QUANTITATIVE ANALYSIS OF CAFFEINE, EPHEDRINE, AND YOHIMBINE IN STIMULANT-BASED FAT BURNER SUPPLEMENTS

QUANTITATIVE ANALYSIS OF CAFFEINE, EPHEDRINE, AND YOHIMBINE IN STIMULANT-BASED FAT BURNER SUPPLEMENTS

M. S. Nithish Kumar, S. Kalai Vani, S. Deepa * and K. Sathesh Kumar

Sri Ramachandra Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India.

ABSTRACT: Introduction: The use of supplements in the fitness industry has experienced a notable surge due to the growing awareness of their potential health benefits. Fat burners, a popular type of supplement, are commonly employed to assist in weight loss. However, it is important to acknowledge that fat burners can have adverse effects on the body. Common side effects include increased heart rate, elevated blood pressure, sleep disturbances, restlessness, heightened anxiety, and digestive issues. They often contain ingredients like green tea extract, herbs, and minerals, which can enhance energy levels and speed up metabolism. Some fat burners also include stimulants like caffeine, ephedrine, and yohimbine, which can lead to additional problems like rapid heart rate, dehydration, and agitation. In certain cases, fat burners may even pose a risk to liver health. Objective: To ensure the safety and accuracy of fat burner supplements, a method known as RP-HPLC (Reversed Phase High-Performance Liquid Chromatography) was developed and validated according to the guidelines established by ICH of Technical Requirements for Pharmaceuticals for Human Use. This method enables the precise quantification of caffeine, ephedrine, and yohimbine in fat burner supplements that contain stimulants. Results and Discussion: The study's results demonstrated that the developed RP-HPLC method was suitable, specific, and accurate for analyzing caffeine, ephedrine, and yohimbine. The linear regression data revealed that the method-maintained linearity across a concentration range of 150μg to 900μg for caffeine, 2.5μg to 15μg for yohimbine, and 25μg to 150μg for ephedrine, effectively fulfilling its intended purpose. Analysis of samples indicated that unbranded fat burner products, including those not labeled as containing caffeine, contained varying amounts of caffeine ranging from 199.8 mg to 297.3 mg. Ephedrine was absent in all ephedra-based supplements, while yohimbine exceeded the labeled quantities. These findings emphasize the importance of implementing analytical controls for fat burners to ensure consumer safety. Conclusion: The use of fat burner supplements has gained popularity in the fitness industry as a means to aid weight loss. However, it is crucial to be aware of the potential side effects associated with these supplements. The development and validation of the RP-HPLC method have provided a reliable approach to analyze the components of fat burners, specifically caffeine, ephedrine, and yohimbine. The study's findings underscore the necessity for proper regulation and quality control measures to ensure the safety and accuracy of fat burner supplements for consumers.

Keywords: Fat burner supplements, Caffeine, Ephedrine, Yohimbine, RP-HPLC

INTRODUCTION: The use of supplements in the fitness industry is on the rise, as more people have become aware of their potential health benefits ¹. Supplements can be used to improve overall health and performance, as well as to supplement a healthy diet and exercise program ².

Supplements can make it easier for people to achieve their fitness goals and can also be used to enhance the results of exercise and nutrition ³. Many people choose to use supplements to support their fitness goals, such as increasing muscle mass or losing weight ^{4, 5}.

Additionally, there are a variety of supplements available to assist with performance, such as pre-workout drinks, post-workout shakes, and energy boosters ⁶. Fat burners are one of the supplements that are frequently utilized to shed weight. Supplements called "fat burners" aid in the burning of fat and weight loss ⁷. They are available as pills, powders, or liquids that can be consumed. Caffeine, yohimbine, green tea extract, and different herbs and minerals are common components of fat burners and may assist to increase energy and speeding up metabolism ⁸. Some fat burners also include appetite suppressants to aid in lowering food cravings ⁹. Fat burners can have significant consequences for your health ¹⁰. The most prevalent negative reactions include increased heartbeat and elevated blood pressure, insomnia, jitteriness, higher anxiety levels, and digestive difficulties ^{11, 12}.

