AACB NSW MAIN

THERAPEUTIC DRUG MONITORING IN OLDER PATIENTS

AJ McLachlan

Faculty of Pharmacy, University of Sydney, NSW 2006 and Centre for Educational and Research on Ageing, Concord Hospital, Concord, NSW 2139, Australia andrewm@pharm.usyd.edu.au

Older people (> 65 yrs) and especially the oldest of old (> 85 yrs) display significant inter-patient variability in response to medicines which is a result of an age-associated increase in medical co-morbidities, changes in organ function, loss of homeostatic control, frailty, nutritional deficits and multiple medications (with the risk of interactions). Age-related changes in physiology and polypharmacy lead to significant changes in the pharmacokinetics and pharmacodynamics of medicines (1) which leads to an increase risk of medication misadventure related to adverse drug effects. Therapeutic drug monitoring (TDM) is a system of care which can be used to optimise quality use of medicine in older people by individualising drug and dose selection. Key issues for TDM in this population relate to the need to refine concentration targets (due to possible changes in pharmacodynamics), account for the impact of co-administered medicines, regularly review the clinical status of patients and simultaneously monitor organ function and homeostatic control. Chronological age is a relatively insensitive metric of drug dosing, whereas frailty is emerging as an influential covariate in drug dosing and adjustment independent. TDM also has a pivotal role in assessing possible drug-related problems such as drug induced delirium and changes in cognition.

1. Hilmer SN, McLachlan A, Le Couteur DG. Clinical pharmacology in geriatric patients. Fund Clin Pharmacol 2007; 21:217-230.

HPLC METHODS DEVELOPMENT

JW Earl,

Clinical Biochemistry, The Children’s Hospital at Westmead, Sydney, Australia johne@chw.edu.au

Establishing a published method from a journal article can be quite challenging, as there is usually a need to “fine tune” the assay to local conditions.

De Novo method development requires good technological skills and a thorough understanding of the chemical and physical processes which contribute to chromatographic separation, detection and analysis. Retention is governed by size, shape, polarity, charge, hydrogen bonding, dipole interaction, aromaticity, hydrophobic interaction, and the electron orbital configuration of the analyte and the way all these affect its binding to the stationary phase, as well as the structure, composition and geometry of the stationary phase. HPLC separation of different molecules usually involves choosing an appropriate stationary phase and then making subtle modifications to the composition of the mobile phase which exploit chemical structure differences between the molecules. HPLC is particularly useful for analysing unstable molecules.

Sensitivity is also governed by chemical structure and although a variety of detectors can be used including mass spectrometry, many important biological chemicals still cannot be measured at physiological levels. The most sensitive methods include HPLC with fluorescence or electrochemical detection, or GLC with electron capture detection, but the most specific methods usually involve mass spectrometry. Derivatisation or chemical modification can be used in some circumstances to increase sensitivity and selectivity and there is still much room for improvement in this area.

Method validation, determination of reference intervals and understanding the physiology of the analyte are all important additional components in developing and establishing a new method as changing physiology may alter the sample matrix in a way which impacts upon the analytical process.

A single point measurement produces limited information on biological systems. A program of pharmacokinetic and pharmacodynamic analysis or physiological mathematical modelling adds considerable value to a newly developed method.

IMMUNOSUPPRESSANT DRUG ANALYSIS: AN AUSTRALIAN PERSPECTIVE ON CHANGING FROM IMMUNOASSAYS TO LC-MS

BC Sallustio

Department of Cardiology and Clinical Pharmacology, The Queen Elizabeth Hospital, Woodville, SA 5011 and Discipline of Pharmacology, University of Adelaide, Adelaide, SA 5000

benedetta. sallustio@nwahs.sa.gov.au

Second generation immunosuppressant drugs (cyclosporin, tacrolimus, sirolimus, everolimus and mycophenolate) have had a large impact on transplant outcomes. However, due to narrow therapeutic indices and highly variable pharmacokinetics, therapeutic drug monitoring is necessary to individualise patient doses. Worldwide, immunoassays still form the most widely employed methods for quantitation of immunosuppressants in whole blood or plasma. Although convenient in terms of labour and turn-around times, these methods have several limitations including high costs, significant cross-reactivity with metabolites and poor sensitivity for the newer highly potent agents. Chromatographic techniques, such as HPLC offer significant cost-savings and increased specificity but often still lack sensitivity for clinical applications. The increasing affordability of LC-MS and LC-MS/MS allows the opportunity to provide sensitive and specific analysis of immunosuppressants with significant cost-savings compared to immunoassays.

