Jenna Waldron and Craig Webster.

Department of Clinical Biochemistry and Immunology, Birmingham Heartlands Hospital.

Mevalonic acid (MVA) is synthesised at an early and rate-limiting step in the biosynthesis of cholesterol by the enzyme HMG-CoA reductase, and is a useful measure of statin efficacy or treatment. We have developed a specific and sensitive high performance liquid chromatography-tandem mass spectrometry for the measurement of MVA. Following its conversion to mevalonic acid lactone (MVAL), MVA, and a deuterated internal standard, were extracted from human plasma samples using solid-phase extraction. Chromatographic separation was achieved using a Luna PFP column (Phenomenex), which enables enhanced selectivity and improved resolution for polar compounds. An isocratic system was used with a mobile phase consisting of 15% methanol and ammonium formate buffer (5mM, pH 2.5), and a flow rate of 0.5 ml/min. Analysis was performed using an API 5000 tandem mass spectrometer (Applied Biosystems) and using an electrospray ESI source in positive mode. The instrument was tuned for the mass transitions of m/z 131?113 and 131?69. The retention times achieved for each transition allows for a total run time of 5 minutes. In conclusion, we have developed a method that is analytically specific and sensitive, and could potentially be used to determine the endogenous levels of MVA in normal individuals, and in patients undergoing statin treatment to aid in their lipid management.

Hardy KJ, Gossain S and Penny A

Objectives

To develop an on-line tool that allows timely and targeted completion of a root cause analysis investigation (RCA) for MRSA bacteraemia cases within an acute hospital setting.

Authors: Jason T. Evans (1), Beck Taylor (2), Desmond Estephane (1), Sarah Gardiner (1), E. Grace Smith (1), Peter M. Hawkey (1).

Objectives: The utility of DNA fingerprinting of M. tuberculosis strains has been enhanced by MIRU-VNTR typing. An enhanced set of 24 loci that offers greater discrimination over the original 12 loci and aims to improve the concordance of strain typing data with epidemiological data has been published. The aim of this study was to evaluate the optimal MIRU-VNTR loci set in discriminating clusters defined by the original MIRU-VNTR loci that have varying levels of epidemiological links.

J Duffy, C Webster

Department of Clinical Biochemistry and Immunology, Heartlands Hospital, Bordesley Green East, Birmingham, UK.

Introduction:  Vitamin D status is commonly assessed by measuring the concentration of 25OH vitamin D in serum.  LC-MS/MS has recently become the method of choice for its measurement.

The method described here is a gradient LC-MS/MS method which is both quick and precise and has excellent sensitivity.

Method:  Serum samples were initially precipitated with methanol containing deuterated vitamin D3 (internal standard).  The samples were then submitted to a single hexane extraction and evaporated to dryness.  Samples were reconstituted in 50:50 Methanol:water and 60uL injected onto a Luna C8(2) Mercury Cartridge 20x2mm (Phenomenex).  HPLC was performed using a gradient elution starting with 70% B and increasing to 95% B over 0.5 minutes.  Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.2% formic acid in methanol and the flow rate was 0.8ml/min.  Sample analysis was in positive mode on an API 3200 Q Trap(r) tandem mass spectrometer (Applied Biosystems, Warrington, UK) using an APCI ion source and transitions m/z 413.2>355.4, m/z 401.3>365.4 and m/z 407.4>371.4 for vitamin D2, D3 and deuterated D3 respectively.

Results:  Run time was 2.5 minutes in total with D2 eluting at 1.62 minutes and D3 at 1.60 minutes.  The method was linear up to 309nmol/L for D2 (r=0.9977) and 314nmol/L for D3 (r=0.9976).  Intra-assay variation at 5nmol/L was 25 % for D2 and 21% for D3.  Inter-assay variation was 10.2% and 11.0% for D2 and 4% and 11.2% for D3 at 15.6nmol/L and 43.9nmol/L respectively.

Conclusion:  The method described above is a rapid and sensitive gradient method for quantitation of 25 OH Vitamin D using APCI ionisation to increase sensitivity and decrease background noise.