CONFIDENTIALSmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com ANALYTICAL PROCEDURES_SUMMARY The anal%tical metho&s use& to test 'u(irocin )ree *ci& to s(eci+ication are the metho&s o+ the ,uro(ean Pharmaco(oeia- metho&s e.ui/alent to the ,uro(ean Pharmaco(oiea metho&s or in house metho&s# Table 1 Methods Used to Test Mupio!i" Fee A!id to Spe!i#i!atio" A"al$ti!al Method Method Details 0escri(tion 0escri(tion 1&enti+ication 1n+rare& a2sor(tion s(ectro(hotometr% 'u(irocin content 2% HP34 'u(irocin )ree *ci& 4ontent 2% HP34 (H Potentiometric &etermination o+ (H 5elate& Su2stances content 2% HP34 0rug 5elate& 1m(urities 2% HP34 S(eci+ic 6PT14*3 rotation 6(tical rotation Water content Water semi7micro &etermination 5esi&ual Sol/ents 2% 84 5esi&ual Sol/ent 2% 84 SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com DESCRIPTION O##i!ial e#ee"!e i" Peu% Ph& Eu& *s (er current Ph# ,ur monogra(h +or 'u(irocin# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com EUROPEAN P'ARMACOPOEIAL MONO(RAP' FOR INFRARED A)SORPTION SPECTROP'OTOMETRY O##i!ial e#ee"!e i" Peu% 4urrent Ph# ,ur *&*&*+& A)SORPTION SPECTROP'OTOMETRY, INFRARED 1n+rare& s(ectro(hotometers are use& +or recor&ing s(ectra in the region o+ 400079:0 cm ;1 <!#:71:#4 =m> or in some cases &o"n to !00 cm ;1 <:0 =m># APPARATUS S(ectro(hotometers +or recor&ing s(ectra consist o+ a suita2le light source- monochromator or inter+erometer an& &etector# )ourier trans+orm s(ectro(hotometers use (ol%chromatic ra&iation an& calculate the s(ectrum in the +re.uenc% &omain +rom the original &ata 2% )ourier trans+ormation# S(ectro(hotometers +itte& "ith an o(tical s%stem ca(a2le o+ (ro&ucing monochromatic ra&iation in the measurement region ma% also 2e use&# Normall% the s(ectrum is gi/en as a +unction o+ transmittance- the .uotient o+ the intensit% o+ the transmitte& ra&iation an& the inci&ent ra&iation# 1t ma% also 2e gi/en in a2sor2ance# The a2sor2ance <*> is &e+ine& as the logarithm to 2ase 10 o+ the reci(rocal o+ the transmittance <T>? SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com T @ 1 1o 1 1o @ intensit% o+ inci&ent ra&iation- 1 @ intensit% o+ transmitte& ra&iation# PREPARATION OF T'E SAMPLE FOR RECORDIN( )Y TRANSMISSION OR A)SORPTION Pre(are the su2stance 2% one o+ the +ollo"ing metho&s# Li-uids# ,xamine a li.ui& either in the +orm o+ a +ilm 2et"een ! (lates trans(arent to in+rare& ra&iation- or in a cell o+ suita2le (ath length- also trans(arent to in+rare& ra&iation# Li-uids o solids i" solutio"# Pre(are a solution in a suita2le sol/ent# 4hoose a concentration an& a (ath length o+ the cell "hich gi/e a satis+actor% s(ectrum# 8enerall%- goo& results are o2taine& "ith concentrations o+ 107100 gAl +or a (ath length o+ 0#:70#1 mm# *2sor(tion &ue to the sol/ent is com(ensate& 2% (lacing in the re+erence 2eam a similar cell containing the sol/ent use&# 1+ an )T715 instrument is use&- the a2sor(tion is com(ensate& 2% recor&ing the s(ectra +or the sol/ent an& the sam(le successi/el%# The sol/ent a2sor2ance- correcte& 2% a com(ensation +actor- is su2tracte& using calculation so+t"are# Solids# ,xamine soli&s &is(erse& in a suita2le li.ui& <mull> or in a soli& <hali&e &isc>- as a((ro(riate# 1+ (rescri2e& in the monogra(h- ma$e a +ilm o+ a molten mass 2et"een ! (lates trans(arent to in+rare& ra&iation# A& Mull Triturate a small .uantit% o+ the su2stance to 2e examine& "ith the minimum .uantit% o+ li.ui& (ara++in 5 or other suita2le li.ui&B :710 mg o+ SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com the su2stance to 2e examine& is usuall% su++icient to ma$e an a&e.uate mull using one &ro( o+ li.ui& (ara++in 5# 4om(ress the mull 2et"een ! (lates trans(arent to in+rare& ra&iation# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com )& Dis! Triturate 17! mg o+ the su2stance to 2e examine& "ith 007400 mg- unless other"ise s(eci+ie&- o+ +inel% (o"&ere& an& &rie& (otassium 2romi&e 5 or (otassium chlori&e 5# These .uantities are usuall% su++icient to gi/e a &isc o+ 1071: mm &iameter an& a s(ectrum o+ suita2le intensit%# 1+ the su2stance is a