USPXXII
`
`Chemical Tests / Antimicrobial Agents-Content
`
`(341)
`
`1531
`
`of the amount added and which is not exceeded. An example of
`such a label statement i.s "-- (unit) added as preservative."
`[NOTE-"- - (unit)" would be a number followed by the unit
`of measurement, e.g., 0.015 mg per mL or 0.1%.J
`The most commonly used aients include the two mercurials,
`phenylmercuric nitrate and thimcrosal, the four homologous es(cid:173)
`ters of p-hydroxybenzoic acid, phenol, benzyl alcohol, and chi~
`robut.anol The methods for the first two named are polar~
`graphic, while quantitative gas chromatography is employed in
`tbe determination of the other ageots.
`
`GENERAL GAS CHROMATOGRAPHIC
`METHOD
`The general procedure set forth in 'the following paragraphs is
`applicable to the quantitative determination of benzyl alcohol,
`chlarobutanol, phenol, and the methyl, ethyl, propyl, and butyl
`esters of p-hydroxybenzoic acid, the latter being treated as a
`group, the individual members of which, if present, are capable
`of separate determination. Prepare the Internal Standard Sa(cid:173)
`lution and the Standard PreparaJion for each agent as directed
`individually below. Unless otherwise directed below, prepare the
`Test Preparar/Qn from accurately mea,ured portions of the In(cid:173)
`ternal Standllrd Solution a.nd the sample under test, of such size
`that the concentration of the agent and the composition of the
`solvent correspond closely to the concentration and composition
`of the Standard Preparation. Suggested operating parameters
`of the gas chromatograph apparatus are given in the accompa(cid:173)
`nying table, the carrier gas being helium or nitrogen, and the
`detector being the flame-ioni.zation type.
`
`Benzyl Alcohol
`lllteraal Standard Solution-Dissolve about 380 rng of phenol
`in 10 mt of methanol contained i:11 is 200-mL volumetric flask.
`Add water to volume, aod mix.
`Standard Preparatioo-Dissolve about 180 mg of benzyl al(cid:173)
`cohol, accurately weighed, in 20.0 mL of methanol contained in
`a 100-mL volumetric flask. Add Imernal Standard Solution to
`volume, and mix.
`Proce4ure-Using 5-µL portions of the Standard Preparation
`and the Te.rt Preparation, record their gas chromatograms w:itb
`the apparatus adJwted to the parameters set forth In the accom(cid:173)
`panymg table. Measure the areas under the peaks for benzyl
`aloo~ol and _phen?l of the chromatogram for _the St01f,d'!f'd Prep(cid:173)
`aration, de.ugnatmg them P 1 and·P 1, respectively. S1milarly, do(cid:173)
`termine the corresponding values p 1 and p2 for the Test Prepa(cid:173)
`ration. Calculate the content, in mg per mL, of bcnzyl aleobol
`(C7H80) in the specimen taken by the formula:
`lOO(C/Y)(pif pu(P,J Pi),
`in which C is the concentration, in mg per mL, of benzyl alcohol
`in the Standard Preparation, and V is the volume, in mL, of the
`specimen under test used in preparing each 100 mL of the Test
`PreparaJion.
`
`Cblorobutanol
`Internal Standard Solution-Dissolve about 130 mg of benz(cid:173)
`aldebydc in S mL of methanol contained in a 100-mL volumetric
`flask. Add water to volume, and mix.
`Standard Preparatiott-Dissolvc about 500 mg of anhydrous
`chlorobutanol, accurately weighed, in 5 mL of methanol con(cid:173)
`tained in a 100-mL volumetric ilask. Alld water to volume, and
`mill- Pipet 2 mL or I.his solution and : mL of the Internal Stan(cid:173)
`dard Soluzion into a So-mL volumetric flask, add dilute methanol
`( 1 in 20) to volume, and mix.
`Procedure-Using 5-µ.L portions of the Standard Preparation
`and thci Te1t Preparation, record their gas chromatograms with
`tbe apparatll8 adjusted to the parameters set forth in the accom(cid:173)
`panymg table. Measure the areas under the peaks for chloro(cid:173)
`butanol and benzaldehyde of the chromatogram for the Standard
`Preparation, designating t!tem PI and P1, respectively. Similarly,
`determine the corresponding values p 1 and p. for the Test Prep-
`
`Suggested Opentiog Parameters of Gas
`Chromatograph Apparatus
`Flow
`Column
`Rate,
`Column
`Packing
`Column Size
`Phases and mLper Temper-
`ID
`Lens_th
`min.
