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HPLC Analysis of Garlic Clove Homogenates

Introduction

Although pyruvic acid determination affords a relatively simple means of quantifying total thiosulphinate in garlic clove homogenates it is unable to provide the separation and quantitation of individual thiosulphinates available by modern analytical techniques. Ever since the identification of allicin (diallyl thiosulphinate) considerable effort has been directed towards the isolation and identification of the individual compounds responsible for the flavours and odours of freshly cut or crushed garlic. Thiosulphinates other than allicin were originally found to be present in garlic homogenates after separation by thin layer chromatography followed by reaction with cysteine or with N-ethylmaleimide but these methods lacked resolution and sensitivity and so GC and HPLC analysis became the methods of choice throughout the 1980ís and 1990ís.
Because of their excellent resolution and mass identification capabilities, GC and GC-MS have been used extensively in the effort to characterise allium volatiles despite the early cautionary note that many of the compounds seen by GC may be Ďartifacts of analysisí.
The instability of thiosulphinates leads to rapid decomposition in the oven of a gas chromatograph, even at moderate temperatures, and while indirect quantitation has been reported, the methods suffer from significant limitations.
HPLC analysis of allicin in garlic was first reported in 1985 by Miething who analysed diethyl ether extracts of garlic and garlic products by silica-HPLC. A number of workers have since reported on HPLC analysis of alliums and whilst there are significant differences in the techniques employed, HPLC has been shown to provide a reliable method of measuring what is actually present in garlic tissue and of avoiding the problems of erroneous compounds associated with GC.
The recent work of Block and Naganathan at the Department of Chemistry, State University of New York is part of an extensive HPLC study of allium thiosulphinates and the techniques described have been adapted here to provide a reliable method of analysis of a number of diverse garlic clones

Materials and Methods

Materials:
Garlic samples were obtained from twenty-one different garlic clones grown under controlled conditions in the UK.
Solvents used for HPLC were obtained from Sigma and Philip Harris (HPLC grade) and used without further purification.
The method employed in this study is based on the work of Block et al although modifications to the method of extract preparation have been made. Whereas Block used a method of room temperature distillation after extraction this method employs a technique of centrifugation and concentration with nitrogen. The low temperatures and rapidity associated with this method of concentration ensured minimal degradation of the extracted thiosulphinates in the dichloromethane and the extracts remained stable for periods in excess of one hour following concentration.

Preparation of extracts:
Fresh garlic cloves were peeled, coarsely chopped with a knife and a 10 g sample homogenised for 2 min with 100 ml distilled water in a food blender (Kenwood). The homogenate was allowed to stand at room temperature for 15 min to ensure the complete enzymatic transformation of thiosulphinates before being filtered through 4 layers of cheesecloth.
A 50 ml sample of the filtrate was pipetted to a 500 ml conical flask and 15 g sodium chloride (NaCl) added to saturate the solution. After ensuring complete dissolution of the NaCl, 50 ml cold dichloromethane (CH2Cl2) was added and the stoppered flask agitated vigorously for 3 min. The resultant emulsion was centrifuged at 4000 rpm for 15 min, the lower solvent layer removed and dried twice with anhydrous sodium sulphate (Na2SO4).
A 10 ml sample of the dry extract was then pipetted to a 10 µl measuring cylinder. This was placed in a water bath at 25ºC ± 5ºC, and the extract was rapidly concentrated by bubbling dry nitrogen through it. The solvent was evaporated to < 1 ml final volume and then adjusted accurately to 1 ml with dichloromethane. The concentrated extract was taken up in a glass syringe and injected directly through a 0.2 µm in-line nylon filter into a 10 µm fixed sample loop.
Due to the instability of thiosulphinates in organic solvents all HPLC runs were undertaken within 45 min of extraction.

Equipment
Si-HPLC analyses were performed on a Rainin Microsorb silica gel column ( 5 µm, 250 mm x 4.6 mm with a 15 mm x 4.6 mm guard ). Solvent delivery and gradient control were achieved using a Merck-Hitachi L-6200A Intelligent Pump and detection at 254 nm with a Merck-Hitachi L4500 Diode Array Detector. A Merck-Hitachi D-6500 DAD System Manager was used for data collection and processing.
The program used throughout the experiments is shown in the table below.

Time (min)
Flow (ml/min)
Composition (%)
Remarks
   
2-Propanol
Hexane
 
0 1.70
2 98  
6 1.70 2 98 Linear increase
16 1.70 10 90  
25 1.70 10 90 Linear decrease
30 1.70 2 98  

 

 


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