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Ann Transplant, 2009; 14(2): 61-72 Review Paper

Received: 2008.10.28
Review of the newest HPLC methods with mass
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Accepted: 2009.01.27
Published: 2009.05.08
spectrometry detection for determination of
immunosuppressive drugs in clinical practice

SE
Magdalena Korecka, Leslie M. Shaw
University of Pennsylvania, Department Pathology and Laboratory Medicine, Philadelphia, PA, U.S.A.
This work was presented at the International Conference Therapeutic Drug Monitoring in Optimizing the

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Immunosuppressive Therapy, Warsaw, Poland, 26th27th September 2008

Summary

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High performance liquid chromatography coupled with electrospray mass spec-

N A
trometry is widely used for quantitative determination of immunosuppressive
drugs (sirolimus, tacrolimus, everolimus, CsA and MPA) in biological uids. The
growth in volume for testing these drugs and economic constraints in clinical lab-
O N
oratories has led to heightened demand for high throughput methods.
Fast-ow on-line extraction with switching valve technique and implementation of
automation accelerates sample preparation. For on-line purication the combina-
SO

tion of fast ow of washing solution and narrow-bore extraction column provides

a clean sample in a very short time without compromising precision and accura-
cy. The unique feature of multireactant monitoring tandem mass spectrometry
reduces signicantly the need for chromatographic separation, as long as matrix
effects are not detected, and permits simultaneous measurement of several drugs
R

in one run when they are present in the same specimen. Additionally, the same
method together with the identical sample preparation and HPLC-MS conditions
and setting can be used for measurement of all ve immunosuppressants, four
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PE

of them in blood, MPA in plasma. Thanks to the high sensitivity of LC-MS only a
small volume of biological sample is required for analysis. However for MPA quan-
titation, mass interference attributable to in-source fragmentation of its glucuro-
nide metabolite can be a problem if the latter is not chromatographically separat-
ed from the parent drug before introduction of the sample into the ion source.
Thus, chromatographic separation is extremely important for MPA analysis.
In conclusion, important features of LC-MS methodology for immunosuppres-
sive drugs include: shortened analysis time, increased throughput, selectivity and
lower cost of analysis.
Key words: immunosuppressive drugs therapeutic drug monitoring high-pressure liquid
chromatography with mass spectrometry

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Word count: 4649

Tables: 3
Figures: 2
References: 73
Authors address: Leslie M. Shaw, University of Pennsylvania, Department Pathology and Laboratory Medicine, 3400 Spruce
Street, Philadelphia, PA 19104, U.S.A., e-mail: shawlmj@mail.med.upenn.edu

