Establishing metrological traceability of measurement is essential to improve the accuracy and comparability of measurement results. With increasing recognition of the importance of traceability, some regulatory policies have been applied to enforce its implementation. Technology advancement also provides more tools for improving measurement traceability.  During the assay development on the Mindray CL-2000i Chemiluminescence Immunoassay System,  well recognized highest reference methods or reference materials were used in assigning the values of master calibrators; the accuracy of product calibrators was guaranteed through an unbroken metrological traceability chain.

by Xiang Yu and Ke Li

Introduction
With the advancement in automation over the past 20 years, most of the immunoassays have been shifted from traditional manual assays to fully automatic systems leading to an overall improvement of the quality of measurements. The accuracy and comparability of testing results have been emphasized, since they are the keys to defining and using common clinical decision values and reference intervals, following constant standards and practice guidelines, pooling data from different studies based on different analytical systems to facilitate clinical research.

One critical mechanism to improve the accuracy and comparability of clinical testing results is to make the testing results traceable to higher reference materials or methods in calibration hierarchy. Briefly, the testing results should have metrological traceability. The general principles and features have been described in the document of the International Organization for Standardization (ISO) 17511:2003 [1].

Ideally, results produced by different routine methods for the same measurand should be metrologically traceable to the highest level of calibration hierarchy – the International System of Units (SI units), with an estimated measurement uncertainty. However, only a limited number of analytes, including some metabolites, electrolytes, steroid hormones, has reference materials available with traceability to the SI unit. Most of the clinical analytes still have no primary and secondary reference measurement procedures and are not traceable to the SI unit. They are not well defined and have only traceability to an international conventional standard or manufacturers’ internal standard, such as tumour markers and viral antigens [2].

The EU directive on in vitro diagnostic devices (IVDD) enacted in 1998 stated “The traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and/or available reference materials of a higher order” [3]. Therefore, for all the IVD analytical system (reagents), manufacturers must ensure their products are standardized against available reference materials or methods in order to be distributed in the EU market.

Traceability chain and value assignment procedure on Mindray CL-2000i System
Mindray CL-2000i system is a closed system composed of a fully automatic immunoanalyser, related reagents and calibrators. The calibration hierarchy was established and documented strictly based on EN ISO 17511:2003 [1]. Mindray’s traceability procedure is indicated in figure 1, ensuring the establishment of metrological traceability between the testing results and the highest standard available. Based on the characteristics of different analytes, three major traceability chains have been used: traceable to an SI unit, traceable to an international conventional calibrator, and traceable to manufacturers’ selected procedure.

Measurements traceable to the SI unit
If the chemical and physical properties of an analyte are well defined, there should be a primary reference measurement procedure with the measurement traceable to the SI unit (mole). CL-2000i total T3, total T4, progesterone, testosterone and estradiol are traceable to this highest level of calibration hierarchy. Mindray has performed the traceability of the above measurements in collaboration with the Reference Institute for Bioanalytics (RfB), a German reference laboratory certified by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) [4]. Thirty Mindray master calibrators at different levels covering the whole detection range were assigned values for each analyte at RfB with the reference measurement procedure of Isotope dilution mass spectrometry (ID-MS). The calibrator values with uncertainty were then applied to define the values of Mindray working calibrators and product calibrators, and the metrological traceability between the testing results of CL-2000i end-users’ routine measurement procedure and the SI unit was finally established. The assays that are traceable to the SI unit are indicated in Table 1.

Measurements traceable to an international conventional calibrator
The reference materials, such as WHO standards and some national standard materials are defined by convention or consensus, without traceability to the SI unit; the assigned values are in arbitrary units (e.g. WHO international unit). Most of assays for tumour markers, hormones, and viral antigen/antibody of the CL-2000i system are traceable to this kind of reference materials, indicated in Table 1.

Measurements traceable to manufacturers’ selected procedure
For analytes that are either not traceable to the SI unit, or for which no reference method and reference material are available, a commercial certified measurement procedure with traceability, high accuracy and analytical specificity was selected for Mindray master calibrator value assignment; the measurement accuracy of the Mindray routine measurement procedure is ensured and also indicated in Table 1.

Principle of the traceability of Mindray CL-2000i end-user’s measurement results
The immunoanalyser is calibrated by measuring three levels of product calibrators and relative light units (RLUs) generated. The corresponding concentration of each calibrator was used to adjust the master calibration curve stored in the barcode of each lot of reagents.

The value of end-user’s product calibrators and the master curve stored in the barcode are both defined by the Mindray routine measurement procedure that is calibrated by Mindray working calibrators in the manufacturer’s laboratory. The working calibrators have roughly 12 concentration levels and have the same matrix as the end-user’s product calibrators.

