Ephedrine In Urine Research Articles

Quantitative analysis of clonidine and ephedrine by a microfluidic system: On-chip electromembrane extraction followed by high performance liquid chromatography.

In this work, a microfluidic device was developed for on-chip electromembrane extraction of trace amounts of ephedrine (EPH) and clonidine (CLO) in human urine and plasma samples followed by HPLC-UV analysis. Two polymethylmethacrylate plates were used as substrates and a microchannel was carved in each plate. The microchannel channel on the underneath plate provided the flow pass of the sample solution and the one on the upper plate dedicated to a compartment for the stagnant acceptor phase. A piece of polypropylene sheet was impregnated by an organic solvent and mounted between the two parts of the chip device. An electrical field, across the porous sheet, was created by two embedded platinum electrodes placed in the bottom of the channels which were connected to a power supply. The analytes were converted to their ionized form, passed through the supported liquid membrane, and then extracted into the acceptor phase by the applied voltage. All the effective parameters including the type of the SLM, the SLM composition, pH of donor and acceptor phases, and the quantity of the applied voltage were evaluated and optimized. Several organic solvents were evaluated as the SLM to assess the effect of SLM composition. Other parameters were optimized by a central composite design. Under the optimal conditions of voltage of 74V, flow rate of 28μLmin-1, 100 and 20mM HCl as acceptor and donor phase composition, respectively, the calibration curves were plotted for both analytes. The limits of detection were less than 7.0 and 11μgL-1 in urine and plasma, respectively. The linear dynamic ranges were within the range of 10-450 and 25-500μgL-1 (r2˃0.9969) for CLO, and within the range of 20-450 and 30-500μgL-1 (r2˃0.9907) for EPH in urine and plasma, respectively. To examine the capability of the method, real biological samples were analyzed. The results represented a high accuracy in the quantitative analysis of the analytes with relative recoveries within the range of 94.6-105.2% and acceptable repeatability with relative standard deviations lower than 5.1%.

Application of Surfactant Micellar Solutions as Extractants and Mobile Phases for TLC-Determination of Purine Bases and Doping Agents in Biological Liquids

Separation of caffeine and its metabolites (theophylline and theobromine) and doping agents (spironolactone, propranolol, and ephedrine) and determination of caffeine in serum sample and propranolol and ephedrine in urine were studied on normal-phase thin layers (“Sorbfil-UV-254”). Aqueous organic solvents and aqueous micellar surfactant solutions were compared as the mobile phases for separation. The acceptable separation of purine bases and doping agents was achieved by micellar Thin Layer Chromatography and normal-phase Thin Layer Chromatography. Anionic surfactant solution with added 1-propanol was the best eluent as for caffeine, theophylline, and theobromine separation, as for doping agents. The best characteristics of caffeine extraction from serum, and propranolol and ephedrine from urine were achieved when micellar eluent based on non-ionic Tween-80 surfactant was used. DOI: http://dx.doi.org/10.17807/orbital.v7i1.632

Electrochemical applications and computational studies on ephedrine drug

A novel, simple, sensitive and highly selective pseudo-carbon paste electrode modified with poly(acrylic) acid (PCPE-PAA) was described to be useful for the electrochemical determination of ephedrine substance. The PCPE-PAA electrode was characterized using scanning electron microscope (SEM) and cyclic voltammetry. Cyclic voltammogram for ephedrine shows only one anodic oxidation peak at 1.15 V (vs. SCE) using Britton–Robinson buffer (pH 9). Theoretical calculations were performed using PM3 method to be helpful in studying the electrochemical behavior of ephedrine. The highest occupied orbitals (HOMO) of ephedrine and the lowest unoccupied orbitals (LUMOs) of its oxidation product (methcathinone) are mainly localized in the side chain of benzene ring. The values of HOMOs, LUMOs and atomic charges clearly indicate that the oxidation process occurs on the hydroxyl group of ephedrine forming ketone group, supporting the proposed electrochemical mechanism. Square wave voltammetry was used for the direct electrochemical determination of ephedrine. Ephedrine gives a linear range from 6 × 10−5 to 1 × 10−3 M with a correlation coefficient of 0.998 and a relative standard deviation of 2.165 × 10−7. A lower detection limit of 3.5 × 10−7 M was obtained. The effect of some interferences such as ascorbic acid, uric acid, urea, glucose and glycine on the peak height of ephedrine was examined. The suggested method has been applied successfully for the direct electrochemical determination of ephedrine in urine and different pharmaceutical formulations.

