Determination of benzethonium chloride in anthrax vaccine adsorbed by HPLC

Biologicals 34 (2006) 257e263 www.elsevier.com/locate/biologicals

Determination of benzethonium chloride in anthrax vaccine adsorbed by HPLC Hsiaoling Wang*, Alfred V. Del Grosso, Joan C. May Laboratory of Analytical Chemistry, Office of Vaccine Research and Review, Center of Biological Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike HFM-406, Rockville, MD 20852, USA Received 16 November 2005; accepted 18 November 2005

Abstract A novel and sensitive HPLC method for the determination of benzethonium chloride (BZC) in anthrax vaccine was developed. Adjuvant Alhydrogel was removed by syringe filter after a simple sample pretreatmentdacidification prior to injection. Chromatography was performed by isocratic reverse phase separation with methanol/262 mM ammonium acetate (80/20, v/v) on an endcapped C18 column with diode array detector (DAD). The method showed excellent recovery (100  1.5%). The results indicated that this method could accurately determine BZC at the limit of detection (LOD) of 0.5 ppm and the limit of quantitation (LOQ) of 1.5 ppm with dynamic range up to 100 ppm. The comparison of analysis between new HPLC and old titrimetric methods is also reported. The HPLC method is proven to be more accurate and precise with much less vaccine sample and human labor required. Published by Elsevier Ltd on behalf of The International Association for Biologicals. Keywords: Benzethonium chloride; HPLC; Anthrax vaccine

1. Introduction Anthrax is an acute infectious disease caused by the rodshaped bacterium Bacillus anthracis. Anthrax only occurs in warm-blooded animals and can also infect human beings, though in extremely low rate. However, Anthrax is considered to be one of the biological weapons most likely for terrorism or warfare. Anthrax spore laced letters caused five deaths in the fall of 2001 in Untied States. Anthrax vaccine for use in human has been in existence for more than 60 years [1]. The current vaccine in use in the Untied States, Anthrax Vaccine Adsorbed (AVA), was licensed in 1970. The development of HPLC methods for analyses of anthrax vaccine is a part of the research effort supported by FDA to improve the quality control of the vaccine for the purpose of countering bioterrorism.

* Corresponding author. Tel.: þ1 301 496 4570; fax: þ1 301 435 4991. E-mail address: [email protected] (H. Wang).

The final product of AVA contains protective antigen (PA), aluminum hydroxide, formaldehyde, benzethonium chloride (BZC) and sodium chloride. BZC is used as an anti-microbial agent in anthrax vaccine [2]. It is also a common component in other injectable and nasal medications, such as thrombin, ketamine, orphenadrine, and butorphanol [3]. The content of BZC in anthrax vaccine is determined by a traditional two-phase titrimetric method, which is adapted from the standard method used for the quaternary ammonium compound (QAC) analysis in milk [4]. This method not only uses a toxic chemical, acetylene tetrachloride, as organic solvent, but also takes days for a skilled chemist to complete the analysis because it involves tedious procedures like organic phase extraction, centrifugal separation, two-phase titration, in addition to preparation of numerous chemical solutions. Due to the nature of the phase extraction and titration, the results of analysis do not have 100% recovery and usually depend on analysts’ own judgment. In this study, we have proven that BZC content in anthrax vaccine can be conveniently determined by HPLC methodology with a simple pretreatment of the vaccine

1045-1056/05/$32.00 Published by Elsevier Ltd on behalf of The International Association for Biologicals. doi:10.1016/j.biologicals.2005.11.004

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H. Wang et al. / Biologicals 34 (2006) 257e263

sample. The data presented and conclusions made are research findings.

through a syringe filter having porosity of 0.45 mm with a 1-mL polypropylene syringe.

2. Experimental

2.4. Sample treatment and preparation

2.1. Chemicals and samples

Anthrax vaccine was stored at 2e8  C in a refrigerator before analysis. Vaccine sample was set at room temperature for more than half an hour. Shake the vaccine sample to form a uniform suspension solution. Accurately transfer 0.3 mL of this solution to 4 mL glass vials. Accurately add 0.7 mL 60% acetic acid, and vortex for about 10 s. Filter through a syringe filter having porosity of 0.45 mm with a 1-mL polypropylene syringe.

