In competitive sports, doping refers to the use of banned athletic performance—enhancing drugs/materials by athletic competitors. The use of doping agents is generally considered both unhealthy and contrary to the ethics of sport. Accordingly, the World Anti-Doping Agency (WADA) has been established with the mission of leading a collaborative worldwide movement for doping-free sport, and its activities focus on the responsibilities provided by the World Anti-Doping Code. One such responsibility is to publish a prohibited list, which identifies the substances and methods prohibited in- and out-of-competition, particularly in sports [1
]. Since 2004, WADA has annually updated the prohibited list. If a substance or method is deemed to meet two of the following three criteria, it can be added to the list: (1) It has the potential to enhance or enhances sport performance, (2) it represents an actual or potential health risk to the athlete, and (3) it violates the spirit of sport described in the introduction to the code. The presence of a prohibited substance or its metabolites or markers (including elevated quantities of endogenous substances) in a specimen or evidence of the use of a prohibited method will be considered an adverse analytical finding (AAF) [2
Dietary supplements are used by athletes in every aspect of the sport, reflecting their popularity. Approximately half of adults in the United States regularly consume different types of dietary supplements [3
]. Regardless of regional, cultural, or economic differences, a similar prevalence is likely to be observed in many other countries. In sports, products described as “supplements” can target various roles in an athlete’s performance plan [4
]. Athletes may consume specific nutrients to maintain optimal health, manage micronutrient deficiencies, and meet energy and macronutrient requirements, which may be difficult to achieve by daily diets alone. Other motivations that supplement consumption as reported by athletes include direct enhancement of performance, manipulation of physique, mitigation of musculoskeletal pain, acceleration of recovery from injury, and improvement of mood. However, some products may contain doping substances, and some herbal extract-based products may be contaminated with agents prohibited in sport. In 1999, Ros et al. reported that the urine of a Dutch professional cyclist who had consumed a herbal food supplement called “Limiet 65 slankheidsdruppels” was discovered to be positive for norpseudoephedrine (20.2 µg/mL) during a doping control [5
]. Both ephedrine and its derivatives such as cathine, methylephedrine, and pseudoephedrine are considered doping substances, and relatively high doses of these substances would exert several harmful effects on the body’s health [6
]. In addition, consuming Papaver somniferum
] and a Chinese herbal medicine called “LiDa Dai Dai Hua Jiao Nang” [8
] could cause athletes to fail a doping test. An AAF resulting from the intake of herbal medicine can be caused by an athlete’s poor knowledge of banned substances indicated on a product label, due to the fact that the labeled ingredients indeed contain banned substances, or due to the athlete’s limited investigation of herbal ingredients.
Lotus plumule, the green embryo of lotus (Nelumbo nucifera
Gaertn) seeds with a bitter taste, has been widely consumed as a tea by Asian people. As a traditional medicine, lotus plumule is used for treating nervous disorders, insomnia, high fever (with restlessness), and cardiovascular disease [9
]. Lotus plumule possesses several pharmacological properties, which are generally considered to be related to its active components, especially flavonoids and alkaloids [10
]. Moreover, lotus plumule contains higenamine, liensinine, dauricine, isoliensinine, neferine, and nuciferine, which exhibit high bioactivity and favorable health care function [12
]. In particular, higenamine was added to the WADA prohibited list in 2017 under the S3 category as a nonselective β2-agonist. Higenamine is a natural constituent of several traditional botanical remedies and is listed as an ingredient in over-the-counter weight loss and sports supplements sold in the United States [14
]; therefore, different dietary supplements used as fat burners could potentially contain this ingredient. WADA established a criterion for higenamine as a banned substance, according to which its analytical finding should not be reported at levels below 10.0 ng/mL (i.e., 50% of the minimum required performance level for β2
]. However, concerns have been raised regarding the potential cause of increased cases of unintentional higenamine doping in the Asian region. Masato et al. [16
] investigated higenamine levels in human urine after the administration of a throat lozenge containing Nandina domestica fruit. They observed that urinary concentrations of higenamine after intake did not reach the cut-off level of 10.0 ng/mL. In the Asian market, lotus plumule is usually available as a concentrated powder, especially in Taiwan; therefore, athletes can easily obtain such products. However, the literature contains only preliminary data regarding this topic; moreover, only a few studies have addressed the topic. Accordingly, the present study assessed the potential risk of lotus plumule consumption by athletes.
