Hair analysis for forensic diagnostics is gaining popularity in both research and applied settings. Commercially available dynamic multiple reaction monitoring (Dyn-MRM) software applied to hair samples can provide drug-use history for several months. The cost-effective drug test using Dyn-MRM software facilitates analysis of over 200 analytes in a 10-minute chromatographic run.

by Professor D. P. Naughton and Professor A. Petróczi

Background
Considerable global efforts are expended to address substance abuse which has major effects on public health and quality of life, as well as on economic and societal prosperity. This grand challenge impacts on a wide range of healthcare, regulatory and research endeavours. Key examples include destruction of lives through abuse of class A drugs, efforts to reduce doping in sport, attempts to address alcohol abuse and the lethal dangers of ‘legal highs’ (novel psychoactive substances).

Healthcare and regulatory officials engage in a wide range of activities to combat substance abuse. These include criminalization, banning substances in sport, education programs to prevent and efforts to understand and alter drug-related behaviour. However, in many cases there is an unmitigated failure to address the issues around substance misuse or abuse.

For example, for doping in sports, where vast efforts and resources are expended, indirect assessment of doping produces prevalence figures some 10-fold higher than positive doping test rates [1]. These frequent prevalence reports, at odds with figures from analytical tests, corroborate the belief that current anti-doping testing regimes are far from adequate [1]. Using doping in sport as an exemplar, major improvements in approaches to test for prohibited substances are needed. The advent of more advanced instrumentation aids testing in a number of ways. Increased sensitivity and affordability are very important but so are software developments that provide capability to monitor several hundred substances in one liquid chromatography-tandem mass spectrometry (LC-MS/MS) cycle of less than 10 minutes. These advances bring opportunities that require both instrumentation updates and frequent training updates for staff.

Hair analysis
Despite major advances in instrumentation and software, there are still obstacles to performing successful drug tests that benefit the drug taker. In sport, doping practices are frequently highly advanced, with some athletes taking heed of in-depth knowledge about most key parameters including generic testing methods, masking drugs and advances in detection for specific substances. The burden of proof has shifted to acquiring samples both in and out of competition as well as ensuring that appropriate tests are performed on each sample [2]. Owing to varied pharmacokinetics, analyte distribution and analytical procedures used, testing for a wide range of drugs in all samples is prohibitive [3]. Current testing approaches using biofluids impart considerable practical and financial consequences for the testing regime. Furthermore, cases of microdosing, masking and using novel substances make life challenging for the anti-doping officials.

Drug testing based on biofluids presents a number of issues that add considerably to cost but also are restrictive in terms of the number of tests required to cover a suitable duration owing to the pharmacokinetic profiles of many drugs. Where drugs or their metabolites are washed out efficiently after cessation of use, detection is less viable. The relatively short half-lives of many substances means the window for detection can be limited, which affects the success of occasional testing. The cost of supervised sampling along with the requirement for biofluid storage and handling to avoid sample corruption or infection is prohibitive for major levels of testing. Focusing on doping in sport, further complexities arise through variations in the lists of prohibited substances for testing in and out of competition [4]. The advantage of a longer window of detection via hair-analysis is suited to out of competition testing where a cumbersome system currently exists for sampling which is intrusive and controversial [5]. Thus, new approaches that allow a single test to be conducted simultaneously for (i) a wide range of substances and (ii) covering a prolonged period such as a 3-month window, would be valuable in sport for out of competition testing but also, beyond sport, for social drugs and new psychoactive substances.

In contrast to drug tests based on biofluids, hair analysis provides a range of advantages including: ease of sampling, ability to conduct multiple tests on one cut-hair sample to cover a prolonged duration (a typical 3-cm hair sample is equivalent to approximately 3 months’ growth), lack of issues with infection risk, facile storage at room temperature, lack of requirement to process tissue containing genetic data, and good stability of many drugs and metabolites in the hair matrix.

