Synthesis, spectroscopic characterization, stability assessment and DNA-binding of new 2,6-piperidinedione derivatives.

This work reports on structural characterization of new antineoplaston (ANP) representatives, namely 3-(benzoylamino)-2,6-piperidinedione (BPD), 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) and 3-(p-nitrobenzoylamino)-2,6-piperidinedione (NPD). These compounds were prepared by reacting N-(4-substituted benzoyl)-glutamines with N-hydroxysuccinimide to afford the corresponding esters, which were heated to produce the corresponding 2,6-piperidinedione (PD) compounds. Non-destructive analytical procedures such as 1H NMR and NIR analyses confirmed the postulated chemical structures of these PD compounds. HPLC chromatograms at an ambient temperature or from solutions preheated at 30, 40 or 60 degrees C displayed only a single peak for each compound. Combination of heat with pH modification had virtually no effect on the obtained peaks, thus attesting to the stability and purity of these compounds. MS analysis displayed molecular mass ions indicative of BPD, MPD and NPD at m/z 233.4, 263.2 and 278.3, respectively. The fragmentation patterns using MS/MS analyses conformed to the structural and molecular formulae of the prepared compounds. Furthermore, preliminary biological assessments showed the capacity of these compounds to bind to the DNA. NPD, but not BMP or MPD, had a superior affinity to the DNA than the prototype ANP-A10.

Determination of linezolid in human plasma by LC-MS-MS.

A rapid, sensitive and selective LC-atmospheric pressure-chemical ionization-MS-MS method for the determination of the new antimicrobial agent, linezolid, in human plasma using selected reaction monitoring (SRM) was developed. Linezolid and the internal standard were extracted from the biological samples by solid phase extraction (SPE) and analyzed on a reversed-phase Shim Pack CLC-CN, C18 column with the mobile phase of acetonitrile and 20 mM ammonium acetate solution (4 + 1 v/v). Detection was accomplished using an LCQ mass spectrometer (Finnigan), which was programmed in positive MS-MS mode to permit measurement of the fragment ions of linezolid and internal standard at m/z 296.2 and 223.2, respectively. The assay run-time was less than 3.5 min. Quantitative analysis was based on peak area ratio of linezolid to the internal standard. Calibration plots were established over the concentration range of 0.1-20 micrograms ml-1 of linezolid with the lowest detection limit of 0.05 microgram ml-1 using 10 microliters sample volume. The SPE technique quantitatively recovered linezolid and the internal standard from the plasma samples at a percentage range of 89.1-93.7%. Determination of control samples of linezolid in plasma validated the LC-MS-MS-SRM method. Intra-assay and inter-assay precision were in the range of 5.1-11.4% relative standard deviation, whereas the intra- and inter-accuracy were in the range of 97.5-114.0% of the nominal concentrations of linezolid added. The data confirmed that the plasma samples of linezolid were stable at room temperature and when stored at -20 degrees C for at least 10 d. The developed LC-MS-MS-SRM method is recommended for the determination of linezolid in human plasma.

Determination of diclofenac sodium, flufenamic acid, indomethacin and ketoprofen by LC-APCI-MS.

A sensitive, selective and accurate high-performance liquid chromatography-mass spectrometry (LC-MS) assay for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs), namely diclofenac sodium (DIC), flufenamic acid (FLU), indomethacin (IND) and ketoprofen (KET), either individually or in mixtures, was developed. The examined drugs were injected onto Shim-pack GLC-CN column and were eluted with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate solution (5:1 v/v)/pH 7.4 at a flow rate l ml min(-1). The mass spectrometer, operated in the single ion monitoring mode, was programmed to admit the negative ions [M-H] at m/z 295.9 (DIC), 280.1 (FLU), 355.8 (IND) and 252.9 (KET), respectively. The calibration curves were linear (r > or = 0.9993) over the concentration range 50-300 ng ml(-1) (FLU, DIC) and 100-500 ng ml(-1) (KET, IND) with detection limits of 0.5-4.0 ng. The mean predicted concentrations for the analytes were in the range -5.9 and 5.2% of the nominal concentrations. Within-day and between-day precision were in the range of 0.8-9.1% of the R.S.D. Mean recovery percentages of the individual compounds from laboratory-made mixtures and pharmaceutical formulations were (99.5-101.5%) and (100.6-102.2%), respectively.

Comparative LC-MS and HPLC analyses of selected antiepileptics and beta-blocking drugs.

