Special Issue: Review

Received: 9 March 2009, Revised: 21 April 2009, Accepted: 22 April 2009 Published online in Wiley Interscience: 15 July 2009

(www.interscience.wiley.com) DOI 10.1002/bmc.1271

High-performance liquid chromatography

John Wiley & Sons, Ltd.

coupled with mass spectrometry for the

quantitative analysis of vinca-alkaloids in
biological matrices: a concise survey from
the literature
Carola W. N. Damena*, Hilde Rosinga, Jan H. M. Schellensb,c and Jos H. Beijnena,c
Quantitative analysis of vinca-alkaloids in biological matrices

ABSTRACT: The bioanalysis of vinca-alkaloids has been investigated extensively. High-performance liquid chromatography
coupled to ultraviolet, fluorescence or electrochemical detection have been described. During recent years liquid chromato-
graphy coupled with mass spectrometry (LC-MS) has become the first choice for the quantitative bioanalysis of the vinca anti-
cancer agents. This paper reviews recent methods for the bio-analysis of vinca-alkaloids using LC-MS, supplemented with our
own experience. We will focus on sample pre-treatment, chromatography and MS detection and pay attention to problems
which can occur during the bioanalysis of vinca-alkaloids. These problems encounter carry-over and absorption effects and
solutions will be provided how to circumvent these problems. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: vinca-alkaloids; bioanalysis; LC-MS; vinorelbine; vincristine; vinflunine; vinblastine

Introduction
The vinca-alkaloids vincristine, vinblastine and vinorelbine
[Fig. 1(A–C)] are currently registered for the treatment of a wide
variety of cancer types. Vinflunine [Fig. 1(D)] is a relatively new
vinca-alkaloid, which is currently being investigated in phase III
clinical trials. Vinblastine and vincristine are naturally occurring
alkaloids derived from the Catharanthus roseus plant; vinorelbine
and vinflunine are semi-synthetic derivatives. All vinca-alkaloids
bind to beta-tubulin and disrupt microtubule function during
mitosis, which in turn leads to mitotic arrest and cell death (Gan
and Kavallaris, 2008; Kavallaris et al., 2008; Pasquier and Kavalla-
ris, 2008). Additionally, they can inhibit angiogenesis (Pasquier
and Kavallaris, 2008). Vinca-alkaloids are prescribed for a wide
variety of cancers, including non-small cell lung cancer, breast
cancer, bladder cancer, lymphomas and leukemias. Additionally,
vincristine is used in combination with actinomycin-D for the
treatment of Wilms’s tumor.
Although vinca-alkaloids are used in clinical practice since the
1960s, there is still a great deal of interest into the pharmacokinetic
properties of these drugs. Van Tellingen et al. (1991) reviewed
analytical methods for the determination of vinca-alkaloids
in biological specimens. This review mainly dealt with methods
using high-performance liquid chromatography (HPLC) coupled
with ultraviolet (UV), fluorescence or electrochemical (ECD) detec-
tion. Nowadays however, HPLC coupled with mass spectrometry
(MS) has become the first choice for the quantitative bioanalysis
of small molecules including the vinca-alkaloids (Stokvis et al.,
2005).
We now present an overview of published methods using LC-
MS for the bioanalysis of the licensed vinca-alkaloids vincristine,
vinblastine, vinorelbine and vinflunine. The vinca-alkaloids
vindesine and vintriptol have been withdrawn from the market
since several years. As no LC-MS methods of these drugs are
described, they are not discussed here.
Bioanalytical LC-MS Assays for Vinca-alkaloids
Bioanalytical quantitative LC-MS assays consist of three compo-
nents: sample pre-treatment, chromatography and detection.
All influence the accuracy, precision, selectivity and sensitivity of
the analytical method. In the following paragraphs, the three
steps of a bioanalytical method will be discussed separately. The
pros and cons of the different approaches for the analysis of
vinca-alkaloids will be considered and supplemented with our
own experience in this field. Table 1 gives an overview of the
published LC-MS methods for vinca-alkaloids.
* Correspondence to: C. W. N. Damen, Department of Pharmacy and Pharma-
cology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg
6, 1066 EC Amsterdam, The Netherlands. E-mail: carola.damen@slz.nl
a
Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Nether-
lands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands
b
Science faculty, Department of Pharmaceutical Sciences, Division of
Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The
Netherlands
c
Department of Medical Oncology, The Netherlands Cancer Institute,
Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
Abbreviations used: LLE, liquid–liquid extraction; PP, protein precipitation;
SPE, solid-phase extraction; TFA, trifluoroacetic acid.
83

Biomed. Chromatogr. 2010; 24: 83–90 Copyright © 2009 John Wiley & Sons, Ltd.

