Journal of Pharmaceutical and Biomedical Analysis
`19 (1999) 877–882
`
`Analysis of benzalkonium chloride and its homologs: HPLC
`versus HPCE1
`
`Shelly J. Prince a,*, Hei-Jen McLaury b, Loyd V. Allen c, Phil McLaury d
`a Southwestern Oklahoma State Uni6ersity School of Pharmacy, 100 Campus Dri6e, Weatherford, OK 73096, USA
`b Parke-Da6is Pharmaceuticals, 170 Tabor Road, Morris Plains, NJ 07950, USA
`c Uni6ersity of Oklahoma College of Pharmacy, 1110 N. Stonewall, Oklahoma City, OK 73117, USA
`d No6artis Pharmaceutical Corporation, 59 Route 10, East Hano6er, NJ 07936, USA
`
`Received 18 November 1996; accepted 23 June 1998
`
`Abstract
`
`Benzalkonium chloride (BAK) is a mixture of alkylbenzyldimethylammonium chloride homologs with n-C12H25,
`n-C14H29, and n-C16H33 comprising a major portion of the alkyl groups present. An analytical method for BAK must
`differentiate and quantitate the homologs in the BAK mixture. Reversed-phase high performance liquid chromatog-
`raphy (HPLC) separates compounds based on their affinity for a nonpolar column, which is a direct correlation to
`the compounds’ polarity. High performance capillary electrophoresis (HPCE), however, separates compounds in an
`electric field according to their charge and size. The BAK homologs are suitable for separation by either of these
`methods because their polarity and sizes differ significantly. The HPLC method employed a mobile phase of 60%
`acetonitrile and 40% 0.1 M sodium acetate buffer pH 5 pumped at 1.0 ml min (cid:28) 1, a 4.6(cid:29)250 mm cyano column with
`5 mm packing, and UV detection at 254 nm. The HPCE method utilized a run buffer of 30% acetonitrile and 70%
`0.05 M sodium phosphate pH 3.06, a 50 mm(cid:29)20 cm open silica capillary, 7.5 kV electric field and UV detection at
`214 nm. Both HPLC and HPCE demonstrated good linearity in the range of 0.025 to 0.8 mg ml (cid:28) 1 with r 2 values of
`approximately 0.99. The HPLC method produced good separation of the homolog peaks with a total analysis time
`of 25 min. HPCE run time was less than 5 min and demonstrated good separation of the three homologs. The HPLC
`method, however, was superior to HPCE in the areas of sensitivity and precision. The HPLC has been extensively
`used in the routine quantitation and qualitation of benzalkonium chloride concentrations in various products;
`however, long analysis times make this method inefficient. The HPCE method produced comparable results to the
`HPLC method but with much shorter analysis times. An HPCE analysis method, as presented here, may prove to be
`a much more useful and efficient method for the analysis of benzalkonium chloride and its homologs. © 1999 Elsevier
`Science B.V. All rights reserved.
`
`Keywords: Benzalkonium chloride; High performance liquid chromatography; High performance capillary elec-
`trophoresis
`
`* Corresponding author. Tel.: (cid:27)1 580 7743105; fax: (cid:27)1 580 7747020; e-mail: princes@swosu.edu
`1 Presented at the 1997 AAPS Annual Meeting.
`
`0731-7085:99:$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
`PII S0731-7085(98)00187-3
`
`Innopharma EX1018, Page 1
`
`

