ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2013, том 68, № 12, с. 1160-1170
ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
ONE-STEP SOLID-PHASE UV SPECTROPHOTOMETRIC METHOD FOR PHENOL DETERMINATION IN VACCINES: DEVELOPMENT
AND QUALITY ASSESSMENT © 2013 J. Vukovic*, R. Jurisic Grube sic*, D. Kremer**, A. Spaic*
*Department of Analytics and Control of Medicines, Faculty of Pharmacy and Biochemistry Ante Kova Сica 1, HR-10000 Zagreb, Croatia **Department of Pharmaceutical Botany with Fran Kusan botanical garden, Faculty of Pharmacy and Biochemistry, University of Zagreb Schrottova 37, HR-10000 Zagreb, Croatia Received 29.02.2012; in final form 20.12.2012
A solid-phase spectrophotometric technique was used to develop very sensitive, inexpensive, one-step procedure for determination of phenol (UV-SPS procedure). The proposed procedure is based on simultaneous sorption of phenol on an anion exchanger QAE Sephadex A-25 (40 mg), within 10 min at pH 11 and measurement of the intrinsic absorbance of the solid phase in a 1-mm cell at 289 nm and 500 nm, without previous derivatization. The optimum experimental conditions were investigated and comprehensive quality control of the new procedure was carried out using a prevalidation strategy accompanied by a very informative system of diagnosis. The UV-SPS procedure is characterized with both ideal calibration and analytical evaluation function within analyte working range from 0.3 to 3.0 prnol (6.0 to 60.0 nmol/mL). Random deviations (from ±0.3 to ±5.0%) and systematic deviations (from —3.0 to +4.9%) confirmed the favourable precision and accuracy of the UV-SPS method. Evaluated limiting values (LD = 1.0 цМ, Lq = 6.0 цМ) showed that this method enables determination of very low levels of phenol. The sensitivity of UV-SPS procedure is 50 times higher than that provided by the corresponding method in solution. The UV-SPS method was successfully applied to the determination of phenol in vaccines (recovery 95.6—103.4%).
Keywords: solid-phase spectrophotometry, phenol determination, prevalidation, vaccines.
DOI: 10.7868/S0044450213120141
Phenols are considered to be priority pollutants because of their harmful effect on organisms and possible accumulation in the environment. From the pharmaceutical point of view, monitoring of phenols is very important and demanding task either as a pharmaco-poeial phenol assay or as determination of phenol impurity in pharmaceutical substances. Many drugs are phenols of different type (antiseptics, germicides, analgesics, antipyretics, etc.); phenols are used as preservatives in pharmaceutical formulations (ointments, ear and nose drops, mouthwashes, antiseptic lotions, etc.). In addition to their medicinal use, phenols can occur as related substances and/or impurities in many pharmaceutical substances during the manufacturing process and/or storage (e.g., guaiacol, salicylic acid, hexylresorcinol, and immunosera and vaccines) [1]. According to ICH Guidelines [2—4], an impurity is any component of the new drug substance which is not the chemical entity defined as the new drug substance and it may be classified as organic, inorganic, and residual solvents.
According to the most pharmacopoeias [1, 5, 6], phenol contains not less than 99.0% and not more than 100.5% of C6H6O, calculated on the anhydrous basis, except in the Japanese Pharmacopoeia [7] where the content of phenol should not be less than 98%. Pharmacopoeial assay for determination of phenol content [1, 5—7] is based on well-known Koppeschaar reaction which employs a measured quantity of bromide-bromate solution in acidic medium to generate bromine which reacts with phenol. After completion of the phenol bromination, potassium iodide is added to the mixture to react with the excess of bromine. The quantity of iodine formed in this reaction is determined by titration with thiosulfate [8, 9]. This procedure is complicated and questionable because of the use of hepatotoxic and environmentally hazardous reagents and solvents.
Determination of phenol impurity in immunosera and vaccines is based on the reaction of phenol with 4-aminoantopyrine [1]. This reaction is widely used in conventional spectrophotometric methods in analytical practice [10—13]. The content of phenol is calcu-
lated from a calibration curve obtained using reference solutions of phenol.
