The Laboratory Notebook

Answering important questions about quality.

By Robert Green

This month we have interesting questions (hopefully with equally interesting answers) on measuring nutritional supplement analytes in the bloodstream, the selection of HPLC detectors and the analysis of chicken collagen type II.

Q. I would like to obtain clinical data demonstrating that by taking my nutritional supplement the analytes of interest are absorbed into the bloodstream. Can these studies be done?

A. With the continuing crackdown on marketing claims, the desire to differentiate competing products and the increasing professionalism of the industry, we have seen a dramatic increase in the demand for nutritional supplement blood studies and are involved with several studies now. Here is an overview of the subject.

Pharmacology is the study of the mechanisms by which drugs work on living systems. There are two general areas of pharmacology: pharmacodynamics and pharmacokinetics. Pharmacodynamics is the study of the molecular and physiological effects of drugs on cellular systems and the mechanisms of action involved. For example, if you wanted to determine if taking chondroitin sulfate reduces inflammation of the joints, it would be a pharmacodynamic study. Pharmacokinetics is the study of the way in which the body handles the drugs. This includes absorption, distribution and excretion of the drug. So if you wanted to determine if chondroitin sulfate enters the bloodstream after it is ingested orally, this is a pharmacokinetic study. Simple pharmacokinetic studies are manageable and relatively affordable. Pharmacodynamic studies are effectively clinical trials and, depending upon the subject of the study, can be a major undertaking.

Your question involves the issue of absorption. All forms of treatment except intravenous require absorption and this process is directly related to the chemical nature of the drug and its size. For nutritional supplements, the route of administration is primarily oral. Absorption of orally administered drugs can be extremely difficult. Gastrointestinal enzymes, inhospitable pH and bacteria present in the body can destroy or modify some drugs before they have a chance to be absorbed into the bloodstream.

A term frequently used when discussing the effect of ingesting a drug is “bioavailability,” or the degree to which the unchanged drug reaches the bloodstream. When orally administered, a drug may be much less available, or it may not be available at all, due to its failure to cross the gastrointestinal barrier or its destruction in the gut or liver.

If you think analyzing a raw material or finished product can be complicated (and it can), just think of the increased complexity analyzing the same material in a blood sample! To conduct the analysis, the blood specimen must be properly treated so that the analyte(s) of interest can be measured without damaging them. Extra care must be taken because blood samples are considered biohazards capable of transmitting disease.

Blood work involving nutritional sup­­­plement studies is a distinct specialty. While there are perhaps thousands of c­­linical chemistry labs conducting blood tests every day, they are unfamiliar with nutritional supplements (other than the typical vitamins and minerals) and the methods used to analyze them. Moreover, most clinical chemistry is conducted using automated instrumentation, which was not designed to accommodate nutritional supplement products. Anticipating a demand for these studies, we have developed accurate and cost-effective protocols to conduct them.

Q. I thought there was one type of analysis known as “HPLC,” referring to the instrument used, yet I see you use different detectors depending upon the product analyzed. Can you explain what this refers to?

A. We have often discussed the fact that there is no one analytical instrument in existence with which all products can be analyzed. Rather, there are a multitude of instruments used and part of an analytical lab’s skill is to select the appropriate instrumentation for each product tested. It actually is more difficult than that. Once you select the instrumentation, in most cases it has numerous variables requiring the selection of the optimum configuration for the analysis at hand. This is particularly true with HPLC.

HPLC refers to very sophisticated instrumentation, which first separates the items of interest (called “analytes”) and then measures them. To do this there are several steps in the process which must be fine-tuned for the particular analyte(s). One such step is the selection of the detector used.

The detector for an HPLC is the component that actually detects the analyte (which is why it is called a detector) and emits a response. This response then results in the peaks on a chromatogram and forms the basis of the analytical report. There are many types of detectors that can be used with HPLC. Some of the more common detectors include: Refractive Index (RI), Fluorescent and Ultra-Violet (UV).

RI detectors measure the ability of molecules in the sample to bend or refract light. This property for each compound is called its “refractive index.” Detection occurs when a beam of light directed at the sample is bent. Most sugars, triterpenes and MSM are analyzed with an RI detector.

Fluorescent detectors measure the ability of a compound to absorb and then re-emit light at given wavelengths. This method is particularly useful for compounds found in minute quantities (such as pesticides) where we can make a fluorescent derivative, which can emit at a 100-fold increase. This enables us to detect quantities so tiny they might otherwise fall under our limit of detection.

By far the most commonly used detector for nutritional supplements is the UV detector. This detector measures the ability of a sample to absorb light. Today there are two types of UV detectors in use: variable wavelength and diode array detectors. A variable wavelength detector measures one wavelength at a time, but can detect over a wide range of wavelengths. A newer innovation, the diode array detector measures a spectrum of wavelengths simultaneously. Because of the increased data collected and speed in which it does it, we now rely exclusively on diode array detectors for UV analyses.

So you see, an essential skill of an analytical chemist is to match the analyte of interest to the most appropriate detector in order to obtain the most accurate analysis possible.

Q. I would like a chicken collagen type II sample analyzed to confirm it is indeed what it is purported to be. How do you do it?

A. Collagens are a family of closely related proteins with a triple helix protein structure. Over 10 collagen types have been identified, but in our industry there is increased interest in type II collagen, which is the major type found in cartilage. Type II contains a family of compounds know as glycosaminoglycans, which include chondroitin sulfate, hyaluronic acid and glucosamine.

Because of the complex nature and unique characteristics of collagen type II, its analysis cannot be conducted by HPLC or another method we generally employ, so we went back to the books. Taking a cue from clinical chemistry, we determined that collagen type II could be analyzed by an enzyme assay known as ELISA (pronounced e-liza), which is short for enzyme linked immunosorbent assay.

ELISA is based on an antigen-antibody interaction followed by an enzymatic action. In short, the immune reaction in mammals is triggered by the recognition of a foreign body (antigen) by a particular subset of cells (B cells). Antigen-specific B cells multiply and produce specific proteins (antibodies), which bind to the foreign antigens, aiding the subsequent elimination of the antigen. Immunodiagnostic assays use these antibodies to detect specific antigens. In ELISA, the final step is an oxidation reaction of the bound enzyme-labeled antibody, producing a color that is proportional to the amount of antigen or antibody present in the sample. Spectrophotometric methods are then used to detect and quantify the enzyme-linked antibody complex.

ELISA is used for the detection of HIV and Hepatitis C, and now, to qualify and quantify type II collagen. And that’s how we do it.NW