Metrics EX1034, Page 1
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`Metrics EX1034, Page 1
`
`

`
`Journal of
`Pharmaceutical
`Sciences
`
`Edward G. Feldmann, Ph.D.
`Editor
`Pharmaceutical Consultant
`Services, Falls Church, Virginia
`
`EDITORIAL ADVISORY BOARD
`James E. Axelson, Ph.D., University of
`British Columbia, Canada
`
`Meir Bialet, Ph.D., The Hebrew University
`of Jerusalem, Jerusalem, Israel
`
`Davi(i J. Cutler, Ph.D, University of
`Sydney, Sydney, Australia
`
`Andrew T. McPhail, Ph.D., Duke University,
`Durham,NC
`
`William E. Evans, Pharm.D., St. Jude
`Children's Research Hospital, Memphis, TN
`
`Kamal K. Midha, D.Sc., University of
`Saskatchewan, Saskatoon, Canada
`
`William 0. Foye, Ph.D., Massachusetts
`College of Pharmacy and Allied Health
`Sciences, Boston, MA
`
`Emilio Gelpi, Ph.D., CID-CSIC, Barcelona,
`Spain
`
`David J. W. Grant, D.Sc., University of
`Mirmesota, Minneapolis, MN
`
`DetlefGrtiger, Ph.D., Institute for Plant
`Biochemistry, Halle, Germany
`
`Tsuneji Nagai, Ph.D., Hoshi University,
`Tokyo, Japan
`
`MichaelS. Roberts, Ph.D., University of
`Queensland, Brisbane, Australia
`
`Malcolm Rowland, Ph.D., University of
`Manchester, Manchester, UK
`
`Jilriiang Shen, Ph.D., The State
`Pharmaceutical Administration of
`China, Beijing, China
`
`:/ I
`
`!
`
`II
`li
`
`David W. A. Bourne, Ph.D., University of
`Oklahoma, Oklahoma City, OK
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`Robert L. Bronaugh, Ph.D., Food and Drug
`Administration, Washington, DC
`
`Iris H. Hall, Ph.D., University of North
`Carolina, Chapel Hill, NC
`
`Mikl6s Simonyi, D.Sc., Hungari'anAcademy
`of Sciences, Budapest, Hungary
`
`Manabu Hanano, Ph.D., University of
`Toky~ Toky~Japan
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`Felix Theeuwes, D.Sc.,Alza Corporation,
`Palo Alto, CA
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`Win L. Chiou; Ph.D., University of Illinois,
`Chicago, IL
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`William J. Jusko, Ph.D., State University of
`New York at Buffalo, Buffalo, NY
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`DanielL. Weiner, Ph.D., Syntex Research,
`Palo Alto, CA
`
`James C. Cloyd, Pharm.D., University of
`Minnesota, Minneapolis, MN
`
`Stanley A. Kaplan, Ph.D., A.L. Laboratories,
`Inc., Baltimore, MD
`
`Joei L. Zatz, Ph.D., Rutgers University,
`Piscataway, NJ
`
`JOURNAL STAFF
`Maralyn E. Kaufman, Ph.D.
`Associate Editor
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`Sharon Boots, Ph.D.
`Consultant
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`Susan E. Ysais
`Administrative Assistant
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`Kathleen Nash
`Proofreader
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`THE AMERICAN PHARMACEUTICAL ASSOCIATION
`
`President:
`Lowell J. Anderson, Pharmacist
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`Executive Vice President:
`John A. Gans, Pharm.D.
`
`President-elect:
`Tim L. Vordenbaumen, Pharmacist
`
`Immediate Past President:
`Robert J. Osterhaus, Pharmacist
`
`Treasurer:
`Jean Paul Gagnon, Ph.D.
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`Board of Trustees:
`Tery Baskin, Pharmacist; Leonard N.
`Camp, Pharmacist; Thomas S. Foster,
`Pharm.D.; Ro"bert D. Gibson; Pharm. D.;
`Clark H. Gustafson, Phar:ii:J.acist; John
`Hasty, Pharmacist; Gary W. Kadlec,
`Pharmacist; Calvin H. Knowlton, Ph.D.;
`Shirley P. McKee, Pharmacist; Martha
`M. Rumore, l'harm.D.
