Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

Determination of amino acids and catecholamines derivatized with 3-(4-chlorobenzoyl)-2-quinolinecarboxaldehyde in PC12 and HEK293 cells by capillary electrophoresis with laser-induced fluorescence detection

An effective micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF) method has been proposed for the separation and the determination of 16 amino acids and two catecholamines using a new fluorogenic reagent, 3-(4-chlorobenzoyl)-2-quinolinecarboxaldehyde (Cl-BQCA), as the derivatizing reagent. The highest derivatization efficiency was achieved in pH 8.0 borate buffer at 50 °C for 50 min. The optimal separation of Cl-BQCA-labeled amines was obtained with a running buffer (pH 9.15) containing 120 mM boric acid, 38.5 mM sodium dodecyl sulfate, and 17% acetonitrile. The detection limit (S/N = 3) was found to be as low as 1.4 nM. The present method has been successfully used to detect amino acids and catecholamines in HEK293 and PC12 cell samples. This study explores the potential of MEKC-LIF with Cl-BQCA labeling as a tool for monitoring amino acids and catecholamines during the complex physiological and behavioral processes in various matrices.     

Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method based on chemical derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20 °C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concentration, buffer solution, reaction temperature and reaction time. Separation of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liquid chromatography (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quantitative analysis of biogenic amines involved in various matrices.     

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein as a new fluorescent labeling reagent for aliphatic amines in environmental and food samples using high-performance liquid chromatography

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein (SAMF), a new fluorescein-based amine-reactive fluorescent probe was well designed, synthesized and used as a pre-column derivatizing reagent for the determination of aliphatic amines in HPLC. It exhibited relatively pH-independent fluorescence (pH 4-9) and excellent photostability. The derivatization was performed at room temperature in 6min. On a C18 column, the derivatives of SAMF with eight aliphatic amines were baseline separated in 28 min with a mobile phase of methanol-water (57:43, v/v) containing 10 mmol l(-1) pH 5.0, H3Cit3-NaOH buffer. With fluorescent detection at lambda(ex)/lambda(em) = 484/516 nm, the detection limit could reach 2-320 fmol (signal-to-noise = 3), which was equivalent to or better than the detection limits obtained from other analytical methods of aliphatic amines. The proposed method has been applied to the determination of the aliphatic amines in environmental and food samples such as lake water, red wine, white wine, and cheese with satisfying recoveries varying from 95 to 106%.     

Sensitive determination of biogenic amines by capillary electrophoresis with a new fluorogenic reagent 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

An effective approach was proposed to the derivatization of seven biogenic amines using 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA) as a fluorogenic reagent. The sensitive determinations of these derivatives were achieved by micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection. The derivatization and electrophoretic conditions have been optimized. A running buffer was composed of mixtures of 25 mM pH 9.5 boric acid, 25 mM SDS, and 27% ACN. At 25 °C and 22.5 kV, the baseline separation of the derivatives was accomplished in 13 min. The detection limit (S/N = 3) was found as low as 0.4 nM. The proposed method was validated by the linearity of two orders magnitude and correlation coefficient in the range 0.9969–0.9998. Also, the procedure was successfully applied to the determination of biogenic amines in soy sauce, fish and wine samples.     

Determination of primary and secondary aliphatic amines with high performance liquid chromatography based on the derivatization using 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene

In this article, the simultaneous determination of primary and secondary aliphatic amines including dimethylamine (DMA), diethylamine and eleven primary aliphatic amines by high performance liquid chromatography (HPLC) with fluorescence detection has been achieved using a BODIPY-based fluorescent derivatization reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su). The derivatization reaction of TMBB-Su with aliphatic amines was optimized with orthogonal design experiment and the derivatization reaction proceeded at 15 °C for 25 min. The baseline separation of these derivatives was carried out on a C₈ column with methanol-tetrahydrofuran-50 mM pH 6.50 HAc-NaAc buffer (55/5/40, v/v/v) as a mobile phase. Detected at the excitation and emission of 490 and 510 nm, respectively, the detection limits were obtained in the range of 0.01-0.04 nM (signal-to-noise ratio = 3). The proposed method has been applied to the determination of trace aliphatic amines in viscera samples from mice without complex pretreatment or enrichment method. The recoveries ranged from 95.1% to 106.8%, depending on the samples investigated.     