h%&rochlori&e- it is recommen&e& to use (otassium chlori&e 5# 4are+ull% grin& the mixture- s(rea& it uni+orml% in a suita2le &ie- an& su2mit it to a (ressure o+ a2out 800 'Pa <8 tCcm ;! ># )or su2stances that are unsta2le un&er normal atmos(heric con&itions or are h%grosco(ic- the &isc is (resse& in vacuo# Se/eral +actors ma% cause the +ormation o+ +ault% &iscs- such as insu++icient or excessi/e grin&ing- humi&it% or other im(urities in the &is(ersion me&ium or an insu++icient re&uction o+ (article siDe# * &isc is reEecte& i+ /isual examination sho"s lac$ o+ uni+orm trans(arenc% or "hen transmittance at a2out !000 cm ;1 <: =m> in the a2sence o+ a s(eci+ic a2sor(tion 2an& is less than 90 (ercent "ithout com(ensation- unless other"ise (rescri2e&# (ases# ,xamine gases in a cell trans(arent to in+rare& ra&iation an& ha/ing an o(tical (ath length o+ a2out 100 mm# ,/acuate the cell an& +ill to the &esire& (ressure through a sto(coc$ or nee&le /al/e using a suita2le gas trans+er line 2et"een the cell an& the container o+ the gas to 2e examine&# 1+ necessar% a&Eust the (ressure in the cell to atmos(heric (ressure using a gas trans(arent to in+rare& ra&iation <+or exam(le nitrogen 5 an& argon 5># To a/oi& a2sor(tion inter+erences &ue to "ater- car2on &ioxi&e or other atmos(heric gases- (lace in the re+erence 2eam- i+ (ossi2le- an i&entical cell that is either e/acuate& or +ille& "ith the gas trans(arent to in+rare& ra&iation# FOR RECORDIN( )Y DIFFUSE REFLECTANCE SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com Solids# Triturate a mixture o+ the su2stance to 2e examine& "ith +inel% (o"&ere& an& &rie& (otassium 2romi&e 5 or (otassium chlori&e 5# Fse a mixture containing a((roximatel% : (er cent o+ the su2stance- unless other"ise s(eci+ie&# 8rin& the mixture- (lace it in a sam(le cu( an& examine the re+lectance s(ectrum# The s(ectrum o+ the sam(le in a2sor2ance mo&e ma% 2e o2taine& a+ter mathematical treatment o+ the s(ectra 2% the Ku2el$a7'un$ +unction# FOR RECORDIN( )Y ATTENUATED TOTAL REFLECTION *ttenuate& total re+lection <inclu&ing multi(le re+lection> in/ol/es light 2eing re+lecte& internall% 2% a transmitting me&ium- t%(icall% +or a num2er o+ re+lections# Ho"e/er- se/eral accessories exist "here onl% one re+lection occurs# Pre(are the su2stance as +ollo"s# Place the su2stance to 2e examine& in close contact "ith an internal re+lection element <15,> such as &iamon&- germanium- Dinc seleni&e- thallium 2romi&e7thallium io&i&e <K5S7:> or another suita2le material o+ high re+racti/e in&ex# ,nsure close an& uni+orm contact 2et"een the su2stance an& the "hole cr%stal sur+ace o+ the internal re+lection element- either 2% a((l%ing (ressure or 2% &issol/ing the su2stance in an a((ro(riate sol/ent- then co/ering the 15, "ith the o2taine& solution an& e/a(orating to &r%ness# ,xamine the attenuate& total re+lectance <*T5> s(ectrum# IDENTIFICACION USIN( REFERENCE SU)STANCES Pre(are the su2stance to 2e examine& an& the re+erence su2stance 2% the same (roce&ure an& recor& the s(ectra 2et"een 400079:0 cm ;1 <!#:71:#4 =m> un&er the same o(erational con&itions# The transmission minima <a2sor(tion maxima> in the s(ectrum o2taine& "ith the su2stance to 2e examine& corres(on& in (osition an& relati/e siDe to those in the s(ectrum o2taine& "ith the re+erence su2stance <CRS). SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com When the s(ectra recor&e& in the soli& state sho" &i++erences in the (ositions o+ the transmission minima <a2sor(tion maxima>- treat the su2stance to 2e examine& an& the re+erence su2stance in the same manner so that the% cr%stallise or are (ro&uce& in the same +orm- or (rocee& as (rescri2e& in the monogra(h- then recor& the s(ectra# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com IDENTIFICATION USIN( REFERENCE SPECTRA Co"tol o# esolutio" pe#o.a"!e# )or instruments ha/ing a monochromator- recor& the s(ectrum o+ a (ol%st%rene +ilm a((roximatel% : =m in thic$ness# The &i++erence x <see )igure !#!