2006 5 M ay 2006 C h inese J ou rna l of C h rom a tog raphy Vo l. 24 N o. 3 213 217 2 12 1, 2, 2, 2, 2, 2 (1., 300072; 2., 100013) : 2 ( ) (M RM ) 12 ph 5120,,,, HLB, W a te rs ACQU ITY U PLC TM B EH C18, 011% 2 ( ), 0102 0138 g / kg, 0107 1127 g / kg 2 g / kg 014 g / kg, 12 6913% 9413%, 315% 1617% : 2 ; ; ; ; : O 658 : A : 100028713 (2006) 0320213205 : S im u lta n e o u s D e te rm in a t io n o f Tw e lve G lu co co r t ico id s R e s id u e s in M ilk b y U lt ra P e rfo rm a n ce L iq u id C h rom a to g rap h y2 E le c t ro sp ra y T a n d em M a s s Sp e c t rom e t ry CU I X iao liang 1, 2, SHAO B ing 2, ZHAO R ong 2, M EN G J uan 2, TU X iaom ing 2 ( 1. D ep a r tm en t of Chem is try, Tia n jin U n ivers i ty, Tia n jin 300072, Ch in a; 2. In s ti tu te of N u tr i tion & Food H ygien e, B ei jin g Cen ter for D isea se P reven tion &Con trol, B ei jin g 100013, Ch in a ) A b s t ra c t: A com p rehens ive ana lytica l m e thod based on u ltra p e rfo rm ance liqu id ch rom a togra2 p hy2e lec trosp ray tandem m ass sp ec trom e try (U PLC 2M S /M S ) w ith nega tive m ode has been de2 ve lop ed fo r the s im u ltaneous de te rm ina tion of tw e lve glucoco rtico ids res idues in m ilk. The m u lti2reac tion m on ito ring m ode w as em p loyed fo r the de te rm ina tion. M ilk sam p les w e re ex trac2 ted by son ica tion in a m e thano l/ ace ta te buffe r ( ph 5120) so lu tion, and then defa tted w ith n 2 hexane. Sam p le concen tra tion and p u rifica tion w e re p e rfo rm ed us ing O as is HLB, Sep 2p ak s ilica and Sep 2p ak am ino2p rop y l so lid p hase ex trac tion ca rtridges. a W a te rs ACQU ITY U PLC TM The sep a ra tion w as p e rfo rm ed on B EH C18 co lum n (100 m m 110 m m i1d1, 117 m ) w ith grad ien t e lu tion us ing m e thano l and w a te r ( con ta in ing 011% fo rm ic ac id ) a t a flow ra te of 011 m L /m in. Iden tifica tion of the glucoco rtico ids w as done us ing re ten tion tim es and the d is tribu tion of d iag2 nos tic ion p a irs. Q uan tifica tion of the glucoco rtico ids w as based on the p eak a reas of the p a ren t ion and a fragm en t ion w ith a h ighe r s igna l. The lim its of de tec tion (LOD ) of the m e thod w e re from 0102 to 0138 g / kg and the lim its of quan tifica tion (LOQ ) ranged from 0107 to 1127 g / kg. A ve rage recove ries fo r the tw e lve glucoco rtico ids ( sp iked a t the leve ls of 2 and 014 g / kg) ranged from 6913% to 9413%, w ith re la tive s tanda rd dev ia tions be tw een 315% and 1617%. Rou tine tes ts show ed tha t the m e thod is fas t, sens itive and sp ec ific fo r the de te rm ina2 tion of g lucoco rtico ids res idues in m ilk. Ke y w o rd s: u ltra p e rfo rm ance liqu id ch rom a tograp hy2e lec trosp ray tandem m ass sp ec trom e try (U PLC 2M S /M S ) ; so lid2p hase ex trac tion; glucoco rtico ids; res idues; m ilk : 2005211216 :,,,,, Te l: (022) 27406036, E 2m a il: x iao liangc@163. com. :,,,, Te l: (010) 64407191, E 2m a il: shaob ingch@ s ina. com. : (N o. 7041004).
