Fluka Reference Standards for Analysis of Herbal Drugs

By: Matthias Nold, AnalytiX Volume 10 Article 1

Gerrit Foerster, Head of Isolation Lab, HWI ANALYTIK GmbH, Rheinzaberner Str. 8, 76761 Ruelzheim, Germany
FrankMichel, Head of Marketing, HWI ANALYTIKGmbH, Rheinzaberner Str. 8, 76761 Ruelzheim, Germany f.michel@hwi-analytik.de
Matthias Nold, Product Manager Analytical Standards matthias.nold@sial.com

Figure 1 Hawthorn (crataegus monogyna)

Figure 1 Hawthorn (crataegus monogyna)

In collaborationwith HWI ANALYTIK, Sigma-Aldrich is launching a new product line of primary reference standards for use in the quality control, in-process control and stability testing of herbalmedicinal products.

These standards are produced and qualified byHWI ANALYTIK in Ruelzheim (Germany), and are exclusively distributed by Sigma-Aldrich. A first series of 23 standards is listed in Table 1. The unique and novel certification concept of these reference standards is based on the quantitative NMR technique (qNMR) for content assignment. The application of qNMR, a relative primary method of measurement, is advantageous compared to the usual combination of two chromatographic methods, Karl Fischer titration and residual solvent determination due to higher reliability of the certified content value. The analytical report delivered with these products contains two identity proofs, the content by qNMR and the chromatographic purity. Comprehensive documentation on further parameters, detailed method descriptions and validation data can be ordered on request directly from HWI ANALYTIK.

While the following article in this issue (page 14 f) focuses on the content assignment of these standards by quantitative NMR, this article addresses the applications of reference standards in the analysis of herbal medicinal products.

Reference standards used for pharmaceutical analysis in a GMP environment are standards for qualitative and/or quantitative determinations within the scope of in-process controls, batch release analyses and stability studies of herbal drugs, herbal preparations and finished products. In case of herbal medicinal products, the reference standard may be a standard specimen of the drug, the drug preparation (e. g. extract or tincture) or a chemically defined substance. Regarding the latter case, only a limited number of herbal substances and herbal preparations possess constituents which are generally accepted to contribute substantially to their therapeutic activity. These are defined as ‘constituents with known therapeutic activity’, e. g. sennosides in preparations from senna leaves or fruits. However, for the majority of herbal substances and herbal preparations, the constituents or groups of constituents responsible for the therapeutic activity are not yet known. In some cases, certain constituents or groups of constituents may be generally accepted to contribute to the therapeutic activity but are not responsible for the full therapeutic effect. Such constituents or groups of constituents are useful for control purposes and are defined as ‘active markers’. Distinct from them, analytical markers are constituents or groups of constituents that serve solely for analytical purposes [1].

Product Name Prod.No. Pack Size
Bisabolol oxide A, primary reference standard 00630590 25 mg
Chlorogenic acid, primary reference standard 00500590 10 mg
Coumarin, primary reference standard 01260595 25 mg Ginkgolide A, primary reference standard 00770590 10 mg
* Ginkgolic acid C15:1, primary reference standard 02580185 10 mg
Ginkgolic acid C17:1, primary reference standard 01390590 10 mg
Ginsenoside Rb1, primary reference standard 00170580 10 mg
Ginsenoside Rg1, primary reference standard 00370580 10 mg
Harpagoside, primary reference standard 00420580 10 mg Hulupinic acid, primary reference standard 01090595 10 mg
* Hypericin, primary reference standard 00190180 10 mg Hyperoside, primary reference standard 00180585 10 mg Isoquercitrin, primary reference standard 00140585 10 mg Quercetin dihydrate, primary reference standard 00200595 50 mg
Quercitrin, primary reference standard 00740580 10 mg Rosmarinic acid, primary reference standard 00390580 10 mg
Rutin trihydrate, primary reference standard 00300590 50 mg
Sennoside A, primary reference standard 01870575 10 mg
Sennoside B, primary reference standard 00530580 10 mg
Silibinin, primary reference standard 02000585 10 mg
Valerenic acid, primary reference standard 02010595 10 mg
Vitexin-2"-O-rhamnoside, primary reference standard 00660585 10 mg
Xanthohumol, primary reference standard 01130595 10 mg

Table 1 Primary Reference Standards fromHWI ANALYTIK exclusively available at Sigma-Aldrich
* for Ginkgolic acid C15:1 and Hypericin, the content determination by qNMR was not applicable due to overlapping signals. For these two products, the content has been assigned by the usual mode combining two chromatographic methods, water content by Karl Fischer and determination of residual solvents and of inorganic compounds.

