Anal Bioanal Chem (2011) 400:26632670

DOI 10.1007/s00216-011-5048-6

ORIGINAL PAPER

Quantitative determination of methylphenidate in plasma

by gas chromatography negative ion chemical ionisation mass
spectrometry using o-(pentafluorobenzyloxycarbonyl)-benzoyl
derivatives
Hans J. Leis & Helmut Schtz & Werner Windischhofer

Received: 22 December 2010 / Revised: 23 March 2011 / Accepted: 19 April 2011 / Published online: 11 May 2011
# Springer-Verlag 2011

Abstract The use of a novel electrophoric derivatisation

reagent, o-(pentafluorobenzyloxycarbonyl)-benzoyl chloride,
for the quantitative determination of methylphenidate in
plasma is described. The drug can be quantitatively measured
down to 72 pg/mL plasma using only 250 L of sample due
to the extraordinary sensitivity of the derivatives under
negative ion chemical ionisation mass spectrometry. Plasma
samples were made alkaline with carbonate buffer and treated
with extraction solvent n-hexane and reagent solution for
30 min, which, after concentration, was measured by GCNICI-MS. The method is rapid as extraction and derivatisation
occur in one single step. A stable isotope-labelled internal
standard was used and its synthesis described. Full validation
data are given to demonstrate the usefulness of the assay,
including specificity, linearity, accuracy and precision, longterm stability, short-term stability, freezethaw stability, stock

Published in the special issue on Analytical Sciences in Austria with

Guest Editors G. Allmaier, W. Buchberger and K. Francesconi.
H. J. Leis : W. Windischhofer
Division of Analytical Biochemistry and Mass Spectrometry,
University Childrens Hospital,
Auenbruggerplatz 34/2,
8036 Graz, Austria
H. Schtz
Consultancy Services for Bioequivalence and Bioavailability
Studies, BEBAC,
Neubaugasse 36/11,
1070 Vienna, Austria
H. J. Leis (*)
Research Unit of Osteology and Analytical Mass Spectrometry,
University Childrens Hospital,
Auenbruggerplatz 34/2,
8036 Graz, Austria
e-mail: hans.leis@medunigraz.at

solution stability, autosampler stability, aliquot analysis,

robustness, matrix effect, and prospective analytical batch
size accuracy. The method has been successfully applied to
pharmacokinetic profiling of the drug after oral application.
Keywords Methylphenidate . GC-MS . Negative ion
chemical ionisation . PBBCl . Derivatisation

Introduction
Attention-deficit hyperactive disorder (ADHD) is a neurobehavioural problem mostly encountered with school-aged
children at a high prevalence of 510% of the general
population [1, 2]. The cyclic amphetamine analogue
methylphenidate [dl-threo-methyl 2-phenyl-2-(piperidyl)
actetate] (MPH) is widely used for the management of
children having ADHD, with or without hyperactivity [3].
The psychostimulant drug displays a high intrinsic clearance
due to the rapid hydrolysis of the methyl ester function [4]
occurring in a stereoselective manner [5]. As a consequence,
the plasma concentrations after oral therapeutic doses
encountered are low, typically within cmax of 1020 ng/mL.
There is an individual variability in the response to MPH
concentrations that makes it necessary to adjust for optimal
medication and elimination of toxicological side effects. This
is of particular importance as children are the main target
group of this medication [6]. Quantification in the lower
picogram per millilitre range is hence desirable for reliable
pharmacokinetic studies with MPH. Besides the treatment of
ADHD, MPH improves attention, concentration, fine motor
coordination and balance, and due to these sport-related
benefits, the drug is considered as a doping agent [7, 8].
MPH is thus not permitted for use in competition by the
International Olympic Committee.

