DIALOG
C hromSystemS
®
2014/2
MassTox TDM Series A for LC-MS/MS:
Install and Measure the Same Day
®
Dr habil Richard Lukačin, Chromsystems GmbH
Easy access to a complete test menu for therapeutic
drug monitoring
Determination of the active substance concentration of a
drug in blood, serum, plasma or urine (Therapeutic Drug
Monitoring, TDM) is an inherent part of medical laboratory
analyses. TDM is particularly useful in the case of drugs
that exhibit only a narrow therapeutic range and where
underprovision as well as overdosing must be avoided, such
as some antiepileptics, antiarrhythmics and certain psychotropic drugs. The treating physician is thereby provided with
a point of reference for the appropriate dosage of these
substances, with a particular focus on the patient-specific
pharmacokinetics. Also not to be forgotten in this context are
drug interactions, which play an important role in geriatrics,
as such patients are frequently treated with a large number
of medications at the same time.
Various techniques are available for the quantification of
drugs in the previously mentioned matrices, though mass
spectrometry has been established as the “gold standard”
and is recognised as such among experts. In practice, however, only few drugs can be isolated through a single sample
preparation and then be determined by mass spectrometry.
Furthermore, and particularly in the case of multiple drug
administration, characterised by the occurrence of a large
number of metabolites, interferences must be expected.
Although considerably minimised through the technology
applied, interferences can nevertheless occasionally occur
potentially resulting in false estimations of the drug concentration in the investigated matrix. This in term can lead to
incorrect treatment of patients.
In order to satisfy the demands of an overall pharmacotherapeutic concept, within a routine laboratory setting, a
universal method for the determination of drug concentrations
should be available that fulfils the following requirements:
1)It must be possible to process the large majority of laboratory drug determinations using a universal sample
preparation, followed by sample analysis using the same
method aligned with the “gold standard“. Maintaining
identical conditions leads to a maximum reduction of
pressure on laboratory personnel.
2)The method used must be verified and validated in
accordance with the IVD directives of various countries,
including verification of the method robustness and
reliability. Whenever possible the validation should be
carried out externally. Consequently, customers’ work for
a performance check of the method can be reduced to
a minimum, if required at all.
3)Measurement errors caused by interfering components
must be excluded as far as possible through the use of
multi-point calibrators and controls of human origin [1]
k readers
o
o
-b
e
e
v
fi
f
o
e
Win on
as well as isotope-labelled internal standards. To enable
assessment of method accuracy and precision, enrolment
in quality control schemes from external providers should
be undertaken, as well as internal and external method
comparisons made. For establishment of target values
and method performance, certified reference material
should be used whenever possible.
4)Further development of the method should be ensured, i.e.
the integration of additional new parameters. However,
development must not lead to impairment of the overall
system and the general performance of the applied
method in the process. Ongoing maintenance of the
methods and the technology with short reaction times
must be ensured at any time.
Conclusion
The concept demanded in the foregoing is currently fulfilled
by only one single mass spectrometry method that is used
worldwide in various private and hospital laboratories.
MassTox® TDM Series A, the three-component system from
Chromsystems, is able to measure 158 drugs of various
classes. This complete solution consists of the BASIC Kit A,
which contains all necessary materials and reagents for
the sample preparation as well as the mobile phases,
MasterColumn® A, a single analytical column for the chromatography and finally parameter sets, which specify
the drug category to be analysed (Fig. 1).
Each parameter set, of which there are 13, consists of
multi-point calibrators, controls and internal standards.
To fulfil the requirement for universal methodology, the
sample preparation procedure for all parameter sets is
identical. Furthermore, there are only marginal changes
for the different analytes required. For example, injection
volume or gradient may need to be modified just once for
the specific parameter set and can then remain unchanged.
Unique is also the fact that during set-up and installation
of the method, experts from Chromsystems Scientific Support Team are available whose success rate with regard
to installations and fault rectification is almost 100 %. It
is also worth mentioning that further development of the
recently introduced parameter sets has already begun in
accordance with customer requests, but also with regard
to improvements in the performance of the method. A new
version of the PARAMETER Set Neuroleptics 2 has just been
released and renamed Neuroleptics 2/Extended (NL2/XT)
in order to avoid confusion. The NL2/XT Set has been extended by 6 analytes to 13, and a considerable increase
in the security of quantification has been achieved through
the implementation of 9 additional isotope-labelled internal
standards, these rising now to 11 from previously 2 (Fig. 2).
Further extensions and additions to the parameter sets are
planned, and the sets Antidepressants 1 and Neuroleptics 1
Page 1–2
MassTox® TDM Series A for LC-MS/MS:
Install and Measure the Same Day
Dr habil Richard Lukačin, Chromsystems GmbH
Page 3–5
Therapeutic Drug Monitoring of Antiepileptic Drugs during Pregnancy
Prof Dr James C. Ritchie, Emory University
School of Medicine, Atlanta, USA,
Dr habil Richard Lukačin, Chromsystems GmbH
Page 6–8
Determination of Methylphenidate and
Ritalinic Acid in Serum and Saliva of
Patients with ADHD
Cand med Sophie Studer,
Prof Dr Hans-Willi Clement,
Prof Dr Christian Fleischhaker,
Prof Dr Eberhard Schulz,
University Hospital Freiburg, Neuropharmacological Research Laboratory, Freiburg
Page 9–10
TDM on a Shimadzu 8040 LC-MS/MS
Instrument: An Installation Report
Assist Prof Željko Debeljak, University Clinical
Hospital Center Osijek, Croatia,
Faculty of Medicine, J. J. Strossmayer University of Osijek, Croatia
Page 11
Product News: MassChrom® Steroid Kit
Page 12
News/Calendar/Quiz/Imprint
DIALOG 2014/2
Page 2
are currently being extended by further important metabolites. In order to enable continuous refinement of the concept
even further, customer feedback is necessary and highly
regarded. Concept development also includes a solution for
automation of the MassTox® TDM Series A, which has already
been realised for the PARAMETER Sets Antiepileptics and
Mycophenolic Acid. The following pages include customer
reports that illustrate their experiences with the described
system in finer detail.
BASIC Kit A
Reference
[1] Yüksekdağ N, Lukačin R. (2013) Labor: Tierisches Ausgangsmaterial ist nur zweite Wahl. Dtsch Ärztebl 110(18): A–856.
MasterColumn® A
PLUS
BASIC Kit A consists of:
•
•
•
•
Mobile Phase 1
Mobile Phase 2
Precipitation Reagent
Extraction Buffer
•
•
•
•
Analytical column:
equilibrated, with test chromatogram
Dilution Buffer 1
Dilution Buffer 2
Rinsing Solution
Reaction vials
PLUS
Single
Single PARAMETER
PARAMETER Sets
Sets
Antiarrhythmic Drugs
Antidepressants 2/
Psychostimulants
Antiepileptic Drugs
Antidepressants 1
Benzodiazepines 1
Anti-HIV Drugs
Antimycotic Drugs
Neuroleptics 2/EXTENDED
Mycophenolic Acid
Benzodiazepines 2
Tricyclic
Antidepressants TCA 2
Tricyclic
Antidepressants TCA 1
Neuroleptics 1
Components of each PARAMETER Set: Multilevel Plasma Calibrator Set (3PLUS1® or 6PLUS1®)
+ MassCheck® Plasma Control, Level I and Level II
+ Internal Standard
Figure 1: MassTox® TDM Series A.