Instrumentation advances
We recently reported a hair-based method, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), for the analysis of substances of forensic nature [6]. The multi-drug/metabolite assay employs a dynamic multiple reaction monitoring (Dyn-MRM) method using proprietary software [7, 8]. It allows both screening and validated confirmatory analysis depending on the focus of the investigation. This approach has several benefits: (a) the Dyn-MRM software is suited to screen over 200 compounds on a single chromatographic run of under 10 minutes, (b) full validated methods for compounds can be incorporated into the software, (c) hair samples provide the opportunity to cover longer windows for detection in one test (e.g. approximately 3-month history covered in a 3-cm sample), and (d) the software is designed to allow ready adoption of new compounds of interest. The advantage of Dyn-MRM is that multiple reaction monitoring is employed with a focus on scanning for specific peaks at their selected elution times. This efficient method allows the analysis of large numbers of analytes simultaneously in a short run (Fig. 1). In our report, the proprietary software has been extended and applied to cover a range of drugs and metabolites of interest to forensic investigations including cognitive enhancers, amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opioids, steroids and sedatives. The chromatographic run is calibrated by a test mixture containing approximately 20 substances and further tailoring would be required to match a specific remit such as the WADA (World Anti-Doping Agency) prohibited list more closely [4].
 
Conclusion and future perspectives
The application of Dyn-MRM software to screen for a large range of drugs brings considerable advantages to laboratories involved in drug testing. The ease of use and ability to add new compounds to the screening database are noteworthy. Coupling this commercially available software to hair analysis adds the extra dimension of being able to screen for drug use over several months in one hair sample. This advance will add considerably to the efficiency of drug testing but will remain as an adjunct to other testing methods for out of competition testing in sport as it will not cover all analytes of interest to anti-doping officials [4]. Some substances are unlikely to be found in hair (e.g. performance enhancing peptides) and for other substances there will be issues with establishing a threshold – either for endogenous substances (e.g. testosterone) or for substances consumed through diet (e.g. drugs used in farming). Further limitations are that (i) a single use of a drug may be undetectable owing to the low levels deposited in hair, and (ii) more research is warranted to ascertain the effects of hair type and colour on analyte uptake and stability. In spite of these limitations, hair analysis coupled to modern advances in instrumentation sensitivity and software capabilities is promising in many scenarios especially to obtain a prolonged history of abuse and where ‘zero tolerance’ is applied (e.g. for synthetic steroids). In addition, hair analysis may have a role in support of the Athlete Biological Passport through analysis of indirect biomarkers of doping [9].

References
1. de Hon O, Kuipers H, van Bottenburg M. Prevalence of doping use in elite sports: A review of numbers and methods. Sports Med. 2015; 45(1): 47–69.
2. World Anti-Doping Agency (WADA). International Standards. 2015; https://www.wada-ama.org/en/what-we-do/international-standards.
3. Maennig W. Inefficiency of the anti-doping system: Cost reduction proposals. Subst Use Misuse 2014; 49(9): 1201–1205.
4. WADA. List of prohibited substances and methods. 2015; http://list.wada-ama.org/.
5. Hanstad DV, Loland S. Elite athletes’ duty to provide information on their whereabouts: Justifiable anti-doping work or an indefensible surveillance regime? Eur J Sport Sci. 2009; 9(1): 3–10.
6. Shah I, Petroczi A, Uvacsek M, Ranky M, Naughton DP. Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS. Chem Cent J. 2014; 8(1): 73.
7. Agilent Technical Overview. Ion optics innovations for increased sensitivity in hybrid MS systems. Agilent Technologies USA 5989-7408EN. 2007; http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-7408EN_HI.pdf.
8. Stone P, Glauner T, Kuhlmann F, Schlabach Tim, Miller K. New dynamic MRM mode improves data quality and triple quad quantification in complex analyses. Agilent Technologies USA 5990-3595EN. 2009; http://www.chem.agilent.com/Library/technicaloverviews/Public/5990-3595en_lo%20CMS.pdf.
9. Vernec AR. The athlete biological passport: an integral element of innovative strategies in antidoping. Br J Sports Med. 2014; 48(10):817–819.

