Cleaning validation using UV–VIS spectrophotometry
Robert H. Clifford, PhD, industrial business unit manager, Shimadzu Scientific Instruments
Maintaining quality control and product safety is key to pharmaceutical manufacturing. Cleaning manufacturing equipment is
essential for preventing contamination and cross-contamination. Contaminants from the environment must not get mixed with
product ingredients, and residual substances adhering to manufacturing equipment cannot contaminate the next product in line
for processing.
To verify these requirements, the cleaning process must be validated. The quantitation limit of an analytical instrument is
the value at which residual samples can be quantitated. To determine whether the instrument to be used for cleaning validation
has the resolution or sensitivity to detect to the permissible level of residual substance, it is important to determine the
quantitation limit. In this example, the quantitation limit was determined using ultraviolet–visible (UV–VIS) spectrophotometry
using samples consisting of one sample of an undisclosed detergent, Detergent A, often used for cleaning in the pharmaceutical
field, and samples of acetylsalicylic acid and isopropylantipyrine.
Figure 1 (UV–VIS): Absorption spectra of detergent A with sample concentrations of 100 mg/L and 10 mg/L. (FIGURES 1 (UV-VIS)
IS COURTESY OF THE AUTHOR)
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One method of obtaining the quantitation limit is to determine the concentration value that corresponds to the absorbance
(10 times the noise level). This method involves measuring the absorption spectrum of a standard sample and noting the wavelength
of the greatest absorption peak. The next step is to measure the absorbance values at the wavelength of the greatest absorption
peak using several samples of known concentration. The slope of the calibration curve is determined from the relationship
between the concentrations of the samples and the respective absorbance values. Finally, repeat measurement of a blank sample
(i.e., dilute solvent) is conducted to obtain the standard deviation. The quantitation limit is calculated from the slope
of the calibration curve and the value equivalent to 10 times the standard deviation. Determination of the quantitation limits
of Detergent A, acetylsalicylic acid, and isopropylantipyrine according to this method are presented in Figures 1–4 (UV-VIS).
Figure 2 (UV–VIS): Calibration curve at a measurement wavelength of 225 nm.
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Figure 1 (UV–VIS) shows the absorption spectra of detergent A with sample concentrations of 100 mg/L and 10 mg/L. Figure 2
(UV–VIS) indicates the calibration curve at a measurement wavelength of 225 nm. The quantitation limit for detergent A is
determined to be approximately 0.16 mg/L.
Figure 3 (UV–VIS): Absorption spectrum of acetylsalicylic acid methanol solution.
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Figure 3 (UV-VIS)shows the absorption spectrum of acetylsalicylic acid methanol solution. The sample concentrations from higher
to lower absorbance values are 400, 160, 80, 40, 20, and 8 mg/L. After 10 repeat measurements of a blank sample, the quantitation
limit for acetylsalicylic acid is determined to be 0.42 mg/L.
Figure 4 (UV–VIS): Absorption spectrum of isopropylantipyrine methanol solution.
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In Figure 4 (UV-VIS), the absorption spectrum of isopropylantipyrine methanol solution is shown. The sample concentrations
from higher to lower absorbance values are 80, 32, 16, 8, 4, and 1.6 mg/L. After 10 repeat measurements, the quantitation
limit for isopropylantipyrine is determined to be 0.092 mg/L.
In conclusion, the measurement results for detergent A, acetylsalicylic acid, and isopropylantipyrine illustrate the method
of calculating quantitation limits based on measurement conducted using a UV-VIS spectrophotometer. Obtaining the quantitation
limit makes it possible to verify the lower limit of residual substances and detergent that can be quantitated. UV-VIS spectrophotometry
is a justifiable addition to cleaning validation tools alongside total organic carbon analyzers and high-performance liquid
chromatographs.
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