Focusing Review

CHROMATOGRAPHY, Vol.32 No.1 (2011)

Focusing Review

Development and practical application of HPLC methods
for medicaments and related compounds
Mitsuhiro Wada
Graduate School of Biomedical Sciences, Nagasaki University
1−14 Bunkyo−machi, Nagasaki, 852−8521, Japan

Abstract
HPLC methods for determination of medicaments and related compounds were developed to promote the rational use of these compounds.
According to the aim of analysis, suitable hyphenated detections such as UV, fluorescence and chemiluminescence with HPLC were used.
Labeling, a powerful tool to provide desirable chromatographic properties to the analytes was also utilized for sensitive determination of medicaments and related compounds in biological samples such as urine, plasma, hair and microdialysate. Furthermore, the methods could be
successfully applied to evaluate drug−drug interaction or quality control of healthy foods.
These developed HPLC methods could be contribute to the practical application studies on the rational use of medicaments.
Keywords: Rational use of medicaments; HPLC; Biological sample; Hyphenated detection; Labelingas endogenous components in biological samples are commonly

1. Introduction“Rational use of medicaments” is one of the most consider-non−volatile and polar compounds. And HPLC is become wide-

able paradigms in the pharmaceutical region. All of pharmaceuticalspread, thus it is the most suitable among chromatographic tech-researches might be performed to promote this. Chromatographic

niques for their analysis.

analysis of medicaments in biological fluids is often required to ac-

If the target molecule does not have desirable properties forcomplish the rational use of medicaments and related compounds

spectrometric or luminometric analysis, a labeling reaction is[1]. Nowadays, any useful information such as quality control,

needed to introduce the properties in these molecule. A labeling re-pharmacokinetics, pharmacodynamics, pharmacology and toxicol-

action is commonly achieved by the selective reaction between theogy of medicament are always obtained with chromatographic

functional group of the analyte and that of the labeling reagent. La-

analysis. Therapeutic drug monitoring based on determination ofbeling serves to improve the sensitivity, selectivity and the separa-

medicaments is one of the representative examples.

tion behavior. In our previous studies, 4−(4, 5−diphenyl−1H −imi-Among the separation techniques employed for medicamentdazol−2−yl)benzoyl chloride (DIB−Cl) was one of the most power-

or related compounds analyse such as thin−layer chromatography,

ful fluorescent labeling reagents and has been applied to determine

gas chromatography, high−performance liquid chromatography

the varied medicaments such as 3,4−methylenedioxymethampheta-(HPLC), capillary electrophoresis and capillary electrochroma-mine (MDMA) and its metabolite [2, 3], and morphine (Mor) [4].

tography, HPLC and GC have been proven to be the most popular,

Especially, the methods for MDMA were successfully showed ap-

because these can offer better sensitivity, selectivity and applicabil-plicability to the several biological samples such as human hair [3]ity. In addition, most medicaments and related compounds as welland plasma, rat brain and blood microdialysates [2]. To date, many

Correspondence: Graduate School of Biomedical Sciences, Nagasaki University, 1−14 Bunkyo−machi, Nagasaki, 852−8521, Japan
Phone: +81−95−819−2451
Fax: +81−95−819−2450
E−mail: m−wada@nagasaki−u.ac.jp―１―

CHROMATOGRAPHY, Vol.32 No.1 (2011)labeling reagents have been developed for each reactive functionalratio (S/N) = 3) and 0.04µg/mL (SA), respectively. This method

group. Several reviews describing the labeling reagents for HPLC

could be applied to determine analytes in patients’ sera adminis-

have been published [5−7].

tered with low−dose enteric−coated Asp. A simple HPLC−UVOn the other hands, microdialysis technique is often utilized

method was developed for determination of paclitaxel, an anti−tu-

as a sampling tool to monitor concentrations of free−form com-

mor agent in human and rat blood samples [11]. Paclitaxel inpounds or neurotransmitters in organs, and is also applied for neu-

plasma was extracted by a simple liquid−liquid extraction with tertropharmacological and pharmacokinetic studies [8]. A microdialy-−butylmethyl ester. The LOD of paclitaxel in human plasma wassis probe consists of a small semipermeable membrane, on which