`ature
`SuEport
`so
`1.8 m 3mm 5 percent
`140°
`Gl6/Sl
`40
`1.2m 3mm S percent
`G16/Sl
`1.2 m 3mm 5 percent
`Gl6/S1
`1.8 m 2mm 5 percent
`G2/Sl
`
`110°
`
`145°
`
`150°
`
`Agent
`Benzyl
`Alcohol
`Chlorobu-
`tano[
`Phenol
`
`Parabens
`
`50
`
`20
`
`aration. Calculate the content, in mg per mL, of cblorobutanol
`(C4H 7C l30) in the specimen taken by the formula:
`lOO(Cf V)(pif p.)(Pz/ Pi),
`in which C is tbe concentration, in mg per mL, of ehlorobutanol
`in the Standard Preparation, and Vis the volume, in ml.. of the
`specimen. under test used in preparing each 100 mL of the Test
`Preparation.
`
`Phenol
`hternal Studarcl Soludon-Pipet l mL of benzyl alcohol into
`a 500-mL volumetric flask. add methanol to volume, and mix.
`Standarcl Preparation-Dis.wive about 75 mg of phenol, ac(cid:173)
`curately wejghed, in 7.5 mL of methanol contained in a 100-mL
`volumetric ff ask. Add 20.0 mL of lnJernal. Standard Solution,
`then add water to volume, and ~ix.
`Procedore--Using 3-µL portions of the Standard Preparation
`and the Test Preparation, record their gas chromatograms with
`the apparatus adJUstcd to the parameters set forth in the 111,;1,;010-(cid:173)
`panying table. Measure the areas under the peaks forJhenol
`and benzyl alcohol of the chromatogram for the Standar Prep(cid:173)
`aration, designating them P1 and P2, respectively. S~arly, dc(cid:173)
`ternrine the corresponding values p 1 and P2 for the Test Prepa(cid:173)
`ration. Calculate the content, in mg per mL, of phenol (C~O)
`in each ml of the specimen taken by the formuJa:
`100{ C/ VJ(p1/ P2)(P2/ Pi) ,
`in which C is the concentration, in mg per mL, of phenol in the
`Srandard Preparation, and Vis the volume, in mL, of the spec(cid:173)
`imen under test used in preparing each 100 mL of the Te.st
`Preparation.
`Metbylparaben and Propylparaben
`Internal Stanclarcl Solutio~Place about 200 mg of be~
`phenone in a 250-mL volumetric flask, .add ether to volume, and
`milt.
`Standard PreparatiOII- Place 100 mg of methylparaben and
`10 mg of propylparabcn, each aC<.."Urately weighed, in a 200-mL
`volumetric flask. add Internal Standard Solution to volume, and
`milt. Place 10 mL of this solution in a 25-mL conical flask, and
`proceed as directed under Test Preparation, beginning with " Add
`3 mL of pyridine."
`Test Preparation-Pipet 10 mL of the specimen under test and
`10 mL of the Intern.al Standard Solution into a small separator.
`Shake vigorously, allow the layers to separate, draw off the aqueous
`layer into a second separator, and tranrler the ether layer into a
`small flask through a fuonel containing anhydro~ sodium sulfate.
`Extract tile aqueous layer with iwo I 0-mL portiom1 of ether, also
`filtering the extracts through the anhydrous sodi1t111 sulfate.
`Evaporate the combined extracts under a stream of dry air until
`the volume is reduced lo about 10 mL, then transfe.r the residue
`to a 25-mL conical flask. Add 3 mL of pyridine, complete the
`evaporation of the ether, and boil on a hot plate until the volume
`is reduced to about l ml. Cool, and add 1.0 mL of a suitable
`silylation agent, such as hexamethyldisilazane to which has been
`added trimethylchlorosilane, bis(trimethylsilyl)acetamide, or
`bis(trimethylsilyl)trifluoroacetamide. Mix, and allow to stand for
`not Jess than 1 S minutes.
