Direct and rapid quantitation of ephedrines in human urine by paper spray ionization/high resolution mass spectrometry.

A rapid and direct paper spray ionization/mass spectrometry (PSI/MS) method was developed for quantitative analysis of ephedrine, pseudoephedrine, norpseudoephedrine, and methylephedrine in human urine. This method involves the use of a triangular filter paper and high-resolution mass spectrometry, where the molecular ions of ephedrines were generated by applying high voltage after loading the spray solvent to the paper which urine sample was pre-loaded. Small amounts (2µL) of urine spiked with an internal standard were directly analyzed for ephedrines. The PSI/MS method was validated for linearity, within- and between-run precision, accuracy, and limit of detection. The results showed good linearity (R(2)≥0.9928) and acceptable precision and accuracy. Furthermore, the accuracy of the method was assessed by analyzing a blind urine sample from World Anti-Doping Agency and comparing the measured concentrations with the nominal concentrations. This test resulted in accuracies ranging from 96.4 to 106.1%, indicating that the PSI/MS method has the potential to be an alternative technique for the fast quantitation of ephedrines in doping control analysis.

Magnetic nano graphene oxide as solid phase extraction adsorbent coupled with liquid chromatography to determine pseudoephedrine in urine samples.

This paper reports on a method based on magnetic solid phase extraction (MSPE) for the determination of pseudoephedrine. Magnetic nanographene oxide (MNGO) was applied as a new adsorbent for the extraction of pseudoephedrine from urine samples. Synthesis of MNGO was characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). The main factors influencing extraction efficiency, including the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, ionic strength of extraction medium, and agitation rate, were investigated and optimized. Under optimized extraction conditions, a good linearity was observed in the range of 100-2000ng/mL with a correlation coefficient of 0.9908 (r(2)). Limit of detection (LOD) and limit of quantification (LOQ) were 25 and 82.7ng/mL, respectively. Inter-day and intra-day precision and accuracy were 6.01 and 0.34 (%), and 8.70 and 0.29 (%), respectively. The method was applied for the determination of pseudoephedrine in urine samples of volunteers receiving pseudoephedrine with the recovery of 96.42. It was concluded that the proposed method can be applied in diagnostic clinics.

Determination of the pseudoephedrine content in pharmaceutical formulations and in biological fluids using a microbore HPLC system interfaced to a microfluidic chemiluminescence detector.

A novel automated precolumn derivatization followed by separation using liquid chromatography for the determination of pseudoephedrine (PSE) by a microfluidic chemiluminescence detector has been developed. An on-line derivatization procedure was utilized by converting PSE into a highly light emitting species in a Ru(bipy)3(2+)-peroxydisulphate chemiluminescence (CL) system by derivatizing it with a 1.0 M formaldehyde solution. The derivatized analyte was directly injected into a microbore high-performance liquid chromatography (HPLC) system coupled to an on-chip chemiluminescence detector. The newly developed highly selective, sensitive and fast HPLC-CL method was validated and successfully applied for the analysis of PSE in pharmaceutical formulations and a human urine sample. The selectivity of the method is not only due to the HPLC separation but is also due to the highly selective detection principle of the Ru(bipy)3(2+)-peroxydisulphate CL system used. There was no interference observed from the common preservatives and excipients used in pharmaceutical preparations, which did not show any significant CL signal. The retention time of PSE was less than 3 min, and the detection limits and quantification limits were found to be 5.7 and 26.0 µg L(-1), respectively.

Hydration and urinary pseudoephedrine levels after a simulated team game.

This study investigated the influence of dehydration on urinary levels of pseudoephedrine (PSE) after prolonged repeated effort activity. Fourteen athletes performed a simulated team game circuit (STGC) outdoors over 120 min under three different hydration protocols: hydrated (HYD), dehydrated (DHY) and dehydrated + postexercise fluid bolus (BOL). In all trials, a 60 mg dose of PSE was administered 30 min before trial and at half time of the STGC. Urinary PSE levels were measured before drug administration and at 90 min postexercise. In addition, body mass (BM) changes and urinary specific gravity (USG), osmolality (OSM), creatinine (Cr), and pH values were recorded. No differences in PSE levels were found 90 min postexercise between conditions (HYD: 208.5 ± 116.5; DHY: 238.9 ± 93.5; BOL: 195.6 ± 107.3 µg · ml(-1)), although large variations were seen within and between participants across conditions (range: 33-475 µg · ml(-1): ICC r = .03-0.16, p > .05). There were no differences between conditions in USG, OSM, pH or PSE/Cr ratio. In conclusion, hydration status did not influence urinary PSE levels after prolonged repeated effort activity, with ~70% of samples greater than the WADA limit (>150 µg · ml(-1)), and ~30% under. Due to the unpredictability of urinary PSE values, athletes should avoid taking any medications containing PSE during competition.

