2000
`
`u
`2
`F 19
`
`THE UNITED STATES PHA ACOPEIA
`
`THE NATIONAL FORMULARY
`
`By authority of the United States Pharmacopeia[
`Convention, Inc., meeting at Washington, D.C.,
`March 9-12, 1995. Prepared by the Committee of
`Revision and published by the Board of Trustees
`
`Official from January 1, 2000
`
`UNITED STATES PHARMACOPEIAL CONVENTION, INC.
`12601 Twinbrook Parkway, Rockville, MD 20852
`
`Page 1
`
`NPS EX. 2047
`CFAD v. NPS
`IPR2015-00990
`
`

`
`dc:il
`o n
`
`tte · Ir
`Nat1
`mai1
`1s n
`now
`
`h
`ume
`COp I
`are
`visi(.
`subs
`USI
`sion
`COfJ=
`
`NOTICE AND WARNING
`
`Concerning U.S. Patent or Trademark Rights
`
`The inclusion in the Pharmacopeia or in the National Formulary of a monograph on any drug in
`respect to which patent or trademark rights may exist shall not be deemed, and is not intended
`as, a grant of, or authority to exercise, any right or privilege protected by such patent or
`trademark. All such rights and privileges are vested in the patent or trademark owner, and no
`other person may exercise the same without express permission, authority, or licen.se secured
`from such patent or trademark owner.
`
`Concerning Use of USP or NF Text
`Attention is called to the fact that USP and NF text is fully copyrighted. Authors and others
`wishing to use portions of the text should request permission to do so from the Secretary of the
`USPC Board of Trustees.
`
`© 1999
`The United States Pharmacopeia! Convention, Inc.
`12601 Twinbrook Parkway, Rockville, MD 20852.
`All rights reserved
`ISSN 0195-7996
`ISBN 1-889788-03-1
`
`Printed by National Publishing, Philadelphia, PA
`
`Page 2
`
`

`
`Globulin I Official Monographs
`
`USP 24
`
`Packaging and storage-Preserve at a temperature between 2° and
`so.
`Expiration date-The expiration date is not later than 3 years after
`date of issue from manufacturer's .cold storage (5°, 3 years).
`Labeling-Label it to state that passive immunization with Immune
`Globulin modifies hepatitis A, prevents or modifies measles, and
`provides replacement therapy in persons having hypo- or agam(cid:173)
`maglobulinemia, that it is not standardized with respect to antibody
`titers against hepatitis B surface antigen and that it should be used
`for prophylaxis of viral hepatitis type B only when the specific
`Immune Globulin is not available, that it may be of benefit in
`women who have been exposed to rubella in the first trimester of
`pregnancy but who would not consider a therapeutic abortion, and
`that it may be used in immunosuppressed patients for passive im(cid:173)
`munization against varicella if the specific Immune Globulin is not
`available. Label it also to state that it is not indicated for routine
`prophylaxis or treatment of rubella, poliomyelitis or mumps, or for
`allergy or asthma in patients who have norm~l levels of immuno(cid:173)
`globulin, that the plasma units from which it has been derived have
`been tested and found non-reactive for hepatitis B surface antigen,
`and that it should not be administered intravenously but be given
`intramuscularly, preferably in the gluteal region.
`
`Rho (D) Immune Globulin
`» Rho (D) Immune Globulin conforms to the regula(cid:173)
`tions of the FDA concerning biologics (see Biologics
`(1041)). It is a sterile, nonpyrogenic solution of glob(cid:173)
`ulins derived from human blood plasma containing an(cid:173)
`tibody to the erythrocyte factor Rho (D). It contains
`not less than 10 g and not more than 18 g of protein
`per 100 mL, not less than 90.0 percent of which is
`gamma globulin. It has a potency, determi.ned by a
`suitable method, not less than that of the U. S. Refer(cid:173)
`ence Rho (D) Immune Globulin. It contains 0.3 M gly(cid:173)
`cine as a stabilizing agent and contains a suitable
`preservative.
`Packaging and storage-Preserve at a temperature between 2° and
`so.
`Expiration date-The expiration date is not later than 6 months
`from the date of issue from manufacturer's cold storage, or not later
`than 1 year from the date of manufacture, as indicated on the label.
