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Featured Articles
External Quality Assessment (EQA) for trace element measurements in clinical laboratories
External Quality Assessment (EQA) is the cornerstone of quality assurance and method validation in clinical testing labs in the UK, ensuring that the results of patient investigations are reliable and comparable wherever they are produced. In this article we focus specifically on EQA for laboratories performing trace element measurements, although many of the points are applicable to the wider pathology areas.
by S.-J. Bainbridge and Dr C. F. Harrington
Introduction
External Quality Assessment (EQA), also termed proficiency testing (PT), involves the regular distribution of test materials to participating laboratories so that they may evaluate their analytical performance against a peer-group, detect any accuracy or other problems that may develop with the assay and so improve the results that they produce. The key elements that differentiate EQA from PT include: education and support; identification of method poor performance; and method evaluation [1].
Historically clinical science was one of the first disciplines to realize the usefulness of EQA and take steps to implement schemes that would be of use in the hospital laboratory. The first proficiency survey of UK clinical pathology laboratories was reported in 1953 and revealed a wide spectrum of results for the common tests [2]. Further surveys in the 1950s and 60s confirmed the need for regular PT. In 1969, the National Quality Control Scheme was initiated by the Wolfson Research Laboratories, Birmingham and involved the distribution of specimens every 14 days [2]. This is now known as the UK National External Quality Assessment Scheme (UKNEQAS) and is responsible for about 30 different schemes.
In 2013, the importance of EQA in the NHS pathology services was emphasized by Dr Ian Barnes in a Department of Health review into quality assurance [3]. The review assessed current NHS quality assurance frameworks and governance mechanisms for pathology services. It gathered a diverse range of evidence: examining expectations of pathology services; identifying areas for improvement; and recommending a system-wide way forward. It recommended strengthening and standardizing the current quality assurance structures that are in place, which are based on the Royal College of Pathologists (RCPath) Joint Working Group for Quality Assessment (JWGQA), which co-ordinates and oversees the standards and performance of EQA schemes for all schemes regardless of provider.
EQA for trace elements
The Trace Elements External Quality Assessment Scheme (TEQAS), which is part of UKNEQAS but based in Guildford, UK, was established in 1979 with distribution of specimens on a monthly schedule to UK hospital laboratories measuring copper and zinc in serum. During the next five years the scheme developed with inclusion of other participants and the introduction of additional analytes and specimen types. Following an international conference on Aluminium and Renal Disease in 1986, a two year arrangement was established with the EU Commission to fund participation in the serum aluminium programme for European laboratories involved with the monitoring of patients with chronic renal failure. An outcome of this work was the realization that analytical standards of performance for this measurement were very poor. In collaboration with the UK Department of Health it was proposed that the scheme should be linked to UKNEQAS in order to provide a mechanism for referral of poor performers to the Clinical Chemistry Advisory Panel. This link was formally established by the Advisory Committee on Analytical Laboratory Standards in 1988. The aims of TEQAS are consistent with the intentions of UK NEQAS, to:
- Provide professionally-led and scientifically-based schemes with a primarily educational objective.
- Provide regular distributions of specimens.
- Provide rapid feedback of performance.
- Support participants where problems occur.
- Stimulate the overall improvement in performance among all participating laboratories.
The main TEQAS scheme provides EQA for: Al, Cr, Co, Cu, Se and Zn in serum; As, Cd, Cr, Co, Pb, Mg, Mn, Hg, Se, Tl and Zn in whole blood; and As, Cd, Cr, Co, Cu, Fe, Pb, Mn, Hg, Ni, Tl and Zn in urine. This operates on a monthly cycle, with the results from the measurement of two specimens being returned on-line on the last day of each month. The report is then available five days later to download. Two smaller schemes providing Al in dialysis fluid and Cu and Fe in solid matrices are also available, but have a more limited number of participants.
The main steps in the EQA process
For a better appreciation of the overall EQA scheme it is useful to divide it into a number of process-based areas as shown in Figure 1. The activities that comprise these areas are also shown. Some of these areas come under particular focus as part of ISO 17043:
Design
Appropriate design of the PT scheme ensures that participants will have paid for a service that provides high quality comparable test items that are representative of patient samples they would normally expect to analyse. These test items will have undergone thorough assessment procedures in accordance with acceptable statistics to ensure a homogenous and stable test item.