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`House of Delegates:
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`Speaker:
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`Secretary:
`John A. Gans, Pharm.D.
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`Senior Director, Programming and
`Publicll,tions:
`
`James P. Caro, Pharmacist
`
`~
`ii
`'I
`I
`li
`
`The Journal of Pharmaceutical Sciences (ISSN 0022-3549) is published monthly by the American Pharll).aceutical Association (APhA) at 2215 Constitution Ave., NW,
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`Copyright© 1993, American Pharmaceutical Association, 2215 Constitution Ave., NW, Washington, DC 20037-2981: all rights reserved.
`
`Metrics EX1034, Page 2
`
`

`
`Determination of Benzalkonium Chloride in Ophthalmic
`Solutions Containing Tyloxapol by Solid-Phase Extraction and
`Reversed-Phase High-Performance Liquid Chromatography
`
`TONY Y. FANx AND G. MICHAEL WALL
`Received September 3, 1992, from Analytical Chemistry, Alcon Laboratories, Inc., 6201 South Freeway, Fort Worth, TX 76134.
`publication February 25, 1993.
`
`Accepted for
`
`Abstract D A procedure using solid-phase extraction (Supelcoclean
`CN) followed by HPLC [Beckman Ultrasphere CN, acetonitrile:phos(cid:173)
`phate solution (60:40, v/v)] was developed and validated to quantitate the
`quaternary ammonium preservative benzalkonium chloride in an exper(cid:173)
`imental ophthalmic formulation containing the polymeric material tylox(cid:173)
`apol. This procedure makes routine determinations of benzalkonium
`chloride at concentrations of 0.0035 to 0.01% simpler than the traditional
`ion-pairing colorimetric methods. This method is quick, specific, and
`especially useful for drug product stability studies. In addition, because
`the method distinguishes each homologue, it can be extended to
`routinely determine the homologue ratio for quality control purposes.
`
`Benzalkonium1 chloride (BAC; 1) is widely used as an
`antimicrobial preservative in aqueous pharmaceutical prep(cid:173)
`arations; especially in ophthalmic solutions. BAC is actually
`a mixture of n-alkylbenzyldimethyl ammonium chlorides
`with n-alkyl chain lengths varying from C8 to C18.1 Because
`the homologues present different bactericidal activity,2 it is
`· sometimes necessary to determine not only the total amount
`of BAC but also the ratio of its homologues in the formula(cid:173)
`tions. Among the European,3 British,4 and United Statesl
`pharmacopoeias, only the USP specifies the percentage of
`individual homologues: (1) the content of the n-C12H25 ho(cid:173)
`mologue is not <40.0%, (2) the content of the n-C14H29
`homologue is not <20.0%, and (3) the total content of the
`C12H25 and C14H29 homologues comprise together not
`
`1
`
`[R is CH2CHzO(CH2CH20)mCH2CH20H;
`m is 6 to 8; n is not more than 5]
`
`<70.0% of the total alkylbenzyldimethylammonium chloride
`content. 1
`A quick and easy method for the determination of both the
`total and relative homologue ratio is desirable. HPLC with
`UV detection is a desirable technique because of its separa(cid:173)
`tion capability and suitability for automated routine analysis.
`For ophthalmic preparations, such determinations are not
`trivial because BAC is usually present in very low concen(cid:173)
`trations (0.007-0.01 %, w/v), requiring low UV detection
`wavelengths (210-215 nm) for good sensitivity. Because other
`excipients are usually present in much higher concentrations,
`interference at these low wavelengths is a common problem.
`Over the years, various specific and nonspecific methods
`have been developed for the determination of BAC. These
`have included extraction by complexing BAC with various
`dyes,G-9 titration of quarternary ammonium compounds with
`iodate10 or tetraphenylboron, 11 pyrolysis and subsequent gas
`chromatography,l2 chemical ionization mass spectrometry,l3
`and HPLC of simple aqueous solutions.14-16 In addition, the
`USP monograph describes a reversed-phase HPLC method to
`determine the homologue ratio of BAC raw material at a
`relatively high concentration (4 mg/mL) and a titration
`method to determine the total content of BAC in solution
`based on potassium iodate equivalents.1 None of these meth(cid:173)
`ods could be directly used for the analysis of BAC in complex
`ophthalmic solutions because either they do not have the
`required specificity and sensitivity or they can not completely
`separate BAC from the matrix. Interferences have been
`observed by the presence of polymeric material, -suspended
`particles, and active ingredients. These kinds of samples are
`not suitable for direct HPLC injection and therefore require
`some kind of sample preparation prior to HPLC.
