Method for derivatization of ephedrine and pseudoephedrine in nonaqueous media and determination by nonaqueous capillary electrophoresis with laser induced fluorescence detection

A nonaqueous capillary electrophoresis with laser-induced fluorescence detection method was developed for the quantification of ephedrine and pseudoephedrine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol in nonaqueous media. The derivatization was made in off-line mode. By a series of optimizations, a derivatization buffer composed of 40 mm ammonium acetate and 20% acetonitrile and a running buffer composed of 80 mm ammonium acetate and 3% acetic acid were applied for the derivatization and separation of ephedrine and pseudoephedrine, respectively. Linear relationships for ephedrine and pseudoephedrine were obtained in the range 1.23-19.60 mg/L (correlation coefficients 0.9970 for ephedrine and 0.9994 for pseudoephedrine), and the detection limits for ephedrine and pseudoephedrine were 0.014 and 0.011 mg/L, respectively. The method was applied to the analysis of ephedrine and pseudoephedrine in four preparations with recoveries in the range 93.9-105.1%.     

Micellar liquid chromatographic method for the determination of ephedrine and pseudoephedrine in human serum by direct inject of the sample with simple dilution

A micellar high-performance liquid chromatographic method was developed to simultaneously determine ephedrine and pseudoephedrine in human serum. The serum sample pretreatment was a simple dilution in a micellar solution, filtration, and direct injection, thus avoiding time-consuming and tedious steps. Hence, there is no need to use an internal standard. The serum samples were analyzed using a mobile phase containing 1.50?×?10^?1?mol/L sodium dodecyl sulfate and 0.02?mol/L sodium dihydrogen phosphate with 7.5% (v/v) 1-propanol at pH 3.0, running at 1.0?mL/min by an Inertsil C18 (150?×?4.6?mm, 5?µm) column at 30°C. The UV wavelength was set at 210?nm. The developed method was validated by linearity (r?>?0.9990) and intra- and inter-day precisions of ephedrine and pseudoephedrine (relative standard deviation; RSD%, 0.04–10.40, and RSD %, 0.30–10.25, respectively), LODs for ephedrine and for pseudoephedrine was 2.63 and 2.70?µg/mL, respectively; lower limit of quantification for ephedrine and for pseudoephedrine was 4.38 and 4.51?µg/mL, respectively. Finally, the proposed method was applied to investigate ephedrine and pseudoephedrine in real human serum samples after oral administration of Kechuanning Koufye including Ephedra herb. It is environmentally friendly, easy-to-handle, and feasible method for routine analysis in clinical laboratory.     

Determination of ephedrine and related compounds in pharmaceutical preparations by ion chromatography with direct conductivity detection

An ion chromatographic method with conductivity detection for the simultaneous determination of ephedrine, pseudoephedrine and norephedrine was developed. A mixture of 2.0 mmol/L HNO3 and 2% (v/v) acetonitrile was used as eluent. The three ephedrine-like compounds were separated and determined within 20 min. The linear ranges were 0.08-50 microg/mL for ephedrine, 0.08-40 microg/mL for pseudoephedrine and 0.06-40 microg/mL for norephedrine. The detection limits were 0.03 microg/mL for ephedrine and pseudoephedrine, and 0.02 microg/mL for norephedrine. The method has been applied successfully to the determination of these sympathomimetics in pharmaceutical preparations and in Ephedra herbs.     

中空纤维液-液-液微萃取/HPLC分析人尿液中麻黄碱及伪麻黄碱

建立了中空纤维液-液-液微萃取高效液相色谱对人尿液中的麻黄碱和伪麻黄碱进行纯化、分离、富集以及测定的方法。采用中空纤维三相微萃取装置,考察了影响萃取的因素,确定了萃取条件:中空纤维壁上的有机相为正辛醇,以50μL盐酸溶液(pH 2.0)为接受相,在室温下萃取60 min。该条件下麻黄碱和伪麻黄碱的富集倍数分别为180倍和220倍,两者的线性范围分别为0.01～5 mg/L和0.005～0.75 mg/L,相关系数(r)分别为0.998 2、0.997 8,定量下限分别为0.01、0.005 mg/L。该方法使用极少量的有机溶剂,便可有效地对尿样中麻黄碱和伪麻黄碱进行纯化、分离和富集,萃取效率高,可用于尿液中麻黄碱和伪麻黄碱的同时测定。     