The Queen Elizabeth Hospital is the second largest renal transplantation centre in Australia, performing an average of 70 transplants per year. Our laboratory introduced LC-MS/MS in December 2004 and in 2005/06 performed approximately 6200 patient immunosuppressant assays comprising 41% tacrolimus and 21% sirolimus (by LC-MS/MS), 12% mycophenolic acid (by HPLC-UV), 20% cyclosporin (by CEDIA) and 5% everolimus (by FPIA). Tacrolimus and sirolimus samples are processed together and are measured simultaneously using ascomycin and 32-desmethoxy rapamycin, respectively, as internal standards. Using 100 mL of whole blood, the lower limit of quantitation for both drugs is 1.5 mg/L, with intra- and inter-assay accuracies of 94.2% and 95.9%, respectively, for tacrolimus, and 84.8% and 97.9%, respectively, for sirolimus. Corresponding intra- and inter-assay coefficients of variation were <15% for tacrolimus, and <10% for sirolimus. Using LC-MS/MS our laboratory has maintained a daily service with same day turn-around, whilst significantly increasing assay sensitivity so that monitoring is now also useful following paediatric liver transplantation, where a target tacrolimus whole blood therapeutic concentration of 2 mg/L is increasingly used.

HPLC WITH COULOMETRIC DETECTION

BC McWhinney

HPLC Section, Department of Chemical Pathology, Queensland Health Pathology Service, Royal Brisbane Hospital, Herston, Brisbane Qld. 4029, Australia brett_mcwhinney@health.qld.gov.au

HPLC coupled with Electrochemical detection has been used in the Clinical Laboratory for several decades. The analysis of urine catecholamines was one of the first applications that utilised the sensitivity and selectivity of this technique other assays have gradually come on-line for both urine and plasma.

While the Electrochemical detector (ECD) has changed its design, configuration and features over the years, the fundamentals of analyte detection remain the same. Compounds that are electrochemically active will oxidise when an appropriate positive potential is applied, with the resultant generation of an electrical current that is recorded and the peaks quantitated.

There are two different ECD configurations. In the Amperometric detector, the mobile phase passes over the surface of the working electrode, analytes of interest are either oxidised or reduced depending on the applied potential. Approximately 10% analyte conversion occurs. The second configuration involves a highly porous working electrode, in which the mobile phase percolates through the electrode. Due to the significantly increased contact surface area, approximately 100% analyte conversion occurs, relative to Amperometric detection.

Each configuration requires precautions to ensure smooth operation and minimal user intervention. Mobile phase components, especially the water quality must be of the highest level possible, particularly if high sensitivity detection is required. Buffers and ion pairing agents will increase the background current generated, therefore their concentrations must be optimised. HPLC hardware especially the pump will also impact on the signal generated since ECDs are extremely flow sensitive. Pumps that produce pressure pulses due to design or faulty parts will cause extremely noisy baselines and thus make quantitation of peaks difficult.

The ECD for a number of clinical assays produces the sensitivity and selectivity that no other HPLC detector can offer.

DRUG MEASUREMENTS IN CHILDREN

CE Nath
Department of Biochemistry, The Children’s Hospital at Westmead, Westmead, NSW, 2145, Australia christan@chw.edu.au

Children are different from adults. They undergo periods of growth and development that are characterized by changes in the size and function of various systems within the body, including the hepatic and renal systems. Body composition also changes with age. As children become older there is a decrease in the extracellular fluid, a decrease in total body water and an increase in total body fat. These maturational changes can affect drug disposition and drug action enormously, making it very important that we perform therapeutic drug monitoring in children.
We use either gas chromatography or high performance liquid chromatography to measure the plasma concentrations of a number of drugs that are administered to children with cancer. These include drugs used in blood or marrow transplantation (busulphan, melphalan, carboplatin, fludarabine-des-phosphate), antifungal agents (amphotericin B, ambisome), antiviral agents (acyclovir), immunosuppressants (mycophenolic acid) and enzymes (L-asparaginase activity). Drug measurements using chromatographic analysis methods are ideal, as it is possible to separate the parent drug from its metabolites and to obtain highly accurate and precise concentration determinations, even at very low concentrations.
After obtaining the drug concentration measurements, it is still necessary to understand what these results mean and how they relate to children. Modelling the pharmacokinetics (dose versus concentration relationship) and pharmacodynamics (concentration versus therapeutic or toxic effect relationship) adds value to the chromatographic test by providing clinical insights into drug dosing. The optimal dose of any drug can be determined for children of all ages, taking into account their renal function, liver function, disease, concomitant medication and other factors. Optimal blood sampling times and corresponding therapeutic concentration ranges can then be identified and used for therapeutic drug monitoring and dose adjustments.