`
`specific for immunoglobulin IgG; and at least the C3d
`component of human complement (for use in the direct
`antiglobulin test, it contains this Anti-C3d and Anti(cid:173)
`IgG activity) and which may be artificially colored
`green; (2) a reagent containing antibodies only against
`immunoglobulin IgG (not heavy chain specific) in(cid:173)
`tended for use in the indirect antiglobulin test, and
`which may be artificially colored green; and (3) rea(cid:173)
`gents containing antibodies specific for individual or
`selected components of human complement, such as
`Anti-C3, and Anti-C3b-C3d-C4, or a single class of
`immunoglobulins, such as Anti-IgG (heavy chain spe(cid:173)
`cific), used only to identify plasma components coated
`on the surface of red blood cells. Anti-Human Glob(cid:173)
`ulin Serums containing Anti-IgG meet the requirements
`of the test for potency, in parallel with the U. S. Ref(cid:173)
`erence Anti-Human Globulin (Anti-IgG) Serum (at a
`1 :4 dilution) when tested with red cells suspended in
`isotonic saline sensitized with decreasing amounts of
`non-agglutinating Anti-D (Anti-Rho) serum, and with
`cells sensitized in the same manner with an immuno(cid:173)
`globulin IgG Anti-Fya serum of similar potency. Anti(cid:173)
`Human Globulin Serum containing one or more Anti(cid:173)
`complement components meets the requirements of the
`tests for potency in giving a 2+ agglutination reaction
`(i.e., agglutinated cells dislodged into many small
`clumps of equal size) by the low-ionic sucrose or su(cid:173)
`crose-trypsin procedures when tested as recommended
`in the labeling. Anti-Human Globulin Serum contain(cid:173)
`ing Anti-3Cd activity meets the requirements for sta(cid:173)
`bility, by potency testing of representative lots every 3
`months during the dating period.
`Packaging and storage-Preserve at a temperature between 2° and
`so.
`Expiration date-Its expiration date is not later than 1 year after
`the date of issue from manufacturer's cold storage (5°, 1 year; or
`0°, 2 years).
`Labeling-Label it to state the animal source of the product. Label
`it also to state the specific antibody activities present; to state the
`application for which the reagent is intended; to include a caution(cid:173)
`ary statement that it does not contain antibodies to immunoglobu(cid:173)
`lins or that it does not contain antibodies to complement compo(cid:173)
`nents, wherever and whichever is applicable; and to state that it is
`for in vitro diagnostic use.
`[NOTE-The lettering on the label of
`the general-purpose polyspecific reagent is black on a white back(cid:173)
`ground. The label of all other Anti-Human Globulin Serum con(cid:173)
`tainers is in white lettering on a black background.]
`
`Anti-Human Globulin Serum
`)) Anti-Human Globulin Serum conforms to the reg(cid:173)
`ulations of the FDA concerning biologics (see Biolog(cid:173)
`ics (1041)). It is a sterile, liquid preparation of serum
`produced by immunizing lower animals such as rabbits
`or goats with human serum or plasma, or with selected
`human plasma proteins. It is free from agglutinins and
`from hemolysins to non-sens.itized human red cells of
`all blood groups. It contains a suitable antimicrobial
`preservative. Three varieties of Anti-Human Globulin
`Serum are recognized: (1) a general-purpose polyspe(cid:173)
`cific reagent which, as a minimum, contains antibodies
`
`Glucagon
`
`His-Ser- Gin -Giy- Thr- Phe- Thr- Ser- Asp-Tyr- Ser- Lys-Tyr- Leu-Asp-Ser-
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`
`Arg- Arg- Alu-Gin- Asp- Phe- Vul- Gin-Trp- Leu-Met -Asn- Thr
`17
`18
`19
`20
`21
`22
`23 24
`25
`26 27
`28
`29
`
`CmHmN43049S
`Glucagon (pig).
`Glucagon
`[16941-32-5].
`
`34S2.S2
`
`» Glucagon is a polypeptide hormone that has the
`property of increasing the concentration of glucose in
`
`Page 3
`
`

`
`USP 24
`
`Official Monographs I Glucagon
`
`775
`
`the blood. It is obtained from porcine and bovine pan(cid:173)
`creas glands.
