ANALYST, NOVEMBER 1987, VOL. 112 1619

Spectrop hotometric Determination of Polyquaterniu m-I with Trypan Blue by a Difference Procedure

Larry E. Stevens and Jill 1. Eckardt Analytical Chemistry, Alcon Laboratories, 6201 South Freeway, Fort Worth, TX 76134, USA

A spectrophotometric method is described for the determination of polyquaternium-1 in a pharmaceutical solution. This method is based on a bathochromic shift of the trypan blue absorption which occurs when it is complexed with polyquaternium-I. The difference in absorption is measured at 680 nm with an apparent average molar absorptivity of 1.5 x 105 I mol-1 cm-1. Linearity is observed over the range 5-15 pg ml-l. Keywords: Polyquaternium- 1 determination; trypan blue; difference spectrophotometry; pharmaceutical preparations

Polyquaternium-1 , co- { 4-[ tris( 2-hydroxyethyl)ammonio]- but-2-enylpoly(dimet hylammoniobut-2-enyl) } tris( 2-hydroxy- ethy1)ammonium polychloride (I), is a polycationic antimicro- biological agent used in a variety of applications ranging from water treatment to pharmaceutical preparations . 1 The mole- cular mass of polyquaternium-1 ranges from 5000 to 10 000.

Trypan blue,2 tetrasodium 3,3’-(3,3’-dimethyl)-1,l’-bi- phenyL4,4’-diylbis( azo)bis( 5-amino-4-hydroxy-naphthalene- 2,7-disulphonate) (II), is an anionic dye that forms a water- soluble ion pair with polyquaternium-1 and is a good chromophore. This paper describes a spectrophotometric method which uses this complex for the determination of polyquaternium-1 in a pharmaceutical preparation by taking advantage of the bathochromic shift that occurs in the absorbance of trypan blue from 658 to 700 nm. The concentration is determined from the absorbance at the wavelength where the maximum in the difference spectrum occurs (680 nm).

Experimental Reagents and Solutions

Analytical-reagent grade or spectroscopic-reagent grade materials were used.

Trypan blue solution, 0.05% mlV. Prepared in distilled water.

Polyquaternium-1 standard solutions. Approximately 0.0005, 0.001 and 0.0015% mlV in distilled water.

Buffer solution. Prepared by dissolving 30 g of sodium chloride in 1000 ml of 0.5 M sodium phosphate solution (pH

Sample solution. OPTI-SOFT brand contact lens soaking 3.5).

solution.

Apparatus

A Perkin-Elmer 559 A double-beam spectrophotometer with 1-cm path length cuvettes was used.

Procedure

Determination of polyquaternium-l in a pharmaceutical soh- tion by difference spectrophotometry Pipette 5.0-ml portions of each of the polyquaternium-1 standard solutions, distilled water blank and samples into separate 15-ml test-tubes. To each of these test-tubes add 3.0 ml of the buffer solution and vortex to mix well. Add 1.0 ml of the trypan blue solution and mix well again. Measure the absorbance of each solution at 680 nm, subtracting the blank in each instance. Calculate the sample concentrations based on the slope and intercept obtained from the three standard concentrations.

Results and Discussion Polyquaternium-1, which does not have a chromophore that absorbs above 200 nm, can be determined spectrophoto- metrically by the formation of a water-soluble complex with trypan blue. The formation of an ion pair between the quaternary ammonium and sulphate ions increases the elec- tron delocalisation in trypan blue and produces a batho- chromic shift of about 40 nm (Fig. 1). The absorbance of the complex is then measured at its maximum difference (AA) from a blank at 680 nm.

Effect of Concentration of Reagent and Time

The amount of trypan blue necesary to obtain a linear graph for a sample concentration range of 5-15 pg ml-1 was studied.

(HOCH2CH213& - CH2CH= CHCH2

( n + 2)CI-

N a 0 , S S 0 , N a

NH2 O H O H NH2 CH, CH,

II

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1620 ANALYST, NOVEMBER 1987, VOL. 112

0.5

0.4

6 0.3 0.2

0.1

0

F -

Fig. 1. Absorption spectra for 5.8 X 10-5 M trypan blue in (A) buffer and (B) buffer with 1.1 X 10-6 M polyquaternium-1

0.4

q 4 0.3

0.2

0.1

0 10 20 30 40 50 Polyquaternium-1/pg ml-1

Fig. 2. Effect of trypan blue polyquaternium-1 concentration ratio. (A) 0.02 mg ml-* trypan blue; (B) 0.05 mg ml-1 trypan blue; and (C) 0.1 mg ml-1 trypan blue

Solutions which contained from 0 to 500 pg of poly- quaternium-1 were prepared and mixed with various amounts of trypan blue in 10 ml of buffer. Absorbance values obtained at 680 nm are shown in Fig. 2, from which it can be seen that 0.05 mg ml-1 of trypan blue is necessary to achieve linearity over the concentration range 2.5-7.5 pg ml-1. This corre- sponds to a range of 5-15 pg ml-1 for a 5-ml sample aliquot. A slight excess over this amount can be used; however, this also raises the blank absorbance and decreases the sensitivity.

