Determination of aristolochic acids in medicinal plants (Chinese) prepared medicine using capillary zone electrophoresis

Aristolochic acids (I and II) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. The aim of this work was to develop a method for the analysis of aristolochic acids in medicinal plant/Chinese prepared medicine (CPM) using (CZE). The buffer used was 30 mM sodium tetraborate at pH 9.5, detection was at 254 nm, applied voltage at 18 kV and the temperature was set at 25 degrees C. The effect of ionic strength, pH, and applied voltage on the separation was investigated. The precision values (relative standard deviation, RSD, %) for the relative migration time and peak area or peak height for aristolochic acids I and II were found to be less than 0.3% and between 2.6 to 4.0%, respectively. The limit of detection for aristolochic acids I and II was found to be 1.2 and 0.9 mg/L, respectively. The proposed method using pressurized liquid extraction (PLE) with CZE was used to determine the amount of aristolochic acids in medicinal plants or CPM samples with complex matrix and the results were compared with high-performance liquid chromatography (HPLC). Method precision (RSD, n = 6) was found to be less than 4% when those from applied to medicinal plants and CPM samples.     

Assessment of Chinese medicinal herb metabolite profiles by UPLC-MS-based methodology for the detection of aristolochic acids

In this study, aristolochic acid in different herbal medicines containing a mixture of varying herb species was identified through fingerprint pattern similarities. Aristolochic acid I and II are nephrotoxic compounds naturally present in the Aristolochia plant species that are commonly used in Chinese herbal medicines. Twenty-four commercially available herbal formulations were extracted into an aqueous solution and injected into a UPLC-MS system. All the samples were analysed by multiple reaction monitoring (MRM) to check for the presence of aristolochic acids I and II. The same samples were then fingerprinted using two different gradient methods and the chromatograms deconvoluted into retention time (RT) and masses for the chemicals present taking concentration into account. Statistical analysis of this data revealed that samples were highly heterogeneous, and that the main differences between the preparations were concentrations of polar compounds. A model was constructed where the samples could be separated into two groups differentiated by the presence of the two forms of aristolochic acid.     

Rapid determination of aristolochic acid I and II in medicinal plants with high sensitivity by cucurbit[7]uril-modifier capillary zone electrophoresis

Aristolochic acids (AAs) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. Hence, quantitative analysis and quality control for the plants containing AAs is of great importance. In this study, a novel macrocylcic molecule, cucurbit[7]uril (CB[7]) was employed as a modifier in capillary zone electrophoresis (CZE) for rapid determination of aristolochic acid I and II in medicinal plants. In similarity to other macrocyclic molecules, such as cyclodextrins (CDs), CB[7] can be used to manipulate selectivity in CE because it can form inclusion complexes with a variety of guest molecules. During the running process, CB[7] bears a positive charge in the pH range of 2.5-7.5 and can be adsorbed onto the inner wall of a fused-capillary, leading to a reversal of the electroosmotic flow (EOF). By applying a negative polarity, a rapid separation of AA-I and AA-II was achieved within 7min using 100mM phosphate buffer (pH 7.5) containing 3mM CB[7] and 10% acetonitrile (v/v) as modifiers, due to the same directions of the EOF and the electrophoretic mobilities of the analytes. By applying electrokinetic injection with field-enhanced sample stacking, two kinds of aristolochic acids in four medicinal plants were successfully determined with high sensitivity, high separation efficiency, repeatability and recovery. The proposed method was also used to determine AA-I and AA-II in two slimming pills with complex matrix.     

Simultaneous determination of nine aristolochic acid analogues in medicinal plants and preparations by high-performance liquid chromatography

By optimizing the extraction, separation and analytical conditions, a simple, reliable and effective high-performance liquid chromatography method coupled with photodiode array detector (HPLC-DAD) is presented for simultaneous determination of nine aristolochic acid (AA) analogues, i.e., AA I, AA II, AA C, AA D, 7-OH AA I, aristolic acid, AL II, AL III and AL IV, in twelve medicinal herbs and two preparations. The separation was completed on a C18 column with aqueous methanol containing 0.2% (V/V) acetic acid as mobile phase. Linearities of around two orders of magnitude were obtained with correlation coefficients exceeding 0.9950. Satisfactory intra-day and inter-day precisions were achieved with R.S.D.s less than 4.35%, and the average recovery factors obtained were in the range of 88.4-98.8%. The proposed method appears to be suitable for use as a tool for safety assurance and quality control for commercially available suspect samples containing aristolochic acid analogues.     