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Review Paper
BACKGROUND
As of today 5 major maintenance immunosup-
pressive drugs are used in modern transplan-
tation: cyclosporine A (CsA Novartis), tac-
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rolimus (TAC Astellas), sirolimus (SIR Wyeth),
everolimus (RAD Novartis) and mycophenol-
ic acid (CellCept Roche, or Myfortic (sodium
salt) Novartis).
CsA (a cyclic undecapeptide) and tacrolimus
(macrolide lactone) are the calcineurin inhibitors
which by blocking interleukin-2 production lead-
ing to suppression of T lymphocyte proliferation
[1]. These two drugs are the basis of most main-
U
tenance immunosuppressive protocols follow-
ing organ transplantation. Sirolimus (a 31-mem-
bered triene macrolide lactone) and everolimus
(semi-synthetic 40-O-(2-hydroxyethyl) derivative
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of rapamycin) has immunosuppression mecha-
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nisms distinct from those of CsA and TAC. SIR
N A
and RAD inhibit the functioning of the mamma-
lian target of rapamycin (mTOR), a kinase that
induces intracellular signal transduction at the
O N
G1-S interface, leading to an inhibition of T-cell
cycle progression [2,3]. Mycophenolic acid is a
potent, selective and uncompetitive reversible
SO
inhibitor of inosine monophosphate dehydroge-
nase (IMPDH), which is the rate limiting enzyme
in the de-novo synthesis of guanosine nucleotides
and consequently DNA production [4].
R
Due to their complementary mechanism of ac-
tion calcineurin inhibitors and inhibitors of
the mTOR are often combined in the clinic. It
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PE
was shown that this co-administration provides
synergistic immunosuppression that is a major
benet for transplant patients [3,5]. The exist-
ing drugs have a few common features that are
very important for their simultaneous analysis.
They are soluble in organic solvents like alco-
hols, acetonitrile and they are practically insol-
uble in water [3]. They all, except for MPA, can
be measured in whole blood due to high distri-
bution in erythrocytes, between 4060% for CsA,
about 95% for SIR, 9598% for TAC and 75%
for everolimus [68].
Therapeutic drug monitoring (TDM) of immuno-
py is for personal use only - distribution prohibited.
suppressive drugs helps to achieve optimal thera-
peutic efcacy while minimizing toxicity. All the
drugs have narrow therapeutic ranges for exam-
ple 515 ng/mL for SIR [9] and 13 g/L for rected into a mass analyzer, through the series
MPA [10]. Variation in concentration outside the
narrow therapeutic index can result in adverse
clinical outcomes: toxicity when the concentra-
62
Ann Transplant, 2009; 14(2): 61-72
tion is too high or rejection when the concen-
tration is too low. The variable pharmacokinet-
ics of immunosuppressants within and between
patients as a result of variations in absorption,
distribution and/or elimination makes it impos-
sible to reliably predict the best dose for each
patient [10]. The resulting variability in trough
blood concentration can reach up to 50% [11].
For TAC, SIR and RAD trough samples are pri-
marily used for drug monitoring as an estimate
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of drug exposure. However, for CsA and MPA ad-
ditional samples collected at several additional
time points over the 12 hour dose interval can
provide a more accurate estimation of drug ex-
posure than the trough sample alone and there-
by provide for better clinical outcome [10]. In
busy transplant centers the number of samples
sent to the laboratory for this essential test can
be considerable. Thus there is a need for robust,
sensitive and high throughput methods, with sim-
ple sample preparation, that allows measuring a
few drugs in one blood sample thereby saving
time and reagents.
Mass spectrometry is already well established
as a quantitative tool for small molecules, and
is based on producing, differentiating and de-
tecting ions in the gas phase. Conversion of dis-
solved analyte eluting from a separation system
into gas-phase ions occurs in the ion source and
is generally associated with evaporation, pres-
sure reduction and an ionization process. From
the ve currently available ion sources, electro-
spray (ESI) is the most widely used for mass spec-
trometric quantication of immunosuppressive
drugs. This ion source has the ability to produce
singly or multiply charged gaseous ions directly
from an aqueous/organic solvent system by cre-
ating a ne spray of highly charged droplets in
the presence of a strong electric eld [12,13].
ESI offers many advantages over other ioniza-
tion techniques, including the ability to analyze
low and high-mass compounds, excellent quanti-
tative capabilities and reproducibility, high sensi-
tivity, simple sample preparation, amenability to
automation, soft ionization and minimal amount
of in-source fragmentation. In selected circum-
stances ESI can be quite effective even without
separation, especially when combined with tan-
dem mass spectrometry [14].
From the ion source the gas phase ions are di-
of lenses. The mass analyzer differentiates these
ions according to their mass-to-charge ratio (m/z).
The triple quadrupole mass analyzer is the most
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Ann Transplant, 2009; 14(2): 61-72
common mass analyzer for compounds with
m/z <1000, but also can operate at upper mass-
es above 4000 m/z [13]. The majority of methods
for quantication of immunosuppressive drugs
employ a triple quadrupole as the mass analyz-
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er. Quadrupoles consist of four precisely paral-
lel rods (poles) equally spaced around a central
axis. By applying precisely controlled voltages to
opposing sets of poles, we create what is known as
a mass lter. Only ions with a particular mass-
to-charge ratio will pass through the lter to be
detected at a particular applied voltage [13].
In the past few years, high-pressure liquid chro-
matography with mass spectrometry (HPLC-MS)
U
has been popularly utilized in drug quantitation
and pharmacokinetics studies and now is consid-
ered to be the gold standard analytical method in
therapeutic drug monitoring [6]. Immunoassays
LY L
and HPLC methods with UV detection for meas-
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urement of immunosuppressive drugs reveal too
N A
many disadvantages compared to HPLC-MS such
as cross reactivity of metabolites with the antibod-
ies for immunoassays [15] and long, laborious
O N
