Limit of residual solvents
Standard solution I
Transfer 3.0 mL of acetonitrile, 3.0 mL of methanol, 3.0 mL of isopropyl acetate, and 3.0 mL of heptane to a 50-mL volumetric flask. Dilute with dimethylacetamide to volume, and mix well.
Standard solution II
Transfer 1.0 mL of Standard solution I to a 100-mL volumetric flask, dilute with dimethylacetamide to volume, and mix well. Further dilute 10.0 mL of this solution with dimethylacetamide to 50.0 mL, and mix well
Test solution
Transfer 1 g of Eprinomectin in dimethylacetamide to a 10-mL volumetric flask. Dilute with dimethylacetamide to volume, and mix well.
Sensitivity solution I
Transfer 3.0 mL each of methanol, isopropyl acetate, and heptane to a 50-mL volumetric flask. Dilute with dimethylacetamide to volume, and mix well. Further dilute 50 µL of this solution with dimethylacetamide to 25 mL, and mix well.
Sensitivity solution II
Transfer 3.0 mL of acetonitrile to a 50-mL volumetric flask, dilute with dimethylacetamide to volume, and mix well. Further dilute 50 µL of this solution with dimethylacetamide to 25 mL.
Sensitivity solution III
Transfer 5.0 mL of Sensitivity solution I and 1.0 mL of Sensitivity solution II to a 50-mL volumetric flask, dilute with dimethylacetamide to volume, and mix well. [NoteThis solution contains 100 ppm (m/m) of methanol, isopropyl acetate, and heptane and 20 ppm (m/m) of acetonitrile.]
Chromatographic system (see Chromatography 621)
The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 25-m fused-silica analytical column coated with a 20-µm S3 stationary phase. The carrier gas is helium with a flow rate of 20 mL per minute. The chromatograph is programmed as follows: the column temperature is increased from 110
at a rate of 5
per minute to 160
and maintained at 160
for 5 minutes. The column temperature is then increased at a rate of 30
per minute to 220
and maintained at 220
for 25 minutes. The injection port temperature is maintained at 200
, and the detector temperature is maintained at 220
. Chromatograph
Sensitivity solution III and
Standard solution II as directed for
Procedure: in the chromatogram obtained from
Sensitivity solution III, the peaks for methanol, acetonitrile, isopropyl acetate, and heptane are detectable and elute at relative retention times of 1, 2.1, 7.6, and 8.6, respectively. In the chromatogram obtained from
Standard solution II, the relative standard deviations for the areas of the solvent peaks are not more than 5.0% for six injections.
Procedure
Separately inject equal volumes (about 1 µL) of
Standard solution II and the
Test solution. Reinject
Standard solution II in duplicate after every six sample injections. The individual values for the area response of the two injections agree within ±5% of their corresponding average response. Calculate the percentage of each solvent present using the following formula:
0.12D(rU / rS)
in which
D is the density, in mg per mL, of acetonitrile (0.787), isopropyl acetate (0.870), methanol (0.796), and heptane (0.684);
rU is the solvent peak area in the chromatogram obtained from the
Test solution; and
rS is the solvent peak area in the chromatogram obtained from
Standard solution II. Not more than 0.005% of acetonitrile is found, and the sum of all solvents is not more than 0.5%.
Limit of 8a-oxo-B1a
Solution A, Solution B, Diluent, and System suitability solution
Proceed as directed in the Assay.
System suitability mobile phase
Use the Mobile phase as directed in the Assay.
Mobile phase
Use a mixture of acetonitrile and
Solution A (13:7). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard solution
Use the Standard preparation, prepared as directed in the Assay.
Test solution
Use the Assay preparation, prepared as directed in the Assay.
Butylated hydroxytoluene solution
Transfer 50 mg of butylated hydroxytoluene to a 100-mL volumetric flask, and dilute with methanol to volume. Sonicate, if necessary, and mix well. Dilute 2 mL of the resulting solution with Diluent to 100 mL.
System suitability determination
Use the conditions as directed for Chromatographic system in the Assay.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is 1.5 mL per minute, and the column temperature is 40
. Chromatograph the
Butylated hydroxytoluene solution and the
Test solution, and record the peak responses as directed for
Procedure: the retention time for the peak corresponding to butylated hydroxytoluene is approximately 12 to 17 minutes, and the relative standard deviation of the peak area is not more than 3.0% for six injections. In the chromatogram obtained from the
Test solution, the retention time for the peak corresponding to 8a-oxo-B
1a is approximately 4 to 9 minutes.