2664

Several analytical methods have been reported for the

determination of MPH in plasma and urine. Highperformance liquid chromatography has been used with
fluorescence detection [9] as well as in combination with
mass spectrometry (MS) [1014] and tandem MS [15].
Heptafluorobutyryl-L-prolyl and trifluoroacety-L-prolyl
derivatives were used for enantioselective measurement of
the drug by GC-MS using either electron ionisation (EI)
[16], positive ion chemical ionisation [17] or negative ion
chemical ionisation (NICI) [17, 18]. Besides that, GC-MS
of pentafluoropropyl derivatives have been used with EI
[19, 20] and positive ion chemical ionisation [21] and, more
recently, capillary electrophoresis ion trap mass spectrometry
[22]. Besides the advantage of enantioselective detection,
none of the methods achieved a limit of quantification
(LOQ) below 0.4 ng/mL. A sensitive method has been
described to detect 70 pg/mL plasma using GC-NICI-MS of
the heptafluorobutyrate [23].
Recently, we have introduced a new derivatisation
reagent, o-(pentafluorobenzyloxycarbonyl)-benzoyl chloride (PBBCl) that readily reacts with primary and secondary
amines [24]. The obtained derivatives display the beneficial
characteristics of a pentafluorobenzyl ester during NICI
detection, resulting in striking sensitivity due to reduced
fragmentation and high efficacy of the dissociative resonance electron capture process. The potential usefulness of
this derivative for ultrasensitive determination of MPH has
been preliminarily demonstrated [24].
The use of stable isotope-labelled analogues as internal
standards comprises a major advantage of mass spectrometric
detection. This standardisation method has previously been
applied to the analysis of MPH [23].
For pharmacokinetic applications, robustness and short
analysis time is a major concern since they involve the
processing of a large a number of samples. Additionally, as
the target group for MPH medication are children,
reduction of sample size is of critical importance. It was
therefore the aim of this study to elaborate a method for the
determination of MPH in human plasma that meets the
requirements of sensitivity, specificity, speed and ruggedness
for pharmacokinetic applications and drug monitoring.

Experimental
Chemicals and reagents
Pentafluorobenzyl alcohol was purchased from ABCR
(Karlsruhe, Germany). Phtalic anhydride was supplied by
Sigma-Aldrich (Vienna, Austria). Methylphenidate was
from Cerilliant (USA). All other substances, solvents and
reagents of analytical grade were from Merck (Darmstadt,
Germany). PBBCl reagent and isotope-labelled standard

H.J. Leis et al.

may be obtained from the corresponding author or via

www.bpa-lab.com. Plasma samples were collected as part
of a pharmacokinetic study and hence under approval of the
corresponding authorities.
Synthesis of o-(pentafluorobenzyloxycarbonyl)-benzoyl
chloride
Synthesis of the PBBCl reagent was accomplished as
previously described [24]. Briefly, phtalic anhydride
(148 mg, 1 mmol) and pentafluorobenzyl alcohol (198 mg,
1 mmol) were dissolved in benzene and allowed to react at
100 C for 2 h in the presence of pyridine. The intermediate
acid was treated with thionyl chloride at room temperature
for 1 h. Excess reagent was removed under nitrogen and the
oily residue dissolved in 10 mL of dichloromethane, yielding
a 10-mM solution of the PBBCl reagent.
Preparation of [18O2H3]-MPH
MPH HCl (10 mg) was dissolved in 800 L H218O and
20 L fuming HCl was added. The vial was closed under
nitrogen and kept at 100 C for 72 h. After cooling, the
mixture was evaporated to dryness under nitrogen. The dry
residue was dissolved in 300 L of [2H3]-methanolic HCl
(prepared by adding 20 L of acetyl chloride to 0.5 mL of
[2H3]-methanol) and left for 1 h at room temperature. The
solvent and reagent were removed under nitrogen and the
dry residue dissolved in acetonitrile. Isotope distribution
was checked by GC-NICI-MS.
Plasma sample preparation and derivatisation
One nanogram of the internal standard [18O2H3]-MPH
(50 L of an acetonitrile working solution) was pipetted
into a 5-mL glass tube and 0.25 mL of plasma was added.
After vortexing, 0.25 mL carbonate buffer (pH 9.0) was
added along with 1 mL of n-hexane and 100 L of PBBCl
reagent solution (1 mM in dichloromethane). It is crucial to
add the internal standard immediately after thawing of the
samples. The mixture was shaken on a rotary shaker for
30 min and phases were allowed to separate upon standing.
The (upper) n-hexane phase was transferred to a fresh glass
tube and solvent-evaporated under nitrogen. The dry residue
was reconstituted in 80 L of ethyl acetate and transferred to
autosampler vials. Two microlitres were used for GC-NICIMS analysis using m/z 380 and m/z 385 for the detection of
MPH and internal standard (IS), respectively.
Gas chromatographymass spectrometry
An ISQ quadrupole mass spectrometer coupled to a
TRACE GC Ultra (Thermo Scientific, Vienna) was used.