甲基苯丙胺检测方法研究进展

通过调研和搜集国内外发表的重要文献,对毒品甲基苯丙胺的检测方法进行全面的研究分析,对于传统检测技术、电子微芯片竞争免疫法、局域表面等离子共振技术、有机场效应晶体管检测冰毒、电化学发光方法、快速傅立叶变换方波伏安法、无标记安培免疫、酶联免疫吸附等技术进行了详细论述,形成最新的研究进展,并介绍课题组检测甲基苯丙胺的最新研究成果,以期为检测人员和科研人员开展毒品甲基苯丙胺的检测时提供参考和借鉴,从而推动分析检测技术的不断更新与发展。     

Microemulsion electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive microemulsion electrokinetic chromatography with laser-induced fluorescence detection method was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. By a series of optimization, a running buffer composed of 20 mM borate + microemulsion (23.3 mM Sodium dodecyl sulfate/180.85 mM 1-butanol/16.4 mM n-heptane) +8% acetonitrile was applied for the separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.058-11.58 microg.mL(-1) (correlation coefficient: 0.9993 for E, 0.9995 for PE), and the detection limits for E and PE were 5.3 and 3.9 ng.mL(-1). The method was applied to the analysis of the two alkaloids in Chinese traditional herbal preparations with recoveries in the range of 96.9-105.4%.     

Determination of methamphetamine and amphetamine in abusers' plasma and hair samples with HPLC-FL

This paper describes highly sensitive HPLC methods for the determination of amphetamine (AP) and methamphetamine (MP) in abusers' plasma and hair samples. AP and MP were derivatized with the fluorescent reagent, DIB-Cl, to yield a highly fluorescent DIB-derivatives of AP and MP, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 and 430 nm, respectively. The separation was achieved on an ODS column with isocratic mobile phases composed of acetoniltrile and citrate buffer (55:45, v/v) for plasma samples and of acetonitrile-methanol-citrate buffer (45:20:37.5, v/v/v) for hair samples. The limits of detection were less than 0.87 ng/mL and 0.12 ng/mg in plasma and hair samples, respectively, for both AP and MP. The methods were then applied to the determination of MP and its metabolite AP in plasma obtained from two cases of illegally ingested MP and in one of the cases' hair received later. Case I was treated with dialysis; samples before and after dialysis were analyzed by the described method. After dialysis for 5 h, the total plasma levels of AP and MP decreased from 720 to 190 ng/mL. For case II, MP and AP levels were monitored for 3 days after digestion. Total plasma levels decreased from 57 ng/mL in the day of digestion to 11 ng/mL after 3 days. In hair samples, AP and MP could also be detected in very low concentrations.     

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%.     

A validated HPLC‐PDA method for identification and quantification of two bioactive alkaloids,ephedrine and cryptolepine,in different Sida species

A simple, rapid, accurate and reproducible reverse‐phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP‐18 X‐terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10–250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra‐day and inter‐day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species. Copyright © 2013 John Wiley & Sons, Ltd.     

离子对色谱法测定麻杏石甘汤中的麻黄碱和伪麻黄碱

麻杏石甘汤出自《伤寒论》,由麻黄、杏仁、甘草和石膏四味中药组成。麻黄主要含麻黄碱和伪麻黄碱,两者是麻杏石甘汤中的主要有效成分。对麻黄及其制剂中的麻黄碱和伪麻黄碱含量测定的方法主要有高效液相色谱法（HPLC）、高效毛细管电泳法和气相色谱-质谱联用法等。由于麻黄碱和伪麻黄碱结构相似,而且生物碱容易产生拖尾现象,采用反相高效液相色谱法一般难以使两者达到良好的分离。本文采用反相离子对HPLC使两者获得了良好的分离,并以此法对麻杏石甘汤中的麻黄碱和伪麻黄碱进行了测定。     

