Assay for vitamin A

[N OTE Use low-actinic glassware throughout this procedure.]

Diluting solutionPrepare a mixture o tetrah!drouran and acetonitrile
"#$#%& and mix.
'o(ile phasePrepare a )ltered and degassed mixture o methanol&
acetonitrile& and n-hexane "*+.,$*+.,$-..%.
'a/e ad0ustments i necessar! "see 1!stem 1uita(ilit! under
2hromatograph! 3 +4#5%.
1tandard preparation[NOTEThe U1P 6itamin 7 81 is all-trans retin!l
acetate. Use it to anal!9e Oral 1olution that contains :itamin 7 alcohol
"retinol% :itamin 7 acetate& or :itamin 7 palmitate.] Dissol:e
;uantitati:el! an accuratel! weighed ;uantit! o U1P 6itamin 7 81 in
Diluting solution to o(tain a solution ha:ing a /nown concentration o
a(out ..<< mg o U1P 6itamin 7 81 per m=.
7ssa! preparationTranser an accuratel! measured :olume "##ml% o
Oral 1olution& e;ui:alent to a(out <.< mg o :itamin 7 to a ,..-m=
separator! unnel containing #. m= o water and 4. m= o deh!drated
alcohol. 7dd #,. m= o sol:ent hexane& insert the stopper& and sha/e
or # minute. 7dd another #,. m= o sol:ent hexane& insert the
stopper& sha/e& and allow the la!ers to separate. Discard the a;ueous
la!er& and )lter the sol:ent hexane extract through anh!drous sodium
sulate into a ,..-m= round-(ottom >as/. E:aporate the solution with
the aid o a rotar! e:aporator o:er a water (ath maintained at a(out
+,? to dr!ness. @mmediatel! add #... m= o Diluting solution& swirl to
dissol:e the residue& and )lter.
2hromatographic s!stem "see 2hromatograph! 3 +4#5% The li;uid
chromatograph is e;uipped with a 4+,-nm detector and a *.+-mm A
,.-cm column "prepared rom two concatenated *.+-mm A 4,-cm
columns% that contains pac/ing =#. The column temperature is
maintained at a(out *.?& and the >ow rate is a(out #., m= per minute.
2hromatograph the 1tandard preparation& and record the pea/
responses as directed under Procedure$ the relati:e standard de:iation
or replicate in0ections is not more than ,..B.
Procedure[NOTEUse pea/ areas where pea/ responses are
indicated.] 1eparatel! in0ect e;ual :olumes "a(out 4. m=% o the
1tandard preparation and the 7ssa! preparation into the
chromatograph& record the chromatograms& and measure the pea/
responses. 2alculate the ;uantit!& in mg& o :itamin 7 as the retinol
e;ui:alent "24.C<.O% in each m= o the Oral 1olution ta/en (! the
ormula$
#."..D-2 E 6%"r U E r 1%&
in which ..D- is the actor used to con:ert retin!l acetate to its retinol
e;ui:alent& 2 is the concentration& in mg per m=& o U1P 6itamin 7 81
in the 1tandard preparation& 6 is the :olume& in m=& o Oral 1olution
ta/en or the 7ssa! preparation& and r U and r 1 are the pea/
responses or retinol or retin!l ester o(tained rom the 7ssa!
preparation and the 1tandard preparation& respecti:el!.
Assay for vitamin D
[N OTE Use low-actinic glassware throughout this procedure.]
Diluting solutionPrepare a mixture o tetrah!drouran and acetonitrile
"#$#%& and mix.
'o(ile phasePrepare a )ltered and degassed mixture o methanol&
acetonitrile& and n-hexane "*+.,$*+.,$-..%.
'a/e ad0ustments i necessar! "see 1!stem 1uita(ilit! under
2hromatograph! 3 +4#5%.
1tandard preparationDissol:e ;uantitati:el! an accuratel! weighed
;uantit! o U1P 2holecalcierol 81 or U1P Ergocalcierol 81 in Diluting
solution to o(tain a solution ha:ing a /nown concentration o a(out ,
Fg per m=.
7ssa! preparationTranser an accuratel! measured :olume "4,ml% o
Oral 1olution& e;ui:alent to a(out ,. Fg o cholecalcierol or
ergocalcierol& to a ,..-m= separator! unnel containing #. m= o
water and 4. m= o deh!drated alcohol. 7dd #,. m= o sol:ent hexane&
insert the stopper& and sha/e or # minute. 7dd another #,. m= o
sol:ent hexane& insert the stopper& sha/e& and allow the la!ers to
separate. Discard the a;ueous la!er& and )lter the sol:ent hexane
extract through anh!drous sodium sulate into a ,..-m= round-(ottom
>as/. E:aporate the solution o:er a water (ath maintained at a(out
+,? to dr!ness. @mmediatel! add #... m= o Diluting solution& swirl to
dissol:e the residue& and )lter.
2hromatographic s!stem$ "see 2hromatograph! 3 +4#5% The li;uid
chromatograph is e;uipped with a 4+,-nm detector and a *.+-mm A
,.-cm column "prepared rom two concatenated *.+-mm A 4,-cm
columns% that contains pac/ing =#. The column temperature is
maintained at a(out *.?& and the >ow rate is a(out #., m= per minute.
2hromatograph the 1tandard preparation& and record the pea/
responses as directed under Procedure$ the relati:e standard de:iation
or replicate in0ections is not more than ,..B.
