47.2.02
AOAC Of f i c ial Method 983.15
Phe no lic An ti ox i dants
in Oils, Fats, and But t er Oil
Liq u id Chro m ato g raphic Method
First Ac t ion 1983
Fi nal Ac tion
Re v id First Ac t ion 1992
Fi nal Ac tion1994
蚂蚁搬骨头IUPAC–AOAC Method
(Ap p li c a b le to propyl gallate [PG], 2,4,5-trihydroxybutyrophenone [THBP], tert-butylhydroquinone [T
B HQ], nordihydroguaiaretic acid [NDGA], 2- and 3-tert-butyl-4-hydroxyanisole [B HA], 2,6-di-tert-butyl-4-hydroxymethylphenol [Ionox-100], and 3,5-di-tert-butyl-4-hydroxytoluene [BHT] at 20–200 m g/g in oils and fats, 10–100 m g/g in but t er oil, and to octyl and dodecyl gallate [OG and DG] at 10–100 m g/g in but t er oil.)
See Ta b les 983.15A–C for the re s ults of the interlaboratory study sup p ort i ng ac c ep t ance of the method.A.Prin ci ple
An t i o x i d ants are ex t racted into acetonitrile. Ex t ract is con c en t rated and di l uted with 2-propanol. An t i o x i d ants are p a r ated by liq u id chromatography and mea s ured by ul t ra v i o l et de t ec t ion at 280 nm.
B. Ap p a r a t us
(a) Gra d i e nt liq u id chromatograph.—With 10 mV re c order or in te gra tor to elec tron i cally mea sure peak heights,10m L loop in j ec t ion valve, and de t ec t or to mea s ure absorbance at 280 nm. Typ i cal op er at ing con di tions:de tec tor n si tiv ity,0.05AUFS; tem per a ture,am bi ent;flow rate,2.0mL/min.
(b) LC col u mn.—Packed with C18-bonded spher i c al (pre f erred) sil i ca, or equiv a l ent. U guard col u mn if de s ired. Must be ca p a b le of ba l ine res o l u t ion of all 9 an t i o x i d ants as shown in Fig u re 983.15. Ver i fy peak iden ti ties, if nec e s s ary, by in j ec t ion of in di vid ual standards.
(c) Glass w are.—Rin all glass w are with CHCl3,ac e tone,and meth a n ol, suc c es s ively, and blow dry with N2.
C. Re a gents
(a) Sol vents.—Acetonitrile, 2-propanol, and hex a ne. Dis tilled-in-glass grade.
(b)Mo b ile pha.—(1) 5% Ace t ic acid in H2O.—LC grade.
(2) Acetonitrile–meth a n ol (1 + 1, v/v).—LC grade.
Ta ble983.15A.Interlaboratory study results for phe no lic an ti ox i dants in oils,liq uid chro mato graphic method
An ti ox i dant a Added, mg/g b Re cov ered,mg/g
Results
bad on
mean
re cov ery,%
Re peat ab il ity Reproducib ility
HorRat s r RSD r, %s R RSD R, %
PG193.7184 95.216.0 8.6616.0 8.66 1.20 PG 96.7 93.8 96.9 4.50 4.80 4.50 4.800.60 PG 19.4 17.6 90.9 2.0111.5 2.5214.3 0.25 THBP c203.2199 98.1 9.99 5.01 9.99 5.010.70 THBP101.6 99.1 97.5 5.25 5.30 5.58 5.630.71 THBP 20.3 19.6 96.7 0.97 4.94 1.53 7.770.77 TBHQ196.1201 103 8.77 4.3622.8 11.4 1.58 TBHQ 98.1100 102 3.63 3.6321.5 21.5 2.69 TBHQ 19.6 19.1 97.5 1.50 7.85 3.1716.6 1.63 NDGA 93.8 91.7 97.8 5.06 5.51 6.39 6.970.87 NDGA 18.8 18.4 98.3 0.34 1.83 0.60 3.240.32 BHA198.5197 99.1 6.54 3.32 6.61 3.360.47 BHA 99.2 99.7101 5.43 5.45 6.15 6.170.77 BHA 19.9 19.5 98.0 0.43 2.19 0.76 3.920.39 IONOX-100208.2198 95.317.7 8.9219.4 9.76 1.37 IONOX-100104.1
98.7 94.810.2 10.4 10.5 10.6 1.34 IONOX-100d 20.8 20.2 97.0 0.71 3.54 1.14 5.630.56 BHT d202.9170 83.8 3.50 2.06 4.54 2.670.37 BHT d101.5 84.3 83.1 2.36 2.80 2.36 2.800.35 BHT 20.3 17.2 85.1 0.90 5.22 1.06 6.180.61 a
PG = Propyl gallate, THBP = 2,4,5-trihydroxybutyrophenone, TBHQ = tert-butylhydroquinone, NDGA = nordihydroguaiaretic acid, BHA = 2- and
3-tert-butyl-4-hydroxyanisole, Ionox-100 = 2,6-di-tert-butyl-4-hydroxymethylphenol, BHT = 3,5-di-tert-butyl-4-hydroxytoluene.