`·
`
`FRESENIUS EXHIBIT 1068
`Page 66 of 158
`
`

`

`1532
`
`(351) Assay for Steroids / Chemical Tests
`
`USP XXII
`
`Proced11re-Using a 2-µ.L portion of the silanized solution from
`the Standard Preparation, record the gas chromatogram with the
`apparatus adjusted to the parameters set forth in the accompa(cid:173)
`nying table. Measure the areas under the peaks for methylpar(cid:173)
`aben, propylparaben, and benz.ophenone, designating them P.,
`P2, and P3, respectively. Similarly, measure the corresponding
`areas for the silanized solution from the Test Preparation, des(cid:173)
`ignating them p,, P2, and p3, respectively. Calculate the content,
`in µg per ml, of methylparaben (CaH8O3) in the sample under
`test by the formula:
`l0(CM/V)(pJ/p3)(P3/P1),
`in which CM is the concentration, in µ.g per mL, of rocthylpara ben
`in the Standard Preparation, and Vis the volume, in mL, of the
`specimen taken. Similarly, calculate the content, in µ,g per mL,
`of propylparaben (C1oH120 3) in the specimen under test by the
`formula:
`
`I0(Cp/V)(p,,/p;)(P1/P~,
`in which Cp is the concentration, in µg per mL, of propylparaben
`fo the Standard Preparation.
`Ethylparaben and Butylparaben may be determined in a sim(cid:173)
`ilar manner.
`
`POLAROGRAPWC ME1HOD
`Pbenylmercuric Nitrate
`Standard Preparation-Dissolve about 100 mg of phenylmer(cid:173)
`curic nitrate, accurately weighed, in sodium hydroxide solution
`(l in 250) oontained in a 1000-mL volumetric flask, warming if
`necessary to effect solution, add the sodium hydroxide solution
`to volume, and mix. Pipet 10 mL of this solution into a 25-mL
`volumetric flask, and proceed as directed under Test Preparation,
`beginning with "add 2 mL of potassium nitrate solution (1 in
`100).'.'
`Test Preparation-Pipet 10 mL of the specimen under tC$t into
`a 25-mL volumetric flask, add 2 mL of potassium nitrate solution
`(1 in JOO) and IO mL of pH 9.2 alkaline borate buffer (see under
`Buff er Solutions in the section, R.eagen1s, Indicators, and So(cid:173)
`lu.tions), and adjust to a pH of 9.2, if necessary, by the addition
`of 2 N nitric acid. Add 1.5 mL of freshly prepared gelatin s~
`lution (I in 1000), then add the pH 9.2 alkaline borate buffer to
`volume, and mix.
`Procedure-Pipet a portion of the Test Preparation into the
`polarographic cell, and deaerate by bubbling nitrogen through
`the solution for 15 minutes. Insert the dropping mercury elec(cid:173)
`trode of a suitable polarograph (see Polarowaphy (801) ), and
`record the polarogram from -0.6 to - 1.5 volts versus the sat(cid:173)
`urated calomel electrode. Determine the diffusion current of the
`Test Preparation, (id)u, as the difference hetwC".en the residual
`current and the limiting current. Similarly and concomitantly
`determine tbc diffusion current, ((d)s, of the Standard Prepa(cid:173)
`ration. Calculate the quantity, in µg, of phenylmercuric nitrate
`(CJi)HgN03) in each mL of the &-pecimen taken by the formula:
`2. 5C[(i.Ju/ (id)s].
`in which C is the concentration, in p,g per mL, of phenylmercuric
`nitrate in the Standard Preparation.
`
`TbimerosaJ
`. Standard Preparation-On the day of use, place about 25 mg
`of thimerosal, accurately weighed, in a 250-mL volumetric flask,
`add water to volume, and mix. Protect from light. Pipet 15 ml
`of this solution into a 25-mL volumetric flask, add 1.5 mL of
`t?elatin solution (I in 1000), the» add potassium nitrate Mlution
`(l in l 00) to volume, and mix.
`Test Preparation---Pipet 15 mL of the test specimen into a 25-
`mL volumetric flask, add 1.5 mL of gelatin solution (l in 1000),
`add potassium nitrate solution (1 in 100) to volume, and mix.
`Procedure-Transfer a portion of the Test Preparation to a
`polarographic cell, and deaerate by bubbling nitrogen through
`the S-Olution for 15 minutes. Insert the dropping mercury elec(cid:173)
`trode of a suitable polarograph (sec Polarography (801) ), and
`,ecord the polarogram from -0.2 to - 1.4 volts versus the sat-
`
`urat.ed calomel electrode. Determine the diffusion current, (!.Ju,
`as the difference between the residual current and the linuting
`current. Similarly and concomitantly determine the diffusion
`current, (id)s, of the Standard Preparation. Calculate the quan(cid:173)
`tity, in µg, of thimerosal (<;liglfgNaO2S) in each mL of the test
`specimen taken by the formula:
`l.667C[(id)u/(iJ)sJ,
`in which C is the concentration, in 14g per mL, of thimerosal in
`·
`the Standard Preparation.
