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ASSAY OF HYDROXYPROPYL METHYLCELLULOSE IN AN OPHTHALMIC

FORMULATION BY SIZE EXCLUSION CHROMATOGRAPHY

Steven W. Shea, Erlinda P. Murray, Bhavan V. Mehta, and Laurel S. Hacche,

Allergan Research& Development,2525 Dupont Dr., Irvine, CA 92715, USA

INTRODUCTION
Gas chromatography (GC)I, colorimetric2, and size exclusion chromatography
(SEC)3 techniques have been applied to the analysis of hydroxypropyl
methylcellulose. Boththe GC and colorimetricassay solutionsoffer indirectdetection
of HPMC. Hence, no conformational or molecular weight characterizations can be
observedconcurrently. Studies have also shown exceptionally poor precision using
the colorimetricprocedure with relative standard devi_itions(RSDs) often exceeding
40% thus preventingadequate transfer of methodologyto an altemate QC site. Both
indirect methods additionally suffer from potential matrix interference which may
requirespikingof standard material in sample matrix as a control.

The purpose of this work is to examine an SEC methodology which meets the
followingobjectives in the analysis and characterizationof HPMC:

To develop a viable stabilityindicatingSEC method for the assay of HPMC

in an ophthalmicformulation.

To establish criteria for the successful transfer of SEC methodology to

altemate QC sites.

To characterizethe HPMC raw materialfor weight, number, and z-average

molecular weights.

H. OH r _C]'I2OCH3 1 H .OCH3

H H H H

t----o o-r-., t--i i t---o- o.

C8H1506 - (C10H1806)n - C8H1505

Formula Weight Approximately 86000

Figure 1: Structure of HydroxypropylMethylcellulose

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MATERIALS AND METHODS

HydroxypropylMethylcellulose

HPMC raw materialswere supplied by Dow USA Chemical Corporation,F4M

premium grade.

High Performance Liauid Chromatography

Hydroxypropylmethylcellulose(HPMC) is determined in an ophthalmicformulationby

modificationof a method publishedby D.D. MacLaren et al.3 This procedureutilizes
a Waters Linear SEC column with a stationary phase consisting of cross-linked
hydroxylated methacrylate gel with residual carboxyl functionality which provides
sufficient selectivity for HPMC. HPLC analysis was performed utilizingthe following
instrumentalparameters:

Autosampler = Waters WISP 715

Pump = Waters 600E
Detector = Waters 410 Refractive Index @ 35C
Column = Waters UltrahydrogelLinear, 300 x 7.8 mm, 6-13 mm
Guard Column = Waters Ultrahydrogel
Mobile Phase = 90/10 (v/v) Water/Acetonitrile
InjectionVolume = 100 mL
Flow Rate = 0.7 mL/min
Column Temperature = 35C
Data AnalysisSystem = Digital EquipmentCorp Vax 6210 computerrunning
PE Nelson Access*Chromsoftware, version 1.9

The above procedure was also performed substitutingthe RI detector with a Sedex
model 55 evaporative lightscatteringdetector to aid in characterizationof the polymer
and to determine response relative to refractive index detection. Additionalmaterial
characterization was performed utilizing triple detectors connected in series.
Calculationsand data capture were performed usingViscotekTriSEC software.

Triple Detector = Viscotek H502 Inline Viscometer

Viscotek Right Angle Light Scattering (RALLS)
Waters 410 Refractive Index @ 35C

Statistical Analysis

One-Way Analysis of Variance (ANOVA) was performed by application of RS1

software (BBN Software Product Corp.) using a VAX 6210 computer (Digital
EquipmentCorp.).

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CALCULATIONS
An x Cs x DF
Assay of HPMC: %w/v HPMC =
As

where:
An = Peak area of compoundin sample*.
As = Peak area average of HPMC in bracketingstandards
Cs = Concentrationof the workingstandard(%w/v)
DF = Dilutionfactor

*The sum of contributionsfrom all three peak areas for HPMC are usedto calculatethe
assay value,

MolecularWeight Characterizationof HPMC 5:

where:

MW = T_,n=
I(hM,)/,T.,n=
- l(h,) Mw = Weight-average
molecular weight
Mn = Number-average
molecular weight
Mn = T_,n=
l(h,)/,T_,n=
l(h,,M,) Mz = z-average molecular
weight
hi = SEC curve heightat the
ith volume increment
Mz = T"n=l(h)M"/'T-'n=l(h'M') Mi = Molecularweight of the
species eluted in the ith
[h] = (2P1/P2)/c retentionvolume
increment
[h] = IntrinsicViscosity
P = MW/Mn P1 = DifferentialPressure
P2 = Reference Pressure/2
c = Concentration
P = Polydispersity

RESULTS AND DISCUSSION

ASSAY METHOD DEVELOPMENT

Analysis of HPMC with RI detection produces tri-modal chromatographicseparation

as seen in Figures 1-3. The additionalformulationcomponentsof dextrose (a tonicity
adjuster) and chlorobutanol(a preservative) were also adequately resolved such that
they may be concurrently assayed, as desired, and utilized to determine system
suitability. Chlorobutanolexhibitedan extended retentiontime due to interactionswith
the columnstationary phase.