Based on this background information, the aims of this study were to investigate both the constituents present in lotus plumule and its implications on doping violation. We first quantitatively analyzed the concentrations of higenamine and related alkaloids in selected products of lotus plumule in Taiwan. For this quantitative analysis, we applied the design of experiment (DOE) method to optimize a microwave-assisted extraction (MAE) process by using response surface methodology (RSM). In addition, chemometric tools, namely hierarchical cluster analysis (HCA) and principal component analysis (PCA), were applied to analyze quality variations and multivariate associations in the studied products. Finally, we conducted a human study with a multiple-administration design for three consecutive days to determine whether supplementation with concentrated herbal extract products (HEPs) of lotus plumule could cause a urinary concentration higher than 10.0 ng/mL and result in an AAF as defined by WADA.
2. Materials and Methods
2.1. Chemicals and Reagents
Higenamine hydrochloride (6,7-dihydroxy-1-(4-hydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline hydrochlorid; purity ≥ 95%) was obtained from Combi-Blocks Inc. (San Diego, CA, USA). Liensinine, dauricine, isoliensinine, neferine, and nuciferine (purity ≥ 98%) were purchased from Grand Chemical Co. Ltd. (Miaoli, Taiwan). Analytical-grade acetonitrile was supplied by J.T. Baker Avantor Performance Materials, Inc. (Center Valley, PA, USA). Ethanol 95% (v/v) was obtained from Echo Chemical (Miaoli, Taiwan). Sodium phosphate monobasic was supplied by Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used in the study were of analytical grade. Pure water was obtained by using the Milli-Q system (Millipore, Bedford, MA, USA).
2.2. Extraction Procedure
2.2.1. MAE Process
MAE was performed using a MARS 5 microwave system (CEM, Matthews, NC, USA). During extraction, time, power, and temperature could be controlled. A preliminary study was performed to determine the effect of the solid-to-solvent ratio on the percent yield of total alkaloids from lotus plumule. A solid-to-solvent ratio of 1:20 provided the maximum concentration of alkaloids (data not shown). Therefore, the extraction was performed by placing 1.0 g of a ground sample of lotus plumule in a vessel with 20 mL of extraction solvent.
2.2.2. DOE Study for MAE
The Box–Behnken design (BBD) was used as the model to determine the polynomial relationship between variables and selected responses (dependent variables, e.g., higenamine concentration). RSM was used to determine the optimal conditions for effectively extracting alkaloids from lotus plumule. The effects of three independent variables, namely extraction time (5–30 min), microwave power (500–1500 W), and temperature (60–120 °C), on the dependent variables were investigated to determine the optimal conditions for maximizing the percent yield of the selected alkaloids from lotus plumule. For each variable, the corresponding low, middle, and high ranges were designated as −1, 0, and +1, respectively (Table 1
). After the variables and their ranges were determined, experiments were established based on the BBD. The complete design comprised 17 experiments with five replicates of the central points to fit the full quadratic equation model. For the polynomial equation, the dependent variables were the extraction yields of higenamine, liensinine, daurcine, isoliensinine, and neferine. After the extraction process, the extracts were immediately cooled to room temperature, filtered through filter paper (Advantec, Tokyo, Japan), and quantitatively adjusted to 20 mL with 95% (v/v) ethanol. The samples were filtered through a 0.45 μm syringe filter and diluted up to 20-fold with 50% (v/v) methanol, after which high-performance liquid chromatography (HPLC) analysis was performed.
2.2.3. Comparison with Other Extraction Methods
The MAE method was compared with the following three extraction methods: Soxhlet extraction (SE), heat reflux extraction (HRE), and ultrasound-assisted extraction (UAE). All relevant experiments were performed in triplicate.
SE: The extraction process was performed using a Soxhlet apparatus. Specifically, 10.0 g of a ground sample of lotus plumule was placed in a paper thimble (28 × 100 mm; Advantec). The extraction was performed for 8 h with 200 mL of 95% (v/v) ethanol at 90 °C. After cooling, the extract was filtered through filter paper and quantitatively adjusted to 200 mL with 95% (v/v) ethanol. The subsequent processes were the same as those in the methods described in the preceding section.
HRE: In this extraction process, 5.0 g of a ground sample of lotus plumule was mixed with 100 mL of 95% (v/v) ethanol in a round-bottom flask and boiled at 90 °C for 4 h. After cooling, the extract was filtered through filter paper and quantitatively adjusted to 100 mL with 95% (v/v) ethanol. The subsequent processes were the same as those in the methods described in the preceding sections.
UAE: An ultrasonic bath (Branson 3210) was used to perform UAE. In this process, 1.0 g of a ground sample of lotus plumule was mixed with 20 mL of 95% (v/v) ethanol in a tube and ultrasonicated for 30 min. Subsequently, the extract was filtered through filter paper and quantitatively adjusted to 20 mL with 95% (v/v) ethanol. The subsequent processes were the same as those in the methods described in the preceding sections.