Pseudoephedrine and preexercise feeding: influence on performance.

PURPOSE: This study examined the influence of preexercise food intake on plasma pseudoephedrine (PSE) concentrations and subsequent high-intensity exercise. In addition, urinary PSE concentrations were measured under the same conditions and compared with the present threshold of the World Anti-Doping Agency (WADA). METHODS: Ten highly trained male cyclists and triathletes (age = 30.6 ± 6.6 yr, body mass [BM] = 72.9 ± 5.1 kg, and V˙O2max = 64.8 ± 4.5 mL·kg·min; mean ± SD) undertook four cycling time trials (TT), each requiring the completion of a set amount of work (7 kJ·kg BM) in the shortest possible time. Participants were randomized into a fed or nonfed condition and orally ingested 2.8 mg·kg BM of PSE or a placebo (PLA) 90 min before exercise; in the fed trials, they consumed a meal providing 1.5 g·kg BM of CHO. Venous blood was sampled at 30, 50, and 70 min and pre-warm-up and postexercise for the analysis of plasma PSE and catecholamine concentrations, and urine was also collected for the analysis of PSE concentration. RESULTS: Independent of the preexercise meal, 2.8 mg·kg BM of PSE did not significantly improve cycling TT performance. The fed trials resulted in lower plasma PSE concentrations at all time points compared with the nonfed trials. Both plasma epinephrine and blood lactate concentrations were higher in the PSE compared with the PLA trials, and preexercise and postexercise urinary PSE concentrations were significantly higher than the threshold (150 µg·mL) used by WADA to determine illicit PSE use. CONCLUSION: Irrespective of the preexercise meal, cycling TT performance of approximately 30 min was not improved after PSE supplementation. Furthermore, 2.8 mg·kg BM of PSE taken 90 min before exercise, with or without food, resulted in urinary PSE concentrations exceeding the present WADA threshold.

Interconversion of ephedrine and pseudoephedrine during chemical derivatization.

Gas chromatography-mass spectrometry (GC-MS) analysis after heptafluorobutyric anhydride (HFBA) derivatization was one of the published methods used for the quantification of ephedrine (EP) and pseudoephedrine (PE) in urine. This method allows the clear separation of the derivatized diastereoisomers on a methyl-silicone-based column. Recently the authors came across a human urine sample with apparently high levels (µg/ml) of EP and PE upon initial screening. However, duplicate analyses of this sample using the HFBA-GC-MS method revealed an unusual discrepancy in the estimated levels of EP and PE, with the area response ratios of EP/PE at around 29% on one occasion and around 57% on another. The same sample was re-analyzed for EP and PE using other techniques, including GC-MS after trimethylsilylation and ultra-high-performance liquid chromatography-tandem mass spectrometry. Surprisingly, the concentration of EP in the sample was determined to be at least two orders of magnitude less than what was observed with the HFBA-GC-MS method. A thorough investigation was then conducted, and the results showed that both substances could interconvert during HFBA derivatization. Similar diastereoisomeric conversion was also observed using other fluorinated acylating agents (e.g. pentafluoropropionic anhydride and trifluoroacetic anhydride). The extent of interconversion was correlated with the degree of fluorination of the acylating agents, with HFBA giving the highest conversion. This conversion has never been reported before. A mechanism for the interconversion was proposed. These findings indicated that fluorinated acylating agents should not be used for the unequivocal identification or quantification of EP and PE as the results obtained can be erroneous.

Determination of urinary concentrations of pseudoephedrine and cathine after therapeutic administration of pseudoephedrine-containing medications to healthy subjects: implications for doping control analysis of these stimulants banned in sport.

Due to its stimulatory effects on the central nervous system, and its structural similarity to banned stimulants such as ephedrine and methamphetamine, pseudoephedrine (PSE) at high doses is considered as an ergogenic aid for boosting athletic performance. However, the status of PSE in the International Standard of the Prohibited List as established under the World Anti-Doping Code has changed over the years, being prohibited until 2003 at a urinary cut-off value of 25 µg/ml, and then subsequently removed from the Prohibited List during the period 2004-2009. The re-consideration of this position by the World Anti-Doping Agency (WADA) List Expert Group has led to the reintroduction of PSE in the Prohibited List in 2010. In this manuscript, we present the results of two WADA-sponsored clinical studies on the urinary excretion of PSE and its metabolite cathine (CATH) following the oral administration of different PSE formulations to healthy individuals at therapeutic regimes. On this basis, the current analytical urinary threshold for the detection of PSE as a doping agent in sport has been conservatively established at 150 µg/ml

Monolithic molecular imprinted polymer fiber for recognition and solid phase microextraction of ephedrine and pseudoephedrine in biological samples prior to capillary electrophoresis analysis.