Many fat burners also contain stimulants like caffeine ephedrine and yohimbine, which can lead to additional issues like increased heart rate, dehydration, and agitation. Fat burners can even harm the liver in some cases ¹³. Supplements are not regulated by the FDA, so it is difficult to determine which ingredients are safe and which may be harmful ¹⁴. Most of the supplements contain undeclared contents of caffeine like stimulant-based fat burners¹⁵ Caffeine's maximum recommended daily dose varies according to age and health. Adults shouldn't ingest in excess of 400 milligrams (mg) of caffeine per day, according to the FDA ¹⁶. This is approximately four cups of coffee. Pregnant women, people with certain medical disorders, and people taking certain drugs, on the other hand, ought to restrict their caffeine intake to 200 mg per day or less ¹⁷.

Ephedra-based fat burners have been prohibited in the United States since 2004 due to safety concerns ¹⁸. The active ingredient, ephedrine, has been linked to serious side effects such as heart attack and stroke ¹⁹. As a result, these products are no longer available for purchase in the United States and should be avoided ²⁰. The Ministry of Health and Family Welfare outlawed Ephedra in India. The decision to ban ephedra was made in response to a report from the Drugs Technical Advisory Board, which concluded that there were no therapeutic benefits associated with its use and that its side effects posed serious health risks ²¹. The main issues with yohimbine as a dietary supplement are incorrect labeling and potentially serious side effects ²². Yohimbine supplements are therefore prohibited in many countries, including Australia, Canada, and the United Kingdom, for this reason ²³. This research is intended to quantify the amount of caffeine, yohimbine, and ephedrine in marketed fat burner supplements by HPLC analysis.

MATERIALS AND METHODS:

Chemicals used: Acetonitrile HPLC, methanol HPLC AR Grade, Sodium hydrogen phosphate dehydrate AR Grade, ortho-phosphoric acid AR grade, Millipore Water HPLC Grade, Ephedrine Working Reference Standard, Caffeine working reference standard and yohimbine working reference standard.

Preparation of Buffer: 3.9g of sodium dihydrogen phosphate dehydrate was added into a 500ml-volumetric flask. 250ml of water was added and sonicated for 5 min made up the volume with water and the pH was adjusted to 3.6 with 10%v/v orthophosphoric acid.

Preparation of Stock Solutions:

Standard Preparation:

Caffeine Stock Solution: Accurately weigh and transfer about 40mg of Caffeine into a 20ml volumetric Flask and add 5ml of water and sonicate and makeup to the volume with water.

Ephedrine Stock Solution: Accurately weigh and transfer about 20mg of ephedrine into a 20ml volumetric Flask and makeup up the volume with water.

Yohimbine Stock Solution: Accurately weigh and transfer about 10mg of yohimbine into a 50-volumetric Flask and makeup up the volume with water.

Working Standard Solution of Caffeine, Ephedrine, and Yohimbine: Take 3ml of caffeine stock solution, 0.5ml of yohimbine stock solution and 1 ml of ephedrine sock solution respectively transferred into 10ml volumetric flask and makeup to the volume with water.

Liquid Chromatography Conditions: The HPLC system consisted of a binary pump Separation module fitted with a C18 column (250 mm × 4.6 mm). The injection volume used was 10 μL. A Shimadzo HPLC system equipped with a Shimadzo PDA detector was used ^{24, 25, 26}. The gradient flow via a symmetric C 18 column (250 mm 4.6 mm, 5 m spherical particles) was operated by the solvent delivery system with mobile phase A 50mm sodium dihydrogen phosphate dehydrate buffer calibrated to pH 3.6 and mobile phase B as acetonitrile, respectively ²⁷. The mobile phase flow rate was 1 mL/min with gradient flow of 90% buffer for 3-10 min, 60% buffer for 10-15 min, and 90% buffer for 15 to 20min respectively, and the run time was 20 minutes. Filtration via a 5 m Millipore membrane filter and sonication for 10 minutes were used to degas the mixture. The HPLC system was operated at 25 °C and detection was at 211 nm.