sample preparation for HPLC-UV [16]. The main
attraction of HPLC-MS is high selectivity and sen-
sitivity. This technique permits the quantication
SO
of the main drug independently from its metab-
olites. Since the immunosuppressive agents are
used in combined regimens and their amenabil-
ity to analysis by HPLC-ESI- mass spectrometry,
a very important advantage of HPLC-MS quanti-
R
tation methods is the possibility of simultaneous
analysis of several compounds in one short run.
For four out of ve major immunosuppressants
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PE
(CsA, TAC, SIR and RAD) the desired specicity
and sensitivity can be achieved without chromato-
graphic separation in time, which shortens anal-
ysis time, increases throughput and also makes
a big impact on the laboratory budget. One of
the most recent studies has shown that the same
chromatographic system used for analysis of 4
immunosuppressive drugs in blood can also be
used for the analysis of MPA in plasma [17]. All
HPLC-MS methods need less laborious sample
preparation when compared with HPLC with
UV detection. All these advantages of HPLC-MS
methods can shorten total cycle time and save
reagent usage.
py is for personal use only - distribution prohibited.
The purpose of this paper is to review the recent-
ly published HPLC-MS methods for analysis of
immunosuppressive drugs and to provide a use-
ful resource for those who are planning to use
mass spectrometry in their laboratories for the
analysis of immunosuppressants.
Korecka M et al Review of the newest HPLC methods with mass spectrometry
SAMPLE PREPARATION
For all immunosuppressive drugs the matrix is-
sue has been already resolved. Cyclosporine, tac-
rolimus, sirolimus and everolimus are measured
in whole blood while for total MPA analysis plas-
ma or serum is the matrix of choice [10] and
the ultra-ltrate obtained from plasma is used
for analysis of free MPA [18,19]. Dried blood
spot (DBS) sampling could be an alternative
SE
and two methods were published that use DBS
capillary blood obtained from nger prick for
analysis CsA and TAC [20,21]. The usefulness of
dried blood spot sampling for therapeutic drug
monitoring of tacrolimus was investigated in re-
nal transplant patients. There was no signicant
difference between the concentrations (ranging
3.3353.9 g/L) of 34 samples from 26 stable re-
nal transplant outpatients, measured both after
venous and DBS sampling. DBS seems promis-
ing for routine patient monitoring [22]. TAC can
be additionally measured in tissue biopsy sam-
ples [23], MPA in saliva [24] and CsA in T-lym-
phocytes [1].
Mass spectrometry detection, since more sensi-
tive when compared with UV detection, allows
decreased sample volume used for analysis of
immunosuppressive agents. For the majority of
HPLC-MS methods the volume of whole blood
used ranged from 0.01 [25] to 0.5 mL [26,27],
most often 0.1 mL [3,28,29], and for MPA anal-
ysis 0.05 [30,31] 0.2 mL [18] of plasma.
The high specicity of mass spectrometry allows
for a less intensive sample preparation compared
to the HPLC-UV technique [26]. The three most
widely used methods of sample preparation for
the immunosuppressive drugs quantication
are: 1) on-line sample clean-up with switching
valve and 2-dimentional chromatography and
off line clean-up with 2) solid phase extraction
columns (SPE) or with 3) liquid-liquid extrac-
tion (LLE).
On line sample clean-up
Several HPLC-MS methods employed on-line
sample clean-up followed by protein precipita-
tion generally with the mixture of ZnSO4 and
methanol, acetonitrile or acetone with internal
standard, as an approach for shortening sample
preparation and increasing sample throughput
[3,25,28,29,3236]. Addition of water to blood
before adding ZnSO4 and organic solvents pro-
tects the sample from clumping, gives cleaner
63
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Review Paper
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U
LY L
extracts with less ion suppression and improves
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the extraction efciency [37]. Additionally, this
N A
extraction with water could be adapted to an au-
todiluter for specimen delivery because water
allows the specimen to be ushed from the dis-
O N
penser with no precipitation in the line. All pi-
petting during the sample preparation procedure
can be automated using a commercially availa-
SO
ble robotic pipetting system from Tecan Freedom
EVO (Tecan, Switzerland) [38]. Supernatant ob-
tained from the protein precipitation step is in-
jected into the system (injection volume ranged
from 5400 L [1,17,18,25,28,3234,3944] and using highly organic, acidied eluting solvent at
R
two-dimensional chromatography is performed
through a split arrangement of two pumps and
6- or 10-port switching valve plumbed for exam-
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PE
ple as shown in Figure 1. In this system two col-
umns (extraction and analytical) and two solvents
(extraction and eluting) are used. First dimen-
sion chromatography is accomplished through-
out the extraction column. The pump that is ded-
icated for extraction solution (highly aqueous,
for example water [32,34,41], ammonium acetate
buffer with or without low (2040%) addition of
methanol [25,33,42] is connected to the sample
introduction unit. Sample is injected with the ex-
traction solution and cleaned on extraction col-
umn, all debris and contaminants are ushed out
to waste to protect the mass spectrometer from
contamination. Recently it is advised that as an
py is for personal use only - distribution prohibited.
extraction column a narrow-bore LC column (ID
0.51.0 mm) packed with large particles of sta-
tionary phase material (3050 m), with a very
high ow of extraction solution (35 mL/min for
column with ID 1mm) should be used. The com-
bination of the fast ow and large particle size
provides the desired chromatographic behavior
64
Ann Transplant, 2009; 14(2): 61-72
Figure 1. Example of setting the switching valve
for quantitation of immunosuppressive
drugs by HPLC with mass spectrometry
detection.
SE
(turbulent ow behavior) that allows for the rap-
id passage of large biomolecules of the biological
sample (mainly phospholipids and carbohydrates)
with the simultaneous retention of the small-mol-
ecule analyte(s) of interest [45]. This sample ex-
traction stage lasts for less than 1.0 min. Second
dimension chromatography is performed using a
standard analytical column that will be discussed
later. After switching the valve from position A
to position B the compound(s) of interest is/are
