Establishing The Purity of Nutraceuticals and Energy Drinks with NMR

Andy McAlister

Editor of 'the resonance'. With a multi-industry background in communications, including writing and editing, Andy McAlister has worked with Bruker for more than 11 years.

 

Nutraceuticals and energy drinks are burgeoning billion dollar industries that make use of natural products. To ensure the end product is consistent with labeling and complies with legislation, manufacturers rely on sensitive and specific analytical methods such as nuclear magnetic resonance – NMR spectroscopy – to guarantee freedom from contaminants and safeguard quality.

Introduction

The international herb supplement and remedies market has become a multi-billion dollar industry that is anticipated to reach the $93 billion level by 2015[1], with a corresponding rise in sales of the constituent botanical compounds. The growing use of natural health products, largely driven by the health benefits and cost savings offered in comparison with conventional pharmaceuticals, has given cause for concern, with the possibility that nutraceuticals may be adulterated to enhance efficacy or for economic gain. Such adulteration may also occur inadvertently.
Another booming economy in the United States is the energy drinks market, worth nine billion dollars in 2011. Many energy drinks contain natural product extracts such as ginseng, ginkgo and milk thistle, as well as vitamins, for example niacin and ascorbic acid. As a result, they are classified as dietary supplements and must comply with the 2007 FDA cGMP ruling which requires manufacturers to ensure the identity, purity, strength and composition of their products.
There is a clear need for analytical methods capable of addressing these issues, and NMR-based screening tools have proved very effective. This article describes the application of NMR to the detection of adulteration of the nutraceuticals RegenErect™, Zi Xiu Tang Bee Pollen and borage oil, evaluating product authenticity, and screening of Monster Energy® Drink.
 

Sample selection

The nutraceuticals and energy drink studied were selected for a variety of reasons. RegenErect, a natural sexual enhancement product stated to contain silkworm, oyster, goji and white willow bark extracts, had been the subject of an FDA recall due to the reported presence of sulfoaildenafil (Figure 1), an unapproved, undeclared analog of sildenafil (Figure 2), the active compound of Viagra®.

Figure 1: The structure of sulfoaildenafil.

Figure 2: The structure of sildenafil.

An FDA product recall was also issued for Zi Xiu Tang Bee Pollen, which was said to be adulterated with the controlled substance sibutramine (Figure 3), presenting a significant risk for patients with a history of coronary artery disease, congestive heart failure, arrhythmias or stroke.

Figure 3: The structure of sibutramine.

Borage oil is a major source of ɣ-linolenic acid (GLA), and is used for the prevention and treatment of a number of clinical conditions including osteoporosis, diabetes and cancer. However, cultivation of borage plants is expensive and, as a result, there is a temptation to adulterate the product with the cheaper safflower oil that does not contain GLA. In the case of Monster Energy Drink, a complex mixture of many different compounds must be verified to ensure compliance with the 2007 FDA cGMP regulations for dietary supplements.

NMR analysis

Nutraceuticals

Zi Xiu Tang Bee Pollen and RegenErect were purchased online immediately after the FDA adulteration notices were issued, and borage oil was adulterated with 0.1 to 10 % safflower oil in the laboratory. The Zi Xiu Tang Bee Pollen and RegenErect NMR samples were prepared by dissolving 25 mg of each material in 1 ml of dimethyl sulfoxide-d6 (DMSO-d6) containing 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). The borage oil NMR samples were prepared using chloroform (CDC1³).
NMR data was acquired using the workflow-oriented TopSpin™ 3.2 user interface (Bruker) on an AVANCE™ III 400 MHz NMR spectrometer (Bruker) at 298 K. CPMG1d (Carr-Purcell-Meiboom-Gill sequence in 1D mode) and NOESY1d (nuclear Overhauser effect spectroscopy in 1D mode) pulse sequences were performed with 64 scans, 64K points, and a relaxation delay of 4 and 10s respectively. Assure™ – Raw Material Screening (RMS) v1.5 software was used for automated evaluation of the NMR data, enabling identification of sample components through spectral library matching and chemometric analysis. Quantification was performed with Assure-RMS software, using PULCON[2] (pulse length based concentration measurements) to provide a ‘calibrated NMR spectrometer’. The linearity of the NMR spectrometer and the proportionality of the NMR signal to the number of atoms giving rise to the signal allows for the use of calibration to a single quantification standard sample. Freedom from the need to match extinction coefficients or ionization efficiency enables the quantification calibration to be applied to other compounds.