Rapid Determination of Aristolochic Acids I and II in Some Medicinal Plants by High-Performance Liquid Chromatography

Aristolochic acids (AA) are toxic components of Aristolochia plants which result in diseases of the kidney such as urothelial cancer. It is, therefore, essential to monitor the amount of aristolochic acid in herbal medicines. In this study a reversed-phase high-performance liquid-chromatographic (HPLC) method has been developed for rapid determination of aristolochic acids I and II. Baseline separation was achieved within five minutes by use of an ODS C₁₈ column with methanol–water, 60:40, as mobile phase. Two kinds of aristolochic acid were successfully determined in 31 herbal samples of Aristolochia fangchi Wu and Caulis Aristolochiae Manshuriensis. The results indicated that in most samples the aristolochic acid I content is much higher than that of aristolochic acid II. The two kinds of aristolochic acid were not detected in Aristolochia fangchi from the Guangdong region, so Aristolochia fangchi from this region is recommended for use in herbal remedies.     

Evaluation of Chinese medicinal herbs fingerprinting by HPLC-DAD for the detection of toxic aristolochic acids

Aristolochic acids are known to contribute to various renal disorders; therefore, expanding the availability of analytical methodology to detect these compounds is important in order to assess the quality of Chinese herbal medicines in which they can be found. Twelve medicinal herbal samples were procured from various sources and extracted in duplicate prior to a "fingerprint" analysis using conventional HPLC-DAD. Multivariate analysis was performed on the entire chromatographed fingerprints. The resulting output was a partial least-square discriminant analysis model, which was able to evaluate the potential presence of aristolochic acids I and II as well as providing an individual herbal "fingerprint". The results of this study provide evidence that the presence of aristolochic acids contained within certain herbal extractions could be detected using a simple method, although some limitations apply to this method for quality control, since newly detected samples for aristolochic acid (positives) will need further confirmation with purity checks or MS hyphenation.     

Selective analysis of aristolochic acid I in herbal medicines by dummy molecularly imprinted solid‐phase extraction and HPLC

In this study, surface molecularly imprinted polymers were prepared as the selective sorbents for separation of aristolochic acid I in herbal medicine extracts by a facile approach. A less toxic dummy template, ofloxacin, was used to create specific molecule recognition sites for aristolochic acid I in the synthesized polymers. The polymers were characterized by Fourier‐transfer infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, elemental analysis, and nitrogen adsorption–desorption test. The adsorption capacity was calculated using adsorption kinetics, selectivity, and recycling experiments. The obtained polymers exhibited high thermostability, fast equilibrium time, and excellent binding ability. Subsequently, the polymers applied as the solid‐phase extraction absorbent was proposed and used for the enrichment and analysis of aristolochic acid I in herbal plants. The result showed that the aristolochic acid I was enriched up to 16 times after analysis by using high‐performance liquid chromatography. The good linearity for aristolochic acid I was obtained in the range of 0.1–200 μg/mL (R ² = 0.9987). The recovery and precision values were obtained (64.94–77.73%, RSDs% ≤ 0.8%, n  = 3) at three spiked concentration levels. This work provided a promising method for selective enrichment, extraction, and purification of aristolochic acid I from complex herbal plants.     

