Determination of Inositol in Royal Jelly

Pranee Nandhasri, Aung Kyaw Htoon and Chaveewon Phunchaisri

Among seven optically inactive and one pair of The present work compares the HPLC and GC methodsoptically active forms of inositol, only the optically for the determination of inositol in RJP using the criteria.inactive meso form (meso-inositol) is nutritionally of minimal sample preparation and simplicity.active and occurs in nature in the free form. It actsas a lipotropic agent and prevents the development of HPLC Procedureacute fattv livers and the accumulation of cholesterol

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Th Dissolve 50 mg of inositol reference standard (E. Merck,reeze- ne rova J€ Y IS fa er vgrosCOplC. e pro- . .d th f ' dd b h ' d t l

.k W. Germanv) m LC grade water. The fmal volume wasucers ere ore may a car 0 Y fa es, I e sugars, . . .

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t t ' h. . tu b t

.b made UP to 25 mL to gIve a concentratIOn of 2 mg/0 pro ec agams a mosp enc mOlS re a sorp IOn y

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dried royal jelly. The so called "Royal jelly product" mL (stoc so utwn).

(RJP) is dried rovaljellv with somecarbohvdrates added.P t

.f t d d." " repara IOn 0 5 an ar curve

Freeze-dried royal jelly and RJP are regarded as A standard curve was prepared in the range of 0.5 toimportant nutritive supplements because of then 2 mg/mL by diluting appropriate volumes of the stockbalanced content of water soluble and fat soluble Vl- solution with LC grade water. Standard solutions weretamins, essential amino acids, enzymes, minerals, sugar filtered, injected into HPLC and the correspondingand sugar alcohols, including inositol. The amount peak area recorded. The result factor was calculatedof inositol in royal jelly is generally about 100 fJg/g from the standard curve.(Pongsakorn, 1988).

Preparation of samplesA high performance liquid chromatograph (HPLC) . ..procedure for the separation of sugar and sugar alcohols 200 mg of freeze dned and homogemzed royal Jellyin coconut water has been reported. Positive identi- or RJP was tr:nsferred mto 15 mL graduated tubes and

fication was made only for glucose, fructose, sucrose m~de up to ='~L. The soluhon was ~ortexed for oneand sorbitol (Bergonia, Flavier and Del Rosario, 1984). mmute, centnfuged at 2,000 rpm, fIltered throughManitol and inositol, with close relative retention times 0.45 ilm membrane fllter and 10 ilL mJected mto the(RRT) of 1.20 and 1.25, respectively, were eluted after HPLC.fructose, RRT 1.14, when chromatographed on a Waters

HPLC d. .

. con IhonsSugar-Pak column at 90°C usmg water as eluent.A Varian, Vista 5500 LC svstem with Waters 410

Some HPLC procedures for the separation of sugar and Differential refractometer d~tector which was moni-sugar alcohols have been reported, generally based on to red at sensitivity 256 and scale factor 1. The Sugar-fJ-Bondapak Carbohydrate Analysis column (Gum and Pak column (Waters, S.5., 300 x 6.5 mm) was main-Brown, 1977), Lichrosorb Si 60 (Merck) (Rocca and tained at 90'C and LC grade water was used as mobileRouchouse, 1976; Nachtmann and Budna, 1977). phase at a flow rate of 1.0 mL/rnin.Aminex A-5 (Bio-Rad) column (Goulding, 1975) using'reversed phase, normal phase and ion exchange GC Proceduretechniques, respectively. However, they lack infor- .mation on inositol. Standard preparation and standard curve

. About 10 mg of inositol reference standard \vas trans-Successful separaho~ was ~eported by gas. chroma- ferred accurately into a 5 mL capacity micro reactiontography (GC) usmg tnmethylchlorosllane as vessel with solid screw cap seal and derivatized. ThedenvatIzatwn agent (Sweely, Bentley, Maklta and Wells derivatized product was dissolved in 1 mL of n-hexane1963).

to give a concentration of 10 mg/mL. The injectionof 0.2 to 1 fJL of this standard preparation resulted

Based on a paper presentedat the Asia Pacific Regional Seminal in a standard curve for the range of 2 to 10 !lg. Theon Analysis of Trace Constituents in Foods, Penang, Malaysia, corresponding peak areas were recorded and the result15-17 November, 1990. factor was calculated from the standard curve.