Pharmacopoeias and other sources [14, 15] usually recommend instrumental techniques (HPLC, GC, CE) for determination of phenol impurity in drug substances. Analytical techniques used for determination of phenol impurities are mainly liquid chromatogra-phy (LC) [16—19], high performance liquid chromatography (HPLC) [20—23], capillary electrophoresis (CE) [24—29], and gas chromatography/mass spectrometry (GC/MS) [30—33]. To improve the sensitivity, solid phase extraction as an enrichment step has successfully been combined with different techniques to determine phenol [34—36]. Also, many synthetic resins and low-cost natural adsorbents were used for removal of phenol and its derivatives from wastewater [37, 38]. The need to characterize impurities in drug substances and drug products at trace levels, as well as ever increasing demand to speed up pharmaceutical development has led to an extensive use of so-called hyphenated techniques [39—43]. In spite of many advantages of instrumental techniques, certain limitations arise like absence of simplicity and protraction. Therefore, introduction of a very simple and low-cost one-step UV-spectrophotometric procedure for phenol analysis on reactive carrier using no chromogenic agent and pre-treatment of the analyte could be useful for fast determination of phenol in very low concentrations.
Solid-phase spectrophotometry (SPS) technique enables direct measurement of an analyte concentrated on an appropriate solid support. This technique is based on different methodologies, types of solid supports and reaction mechanisms. The first application of SPS was for determination of traces of metal ions in water [44]. The main advantage of this method is that it could be over a few hundred times more sensitive than the conventional corresponding solution method, without using any expensive apparatus. Although the majority of SPS methods are devoted to the environmental pollution control [45—47], a substantial number of papers deal with application to drug [48—50] and food control [51, 52], and to clinical chemistry and medicine [53, 54]. Although phenol is very often determined by conventional spectropho-tometry [55, 56], there are few studies published concerning determination of phenol by SPS [57—59]. These SPS methods are based on the reaction of phenol with 4-aminoantipyrine and preconcentration of antipyrine dye formed on a solid support. The use of chromogenic agent, strict monitoring of experimental conditions, and mode of dye collection undoubtedly makes these methods more complicated and could influence the efficiency of the product adsorption on the solid support.
The intention of this work was to develop simple, sensitive, one-step solid-phase spectrophotometric method for determination of phenol without using ex-
pensive instrumentation and chromogenic agents. Since pharmaceutical analysis requires an unambiguous evaluation of the advantages and disadvantages of the analytical procedure/method, a substantial part of this work is devoted to quality assessment of the UV-SPS procedure using comprehensive and very informative prevalidation strategy [60]. The presented analytical system may be a suitable alternative to pharmacopoe-ial assay for phenol content determination. The high speed, easy use, high level of sensitivity and relative freedom from random contamination during sample handling recognized this system as a valuable procedure for the phenol monitoring important in the pharmaceutical analysis, and this procedure could offer wider applications.
EXPERIMENTAL
A double beam UV-Visible Spectrophotometer Cary 50 Bio (Varian, Inc., Palo Alto, USA) with 1-mm quartz cells was used for all absorbance measurements.
All chemicals were of analytical-reagent grade; highly purified water was prepared with a Mili-Q SP system (Milipore, Milford, MA). For preparation of the phenol stock standard solution, 10.0 mg of phenol (Kemika, Zagreb, Croatia) was dissolved in 100.0 mL water using a volumetric flask. Working standard solutions of desired concentrations were prepared daily by appropriate dilution of the stock standard solution. Buffer solution H3BO3/NaOH, pH 11.0 (cT = 0.4 M) was prepared by dissolving 24.64 g of H3BO3 (Kemika, Croatia) in 900.0 mL of water. The final pH was adjusted to 11.0 by adding 10.0 M NaOH (Kemika), and the solution was diluted to 1000.0 mL.
Anion-exchange gel QAE-Sephadex A-25 (Pharmacia Fine Chemicals, Sweden) with particle size of 40—120 |m in the Cl- form was used. The gel was used in its original dry state to avoid any contamination.
To determine the concentration ofphenol a general procedure was applied. Appropriate volumes of phenol standard stock solution, containing 0.3 to 3.0 |mol (6.0 to 60.0 nmol/mL) were transferred to a 100.0-mL container. Then 1.0 mL of borate buffer solution (pH 11.0) was added and the volume was made up to 50.0 mL with water. To the prepared solution, 40.0 mg of QAE Sephadex A-25 ion-exchange gel was added and the mixture was stirred for 10 min. The gel beads were allowed to settle and the suspension was transferred to a 1-mm quartz cell using a pipette. Blank samples were prepared in the same manner but without analyte. The absorbance of analyte sorbed on the resin was measured in a 1-mm optical path length quartz cell at two wavelengths, at 289 nm (the absorption maximum wavelength of phenol) and at 500 nm (the wavelength at which only the gel absorbs light). The absorbances of blanks were measured in the same cell packed with resin previously equilibrated with a blank solution. The net absorbance for the phenol in
1162 Table 1.
VUKOVIC h gp. Strateg
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