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
[Caution2,3,7,8-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.