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.

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.

Nano graphene based sensor for antiarrhythmic agent quinidine in solubilized system.

An ultrasensitive electrochemical sensor based on the electrocatalytic properties of nano graphene (nGN) and multiwalled carbon nanotubes (MWCNTs) for the detection of quinidine (QD) in solubilized systems has been developed. This nano graphene-multiwalled carbon nanotube (nGN-MWCNT) composite film modified sensor shows the higher stability and stronger catalytic activity towards oxidation of quinidine and the over-potential decreased significantly compared with the bare glassy carbon electrode (GCE). The electrochemical characterization of the sensor was done by scanning electron microscopy (SEM) and cyclic voltammetry (CV) with working electrode surface area 0.56 cm(2) and diffusion coefficient 2.15×10(-3)cm(2)s(-1). Under the optimized conditions, the oxidation peak current of QD is found to be proportional to its concentration in the range of 60 ng-50 µg with a detection limit of 0.186 ng. The sensor was successfully employed for the detection of quinidine in bulk drugs and in its commercial pharmaceutical formulation.

Templating effect in DNA proximity ligation enables use of non-bioorthogonal chemistry in biological fluids.

Here we describe the first example of selective reductive amination in biological fluids using split aptamer proximity ligation (StAPL). Utilizing the cocaine split aptamer, we demonstrate small-molecule-dependent ligation that is dose-dependent over a wide range of target concentrations in buffer, human blood serum and artificial urine medium. We explore the substrate binding preferences of the split aptamer and find that the cinchona alkaloids quinine and quinidine bind to the aptamer with higher affinity than cocaine. This increased affinity leads to improved detection limits for these small-molecule targets. We also demonstrate that linker length and hydrophobicity impact the efficiency of split aptamer ligation. The ability to carry out selective chemical transformations using non-bioorthogonal chemistry in media where competing reactive groups are present highlights the power of the increased effective molarity provided by DNA assembly. Obviating the need for bioorthogonal chemistry would dramatically expand the repertoire of chemical transformations available for use in templated reactions such as proximity ligation assays, in turn enabling the development of novel methods for biomolecule detection.

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.

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.

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).

Improving resolution in single-scan 2D spectroscopy.

New schemes are introduced that allow one to improve the resolution in the indirect dimension of single-scan 'ultrafast' two-dimensional NMR spectra. The methods combine undersampling with band-selective pulses to recover signals that lie outside the detection bandwidth. The efficiency is illustrated for homonuclear total correlation spectroscopy (TOCSY) of quinidine.

Application of multivariate methods to the simultaneous Fourier Transform Infrared (FT-IR) spectrometric determination of stereo-isomers; quinine and quinidine.

The two stereo-isomers; quinine and quinidine have been determined in their mixtures in the IR region using chemometric multivariate methods, principal component regression (PCR) and partial least squares (PLS). A training set of thirty synthetic binary mixture solutions in the possible combinations containing 0.0 - 4.0 and 4.0 - 0.0% w/v quinine and quinidine, respectively in chloroform was used to develop the multivariate calibrations. A validation set containing thirty synthetic binary mixtures of variable ratios in the range of 0.2 - 4.0 and 4.0 - 0.2% w/v for quinine and quinidine, respectively in chloroform was used to validate the developed calibrations. The results of analysis of the validation synthetic mixtures were found to be 100.5+/-0.44% (R.S.D.%=0.44) and 100.5+/-0.38% (R.S.D.%=0.38) for quinine and 100.1+/-0.67% (R.S.D.%=0.67) and 100.1+/-0.68% (R.S.D.%=0.68) for quinidine using PCR and PLS models, respectively.

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.

Some fundamental and technical aspects of the quantitative analysis of pharmaceutical drugs by matrix-assisted laser desorption/ionization mass spectrometry.

