Analytical Biochemistry 610 (2020) 113891

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Review article

HPLC methods for quantifying anticancer drugs in human samples: A

systematic review
Reyhaneh Sabourian a, b, Seyedeh Zohreh Mirjalili a, b, Negar Namini a, Fateme Chavoshy a,
Mannan Hajimahmoodi a, **, Maliheh Safavi c, *
a
Drug and Food Control Department, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b
Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran
c
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Pharmacokinetic (PK) study of anticancer drugs in cancer patients is highly crucial for dose selection and dosing
HPLC intervals in clinical applications. Once an anticancer drug is administered, it undergoes various metabolic
UV pathways; to determine these pathways, it is necessary to follow the administered drug in biological samples via
Mass
different analytical methods. In addition, multi-drug quantification methods in patients undergoing multi-drug
Anticancer
Multi-drugs
regimens of cancer therapy can have several benefits, such as reduced sampling time and analysis costs. In
Metabolite order to collect and categorize these studies, we conducted a systematic review of HPLC methods reported for the
analysis of anticancer drugs in biological samples. A systematic search was performed on PubMed Medline,
Scopus, and Web of Science databases, and 116 studies were included. In summary of included studies, when the
objective of a method was to quantify a single drug, MS, or UV detectors were utilized equivalently. On the other
hand, in methods with the aim of quantifying drug and metabolite(s) in a single run, MS detectors were the most
utilized. This review can provide a comprehensive insight for researchers prior to developing a quantification
method and selecting a detector.

1. Introduction Moreover, metal-based drugs, such as carboplatin and oxaliplatin, are

After cardiovascular diseases, cancer is the second most deadly dis­
ease and responsible for millions of global deaths [1]. In 2014, the World
Health Organization (WHO) reported that the worldwide cancer inci­
dence increased from 12.7 million in 2008 to 14.1 million in 2012 [2]. In
addition, cancer grew to 18.1 million new cases and 9.6 million deaths
in 2018 [3]. In cancer disease, control of cell proliferation is lost, leading
either to a solid tumor or to liquid tumors. The main treatments for
cancer are surgery, chemotherapy, and/or radiotherapy [4]. The pur­
pose of chemotherapy is to selectively annihilate tumor cells or at least
limit their growth [5]. Anticancer drugs were categorized according to
their action mechanism, such as alkylating agents, antibiotics, micro­
tubule inhibitors, antimetabolites, hormones, and hormone antagonists
[6]. In another category, these drugs were divided into molecular tar­
geting agents, including monoclonal antibodies, small molecule in­
hibitors, gene delivery agents, and histone deacetylase inhibitors [7].
currently used in the treatment of cancer. Drugs of this ilk can be
categorized as metal-DNA or metal-protein adducts [8].
High reactivity of conventional anticancer drugs is responsible for
their expected effect, cell toxicity, and responsible for their main side
effects. Furthermore, this characteristic makes them significantly un­
stable and toxic. Special considerations have been made about their
safety and quality [9]. Therefore, the necessity of analytical methods for
therapeutic drug monitoring (TDM) of anticancer drugs is of great
importance [5]. In addition, pharmacokinetic (PK) study of anticancer
drugs in cancer patients is essential for selecting doses and dosing in­
tervals in clinical applications [10].
Following the administration of an anticancer drug, it undergoes
various metabolic pathways, either inactivating drug substance to pro­
duce by-products, or producing the active metabolite for its pharma­
ceutical actions. To specify these pathways, it is necessary to follow the
administered drug in biological samples via different analytical methods

* Corresponding author. P. O. Box 33535, Sh.Ehsani Rad., Enqelab St., Parsa Sq., Ahmadabad Mostoufi Rd., Azadegan Highway, Tehran, 3313193685, Iran
** Corresponding author. P. O. Box: 14155-6451, Tehran, 14174, Iran.
E-mail addresses: Hajimah@sina.tums.ac.ir, Hajimah@sina.tums.ac.ir (M. Hajimahmoodi), m.safavi@irost.ir (M. Safavi).

https://doi.org/10.1016/j.ab.2020.113891
Received 17 April 2020; Received in revised form 9 July 2020; Accepted 24 July 2020
Available online 5 August 2020
0003-2697/© 2020 Elsevier Inc. All rights reserved.
R. Sabourian et al.
[11]. Anticancer drug monitoring in biological samples has three pur­
poses: developing new drugs, TDM, and monitoring the exposed
healthcare professionals [5]. To achieve the mentioned goals, the most
important human biological samples for monitoring anticancer drugs
are blood, urine, tissue, saliva, and cerebrospinal fluid (CSF), and the
same goes for anticancer drugs [11]. Drug analysis, which is a major
subdivision of analytical chemistry, is the basis of drug quality control.
Literature includes many different analytical methods for quantifying
anticancer drugs in biologic samples, such as high performance liquid
chromatography (HPLC), gas chromatography (GC) [12], and capillary
electrophoresis (CE). This comprehensive review describes the appli­
cation of HPLC methods for determining anticancer drugs in biological
samples.
2. Material and methods
2.1. Eligibility criteria
In order to achieve the objective of this study, we reviewed the HPLC
methods reported for the analysis of anticancer drugs in biological
samples. As inclusion criterion, at least one anticancer drug should be
assayed in the included primary studies. We discriminated against
approved drugs for different types of cancer based on the classification
of the US National Cancer Institute [13]. All primary studies including
“high performance liquid chromatography” systems for the separation of
anticancer drugs in human biological samples were entered in this re­
view without any language restriction. As a result, the articles using
other chromatography systems were excluded. In vitro and animal
studies were further excluded. There was no restriction on the age and
sex of patients entered in the primary studies. Biological and radio­
labeled drugs were excluded from this study because of the different
structure and separation methods.
2.2. Search strategy
An electronic and manual search on PubMed Medline, Scopus, and
Web of Science databases and the reference list of the included studies
was conducted. Titles and abstracts were then examined to remove
obviously irrelevant studies. Ultimately, full-texts were separately
examined for compliance of studies with inclusion criteria of the review;
afterwards, cases of disagreement were judged (Supplementary data 1.).
3. Theory: bioanalytical assay steps
In general, quantitative and bioanalytical assays include three steps:
the first step is sample preparation for analysis, the second is the sepa­
ration of analytes in a chromatographic column, and the third is the
detection of separated analytes in a suitable and sensitive detector.
Finally, the developed method is useable in clinical studies if it has been
validated and the ensued results are accurate, precise, and reproducible.
All these steps are effective on validation parameters, such as precision,
selectivity, accuracy, and linearity of bioanalytical methods.
3.1. Sample preparation
Sample preparation is an inevitable step of drug analysis in biological
samples, and development of these methods has recently become more
important due to the increased demand for accuracy, precision,
repeatability, and reliability of the analysis methods [14].
A biologic sample consists of thousands of components, including
carbohydrates, proteins, lipids, salt, and other endogenous and back­
ground compounds. In most biological samples, these components are
present in large amounts and can interfere with the desired trace analyte
that can ensure a matrix effect, where its removal is the first objective of
sample preparation steps prior to sample injection. In addition, low
analyte concentration increases the demand of sample preparation for
2
Analytical Biochemistry 610 (2020) 113891
improving the sensitivity. Moreover, some analytes are unstable in
biologic samples, and through changing the solvent during these
methods, the stability of the target analyte can be enhanced [15]. In the
studies included in this article, the most commonly used samples were
plasma, serum, and urine.
Urine is made of water, ions, creatinine, urea, certain components
generated in the liver and kidney, such as drugs and their metabolites
[16]. It has some advantages over serum. For instance, urine sampling is
non-invasive, and a sufficient number of samples is available. In addi­
tion, it is simple and does not require complicated sample preparation
steps. Therefore, urine can be utilized extensively during drug evalua­
tion studies. Another important sampling method, particularly for
quantitative analysis, is blood, plasma, or serum owing to the excellent
correlation between drug concentration and its pharmacologic action. In
addition to venipuncture blood sampling, the use of dried blood spot
(DBS) biosampling has recently increased for quantitative purposes. In
this method, blood is deposited in a special card and let to dry; more­
over, before analysis, a circle is punched out from the DBS and extracted
for analysis. This approach has several benefits, such as requiring low
sample volume, no need for special equipment to store samples, and
more stability of samples. Moreover, extraction of analyte from these
samples requires low volumes of solvents, making it a cost-effective and
green method [17]. Other such samples as saliva, feces, and CSF were
used in this article.
During sample preparation for chromatographic analysis, some ac­
tions of the former step can be considered as sample pretreatment, such
as dilution, centrifugation and filtration, pH adjustment. The sample
preparation steps, including SPE, LLE, PPT, SPME, LPME, and other
methods should follow the mentioned steps.
Promoting chromatographic techniques and reducing the run time of
chromatography, sample preparation is the rate-limiting step of bio­
analysis. To overcome this issue, several methods are available such as
solid-phase extraction (SPE), liquid-liquid extraction (LLE), and protein
precipitation (PPT).
In LLE, two immiscible liquid phases and in SPE, a liquid and a solid
phase are used to separate the analyte from other interferes. These
sample preparation techniques facilitate the concentration of analyte in
ready-to-inject samples. SPE reduces the solvent usage and extraction
time in comparison with LLE [18]. Different solid phases (or matrixes)
and solvents can be employed in SPE, hence its adaptability to several
drugs; however, it is often a complex procedure comprising three or four
sequential steps. LLE is preferred to lipophilic drugs since the analyte
migrates from usually aqueous biologic samples to the organic solvent.
Another method, which is a straightforward and the simplest sample
preparation technique, is PPT. This method includes the addition of a
precipitating solvent (methanol (MeOH), acetonitrile (ACN), or a
perchloric acid solvent), followed by homogenization and centrifugation
[19]. Because of the high protein content of biologic samples, protein
precipitation is applied extensively to remove and precipitate the
endogenous proteins while this method is not highly selective. However,
PPT is fast and simple and does not need any special equipment; it is still
a widespread method in sample preparation steps [14]. In addition,
many examples of metabolite determination using PPT can be mainly
found in urine, serum, and PK studies, such as microsomes and hepa­
tocytes [20–22]. Semi-automated combination of PPT and SPE proced­
ure in plate format has been applied in the metabolism studies of various
drugs [23].
Due to the mentioned complicated extraction steps during sample
preparation of biologic samples, it is suggested that an internal standard
(IS) be utilized. A proper IS has certain characteristics: it is not an
endogenous compound and has physicochemical properties similar to
the target analyte.
3.2. High performance liquid chromatography (HPLC)
Liquid chromatographic (LC) methods have the benefit of high
R. Sabourian et al.
separation capacity, hence their repeated use for the analysis of anti­
cancer drugs [24]. HPLC is the advanced form of LC employed for
separating specific molecules in complex mixtures such as biological
fluids. Owing to its remarkable selectivity and sufficient precision, HPLC
is the most applied LC method for the analysis of drugs; reversed-phase
HPLC (RP-HPLC) is certainly the most common technique in pharma­
ceutical drug development such as analysis of drug substances in bio­
logical samples [25,26]. Of note, most of the methods reviewed in this
study employed reversed-phase (RP) columns such as C18 or C8.
Furthermore, HPLC columns are usually filled with 3–5 μm particles;
however, the ultra performance liquid chromatography (UPLC) tech­
nique is based on the use of a stationary phase comprised of particles less
than 2.0 μm [27]. Accordingly, we concentrated on HPLC separation
methods, and other separation techniques such as UPLC were excluded
due to the different required backpressures and the ensued resolution.
Frequently used mobile phases for analysis contain volatile buffers or
acids, such as ammonium acetate, ammonium formate, and acetic acid
(AA) or formic acid. Organic solvents, such as ACN and MeOH, are often
utilized with either gradient or isocratic elution modes in HPLC sepa­
ration methods.
3.3. Detection
In this article, most of the reviewed methods employed mass spec­
trometry (MS) detection for quantitative assays. Tandem mass spec­
trometry (MS/MS) involves two or more MS detectors coupled to
achieve better sample analysis and consists of triple quadrupole mass
spectrometer (QqQ), quad time of flight (Q-TOF), and hybrid mass
spectrometer. Here, most of the reviewed studies used QqQ mass spec­
trometry. When a QqQ system is operated in the multiple reaction
monitoring mode (MRM), the analyte is detected by either its molecular
ion or a typical fragment ion. This results in better sensitivity and higher
selectivity compared with any other MS systems. The second most used
methods in the present review were applied Ultraviolet (UV) detectors
and validated methods using UV detectors, which are the most feasible
methods worldwide. The photodiode array (PDA) detector is a special
form of UV detectors that record full UV to visible spectra. Other
detection methods including fluorescence detectors were further
included.
3.4. Validation
After a bioanalysis method is developed, it should be validated
before application into clinical pharmacological studies and routine use.
Validation is necessary for confirming the accuracy and precision of the
obtained data [28]. Food and Drug Administration (FDA) published
guidelines for the validation of bioanalytical assays in 2001, and the last
available version of this guideline was published in May 2018 [29,30].
Moreover, international conferences for harmonization (ICH) endorsed
the bioanalytical method validation in October 2016. The final edition
of this guideline can be applied to the validation of bioanalytical
methods in clinical and non-clinical studies; this edition involves vali­
dation parameters such as specificity, selectivity, range, accuracy, pre­
cision, carry over, dilution linearity, hook effect, and stability. Based on
the ICH guideline, a full validation should be performed to develop a
bioanalytical method for assaying an analyte in clinical and critical
nonclinical studies. In addition, when an analytical method is developed
for report in the literature, it should be fully validated. The guidelines
delineate which parameters should be evaluated, based on the aim of
developed bioanalytical method, how they should be assessed, and the
requirements to be met. Generally, one analyte has to be measured, but
it is preferable that more than one analyte be measured if possible. This
includes the enantiomers or isomers of a drug, a drug with its metabolite
(s) or two different drugs. In these circumstances, the principles of
method validation apply to all the selected analytes [31].
3
Analytical Biochemistry 610 (2020) 113891
4. Results and discussion
The analysis methods developed for the quantification of anticancer
drugs, were reviewed in this study. We categorized the obtained studies
based on the number of analytes simultaneously quantified in a single
run. Of the 116 included studies, 47 studies determined a single anti­
cancer drug, 38 studies measured an anticancer drug with its metabolite
(s) simultaneously, and 32 studies quantified multi-drug in a single run
(Supplementary data 2.).
4.1. Single drug quantification methods
Anticancer drugs have various structures and physicochemical
properties. Table 1 shows the structure of anticancer drugs. Because of
the structural variations in these drugs, it is not possible to find the most
optimal approach to their extraction, separation, and detection. Partic­
ularly when a method aims to quantify the biologic samples, it is critical
that the method be able to quantify the analyte with high selectivity and
sensitivity.
4.1.1. Mass spectrometer detection methods: HPLC-MS(/MS)
Methods that have used mass detectors comprise a huge part of
analysis methods owing to high sensitivity and selectivity. HPLC-MS
methods for the quantification of anticancer drugs are summarized in
Table 2.
Geng et al. [32] developed a sensitive and simple method for
measuring docetaxel (DTX) concentrations for clinical TDM in human
plasma with HPLC-MS/MS method and paclitaxel (PCT) as internal
standard (IS). In similar studies, HPLC-MS/MS methods were reported
for the quantification of sorafenib, cabazitaxel, unbound vismodegib,
exemestane, vincristine, lenalidomide, lenvatinib, busulfan, imatinib,
crizotinib, olaparib, and methotrexate (MTX) in human plasma samples
[33–44]. Dubbelman et al. [45] developed and validated a sensitive
HPLC-MS/MS method to measure eribulin in human plasma, whole
blood, urine, and feces.
DBS sampling method is less invasive than venipuncture and is more
likely to be preferred and accepted among patients, especially pediatric
and geriatric patients [46]. Thus, the ability of DBS sampling method to
identify and quantify anticancer drugs is of high value. In DBS assay
methods, the sensitivity of detectors is very important due to the low
quantity of samples. PCT quantification of DBS samples obtained from
34 patients under PCT chemotherapy was performed, and the developed
method was validated [47]. PCT was extracted from DBS with a mixture
of MeOH and ACN. The drug was stable in DBS at 45 ◦ C and 25 ◦ C for
three weeks. This method can be used instead of routine blood sampling
methods. Ansari et al. [48] quantified busulfan in DBS and plasma
samples. Busulfan was detected with tandem mass spectrometry in MRM
mode and D8-busulfan was employed as IS. Samples were obtained from
children undergoing stem cell transplantation. The busulfan level in DBS
was correlated with conventional methods. Quantification of metho­
trexate polyglutamates (MTXPGs), as a potential marker for MTX, was
developed and validated by Hawwa et al. [49]. Due to the metabo­
lization of MTX to MTXPGs during the 24 h of administration, MTX
measurement in serum was of no value. The results were confirmed with
the matched red blood cell samples using validation methods. In two
different studies, Nijenhuis et al. [50] developed a quantification
method for vemurafenib in a DBS sample and investigated the correla­
tion between DBS and plasma concentration of vemurafenib in paired
samples obtained from 43 patients. 13C6-vemurafenib was utilized as IS.
Measurement of gefitinib in DBS punch following simple extraction with
MeOH was successfully correlated with plasma samples. Paired samples
were collected from 10 non-small cell lung cancer (NSCLC) patients, and
erlotinib was used as IS. Gefitinib stability in DBS cards at room tem­
perature and − 20 ◦ C was approximately five month [51]. In another
study, quantification of capecitabine (Cape) in DBS was developed and
validated, and Cape-D11 was utilized as IS [52]. During a very simple
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 1
Structure of anticancer drugs..

