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%.     

Determination of diastereoisomeric alkaloids in urine by capillary electrophoresis with electrochemiluminescence detection

A new and simple capillary electrophoresis with electrochemiluminescence detection was developed for the separation and the quantification of a pair of diastereoisomenc alkaloids（ephedrine and pseudoephedrine）.The limits of detection（S/N = 3） were 4.5×10^-8 mol/L for ephedrine and 5.2×10^-8 mol/L for pseudoephedrine,respectively.The RSDs of migration time and peak area were less than 1.3 and 2.5%（n = 5）,respectively.The applicability of the propose method was illustrated in the determination of ephedrine and pseudoephedrine in human urine,ephedrine in nasal drops,and the monitoring of pharmacokinetics for pseudoephedrine.     

毛细管电泳/发光二极管诱导荧光法测定麻黄中麻黄碱与伪麻黄碱含量

基于麻黄碱及伪麻黄碱衍生物的光谱及化学性质,设计并构建了毛细管电泳/发光二极管诱导荧光检测系统.对关键光学元件进行组合选择,以蓝光发光二极管为光源,BP 470和BP 530分别为光源滤光片和荧光滤光片,光电倍增管检测信号,并对电泳分离系统的缓冲溶液、分离电压等参数进行优化;以FITC为衍生试剂,10 mmol/L Na2B4O7+ 16 mmol/L SDS为缓冲溶液,12 kV电压下可实现麻黄中麻黄碱和伪麻黄碱的基线分离.在0.25～10 mg/L范围内,麻黄碱和伪麻黄碱标准溶液的质量浓度与荧光响应的峰高之间呈较好的线性关系,相关系数(r)均大于0.99,其检出限分别为0.38 μg/L和0.29 μg/L,峰高的日内重复性(RSD)分别为2.0％和2.2％,日间重复性(RSD)分别为5.4％和5.1％.将该方法用于中药麻黄中麻黄碱和伪麻黄碱的测定,加标回收率分别为94％和107％.     

Chiral determination of amphetamine and related compounds using chloroformates for derivatization and high-performance liquid chromatography

The enantiomeric determination of amphetamine and various amphetamine-type compounds by liquid chromatography after chiral derivatization with 9-fluorenylmethyl chloroformate-L-proline (FMOC-L-Pro) is reported. The results obtained were compared with those achieved after achiral derivatization with 9-fluorenylmethyl chloroformate and subsequent separation of the derivatives on a beta-cyclodextrin chiral stationary phase. Conditions for the derivatization of amphetamines with FMOC-L-Pro were investigated, including the effect of the derivatization reagent concentration, pH and reaction time, using amphetamine, ephedrine and pseudoephedrine as model compounds. On the basis of these studies, possible conditions for the determination of each amphetamine are indicated. To demonstrate the utility of the proposed procedures, data on linearity, repeatability and sensitivity are given. Results of the determination of ephedrine enantiomers in different pharmaceutical samples are also presented.     

Rapid and ultrasensitive determination of ephedrine and pseudoephedrine derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper presents a micellar electrokinetic chromatography method with laser-induced fluorescence detection to analyze ephedrine (E) and pseudoephedrine (PE) after derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein. The optimum derivatization conditions were: 0.05 M Na2CO(3/NaHCO3 (pH 9.5), reaction time 30 min at 45 degrees C, molar ratio of DTAF to E and PE mixture 20:1. The baseline separation was achieved within 8 min with running buffer composed of 20 mM borate+20 mM SDS+15% acetonitrile (v/v) (adjusted pH 9.8), and applied voltage of 20 kV. Good linearity relationships (correlation coefficients: 0.9906 for E and 0.9941 for PE) between the peak heights and concentration of the analytes were obtained (2.5-50 ngmL(-1)). The detection limits for E and PE were 3.85 x 10(-4) and 1.41 x 10(-4)ngmL(-1), respectively, which indicated that the proposed method surpassed other chromatographic alternatives in terms of limit of detection by at least 10(3) folds. The method was applied to the analysis of the two alkaloids in ephedra herb plants and its preparations with recoveries in the range of 89.6-107.0%.     

In-capillary derivatization and analysis of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5 kV, 15s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1 min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL(-1) for E and 1.6 ng mL(-1) for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.     

