Received: 9 December 2019 Revised: 20 March 2020 Accepted: 2 April 2020

DOI: 10.1002/bmc.4843

RESEARCH ARTICLE

Pre-column derivatization method for determining

phenylephrine in human plasma and its application in a
pharmacokinetic study

Li Li1,2 | Yong Wang3 | Guanglei Chen3 | Kelly Dong2 | Haifeng Song1

1
Institute of Lifeomics, Academy of Military
Medical Science, Military Academy of
Sciences, Beijing, China
2
Beijing United-Power Pharma Tech Co.Ltd,
Beijing, China
3
China Resources Sanjiu Medical and
Pharmaceutical Co.Ltd, Shenzhen, China
Correspondence
Haifeng Song, Institute of Lifeomics, Academy
of Military Medical Science, Military Academy
of Sciences, Beijing 100850, China.
Email: songhf6811@163.com
Funding information
National Program on Key Research Project of
New Drug Innovation, Grant/Award Number:
No. 2015ZX09501008
Abstract
In the present study, a rapid derivatization liquid chromatography–tandem mass
spectrometry (LC–MS/MS) method was developed and validated to evaluate phenyl-
ephrine in human plasma. The plasma samples were processed to precipitate the pro-
teins, followed by derivatization of the phenylephrine in the plasma with dansyl-
chloride solution and extraction with methyl tert-butyl ether–n-hexane (2:1, v/v). The
treated samples were analyzed on a Gemini C18 column with 3 min gradient elution,
and sensitive detection was achieved with a Waters TQ-s. The method gave linear
results over a concentration range from 0.020 to 10.0 ng/ml. The lower limit of quan-
tification was 0.020 ng/ml. Intra- and inter-day precision was <15%, and accuracy
was 95.0–105.3%. The validated LC–MS/MS method was successfully applied in the
pharmacokinetic analysis of phenylephrine in Chinese subjects with common cold
after a single-dose administration of 5, 10 or 20 mg phenylephrine. This pre-column
derivatization method may also be applied for the analysis of endogenous hormones
such as norepinephrine and adrenaline in a biological matrix.

KEYWORDS

derivatization, liquid chromatography–tandem mass spectrometry, pharmacokinetics,

phenylephrine

1 | I N T RO D UC TI O N
Phenylephrine is a selective α1-adrenergic receptor agonist in the
phenethylamine class used primarily to induce decongestion and pupil
dilation and increase blood pressure. Phenylephrine has been
marketed as an alternative to the decongestant pseudoephedrine as
an over-the-counter medicine for over 10 years (Horak et al., 2009;
Gelotte, 2018).
Phenylephrine {(R)-3-hydroxy-α-[(methylamino)methyl]-benz-
enemethanol} is a structural analog of adrenaline but exhibits different
pharmacological activity. Phenylephrine is a small molecule with high
polarity. It shows very limited retention on reverse-phase columns,
although it can be retained on an amino column; however, the sensi-
tivity is not sufficient for clinical sample analysis. Few methods have
been reported for the determination of phenylephrine in human
plasma or serum (Feng, Zhao, Jiang, & Hu, 2013; Gumbhir &
Mason, 1996; Ptácek, Klíma, & Macek, 2007). Feng et al. reported an
ultra-performance liquid chromatography–tandem quadrupole mass
spectrometry (UPLC–MS/MS) method coupled with a hydrophilic
interaction chromatography column and achieved a lower limit of
quantitation (LLOQ) of 10 pg/ml using a 250 μl plasma sample. How-
ever, the separation was performed on a hydrophilic interaction chro-
matography column, even though solid-phase extraction pretreatment
was performed to clean the sample. A strong ion suppression was
observed using this method (absolute matrix effect ranged from 46.5
to 60.4%), which may lead to high variability in assay performance.
Compound derivatization can improve chromatographic behavior and
enhance instrument sensitivity. As a pre-column derivatization
reagent for liquid chromatography, dansyl-chloride(DNS-Cl) is advan-
tageous because of its simple reaction setup, stable reaction product,