`The purpose of this study was to develop an HPLC method
`appropriate for measuring BAC in an experimental oph(cid:173)
`thalmic solution containing BAC (0.007%, w/v) and the
`polymeric material tyloxapol (0.25%, w/v). Tyloxapol (2), a
`polymeric alkyl aryl polyether alcohol commonly used as an
`emulsifier or surfactant, presented a problem for HPLC
`analysis of BAC because it produced a large solvent front that
`partially masked the BAC peaks. The USP HPLC method for
`BAC could not be used because of the interference oftyloxapol
`and the lack of sensitivity at 254 nm. To solve this problem,
`a combination solid-phase extraction (SPE)/HPLC procedure
`was developed. The combination of SPE sample clean-up with
`the resolving capability of HPLC provided a powerful tool for
`the routine analysis of complex ophthalmic solutions. This
`paper describes a SPE/HPLC method suitable for the deter(cid:173)
`mination of the total BAC content as well as each homologue
`ratio in an experimental ophthalmic solution containing
`tyloxapol. This technology should be applicable to other types
`of complex formulations.
`
`!
`
`1172 I Journal of Pharmaceutical Sciences
`Vol. 82, No. 11, November 1993
`
`0022-3549/91/1100-1172$02.50/0
`© 1993, American Pharmaceutical Association
`
`Metrics EX1034, Page 3
`
`

`
`Experimental Secti.on
`Apparatus-An HPLC system that consisted of a Hewlett-Packard
`1090 quaternary pump (Hewlet-Packard, Fullerton, CA), a Waters
`Associates (Waters, Milford, J'4A) WISP 710B autoinjector, 490
`programmable multiwavelength detector, and a Spectra-Physics
`ChromJet Integrator (Spectra-Physic$, San Jose, CA) was used. All
`HPLC separations were performed isocratically on a 5 p,m (150 x 4.6
`mm, i.d.) Ultrasphere cyano (nitrile-bonded silane, CN) column
`(Beckman, San Ramon, CA). A Burdick & Jackson, 12-port solid(cid:173)
`phase extraction manifold (Burdick & Jackson, Muskegon, MI) was
`used for the sample extractions. The manifold was connected to an
`in-house vacuum source, and a control valve was used to regqlate the
`magnitude of vacuum applied. The container itself was large enough
`to allow 12 10-mL volumetric flasks to be attached to the rack at the
`same time for sample collection. A stop valve was also provided on
`each port for individual flow stoppage. All extractions were performed
`with Supelcoclean (Supelco, Bellefonte, P A) disposable cy,ano · SPE
`columns with 1-mL capacity.
`'
`Reagents and Solutions-All reagents and solvents were reagent
`or HPLC grade and purchased from J. T. Baker (Phillipsburg, NJ).
`The phosphate solution was prepared by dissolving 6 mL of concen(cid:173)
`trated phosphoric acid (reagent grade) in 1950 mL of distilled water.
`The pH was adjusted to 5.0 by the addition of 50% NaOH solution, and
`the total volume was adjusted to 2 L with distilled water. The mobile
`phase was acetonitrile:phosphate solution (60:40, v/v), and the wash
`solvent was acetonitrile:phosphate solution (30:70, v/v). An experi(cid:173)
`mental ophthalmic formulation was used for ,this study that con- .
`tained proprietary drug (0.1%), mannitol (4,7%), sodium citrate
`(0.04%), citric acid (0.02%), tyloxapol (0.!;!5%), BAC (0.007%), and
`edetate disodium (0.01%; all w/v). BAC was deleted for validation
`purposes, making this formulation a BAC vehicle.