A highly sensitive and specific assay procedure based on the combination of liquid chromatography and mass spectrometry (LC-MS) has been developed for the quantitative analysis of selected antiepileptics (carbamazepine and phenytoin) and beta-blocking drugs (acebutolol, atenolol, pindolol and propranolol) using APCI as an ionization process. The measured concentration range was 100-300 ng ml-1 for all drugs except phenytoin (0.5-1.5 micrograms ml-1). Analysis was based on direct injection of methanolic solutions of drugs into the mass spectrometer with the subsequent elution with a mobile phase consisting of methanol and 1% acetic acid solution (4:1) at a flow rate 1 ml min-1. The mass spectrometer was programmed to permit detection and determination of either fragment or molecular ions of carbamazepine, phenytoin, acebutolol, atenolol, pindolol and propranolol at m/e 194.3, 252.9, 337.2, 267.1, 249.1 and 260.1, respectively. The recorded chromatograms exhibited well-resolved peaks at retention times < 1 min. The peak area was correlated linearly to the drug concentration. Intraday precision gave relative standard deviations in the range 1.75-4.02%. Compared to HPLC, the described LC-MS was faster, more sensitive and specific. Unlike HPLC, LC-MS could be applied to analyze incompletely resolved mixtures. The absolute detection limits for LC-MS and HPLC were 0.2-0.5 and 10-25 ng, respectively. Recovery studies of the investigated compounds in pharmaceutical products using LC-MS and HPLC gave mean percentages of 97.5-102.0 and 98.4-103.3, respectively. Statistical analysis of the data using t- and F-tests showed insignificant differences between both methods for the analysis of carbamazepine, phenytoin, acebutolol and atenolol in pharmaceutical formulations. However, LC-MS gave more accurate results than HPLC for determination of pindolol in tablets. Propranolol could only be determined in tablets using LC-MS.

High-performance liquid chromatography-mass spectrometric analysis of furosemide in plasma and its use in pharmacokinetic studies.

This study presents a rapid, specific and sensitive high-performance liquid chromatography-mass spectrometric (LC-MS) assay for the determination of furosemide in human plasma using diclofenac as an internal standard (IS). Both compounds were extracted from human plasma with ethyl acetate at pH 1 and were chromatographed using Shim-Pack GLC-CN column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer solution pH 7, 4:1 (v/v) at a flow rate 1 ml min(-1). Furosemide and IS were detected by mass spectrometer operated in the negative single ion monitoring mode using APCI as an ionization process at m/z 329.2 and 294.1, respectively. The assay linearity of furosemide was confirmed over the range 50-2,000 ng ml(-1). Detection limit for furosemide in plasma was 10 ng ml(-1). The selected concentration range corresponds well with the plasma concentrations of furosemide for pharmacokinetic study. Intraday and interday relative standard deviations were 1.3-4.7 and 2.7-11.5%, respectively. The extraction recovery percentages of furosemide and IS from plasma were in the range 89.3-97.1%. The developed LC-MS procedure was applied for the determination of the pharmacokinetic parameters of furosemide after an oral administration of tablet formulation (40 mg) to two healthy male volunteers. The calculated parameters were in good agreement with the reported values.

Total salivary gland proteins of female Culex pipiens and Aedes caspius (Diptera: Culicidae) and their fractionation during adult development and after blood sucking.

Salivary glands of Culex pipiens and Aedes caspius were analyzed to determine total protein content and its fractionation during adult female development and after blood sucking. In both species, the molecular weight of proteins ranged between 26.000 and 84.000 Daltons. These proteins were not identical in the two species. In Cx. pipiens, the total protein level increased during the first 3 days of adult development from 4.94 +/- 0.84 to 6.6 +/- 0.37 micrograms/gland. During this period, the salivary gland proteins were separated into 35, 34 and 37 fractions respectively. Cx. pipiens released in the human host 64% of the total proteins while taking a blood meal compared to unfed females. This decrease in protein level was proportional to protein fractions. Over the next 6 days, the protein level increased again to attain values comparable to those obtained prior to blood sucking. In Ae. caspius, the total protein level of the salivary glands did not change during the first 4 days of adult development (range between 3.13 +/- 0.27 and 3.91 +/- 0.36 micrograms gland), but on the fifth day, 2-fold increase was observed. The total salivary gland protein increased during the next 3 days after blood sucking to reach 15.5 +/- 0.98 micrograms/gland. During this period, a tremendous change in protein patterns was observed. After oviposition, on the fourth day, a significant reduction in the total protein level was observed (4.13 +/- 0.56 micrograms/gland), but over the next 3 days the level increased again (range between 4.13 +/- 0.66 and 7.13 +/- 0.66 micrograms/gland).

Nutritional reserves in autogenous and anautogenous populations of Culex pipiens and Aedes caspius (Diptera: Culicidae).

