Substantial renal conversion of L-threo-3,4-dihydroxyphenylserine (droxidopa) to norepinephrine in patients with neurogenic orthostatic hypotension.

BACKGROUND: The pressor effect of L-threo-3,4-dihydroxyphenylserine (L-DOPS, droxidopa, Northera™) results from conversion of L-DOPS to norepinephrine (NE) in cells expressing L-aromatic-amino-acid decarboxylase (LAAAD). After L-DOPS administration the increase in systemic plasma NE is too small to explain the increase in blood pressure. Renal proximal tubular cells abundantly express LAAAD. Since NE generated locally in the kidneys could contribute to the pressor effect of L-DOPS, in this study we assessed renal conversion of L-DOPS to NE. METHODS: Ten patients who were taking L-DOPS for symptomatic orthostatic hypotension had blood and urine sampled about 2 h after the last L-DOPS dose. L-DOPS and NE were assayed by alumina extraction followed by liquid chromatography with electrochemical detection. Data were compared in patients off vs. on levodopa/carbidopa. RESULTS: In patients off levodopa/carbidopa the ratio of NE/L-DOPS in urine averaged 63 times that in plasma (p = 0.0009 by t test applied to log-transformed data). In marked contrast, in the three patients on levodopa/carbidopa the ratio of NE/L-DOPS in urine did not differ from that in plasma. CONCLUSION: There is extensive renal production of NE from L-DOPS. Carbidopa seems to attenuate the conversion of L-DOPS to NE in the kidneys. Further research is needed to assess whether the proposed paracrine effect of L-DOPS in the kidneys contributes to the systemic pressor response.

Sensitivity improvement of the LC-MS/MS quantification of carbidopa in human plasma and urine by derivatization with 2,4-pentanedione.

The reliable quantification of carbidopa in biological samples at low concentrations is challenging because of the polar and highly unstable nature of the compound. In this paper, LC-MS/MS methods are described for the determination of carbidopa in 50µL of human plasma and 25µL of human urine in the concentration ranges 1-1,000ng/mL and 100-50,000ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40°C. The product is a relatively non-polar molecule that is suitable for reversed-phase liquid chromatography (3.5min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalization. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5mg/mL sodium metabisulfite and 1mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5mg oral dose.

Sensitive determination of carbidopa through the electrochemiluminescence of luminol at graphene-modified electrodes.

Using the concept of electrogenerated chemiluminescence (ECL), a sensitive analytical method for the determination of carbidopa is described. Electro-oxidation of carbidopa on the surface of a graphene oxide (GO)-modified gold electrode (GE) leads to enhancement of the weak emission of oxidized luminol. Under optimum experimental conditions, the ECL signal increases linearly with increasing carbidopa concentrations over a range of 1.0 × 10(-9) -1.7 × 10(-7) M, with a detection limit of 7.4 × 10(-10) M. The proposed ECL method was successfully used for the determination of carbidopa in urine samples.

Simultaneous determination of levodopa and carbidopa by synchronous fluorescence spectrometry using double scans.

A synchronous fluorescence spectrometric method is described for the simultaneous determination of binary mixtures of levodopa and carbidopa in pharmaceutical formulation and urine sample, without prior separation steps, using two scans. At Delta lambda = 30 nm, only carbidopa yields a detectable signal that is independent of the presence of levodopa. Similarly, at Delta lambda = 65 nm the signal of levodopa is not influenced by the presence of carbidopa. Signals at two wavelengths, 288 nm (Delta lambda = 30 nm) and 281 nm (Delta lambda = 65 nm), vary linearly with carbidopa and levodopa concentrations over the range 0.019-1.971 microg mL(-1) (for levodopa) and 0.022-2.262 microg mL(-1) (for carbidopa), respectively. The correlation coefficients for the standard calibration graphs were 0.9962 and 0.9951 (n=10) for carbidopa and levodopa, respectively. The limits of detection (LOD estimated as per IUPAC recommendations) were 0.01 and 0.006 microg mL(-1) for carbidopa and levodopa, respectively. The method was successfully applied to the determination of levodopa and carbidopa in pharmaceutical formulation and urine sample. The recovery results were satisfactory.

Simultaneous determination of levodopa, carbidopa and their metabolites in human plasma and urine samples using LC-EC.