All solvents and acetic acid were HPLC grade from Fisher Scientific (Fairlawn, NJ, USA). BZC was purchased from Aldrich Chemical Corp (Milwaukee, WI, USA) with purity of 97%. Deionized water was obtained from a Milli-Q system by Millipore Corp (Bedford, MA, USA). Ion pair reagents (IPRs)daliphatic sulfonic acid, sodium salts were purchased from Fluka Chemical Corp (Milwaukee, WI, USA). Anthrax vaccine samples were produced by BioPort Corporation (Lansing, MI, USA). 2.2. Apparatus The HPLC instrument used for HPLC method development was an HP1050 equipped with a diode array detector (DAD) from HewlettePackard (Palo Alto, CA, USA). Chromatographic columns were Luna C18(2) with 3 mm particles from Phenomenex (Torrance, CA, USA) and Discovery HS C18 with 3 mm particles from Supelco (Bellefonte, PA, USA). One-millilitre polypropylene syringes were from Scientific Resource Inc (Duluth, GA, USA). Syringe filtersdAgilent 5064-8221 have 13 mm diameter with 0.45 mm pore size that were purchased from Agilent Technologies (Wilmington, DE, USA). 2.3. Stock solutions and standards Dissolve an accurately weighted quantity of benzethonium chloride quantitatively in water to obtain a solution having a known concentration of about 5000 mg/mL to be standard stock solution. Dilute three accurately measured volumes of this solution to a known set of calibration stock solutions with concentrations of about 10, 50 and 100 mg/mL. Mix accurately measured volume of calibration stock solutions (0.12e 0.3 mL), 1.4 mL 60% acetic acid (v/v) and extra accurately measured volume of water to form a set of calibration solutions with total volume of 2.0 mL. This set of calibration solutions has known concentrations of about 1.5, 3.0, 5.0, 8.0, and 12.0 mg/mL, respectively. Filter 1 mL of each calibration solution through a syringe filter having porosity of 0.45 mm with a 1-mL polypropylene syringe (recommend the Agilent 5064-8221 syringe filter for least contamination and best recovery). Dissolve an accurately weighted quantity of benzethonium chloride quantitatively in water to obtain a solution having a known concentration of 6000 mg/mL. Dilute an accurately measured volume of this solution to a known concentration of 30 mg/mL to be QC working standard. Mix accurately measured volume of 0.3 mL of this solution with accurately measured volume of 0.7 mL of 60% acetic acid in a 4-mL glass vial to form a known concentration of 9.0 mg/mL. Filter

2.5. Chromatography and quantitation The liquid chromatogram is recorded at wavelength of 275  4 nm with reference of 350  50 nm. A 50 mm  4.6 mm, 3 mm Luna C18(2) column from Phenomenex along with a 4 mm  3 mm C18 AJO-4287 guard column from the same company is used and maintained at 40  1  C. Mobile phase is the mixture of methanol/262 mM ammonium acetate with pH value of 3.82  0.02 (80/20, v/v). The flow rate is set at 1 mL/min. Separately inject equal volumes (50 mL) of the standard solutions (at least with three replicates), the QC check solution and the vaccine mixtures into the chromatograph, record the chromatograms, and measure the peak areas of BZC response. Plot the values of the standard solutions versus their concentrations in mg/mL, and perform a linear regression on five plotted points. Use the slope and y-intercept to calculate the concentrations of the QC check solution and the vaccine mixtures, in mg/mL (ppm). The correlation coefficient of the linear regression should be no less than 0.999. The QC check solution should have less than 3% error. The dilute factor of the vaccine mixture comparison to original vaccine sample is 10/3. 3. Methods development and discussion 3.1. HPLC method development BZC belongs to the category of QAC (Fig. 1). This type of chemicals is considered to be difficult to separate owing to its polarity and ability to form micelles at low concentration. However, a great number of chromatographic methods have been developed over the years with the rapid advancement

Fig. 1. BZC molecular structure.