`
`(351} ASSAY FOR STEROIDS
`The following procedure is applicable for delennination of those
`PharmacopeiaJ steroids that possess reducing functional groups
`such as a-ketols.
`Standard Preparation-:Dissolve in alcohol a suitable quantity
`of the USP Reference Standard specified in the individual mono(cid:173)
`graph, previously dried UJ1der the conditions specified in the in(cid:173)
`dividual monograph and accurately weighed, and dilute quanti(cid:173)
`tatively and stepwise with alcohol to obtain a solution havini a
`concentration of about l 0 p.g per mL. Pi pet 20 mL of this solution
`into a glass~toppered, 50-mL conical flask.
`·
`·
`Assay Pre~tion-Prepare as directed in .the individual
`mo.nograph.
`·
`·
`•
`·
`. Procedure-To each of the two flasks oontaini~ the Assay
`Preparation and the Standard Preparalion, respectively, and to
`a similar flask containing 20.0 mL of alcohol to serve as the bJank,
`add 2.0 mL of a solution prepared by dissol'l'ing 50 mg of blue
`tetramlium in 10 mL of methanol, and mix. Then to each flask
`add 2.0 mL of a mixture of alcohol and tetramethylammoruum
`hydroxide TS (9: 1). mix, and allow to stand in the dark for 90
`minutes. Without delay, concomitantly determine the absorb(cid:173)
`ances of the solutions from the Assay Preparation.and the Stan(cid:173)
`dard J>reparatwn at about 525 run, with a suitable ::pectropho(cid:173)
`tometer, against the blank. Calculate the result by tlie formula
`given in ).be individual monograph, in which C is the concentra(cid:173)
`tion, in µg per mL, of the Refetence Standard in the.Standard
`Preparation. and Au and As are the absorbances of the solutions
`from the Assay Preparation and the Standard Preparatton, re-
`spectively.
`·
`·
`
`{361} BARBITURATE ASSAY
`ime;nal Standard, Internal Standard Solutwn, Staru/ard
`Pref>'!ratwn, and Assay Preparation-Prepare as directed in the
`individual monograph. .
`·
`Chromst~bic Syste11r-Under typical conditions, the gas
`chromatograph lS equipped with a flame-ionization detector and
`contains a 0.9-m X 4-1nm glass column packed with 3 petcent
`liquid phase GlO on support 80- to 100-mesh SlA. The colwnn
`is maintained at a temperature of 200 ± 10°, and the injection
`port and detector are maintained at about 225°, the column tem(cid:173)
`perature being varied within the designated tolerance, as neces(cid:173)
`sary, to meet System Suitability specifications and provide suit(cid:173)
`able retention times. Use a suitable carrier gas, such as dry
`nitrogen, at an appropriate flow rate, such as 60 to 80 mL per
`minute. Use on-column injection. (N011!-Jf the instrument is
`not equipped for on-column injection, use an injection port lined
`with glass that has been washed successively with chromic acid
`cleansing solution, water, methanol, chloroform, a 1 in 10 solution
`of trimethylchlorosilane.in chloroform, and chloroform.]
`System Suitability (see Chromatography (621))-Chromato(cid:173)
`graph five replicate injections of the Siandard Preparation, and
`r«ord•peak ~ponses as directed under Procedure. The relative
`standard deviation for the ratio Rs is not more than 1.5%. In a
`suitable chromatogram, the resolution, R, between the barbituric
`acid and the Internal Standard is not less than the value given
`in the mdividual monograph, and the tailing factor, T, for each
`of the two peaks is not more than 2.0.
`Procedare-Inject a suitable portion (about 5 µL) of the Stan(cid:173)
`dard Preparation into a suitable gas chromatograph, and record
`the chromatogram. Similarly inject a suitable portion of the As(cid:173)
`say Preparation, and record the chromatogram. Calculate the
`
`FRESENIUS EXHIBIT 1068
`Page 67 of 158
`
`

`

`USPXXII
`
`Chemical Tests / EJastomeric Oosures for Injedions
`
`(381}
`
`1533
`
`content of the barbiturate or barbituric acid in the assay specimen
`by the formula given in the individual mooogra_ph, in which Ru
`is the ratio of the peak response of the barbitunc acid to that of
`the Internal Standard obtained for the A.rsay Preparation, ~ is
`the ratio of the weight of the barbituric acid to that of the lnlernaJ
`Standard in the Standard Preparation, C1 is the ooncentration,
`in mg per mL, of fnternaJ Standard in the Internal Standard
`Solution, and Rs is the ratio of the peak response of the barbituric
`acid -to that of the Internal Standard in the Standard Prepa(cid:173)
`ration.