Figure 1. Chemical structures of vinca-alkaloids. Vincristine (A), vinblastine
(B), vinorelbine (C) and vinflunine (D).
Sample Pre-treatment
The sample pre-treatment is an important part for the quantitative
bioanalysis using LC-MS. During sample pre-treatment endo-
genous compounds such as proteins, salts and lipids are removed
from the sample. These compounds can influence the ionization
efficiency of the mass spectrometer and therefore the sensitivity
of the method. The most widely applied techniques are solid-
84
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C. W. N. Damen et al.
phase extraction (SPE), liquid–liquid extraction (LLE) and protein
precipitation (PP). For the bioanalysis of vinca-alkaloids all these
methods are described.
SPE is a chromatographic procedure, based on the same
principles as LC. Because of the availability of a wide range of
cartridges and solvents, SPE is suitable for many analytes with
various properties. For vinca-alkaloids SPE using different mate-
rials is employed. Reversed-phase applications are most often
described. Schmidt et al. (2006) used Strata-X columns for the
bioanalysis of vincristine in plasma. Strata-X is a polymeric mate-
rial with reversed-phase properties. Skolnik et al. (2006) and Lee
et al. (2007) also utilized a polymeric reversed-phase material in
the form of Oasis HLB columns for the simultaneous bioanalysis
of vincristine and actinomycin-D. Oasis HLB columns are also
described in a study about the metabolism of vinorelbine (De
Graeve et al., 2008). Vinorelbine and metabolites are extracted
from urine and bile using these columns. The major advantage
of the polymeric reversed-phase materials is the ease of use,
since there is no need to keep the phases moisturized to main-
tain their interaction capacities. Additionally, they have both
hydrophilic and lipophilic properties and are capable of captur-
ing polar analytes. This is advantageous in the search for meta-
bolites, which are usually more polar, as shown by De Graeve
et al. (2008) for vinorelbine.
Other reversed-phase cartridges used for sample pre-treatment
are Bond-Elut C2 cartridges. Guo et al. (2004) extracted vincristine
from mouse plasma and brain tissue and in our own department
we also used the C2 cartridges for the extraction of vinorelbine
from plasma (Damen et al., 2008). The only described SPE method
not based on a reversed-phase mechanism uses Extrelut-3 car-
tridges (Ragot et al., 2001). Extrelut cartridges contain kieselguhr
as stationary phase and extraction is based on a normal-phase
mechanism (Walker and Mills, 2002). The cartridges are used to
extract vinorelbine, 4-O-deacetylvinorelbine and vinorelbine-
6′-oxide from human serum. We have tested several SPE materials
for the extraction of vinorelbine from plasma (Damen et al., 2008).
The highest recoveries were obtained with Bond-Elut C2, Isolute-CN,
Oasis MAX and Oasis-HLB stationary phases. The reproducibility,
however, was variable. The results with Bond-Elut C2 cartridges
provided the best reproducibility and therefore these were used
for further method development. A major disadvantage of SPE
is, however, that it is a labour-intensive and often complex pro-
cedure. This can be simplified by using an on-line SPE strategy.
Corona et al. (2008) used this approach for sample clean-up. The
on-line SPE cartridge is also based on a reversed-phase mechanism.
A POROS-R1 perfusion column was selected. Unfortunately, this
setup is still labour-intensive as the plasma samples are not injected
directly onto the on-line SPE cartridges, but are first subjected to
a protein precipitation procedure which is performed manually.
Liquid–liquid extraction has been investigated extensively for
vinca-alkaloids. Van Tellingen et al. (1991) described that the
recovery is critically dependent on the pH of the matrix. Plasma
was adjusted to the appropriate pH, which was for vincristine
pH 4–5 and for vinblastine pH 2.5–3.5 followed by extraction
with chloroform. For vinorelbine, however, Van Tellingen reported
that adjusting the pH and extracting with chloroform led to very
poor recoveries. For vinorelbine diethylether appeared to be a
suitable extraction solvent. For LLEs of vincristine, vinblastine
and metabolites, plasma was acidified and extracted with chlo-
roform (Ramirez et al., 1997) or methylene chloride (Dennison
et al., 2008). For vinflunine a different approach is described.
Plasma is not acidified, but alkalinized with sodium hydroxide
Biomed. Chromatogr. 2010; 24: 83–90
 Table 1. Overview of bioanalytical LC-MS methods for vinca-alkaloids

