ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21 1019 2005 © The Japan Society for Analytical Chemistry

Reviews Separation Methods for Toxic Components in Traditional Chinese Medicines

Wei LI, Zheng CHEN, Yiping LIAO, and Huwei LIU†

The Key Lab of Bioorganic Chemistry and Molecular Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R.

Traditional Chinese medicines (TCMs) with many unique functions for treating diseases have attracted the interest of people worldwide. They have been popularly utilized for therapy and health promotion in most Asian countries and even in many European and North American countries. However, it should be clearly noted that TCMs are mixtures with complicated composition usually containing hundreds, even thousands of chemically different constituents, and it is the multiple constituents that work synthetically to determine the ultimate effect of a formula of TCM. Meanwhile, some components with toxicity in some TCMs, having various negative effects on different parts of body, may do serious harm to people’s health; such harm in particular requires our attention. In this article, applications of different chromatographic and electrophoretic techniques in the analysis of toxic components in TCMs in recent decades have been comprehensively reviewed and some hyphenated procedures (combinations of two kinds of measurement) applied in this field are also summarized.

(Received February 28, 2005; Accepted March 11, 2005)

1 Introduction 1019 3 Separation and Determination Methods for 2 Quantitative Analysis of Nephrotoxic Toxic Alkaloids in TCMs 1024 Components in TCMs 1020 3·1 Analysis methods of ephedrine 2·1 Thin layer chromatography (TLC) 3·2 Analysis methods of other alkaloids in TCMs 2·2 High-performance liquid chromatography 4 Prospects for Future Study on the Analysis of (HPLC) Toxic Components in TCMs 1026 2·3 Hyphenation procedures of HPLC 5 Acknowledgements 1027 2·4 Capillary electrophoresis (CE) techniques 6 References 1027 2·5 Comparison between HPLC and CE