Determination of Phosphoamino Acids by 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein and MEKC with LIF Detection

The fluorescent reagent 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein (SAMF), has been newly synthesized for use as a label for characterization of phosphoamino acids by micellar electrokinetic capillary electrophoresis (MEKC) with laser-induced fluorescence (LIF) detection. The conditions for derivatization and separation of the phosphoamino acids, including pH, concentration of electrolyte, and Brij-35 concentration were optimized in detail. Derivatization was performed at 35 °C for 10min in borate buffer (pH 8.0). The derivatives were separated to baseline by use of running buffer containing 50 mM borate and 20 mM Brij-35 at pH 9.3. Detection limits ranged from 5 × 10^?11 to 1 × 10^?9 mol L^?1 (signal to noise ratio = 3). The method was used for characterization of the phosphoamino acids in a sample from hydrolysis of a novel protein kinase from tobacco cells.     

Determination of primary biogenic amines in various food products using isocratic HPLC with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde as a new precolumn fluorogenic reagent

A simple HPLC approach has been successfully established for the sensitive determination of six biogenic amines (BAs) in food samples. The method involves derivatization with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde newly synthesized as a new fluorogenic reagent followed by LC isocratic elution mode. The optimization of both derivatization and separation conditions is carefully studied. Related analyses of the eluted compounds, in the presence of MeOH/THF/H(2)O (78:2.5:19.5 v/v/v) as a mobile phase containing 8 mM, pH 6.0 phosphate buffer solution, have been carried out on a C(18) column. The LOD (S/N = 3) of 2.5 nM, RSD value from 1.0 to 5.1% in peak areas, and good response linearity (R(2) >0.9936) are provided with fluorescence detection at lambda(ex)/lambda(em) = 480/545 nm. Obviously, recovery ranging from 95 to 107% in this method demonstrates its accuracy for determination of histamine, tyramine, 2-phenylethylamine, putrescine, cadaverine, and spermidine in the storage fish sample. Thus, the present method could be developed to monitor BAs in fish, cucumber, and spinach samples.     

毛细管电色谱-激光诱导荧光检测法分析食品中的生物胺

以4-氟-7-硝基-2,1,3-苯并氧杂恶二唑(NBD-F)为衍生化试剂,建立了食品中5种痕量生物胺(色胺、组胺、酪胺、亚精胺、精胺)的毛细管电色谱-激光诱导荧光检测(CEC-LIF)分析方法。采用50 mmol/L硼酸盐缓冲溶液(pH 8.0)作为衍生介质,在75℃条件下对生物胺进行衍生化反应25 min。生物胺衍生产物的最优色谱条件:固定相为C18毛细管电色谱柱,流动相为乙腈-乙酸铵(20 mmol/L,pH 8.0)(75∶25,v/v),辅助压力为6.9 MPa,分离电压为-8 kV,流速为0.03 mL/min。实验结果表明,生物胺的检出限(LOD,S/N=3)为0.1~1.0μg/L,加标回收率为78.3%~113.9%。该方法可成功用于加工和发酵食品中生物胺的测定,结果与传统HPLC法的检测结果无显著性差异,且检出限更低、分析速度更快,对于食品中痕量污染物的残留监测具有应用价值。     

Poly(dimethylsiloxane) microchip for precolumn reaction and micellar electrokinetic chromatography of biogenic amines

We have demonstrated that precolumn derivatization and capillary electrophoresis separation on a poly(dimethylsiloxane) (PDMS) microchip can be realized as efficient as those on glass microchips. In an optimized condition of micellar electrokinetic chromatography (MEKC), using 25 mM sodium borate buffer (pH 10.0) with 25 mM sodium dodecyl sulfate (SDS) and 5% v/v methanol, the electroosmotic flow in an oxidized PDMS microchip is stabilized within 3% for days. By employing a fluorometric derivatization with o-phthaldialdehyde (OPA) in an optimally designed reaction chamber, four most important biogenic amines occurring in foods, histamine, tyramine, putrescine, and tryptamine, are quantitatively determined in less than 1 min at the levels applicable to real samples. The migration behaviors of anionic OPA-derivatized biogenic amines under the MEKC conditions are analyzed, and it has been found that under our separation conditions, the electrophoretic mobility of the SDS micelles is significantly greater than those of the anions in the aqueous phase. The channel manifold in a PDMS substrate is fabricated using replica molding against a thick photoresist, SU-8, pattern generated by photolithography. The plate with the microchannel pattern is strongly, irreversibly bonded to another PDMS plate by using a new bonding technique, which employs surface oxidation by corona discharge generated from a cheap, handy source, Tesla coil.     