The authors are researchers at the Industrial Metrology and Testing Sample preparationService Centre (MTC), Thailand Institute of Scientific and Tech-nological Research iTISTR), 196 Phaholyothin Road,Chatllchak, About 50 mg of freeze dried and homogenized royalBangkok 10900, Thailand. jelly was used to prepare the derivatized product by

ASEAI\' Food Journal Vol. 6, No.4, 1991 165

",the same procedure as the standard. The derivatized ~ gsample was dissolved in 1 mL of n-hexane and 1 ~L ~ ~was injected into the GC for inositol determination. g

""Preparation of trimethylsilyl ester derivatives ~

" M.~

Trimethylsilyl is the most frequently used method of .J ~derivatization for GC of carbohydrates, owing to the

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rapidity and relative simplicity of the technique. The ~:~~ ~,method originally proposed by Sweeley, et a!. (1963),

)l-' Pistill widely use'd, can be summarized as follows. .J~~L

The carbohydrate sample, 10 mg, is dissolved in 1.0 mL iJIof anhydrous pyridine (dried over KOH pellets) and

~ 5 10 m;n 0 5 10 mln 0 5 10 m,ntreated successively with 2.0 mL of hexamethvldisila-.I . 1a 11: 1czane (HMOS) and 0.1 mL of trimethylchlorosllane(TMCS). The mixture, in a stoppered vial, is shaken Figure 1. H:LC Chromatogr~m of inositol standar~ in \vater

vigorouslv for about 30 sec and'then allowed to stand (FIgure la), royal Jelly product (RJP) (FIgure 1b)

f t I.t - .

f th I t.

f tl t. and RJP splkmg with standard mosltol (Figure Ie).or a cas :J mll1 or e comp e IOn 0 1e reae Ion.

The pyridine is removed by evaporation at 40"'C underreduced pressure after the reaction with,TMCS and reported by Bergonia, et a!. (1984) and also cautionedthe residue is dissolved in n-hexane. The hexane extract by the manufacturer.is injected directly into the Gc.

A number of variations on column temperature andGC conditions mobile phase fIO'iNrate were made to achieve better

resolution between fructose and inositol on the Sugar-The Gas Chromatograph (GC), Perkin Elmer, Sigma Pak column (Table 1).3 with flanle ionization detector consisted of a stainlesssteel column, 12' x 1/8'" with 3% OV 101 Chromosorb It was concluded that the HPLC method was unsat-W-AW on 100/120 mesh support. The oxygen free isfactory for the separation of inositol from fructose inpurified nitrogen (99.9%) was used as carrier gas witha flow rate of 20 mL/min. The injector and detector Table 1. HPLC data from fructose and inositol elutedtemperature were maintained at 2S0"C. The column on Sugar-Pak columnoven tem p erature was set isothermal at 220°C.

C I R , ,. ,. ('

)0 umn e en Ion Ime mmTemp. Flow rate

Evaluation of existing HPLC method (OC) (mUmin) Fructose Inositol Fructose + inositol mixture

A Sucrar-Pak column, claimed bv the manufacturer to 90 0.5 10.24 10.60 10.55 (Merged single peak)be id;al for rapid separation of ohgosaccharides sugars 80 0.5 10.39 10.63 10.66 (Merged single peak),

60 0.5 11.14 10.83 10.37 (Shouldered peak)and sugar alcohols (Waters, 1987), was selected for the 33 0.4 14.04 13.38 13.62 (Shouldered peak)determination of inositol in royal jelly and RJP. When 30 0.3 19.93 19.69 18.69,19.93(Shouldered peak)this column was run at sooe using water as mobilephase, the chromatogram of this standard shown in

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hwith serious baseline drift.meanty stu y s owe camp ete meanty WIt In t e

concentration range selected of 0.0 - 2.0 mg/mL.A Alt ' GC 5

. M h dn ernahve eparahon et 0

The application of the method to royal jelly and RJP The methyl silicone (OV 101) stationary phase wasga.ve good s:para.hon for sucrose, glucose and fruct~se s~lected because of its excellent stability at highwIth retentIOn times (RT) 3.62, 4.46 and 5.39 mm, temperature (maximum operating temp. 3S0"C) andrespectively (Figure 1b). The peaks m the elutIOn being free from active hydrogens. It gives relativelyprofIles were Identllted by spiking. Splkmg of the RJP short retention times for sugar and sugar alcoholswIth a standard mosltol gave the chromatogram shown because of its small attached methyl group on silica.in Figure 1c, where the co-elution of fructose and particles.inositol (RT = 5.46) was observed.