chemically different constituents, but only a few compounds are 1 Introduction responsible for the beneficial effects and there also exist many toxic ingredients.2–4 These multiple constituents work Traditional Chinese medicine (TCM) is a remarkable medicine synthetically to determine the ultimate effect of each kind of resource that has played a significant role in the therapy field. TCM.5,6 In many cases, the toxic components also act as active Especially in oriental countries such as China, Korea, and ingredients in some specific therapy, but at the same time there Japan, TCMs have been widely used in clinical treatment for are inevitably various hazardous effects on people’s health, thousands of years and have gained increasable popularity which mainly focus on the damage to kidney and liver. It can worldwide, even in European and North American countries. be said that these two impacts of the same ingredient conflict in Nowadays, there are more than 500 species of medicinal any therapy process. recorded in the Chinese Pharmacopoeia1 and the various active With the objectives of ensuring the safe use of a medicine and components in TCMs have deep effects on peoples’ health. For reducing the side effects of the toxic components as much as some severe diseases which are difficult to cure completely, possible, further investigation of pharmaceutical toxicology and TCM can effectively control the state of illness and can improve effective detection methods are required for quality assurance of the resistance of patients. Furthermore, there are many distinct the contents of toxic compounds in TCMs. The contents of the functions of some kinds of Chinese herbal medicines on therapy toxic compounds are related with the species, region, growing of certain disease such as rheumatism, which cannot be replaced condition, storage process and many other factors of the by other medicines. However, it is necessary to note that these medicinal plants. In addition, different extraction and TCMs are complex mixtures usually containing hundreds of preparation methods of herbs can also influence the analytical results. How to critically confirm the composition of a certain † To whom correspondence should be addressed. kind of TCM and how to determine the contents of active E-mail: [email protected] components, therefore, are always challenging. Up to now, 1020 ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21 much attention has been paid to the study of the standardization of quality control for TCM and there have been many reviews on the methodology for evaluating active components in TCM.3,6–10 Many chromatographic and electrophoretic techniques, including thin layer chromatography (TLC), high- performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE) have been applied to the analysis of active components in TCM.11–16 Besides, some on-line hyphenated techniques such as liquid chromatography-mass spectrometry (LC-MS) and LC-NMR, CE-MS have been developed and strongly recommended for Fig. 1 Structures of aristolochic acid I and II. quality control of TCM in recent years.17–19 These approaches can provide more reliable information about the structures of compounds, which is more helpful in the evaluation of TCM medicines in the past two years,24 so that further cases can be composition. In addition, some hyphenated methods show prevented. obviously higher sensitivity compared with those of The Aristolochia genus contains more than 800 herbaceous or conventional approaches, and the improvement is very crucial shrubby species growing in both temperate and tropical to the analysis of a trace compound that is difficult to be regions.40 There are more than 40 kinds of Aristolochia species detected by other methods. Different from conventional study, in China such as Aristolochia fangchi Wu and Caulis a chromatographic fingerprint of a TCM is a chromatographic Aristolochiae Manshuriensis, which are found mainly in pattern of the extract of some common components with Jiangsu, Guangdong, Guangxi provinces.41 Although the names pharmacologically active or chemically characteristics;6,20,21 this of these TCMs are very similar, they may exert quite different has been proved to be an effective methodology for the effects on people’s health due to their different species and assessment of TCM. In this review, some typical toxic regions. For example, Caulis Akebiae from Akebia quinata components in TCM are outlined and different chromatographic (Thunb.) Decne. or Akebia trifoliata (Thunb.) Koidz. is more and electrophoretic methods commonly used in the analysis of safe compared with those from Aristolochia such as Caulis these compounds in recent decades are summarized. Aristolochiae Manshuriensis.42 What is more, the cultivating methods and different parts of the medicinal plants can also influence the contents of AAs. So it is necessary to develop 2 Quantitative Analysis of Nephrotoxic effective methods for analysis of AAs and to systematically Components in TCMs investigate the distribution of AAs in Aristolochia plants. Many reports on the analysis of AAs with different methods including Many researches have indicated that the majority of kidney TLC, HPLC, CE and other means have been published already. intoxication accidents result from some kinds of TCMs containing aristolochic acid (AA), a mixture of AA-I and AA-II 2·1 Thin layer chromatography (TLC) (see Fig. 1 for the chemical structures of AAs). These Owing to their similar structures and identical medicinal compounds are known nephrotoxin and potential carcinogens activities, these two acids are rather difficult to separate. that can be found in Aristolochia species; they are often used in Preliminary detection of AA-I was made by TLC43–45 and the TCMs.22,23 Because of the similarity of Chinese names for total content of AAs was usually determined by ultraviolet several herbs, the possibility of the inadvertent substitution of spectrophotometry.46 TLC has the advantages of simplicity, innocuous herbs by Aristolochia species exists not only in TCM versatility, high speed, specific sensitivity and simple sample but also in other -containing dietary supplements. In preparation. It can be employed for multiple sample analysis. Belgium, there have been approximately 100 women patients For these reasons, it is still confirmed by many pharmacopoeias who had followed a weight-reducing treatment that usually as a convenient approach to provide the first characters of consisted of Chinese herbs and appetite suppressants. Half of TCMs including the Aristolochia medicinal plants.6 A TLC them suffered a severe kidney disease because a component in method was developed using a silicagel 60 F254 TLC glass- these slimming regimens called Stephania tetrandra was backed plate for the detection of AA-I.45 The sample was eluted 24,25 mistakenly substituted by Aristolochia fangchi. This with a mixture composed of CHCl3:MeOH:acetic acid disease, so-called Chinese herbs nephropathy (CHN), is (65:20:2). After evaporation of the solvents, the plate was characterized by a progressive interstitial fibrosis leading to a sprayed with a solution of 0.5% diphenylamine in H2SO4 60% severe atrophy of the proximal tubules.26 Such damage caused and heated for 10 min at 100˚C (blow-dryer or oven). Under by AA is currently incurable because AA will be converted into these conditions, AA-I was detected under visible light as a dark aristololactam (AL) in the body, and then the forming of AA- blue or black spot, which developed a yellow fluorescence when DNA adducts is not only responsible for the tumor development observed at 366 nm. Zhu and Phillipson44 determined the but also for the permanent destructive fibrotic process in the content of AA in some samples containing Fang ji purchased kidney.27–34 Since CHN was firstly reported in Belgium, it has from herbal shops in Hong Kong by TLC, finding that AA was been reported in many other areas including France,35 Spain,28 present in each of the nine purchased samples. Xu et al.47 used UK,36 Japan37,38 and China Taiwan.39 Considering the safe use TLC-scanning method to analysis AA-I in Aristolochia plants of medicines containing TCM, many health institutions such as including Aristolochia fangchi Wu, A. heterophylla, A. Food and Drug Administration (FDA) and Medicines Control moupinensis, A. austoszechuanica and A. tagala. High Agency (MCA), have warned people to pay attention to the performance thin-layer chromatography (HPTLC) was safety information about botanical products containing successfully employed for quality control of Stephania aristolochic acid. Indeed, many countries including the UK, tetrandra.48,49 This method allows detection of 1 ppm of AA Canada, Australia, and Germany have announced bans to that may be present due to adulteration with toxic species. restrict the importation, sale and use of AA-containing Compared with other TLC methods, this HPTLC method on ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21 1021

Table 1 Applications of HPLC in analysis of AAs Analytes Column Mobile phase Detector Ref.

