Pure & Appl. Chern., Vol. 60 , N o . 6 , pp . 871-876, 1988. Printed in G reat Britain. @ 988 IUPAC INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY APPLIED CHEMISTRY DIVISION COMMISSION ON FOOD CHEMISTRY* A Collaborative Study of HPLC METHODS FOR THE DETERMINATION OF PATULIN IN APPLE JUICE Prepared f o r publication by STANISLAW J . KUBACKI and HALINA GOSZ CZ Department o f Analysis, Institute o f the Fermentation Industry, Rakowiecka 36,02-532, Warsz awa 12, Poland * Membership of the Commission during the preparation of the report (1985-87) was as follows: Chairman: A . E. Pohland (USA); Vice-Chairman: P. S . Steyn (Republic of South Africa); Secretary: D. L. Park (USA); Titular Members: R. Battaglia (Switzerland); P. Krogh (Denmark); H. A. M . G. Vaessen (Netherlands); Associate Members: M. J.-J. Castegnaro (France); H. B. S . Conacher (Canada); R. L. Ellis (USA); J. Fremy (France); J. Jacob (FRG); M. Jemmali (France); S . J . Kubacki (Poland); R. Livingston (USA); P. G. Thiel (Republic of South Africa); Y. Ueno (Japan); T. Yasumoto (Japan); National Representatives: M. M. Abdel Kader (Arab Republic of Egypt); A. Calvelo (Argentina); P. B. Czedik- Eysenberg (Austria); J. Davidek (Czechoslovakia); E. Luck (FRG); R. LBsztity (Hungary); Z. Berk (Israel); S. S . A. Marina Miraglia (Italy); T. Kato (Japan); C. L. Lim (Malaysia); A. Rutkowski (Poland); L. E. Coles (UK). Republication of this report i s permitted without the need for forma l IUP AC perm ission o n condition that an acknowledgement, with full reference together with IUPAC copyright symbol ( 1988 IUPAC), is printed. Publication of a translation into another languag e is subject to the additional condition of prio r approval from the relevant IUP AC National Adhering Organization.
Pure & Appl. Chern., Vol. 60, No. 6, pp . 871-876, 1988.Printed in G reat Britain.@ 1988 IUPAC
INTERNATIONAL UNION OF PUREAND APPLIED CHEMISTRY
APPLIED CHEMISTRY DIVISIONCOMMISSION ON FOOD CHEMISTRY*
A Collaborative Study of
HPLC METHODS FOR THEDETERMINATION OF PATULIN IN
APPLE JUICE
Prepared for publication by
STANISLAW J . KUBACKI and HALINA GOSZCZ
Department of Analysis, Institute of the Fermentation Industry,Rakowiecka 36,02-532, Warszawa 12, Poland
* Membership of the Commission during the preparation of the report (1985-87) was asfollows:
Chairman: A . E. Pohland (USA); Vice-Chairman: P. S. Steyn (Republic of South Africa);Secretary: D. L. Park (USA); Titular Members: R. Battaglia (Switzerland); P. Krogh(Denmark) ; H. A . M . G. Vaessen (Netherlands); Associate Members: M. J.-J. Castegnaro(France); H. B. S. Conacher (Canada); R. L. Ellis (USA); J. Fremy (France); J. Jacob(FRG); M. Jemmali (France); S. J . Kubacki (Poland); R. Livingston (USA); P. G. Thiel(Republic of South Africa); Y. Ueno (Japan); T. Yasumoto (Japan); National Representatives:M. M. A bdel Kader (A rab Republic of Egypt); A . Calvelo (Arg entina ); P. B. Czedik-Eysenberg (A ustria); J. Davidek (Czechoslovakia); E . Luck (F RG ); R . LBsztity (Hungary);Z. Berk (Israel); S. S . A . M arina Miraglia (Italy); T. Kato (Ja pan); C. L . Lim (Malaysia); A .Rutkowski (Poland); L . E. Coles (UK ).
