Methadone maintenance therapy is central to the treatment of opiate dependence. Assessment of adherence is essential to ensure success and to prevent misuse of prescribed medications. A variety of specimen types can be tested for methadone and its main metabolite using a number of different analytical methods. The benefits and limitation associated with each are discussed.
by Dr Elizabeth Fox and Dr Deepak Chandrajay
Introduction
Opiate dependence is an important problem worldwide. In the UK, individuals seeking help with their addiction are referred to substance misuse services where they are usually offered methadone or buprenorphine substitution therapy [1]. Methadone is a synthetic opioid with pharmacological actions similar to opiates mediated through the mu receptor. Treatment is initiated at a dose of 10–40 mg daily and gradually increased by 10–20 mg weekly. The usual maintenance dose is 60–120 mg daily, but some clients require a higher dose for symptomatic relief [2]. Its long half-life allows for a once-daily dosing schedule and the accumulation in the body means that steady-state plasma concentrations are easily achieved after repeated administration.
Methadone reduces or eliminates withdrawal symptoms and helps the subject reach a drug-free state in a controlled way. There is evidence of reduced illicit opiate misuse, criminal activity and mortality when patients are on maintenance therapy [2, 3]. Injecting behaviours and incidence of HIV infection are also reduced [4].
Methadone is a lipid soluble drug with an oral bioavailability of approximately 95%. It is metabolized by cytochrome P-450 (CYP) enzymes and demethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Both parent drug and metabolite are excreted in the urine and can also be detected in blood, oral fluid, sweat and hair. Co-administration of CYP enzyme inducing drugs such as rifampicin, phenytoin, and zidovudine can precipitate opiate withdrawal symptoms. Fluoxetine and fluvoxamine can inhibit CYP enzymes and have an opposite effect on methadone metabolism [5].
In contrast to most other forms of therapeutic drug monitoring where blood concentration is maintained within a narrow therapeutic window, methadone is monitored almost exclusively to confirm adherence with the treatment regimen. The client may seek to falsify the drug test to feign adherence when the drug is actually being sold to others, or simply to mask illicit drug use. Such individuals will submit a specimen spiked with methadone mixture, therefore effective methods for methadone testing should use matrices which are resistant to tampering and/or include measures to detect falsified samples. Absence of EDDP from a methadone-positive urine sample strongly suggests that it has been spiked with medication. Measurement of urine creatinine will identify samples which have been diluted or substituted, for example with tea. Methadone mixture is green so a green tinge to urine should raise suspicions of sample spiking. The temperature and pH of fresh urine specimens can be recorded to assess reliability. Salivary IgG is useful to confirm integrity of oral fluid specimens.
Analytical methods used for methadone testing
The key analytical methods used to measure methadone and EDDP are immunoassay, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). The benefits and limitations of each are summarized in Table 1. Immunoassay is rapid, high throughput when automated and low cost. Sensitivity is determined by the detection cut-off concentration of the test kit and specificity by the specific antibody used in the kit. Point-of-care test (POCT) kits are available for use with urine and oral fluid in the clinic and require little expertise or training. POCT offers the significant advantage of producing instant results that can be discussed during the consultation. Laboratory-based immunoassays can be run on multichannel clinical chemistry analysers and do not require additional staff training. All immunoassay-based techniques are prone to interference from unrelated compounds due to cross-reaction with the specific antibody. Cross reactivity data are available from the manufacturer and should be borne in mind when interpreting results. False-positive methadone results have been documented with diphenhydramine, doxylamine and phenothiazines [6]. Immunoassay-based tests, whether designed for POCT or laboratory use, are sold as screening tests. The manufacturers recommend the confirmation of positive results using an alternative methodology such as LC-MS/MS or GC-MS. That said, not all laboratories and substance misuse clinics routinely confirm methadone and EDDP positive results. UK Department of Health guidance on adherence testing recommends only that positive screen tests are confirmed ‘if appropriate’[1].
Mass spectrometric techniques offer the best possible sensitivity and specificity and are considered the ‘gold standard’. Test menus are user-defined which allows simultaneous detection of methadone and EDDP and any other drug as required. Disadvantages of these techniques are that they require expensive specialist instrumentation, labour intensive sample preparation, and complex data interpretation. Turnaround times can be lengthy and they are not amenable to POCT. LC-MS/MS methods require considerably less sample preparation than GC-MS and are now used in many clinical laboratories. A few labs including our own have adopted LC-MS/MS for first-line drug screening of urine and oral fluid specimens.