Detecting aristolochic acids in herbal remedies by liquid chromatography/serial mass spectrometry

Targeted liquid chromatography/serial mass spectrometry (LC/MS/MS) analysis, using a quadrupole ion-trap mass spectrometer, permitted the detection of aristolochic acids I and II in crude 70% methanol extracts of multi-component herbal remedies without any clean-up or concentration stages. The best ionisation characteristics were obtained using atmospheric pressure chemical ionisation (APCI) and by including ammonium ions in the mobile phase. Limits of detection for aristolochic acids were influenced by the level of interference created by other components in the sample matrix. They were determined to be between 250 pg and 2.5 ng on-column within a matrix containing compounds extracted from 2 mg of herbal remedy. With a herbal remedy that only permitted the higher limit of detection, this sensitivity was sufficient to detect the aristolochic acids extracted from 0.1% dry weight of Aristolochia manshuriensis included in the preparation.     

Differentiation of herbs linked to "Chinese herb nephropathy" from the liquid chromatographic determination of aristolochic acids

A HPLC method was developed and applied to analyze aristolochic acids (AA-I and AA-II) in Chinese medicinal herbs. The herb samples were extracted by using ultrasonication with the extraction efficiency of better than 82%. Extracts were then filtered and injected onto a C18 column eluting under a gradient program using methanol and water-containing 0.5% acetic acid. The method with the detection limits of 1.33 ng for AA-I and 7.29 ng for AA-II per injection was successfully applied for the analysis of traditional Chinese medicine (TCM) and related products and differentiation of Chinese medicinal herbs that have previously been misused and caused toxicological effects. The developed protocol provided an example that analysis of selected component markers could serve for health security and quality control of TCM consumption.     

Rapid determination of aristolochic acids I and II in herbal products and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs) are a mixture of structural-related compounds, in which aristolochic acid I (AA I) and aristolochic acid II (AA II) are reported to be correlated with Aristolochic acid nephropathy (AAN). In this work, a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine AA I and AA II in herbal products and biological fluids. By using gradient elution with a mobile phase composed of a mixture of 10 mM ammonium formate buffer (pH 3.0) and acetonitrile, AAs could be determined within 10 min. Under optimum UHPLC-MS/MS conditions, the limit of detections was 0.14 and 0.26 ng mL⁻¹ for AA I and AA II, respectively. Run-to-run repeatability and intermediate precision of peak area for AA I and AA II were less than 5.74% relative standard deviation (RSD). Accuracy was tested by spiking 10, 100 and 1000 ng mL⁻¹ in rat serum and the recoveries were within 76.5-92.9%. Matrix effects were within 78.8-127.7%. The developed method was successfully applied to determine AA I and AA II in several herbal products and to investigate their pharmacokinetic behavior in female Wister rats. The result shows that the developed UHPLC-MS/MS method is efficient, sensitive, and accurate for the determination of AA I and AA II in herbal products and biological samples.     

超高效液相色谱-电喷雾三重四极杆质谱法测定中药中马兜铃酸A和B的含量

利用超高效液相色谱-电喷雾三重四极杆质谱仪建立了中药中马兜铃酸A和B的定性定量分析方法。选取柴胡、生甘草、桔梗、龙胆泻肝丸、消胖丸、减肥茶等14种代表性样品,用甲醇-水(70:30, v/v)溶液加热回流提取,经Oasis MAX固相萃取柱富集净化后,在Eclipse RP HD C18反相柱(150 mm×2.1 mm, 1.8 μm)上进行分离;流动相为5 mmol/L乙酸铵水溶液(pH 7.5)-乙腈(75:25, v/v)。采用电喷雾离子源正离子模式(ESI+)和多反应监测模式(MRM)进行质谱分析。马兜铃酸A和B的线性范围分别为0.5～200 μg/L和1～200 μg/L,相关系数(r2)均大于0.995;检出限(LODs)分别为5 μg/kg和7.5 μg/kg;定量限(LOQs)分别为12.5 μg/kg和25 μg/kg。在100 μg/kg和500 μg/kg添加水平下,马兜铃酸A和B的回收率(n=6)范围分别为60.3%～96.4%和61.3%～94.7%,相对标准偏差均不大于10.2%。该方法灵敏度高,重复性好,操作简便,适用于中药材、饮片及中成药中马兜铃酸A和B的痕量检测。     