Republication of this report is permitted w ithout the need fo r for ma l IUP AC perm ission o n condition that anacknowledgement, with full reference together with IUPAC copyright symbol (01988 IUPAC), is printed.Publication of a translation into another languag e is subject to the additional condition of prio r approval from the
A collaborative study of HPLC (high performanceliquid chromatography) m ethods for thedeterm ination of patulin in apple juice
Abstract - Two HPLC methods f or t he determinati on of pa tul in i n applejuice were collabo rativel y tes ted i n 12 laboratories from 10 coun-tr ie s. The coll abo rat ors themselves spiked the previously pasteurizedapple juice samples wi th pat uli n standard sol uti on before analysi s.Two samples a t thr ee le ve ls of f o r t i f i c a t i o n and one blank sample wereana lymd f o r each tes te d method. Although both of t he methods werebased on reversed phase HPLC they employed di ff er en t clew-up proce-dures: pa rt it io ni ng ex tra ct io n and column chromatography. Mean reco-ve ri es of pa tu li n ranged from 77 t o 85 % and mean coefficients ofva ri at io n were 7.3 7 and 15 k for the two methods respectively.
I N T R O D U C T I O N
Oswald e t a l . ( re f . 1) found no tumorigenicitywhen patul in was administered oral ly t omice and rats; nevertheless there i s considerable interest in t h i s myootoxin f o r tworeasons:
1 P a t u l i n i s to xic and produces tumors i n ra t s a t the place of in jec t ion
2
when in je ct ed subcutaneously (ref . 2 ) .
Patulin i s a good quali ty indic ator of f ru it s used i n the processing ofapple juice, since the p a t u l i n concentration i s reduced by only about20 % during processing (ref. 3).
The absence of carci noge nic ity of pat ul in res ul te d i n a lack of regulatory action in
50,ug.L of juice (ref. 4).
The most widely used qu an ti ta ti ve to ol s f o r pa tu li n determination have been T L C and,more recently, HPLC ( re f . 5 ) . Scot t ( ref . 6 ) organized and described re s u l t s of a col-laborative study of a TLC method for the determination of p a t u l i n in apple juice. T h emethod has been adopted by the A.O.A.C. as an o f f i c i a l f i rs t action method for the semi-quantit ative analysi s of pat ulin in apple juice. Although T L C methods predominated i nthe early seventies they later gave way t o those based on HPLC. The fo llowing four rea-sons were responsible for th is :
most co t r i e s , although Sweden, Norway and Switzerland have act ion leve l s of-yn
1 TL C i s tedious and time consuming.
2 Confirmation i s needed because of poor resolution from coextractants,
3 The methods provide semiquant ita tive res ul t s.
4
especially from 5-hydroxymethylfurfural (HMF).
The methods ar e not s uf fi ci en tl y se ns it iv e (20,143.L'~, ref. 7).
For these reasons and because of r ecen t advances in H-SlrC technology, HPLC soon becamenot only an att rac ti ve alt erna tiv e t o conventional TLC or GC methods but i s a t presentthe method of choice f o r the determination of pat ul in i n food products. Therefore ithas been decided by the NPAC Commission on Food Chemistry to es ta bl is h an i nt er na ti o-
were sel ec te d for the col la bor at ive study. Although both of them are based on reversedphase HPLC they employ en ti re ly di ff er en t clean-up procedures.
nally recommended method of analysis f o r p a t u l i n based on HPLC. Two analyt ica l methods
O R G A N I Z A T I O N O F T H E S T U D Y
Due t o the decomposition of pa tu li n i n apple ju ic e over th e several weeks time periodof a collaborative study (ref. a), and the formation of an interfering substance (HMF)in apple
bora tor s themselves would spike the previously pasteurized apple ju ic e samples j u s tbefore analysis.