Specimen types for methadone testing
The main sample types are summarized in Table 2.
Urine
Regular urine testing is the most commonly used means of confirming adherence with methadone prescription. The advantages of urine are that both methadone and EDDP are readily detected and collection is easy and non-invasive. Presence of EDDP provides proof that methadone has been ingested and not spiked into the sample. A disadvantage of urine is that it is easy to manipulate. A sample of donor urine, if the donor is taking methadone, submitted in place of the patient’s own is difficult to recognize. Supervised collection is not always desirable as it is an invasion of privacy and subjects may suffer from ‘shy bladder’. The concentrations of methadone and EDDP do not correlate well with dose (because of the variability of untimed urine samples), so qualitative urine analysis is only suitable for confirming use.
Commercially available methadone and EDDP immunoassays typically have a fixed cut-off or detection threshold of between 100 and 300 µg/L. Multidrug panel tests usually include either methadone or EDDP. Choosing a test which specifically detects EDDP will minimize the chance that a spiked urine is passed off as positive. An estimated 4% of methadone-positive samples submitted to our laboratory lack detectable EDDP; a methadone-only assay would not identify these specimens (unpublished observation). Our approach to urine testing is to measure both methadone and EDDP by LC-MS/MS in all samples. The testing strategy in an increasing number of substance misuse clinics is to use POCT as a first-line test, then to refer suspicious or disputed samples to the lab for confirmation. A laboratory immunoassay would offer no further information for samples that have already been tested at the point of care and could theoretically suffer the same interference.
Oral fluid
An alternative matrix for methadone testing is oral fluid. The advantage of oral fluid is that collection is simple, easily observable and can be done in the consultation room. POCT devices are available for instant results or samples can be sent for laboratory analysis. Both immunoassay and mass spectrometric analytical methods are available. The amount of methadone and EDDP present in oral fluid is dependent upon salivary pH. Methadone is a basic drug and under acidic conditions it becomes ionized and ‘trapped’ in the saliva. Unstimulated saliva is more acidic than stimulated saliva so false negatives can be avoided by asking the subject to abstain from eating, drinking or chewing for 10 minutes prior to collection. A recent study involving subjects on daily methadone doses found that the concentration of methadone in saliva correlated poorly with dose and that EDDP was below detection in 12% of samples [7]. However, methadone was readily detectable in all samples suggesting that oral fluid is a useful specimen for confirming adherence. Oral fluid methadone does not reflect the plasma concentration so would not be useful for assessing dose adequacy. Contamination with methadone from the oral cavity is a problem and absence of EDDP, if measured, should be interpreted with caution as it does not necessarily equate to sample adulteration.
Blood
The main advantage of using blood to monitor methadone therapy is that it’s virtually impossible to falsify the sample. Plasma concentration correlates with methadone dose but the concentrations at which therapeutic effect is achieved have not been well defined. Several studies have suggested target concentrations; other studies have found no correlation between plasma concentration and either heroin use or opiate withdrawal symptoms [8]. A further study suggested that the pharmacodynamics of methadone can be altered by the presence of other drugs therefore altering the relationship between plasma methadone and effect [9]. There is debate in the literature as to whether plasma concentration is any more useful than daily dose for predicting response to treatment [10]. Given the polypharmacy present in the majority of subjects receiving methadone, routine use of plasma methadone to titrate dose is likely to need further evaluation. Intravenous drug users tend to have poor venous access so collecting samples may be challenging. Methods using dried blood-spot samples to circumvent this problem have been described but skin contamination with methadone is likely to be an issue [11]. Blood is not the ideal specimen to assess use of other substances because of the very short detection window, so additional testing may be required. In conclusion, blood testing is best reserved for difficult cases where knowledge of the plasma concentration may be helpful.
Other matrices
Monitoring of methadone therapy using sweat analysis has been evaluated. Patches are typically worn for up to 7 days then dispatched to the laboratory for analysis. They are tamper-evident and claim to be difficult to adulterate. Large inter- and intra-individual variations in sweat methadone concentration have been observed and there is only a weak correlation between patch concentration and dose. Sweat testing is, however, useful for detecting exposure to other substances so may be applicable to some cases. Hair analysis can be used to retrospectively confirm adherence with methadone treatment but is not useful for real-time assessment.
Concluding remarks
The current trend is for substance misuse services to perform methadone adherence testing in the clinic and refer samples to the laboratory for confirmation where necessary. Substance misuse clinic personnel are not laboratory scientists, therefore a key role of the laboratory that performs confirmatory testing is to develop a good working relationship and ensure all aspects of testing are fully understood.
