Myo-inositol is a 6-carbon cyclic polyalcohol
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1 Schimpf et l.: Journl of AOAC Interntionl Vol. 95, No. 4, INFANT FORMULA AND ADULT NUTRITIONALS Determintion of Myo-Inositol (Free nd Bound s Phosphtidylinositol) in Infnt Formul nd Adult Nutritionls by Liquid Chromtogrphy/Pulsed Amperometry with Column Switching: First Action Kren Schimpf nd Lind Thompson Abbott Lbortories, 3300 Stelzer Rd, Columbus, Steve Bugh AOAC INTERNATIONAL, 481 N. Frederick Ave, Suite 500, Githersburg, MD Myo-inositol is 6-crbon cyclic polylcohol lso known s meso-inositol, met sugr, inosite, nd i-inositol. It occurs in nture in both free (myo-inositol) nd bound (inositol phosphtes nd phosphtidylinositol) forms. For the determintion of free myo-inositol, smples re mixed with dilute hydrochloric cid to extrct myo-inositol nd precipitte proteins, diluted with wter, nd filtered. For the determintion of myo-inositol bound s phosphtidylinositol, smples re extrcted with chloroform, isolted from other fts with silic SPE crtridges, nd hydrolyzed with concentrted cid to free myo-inositol. Prepred smples re first injected onto Dionex CrboPc PA1 column, which seprtes myo-inositol from other lte-eluting crbohydrtes. After column switching, myo-inositol is further seprted on CrboPc MA1 column using 0.12% sodium hydroxide mobile phse; strongly retined crbohydrtes re eluted from the PA1 column with 3% sodium hydroxide mobile phse. Elunt from the CrboPc MA1 nlyticl column psses through n electrochemicl detector cell where myo-inositol is detected by pulsed mperometry using gold electrode. The method showed pproprite performnce chrcteristics versus selected estblished stndrd method performnce requirement prmeters for the determintion of myo-inositol: liner response; repetbility (RSD r ) of 2%; nd intermedite precision (RSD ir ) of 2.5%. Instrument LOD nd LOQ Submitted for publiction Jnury 24, The method ws pproved by the Expert Review Pnel on Infnt Formul nd Adult Nutritionls s First Action. See Stndrds News, (2011) Inside Lbortory Mngement, September/October issue. The AOAC Stkeholder Pnel on Infnt Formul nd Adult Nutritionls (SPIFAN) invites method users to provide feedbck on the First Action methods. Feedbck from method users will help verify tht the methods re fit for purpose nd re criticl to gining globl recognition nd cceptnce of the methods. Comments cn be sent directly to the corresponding uthor. Corresponding uthor s e-mil: kren.schimpf@bbott.com DOI: /jocint.CS2011_18 were nd mg/100 ml, respectively, nd correspond to free myo-inositol quntittion limit of mg/100 g nd phosphtidylinositol quntittion limit of mg/100 g. Correltion with the reference microbiologicl ssy ws good. The proposed method hs been ccepted by the Expert Review Pnel s n AOAC First Action Method, suitble for the routine determintion of myo-inositol in infnt formul nd dult nutritionls. Myo-inositol is 6-crbon cyclic polylcohol (Figure 1) tht occurs s nhydrous, hydroscopic crystls. Myo-inositol hs sweet tste, is soluble in wter, nd is slightly soluble in lcohol. It is insoluble in ether nd other orgnic solvents. Aqueous solutions re neutrl. It hs moleculr weight of , melting point of C, nd is opticlly inctive. There re nine possible stereoisomeric forms of inositol. Seven re opticlly inctive or meso. Two opticlly ctive forms, the rcemic form, nd severl cis, trns-isomers occur nturlly. Myo-inositol is the most widely distributed inositol isomer nd is present in ll living cells. It occurs in nture in both free (myo-inositol) nd bound (inositol phosphtes nd phosphtidylinositol) forms. Fruits, bens, grins, nd nuts re ll good sources