`packaging and storage-Preserve in tight, glass containers, under
`nitrogen, in a refrigerator.
`USP Reference standards (11)-USP Glucagon RS. USP Dextrose
`RS.
`Identification-The retention time of the major peak in the chro(cid:173)
`matogram of the Test solution obtained as directed in the test for
`Chromatographic purity corresponds to that in the chromatogram
`of the Standard solution.
`Chromatographic purity-
`Mobile phase-Prepare a solution containing 9.8 g of monobasic
`sodium phosphate and 170 mg of cysteine in 710 mL of water.
`Prepare a filtered and degassed mixture· of this solution and 290
`mL of acetonitrile, and adjust with phosphoric acid to a pH of 2.6.
`Make adjustments, if necessary (see System Suitability under Chro(cid:173)
`matography (621)).
`Standard solution-Dissolve an accurately weighed quantity of
`USP Glucagon RS in 0.01 N hydrochloric acid to obtain a solution
`having a known concentration of about 1 mg per mL.
`Test solution-Dissolve an accurately weighed quantity of Glu(cid:173)
`cagon in 0.01 N hydrochloric acid to obtain a solution containing
`about 1 mg per mL.
`Chromatographic system (see Chromatography (621))-The liq(cid:173)
`uid chromatograph is equipped with a 214-nm detector and a 4.6-
`mm X 25-cm column that contains packing L7 with a 150 A pore
`size. The column temperature is maintained at 35° and the flow rate
`is about 1.0 mL per minute. Chromatograph the Standard solution,
`and record the chromatograms as directed for Procedure: the res(cid:173)
`olution, R, is not less than 1.8; the retention time for the major
`glucagon peak is between 16 and 20 minutes; and the tailing factor
`is not more than 2.0.
`Procedure-Separately inject equal volumes (about 20 1-1L) of
`the Standard solution and the Test solution into the chromatograph,
`and record the peak responses. Calculate the percentage of each
`impurity in the portion of Glucagon taken by the formula:
`
`1 OO(r; I r1),
`
`in which r; is the peak response for an individual impurity, and r1
`is the sum of the responses of all of the peaks: not more than 2.5%
`of any individual impurity is found, and not more than 10.0% of
`total impurities is found.
`Water, Method I (921): not more than 10.0%.
`Residue on ignition (281 ): not more than 2.5%.
`Nitrogen content, Method II (461): between 16.0% and 18.5%,
`calculated on the anhydrous basis.
`·
`Zinc content (591): not more than 0.05%.
`Assay-
`NoTE-All buffers have a final pH of 7.4, unless otherwise
`indicated.
`Hepatocyte preparation-
`CALCIUM-FREE PERFUSION BUFFER WITH DEXTROSE-Prepare a
`solution containing, in each liter, 7.92 g of sodium chloride, 0.35
`g of potassium chloride, 1.80 g of dextrose, 0.15 g of edetic acid,
`and 2.38 g of N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid.
`Oxygenate prior to circulation.
`COLLAGENASE BUFFER-Prepare a solution containing, in each
`liter, 3.62 g of sodium chloride, 23.83 g of N-2-hydroxyethylpiper(cid:173)
`azine-N' -2-ethanesulfonic acid, 0.35 g of potassium chloride, 0.52
`g of calcium chloride, and 1.8 g of dextrose. Adjust to a pH of 7.6,
`and oxygenate. Immediately before perfusion, dissolve a quantity
`of collagenase in this solution to obtain a concentration of 0.03%
`to 0.05%.
`WASH BUFFER-Prepare a solution containing, in each liter, 7.92
`g of sodium chloride, 0.35 g of potassium chloride, 0.15 g of edetic
`acid, 2.38 g of N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic
`acid, 22 g of calcium chloride, and 0.12 g of magnesium sulfate.
`INCUBATION BUFFER-Prepare a solution containing, in each liter,
`6.19 g of sodium chloride, 0.35 g of potassium chloride, 0.22 g of
`calcium chloride, 0.12 g of magnesium sulfate, 0.16 g of monobasic
`
`potassium phosphate, 11.915 g of N-2-hydroxyethylpiperazine-N'-
`2-ethanesulfonic acid, and 1% bovine serum albumin (BSA). Adjust
`to a pH of 7.5.