`
`(371) COBALAMIN
`RADIOTRACER ASSAY
`All radioactive determinations required by this method should
`be made with a suitable oounting assembly over a period of time
`optimal for the particular counting assembly used. All proce(cid:173)
`dures should be performed in replicate to obtain the greatest
`accuracy.
`Refereace Standard-USP Cyanocobalamtn &ference Stan(cid:173)
`dard- Dry over silica g&l for 4 hours before using.
`Cyaoocobalamie Tracer Raeeat-Dilutc an accurately mea(cid:173)
`sured volume of a solution of radioactive cyanocobalamin* with
`water to yield a solution having a radioactivity between S00 and
`S000 oounts per minute/er m.L. Add 1 drop of cresol per liter
`of solution prepared, an store in a refrigerator.
`Standardization-Prepare a solution of a weighed quantity of
`USP Cyanocobalamin RS in water to contain 20 to 50 I:'$ per
`mL. Perform the entire assay on a 10.0-mL portion of this s&(cid:173)
`lution, proceeding as directed under Assay Preparation, bcgii>(cid:173)
`ning with "Add water to make a measured volume.''
`Cresol-Culon Tetrachloride Solutioa-Mix equal volumes of
`carbon tetrachloride and freshly distilled ctesol.
`P!!MpJJate-◊.,AAid.P. &ilution-Dissolve 100 m~ of potassium
`cyanide in JOO() mL of a saturated llOlutiott of dibasic sodium
`phosphate, and mix.
`Butall0l-Benzallr.oniu111 Cbloride SolutioD--Dilute benzalko(cid:173)
`nium chloride solution (17 in 100) with water (3: !), and mix with
`3'6 volumes of butyl aloobol.
`. Alwnilla-Reshl Col111DD-Place a plcdget of glass wool in the
`·bottom of a constricted glass tube such as a 50-mL buret. With
`the tube held in an upri~t position, add a volwne of a slurry of
`ion~xchange resin (sec 1n the section, Reagents, Indicators, and
`Solutions); in water, sufficient to give a column of S;Cttlcd resin
`7 cm i,!I height. When. the solid has settled somewhat, allow the
`water to drain so that there is only 1 cm of liquid above the resin
`column, and tamp the resin lightly. Theo add a volume of a
`slurry of anhydroUB alumina (not acid-washed} iii water sufficient
`to increase the height of the settled column to 10 cm, and allow
`the water to drain to about 1 cm from the top of the alumina.
`Add a pledget of glass wool, and wash the column, using a total
`of SO mL of water, and again drain to within l cm of the top of
`the coJwnn. Prepare a fresh column for each determination.
`Assay Preparation-Transfer to a beaker a weighed quantity
`or measured volume of the preparation to be assayed, equivalent
`in vitamin B12 activity to that of 200 to 500fg of cyanocobala.min.
`Add water to make a measured volume o not less than 25 mL,
`then add S.O mL of Cyanocobalamin Tracer Reagent. Add, while
`working under a hood, 5 mg of sodium nitrite and 2 mg of po(cid:173)
`tassium cyanide for each mL of the resulting solution. Adjust
`the solution•with diluted hydrochloric acid to a pH of apf.roxi(cid:173)
`mately 4, and heat on a steam bath for 15 minutes. Coo, and
`adjust the solution with 1 N sodium hydroxide to a pH between
`7.6 and 8.0. Centrifuge or filter to remove any undi4solved solids.
`Procedare-Transfer the A.ssav heoarat~on to a 250-mL cen(cid:173)
`trifuge bottle, add 10 mL of c;esol.-:Carbon Tetrachloride So(cid:173)
`lutwn, suitably close the bottle with a glass, polyethylene, or foil(cid:173)
`wrapped rubber stopper, shake vigorously for 2 to 5 minutes, and
`centrifuge. Remove and aave the lower, solvent layer. Repeat
`the extraction using a S·mL portion of Cresol-Carbon Tetra(cid:173)
`chloride Solution., and oombine the lower, solvent-layer extracts
`• A solution of cyanooobalamin made radioactive by the i.1)(cid:173)
`corporation of 60Co is available from Merck and .co., Inc., Rah(cid:173)
`way, NJ 0706S.
`
`in a centrifuge bottle or separator of 50- to 100-mL capacity.