back-ushed from the extraction column into the
analytical one and then to the mass spectrometer
a ow rate compatible with the analytical column
and ion source of mass spectrometer. At the time
of eluting the rst dimension chromatography is
in equilibration stage to prepare the extraction
column for next injection, so the equilibration
time does not add to the run time that is usually
23 min. All of these unique features of on-line
sample clean up increase the sample throughput
save on reagent costs, and make the two step sam-
ple preparation a very simple and fast procedure
and less likely to introduce errors. Based on a few
years of experience with using this technique in
our laboratory we can assert that the lack of con-
tamination by samples is comparable to that ob-
tained using the SPE sample preparation proce-
dure. This observation is additionally supported
by several published methods that directly use ex-
tract from protein precipitation without on-line
cleaning [31,37,39].
Solid phase extraction
Off line cleaning is another kind of sample prep-
aration widely used in methods for quantitation
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Ann Transplant, 2009; 14(2): 61-72
of immunosuppressive drugs. Solid phase extrac-
tion (SPE) is a technique designed for rapid, se-
lective sample preparation and purication prior
to chromatographic analysis. Using liquid chro-
matography principles to control selectivity, SPE
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provides sample clean-up, recovery, and concen-
tration necessary for accurate quantitative anal-
ysis. The SPE procedure typically follows a pro-
tein precipitation step, similar to what is done
for on-line clean-up procedures. The superna-
tant is applied to a column previously equilibrat-
ed with methanol and then water. The most wide-
ly used columns are those with C18 as a matrix.
The columns are washed sequentially with wa-
ter, 50% methanol in water, and heptane. Then
U
compounds of interest are eluted from the ma-
trix with a mixture of isopropyl alcohol and hep-
tane and dried under a stream of air. The dried
extract residue is usually dissolved in a small vol-
LY L
ume (50 L) of methanol [27] or of mobile phase
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[26] and injected onto the column. Injection vol-
N A
ume ranged from 5 to 25 L [19,26,27,30,46].
This whole procedure is more time, effort and
reagent consuming compared to the on-line ex-
O N
traction procedure. However, studies have shown
that samples obtained from SPE columns are less
likely to give rise to matrix effects [37].
SO
Automated versions of the SPE procedure are
based on the use of the 96-well format for ex-
traction together with a robotic liquid handling
system. This approach automates most of the te-
R
dious steps encountered in the traditional man-
ual extraction procedure and eliminates some of
the time consuming steps like tube labeling and
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PE
most capping/uncapping steps offering a desir-
able short analysis time, which is important for
high throughput in clinical applications [47].
Liquid-liquid extraction
Liquid-liquid extraction (LLE) is an attractive al-
ternative to SPE because of its relative simplicity
and ease of development. The extraction is per-
formed mainly with tert-butyl ether or 1-chlorobu-
tane. LLE can be performed manually [48] or this
process can be fully or semi-automated [49].
In a typical automated 96-well LLE, biological sam-
py is for personal use only - distribution prohibited.
ples are transferred, using a robotic liquid handling
system, into separate wells of a 96-well plate con-
sisting of a racked collection of microtubes. Then
the internal standard solution and the extracting
organic solvent are added using the robotic sys-
tem. The microtubes are capped with strip caps
and then the plate is shaken ofine. The aqueous
Korecka M et al Review of the newest HPLC methods with mass spectrometry
and the organic layers are then separated by cen-
trifugation. The robot is used to transfer the or-
ganic layers from the microtubes to a clean deep
96-well collection plate. The extracts in the 96-well
plate are evaporated ofine and then the dried res-
idues in the 96-well plate are dissolved by adding
the reconstitution solution using the robotic sys-
tem. The 96-well plate is sealed with a cover mat
and placed on the autosampler, ready for injec-
tion [45]. The Tomtec Quadra 96 station (Tomtec,
SE
Hamden, CT, USA) can be used for automation of
LLE as described in the published method for si-
multaneous analysis of CsA and everolimus [49].
The automation increases the sample through-
put, shortens preparation time by approximately
3-fold when compared to manual LLE and gives
the analyst more time for other tasks. However it
has been found that there is no appreciable dif-
ference in extraction efciency between manual
and automated LLE [50].
When comparing these three methods present-
ed here for sample preparation, protein precip-
itation with on line cleaning, SPE and LLE, the
latter provides cleaner extracts than SPE, and
SPE provides extracts with less matrix effect com-
pared to protein precipitation extract. However,
the main attraction to the protein precipitation
with on-line cleaning technique, compared with
manual LLE or SPE, is its speed, simplicity and
universality in that the same basic procedure can
be applied to extract almost any analyte [45].
INTERNAL STANDARD SELECTION
Selection of an appropriate internal standard
is critical to the success of a quantitative meth-
od. Internal standards are used in high-per-
formance liquid chromatography mass spec-
trometry methods to compensate for sample
to sample differences not only in extraction ef-
ciency during sample preparation, as for clas-
sic HPLC with UV detection, but also for ioniza-
tion variability during the transfer of the analyte
from the liquid phase to the gas phase and ma-
trix effect [51]. Stable isotope labeled internal
standards should be the rst choice of internal
standards for the measurement of immunosup-
pressive drugs. An isotopically labeled internal
standard will have a similar extraction recovery,
ionization response in ESI mass spectrometry,
and a similar chromatographic retention time.
It will be affected by matrix effect in the same
way like analyte of interest. Unfortunately, such
chemicals are not readily available for all immu-
nosuppressive agents and thus structural ana-
65
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Review Paper Ann Transplant, 2009; 14(2): 61-72