AA-I in herb mixtures C-18 Symmetry column (4 µm, 250 MeOH (0.5% acetic acid) and H2O UV 45 × 4.6 mm i.d.) (0.5% acetic acid), gradient (60:40 254 nm, to 100:0) 224 nm AA in Aristolochia plants LiChrospher 100 RP-18e column Acetonitrile water and 2% (v/v) UV 56 acetic acid (45:55, v/v) 395 nm AA-I and AA-II in Kampo TSKgel ODS-80TM (5 µm, 150 × 4.6 Water/acetonitrile/phosphoric acid PDA 200 – 57 medicines mm i.d. TOSOH) (595:405:1) 600 nm AA-I and AA-II in TCMs Zorbax Extend-C18 column (3.5 µm, Mixture of methanol and water MS 60 150 × 2.1 mm i.d.) containing 1.0 mM ammonium acetate and 0.2% (v/v) acetic acid. AA in Aristolochia plants ODS Hypersil C18 column (5 µm, Methanol/water (60:40) UV 61 250 × 4.6 mm i.d.) 254 nm AA-I and AA-II in herbal Cosmosil 5C18-MS column Phosphate buffer-acetonitrile, UV 62 medicines gradient elution 390 nm AA-I and AA-II in Radix Hypersil Elite C18 column (5 µm, 1% Acetic acid in water and UV 63 Aristolochiae 250 × 4.6 mm i.d.) methanol, gradient elution 270 nm AA in Aristolochia plants LichroCART Methanol/water/glacial acetic acid UV 64 from Sudan and China (250 × 4.0 mm i.d.) (72:28:1) 310 nm

silica gel, with toluene–ethyl acetate–water–formic acid (20:10:1:1) as the eluent, followed by derivatization with tin(II) chloride, is a more rapid and powerful screening tool. A combination of two independent chromatographic systems of ion-exchange TLC and reversed-phase HPLC was also applied in the identification of AA and AA-adducts formed by AL-I.50

2·2 High-performance liquid chromatography (HPLC) Over the past decades, HPLC with powerful separation ability has been popularly applied for herbal medicine analysis. In general, it can be used to analyze almost all the compounds in the herbal medicines through suitable optimization procedures involving the composition of mobile phase, pH, and analytical columns.51–53 The method has been widely accepted as a routine method for the separation of two AAs and many reports have been published.54-62 Some of the applications and typical separation conditions are listed in Table 1. A facile reversed- phase HPLC method for the analysis of AA and AL analogues was developed for the quantitative determination of AA and AL in 12 species of Aristolochia (A. elegans, A. zollingeriana, A. cucurbitifolia, A. mollis, A. kaempferi, A. shimadii, A. heterophylla, A. debilis, A. foveolata, A. contorta, A. trilobata and A. odoratissima).65 The chromatograms obtained in this study can serve as fingerprints to identify plant species in the Aristolochia genus. For most cases, the separation was achieved on a C18 or C8 column with a UV detector; MS was often applied to create hyphenation techniques giving more satisfactory sensitivity. A mixture of acetonitrile and water was often utilized as the mobile phase with some special modifiers, Fig. 2 HPLC profiles of Chinese medicinal slimming powder and such as ammonium carbonate,24 TFA,59 or acetic acid55,56 to AA-I and AA-II.24 Conditions: column, LiChrospher 100 RP-18 control pH. Lee established a simple, sensitive and usable reversed-phase column (5 µm, 25 × 0.3 cm i.d., E. Merck); mobile HPLC procedure with a RP-C18 column for the determination phase, 0.3% ammonium carbonate solution: acetonitrile (75:25, v/v), of AA-I and AA-II in medicinal plants and slimming products.24 pH 7.5; flow rate, 1.0 ml/min; UV detection, 254 nm. The mixed solvent of 0.3% ammonium carbonate–acetonitrile (75:25, v/v) at pH 7.5 was believed to be the optimal condition, under which two kinds of AA can be baseline separated within retention time of AA analogues. Increasing the pH of mobile 20 min, as shown in Fig. 2. A facile reversed-phase HPLC phase decreased the retention time of AA analogues. However, method for simultaneous resolution and quantitative it increased the retention times of AL analogues slightly at determination of 17 analogues of AA and AL using a C18 higher pH (> 5.0); the elution reversal between AA and AL is column and a mixture of sodium acetate and acetonitrile as observed because of the ionization of carboxyl groups on AA. mobile phase was described by Jon.55 It was demonstrated that Another choice of mobile phase, one composed of methanol and even a slight change of the pH may have a great effect on the water, was also frequently used in this analysis; it is more 1022 ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21

(a)

(b)

Fig. 3 HPLC chromatogram of Aristolochia manshuriensis (‘Kanmokutsu’).40 Conditions: column, Symmetry C18, (150 × 4.6 mm i.d., Waters); mobile phase, 1% acetic acid: methanol (1:1); temperature, 35˚C; flow rate, 1.0 ml/min; UV detection, 250 nm.

Fig. 5 Effect of β-CD concentration on resolution (a) and theoretical plate number (b) of AAs.79 Conditions: borate buffer (pH 9.2), 50 µm i.d. × 48.5 cm (40.0 cm effective) fused silica capillary, applied voltage of 30 kV, capillary temperature of 10˚C, UV detection at 254 nm, injection at 50 mbar for 5 s, β-CD concentration varied in a range of 5 to 15 mM.