`Wash the combined extracts with successive 10-mL portions
`of S N sulfuric acid until the last washing is practically colorless
`(two washings usually suffice). During each washing, shake for
`2 to 5 minutes, allow the layers to separate, centrifuge, if nec(cid:173)
`essary, and discard the acid layer. Wash further with two suc(cid:173)
`cessive 10-mL portions of Phosphate-Cyanide Solution. Finally,
`wash with JO mL of water. Discard all of the washings.
`To the washed extract add 30 mL of a mbtture of Butanol(cid:173)
`Benzallronium Chloride Solution and carbon tetrachloride (2:
`1). Extract with two 5-mL portions of water, each time shaking
`vigorously for l minute, centrifuging, and removing and saving
`the upper, aqueous layer.
`.
`Pass the combined aqueous extracts through the Alumina-Resin
`Column at a rate of about 1 mL per minute, maintaining a 1-
`cm layer of liquid on the head of the column by adding water as
`needed. Discard as much of the forerun as is colorless (usUJ1lly
`about 5 mL), and collect the colored eluate (usually about 10
`mL) in a 50-mL centrifuge tube or separator containing 500 µL
`of diluted acetic acid. Extract the eluate by shaking for 2 to 5
`minutes with 5 mL of Cresol-Carbon Tetrachlon'de Solution,
`and discard the upper, aqueous layer. To the extract add 5.0 mL
`of water, S mL of carbon tetrachloride, and 10 mL of butyl
`alcohol. Shake, allow to separate until the upper layer is clear,
`and remove the ·upper, aqueous layer.
`·
`Determine the absorbances of the aqueous extract, in a 1-cm
`cell, at 361 run and 550 run, with a suitable spectrophotometer,
`using a tungsten light source. Make the 361-nm reading using
`a filter capable of reducing stray light. Calculate the ratio Am}
`Asso: the purity of the aqueous extract is acceptable if the ratio
`is between 3.10 and 3.40. If a ratio outside this ~e is .observed,
`purif_y the aqueous extract by repeating the extraction cycle, pro(cid:173)
`ceeding as directed in the foregoing paragraph.
`If an acceptable absorbance ratio is observed in the aqueous
`extract, determine the radioactivity, in counts per minute, using
`a suitable counter over a period optimal for the particular oount(cid:173)
`ing assembly used. Average the results, and correct the average
`ror t11e o·t,s,erved bac:itgronnu radioactivity determined ;;.er two
`or more 30-minute periods.
`Calculation-Calculate the. cobalru.p.in content, expre.ssed in µg
`of cyanocobalamio, of the por,tion talccn for assay by the formula:
`· R(Cs/CuXAr/As),
`in which R is the quantity, in µg, of cyanocobalamin in the portion
`of the standard solution taken, Cs and Cu are the corrected av(cid:173)
`erage radioactivity values, expressed in counts per minute per
`mL, of the standard and assay solutions, respectively, and Au
`and As !lfC the absorbances determined at 361 nm of the assay
`and standard solutions, respectively.
`
`· (381) ELASTOMERIC
`CLOSURES FOR INJECTIONS
`An elastomeric closure may be of synthetic or natural origin.
`It is generally a complex mixture of many ingredients. These
`include the basic polymer, fillers, accclerators,.vulcanizing agents,
`.and pigments. The properties of the elastomeric closure are de(cid:173)
`pendent not only upon these ingredients, but also on the pro(cid:173)
`cessing procedure, such as !Pixing, milling, dusting agents used,
`molding, and curing. ·
`'
`Factors such as cleansing procedures, contacting media, and
`conditions of storage may also affect the suitability of an elas(cid:173)
`tomeric closure for a spceific use. Evaluation of such factors
`should be made b>.'. appropriate additional specific tests to de(cid:173)
`termine the suitability of an elastomeric clooure for its intended
`use. Cnteria for the selection of an elastomeric closure should
`also include a careful review of a.H the ingredients to assure r.'IJ~ t.
`no known or suapected carcinogens, or other toxic substances are
`added.
`Defmitioa.:....An elastomeric closure is a packaging component
`that is, or uiay be, m direct contact with the drug.
`.
`Biological Test Procedures
`Two stages of testing are indicated. The fl18t stage is the per(cid:173)
`formance of in-vitro tests according to the procedures set forth
`
`FRESENIUS EXHIBIT 1068
`Page 68 of 158
`
`

`

`7
`
`1534
`
`(391) Epinephrine Assay / Chemical Tests
`
`USPXXII
`
`ia chapter (87), Biological Reactivity Test,, In,-vitro. Materials
`that meet the requirements of the in-vitro tests are not required
`to undergo further testing. Materials that do not meet the re(cid:173)
`quirements of the in-vitro tests are subjected to the second stage
`of testing which is the performance of in-vivo tests, i.e., the Sys(cid:173)
`temic Injection Test and /n1racutaneous Tesi, according to the
`procedures set forth in chapter. (88), Biological Reactivity Tests,
`·
`In-vivo.
