Bitter Orange Standard Reference Materials

Three bitter orange-containing Standard Reference Materials have been developed as part of the Analytical Chemistry Division's program to produce SRMs related to dietary supplements. Levels of five citrus alkaloids and caffeine were determined, and the materials were screened for pesticide residues and toxic heavy metals. Multiple analytical methods with measurements made at NIST and by collaborating laboratories were used to assign values. The methods are independent, with two types of extractions, two separation methods, and four detection methods. The bitter orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods.

The development of bitter orange-containing dietary supplement SRMs is part of a collaborative effort between NIST and the National Institutes of Health, Office of Dietary Supplements (NIH-ODS). In December 2003 the Food and Drug Administration ruled that dietary supplements that contain ephedrine alkaloids are adulterated, based on potential health risks associated with their use. This ruling, which became effective in April 2004, prohibits the sale of ephemera-containing dietary supplements in the United States. As a result of this action, many manufacturers of dietary supplements reformulated weight loss products with ingredients that are considered to be safer alternatives to ephemera, such as bitter orange, and reference materials are needed to support Good Manufacturing Practices (GMPs) for these products.

As with other complex-matrix reference materials produced by NIST, value assignment of the bitter orange SRMs was accomplished by using multiple independent analytical methods with measurements performed at NIST and by collaborating laboratories. Data from the different methods and sources were evaluated statistically and combined to yield a consensus value and expanded uncertainty interval. As many as six data sets were utilized in value assignment of the citrus alkaloids. The corresponding methods are substantially independent; at NIST at least two extraction approaches, two chromatographic separation methods, and four modes of detection were used. Extraction studies were carried out to develop approaches for quantitative recovery of the bitter orange alkaloids from the various types of samples. Three extraction methods were studied: sonication extraction, pressurized-fluid extraction, and Soxhlet extraction. In general, higher levels of the alkaloids were extracted with longer solvent contact times, particularly for the ground plant material. Multiple extraction cycles were also useful in maximizing analyte recovery. Instrumental analytical methods included the use of ion-pairing liquid chromatography with fluorescence detection, and liquid chromatography with a pentafluorophenyl column in combination with tandem mass spectrometry. These robust methods offered substantial independence, and provide enhanced sensitivity and selectivity resulting in accurate and precise measurements.  

• Multiple analytical methods have been developed for the measurement of citrus alkaloids in plant, extract, and finished product samples.
• Three bitter orange containing SRMs have been produced with certified, reference, or information values for citrus alkaloids and toxic elements and a certified value for caffeine (in one SRM):

SRM 3258 Bitter Orange (Fruit)
SRM 3259 Bitter Orange Extract
SRM 3260 Bitter Orange-Containing Solid Oral Dosage Form
SRM 3261 Bitter Orange Dietary Supplement Suite.

Click on an SRM name to be transferred to a page where you can view its Certificate of Analysis.