Macromolecular crowding-assisted fabrication of liquid-crystalline imprinted polymers.

A macromolecular crowding-assisted liquid-crystalline molecularly imprinted monolith (LC-MIM) was prepared successfully for the first time. The imprinted stationary phase was synthesized with polymethyl methacrylate (PMMA) or polystyrene (PS) as the crowding agent, 4-cyanophenyl dicyclohexyl propylene (CPCE) as the liquid-crystal monomer, and hydroquinidine as the pseudo-template for the chiral separation of cinchona alkaloids in HPLC. A low level of cross-linker (26%) has been found to be sufficient to achieve molecular recognition on the crowding-assisted LC-MIM due to the physical cross-linking of mesogenic groups in place of chemical cross-linking, and baseline separation of quinidine and quinine could be achieved with good resolution (R(s) = 2.96), selectivity factor (α = 2.16), and column efficiency (N = 2650 plates/m). In contrast, the LC-MIM prepared without crowding agents displayed the smallest diastereoselectivity (α = 1.90), while the crowding-assisted MIM with high level of cross-linker (80%) obtained the greatest selectivity factor (α = 7.65), but the lowest column efficiency (N = 177 plates/m).

Comparison of information-dependent acquisition, SWATH, and MS(All) techniques in metabolite identification study employing ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) analysis is a powerful and essential tool for metabolite identification in drug discovery and development. An MS(2) (or tandem, MS/MS) mass spectrum is acquired from the fragmentation of a precursor ion by multiple methods including information-dependent acquisition (IDA), SWATH (sequential window acquisition of all theoretical fragment-ion spectra), and MS(All) (also called MS(E)) techniques. We compared these three techniques in their capabilities to produce comprehensive MS(2) data by assessing both metabolite MS(2) acquisition hit rate and the quality of MS(2) spectra. Rat liver microsomal incubations from eight test compounds were analyzed with four methods (IDA, MMDF (multiple mass defect filters)-IDA, SWATH, or MS(All)) using an ultrahigh-performance liquid chromatography-qudrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) platform. A combined total of 227 drug-related materials (DRM) were detected from all eight test article incubations, and among those, 5% and 4% of DRM were not triggered for MS(2) acquisition with IDA and MMDF-IDA methods, respectively. When the same samples were spiked to an equal volume of blank rat urine (urine sample), the DRM without MS(2) acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS(2) acquisition with either the SWATH or MS(All) method. However, the quality of the acquired MS(2) spectra decreased in the order of IDA, SWATH, and MS(All) methods. An average of 10, 9, and 6 out of 10 most abundant ions in MS(2) spectra were the real product ions of DRM detected in microsomal samples from IDA, SWATH, and MS(All) methods, respectively. The corresponding numbers declined to 9, 6, and 3 in the urine samples. Overall, IDA-based methods acquired qualitatively better MS(2) spectra but with a lower MS(2) acquisition hit rate than the other two methods. SWATH outperformed the MS(All) method given its better quality of MS(2) spectra with an identical MS(2) acquisition hit rate.

Monitoring disopyramide, lidocaine, and quinidine by micellar liquid chromatography.

A micellar liquid chromatography (MLC) method using a C18 column was developed to determine three antiarrhythmic drugs--disopyramide, lidocaine, and quinidine--that are most usually monitored in serum samples. After the application of an interpretative strategy for optimization of sodium dodecyl sulfate (SDS) and modifier concentrations in order to ensure the minimum analysis time, maximum sensitivity, and good resolution, the optimum chromatographic conditions for the determination of the three antiarrhythmics were flow rate, 1 mL/min; injection volume, 20 microL; separation temperature, 25 degrees C; mobile phase, 150 mmol/L SDS-7% (v/v) butanol-phosphate buffer, 10 mmol/L, pH 7-0.9% (w/v) NaCl; and detection at 214 nm. The calibration curves for the drugs were linear (r2 > 0.999). The intraday and interday precisions were lower than 3.9% (CV). Recoveries were 100 +/- 0.6% when the method was applied to both serum samples spiked with the antiarrhythmics (n = 10) and real serum samples. In all cases, the results were similar to those obtained using the reference method (fluorescence polarization immunoassay) usually used in the Spanish hospital. The proposed method is useful for hospital monitoring of the antiarrhythmics by direct injection into the chromatograph.

