Hydrolysis kinetics and stability predictions for a mixture of R and S temocillin isomers

Abstract

The hydrolysis kinetics of a temocillin isomer mixture (~68% R and ~32% S) were studied as a function of pH, temperature and buffers. Temocillin concentrations were determined from HPLC analyses of the individual isomers where [TEM]=[R]+[S]. Rate constants ( k R, k S and k SUM) were determined from linear first-order plots for [R], [S] and [TEM], respectively. The true time for 10% loss of temocillin ( T true) was determined by examining the time-course for the sum, [R]+[S]. Although the k R and k S values were generally similar, they differed by 30–40% under some conditions. Nevertheless, the calculated time for 10% loss ( T 90=−ln 0.9/ k SUM) agreed with T true for all experimental conditions. This was explained using computer simulations that indicated epimerization was faster than hydrolysis. Under these circumstances, reliable predictions of temocillin stability were achieved with k SUM= k H1( a H + ) f 1+ k H2( a H + ) f 2+ k H3( a H + ) f 3+ k H 2O + k OH( a OH − )+ k B 4O 7 [B 4O 7 2−], where the catalytic constants were k H1, k H2 and k H3 for hydrogen ion activity, k OH for hydroxyl ion activity, k B 4O 7 for B 4O 7 2− (in borate buffer only); k H 2O was the first-order rate constant for spontaneous hydrolysis and f 1, f 2, f 3 were the fractions of temocillin in three stages of dissociation. The Arrhenius expression, k= Ae − E/ R T , and the experimentally determined A and E values were substituted for k H1, k H2 k H3, k OH and k B 4O 7 . This equation successfully calculated 92% of the observed k SUM values with <10% error. A shelf-life of ~1.8 days was predicted and experimentally verified at 30°C in the region of maximum stability, pH 6.5–7.5.