Using cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography to determine selective serotonin reuptake inhibitors

We have employed a rapid and highly efficient on-line preconcentration method, cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC), for the analysis of selective serotonin reuptake inhibitors (SSRIs) of antidepressant drugs. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of high-conductivity buffer (HCB), sodium dodecyl sulfate (SDS), and organic modifier, the injection length of the HCB, and the injection time of the sample - to optimize the separation process. The optimal background electrolyte was 50 mM citric acid/disodium hydrogenphosphate buffer (pH 2.2) containing 100 mM SDS and 22% isopropyl alcohol. The sensitivity enhancements of the SSRIs sertraline, fluoxetine, paroxetine, fluvoxamine, and citalopram ranged from 5.7 x 10(4) to 1.2 x 10(5); the coefficients of determination exceeded 0.9938 and the relative standard deviations of the peak heights were less than 3.2%; the detection limits ranged from 0.056 to 0.22 ng/mL. We employed the optimal conditions to analyze these five SSRIs in a plasma sample prepared using solid-phase extraction (SPE) to minimize the influence of the matrix. Although the limits of detection of the SSRIs in human plasma were higher than those in pure water, this present technique is more sensitive than other, more-conventional methods. The recovery of the SPE extraction efficiency was satisfactory (up to 89%). Our findings suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used successfully to determine these five SSRIs in human plasma.     

Simultaneous analysis of cocaine and its metabolites in urine by capillary electrophoresis-electrospray mass spectrometry using a pressurized liquid junction nanoflow interface

A new method for the simultaneous separation of cocaine and four metabolites in urine by CE-ESI-MS via a pressurized nanoliquid junction interface was developed. The resolution of cocaine, cocaethylene, benzoylecgonine, norcocaine, and ecgonine methyl ester was achieved in a polyvinyl-alcohol-coated capillary with 75 μm id × 50 cm total length, using a 15 mM ammonium formate electrolyte solution (pH 9.5) in less than 15 min. In addition, to enhance sensitivity, a field-amplified sample injection (FASI) was evaluated in terms of injection time and sample solvent composition. The limits of detection achieved with the FASI method ranged from 1.5 to 10 ng/mL for all the compounds. The detection of the studied compounds was performed using an ion-trap mass spectrometer in a positive ionization mode. A mixture of methanol:water (80:20 v/v) containing 0.1% v/v of formic acid was employed as spray liquid and delivered at ~200 nL/min. Under optimal CE-MS conditions, linearity was assessed in the concentration range of interest for all analytes with correlation coefficients r2 ≥ 0.9913. Intra- and inter-day precision provided a relative standard deviation lower than 1.54% for migration times and lower than 12.15% for peak areas. Finally, urine samples, spiked with the standard mixture, were extracted using a solid-phase extraction procedure and injected under FASI conditions, providing recoveries from 80% to 94% for all analytes.     

Determination of malachite green in fish water samples by cloud‐point extraction coupled to cation‐selective exhaustive injection and sweeping‐MEKC

We have employed a high‐sensitivity off‐line coupled with on‐line preconcentration method, cloud‐point extraction (CPE)/cation‐selective exhaustive injection (CSEI) and sweeping‐MEKC, for the analysis of malachite green. The variables that affect CPE were investigated. The optimal conditions were 250 g/L of Triton X‐100, 10% of Na₂SO₄ (w/v), heat‐assisted at 60°C for 20 min. We monitored the effects of several of the CSEI‐sweeping‐MEKC parameters – including the type of BGE, the concentrations of SDS, the injection length of the high‐conductivity buffer, and the injection time of the sample – to optimize the separation process. The optimal BGE was 50 mM citric acid (pH 2.2) containing 100 mM SDS. In addition, electrokinetic injection of the sample at 15 kV for 800 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancement for malachite green was 1.9×10⁴ relative to CZE; the coefficients of determination exceeded 0.9928. The LOD, based on an S/N of 3:1, of CSEI‐sweeping‐MEKC was 0.87 ng/mL; in contrast, when using off‐line CPE/CSEI‐sweeping‐MEKC the sensitivity increased to 69.6 pg/mL. This proposed method was successfully applied to determine trace amounts of malachite green in fish water samples.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

