Keisuke Kitamura

Second derivative spectrophotometric determination of partition coefficients of chlorpromazine and promazine between lecithin bilayer vesicles and water

Abstract Absorption spectra of chlorpromazine and promazine in the presence of lecithin (egg yolk) bilayer vesicles were measured against the reference solutions containing the same amount of lecithin vesicles alone and their second derivative spectra were obtained by the Savitzky-Golay method. The residual background signal effects by the lecithin vesicles observed in the absorption spectra were entirely eliminated and both drugs clearly showed three derivative isosbestic points. The derivative intensity change (δ D ) induced by the addition of the lecithin vesicles was measured at the λ max of each drug, and from the relationship between the δ D value and the lipid concentration, the partition coefficients ( K p s) were calculated. The fractions of partitioned chlorpromazine and promazine calculated by using the obtained K p values agreed well with the experimental values. For the drugs which have the nature of metachromasy between the lipid vesicles and water, and even whose absorption bands overlap with the background signals by the lipid vesicles, the proposed method can be applied to the determination of their partition coefficients without disturbing the equilibrium conditions by separation procedures.

Determination of partition coefficients of diazepam and flurazepam between phosphatidylcholine bilayer vesicles and water by second derivative spectrophotometric method.

Second derivative spectrophotometry allowed the establishment of a simple and accurate method for the determination of partition coefficients of benzodiazepine drugs in a liposome/water system. The absorption spectra of diazepam (DZ) and flurazepam (FZ) in phosphatidylcholine (egg yolk) bilayer vesicle suspensions showed small spectral changes depending on the concentration of phosphatidylcholine vesicles. However, the intense background signals caused by the light scattering of the phosphatidylcholine vesicles made it difficult to yield a correct base line, thus the quantitative spectral data could not be obtained. In the second derivative spectra, the spectral changes were enhanced and three derivative isosbestic points were observed for each drug indicating the entire elimination of the residual background signal effects. The derivative intensity change of each drug (DeltaD) induced by its interaction with phosphatidylcholine bilayers was measured at a specific wavelength. From the relationship between the DeltaD value and the lipid concentration, the molar partition coefficients (K(p)s) of DZ and FZ were calculated and obtained with a good precision of R.S.D below 10%. The fractions of the partitioned DZ and FZ calculated by using the obtained K(p) values agreed well with the experimental values. The results prove that the derivative method can be usefully and easily applied to the determination of partition coefficients of benzodiazepines in the liposomes/water system without any separation procedures.

Second derivative spectrophotometric determination of partition coefficients of phenothiazine derivatives between human erythrocyte ghost membranes and water

The absorption spectra of six phenothiazine derivatives, chlorpromazine, triflupromazine, promazine, promethazine, trifluoperazine and prochlorperazine, measured in the solutions containing various amounts of human erythrocyte ghosts (HEG) showed bathocromic shifts according to the amount of HEG. Due to the strong background signals caused by HEG, the baseline compensation was incomplete, even though the sample and the reference solutions contained the same amount of HEG, hence further spectral information could not be obtained. The second derivative spectra of these absorption spectra clearly showed the derivative isosbestic points, indicating that the residual background signal effects were entirely eliminated. The derivative intensity differences of the phenothiazines (DeltaD values) before and after the addition of HEG were measured at a specific wavelength. Using the DeltaD values, the partition coefficients (K(p)) of these drugs were calculated and obtained with R.S.D. of below 10 %. The fractions of partitioned phenothiazines calculated from the K(p) values agreed well with the experimental values. The results indicate that the derivative method can be applicable to the determination of partition coefficients of drugs to HEG without any separation procedures.

Determination of dissociation constants of sparingly soluble phenothiazine derivatives by second-derivative spectrophotometry

The dissociation constants (pKa) for sparingly soluble phenothiazines (promazine, chlorpromazine, trifluoropromazine) in water were measured by second-derivative spectrophotometry. The intense background signals in the absorption spectra due to the turbidity caused by the precipitation of insoluble free base of the phenothiazine derivatives were eliminated in the second-derivative spectra, and the solubilities of the phenothiazine derivatives could be easily determined from the peak-to-trough lengths (D values) of the second-derivative signals. The pKa values were calculated from the pH dependence of the D values. The pKa values obtained agreed well with reported values and the standard deviations for 6–10 determinations were ⩽ 0.02. The solubilities of the free bases of the phenothiazines were also determined.