10ng/mL (at S/N=3). This HPLC method was successfully applied

analytes are recovered by passive diffusion. As the advantages ofto pharmacokinetic studies of paclitaxel administered to rats.

microdialysis, long−term sampling is possible with minimal dam-

FL detection counts the increase in photon number occurred,

age for organs or tissues and the clean−up procedure is not ordinar-

while UV detection measures the ratio of transmitted light that

ily required. This tool played important role in our application

passes out through the sample solution and the original irradiation

studies on pharmacokinetics and drug−drug interactions [2, 4, 9].

light. Therefore an HPLC−FL detection method has been favorably

The aim of this review is to overview our current HPLC meth-comparable to UV detection in regard to sensitivity and selectivity,

ods for determination of medicaments and related compounds (e.g.

and extensively employed for the determination of trace amounts

drugs of abuse and food components) in biological samples. Fur-of compounds. Additionally, for FL detection, two wavelengths forthermore, their advanced applications for evaluation of pharma-

excitation and emission should be selectively used; this is the ma-

cokinetic behavior on drug−drug interaction of medicaments are

jor reason why FL detection is more selective than UV detection.

described.

Medicaments with their intrinsic FL can be detected by HPLC−FL
without pretreatment such as fluorescent labeling. In our previous
report, by utilizing intrinsic FL of donepezil (DP) hydrochloride,

2. HPLC methods for determination of medicaments and theirrapid and sensitive determination of DP in human plasma, rat

applications
Representative medicaments described in this chapter were in-plasma and microdialysis was achieved with a short C30 column

dicated in Figure 1. The HPLC−UV detection has wide adaptability

and an isocratic elution [9]. The method provided the LOD of DP

and simplicity with a relatively inexpensive instrument, althoughin human plasma, rat plasma and microdialysis ranged from 0.2 tothe sensitivity of the UV detection inferior to those of MS, FL or

2.1ng/mL (S/N =3), and was successfully applied to plasma sam-electrochemical detection. However, since the performance of UV

ples from Alzheimer’s patients or rat brain and blood mi-

detector has been recently improved and also the levels of medica-crodialysates after a single administration of DP. Furthermore, thements in biological samples are relatively high (sub−micro g/mL

possibility of interaction of Asp or clopidogrel on DP hydrochlo-level), these determination can be achieved by an HPLC−UVride in rats was evaluated [12]. Pharmacokinetic parameters of DP

method. Among the medicaments, aspirin (Asp) and its metabolite,

for administration of DP (5mg/kg, i.p.) and co−administration ofsalicylic acid (SA), in human serum were determined by an HPLC

Asp (200mg/kg, p.o. or 200mg/kg, i.p.) or clopidogrel (5mg/kg, p.

−UV method [10]. A relatively small volume of serum (100 µL)o.) groups were calculated and compared among them; no signifi-

cleaned up by solid phase extraction was required for determina-

cant difference was observed.

tion with limit of detections (LODs) of 0.11 (Asp, signal−to−noise

Morphine (Mor) is most frequently used as an analgesic drug
for both postoperative and cancer pain. The World Health Organi-O

UV detection

H

O

H

O

O

H

H
H
O

N
H

COOH
O

O
HO

H

FL detection with labeling
NCH3

O

nonsteroidal anti−inflammatory drugs (NSAIDs). Several studies

O

HO

CH3

have demonstrated the benefits of the combined use of Mor withHO

Aspirin (Asp)

Paclitaxel

OH

O

CH2CH

O

C

H N

H3 CO

NSAIDs in comparison with Mor alone [13]. These reports detailed

OH

Morphine (Mor)FL detection

pain, in which opioids including Mor are used in combination with

H

O

O

zation proposed a three−stage approach for the treatment of chronicOHO

the additive effect on pain relief caused by the different pharma-

CH3

codynamics of Mor and NSAIDs. On the other hand, although

CH3

pharmacokinetic interactions between Mor and NSAIDs cannot be

H

N

CH3

H3 CO

Donepezil (DP)HO

neglected, only limited knowledge is available [14]. This might beCH3caused by the lack of a simple and sensitive method for Mor deter-

