Allicin by High-Performance Liquid Chromatography
INA Method 110.001

Assay Title: Determination of allicin in garlic by high-performance liquid chromatography.

Scope: This assay can be used to determine allicin content in Allium sativum
(garlic) powdered extracts and cloves.

References: Garlic. The Science and Therapeutic Application of Allium sativum L. and Related Species; Second Edition; Heinrich P. Koch, and Larry D. Lawson, editors; Williams & Wilkins, Baltimore

"HPLC Analysis of Allicin and Other Thiosulfinates in Garlic Clove Homogenates," Lawson, L.D., Wood, S.G., and Hughes, B. Planta Medica, 57, 1991, pp 263 – 270

"Characterization of Formation of Allicin and Other Thiosulfinates from Garlic," Lawson, L.D., Hughes, B. Planta Medica, 58, 1991, pp. 345 – 350

"A Spectrophotometric Method for Quantitative determination of Allicin and Total Garlic Thiosulfinates." Han, J., Lawson, L., Hand, G., and Han, P. Anal. Biochem. 225, 1995, pp 157 - 160

Safety Precautions: Consult the Material Safety Data Sheet (MSDS) for any chemical used that is unfamiliar. All chemicals should be considered hazardous - avoid direct physical contact. For more safety information, see http://hazard.com/msds/

Background: Allicin (diallylthiosulfinate) is responsible for the usual odor of fresh-cut garlic. It is produced in crushed garlic cloves or in wetted garlic powder through the rapid lysis of alliin by alliinase. Allicin has a water solubility of about 2%, is moderately soluble in hexane, and is very soluble in organic solvent more polar than hexane. Allicin yield is commonly used as a measure of garlic quality. Preparation of a pure standard is necessary since its instability precludes its commercial availability.

Allicin is most stable in water (especially if acidified), with a half-life of about 30 days at room temperature, and least stable in hexane where its half-life is only 2 hours. Allicin's greater stability in water is due to hydrogen bonding which reduces the rate of self-reaction. The stability of allicin is also very temperature-dependent. Refrigeration increases its stability about 20-fold, but the only sure way to store allicin is in water at -70°C.

Principle: Allicin reference standard is isolated from an aqueous extract of garlic powder using solid phase extraction (SPE) chromatography. The concentration of allicin in the isolated fraction is standardized by spectrophotometry and HPLC analysis. Sample analysis is carried out by extraction of garlic powder or cloves with water and quantified against the isolated allicin external standard. The HPLC column is Phenomenex Prodigy™ ODS (3) C18, 5µm, 100Å, 4.6 x 250 mm; the mobile phase is methanol/water (50/50) at 0.9 mL/min and detection is at 240 nm.

Apparatus

• Balance, analytical, accurate to ±0.1 mg
• Centrifuge tubes, glass, 50-mL, with screw cap
• Pipettes, Class A, assorted sizes
• SPE Columns, Bakerbond SPE C18, 1.0 g, 6-mL, Cat. #7020-07
• Solid Phase Vacuum Manifold and vacuum pump
• Volumetric flasks, Class A, assorted sizes
• HPLC system, HP Model 1050, or equivalent
• High-Performance Liquid Chromatography System as described in USP chapter <621>. Verify and document that apparatus, software, and subsystems meet performance requirements for Installation Qualification/Operation Qualification (IQ/OQ).
• Spectrophotometer
• Garlic Press

Reagents:

• Methanol, 99.99%HPLC, Spec. and GC suitable
• Water, HPLC grade or NanoPure
• Garlic Powder having high allicin yield (≥12 mg/g)

HPLC and SPE Mobile Phase Preparation:

Prepare by combining equal volumes (1:1) of methanol and water.

Standard Preparation:
Condition SPE column by rinsing with 6 mL of methanol, discarding the methanol.

Equilibrate the column with three column volumes (18 mL) of SPE mobile phase. Stop the flow when the liquid meniscus contacts the top of the column packing. Discard the collected eluent.

Create an allicin solution by shaking 0.4 g of garlic powder with 10 mL of water. Allow solution to settle and apply 1 mL of the solution to the column head. Initiate flow and adjust flow rate to about 1 drop/second. Bring meniscus to top of column packing.

Add 5.2 mL of mobile phase and initiate flow. Bring meniscus to top of column packing and
discard the 5.2 mL of eluent.

Cool a 5-mL volumetric flask containing 1.5 mL of water in a refrigerator at 4°C for
approximately 10 minutes.

