Anal Bioanal Chem (2008) 391:1063–1071

DOI 10.1007/s00216-008-2054-4

ORIGINAL PAPER

A method for quantifying the unstable and highly polar drug

nafamostat mesilate in human plasma with optimized
solid-phase extraction and ESI-MS detection: more
accurate evaluation for pharmacokinetic study
Yan-guang Cao & Ming Zhang & Dan Yu &
Jing-ping Shao & Yuan-cheng Chen & Xiao-quan Liu

Received: 13 December 2007 / Revised: 19 February 2008 / Accepted: 5 March 2008 / Published online: 6 April 2008
# Springer-Verlag 2008

Abstract An advanced quantification method was developed nafamostat mesilate pharmacokinetics in 30 Chinese healthy
with solid-phase extraction (SPE) and mass spectrometry volunteers was investigated with three doses via intravenous-
(MS) determination for nafamostat, an unstable and highly drip infusion. The pharmacokinetic parameters were also
polar drug, in human plasma. For unstable drugs with an ester estimated and compared with those of Japanese volunteers
group, the main analytical challenge is how to avoid the ester (slightly lower plasma concentration and longer terminal
hydrolysis, and strong acid or alkaline conditions should be elimination half-life for Chinese volunteers). The difference
excluded during sample preparation. Considering that, we in the pharmacokinetics may be ascribed to the quantification
developed a relatively mild method with SPE for sample method, because previous isotope labeling may have over-
preparation without strong acid and alkaline treatment, which estimated the parent drug.
was optimized with different pHs and salt concentrations in
phosphate-buffered saline treatment. The results indicated that Keywords Nafamostat mesilate . Unstable drug .
pH 5 gave the most efficient extraction and 0.1 M salt con- Highly polar drug . Solid-phase extraction .
centration enhanced the extraction the most, with a minor Clinical pharmacokinetics
effect on MS monitoring. The extraction method effectively
avoided drug hydrolysis and achieved good drug enrichment
over 82.2%. The linear range of quantification was 1.25–
160 ng mL−1. The stability of the drug in sample treatment Introduction
was fully validated according to the sample processing
procedure, including the stability in fresh blood, mobile phase, Nafamostat mesilate, a strong synthetic protease inhibitor,
plasma and acidic methanol, and the results indicated that the is mainly used for treatment of disseminated intravascular
drug remained stable during the whole sample preparation. coagulation and acute pancreatitis [1, 2], and is also used as
Compared with a previous isotope-labeling method, more an anticoagulant in extracorporeal circulation [3, 4]. Many
accurate and specific quantification of plasma concentration recent papers have indicated that nafamostat exhibits lots of
was achieved with liquid chromatography–electrospray other pharmacological activities and many of those have
ionization MS determination. With use of our method, been applied to clinical therapy, such as an antitumor agent
[5, 6] and for liver protection [7, 8]. Nafamostat, which is
an ester conjugate of 6-amidino-2-naphthol (AN) and p-
guanidinobenzoic acid (p-GBA), can rapidly hydrolyze into
Y.-g. Cao : M. Zhang : D. Yu : J.-p. Shao : Y.-c. Chen : AN and p-GBA in vivo and the esterase responsible for
X.-q. Liu (*) hydrolysis distributes all over the body [9]. The system
Key Laboratory of Drug Metabolism & Pharmacokinetics,
China Pharmaceutical University, retention time of the parent drug is very short; however,
Nanjing 210009, China many papers have pointed out that the nafamostat parent
e-mail: [email protected] drug was the only active form and the ester structure was an
1064 Anal Bioanal Chem (2008) 391:1063–1071