Analytical Method Validation: The method used for the quantification of caffeine, ephedrine, and yohimbine is validated in terms of system suitability, accuracy, reproducibility, linearity, specificity, LOD, LOQ, and robustness according to ICH Q₂ (R2) quality guidelines ^{28, 29}.

System Suitability: The system suitability of the analytical method is verified by five replicate injections of a working standard solution of Caffeine, ephedrine, and yohimbine was made and the system suitability parameter was evaluated by calculating %RSD ³⁰.

Specificity: It was performed by analyzing the placebo interference and comparing it with a standard chromatogram. In a 50 ml volumetric flask, 200 mg of placebo were weighed, transferred, and mixed with 25 ml of water before being sonicated for 5 minutes to make up the volume. Further dilution was made by taking 1ml from the placebo stock solution and transferring it into a 10ml volumetric flask and made up to the volume with water and transferred to a vial and subjected to injection ^{31, 32}.

Linearity and Range: The linearity of the detector response of the assay method was demonstrated by injecting five standard solutions with concentrations ranging from 25% to 150% of the target test concentration. Plot a graph that shows peak area versus concentration. The five levels of linearity chosen were 25%, 30%, 50%, 100%, and 150%. The regression line's slope, Y-intercept, and correlation coefficient (r) were all calculated ³³.

Accuracy: The accuracy of the assay method can be validated by accurately quantifying the active ingredient in the product by spiking the active ingredient in a placebo at various concentrations ranging from 50% to 150% of the target test concentration. Weigh accurately 200mg of placebo and a suitable amount of caffeine, yohimbine, and ephedrine standard as per specified accuracy levels of 50%, 100%, and 150% and transfer it to a 50 mL volumetric flask. Add 25 ml of diluent; sonicate it for 10 minutes and makeup to volume with Diluent. Further dilution was made by taking 1ml from the placebo stock solution and transferring it into a 10ml volumetric flask and made up to the volume with water. Accuracy samples were prepared in duplicates.

Precision (Repeatability): The precision of the assay method can be validated by using artificially prepared samples by spiking 100% concentration of the analyte in the sample matrix. Six artificially prepared samples were prepared by spiking 100% concentration of caffeine, ephedrine, and yohimbine in the sample matrix and all the samples were injected subsequently. The assay and relative standard deviation of assay results were determined.

Intermediate Precision: The intermediate precision was established by preparing six sample solutions on the product, of the same batches used for repeatability, as per the test method on different days by different analysts and injected on HPLC.  Assay and relative standard deviation of assay results and also the overall relative standard deviation of % assay results from the total of twelve determinations (Six from precision study and another six from intermediate precision) were determined ³⁴.

Robustness:

Effect of Variation in Mobile Phase Composition: To demonstrate the robustness, check the system suitability parameters by injecting standard preparation by using two mobile phases with variation in +10% and -%10 of acetonitrile phase from the actual composition and evaluate the system suitability parameters. Also, analyze the artificially prepared sample solutions in duplicate and calculate the % difference between assay values from such method parameters and variation parameters.

Effect of Variation in Flow Rate: To demonstrate the robustness of the test method, check the system suitability parameters by injecting standard preparation into the HPLC system with a flow rate of ±10%. Evaluate the system suitability parameters. Also, analyze the artificially prepared sample solutions in duplicate and calculate the % difference between assay value from such method parameters and variation parameters ³⁵.

Real-time Sample Analysis: Five types of stimulant-based fat burners were purchased online. The selected fat burners supplements were said to contain a combination of caffeine, ephedra extract, and yohimbine, and also supplements containing a single compound of caffeine, ephedra extract, and yohimbine respectively

Sample Preparation: Five capsules were taken and the contents were emptied and the average weight was taken. The amount to be taken was calculated from the average weight and label claim of the sample and it’s transferred into a 50ml volumetric flask. 25ml of water was then added and sonicated for 30min. Further dilution was made by taking 1ml of the primary stock solution and transferring it into a 10ml volumetric flask and making up the volume with water.