`

`878
`
`S.J. Prince et al. :J. Pharm. Biomed. Anal. 19 (1999) 877–882
`
`1. Introduction
`
`Benzalkonium chloride (BAK) is a bactericidal
`antimicrobial agent used as a preservative in over
`65% of the ophthalmic products currently avail-
`able on the market [1]. It is used to preserve otic,
`nasal and parenteral formulations as well. It pos-
`sesses surfactant properties and is also used as a
`topical antiseptic and medical equipment disinfec-
`tant. BAK is an unusual compound in that it is
`composed of a mixture of straight chain homologs
`which possess different physical, chemical and
`microbiological properties. Therefore, the propor-
`tions of these homologs in the mixture determine
`its effectiveness as a preservative and disinfectant.
`The BAK is a mixture of alkylbenzyldimethyl-
`ammonium chlorides with the general formula
`[C6H5CH2N(CH3)2R]Cl where R(cid:30)n-C8H17
`to
`n-C19H39. The C12, C14 and C16 chains comprise
`the major portion of the alkyl mixture. The ho-
`mologs do not possess identical bactericidal activ-
`ity; therefore, the USP:NF sets forth guidelines
`for the content of Benzalkonium Chloride NF as
`follows [2]:
`
`On the anhydrous basis, the content of the
`n-C12H25 homolog is not less than 40%, and the
`content of the n-C14H29 homolog is not less
`than 20%, of
`the total alkylbenzyldimethyl-
`ammonium chloride content. The amounts of
`the n-C12H25 and n-C14H29 homolog compo-
`nents comprise together not less than 70% of
`the total alkylbenzyldimethylammonium chlo-
`ride content.
`
`In general, the C12 homolog is most effective
`against yeast and fungi, the C14 homolog against
`gram-positive bacteria and the C16 homolog
`against gram-negative bacteria [3].
`The BAK is a difficult compound to analyze
`due to its multicomponent nature. The assay used
`must be both quantitative and qualitative to be
`able to identify and distinguish the different ho-
`molog components and the level of each in the
`mixture. The method should be sensitive and effi-
`cient enough to detect all of the homologs present
`in a reasonable amount of time. High perfor-
`mance liquid chromatography (HPLC) has been
`
`used extensively and successfully to separate and
`quantitate the benzalkonium chloride homologs
`[2,4–14]. The analysis times, however, typically
`range from 15 to 30 min. These lengthy analysis
`times can be quite troublesome to those doing
`routine QA:QC checks of ophthalmic products
`containing BAK where there is a large number of
`samples. High
`performance
`capillary
`elec-
`trophoresis (HPCE) is an analytical method which
`separates compounds on the basis of their charge
`and size. This method will produce adequate sepa-
`ration of the homologs because of their incremen-
`tal sizes and because the quaternary ammonium
`molecules carry a positive charge, as shown by
`Althia et. al. in a study of the analysis of BAK
`and histamine acid phosphates mixture by using
`HPCE [15]. The purpose of this study was to
`compare the analysis of BAK using HPLC to
`analysis using HPCE.
`
`2. Materials and methods
`
`2.1. Sol6ents and chemicals
`
`The BAK was supplied as a 50% aqueous solu-
`tion from Spectrum (Gardena, CA). The C12, C14,
`and C16 homologs were purchased from Sigma
`(St. Louis, MO). The acetonitrile and sodium
`acetate were purchased from Fisher Scientific
`(Fair Lawn, NJ), and the sodium phosphate,
`monobasic was purchased from Mallinckrodt
`(Paris, KY). All other chemicals used in the ana-
`lytical methods were HPLC grade. Water was
`purified by using a Milli-Q water system from
`Millipore (Bedford, MA).
`The composition of
`the BAK sample from
`Spectrum Chemicals used for HPLC analysis was
`64.64% C12, 27.49% C14, and 7.86% C16 homolog.
`In comparison, the BAK sample prepared in the
`laboratory from the individual homologs used for
`HPCE analysis consisted of 41.84% C12, 32.65%
`C14, and 25.51% C16 homolog. A sample concen-
`tration of 0.1 mg ml(cid:28) 1 was used for determina-
`tion of precision, and the concentrations for
`determination of linearity were 0.025, 0.05, 0.1,
`0.2, and 0.4 mg ml(cid:28) 1, with the exception of the
`BAK sample used in the HPLC method. The
`
`Innopharma EX1018, Page 2
`
`

`

`S.J. Prince et al. :J. Pharm. Biomed. Anal. 19 (1999) 877–882
`
`879
`
`BAK concentration used for HPLC precision was
`0.2 mg ml (cid:28) 1, and the concentrations used for
`linearity were 0.05, 0.1, 0.2, 0.4, and 0.8 mg ml(cid:28) 1
`due to the low concentration of the C16 homolog
`in the BAK mixture.
`
`2.2. Equipment
`
`The HPLC system consisted of a Waters M-
`6000A Solvent Delivery System, Micromeritics
`728 Autosampler, Rheodyne 7010 Electrically Ac-
`tuated Valve, Waters Model 441 Fixed Wave-
`length Absorbance Detector and Waters 745 Data
`Module. A Beckman P:ACE System 2100 was
`used for the HPCE analysis and Beckman System
`Gold software was used for data collection.
`
`2.3. Experimental conditions
`
`The column used for the HPLC method was a
`250(cid:29)4.6 mm i.d. stainless steel column filled with
`Bakerbond cyano 5 mm packing and was kept at
`room temperature. The mobile phase consisted of
`acetonitrile–sodium acetate (pH 5.0; 0.1 M)
`(60:40, v:v), and the pH of the buffer was ad-
`justed by adding glacial acetic acid. Elution was
`performed at a flow rate of 1.0 ml min(cid:28) 1 and the
`injection volume was 20 ml. The absorbance was
`monitored at 254 nm.
`The capillary used for the HPCE method was a
`Beckman P:ACE System:eCAP 50 mm i.d.(cid:29)26
`cm open silica capillary with a 20 cm effective
`length maintained at 20°C. The run buffer was
`acetonitrile-sodium phosphate, monobasic (pH
`3.06 adjusted using phosphoric acid; 0.05M)
`(30:70, v:v), and the applied voltage was 7.5 kV.
`All samples were injected into the silica capillary
`by 5 s of pressure at 0.5 c, a volume of approxi-
`mately 12.5 nl, and detected by absorbance at 214
`nm.
`
`3. Results and discussion
`
`Both the HPLC and HPCE methods used for
`analysis of benzalkonium chloride solutions pro-
`duced adequate resolution of
`the three major
`homolog peaks corresponding to the C12, C14, and
`
`C16 homologs (Figs. 1 and 2). The HPLC method
`produced an analysis time of 25 min for all three
`homologs, whereas the HPCE method produced
`an analysis time of only 5 min. The sensitivities of
`the HPLC and HPCE methods were approxi-
`mately 2 and 5 mg ml (cid:28) 1, respectively. The HPCE
`method was less sensitive due to a shorter path
`length in the detector and a smaller injection
`volume (12.5 nl for HPCE vs. 20 ml for HPLC).
`
`Fig. 1. Chromatogram of benzalkonium chloride 0.2 mg ml (cid:28) 1.
`
`Innopharma EX1018, Page 3
`
`