Procedure[NOTEUse pea/ heights where pea/ responses are
indicated.] 1eparatel! in0ect e;ual :olumes "a(out 4. m=% o the
1tandard preparation and the 7ssa! preparation into the
chromatograph& record the chromatograms& and measure the pea/
responses or :itamin D. 2alculate the ;uantit!& in mg& o
cholecalcierol "24-C**O% or o ergocalcierol "24DC**O% in each m= o
the Oral 1olution ta/en (! the ormula$
#..G"#.2 E 6%"r U E r 1%&
in which #..G is a correction actor to account or the a:erage amount
o pre:itamin D in the Oral 1olution& 2 is the concentration& in mg per
m=& o U1P 2holecalcierol 81 or Ergocalcierol 81 in the 1tandard
preparation& 6 is the :olume& in m=& o Oral 1olution ta/en or the
7ssa! preparation& and r U and r 1 are the :itamin D pea/ responses
o(tained rom the 7ssa! preparation and the 1tandard preparation&
respecti:el!.
Assay for ascorbic acid
Transer ,ml o Oral 1olution& e;ui:alent to +. mg o ascor(ic acid& to a
conical >as/. 7dd ,. m= o water& #.. m= o ..# N suluric acid 61& and
#,.. m= o ..# N iodine 61. 1tir the contents or <. seconds& add , m=
o starch T1& and immediatel! titrate with ..# N sodium thiosulate 61
to the disappearance o the color. Each m= o ..# N iodine is e;ui:alent
to D.D.+ mg o 2 +C DO +.
Assay for Dexpanthenol
7ssa! or dexpanthenol [N OTE The ollowing procedure is
applica(le also to the determination o the dextrorotator! component
o racemic panthenol in preparations containing panthenol.]
Deh!drated mixtures !ielding ormulations similar to the media
descri(ed herein ma! (e used pro:ided that& when constituted as
directed& the! ha:e growth-promoting properties e;ual to or superior
to those o(tained with the media prepared as descri(ed herein.
1tandard stoc/ solutionDissol:e an accuratel! weighed ;uantit! o
U1P Dexpanthenol 81 in water& and dilute with water to o(tain a
solution ha:ing a /nown concentration o a(out D.. Fg per m=. 1tore
in a rerigerator& protected rom light& and use within <. da!s.
1tandard preparationOn the da! o the assa!& dilute an accuratel!
measured :olume o 1tandard stoc/ solution with water to o(tain a
solution ha:ing a /nown concentration o a(out #.4 Fg o dexpanthenol
per m=.
7ssa! preparationTranser an accuratel! measured :olume "4ml% o
Oral 1olution& e;ui:alent to a(out #.4 mg o dexpanthenol& to a #..-m=
:olumetric >as/& dissol:e in and dilute with water to :olume& and mix.
Dilute a portion o this solution ;uantitati:el!& and stepwise i
necessar!& with water to o(tain a solution ha:ing a concentration o
a(out #.4 Fg o dexpanthenol per m=.
7cid-h!drol!9ed casein solution'ix #.. g o :itamin-ree casein with
,.. m= o + N h!drochloric acid& and re>ux the mixture or D to #4
hours. 8emo:e the h!drochloric acid rom the mixture (! distillation
under reduced pressure until a thic/ paste remains. 8edissol:e the
resulting paste in a(out ,.. m= o water& ad0ust the solution with # N
sodium h!droxide to a pC o <., H ..#& and add water to ma/e #...
m=. 7dd 4. g o acti:ated charcoal& stir or # hour& and )lter. 8epeat
the treatment with acti:ated charcoal. 1tore under toluene in a
rerigerator at a temperature not (elow #.?. Iilter the solution i a
precipitate orms during storage.
2!stine-tr!ptophane solution1uspend *.. g o =-c!stine and #.. g o
=-tr!ptophane "or 4.. g o D&=-tr!ptophane% in -.. m= to D.. m= o
water& heat to -, H ,?& and add dilute h!drochloric acid "# in 4%
dropwise& with stirring& until the solids are dissol:ed. 2ool& add water to
ma/e #... m=& and mix. 1tore under toluene in a rerigerator at a
temperature not (elow #.?.
7denine-guanine-uracil solutionDissol:e 4.. mg each o adenine
sulate& guanine h!drochloride& and uracil& with the aid o heat& in #.
m= o * N h!drochloric acid& cool& add water to ma/e 4.. m=& and mix.
1tore under toluene in a rerigerator.
Pol!sor(ate D. solutionDissol:e 4, g o pol!sor(ate D. in alcohol to
ma/e 4,. m=& and mix.
8i(o>a:in-thiamine h!drochloride-(iotin solutionPrepare a solution o
ri(o>a:in& thiamine h!drochloride& and (iotin in ...4 N acetic acid
containing 4. mg o ri(o>a:in& #. mg o thiamine h!drochloride& and
...* mg o (iotin per m=. 1tore under toluene& protected rom light& in
a rerigerator.
p-7mino(en9oic acid-niacin-p!ridoxine h!drochloride solutionPrepare
a solution in neutral 4,B alcohol containing #. mg o p-amino(en9oic
acid& ,. mg o niacin& and *. mg o p!ridoxine h!drochloride per m=.
1tore in a rerigerator.