EXPERIENCE IN CYCLOSPORIN A TDM:
FROM IMMUNOASSAY TO TANDEM MASS SPECTROMETRY

CS Ho1, EYK Luk1, CB Leung2, CC Szeto2, PKT Li2, CWK Lam1,3
1Biomedical Mass Spectrometry Unit, Department of Chemical Pathology, 2Renal Unit, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, 3Macau Institute of Applied Research for Medicine and Health, Macau University of Science and Technology, Taipa, Macau chungshunho@cuhk.edu.hk

Introduction
Therapeutic drug monitoring (TDM) of whole blood cyclosporin A (CsA) was routinely performed using Abbott TDx immunoassay, which was expensive and suffered from interference by cross-reacting metabolites. To overcome these shortcomings, measurement of CsA was transferred to a liquid chromatography electrospray ionization tandem mass spectrometry (LCTMS) method.
Methods
Analytical performance of a LCTMS method was compared with that of TDx method. To familiarize transplant clinicians with the new CsA results, results from both methods were parallel-reported for 6 months. To establish new therapeutic ranges, pharmacokinetic study was conducted with 158 stable renal transplant patients.
Results
The new LCTMS method had improved analytical performance and lower consumable cost over the TDx method. Establishing new C2 sampling therapeutic range using the initial sample correlation data was unsuccessful as 40% of the C2 samples were suspected to be collected outside the appropriate sampling window. The subsequent pharmacokinetic study showed that the regression characteristics between the 2 methods were significantly different for C0 and C2 samples, suggesting varying CsA drug/metabolites ratio for these 2 sampling periods. Through close collaboration with transplant clinicians, new C0 and C2 therapeutic ranges for the LCTMS method were adopted for the routine service.
Conclusions
The LCTMS method has improved the CsA TDM service with improved analytical performance plus reduction in laboratory expense. It was essential to establish appropriate therapeutic ranges for the new method with collaboration with the transplant clinicians.

LIQUID CHROMATOGRAPHY MASS SPECTROMETRY IN THE CLINICAL BIOCHEMISTRY LABORATORY

CS Ho, CWK Lam
Biomedical Mass Spectrometry Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong. chungshunho@cuhk.edu.hk

Over the last 12 years, our Department has experienced the evolution of using liquid chromatography mass spectrometry (LCMS) technology from a specialized research laboratory to a routine clinical biochemistry laboratory. This presentation will focus on such a development in the clinical biochemistry laboratory. Electrospray ionization tandem quadrupole mass spectrometers have been used for quantitation. This mass spectrometry configuration is able to replace expensive and poorly performed immunoassays, providing cost effective services together with improved analytical performance and turnaround time. Examples of these analytes are whole blood immunosuppressants (Cyclosporin A, Tacrolimus, Sirolimus and Everolimus), and serum/urine steroids (17-hydroxyprogesterone, androstenedione, testosterone, cortisol and cortisone). The same technique has been used to provide target toxicology confirmation after an initial screening by high performance liquid chromatography with ultra-violet detector or immunoassays, for example, ketamine and benzodiazepines. Furthermore, this technique has also been used to measure serum free carnitine and acylcarnitines, assessing fatty acid oxidation status in patients suspected with inborn errors of metabolism. Recently, electrospray ionization time of flight mass spectrometry has been introduced for general unknown drug screening. Practical considerations in acquiring LCMS technology into the clinical biochemistry will be discussed. Finally, future service development using such technology will be explored.

PLASMA METANEPHRINES

DN Pillai
Clinical Chemistry, SEALS, The Prince of Wales Hospital, Randwick NSW 2031, Australia.

dilo.pillai @sesiahs.health.nsw.gov.au

The biochemical diagnosis of phaeochromocytoma has relied traditionally, on elevations in urinary catecholamines and metabolites HMMA and total metanephrines. The advent of the coulometric detector with its enhanced sensitivity has enabled measurement of plasma free metanephrines.

There is evidence that plasma metanephrines have superior sensitivity to urinary catecholamines

for diagnosis of phaeo. However, diagnostic specificity remains an issue. The absence of a quality assurance program for plasma metanephrines may have limited general application of plasma metanephrines as a diagnostic tool.
Clean up for HPLC methods involve solid phase extraction with weak cation exchange resin. Chromatography is on C18 reverse phase columns. Analysis of nanomolar concentrations of metanephrines in plasma, presents problems that are not evident when measuring micro molar concentrations in urine. Although extraction and chromatography appears to be straight forward, the assay requires appropriate care in the choice and maintenance of instrumentation, use of high quality reagents and good chromatographic techniques. Current HPLC methods are however of limited value in laboratories processing large sample numbers.
In the recent past, LC MS as well as RIA and ELISA methods have been described. The take-up

rate for these methods has been limited. However they may become viable and supersede current HPLC methods for high throughput situations. The convenience of collection as well as the

enhanced sensitivity of plasma metanephrines does suggest that they may well become the test

of choice in screening for phaeochromocytoma.