`

`880
`
`S.J. Prince et al. :J. Pharm. Biomed. Anal. 19 (1999) 877–882
`
`Fig. 2. Electropherogram of benzalkonium chloride 0.1 mg ml (cid:28) 1.
`
`The sensitivity for both methods is adequate,
`however, because the minimum concentration of
`BAK in ophthalmic formulations is 40 mg ml(cid:28) 1
`(0.004%).
`For both HPLC and HPCE systems, the preci-
`sion was determined by the intraday variation of
`five injections of each homolog and BAK mixture
`and the interday variation for 5 days (Table 1).
`The precision for HPLC was higher than for
`HPCE in most instances. This was probably due
`to problems encountered with capillary degenera-
`tion, which was somewhat resolved by recondi-
`tioning the column after every five sample runs.
`The day 5 HPCE results for the C16 homolog and
`BAK samples are not reported because the capil-
`lary became unusable and reconditioning efforts
`failed; therefore, the data could not be obtained.
`This problem may have been due to adsorption of
`the C16 homolog to the silica capillary. The main
`causes of adsorption to the silica capillary are
`ionic interactions between cationic solutes and the
`negatively charged wall and hydrophobic interac-
`
`tions [16]. Because the C16 homolog is more hy-
`it
`drophobic than the C12 or C14 homologs,
`exhibited a higher degree of hydrophobic interac-
`tions. Furthermore, all benzalkonium chloride ho-
`mologs are large cations, so all demonstrated
`ionic interactions with the negatively charged wall
`to various degrees. Adsorption to the silica capil-
`lary walls can be minimized by increasing buffer
`concentration which will reduce solute interac-
`tions, by using buffers at the extremes of pH and
`also by coating the capillary wall [16]. The 0.05 M
`sodium phosphate buffer system used in this study
`was at low pH (3.06), but a higher buffer concen-
`tration exceeded the maximum suggested operat-
`ing current.
`The results for the standard curves obtained by
`the HPLC and HPCE methods are given below:
`HPLC
`C12 Homolog: peak area(cid:30)5132(cid:27)314897 con-
`centration (r 2(cid:30)0.9988)
`C14 Homolog: peak area(cid:30)5230(cid:27)330413 con-
`centration (r 2(cid:30)0.9991)
`
`Innopharma EX1018, Page 4
`
`