高效毛细管电泳分离/电导检测麻黄碱和伪麻黄碱

采用高效毛细管电泳电导检测法分离麻黄碱和伪麻黄碱，初步探讨了分离机理，建立了检测方法。以柠檬酸－柠檬酸钠为缓冲体系，铜盐为络合剂，在pH值为4．5、电压13.5kV的条件下，盐酸麻黄碱和伪麻黄碱得到了较好的分离，加入适量乙醇可改善峰形和分离效果。用该法以水杨酰胺为内标，对含盐酸麻黄碱和伪麻黄碱的实际样品进行检测，回收率为97.3%-101.1%，结果令人满意。     

基于中空纤维液相微萃取的麻黄碱和伪麻黄碱优势构象的确定及含量测定

利用中空纤维液相微萃取方法(HF-LPME)分析麻黄碱和伪麻黄碱在不同基质中的优势构象,阐明了麻黄碱和伪麻黄碱的萃取机理;结合高效液相色谱(HPLC)建立了微量麻黄碱和伪麻黄碱的分离测定方法。以聚偏氟乙烯中空纤维为有机溶剂载体,正己醇为萃取溶剂,麻黄碱和伪麻黄碱的NaOH(5 mol/L)溶液为样品相,0.01 mol/L H2SO4溶液为接收相,在1200 r/min转速下萃取35 min,收集萃取液直接进行HPLC分析。麻黄碱和伪麻黄碱在水溶液中的线性范围为5～100 μg/L,检出限分别为1.9 μg/L和1.2 μg/L,富集倍数分别为38和61倍,平均回收率分别为100.6%±1.2%和103.2%±3.5%;在鼠尿液中的线性范围为100～5×104 μg/L,检出限分别为30 μg/L和42 μg/L,富集倍数分别为20和17倍,平均回收率分别为108.4%±4.4%和106.1%±5.4%。研究表明该方法操作简单,选择性高,适用于微量麻黄碱的含量测定和分析。     

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high-purity methamphetamine

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d‐methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d‐methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C₁₈ MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH₂PO₄–acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d‐methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high‐purity d‐methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high‐purity methamphetamine profiling. Copyright © 2011 John Wiley & Sons, Ltd.     

Separation and determination of ephedrine and pseudoephedrine in Ephedrae Herba by CZE modified with a Cu(II)-L-lysine complex

A CZE method using a complex of 2.5 mM Cu(II)-L-lysine (molar ratio is 1:2) as additive in a run buffer solution composed of Tris-H(3)PO(4) (pH 4.5) was developed for the simultaneous determination of ephedrine and pseudoephedrine within 4 min. The effects of pH, composition, and concentration of run buffer as well as the composition and concentration of the Cu(II)-L-lysine complex on the separation were investigated. The linear ranges for the determination of ephedrine and pseudoephedrine were 15.0-225.0 and 20.0-250.0 mg/L with LODs both of 5.0 mg/L. Satisfactory result for the determination of ephedrine and pseudoephedrine in Ephedrae Herba from different producing area was obtained by the proposed method. Ephedrine and pseudoephedrine were separated effectively with each other and with the other compounds in the sample. The RSD for the determination of the two constituents in the samples varied from 1.82 to 2.76%, and the recovery ranged between 95.0 and 104.0%.     

Quantification of glutathione and glutathione disulfide in human plasma and tobacco leaves by capillary electrophoresis with laser-induced fluorescence detection

A new capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the rapid separation and sensitive detection of glutathione (GSH) and glutathione disulfide (GSSH) after derivatization by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl). The derivatization and separation conditions were investigated in detail and the optimums were obtained. Under the optimum experiment conditions, linear relationships between the peak height and concentrations of the analytes in normal and second-derivative electrophoregrams were obtained (0.22-45.00 μM). The detection limits for glutathione and glutathione disulfide in normal and second-derivative electrophoregrams were 0.046 and 0.012 μM and 0.046 and 0.014 μM, respectively. The method was applied to the analysis of glutathione and glutathione disulfide in human plasma and tobacco leaves with satisfactory results.     

