Proceedings of the 7 th lnternatumal Working Conference on Stored-product Protection - Volume 2

Development of a simplified analytical method for ergosteroldetermination in paddy rice

S. Wattananon1 & G. Srzedmck12

Abstract

Moulds are considered as one of the principal causes ofstorage losses in grain. High temperature and moisturefavour their reproduction and growth. Ergosterol IS the mamsterol found m most fungi, mostly as a component of cellmembrane. Hence, ergosterol appears to be a suitableindicator of mould activity and thus of spoilage m storedgram.

Introduction

Previous work on ergosterol has shown that the HPLCmethod was the most accurate one for determination of thiscompound in paddy rice. However, given the complexity ofsolvent extraction methods, attempts have been made atmodifying the HPLC method according to a matnx includingcnteria such as recovery, hnearity of the range, cost,operator safety, userfriendliness and environmentalacceptability, especially in terms of recyclmg of solvents. Inorder to achieve these objectives sohd phase extraction(SPE) method has been tested by usmg a wide range ofclean-up and HPLC columns as well as mobile phases. SPEappears to fulfill most of the acceptability cnteria whencompared with the conventional solvent extraction methods.

PRINCIPAL STEPS OF THE METHOD

Rice sample~

Extraction of total lipid~

Saponifica tion~

Extraction by SPE~

HPLC

1 Rajabhat Institute, Lampang, Thailand

2 The University of NSW, Sydney, Australia

Procedure

Extraction of total lipid from rice

The total lipid is extracted from rice by using the mixtureof methanol chloroform water (2 . 1 0.8).

Saponification of the lipid extract

Supernatant from lipid extraction IS saponified by boilinggently under reflux for 4h.

Extraction of ergosterol by solid phase extraction

Method: Gessner & Schmitt (1996).Chemicals :Pure methanolPotassium hydroxide, 1.0 M m pure methanolPotassium hydroxide, 0.4 M in 60 % methanolPotassium hydroxide, O. 1 M in pure isopropanolPotassium hydroxide, O. 14 M m pure methanolPotassium hydroxide, O. 12 M in pure methanolHydrogen chloride, 0.75 M

Glassware:5 ml glass syringes25 and 50 ml beakersSmall sample tubes for HPLC analysis

SPE by modified gessner & schmitt method

Step 1: Condittoning of SPE cartridgeThe solid phase extraction cartridges (CI8) are

conditioned by three 2.5 ml volumes of methanol, followedby three 2.5 ml volumes of conditioning solvent which is themixture of 6 volumes of O. 12 M KOH and 1 volume of 0.75M HCl. An additional 2 ml of conditioning solvent must beleft above the column packing to prevent the column frombecoming dry dunng conditioningStep2: Extraction of ergosterolAfter the total lipid extraction of rice followed by

saponification, the sample was loaded into the previouslyconditioned column at a flow rate of 1 ml/min, Afterintroduction of the sample, the column was washed with 2.5ml of 0.4 M KOH in 60 % methanol. Then, ergosterol waseluted into the HPLC sample tube with 1.5 ml of 0.1 MKOH m pure isopropanol and 0.3 ml of 1 M KOH m puremethanol. The sample tube should be stored at low

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Proceedmqe of the 7th International Worki'ng Conference on Stored-product Protection - Volurn.e 2

temperature until ready for injection to HPLC.

Conclusion

The method has been tested on spiked nee samples andproved to be meeting most of the cntena for use asalternative to the currently used solvent extractionprocedure. The validation on a larger number of ricesamples from storage tests IS currently in progress

References

Gessner, M. and Schmitt, A.1., 1996 Use of Solid-Phase

Extraction to Determine Ergosterol Concentrations m PlantTissue Colonized by Fungi. Applied and EnvironmentalMicrobiology, 62, 2, 415-419.Wattananon, S., Photchanachai, S., Mulholland, M. &Srzedmcki, G., 1997 Determination of Ergosterol as anIndicator of Fungal Growth in Paddy RIce. Part 2. Paperto be presented at the 18th ASEANTechmcal Seminar onGrain Postharvest Technology, 12 - 14 March 1997,Manila, Philippines (Proceedings in print).

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