Table 1. Compounds used as internal standards for measurement

of immunosuppressive agent in published HPLC-MS
methods.

Drug Internal standard

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CsA CsD, CsA-d12

Tacrolimus Ascomycin, FR 900520
Sirolimus SIR-d3, Ascomycin, 32-O-desmethoxy-rapamycin,

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Everolimus Ascomycin, SDZ RAD 223-756
Mycophenolate Carboxybutoxy ether, indomethacine
mofetil
Figure 2. Amount of internal standard used in any given analysis
should be in the 1/3 lower end of calibration curve [52].

U
logues or another related molecule that close-
ly matches the chromatographic and ionization
characteristics of the analyte are preferred. The
de-methylated (14) or hydroxylated (+16) ana-
the CsD [55] and ascomycin [42] mass transition
that come from patient samples, CsA metabolites
or CsD that increase falsely area under the inter-

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logs should not be used as internal standards nal standard peak (ascomycin or CsD) leading to
This copy is for personal use only - distribution prohibited.

since these are the most common mass shifts ob-
N A
served in naturally occurring metabolites of the
parent compound [52].The most widely used
internal standards for quantication of immu-
O N
nosuppressive drugs are presented in Table 1.
Deuterated everolimus can be obtained from
Novartis Pharma but not for all scientist [53]
SO
lower RAD or CsA results, respectively.
Another paper published in 2008 has shown that
the use of SIR-d3 as the internal standard in the
assay for quantitation of SIR, provides results with
lower inter-patient variation compared with the
results obtained from the assay that used 32-O-

and as of today there is no report that this IS desmethoxyrapamycin [56].