Consequences of the formation of 3,4-dimethyl-5-phenyl-1,3-oxazolidine on the analysis of ephedrines in urine by gas chromatography and a new method for confirmation as N-trifluoroacetyl-O-t-butyldimethylsilyl ether derivatives

The compound 3,4-dimethyl-5-phenyl-1,3-oxazolidine can appear as an artifact during the gas chromatographic analysis of ephedrines. Its presence is a risk for doping control and forensic analyses. An evaluation about the consequences of its formation showed the possibility of a false positive for ephedrine, a false negative for pseudophedrine and increased uncertainty in the quantitative approach. Misinterpretations can be avoided with the observation of fragments m/ z 56 and 71 in the ephedrine mass spectrum during GC–MS analysis and also by the formation of N-TFA-O-TBDMS derivatives prior to GC analysis. These N-TFA-O-TBDMS derivatives lead to an increase in the number and mass of diagnostic ions, meet the identification criteria, and provide an improvement in chromatographic resolution, allowing the separation of the ephedrines.

Elimination of Ephedrines in Urine Following Administration of a Sho-seiryu-to Preparation

Sho-seiryu-to is one of the most common Traditional Chinese Medicine preparations for the attenuation of colds. Ephedrae Herba is one of the prescriptions of Sho-seiryu-to. The major ingredients of Ephedrae Herba, ephedrines, are banned substances on the World Anti-Doping Agency (WADA) list. The purpose of this study was to investigate the elimination of urinary ephedrines after administering Sho-seiryu-to preparation and to determine the possibility of positive ephedrines test results in urine. Six healthy volunteers took a single 2.5-g dose of concentrated Sho-seiryu-to preparation. All urine was collected for 48 h. The concentrations of urinary ephedrines were analyzed by high-performance liquid chromatography and the elimination half-life of the ephedrines was estimated. The results show that ephedrine and cathine (norpseudoephedrine), the prohibited substances of the WADA, were excreted in the urine after taking a single dose of Sho-seiryuto preparation. The peak concentration of ephedrine was 3.88 +/- 1.87 mg/mL (mean +/- SD), which was lower than the WADA permitted value (10 mg/mL). The estimated elimination half-lives of ephedrine, norephedrine, pseudoephedrine, and norpseudoephedrine following administration of this preparation were 5.3 +/- 1.2, 4.9 +/- 0.9, 4.4 +/- 1.0, and 5.4 +/- 1.8 h, respectively. This study concluded that the urine would not violate the antidoping rules after administering a single dose of Sho-seiryu-to preparation. Nevertheless, an applied multiple-dose study upon administering the preparation for three times per day for three days showed a positive urine ephedrine result (13.7 mg/mL). Athletes should be careful when taking more than a single dose of Sho-seiryu-to preparation.

Simultaneous quantification of amphetamines and ephedrines in urine by GC/MS using analytical-grade acetic anhydride/pyridine as derivatizing reagents: a suitable approach to reduce costs of routine analyses

A GC/MS method for the simultaneous analyses of different amphetamines and ephedrines in urine employing analytical-grade acetic anhydride/pyridine as derivatizing reagents was developed and validated. Solid-phase extraction was performed on the samples, which were then derivatizated and analyzed by GC/MS. The method showed a broad linear dynamic range (25-1000 ng mL-1 with R² > 0.99), high sensitivity (LODs of 0.0140 to 15.33 ng mL-1 and LOQs of 0.0466 to 51.10 ng mL-1), good precision (CV < 6% for intra- and inter-assays), and excellent extraction recovery (87 to 96%) for all the compounds studied. After validation, the method was applied in the analyses of real samples of human urine which were previously determined to contain at least one of such drugs. In all the samples, the amphetamines and ephedrines were promptly quantified, showing that the association of acetic anhydride and pyridine can be conveniently employed as a derivatizing agent.

