Analytica Chimica Acta. 86 (1976) 69-77 OElsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

AUTOMATED NEPHELOMETRIC DETERMINATION OF POLYVINYL- PYRROLIDONE IN SALAZOPYRIN

L. HAGEL* and R. ANDERSSON

Pharmacia AB. Box 604. S-541 25 Uppsala (Sweden)

(Received 13th April 1976)

SUIvmARY

An automatic nephelometric method is described for the determination of polyvinyl- pyrrolidone (PVP) in Salazopyrin. PVP is separated from the other constituents of Salazopyrin on a two-layer anion and cation exchanger in 96-99 % yield. The PVP is determined nephelometrically after precipitation with perchloric acid, in an AutoAnalyzer II system at 30 samples per hour with a relative standard deviation of 0.5 %. The nephelometric response of the precipitation reaction is strongly dependent upon reaction time and, because of the precipitation reagent used, on the molecular weight of the sample. An investigation of the influence of these factors is presented.

Salazopyrin (Azulfidine), a registered drug for the treatment of ulcerative colitis, contains about 3 % of polyvinylpyrrolidone (PVP) as a binder and coating substance. The aim of this investigation was to find a reliable method of high capacity for routine analyses of Salazopyrin for PVP.

Three approaches to the determination of PVP have been described. One of the methods involves the precipitation of PVP with trichloroacetic acid and then determination of the nitrogen content of the precipitate [ 11; this method lacks sensitivity because the precipitation is incomplete [ 21. Another approach involves the affinity of certain colour agents for PVP; e.g. PVP can be added to an iodine-iodide solution and the increase of absorption at 500 nm is related to the PVP concentration; the sensitivity of the method (referred to below as the “Iodine Method”) is 1 pg ml-’ [2]. In a similar method, PVP is adsorbed on a layer of silica gel and stained with Vital Red. The PVP-Vital Red complex is extracted with dimethylformamide and determined calorimetrically. The limit of determination is 0.6 pg PVP ml-’ [33. In a third method a dye spot placed on chromatographic paper is mobilized with the liquid phase by it-s PVP content. The distance travelled by the spot is related to the PVP concentration in the liquid phase. The detection limit is about 5 pg PVP ml-’ [4]. PVP can also be determined by precipitation with perchloric acid, followed by a turbidimetric or nephelometric determination of the precipitate.

*Present address: Institute of Chemistry, Analytical Department, Box 531, S-751 21 Uppsala, Sweden.

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The turbidity given by 1 pg PVP ml-’ is measurable 141. This method is referred to below as the “PA Method”.

After separation from the other constituents of Salazopyrin, PVP can be determined by any of the methods mentioned above. However, the Iodine Method and the PA Method seem to have the greatest advantages with regard to simplicity and sensitivity. Furthermore, they are the only methods suitable for automation, and have been adapted to Technicon AutoAnalyzer II systems. The PA Method is:preferred.because it is simple and not affected by the preceding separation step. This paper gives a detailed description of a fully automated nephelometric method for the determination of PVP and reports the results of an investigation of some basic factors (i.e. reaction time and sample molecular weight) which influence the nephelometric response. The Iodine Method is also described briefly.

In the proposed method, PVP is separated from Salazopyrin on a two-layer ion exchanger (QAESephadex stratified on SP-Sephadex) with water as eluant. The effluent is analyzed in the automatic system, where perchloric acid is added to the sample and PVP is precipitated. The solution is mixed in a coil and the light scattered at 90” from the incident beam is measured in a fluoronephelometer at 360 nm.

EXPERIMENTAL

Appura tus The flow chart for the nephelometric determination is shown in Fig. 1.

The parts include a Technicon proportioning pump III (Tygon, Acidflex pump tubes), ;i Technicon fluoronephelometer II with a 360 i 32 nm filter, a recorder (W + W 1100, LKB-Beckman) and a Sampletron PB 1000 sampler (Staprodukter, Uppsala) Millipore Swinnex-25 adaptors with 0.22~Mm Millipore filters are needed.

The equipment for the separation step is shown schematically in Fig. 2 and will be presented in detail elsewheie [5]. The thickness of the layers was optimized to separate PVP with a yield of almost 100 % from the other constituents of Salazopyrin.

