Dear all,
I just found this "survey" article on preservatives and their test methods.
Analysis of preservatives in pharmaceutical products
at:
http://www.pharmainfo.net/reviews/analy ... l-products.
In:
http://www.pharmainfo.net/volumes-and-i ... e-1?page=1
Author: Khairi M.S. Fahelelbom. 01/28/2007
Check it out. It has large section on BKC/BAK/BAC.
I am copying the relevant text below, just in case the content is deleted on that server. Sorry that I copy with no permission from the author (Khairi M.S. Fahelelbom).
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Quaternary ammonium compounds have been in clinical as antimicrobial additives use since 1935. They have been used to maintain sterility of a variety of prescriptions and OTC products such as cosmetics, infant care products; pharmaceutical nasal sprays ophthalmic solutions and otic drops 89 . Marpel et al 90 prepared a review article about the safety of benzalkonium chloride in intranasal solutions .
Several chemical derivatives of the quaternary ammonium ion have been used as preservatives. The most frequently used individuals are benzalkonium chlorides (BKCs), cetylpyridinium bromide (CPB), cetylpyridinium chloride (CPC) cetrimide, and benzaethonium chloride (BZC) (Table I).
BKCs are a mixture of alkylbenzyldimethyl ammonium chlorides with the general formula [C 6 H 5 CH 2 N (CH 3 ) 2 R] + Cl - , where R = n-C 8 H 17 to n-C 19 H 39 . The n-C 8 H 17, n-C 12 H 25 and n-C 16 H 33 homologues comprise the major portions of the alkyl mixture. The C12 homologue is most effective against moulds, yeast and fungi, the C14 homologue is most effective against gram-positive bacteria, and the C16 homologue is most effective against gra m- negative bacteria 91 . The ratio of alkyl homologues must meet specific USP 4 requirements. These requirements state that the C12 homologue must comprise at least 40% of the total BKCs content, and that the C14 homologue must be at least 20%. Furthermore, these two homologues together must comprise at least 70% of the total content. Cetrimide is the tetra-decyl tri-methyl ammonium bromide with a small amount of dodecyl and hexadecyl derivatives 92 .
Quaternary ammonium salts are chemically stable in aqueous solutions, will withstand autoclaving, and compatible with heavy metals, alkalis, oxidants and anionic surfactants 21 . The reported methods of analysis can be classified as follows:
III. A. 3. a. Capillary electrophoresis methods:
The developed analytical procedure demonstrates that CE is a reliable and sensitive method, and offers a convenient analytical technique to determine quaternary ammonium compounds, due to the presence of a positive charge. For these reasons, several analytical methods based on utilization of capillary electrophoresis (CE) technique have been reported for the determination of this group of preservatives. Hou et al 93 , developed a capillary zone electrophoresis (CZE) method to determine BKCs in ophthalmic solutions; the sample was separated at a potential of 15 kV and detected with UV-VIS detector at 200 nm. CZE was superior over HPLC in terms of sensitivity and precision.
Mixtures of cationic surfactants; BKCs and CPC were resolved and determined using CZE and MEKC-CZE in industrial house hold surfactants. The MECK method has an excellent resolution for all house hold solutes including C12-C18 homologues 94 . Prince et al 95 , utilized HPCE for the separation and determination of BKCs and their homologues on silica capillary column and UV detection at 214 nm. However, HPCE was more sensitive and precise compared to the HPLC. Also, BKCs in nasal drops containing naphazoline, dexamethasone were determined by a similar procedure developed by Raith et al 96 .
Comparative studies for the determination of BKCs utilizing HPLC coupled with UV detection at 250 nm, CE coupled with UV detection at 254 and 214 nm, and first derivative spectroscopy was developed by Bernal et al 97 . These methods were successfully applied for the determination of BKCs in the presence of beclomethasone dipropionate (or fluticasone propionate) active ingredients and excipients in nasal sprays.
BKCs, 2-phenylethanol and beclomethasone dipropionate in nasal spray products were separated and determined with HP-CE. Fused-silica tubes were used at 20 0 C and 50 0 C with UV detection at 214 and 254 nm, respectively 98 .
Jimidar et al 99 , utilized CE coupled with UV detection at 215 nm for the determination of BKCs in pharmaceutical products. C12 and C14 -BKCS derivatives were separated and quantified. BKCs were also separated from histamine acid phosphate and determined using imidazole as an internal standard by CE where the separation was conducted at 30 0 C with detection at 200 nm 100 .
Cetylpyridinium chloride in mouth wash was determined by CE with indirect UV detection or by HPLC with conductometric detection 101 .
III. A. 3. b. High performance liquid chromatographic methods:
HPLC methods with different impacts of column packing type, mobile phase composition, buffer, ionic strength and column temperature, were described for the determination of quaternary ammonium compounds in various pharmaceutical products. Amino functional group 102 , reversed phase 103,104 and cyano 105-106 packing columns were developed. The European Pharmacopoeia 22 specifies HPLC for the determination of BKCs in pure form. In addition to the methods mentioned before, the following methods have been reported for their determination in pharmaceutical products. BKCs, in aerosol preparations, were determined by HPLC with cyano-column and the mobile phase was aqueous acetonitrile at pH 5, and the detection was performed at 262 nm 103 .
Bernal et al 104 , utilized the HPLC with reverse phase column and acetonitrile-water mixture as a mobile phase for the determination of BKCs in pharmaceutical products. The detection was performed at 210 nm. In ophthalmic solutions, Fan and Wall 105 and Kummerer et al 106 , determined BKCs by HPLC with cyano column after the sample has been extracted to solid phase. Ambrus et al 107 , described a direct RP-HPLC method with UV-VIS detector for the determination of BKCs in ophthalmic systems.