The authors
Declan P. Naughton* PhD, Andrea Petróczi PhD
School of Life Sciences, Kingston University, London, UK

*Corresponding author
E-mail: D.Naughton@kingston.ac.uk

New psychoactive substances (NPS) reach the recreational drugs market at a fast pace and are of concern because of potential health risks. In addition to not being legally regulated, NPS escape detection in standard drug tests. Drug testing laboratories, therefore, must adapt their analytical methods to also cover these new substances. For screening and confirmation of NPS, mass-spectrometric multicomponent methods are useful.

by Prof. Olof Beck and Prof. Anders Helander

New psychoactive substances
The emergence of new drugs of abuse that are designed to circumvent narcotics legislation by slight chemical structural modifications of already classified drugs represents an ever increasing problem [1, 2]. Nowadays, this phenomenon is commonly termed ‘new psychoactive substances’ or ‘NPS’, but also other names such as designer drugs, legal highs, research chemicals, smart drugs, bath salts, and spice have been and are used. The NPS problem is of global concern but may vary in extent between countries, partly due to national differences in legislation and drug culture. Statistics from the EU Early Warning System operated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol on the number of NPS reported for the first time in Europe on a yearly basis gives a good insight on the progress of this phenomenon (Fig. 1) [2]. Over the past 6 years particularly, it has escalated to the level of more than 100 new substances in 2014 (i.e. about two new substances each week on average). The NPS market was long dominated by stimulants and synthetic cannabinoids but currently comprises all classes of abused substances [2].

Problems related to NPS
NPS are of particular concern because they can be sold openly in web-based shops and elsewhere and thereby reach new drug users that are attracted by their ‘legal’ status. Of public concern are the unforeseen toxic effects of NPS, as using these uncontrolled and unsafe substances and products may lead to severe intoxication and even death [1, 3]. In Sweden, the progress of the NPS phenomenon and associated harmful effects has been followed in a collaborative project between the Department of Laboratory Medicine at the Karolinska University Hospital and the Karolinska Institutet, and the Swedish Poisons Information Center [3, 4]. This project, named STRIDA, enrolls patients with suspected NPS intoxication presenting in emergency departments all over the country. By combining the results from laboratory investigations of serum and urine samples with clinical information, new knowledge about NPS prevalence and toxicity is compiled. Since the start in 2010, the STRIDA project has documented over 2000 non-fatal but often severe acute intoxication cases involving a large number of different NPS. Polydrug use is commonly seen in these cases [3].

NPS in drug screening
One reason for using NPS instead of conventional drugs of abuse may be that NPS often remain undetected in standard drug testing procedures. Accordingly they are especially attractive alternatives for individuals who want to minimize the risk of being detected, such as in workplace drug testing and drug rehabilitation programmes.

The established procedure for drug testing is to use initial screening by immunoassays and then to confirm positive samples using methods based on the more sensitive and selective mass spectrometry (MS) technique. On one hand, the NPS present a challenge for the immunoassay screening, as available methods are typically directed only towards the conventional substances, e.g. amphetamines (amphetamine and methamphetamine), tetrahydrocannabinolcarboxylic acid (THC, cannabis), morphine (heroin), and benzoyl ecgonine (cocaine). On the other hand, as NPS are often designed to mimic and are chemical derivatives of conventional drugs, there is a possibility that certain NPS will also bind to (i.e. cross-react with) the antibodies used in immunoassay screening methods. And this is indeed the case. However, when these ‘false-positive’ screening results are subjected to confirmatory analysis by methods based on MS detection, they will turn out negative (i.e. ‘false-negative’ for drug use), if the MS method is only directed toward the standard set of abused drugs.

Cross-reactivity of NPS in immunoassays
When ecstasy (3,4-methylenedioxymethamphetamine, MDMA) became established as a street drug, interest emerged to detect it in immunoassay screening. MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) were found to be detectable in existing assays for amphetamine and methamphetamine, due to a high degree of cross-reactivity for these compounds [5]. Likewise, also other new amphetamine-like substances were detectable [6].