高效液相色谱法测定麻黄浸膏粉中麻黄类生物碱

采用高效液相色谱法分离了麻黄浸膏粉中去甲基麻黄碱、去甲基伪麻黄碱、麻黄碱、伪麻黄碱和甲基麻黄碱等 5种主要麻黄类生物碱。所用分离柱为HIQSILC18,流动相为含少量 (体积分数为 0 .2 % )甲醇的 0 .0 2mol L磷酸二氢钠溶液 (用HCl和三乙胺调至pH 3.2 )。 5种麻黄类生物碱在 2 5min内得到了有效分离、峰型对称且尖锐。     

Bufferized solvent extraction and HPLC fluorometric detection method for sarafloxacin in pig and chicken muscles

In this study, a method for the detection of sarafloxacin in pig and chicken muscles was developed using HPLC‐FLD as a regulatory residue technique. Good extraction efficiency was achieved using a mixture of 1% orthophosphoric acid–0.2 m MgCl₂ in water and acetonitrile as an extraction solvent, and n‐hexane partitioning and centrifugation for cleanup was used in the absence of dehydration. Specificity, linearity, detection and quantification limits, recovery, accuracy and precision were all validated, and all results were sufficient for the SARA regulatory residue method in pig and chicken muscles. The method developed and described herein was not only simple but also reliable, and was applied to market samples to determine their residue contents. Copyright © 2010 John Wiley & Sons, Ltd.     

Simple column‐switching HPLC method for determining levels of the antifungal agent micafungin in human plasma and application to patient samples

Micafungin is an echinocandin antifungal agent that is important for treating candidiasis in emergency and intensive care medicine; however, current methods for measuring micafungin plasma levels are lengthy and complicated. We report a simple quantitative method using column‐switching high‐performance liquid chromatography (HPLC) for determining micafungin in human plasma samples. Human plasma was directly injected into a column‐switching HPLC system with a MAYI‐ODS pre‐column to remove the plasma matrix. The calibration curve for micafungin showed good linearity in the range 0.1–10 µg/mL in human plasma. The mean relative standard deviation value of the intra‐day and inter‐day precision was less than 7.3%. More than 450 successive, accurate measurements were made before the system had to be washed with ammonium acetate solution. The therapeutic micafungin level in patients' plasma was successfully measured using this method. Because the pretreatment is simple and reproducible, our method can be used for routine therapeutic monitoring. Copyright © 2012 John Wiley & Sons, Ltd.     

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, BMIMBF₄) 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 × 10^–8 mol/L for methylephedrine (ME) and 9.2 × 10^–9 mol/L for pseudoephedrine (PSE). The limits of quantitation (signal‐to‐noise ratio = 10) in human urine samples were 2.6 × 10^?7 mol/L for ME and 3.6 × 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. Copyright © 2009 John Wiley & Sons, Ltd.     

Simple method for determination of five terpenoids from different parts of Tripterygium wilfordii and its preparations by HPLC coupled with evaporative light scattering detection

By optimizing the extraction, separation, and analytical conditions, a reliable and accurate high-performance liquid chromatography method coupled with evaporative light scattering detection (ELSD) was developed for simultaneous determination of five terpenoids, i.e., triptolide, tripchlorolide, demethylzelastral, wilforlide B, and wilforlide A, in root, stem, leaves, root bark, twig, and root without bark of Tripterygium wilfordii Hook. f and six of its herbal preparations. This approach would thus provide a more accurate and general method for evaluating the quality of the herb and its preparations. Separation of these five terpenoids was achieved on a ZORBAX Eclipse XDB-C8 column with gradient elution using water and acetonitrile as solvents, both containing 0.05% formic acid, at a temperature of 30 degrees C and a flow rate of 0.8 mL/min. The drift tube temperature of ELSD was set at 100 degrees C, and the nitrogen flow rate at 1.5 L/min. Good linear relationships were obtained with correlation coefficients for the analytes exceeding 0.992, and the LOD and LOQ were less than 0.149 microg and 0.297 microg on column, respectively. Intra-day and inter-day precision of the analytes were less than 1.25% and 5.97%, respectively, and the average recovery rates obtained were in the range of 95.9 +/- 3.7% to 100.4 +/- 5.0% for all terpenoids with RSDs below 4.99%. Quantitative analysis of the five terpenoids in different parts of Tripterygium wilfordii and its six preparations showed that the contents of the terpenoids varied significantly. The tender root contained higher concentrations of triptolide, tripchlorolide, demethylzelastral, and wilforlide B than any other part of the herb. Correspondingly, the root bark contained the greatest concentration of wilforlide A, and the stem and twig came in second and third. This suggested that we could infer whether the medicinal materials were absolute roots without bark or not from the comparative contents of these terpenoids in the tablets in view of the fact that only the roots without bark are the valid officinal part of the plant. This method and the quantitation results obtained can provide a scientific and general as well as simple and convenient approach for the product manufacturers to set up quality control standards and for informing the public about the quality and safety of the preparations.     