H. Wang et al. / Biologicals 34 (2006) 257e263

5.0

A

NaAc/HAc 4.5

Rt (min)

4.0 3.5 3.0 2.5

MeOH:100 mM buffer = 80:20

2.0 3.5

4.0

4.5

5.0

5.5

6.0

pH value 8.0 HAc/NH4OH 7.5

Rt (min)

7.0 6.5 6.0 5.5 MeOH:262 mM buffer = 75:25

5.0 4.5 3.5

4.0

4.5

5.0

5.5

6.0

pH value 4.5

B

HAc/NH4OH (pH 3.82) 4.0

Rt (min)

3.5 3.0 2.5 MeOH:Buffer = 80:20 2.0 1.5

0

100

200

300

400

500

Buffer Concentration (mM)

C

12.0 10.0

MeOH:262 mM buffer

8.0

Rt (min)

of chromatographic techniques. These methods are generally utilized for environmental monitoring and quality control of products. Ion pair chromatography and capillary electrophoresis, to name a few, are among the most commonly used techniques [5e8]. In our study, reverse phase HPLC was chosen for the method development to determine BZC content in anthrax vaccine simply because of its convenience and instrumental availability in most research and industrial laboratories. The HPLC method used for the measurement of BZC content in grapefruit seed extracts [9], which involves inconvenient organic solvent extraction and evaporation, is considered too cumbersome to be adapted for this application. The method development started with ion-paired chromatography, which is typically used for positive ion compound, and a 3-mm 100  4.6 Luna C18(2) column. Mobile phase was the mixture of NH4Ac (pH w4.0) buffer and methanol. Standard BZC solution was used as the test sample because no other substance in the vaccine was expected to cause potential interferences in such HPLC separation except protein. Preliminary results demonstrated that BZC positive ion (BZþ) could not be detected unless the vaccine sample was acidified to a pH value no greater than 2.5. Variables of DAD such as wavelength, bandwidth and reference wavelength were optimized for signal sensitivity, signal to noise ratio (S/N) and linearity from a set of standard BZC solutions with different concentrations. Column temperature was varied from 30  C up to 65  C to detect its impact on BZþ elution. Though high temperature has the advantage of quick elution, it is better to use relatively low temperature for the lifetime of column and for minimizing potential side reactions in complicated biological sample such as vaccine containing proteins. Keeping column temperature around 40  C was considered to be a good choice for the BZC detection. Most of the effort was spent in searching for a suitable mobile phase. Many anionic sulfonate ion pair reagents (IPRs) and their concentrations in mobile phase were tested for their effects on the BZþ retention. Retention time increases with the increase of alkyl chain length in the sulfonic acid and the concentration of IPR. Sodium perchlorate [10] was also tested as IPR and BZþ signal had a fairly good shape in the chromatogram. This led to further testing on the BZþ elution without using any IPR in the mobile phase. The result indicated that BZþ ions had enough hydrophobic interaction with stationary phase to let them be eluted in a quite reasonable retention time of 2.90 min with the k# value of 1.6. It has been proved that such hydrophobic interaction has made BZC a good displacer in displacement chromatography for protein or peptide purification [11]. Based on this finding, it was decided that mobile phase would not use any sort of IPR. The obvious advantages would be the shorter equilibrium time, the longer lifetime of the column and more reproducible BZþ elution. Buffer pH value, its concentration, its composition and the percentage of organic modifier were also tested as variables for mobile phase optimization. Some important results are shown in Fig. 2. The characteristics of peak shape were monitored with the change of mobile phase parameters. The peak width generally increases with the retention time. However,

259

6.0 4.0 2.0 0.0 68

HAc/NH4OH (pH 3.82)

70

72

74

76

78

80

82

MeOH Fig. 2. Variables tested for mobile phase optimization (40  C) with column of 100 mm  4.6 mm, 3 mm Luna C18(2). (A) pH value and their compositions effect on BZC retention time. (B) Buffer concentration effect on retention. (C) Methanol percentage effect on retention.

