Currently, the diagnosis of bowel diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) relies on invasive and expensive procedures. Identification of biomarker-based tests to aid diagnosis is an important area of research. Here we review the use of mass spectrometry in this search and discuss recent findings.
by Dr B. De Lacy Costello, Professor N. M. Ratcliffe and S. Shepherd
Inflammatory bowel disease (IBD) is an inflammatory autoimmune disease caused by an inappropriate response of the immune system to commensal gut microbes [1]. There are two types of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). UC affects the large bowel only, affecting variable lengths of the colon continuously from the rectum, primarily affecting the mucosa [Fig. 1]. CD can affect any part of the GI tract, and is a transmural disease [2]. Common symptoms of IBD are severe abdominal pain, defecation urgency and diarrhoea, which can contain blood.
Irritable bowel syndrome (IBS) is a functional disorder of the digestive tract. It is characterized by its symptoms, with no physiological changes in the GI tract. IBS can be diarrhoea predominant (IBS-D), constipation predominant (IBS-C) or symptoms can alternate between the two (IBS-A). Common symptoms include abdominal pain and cramps, bloating and flatulence, and unusual bowel habit. IBS has, as yet, no known cause. People with IBS show abnormal gut motility and hypersensitivity to pain in the GI tract. Stress and anxiety are known to cause changes in gut motility [3] with stress and anxiety being common symptoms of IBS. When under physical or psychological stress IBS patients showed increased gastro-intestinal sensitivity when compared to healthy controls [4]. Recently it has been thought that there may be changes in the gut microbiota in patients with IBS, the evidence being that IBS symptoms often occur after infective gastroenteritis or in patients in remission from IBD or diverticulitis. SIBO (small intestinal bowel overgrowth) has also been implicated in IBS and other function bowel disorders. One current hypothesis is that an altered microbiota activates the immune system within the mucosa, leading to an increase in epithelial permeability, causing dysregulation of the enteric nervous system [5]. Genome-wide association studies have successfully identified many genetic loci involved in susceptibility to IBD, and it is thought that genetic factors may also play a role in IBS [1].
Diagnosis of GI disease
IBS-D can present with symptoms similar to IBD and other non-functional bowel conditions. The diagnosis of IBS is often one of exclusion, where more serious bowel diseases, such as IBD or colon cancer which present with common symptoms, are ruled out. The current gold standard for diagnosis of IBD is endoscopic and histological testing; however, these investigations are both invasive and costly, and have associated risks. Of the patients referred for endoscopy few actually have organic bowel disease [6]. The costs associated with functional bowel disease are significant, with healthcare costs for IBS patients being significantly higher than non IBS controls [7].
There are currently no known biomarkers of IBS. There are various biomarkers that have potential in the differentiation of functional from inflammatory gastrointestinal disease, but there is still a need to identify biomarkers and to develop quicker, lower cost and less invasive testing for diagnosis of gastro-intestinal disease.
Biomarkers such as lactoferrin, calprotectin, c-reactive protein (CRP) and erythrocyte sedimentation rate (ECR) have all been used to help distinguish functional from inflammatory bowel disorders and to diagnose IBD. Serological markers such as antibodies to bacterial and fungal antigens that can indicate an abnormal response to commensal microbes can also be useful in identifying IBD.
Fecal calprotectin and lactoferrin are protein biomarkers of inflammation. In 2010 a meta-analysis of six studies (n=670) in adults by Van Rheenen et al. [8] found that screening patients by testing fecal calprotectin levels would have reduced the number of endoscopies performed by 67%, although its diagnosis would have been delayed in 6% of patients. When taking a weighted mean of 19 studies including 1001 patients, where IBD patients were compared with controls of IBS and other colonic diseases, fecal lactoferrin has a sensitivity and specificity of 80% and 82%, respectively [9].
Although these biomarkers can be useful as part of the screening process when establishing a diagnosis [6, 8], there is currently no biomarker or test that can replace the need for endoscopic and histological investigations. Mass spectrometry techniques are at the forefront of research for biomarker prospecting for IBS/IBD.
Mass spectrometry
Mass spectrometry (MS) has the ability to identify numerous compounds in a single sample. It is also high throughput allowing rapid analysis of many samples, which is especially useful for large studies or for the diagnosis of many samples. The ability to obtain results quickly, usually in less than 1 hour makes it attractive for clinical use.
Proteomic approach
Although MS (with associated sample vaporisation methods) was originally limited to low molecular weight volatile compounds, in the last 2 decades advances in MS technology have enabled its use with high molecular weight compounds, changing the way proteins are analysed. The soft ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) allow for the analysis of proteins and other macromolecules [10]. The identification of proteins through peptide mass fingerprinting, or peptide sequencing using MS is more rapid than techniques such as de novo protein sequencing and data can be analysed automatically. MS can also be used to determine the abundance of a molecule in a sample [10].
Differential protein expression can identify different diseases, and can indicate the degree of the disease state, or be used to assess the effects of treatment – for example the response of IBD patients to anti-TNF alpha antibodies (infliximab) [11]. It also has applications in the identification of protein biomarkers.
In 2011 MALDI-MS was used by M’koma et al. for tissue analysis; through profiling of the proteome of the colonic submucosa they were able to distinguish UC from CD by comparing proteomic spectra. Definitive diagnosis of either UC or CD is important as people with UC also have an increased risk of colon cancer [12].
Goo et al. have investigated protein biomarkers for IBS. ESI with LC-MS was used on protein fragments from the urine of women with IBS. They found differences in some specific components of the urinary proteome, and demonstrated that there is a possibility for future biomarker studies for IBS [13].
There are still limitations to mass spectrometric protein analysis, for example the difficulty in detecting hydrophobic membrane proteins. However, it seems promising that, with the advances in mass spectrometry technology, there will be an increase in the discovery of protein biomarkers and key pathogenic factors of gastro–intestinal disease, and improved diagnosis and therapy.
Metabolomic approach
The metabolome is the set of small molecule metabolites found in a biological sample. Unlike proteomics, metabolomics can be a direct measure of production of compounds and activity of cells or systems in an organism. This can be especially useful when looking for disease biomarkers in IBS and other bowel diseases as it can be used to understand the environment of the GI tract, as well as factors such as digestion and absorption of dietary products and gut microbial activity [14], which are implicated in IBS pathogenesis.