It is the Mindray standard measurement procedure that determines the values of Mindray working calibrators. The Mindray standard measurement procedure makes use of the Mindray standard CL-2000i automatic immunoassay analyser, standard reagents, and Mindray master calibrators. Mindray master calibrators are composed of a series of human serum at different concentration levels. They are stored at -70°C and represent the highest accepted standard available.

The values of the Mindray master calibrators are fixed, and the measurement standard established by the Mindray standard measurement procedure is preferably not variable and should be kept as consistent as possible. On the other hand, the value of working calibrators and end-user’s product calibrators can be flexible within a certain range. The assigned values of calibrators will be adjusted according to the results of internal QC and method comparison so as to ensure the traceability between the reference and end-user’s routine measurement procedures.

Discussion
We have made our best efforts for the traceability of the Mindray CL-2000i system, eventhough the implementation of traceability is challenging, especially the traceability in immunoassays.

Firstly, majority of analytes lack a primary reference measurement procedure and thus are not traceable to the SI units. The chemistry and physical properties of these analytes still require more accurate definition.

Secondly, the international conventional calibrators have played an important role in harmonizing testing results. However, there are still some issues with using the international standards, such as the long term stability of WHO standards, the matrix effect, the difference between different generations of the standards, and difference between the source of the standards and the real sample in the clinic.

Thirdly, some of the analytes have neither reference materials nor reference methods available, and are only traceable to manufacturers’ in-house standards. The harmonization of clinical results could not be fully implemented [5].

References
1. ISO 17511:2003. In vitro diagnostic medical devices –measurement of quantities in biological samples – metrological traceability of values assigned to calibrators and control materials. Geneva, Switzerland: ISO
2. Database of higher-order reference materials and reference measurement methods/procedures. http://www.bipm.org/en/committees/jc/jctlm/jctlm-db
3. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. Off J Eur Union 7 December 1998; L 331:1–37.
4. JCTLM: Joint Committee for Traceability in Laboratory Medicine. http://www.bipm.org/en/committees/jc/jctlm/
5. Danni L. Meany and Daniel W. Chan Comparability of tumor marker immunoassays: still an important issue for clinical diagnostics? Clin Chem Lab Med 2008; 46(5):575–576.

The authors
Xiang Yu*, MSc and Ke Li, PhD
Immunoassay Department, Shenzhen
Mindray Bio-Medical Electronics Co. Ltd., Nanshan, Shenzhen, 518057 China

*Corresponding author
Email: yuxiang@mindray.com

Therapeutic drug monitoring of anti-epileptic drugs has greatly advanced since the development of colorimetric assays for the measurement of phenytoin and phenobarbital in the mid-1950s. Today, not only have laboratory technology and assay development advanced, but so have the pharmaceutical agents available for the treatment of epilepsy disorders. However, under UK National Institute for Health and Clinical Excellence (NICE) Guidelines, therapeutic drug monitoring is still justified for newer anti-epileptic drugs like levetiracetam and pregabalin, for which we have developed quick and robust LC-MS/MS assays.

by Jonathan C. Clayton, Katherine Birch and Carrie A. Chadwick

Background
Therapeutic drug monitoring (TDM) is an important consideration in the treatment of epilepsy. It has long been known that a dose of a given drug may be effective in one patient but not in another [1]. This is of particular importance when too high a concentration of drug can have toxic effects, and too low a concentration has no therapeutic effect. Problems arise when, in different patients, a specific dosage leads to a therapeutically significant concentration in one, but could be ineffective or even toxic in another. Understanding the relationship between dosage and the concentration of the active drug at receptor sites has long been a topic for research [2], which has led to the development of assays to measure the plasma concentration of anti-epileptic drugs (AEDs). TDM of AEDs has advanced since colorimetric assays for phenytoin and phenobarbital were developed in the mid-1950s [3]. Older AEDs such as phenytoin and valproate have narrow therapeutic ranges (the plasma drug concentration range below which the drug may be ineffective and above which the patient may experience toxic effects). However, even the plasma concentration at which a given drug is effective may vary from individual to individual, depending on a number of factors known as pharmacokinetics [4]. Many newer AEDs, such as lamotrigine and topiramate do not have the narrow therapeutic range as seen with the older AEDs, however, TDM is still applicable [5]. Today both older AEDs such as phenytoin, phenobarbital and sodium valproate as well as newer AEDs such as lamotrigine and topiramate are subject to TDM [4]. This has led to the development of new assays for monitoring the serum concentration of these drugs. Methods include immunoassays such as enzyme multiplied immunoassay technique (EMIT) and cloned enzyme donor immunoassay (CEDIA), kinetic interaction of microparticles (KIMS) and chemiluminescent assays (CLIA) [6]. However, more liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are being developed for newer AEDs, which can detect a number of AEDs in a single assay [7].
Best Practice Guidelines for TDM published in 2008 [1], along with a review discussing TDM of the newer AEDs [8] have provided a rationale for developing methods for two second generation AEDs, levetiracetam and pregabalin. These drugs are becoming increasingly popular with levetiracetam being used as an adjunct for partial and generalized tonic–clonic seizures, and pregabalin used as an adjunct for partial seizures [9]. Pregabalin, and to a lesser extent levetiracetam, is also used in the treatment of non-epileptic disorders such as neuropathic pain [9]. The increasing popularity of these drugs with clinicians has led to an increasing demand for determination of plasma concentrations of these drugs. TDM is justified for determining compliance with treatment with either drug, but also for determining overdosing, and dosing in renal failure, of levetiracetam.

