Dean Duxbury

What is a hyphenated analytical technique? The answer, according to a chapter on “Hyphenated Techniques” in Analytical Chemistry (Wiley-VCH, 1998), is “the marriage … of two separate analytical techniques via appropriate interfaces, usually with the backup of a computer tying everything together.”

Lab technicians compare analysis of pesticide residues using Waters’ Acquity UPLC™-MS-MS and HPLC-MS-MS. The narrower peak widths obtained by UPLC resulted in a signifi cant increase in signal response over that achieved under HPLC conditions.

In general, the term hyphenated techniques ranges from the combination of spectroscopic techniques (e.g., mass spectrometry) with chromatographic techniques (e.g., liquid chromatography or gas chromatography), or the coupling of separation techniques with spectroscopic detection techniques.

Jon DeVries, Technical Manager, Medallion Laboratories, St. Paul, Minn. (phone 800-245-5615, explains the concept and rise of hyphenated techniques. “While it is possible to determine that an analyte is not present [at a known detection level] with certainty, the analyst cannot always be certain of the presence and quantity of an analyte. If the analytical device being used is the correct one, and is working properly and no signal (e.g., color change, titration, digital, chromatographic peak, analog meter response) is observed, then obviously there is none of the analyte present.”

On the other hand, just because one observes a positive signal (color change, titrant consumed, chromatographic peak, etc.), that signal is not absolute proof that the analyte is present, DeVries says. While each chemical compound will give a specific response, the caveat is that more than one chemical compound may give the same response. Therefore, the more specific an analytical method is, the higher the level of confidence. Many analysts desire a higher degree of specificity in their determinations than can necessarily be obtained by a single technique or instrument.

Vitamin Analysis
“By combining techniques or instruments for analysis of an analyte, one increases method specificity, very often in a synergistic fashion,” DeVries says. For example, look at the analysis of a vitamin in a solid, he adds. The vitamin is extracted with a solvent and can then be injected onto a gas chromatograph or into a mass spectrometer. The first degree of specificity will be the extracting solvent (chosen to extract the vitamin, but other compounds are extracted as well). The gas chromatograph separates the vitamin from other compounds and gives a peak (i.e., a volatile compound with a specific retention time based on interaction of the column and the analyte and temperature), but additional compound(s) may have the same or similar retention time.

In the mass spectrometer, the vitamin will yield a parent ion and a number of ion fragments in a specific pattern, but other compounds will also be adding ions to the ion pattern of the mass spectrum, DeVries says. When the techniques are combined into an extraction GC-MS method of analysis, a very high degree of specificity is obtained.

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“The vitamin needs to be extractable into the solvent, to be volatile to pass through the GC, and to elute at a specific retention time on the GC. When the GC feeds into the MS, the mass spectra generated is now only that of the GC eluent at that retention time, and the parent ion and ion fragmentation pattern need to match for the vitamin. Further, the ratio of intensities of the parent ion to the ion fragments is constant for a given compound and can be used as further proof of identity, yet another degree of specificity. Thus, the overall method specificity exceeds the sum of the individual technique specificities, and the analyst can be very confident in his or her results,” DeVries says.

Technique Combinations
He notes that the most common combinations used for improved analytical specificity are gas chromatography coupled with mass spectrometry (GC-MS), liquid chromatography coupled with mass spectrometry (LC-MS) and in some cases, LC with two stages of mass spectrometry (LCMS-MS). The latter is particularly effective for water-soluble, minimally volatile compounds in complex matrices such as acrylamide in foods or perchlorate in aqueous solutions. The high sensitivity of these instruments combined with the high selectivity allows detection and quantitation at very low levels.

“GC-MS is particularly effective for residue analysis, (e.g., pesticides and natural toxins, wherever it is possible to volatilize the analyte) due to the high selectivity, efficiency, and specificity of the GC column combined with the specificity of the MS typically giving very distinct ion patterns. As a general rule, analysts consider a match of retention time and four MS peaks of matching mass and intensity as fully confirmatory,” DeVries says.

Other hyphenated techniques available include GC coupled with Fourier transform infrared spectroscopy (GC-FTIR), microscopy coupled with FTIR (for samples that are very small physically), LC coupled with scanning ultraviolet-visible spectroscopy (LC-UV-Vis), LC coupled with refractive index (LC-RI), and inductively coupled argon plasma analysis coupled with mass spectrometry (ICP-MS), DeVries adds.

“All of the techniques have important and varied application with relatively little overlap between techniques regarding applicability. A thorough discussion with your analyst before initiating any new analysis is generally in order to determine whether the high degree of specificity achievable with the hyphenated techniques is necessary, or whether more traditional methodologies will suffice,” DeVries says.

Food Safety
“Two main regulated areas in food safety are the detection of pesticide residues in fruit, vegetables, and animal products, and veterinary drug residues in animal products. The technology of choice for this type of analysis is liquid or gas chromatography combined with mass spectrometry [for the study of known compounds],” says Ramesh Rao, Business Manager, Waters Corp., Milford, Mass. (phone 800-252-4752,

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Liquid chromatography is a chemistry-based tool used for separating mixtures of chemical compounds, Rao explains. Once separated, the amount of a chemical compound within the mixture may be determined by mass spectrometry.

A mass spectrometer is an instrument designed to separate gas phase ions according to their mass-to-charge ratio and is used to determine a compound’s molecular weight. The molecular weight value can be used to confirm, quantify, identify, or characterize compounds of interest.

LC-MS is a hyphenated technique that combines the separation capability of high performance LC with the detection power of MS.

“For more advanced experimental needs, two or more MS experiments can be combined sequentially. This technique, referred to as MS-MS, can provide additional sensitivity and selectivity. In many cases related to food safety, MS-MS is the only technique to provide detection to the required levels and a one-step approach in quantifying and confirming the presence of target contaminants,” Rao says.

Traditionally, analytical methods used for food safety were compound class-specific, Rao notes. Recently, the focus has been on multi-residue methods, some that identify more than 100 distinct compounds in one experiment. These emerging methods can result in marked gains in laboratory productivity.

“There is also significant interest in first screening methods for samples containing large numbers of contaminant compounds. First screening can rapidly eliminate negative samples and identify potentially positive samples for further analysis.

Waters’ Acquity UPLC System™ along with time-of-flight mass spectrometry [for the study of unknown compounds] provides a unique tool to address this need. This system’s ability to use novel 1.7-micron column packings significantly improves the separation of chromatographic peaks and reduces analysis times,” Rao says.

A future trend in food technology is detecting unexpected contaminants which do not fall under routine surveillance testing or for which there is no current legislation, Rao adds. Examples of contaminants of interest are mycotoxins (in a variety of agricultural products including nuts, rice, and spices), marine biotoxins (in fish and shellfish), and Sudan red dyes (in chilies and other spices).

by Dean Duxbury,
Contributing Editor
Consultant, Oak Brook, Ill.
[email protected]