RESULTS AND DISCUSSION:

Analytical Method Validation:

System Suitability: The %RSD of caffeine, yohimbine, and ephedrine was found to be 0.3%, 0.5%, and 0.1% respectively. From the system suitability studies, it is evident that the measurement system and the analytical operations associated with the analytical procedure are adequate for the intended analysis.

TABLE 1: SYSTEM SUITABILITY PARAMETER

Specificity: The chromatograms of the placebo showed no peak at the retention time of the principal peak. Hence, it is concluded that the method is found to be specific.

FIG. 1: HPLC CHROMATOGRAM OF EPHEDRINE, CAFFEINE, YOHIMBINE

Linearity: A linearity graph of the area at each level against the concentration (μg) of caffeine, yohimbine, and ephedrine was plotted and was found to be linear. The correlation coefficient of caffeine, yohimbine, and ephedrine was found to be 0.9945,0.9919, and 0.9999 respectively.

The linear regression data shows that the method is linear over the entire concentration range of 150μg to 900μg for caffeine, 2.5μg to 15μg for yohimbine, and 25μg to 150μg for ephedrine and it is adequate for its intended concentration range.

 TABLE 2: LINEARITY DATA OF CAFFEINE

FIG. 2: LINEARITY GRAPH OF CAFFEINE

TABLE 3: LINEARITY DATA OF YOHIMBINE

FIG. 3: LINEARITY GRAPH OF YOHIMBINE

TABLE 4: LINEARITY DATA OF EPHEDRINE

FIG. 4: LINEARITY DATA OF EPHEDRINE

Accuracy: The percentage recovery of Caffeine, ephedrine, and yohimbine from the placebo at each of the levels is more than 90.0%. Therefore, the method is considered accurate and precise concerning measuring the Concentration of caffeine, ephedrine, and yohimbine in placebo-spiked samples.

TABLE 5: ACCURACY DATA OF CAFFEINE

TABLE 6: ACCURACY DATA OF YOHIMBINE

TABLE 7: ACCURACY DATA OF EPHEDRINE

Precision (Repeatability): The %RSD of the assay method was found to be less than 2% for caffeine, ephedrine, and yohimbine respectively.

The analytical method meets the pre-established acceptance criteria and hence considered that the method is precise.

TABLE 8: REPEATABILITY DATA OF CAFFEINE

TABLE 9: REPEATABILITY DATA OF EPHEDRINE

TABLE 10: REPEATABILITY DATA OF YOHIMBINE

Intermediate Precision: The %RSD of the assay method was found to be less than 2% for caffeine, ephedrine, and yohimbine respectively.

The analytical method meets the pre-established acceptance criteria of intermediate precision.

TABLE 11: INTERMEDIATE PRECISION OF CAFFEINE

TABLE 12: INTERMEDIATE PRECISION OF EPHEDRINE

TABLE 13: INTERMEDIATE PRECISION OF YOHIMBINE

Robustness: After individually modifying the flow rate and organic phase conditions from the suggested technique, no appreciable variation in retention time was seen. All other system suitability parameter calculations were made following the accepted standards, and the data produced is equivalent to the real conditions. According to the abovementioned findings, it can be said that the approach is unaffected by slight, intentional changes in flow rate and organic phase composition.

