months of storage at −20, 4 or 22 °C, three bags were randomly sampled from each treatment group, the contents opened and thoroughly mixed, and duplicate 50 g samples collected from each bag. The samples were stored frozen at −40 °C until
Fig. 2. Representative chromatograms of fish feed extract and fish feed spiked with 60 mg/kg of MT stored at room temperature (22 ± 4 °C) for 3 months. Concentration of MT after 3 months was found to be 42.11 mg/kg. HPLC analysis was carried out on a C18 column (3.0 × 150 mm, 3.5 μm) using water–acetonitrile gradient (20% to 80% acetonitrile in 15 min at 0.5 ml/min; UV detection at 245 mm and 255 mm).
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Fig. 3. (A) Mass spectrum of MT extracted from the fish feed. m/z 325.1: [M + Na]+; 303.1: [M + H]+; 285.1: [M–H2O + H]+; 267.1: [M–2H2O + H]+. The mass spectrum was found to be identical with that of the authentic sample. (B) Mass spectrum of the internal standard extracted from the fish feed. m/z 341.1: [M +Na]+; 319.1: [M+H]+; 301.1: [M–H2O+H]+; 287.1: [M–CH3OH +H]+; 269.1: [M–CH3OH–H2O+H]+; 251.1: [M–CH3OH–2H2O +H]+. The mass spectrum was found to be identical with that of the authentic sample.
analysis. MT concentrations in the collected feed samples were measured by HPLC. A higher temperature (40± 2 °C) was also tested (i.e., accelerated testing) with samples collected on weeks 0, 1, 2, 3, and 4. Temperature and humidity conditions were recorded using a HOBO (Onset Computer Corp, Pocasset, MA) data logger. 2.6. Statistics The data from the homogeneity study were analyzed using a randomized complete block design. There were 2 blocks (commercial-sized feed batches), 3 sampling periods within block (beginning, middle, and end of run), and 2 subsamples within each sampling period for a total of 12 samples. Means comparisons were made using the least squares difference test (LSD). The data from the segregation study were analyzed using a randomized complete block design. There were 3 bags of feed per sampling time (replicates), 2 sampling periods (1 and 3 months), 3 sampling locations within each bag (top, middle and bottom of the bag), and 2
subsamples within each sampling location for a total of 36 samples plus 2 time-0 control samples = 38 total samples. Means comparisons were made using the least squares difference test (LSD). The data from all of the storage experiments (longterm storage, accelerated testing, and freezer storage validation) were analyzed by ANOVA using a randomized design, and means were compared using the least squares difference test (LSD). For the long-term storage experiment, there was 1 batch of feed, 3 environmental conditions (− 20 °C, 4 °C, and 22 °C), 3 bags of feed per sampling period (replicates), 3 sampling periods (1, 2, and 3 months), and 2 subsamples from each bag for a total of 54 samples plus 2 control samples = 56 total samples. For the accelerated testing, there was 1 environmental condition (40 °C), 3 bags of feed per sampling period (replicates), 4 sampling periods (1, 2, 3, and 4 weeks), and 2 subsamples from each bag for a total of 24 experimental samples plus 2 control samples = 26 total samples. For the freezer storage validation, there were 2 temperatures (− 40 °C and− 20 °C), three bags of
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feed per sampling period (replicates), and 2 subsamples per bag. 3. Results 3.1. Freezer storage validation experiment All of the samples for the homogeneity, segregation and storage studies had to be stored at −40 °C for up to several months before analysis. Therefore, an experiment was conducted to determine if frozen storage had an effect on the concentrations of MT in fish feed. There was no significant effect of storage at −40 °C on MT concentrations in fish feed stored for 3, 5, and 11 months, or feed stored at −20 °C for 6 months (Table 1). The concentration of MT significantly declined by 4.3%, however, when stored at −20 °C for 18 months (P b 0.01, Table 1). Representative chromatograms of fish feed extract (negative control) and fish feed spiked with 60 mg/kg of MT are shown in Fig. 2. Mass spectral data of MT and IS extracted from fish feed are shown in Fig. 3A and B. The mass spectra of the extracted chemicals were identical to those of the authentic MT and IS. 3.2. Homogeneity Study There was no significant difference in MT concentrations among feed samples collected at the beginning, middle or end of standard, commercial-scale production runs (Fig. 4).