A novel capillary electrophoresis (CE) method coupled with monolithic molecular imprinted polymer (MIP) fiber based solid phase microextraction (SPME) was developed for selective and sensitive determination of ephedrine (E) and pseudoephedrine (PE). With in situ polymerization in a silica capillary mold and E as template, the MIP fibers could be produced in batch reproducibly and each fiber was available for 50 extraction cycles without significant decrease in extraction ability. Using the MIP fiber under optimized extraction conditions, CE detection limits of E and PE were greatly lowered from 0.20 to 0.00096 µg/mL and 0.12 to 0.0011 µg/mL, respectively. Analysis of urine and serum samples by the MIP-SPME-CE method was also performed, with results indicating that E and PE could be selectively extracted. The recoveries and relative standard deviations (RSDs) for sample analysis were found in the range of 91-104% and 3.8-9.1%, respectively.

Rapid quantitative determination of ephedra alkaloids in tablet formulations and human urine by microchip electrophoresis.

Microchip electrophoresis with fluorescence detection has been applied for fast separation and determination of ephedra alkaloids in pharmaceutical formulations and body fluids. A custom epifluorescence microscope setup was employed and the compounds were separated within 40 s, allowing the detection of less than 200 ng/L for both analytes. Quantitation of the two stimulants was performed via a derivatization step using FITC without any extraction or preconcentration steps. The effects of different microchip types and excitation light sources were investigated and the method was successfully applied for the analysis of these compounds in tablet formulations, yielding recovery rates from 100.2 to 101.1% and relative standard deviations from 1.5 to 3.4%. Analysis of ephedrines was also carried out with human urine samples at detection limits of 500-1000 ng/L and relative standard deviations from 2.2 to 3.3% using argon ion LIF detection.

Analysis of stimulants in oral fluid and urine by gas chromatography-mass spectrometry II: pseudophedrine.

This study was designed to optimize a method for the identification and quantification of ephedrines in oral fluid (OF) and for its application to subjects taking different doses of pseudoephedrine. Ephedrines use by athletes is banned by World Anti-Doping Agency (WADA), only "in competition" if their concentration in urine exceeds the cutoff limit. The study aimed to establish if there is a correlation in terms of times of elimination and of concentration trends of ephedrine in OF and urine after administration of therapeutic doses of pseudoephedrine to various subjects. Results obtained from excretion studies performed on eight subjects showed reproducible times of disappearance of ephedrines from OF. Pseudoephedrine was generally at low concentrations or undetectable in oral fluid samples 12 h after administration, whereas urine samples collected in the same period of time showed higher ephedrine concentrations and exceeding cutoff values generally between 8 and 24 h after administration of the drug. Within- and between-individual variability was observed in terms of concentrations of pseudoephedrine in OF following the administration of the same dose. Only in the case of sustained-release drugs were constant pseudoephedrine concentrations achieved in OF.

Simultaneous determination of eight sympathomimetic amines in urine by gas chromatography/mass spectrometry.

A GC method was developed for the identification and quantitation of eight sympathomimetic amines in urine, i.e., amphetamine, methamphetamine, mephentermine, ephedrine, pseudoephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine. Methoxyphenamine was used as the internal standard (IS). The assay is rapid, sensitive, and simple to perform. It involves a liquid-liquid extraction procedure with simultaneous in-solution derivatization of the organic layer with pentafluorobenzoyl chloride (PFB-CI), followed by GC/MS analysis. These derivatives and the IS were extracted from 1 mL alkaline urine into hexane before derivatization with PFB-CI. The organic layer was then removed and evaporated to dryness before dissolution with hexane for GC/MS analysis. Calibration curves for each analyte showed linearity in the range of 25-5000 ng/mL (r2 > or = 0.997). Recoveries ranged from 88 to 99%, with the precision of recoveries typically < or = 5%. The LOD values ranged from 7 to 28 ng/mL, and the LOQ values ranged from 23 to 94 ng/mL. At least four ions were available for each analyte for confirmation of identity by MS.

