Simultaneous determination of levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma by an HPLC-MS/MS method.

BACKGROUND: In this study, we developed and validated a HPLC-MS/MS method capable of simultaneously determining levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma. RESULTS & METHODOLOGY: Chromatographic separation was achieved using a C8 column with a mobile phase consisting of a gradient of water and acetonitrile:methanol (90:10 v/v), both containing 0.1% formic acid. The developed method was selective, sensitive (LD<7.0 ng ml(-1)), linear (r>0.99), precise (RSD<11.3%), accurate (RE<11.8%) and free of residual and matrix effects. The developed method was successfully applied in plasma patients with Parkinson's disease using Stalevo®. CONCLUSION: The new method can be used for the clinical monitoring of these substances and applied to adjustments in drug dosages.

Insulin enhances L-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance.

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on L-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 microU/ml) increased L-dopa uptake into PT cells by about 50% (705+/-186 vs.1117+/-140 pmol L-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity L-dopa transport sites (L-dopa 0.2 microM) (0.59+/-0.05 vs. 0.82+/-0.09 pmol L-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1-10 microM) or colchicine (50-100 microM), whereas it was abolished by cytochalasin D or latrunculin B (both 1 microM). This suggests that the process is independent of Na(+),K(+)-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. L-dopa transport by the low-affinity transport sites (L-dopa 5 microM) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal L-dopa reabsorption as compared to control animals (81+/-4 vs. 51+/-9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal L-dopa reabsorption.