of myo-inositol. Myo-inositol is essentil for the growth of mny mmmlin cells. Although free myo-inositol is not essentil for humns, the bnorml metbolism of myo-inositol by the body hs been ssocited with dibetes mellitus nd chronic renl filure. AOAC Officil Method Myo-Inositol (Free nd Bound s Phosphtidylinositol) in Infnt Formul nd Adult Nutritionls Liquid Chromtogrphy/Pulsed Amperometry with Column Switching First Action 2011 The liquid chromtogrphy method with electrochemicl (pulsed mperometry) detection (PAD) llows for the quntittion of myo-inositol in infnt, peditric, nd dult nutritionl formuls. The concentrtion of myo-inositol
2 2 Schimpf et l.: Journl of AOAC Interntionl Vol. 95, No. 4, 2012 Pump 1 PA1 Gurd Column HO Electrochemicl Detector MA1 Gurd nd Anlyticl Columns Pump 2 Figure 1. Myo-inositol. is clculted by comprison with stndrds of known concentrtion. Totl myo-inositol, s defined by AOAC stndrd method performnce requirement (SMPR) (1), cn be clculted by dding the free myo-inositol nd myo-inositol bound s phosphtidylinositol dt. The method ws vlidted for the quntittion of free myo-inositol only. Repetbility ws determined from triplicte nlyses performed on the sme dy. Intermedite precision ws determined from replicte determintions performed over multiple dys. Accurcy ws determined by comprison to reference microbiologicl ssy nd expressed s the percentge of HPLC results reltive to microbiologicl results. Instrument LODs nd LOQs were determined sttisticlly from injections of low-level stndrds. Cution: Refer to Mteril Sfety Dt Sheets (MSDS) of chemicls prior to use nd follow sfe hndling procedures nd the suggested personl protective equipment. A. Apprtus () Anlyticl blnce. Minimum weighing cpcity of t lest g. (b) Centrifuge. (c) Desicctor. (d) N-evp. With wter bth or equivlent. (e) Oven. Cpble of mintining 110 C. (f) ph meter. With ph 4 nd 7 buffers. (g) Stir plte. Multiposition with stir brs. Tble A. Instrument operting conditions Pump 1 pressure limit 2000 psi Pump 1 mobile phse 0.12% (30 mm) N Pump 1 flow rte 0.4 ml/min Pump 2 pressure limit 2000 psi Pump 2 mobile phse 3% (750 mm) N Pump 2 flow rte 0.4 ml/min Injection volume 20 µl Myo-inositol retention time min Run time 25 min Switching vlve configurtion time tble t, min Configurtion (see Figure A) (see Figure B) (see Figure A) (h) Tubing. PEEK or Teflon in. id. (i) Vcuum mnifold. (j) Vortex mixer. (k) HPLC system. Corrosion-resistnt utosmpler, isocrtic pump (2), 6-port switching vlve, nd pulsed mperometry detector with gold electrode. Autosmpler cpble of injecting 20 µl. (l) Columns. Dionex CrboPc MA1 (4 250 mm) P/N 44066, MA1 (4 50 mm) P/N 44067, nd PA1 (4 50 mm) P/N 43096, or equivlent ( B. Mterils () Bekers. Assorted sizes. (b) Centrifuge tubes. 50 ml with Teflon-coted cps. (c) Syringe filters. Nylon, 0.45 nd 0.2 µm. (d) Filter pper. Whtmn 2V or equivlent (www. whtmn.com). (e) Erlenmeyer flsks. 50 or 125 ml or equivlent. (f) Volumetric flsks. Assorted sizes. (g) Funnels. Suitble for use with filter pper. (h) Pipets. Volumetric (Clss A); ssorted sizes. (i) Solid-phse extrction (SPE) crtridges. Silic, 1 g (J.T. Bker P/N , or equivlent. (j) Syringes. 1 ml disposble nd 25 ml gs-tight glss with 4 in. stinless steel needles. C. Chemicls nd Solvents () Acetic cid. Glcil, ACS. (b) Chloroform. High-purity, HPLC grde. (c) Diethyl ether. Anhydrous, HPLC grde. (d) Drierite (desiccnt). Anhydrous clcium sulfte, 8 mesh. (e) Helium. Zero grde or equivlent. (f) Hexne. HPLC grde. (g) Hydrochloric cid. Concentrted (36 38%), ACS. (h) Distilled or deionized lbortory wter. Wste Figure A. Switching vlve configurtion 1. Figure B. Switching vlve configurtion 2.