`Sample Preparation-Test solutions were prepared by the addi(cid:173)
`tion of appropriate amounts of BAC to an ophthalmic solution BAC
`vehicle (an ophthalmic solution containing all ingredients except
`BAC) that contained tyloxapol (0,025%, w/v} as one of the ingredients.
`A flow control valve was attached to each SPE column, and the whole
`unit was placed onto the female luer fitting of the vacuum manifold.
`Reduced pressure (~ 10 mmHg) was applied to the manifold with an
`in-house vacuum line. The SPE columns were conditioned with
`acetonitrile (2 mL) followed by distilled water (2 mL). When the level
`of the distilled water had reached ~ 1 mm above the top of the column
`packing, slow addition of the test sample (4 mL) was initiated.
`(Caution was taken not to disturb or dry out the column packing bed).
`After the sample had passed through, the column was washed with
`wash solvent (2 mL). The vacuum was disconnected, a 10-mL
`volumetric flask was placed under each SPE column, then the reduced
`pressure was applied again. The retained BAC was eluted from the
`column with mobile phase (5 mL), the vacuum was then disconnected,
`and the fla.sks were removed and diluted to volume with distilled
`water. These samples were directly analyzed by HPLC.
`HPLO Assay Procedure-The mobile phase was mixed and
`filtered before use. The chromatographic system employed a flow rate
`of2 mL/min, 100cp,L injection voluJUe, 10-min run time, UV detection
`(210 nm) at 0.01 AUFS, a recorder attenuation of xs, and a chart
`speed of 0.5 em/min. After a stab.le baseline was established, replicate
`standards were injected to ensure reproducibility prior to sample
`analysis. System suitability criteria were established: relative stan(cid:173)
`dard deviation of six replicate injections, 52.0%; resoh1tion between
`the 0 12 and 0 14 pealts, ;:;:2; tailing factor for the 0 12 peak, s2; and
`number of theoretical plates, > 3000 plates/column. A standard was
`in&erted between every six samples. The BAO homologues were
`quantitated by the calculation described in the USP HPLC method, 1
`taking into consideration the molecular weight of each homologue.
`The percentage of each BAC homologue and the percent recovery of
`total BAC were calculated as follows: %of each ho1Ilologue = 100 A/B,
`and % recovery = 100 Bsamp!JBstandard> where A is the product of the
`area obtained from each homologue multiplied by its molecular
`weight and B is the sum of all of these products. The molecular
`weights of the 0 10, 0 12, 0 14, 0 16, and 0 18 BAC homologues (most
`common) are 312, 340, 368, 396, and 424, respectively.
`
`Results and Discussion
`A combination SPE/HPLC method was developed for de(cid:173)
`termination of BAC in ophthalmic solutions containing the
`
`polymeric material tyloxapol. The low concentration of BAC
`in this experimental ophthalmic formulation (0.007%, w/v)
`necessitated using low UV wavelength (210 nm) detection for
`increased sensitivity. However, this low UV wavelength
`magnified interference problems encountered with direct
`HPLC analysis: tyloxapol eluted as a large peak after the
`solvent front, making quantitation of the BAC C12 homologue
`difficult and the BAC C 10 homologue impossible (Figure 1).
`Solid-phase extraction was employed prior to HPLC to remove
`most of the interference by tyloxapol and, thereby, reduce
`excessively long run tim~s (Figure 2). Only SPE columns from
`one manufacturer were used to obtain the data herein because
`vendor-to-vendor variability in SPE columns has beenprevi-
`ously reported for cyano cartridges.l 7
`.
`·
`Validation data were generated for this method with the
`experimental formulation. Linearity was satisfactory (Table
`1). Three six-point vehicle standard curves (duplicate samples
`at three different concentrations) were generated for the
`experimental ophthalmic formulation with concentrations of
`BAC ranging from 50 to 150% Label [0.007% (w/v) BAC =
`100% Label; the concentration range for injected samples was
`0.014-0.042 mg/mL]. The curves obtained were linear (r2 =
`0.999) and the y-intercepts, ranging from 1.2 to 3.1 %, were
`small enough to justify the use of a single-point standard
`(Table I). Total recoveries were acceptable and in the range
`97-103% (Table 1). The precision was also satisfactory (Table
`II). The injection of three sets of six vehicle standard repli(cid:173)
`cates [0.007% (w/v) BACJ gave acceptable values for relative
`standard deviations (ranging from 0.74 to 1.52%).