Materials
The participants can be assured that the materials used in the production of the PT samples have been ethically and legally obtained and that the competence of the suppliers have been evaluated and verified to ensure their products or services do not affect the quality of the PT scheme.
Evaluation
ISO 17043:2010 ensures the evaluation of the participants performance is conducted fairly and consistently guaranteeing they receive an accurate evaluation calculated from the use of robust statistical methods.
Knowledge and Experience
In addition to all this ISO 17043:2010 ensures that the operations of the PT scheme are carried out by personnel that have the training, skills and competence necessary to professionally carry out their assigned tasks. Participants can feel secure in knowing that they have access to the specialist knowledge and expertise in the field of trace element testing to be able to discuss any concerns they may have.
Establishment of performance criteria
Measurements of performance are based on deviations of results from target values, which are used to calculate a Z-score. As EQA has developed, various organizations have produced documents that summarize best practice. Those from authoritative international bodies include:
- ISO 17043 (Conformity assessment – General requirements for PT).
- ISO 13528 (Statistical methods for use in PT by interlaboratory comparisons).
- IUPAC (The international harmonized protocol for the PT of analytical chemistry laboratories) [4].
All these documents recommend that assessment of performance should be based upon calculation of a Z-score (or a derivative which takes uncertainty into consideration).
The Z-score is calculated as:
x-X/ SDPT
where x = laboratory result,
X = target value, and
SDPT = standard deviation for PT (also represented as σ)
The ‘standard deviation for PT’ is set by the scheme organizer but should ideally be a value that will allow the score to demonstrate whether or not the performance is fit for the purpose for which the assay is being used. It is recommended that this value be set so that a Z-score of up to ±2 indicates acceptable performance and a score of more than ±3 indicates unsatisfactory performance.
In the TEQAS scheme, we have used quality specifications based on biological variation for the ‘standard deviation for PT’ and the determination of these quality specifications has been published [5]. For assays where there is insufficient data to prepare specifications in this way we have produced values that are related to performance within the scheme during recent years.
The quality specifications and their corresponding SDPT for some illustrative elements are shown in Table 1. These are presented as either a percentage of the target value or a fixed value depending on the concentration of the target value, and the one used is whichever is the greater. This allows for the increase in imprecision at low concentrations and conforms to a ‘funnel’ shape.
Scheme accreditation
Accreditation is fast becoming a preferred mechanism for delivering confidence in UK Healthcare and with the application of BS EN ISO 15189:2012 into Medical Laboratories and its requirement for the laboratories to seek confirmation for confidence in their results, the need for EQA schemes in the relevant fields of medical laboratories is ever increasing. Participation in a suitable scheme can be an effective way of demonstrating the laboratories’ technical competence. ISO 15189:2012 requires laboratories to evaluate their PT providers and a recognized acceptable basis of their evaluation recommended by the UK Accreditation Service (UKAS) is the participation in PT schemes with those providers that have been accredited to ISO/IEC 17043:2010. This International Standard specifies criteria and the general requirements for the competence of the PT providers and their responsibility for all tasks in the development and operation of the PT scheme. Some of the main differences introduced with ISO 17043 are summarized in Table 2.
Assessment of conformance
When conducting an assessment of a PT scheme for conformance to ISO 17043:2010, the assessors will take a holistic approach looking at the management system as a whole. The assessment will include areas such as scheme organization, scheme management, evaluation processes, technical competence and impartiality and integrity. Each separate area of the PT scheme are all interlinked and therefore when accreditation is granted by the accreditation body (UKAS in the UK) it will not be given on a single fact but the overall competence of the PT provider. Accreditation to ISO 17043:2010 can be a hard and thorough task for PT providers to undertake but once accreditation is granted it provides the necessary assurance of a competent and professional scheme which can provide an open and honest service whilst maintaining confidentiality for all those participants enrolled in the scheme.