Pentazocine (PZ)

mination. Therefore, a sensitive HPLC−FL detection method cou-

Figure 1. Target medicaments in this study.

pled with a microdialysis technique was developed for the determi-―２―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Mitsuhiro Wada

nation of Mor in brain and blood microdialysates [4]. Mor was eas-

incorporation rate values of PZ were calculated from the following

ily labeled with DIB−Cl under the mild reaction conditions (atequation;
The incorporation rate = PZ concentration in black or whiteroom temperature for 10min, Figure 2). The separation of DIB−

hair/AUC0−inf of PZ in plasma

Mor was carried out on a C18 column with CH3CN/0.1 M acetate
buffer (pH 5.4) within 14 min as shown in Figure 3. The LODs of

The ratios for black and white hair were 0.053 and 0.007, re-

Mor in brain and blood microdialysates at S/N=3 were 0.4 and 0.6

spectively.

ng/mL, respectively. The proposed method was successfully applied to the preliminarily study of potential pharmacokinetic inter-

3. HPLC methods for determination of abused drugs and theiraction between Mor and diclofenac which was one of the most fre-applications

quently used NSAIDs [15]. No significant difference was observed

Abuse of drugs has been widespread worldwide and nowa-

for any pharmacokinetic parameters of Mor between rats adminis-

days, drugs of abuse involving medicaments have been diversified.

tered Mor with/without diclofenac. Pentazocine (PZ) which is

Therefore, development of determination methods for drugs might

structurally similar to Mor, is a shortacting narcotic−antagonist an-lead to promote “rational use of medicaments”. Among drugs ofalgesic and was determined by an HPLC−FL method with DIB−Cl

abuse, 3,4−methylenedioxymethamphetamine (MDMA), com-labeling [16]. PZ extracted from hair or plasma sample was labeledmonly called ‘ecstasy’, is an amphetamine analog used as a recrea-

and the resulted solution was cleaned up with solid phase extrac-

tional drug among the young. It is usually used as an ecstasy tablet,tion. The LODs of PZ at S/N=3 for rat hair and plasma were 0.18

and the concentration of MDMA in the tablets is variable and un-

ng/mg and 0.57ng/mL, respectively. The method was successfully

certain, and thus it is possible to take an overdose that serves toxic

applied for the monitoring of PZ levels in Zucker rat hair andeffects. The toxicology of MDMA and related drugs has been re-plasma samples after a single administration of 25mg/kg PZ. The

viewed in detail [17]. Additionally, since an ecstasy tablet includes
other components such as caffeine, ketamine, ephedrine and methamphetamine, unexpected toxicity may appear owing to drug−drug

pH 10

N

10 min at r. t.

N
H

OH

NCH3

O
C

O

O

DIB-MDAO

OH

λex= 355 nm
λem= 486 nm

DIB-Mor

DIB-MDMAHO

DIB-MPPADIB-ClDIB-MDA

N
H

Mor

+

DIB-MDMA

O
C Cl

N

DIB-MPPA

NCH3

(B)

(D)

(A)

(C)

Figure 2. Labeling reaction of Mor with DIB−Cl.
(B)Mor
150 min afteradministration

FI, arbitrary unitFFI, arbitrary unit
F

Mor

(A)

150 min afteradministration0

Beforeadministration

0
10
20
Retention time, min

8
16
24 32
Retention time, min.

0

8
16
24 32
Retention time, min.

Figure 4. Chromatograms of rat blood and brain dialysates.
Samples: (A) control rat blood microdialysate spiked with 100 ng/
mL of IS; (B) spiked blood microdialysate with 50 ng/mL of
MDA, 100 ng/mL of MDMA and 100 ng/mL of IS; (C) control rat
brain microdialysate spiked with 100 ng/mL of IS; and (D) spiked
brain microdialysate with 50 ng/mL of MDA, 100 ng/mL of
MDMA and 100 ng/mL of IS.
Printed from Ref. 2 with permission from John Wiley & Sons, Ltd.

