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Determination of taurine in plasma by high-performanceliquid chromatography using

4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl

chloride as a fluorescent labeling reagent

Hirofumi Inoue, Keiko Fukunaga, and Yasuto Tsuruta*

Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0092, Japan

Received 10 March 2003

Abstract

A sensitive high-performance liquid chromatography method for the determination of taurine in human plasma was developed.

Taurine and N-methyltaurine (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxy-

phenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 70 C for 20 min at pH 7.5.

The fluorescent derivatives were separated on a reversed-phase column by a stepwise elution using (A) acidic phosphate buffer/

acetonitrile (83/17) and (B) acetonitrile and detected by fluorescence measurement at excitation and emission wavelengths of 318 and

392 nm, respectively. The detection limit (signal-to-noise ratio 3) of taurine was 3 fmol per injection. The within-day and day-to-

day relative standard deviations were 3.04.8 and 2.54.7%, respectively. The concentration (means) of taurine in normal human

plasma was 48.9 7.5lM.

2003 Elsevier Science (USA). All rights reserved.

Keywords: HPLC; Fluorescent labeling reagent; 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; Taurine; Plasma

Taurine is a free b-amino acid containing sulfur and

is widely distributed in biological fluids and tissues

without being incorporated into protein. Taurine has

many physiological functions such as being a neuro-

transmitter [1], an antioxidant [2], a modulator of in-

tracellular calcium levels [3], a membrane stabilizer [4],

and an osmolyte [5]. The concentration of taurine inplasma varies in association with various diseases such

as psychosis (trauma [6], depression [7], schizophrenia

[8], epilepsy [9]), sepsis [10], retinitis pigmentosa [11],

and cancer [12]. In addition, it is reported that taurine

depletion is related to cardiomyopathy [13] and the

taurine levels in blood increase after myocardial in-

farction [14]. Therefore, the determination of taurine

could provide useful information for understanding the

conditions of these disorders.

At present, some methods for the determination of

taurine in plasma by gas chromatography (GC) [15], gas

chromatographymass spectrometry (GC-MS) [16],

high-performance liquid chromatography (HPLC) [17

24], and capillary electrophoresis (CE)1 [25] have been

reported. However, the GC and GC-MS methods re-

quire a two-step derivatization procedure to converttaurine into volatile derivatives and the CE method

requires a complex adjustment for analysis of tau-

rine.In the HPLC method, a precolumn derivatization

technique using derivatization reagents such as

4-dimethylaminoazobenzene-40-sulfonyl chloride [17],

7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) [18],

1-dimethylaminonaphthalene-5-sulfonyl chloride (Dan-

Analytical Biochemistry 319 (2003) 138142

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

*Corresponding author. Fax: +81-84-936-2024.

E-mail address: tsuruta@fupharm.fukuyama-u.ac.jp (Y. Tsuruta).

1 Abbreviation used: DMS-Cl, 4-(5,6-dimethoxy-2-phthalimidinyl)-

2-methoxyphenylsulfonyl chloride; IS, internal standard; CE, capillary

electrophoresis; NBD-Cl, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole;

Dansyl-Cl, 1-dimethylaminonaphthalene-5-sulfonyl chloride; OPA-

ME, o-phthalaldehyde-mercaptoethanol.

0003-2697/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0003-2697(03)00277-X

http://mail%20to:%20tsuruta@fupharm.fukuyama-u.ac.jp/http://mail%20to:%20tsuruta@fupharm.fukuyama-u.ac.jp/

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syl-Cl) [19], o-phthalaldehyde-mercaptoethanol (OPA-

ME) [2022], and fluorescamine [23,24] is employed.

Although fluorescamine is utilized for both UV and

fluorescence detection, the fluorescence intensity of the

derivative may decrease over a few hours and the sen-

sitivity of UV detection is relatively low. The fluoro-

metric detection methods using NBD-Cl, Dansyl-Cl,and OPA-ME are fairly sensitive in comparison with the

absorbance detection [17,24], however, the methods us-ing those reagents still require a relatively large sample

volume. Furthermore, the derivatives with NBD-Cl and

Dansyl-Cl are light sensitive and the derivative with

OPA-ME is unstable.

We previously developed an extremely sensitive

fluorescent derivatization reagent, 4-(5,6-dimethoxy-

2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

(DMS-Cl), for the determination of amino acids. This

reagent reacts quantitatively with amino acids to form

stable and highly fluorescent sulfonamides with a la-beling yield of about 100% [26]. In this paper, a highly

sensitive, simple HPLC method for the determination of

taurine in plasma with fluorescence detection after pre-

column derivatization with DMS-Cl is described.

