(non ox' i nol).
» Nonoxynol 9 is an anhydrous liquid mixture consisting chiefly of monononylphenyl ethers of polyethylene glycols corresponding to the formula:
in which the average value of n is about 9. It contains not less than 90.0 percent and not more than 110.0 percent of nonoxynol 9.
Packaging and storage Preserve in tight containers.
USP Reference standards 11
A: Its IR absorption spectrum, obtained by spreading a capillary film of it between sodium chloride plates, exhibits maxima at 1117 cm1 (strong); at 1512, 1582, and 1610 cm1 (medium, sharp); at 2871, 2928, and 2956 cm1 (strong, unresolved); at 831 cm1 (medium, broad); and at 1250 cm1 (medium, sharp).
B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation as obtained in the Assay.
Cloud point Transfer 1.0 g to a 250-mL beaker, add 99 g of water, and mix to dissolve. Pour about 30 mL of the solution into a 70-mL test tube. Support the test tube in a hot water bath, and stir the contents with a thermometer constantly until the solution becomes cloudy, then remove the test tube from the bath immediately, so that the temperature rises not more than 2 further, and continue stirring. The cloud point is the temperature at which the solution becomes sufficiently clear that the entire thermometer bulb is seen plainly: it is between 52 and 56.
Acid value 401: not more than 0.2.
Water, Method I 921: not more than 0.5%.
Polyethylene glycol Transfer about 10 g, accurately weighed, to a 250-mL beaker. Add 100 mL of ethyl acetate, and stir on a magnetic stirrer to effect solution. Transfer, with the aid of 100 mL of 5 N sodium chloride, to a pear-shaped, 500-mL separator fitted with a glass stopper. Insert the stopper, and shake vigorously for 1 minute. Remove the stopper carefully to release the pressure. Immerse a thermometer in the mixture, and support the separator so that it is partially immersed in a water bath maintained at 50. Swirl the separator gently while letting the internal temperature rise to between 40 and 45, then immediately remove the separator from the bath, dry the outside surface, and drain the salt (lower) layer into another pear-shaped, 500-mL separator. In the same manner, extract the ethyl acetate layer a second time with 100 mL of fresh 5 N sodium chloride, combining the two aqueous extracts. Discard the ethyl acetate layer. Wash the combined aqueous layers with 100 mL of ethyl acetate, using the same technique, and drain the salt (lower) layer into a clean pear-shaped, 500-mL separator. Discard the ethyl acetate layer. Extract the aqueous layer with two successive 100-mL portions of chloroform, draining the chloroform (lower) layers through Whatman folded filter paper 2V, and combining them into a 250-mL beaker. Evaporate on a steam bath to dryness, and continue heating until the odor of chloroform is no longer perceptible. Allow the beaker to cool. Add 25 mL of acetone, and dissolve the residue on a magnetic stirrer. Filter through Whatman folded filter paper 2V into a tared 250-mL beaker, rinsing with two 25-mL portions of acetone. Evaporate on a steam bath to dryness. Dry in vacuum at 60 for 1 hour. Allow the beaker to cool, and weigh: not more than 1.0% of polyethylene glycol is found.
Free ethylene oxide
Stripped nonoxynol 9 Maintain Nonoxynol 9 at a temperature of 150 with constant stirring in an open vessel until it no longer displays a peak for ethylene oxide when chromatographed as directed below.
Standard solutions [noteEthylene oxide is toxic and flammable. Prepare these solutions in a well-ventilated hood, using great care. ] Chill all apparatus and reagents used in the preparation of standards in a refrigerator or freezer before use. Fill a chilled pressure bottle with liquid ethylene oxide from a lecture bottle, and store in a freezer when not in use. Use a small piece of polyethylene film to protect the liquid from contact with the rubber gasket. Transfer about 100 mL of chilled isopropyl alcohol to a 500-mL volumetric flask. Using a chilled graduated cylinder, transfer 25 mL of ethylene oxide to the isopropyl alcohol, and swirl gently to mix. Dilute with additional chilled isopropyl alcohol to volume, replace the stopper, and swirl gently to mix. This stock solution contains about 43.6 mg of ethylene oxide per mL. Pipet 25 mL of 0.5 N alcoholic hydrochloric acid, prepared by mixing 45 mL of hydrochloric acid with 1 L of alcohol, into a 500-mL conical flask containing 40 g of magnesium chloride hexahydrate. Shake the mixture to effect saturation. Pipet 10 mL of the ethylene oxide solution into the flask, and add 20 drops of bromocresol green TS. If the solution is not yellow (acid), add an additional volume, accurately measured, of 0.5 N alcoholic hydrochloric acid to give an excess of about 10 mL. Record the total volume of 0.5 N alcoholic hydrochloric acid added. Insert the stopper in the flask, and allow to stand for 30 minutes. Titrate the excess acid with 0.5 N alcoholic potassium hydroxide VS. Perform a blank titration, using 10.0 mL of isopropyl alcohol instead of ethylene oxide solution, adding the same total volume of 0.5 N alcoholic hydrochloric acid, and note the difference in volumes required. Each mL of the difference in volumes of 0.5 N alcoholic potassium hydroxide consumed is equivalent to 22.02 mg of ethylene oxide. Calculate the concentration, in mg per mL, of ethylene oxide in the stock solution. Standardize daily. Store in a refrigerator. Prepare a 1000-ppm standard by pipeting the calculated volume (about 2 mL) of cold stock solution which, on the basis of the standardization, contains 88.6 mg of ethylene oxide, into a container and adding 87.0 g of Stripped nonoxynol 9. Prepare 10-, 5-, and 0.5-ppm standards by quantitatively diluting the 1000-ppm standard with additional Stripped nonoxynol 9.