The system works on the principle of a three-part modular kit system:
1) The BASIC Kit A provides all basic components for the sample preparation and the mobile phases for the chromatography.
2) MasterColumn® A, a single analytical column for analysis of all 158 analytes.
3) 13 individual parameter Sets, defining the drugs to be measured with the specific multi-point calibrators, controls and internal standards.
Neuroleptics 2/EXTENDED
Group 1
  Neuroleptics 2/EXTENDED
Group 2



 


Figure 2: Chromatograms for the MassTox® TDM Series A PARAMETER Set Neuroleptics 2/EXTENDED.
The specific set comprises the 13 analytes amisulpride, chlorprothixene, levomepromazine, melperone, perazine, pipamperone, promethazine, sertindole, sulpiride, thioridazine, ziprasidone, zotepine and zuclophenthixol.
The two chromatograms show as an example all 13 analytes as well as the 11 internal standards.
DIALOG 2014/2
Page 3
Therapeutic Drug Monitoring of Antiepileptic Drugs
during Pregnancy
Prof Dr James C. Ritchie1, Dr habil Richard Lukačin2
1Emory University School of Medicine, Department of Pathology & Laboratory Medicine, Atlanta, USA; 2Chromsystems GmbH
Introduction
Epilepsy is the most frequent neurological disorder worldwide with a prevalence of approximately 0.5 % in Western countries. Around one quarter of the individuals with
epilepsy are women of reproductive age, with majority of
neuropsychiatric illnesses having onset prior to family planning. In the United States there are > 4,000,000 deliveries
annually and it is estimated that > 50 % of these births are
due to inadvertent conception. This means that every year
> 20,000 of the pregnant women have epilepsy, and most of
them are taking antiepileptic drugs (anticonvulsants, AEDs)
for adequate control of their seizures.
In general, women with epilepsy have decreased fertility
resulting from lower pituitary & ovarian hormone levels,
elevated LH/FSHα levels with inadequate ovarian responses,
and increasing SHBGβ concentrations due to the enzymeinducing effects of some AEDs [1]. Paradoxically some
AEDs can also diminish the efficacy of oral contraceptives
and increase the risk of unplanned pregnancy by lowering
ethinyl estradiol and levonorgestrel levels, increasing the
binding by SHBG, and decreasing hyperandrogenism in
women (Table 1).
concentrations during pregnancy can lead to changes in
free drug concentrations and increased hepatic extraction
of drugs. Elevated cardiac output during pregnancy can
increase hepatic blood flow accompanied by enhanced
drug elimination. Furthermore, increased renal blood flow
can result in a higher glomerular filtration rate, which can
cause an increased renal clearance of unchanged drug.
Finally, pregnancy can alter the activity of Cytochrome P450
3A4 (CYP3A4), which can affect the systemic absorption
and/or hepatic elimination of up to 50 % of drugs.
extraction procedure as well as common mobile phases.
We also performed assay comparisons with five commercially available immunoassays (Fig. 3). Finally, for each
drug we plotted the dose to plasma concentration curve
and calculated the apparent clearance and relative clearance. Examples for lamotrigine and topiramate are shown
in Figure 4.
Indeed, recent clinical studies have revealed that physiologic changes during the different stages of pregnancy
may lead to altered pharmacokinetics (especially altered
clearance) for the AEDs and broad individual variations,
which can result in difficulty predicting appropriate drug
dosages throughout pregnancy [5] (Fig. 1).
The precision of the Chromsystems LC-MS/MS method was
investigated in our laboratory (Table 2). We were unable to
obtain samples for all AEDs, as they were not in common
use in our clinic or there were not comparable commercial
assays available. Both the intra- and interassay variance were
in acceptable range for all the compounds tested. Figure 4
shows the comparison of the LC-MS/MS assay results for
levetiracetam, lamotrigine, 10-OH-carbazepine, topiramate
and lacosamide to those of our reference laboratory, which
use various commercial immunoassays. Correlation coefficients (R2) were in our acceptable range and no values were
under 0.8. There was some constant bias in the comparisons
for 10-OH carbazepine and topiramate, which is most likely
due to differences in the standardisation of the respective
For all of the above reasons, TDM for AEDs should play an
important role in the management of patients who must
take these medicines and who become pregnant. Here,
we describe the measurement of a wide variety of AEDs
in two groups of pregnant women (epileptics and bipolar
patients) using the MassTox® TDM Series A Modular System
from Chromsystems [6].
Results
Table 1: Several AEDs effect hormonal contraceptive agents.
AED Effects on Hormonal Contraceptive Agents
No Significant Effects
• Phenobarbital
• Phenytoin
• Carbamazepine
• Primidone
• Topiramate (> 200 mg)
• Oxcarbazepine (> 1200 mg)
• Ethosuximide
• Gabapentin
• Valproate
• Lamotrigine
• Levetiracetam
• Zonisamide
Drugs are generally contraindicated in pregnancy. However, pregnant women are often unable to stop AEDs
due to increased risk of seizures, risk of self-injury, high
risk of miscarriage, cognitive decline, and possible loss of
driving privileges. Status epilepticus has a 30 % maternal
mortality rate and a 50 % fetal mortality rate [2, 3]. On
the other hand, it is also well known that fetal drug exposure to some older AEDs (e.g. valproic acid) increases the
risk of congenital malformations. AEDs may also cause
decreased fetal growth, neonatal hemorrhage, decreased
viability, increased fetal loss and infant mortality as well
as developmental delays. The above concerns often lead
to non-adherence with medications, which can increase
seizure frequency and put the mother’s life at risk [4].
Thus, expectant mothers and their physicians are faced with a
clinical dilemma: In pregnant women with epilepsy, seizures
must be prevented as they carry high risks for mother and fetus. However, fetal exposure to AEDs must also be minimised.
Additionally it should be noted that AEDs are also used for
the treatment of a broad range of other medical conditions
such as bipolar disorders, cancer, neuropathic pain, anxiety
disorders and migraines. Young women with these disorders face similar decisions should they become pregnant.
It is well known that pregnancy produces many physiological
changes, which can alter drug ADMEγ profiles. Changes in
total body water and extracellular fluid can lead to altered
drug distribution. Moreover, decreases in serum albumin
500
Apparent Clearance (mg/mg/l)
Lower Hormone Levels
400
300
200
100
0
0
5
10
15
20
25
30
35
0
5
10
Pregnancy –––––––– WEEKS –––––––– Postpartum
Figure 1: Maternal apparent clearance of lamotrigine during pregnancy.
The data show a progressive increase in lamotrigine clearance throughout pregnancy, reaching a peak of more than 330 % of baseline clearance
by week 32 gestational age. Clearance then declines and returns rapidly to preconception baseline values in the postpartum period [5].
Methods
We measured serum AED levels once per month throughout
pregnancy in both groups using the commercially available mass spectrometry kit (MassTox® TDM Series A) from
Chromsystems. The assay system is capable of measuring
26 different AEDs utilizing a single set of standards and a
common extraction protocol. The assay was set up on both
a Waters TQD and an AB Sciex 4000 Q Trap LC-MS/MS.
The method features a four-point calibration curve for each
analyte and two mass transitions for each drug (except 2).