H. Wang et al. / Biologicals 34 (2006) 257e263

260 0.17

Peak width (min)

0.15 MeOH:Buffer = 80:20 0.13 0.11 0.09 0.07

0

100

200

300

400

500

Buffer Concentration (mM) Fig. 3. Buffer concentration effect on BZC peak width (40  C).

there was one exception. The peak width narrowed with the increment of buffer concentration while the retention time was increased (Figs. 2B and 3). Buffer capacity was enhanced by increasing the concentration of the buffer. Higher buffer concentration not only improved peak width, but also resulted in more reproducible separation by reducing local perturbation of the pH of the migrating BZþ ions. The buffer concentration within 200e300 mM was considered good range under the tested separation conditions. The tailing factor of BZþ did not change dramatically and remained near 1.5. All sorts of HPLC conditions tested (i.e. buffer components, buffer pH value, buffer concentration, and methanol percentage in mobile phase, etc.) gave very consistent peak area for the BZC standard solution with same concentration. There are a variety of HPLC columns available to improve speed and resolution for the separation. Preliminary tests with the vaccine sample did not show any interference from other co-existing small molecules in the vaccine. In order to further reduce equilibrium time, the overall analysis time and solvent consumption, columns with shorter lengths were tested. A column with a length of 50 mm allowed the separation to complete within 3 min for a single run. With the shorter retention time of BZþ ions, the signal had better sensitivity and peak shape (tailing factor of 1.1). 3.2. Vaccine sample pretreatment Anthrax vaccine is a sterile, milky-white suspension. The BZC molecular in anthrax vaccine has its special chemical environment. Table 1 gives the main compositions in the vaccine. The adjuvant, Alhydrogel, in the vaccine has an isoelectric point (pI ) of 11 and a wide range of particle size from Table 1 Main composition of Anthrax Vaccine Adsorbed Composition

Product specification

Protective antigen (PA) Alhydrogel Formaldehyde Sodium chloride Benzethonium chloride (BZC)

Tested on final bulk 0.8e1.5 mg Al/mL Less than 0.002% (w/v) 0.75e0.95% (w/v) 0.0015e0.0030% (w/v)a

a

Equivalent to 15e30 ppm.

0.5 mm to 10 mm. While the pH value of the final vaccine is between 7.5 and 8.5, the surfaces of Alhydrogel are positively charged and can readily adsorb PA, with pI 5.5, that is predominantly negatively charged [12]. These negatively charged sites on the PA surface are the result of the existence of many acidic amino acid residues such as aspartate (pKa 3.9), glutamine (pKa 4.3) and histidine (pKa 6.0) in PA protein chains and their tertiary structures. Some of these sites could also attract BZþ through the same electrostatic attraction. Hydrophobic interaction may also contribute to the interaction between BZþ and PA surfaces. Such interactions have been used for determination of total protein in urine [13]. No BZþ signal was detected for the non-acidified vaccine sample. Clearly BZþ exists in a binding form rather than a free form in the vaccine suspension. Hence, before the vaccine sample could be injected for HPLC analysis, pretreatment was needed. Pretreatment would remove large particles in the sample solution to prevent column clogging and release BZþ efficiently from its binding form. Theoretically, BZþ ions can be released from their binding form by adding enough acid to the vaccine solution if electrostatic attraction is the main factor for the binding between BZþ and PA. Acidification was used as one of the vaccine pretreatment steps for releasing BZþ and dissolving Alhydrogel. In the experiment, apparently not all the adjuvant gel dissolved quickly because the solution did not turn completely clear. Then centrifugation was used to separate the solution containing free BZþ and the undissolved adjuvant. Only the supernatant portion was sampled for HPLC injection. However, after several injections of these samples, the back-pressure of HPLC system gradually increased due to the existence of some Alhydrogel particles in the transparent supernatant. To solve the problem, syringe filter was used as another step for pretreatment to get rid of adjuvant particles. More than two dozens of hydrophilic, acetic acid compatible syringe filter samples were requested from different manufacturers. Tests were conducted to check for possible contamination and to measure the recovery from these filters with standard BZC solution of 10 ppm. Filters with 0.2 mm pore size gave very low recovery except Agilent syringe filter (recovery w98%). Thus the 0.2 mm pore size filters were eliminated. The chromatograms were considered not acceptable if (1) the contamination peak was huge comparison to the BZC signal (more than twice the size of 10 ppm BZþ signal). High contamination might not cause difficulty with BZþ signal detection but would affect column lifetime and complicate the separation; (2) the contamination peaks eluted too closely or co-eluted with BZþ signal. Two types of syringe filters with least contamination and more than 99% recoveries were further tested for their recoveries over the wide concentration range of BZC standard solutions. It is an easy decision to choose Agilent 5064-8221 syringe filter (Table 2). The poor recoveries from other syringe filers were due to the adsorption of BZC by the filter membranes. The result proved that acidifying the vaccine sample could effectively release BZC from its binding form. It is critical to find out how long it takes for this reaction to complete and