Standard preparations Transfer 5 ± 0.01 g of each Standard solution to suitable serum vials equipped with pressure-tight septum closures designed to relieve any excessive pressure, and seal them.
Test preparation Transfer 5 ± 0.01 g of Nonoxynol 9 to a serum vial of the same kind as the vials used for the Standard preparations.
Chromatographic system (see Chromatography 621)Use a gas chromatograph equipped with a flame-ionization detector. Under typical conditions, the instrument contains a 6.4-m × 2.1-mm (ID) nickel column packed with 60- to 80-mesh support S9, the column is maintained at 100, the injection port is maintained at 160, the detector is maintained at 200, and helium is used as the carrier gas at a flow rate of 30 mL per minute. The resolution, R, of ethylene oxide and acetaldehyde, upon chromatographing a solution containing 10 µg per mL of each in Stripped nonoxynol 9, is not less than 1.5. None of the points used for constructing the straight line Calibration curve deviates from the line by more than 10%.
Calibration Place the vial containing the 10-ppm ethylene oxide Standard preparation in an oven, and heat at 90 for 30 minutes. Remove the vial from the oven. Using a gas-tight syringe, immediately inject a 100-µL aliquot of the headspace gas into the gas chromatograph. Obtain the area for the ethylene oxide peak (retention time approximately 8 minutes). Raise the temperature of the column to 200 after ethylene oxide elutes to volatilize heavy components. Re-equilibrate the column at 100. Repeat the foregoing steps, using the vials containing the 5- and 0.5-ppm Standard preparation. Plot area units versus ppm ethylene oxide for the standards on linear graph paper, and draw the best straight line through the points.
Procedure Place the vial containing the Test preparation in an oven, and heat at 90 for 30 minutes. Remove the vial from the oven. Immediately inject a 100-µL aliquot of the headspace gas into the gas chromatograph, and obtain the area for the ethylene oxide peak. Calculate the concentration of ethylene oxide in the test specimen, in ppm, by the formula:
rUSin which rU is the peak area obtained from the Test preparation, and S is the slope of the standard curve, in ppm per area unit. Not more than 1 ppm is found.
Limit of dioxane
Apparatus Assemble a closed-system vacuum distillation apparatus, employing glass vacuum stopcocks (A, B, and C), as shown in the accompanying diagram. The concentrator tube (D)* is made of borosilicate or quartz (not flint) glass, graduated precisely enough to measure the 0.9 mL or more of distillate collected and marked so that the analyst can dilute accurately to 2.0 mL.
Standard solution Prepare a solution of dioxane in water having a known concentration of about 100 µg per mL. Use a freshly prepared solution.