Compound/ Species Sample Sample LC Interface MS IS LLQ Runtime Remarks Reference

matrix (μL) preparation mode (ng/mL) (min)
Mobile phase Column
Vinorelbine

Biomed. Chromatogr. 2010; 24: 83–90

Plasma Human 200 SPE 1 mM NH4Ac, Gemini C18 (5 μm, 50 mm × 2 mm) ESI MRM Vintriptol 0.1 5 (Damen et al., 2008)
pH 10.5:ACN:MeOH
Plasma Mouse 25 SPE 1 mM NH4Ac, Gemini C18 (5 μm, 50 mm × 2 mm) ESI MRM Vintriptol 0.8 5 Mouse plasma (Damen et al., 2008)
pH 10.5:ACN:MeOH diluted with
human plasma
Plasma Human 50 PP 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) H-ESI MRM Vinorelbine-d3 0.1 5 (Damen et al., 2009b)
pH 10.5:ACN:MeOH
Plasma Mouse 50 PP 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) H-ESI MRM Vinorelbine-d3 0.1 5 Cal samples in (Damen et al., 2009b)
pH 10.5:ACN:MeOH human plasma
Serum Human 500 SPE 2 mM NH4For, pH 3:ACN Nucleosil C18 (5 μm, 150 mm × 1 mm) ESI SIM Vinblastine 0.5 15 (Ragot et al., 2001)
Whole blood Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 0.25 20 (Van Heugen et al., 2001)
Plasma Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 0.5 20 (Van Heugen et al., 2001)
Quantitative analysis of vinca-alkaloids in biological matrices

Urine Human 1000 Dilution 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2.5 20 (Van Heugen et al., 2001)
Feces Human 0.1 gr SLE 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2 μg/g 20 (Van Heugen et al., 2001)

4-O-Deacetylvinorelbine
Plasma Human 50 PP 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) H-ESI MRM Vinorelbine-d3 0.1 5 (Damen et al., 2009b)
pH 10.5:ACN MeOH
Plasma Mouse 50 PP 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) H-ESI MRM Vinorelbine-d3 0.1 5 Cal samples in (Damen et al., 2009b)
pH 10.5:ACN MeOH human plasma
Serum Human 500 SPE 2 mM NH4For, pH 3:ACN Nucleosil C18 (5 μm, 150 mm × 1 mm) ESI SIM Vinblastine 1 15 (Ragot et al., 2001)
Whole blood Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 0.25 20 (Van Heugen et al., 2001)

Copyright © 2009 John Wiley & Sons, Ltd.

Plasma Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 0.5 20 (Van Heugen et al., 2001)
Urine Human 1 dilution 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2.5 20 (Van Heugen et al., 2001)
Feces Human 0.1 gr SLE 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2 μg/g 20 (Van Heugen et al., 2001)

20′-Hydroxyvinorelbine
Whole blood Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 0.5 20 (Van Heugen et al., 2001)
Plasma Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 0.5 20 (Van Heugen et al., 2001)
Urine Human 1000 Dilution 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2.5 20 (Van Heugen et al., 2001)

Vinorelbine-6′-Oxide
Whole blood Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2.5 20 (Van Heugen et al., 2001)
Plasma Human 500 PP 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 2.5 20 (Van Heugen et al., 2001)
Urine Human 1000 Dilution 40 mM NH4Ac, pH 3:ACN Spherisorb CN (3 μm, 100 mm × 4.6 mm) ESI MRM Vinblastine 12.5 20 (Van Heugen et al., 2001)
Serum Human 500 SPE 2 mM ammonium formate, Nucleosil C18 (5 μm, 150 mm × 1 mm) ESI SIM Vinblastine 0.5 15 (Ragot et al., 2001)
pH 3:ACN

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85
 86

Table 1. (Continued)

Compound/ Species Sample Sample LC Interface MS IS LLQ Runtime Remarks Reference

matrix (μL) preparation mode (ng/mL) (min)
Mobile phase Column
Vinblastine
Plasma Human 1500 LLE 15 mM NH4Ac:ACN Ultrasphere C18 (5 μm, 150 mm × 2 mm) APCI SIM Vinorelbine 0.51 4 (Ramirez et al., 1997)

4-Deacetylvinblastine
Plasma Human 1500 LLE 15 mM NH4Ac:ACN Ultrasphere C18 (5 μm, 150 mm × 2 mm) APCI SIM Vinorelbine 0.74 3 (Ramirez et al., 1997)