Proteins, carbohydrates, and lipids were compared in larval, pupal, and adult stages of autogenous and anautogenous populations of Culex pipiens and Aedes caspius. All developmental stages of autogenous mosquitoes accumulated significantly more proteins and carbohydrates than did anautogenous sibligns. For lipids, the same pattern was observed with the exception that adult females of both autogenous and anautogenous Ae. caspius had comparable quantities (170-176 micrograms/mg). In general, mosquito larvae contained the highest quantities of the nutrients which then started to decrease in pupae and adults. In addition, autogenous Ae. caspius accumulated more carbohydrates and lipids but less proteins than did Cx. pipiens. The results suggest that autogeny is associated with an inherent ability of autogenous mosquitoes to store more nutritional reserves than anautogenous counterparts. Those reserves are the precursors for oogenesis to produce the first egg clutch in autogenous femles.

A rapid first-derivative spectrophotometric analysis of cephalexin and cephradine in human urine.

A rapid, simple, and accurate first-derivative spectrophotometric method has been established for the determination of either cephalexin or cephradine in urine. Quantitative analysis of each antibiotic was achieved by measuring the peak amplitude at 268 nm. The relative and absolute recoveries ranged from 98.90 to 104.00% for cephalexin and from 97.85 to 105.10% for cephradine. The within-day coefficient of variation varied from 3.27 to 6.45%. The applicability of the method for the determination of the cumulative amount of either cephalexin or cephradine excreted unchanged in urine, following an oral dose containing 500 mg of the drug to a human male volunteer, is demonstrated.

Physico-chemical characterization of a new salt of ibuprofen.

A new salt of ibuprofen was prepared by reaction with t-butylamine; its formation was confirmed by IR and 1H-NMR spectroscopy. The salt was characterized by thermoanalytical, X-ray powder diffraction and solubility studies. The salt was found to be 1.5 times more soluble in water than was ibuprofen, with an enthalpy of solution of -8.84 kcal mol-1.

Comparison of the pharmacokinetics and pharmacodynamics of two commercial products containing glibenclamide.

This investigation was carried out to evaluate the in vitro dissolution as well as the pharmacokinetic and pharmacodynamic properties of two tablet oral dosage forms of glibenclamide, Daonil (product A) and Glucomid (product B). The two products were found to comply with the compendial requirements for both disintegration and content uniformity. Further, the in vitro dissolution characteristics of the two products are similar. The bioavailability and pharmacodynamic studies were carried out on 16 healthy male adult volunteers who received a single dose of each product in a double-blind crossover design. Blood samples were obtained over a 12-h interval and analyzed for serum glucose by glucose-oxidase method, insulin by radioimmunoassay and glibenclamide by a sensitive HPLC assay. The two products were not found to be significantly different with respect to peak serum concentrations (187.9 +/- 13.3 and 167.6 +/- 9.1 ng.ml-1 for A and B, respectively) or to the corresponding peak times (4.2 +/- 0.2 and 4.1 +/- 0.2 h for A and B, respectively). Furthermore, the two products were not found significantly different in the extent of absorption as indicated by the area under serum concentration-time curve (1,118.0 +/- 86.7 and 986.5 +/- 75.1 ng.h.ml-1 for A and B, respectively). The two products were also found to be pharmacodynamically equivalent. This was reflected by the comparable serum glucose and insulin levels. These levels correlated very well with glibenclamide concentrations after the administration of each product. These findings indicate that the two products are bioequivalent in terms of bioavailability and pharmacodynamic effects in normal healthy males.

A rapid high-performance liquid chromatography assay of glibenclamide in serum.

A rapid high-performance liquid chromatography (HPLC) determination of glibenclamide in human serum is described. Serum samples to which flufenamic acid has been added as internal standard were treated with acetonitrile as a protein precipitant. After centrifugation, separation and reconstitution, the redissolved residue was eluted from 5 mu Spherisorb C-8 reversed phase column at ambient temperature using a mobile phase consisting of acetonitrile-water (45:55 v/v) at pH 3.7-3.8 and pumped at a flow rate 2 ml/min. The effluent was monitored at 230 nm. The analysis time was no longer than 12 min. A linear relationship between the peak height ratio (glibenclamide/flufenamic acid) and concentration was obtained in the range 20-400 ng/ml. A typical calibration curve has a regression equation y = 0.0035x + 0.015 (r = 0.9999). The detection limit of glibenclamide in serum was 20 ng/ml. The mean recovery of drug from serum samples spiked with known amounts of glibenclamide was 96.77%. Within-day and between-day coefficients of variation were 1.6-4.0% and 1.4-3.5%, respectively. Stability testing indicated that glibenclamide was stable for at least 10 days in serum -20 degrees C. The method developed was applied to determine some pharmacokinetic parameters after the oral administration of 5 mg glibenclamide tablets to a human volunteer.