4
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 2
HPLC-MS(/MS) methods for quantification of single anticancer drug.
Parent drug Chromatography Method/Interface Extraction method Run time/ Range Reference
Method
application

Docetaxel RP C8/isocratic 0.1 M phosphate buffer (pH, 4): ACN HPLC-MS/MS-ESI/ PPT 10 min/TDM 10-2000 ng/mL [32]
150 × 4.6 mm, 5 μm QqQ/MRM+ MeOH
Sorafenib RP C18/isocratic ACN: 0.1% formic acid (80:20 v/v) HPLC-MS/MS-ESI/ SPE 2 min/clinical 10-10,000 ng/ [33]
100 × 2 mm, 2.8 μm simple quadrapole/ MeOH study mL
+
Cabazitaxel RP C18/gradient/ammonium formate: ACN HPLC-MS/MS-ESI/ LLE 5 min/clinical 1.00–100 ng/mL [34]
50 × 2.1 mm,3 μm QqQ/MRM+ 4% ammonium study
hydroxide/ACN/n-
butylchloride
Vismodegib RP C18/isocratic ACN: water, 50 × 2 mm, 5 μm HPLC–MS/MS-ESI/ SPE 4 min/clinical 0.100–100 ng/ [35]
+ Formic acid: water studies mL
Vincristine RP C8 gradient HPLC-MS/MS-ESI/ SLE 8 min/PK study 0.25–50 ng/mL [36]
Acetic acid: water, 50 × 2.0 mm, 3.0 μm Q/MRM+ MeOH: water (1:1 v/v)
lenalidomide RP/isocratic HPLC/MS/MS SPE 3 min/clinical 2–1000 ng/mL [37]
Ammonium Formate: ACN (15:85 v/v) 5 μm ISI/QqQ/MRM+ ACN: water (1:1 v/v) studies
Lenvatinib RP C8/isocratic ammonium acetate: ACN (3:2 v/v), HPLC-MS/MS organic solvent 4 min/clinical 0.0800–400 ng/ [38]
150 mm × 2.1 mm ESI/QqQ MeOH: water studies mL in serum
Busulfan RP C18/gradient formic acid: ammonium acetate, HPLC-MS/MS ESI/ LLE 10 min/clinical 0–5000 ng/mL [39]
100 × 2.1 mm, 3.5 μm QqQ/MRM+ EDTA studies
Imatinib RP C18/isocratic ammonium formate: ACN HPLC–MS/MS SPE 1.2 min/clinical 10–5000 ng/mL [40]
50 × 2.1 mm, 1.8 μm ESI/QqQ/SRM+ MeOH: water studies
Olaparib RP C18/gradient HPLC–MS/MS LLE 2 min/clinical 10–5000 ng/mL [41]
Ammonium acetate: MeOH ESI/QqQ/MR M+ MeOH studies
50 × 2.0 mm, 5.0 μm
Crizotinib RP C18/gradient HPLC-MS/MS SPE 7 min/PK study 5–5000 ng/mL [42]
formic acid: water: MeOH ESI/Q-Trap/MRM+ MeOH: water
50 × 2.1 mm, 5.0 μm
Busulfan RP C8/gradient formic acid: water, formic acid: HPLC- MS/MS LLE 3 min/PK study 14–4274 ng/mL [43]
ACN, 50 × 2 mm, 3 μm ESI/QqQ/MRM+
Methotrexate RP C18/gradient acetic acid: ACN, 50 × 2.1 mm, 3 HPLC- MS/MS PPT 5 min/TDM 0.05–25.0 μM [44]
μm ESI/QqQ/MRM+ ACN: water (70:30 v/
v)
Eribulin RP C18/gradient formic acid: water HPLC-MS/MS-ESI/ LLE 10 min/clinical 0.2–100 ng/mL [45]
30 × 2.0 mm, 3.0 μm QqQ/MRM+ MeOH: water (1:1) studies for plasma
Paclitaxel Isocratic/water: MeOH (25:75, v/v), both with 0.1% HPLC-MS/MS- ESI/ MeOH: ACN (90:10, v/ 2.3 min/TDM 2.5–400 ng/mL [47]
formic acid and 2 mM of ammonium formate, 50 × QqQ/MRM+ v)
4.6 mm, 2.6 μm
Busulfan RP C18/gradient acetic acid: ACN, 50 × 2.1 mm, 2.6 HPLC-MS/MS- ESI/ LLE 7 min/Clinical 100–2000 ng/ [48]
μm QqQ/MRM+ MeOH study mL
Methotrexate RP 18/gradient 10 mM NH4HCO3 (pH, 7.5): ACN HPLC-MS/MS- ESI/ PPT and SPE 10 min/Clinical 5–400 nM [49]
polyglutamates 150 × 2.1 mm, 3 μm QqQ/MRM+ study
Vemurafenib RP 18/gradient 10 mM ammonium acetate in water HPLC-MS/MS- ESI/ LLE 7 min/TDM 1–100 μg/mL [50]
(pH, 7) and MeOH QqQ/MRM+ MeOH: ACN (50:50, v/
50 × 2 mm, 5 μm v)
Gefitinib RP C18/isocratic 10 mM ammonium acetate in HPLC-MS/MS- ESI/ LLE 3 min/Clinical 37.5–2400 ng/ [51]
water: ACN, 50 × 2.1 mm, 3 μm QqQ/MRM+ MeOH PK study mL
Capecitabine RP C18/isocratic ACN: 2 mM ammonium formate HPLC-MS/MS- ESI/ LLE 2.5 min/TDM 10–10000 ng/ [52]
(pH, 3), 150 × 4.6 mm, 5 μm QqQ/MRM+ Ethyl acetate mL
Osimertinib C18/gradient, 2 mM ammonium acetate in water HPLC-MS/MS PPT 3.8 min/ 25–500 ng/mL [53]
(+0.1% formic acid): 2 mM ammonium acetate in QqQ/ESI/MRM MeOH Clinical study
MeOH (+0.1% formic acid), 50 × 2.1 mm, 3 μm

RP, Reverse phase; ACN, acetonitrile; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; ESI, electrospray ionization source; QqQ,
triple quadrupole mass spectrometer; MRM+, positive multiple reaction monitoring; PPT, protein precipitation; MeOH, methanol; TDM, therapeutic drug monitoring;
SPE, solid-phase extraction; SLE, solid-liquid extraction; LLE, liquid-liquid extraction; EDTA, Ethylenediaminetetraacetic acid; SRM, selected reaction monitoring; Q-
Trap, quadrupole ion trap; PK, pharmacokinetic.

extraction method, Cape was extracted from DBS cards with ethyl ace­
tate, which is a robust method with several advantages over previous
reported methods. Osimertinib level in human plasma was investigated
via developing and validating a simple HPLC-MS/MS method [53]. Most
of the previous studies made use of UPLC methods for this analysis;
however, this study provided a short-run time that was comparable to
UPLC methods, which is valuable owing to the more availability of HPLC
in laboratories.
4.1.2. UV detection methods (HPLC-UV and HPLC-PDA)
As previously mentioned, after MS, UV detection methods were the
most included in the present review. UV detectors are more feasible and
less complicated in comparison with MS detectors. In this section, we
summarized the qualification methods that utilized UV or PDA de­
tectors. Table 3 depicts the quantification methods that use HPLC-UV or
HPLC-PDA detectors.
HPLC-MS method was the most used method for quantifying the
plasma level of imatinib; however, Miura et al. [54] developed a simple
and sensitive HPLC-UV method comprised of a one-step SPE with an
Oasis HLB® cartridge. These modifications made it faster than previous
methods and provided high recovery with small sample volumes.
Optimization of the mobile phase provided good separation, sharp
peaks, and proper retention time. This method showed the same result as
HPLC-MS/MS regarding imatinib plasma concentration; so, it could be
used alternatively for TDM and PK clinical studies with low costs. Awidi
et al. [55] compared the plasma concentration of imatinib by HPLC-UV

5
 Table 3

R. Sabourian et al.
HPLC-UV or HPLC-PDA detectors for quantification of single anticancer drug.
Parent drug Chromatography Method/Wavelength Extraction method Run time/Method Range Reference
application

Imatinib RP C18/0.5% KH2PO4 (pH, 3.5): ACN: MeOH (55:25:20, v/v/v) HPLC-UV SPE 12.3 &14.5 min/ 10–5000 ng/mL [54]
250 × 4.6 mm λmax = 265 nm TDM &
Clinical PK study
Imatinib HPLC-UV: RP C18/ACN: phosphate buffer HPLC-UV λmax = 265 PPT MeOH 3 min/TDM 100–4000 ng/mL [55]
(30:70, v/v), 150 × 3.9 mm, 5 μm nm LLE hexane: ethyl acetate (30:70, v/v) Method comparison 10–4000 ng/mL
LC-MS/MS: RP C18/ACN: water: formic acid (30:70:0.1, v/v/v), 50 × and
2.1 mm, 3.5 μm LC-MS/MS-QqQ/ESI/
SRM+
Imatinib HPLC-UV trend solvent bar microextraction and hollow /TDM 20.0–6000.0 ng/mL [56]
fiber liquid phase microextraction technique
Imatinib RP-C8/isocratic HPLC-UV PPT MeOH 15 min/TDM 500–4000 ng/mL [57]
0.02 M dipotassic hydrogen phosphate buffer: ACN (73:27, v/v) 250 λmax = 265 nm
× 4 mm, 5 μm
Imatinib HPLC-PDA: C18/0.06 M KH2PO4: ACN (72–28 v/v), 250 × 4.6 mm, 5 HPLC-PDA: λmax = PPT 12 min/TDM 80-4000 & 10–5000 ng/mL [58]
μm 265 nm LC-MS/MS- perchloric acid & LLE
LC-MS/MS: RP C18/gradient ACN: 4 mM ammonium formate (pH, ESI/MRM
3.2)
Ponatinib RP C18/ACN: 0.037 M KH2PO4 (pH 4.5) (39:61, v/v), 250 × 4.6 mm, HPLC-UV PPT 15 min/TDM 5–250 ng/mL [59]
5 μm λmax = 250 nm HLB cartridge
Sorafenib C18/isocratic HPLC-UV PPT with ACN 12 min/TDM 0.1–20 μg/mL [60]
ACN: 20 mM ammonium acetate (53:47, v/v), 150 × 4.6 mm, 5 μm λmax = 260 nm
.
Lapatinib C18/isocratic ACN: 20 mM ammonium acetate (53:47, v/v), 150 × HPLC-UV PPT 12 min/TDM 0.2–10 μg/mL [61]
4.6 mm, 5 μm λmax = 260 nm ACN