#!4#7 1> 2et"een the (ercentage transmittance at the transmission maximum * at !8G0 cm ;1 <#48 =m> an& that at the transmission minimum B at !849#: cm ;1 <#:1 =m> must 2e greater than 18# The &i++erence % 2et"een the (ercentage transmittance at the transmission maximum 4 at 1:89 cm ;1 <9#!9 =m> an& that at the transmission minimum 0 at 1:8 cm ;1 <9#! =m> must 2e greater than 10# )or )ourier7trans+orm instruments- use suita2le instrument resolution "ith the a((ro(riate a(o&isation (rescri2e& 2% the manu+acturer# The resolution is chec$e& 2% suita2le means- +or exam(le 2% recor&ing the s(ectrum o+ a (ol%st%rene +ilm a((roximatel% : =m in thic$ness# The &i++erence 2et"een the a2sor2ances at the a2sor(tion minimum at !8G0 cm ;1 an& the a2sor(tion maximum at !849#: cm ;1 is greater than 0## The &i++erence 2et"een the a2sor2ances at the a2sor(tion minimum at 1:89 cm ;1 an& the a2sor(tion maximum at 1:8 cm ;1 is greater than 0#08# # SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com Fi/ue *&*&*+&01& H T%(ical s(ectrum o+ (ol%st%rene use& to /eri+% the resolution (er+ormance Ieri+ication o+ the "a/e7num2er scale# The "a/e7num2er scale ma% 2e /eri+ie& using a (ol%st%rene +ilm- "hich has transmission minima <a2sor(tion maxima> at the "a/e num2ers <in cm J1 > sho"n in Ta2le *&*&*+&01& Ta2le !#!#!4#71# H Transmission minima an& acce(ta2le tolerances o+ a (ol%st%rene +ilm SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com Transmission minima <cm ;1 > *cce(ta2le tolerance <cm 71 > 'onochromator instruments )ourier7trans+orm instruments 090#0 K 1#: K 1#0 !849#: K !#0 K 1#0 1901#! K 1#0 K 1#0 1:8#0 K 1#0 K 1#0 11:4#: K 1#0 K 1#0 10!8# K 1#0 K 1#0 Method# Pre(are the su2stance to 2e examine& accor&ing to the instructions accom(an%ing the re+erence s(ectrumAre+erence su2stance# Fsing the o(erating con&itions that "ere use& to o2tain the re+erence s(ectrum- "hich "ill usuall% 2e the same as those +or /eri+%ing the resolution (er+ormance- recor& the s(ectrum o+ the su2stance to 2e examine&# The (ositions an& the relati/e siDes o+ the 2an&s in the s(ectrum o+ the su2stance to 2e examine& an& the re+erence s(ectrum are concor&ant in the ! s(ectra# Co.pe"satio" #o 1ate 2apou a"d at.osphei! !abo" dio3ide# )or )ourier7trans+orm instruments- s(ectral inter+erence +rom "ater /a(our an& car2on &ioxi&e is com(ensate& using suita2le algorithms accor&ing to the manu+acturerLs instructions# *lternati/el%- s(ectra can 2e ac.uire& using suita2le (urge& instruments or ensuring that sam(le SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com an& 2ac$groun& single 2eam s(ectra are ac.uire& un&er exactl% the same con&itions# IMPURITIES IN (ASES )or the anal%sis o+ im(urities- use a cell trans(arent to in+rare& ra&iation an& o+ suita2le o(tical (ath length <+or exam(le- 17!0 m># )ill the cell as (rescri2e& un&er 8ases# )or &etection an& .uanti+ication o+ the im(urities- (rocee& as (rescri2e& in the monogra(h# MUPIROCIN FREE ACID CONTENT )Y 'PLC O##i!ial e#ee"!e i" Peu? 1n7house metho& Pepaatio" o# Solutio"s Mobille Phase Mobile Phase Potassium &ih%&rogen (hos(hate 0#0!: ' + &iso&ium h%&rogen (hos(hate 0#0!: '# 0issol/e #9 g o+ KH!P64 an& #: g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at 1!0 K ! M4- in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Potassium &ih%&rogen (hos(hate 0#008G ' + &iso&ium h%&rogen (hos(hate 0#00 '# 0issol/e 1#18 g o+ KH!P64 an& 4#0 g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at 1!0 K ! M4- in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# 0iso&ium tetra2orate 0#01 '# 0issol/e #80 g o+ Na!B46G-10H!6 in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Store (rotecte& +rom atmos(heric car2on &ioxi&e# So&ium car2onate 0#0!: ' + so&ium h%&rogen car2onate 0#0!: '# 0issol/e !#94 g o+ Na!46 an& !