The goal of this study was to compare the performance of

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1 676 WHEELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 FOOD COMPOSITION AND ADDITIVES Isoltion of Light Filth from Ground Oregno nd Ground Mrjorm: A Modifiction Using Isopropnol s Deftting Agent: In-House Study MARK WHEELER U.S. Deprtment of Agriculture, Food Sfety nd Inspection Service, OPPDE, th St, SW, Wshington, DC BARBARA BENNETT U.S. Food nd Drug Administrtion, Center for Drug Evlution nd Reserch, 9201 Corporte Blvd, Rockville, MD HARRY MARKS U.S. Deprtment of Agriculture, Food Sfety nd Inspection Service, OPPDE, th St, SW, Wshington, DC A procedurl modifiction of the AOAC Officil Method for extrcting light filth from ground oregno nd ground mrjorm ws tested in n intrlbortory study. The modified method specifies isopropnol deftting, A(), rther thn chloroform isopropnol deftting, A(b), followed by direct flottion s directed in AOAC Officil Method, B(b). The modified method provided comprble results in less time while lso providing sfety, helth, nd finncil benefits. Submitted for publiction December The recommendtion ws pproved by the Methods Committee on Microbiology nd Extrneous Mterils s First Action. See Officil Methods Progrm Actions, (2001) Inside Lbortory Mngement, My/June issue. Corresponding uthor s e-mil: mrk.wheeler@usd.gov. The gol of this study ws to compre the performnce of the proposed method with tht of the AOAC Officil Method in n intrlbortory study. The proposed method contins procedurl modifiction of the officil method for extrcting light filth from ground oregno nd ground mrjorm. The AOAC Officil Method A(b) specifies deftting the product 3 times with chloroform. The residue is then rinsed with isopropnol, which is followed by deftting once with isopropnol. After the deftting steps, the residue is wshed with hot wter, nd the light filth is isolted from n queous solution with minerl oil. The proposed method follows the deftting steps s directed in A(), which requires deftting the product 3 times with isopropnol. As in the officil method, fter the deftting steps the residue is wshed with hot wter, nd the light filth is isolted from n queous solution with minerl oil. The use of chloroform in the deftting steps of the officil method presents serious helth nd sfety concerns nd is costly nd time consuming. Chloroform is nrcotic nd suspected humn crcinogen (1), nd chloroform spill is cuse for building evcution nd professionl clenup. All deftting steps nd the hndling of this regent, such s mesuring the volume to dd to the test portion, must be performed in fume hood. Although isopropnol, like chloroform, should be hndled in fume hood nd disposed of s hzrdous wste, specific helth concerns ssocited with isopropnol exposure re not s severe. Also, chloroform is considerbly more expensive thn isopropnol. Finlly, becuse the officil method specifies 4 deftting steps nd rinsing step, wheres the proposed method specifies only 3 deftting steps, the time required to nlyze test smple by the proposed method is reduced by pproximtely 1 h, from 4.5 to 3.5 h. The officil method ws dopted Officil First Action fter collbortive study with ground oregno nd n intrlbortory study with ground mrjorm (2). This method replced method tht specified petroleum ether s the deftting gent. In the collbortive study, the verge recoveries of elytrl frgments nd rodent hirs from ground oregno were 93.5 nd 91.0%, respectively. On the bsis of the rw dt reported by Glze (2), the verge recoveries of elytrl frgments nd rodent hirs from ground mrjorm in the intrlbortory study were 98 nd 94%, respectively. The proposed method should perform s well s or better thn the officil method. Study Design The study described in this pper ws n intrlbortory study, with 2 nlysts. The mtrixes used were freshly ground oregno nd mrjorm obtined from spice compny. Ech spice ws from single lot. Test portions were numbered from 1 to 40 for ech spice. Test portions 1 20 were nlyzed by the proposed method. Test portions were nlyzed by the officil method. Ech test portion consisted of 10 g spice tht ws weighed into tin cn nd then spiked with elytrl squres of Tribolium sp. nd mouse hirs. The elytrl squres were 0.5 mm in size nd the mouse hirs were 1.5 mm in length. Ech test portion ws spiked with 5, 15, or 30 contminnts of ech type, providing low, medium, nd high spiking levels, respectively. In ddition, for ech set of 20 test portions to be nlyzed by single method there were 6 test portions t the low level nd the high level nd 8 test portions t the medium

