Valerie Walker

Headspace solid-phase microextraction procedures for gas chromatographic analysis of biological fluids and materials

Solid-phase microextraction (SPME) is a new solventless sample preparation technique that is finding wide usage. This review provides updated information on headspace SPME with gas chromatographic separation for the extraction and measurement of volatile and semivolatile analytes in biological fluids and materials. Firstly the background to the technique is given in terms of apparatus, fibres used, extraction conditions and derivatisation procedures. Then the different matrices, urine, blood, faeces, breast milk, hair, breath and saliva are considered separately. For each, methods appropriate for the analysis of drugs and metabolites, solvents and chemicals, anaesthetics, pesticides, organometallics and endogenous compounds are reviewed and the main experimental conditions outlined with specific examples. Then finally, the future potential of SPME for the analysis of biological samples in terms of the development of new devices and fibre chemistries and its coupling with high-performance liquid chromatography is discussed.

Headspace solid-phase microextraction profiling of volatile compounds in urine: application to metabolic investigations

Volatile compounds contribute substantially to the metabolic pool in man. Their analysis in body fluids is problematic. We investigated headspace solid-phase microextraction (HS-SPME) with Carboxen-polydimethylsiloxane fibres and gas chromatography-mass spectrometry for profiling urinary volatile components. These fibres were more sensitive for very volatile and sulfur compounds than three other phases tested. We detected a wide range of compounds in normal urine at acid and alkaline pH. Profiles presented for five individuals with metabolic disturbances demonstrate abnormal accumulation of sulfur compounds, fatty acids and plasticisers. HS-SPME can complement profiling of non-volatile compounds in metabolic investigations and could be a useful extension of the diagnostic repertoire.

Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography–mass spectrometry

Trimethylaminuria (fish odour syndrome) is diagnosed from an increase in urinary excretion of trimethylamine with decreased trimethylamine oxide. We report a new quantitative stable isotope dilution gas chromatography-mass spectrometry procedure for the analysis of these metabolites using solid-phase microextraction (SPME). Both polydimethylsiloxane and mixed Carboxen-polydimethylsiloxane SPME fibres were found to be suitable for the headspace extraction of TMA. This new sampling technique could have wide application for the analysis of volatile and semi-volatile compounds by metabolic screening laboratories.

Solid-phase extraction in clinical biochemistry:

In order to measure low concentrations of analytes in plasma and urine, it is often necessary to extract and concentrate them. With solid-phase extraction (SPE), this is achieved by partitioning the analytes between a solid and a liquid or headspace vapour. A wide range of high-quality materials is now available to do this, offering a variety of separation modes for different applications. These include partitioning using reversed-phase, normal-phase, ion-exchange, restricted-access and immunoaffinity sorbents or molecularly imprinted polymers and, increasingly, combinations of these processes. Solid-phase microextraction was introduced to analyse volatile and semi-volatile compounds. The range of sampling formats has expanded from simple packed syringes to cartridges, disks, SPE pipette tips and 96-well plates. These developments have facilitated automated off- and on-line sample processing. The basic principles of SPE and the recent innovations are reviewed here. This is a technological growth area. Some of the developments are finding application in clinical toxicology. However, they could also be of wider value in clinical chemistry - for example, for analyses of volatile and non-volatile metabolites, peptides, radioactive elements and trace metal speciation.

Headspace solid-phase microextraction with 1-pyrenyldiazomethane in-fibre derivatisation for analysis of faecal short-chain fatty acids

Short chain fatty acids (SFCAs) are nutritionally important products of colonic bacteria. Their analysis in faeces is problematic. We report a headspace solid-phase microextraction procedure in which faecal SCFAs are derivatised on the fibre in-situ with 1-pyrenyldiazomethane. With this method sharp, well-resolved chromatographic peaks were obtained with no interferences. Inclusion of deuterated analogues enabled accurate quantification. Good linearity, recoveries and precision were achieved. Differences observed between the SCFA profiles of normal subjects and patients with cystic fibrosis indicate the potential of this new technique for clinical studies. 2-Methylbutyric acid was found in all faecal samples. Few have reported this before.

Urine 4-heptanone : a β-oxidation product of 2-ethylhexanoic acid from plasticisers

4-Heptanone is a common volatile constituent of human urine and is of unknown origin. We hypothesised that it arises from in vivo beta-oxidation of 2-ethylhexanoic acid (EHA) from plasticisers, similar to formation of 3-heptanone from valproic acid. We investigated urine from individuals with normal and increased plasticiser exposure. Using GC/MS, solvent-extracted organic acids were analysed as trimethylsilyl (TMS) derivatives and heptanone with headspace solid-phase microextraction. We identified 3-oxo-2-ethylhexanoic acid, the beta-oxidation product of EHA, as an enol in all samples. This is the first report of its TMS mass spectrum. We also found 2-ethyl-1,6-hexanedioic acid and 5-hydroxyEHA, omega- and omega-1-oxidation products of EHA, respectively, and 2-ethylhexanoylglucuronide, but only in trace amounts in some plasticiser samples. These compounds have not been reported in human urine, nor has the TMS mass spectrum of 5-hydroxyEHA. The median concentrations of 3-oxoethylhexanoic acid and total 4-heptanone of seven plasticiser samples were around 30--175-fold higher than normal samples. 4-Heptanone was barely detectable and 3-oxoethylhexanoic acid was not increased in an eighth plasticiser sample, from a baby with deficiency of 2-methylbranched-chain acyl-CoA dehydrogenase. beta-Oxidation is a major catabolic pathway of EHA in man, and might be involved in the metabolism of other branched-chain drugs and environmental pollutants.