Pazopanib C18/isocratic HPLC-UV LLE diethyl ether 9.5 min/TDM 0.5–100 [62]
6

0.02 M ammonium acetate (pH, 7): ACN/MeOH (70:30, v/v), (47:53, λmax = 260 nm μg/mL
v/v), 150 × 4.6 mm, 5 μm
Sorafenib C18/isocratic ACN: water (pH, 4.1) (65:35, v/v), 250 × 4.6 mm, 4 μm HPLC-UV λmax = 265 PPT 7 min/Clinical 0.1–50 μg/mL [63]
tosylate nm study
LC-MS/Q-TOF/ESI(+)
Gefitinib C18/gradient elution of 0.1% triethylamine solution and ACN, 150 × HPLC-PDA λmax = 340 SPE for CSF 10 min/BBB 23.3–224.7 [64]
4.6 mm, 5 μm nm LLE for plasma penetration study, ng/mL in plasma and
clinical study 0.398–3.554 ng/mL in CSF
HU C18/gradient 20 mM ammonium acetate: ACN, 150 × 4.6 mm, 5 μm HPLC-UV PPT 18 min/TDM 5–400 μM [65]
λmax = 240 nm MeOH &
PK
Mitomycin C RP C18/isocratic HPLC-UV λmax = 365 PPT 12 min & 30 min/ 0.05–5 to 5–100 μg/mL [66]
MeOH: 20 mM ammonium acetate buffer (pH 6.5) (23:73, v/v) and nm ACN PK study
ACN: 20 mM phosphate buffer (pH, 6.5) (9:91, v/v), 150 × 3.0 mm, 3
μm
5-fluorouracil RP C18/isocratic water, (pH, 3.2) 250 × 4.6 mm, 5 μm HPLC-UV λmax = 260 PPT 10 min/TDM and 0.05–100 μg/mL [67]

Analytical Biochemistry 610 (2020) 113891

nm perchloric acid PK
5-fluorouracil RP C18/isocratic ACN: ammonium acetate (pH 3.5) (2.5:97.5 v/v) HPLC-UV λmax = 265 LLE isopropanol/ethyl acetate (15:85 v/v) 7 min/PK 0.01–10 μg/mL [68]
250 × 4.6 mm, 5 μm nm
Cape C18/gradient, 0.1% formic acid: MeOH HPLC-UV LLE 8 min/PK and TDM 0.05–10.00 μg/mL [69]
150 × 4.6 mm, 3.5 μm λmax = 305, 265 nm ethyl acetate: ACN (4:1, v/v)
Cisplatin RP C18/isocratic, MeOH: water (80:20, v/v), 250 × 4.6 mm, 5 μm HPLC-UV PPT MeOH 15 min/TDM 0.025–2.00 μg/mL [70]
λmax = 254 nm and PK
TMZ RP ACN: 0.1% v/v acetic acid (10:90, v/v), 250 × 4.6 mm, 5 μm HPLC-UV λmax = 225 SALLE 10 min/PK 0.47–20 μg/mL [71]
ACN
MTX RP C18/Isocratic HPLC-UV SPE 12 min/TDM 0.025–5.00 μM [72]
50 mM sodium acetate buffer (pH 3.6): ACN (89:11, v/v), 250 × 4.6 λmax = 307 nm
mm, 5 μm
(continued on next page)
 R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

and HPLC-MS/MS methods. Risperidone was utilized as IS in HPLC-UV

RP, Reverse phase; ACN, acetonitrile; MeOH, methanol; HPLC, high performance liquid chromatography; UV, ultraviolet; SPE, solid-phase extraction; TDM, therapeutic drug monitoring; PK, pharmacokinetic; LC-MS/MS,
liquid chromatographic tandem mass spectrometry; PPT, protein precipitation; LLE, liquid-liquid extraction; QqQ, triple quadrupole mass spectrometer; ESI, electrospray ionization source; SRM+, positive selected re­
Reference

method and imatinib-D8 for HPLC-MS/MS method. Both methods were

[73]
reliable, fast, and easy with good accuracy, precision, selectivity, and

[74]

[75]
sensitivity. Insignificant differences were observed between these two
methods and this method can be used as a substitute. This is valued
owing to the more availability of HPLC-UV in contrast to the expensive
mass spectrometer. However, HPLC-UV had a longer run time and
required a large volume of samples due to its lower sensitivity.
0.05–10.0 μg/mL

Faridi et al. [56] determined the urinary and plasma levels of ima­
0.0263–0.6312

5–750 ng/mL

tinib with a novel microextraction technique coupled with a HPLC-UV

method. Gradients of pH played an important role in the results.
μg/mL
Range

Trabelsi et al. [57] monitored the plasma levels of imatinib by use of

action monitoring; PDA, photodiode array; Q-TOF: quadrupole time of flight; CSF, cerebrospinal fluid; BBB, blood brain barrier; SALLE, salting out assisted liquid-liquid extraction.

a HPLC-UV method. Because of the hard solubility of imatinib mesylate,

the pH of aqueous media was important and the volume of MeOH was
twice that of plasma. Roth et al. [58] developed and validated a
Run time/Method

9 min/PK study

cost-effective, available, and specific HPLC-PDA method to determine

30 min/TDM

10 min/TDM
and Method
comparison

imatinib in three different doses in human plasma in chronic myeloid

application

leukemia (CML) patients and compared it to a LC-MS/MS method. Due

to the high resolution following efficient direct PPT, the study was
performed without IS. After pretreatment with LLE method,
HPLC-MS/MS was employed to separate imatinib and its deuterated IS.
In this study, no significant differences were observed between the re­
sults of these two methods. Yasu T et al. [59] developed a simple
HPLC-UV method for quantifying ponatinib in human plasma with the
aim of TDM. In this method, ponatinib and bosutinib (as IS) were
extracted by a PPT method using an Oasis HLB® cartridge. The obtained
limit of quantification (LOQ) in this HPLC-UV method was higher than
40% w/v silver nitrate

that obtained from LC-MS/MS method; however, this limit was adequate
Extraction method

in clinical applications.
Escudero-Ortiz et al. [60,61] developed and validated selective
HPLC-UV methods for the quantification of sorafenib and lapatinib in
two separate studies for TDM purposes. In each study, one of the drugs
MeOH
PPT

PPT

was used as the main analyte, and the other was considered as IS.
Optimization of the stationary and mobile phases provided sharp and
symmetrical peaks with appropriate retention times. The lower limit of
HPLC-UV λmax = 310

HPLC-UV λmax = 235

Method/Wavelength

quantification (LLOQ) in this method was sufficient for determining

these drugs at therapeutic ranges in plasma samples. These methods
λmax = 302 nm

provided acceptable sensitivity that was useful in clinical laboratories

HPLC-UV

with no available MS detectors. In another study [62], this group

developed and validated a fast and sensitive HPLC-UV method for the
nm

nm

quantification of pazopanib in human plasma. They used gefitinib as the

IS due to its similarity to the main analyte. In this study, the stationary
RP C18/10 mM sodium phosphate buffer (pH, 6.40) MeOH (78:22%,

RP C18/isocratic, acetate buffer (pH, 5.9): MeOH (85:15, v/v), 250 ×

and mobile phases were optimized, providing sharp and symmetrical

peaks with a shorter run time. Sharma et al. [63] determined the plasma
level of sorafenib in the existence of stress-induced decomposition
products through developing and validating a fast, cost-effective, and
sensitive HPLC-UV method and using analytical quality by design
approach to provide the most optimal resolution and least peak tailing.
It was found that higher column temperature and acidic pH provided
C8/diluent buffer (pH, 6.0): ACN (93:7, v/v)

higher selectivity; in this regard, orthophosphoric acid and formic acid

were employed in the mobile phase of HPLC-UV and LC-MS, respec­
tively, to adjust the pH (at 4.1). To determine the degradation products,
a Q-TOF mass spectrometer was applied in the positive ion mode of
electrospray ionization source (ESI). Fang et al. [64] described a sensi­
v/v) 250 × 4.6 mm, 5 μm
150 × 4.6 mm, 3.5 μm

tive HPLC-PDA method to monitor gefitinib level in plasma and CSF

samples of NSCLC patients. During the CSF sample preparation phase,
Chromatography

use of formic acid to acidify the elution significantly increased the eluted
4.6 mm, 5 μm

power. On the contrary, increasing the pH of loading solution led to

better sample purification but reduced the elution efficacy. Legrand et al.
[65] measured the plasma levels of hydroxyurea with a HPLC-PDA
Table 3 (continued )

method. Following PPT with MeOH, derivatization was performed by

adding xanthydrol, and methylurea was used as IS. This method was
Vinorelbine

able to detect hydroxyurea in small volume samples. Lemoine et al. [66]

Parent drug

described and validated HPLC-PDA method to specify mitomycin C in

urine, peritoneal fluid, and plasma. Porfiromycin was used as IS.
MTX

MTX

Minhas et al. [67] monitored the plasma levels of 5-fluorouracil

7
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

(5-FU) with an accurate and reproducible HPLC-UV method and vali­
dated that procedure. Simple composition of the mobile phase and rapid
sample preparation were the upsides of this method. Optimization of the
pH value by adding perchloric acid, solved early 5-FU elution and pro­
vided a good resolution. In another study, Pi et al. [68] developed and
validated a sensitive HPLC-UV method for 5-FU monitoring in plasma.
Piorkowska et al. [69] analyzed the plasma level of Cape with a rapid,
simplified, and cost-effective HPLC-UV method. In their method,
selecting voriconazole as IS shortened the run time but required online
wavelength switching as their different optimal wavelength to monitor
the Cape.
Tezcan et al. [70] described a HPLC-PDA method for monitoring the
plasma concentration of free cisplatin in different types of cancer.
Deproteinization with cold MeOH was used for sample extraction of
cisplatin and NiCI2.6.H2O as IS. A significant difference was observed
between different cancer types regarding cisplatin concentration, and
lung cancer patients had high concentrations. To analyze temozolomide
(TMZ) in plasma and biologic samples, Jain et al. [71] developed and
validated a cost-effective and robust double salting out assisted
liquid-liquid extraction (SALLE) HPLC method and a HPLC-UV method
with a PDA detector. Using SALLE with ACN increased the extraction
efficacy of low quantities of TMZ from plasma and caused low inter­
ference from plasma in contrast to LLE. Acetaminophen was used as IS.
Begas et al. [72] described a HPLC-UV method to quantify MTX in
serum. After SPE, samples were separated on the Kromasil® C18 col­
umn, and 1,3,7-trimethyluric acid was used as IS. In another study, De
Abreu et al. [73] monitored MTX in the plasma samples collected from
pediatric patients with a validated HPLC-UV method. Next, they
compared the obtained data with the results of ELISA method. Li et al.
[74] developed and validated a simple, fast, and sensitive HPLC-UV
method to quantify MTX in serum samples, and ferulic acid was used
as IS. Optimizing the pH and mobile phase provided appropriate
retention time and symmetric sharp peaks with good separation and
resolution. Mallu et al. [75] validated and developed a method for the
quantification of vinorelbine in spiked human plasma. They reported no
RP-HPLC method for vinorelbine determination in spiked samples, and
this method was accurate, precise, and sensitive and suitable to PK
studies.
4.1.3. Fluorescence and other detection methods (HPLC-FLD and RLS-
HPLC)
HPLC coupled with fluorescence detector (FLD) is an excellent
analytical method owing to its selectivity, robustness, and lower appli­
cation costs compared to HPLC coupled with mass spectroscopy (HPLC-
MS). FLD sensitivity is 10–1000 times higher than that of a UV or PDA
detector, which can be coupled with HPLC separation technique [46].
Etoposide in DBS and plasma samples of lung cancer patients was
quantified in the presence of dansyl-threonine as IS. Extraction of ana­
lyte with MeOH:ACN:water resulted in high recovery and reproducible
extraction of etoposide from DBS cards.
A significant correlation was observed between two different sam­
ples [76]. This method was cheaper and patient-friendly and can be used
for the measurement of etoposide. HPLC coupled with FLD was used to
quantify the plasma level of ruxolitinib [77]. Following sample extrac­
tion with MeOH and ACN, the chromatographic conditions were opti­
mized to achieve separation and symmetric peaks with a short run time.
This method can replace the previous ones in clinical applictions.
Another HPLC-FLD method for measuring the plasma and urine con­
centration of epirubicin (Epi); it was the first LC-FLD method that could
simultaneously analyze both of these biologic matrices [78]. They
optimized the analysis condition and achieved a lower limit of detection
(LLOD) and LOQ with less requirement of urine samples for Epi analysis
in contrast to previous studies with fluorescence detector. Li et al. [79]
described a simple, fast, and sensitive resonance light scattering
(RLS)-HPLC method to quantify the urinary levels of 6-mercaptopurine
(6-MP); a complex was formed between 6-MP and palladium (II) to in­
crease the RLS intensity of the system in acid medium. In this study,
adenine was utilized as IS. The obtained LOD was lower than fluores­
cence and electrochemical methods. In spite of not having a better
sensitivity compared with some chemiluminescence methods, it was
sufficient for this aim. The above-noted studies are summarized in
Table 4.
4.2. Drug and metabolite(s) determination methods
As a matter of fact, drugs and metabolites are mainly similar to each
other in terms of chromophores and structure; therefore, high resolution
chromatography system could be very important in separating the ob­
tained peaks. To predict the efficacy and toxicity of the drugs and their
metabolites, it is important to assess their PK profile. Determination of
metabolites can be beneficial for assessment and explanation of (pre)
clinical data.
4.2.1. Mass spectrometer detection methods: HPLC-MS(/MS)
Similar to the previous section, most of the methods were developed
to assay an anticancer drug and its metabolites with an HPLC system
coupled with an MS detector. HPLC-MS/MS method is an efficient
method for TDM owing to its sensitivity and selectivity. Table 5 sum­
marizes the methods with HPLC-MS systems to simultaneously quantify
anticancer drugs and their metabolites.