3 Schimpf et l.: Journl of AOAC Interntionl Vol. 95, No. 4, Tble B. PAD settings with gold electrode Anlog rnge 1 uc Detector progrm: Dionex PED t, s E, V Integrtion period s Detector progrm: Dionex ED 50 with specil gold electrode cell SP4270 t, s E, V Integrtion period s (i) Met-phosphoric cid. ACS. (j) Methnol. HPLC grde. (k) Myo-inositol. USP reference stndrd, officil lot; store desiccted. See stndrd lbel for purity. (l) Sodium cette trihydrte or sodium cette. Anhydrous, ACS. (m) Sodium chloride. (n) Sodium hydroxide. 50% (w/w), low crbonte form. D. Preprtion of Regents nd Stndrd Solutions All solutions cn be scled up or down for convenience provided good lbortory prctices re observed. Solutions cn be stored t 2 30 C in tight, inert continers otherwise noted. () Myo-inositol stock stndrd solution (pproximtely 2000 mg/l). Accurtely weigh pproximtely g myo-inositol nd quntittively trnsfer to 50 ml volumetric flsk. Dilute to volume with wter. Mix well. Store refrigerted. Expirtion: 3 months. (b) Myo-inositol intermedite stndrd solution (pproximtely 200 mg/l). Dilute 10.0 ml stock stndrd to 100 ml with lbortory wter nd mix well. Discrd fter use. (c) Myo-inositol working stndrd solutions (pproximtely 4, 2, 1, 0.5, 0.2, nd 0.05 mg/l). Dilute 2.0, 1.0, nd 0.5 ml myo-inositol intermedite stndrd to 100 ml with lbortory wter (4, 2, nd 1 mg/l). Dilute 0.5 ml myo-inositol intermedite stndrd to 200 ml with lbortory wter (0.5 mg/l). Dilute 4 nd 1 ml of the 0.5 mg/l myoinositol working stndrd to 10 ml with lbortory wter (0.2 nd 0.05 mg/l). Expirtion: 2 weeks. (d) Hydrochloric cid, 0.5%. Add 1.25 ml concentrted hydrochloric cid to pproximtely 200 ml wter in 250 ml volumetric flsk. Dilute to volume with wter nd mix well. Expirtion: 6 months. (e) Sodium chloride, 1 N. Dissolve 5.8 g sodium chloride nd dilute to 100 ml with wter. Expirtion: 1 month. (f) Sodium hydroxide, 0.12% or 30 mm (Pump 1). Quickly weigh 4.8 (±0.1) g of 50% sodium hydroxide into 2000 ml volumetric flsk contining pproximtely 1900 ml wter. (Note: It is importnt tht the sodium hydroxide does not bsorb crbon dioxide from the ir.) Swirl to mix well. Dilute to volume with wter nd mix well. Expirtion: 1 month. (g) Sodium hydroxide, 3.0% or 750 mm (Pump 2). Quickly weigh 120 (±3) g of 50% sodium hydroxide into 2000 ml volumetric flsk contining pproximtely 1900 ml wter. (Note: It is importnt tht the sodium hydroxide does not bsorb crbon dioxide from the ir.) Swirl to mix well. Dilute to volume with wter nd mix well. Expirtion: 1 month. (h) 6% Metphosphoric cid. Weigh 6.0 g metphosphoric cid into 100 ml volumetric flsk. Dissolve nd dilute to volume with lbortory wter. Mix well. Store refrigerted. Expirtion: 1 week. (i) Phosphtidylinositol extrction solutions. (1) Chloroform:methnol (2:1). Mix 60 ml chloroform nd 30 ml methnol. (2) Hexne:diethyl ether (80:20). Mix 80 ml hexne nd 20 ml diethyl ether. (3) Hexne:diethyl ether (50:50). Mix 50 ml hexne nd 50 ml diethyl ether. (4) Methnol:chloroform:wter (75:15:10). Mix 75 ml methnol, 15 ml chloroform, nd 10 ml wter. E. Smple Preprtion nd Extrction () Smple preprtion for free myo-inositol determintions. Prepred smples tht re constntly stored t 1 8 C in closed continers re stble for up to 5 dys. After 5 dys, smples must be prepred gin. Figure C. Exmples of typicl chromtogrms of () myo-inositol stndrd, (b) free myo-inositol in SRM 1849, (c) myo-inositol from phosphtidylinositol in SRM () (b) (c)
