The Use of a Multi Purpose Sampler for Headspace GC-MS Analysis of Volatile Organic Compounds in Human Urine

A multi purpose sampler (Gerstel MPS), designed for liquid large volume, gaseous and headspace samples was tested for its suitability in the GC-MS analysis of organic volatiles in human urine. Headspace sampling with a volume, temperature and speed controlled gas tight syringe was combined with a temperature controlled cold injection system for cold trapping, enrichment and focussing of analyte. Regular 2 ml GC-vials filled with 1 ml urine were used as headspace sampling vials. A 100 vial autosampler tray was equipped with an additional temperature and heating time controlled pre-heating module for 5 vials. Preliminary experiments were done with urine samples from our clinical laboratory with a ketone or glucose positive test result. In addition urine samples from healthy volunteers were taken and analysis were done with and without acidifying the urine. The promising results in regard to sensitivity and practicality in a day to day routine together with the cost cutting philosophy of a multi purpose sampler makes this highly automated system very attractive for clinical routine use. INTRODUCTION The concept of "metabolic profiling" has been widely applied in general to all different kinds of biological fluids such as urine, serum, cerebrospinal fluid, amniotic fluid, breast milk and to tissue homogenates [1-3]. Next to the organic acid fraction in urine and serum the profiles of organic volatiles have been intensively studied and linked to metabolic disorders [4-10]. The profile of organic volatiles in urine covers a diverse group of different polarity: alcohols, aldehydes, ketones, Oand N-heterocycles, sulfur containing compounds (isocyanates, sulfides) and hydrocarbons are found regularly and may be derived from nutrients, intermediates or environmental contaminants [11]. Patternrecognition of profiles [5] and especially the concentration of several ketones [6,12], such as 4-heptanone [8,13] were related to diabetes. A possible relationship was found between endogenous volatile urinary metabolites with structures similar to certain neurotoxins [14] and the development of the diabetic polyneuropathy [10,15]. The sampling techniques used for the analysis of organic volatiles include static and dynamic headspace with condensation in a cryogenic trap [16,17] or adsorption onto the hydrophobic porous polymer Tenax (poly 2,6-diphenyl-p-phenylene oxide) [5,8,18,19], solvent extraction [4,13] and the use of a transevaporator [20-22]. Modifications have also be done concerning the instrumentation [23,24]. Next to the GC-MS other selectiv detectors for complex sulfur, nitrogen, phosphorous or halogen can be very useful in headspace analysis [25]. Changes in the composition of the volatile sulfur containing compounds in the urine of diabetic persons can reliable be registered by use of a sulfur detector [25]. Mercaptanes such as methanthiole, ethanthiole, dimethylsulfide and dimethyldisulfide can result from the enterobacterial degradation of methionine in the state of hepatic encephalophaty, but may also in some extent be due to sulfur compounds (methanthiole, dimethyldisulfide) found in coffee [26]. EXPERIMENTAL Sample preparation. The urine samples, including those with a ketone and glucose positive test result, were collected from clinical routine laboratory. 2ml GC vials were filled with aliquots of 1 ml acidified urine and analyzed. Headspace GC-MS with Gerstel MPS Multi Purpose Sampler. Each sample is heated for the same period of time at the same temperature in the pre-heating module (Figure 1, 3). Solvent flushing of the MPS with helium is done by injecting the special designed syringe into the CIS-3 for 8 min. The heated syringe can then be filled with a defined volume of helium and injected into the headspace vial. The depht of injection is controlled for both the position in the vial and in the injector. The sample is injected into the cooled CIS-3 for focussing and enrichment (Figure 1, 6)and after heating up to the desired temperature transferred to the capillary column in either split or splitless mode. Instrumentation. The applied system consists of a Multi Purpose Sampler (Gerstel GmbH, Mülheim an der Ruhr, Germany), operated in headspace-mode and equipped with a 1000 μl gas tight syringe, a HP7673 tray for 100 2ml standard vials (Hewlett-Packard, Avondale, USA) plus an additional pre-heating module for 5 vials with control of temperature and heating-time (Gerstel GmbH, Mülheim an der Ruhr, Germany), a temperature controlled cold injection system CIS-3, (Gerstel GmbH, Mülheim an der Ruhr, Germany) used as interface, cold trap and injection system for the subsequently following GC-MSD combination (HP 5890/5972, Hewlett-Packard, Avondale, USA). Figure 1. Gerstel Multi Purpose Sampler in standby (left) and injection mode (right). CIS Heatable Syringe