Here, we describe methods for the detection and quantification of levetiracetam or pregabalin in serum using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The methodology is identical for both levetiracetam and pregabalin and so, should demand for TDM of these drugs increase in the future, there is scope for them to be combined into one assay.

Materials and methods
Levetiracetam (1 mg/mL in MeOH) and pregabalin (1 mg/mL in MeOH) stock solutions, levetiracetam-D6 (100 µg/mL in MeOH) and pregabalin-D6 (100 µg/mL in MeOH) were purchased from Cerilliant (distributed by LGC Standards, Middlesex, UK). EQA materials used for accuracy assessment were kindly supplied by the LGC Heathcontrol EQA scheme. HPLC grade water and methanol were purchased from Sigma-Aldrich Ltd (Poole, Dorset, UK). All other chemicals were purchased from Sigma-Aldrich Ltd or VWR Ltd. ClinChek® Control Levels 1 and 2 were purchased from RECIPE (Munich, Germany).

Standards
Standard solutions were made by preparing serial dilutions of stock solution in PBS/BSA (phosphate buffered saline containing 0.5% bovine serum albumin) (137 mmol/L NaCl, 2.7 mmol/L KCl, 5.4 mmol/L Na2HPO4•7H2O, 1.8 mmol/L KH2PO4, 0.5% BSA). The standards were stored at –20°C until use.

Internal standards
Each internal standard was prepared to a final concentration of 10 mg/L in HPLC grade methanol containing 50 mmol/L ZnSO4∙7H2O. The internal standards were stored at room temperature until use.

Sample preparation
For assay purposes, standards, quality control (QC) and serum samples were prepared in an identical fashion. In a 96-well plate, 80 μL internal standard solution (in ZnSO4 in MeOH) are added to 20 μL sample followed by agitation and centrifugation. Eighty microlitres of H2O was then added to each well, the plate heat sealed, agitated and centrifuged.

Chromatography and mass spectrometry
Chromatography was performed on a Waters Acquity UPLC system equipped with a Waters Acquity UPLC BEH C18 1.7 μm 2.1 x 50 mm column. Mobile phase A consisted of 10 mmol/L ammonium acetate and mobile phase B consisted of MeOH.
A flow rate of 0.5 mL/min was maintained for the run time of 2.5 minutes. A linear gradient of mobile phase B from 2% to 50% was run between 0 and 1 minutes, followed by a constant concentration of 50% mobile phase B. Ninety-eight per cent mobile phase B was run from 1.75 to 2.5 minutes. The injection volume was 5 μL.
Mass spectrometric determination was carried out using a Waters TQD in ESI+ mode. The source temperature was 130 °C, desolvation temperature was 400 °C, cone gas flow was 50 L/hr and the desolvation gas flow was 800 L/hr. Targetlynx™ software was used to process the data and quantify the drugs in the standards, controls and patient samples.

Method validation
Validation of the assays was carried out according to Honour [10]. Precision and bias were determined by measuring QC samples over 5 batches with 5 samples in each batch. The coefficients of variance (CVs) were calculated for intra-batch and inter-batch precision. Bias was calculated from the nominal target values for each of the QC materials.
Accuracy was assessed using EQA materials from the LGC Heathcontrol AE1 Anti-epileptic drug EQA scheme.
Matrix effects were determined by running a water blank, extracted water and extracted drug-free serum against a background infusion of each drug.
The limit of blank (LOB) was determined by running 10 extracted water samples and was quantified as the highest concentration measured in the absence of analyte.
The lower limit of quantitation (LLOQ) was determined by spiking drug-free serum with known quantities of each drug, and was quantified as the lowest detectable concentration whose CV was <15% and bias <20%.
Specificity was determined by spiking PBS/BSA with high concentrations of six more commonly used AEDs (carbamazepine, carbamazepine epoxide, phenobarbital, phenytoin, primidone and sodium valproate.
Carry-over was determined by spiking drug-free serum with high concentrations of each drug, and analysing followed by drug-free serum.

