Limit of monochlorophenols and 2,4-dichlorophenol—
Phosphate buffer—
Transfer about 1.38 g of anhydrous monobasic sodium phosphate and about 1.42 g of dibasic sodium phosphate to a 1-liter volumetric flask, dissolve in and dilute with water to volume, and mix.
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile and
Phosphate buffer (1:1). Make adjustments if necessary (see
System Suitability under
Chromatography
621
).
Standard solution—
Quantitatively dissolve accurately weighed quantities of 4-chlorophenol and 2,4-dichlorophenol in acetonitrile, dilute with an equal volume of water, and mix. Transfer a portion of this solution to a suitable container, and dilute quantitatively, and stepwise if necessary, with a mixture of acetonitrile and water (1:1) to obtain a solution having known concentrations of about 0.5 µg of 4-chlorophenol and 0.1 µg of 2,4-dichlorophenol per mL.
Test solution—
Transfer about 250 mg of Triclosan, accurately weighed, to a 25-mL low-actinic volumetric flask, dissolve in 20 mL of acetonitrile, dilute with water to volume, and mix.
Chromatographic system (see Chromatography
621
)—
The liquid chromatograph is equipped with a coulometric electrochemical detector with electrode 1 set at 0.45 V and electrode 2 set at 0.75 V, both having a positive (oxidative) polarity and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 9.0% for 2,4-dichlorophenol.
Procedure—
Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. The peak responses for 4-chlorophenol and 2,4-dichlorophenol in the chromatogram of the Test solution are not greater than the corresponding peaks in the chromatogram of the Standard solution.
Limit of 1,3,7-trichlorodibenzo-p-dioxin, 2,8-dichlorodibenzo-p-dioxin, 2,8-dichlorodibenzofuran, and 2,4,8-trichlorodibenzofuran—
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile, water, and glacial acetic acid (70:30:0.1). Make adjustments if necessary (see
System Suitability under
Chromatography
621
).
Standard solution—
Transfer accurately weighed quantities of 2,8-dichlorodibenzofuran, and 2,4,8-trichlorodibenzofuran to a volumetric flask, add accurately measured volumes of 1,3,7-trichlorodibenzo-p-dioxin and 2,8-dichlorodibenzo-p-dioxin, and dissolve in methanol. Dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having concentrations of about 0.5, 1.0, 0.5, and 1.0 µg per mL, respectively.
Test solution—
Transfer about 2.0 g of Triclosan, accurately weighed, to a screw-capped centrifuge tube, add 5 mL of 2 N potassium hydroxide, and shake for 10 minutes to dissolve. Add 3 mL of n-hexane, shake for 10 minutes, and allow the phases to separate. Transfer the organic layer to a suitable container, add another 3 mL of n-hexane to the aqueous layer, shake for 10 minutes, and allow the phases to separate. Transfer the organic layer to the previous extract, discard the aqueous layer, add 3 mL of 2 N potassium hydroxide to the combined organic layers, shake for 10 minutes, and allow the phases to separate. Discard the aqueous layer, add another 3 mL of 2 N potassium hydroxide to the combined organic layers, shake for 10 minutes, and allow the phases to separate. Transfer the organic layer to a suitable container, and evaporate with the aid of a stream of nitrogen to dryness. Dissolve the residue in 1.0 mL of methanol, and mix.
Chromatographic system (see Chromatography
621
)—
The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.59 for 2,8-dichlorodibenzofuran, 0.71 for 2,8-dichlorodibenzo-
p-dioxin, 0.88 for 2,4,8-trichlorodibenzofuran, and 1.0 for 1,3,7-trichlorodibenzo-
p-dioxin; and the relative standard deviation for replicate injections is not more than 15.0%, determined from the 2,8-dichlorodibenzo-
p-dioxin peak.
Procedure—
Inject a volume (about 20 µL) of the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. The peak responses for 2,8-dichlorodibenzofuran, 2,8-dichlorodibenzo-p-doxin, 2,4,8-trichlorodibenzofuran, and 1,3,7-trichlorodibenzo-p-dioxin obtained from the Test solution are not greater than the corresponding peaks obtained from the Standard solution.
Limit of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran—
[Caution—2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran are extremely toxic substances. Exercise all necessary precautions in the conduct of this procedure.
]
Stationary phase A—
Transfer about 10 g of silica gel to a suitable container, add about 3 mL of 1 N sodium hydroxide, and mix.
Stationary phase B—
Transfer about 60 g of silica gel to a suitable container, add about 74 mL of concentrated sulfuric acid, and mix.
Chromatographic column A—
Transfer 5.1 g of Stationary phase A, 0.5 g of silica gel, 6.2 g of Stationary phase B, and 3.2 g of sodium sulfate to a glass chromatographic column having an internal diameter of 10 mm. Wash the column with 50 mL of n-hexane, and discard the eluate.
Chromatographic column B—
Transfer 2.5 g of alumina and 2.5 g of sodium sulfate to a glass chromatographic column having an internal diameter of 6 mm. Wash the column with 30 mL of n-hexane, and discard the eluate.
Internal standard solution—
Transfer accurately measured quantities of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 13C-labeled, and 2,3,7,8-tetrachlorodibenzofuran, 13C-labeled, in nonane, and dilute quantitatively, and stepwise if necessary, with 2,2,4-trimethylpentane to obtain a solution having known concentrations of about 1.0 pg of each per µL.
Test solution—
Transfer about 30 g of Triclosan, accurately weighed, to a separatory funnel, add 30 µL of Internal standard solution, dissolve in 200 mL of 1 N sodium hydroxide, extract with four 30-mL portions of n-hexane, and combine the extracts. Wash the combined extracts with 20 mL of water, extract the washing with 15 mL of n-hexane, and add the extract to the other combined extracts. Add about 3 g of anhydrous sodium sulfate to the combined extracts, allow to stand for 30 minutes, quantitatively transfer to an appropriate round-bottom flask, and distil, using a distillation apparatus with a vigreux column, until about 1 mL remains. Transfer this solution to the top of Chromatographic column A, and elute with 50 mL of n-hexane. Collect the eluate on top of Chromatographic column B, and elute with 30 mL of a mixture of n-hexane and methylene chloride (98:2), discarding the eluate. Elute with 40 mL of a mixture of n-hexane and methylene chloride (1:1), collecting the eluates in a round-bottom flask. Distill the combined eluates, using a distillation apparatus with a vigreux column, until about 1 mL remains. Further concentrate this solution with the aid of a stream of nitrogen to about 50 µL, evaporate at room temperature to dryness, and dissolve in 10 µL of 2,2,4-trimethylpentane.
Chromatographic system (see Chromatography
621
and Mass Spectrometry
736
)—
The gas chromatograph is equipped with a high-resolution mass spectrograph with an electron-impact ionization source and a 0.25-mm × 60-m capillary column coated with phase G48. The carrier gas is helium. The chromatograph is programmed as follows. Initially the temperature of the column is equilibrated at 80

