JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 34, 93È97 (1999)

Quantitative Determination of E5880 in RatPlasma by High-performance LiquidChromatography/Electrospray Ionization TandemMass Spectrometry

Kiyomi Kikuchi, Yoshihisa Sano,* Sachie Taniguchi, Kenji Matsui, Masayuki Namiki, Hatsue Ito,Hideki Sakurai and Tsutomu YoshimuraTsukuba Research Laboratories, Eisai Co. Ltd., 1È3 Tokodai 5-chome, Tsukuba-shi, Ibaraki 300-2635, Japan

A simple and sensitive method is described for the determination of E5880 in rat plasma. The method is based onhigh-performance liquid chromatography/electrospray ionization mass spectrometry, using deuterated E5880 as aninternal standard. Selected reaction monitoring is employed for selectivity and sensitivity, this in turn enablesquantiÐcation in a short period of time (within 7 minutes) over the extended range of 0.1–1000 ng/ml with accept-able precision and accuracy. The method demonstrated to be suitable for the quantitative analysis of E5880 in ratplasma. The pharmacokinetic proÐle of E5880 after a single intravenous administration of E5880 was elucidated.Copyright 1999 John Wiley & Sons, Ltd.(

KEYWORDS: E5880, platelet activating factor, mass spectrometry, rat, pharmacokinetics

INTRODUCTION

Platelet activating factor (PAF) is a potent lipid medi-ator of various inÑammatory and allergic reactions.1h3E5880 has been reported to have certain antagonisticactivity to PAF in vitro and inhibitory e†ects in in vivoshock models, and is now being developed as a treat-ment for disseminated intravascular coagulation (DIC)shock.4 Since the circulating levels of E5880 afteradministration of therapeutic doses are low, it is neces-sary to develop a highly sensitive and speciÐc analyticaltechnique for the determination of the E5880 in bio-logical Ñuids.

A sensitive assay method has been reported tomeasure E5880 in dog plasma using radioimmunoassay(RIA).5 However, cross-reactivities with related com-pounds such as the unknown metabolites of E5880 inthe RIA have not been satisfactorily elucidated. High-performance liquid chromatography is generally moreselective for the target compound than RIA, althoughE5880 does not show a marked absorbance in the UVrange.

Recently, high-performance liquid chromatography/tandem mass spectrometry has been pronounced as theassay method for drugs in biological samples with greatadvantages especially in selectivity,6h8 and we have

* Correspondence to : Y. Sano, Tsukuba Research Laboratories,Eisai Co. Ltd, 1È3 Tokodai 5-chome, Tsukuba-shi, Ibaraki 300-2635,Japan

already succeeded in the measurement of menaquinone-4(vitamin K), an agent for osteoporosis, in osteoblastsusing this technique.9

In this study, we report the development of a novelassay method for E5880 in rat plasma, using high-performance liquid chromatography/electrospray ion-ization tandem mass spectrometry. The method wassufficiently simple and sensitive for the quantitativeanalysis of E5880 in plasma after a single intravenousadministration to rats.

EXPERIMENTAL

Chemicals and reagents

E5880, (])-1-ethyl-2-MN-(2-methoxybenzoyl)-N-[(2R)-2-methoxy-3-[4-(N-octadecylcarbamoyloxy) piperidino-carbonyloxy]propyloxycarbonyl]aminomethylN pyri-dinium chloride, was synthesized by Eisai Chemical Co.,Ltd. (Ibaraki, Japan). (deuterated E5880) was[2H5]synthesized by Eisai Co., Ltd. (Ibaraki, Japan) as theinternal standard (IS) for the assay. The isotopic purityof deuterated E5880 was 99.9%. The structures of thesecompounds are shown in Figure 1. HPLC solvents wereof HPLC grade and were obtained from Wako PureChemical Industries, Ltd. (Osaka, Japan), except TFA,which was of Kanto Chemical special grade and wasobtained from Kanto Chemical Co., Inc. (Tokyo,Japan).

CCC 1076È5174/99/020093È05 $17.50 Received 29 June 1998Copyright ( 1999 John Wiley & Sons, Ltd. Accepted 29 October 1998

94 K. KIKUCHI ET AL.

Figure 1. Chemical structures of E5880 and Í2H5ËE5880

(deuterated E5880). The ions m/z 825.6 and 511.5 correspond tothe intact molecular cation and the prominent product ion forE5880 and the ions m/z 830.6 and 511.4 for deuterated E5880,respectively.

