Development and certification of reference standards for phenolic content in biologicals, based on comparison of results obtained by GLC, HPLC, spectrophotometric, and colorimetric methods

Phenol is used as a preservative in a number of biological products. Methods that are used for quantitative determination of phenol differ a lot. Current requirements for accredited laboratories include continuous internal quality control. Reference standards with a certified content of the analyte are an effective metrological tool for ensuring such control. The aim of the study was to develop and certify reference standards for phenolic content in biological products, based on comparison of results obtained by GLC, HPLC, spectrophotometric, and colorimetric methods. Materials and methods: diluent for allergens by (candidate reference standard), 2.5 and 5 mg/mL phenol solutions, and 2.5 mg/mL 2-phenoxyethanol solution were used in the study. The experiments were performed using spectrophotometric, colorimetric, HPLC, and GLC procedures. The statistical analysis of results included calculation of the arithmetic mean, standard deviation, coefficient of variation, and analysis of variance with Student’s t-test and Fisher’s F-test. Results: the results of phenolic content determination by the spectrophotometric, colorimetric, and HPLC methods were statistically comparable. The F value obtained for equal sample sizes (n = 40) was F = 0.9343, given the critical value F_crit = 3.96. A reference standard certified by one of these methods can be used to control the consistency of phenol determination by a relevant method. The results of phenolic content determination by the GLC method showed statistically significantly differences: F = 17.47, given F_crit = 3.96, which demonstrated the need for certification of another reference standard. Conclusions: two reference standards were certified in the study: reference standard 42-28-449 with the certified phenolic content of 2.56‒3.32 mg/mL, to be used with the spectrophotometric, colorimetric, and HPLC methods; and reference standard 42-28-451 with the certified phenolic content of 2.92‒3.28 mg/mL, to be used with the GLC method.

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