Fig. 4. Concentrations of MT in bags of fish feed collected from the beginning, middle, and end of standard commercial-scale production runs of Masculinizing Feed for Tilapia®. Specifically, duplicate samples were collected from the first, fifth and tenth bags of two normal 400–500 lb production runs by Rangen, Inc. The average MT concentrations in the two production runs were 57.9 and 58.7 mg/kg, respectively. Data shown are the mean concentrations of MT from two independent production runs + SEM (n = 2).
Fig. 5. Concentrations of MT in fish feed stored at room temperature (22 ± 2°C) for 1 and 3 months. At each time point, duplicate samples were collected from the top, middle, and bottom of three standard 20-kg bags of MT-treated fish feed (initial MT concentration was 59.9 ± 0.6 mg/kg). Values shown are the mean ± SEM of the three replicate bags (n = 3). Means with different superscripts are significantly different at p b 0.05.
3.3. Segregation study There was no significant difference in MT concentrations among the samples collected from the top, middle, and bottom of the bags after either 1 or 3 months of storage at room temperature (Fig. 5). MT concentrations did change with time, however, during the segregation experiment. MT concentrations in the feed declined from 59.9 ± 0.6 mg/kg to an average of 55.7 mg/kg after one month of storage (Fig. 5). This was a decline of 7% from initial levels. Concentrations of MT declined further to an average of 43.5 mg/kg after three months of storage (Fig. 5). This was a decline of
Fig. 6. Effect of storage at 40 °C on the concentration of MT in fish feed stored. Values shown are the mean ± SEM of three replicate bags (n = 3). Means with different superscripts are significantly different at p b 0.05.
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3 months of storage at room temperature were 53.3 + 0.1, 46.6 + 0.4, and 41.2 + 0.3, respectively (Fig. 7). A linear regression line was fitted to this data that had an r2 value of 0.99. The equation describing the decline in MT concentration with time at room temperature was: MT concentration = − 6.0x + 59.0, with x being time in months. Using this equation, it was calculated that MT levels would decline 5% from an initial concentration of 60 mg/kg in approximately 10 days at room temperature. MT concentrations did not change significantly over the 3-month experimental period when the feed was stored in the refrigerator (4 °C) or freezer (−20 °C). 4. Discussion Fig. 7. Effects of temperature on MTconcentrations in fish feed stored for 1, 2 or 3 months. Triplicate 1 kg bags of fish feed containing an initial concentration of 59.0 + 0.3 nmg/kg MT was stored at room temperature (22 ± 4 °C), in a refrigerator (4± 2 °C), or in a freezer (−20± 2 °C). After 1, 2, and 3 months of storage, duplicate 50 g samples were collected from each bag, and MT concentrations were measured by HPLC. Values shown are the mean + SEM of three replicate bags (n = 3). Means with different superscripts are significantly different at p b 0.05.
27.4% from initial levels. The 21.9% decline in the concentration of MT from 1 to 3 months was significant at p b 0.05. 3.4. Short-term storage at 40 °C The initial MT concentration in the long-term storage study was 59.0 ± 0.3 mg/kg. MT concentrations declined linearly with time during the short-term, accelerated storage experiment conducted at 40 °C (Fig. 6). At each time point, MT concentrations were significantly lower than the levels the previous week. A linear regression line was fitted to the data that had an r2 value of 0.99. The equation describing the decline in MT concentrations with time was: MT concentration = − 6.1x + 57.9, with x being time in weeks. Using this equation, it was calculated that MT levels would decline 5% from an initial concentration of 60 mg/kg (i.e., to 57 mg/kg) in 1 day at 40 °C. The MT levels would decline 50% in approximately 4.5 weeks at 40 °C. 3.5. Long-term storage under three temperature conditions The initial MT concentration in the long-term storage study was 59.0 + 0.3 mg/kg. MT concentrations declined linearly with time when the feed was stored at room temperature (22 + 4 °C). MT concentrations after 1, 2 and
Prolonged storage for up to 6 months at − 40 °C had no effect on the concentration of MT in fish feed. Inasmuch as the samples collected in the homogeneity, segregation, and storage studies were all stored at − 40 °C for less than 6 months, it is concluded that MT concentrations in the samples were not affected by the temporary storage at − 40 °C subsequent to analysis. Feed stored at − 20 °C showed no change in MT concentrations in 6 months, and only a 4.3% decline after 18 months. This suggests that MT-treated feed may be stored for at least six months, and possibly up to 18 months, in a standard freezer and still retain sufficient potency for sex-reversing fish within 5% of the product label levels of MT. The evidence from the homogeneity study indicated that the MT-treated feed is homogenously mixed during the manufacturing and bagging process, as there were no differences in MT concentrations among bags sampled at the beginning, middle, or end of a standard production run. The results of the segregation study indicated that MT does not migrate in a standard 20 kg plastic bag stored upright at room temperature for 1 or 3 months. The results eliminate the concern that MT may associate with oils in the fish feed and migrate to the bottom of the bag during storage. During the segregation experiment, the concentrations of MT declined significantly by 7% (from 59.9 to 55.7 mg/kg) after 1 month of storage, and by 27.4% (from 59.9 to 43.5 mg/kg) after 3 months of storage. These drops in MT concentrations were very similar to those observed after 1 month (9.7% decline) and 3 months (30.1% decline) of storage at room temperature in the actual storage experiment. The slight differences between the experiments may be due to the size of the storage bags (1 kg bags were used in the storage experiment), and/or differences in “room temperature” that varied by 4 °C.