The p ar ti ci pa nt s were each provided with two (one f o r each method) 125 ml hypo-vials ofpasteu rised apple juic e concentrate, twelve (six for each method) 6 ml hypo-vials con-taining aqueous s p i k i n g solutions of p a t u l i n in acetate buffer a t pH = 4, two (one f o reach tested method) 6 ml vials containing acetate buffer solution and one 6 ml v i a lcontaining standard solution of patdin in acetate buffer (1.25 &a). The collabora-t or s were asked t o s to re a l l the v ia l s in the refr igera tor until needed.
juice after the container was opened (r ef , 7) , i t was decided t h a t the colla-
Determination of patulin in apple juice by HPLC methods a7 3
Each laboratory was asked f i r s t to determine the concentration of the standard solutionby means of reversed phase HPLC. Then, t o c a m out analyses of six samples of apple ju-ice for each method followed by one ana lys is of sample of acetate buffer. In order toge t apple ju ic e ready t o be analyzed by the method of Tanner and Zanier 10 g of the con-centrate (sample S1) was di lu ted to 50 m l with di s ti ll ed water and seven 5 ml portionswere transferred e i t he r to centr ifuge tubes or t o separatory funnels fo r fur ther analy-sis. The entire contents of vials A , B, C , D, E , F (containing spiking solut ions ) wereadded t o six of the above mentioned portions of the apple juice. To reduce the losses
of the spiking substance each hypo-vial was rinsed twice with 2.5 ml of ethyl acetate.In a l l cases bath ri nsi ngs were al so added t o the samples followed by partitionongextraction. One sample of dil ut ed apple ju ic e was spiked with the acetate buffer (sampleC). For the method of Stray the colla borat ors were instr ucte d t o di lu te 100 g of theapple jui ce con oentrate (sample S2) t o 500 ml with di s ti ll e d water. Seven 50 m l portionsof the di luted juice were transferred t o individ ualmu lsepa ratory funnels fo r further
analysis. Six of them were fo rt if ie d with the spiked solut ions in V i a l s H, I , K, L, Mand N. The acetate buffer sample was added t o t he l a s t portion of the diluted juioe.All the vials containing spiking solutions and buffer acetate were rinsed with two 5 m l
portions of ethyl acetate taken from the volume (50 ml) dedicated f or the f i r s t p e r t i -
tioning eXtZ%CtiOR. The pa rt ic ip an ts were asked t o complete the analyses for each me-thod in one working day.
M E T H O D S
The following two HPLC methods were collabcrativelr studied on the basis of the previo-usly prepared literature survey(ref. 13):
1
2
method developed by S tr ay ( r e f . 3 1 ,
method by Tanner and Zanier(ref. 9). T h i s method had been published bythe same authors ear l i er ( re f . 10) before i t was adopted by the FederalCommission f o r the Swiss Food Manual as o f f i c i a l ( 1984).
Fig. 1 and Fig. 2 show flow diagrams of the method of Tanner and Zanier and the methodof Stray respectively .
The s ta t is t ic a l analyses of th e r e s u l t s were ca rr ie d out following gui de lin es recommen-
ded by the International Standards Organization (ref. 11). Additionally, analyses ofvariances according t o St ein er (r e f . 12)were a ls o performed.
Sample ( 5 m l)
Extract ion with eth yl a cetat e
Partitioning extraction by aqueoussodium carbonate solution 7
discardedConoe tration
HPLC ( ODS oolumo)
1t
!B i g . 7 . Schematic representation o f Tanner
and Zanier procedure
Sample (50 m l )
Extract ion with eth yl acetate
Ccncentrat ion
Adsorption chromatography of patu-l in
on s i l i c a gel, solvent;toluene-ethyl acetate ( 7 t 3 )
Concentration
HPLC (ODs column 1
41
i
t
t
Fig. 2. Schematic representation of s t r a yprocedure
TAIILZ 1. The collaborat ive re su lt s f or thedetermination of patul in i n the standardsolution
The co ll ab or at or s were provided with the samples in July, 1 9 8 6 and the r e s u lt s werereturned by October 15 , 1986 . O f th e 2 1 laborator ies invi ted to par t ic ip ate in the stu-dy, 13 agreed t o take part. Final ly 1 2 laboratories from 10 countries submitted results( se e Acknowledgments).
From the c oll abo rat ors re s u lt s for the concentration of the standard sol uti on sample (ST),
determined by reversed phase (ODs column) HPLC, the 1.25,ug/ml solut ion in ac eta te buf-f e r ( pH = 4 ) appears t o be sta ble (Table 1). Some labo ratori es (nos. 2 , 3, 10 and 11)
fa il ed t o reproduce the tru e concentration with suffi cie nt accuracy [< 10 $6).