Biomedical Chromatography. 2020;e4843. wileyonlinelibrary.com/journal/bmc © 2020 John Wiley & Sons, Ltd. 1 of 7
https://doi.org/10.1002/bmc.4843
2 of 7
quantitative dansylation, strong UV absorption and fluorescence with
diminished interference. It is often used to quantitatively analyze
amino acids (Huo et al., 2014; Timperio, Fagioni, Grandinetti, &
Zolla, 2007; Tuberoso, Congiu, Serreli, & Mameli, 2015; Zhang
et al., 2012) and amines (Jing, Li, & Jiang, 2016; Salomonsson, Hans-
son, & Bondesson, 2013; Yang, Mu, Zhang, & Zhu, 2014). In this
study, we established and validated an LC–MS/MS method for the
quantitative analysis of phenylephrine in human plasma following
pre-column derivatization with DNS-Cl. This novel robust method
requires only 100 μl of plasma sample for analysis, and no matrix
effect was observed. This method was successfully applied to the
pharmacokinetic evaluation of phenylephrine in a cohort of
Chinese patients with common cold after oral administration of an
over-the-counter drug.
2 | EXPERIMENTAL
2.1 | Materials and reagents
Phenylephrine hydrochloride was obtained from USP (100% purity,
Rockville, MD, USA). (R)-(−)Phenylephrine-2,4,6-d3 HCl (99% purity,
internal standard, IS) was purchased from C/D/N Isotopes Inc.
(Pointe-Claire, Quebec, Canada). HPLC-gradeDNS-Cl was purchased
from Sigma-Aldrich(St Louis, MO, USA). Acetonitrile, formic acid,
2-propanol, methyl tert-butyl ether and methanol of HPLC-grade
were purchased from Thermo Fisher Scientific (Waltham, MA,
USA). HPLC-graden-hexane and analytical-grade sodium bicarbonate
(NaHCO3) were produced by Sinopharm Chemical Reagent Co. Ltd
(Shanghai, China). Dimethyl sulfoxide (99.8% purity) was purchased
from J&K Scientific Ltd (Beijing, China). Deionized water was col-
lected from a Milli-Q water purification system (Millipore, Billerica,
MA, USA). Human plasma (K2EDTA) was purchased from
BioreclamationIVT (Westbury, NY, USA).
2.2 | Chromatography and mass spectrometry
Chromatographic analysis was carried out on an Acquity UPLC I-Class
system (Waters Corporation, Milford, MA, USA) equipped with a
96-well autosampler. Chromatographic separation was performed
with a Phenomenex Gemini C18 column (2.0 × 50 mm, 5 μm; Torrance,
CA, USA). The analytical column temperature was maintained at room
temperature. The mobile phase consisted of 0.1% formic acid in water
(A) and 0.1% formic acid in acetonitrile (B); the total runtime gradient
was 3 min with a flow rate of 0.5 ml/min. The autosampler was
maintained at 4
C, and the injection volume was 5 μl.
Mass spectrometry detection was achieved with a Waters Xevo™
TQ-S mass spectrometer equipped with an electrospray ionization
source in positive ion mode. Analytes were quantified in multiple-
reaction monitoring mode with the transitions of m/z 634.05 !
537.01 for dansylated phenylephrine and m/z 637.07 ! 540.07 for
dansylated phenylephrine-d3(IS). The electrospray voltage, source
LI ET AL.
temperature, desolvation temperature and desolvation gas flow rate
were set to 3.0 kV, 150
C, 500
C and 650 L/h, respectively. All data
were acquired using UNIFI software (Waters Corp.) and quantification
was performed with Watson LIMS (Thermo Fisher Scientific) using lin-
ear regression.
2.3 | Preparation of stock and working solutions,
calibration standards and quality control samples
Stock solutions of phenylephrine (1 mg/ml) and phenylephrine-d3(IS,
1 mg/ml mg/ml) were prepared in dimethyl sulfoxide. All stock solu-