2.3. Quantitative Determination of Lotus Plumule Products
2.3.1. Chromatographic Conditions and Validation
A Hitachi Chromaster HPLC system (Tokyo, Japan) equipped with a 5160 pump, 5260 autosampler, and 5430 photodiode array ultraviolet (UV) detector was used to validate the chromatographic conditions. All analytical samples were separated using a Luna phenyl-hexyl column (250 × 4.6 mm, i.d. 5 μm; Phenomenex, Torrance, CA, USA). Gradient elution was performed using acetonitrile as solvent A and 0.01 M Na2HPO4 (pH 2.5 adjusted with orthophosphoric acid) as solvent B filtered through a membrane filter (0.45 μm, Millipore) and sonicated before use. The gradient program was as follows: 0–10 min, 10% A; 10.1–35 min, 15–25% A; 35–40 min, 25–30% A; 40.1–45 min, 90% A; and 45.1–55 min, 10% A. The flow rate of the mobile phase was 1.0 mL/min, and the sample injection volume was 20 μL.
Stock solutions of higenamine, liensinine, dauricine, isoliensinine, neferine, and nuciferine were prepared in methanol (1.0 mg/mL) and stored at −70 °C; they were warmed to room temperature before use. Calibration standards were serially diluted with 50% (v/v) methanol to form the working standard solutions (0.1–25.0 μg/mL). Quality control (QC) samples were also prepared in the same manner. Using least squares linear regression, we obtained a calibration curve (y = ax + b) by plotting the peak area (y) against the concentration (x) of the calibration solution. Limit of quantification (LOQ) and limit of detection (LOD) were defined as peaks’ height yielding signal-to-noise (S/N) ratios of 10 and 3, respectively. Moreover, intra- and inter-day precision and accuracy were evaluated through an analysis of variance (ANOVA) based on replicate analysis of QC samples; a standard calibration curve was used for each analytical run. Precision is presented as the relative standard deviation (RSD%) at each concentration level, and accuracy is presented as relative error (RE%).
2.3.2. Preparation of Lotus Plumule Products
Eleven commercially available products in Taiwan containing herbal extract product (HEP) and crude lotus plumule (CLP) were purchased and stored at room temperature until use. All purchased products were well within their expiration dates as provided by the manufacturer. Samples were prepared using the optimized MAE condition and analyzed through the developed HPLC analytical method. Each sample (1.0 g) was mixed with 20 mL of 95% (v/v) ethanol in a vessel and irradiated with microwave power (1046 W) at 120 °C for 26 min. After cooling, the extract was filtered through filter paper and quantitatively adjusted to 20 mL with 95% (v/v) ethanol. The subsequent processes before HPLC analysis were the same as those in the methods described in the preceding sections. All commercial products were subjected to HCA and PCA using R language version 3.1.2 as described previously [17
]. Cluster analysis is a multivariate technique that arranges components on the basis of their characteristics. It classifies components on the basis of their similarity in space. As a result, cluster exhibits high homogeneity in the intergroup and high heterogeneity among different groups [18
]. Regarding HCA, the Euclidean distance was used to calculate the similarity between the vectors of the log-transformed concentrations of the alkaloids that were extracted from all tested products. Subsequently, using the complete-linkage method, we created a hierarchical clustering tree by iteratively merging the most similar clusters. PCA is used to reduce the dimensionality of a dataset consisting of a large number of interrelated variables, while retaining the variation present in the data set as much as possible. This is achieved by converting to a new set of variables which are irrelevant and ordered, so the first few components retain most of the variation in all the original variables [19
]. Regarding PCA, the vectors of the log-transformed concentrations of all tested products were converted into a set of linearly uncorrelated principal components. The top two principal components with the highest explained variance percentage were then used for visualization and analysis.
2.4. Human Study
2.4.1. Enrollment Criteria and Dosage Regimen
The study protocol was approved by the Institutional Review Board of E-Da Hospital (No. EMRP63107N). A human study was conducted over a period of three days to examine the concentration of higenamine in urine after product administration; the product was administered to six healthy men aged between 25 and 35 years. HEP-3 was selected as the study model because it was determined to have the highest higenamine concentration. Participants who consumed any supplements or medications regularly, had a body mass index that was outside the range of 18.5–24 kg/m2, smoked, or consumed alcohol were excluded from the study. Before the study, all participants provided written informed consent. The participants were requested to consume 0.8 g powder of HEP-3 (taken with warm drinking water) three times a day at 8:00, 13:00, and 18:00 for three days consecutively (i.e., days 1, 2, and 3). On the first day, urine specimens were collected before the administration, 1, 2, and 3 h after each administration of the product (i.e., 9:00, 10:00, 11:00, 14:00, 15:00, 16:00, 19:00, 20:00, and 21:00). On the second and third days, urine specimens were collected 1, 2, and 3 h after the last administration of the product (i.e., 19:00, 20:00, and 21:00). The collected urine specimens were stored at −20 °C before analysis.
