23.1.03A AOAC Of fi cial Method 2003.04 Clopidol in Chicken Tis sues

23.1.03A
AOAC Of fi cial Method 2003.04
Clopidol in Chicken Tis sues
Liq uid Chro ma tog ra phy
First Ac tion 2003
Final Action 2006
(Ap pli ca ble to the de ter mi na tion of res i dues of clopidol in
chicken mus cle at con cen tra tions �0.1 mg/kg.)
See Ta bles 2003.04A and 2003.04B for the re sults of the
interlaboratory study sup port ing ac cep tance of the method.
A. Prin ci ple
Clopidol is ex tracted from chicken mus cle with acetonitrile. The
ex tracts are cleaned up on an alu mina col umn and then on an an ion
ex change col umn. Clopidol is sep a rated from co-ex tract ives by
re versed-phase LC and quantitated at 270 nm.
B. Re agents
(a) Clopidol an a lyt i cal stan dard.—Pu rity >99.
(b) Alu mina.—Neu tral, 100–200 mesh (Merck KgaA, 64271
Darmstadt, Ger many), or equiv a lent. Ac ti vate at 300�C for 4 h and then
cool in des ic ca tor.
(c) An ion ex change resin.—AG ® 1-X8 Resin 100–200 mesh,
ac e tate form (Bio-Rad Lab o ra to ries, Her cu les, CA, USA), or
equiv a lent. Im merse in meth a nol for 2 h be fore use.
(d) Sol vents.—Acetonitrile and meth a nol, LC grade, and ace tic
acid (gla cial), AR grade.
(e) Meth a nol–am mo nia (sp.gr. 0.88) so lu tion.—98 + 2 (v/v).
(f) So dium sul fate, an hy drous.—Heat at 650�C for 4 h. Cool in
des ic ca tor.
(g) Mo bile phase.—Acetonitrile–deionized wa ter (20 + 80).
Fil ter mo bile phase un der vac uum through 0.45 �m fil ter be fore use.
(h) Eluting sol vent.—0.5% Ace tic acid (gla cial) in meth a nol.
Mix 2.5 mL ace tic acid (gla cial) with 497.5 mL meth a nol.
(i) Wa ter.—Deionized, �18.0 meg ohm-cm.
(j) Fil ter mem brane.—Ny lon 66 mem brane, 0.45 �m � 47 mm
(Supelco, Bellefonte, PA, USA; www.sigmaaldrich.com), or
equiv a lent.
(k) Glass wool.—Must be free of in ter fer ence with UV
de tec tion. Store by im mers ing in meth a nol.
C. Ap pa ra tus
(a) Liq uid chromatograph.—LC sys tem con sist ing of sol vent
de liv ery pump, uni ver sal LC in jec tor with 10 �L sy ringe, UV
de tec tor mon i tor ing 270 nm, and data out put de vice (re corder,
in te gra tor, or data sys tem for mea sur ing peak heights). If LC is not
equipped with de-gas mod ule, gently sparge mo bile phase with
he lium for 5 min im me di ately be fore use. Set flow rate of mo bile
phase at 1.0 mL/min; set de tec tor wave length at 270 nm with
sen si tiv ity of 0.01 absorbance unit full scale. Peak res o lu tion should
be as in di cated in Fig ure 2003.04A.
(b) LC col umn.—30 cm � 3.9 mm id of �Bondapak C18, 10 �m
(Wa ters Corp., Milford, MA, USA; www.wa ters.com), or
equiv a lent.
(c) Blender.—Suitable for mincing chicken.
(d) Ho mog e nizer.—Overhead probe high-performance
dispenser for volumes from 1 to 2000 mL.
(e) Cen tri fuge.—Capable of centrifuging 80 mL capacity glass
tubes with Teflon-lined screw caps at 1400 � g.
(f) Ro tary evap o ra tor.—With water bath, tap water cooled
condenser, and vacuum aspirator.
(g) Alu mina cleanup col umn.—Chro mato graphic tubes (400 �
20 mm id), fit ted with draw-off valve. Place small plug of glass wool
at bot tom of col umn; fill with 40 mL meth a nol, pour in 15 g alu mina,
and tap on sides of col umn to pro duce even set tling of bed; drain
sol vent. Prerinse col umn with 10 mL meth a nol to con di tion; drain
sol vent to top of pack ing ma te rial.
Ta ble 2003.04A. Interlaboratory study re sults for the de ter mi na tion of clopidol res i dues (for ti fied) in chicken mus cle (n = 16)
Added, mg/kg Avg. rec., % s r s R RSD r , % RSD R , % r R HorRat
0.10 82.8 0.010 0.019 11.7 22.6 0.028 0.054 1.0
0.20 83.0 0.017 0.027 10.2 15.9 0.048 0.076 0.8
0.50 81.8 0.062 0.067 15.1 16.4 0.173 0.190 0.9
1.00 85.4 0.119 0.102 13.9 11.9 0.333 0.288 0.7
2.00 82.0 0.181 0.261 11.0 15.8 0.507 0.737 1.1
5.00 82.4 0.409 0.575 9.9 13.9 1.146 1.626 1.1
Ta ble 2003.04B. Interlaboratory study re sults for the de ter mi na tion of clopidol res i dues (in curred) in chicken mus cle (n = 9)
In curred sam ples, mg/kg Avg. de ter mined, mg/kg sr sR RSDr , % RSDR , % r R HorRat
0.100 ± 0.009 a,b
0.099 0.008 0.020 8.5 19.8 0.024 0.055 0.9
0.191 ± 0.008 a,c
0.172 0.008 0.026 4.9 15.2 0.024 0.073 0.7
0.687 ± 0.062 a,d
0.659 0.020 0.082 3.1 12.6 0.057 0.231 0.7
a
Tar get con cen tra tion from ap proved pro to col.
b Up per breast mus cle.
c Lower breast mus cle.
d Leg mus cle.
� 2006 AOAC IN TER NA TIONAL