4 4 Schimpf et l.: Journl of AOAC Interntionl Vol. 95, No. 4, 2012 Tble 1. Free myo-inositol vlidtion dt intermedite precision, mg/100 g HPLC method Dy Product type Hypollergenic infnt formul RTF RSD, % Soy-bsed infnt formul powder RSD, % Specilty infnt formul RTF RSD, % Milk-bsed infnt formul RTF RSD, % Very low-ft infnt formul powder RSD, % Free mino cid-bsed infnt formul powder RSD, % Peditric nutritionl beverge RSD, % Adult nutritionl beverge NA n 4 4 RSD, % NA = No dt generted. Tble 2. Free myo-inositol vlidtion dt intermedite precision, mg/100 g Product type n Avg. HPLC method RSD, % Hypollergenic infnt formul RTF Soy-bsed infnt formul powder Specilty infnt formul RTF Milk-bsed infnt formul RTF 20 b Very low-ft infnt formul powder Free mino cid-bsed infnt formul powder Peditric nutritionl beverge Adult nutritionl beverge 8 c b c Intrlbortory triplicte nlyses on 3 dys. Intrlbortory single, duplicte, or triplicte nlyses on 9 dys. Intrlbortory qudruplicte nlyses on 2 dys. Thoroughly mix or stir products prior to smpling. For liquid products, ccurtely weigh 0.5 to 5 g (±10%) of product into 100 ml volumetric flsk nd record the weight to the nerest g. For powdered products tht do not require reconstitution, ccurtely weigh 0.25 to 1.5 g powder into 100 ml volumetric flsk nd record the weight to the nerest g. Add pproximtely 10 to 15 ml lbortory wter to the volumetric nd swirl or stir to completely dissolve the powder. For powdered products tht re not homogeneous t the subgrm level, reconstitute following the product lbel instructions nd ccurtely weigh 0.5 to 5 g reconstituted product into 100 ml volumetric flsk. Record the weight to the nerest g. Add enough 0.5% hydrochloric cid to ech smple to djust the smple ph to 4.5 ± 0.2 nd swirl to mix. Allow the volumetric flsks to set minimum of 2 min. Dilute to volume with lbortory wter nd mix well. Filter smples through Whtmn 2V filter pper into 125 ml Erlenmeyer flsks. (Note: Although some smples will filter cloudy, the filtrtes cn still be used.) Filter n liquot of smple filtrte through 0.45 µm syringe filter into n utosmpler vil. (b) Smple preprtion for phosphtidylinositol determintions. (1) Extrction. Weigh 4 g (±10%) liquid Tble 3. Free myo-inositol vlidtion dt ccurcy dt Product type HPLC method, mg/100 g Microbiologicl method b, mg/100 g HPLC/micro, % Hypollergenic infnt formul RTF Soy-bsed infnt formul powder Specilty infnt formul RTF Milk-bsed infnt formul RTF Very low-ft infnt formul powder Free mino cid-bsed infnt formul powder Peditric nutritionl beverge Adult nutritionl beverge b Multiple intrlbortory nlyses on multiple dys. Single nlyses.