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Vincristine
Plasma Human 100 PP-on- 25 mM NH4Ac:0.3% Luna Phenyl-Hexyl (5 μm, 50 mm × 2.1 mm) APCI MRM Vinblastine 0.1 5 (Corona et al., 2008)
line SPE HCOOH:MeOH
Plasma Human 30 PP 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) ESI MRM Vinorelbine 0.25 6 Simultaneous (Damen et al., 2009a)
pH 10.5:ACN:MeOH actinomycin-D
Plasma Human 100 Diltution 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) ESI MRM Vinorelbine 1 6 Simultaneous (Damen et al., 2009a)
Ultrafiltrate pH 10.5:ACN:MeOH actinomycin-D
Dried Blood Human 15 SLE 1 mM NH4Ac, Xbridge C18 (5 μm, 50 mm × 2.1 mm) ESI MRM Vinorelbine 1 6 Simultaneous (Damen et al., 2009c)
Spot pH 10.5:ACN:MeOH actinomycin-D
Plasma Human 500 LLE 0.2% HCOOH:MeOH Inertsil ODS-3 C18 (5 μm, 150 mm × 2.1 mm) TIS MRM Vinblastine 0.012 20 (Dennison et al., 2008)
Plasma Mouse 10 SPE 15 mM NH4Ac:0.02% Luna C8 (3 μm, 50 mm × 2 mm) ESI SIM Vinblastine 10 6 (Guo et al., 2004)
HCOOH:MeOH
Brain Mouse 0.1 gr SPE 15 mM NH4Ac:0.02% Luna C8 (3 μm, 50 mm × 2 mm) ESI SIM Vinblastine 10 ng/g 6 (Guo et al., 2004)
HCOOH:MeOH
Plasma Human 500 SPE 5 mM NH4Ac, pH3.5:MeOH YMC ODS-AQ C18 (3 μm, 100 mm × 2 mm) TIS MRM Vinorelbine 0.05 8 Simultaneous (Lee et al., 2007)
actinomycin-D
Plasma Human 2000 LLE 15 mM NH4Ac:ACN Ultrasphere C18 (5 μm, 150 mm × 2 mm) APCI SIM Vinorelbine 0.30 3 (Ramirez et al., 1997)
Plasma Human 1000 SPE 0.1% TFA:MeOH:ACN Polar RP (4 μm, 250 mm × 2 mm) APCI SIM Vinblastine 0.18 10 (Schmidt et al., 2006)
Plasma Human 500 SPE 1% HAc, pH 4:MeOH Luna C8 (3 μm, 50 mm × 2 mm) TIS MRM Vinblastine 0.5 18 Simultaneous (Skolnik et al., 2006)
actinomycin-D
Vincristine-amide

Copyright © 2009 John Wiley & Sons, Ltd.

Plasma Human 500 LLE 0.2%HCOOH:MeOH Inertsil ODS-3 C18 (5 μm, 150 mm × 2.1 mm) TIS MRM Vinblastine 0.012 20 (Dennison et al., 2008)

Vinflunine
Plasma Rat 100 LLE 10 mM NH4Ac:MeOH Shim-pack C18 (5 μm, 150 mm × 4.6 mm) ESI SIM Finasteride 2.5 8 (Zhao et al., 2006)
Whole Blood Human 500 PP 40 mM NH4Ac:, pH3:ACN Spherisorb CN (5 μm, 10 mm × 2 mm) ESI MRM Vinblastine 0.25 25 (Zorza et al., 2007)
Urine Human 10,000 Dilution 40 mM NH4Ac:, pH3:ACN Spherisorb CN (5 μm, 10 mm × 2 mm) ESI MRM Vinblastine 50 25 (Zorza et al., 2007)
Feces Human 0.1 gr SLE 40 mM NH4Ac:, pH3:ACN Spherisorb CN (5 μm, 10 mm × 2 mm) ESI MRM Vinblastine 50 μg/g 25 (Zorza et al., 2007)

4-O-deacetylvinflunine
Whole blood Human 500 PP 40 mM NH4Ac:, pH3:ACN Spherisorb CN (5 μm, 10 mm × 2 mm) ESI MRM Vinblastine 0.20 25 (Zorza et al., 2007)
Urine Human 10,000 dilution 40 mM NH4Ac:, pH3:ACN Spherisorb CN (5 μm, 10 mm × 2 mm) ESI MRM Vinblastine 50 25 (Zorza et al., 2007)
Feces Human 0.1 gr SLE 40 mM NH4Ac:, pH3:ACN Spherisorb CN (5 μm, 10 mm × 2 mm) ESI MRM Vinblastine 50 μg/g 25 (Zorza et al., 2007)

ACN, acetonitrile; APCI, atmospheric pressure chemical ionization; Cal samples, calibration samples; ESI, electrospray ionization; HAc, acetic acid; HCOOH, formic acid; H-ESI, heated electrospray ionization; IS, internal standard; LC,
liquid chromatography; LLE, liquid–liquid extraction; LLQ, lower limit of quantification; MeOH, methanol; MRM, multiple reaction monitoring; NH4Ac, ammonium acetate; NH4For, ammonium formate; PP, protein precipitation; SIM,
selective-ion monitoring; SLE, solid–liquid extraction; SPE, solid-phase extraction; TFA, trifluoroacetic acid; TIS, turbo ionspray.