Table 4
HPLC-FLD methods for single anticancer drug quantification.
Parent drug Chromatography Method Extraction method Run time/Method Range Reference
application

Irinotecan RP C8/isocratic 0.1 M phosphate buffer HPLC-FLD LLE 17 min/TDM 10–3000 ng/mL [115]
(pH 4) ACN, 150 × 4.6 mm, 5 μm λex = 370 nm; ACN: MeOH (1:4 v/v)
λem = 420
and 540 nm
Etoposide RP C18/isocratic phosphate buffer HPLC-FLD LLE 12 min/TDM 0.5–20 μg/mL [76]
(pH, 4): ACN, 150 × 4.6 mm, 3 μm λex = 230 nm; ACN: MeOH: water
λem = 330 nm (33:35:30, v/v)
Ruxolitinib RP C18/isocratic water: ACN (pH, 4.8) HPLC-FLD PPT 5 min/ 0.2–500 ng/mL [77]
(67:33, v/v), 75 × 4.6 mm, 3.5 μm λex = 320 nm; MeOH TDM
λem = 386 nm
Epirubicin Hydro-RP/isocratic 40 mM phosphate buffer HPLC-FLD SPE dichloromethane: 8 min/TDM Plasma: 1–1500 ng/mL [78]
(pH, 4.1): ACN (69:31, v/v), λex = 497 nm; 2-propanol: MeOH urine: 1–10000 ng/mL
150 × 4.6 mm, 4 μm λem = 557 nm (2:1:1, v/v/v)
6-mercaptopurine RP MeOH: water (25:75, v/v) RLS-HPLC-FLD PPT 15 min/Clinical 0.0615–2.40 μg/mL [79]
250 × 4.6 mm, 4 μm λex = λem = ACN study
315 nm

RP, Reverse phase; ACN, acetonitrile; HPLC, high performance liquid chromatography; FLD, fluorescence detector; LLE, liquid-liquid extraction; MeOH, methanol;
TDM, therapeutic drug monitoring; PPT, protein precipitation; SPE, solid-phase extraction; RLS, resonance light scattering.

8
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 5
HPLC-MS(/MS) methods for quantification of drug and metabolites.
Parent drug Chromatography Method/ Extraction method Run time/Method Range of different analytes Reference
Interface application

Abiraterone RP C18/isocratic, water with 1 HPLC-MS/ SPE 10 min/PK Abiraterone and metabolites (0.5–100 [80]
mM ammonium formate: MS Q-trap/ ng/mL) and glucuronides (0.05–10.00
MeOH (80:20 v/v), 100 × 2.1 TIS/MRM+ ng/mL)
mm, 2.7 μm
Tamoxifen RP C18/gradient, 5 mM HPLC-MS/ PPT 10 min/clinical study Tamoxifen, N-desmethyl tamoxifen [81]
ammonium formate buffer (pH, MS-QqQ/ ACN (5–1000 ng/mL), N-desmethyl-4 (4′ )-
3.5), 150 × 2.1 mm, 2.6 μm APCI/ hydroxy tamoxifen (1–200 ng/mL), 4-
MRM+ hydroxy tamoxifen (0.4–80 ng/mL),
4′ -hydroxy tamoxifen (0.2–40 ng/mL)
Docetaxel HPLC-UV: RP C8/ HPLC/UV – 18 min/clinical MS: 0.25–500 ng/mL [82]
20 mM ammonium acetate in and study UV: 0.05–100 μg/mL
water (pH, 5): ACN (40:60, v/v) HPLC/MS/
150 × 2.1 mm, 3.5 μm MS/QqQ/
HPLC-MS/MS: ESI
C18/gradient
10 mM ammonium hydroxide
in water: MeOH, 150 × 2.1 mm,
5 μm
Paclitaxel C18/gradient HPLC/MS/ PPT 21 min/TDM PCT (1–10000 ng/mL), 6α-hydroxy- [84]
0.1% formic acid: water and MS-QqQ/ MeOH: water with PCT (1–1000 ng/mL)
0.1% formic acid: ACN, 150 × TIS/ESI/ 0.1% formic acid
2.1 mm, 3.5 μm SRM+
Paclitaxel Synergi Polar-RP/gradient HPLC-MS/ LLE 8 min/TDM PCT (10–10000 ng/mL), 6-alpha-OH [83]
0.1% (v/v) formic acid in MS-QqQ/ and 3-para-OH
water: ESI/MRM+ metabolites (1–1000 ng/mL)
0.1% (v/v) formic acid in ACN,
(50 × 2 mm, 4 μm)
Omacetaxine XBridge BEH Phenyl column/ HPLC-MS/ PPT & SPE 5.6 min/PK Plasma: (0.1–100 ng/mL) urine: [85]
gradient MS- QqQ/ (0.1–50 ng/mL)
0.1% formic acid in water: TIS/MRM+
MeOH
50 × 2.1 mm, 5 μm
Irinotecan C18/gradient ACN 0.1% formic HPLC-MS/ PPT 10.92 min/TDM IRI (25–2500 ng/mL), SN-38 (5–500 [86]
acid, 50 × 2.1 mm, 3 μm MS-QqQ 10 mM ammonium ng/mL)
acetate (pH, 9)
Irinotecan C18/gradient 0.1% acetic acid: HPLC-MS/ PPT 18 min/TDM/ IRI (10–10000 ng/mL), SN-38 (1–500 [87]
water and 0.1% acetic acid: MS-QqQ/ Clinical ng/mL), SN-38G and APC (1–5000 ng/
ACN, 100 × 2.0 mm, 3 μm TIS/ESI/ PK Study mL)
SRM+
Gemcitabine C18/formic acid: ACN, 100 × HPLC-MS/ PPT 20.3 min/ Gemcitabine (0.125–40.0 μg/mL), [88]
2.1 mm, 3 μm MS-QqQ/ individualized dFdU (1.25–80.0 μg/mL)
ESI/ treatment strategies
MRM+
Erlotinib RP C8/gradient: 0.1% formic HPLC-MS/ PPT 10 min/PK plasma: 5–2500 ng/mL [89]
acid in water: 0.1% formic acid MS- QqQ/ lung tumor tissue homogenate:
in MeOH, 150 × 2.0 mm, 4.0 ESI(+) 5.0–500 ng/mL
μm lung tumor: 50–5000 ng/g
Sorafenib C18/isocratic 10 mM HPLC-MS/ PPT 4 min/PK sorafenib (50–10000 ng/mL), [90]
ammonium acetate (pH 3.8 MS-QqQ sorafenib N-oxide (10–2500 ng/mL)
with formic acid): 0.1% formic /ESI(+)
acid in ACN (35:65, v/v),
50 × 2.1 mm, 3.5 μm
Sunitinib C18/gradient HPLC-MS/ PPT 4 min/PK 0.10–100 ng/mL [91]
water containing 0.1% formic MS/Q-trap/ MeOH: ACN
acid: ACN containing 0.1% TIS/MRM+
formic acid 50 × 2.1 mm, 3.5
μm
Gefitinib C18/isocratic HPLC-MS/ PPT 3 min/Clinical gefitinib (5–1000 ng/mL), O- [92]
ACN with 0.1% formic acid: MS-QqQ/ PK Study desmethyl gefitinib (5–500 ng/mL)
water (30:70, v/v) ESI/
150 × 2.1 mm, 5 μm MRM+
Sunitinib RP 80/gradient HPLC-MS/ PPT 10 min/TDM 2.5–500 ng/mL [93]
0.1% formic acid in water: MS- ESI/ cold ACN
0.1% formic acid in MeOH, 150 QqQ/
× 2.0 mm, 4.0 μm MRM+
Imatinib mesylate C18/gradient HPLC-API- PPT 13 min/TDM 0.500–10.0 μg/mL [94]
water: MeOH (with MS/ESI/
10 mM ammonium acetate and SIM+
0.1% formic acid, 50 × 2 mm, 3
μm
Imatinib C18/gradient HPLC-MS/ PPT 6 min/TDM 10–2000 ng/mL [95]
0.05% formic acid: MS- ESI/
MeOH, 50 × 2.0 mm, 3 μm
(continued on next page)

9
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 5 (continued )
Parent drug Chromatography Method/ Extraction method Run time/Method Range of different analytes Reference
Interface application

QqQ/
MRM+
Imatinib RP C18/isocratic HPLC-MS/ PPT 6.0 min/PK and imatinib (8–5000 ng/mL), NDI [96]
MeOH: water (55:45, v/v) MS- ESI/ MeOH TDM (3–700 ng/mL)
containing 0.1% formic acid QqQ/
and 0.2% ammonium acetate, MRM+
100 × 4.6 mm, 2.4 μm
Imatinib RP C18/MeOH: ACN: water HPLC-MS/ LLE 3.5 min/TDM Imatinib and NDI (0.01–10 μg/mL) [97]
(65:20:15, v/v/v) with 0.05% MS- ESI/
formic acid QqQ/
50 × 2.1 mm, 3.5 μm MRM+
Cyclophosphamide C18/ACN: water (50:50, v/v) HPLC-MS/ PPT 3 min/ CP (5–5000 ng/mL), 4-Hydroxyl CP [98]
with 0.1% formic acid 50 × 2.1 MS- ESI/ cold ACN pharmacogenomics (5–500 ng/mL)
mm, 3.5 μm QqQ/ & LLE study
MRM+
Cisplatin RP C18/isocratic water: 0.1% HPLC-MS/ LLE 2.6 min/PK 1.0–100.0 ng/mL [99]
AA in aqueous ACN (ACN: H2O: MS- ESI/
AA, (95:5:0.1, v/v/v), 150 × QqQ/
2.1 mm, 3 μm MRM+
Niraparib C18/gradient 20 mM HPLC-MS/ Plasma: PPT urine: 7 min/pharmacological Plasma: 1–500 ng/mL [100]
ammonium acetate in water: MS- ESI/ dilution clinical studies urine: 1–100 ng/mL
0.1% formic acid in ACN: QqQ/ ACN: MeOH (50:50,
MeOH (50:50, v/v), 50 × 2.1 MRM+ v/v)
mm, 5 μm
5-fluouracil dC18/isocratic HPLC-MS/ LLE 10 min/TDM and U (0.01–10 μM), UH2 (0.1–10 μM), 5- [101]
1 mM ammonium acetate in MS-Q-trap/ ethyl acetate: 2- toxicity FU (0.1–75 μM), DHFU (0.75–75 μM)
water: 0.5 mM formic acid: TIS/MRM+ propanol (10:1, v/ prediction
3.3% MeOH, 150 × 2.1 mm, 3 v)
μm
5-fluouracil Luns PFP/gradient, acetic acid/ HPLC-MS/ SALLE 8 min/TDM FβAL & DHFU (50–10000 ng/mL), [102]
formic acid/water MS- ESI/ FUPA (50–5000 ng/mL), 5-FU
(0.25:0.05:99.7, QqQ/ (50–100000 ng/mL), U (5–200 ng/
v/v/v): ACN, 150 × 2.0 mm, 3 MRM+ mL), UH2 (10–400 ng/mL)
μm
Capecitabine C18/gradient 0.05% formic HPLC-MS/ PPT 9 and 5 min/PK Cape (50–6000 ng/mL), 5-FU [103]
acid in water: 0.05% formic MS- TIS/ (50–5000 ng/mL)
acid in MeOH, 50 × 2.1 mm, 5 QqQ/
μm MRM+
Capecitabine dC18/gradient 0.025% AA: HPLC-MS/ PPT 10.5 min/ Cape, 5′ -dFCR, and 5α-dFUR [104]
0.0025% ammonium hydroxide MS- TIS/ bioequivalence study (10.0–5000 ng/mL), 5-FU (2.00–200
solution (pH 3.8): MeOH, 100 QqQ/ ng/mL)
× 4.6 mm, 3 μm MRM+,-
Capecitabine RP C18/gradient HPLC-MS/ 15 min/TDM Cape (50–6000 ng/mL), 5-FU [105]
water, 10 mM MS-APCI+,- (50–5000 ng/mL)
ammonium acetate
(pH, 6.8):water, 0.1%
Formic Acid: ACN, 100 × 2.0
mm, 3 ìm
Capecitabine Hypercarb (porous graphitic HPLC-MS/ PPT 15 min/clinical PK Cape (10–1000 ng/mL), 5′ -dFCR & 5α- [106]
carbon) column/gradient MS- ESI/ 10% trichloroacetic study dFUR (10–5000 ng/mL), 5-FU &
10 mm ammonium acetate in QqQ/ acid in water: DHFU (50–5000 ng/mL)
ACN: 2-propanol: MRM+,- MeOH: ACN
tetrahydrofuran (1:3:2.25, v/v/
v) 30 × 2.1 mm, 5 μm
Exemestane C18/gradient HPLC-MS/ PPT 5 min/PK & dynamic exemestane (0.4–40 ng/mL), 17β- [107]
0.1% aqueous formic acid: MS- ESI/ ACN studies Hydroxy exemestane
ACN, 100 × 2.1 mm, 5 μm QqQ/ and 17β-dihydro exemestane-17-O-
MRM+ β-D-glucuronide (0.2–15.0 ng/mL)

RP, Reverse phase; MeOH, methanol; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; Q-Trap, quadrupole ion trap; TIS: turbo ion
spray; MRM+, positive multiple reaction monitoring; SPE, solid-phase extraction; PK, pharmacokinetic; QqQ, triple quadrupole mass spectrometer; APCI: atmosphere
pressure chemical ionization; PPT, protein precipitation; ACN, acetonitrile; UV, ultraviolet; ESI, electrospray ionization source; LLE, liquid-liquid extraction; TDM,
therapeutic drug monitoring; PCT, paclitaxel; IRI, irinotecan; dFDU, 2′ ,2′ -difluoro-2′ -deoxyuridine; SIM+, positive single ion monitoring; API, atmospheric pressure
ionization; NDI, N-desmethyl imatinib; CP, cyclophosphamide; U, uracil; UH2, 5,6-dihydrouracil; 5-FU, 5-fluouracil; DHFU, 5-fluoro-5,6-dihydrouracil; SALLE, salting
out assisted liquid-liquid extraction; FβAL, α-fluoro- β-alanine; FUPA, α-fluoro-β-ureidopropionic acid; Cape, capecitabine; 5α-dFUR, 5′ -deoxy-5-fluorouridine; 5′ -dFCR
5′ -deoxy-5-fluorocytidine.