THE USE OF MASS SPECTROMETRY IN INVESTIGATION OF THE KYNURENINE PATHWAY

GA Smythe, S Bustamante, R Pickford
Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW 2052, Australia
g.smythe@unsw.edu.au

The vital coenzyme nicotinamide adenine dinucleotide (NAD) is critically involved in the production of ATP and energy metabolism and the DNA repair enzyme PARP. The de novo synthesis of NAD from tryptophan occurs via the kynurenine pathway (KP) which generates important acid intermediates. Of particular interest are kynurenic acid (KA), quinolinic (QUIN), picolinic (PIC) and nicotinic (NA) acids. These compounds are involved in complex inter-relationships with inflammatory and apoptotic responses associated with neuronal damage and cell death in the central nervous system. To facilitate these products of the KP we have utilized an Agilent 5973B GC-MS system in electron capture negative ionization mode for their concurrent trace quantification. Either deuterium (2H-) or 13C-labelled isotopomers of KA, QUIN, PIC and NA were synthesized and used as internal standards. The compounds were converted to their hexafluoroisopropyl esters prior to chromatography. Nicotinamide also was readily quantified after conversion to nicotinic acid using gas-phase hydrolysis. The on-column limit of quantification was less than 1 fmol for each of the analytes and calibration curves were linear. A packed column liner was developed and used in the gas chromatograph inlet to effectively eliminate sample interference effects in the analysis of trace (femtomolar) levels of quinolinic acid. While this method enables rapid and specific concurrent quantification of these major KP acids in tissue extracts, physiological and culture media, it is not amenable to the analysis of the polar molecule NAD per se. In order to achieve the analysis of this final product of the KP, NAD itself, we developed LC-MS and LC-MS/MS methods using a Finnigan LCQ Deca XP Plus ion trap. This system has enabled us to follow the NAD synthetic pathway through the addition of isotopically labelled tryptophan (13C- and 15N- total label) to cell growth media. The mass spectrometer is then used to detect the labelled precursor and products. Tracking the incorporation of 13C and 15N into kynurenine pathway metabolites has allowed us to demonstrate, for the first time, the biosynthesis of NAD from tryptophan in both primary foetal human neurons and primary foetal human astrocytes. Ongoing work is focussing on the quantitative aspect of this assay, which will allow us to look at changes in the relative amounts of labelled and unlabelled kynurenine pathway metabolites during both precursor loading and cytokine (especially IFN-γ) activation of the rate limiting enzyme indoleamine 2,3-dioxygenase (IDO).

SERUM CAROTENOIDS

GA Woollard, A Hammer-Plecas
Department of Chemical Pathology, LabPlus, Auckland City Hospital, Auckland, New Zealand

Carotenoids are not synthesized de novo by humans and the level in serum is a reflection of dietary exposure. The most significant carotenoids are lutein/zeaxanthin, β-cryptoxanthin, cis & trans-lycopenes, α-carotene and β-carotene. Interest in these compounds is centred on their utility as nutritional markers, precursors for retinol, markers of malabsorption syndromes and their relationship to heart disease and certain cancers.
HPLC with UV detection on a reversed phase column is the preferred method of measurement of carotenoids. There are a large number of different carotenoids in serum and the chromatography must be capable of resolving each component from its various positional and geometric isomers. This difficult to achieve and the success of the separation is dependent on the selection of mobile phase and on choice of column, especially its carbon loading. This presentation will discuss some of these topics.

APPLICATION OF DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC) IN MOLECULAR GENETICS.

IE Piatkov
Diversity Health Institute, Sydney-West Area Health Service, NSW, Australia irinap@icpmr.wsahs.nsw.gov.au
Over the last few years DHPLC has successfully found its way into various molecular biology applications. New developments in column chemistry and technology have significantly improved the separation and analysis of Nucleic Acids. DHPLC is extensively used in basic research and clinical laboratories to investigate and diagnose genetic disorders and susceptibility to disease.
The high sensitivity of DHPLC combined with the accuracy of the heteroduplex approach and instrument mode of operation has allowed the development of a variety of applications detecting structural variations in DNA and RNA. This highly versatile technology makes it an attractive tool for research in characterising novel and already known mutations, DNA methylation and single-nucleotide polymorphism analysis.
Depending upon the particular nucleic acid and the assay being performed a variety of analytical columns can be used including: DNASep, OligoSep, RNASep, Transgenomic, Omaha, NE. Also three modes of instrument operation can be utilized to achieve different goals in separation and detection. By adjusting the temperature of the column, Nucleic Acids can be analysed under non-denaturing, partially denaturing and fully denaturing conditions. UV or fluorescence detectors are applied for signal detection. In addition the fraction of interest can be collected for downstream analysis such as sequencing or cloning.
Above all, DHPLC provides a non-intensive, low cost alternative to current molecular biology procedures for DNA, oligonucleotide and RNA sizing, quantification, purification and QC experiments to be performed on a single platform instrument. This methodology is already very popular in epigenetic and mitochondrial DNA studies. Since it has been introduced to the research community, it has been moved from being performed by very small numbers of practitioners to being performed in hundreds of laboratories.