There are 18 stable labeled internal standards and three
gradient protocols all with 3.5 minutes run time. The drugs
are separated into 5 different groups based on their chromatographic and ionization characteristics (Fig. 2). For all compounds 50 µl of sample is used and there is a single common
assays. Dose (mg/day) to serum concentration curves for
lamotrigine and topiramate seem to be reasonably linear
with inter-individual variations (Fig. 4). Additionally Figure 4
shows the graphs of the relative clearance of these two
compounds versus the gestation period. For lamotrigine it is
obvious that relative clearance increases in some individuals
starting at approximately the 25th week of gestation and
peaks just after birth. Recently investigations have shown
that approximately 28 % of women have little or no change
in the clearance of lamotrigine during pregnancy [7].
However, the majority of women may experience up to a
234 % increase in clearance during their pregnancy. The
authors speculate that genotypic variations in the activity or
induction of UGT1A4δ could partly explain the varying degrees of enhanced clearance between the two populations.
Although we had a smaller number of subjects, topiramate
DIALOG 2014/2
Page 4
relative clearance also appears to increase in the second
and third trimester. This finding confirms that of Westin et
al. [8]. These Norwegian researchers found a significant
decline of the dose-corrected topiramate serum concentrations during pregnancy using a commercial immunoassay.
Table 2: Intra- and Interassay.
Precision of the Chromsystems AED-assays. Drugs which are included in MassTox® TDM AEDs group 4 were not investigated.
Precision
Group 1
Concentration (µg/ml)
Intraassay (% CV)
Interassay (% CV)
Carbamazepine
3.20
2.50
4.00
Carbamazepine-10,11-epoxide
0.95
4.50
7.20
Carbamazepine-diol
1.10
6.90
8.30
10-OH-Carbamazepine
8.25
4.00
7.50
Oxcarbazepine
0.30
6.25
15.10
Lacosamide
1.98
7.20
11.00
Lamotrigine
3.05
3.10
6.80
Levetiracetam
16.00
3.60
6.65
Gabapentin
4.30
2.52
5.20
Topiramate
3.28
4.60
6.85
Conclusion
This work builds on previous work by our group and others
showing that pregnancy constitutes a special population
when it comes to therapeutic drug monitoring. It clearly
demonstrates that clearance changes during pregnancy
can lead to sub-therapeutic plasma levels of the AEDs.
Additionally AEDs have recently become a major target for
big pharma and the numbers of these drugs is increasing
rapidly. Thus, a multiplexed assay strategy like the Chromsystems MassTox® TDM Series A modular system is ideally
suited for the accurate and timely measurement of these
important therapeutic agents.
Group 2
Group 3
Abbreviations
α
LH
= Luteinizing hormone
FSH
= Follicle-stimulating hormone
β SHBG
= Sex hormone-binding globulin: a glycoprotein that binds to androgen and
estrogen
γ
ADME = absorption, distribution, metabolism, and excretion
δ
UGT1A4 = an isozyme of the UDP glucuronosyltransferase 1 Family, Polypeptide A4
Group 5
Zonisamide


­
9.30
2.50








5.10





1
2
3
4
5
6
7
8
9
10
Gabapentin
Pregabalin
Sultiam
Tiagabine
Topiramate
Vigabatrin
Internal Standard 9
Internal Standard 10
Internal Standard 11
Internal Standard 12
5
10
2
1

7
3

­

 

4
6
8
9



Figure 2: Chromatograms for the MassTox® TDM Series A PARAMETER Set Antiepileptic Drugs.
The specific Parameter Set for AEDs encompasses 26 drugs in
5 groups. The chromatograms show the peaks for each of the drugs
and the 18 internal standards.
Epilepsy – a Complex Imbalance in the Brain
Epilepsy is a collective term for functional disorders of the brain, with spontaneous bursts of
electrical activity occurring spasmodically in the cortical neurons [1]. Alongside diabetes mellitus and rheumatic disorders, this chronic disease of the central nervous system is one of the
most widespread neurological illnesses and can occur in any human population and at any
age. Approximately 1 % of the entire population suffers from epilepsy. It is also estimated that
there are 2.4 million additional new cases every year [2]. Across species, epilepsy has also
been observed in dogs, cats and other mammals. There are multiple underlying causes for the
occurrence of this disease; however, they are not yet fully understood. Genetic causes have
been observed, as well as brain damage caused by an accident which, in spite of apparent
healing, can result in epileptic seizures.
Regardless of the actual cause, an existing imbalance between neuronal excitation and
inhibition in the brain can lead to one of the many various forms of epilepsy. The disease
is generally seen as a dysfunctional interplay between the neuronal membrane and the
neurotransmitters that are responsible for the signal transmission. Various processes in the
nervous system can deviate from the “normal” and may affect, for example, the neuronal ion channels or the concentrations of endogenous transmitters and modulators.
The result is that the propagation and duration of the neuronal excitation can no longer be
controlled. This manifests itself mostly as excessive firing activity of the neurons, thereby leading
to epileptic seizures.
In order to suppress or avoid this excessive electrical activity in the brain, a wide variety of
medications are now prescribed for treating the various forms of epilepsy, thus allowing many
patients to live a normal everyday life. There are, for example, medications that are used to block
the ion channels, preventing the neurons from firing high-frequency action potentials. Other drugs
inhibit the γ-aminobutyric acid (GABA) receptors by increasing the channel opening frequency
that causes a hyperpolarisation of the neuronal receptors, thereby inhibiting the excitation of
the postsynaptic cells. However, monotherapy fails in at least one third of patients, leading to
the essential use of one or more additional therapeutics. This is particularly true for severe and
more peculiar forms of epilepsy, and above all in the case of children.
References
[1] http://www.gesundheit.de/lexika/medizin-lexikon/epilepsia
[2] http://www.who.int/mental_health/neurology/epilepsy/en/
DIALOG 2014/2
Page 5
Levetiracetam
20
60
Emory Lab (µg/ml)
y = 0.9724x + 1.0297
R² = 0.9901
50
40
30
20
10
0
0
10
20
30
40
50
60
60
y = 1.0612x + 0.4766
R² = 0.9479
15
10
5
0
70
10-OH-Carbazepine
Emory Lab (µg/ml)
70
Emory Lab (µg/ml)
Lamotrigine
0
5
Reference Lab (µg/ml)
10
15
40
30
20
10
0
0
20
10
Reference Lab (µg/ml)
Emory Lab (µg/ml)
15
10
5
0
0
10
40
50
60
y = 0.8779x + 1.2942
R² = 0.9813
y = 1.0495x + 0.8619
R² = 0.8182
20
30
Lacosamide
20
25
20
Reference Lab (µg/ml)
Topiramate
Emory Lab (µg/ml)
y = 1.1265x + 0.3817
R² = 0.9523
50
15
10
5
0
20
0
Reference Lab (µg/ml)
5
10
15
20
Reference Lab (µg/ml)
Figure 3: Assay comparisons.
Regression analysis for five AEDs, comparing the commercial immunoassay undertaken by a reference laboratory with the Chromsystems method at Emory Laboratory.
Correlation coefficients (R 2) were in an acceptable range. Some constant bias for 10-OH-carbazepine and topiramate was observed, most likely due to differences in the assay standardisation.
References
40
y = 0.0121x + 1.4448
R² = 0.2124
30
20
10
0
0
500
[1] Kaplan PW. (2004) Reproductive health effects
and teratogenicity of antiepileptic drugs.
Neurology 63(10 Suppl 4): S13–23.