Apparatus for LC/ESI/MS/MS

HPLC was performed on an ODS column (L-columnODS, 150 mm] 1.5 mm i.d., Chemicals Inspection andInstitute, Tokyo, Japan) usng an LC-10A system(Shimadzu, Kyoto, Japan). The mobile phase was

(970 : 30 : 1) at a Ñow rate of 0.2MeOH/H2O/TFAml/min.

Mass spectrometric detection was carried out with aFinnigan TSQ 7000 triplequadrupole instrument (SanJose, CA, USA) using positive ion electrospray ioniza-tion (ESI) as an LC/MS interface. The temperature ofthe heated capillary was set at 275 ¡C. The sheath gaspressure and auxiliary gas Ñow were set at 80 psi and 5units, respectively. Selected reaction monitoring (SRM)was employed using argon as the collision gas at 1.7mTorr, with collision energy of [35 eV for both E5880and deuterated E5880 during collision induced disso-ciation (CID). Precursor to product transitions weremonitored from m/z 825.6 to m/z 511.5 for E5880, andfrom m/z 830.6 to m/z 511.4 for deuterated E5880. Totalscan time was set at 2 seconds.

Preparation of standard solutions

The stock solutions of E5880 (100 ng/20 ll) and deuter-ated E5880 (100 ng/50 ll) were prepared in

(970 : 30 :1) and stored at 4 ¡C. UnderMeOH/H2O/TFAthese conditions, they were stable for a period of up to63 days. A series of working E5880 solutions were pre-pared by dilution of the stock solution just prior to use :0.01, 0.02, 0.1, 0.2, 1, 2, 10 and 20 ng/20 ll. A workingdeuterated E5880 solution was prepared in

(970 : 30 :1) at 0.1 ng/50 ll. Cali-MeOH/H2O/TFAbration samples were prepared for each assay by adding20 ll of each E5880 solution to 0.1 ml of plasma andtreated as described in the section of ““Sample prep-arationÏÏ.

Administration and sample collection

Pharmacokinetic study was performed using maleSprague Dawley rats of 8 weeks age purchased fromJapan SLC Inc. (Shizuoka, Japan). E5880 as a 0.01NHCl solution was administered intravenously into thefemoral vein at a dose of 0.2 mg/kg. Blood samples ofapproximately 0.25 ml were drawn from the jugularvein at 5, 15 and 30 minutes and 1, 2, 4, 6, 8 and 24hours after dosing, using heparin as an anticoagulant.After centrifugation (3000 rpm, 10 minutes, 4 ¡C,KUBOTA 8800, KUBOTA MANUFACTURINGCORPORATION, Tokyo, Japan), 0.1 ml of plasma wascollected in a polypropylene tube. Plasma samples at 5and 15 min were diluted 5 times with blank plasma.Samples were stored frozen (at [25 ¡C) until analysis.

Sample preparation

Twenty ll of (970 : 30 :1) as a substi-MeOH/H2O/TFAtute for E5880 solution, 50 ll of IS solution and 1 ml ofacetonitrile were added to 0.1 ml plasma. The samplewas mixed and centrifuged at 3000 rpm for 5 minutes(KUBOTA KR/600, KUBOTA MANUFACTURINGCORPORATION, Tokyo, Japan) at 4 ¡C. The upperlayer was collected and evaporated under a nitrogenstream at 30 ¡C. The residue was dissolved in 0.1 ml of

(970 : 30 :1), and Ðltered through aMeOH/H2O/TFAMillipore Ðlter (HYC PTFE, 0.2 lm). A 10 ll aliquot ofthe Ðltrate was injected into the LC/MS/MS system.

Calibration curve

Calibration curves were constructed by plotting thepeak area ratios of E5880 to deuterated E5880 (IS)against the concentration of E5880. A weighted (1/X2)linear regression line was Ðtted to the 0.1È1000 ng/mlconcentration range. The concentrations of E5880 intest samples were calculated from the peak area ratiosusing this line and expressed as ng/ml.