MASS SPECTROMETRY METHOD DEVELOPMENT: LESSONS FROM BIOCHEMICAL GENETICS

JJ Pitt
VCGS Pathology, Murdoch Children’s Research Institute, Melbourne, Vic. 3052, Australia
james.pitt@ghsv.org.au

Most biological molecules are moderately polar and exist in an aqueous environment making them suitable for analysis by electrospray mass spectrometry. For urinary metabolites, the formation of conjugates such as glucuronides and sulphates enhances their detection through the introduction of a charge-carrying group. In many cases samples can be analysed by direct injection into the mass spectrometer with minimal sample pre-treatment. Derivatisation can be useful to improve sensitivity or to emphasise particular classes of compounds. However, the quality of data obtained by direct injection techniques can be limited by ion suppression and the occurrence of isomers with identical molecular weights and similar fragmentation patterns. LC/MS or LC/MSMS can be used to circumvent many of these issues. The use of MS compatible buffers at low concentration and matching column flow rates with the ion source are important factors in ensuring good LC/MS results. Columns with 1 to 2.1 mm diameter are good choices for general clinical work. Complete separation of all the components in a mixture may not be necessary because of the selectivity of MS and short columns and run-times can often be used. A variety of MS operating modes are available for different applications eg a small number of analytes can be detected by monitoring the relevant ions or the MS can be operated in scanning mode to detect a large number of analytes in screening type tests. Absolute MS responses are more variable than conventional LC detector responses and quantitation is best achieved through the use of internal standards, preferably stable isotope analogues. Analyses of phospholipids, methylmalonate, acyl carnitines and intact steroid and bile acid conjugates will be used to illustrate these principles.

VITAMIN D2 AND D3

JA Grant
Biochemistry Department, Royal Melbourne Hospital, Parkville Vic 3050
janine.grant@mh.org.au

Originally identified as the dietary element able to prevent Rickets, “vitamin” D is more correctly classified as a prohormone. Two main forms exist: cholecalciferol (vitD3) obtained via endogenous synthesis and dietary intake, and ergocalciferol (vitD2) obtained from dietary sources only. Both are transported by vitamin D binding protein and further metabolised by specific hydroxylase enzymes in the liver, kidney and other tissues.
Deficiency of Vitamin D is a major risk factor for bone loss and fracture and may also predispose to a wide range of proliferative and immune diseases. In humans, in vivo synthesis is normally the main source, with dietary supply important only when UV exposure is inadequate. Deficiency is common and supplementation is a major strategy in reducing osteoporotic fractures. Accurate assessment of vitamin D status is essential, both to identify patients at risk and to monitor safe and effective treatment.
Of the circulating metabolites recognised, 25-hydroxyvitamin D provides the best estimate of clinical vitamin D status. A number of different analytical techniques have been applied to its measurement and a range of commercial assays is available. Most Australian laboratories use immunoassay-based methods. Currently manual radioimmunoassays predominate, however automated chemiluminescent and enzyme immunoassays are becoming more popular as request numbers increase. Variable reactivity with 25-hydroxyvitaminD2 (25OHD2) demonstrated by such assays may, however, compromise accurate assessment of vitamin D status in patients undergoing replacement therapy with ergocalciferol. Chromatographic techniques have the advantage of being able to separately quantitate 25-hydroxyvitaminD3 and 25OHD2. Many high-performance liquid chromatography methods using varying extraction, separation and detection techniques have been described, although few have been used routinely. Recently, isotope-dilution liquid chromatography-tandem mass spectroscopy has emerged with potential as both a reference and routine method for 25OHD analysis.

RECENT DEVELOPMENTS IN HPLC COLUMN TECHNOLOGY

JJ Pesek, MT Matyska
Department of Chemistry, San Jose State University, San Jose, CA 95192 USA pesek@sjsu.edu

Each year as many as 100 new columns are introduced into the market place. They include new varieties of reversed phase materials as well as stationary phases for normal phase, chiral separations, ion-exchange and preparative processes. An overview will be presented about recent developments in commercial stationary phases for HPLC. The main focus of the presentation will be on new stationary phases that are based on silica hydride.
The surface of hydride-based stationary phases creates a separation medium that possesses unique properties when compared to ordinary silica. The modes of separation which are possible include reversed phase, aqueous normal phase and organic normal phase. The three modes are generally found to some degree on all hydride stationary phases encompassing a range of organic moieties attached to the surface. Another feature is the ability to utilize mobile phases ranging from 100% aqueous to completely organic for the analysis of ionic/polar compounds as well as solutes that are highly hydrophobic. Most stationary phases that are based on ordinary silica normally are capable of providing only a single retention mechanism depending on the organic group bonded to the surface. For hydride materials in the aqueous normal phase bases are retained under acidic conditions in high organic content mobile phases so it is not necessary to use high pH eluents that can damage many instrumental components such as pumps and valves. Another property of stationary phases with hydride surfaces is their rapid equilibration after a gradient. Because of the multimodal capabilities of the hydride materials, inverse gradients (from high organic content to a more polar composition in the mobile phase) can be run for selective retention and elution of polar compounds.