Using deuterated derivatives as IS, Caron et al. [80] analyzed the
human plasma levels of abiraterone (Abi), D (4)-abiraterone, 3-keto-5
α-abiraterone, and their inactive derivatives by a HPLC-MS/MS method
with a triple-quadrupole linear ion trap (Q-Trap) mass analyzer in MRM
mode. This method was validated in accordance with the FDA. The
objective of this method was to analyze polar and non-polar metabolites
of the abiraterone acetate. Since they are isomeric molecules, they
cannot be separated, hence the fact that the chromatographic conditions
were optimized through selecting the best column and mobile phase to
improve separation, which had good selectivity and sensitivity.
Teunissen et al. [81] described a quantification method for tamoxifen
and its five metabolites in samples of patients under 20 mg tamoxifen
daily. This method reduced the run time and increased the sensitivity in
comparison with the previous methods.

10
R. Sabourian et al.
Hendrikx et al. [82] quantified DTX and its metabolites by a
HPLC-UV method at 227 nm wavelength; they also identified the ob­
tained peaks by a multistage accurate mass spectrometry method.
Quantification and validation were performed by LC-MS/MS, conducted
in a QqQ MS.
PCT is a widely used drug in several cancer treatments. PCT clear­
ance is mostly dependent on metabolism, hence the importance of the
quantification of its metabolites. Christner et al. [83] reported a vali­
dated method for PCT and the quantification of its 6-alpha- hydroxy and
3-para-hydroxy metabolites in human plasma. This method was devel­
oped for clinical trials. In another study, PCT and its main metabolite,
6-alpha-hydroxy PCT, was determined in patients with analytical
reference standard of PCT and DTX as IS [84]. The developed
HPLC-MS/MS method was coupled with an atmospheric pressure
interface (API) QqQ MS via a turbo ion spray (TIS) ionization interface.
This method required a small volume of plasma and was characterized
by a wide range of concentrations.
Nijenhuis et al. [85] investigated the PK of omacetaxine via
measuring the plasma and urine levels of omacetaxine mepesuccinate
and its 4′ -des-methyl and cephalotaxine metabolites using HPLC-MS/MS
method with deuterated analogues of all compounds as IS.
During the two different studies, irinotecan (IRI) and its metabolites
were analyzed. Herviou et al. [86] developed and validated an
HPLC-MS/MS method. They developed a QqQ MS detector with turbu­
lent flow technology that provides a short time preparation for deter­
mining IRI and its active metabolite, SN38, in plasma for TDM purpose.
This study introduced a novel approach for the simultaneous and fast
assessment of IRI and SN38. TurboFlow™ technology accelerates the
preparation and enables testing with low sample volumes; combining it
with an HPLC-MS/MS method makes the PK study fast, robust, and
economical. Marangon et al. [87] studied the TDM of IRI and through
developing and validating a HPLC-MS/MS method that was attached to
an API QqQ MS and was prepared with a TIS source in positive mode for
simultaneous quantification. The selected reaction monitoring (SRM)
mode assay provided high selectivity and sensitivity. Using campto­
thecin the IS, they investigated the plasma of metastatic colorectal
cancer patients receiving IRI in combination with 5-FU, leucovorin, and
bevacizumab.
Two methods were described to determine gemcitabine and its
metabolite. Bjanes et al. [88] examined the stability of gemcitabine and
its metabolite in blood with HPLC-MS/MS coupled with QqQ MS that
was equipped with an ESI with positive ions in MRM mode. DFdC (2′ ,
2′ -difluoro-2′ -deoxycytidine) and dFdU (2′ ,2′ -difluoro-2′ -deoxyuridine)
were used as IS.
This part reports different tyrosine kinase inhibitors and their me­
tabolites that were quantified during several developed methods.
Lankheet et al. [89] described an HPLC-MS/MS method combined with
QqQ MS equipped with positive ion mode ESI to quantify O-desmethyl
erlotinib and erlotinib in human EDTA-treated plasma and lung tumor
tissue samples. Li et al. [90] specified the plasma levels of sorafenib and
its active metabolite, sorafenib N-oxide, with HPLC-MS/MS and
Micromass Quattro LC QqQ MS in the positive ion mode of ESI. This
method was developed and validated for PK monitoring. Quantification
of major metabolites is conducive to finding and preventing the adverse
effects. Qiu et al. [91] validated and developed a highly selective
HPLC-MS/MS method in combination with an API Q-Trap MS with a TIS
ionization interface in positive mode for the simultaneous measurement
of sunitinib and its two metabolites in the plasma of Chinese patients. To
reduce the E/Z-isomerism of sunitinib, complete tests, such as plasma
gathering, sample pretreatment, and instrumental analyses were ach­
ieved under feeble yellow sodium light. This method was appropriate for
clinical trials and made it possible to analyze 120 to 150 samples per
day. In this method buspirone hydrochloride was utilized as IS. Wang
et al. [92] described HPLC-MS/MS to simultaneously quantify the
plasma concentration of gefitinib and its main metabolite (O-desmethyl
gefitinib). Chromatographic detection was performed by use of API QqQ
11
Analytical Biochemistry 610 (2020) 113891
MS along with positive ESI mode. This assay method was employed to
investigate the effect of hydroxychloroquine on the PK of gefitinib.
O-methyl gefitinib-D3 was used as the IS. It was demonstrated that PPT
with ACN resulted in the most optimal efficacy (>99.9%); therefore, this
simple and cost-effective procedure was selected for pretreatment. Using
0.1% formic acid in the mobile phase enhanced the peak’s sharpness and
signal intensity; however, it should be used in low concentration to
protect the column and avoid opposing effects on the peak as the acid
may produce protons that possibly change the charge of the ions. Some
factors such as flow rate and mobile phase composition should also be
optimized to achieve a short run time. Lankheet et al. [93] reported an
HPLC-MS/MS method for quantifying sunitinib and N-desethyl sunitinib
in plasma samples of 58 patients under treatment with sunitinib. This
method benefits from reliable results and a short run time, hence suc­
cessfully applied for the routine TDM of sunitinib.
Several studies have recently developed assay methods to quantify
imatinib and its metabolites, some of which were eligible, thus reviewed
here. Using HPLC-MS, Rezende et al. [94] examined the level of imatinib
and its metabolite, N-desmethyl imatinib (NDI), in the serum of patients
with CML and validated it to consider TDM. This method had certain
advantages such as short run time, simple pretreatment with PPT, and
being economical. These advantages made the method suitable for the
determination of hundred samples per day, and deuterated Imatinib-D8
was used as IS. A study with a similar method was reported by Zhang M
et al. [95], and a study was performed by Zhang Y et al. [96], using
palonosetron as IS. Finally, Zhuang et al. [97] developed a more stable,
sensitive, and specific HPLC-MS/MS method for the simultaneous
quantification of the plasma levels of imatinib and its metabolite NDI in
gastrointestinal stromal tumor patients.
Shu et al. [98] described an HPLC-MS/MS method with a QqQ
spectrometer equipped with positive ESI in SRM mode simultaneously
quantify cyclophosphamide (CP) and 4-hydroxy CP in human plasma.
Since the CP dose was low in this study, the method was developed by
dansyl hydrazine derivative reaction and LLE. Evaluation of the reaction
time and temperature optimized the derivatization condition. Among
the derivative reagents, dansyl hydrazine was chosen owing to less
toxicity with fast reaction and simple preserve condition, hence the use
of Ifosfamide (IF) as IS. Tang et al. [99] examined the plasma levels of
platinum derived from cisplatin through the use of HPLC-MS/MS
coupled with an API QqQ MS in the positive mode of ESI. Cisplatin
and IS, trans-diammine dichloro palladium (II), were chelated with
diethyl dithiocarbamate to study PK and cisplatin distribution. Van
Andel et al. [100] assayed the plasma and urinary levels of niraparib and
its carboxylic acid metabolite. The HPLC system was coupled with API
MS/MS in the positive mode of ESI.
Buchel et al. [101] reported an HPLC-MS/MS method with a simple
sample preparation that was able to simultaneously quantify uracil (U),
5,6-dihydrouracil (UH2), 5-FU, and 5-fluoro-5,6-dihydrouracil (DHFU)
and applicable to routine clinical use. Stable isotope-labeled of the
analytes was used as IS. The new method was good for analyzing clin­
ically relevant levels of 5-FU and studying the TDM and toxicity of this
drug in cancer patients. In another study, Chavani et al. [102] developed
an HPLC-MS/MS method with a QqQ MS in positive ion mode ESI to
measure the plasma levels of U and its derivatives and metabolites (UH2,
5-FU, α-fluoro- β-alanine (FβAL), α-fluoro-β-ureidopropionic acid
(FUPA) and DHFU) with the aim of the TDM of 5-FU and its metabolites.
In this study, for the first time, an HPLC-MS/MS method was developed
and was able to simultaneously quantify the plasma levels of all the
mentioned analytes; thus, it had many advantages, such as reduced
required labor, run time, and volumes of the sample and reagents.
Deenen et al. [103] measured the plasma levels of Cape and its metab­
olites, 5-FU, FUH2, FUPA, 5′ -deoxy-5-fluorocytidine (5′ -dFCR),
5′ -deoxy-5-fluorouridine (5α-dFUR), and FβAL in two parts. One part
was allocated to simultaneously measuring the level of Cape, 5′ -dFCR,
and 5′ -dFUR; the second part concerned thequantification of 5-FU,
FUH2, FUPA, and FβAL with hydrophilic chromatography due to the
R. Sabourian et al.
difference in the physicochemical properties of these two groups. Both
assays were validated and then widely used for the clinical studies of
Cape and 5-FU. In the present study, the IS was the stable labeled iso­
topes for each analyte to increase the sensitivity and accuracy. Deng
et al. [104] reported an HPLC-MS/MS method on API QqQ MS with a TIS
interface in the ESI mode by a polarity-switching strategy conducted in
two parts through dividing the analytes into two groups to avoid
sensitivity loss. Stable isotope-labeled compounds 13C and 15N2-DFCR
were used as IS. Montange et al. [105] described an HPLC-MS/MS
method with atmospheric pressure chemical ionization (APCI) with
both positive and negative modes for a simultaneous determination of
the plasma levels of Cape and its metabolites, such as 5-FU, the active
metabolite of Cape, with the aim of TDM. Two or three separate ex­
tractions and chromatographic phases were required for TDM of Cape;
however, with the proposed HPLC-MS/MS method, it was possible to
perform TDM for Cape and its metabolites with only one extraction step
for simultaneous measurement. Ultimately, using an innovative vali­
dated HPLC-MS/MS method, Vainchtein et al. [106] specified the
plasma levels of Cape and its metabolites, including 5′ -dFCR, 5α-dFUR,
FUH2, and 5-FU. The purpose of the assay was to assess the PK of the
Cape and its metabolites, detected using QqQ MS in the positive and
negative ion modes of ESI, and a mixture of fludarabine and 5-chlorour­
acil was used as IS. Due to the different polarities of Cape, 5-FU and
FUH2, using a Hypercarb column was the best choice and gradient
elution.
To investigate the PK of exemestane in patients with breast cancer,
Wang et al. [107] reported a rapid HPLC-MS/MS method able to quan­
tify the plasma levels of exemestane, its main metabolite, 17β-Hydroxy
exemestane and its inactive metabolite, 17β-dihydro exemesta­
ne-17-O-β-D-glucuronide, Denatured isotopes of these three analytes
were used as IS.
4.2.2. UV detection methods (HPLC-UV and HPLC-PDA)
As mentioned before, UV detectors have several advantages in
contrast to complicated MS detectors. UV detectors are more available
and easy to handle. Therefore, the developed methods that use this
detector and can be validated are globally preferred. Table 6 lists the
developed methods using UV detectors.
Zakrzewski et al. [108] measured the urinary levels of 6-MP and its
three metabolites, namely 6-thioguanine, 6-MP riboside, and 6-thiogua­
nine riboside in patients by an economical, rapid, and simple HPLC-UV
method coupled with iodine-azide reaction. The iodine-azide reaction
showed superior selectivity because only the peaks of thiols were
observed on the chromatogram. Parameters were optimized to generate
Table 6
HPLC-UV methods for quantification of drug and metabolites.
Parent drug Chromatography Method/Wavelength
6-mercaptopurine RP C18/sodium azide and sodium HPLC-UV coupled
heptane sulfonate: ACN: water with the iodine-azide
(50:1:49, v/v/v), 150 × 3.9 mm, reaction
5 μm
Erlotinib RP C18/isocratic HPLC-UV
MeOH: 20 mM phosphate buffer λmax = 258 nm
(pH, 5) (60:40, v/v), 150 × 4.6
mm, 5 μm
Sorafenib Inertsil ODS-3 column/20 Mm HPLC-UV
ammonium acetate buffer (pH, λmax = 265 nm
4.0): ACN (30:70, v/v) 150 × 2.1
mm, 5 μm
Tamoxifen C18/isocratic 5 mM HPLC-PDA
triethylammonium phosphate λmax = 280 nm
buffer (pH, 3.3): ACN (57:43, v/
v), 150 × 4.6 mm, 5 μm
RP, Reverse phase; ACN, acetonitrile; HPLC, high performance liquid chromatography; UV, ultraviolet; TDM, therapeutic drug monitoring; 6-MP, 6-mercaptopurine; 6-
TG, 6-thioguanine; MeOH, methanol; PK, pharmacokinetic; SPE, solid-phase extraction; PDA, photodiode array; N-DMT, N-desmethyltamoxifen; 4-HT, 4-
hydroxytamoxifen.
Analytical Biochemistry 610 (2020) 113891
the highest peak area and efficacy of the post-column reaction; for
example, at low iodine levels and high levels of iodide ions, a higher
peak area was seen. Urine samples did not need any pre-clean-up step
and addition of stabilizing agents prior to analysis.
In another study, Rodriguez et al. [109] quantified the urine and
serum concentrations of both erlotinib and its metabolites by a validated
HPLC-UV method without sample treatment steps. Exemestane was
selected as the IS and determinations were carried out in NSCLC pa­
tients. This method could indicate the effect of simultaneously receiving
other drugs on the metabolism of erlotinib. In addition, Shimada et al.
[110] developed an HPLC-UV method that could quantitate sorafenib
and sorafenib N-oxide in serum samples. Because these two analytes are
lipophilic and rarely soluble in water, in this study, SPE was used with
octadecyl silyl-silica gel to eliminate the contaminate interferences;
liranaftate was used as IS.
Finally, Antunes et al. [111] reported a sensitive HPLC-PDA method
to specify tamoxifen and its three metabolites in the plasma samples of
breast cancer patients. Following development, they fully validated the
new method, which was adequately precise and sensitive to be applied
in the TDM of tamoxifen and PK studies.
4.2.3. Fluorescence detection methods (HPLC-FLD)
This part of the review describes the HPLC-FLD methods for the
simultaneous quantification of anticancer drugs and their metabolites.
As mentioned before, the advantages of FLD include more sensitivity
and selectivity; however, it typically requires derivatization steps,
limiting its usage. HPLC-FLD methods for the quantification of anti­
cancer drugs and their metabolites are listed in Table 7.
Rangel-Mendez et al. [112] simultaneously measured tamoxifen and
its metabolites in the plasma samples of patients under adjuvant
tamoxifen therapy with an HPLC-FLD method. Contrary to UV detectors,
FLD is highly sensitive for the detection of tamoxifen and its metabolites.
In this method, n-propranolol was used as IS. In addition, Heath et al.
[113] compared HPLC-MS/MS and HPLC-FLD to monitor cancer pa­
tients via measuring tamoxifen and its metabolites in plasma samples.
The upper limit in HPLC-FLD method was higher than HPLC-MS/MS
method, ascribed to the difference in the sample extraction column
used in these two methods.
Prijovich et al. [114] developed and validated a versatile online SPE
extraction coupled with an HPLC method for monitoring the plasma
levels of IRI and its metabolites; this method was tested in healthy
volunteers. The method of this study provided a simple isocratic mobile
phase without ion-pairing agents; it is possibly the most rapid and sen­
sitive method for assaying IRI and its related metabolites. This method
Extraction method Run time/Method Range Reference
application
Dilution with water 20 min/TDM 6-MP (0.4–5.0 μM), 6-TG (0.6–5 [108]
μM), 6-MP riboside (0.5–5 μM), 6-
TG riboside (0.9–5 μM)
Direct injection 15 min/ erlotinib (380–6510 ng/mL), [109]
PK clinical study desmethyl erlotinib (66–558 ng/
mL)
SPE 15 min/TDM 0.03–30 μg/mL [110]
LLE 16 min/TDM and Tamoxifen (38.42–83.69 ng/mL), [111]
PK study N-DMT (86.81–204.8 ng/mL), 4-
HT (0.76–1.53 ng/mL), endoxifen
(4.17–8.22 ng/mL)
12
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 7
HPLC-FLD methods for quantification of drug and its metabolites.
Parent drug Chromatography Method/Interface Extraction method Run time/ Range Reference
Method
application