Topiramate
1000
Concentration (µg/ml)
Concentration (µg/ml)
Lamotrigine
[2] Teramo K, Hiilesmaa V, Bardy A, Saarikoski S.
(1979) Fetal heart rate during a maternal grand
mal epileptic seizure. J Perinat Med 7(1): 3–6.
50.0
y = 0.0341x + 2.1317
R² = 0.5651
40.0
[3]
30.0
20.0
[4] Barrett C, Richens A. (2003) Epilepsy and preg
nancy: Report of an epilepsy research foundation
workshop. Epilepsy Res 52(3): 147–87.
10.0
0.0
1500
Vajda FJ, O‘brien TJ, Hitchcock A, Graham J, Cook M,
Lander C, Eadie MJ. (2004) Critical relationship
between sodium valproate dose and human
teratogenicity: results of the Australian register
of anti-epileptic drugs in pregnancy.
J Clin Neurosci 11(8): 854–8.
0
200
Dose (mg/day)
400
600
800
1000
Dose (mg/day)
[5]Pennell PB, Newport DJ, Stowe ZN, Helmers SL,
Montgomery JQ, Henry TR. (2004) The impact
of pregnancy and childbirth on the metabolism
of lamotrigine. Neurology 62(2): 292–5. Erratum
in: Neurology 2010, 74(24): 2028.
Lamotrigine-Clearance
60.0
50.0
y = 0.1007x + 2.9226
R² = 0.0505
40.0
30.0
20.0
10.0
0.0
-15
-5
5
15
EGA-Weeks
25
35
45
Relative Clearance (L/Kg/Hr)
Relative Clearance (L/Kg/Hr)
[6] Chromsystems Instruction Manual. (2013)
MassTox® TDM Series A Modular System.
Topiramate-Clearance
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
y = 0.0679x 2 + 0.2381x + 1.1512
R² = 0.0479
0
1
2
3
4
5
Trimester
Figure 4: Clearance changes of lamotrigine and topiramate during pregnancy. The dose to plasma concentrations are reasonably linear with inter-individual variations.
For lamotrigine, relative clearance increases in some individuals at approximately week 25 and peaks just after birth. EGA = Estimated Gestational Age.
[7]Polepally AR, Pennell PB, Brundage RC, Stowe ZN,
Newport DJ, Viguera AC, Ritchie JC, Birnbaum
AK. (2014) Model-based lamotrigine clearance
changes during pregnancy: clinical implication.
Ann Clin Transl Neurol 1(2): 99–106.
[8] Westin AA, Nakken KO, Johannessen SI, Reimers A,
Lillestølen KM, Brodtkorb E. (2009) Serum con
centration/dose ratio of topiramate during pregnancy. Epilepsia 50(3): 480–5.
DIALOG 2014/2
Page 6
Determination of Methylphenidate and Ritalinic
Acid in Serum and Saliva of Patients with ADHD
Cand med Sophie Studer, Prof Dr Hans-Willi Clement, Prof Dr Christian Fleischhaker, Prof Dr Eberhard Schulz
University Hospital Freiburg, Department of Child and Adolescent Psychiatry, Psychotherapy and Psychosomatics,
Neuropharmacological Research Laboratory, Freiburg
Attention deficit hyperactivity disorder (ADHD)
What do fidgets and Johnny Head-in-the-Clouds (a fictional
character from a traditional German tale) have in common
with Alexander the Great, Winston Churchill or Benjamin
Franklin? For all of these, a diagnosis of attention deficit
hyperactivity disorder (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 paediatrician 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, and it is
still valid today – hyperactivity, inattentiveness and impulse
control disorder. In order to be able to evaluate these characteristics in a standardised way, Conners developed parent
and teacher questionnaires at the end of the 1960s that are
still used today to investigate hyperactivity symptoms [3].
A vast amount of results from numerous neurophysiological
and neuropsychological research studies on the aetiology
and pathogenesis of ADHD have been published to date.
However, 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.
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), which demonstrably improves the
core symptoms of ADHD [4], is one of the best-researched
paediatric psychopharmaceuticals with long-term clinical
experience. Nevertheless, the “pill for the troublemaker”
is one of the most criticised and controversially discussed
pharmacological products. A frequently mentioned point is
the possible addiction potential of Ritalin®, for which reason
Methylphenidate
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, which can lead to addiction-creating conditions, such as euphoria and hallucinations.
The story of Ritalin® begins at the Swiss company Ciba,
where the psychostimulant was successfully synthesised
and the effectiveness of the substance proven in a selfexperiment. 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
Ritalinic acid
(R)-Amphetamine
O
HO
H
N
H
NH2
Figure 1a: Structural formulas of methylphenidate [(methyl 2-phenyl-2-piperidin-2-yl) acetate], ritalinic acid [(2-phenyl-2-piperidin-2-yl)
acetic acid] and (R)-amphetamine [(2R)-1-phenylpropan-2-amine].
(2S,2‘R)-Methylphenidate
2'
H
NH
H
(2R,2‘S)-Methylphenidate
O
2
HN
H 2'
O
OCH3
H3CO
2
H
(2R,2‘R)-Methylphenidate
NH
H
O
2'
H
(2S,2‘S)-Methylphenidate
2
HN
H 2'
O
OCH3
H3CO
Methylphenidate (Ritalin®)
Today, stimulants such as Ritalin® in combination with psychotherapy and psychoeducation represent the method of
CH3
Figure 1b: Methylphenidate possesses two chiral centres (C2, C2‘). This results in four possible configuration isomers.
2
H
DIALOG 2014/2
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
MPH is derived from amphetamine. Its fundamental structure is based on the phenylethylamine skeleton (Fig. 1a).
Both substances exhibit no hydroxyl groups on the phenyl
ring, facilitating diffusion into the central nervous system.
MPH exhibits two chiral (asymmetric) 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
isomer dexmethylphenidate (Focalin®) is approved, which
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 metabolised renally by carboxylesterase
CES1A1 to pharmacologically inactive 2-phenyl-2-(piperidin2-yl) acetic acid (ritalinic acid). 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
ritalinic acid (RA) possesses only a small pharmacodynamic
activity, or none at all, this fact is of minor significance.
Therapeutic drug monitoring 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, in order to
ensure a high degree of drug safety, a method based on
alternative body fluids for TDM is desirable. Saliva is becomTable 1: Sample preparation.
Page 7
After a brief interim 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. The calibrators and
control materials for the determination of MPH in serum or
plasma were also from Chromsystems. To produce a series
of MPH standards in saliva, saliva from IBL Hamburg was
spiked with MPH hydrochloride from Sigma-Aldrich.
After sample preparation (Table 1), the eluates obtained were
separated chromatographically in an analytical column at a
flow rate of 0.6 ml/min (MasterColumn® A, Chromsystems
GmbH) 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 and 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 [12] (Fig. 3b). 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 interassay study have already
been carried out. The results are summarised 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.
ing 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 under MPH
therapy, 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 the 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.
Conclusions
Materials and methods
In order to carry out an effective pharmacotherapy with few
side effects, it is necessary to establish less-invasive TDM
methods. This applies in particular to sensitive patient groups,
such as children and adolescents who, in comparison to
adults, 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
and less-painful 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 – from 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.
Reagent kit for LC-MS/MS analysis: MassTox® TDM Antidepressants 2/Psychostimulants (atomoxetine, methylphenidate,
mianserin, reboxetine, ritalinic acid, trazodone; Chromsystems Instruments & Chemicals GmbH), methylphenidate
hydrochloride C-II (Sigma-Aldrich), saliva (IBL Hamburg).
