Related compounds
Mobile phase and Diluent
Prepare as directed in the
Assay.
System suitability solution
Prepare as directed for Resolution solution in the Assay.
Methacycline standard stock solution
Dissolve an accurately weighed quantity of
USP Methacycline Hydrochloride RS in
Diluent, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 1.2 mg per mL.
Standard solution 1
Prepare as directed for the
Standard preparation in the
Assay.
Standard solution 2
Transfer 2.0 mL of
Standard solution 1 and 2.0 mL of the
Methacycline standard stock solution to a 100-mL volumetric flask, dilute with
Diluent to volume, and mix. This solution contains about 0.024 mg each of
USP Doxycycline Hyclate RS and
USP Methacycline Hydrochloride RS per mL.
Test solution
Use the Assay preparation, prepared as directed in the Assay.
Chromatographic system
(see
Chromatography 621) Prepare as directed in the
Assay. Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.4 for 4-epidoxycycline (the main degradation product), 0.6 for methacycline, 0.7 for 6-epidoxycycline, and 1.0 for doxycycline; the resolution,
R, between 4-epidoxycycline and doxycycline is not less than 3.0; and the tailing factor is not more than 2.0. Chromatograph
Standard solution 1, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 20 µL) of
Standard solution 2 and the
Test solution into the chromatograph, record the chromatograms for a period of time that is 1.7 times the retention time of doxycycline, and measure the peak areas. Calculate the percentage of methacycline in the portion of Doxycycline Hyclate taken by the formula:
10,000(CM / W)(rU / rM),
in which
CM is the concentration, in mg per mL, of
USP Methacycline Hydrochloride RS in
Standard solution 2; W is the weight, in mg, of Doxycycline Hyclate taken to prepare the
Test solution; and
rU and
rM are the methacycline peak responses obtained from the
Test solution and
Standard solution 2, respectively. Not more than 2% of methacycline is found. Calculate the percentage of each related compound, other than methacycline, in the portion of Doxycycline Hyclate taken by the formula:
10,000(CS / W)(ri / rS),
in which
CS is the concentration, in mg per mL, of
USP Doxycycline Hyclate RS in
Standard solution 2; W is the weight, in mg, of Doxycycline Hyclate taken to prepare the
Test solution; ri is the peak response for each impurity obtained from the
Test solution; and
rS is the doxycycline peak response obtained from
Standard solution 2. Not more than 0.5% of any impurity eluting before methacycline is found; not more than 2% of 6-epidoxycycline is found; and not more than 0.5% of any impurity eluting after the main doxycycline peak is found.
Assay
Mobile phase
Transfer 2.72 g of monobasic potassium phosphate, 0.74 g of sodium hydroxide, 0.50 g of tetrabutylammonium hydrogen sulfate, and 0.40 g of edetate disodium to a 1000-mL volumetric flask. Add about 850 mL of water, and stir to dissolve. Add 60 g of tertiary butyl alcohol with the aid of water, dilute with water to volume, and adjust with 1 N sodium hydroxide to a pH of 8.0 ± 0.1. Pass this solution through a filter having a porosity of 0.5 µm or finer, and degas before using. Make any necessary adjustments (see
System Suitability under
Chromatography 621). Decreasing the proportion of tertiary butyl alcohol results in a longer retention time of doxycycline and improved separation of doxycycline from the related compounds.
Diluent
Use 0.01 N hydrochloric acid.
Resolution solution
Prepare a solution of
USP Doxycycline Hyclate RS in
Diluent containing about 6 mg of doxycycline per mL. Transfer 5 mL of this solution to a 25-mL volumetric flask, heat on a steam bath for 60 minutes, and evaporate to dryness on a hot plate, taking care not to char the residue. Dissolve the residue in 0.01 N hydrochloric acid, dilute with
Diluent to volume, and mix. Pass a portion of this solution through a filter having a porosity of 0.5 µm or finer, and use the filtrate as the
Resolution solution. This solution contains a mixture of 4-epidoxycycline, 6-epidoxycycline, and doxycycline. When stored in a refrigerator, this solution may be used for 14 days.
[NOTEThroughout the following sections, protect the
Standard preparation and the
Assay preparation from light.
]
Standard preparation
Transfer about 12 mg of
USP Doxycycline Hyclate RS, accurately weighed, to a 10-mL volumetric flask, add about 6 mL of
Diluent, sonicate for about 5 minutes or until dissolved, dilute with
Diluent to volume, and mix.
Assay preparation
Transfer about 120 mg of Doxycycline Hyclate, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix. Pass through a membrane filter having a porosity of 0.5 µm or finer.
Chromatographic system
(see
Chromatography 621)The liquid chromatograph is equipped with a 270 nm detector, and a 4.6-mm × 25-cm column that contains packing L21 and is maintained at 60 ± 1
. The flow rate is about 1 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.4 for 4-epidoxycycline (the main degradation product), 0.7 for 6-epidoxycycline, and 1.0 for doxycycline, the resolution,
R, between the 4-epidoxycycline peak and the doxycycline peak is not less than 3.0, and the tailing factor for the doxycycline peak is not more than 2.0. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms for a period of time that is 1.7 times the retention time of doxycycline, and measure the responses for the major peaks. Calculate the potency, in µg of doxycycline (C
22H
24N
2O
8) per mg, of the Doxycycline Hyclate taken by the formula:
100(CP / W)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Doxycycline Hyclate RS in the
Standard preparation; P is the designated potency, in µg of doxycycline (C
22H
24N
2O
8) per mg, of
USP Doxycycline Hyclate RS;
W is the quantity, in mg, of Doxycycline Hyclate taken to prepare the
Assay preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.