Preliminary tests In turbidimetric measurements, 5.5-9 % perchloric acid gives an optimal

response [4]. This was confirmed in nephelometric detenninations of PVP; 6 %I perchloric acid in the final solution gives a better response than 11% or 3 7% acid.

Tween 20, Brij 35 and Triton X-100 were tested as wetting agents. Aged solutions of Tween and acid cannot be used, probably because of hydrolysis of the ester; the other two agents are not affected by the acid. Triton was chosen because it gave a slightly better base line than Brij.

The reaction time from adding perchloric acid to the final detection was varied by means of different sizes of coils. For 20-50 pg PVP ml-’ the best

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Millipore-

HCIOI *‘%5 S fiN22fl C5

W

1

refurn HCIOL - bottle

eo.92 ~ ” *waste

L ----- A Fluoronepheiometer(h=360nm)

H,O “’ 3

M$pore - JqyJ

to sampfer

-QScm ClAE Sephadex A25

lcm SP Sephadex C25

Fig. 1. Flow chart for nephelometric determination of PVP (figures represent flow rates of pump tubes in ml mine’,* acidflex pump tubes).

Fig. 2. Equipment for separation of PVP from Salazopyrin.

characteristics of the curves were found at reaction times of 40-100 s. For shorter times, mixing was not effective; with longer times, sensitivity was poor. A reaction time of 92 s was chosen as optimal.

In order to avoid disturbances from dust particles, all solutions except the sample must be filtered through 0.22~pm Millipore filters. This purifi- cation step is included in the system by inserting Millipore Swinnex-25 adaptors after the pump for acid and rinsing water. For the acid, one fraction of the filtered stream is re-pumped into the reaction stream, while the other is allowed to drain off to create a smooth flow.

Reagents The ion exchangers, SP-Sephadex C-25 (Sulphopropyl cation exchanger,

Pharmacia Fine Chemicals AB), and QAE-Sephadex A-25 (Quaternary Aminoethyl anion exchanger, Pharmacia Fine Chemicals AB), are allowed to swell for 1 h in distilled water before use_

The perchloric acid (9 %) is prepared by mixing 125 ml of 70 % HClO+ 800 ml of HzO, and 0.5 ml of Triton X-100, and adjusting the volume to 11.

The bulk standard is prepared by dissolving 1 g of PVP, exactly weighed, and with the same molecular weight distribution as the sample, in 500 ml of

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distilled water. For working standards, 10,15,20 and 25 ml of bulk standard are diluted to 1 1 with distilled water ( + 20, 30, 40 and 50 pg PVP ml-‘).

Procedure Salazopyrin (0.5 g, exactly weighed) is dissolved in 50 ml of 0.1 M sodium

hydroxide_ In a column, 1 cm of SP-Sephadex is placed, and above this layer 3 cm of QAESephadex is stratified (Fig. 2). The column is washed with 3-5 ml of water and a sample containing 200-500 pg PVP ml-’ is applied to the bed surface with a 500~~1 Eppendorf pipette. The column is eluted with approximately 5 ml of water and the exact volume is determined by weighing.

The samples are analyzed at a speed of 30 samples per hour with a sample- to-wash ratio of 18/2. The sample time makes it possible to measure the curves at a steady state. It is advisable not to run more than 10 samples between each group of calibration samples consisting of 4 single standards. The standards before and after every 10 samples are approximated to a second degree function (Fig. 3).

RESULTS

The separation step gives a yiald of 96-99 % of PVP and is independent of small variations (k 0.5 cm) in the ion-exchange layers. This step shows no interference with the subsequent nephelometric determination.

The AutoAnalyzer step gave a relative standard deviation (s,) of 0.5 % (N = 10). The random error of the whole method (separation, determination and calculations) gave s, = 1.0 % (N = 10). A reference substance was

Fig. 3. Standard curves for the nephelometric determination of PVP. The dotted curve in the main figure represents standard curve constructed from kinetic curves on AutoAnalyzer for a reaction time of 92 s. The dotted curve in the expanded part represents the standard curve constructed from manual kinetic curves for a reaction time of 110 s (;\ = 546 nm).