Simultaneous determination of five aristolochic acids and two aristololactams in Aristolochia plants by high-performance liquid chromatography

An HPLC method was developed for the simultaneous determination of five aristolochic acids (AAs) and two aristololactams (ALs) in the following six Chinese drugs derived from Aristolochia species. Samples were analyzed on a C(18) column with acetonitrile and 3.7 mm phosphoric acid buffer gradient elution, detected at 260 nm. Assay was linear over the range (microg/mL) 0.386-38.6 for aristolochic acid Va, 0.632-63.2 for aristolochic acid IVa, 0.200-20.0 for 9-hydroxy aristolochic acid I, 0.352-35.2 for aristololactam II, 0.296-29.6 for aristolochic acid II, 0.274-27.4 for aristololactam I and 3.12-312 for aristolochic acid I. Average recoveries (%) of samples were 102.0, 95.9, 99.2, 102.2, 97.2, 97.1 and 97.8 for these seven constituents, respectively. The detection limit and retention time for the seven constituents ranged from 10.0 to 15.8 ng/mL and from 12 to 21 min. As a result of drug determination, contents (in mg/g) were as follows: AA-I, 0.69-1.77; AA-II, 0.02-0.18; 9-OH AA-I, 0.04-0.12; AA-IVa, 0.76-3.36; AA-Va, 0.04-0.31; AL-I, 0.07-0.36; and AL-II, 0.01-0.09 in Madouling; AA-I, 0.03-0.41; AA-II, 0.01-0.11; 9-OH AA-I, 0.00-0.60; AA-IVa, 0.00-0.77; AA-Va, 0.00-0.14; and AL-I, 0.00-0.04 in Tianxianteng; AA-I, 1.19-4.71; and AA-II, 0.24-1.69 in Qingmuxiang; AA-I, 2.79-5.48; AA-II, 1.06-1.86; 9-OH AA-I, 0.01-0.09; AA-IVa, 0.38-0.69; AA-Va, 0.00-0.61; AL-I, 0.00-0.02; and AL-II, 0.00-0.02 in Bei-madouling-gen; AA-I, 0.64-4.23; AA-II, 0.06-0.40; and AA-IVa, 0.08-0.25; in Guangfangji; and AA-I, 1.88-9.72; AA-II, 0.26-1.88; and AA-IVa, 0.09-0.52 in Guanmutong. The other constituents were not detected in Tianxianteng, Qingmuxiang, Guangfangji and Guanmutong.     

Determination of aristolochic acid I in botanicals and dietary supplements potentially contaminated with aristolochic acid I using LC-UV with confirmation by LC/MS: collaborative study

An interlaboratory study was conducted to evaluate a method for the determination of aristolochic acid I, also known as aristolochic acid A, at levels > 2.00 microg/g in botanical species and dietary supplements potentially contaminated with aristolochic acid I. Aristolochic acid I was extracted from various matrixes with aqueous acetonitrile. The amount of aristolochic acid I present was determined by liquid chromatography (LC) using an ultraviolet (UV) detector with confirmation by LC/mass spectrometry (MS). Thirteen blind duplicates were successfully analyzed by 10 collaborators, and aristolochic acid I was successfully confirmed in 1 blind duplicate by 8 collaborators. For repeatability, the relative standard deviation (RSD(r)) ranged from 1.72 to 16.3% and for reproducibility, the RSDR ranged from 5.42 to 19.8%. HorRat values were not applicable for 2 materials but varied from 0.7 to 1.8 for 11 materials. Each collaborating laboratory had calibration curves with correlation coefficients > 0.998. In addition, all of the collaborators that conducted the confirmation were able to verify the identity of aristolochic acid I using LC/MS/MS (using either ion trap or triple quad).     