H. Wang et al. / Biologicals 34 (2006) 257e263 Table 2 BZC recovery on two filters 1 ppm

2 ppm

4 ppm

8 ppm

16 ppm

SRi 44513-NN Agilent 5064-8221

87.7% 99.8%

91.2% 101.1%

93.5% 100.4%

95.8% 98.7%

98.9% 99.2%

how much acid is enough to release BZC in the vaccine. A series of experiments were carried out for those purposes including the dynamic test for vaccine acidifying reaction, the BZC signal stability test for standard BZC solution and vaccine samples, and the BZC recovery test by spiking the standard BZC solution into the vaccine sample. Fig. 4 shows the dynamics of the acidifying reaction with various amount of acetic acid added to the vaccine mixtures with same amount of vaccine in all sample solutions. It is clear that BZþ ions were released from PA surfaces instantly after the addition of the acid because BZC and formaldehyde were added at the final step as preservative and stabilizer in anthrax vaccine processing [4]. Therefore, BZC ions interacted with PA on the direct surface. BZþ ions would not be buried in the depth of the adjuvant gel because of their electrostatic repulsive interactions. However, not all BZCs were released from their binding form when the acid amounts were at 10% and 20%. Their signals decreasing with reaction time can be explained by the existence of two competitive reactions. One is the Alhydrogel acidifying reaction that is a slow reaction. Another reaction is protein hydrolysis that will not only consume acid, but also generate very acidic peptide terminals that might create new negative sites on the peptide chains to attract the free BZþ again. The pH value of the reaction solution for a high percentage acid addition remained steady, but increased for the low percentage acid additions over time. The progress of the protein hydrolysis could easily be monitored in the chromatograms. The progress was indicated by the increase in intensity of the peptide peaks eluting before BZþ (Fig. 5). The small peak which elutes after BZþ is the sole contamination peak from the selected Agilent syringe filter. Its peak area was independent of the BZC concentration. It was concluded from the tests that 40% or more acetic acid had to be added to the anthrax vaccine sample in order to assure the effective release and maintenance of BZþ in free form. The pH value of the acidified vaccine sample is equivalent to 1.5.

BZC Peak Area (mAU*2)

25 20 15

60 40 20 10

5 0

0

10

20

HAc (pH 1.1) HAc (pH 1.5) HAc (pH 1.9) HAc (pH 2.2) 30

Intensity (mAU)

A

Filter

10

261

B C

0

0.5

1

1.5

D

2

2.5

3

Time (min) Fig. 5. Effect of protein hydrolysis on chromatogram (acidifying at time of 0 and 24 h). (A) and (B) Peaks of hydrolysis products. (C) BZC signal. (D) Contamination peak from Agilent syringe filter.

Both standard BZC solution and vaccine sample have stable signals of BZþ for over three days after their acidification. The relative standard deviations of their peak area are less than 2.2%. The reaction of protein hydrolysis progresses faster while stored at room temperature than at 4  C. The recovery of BZC was tested by spiking standard BZC solution into the vaccine sample because vaccine matrix without BZC was not available. The BZC concentration in prepared vaccine samples was between 7.0e7.5 ppm and the 0.1 mL of three levels of standard BZC solutions (10 ppm, 20 ppm and 30 ppm, respectively) were added to the vaccine samples for the measurement of the recovery. These spiked samples were vortexed and balanced in room temperature for over half an hour before acidification treatment. The excellent recovery and precision results were obtained for three consecutive days (Table 3). These results demonstrated that pretreatment of the vaccine simply with acetic acid could effectively released BZþ bound in the vaccine. Electrostatic repulsive forces between BZþ and PA surfaces in the acid treated sample were strong enough to prevent substantial adsorption by other attractive forces. 3.3. Vaccine analyses with both titrimetric and HPLC methods

40

50

60

Reaction time (min) Fig. 4. Dynamics of acidifying reaction of vaccine sample.