Materials and methods

Chemicals and solvents

All chemicals were of analytical-reagent grade, unless

stated otherwise. DMS-Cl was prepared as described in

a previous paper [26]. Taurine and N-methyltaurine

were purchased from Nacalai Tesque (Kyoto, Japan)and Merck (Darmstadt, Germany), respectively. HPLC-

grade acetonitrile was obtained from Wako Pure

Chemicals (Osaka, Japan). Deionizeddistilled water

was purified with the Milli-QII system (Yamato, Tokyo,

Japan) prior to use.

Instrumental conditions

The HPLC system (Shimadzu, Kyoto, Japan) con-

sisted of two LC-10AD HPLC pumps, a CTO-10AC

column oven, a DGU-14A on-line degasser, an SIL-10AXLXL auto injector, an RF-10AXLXL fluorescence de-

tector, and a CLASS-LC10 LC workstation with a

CBM-10A communications bus module. A Nova Pak

C18 column (150 3.9 mm, i.d., 4lm, Waters) con-

nected to a TSK Guardgel ODS-80TMM (15 3.2 mm,

i.d.; Tosoh, Tokyo, Japan) as a guard column was used

with a stepwise system of (A) phosphate buffer/aceto-

nitrile (83/16) and (B) acetonitrile at 20 C. The stepwise

elution program was an isocratic elution of 0% B for

20 min, followed by a stepwise increase in B to 80% to

wash the column for 5 min and then a stepwise decrease

to 0% to reequilibrate the column for 5 min. Phosphate

buffer was prepared by 500-fold dilution of KH2PO4(1 M, adjusted to pH 2.5 with phosphoric acid). The

flow rate was 1 ml/min. The fluorescence intensities were

monitored at excitation and emission wavelengths of

318 and 392 nm, respectively.

Preparation of plasma sample

Blood was collected from 12 healthy volunteers(Japanese staff and students in our laboratory) at 10:00

a.m. and plasma was prepared according to the method

of Trautwein and Hayes [27] to ensure that minimal

plasma contamination occurs from the release of intra-

cellular taurine. Venous blood was collected from the

antecubital vein into a dry, sterile, disposable plastic

syringe and immediately transferred into a plastic tube

containing 10% ethylenediaminetetraacetic acid tetra-

sodium salt (10 ll/ml blood). Blood was kept at room

temperature prior to centrifugation. Plasma was sepa-rated by centrifugation for 15 min at 1500g at room

temperature and stored in a freezing chamber until

analysis. For the determination of taurine, plasma

sample was prepared by 10-fold dilution of plasma with

water.

Analytical procedure

To plasma sample (20ll), N-methyltaurine (5lM,

20ll) as an internal standard (IS), borate buffer (0.1 M,

pH 7.5, 60ll), and DMS-Cl (5 mM, in acetonitrile,

100ll) were successively added and mixed well. The

labeling reaction was carried out at 70 C for 20 min and

then proline (0.1 M, in borate buffer (0.1 M, pH 7.5),100ll) was added to the reaction mixture. After stand-

ing for more than 2 min at room temperature, the mix-

ture was acidified with phosphoric acid (1 M, 200ll) and

then centrifuged at 2000g for 10 min. An aliquot of the

supernatant (20ll) was subjected to HPLC.

Results and discussion

HPLC separation

The derivatives of taurine and IS labeled with DMS-

Cl were successfully separated on a reversed-phase col-

umn. Typical chromatograms obtained from a standard

solution and human plasma are shown in Fig. 1. The

peaks due to taurine and IS were eluted at 12.2 and

17.4 min, respectively, and were completely separated

from the peaks of the reagent blank and other plasma

components under the described conditions. The

maximum fluorescence wavelengths of the eluate corre-

sponding to peak due to taurine were 318 nm (excita-

tion) and 392 nm (emission). The peak due to the

fluorescent derivative of taurine in human plasma was

H. Inoue et al. / Analytical Biochemistry 319 (2003) 138142 139

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identified by comparing the retention time with that of

the standard solution and by cochromatography of the

standard and plasma.

Reaction conditions

Standard solutions of taurine and IS (5lM each,

20ll each) were used to determine the optimum labeling

conditions. As the labeling reaction of taurine and IS

with DMS-Cl proceeded in a basic medium, the effect of

the pH of borate buffer (0.1 M) was examined. The peak

area reached the maximum at the pH range 7.59.0, as

shown in Fig. 2.