2.4.2. Urine Sample Preparation and Analysis
Urine samples were collected and underwent enzymatic hydrolysis to evaluate the total amount of higenamine [16
]. An aliquot (1 mL) of each urine sample was pipetted into a glass tube, followed by the addition of 12.5 μL of internal standard (IS, dobutamine-d4
, 500 ng/mL). Moreover, an aliquot (1 mL) of 0.1 M phosphate buffer (pH = 6.0) and 25 μL of β
-glucuronidase (from Escherichia coli
) were added and mixed thoroughly under heating at 50 °C for 60 min for hydrolysis. Subsequently, the hydrolyzed sample was applied onto a solid-phase extraction cartridge (DAU, 3 mL, Bond Elut, Agilent). The solid-phase cartridge was washed with 2 mL of deionized water, 2 mL of 0.1 M HCOOH, and 2 mL of methanol. The eluate was collected from 2 mL of a mixture of dichloromethane, isopropyl alcohol, and NH4
OH (80:20:2, v/v/v). The supernatant organic phase was obtained and dried under nitrogen at 60 °C. The residue was reconstituted using 200 μL of a mixture of 0.02% (v/v) formic acid and methanol (85:15, v/v), and 20 μL was injected into an HPLC tandem mass spectrometry (HPLC-MS/MS) system.
The urine samples were analyzed on an Agilent 1260 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) and an Agilent 6470 triple-quadruple mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) operated in positive ion mode. The analytes were separated using a Supelco Ascentis HP-C18 column (100 × 2.1 mm, id. 3μm; Sigma–Aldrich) and a Supelco Discovery HS-C18 guard column (20 × 2.1 mm; id, 3μm; Sigma–Aldrich). Gradient elution was employed using 0.1% (v/v) formic acid as solvent A and acetonitrile as solvent B. The gradient program was as follows: 0–0.5 min, 95% A; 0.5–6.0 min, 95–5% A; 6.0–6.1 min, 5–95% A; and 6.1–15.0 min, 95% A. The flow rate was set at 0.3 mL/min, and the injection volume was 20 μL. The total run time was 15 min for each sample. In multiple reaction monitoring, the detector response is due to a specific transition of molecular ions to fragments (molecular ions ⟶ fragment). The fragmentations of 272.3 ⟶ 107.0 m/z (CE: 24 eV) and 272.3 ⟶ 161.0 m/z (CE: 16 eV) ion were selected for higenamine, and that of the IS were 306.3 ⟶141.0 m/z (CE: 24 eV) and 306.3 ⟶ 107.0 (CE: 36 eV). The linearity, ranging from 0.5 to 30.0 ng/mL, was validated for higenamine, and the coefficient of determination (R2
) for calibration was determined to be 0.9989. The observed intra- and inter-day accuracy ranged from 89.3% to 96.2%, and the observed precision ranged from 0.6% to 6.3%. The mean percent recovery of higenamine varied from 73.8% to 82.7%. Analysis data are presented in Table S1
2.5. Statistical Analysis
The obtained data are expressed as mean ± standard deviation. ANOVA for RSM was performed using Design-Expert 6.0.3 software (StatEase Inc., Minneapolis, MN, USA). The effects of the different extraction methods on alkaloid yield were compared statistically using ANOVA followed by a post hoc Scheffe test using SPSS v14.0 (SPSS Inc., Chicago, IL, USA). A p-value of < 0.05 was considered to indicate a significant difference.
This study presents a validated HPLC-DAD method for identifying and quantifying higenamine and other major bioactive alkaloids, namely liensinine, dauricine, isoliensinine, neferine, and nuciferine, in lotus plumule. Furthermore, an optimized MAE method was used to extract alkaloids from lotus plumule using RSM to screen lotus plumule products in Taiwan. HCA and PCA provided a useful basis for overall evaluation of the quality differences among different lotus plumule products. Results obtained from comparing multiple extraction methods indicate that MAE achieved the highest extraction yields for all alkaloids in lotus plumule. Moreover, high concentrations of higenamine could be found in lotus plumule. Results obtained from a human study demonstrate that urinary concentrations of higenamine in all participants with a common consumption pattern exceeded the cut-off of 10.0 ng/mL. We suggest that athletes should avoid consuming lotus plumule during in- and out-of-competition periods and pay special attention to the use of herbal products. When athletes need to use lotus plumule containing products, they should consult health professionals, such as a physician or pharmacist, to lower the misuse risk of higenamine.