70

The procedure for sample preparation for HPLC analysis is summarized in Fig. 6. HPLC conditions are given in Section

H. Wang et al. / Biologicals 34 (2006) 257e263

262

Table 3 Recovery results by spiking standard BZC solution into vaccine sample (n ¼ 6)

Table 4 Intraday and interday precision and accuracy of BZC control samples

Day

BZC added (ppm)

BZC found (ppm)

C.V. (%)

Recovery (%)

Interday (n ¼ 18)

1

0.909 1.819 2.728

0.909 1.805 2.704

1.00 0.35 0.79

100.0 99.3 99.1

Average relative Average CV (%) Maximum relative Maximum CV (%) error (%) error (%) 1.27 0.29 2.66 0.57

2

0.909 1.819 2.728

0.919 1.835 2.710

0.93 0.74 1.21

101.1 100.9 99.3

0.909 1.819 2.728

0.919 1.815 2.693

1.28 1.18 1.19

101.1 99.8 98.7

3

Intraday (n ¼ 6)

experimental error than organic extraction. Organic extraction used for BZC analysis not only involves a non-quantitative phase transfers, but also has to deal with more complicated chemical environment and even less quantitative situation in vaccine sample in which the BZC is adsorbed on gelatinous precipitate with high surface area. The recoveries of several spiked AVA samples by titrimetric method were all less than 96%. The simplicity in the sample preparation procedure in this HPLC analysis also helps to improve the accuracy and precision of the BZC determination. Thus, we believe that the BZC content determined by the titrimetric method is generally lower than its actual content, while the HPLC method gives more accurate values of the BZC content of the vaccine. Other very important advantages of using HPLC to determine BZC in anthrax vaccine are the smaller amount of vaccine sample required and a less time consuming procedure. Titrimetric method needs 12 mL vaccine sample for three replicates and takes more than 12 h for a skilled chemist to complete the analytical procedure. The HPLC method only needs 0.9 mL vaccine sample for three replicates and takes at most 4 h for a chemist or technician with minimum training to perform the task.

2.5. LOD and LOQ were estimated as 0.5 ppm and 1.5 ppm at S/N of 3 and 10, respectively, with standard BZC solutions. The detector response was linear to a series of BZC standards in the range of 1.5e100 ppm concentrations with the correlation coefficient of 0.9996 (n ¼ 6) or better. The inter-assay precision and accuracy were determined by analyzing eighteen calibration curves with quality control samples on eighteen different days. The intra-assay precision and accuracy were determined by analyzing six replicates of the quality control samples on the same day. The result is shown in Table 4. Twelve vaccine samples were analyzed by both titrimetric and HPLC methods. The results are shown in Table 5. It is clear that the HPLC results had better precision than the titration results. The content of BZC determined by HPLC method is about 3.6% higher than the value determined by the titrimetric method of the same vaccine. In the two-tailed T-test, the calculated t-value of the data sets is 3.0521 and the critical t-value is 2.0739. The difference is statistically significant, since the two-tailed probability is 0.005843, which is much lower than alpha value 0.05. Chemically acidifying the vaccine sample to release BZþ ions in HPLC involves less

4. Conclusions An HPLC method has been developed for the determination of benzethonium chloride in anthrax vaccine. Benzethonium

Calibrants

QC

Vaccine sample

5000 ppm stock BZC solution

6000 ppm stock BZC solution

Shake well

30 ppm QC working standard

0.3 ml vaccine sample

10, 50, 100 ppm calibration stock solutions

2ml 2~15 ppm calibration working standards with 1.4 ml 60 HAc

Add 0.7 ml 60 Hac to 0.3 ml QC check solution

Add 0.7 ml 60 HAc (pH < 1.5)

Vortex, Filter through Agilent 5064-8221 syringe filter for HPLC Fig. 6. Sample treatment for HPLC analysis.