1alt solution 7Dissol:e 4, g o mono(asic potassium phosphate and
4, g o di(asic potassium phosphate in water to ma/e ,.. m=. 7dd ,
drops o h!drochloric acid& and mix. 1tore under toluene.
1alt solution JDissol:e #. g o magnesium sulate& .., g o sodium
chloride& .., g o errous sulate& and .., g o manganese sulate in
water to ma/e ,.. m=. 7dd , drops o h!drochloric acid& and mix.
1tore under toluene.
P!ridoxal-calcium pantothenate solutionDissol:e *. mg o p!ridoxal
h!drochloride and <-, mg o calcium pantothenate in #.B alcohol to
ma/e 4.. m=& and mix. 1tore in a rerigerator& and use within <. da!s.
Pol!sor(ate *.-oleic acid solutionDissol:e 4, g o pol!sor(ate *. and
..4, g o oleic acid in 4.B alcohol to ma/e ,.. m=& and mix. 1tore in a
rerigerator& and use within <. da!s.
'odi)ed pantothenate medium
7cid-h!drol!9ed casein solution 4, m=
2!stine-tr!ptophane solution 4, m=
Pol!sor(ate D. solution ..4, m=
Dextrose& anh!drous #. g
1odium acetate& anh!drous , g
7denine-guanine-uracil solution , m=
8i(o>a:in-thiamine h!drochloride-(iotin solution , m=
p-7mino(en9oic acid-niacin-p!ridoxine h!drochloride solution , m=
1alt solution 7 , m=
1alt solution J , m=
P!ridoxal-calcium pantothenate solution , m=
Pol!sor(ate *.-oleic acid solution , m=
Dissol:e the anh!drous dextrose and sodium acetate in the solutions
pre:iousl! mixed& and ad0ust with # N sodium h!droxide to a pC o +.D.
Iinall!& dilute with water to 4,. m=& and mix.
Dou(le-strength modi)ed pantothenate mediumPrepare as directed
under 'odi)ed pantothenate medium& (ut ma/e the )nal dilution to
#4, m= instead o 4,. m=. Prepare resh.
1toc/ culture o pediococcus acidilacticiDissol:e in a(out D.. m= o
water& with the aid o heat& +.. g o peptone& *.. g o pancreatic digest
o casein& <.. g o !east extract& #., g o (ee extract& #.. g o
dextrose& and #,.. g o agar. 7d0ust with ..# N sodium h!droxide or ..#
N h!drochloric acid to a pC o (etween +., and +.+& dilute with water to
#... m=& and mix. 7dd #.-m= portions o the solution to culture tu(es&
place caps on the tu(es& and sterili9e in an autocla:e at #4#? or #,
minutes. 2ool on a slant& and store in a rerigerator. Prepare a stoc/
culture o Pediococcus acidilacticiK on a slant o this medium. @ncu(ate
at <,? or 4. to 4* hours& and store in a rerigerator. 'aintain the stoc/
culture (! monthl! transer onto resh slants.
@noculum@noculate three 4,.-m= portions o 'odi)ed pantothenate
medium rom a stoc/ culture slant& and incu(ate at <,? or 4. to 4*
hours. 2entriuge the suspension rom the com(ined portions& and
wash the cells with 'odi)ed pantothenate medium. 8esuspend the
cells in suLcient 'odi)ed pantothenate medium so that a #$,.
dilution& when tested in a #<-mm diameter test tu(e& gi:es D.B light
transmission at ,<. nm. Transer #.4-m= portions o this stoc/
suspension to glass ampuls& seal& ree9e in li;uid nitrogen& and store in
a ree9er. On the da! o the assa!& allow the ampuls to reach room
temperature& mix the contents& and dilute # m= o thawed culture with
sterile saline T1 to #,. m=.
[NOTEThis dilution ma! (e altered when necessar! to o(tain the
desired test response.]
ProcedurePrepare in triplicate a series o eight culture tu(es (!
adding the ollowing ;uantities o water to the tu(es within a set$ ,..
m=& *., m=& *.. m=& <., m=& <.. m=& 4.. m=& #.. m=& and ... m=. To
these same tu(es& and in the same order& add ...& ..,& #..& #.,& 4..&
<..& *..& and ,.. m= o the 1tandard preparation.
Prepare in duplicate a series o ):e culture tu(es (! adding the
ollowing ;uantities o water to the tu(es within a set$ *.. m=& <., m=&
<.. m=& 4.. m=& and #.. m=. To these same tu(es& and in the same
order& add #..& #.,& 4..& <..& and *.. m= o the 7ssa! preparation. 7dd
,.. m= o Dou(le-strength modi)ed pantothenate medium to each
tu(e& and mix. 2o:er the tu(es with metal
caps& and sterili9e in an autocla:e at #4#? or , minutes. 2ool to room
temperature in a chilled water (ath& and inoculate each tu(e with ..,
m= o the @noculum. 7llow to incu(ate at <-? or #+ hours. Terminate
growth (! heating to a temperature not (elow D.?& such as (!
steaming at atmospheric pressure in a sterili9er or , to #. minutes.
2ool& and concomitantl! determine the percentage transmittance o
the suspensions& in cells o e;ual pathlength& on a suita(le
spectrophotometer& at a wa:elength o ,<. nm.
2alculationDraw a dose-response cur:e on arithmetic graph paper (!
plotting the a:erage response& in percent transmittance& or each set o
tu(es o the standard cur:e against the standard le:el concentrations.