b
An t i o x i d ant val u es mea s ured to 4 sig n if i c ant fig u res, m g/g, but rounded to near e st 0.1 m g/g.
c
Two of 7 lab o r a t o r ies re j ected as out l i e rs by the pair Grubbs test.
d
One of 7 lab o r a t o r ies re j ected as out l ier by the sin g le Grubbs test.
26个英文字母大小写的规范书写Ta ble983.15B.Interlaboratory study results for phe no lic an ti ox i dants in lard,liq uid chro mato graphic method
An ti ox i dant a Added b, mg/g Re cov ered,
mg/g
Results
bad on
mean,
re cov ery,%
Re peat ab il ity Reproducib ility
HorRat
s r RSD r, %s R RSD R, %
PG 96.990.193.0 3.18 3.53 3.18 3.530.44 PG 38.734.689.4 1.55 4.48 1.55 4.480.49 THBP101.697.996.4 6.75 6.8914.3 14.6 1.84 THBP c 40.736.489.5 1.35 3.71 1.47 4.060.45 TBHQ 98.195.697.57.71 8.0722.1 23.2 2.91 TBHQ 39.335.089.0 6.0417.3 11.6 33.2 3.62 NDGA 93.887.893.6 2.65 3.02 4.74 5.400.67 NDGA 37.535.394.2 1.06 3.01 1.79 5.060.55 BHA 99.297.498.2 2.49 2.56 3.72 3.820.48 BHA 39.738.396.6 1.90 4.97 1.90 4.970.54 IONOX-100104.199.395.4 4.83 4.87 5.45 5.490.69 IONOX-100 41.740.797.7 3.49 8.56 4.9012.0 1.32 BHT101.587.986.6 4.63 5.27 4.90 5.580.70 BHT 40.634.685.2 1.11 3.22 1.17 3.390.37
a约翰希金斯
See Ta b le 983.15A for iden t i f i c a t ion of an t i o x i d ants.
b
An t i o x i d ant val u es mea s ured to 4 sig n if i c ant fig u res, m g/g, but rounded to near e st 0.1 m g/g.
c
培风塔
One of 7 lab o r a t o r ies re j ected as out l ier by the sin g le Grubbs test.
Ta b le 983.15C. Interlaboratory study results for phe n o l ic an t i o x i d ants in but t er oil, liq u id chro m ato g raphic method
An ti ox i dant a Mean added,b
mg/g
Mean recovered,
mg/g
军训入场词Re cov ery,
平方怎么输入%s r RSD r,%s R RSD R,%HorRat
PG 92.1 89.3 97.0 4.76 5.33 6.08 6.810.84 PG 46.0 46.9 102 3.86 8.23 4.54 9.67 1.08 PG 9.20 9.53104 0.450 4.72 0.875 9.170.81 THBP 87.0 82.3 94.6 5.34 6.4811.2 13.6 1.67 THBP 43.6 42.8 98.1 3.77 8.81 6.68 15.6 1.73 THBP 8.69 8.7 101 1.39 15.9 2.0
0 22.9 1.99 TBHQ105.9 111 105 10.9 9.7824.3 21.8 2.76 TBHQ 52.8 51.8 98.2 2.01 3.8711.0 21.2 2.42 TBHQ 10.5 11.3 107 1.55 13.8 3.82 34.0 3.05 NDGA c 96.5 93.0 96.4 6.27 6.74 6.27 6.740.84 NDGA 48.3 47.0 97.3 3.22 6.86 4.66 9.91 1.12 NDGA 9.63 8.9 92.5 2.11 23.7 2.39 26.8 2.37 BHA101.3 96.3 95.2 8.49 8.81 8.49 8.81 1.11 BHA 50.6 48.8 96.5 2.29 4.70 2.50 5.120.58 BHA 10.1 10.2 101 0.515 5.06 0.597 5.870.52 IONOX-100105.7 103 97.4 4.79 4.66 7.52 7.310.93 IONOX-100 52.9 50.2 94.9 3.77 7.51 4.13 8.240.94 IONOX-100 10.6 9.36 88.6 1.22 13.0 1.25 13.4 1.20 OG 89.2 86.3 96.8 3.80 4.40 4.37 5.060.62 OG 43.7 42.0 96.2 2.89 6.87 2.89 6.870.76 OG 8.76 8.19 93.5 1.69 20.6 1.69 20.6 1.80 BHT 96.5 76.7 79.4 9.16 12.0 9.51 12.4 1.55 BHT c 48.4 38.8 80.2 2.58 6.64 3.16 8.120.91 BHT d 9.65 7.47 77.4 0.443 5.94 1.03 13.8 1.22 DG101.1 96.7 95.7 4.02 4.16 7.94 8.21 1.03 DG 50.6 48.8 96.5 2.98 6.12 3.05 6.240.71 DG 10.1 9.76 96.4 0.468 4.80 0.742 7.610.68 a
See Ta b le 983.15A for iden t i f i c a t ion of an t i o x i d ants.