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The storage experiments indicated that MT concentrations in fish feed are stable and show little or no decline when stored at 4 °C or below. MT concentrations decline linearly, however, if the feed is stored at room temperature or above; the rate of MT disappearance is temperature dependent. Linear regression analysis indicated that the half-life of MT (i.e., the time it takes for T concentrations to fall from 60 to 30 mg/kg) at 40 °C is approximately 1.1 month, and the half-life of MT at 22 °C is approximately 4.8 months. The results for the present investigation corroborate previous research that demonstrated that there was no degradation of MT in fish feed stored at − 18 °C for up to 6 months (Teichert-Coddington et al., 2000). In another feed storage experiment, Smith and Phelps (2001) showed that MT levels in feed decreased from 60.4 mg/kg to 54.8 mg/kg (∼ 10%) when feed was stored for 2 months in the refrigerator followed by 26 days at 28 + 1.5 °C. Our results suggest that all of the MT degradation occurred at 28 °C. In conclusion, the results from the present study indicate that (1) MT can be mixed homogenously during manufacture, (2) MT does not migrate to the bottom of the bag during storage, (3) MT degrades linearly when stored at room temperature or above, but is stable for at least three months when stored in a refrigerator (4 °C), and for at least 6 months when stored in the freezer (− 20 °C or − 40 °C). Acknowledgements This research was supported by the North Central Regional Aquaculture Center under a grant from the
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United States Department of Agriculture (USDA Grant No. 2003-38500-12995) to Michigan State University (agreement 61-4063B between Michigan State University and the University of Wisconsin–Madison). The authors thank the following for their help and support. Dr. Ted Batterson, Michigan State University, David Brock, Rangen, Inc., Rosalie Schnick, National Coordinator for Aquaculture New Animal Drug Applications, Dr. Henry Lardy, University of Wisconsin, Dept. of Biochemistry, Dr. James Nitao, FDA Centers for Veterinary Medicine, and Dr. Thomas Bell, U.S. Fish and Wildlife, Aquatic Animal Drug Approval Partnership. References Beardmore, J.A., Mair, G.C., Lewis, R.I., 2001. Monosex male production in finfish as exemplified by tilapia: applications, problems, and prospects. Aquaculture 197, 283–301. Gale, W.L., Fitzpatrick, M.S., Lucero, M., Contreras-Sanchez, W.M., Schreck, C.B., 1999. Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in androgens. Aquaculture 178, 349–357. Marwah, A., Marwah, P., Lardy, H., 2005. Development and validation of a high performance liquid chromatography assay for 17amethyltestosterone in fish feed. J. Chromatogr., B, Biomed. Sci. Appl. 824, 107–115. Smith, E.S., Phelps, R.P., 2001. Impact of feed storage conditions on growth and efficacy of sex reversal of Nile tilapia. N. Am. J. Aquac. 63, 242–245. Teichert-Coddington, D., Manning, B., Eya, J., Brock, D., 2000. Concentration of 17a-methyltestosterone in hormone-treated feed: effects of analytical technique, fabrication, and storage temperature. J. World Aquac. Soc. 31, 42–50. Wassermann, G.J., Afonso, L.O.B., 2003. Sex reversal in Nile tilapia (Oreochromis niloticus Linnaeus) by androgen immersion. Aquac. Res. 34, 65–71.