 

Energy drink

A 400 µl aliquot of a 16 oz. can of Monster Energy Drink was mixed with 200 µl of D₂O (deuterium oxide) phosphate buffer and placed in an NMR tube with 1 µl of 300 mM DSS in D₂O. NMR spectra of the known constituents of the drink were obtained from the Bruker Biofluid Reference Compound Database or in-house spectral databases.
All NMR experiments were performed using an AVANCE III 600 MHz spectrometer at 298 K with a 5 mm TCI CryoProbe™ (Bruker). ¹H NMR spectra were referenced to DSS at 0.00 ppm. Standard ¹H NOESY spectra with presaturation were acquired with 32 scans, 64K points and a 20s relaxation delay, at a set temperature of 313 K. The acquisition time was 13 minutes. All spectra were acquired, processed and analyzed automatically, screening samples using Assure-RMS v1.5. Evaluation of the targeted screening method for Monster Energy Drink was carried out using the interactive method builder interface; a spectral database was created using the spectral base builder for the individual compounds in Monster Energy Drink.

Results and discussion

Nutraceuticals

Automated, targeted screening was used to analyze RegenErect, using an NMR spectral database containing sulfoaildenafil to establish the presence or absence of the undeclared drug compound. In addition to the constituents listed in the RegenErect supplement facts – silkworm, white willow bark, goji and oyster extracts – as determined by the FDA in 2011, large quantities of sulfoaildenafil were detected. Quantification was based on the peaks at 8.00, 7.92 and 7.44 ppm (Figure 4, Table 1).

Figure 4: NMR spectrum of RegenErect. Peaks marked * are sulfoaildenafil.

Table 1: Quantification of sulfoaildenafil in a pure RegenErect standard and the adulterated product. Large quantities of the undeclared drug were detected.

Zi Xiu Tang Bee Pollen was similarly analyzed by targeted NMR screening using Assure-RMS. Six samples from two different

lot numbers were analyzed for sibutramine by comparison to a spectral database of potential product constituents. Despite the packaging being marked as ‘GMP’ and ‘Anti-counterfeit’, the NMR spectra clearly show the presence of large quantities of materials that were not declared in the supplement facts (Figure 5).

Figure 5: NMR spectrum of Zi Xiu Tang Bee Pollen spectrum showing declared constituents (Chinese yam, bee pollen, wolfberry and lotus seed) and adulterants. Large quantities of materials in the aromatic and aliphatic regions, highlighted by the red boxes, were not identified in the supplement facts.

Chemical shift and lineshape analysis of the aromatic signals were used to identify and quantify two adulterants; sibutramine and phenolphthalein (Figure 6). A spectral database of sibutramine and phenolphthalein was generated from the adulterated sample and available literature[3,4].

Figure 6. Sibutramine and phenolphthalein were identified and quantified using chemical shift and lineshape analysis.

The targeted NMR screening approach was also applied to the detection of safflower oil adulteration of borage oil, using Assure-RMS with multivariate chemometric tools to quantify the extent of contamination. From figure 7 the NMR spectra of 100 % borage oil and 100 % safflower oil show remarkable similarity, but have specific differences in some of the chemical shift regions, such as 1.7, 2.0 and 2.8ppm. However, in the industry, products sold as borage oil are rarely 100 % safflower oil adulterant; a mixture containing, for example, 10 % adulterant is more common.

Figure 7: NMR spectra of borage and safflower oils.

Replicate samples (n = 5) of each of the solutions in Table 2 were used to calibrate an Assure-RMS partial least squares (PLS) model, and internal cross-validation of the borage/safflower mixtures (Figure 8) was performed (12 PLS components, R2 = 0.99, root mean square error of cross-validation (RMSECV) = 0.017).

Table 2: Calibration solutions for the analysis of adulterated borage oil samples.

Figure 8: Automated validation tests were used for PLS model validation.

Automated results from two adulterated samples of borage oil (Table 3) show good correlation between the actual and predicted amount of safflower oil present.

Table 3: Good correlation was achieved between the actual and predicted amounts of safflower oil present. The percentage error is calculated using a 95 % confidence interval from the RMSECV.

Energy drink

Figure 9 shows the ¹H NMR spectrum of Monster Energy Drink, with many spectral resonances observed. The specific constituents – sugars, stimulants, organic acids and herbal extract – were identified by an NMR spectral database created using Assure-RMS. Each sample was automatically screened for the compounds in the spectral database, with verification of those substances identified. Figure 10 shows the chemical shift regions of the individual compounds overlaid on the Monster Energy Drink spectrum.

Figure 9: ¹H NMR spectrum of Monster Energy Drink showing its individual constituents recorded in the NMR spectral database. The dotted lines indicate the regions used for quantification.

Figure 10. Overlay of the standard spectra with the spectrum of Monster Energy Drink.

Detailed study of the peak multiplets and lineshape analysis (Figure 11) help to confirm compound identity and enable quantification (Table 4). Ginseng extract was identified, but not quantified, in this study.

Figure 11: Detailed study of the peak multiplets and lineshape analysis help to confirm compound identification and enable quantification.

