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Microsoft word - t108-quality control of small molecule pharmaceuticals using spectrophotometry.docx

APPLICATION NOTE                                                        NanoDrop 2000/2000c  Quality Control of Small Molecule
Pharmaceuticals using Spectrophotometry

Experimental Procedures
The control of pharmaceutical product quality is essential to Test samples were analyzed per the US Pharmacopeia ensure safe and effective use in patients. The Thermo specifications that unknown concentrations of acetaminophen Scientific NanoDrop 2000 Spectrophotometer offers a rapid be quantified using absorbance at 244 nm against an and cost-effective method for spot-checking batch quality at acetaminophen standard curve prepared from a known various points in the pharmaceutical production line. The standard of ≥ 99.9% purity. Prior to experimentation, the concentrations of many compounds can be determined based absorbance maximum of acetaminophen was confirmed as upon UV-Visible absorbance, and, in some instances, purity can also be monitored by analyzing spectral data. In this study, UV absorbance spectra measured using a NanoDrop™ 2000 Spectrophotometer were used to compare several acetaminophen samples from various sources with a known standard. In addition, reproducibility was assessed within each sample to determine the affects of aggregates or light scattering in the unknown samples. The auto-ranging pathlength capabilities (fig. 1) of the NanoDrop 2000 allow this instrument to quantify pharmaceutical samples across a much broader concentration range than is possible using a conventional cuvette-based spectrophotometer. By automatically selecting the optimum Figure 2: Absorbance spectrum of 6 mg/mL acetaminophen pathlength (ranging from 1.0 mm to 0.05 mm) the NanoDrop standard. Absorbance maximum at 244 nm was used for 2000 can accurately measure the absorbance of a sample across a dynamic range nearly 300 fold greater than that of a cuvette- Six samples of acetaminophen were compared to the standards. Each of these samples was dissolved in 100 mL deionized H2O by incubating for 4 hr at room temperature on an orbital shaker (~100 rpm), resulting in ~5 mg/mL solutions. A 6 mg/mL standard was prepared using 600 mg acetaminophen (Sigma-Aldrich, A7085) dissolved in 100 mL deionized H2O. A standard curve was prepared by performing a serial dilution of this standard. Absorbance of triplicate, 1 μL aliquots of each dilution were measured at 244 nm using the NanoDrop 2000 UV-Vis application. A linear relationship Figure 1: Sample liquid column during measurement, showing between absorbance and concentration was observed the 1.0 mm (left) and 0.1 mm (right) pathlengths. throughout the entire standard curve range (fig. 3). Test acetaminophen solutions were measured against this standard curve in order to experimentally determine concentrations and calculate the total acetaminophen content. Thermo Fisher Scientific – NanoDrop Products Wilmington, Delaware USA Technical support: 302-479-7707 APPLICATION NOTE                                                     NanoDrop 2000/2000c   equiv 20
Concentration (mg/mL)
Figure 3: Standard curve of acetaminophen absorbance at 244 Figure 4: Absorbance spectrum of unknown acetaminophen sample “B.” Nearly identical absorbance spectrum to that observed from measurements of the standard (fig. 2) suggests a Three separate solutions for each test acetaminophen sample Conclusion
were measured in triplicate to assess reproducibility (table 1). Reproducibility was very good; CV values of less than 1 % The linear relationship between acetaminophen concentration and absorbance at 244 nm makes spectrophotometry an ideal method to verify batch concentrations. The Thermo Scientific Replicate
NanoDrop 2000 Spectrophotometer can be employed to A244 (n=3)
provide a rapid and accurate spot-check of pharmaceutical products. The instrument’s auto-ranging pathlength greatly reduces the need for sample dilution. The full spectrum display makes this instrument ideal for providing insight into the purity of various batches of product. Utilizing a traditional cuvette-based system, a greater number of large-volume serial dilutions would be required in order to dilute the samples from their stock concentrations to a measureable concentration range. By reducing the number of dilutions required, the NanoDrop 2000 greatly reduces this potential source of error. In addition, the instrument’s short measurement cycle and general ease of use greatly increases the rate at which samples can be processed, making it possible to implement multiple quality control checks throughout the production process. Table 1: Acetaminophen content of six unknown samples References
Absorbance spectra of the test acetaminophen samples (fig. 4) showed a nearly identical profile to the standard solution, 1. United States Pharmacopeia and Natural Formulary (USP 29 NF 24). Supplement No. 2. Rockville, MD: United States Thermo Fisher Scientific – NanoDrop Products Wilmington, Delaware USA Technical support: 302-479-7707


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