THERAPEUTIC DRUG MONITORING FOR HIV PATIENTS

JE Ray
Division of Clinical Pharmacology & Toxicology
St. Vincent’s Hospital, Sydney, NSW 2010
jray@stvincents.com.au

The human immunodeficiency virus type 1 (HIV-1) was identified and characterised 20 years ago and as an infectious disease-causing agent, HIV-1 is now one of the most common causes of death worldwide. Four classes of pharmacological agents are now available for the treatment of HIV: nucleoside/nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors (PI) and entry inhibitors. Current treatment of HIV involves the use of a combination of two or more of these classes to suppress viral replication known as highly active antiretroviral therapy or HAART. Treatment of human immunodeficiency virus (HIV) with antiretroviral therapy (ART) reduces morbidity and mortality, suppresses plasma viral load, and restores immune function, but numerous obstacles can limit the success of this therapy. After starting triple drug regimens (ART) approximately 40% of people treated in the clinic setting will experience therapeutic failure (viral rebound) within two years of starting treatment. Furthermore, drug-related toxicity has been shown to be the dominant factor in discontinuation of ART where 36% of 862 patients discontinued their first antiretroviral regimen after 45 weeks of treatment and 58% of the discontinuations were due to drug-related toxicity. The therapeutic strategy of giving the same dose to all patients ignores the striking and well known inter-patient pharmacokinetic variability seen for these agents in this population. Substantial drug-drug interactions in these people and the significant impact of the disease itself on drug absorption, distribution and elimination mean that the potential for people to receive suboptimal or toxic concentrations of these drugs cannot be overlooked. It is therefore no longer acceptable to assume that one dose fits all. Therapeutic drug monitoring (TDM) has the goal of promoting optimal drug treatment by maintaining drug concentrations within a “therapeutic range”, above which there is an increased risk of toxicity and below which there is a high probability that the drug will be ineffective. TDM of ART remains controversial, but is clearly undervalued and misunderstood by many clinicians.

PLATELET SEROTONIN AND CARCINOID SYNDROME

L A Johnson, B McWhinney, A Carter, A Clague
Chemical Pathology, Queensland Health Pathology Service, Brisbane, 4029, Australia. lambro_johnson@health.qld.gov.au

Evidence presented over a decade ago1, 2 showed that platelet serotonin was a more discriminating test for carcinoid syndrome than urine 5-HYDROXY INDOLE-ACETATE (5-HIAA), but the test has not been widely applied, possibly on account of assay difficulties. We developed a simple assay for serotonin in whole blood which is related to the platelet count, and designated "platelet serotonin" 3. We now compare the two tests over a seven year period.
EDTA whole blood (250 µL) with internal standard (50 µL 10 µM N-methyl serotonin), is deproteinized with acetonitrile (1.5 mL), the supernatant diluted with water (0.5 mL) and extracted with chloroform (3 mL). The upper aqueous layer (30µL) is subjected to ion pair (octane sulphonate) C18 RP-HPLC with electrochemical detection. Recovery is > 80%, between run CV <7% and the linear range is 50 to 2000 nM. No chromatographic interferences were observed in >2000 blood samples assayed. However platelet serotonin levels may be lowered by SSRIs and raised by monoamine oxidase inhibitors.
Unlike urine 5-HIAA, platelet serotonin is unaffected by diet, incomplete collections, and variable creatinine excretion. Histograms of 330 patients who had both tests clearly show better separation of normal and abnormal results for serotonin and strongly suggest better identification of patients with carcinoid syndrome.
Our results support the findings of Kema et al1 that platelet serotonin is a more sensitive, more specific, and far more convenient test than urine 5-HIAA, and is recommended as the preferred screening test for carcinoid syndrome.
1. Kema IP, de Vries EGE et al Clin Chem 1992; 38 (4):534-540
2. Pussard E et al Clin Chem 1996; 42:1086-1091
3. Johnson LA and Clague A Clin Biochem Revs 1999; 37:93

USE OF ISOTOPE DILUTION-TANDEM MASS SPECTROMETRY (ID-MS-MS) AS A REFERENCE METHOD

M.J. Whiting
SouthPath Laboratories, Flinders Medical Centre, Bedford Park, South Australia 5042
Malcolm.Whiting@flinders.edu.au