`Pbysicochemical Test Procedures
`The following tests arc designed to detennine pertinent physi(cid:173)
`cochemical extraction characteristics of elastomeric cl~ures.
`Since the tests are based oo the extrc!.Ction of the elastomer, it is
`essential that the designated amount of surface area of sample
`be available. In each case, the specified surface area is available
`for extraction at the designated temperature. The test methods
`are devised to detect the majority of expected variations.
`Extraction Solveila(cid:173)
`A: Purified water.
`B: Drug product vehicle (where applicable).
`lsopropyl alcohol.
`·
`C:
`Apparatus-
`Autocfave-Use an autoclave. capable of maintaining a tem(cid:173)
`perature of 121 ± 2°, equipped with a thermometer, a pressure
`gauge, and a rack adequate to accommodate the test containers
`above the water levcL
`Oven-Use an oven, preferably a forced-draft model, that will
`maintain an operating temperature of 10S0 ± 2°.
`.
`Reflux Apparatwr-Use a suitable reflux apparatus having a
`·
`·
`capacity of about 500 mL.
`Procedure-
`Preparation of Sample-Place in a suitable extraction con(cid:173)
`tainer a sufficient number of clastomeric closures to provide 100
`cm.2 of exposed surface area. Add 300 m.L of purified water to
`each container, cover with a suitable inverted beaker, anci au(cid:173)
`toclave at 121 ± 0.5° for 30 minutes. [NOTE-Adjust so.that
`the temperature rises rapidly, preferably within 2 to 5 minutes.]
`Decant, usµig a stainJess steel screen to hold the closures in the
`containers. Rinse with 100 mL of _purified water, gently swirl,
`and discard the rinsing$. Repeat with a second 100-mL portion
`of purified water; Treat all blank containers in a similar manner.
`Extracts (with use of Extraction Solvent A)--Place a property
`prepared sample, having an exposed surface area of 100 cm2, in
`a suitable container, and add 200 mL of purified water. Cover
`with a suitable inverted beaker, and extract by heating in an
`autoclave at 121 ° for 2 hours, allowing adequate time for the
`liqwd within the container to reach the extraction temperature.
`Allow the autoclave to cool rapidly, and cool to room temperature.
`Treat the blank container in a similar manner.
`Extracts (with use of Extraction Solvent B or lj-Place a
`pro1>Crly prepared sa~le, having an. exposed surface area of 100
`cm2, in a suitable Re ux Apparatus containing 200 mL of Ex(cid:173)
`traction Solvent, an reflux for 30 minutes. Treat the blank in
`a similar manner.
`·
`·
`Turbidity-[Norn-Use Extracts prepared with Extraction
`Solvent A, B, or C.J Agitate the. container, and transfer a suf(cid:173)
`ficient quantity of Extract, diluted with Extraction Solvent, if
`necessary, to a cell. Measure the turbidity in a suitable. nephe(cid:173)
`lometer (see Spectrophotometry and Light-scatterini (851}),
`against fixed. reproducible standards.* The tutbidity is the. dif·
`ferencc between the values obtained for the blank and the sample
`expressed in Nephelos units, an arbitrary linear numerical scale
`expressing a. haze range from absolute clarity to the zone of
`turbidity.
`·
`· Reducing Agents-[Norn-Use Extracts prepared with Ex(cid:173)
`tr.action Solvent A.} Agitate .the container, transfer 50. mL of
`sample extract to a suitable container, and titrate with 0.01 N
`iodine VS, using 3 mL of starch TS as th.e indicator. Treat the
`blank extract in a similar manner. The difference bctw~n the
`blank and the sample titration is expressed in µiL of 0:01 N
`iodine.
`·
`Hea,y Metals (231 }-[NOTE-Use Extracts prepared with
`Extraction Solvent A or B.) Transfer 20 mL of the blank and
`• A suitable Nephelos Standard is available from C9lcman
`Instruments, Inc., Maywood, IL 60153.
`·
`
`the sample extracts to separate color-comparison tubes. Transfer
`2, 6, and 10 mL of Standard Lead Solution into separate color(cid:173)
`comparison tubes, add 2 mL of I N acetic acid to each tube, and
`adjust the volume to 25 mL with purified water. Add 10 mL of
`freshly prepared hydrogen sµlfide TS w each tube, mix, allow to
`stand for 5 minutes, and view downward over a white surface.