active and essential group [10, 11]. Thus, it is essential and Experimental
necessary to quantify the parent drug for pharmacokinetic
study. Chemicals and reagents
Nafamostat mesilate has been on the market for a long
time. However, there were few practical methods for its Nafamostat mesilate (99.68%) and AN (99.50%) standard
quantification in vivo. The radioisotope-labeling method has were obtained from HuaMai (QingDao, ShanDong, China),
been described [12], which often overestimates the parent the internal standard gabapentin was supplied by EnHua
drug because the metabolites also contain radioisotope, (XuZhou, JiangSu, China), methanol was of HPLC grade
especially for unstable drugs. Another limitation of the and was obtained from Merck (Darmstadt, Germany) and
method is the harmful effect of the radioactive isotope on the other chemicals used were of analytical grade. Ultrapure
the human body and its use is forbidden for Chinese clinical water was obtained from a Milli-Q water-purification system.
studies. Besides, spectrofluorometric determination of
nafamstat mesilate in blood based on trypsin-inhibitory Chromatographic and detection conditions
activity has been developed [13]; it is time-consuming and
not suitable for the preparation of batch of samples in A Shimadzu 2010 LC-MS system including two LC-
pharmacokinetic studies. High-performance liquid chroma- 20ADvp pumps, an online vacuum deaerator, a constant-
tography (HPLC) with UV detection has been reported for temperature automatic sampler and a quadruple mass spec-
nafamostat analysis in vitro, but the sensitivity was poor, trometer equipped with an ESI interface source was used.
and was not sufficient for plasma concentration analysis [9]. The chromatographic separation was accomplished using
In the work reported here, we tried to develop a practical a Shimadzu C18 column (150 mm×2.0 mm, 5 μm). The
and highly sensitive method for its quantification in human column and autosampler tray temperature were kept
plasma. constant at 30 and 4 °C, respectively. The mobile phase
For analysis of unstable drugs, the main challenge is how consisted of methanol–water–formic acid (15:85:0.001, v/v/v)
to keep the drug stable during sample preparation. Because at a flow rate of 0.2 mL min−1. The sample injection volume
of the ester group, nafamostat is prone to hydrolysis, was 10 μL.
especially in strong acidic and alkaline conditions. Thus,
the sample preparation method should exclude the possible MS conditions
unstable factors and the procedure should also be strictly
controlled. In addition, to develop a practical quantification Samples were ionized in positive-ion ESI mode under the
method for nafamostat, another challenge should be con- following source conditions: gas flow 1.5 L min−1; curve
fronted. The sensitivity must be high enough to quantify low dissolution line (CDL) voltage fixed as in tuning; CDL
plasma concentration in vivo; thus, an effective enrichment temperature 250 °C; block temperature 200 °C. Analysis was
method should be developed for improvement of sensitivity. carried out using selected ion monitoring for specific m/z
However, nafamostat is a highly polar compound with both 174.55 for nafamostat [(M+2H)/2]+, 186.90 for AN [M+H]+
highly polar guanidino and amidino groups (Fig. 1), which and 172.05 for gabapentin [M+H]+.
makes it difficult to extract it from biological fluids with
organic solvents, especially not under extremely acidic con- Standard solutions
ditions. How to effectively extract the sample without drug
hydrolysis must be solved during method development. Standard solutions were prepared and were serially diluted
In the present study, we developed and fully validated a with methanol before each experiment. They were used to
relatively mild method for plasma nafamostat quantification spike the blank plasma samples prior to extraction. A
with solid-phase extraction (SPE) and liquid chromatogra- standard solution of the internal standard gabapentin was
phy (LC)–electrospray ionization (ESI) mass spectrometry prepared in methanol at a concentration of 1 mg mL−1 as a
(MS) determination. In the method, strong acidic and stock solution.
alkaline conditions were avoided for drug stability, and the
SPE procedure was optimized with different pHs and salt Sample preparation
concentrations of phosphate-buffered saline (PBS) for
cleanup. In order to closely monitor the possible hydrolysis Spiked plasma samples (1 mL) were extracted by SPE on a
during the sample preparation procedure, we simultaneously C18 cartridge (Sep-Pak Cartridges, Waters, USA) using the
determined the main hydrolysis metabolites of AN. With use following procedure: (1) conditioning with 1 mL methanol
of the method, nafamostat mesilate pharmacokinetics in followed by 1 mL 95:5 (v/v) water–methanol; (2) loading
Chinese healthy volunteers was investigated with three doses the plasma sample diluted with 1 mL (1:1, v/v) pH 7.4 PBS
via intravenous drip infusion. (0.1 M) containing 1 μg mL−1 of internal standard; (3)
Anal Bioanal Chem (2008) 391:1063–1071 1065