Analysis of amino acid neurotransmitters by capillary electrophoresis and laser-induced fluorescence using a new fluorescein-derived label

A capillary electrophoresis laser-induced fluorescence detection method (CE-LIF) was developed for the separation of eight neurotransmitters tagged on their amino function with 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein (SAMF), a new fluorescent reagent synthesized in our lab. Derivatization was performed in boric acid buffer (pH = 7.75) at 37 °C over 15 min. The pH-independent fluorescence of SAMF (pH 4–9) permits background buffers over a wide range of pH. It was demonstrated that an acidic running buffer offers a better resolution compared to basic medium in terms of resolution and peak shapes. Employing Cu²⁺ as the additive, the molecules were baseline-separated using a running buffer consisting of 40 mM sodium acetate and 2 mM Cu²⁺ (pH 6.0). The detection limits ranged from 1 to 2 × 10⁻¹⁰ M. The method has been validated for the characterization of lymphocyte samples. The results obtained illustrate the advantages of combining SAMF derivatization with CE-LIF for determining neurotransmitters.     

Urine polyamines determination using dansyl chloride derivatization in solid-phase extraction cartridges and HPLC

The derivatization of biogenic amines such as putrescine, cadaverine, spermidine and spermine with dansyl chloride in solid phase extraction cartridges is described. Different types of filling materials were tested in order to have the highest retention of the different analytes. The best results were obtained by using C18 cartridges. The optimal conditions were: amine solution buffered at pH 12, 2 mM dansyl chloride (acetone-bicarbonate solution 20 mM (pH 9-9.5), 2 + 3 v/v) as reagent concentration, room temperature and 30 min reaction time. The developed procedure was applied to the determination of these polyamines in urine samples from healthy controls and cancer patients using HPLC with 1,7-diaminoheptane as internal standard. The concentrations ranged from 0.5 to 5 micrograms mL-1 and the detection limits were 10 ng mL-1 for all polyamines. By concentrating the urine extracts, the detection limits were improved down to 2 ng mL-1. The accuracy and the precision of the method were tested. The proposed dansylation method is advantageous with respect to solution dansylation. It improves the total analysis time, avoids high temperatures that can affect the thermal stability of the derivatives and could make possible the automation of the procedure.     

4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin-2-yl)benzoic acid N-hydroxysuccinimide ester as a highly sensitive chemiluminescence derivatization reagent for amines in liquid chromatography

4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin-2-yl)benzoic acid N-hydroxysuccinimide ester was synthesized as a highly sensitive and selective chemiluminescence derivatization reagent for primary and secondary amines in liquid chromatography. Methyl-n-octylamine, n-nonylamine and n-decylamine were used as model compounds to optimize the derivatization, separation and chemiluminescence reaction conditions. This reagent reacts selectively with amines in the presence of triethylamine to give the highly chemiluminescent derivatives, which produce chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The chemiluminescent derivatives of the three amines can be separated within 20 min by reversed-phase liquid chromatography with isocratic elution, followed by chemiluminescence detection. The detection limits (signal-to-noise ratio=3) for primary and secondary amines are at sub-fmol levels for a 20-microl injection. Furthermore, this method was applicable to the determination of amantadine in human plasma.     

R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.     

Determination of catecholamines and serotonin by micellar electrokinetic chromatography with laser-induced fluorescence detection

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein, a new synthesized fluorescent reagent, was established for the first time as a label for the sensitive analysis of catecholamines (CAs) and serotonin (5-HT) by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. After careful study on the derivatization conditions such as pH value, reagent concentration, temperature and reaction time, the labeling reaction was accomplished as quickly as 7 min with stable yield. The separation parameters for the CAs and 5-HT were also optimized in detail. The derivatives were baseline separated in a running buffer containing 30 mM boric acid and 15 mM sodium dodeculsulfate at pH 9.0. The detection limits ranged from 5 x 10(-10) to 2 x 10(-9) M (signal-to-noise ratio = 3). The rapid and sensitive method was also applied to the determination of the CAs and 5-HT of urine samples.     