References
1. Department of Health (England) and the devolved administrations. Drug Misuse and Dependence: UK Guidelines on Clinical Management. London: Department of Health (England), the Scottish Government, Welsh Assembly Government and Northern Ireland Executive. 2007; www.nta.nhs.uk/uploads/clinical_guidelines_2007.pdf.
2. National Institute for Health and Clinical Excellence. Methadone and buprenorphine for the management of opioid dependence. Technology appraisal guidance 114. NICE 2007; http://guidance.nice.org.uk/TA114.
3. Advisory Council on the Misuse of Drugs. Reducing drug-related deaths: a report by the Advisory Council on the Misuse of Drugs. ACMD, Home Office 2000; ISBN 0-11-341239-8.
4. NTORS, The National Treatment Outcome Research study. 2001; http://webarchive.nationalarchives.gov.uk/+/www.dh.gov.uk/en/publicationsandstatistics/publications/publicationspolicyandguidance/dh_4084908.
5. McCance-Katz EF, Sullivan L and Nallani S. Drug interactions of clinical importance among the opioids, methadone and buprenorphine and other frequently prescribed medications: a review. Am J Addict. 2010; 19(1): 4–16.
6. Lancelin F, Kraoul L, Flatischler N, Brovedani-Rousset S, Piketty ML. False-positive results in the detection of methadone in urines of patients treated with psychotropic substances. Clin Chem. 2005; 51(11): 2176–2177.
7. Gray TR, Dams R, Choo RE, Jones HE, Heustis MA. Methadone disposition in oral fluid during pharmacotherapy for opioid-dependence. Forensic Sci Int. 2011; 206 (1–3): 98–102.
8. Shiu JR, Ensom MHH. Dosing and monitoring of methadone in pregnancy: literature review. Can J Hosp Pharm. 2012; 65(5): 380–386.
9. Kharasch ED, Walker A, Whittington D, Hoffer C, Sheffels Beynek P. Methadone metabolism and clearance are induced by nelfinavir despite inhibition of cytochrome P4503A (CYP3A) activity. Drug Alcohol Depend. 2009; 101(3): 158–168.
10. Hallinan R, Ray J, Byrne A, Agho K, Attia J. Therapeutic thresholds in methadone maintenance treatment: a receiver operating characteristic analysis. Drug Alcohol Depend. 2006; 81(2): 129–136.
11. Saracino MA, Marcheselli C, Somaini L, Pieri MC, Gerra G, et al. A novel test using dried blood spots for the chromatographic assay of methadone. Anal Bioanal Chem. 2012; 404(2): 503–511.
The authors
Liz Fox* PhD, FRCPath and Deepak Chandrajay MBBS, MRCP
Specialist Laboratory Medicine, St James’s University Hospital, Leeds, UK
*Corresponding author
E-mail: Elizabeth.fox@leedsth.nhs.uk
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
The kidneys play an important role in homeostasis, they regulate the amount of water and salts present in the body by filtering blood through the nephrons. Waste products are filtered out and eliminated from the body in the urine, which is made up of the excess water, salts and waste products.
When the kidneys are not functioning efficiently, waste products and fluids begin to accumulate instead of being excreted which can cause serious health problems. Furthermore, kidney disorders can often develop and advance over a period of time without showing any signs; alternatively, symptoms are not recognized as being associated with kidney problems. Kidney function testing is therefore relevant for diagnosing and monitoring disease and assists in the development of appropriate treatment plans. Laboratory automation facilitates the efficiency and productivity of clinical laboratories. The determination of parameters related to kidney function by using tests incorporating reagents applicable to a variety of automated analysers facilitates clinical effectiveness and patient outcomes when managed by qualified laboratory professionals.
Kidney function assessment
Many conditions can affect the ability of the kidneys to carry out their vital functions. Some conditions can lead to a rapid (acute) decline in kidney function; other conditions lead to a gradual (chronic) decline. A number of clinical laboratory tests in blood and urine can be used to assess renal function. The unit measure of kidney function is the glomerular filtration rate (GFR), which can be defined as the volume of plasma cleared of an ideal substance –freely filtered at the glomerulus and neither secreted nor reabsorbed by the renal tubules- per unit of time. The normal range is 80-120 ml/min. Measuring this rate is a laborious process. Creatinine is the closest to an ideal endogenous substance for measuring GFR.[1,2] Creatinine is derived from creatine and creatine phosphate in muscle tissue and is defined as a nitrogenous waste product. Creatinine is not reutilized but is excreted from the body in the urine via the kidney. As a consequence of the way in which creatinine is excreted by the kidney, its measurement is used almost exclusively in the assessment of kidney function.