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Y

l;I '
, JJ i o, I | I

O" 20" 40- SO* O0 tile" [

Time (oinl 10" 20 30" 40"
Tlae (mini

Figure 2: Chromatogram of HPMCinOphthalmic Figure 3: Chmmatogram ofHPMCin

FormulationMatr_ OphthalmicFormulation- Expanded
Baseline

rtxJ= 1688157"x + 28962.19

R^2 = 0.9994450
1400(XX),

1200000,' /

1000000.' #/
@

_ 600000." /

411 61 88 iSO-
Tile lllnl 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
%w/v HPMC

Figure 4: Chromatogramof HPMC Standard Figure 5: HPMC Standard Linearity

1'
/

Criteria for Stability

Samples of full formulation and formulation placebo containing no HPMC were
exposed to intense light, kept at 45C, adjusted to pH 2 with 37% HCI, adjusted to pH
12 with 50% NaOH, treated with 30% H202, or left untreated. All samples, treated and
untreated, were stored at their respective conditions for 8 days. Each stressed
placebo was analyzed by the proposed analysis procedure and the resulting
chromatograms were compared with that of an unstressed placebo. There was no
evidence of interfering peaks in any of the placebos. Based on these data, it is
concluded that the method is specific and stability indicating for the determination of
HPMC.

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Standard Linearity
To determine linearity, a standard curve was generated from 20% to 120% of
formulation label claim after dilutions. Because there is no significant difference
between the Y-intercept and the origin (a=0.05, two-tailed t-test), a single point
standard is used. A summary of the data is shownin figure 5:

Accuracy Data
A sample of the formulationwas spiked at 80%, 100%, and 120% of formulation label
claim with HPMC to provide information about the accuracy of the method. A
summaryof the recovery data follows:

Table h Accuracyat 80%, 100%, and 120% of FormulationLabel Claim

Accuracyat 80% Accuracyat 100% Accuracyat 120%

Label Claim Label Claim Label Claim
ParameterPeak Peak Area . Peak Area
Area
Mean (%) 102.4 98.5 99.1
SD 0.9 1.8 1.7
% RSD _ 1.0 1.8 1.7
n 3 6 3

Day-to-Day and Person-to-Person Sample Precision

Six replicates of formulationplacebo were spiked to a concentration of 0.5000 %w/v
HPMC. The percent w/v was calculated by two analystsusingdifferent systems. On a
second day, six replicates of the same sample were obtained to provide information
concerning day-to-day reproducibility. A one-way analysis of variance (ANOVA) has
shown that the mean HPMC values obtained for Day 1, Day 2, and Person 2 are
equivalent [p-value = 0.754]. A summary of precisionfor peak area data follows:

Table Ih Method Precisionfor Day-to-Day and Person-to-Person

Parameter Day 1 _ person 2

Mean (%, w/v) 0.4918 0.4906 0.4886
SD 0.009 0.006 0.006
% RSD (.+.) 1.8 1.3 1.2
n ! 6 6 6

TECHNOLOGY TRANSFER

Determining the ruggedness of an SEC procedure provides an important measure of

assurance in correlating data produced at alternate testing sites. Transfer of the
methodology was validated by statistically evaluating accuracy results obtained for the
same sample tested by two different analysts at one facility and by a third analyst at a
remote QC site. A one-way analysis of variance (ANOVA) has shown that the mean
values of HPMC obtained by both sites are statistically equivalent [p-value = 0.28].
See Table III below for the recovery data.

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Table II1: Accuracy Data for Method Transfer

%w/v HPMC
Person 1 Person 2 Lab 2
0.5013 0.4976 0.5126
0.4954 0.4982 0.5146
0.5109 0.5013 0.4976
0.5006

Criteriaestablishedfor standard precision(%RSD < 2.5, n=3), linearity(R2 > 0.997),

and systemsuitability(Resolutionbetween last HPMC peak and dextrosepeak > 1.4)
were met for analyses performedat bothsites.

(_HARAGTERIZATIQN

HPMC raw material has been characterized utilizing triple detection of the
chromatographic eluent. Refractive Index clearly is the most sensitive means of
detecting HPMC of different molecular weights. Light scattering appears least
sensitive for low molecular weight species. An overlay of a triple detector
chromatogramfor an HPMC standard is shown in figure 6 below

Mw = 1.82M
Mz = 2.95M
P = 2.01
[h] = 6.54 dl/g

VE INDEX

P_mIi==V_tme(=L)

Figure 6: Triple Chromatogram of HPMC Standard

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Preliminary SEC work utilizing Evaporative Light Scattering detection (figure 7) has
revealed a single homogeneouspeak for HPMC elution. It is possiblethat this is due
to solute/solventinteractionsaffecting HPMC conformation and/or poor resolutionof
the high molecular weight component. Evaporative light scattering was found to be
approximately16 times as sensitivefor detection of HPMC relative to refractive index
detection.

18 29 38" 4e"
Time (min) -

Figure 7: Chromatogramof HPMC in Ophthalmic FormulationUsing

EvaporativeLight Scattering Detection.

CONCLUSIONS

Size exclusion chromatographicanalysis of HPMC is the preferred raw

material and formulationanalysis procedure because:

It is specificfor HPMC, unlikethe GC (USP) and colorimetricprocedures.

It yields significantly improved assay precision vs. the colorimetric
method.
It successfullyresolvesHPMC from formulationmatrixcomponents.

The SEC method is viable for the assay of hydroxypropylmethylcellulose

in ophthalmicformulations.

The procedure was successfullytransferred to an altemate QC facility.

The method isthereforesufficientlyrugged.

Triple detection of the HPMC peak has produced preliminary values for
Mw, Mn, Mz, polydispersity,and intrinsicviscosity.

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