Biomed. Chromatogr. 2010; 24: 83–90

C. W. N. Damen et al.
Quantitative analysis of vinca-alkaloids in biological matrices
and then extracted with ethyl acetate (Zhao et al., 2006). We have
also tested LLE with diethylether for the bioanalysis of vinorel-
bine (Damen et al., 2008), but experienced large batch-to-batch
variations in extraction recovery and MS ion suppression between
plasma samples of different volunteers.
The major advantages of both SPE and LLE are that relatively
clean samples are obtained, which prevents ion suppression in
the mass spectrometer. Additionally, the analytes are often present
in an organic solvent at the end of the extraction procedure. After
evaporation the residue is redissolved in a solvent compatible
with the LC-MS system. The analyte can be concentrated in the
final extract, resulting in a lower limit of quantification. However,
both SPE and LLE are very time-consuming.
Protein precipitation is the simplest means of bioanalytical
sample pre-treatment. It only involves the addition of a precipi-
tating solvent, subsequent homogenizing and centrifugation.
The clear supernatant can be injected on to the LC-MS system.
Van Heugen et al. (2001) and Zorza et al. (2007) both use the same
protein-precipitation procedure for the extraction of vinorelbine,
vinflunine and metabolites from blood and plasma. These plat-
forms however use 6 mL of a methanol-acetonitrile mixture for
the precipitation of 500 μL plasma or whole blood. This large
dilution step requires a time-consuming evaporation step. We
have also investigated protein-precipitation procedures and
for vinorelbine we found that multiple injections of these final
extracts resulted in rapid decline of the performance of the
analytical column (Gemini C18) (Damen et al., 2008). After using a
more robust HPLC column (Xbridge C18) and a stable isotopically
labeled internal standard a fast protein precipitation method for
vinorelbine and the major metabolite with very low quantification
limits could be designed (Damen et al., 2009b). This procedure
was also applied for the simultaneous bioanalysis of vincristine
and actinomycin-D from plasma (Damen et al., 2009a).
Urine can be diluted with an appropriate buffer and directly
injected on the LC-MS system (Van Heugen et al., 2001; Zorza
et al., 2007). Extraction of vinorelbine, vinflunine and metabolites
from feces has also been described. To perform a solid–liquid
extraction (SLE), feces was homogenized and sonicated in
methanol–ammonium acetate buffer pH 3 and centrifuged. The
supernatant was diluted and injected on to the HPLC system
equipped with MS detection.
All vinca-alkaloids possess a high protein binding. With the
use of protein precipitation, LLE or SPE procedures, the total
concentration in plasma, serum or blood is determined. However,
only the protein-unbound fraction of the drug is considered
pharmacologically active, thus it may be essential to determine
selectively the unbound drug concentration (Liu and Huang, 1999).
A bioanalytical method for the quantification of the protein
unbound fraction of vincristine was developed by us (Damen
et al., 2009a). A plasma-ultrafiltrate using centrifugal filter units
was prepared. Molecules with a size smaller than the cut-off of
the filter can pass though the column, while larger molecules are
retained on top of the filter. Therefore, free vincristine can pass
through the filter while vincristine bound to plasma proteins will
be retained. The obtained plasma-ultrafiltrate was diluted with
methanol to prevent loss of the drug by absorption effects and
injected on to the LC-MS system. The lower limit of quantification
(LLQ) of this method was 1 ng/mL for vincristine, which is a factor
of 10 higher than the LC-MS method with plasma as sample
matrix (Damen et al., 2009a). This difference can be explained by
the fact that for plasma ion-enhancement in the MS was observed