`TEST ANIMALS-Male Sprague-Dawley rats are maintained on a
`standard rat chow diet and freely given water. On the morning of
`the test, select a healthy rat weighing approximately 300 g, and
`administer 100 Units of Heparin Sodium subcutaneously.
`PROCEDURE-Anesthetize the rat with an appropriate gas anes(cid:173)
`thetic. Open the abdominal cavity and isolate the portal vein. Insert
`an angiocatheter connected to a perfusion pump, and tie into the
`portal vein at the general location of the lienal branch. Start the
`perfusion (25 mL per minute) in situ with Calcium-free perfusion
`buffer with dextrose, equilibrated with oxygen, at a temperature of
`37°. As the liver enlarges, cut the inferior vena cava to allow pres(cid:173)
`sure equilibrium. [NOTE-About 300 mL of the perfusate is needed
`to clear the liver of red blood cells at a flow rate of 40 to 50 mL
`per minute.] Then circulate Collagenase buffer at a flow rate of 40
`to 50 mL per minute for about 10 minutes. The exact concentration
`of collagenase (within the range of 0.03% to 0.05%) is determined
`empirically for each lot of enzyme. The concentration of collage(cid:173)
`nase is that necessary to cause consistently a breakdown of the liver
`about 10 minutes after initial entry of the Collagenase buffer into
`the liver. When the liver significantly increases in size, changes
`color and consistency, and starts to leak perfusate out of the lobes,
`change the system to the oxygenated prewarmed Wash buffer.
`About 100 mL of Wash buffer is needed to wash the liver of col(cid:173)
`lagenase at a flow rate of 25 mL per minute. Surgically remove the
`liver from the animal and place in a prewarmed tray containing
`oxygenated Wash buffer (37°). Gently comb the liver with a stain(cid:173)
`less steel, fine-toothed comb to free the hepatocytes. Wash the he(cid:173)
`patocytes with Wash buffer, and filter through cheesecloth (or a
`150-1-Lm mesh polyethylene net) into a plastic beaker. Centrifuge
`the cell suspension for about 2 minutes at 40 X g to form a loosely
`packed pellet. Discard the supernatant liquid, and resuspend the
`pellet in Wash buffer. Repeat the washing procedure twice for a
`total of three washes. Resuspend the final pellet in 100 to 200 mL
`of Incubation buffer depending on cell yield. [NoTE-If the Assay
`procedure is interrupted, cool the cells by collecting the cells in a
`beaker placed in ice. The cells are washed with ice cold Wash buffer
`and stored on ice until ready for use. At that point the cells are
`pelleted once more and resuspended in ice cold Incubation buffer.]
`SUITABILITY-The concentrations of cells may vary due to the
`collagenase activity and the viability of the hepatocytes. To check
`cell viability and to determine viable cell concentration, dilute du(cid:173)
`plicate 100-1-LL aliquots of cell suspension with 400 1-1L of Wash
`buffer and 500 1-1L of isotonic 0.4% trypan blue. The aliquots are
`counted in a hemocytometer. The cells are suspended in Incubation
`buffer to obtain a cell concentration of 4 X 106 per mL. Count
`several distinct fields: the viability is greater than 90%. [NOTE(cid:173)
`Viable cells are those cells that exclude the trypan blue.]
`Negative control solution-Prepare a solution containing 0.5%
`bovine serum albumin in sterile water.
`Incubation flasks-Use 25-mL conical flasks, the bottoms of
`which have been heated and pushed inward to form a conically
`raised center.
`Standard preparations-Prepare two solutions by dissolving in
`each a vial of USP Glucagon RS in 0.01 N hydrochloric acid or
`other appropriate diluent to obtain a final concentration of 1.0 USP
`Glucagon Unit per niL. All dilutions thereafter are made using 0.5%
`BSA (w/v) in water. Dilute accurately measured volumes of each
`solution with Negative control :Solution to obtain six concentra(cid:173)
`tions-200, 100, 50, 25, 12.5, and 6.25 micro-Units per mL-of
`each solution (Standard preparations). Pipet 0.2 mL of each Stan(cid:173)
`dard preparation into separate Incubation flasks. Pipet 0.2 mL of
`Negative control solution into each of 2 flasks (Negative control
`solution I and 2). Then add the hepatocytes into each of the eight
`flasks. [NoTE-Hepatocytes incubated with a saturating concentra(cid:173)
`tion of 500 X 10-6 USP Glucagon Units demonstrates at least 165%
`stimulation of the control glycogenolysis rate (0.2 mL of a 12.5
`USP Glucagon Units per mL solution will give a concentration of
`481 X 10-6 USP Glucagon Units per mL in the incubation flask).]