Pharmacokinetic analysis

Pharmacokinetic parameters were calculated from theplasma concentrations of E5880 using a model indepen-dent method. The was obtained from theAUC(0h24 hr)plasma concentrations for 24 hours after administrationusing the trapezoidal method. For the purpose of AUCcalculation, the plasma concentration at time zero wasestimated to be equal to the concentration at the Ðrsttime-point measured (5 minutes). Half life was esti-(t1@2)mated from the slope of the plasma concentrations atthe last two time points (8 and 24 hours afteradministration). was calculated using theAUC(0~=)equation

AUC(0~=) \ AUC(0h24 hr)] C(24 hr) ] t1@2/ln2

where was the plasma concentration at 24 hoursC(24 hr)after administration.

Copyright ( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 93È97 (1999)

LC/ESI/MS/MS OF E5880 IN RAT PLASMA 95

RESULTS AND DISCUSSION

LC/MS/MS conditions

Figure 2 shows the ESI positive mass spectrum forE5880. The intact molecular cation for the compound(m/z 825.6) was observed as the base peak ion.

At Ðrst we attempted to improve the intensity of themolecular cation of E5880 on LC/MS to achieve highsensitivity detection. As the heated capillary tem-perature had an inÑuence on the intensity of the molec-ular cation of E5880, it was set at 275 ¡C, the maximumtemperature permitted on the instrument, taking intoconsideration the signal-to-noise (S/N) ratio of the peakintensity. Sheath gas pressure also a†ected the sensi-tivity. It was observed that intensity increased withincreasing gas pressure up to 80 psi. The auxiliary gasÑow, however, did not a†ect markedly the sensitivityand was set at 5 units. Under these conditions, E5880produced the molecular cation most e†ectively with noapparent fragmentation.

We decided to use selected reaction monitoring(SRM) for the determination of E5880 to achieve highselectivity and sensitivity. Figure 3 shows the production mass spectrum of the molecular cation of E5880.Fragmentation of the precursor ion was apparent, withm/z 511.5 being the prominent product ion for the com-pound. The e†ects of the collision energy and collision

Figure 2. ESI positive mass spectrum of E5880.

Figure 3. Product ion mass spectrum of the intact molecularcation of E5880.

gas pressure on the intensity were investigated in orderto obtain a maximum response for the product ion.Based on this result, the energy level was set at [35 eV.The argon gas pressure was Ðxed at 1.7 mTorr becauseit was observed that it had a slight e†ect on peak inten-sity.

The LC/MS/MS conditions for deuterated E5880, theinternal standard, were identical to those for E5880except for the mass set (see EXPERIMENTAL section).

For the HPLC mobile phase, we found that solventscontaining ammonium acetate caused signiÐcant con-tamination between samples. The use of

(970 : 30 :1) solved this problem andMeOH/H2O/TFAseparated the analytes from the biological componentsin a shorter analysis time (7 minutes).

Calibration range, precision and accuracy

Figure 4 presents typical LC/ESI/MS/MS chromato-grams for E5880 and deuterated E5880 obtained fromthe calibration samples. The chromatograms wereobtained in a short analysis time and showed no otherpeaks, indicating the high selectivity of this method.Furthermore, no interfering peaks at the retention timeof the analyte were observed in the blank samples.Response was linear over the range of 0.1 to 1000 ng/mlfor E5880 (r\ 0.998, S/N ratio [ 10 at 0.1 ng/ml). Theaccuracy of the calibration samples was within ^12.5%(Table 1). The linear range improved greatly comparedwith the previous method using RIA (0.2 to 10 ng/ml),5allowing the determination of a wider concentrationrange of plasma samples.

The intra- and inter-day precision and accuracy weredetermined for the present assay method. The resultsare summarized in Table 2. The accuracy results of theintra- and inter-assay variation studies were satisfacto-rily, within ^13.7%, except for the lowest concentrationin the intra-assay study, which was 20.0%. The intra-and inter-day precision (5 and 3 replicates, respectively)was also acceptable. The RSD values were less than14.3% over the calibration range. Based on theseresults, it was concluded that the quantiÐcation limit forE5880 was 0.1 ng/ml (1 pg per injection). This methodwas 200 times and 2 times more sensitive than the pre-vious HPLC method and the radioimmunoassaymethod,5 in which the quantiÐcation limit was 20 ng/mland 0.2 ng/ml, respectively.

