Extraction column— Use a solid-phase extraction column that contains benzenesulfonylpropyl bonded to silica gel in the hydrogen form having a sorbent mass to column volume ratio of 1 g per 6 mL, or equivalent. Condition the column prior to use by washing with 10 mL of methanol and with 10 mL of water. [NOTE—Do not allow the column to dry.]

Test solution— Mix 1 mL of Fluidextract with 5 mL of water, and transfer to the Extraction column. Allow to drain, and discard the eluate. Wash the column with 10 mL of water and 10 mL of methanol, discarding the eluates. Elute the amino acid fraction with 3 mL of ammonium hydroxide solution in methanol (7 in 100), and collect the eluate.

Standard solution: 0.5 mg of USP S-Allyl-L-Cysteine RS per mL.

Application volume: 5 µL.

Developing solvent system: a mixture of ethyl acetate, methanol, water, acetone, and glacial acetic acid (10:4:3:3:1).

Procedure— Proceed as directed in the chapter. Spray with iodoplatinate TS, and examine the plate: the chromatogram of the Test solution exhibits, among several yellow spots on the purple plate, a yellow spot at an RF value of about 0.4 corresponding to that of the yellow spot obtained in the chromatogram of the Standard solution (presence of S-allyl-L-cysteine).

Mobile phase— Transfer 15.8 g of sodium citrate dihydrate to a 1000-mL volumetric flask containing 250 mL of water, carefully add 10.5 mL of hydrochloric acid, and mix. Using a pH meter, adjust with 6 N sodium hydroxide to a pH of 4.0. Dilute with water to volume, and mix.

Derivatizing reagent— Dissolve 0.8 g of o-phthalaldehyde in 2 mL of 2-mercaptoethanol, add to a solution containing 24.70 g of boric acid and 22.35 g of potassium hydroxide in 1000 mL, and mix.

Reactivating solution— Prepare 0.2 N sodium hydroxide by dissolving 0.8 g of sodium hydroxide in 100 mL of water.

Standard solution— Dissolve an accurately weighed quantity of USP S-Allyl-L-Cysteine RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.01 mg per mL.

Test solution— Transfer about 2.0 g of Fluidextract, accurately weighed, to a 100-mL volumetric flask, dilute with trichloroacetic acid solution (5 in 100) to volume, and mix. Centrifuge for 5 minutes, and filter the supernatant.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 4.6-mm × 12-cm column that contains packing L17. The column temperature is maintained at 40. The Mobile phase and the Reactivating solution are pumped separately, each at the rate of about 0.4 mL per minute, by pumps connected to the opposing arms of a tee. The outlet of the tee is connected to the injector and the chromatographic column. The outlet of the column is attached to a tee, the opposing arm of which is attached to a tube from which the Derivatizing reagent is constantly pumped through the system at a rate of about 0.6 mL per minute. The outlet of the tee is connected to a 0.5-mm × 2.0-m postcolumn polytef reaction coil maintained at 40. The outlet of the reaction coil is connected to a fluorometric detector set at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The system is programmed to deliver the Mobile phase for 10 minutes, the Reactivating solution for the next 6 minutes, and the Mobile phase for the 24 minutes remaining before the next injection. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the capacity factor, k¢, is between 2.5 and 4.5; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the heights of the major peaks. Calculate the quantity, in mg, of S-allyl-L-cysteine (C6H11SN) in the portion of Fluidextract taken by the formula: in which C is the concentration, in mg per mL, of USP S-Allyl-L-Cysteine RS in the Standard solution; W is the weight, in g, of Fluidextract taken to prepare the Test solution; and rU and rS are the peak heights for S-allyl-L-cysteine obtained from the Test solution and the Standard solution, respectively: not less than 0.05% is found, calculated on the dried basis.

(Official January 1, 2007)