Table 2: Saliva was spiked with 25, 30 and 40 ng/ml MPH.
The following interassay values were obtained.
Target
(ng/ml)
Mean
(n = 10)
SD
CV (%)
25
25.4
1.2
4.89
30
30.0
1.7
5.69
40
40.8
2.5
6.08
>Reconstitution of the Internal Standard Mix
>Add 800 µl of Internal Standard Mix to 12 ml of
Precipitation Reagent (= Mixture A).
>Place 50 µl of sample/calibrator/MassCheck® control
into a 1.5 ml reaction vial.
>Pipette 25 µl of Extraction Buffer into the vial
>Briefly mix and incubate for 2 min at room tempera ture (do not centrifuge).
>Add 250 µl of Mixture A and mix for at least 30 s
(vortex).
>Centrifuge for 5 min at 15 000 x g.
>Dilute the supernatant with Dilution Buffer according
to instrument sensitivity.




Figure 2: Example chromatogram of an LC-MS/MS measurement.
The eluates were obtained using the MassTox® TDM Series A PARAMETER Set Antidepressants 2/Psychostimulants in serum/plasma. MPH elutes with a retention time of about 1.25 min and RA of about 1.15 min.
DIALOG 2014/2
Page 8
3A
Serum methylphenidate, 5.50 µg/l
References
Serum ritalinic acid, 195 µg/l
[1] Krause J, Krause KH. ADHS im Erwachsenenalter. Die Aufmerksamkeitsdefizit-/
Hyperaktivitätsstörung bei Erwachsenen. 3. Aufl, Schattauer Verlag Stuttgart (2009).
[2] Kramer F, Pollnow H. (1932) Über eine hyperkinetische Erkrankung im Kindesalter.
Monatsschrift für Psychiatrie und Neurologie 82(1–2): 1–40.
12000
40000
11500
38000
11000
10500
36000
10000
34000
9500
32000
9000
[3] Steinhausen HC. Der Verlauf hyperkinetischer Störungen. In: Steinhausen HC (Hrsg).
Hyperkinetische Störungen im Kindes- und Jugendalter. Kohlhammer Verlag Stuttgart (1995).
[4]Riederer P, Batra A. Neuro-Psychopharmaka. Ein Therapie-Handbuch. 2. neu bear
beitete Aufl, Springer Verlag Berlin, Heidelberg (2006).
30000
8500
28000
7500
26000
7000
24000
Intensity
Intensity
8000
6500
6000
5500
[5] Kappeler T. (2007) Methylphenidat: Basics für die Apotheke. pharmaJournal 10: 4–7.
[6] Kavale K. (1982) The efficacy of stimulant drug treatment for hyperactivity: a metaanalysis. J Learn Disabil 15(5): 280–9.
22000
20000
[7] Schachter HM, Pham B, King J, Langford S. (2001) How efficacious and safe is short
acting methylphenidate for the treatment of attention-deficit. CMAJ 165(11): 1475–88.
18000
5000
16000
4500
4000
14000
3500
12000
3000
10000
2500
[8] Spencer T, Biederman J, Wilens T, Harding M, O‘Donnell D. (1996) Pharmacotherapy
of attention-deficit hyperactivity disorder across the life cycle. J Am Acad Child
Adolesc Psychiatry 35(4): 409–32.
8000
[9] Chiappin S, Antonelli G, Gatti R, De Palo EF. (2007) Saliva specimen: A new labo
ratory tool for diagnostic and basic investigation. Clin Chim Acta 383(1–2): 30–40.
2000
6000
1500
4000
1000
2000
500
0
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
0.0
Time, min
0.2
0.4
0.6
0.8
1.0
1.2
1.6
1.4
1.8
2.0
2.2
2.4
2.6
2.8
Time, min
[10] Marchei E, Farrè M, Pellegrini M, Rossi S, García-Algar Ó, Vall O, Pichini S. (2009)
Liquid chromatography–electrospray ionization mass spectrometry determination
of methylphenidate and ritalinic acid in conventional and non-conventional biological
matrices. J Pharm Biomed Anal 49(2): 434–9.
[11] Marchei E, Farrè M, Garcia-Algar O, Pardo R, Pellegrini M. (2010a) Correlation
between methylphenidate and ritalinic acid concentrations in oral fluid and plasma.
Clin Chem 56(4): 585–92.
3B
Saliva methylphenidate, 19.9 µg/l
[12] Marchei E, Farrè M, Pellegrini M, Rossi S, García-Algar Ó, Vall O, Pacifici R, Pichini S.
(2010b) Pharmacokinetics of methylphenidate in oral fluid and sweat of a pediatric
subject. Forensic Sci Int 196(1–3): 59–63.
Saliva ritalinic acid, 5.68 µg/l
44000
[13] van den Anker JN, Schwab M, Kearns GL. (2011) Developmental pharmacokinetics.
Handbook of experimental pharmacology 205: 51–75.
1300
42000
1250
40000
1200
38000
1150
1100
36000
1050
34000
1000
32000
950
30000
900
28000
850
800
750
24000
Intensity
Intensity
26000
22000
20000
700
650
600
550
18000
500
16000
450
14000
400
12000
350
10000
300
8000
250
200
6000
150
4000
100
2000
50
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
0
0.0
0.2
0.4
Time, min
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Time, min
Figure 3: (A) Chromatogram of a serum sample from a patient with methylphenidate treatment.
(B) Chromatogram of a saliva sample from the same patient.
Extended Parameter Set for the Analysis of Neuroleptics
Chromsystems has improved the TDM Series A PARAMETER
Set Neuroleptics 2. Hereafter, results are ensured by custommade internal standards for virtually all parameters.
The number of analytes has additionally been increased from
6 to a total of 13. Currently, the Neuroleptics 2/EXTENDED
PARAMETER Set (order no. 92914/XT) incorporates the
following analytes: amisulpride, chlorprothixene, levomepromazine, melperone, perazine, pipamperone, promethazine, sertindole, sulpiride, thioridazine, ziprasidone,
zotepine and zuclophenthixol.
The parameter set is part of the modular system MassTox® TDM
Series A that consists of 3 components:
> the MasterColumn® A for the analysis of all 158 parameters,
> the Basic Kit A with mobile phases and sample preparation
material,
> the specific parameter sets.
Careful optimisation of all kit reagents and chromatographic
separation, reduces matrix effects (“ion suppression”) and
increases the robustness of the method.
The use of 11 isotopically labelled co-eluting internal
standards and 3PLUS1® multilevel calibrators ensures
reproducible and reliable measurement of the analytes.