Stability of 17α-methyltestosterone in fish feed Terence P. Barry a,⁎, Ashok Marwah b , Padma Marwah b a
University of Wisconsin — Madison, Department of Animal Sciences, Laboratory of Fish Endocrinology and Aquaculture, 660 North Park Street, Madison, WI 53706, United States b Department of Biochemistry, Enzyme Institute, 1710 University Avenue Madison, WI 53726, United States Received 14 January 2007; received in revised form 27 April 2007; accepted 1 May 2007
Abstract The synthetic steroid 17α-methyltestosterone (MT) is used as a feed additive to produce predominately male populations of tilapia (Oreochromis sp.). The legal use of MT by the U.S. tilapia industry requires an approved New Animal Drug Application (NADA) from the U.S. Food and Drug Administration (FDA). The present study was conducted to satisfy the remaining FDA data requirements in the area of product chemistry. The results indicated that (1) MT-treated feed is homogenously mixed during the manufacturing and bagging process, (2) MT remains uniformly distributed throughout the feed during prolonged storage at room temperature, and (3) MT concentrations in fish feed are stable for at least several months when feed is stored at 4 °C or lower, but decline linearly with time at higher temperatures. The half-life of MT (i.e., the time required for the MT concentration to fall from 60 mg/kg to 30 mg/kg) was 1.1 and 4.8 months for feed stored at 40 °C and 22 °C, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: 17α-methyltestosterone; MT; Tilapia; Drug; FDA; NADA
1. Introduction The synthetic steroid 17α-methyltestosterone (MT, Fig. 1) is used as a feed additive to produce predominately male populations of tilapia (Oreochromis sp.). Mono-sex male production is superior to mixed-sex production because the absence of females prevents precocious spawning and eliminates behaviors that limit fish growth in mixed-sex populations (Beardmore et al., 2001). Allmale populations of tilapia are produced as follows. Tilapia in the early stages of sexual differentiation (i.e., 7 to 12 days post-hatch, 9 to 11 mm total length, 10 to 15 mg) are fed a diet containing 60 mg MT per kilogram ⁎ Corresponding author. Tel.: +1 608 262 6450; fax: +1 608 262 0454. E-mail address: [email protected] (T.P. Barry). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.05.001
of feed. The MT-treated feed is offered to the fish at an average of 15–20% body weight per day for 28 consecutive days. The efficacy of this treatment regime has been verified in numerous studies conducted under Investigational New Animal Drug (INAD) exemptions that allowed limited use of MT for sex-reversing fish (e.g., Gale et al., 1999; Wassermann and Afonso, 2003). The legal use of MT by the U.S. tilapia aquaculture industry will depend on the U.S. Food and Drug Administration (FDA) approving a New Animal Drug Application (NADA) for this product. The present study was conducted to satisfy the remaining FDA requirements in the area of product manufacturing. Specifically, the FDA required studies to: (1) document the stability of MT in feed under different storage conditions; (2) determine if MT is
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Fig. 1. Chemical structures of 17α-methyltestosterone (MT) and 3β-methoxy-17β-hydroxyandrost-5-en-7-one as internal standard (IS).
mixed homogeneously during the manufacturing process (i.e., verify that all bags from a given production run contain the same concentration of MT); and (3) address the concern raised by the FDA that MT might become associated with lipids in the fish feed and migrate to the bottom of the bag during prolonged storage. In all studies MT was measured using an FDAapproved high performance liquid chromatography (HPLC) method developed specifically for measuring MT in fish feed (Marwah et al., 2005).