The hierarchy of analytical methods in the clinical laboratory places reference methods in-between definitive methods and everyday routine methods. In the past, isotope-dilution mass spectrometry (ID-MS) has been reserved for definitive methods, due to its highest-order accuracy based on certified reference materials and metrological traceability. Since tandem mass spectrometers are now affordable bench-top instruments, which can be operated by experienced clinical chemists after basic training, this technology has become within the reach of many special chemistry laboratories. Furthermore, when interfaced to HPLC, tandem mass spectrometry (MS-MS) can be used as a reference, and even routine, analytical technique that is versatile, robust and highly specific.
A recent example of the application of ID-MS-MS as a reference method concerns the analyte creatinine in serum or plasma. With the reporting of estimated glomerular filtration rates (eGFR) from equations utilizing serum creatinine concentrations, the issue of standardization of creatinine measurements was raised by the Laboratory Working Group of the National Kidney Disease Education Program of the USA. One of the recommendations of this group was that serum creatinine methods should be recalibrated so that they are traceable to a reference procedure using ID-MS. Although gas chromatography-ID-MS is the accepted reference method for creatinine, LC-ID-MS offers easier preparation and higher throughput since sample derivatization is not required. In our laboratory, we developed an ID-MS-MS method as a reference procedure using 914a, 909b and 967 as certified reference materials (SRM) from the National Institute of Standards and Technology, USA. The availability of reference methods provides regional laboratories with a useful means to check the accuracy base of their routine methods, and helps with the troubleshooting of problem methods and samples.

INDUCTION COUPLED PLASMA MASS SPECTROMETRY

RC McQuilty
Biochemistry Department, Royal Prince Alfred Hospital, Camperdown, NSW.2050, Australia
robert.mcquilty@email.cs.nsw.gov.au

Induction coupled plasma mass spectrometry (ICPMS) is a powerful technique in the field of elemental analysis. It allows for the simultaneous determination of many elements in a wide variety of applications. The Biochemistry department at RPA has used ICPMS for the analysis of trace and toxic elements in whole blood, plasma, urine, red cells, water and various fluids for over 15 years.
Its strengths are its broad multi element coverage, sensitivity, rapid analysis time, wide analytical working range and the ability to provide isotopic information
Since its commercial introduction in the 1980s there have been major developments in sample introduction, plasma efficiency, ion transmission, interference removal and dynamic range making ICPMS a major addition to a large trace element laboratory.

MEASURING COENZYME Q IN PLASMA BY HPLC

M Lever, SL Molyneux
Biochemistry Unit, Canterbury Health Laboratories, and Department of Chemistry, University of Canterbury, Christchurch, New Zealand
michael.lever@chmeds.ac.nz

Introduction
Coenzyme Q (CoQ) is a lipophilic quinone. It is an essential component of the mitochondrial electron transport chain, and may function as a lipid soluble antioxidant. Its biosynthesis is inhibited by statin drugs and this may contribute to muscular complications of cholesterol lowering therapy. Circulating levels are homeostatically controlled and low levels may be associated with a poorer prognosis in vascular disease.
Why measure CoQ?
We measure plasma total CoQ (reduced plus oxidized) to monitor patients receiving statin therapy, and for clinical research into its significance in vascular disease. Measuring the ratio of oxidized to reduced CoQ is not analytically demanding, but collecting and transporting specimens is difficult.
Extraction of CoQ
CoQ is more soluble in lipids than in lower alcohols such as ethanol (which do not mix with triacylglycerols). Two-phase extraction, evaporation, and redissolution in ethanol gives low yields. Single phase extraction into propan-1-ol is more successful.
Separation of CoQ
Separation from other hydrophobic plasma components is best on a high-carbon load reverse phase column (or a C30 column) using a highly hydrophobic mobile phase such as alcohol mixtures containing some heptane. The polarity of the injection solvent needs to be matched to ensure reliable retention. This last consideration renders normal-phase chromatography impractical.
Detection of CoQ
Detection can be by UV absorbance of the oxidized form, by fluorescence of the reduced form, or electrochemically. Fluorescence detection does not have the necessary sensitivity for measurement of plasma levels because of the poor quantum yield of reduced CoQ. UV detection poses specificity problems. Electrochemical detection is preferred, and can be used to simultaneously measure both oxidized and reduced CoQ.

ASSAY OF VITAMINS A AND E IN SERUM

R Greaves
Department of Complex Biochemistry, The Royal Children’s Hospital, Parkville, VIC, 3052 Australia.
Chair, AACB Vitamin Working Party.
ronda.greaves@rch.org.au