Profiling of cocaine by micellar electrokinetic chromatography

The potential of micellar electrokinetic chromatography (MEKC) for the profiling of cocaine samples is described. An MEKC system containing sodium dodecyl sulfate (SDS) and methanol was optimized using a test mixture of cocaine, its common impurities (benzoylecgonine, norcocaine, tropacocaine, and trans-cinnamoylcocaine), and several degradation products. The effect of pH, percentage modifier, and concentration surfactant on the separation has been investigated. The optimal separation buffer for cocaine samples consisted of 75 mM SDS, 17.5% methanol, and 25 mM borate (pH 8.3) and was well suited to separate components of diverse polarity in one run. Various cocaine seizures have been analyzed with the MEKC system and their signatures were compared. The electrokinetic chromatograms obtained were characteristic, and differences and similarities among the samples could easily be observed. Several impurities were identified in the samples by means of migration times and comparison of recorded and library UV spectra. The composition of the samples was determined semiquantitatively using relative corrected peak areas.     

Determination of cypromazine and its metabolite melamine in milk by cation-selective exhaustive injection and sweeping-capillary micellar electrokinetic chromatography

In this study, we described a high‐sensitive on‐line preconcentration method for cypromazine (CYP) and melamine (MEL) analysis using cation‐selective exhaustive injection (CSEI) combined with sweeping‐MEKC. The optimum conditions of on‐line concentration and separation were discussed. The BGE contained 100 mM SDS, 50 mM phosphoric acid (pH=2.0) and 15% acetonitrile (v/v). The sample was injected at 10 kV for 600 s, separated at ?20 kV, and detected at 210 nm. The sensitivity enhancements were 6222 for CYP and 9179 for MEL. The linear dynamic ranges were 0.4?25 ng/mL for CYP (r=0.9995) and 0.2?12 ng/mL for MEL (r=0.9991). The LODs (signal‐to‐noise ratio, 3) were 43.7 and 23.4 pg/mL for CYP and MEL, respectively. The proposed method was applied to analyze CYP and MEL in dairy products pretreated using off‐line SPE to minimize the influence of the matrix. The recoveries of CYP and MEL were satisfactory (ca. 74–83%). The experimental results suggest that the CSEI‐sweeping‐MEKC method is feasible for the application to simultaneously detect trace levels of CYP and its metabolite MEL in real milk samples.     

Using sweeping micellar electrokinetic chromatography to analyze Delta9-tetrahydrocannabinol and its major metabolites

We have applied sweeping micellar electrokinetic chromatography (sweeping-MEKC) to the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH). We monitored the effects of several of the sweeping-MEKC parameters, including the proportion of organic modifier, the concentration of sodium dodecyl sulfate (SDS), the pH, and the sample injection volume, to optimize the separation process. The optimal buffer for the analysis of the three analytes was 25 mM citric acid/disodium hydrogenphosphate (pH 2.6) containing 40% methanol and 75 mM SDS. Under the optimized separation parameters, the enrichment factors for THC, THC-COOH, and THC-OH when using sweeping-MEKC (relative to MEKC) were 77, 139, and 200, respectively. The limits of detection (LODs) for the three compounds in standard solutions ranged from 3.87 to 15.2 ng/mL. We combined the sweeping-MEKC method with solid-phase extraction to successfully detect THC, THC-COOH, and THC-OH in human urine with acceptable repeatability. The LODs of these analytes in urine samples ranged from 17.2 to 23.3 ng/mL. Therefore, this sweeping-MEKC method is useful for determining, with high sensitivity, the amounts of THC and its metabolites in the urine of suspected THC users.     