Procedure
Separately inject equal volumes (about 15 µL) of the
Butylated hydroxytoluene solution and the
Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the content of 8a-oxo-B
1a, in mg, on the anhydrous, solvent-free, and antioxidant-free basis taken by the formula:
DCPF(rU / rS)
in which
D is the dilution factor, in mL, used to prepare the
Test solution; C is the concentration, in mg per mL, of butylated hydroxytoluene in the
Butylated hydroxytoluene solution; P is the purity of butylated hydroxytoluene used to prepare the
Butylated hydroxytoluene solution; F is equal to 0.4 and is the relative response factor for butylated hydroxytoluene with respect to 8a-oxo-B
1a; and
rU and
rS are the peak areas for 8a-oxo-B
1a and butylated hydroxytoluene in the chromatograms obtained from the
Test solution and the
Butylated hydroxytoluene solution, respectively. Not more than 0.5% of 8a-oxo-B
1a is found.
Related compounds
Solution A, Solution B, Mobile phase, Diluent, System suitability solution, and Chromatographic system
Proceed as directed in the Assay.
Standard solution
Use the Standard preparation, prepared as directed in the Assay.
Test solution
Use the Assay preparation, prepared as directed in the Assay.
Procedure
Inject a volume (about 15 µL) of the
Test solution into the chromatograph, record the chromatogram, and measure the peak areas. Calculate the percentage of eprinomectin related compounds in the portion of Eprinomectin taken by the formula:
100(ri / rs)
in which
ri is the peak area of each individual related substance obtained from the
Test solution, and
rs is the sum of the responses of all the peaks: for related compounds with relative retentions of 0.23, 0.93, and 1.16 with respect to the B
1a peak, not more than 1.0%; for impurity A, not more than 1.0%; for impurity E, not more than 1.0%; for all other known impurities, not more than 0.5%; for total unknown impurities, not more than 1.0%; and for total impurities, not more than 5.0% is found.
Assay
Solution A:
0.1% (v/v) solution of perchloric acid in water.
Solution B:
acetonitrile.
Mobile phase
Use variable mixtures of
Solution A and
Solution B as directed for
Chromatographic system. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Diluent
Prepare a solution of four volumes of methanol and one volume of water.
Standard preparation
Dissolve an accurately weighed quantity of
USP Eprinomectin RS in
Diluent to prepare a solution having a known concentration of about 0.500 mg per mL.
System suitability solution
Transfer 4 mL of Standard preparation to an HPLC vial. Add 2 drops of 1 M sodium hydroxide and let stand for 20 minutes prior to injecting into the chromatograph.
Assay preparation
Dissolve an accurately weighed quantity of Eprinomectin in Diluent to prepare a solution having a known concentration of about 0.500 mg per mL.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 245-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is 1.5 mL per minute, and the column temperature is 40
. The chromatograph is programmed as follows:
Time (minute) |
Solution A
(%) |
Solution B
(%) |
Elution |
015 |
45 |
55 |
isocratic |
1525 |
45®5 |
55®95 |
linear gradient |
2530 |
5®45 |
95®55 |
linear gradient |
3035 |
45 |
55 |
isocratic |
Chromatograph the
System suitability solution and the
Standard preparation as directed for
Procedure: the relative retention times are about 0.55, 0.77, 1.00, 1.05, and 1.28 for impurity A, component B
1b, component B
1a, impurities C + D, and impurity E, respectively; the resolution,
R, between components B
1b and B
1a is not less than 3; the resolution,
R, between component B
1a and impurities C + D is at least 1; the symmetry factor for the B
1a peak is not more than 1.5; and the theoretical plate count for the B
1a peak is greater than 4,500. In the chromatogram of the
Standard preparation, the relative standard deviation for the peak corresponding to B
1a is not more than 1.0% for five injections.
Procedure
Separately inject equal volumes (about 15 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the percentage of component B
1a by the formula:
100(rU / rT)
in which
rU is the peak area of component B
1a in the chromatogram obtained from the
Assay preparation; and
rT is the sum of the peak areas of components B
1a and B
1b.
Calculate the quantity, in mg, of C50H75NO14 (component B1a) and C49H73NO14 (component B1b) in the portion of Eprinomectin taken by the formula:
DC(rU / rS)
in which
D is the dilution factor, in mL, used to prepare the
Assay preparation; C is the concentration, in mg per mL, of component B
1a or component B
1b in the
Standard preparation; and
rU and
rS are the peak areas of component B
1a or component B
1b in the chromatograms obtained from the
Assay preparation and the
Standard preparation, respectively.
1S (USP31)