(h) An ion ex change col umn.—Chro mato graphic tubes (250 �
10 mm id) with scintered glass disk, fit ted with draw-off valve. Place
small plug of glass wool at bot tom of col umn, and then fill col umn
with 20 mL meth a nol. Add suf fi cient an ion ex change resin to
col umn to form bed height of 2–3 cm af ter set tling by tap ping gently
on sides of col umn. Drain meth a nol; prerinse col umn with 10 mL
meth a nol to con di tion, drain ing sol vent to top of col umn bed.
(i) Vol u met ric flask.—Grade A, 100 mL, glass vol u met ric flask
with stop per.
(j) Pipets.—Grade A, glass pipets, 1, 2, 5, 10, and 15 mL.
(k) Bal ance.—Elec tronic bal ance, ca pac ity 200 g, readability
0.002 g.
(l) Vor tex mixer.
D. Prep a ra tion of Stan dard So lu tions
(a) Stock stan dard so lu tion.—2.0 mg/mL. Dis solve 200.00 mg
clopidol (cor rected for po tency) in meth a nol–am mo nia so lu tion in
100 mL vol u met ric flask, di lute to vol ume with meth a nol–am mo nia
so lu tion, and mix.
(b) In ter me di ate stan dard so lu tion.—0.020 mg/mL. Add 1.0 mL
stock stan dard so lu tion, D(a), to 100 mL vol u met ric flask, di lute to
vol ume with meth a nol, and mix.
(c) Work ing stan dard so lu tions.—0.20, 0.40, 1.00, 2.00, and
3.00 �g/mL. Add 1, 2, 5, 10, and 15 mL in ter me di ate stan dard
so lu tion, D(b), to five 100 mL vol u met ric flasks in proper or der,
ap pro pri ately la beled. Di lute to vol ume with meth a nol, and mix.
E. Prep a ra tion of Test Ex tracts
Ac cu rately weigh 10.0 g rep re sen ta tive minced chicken mus cle
into 80 mL glass cen tri fuge tube. Add 20 g an hy drous so dium sul fate
and 35 mL acetonitrile, and ho mog e nize for 1.5 min at 15 000 rpm.
Cen tri fuge for 3 min at 1400 � g. Use holder to po si tion
pre con di tioned alu mina col umn, C(g), above pre con di tioned an ion
ex change col umn, C(h), as shown in Fig ure 2003.04B, while
en sur ing that meth a nol lev els in col umns do not drop be low level of
re spec tive pack ing ma te ri als. De cant clear tis sue ex tract into
alu mina col umn and let it run through both col umns to waste at
ap prox i mately 3 mL/min, mak ing sure that tis sue ex tract lev els in
col umns do not drop be low level of re spec tive pack ing ma te ri als.
Re-ex tract by add ing 35 mL acetonitrile to tis sue sed i ment plug, and
mix on a Vor tex mixer to break up tis sue sed i ment. Dis rupt plug with
a spat ula, if re quired. Recentrifuge and ap ply tis sue ex tracts to
as sem bled col umns. Rinse as sem bled col umns with 20 mL
meth a nol. Re move alu mina col umn and rinse sep a rately with
ad di tional 20 mL meth a nol. Elute clopidol off an ion ex change
col umn with 20 mL 0.5% ace tic acid–meth a nol mix ture into 100 mL
evap o rat ing flask. Evap o rate to dry ness on ro tary evap o ra tor at
60�C. Dis solve res i due in proper vol ume of meth a nol for LC
anal y sis (1.0 mL, or as ap pro pri ate for the ex pected con cen tra tion of
analyte).
F. Prep a ra tion of Cal i bra tion Curve
Check sys tem suit abil ity be fore de ter mi na tion by in ject ing
stan dard so lu tion (such as 1.00 �g/mL), then 10 �L of 0.20, 0.40,
1.00, 2.00, and 3.00 �g/mL clopidol work ing stan dard so lu tion,
D(c), in du pli cate un der LC con di tions. Pre pare 5-point stan dard
curve (peak height re sponse, in mil li me ters, vs clopidol
con cen tra tion, �g/mL), which should be lin ear be tween 0.20 and
3.00 �g/mL. The r 2 should be �0.99.
� 2006 AOAC IN TER NA TIONAL
Fig ure 2003.04A. Liq uid chromatograms for clopidol
de ter mi na tion. (A) Con trol; (B) stan dard so lu tion
(0.50 �g/mL); (C) for ti fi ca tion chicken mus cle
(0.10 mg/kg).
G. LC De ter mi na tion
In ject in du pli cate 10 �L test ex tracts into LC sys tem. Peak
heights should agree within 5% for du pli cate in jec tions. Cal cu late
res i due of clopidol in orig i nal chicken test portion us ing the
fol low ing for mula:
Clopidol, mg/kg =
P � b v
�
m w
where P = peak height (mm) at clopidol re ten tion time of test
ex tracts, b = in ter cept at or di nate, m = slope, from cor re spond ing
lin ear re gres sion equa tion, v = fi nal ex tract vol ume of test por tion
(mL), w = test por tion mass (g).

Fig ure 2003.04B. Alu mina and ion-ex change resin
as sem bled col umns.
Reference: J. AOAC Int. 84, 685(2003).
CAS 2971-90-6 (3,5-dichloro-2,6-dimethyl-4-pyridinol)
� 2006 AOAC IN TER NA TIONAL