was used for any method.
The amount of added internal standard should
be well above the limit of quantication but not
R
It was also reported that choice of appropriate
solvent can affect performance of internal stand-
ard and for example methanolic solutions of as-

so high as to suppress the ionization of the an- comycin should be avoided [57]. Another inter-
alyte. A good rule of thumb is to target the in- esting observation is that ascomycin degraded
ternal standard to the lower 1/3 of the working quickly in the poor quality acetonitrile [57]. Nine
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PE

standard curve [52] (Figure 2).
For the method with simultaneous analysis of few
drugs one common or a few separate, one for each
immunosuppressant, internal standards can be
used. Ascomycin is the most popular internal stand-
ard for macrolides: TAC, SIR and RAD and CsD
for cyclosporine A. The effectiveness of these two
internal standards was discussed in several papers
[42,51,54]. It was shown that the precision for CsA
quality control samples obtained from the assay that
used ascomycin as an internal standard is poorest
when compared to the results obtained from the
assay that employed CsA-d12 or CsD as the internal
py is for personal use only - distribution prohibited.
commercial methanol used in HPLC eluting sol-
vents were evaluated for signal suppression of
sirolimus, tacrolimus, and MPA in MS/MS detec-
tion [58]. Product ion intensity was found to vary
by 10 folds among the methanol tested. Though
appropriate internal standards could compen-
sate for the signal loss, performance of the as-
say (e.g., LLOQ) could be compromised. A rig-
orous assessment of an internal standard should
be a part of method validation.
CHROMATOGRAPHIC SEPARATION
The most commonly used type of stationary

standard [54]. Also comparison of RAD results be-
tween the assays that used ascomycin or SDZ RAD
223-756 internal standard and CsA results between
the assays that used CsD or CsA-d12 internal stand-
ard showed the lower results for runs that used
ascomycin and CsD as internal standard [42,51].
Studies revealed the presence of interferences in
phase for all immunosuppressive drug analy-
ses is a reversed-phase C18 or C8 column, for ex-
ample Novopak-C18 [27] or Zorbax-C8 [3], with
column heating that ranged from 35C to 75C.
Column heating generally reduces peak broad-
ening and allows to use higher ow rate of mo-
bile phase by decreasing the pressure on the col-