The effect of reaction time on the labeling reaction

was tested at various temperatures. As shown in Fig. 3,

the maximum peak area due to taurine was obtainedfrom the reaction for 10 min at 70 C, while the labeling

reaction of IS proceeded rapidly and was completed

within 5 min at 70 C. Therefore, the labeling reaction

was carried out at 70 C for 20 min.

The concentration of DMS-Cl in acetonitrile was

determined using diluted plasma spiked with standard

taurine (0.4 nmol). The most intense and constant peak

areas were obtained when the concentration of the re-

agent solution was more than 2.5 mM.

After the labeling reaction, proline was added to

convert the excess of DMS-Cl to the proline derivative,

because the excess of DMS-Cl was suspected of causing

the guard column to degrade. Incidentally, the reaction

took place at room temperature within 2 min and the

derivative of proline was eluted by washing the column

with 80% B.

When the reaction mixture was directly subjected toHPLC without acidification, both peaks due to taurine

and IS were eluted as broad peaks. However, those

peaks became sharp by the acidification of the reaction

mixture with phosphoric acid.

Influence of amino acids

The influence of amino acids on the determination oftaurine was examined using diluted plasma spiked with

27 species of amino acids (Ala, Arg, Asn, Asp, Cit, Cys-

Cys, Cys, Glu, Gln, Gly, His, Hse, Hyp, Ile, Leu, Lys,

Met, Orn, Pro, Phe, Ser, Thr, Try, Tyr, Val, Abu, and

Fig. 2. Effect of pH of borate buffer (0.1 M) on the labeling reaction of

taurine and IS with DMS-Cl. Curves: 1, taurine; 2, IS.

Fig. 3. Effect of reaction time and temperature on the labeling reaction

of taurine and IS with DMS-Cl. Curves: 1, Taurine at 25 C; 2, Taurine

at 50 C; 3, Taurine at 70 C; 4, IS at 25 C; 5, IS at 50 C; 6, IS at

70 C.

Fig. 1. Chromatograms obtained from (A) a standard solution and (B)

a plasma sample according to the procedure described under Materials

and methods. Peaks: 1, Taurine; 2, IS. Concentration of taurine:

(A) 5lM; (B) 3.37 lM.

140 H. Inoue et al. / Analytical Biochemistry 319 (2003) 138142

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eAhx, 0.2 nmol each). These amino acids were eluted by

washing the column with 80% B and did not interfere

with the determination of taurine.

Precision

The within-day and day-to-day precisions were tested

using plasma from normal subjects. The within-day

precision was examined with eight replicate assays in

1 day and the day-to-day precision by assays on 5 days.

As shown in Table 1, the within-day and day-to-day

relative standard deviations were 3.04.8 and 2.54.7%,

respectively.

Linearity, recovery, and detection limit

The linearity and the recovery were examined using

diluted plasma (concentration of taurine: 5.20lM)

spiked with various amounts of standard taurine (con-

centration in diluted plasma: 1.0, 2.5, 5, 10, and 20 lM

each). The relationships between the peak area ratios of

taurine to IS and the concentrations of taurine were

linear (Fig. 4). When the recovery was obtained from the

slope ratio of regression equations with/without plasma,the recovery was 100.5%.

The detection limit (signal-to-noise ratio 3) of

taurine was 3 fmol per injection.

Determination of taurine in human plasma

The concentrations of taurine in plasma from 12

healthy volunteers who were eating self-selected diets

were determined by the present method. The concen-

trations of taurine in plasma are given in Table 2. The

mean value (meanSD) was 48.9 7.5lM. The mean

value was similar to the values reported previously [15,

17,18,22,27].

In conclusion, we have established a precolumn

HPLC method for the determination of taurine in

plasma using a fluorescent labeling reagent. As the

proposed method is highly sensitive and reproducible

and requires only a small amount of plasma, it may be

useful for clinical and biochemical research including

studies using small animals.

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Table 1

Precision of determination of taurine in plasma

Within-day (n 8)a Day-to-day (n 5)b

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R.S.D.c R.S.D.c

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3.6 4.3

a Within-day precision is tested on 10 replicates in 1 day.bDay-to-day precision is tested on 5 days.c R.S.D., relative standard deviation.

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Concentration of taurine in normal human plasma

Age Sexa Concentration (lM)

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23 M 52.8

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