to analysis by an online single quadruple mass detector to ensure the homogeneity of peaks (MT and IS) and integrity of the data. In brief, the samples were analyzed as follows. Feed samples were extracted with methanol and MT and the IS were partitioned between 80% methanol and hexane. The methanol layer was evaporated, purified by solid phase extraction, and subjected to chromatography on a C18 column using a water: acetonitrile gradient (Marwah et al., 2005). 2.2. Freezer storage validation experiment
2. Methods 17α-methyltestosterone (USP, cGMP, N99% pure) was purchased from Spectrum Chemical (Gardena, CA). The internal standard (IS) was supplied by Steraloids, Inc. (Newport, USA). All other solvents and reagents were HPLC grade and purchased from Sigma Chemical Company (St. Louis, MO) or Fischer Scientific (Hampton, NH). Control and MT-treated feeds were obtained from Rangen, Inc. (Buhl, ID). The MT-treated feed has a MT concentration of approximately 60 mg MT per kg of feed, and goes by the brand name, Masculinizing Feed for Tilapia®. The control diet was identical to the experimental diet but contained no MT. Both the control and MT-treated feeds are 40 to 50% crude protein with a particle size of 400 to 1000-μm in diameter. The feed was initially packaged in commercial-sized plastic bags, 46 cm wide by 86 cm high, and containing 20 kg of feed. 2.1. Analytical procedures Concentrations of MT in feed samples were measured by high performance liquid chromatography-mass spectrometric analysis (LC-MS) using 3β-methoxy17β-hydroxyandrost-5-en-7-one as an internal standard (IS). The analytical method was developed specifically for measuring MT in fish feed, and is highly accurate, reproducible, and robust (Marwah et al., 2005). Representative HPLC chromatograms were subjected
All feed samples for the homogeneity, segregation, and storage studies were temporarily stored in a − 40 °C freezer before analysis. Thus, it was necessary to determine if frozen storage had an effect on the concentration of MT in the samples. Feed samples containing known concentrations of MT (both feed obtained from Rangen, and “fortified” feed samples prepared by adding 60 mg/kg MT to control feed in the laboratory) were prepared and stored at −20 °C or −40 °C for various times up to 18 months. At the end of the storage periods the MT levels in the samples were measured. Fresh “fortified” feed samples were also prepared and analyzed for MT at the end of the storage period for comparison with MT levels in the stored samples. 2.3. Homogeneity study The homogeneity study was conducted using two separate batches of feed produced at Rangen on a commercial scale (i.e., minimum 180 kg). Duplicate 50 g samples were collected from bags of feed at the (1) beginning, (2) middle and (3) end of the batch process as per FDA-Center for Veterinary Medicine (CVM) Guideline No. 5. Specifically, feed was sampled from the first, fifth and tenth bags of a normal 180–225 kg production run. The samples were collected in 50 ml polypropylene centrifuge tubes from the top of each bag by personnel at Rangen, and shipped by overnight mail on dry ice to researchers at UW-
T.P. Barry et al. / Aquaculture 271 (2007) 523–529 Table 1 Effect of freezing on the concentration of MT in fish feed Storage temperature
Storage time (months)
Initial MT concentration before storage (mg/kg feed)
MT concentration after storage (mg/kg feed)
− 20 °C − 20 °C − 40 °C − 40 °C − 40 °C
6 18 3 5 11
59.9 + 0.6 56.0 + 0.1 a 59.9 + 0.6 59.9 + 0.6 59.9 + 0.6
59.6 + 0.7 53.6 + 0.1⁎⁎ 60.5 + 0.3 58.8 + 1.5 58.3 + 0.7
⁎⁎Significantly different than initial MT levels at p b 0.01. a The batch of MT-treated feed used in the 18-month sample was different than the batch used in the other samples.
Madison for analysis of MT concentrations. All samples from both batches were analyzed at the same time. The samples were stored at −40 °C until analysis. 2.4. Segregation study The segregation study was designed to evaluate the potential of MT to segregate in different parts of the feed or storage bag during transport and storage. It is possible, for example, that MT may associate with oils in the feed, and the oils may migrate to the bottom of the bag during transportation and prolonged storage. The experiment was conducted using one commercialsized batch of feed as follows. Three full 20 kg bags of feed from the middle of a production run were shipped by semi-truck from Buhl, ID to Madison, WI where they were stored upright at room temperature. Each bag was sampled and tested after 1 and 3 months of storage. The samples were collected using a 20-cm metal feed
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probe. Duplicate ∼ 50 g samples were collected from the top, middle, and bottom of each bag. The holes from which the samples are taken on month 1 were sealed with tape, and the bags were stored for an additional 2 months at which time the month-3 samples were collected from the top, middle, and bottom of each bag using the feed probe. All samples were stored at − 40 °C until analysis. 2.5. Storage studies Long-term storage experiments were conducted at three different temperatures: “Freezer” (−20 ± 2°C), “Refrigerator” (4± 2 °C), and “Room Temperature” (22± 2 °C). The experiment was conducted using a standard commercial refrigerator/freezer (− 20 and 4 °C). Samples were collected at times 0, 1, 2, 3 months. The experiment was conducted as follows. A 20 kg bag of feed was shipped from Rangen in Buhl, ID to Madison, WI. The feed was thoroughly mixed by pouring the feed back and forth 25 times between two large plastic containers. Duplicate 50 g time-zero samples were collected and stored frozen at −40 °C. The remaining feed was then placed into 1 kg plastic bags and sealed with tape. The 1 kg bags were made by cutting and heat-sealing 20 kg feed storage bags obtained from Rangen. For each of the three environmental conditions, there were three replicate bags per sampling period. After 1, 2 and 3 months of storage at −20, 4 or 22 °C, three bags were randomly sampled from each treatment group, the contents opened and thoroughly mixed, and duplicate 50 g samples collected from each bag. The samples were stored frozen at −40 °C until
Fig. 2. Representative chromatograms of fish feed extract and fish feed spiked with 60 mg/kg of MT stored at room temperature (22 ± 4 °C) for 3 months. Concentration of MT after 3 months was found to be 42.11 mg/kg. HPLC analysis was carried out on a C18 column (3.0 × 150 mm, 3.5 μm) using water–acetonitrile gradient (20% to 80% acetonitrile in 15 min at 0.5 ml/min; UV detection at 245 mm and 255 mm).