Researchers have explored the use of various techniques incorporating MS on breath [15], urine [16] and stool [17] samples in search of metabolic biomarkers of bowel disease for non-invasive testing and many possible candidates have been identified.
The commonly used analytical techniques in metabolomics are GC-MS (gas chromatography-mass spectrometry) or LC-MS (liquid chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) spectrometry. NMR has the advantage that there is no need to have the compounds in the vapour phase, although the limit of detection using NMR is much poorer than MS.
LC-MS metabolomic studies have been recently undertaken using urine to identify putative colon inflammation biomarkers [18]. The authors note that urinary biomarkers would be preferable to sampling intestinal tissue or blood as the collection of urine samples is non-invasive and multiple samples are more
readily obtained.
The analysis of volatile organic compounds (VOCs) or metabolites (VOMs) is an emerging area of disease diagnosis. VOCs are small molecules that are readily analysed by GC-MS. Other commonly used methods of VOC detection are selected ion flow tube mass spectrometry (SIFT-MS) [Fig. 2], and the similar technique of PTR-MS (proton transfer MS).
There are already several FDA approved tests using volatiles from breath. These include testing for heart transplant rejection, hemoglobin breakdown in children and measurement of hydrogen or methane to diagnose GI lactose or fructose malabsorption. The measurement of breath hydrogen has also been used to diagnose SIBO. Recent work by Španĕl et al. using SIFT-MS quantified the breath pentane concentration of study subjects using the reaction of O2+ with pentane. It was found that patients with CD and UC had significantly elevated breath pentane levels compared to healthy controls [15].
Testing for fecal biomarkers of bowel disease is facile as samples are easily obtained and have been in contact with the gastro intestinal tract. The changes in the odour of feces and flatus reported in many bowel conditions are due to changes in the VOC profile. This altered VOC profile could lead to identification of biomarkers of disease state. A recent pilot study carried out by Ahmed et al. using GC-MS on fecal samples from IBD and IBS patients identified a key set of VOMs which were able to distinguish IBS-D from Active IBD with a sensitivity of 96% and a specificity of 80% [19].
Conclusions
MS techniques show promise for the identification of biomarkers of various GI disease states, which have the potential to reduce invasive testing, improve patient care and reduce healthcare costs.
Instrumentation is still expensive and relatively large, limiting its use in hospital settings and particularly limiting its use for near-patient testing. Also biomarker discovery is still in its infancy and much remains to be clarified in relation to the significance of markers to disease and the underlying metabolic pathways.
However, work to reduce the size and cost of mass spectrometers is well advanced and would open up the possibility of instruments being deployed for point-of-care detection and monitoring of diseases including IBS and IBD.
References
1. Khor B, Gardet A, Xavier RJ. Nature 2011; 474(7351): 307–317.
2. Geboes K. Churchill Livingstone Elsevier 2003; 255–276.
3. Drossman DA, Camilleri M, Mayer EA, Whitehead WE. Gastroenterology 2002; 123(6): 2108–2131.
4. Murray CD, Flynn J, Ratcliffe L, Jacyna MR, et al. Gastroenterology, 2004; 127(6): 1695–1703.
5. Simrén M, Barbara G, Flint HJ, Spiegel BM, Spiller RC, et al. Gut 2013; 62(1): 159–176.
6. Kok L, Elias SG, Witteman BJ, Goedhard JG, Muris JW, et al. Clinical chemistry 2012; 58(6): 989–998.
7. Maxion-Bergemann S, Thielecke F, Abel F, Bergemann R. Pharmacoeconomics 2006; 24: 21–37.
8. Van Rheenen PF, Van de Vijver E, Fidler V. BMJ 2010; 341: doi 10.1136/bmj.c3369.
9. Gisbert JP, McNicholl AG, Gomollon F. Inflammatory bowel diseases 2009; 15(11): 746–1754.
10. Alberici RM, Simas RC, Sanvido GB, Romão W, Lalli PM, Benassi M, Eberlin MN. Analytical and bioanalytical chemistry 2010; 398(1): 265–294.
11. Han NY, Kim EH, Choi J, Lee H, Hahm KB. Journal of Digestive Diseases 2012; 13(10): 497–503.
12. M’Koma AE, Seeleyv EH, Washington MK, Schwartz DA, Muldoon RL, Herline A, Caprioli RM. Inflammatory bowel diseases 2011; 17(4): 875–883.
13. Goo YA, Cain K, Jarrett M, Smith L, et al. Journal of Proteome Research 2012; 11(12): 5650–5662.
14. Collino S, Martin FPJ, Rezzi S. British journal of clinical pharmacology 2013; 75(3): 619–629.
15. Hrdlicka L, Dryahina K, Spanel P, Bortlik M, et al. Gastroenterology 2012; 142(5): S-784.
16. Rao AS, Camilleri M, Eckert DJ, Busciglio I, Burton DD, Ryks M, Zinsmeister AR. Am J Physiol Gastrointest Liver Physiol 2011; 301(5): G919–G928.
17. Garner CE, Smith S, de Lacy Costello B, White P, Spencer R, Probert C, Ratcliffe NM. FASEB J. 2007; 21(8): 1675–1688.
18. Otter D, Cao M, Lin H-M, Fraser F, Edmunds S, et al. J Biomed Biotechnol. 2011; 2011: 974701
19. Ahmed I, Greenwood R, de Lacy Costello B, Ratcliffe NM, Probert CS. PloS one, 2013; 8(3): e58204.
The authors
Ben De Lacy Costello PhD, Norman M. Ratcliffe*PhD and Sophie Shepherd BSc
Institute of Bio-Sensing Technology, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY
*Corresponding author
E-mail: Norman.Ratcliffe@uwe.ac.uk
Autoantibodies against Phospholipase A2 Receptor
, /in Featured Articles /by 3wmediaFlexibility and Reliability in Nephelometry
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, /in Featured Articles /by 3wmediaChronic low back pain: could an anaerobic infection be responsible?