However, although many NPS showed low cross-reactivity in commercial immunoassays [7, 8], the stimulant methylenedioxypyrovalerone (MDPV) was reported to cross-react in the CEDIA phencyclidine test [9]. A study from the authors’ laboratory comprising 45 NPS confirmed that several possessed chemical similarities leading to high cross-reactivity in the immunochemical screening tests commonly employed in routine urine drug testing [10]. The detectability of NPS observed to possess cross-reactivity was further confirmed by analysis of urine specimens from authentic intoxication cases included in the STRIDA project (Table 1). Given a more widespread use of new drugs among individuals subjected to drug testing, an increased number of unconfirmed positive screening results may occur.
The cross-reactivity for NPS in current screening assays may be seen as a problem or as a possibility to detect more substances. One possibility for improved drug testing is to include the most common new substances in the confirmation methods. As ecstasy became established as an illicit drug, new immunochemical screening tests for amphetamine/methamphetamine were developed that also included MDMA and MDA. Authentic case samples were used to demonstrate the capability of several commercial amphetamine class screening tests to detect MDMA/MDA. At that time, cross-reactivity towards the new ‘amphetamine’ analytes was wanted [5]. With the advent of the large number of NPS, both legal and illegal, the strategy to also cover new substances in the screening assays for classical narcotic drug substances may not be feasible. For example, the multitude of new synthetic cannabinoids (‘spice’) have not been incorporated in screening tests for THC, but resulted in the development of new independent tests [11].

One approach put forward to understand the potential of immunoassays to detect NPS is to use molecular similarity models [12]. Interestingly, the work of Petrie and co-workers [13] included such a molecular modelling method to predict the cross-reactivity of 261 amphetamine-like compounds. However, when comparing the theoretical data with our experimental data for one compound, the predicted reactivity for butylone was 10 times lower than that observed. In a more recent publication, it was proposed that molecular similarity models could be used to design new immunoassays with sensitivity for a larger number of target compounds [14].

NPS analysis by mass spectrometry
Another analytical strategy to cover NPS in drug testing is to employ MS-based ‘screening’ methods. As part of the STRIDA project, a multicomponent analytical MS method for NPS analysis in urine and serum specimens has been developed [15]. The method uses MS in combination with liquid chromatography (LC-MS/MS in selected-reaction monitoring mode) and is continuously updated as new NPS appear. There are also other methods for multicomponent screening of drugs in urine and plasma/serum, which proves that this technology can be employed in routine drug testing [16].

The LC-MS/MS technique has great potential for drug testing and for clinical laboratories in general. There are examples of laboratories that have already successfully replaced immunoassay screening by MS methods, also for the conventional drugs of abuse [17]. One way to make this possible and cost-effective is to use simple sample preparation procedures, e.g. a simple dilution of urine with internal standards [16]. When studying the cross-reactivity of 30 NPS in commercial ELISA tests for serum and blood, only a few were found to display cross-reactivity, and it was therefore proposed that MS methods should be used in future drug screening [18]. One attraction of MS-based screening is that accurate results are already obtained from the initial analytical step, which may be especially important in cases of acute intoxication (Fig. 2).

Potential of high-resolution MS
One promising technique for drug screening is high-resolution MS (HRMS) [19]. In the HRMS technique, the acquisition of data can be made with an untargeted design. Thousands of substances can be monitored at the same time without the need for optimizing MS parameters for each compound. In addition, new compounds can be searched for retrospectively.

Conclusion
The NPS present a challenge for drug testing laboratories and calls for novel drug screening strategies. It is likely that the current broader spectrum of abused psychoactive drugs will persist in at least in the foreseeable future. This new drug situation has put the performance of drug testing into focus and indicates that drug testing laboratories will play a more important role, as on-site drug screening using dipsticks is likely to lose significance.