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Fig. 3. (A) Mass spectrum of MT extracted from the fish feed. m/z 325.1: [M + Na]+; 303.1: [M + H]+; 285.1: [M–H2O + H]+; 267.1: [M–2H2O + H]+. The mass spectrum was found to be identical with that of the authentic sample. (B) Mass spectrum of the internal standard extracted from the fish feed. m/z 341.1: [M +Na]+; 319.1: [M+H]+; 301.1: [M–H2O+H]+; 287.1: [M–CH3OH +H]+; 269.1: [M–CH3OH–H2O+H]+; 251.1: [M–CH3OH–2H2O +H]+. The mass spectrum was found to be identical with that of the authentic sample.
analysis. MT concentrations in the collected feed samples were measured by HPLC. A higher temperature (40± 2 °C) was also tested (i.e., accelerated testing) with samples collected on weeks 0, 1, 2, 3, and 4. Temperature and humidity conditions were recorded using a HOBO (Onset Computer Corp, Pocasset, MA) data logger. 2.6. Statistics The data from the homogeneity study were analyzed using a randomized complete block design. There were 2 blocks (commercial-sized feed batches), 3 sampling periods within block (beginning, middle, and end of run), and 2 subsamples within each sampling period for a total of 12 samples. Means comparisons were made using the least squares difference test (LSD). The data from the segregation study were analyzed using a randomized complete block design. There were 3 bags of feed per sampling time (replicates), 2 sampling periods (1 and 3 months), 3 sampling locations within each bag (top, middle and bottom of the bag), and 2
subsamples within each sampling location for a total of 36 samples plus 2 time-0 control samples = 38 total samples. Means comparisons were made using the least squares difference test (LSD). The data from all of the storage experiments (longterm storage, accelerated testing, and freezer storage validation) were analyzed by ANOVA using a randomized design, and means were compared using the least squares difference test (LSD). For the long-term storage experiment, there was 1 batch of feed, 3 environmental conditions (− 20 °C, 4 °C, and 22 °C), 3 bags of feed per sampling period (replicates), 3 sampling periods (1, 2, and 3 months), and 2 subsamples from each bag for a total of 54 samples plus 2 control samples = 56 total samples. For the accelerated testing, there was 1 environmental condition (40 °C), 3 bags of feed per sampling period (replicates), 4 sampling periods (1, 2, 3, and 4 weeks), and 2 subsamples from each bag for a total of 24 experimental samples plus 2 control samples = 26 total samples. For the freezer storage validation, there were 2 temperatures (− 40 °C and− 20 °C), three bags of
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feed per sampling period (replicates), and 2 subsamples per bag. 3. Results 3.1. Freezer storage validation experiment All of the samples for the homogeneity, segregation and storage studies had to be stored at −40 °C for up to several months before analysis. Therefore, an experiment was conducted to determine if frozen storage had an effect on the concentrations of MT in fish feed. There was no significant effect of storage at −40 °C on MT concentrations in fish feed stored for 3, 5, and 11 months, or feed stored at −20 °C for 6 months (Table 1). The concentration of MT significantly declined by 4.3%, however, when stored at −20 °C for 18 months (P b 0.01, Table 1). Representative chromatograms of fish feed extract (negative control) and fish feed spiked with 60 mg/kg of MT are shown in Fig. 2. Mass spectral data of MT and IS extracted from fish feed are shown in Fig. 3A and B. The mass spectra of the extracted chemicals were identical to those of the authentic MT and IS. 3.2. Homogeneity Study There was no significant difference in MT concentrations among feed samples collected at the beginning, middle or end of standard, commercial-scale production runs (Fig. 4).
