Journal of Clinical and Hospital Pharmacy (1985) 10,73-77.
`
`.
`HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF
`BENZALKONIUM CHLORIDES-VARIATION IN COMMERCIAL
`PREPARATIONS
`
`Melvin R. Euerby*
`Quality Control Laboratory, Deparrment of Pharmacy, Leicester Royal Infirmary, Leicesrer LEI 5 WW, U . K.
`
`SUMMARY
`High performance liquid chromatography (HPLC) has been used to iden-
`tify and determine the various alkyl benzyldimethylammonium chloride
`homologues (Cro to C, 6 ) present in commercial benzalkonium chloride
`preparations. The assay is especially suited for routine quality control work
`since it has proved to be quick and easily reproducible. It has been found
`that there can be a large degree of variation in the quality of differing
`benzalkonium chloride sources.
`
`I N T R O D U C T I O N
`Benzalkonium chloride is a mixture of n-alkyl benzyldimethylammonium chlorides
`with n-alkyl varying from C, to CIS. It is apparent that, depending on the source
`of origin, batches of benzalkonium chloride can contain differing amounts of each
`homologue. This has severe implications in that the relative amounts of the different
`alkyl benzyldimethylammonium chloride homologues can greatly affect both the anti-
`microbial spectrum and activity of the benzalkonium chloride (1-4). It is therefore
`important to be able to determine the proportion of the homologues, in order to
`standardize the preservative activity of benzalkonium chlorides. Looking at the
`British, European and United States Pharmacopoeias, only the latter sets limits on the
`percentage of C,, and C,, homologues (not less than 400, and 20y0 of the C,, and
`C,, homologues respectively and together they must comprise at least 70°0 of the
`total benzalkonium chloride content). All three Pharmacopoeias determine the
`benzalkonium chloride total content by the addition of a known excess of iodide to the
`sample. The quaternary ammonium iodide is removed by shaking with chloroform
`and the excess iodide is then titrated by an iodate method. The calculation makes the
`assumption that the average molecular weight for the mixtures is equivalent to that of
`the C, homologue. However, since the average molecular weight of some commercial
`samples has been shown to correspond more closely to the C,, or C , homologues ( 5 ) ,
`this method does not give sufficient accuracy.
`
`'Present address: The School of Pharmacy, The University of London, Brunswick Square, London.
`
`73
`
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`M. R . Euerby
`74
`It is extremely likely that samples may pass the BP and EP specifications but have
`widely differing preservative activity. The disadvantage with the current USP specifi-
`cation for determining the percentage of the homologues is that it employs a compli-
`cated microhydrogenation followed by gas chromatography of the resulting n-alkyl
`dimethy lamines.
`It was obvious that a quick and easy determination of both the total content and
`relative proportions of the homologues was required for routine quality control pur-
`poses. There are many examples in the literature of non-official methods for assaying
`benzalkonium chlorides, though many are unable to assess the proportion of homo-
`logues (6-9) and many others tend to be unsuitable for routine work ( 5 , lo), the only
`exceptions are the two HPLC methods. The first method was designed specifically for
`determining benzalkonium chlorides in the presence of alkaloids and polymeric
`substrates (1 1) in contrast to the second method which simply determined them in
`very dilute aqueous solutions (12).
`It was therefore decided to re-examine the second HPLC method (12) with a view to
`comparing it to the BP and EP official method and to assess the variability of the
`relative proportions of the homologues from differing suppliers/manufacturers of
`dilute ophthalmic solutions O.O29bw/v, strong 500; w/v solutions and concentrated
`solid forms.
`
`M E T H O D
`
`Apparatus and materials
`A Spectra-Physics SP8700 solvent delivery system, Pye-Unicam PU 4020 uv
`detector, Spectra-Physics SP 4270 computing integrator and Rheodyne 7125 valve
`injector fitted with a 100 p1 loop were used.
`The alkyl benzyldimethylammonium chloride homologues used in constructing the
`calibration curves were obtained from the United States Pharmacopoeia Commission.
`
`HPLC conditions
`Mobile phase-acetonitrile/O-lM sodium acetate (adjusted to pH5 with glacial acetic
`acid) 40:60, continuous degassing with helium
`Flow rate-1.5 ml/min
`absorption at 262 nm and 0.32 AUFS
`Detection-UV
`Spherisorb 5CN (125 cm, 4.6 mm i.d.) 5 p
`Column-'Hichrom'
`Sample and standard preparation. The standard reference solution 0.02°,w/v was
`made up as follows: USP Benzalkonium chloride standard lO",w/v (1 ml) was made
`up to 500ml with HPLC water and stored in the dark. Commercial samples were
`diluted with HPLC water to a h a 1 concentration of 0~02°,w/v and subjected to
`HPLC analysis.
`
`R E S U L T S AND D I S C U S S I O N
`Slight modifications of the method, as described in the literature (12), gave excellent
`separation of the Cloy ClZ, C,, and C,, homologues in an analysis time of 11 min (Fig.
`la). The relative SDs were found to be _+4.2700, _+0.62",, _+0.78", and _f2.42",,
`
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`Vuriarion in benzalkonium chlorides
`b
`
`C
`
`75
`
`- c
`
`0
`
`12
`
`0
`
`16
`
`0
`
`16
`
`M l n
`Fig. 1. Typical chromatogram of the separation of homologues in benzalkonium chlorides: (a) USP
`reference standard (b) Sample E (c) Co-chromatogram of (a) and (b).
`
`for the C,o, C,,, C,, and C,, homologues respectively, quantification being by
`comparison of the sample and standard peak areas. As expected, the C,, and C,,
`homologues gave lower relative standard deviations compared to the C,, and C,,
`because C,, and C,, contribute to a larger extent to the total benzalkonium
`chloride content. T h e area under the peak for each homologue responded linearly with
`concentration within the range examined.