while for plasma-ultrafiltrate ion-suppression was observed. This
Biomed. Chromatogr. 2010; 24: 83–90 Copyright © 2009 John Wiley & Sons, Ltd.
result is surprising as plasma-ultrafiltrate is a much cleaner
matrix than a protein-precipitate plasma sample. The vincristine
in the analysed ultrafiltrate samples was approximately 30%
compared with the concentration in plasma samples, leading
to the conclusion that a protein binding of approximately 70%
was observed (Damen et al., 2009a).
A promising new development in bioanalysis of small mole-
cules is the use of dried blood spots (Spooner et al., 2009; ter
Heine et al., 2008). This procedure using sampling on collection
cards has many advantages. Samples are obtained from a finger-
prick or heelprick with an automatic lancet, which is less invasive
to a patient compared with a venipuncture. This maybe preferable
in populations where intensive sampling by means of venipuncture
may be difficult, for example, in children, neonates and patients
who are suffering from phlebitis or abscesses at injection sites.
Additionally, patients can obtain samples themselves at home
and send the samples by mail (Knudsen et al., 1995). Sample
pre-treatment is fast and easy. Punched out disks the dried blood
spots are sonicated in extraction solvent. This extract can be
directly injected on to the LC-MS system. In our department
we developed such a bioanalytical method for vincristine and
actinomycin-D in dried blood spots (Damen et al., 2009c). This
method is now used to support a pharmacokinetic study in
malnourished children in Malawi. The stability of the compounds
in dried blood spots has been demonstrated for at least 3 months
at ambient temperatures and at 40–45°C. For pharmacokinetic
studies of other vinca-alkaloids this dried blood spot sampling
method and analysis may also be applicable.
Chromatography
The major goal of chromatography in bioanalysis is the separation
of analytes from endogenous compounds. These endogenous
compounds can cause matrix effect in MS detection. Separation
of the analyte, possible metabolites and the internal standard
from each other is in most cases not necessary due to the selec-
tivity of the mass spectrometer. This allows short run times, which
promote high throughput analysis. Ion-exchange chromatography
on unmodified silica has been reported (Van Tellingen et al., 1991)
as the most favorable LC system for the bioanalysis of vinca-alkaloids
with ECD and fluorescence detection. MS, however, limits the
choice of eluents to volatile solvents and therefore ion-exchange
chromatography is usually not suitable for LC-MS platforms. Fur-
thermore, it has to be taken into account that mobile phase
additives such as trifluoroacetic acid (TFA), which is often used
as ion-pairing agent, reduces the MS signal dramatically (Stokvis
et al., 2005).
In order to maintain stable retention times on a HPLC column
the pH of the eluent should be two units above or below the
pKa of the analytes. Additionally, the general thought is that for
positive ionization mass spectrometry, acidic eluents are most
appropriate as they are able to donate a proton. Therefore,
acidic eluents with ammonium acetate, ammonium formate,
formic acid, acetic acid and even TFA are often described for the
bioanalysis of vinca-alkaloids (see Table 1). However, alkaline
mobile phases in combination with positive ionization also appeared
well suited for the bioanalysis of weak basic drugs and can lead
to improved chromatography (asymmetry factors) and therefore
to higher signal-to-noise ratios (Stokvis et al., 2005). Analyte
protonation in the MS occurs because the vinca-alkaloids (and
metabolites) are stronger gas-phase bases than NH3. Conse-
quently, in the gas-phase MH+ and NH3 are present instead of
87
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 C. W. N. Damen et al.