Beforeadministration

0
10
20
Retention time, min

Figure 3. Chromatograms of (A) brain dialysate and (B) blood dialysate 150 min after administration of Mor.
Printed from Ref. 4 with permission from Springer.

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CHROMATOGRAPHY, Vol.32 No.1 (2011)

Table 1. Pharmacokinetic parameters of MDMA and MDA in rat
blood and brain after single administration of MDMA (5mg/kg) with or without caffeine (20mg/kg).

interactions [18]. Therefore, simultaneous determination of
MDMA and its metabolite 3,4−methylenedioxyamphetamine(MDA) in rat blood and brain microdialysates by HPLC−FL was

Parametersdeveloped [2]. Microdialysates were directly subjected to labelingwith DIB−Cl. The DIB−derivatives of MDMA, MDA and the in-

MDMA alone(n=5)

MDMA withcaffeine (n=3)

ternal standard, 1−methyl−3−phenylpropylamine (MPPA), wereIn blood

isocratically separated on an ODS column using a mixture of 50

Cmax, ng/mL

1447±501550±45mM phosphate buffer (pH 7.0)/CH3CN/CH3OH/2−propanol (50:45:

Tmax, min1138±51177±24

5:2, v/v/v/v %) as a mobile phase (Figure 4). The LODs (S/N=3)

T1/2, min1143±81157±6for MDA and MDMA were 1.2 and 4.2 for blood microdialysate

AUC0−300, mg•min/mL

1153±51191±5band 1.3 and 4.8ng/mL for brain microdialysate, respectively. More-

MRT0−300, min

1185±41109±11

over, the proposed method was successfully applied for the moni-CL, mL/min/kg

196±101151±4atoring of MDMA and MDA in rat blood and brain microdialysates,

In brainand the pharmacokinetic parameters of MDMA in the mi-Cmax, ng/mL

1009±281496±80b

crodialysates after administration of MDMA (5mg/kg, i.p.) with or

Tmax, min1146±41197±24

without caffeine (20mg/kg, i.p.) were evaluated (Figure 5). The

T1/2, min1144±51154±9pharmacokinetic parameters of MDMA in blood and brain are

AUC0−300, mg•min/mL

1118±81193±10summarized in Table 1. Following the administration of MDMA

MRT0−300, min

1192±31112±15

with caffeine, the AUC0−300 (53±5µg•min/mL) of MDMA in blood

CL, mL/min/kg

1142±41150±7significantly increased to 91±5µg•min/mL (p < 0.01), and clear-

Data are expressed as mean ± SEM.
ap < 0.05, significantly different from MDMA alone.
b
p < 0.01, significantly different from MDMA alone.
Printed from Ref. 2 with permission from John Wiley & Sons, Ltd.

ance (CL) of MDMA (96±10mL/min/kg) decreased by half to 51±
4mL/min/kg. No change of Cmax and AUC for MDA was observed(data was not shown), which suggested that caffeine may interferewith renal clearance of MDMA. On the other hand, the Cmax of
MDMA (1009±28ng/mL) in brain significantly decreased to 496±

unchanged. These results suggested that caffeine inhibits the trans-

80ng/mL (p < 0.01) and 93±21ng/mL (p < 0.05). Moreover, theportation of MDMA to brain via the blood−brain barrier, although

Tmax and the mean residence time (MRT) of MDMA were consider-

the exclusions of MDMA from brain to blood were not influenced

ably prolonged, but the elimination half−life (T1/2) of MDMA wasby caffeine. The inhibition of P−glycoprotein by caffeine was expected to be the reason for the increased level of MDMA in brain[19]. The proposed method coupled with a microdialysis techniquecould be concluded to be a powerful tool for pharmacokinetic interaction studies of MDMA with other drugs. Furthermore, MDMAs
in several biological matrices such as urine [20] and hair [3, 21]
were also determined by HPLC−FL or −CL detections.
4. HPLC methods for determination of food components andtheir applications
Analysis of components in healthy foods or functional foodsis also important by the reasons as follows: 1) food−drug interaction has been noticed as well as drug−drug interaction. Food component analysis could provide available information to prevent
such interaction. 2) There is no regulation on the quality of healthy
foods in Japan. So analysis of components of these foods might

