DETERMINATION OF NATURAL AND SYNTHETIC STEROIDS IN ANIMAL URINE SAMPLES TWO SOLID-PHASE EXTRACTIONS IN ONE METHOD: ROBOTIC SYSTEM SIGNIFICANTLY REDUCES PROCESSING TIME


The Chemical and Veterinary Investigations Offi ce Münsterland-Emscher-Lippe (CVUA-MEL), a governmental laboratory in Germany, has so far carried out all steps required for sample preparation for the analysis of steroids in animal urine manually. This is a lengthy and very time-consuming activity as two solid phase extractions have to be carried out successively. In order to lighten the workload of the laboratory staff and to be more time-effi cient, the CVUA-MEL has recently implemented the LCTech GmbH modular robotic system FREESTYLE for automated sample preparation. In conjunction with the MACHEREY-NAGEL’s CHROMABOND SPE columns, good quality and reproducible results are obtained and, moreover, a high sample throughput can be achieved. The system operates under a positive pressure of up to 4 bar, and makes it possible to process samples with different viscosities and reliably excludes cross-contamination.


The National Residue Control Plan (NRKP) for food of animal origin is a program for preventative consumer health protection, which has been implemented across the EU since 1989 applying uniform standards. Within the framework of this program, live livestock is also investigated for residues of unwanted substances. In this context, the CVUA-MEL regularly checks animal urine samples for steroids. “The use of these substances as growth promoters is prohibited in the EU. Synthetic steroids must therefore be detectable in urine even at very low concentrations,” explains Dr. Thorsten Bernsmann, Head of Division at CVUA-MEL. “Minimum detection limits exist for individual substances of this substance group, i.e. a so-called “recommended concentration” of 1 µg/l urine has been set”. In order to achieve this very low detection limit, it is necessary to carry out sample preparation before analysis.


Laborious Manual Clean-up


The sample preparation for the determination of steroids in animal urine is complex, since two solid phase extractions have to be carried out successively: The urine samples are fi rst centrifuged to separate solid impurities. The pH is then adjusted to a value of about 5 using a buffer in order to be able to hydrolyse the samples enzymatically with glucuronidase or arylsulfatase.


By hydrolysis, for example, sugar bound steroids are converted into their free form so that they can be detected during the fi nal measurement. “The fi rst clean-up of the hydrolysed sample extract, which is a retentive solid phase extraction, is carried out with a C18-SPE column. A wash with a 5% methanol solution and elution with pure methanol follows, discarding the fi rst millilitre. The second purifi cation is carried out using an NH2-SPE


IET May / June 2017 www.envirotech-online.com


FREESTYLE Software Allows for Method Set-up with Just a Few Mouse Clicks


Since February 2015, the CVUA-MEL changed their most complex, so far manually performed methods over to automated sample preparation using the LCTech FREESTYLE system. One of the recently automated methods is the analysis of urine samples for steroids. The software of the modular robotic system was specifi cally adapted for this special type of sample preparation: “The special feature was the integration of two successive solid phase extractions using different column formats into one single method (“Dual SPE”),” explains Sebastian Wierer, process developer at LCTech. For this purpose, we have developed a


column. This is a non-retentive solid phase extraction,” explains Dr. Bernsmann. The eluates are collected, evaporated, re-dissolved with solvent and subsequently measured.


“Up to the beginning of 2015, we carried out the described clean-up procedure manually at the CVUA-MEL, which was very time-consuming: Around 24 samples keep one employee busy for a whole working day,” explains Dr. Bernsmann. The samples often behave differently on the SPE columns, because they have, for example, different viscosities. “During manual processing, the lab staff are able to regulate the negative pressure, however, they have to pay attention to each sample individually, since several samples are processed at the same time,” says the scientist. “While one sample runs slowly through the SPE column and requires some negative pressure, the other runs smoothly, and this column must be prevented from running dry”.


special system software expansion, which allows for the combined processing of two columns within one method.” Owing to this modifi cation, the processing method for the “Dual SPE” can be easily set up in the software: In a special software window, all parameters required for the processing steps are stored.


In order to lighten the work load of the lab staff and to reduce the effort in- volved in two successive solid phase extractions, the CVUA-MEL has recently introduced automated sample preparation using the modular FREESTYLE robotic system by LCTech GmbH for the detection of steroids in animal urine samples.


Source: LCTech GmbH


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56