#09 g o+ NaH46 in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Store (rotecte& +rom atmos(heric car2on &ioxi&e# 4alcium h%&roxi&e- saturate& at !: M4# Sha$e an excess o+ SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com calcium h%&roxi&e 5 "ith car2on &ioxi&e7+ree "ater 5 an& &ecant at !: M4# Store (rotecte& +rom atmos(heric car2on &ioxi&e# ST65*8, Store 2u++er solutions in suita2le chemicall% resistant- tight containers- such as t%(e 1 glass 2ottles or (lastic containers suita2le +or a.ueous solutions Pepaatio" o# Sa.ple Solutio" *ccuratel% "eigh 4:7:: mg 'u(irocin )ree *ci& into a !00 m3 /olumetric Nas$# 0issol/e the material in : m3 o+ methanol# ,nsure com(lete &issolution- using sonication i+ necessar%# 0ilute to /olume using 0#1 ' ammonium acetate solution (H :#G an& mix thoroughl%# Pepaatio" o# Sta"dad Solutio" *ccuratel% "eigh 4:7:: mg 'u(irocin )ree *ci& <or :0790 mg calcium salt> into a !00 m3 /olumetric Nas$# 0issol/e the material in : m3 o+ methanol# ,nsure com(lete &issolution- using sonication i+ necessar%# 0ilute to /olume using 0#1 ' ammonium acetate solution (H :#G an& mix thoroughl%# Cho.ato/aphi! Paa.etes 4olumn? *((rox !: cm containing a suita2le 2on&e& 48 silica (ac$ing <e#g# Oor2ax 48- 0u(ont 3t&> 'o2ile (hase? 0#1 ' ammonium acetate <(H :#G>? TH) <98?!> )lo" rate? 1#0 m3Amin 0etection? FI at !0 nm *ttenuation? *2out 0#: *F)S 1nEection Iolume? 10 =3 Po!edue Set u( the HP34 s%stem using the instrument (arameters &escri2e& an& ensure the s%stem suita2ilit% criteria are met# 1nEect 10 =3 ali.uots o+ sam(le an& stan&ar& solutions an& measure the (ea$ areas# The SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com retention time o+ 'u(irocin an& &rug7relate& im(urities are gi/en in Ta2le ! Table *& T$pi!al Rete"tio" Ti.es Co.po"e"t Mi"utes 0ea& /olume 1#0 B53 :9G98 <Pseu&omonic aci& )> #8 B53 !!89 <Pseu&omonic aci& 0> 9#G 'u(irocin 8#9 Cal!ulatio" 'u(irocin content <P> @ Sam(le area x Stan&ar& Wt x P St& Purit% Stan&ar& *rea x Sam(le Wt S$ste. Suitabilit$ • )rom six re(licate inEections o+ the stan&ar&- calculate the relati/e stan&ar& &e/iation o+ the (ea$ area# RSD @ Stan&ar& &e/iation o+ the (ea$ area x 100 *ritmetic mean o+ the (ea$ area The 5S0 shoul& not 2e greater than !P# • 4alculate the resolution +actor 2et"een (seu&omonic aci& 0 <B53 !!89> an& 'u(irocin- the tailing +actor o+ 'u(irocin an& the num2er o+ theoretical (lates using a solution o+ the stan&ar& <Note? 1n the a2sence o+ a la2orator% stan&ar& containing a measura2le amount o+ (seu&omonic aci& 0- a reasona2l% (ure s(ecimen o+ 'u(irocin containing a2out P (seu&ominic aci& 0 ma% 2e su2stitute& in the s%stem suita2ilit% tests># SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com 5esolution +actor <5> @ ! <T ! J T 1 > W ! + W 1 Where T 1 - T ! @ 5etention time o+ the ! (ea$s W 1 - W ! @ Basal (ea$ "i&ths o+ the ! (ea$s The resolution +actor shoul& not 2e less than #:# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com Tailing +actor <T> @ W 0#0: !) Where? W4&45 @ Pea$ "i&th o+ 'u(irocin at :P (ea$ height# ) @ Wi&th along lea&ing e&ge o+ (ea$ to (oint 2eneath (ea$ a(ex along :P (ea$ height time# The tailing +actor shoul& not 2e greater than 1#:# Num2er o+ theoretical (lates <N> @ :#:4: 1 ! W 0#: Where? T @ 5etention time o+ 'u(irocin W 0#: @ Pea$ "i&th o+ 'u(irocin at hal+ (ea$ height# Num2er o+ theoretical (lates shoul& not 2e less than 4000# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com EUROPEAN P'ARMACOPOEIAL MONO(RAP' FOR POTENTIOMETRIC DETERMINATION OF P' O##i!ial e#ee"!e i" Peu? 4urrent Ph# ,ur *&*&6 POTENTIOMETRIC DETERMINATION OF P' The (H is a num2er "hich re(resents con/entionall% the h%&rogen ion concentration o+ an a.ueous solution# )or (ractical (ur(oses- its &e+inition is an ex(erimental one# The (H o+ a solution to 2e examine& is relate& to that o+ a re+erence solution <(H> 2% the +ollo"ing e.uation? (h @ (H J , 7 ,: $ in "hich , is the (otential- ex(resse& in /olts- o+ the cell containing the solution to 2e examine& an& Es is the (otential- ex(resse& in /olts- o+ the cell containing the solution o+ $no"n (H <(Hs>- $ is the change in (otential (er unit change in (H ex(resse& in /olts- an& calculate& +rom the Nernst e.uation# Table *&*&6&01& 7 8alues o# 9 at di##ee"t te.peatues Te.peatue :;C< 9 :8< 1: 0#0:G! !0 0#0:8! !: 0#0:9! 0 0#0901 : 0#0911 SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com The (otentiometric &etermination o+ (H is ma&e 2% measuring the (otential &i++erence 2et"een ! a((ro(riate electro&es immerse& in the solution to 2e examine&? 