214 24,,,,, ( [ 1999 ] 17 ) 0175 g / kg, 10 g / kg ( h ttp : / / www. em ea. eu. in t / index / indexv1. h tm, ( bec lom e thasone ) ( flum e thasone ) ( flud roco rtisone ace ta te ) 2005205218) ( budeson ide ) ( triam c ino lone ace2 0175 g / kg, 2 g / kg, 013 g / kg; ton ide ) ( fluoc ino lone ace ton ide ) ( 98%, S igm a ) : 10 g / kg; 1010 m g, 10 4 g / kg m L,, - 20 10 g / kg 10 g / kg 6 g / kg ( GC ) [ 1-2 ] ( H PLC ) [ 3-4 ] 2 ( GC 2M S ) [ 5 ] 2,, 1. 2 : W a te rs ACQU 2 (LC 2M S ) [ 6-13 ] GC H PLC, ITY U PLC TM B EH C18 (100 m m 110 m m i1d1, GC 2M S 117 m ) ; : 40 ; : 10 ; : 2 L A 011% ( ),,, B,, 65%A H PLC 2M S 6100 m in 60%A 6100 m in 20% A 3100 m in 510%A,, 0110 m in,, 65% A ( 4 m in ), 011 m L /m in ; : ( ES I ( - ) ) ;, 10, (U PLC ) HLB, 12 2 / (U PLC 2M S /M S ),, 1 1. 1 ACQU ITY TM (W a te rs ) M ic rom ass λ 2Q ua ttro U ltim a TM P t (W a te rs ) ; (A llegra TM X222R, B eck2 m an ) ; (LABO RO TA 4000, H e ido l2 p h ) O as is HLB ( SPE ) Sep 2p ak am ino2p rop y l ( ), Sep 2p ak s ilica ( ) ( 6 m L, 500 m g, W a te rs ) (D ikm ap u re ), ( 99%, A c ros O rgan ics ), (M illip o re, 1812 M cm ), 2 ( 012 m o l/l, ph 5120) : (p redn isone ) (p redn iso lone ) ( co rtisone ) ( hyd roco rtisone ) (m e thylp red2 n iso lone ) ( dexam e thasone ) : 3100 kv; : 45 V; 1 (R F lens 1) 2 ( RF lens 2 ) 2710 V 010 V; : 99 ; : 350 ; : 500 L / h; : 210 1 ( ) 1. 3 510 g, 50 m L, 10 m L 012 m o l/l ph 5120 2 35 m L, 5 m in, 0 10 000 r /m in 10 m in,, 20 m L,,, 5 m L, 45,
3, : 2 12 215 8 m L 32 m L, HLB SPE 6 m L 6 m L,, 6 m L, 6 m L, 3 m L, ( 6 m L ), 6 m L, 6 m L, 3 m L 2 ( 40 60 ), ( 6 m L 40 60 2 6 m L ),, 3 m L 2 ( 40 60),,, 1 m L, 1 m in, U PLC 2M S /M S 2 1 12 T a b le 1 M S p a ram e te rs fo r g lu coco r t ico ids a n a lys is Com p ound t R / m in [M +HCOO ] - (m / z ) D augh te r ions (m / z ) P redn isone 6112 40313 32712, 35712 8 P redn iso lone 6153 40513 32911, 35910 13 Co rtisone 7170 40513 32911, 35910 8 Hyd roco rtisone 7162 40713 33018, 36111 15 M e thylp redn iso lone 9157 41913 34311, 37311 11 D exam e thasone 9140 43713 36111, 39110 12 B ec lom e thasone 9165 45314 37618, 40618 14 F lum e thasone 9104 45510 37817, 40910 15 F lud roco rtisone ace ta te E c / ev 9153 46711 42111, 40313 11 B udeson ide 11142 47512 35711, 33911 13 Triam c ino lone ace ton ide F luoc ino lone ace ton ide E c : co llis ion ene rgy. 9162 47911 37510, 35617 19 9171 49711 43110, 35510 16 te r ions w e re used fo r quan tita tive ana lys is. The p recu rso r and unde rlined daugh2 2. 1 [M + H ] + [ 7 ] [M - H ] - [ 9 ] [M + HCOO ] - [ 13 ],, :, [M + HCOO ] -, [M + HCOO ] -, 1, 1,, 8 [M - H ] - [M - H - CH 2 O ] -, 8, [M + HCOO ] - [M - H ] -, C 221 [M - H - CH 2 O ] - [M - H ] -, C 221, C 220 C 221 C C, [M - H - H 2 O ] - C 216 C 217 C 9,, C 216, [M + HCOO ] -,, 1 1 ( a ) ( b ) ( c ) [M + HCO O ] - F ig. 1 P rop o se d f ra gm e n ta t io n sch em e fo r co llis io n 2in d u ce d d isso c ia t io n o f ( a ) b u d e so n id e, ( b ) t r iam c in o lo n e a ce to n ide a n d ( c ) f lu oc in o lo n e a ce to n id e