tions were stored at −60 to −90
C. IS stock solution was serially
diluted with acetonitrile–water (1:1, v/v, 0.1% formic acid) to achieve
IS working solution at 20 ng/ml. The IS working solution was stored at
room temperature.
Calibration standards were freshly prepared each day by serial
dilution with blank pooled plasma to final concentration levels at
0.0200, 0.0400, 0.100, 0.500, 1.00, 2.00, 5.00 and 10.0
ng/ml. Similarly, quality control (QC) samples were prepared using
the blank pooled plasma at concentrations of 0.0600 (low concen-
tration quality control, LQC), 0.600 (middle concentration quality
control, MQC) and 8.00 (high concentration quality control, HQC)
ng/ml.
2.4 | Plasma sample preparation
Aliquots of 100 μl of human plasma samples and 15 μl of IS working
solution (20.0 ng/ml) were added to 1.1 ml plastic tubes, followed by
the addition of 400 μl of methanol (containing 0.1% formic acid). The
mixture was then vortexed for 2 min and centrifuged at 4816g for 15
min. The supernatant was transferred into a clean tube and evapo-
rated to dryness under a gentle stream of nitrogen. The residue was
dissolved in 100 μl NaHCO3 (100 mM; pH adjusted to 10.5 using 1 M
NaOH) and then mixed with an equal volume of DNS-Cl solution (1
mg/ml in acetonitrile). The derivatization reaction shown in Figure 1
was conducted in a water bath at 60
C for 6 min. The reaction mix-
ture was extracted with 600 μl of methyl tert-butylether–hexane (2:1,
v/v). After mixing and centrifugation, the upper organic layer was
transferred into a clean plastic tube and evaporated under nitrogen.
Finally, the residue was dissolved in 100 μl acetonitrile–water (1:1,
v/v, containing 0.1% formic acid). The reconstituted sample (5 μl) was
injected for LC–MS/MS for analysis.
2.5 | Method validation
Our method was validated based on regulatory guidelines (Chinese
Pharmacopoeia 2015, appendix 9,012, part IV 2015; Guideline on
bioanalytical method validation, EMA, 2011) for the following parame-
ters: selectivity, matrix effect, recovery, accuracy and precision, carry-
over and stability.
LI ET AL.
F I G U R E 1 Derivatization reaction and the
chemical structures of phenylephrine and
phenylephrine-d3(IS)
The selectivity of the method was assessed by comparison of the
chromatograms from six individual blank human plasma samples with
mixed-gender samples to evaluate potential interference at the reten-
tion times of the derivatized phenylephrine and IS.
The calibration curve of phenylephrine was constructed by plot-
ting the peak area ratios of derivatized phenylephrine to IS against the
nominal concentration of calibration standards at eight levels in the
range from 0.0200 to 10.0 ng/ml with a weighting factor 1/x2. The
plasma concentrations of the study samples were back-calculated by
linear regression.
Intra- and inter-day precision and accuracy were determined by
assaying six QC replicates at the LLOQ and low, middle and high con-
centrations in three consecutive batches. The accuracy and precision
of the method were evaluated by calculating the relative error (RE, %)
and coefficient of variance (CV, %), respectively.
Matrix effects were assessed by comparing the peak area of the
derivatized phenylephrine and IS in six pretreated blank plasma lots
(from equal numbers of male and female subjects) with those of the
derivatized phenylephrine and IS in neat solution at equivalent concen-