The assay of the fat soluble vitamins A and E is of clinical utility for the monitoring of adequate fat absorption in pancreatic insufficiency disorders and in patients receiving total parenteral nutrition. Requests for Vitamin A analysis also originate from the immigration clinic at The Royal Children’s Hospital because vitamin A deficiency has been identified by the WHO as a significant health issue in developing countries.
HPLC is the methodology used by NATA accredited medical testing laboratories in Australia. Sample preparation usually involves precipitation of proteins with methanol or ethanol followed by extraction of the aqueous phase by hexane. The organic layer is either injected directly onto the HPLC or evaporated under nitrogen and the residue redissolved in a variety of solvents for subsequent chromatographic analysis. The separation of the analytes is usually performed with an alkane bonded silica (typically, Octadecylsilane) column. The analytes are quantitated spectrophotometrically by their absorption; Vitamin A (retinol) at λmax 325 nm and Vitamin E (α-tocopherol) at λmax 292 nm.
Whilst the methodology used by different Australian laboratories is similar, there is a large dispersion of results for both Vitamin A and E in the RCPA-QAP program. This dispersion is of concern for the clinical interpretation of assay results.
There needs to be a re-assessment of the robustness of the assay for these vitamins. Aspects that should be considered critically include: 1) Protection of samples from light (including fluorescent light) during all sample preparation steps; 2) Adequate volume of protein precipitating agent; 3) Separate internal standards for vitamin A and E; 4) Calibrators traceable to NIST; 5) Technique used to evaporate extraction solvent and reconstitute the residue; 6) Avoidance of exposure to heat and oxidizing reagents; 7) Efficient chromatography with adequate retention and resolution from interferences including carotenoids and other tocopherols; and 8) Age related reference intervals.

TANDEM MASS SPECTROMETRY – TO DERIVATISE OR NOT TO DERIVATISE

V Wiley, T Wotton, R Pankanjanato, T Hill, B Wilcken

NSW Newborn Screening Programme, The Children’s Hospital at Westmead, Sydney, NSW, 2114, Australia

veronicw@chw.edu.au

Since April 1998 electrospray tandem mass spectrometry has allowed us to screen 850,000 babies born in NSW and ACT for 30 disorders providing enormous clinical benefit. The disorders were detected by measuring selected amino acids (Gly, Ala, Leu/Ile, Met, Cit, Phe, Tyr) and acyl carnitines (Carnitine,C2,C3,C4,C5,C5D,C5-OH,C6,C8,C10,C10:1,C12,C14,C14:1,C16, C16OH,C18OH). During this time all aspects of the method have been under review to ensure optimal reliability determining sensitivity, specificity, positive predictive value and cost with these criteria having analytical as well as population definitions. Modifications have included reducing the sample volume, incorporating the use of matrix matched calibrators, drying with warmed air rather than nitrogen, computer manipulation of the data with development and modifications of action level algorithms as well as upgrading instrumentation. The sample preparation until recently included derivatisation with a strongly corrosive chemical, butanolic HCl, to enhance analytical sensitivity and required 2 hours of person time (6 hours actual time) for the average 5x96 well microtitre plates per day.

In order to investigate the effect of eliminating derivatisation of samples, 20,000 samples collected as part of routine newborn screening as well as 1100 samples from the archival storage previously determined to require further investigation were analysed by each of the two sample preparation procedures. Eliminating the derivatisation step of the sample preparation procedure reduced the person time and actual time requirements as well as improved occupational health and safety. The measurement of uncertainty for some analytes, notably glutaryl carnitine and citrulline, was worse but not to a level leading to misclassification. The correlation coefficient for comparison of the sample preparation procedures was greater than 0.95 for all analytes.

EXPERIENCE WITH ELSD IN THE CLINICAL LAB

MA Gruca
James Fairfax Institute of Paediatric Nutrition, The Children’s Hospital at Westmead, Westmead NSW

Quantitative perfusion marker tests are performed to assess pancreatic function in patients with Cystic Fibrosis. Aspirated fluids are analysed for the marker (which enables calculation of secretion rates of different analytes), bile acids and the pancreatic enzymes lipase and colipase. The marker used, gentamicin, is measured by a micro particle enzyme immunoassay. Ten conjugated bile acids are measured using a gradient elution with the pH optimized for the separation of the glycine and taurine conjugates. Detection is with a UV detector.
Our aim was to develop a simple HPLC method that would also measure the five free bile acids in the duodenal aspirates from the “quantitative perfusion test”. Free bile acids (those not conjugated with glycine or taurine) may have a role as damaging agents in the colon, or as an indicator of liver dysfunction, but they are UV transparent.
UV detection lacks sensitivity for some compounds and is further compromised by different responses for different sample components of equal concentrations. An ELSD (evaporative light scattering detector) gives a response for almost all sample components and with less variation amongst the different components. An ELSD can be used with gradient elution, but is restricted to volatile mobile phases.
ELSD suitable method for separating all 15 bile acids was developed by changing our original phosphate buffers to volatile acetate buffers. We were also able to substitute methanol, a cheaper and safer alternative, for the UV transparent acetonitrile solvent in our gradient elution.
In addition, using the ELSD, we were able to develop a method to measure mannitol in the duodenal aspirate as a possible marker instead of adding gentamicin. This would be an additional time and resource savings for our department.