The re su lt s obtained by the part ici pan ts fo r the concentration of patu lin in the spikedapple juice (samples A through F and samples H through N) as well as i n the blank applejuice (samples 0 and 0 ) are tabulated in Table 2 f o r the method of Tanner and Zanierand i n Table 3 for the method of Stray. The samples were spiked in duplicate a t threeknown ooncentrations; 5 , 50 and 250 ,ug .L- l . The f i r s t spiking concentration was e i t h e rat the limit of de te ct io n (method o Tanner and Zanier) or very close to i t (method of
indicate that the limits of de te ct io n reported by the aut ho rs of t he methods are beyondthe reac h of most lab ora tor ies . Because of the presence of in te rf er in g substances i next racts as analyeed by HPLC the real limits of det ec tio n are between 10 and 20 ug.L'l.
Occassionally fal se p osit ives estimated t o be a t the le ve l of 10,ug.L" were recordedfor the blank samples G and 0.
It i s easily seen from a cursory examination of the data i n Table 2 and Table 3 t ha tsome lab ora tor ies ar e outl ier s. The re s u l t s provided by the lab or at or ie s nos. 5, 8, 10and 11 f o r the method of Tanner and Zanier and th e la bo ra to ri es nos. 3 , 4 and 1 1 f o rthe method of St ra y deviate so much from comparable entries from other laboratories that
they may be considered as i r reconcilable with the other data without apply ing Dixon'so u t l i e r t e s t. 1% was apparent that these laborator ies d i d not have the methods undercontrol. Additional enquiry sent t o the part icipa nts revealed that a l l the laborator iesexcept one identified as ou t l i e r s had never used the methods before. I n most oases poorHPLC resolut ion of p a t u l i n from concurrent interfering substances was responsible forthe erroneous res ult s. An apple juic e concentrate rel at iv el y ric h i n inter fer ing substan-ces was del iberate ly se lected for t h i s study. The rej ect ed o ut li er s i n Table 2 and inTable 3 are put i n parenthesis.
Average recoveries obtained by $he collabmrators fo r the concentration of p atu lin i n th espiked apple juice were 85 $% (samples B and E) and 83 % (samples C and F ) for the methodof Tanner and Zanier and 80 $% (samples I and $4) and 77 $ (samples K and I?) f o r th e me-thod of Stray fo r the spiking level s 50,ug.L' an8 2 5 0 p . L W 1 respectively. The diffe-rences between t he mean reco ver ies found f o r both th e spiking l ev el s and the methodswere not s t a t i s t i ca l l y d is tinguishable ( t - t e s t ) .
The results showed the low variation anticipated for a method that i s based on HPLC com-pared with TL C (mf . 6 ) . (samples B and E ) and
Stray, l imit of detect ion = 1pg.L- Q1. Most of the fin din gs obtained fo r the se samples
Coeff icients o f variation were 7.5
TABLE 2. R e s u l t s reported by participants (method by Tanner and Zanier)
Determina tion ofpatulin in apple juice by HPLC methods a7 5
7.2 [samples C and F) f o r t h e m eth od of Tanner and Zanie r and 12 % ( s a mp le s I an d M )
and 18 ii ( s m l e s K and N) f o r t h e m ethod o f S t r a y f o r t h e s p i k i n g l e v e l s o f 50,ug.L-’and 2 5 O p g . L - 7 r e s p e c t i v e l y . The mean c o e f f i c i e n t s o f v a r i a t i o n w ere 7.3 % f o r t h e m e -th o d o f T a n n e r a n d Z a n ie r and 15 % f o r t h e m eth od o f S t r a y , C a l c u l a t i o n of w i t h i n a n db e twe e n - la b o ra to ry c o mp o n e n t s o f t o t a l v a r ia n c e s ( re f . 1 2 ) f o r t h e tw o sam ple s e t s ( s p i -k ing l e v e l s ) f o r e a c h m ethod r e v e a le d t h e l a r g e random e r r o r c o n t r i b u t i o n t o t h e t o t a lv a r i a n c e ( T a b le 4 and Table 5 ) . F - r a t i o s Sd2/Sr2 g a ve n o e v i d e n c e f o r t h e p r e s e n c e o fs i g n i f i c a n t s y s t e m a t i c e r r o r s among t h e l a b o r a t o r i e s . The v a l u e s o f r ( r e p e a t a b i l i t y )and R [ r e p r o d u c i b i l i t y ) c o m p u t e d a c c o r d i n g t o t h e IS0 g u i d e l i n e s a r e show n i n T a b le 6.They mean t h a t t h e d i f f e r e n c e be tw e en tw o s i n g l e d e t e r m i n a t i o n s f ou nd e i t h e r i n one la -
b o r a t o r y o r i n t w o d i f f e r e n t l a b o r a t o r i e s on i d e n t i c a l t e s t m a t e r i a l w i l l e x c e e d th er e p e a t a b i l i t y ( r 1 o r r e p r o d u c i b i l i t y ( R 1 r e s p e c t i v e l y n o t more t h a n in 5 % of t h e c a s e s( 9 5 k p r o b a b i l i t y ) .