The cur:e is drawn (! connecting each ad0acent pair o points with a
straight line. Irom this standard cur:e& determine (! interpolation the
potenc!& in terms o dexpanthenol& o each tu(e containing portions o
the 7ssa! preparation.
Di:ide the potenc! o each tu(e (! the amount o the 7ssa!
preparation added to it& to o(tain the indi:idual responses. 2alculate
the mean response (! a:eraging the indi:idual responses that :ar!
rom their mean (! not more than #,B& using not less than hal the
total num(er o tu(es. 2alculate the potenc! o the portion o the
material ta/en or assa!& in terms o dexpanthenol& (! multipl!ing the
mean response (! the appropriate dilution actor.
Assay for niacin or Niacinamide
[N OTE Use low-actinic glassware throughout this procedure.]
Diluting solutionDissol:e 4, g o edetate disodium in #... m= o
water& and mix.
'o(ile phase'ix ..* m= o trieth!lamine& #,.. m= o glacial acetic
acid& and <,. m= o methanol& and dilute with ....D ' sodium #-
hexanesulonate to 4... m=. Iilter& and degas. 'a/e ad0ustments i
necessar! "see
1!stem 1uita(ilit! under 2hromatograph! 3 +4#5%.
1tandard preparation[NOTEUse U1P Niacin 81 or Oral 1olution that
contains niacin and U1P Niacinamide 81 or Oral 1olution that contains
niacinamide.] Dissol:e an accuratel! weighed ;uantit! o U1P Niacin
81 or U1P Niacinamide 81 in Diluting solution in a :olumetric >as/& and
dilute ;uantitati:el! and stepwise i necessar!& with Diluting solution to
o(tain a solution ha:ing a /nown concentration o a(out ...4, mg per
m=.
7ssa! preparationTranser an accuratel! measured :olume "#.4,ml%
o Oral 1olution to a #..ml :olumetric >as/& dissol:e in Diluting
solution& and dilute to the mar/ with Diluting solution to o(tain a
solution ha:ing a /nown concentration o a(out ...4, mg o niacin or
niacinamide per m=.
2hromatographic s!stem "see 2hromatograph! 3 +4#5%The li;uid
chromatograph is e;uipped with a 4-.-nm detector and a *.+-mm A
4,-cm column that contains pac/ing =-. The >ow rate is a(out 4.. m=
per minute.
2hromatograph the 1tandard preparation& and record the pea/
responses as directed under Procedure$ the relati:e standard de:iation
or replicate in0ections is not more than 4..B.
Procedure[NOTEUse pea/ areas where pea/ responses are
indicated.] 1eparatel! in0ect e;ual :olumes "a(out 4. F=% o the
1tandard preparation and the 7ssa! preparation into the
chromatograph& record the chromatograms& and measure the pea/
responses. 2alculate the ;uantit!& in mg& o niacin "2+C,NO4% or
niacinamide "2+C+N4O% in each m= o the Oral 1olution ta/en (! the
ormula$
2"= E D%"r U E r 1%&
in which 2 is the concentration& in mg per m=& o U1P Niacin 81 or U1P
Niacinamide 81 in the 1tandard preparation& = is the la(eled amount&
in mg per m=& o niacin or niacinamide in the Oral 1olution ta/en& D is
the concentration& in mg per m=& o niacin or niacinamide in the 7ssa!
preparation& (ased on the la(eled ;uantit! and the extent o dilution&
and r U and r 1 are the pea/ responses or niacin or niacinamide
o(tained rom the 7ssa! preparation and the 1tandard preparation&
respecti:el!.
Assay for Pyridoxine Hydrochloride
[N OTE Use low-actinic glassware throughout this procedure.]
Diluting solutionDissol:e 4, g o edetate disodium in #... m= o
water& and mix.
'o(ile phase'ix ..* m= o trieth!lamine& #,.. m= o glacial acetic
acid& and <,. m= o methanol& and dilute with ....D ' sodium #-
hexanesulonate to 4... m=. Iilter& and degas. 'a/e ad0ustments i
necessar! "see
2hromatographic s!stem "see 2hromatograph! 3 +4#5%The li;uid
chromatograph is e;uipped with a 4-.-nm detector and a *.+-mm A
4,-cm column that contains pac/ing =-. The >ow rate is a(out 4.. m=
per minute.
2hromatograph the 1tandard preparation& and record the pea/
responses as directed under Procedure$ the relati:e standard de:iation
or replicate in0ections is not more than 4..B.
1tandard preparationDissol:e an accuratel! weighed ;uantit! o U1P
P!ridoxine C!drochloride 81 in Diluting solution& and dilute
;uantitati:el!& and stepwise i necessar!& with Diluting solution to
o(tain a solution ha:ing a /nown concentration o a(out ....+ mg per
m=.
7ssa! preparationTranser an accuratel! measured :olume "+ml% o
Oral 1olution to a :olumetric >as/& dissol:e in Diluting solution& and
dilute with Diluting solution to o(tain a solution ha:ing a concentration
o a(out ....+ mg o p!ridoxine h!drochloride per m=.