`
`Page 4
`
`

`
`776
`
`Glucagon I Official !Honographs
`
`VSP
`
`Assay preparations-Using accurately weighed quantities of
`Glucagon, proceed as directed for Standard preparations.
`D-Glucose determination-
`STOCK STANDARD SOLUTION-Transfer 2.0 g of USP Dextrose
`RS, accurately weighed, to a 200-mL volumetric flask, dissolve in
`and dilute with saturated benzoic solution to volume.
`STANDARD SOLUTIONS-Transfer suitable quantities of Stock
`standard solution to three flasks and dilute with saturated benzoic
`acid solution to obtain known solutions having known concentra(cid:173)
`tions of 0.5, 1.0, and 1.5 times the typical sample glucose concen(cid:173)
`tration.
`POTASSIUM FERROCY ANIDE SOLUTION-Dissolve 1.25 g of tri(cid:173)
`hydrate potassium ferrocyanide in 125 mL of Sterile Water for
`Injection.
`SYSTEM SUITABILITY-Analyze the Potassium ferrocyanide so(cid:173)
`lution, the Standard solutions, and five replicates of the middle
`Standard solution. Prepare a standard curve using the Standard so(cid:173)
`lutions as directed for Procedure: the relative standard deviation of
`the standard curve is not more than 2.0%; the response of the Po(cid:173)
`tassium ferrocyanide solution is not more than 30 mg per liter; and
`the relative standard deviation is not more than 2.0% for the rep(cid:173)
`licate analyses of the middle Standard solution.
`Procedure-Dispense 5 mL of Hepatocytes preparation into the
`special incubation flasks in sequence from high glucagon concen(cid:173)
`tration to low glucagon concentration alternating the Standard prep(cid:173)
`arations with the Assay preparations. The flasK.s are swirled in an
`orbiting water bath at 125 rpm at 30° for approximately 30 to 60
`minutes. [NoTE-The exact incubation time must be determined to
`optimize the signal-to-noise.] Following incubation place 0.5- to
`1.0-mL aliquots, in duplicate, from each incubation flask into la(cid:173)
`beled tubes, and centrifuge at 100 X g. Determine the percentage
`of glucose concentration in each flask's supernatant liquid.
`To conform to the linear range of the instrument being used, it
`may be necessary to adjust by dilution each of the preparations. Use
`a glucose analyzer that has demonstrated appropriate specificity,
`accuracy, precision, and linear response over the range of concen(cid:173)
`trations being determined. [NOTE-A suitable analyzer may use an
`immobilized, oxidase-enzyme membrane or jacket-generating hy(cid:173)
`drogen peroxide, which is then detected at the electrode.] Perform
`the glucose analysis in the following sequence: Negative control
`solution I, Standard preparations, Assay preparations, and Nega(cid:173)
`tive control 2. Determine the percentage of glucose against the Neg-
`ative control solution for each preparation.
`·
`Calculations-
`LINEARITY TEST-Use an analysis of variance.·(ANOVA) with
`one sample assayed against a standard, and using two replicates
`each, construct a table (see Table 1 ). Compare the value of the ratio
`MSNLIMSRES 1 to a critical value obtained from a table for an F
`distribution with m - 2 and 3m - 3 degrees of freedom (df), where
`m is the number of dose levels for each preparation. If the ratio
`MSNL/MSRES does not indicate the presence of significant non(cid:173)
`linearity (ratio value is lower than the critical value), then proceed
`to the test for parallelism. If the ratio exceeds the critical value
`(significance level of 0.05), the nonlinearity is statistically signifi(cid:173)
`cant and the test is repeated, discarding the results from either the
`highest or lowest dose of both the Standard preparations and the
`Assay preparations (four dose levels). If the ratio MSNLIMSRES
`does not indicate the presence of significant nonlinearity, then pro(cid:173)
`ceed to the test for parallelism.