[Analysis of the preferred conformations and determination of the concentrations of ephedrine and pseudoephedrine based on hollow fiber liquid-phase microextraction].

The preferred conformations of the ephedrine and pseudoephedrine in Ephedra sinica Stapf and rat urine were analyzed by the hollow fiber liquid-phase microextraction (HF-LPME) and their extraction mechanisms were illuminated. The method of the separation of the ephedrine and pseudoephedrine and the determination of their concentrations with high performance liquid chromatography (HPLC) were established. The optimal experimental conditions were as follows: the organic phase carrier was the hollow fiber of polyvinylidene fluoride (MOF-503), organic solvent was n-hexanol, the extraction time was 35 min, the stirring rate was 1200 r/min, the sample phase was the NaOH solution (5 mol/L) of the analyte, the acceptor was 0.01 mol/L H2SO4 solution. The extracts were analyzed by HPLC. Under the optimal conditions, the method is convenient and highly sensitive. In Ephedra sinica Stapf, the linear ranges of ephedrine and pseudoephedrine were 5-100 microg/L, the detection limits were 1.9 microg/L and 1.2 microg/L and the enrichment factors were 38 and 61, respectively. The average recoveries of ephedrine and pseudoephedrine were 100.6% +/- 1.2% and 103.2% +/- 3.5%, respectively. In rat urine, their linear ranges were 100 - 5 x 10(4) microg/L, the detection limits were 30 microg/L and 42 microg/L and the enrichment factors were 20 and 17, respectively. In rat urine, their average recoveries were 108.4% +/- 4.4% and 106. 1% +/- 5.4%, respectively. The obtained results indicated that the method can be successfully applied for the extraction and determination of the ephedrine and pseudoephedrine in Ephedra sinica Stapf and rat urine.

The relevance of the urinary concentration of ephedrines in anti-doping analysis: determination of pseudoephedrine, cathine, and ephedrine after administration of over-the-counter medicaments.

This article describes a method for the detection and quantitation of cathine, pseudoephedrine, ephedrine, and methylephedrine in urine, using their deuterated analogues as internal standards and derivatization to form the corresponding trimethylsilyl derivatives after a simple liquid-liquid extraction. The study was designed to evaluate whether the urinary cutoff values set by the World Anti-Doping Agency for the banned ephedrines (cathine >5 microg/mL, ephedrine and methylephedrine >10 microg/mL) can be exceeded after the normal self-administration of common over-the-counter medicaments containing nonbanned ephedrines. The present method, after validation, has been applied on real urine samples obtained from 9 healthy volunteers taking different doses of over-the-counter preparations containing ephedrines. Results obtained from excretion studies show high interindividual differences in the urinary concentrations of both pseudoephedrine and cathine, not dependent on body weight or sex nor, in some instances, on the administered dose. The same typical therapeutic dose of pseudoephedrine (60 mg) produced a urinary concentration of more than 5 microg/mL for cathine and of more than 100 microg/mL for pseudoephedrine in 2 of 9 subjects only. When a dose of 120 mg was administered, cathine concentration exceeded 5 microg/mL in 4 of 7 subject, and also concentrations of pseudoephedrine above 100 microg/mL. After administration of 5 x 120 mg of pseudoephedrine (120 mg administered every 7 days for 5 weeks) to one of the subjects exceeding the urinary threshold values, the urinary concentration of cathine and pseudoephedrine exceeded 5 microg/mL (peak concentration 14.8 microg/mL) and 100 microg/mL (peak concentration 275 microg/mL), respectively. When the same subject took 180 mg of pseudoephedrine, the urinary concentration values were below 5 microg/mL for ephedrine and 100 microg/mL for pseudoephedrine. In the case of ephedrine administration in a sustained-release formulation containing 12 mg of ephedrine, 2 of 3 subjects exceeded the urinary cutoff value of 10 microg/mL. The high interindividual variability is still significant even if the urinary concentration values are adjusted by specific gravity and/or creatinine. These results confirm a high interindividual variability in the urinary concentration of ephedrines after the administration of the same therapeutic dose of a preparation.

Simultaneous determination of methylephedrine and pseudoephedrine in human urine by CE with electrochemiluminescence detection and its application to pharmacokeinetics.