Fig. 4. Concentrations of MT in bags of fish feed collected from the beginning, middle, and end of standard commercial-scale production runs of Masculinizing Feed for Tilapia®. Specifically, duplicate samples were collected from the first, fifth and tenth bags of two normal 400–500 lb production runs by Rangen, Inc. The average MT concentrations in the two production runs were 57.9 and 58.7 mg/kg, respectively. Data shown are the mean concentrations of MT from two independent production runs + SEM (n = 2).
Fig. 5. Concentrations of MT in fish feed stored at room temperature (22 ± 2°C) for 1 and 3 months. At each time point, duplicate samples were collected from the top, middle, and bottom of three standard 20-kg bags of MT-treated fish feed (initial MT concentration was 59.9 ± 0.6 mg/kg). Values shown are the mean ± SEM of the three replicate bags (n = 3). Means with different superscripts are significantly different at p b 0.05.
3.3. Segregation study There was no significant difference in MT concentrations among the samples collected from the top, middle, and bottom of the bags after either 1 or 3 months of storage at room temperature (Fig. 5). MT concentrations did change with time, however, during the segregation experiment. MT concentrations in the feed declined from 59.9 ± 0.6 mg/kg to an average of 55.7 mg/kg after one month of storage (Fig. 5). This was a decline of 7% from initial levels. Concentrations of MT declined further to an average of 43.5 mg/kg after three months of storage (Fig. 5). This was a decline of
Fig. 6. Effect of storage at 40 °C on the concentration of MT in fish feed stored. Values shown are the mean ± SEM of three replicate bags (n = 3). Means with different superscripts are significantly different at p b 0.05.
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3 months of storage at room temperature were 53.3 + 0.1, 46.6 + 0.4, and 41.2 + 0.3, respectively (Fig. 7). A linear regression line was fitted to this data that had an r2 value of 0.99. The equation describing the decline in MT concentration with time at room temperature was: MT concentration = − 6.0x + 59.0, with x being time in months. Using this equation, it was calculated that MT levels would decline 5% from an initial concentration of 60 mg/kg in approximately 10 days at room temperature. MT concentrations did not change significantly over the 3-month experimental period when the feed was stored in the refrigerator (4 °C) or freezer (−20 °C). 4. Discussion Fig. 7. Effects of temperature on MTconcentrations in fish feed stored for 1, 2 or 3 months. Triplicate 1 kg bags of fish feed containing an initial concentration of 59.0 + 0.3 nmg/kg MT was stored at room temperature (22 ± 4 °C), in a refrigerator (4± 2 °C), or in a freezer (−20± 2 °C). After 1, 2, and 3 months of storage, duplicate 50 g samples were collected from each bag, and MT concentrations were measured by HPLC. Values shown are the mean + SEM of three replicate bags (n = 3). Means with different superscripts are significantly different at p b 0.05.
27.4% from initial levels. The 21.9% decline in the concentration of MT from 1 to 3 months was significant at p b 0.05. 3.4. Short-term storage at 40 °C The initial MT concentration in the long-term storage study was 59.0 ± 0.3 mg/kg. MT concentrations declined linearly with time during the short-term, accelerated storage experiment conducted at 40 °C (Fig. 6). At each time point, MT concentrations were significantly lower than the levels the previous week. A linear regression line was fitted to the data that had an r2 value of 0.99. The equation describing the decline in MT concentrations with time was: MT concentration = − 6.1x + 57.9, with x being time in weeks. Using this equation, it was calculated that MT levels would decline 5% from an initial concentration of 60 mg/kg (i.e., to 57 mg/kg) in 1 day at 40 °C. The MT levels would decline 50% in approximately 4.5 weeks at 40 °C. 3.5. Long-term storage under three temperature conditions The initial MT concentration in the long-term storage study was 59.0 + 0.3 mg/kg. MT concentrations declined linearly with time when the feed was stored at room temperature (22 + 4 °C). MT concentrations after 1, 2 and
Prolonged storage for up to 6 months at − 40 °C had no effect on the concentration of MT in fish feed. Inasmuch as the samples collected in the homogeneity, segregation, and storage studies were all stored at − 40 °C for less than 6 months, it is concluded that MT concentrations in the samples were not affected by the temporary storage at − 40 °C subsequent to analysis. Feed stored at − 20 °C showed no change in MT concentrations in 6 months, and only a 4.3% decline after 18 months. This suggests that MT-treated feed may be stored for at least six months, and possibly up to 18 months, in a standard freezer and still retain sufficient potency for sex-reversing fish within 5% of the product label levels of MT. The evidence from the homogeneity study indicated that the MT-treated feed is homogenously mixed during the manufacturing and bagging process, as there were no differences in MT concentrations among bags sampled at the beginning, middle, or end of a standard production run. The results of the segregation study indicated that MT does not migrate in a standard 20 kg plastic bag stored upright at room temperature for 1 or 3 months. The results eliminate the concern that MT may associate with oils in the fish feed and migrate to the bottom of the bag during storage. During the segregation experiment, the concentrations of MT declined significantly by 7% (from 59.9 to 55.7 mg/kg) after 1 month of storage, and by 27.4% (from 59.9 to 43.5 mg/kg) after 3 months of storage. These drops in MT concentrations were very similar to those observed after 1 month (9.7% decline) and 3 months (30.1% decline) of storage at room temperature in the actual storage experiment. The slight differences between the experiments may be due to the size of the storage bags (1 kg bags were used in the storage experiment), and/or differences in “room temperature” that varied by 4 °C.