216 24 2. 2,,, Van den H auw e [ 13 ], [M + HCOO ] -, 2 ( 90 10, 013% ) (0105% 013%), [M + HCOO ] - 0105% 011%,, 12 ; 011% 013%,, ; 011% (0105% 013% ), 011% 011% 011%, ( ),, 011% [M + HCOO ] -,, ;,,, 011%, 011%, 2. 3,,, ( 117 m ), U PLC 2M S /M S, HLB 2,, 2. 4 12 1 500 g /L 2 ( 2 g / k g) F ig. 2 C h rom a tog ram s of sp ik e d sam p le co n ta in in g 2 g / k g o f e a ch g lu co co r t ico id y x ( g /L ), ( r) 0199,
3, : 2 12 217 1 500 g /L 6, 211% 419%; ( 5 d ) 316% 611%, [M + HCOO ] - 510 g, 014 g / kg 2 g / kg, (RSD ) (LOD ) ( S /N = 3) (LOQ ) ( S /N = 10) ( n = 6) 2, 2 5,, 0115 g / kg, 3 ( 10 g /L ) Com p ound 2 12 ( n = 6) T a b le 2 Sp ik e d re co ve r ie s, RSD, LOD a n d LOQ o f 12 g lu co co r t ico id s ( n = 6) 014 g / kg recove ry / % RSD / % Sp iked leve ls 2 g / kg recove ry / % RSD / % LOD / ( g / kg) ( S /N = 3) LOQ / ( g / kg) ( S /N = 10) P redn isone 7813 819 7612 819 0108 0127 P redn iso lone 8816 810 8113 913 0116 0153 Co rtisone 8614 418 8015 1319 0110 0133 Hyd roco rtisone 8719 1316 9012 716 0107 0123 M e thylp redn iso lone 8212 813 8411 514 0106 0120 D exam e thasone 6913 815 7013 517 0103 0110 B ec lom e thasone 8712 1118 8516 519 0115 0150 F lum e thasone 8316 811 8412 614 0102 0107 F lud roco rtisone ace ta te 8516 419 7913 1613 0136 1121 B udeson ide 7615 1617 7211 917 0122 0173 Triam c ino lone ace ton ide 8314 1013 8412 618 0138 1127 F luoc ino lone ace ton ide 9413 315 9315 515 0113 0143 : 3 ( a ) ( b ) ( 20 p g) ( c d ) F ig. 3 C h rom a tog ram s of ( a ) p redn iso lon e a nd co r t isone, ( b ) h yd ro co r t ison e a n d ( c a n d d ) a m ilk b la n k sam p le [ 1 ] S im p son P M. J Ch rom a togr, 1973, 77: 161 [ 2 ] D e lahau t P H, J acquem in P, Co lem on ts Y, D ubo is M, D e2 G raeve J, D e luyke r H. J Ch rom a togr B, 1997, 696: 203 [ 3 ] D e rendo rf H, Rohdew a ld P, Hochhaus G, M o llm ann H. J Pha rm B iom ed Ana l, 1986, 4: 197 [ 4 ] Sh iba ta N, Hayakaw a T, Takada K, Hosh ino N, M inouch i T, Yam a ji A. J Ch rom a togr B, 1998, 706: 191 [ 5 ] B agna ti R, Ram azza V, Zucch i M, S im one lla A, L eone F, B e llin i A, Fane lli R. A na l B iochem, 1996, 235: 119 [ 6 ] F io ri M, P ie rdom in ic i E, Longo F, B ram b illa G. J Ch rom a2 togr A, 1998, 807: 219 [ 7 ] Van den Hauw e O, D um ou lin F, A n tignac J P, B ouche M P, E llio tt C, Van Pe teghem C. A na l Ch im A c ta, 2002, 473: 127 [ 8 ] Tang P W, Law W C, W an T S M. J Ch rom a togr B, 2001, 754: 229 [ 9 ] B ram b illa G, B u ia re lli F, Ca rton i G P, Cocc io li F, Co lam 2 on ic i C, Fagio lo A, G iann in i C, N e ri B. J Ch rom a togr B, 2001, 755: 265 [ 10 ] S to lke r A A M, Schw illens P L W J, Van G inke l L A, B rink2 m an U A Th. J Ch rom a togr A, 2001, 893: 55 [ 11 ] O Keeffe M J, M a rtin S, Regan L. A na l Ch im A c ta, 2003, 483: 341 [ 12 ] A n tignac J P, L e B izec B, M on teau F, Pou la in F, A nd re F, J Ch rom a togr B, 2001, 757: 11 [ 13 ] Van den Hauw e O, D um ou lin F, E llio tt C, Van Pe teghem C. J Ch rom a togr B, 2005, 817: 215