trations. The matrix effect of derivatized phenylephrine was deter-
mined at two-level QC samples (0.0600 ng/ml for LQC and 8.00 ng/ml
for HQC), and the matrix effect of the IS was assessed at 15
ng/ml. The absolute matrix effect was defined by the ratio of peak area
to the matrix/peak area in neat solution. Variation in the matrix effects,
which was expressed as the RSD, was evaluated by direct comparison
of the peak area with the matrix between six different lots of plasma.
The inter-subject variability of the matrix effects at each concentration
should be <15%. In this study, the matrix effects of normal plasma,
hemolytic plasma and hyperlipidemia plasma were evaluated.
Recovery of the derivatized phenylephrine was evaluated using
three QC samples (0.0600 ng/ml for LQC, 0.600 ng/ml for MQC and
8.00 ng/ml for HQC), whereas the IS was also evaluated at 15 ng/ml.
Carryover was evaluated by injecting a double blank sample
above the upper limit of quantification (ULOQ) at 10 ng/ml. The peak
area of the double blank sample was compared with that of
the LLOQ.
The stability of phenylephrine in human plasma was evaluated by
analyzing the LQC and HQC samples (three replicates at each level)
during the sample storage and processing procedures. In this study,
3 of 7
the stability of whole blood at room temperature for 4 h was evalu-
ated. The benchtop stability was evaluated by incubating the QC sam-
ples at room temperature for 4 h, which is the routine preparation
time of the samples. The freeze–thaw stability was measured after
four freeze–thaw cycles. The autosampler stability for the derivatized
product was tested for 48 h. Long-term stability was assessed by ana-
lyzing QC samples stored at −20
C and −70
C for at least 1 month.
All stability QC samples were evaluated with a freshly prepared cali-
bration curve.
2.6 | Plasma sample collection
The purpose of the clinical study was to study the tolerance level and
the safety profile phenylephrine in Chinese patients with common
cold after single oral administration of different doses of phenyleph-
rine on an empty stomach. We also preliminarily evaluated the possi-
ble pharmacokinetic and pharmacodynamic characteristics. This study
was approved by the ethics committee of the Beijing People’s Hospi-
tal. Blood samples were collected at 10, 15, 20, 25, 30, 40, 60, 90,
120, 240, 360 and 480 min after drug administration and immediately
centrifuged to obtain the plasma samples. The plasma samples were
transferred to new tubes and then stored in a −70
C freezer until
analysis. The plasma concentrations of phenylephrine in the patient
plasma were determined using a fully validated LC–MS/MS method.
3 | RESULTS AND DISCUSSION
3.1 | Optimization of derivation and extraction
conditions
As phenylephrine and IS have two derivatization sites on the second-
ary amine and phenolic hydroxyl groups, optimization of the pH in the
derivatization mixture yielded a stable double derivative product with
higher mass spectrometric sensitivity. Different pH values of 9, 10,
10.5, and 11 were evaluated, and the results (Figure 2) showed the
highest yield at pH 10.5, demonstrating the reproducibility of this
reaction condition. Other reaction conditions, such as reaction time,
4 of 7
F I G U R E 2 Mass spectrometry response at different pH values
(mean ± SD, n = 3); the reaction was performed at 60
C for 6 min
temperature and amount of DNS-Cl, were optimized. In the presence


