Research Article Received: 24 September 2009 Revised: 25 January 2010 Accepted: 19 February 2010 Published online in Wiley Interscience: (www.interscience.wiley.com) DOI 10.1002/jsfa.3966 Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders Ronald L Prior,a∗ Ellen Fan,b Hongping Ji,c Amy Howell,d Christian Nio,e Mark J Paynef and Jess Reedg Abstract BACKGROUND: The objective of this study was to validate an improved 4-dimethylaminocinnamaldehyde (DMAC) colorimetric method using a commercially available standard (procyanidin A2), for the standard method for quantification of proanthocyanidins (PACs) in cranberry powders, in order to establish dosage guidelines for the uropathogenic bacterial anti-adhesion effect of cranberry. RESULTS: Commercially available cranberry samples were obtained (five from U.S. sources and six from European sources) for PAC quantification in five different analytical laboratories. Each laboratory extracted and analyzed the samples using the improved DMAC method. Within-laboratory variation (mean ± SD) was 4.1 ± 1.7% RSD (range, 2.3–6.1% RSD) and the between laboratory variability was 16.9 ± 8.5% RSD (range, 8–32% RSD). For comparative purposes, the cranberry samples were alternatively quantified using weights of extracted PACs (gravimetric). The correlation coefficient between the two methods was 0.989. CONCLUSION:ThisimprovedDMACmethodprovidesasimple,robust and relatively specific spectrophotometric assay for total PACs in cranberry samples using commercially available procyanidin A2 dimer as a standard. DMAC is most useful within a given type of food such as cranberries, but may not be appropriate for comparing concentrations across different food types, particularly in those cases where large differences exist among the relative amounts of each oligomer and polymer. c 2010 Society of Chemical Industry Keywords: proanthocyanidins; cranberry; DMAC; BL-DMAC; urinary tract infection; bacterial adhesion INTRODUCTION juice preparation made by Ocean Spray Cranberries, Inc., contain- Cranberry (Vaccinium macrocarpon Ait.) has been utilized tradi- ing 36 mg PACs, measured by the DMAC colorimetric method tionally for the prevention of urinary tract infections (UTIs), and clinical research supports this claim.1,2 An important mechanism of action may be the bacterial anti-adhesion activity attributed ∗ Correspondence to: Ronald L Prior, USDA, Arkansas Children’s Nutrition Center, to ingesting cranberry products.3 Proanthocyanidins (PACs) in 15 Children’s Way, Little Rock, AR 72202, USA. cranberry have been shown to inhibit primarily P-fimbriated Es- E-mail: [email protected] 4–7 8 cherichiacoli adhesion to uroepithelial cells in vitro and ex vivo, a USDA-ARS, Arkansas Children’s Nutrition Center, 15 Children’s Way, Little Rock, interfering with this primary step in the infection process.9 PACs, AR 72202, USA also referred to as condensed tannins, are oligomers and poly- b Brunswick Laboratories, 50 Commerce Way, Norton, MA 02766, USA mers of monomeric flavan-3-ols, such as catechin and epicatechin (Fig. 1). The PACs in cranberry contain a high proportion of the c Brunswick Laboratories (China), 320, A3 Building, 218 Xing Hu Rd, Suzhou unusual A-type double linkages which may be important struc- Industrial Park, Suzhou, Jiangsu, China tural features in the anti-adhesion process.5 In one study, other d Rutgers University, Marucci Center for Blueberry Cranberry Research, 125A Lake food sources of PACs that contain only B-type linkages (chocolate, Oswego Rd., Chatsworth, NJ 08019, USA grape, apple and green tea) were consumed. However, they did not elicit ex vivo bacterial anti-adhesion activity in urine follow- e Agrobio, ZAC des Trois Marches, 3, impasse de la jonchee,´ F-35132 Vezin Le Coquet, France ing ingestion. Only cranberry juice with A-type PACs prevented 10 bacterial adhesion. f Hershey Center for Health and Nutrition, The Hershey Company, 1025 Reese The current recommended daily dosage of cranberry for UTI Avenue, Hershey, PA 17033, USA prevention is based on the efficacious levels that have been g Department of Animal Sciences, Environmental Toxicology Center, Interna- administered in human intervention trials. Often, the daily con- tional Agriculture, 256 Animal Science Bldg., 1675 Observatory Drive, Madison, sumption of 300 mL of Cranberry Juice Cocktail (a 27% cranberry WI 53706, USA J Sci Food Agric (2010) www.soci.org c 2010 Society of Chemical Industry www.soci.org RL Prior et al. of colorimetric quantification methods for cranberry PACs can be found in the paper by Howell.23 DMACisacolorimetricmethodthatappearstobemoreaccurate than other methods and has been successfully used to quantify cranberry PACs.15 It is less likely to have interferences from sample