The purpose of the present paper was to study some of the underlying physical and technical aspects of high-throughput quantitative matrix-assisted laser desorption/ionization (MALDI) of small drug molecules. A prototype MALDI-triple quadrupole instrument equipped with a high repetition rate laser was employed. Initially, the detection limits and dynamic ranges for the quantitation of four drugs (quinidine, danofloxacin, ramipril and nadolol) were determined. Internal standards were carefully chosen for each of these analytes in terms of structure similarity and fragmentation pathways. Three organic matrices were tested for these assays, resulting in different crystallization behaviors and measurement reproducibilities. alpha-Cyano-4-hydroxycinnamic acid yielded the best results and was subsequently employed for the quantitative determination of all four analytes. Further experiments considered the role of laser energy and pulse rate on the ablated areas as well as ion signals. Light microscope and scanning electron microscope images allowed the examination of the ablated area of the MALDI spots. The images showed convincing evidence that the ablated area was virtually void of crystals after analysis, with no preferential removal of material in the center of the laser's path. Average values for the amount of material ablated were determined to be 3.9+/-0.5% of the total spot size, and as low as 19.5 attomoles of analyte were detectable for our most sensitive analyte, ramipril. It was calculated that, under these assay conditions, it was possible to accurately quantify less than 1 femtomole of all analytes with the use of appropriately pure internal standards. These studies showed very promising results for the quantitative nature of MALDI for small molecules with molecular weights less than 500 Da.

High-throughput solution-based medicinal library screening against human serum albumin.

High-throughput screening of combinatorial libraries has evolved from studying large diverse libraries to analyzing small, structurally similar, focused libraries. This paradigm shift has generated a need for rapid screening technologies to screen both diverse and focused libraries in a simple, efficient, and inexpensive manner. We have proactively addressed these needs by developing a high-throughput, solution-based method combining size exclusion (SEC), two-dimensional liquid chromatography (2-D LC), and mass spectrometry (MS) for determining the relative binding of drug candidates in small, focused medicinal libraries against human serum albumin (HSA). Two types of libraries were used to evaluate the performance of the system. The first consisted of five diverse ligands with a wide range of hydrophobicities and whose association constants to HSA cover 3 orders of magnitude. A beta-lactam library composed of structurally similar compounds was used to further confirm the validity of the methodology. The ability to distinguish site-specific interactions of drugs competing for individual domains of the HSA receptor is also demonstrated. Comparison of chromatographic profiles of the library components before and after incubation with the receptor using multiple reaction monitoring allowed a ranking of the ligands according to their relative binding affinities. The observed rankings correlate closely with literature values of the association constants between the respective ligands and HSA. This simple, rugged methodology can screen a wide spectrum of chemical entities from combinatorial mixtures in less than 6 min.

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.

Effect of capillary efflux transport inhibition on the determination of probe recovery during in vivo microdialysis in the brain.

Intracerebral microdialysis probe recovery (extraction fraction) may be influenced by several mass transport processes in the brain, including efflux and uptake exchange between brain and blood. Therefore, changes in probe recovery under various experimental conditions can be useful to characterize fundamental drug transport processes. Accordingly, the effect of inhibiting transport on probe recovery was investigated for two capillary efflux transporters with potentially different membrane localization and transport mechanisms, P-glycoprotein and an organic anion transporter. Fluorescein/probenecid and quinidine/LY-335979 were chosen as the substrate/inhibitor combinations for organic anion transport and P-glycoprotein-medicated transport, respectively. Probenecid decreased the probe recovery of fluorescein in frontal cortex, from 0.21 +/- 0.017 to 0.17 +/- 0.020 (p < 0.01). Quantitative microdialysis calculations indicated that probenecid treatment reduced the total brain elimination rate constant by 3-fold from 0.37 to 0.12 (ml/min. ml of extracellular fluid). In contrast, the microdialysis recovery of quinidine, delivered locally to the brain via the probe perfusate, was not sensitive to P-glycoprotein inhibition by systemically administered LY-335979, a potent and specific inhibitor of P-glycoprotein. Recovery of difluorofluorescein, an analog of fluorescein, was also decreased by probenecid in the frontal cortex but not in the ventricle cerebrospinal fluid. These experimental observations are in qualitative agreement with microdialysis theory incorporating mathematical models of transporter kinetics. These studies suggest that only in certain circumstances will efflux inhibition at the blood-brain barrier and blood-cerebrospinal fluid barrier influence the microdialysis probe recovery, and this may depend upon the substrate and inhibitor examined and their routes of administration, the localization and mechanism of the membrane transporter, as well as the microenvironment surrounding the probe.

A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe.