Fig. 1 Full-scan mass spectra

for nafamostat (a), 6-amidino-2-
naphthyl (AN) (b) and the in-
ternal standard (c)

cleanup in two steps—1 mL pH 5.0 PBS (0.1 M), then Calibration curve and quality control samples
4 mL purified water; (4) elution of the analytes with 0.5 mL
acid methanol (1% formic acid, v/v). Calibration curves were prepared by spiking blank plasma
The methanolic eluate was evaporated to dryness under a with 20 μL nafamostat or AN working solutions to produce
stream of nitrogen at 30 ◦C. The residue was reconstituted concentrations of 1.25, 2.5, 5, 10, 20, 40, 80 and 160 ng mL−1.
in 100 μL mobile phase. A 10-μL aliquot of the sample The following steps were the same as those described in
was injected into the HPLC-MS system for analysis. “Sample preparation.” The blank plasma sample was also
1066 Anal Bioanal Chem (2008) 391:1063–1071

analyzed in each run to confirm the absence of interferences. conducted by comparing the analytic results of the sample
Quality control samples, which were analyzed at the same incubation at 30 °C for 30 min with those obtained without
time as test samples, were prepared at concentrations of incubation at the same concentration. The stability in the
5 ng mL−1 (low level), 20 ng mL−1 (medium level) and mobile phase was evaluated by comparing the result for
80 ng mL−1 (high level) for both analytes in the same the same sample dissolved in the mobile phase lying on the
manner. The total amount of the quality control samples was autosampler plate at different times. During the whole
about 15% of the test samples in the same batch. process of the stability validation, AN was simultaneously
monitored to evaluate possible hydrolysis.
Method validation
Possible hydrolysis proportion estimated with AN
The method was validated for specificity, sensitivity,
extraction recovery, precision and accuracy. The specificity In addition to the validation of the stability, we also
of this method was assessed by comparing the chromato- monitored ion 186.90 for AN qualification without adding
grams obtained from the sample spiked with a concentration AN in order to evaluate the proportion of hydrolysis. The
of nafamostat or AN at the lowest limit of quantification proportion of hydrolysis
 was calculated using the following
(LLOQ) and the medium-level concentration (20 ng mL−1) formula: HP ¼ NAN N nafamostat
100%, where HP is the
with those obtained from blank samples. proportion of hydrolysis, and NAN and Nnafamostat represent
The potential matrix effect was evaluated by comparing the number of moles of AN and nafamostat, respectively.
the peak areas of the analytes dissolved in the supernatant
of the processed blank plasma with that of ultrapure water Pharmacokinetic study
at the same concentration. The matrix effect of the internal
standard was evaluated in the same way. The possible mutual Thirty adult volunteers were randomly classified into three
effects on the ionization processes were also evaluated by groups with five men and five women in each, aged
comparing the signal intensities of each compound separately. between 20 and 26 years (mean 24 years). Written informed
Recoveries of nafamostat and AN were determined by consent was obtained from each adult prior to the study.
comparing the response of pretreated quality control plasma Subjects were allowed to enroll in the study, with the
samples with the response of identical standards prepared in exception of those with existing hepatic impairment
the mobile phase which did not undergo sample pretreatment. (confirmed by a previous liver biopsy specimen or liver
The within-batch precision and accuracy were determined spleen scan). Subjects were excluded if they had a history
by repeated analysis of five spiked samples of nafamostat (within the past 12 months) of alcohol and/or drug abuse, or
and AN at each quality control level on 1 day (n=5). had a positive drug or alcohol screening result at the
Between-batch precision and accuracy were determined by screening visit or on the day before the study started. The
repeated analysis on three different days (n=3 series per drug was dissolved in 250 mL glucose solution and was
day, n=45 in total). given via constant intravenous-drip infusion within
The stability of nafamostat was assessed by analyzing 120 min. The three doses were 10, 20 and 40 mg.
samples under different conditions according to the sample During drug infusion, 3 mL blood was collected at 0, 30,
preparation procedure. The stability was validated as 60 and 120 min and after infusion, blood was collected at 0,
follows: (1) the incubation stability in human plasma for 2, 5, 10, 30, 60, 120, 180, 240 and 360 min through an
30 min at 4 °C; (2) the incubation stability in fresh blood at indwelling catheter. Blood was collected into heparinized
4 °C; (3) the stability in acidic methanol for 30 min at 30 °C; tubes and immediately centrifuged at 4 °C to obtain plasma;
(4) the stability in the mobile phase at 4 °C (autosampler the prepared plasma was stored on ice and after a batch of
temperature) for 6, 12 and 24 h. All the assays were con- samples had been collected (about 5 min), the plasma was
ducted at three concentrations (Table 3). Assay 1 was analyzed as described above.
evaluated by comparing analytes with 30-min incubation in
human plasma and those without incubation. Assay 2 was
conducted as follows. The same amount of analytes was Results and discussion
added to human plasma and freshly collected human blood
separately. The blood sample was centrifuged immediately Optimization of chromatographic condition and MS
for about 5 min at 4 °C and the plasma obtained was kept detection
on ice for about 10 min. Then both samples were prepared
using the same procedure; the fresh blood stability was MS parameters were optimized to achieve the maximum
evaluated by comparing the analytic results of the blood abundance of the product ions of the compounds analyzed.
sample with those of the plasma sample. Assay 3 was Full-scan mass spectra and product ion scan spectra of
Anal Bioanal Chem (2008) 391:1063–1071 1067

