Quantification Of Ephedrine Research Articles

Magnetic dispersive solid phase microextraction technique coupled with LC–MS/MS for evaluating content versus label claims in ephedrine-free food supplements

Sport supplements and weight management supplements are prime targets for adulteration. In response to concerns regarding the safety of ephedra-containing food supplements, manufacturers have marketed “ephedra-free” products. Therefore, a rapid and convenient magnetic dispersive solid phase microextraction method is proposed in order to extract ephedrine from complex food supplements. Quantification of ephedrine is accomplished by ion-spray LC–MS/MS using multiple reaction monitoring (MRM) mode. The LC–MS/MS method is validated in the range of 5–100 ng/mg. The proposed procedure is applied to the determination of ephedrine in various food supplements. Sixteen food supplements including sport and weight loss supplements are provided from two local drugstores (Tabriz, Iran). The mean content of the ephedrine, identified in five products, amounted to 1055 ng/mg. The proposed method is reliable and fast for determination of ephedrine content in food supplements.

Trends in accumulation of ephedrine in callus cultures of Ephedra major Host

Ephedra major Host, a medicinal plant, belongs to the family of Ephedraceae. Ephedrine is the main alkaloid in Ephedra, which has different medicinal properties. However, the amount of ephedrine in plant material is low and callus culture can be a way to increase the alkaloid content. The aim of this research was to compare Murashige and Skoog (MS) and Gamborg’s B5 culture media for callus induction and ephedrine production. For this purpose, stem explants were cultured on MS or B5 media containing 0.0, 0.5, 1.0, 2.0, or 3.0 mg L−1 of kinetin (Kin) either alone or in combination with 0.0, 0.5, 1.0, or 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and/or naphthalenacetic acid (NAA), in five replicates. MS medium containing 1.0 or 2.0 NAA and 0.5 mg L−1 Kin were the most effective for callus induction. The highest percentage of callus induction (100%) on B5 culture medium was obtained with 2.0 2,4-D and 0.5 mg L−1 Kin treatments. The results showed that there was no significant difference between MS and B5 media for callus induction, and fresh and dry weight production. High-performance liquid chromatography was conducted for the identification and quantification of ephedrine in the obtained callus. The highest level of ephedrine (7.38 mg g−1 DW) was found in callus grown on MS medium containing 0.5 mg L−1 of 2,4-D. The results revealed that ephedrine can accumulate in callus cultures to levels much higher than in E. major wild plants.

Validated quantification method for five ephedrines in dietary supplements using LC-MS/MS: Application to 503 cases.

Ephedrines in dietary supplements can arise from herbs or illegal adulteration so a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for separation and quantification of ephedrine, pseudoephedrine, norephedrine, norepseudoephedrine, and methyl-ephedrine, some of which are isomer pairs of pseudo-structures. This method includes liquid-liquid extraction of ephedrines from three typical dietary supplement matrices-solid, liquid, and oil-as well as liquid chromatographic separation. After liquid chromatographic separation, ephedrines are qualitatively and quantitatively analyzed using triple quadrupole mass spectrometry with positive electrospray ionization in multi-reaction monitoring (MRM) mode. Ephedrine recoveries in a solid matrix ranged from 53.3-91.5%, in a liquid matrix from 56.4-102.3%, and in an oil matrix from 51.7-01.2%. Linearity ranges were 10-1000ng/g in solid and oil matrix and 1-100ng/ml in liquid matrix. Accuracy was -11.5-16.3%. Intra-day and inter-day variation are less than 5.9% and 7.3%, respectively. Expanded uncertainty of quantification is less than 0.123ng/g in a solid matrix, less than 0.139ng/ml in a liquid matrix, and less than 0.158ng/g in an oil matrix. Data collected for more than 500 routine samples are presented and discussed.

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.

