5


very difficult to retrieve with sufficient certainty at reasonable costs using traditional chromatographic methods. HILIC will certainly continue to expand strongly in this field and it is even likely that a majority of the newly developed methods for clinical routine analysis will be based on HILIC in the future.


Food Safety


Figure 2. Separation of methylmalonic acid (MMA, m/z 117) from a precipitation-treated sample of patient plasma containing 0.137 mol/L MMA, using a HILIC column and an isocratic eluent [7]


.


Figure 3. HILIC-ELSD separation of milk hydrolysate for detection of L- hydroxyproline (peak no. 4). Injection of 2 µL on ZIC-HILIC 150x2.1, 5 µm, 200 Å column operated at 0.8 mL/min using isocratic elution during 10 minutes with 85% acetonitrile in 50 mM ammonium acetate (pH 5.56) followed by acetonitrile gradients 10-15min 85%-65% and 15-20min 65%. Courtesy of Jeffery Lei, Merck China.


Other biomarker analysis where HILIC already have been put to use are; amino acids (S- adenosyl-methionine [8]


, ornithine [9] , 4-amino-


3-hydroxyphenylalanine and 3-amino-4-hydroxyphenylalanine [10] free metanephrines [11] metabolites [12]


; plasma- ; morphine and its [13]; sulfated and glucurunated steroids [14]


; cocaine and its metabolites ;


quaternary ammonium compounds like acetylcholine, choline, and butyrobetaine [15]


acetylcholine precursor betaine and phospholipid precursor dimethylglycine [16] nitrosamine metabolites [17]


, lung disease


biomarkers hydroxyproline, nitrotyrosine, proline and tyrosine [18]


; cardiovascular


disease biomarker asymmetric dimethylarginine (ADMA) [19,20]


.


Evidently metabolomics and clinical analysis are major growth fields in HILIC separations. The reason for this is fundamentally simple; most metabolites and biomarkers found in body fluids are per se of polar and hydrophilic nature. HILIC is thus an ideal technique for separating compounds from biological samples such as serum, plasma, cerebrospinal fluid, urine, dialysis fluid, hair, sweat, saliva, tissue, and breath condensates. Using a separation technique fit-for-the- purpose enables finding information that never previously was revealed, or at least was


; ;


In September 2008 the world witnessed how thousands of children were poisoned by Chinese milk products that had been manipulated by addition of melamine to increase the nitrogen content and mislead the Kjeldahl nitrogen analyzers into reporting high protein content. Within a few weeks the Food and Drug Administration in USA had published recommended methods [21] based on HILIC separations and MS/MS detection for analyzing melamine and its decomposition product cyanuric acid which together form an insoluble complex precipitating in the kidney.


Recently it was discovered that hydrolyzed protein extracts from old leather scraps have been added to milk for the same nitrogen- rising purpose. HILIC separation approaches with detection by ELSD or MS/MS have been developed to screen for and determine L- hydroxyproline, which is an amino acid not naturally occurring in milk, see Figure 3.


Also this time the scene for the tampering is China, but it would be naïve to think that this problem is only limited to dairy products in a specific country. Potentially any compound rich in nitrogen can be used to fool Kjeldahl nitrogen analyzers in any food product where protein content is important for price and sales. Analysis of native amino acids [22]


or


other small molecular indicators for changed composition of food products are thus emergent areas where HILIC will continue to make a difference for ensuring food safety.


Diethylene glycol is another contaminant that keeps coming back in different consumer products causing hundreds of human deaths with a frightening regularity. It is typically used in place of glycerin or propylene glycol and there have been several epidemics due to the substitution by diethylene glycol into products like medicine and toothpaste in the past fifteen years. Development of rapid protocols


based on HILIC-MS [23]


have been vital to find and stop these poisoned products.


Aminoglycoside antibiotics have until recently been very difficult to analyze but there have been a large need due to the extensive use in veterinary medicine and animal husbandry. Utilizing simultaneous HILIC and ion exchange separation mechanisms on a zwitterionic column, these compounds can now readily be separated and detected by MS [24]


.


All in all we can conclude that with the ever increasing food prices and amounts of industrially processed food the incentives and possibilities for food tampering increase. Many of the potential compounds in question here are polar and hydrophilic, wherefore HILIC would be the most obvious separation technique to choose when solving these analysis problems.


Simultaneous Anion and Cation Analysis The possibility to simultaneously quantify inorganic anions and cations in one chromatographic run is an attractive feature of HILIC. It is straightforward when used with a zwitterionic column in combination with detection techniques like evaporative light scattering (ELSD) [25]


, charged aerosol (CAD) [26]


,


or potentially also condensation nucleation light scattering (CNLSD). The pharmaceutical industry quickly realized the benefits of having one generic HILIC-based analysis method for determination of several counter ions in a range of different pharmaceutical preparations. The organic-rich eluents used in HILIC separations also proved to be beneficial for solubility of the often poorly water-soluble drugs.


Quite a substantial part of the analysis traditionally performed with ion chromatography can thus be transferred to HILIC. Applications where low detection limits and very complex samples make analysis difficult, will however not likely be performed with HILIC in the foreseeable future. It is thus not superior performance that will make ion analysis by HILIC a success, but ease of use and the possibility to run both anions and cations in one run.


Environmental Analysis Environmental analysis literally exploded with the realization that a compound that was incredibly useful in industry or households was a potential disaster agent when finally ending up in nature. Hydrophobic compounds like DDT, PBC and other chlorinated organic compounds were quickly banned since they could be detected after enrichment and biomagnification in the food chain.


Today environmental analysis faces other, but potentially as dangerous compounds, many of


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