Results
Chromatography and mass spectrometry
Levetiracetam and levetiracetam-D6 had a retention time of 0.88 minutes and the cycle time from injection to injection was 3 minutes. Pregabalin and pregabalin-D6 had a retention time of 0.82 minutes and the cycle time from injection to injection was 3 minutes. The chromatography profile is identical for both of the drugs. The profile produced clean, sharp peaks with no co-eluting elements. The quantification transition for levetiracetam was m/z 170.90>69.16 and the confirmation transition was m/z 170.90>98.17. For pregabalin, the quantification transition was m/z 159.90>55.12 and the confirmation transition was m/z 159.90>83.08. For the internal standards, levetiracetam-D6 had the transition m/z 177.00>132.00 and pregabalin-D6 had the transition m/z 166.10>102.90.

Method validation
The intra- and inter-assay CVs are <8% for both drugs suggesting good precision of the assay. The inter- and intra-assay bias for levetiracetam was acceptable at <6%, while for pregabalin the inter- and intra-assay bias was <10% apart from the inter-assay bias at 10 mg/L (Table 1). External quality assessment materials were analysed as per patient samples. The results (Table 2) were compared with the target value supplied by LGC Heathcontrol, and with the returns of other laboratories using similar methods (LC-MS and LC-MS/MS) in order to determine the accuracy of the assay. Matrix effects were investigated using injections of drug-free serum, extracted water and blank water against a constant background infusion of each drug in methanol (50 mg/L levetiracetam, 25 mg/L pregabalin). No matrix effects are seen around the relevant retention times for either drug (Fig. 1). The LOB was quantified as the highest apparent analyte concentration in the absence of analyte. The LLOQ was quantified as the lowest level of analyte detectable whose CV was <15% and whose bias was <20% (Table 3). The methods for both levetiracetam and pregabalin showed no interference from any other commonly prescribed AEDs, with responses of ‘0’ to the interference samples from both methods. Blank serum samples and extracted water samples run immediately after samples containing either ~200 mg/L levetiracetam or 100 mg/L pregabalin gave responses of ‘0’, indicating no problems with carry-over. Discussion
We have developed and validated LC-MS/MS assays for the quantification of levetiracetam and pregabalin in serum.
Two optimal transitions were identified for both drugs, thus providing a ‘quantifier’ transition and a ‘confirmation’ transition in order to increase confidence of identification owing to the risk of misidentification of analytes with the same molecular weights as the drugs of interest.
The chromatography method is identical for both levetiracetam and pregabalin, and with the two drugs having different retention times (0.88 and 0.82 minutes respectively), should there ever be a wish to combine these assays into one single run, this should be straightforward. Additionally, should assays for any other AEDs be developed, this chromatography method would be an appropriate starting point. Serum proteins are precipitated by the addition of ZnSO4 in methanol, which also aids the retained solubility of the drug. Following centrifugation, an equal volume of H2O is added so the drug is in 50 : 50 methanol/water. Following a further centrifugation, 5 µl of supernatant is injected onto the column. The method is quick and robust. The assay has acceptable precision and bias. All the EQA materials ran well within their acceptable ranges, close to the target value.
Other LC-MS/MS methods for the detection of levetiracetam [11, 12] and pregabalin [13] have been described, all of which have longer cycle times between injections, larger sample volume requirements, and, in some cases, have more complex sample preparation. The method described here benefits from being quick, with a simple sample preparation procedure.
Methods for the measurement of levetiracetam in saliva have been described [11] and it has been shown that there is good correlation between saliva, plasma and serum, meaning saliva would be a suitable alternative to serum [14]. To date, no such method has been described for pregabalin, but cases of pregabalin toxicity have been described which would advocate the development of further methods for the TDM of pregabalin [14].
The monitoring of levetiracetam and pregabalin is justified [1, 5] to monitor compliance and overdosing, and quick and robust methods for their measurement in serum have been described here. Further work could include development of assays for the measurement of these drugs in saliva, with comparison studies required.