Summary
The 2013 Barnes review into quality assurance in the NHS pathology services reinforced the importance of quality assurance and this article has discussed the implications of recently introduced ISO standards for clinical pathology departments (ISO 15189:2012) as well as for EQA scheme providers (ISO 17043:2010). This strengthens and standardizes the systems used in clinical testing laboratories and ensures high quality and comparable results for patient tests.
References
1. James D, Ames D, Lopez B, Still R, Simpson W, Twomey. External quality assessment: best practice. J Clin Pathol. 2014; doi: 10.1136/jclinpath-2013-20621.
2. Bullock DG. External quality assessment schemes for clinical chemistry in the United Kingdom. Ann Ist Super Sanita 1995; 31: 61–69.
3. Barnes I. Pathology Quality Assurance Review 2014. www.england.nhs.uk/wp-content/uploads/2014/01/path-qa-review.pdf
4. Thompson M, Ellison SLR, Wood R. The International Harmonized Protocol for the proficiency testing of analytical chemistry laboratories (IUPAC Technical Report). Pure Appl Chem. 2006; 78: 145–196.
5. Arnaud J, Weber J-P, Weykamp CW, Parsons PJ, Angerer J, Mairiaux E, Mazarrasa O, Valkonen S, Meditto A, Patriarca M, Taylor A. Quality specifications for the determination of copper, zinc, and selenium in human serum or plasma: evaluation of an approach based on biological and analytical variation. Clin Chem. 2008; 54(11): 1892-1899.
6. Summary of ISO 15189 additional requirements. CPA UK Ltd, 2012. http://www.ukas.com/Library/Services/CPA/Summary%20of%20Idifferences%20betwen%20ISO%2015189%20&%20CPA.pdf
The authors
Sarah-Jane Bainbridge and Chris F. Harrington* PhD
TEQAS, Trace Element Centre, Surrey Research Park, Guildford GU2 7YD, UK
*Corresponding author
E-mail: Chris.harrington1@nhs.net
Metabolic syndrome: definition, grading and treatment
Metabolic syndrome is characterized by a collection of disorders, making it difficult to diagnose and stage. This article describes the criteria used for diagnosis as well as discussing treatment strategies.
by Prof. Giuseppe Derosa and Dr Pamela Maffioli
Definition and grading
Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease; it affects one in five people in the United States, and prevalence increases with age. There are different definitions of metabolic syndrome; according to the Adult Treatment Panel (ATP) III [1], metabolic syndrome requires the presence of at least three of the listed criteria (Table 1).
Recently insulin resistance has been cited to be associated with other metabolic risk factors and correlates with cardiovascular risk. The pro-inflammatory state has also been developed and used as a marker to predict coronary vascular diseases in metabolic syndrome: it is identified by higher C-reactive protein (CRP) levels, commonly present in people with metabolic syndrome. One cause of elevated CRP is obesity, because adipose tissue releases inflammatory cytokines that may elicit higher CRP levels. Also, the pro-thrombotic state has been recently considered for the definition of metabolic syndrome, characterized by increased plasma plasminogen activator inhibitor-1 (PAI-1) and fibrinogen. However, the ATP III panel did not find adequate evidence to recommend routine measurement of insulin-resistance, pro-inflammatory state (e.g. high-sensitivity C-reactive protein), or pro-thrombotic state (e.g. fibrinogen or PAI-1) in the diagnosis of the metabolic syndrome.
The World Health Organization (WHO) criteria, instead, emphasized insulin resistance as the major underlying risk factor and required evidence of insulin resistance for diagnosis (Table 2) [2, 3].
The International Diabetes Federation (IDF), instead, dropped the WHO requirement for insulin resistance, but made abdominal obesity necessary for the diagnosis, with particular emphasis on waist measurement as a simple screening tool [4]; the other criteria (Table 3) were essentially identical to those provided by ATP III [1].
The American Association of Clinical Endocrinologists (AACE) proposed a third set of clinical criteria for the insulin resistance syndrome [5]. These criteria appear to be a hybrid of those of the ATP III and WHO metabolic syndrome. However, no defined number of risk factors is specified and diagnosis is left to clinical judgment (Table 4).