`PARALLELISM TEST-Compare the ratio MSNP/MSRES 2 to a crit(cid:173)
`ical value obtained from an F distribution having 1 and 4m - 5 df.
`If the ratio MSNPIMSRES 2 does not indicate the presence of sig(cid:173)
`nificant nonparallelism, then the assay is considered valid. Use the
`appropriate dose levels for the estimation of the relative potency.
`RELATIVE.POTENCY-Calculate the relative potency, R, of the As(cid:173)
`say preparations as compared to the Standard preparations as
`follows.
`(1) Xj is defined as the log 10 of the jth dose of the Standard prep(cid:173)
`arations or the Assay preparations. The glucagon dose varies from
`12.5 to 200 X 10- 6 USP Glucagon Units per mL. For ease in the
`
`subsequent calculations, these doses are respectively represented b.
`1 through 5 as shown in the table below.
`Y
`j
`5
`4
`1
`2
`3
`200
`100
`Dose
`12.5
`25
`50
`2.3o
`2.oo
`xj
`1.10
`1.40
`1.70
`(2) To differentiate between the Standard preparations and th
`Assay preparations in the calculations, the subscript ''i'' will b:
`used with i = 1 to designate the Standard preparations and i ::::: 2
`to designate the Assay preparations. Yijk will denote the glucose
`concentration associated with the klh replicate of the jlh dose of the
`ith preparation. For example, Y1jk is the glucose concentration asso,
`ciated with the k1h replicate of the jlh dose of the appropriate Stan.
`dard preparation; Y11k is the glucose concentration associated with
`the klh replicate of dose 1 of the Standard preparation and y
`would denote the glucose concentration associated with the kth re~~
`licate of dose 1 of the Assay preparation. Dose 1 represents a glu.
`cose dose of 12.5 X 10-6 USP Glucagon ~nits per. mL. Finally,
`Y132 would represent the glucose concentration associated with the
`2nd replicate of dose 3 for the Standard preparation.
`(3) Ys and Y1 denote the average glucose concentrations for the
`Standard preparation and the Assay preparation, respectively.
`(4) Calculate the least-squares slope estimate, b, for a linear re(cid:173)
`gression relating the yijk' s to the X/ s as follows: b = sxy IS XX With
`Sxy and Sxx calculated using the equations in Table 2.
`(5) The log potency, M, is calculated using M = -1[(Ys - Y
`I b].
`(6) R = antilog (M).
`(7) Calculate the confidence limits (upper and lower) for the rel(cid:173)
`ative potency, R, using the value s2 = MSRES 3 (see Table 1 and
`Table 2) as follows. Obtain t from a table for a t distribution having
`4m - 4 df. For the 95% limits, the t values can be obtained from
`Table 9 under Design and Analysis of Biological Assays (111).
`[NoTE-For confidence limits having other probability levels
`(i.e., 100(1 - a) %), the right tail t critical value having al2 area
`to its right is used.]
`Calculate g = t2S 2 I b2Sxx
`
`)
`1
`
`and I' = (ts I b) J(l/m)(l - g) + (M'/S,.),
`
`and calculate ML = (M
`F)l(l - g)
`(M + F)/(1 - g)
`and Mu
`where M is the log potency and ML and Mu are the log potency
`lower and upper confidence limits. The lower and upper confidence
`limits for the relative potency, R, are given by
`RL = antilog (ML)
`RU = antilog (Mu)
`It meets the requirements if the potency is between 0.9 to 1.3
`USP Glucagon Units per mg, and the confidence interval width at
`P = 0.95 does not exceed 45% of the computed potency.
`Table 1. ANOV A for the Rat Hepatocyte
`Assay for Glucagon.
`SS (Sum
`Source
`of Squares)
`df
`SS.PREP
`1
`Preparations
`1
`Replicates
`SSREP
`Linear Slope
`SSLIN
`1
`Residual 3
`4m-4
`SSRES 3
`1
`Nonparallelism
`SSNP
`4m-5
`Residua12
`SSRES 2
`m-2
`Nonlinearity
`SSNL
`3m-3
`Residual 1
`SSRESI
`4m-1
`TOTAL
`SST
`NOTES-This analysis pertains to one sample assayed against a
`standard, using two replicates each.