, /in Featured Articles /by 3wmediaAround 80% of people in Western countries experience low back pain at some point in their life; indeed during a single year up to half of the adult population will experience back pain. In the majority no single clear cause can be identified, and the condition is self limiting. However for the approximately 7% of patients who develop chronic low back pain, quality of life can be significantly impaired. Chronic low back pain also has a serious financial impact in terms of healthcare costs and lost working days; in most industrialized countries it is the most common reason for workplace absence.
Now researchers at the Spine Centre of Southern Denmark have published the results of two very interesting and potentially far-reaching studies. The first involved sixty-one patients who had been suffering from low back pain for more than six months. Lumbar disc herniation was confirmed by MRI and the patients underwent primary surgery for removal of nucleus material. When this material was cultured for micro-organisms, forty-six percent of patients had positive cultures, predominantly with the normally commensal anaerobic bacterium Propionibacterium acnes. A significantly higher number of patients with anaerobic infections developed new Modic changes (MC), MRI-visible bone edema associated with low back pain, in vertebrae adjacent to the previous disc herniation compared with patients with negative cultures or cultures positive for aerobic organisms.
The second study was a double blind randomized controlled trial involving 162 patients who had been suffering from low back pain for more than six months and with MC in a disc adjacent to a previous disc herniation. Patients received either placebo or the antibiotic Bioclavid for a hundred days and were followed up at the end of treatment and after one year; outcome measures included both pain and workplace absence. Improvement was highly significant in the group treated with the antibiotic.
The authors suggest that when the lumbar disc is herniated, the anaerobic bacteria penetrate it and precipitate an insidious infection and chronic low back pain. Although they stress that antiseptic techniques were rigorously followed when the nucleus material was removed, it is surely still necessary to find a method of demonstrating anaerobic infection in patients who have low back pain and relevant MC but who have not had surgery. If this could be done many desperate chronic lower back pain sufferers might finally be able to stop taking analgesics or visiting osteopaths, chiropractors and acupuncturists, and get relief from a course of antibiotics instead.
Mass spectrometry: exciting perspectives for clinical labs
, /in Featured Articles /by 3wmediaMass spectrometry is poised for a new era, as clinical labs and researchers, hospital managers and industry prepare themselves for expansion in its use. Fuelling growth are trends towards personalized healthcare, the identification of novel biomarkers for translational medicine, large-scale epidemiological screenings as well as everyday clinical chemistry tests beyond just toxicology and endocrinology. There is room for such growth. At present, clinical lab applications of mass spectrometers account for only about 5% of the market.
Superior sensitivity and specificity, samples reusable
Mass spectrometry identifies a molecule by its unique mass-to-charge ratio, and is both highly sensitive and specific. In spite of concerns about cost and steep learning curves, the superiority of mass spectrometry versus immunoassays has never been disputed. Indeed, a study by the US National Cancer Institute (NCI) in 2008 focused on using mass spectrometry to distinguish between breath samples from patients with ovarian epithelial cancer versus those with polycystic ovarian syndrome or endometriosis.
Another advantage of mass spectrometry is its ability to use the same serum for multiple analyte profiling. This makes it useful in large-scale clinical studies, where samples have often been archived. Another NCI study, for instance, used mass spectrometry to identify biomarkers in blood from patients with acute myeloid leukemia; some of the samples were almost 10 years old. Dated samples have also been used for a range of other biomarkers, including malignant melanoma, soft tissue sarcomas and non-small cell lung cancer.
Gas chromatography and liquid chromatography
As a technology, mass spectrometry is not new in a lab setting. Gas chromatograph MS (GC-MS) has been used for ages in the diagnosis of organic metabolic disorders. More recently, liquid chromatograph mass spectrometry (LC-MS) has become a recommended resource for screening newborns.
The longer use of GC-MS means a bigger user base, as well as a more extensive legacy database, richer software libraries and advanced algorithms. Although GC-MS requires more complex processes for sample preparation (discussed below), it is relatively inexpensive compared to LC-MS systems, and has been considered effective enough for the bulk of applications.
The challenge of standardization
However, there is still some way to go before mass spectrometry attains wider use. One key barrier is a lack of standardization, above all in the preparation of samples. Clinical labs have different approaches to this issue, especially in terms of purification. This leads to sometimes-significant differences in results. Confounding the problem are continuing changes in the methods used for sample preparation, over time even within individual laboratories.
In the US, the Clinical and Laboratory Standards Institute has published two sets of recommendations on the use of MS. However, these leave quite a bit of room for interpretation and are considered no more than broad guidelines.
Preparation of samples for mass spectrometry
Typically, two steps are involved in preparing a sample: the concentrating of analytes, followed by ionization. The sample itself consists of two parts: the analytes of interest, and other components which are collectively known as the sample matrix. Sample preparation is considered the most difficult when whole blood or fractions are involved, given a relatively low density of analytes. Urine lies at the the other end of the spectrum, since the kidneys have already done most of the job of concentrating analytes.
Techniques for preparing samples include solid-phase extraction (SPE), immunoextraction (or immunoaffinity purification) and so-called ‘dilute-and- shoot’. In SPE, analytes and other matrix compounds are separated on the basis of their physical and chemical properties, among them charge and polarity. SPE systems consist of a liquid, mobile phase and a solid stationary phase (usually disposable cartridge-based). The liquid phase uses two different solvents, one for binding and washing, and another for elution.
Immunoextraction separates antibodies bound to the analytes from ‘free’ matrix components, by immobilizing them to a chromatographic column or polystyrene beads. After incubation with an immobilized antibody, unwanted components are washed away, and the enriched analyte is then eluted; another method is to concentrate the sample by drying, followed by re-suspension and injection into the chromatography system.
The third mechanism for preparing MS samples, dilute-and-shoot, is generally used in samples with a relatively high concentrations of analytes (e.g. urine). Here, dilution is usually effective enough to reduce matrix components to a
manageable level.
Successful ionization essential
The process of analysis relies wholly on successful ionization, as mass spectrometers can only detect charged analytes in a gaseous phase. Ionization can be either positive (cationic) or negative (anionic). The most common techniques for ionization in a clinical lab consist of chemical ionization and electrospray.