Study of polyethylene glycol as a green solvent in the microwave-assisted extraction of flavone and coumarin compounds from medicinal plants

In this paper, the application of polyethylene glycol (PEG) aqueous solution as a green solvent in microwave-assisted extraction (MAE) was firstly developed for the extraction of flavone and coumarin compounds from medicinal plants. The PEG solutions were optimized by a mono-factor test, and the other conditions of MAE including the size of sample, liquid/solid ratio, extraction temperature and extraction time were optimized by means of an orthogonal design L(9) (3(4)). Subsequently, PEG-MAE, organic solvent-MAE, and conventional heating reflux extraction (HRE) were evaluated with nevadensin extraction from Lysionotus pauciflorus, aesculin and aesculetin extraction from Cortex fraxini. Furthermore, the mechanism of PEG-MAE was investigated, including microwave-absorptive property and viscosity of PEG solutions, the kinetic mechanism of PEG-MAE and different microstructures of those samples before and after extraction. Under optimized conditions, the extraction yields of nevadensin from L. pauciflorus, aesculin and aesculetin from C. fraxini were 98.7%, 97.7% and 95.9% in a one-step extraction, respectively. The recoveries of nevadensin, aesculin and aesculetin were in the range of 92.0-103% with relative standard derivation lower than 3.6% by the proposed procedure. Compared with organic solvent-MAE and conventional extraction procedures, the proposed methods were effective and alternative for the extraction of flavone and coumarin compounds from medicinal plants. On the basis of the results, PEG solution as a green solvent in the MAE of active compounds from medicinal plants showed a great promising prospect.     

Determination of aristolochic acids in medicinal plant and herbal product by liquid chromatography-electrospray-ion trap mass spectrometry

This paper describes a liquid chromatography-electrospray-ion trap mass spectrometry (LC-ES-ITMS) method for the determination of aristolochic acid I and II (AA-I and AA-II) in medicinal plants and Chinese herbal remedies. A reversed phase C₁₈ column with gradient elution was utilized. The effects of mobile phase additives, acetic acid and ammonium acetate, on LC separation and ES ionization were investigated. For both AA-I and AA-II, the [M+NH₄]⁺ ion was found to be the precursor ion for target MS/MS analysis. The MS/MS product ion, [M+H−44]⁺, was used for the quantitative measurement of AA-I and AA-II. The linearity was good from 0.03 to 5 μg ml⁻¹ and good correlation (r²=0.999) over the range examined was determined for both AA. The detection limit based on a signal-to-noise ratio of three was 0.012 and 0.015 μg ml⁻¹ for AA-I and AA-II, respectively. Various Chinese herbal remedies obtained from renal failure patients and medicinal plants were examined by this newly developed method.     

Determination of berberine and strychnine in medicinal plants and herbal preparations by pressurized liquid extraction with capillary zone electrophoresis.

A simple and rapid method for the determination of berberine and strychnine in medicinal plants and herbal preparations for regulatory purposes using a home-made pressurized liquid extraction (PLE) system with capillary zone electrophoresis (CZE) using ultraviolet detection at 254 nm was developed. The effects of pH, concentration of buffer, and organic modifiers in the electrophoretic separation were investigated. The buffer used for CZE contained 50 mM ammonium acetate, pH 3.1. The effect of temperature on the extraction efficiency of strychnine in medicinal plants by PLE was demonstrated. Comparable or higher extraction efficiency was achieved with PLE for strychnine in medicinal plants and berberine in herbal preparations compared to soxhlet extraction. The effect of matrix interference in medicinal plants and herbal preparations containing a number of medicinal plants samples using CZE was investigated by standard additional experiments. The reproducibility of the method using PLE with CZE was found to vary between 2.4 and 10.7% (n = 5/6) for different types of samples on different days.     