Urea, a byproduct of protein metabolism, is produced in the liver and then is filtered from the blood and excreted in the urine by the kidneys. The blood urea nitrogen test (BUN) measures the amount of nitrogen contained in the urea, high levels can indicate kidney dysfunction. As these levels are also affected by protein intake and liver function, this test is usually done together with a blood creatinine test.
Cystatin C is a small cysteine proteinase inhibitor that is steadily produced by all nucleated cells. The small molecular weight of cystatin C allows it to be freely filtered by the glomerular membrane and therefore cystatin C levels in the blood are indicative of a normal or impaired GFR. Levels of cystatin C in serum/plasma are almost entirely dependent on GFR.[3]
Other tests for the measurement of other parameters regulated in part by the kidneys can also be useful for the evaluation of kidney function; these tests include electrolytes (sodium, potassium, chloride, bicarbonate), protein, uric acid and glucose:
Application of kidney function tests to automated systems
In clinical settings the application of tests for the determination of parameters related to kidney function to automated systems, facilitates clinical effectiveness and productivity. There are currently tests available for the determination of creatinine, BUN, cystatin C, electrolytes, protein, uric acid and glucose among others. If a variety of these tests could be applied to one system, the result output for each system would increase, which would maximize efficiency. The use of tests incorporating reagents applicable to a variety of automated analysers is beneficial as it increases the testing capacity of one system. This is further enhanced by the analyser’s capability to employ different methodologies with different reagents. The combination of automation and the use of stable, high performance reagents, lead to optimal analytical performance, extensive measuring ranges to ensure detection of abnormal values and reduced interference to produce more accurate results. For instance, a study using a creatinine test reported no interference with bilirubin and metamizol.[6]
The application of other kidney function related tests to studies in patients with nephrotic syndrome, chronic liver diseases and diabetes have also been reported.[7-8]
The automation of laboratory testing still requires qualified laboratory professionals for the evaluation of the results but reduces errors, staffing concerns and safety issues. This facilitates the diagnosis and the monitoring of kidney function, which is of great importance in clinical practice and in research.
Conclusion
The kidneys are the body’s natural filtration system and perform many vital functions. Kidney function tests is a collective term for a variety of individual tests and procedures for the evaluation of how well kidneys are functioning. The determination of parameters related to kidney function (i.e.creatinine, BUN, cystatin C, electrolytes, protein, uric acid, glucose) by using tests incorporating reagents applicable to a variety of automated analysers, increases the testing capacity of the systems and facilitates clinical effectiveness and patient outcomes when managed by qualified laboratory professionals.
References
1. Berger A. Renal function – and how to assess it. BMJ. 2000; 321: 1444.
2. Traynor J, Mactier R, Geddes CC, Fox JG. How to measure renal function in clinical practice. BMJ. 2006; 333 (7571): 733-737.
3. Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? Clin. Chem. 2002; 48(5): 699-707.
4. Kirby M. Screening for microalbuminuria. The British Journal of Diabetes and Vascular Disease. 2002; 2(2): 106-109.
5. Sechi LA, Catena C, Zingaro L., Melis A, De Marchi S. Abnormalities of glucose metabolism in patients with early renal failure. Diabetes. 2002; 51: 1226-1232.
6. Harmonien AP. Bilirubin and metamizol do not interfere with the Randox enzymatic creatinine test. An evaluation of a new enzymatic creatinine determination method. Eur. J. Clin. Chem. Clin. Biochem. 1996; 34(12): 975-976.
7. Mula-Abed W-AS and Hanna BE. Measurement of serum fructosamine as an index of glycated protein in patients with nephrotic syndrome and chronic liver diseases. Bahrain Medical Bulletin 2001; 23(4).
8. Hirnerova E, Krahulec B, Strbova L, Stecova A, Dekret J, Hajovska A, Ch A Dukat A. Effect of vitamin E supplementation on microalbuminuria, lipid peroxidation and blood prostaglandins in diabetic patients. Bratisl. Lek. Listy 2004; 105(12): 408-413.
Author
María Luz Rodríguez
Randox Laboratories Limited,
55 Diamond Road, Crumlin,
County Antrim, N. Ireland, BT29 4QY,
United Kingdom
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.
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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
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