66

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Ann Transplant, 2009; 14(2): 61-72
umn. Various analytical column dimensions are
used across the published method. The length
ranged from 3.3 to 250 mm and the outside di-
ameter from 2 to 4.6 mm. A few reported meth-
ods employ only a short guard column (3 or 4
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mm length) as an analytical one avoiding a chro-
matographic separation step [25,33,37,39]. For
this kind of method matrix effects must be as-
sessed very careful since chromatographic sepa-
ration can be a key step for elimination of matrix
effects. The features of the extraction columns
used for the methods with switching valve and 2
dimensional chromatography were already pre-
sented in the section on Sample preparation.
U
The acidied mixture of methanol (usually more
than 80%) and water with the addition of am-
monium acetate [18,2530,39,4244,46,48,49],
and/or formic acid [1,44] or sodium/ammoni-
LY L
um formate [3,17] is usually used as an eluting
This copy is for personal use only - distribution prohibited.
solvent. A few methods for MPA and CsA analysis
N A
introduced acetonitrile with or without methanol
into the mobile phase [31,44,49,59]. The addi-
tives in mobile phase (for example ammonium
O N
acetate and/or formic acid) increase the ioniza-
tion of analyzed compounds and improve meth-
od sensitivity. Adduct formation together with mo-
SO
bile phase pH adjustment has been commonly
used to optimize detection conditions [60]. The
ow of eluting solvent is usually isocratic, howev-
er a few methods use gradient [3,25,37,39]. Flow
rate of mobile phase varied from 0.1 mL/min
R
[61] up to 0.8 mL/min [28,38,42] for the meth-
od with ESI and 1 mL/min for the method with
APCI [33] managing the total run time in the
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PE
range from 2.5 [29,39] to 20 min [34].
Mass spectrometry detection in the analysis of
macrolides and CsA allows to achieve the de-
sire selectivity and sensitivity without chromato-
graphic separation. All compounds can be elut-
ed with the same retention time with no effect on
selectivity and sensitivity as long as no matrix ef-
fect is observed for any of the analytes involved.
However for MPA quantication adequate sepa-
ration on an analytical column is a crucial step of
the analysis. Two metabolites of MPA, MPA-glu-
curonide (MPAG) and the acyl glucuronide of
MPA (AcMPAG) and the generally used internal
py is for personal use only - distribution prohibited.
standard, carboxybutoxy ether of MPA (MPAC),
undergo in-source fragmentation and produce
additional peaks of the ammonium or protonat-
ed adduct ions of MPA and can falsely increase
the area under the MPA peak when chromato-
graphic separation is omitted [18,31,62]. In one
paper published in 2000 [45] attention was drawn
Korecka M et al Review of the newest HPLC methods with mass spectrometry
to such in-source transformation of drug conju-
gate metabolites to the respective target analytes.
The specicity of LC/MS/MS analyses must be
assessed critically whenever drug metabolites
with a higher molecular mass than the target an-
alytes must be assumed in patients samples. The
in-source fragmentation of MPAG, AcMPAG and
MPAC to MPA can varied depend on the mobile
phase composition, instrument settings and de-
sign and some mass spectrometer parameters like
SE
capillary or cone voltage or temperature in the
ion source [30,36]. Since MPA analysis has two
requirements not needed for the others immuno-
suppressive drugs (plasma as a matrix and neces-
sity of chromatographic separation) it is usually
analyzed in a separate assay, not simultaneously
with the co administered immunosuppressants.
However one method that was published in 2007
[17] described the LC and MS conditions that can
be used both for MPA and the macrolides and
CsA quantication without any changes. Since the
sample matrix for these two groups of drugs dif-
fers there is a requirement to prepare two differ-
ent samples for analysis, but the LC/MS setting is
the same and assures full separation of MPA from
the metabolites and internal standard.
MASS SPECTROMETRY DETECTION
Ionization process and matrix effect
Ionization procedure refers to the two processes
leading to the formation of gas-phase ions sampled
by the mass spectrometer: (1) transfer of the an-
alyte into the gas phase and (2) the addition of a
charge to form an ion [63]. In the presence of oth-
er sample components the ionization process can
be changed and will be mostly observed as a loss in
response and referred to as matrix effect that can
inuence the accuracy, precision and lower lim-
it of quantication of the assay. The matrix effect
can occur with any biological matrix (plasma, se-
rum, blood or urine), and it may vary depending
on the source of matrix [45]. It is also compound
dependent [63] and it was shown that the meth-
ods based on electrospray ionization, mainly used
for quantication of immunosuppressive drugs, are
more likely to experience a matrix effect than that
based on heated nebulizer [6466]. Assessment of
matrix effect is a very important step during meth-
od development/validation and can not be omit-
ted. There are two commonly used tests for evalu-
ation of the matrix effect: post-extraction addition
and post-column infusion, and they have been de-
scribed in numerous of publications [64,66,67].
The probability of the occurrence of the matrix
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Table 2. MRM transitions (quantifier and qualifier pairs of ions)
employed for tandem mass spectrometry detection of
immunosuppressive drugs.
MRM-transition MRM-transition
This copy is for personal use only - distribution prohibited.
dem mass spectrometry for detection of ions.
Single quadrupole mass spectrometry detects
mainly sodium adducts which gives the strong-
est signal when compared with proton or potassi-
um adduct ions [3]. However only one published

quantifier qualifier method with single quadrupole mass spectrom-

Sirolimus [M+NH4]+ 931/864 931/882 etry detection uses an additive in the mobile
phase (sodium formate) [3], the rest of them
Tacrolimus [M+NH4]+ 821/768 975/908 do not use any additives [32,34,41] and forma-
Everolimus [M+NH4] +
975/908 975/858 tion of sodium adduct is connected with com-

SE
mon presence of sodium ions in water. The pos-
MPA [M+NH4]+ 338/207 Not reported itive mode is generally more sensitive than the
MPA [M+H]+ 321/207 Not reported negative mode for single quadrupole mass spec-

trometry detection [32].
MPA [M-H] 318/191 Not reported

U
CsA [M+NH4]+ 1219/1203 1219/1185 The majority of methods that use tandem mass
spectrometry detection operate in the positive
CsA [M+H]+ 1202/425 Not reported
mode while detecting primarily ammonium ad-
duct [18,2729,33,3739,48] and its fragment.