Procedure[NOTEUse pea/ areas where pea/ responses are
indicated.] 1eparatel! in0ect e;ual :olumes "a(out 4. F=% o the
1tandard preparation and the 7ssa! preparation into the
chromatograph& record the chromatograms& and measure the
responses or the ma0or pea/s. 2alculate the ;uantit!& in mg& o
2DC##NO<MC2l "p!ridoxine h!drochloride% in each m= o the Oral
1olution ta/en (! the ormula$
2"= E D%"r U E r 1%&
in which 2 is the concentration& in mg per m=& o U1P P!ridoxine
C!drochloride 81 in the 1tandard preparation& = is the la(eled amount&
in mg per m=& o p!ridoxine h!drochloride in the Oral 1olution ta/en& D
is the concentration& in mg per m=& o p!ridoxine h!drochloride in the
7ssa! preparation& (ased on the la(eled ;uantit! and the extent o
dilution& and r U and r 1 are the pea/ responses or p!ridoxine
h!drochloride o(tained rom the 7ssa! preparation and 1tandard
preparation& respecti:el!.
Assay for thiamine hydrochloride
Diluting solution& 'o(ile phase& and 2hromatographic s!stemProceed
as directed in the 7ssa! or niacin or niacinamide.
[N OTE Use low-actinic glassware throughout this procedure.]
Diluting solutionDissol:e 4, g o edetate disodium in #... m= o
water& and mix.
'o(ile phase'ix ..* m= o trieth!lamine& #,.. m= o glacial acetic
acid& and <,. m= o methanol& and dilute with ....D ' sodium #-
hexanesulonate to 4... m=. Iilter& and degas. 'a/e ad0ustments i
necessar! "see 1!stem 1uita(ilit! under 2hromatograph! 3 +4#5%.
2hromatographic s!stem "see 2hromatograph! 3 +4#5%The li;uid
chromatograph is e;uipped with a 4-.-nm detector and a *.+-mm A
4,-cm column that contains pac/ing =-. The >ow rate is a(out 4.. m=
per minute.
2hromatograph the 1tandard preparation& and record the pea/
responses as directed under Procedure$ the relati:e standard de:iation
or replicate in0ections is not more than 4..B.
1tandard preparationDissol:e an accuratel! weighed ;uantit! o U1P
Thiamine C!drochloride 81 in Diluting solution& and dilute
;uantitati:el!& and stepwise i necessar!& with Diluting solution to
o(tain a solution ha:ing a /nown concentration o a(out ....+ mg o
U1P Thiamine C!drochloride 81 per m=.
7ssa! preparationDissol:e an accuratel! measured :olume "<ml% o
Oral 1olution in Diluting solution& and dilute with Diluting solution to
o(tain a solution ha:ing a concentration o a(out ....+ mg o thiamine
h!drochloride per m=.
Procedure[NOTEUse pea/ areas where pea/ responses are
indicated.] 1eparatel! in0ect e;ual :olumes "a(out 4. F=% o the
1tandard preparation and the 7ssa! preparation into the
chromatograph& record the chromatograms& and measure the pea/
responses. 2alculate the ;uantit!& in mg& o 2
#4
C
#-
2lN
*
O1MC2l "thiamine
h!drochloride% in each m= o the Oral 1olution ta/en (! the ormula$
2"= E D%"r U E r 1%&
in which 2 is the concentration& in mg per m=& o U1P Thiamine
C!drochloride 81 in the 1tandard preparation& = is the la(eled amount&
in mg per m=& o thiamine h!drochloride in the Oral 1olution ta/en& D is
the concentration& in mg per m=& o thiamine h!drochloride in the
7ssa! preparation& (ased on the la(eled ;uantit! and the extent o
dilution& and r U and r 1 are the pea/ responses or thiamine
h!drochloride o(tained rom the 7ssa! preparation and 1tandard
preparation& respecti:el!.
[NOTE2ommerciall! a:aila(le atomic a(sorption standard solutions
or the minerals& where applica(le& ma! (e used where preparation o a
1tandard stoc/ solution is descri(ed in the ollowing 7ssa!s. Use
deioni9ed water where water is speci)ed. Nhere atomic a(sorption
spectrophotometr! is speci)ed in the 7ssa!& the 1tandard preparations
and the 7ssa! preparation ma! (e diluted ;uantitati:el! with the
sol:ent speci)ed& i necessar!& to !ield solutions o suita(le
concentrations adapta(le to the linear or wor/ing range o the
instrument.]
Assay for Ribofavin
[N OTE Use low-actinic glassware throughout this procedure.]
Diluting solutionDissol:e 4, g o edetate disodium in #... m= o
water& and mix.
'o(ile phase'ix ..* m= o trieth!lamine& #,.. m= o glacial acetic
acid& and <,. m= o methanol& and dilute with ....D ' sodium #-
hexanesulonate to 4... m=. Iilter& and degas. 'a/e ad0ustments i
necessar! "see 1!stem 1uita(ilit! under 2hromatograph! 3 +4#5%.
1tandard preparationDissol:e an accuratel! weighed ;uantit! o U1P
8i(o>a:in 81 in Diluting solution& (! heating i necessar!& in a
:olumetric >as/& and dilute ;uantitati:el!& and stepwise i necessar!&
with Diluting solution to o(tain a solution ha:ing a /nown
concentration o a(out 4 Fg per m=.
7ssa! preparationTranser a :olume o Oral 1olution "#ml%& accuratel!
measured& to a #..ml :olumetric >as/& dissol:e in Diluting solution&
and dilute with Diluting solution to o(tain a solution ha:ing a /nown
concentration o a(out 4 Fg o ri(o>a:in per m=.