`The number of dose levels for each preparation is denoted by m.
`Table 2 gives the equations for calculating the SS terms.
`
`MS (Mean
`Square)
`MSPREP
`MSREP
`MSLIN
`MSRES 3
`MSNP
`MSRES 2
`MSNL
`MSRESI
`
`Page 5
`
`

`
`vsP 24
`
`Official Monographs I Gluconolactone
`
`777
`
`In each row of the ANOV A table, the MS is obtained by dividing
`the ss term by the df term.
`
`Table 2. Equations for Calculating the Sums
`of Squares in the Analysis of Variance.* (continued)
`
`Table 2. Equations for Calculating the Sums
`of Squares in the Analysis of Variance. •
`
`Yi .. =II Yijk
`jk
`
`Y.j. =II Yijk
`ik
`
`Y.k =II Yijk
`ij
`
`( I~I y,,,)'
`CF= - - - -
`4m
`
`( ~ x,)(Y, .. )
`ss = II x.yJjk - - - - -
`jk
`m
`
`xy
`
`J
`
`s~ = 2 I xf
`
`m
`
`IY\.
`SSPREP = _;-
`2m
`
`CF
`
`SSREP
`
`IY~k
`_k_ - CF
`2m
`
`(s.,)'
`
`SSLIN = - -
`Sxx
`
`SST = IIIyijk - CF
`ij k
`SSRES3 = SST - SSPREP - SSREP
`
`SSLIN
`
`IY3.
`SSNL = ~i -
`4
`
`- SSLIN - CF
`
`SSRES 1 = SSRES2 - SSNL
`
`Glucagon for Injection
`» Glucagon for Injection is a mixture of the hydro(cid:173)
`chloride of Glucagon: with one or more suitable, dry
`diluents. It contains not less than 80.0 percent and not
`more than 125.0 percent of the labeled amount of
`glucagon.
`Packaging and storage-Preserve in Containers for Sterile Solids
`as described under Injections (1).
`USP Reference standards (11)-USP Glucagon RS. USP Dextrose
`RS. USP Endotoxin RS.
`Constituted solution-At the time of use, it meets the requirements
`for Constituted Solutions under Injections (1).
`Bacterial endotoxins (85)-It contains not more than 125.0 USP
`Endotoxin Units per mg of glucagon.
`pH and Clarity of solution-Dissolve it in the sol vent and in the
`concentration recommended in the labeling: the pH of the solution
`is between 1.7 and 3.0, and the solution is clear.
`Other requirements-Both Glucagon for Injection and the accom(cid:173)
`panying solvent meet the requirements for Sterility Tests (71) and
`Labeling under Injections (1). Glucagon for Injection meets there(cid:173)
`quirements for Uniformity of dosage units (905).
`Assay-Proceed as directed for the Assay under Glucagon. The
`potency is not less than 80.0% and not more than 125.0% of the
`labeled amount of glucagon, and the confidence interval width at P
`0.95 does not exceed 45% of the computed potency. Repeat the
`assay if the confidence interval width exceeds 45% of the computed
`potency or the potency is less.than 80.0% or more than 125.0% of
`the labeled amount of glucagon.
`
`Gluconolactone
`
`HO'}od
`\-YbH
`OH
`
`178.14
`C6H100 6
`D-Gluconi.c acid a-lactone.
`Glucono delta-lactone
`[90-80-2].
`
`SSRES2 = SSRES3 - SSNP
`
`• Refer to the Calculations for section on Relative Potency for
`the definitions of xj and Yijk·
`
`>> Gluconolactone contains not less than 99.0 percent
`and not more than 101.0 percent of C6H 100 6 •
`Packaging and storage-Preserve in well-closed containers.
`Identification-Dissolve about 0.5 g in 5 mL of warm water in a
`test tube. Add 1 mL of freshly distilled phenylhydrazine, and heat
`on a steam bath for 30 minutes. Cool the solution, induce crystal(cid:173)
`lization by scratching the inner surface of the test tube with a glass
`rod, and collect the crystals of the phenylhydrazide of gluconic acid.
`Dissolve the crystals in 10 mL of hot water to which a small amount
`of activated charcoal has been added, filter, recrystallize, wash the
`
`Page 6