2 WHEELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, Tble of product Methods for spices, herbs, nd botnicls; for those not listed, use () nd (b) for ground form Spice Form Smple, g Pretretment A Isoltion B Hevy Method Light filth Mrjorm Ground (5) 10 b Unground (1) Oregno Ground (5) 10 b Unground (1) Refs.: JAOAC (1): 58, 447(1975); (5): J. AOAC Int. 85, 676(2002). Tble 1. Intrlbortory study results for the isoltion of light filth from ground oregno nd ground mrjorm Filth type Spiking level Method Spice No. of replictes nlyzed b Men No. of contminnts recovered Men rec., % Repetbility c Anlyst reproducibility d s r RSD r s R RSD R Insect frgments Low (5) AOAC Oregno Mrjorm 5 e Proposed Oregno Mrjorm Medium (15) AOAC Oregno Mrjorm Proposed Oregno Mrjorm High (30) AOAC Oregno Mrjorm Proposed Oregno Mrjorm Rodent hirs Low (5) AOAC Oregno Mrjorm Proposed Oregno Mrjorm Medium (15) AOAC Oregno Mrjorm Proposed Oregno Mrjorm High (30) AOAC Oregno Mrjorm Proposed Oregno Mrjorm b c d e Number in prentheses is number of contminnts of ech type dded. Two nlysts nlyzed equl numbers of test portions, i.e., 3 test portions per nlyst for the low nd high spiking levels nd 4 per nlyst for the medium spiking level. s r nd RSD r = repetbility stndrd devition of the percent recovery nd repetbility reltive stndrd devition, respectively [RSD x is the coefficient of vrition (CV), which equls 100 times the stndrd devition divided by the men]. s R nd RSD R re the reproducibility stndrd devition of the percent recovery nd the reproducibility reltive stndrd devition, respectively. One outlier result (zero frgments recovered) ws deleted.

3 678 WHEELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Tble 2. Summry of percent recoveries weighted by number of contminnts Anlyst Contminnt Spice Mrjorm Oregno Combintion Isopropnol CHCl 3 Isopropnol CHCl 3 Isopropnol CHCl 3 1 Insect frgment Hir frgment Combintion Insect frgment Hir frgment Combintion Pooled Insect frgment Hir frgment Combintion level. The Study Director prepred ll the spikes nd spiked ll the test portions in the lbortory before ny testing. All of the test portions were prepred t the sme time. The nlysts knew the spiking levels used, but they did not know the spiking levels of the individul test portions. All the test portions of spice were nlyzed, nd recoveries were counted before ny dt nlysis ws performed. METHOD Procedure Form filter pper cup, 400 ml 1 L, B(j), nd weigh test portion into cup. Add 400 ml isopropnol to cup in beker, nd boil gently on hot plte in fume hood for 10 min. Trnsfer cup to Büchner funnel, nd spirte to slow drip. Repet boiling extrction twice with 400 ml isopropnol. Proceed with isoltion step specified in Tble Sfety Precutions (1) Use chemicl fume hood during the deftting steps. (2) Dispose of isopropnol s hzrdous wste. Criticl Control Points The lbortory sink should be lrge enough for the test portion to be wshed properly without splshing. Some residues tend to cling to the filter pper cup. This residue should be removed from the filter pper cup by thorough wshing. Sttisticl Anlysis Method performnce is described by clculting the men recovery nd the between-smple stndrd devition. The between-smple stndrd devition is mesure of the increse in the vribility of results over wht would be expected if results for given smple were rndomly distributed s bino- Tble 3. Summry of percent recoveries weighted by number of contminnts excluding the outlier Anlyst Contminnt Spice Mrjorm Oregno Combintion Isopropnol CHCl 3 Isopropnol CHCl 3 Isopropnol CHCl 3 1 Insect frgment Hir frgment Combintion Insect frgment Hir frgment Combintion Pooled Insect frgment Hir frgment Combintion

4 WHEELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, Tble 4. Summry of men percent recovered by type of spice nd nlysts Spice Mrjorm Oregno Combintion Anlyst Isopropnol CHCl 3 Isopropnol CHCl 3 Isopropnol CHCl (98.0) (97.0) Pooled (97.9) (97.7) 97.7 Outlier result (23 recovered contminnts) ws removed. mil distribution. Assuming n expected recovery of p, if σ p is the between-smple stndrd devition, then for test portions with n contminnts, the vrince of the recoveries is σ 2 = [p(1 p)/n] + σ p 2. For ech type of contminnt, the contminnt smple percent recovery is clculted s 100 times the number of contminnts found divided by the number tht were used to spike the smple. The contminnt men recovery for method is the weighted verge of the contminnt smple recoveries where the weight for ech smple is equl to the number of contminnts tht were used to spike the smple. To chrcterize the between-smple stndrd devition of the results for the methods, n nlysis of vrince (ANOVA) pproch is used. In this pproch, it is ssumed tht within smple the distribution of the number of recovered contminnts for given type of contminnt is binomil distribution. Designte E(p) s the expected vlue of the percent recovery nd σ p s the between-smple stndrd devition. If p i is the men recovery for the ith smple nd n i is the number of contminnts used to spike the ith smple, i =1,...,i, then the ANOVA model ssumes tht n i vr(p i ) = E(p)(1 E(p)) + n i σ p 2. Define the reltive weighted men squre error (RMSE) to be equl to n i (p i p) 2 /(i 1) divided by p(1 p), where p is the men recovery, n i p i / i. By computing expected vlues, it cn be shown (3) tht the expected vlue of RMSE is pproximtely equl to the following: E(RMSE) 1 + σ p 2 2 ( ni ni / ni) p( 1 p)( i 1) An estimte of the reltive between-smple stndrd devition, σ p / (E(p)(1 E(p)) ½, is thus obtined by using the following eqution: σ p ( E( p)( 1 E( p)) 12 / RMSE 1 = (mx ( 0, )) n vr ( n)/ In where is the verge of n i, nd vr(n) is the vrince of n i over the i test portions. Anlysts Comments As prt of the preliminry study, the lbortory performed ruggedness test on the spices with retil test portions. The verge recoveries from the ground oregno were 96.8% of the elytrl frgments nd 97.5% of the rodent hirs. The verge recoveries from the ground mrjorm were 99.3% of the elytrl frgments nd 91.2% of the rodent hirs. By eliminting one step, the nlyticl time ws reduced by bout 1 h, from 4.5 to 3.5 h. Overll, the pltes from both methods were free of spice mteril, i.e., the grid lines on the pper were clerly visible. The time needed to red the pltes rnged from 4.0 to 6.5 min. 12 / Tble 5. Summry of men percent recovered by type of contminnt nd nlysts Contminnt Frgment Hir Combintion Anlyst Isopropnol CHCl 3 Isopropnol CHCl 3 Isopropnol CHCl (96.1) (97.0) Pooled (96.5) (97.7) 97.7 Outlier result (23 recovered contminnts) ws removed.

5 680 WHEELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Tble 6. Summry of reltive stndrd devition of popultion percent recoveries weighted by number of contminnts Mrjorm Oregno Combintion Anlyst Contminnt Isopropnol CHCl 3 Isopropnol CHCl 3 Isopropnol CHCl 3 1 Insect frgment Hir frgment Combintion Insect frgment Hir frgment Combintion Pooled Insect frgment Hir frgment Combintion Results nd Discussion From the outset, one test smple ws eliminted from the dt in the subsequent nlyses. For this product, none of the 5 elytrl frgments were recovered by the nlyst when the officil method ws used. The likelihood of this hppening, if the product were properly hndled, is virtully zero, nd thus it ws ssumed tht this product ws improperly hndled or ws mistkenly left unspiked. As requested by AOAC INTERNATIONAL, mens of the recoveries, repetbility nd reproducibility stndrd devitions (s r nd s R, respectively), nd the corresponding coefficients of vritions (RSD r nd RSD R, respectively) were computed for ggregtions of test portions defined by spike level, method, spice, nd type of contminnt. These dt re presented in Tble 1. The repetbility mesure includes the between-smple within-nlyst effects, wheres the reproducibility mesure lso includes this effect in ddition to the between-nlyst effect. Becuse only 2 nlysts were in the study, the mesure of reproducibility is not sttisticlly relible. An ANOVA model of ctegoricl dt (PC-SAS ed. 6.12, PROC CATMOD) indicted tht the spiking level ws not sttisticlly significnt (P-vlue of bout 0.20 for the vrious models). Consequently, sttisticl nlysis ws performed by pooling dt cross the different spiking levels. Mens of recoveries for different ggregtions of dt re provided in Tble 2. Tble 2 shows tht the men recoveries for both contminnts were well bove the proposed 90%; the men for the elytrl squres of Tribolium sp. ws pproximtely 99%, nd tht for mouse hirs, 95%. Over ll test portions, the men recovery ssocited with the officil method (97.7%) ws similr to the men recovery ssocited with the Tble 7. Summry of reltive stndrd devition of popultion percent recoveries weighted by number of contminnts excluding the outlier Mrjorm Oregno Combintion Anlyst Contminnt Isopropnol CHCl 3 Isopropnol CHCl 3 Isopropnol CHCl 3 1 Insect frgment Hir frgment Combintion Insect frgment Hir frgment Combintion Pooled Insect frgment Hir frgment Combintion