Chemical ionization generates ions by combining heat and plasma (produced by high-voltage electricity), at atmospheric pressure. While high temperatures vaporize the sample, the plasma (also known as a corona discharge) ionizes the evaporated solvent. Following this, mechanical interaction of the sample components (including analytes of interest) leads to the formation of negative or positive ions.
On its part, electrospray ionization uses electricity, heat and air to successively reduce the size of droplets that elute off the chromatographic column and sharply increase their charge. Ions (above all, proteins) desorb from the liquid droplet surface into a gas phase and then enter the mass spectrometer.
Challenges for vendors
Until recently, industry has focused on process improvements, while researchers have concentrated on improving the specificity and sensitivity of mass spectroscopy. Innovations from vendors have aimed at increasing the efficiency of ionization and of ion transfer, and accelerating discovery of biomarkers by combining size exclusion and affinity capture to enrich low molecular weight proteins, and more quickly separate diseased from clear samples. Some companies have also coupled reference databases of micro-organisms to their mass spectroscopy systems.
The greatest challenge for industry, however, has been to increase user acceptability. Research scientists rather than clinical lab technologists have been the traditional target for mass spectrometry manufacturers. The former, typically, have more interest in top-of-the-line technical specifications and performance than user-friendliness. The potential demand from clinical labs is forcing vendors to change approach. As a result, several are now beginning to package equipment sales with training and support.
Industry is also paying attention to systems integration, to bundle sample preparation instrumentation into a mass spectrometry suite and control its findings. Indeed, software has so far proved to be one of the biggest impediments to the growth of mass spectrometry, once again given the delicate balance between enabling new users to operate a system on the one side, while permitting complex adjustment of performance parameters on the other. OEMs have sought to plug this gap with bespoke add-ons but, as all IT systems designers know, this adds to system cost.
Researchers aim for more precision, ease of use
On the R&D side, a potentially promising area consists of so-called time-of-flight (TOF) mass spectrometers. TOF provides accuracy of 1 part per million by accelerating gas phase ions toward a detector via an electric field. Other initiatives are focused on robotic assistance, turbulent-flow chromatography and ion mobility – with considerable potential seen in linear ion traps. Scientists are also exploring the use of nanospray interfaces as well as microfluidics, though most successes to date have been at bench scale. In the future, such improvements will permit a reduction of detection thresholds, along with greater precision, ease of use and efficiency.
Some trade-offs inevitable
For both researchers and industry, the Holy Grail is to devise adequate user-configurability for trade-offs between high throughput on one side (required, for example, in epidemiological studies or newborn screening), and sensitivity and specificity on the other. Even now, detection of steroids such as cortisol, estradiol and testosterone remain a challenge at the lower end of their reference range, but require high precision in certain categories of patients, for example elderly female patients.
Lab use of mass spectrometry still minor, room for growth
No one doubts that the market for mass spectrometry is potentially huge. Globally, sales have been rising briskly, after falling due to the recession. A study from Los Angeles-based Strategic Directions International estimates the 2011 mass spectrometer market at USD 3.9 billion, with projections of USD 4.8 billion by 2014. The US and Canada hold the largest share of the market (38%) followed by Europe (31%) and Japan (13%), with other countries accounting for the remainder. Leaders in the mass spectrometer market include AB Sciex, Thermo Fisher Scientific, Waters and Agilent Technologies (all from the US), along with Hitachi and Shimadzu. European companies have a smaller presence, and include Germany’s GSG, Spectromat and Thermolinear.
As mentioned before, the clinical lab segment accounts for a very small share of total sales. The biggest users are pharmaceutical companies (a share of 20% of sales, with mass spectrometers increasingly used for metabolomic screening and drug discovery). Government follows closely (with an 18.5% share), universities (12.6%) and environmental/general testing services (9.4%). Electronics, the food and chemical industries also buy more mass spectrometers than clinical laboratories or hospitals.
However, the hope is that continuing growth in this entrenched base of other users will drive down unit costs of mass spectrometers, just as clinical labs get ready to increase their own requirements.
The evolution of mass spectrometry for endocrine medicine
, /in Featured Articles /by 3wmediaLiquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is rapidly emerging as the technology of choice for measuring steroid hormones. This review will focus on the utility of clinical mass spectrometry for the assessment of endocrine disorders.
by Dr P. Monaghan, L. Owen, Prof. P. Trainer and B. Keevil
Mass spectrometry or immunoassay?
The technological armamentarium of the modern day clinical laboratory has been greatly enhanced by the introduction and continued evolution of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This technique is almost universally applicable to the measurement of small molecule compounds such as steroid hormones and is proving to be invaluable for the endocrinologist towards diagnosing and managing complex endocrine disorders [1]. Furthermore, LC-MS/MS is rapidly expanding for the application of quantitative peptide hormone and protein measurement. LC-MS/MS offers a number of considerable advantages over conventional immunoassay (IA) technology: greater analytical sensitivity and specificity, lack of susceptibility to interference from anti-reagent antibodies and cross-reacting compounds, multiplexing capability for steroid profiles, and low running costs for consumables in comparison to antibody-based reagents. However, like IA, LC-MS/MS is also vulnerable to interference that can compromise the analytical integrity of the method. The potential sources of analytical interference and inaccuracy to consider for IA and LC-MS/MS methodologies are summarized in Table 1.
Improved specificity: safer medical management of Cushing’s syndrome
The use of the 11β-hydroxylase inhibitor metyrapone generally has an adjunctive role in the medical management of Cushing’s syndrome with the aim of improving the medical status of patients prior to surgery or radiotherapy. Patients receiving adrenal-directed anti-steroidogenic drugs such as metyrapone require frequent clinical and biochemical monitoring to minimize the risk of treatment-induced hypoadrenalism.
Current clinical guidance advocates that metyrapone dose is titrated against serum cortisol concentration and some centres, including our own, assess normalization of cortisol production via the measurement a day curve with a mean serum cortisol target between 150–300 nmol/L. The monitoring of metyrapone therapy relies on the measurement of serum cortisol that by the vast majority of laboratories is performed by routine IA. However, metyrapone treatment causes altered steroid metabolism and therefore serum cortisol measurement is susceptible to positive interference when performed by IA due to cross-reactivity with precursor steroids such as 11-deoxycortisol (11DOC) that build up in the circulation as a result of the metyrapone blockade of the adrenal steroidogenic pathway.