Fig. 4 Schematic illustration of a pressurized liquid extraction system.63 2·3 Hyphenation procedures of HPLC In order to further confirm the structural identification of economical than the system of acetonitrile and water. The compositions of Chinese herbs, researchers found that amounts of AA-I and AA-II in four groups of medicinal plants combining a chromatographic separation system on-line with a from the Aristolochiaceae and some related plants were special detector is the most important approach for the successfully determined by HPLC with mobile phase composed identification of target and unknowns. Especially, this of 1% acetic acid and methanol (1:1).40 A typical hyphenation instrument can effectively enhance the method chromatogram of A. manshuriensis is shown in Fig. 3. In order sensitivity in many trace-level analytical cases. The LC-MS to achieve satisfactory separation, researchers found that method was also used in determination of the contents of AAs, gradient elution was necessary in some methods.45,63 Ong but the operation procedure is more complicated than that of developed a pressurized liquid extraction (PLE) method in the HPLC.59,60 A method has been developed using RP-HPLC dynamic mode to extract AA in some medicinal plants.63 The coupled with atmospheric pressure chemical ionization (APCI) extraction cells were made of stainless steel, with 150 × 4.6 mm tandem mass spectrometry under the positive ion detection i.d. (2.50 ml) and 100 × 10 mm i.d. (7.85 ml); each cell was mode [LC/(+)APCI/MS/MS] to determine the amount of AA-I heated in a GC oven (see Fig. 4). Extraction was finished in methanol extracts of Xixin.59 The limit of detection of AA-I, through a HPLC pump within 15 – 20 min and the complete estimated by monitoring with LC/MS/MS, was at the low ppm analysis was achieved with a gradient elution. The obvious level. A solvent system consisted of a step-gradient was disadvantage of these methods with gradient elution is that they established, starting at 20% (v/v) methanol (containing 0.005% are time-consuming. In generally, the analytical times reported TFA) in water (0.005% TFA, pH 3.42), increasing to 100% were usually above 15 min, even more than 40 min, which MeOH over 15 min, holding at 100% MeOH for another 6 min, prevents the achievement of high throughput analysis. In our and then quickly returning to 20% MeOH over 2 min. Chan previous work, a rapid HPLC method using methanol and water described a liquid chromatography–electrospray-ion trap mass as mobile phase was developed for the analysis of AA-I and spectrometry (LC-ES-ITMS) method for the determination of AA-II in Aristolochia plants.61 Different ratios of methanol to AAs in medicinal plants and Chinese herbal remedies utilizing a water, ranging from 30:70 to 90:10 (v/v), were attempted; the reversed phase C18 column and gradient elution.60 In another ratio of 60:40 was the optimal one for the fast separation of AA- study, AA-I and AA-II were successfully detected using a TLC I and AA-II. Under the optimal conditions, the two AAs were and a HPLC equipped with a photodiode array detector (PDA) baseline separated within 5 min, showing satisfactory sensitivity and EI-MS detector.57 With the HPLC-PDA, it was possible to and repeatability. quickly detect AA-I and AA-II in ten kinds of Kampo ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21 1023

Table 2 Contents of AA-I and AA-II determined in some real samples of herbal medicinal plants by CZE79 (% w/w, dry plants)