b
An t i o x i d ant val u es mea s ured to 4 sig n if i c ant fig u res, m g/g, but rounded to near e st 0.1 mg/g.
c
Two of 9 lab o r a t o r ies re j ected as out l i e rs by the pair Grubbs test.
d
Two of 9 lab o r a t o r ies re j ected as out l i e rs; by the Cochran test, then by sin g le Grubbs test.
Run lin e ar gra d i e nt, from 30% of (2) in (1) to 100% (2), over 10min with hold un t il last an t i o x i d ant (DG) is eluted. For test so l u t ion only, in c rea flow rate to 4 mL/min at 100% (2) over 6 min or un t il nonpolar lipids are eluted. For test so l u t ions and stan d ards, re t urn to 30% (v/v) (2) in (1) over 1 min at 2 mL/min and let ba l ine and pres s ure sta b i l ize (ca 6 min). (Note : Re d uce flow rate and pro p or t ion a tely in c rea rins i ng and equil i b ra t ion times if ex c es s ive back pres s ure re s ults.) Run blank sol v ent gra d i e nt (no in j ec t ion) to en s ure that no peaks in t er f er i ng with any an t i o x i d ant are pres e nt. To re m ove or re d uce peaks aris i ng from elu t ion sol v ent (1), re p lace in l et fil t er with prerind solid-pha C18
ex trac tion car tridge and u in-line fil t er. If small in t er f er i ng peaks are not elim i n ated,sub t ract peak height of gra d i e nt in t er f er e nce from that of rel e v ant stan d ard or test so l u t ion.(c ) An t i o x i d ants .—BHA (2- and 3-BHA mix t ure), BHT, TBHQ,Ionox-100, THBP, and PG (PolyScience, 6600 W. Touhy Ave, Niles, IL 60714, USA, is suit a ble source [Ionox-100 is no lon g er avail a ble]); NDGA, OG , and DG (Aldrich Chem i c al Co., Inc.,Mil w au k ee, WI, USA, is suit a ble source), pu r ity ³97% (w/w).(d ) Stan dard so lu tions .—Pre p are in 2-propanol–acetonitrile (1 + 1, v/v). (Cau t ion : TBHQ is readily ox i d ized, es p e c ially in light. Re f rig e r a te all an t i o x i d ant so l u t ions and store out of di r ect light.)Mon i t or TBHQ re s pon rel a t ive to PG or THBP and pre p are fresh stan d ards if re s pon de c reas more than 5%. (1) Stock stan d ard so lu tion .—1 mg/mL. Ac c u r ately weigh ca 50 mg to near e st 0.1 mg each an t i o x i d ant and trans f er into sin g le 50 mL vol u m et r ic flask.Dis s olve, di l ute to vol u me, and mix. (2) Working stan d ard so lu tion .—0.01 mg/mL (10 m g/mL). Pipet 1 mL stock stan d ard so l u t ion into 100 mL vol u m et r ic flask, di l ute to vol u me, and mix.
(e ) Ex trac tion sol vents.—(1)Sat u rated hex ane .—Sat u rate ca 300 mL hex a ne in pa r a t ory fun n el by add i ng acetonitrile un t il 2 lay e rs re m ain af t er shak i ng 2 min. Dis c ard acetonitrile lower layer. (2)Sat u r ated acetonitrile.—Sat u r ate ca 300 mL acetonitrile in pa r a t ory fun n el
by add i ng hex a ne un t il 2 lay e rs re m ain af t er shak i ng 2 min. Re m ove and dis c ard hex a ne up p er layer.
