Abstract
Phenol is commonly used as an antimicrobial agent with an initial concentration of 0.35% (w/v) in injectable diluted tuberculin purified protein derivative (TPPD) solution. The anti-microbial action of phenol in TPPD is directly concentration dependent. Furthermore, high phenol content (>0.5%) may have a negative effect on the stability and clinical effectiveness of TPPD solution. Therefore, simple, rapid and reliable reversed phase liquid chromatographic (RPLC) and capillary zone electrophoretic (CZE) methods were firstly developed and validated for phenol quantification in Connaught tuberculin (CT68) PPD diluted preparations at 5 TU per test dose of 0.1 mL. In RPLC, the elution was carried out by 80% (v/v) ACN mixed with 20% (v/v) phosphate buffer containing 0.05% (v/v) triflouroacetic acid (pH 3.2) at 0.2 mL min−1 flow rate and 20.0 °C column temperature. In addition, phenol was separated from tuberculin (CT68) protein with a resolution of (R = 2.81) and was quantified within 3 min.
In CZE, the migration of phenol was performed by 50 mmol L−1 borate buffer (pH 9.8) at −20 kV applied voltage and 25.0 °C capillary temperature. Furthermore, excellent linearity was achieved within 0.17–0.53% (w/v) for the phenol content with coefficients of determination (r2) higher than 0.9995. Moreover, the detection and quantification limits were found to be 0.046 & 0.153% and 0.051 & 0.171% (w/v) with RPLC and CZE respectively. Additionally, the intraday precision (RSD%, n = 9) was ranged between 0.18 and 0.39 and 0.33–54 with RPLC and CZE respectively. Moreover, the interday precision (RSD%, n = 27) was varied between 2.06 and 2.99 and 2.25–3.40 by RPLC and CZE, respectively. Furthermore, the obtained mean recoveries were ranged between 91.32 and 107.51% with RPLC and 90.71–108.92% with CZE. In addition, the effect of different storage temperatures at 4, 25 and 37 °C over storage periods of 2, 7, 14, 21 and 30 days was also studied on the TPPD product. The obtained results have revealed that the phenol content was effectively decreased about 37% of its original content after 30 days at storage temperatures of 25 and 37 °C. However, the phenol content did not change and was stable up to 21 days at storage temperature of 4 °C. Therefore, the simple and rapid proposed analytical methods could be used for a rapid expiry investigation of TPPD products based on phenol quantification, as a marker.
•Rapid phenol detection as a marker for the stability of tuberculin PPD solution.•Reliable HPLC and CZE methods for phenol quantification in tuberculin PPD solution.•Study the effect of storage temperature and time on phenol quantification.