Test solution Transfer 20.0 g to a 50-mL round-bottom flask (E) having a 24/40 ground-glass neck joint. Add 1.0 mL of water. Place a small polytef-covered stirring bar in the flask, insert the stopper, and stir to mix. Immerse the flask in an ice bath, and chill for about 1 minute. Wrap heating tape around the tube connecting the concentrator tube (D) and the round-bottom flask, and apply about 10 V to the tape. Apply a light coating of high-vacuum silicone grease to the ground-glass joints, and connect the concentrator tube to the 10/30 joint and the round-bottom flask to the 24/40 joint. Immerse the vacuum trap in a Dewar flask filled with liquid nitrogen, close stopcocks A and B, open stopcock C, and begin evacuating the system with a vacuum pump. Prepare a slurry bath from powdered dry ice and methanol, and raise the bath to the neck of the round-bottom flask. After freezing the contents of the flask for about 10 minutes, and when the vacuum system is operating at 0.05-mm pressure or lower, open stopcock A for 20 seconds, then close it. Remove the slurry bath, and allow the flask to warm in air for about 1 minute. Immerse the flask in a water bath maintained at a temperature of between 20 and 25, and after about 5 minutes warm the water bath to between 35 and 40 (sufficient to liquefy most specimens) while stirring slowly but constantly with the magnetic bar. Cool the water in the bath by adding ice, and chill for about 2 minutes. Replace the water bath with the slurry bath, freeze the contents of the round-bottom flask for about 10 minutes, open stopcock A for 20 seconds, and then close it. Remove the slurry bath, and repeat the heating steps as before, this time reaching a final temperature of between 45 and 50 or a temperature necessary to melt the specimen completely. If there is any condensation in the tube connecting the round-bottom flask to the concentrator tube, slowly increase the voltage to the heating tape, and heat until condensation disappears.
Stir with the magnetic stirrer throughout the following steps. Very slowly immerse the concentrator tube in a Dewar flask containing liquid nitrogen.
[CautionWhen there is liquid distillate in the concentrator tube, immerse the tube in the liquid nitrogen very slowly, or the tube will break. ] Water will begin to distill into the concentrator tube. As ice forms in the concentrator tube, raise the Dewar flask to keep the liquid nitrogen level only slightly below the level of ice in the tube. When water begins to freeze in the neck of the 10/30 joint, or when liquid nitrogen reaches the 2.0-mL graduation mark on the concentrator tube, remove the Dewar flask, and allow the ice to melt without heating. After the ice has melted, check the volume of water that has distilled, and repeat the sequence of chilling and thawing until not less than 0.9 mL of water has been collected. Freeze the tube once again for about 2 minutes, and release the vacuum first by opening stopcock B, followed by opening stopcock A. Remove the concentrator tube from the apparatus, close it with a greased stopper, and allow the ice to melt without heating. Mix the contents of the concentrator tube by swirling, note the volume of distillate, and dilute with water to 2.0 mL, if necessary.
Chromatographic system (see Chromatography 621)Use a gas chromatograph equipped with a flame-ionization detector. Under typical conditions, the instrument is equipped with a 2-mm × 1.8-m glass column that contains support S10. The column is maintained at a temperature of about 140, the injection port at 200, and the detector at 250. Nitrogen or helium is the carrier gas, flowing at a rate of about 35 mL per minute. Install an oxygen scrubber between the carrier gas line and the column. Condition the column for about 72 hours at 230 with 30 to 40 mL per minute carrier flow. [noteSupport S10 is oxygen-sensitive. Each time a column is installed, flush with carrier gas for 30 to 60 minutes before heating. ]
Procedure Separately inject equal volumes (about 2 to 4 µL) of the Standard solution and the Test solution. The height of the peak in the chromatogram of the Test solution is not greater than that in the chromatogram of the Standard solution not more than 10 µg per g is found.
Mobile phase Prepare a degassed solution containing a mixture of methanol and water (80:20), making adjustments if necessary (see System Suitability under Chromatography 621).
Resolution solution Dissolve octoxynol 9 and USP Nonoxynol 9 RS in Mobile phase to obtain a solution containing about 25 mg of each per mL.
Standard preparation Dissolve an accurately weighed quantity of USP Nonoxynol 9 RS in Mobile phase to obtain a solution having a known concentration of about 25 mg per mL.
Assay preparation Dissolve about 2.5 g of Nonoxynol 9, accurately weighed, in Mobile phase in a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 25-cm column that contains 10-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution: the resolution, R, is not less than 2.0. Chromatograph replicate injections of the Standard preparation: the nonoxynol oligomers elute as a major peak, usually with shoulders and bumps. Include these in the peak response for Nonoxynol 9. The relative standard deviation is not more than 2.0%.
Procedure Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for Nonoxynol 9, including any shoulders and bumps. Calculate the quantity, in mg, of Nonoxynol 9 in the portion of specimen taken by the formula:
100C(rU / rS)in which C is the concentration, in mg per mL, of USP Nonoxynol 9 RS in the Standard preparation, and rU and rS are the peak responses of Nonoxynol 9 obtained from the Assay preparation and the Standard preparation, respectively.
* A suitable tube is available as Chromaflex concentrator tube, Kontes Glass Co., Vineland, NJ (Catalog No. K42560-0000).
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USP35NF30 Page 4071