Given that multiple definitions of the same disease can generate confusion among physicians, the major organizations made an attempt to unify the various criteria for the definition of metabolic syndrome [6]. It was agreed that there should not be an obligatory component, but that waist measurement would continue to be a useful preliminary screening tool. Three abnormal findings out of five would qualify a person for the metabolic syndrome according to the unified definition shown in Table 5.
As readers can easily understand, individuals with metabolic syndrome are at increased risk for coronary heart disease (CHD) [7]. In particular, in the absence of diabetes, the metabolic syndrome generally did not raise the 10-year risk for CHD by more than 20% [8], in particular 10-year risk generally ranged from 10% to 20% for men and did not exceed 10% for women. However, in the presence of diabetes, the risk increases. Obviously, patients fulfilling all or almost all of the metabolic syndrome potential criteria, have earlier and more serious organ damage, at both cardiac and vascular levels, than patients with only three out of five components of the metabolic syndrome definition.
Treatment
Despite the grade of metabolic syndrome, however, there are two general approaches to its treatment. The first strategy modifies root causes, overweight/obesity and physical inactivity, and their closely associated condition, insulin resistance. The second approach directly treats the metabolic risk factors such as atherogenic dyslipidemia, hypertension, the pro-thrombotic state, and underlying insulin resistance. ATP III recommended that obesity be the primary target of intervention for metabolic syndrome [9]. First-line therapy should be weight reduction; the current recommendations for the treatment of overweight and obese people include increased physical activity and reduced calorie intake [10, 11]. Pharmacological treatment with orlistat can be another option, and when it is not tolerated, bariatric surgery should be considered. However, surgery irreversibly changes the overall architecture of the digestive tract; in this regard, the endoscopic duodenal–jejunal bypass liner can be another option. It consists of a sheath that is inserted endoscopically through the mouth into the digestive tract of the obese patient creating a physical barrier between the intestinal wall and the food ingested. The device can be considered as an alternative to bariatric surgery because of the minimal adverse events and the possibility to easily remove the device when the desired weight has been achieved [12]. Weight loss is important because it lowers serum cholesterol and triglycerides, raises HDL-cholesterol, lowers blood pressure and glucose, and reduces insulin resistance. Published data further show that weight reduction can decrease serum levels of CRP and PAI-1 [13–16]. In addition, other lipid and non-lipid risk factors associated with the metabolic syndrome should be appropriately treated. Atherogenic dyslipidemia includes elevated serum triglycerides and apolipoprotein B, increased small LDL particles, and reduced level of HDL-cholesterol. The treatment strategy for atherogenic dyslipidemia in metabolic syndrome focuses on triglycerides. If triglycerides are ≥150 mg/dL and HDL-cholesterol is <40 mg/dL, a diagnosis of atherogenic dyslipidemia is made. If triglycerides are <200 mg/dL, and specific drug therapy to reduce triglyceride-rich lipoproteins is not indicated. However, if the patient has CHD or CHD risk equivalents, LDL-cholesterol goal has to be considered together with the use of a drug to raise HDL-cholesterol (fibrate). On the other hand, if triglycerides are 200–499 mg/dL, non-HDL cholesterol becomes a secondary target of therapy. Goals for non-HDL cholesterol are 30 mg/dL higher than those for LDL-cholesterol. First the LDL-cholesterol goal is attained, and if non-HDL remains elevated, additional therapy may be required to achieve the non-HDL goal. Alternative approaches for treatment of elevated non-HDL cholesterol that persists after the LDL goal has been achieved are (a) higher doses of statins, or (b) moderate doses of statins + triglyceride-lowering drug (fibrate). If triglycerides are very high (≥500 mg/dL), attention turns first to prevention of acute pancreatitis, which is more likely to occur when triglycerides are >1000 mg/dL. Triglyceride-lowering drugs (fibrate) become the first line therapy; although statins can be used to lower LDL-cholesterol to reach the LDL-cholesterol goal, in these patients it is often difficult (and unnecessary) to achieve a non-HDL cholesterol goal of only 30 mg/dL higher than for LDL-cholesterol [9].
