Advances in nano-ultrahigh-performance liquid chromatography (nUHPLC) and "plug-and-play" micro-UHPLC (µUHPLC) for the detection of veterinary drugs and steroids in porcine meat and urine respectively are described. Recent developments in "plug-and-play" µUHPLC devices offer several advantages compared to earlier micro-LC systems. As well as the ease of use, solvent consumption can be reduced by more than 95% and the amount of sample required can be reduced 10-fold. In addition, the performance and the robustness of the µUHPLC system described is comparable to conventional UHPLC, and can be successfully applied for the routine analysis of residues and contaminants in food.
Residues and contaminants that can affect consumer safety (such as veterinary drugs, growth promoters, pesticides, or natural toxins) are continually monitored to ensure product safety and compliance with legislation (1). Residues and contaminants may be present at low concentrations in the ng/kg to µg/kg range, and so most laboratories use liquid chromatography coupled to tandem mass spectrometer (LC–MS–MS). Significant improvements have been made over the last decade, with LC methods shifting from traditional particles (3–5-µm) to ultrahigh-performance liquid chromatography (UHPLC) using smaller particles (1.7–2-µm). This has led to narrower peaks, which has improved peak capacity and detectability. Improvements in MS instruments have increased sensitivity and acquisition speeds (required for quantitative measurement of the narrow peaks). Furthermore, MS instruments have become compatible with the higher flow rates typically used in UHPLC. Many laboratories have transferred existing HPLC methods to UHPLC in combination with highly sensitive and fast scanning MS–MS instruments.
The currently applied UHPLC techniques in the field of routine food analysis can be divided in different classes: low flow UHPLC (flow rate = 10–100 µL/min); conventional UHPLC (100–600 µL/min); and high flow UHPLC (>600 µL/min). Nano- or micro-liquid chromatography [nLC (<1 µL/min) or µLC (1–10 µL/min)] are missing from this list because they have not been routinely used in the field of routine food safety analysis for a number of reasons.Variable flow rates were one major issue with nLC and µLC attributed to nano- or micro-flows that were attained by splitting regular flow rates using capillaries; the viscosity of the mobile phase changes during analysis; and injection of dirty samples that could cause clogging. This hampered the implementation in routine laboratories because unstable flow rates caused unstable retention times that meant the methods could not be used for official control purposes where it is mandatory that retention times are consistent to allow proper identification (2,3). This was especially true in laboratories where a technique is performed on a daily and routine basis.
Another drawback of early nLC or µLC techniques were issues associated with the connections between the autosampler, trapping columns, analytical columns, and electrospray emitters. Each connection would introduce void volumes that eventually resulted in serious peak broadening. Moreover, detecting a small leakage with all these connections was a challenging task at low nano or micro flow rates. This was almost impossible for routine applications where various applications would be performed on a single instrument and columns would have to be frequently changed. For these reasons outlined, conventional nLC and µLC were not really applicable for routine applications (4).
Nowadays, there are several commercial nLC and µLC instruments available, without the flow rate issues discussed above, that are able to produce stable nano- or micro-flow without using flow splitting. The most recent nano pumps can produce stable low flow rates of 10–4000 nL/min and handle a pressure of 10,000 psi (approximately 700 bar). This high-pressure tolerance made it possible to use nano or microbore columns packed with sub-2-µm particles. nUHPLC and µUHPLC are now becoming the standard in nano and microLC separations.
A major issue remaining is the connection of pre-columns and analytical columns to both the nUHPLC and nano electrospray emitter, restricting the implementation of the technique in the field of routine food control. In response to this, n(U)HPLC and µ(U)HPLC devices have been developed that can be directly connected to the electrospray source or LC instrument without manual connections. These solutions lead to more robust equipment as no manual connections have to be made. Potential advantages of the nLC and µLC are reduced use of high-grade solvents and reference standards. Furthermore, at these lower flow rates in theory the detectability is improved and ionization suppression is reduced (5–7). To explore the applicability of nUHPLC and µUHPLC in food safety, the levels of veterinary drugs and steroids in porcine meat and urine were determined.