5 Schimpf et l.: Journl of AOAC Interntionl Vol. 95, No. 4, or reconstituted powder product or 1 g (±10%) homogeneous powder into 50 ml centrifuge tube nd record the weight to the nerest g. Add 10 ml methnol nd stir for t lest 20 min. Add 20 ml chloroform nd stir for t lest 5 min. If lrge clumps form when chloroform is dded, cp tube nd shke well to mix smple. Add 5 ml 6% metphosphoric cid nd 1 ml 1 N NCl nd mix well. Centrifuge. Remove the bottom chloroform lyer nd evporte with nitrogen in 60 C wter bth. (2) Smple clenup. Condition 1 g silic SPE crtridge with 6 ml hexne. Dissolve residue in bottom of centrifuge tube in 1 ml chloroform:methnol (2:1). Quntittively trnsfer dissolved residue to the conditioned silic SPE crtridge. Rinse SPE crtridge with 3 ml hexne:diethyl ether (80:20) nd discrd the elunt. Rinse SPE crtridge with 3 ml hexne:diethyl ether (50:50), 4 ml methnol, nd 4 ml methnol:chloroform:wter (75:15:10) nd collect ll elunts in single 50 ml centrifuge tube. Evporte elunts collected from SPE crtridge with nitrogen in 60 C wter bth. (3) Hydrolysis. Add 40 µl concentrted cetic cid nd 2 ml concentrted hydrochloric cid to residue in centrifuge tube from the smple clenup step. Tightly cp tube. Het in 110 C oven for 2 h. Cool. Dilute mixture with pproximtely 10 ml H 2 O nd then dd 1.25 ml 50% (w/w) sodium hydroxide. Trnsfer smple to 50 ml volumetric flsk nd dilute to volume with wter. Filter n liquot of smple filtrte through 0.45 µm syringe filter into n utosmpler vil. (c) HPLC nlysis. (1) See Tbles A nd B for instrument operting conditions nd PAD settings, respectively. (2) Instrument strtup. The HPLC system should be locted in n re where temperture fluctutions will be miniml throughout the run. Prepre mobile phses. Helium sprge mobile phses nd/or pressurize mobile phse reservoirs. If necessry, clen nd polish the gold working electrode. Turn on the detector nd pump mobile phse over the columns t flow rte of 0.40 ml/min for t lest ½ h to equilibrte the system. Verify tht the detector is stble before beginning n nlysis. Inject 20 μl of the most concentrted stndrd t lest 5 times nd note the pek res or heights. If the system is equilibrted, the RSD of the pek res or heights of the lst three stndrd injections should be 2.0%. (3) Stndrd nd smple nlysis. Once the system hs equilibrted, inject one stndrd t ech concentrtion. After set of stndrds hs been injected, control smple nd up to 14 smples cn be injected before nother set of stndrds should be injected. (4) System shutdown. After ll smples nd stndrds hve been nlyzed, inject one vil of wter to clen out the utosmpler needle nd tubing. Store the nlyticl columns in mobile phse [0.12% (30 mm) sodium hydroxide]. Turn off the electrochemicl cell. Flush the pump heds with wter to remove sodium hydroxide. F. Clcultions Before clculting myo-inositol concentrtions in smples, compre the myo-inositol stndrd peks with the myo-inositol smple peks nd confirm tht there re not ny interfering compounds nd tht the myo-inositol smple pek res or heights re within the rnge of the myo-inositol stndrd pek res or heights. See Figure C for exmples of typicl chromtogrms. The concentrtion of myo-inositol cnnot be clculted if there re interferences or if the seprtion is poor. The myo-inositol retention time should be 11 to 13 min (depending on the individul nlyticl column). () Concentrtion of working stndrds. Tble 4. Free myo-inositol nd myo-inositol from phosphtidylinositol dt, mg/100 g Product type n Myo-inositol from phosphtidylinositol Free myo-inositol Totl (free + phosphtidyl bound) myo-inositol Myo-inositol from phosphtidylinositol, % RSD, % SRM Hypollergenic infnt formul RTF Not detected Hypollergenic infnt formul powder Not detected Milk-bsed infnt formul RTF 3 b Milk-bsed infnt formul powder 3 b Soy-bsed infnt formul RTF 3 b Soy-bsed infnt formul powder 3 b Specilty infnt formul RTF 3 b Specilty infnt formul powder 3 b 1.67 mg/100 g Lctose-free infnt formul RTF 3 b Lctose-free infnt formul powder 3 b Specilty infnt formul powder 3 b 1.51 mg/100 g Rice strch infnt formul RTF 3 b Rice strch infnt formul powder 3 b Milk-bsed infnt formul RTF 3 b Milk-bsed infnt formul powder 3 b Peditric nutritionl beverge 3 b b Intrlbortory triplicte nlyses on 3 dys. Intrlbortory triplicte nlyses on 1 dy.