`

`S.J. Prince et al. :J. Pharm. Biomed. Anal. 19 (1999) 877–882
`
`881
`
`Table 1
`Intraday and interday variation for benzalkonium chloride and its homologs using HPLC and HPCE
`
`Day 1
`
`HPLC
`2.38a
`3.31
`4.87
`2.59
`
`HPCE
`11.06
`0.96
`6.63
`4.67
`
`C12
`C14
`C16
`BAK
`
`C12
`C14
`C16
`BAK
`
`a RSD%, n(cid:30)5.
`
`Day 2
`
`Day 3
`
`Day 4
`
`Day 5
`
`Interday
`
`2.26
`2.77
`3.73
`1.97
`
`12.46
`1.82
`6.47
`2.30
`
`2.78
`5.91
`2.99
`0.99
`
`3.37
`4.02
`6.55
`3.40
`
`1.74
`2.92
`2.53
`3.30
`
`8.10
`5.19
`9.20
`5.90
`
`3.85
`3.86
`3.34
`1.13
`
`8.30
`9.37
`—
`—
`
`0.50
`1.16
`1.62
`0.93
`
`3.57
`19.86
`9.01
`21.01
`
`C16 Homolog: peak area(cid:30)6768(cid:27)310245 con-
`centration (r 2(cid:30)0.9942)
`BAK mixture: peak area(cid:30)2874(cid:27)303956 con-
`centration (r 2(cid:30)1.0000)
`
`HPCE
`C12 Homolog: peak area(cid:30) (cid:28)6.371(cid:29)10 (cid:28) 3(cid:27)
`6.205(cid:29)10(cid:28) 3 concentration (r 2(cid:30)0.9995)
`C14 Homolog: peak area(cid:30) (cid:28)1.795(cid:29)10 (cid:28) 3(cid:27)
`5.062(cid:29)10(cid:28) 3 concentration (r 2(cid:30)0.9981)
`C16 Homolog: peak area(cid:30) (cid:28)2.654(cid:29)10 (cid:28) 2(cid:27)
`5.400(cid:29)10(cid:28) 3 concentration (r 2(cid:30)0.9960)
`BAK mixture: peak area(cid:30)4.537(cid:29)10 (cid:28) 3(cid:27)
`5.125(cid:29)10(cid:28) 3 concentration (r 2(cid:30)0.9997)
`The linearity for both the HPLC and HPCE
`methods is comparable; therefore, both methods
`are useful for the accurate quantitation of benzal-
`konium chloride as well as the individual ho-
`mologs in the mixture.
`
`4. Conclusion
`
`A high performance capillary electrophoresis
`assay for qualitation and quantitation of benzal-
`konium chloride and its homologs has been inves-
`tigated. The primary advantage of the HPCE
`method, when compared to HPLC,
`is a much
`shorter analysis time. The linearity and sensitivity
`of both methods is comparable; however, the
`precision for the HPLC method was better than
`
`for HPCE. This problem may have been due in
`part to sorption of the C16 homolog to the capil-
`lary wall.
`In summary, HPCE has the potential to be a
`more efficient and useful method for BAK analy-
`sis than the established HPLC methods.
`
`References
`
`[1] G. Hecht, in: A.R. Gennaro (Ed.), Remington: The Sci-
`ence and Practice of Pharmacy, Mack, PA, 1995, pp.
`1563–1573.
`[2] United States Pharmacopeia 23:National Formulary 18,
`United States Pharmacopeial Convention, Inc., Mary-
`land, 1995, pp. 2218–2219.
`[3] J.J. Merianos, in: S.S. Block (Ed.), Disinfection, Steriliza-
`tion, and Preservation, Lea and Febiger, PA, 1991, pp.
`225–255.
`[4] M.R. Euerby, J. Clin. Hospital Pharm. 10 (1985) 73–77.
`[5] A. Bettero, A. Semenzato, C.A. Benassi, J. Chromatogr.
`507 (1990) 403–407.
`[6] L.J. Cohn, V.J. Greely, D.L. Tibbetts, J. Chromatogr.
`321 (1985) 401–405.
`[7] T.Y. Fan, G.M. Wall, J. Pharm. Sci. 82 (1993) 1172–
`1174.
`[8] A. Nakae, K. Kunihiro, G. Muto, J. Chromatogr. 134
`(1977) 459–466.
`[9] D.F. Marsh, L.T. Takahashi, J. Pharm. Sci. 72 (1983)
`521–525.
`[10] G. Ambrus, L.T. Takahashi, P.A. Marty, J. Pharm. Sci.
`76 (1987) 174–176.
`[11] R.C. Meyer, J. Pharm. Sci. 69 (1980) 1148–1150.
`[12] L. Elrod Jr., T.G. Golich, J.A. Morley, J. Chromatogr.
`625 (1992) 362–367.
`
`Innopharma EX1018, Page 5
`
`

`

`882
`
`S.J. Prince et al. :J. Pharm. Biomed. Anal. 19 (1999) 877–882
`
`[13] A. Gomez-Gomar, M.M. Gonzalez-Aubert, J. Garces-
`Torrents, J. Costa-Segarra, J. Pharm. Biomed. Anal. 8
`(1990) 871–876.
`[14] G. Parhizkari, R.B. Miller, C. Chen, J. Liq. Chromatogr.
`18 (1995) 553–563.
`
`[15] K.D. Altria, J. Elgey, J.S. Howells, J. Chromatogr. B 686
`(1996) 111–117.
`[16] D.N. Heiger, High Performance Capillary Electrophore-
`sis—An Introduction, Hewlett-Packard, France, 1992,
`pp. 32–35.
`
`.
`
`Innopharma EX1018, Page 6
`
`