The Use of a Multipurpose Sampler for Headspace GC-MS Analysis of Volatile Organic Compounds in Human Urine and Plasma

A multi-purpose-sampler (Gerstel MPS), designed for liquid large volume, gaseous and headspace samples was used for the GC-MS analysis of organic volatiles in human urine and plasma. Headspace sampling with a volume, temperature and speed controlled gas tight syringe was combined with a temperature controlled cooled injection system for cold trapping, enrichment and focussing of analyte. Regular 2 ml GC-vials filled with 1 ml acidified urine or plasma were used as headspace sampling vials. A 100 vial autosampler tray was equipped with an additional temperature and heating time controlled preheating station for 5 vials. Profiles of organic volatiles were determined and 4-heptanone as a ketone of medical interest was quantified. Calibration curves and imprecision of the urine method for 4-heptanone concentrations in the range from 40 to 800 ng/ml showed a correlation coefficient of r = 0,9980 and a coefficient of variation (CV) between 3.0 and 3.4% respectively. In this pilot study including 92 patients with diabetes mellitus (Type I and II) and 51 controls the median for the diabetic group was 179 ng/l compared to 188 ng/l in the control group. Further studies have to show if there actually exsists a relationship between 4-heptanone and diabetes mellitus or if the origin of 4heptanone is solely environmental. A possible source could be the widespread plasticizer Di-(2-ethyl hexyl)phthalate (DEHP), which in vivo could be hydrolyzed and oxidized to the corresponding β-ketoacid. Spontaneously and upon heating this acid would yield 4-heptanone. All major intermediates have now been found and identified in serum and urine. First results from human studies under current investigations show in fact that the plasticizer DEHP is the origin of elevated 4-heptanone concentrations in urine and plasma of patients receiving intravenously applied infusions. 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 Nheterocycles, sulfur containing compounds (isocyanates, sulfides) and hydrocarbons are found regularly and may be derived from nutrients, intermediates or environmental contaminants [11]. Pattern recognition of profiles [5] and especially the concentration of several ketones [6, 12], such as 4heptanone [8, 13] were related to diabetes mellitus. In diabetic patients elevated levels of 4-heptanone in urine were found and tentatively related to more specific stages of the disease [8, 13]. A possible relationship also 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 selective 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, dimethyl sulfide and dimethyl disulfide 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, dimethyl disulfide) found in coffee [26]. EXPERIMENTAL Sample preparation. A total of 189 urine samples (spontaneous and 24h collecting period) were taken from 51 healthy controls and 92 diabetic patients. 2ml GC vials were filled with aliquots of 1 ml acidified (30μl conc. HCL) urine and analyzed in duplicates. Serum samples were collected from patients with diabetes, liver diseases and on hemodialysis. 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 HP-7673 tray for 2ml standard vials (Hewlett-Packard, Waldbronn, Germany) 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, Waldbronn, Germany). Figure 1. Gerstel Multi Purpose Sampler in standby (left) and injection mode (right). CIS Heatable Syringe