Different strategies for the preconcentration and separation of parabens by capillary electrophoresis

Several strategies, namely, large volume sample stacking (LVSS), field‐amplified sample injection (FASI), sweeping, and in‐line SPE‐CE, were investigated for the simultaneous separation and preconcentration of a group of parabens. A BGE consisting of 20 mM sodium dihydrogenphosphate (pH 2.28) and 150 mM SDS with 15% ACN was used for the separation and preconcentration of the compounds by sweeping, and a BGE consisting of 30 mM sodium borate (pH 9.5) was used for the separation and preconcentration of the compounds by LVSS, FASI, and in‐line SPE‐CE. Several factors affecting the preconcentration process were investigated in order to obtain the maximum enhancement of sensitivity. The LODs obtained for parabens were in the range of 18–27, 3–4, 2, and 0.01–0.02 ng/mL, and the sensitivity evaluated in terms of LODs was improved up to 29‐, 77‐, 120‐, and 18 400‐fold for sweeping, LVSS, FASI, and in‐line SPE‐CE, respectively. These preconcentration techniques showed potential as good strategies for focusing parabens. The four methods were validated with standard samples to show the potential of these techniques for future applications in real samples, such as biological and environmental samples.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine using reversed-phase high-performance liquid chromatography with ultraviolet detection is described. Following solid-phase extraction, chromatography was performed using a column containing an octadecylsilica-coated packing, eluted with 6% acetonitrile in phosphate buffer, pH 2.1, and detected at 233 nm. Using 80-microliters samples, the detection limit is 18 ng/ml for benzoylecgonine and benzoylenorecgonine and 35 ng/ml for cocaine and norcocaine. The coefficients of variation range from 3.5% (benzoylecgonine) to 7.0% (norcocaine). The procedure has been applied to samples of guinea pig plasma, urine and amniotic fluid and human urine.     

Comparison of two capillary electrophoresis online stacking modes by analysis of polycyclic aromatic hydrocarbons in airborne particulates

Naphthalene, fluorene, pyrene, anthracene, phenanthrene, and chrysene were successfully separated by CD-modified MEKC (CD-MEKC) using 20 mM borate (pH 9.0) containing 90 mM SDS and 75 mM beta-CD. Two online stacking methods, i.e., sweeping and field-enhanced sample injection (FESI), were explored to enhance the detection sensitivity. The influences of some crucial parameters in sweeping and FESI procedures were investigated. For FESI method, a plug of water and low-conductivity sample matrix was used to increase the stacking efficiency. Compared with the sweeping method, FESI can increase the sensitivity in the range of 10-20-fold. The proposed method was used for the analysis of polycyclic aromatic hydrocarbons in airborne particulates.     

Using the cationic surfactants N-cetyl-N-methylpyrrolidinium bromide and 1-cetyl-3-methylimidazolium bromide for sweeping–micellar electrokinetic chromatography

This paper describes a sweeping–micellar electrokinetic chromatography (sweeping–MEKC) technique for the determination of seven benzodiazepines, using, as sweeping carriers, the ionic liquid-type cationic surfactants 1-cetyl-3-methylimidazolium bromide (C₁₆MIMBr) and N-cetyl-N-methylpyrrolidinium bromide (C₁₆MPYB). These surfactants resemble the commonly employed cationic surfactant cetyltrimethylammonium bromide (CTAB), but they provide different separation efficiencies. We optimized the separation and sweeping conditions, including the pH, the concentrations of organic modifier and surfactant, and the sample injection volume. Adding C₁₆MIMBr or C₁₆MPYB to the background electrolyte enhanced the separation efficiency and detection sensitivity during the sweeping–MEKC analyses of the benzodiazepines. C₁₆MIMBr enhanced the sensitivity for each benzodiazepine 31–59-fold; C₁₆MPYB, 86–165-fold. In the presence of C₁₆MPYB, the limits of detection for the seven analytes ranged from 4.68 to 9.75 ng/mL. We adopted the sweeping–MEKC conditions optimized for C₁₆MPYB to satisfactorily analyze a human urine sample spiked with the seven benzodiazepines. To minimize the matrix effects, we subjected this urine sample to off-line solid phase extraction (SPE) prior to analysis. The recoveries of the analytes after SPE were satisfactory (ca. 77.0–88.3%). Our experimental results reveal that the cationic surfactant C₁₆MPYB exhibits superior sweeping power relative to those of C₁₆MIMBr and CTAB and that it can be applied in sweeping–MEKC analyses for the on-line concentrating and analyzing of benzodiazepines present in real samples at nanogram-per-milliliter concentrations.     