Pharmacokinetic determination of ephedrine in Herba Ephedrae and Wu Tou Tang decoctions in rats using ultra performance liquid chromatography tandem mass spectrometry

A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and validated for the determination and quantification of ephedrine in rat plasma samples. An Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm) was used for chromatographic separation. Electrospray ionization in the positive mode was used, and the precursor-fragment ion pairs of m/z 166/148 and m/z 289/97 were adopted to characterize ephedrine and testosterone (internal standard), respectively. The method was validated using 10, 100 and 500 ng/mL of ephedrine. It demonstrated adequate levels of precision and accuracy, matrix effect, extraction recovery and stability. Linearity over the concentration range of 0.5–2000 ng/mL was acceptable with a correlation coefficient (r2) better than 0.990.To determine the pharmacokinetic behaviour of this sympathomimetic compound in the Sprague-Dawley rats, ephedrine hydrochloride, Herba Ephedrae single-herb and Wu Tou Tang decoctions were administered orally, and ephedrine hydrochloride was also administered by intravenous injection, and blood samples were collected over 24 h. Ephedrine was measured in plasma and pharmacokinetic parameters were determined by using the standard non-compartmental method and calculated by using Practical Pharmacokinetic Program-Version 87/97.The AUC(0–t) and Tmax values were significantly different (p < 0.05). Ephedrine AUC(0–t) values were significantly lower following the Wu Tou Tang decoction compared to the other oral treatments, suggesting that some components in the decoction may reduce the bioavailability of ephedrine

A surface plasmon resonance-based biochip to reveal traces of ephedrine

Ephedrine is a crucial chemical precursor for clandestinely produced amphetamine and its related drugs. Also, ephedrine is intentionally added to illegal preparations of methamphetamine or amphetamine because it is cheaper than the other two drugs. Consequently there is a pressing need to prevent the entry of ephedrine into commerce. Therefore the challenge calls for non-invasive techniques that provide data on the presence of ephedrine even in trace amounts. In this paper we describe the synthesis of a new ephedrine derivative with a carbon linker featuring an amino reactive group, and its conjugation to the glutamine binding protein (GlnBP) from E. coli as a carrier protein for the production of polyclonal antibodies against ephedrine. Proof-of-principle results of an efficient SPR-based indirect competitive immunoassay for the detection and quantification of ephedrine are presented. The detection limit of the assay was about 33 ng ml−1.

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.

Mono-Brominated-Propyphenazone as a New Derivatization Reagent for High Performance Liquid Chromatography of Ephedrine with UV-Detection

mono-brominated-propyphenazone was used as a derivatization reagent for the detection and quantification of ephedrine by high performance liquid chromatography using Zorbax-XDB C18 column, and 0.02 M sodium acetate (pH 4.4): acetonitrile (52:48 v/v) as mobile phase and UV detection at 280nm. The cited reagent reacts with the secondary amino group of ephedrine in acetone using anhydrous potassium carbonate as hydrobromide acceptor. The reaction was completed within 20 minutes in 100 µL acetone with heating at 90 ± 5°C in a capped mini reaction vial. The stoichiometry of the reaction was one-to-one mol from each mono-brominated-propyphenazone and ephedrine. The linear concentration range of ephedrine was 30 to 900 ng per injection (50 µL). The detection limit was 9 ng/injection (50 µL). The derivatized ephedrine was synthesized and confirmed with spectral analysis. This method was applied for determination of spiked ephedrine in human urine and after extraction with charcoal column using methanol: 0.2 M NaOH (20:0.2, mL:mL) as eluant.

Highly Sensitive Spectrofluorimetric Determination of Ephedrine Hydrochloride in Pharmaceutical Preparations

A sensitive and specific spectrofluorimetry method was developed and validated for the quantification of ephedrine (EP) in pharmaceutical preparations. The method is based on the fluorescent enhancing reaction of EP with 7-chloro-4-nitrobenzofurazan (NBD-C1; derivatization reagent), in borate buffer of pH 9 to yield a yellow, fluorescent product. Under these experimental conditions, the derivatized product of EP had excitation and emission wavelength maxima at 458 and 516 nm, respectively. The linear range of this method was 20-2500 ng/mL. The detection limit was 7.3 ng/mL EP. Intra- and interday precisions of the assay at 3 concentrations within this range were 0.037-1.77%. The low relative standard deviation values indicate good precision, and high recovery values indicate excellent accuracy of the method. The proposed method was applied to the determination of the examined drugs in pharmaceutical formulations, and the results indicate that the method is equally as accurate, precise, and reproducible as the official method.

Rapid simultaneous determination of ephedrines, amphetamines, cocaine, cocaine metabolites, and opiates in human urine by GC–MS

This paper presents a simple and sensitive chromatographic procedure for the simultaneous determination and quantification of ephedrines, amphetamines, cocaine, cocaine metabolites, and opiates in human urine by gas chromatography–mass spectrometry. This method involves enzyme hydrolysis in the presence of a deuterated internal standard, liquid–liquid extraction, and derivatization with pentafluoropropionic anhydride and pentafluoropropanol. The recovery of each compound averaged at 65.8% or more. The limits of detection determined for each compound by using a 2-mL sample volume ranged from 5 to 50 ng/mL. The calibration curves were linear to 1000 ng/mL for all compounds when determined using methamphetamine- d 4 and MDMA- d 5 as internal standards. This method was successfully applied for the analysis of urine samples suspected to contain intoxicants such as methamphetamine and heroin.

