Author: Dr. Kim Baughman

During the past ten years, High Performance Liquid Chromatography/ Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) has become one of the fastest growing analytical techniques for the determination of trace level compounds.  This technique involves using two mass spectrometers, in tandem, as the detector for an HPLC.  The ion fragments from the first mass spectrometer (parent ions) are accelerated into the second mass spectrometer, where secondary ionization occurs (daughter ions).  This results in a highly sensitive and highly selective analysis.

The evolution of LC/MS/MS from the early LC/MS instruments has revolutionized vast segments of analytical chemistry that deal with trace level contamination.  The initial problems of interferences are reduced significantly by the tandem arrangement.  This has resulted in significant growth in published applications.

The growth encompasses a wide range of research areas, including: Food Safety, Environmental Protection, and Pharmaceutical Development.  Much of this is due to the advancements in the instrumentation (primarily in the interface between the LC and the MS and the implementation of atmospheric pressure ionization), which have improved the sensitivity and the selectivity of the technique to that unmatched by most other analysis methods.  Also, the newer generation of instruments has become more rugged, allowing them to be used routinely in commercial analytical testing laboratories. Until recently, HPLC/MS/MS instruments were found almost exclusively in research laboratories; however, more and more commercial laboratories are adding this capability.  Regulatory methods are being published and routine monitoring programs are being developed for which HPLC/MS/MS is the method of choice.

Environmental Applications of HPLC/MS/MS

The EPA has published several methods for environmental testing using HPLC/MS/MS.  Method 535 for the determination of chloroacetanilide and acetamide herbicide degradants and metabolites was included in the UCMR2 rule and will be required testing at hundreds of public water systems over the next three years.

Both the EPA and the US Geological Survey are using HPLC/MS/MS for major research programs into the occurrence of trace levels of emerging contaminants in the environment (also referred to as endocrine disruptors).  These compounds include: pharmaceutical residues, veterinary medicine residues, and pesticides as well as metabolites of these products.

Trace levels of the emerging contaminants are being studied in surface waters and in solid waste.  Because of the concern at even part per trillion levels for some of these compounds, HPLC/MS/MS has factored heavily in this research area, which is expected by many to grow rapidly in the next decade.  The EPA published Method 1694 in December of 2007 for the determination of some of these compounds.

Food Safety Applications of HPLC/MS/MS

The FDA has published several methods using LC/MS/MS for the determination of trace residues of contaminants in food products, many of which have been traced to imported foods.  One of the first methods published was for the determination of acrylamide in potatoes, which have been sometimes found to contain the chemical when prepared at high temperature.  Because of the need to detect very low levels of the compound, LC/MS/MS is the preferred analytical technique.

There are also several FDA methods for the analysis of various seafood products for trace levels of contaminants.  This issue has been visible in media reports recently and applies to shrimp, crabmeat, crawfish, and fish.  The methods include determination of the following residues:

• Chloramphenicol
• Nitrofuran metabolites
• Gentian violet (also known as crystal violet)
• Leucocrystal violet (LCV)
• Leucobrilliant green (LBG)
• Brilliant green (BG)
• Malachite green and Leucomalachite Green
• Fluoroquinolones

Some of these compounds are also found in honey, milk and other food sources.  There are published methods for determining veterinary residues (mainly antibiotics), food dyes, and pesticide residues in food products.  LC/MS/MS can also be used to determine melamine and cyanuric acid which were at the center of the recent pet food poisoning incident.

HPLC/MS/MS is also becoming a powerful tool in the determination of pesticides in a variety of matrices.  Many residues can now be detected at trace levels which have proved problematic by other techniques.  This method has also proved to be valuable for the determination of nitrosamine compounds.

Pharmaceutical Applications of HPLC/MS/MS

The main use for LC/MS/MS in the pharmaceutical industry has been in biological samples.  The determination of target drug and metabolites in plasma and urine are critical for the evaluation of new drugs.  These analyses are used for pharmacokinetic studies to evaluate residence time in the body as well as toxicology analyses.  The high sensitivity and selectivity and the high throughput of LC/MS/MS make it effective for determining trace levels in complex biological matrices.

The LC/MS/MS is also used for the analysis of impurities in drug substances, and it has become valuable for the detection of extractables and leachables in the packaging industry.  These are compounds that may leach into the product from the package and are of concern, both in the pharmaceutical industry and in the food industry.

The Continued Growth of HPLC/MS/MS in the Analytical Laboratory

Because scientists are concerned about lower and lower levels of detection for compounds in complex matrices, an expansion in the prevalence of the development of LC/MS/MS methods can be expected as can an increase in the availability of the service throughout the analytical industry.  As is often the case in the history of analytical instrumentation, the maturing of the technique will likely continue to result in a number of additional applications.  Because of the advancements, both in the sensitivity and in the ruggedness of the instruments, applications are being developed at an ever increasing rate.  Many commercial analytical laboratories have added this capability and the time when this technique is included as one of the routine capabilities in the analytical testing industry may not be far away.

And the expansion will continue; new methods will be developed for specific applications.  Once commercial laboratories have the capability of HPLC/MS/MS, as some already do, they will be able to respond to specific needs with customized method development.  The envelope of detection will be pushed, as will the ability to determine ultra-trace level contaminants in complex matrices.

The ultimate benefit of this instrument will be realized by all of us.  The enhanced ability to detect compounds in complex matrices at ever decreasing concentrations will result in safer products and a safer environment.

Microbac Laboratories Inc. purchased our first HPLC/MS/MS in 2005 and purchased our fourth in 2008.  Current applications include low level pesticide determination and drug and metabolite determination in biological samples (bioanalytical).  We are currently adding capabilities for the determination of contaminants in imported seafood and the determination of pharmaceuticals and endocrine disruptors in drinking water and surface water.