GC-NICI-MS of methylphenidate

The GC was fitted with a BPX5 fused silica capillary

column (15 m0.25 mm i.d., SGE). The injector was
operated in the splitless mode at 280 C. Helium was used
as a carrier gas at a constant flow rate of 1.5 mL/min. Initial
column temperature was 160 C for 1 min, followed by an
increase of 40 C/min to 310 C and an isothermal hold of
4 min. The mass spectrometer transfer line was kept at
315 C. NICI was performed with methane as a moderating
gas at an electron energy of 70 eV and an emission current
of 0.250 A.
Analytical method validation
Calibration graphs were established in the range of 0.072
18.25 ng/mL plasma with nine calibration points in
duplicate. For this purpose, blank plasma was spiked with
the appropriate amounts of MPH and serial dilution of the
highest calibrant with blank plasma. Standard solutions of
MPH were prepared in acetonitrile and stored at 20 C.
Calibration curves were calculated by polynomial regression
analysis (quadratic fit) weighting for 1/s (s = standard
deviation of duplicates). For linearity check, five individual
calibration curves were measured and the coefficients of
regression evaluated. Back-calculated values of all measured
calibrants were correlated to their nominal values and the
correlation coefficients calculated.
Inter-day precision was determined at 0.072- (LOQ),
0.210-, 1.5- and 15-ng/mL concentration levels by carrying
five identical samples at each concentration level throughout
the analytical sequence. Spiked samples were prepared from
blank plasma. Intra-day precision was determined at 0.072(LOQ), 0.210-, 1.5- and 15-ng/mL concentration levels by
carrying five identical samples at each concentration level
throughout the analytical sequence. Spiked samples were
prepared from blank plasma. Accuracy of the methods was
also tested at the aforementioned concentrations. Thus, the
data from inter- and intra-day precision measurements were
used to calculate the deviation of the values measured from
the actual spiked values. Specificity was tested by analysing
six different blank plasma samples. Stock solution stability
was tested in acetonitrile and methanol after storage at 20 C.
To measure freezethaw stability, the samples were analysed
immediately after spiking with the indicated amounts of
MPH and after three freezethaw cycles. The long-term
stability of stored samples was also investigated at the
0.210- and 15-ng/mL concentration levels after storage
for 6 months at 20 C. Short-term (benchtop) stability
was assessed at the 1.5-ng/mL concentration level after
3 h at room temperature. Autosampler stability was
determined by analysing a set of spiked samples at
different concentrations together with the corresponding
calibration curve at two different days. The samples were
thereby left at ambient temperature until reanalysis.

2665

Aliquot analysis was validated by analysing 50% aliquots

(250 L sample, 1:1 dilution). Robustness towards GC
temperature programming was determined by analysing
one set of samples under different GC programme
settings.
Matrix effect was investigated using six lots of matrix,
including the haemolysed and hyperlipidaemic sample
matrix. For each analyte and the internal standard, the
matrix factor (MF) was calculated in each lot of matrix by
calculating the ratio of the peak area in the presence of
matrix (measured by analysing blank matrix spiked with
analyte at a concentration of 0.210 ng/mL after extraction)
to the peak area in the absence of matrix (pure solution of
the analyte). The IS normalised MF was also calculated by
dividing the MF of the analyte by the MF of the IS. The IS
was added to the matrix or buffer together with the
unlabelled target. Normalised MFs were calculated for each
sample separately. For the assessment of accuracy and
precision at prospective analytical batch size, five replicates
of spiked samples at the 0.210-, 1.5- and 15-ng/mL
concentration levels together with at least 90 blank plasma
samples were extracted and chromatographed with a set of
calibration standards in one single run. Quality control
(QC) samples were analysed once before the blank plasma
samples and again after analysis of 90 blank plasma
samples. Accuracy was measured as bias (per cent
deviation of the calculated versus the nominal values) and
precision was expressed as coefficient of variation (%).