References
1. Patsalos PN, Berry DJ, Bourgeois BF, Cloyd JC, Glauser TA, et al. Antiepileptic drugs—best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission of therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia 2008; 49: 1239–1276.
2. Eadie MJ. Therapeutic drug monitoring—antiepileptic drugs. Br J Clin Pharmacol. 1998; 46: 185–193.
3. Theodore WH. Rational use of antiepileptic drug levels. Pharmac Ther. 1992; 54: 297–305.
4. Glauser TA, Pippenger CE. Controversies in blood-level monitoring: reexamining its role in the treatment of epilepsy. Epilepsia 2000; 41(Suppl. 8): S6–S15.
5. National Institute for Health and Clinical Excellence. The epilepsies: the diagnosis and management of the epilepsies in adults and children in primary and secondary care. Clinical guidelines 137. NICE 2012; http://guidance.nice.org.uk/CG137 (accessed 15 October 2013).
6. Aldaz A, Ferriols R, Aumente D, Calvo MV, Farre MR, et al. Pharmacokinetic monitoring of antiepileptic drugs. Farm Hosp. 2011; 35: 326–329.
7. Shibata M, Hashi S, Nakanishi H, Masuda S, Katsura T, Yano I. Detection of 22 antiepileptic drugs by ultra-performance liquid chromatography coupled with tandem mass spectrometry applicable to routine therapeutic drug monitoring. Biomed Chromatogr. 2012; 26: 1519–1528.
8. Krasowski MD. Therapeutic drug monitoring of the newer anti-epilepsy medications. Pharmaceuticals 2010; 3: 1909–1935.
9. Wahab, A. Difficulties in treatment and management of epilepsy and challenges in new drug development. Pharmaceuticals 2010; 3: 2090–2110.
10. Honour JW. Development and validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem. 2011; 48: 97–111.
11. Guo T, Oswald LM, Mendu DR, Soldin SJ. Determination of levetiracetam in human plasma/serum/saliva by liquid chromatography-electrospray tandem mass spectrometry. Clin Chim Acta 2007; 375: 115–118.
12. Blonk MI, van der Nagel BC, Smit LS, Mathot RA. Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2010; 878: 675–681.
13 Nirogi R, Kandikere V, Mudigonda K, Komarneni P, Aleti R. Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma. J Chromatogr B. 2009; 877: 3899–3906.
14. Patsalos PN, Berry DJ. Therapeutic drug monitoring of antiepileptic drugs by use of saliva. Ther Drug Monit. 2013; 35: 4–29.

The authors
Jonathan Clayton* MPhil, MSc; Katherine Birch DipRCPath; and Carrie Chadwick FRCPath
The Buxton Laboratories, The Walton Centre NHS Foundation Trust, Liverpool, UK

*Corresponding author
E-mail: Jonathan.clayton@nhs.net

Alzheimer’s disease (AD), a progressive and eventually fatal neurodegenerative condition, was first described over a century ago. The prevalence of the disease has greatly increased since then: indeed the World Health Organization estimates that around 36 million people are living with dementia, the majority of whom are suffering from AD. This number is expected to double by 2030 and triple by 2050, mostly due to increased human longevity: the incidence of AD increases exponentially after the age of 65, with nearly 50% of people over 85 affected. Very early diagnosis and timely and effective therapy are urgently needed if health and social services are not to be totally overwhelmed catering for the needs of both patients and their frequently elderly carers.
Changes in the brains of AD patients may commence up to two decades before clinical symptoms become apparent. The two major abnormalities, beta-amyloid plaques (Aβ) and neurofibrillary tangles (NFT), are very visible at autopsy and continued improvements in medical imaging technologies may allow eventual visualization in the brains of living patients. A definitive diagnosis of AD, though, is usually still based on neuropsychological testing and MRI and/or CT scans to rule out other causes of cognitive decline at a stage of the disease when the drugs currently available, which regulate neurotransmitters, are no longer very effective.
Ongoing research to allow earlier diagnosis has found that gradually increasing concentrations of both Aβ and NFT can be detected in the cerebrospinal fluid of AD patients. And two very recently published studies give additional cause for optimism. The first, published in Nature Genetics, was a large international study that scanned the DNA from more than 74,000 AD patients and healthy controls from 15 different countries to find novel genetic risk factors. As well as the genes already implicated in the disease, such as APOE4, which is strongly linked to late-onset AD, eleven new genes were discovered that had previously not been linked to the condition. This work could facilitate very early diagnosis in individuals at risk. And a smaller British Medical Research Council study discovered a compound that actually prevents further neurodegeneration in animal models.
It has been recognized, however, that an international approach would be most effective in reducing the impact of AD and other types of dementia. To this end health ministers from the G8 countries will be meeting in London in December to develop a coordinated plan of action. It is to be hoped that the result of their deliberations will be global cooperation between companies, researchers and clinicians, and ultimately timely diagnosis and therapy for this appalling condition.