Figure 5. Concentration−time profiles of MDMA in rat blood (A)and brain (B) microdialysates after a single administration of MDMA with or without caffeine.
Data are expressed as mean±SEM (MDMA alone group, n = 5;
MDMA with caffeine group, n = 3).
Printed from Ref. 2 with permission from John Wiley & Sons, Ltd.

contribute the preservation of their quality.
Morinda citrifolia commonly known as Noni is in high demand as an alternative medicine due to its possibilities for anti−microbial, anticancer, anti−inflammatory, antioxidant effects [22].
However, there is few available information to evaluate the effects―４―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Mitsuhiro Wada

with quantitative data of ingredient. Scopoletin, a coumarin deriva-

tected by both detectors. However, 7−HC was detected by only FL

tive, is one of the representative ingredients contributing to the de-detector (Figure 6C). Amounts of scopoletin in Noni juices was

sirable effects in Noni. Therefore, quantification of coumarin de-ranging from 5.1 to 231mg/mL (n = 12) (Table 2). And 7−HC wasrivatives such as scopoletin, 7−hydroxycoumarin (7−HC) and 4−

from 0.04 to 0.45mg/mL, whereas no 4−HC was detected in anyhydroxycoumarin (4−HC) in Noni by HPLC−UV or −FL detection

Noni samples examined. By a multi−wavelength FL detector, more

was demonstrated for quality control purposes [23]. The eluent wasthan 95% of peak purity for coumarin derivatives in Noni sample

monitored by UV and FL detectors connected succeedingly as

was confirmed. Furthermore, the quenching effects of Noni prod-shown in Figure 6, three standard coumarin derivatives (scopoletin,

ucts and coumarin derivatives on reactive oxygen species (ROS)

7−HC and 4−HC) were separated within 30min (Figure 6A). The

were evaluated by a luminol chemiluminescent assay. Both Noni

retention times for scopoletin, 7−HC and 4−HC were 28.6, 27.2

samples and coumarin derivatives dose−dependently quenchedand 10.8min, respectively. In Figure 6B, scopoletin could be de-

ROS such as superoxide (O2−), singlet oxygen (1O2), hydroxyl radical (.OH) and peroxynitrite (ONOO−). The profiling of quenchingeffect on ROS might be also useful for the quality control of Noni
products, and due to these data, the contribution ratio of quenching

FL detection

effect for scopoletin could be evaluated. The EC50 of scopoletin for
(B)

(A)

O2−, 1O2, .OH and ONOO− were 1.27±0.22mg/mL, 0.68±0.04mg/(C)mL, >4.00mg/mL, and 0.042±0.002mg/mL, respectively. Based onthese data, the contribution ratio of scopoletin for ROS in Noni

7-HC

juices was also evaluated (Table 2). The scopoletin was found to bescopoletin

4-HC

a potent ONOO− quencher in this study. These findings might bescopoletinavailable for the quality evaluation to control Noni products.
Curcuminoids, such as curcumin (C), demethoxycurcumin(DMC) and bisdemethoxycurcumin (BDMC), are derived from the7-HCturmeric plant (Curcuma longa L.). These are commonly used as a
UV detection

dietary spice and a natural coloring agent in South and Southeast
Asia. Over the years, curcumin was reported to have a wide rangeof pharmacological activities, including anti−inflammatory, anti-

Retention time, mincancer, anti−oxide, anti−angiogenic and immunomodulatory [24].

Figure 6. Chromatograms of coumarin derivatives in Noni samples.
Sample: (A) 0.1µg/mL of standards; (B) sample K; (C) sample F.
Detection: (A) 286 nm (UV) and 450nm with 320nm of excitation(FL); (B) and (C) 322nm (UV) and 450 nm with 320nm of excitation (FL).
Printed from Ref. 23 with permission from Elsevier Science Publishers B.V..