Results and discussion

Preparation of [18O2H3]-MPH
We have previously described the synthesis of [18O2]-MPH
[23]. The labelling procedure described here offers a fast
and inexpensive way to incorporate a specific label at the
carboxylic acid group with an even higher isotopic label
(M+5). After hydrolysis in H218O and re-esterification with
[2H3]-methanolic HCl, the 18O2H3-labelled product is
obtained in quantitative yield. Unlabeled species were
below 0.1%. No back exchange of label was observed
under the conditions employed for the sample preparation
and analysis. The increase in mass by 5 amu is also of
advantage compared with the commercially available [2H3]MPH labelled at the methyl ester carbon.
NICI mass spectrometry of MPH-PBB derivatives
As previously described, heptafluorobutyrate derivatives of
MPH show a prominent fragment ion series at m/z 409/389/
369/349, resulting from sequential loss of HF from the
molecular ion [23]. Reduced fragmentation and production

2666

H.J. Leis et al.

of more high-mass ions by NICI, however, occurs only

to a certain extent when perfluoroacyl derivatives are
measured. These derivatives produce molecular anions by
resonance electron capture (REC), but fragmentation is
quite intense, leading to the aforementioned ion series.
Thus, the proportion of the total ion current is very low,
no matter which fragment ion is chosen for quantification. Fragmentation under NICI conditions can, however,
be drastically reduced to only a few fragment ions with
striking intensity, as for pentafluorobenzyl derivatives of
carboxylic acids undergoing dissociative REC that leaves
Fig. 1 NICI mass spectra of the
PBB derivatives of methylphenidate (a) and [18O2H3]methylphenidate (b)

the carboxylate anion with high stability and high

abundance. PBB derivatives also show this fragmentation
mechanism and yield similar results [24]. The NICI
spectra of the PBB derivative of MPH and [18O2H3]MPH are shown in Fig. 1a, b, respectively. The carboxylate
anion is present with striking abundance and fragmentation
is extremely reduced, resulting in more than 90% of the total
ion current for the diagnostic fragment ions at m/z 380
(MPH) and m/z 385 (IS), respectively. This extremely
efficient process accounts for the extraordinary sensitivity
of these derivatives.
380

100

A
90

Relative Abundance

80
F

70

60

m/z 380
F

50
O
O

40

30

COOCH3

20

381

10
0
100

200

300

400

500

600

500

600

m/z
100

385

90
F

80
F

Relative Abundance

70

m/z 385
F

60

O
O

50
N 18

40

O
2

30

2
2

386
20
10
0
100

200

300

400

m/z

GC-NICI-MS of methylphenidate

2667

Fig. 2 Typical chromatogram

obtained after analysis of
plasma containing 0.072 ng/mL
of methylphenidate. Samples
were converted to the PBB
derivatives and analysed by
GC-NICI-MS

5,75

100
90
80
70

methylphenidate

60

m/z 380

50
40

Relative Abundance

30
20
10
0
5,74

100
90
80
70
60

[18O2H3]-methylphenidate IS

50

m/z 385

40
30
20
10
0
4,0

4,5

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

Time (min)

Gas chromatography of MPH-PBB derivatives

Plasma sample preparation and derivatisation

In comparison to heptafluorobuyrate derivatives, retention

times are shifted dramatically due to the bulky PBB group,
thus allowing detection at less interference from volatile
matrix components. In Fig. 2, a typical chromatogram
obtained after the analysis of plasma containing 0.072 ng/mL
MPH is shown. There are no interfering peaks from matrix
or reagent.