BKCs and their homologues were separated and determined by HPLC using cyano-column and UV detection at 254 nm. BKCs and nonoxynol-9 in preparations were determined by RP-HPLC during a pre-formulation study on cyano column and UV detection at 214 nm 108 .
Parhizkari et al 109, 110 , developed two stability indicating ion-pair RP-HPLC methods for the determination of BKCs in ophthalmic solutions where C12- and C14-homologues were separated and quantified. Also, BKCs were determined in eye care products by HPLC; they were separated from various products and concentrated by either solid-phase extraction onto C18 cartridges or by an online column-switching technique 111 . Other HPLC procedures with UV detection were reported for the analysis of quaternaries include determination of BKCs in ophthalmic solutions using phenyl column 112 and cyano-column 113 in addition to the determination of CPC in pharmaceutical and cosmetic products 114 .
III. A. 3. c. Gas chromatographic methods:
This class of compounds is not amenable to direct GC analysis due to volatility problems. However, the volatility is greatly enhanced by pyrolysis to tertiary amines and subsequent GC. This procedure was described by Suzuki et al 115 . In that procedure, BKCs were reacted with potassium t-butoxide, and extracted into benzene. The extract was analyzed on SE-30 column and the produced alkyl homologues were quantified. These procedures were similar to the procedure developed by Cybulski 116 where, BKCs were converted to two tertiary amines: alkyldimethyl amine and alkylbenzylmethyl amine. The pyrolysis products of BKCs were identified by GC-MS.
III. A. 3 .d. Thin layer chromatographic methods:
The chain homologues of BKCs, cetrimide, CPB and CPC were separated on TLC and detected by the yellowish-brown spots on a colorless background. They were quantified by UV densitometry 117,118 .
III. A. 3. e. Flow injection analysis:
Owing to the presence of positive charges, quaternary ammonium compounds have higher ability towards ion-pair reactions with acidic dyes. The formed colored ion pair products were extracted into a suitable organic solvent and measured by the spectrophotometric technique. Sakai 119 reported a FIA procedure for the analysis of BKCs after ion-pair formation with tetrabromophenolphthalein ethyl ester reagent in CH 2 Cl 2 followed by phase separation and detection at 610 nm with the flow-cell operated at 45 0 C. Halvax et al 120 , developed a FIA method for the determination of BKCs in pharmaceutical products containing xylometazoline, timolol, phenylephrine or carbachol by on-line ion-pair extraction of BKCs with picrate into CHCl 3 and detection at 370 nm.
The determination of BKCs after the reaction with quinine - bromophenol blue present in the carrier stream, followed by on-line extraction and measurement at 610 nm. This method was reported by Miyaji et al 121 .
BZC in pharmaceutical products was determined by FIA where the sample was injected in stream containing quinidine and bromochlorophenol blue in 1,2-dichloroethane. The product in the organic layer was measured at 605 nm 122 .
The interference of thiomersal and BKCs in the simultaneous determination of phenylephrine HCl and pheniramine maleate products was eliminated by passage through beds of anion-exchange resin and then through cation-exchange resin, using two porous-membrane phase separators and determined by FIA with photometric detector 123 .
III. A. 3. f. Spectrophotometric methods:
Several spectrophotometric procedures have been reported in the literature for the determination of quaternary ammonium compounds in pharmaceutical products. Gorog 124 , in his text book reviewed spectrphotmetric method for the determination of quaternary ammonium preservatives.
Preservatives including cetyltrimethyl ammonium bromide, CPB, and dimethyldodecylbenzyl ammonium bromide react with 1-(4-nitrophenyl)-3-(4-phenylazophenyl) triazene and the produced colors are measured at 530, 540 and 590 nm, respectively 125 .
A colorimetric method for the determination of BKC in eye-drops based on ion-pair formation between BKC and eosin was developed by Hadady and Fabian 126 . The method is based on measurement of the decrease in absorbance of eosin . A colorimetric method based upon ion pair association between quaternary ammonium salts, such as CPC, and tetraiodofluorescein and quinine at pH 7 in the presence of 1,2-dichloroethane was developed by Sakai et al 127 .
Two spectrophotometric methods were developed by Belal et al , for the determination of BKC in ophthalmic preparations based on the formation of highly colored charge-transfer complexes with 7,7,8,8-tetracyanoquinodimethane and 2,3-dichloro - 5,6-dicyano -p- benzo-quinone BKC in pharmaceutical products 128 .
Colorimetric procedures for the determination of BKCs were developed through reaction with bromophenol blue followed by organic solvent extraction and measurement at 610 nm 129 or reaction with 4-(4-nitrophenylazo) resorcinol and NaOH and measuring the absorbance at 630nm. 130 . Also, a direct UV measurement of this group of compounds was also reported 131-133 .
III. A. 3. g. Titrimetric methods :
Titrimetric methods were also reported for the determination of quaternary ammonium compounds. These methods are based on coupling of the positive charge with negatively charged titrants. End points were detected potentiometrically or using visual indicator.
BZC in pharmaceutical preparations was determined by titration with sodium tetraphenylborate or sodium dodecyl sulfate and the end point was detected by potentiometer using ISE 134 . A similar procedure was reported by Satake et al 135 , where BKCs and zephiramine in a bactericidal solution were determined by titration with sodium tetraphenylborate using ISE sensitive to tetraphenylborate.
CPC, BKCs, acrinol (ethacridine lactate), and methylephedrine were determined, using sodium tetraphenylborate as titrant and tetrabromophenol-phthalein ethyl ester as indicator 136 . Other titrimetric procedures for the determination of quaternary ammonium compounds are also available 137-140 .
It would be useful to see a couple of chromatograms with baselines indicated.