Species Cultivation region AA-I, % AA-II, % Aristolochia Zhaoqing, Guangdong —a — fangchi Wu Zhongshan, Guangdong — — Luoding, Guangdong — — Qujiang, Guangdong — — Gaoyao, Guangdong — — Cangwu, Guangxi 0.072 0.018 Cenxi, Guangxi 0.12 — Caulis Tumen, 0.30 0.063 Aristolochiae Antu, Jilin 0.073 0.022 Manshuriensis Dunhua,Jilin 0.17 0.043 Huanren, — — Dandong, Liaoning 0.049 0.021 Shanghai 0.20 0.069 Mudanjiang, 0.13 0.031 Stephania Shaotong, Yunnan 0.065 0.014 tetrandra S. Moore Fig. 6 Typical electropherograms of AA-I and AA-II by CZE.79 a. “—” means that the content of AA is lower than the detection limit. (a) Standard sample and (b) a real sample of Caulis Aristolochiae Manshuriensis from Dunhua, Jilin region. Conditions: 120 mM sodium borate buffer (pH 8.8) containing 10 mM β-CD, 50 µm i.d. × 48.5 cm (40.0 cm effective) fused silica capillary, applied voltage of compositions and concentrations, capillary temperature and 30 kV, capillary temperature of 10˚C, UV detection at 254 nm, applied voltage. Results indicated that β-CD as additive is the injection at 50 mbar for 5s. key parameter in the separation of AA-I and AA-II. Great improvement of the resolution was obtained by adding β-CD into the buffer, which results from the special inclusion ability medicines in which the Aristolochia fangchi root or Aristolochia of AA into the hydrophobic cavity of CD. The effects of other manshuriensis stem may be misused. kinds of CD such as α-CD, γ-CD and HP-CD, were also studied, but no obvious improvement was observed comparing 2·4 Capillary electrophoresis (CE) techniques with β-CD. This can be explained by the fact that the cavity As a micro-column separation technique, CE has been proved diameter of β-CD is in concordance of the molecular sizes of to be an effective and convenient technique in various fields, AA-I and AA-II with three-cyclic structures. Based on this, the including the analysis of herbal medicines.66–73 From the concentration of β-CD was further optimized, illustrating that practical point of view, CE shows more advantages than other the resolution and theoretic plate number fluctuated with the separation techniques. The capillaries attain equilibrium more increase in β-CD concentration as shown in Fig. 5. It was easily compared with HPLC columns and the contamination concluded that 10 mM β-CD was the obvious optimal point problem is effectively avoided. Various separation patterns of with highest resolution and high theoretic plate numbers of AA- this convenient technique can realize highly-effective separation I and AA-II. Through the further optimization of other of both ionic and neutral compounds in complicated matrices. parameters, the separation conditions of 120 mM borate buffer However, there are only a few publications on the analysis of (pH 8.8) containing 10 mM β-CD under 10˚C temperature and AA-I and AA-II by CE at present. In our laboratory, two kinds 30 kV applied voltage was confirmed to be optimal. Under of CE methods have been established for the separation and these conditions, baseline separation of AA-I and AA-II was determination of AA-I and AA-II from Aristolochia plants, achieved within 4 min with excellent separation efficiency, as which were proved to have great application values. shown in Fig. 6. The linearity was satisfactory, with a 2·4·1 b-cyclodextrin-modified capillary zone electrophoresis correlation coefficient (r) greater than 0.99. The detection (CZE) limits for both compounds were found to be 1.0 µg mL-1 and Since its first introduction by Jorgenson and Lukacs,74 CZE recoveries were 96.7 and 77.8%, respectively. Furthermore, has become an important method for the separation of charged good repeatability of <0.3% (RSD) for migration time and natural products, especially for the natural products with <4.7% (RSD) for peak area was obtained. Real samples polyphenolic structures such as flavonoids, coumarin and including 10 kinds of Aristolochia fangchi Wu, 21 kinds of organic acids.75–77 Kvasnicka et al. used a CZE method to detect Caulis Aristolochiae Manshuriensis and 6 kinds of other AA in dietary supplements and selected herbs with a mixture of Aristolochia medicinal plant samples collected from various morpholinethanesulphonic acid, bistrispropane and regions in China have been analyzed by this method; some of hydroxyethylcelullose in 10% methanol as the background the results which can show the distribution of AA-I and AA-II electrolyte.78 A clear separation of AA from other sample among different samples are listed in Table 2. From them, we constituents was achieved within 5 min without any sample can conclude that the species of medicinal plant samples has an clean-up. In our previous work, a fast CZE method with β- important impact on the distribution of the contents of AAs and cyclodextrin (β-CD) as modifier was developed for the analysis that AA-I is the major component in Aristolochia plants. of AA-I and AA-II.79 Separation conditions were systematically 2·4·2 Micellar electrokinetic chromatography (MEKC) optimized by investigating the effects of buffer pH, the As another separation pattern of CE, MEKC exhibits a concentration of sodium borate, organic modifiers with different broader application prospect than CZE does because it enables 1024 ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21

as low as that of HPLC which ranges from 0.01560 to 1.0 µg/mL,40 because of the relative short detection window and very small amount of injection in CE. Therefore, it will be interesting to investigate on-line preconcentration techniques for CE to improve its detection sensitivity.

