Identification—
B:
Thin-Layer Chromatographic Identification Test 
201
—
Test solution:
Dissolve 10 mg of Metrifonate in methanol, and dilute with methanol to 10.0 mL.
Developing solvent sytstem:
a mixture of toluene, dioxane, and glacial acetic acid (70:25:5)
Procedure—
Proceed as directed in the chapter. After allowing the plate to air-dry, spray the plate with a 5% solution of 4-(
p-nitrobenzyl)pyridine in acetone, and heat at 120
for 15 minutes. Before the plate cools, spray it with a 10% solution of tetraethylenepentamine in acetone, and immediately examine the plate: the principal spot in the chromatogram obtained from the
Test solution corresponds in
RF value, size, and blue color to that in the chromatogram obtained from the Standard solution.
C:
Dissolve 20 mg of Metrifonate in 1 mL of 2 N sodium hydroxide, add 1 mL of pyridine, shake, and heat on a water bath for 2 minutes: a red color develops in the pyridine layer.
D:
To 100 mg of Metrifonate add 0.5 mL of nitric acid, 0.5 mL of a 50% solution of ammonium nitrate, and 0.1 mL of 30 percent hydrogen peroxide, and heat on a water bath for 10 minutes. Heat to boiling, and add 1 mL of
ammonium molybdate TS: a yellow color precipitate is formed.
Acidity—
Dissolve 2.5 g of it in carbon dioxide-free water, dilute with carbon dioxide-free water to 50 mL, and add 0.1 mL of
methyl red TS. Not more than 1.0 mL of 0.1 N sodium hydroxide is required to change the color of the indicator.
Chromatographic purity—
Solution A—
Dissolve 1.36 g of monobasic potassium phosphate in water, and dilute with water to 1000 mL. Adjust with phosphoric acid to a pH of 3.0.
Solution B—
Use 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 mixture of acetonitrile and water (1:1).
Standard preparation—
Prepare a solution of USP Metrifonate RS in Diluent containing 20 mg per mL.
Test solution—
Transfer 500 mg of Metrifonate, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 210-nm detector and a 4-mm × 25-cm column that contains 5-µm packing L7. The column is maintained at a constant temperature of about 40
. The flow rate is about 1.5 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0 |
90 |
10 |
equilibration
(10 minutes) |
| 0–5 |
90 |
10 |
isocratic |
| 5 |
90®85 |
10®15 |
step gradient |
| 5–25 |
85 |
15 |
isocratic |
| 25 |
85®45 |
15®55 |
step gradient |
| 25–end* |
45 |
55 |
isocratic |
|
*
The elution concludes at 3 times the retention time of metrifonate.
|
Procedure—
Separately inject equal volumes (about 50 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the percentage of each impurity taken by the formula:
100F(ri / rS),
in which
F is a response factor, being 0.38 for the desmethylmetrifonate peak, if present at a retention time of 0.5 relative to that of Metrifonate, 0.03 for the dichlorvos peak, if present, at a retention time of 1.9 relative to that of Metrifonate, and 1.0 for any other impurity;
ri is the peak area for the individual impurity obtained from the
Test solution; and
rS is the peak area for Metrifonate obtained from the
Standard solution: not more than 1.0% of desmethylmetrifonate, 0.2% of dichlorvos, and 0.5% of any other impurity are found; and a total of not more than 1.0% of impurities other than desmethylmetrifonate and dichlorvos is found.
Assay—
Dissolve about 300 mg of Metrifonate, accurately weighed, in 30 mL of alcohol. Add 10 mL of monoethanolamine, and allow to stand for 1 hour at 21 ± 1
. Cool while adding a mixture of 100 mL of water and 15 mL of nitric acid. While maintaining the temperature at 21 ± 1
, titrate with 0.1 N silver nitrate VS, determining the endpoint potentiometrically using a silver electrode. Each mL of 0.1 N silver nitrate is equivalent to 25.74 mg of C
4H
8Cl
3O
4P.
;)