Application of ionic liquids in the microwave-assisted extraction of polyphenolic compounds from medicinal plants

Ionic liquids (ILs) solutions as solvents were successfully applied in the microwave-assisted extraction (MAE) of polyphenolic compounds from medicinal plants. ILs, its concentration and MAE conditions were investigated in order to extract polyphenolic compounds effectively from Psidium guajava Linn. (P. guajava) leaves and Smilax china (S. china) tubers. The results obtained indicated that the anions and cations of ILs had influences on the extraction of polyphenolic compounds as well as the ILs with electron-rich aromatic π-system enhanced extraction ability. Under the optimized conditions, the extraction yields of the polyphenolic compounds were in the range of 79.5-93.8% with one-step extraction, and meanwhile the recoveries were in the range of 85.2-103% with relative standard deviations (R.S.D.s) lower than 5.6%. Compared to conventional extraction procedures, the results suggested that the proposed method was effective and alternative for the extraction of polyphenolic compounds from medicinal plants. In addition, the extraction mechanisms and the structures of samples before and after extraction were also investigated. ILs solutions as green solvents in the MAE of polyphenolic compounds from medicinal plant samples showed a great promising prospect.     

Pressurized hot water extraction of bioactive or marker compounds in botanicals and medicinal plant materials

To reduce the use of organic solvent, pressurized hot water extraction (PHWE) has been shown to be a feasible option for the extraction of bioactive and marker compounds in botanicals and medicinal plants. The parameters that may affect the extraction efficiencies in PHWE include temperature, extraction time and addition of small percentage of organic solvent or surfactants. Currently, applications of PHWE for the extraction of thermally labile compounds in botanicals are still rather limited. PHWE with and without the additional of a small percentage of organic solvent such as ethanol is highly suited for the chemical standardization and quality control of medicinal plants. At the same time, it can be applied at the pilot scale as a manufacturing process for medicinal plants. Surfactant assisted PHWE was found to enhance the extraction of thermally labile and more hydrophobic species in medicinal plants at a lower temperature. The addition of small amount of surfactants in PHWE is highly suited for the determination of bioactive or marker compounds in medicinal plants. With proper optimization, PHWE was observed to have good extraction efficiency and precision when compared to other reference methods of extraction.     

Simultaneous analysis of six aristolochic acids and five aristolactams in herbal plants and their preparations by high-performance liquid chromatography-diode array detection-fluorescence detection

Aristolochic acid analogues, including aristolochic acids (AAs) and aristolactams (ALs), are known to be nephrotoxic, carcinogenic and mutagenic. In this paper, a high-performance liquid chromatography-diode array detection-fluorescence detection (HPLC-DAD-FLD) method was developed for the simultaneous determination of six AAs together with five ALs. Baseline separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. The hyphenation of DAD and FLD allows the method to directly meet the analysis requirements of most herbal plants with high sensitivity and selectivity. For trace analysis, aristolochic acids were reduced to their corresponding aritstolactams in acidic solution containing iron powder, and then high sensitive detection and quantification were carried out. The method was successfully validated in the matrices of various Aristolochiaceae plants and their preparations. Linearities of around 3-4 orders of magnitude were obtained with correlation coefficients exceeding 0.9970. The detection limits were decreased to 0.2ng/ml. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 5.74%, and the average recovery factors were in the range of 94.5-99.2%.     

Three‐phase hollow fiber liquid‐phase microextraction based on a magnetofluid for the analysis of aristolochic acids in plasma by high‐performance liquid chromatography

A new and fast sample preparation technique based on three‐phase hollow fiber liquid‐phase microextraction with a magnetofluid was developed and successfully used to quantify the aristolochic acid I (AA‐I) and AA‐II in plasma after oral administration of Caulis akebiae extract. Analysis was accomplished by reversed‐phase high‐performance liquid chromatography with fluorescence detection. Parameters that affect the hollow fiber liquid‐phase microextraction processes, such as the solvent type, pH of donor and acceptor phases, content of magnetofluid, salt content, stirring speed, hollow fiber length, extraction temperature, and extraction time, were investigated and optimized. Under the optimized conditions, the preconcentration factors for AA‐I and AA‐II were >627. The calibration curve for two AAs was linear in the range of 0.1–10 ng/mL with the correlation coefficients >0.9997. The intraday and interday precision was <5.71% and the LODs were 11 pg/mL for AA‐I and 13 pg/mL for AA‐II (S/N = 3). The separation and determination of the two AAs in plasma after oral administration of C. akebiae extract were completed by the validated method.