3 Separation and Determination Methods for Toxic Alkaloids in TCMs

82 Fig. 7 Typical electropherograms of AA-I and AA-II by MEKC. In recent years, the damage of liver caused by Chinese herbal Conditions: 100 mM sodium borate containing 20 mM SDS, 50 µm medicine has attracted more attention, and the proportion of this I.D. × 48.5 cm (40.0 cm effective) fused silica capillary, applied voltage of 30 kV, capillary temperature of 25˚C, UV detection at 254 kind of disease among all of the medical liver diseases has nm, injection at 50 mbar for 5 s. increased to 30%. It was demonstrated that these chronic liver diseases can be mainly classified into two categories. One kind of these diseases is related to the specific immunoreactions in which toxic components in TCMs react as haptens, and the good separation of both charged and electrically neutral other is led by hepatotoxicity caused by some herbal remedies analytes.80,81 Neutral compounds can be separated due to their and their metabolites, which ranges from mild liver enzyme different partition between the micelle and aqueous phase, and alterations to chronic liver disease and liver failure.83 It has the separation mechanism of MEKC is a combination of both been reported that many intoxications may result from some electrophoretic and chromatographic ability. In order to further toxic alkaloids, which can cause various harms to people, so realize the separation of some other toxic AA-related neutral many studies have been focused on the development of suitable components such as AL that can not be achieved by CZE separation and determination methods for the analysis of toxic method, we developed a MEKC method using sodium dodecyl alkaloids in TCMs. sulfate (SDS) as surfactant. A suitable surfactant, which affects the direction and level of EOF and the configuration of 3·1 Analysis methods of ephedrine micelles, is essential to the separation efficiency in MEKC. Ephedrine, the major bioactive component in Ma-Huang, Two kinds of anionic surfactants including SDS, sodium cholate which is obtained from the stems of plants of sinica, (SC) and CTAB, which is a cationic surfactant, were applied in has been popularly used as a natural product in therapy of the experiments. Results illustrate that SDS is the key asthma, weight loss, energy enhancement and many other parameter for the separation through manipulating the areas.84 The contents of different kinds of ephedrines have been interaction between AA and the micelle. Figure 7 illustrates82 regarded as quantitative standards in the evaluation of TCMs that some neutral components of AA-related compounds were containing Ma-Huang. However, they are classified as not overlapped by EOF, as in the result obtained from CZE. prohibited substances according to the International Olympic Furthermore, it also indicates that, in parallel with AA-I and Committee (IOC)’s list of prohibited classes of substances, due AA-II, neutral compounds can be separated by this method, to their potency to stimulate the central nervous system85 and although the further optimization is still required. the concentration limits of five ephedrine derivatives in urine have been established.86 Furthermore, high doses of ephedrines 2·5 Comparison between HPLC and CE are also responsible for some incidents of acute hepatitis and As a convenient separation tool, the HPLC method has been nephrolithiasis.84 commonly applied in analysis of AAs in TCMs and other Many chromatographic methods have been used in this field. products. Although it has with some advantages, such as low GC is a popular and useful analytical tool for the determination detection limit, it exhibits the obvious disadvantages of high of ephedrines in pharmaceutical preparations and in biological cost due to the magnitude of the mobile phase and slow fluids after proper derivatization.87–90 With the use of a processing, which would not meet the needs of high throughput hyphenated GC-MS instrument, reliable information on the analysis. In our previous HPLC method, the resolution between identity of the chemical structure of analytes, especially two components is 2.37 and theoretic plate numbers lower than unknown compounds, is available, which is very helpful to the 15000/m were obtained, so even if the analytical time is safety control of quantity of TCM. El-Haj89 applied a GC-MS shortened, the separation efficiency needs to be further method to detect methamphetamine and ephedrine in urine improved, especially for the analysis of real samples. based on derivatization with cyclohexanone. The method was Compared with HPLC, the proposed CE methods show great found to be superior to the methods based on acyl and separation efficiency with resolution between two components trimethylsilyl (TMS) derivatization because of its higher of higher than 4 and theoretic plate numbers greater than stability and selectivity. However, the most serious 170000/m for AA-I and AA-II, which has not been reported in disadvantage of GC lies with the tedious derivatization sample HPLC. In the analysis of AAs in TCMs, not only the AA-I and work-up. As far as the operation procedure is concerned, the AA-II but also the other AA-related components were separated HPLC method is more convenient. completely, so the contents of AAs could be exactly determined Choosing a suitable column or stationary phase is very without interference from other components. In addition, the important for HPLC methods that are commonly used in the baseline separation of AA-I and AA-II can be achieved within 4 separation of ephedrines.91 The analysis of six alkaloids min, which is much shorter than the times needed by most of including ephedrine, pseudoephedrine, norephedrine, the HPLC methods. As described above, the CE method shows norpseudoephedrine, methylephedrine, and more advantages including simplicity, high-speed and low-cost methylpseudoephedrine included in ground botanical raw compared with HPLC, and it is practical for the determination material of Ephedra sinica was performed on a polar-embedded of AA-I and AA-II. However, the detection limit of CE is not phenyl column using UV detection at 210 nm.92 A simple, ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21 1025

Table 3 Applications of CE in the analysis of some toxic alkaloids in TCMs

Analytes Mode Separation conditions Ref. Ephedrine from Ma-Huang NACE 50 mM ammonium acetate, 30 mM acetate acid, 900 mM 98 acetonitrile Ephedrine alkaloids in Ephedra sinica Stapf NACE 50 mM ammonium acetate in methanol 99 Aconitine alkaloids in TCM NACE 20 mM sodium tetraborate, 70% methanol, pH = 8.5 100 Nine kinds of morphine analogues NACE acetonitrile: methanol: acetate acid = 49:50:1, 20 mM 101 ammonium acetate Tetrahydropalmatine in Rhizoma Corydalis NACE 50 mM sodium acetate, 2 mM acetate acid in methanol 102 Quinolizidine alkaloids in the roots of Sophora CZE 75% 130 mM sodium diphosphate, pH = 3.5, 25% acetonitrile 103 subprostata Ephedrine CZE 5 mM Tris, 5 mM sodium borate, pH = 6.5 104 Chiral ephedrine derivatives in unregulated drug CZE 50 mM potassium dihydrogenphosphate containing 20 mM 105 products DM-CD, pH 2.6, 0.6% (w/w) SDS, 1.2% (w/w) n-butanol, 80 mmol/L n-octanol, 0.5% (w/w) 106 Ephedrine enantiomers MEEKC (2R,3R)-di-n-butan-1-(2R,3R)-di-n-butyl tartrate, 97.7% (w/w) Tris buffer, pH 8.1 Ephedrines CZE 1.0 mM citric acid–sodium citric acid, 4.0 mM Cu2+, 9% 107 ethanol, using conductivity detector Aconitum alkaloids in herbal medicines NACE 40 mM ammonium acetate, 0.1% acetic acid, 80% methanol 108 Ephedrines in TCM CZE 100 mM borate buffer, pH 9.80 109 Ephedrine from TCM containing Ma-Huang CZE 20 mM sodium acetate, 50% acetonitrile 110 Strychnine and brucine in the extracts of Strychnos CZE 10 mM phosphate buffer–methanol (9:1), pH 2.5 111 nux-vomica seeds

