Nawal A. El-Rabbat

Stereoselective metabolic study of famprofazone

Famprofazone (1) metabolites were studied in human urine after medication by 50 mg oral dose. The human urine was collected over 48 h from six volunteers at time intervals of 6, 12, 24 and 48 h. The amount of famprofazone metabolites were recovered from the urine samples by application of Extrelut extraction method. The resultant extracts were derivatized using N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA) for trimethylsilylation followed by N-methyl-bis-trifluoroacetamide (MBTFA) for trifluoroacetylation. Methamphetamine (2) and 3-hydroxymethyl-propyphenazone (3), excreted in human urine, were identified as famprofazone metabolites by gas chromatography-mass spectrometry (GC-MS). The quantitative results revealed that the average amounts of 2 and 3, excreted in human urine were equal to 2.6 and 4 mg, respectively, through 48 h. However, 3 was analysed after enzymatic hydrolysis of the urine samples using beta-glucuronidase/arylsulphatase. The excreted methamphetamine enantiomers could be separated by application of indirect GC-technique using S-(-)-N-trifluoroacetylprolyl chloride (TPC) as a chiral derivatizing agent. The average amount of (-)-methamphetamine isomer excreted in the urine was found to be three fold those of the (+)-isomer.

Rapid spectrophotometric assay of phenothiazine drugs in pharmaceutical preparations

A simple, accurate and rapid method for the quantitative determination of ten phenothiazine drugs in either the pure form or in pharmaceutical formulations is proposed. The method is based on the development of pink or violet products with N-bromosuccinimide in a strong sulphuric acid medium. The reaction is suggested to proceed via oxidation of the phenothiazine nucleus into a semiquinonoid radica. The wavelengths of maximum absorption range from 512 to 565 nm. Molar absorptivities range from 6 × 103 to 20 × 103 l mol–1 cm–1. A linear correlation was found between absorbance (at the λmax.) and concentration. The resulting colours are well developed within 20–30 min and are stable for at least 10 h. Results of analyses of pure drugs and their dosage forms by the proposed method are in good agreement with those of the official BP 1980 and USP XX procedures.

Simultaneous determination of free and bound adriamycin, adriamycinol, adriamycinone and duanorubicin in plasma using column-switching technique and protein-coated pre-columns☆

Adriamycin, adriamycinol, adriamycinone and duanorubicin were simultaneously determined by the development of an on-line plasma clean-up system. A short protein-coated Lichrosorb, RP-8, RP-2, CN and muBondapak phenyl as well as ODS silica have been examined for their performance as pre-columns. The drugs and metabolites were separated from weakly retained plasma components through two steps; phosphate buffer saline, pH 7.4 and 15% acetonitrile in 0.1 M sodium dihydrogen phosphate, pH 3. The chromatographic conditions were: ODS/TM column, flow rate 1 ml/min, 35% acctonitrile in 0.1 M sodium dihydrogen phosphate (pH 3) containing 0.3% heptafluorobutyric acid as mobile phase. The detection was carried out using fluorescence monitor operated at an emission 555 nm and excitation 460 nm. Good resolution was obtained within 13 min. This method is reproducible for analysis of drugs and metabolites (99.3-100.1%, CV < 2%) in plasma.

Preparation of a specific monoclonal antibody against 2′-deoxycytidine

To evaluate the plasma 2 0 -deoxycytidine (2 0 dCyd) level as a prognostic marker for breast cancer patients, a specific monoclonal antibody was required to develop a immunoassay system for the accurate determination of plasma 2 0 dCyd concentration. In this study, a highly specific monoclonal antibody against 2 0 dCyd has been prepared. 3 0 -Hemisuccinyl2 0 dCyd-Keyhole limpet hemocyanin protein conjugate (3 0 sdCyd-KLH) was used as an immunogen. WKY/NCrj rats were immunized with an emulsion of 3 0 sdCyd-KLH and Freund’s complete adjuvant. A fortnight after single immunization, the medial iliac lymph-node cells were taken and fused with SP2/0 mouse myeloma cells. From the positive high titer clones, 4 clones that secreted specific anti-2 0 dCyd antibodies were selected. The four clones showed slight cross reactivity with 5methyl-2 0 -deoxycytidine (5MedCyd) and 3 0 -deoxycytidine (3 0 dCyd). The most selective monoclonal antibody which belonged to IgG2b, type was termed RH-4. The specificity of RH-4 antibody in hybridoma culture supernatant was determined. Among 39 different nucleosides, nucleotides and bases tested by enzyme immunoassay (EIA), only 3 0 dCyd and 5MedCyd showed very slight cross reactivity. RH-4 secreting clone was grown in BALB/c slc-nude mice in vivo. After the purification of the antibody by protein A column chromatography and choice of a proper concentration, the slight cross reactivity was completely overcome. This monoclonal antibody will be useful for the determination of plasma 2 0 dCyd concentrations. We are now developing a sensitive and conventional method for the application of RH-4 antibody. # 1998 Elsevier Science B.V.

Redox properties of isradipine and its electrochemical detection in the HPLC determination of the compound in human serum.