TABLE 14: ROBUSTNESS OF CAFFEINE

TABLE 15: ROBUSTNESS OF EPHEDRINE

TABLE 16: ROBUSTNESS OF YOHIMBINE

Real-Time Sample Analysis:

TABLE 17: REAL-TIME SAMPLE ANALYSIS

FIG. 5: FAT BURNERS- 1

FIG. 6: FAT BURNERS- 2

FIG. 7: FAT BURNERS- 3

FIG. 8: FAT BURNERS- 4

FIG. 9: FAT BURNERS- 5

The analysis of five fat burner samples yielded noteworthy findings. Firstly, all five samples, including those (3, 4, and 5) as mentioned in Table 17 lacking a caffeine declaration on the label, contained caffeine. The quantity of yohimbine present exceeded the declared amounts (1,4) as mentioned in Table 17, while ephedrine was absent from all fat burners containing ephedra. Consequently, ephedra extract devoid of ephedrine has been utilized in these products (1,3) as mentioned in Table 17. However, the efficacy of ephedrine-free ephedra extract for weight loss remains unknown. Three of the fat burner samples exhibited adulterated caffeine, a common occurrence (3,4,5) as mentioned in Table 17. While caffeine has been associated with weight loss benefits, it is essential to adhere to the maximum safe dosage of 400mg. The caffeine content in these fat burners ranged from 199.8 mg to 297.3 mg, raising concerns about potential excessive caffeine consumption. This highlights the importance of cautious caffeine intake. Furthermore, the analysis revealed higher concentrations of yohimbine than indicated on the labels, emphasizing the significance of implementing rigorous analytical controls for fat burners to ensure consumer safety.

CONCLUSION: In conclusion, the quantitative analysis of caffeine, ephedrine, and yohimbine in stimulant-based fat burner supplements plays a crucial role in determining the composition and safety of these products. The analysis provides valuable information regarding the concentration levels of these compounds, which are known to have stimulant and thermogenic effects. By employing reliable analytical techniques such as high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS), researchers can accurately measure and quantify the amounts of caffeine, ephedrine, and yohimbine present in fat burner supplements. This information is essential for regulatory purposes and consumer safety, as it helps ensure that the labeled contents of the supplements align with their actual composition. An RP-HPLC/DAD method was developed to efficiently analyze caffeine, ephedrine, and yohimbine in stimulant-based fat burner supplements, providing results within a 15-minute timeframe. The application of RP-HPLC/DAD in the analysis of five fat burner supplements revealed discrepancies between the labeled and actual amounts of yohimbine, with higher concentrations detected than indicated on the labels. Furthermore, most of the fat burner samples exhibited adulterated caffeine content, indicating the presence of additional caffeine sources not disclosed on the labels. The analysis allows for monitoring the concentration levels of these compounds, enabling manufacturers to comply with regulatory guidelines and avoid potentially harmful or adulterated products.

Additionally, it provides consumers with valuable information, enabling them to make informed decisions about the supplements they choose to use. The inclusion of these substances in fat burner supplements is a matter of concern due to their status as controlled substances and associated toxicological considerations. It is crucial to acknowledge the potential adverse consequences that may arise from the consumption of these compounds. However, the study did not explore the possible interactions between these substances and other medications, leaving a gap in our understanding of their combined effects. Furthermore, the quantitative analysis of these stimulants can also contribute to scientific research in the field of fat-burning and weight loss. It provides insights into the effectiveness of these compounds and their potential synergistic or adverse effects when combined in fat burner supplements. Expanding the scope of this research would be beneficial, encompassing a quantitative analysis of daily caffeine intake and a comprehensive examination of individuals who regularly use fat burners for weight loss. Such an extension would provide valuable insights into the overall caffeine exposure and the potential health implications associated with long-term fat burner usage. In summary, the quantitative analysis of caffeine, ephedrine, and yohimbine in stimulant-based fat burner supplements is crucial for regulatory compliance, consumer safety, and advancing scientific knowledge. It ensures accurate labeling, monitors concentration levels, and helps promote the responsible use of these supplements.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Kumar MSN, Vani SK, Deepa S and Kumar KS: Quantitative analysis of caffeine, ephedrine, and yohimbine in stimulant-based fat burner supplements. Int J Pharm Sci & Res 2025; 16(4): 1115-26. doi: 10.13040/IJPSR.0975-8232.16(4).1115-26.

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