of excessive DNS-Cl, the reaction was completed in 6 min at 60 C.
Derivatized products were further extracted, and different
organic solvents such as methyl tert-butyl ether, ethyl acetate, n-
hexane and organic solvents at different ratios were examined.
Methyl tert-butylether–n-hexane (2:1, v/v) was used as the extraction
solvent to obtain the highest recovery rate. In this study, the derivati-
zation processes and extraction were carried out in 96-well plates to
maximize the throughput.
3.2 | LC–MS/MS condition optimization
After double site derivatization, the molecular mass was increased by
233 Da for each dansyl part added, resulting in [M + H]+ values of
634 and 637 for phenylephrine and IS, respectively (Figure 3). The
highest response daughter ion was 170, but the noise was too high; a
value of 537 was chosen as the daughter ion to obtain the best signal-
to-noise ratio. After derivatization, the signal-to-noise ratio of the
analyte at 20 pg/ml in human plasma was >50, showing a significantly
increased response. Owing to the increased sensitivity, the sample
volume could be reduced to as low as 100 μl.
A range of analytical columns was tested, including the Thermo
Betabasic-18 and Biobasic-18, Phenomenex Luna C18, Gemini and
Waters CSH C18, to achieve the best peak shape and resolution from
the noise. Compared with the methanol, water and formic acid sys-
tem, the acetonitrile, water and formic acid system produced lower
noise. The LC gradient was optimized to obtain the best signal-to-
noise ratio and minimum matrix effect compared with the method
reported by Feng et al.
3.3 | Method validation
The selectivity of the method was investigated by comparing the
chromatograms of blank plasma and spiked plasma (n = 6). The
LI ET AL.
results revealed no endogenous interference at the corresponding
retention times of dansylated phenylephrine or dansylated
phenylephrine-d3.
The calibration curves calculated in the range from 0.0200 to
10.0 ng/ml were linear and were used to quantify dansylated phenyl-
ephrine in the extracted human plasma. The correlation coefficients (r)
(Gelotte, 2018) of the calibration curves were between 0.9962 and
0.9988. The representative linear equation of one calibration curve
was: y = 0.355x + 0.0019. The precision (CV) and accuracy (RE) of the
LLOQ (0.0200 ng/ml) of six standard curves were 5.0 and 0.0%,
respectively.
Intra- and inter-day precision values (CV) were 1.1–5.3% and the
accuracies (RE) were −5.0–5.3% (Table 1). The results indicated that
the accuracy and precision of the method were acceptable for deter-
mining the phenylephrine concentration in human plasma.
The absolute matrix effect (ratio of peak area in the matrix to neat
solution, shown in Table 2) of the analyte in normal plasma (n = 6) at
the LQC and HQC were 99.3 and 98.8%, respectively. These values in
hyperlipidemia plasma (n = 6) at the LQC and HQC were 101.5 and
100%, respectively. No differences among different types of matrices
were observed.
The extraction recovery of phenylephrine (dansylated phenyleph-
rine) and the IS (dansylated phenylephrine-d3) in human plasma was
66.0–69.0%, with an overall CV of <1.3% (Table 2).
The carryovers from 12 validation runs, assessed by determining
the peak area from the double blank immediately after injection of
the ULOQ at 10 ng/ml, were all <20% of the LLOQ response, meet-
ing the validation acceptance criteria of recent guidelines from the
US Food and Drug Administration and European Medicines Agency
for bioanalytical method validation.Using the validated method, we
investigated the stability of phenylephrine under various conditions.
The stability data (Table 3) indicated that phenylephrine in human
blood samples was stable at room temperature for 4 h (RE: −2.1%
for LQC and −0.1% for HQC), at room temperature for 4 h (RE:
1.7% for LQC and −1.9% for HQC), for four freeze–thaw cycles (RE:
0.0% for LQC and −4.7% for HQC). Phenylephrine in human plasma
samples was stable at −20
C for 1 month (RE: 1.7% for LQC and
−6.0% for HQC) and at −70
C for 1 month (RE: 0.0% for LQC and
−6.4% for HQC). The processed samples were stable in the auto-
sampler for 48 h (RE: 3.3% for LQC and −6.0% for HQC). No carry-
over was observed in the blank sample injected after injecting
the ULOQ.
The incurred sample reanalysis results showed a repeatability of
97.9% (47 of the 48 samples met the acceptance criteria).
3.4 | Pharmacokinetic study
The LC–MS/MS method described above was successfully applied to
evaluate the pharmacokinetic profiles of phenylephrine in human
plasma after a single-dose oral administration. The obtained mean
plasma concentration–time profiles of phenylephrine are shown in
Figure 4. The plasma level reached their maximum (Tmax) 35.0 ± 21.9
LI ET AL. 5 of 7