NH4+ and M (Zhou and Cook, 2000). Vinca-alkaloids are weak

basic compounds and therefore the use of alkaline mobile
phases was used for analysis of vincristine, vinorelbine and its
metabolite 4-O-deacetylvinorelbine (Damen et al., 2008, 2009a).
With an RP C18 HPLC column coupled to a mass spectrometer,
higher signal-to-noise ratios were obtained using an alkaline
mobile phase than an acidic mobile phase (Damen et al., 2008,
2009a).
Different stationary phases have been employed for the bio-
analysis of vinca-alkaloids. Most platforms use reversed-phased
columns with either C8 or C18 material. Additionally, a CN column,
a phenyl-hexyl column and a polar reversed-phase are described
(see Table 1). For high-throughput analysis a short run-time is of
paramount importance. Run times above 10 min are usually not
necessary as proved by many reports discussed in this review
with excellent quantification performances (Table 1).

Detection
For quantitative bioanalysis of small molecules triple quadrupole
mass spectrometry is the detector of first choice due to its high
sensitivity and selectivity (Stokvis et al., 2005). For mass spectro-
metric detection, the analytes have to be ionized first. For
vinca-alkaloids electrospray ionization (ESI) is the most often
used technique. A variant of ESI using a heated gas for desolvation
of the eluent is called turbo ionspray (TIS) or heated electrospray
ionization (H-ESI). These ionization methods can handle LC eluent
flow rates up to 1 mL/min without the need for a prior eluent
split. Atmospheric pressure chemical ionization (APCI) is only
described for vincristine and vinblastine (Corona et al., 2008;
Ramirez et al., 1997; Schmidt et al., 2006). APCI ionization is based
on a principle generating ions via chemical ionization in the gas
phase (Bruins, 1994). We have compared the signal-to-noise ratios
(S/N) for vincristine and vinorelbine using ESI and APCI (Damen
et al., 2009a). For both compounds ESI was slightly more sensi-
tive than APCI. The major gain was, however, reached by using
H-ESI. For the quantification of vinorelbine and its metabolite
4-O-deacetylvinorelbine high LC eluent flow (0.4 mL/min) was
used and therefore ionization efficiency improved using a heated
gas (Damen et al., 2009b). The use of this heated gas enhances
desolvation of the eluent and liberates more analyte ions, leading
to improvement of the ionization efficiency resulting in higher Figure 2. MS/MS product ion scans of vinblastine (A, precursor ion m/z
S/N ratios (Cech and Enke., 2001; Page et al., 2007). However, abso- 811), vincristine (B, precursor ion m/z 825), and vinorelbine (C, precursor
lute signals using H-ESI are variable and it is advised to use stable ion m/z 779).
isotopically labeled internal standard, if available, to improve the
precision of the method.
Once the ions have entered the mass spectrometer, detection 2008, 2009b; Van Heugen et al., 2001) and corresponds to the open-
takes place. This can be accomplished by means of selecting the ing of the large ring with seven carbon atoms and one nitrogen
molecular ion in selective ion monitoring mode (SIM), but pref- atom at the Catharantine part of the molecule (Fig. 2). There is
erably a typical fragment ion is selected in multiple reaction much tension in this ring system and this explains why this part
monitoring (MRM). This leads to higher selectivity and sensitivity of the molecule will fragment first. All other fragments are minor
than in SIM mode (Table 1). The methods using MRM describe a fragments with a relative abundance less than 30% compared
lower LLQ than the methods using SIM. Only Schmidt et al. (2006) with the abundance at m/z 122 (De Graeve et al., 2008).
report a method for the quantification of vincristine in plasma The fragmentation of vinflunine and metabolites occurs in a
with a LLQ of 0.18 ng/mL using SIM. This method, however, uses similar way as the fragmentation of vinorelbine as the compounds
a large sample volume of 1 mL plasma and time-consuming SPE differ only two fluor atoms vs two hydrogen atoms [Fig. 1(C, D)].
where the analyte is concentrated 10 times. For vinflunine the major MS/MS fragment is found at m/z 160
When MRM is applied, fragmentation of the analytes takes place. instead of m/z 122 (Zorza et al., 2007).
Figure 2 shows the MS/MS fragmentation pattern of the vinca- Zhou et al. (2005) described a method for the identification of
alkaloids. The MS fragmentation for vinorelbine and metabolites vinca-alkaloids from a crude extract of the Catharanthus roseus
was studied extensively by De Graeve et al. (2008). The major plant. The MS/MS fragmentation of the natural occurring vinca-
fragment is found at m/z 122 (Damen et al., 2008; Damen et al., alkaloids vinblastine and vincristine is discussed in that paper.
88