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The storage experiments indicated that MT concentrations in fish feed are stable and show little or no decline when stored at 4 °C or below. MT concentrations decline linearly, however, if the feed is stored at room temperature or above; the rate of MT disappearance is temperature dependent. Linear regression analysis indicated that the half-life of MT (i.e., the time it takes for T concentrations to fall from 60 to 30 mg/kg) at 40 °C is approximately 1.1 month, and the half-life of MT at 22 °C is approximately 4.8 months. The results for the present investigation corroborate previous research that demonstrated that there was no degradation of MT in fish feed stored at − 18 °C for up to 6 months (Teichert-Coddington et al., 2000). In another feed storage experiment, Smith and Phelps (2001) showed that MT levels in feed decreased from 60.4 mg/kg to 54.8 mg/kg (∼ 10%) when feed was stored for 2 months in the refrigerator followed by 26 days at 28 + 1.5 °C. Our results suggest that all of the MT degradation occurred at 28 °C. In conclusion, the results from the present study indicate that (1) MT can be mixed homogenously during manufacture, (2) MT does not migrate to the bottom of the bag during storage, (3) MT degrades linearly when stored at room temperature or above, but is stable for at least three months when stored in a refrigerator (4 °C), and for at least 6 months when stored in the freezer (− 20 °C or − 40 °C). Acknowledgements This research was supported by the North Central Regional Aquaculture Center under a grant from the
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United States Department of Agriculture (USDA Grant No. 2003-38500-12995) to Michigan State University (agreement 61-4063B between Michigan State University and the University of Wisconsin–Madison). The authors thank the following for their help and support. Dr. Ted Batterson, Michigan State University, David Brock, Rangen, Inc., Rosalie Schnick, National Coordinator for Aquaculture New Animal Drug Applications, Dr. Henry Lardy, University of Wisconsin, Dept. of Biochemistry, Dr. James Nitao, FDA Centers for Veterinary Medicine, and Dr. Thomas Bell, U.S. Fish and Wildlife, Aquatic Animal Drug Approval Partnership. References Beardmore, J.A., Mair, G.C., Lewis, R.I., 2001. Monosex male production in finfish as exemplified by tilapia: applications, problems, and prospects. Aquaculture 197, 283–301. Gale, W.L., Fitzpatrick, M.S., Lucero, M., Contreras-Sanchez, W.M., Schreck, C.B., 1999. Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in androgens. Aquaculture 178, 349–357. Marwah, A., Marwah, P., Lardy, H., 2005. Development and validation of a high performance liquid chromatography assay for 17amethyltestosterone in fish feed. J. Chromatogr., B, Biomed. Sci. Appl. 824, 107–115. Smith, E.S., Phelps, R.P., 2001. Impact of feed storage conditions on growth and efficacy of sex reversal of Nile tilapia. N. Am. J. Aquac. 63, 242–245. Teichert-Coddington, D., Manning, B., Eya, J., Brock, D., 2000. Concentration of 17a-methyltestosterone in hormone-treated feed: effects of analytical technique, fabrication, and storage temperature. J. World Aquac. Soc. 31, 42–50. Wassermann, G.J., Afonso, L.O.B., 2003. Sex reversal in Nile tilapia (Oreochromis niloticus Linnaeus) by androgen immersion. Aquac. Res. 34, 65–71.