. . When TMCS inositol in n-hexane was injected into thisThe lugher content of glucose and fructose m RJP and column with flame ionization detection, theroyal jelly could be attributed to the inversion of sucrose chromatogram sho\'.:n in Figure 2a was obtained.in the HPLC column. This conversion caused thebroadening/splitting of the sucrose peak and was The complete linear calibration plot of TMCS-inositol

166 ASEAN Food Journal Vol. 6, :\'0. 4, 1991

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Figure 2.GC Chromatogram of TMCS-derivativesin n-hexane of the standard inositol (Figure 2a); mixture of fructose,glucose and inositol at RT 4.92, 5.99 and 8.74 min, respectively (Figure 2b); royal jelly (Figure 2c); androyal jelly spiked \vith 13.7 !lg of inositol (Figure 2d).

was in the range of 2-10 ~g. The detection limit was0.5 ~g for TMCS-inositol, ten times better than by HPLC J. Chromatograph 103: 229; 1975.v'lith refractive index detection.

Gum, E.K. and Brown, RD. Two alternative HPLCTMCS sugar and sugar alcohols on the column gave separation methods for reduced and normalgood separahon for fructose, glucose and InosItol wIth cell oligo saccharides. Anal. Biochem. 82: 372; 1977.RT 4.92, 5.99 and 8.74 min, respectively. A typicalchromatogram of the mixture is shown in Figure 2b. Heinz Nutrition Research Division. The water-solubleThe analysis of inositol in royal jelly and a royal jelly vitamin: Inositol. Nutritional data. 3rd ed. 29: 1956.sample spiked with 13.7 ~g inositol are shown in Figure Mellon Institute, Pittsburgh, Penn. USA.2c and Figure 2d, respectively.

Nachtmann, F. and Budna, K.W. Sensitive determina-The results obtained for inositol in royal jelly and RJP tion of derivatized carbohvdrates by HPLC. J.were492and54mg/100g, respectively. Percent recovery Chromatograph 140: 304; 1977.studies on both samples were performed by addingknown quantities of inositol to the sample prior to Pongsakorn, S. Royal jelly. A family doctor magazine.derivatization with satisfactory recoveries of 100-102%. Bangkok, Thailand. 7: 72-73; 1988.

This work indicates that inositol in royal jelly and RJP Rocca, J.L. and Rouchouse, A. Separation of sugarscannot be reliably determined by HPLC using the Sugar- on microparticulatesilica by HPLC.J. ChromatographPak column. The alternative GC method based on 117: 216; 1976:trimethylchlorosilane derivatization and flame ionization

.detection is rapid, accurate and can probably be suc- Sweely, c.c., Bentley, R., Makita, M. and Wells, W.W.cessfully applied to other bee products like honey and GC oftrimethylsilyl derivatives of sugars and relatedbee pollen. substances. J. Am. Chem. Soc. 85: 2497; 1963.

Waters. Rapid separation of similar sugar and sugarREFERENCES alcohol pairs. Notes on food and beverages. Waters

Bergonia, H.A., Flavier, M.F. and Del Rosario, E.J. HPLC Chromatography Division, Milipore Corporation

analysis of carbohydrates in coconut water. Pro- PublIsher. Vol. 2. Sept. 1987.

ceedings of the 2nd ASEAN Workshop on FoodAnalytical Techniques. March 18-24, 1984. pp 203-221

Goulding, RW. Liquid chromatography of sugars andrelated poly hydric alcohols on cation exchangers.

ASEA:-\ Food Journal Vol. 6, No.4, 1991 167