H. Wang et al. / Biologicals 34 (2006) 257e263 Table 5 Results of vaccine sample analyses by titration and HPLC Calibration curve

Titration Y ¼ 0.1516X  0.2132 (r ¼ 0.9999)

HPLC Y ¼ 5.0971X  0.0814 (r ¼ 0.9998)

A B C D E F G H I J K L

19.48  0.80 20.36  0.81 19.92  0.87 20.83  0.57 20.56  0.55 20.88  0.60 19.52  0.61 19.83  0.77 20.98  0.79 20.62  0.90 20.06  0.79 19.44  0.79

20.43  0.35 20.85  0.37 20.29  0.46 21.97  0.17 21.22  0.01 21.70  0.44 20.30  0.31 20.36  0.17 21.86  0.21 21.23  0.32 20.76  0.08 20.48  0.31

ions in the vaccine are released as free form for HPLC detection by a simple acidification treatment. The acidification was shown to be effective by recovery studies on vaccine sample. Acidified standard BZC solution and vaccine samples remained stable for more than three days. The method gave LOD and LOQ of 0.5 ppm and 1.5 ppm, respectively, and had excellent linearity in the range of 1e100 ppm. In comparison to the two-phase titrimetric method used currently, the HPLC analysis yielded better accuracy and precision with much less sample and time in addition to the environmental benefit.

Acknowledgement This project was supported in part by an appointment to the Research Fellowship Program at the Center for Biologics Evaluation and Research administered by Oak Ridge

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Associated Universities through a contract with the U.S. Food and Drug Administration. References [1] Nass M. Anthrax vaccine: model of a response to the biologic warfare threat. Infect Dis Clin North Am 1999;13(1):187e208. [2] Puziss M, Manning LC, Lynch JW, Barclay E, Abelow I, Wright GG. Large-scale production of protective antigen of Bacillus anthracis in anaerobic cultures. Appl Microbiol 1963;11:330e4. [3] Montvale NJ, editor. Physician desk reference. 54th ed. Medical Economics Company, Inc.; 2000. p. 853. [4] 954.06 Quaternary ammonium compounds in aqueous solutions and milk eosin yellowish method. In: AOAC official methods of analysis. 15th ed. Washington, DC: Association of Official Chemists; 1990. p. 1154e5. [5] Haruyama M, Okaya Y. Determination of cationic preservatives in cosmetics by high performance liquid chromatography. Jpn J Toxicol Environ Health 1995;41(5):367e74. [6] Bluhm LH, Li T. Chromatographic purification of quaternary ammonium and pyridinium compounds on normal phase silica gel. Tetrahedron Lett 1998;39(22):3623e6. [7] Castro R, Moyano E, Galcerabn MT. Ion-pair liquid chromatographye atomspheric pressure ionization mass spectrometry for the determination of quaternary ammonium herbicides. J Chromatogr A 1999;830(1): 145e54. [8] Tayor RB, Toasaksiri S, Geid RG. Determination of antibacterial quaternary ammonium compounds in lozenges by capillary electrophoresis. J Chromatogr A 1998;798(1e2):335e43. [9] Takeoka G, Dao L, Wong RY, Lundin R, Mahoney N. Identification of benzethonium chloride in commercial grapefruit seed extracts. J Agric Food Chem 2001;49:3316e20. [10] Ishikawa A, Shibata T. Cellulosic chiral stationary phase under reversedphase condition. J Liq Chromatogr 1993;16:859e78. [11] Ramanan S, Velayudhan A. Displacement chromatography of chemotactic peptides. J Chromatogr A 1999;830(1):91e104. [12] Ragheb HA, Regnier FE, White JL, Hem SL. Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminiumcontaining adjuvants. Vaccine 1995;13(1):41e4. [13] Iwata J, Nishikaze O. New micro-turbidimetric method for determination of protein in cerebrospinal fluid and urine. Clin Chem 1979;25(7): 1317e9.