Measurement of benzoylecgonine and cocaine in urine, separation of various cocaine metabolites using reversed-phase high-performance liquid chromatography

The application of reversed-phase high-performance liquid chromatography to the measurement of benzoylecgonine and cocaine in urine is described. Following a simple extraction and clean-up procedure, chromatography is performed using a column containing an octadecylsilica coated packing, elution with 17% acetonitrile in pH 2.7 phosphate buffer and ultraviolet detection at 200 or 235 nm. The detection limit is ca.0.1 microgram of drug per ml urine, and using the ethyl ester of benzoylecgonine as an internal standard, benzoylecgonine and cocaine are quantified with coefficients of variation of 7.0 and 2.8%, respectively. The procedure has been applied to urines from subjects receiving intranasal cocaine, and compared to the enzyme multiplied immunoassay technique. The chromatography procedure also permits the separation of norcocaine and benzoylnorecgonine.     

A chemometric approach for the elucidation of the parameter impact in the hyphenation of field-enhanced sample injection and sweeping in capillary electrophoresis

The aim of this work was to elucidate the impacts of parameters influencing cation-selective exhaustive injection coupled to sweeping and micellar electrokinetic chromatography (MEKC). A chemometric approach using cationic compounds, acidic conditions (phosphate buffer, pH 2.3) and polyacrylamide-coated capillaries to suppress electroosmotic flow were used. It was demonstrated that the water plug was not useful because of long electrokinetic injections. If conductivity of the high conductivity buffer (HCB) and the HCB to sample conductivity ratio are sufficiently high (>1.66 S/m and >30, respectively), variations of HCB conductivity do not impact sensitivity. The length of the HCB must be long enough so that the most mobile cation remains stacked in this zone for a given injection time. SDS concentration should be as high as possible (the maximum concentration is dictated by MEKC, here 90 mM), so sensitivity is not impacted. We have shown analytes can be lost after electrokinetic injection, when the polarity of the voltage is reversed. Introducing a plug of micellar electrolyte before polarity reversal avoids these losses. Following these recommendations only injection time and sample conductivity impacted sensitivity enhancement. Sample conductivity had to be the lowest as possible and controlled in real case analyses to obtain repeatable enrichment factors.     

SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata-X and X-C). DOX was determined on a 56 cm (effective length 50 cm) x 50 microm id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 sx50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 microg/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.     

Use of SDS micelles for improving sensitivity, resolution, and speed in the analysis of beta-lactam antibiotics in environmental waters by SPE and CE

This study dealt with the potential of MEKC with LIF detection involving derivatization with sulfoindocyanine succinimidyl ester (Cy5) for the separation and determination of beta-lactam antibiotics (ampicillin, amoxicillin, cephradine, and cephalexin) in environmental water samples. Water samples of 50 mL were enriched by SPE by passage through a weak base-cation Amberlite(R) IRA-93 exchange column. SDS micelles play important roles in the whole analytical process by improving the yield (sensitivity) and the kinetics of the labeling reaction, the elution of the retained antibiotics from the SPE preconcentration system and the electrophoretic resolution of their Cy5-derivatives. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 10 min and direct injection for MEKC analysis, which is conducted within about 15 min using 15 mM SDS in the running buffer (35 mM sodium borate at pH 9.3). LODs from 30 to 45 ng/L and RSDs (within-day precision) from 3.5 to 5.9% were obtained for the antibiotics in water samples with average recoveries ranging from 96.4 to 99.4%. These results indicate that the method proposed is a straightforward and sensitive tool for the determination of these antibiotics in environmental water samples providing similar quantitative results to those using more expensive equipment like LC-electrospray MS/MS.     

