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A new, sensitive, rapid, and precise flow injection spectrophotometric method for the determination of penicillamine (PEN) in pharmaceutical formulations has been developed and validated. Penicillamine is a synthetic amino acid that contains an additional SH group and can form non-toxic, water-soluble chelates with heavy metals, which are then excreted in the urine. PEN was the first chelator used for Wilson's disease, and it can also be used for lead, mercury and arsenic poisoning. The method is based on a fast one-step redox reaction wherein PEN reduces the Cu(II)-bathocuproine complex to the Cu(I)-bathocuproine complex, which exhibits absorption maxima at 483 nm. Therefore, a simple two stream single reagent manifold was used. By decreasing the inner volume of the flow cell (from 160 μL to 30 μL), the miniaturization of other parts of the manifold was enabled. Therefore the sensitivity of PEN determination was advanced, and sampling frequency increased. Optimization of experimental conditions and manifold parameters was carried out through the univariate method. The reagent, prepared in acetic buffer solution (pH = 5), was merged with a sample (200 μL) and injected into a carrier stream of distilled water at a confluence point, where streams combined. The flow rate of the carrier stream was 6 mL/min, and the reagent was pumped at the rate of 2 mL/min. The reaction coil, 30 cm long and 0.8 mm I.D. (volume 150 μL), was kept at room temperature. Since the flow rate through the reactor was 133 μL/sec, the incubation time was just over a second, yet sufficiently long for the reaction to reach a steady state. A linear calibration curve was established in a concentration range of PEN from 2×10−7 to 4×10−5 mol L−1 with regression equation y = 8.48×103 x – 0.0011 (R2 = 0.9996). The novel FI-method is sensitive enough to enable LOD below 8 ppb of PEN at a sampling rate of 180 s/hr without expensive instruments. In addition, method is precise (RSD 0.9%, n = 100) and accurate (recovery in the range from 98.7% to 101.6%). The proposed method can be applied for the determination of PEN in pharmaceutical formulations since usual excipients and other present active substances in pharmaceuticals do not interfere.

A flow-injection indirect spectrophotometric method for the determination of ascorbic acid (AA) is proposed. The method is based on the coupled redox-complexation reaction, the first step of which is the reduction of iron (III) by ascorbic acid ; the second one includes the complexation of the reduced iron by using 2, 4, 6-tripyridyl-s-triazine (TPTZ) as chromogenic reagent in buffered medium (pH = 3.6). The stable Fe(TPTZ)22+ complex exhibits an absorption maximum at λ = 593 nm. The three-line manifold with one reaction coil was used. Optimization of manifold parameters and experimental conditions were carried out by means of univariate method. A linear calibration curve is established in a concentration range of 8×10−8 to 1×10−5 mol L−1 AA with regression equation y = 3.14 × 104 x  0.0007 (R2 = 0.9997) (n = 11) and detection limit of 2.4×10−8. The analytical frequency was 180 h−1. Also, the reagent consumption is minimal, only 0.5 mL per sample. The proposed method is simple, rapid, sensitive and reproducible (RSD 0.8%, n = 100). The proposed method with TPTZ as cromogenic reagent is very selective, because only the reducing substances with standard (formal) potentials lower than 0.6 V would have the thermodynamic prediction to interfere in the proposed method. In addition, although some reducing substances have the thermodynamic prediction to interfere in the proposed method, because of the specifity of flow injection analysis, the slow reactions are kinetic discriminated, because the main reaction is allowed to proceed for only few seconds. Tested reducing substances (thiol compounds) did not give serious errors when present at same concentrations as the ascorbic acid. The proposed method can be applied for the determination of AA in pharmaceutical preparations, down to picomole quantity. Usual excipients and other present active substances in pharmaceuticals do not interfere with the proposed method.