components, such as anthocyanins, since the reaction is read at 640 nm. The DMAC method was used to substantiate French health claims for the bacterial anti-adhesion activity of cranberry juice concentrate and juice concentrate extract powder granted in 2004, cranberry juice cocktail in 2005, and fresh frozen and pureed´ cranberry in 2007 by the French food safety authority, AFSSA (Agence franc¸aisedesecurit´ e´ sanitaire des aliments), which concluded that the daily intake of 36 mg of PAC in cranberry contributes to decreasing adhesion of certain uropathogenic P- fimbriated E. coli to the walls of the urinary tract. However, the quantification method used in the 2002 petition, the ‘old DMAC (ref. PAC 003)’ involves using a proprietary standard which is not commercially available. This limits the widespread use of Figure 1. Numbering system for flavanoid structure (epicatechin) and this method for quantification of PACs in cranberry products and structure of procyanidins dimer A2. justifies the need to develop an improved method. Accurate standardization of PAC content is vital to establishing dosage guidelines for consumers, monitoring efficacy and shelf- (an aldehyde condensation of 4-dimethylaminocinnamaldehyde), life of dietary supplements and other products, and to formulate has been recommended. This dose has been shown to reduce standardized test materials for use in research studies. Currently, bacteriuria and pyuria in a large clinical trial.11 Other types of there is no universally accepted method for quantification of cranberry products, including encapsulated powders, have also cranberry PACs. The objective of this study was to validate an demonstrated activity in vitro,6 ex vivo12,13 and in vivo.14 improved ‘DMAC’ method (originally modified and improved by A number of analytical procedures including colorimetric, Brunswick Labs, Norton, MA, USA) which uses a commercially gravimetric, chromatographic and mass spectrometric methods available A2 dimer standard (Fig. 1). This method could be utilized are currently being employed for the measurement of cranberry worldwide as an accurate, reproducible, inexpensive and rapid PACs;15–17 however, due to the complexities of the PAC structures standard method for quantification of PACs in cranberry powders and A-type linkages, the results can often be erroneous and may with an outcome similar to that obtained using the previous DMAC not be reproducible.18 Gravimetric methods involve fractionating method for the reference quantity of 300 mL of Cranberry Juice PACs and weighing the total content. This technique can be Cocktail (36 mg PAC). Under these circumstances, the existing expensive and labor intensive. Mass spectrometry may be useful health claims in France can be upheld with the current value of for authentication, but it is not suitable for quantification unless 36 mg PAC and also be validated by competent authorities. standards are available and response factors for the individual oligomers are known. Normal-phase high-performance liquid chromatography methods are effective at quantifying PACs with EXPERIMENTAL B-type linkages, such as those in chocolate, grapes and other Chemicals 19,20 foods, but may be less effective at estimating total PACs for 1. HPLC grade water was from a Millipore MilliQ R/O water cranberry due to the heterogeneous A-linked PACs and lack of purification system (Millipore, Millipore, Bedford, MA) appropriate standards. 2. HPLC grade methanol (99.9%) was from Fisher Scientific, Colorimetricassaysareadvantageousbecausetheyarenormally Houston, TX; cat. #A452-4 inexpensive, rapid, and simple to perform. Certain colorimetric 3. HPLC grade reagent alcohol (91%) was from Fisher Scientific; assays are specific for flavan-3-ols and PACs and depend on the cat. #A995-4 reaction of chemical reagents with sites on the PAC molecule. 4. HPLC grade acetone was from Burdick & Jackson, Morris However, a lack of appropriate standards and interferences from Township, NJ; cat. #AH010-4 other sample components, such as anthocyanins and extraction 5. 4-Dimethylaminocinnamaldehyde (DMAC; F.W. 175.23) was solvents, can lead to over- or underestimation of PACs. One from Sigma, St Louis, MO, USA; cat. #EC-228-267-0 such method that is subject to overestimation of PACs is the 6. Hydrochloric acid (37%; 12.5 mol L−1) was from Sigma; cat. European Association for the Valorization of Cranberry (EuraCran) #320331-500 method, a modification of the acid catalyzed autoxidation reaction 7. Acetic acid (glacial) was from Fisher Scientific; cat. #A35-500 that leads to the formation of anthocyanidins from PACs. The 8. ProcyanidinA2(HPLC;purity>99%) was from Extrasynthese,` resulting anthocyanidins are red in color and are then quantified Genay Cedex, France; cat.