Transformation of cocaine during water chlorination

The stability of cocaine and its two main human metabolites, benzoylecgonine and ecgonine methyl ester, in chlorine-containing waters has been investigated by direct injection of different reaction time aliquots in a liquid chromatograph (LC) coupled to a quadrupole-time-of-flight mass spectrometer (QTOF-MS). Factors potentially affecting cocaine degradation (the only compound showing a significant decrease in the preliminary study) were evaluated in detail by means of a Box–Behnken experimental design. Sample pH resulted to be the most important variable, increasing both the rate of chlorination-mediated reactions and the ester hydrolysis process. From these reactions, and due to the high mass accuracy measurements obtained with the QTOF system, four by-products could be positively identified: benzoylecgonine, norcocaine, norbenzoylecgonine and N-formylnorcocaine. Finally, their formation and cocaine degradation yields were assessed under chlorination experiments with two real surface water samples. In one of them, showing a low anthropogenic impact, benzoylecgonine and norcocaine were notably generated even after only 1?h of reaction, whereas at higher contact times also norbenzoylecgonine and N-formylnorcocaine could be determined with a lower yield. On the other hand, the second sample, with a higher organic matter content, consumed rapidly the chlorine, so that only benzoylecgonine was produced. These findings point out the convenience of monitoring the described transformation products, in addition to the precursor illicit drug, during drinking water production, taking into account that cocaine traces might be present in water catchments and particularly in areas with high population densities.     

Analysis of the herbicides paraquat,diquat and difenzoquat in drinking water by micellar electrokinetic chromatography using sweeping and cation selective exhaustive injection

Optimum conditions for the determination of the herbicides paraquat, diquat and difenzoquat by micellar electrokinetic chromatography (MEKC) using sweeping and cation-selective exhaustive injection (CSEI) as on-line concentration methods were developed. Sodium dodecyl sulfate (80 mM) in 50 mM phosphate buffer (pH 2.5) with 20% acetonitrile was used as a background electrolyte for the methods studied. The limits of detection, based on a signal-to-noise ratio of 3:1, were about 2.6-5.1 mg 1(-1) in purified water when MEKC was applied for the standards. By using an on-line preconcentration method known as sweeping-MEKC, up to a 500-fold increase in detection sensitivity was obtained whereas up to a 50 000-fold increase for CSEI-sweeping-MEKC was achieved. The limits of detection using optimum CSEI-sweeping-MEKC were lower than 1 microg 1(-1) and the method was validated obtaining good reproducibility (relative standard deviation lower than 22%) and linearity. CSEI-sweeping-MEKC was successfully applied to the determination of the three herbicides in spiked tap water below the levels established by the US Environmental Protection Agency.     

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.     

Determination of cocaethylene, cocaine and their metabolites in rat serum microsamples by high-performance liquid chromatography, and its application to pharmacokinetic studies in rodents.

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaethylene in rat serum microsamples (50 microliters), a substance formed in vivo when cocaine and ethanol are present concurrently. The separation used a 2 mm I.D. reversed-phase Nova-Pak C18 column with a mobile phase of acetonitrile-phosphate buffer containing an ion-pairing reagent. With an ultraviolet detector operated at 230 nm, a linear response was observed from 0.05 to 2.0 micrograms/ml with a detection limit of 5 ng/ml for cocaethylene, cocaine and norcocaine. The method showed a longer half-life for cocaethylene than for cocaine in rat.