Conclusion
In conclusion, metabolic syndrome increases cardiovascular risk; a multifactorial approach is necessary in order to prevent the development of the various components of this disease.
References
1. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report. Circulation 2002; 106: 3143–3421.
2. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus: provisional report of a WHO consultation. Diabet Med. 1998; 15: 539–553.
3. World Health Organization. Definition, diagnosis and classification of diabetes mellitus and its complications: report of a WHO Consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva, Switzerland: World Health Organization; 1999. Available at: http:// whqlibdoc.who.int/hq/1999/WHO_NCD_NCS_99.2.pdf.
4. Alberti KG, Zimmet P, Shaw J; IDF Epidemiology Task Force Consensus Group. The metabolic syndrome: a new worldwide definition. Lancet 2005; 366: 1059–1062.
5. Einhorn D, Reaven GM, Cobin RH, Ford E, Ganda OP, Handelsman Y, Hellman R, Jellinger PS, Kendall D, Krauss RM, Neufeld ND, Petak SM, Rodbard HW, Seibel JA, Smith DA, Wilson PW. American College of Endocrinology position statement on the insulin resistance syndrome. Endocr Pract. 2003; 9: 237–252.
6. Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, Fruchart JC, James WP, Loria CM, Smith SC Jr; International Diabetes Federation Task Force on Epidemiology and Prevention; Hational Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; International Association for the Study of Obesity. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009; 120 (16): 1640–1645.
7. Lakka HM, Laaksonen DE, Lakka TA, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. JAMA 2002; 288: 2709–2716.
8. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbrshatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation 1998; 97: 1837–1847.
9. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation. 2002; 106 (25): 3143–3421.
10. American Diabetes Association. Nutrition principles and recommendations in diabetes. Diabetes Care 2004; 27(S1): 36–46.
11. American Diabetes Association. Physical activity/exercise and diabetes. Diabetes Care 2004; 27(S1): 58–62.
12. Derosa G, Maffioli P. Possible therapies for obesity: focus on the available options for its treatment. Nutrition 2014; doi: 10.1016/j.nut.2014.09.005.
13. Dengel DR, Galecki AT, Hagberg JM, Pratley RE. The independent and combined effects of weight loss and aerobic exercise on blood pressure and oral glucose tolerance in older men. Am J Hypertens. 1998; 11: 1405–1412.
14. Ahmad F, Considine RV, Bauer TL, Ohannesian JP, Marco CC, Goldstein BJ. Improved sensitivity to insulin in obese subjects following weight loss is accompanied by reduced protein-tyrosine phosphatases in adipose tissue. Metabolism 1997; 46: 1140–1145.
15. Su HY, Sheu WH, Chin HM, Jeng CY, Chen YD, Reaven GM. Effect of weight loss on blood pressure and insulin resistance in normotensive and hypertensive obese individuals. Am J Hypertens. 1995; 8: 1067–1071.
16. Derosa G, Limas CP, Macías PC, Estrella A, Maffioli P. Dietary and nutraceutical approach to type 2 diabetes. Arch Med Sci. 2014; 10(2): 336–344.
The authors
Giuseppe Derosa1,2 MD, PhD, Pamela Maffioli1,3 MD
1Department of Internal Medicine and Therapeutics, University of Pavia, Fondazione IRCCS Policlinico S. Matteo, PAVIA, Italy.
2Center for the Study of Endocrine-Metabolic Pathophysiology and Clinical Research, University of Pavia, PAVIA, Italy.