1 o+ these electro&es is sensiti/e to h%&rogen ions <usuall% a glass electro&e> an& the other is the re+erence electro&e <+or exam(le- a saturate& calomel electro&e># Appaatus# The measuring a((aratus is a /oltmeter "ith an in(ut resistance at least 100 times that o+ the electro&es use&# 1t is normall% gra&uate& in (H units an& has a sensiti/it% such that &iscrimination o+ at least 0#0: (H unit or at least 0#00 I ma% 2e achie/e&# Method# Fnless other"ise (rescri2e& in the monogra(h- all measurements are ma&e at the same tem(erature <!07!: M4># Table *&*&6&0* sho"s the /ariation o+ (H "ith res(ect to tem(erature o+ a num2er o+ re+erence 2u++er solutions use& +or cali2ration# )or the tem(erature correction- "hen necessar%- +ollo" the manu+acturerLs instructions# The a((aratus is cali2rate& "ith the 2u++er solution o+ (otassium h%&rogen (hthalate <(rimar% stan&ar&> an& 1 other 2u++er solution o+ &i++erent (H <(re+era2l% one sho"n in Ta2le !#!##7!># The (H o+ a thir& 2u++er solution o+ interme&iate (H rea& o++ on the scale must not &i++er 2% more than 0#0: (H unit +rom the /alue corres(on&ing to this solution# 1mmerse the electro&es in the solution to 2e examine& an& ta$e the rea&ing in the same con&itions as +or the 2u++er solutions# When the a((aratus is in +re.uent use- chec$s must 2e carrie& out regularl%# 1+ not- such chec$s shoul& 2e carrie& out 2e+ore each measurement# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com *ll solutions to 2e examine& an& the re+erence 2u++er solutions must 2e (re(are& using car2on &ioxi&e7+ree "ater 5# PREPARATION OF REFERENCE )UFFER SOLUTIONS Potassiu. tetao3alate 4&45 M# 0issol/e 1!#91 g o+ 4 4 H K6 = -!H ! 6 in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Potassiu. h$do/e" tatate, satuated at *5 >C# Sha$e an excess o+ 4 + H : K6 9 /igorousl% "ith !abo" dio3ide0#ee 1ate R at !: M4# )ilter or &ecant# Pre(are imme&iatel% 2e+ore use# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com Potassiu. dih$do/e" phosphate 4&4*5 M ? disodiu. h$do/e" phosphate 4&4*5 M# 0issol/e #9 g o+ KH!P64 an& #: g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at 1!0 K ! M4- in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Potassiu. dih$do/e" phosphate 4&44=@ M ? disodiu. h$do/e" phosphate 4&4646 M# 0issol/e 1#18 g o+ KH!P64 an& 4#0 g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at 1!0 K ! M4- in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Disodiu. tetaboate 4&41 M# 0issol/e #80 g o+ Na!B46G- 10H!6 in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Store (rotecte& +rom atmos(heric car2on &ioxi&e# Sodiu. !abo"ate 4&4*5 M ? sodiu. h$do/e" !abo"ate 4&4*5 M# 0issol/e !#94 g o+ Na!46 an& !#09 g o+ NaH46 in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent# Store (rotecte& +rom atmos(heric car2on &ioxi&e# Cal!iu. h$do3ide, satuated at *5 >C# Sha$e an excess o+ calcium h%&roxi&e 5 "ith car2on &ioxi&e7+ree "ater 5 an& &ecant at !: M4# Store (rotecte& +rom atmos(heric car2on &ioxi&e# STORA(E Store 2u++er solutions in suita2le chemicall% resistant- tight containers- such as t%(e 1 glass 2ottles or (lastic containers suita2le +or a.ueous solutions SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com DRU( RELATED IMPURITIES )Y 'PLC Pepaatio" o# Solutio"s Pepaatio" o# Mobile Phase Pre(are a 0#1 ' ammonium acetate solution an& a&Eust to (H :#G "ith small amounts o+ acetic aci&# 'ix the 2u++er "ith tetrah%&ro+uran in the ratio G0P 2u++er? 0P tetrah%&ro+uran# )ilter an& &egas 2e+ore use# Pepaatio" o# Dilue"t Pre(are a 0#1 ' so&ium acetate solution an& a&Eust to (H 4#0 "ith small amounts o+ acetic aci&# 'ix the 2u++er "ith methanol in the ratio <:0?:0># Pepaatio" o# Sa.ple Solutio" *ccuratel% "eigh a2out 0#1 g o+ sam(le into a 10m3 +las$# 0issol/e in an& &ilute to /olume "ith &iluent# Pepaatio" o# Sta"dad Solutio" *ccuratel% "eight a2out 0#0: g o+ the la2orator% re+erence stan&ar& into a !00 m3 /olumetric +las$# 0issol/e in an& &ilute to /olume "ith &iluent# Cho.ato/aphi! Paa.etes 4olumn? *((rox !: cm containing a suita2le 2on&e& 48 silica (ac$ing <e#g# Oor2ax 48- 0u(ont 3t&> 'o2ile (hase? 0#1 ' ammonium acetate <(H :#G>? TH) <G0?0> )lo" rate? 1#0 m3Amin SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com 0etection? FI at !40 nm *ttenuation? 