TABLE 3. Results reported by participants (method by Stray)
K, N( 2 5 0 p g . L -' ) 3 4 . 6 9 6 . 8 2 7 . 7 7 7 . 7
s.d. stan dard de via tio n: Sr with in-lab orato ry s.d.: sb between-la boratory s.d.
R E C O M M E N D A T I O N SB o t h the t e s t e d m e th od s f o r the d e t e r m i n a t i o n o f p a t u l i n i n a p p l e j u i c e b y means of H i 5 C
a h o u l d be o f f i c i a l l y recommended by IUPAC.
A C K N O W L E D G E M E N T S
Tho au thors a re g r ea t ly indeb ted t o D r . A.E. Pohland, US Food and D r u g Adminis t ra t ion,Cente r fo r Food Sa fe ty and Applied Nu tr it io n, Divi sion of Contaminants Chemistry, Natu-ra l Products snd Ins tru me nta tio n Branch, Washington, D.C. 20204 fo r p rov id ing the pa tu -l i n s tandard and k ind ass i s t ance i n prepar ing t h i s a r t i c le . Many thanks a r e due t o thef o l l o w i w co l l a b o r a to r s f o r t h e i r p a r t i c i p a t i o n :
I.B. Agater , Laboratory o f the Government Chemist, London, England.
J. Anderson, Counci l fo r Sc ie n t i f i c and Indu s t r i a l Resea rch, Natural
Food Resea rch In s t i t u t e , P re t o r ia , Repub l ic of Sou th Af r ica .R. Ba tt ag l i a , Xantonales Laborator ium Zurich, Zurich, Switzer land.
J.R. Beali ng, Food Ins pec tio n Ser vic e, Rotterdam, The Netherland s.
J.M. Fremy, L abo rat oir e Cent ral d 'Hygiene A lima ntair e, Di rec tio n de l aQu a l i t e S e r v i c e s Ve t e r i n a r i e s , P a r i s , F ra nc e.
H. GOSZCZ , I n s t i t u t e of the Ferm enta tion In du st ry , Warszawa, Poland,
W. iKronert, Max von Pet t enk ofe r-I nst i tu t des Bundesgesundhei tsamtes ,
B e r l i n , F RG .
C. La i r ie , Di rec t i on du Labora to i re du Cont ro le de l a Qu al i t e , Rennes,France.
C.W. Thorpe, Food and Drug Administration, Washington, Dc, USA.
P. T h i e l , Na t i o n al R e se a rc h I n s t i t u t e fo r Nut r i t io na l Diseases , SA Ide-
di ca l Researoh Council , Tygerberg, Republ ic of South Africa.
A. Vidnes, Nat ional Control Authori ty fo r Processed F ru i t s and Vegeta-b l e s , Oslo, Norway.
A. V i s c o nt i , I n s t i t u t o T os si ne e l i c o t o s s i n e CNR, Bari , I t a l y .
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