6 6 Schimpf et l.: Journl of AOAC Interntionl Vol. 95, No. 4, 2012 C W = W 1/0.05 1/10 A 1 /V 1 A 2 /V 2 = W 2 A 1 /V 1 A 2 /V 2 where C W is the concentrtion of the working stndrd solution in milligrms per liter; W is the weight, in milligrms, of myo-inositol stndrd weighed; 0.05 is the dilution volume of the stock stndrd in liters; 1/10 is the intermedite stndrd dilution (10 to 100 ml); A 1 is the liquot of intermedite stndrd used, in milliliters; V 1 is the dilution volume of the working stndrd in milliliters; A 2 is the liquot of working stndrd used, in milliliters, if pplicble; nd V 2 is the dilution volume of the working stndrd in milliliters, if pplicble. (b) Preprtion of stndrd curve. For ech working stndrd concentrtion, verge the pek res or heights from ech two consecutive sets of stndrds. Prepre stndrd curve by performing liner lest squres (regression) on the concentrtions versus the verged pek res or heights. A stndrd curve must hve correltion of t lest to be considered cceptble for smple clcultions. At ech working stndrd level, the pek res or heights of stndrds injected before nd fter set of smples must not increse or decrese by more thn 7%. (c) Clcultion of myo-inositol in smples nd controls. The concentrtion of myo-inositol in prepred smple or control is extrpolted from the stndrd curve prepred bove. From the diluted, prepred smple concentrtion, the product concentrtion cn be clculted: C p = (C d D 1 )/S where C p is the concentrtion of myo-inositol in the product smple or control in milligrms per kilogrm; C d is the concentrtion of myo-inositol in the prepred smple or control in milligrms per liter; D 1 is the dilution volume in milliliters; nd S is the smple weight in grms. Totl myo-inositol, s defined by AOAC SMPR (1), cn be clculted by dding the free myo-inositol nd myoinositol bound s phosphtidylinositol dt. Note: For ech set of smples, the control result must be within three stndrd devitions of the control men. Reference: J. AOAC Int. 95, (future issue) Results nd Discussion During vlidtion, the verge repetbility ws 2.0% RSD nd the intermedite precision ws 2.5% RSD, s summrized in Tbles 1 nd 2. Accurcy ws determined by comprison to reference microbiologicl ssy s detiled in Tble 3 (2). The instrument LOD nd LOQ of the method were estimted to be nd mg/100 ml, respectively, nd correspond to free myo-inositol quntittion limit of mg/100 g. Although this method hs not been fully vlidted for the quntittion of myo-inositol bound s phosphtidylinositol, some dt hve been generted nd re summrized in Tble 4. The phosphtidylinositol quntittion limit hs been estimted to be mg/100 g. References (1) AOAC SMPR (2012) J. AOAC Int. 95, dx.doi.org/ /joc.int (2) Atkin, L., Schultz, A., Willims, W., & Frey, C. (1943) Ind. Eng. Chem. Anl. Ed., 15, i
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