2hromatographic s!stemThe li;uid chromatograph is e;uipped with
a 4-.-nm detector and a *.+-mm A 4,-cm column that contains
pac/ing =-. The >ow rate is a(out 4.. m= per minute.
2hromatograph the 1tandard preparation& and record the pea/
responses as directed under Procedure$ the relati:e standard de:iation
or replicate in0ections is not more than 4..B.
Procedure 1eparatel! in0ect e;ual :olumes "a(out 4. F=% o the
1tandard preparation and the 7ssa! preparation into the
chromatograph& record the chromatogram& and measure the responses
o pea/s or ri(o>a:in and ri(o>a:in-,O-phosphate. The relati:e
retention times are a(out ..#D or ri(o>a:in-,P-phosphate and #.. or
ri(o>a:in. 2alculate the ;uantit!& in mg& o ri(o>a:in "2
#-
C
4.
N
*
O
+
% in
each m= o the Oral 1olution ta/en (! the ormula$
#.*G< 2"= E D%"r U E r 1%&
in which #.*G< is the actor or con:erting ri(o>a:in-,P-phosphate to
8i(o>a:in& 2 is the concentration& in mg per m=& o U1P 8i(o>a:in 81 in
the 1tandard preparation& = is the la(eled amount& in mg per m=& o
ri(o>a:in in the Oral 1olution ta/en& D is the concentration& in mg per
m=& o ri(o>a:in in the 7ssa! preparation& (ased on the la(eled
;uantit! and the extent o dilution& r U is the pea/ response or
ri(o>a:in-,O-phosphate o(tained rom the 7ssa! preparation& and r 1
is the pea/ response or ri(o>a:in o(tained rom the 1tandard
preparation.
Assay for Cyanocobalamin
7ssa! preparationTranser an accuratel! measured :olume o Oral
1olution& e;ui:alent to a(out #.. mg o c!anoco(alamin& to an
appropriate :essel containing& or each m= o the Oral 1olution ta/en&
4, m= o an a;ueous extracting solution prepared 0ust prior to use to
contain& in each #.. m=& #.4G g o di(asic sodium phosphate& #.# g o
anh!drous citric acid& and #.. g o sodium meta(isul)te. 7utocla:e the
mixture at #4#? or #. minutes. 7llow an! undissol:ed particles o the
extract to settle& and )lter or centriuge i necessar!. Dilute an ali;uot
o the clear solution with water to o(tain a )nal solution containing
:itamin J#4 acti:it! approximatel! e;ui:alent to that o the 1tandard
preparation.
1tandard c!anoco(alamin stoc/ solutionDissol:e an accuratel!
weighed ;uantit! o U1P 2!anoco(alamin 81 in 4,B alcohol to o(tain a
solution ha:ing a /nown concentration o a(out #.. mg o
c!anoco(alamin per m=. 1tore in a rerigerator.
1tandard preparationDilute a suita(le :olume o 1tandard
c!anoco(alamin stoc/ solution with water to a measured :olume such
that ater the incu(ation period as descri(ed under Procedure the
diQerence in transmittance (etween the inoculated (lan/ and the ,..-
m= le:el o the 1tandard preparation is not less than that which
corresponds to a diQerence o #.4, mg in dried cell weight. This
concentration usuall! alls (etween ...# ng and ...* ng per m= o
1tandard preparation. Prepare this solution resh or each assa!.
7cid-h!drol!9ed casein solutionPrepare as directed under 7ssa! or
calcium pantothenate "'ethod 4%.
7sparagine solutionDissol:e 4.. o =-asparagine in water to ma/e
4.. m=. 1tore under toluene in a rerigerator.
7denine-guanine-uracil solutionPrepare as directed under 7ssa! or
calcium pantothenate "'ethod 4%.
Ranthine solution1uspend ..4. g o xanthine in <. to *. m= o water&
heat to a(out -.?& add +.. m= o + N ammonium h!droxide& and stir
until the solid is dissol:ed. 2ool& and add water to ma/e 4.. m=. 1tore
under toluene in a rerigerator.
1alt solution 7Dissol:e #. g o mono(asic potassium phosphate and
#. g o di(asic potassium phosphate in water to ma/e 4.. m=& and
add 4 drops o h!drochloric acid. 1tore this solution under toluene.
1alt solution JDissol:e *.. g o magnesium sulate& ..4. g o sodium
chloride& ..4. g o errous sulate& and ..4. g o manganese sulate in
water to ma/e 4.. m=& and add 4 drops o h!drochloric acid. 1tore this
solution under toluene.
Pol!sor(ate D. solutionDissol:e 4. g o pol!sor(ate D. in alcohol to
ma/e 4.. m=. 1tore in a rerigerator.
6itamin solution @Dissol:e #. mg o ri(o>a:in& #. mg o thiamine
h!drochloride& #.. mg o (iotin& and 4. mg o niacin in ...4 N glacial
acetic acid to ma/e *.. m=. 1tore under toluene& protected rom light&
in a rerigerator.
6itamin solution @@Dissol:e 4. mg o p-amino(en9oic acid& #. mg o
calcium pantothenate& *. mg o p!ridoxine h!drochloride& *. mg o
p!ridoxal h!drochloride& D mg o p!ridoxamine dih!drochloride& and 4
mg o olic acid in dilute neutrali9ed alcohol "# in *% to ma/e *.. m=.