Rapid and green analytical method for the determination of quinoline alkaloids from Cinchona succirubra based on Microwave-Integrated Extraction and Leaching (MIEL) prior to high performance liquid chromatography.

Quinas contains several compounds, such as quinoline alkaloids, principally quinine, quinidine, cinchonine and cichonidine. Identified from barks of Cinchona, quinine is still commonly used to treat human malaria. Microwave-Integrated Extraction and Leaching (MIEL) is proposed for the extraction of quinoline alkaloids from bark of Cinchona succirubra. The process is performed in four steps, which ensures complete, rapid and accurate extraction of the samples. Optimal conditions for extraction were obtained using a response surface methodology reached from a central composite design. The MIEL extraction has been compared with a conventional technique soxhlet extraction. The extracts of quinoline alkaloids from C. succirubra obtained by these two different methods were compared by HPLC. The extracts obtained by MIEL in 32 min were quantitatively (yield) and qualitatively (quinine, quinidine, cinchonine, cinchonidine) similar to those obtained by conventional Soxhlet extraction in 3 hours. MIEL is a green technology that serves as a good alternative for the extraction of Cinchona alkaloids.

An electrochemical microfluidic platform for human P450 drug metabolism profiling.

This paper is the first report of a P450-electrode in a microfluidic format. A 30 µL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(M) values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron > nifedipine > quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested.

Development and validation of RP-HPLC-fluorescence method for quantitative determination of quinidine, a probe substrate for P-glycoprotein inhibition assay using Caco-2 cell monolayer.

A simple, sensitive and specific reverse-phase high-performance liquid chromatographic (RP-HPLC) method with fluorescence detection was developed for quantitation of quinidine from HBSS buffer. The method was applicable in the bi-directional transport assay for evaluation of the inhibitory effect of test compounds on P-glycoprotein-mediated quinidine transport; quinidine was used as a probe P-glycoprotein substrate. The calibration curve was linear (correlation coefficient >/=99) in the range 0.30-100.00 nm. The method was validated and is specific and sensitive with limit of quantitation of 300 pm for quinidine. The method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions where the analyte was found to be stable. The applicability and reliability of the analytical method was evaluated by successful demonstration of efflux ratio (P(app)B --> A/P(app)A --> B) in the Caco-2 cell monolayer efflux assay. The efflux ratio for quinidine (100 nm) alone was 10.8, which reduced to less than 2 in the presence of the classical P-gp inhibitors verapamil and ketoconazole (100 mum each).

Detection of Cutting Agents in Drug-Positive Seized Exhibits within the United States.

The following report summarizes a study performed on seized drug exhibits collected in two U.S. states to evaluate the presence and identification of cutting agents. Aliquots of seized drug materials from Kentucky (n = 200) and Vermont (n = 315) were prepared using a dilute-and-shoot procedure. Initial analysis was performed using gas chromatography-mass spectrometry (GC-MS) followed by analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Active compounds detected overall included caffeine (31.0%), quinine/quinidine (24.7%), levamisole (11.6%), acetaminophen, (8.2%) and procaine (8.2%). These compounds were found with several drugs of abuse, such as heroin, fentanyl, methamphetamine, and cocaine. This novel information about cutting agents used to dilute or alter drugs of abuse is important to criminal investigations and in the management of acute intoxications at health centers. However, common methodologies for analysis and standard reporting practices frequently do not include cutting agents, resulting in lacking or inadequate information regarding prevalence of these substances.

Quantitative determination of major alkaloids in Cinchona bark by Supercritical Fluid Chromatography.