`Though good results were obtained with this method most
`of the time, spurious results were infrequently observed:
`occasionally, an unexpected value (low or high by ~2%) was
`obtained for BAC. A significant amount of effort was expended
`trying to track down spurious data that. might have been the
`
`• b
`
`I I I I
`
`d
`
`0
`
`• .. c:
`Q. .. • a:
`...
`~ • ;
`
`Q
`
`0
`
`5
`
`10
`
`Time
`
`(minutes)
`
`Figure 1-A typical HPLC chromatogram of BAC sample prior to
`extraction: (a) drug; (b) tyloxapol; (c) BAC 0 10; (d) BAC 0 12; (e) BAC C14;
`(f) BAC C1s; (g) BAC C1s·
`
`Journal of Pharmaceutical Sciences I 1173
`Vol. 82, No. 11, November 1993
`
`Ill
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`Metrics EX1034, Page 4
`
`

`
`......... ~~--,
`
`Table 11-EJAC Vehicle Stanclard Replicates 8
`
`Concentration,
`mg/mL
`
`Area Counts (Recovery %)
`
`Replicate Set 1 Replicate Set 2 Replicate Set 3
`
`0.028
`0.028
`0.028
`0.028
`0.028
`0.028
`Rei. Std. Dev., %
`Avg. Recovery, %
`
`260 543 (101)
`259 225 (101)
`. 256 120 (99)
`260 979 (101)
`256 638 (1 00)
`257 997 (1 00)
`0.78
`.
`100
`
`259 993 (1 01)
`260 055 (1 01)
`256 736 (1 00)
`260 260 (1 01)
`257 514 (100)
`261 950 (1 02)
`0.74
`101
`
`258 516 (100)
`256 329 (1 00)
`253 007 (98) .
`263 153 (102)
`263 088 (1 02)
`258 469 (1 00)
`1.52
`100
`
`a Samples of 1 00% target concentration (0.028 mg/mL) were prepared
`by SPE and were analyzed by HPLC (see Experimental Section for
`conditibns).
`
`tyloxapol. The sample clean-up step (i.e., SPE extraction) and
`lower wavelength detection represent improvements over
`existing methods (e.g., the USP HPLC BAC method) that
`allow for analysis of BAC at low concentrations in tyloxapol(cid:173)
`containing formulations. The method should be easily
`adapted to other solutions arid suspensions containing poly(cid:173)
`meric material. The disadvantage of an infrequent spqrious
`result was easily remedied by reassay of the suspect sample.
`It is thought that the occasional lack of precision was a result
`of variability between the SPE columns. None of these
`problems was deemed significant enough to preclude the use
`of this method because the magnitude of error for total BAC
`content was seldom > 2%. Improvements in the commercially
`available SPE columns or alterations in the HPLC conditions
`may render this technique even more reliable, but until then,
`it is still an acceptable method for the analysis of BAC in
`complex ophthalmic solutions.
`
`References and Notes
`1. United States Pharmacopeia, 22nd rev.; U.S. Pharmacopeia!
`Convention: Rockville, MD, 1990; p 1905. ·
`2. Giles, R.; Daoud, N. N.; Gilbert, P.; Dickson, N. A. J. Pharm.
`Pharmacal. 1983, 34(suppl.), 110.
`3. European Pharmacopeia, 2nd. ed.; Council of European: France,
`1985, Part II-9, p 371.
`4. British Pharmacopeia, vol. 1; British Pharmacopeia Commission:
`·
`U.K., 1988; p 63.
`.
`~· Auerbach M. E. Anal. Chern. 1943, 15, 492.
`'
`6. Colichtnan, E. L. Anal. Chern. 194 7, 19, 430.
`7. Ballard, C. W.; Isaacs, J.; Scott, P. G. W. J. Pharin: -Fharrnacol.
`~954, 6, 971.
`8. Chatten, L. G.; Okamura, K. 0. J. Pharm. Sci. 1973, 62, 328.