0#0! *F)S 1nEection Iolume? 10 =3 Po!edue Set u( the HP34 s%stem using the instrument (arameters &escri2e& an& ensure the s%stem suita2ilit% criteria are met# 1nEect six ali.uots o+ the stan&ar& solution +ollo"e& 2% an ali.uot <10 =3> o+ each sam(le# 'easure the (ea$ areas o+ all com(onents- exclu&ing those &ue to &iluent e++ects- in the sam(le solutions# 'easure the (ea$ areas o+ 'u(irocin in the stan&ar& solutions# T%(ical retention times are gi/en in Ta2le # Table 6& T$pi!al Rete"tio" Ti.es 4om(onent 'inutes 5elati/e to 'u(irocin B53 :9G98 <Pseu&omonic aci& )> B53 4:!94 <5earrangement 1> B53 4:!9: <5earrangement !> B53 !!89 <Pseu&omonic aci& 0> B53 !0!9G <Pseu&omonic aci& B> 'u(irocin <Pseu&omonic aci& *> B53 4:!99 B53 4:!9G B53 4:!98 B53 !0!8G <Pseu&omonic aci& 4> : G#: 8 9 10 1! 14 1: !: !G 0#4! 0#9 0#9G 0#G: 0#8 7 1#1G 1#!: !#08 !#!: Cal!ulatio" SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com P 1n&i/i&ual im(urit% @ * x St& Wt x S(l Iol x P St& Purit% *1 x St& Iol x S(l Wt P Total im(urities @ Q * x St& Wt x S(l Iol x P St& Purit% *1 x St& Iol x S(l Wt Where? * @ *rea o+ an in&i/i&ual im(urit% in the sam(le * 1 @ is the area o+ mu(irocin in the stan&ar& Q * is the sum o+ the areas o+ the in&i/i&ual im(urities in the sam(le# S$ste. Suitabilit$ • )rom at least six inEections o+ the stan&ar& calculate the relati/e stan&ar& &e/iations o+ the (ea$ area o+ 'u(irocin# 5S0 @ Stan&ar& &e/iation o+ the (ea$ area x 100 *rithmetic mean o+ the (ea$ area The 5S0 shoul& not 2e greater than :P# • 4alculate a resolution +actor 2et"een (seu&omonic aci& 0 <B53 !!89> an& 'u(irocin- the tailing o+ 'u(irocin an& the num2er o+ theoretical (lates using a solution o+ the stan&ar&# <Note? 1n the a2sence o+ a la2orator% stan&ar& containing a measura2le amount o+ (seu&ominic aci& 0- a reasona2l% (ure s(ecimen o+ 'u(irocin containing a2out P (sue&omonic aci& 0 ma% 2e su2stitute& in the s%stem suita2ilit% tests># SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com 5esolution +actor <5> @ ! <T ! J T 1 > W ! + W 1 Where? T 1 - T ! @ 5etention time o+ the ! (ea$s W 1 - W ! @ Basal (ea$ "i&ths o+ the ! (ea$s The resolution +actor shoul& not 2e less than #:# Tailing +actor <T> @ W 0#0: !) Where? W 0#0: @ Pea$ "i&th o+ 'u(irocin at :P (ea$ height ) @ Wi&th along lea&ing e&ge o+ (ea$ to (oint 2eneath (ea$ a(ex along :P (ea$ height line# Num2er o+ theoretical (lates <N> @ :#:4: 1 ! W 0#: Where? T @ retention time o+ 'u(irocin W 0#: @ (ea$ "i&th o+ 'u(irocin at hal+ (ea$ height# The tailing +actor shoul& not 2e greater than !#0# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com Num2er o+ theoretical (lates shoul& not 2e less than 000# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com EUROPEAN P'ARMACOPEIAL MONO(RAP' FOR OPTICAL ROTATION O##i!ial e#ee"!e i" Peu? 4urrent Ph# ,ur 6(tical rotation is the (ro(ert% &is(la%e& 2% chiral su2stances o+ rotating the (lane o+ (olarisation o+ (olarise& light# 6(tical rotation is consi&ere& to 2e (ositi/e <+> +or &extrorotator% su2stances <i#e# those that rotate the (lane o+ (olarisation in a cloc$"ise &irection> an& negati/e <;> +or lae/orotator% su2stances# The s(eci+ic o(tical rotation is the rotation RSmT t is the rotation- ex(resse& in ra&ians <ra&>- measure& at the tem(erature t an& at the "a/elength U gi/en 2% a 1 m thic$ness o+ li.ui& or a solution containing 1 $gAm o+ o(ticall% acti/e su2stance# )or (ractical reasons the s(eci+ic o(tical rotation RSmT t is normall% ex(resse& in millira&ians metre s.uare& (er $ilogram <mra&Cm ! C$g ;1 ># The Pharmaco(oeia a&o(ts the +ollo"ing con/entional &e+initions# The angle o+ o(tical rotation o+ a neat li.ui& is the angle o+ rotation S- ex(resse& in &egrees <M>- o+ the (lane o+ (olarisation at the "a/elength o+ the 07line o+ so&ium <U @ :89# nm> measure& at !0 M4 using a la%er o+ 1 &mB +or a solution- the metho& o+ (re(aration is (rescri2e& in the monogra(h# The s(eci+ic o(tical rotation RSmT !0 0 o+ a li.ui& is the angle o+ rotation S- ex(resse& in &egrees <M>- o+ the (lane o+ (olarisation at the "a/elength o+ the 07line o+ so&ium <U @ :89# nm> measure& at !0 M4 in the li.ui& su2stance to 2e examine&- calculate& "ith re+erence to a la%er o+ 1 &m an& &i/i&e& 2% the &ensit% ex(resse& in grams (er cu2ic centimetre# SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com The s(eci+ic o(tical rotation RSmT !0 0 o+ a su2stance in solution is the angle o+ rotation S- ex(resse& in &egrees <M>- o+ the (lane o+ (olarisation at the "a/elength o+ the 07line o+ so&ium <U @ :89# nm> measure& at !0 M4 in a solution o+ the su2stance to 2e examine& an& calculate& "ith re+erence to a la%er o+ 1 &m containing 1 gAml o+ the su2stance# The s(eci+ic o(tical rotation o+ a su2stance in solution is al"a%s ex(resse& "ith re+erence to a gi/en sol/ent an& concentration# 1n the con/entional s%stem a&o(te& 2% the Pharmaco(oeia the s(eci+ic o(tical rotation is ex(resse& 2% its /alue "ithout unitsB the actual units- &egree millilitres (er &ecimetre gram R<M>CmlC&m ;1 Cg ;1 T are un&erstoo&# The con/ersion +actor +rom the 1nternational S%stem to the Pharmaco(oeia s%stem is the +ollo"ing? RSmT t @ RST t x 0#1G4: 1n certain cases s(eci+ie& in the monogra(h the angle o+ rotation ma% 2e measure& at tem(eratures other than !0 M4 an& at other "a/elengths# The (olarimeter must 2e ca(a2le o+ gi/ing rea&ings to the nearest 0#01M# The scale is usuall% chec$e& 2% means o+ certi+ie& .uartD (lates# The linearit% o+ the scale ma% 2e chec$e& 2% means o+ sucrose solutions# Method# 0etermine the Dero o+ the (olarimeter an& the angle o+ rotation o+ (olarise& light at the "a/elength o+ the 07line o+ so&ium <U @ :89# nm> at !0 K 0#: M4# 'easurements ma% 2e carrie& out at other tem(eratures onl% "here the monogra(h in&icates the tem(erature correction to 2e ma&e to the measure& o(tical rotation# 0etermine the Dero o+ the a((aratus "ith the tu2e close&B +or li.ui&s the Dero is SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com &etermine& "ith the tu2e em(t% an& +or soli&s +ille& "ith the (rescri2e& sol/ent# 4alculate the s(eci+ic o(tical rotation using the +ollo"ing +ormulae# )or neat li.ui&s? RSmT !0 0 @ SVV l ρ!0 )or su2stances in solution? RSmT !0 0 @ 1000S l c "here c is the concentration o+ the solution in grams (er litre 4alculate the content c in grams (er litre or the content cWin (er cent mAm o+ a &issol/e& su2stance using the +ollo"ing +ormulae? S @ angle o+ rotation in &egrees rea& at !0 K 0#:M4- l @ length in &ecimetres o+ the (olarimeter tu2e- X!0 @ &ensit% at !0 M4 in grams (er cu2ic centimetre# )or the (ur(oses o+ the Pharmaco(oeia- &ensit% is re(lace& 2% relati/e &ensit% <!#!#:>- SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com *&5&1* AATER% SEMI0MICRO DETERMINATION The semi7micro &etermination o+ Water is 2ase& u(on the .uantitati/e reaction o+ "ater "ith sul+ur &ioxi&e an& io&ine in a suita2le anh%&rous me&ium in the (resence o+ a 2ase "ith su+Ycient 2u++ering ca(acit%# Appaatus The a((aratus consists o+ a titration /essel "ith? H ! i&entical (latinum electro&esB H tight inlets +or intro&uction o+ sol/ent an& titrantB H an inlet +or intro&uction o+ air /ia a &esiccantB H a sam(le inlet Ytte& "ith a sto((er or- +or li.ui&s- a se(tum# 1nlet s%stems +or intro&uction o+ &r% nitrogen or +or as(iration o+ sol/ents ma% also 2e Ytte&# The titration is carrie& out accor&ing to the instrument su((lierLs instructions# 4are is ta$en throughout the &etermination to a/oi& ex(osure o+ reagents an& sol/ents to atmos(heric moisture# The en&7 (oint is &etermine& using ! i&entical in&icator electro&es comrecte& to an electrical source that maintains 2et"een the electro&es either a constant current <!#!#9:# Ioltametric titration> or a constant /oltage <!#!