Application of a rapid and selective method for the simultaneous determination of carebastine and pseudoephedrine in human plasma by liquid chromatography–electrospray mass spectrometry for bioequivalence study in Korean subjects

We describe a simple, rapid and sensitive high‐performance liquid chromatography–electrospray ionization tandem mass spectrometric method that was developed for the simultaneous determination of carebastine and pseudoephedrine in human plasma using cisapride as an internal standard. Acquisition was performed in multiple‐reaction monitoring mode by monitoring the transitions: m/z 500.43 > 167.09 for carebastine and m/z 166.04 > 147.88 for pseudoephedrine. The devised method involves a simple single‐step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a C₁₈ reversed‐phase chromatographic column at 0.2 mL/min by isocratic elution with 10 mM ammonium formate buffer–acetonitrile (30:70, v/v; adjusted to pH 3.3 with formic acid). The devised method was validated over 0.5–100 ng/mL of carebastine and 5–1000 ng/mL of pseudoephedrine with acceptable accuracy and precision, and was successfully applied to a bioequivalence study involving a single oral dose (10 mg of ebastine plus 120 mg of pseudoephedrine complex) to healthy Korean volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7‐hydroxymethotrexate (7‐OH‐MTX) and 2,4‐diamino‐N^10‐methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7‐OH‐MTX, DAMPA and 2,4‐diaminopteroic acid (DAPA) as an internal standard were separated on a reversed‐phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7‐OH‐MTX and DAMPA from plasma at 100 pmol/mL were 81.5 ± 5.4, 82.5 ± 5.3 and 56.2 ± 7.0%, respectively. The limits of quantification for MTX, 7‐OH‐MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within‐ and between‐day variations for MTX, 7‐OH‐MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy. Copyright © 2011 John Wiley & Sons, Ltd.     

High-performance liquid chromatography quantitative analysis of ephedrine alkaloids in Ephedrae Herba on a perfluorooctyl stationary phase

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15 mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R² > 0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87–110.99%), limits of detection (5.35–5.76 µg/mL), and limits of quantification (20 µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents of three batches of Ephedrae Herba from three pharmaceutical companies.     

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.     

Simultaneous determination of chlorpheniramine and pseudoephedrine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific procedure for simultaneous quantitation of chlorpheniramine and pseudoephedrine in human plasma has been developed and validated. Analytes were extracted from plasma samples by liquid-liquid extraction, separated on a Diamonsil C18 column (250 x 4.6 mm i.d.) and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Diphenhydramine was used as the internal standard. The method has a lower limit of quantitation of 0.2 and 2.0 ng/mL for chlorpheniramine and pseudoephedrine, respectively. The intra- and inter-day relative standard deviation, calculated from quality control (QC) samples were below 4.3% for chlorpheniramine and below 9.5% for pseudoephedrine. The inter-day relative error as determined from QC samples was within 4.7% for each analyte. The overall extraction recoveries of chlorpheniramine and pseudoephedrine were 77 and 61% on average, respectively. The method was successfully applied to pharmaockinetic study of chlorpheniramine and pseudoephedrine in volunteers receiving formulations containing 4 mg of chlorpheniramine maleate and 60 mg of pseudoephedrine hydrochloride.