High-performance liquid chromatography (HPLC).
High-performance liquid chromatography (HPLC) is another separating technique applied by PhytoLab. We are currently working with 30 high-pressure gradient systems and are not only using the classical UV-VIS absorbance detectors, but also dual wavelength, fluorescence, conductivity, mass and photodiode array detectors, as well as electrochemical and refractive index detectors. All of the systems are integrated into a so-called client-server network, ensuring a high level of data integrity at all times. Modern HPLC software enables us to draw up reports completely in accordance with your own individual requirements. Our extensive routine tests include quantitative analyses of various plant constituents and residual mycotoxins. We also have a liquid chromatography-mass spectrometry system (LC-MS) at our disposal for special applications and validation procedures.
HPLC at PhytoLab
Working as a laboratory that has been accredited in accordance with EU Directive 93/99/EEC not only means having highly qualified personnel at our disposal, but also state-of-the-art instruments and equipment. As far as routine HPLC is concerned, we work in cooperation with Waters of Milford in MA, USA, the world leader of the HPLC market (www.waters.com).
We are currently using 30 high-pressure gradient systems with various detection techniques to carry out routine analyses in the fields of food, food supplements, cosmetics, phytopharmaceuticals and the plant-based starting materials that go into these. Apart from the classical UV-Vis detectors, we also use dual wavelength detectors, fluorescence detectors, photodiode array detectors, conductivity detectors, electrochemical detectors, refractive index (RI) detectors and evaporative light scattering detectors (ELSD). Mass spectrometers are being used for selection detection to an increasing extent in modern HPLC analysis, particularly in the field of residue analysis. All of the systems are integrated into a so-called client-server network, ensuring a high level of data integrity. Modern HPLC software (Empower®) software enables us to draw up HPLC reports completely in accordance with your own individual requirements. We not only apply the official methods laid down in all of the usual pharmacopoeias, in § 35 of the German Food and Commodity Goods Law, AOAC methods and DIN/ISO methods, but also test specifications which we have developed and validated in accordance with the ICH Guidelines.
Quantitative analysis of plant constituents and residual mycotoxins
Our large-scale routine tests also include quantitative analyses of various plant constituents or residual mycotoxins. The HPLC techniques most frequently used at PhytoLab include the following methods and tests:
- Content quantitation of plant constituents as per the relevant European Pharmacopoeia monographs (www.pheur.org).
- Content quantitation of plant constituents as per the relevant monographs of the German Pharmacopoeia (DAB) and other European and international monographs.
- Official methods of AOAC International (www.aoac.org) and the American Herbal Pharmacopoeia (www.herbal-ahp.org)
Cooperation with the AOAC
As the result of intensive cooperation with the American Association of Analytical Chemists (AOAC), we have played an important role in the development and validation of numerous methods and procedures relating to food supplements, including participation in pooled testing schemes. These activities include the following quantitative analyses:
- Quantitation of phytosterols and fatty acids in saw palmetto fruit and in preparations and finished products made from saw palmetto fruit
- Quantitation of constituent substances in Ginkgo biloba, such as terpene lactone (ginkgolide and bilobalide), ginkgoflavon-glycosides and ginkgolic acid
- Glucosamine in food supplements
HPLC method for quantitation of the genotoxic anthraquinones rubiadin and lucidin
As a result of the EU Commission's decision of June 5, 2003, regarding the marketing authorisation of Noni products in accordance with the regulation concerning novel foods 258/97, PhytoLab has developed and validated a selective HPLC method for quantitative determination of the genotoxic anthraquinones rubiadin and lucidin. This method has not only been filed with the German Federal Office of Consumer Protection and Food Safety (BVL, www.bvl.bund.de) but also with the corresponding agency in the Netherlands - MEB agency, Novel Food Unit (up to the end of 2004: Committee on Safety Assessment of Novel Foods at the Health Council of the Netherlands).
The tests most frequently performed include quantitative determination of the following:
- Isoflavones in soya and soya products
- Isoflavones in red clover
- Isoflavones in Noni products
- Vitamins
- Caffeine
- Catechines (particularly including epigallo catechin gallate in green tea)
- Aspalathin in rooibos
- Alkaloids in various plants and mother tinctures
- Artificial colourings (Sudan I to IV, Sudan Orange, Para Red, Rhodamine B, Orange II and Butter Yellow)
- Aflatoxins
- Ochratoxin A
- Patulin
- Trichothecene
- Fumonisins
- Zearalenone
- Deoxynivalenol (DON)
- Nivalenol (NIV)
Pooled testing schemes (FAPAS®)
PhytoLab takes part in international pooled testing schemes on a regular basis in order to validate our own results. These are primarily focused on the FAPAS® international proficiency testing schemes (www.fapas.com), involving more than 100 laboratories from all over the world. The results of these schemes show us that we are in a position to be able to certify the accuracy and precision of analytical results, particularly with respect to the analysis of mycotoxins.
A large proportion of our HPLC analyses are also carried out within the framework of stability studies on finished medicinal products and the pertinent active constituents, which usually take the form of herbal extracts. All methods have been validated and constitute an element of our accreditation.
Brief description of the HPLC technique
HPLC is a chromatographic separating technique in which high-pressure pumps force the substance or mixture being analysed (analyte) together with a liquid solvent - the mobile phase (also referred to as the eluant) - through a separating column containing the stationary phase. There is usually a preliminary column upstream of the separating column, which serves to filter out any impurities. A separating column in an HPLC unit is usually between 5 and 25 cm long and has an inside diameter measuring between 2 and 4.6 mm.
If a constituent substance interacts strongly with the stationary phase, it remains in the column for a relatively long time, whereas a substance that does not interact with the stationary phase as strongly leaves the column sooner. Depending on the strength of the interactions, the various constituents of the analyte appear at the end of the separating column at different times - retention times - where they can be identified by means of a suitable detector.
A distinction is made between two methods of HPLC: normal phase chromatography (NP) and reversed phase chromatography (RP). The NP-HPLC method uses a polar stationary phase (e.g. unmodified, pure silica gel). The strength of the mobile phase's elution capacity is generally determined by the polarity. The various solvents are arranged in the elutropic series in order of increasing polarity. The speed at which a substance elutes increases with the polarity of the mobile phase. The polar molecules have a longer retention time than the nonpolar molecules and therefore leave the column later.
RP-HPLC is the most commonly used method in practice. The stationary phase is produced by allowing silanes that have been substituted by long-chain hydrocarbons to react with silica gel. The polar surface of the silica gel particles is coated with a nonpolar layer of alkanes, thereby "reversing" the polarity. The most frequently used mobile phases consist of mixtures of water or buffer and acetonitrile, tetrahydrofuran (THF) or methanol as the organic modifier. The composition of the mobile phase remains the same throughout the entire period in isocratic HPLC. If the gradient method is used, the polarity of the solvent mixture changes continuously throughout the analysis as the organic component in the eluant increases.
One special application of RP-HPLC is the separation of polar analytes that would demonstrate excessively long retention times in normal phases. A C18 column (octadecyl silanised silica gel) is usually used for this. Once separated by means of HPLC, the analytes are detected using standard commercial detectors, such as UV-Vis detectors, fluorescence detectors, photodiode array detectors (DAD), conductivity detectors, electrochemical detectors, refractive index (RI) detectors and evaporative light scattering detectors (ELSD). Mass spectrometers are also being used to an increasing extent for selective detection in modern HPLC applications.