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analyzed under routine conditions for more than a year; 85 analyses gave s, = 3.2 %.

The standard curve is non-linear, but the linearity is improved at higher concentrations (Fig. 3). The shape of the curve can be predicted from the dependence of the nephelometric response on concentration and reaction time. The detection limit depends on the pre-treatment of the sample to remove disturbing dust particles and on the final design of the system. With the present system, 0.5 pg PVP ml-’ in the effluent is easily detectable.

DISCUSSION

The yield of less than 100 o/o probably results from occlusion of PVP in the precipitation reaction of salicylazosulfapyridine when the elution starts. This effect is pronounced at higher concentrations of Salazopyrin (>1.2 70 (w/v))_ The capacity of the automatic system can be raised to 60 samples per hour with a relative standard deviation of 1 %, but 55 samples per hour appears to be a critical limit because of contamination effects. The nephelo- metric response to precipitation of PVP with perchloric acid is strongly time-dependent (Fig. 4). Automatic kinetic curves were obtained by pumping the reaction mixture continuously from a beaker directly into the nephelometer. Manual determinations permitting the kinetic curves to be studied after 10 s were performed on a Zeiss PM &II with fluorescence assembly at 546 nm. The curves are surprisingly similar in nature although the reaction mixture was not mixed continuously in the manual determinations (as in the automatic) and another wavelength was used.

A A

-100

-90

-80 Zj 5 -70 s

5f$ -60 2%’

-50 -2 +I

-$O F z S&

-30-&g

-203s

-70 -

80

;3 i-Ad-w* 160

I I LO 240 320

Reaction time (9 Fig. 4. Kinetic curves for the nephelometric determination of PVP a) 100 pg b) 60 pg c) 20 pg and d) 5 yg PVP ml-l. The filled symbols represent results from automatic determination. The open symbols represent manual determination.

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The time-dependence of the nephelometric response for precipitation of PVP can be explained by the influence on the scattered light of the con- tinuously growing particles. A theoretical interpretation of the mechanism is intricate and lies beyond the scope of this investigation. However, for particles small in comparison with wavelength the nephelometic response is a first or second degree function of time [6] and for particles larger than X/20 the amount of light scattered at 90” decreases rapidly with increasing particle size [7] _ These two factors appear to compete to give the shape of the kinetic curve. Similar kinetic curves have been reported for the nephelo- metric determination of rubber formulations 183 and for a nephelometric immunological method [9] .

The precipitation rate depends n_ot only on the PVP concentration (Fig. 4), but also on the molecular weight (M_ ) of the sample (Fig. 6). This dependence is noticed particularly in the range M, = 12 000-50 000 (Fig. 7). It is therefore essential that t&e sample and standard have the same molecular weight distribution (for RI,” < 50 000) and not only the same K-value (a molecular weight estimate calculated from the intrinsic viscosity according to the Mark-Houwink relation [q] = K(IM)~ ).

As shown in Fig. 3, the calibration curve can be explained from the kinetic curves for different concentrations. Furthermore, calibration curves at different reaction times can be predicted from manual kinetic curves at roughly equal reaction times. This is shown in Fig_ 5 where, in order to separate the curves, the relative response is plotted against concentration. This plot also gives some information about the shape of the calibration curve for a particular PVP-range at different reaction times. It can be shown

I I I I I I I I I I- 20 40 60 80 700

PVP (m ml“/ Fig. 5_ Comparison of relative nephelometric response as a function of PVP concentration at different reaction times for manual and automatic determination. Solid curves show the relative response in the AutoAnalyzer system. Different reaction times from the use of different coils: - = 32 s, l = 92 s and A = 182 s reaction time. (The amplification of the signal is not equal for different times). Dotted curves show the relative response calculated from manual kinetic curves. Reaction time: 0 = 35 s, 0 = 90 s and a = 180 s.