nafamostat were obtained by direct infusion into the mass

spectrometer of 2 μg mL−1 diluted in methanol at a flow
rate of 0.2 mL min−1. Two protonated species were found
for nafamostat, at [M+H]+ m/z 348.0 and [(M+2H)/2]+ m/z
174.55, respectively. The [M+H]+ ion was relatively
abundant in neutral conditions; however, the [(M+2H)/2]+
ion was found to be more abundant in the acidic mobile
phase. Thus, we the chose the [(M+2H)/2]+ ion as the
quantification species. The protonated species for AN and
the internal standard were [M+H]+ at m/z 186.90 and m/z
172.05, respectively. The full-scan mass spectra for
nafamostat, AN and the internal standard are shown in
Fig. 1.
Nafamostat is very hydrophilic, which means it is rapidly
eluted in reversed-phase chromatography. Only adjusting the
ratio of water and organic solvent in the mobile phase cannot
obviously improve the retention. Because of the proneness to
hydrolysis, a minor amount of formic acid was added to the
mobile phase, and fortunately the retention was significantly
enhanced. We also validated the stability of the acidic mobile
phase to ensure there was no hydrolysis in such an acidic
condition. Finally, the chromatographic conditions were deter-
mined as follows, The system was operated using isocratic
conditions on a C18 column (150 mm×2.0 mm, 5 μm) with a
mobile phase consisting of methanol–water–formic acid
(15:85:0.001, v/v/v) at a flow rate of 0.2 mL min−1. Typical
chromatograms are shown in Fig. 2.

Sample preparation

Liquid–liquid extraction was performed with various extrac-

tion solvents (ethyl ether, dichloromethane, methyl-t-butyl
ether and ethyl acetate) and also with pretreatment with
PBS of different pH to improve the extraction efficiency.
Unfortunately, no solvent allowed good extraction recovery.
Since nafamostat is a highly polar compound and nearly
insoluble in most organic solvents, protein precipitation
was also tried with methanol or acetonitrile; however, the
sensitivity was significantly reduced owing to dilution of
the samples after deproteinization. In addition, injection of
the supernatant after precipitation of the protein led to
numerous compounds being eluted late and significant reduc-
tion of the lifetime of the analytical column.
Then we tried SPE. PBS of different pH for cleanup was Fig. 2 Chromatograms of injection blank plasma (a), calibration
standard containing 80 ng mL−1 of nafamostat and AN standard in
tested in order to get a more efficient recovery and less drug human plasma (b). Chromatogram of the sample after intravenous
hydrolysis. The relationship between the extraction recov- dripping infusion for 1.5 h at a dose of 20 mg in a healthy volunteer (c)
ery and the pH of the PBS for cleanup is shown in Fig. 3.
The results indicated that the extraction efficiency was
highest at pH 5 for our tested pH range of 3–8. A relatively efficiency, however, high-salt PBS would interfere with MS
low pH may promote drug hydrolysis and apparently monitoring, especially for this hardly volatile salt. So we
decrease the extraction recovery. Besides pH, the salt subsequently cleaned up the solid column with 4 mL of pure
concentration of PBS was also closely related to extraction water in order to completely wash out the large amount of
recovery. High salt concentration may improve the extraction phosphate. Although we tried other volatile salts, such as
1068 Anal Bioanal Chem (2008) 391:1063–1071