Prostaglandins, Thromboxane, Leukotrienes and the Cerebral Circulation in Health and Disease

Arachidonic acid is ubiquitously distributed throughout the body and its derivatives form an extraordinary and bewildering array of compounds of widely differing properties. It is most unwise to generalize about how a given cell or tissue will utilize each pathway. This system responds to both physiological and pathological stimuli but the respective roles of the various products can be very difficult to unravel as will become all too apparent. The recent British Medical Bulletin (1983) provides an excellent overview of the subject. Von Euler (1936) created the term “prostaglandin” to christen the depressor, smooth muscle–stimulating acidic lipid that he and Goldblatt had demonstrated in human seminal plasma. Bergstrom began to determine the structure of this group of compounds in 1947 with publication in the early 1960s (for review, see Bergstrom et al. 1968). Following the isolation and description of the effects of the primary and relatively stable prostaglandins (PGF2 α, PGE1,PGE2, PGD2, etc.) came the discovery that prostaglandin synthesis was inhibited by the non–steroidal antiinflammatory agents such as indomethacin and aspirin (Vane 1971). Certain discrepancies became apparent in the period 1970–1976 between the effects of prostaglandin synthesis inhibition and the effects of the known endogenous prostaglandins. Then, in rapid succession, came the description of the short-lived derivatives of arachidonic acid metabolism, including rabbit aortacontracting substance (Palmer et al. 1973), the cyclic endoperoxides, PGG2 and PGH2, and thromboxane A2 and its inactive metabolite thromboxane B2 (Samuelsson et al. 1978, for review).

Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h.

Parathyroid hormone (PTH) concentrations were compared in serum and EDTA plasma from 36 patients attending a renal stone clinic. Serum PTH concentrations ranged from 0·9 to 10·9pmol/L, with a mean of 4·6pmol/L. When serum and EDTA plasma results were compared, in samples frozen within 30 min of collection, EDTA plasma results were found to be significantly higher than those in serum (P <0·0001; Wilcoxon test), with an average increase of 19·5% over the serum result. Results from EDTA-preserved blood left to stand at room temperature for 48 h were on average 14·8% lower than results from the corresponding EDTA plasma samples frozen within 30 min, with a highly significant difference (P <0·0001). Freshly frozen serum and 48h EDTA plasma PTH results were not significantly different. Parathyroid hormone in EDTA-preserved blood is not completely stable, and this could lead to misclassification of results for samples which are not frozen quickly.

Association of hypovolemia after subarachnoid hemorrhage with computed tomographic scan evidence of raised intracranial pressure.

Hypovolemic patients are more likely to suffer delayed cerebral ischemia and infarction after a subarachnoid hemorrhage (SAH). Prompt recognition and correction of hypovolemia may improve the outcome. We have identified computed tomographic (CT) scan findings that increase the probability of a patient presenting with hypovolemia soon after an SAH. The plasma volume (PV) of 25 patients admitted within 96 hours of an SAH was measured using radioiodinated serum albumin. The normal PVs were measured in an outpatient setting 6 months later or predicted from their total body water. Nine patients (36%) were found to be hypovolemic, defined as a fall in PV exceeding 10% of the normal PV (mean fall, 18 +/- 2%). Sixteen patients were normovolemic or hypervolemic (mean PV, +9 +/- 2%). The basal cisterns were compressed or obliterated on the CT scans of all hypovolemic patients compared with 12.5% of normovolemic patients (chi-square, 14.52; P less than 0.01). The probabilities of a patient being hypovolemic if the CT scan indicated raised intracranial pressure were high: hydrocephalus, P = 0.80; compression of the basal cisterns, P = 0.82; and compression of the basal cisterns associated with intracerebral hematoma or midline shift, P = 1.00. Patients with an SAH and radiological evidence of raised intracranial pressure should be considered at particular risk for systemic hypovolemia.

Automated headspace gas chromatographic analysis of faecal short-chain fatty acids

A method was developed and validated for analysis of faecal short-chain fatty acids using automated headspace gas chromatography. Quantification was by standard addition. Ghosting was minimized by lining the transfer tube from the headspace sampler to the gas chromatograph with deactivated fused silica and addition of formic acid to sample vials. Saturation of samples with lithium sulphate increased recoveries. The method was used to analyse small amounts of faecal matter collected from premature babies. Advantages of the technique are rapid, accurate, analysis of faecal specimens in batches, with minimum sample preparation.