Development and validation of an LC–MS/MS method for the quantification of ephedrines in urine

The objective of this study was to develop a simple and robust LC–MS/MS method for the quantification of ephedrine type substances in urine. Sample preparation consisted of a 10-fold dilution step of the samples into the internal standard solution (ephedrine-d 3, 4 μg/mL in water). Baseline separation of the diastereoisomers norpseudoephedrine–norephedrine and ephedrine–pseudoephedrine was performed on a C8-column using isocratic conditions followed by positive electrospray ionisation and tandem mass spectrometric detection. The mobile phase consisted of 98/2 (H 2O/ACN) containing 0.1% HAc and 0.01% TFA. Calibration curves were constructed between 2.5 and 10 μg/mL for norephedrine and norpseudoephedrine and 5 and 20 μg/mL for ephedrine, pseudoephedrine and methylephedrine. The bias ranged from −5.5 to 12% for norephedrine, −4.1 to 8.0 % for norpseudoephedrine, 0.3 to 2.1 % for ephedrine, 1.6 to 2.6 % for pseudoephedrine and 2.9 to 5.0 % for methylephedrine. Precision of the method varied between 2.8 and 10.4% for all compounds and the matrix effect was less than 15%. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC–NPD method. Results show a good correlation between both methods with correlation coefficients higher than 0.95 for all analytes.

Urinary Elimination of Ephedrines Following Administration of the Traditional Chinese Medicine Preparation Kakkon-to

Kakkon-to is one of the most common Traditional Chinese Medicine preparations for the attenuation of colds. Ephedrae Herba is one of the prescriptions of Kakkon-to. The major ingredients of Ephedrae Herba, ephedrines, are banned substances on the World Anti-Doping Agency (WADA) list. The purpose of this study was to investigate the elimination of urinary ephedrines after administering Kakkon-to and to determine the possibility of urinary positive ephedrine test results. Six healthy volunteers took one single dose of 2.5 g Kakkon-to extract granules. The concentrations of urinary ephedrines were analyzed by high-performance liquid chromatography. The result showed that ephedrine and norpseudoephedrine were excreted in the urine after taking one single dose of Kakkon-to. However, the highest amount of ephedrines in urine was ephedrine and the peak concentration was 4.35 +/- 1.82 microg/mL (mean +/- standard deviation), which was lower than the WADA permitted value (10 microg/mL). The estimated elimination half-lives of ephedrine, norephedrine, pseudoephedrine, and norpseudoephedrine following administration of this preparation were: 5.2 +/- 1.2, 4.2 +/- 1.3, 4.2 +/- 0.9, and 6.5 +/- 2.8 h, respectively. This study concluded that the urine would not violate the rule of doping after administering a single dose of Kakkon-to. Nevertheless, a further study on administering the preparation for 3 times per day for 3 days showed a positive ephedrine result. Athletes should be careful when taking more than a single dose of Kakkon-to.

Liquid–liquid–liquid microextraction followed by HPLC with UV detection for quantitation of ephedrine in urine

Liquid-liquid-liquid microextraction (LLLME) in combination with HPLC and UV detection has been used as a sensitive method for the determination of ephedrine in urine samples. Extraction process was performed in a homemade total glass vial without using a Teflon ring, usually employed. Ephedrine was first extracted from 3.5 mL of urine sample (pH 12) into a microfilm of toluene/benzene (50:50). The analyte was subsequently back extracted into an acidic microdrop solution (pH 2) suspended in the organic phase. The extract was then injected into the HPLC system directly. An enrichment factor of 137 along with a good sample clean-up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 0.01-50 mg/L with regression coefficient corresponding to 0.998. The LODs and LOQs, based on a S/N of 3 and 10, were 5 and 10 microg/L, respectively. The method was eventually applied for the determination of ephedrine in urine sample after oral administration of 5 mg single dose of drug.

Near Infrared Raman Spectroscopy (NIRS): A technique for doping control

This paper reports the application of near‐infrared Raman spectroscopy to detect ephedrine in biological medium. At present time, the doping control for ephedrine in athletes uses the urinalysis by the gas chromatography/mass spectrometry, with main disadvantage the need of collecting urine and the time delay to obtain results. This work aims to develop a noninvasive technique that will allow to evaluate the concentration of the ephedrine in a real time diagnosis. A Raman system composed by a Ti:Saphire laser pumped by an Argon laser was used, operating at the wavelength of 785 nm, with a laser power of 70 mW at sample position. Raman scattered photons were collected by a f/l.8 spectrometer and a N2‐cooled CCD detector. Ephedrine Raman peaks at 1002 and 1603 cm−1 were studied, opening possibility for the identification and quantification of ephedrine. Raman spectra of ephedrine with different concentrations in human urine were taken, and the intensity of the ephedrine peak at 1002 cm−1 was measured as a function of its concentration. It was also studied the Raman spectrum of an urine sample from a Wistar rat, after a subcutaneous inoculation of an ephedrine solution in physiologic serum, at the concentration of 5 mg/ml. It was found that Raman spectroscopy could detect ephedrine in urine at concentrations lower than the doping limit legally permitted by the International Olympic Committee.