References
1. Lewin AH, Seltzman HH, Carroll FI, Mascarella SW, Reddy PA. Emergence and properties of spice and bath salts: A medicinal chemistry perspective. Life Sci. 2014; 97: 9–19.
2. EMCDDA. New psychoactive substances in Europe. An update from the EU Early Warning System (March 2015). 2015. Available at: http://www.emcdda.europa.eu/attachements.cfm/att_235958_EN_TD0415135ENN.pdf.
3. Helander A, Bäckberg M, Hultén P, Al-Saffar Y, Beck O. Detection of new psychoactive substance use among emergency room patients: results from the Swedish STRIDA project. Forensic Sci Int. 2014; 243: 23–29.
4. Helander A, Bäckberg M, Beck O. MT-45, a new psychoactive substance associated with hearing loss and unconsciousness. Clin Toxicol. 2014; 52(8): 901–904.
5. Hsu J, Liu C, Hsu CP, Tsay WI, Li JH, Lin DL, Liu RH. Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens. J Anal Toxicol. 2003; 27: 471–478.
6. Apollonio LG, Whittall IR, Pianca DJ, Kyd JM, Haher WA. Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using Bio-Quant direct Elisa presumptive assays for amphetamine and methamphetamine. J Anal Toxicol. 2007; 31: 208–213.
7. Kerrigan S, Mellon MB, Banuelos S, Arndt C. Evaluation of commercial enzyme-linked immuno assays to identify psychedelic phenethylamines. J Anal Toxicol. 2011; 35: 444–451.
8. Bell C, George C, Kicman AT, Traynor A. Development of a rapid LC-MS/MS method for direct urinalysis of designer drugs. Drug Test Anal. 2011; 3: 496–504.
9. Macher AM, Penders TM. False-positive phencyclidine immunoassay results caused by 3,4-methylenedioxypyrovalerone (MDPV). Drug Test Anal. 2012; 5: 130–132.
10. Beck O, Rausberg L, Al-Saffar Y, Villen T, Karlsson L, Hansson T, Helander A. Detectability of new psychoactive substances, ‘legal highs’, in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse. Drug Test Anal. 2014; 6: 492–499.
11. Arntson A, Ofsa B, Lancaster D, Simon JR, McMullin M, Logan B. Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens. J Anal Toxicol. 2013; 37: 284–290.
12. Krasowski MD, Pizon AF, Siam MG, Giannoutsos S, Iyer M, Ekins S. Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine. BMC Emerg Med. 2009; 9: 5.
13. Petrie M, Lynch KL, Ekins S, Chang JS, Goetz RJ, Wu AHB, Krasowski MD. Cross-reactivity studies and predictive modeling of “Bath Salts” and other amphetamine-type stimulants with amphetamine screening immunoassays. Clin Toxicol. 2013; 51: 83–91.
14. Krasowski MD, Ekins S. Using cheminformatics to predict cross reactivity of “designer drugs” to their currently available immunoassays. J. Cheminform. 2014; 6: 22.
15. Al-Saffar Y, Stephanson NN, Beck O. Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine – experience from the Swedish population. J Chromatogr B 2013; 930: 112–120.
16. Beck O, Ericsson M. Methods for urine drug testing using one-step dilution and direct injection in combination with LC-MS/MS and LC-HRMS. Bioanalysis 2014; 6 : 2229–2244.
17. Eichhorst JC, Etter ML, Rousseaux N, Lehotay DC. Drugs of abuse testing by tandem mass spectrometry: A rapid, simple method to replace immunoassays. Clin Biochem. 2009; 42: 1531–1542.
18. Swortwood MJ, Hearn WL, DeCaprio AP. Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays. 2014; 6: 716–727.
19. Maurer HH. What is the future of (ultra) high performance liquid chromatography coupled to low and high resolution mass spectrometry for toxicological drug screening? J Chromatogr A 2013; 1292: 19–24.

The authors
Olof Beck*^1,3 PhD and Anders Helander^2,3 PhD
¹Department of Clinical Pharmacology, Karolinska University Laboratory Huddinge, Sweden
²Department of Clinical Chemistry, Karolinska University Laboratory Huddinge, Sweden
³Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden

*Corresponding author
E-mail: olof.beck@karolinska.se

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.

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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