3PhD School in Experimental Medicine, University of Pavia, PAVIA, Italy
E-mail: giuseppe.derosa@unipv.it
Determination of methylphenidate and ritalinic acid in serum and saliva of patients with ADHD
by Sophie Studer, Hans-Willi Clement, Christian Fleischhaker, Eberhard Schulz
Attention deficit hyperactivity disorder (ADHD)
What do fidgets and Johnny Head-in-the-Clouds (a fictional character from a German tale) have in common with Alexander the Great, Winston Churchill or Benjamin Franklin? For all of these, a diagnosis of ADHD would be made today [1]. The first indications of behavioural abnormalities in childhood date back to the mid-19th century. However, clear descriptions of the medical condition were first found in 1902 in the notes of the English pediatrician George Still. The characteristics he described were extreme motor unrest and “the abnormal inability to maintain concentration”, which led to failure to achieve at school. In 1932, two neurologists at the Berlin Charité Hospital, Kramer and Pollnow, described the symptoms of an illness they termed a “hyperkinetic disease”, which included the inability to appreciate danger, to follow rules, to control impulses and a lack of planning skills [2], as well as being easily distracted and showing motor hyperactivity. This was the first description of the leading symptoms of ADHD in German language, which is still valid – hyperactivity, inattentiveness and impulse control disorder. In order to be able to evaluate these characteristics and to investigate hyperactivity symptoms in a standardized way, Conners developed parent and teacher questionnaires at the end of the 60s that are still used today [3].
Most scientific papers are merely limited to attempts to explain the origin and course of the disease portrayed. Whereas these hyperactivity symptoms are actually seen to be the interaction of morphological changes already present at birth with external factors that affect the organism.
Methylphenidate (Ritalin®)
Today, stimulants such as Ritalin® in combination with psychotherapy and psychoeducation represent the method of choice for the treatment of hyperkinetic disorders. When a definite diagnosis has been made, pharmacotherapy is always indicated if the ADHD symptoms are marked, occur in many situations and when the effectiveness or practicability of psychoeducative and behavioural therapy measures are lacking. In addition, no contraindications for the individual psychostimulants must exist.
Methylphenidate (MPH) demonstrably improves the core symptoms of ADHD [4] and is one of the best-researched pediatric psychopharmaceuticals with long-term clinical experience. Nevertheless, the “pill for the troublemaker” is one of the most controversially discussed pharmacological products. A frequently mentioned point is the possible addiction potential of Ritalin®, for which reason the drug is also subject to the German controlled substances act. However, one must differentiate here between oral administration in therapeutic doses and “snorting” or intravenous application in excessive amounts.
The story of Ritalin® begins at the Swiss company Ciba, where the psychostimulant was successfully synthesized and the effectiveness of the substance proven in a self-experiment. When the drug was taken by Leandro Panizzon’s wife Marguerite (“Rita”), she made considerable progress. Ritalin®, probably the best-known MPH today, is named after her. Ciba introduced it to the market in 1954, 10 years after its development, for the treatment of psychoses, chronic tiredness and lethargy [5]. A short time later, meta-analyses became possible based on numerous study results. A distinct alleviation of symptoms was shown in about 75 % of all children treated with Ritalin® for ADHD. Alongside the reduction of hyperactivity and impulsiveness in the mentioned group, the ability for concentration and attentiveness increased considerably, also manifested in improved school achievements [6-8].
Structure and metabolism of methylphenidate
The fundamental structure of MPH is based on the phenylethylamine skeleton (Fig. 1a) and exhibits no hydroxyl group on the phenyl ring, facilitating diffusion into the central nervous system. It exhibits two chiral centres, consequently there are four configuration isomers (Fig. 1b). In practice, only the D- and L-threo forms find use in the treatment of ADHD. In the USA and in Switzerland the pure D- threo dextromethylphenidate isomer (Focalin®) is approved, and is regarded as the main pharmacologically active form. In comparison, the original Ritalin® consists of a mixture of the enantiomeric D- and L-threo forms. MPH is always manufactured in the protonated form as the hydrochloride salt [5].
The oral bioavailability of MPH is about 30 % (D-enantiomer > L-enantiomer), whereby foodstuffs have no relevant influence on the resorption. Generally available preparations reach their maximum plasma level within 1.5-2 hours. The effect is already shown after 15-30 minutes and reaches its highest level after 2-3 hours. In contrast, retard preparations such as Concerta® have a considerably longer duration of effect, which can be around 10-12 hours.