1tore& protected rom light& in a rerigerator.
Jasal medium stoc/ solutionPrepare the medium according to the
ollowing ormula and directions. 7 deh!drated mixture containing the
same ingredients ma! (e used pro:ided that& when constituted as
directed in the la(eling& it !ields a medium compara(le to that
o(tained rom the ormula gi:en herein.
7dd the ingredients in the order listed& careull! dissol:ing the c!stine
and tr!ptophane in the h!drochloric acid (eore adding the next eight
solutions in the resulting solution. 7dd #.. m= o water& mix& and
dissol:e the dextrose& sodium acetate& and ascor(ic acid. Iilter& i
necessar!& add the Pol!sor(ate D. solution& ad0ust the solution with # N
sodium h!droxide to a pC o (etween ,., and +..& and add puri)ed
water to ma/e 4,. m=.
=-2!stine ..# g
=-Tr!ptophane ..., g
# N C!drochloric acid #. m=
7denine-guanine-uracil solution , m=
Ranthine solution , m=
6itamin solution @ #. m=
6itamin solution @@ #. m=
1alt solution 7 , m=
1alt solution J , m=
7sparagine solution , m=
7cid-h!drol!9ed casein solution 4, m=
Dextrose& anh!drous #. g
1odium acetate& anh!drous , g
7scor(ic acid # g
Pol!sor(ate D. solution , m=
Tomato 0uice preparation2entriuge commerciall! canned tomato
0uice so that most o the pulp is remo:ed. 1uspend a(out , g per liter
o anal!tical )lter-aid in the supernatant li;uid& and )lter& with the aid
o reduced pressure& through a la!er o the )lter-aid. 8epeat& i
necessar!& until a clear& straw-colored )ltrate is o(tained. 1tore under
toluene in a rerigerator.
2ulture medium[NOTE7 deh!drated mixture containing the same
ingredients ma! (e used pro:ided that& when constituted as directed in
the la(eling& it !ields a medium e;ui:alent to that o(tained rom the
ormula gi:en herein.] Dissol:e ..-, g o !east extract& ..-, g o dried
peptone& #.. g o anh!drous dextrose& and ..4. g o mono(asic
potassium phosphate in +. to -. m= o water. 7dd #. m= o Tomato
0uice preparation and # m= o Pol!sor(ate D. solution. 7d0ust with # N
sodium h!droxide to a pC o +.D& and add water to ma/e #.. m=. Place
#.-m= portions o the solution in test tu(es& and plug with cotton.
1terili9e the tu(es and contents in an autocla:e at #4#? or #, minutes.
2ool as rapidl! as possi(le to a:oid color ormation resulting rom
o:erheating the medium.
1uspension mediumDilute a measured :olume o Jasal medium
stoc/ solution with an e;ual :olume o water. Place #.-m= portions o
the diluted medium in test tu(es. 1terili9e& and cool as directed or
2ulture medium.
1toc/ culture o lacto(acillus leichmanniiTo #.. m= o 2ulture
medium add #.. to #., g o agar& and heat the mixture on a steam
(ath& with stirring& until the agar dissol:es. Place #.-m= portions o the
hot solution in test tu(es& co:er the tu(es& sterili9e at #4#? or #,
minutes in an autocla:e "exhaust line temperature%& and allow the
tu(es to cool in an upright position. @noculate three or more o the
tu(es (! sta( transer o a pure culture o =acto(acillus leichmannii.4
[NOTEJeore )rst using a resh culture in this assa!& ma/e not ewer
than #. successi:e transers o the culture in a 4-wee/ period.]
@ncu(ate or #+ to 4* hours at a temperature (etween <.? and *.?
held constant to within H..,?. 1tore in a rerigerator.
Prepare resh sta( cultures at least three times each wee/& and do not
use them or preparing the @noculum i more than * da!s old. The
acti:it! o the microorganism can (e increased (! dail! or twice-dail!
transer o the sta( culture& to the point where de)nite tur(idit! in the
li;uid @noculum can (e o(ser:ed 4 to * hours ater inoculation. 7 slow-
growing culture seldom gi:es a suita(le response cur:e and ma! lead
to erratic results.
@noculum[NOTE7 ro9en suspension o =acto(acillus leichmannii
ma! (e used as the stoc/ culture& pro:ided it !ields an inoculum
compara(le to a resh culture.] 'a/e a transer o cells rom the 1toc/
culture o lacto(acillus leichmannii to 4 sterile tu(es containing #. m=
o the 2ulture medium each. @ncu(ate these cultures or #+ to 4* hours
at a temperature (etween <.? and *.? held constant to within H..,?.
Under aseptic conditions centriuge the cultures& and decant the
supernatant li;uid. 1uspend the cells rom the culture in , m= o sterile
1uspension medium& and com(ine. Using sterile 1uspension medium&
ad0ust the :olume so that a # in 4. dilution in saline T1 produces -.B
transmittance when read on a suita(le spectrophotometer that has
(een set at a wa:elength o ,<. nm& e;uipped with a #.-mm cell& and
read against saline T1 set at #..B transmittance. Prepare a # in *..
dilution o the ad0usted suspension using Jasal medium stoc/ solution.
The cell suspension so o(tained is the @noculum. [NOTEThis dilution
ma! (e altered& when necessar!& to o(tain the desired test response.]