Our group has recently quantified the level of positive interference in serum cortisol IA for patients receiving metyrapone therapy by employing a direct quantitative comparison with LC-MS/MS [2]. A modest correlation between plasma adrenocorticotropic hormone (ACTH) concentration and the extent of positive interference in the IA for serum cortisol was also observed as 90% of patients in our study had ACTH-driven Cushing’s syndrome [3]. Our study concluded that for patients receiving metyrapone therapy, cortisol analysis by LC-MS/MS mitigates the potential for erroneous clinical decisions concerning dose titration [Figure 1] and is likely to reduce the risk of unrecognized hypoadrenalism which may result in symptoms that mimic the side-effects of metyrapone treatment, or at worst be fatal.
Improved sensitivity: estradiol measurement
Progress in both LC-MS/MS and online sample preparation technology (pre-analytics) has advanced the analytical sensitivity of this methodology to the extent that for the measurement of many steroid hormones, modern MS applications have now transcended conventional IA methods in this regard. An example of this is the high sensitivity measurement of serum estradiol. External quality assurance data reveals that a wide range of concentrations can be obtained by immunoassay when measuring samples for estradiol at lower concentrations. Furthermore, a recent position statement from the Endocrine Society has stressed the need for better analytical methods to address the current poor performance of assays for measuring low concentrations of estradiol [4]. To this end, our group has developed a novel direct assay that is applicable to routine clinical use for the measurement of estradiol and estrone (therefore permitting calculation of total estrogen status) in male patients and patients on aromatase inhibitors [5]. This high sensitivity assay uses ammonium fluoride in the mobile phase to facilitate more efficient ionization and thereby increase analytical sensitivity. Additionally, an on-line solid phase extraction (OSM) system [Figure 2 (Waters, Manchester, UK)] allows a large volume of extract to be loaded and this coupled with a XEVO™TQS tandem mass spectrometer enables unprecedented analytical sensitivity to be achieved.
Conclusions and future prospects
LC-MS/MS is a very powerful tool which is enabling substantial innovations in the endocrine laboratory. Indeed, it is likely that the majority of emerging small molecules will be addressed by LC-MS/MS analysis. There are two keys areas in which future research and development for LC-MS/MS ought to be directed. Firstly, the utility of LC-MS/MS for the quantification of peptide hormones and proteins is already becoming a reality with published methods available for measurement of renin activity [6], parathyroid hormone [7] and insulin-like growth factor-1 [8] amongst others. These current methods require the skills of highly trained personnel in order to develop and run these assays, and it is hoped that continued innovation in this area will culminate in the development of rapid protein assays that are applicable to routine clinical use. Secondly, it seems feasible with existing technology to develop fully automated random-access LC-MS/MS analysers that will enable greater ease of use in non-specialist laboratory settings. However, the automation of mass spectrometry will not be achieved without a concerted effort from the in vitro diagnostics industry to fully realize the potential of LC-MS/MS across clinical medicine.
References
1. Monaghan PJ, Keevil BG, Trainer PJ. The use of mass spectrometry to improve the diagnosis and the management of the HPA axis. Rev Endocr Metab Disord 2013 Mar 15. [Epub ahead of print].
2. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone. Ann Clin Biochem 2011; 48: 441–446.
3. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Response to ‘Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone’ by Halsall DJ and Gurnell M. Ann Clin Biochem 2012; 49: 204–205.
4. Rosner W, et al. Challenges to the measurement of estradiol: An Endocrine Society Position Statement. J Clin Endocrinol Metab 2013; 98: 1376–1387.
5. Owen LJ, Wu FC, Labrie F, Keevil BG. A rapid direct assay for the routine measurement of oestradiol and oestrone by LC-MS/MS. Ann Clin Biochem [In press].
6. Carter S, Owen LJ, Kerstens MN, Dullaart RP, Keevil BG. A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solid-phase extraction. Ann Clin Biochem 2012; 49: 570–579.
7. Kumar V, Barnidge DR, Chen LS, Twentyman JM, Cradic KW, Grebe SK, Singh RJ. Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry. Clin Chem 2010; 56: 306–313.
8. Kay R, Halsall DJ, Annamalai AK, et al. A novel mass spectrometry-based method for determining insulin-like growth factor 1: assessment in a cohort of subjects with newly diagnosed acromegaly. Clin Endocrinol 2013; 78: 424–430.
9. Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: Minimizing risk. Ann Clin Biochem 2011; 48: 418–432.
10. Vogeser M, et al. Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory. Clin Chem 2010; 56: 1234–1244.
11. Duxbury K, Owen LJ, Gillingwater S, Keevil BG. Naturally occurring isotopes of an analyte can interfere with doubly deuterated internal standard measurement. Ann Clin Biochem 2008; 45: 210–212.
12. Davison AS, Milan AM, Dutton JJ. Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry. Ann Clin Biochem 2013; 50: 274.
13. Twentyman JM, Cradic KW, Singh RJ, Grebe SK. Ionic cross talk can lead to overestimation of 3-methoxytyramine during quantification of metanephrines by mass spectrometry. Clin Chem 2012; 58: 1156–1158.
The authors
Phillip J. Monaghan*1 PhD, Laura J. Owen2 MSc, Peter J Trainer3 MD, and Brian G Keevil2 MSc
1Department of Clinical Biochemistry, 3Department of Endocrinology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, UK.
2Department of Clinical Biochemistry, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK.
*Corresponding author
E-mail: Phillip.Monaghan@nhs.net
LC-MS/MS in clinical diagnostic laboratories: screening for catecholamine-producing tumours
, /in Featured Articles /by 3wmediaAccurate quantitative targeted analysis of low molecular weight compounds is one of the most important needs in clinical diagnostic laboratories. The enhanced analytical specificity and sensitivity of modern liquid chromatography–tandem mass spectrometry based methods satisfy this requirement for screening of endocrine related disorders, including those affecting steroidogenic systems or overproduction of catecholamines.
by Dr M. Peitzsch and Professor G. Eisenhofer
Liquid chromatography – tandem mass spectrometry (LC-MS/MS)
The development of electrospray ionization (ESI) enabled the introduction of aqueous chromatographic eluates into mass spectrometers, an advance for which John Bennett Fenn was awarded the Nobel Prize in chemistry in 2002. Subsequent refinements in liquid chromatography coupled with ESI mass spectrometry led to analytical applications directed at a broad range of macromolecules from peptides, proteins, glycoproteins and glycolipids to lower molecular weight polar and non-polar compounds, including fatty acids, vitamins, nucleic acids, steroids, amino acids and biogenic amines.