A capillary electrophoresis (CE) method has been developed for the determination of quinidine sulfate (QS) and (3S)-3-hydroxyquinidine (3-OHQ) by direct injection of microsomal incubation mixtures. 3-OHQ is the CYP 3A4 metabolite of QS and hence useful for metabolism screening studies. The method was validated analytically and tested for its effectiveness as a metabolic inhibition model. A linear calibration was found to provide the best fit for the standard curve with an r of 0.9966 and all residuals less than 12%. The percent relative standard deviations (RSDs) of the two controls, 2 and 8 microg/ml were 5.27 and 2.90% and the percent difference from normal (% DFN) were -12.58 and -0.31% respectively. The limit of quantitation (LOQ) in the incubation matrix was 0.5 microg/ml. 3-OHQ formation complied with Michaelis-Menten kinetics and the mean values+/-S.D. of Km and Vmax were 36.98+/-4.62 microg/ml and 321.39+/-3.88 ng/mg/h respectively. Preliminary inhibition studies suggest that the method has adequate sensitivity to screen for high and medium inhibitors of the CYP 3A4 isozyme. The lack of sample preparation coupled with the small sample size capability of CE would enable the direct injection technique to aid in miniaturized high-throughput screening.

Grapefruit juice has no effect on quinine pharmacokinetics.

OBJECTIVE: As quinine is mainly metabolised by human liver CYP3A4 and grapefruit juice inhibits CYP3A4, the effect of grapefruit juice on the pharmacokinetics of quinine following a single oral dose of 600 mg quinine sulphate was investigated. METHODS: The study was carried out in ten healthy volunteers using a randomised cross-over design. Subjects were studied on three occasions, with a washout period of 2 weeks. During each period, subjects received a pretreatment of 200 ml orange juice (control), full-strength grapefruit juice or half-strength grapefruit juice twice daily for 5 days. On day 6, the subjects were given a single oral dose of 600 mg quinine sulphate with 200 ml of one of the juices. Plasma and urine samples for measurement of quinine and its major metabolite, 3-hydroxyquinine, were collected over a 48-h period and analysed by means of a high-performance liquid chromatography method. RESULTS: The intake of grapefruit juice did not significantly alter the oral pharmacokinetics of quinine. There were no significant differences among the three treatment periods with regard to pharmacokinetic parameters of quinine, including the peak plasma drug concentration (Cmax), the time to reach Cmax (tmax), the terminal elimination half-life (t1/2), the area under the concentration-time curve and the apparent oral clearance. The pharmacokinetics of the 3-hydroxyquinine metabolite were slightly changed when volunteers received grapefruit juice. The mean Cmax of the metabolite (0.25+/-0.09 mg l(-1), mean +/- SD) while subjects received full-strength grapefruit juice was significantly less than during the control period (0.31+/-0.06 mg l(-1), P < 0.05) and during the intake of half-strength grapefruit juice (0.31+/-0.07 mg l(-1), P < 0.05). CONCLUSION: These results suggest that there is no significant interaction between the parent compound quinine and grapefruit juice, so it is not necessary to advise patients against ingesting grapefruit juice at the same time that they take quinine. Since quinine is a low clearance drug with a relatively high oral bioavailability, and is primarily metabolised by human liver CYP3A4, the lack of effect of grapefruit juice on quinine pharmacokinetics supports the view that the site of CYP inhibition by grapefruit juice is mainly in the gut.

Metabolism of quinine in man: identification of a major metabolite, and effects of smoking and rifampicin pretreatment.

Our previous studies have shown that cigarette smoking and rifampicin pretreatment enhance the elimination of quinine, metabolism assumed, by analogy with quinidine, to be carried out by CYP3A (P450IIIA). This finding is unexpected since it has been shown that smoking induces the CYP1A rather than the CYP3A enzyme family, suggesting that the metabolism of quinine may be catalysed by CYP1A. Therefore, we conducted this study to identify possible quinine metabolites in human urine and to determine which metabolic pathway is induced by cigarette smoking and rifampicin pretreatment. A specific HPLC procedure was employed to identify metabolites of quinine in urine samples collected from healthy volunteers after an oral dose of 600 mg quinine sulphate. The results showed that there were at least seven possible metabolites of quinine detected in human urine. Three of these were identified as 2'-oxoquininone, quinine glucuronide and 3-hydroxyquinine. The 3-hydroxyquinine appeared to be a major metabolite of quinine in urine samples from every subject who took an oral dose of quinine. Although cigarette smoking and rifampicin pretreatment were shown to cause a marked increase in the elimination of quinine there were no significant changes in the formation of 3-hydroxyquinine as measured in the urine samples. This suggests that the effects of smoking and rifampicin are more complicated than we expected and require further investigation.