Table 1. Calibration results for E5880 in ratplasma

Concentration Concentration

added found Accuracy

(ng mlÉ1) (ng mlÉ1) (%)

0.1 0.10 0.0

0.2 0.21 5.0

1 1.09 9.0

2 2.04 2.0

10 10.40 4.0

20 19.77 É1.2

100 99.25 É0.8

200 193.55 É3.2

1000 875.38 É12.5

( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 93È97 (1999)

96 K. KIKUCHI ET AL.

Figure 4. Typical LC/ESI/MS/MS chromatograms of (a) a plasma sample added with deuterated E5880, (b) a plasma sample spiked withE5880 (1 ng/ml) and deuterated E5880.

Stability in plasma and extracted samples

The samples for the stability test were prepared usingpooled rat plasma at concentrations of 0.2 ng/ml, 10ng/ml and 200 ng/ml.

E5880 was shown to be stable in rat plasma duringtwo freeze ([25 ¡C)/thaw cycles (more than 94.6% forthe three concentrations) and when stored at [25 ¡Cfor 7 days (more than 88.4%). E5880 was also stable inthe injection Ñuid at 4 ¡C for 7 days (more than 91.3%).

Pharmacokinetic proÐle in rats

The method was applied to determine the phar-macokinetic proÐle of E5880 in rats. Quality controlsamples at concentrations of 0.2, 10 and 200 ng/ml wereassessed for accuracy and were all within ^15.5%. Thisresult was adequate and indicated that the measurementwas successfully performed.

Figure 5 shows plasma concentration proÐle ofE5880 after a single intravenous administration ofE5880 to rats at a dose of 0.2 mg/kg. The plasma con-centrations could be monitored for 24 hours afteradministration. E5880 in plasma decreased rapidly until

1 hr, and was eliminated slowly thereafter. Terminalphase half-life was 10.3 hr, and this value was approx-imately half of that obtained in a previous study (20.1hr) using RIA as the assay method. (310.38AUC(0~=)

Figure 5. Plasma concentration of E5880 after a single intra-venous administration of E5880 to rats at a dose of 0.2 mg/kg.Each point represents the mean ÀSEM of three animals.

Table 2. Intra- and inter-assay variation for E5880 in rat plasma

Intra-assay (n ¼5) Inter-assay (n ¼3)

Mean Mean

Concentration concentration concentration

added found ÀSD RSD Accuracy found ÀSD RSD Accuracy

(ng mlÉ1) (ng mlÉ1) (%) (%) (ng mlÉ1) (%) (%)

0.1 0.12 0.01 8.3 20.0 0.11 0.01 9.1 10.0

0.2 0.22 0.01 4.5 10.0 0.21 0.03 14.3 5.0

10 10.34 0.36 3.5 3.4 9.90 0.61 6.2 É1.0

200 190.42 2.01 1.1 É4.8 186.35 10.52 5.6 É6.8

1000 866.85 23.68 2.7 É13.3 863.14 22.50 2.6 É13.7

Copyright ( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 93È97 (1999)

LC/ESI/MS/MS OF E5880 IN RAT PLASMA 97

ng É hr/ml) was also lower than in the RIA study(unpublished data). One possible explanation for thisdiscrepancy is that the antiserum used in the RIA assaymight exhibit cross-reactivity with E5880 related com-pounds such as unknown metabolites in rat plasma.

In conclusion, an LC/MS/MS method for the deter-mination of E5880 with minimum pretreatment of bio-logical samples and a short analysis time per sample hasbeen established. The linear calibration range (104-foldrange of concentrations) of the LC/ESI/MS/MS methodfor E5880 is larger than that of the RIA method5 pre-viously used in the pharmacokinetic analysis, for several

other drugs in which the calibration range was several10 or 102-fold concentration.7,8,10,11 Moreover, thesensitivity, accuracy and precision of the method areadequate for the determination of E5880 concentrationsin samples of pharmacokinetic studies.

Acknowledgements

The authors wish to thank Dr. Mannen Mishima and Mr. MitsumasaShino for helpful discussions along the way, and Mr. Kenji Hayashifor the synthesis of E5880.[2H5]

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( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 93È97 (1999)