High-performance liquid chromatographic determination of propranolol and 4-hydroxypropranolol in serum.

A simple, sensitive and selective high performance liquid chromatographic method for the analysis of propranolol and its major metabolite 4-hydroxypropranolol is developed. The drugs and the internal standard, quinidine, were extracted from serum with ether at pH 10. The latter was evaporated and the residue was dissolved into phosphoric acid solution. Propranolol, 4-hydroxypropranolol and quinidine were eluted from 5 microns, C-18 reversed phase column with a mobile phase consisting of acetonitrile-methanol-phosphoric acid at pH 4 and detected with fluorescence detector. Quantification was achieved by measuring the peak height ratio of each drug to the internal standard. Interference due to either biological constituents of serum or other propranolol metabolites such as n-desisopropyl propranolol and propranolol glycol was absent. The mean percentage recoveries for serum samples spiked with propranolol and 4-hydroxypropranolol were 94.7 and 98.4%, respectively. Detection limits were 10 ng/ml for propranolol and 5 ng/ml for 4-hydroxypropranolol. Within-day coefficients of variation ranged from 3.2-6.9% for propranolol and 0.8-6.2% for 4-hydroxypropranolol.

Simultaneous high-pressure liquid chromatographic analysis of ampicillin and cloxacillin in serum and urine.

A rapid, specific and sensitive high pressure liquid chromatographic (HPLC) assay for the simultaneous determination of ampicillin and cloxacillin in serum and urine is developed. Ampicillin, cloxacillin and cephalexin (internal standard) were eluted from a 6.5 mu Synchropack RPP reversed phase column at ambient temperature using a mobile phase comprised of methanol:water (3/7v/v) and containing 0.011 M sodium-n-octane sulphonate, 0.005 M NaH2PO4 and 1.3% v/v of 0.5 M HCl (pH 2.7). The analysis time required no longer than 11 min. Equations are presented for the linear relationships between the peak height ratios of ampicillin/cephalexin and cloxacillin/cephalexin over the range 10-80 micrograms/ml (ampicillin) and 5-25 micrograms/ml (cloxacillin), respectively. The sensitivity limits for ampicillin and cloxacillin in serum and urine were 5 micrograms/ml and 1 microgram/ml, respectively. Quality criteria such as accuracy, precision and specificity were studied extensively. We investigated the applicability of the HPLC assay for the developed simultaneous determination of the cumulative amounts of ampicillin and cloxacillin, excreted unchanged in urine after an oral dose containing 500 mg ampicillin and 500 mg cloxacillin to a human volunteer.

Utility of iodine and 7,7,8,8-tetracyanoquinodimethane for determination of terfenadine.

Simple and sensitive spectrophotometric methods for the assay of terfenadine are described. The first is based on the reaction of terfenadine with iodine to give a molecular charge-transfer complex, the terfenadine acting as an n-electron donor and iodine as a sigma-electron acceptor. The second depends on the formation of a highly coloured stable radical anion between terfenadine and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a pi-electron acceptor. Beer's law is obeyed over the terfenadine concentration range 0.2-1.2 mg/100 ml. The proposed methods have been successfully applied to the analysis of commercial terfenadine tablets.

Utility of 2,3-dichloro-5,6-dicyano-p-benzoquinone in assay of codeine, emetine and pilocarpine.

A simple and sensitive spectrophotometric method for the assay of codeine, emetine and pilocarpine is described, based on the interaction of these drugs (as n-electron donors) with 2,3-dichloro-5,6-dicyano-p-benzoquinone (as pi -acceptor) to give a highly coloured radical anion which exhibits maximum absorption at 460 nm. Formation of the radical anion has been established by electron spin resonance measurements. Beer's law is obeyed for the alkaloids investigated. The assay results are in accord with pharmacopoeial assay results. The procedure is sufficiently sensitive to permit unit dose assay of the individual alkaloids in pharmaceutical formulations.

Colorimetric determination of two fenamates in capsule dosage form.

The colorimetric determination of mefenamic acid and flufenamic acid with potassium ferricyanide in sodium hydroxide medium is described. The orange product is measured at 464 nm. The molar absorptivities are 1.9 x 10(3) and 2.9 x 10(3) 1.mole(-1).cm(-1) for mefenamic acid and flufenamic acid, respectively. The method has been applied successfully to the determination of these drugs in capsules.