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140 180 Reaction time Is)

Fig. 6. Kinetic curves for 40 pg PVP ml-’ with different molecular weights. E,” = a) 189 000 b) 48 000 c) 12 200. Fractions obtained from [ 121.

o!! ( , I 1 w

010 30 so loo 150 iGwx 10-J

Fig. 7. Nephelometric response as a function of M, for 40 pg PVP ml-‘.

that the relative response curve at a reaction time of 90 s implies a second- degree calibration curve, while a reaction time of several minutes gives a first-degree calibration curve for 20-50 pg PVP ml-‘. The linearity of the calibration curve is thus increased, while the response decreases with increasing reaction time.

Recent investigations have shown that, by using 14 ‘3% (W/W) trichloro- acetic instead of perchloric acid as precipitation reagent, the nephelometric response is not only independent of molecular weight (12 OOOlic;r,L 189 000), but is also about four times larger than that obtained with perchloric acid. This reagent will be studied further. Molecular weight dependences have also been reported both for the Iodine Method [lo] and for the staining of PVP

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with Vital Red [ll] . For the latter method an approximately linear relationship between response and M over the whole molecular weight range is reported.

To attempt to establish its general applicability, this method was used for the determination of 1 pg PVP ml-’ in beer, for which another method has been described [3] _ The low concentration of PVP after separation (0.1 pg ml-‘) made it necessary to amplify the signal so much that dust particles in the sample interfered severely_ To improve the sensitivity, the amount of eluant can be decreased and the response can be increased by prolonging the reaction time or by using another precipitation reagent (i.e. trichloroacetic acid). Furthermore, the solutions for elution and preparation of standards must be dust-free. Thus, the present method cannot be used to determine PVP in beer.

Since the separation step is not specific for PVP, but merely separates charged from non-charged solutes, the method is not generally applicable to the analysis of PVP in all materials. However, the content of PVP (22 pg ml-’ ) found in a complex coating substance by this method compared well with the expected value (20 pg ml-‘).

The formation of a coloured complex between PVP and triiodide [2] has also been adapted to an AutoAnalyzer II system (Fig. 8). Iodine is generated in the system from iodate and iodide at pH 2.2. Citric acid buffer is added to ensure a suitable pH, the sample is added, and the absorbance at 420 nm is measured. The reproducibility and capacity are comparable with those

air

sample 0160

I :s_c-c&ii+A

Q80 *waste

H20 zoo h to smpkr

Fig. 8. Flow chart for determination of PVP by reaction with iodine-iodide (figures represent flow rates of pump tubes in ml min-‘).

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of the nephelometric method. The standard curve (Z-20 Hg PVP ml-‘) is linear, but the separation step contributes a “blank” corresponding to approximately 0.5 fig PVP ml- ‘_ The last coil in the system also visibly adsorbs iodine.

The authors express their gratitude to Professor Bengt Nyg&d for most valu- able discussions and constructive criticism. The encpuragement and interest of Professor Carl-Olof Bjijrling and Dr. Adolf Berggren isalso appreciated- Lars Hagel is indebted to Pharmacia AB for their sup@ort of this work.

REFERENCES

1 K. Zipf, KIin. Wochenschr., 23 (1944) 340. 2 G. B. Levy and D. Fergus, Anal. Chem., 25 (1953) 1408. 3 W. Postel, Brauwissenschaft, 26 (1973) 337. 4 D. A. Shiraeff, J. Ass. Offic. Anal. Chem., 47 (1964) 724. 5 R_ Andersson, to be published. 6 H.-G. E&s, in M. 3. Huglin (Ed.), Light Scattering from Polymer Solutions, Academic

Press, London and New York, 1972. p- 434. 7 J. Springer, Einfiihrung in die Theorie der Lichtstreuung verdiinnter LSsungen grosser

Molekitile, Applied Research Laboratories, 1970, p. 29. 6 A R. Matz, Bull. Parenter. Drug Assoc., 19 (1965) 3. 9 H. von Ebeling, 2. Klin. Chem. Klin. Biochem, 11 (1973) 209.

10 C. Fioretti, Arch. Vet. Ital., 11 (1960) 189. 11 ‘L. J. Frauenfeider, J. Ass. Offic. Anal. Chem., 57 (1973) 796. 12 K. Granath and R. Stromberg, Der Anaesthesist, 17 (1968) 224.