Fig. 3 Relationship between extraction efficiency and pH values of Fig. 4 The mean standard curves for nafamostat and AN (n=5). As
0.1 M phosphate-buffered saline (PBS) for cleanup in solid-phase and Ai refer to the areas of the drug and the internal standard,
extraction (SPE). Each point is the mean of three determinations at respectively
20 ng mL−1

ammonium acetate and ammonium formate, the recovery control level and the internal standard extraction recovery
was very low and the mechanism was not clear. After was more than 70% for the 20 ng mL−1 level.
optimization, we choose pH 5 and 0.1 M PBS for cleanup in
sample treatment. Accuracy and precision

Method validation The intrabatch and interbatch accuracy and precision data
of both compounds are summarized in Table 2. In this
Specificity and sensitivity assay, the interbatch precision was less than 11.97% for
each quality control level. The interbatch precision was less
No additional peaks were observed for endogenous sub- than 10.79%.
stances that could interfere with the detection of the analytes.
Representative chromatograms for nafamostat and AN in Matrix effect
plasma samples are presented in Fig. 2.
The LLOQ of the assay, defined as the lowest concen- The effects of the plasma matrix on ionization efficiency
tration on the standard curve that can be quantified with were expressed as the ratio of the mean peak area of
accuracy within 15% of the nominal value and precision analytes spiked after extraction from five different lots of
not exceeding 15%, was 1.0 ng mL−1 both for nafamostat plasma to that of the neat standards at different concen-
and AN. trations. By analysis of the samples at three levels, matrix
effect values were calculated. The average matrix effect
Linearity values obtained were 107.3% (coefficient of variation,
CV, of 4.5%, n=5), 93.8% (CV=7.5%, n=5) and 96.9%
The five standard curves were linear over a concentration
range of 1.25–160 ng mL−1 both for nafamostat and AN.
Table 1 Summary of solid-phase extraction recovery from quality
The average correlation coefficient was higher than 0.998 control samples in human plasma (n=5)
in both cases, with a mean slope (± standard deviation) of
0.028±0.031 and a mean y-intercept of −0.031±0.017 for Concentration (ng mL−1) Percentage extraction recovery (CV, %)
nafamostat and a mean slope of 0.100±0.034 and a mean Nafamostat AN
y-intercept of −0.034±0.042 for AN. The mean standard
curve is show in Fig. 4. 5 82.2 (3.21) 85.4 (5.20)
20 88.4 (2.50) 87.5 (3.89)
Extraction recovery 80 89.4 (3.20) 90.1 (2.10)
Mean ± SD 86.7±3.90 87.7±2.35

The extraction recovery is summarized in Table 1 and in the CV coefficient of variation, AN 6-amidino-2-naphthyl, SD standard
assay, the recovery was not less that 82.2% for each quality deviation
Anal Bioanal Chem (2008) 391:1063–1071 1069

Table 2 Summary of precision and accuracy from quality control samples in human plasma (n=5)

Nominal concentration Interbatch Intrabatch

(ng mL−1)
Measured concentration (ng mL−1) CV RE Measured concentration (ng mL−1) CV RE
(mean ± SD) (%) (%) (mean ± SD) (%) (%)

Nafamostat
5 4.6±0.3 5.62 −7.48 4.9±0.4 8.88 −3.67
20 20.0±2.4 11.97 −0.16 19.5±1.7 8.94 −3.18
80 80.4±4.3 5.32 0.49 82.9±6.6 8.02 2.89
AN
5 4.7±0.5 10.79 −4.69 5.2±0.4 7.58 2.83
20 19.9±2.0 10.17 −0.52 20.6±1.1 5.26 2.50
80 83.4±5.5 6.63 4.20 77.7±3.8 4.87 −3.24