#19# *m(erometric titration># Where &irect titration is use& <metho& *>- a&&ition o+ titrant causes either a &ecrease in /oltage "here constant current is maintaine& or an increase in current "here constant /oltage is maintaine&- until the en&7(oint is reache&# 1nstrmnents "ith automatic en&7(oint &etection are commonl% use&# Stan&ar&isation# To the titration /essel- a&& methanol 5- &rie& i+ necessar%- or the sol/ent recommen&e& 2% the su((lier o+ the titrant# Where a((lica2le +or the a((aratus use&- eliminate resi&ual "ater Yrom the measurement cell or carr% out a (re7titration# 1ntro&uce a suita2le amount o+ "ater in an a((ro(riate +orm <"ater 5 or a certiYe& SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com re+erence material> an& carr% out the titration- stirring +or the necessar% time# The "ater e.ui/alent is not less than 80 (er cent o+ that in&icate& 2% the su((lier# Stan&ar&ise the titrant 2e+ore the Yrst use an& at suita2le inter/als therea+ter# Fnless other"ise (rescri2e&- use 'etho& *# Method A# 1ntro&uce into the titration /essel methanol 5- or the sol/ent in&icate& in the monogra(h or recommen&e& 2% the su((lier o+ the titrant# Where a((lica2le +or the a((aratus use&- eliminate resi&ual "ater +rom the measurement cell or carr% out a (re7titration# 1ntro&uce the su2stance to 2e examine& ra(i&l% an& carr% out the titration- stirring +or the necessar% extraction time# Method )# 1ntro&uce into the titration /essel methanol 5- or the sol/ent in&icate& in the monogra(h or recommen&e& 2% the su((lier o+ the titrant# Where a((lica2le +or the a((aratus use&- eliminate resi&ual "ater +rom the measurement cell or carr% out a (re7titration# 1ntro&uce the su2stance to 2e examine& ra(i&l% an& in a suita2le state o+ &i/ision# *&& an accuratel% measure& /olume o+ the titrant- su+Ycient to gi/e an excess o+ a2out 1 m3 or the (rescri2e& /olume# *llo" to stan& (rotecte& +rom light +or 1 min or the (rescri2e& time- "ith stirring# Titrate the excess o+ reagent using methanol R or the (rescri2e& sol/ent- containing an accuratel% $no"n .uantit% o+ "ater# Suitabilit$ The accurac% o+ the &etermination "ith the chosen titrant must 2e /eri+ie& +or each su2stance to 2e examine&# The +ollo"ing (roce&ure- gi/en as an exam(le- is suita2le +or sam(les containing !#:7 !: mg o+ "ater# The "ater content o+ the su2stance to 2e examine& is &etermine& using the reagentAsol/ent s%stem chosen# Therea+ter- se.uential $no"n amounts o+ water R are a&&e& in an a((ro(riate +orm <at least : a&&itions> an& the cumulati/e "ater content &etermine& a+ter each SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com a&&ition# 4alculate the (ercentage reco/er% <r> at each (oint using the +ollo"ing ex(ression? W 1 @ amount o+ "ater a&&e&- in milligramsB W ! @ amount o+ "ater +oun&- in milligrams# 4alculate the regression line o+ the cumulati/e "ater &etermine& against the "ater a&&e&# 4alculate the slo(e <b>- the interce(t "ith the y7axis <a> an& the interce(t o+ the extra(olate& cali2ration line "ith the x7axis <d># 4alculate the (ercentage mean reco/er% <># 4alculate the (ercentage errors <e 1 an& e ! > using the +ollo"ing ex(ressions? a @ the y7axis interce(t- in milligrams o+ "aterB d @ the x7axis interce(t- in milligrams o+ "aterB M @ "ater content o+ the su2stance- in milligrams o+ "ater# The reagentAsol/ent s%stem is consi&ere& to 2e acce(ta2le i+? H Ze 1 Z an& Ze ! Z are not greater than !#: (er centB H b is 2et"een 0#9G: an& 1#0!: SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3 CONFIDENTIAL SmithKline Beecham Pharmaceuticals Worthing West Sussex BN14 8QH T +44 190 8!! 000 """#gs$#com RESIDUAL SOL8ENTS )Y (C O##i!ial e#ee"!e i" Peu% I"0house .ethod Sa.ple a"d Sta"dad Pepaatio" Sta"dad Pepaatio" To a :0 m3 /olumetric +las$ a&& a2out 40 m3 o+ !7methox%ethanol# *&& !: =3 o+ each o+ the +ollo"ing sol/ents? He(tane 'eth%l iso2ut%l $etone ,th%l acetate *cetone 'a$e to /olume "ith ! methox%ethanol# Sa.ple Pepaatio" Weigh a2out 1g o+ sam(le into a 10m3 /olumetric +las$- &issol/e an& &ilute to /olume "ith ! methox%ethanol# Po!edue SmithKline Beecham Limited Registered in England and Wales No. 2337959 Registered Ofce 9! "reat West Road# Brent$ord %iddlese&# 'W 9"3