NMR analysis indicated that the nitrocefin
purchased from BioVision Inc. and Oxoid Limited was significantly
purer than that from Cayman Chemicals. The BioVision nitrocefin was
used in this study. Several values have been reported for the change
in extinction coefficient upon hydrolysis of nitrocefin (e.g., refs (15 (link), 21 (link))). To determine this value independently,
a stock solution containing 5.0 mg of nitrocefin was diluted to a
range of seven concentrations from 10 to 75 μM in 100 mM sodium
phosphate buffer, pH 6.4. The A486 values
before and after complete hydrolysis by 20 min incubation with 5 nM
BlaC were determined. The slope of a linear fit of ΔA486 against nitrocefin concentration yielded
Δε486. The procedure was performed in duplicate,
yielding a Δε486 of 17 ± 1 mM–1 cm–1. Clavulanic acid powder is hygroscopic, so
its concentration was determined by the absorbance at 256 nm in NaOH.
The extinction coefficient was determined by quantitative NMR versus
a standard of trimethylsilylpropanoic acid. For ChemCruz and Matrix
clavulanic acid, which are sold in a cellulose matrix, we found identical
UV–vis spectra, yielding an absorbance at 256 nm of 20.0 ±
0.1 mM–1 cm–1. For TRC clavulanic
acid, which is a pure powder, the UV–vis spectrum is clearly
different, and the NMR spectrum shows impurities. The extinction coefficient
at 256 nm is 18.7 mM–1 cm–1.
purchased from BioVision Inc. and Oxoid Limited was significantly
purer than that from Cayman Chemicals. The BioVision nitrocefin was
used in this study. Several values have been reported for the change
in extinction coefficient upon hydrolysis of nitrocefin (e.g., refs (15 (link), 21 (link))). To determine this value independently,
a stock solution containing 5.0 mg of nitrocefin was diluted to a
range of seven concentrations from 10 to 75 μM in 100 mM sodium
phosphate buffer, pH 6.4. The A486 values
before and after complete hydrolysis by 20 min incubation with 5 nM
BlaC were determined. The slope of a linear fit of ΔA486 against nitrocefin concentration yielded
Δε486. The procedure was performed in duplicate,
yielding a Δε486 of 17 ± 1 mM–1 cm–1. Clavulanic acid powder is hygroscopic, so
its concentration was determined by the absorbance at 256 nm in NaOH.
The extinction coefficient was determined by quantitative NMR versus
a standard of trimethylsilylpropanoic acid. For ChemCruz and Matrix
clavulanic acid, which are sold in a cellulose matrix, we found identical
UV–vis spectra, yielding an absorbance at 256 nm of 20.0 ±
0.1 mM–1 cm–1. For TRC clavulanic
acid, which is a pure powder, the UV–vis spectrum is clearly
different, and the NMR spectrum shows impurities. The extinction coefficient
at 256 nm is 18.7 mM–1 cm–1.