A novel method for the determination of ephedra alkaloids (methylephedrine and pseudoephedrine) was developed by electrophoresis capillary (CE) separation and electrochemiluminesence detection (ECL). The use of ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate, BMIMBF(4)) improved the detection sensitivity markedly. The conditions for CE separation, ECL detection and effect of ionic liquid were investigated in detail. The two ephedra alkaloids with very similar structures were well separated and detected under the optimum conditions. The limits of detection (signal-to-noise ratio = 3) in standard solution were 1.8 x 10(-8) mol/L for methylephedrine (ME) and 9.2 x 10(-9) mol/L for pseudoephedrine (PSE). The limits of quantitation (signal-to-noise ratio = 10) in human urine samples were 2.6 x 10(-7) mol/L for ME and 3.6 x 10(-7 )mol/L for PSE. The recoveries of two alkaloids at three different concentration levels in human urine samples were between 81.7 and 105.0%. The proposed method was successfully applied to the determination of ME and PSE in human urine and the monitoring of pharmacokinetics for PSE. The proposed method has potential in therapeutic drug monitoring and clinical analysis.

Problems of the use of pseudoephedrine by athletes.

Pseudoephedrine (PSE) as a sympathomimetic is an ingredient of many proprietary medicines which are available on the medical market over the counter (OTC drugs). It can be converted to cathine (CATH, norpseudoephedrine) inside the body. Until the end of 2003, PSE had been a banned substance in sport in case its urinary concentration was greater than 25 mircog/ml. Then the World Anti-Doping Agency (WADA) removed PSE from the prohibited list. Prior to 2004 CATH was a forbidden substance and it is still one. CATH is included on the WADA prohibited list in the group of stimulants. The results of a doping control concerning PSE conducted in the Department of Anti-Doping Research of Institute of Sport in Warsaw in the years 2001-2003 and 2004-2007 have been compared. Moreover, several dozen of urine samples collected from the patients taking OTC drugs with PSE have been analysed. In these samples the concentration of PSE and CATH has been estimated. The results of this study have shown that athletes were using PSE frequently and in high doses between 2004 and 2007 when this substance was permitted by WADA. It is possible that athletes can obtain a positive result of doping control with CATH after the use of PSE.

Influences of urinary pH on the pharmacokinetics of three amphetamine-type stimulants using a new high-performance liquid chromatographic method.

A simple and sensitive high-performance liquid chromatographic (HPLC) method after precolumn derivatization with 9,10-anthraquinone-2-sulfonyl chloride (ASC) has been developed and validated for the analysis of amphetamine-type stimulants (ATS) in biological samples. Based on this method, we investigated the impact of urinary pH on the pharmacokinetics of phenylpropanolamine (PPA), pseudoephedrine (PSE), and fenfluramine (FEN) in rats. The drugs were orally administrated to rats, which had been induced, by repeated oral doses of ammonium chloride or sodium bicarbonate, to excrete urine at lower or higher pH than the normal value, respectively. Results revealed that as the increase of urinary pH, the mean elimination half-life (t(1/2)), the mean residence time (MRT) and the area under the plasma concentration-time curve (AUC) of PPA, PSE, and FEN were greatly raised, while the total plasma clearance (CL/F) decreased considerably. These findings have important clinical implications.

Interpretation of urinary concentrations of pseudoephedrine and its metabolite cathine in relation to doping control.

Until the end of 2003 a urinary concentration of pseudoephedrine exceeding 25 microg/mL was regarded as a doping violation by the World Anti-Doping Agency. Since its removal from the prohibited list in 2004 the number of urine samples in which pseudoephedrine was detected in our laboratory increased substantially. Analysis of 116 in-competition samples containing pseudoephedrine in 2007 and 2008, revealed that 66% of these samples had a concentration of pseudoephedrine above 25 microg/mL. This corresponded to 1.4% of all tested in competition samples in that period. In the period 2001-2003 only 0.18% of all analysed in competition samples contained more than 25 microg/mL. Statistical comparison of the two periods showed that after the removal of pseudoephedrine from the list its use increased significantly. Of the individual sports compared between the two periods, only cycling is shown to yield a significant increase.Analysis of excretion urine samples after administration of a therapeutic daily dose (240 mg pseudoephedrine) in one administration showed that the threshold of 25 microg/mL can be exceeded. The same samples were also analysed for cathine, which has currently a threshold of 5 microg/mL on the prohibited list. The maximum urinary concentration of cathine also exceeded the threshold for some volunteers. Comparison of the measured cathine and pseudoephedrine concentrations only indicated a poor correlation between them. Hence, cathine is not a good indicator to control pseudopehedrine intake. To control the (ab)use of ephedrines in sports it is recommended that WADA reintroduce a threshold for pseudoephedrine.