It is believed that these desirable effects may appear upon the intake of curcuminoids or turmeric products. Since the quality of turmeric products is directly based on the amount of the curcuminoids, a reliable method for the quantitative analysis of curcuminoids in turmeric products is required. Therefore, an HPLC method
using FL detection for the quantitation of curcuminoids, such as C,

Table 2. Amount and contribution ratios of quenching effect of scopoletin in Noni samples.
Contribution ratio%

SampleConcentration of
scopoletin, µg/mL

O2−

1

A

39.8

0.9

B

39.0

2.4

.

OH

ONOO−

0.1

1.3

15.0

0.4

6.5

48.6

O2

C

35.7

2.3

0.3

5.5

44.1

D

35.2

0.2

0.1

0.4

36.1

E

11.0

4.0

0.9

6.1

75.2

F

54.4

1.7

0.4

2.8

41.8

G

33.3

1.6

0.4

3.0

48.7

H

41.1

0.5

0.1

0.9

14.8

Printed from Ref. 23 with permission from Elsevier Science Publishers B.V..

―５―

CHROMATOGRAPHY, Vol.32 No.1 (2011)(A)(Graduate School of Biomedical Sciences, Nagasaki University)(B)

for his beneficial suggestions. The author greatly acknowledges all
collaborators and coworkers of his studies.
Financial supports from the Ministry of Education, Culture,
Sports, Science and Technology of Japan are gratefully acknowledged.
Finally, the author is very grateful to the Japan Society for
Chromatographic Sciences, which selected him as a recipient of the
Encouragement Award in 2010 and give him the opportunity to
publish this focusing review.
References

Figure 7. Chromatograms of a standard solution (A) and turmeric
powder G (B).
The peak number: 1, BDMC; 2, DMC; 3, C. The arrow indicateschanging of the detection wavelengths from 287 nm (λex) and 303
nm (λem) for I.S. to 426nm (λex) and 539nm (λem) for curcuminoids.
Printed from Ref. 25 with permission from The Japan Society for
Analytical Chemistry.

[1]

Aboul−Enein H. Y.; Hefnawy M. M.; and Nakashima K. In
drug monitoring and clinical chemistry handbook of analytical separations Vol.5. Hempel G., Ed; Elsevier, Amsterdam
2004, Chapter 2. pp 15−75.

[2]Tomita M.; Nakashima M. N.; Wada M.; Nakashima K. Biomed. Chromatogr.; 2007, 21. 1016−1022.

[3]

Nakamura S.; Tomita M.; Wada M.; Chung H.; Kuroda N.;
Nakashima K. Biomed. Chromatogr.; 2006, 20. 622−627.

DMC and BDMC in turmeric products was developed [25]. This[4]

method involves a simple ultrasonic extraction with methanol as a

Wada M.; Yokota C.; Ogata Y.; Kuroda N.; Yamada H.;
Nakashima K. Anal. Bioanal. Chem.; 2008, 391. 1057−62.

pretreatment of turmeric products. The separation of curcuminoids[5]

and 2,5−xylenol as an internal standard was achieved within 30

Nakashima K.; Ikeda R.; Wada M. Anal. Sci.; 2009, 25. 21−
31.

min on a Cadenza CD−C 18 column (250×4.6mm; i.d., 3µm) with[6]

a mixture of acetate buffer and CH3CN (Figure 7). The instrumen-

Yamaguchi M.; Yoshida H.; Nohta H. J. Chromatogr. A;
2002, 950. 1−19.

tal LODs for C, DMC and BDMC (S/N ratio = 3) were 1.5, 0.9 and[7]

0.09ng/mL, respectively. The proposed method was successfully

Fukushima T.; Usui N.; Santa T.; Imai K. J. Pharm. Biomed.
Anal.; 2003, 30. 1655−1687.

applied to determine curcuminoids in commercial turmeric prod[8]ucts, such as turmeric powders, a tablet, a dressing, a beverage, tea,

Nakashima M. N.; Wada M.; Nakashima K. Current Pharm.
Anal.; 2005, 1. 127−133.

and crude drugs.
[9]Nakashima K.; Itoh K.; Kono M.; Nakashima M. N.; Wada
M. J. Pharm. Biomed. Anal.; 2006, 41. 201−206.