The electrochemical behaviour of isradipine in a mixed solution of Britton-Robinson buffer (pH 11.8):acetonitrile:methanol (6:3:1, v/v) was studied by cyclic voltammetry and spectroelectrochemistry using an optically transparent thin layer electrode of carbon cloth. The cyclic voltammogram showed several peaks whose shape and potentials depended on the pH. The peak at 330 nm, corresponding to the absorbance of the dihydropyridine ring, disappeared after electrolysis at a potential that was more positive than the oxidation peak. The oxidation peak corresponds to the oxidation of the dihydropyridine ring. Peak height at pH 11.8 was proportional to isradipine concentration. On the basis of the redox properties of isradipine, HPLC was conducted applying electrochemical detection on a polybutadiene coated alumina column using an alkaline mobile phase. The method was applied for the determination of isradipine content in human serum. A good linear relationship between isradipine concentration and peak height was found in the concentration range of 2-200 ng/mL with a correlation coefficient of 0.9924. The detection limit was 0.5 ng/mL. The within-day and day-to-day variation were examined for control human serum and percentage relative standard deviation ranged from 0.5 to 6.7. Interference from many other coadministered drugs was studied in the specified experimental conditions. Photo and heat stabilities of the compound were also studied.

Enzyme-linked immunosorbent assay for 2-deoxycytidine

An enzyme-linked immunosorbent assay (ELISA) method that can be used to quantify 2 0 -deoxycytidine (dCyd) in plasma is described. In this method, 3 0 -succinyl-dCyd bovine serum albumin conjugate (3 0 sdCyd‐BSA) adsorbed on the microtiter plate wells is bound to anti-dCyd monoclonal antibody in inverse proportion to free dCyd in the sample or standard. Bound antibody is quantified with alkaline phosphatase-labeled anti-immunoglobulin (Alp-IgG) antibody and p-nitrophenylphosphate (PNPP) substrate solution. A linear relationship with good correlation coefficients was obtained in the linear range of 10‐1000 m M. There was no cross-reactivity from structurally related compounds with the antibody. In addition, the constituents of plasma do not affect the assay. The intra- and inter-assay reproducibilities are satisfactory at 2‐3% relative standard deviation. A short period of incubation at 37C is required for equilibration of the antigen‐antibody reaction. There is a good correlation between the results obtained with the proposed ELISA and a liquid chromatographic method over the entire linear range. The analytical procedure is convenient, and one can analyze 150 samples per working day, facilitating the processing of serial samples. The present method is expected to contribute to further elucidation of the role of dCyd in various biological and biochemical systems. ©2000 Elsevier Science B.V. All rights reserved.

Effect of cyclodextrins on the stability of adriamycin, adriamycinol, adriamycinone and daunomycin

The stability of adriamycin (ADR), adriamycinol, adriamycinone (ADR-ONE) and daunomycin in the presence of alpha-, beta- and gamma-cyclodextrins (CDs) was studied using high-performance liquid chromatography. It was found that alpha-CD did not affect the degradation of tested compounds, beta-CD caused a little effect and gamma-CD resulted in pronounced stabilizing effect. The formation of complexes between ADR and ADR-ONE with CDs was monitored by fluorescence spectroscopy. The fluorescence spectrum of ADR-gamma-CD complex had an activation maximum at 460 nm, emission maximum at 555 nm and a shoulder at 585 nm. A similar finding was observed in case of alpha-CD. In case of beta-CD, the fluorescence intensity at 580 nm peak enhanced less than in case of gamma-CD. With ADR-ONE, alpha-CD did not cause any significant change compared with the spectrum of free molecule. On the other hand, it was noticed that, the fluorescence spectra of ADR-ONE with both beta- and gamma-CD were the same but showed a significant difference to the spectrum of free molecule, especially the molar fluorescence of the 585 nm emission peak.

High‐performance liquid chromatographic determination and pharmacokinetic study of cefepime in goat plasma and milk after pre‐column derivatization with Hg(I)

A highly sensitive and selective HPLC method with UV detection was developed for the determination of cefepime in goat plasma and milk. The proposed method was based on the complexation of cefepime with Hg(I) ions that imparts the high selectivity of the proposed method with enhancement of the sensitivity which enabled the analysis of cefepime in complex matrices such as plasma and milk. Detection was performed at 263 nm, using cefuroxime sodium as an internal standard. Chromatographic separation of cefepime and the internal standard was achieved with Aqua RP-C(18) column using methanol/triethylamine-acetate buffer, pH 3.5 (18:82, v/v) as mobile phase at a flow rate of 1 mL/min. Linear detector responses were observed spanning the range of 1.3-20 μg/mL. The LOD for standard cefepime was 0.43 μg/mL, whereas the LOD for cefepime in goat plasma was 0.84 μg/mL and the corresponding value in goat milk was 1.1 μg/mL. No interference from endogenous substances in plasma and milk was observed. The developed HPLC method has been successfully applied for the pharmacokinetic study of cefepime in goat plasma and milk, for the first time, after a single intramuscular injection of 50 mg cefepime/kg body weight.

HPLC Determination of some antibilharzial antimonials by extraction of antimony.

The antimonial drug (antimony potassium tartrate, antimony piperazine tartrate or antimony lithium thiomaleate) in aqueous solution or biological fluid is treated with sodium diethyldithiocarbamate in the presence of a suitable masking reagent, the pH is adjusted to 9 +/- 0.5. and the antimony complex extracted with n-hexane and determined by reversed-phase HPLC with an ODS column and detection at 254 nm. The limits of detection are 20 ng (for antimony potassium tartrate and antimony lithium thiomaleate) and 16 ng (for antimony piperazine tartrate).

HPLC determination of niridazole

A high-performance liquid chromatographic method has been developed for the determination of niridazole in bulk form and in pharmaceutical dosage form. A reversed-phase system, based on an octadecylsilane-bonded stationary phase and a 60:40 v/v methanol/water mobile phase, is used. The detector response at 370 nm is linearly related to the amount injected, over a wide range. The method is sensitive, simple, rapid and precise.