Tamoxifen RP C18/gradient ACN: 35 mM KH2PO4 HPLC-FLD λex = PPT/ACN 10 min/clinical Endoxifen & 4-HT (3–20 [112]
(pH, 3.0) (38:62, v/v) and ACN, 100 × 260 nm, λem = LLE/hexane: isobutanol study ng/mL), tamoxifen
4.6 mm, 5 μm 375 nm (68:32, v/v) (50–1000 ng/mL)
Tamoxifen RP C18/isocratic 20 mM potassium HPLC-FLD SPE 20 min/clinical Tamoxifen (1.8–90 ng/mL) [113]
trihydrate: ACN (65:35, v/v), 250 × 4.6 λex = 256 nm, study Endoxifen (1.95–98 ng/mL)
mm, 5 μm λem = 380 nm 4-HT (2–101 ng/mL)
LC-MS/MS/Q-trap N-DMT (4–200 ng/mL)
Irinotecan C18/ACN in 0.1 M KH2PO4 (pH, 2.9), HPLC-FLD Online SPE/MeOH in 25 10 min/TDM 2.0 pg/mL-200 ng/mL [114]
300 × 3.9 mm, 10 μm λex = 375 nm, mM KH2PO4 (pH 2.9)
λem = 430 & 540 (20:80, v/v) and PPT
nm
MTX RP C18/isocratic 50 mM phosphate HPLC-FLD PPT & SPE 60 min/TDM 0.1–10 nM [116]
buffer (pH, 5.3): ACN (9:1, v/v), 50 × λex = 368 nm,
4.6 mm, 5 μm λem = 452 nm
Bendamustine RP C18/isocratic ACN: 10 mM KH2PO4 HPLC-FLD PPT 14 min/PK study BM: plasma (8.192–10000 [117]
(pH, 2.5) (32:68, v/v), 250 × 4.6 mm, 5 perchloric acid: MeOH ng/mL), urine (5–1000
μm (10:90, v/v) ng/mL)
Ɣ–OH–BM: plasma
(10–1000 ng/mL), urine
(5–1000 ng/mL)

RP, Reverse phase; ACN, acetonitrile; HPLC, high performance liquid chromatography; FLD, fluorescence detector; PPT, protein precipitation; LLE, liquid-liquid
extraction; 4-HT, 4-hydroxytamoxifen; LC, LC: liquid chromatographic; MS/MS, tandem mass spectrometry; Q-Trap: quadrupole ion trap; SPE, solid-phase extrac­
tion; N-DMT, N-desmethyltamoxifen; TDM, therapeutic drug monitoring; MeOH, methanol; BM, bendamustine; Ɣ–OH–BM, gamma-hydroxy- bendamustine.

can be used in the clinical and PK studies of IRI. In a similar study, IRI
and its active metabolite SN-38 was quantified in DBS sample [115].
Camptothecin was used as IS, and IRI quantification was highly corre­
lated with plasma and DBS assay methods.
Using an HPLC-FLD method, Uchiyama et al. [116] quantified MTX
and its main metabolites in the plasma samples of patients. The plasma
was deproteinized with aqueous ACN solution including trichloroacetic
acid, and 2,4-diaminobutyric acid was utilized as an IS. To increase
sensitivity, they reported post-column photochemical degradation,
without oxidative reagent, which was simple and reliable for this kind of
analysis. Additionally, Xie et al. [117] developed and validated a sen­
sitive HPLC-FLD method for the simultaneous measurement of plasma
and urine levels of bendamustine (BM) and its active metabolite,
gamma-hydroxy-bendamustine; they further employed fluorescent de­
tector for use in a PK study, and valeric acid was used as IS.
4.3. Multi-drug determination analysis methods
If an analytical method is able to simultaneously quantify several
drugs in a single separation run, it is highly beneficial during TDM or PK
studies. Provided the method has been validated for the quantification of
some drugs administered in a therapy regimen, one patient specimen is
enough for several drug quantifications.
4.3.1. Mass spectrometer detection methods: HPLC-MS(/MS)
As mentioned in the previous sections, HPLC-MS methods benefit
from high sensitivity and selectivity; thus, MS is the best detector,
especially in methods developed to quantify several drugs at one sepa­
ration run. These methods are summarized in Table 8.
Oral bioavailability of taxanes is extremely hampered by drug
metabolism via cytochrome P450 and drug efflux through P-glycopro­
tein. To overcome these issues, co-administration with inhibitors of the
mentioned pathways such as ritonavir can be helpful. In two similar
studies, Hendrikx et al. [118,119] developed the quantification methods
of PCT, DTX, and ritonavir in different human samples. In the first study,
they evaluated PCT, DTX, and ritonavir in feces and urine samples by a
HPLC-MS/MS method. This method is useful for monitoring the ab­
sorption and excretion associated with the co-administration of these
drugs. This was the first assay method to quantify each of the unlabeled
drugs in human feces. The latter is a similar method for plasma samples.
During development, they showed that the alkaline mobile phase could
foster the sensitivity of the method for PCT and DTX.
DTX and temsirolimus are widely co-administered in solid tumors;
therefore, using PCT as the IS, Navarrete et al. developed and validated a
simultaneous HPLC-MS/MS method for the quantification of DTX,
temsirolimus, and its main metabolite, sirolimus [120]. In another
study, Gao et al. [121] reported an HPLC-MS/MS method to determine
PCT, DTX, vinblastine, and vinorelbine in the whole blood and plasma
samples of NSCLC patients for PK studies. This method had a 4.5 min
total run time, which is very fast for a multi-drug detection method. This
method can be applied to both plasma and blood samples with the same
sample treatment, reagents, and analytical technique. In another study,
they reported and fully validated a method to simultaneously determine
11 typically-used anticancer drugs. They investigated PCT, DTX,
vinorelbine, vinblastine, carboplatin, pemetrexed, CP, etoposide, IF,
gemcitabine, IRI, and SN-38 at the same time. Following validation, this
method was successfully utilized in TDM for cancer patients [122].
B’Hymer and Cheever [123] detected CP, 4-keto cyclophosphamide,
and IF in human urine. Urine sample was prepared after LLE with ethyl
acetate. This method could be applied to monitor these analytes in
healthcare workers susceptible to toxicity with these anticancer drugs;
nonetheless, without full validation, it cannot be used in clinical studies.
Chen and Zhou [124] reported and fully validated an HPLC-MS/MS
method to analyze 5-FU and eniluracil, a novel modulator of 5-FU in
human plasma, based on FDA validation guidelines. A 5-FU analogue,
5-bromouracil was used as IS. This method was conducive to deter­
mining the effect of eniluracil on the PK of 5-FU in clinical studies. Liu
et al. [125] validated a method for the simultaneous quantification of
tegafur (FT), 5-FU, gimeracil, and endogenous uracil in the plasma
samples of cancer patients. During this study, three derivatization
methods were compared to achieve the most optimal sensitivity.
Derivatization with p-Bromophenacyl bromide resulted in a sensitive
and specific method suitable for PK studies.
Developments in tyrosine kinase inhibitors (TKIs) have revolution­
ized the therapy of various tumors. Imatinib was the first approved TKI,
after which, several drugs in this category were approved one after
another. Despite the established efficacy, patient-to-patient variations
were reported in therapy outcome. Therefore, the TDM studies to
simultaneously evaluate these drugs were useful in enhancing the clin­
ical outcomes and reducing the side effects of TKIs. Lankheet et al. [126]

13
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 8
Multi-drugs quantification HPLC-MS(/MS) methods.
Parent drug Chromatography Method/ Extraction method Run time/ Range Reference
Interface Object of
study