`Variation in the proportion of homologues is possible from different sources of
`benzalkonium chlorides (5, 10, 12), therefore commercial 0~02",w/v, 509,w/v and
`solid benzalkonium chloride preparations were purchased and subjected to the above
`assay procedure. As can be seen from Table 1, three different batches from the same
`manufacturer (A) contained roughly the same proportion of the homologues, with the
`
`C, , predominating, illustrating good batch to batch consistency. A similar distribu-
`tion of the homologues was noted for manufacturer B, suggesting a common source of
`benzalkonium chloride with manufacturer A. Samples from manufacturers C and D
`show a predominance of the C , , homologue but are qualitatively different from each
`other and from samples A and B. The sample from manufacturer D failed to comply
`with the USP specification since it contained less than 2096 of the C , , homologue.
`In sharp contrast to samples A-D, the commercial sample E was shown to contain
`three peaks which did not correspond to the Cloy C,,, C,,, and c16 homologues (Figs
`Ib & c) hence failing the USP specification, even though it was marketed as USP
`quality. The supplier later admitted that the product contained the C,, and C,,
`
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`IPR2019-00694
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`76
`
`M . R. Euerby
`
`Table 1. Comparison of commercial batches of benzalkonium
`chlorides
`
`Manufacturer
`
`Percentage of homologue Total content
`C , , C , , c,, c,,
`"
`
`0
`
`A (Batch 1)
`(Batch 2)
`(Batch 3)
`
`B
`C
`D:
`Es:
`
`71.1 24.6 -
`4.3
`2.2
`68.1 25.9
`3.8
`69.2 26.0
`1.5
`3.3
`71.6 24.4 -
`4.0
`- 59.0 41.0 -
`48.8 18.0 19.3
`7.7
`- - - -
`
`0.0187*
`0.0195
`0.0216
`49.5t
`99.7
`91.4
`
`*0.0193nn BPC assay.
`t50.60; BP assay.
`:Possibly contains 6.2O, of the C, homologue.
`$44.3?& BP assay.
`
`homologues instead of the required C,, and C14. The remaining peak has yet to be
`identified.
`It can be seen from 'Table 1 that, not unexpectedly, the HPLC results deviated by
`2-30,
`from those of the BPC method for benzalkonium chloride lozenges, which
`employs a titration with sodium lauryl sulphate after complexation with dimethyl
`yellow in the presence of chloroform, and the BP method for total benzalkonium
`chloride content.
`The HPLC analysis for the total content and proportion of the homologues in
`various benzalkonium chloride preparations has been shown to be quick, easy to use,
`reliable and ideally suited to routine quality control uses. In addition the method is
`ideal for differentiating between sources of benzalkonium chlorides using the relative
`proportions of each homologue. From the above findings it is suggested that the BP
`and EP adopt a specification (the USP gas chromatography method or a similar HPLC
`assay as described), to determine the proportion of each homologue in order to
`standardize the widely varying benzalkonium chloride products presently on the
`market.
`
`REFERENCES
`1. Valko, E.I. & DuBois, A.S. (1944) The antibacterial action of surface-active cations. Journal of Bac-
`teriology, 47, 15-25.
`2 . Valko, E.I. & DuBois, A.S. (1945) Correlation between antibacterial power and chemical structure of
`higher alkyl ammonium ions. Journal of Eacteriologv, 50,481490.
`3. Hooperheide, J.C. (1945) The germicidal properties of certain quaternary ammonium salts with
`special reference to ceryltrimethylammonium bromide. Journal of Bacteriology, 49,277-289.
`4. Giles, R., Daoud, N.N., Gilbert, P. & Dickinson, N.A. (1983) Benzalkonium chloride: BP, EP or
`USPlJournal of Pharmacy and Pharmacology, 35,l IOP.
`5. Daoud, N.N., Crooks, P.A., Speak, R. 81 Gilbert, P. (1983) Determination of benzalkonium
`chloride by chemical ionization mass spectroscopy. Journal of Pharmaceutical Science, 72,290-292.
`6. Auerbach, M.E. (1943) Germicidal quaternary ammonium salts in dilute solution. A colorimetric
`assay method. Analytical Chemistry, 15,492493.
`
`Opiant Exhibit 2042
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
`Page 4
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`

`

`Variation in benzalkonium chlorides
`77
`7. Chatten, L.G. & Okamura, K.O. (1973) Assay of quaternary ammonium compounds in various
`dosage forms by acid-dye method. Journal of Pharmaceutical Science, 62, 1328-1 33 1.
`8. Ballard, C.W., Isaacr, J. & Scott, P.G.W. (1954) The photometric determination of quaternary
`ammonium salts and of certain amines by compound formation wirh indicators. Journal of Pharmacy
`and Pharmacology, 67,971-985.
`9. Colichman, E.L. ( 1947) Photocolorimetric method for determination of quaternary ammonium salts.
`Analyrical Chemistry, 19,430-431.
`10. Jennings, E.C. & Mitchner, H. (1967) Modified Hofmann degradation for the analysis of
`n-alkylbenzyldimethylammonium chlorides by gas chromatography 11. Journal of Pharmaceutical
`Science, 56, 1595-1598.
`11. Marsh, D.F. & Takakashi, T. (1983) Determination of benzalkonium chloride in the presence of
`interfering alkaloids and polymeric substrates by reverse-phase high-performance liquid chromaro-
`graphy. Journal of Pharmaceutical Science, 12,521-525.
`12. Meyer, R.C. (1980) Determination of benzalkonium chloride by reversed-phase high-pressure liquid
`chromatography. Journal of Pharmaceutical Science, 69, 1148-1 150.
`
`Opiant Exhibit 2042
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
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