D.De ter mi na tion
(a ) Ex trac tion .—Ac c u r ately weigh to near e st 0.01 g 50 mL beaker con t ain i ng ca 5.5 g liq u id or but t er oil or ca 3.0 g lard or short e n i ng (liq u e f ied in bulk us i ng 60°C wa t er bath or oven, and swirled or shaken to en s ure ho m o g e n e i ty). De c ant as much test por t ion as pos s i b le into 125 mL pa r a t ory fun n el con t ain i ng 20 mL (22.5 mL for lard or short e n i ng) sat u r ated hex a ne. Reweigh beaker to de t er m ine test por t ion weight. Swirl to mix test por t ion with hex a ne, and ex t ract with three 50 mL por t ions of sat u r ated acetonitrile. If emul s ions form, hold pa r a t ory fun n el un d er hot tap wa t er 5–10 s. Col l ect ex t racts in 250 mL pa r a t ory fun n el and let com b ined ex t racts slowly drain into 250 or 500 mL round-bottom flask to aid re m oval of hex a ne–oil drop l ets. (Note: At this point, 150mL acetonitrile ex t ract may be stored over n ight, re f rig e r a ted.)Evap o r ate to 3–4 mL, us i ng flash evap o r a t or with £40°C wa t er bath, within 10 min. [Note : (1) Pro l onged evap o r a t ion time may cau TB HQ loss. To de c rea evap o r a t ion time, u ef f i c ient vac u um source and wa t er–ice con d enr cool i ng. (2) U 500 mL flask to re d uce “bump i ng” loss. Take care to en s ure quan t i t a t ive trans fer of ex tract af ter e
vap o ra tion.]Using dis pos able pipet,trans f er acetonitrile–oil drop l et mix t ure to 10 mL glass-stoppered grad u a ted cyl i n d er. Rin flask with small por t ions nonsaturated acetonitrile. As rin pools in flask bot t om, pipet rin to cyl i n d er un t il 5 mL is col l ected. Rin pipet through top and con t inue to rin flask with small por t ions 2-propanol, trans f er r ing rins to cyl i n d er un t il 10 mL is col l ected. Mix cyl i n d er con t ents. (Note : De l ay in an a l yz i ng ex t racted test portion may cau TBHQ loss.)(b ) Chro m a t og r a p hy .—Using loop in j ec t ion valve, in j ect 10 m L test ex t ract and elute with sol v ent gra d i e nt pro g ram for test extracts,C (b ). Be f ore and af t er ev e ry 3–4 test in j ec t ions, or more fre q uently if dif f er e nces be t ween stan d ard peak heights are found to be >5%,in ject 10m L an t i o x i d ant work i ng stan d ard so l u t ion (10 m L/mL) and elute with sol v ent gra d i e nt pro g ram for stan d ards, C (b ). For analyte peaks off scale or >3´stan dard,quan ti ta tively di lute test ex tract with 2-propanol–acetonitrile (1 + 1) and re i nject. Iden t ify peaks by com p ar i s on with re t en t ion times of stan d ard.
For re a gent blank de t er m i n a t ion, take 25 mL sat u r ated hex a ne and fol low ex trac tion,(a ), from “. . . ex t ract with three 50 mL por t ions of sat u rated acetonitrile.”In ject 10m L re a gent blank ex t ract and elute with sol v ent gra d i e nt pro g ram for analytes. The re a gent blank should have no peaks in t er f er i ng with an t i o x i d ant de t er m i n a t ion.U elec t ron i c ally de t er m ined 太平间的磨牙声
peak height, or mea s ure peak height to 0.1 mm, us i ng blank gra d i e nt chromatogram as guide to fol l ow ba l ine. De t er m ine an t i o x i d ant peak heights and av e r a ge an t i o x i d ant stan d ard peak heights (from du p li c ate in j ec t ions be f ore and af t er test in j ec t ion, cor r ected for gra d i e nt blank).
E.Cal cu la tions
Cal c u l ate con c en t ra t ion of an t i o x i d ant as fol l ows:
Fig ure 983.15.Chro mato graphic p a ra tion of
an ti ox i dant stan dards,ca 0.1m g each an ti ox i dant:1,PG; 2, THBP; 3, TBHQ; 4, NDGA; 5, BHA; 6, Ionox-100;7, OG; 8, BHT; 9, DG.
where R x and R s are peak heights from test por t ion and stan d ard, re spec tively;C s is con c en t ra t ion stan d ard, m g/mL;W x is test por t ion weight, g/mL, in un d i l uted 10 mL test ex t ract; and D is di l u t ion fac t or, if so l u t ion in j ected is di l uted.
Ref er ence:J. AOAC Int. 76, 765(1993).
CAS-25013-16-5 (butylated hydroxyanisole)
CAS-128-37-0 (butylated hydroxytoluene)大国崛起美国
CAS-500-38-9 (nordihydroguaiaretic acid)
CAS-121-79-9 (propyl gallate)
CAS-1034-01-1 (octyl gallate)
CAS-1166-52-5 (dodecyl gallate)
CAS-1421-63-2 (2,4,5-trihydroxybutyrophenone)
CAS-1948-33-0 (tert-butylhydroquinone)
CAS-88-26-6 (Ionox-100)
Re v id: March 1999