Paclitaxel, Docetaxel, Ritonavir C18/isocratic MeOH: 10 mM HPLC-MS/ Dilution with 9 min/ Urine: PCT and DTX (0.5–500 ng/ [118]
ammonium hydroxide in water MS-QqQ/ MeOH Clinical mL), ritonavir (8–8000 ng/mL)
(70:30, v/v), 150 × 2.1 mm, 5 μm ESI study feces: PCT & DTX (2–2000 ng/g),
ritonavir (8–8000 ng/g)
Paclitaxel, Docetaxel, Ritonavir C18/isocratic MeOH: 10 mM HPLC-MS/ LLE 9 min/ PCT & DTX (0.5–500 ng/mL), [119]
ammonium hydroxide in water MS-QqQ/ tertiary butyl Clinical ritonavir (2–2000 ng/mL)
(70:30, v/v), 150 × 2.1 mm, 5 μm ESI methyl ether study
Docetaxel, Temsirolimus, C8/gradient water with 0.1% HPLC-MS/ PPT MeOH/zinc 7 min/ 10–200 μg/L [120]
Sirolimus formic acid: MeOH with 0.1% MS-QqQ/ sulfate (70:30, v/v) TDM
formic acid (95:5, v/v), 150 × ESI and Online SPE
4.6 mm, 5 μm
Paclitaxel, Docetaxel, Vinblastine, C18/isocratic ACN: 10 mM HPLC-MS/ LLE 4.5 min/ PCT (25–2500 ng/mL), DTX, [121]
Vinorelbine ammonium acetate: formic acid MS/ESI+ TDM vinblastine, vinorelbine
(70:30:0.1, v/v/v), 100 × 2.1 (10–1000 ng/mL)
mm, 3.5 μm
Paclitaxel, Docetaxel, Vinblastine, T3-C18/gradient ACN: 10 mM HPLC-MS/ LLE and PPT 10 min/ PCT (25–2500 ng), DTX, SN-38 [122]
Vinorelbine, Pemetrexed, ammonium acetate: 0.1% formic MS-QqQ/ ACN: MeOH TDM (10–1000 ng), vinorelbine,
Carboplatin, Etoposide, acid, 100 × 2.1 mm, 3.0 μm ESI+ pemetrexed (100–10000 ng),
Cyclophosphamide, Ifosfamide, vinblastine, IRI (10–10000 ng),
Gemcitabine, Irinotecan, SN-38 CP, IF (1–1000 ng), carboplatin,
etoposide, gemcitabine (50–5000
ng)
Cyclophosphamide, Ifosfamide, C18/gradient ACN: water: acetic HPLC-MS/ LLE 25 min/ 4-keto-CP (10–625 ng/mL), CP, IF [123]
acid, 250 × 3 mm, 3.5 μm MS-QqQ/ Ethyl acetate TDM (0.5–25 ng/mL)
ESI
5-fluouracil, Eniluracil Synergi Polar-RP/gradient 10 HPLC-MS/ LLE TDM 5-FU (8.61–1080 ng/mL), [124]
mM ammonium acetate and MS-QqQ/ Ethyl acetate: eniluracil (4.13–1030 ng/mL)
0.01% formic acid in water: ACN, TIS isopropanol (85:15,
150 × 4.6 mm, 4 μm v/v) followed by
ammonium acetate
Tegafur, 5-fluouracil, Gimeracil, Zorbax SB-Aq/gradient ACN: 5 HPLC-MS/ LLE 12 min/ FT (5–5000 ng/mL), 5-FU [125]
Uracil mM ammonium acetate with MS-QqQ/ Isopropanol: ethyl TDM (0.6–700 ng/mL), gimeracil
0.1% formic acid, 150 × 2.1 mm, ESI acetate (3–700 ng/mL), U (6–2000 ng/
3 μm mL)
Dasatinib, Erlotinib, Gefitinib, C18/gradient 10 mM ammonium HPLC-MS/ PPT 10 min/ Erlotinib, gefitinib, imatinib, [126]
Imatinib, Lapatinib, Nilotinib, hydroxide in water (pH, 10.5): 1 MS-QqQ/ ACN TDM lapatinib, nilotinib and sorafenib
Sorafenib, Sunitinib mM ammonium hydroxide in ESI+ (20–10000 ng/mL), dasatinib and
MeOH, 50 × 2.0 mm, 5.0 μm sunitinib (5–2500 ng/mL)
Gefitinib, Erlotinib, Afatinib RP C18/isocratic 10 mM HPLC-MS/ LLE 5 min/ 0.05–100 nM [127]
ammonium hydroxide in ACN: 1 MS-QqQ/ Tert-butylmethyl TDM
mM ammonium hydroxide (pH, ESI+ ether
10.5) (70:30, v/v), 50 × 2.1 mm,
3.5 μm
Erlotinib, Imatinib, Lapatinib, RP C18/gradient ACN with HPLC-MS/ LLE 9 min/ sunitinib (10–1000 ng/mL), other [128]
Nilotinib, Sorafenib, Sunitinib formic acid: 10 mM ammonium MS-QqQ/ MeOH TDM TKIs (50–5000 ng/mL)
formiate, 50 × 4.6 mm, 2.6 μm ESI+
Afatinib, Axitinib, Ceritinib, RP C18/gradient 10 mM HPLC-MS/ LLE 8 min/ afatinib, axitinib, dabrafenib, [129]
Crizotinib, Dabrafenib, ammonium bicarbonate in water: MS-QqQ/ ACN TDM trametinib (2–200 ng/mL),
Enzalutamide, Regorafenib, 10 mM ammonium bicarbonate ESI+ ceritinib, crizotinib,
Trametinib in MeOH: water, 50 × 2.0 mm, enzalutamide, regorafenib
5.0 μm (50–5000 ng/mL)
Afatinib, Crizotinib, Osimertinib, C18/gradient 0.1% formic acid in HPLC-MS/ PPT 7 min/ afatinib (5–250 ng/mL), [130]
Erlotinib and Nintedanib water: 0.1 formic acid % ACN, 50 MS-QqQ/ ACN with 1% TDM crizotinib, osimertinib (5–1000
× 2.1 mm, 2.6 μm ESI+ formic acid ng/mL), erlotinib (20–4000 ng/
mL), nintedanib (1–250 ng/mL)
Dabrafenib, Trametinib, Xselect™ HSS T3/gradient 2 mM HPLC-MS/ PPT 5 min/ Vemurafenib, pazopanib [131]
Vemurafenib, Cobimetinib, ammonium acetate in water with MS-TSQ/ MeOH TDM (200–100000 ng/mL),
Pazopanib, 0.1% formic acid: ACN with 0.1% ESI regorafenib (20–10000 ng/mL), R
R N-Oxide, formic acid, 75 × 2.1 mm, 3.5 μm N-Oxide, N-DM R N-Oxide
N-DM R N-Oxide (20–10000 ng/mL), dabrafenib
(10–5000 ng/mL), cobimetinib
(2–1000 ng/mL), trametinib
(1–500 ng/mL)
Dabrafenib, Trametinib C18/gradient, 10 mM HPLC-MS/ LLE 6.4 min/ dabrafenib (50–5000 ng/mL), [132]
ammonium acetate in water: MS-QqQ/ Tert-butylmethyl TDM trametinib
MeOH, 50 × 2.0 mm, 5.0 μm TIS+ ether (0.5–50 ng/mL)
Bosutinib, Dasatinib, Ibrutinib, C18/isocratic, 10 Mm HPLC-MS/ LLE 7 min/ N-DP (0.25–75 ng/mL), dasatinib [133]
Imatinib, ammonium MS-Q- 1% ammonia TDM and ponatinib (0.5–150 ng/mL),
Nilotinib, Ponatinib formate: ACN each containing Trap/ESI/ aqueous solution imatinib and nilotinib (10–3000
0.1% formic acid, 50 × 2.1 mm, MRM+ and elution with ng/mL), other analytes (1–300
3 μm MTBE () ng/mL)
[134]
(continued on next page)

14
R. Sabourian et al.
Table 8 (continued )
Parent drug Chromatography Method/
Interface
Alectinib, Cobimetinib, RP C18/gradient 10 mM HPLC-MS/
Lenvatinib, Nintedanib, ammonium bicarbonate (pH, MS/QqQ/
Osimertinib, Palbociclib, 10.5) in water: 10 mM TIS/
Ribociclib, Vismodegib, ammonium bicarbonate (pH, MRM+
Vorinostat 10.5) in MeOH-water (1:9, v/v),
50 × 2.0 mm, 5 μm
MeOH, methanol; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; QqQ, triple quadrupole mass spectrometer; ESI, electrospray
ionization source; PCT, paclitaxel; DTX, docetaxel; LLE, liquid-liquid extraction; PPT, protein precipitation; SPE, solid-phase extraction; TDM, therapeutic drug
monitoring; ACN, acetonitrile; IRI, irinotecan; CP, cyclophosphamide; IF, ifosfamide; FT, tegafur; 5-FU, 5-fluouracil; U, uracil; TKIs, tyrosine kinase inhibitors; RP,
Reverse phase; R N-Oxide, Regorafenib N-Oxide; N-DM R-N-Oxide, N-Desmethyl-regorafenib N-Oxide; TIS+, positive timed ion selector; Q-Trap, quadrupole ion trap;
MRM+, positive multiple reaction monitoring; MTBE, tert-butyl methyl ether; N-DP, N-Desmethyl ponatinib.
validated the simultaneous determination of erlotinib, dasatinib, ima­
tinib, lapatinib, gefitinib, nilotinib, sunitinib, and sorafenib in plasma by
a HPLC-MS/MS method. Samples were pretreated by a PPT method
which could be used in the routine TDM of TKIs. Hayashi et al. [127]
reported and validated a fast HPLC-MS/MS method for the quantifica­
tion of gefitinib, erlotinib, and afatinib, co-administrated in NSCLC pa­
tients. This method can be potentially used in TDM of NSCLC patients
under chemotherapy with TKIs. Gotze et al. [128] reported and vali­
dated a HPLC-MS/MS method for the simultaneous analysis of six TKIs
(sunitinib, erlotinib, nilotinib, imatinib, lapatinib, and sorafenib) in
human plasma. The developed method was suitable for the multi-drug
analysis of TKIs in routine clinical study. Herbrink et al. [129] re­
ported a method for the quantification of axitinib, afatinib, trametinib,
ceritinib, dabrafenib, crizotinib, regorafenib, and enzalutamide in
plasma samples. Following successful validation, the method was
applied to the TDM of eight investigated drugs in cancer patients. Reis
et al. [130] reported an HPLC-MS/MS method to analyze crizotinib,
afatinib, nintedanib, osimertinib, and erlotinib used in the treatment of
NSCLC patients. This fully validated method was well-suited to monitor
these TKIs. Cardoso et al. [131] reported HPLC-MS/MS for the simul­
taneous quantification of six anticancer protein kinase inhibitors (PKIs):
trametinib, dabrafenib, cobimetinib, vemurafenib, regorafenib, pazo­
panib, and two active metabolites of regorafenib in human plasma.
Stable isotope-labeled drugs were used as IS. The validated method was
employed in both clinical and PK study programs. Nijenhuis et al. [132]
reported and validated an HPLC-MS/MS method to analyze trametinib
and dabrafenib in plasma and urine. This method seemed to be suitable
for use in the clinical studies of these drugs. Ultimately, Mukai et al.
[133] examined the plasma concentration of nine kinase inhibitors with
their three active metabolites (bosutinib, dasatinib, ibrutinib, dihy­
drodiol ibrutinib, imatinib, NDI, nilotinib, ponatinib, and N-desmethyl
ponatinib). They developed and validated HPLC-MS/MS for the simul­
taneous analysis of these mentioned analytes for the first time. Janssen
et al. [134] developed and validated a HPLC-MS/MS method to simul­
taneously investigate the plasma levels of nine anticancer drugs (alec­
tinib, cobimetinib, lenvatinib, nintedanib, osimertinib, palbociclib,
ribociclib, vismodegib, and vorinostat). This sensitive method was
appropriate for use in clinical routine care and simplified TDM.
4.3.2. UV detection methods (HPLC-UV and HPLC-PDA)
The importance of HPLC-UV methods is not unknown to researchers
owing to their accessibility and ease of use. Table 9 summarizes the
HPLC-UV methods for the simultaneous quantification of anticancer
drugs.
Abd El-Hady et al. [135] employed an eco-friendly HPLC method for
the simultaneous determination of drugs used in the Hodgkin disease,
including MTX, dacarbazine, chlorambucil, and vinblastine. They vali­
dated and compared the method with a CE method for the simultaneous
determination of the analytes in human urine and plasma samples.
15
Analytical Biochemistry 610 (2020) 113891
Extraction method Run time/ Range Reference
Object of
study
4 min/ Alectinib, lenvatinib, nintedanib
TDM and vismodegib (10–200 ng/mL),
cobimetinib and palbociclib
(50–1000 ng/mL), osimertinib
(100–2000 ng/mL), ribociclib
(5.00–100 ng/mL), vorinostat
(25–500 ng/mL)
Bermingham et al. [136] quantified Epi, doxorubicin (Dox), DTX, PCT,
and daunorubicin (Dnr). The detector wavelength was held at 254 nm
for Dox, Dnr, and Epi (for 10 min), then switched to 234 nm for the
detection of DTX and PCT. This method was fully validated and then
utilized to analyze the serum samples of patients. Agrawal et al. [137]
have recently developed a method to determine the blood plasma level
of both Dnr and cytarabine at the same time. They applied and validated
an inexpensive and simple RP-HPLC-UV method, with a short retention
time, to analyze these drugs in blood plasma.
FT is a 5-FU prodrug for treating colorectal, head, and neck cancers.
Peer et al. [138] simultaneously measured FT and 5-FU, setting the
detection wavelength at 272 nm. This method was simple and
eco-friendly and applied for the simultaneous clinical study of low 5-FU
concentrations and high FT concentrations. Gopinath et al. [139]
developed a simple and reliable HPLC-UV method for the quantitative
analysis of 5-FU, CP, Epi, three 5-FU metabolites, and one Epi metabolite
in plasma. Simultaneous analysis of 5-FU, CP, and Epi is important
because the combination of these drugs is routinely applied to breast
cancer; moreover, this method reduces the sampling interval for
different clinical studies. Sanson et al. [140] developed an HPLC-PDA
method for the simultaneous analysis of CP, Dox, 5-FU, and IF, four
structurally-different anticancer drugs. The LOQ obtained was 1 μg/mL
for 5-FU, IF, and CF and 0.5 μg/mL for Dox. The method was simple and
low cost, hence convenient for application in TDM.
Several studies have reported the analysis methods for the quantifi­
cation of TKIs. In the first study, a method for quantifying gefitinib and
erlotinib was reported. These are two TKIs for the treatment of NSCLC
patients. Faivre et al. [141] validated a HPLC-UV method for the con­
current analysis of gefitinib and erlotinib in plasma, and sorafenib was
used as IS. The analysis was done within 15 min over a concentration
range of 20–1000 and 80–4000 ng/mL for gefitinib and erlotinib,
respectively. In another study, erlotinib and lapatinib were specified in
human plasma by an HPLC-UV method developed by Ohgami et al.
[142]. These drugs are not used for patients under cancer chemo­
therapy; therefore, each drug can be used as an IS for quantifying the
other. This method is suitable for the TDM of erlotinib or lapatinib in
clinical institutions that are not equipped with LC-MS/MS. Zhen et al.
[143] developed an HPLC-UV method to simultaneously quantify
vemurafenib and erlotinib in plasma. UV detector was set at 249 nm for
vemurafenib and at 331 nm for erlotinib. The calibrated range was
1.25–100 μg/mL and 50–4000 ng/mL for vemurafenib and erlotinib,
respectively. This method can be utilized in clinical practices. Davies
et al. [144] separated nilotinib and imatinib by an HPLC-UV method in
plasma samples. The validated range was 100–12000 ng/mL. Clozapine
was used as IS owing to its similar structural features to other com­
pounds. Simple separation method simplified the set up process of this
method at laboratories. Pirro et al. [145] developed and validated a
simple and fast HPLC-UV method to quantify major TKIs, dasatinib,
nilotinib, and imatinib in human plasma samples. The LOQ was 50
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 9
Multi-drugs quantification HPLC-UV methods.
Parent drug Chromatography Method/ Extraction method Run time/ Range Reference
Wavelength Object of
study