Figure 2 Lemon balm (Melissa officinalis)

Figure 2 Lemon balm (Melissa officinalis)

Markers are used for both quantitative and qualitative purposes. For all kinds of analytes – constituents with known therapeutic activity, active and analytical markers – there is a need to establish reference standards for quality control and stability testing of herbal preparations and herbal medicinal products.

In the pharmaceutical environment, a primary reference standard initially needs to be established and certified. Its content should be assigned without requiring comparison to another chemical substance. Primary reference standards in pharmaceutical analysis are of a defined purity. A comprehensive documentation needs to be filed for the marketing authorisation dossier. The process of establishment, certification and content of documentation to be filed has recently been described by Veit and Wissel [2, 3].

For routine analysis, working standards may be used.Working standards are secondary standards serving as ready-to-use reference standards for the testing. They are derived from primary reference standards by intercalibration. The extent of characterisation and testing of a secondary chemical reference substance is less than that of a primary chemical reference substance.

Great difficulties may arise when it comes to establishing natural product standards for use in identity testing and assays by the application of constituents with known therapeutic activities or (active) markers. Substances of natural origin usually display a complex molecular structure, which is why they are hardly accessible synthetically and quite frequently have to be isolated from the natural sources. Hence, establishing primary standards for the quality analysis of herbal medicinal products is often very complex and requires special know-how. Additional problems then arise due to the small amounts accessible this way, thus requiring an elaborate concept for the storage and employment of such standards. Special suppliers have therefore started to establish, store and manage the primary standards at a central site and supply their customers with qualified working standards that are ready to use.

Batch-specific control of a herbalmedicinal product containing extracts of lemon balm and hawthorn

In herbal medicinal products containing more than one herbal preparation, the choice of appropriate reference standards becomes far more challenging, because substances usually used as reference standards for a dedicated plant may occur in the other component of the combination drug, too. Due to this challenge, reference standards for a combination product should be selected in such a way that they are characteristic for each individual herbal preparation in the product. In the following example, the batch-specific control of a herbal medicinal product with the two main components, lemon balm and hawthorn, is presented.

For the quantification of hawthorn, the constituent vitexin-2"-O-rhamnoside is used as an analytical marker (chromatogram and the structure of the reference standard vitexin- 2"-O-rhamnoside are shown in Figure 3a). The chromatogram of an extract of hawthorn (Figure 3b), due to its complexity, highlights the need for an appropriate reference standard in the analysis of herbal medicinal products.

One main component of the extract of lemon balm is rosmarinic acid, which is used as an active marker (a chromatogram and the structure of rosmarinic acid are shown in Figure 3c; a chromatogramof an extract of lemon balm is shown in Figure 3d).

In Figure 3e, the chromatogram of the final herbal medicinal product is presented. The content of hawthorn and lemon balm extract in the finalmedicinal product is determined by the use of the reference standards vitexin-2"- O-rhamnoside and rosmarinic acid. This chromatogram highlights the challenge of the analysis of herbal medicinal products due to their complex composition.

Figure 3 reference standards and sample solutions of herbal medicinal products lemon balm and hawthorn

Figure 3a) Reference standard vitexin-2"-O-rhamnoside, c = 0,15 mg/ml; 3b) Sample solution of an extract of hawthorn leaves, flowers and fruits; 3c) Reference standard rosmarinic acid, c = 0,1 mg/ml; 3d) Sample solution of an extract of lemon balm leaves; 3e) Sample solution of the herbal medicinal product, exemplary recorded at 340 nm.

Chromatographic conditions for Fig. 3a-e:
HPLC: Alliance 2695, Waters
Detector: DAD @ 330 nmfor rosmarinic acid
DAD @ 340 nm for vitexin-2"-O-rhamnoside
Column: Synergi Polar RP; 250 x 4,6mm(4 μm) with guard column
Temperature: 25 °C
Mobile Phase: Gradient: Water/TFA (pH 2,0)/acetonitrile
Flow rate: 1,0 ml/min
Injection volume: 20 μl

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  1. European Medicines Agency (2008): Reflection paper on markers used for quantitative and qualitative analysis of herbal medicinal products and traditional herbal medicinal products. Available at: www.emea.europa.eu/pdfs/ human/hmpc/25362907enfin.pdf (05.11.2009)
  2. Veit, M.; Wissel, S. PharmInd 2007, 12, 1475–1480 Available in English at: www.hwi-analytik.de/en/download.html Available in German at: www.hwi-analytik.de/de/download.html
  3. Veit, M.; Wissel, S. PharmInd 2008, 1, 135–138 Available in English at: www.hwi-analytik.de/en/download.html Available in German at: www.hwi-analytik.de/de/download.html

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