`Determine the amount of heavy metals in the blank and in the
`sample. The heavy 1Itetals content is the difference between the
`·
`·
`blank and the sample.
`pH Change---[NOTE-Use Extracts prepared with Extraction
`Solvent A or B, adding to extraczs obtained with So/venl A suf(cid:173)
`ficient potassium chloride to provide a concentration of 0.1%.]
`Determine the pH of sample extracts A and B potentiometrically,
`performing blank determinations with blank extracts A and B,
`and making any necessary corrections. The pH change is the
`difference between the blank and the sample.
`Total Extrac:table&-[NOTB-Use Extracts prepared with Ex(cid:173)
`traction Solvent A, B, or C.] Agitate the containers, and transfer
`100-mL aliquots of the blank and the sample to separate, tared
`evaporating dishes. Evaporate on a steam bath to dryness (Ex(cid:173)
`tracts prepared with Extraction Solvent C) or in an oven at 100°,
`dry at 105° for 1 hour, cool in a desiccator; and weigh. Calculate
`the total extractablcs, io mg, by the fonnula:
`2(Wu - WB},
`in which Wu is the weight, in mg, of residue found in the ~ample
`extract aliquot, and W8 is the weight, in mg, of residue found in
`the blank solution aliquot.
`·
`
`{391) EPINEPHRINE ASSAY
`Refereace Stanllri-USP Epinephrine Bitartrate Reference
`Standard-Keepcont.ainer tightly closed and protected from light.
`Dry in vacuum over silica gel for 18 hours before using.
`1''erro-dtrate Solutlon---On the day needed, dissoive U . g oi
`ferrous sulfate in 200 mL of water to which have been added 1.0
`mL of dilute hydrochloric acid (l in 12) and 1..0 g. of sodium
`bisulfite. Dissolve 500 mg of sodium citrate in 10 mL of this
`solution, and mix.
`Buffer Solution-In a 50-mL volumetric flask mix 4.2 g of
`sodium bicarbonate, 5.0 g of potassium bicarbonate, and 18 mL
`of water (not all of the solids will dissolve at this stage). To
`another 18 mL of water add 3.75 g ofaminoacetic acid and 1.7
`mL of .6 N ammonium hydroxide, mix to dissolve, and transfer
`thi& solution to the 50-mL volumetric flask c.ontaining the other
`mixture. Dilute with water to volume, and mix until solution is
`complete.·
`Standard Prepara~Transfer about 18 mg of USP Epi•
`nephrine Bitartrate RS, accurately weighed, to a !00-mL volu(cid:173)
`metric flask with the aid of 20 mL of sodium bisulfite solution
`(1 in SO), dilute with water to volume, and mix. Transfer 5.0 mL
`of th.is solution to a 50-mL volumetric flask, dilute with sodium
`bi~ulfite solution (I in 500) to volume, and mix. [Norn-Make
`the final dilution when the assay is carried out.J The concentra(cid:173)
`tion of USP Epinephrine Bitartratc RS in the Standard Prepa•
`ration is about I 8 µg per mL.
`Assay .PreparatioU--:.Transfer to a 50-mL volumetric flask an
`accurately measured volume of the Injection under assay, equiv(cid:173)
`alent to about S00 µ,g of epinephrine, dilute with sodium bisulfite
`solution (1 in 500} to volume, if n~sary. and mix. {NOTE-(cid:173)
`The final concentration of sodium bisulfite is in the range of 1
`to 3 mg per mL, any bisulf1tc present in the Injection under assay
`being taken into consideration.]
`Proceda.re-lnto three 50-mL glass-stoppered . conical flasks
`transfer, separately, 20.0-mL aliquots of the Standard Prepa(cid:173)
`ration, the A.r.rqy Preparu11on, and sodium bisulfite solution (1
`in 500) to provide the blank.· To each flask add 200 µL of Ferro(cid:173)
`citrate Solution and 2.0 mL of Buffer Solution, mix, and allow
`the solutions to stand for 30 minutes. Determine the absorbances
`of the solutions in 5-cm cells at the wavelength of maximum
`absorbance at about 530 nm, with a suitable spectrophotometer,
`using the blank to set the instrument. Calculate the quantity, in
`mg, of epinephrine (½H13NO3) in each mL of the Injection taken
`by the formula:
`·
`(I 83.21 /333.29)(0.0SC/J/)(A