MPH is rapidly metabolized renally by carboxylesterase CES1A1 to pharmacologically inactive 2-phenyl-2-(piperidin-2-yl) acetic acid (ritalinic acid, RA). The maximum plasma level of the metabolite is 30-50 times greater than that of the original drug and the half-life is about twice as long. However, as RA possesses only a small pharmacodynamic activity, or none at all, this fact is of minor significance.
TDM of Ritalin® in children
For monitoring pharmacotherapy through concentration measurements, the collection of blood has so far been unavoidable. However, invasive methods present a compliance obstacle, particularly for children. Therefore, to ensure a high degree of drug safety, a method based on alternative body fluids for TDM is desirable. Saliva is becoming increasingly significant in this respect and is already being investigated routinely in immunology and infectious serology diagnostics, in drug and drug-abuse screening and for determining levels of the hormone cortisol [9].
The research group of Marchei et al. has already successfully developed saliva diagnostics for MPH and RA using LC-MS/MS [10]. Further investigations demonstrated almost parallel changes in the MPH and RA concentrations over the time in serum and saliva [11].
These facts, and the availability of the MassTox® TDM Series A Kit from Chromsystems, which permits the determination of the psychostimulant methylphenidate and its metabolites in serum/plasma, were the starting point for an investigation into an LC MS/MS method from Chromsystems for the determination of these analytes in saliva.
For this, serum and saliva samples from 19 ADHD patients (nine children, one adolescent and nine adults) being treated with MPH were collected and investigated. The study participants mainly took long-acting retard products, such as Medikinet retard® or Ritalin LA®. The daily intake ranged from 5 to 60 mg of MPH, corresponding to a dosage of 0.11 to 1.43 mg MPH per kilogram body weight.
As part of the routine follow-up investigations, serum was obtained by blood collection using a serum Monovette, two hours after administration of the drug where possible. In parallel, saliva samples were obtained from the patients using the Salivette system. For this, the subjects chewed on a cotton swab for 2-5 minutes during blood collection. The samples were centrifuged immediately afterwards, aliquoted and then shock frozen in liquid nitrogen to avoid degradation of the substances to be analysed.
Materials and methods
Kit for LC-MS/MS analysis: MassTox® TDM Antidepressants 2/Psychostimulants (atomoxetine, methylphenidate, mianserin, reboxetine, ritalinic acid, trazodone; (Chromsystems GmbH), methylphenidate hydrochloride C-II (Sigma-Aldrich), saliva (IBL Hamburg).
After a brief storage at -80°C, the serum and saliva samples were processed using the parameter set for Antidepressants 2/Psychostimulants for LC-MS/MS analysis and following the manufacturer’s instructions (Table 1). The calibrators and control materials for the determination of MPH in serum/plasma were also from Chromsystems. To produce a series of MPH standards in saliva, saliva (IBL Hamburg) was spiked with MPH hydrochloride (Sigma-Aldrich).
After sample preparation, the eluates obtained were separated chromatographically in an analytical column at a flow rate of 0.6 ml/min (MasterColumn® A, Chromsystems) and then quantified in a mass spectrometer (Thermo TSQ Quantum Ultra) according to their mass-to-charge ratio (Fig. 2).
The Chromsystems test is approved for the determination of psychostimulants in serum/plasma. Figure 3A shows the chromatogram of a patient who has taken Medikinet adult® at a dosage of 30 mg per day. The determination in serum gave a value of 5.5 ng/ml for MPH and 195 ng/ml for RA. As was to be expected from data in the literature, the values for the determination of MHP in saliva were considerably higher – in this case by a factor of 4 – whereas considerably lower values were determined for RA (Fig. 3B) [12].
A comprehensive verification of the determination of MPH and its acid metabolite is still to be performed. Nevertheless, initial experiments to determine the variance within a preliminary inter-assay study have already been carried out. The results are summarized in Table 2.
The values measured for saliva were only slightly poorer than the values for MPH in serum, also determined with the
MassTox® TDM Parameter Set Antidepressants 2/ Psychostimulants.