(CV=2.6%, n=5) for nafamostat (10, 40 and 160 ng mL−1) The assay of the stability in freshly collected blood
and 98.7% (CV=2.4%, n=5) for the internal standard showed that the hydrolysis was less than 8% for a time that
(100 ng mL−1). The possible mutual effects on the ioni- completely exceeded the routine sample preparation period.
zation processes were also evaluated by comparing the Previous reports of nafamostat stability indicated that the
signal intensities of each compound analyzed separately, hydrolytic activity for nafamostat is predominantly located
and no mutual effects were observed. in erythrocytes [9], so immediate plasma separation is an
essential step to minimize the extent of hydrolysis. In addition,
Stability in order to lower the hydrolysis to the greatest extent, plasma
was centrifuged at 4 °C and stored on ice temporarily for a
The results showed that nafamostat was stable in human batch sample collection. Some workers have used esterase
plasma, and no obvious hydrolysis was found for plasma inhibitors for ester drug stability during blood collection
incubation for 30 min. and we also tried the common esterase inhibitors NaF and

Table 3 Stability of nafamostat in the sample preparation procedure

In acidic methanol Concentration With incubation HP (%) Without HP (%) Ratio (%)
at 30 °C for 30 min added (ng mL−1) incubation

10 7.8±1.1 ND 8.0±1.4 ND 97.9

40 34.2±1.4 < 4.2 35.2±0.9 < 3.7 97.0
160 157.4±8.6 < 1.2 165.2±10.2 < 2.5 95.2
In plasma for Concentration Incubation for HP (%) Without incubation HP (%) Ratio (%)
30 min at 4 °C added (ng mL−1) 30 min
5 4.7±0.2 ND 4.7±0.2 ND 99.0
20 18.7±0.9 ND 18.4±1.0 < 4.1 101.6
80 79.5±6.7 < 2.4 79.9±3.2 < 1.7 99.6
In fresh blood Concentration Plasma HP (%) Fresh blood HP (%) Ratio (%)
at 4 °C added (ng mL−1)
2.5 2.7±0.4 ND 2.5±0.1 ND 92.1
10 10.5±0.7 < 3.5 9.8±0.7 < 5.1 93.4
40 41.5±1.9 < 2.0 40.5±3.6 < 1.4 93.7
In mobile phase at 4 °Cb Mean ± SD (ratioa, %)
Concentration 0h 6h 12 h 24 h
added (ng mL−1)
5 5.1±0.3 5.1±0.4 (98.5) 5.1±0.4 (97.5) 5.0±0.3 (97.3)
20 17.5±2.1 17.1±1.7 (97.9) 19.0±1.9 (108.5) 18.1±2.4 (103.5)
80 86.0±3.5 80.3±4.2 (93.4) 78.3±1.5 (91.0) 79.5±3.9 (92.4)

The proportion of hydrolysis (HP) was calculated from the formula HP ¼ NAN =N nafamostat
100%, where NAN and Nnafamostat represent the
number of moles of AN and added nafamostat, respectively.
ND AN under the detectable limit
a
The ratio compared with 0 h at the same concentration. All the HP values are below 2.5%.
b
4 °C is the autosampler temperature.
1070 Anal Bioanal Chem (2008) 391:1063–1071

for nafamostat hydrolysis, and no single inhibitor can result in

the complete inhibition of nafamostat hydrolysis [9, 13].
Thus, we centrifuged the blood immediately at low tem-
perature without addition of any inhibitor. After storage for a
short time at 4 °C for a batch sample collection, the plasma
was prepared using the method described above.
The 30 °C water-bath stability results showed that
nafamostat is stable in acidic methanol for 30-min
incubation. Nafamostat was also stable when kept in the
mobile phase for up to 24 h at 4 °C. All the stability data
are shown in Table 3. The values for the proportion of
hydrolysis for all the samples were all below 5.1%, and are
shown in Table 3.
Considering all the factors for drug stability, the pH and
temperature of the sample or the absence of esterase
(immediate sample treatment), the absence of esterase may
be the critical factor for drug stability; thus, we should
prepare samples immediately after sample collection.