A shaking or vortex time of 5-10 s immediately following the addition of trypan blue to the sample - buffer mixture is necessary for good precision. The complex is stable for 24 h, after which slight precipitation occurs at higher concentra- tions.

Effect of pH The effect on the absorbance values of varying the solution pH from 2.0 to 7.0 was studied. The resulting absorbance values indicated an optimum pH range of 3.5-5.0; however, the values differ by only 3% throughout the pH range 2.5-7.0. The absorbance value at pH 2.0 was 8% less than that obtained at pH 3.5. A buffer concentration of 0.5 M was found to be sufficient to bring the pH into the optimum range.

Effect of Ionic Strength The absorbance of trypan blue increases to a maximum with increasing sodium chloride concentration. Fig. 3 shows that a 3.0% sodium chloride concentration in the buffer solution is necessary to obtain the optimum absorbance. This eliminates any significant difference in absorbance (less than 1%) between a standard and sample with different ionic strengths.

Table 1. Recovery of polyquaternium-1 from product samples with known concentrations

Polyquaternium-l/ pg ml-1

5.0 7.5

10.0 12.5 15.00

Mean+s.d. . . , .

Recovery, 70

Day 1 Day 2 Day 3 101.9, 100.1 100.4, 98.6 99.5, 101.2 98.7, 95.5 - - - - 99.5, 100.7 98.7, 99.7 100.2, 100.8

101.1, 100.6 - - - - 100.4, 99.6 100.2, 99.8 98.7, 100.1 99.8 f 1.76 99.6 f 0.76 100.1 k 0.90

0.15

0.10

q a

0.05

0 0 1 2 3 4 5

NaCI, Yo

Fig. 3. Effect of sodium chloride on absorbance at 680 nm for 10 pg ml-1 of polyquaternium-1

Analytical Data Linearity was demonstrated over the range 5.0-15.0 pg ml-1 of polyquaternium-1. The apparent molar absorptivity was 1.5 X l o 5 1 mol-1 cm-1 at 680 nm. The linearity between the absorbance difference at 680 nm (AA) and concentration (c, pg ml-1) was observed on three different days and was found to be expressed by the equations AA = 0.0326~ - 0.0138, AA = 0.0334~ - 0.0212 and AA = 0.0243~ - 0.0154, with correlation coefficients of 0.999, 0.998 and 0.999, respec- tively. The mean recovery for a set of 20 independent product samples with 10 pg ml-1 of polyquaternium-1 was shown to be 100.3%, with a coefficient of variation of 1.2%. Recovery values for product samples with polyquaternium-1 concentra- tions ranging from 5 to 15 pg ml-1 on three different days are shown in Table 1. The mean recoveries and coefficients of variation for the graphs ranged from 99.6 to 100.1 and from 0.9 to 1.8%, respectively.

Interferences The effect of additional sample components was also studied. These included 1 mg ml-1 of polyvinyl alcohol, 1 mg ml-1 of hydroxyethylcellulose, 1 mg ml-1 of ethylenediamine tetra- acetic acid, 1 mg ml-1 of polysorbate and 10 pg ml-1 of benzalkonium chloride. Of these components, only benzalko- nium chloride showed a slight interference which was ob- served as a 2% increase in recovery for a solution concentra- tion of 10 vg ml-1.

Conclusion Trypan blue was chosen as a complexing agent with the aim of obtaining a water-soluble ion pair which would have a measurable bathochromic shift in a wavelength region above potential solution interferences.

The described procedure is simple, fast, accurate and precise in determining polyquaternium-1 in a pharmaceutical preparation. This method has also been studied in other sample matrices and appears to work well in the presence of

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ANALYST, NOVEMBER 1987, VOL. 112 1621

polyvinyl alcohol, polysorbate, hydroxyethylcellulose and 2. Windholz, M. , Budavari, S. , Stroumtsos, L. Y. , and Fertig, ethylenediaminetetraacetic acid. M. N., Editors, “The Merck Index,” Ninth Edition, Merck,

Rahway, NJ, 1976, p. 1255.

References 1. Petrocci, A . , Clarke, P . , Merianos, J . , and Green, H.,

“Development In Industrial Microbiology,” Volume 20, Society of Industrial Microbiology, Arlington, VA, 1978, p. 11.

Paper A61387 Received October loth, 1986

Accepted June 5th, 1987

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