Doping control analysis in human urine by liquid chromatography–electrospray ionization ion trap mass spectrometry for the Olympic Games Athens 2004: Determination of corticosteroids and quantification of ephedrines, salbutamol and morphine

A new liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI–MS) ( n) ion trap method for the determination of corticosteroids in urine has been developed and validated. Some anabolic agents, such as epitrenbolone, trenbolone, 2-hydroxymethylformebolone, tetrahydrogestrinone, gestrinone and formoterol were included in the LC–ESI–MS method. Matrix interference, specificity, identification capability, carry over and robustness were estimated as validation parameters. Recoveries ranged from 74 to 113% at the minimum required performance limit (MRPL), which is 30 ng mL −1 for corticosteroids and 10 ng mL −1 for anabolic agents. Methods for the confirmation and quantification of norpseudoephedrine, ephedrine, methylephedrine, salbutamol, morphine and morphine glucuronide were also developed and validated and in order to minimize analysis time, direct urine injection was used. These methods proved to be specific, accurate and precise across a calibration range for each substance since matrix interference, specificity, carry over, within and between run precision, limit of detection, limit of quantification, intermediate precision and uncertainty were estimated.

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.

Determination of ephedrine alkaloid stereoisomers in dietary supplements by capillary electrophoresis

Three complementary capillary electrophoresis (CE) methods were developed for the separation and quantification of ephedrine and pseudoephedrine stereoisomers. Either single or dual cyclodextrin-based chiral selector systems provided enantioselective separation of the compounds of interest. The three methods were applied to the analysis of a suite of five standard reference materials (SRMs) containing ephedra. Use of a high-sensitivity UV detection cell enhanced quantification of the analytes of interest over the wide range of concentrations encountered in the SRMs. Results for (−)-ephedrine ranged from 0.31 to 76.43 mg/g, and for (+)-pseudoephedrine ranged from 0.049 to 9.23 mg/g in the materials studied. Results from the three methods agreed well with each other and with the results from other methods of analysis. The addition of known amounts of specific enantiomers was used to confirm the enantiomeric identity of the analytes. The results obtained by the three CE methods were utilized for value assignment of the ephedrine alkaloid content of these five SRMs.

Rapid and sensitive determination of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with on-line regenerating covalent coating.

A rapid, sensitive and reproducible micellar electrokinetic chromatographic method using hexamethyldisilazane as on-line regenerating covalent coating was developed for the quantification of ephedrine (E) and pseudoephedrine (PE). E and PE were derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol for laser-induced fluorescence detection. The on-line regenerating covalent coating formed a combinative double coating with the subsequently produced dynamic SDS coating. The total coating can be easily removed and conveniently regenerated on-line. The simple coating procedure was described. By a series of optimization, a running buffer of 20 mm Na(2)B(4)O(7) + 16 mm SDS was applied for the separation of the derivatives. Linear relationships for E and PE were obtained in the range of 0.044-6.60 microg mL(-1) (correlation coefficients: 0.9975 for E, 0.9981 for PE), and the detection limits for E and PE were 1.71 and 0.67 ng mL(-1), respectively. The separation speed, the reproducibility and the sensitivity were much improved over those of other capillary electrophoresis methods more recently reported. The method was applied to the analysis of the two alkaloids in traditional herbal preparations with recoveries in the range 92.8-104.8%.

Micellar electrokinetic chromatography with laser‐induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive micellar electrokinetic chromatography method with laser-induced fluorescence detection was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. After conducting a series of optimizations, a running buffer of 10 mM sodium borate + 16 mM SDS was used for separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.044-6.6 microg mL(-1) (correlation coefficient: 0.9943 for E, 0.9946 for PE), and the detection limits for E and PE were 0.70 and 0.30 ng mL(-1), respectively. The sensitivity of E and PE was improved by several multiples of ten over those of CZE-LIF method. The method was applied to the analysis of the two alkaloids in ephedra herbal medicine and preparations with recoveries in the range of 98.3-107.1%.

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.

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.