2ali(ration o spectrophotometer2hec/ the wa:elength o the
spectrophotometer periodicall!& using a standard wa:elength cell or
other suita(le de:ice. Jeore reading an! tests& cali(rate the
spectrophotometer or .B and #..B transmittance& using water and
with the wa:elength set at ,<. nm.
ProcedureJecause o the high sensiti:it! o the test organism to
minute amounts o :itamin J#4 acti:it! and to traces o man!
cleansing agents& cleanse meticulousl! (! suita(le means& ollowed
preera(l! (! heating at 4,.? or 4 hours& hard-glass test tu(es& a(out
4. A #,. mm in si9e& and other necessar! glassware.
To test tu(es add& in duplicate& #.. m=& #., m=& 4.. m=& <.. m=& *.. m=&
and ,.. m=& respecti:el!& o the 1tandard preparation. To each o these
tu(es and to our similar empt! tu(es add ,.. m= o Jasal medium
stoc/ solution and suLcient water to ma/e #. m=.
To similar test tu(es add& in duplicate& #.. m=& #., m=& 4.. m=& <.. m=&
and *.. m=& respecti:el!& o the 7ssa! preparation. To each tu(e add
,.. m= o Jasal medium stoc/ solution and suLcient water to ma/e #.
m=. Place one complete set o 1tandard and 7ssa! tu(es together in
one tu(e rac/ and the duplicate set in a second rac/ or section o a
rac/& preera(l! in random order.
2o:er the tu(es to pre:ent (acterial contamination& and sterili9e in an
autocla:e at #4#? or , minutes& arranging to reach this temperature in
not more than #. minutes (! preheating the autocla:e& i necessar!.
2ool as rapidl! as possi(le to a:oid color ormation resulting rom
o:erheating the medium. Ta/e precautions to maintain uniormit! o
sterili9ing and cooling conditions throughout the assa!& since pac/ing
the tu(es too closel! in the autocla:e or o:erloading it ma! cause
:ariation in the heating rate.
7septicall! add .., m= o @noculum to each tu(e so prepared& except
two o the our containing no 1tandard preparation "the uninoculated
(lan/s%. @ncu(ate the tu(es at a temperature (etween <.? and *.?
held constant to within H..,?& or #+ to 4* hours.
Terminate growth (! heating to a temperature not lower than D.? or ,
minutes. 2ool to room temperature. 7ter agitating its contents& place
the container in a spectrophotometer that has (een set at a
wa:elength o ,<. nm& and read the transmittance when a stead!
state is reached. This stead! state is o(ser:ed a ew seconds ater
agitation when the reading remains constant or <. seconds or more.
7llow approximatel! the same time inter:al or the reading on each
tu(e.
Nith the transmittance set at #..B or the uninoculated (lan/& read
the transmittance o the inoculated (lan/. @ the diQerence is greater
than ,B or i there is e:idence o contamination with a oreign
microorganism& disregard the results o the assa!.
Nith the transmittance set at #..B or the uninoculated (lan/& read
the transmittance o each o the remaining tu(es. Disregard the results
o the assa! i the slope o the standard cur:e indicates a pro(lem with
sensiti:it!.
2alculationPrepare a standard concentration-response cur:e (! the
ollowing procedure. Test or and replace an! a(errant indi:idual
transmittances. Ior each le:el o the 1tandard& calculate the response
rom the sum o the duplicate :alues o the transmittances "1% as the
diQerence& ! S 4... - 1. Plot this response on the ordinate o cross-
section paper against the logarithm o the m= o 1tandard preparation
per tu(e on the a(scissa& using or the ordinate either an arithmetic or
a logarithmic scale& whiche:er gi:es the (etter approximation to a
straight line. Draw the straight line or smooth cur:e that (est )ts the
plotted points.
2alculate the response& !& adding together the two transmittances or
each le:el o the 7ssa! preparation. 8ead rom the standard cur:e the
logarithm o the :olume o the 1tandard preparation corresponding to
each o those :alues o ! that alls within the range o the lowest and
highest points plotted or the standard. 1u(tract rom each logarithm
so o(tained the logarithm o the :olume& in m=& o the 7ssa!
preparation to o(tain the diQerence& x& or each dosage le:el. 7:erage
the :alues o x or each o three or more dosage le:els to o(tain (ar"x%
S 'P& the log-relati:e potenc! o the 7ssa! preparation. Determine the
;uantit!& in Fg& o U1P 2!anoco(alamin 81 corresponding to the
c!anoco(alamin in the portion o oral solution ta/en or assa! (! the
e;uation$
antilog ' S antilog "'P T log 8%&
in which 8 is the num(er o Fg o c!anoco(alamin that was assumed to
(e present in each ml in the portion o oral solutino ta/en or assa!.
8eplication8epeat the entire determination at least once& using
separatel! prepared 7ssa! preparations. @ the diQerence (etween the
two log potencies ' is not greater than ...D& their mean& (ar"'%& is the
assa!ed log-potenc! o the test material "see 6itamin J #4 7cti:it!
7ssa! under Design and 7nal!sis o Jiological 7ssa!s 3###5%. @ the
two determinations diQer (! more than ...D& conduct one or more
additional determinations. Irom the mean o two or more :alues o '
that do not diQer (! more than ..#,& compute the mean potenc! o the
preparation under assa!.