Table 4: Quantification of the compounds identified in Monster Energy Drink.

Conclusions

NMR is well suited to the evaluation of herbal supplements; as each botanical material has a unique fingerprint, drug adulterants are typically very obvious from the ¹H NMR spectrum. Quality control is easily performed using an automated NMR protocol, allowing the identity, purity, strength and composition to be reported. The high reproducibility of NMR enables the use of spectral databases, which is key to the automated identification of adulterants using a targeted NMR screening approach, quantifying components through lineshape analysis or PLS regression.
For complex mixtures such as energy drinks, NMR spectroscopy allows visualization of the individual components with minimal sample preparation, and without the need for separation. An automated NMR-based screening tool such as Assure-RMS could be a powerful approach to the verification of the vast number of dietary supplements found in energy drinks, providing the industry and regulatory authorities with a method for product assessment and quality control.

Reference
1. Source: San Jose, CA-based Global Industry Analysts, Inc.
2. Wider, G. and Dreier, L. Measuring Protein Concentrations by NMR Spectroscopy. J Am Chem Soc, 2006. 128(8): p. 2571-2576.
3. Jeffery J. et al. Synthesis of sibutramine, a novel cyclobutylalkylamine useful in the treatment of obesity, and its major human metabolites J Chem Soc Perkin Trans 1, 1996. 21: p. 2583-2590.
4. Vaysse, J. et al. Analysis of adulterated herbal medicines and dietary supplements marketed for weight loss by DOSY 1H-NMR. Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2010. 27(7): p. 903-916.

35 Most Exotic Destinations For Your Next Vacation

We live on a beautiful planet that offers many stunning landscapes, tropical paradises, mist shrouded volcanoes and unbelievable beaches. Raising a toast to the explorer within you, here are a few exotic getaways for you to consider for your next vacation.

1. Bay of Kotor, Montenegro

Simply known as Boka, this winding bay will engulf you with its emerald waters and picturesque mountains. The shoreline houses beautiful medieval towns like Perast, where you can stop for a meal and interact with the locals.

 2. Petra, Jordan

Rising out of a cliff between the Red and Dead Sea, the lost city of Petra still has secrets to reveal. It has several tombs and carved structures spread across a sandy landscape. Many scenes from the Hollywood blockbuster Indiana Jones and the Last Crusade were filmed here.

3. Longsheng Rice Terrace, China

These stunningly beautiful terraced rice fields received the name of Dragon’s Backbone because the rice terraces resemble a dragon’s scales while the summit of the mountain range looks like the backbone of the dragon.

4. Gozo, Malta

The enchanting island of Gozo is an integral part of the Maltese archipelago. Besides being one of the top diving destinations in the Mediterranean, it boasts of mystical backwaters, historical forts and amazing panoramas.

5. The Great Blue Hole, Belize

This submarine sinkhole off the coast of Belize is in every scuba diver’s bucket list. Even if you are not into enjoying a myriad of species underwater, it’s totally worth flying over the Blue Hole for a breathtaking view.

 6. Amer fort, India

A majestic structure sprawling on a hilltop, Amer fort’s architecture is a magnificent blend of Mughal and Hindu influences. Made out of red sandstone and white marble, it consists of a series of courtyards, palaces, halls, and gardens.

7. Forest of Knives, Madagascar

It is truly intriguing that wildlife can happily exist amidst razor sharp vertical rocks in this mesmerizing forest. You can hike around the bizarre pinnacles of limestone and observe different kinds of birds and lemurs.

8. Namje, Nepal

There are no roads to Namje. The only way to get to the Nepalese village is along a series of footpaths with views of Mount Makalu, the world’s fifth-tallest peak.

9. Taha’a, French Polynesia

Most people head to Bora Bora or Tahiti, but why not explore the intimate and exquisite island of Taha’a instead? The landscape is dotted with several vanilla plantations and traditional pearl farm huts.

10. Porto Heli, Greece

From lush olive groves to perfect bays, every scene seems as if it was lifted from a postcard. The calm beaches are ideal for sunbathing and frolicking.

11. Pisco Elqui, Chile

Those who love to star gaze, look no further than the clearest skies in Pisco Elqui. Surrounded by magnificent Andean mountains, this Chilean village enjoys perfect weather year round.

12. Cappadocia, Turkey

Known as Turkey’s most visually striking region, Cappadocia offers caves, clefts, pinnacles and ‘fairy chimneys’ (pointed rock formations). Volcanic eruptions formed this surreal moonscape.

13. Con Dao Island, Vietnam

In addition to hiking and exploring empty coastal roads and deserted beaches, there are some excellent wildlife-watching opportunities in the islands of Con Dao. Most of the land area is part of Con Dao National Park, which protects Vietnam’s most important sea turtle nesting grounds.