Effect of the degree of polynomials in the Savitzky—Golay method for calculation of second-derivative spectra

Abstract The second-derivative spectra of absorption curves simulated by a Gaussian function were obtained by using Savitzky—Golay cubic (CPC) and quintic polynomial convolutions (QPC), based on 17 points assumed to be at 0.25-nm intervals. For data obtained directly from the simulated curves (real-type data), the second derivatives agreed with the true values if the widths at half height of the peaks were > 15 nm for CPC and 5 nm for QPC. But when integer values obtained from the real-type data were used to simulate a 12-bit A/D conversion, considerable noise appeared on the second-derivative spectra of peaks wider than the above values, obtained by both CPC and QPC. This occurred because the rounding errors introduced by the A/D conversion formed small shoulders on the digitally reproduced absorption curves, which were enhanced by the differentiation to generate noise. The noise was more intense in QPC than in CPC, thus CPC is preferable for peaks that are not very narrow.

Determination of isoniazid in tablets by second-derivative ultraviolet spectrophotometry of scraped-spot solutions from thin-layer chromatography

Abstract Isoniazid in solutions of tablets was separated by thin-layer chromatography (TLC). The portion of the precoated plate carrying the isoniazid spot was cut out and the coating was scraped off into water. The isoniazid was quantified in the solution, without separation of suspended adsorbent, by second-derivative spectrophotometry, which eliminated the background in the zero-order spectra. Relative standard deviations (n = 5) were

19F NMR spectrometric determination of the partition coefficients of some fluorinated psychotropic drugs between phosphatidylcholine bilayer vesicles and water

A simple 19F NMR spectrometric method was proposed for the determination of the partition coefficients of fluorinated psychotropic drugs, trifluoperazine (TFPZ), flunitrazepam (FNZ) and flurazepam (FZ) between phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUVs) and water (buffer). Each 19F NMR spectrum of these drugs in the presence of PC SUV showed a single signal accompanying a PC concentration-depending shift change and broadening, which indicated a fast exchange of these drugs between the water phase and the PC bilayer of SUV. From the relationship between the 19F chemical shift change (Deltadelta) of each drug and the PC concentration, the molar partition coefficients (K(p)s) were calculated and obtained with a good precision of RSD below 6%. The fractions of the partitioned drugs calculated by using the obtained K(p)-values were in a good agreement with the experimental values. The results demonstrate that the 19F NMR method can be usefully applied to the determination of partition coefficients of many drugs having fluorine atom(s) without any separation procedure, especially for drugs which do not have absorption in the ultraviolet or visible region, or those having absorption but show insignificant spectral changes according to their incorporation to PC bilayers (e.g. FNZ).

Synthesis of [N‐13CH3] drugs (chlorpromazine, triflupromazine and promazine)

These [N-13CH3] containing drugs (chlorpromazine, triflupromazine and promazine) have been synthesized by means of a two-step demethylation/methylation procedure. Copyright

Determination of p-aminobenzoic acid in the presence of bovine serum albumin by room-temperature phosphorimetry on a poly(vinyl alcohol) substrate

Solid substrate room-temperature phosphorimetry on a poly(vinyl alcohol) (PVA) substrate was applied to the analysis of p-aminobenzoic acid (PABA) in the presence of bovine serum albumin (BSA). The buffer solution containing PABA and BSA was spotted on the PVA substrate and after sufficient drying, room-temperature phosphorescence (RTP) measurements were performed on both the front and the back side of the substrate. The BSA was separated on the front surface of the substrate and it emitted a negligibly weak RTP signal. Therefore, the RTP measurement of PABA on the reverse of the sample spotted side was not interfered by BSA, whereas, impurities in the BSA permeated into the PVA and emitted a weak RTP signal on the back side of the substrate. However, the RTP intensities of the impurities were weak enough as compared to that of PABA under the experimental conditions, so that their interference could be neglected. To improve the RTP intensity and reproducibility of PABA in the presence of BSA, a new substrate that consisted of Millipore-VS and PVA layer was prepared. The analytical figures of merit of this substrate obtained under the best experimental condition for maximum phosphorescence emission are a relative standard deviation of 6.0% (n=5) at a level of 2 nmol/spot and a linear calibration curve from 0.5 to 20 nmol/spot with a correlation coefficient of 0.996 for 25 data points.

Preparation of immobilized glucose oxidase membrane by the plasma polymerization technique

Abstract Glucose oxidase was immobilized on a Millipore (MP) filter by coating with plasma-polymerized propargyl alcohol. The resulting immobilized enzyme membrane was used as a glucose sensor. The properties as a glucose electrode system were evaluated by amperometric response with either the steady-state method or the reaction rate method. The response was proportional to concentrations of the glucose solution up to 2 m M and the sensitivity was dependent on the amount of GOD impregnated into the MP filter.