6 WHEELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, proposed method (97.2%). However, there is one ground oregno, nlyzed by the first nlyst using the proposed method, from which only 23 of the 30 spiked mouse hir contminnts were recovered. This result (23/30) cn be considered n outlier becuse the probbility of recovering 23 contminnts, ssuming 97% probbility of recovering ech contminnt nd binomil distribution, is <1/4000. Becuse there were 24 test portions in the study tht hd 30 contminnts, nd for ech product 2 types of contminnts were recovered, the probbility of result with lower recovery for 30 contminnts occurring in the study, given these ssumptions, is <2%. Furthermore, mong ll results for products spiked with 30 contminnts, the next lowest percent recovery ws 90% (recovery of 27 contminnts), which occurred 3 times mong the 48 nlyses. All the test portions contining 15 contminnts hd recoveries of 80%, nd mong the test portions tht hd 5 contminnts only one hd recovery of <80%. From this one test smple, the nlyst recovered 60% (isolting only 3 of the 5 contminnts). Thus, it ppers tht the test result of recovery of 23 of 30 contminnts is n unusul result nd, for this reson, cn be considered n outlier. When the result for this test smple is deleted, the men recovery for the proposed method is 97.7% (Tble 3). Tbles 4 nd 5 present summries of the men recoveries from the 2 spices for the 2 types of contminnts, by nlyst, for ech of the 2 methods, with nd without the outlier result, respectively. From these tbles, it cn be seen tht there is no consistent nlyst effect over the different types of test portions nd contminnts. When the outlier result is included in the nlyst/method interction effect, controlling for the type of contminnt is sttisticlly significnt t bout the 0.02 level, on the bsis of n ANOVA model of ctegoricl dt (PC-SAS ed. 6.12, PROC CATMOD). However, when the outlier result is removed, the significnce level of this interction is 0.09, indicting just mrginl sttisticl significnce (reltive to the usul cceptnce vlue of 0.05). With the removl of the outlier result, ll men recoveries presented in Tble 5 re >95%, well bove the trget of 90%. Tble 6 presents the results of the estimted reltive between-smple stndrd devition for the vrious ggregtions of dt, similr to those of Tble 2. High vlues of this mesure re ssocited with the first nlyst. Over ll test portions, the estimte of the reltive between-smple stndrd devition for the officil method is 0.04, wheres tht for the proposed method is If in fct the true between-smple stndrd devition were equl to zero, then, s determined by simultion, the expected vlue of the estimte of the reltive between-smple stndrd devition would be pproximtely 0.04, nd the 99th percentile would be pproximtely Therefore, for the officil method, it ppers tht the reltive between-smple stndrd devition is smll, nd there does not pper to be ny significnt systemtic fctor contributing to the vribility of results. However, for the proposed method this cnnot be sid. When the outlier result is deleted, the estimte of the reltive between-smple stndrd devition decreses to 0.14 (Tble 7). This vlue represents the 97th percentile of the distribution of results tht would be obtined if, in fct, the popultion of the between-smple stndrd devition were zero. Thus, these results suggest tht there is n unknown fctor, perhps ssocited with the first nlyst, contributing systemticlly to the vribility of the results obtined for the proposed method. Recommendtions With this number of test portions, nd the high numbers of contminnts recovered by the proposed nd officil methods, it is difficult to scertin the significnce of nlyst effects or interction mong the methods, nlysts, medium, nd contminnts tht might exist. In ny cse, for this study, the 2 methods pper on verge to hve similr recoveries. The stndrd devition of the results obtined by the officil method for test portions with single contminnt is estimted to be (p(1 p)) ½, where p is the expected recovery, wheres for the proposed method the stndrd devition is estimted to be bout 20% higher when ll results re included, nd bout 14% higher fter the one outlier result is excluded. Even with the possible lrger stndrd devition estimted for the proposed method, the recoveries re sufficiently high to enble ccurte nlyses for its intended use. Bsed on the intrlbortory results, the Study Director recommends tht the proposed modifiction be dopted First Action by AOAC INTERNATIONAL for ground oregno nd ground mrjorm. Acknowledgments We thnk Jck Boese for his words of encourgement nd expert dvise. Also, we re grteful to McCormick Spice Co. for providing the spices needed for this study. References (1) Brlow, S.M., & Sullivn, F.M. (1982) Reproductive Hzrds of Industril Chemicls, Acdemic Press, London, UK (2) Glze, L.E. (1975) J. Assoc. Off. Anl. Chem. 58, (3) Scheffe, H. (1959) Anlysis of Vrince, John Wiley nd Sons, New York, NY