5. Conclusion[10] Ohwaki Y.; Yamane T.; Ishimatsu T.; Wada M.; Nakashima

By using hyphenated detections such as UV, FL and CL de-

K. Biomed. Chromatogr.; 2007, 21. 310−317.

tections, sensitive and selective HPLC methods for determinationof medicaments and related compounds were developed. FL label-[11] Yonemoto H.; Ogino S.; Nakashima M. N.; Wada M.; Naka-

ing and microdialysis sampling in these methods could be powerful

shima K. Biomed. Chromatogr.; 2007, 21. 221−224.

tools for sensitive, selective, simple and rapid determination of[12] Wada M.; Nishiwaki J.; Yamane T.; Ohwaki Y.; Aboul−

analytes. Furthermore, applicability of these methods was demon-

Enein H. Y.; Nakashima K. Biomed. Chromatogr.; 2007,

strated to estimate drug−drug interaction. From now on, we will

21. 616−620.
[13] Nagasaki G.; Tanaka M.; Saito A.; Sato M.; Nishikawa T.

perform the research based on the development of analytical meth-

Masui; 2002, 51. 846−850.

ods using chromatographic techniques and practically contribute to[14] Ammon S.; Richter O.; Hofmann U.; Thon KP.; Eichelbaum“rational use of medicaments”.

M.; Mikus G. Drug Metab. Dispos.; 2000, 28.1149−1152.
[15] Kido H.; Sun Y.; Takaba K.; Nakashima M.; Wada M.;

Acknowledgment

Kawada T.; Hara H.; Nakashima K. Jpn. J. Pharm. Health

The author would like to express his sincere gratitude to Pro-

Care Sci.; 2004, 30. 224−230.

fessor Kenichiro Nakashima (Graduate School of Biomedical Sci-

[16] Wada M.; Kurogi R.; Kaddoumi A.; Nakashima M. N.;

ences, Nagasaki University) for his kind supervision on all studies

Nakashima K. Luminescence; 2007, 22. 157−162.

described. The author is also grateful to Professor Naotaka Kuroda―６―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Mitsuhiro Wada[22] Wang M. Y.; West B. J.; Jensen C. J.; Nowicki D.; Su C.;[17] Kalant H.; Can. Med. Assoc. J.; 2001, 165. 917−928.

Palu A. K.; Anderson G. Acta Pharmacol. Sin.; 2002, 23.

[18] Teng S. F.; Wu S. C.; Liu C.; Li J. H.; Chien C. S. Forensic

1127−1141.

Sci. Int.; 2006, 161. 202−208.

[23] Ikeda R.; Wada M.; Nishigaki T.; Nakashima K. Food[19] Kaddoumi A.; Nakashima M. N.; Wada M.; Nakashima K.

Chem.; 2009, 113. 1168−1172.

Eur. J. Pharm Sci.; 2004, 22. 209−216.

[24] Maheshwari R. K.; Singh A. K.; Gaddipati J.; Srimal R. C.

[20] Wada M.; Nakamura S.; Tomita M.; Nakashima M. N.;

Life Sci.; 2006, 78. 2081−2087.

Nakashima K. Luminescence; 2005, 20. 210−215.

[25] Zhang J.; Jinnai S.; Ikeda R.; Wada M.; Hayashida S.; Naka-[21] Nakamura S.; Wada M.; Crabtree B. L.; Reeves P. M.;

shima K. Anal. Sci.; 2009, 25. 385−388.

Montgomery J. H.; Byrd H. J.; Harada S.; Kuroda N.; Nakashima K. Anal. Bioanal. Chem.; 2007, 387. 1983−1990.

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