The method presented here provides a rapid, rugged and

simple way for the analysis of MPH in plasma allowing
processing of large sample batches. As children are the
main target of this medication, a small sample size is
desirable. The extraordinary sensitivity of the PBB derivative allows the use of only 250 L of plasma by keeping a
LOQ of 0.072 ng/mL. Extraction and derivatisation

Table 1 Intra- and inter-day precision and accuracy for methylphenidate determination
Nominal concentration (ng/mL plasma)
Intra-day precision and accuracy
0.072
0.210
1.5
15
Inter-day precision and accuracy
0.072
0.210
1.5
15

Mean

SD

CV (%)

Accuracy (%)

0.071

0.007

9.52

0.99

0.198
1.486
15.282

0.008
0.067
0.206

3.87
4.49
1.35

5.80
0.94
1.88

0.068
0.191
1.453
15.238

0.003
0.006
0.042
0.220

4.00
3.00
2.90
1.44

5.42
8.95
3.11
1.58

2668

H.J. Leis et al.

Table 2 Freezethaw stability of methylphenidate

Nominal concentration (ng/mL plasma)

After 1 freezethaw cycle

After 2 freezethaw cycles
After 3 freezethaw cycles

Mean

SD

0.210
15
0.210
15
0.210

0.202
15.310
0.196
13.939
0.212

0.008
0.268
0.015
0.825
0.019

2.69
8.95
4.80

15
0.210
15

14.034
0.201
13.561

0.237
0.011
0.158

8.33
0.33
11.42

proceeds rapidly and is quantitative in this one-step

procedure. The phase transfer reaction (extractive acylation)
also eliminates excess reagent by hydrolysis in aqueous
buffer and thus minimizes background contamination. As
hydroxyls do not react under these conditions, interference
from co-derivatised matrix components is low [24]. Even
lower detection limits can be obtained, if desired, when
larger plasma samples of 1 mL are extracted and the hexane
phase is dried under nitrogen. Afterwards, derivatisation
can be accomplished as described or with 100 L of the
neat reagent. Using this method, LOQs below 5 pg/mL can
be obtained (results not shown).
Analytical method validation
The calibration graphs established were linear within the tested
range of 0.07218.25 ng/mL plasma with a mean regression
coefficient (r) of 0.99959 (n=5). The mean regression equation
from five calibration curves was Y 0:007
0:01073
0:243460:00483  X 3:04454 2:6774  X 2 . Correlation (r) of back-calculated values with their respective
nominal values was 0.99959.
The coefficients of inter- and intra-day variation (precision)
and accuracy of the spiked samples are presented in Table 1. It
can be seen from these data that the method provides a
highly precise and accurate assay for MPH in human plasma.
This can be attributed at least in part to the use of a stable
isotope-labelled internal standard. Mass spectrometry in
combination with stable isotope dilution is a very powerful
tool in external quality assessment schemes, and assays
based on this technique can be regarded as reference
procedures to validate other analytical methods.
Six different blank matrices were checked for interferences.
In none of the samples was there background contribution
above 25% LOQ.
Short-term stability was determined as the deviation
between the concentrations obtained for samples subjected
to the extraction immediately after thawing and those kept

Deviation (%)

at room temperature for 3 h before extraction. In human

plasma, deviation from immediately analysed samples
was 7.6%. If, however, the internal standard was added
immediately after thawing, the values obtained after 3 h at
room temperature did not deviate substantially (2.9%). With
porcine plasma as a matrix, deviation was significantly higher
(13.2%), but could also be compensated by the immediate
addition of the internal standard (deviation 3.8%). It is well
known that plasma esterases hydrolyse MPH to ritalinic acid;
thus, the internal standard should be added immediately after
thawing. This is another beneficial effect of the stable isotopelabelled standard as it is chemically identical to the target and
thus compensates for loss due to hydrolysis of MPH.
Nevertheless, long benchtop times should be avoided in any
case during sample processing. It should be noted that we
have also investigated the degradation of MPH in alkaline
buffer and found no significant decrease during the time span
of the sample workup.
For autosampler stability, the mean concentrations of
samples chromatographed immediately after sample preparation
16
14