Metabolites of ephedrines in human urine after administration of a single therapeutic dose

Ephedrine (EPH), pseudoephedrine (PEPH), phenylpropanolamine (PPA), methylephedrine (MEPH) and cathine are sympathomimetic amines. These drugs are commonly found in over-the-counter (OTC) cold medicines and some dietary supplements. In Taiwan, the misuse of these drugs has resulted in an increase in athletic violations. Excretion studies of the ephedrine-related drugs have been performed to better understand the metabolic yields of ephedrines in urine. After consuming a single clinical dose of each of these drugs, urine samples from volunteers ( n = 3 for each drug) were subjected to tert-butyl-methyl-ether (TBME) extraction and trifluoroacetic acid (TFAA) derivatization before gas chromatography–mass spectrometry (GC–MS) analysis. Most ephedrines were excreted unchanged in urine, including EPH (40.9%), PEPH (72.2%), and PPA (59.3%). However, only a relatively small amount of MEPH (15.5%) was excreted unchanged in urine. In addition, a trace amount of PPA (1.6%) and cathine (0.7%) was found to be the metabolites of EPH and PEPH, respectively. Urinary EPH, PEPH, and PPA reached peaks at 2–6 h and disappeared in urine at approximately 24–48 h post-administration. For MEPH, the peaks of excretion extended from 4 to 12 h post-administration and were undetectable at approximately 48 h. A single clinical dose of EPH (25 mg) may exceed threshold level (10 μg/mL) in sport drug testing if the urine samples are tested within approximately 8 h post-administration. However, a single dose of MEPH (20 mg) never reached the threshold value (10 μg/mL).

Solid phase micro‐extraction coupled with ion mobility spectrometry for the analysis of ephedrine in urine

Quantitative solid phase micro-extraction (SPME) coupled with ion mobility spectrometry is demonstrated using the analysis of ephedrine in urine. Since its inception in the 1970's ion mobility spectrometry (IMS) has evolved into a useful technique for laboratories to detect explosives, chemical warfare agents, environment pollutants and, increasingly, for detecting drugs of abuse. Ephedrine is extracted directly from urine samples using SPME and the analyte on the fiber is heated by the IMS desorber unit and vaporized into the drift tube. The analytical procedure was optimized for fiber coating selection, extraction temperature, extraction time, sample pH, and analyte desorption temperature. The carryover effects, ion fragmentation characteristics, peak shapes, and drift times of ephedrine were also evaluated based on the direct interfacing of SPME to IMS. A limit of detection of 50 ng/mL of ephedrine in urine and a linear range of 3 orders of magnitude were obtained, showing that SPME-IMS compares well to other techniques for ephedrine and drug analysis presented in the literature.

Quantification of ephedrines in urine by column-switching high-performance liquid chromatography

A method for the quantification of five congener ephedrines in urine samples without sample preparation was developed. The analytes were trapped on a C 18 precolumn and separated on a C 18 BDS analytical column. Baseline separation was achieved for all analytes. The method meets the requirements of the International Olympic Committee (IOC) medical commission regarding cut-off limits for positive doping cases with ephedrines.

Simultaneous quantitation of ephedrines in urine by gas chromatography–nitrogen–phosphorus detection for doping control purposes

A gas chromatographic method for the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine), norpseudoephedrine (cathine) and methylephedrine in urine is described. The method consists of a liquid–liquid extraction with tert.-butyl methyl ether at pH 14. The extracts are analysed on a GC system equipped with an Rtx-5 Amine column and a nitrogen–phosphorus detector. Method validation shows excellent separation, linearity, specificity, accuracy, precision, intra-laboratory repeatability and reproducibility, making the method especially suitable for quantitation of ephedrines in urine samples for doping control purposes. A statistical analysis on the abuse of the different ephedrines in urine from athletes controlled in the Flemish doping control laboratory during the period 1993–2000 is included.