, then, 1 minute after the injection, the temperature is increased at a rate of 20

per minute to 220

, then increased at a rate of 2

per minute to 270

, and maintained at 270

for not less than 20 minutes. The injection port temperature is maintained at 280

. Chromatograph the
Internal standard solution, and record the peak responses as directed for
Procedure: the signal-to-noise ratio at a mass-to-charge ratio of 321.89 is not less than 50.
Procedure—
Inject a volume (about 1 µL) of the Test solution into the chromatograph, record the chromatograms, and measure the peak responses at mass-to-charge ratios of 319.90, 321.89, 331.88, 333.93, 303.90, 305.90, 315.94, and 317.94. The peak response for 2,3,7,8-tetrachlorodibenzo-p-dioxin at a mass-to-charge ratio of 319.90 is not more than the peak response of the associated internal standard at a mass-to-charge ratio of 331.88; the peak response for 2,3,7,8-tetrachlorodibenzofuran at a mass-to-charge ratio of 303.90 is not more than the peak response of the associated internal standard at a mass-to-charge ratio of 315.94.
Assay—
Standard preparation—
Dissolve an accurately weighed quantity of
USP Triclosan RS in dichloromethane, and dilute quantitatively, and stepwise if necessary, with dichloromethane to obtain a solution having a known concentration of about 4.0 mg per mL.
Assay preparation—
Transfer about 40 mg of Triclosan, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with dichloromethane to volume, and mix.
Chromatographic system (see Chromatography
621
)—
The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 15-m capillary column with G3. The carrier gas is helium maintained at about 6 psi. The injector temperature is maintained at 34

and is increased rapidly to 200

immediately after the injection, the column temperature is maintained at 34

, and the detector temperature is maintained at 260

. 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 0.5 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, increase the column temperature by 20

per minute to 140

, then increase column temperature by 4

per minute to 240

, maintain this temperature for not less than 5 minutes, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C
12H
7Cl
3O
2 in the portion of Triclosan taken by the formula:
10C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Triclosan RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.