`9. Marsh, D. F.; Takahashi, L. T. J. Pharm. Sci. 1983, 72, 52i.
`10. Brown, E. R. J. Pharm. Pharmacal. 1963, 15, 379.
`11. Metcalfe,· L. D.; Martin, R. J.; Schmitz, A. A. J. Am. Oil Chern.
`Soc. 1966, 43, 355.
`12. Jennings, E. C.; Mitchner, H. J. Pharm. Sci. 1967, 56, 590.
`13. Daoud, N. N.; Crooks, P. A.; Speak, R.; Gilbert, P. J. Pharm. Sci.
`1983, 72, 290.
`14. Meyer, R. C. J, Pharm. Sc.i. 1980, 69, 148.
`15. Ambrus, G.; Takahashi, L. T.; Marty, P. A. J. Pharm. Sci. 1987,
`76, 174.
`16. Comez-Gomar, A.; Gonzalez-Aubert, M. M.; Garces-Torrents, J.;
`Costa-Segarra; J. J. Pharm. Biomed. Anal. 1990, 8, 871.
`17. Moors, M.; Massart, D. L. Anal. Chim: Acta 1992,262, 135.
`18. Van Horne; K. C. Sorbent Extraction Technology; Analytichem
`· hiternational: Harbor City, CA, 1985; pp 14-16.
`19. Moors, M.; Massart, D. L. J. Pharm. Biomed. Anal. 1991, 9, 129.
`20. Marko, V.; Radova, K.; Novak, I. J. Liq. Chromatogr. 1991, 14,
`1659.
`.
`21. Vendrig, D. E.M. M.; Holthuis, J. J. M. J. Chromatogr. 1987,
`414, 91.
`..
`
`• • c
`& • • a:
`I
`
`d
`
`,i'~"'--·--~·. \ \J \. •
`
`0
`
`5
`
`I
`10
`
`Time
`
`(minute•)
`
`Figure 2-A typical HPLC chromatogram of BAG sample after SPE
`extraction: (a) BAG C10 ; (b) BAG C12; (c) BAG C14; (d) BAG C16; (e) BAG
`C1a·
`
`Table 1-BAC VetlicJe Standard Curvesa
`
`Concentration,
`mg/mL
`
`Area Counts (Recovery %)
`
`Standard
`Curve 1
`
`Standard
`Curve 2
`
`Standard
`Curve 3
`
`0.014
`0.014
`0.028
`0.028
`0.042
`0.042
`f
`Avg. Recovery, %
`
`128 352 (98)
`128 641 (98)
`263 479 (98)
`263 836 {98)
`393 348 (97)
`397 215 (98)
`0.999
`98
`
`128 372 (98)
`125 067 (97)
`264 238 (101)
`26Q 979 (1 00)
`394 036 (1 03)
`389 692 (1 00)
`0.998
`100
`
`123153 (98)
`124 270 (98)
`258 765 (1 00)
`263 762 (1 02)
`397 71 0 (1 03)
`392 404 (1 01)
`0.999
`100
`
`a Samples ranging from 50% to 150% of the target concentration
`(0.028 rilg/mL) were prepared by solid~phase extraction and were
`ana!yzed by high~performance liquid chromatography.
`
`result of one or mor13 possibilities; for example, allowing the
`SPE) column to dry after conditioning and' SPE column-to(cid:173)
`column variability. The drying of a column after conditioning
`probably resulted in desolvation of the column packing and,
`heiiCe, Variable adsorption characteristic8. 18 Also, batch-to(cid:173)
`batch variation has been preViously reported for disposable
`SPE columns for basic drugs on cya:no (BakerbondHi) or Cl8
`(Ba:kerbond~ 9 or Polymer Institute20 brands), and catharan(cid:173)
`thus alkaloids on diol (Analytichem.21) cartddges, suggesting
`the possibility of poor quality control of the SFE column
`packing process.
`/
`In conclusion, this method was proven to be sensitive,
`spt:l'cific, precise, and accurl;lte for the SPE/HPLC analysis of
`BAC in an experimental ophthalmic solution containing
`
`117 4 I Journal of Pharmaceutical Sciences
`Vol. 82, No. 11, November 1993
`
`Metrics EX1034, Page 5