Introduction of tandem mass spectrometry (MS/MS) represented a further breakthrough enabling analyses of relationships between ‘parent or precursor ions’ in the first stage and ‘daughter or product ions’ in the second stage of the instrument [1]. For targeted quantitative analyses, the filtering capabilities and the multiple reaction monitoring (MRM) possible through MS/MS triple quadrupole instruments provide not only high selectivity, but also improved signal-to-noise ratios. In recent years, the increasing commercial availability of stable isotope labelled substances, used as internal standards, has facilitated the application of stable isotope dilution internal standardization as the gold standard for accurate quantitative analyses. Since the physicochemical properties of the target analyte and the stable-isotope-labelled internal standard are similar, this approach compensates for all variations which occur during sample extraction, injection, chromatography, ionization, and ion detection with dow stream improvements in analytical precision and accuracy [2].
The high analytical specificity of LC-MS/MS allows less rigorous sample purification and chromatographic resolution than for standard high performance liquid chromatographic (HPLC) procedures employing ultraviolet, electrochemical or fluorimetric detection. This, and other developments in column chemistry, such as those allowing ultra-high performance liquid chromatography (UPLC), in turn enables higher sample throughput than offered by conventional HPLC procedures. Fusion of LC-MS/MS with other technologies, such as multiplexing parallel LC systems and turbo-flow technology, provides additional advantages for efficient and accurate high-throughput quantitative analyses. Further, automated online sample extraction systems minimize time spent on sample preparation and allow multiple applications to be efficiently handled by one instrument.
All the above possibilities for extending sample throughput, combined with the versatility of a single LC-MS/MS system to take over the jobs of multiple standard HPLC systems, provide advantages that justify the initial high cost of the instrument. Recognized impediments to implementing LC-MS/MS technology include the complexity of the instrumentation associated with the necessity for highly skilled personnel, especially for method development. A lack of standardization combined with a shortage of inter-laboratory comparison programs for quality assurance represent other limitations to acceptance by clinical laboratories.
LC-MS/MS in clinical diagnostics laboratories
The improvements in precision and accuracy offered by LC-MS/MS are now well recognized as offering critical advances over standard HPLC and immunoassay procedures, which are subject to analytical interferences or do not allow precise and accurate identification of structurally-related compounds, such as steroid hormones. Such advances are important to the fields of endocrinology and clinical laboratory medicine where accurate quantitative analysis is crucial for diagnostic purposes [2].
LC-MS/MS applications are now used in clinical and forensic toxicology, such as for drugs-of-abuse testing. In clinical laboratory medicine, LC-MS/MS is used for measurements of endocrine hormones such as steroids, biogenic amines and thyroid hormones, as well as for therapeutic drug monitoring and in new-born screening for assessment of inborn errors of metabolism.
In contrast to commonly used immunoassays, LC-MS/MS enables measurements of multiple analytes for each sample processed. Such determination of analyte profiles includes those for the various thyroid hormones, different vitamin D metabolites and steroid profiles, all available in single analytical runs. Profiles of steroid hormones, although mainly used in research applications, hold considerable promise for the routine clinical assessment of a wide range of
steroidogenic disorders.
LC-MS/MS based screening for catecholamine producing tumours
Pheochromocytomas and paragangliomas (PPGLs) are tumours arising respectively in adrenal and extra-adrenal chromaffin cells that are characterized by an overproduction of catecholamines. Without diagnosis and an appropriate treatment, the excessive secretion of catecholamines by PPGLs can lead to disastrous consequences.
For initial biochemical screening different tests are available, including plasma or urinary measurements of the catecholamines – norepinephrine, epinephrine and dopamine – and their respective O-methylated metabolites – normetanephrine, metanephrine and 3-methoxytyramine. Whereas the free metabolites are usually measured in plasma, analyses in urine are commonly performed after acid hydrolysis in which free metabolites are liberated from sulfate conjugates.
In 2002, Taylor and Singh presented an LC-MS/MS method for the analysis of deconjugated urinary fractionated metanephrines [3]. The outlined advantages of this method over other methods, such as immunoassay and HPLC-ECD (electrochemical detection), included relative freedom from drug interferences, high sample throughput and short chromatographic run times. Subsequently, there has been a plethora of related methods published, including many that enable detection of the much lower concentrations of plasma free metanephrines than urinary deconjugated metanephrines.
Development of new sample preparation procedures, either offline or online to the LC-MS/MS system, have been particularly useful for automated high-throughput procedures [4, 5]. More recent improvements in LC-MS/MS instrumentation have led to improved analytical sensitivity, now even enabling accurate and precise measurements of picomolar plasma concentrations of 3-methoxytyramine, the O-methylated metabolite of dopamine [5–7]. This valuable biomarker not only allows detection of dopamine producing PPGLs, but can also be used to detect malignancy [9]. Using LC-MS/MS, the diagnostic performance of 3-methoxytyramine as a marker of malignancy was characterized by an enhanced diagnostic sensitivity of 86% and specificity of 96% [8] [Fig. 1].
Problems with drug interferences in HPLC-ECD and immunoassay-based methods are largely overcome using LC-MS/MS. For example, problems of acetaminophen (paracetamol) interferences in HPLC-ECD procedures are not a problem for LC-MS/MS [8, 10]. Chromatographic disruptions associated with certain disorders, such as renal insufficiency, are also less of a problem by LC-MS/MS than by HPLC-ECD [8].
Use of plasma free normetanephrine, metanephrine and methoxytyramine for reliable diagnosis of PPGLs requires collection of blood samples after 30 minutes of supine rest and an overnight fast. These conditions pose difficulties for many clinicians, which can result in excessive false-positive results or worse, missed diagnoses when inappropriately high upper cut-offs have been derived from seated sampling. Measurements of urinary metanephrines provide a reasonable alternative test for those situations where blood samples cannot be collected appropriately.