MPH [ng/mL plasma]

No freezethaw cycle

Concentration found (ng/mL plasma)

12
10
8
6
4
2
0
0

10

15

20

Time [hours]

Fig. 3 Pharmacokinetic profile of methylphenidate from a human

volunteer receiving 30 mg of the drug orally

GC-NICI-MS of methylphenidate

and 5 days later were measured and differed between 5.3% at

0.210 ng/mL and 1.2% at 15 ng/mL Thus, MPH-PBB
derivatives are stable at repeated analysis conditions.
Stock solutions of MPH in acetonitrile and methanol
were stable for at least 31 months when stored below 20 C.
The measured difference was 1.08%.
For the assessment of long-term stability, plasma
samples were stored below 20 C in a freezer for 6 months
and compared with the immediately analysed samples.
Deviations ranged from 0.3% at LOQ to 0.7% at 15 ng/mL.
Therefore, the samples can be considered as stable within
6 months of storage.
To determine the freezethaw stability of MPH, spiked
samples were subjected to three freezethaw cycles. The
results are shown in Table 2. Mean concentrations of samples
undergoing three freezethaw cycles did not differ from that
of the reference samples more than the critical 15%.
After analysing sample aliquots of 50% deviation was
0.76%, which shows that samples containing MPH may be
measured with sufficient reliability in 125 L sample
aliquots (1:1 dilution).
Analysis of MPH with three different GC temperature
programmes yielded deviations of 0.21% and 1.31% at
0.210 ng/mL and 0.170.27% at 15 ng/mL The method is
thus robust against variations of the GC temperature
programme.
Evaluation of the matrix effect resulted in an internal
standard-normalised matrix factor of 1.02 with a coefficient
of variation (CV) of 3.7%. This demonstrates that normal,
lipidaemic and haemolytic plasma matrix yield equal
results. This could be expected for a GC-MS method with
appropriate sample preparation as the matrix effects are
usually by far more pronounced in LC-MS applications,
where ion suppression may lead to dramatic changes in
MS response.
Accuracy at prospective analytical batch size has been
estimated for a 90-sample batch. Deviation of samples
analysed after sample batch from early analysed samples
was 1.10% at 0.210 ng/mL, 2.55% at 1.5 ng/mL and 5.74%
at 15 ng/mL. The method is thus suitable for analysing
batch sizes up to 133 samples (18 calibrants + 90 batch
samples + 15 QC samples).
We have applied the method described herein to the
analysis of MPH during pharmacokinetic profiling of the
drug. Figure 3 shows a typical time course from a human
volunteer receiving 30 mg of the drug orally as a retard
formulation. It should be noted here that a promising
challenge is the determination of drugs in oral fluid for drug
level monitoring. However, there are only few data on
MPH levels in saliva, and ongoing studies will support the
use of alternative matrices for noninvasive monitoring.
Nevertheless, the presented method can be used for the
analysis in minute amounts of plasma as well.

2669

Conclusions
Due to the delicate targeting of the drug to children for the
treatment of ADHD, an assay with the highest achievable
sensitivity is desirable to minimize the sample size for
therapeutic drug monitoring, which is crucial for adequate
drug dosage because of the large intra-individual variability
and tolerance. The method described fulfils these criteria
exceptionally by reaching detection limits far below all
assays published so far. The ease of extractive acylation
keeps the sample preparation time to a minimum and allows
large sample batches to be analysed in a short time. The use
of a stable isotope-labelled internal standard adds an
additional dimension of specificity and selectivity to the
mass spectrometric detection, thereby also compensating
ideally for losses during the sample workup procedure and
derivatisation sequence. This is of particular interest for the
analysis of MPH from the plasma matrix as esterase
activities may lead to degradation which is time- and
matrix-dependent. The extraordinary sensitivity of the assay
must be attributed to the new derivatisation reagent,
PBBCl. We have successfully applied this method to the
bulk analysis of plasma samples for a preliminary pharmacokinetic study, demonstrating its ability for routine
measurements.

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