Conclusions
In order to carry out an effective pharmacotherapy with few side effects, it is necessary to establish less-invasive TDM methods. This applies to sensitive patient groups, such as children and adolescents who display distinctly different pharmacokinetic characteristics, indicating the need for a much tighter monitoring of compliance [13]. A similar situation in respect of altered metabolic characteristics can be found in patients with liver or kidney failure, who would also benefit from a less-invasive sample collection method. In summary, the data described here have shown the methodological and analytical suitability of the MassTox® TDM Series A – PARAMETER Set Antidepressants 2/
Psychostimulants in serum/plasma (Chromsystems) – for the determination of MPH and its metabolite RA by LC-MS/MS in both serum/plasma and in saliva. Thus facilitating a much more simplified way of drug monitoring in this special case of pharmacotherapy.
References
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9. Chiappin S, Antonelli G, Gatti R, De Palo EF. (2007) Saliva specimen: A new laboratory tool for diagnostic and basic investigation. Clin Chim Acta 383(1-2): 30-40.
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The authors
Sophie Studer, Hans-Willi Clement, Christian Fleischhaker, Eberhard Schulz
University Hospital Freiburg, Department of Child and Adolescent Psychiatry, Psychotherapy and Psychosomatics, Neuropharmacological Research Laboratory, Freiburg, Germany
The Longitude Prize 2014
The Longitude Prize was originally offered by the British Government to anyone who could come up with a simple method for determining a ship’s longitude when at sea. John Harrison received funding from the Board of Longitude for his work on chronometers, and his name is still the one associated with having solved this problem.
In 2014, the British Prime Minister, David Cameron, launched a new version of the prize, which is being developed and run by Nesta, the UK’s innovation foundation. Six potential themes were announced, including:
- Flight How can we fly without damaging the environment?
- Food How can we ensure everyone has enough to eat?
- Antibiotics How can we prevent the rise of resistance to antibiotics?
- Paralysis How can we restore movement to those with paralysis?
- Water How can we ensure everyone has access to safe and clean water?
- Dementia How can we help people with dementia live independently for longer?
The category chosen (by public vote) to receive the modern prize was that of antibiotic resistance.
The discovery, development and use of antibiotics has been one of the great successes of modern medicine. However, the rise of antibiotic resistance (already warned of by Sir Alexander Fleming in his Nobel Prize winner’s lecture in 1945) threatens to render existing antibiotics useless and no new class of drugs has been discovered since the 1980s. Quotes can easily be found from organizations such as the World Health Organization (WHO), the US Centers for Disease Control and Prevention (CDC) and many senior scientists ranging from the ‘grim future’ to the ‘apocalypse’ of returning to life without antibiotics.
In order to fight antimicrobial resistance, the inappropriate use of antibiotics has to be prevented. To target this, the challenge set by the Longitude Prize committee is to create a ‘cheap, accurate, rapid and easy-to-use point of care test kit’ that will indicate if antibiotics are necessary, and if so which to prescribe. The kit should deliver results within 30 minutes and devices with built-in data recording and transmission capacities will be favoured.
There are of course technical hurdles, such as selecting the right targets for detection, how to detect them (what sample preparation has to happen?) and how to achieve the desired performance (of size/portability, speed, low cost per test, data transmission). However, POC devices do already exist and are in use. Notably, a mobile phone linked test for blindness (the portable eye examination kit or Peek) that allows eye tests to be carried out anywhere on earth. This is particularly advantageous in resource-poor settings. For example in Nigeria, even though less than 50% of the population has access to clean drinking water, 80% of people have mobile phones.
Collaboration between the different fields of medicine, microbiology, biochemistry, engineering and physics will be needed to address these technical issues. This is another aspect where the modern Longitude Prize is echoing the original – the solution can come from anywhere, not necessarily the expected sources. Again, examples of this have been seen in other areas recently. Jack Andraka, born in 1997 has been dubbed the ‘teen prodigy of pancreatic cancer’ for his development of promising early detection test for pancreatic cancer. In 2012, Brittany Wenger, at the age of 17, won the Google Sciences Fair grand prize for a breast cancer diagnosis app. One thing though is certain – innovation will be needed.
For more information see the Longitude Prize 2014 website: https://longitudeprize.org/