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 83–90
Quantitative analysis of vinca-alkaloids in biological matrices
The major fragment obtained for vincristine is m/z 807 (Damen
et al., 2009a, c; Lee et al., 2007; Zhou et al., 2005), which corresponds
to the loss of water from the molecule. The second most intense
fragment is found at m/z 765 (Damen et al., 2009a, c; Lee et al., 2007;
Zhou et al., 2005) and corresponds to the loss of -CO2CH3, which
can occur at three positions in the molecule (Fig. 2). Only Dennison
et al. (2008) report for vincristine that the parent ion and all
fragment ions are doubly charged. Unfortunately, no explanation
is given for this phenomenon in their paper. An API4000 mass
spectrometer was used with TIS and the mobile phase consisted
of 0.2% formic acid in water and methanol. An acidic eluent was
also described in other papers (Lee et al., 2007; Zhou et al., 2005)
where only singly charged ions were found.
For vinblastine fragmentation occurs in the same way as for
vincristine. The major fragment is found at m/z 751 (Trim et al.,
2008; Zhou et al., 2005) and doubly charged at m/z 376.2 (Dennison
et al., 2008), which corresponds with the loss of –CO2CH3. Addi-
tionally, the loss of water has also been described and corresponds
with m/z 793 (Zhou et al., 2005).
Internal Standards
To correct for variations during the extraction procedure and
subsequent LC-MS analysis an internal standard is needed. A
stable isotopically labeled internal standard is favorable in MS
detection. The synthesis of vinca-alkaloids is, however, very com-
plex and therefore until 2008 no stable isotopes of the analytes
were available. In 2008, however, vinorelbine-d3 became com-
mercially available (Toronto Research Chemicals, Canada) and
was successfully used for the bioanalysis of vinorelbine (Damen
et al., 2009b). The major advantage of the deuterated internal
standard was that protein precipitation could be applied instead
of labour-intensive SPE and that H-ESI could be used instead of
ESI (Damen et al., 2008, 2009b). With the H-ESI higher S/N ratios were
obtained than with the ESI leading to more sensitivity. Therefore,
the same detection limit could be reached using 4 times less
sample volume. In view of the chemical similarities, vinflunine
and vinorelbine may serve as potential internal standards for
their quantification.
Almost all papers use a therapeutic vinca-alkaloid for the
quantification of the other vinca-alkaloid (Table 1). Limitations,
however, are reported when using vinblastine as internal stan-
dard for vincristine. Lee et al. (2007) mentioned interference of a
degradation product in the MRM transition of vincristine when
using vinblastine as internal standards. We experienced the same
problem and came to the conclusion that this degradation prod-
uct is vincristine itself (Damen et al., 2009a). When vinblastine
oxidizes, vincristine is formed (Kuboyama et al., 2004; see also
structures A and B in Fig. 1). Vinblastine from different suppliers
was tested, but all contained some vincristine, which interfered
in the development of a very sensitive vincristine assay. There-
fore, vinorelbine was selected as internal standard. (Damen et al.,
2009a; Lee et al., 2007). Dennison et al. (2008) experienced the
same problem and solved it by HPLC purification of vinblastine.
Complications in the Bioanalysis of Vinca-alkaloids
Van Heugen et al. (2001) and Zorza et al. (2007) reported absorption
of vinca-alkaloids to the walls of test tubes and advised to use silicon
coated glass tubes in order to avoid this problem. An alternative
is to store all stock solutions in methanol and prepare all further
dilutions in plasma (Van Tellingen et al., 1991). Using this procedure,
Biomed. Chromatogr. 2010; 24: 83–90 Copyright © 2009 John Wiley & Sons, Ltd.
no absorption effects were observed in this way when using
Eppendorf tubes for vinorelbine (Damen et al., 2008). Also after
SPE and evaporation to dryness, no problems were encountered
re-dissolving vinorelbine during reconstitution. For vincristine,
however, this approach was not successful. In order to develop a
very sensitive vincristine assay an evaporation step was imple-
mented making concentration of the sample possible. However,
when using Eppendorf tubes, vincristine could not be re-dissolved
after evaporation. Sonication, shaking or vortex mixing with many
different organic or aqueous solvents did not yield dissolution. A
protein precipitation procedure without an evaporation step
was then developed (Damen et al., 2009a).
Vinorelbine adheres not only to test tubes, but also interacts
strongly with the stationary phase of the LC column. Using gradient
elution we observed carry-over due to a memory effect on the
LC column. By applying the same gradient every time the vinorel-
bine that remained on the column elutes every time with the
same retention time. Therefore, isocratic elution was applied
(Damen et al., 2008; Damen et al., 2009b). Vinorelbine was also used
as internal standard for vincristine and actinomycin-D (Damen
et al., 2009a, c). In that assay gradient elution was applied and
the vinorelbine concentration was low to minimize the carry-over
of the internal standard.
Conclusion
Although HPLC methods coupled to UV, fluorescence or ECD
detection were successfully applied in the past, LC coupled to
tandem mass spectrometry is now the method of first choice for
the quantification of vinca-alkaloids in biological matrices. The
selectivity and sensitivity of the mass spectrometer enables us
to improve the assessment of the pharmacokinetics of these
compounds. Additionally, because of the improved sensitivity
a smaller sample volume can be used, which is favorable in
pre-clinical studies, but also in studies with, for example, children
with Wilms’s tumor who are treated with the combination of
vincristine and actinomycin-D.
As sample pre-treatments SPE, LLE and PP are described. All
three methods have their positive and negative points. Although
PP is by far the fastest and easiest method, it does not yield very
clean samples, which can be a limitation for ion-suppression and
also for HPLC column lifetime and MS performance in time. Plasma
ultrafiltrate can be used to determine the protein unbound fraction
of vinca-alkaloids, which can be of interest as the drugs are highly
plasma protein bound. A new and promising technique, which
makes easy sampling possible, is the use of dried blood spots.
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