Chinoline alkaloids found in Cinchona bark still play an important role in medicine, for example as antimalarial and antiarrhythmic drugs. For the first time Supercritical Fluid Chromatography has been utilized for their separation. Six respective derivatives (dihydroquinidine, dihydroquinine, quinidine, quinine, cinchonine and cinchonidine) could be resolved in less than 7 min, and three of them quantified in crude plant extracts. The optimum stationary phase showed to be an Acquity UPC2 Torus DEA 1.7 µm column, the mobile phase comprised of CO2, acetonitrile, methanol and diethylamine. Method validation confirmed that the procedure is selective, accurate (recovery rates from 97.2% to 103.7%), precise (intra-day ≤2.2%, inter-day ≤3.0%) and linear (R2 ≥ 0.999); at 275 nm the observed detection limits were always below 2.5 µg/ml. In all of the samples analyzed cinchonine dominated (1.87%-2.30%), followed by quinine and cinchonidine. Their total content ranged from 4.75% to 5.20%. These values are in good agreement with published data, so that due to unmatched speed and environmental friendly character SFC is definitely an excellent alternative for the analysis of these important natural products.

Development and validation of RP-HPLC-UV method for simultaneous determination of buparvaquone, atenolol, propranolol, quinidine and verapamil: a tool for the standardization of rat in situ intestinal permeability studies.

A simple, sensitive and specific reversed phase high performance liquid chromatographic (RP-HPLC) method with UV detection at 251 nm was developed for simultaneous quantitation of buparvaquone (BPQ), atenolol, propranolol, quinidine and verapamil. The method was applicable in rat in situ intestinal permeability study to assess intestinal permeability of BPQ, a promising lead compound for Leishmania donovani infections. The method was validated on a C-4 column with mobile phase comprising ammonium acetate buffer (0.02 M, pH 3.5) and acetonitrile in the ratio of 30:70 (v/v) at a flow rate of 1.0 ml/min. The retention times for atenolol, quinidine, propranolol, verapamil and BPQ were 4.30, 5.96, 6.55, 7.98 and 8.54 min, respectively. The calibration curves were linear (correlation coefficient > or =0.996) in the selected range of each analyte. The method is specific and sensitive with limit of quantitation of 15 microg/ml for atenolol, 0.8 microg/ml for quinidine, 5 microg/ml for propranolol, 10 microg/ml for verapamil and 200 ng/ml for BPQ. The validated method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions and all the analytes were found to be stable. This method is simple, reliable and can be routinely used for accurate permeability characterization.

Adequate tumour quinidine levels for multidrug resistance modulation can be achieved in vivo.

The multidrug resistance (MDR) phenotype can be reversed in vitro by a number of agents thought to interact with P-glycoprotein (P-gp). Although plasma levels, adequate for MDR modulation, can be achieved with certain modulators, concern has been expressed that tumour levels may be inadequate due to high plasma protein binding. Mice bearing an MDR-positive human tumour xenograft were injected intraperitoneally with quinidine (150 mg/kg). After 2 h the mean plasma quinidine level was 1.9 micrograms/ml (5.1 mumol/l) and the mean tumour quinidine effective in vitro. Three tumour biopsy specimens were obtained from patients who had received oral quinidine prior to surgery. Plasma and tumour levels were similar and were comparable with those measured in mice. This study should dispel fears of inadequate tumour levels of this and other modulators due to high plasma protein binding and encourage future clinical trials of modulators in MDR-positive human tumours.

Reduced quinidine clearance in elderly persons.

The influence of age on quinidine pharmacokinetics was assessed in 22 healthy male and female volunteers; 14 of the subjects were young (aged 23 to 34 years) and 8 elderly (aged 60 to 69 years). All subjects received 180 to 300 mg of quinidine base by constant rate intravenous infusion over 10 to 15 minutes. The concentration of total and unbound quinidine in multiple serum samples and in urine collected within 48 hours after the administration of quinidine qas determined with spectrophotofluorometric assay. Mean kinetic values for total quinidine in the young subjects were: elimination half-life (t 1/2 beta), 7.3 hours; total volume of distribution (Vd), 2.39 liters/kg; total clearance, 4.04 ml/min per kg; renal clearance 1.43 ml/min per kg; and percent unbound, 24.6 In the elderly subjects, the values for Vd (2.18 liters/kg) and percent unbound (28.2) did not differ significantly from these values in the young subjects. However, in the elderly subjects t 1/2 beta was significantly longer (9.7 hours, P less than 0.05) and total quinidine clearance significantly less (2.64 ml/min per kg, P less than 0.005) than in the young subjects. Renal clearance of quinidine in the elderly was also significantly less (0.99 ml/min per kg, P less than 0.05) than in the young and was associated with lower rates of creatinine clearance in the elderly (r = 0.66). Reduced clearance of quinidine and prolongation of its elimination half-life could predispose to toxicity in the elderly unless the dose were appropriately adjusted.