LY L
effect can be reduced or eliminated by utilizing a The ammonium adduct is obtained by addition
This copy is for personal use only - distribution prohibited.

more thorough clean-up procedure of the biolog-
N A
ical samples, modifying the chromatographic con-
ditions, or allowing longer run times in order to
enhance the chromatographic separation between
O N
the matrix-effect causing component and the an-
alyte [45]. The choice of internal standard is also
very important for minimizing the inuence of ma-
SO
of ammonium acetate or ammonium formate
to the mobile phase. It was shown that ammoni-
um adduct ions are less stable than sodium ad-
ducts, so more appropriate for fragmentation.
Additionally in positive ionisation mode proton
adduct ions and in negative ionisation mode de-
protonated ions are used for detection.

trix effect on accuracy of the assay. An isotopically

labeled compound used as internal standard will
be affected by any matrix effect in the same way
like analyzed drug so accuracy of the quantitation
should not be impaired provided that adequate sig-
R
The precursor ions give usually more than one
product. The most abundant product ion is cho-
sen as a quantier ion, and one from the less
abundant ions can be used as a qualier ion.

nal from the drug and internal standard occurs so In order to obtain an increase in compound re-
that signal to noise ratio is adequate for both. sponse, the peak area for these two mass transi-
tions can be monitored and summed [27,29].
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PE

Detection The most widely used quantier and qualier

transitions are listed in Table 2.
The analytical methods used for quantitation of
immunosuppressive agents utilize single or tan-

Table 3. Characteristics of HPLC methods with single and tandem mass spectrometry detection for quantification of immunosuppressive
drugs. There is no recently reported method for MPA analysis by HPLC with single mass spectrometry detection.

MPA g/L CsA ng/mL Sirolimus ng/mL Tacrolimus ng/mL Everolimus ng/mL
MS/MS MS MS/MS MS MS/MS MS MS/MS MS MS/MS
Free: 0.5-2.5
LLOQ 1.525.0 5.24-10 0.251.0 0.21.66 0.21.0 0.2-1.66 0.15-0.25 0.252.5
Total: 50-100
Free: 2.5-8.8
Precision* (%) 0.9-10.2 2.5-7.3 1.29.8 0.96-10.8 0.75.8 0.7515 0.99.1 1.9-14.0
py is for personal use only - distribution prohibited.

Total: 1.8-8.5
Free: 95109
Accuracy** (%) 82105 91.3-119 96108 92105 79109 92104 84107 90109
Total: 91110
18, 19, 3, 32, 34, 3, 32, 25, 26, 28, 29, 3, 34, 41, 2729, 33, 28, 29, 42, 43,
References 20, 49 3, 34, 41
30, 44 41, 59, 71 34, 59 33, 37, 39, 48 59, 72 39, 40 46, 49, 73
* Together in- and between day reported for quality control samples; ** reported for quality control samples.

68

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Ann Transplant, 2009; 14(2): 61-72
METHODS CHARACTERISTICS
The lower limit of quantication (LLOQ), preci-
sion and accuracy for all methods that utilize single
or tandem mass spectrometry are shown in Table 3.
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The lowest standard concentration for all present-
ed methods, which dened LLOQ, is lower than
the lowest concentration of each drug expected in
treated patients. It is often the practice that LLOQ
is not investigated at concentrations lower than the
lowest concentration of the observed linear range
[39]. Based on the recommendations of Shah et
al. [68] and the FDA guideline for analytical per-
formance validation [69] the published assays for
analysis of immunosuppressive drugs have excellent
U
between- and within-day analytical recovery and pre-
cision and can be used for TDM test methods.
CONCLUSIONS
LY L
This copy is for personal use only - distribution prohibited.
Liquid chromatography with mass spectrometry
N A
detection is a major breakthrough in therapeutic
drug monitoring of immunosuppressive agents and
is considered as the method of choice in TDM of
O N
immunosuppressants. It provides high specicity,
excellent sensitivity and robustness for drug meas-
urement however the choice of internal standard,
SO
the organic solvents and matrix effect should be
evaluated carefully during method validation. It
has a potential for simultaneous determination of
4 immunosuppressants in blood and additionally
allows analysis of MPA in plasma using the same
R
conditions. Despite the initial high cost for the in-
strumentation, HPLC-MS is more cost effective than
microparticle enzyme immunoassay, as was shown
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PE
for TAC [70]. The reagent cost for immunoassay is
signicantly higher compared with the basic rea-
gents and solvents used for HPLC-MS. Since prices
for mass spectrometers are decreasing more cent-
ers should consider purchase of this equipment
thereby saving money on reagents, potentially im-
proving the quality of results and achieving an in-
crease in the number of reported results.
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