As mentioned above, urinary metanephrines are commonly measured after an acid-hydrolysis deconjugation step. This procedure is based mainly on historical convention, where initially less sensitive instruments did not allow measurements of the much lower urinary concentrations of free rather than deconjugated metanephrines. Improvements in analytical sensitivity now, however, allow analysis of urinary free metanephrines, [11, 12]. Unlike the sulfate-conjugated derivatives, which are produced by a sulfotransferase enzyme located in the gastrointestinal tract, the free metabolites are produced within chromaffin cells. This provides a potential advantage for measurements of the free metabolites. Another advantage is that there are no suitable quality controls or calibrators for measurements of urinary deconjugated metanephrines [12, 13]. Those that are available are almost entirely in the free form so that procedures will always pass quality control even if the deconjugation step is missed and values for patient samples are grossly under-estimated. Measurements of urine free metanephrines avoid this potential pitfall in quality assurance.
Finally, with measurements of urinary free metanephrines it is possible to combine the measurements with urinary catecholamines in a single run [12;14]. This also provides an advantage over measurements of urinary deconjugated metanephrines, where the deconjugation step does not allow measurements of free catecholamines.
The difficulties in applying LC-MS/MS in the clinical chemistry laboratory, such as associated with high initial instrument costs and need for expertise, are easily overshadowed by the analytical advantages. High sample throughput and the analytical versatility offered by LC-MS/MS, which enables rapid method switching, in particular represent important advantages over standard HPLC methods. Nevertheless, such advantages are not easily realized by the small hospital-based laboratory where high sample throughput is not an important consideration. In the US the highly competitive nature of the heath care system is an incentive for centralized testing where efficiency and low operating costs associated with high sample throughput (economy of scale) are more easily realized. In the US the switch from immunoassays or HPLC-based methodology to superior LC-MS/MS technology is therefore likely to remain more advanced than in Europe.
Summary and conclusion
Modern LC-MS/MS systems provide well-recognized accuracy for quantitative targeted measurements of analytes used for clinical diagnostics. The high-throughput capabilities and versatility of LC-MS/MS instrumentation enable multiple applications for rare diseases to be handled by a single instrument. Furthermore, single analyte assays can be extended to accurate profiling by LC-MS/MS assays, providing deeper insight into endocrine metabolic disorders. This, however, remains largely a research-based application and for LC-MS/MS to be readily adapted for routine use in the clinical laboratories, other advantages such as those associated with economy of scale must be appreciated and realized.
References
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14. Whiting MJ. Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Ann Clin Biochem 2009; 46: 129–136.
The authors
Mirko Peitzsch*1 PhD and
Graeme Eisenhofer1,2 PhD
1 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus at the Technical University Dresden, Dresden, Germany
2 Department of Medicine III, University of Dresden, Dresden, Germany
*Corresponding author
E-mail: Mirko.Peitzsch@uniklinikum-dresden.de
The use of MS for the investigation of irritable bowel syndrome and inflammatory bowel disease
, /in Featured Articles /by 3wmediaCurrently, the diagnosis of bowel diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) relies on invasive and expensive procedures. Identification of biomarker-based tests to aid diagnosis is an important area of research. Here we review the use of mass spectrometry in this search and discuss recent findings.
by Dr B. De Lacy Costello, Professor N. M. Ratcliffe and S. Shepherd
Inflammatory bowel disease (IBD) is an inflammatory autoimmune disease caused by an inappropriate response of the immune system to commensal gut microbes [1]. There are two types of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). UC affects the large bowel only, affecting variable lengths of the colon continuously from the rectum, primarily affecting the mucosa [Fig. 1]. CD can affect any part of the GI tract, and is a transmural disease [2]. Common symptoms of IBD are severe abdominal pain, defecation urgency and diarrhoea, which can contain blood.
Irritable bowel syndrome (IBS) is a functional disorder of the digestive tract. It is characterized by its symptoms, with no physiological changes in the GI tract. IBS can be diarrhoea predominant (IBS-D), constipation predominant (IBS-C) or symptoms can alternate between the two (IBS-A). Common symptoms include abdominal pain and cramps, bloating and flatulence, and unusual bowel habit. IBS has, as yet, no known cause. People with IBS show abnormal gut motility and hypersensitivity to pain in the GI tract. Stress and anxiety are known to cause changes in gut motility [3] with stress and anxiety being common symptoms of IBS. When under physical or psychological stress IBS patients showed increased gastro-intestinal sensitivity when compared to healthy controls [4]. Recently it has been thought that there may be changes in the gut microbiota in patients with IBS, the evidence being that IBS symptoms often occur after infective gastroenteritis or in patients in remission from IBD or diverticulitis. SIBO (small intestinal bowel overgrowth) has also been implicated in IBS and other function bowel disorders. One current hypothesis is that an altered microbiota activates the immune system within the mucosa, leading to an increase in epithelial permeability, causing dysregulation of the enteric nervous system [5]. Genome-wide association studies have successfully identified many genetic loci involved in susceptibility to IBD, and it is thought that genetic factors may also play a role in IBS [1].
Diagnosis of GI disease
IBS-D can present with symptoms similar to IBD and other non-functional bowel conditions. The diagnosis of IBS is often one of exclusion, where more serious bowel diseases, such as IBD or colon cancer which present with common symptoms, are ruled out. The current gold standard for diagnosis of IBD is endoscopic and histological testing; however, these investigations are both invasive and costly, and have associated risks. Of the patients referred for endoscopy few actually have organic bowel disease [6]. The costs associated with functional bowel disease are significant, with healthcare costs for IBS patients being significantly higher than non IBS controls [7].
There are currently no known biomarkers of IBS. There are various biomarkers that have potential in the differentiation of functional from inflammatory gastrointestinal disease, but there is still a need to identify biomarkers and to develop quicker, lower cost and less invasive testing for diagnosis of gastro-intestinal disease.