Dihydroquinidine contamination of quinidine raw materials and dosage forms: rapid estimation by high-performance liquid chromatography.

Dihydroquinidine is a commonly encountered contaminant in quinidine raw materials. The USP allows 0-20% dihydroquinidine in quinidine products, but the assays used to quantitate dihydroquinidine have been lengthy or have required sophisticated equipment. The present method separates dihydroquinidine from quinidine and provides rapid, precise quantitation of both dihydroquinidine and quinidine. The clinical importance of dihydroquinidine contamination of quinidine dosage forms remains unanswered.

Assignment of conformation and configuration to potassium permanganate oxidation products of quinidine.

Two epimeric aldehydes [(R)- and (S)-quinidinals] and the corresponding acids[(R)- and (S)-norhydroquinidinoic acids] were prepared by the oxidation of quinidine. The alpha-alpha interactions of the carbonyl group and the aromatic moiety, as reflected in the NMR spectra, were compared with those of quinidine. NMR spectroscopic analyses made it possible to assign both the stable conformation and their configuration at C-3 to these molecules. The free hydroxyl group at C-9 must be present for the chemical shift values to be concentration dependent. These findings provide more information on association in the parent molecules.

Comparison of two spectrofluormetric procedures for quinidine determination in biological fluids.

Different methods for the spectrofluorometric determination of quinidine in plasma and urine were studied. The alkaline washings of plasma and urine extracts remove fluorescent metabolites, as shown by TLC analysis of urine extracts, and do not lead to a significant loss of alkaloids. The spectrofluormetric assays without alkaline washings of the benzene extract averaged 18% higher than the assays with alkaline washings. Since unchanged quinidine and hydroquinidine are responsible for antiarrhythmic activity, the method with alkaline washing is more appropriate for the control of quinidinemia than are other methods. The therapeutic plasma concentration range becomes 0.8-2.5 microng/ml with this methods.

Evaluation of biodegradable poly(lactide) pellets prepared by direct compression.

Biodegradable pellets intended for either parenteral or oral use were successfully prepared from low molecular weight poly(DL-lactide) (low MW PLA, MW' = 2000) or a relatively high molecular weight poly(L-lactide) (L-PLA, MW = 215,000) sample by a simple direct compression technique without the use of heat or organic solvents. The energy imparted during the compression step caused fusion of the low MW PLA particles. Pellets prepared from low MW PLA swelled considerably before eroding in pH 7.4 buffer, but acted as an enteric matrix in 0.1 M HCl. This was attributed to the high carboxyl endgroup:polymer chain ratio which increased with a decrease in molecular weight. Interactions between salts of basic drugs (quinidine sulfate or propranolol hydrochloride) and the polymeric carboxyl endgroups caused retardation in the drug release from low MW PLA pellets. The drug release from L-PLA pellets was independent of the pH of the dissolution media and drug-polymer interactions were absent. The drug release could be increased by admixing sodium chloride prior to compression, or reduced by dipping the pellets into methylene chloride for a short period of time.

Impact of stable conformation of cinchona alkaloids on protonation site.

NMR analyses of quinidine and other cinchona alkaloids and their monoprotonated salts in deuterium oxide and in deuterochloroform revealed that the molecules assume new conformations in polar and nonpolar media, affecting the protonation site and hydrophilic-lipophilic characteristics. The ion-pair feature of the salts is lost and the molecules assume a neutral feature when they are transferred from an aqueous to a lipoid phase. Hydrophobic bonds between the molecules and their environment and within the molecule itself may affect the binding of cinchona alkaloids to membranes in biological fluids.