sensitive, and reliable HPLC method was developed for ephedrines by column-switching without sample preparation. simultaneous determination of Ephedrine alkaloids in Ephedra The analytes were trapped on a C18 precolumn and separated sinica Stapf. The separation was performed on a reversed phase on a C18 BDS analytical column. It successfully resolved the C18 column (150 mm × 3.9 mm i.d.) with a mobile phase problem of time-consuming sample clean-up step and made the composed of methanol and phosphate buffer solution.93 Hood automation possible.85 In order to further enhance the described a rapid HPLC method for quantification of ephedrine sensitivity compared with that of a UV detector, Aymard HCl and other alkaloids carried on a Zorbax XDB C8 column presented a specific HPLC analysis method with fluorometric (150 cm × 4.6 mm i.d.).91 The method was validated and met all detection for quantification of ephedrine and norephedrine in analytical requirements of quality assurance and quality control human plasma after liquid–liquid extraction and derivatization recommended by US FDA. Imaz compared different reversed- with 9-fluorenylmethyl chloroformate.96 The satisfactory phase HPLC columns for basic analysis of ephedrines, such as detection limits of 2 ng/mL for ephedrine and 5 ng/mL for LiChrospher 60 RP-Select B, LiChrospher 100 RP18, Hypersil norephedrine were obtained. Erk applied a ratio-spectra BDS-C18, Inertsil ODS-2, and Spherisorb ODS-Band derivative spectrophotometry in the analysis of a multi- Symmetry Shield RP8.86 Results indicated that Symmetry component system including ephedrine hydrochloride, allowing Shield was the only column, that did not require the use of high an increase in the selectivity of spectrophotometric concentrations of buffer and triethylamine. With this column, a determination.97 good separation of the six ephedrines and the internal standard For the past decade, CE has become a versatile and powerful was achieved using 50 mM phosphate buffer–25 mM separation tool with high separation efficiency and selectivity in triethylamine as a mobile phase. A chiral stationary phase the analysis of herbal drugs.6 Table 3 demonstrated its recent bonded with β-cyclodextrin and the addition of the chiral applications on the analysis of toxic alkaloids in TCMs. A selector to the mobile phase were investigated for chiral number of CE procedures have been developed for the separation of ephedrines,94 showing that the selectivity can be determination of ephedrines in TCM or nutritional supplements improved through the introduction of carboxyalkyl groups in the containing Ma-Huang. Flurer presented a CE method utilizing β-CD rings. Iwanicki achieved the chiral separation through a hydroxypropyl-β-CD as chiral selector at low pH with chiral column (Supelcosil LC-(S) Naphthyl Urea) when the tetramethylammonium chloride (TMAC) added to further analytes were derivatized with both of the following two chiral decrease the EOF. This buffer system permits at least partial derivatization reagents: N-trifluoroacetyl-l-prolyl chloride and resolution of ten members of the ephedrine family.112 Pan 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate. This developed a rapid continuous separation approach by HPLC method was evaluated and compared with CE for its combination of flow injection with CE designed for the analysis application in analyzing the enantiomeric compositions of of ephedrine and pseudoephedrine in TCMs using the double-T ephedrine and pseudoephedrine.95 configuration.109 A CZE method with UV detection was Either analytical method requires an appropriate isolation of optimized by means of an experimental design for the the analyte of interest, in particular from a complex matrix, and separation and the simultaneous quantification of ephedrine, the extraction procedure is always complicated. Roman pseudoephedrine, norephedrine (phenylpropanolamine) and established a solid-phase extraction (SPE) method using a norpseudoephedrine (cathine) in urine without any extraction.113 strong-cation exchange cartridge to remove interference in the Successful results were obtained with a 260 mM Tris–phosphate analysis of ephedrines in Ephedra sinica plants.92 Gmeiner buffer at pH 3.5 in the presence of 13.3 mM dimethyl-β-CD at developed a method for the quantification of five congener 25˚C and 30 kV applied voltage. Under these optimized 1026 ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21

chloro-7-nitrobenzo-2-oxa-1,3-diazol.116 The detection limits were 0.16 µM for ephedrine and 0.17 µM for pseudoephedrine, illustrating that the sensitivities were at least ten times improved over those reported in the literature as obtained by UV detection. Belder developed a CE-MS method using a coupling device that consisted of a modified electrospray (ES) ion source with coaxial sheath liquid.117 The separation performance can be significantly improved by coating the capillaries with poly(ethylene glycol) (PEG) or poly(vinyl alcohol) (PVA), which effectively avoids any resolution decrease or poor peaks caused by the volatile buffers of low ionic strength needed in CE-MS analysis.