F I G U R E 3 Representative chromatograms of phenylephrine (dansylated phenylephrine) and IS (dansylated phenylephrine-d3) in human

plasma by LC–MS/MS. (a) Blank human plasma; (b) blank human plasma spiked with 0.020 ng/ml phenylephrine at LLOQ level (dansylated
phenylephrine) and IS (dansylated phenylephrine-d3); and (c) human plasma sample collected at 90 min after drug administration

min after oral administration of phenylephrine and thereafter the and the areas under concentration–time curve (AUC) were 30.1 ± 6.5,
plasma level declined with an elimination half-time of 94.2 ± 86.6 min. 62.0 ± 15.6 and 164.2 ± 63.6 min ng/ml. The maximum plasma con-
In the low-, medium- and high-dose groups, the maximum plasma con- centration of phenylephrine and drug exposure increased with
centrations (Cmax) were 0.44 ± 0.18, 1.35 ± 0.83 and 3.21 ± 2.60 ng/ml increasing dosage ratios.

TABLE 1 Intra- and inter-day precision and accuracy of the analyte in human plasma (n = 6)

Intra-day Inter-day

Concentration spiked (ng/ml) Mean (ng/ml) RE (%) CV (%) Mean (ng/ml) RE (%) CV (%)
0.020 (LLOQ) 0.020 0.0 5.0 0.019 −5.0 5.3
0.060 (LQC) 0.061 1.7 1.6 0.060 0.0 3.3
0.600 (MQC) 0.632 5.3 1.3 0.623 3.8 1.4
8.000 (HQC) 7.614 −4.8 1.1 7.656 −4.3 1.3

LLOQ, Lower limit of quantitation; LQC, low quality control; MQC; middle quality control; HQC, high quality control.
6 of 7 LI ET AL.

TABLE 2 Extraction recovery of the analyte in human plasma (n = 6)

Concentration spiked Mean recovery Matrix effect (normal, n = 6, Matrix effect (hyperlipidemia n = 3,
Compounds (ng/ml) (%) CV, %) CV, %)
Dansylated 0.060 67.5 101.5, 1.7 101.5, 1.5
phenylephrine 0.600 66.0 — —
8.00 67.6 98.8, 0.9 98.8, 0.2
Dansylated 15.0 68.5 100.4, 2.0 100.7, 1.6
phenylephrine-d3

TABLE 3 Stability results of the analyte in human plasma (n = 3) 4 | C O N CL U S I O N

Nominal
concentration In summary, we developed a novel LC–MS/MS method for quantita-
(ng/ml) tively detecting phenylephrine in human plasma using a pre-column

LQC HQC
derivatization method. The method showed several advantages for
Storage conditions 0.060 8.00 improving chromatographic behavior and mass spectrometric sensitiv-

Three freeze–thaw Mean found 0.060 7.62 ity of the test article in a complex biological matrix. First, the introduc-
cycles concentration (ng/ml) tion of two naphthalene rings reduced the polarity of phenylephrine,
RE (%) 0.0 −4.7 enabling good retention on the reversed-phase column without matrix
CV (%) 1.7 0.4 effects. Second, the derivative containing two dimethylamino groups


Storage at −20 C Mean found 0.061 7.52 significantly improved the signal-to-noise ratio. Third, the derivatiza-
for 1 month concentration (ng/ml) tion method is robust and cost-effective and allows for high-
RE (%) 1.7 −6.0 throughput analysis.

CV (%) 3.3 0.1 This method was successfully applied to detect phenylephrine in
human plasma and characterize the single-dose pharmacokinetics of
Storage at −70
C Mean found 0.060 7.49
for 1 month concentration (ng/ml) 5, 10 and 20 mg phenylephrine in Chinese patients with
RE (%) 0.0 −6.4 common cold.

CV (%) 1.7 0.3

AC KNOWLEDG EME NT S
Room temperature Mean found 0.061 7.85
for 4 h concentration (ng/ml) This study was financially supported by China Resources Sanjiu Medi-

RE (%) 1.7 −1.9 cal and Pharmaceutical Co. Ltd. This work was also supported by a
grant from the National Program on Key Research Project of New
CV (%) 11.5 0.2
Drug Innovation (no. 2015ZX09501008).

CONFLIC T OF INTER E ST STATEMENT

There are no conflicts of interest in this research.

OR CID
Haifeng Song https://orcid.org/0000-0002-0002-5537

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How to cite this article: Li L, Wang Y, Chen G, Dong K,
Song H. Pre-column derivatization method for determining
phenylephrine in human plasma and its application in a
pharmacokinetic study. Biomedical Chromatography. 2020;
e4843. https://doi.org/10.1002/bmc.4843