Chiral discrimination of primary amines by HPLC after labeling with a chiral derivatization reagent, trans-2-(2,3-anthracenedicarboximido)-cyclohexanecarbonyl chloride

Enantiomeric discrimination of chiral primary amines was performed by both reversed-phase HPLC and normal-phase HPLC after labeling with a chiral fluorescent derivatization reagent, (1R,2R)- and (1S,2S)-trans-2-(2,3-anthracenedicarboximido)cyclohexanecarbonyl chloride. Use of HPLC permits separation of diastereomeric derivatives of amines up to C30 which have a primary amino group at the middle of the alkyl chain. The derivatives of primary amines having an anteiso alkyl chain, which has a chiral branched-methyl at the n-3 position of the alkyl chain, were also separated by HPLC, and it was also possible to separate niphatesine D by reversed-phase HPLC after derivatization.     

Validation of a method for the analysis of biogenic amines: Histamine instability during wine sample storage

This paper reports on the development of an optimized method for the simultaneous analysis of eight biogenic amines (histamine, methylamine, ethylamine, tyramine, putrescine, cadaverine, phenethylamine, and isoamylamine). The analytical method thus proposed has the following advantages: the easy derivatization of wine, the quantification of biogenic amines and a complete degradation of excess derivatization reagent during sample preparation in order to preserve the column. It consists in reversed phase separation by HPLC and UV–vis detection of the aminoenones formed by the reaction of amino compounds with the derivatization reagent diethyl ethoxymethylenemalonate (DEEMM). The usefulness of this technique was confirmed by an alternative oenological analytical method for the validation, quality control and uncertainty assessment (OIV Oeno 10/2005). The method was validated and proposed as a reference method to the International Organization of Vine and Wine (OIV). As a specific application of the proposed method, the biogenic amine content of Rhône valley wines was investigated.     

Determination of biogenic amines by RPHPLC with fluorescent detection after derivatization with 2-(9-carbazole)ethyl chloroformate (CEOC)

Summary The use of 2-(9-carbazole)ethyl chloroformate (CEOC) for pre-column derivatization of biogenic amines (BA) has been tested for the first time. The reagent reacts completely with BA within 3 min at ambient temperature in acetonitrile solution to form stable derivatives that are readily analyzed by reversed-phase HPLC. Study of the derivatization conditions revealed derivatization yields to be excellent in borate buffer over the pH range 9.0–10.0. Maximum yields were obtained by use of a three- to fourfold molar excess of reagent. The reaction is extremely tolerant of common buffer salts, no decrease in reaction yield is discernible in well-buffered samples. The emission maximum for the CEOC-derivatives is 360 nm (λ _ex = 293 nm). All the derivatives fluoresced strongly and direct injection of the reaction mixture was possible, with no significant disturbance from the major fluorescent reagent degradation by-products, 2-(9-carbazole)ethanol (CEOC-OH) and bis-(2-(9-carbazole)ethyl) carbonate (CEOC)₂. Separation of the derivatized BA by high-performance liquid chromatography with gradient elution was tested on a Hypersil BDS C18 column. Excellent response linearity was observed over the concentration range from 0.25 to 94.6 μmol L⁻¹ for the labeled BA. Detection limits were 117–840 fmol at a signal-to-noise ratio of 3∶1. Analysis of BA in a shrimp sauce extract was conducted to demonstrate the applicability of the technique to real sample matrixes; results were satisfactory.     

Determination of biogenic amines in lake water by micellar electrokinetic chromatography with fluorescence detection after derivatization with fluorescamine

A simple and rapid method has been developed for the determination of biogenic amines in lake water using micellar electrokinetic chromatography with fluorescence detection. Separation of fluorescamine derivatized biogenic amines was accomplished by using borate buffer of pH 9.5 containing 40 mM of sodium dodecyl sulphate. The method has been optimized with respect to fluorescamine concentration, reaction pH, reaction time, separation voltage and injection time. Detection was performed by using UG-11 excitation filter and 495 nm emission filter. The proposed method for histamine, tyramine and dopamine allowed their separation within 2 min with detection limits in nM range. The interday and intraday reproducibility of peak areas were less than 6.5%. Recovery of spiked samples was 95.76–116.31%.     

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%.