3·2 Analysis methods of other alkaloids in TCMs In addition to ephedrines in Ma-Huang, there are also other toxic alkaloids in TCMs harmful to the health of people. Many analysis methods of these compounds have been introduced. A new approach based on a LC-MS/MS technique was presented for simultaneous qualitative and quantitative analysis of toxic compounds in TCM products including indole alkaloids, quinolizidine alkaloids, diterpenoid alkaloids and bufadienolides. These compounds were successfully determined in extracts of Semen Strychni, Radix Sophorae Tonkinensis, Radix Aconiti Lateralis and Venenum Bufonis, respectively.118 This method was systematically evaluated and good results were obtained with the low detection limits ranging from 0.4 to 1.5 ng. Krenn et al. investigated the separation of the main opium alkaloids morphine, codeine, thebaine, papaverine and noscapine on a non-porous (micropellicular) stationary phase by a HPLC method.119 Miao et al. reported a high-speed countercurrent chromatography (CCC) method for the separation of strychnine and brucine from crude extract of strychnos nux-vomica L. by a two-phase solvent system composed of chloroform/0.07 M sodium phosphate, 0.04 M 120 Fig. 8 Typical electropherograms of (a) standard sample, (b) citric buffer (pH 5.08) (1:1, v/v). Zhou et al. described a positive urine, (c) and (d) dietary supplement extracts (Ephedra 850 method using a silica gel-column quasi-ion exchange and Kwik Energy, respectively) and (e) excretion urine from Kwik chromatography for determination of strychnine content in the Energy113 (Peaks: 1, ephedrine; 2, pseudoephedrine; 3, norephedrine; seeds of Srychnos nux-vomica L. by which the interference of 4, norpseudoephedrine; 5, scopolamine). Conditions: 260 mM other alkaloids can be effectively avoided.121 They also Tris–phosphate at pH 3.5 and 13.3 mM dimethyl-β-CD; applied summarized the extraction approaches of strychnine and related µ voltage, 30 kV (I = 61 A); temperature, 25˚C; uncoated fused-silica compounds in medicinal plants.122 In addition, a non-aqueous capillary, L = 48.5 cm, l = 40 cm, i.d. = 75 µm. CE (NACE) method was developed for the determination of strychnine, brucine and some other highly toxic alkaloids in two commonly used herbal medicines.108 Since its introduction at conditions, a baseline separation of the four compounds was the beginning of the 1990s, NACE exhibits significant achieved in less than 6 min. Figure 8 shows the advantages over aqueous CE for many compounds.123 Organic electropherograms of standard and real samples. With the solvents used in NACE shows greater solubility than water and development of instrument miniaturization, the separation of a wide range of physicochemical properties, which leads to a ephedrines can also be obtained by CE on a chip.114 Detection greater ability to manipulate separation, especially for the by a new two-electrode amperometric detector has the analysis of hydrophobic substances, and is more suitable for the advantage of not requiring chemical derivatization, unlike the realization of CE-MS.124 NACE has been applied to the fluorescence technique that was more commonly used for separation of toxic alkaloids in aconitine root (Radix aconitini microfabricated analytical devices. Satisfactory separation praeparata) and alkaloids in TCM of Maqianzi108 using the performance with theoretic plate numbers up to 20000 and buffer composed of 40 mM ammonium acetate, 80% methanol chiral resolution of 2.5 was achieved. The analysis time was and 0.1% acetic acid; this condition was further optimized to drastically reduced to 3 min by this method. In order to further establish a precise CE-MS method.125 improve method sensitivity and make it suitable for analysis of dilute herb drug extracts, researchers have developed two stacking methods of CE for the separation of very dilute 4 Prospects for Future Study on the Analysis of solutions of ephedra-alkaloids. By such a method, the detection Toxic Components in TCMs limits can be reduced to 10–3 µg/mL level.115 The hyphenated CE instruments, such as CE-LIF and CE-MS, have also been For the analysis of active components in TCMs, it is very applied for the specific and sensitive detection. Ephedrine and crucial to develop effective detection methods with a serious pseudoephedrine were separated by a CE method with laser- consideration of many factors including sample preparations, induced fluorescence (LIF) detection after derivatization by 4- analytical time and detection sensitivity. The exact ANALYTICAL SCIENCES SEPTEMBER 2005, VOL. 21 1027 quantification tools of the toxic compounds in some herbs are Chau, J. Chromatogr., A, 2001, 909, 237. urgent requirements for the quality control of herbal medicines. 18. F. Gong, Y. Z. Liang, Q. S. Xu, F. T. Chau, and A. K. M. Many chromatographic and electrophoretic tools, such as GC, Leung, J. Chromatogr., A, 2001, 905, 193. HPLC, CE and some hyphenation technologies, have been 19. F. Gong, Y. Z. Liang, Q. S. Xu, F. T. Chau, and K. M. Ng, successfully applied in this field, providing valuable Anal. Chim. Acta, 2001, 450, 99. information of the composition of TCM. Of course, it is 20. P. S. Xie, Tradit. Chin. Drug Res. Clin. 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