In this study levodopa (L-DOPA), carbidopa (C-DOPA) and their metabolites were resolved from other endogenous components present in human plasma and urine and determined quantitatively. The developed technique involved the use of a second pump, a switching valve, and a pre-column in the LC system in order to perform on-line sample clean-up and enrichment. This procedure is dependent on an effective removal of the many interfering matrix components that vitiate HPLC analysis. Several unknown endogenous electroactive compounds, present in plasma, were eliminated by the purification step, or suppressed by the pre-treatment or detection conditions. The analyses were separated on an Octyl-bonded reversed-phase column followed by amperometric detection using a carbon fibre microelectrode flow cell operated at +0.8 V versus silver/silver phosphate reference electrode. The cell was compatible with the mobile and the stationary phase used in the flow system without any complex surface reaction. The peak currents obtained for the different analytes were directly proportional to the analyse over the concentration range 0.02-4.0 microg ml(-1). Using this method, the minimum detectable concentration was estimated to be 5 and 8 ng ml(-1) for L-DOPA and C-DOPA, respectively. Recovery studies performed on human plasma samples ranged from 93.83 to 89.76%, with a relative standard deviation of < 6%. The intra- and inter-assay coefficients of variation were < 7%. The accuracy of the assay, which was defined as the percentage difference between the mean concentration found and the theoretical (true) concentration, was 12% or better. The electrochemical pre-treatment regime described in this work permitted a longer application of the same microelectrode. The method showed a good agreement with other available methods described in the introduction and offers the advantages of being simple, less time and labour consuming, does not require additional solvents for extraction, inexpensive and suitable for routine analysis and kinetic purposes.

Simultaneous high-performance liquid chromatographic analysis of carbidopa, levodopa and 3-O-methyldopa in plasma and carbidopa, levodopa and dopamine in urine using electrochemical detection.

Two assay procedures are described for the analysis of levodopa, carbidopa and 3-O-methyldopa in plasma and levodopa, carbidopa and dopamine in urine. The methods are suitable for quantifying the analytes following therapeutic administration of levodopa and carbidopa. Both were based on reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection and with methyldopa as the internal standard. Plasma samples were prepared by perchloric acid precipitation followed by the direct injection of the supernatant. Urine was prepared by alumina adsorption, and the analytes were desorbed with perchloric acid solution containing disodium EDTA and sodium metabisulfite prior to injection into the HPLC system. The methods have been utilized to evaluate the pharmacokinetics and bioavailability of oral dosage forms containing levodopa and carbidopa.

Urinary 5-S-cysteinyldopa in Parkinsonism after DOPA and carbidopa.

The effect of anti-Parkinson therapy on the urinary excretion of 5-S-cysteinyldopa (5SCD), a catechol metabolite of dihydroxyphenylalanine (DOPA) and marker for the melanocyte, was studied by means of high performance liquid chromatography. 5SCD was normal in Parkinson patients treated with anticholinergics. DOPA administration increased 5SCD excretion. Carbidopa and DOPA together elevated 5SCD markedly in a dose-dependent manner to values higher than seen in some patients with metastatic malignant melanoma. The effect of anti-Parkinson therapy should be considered when using 5SCD as a tumor marker or when a Parkinson patient has a melanoma.

Further studies on the metabolism of carbidopa, (minus)-L-alpha-hydrazino-3,4-dihydroxy-alpha-methylbenzenepropanoic acid monohydrate, in the human, Rhesus monkey, dog, and rat.

Major urinary metabolites of carbidopa have been identified. Estimates were made based on the recovery or radio activity or by glc analysis of pooled urine of the amounts of the urinary metabolites II (2-methyl-3'-methoxy-4'-hydroxyphenylpropionic acid), III (2-methyl-3,4-dihydroxyphenylpropionic acid), IV (3,4-dihydroxyphenylacetone), V [2-methyl-3-(3'-methoxy-4'-hydroxyphenyl)lactic acid], VI [2-methyl-3-(3',4-dihydroxyphenyl)lactic acid], and VII (3-hydroxy-alpha-methylphenylpropionic acid). Metabolite II represented similar to 10% of the urinary radioactivity in both man and monkey and 16% in the dog. Metabolite III represented 10, 17, and 19% of the urinary radioactivity in man, monkey, and dog. Metabolite IV represented smaller than 5% of the urinary radioactivity in human and dog. Metabolite VII represented similar to 10% of the urinary radioactivity in man and monkey. The corresponding figure for the rat was similar to 20%. In the dog, compounds V and VI represented smaller than 5% of the urinary radioactivity. It was concluded that the loss of the hydrazine functional group(probably as molecular nitrogen) represents the major metabolic pathway for carbidopa.