DIALOG 2014/2
Page 9
TDM on a Shimadzu 8040 LC-MS/MS Instrument:
An Installation Report
Assist Prof Željko Debeljak, Department of Clinical Laboratory Diagnostics, University Clinical Hospital Center Osijek, Croatia, Department of Pharmacology, Faculty of Medicine, J. J. Strossmayer University of Osijek, Croatia
Introduction
Depending on available instrumentation and chemical
properties of analytes and matrices, bioanalytical method
development is a challenging task that can be met by using
commercial ready-to-use kits. An example is the LC-MS/MS
based MassTox® TDM Series A analytical solution for the
determination of more than 150 drugs and metabolites from
Chromsystems (Gräfelfing/Munich, Germany). It consists of
a basic kit (BASIC Kit A), an analytical column (MasterColumn® A) and the individual TDM parameter sets containing
the specific calibrators, controls and internal standards. In
addition manuals containing analytical method protocols,
verification and validation performance characteristic reports
are also included [1–7]. Clinical laboratories however host
a range of models of LC-MS/MS instruments from different
manufacturers including AB Sciex (Framingham, MA, USA),
Thermo Scientific (Waltham, MA, USA), Waters (Milford,
MA, USA) or Agilent (Santa Clara, CA, USA), just to mention
a few, and dozens of LC-MS/MS instruments with largely
different designs and performance characteristics are
available. Furthermore, the use of a number of HPLC front-end
systems makes it difficult to create analytical methods that
exactly fit any given LC-MS/MS instrument configuration.
Shimadzu’s LC-MS/MS 8040 model (Kyoto, Japan), on
the market since 2012 [8], was installed in the Clinical
Chemistry Department of University Clinical Hospital Centre
Osijek, Croatia (UCHC Osijek) in June 2013. Shortly after,
the ready-to-use TDM LC-MS/MS kit solution from Chromsystems was ordered for use on this instrument. The high
number of LC-MS/MS tuning parameters for the numerous
target analytes makes the installation of the TDM kit a fairly
complex task, although the Chromsystems manuals include
comprehensive information on the generic values for the
tuning variables. This includes the nominal mass transitions
for every analyte, the respective quantifier and qualifier
as well as the nominal mass transitions for all internal standards. Nevertheless, different LC-MS/MS designs require
different instrument-specific tuning variables that cannot
be covered in complete detail. Therefore, Chromsystems
provides comprehensive installation support and on-site
assistance. This is achieved with the help of their scientific
support department to establish the method on the specific
instrumentation that is available in the laboratory. For the
installation at UCHC Osijek, in addition to the standard
install undertaken by a Chromsystems mass spec specialist,
several method optimisation steps were necessary before
and after the actual installation to reach appropriate performance characteristics for the TDM parameter sets on
the Shimadzu 8040 instrument. The general installation
process as well as some obstacles encountered during the
installation of TDM kits and corresponding solutions are
described in the following text.
Installation of the Shimadzu 8040 LC-MS/MS
instrument
The installation of a LC-MS/MS instrument demands some
special requirements. Among those, special attention should
be paid to a stable supply of power, nebulisation and collision gases as well as to a stable laboratory infrastructure
in general. Although each instrument installation ends with
operational qualification/performance verification (OQ/
PV), unfortunately passing these processes might not always
produce acceptable and sufficient performance characte-
Materials and methods
All results given in this text refer to the Shimadzu 8040 LC-MS/MS instrument. Electrospray ionisation (ESI) has been used
in all cases. Among a number of TDM kits available from Chromsystems the following were implemented on the Shimadzu
8040 model.
Drug classes
Chromsystems’ kit labels
Antidepressants
MassTox® TDM PARAMETER Set Antidepressants 1
Antiepileptics (AED)
MassTox® TDM PARAMETER Set Antiepileptic Drugs
Benzodiazepines
MassTox® TDM PARAMETER Set Benzodiazepines 1 & MassTox® TDM PARAMETER Set Benzodiazepines 2
Mycophenolic Acid
MassTox® TDM PARAMETER Set Mycophenolic Acid
Neuroleptics
MassTox® TDM PARAMETER Set Neuroleptics 1
Where possible, results with the LC-MS/MS TDM kits were compared with the HPLC TDM solutions, also from Chromsystems,
that were previously installed on a Shimadzu’s LC-10 series HPLC instrument. A direct method comparison was possible in the
case of Benzodiazepines/Tricyclic Antidepressants and AED kits.
ristics. This was in fact observed during the installation of
the MassTox® Series A Kit on the Shimadzu 8040 instrument
at UCHC Osijek. The instrument failed to pass standard
sensitivity tests as designed by the manufacturer while
other OQ/PV tests provided acceptable results. Sensitivity
was approximately five times lower than anticipated, with
sensitivity being one of the key properties for an LC-MS/
MS instrument. In this described case, reduced sensitivity
would have significantly decreased the number of analytes
that could be analysed with the Chromsystems TDM kit.
Several environmental, chemical or instrumental parameters
may also cause reduced sensitivity. With the help from
Chromsystems’ and Shimadzu’s specialists, potential causes
were systematically checked and it was found that the source
of reduced sensitivity was an unstable power supply with a
fluctuating voltage. The introduction of a stabilised power
supply reduced the observable noise level by a factor of
five and also enabled successful measurements of analytes
with low concentrations.
Tuning and method installation
Ideally, mass transitions found on a specific MS platform
should be transferable to all other MS platforms. LC-MS/MS
manufacturers have developed a range of different architectures for their specific instruments. Unfortunately, this leads
to a number of differences between these systems, including
ion generation, ion guidance through the system and collision
with the inert gas as well as ion detection at the detector.
In addition, the mass axis can be shifted away from an
“ideal” mass calibration. Therefore, it is virtually impossible to directly copy one method established on a certain
instrument to another instrument. Mass axes have to be
adjusted, source parameters optimised and collision energies determined. Furthermore, different manufacturers use
different collision gases that potentially alter the gas phase
chemistry in the collision cell, making it impossible to give
general collision energies for all available collision cells.
These variations call for a careful and critical installation, preferably by manual tuning and method installation.
Within the MassTox® Series A manual Chromsystems provides
2 or 3 MRM options, depending on the TDM parameter
set, that can be installed to meet the user‘s demands. The
use of the quantifier MRM is preferable. In most cases the
first given mass transition yields the highest intensity. In the
unfortunate situation where there is an interference caused
by an instrument contamination, a switch from the quantifier
MRM to another, so called qualifier MRM, may be feasible.
In the UCHC Osijek case, such a change was performed
during the installation of phenytoin. The first given MRM for
phenytoin produced too low signals on the 8040 instrument.
In order to solve this sensitivity issue the second given mass
transition was successfully used as the quantification trace.
Collision energies, Q1 and Q3 biases
Automated optimisation of collision energies, Q1 and Q3
biases that are offered by the Shimadzu 8040 instrument
might be considered as an advantage of this instrument
and its software over others. Simple use of tuning mixes
along with this option provides fast optimisation of these
three factors. However, automated tuning always hampers
the direct control of the instrument’s tuning results. If signal
intensities are low, optimisation results may be dubious or
even completely wrong. High signal intensities might lead
to an overloading of the detector and to falsely determined
settings. Critical inspection of all optimised instrument settings
is therefore mandatory.
Nitrogen flow rates and ESI voltage selection
8040 LC-MS/MS instrument uses nitrogen as the drying and
nebulising gas needed for proper desolvation of molecular
ions and removal of excess mobile phase molecules in the
ESI source. Drying and nebulisation flow rates directly affect
sensitivity and precision. While highest flow rates ensure good
nebulisation and drying, they may lead to a turbulent gas
flow and lower sensitivity and precision. Therefore, optimal
nitrogen flow rate selection is an important part of routine
tuning that is undertaken by the Chromsystems specialists.
During the TDM kit installation at UCHC Osijek, nebulising
gas flows between 2 and 3 l/min and drying gas flows
between 5 and 10 l/min ensured the best performance.
Lower nebulisation flows, although allowed by manufacturer, may lead to disappearance of the analyte signal.