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d 3-ephedrine as internal standard. Calibration curves, with r 2>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.

Comparison of various reversed-phase columns for the simultaneous determination of ephedrines in urine by high-performance liquid chromatography

Different reversed-phase columns for basic analytes were compared for the simultaneous determination of ephedrines in urine, such as LiChrospher 60 RP-Select B, LiChrospher 100 RP18, Hypersil BDS-C18, Inertsil ODS-2, Spherisorb ODS-B and Symmetry Shield RP8. Symmetry Shield was the only column which did not require the use of high concentrations of buffer and triethylamine. With this column, a good separation of the six ephedrines and the internal standard was achieved using 50 m M phosphate buffer–25 m M triethylamine as a mobile phase. Linearity, precision and accuracy were satisfactory for the levels usually found in urine. Due to these all parameters the developed analytical method was found to be suitable for the application in the doping field.

Indirect determination of ephedrine in urine by differential pulse polarography

AbstractThe nitrosation of ephedrine and the electrochemical reduction of N‐nitrosoephedrine were investigated by differential pulse polarography and have been optimized in order to attain the optimum analytical signal. Reduction of the N‐nitrosoephedrine was carried out in one irreversible, diffusion‐controlled step using potassium chloride‐hydrochloric acid. In 0.1 M hydrochloric acid the N‐nitrosoephedrine shows a well‐defined reduction peak at −0.77 V (vs. Ag/AgCl/ 3 M KCl). An indirect differential‐pulse polarographic method is described for the determination of ephedrine at levels higher than 2.5 μg per mL of urine. An extraction procedure with C18 cartridge and a fast and easy nitrosation reaction with sodium nitrite in 0.2 M phthalate potassium hydrogen is also described. Determination of 5 μg/mL of N‐nitrosoephedrine in urine samples of 4 mL was possible using the standard addition method, with a relative error of −4.0% and relative standard deviations no higher than 2.1%.

Evaluation of multicomponent fluorimetric analytical data by use of partial least-squares calibration. Application to the determination of codeine and ephedrine in urine

A partial least-squares calibration (PLS) method for the simultaneous spectrofluorimetric determination of acetylsalicylic acid, codeine and ephedrine is proposed. The use of PLS for the multicomponent spectrofluorimetric determination is demonstrated by analysing synthetic mixtures. Analyte mixtures can be resolved even in the presence of strongly overlapped fluorescence signals. The method was validated by subjecting experimental data to exhaustive statistical analysis. The proposed method was applied to the simultaneous determination of the three drugs in synthetic mixtures with a urine matrix. The concentration ranges considered are 1.56–10.92 and 25–100,μg/ml for the codeine-ephedrine mixture, and 0.8–3.2, 0.1–0.5 and 1.6–4.8 μg/ml for the acetylsalicylic acid-codeine-ephedrine mixture. The analytical results were quite good in all cases.

Simultaneous quantification of ephedrines in urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of ephedrine, pseudo-ephedrine, phenylpropanolamine, norpseudoephedrine and methylephedrine in urine is described. Samples were extracted at basic pH into diethyl ether and then back extracted into a small volume of acetic acid. These extracts were chromatographed on a Phase Sep Spherisorb ODS 1 column with tetraethylammoniumphosphate—methanol mobile phase and the analytes were detected by UV absorbance at 214nm. Calibration curves were linear over the range 0.5–100 μg/ml for each analyte. The selectivity of the method was demonstrated for several drugs found with the ephedrines in pharmaceutical formulations. This method has an excellent accuracy, precision and recovery for all the ephedrine at the cut-off concentrations as set by the IOC for a positive doping case and can thus be used to quantify these ephedrines when present in the urine obtained from sportspersons.

Determination of ephedrines in urine by high-performance liquid chromatography

An improved high-performance liquid chromatographic method with ultraviolet detection for the simultaneous determination of norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine and ethylephedrine in urine is described. The six substances were separated on a reversed-phase column with phosphate buffer-triethylamine (pH 5.5) as the mobile phase. The linearity and reproducibility were satisfactory for the levels usually found in urine (1–30 μg/ml).