Comparative bioavaiability of four commercial quinidine sulfate tablets.

A comparative bioavailability study was performed using four commercially available, chemically equivalent brands of quinidine sulfate tablets. Two 200-mg tablets were administered to 11 different subjects following a completely randomized crossover design. Serum levels, urinary excretion data, and derived pharmacokinetic parameters were compared statistically. There were no statistical differences in the extent of quinidine absorption from the four brands of tablets as evidenced by the cumulative urinary excretion values and the areas under the serum level-time curves. Significant differences in the mean serum levels at 0.5 and 1 hr and differences in the peak times and absorption rate constants indicate that there was a difference in the absorption rate between Treatments A and D and C and D. A significant difference in the peak times also was noted for Treatments B and C. When mean disintegration times for the four tablet formulations were compared with their values for ka, tmax and mean serum levels at 0.5 and 1 hr, rank-order correlations were observed. A considerable degree of variability in quinidine elimination was noted, with half-life values ranging from 2.71 to 8.12 hr (mean half-life of 5.36 hr).

Nonaqueous titration of sulfates of quinine and quinidine using barium acetate.

A nonaqueous titrimetric method is proposed for determining the diastereomeric sulfates of quinine and quinidine. The sulfuric acid content of the alkaloid salts is precipitated, in the form of barium sulfate, with acetous barium acetate solution before the liberated alkaloid is titrated; the necessary calculations are provided. A favorable characteristic of the proposed procedure is the accuracy, speed, and ease of performance. The mean percent recoveries (p = 0.05) obtained with the proposed method for the sulfates of quinine and quinidine were 98.84 +/- 1.00 and 99.74 +/- 1.27, respectively, compared with 100.73 +/- 1.44 and 100.82 +/- 1.16, respectively, when the BP 1968 procedure was applied.

Colorimetric assay of quinine and quinidine in raw materials, formulations, and biological fluids.

The two characteristic erythroquinine and thalleioquin tests for quinine and quinidine were studied to optimize the experimental conditions for quantitative analysis. Both methods were quantitatively sensitive for either quinine or quinidine in a concentration range of 0.1--10 microgram/ml with the erythroquinine method and of 3--50 microgram/ml with the thalleioquin method. A TLC--colorimetric method also is described for the assay of quinine and quinidine in the presence of cinchonine, cinchonidine, and other cinchona alkaloids. The results were compared with those obtained with a spectrophotometric method.

Characterization of quinidine 3-hydroxylation as a probe for the CYP 3A enzyme using a novel capillary electrophoresis technique.

Capillary electrophoresis (CE) with a direct injection technique was used to characterize the formation of (3S)-3-hydroxyquinidine (3-OHQ) as a probe for the CYP 3A isoenzymes in rat liver microsomes. Detection was performed either in the absorbance mode or by employing laser-induced fluorescence (LIF) detection. Michaelis-Menten parameters (mean values+/-S.D.) K(m) and V(max) for the formation of 3-OHQ from the probe drug quinidine sulfate (QS) in rat liver microsomes were 37+/-4.6 micro g/ml (47.1+/-5.9 micro M) and 321+/-4 ng/mg/h (942+/-11.7 pmol/mg/h), respectively. Inhibition studies were performed to evaluate the specificity of 3-OHQ as a probe for the CYP 3A enzyme. Ketoconazole and fluconazole were found to be inhibitors of 3-OHQ formation and exhibited K(i) values of 0.19 and 20.1 micro M, respectively. Inhibition with the weak inhibitor, erythromycin could only be estimated using LIF detection due to lack of sensitivity in the absorbance mode. The formation of 3-OHQ in rat liver microsomes can be used as a model for the screening of the CYP 3A enzyme. Direct injection, ensures faster analysis time due to the lack of sample preparation and the low volume capabilities of the technique makes it attractive for the screening of a large number of compounds.