Add 2.7 mL of mobile phase to the top of the column and place the cold 5-mL volumetric flask for collection of eluent. Collect 2.7 mL by bringing the meniscus to the top of the column packing.

Dilute the collected fraction to volume. Mix.

Note: This allicin solution is stable for about 24 hours at -20°.

Measure, against water, the absorbance of the collected fraction at 240 nm and 254 nm using a 1 cm quartz cuvette. The absorbance ratio (240 nm/254 nm) is typically 1.4 to 1.5.

Calculate the concentration (C) of allicin in solution using the equation below. The extinction coefficient (E) for allicin in water is 145.4 (1 cm cell @ 240 nm).

The chromatographic purity is used as an acceptance criteria for the calibration solution. The % impurity by peak area must be < 12% for the allicin solution to be used as a calibration solution. Because percent impurities may be overestimated using this technique, the actual standard impurity is less than area percent purity data indicates. Do not correct concentration for purity. The standard will pass and may be used, or fails and is rejected.

Dilute the working standard with water to create standards for a linearity curve covering approximately 5 – 80 μg/mL for HPLC analysis. Keep the standards cold and stored at≤0°C until ready for use. This diluted allicins solutions are stable for approximately 30 days at freezer temperature.

Preparation of Sample Solutions:

Garlic Powder

Take a 50-mL centrifuge tube and accurately weigh 400 mg of garlic powder into the tube.

Using a volumetric pipette, deliver 25 mL of cold (refrigerated) water to the sample and immediately cap and shake vigorously for 5 seconds. Good contact between water and powder is essential. Avoid lumps.

Shake vigorously for an additional 30 seconds. Four samples can be held and shaken simultaneously to expedite the agitation process. Avoid heat transfer from hands by holding the tube cap while shaking.

Add an additional 25mL of cold water and shake for 30 more seconds to dilute and mix the solution. Filter each sample through 0.45µm glass filter into a test tube. Transfer the solution to an HPLC vial.

Note: Use of cold autosampler racks and working quickly will minimize allicin loss.
Rotate to new, cold autosampler racks hourly. Alternatively, store samples refrigerated if analysis run time exceeds one hour.

Garlic Clove

Peel the outer skin from at least four garlic cloves. Crush the cloves in a garlic press. Collect the pressed garlic in a beaker and mix thoroughly. Weigh 700-900 mg of the pressed mash into a 50-mL centrifuge tube. Using a volumetric pipette, deliver 25 mL of cold (refrigerated) water to the sample and immediately cap and shake vigorously for 30 seconds. Avoid heat transfer from hands by holding the tube cap while shaking.
Add an additional 25mL of cold water and shake for 30 more seconds to dilute and mix the solution. Filter each sample through 0.45µm glass filter into HPLC vial and cap for injection.

Note: Use of cold autosampler racks and working quickly will minimize allicin loss.
Rotate to new, cold autosampler racks hourly. Alternatively, store samples refrigerated if analysis run time exceeds one hour.

HPLC Conditions:

Column: Phenomenex Prodigy™ ODS (3), 5µm, 100Å, 4.6 x 250mm

Mobile Phase: Methanol:Water (50:50)

Flow Rate: 1.0 mL/min

Detector: 240 nm

Injection Vol.: 25µL

Column Temp: 28°C

Run Time: 16 minutes

Note: Allicin elutes at approximately 9 minutes.

Note: Backflushing, after using the HPLC column, for 10 minutes is recommended.

Procedure:

Procedure: Prepare reference standard solutions and sample preparations as directed.

Prepare an extraction solvent blank of water/methanol.

Make a single injection of the blank.

Make single injections of the reference standard preparations.

Prepare a linearity curve for allicin, with the origin ignored, using peak area vs. concentration (µg/mL). Perform linear regression analysis on the data. The coefficient of determination, r2, must be≥0.999.

Make single injections of sample preparations.

Calculate the percent allicin in the samples against external allicin reference standard.

Quality Assurance:

A duplicate sample preparation and should be analyzed with each set of 20 or less samples.

To monitor method variance, a garlic powder control sample of known composition should be assayed with each batch and the assay result control charted.

Calculations:

Quantify allicin against the allicin reference standard response using the following equation.

Where:
C is the sample's allicin concentration (µg/mL),extrapolated from the calibration curve's linear regression.

FV is the final volume of the sample (mL).

D is the dilution factor of the sample preparation (if needed).

W is the sample weight (mg).