Biomarkers such as lactoferrin, calprotectin, c-reactive protein (CRP) and erythrocyte sedimentation rate (ECR) have all been used to help distinguish functional from inflammatory bowel disorders and to diagnose IBD. Serological markers such as antibodies to bacterial and fungal antigens that can indicate an abnormal response to commensal microbes can also be useful in identifying IBD.
Fecal calprotectin and lactoferrin are protein biomarkers of inflammation. In 2010 a meta-analysis of six studies (n=670) in adults by Van Rheenen et al. [8] found that screening patients by testing fecal calprotectin levels would have reduced the number of endoscopies performed by 67%, although its diagnosis would have been delayed in 6% of patients. When taking a weighted mean of 19 studies including 1001 patients, where IBD patients were compared with controls of IBS and other colonic diseases, fecal lactoferrin has a sensitivity and specificity of 80% and 82%, respectively [9].
Although these biomarkers can be useful as part of the screening process when establishing a diagnosis [6, 8], there is currently no biomarker or test that can replace the need for endoscopic and histological investigations. Mass spectrometry techniques are at the forefront of research for biomarker prospecting for IBS/IBD.
Mass spectrometry
Mass spectrometry (MS) has the ability to identify numerous compounds in a single sample. It is also high throughput allowing rapid analysis of many samples, which is especially useful for large studies or for the diagnosis of many samples. The ability to obtain results quickly, usually in less than 1 hour makes it attractive for clinical use.
Proteomic approach
Although MS (with associated sample vaporisation methods) was originally limited to low molecular weight volatile compounds, in the last 2 decades advances in MS technology have enabled its use with high molecular weight compounds, changing the way proteins are analysed. The soft ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) allow for the analysis of proteins and other macromolecules [10]. The identification of proteins through peptide mass fingerprinting, or peptide sequencing using MS is more rapid than techniques such as de novo protein sequencing and data can be analysed automatically. MS can also be used to determine the abundance of a molecule in a sample [10].
Differential protein expression can identify different diseases, and can indicate the degree of the disease state, or be used to assess the effects of treatment – for example the response of IBD patients to anti-TNF alpha antibodies (infliximab) [11]. It also has applications in the identification of protein biomarkers.
In 2011 MALDI-MS was used by M’koma et al. for tissue analysis; through profiling of the proteome of the colonic submucosa they were able to distinguish UC from CD by comparing proteomic spectra. Definitive diagnosis of either UC or CD is important as people with UC also have an increased risk of colon cancer [12].
Goo et al. have investigated protein biomarkers for IBS. ESI with LC-MS was used on protein fragments from the urine of women with IBS. They found differences in some specific components of the urinary proteome, and demonstrated that there is a possibility for future biomarker studies for IBS [13].
There are still limitations to mass spectrometric protein analysis, for example the difficulty in detecting hydrophobic membrane proteins. However, it seems promising that, with the advances in mass spectrometry technology, there will be an increase in the discovery of protein biomarkers and key pathogenic factors of gastro–intestinal disease, and improved diagnosis and therapy.
Metabolomic approach
The metabolome is the set of small molecule metabolites found in a biological sample. Unlike proteomics, metabolomics can be a direct measure of production of compounds and activity of cells or systems in an organism. This can be especially useful when looking for disease biomarkers in IBS and other bowel diseases as it can be used to understand the environment of the GI tract, as well as factors such as digestion and absorption of dietary products and gut microbial activity [14], which are implicated in IBS pathogenesis.
Researchers have explored the use of various techniques incorporating MS on breath [15], urine [16] and stool [17] samples in search of metabolic biomarkers of bowel disease for non-invasive testing and many possible candidates have been identified.
The commonly used analytical techniques in metabolomics are GC-MS (gas chromatography-mass spectrometry) or LC-MS (liquid chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) spectrometry. NMR has the advantage that there is no need to have the compounds in the vapour phase, although the limit of detection using NMR is much poorer than MS.
LC-MS metabolomic studies have been recently undertaken using urine to identify putative colon inflammation biomarkers [18]. The authors note that urinary biomarkers would be preferable to sampling intestinal tissue or blood as the collection of urine samples is non-invasive and multiple samples are more
readily obtained.
The analysis of volatile organic compounds (VOCs) or metabolites (VOMs) is an emerging area of disease diagnosis. VOCs are small molecules that are readily analysed by GC-MS. Other commonly used methods of VOC detection are selected ion flow tube mass spectrometry (SIFT-MS) [Fig. 2], and the similar technique of PTR-MS (proton transfer MS).
There are already several FDA approved tests using volatiles from breath. These include testing for heart transplant rejection, hemoglobin breakdown in children and measurement of hydrogen or methane to diagnose GI lactose or fructose malabsorption. The measurement of breath hydrogen has also been used to diagnose SIBO. Recent work by Španĕl et al. using SIFT-MS quantified the breath pentane concentration of study subjects using the reaction of O2+ with pentane. It was found that patients with CD and UC had significantly elevated breath pentane levels compared to healthy controls [15].
Testing for fecal biomarkers of bowel disease is facile as samples are easily obtained and have been in contact with the gastro intestinal tract. The changes in the odour of feces and flatus reported in many bowel conditions are due to changes in the VOC profile. This altered VOC profile could lead to identification of biomarkers of disease state. A recent pilot study carried out by Ahmed et al. using GC-MS on fecal samples from IBD and IBS patients identified a key set of VOMs which were able to distinguish IBS-D from Active IBD with a sensitivity of 96% and a specificity of 80% [19].
Conclusions
MS techniques show promise for the identification of biomarkers of various GI disease states, which have the potential to reduce invasive testing, improve patient care and reduce healthcare costs.
Instrumentation is still expensive and relatively large, limiting its use in hospital settings and particularly limiting its use for near-patient testing. Also biomarker discovery is still in its infancy and much remains to be clarified in relation to the significance of markers to disease and the underlying metabolic pathways.
However, work to reduce the size and cost of mass spectrometers is well advanced and would open up the possibility of instruments being deployed for point-of-care detection and monitoring of diseases including IBS and IBD.
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The authors
Ben De Lacy Costello PhD, Norman M. Ratcliffe*PhD and Sophie Shepherd BSc
Institute of Bio-Sensing Technology, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY
*Corresponding author
E-mail: Norman.Ratcliffe@uwe.ac.uk