Application in pharmacokinetic study

The validated method has been successfully applied to

human plasma nafamostat concentration quantification for
pharmacokinetic study in 30 Chinese healthy volunteers.
Compared with pharmacokinetic results based on the
isotope-labeling method, the nafamostat concentration was
lower [12], as shown in Fig. 5. To our knowledge, this may
be a result of the quantitative method, because the isotope-
Fig. 5 Mean plasma concentration against time profiles of nafamostat labeling method often overestimates the parent drug,
during and after intravenous-drip infusion of three doses of nafamostat especially for an unstable drug.
mesilate. 5-1 Data from our assay (n=10 for each dose, ± standard
deviation). 5-2 From Japanese volunteers with isotope-labeling
A two-compartment model could be used to describe the
quantification (cited in [12]) pharmacokinetics of nafamostat in human. Owing to high
sensitivity, plasma nafamostat can be monitored for a long
time and the elimination half-life was longer than that
2-nitrobenzoic acid in blood collection; however, both reported in Japanese volunteers, as summarized and com-
inhibitors do not give an obvious improvement compared pared in Table 4. The area under the curve and the maximum
with the same sample without inhibitor treatment. We concentration increased proportionally to the dose, indicat-
deduced that the inhibitors may interfere with MS monitoring ing the linear pharmacokinetic character. The mean plasma
by an ion-inhibition effect because they are both hardly concentration against time curves of nafamostat are shown
evaporable. Moreover, many types of esterase are responsible in Fig. 5.

Table 4 Estimated pharmacokinetic parameters of nafamostat mesilate after intravenous-drip infusion of three single doses

Parameters 10 mg 20 mg 40 mg

AUC0–t (ng·min·mL−1) 1,438.65±284.84 3,382.25±1,722.54 6,577.34±1,531.13

AUC01 (ng·min·mL−1) 1,655.84±275.64 3,571.14±1,291.37 6,880.46±1,566.92
Cmax (ng·mL−1) 14.49±3.88 (16.5±4.9)a 40.4±12.53 (61.5±7.7)a 60.43±15.82 (93.2±16.7)a
T1/2(α)b (min) 3.65±1.76 3.78±1.89 3.70±1.69 (1.1) a
T1/2(β)b (min) 112.42±53.5 128.19±17.24 122.91±26.63 (23.1)a

AUC area under the curve

a
Parameters estimated with the isotope-labeling method in Japanese volunteers (cited in [12])
b
The elimination half-life in α phase and β phase, respectively
Anal Bioanal Chem (2008) 391:1063–1071 1071

Conclusion 4. Kotani K, Ichiba S, Andou M, Sano Y, Date H, Tedoriya T, Goto

K, Shimizu N (2002) J Thorac Cardiovasc Surg 124:626–627
5. Miyaso H, Morimoto Y, Ozaki M, Haga S, Shinoura S, Choda Y,
The present study developed a practical method for unstable Murata H, Katsuno G, Huda K, Takahashi H, Tanaka N, Iwagaki
ester drug analysis. The whole process not only successfully H (2006) Dig Dis Sci 51:2007–2012
avoided obvious hydrolysis, but also gave higher sensitivity 6. Ohtake Y, Hirasawa H, Sugai T, Oda S, Shiga H, Matsuda K,
Kitamura N (1991) Contrib Nephrol 93:215–217
and specificity. The method was also successfully used for
7. Tsukagoshi S (2001) Gan To Kagaku Ryoho 28:1237–1243
the assessment of the pharmacokinetics of nafamostat 8. Yamashita Y, Ishiguro Y, Sano D, Kimura M, Fujita K, Yoshida T,
mesilate in 30 Chinese healthy volunteers. Horiuchi C, Taguchi T, Matsuda H, Mikami Y, Tsukuda M (2007)
Auris Nasus Larynx 34:487–491
Acknowledgment The authors gratefully acknowledge financial 9. Yamaori S, Fujiyama N, Kushihara M, Funahashi T, Kimura T,
support from the National Natural Science Foundation of the People’s Yamamoto I, Sone T, Isobe M, Ohshima T, Matsumura K, Oda M,
Republic of China (nos. 30572228, 30630076). Watanabe K (2006) Drug Metab Pharmacokinet 21:147–155
10. Yang H, Henkin J, Kim KH, Greer J (1990) J Med Chem
33:2956–2961
11. Aoyama T, Okutome T, Nakayama T, Yaegashi T, Matsui R,
Nunomura S, Kurumi M, Sakurai Y, Fujii S (1985) Chem Pharm
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