Methotrexate, AGP column/isocratic 10 mM HPLC-UV λmax = Centrifuge and 23 min/ MTX (0.1–20 μg/mL), VBL and DTIC [135]
Dacarbazine, phosphate buffer (pH, 6), 150 250 nm dilution TDM (0.3–30 μg/mL), CHL (0.6–60 μg/
Chlorambucil, × 2 mm, 5 μm mL)
Vinblastine
Docetaxel, Paclitaxel, RP C18/gradient, HPLC-UV λmax = Online SPE 23 min/ 0.5–25 mg/mL [136]
Doxorubicin, water: ACN: formic acid 254, 234 nm 30 mM ammonium TDM
Daunorubicin, (90:10:0.1, v/v/v) (pH, 3.5), formate: ACN (98:2,
Epirubicin 150 × 4.6 mm, 5 μm v/v), (pH, 6.8)
Daunorubicin, Cytarabine RP C18/isocratic, 20 mM HPLC-UV PPT 11.2 min/ 5–25 μg/mL [137]
KH2PO4: ACN (pH, 2.5) (20:80, λmax = 254 nm ACN TDM
v/v), 250 × 4.6 mm, 5 μm
Tegafur, 5-fluouracil C16/isocratic MeOH: water HPLC-UV λmax= PPT 17 min/ 5-FU (8–200 ng/mL), FT [138]
(3/97, v/v) 150 × 4.6 mm, 5 272 nm Ammonium sulfate clinical (800–20000 ng/mL)
μm and LLE Isopropanol: study
ethyl acetate (15:85,
v/v)
5-fluouracil, Epirubicin, C18/gradient water (pH, 4.0): HPLC-UV λmax= PPT 80 min/ 5-FU (0.1–10 μg/mL), DHFU, 5α- [139]
Cyclophosphamide MeOH, 150 × 4.6 mm, 5 μm 195, 200, 254, ACN TDM dFUR and Epi (0.1–50 μg/mL), 13-
265, 270, nm dihydro Epi (1–50 μg/mL),
fluorouridine (5–200 μg/mL), CP
(10–500 μg/mL)
Cyclophosphamide, RP C18/isocratic water (pH, HPLC-UV λmax= LLE 20 min/ 0.5–100 μg/mL [140]
Doxorubicin, 5-fluoura­ 4): MeOH, ACN (68:19:13, v/ 195, 265 nm Ethyl acetate TDM and
cil, Ifosfamide v/v) 250 × 4.6 mm, 5 μm PK study
Gefitinib, Erlotinib C8/gradient ACN: 20 mM HPLC-UV λmax= LLE 15 min/ Gefitinib (20–1000 ng/mL), [141]
ammonium acetate (pH, 4.5), 331 nm NaOH, ethyl acetate TDM erlotinib (80–4000 ng/mL)
250 × 3 mm, 5 μm
Lapatinib, Erlotinib C18/isocratic ACN: MeOH: HPLC-UV λmax= PPT 10 min/ 0.125–8.00 μg/mL [142]
water: trifluoro acetic acid 316 nm ACN TDM
(26:26:48:0.1, v/v/v/v), 150
× 4.6 mm, 5 μm
Vemurafenib, Erlotinib C8/100 mM glycine buffer HPLC-UV λmax= LLE 12 min/ Vemurafenib (1.25–100 μg/mL), [143]
(pH, 9.0): ACN (45:55, v/v), 249, 331 nm ACN clinical erlotinib (50–4000 ng/mL)
250 × 4.6 mm, 5 μm study
Nilotinib, Imatinib and its C6-phenyl/isocratic MeOH: 50 HPLC-UV λmax= SPE 35 min/ 12,000-100 ng/mL [144]
metabolite, CGP-74588 mM ammonium acetate (pH, 260 nm TDM
8.0) (65:35, v/v)
Imatinib, RP-C18/isocratic water: HPLC-UV λmax= SPE 10 min/ 0.005–10 μg/mL [145]
Dasatinib, Nilotinib MeOH: ACN: triethylamine 267 nm TDM
Imatinib, NDI, Dasatinib HPLC-UV: 35 mM ammonium HPLC-UV λmax= LLE 5 min/ imatinib (0.05–10 mg/L), NDI [146]
perchlorate in MeOH (pH, 7.5) 270 nm Butyl acetate: butanol TDM (0.01–2 mg/L), dasatinib (1–200
HPLC-MS: 40 mM ammonium HPLC-MS/MS- (4:1, v/v) μg/L)
acetate in MeOH (pH, 6.0), APCI+
100 × 2.1 mm, 5 μm

HPLC, high performance liquid chromatography; UV, ultraviolet; TDM, therapeutic drug monitoring; MTX, methotrexate; VBL, vinblastine; DTIC, dacarbazine; CHL,
chlorambucil; RP, Reverse phase; SPE, solid-phase extraction; PPT, protein precipitation; MeOH, methanol; LLE, liquid-liquid extraction; 5-FU, 5-fluouracil; FT,
tegafur; DHFU, 5-fluoro-5,6-dihydrouracil; 5α-dFUR, 5′ -deoxy-5-fluorouridine; Epi, epirubicin; CP, cyclophosphamide; PK, pharmacokinetic; PDA, photodiode array;
NDI: N-desmethyl imatinib.

ng/mL for dasatinib and 10 ng/mL for nilotinib and imatinib. The
calibration curves ranged from 0.005 to 10 μg/mL. In conclusion, this
method was accurate for the monitoring and PK studies of nilotinib and
imatinib but not sufficient for dasatinib in CML patients. Birch et al.
[146] developed a simple HPLC-UV method for the quantification of
imatinib and its metabolite, NDI in human plasma, or serum. They
further applied the same chromatography procedure coupled with MS
for dasatinib quantification. They validated these methods and
confirmed the HPLC-UV method for TDM but proposed that more studies
should assess the role of TDM in guiding treatment.
4.3.3. Fluorescence detection methods (HPLC-FLD)
In this article, we attempted at covering all detectors coupled with an
HPLC system. Krogh-Madsen et al. [147] developed and validated an
HPLC-UV/FLD method with mixed-mode cation-exchange SPE extrac­
tion method that can be applied for the simultaneous quantification of
all three drugs in spite of their different physicochemical properties. The
method reported in this paper had an improved LLOQ for etoposide and
cytosine arabinoside, but not for Dnr, which is in contrast to previous
studies that are appropriate for simultaneous detection. Pieri et al. [148]
monitored the biological exposure of nurses to Dox and Epi through
quantifying the urinary level of analytes with an HPLC-FLD method that
was less expensive than mass spectrometry and more specific than UV
detection. They validated the method and optimized its characteristics
to result in a good resolution and intense peaks in shorter times despite a
similar structure to analytes. Idarubicin was used as IS. The method was
also appropriate for different applications from TDM to PK studies in
cancer patients. These reported methods are summarized in Table 10.
5. Advantages and disadvantages
Among the existing methods for the quantification of a single anti­
cancer drug, LC-MS methods have resulted in a more sensitive, rapid,
and selective detection compared with other UV-based chromatographic
methods [33,37,39]. Moreover, analyte quantification in certain
matrices requires the use of a specially-sensitive and golden method
such LC-MS/MS [32,34,42]. When using a small amount of sample,
procedures like RED are employed prior to LC-MS/MS quantification

16
R. Sabourian et al. Analytical Biochemistry 610 (2020) 113891

Table 10
Multi-drugs quantification HPLC-FLD methods.
Parent drug Chromatography Method/ Extraction Run time/Object of Range Reference
Interface method study

Cytosine arabinoside, RP C18/gradient 3 g/L KH2PO4 HPLC-FLD mixed-mode 15.5 min/clinical Cytosine arabinoside (13–1500 [147]
Daunorubicin, (pH, 2.0) and phosphoric acid: λex = cation-exchange studies, PK-PD analysis ng/mL), Dnr (15–1000 ng/mL),
Etoposide ACN, 150 × 4.6 mm, 3 μm 230,490 nm SPE etoposide (52.5–3500 ng/mL)
λem = 328,
555 nm
HPLC-UV
λmax = 280
nm
Doxorubicin, C8/gradient 50 mM formic acid: HPLC-FLD SPE 10 min/Nurses 2.0–65.4 ng/mL [148]
Epirubicin ACN, 150 × 4.6 mm, 5 μm λex = 460 nm biological monitoring,
λem = 580 nm TDM and PK study

RP, Reverse phase; ACN, acetonitrile; HPLC, high performance liquid chromatography; FLD, fluorescence detector; SPE, solid-phase extraction; PK, pharmacokinetics;
PD, pharmacodynamics; BM, TDM, therapeutic drug monitoring.

[35]. In addition, LC-MS/MS has replaced complicated pretreatment

procedures [44]. In addition, an LC-MS/MS method with a small plasma
sample compared with chromatographic methods coupled with UV or
fluorimetric detection seems to be more specific, sensitive, and reliable
[36]. Ultimately, LC-MS/MS before SPE procedures seems to minimize
matrix effect compared with precipitation extraction procedures [40].
The ability to simultaneously analyze drugs and their metabolites is
highly important because metabolites can affect the pharmacokinetic of
the drug. Due to the physicochemical properties of the metabolites and
their similar structures, their separation requires the optimization of the
analysis conditions to achieve a proper resolution. Among the four
mentioned studies investigating imatinib and its metabolites with LC-
MS/MS method, one study reached the shortest run time (3.5 min). Fig. 1. Summary of included methods.
They made use of the LLE method for sample preparation [97]. Studies
that have used triple quadrupole mass detectors instead of single
6. Conclusion
quadrupole and LC-MS/MS method instead of LC-MS method could
reach better separation of analytes. Among the mentioned studies, the
In this study, all the HPLC systems, from 2010 to 2020, were
best separation of Cape and its metabolites was achieved in the study of
collected and categorized based on the number of quantifiable analytes
Deng et al. [104]; they were able to quantify the analytes in low LOQ (10
in a single analysis run. This systematic review delineated the devel­
ng) and over a short run time (10.5 min) via optimizing the chromato­
opment of methods for the quantification of anticancer drugs. A sum­
graphic condition and the mobile phase composition. In one study, they
mary of the methods is available in Fig. 1. Totally, in the past decade
could accomplish high sensitivity and short run time with HPLC-FLD
(the time duration of the review), most methods have utilized MS de­
method for analyzing IRI and its metabolites [114]. Contrary to the
tectors owing to their selectivity and sensitivity. Taken together, in all
two other cases [86,87], they conducted this analysis with LC-MS/MS
the included studies, when the objective was to quantify a single drug,
methods. They extracted the analytes on a SPE column, and the anal­
MS or UV detectors were utilized equivalently. Nevertheless, for
ysis was performed on a C18 column with a simple isocratic mobile
methods aimed at the quantification of drug and metabolite(s) in a
phase. Their modifications of chromatographic conditions resulted in a
single run, MS detectors were the most used detectors. Possibly due to
good resolution, rendering this method fast and sensitive to the sepa­
the similar structure of both drugs and their metabolite, MS detector is
ration of IRI and its metabolites. In one study, LC-MS/MS method was
the most optimal alternative and application of UV detectors has been
compared with HPLC-FLD to monitor the plasma levels of tamoxifen and
very limited. Finally, simultaneous multi-drug measurement methods in
its metabolites [113]. The upper limit in the HPLC fluorescence method
patients undergoing multi-drug regimens of cancer treatment can have
was higher than that of the LC-MS/MS method due to the differences in
many benefits, such as the convenience of patients owing to the reduced
the sample extraction column utilized in these methods. This was of
sampling times in addition to reduced analysis costs. Based on the
value because despite the higher sensitivity of the LC-MS/MS method,
structure of the drug, UV detectors can be useful in these methods in
the HPLC fluorescence method was cheaper and more accessible in
addition to MS detectors. This review tried to provide a broad view for
many laboratories with good ruggedness. Simultaneous analyses of MTX
researchers prior to developing a quantification method, hence the de­
and its metabolites requires a tedious extraction process for the removal
cision making about the detectors of the method.
of the interfering compounds and recovery of all the analytes. In addi­
Another aspect of the developed methods is validation of a new
tion, the developed method has a long run time [116], thereby needing
method, which is a necessary step before publishing a newly developed
such modifications to achieve a good sensitivity.
method. In this study, all included methods performed full or at least
Finally, to simultaneously specify several drugs, analysis with LC-
partial validation. In the next study, we are going to collect other
MS/MS method can provide better resolution and shorter run time as
chromatographic methods, such as UPLC and HILIC to compare their
was the case with the study of Gao et al. they were able to achieve 4.5
efficiency and ability to separate several analytes in a single run.
min total run time, which is very fast for a multi-drug detection method
[121]. In another study, CP, Dox, 5-FU, and IF were the four structurally
CRediT authorship contribution statement
different anticancer drugs that Sanson et al. could simultaneously
separate by developing a HPLC-PDA method; the LOQs of the analytes,
Reyhaneh Sabourian: Conceptualization, Methodology, Writing -
on the other hand, was relatively large (1 μg/mL for 5-FU, IF and CF and
original draft, preparation. Seyedeh Zohreh Mirjalili: Methodology,
0.5 μg/mL for Dox) [140].

17
R. Sabourian et al.
Writing - original draft, preparation. Negar Namini: Writing - original
draft, preparation. Fateme Chavoshy: Writing - original draft, prepa­
ration. Mannan Hajimahmoudi: Supervision, Project administration,
Writing - review & editing. Maliheh Safavi: Methodology, Supervision,
Writing - review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
The authors would like to express appreciation to Ayda Ferdowsifard
for critical reading of the manuscript and for her valuable feedback.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ab.2020.113891.
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