DIALOG 2014/2
Another process requiring optimisation during TDM kit installation is the ionisation itself. Too low ionisation voltages
may lead to loss of signal while too high values may cause
discharges and (depending on the polarity of the ionisation
mode) chemical reduction or oxidation of some analytes.
In the long run, too high capillary voltages may also lead
to a deterioration of the ESI capillary and subsequently to
signal losses due to decreased ionisation efficiencies. The
best performing capillary voltages for the 8040 LC-MS/
MS with the different Chromsystems TDM kits fall in the
range of 1 to 4 kV.
Page 10
Table 1: Comparison of dilutions and injection volumes used with the Chromsystems TDM AED Parameter Set on an AB Sciex API 4000 and
our Shimadzu 8040 instrument.
Chromsystems AED kit
Dilutions recommended for
AB Sciex API 4000
at 10 µl injection volume [3]
Optimal dilutions and injection volumes
for Shimadzu 8040
Group 1
1:20
1:5 at 10 µl
Group 2
1:20
1:5 at 10 µl*
Group 3
1:5
1:5 at 5 µl
Group 4
1:5
1:3 at 50 µl
Group 5
1:2.5
1:3 at 50 µl
*Analysis of theophylline requires 1:3 dilution and 50 µl injection volume.
Dwell time
LC-MS/MS combines a continuous (LC) and a discontinuous
(MS/MS) method. While the stream of mobile phase is
continuously coming from the column eluting the analytes,
the standard triple quadrupole mass spectrometer is not
able to measure every single mass – let alone every mass
transition – at exactly the same time. Triple quadrupole
mass spectrometers therefore split their measurement time
into periods. During such a period, all of the instrument’s
parameters are set to a certain set of values allowing a
specific mass transition to be monitored. This so-called dwell
time needs to be adjusted during kit installation. While short
dwell times allow the following of a multitude of MRMs in
a short period of time, at the same time they reduce the
signal-to-noise ratio of an analyte’s peak. Elongated dwell
times lead to an improved signal-to-noise ratio; however,
they also limit the number of MRMs that can be monitored.
Depending on the elution times of the analytes, the right
balance between too short and too long dwell time needs
to be established.
Dilutions and injection volumes
After the installation of a new method, special attention should
be paid to the right choice of injection volume and dilution
of the eluates of the individual MassTox® TDM Series A Parameters. Different analytes have different concentrations as
well as different ionisation efficiencies. Therefore, calibrators,
controls and patient samples should be diluted in a way to
ensure they are within the linear detection range of the mass
spec’s detector, with respect to each analyte’s response. Instrument sensitivity determines the limit of detection (LOD) and
limit of quantitation (LOQ) of a certain analyte, but a high
response, for example of a high level calibrator, might lead
to detector saturation. The right balance between dilution
and injection volume must therefore be found. High dilution
might reduce possible matrix effects and therefore increases
sensitivity [9], however, high dilutions usually go hand in
hand with large injection volumes. Shimadzu’s injectors are
limited to an upper injection volume of 50 µl under standard
settings, limiting the possible degree of dilution. In addition,
overloading the Chromsystems TDM MasterColumn® A with
injection volumes larger than 50 µl is not recommended.
Overloading the column can lead to poor chromatographic
separations. Tailing and/or poor separations of the peak
might be the result of too large injection volume. In instances
where an analyte requires a low dilution, possible adverse
effects of that dilution can be compensated by choosing
a low injection volume. Depending on the injector system,
even with very low injection volumes (e.g. 0.5 µl) it is still
possible to achieve high injection precisions.
When looking at the large number of analytes of the
MassTox® TDM Series A Antiepileptic Drugs Parameter Set,
and with such a broad range of concentrations in real-life
samples, it became clear that with the Shimadzu 8040 instrument it would not be possible to fit all analytes with one
dilution and one injection volume into the linear detection
range. Although Chromsystems’ manuals offer a “dilution
guideline”, the individual scheme of dilutions and injection
volumes had to be determined for the specific instrument
(Table 1). It is interesting to note, that even though the
mass spec manufacturers give some guidance whether
one instrument is comparable to another from a different
manufacturer (e.g. an AB Sciex API 4000 is supposed to
be equally sensitive as a Shimadzu 8040), a direct comparison often leads to different results as can be seen by
the comparison of eluate dilution and injection volumes.
Peak integration and data analysis
Even though automated integration, quantitation and result
processing might be beneficial, data analyses should always
be carefully followed by the user. Automated peak integration
should be adapted for each individual method/kit. Given a
stable kit performance once established, integration settings
usually do not require frequent changes. Integration settings
that need to be manually adjusted during installation of the
TDM parameter set on the Shimadzu 8040 instrument include
slope, minimum area/height, peak width and bandwidth.
Once a set of integration parameters is established data
analysis becomes more efficient as manual peak integration
becomes less frequent.
order to overcome some obstacles with our instrumentation
as it was not possible to “directly copy” the method from the
provided manuals, i.e. due to different instrument architectures
of the LC-MS/MS manufacturers. All Chromsystems TDM parameter sets planned to be installed on our Shimadzu 8040
LC-MS/MS at Osijek hospital also have been implemented.
In order to reach adequate sensitivity, stable signals and
reliable results, several prerequisites had to be fulfilled,
such as a stable voltage and gas supply. Careful method
optimisation including all necessary tuning and instrument
adaptation steps followed. There were no sensitivity problems
with Chromsystems TDM Series A parameter Sets using
our Shimadzu 8040 LC-MS/MS.
Analytical performance: a quick test
Measured factor
Acceptance limit
Additional information
Recovery
80–120 %
2 control levels*
Regression coefficient
> 0.99
4 calibration levels
Relative standard deviation of the
internal standard peak areas
< 20 %
isotopically labelled internal standards
* All calibration and control materials were provided for each Chromsystems parameter set.
Measures given in the table do not represent complete bioanalytical method validation criteria. Rather, they should be considered as a system suitability test. It is generally recommended that such a test is not only performed after an installation of a
new parameter set or a new method, but after each series of daily measurements. In addition to these basic quality indicators,
signal-to-noise-ratios, peak shapes and the stability of analytes’ retention times should always be monitored.
Method comparison as part of an in-house validation:
LC-MS/MS vs. HPLC
At UCHC Osijek, some analytes like AEDs and benzodiazepines can be determined by either LC-MS/MS or HPLC.
Results with RIQAS (Belfast, UK) external quality control
materials obtained by HPLC and LC-MS/MS have been
compared and no major differences were detected. In some
instances, interferences were detected during LC-MS/MS
analysis, presumably caused by different additives. Considering less expensive instrumentation, HPLC at first glance
looks more appealing. However, not all analytes that can
be determined by LC-MS/MS are also achievable by HPLC.
In addition, the level of specificity and selectivity obtained
by LC-MS/MS analyses is by far greater than obtained by
standard HPLC analyses. Furthermore, the speed of the
LC-MS/MS analytical run is much higher, resulting in a
potentially higher throughput in the laboratory.
Conclusion
The method has been successfully established in our laboratory after all installation steps. These steps were necessary in
References
[1] Chromsystems. (2013) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A BASIC Kit.
[2] Chromsystems. (2012) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A Antidepressants 1 in Serum/Plasma.
[3] Chromsystems. (2013) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A Antiepileptic Drugs and Metabolites in Serum/Plasma.