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。     

Separation and determination of ephedrine and pseudoephedrine by combination of flow injection with capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of ephedrine and pseudoephedrine using direct UV absorbance detection has been developed by the combination of flow injection with capillary electrophoresis for the first time. The buffer solution used is a 40 mM borate solution with the pH adjusted to 9.5 using a 2 M NaOH solution. The linear calibration range is 50 to 1000 microg/mL (r = 0.9996) for both analytes, and the recoveries are 91.2-108.2% for ephedrine and 92.6-107.3% for pseudoephedrine, respectively. The relative standard deviation of the peak area is 1.6% for ephedrine and 1.3% for pseudoephedrine (n = 6) at a concentration of 500 microg/mL, respectively. A series of samples is injected repeatedly without current interruption and subsequent rinsing, and the contents of these two alkaloids in three marketed drugs and the medical plant, Ephedra sinica, are determined with satisfactory results by this method.     

Quantitation of oxidized and reduced glutathione in plasma by micellar electrokinetic capillary electrophoresis.

A method for the separation of reduced (GSH) and oxidized (GSSG) glutathione was optimized in terms of buffer concentration, sodium dodecyl sulfate concentration, buffer pH, detection wavelength, run voltage and injection volume. The method demonstrated good linearity (r2 > 0.999) and reproducibility (internal standard corrected peak area RSD < 2.3%) in the range of interest (16-81 microM GSH and 8-40 microM GSSG). A detection limit of less than 1 microM GSH and GSSG was obtained using a high sensitivity flow cell. When the optimized method was applied to plasma samples, concentrations of 1.6 microM GSH and 0.8 microM GSSG were easily detected without the need for derivatization. The on-capillary detection was calculated to be 38.6 fmol of GSH and 18.3 fmol of GSSG.     

芯片毛细管电泳激光诱导荧光快速分离检测麻黄碱类兴奋剂

研究用芯片毛细管电泳激光诱导荧光检测系统分离测定经7-chloro-4-n itrobenzo-2-oax-1,3-d iazole(NBD-C l)衍生的麻黄碱和伪麻黄碱的实验条件。采用胶束毛细管电动色谱分离体系(12 mmol/L SDS 10mmol/L硼砂缓冲液,pH 9.0),在45 mm长的通道上实现了麻黄碱和伪麻黄碱的快速分离,一次分离小于1.5m in。10~100 mg/L范围内,峰高与浓度呈良好的线性关系,麻黄碱、伪麻黄碱的检出限分别是0.83 mg/L和1.10 mg/L。所建立的方法应用于尿中麻黄碱和伪麻黄碱的分离测定,取得满意的结果。     

Head-column field-amplified sample stacking in a capillary electrophoresis-flow injection system

A simple, effective, and continuous online concentration method for the sensitive detection of alkaloids applying CE-flow injection analysis with head-column field-amplified sample stacking was developed. A series of samples was continuously introduced into the capillary by electrokinetic means without interrupting the high voltage. A short water plug was introduced by the EOF at the capillary inlet end prior to sample introduction. Under optimum conditions, 15-fold improvement in concentration sensitivity was achieved, giving an LOD of about 0.67 and 0.73 microg/mL for ephedrine (E) and pseudoephedrine (PE), respectively. The separation could be achieved within 4 min and sample throughput rate could reach up to 7/h. The repeatability (defined as RSD) was 3.62, 1.51% with peak area evaluation and 1.30, 2.58% with peak height evaluation for E and PE, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing E and PE, and the recoveries were 92.3-102.4%.     

Separation of the enantiomers of primary and secondary amphetamines by liquid chromatography after derivatization with (−)-1-(9-fluorenyl)ethyl chloroformate

Summary The chiral reagent (−)-1-(9-fluorenyl)ethyl chloroformate (FLEC) has been evaluated for the enantioselective analysis of amphetamines by liquid chromatography. For separation of the FLEC diastereomers conventional reversed-phase conditions were used. The conditions affording the best enantiomeric resolution and sensitivity were determined for amphetamine, methamphetamine, ephedrine, pseudoephedrine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDE). All the amphetamines assayed could be separated with resolution factors ranging from 0.91 to 1.92. Although FLEC is typically used as a fluorogenic reagent, it was shown that UV detection is the best option for stereospecific analysis of the methylenedioxylated amphetamine derivatives (MDA, MDMA, and MDE), because different fluorimetric responses were obtained for the corresponding diastereomers. The utility of the proposed conditions for stereoselective analysis of amphetamines in different types of sample is discussed.     

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.     

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%.     

Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane

A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.