14. Timgad, Algeria

This Roman colonial town founded by Emperor Trajan in AD100 is a UNESCO world heritage site. Centuries later, it still represents the embodiment of Roman urban planning.

15. The Cook Islands

Many are of the opinion that Cook Islands are the best kept secret in the Pacific Ocean. The 15 islands are scattered over a vast expanse of seductive and sensuous ocean, boasting of idyllic climate and rare beauty.

16. Angkor Wat, Cambodia

Built by the Khymer civilization, Angkor Wat is the most famed Cambodian temple, which even appears on its national flag. The architectural genius unfolds many enduring tales of Cambodian history.

17. Fiji

No list of exotic destinations can be complete without mention of Fiji, an archipelago of over 300 islands in the South Pacific Ocean. Besides pristine white sandy beaches and fish-packed coral reefs, there is ample opportunity to climb a mountain, soak in a hot spring or explore a village.

18. Isle of Skye, Scotland

The stunning scenery in the backdrop of the misty Cuillin hills offers beautiful hikes and spectacular photos.

19. Yellowstone National Park, USA

Home to the famous “Old faithful” geyser, Yellowstone still remains an unspoiled natural beauty. You are bound to see legendary wildlife wherever you go, be it grizzly bears, wolves or antelopes.

20. Bagan, Burma

Bagan boast of the largest and densest concentration of Buddhist temples and stupas in the world. For an unparalleled view, you can take a hot air balloon ride at sunrise.

21. Mount Roraima, Venezuela

Inspiration to Conan Doyle’s Lost World and Avatar, this place is an extraordinary natural geologic formation. It serves as the natural border between Venezuela, Brazil and Guyana in South America.

22. Cano Cristales, Colombia

Known as the most beautiful river in the world and the “Liquid Rainbow”, this Colombian wonder is a must visit if you are heading to South America. The best time to travel is July – November when the river bed is full of gaudy colors like green, yellow, blue, black and red.

23. Salar de Uyuni, Bolivia

The world largest salt flat is breathtaking, especially after rain when it creates a mirror effect. The salt is about 10 meters thick in the center.

24. Panjin Red Beach, China

Red Beach is located in the highest wetland and reed marsh in the world. It is vividly red and not covered in sand at all. The color is from a type of sea weed called Sueda, which stays green during summer and turns red in autumn.

25. Socotra,Yemen

Isolated Socotra looks like a sci-fi movie set. It is home to 800 rare species of flora and fauna, about a third of which is not found anywhere else in the world.

26. Whitehaven Beach, Australia

When you sink your feet into white silica sand that is amongst the purest in the world, you will know why people come here from all over the world.

27. Lake Baikal, Russia

The world’s oldest and deepest lake is an ice-lovers paradise. Adventuresome tourists have driven and walked over the frozen lake, but you can kayak, skate or bike.

28. Giant’s Causeway, Ireland

Resulting from a volcanic eruption 60 million years ago, Giant’s Causeway is an integral part of Ireland’s heritage. The most characteristic feature of the site is the exposure of large, regularly shaped polygonal columns of basalt in perfect horizontal sections.

29. Neuschwanstein Castle, Germany

There is something magical and mystical about castles, especially the Neuschwanstein, which is an inspiration to Disney’s Cinderella castle. It reflects the eccentric life of King Ludwik who withdrew into a world of myth upon losing his sovereignty.

30. Arashiyama Bamboo Forest, Japan

There is a reason why camera-clutching tourists shuffle their way through soaring stalks of bamboo. But the pictures don’t do justice, so add this to your list of “places to see before you die”.

31. Lake Retba, Senegal

Easily mistaken for a pool of strawberry milkshake, the bizarre color of lake Retba is actually a natural phenomenon. Thanks to its high salt concentration, it is a habitat for salt-loving microorganisms that bring out the pink colorization.

32. Badab-e Surt, Iran

The travertine (a sedimentary rock deposited by flowing water from two distinct mineral springs) terrace is a scenic natural wonder in the highlands of northern Iran.

33. Sossusvlei, Namibia

Lifted from a dream, Sossusvlei is full of picturesque sand dunes surrounded by clay pans. The most surreal attraction is the Deadvlei, where the dead acacia trees contrast the white shiny salty floor of the pan.

34. Derweze, Turkmenistan

Wish to stand at “Hell’s door?” Head to Derweze, a giant illuminated hole rich in natural gas, in the middle of the Karakum dessert. The crater is made by geologists more than 40 years ago, and the flames within have been burning ever since.

35. Fregate Island, Seychelles

The private island of Fregate is an ultimate honeymooner’s delight. It is one of the world’s most exclusive resorts with seven pristine beaches to choose from. Simply close your eyes and think idyllic.