[4] Chromsystems. (2012) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A Benzodiazepines 1 in Serum/Plasma.
[5] Chromsystems. (2012) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A Benzodiazepines 2 in Serum/Plasma.
[6] Chromsystems. (2013) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A Mycophenolic Acid in Serum/Plasma.
[7] Chromsystems. (2012) Instruction Manual for the LC-MS/MS Analysis of MassTox® TDM
Series A Neuroleptics 1 in Serum/Plasma.
[8] Shimadzu Corporation. (2012) Liquid Chromatograph Mass Spectrometer, LCMS-8040.
[9] Li M, Alnouti Y, Leverence R, Bi H, Gusev AI. (2005) Increase of the LC–MS/MS sensitivity
and detection limits using on-line sample preparation with large volume plasma injection.
J Chromatogr B Analyt Technol Biomed Life Sci 825(2): 152–60.
DIALOG 2014/2
Page 11
PRODUCT NEWS
New MassChrom Kit for the Steroid Analysis
by Tandem Mass Spectrometry
®
Steroids are a group of compounds with a distinct chemical structure and can be found in animals, plants and fungi.
Lipoproteins and steroid hormones are synthesised from cholesterol, for example, the hormones of the adrenal cortex
(corticosteroids) along with those of the reproductive organs (androgens, estrogens). Furthermore, several doping agents,
called anabolics, are synthetic products of the male sexual hormone testosterone. Anabolic steroids are commonly used
abusively to increase muscle and bone synthesis.
Androstenedione
Aldosterone
Cortisol
11-Deoxycortisol
Cortisone
Corticosterone
Estradiol
Progesterone
17-OH-Progesterone
Testosterone
Dihydrotestosterone (DHT)
Dehydroepiandrosterone (DHEA)
Dehydroepiandrosterone sulfate (DHEA-S)
As steroid hormones serve different functions, they are consequently involved in a variety of diseases. Inborn or acquired
disorders of steroid hormone metabolism like Cushing’s syndrome, adrenal tumours, hyperaldosteronism, and many more,
highlight the importance of steroid analysis in clinical diagnostics. Several methods can be used; however, tandem mass
spectrometry has several advantages. LC-MS/MS is referred to as the gold standard also for the analysis of steroids and
has multiplexing ability, enabling the analysis of several hormones in one single run. The method can also offer higher
sensitivity and specificity in comparison to other methods.
Chromsystems has developed a complete solution aimed at enabling customers to perform steroid analysis efficiently
without the need of high-end instrumentation. The new kit MassChrom® Steroids in serum/plasma (order no. 72000) allows
the determination of 13 steroids by LC-MS/MS with one column and sample preparation procedure.
The parameters that can be analysed with the kit include the clinically most important steroids. Sample preparation is the
same for both steroid panels, using an optimised and efficient clean up procedure in 96 SPE well plates. The use of stable
isotope-labelled internal standards for every single analyte ensures reproducible and reliable quantification of the steroids.
The MassChrom® Steroid kit is available with the analytical column for all parameters together with 6PLUS1® Multilevel
Calibrator Sets and MassCheck® Controls.




Parameters that can be determined with the MassChrom®
Steroid kit.
The 13 analytes of the MassChrom® Steroid kit are divided into 2 panels.
Specifications
Sample Preparation
Steroid Panel 1
Equilibration
Linearity: covering reference range of each steroid
Limit of quantification: 10–200 ng/l
Intraassay: CV = 1–7 %
Interassay: CV = 4–9 %
Analysis time: 10.5 min
→Equilibrate Steroid 96 SPE plate with 0.8 ml
Equilibration Reagent 1 into each well.
→ Centrifuge 1 min at 200 x g.
→Repeat with 0.8 ml Equilibration Reagent 2.
Steroid Panel 2
→Pipette 500 μl sample/calibrator/MassCheck® control, 50 μl Internal Standard Mix and 450 μl Extraction Buffer into each well of the Steroid 96 SPE plate.
→ Vortex for 2 min at 600 rpm.
→ Add 1 ml Wash Buffer into each well, centrifuge for 1 min at 200 x g and repeat.
Linearity: covering reference range of each steroid
Limit of quantification: 10–10000 ng/l
Intraassay: CV = 1–10 %
Interassay: CV = 2–17 %
Analysis time: 12.5 min
SPE Clean up procedure
→ Centrifuge for 2 min at 2000–3000 x g.
→Place plate onto the steroid collection plate.
→ Add 500 μl Elution Buffer and centrifuge for 1 min at 200 x g.
Clean up and injection
→Place steroid collection plate under nitrogen at 45 °C and evaporate the eluates.
→ Add 100 μl Reconstitution Buffer into each well.
→ Vortex collection plate for 2 min at 900 rpm.
→ Centrifuge 5 min at 3000 x g.
→ Seal collection plate and transfer to autosampler.
→ Inject 10–40 µl into the LC-MS/MS system.
DIALOG 2014/2
Page 12
NEWS
The new HPLC catalogue has arrived!
The new catalogue with more than 180 pages is now available. This compendium of our
entire clinical HPLC product portfolio includes our latest products such as the combined
UHPLC method for the analysis of vitamin A and E, automated sample prep solutions and
methods for simplified sample preparations with premixed tubes.
The catalogue also provides valuable and practical information, including chromatograms, HPLC parameters, ordering information, LOQs, recoveries and analysis times
as well as relevant facts on all our available HPLC controls.
For more information and ordering your individual copy of the HPLC catalogue send
us an email: mailbox@chromsystems.com
QUIZ
CALENDAR
Win one of five e-book readers
Visit Chromsystems at national and international
congresses and fairs:
> 11–14 November 2014
ÖGLMKC, Salzburg, Austria
> 26–29 January 2015
ArabHealth, Dubai, UAE
> 4–6 March 2015
APS 29. Jahrestagung/Annual Conference
2015, Fulda, Germany
> 16–18 April 2015
GTFCh Symposium, Mosbach, Germany
For further upcoming events please visit our website
www.chromsystems.com
IMPRINT
Some products are not available in the USA and
Canada.
Join the quiz by answering 5 questions based on this issue of DIALOG and win one of five e-book
readers.
Entry is open until January 15, 2015. Please forward your answers using fax: +49 89 18930-399,
email: mailbox@chromsytems.com or mail: Chromsystems GmbH, Am Haag 12, 82166 Gräfelfing/Germany.
Question 1: The MassTox TDM Series A consists of which 3 components?
®
Question 2: How many analytes can be measured with the specific MassTox® TDM Series A PARAMETER Set for antiepileptic drugs?
Question 3: How many chiral centres does methylphenidate have?
Question 4: What does the abbreviation ESI mean?
Question 5: Name at least 3 PARAMETER Sets of the MassTox® TDM Series A that are not mentioned in this quiz.
Condition of participation
Any recourse to courts of law is excluded. No cash alternative is available. Chromsystems‘ employees, its partner companies/suppliers,
and their relatives are not eligible for entry in the quiz.
Publisher:
Chromsystems
Instruments & Chemicals GmbH
Am Haag 12
82166 Gräfelfing/Germany
Phone: +49 89 18930-300
Fax: +49 89 18930-399
E-Mail: mailbox@chromsystems.com
Editors:
Dr Marc Egelhofer
Dr habil Richard Lukačin
Dr Nihâl Yüksekdağ
Design:
Fred Lengnick Print- & Media Design
Edition November 2014