With the constantly rising population and frequent uncertain weather, the need for high yielding agriculture is paramount. This means that use of plant protection agents, such as herbicides and insecticides is necessary, in order to secure harvests. However, in addition to the positive effects of such agents, severe side effects can be caused if high-level doses are ingested. As a result the use of these substances is strictly controlled, in order to protect the consumer’s health. Nevertheless, recent investigations have shown that restrictions have been violated in the past and sensitive fruit such as strawberries and grapes are frequently in the news for being contaminated.

There are currently about 1300 different active substances in use and only about half of them can be detected. However, they can cause minor irritations, temporary allergic reactions or even substantial damage to the immune system. The number of consumers who exhibit allergic reactions is also rising. Threshold Limit values for pesticides are specified by the European Union commission. However not all pesticides are covered and, in order to fill these gaps, highly efficient, fast analysis methods are required. Since the sample composition can be very complex, it is necessary to develop simple, generic sample preparation methods linked to fast analysis method for pesticides in food. Especially suitable for this purpose are HPLC methods or ultra-fast LC applications.

The analysis of structurally very similar polar substances is frequently unsuccessful, due to the insufficient selectivity of many conventional C18 phases. AS a result, YMC-UltraHT ProC18 was selected as the stationary phase. It is characterised by a balanced phase modification, which allows analysis of both very polar and moderately polar structures successfully.

Figure 1

Figure 2

Method development
The HPLC methods have been carried out on a Jasco X-LC system at ambient temperature with ESI-MS detection. It was found that using acetonitrile/water gradient eluents containing ammonium acetate gave good separation of all components (fi g. 1).
The chromatographic analysis of a mixture consisting of 26 pesticides showed a very good separation when using a YMC-Pack Pro C18 3 μm, 150 x 2.0 mm ID column. The chromatographic results are obtained within 21 minutes.
In figure 2 the method transfer onto a 75 x 2.0 mm ID YMC-UltraHT 2 μm column is shown. The flow rate has been increased from 0.2 ml/min to 0.4 ml/min in order to maintain the gradient profile. The retention pattern has not been altered, but the retention has been decreased to 9 minutes.

Results and discussion
The strict monitoring of pesticides used ever increasingly in agriculture increases the demand for sensitive and fast analytical LC methods. Therefore the need for downscaling of existing methods is one of the challenges that chromatographers meet. The method developed in this study shows the simple method transfer from a conventional HPLC method developed on YMC-Pack Pro C18 3 μm to an ultra-fast LC application. The use of the new YMC UltraHT ProC18, with its particle size of 2 μm enables downscaling without any further method development. The combination of small particles and short column lengths results in shorter retention times without compromising resolution.

Application Courtesy of J. Watanabe, TAKARA BIO INC.

For further information please contact Dr. Friederike Becker, YMC Europe GmbH, Phone: +49 (0)2064 427-261, Fax: +49 (0)2064 427-222 or visit www.ymc.de