JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 35, 354–360 (2000)

Quantitative determination of SC-68328 in dogplasma using flow injection and tandem massspectrometry

Ji Y. Zhang,1* Douglas M. Fast,1 Grant L. Schoenhard,1† Vinod K. Arora, 2‡ Frank J. Belas2§and Ian A. Blair 2

1 Metabolism and Safety Evaluation, Searle R&D, Skokie, Illinois 60077, USA2 Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

A flow injection/tandem mass spectrometric assay was developed to quantitate SC-68328 in dog plasma usingits stable isotopic analog [13C4]SC-68328 as an internal standard (IS). Since SC-68328, a manganese-basedsuperoxide dismutase mimetic, is very unstable, very polar and adheres to silica-based high-performance liquidchromatographic columns, the analyte and IS were derivatized to their bis-isothiocyanate forms followed by aliquid–liquid extraction with methylene chloride and analyzed using positive ion electrospray mass spectrometricdetection. SC-68328 was quantitated using the peak-height ratio of SC-68328 to its IS using MS/MS in themultiple reaction monitoring mode. The lower limit of quantitation of the assay was 0.25 µg ml−1 SC-68328 indog plasma with an inter-day precision of 11.8% and an accuracy of 113% (n = 12). Acceptable precision andaccuracy were also obtained for concentrations in the calibration curve range (0.25–10µg ml−1 SC-68328 indog plasma). Copyright 2000 John Wiley & Sons, Ltd.

KEYWORDS: electrospray ionization; flow injection; tandem mass spectrometry; method development and validation;superoxide dismutase mimetic

INTRODUCTION

SC-68328 is a manganese-based superoxide dismutase(SOD) mimetic (Fig. 1). SOD is a family of enzymesthat rapidly eliminates a biologically generated free rad-ical species, the superoxide anion.O2

ž�/. Since the dis-covery of SOD,1 intensive efforts have been made todevelop the enzyme as a therapeutic agent for the treat-ment of a wide range of diseases and disorders, such asreperfusion injury, ischemic myocardium post-ischemicneuropathies, organ transplation and radiation-inducedinjury.2,3 Because of problems such as cost, bioavailabil-ity, stability and immunogenicity associated with usingan enzyme as a pharmaceutical agent, synthetic lowmolecular mass manganese-based SOD mimetics weredeveloped in an attempt to overcome these problems.These SOD mimetics have been shown to provide signif-icant protection against myocardial ischemia–reperfusioninjury in vitro and in vivo.4–6 The complexes also func-tioned as anti-inflammatory agents and potentiated nitricoxide levels by inhibiting the production of peroxynitrite.7

* Correspondence to: J. Y. Zhang, Metabolism and Safety Evalua-tion, Searle R&D, 4901 Searle Parkway, Skokie, Illinois 60077, USA.E-mail: ji.y.zhang@monsanto.com† Present address: Genentech Inc., 1 DNA Way, South San Francisco,

California 94080, USA.‡ Present address: Bristol-Myers Squibb Company, 5 Research Park-

way, Wallingford, Connecticut 06492, USA.§ Present address: Eli Lilly and Company, Drop Code 0825, Indi-

anapolis, Indiana 46285, USA.

SC-68328 was under development as a therapeutic agentfor diseases mediated by superoxide, particularly for thetreatment of myocardial ischemia–reperfusion injury.

This paper describes the development of an analyticalmethod using liquid–liquid extraction and flow injec-tion/positive ion electrospray ionization tandem massspectrometry (FI/ESI-MS/MS) for the quantitative deter-mination of SC-68328 in dog plasma. The procedure con-sisted of derivatizing SC-68328 and its internal standard totheir bis-isothiocyanate forms followed by a liquid–liquidextraction with methylene chloride and mass spectromet-ric detection. Challenges in developing this assay wereto overcome the problems of stability, excessive polarityand poor chromatographic performance in reversed-phasehigh-performance liquid chromatography (HPLC) for themanganese-based analyte.

EXPERIMENTAL

Chemicals and reagents

All chemicals were of analytical grade and were pur-chased from the following suppliers: methanol, methy-lene chloride from Baxter Healthcare (Muskegon, MI,USA), sodium dihydrogenphosphate and sodium mono-hydrogenphosphate from Aldrich Chemical (Milwaukee,WI, USA) and ammonium thiocyanate from Sigma Chem-ical (St Louis, MO, USA). High-purity water was obtainedusing a Millipore system (Milford, MA, USA). SC-68328

Copyright 2000 John Wiley & Sons, Ltd. Received 10 September 1999Accepted 10 November 1999

FLOW INJECTION AND MS/MS DETERMINATION OF SC-68328 IN DOG PLASMA 355

Figure 1. Structures of SC-68328 and its internal standard[13C4]SC-68328. Asterisks denote the positions labeled with 13C.

and [13C4]SC-68328 standards were obtained from theG. D. Searle compound file (Skokie, IL, USA).

Preparation of standard and quality control samples

Stock solutions of SC-68328 and [13C4]SC-68328(1 mg ml�1) were prepared in a 10 ml volumetric flaskwith methanol and were serially diluted with methanolto obtain the desired concentrations. The stock solutionswere kept refrigerated.4 °C/ and discarded 1 month afterpreparation. The plasma calibration curve concentrationswere 0.25, 0.3, 0.5, 0.8, 1, 4, 8 and 10µg mL�1 withquality control (QC) samples prepared at 0.25, 1, 8 and100 µg mL�1. These standard and QC samples were pre-pared with the appropriate volume of the SC-68328 stocksolution in a 50 ml volumetric flask by diluting to vol-ume with ice-cold dog plasma to achieve the desired finalconcentrations. An aliquot of 400µl was transferred intoa 1.5 ml microvial (Alltech, Deerfield, IL, USA) that wascapped and quickly frozen in a dry ice–acetone bath andstored in a�70°C freezer. The processing time to prepareeach standard and QC pool was limited to less than 5 min.

Derivatization and extraction of samples

The dog plasma standards, QC samples and sampleswere thawed in ice-cold water using a microwave oven,then centrifuged at 3000 rpm (Beckman Instruments, PaloAlto, CA, USA) for 5 min at 4°C. A 200 µl aliquotof each dog plasma standard, QC sample and samplewas transferred into a 1.5 ml polypropylene microvialin an ice-cold water-bath, then 10µl of [ 13C4]SC-68328internal standard solution (100µg ml�1) and 200µl of150 mM ammonium thiocyanate in 10 mM sodium phos-phate buffer (pH 7.05) were added. The mixture wasvortex mixed for 10 s to form the bis-isothiocyanatederivative, which was extracted with 500µl of methy-lene chloride by vortex mixing for 3 min. Following cen-trifugation at 14 000 rpm for 3 min using an Eppendorfcentrifuge (Beckman Instruments) to separate the organicand aqueous layers, the organic layer was transferred toa new 1.5 ml polypropylene microvial and diluted with200 µl of methanol. Finally, the sample was transferredto a clean autosampler injection vial and a 20µl samplewas injected into the FI/ESI-MS/MS system for analysis.

FI/ESI-MS/MS

FI/ESI-MS/MS analyses were performed using a sys-tem comprised of an HP Series 1090 HPLC autosampler

and pump (Hewlett-Packard, Palo Alto, CA, USA) and aFinnigan TSQ-7000 triple quadrupole mass spectrometer(ThermoQuest, San Jose, CA, USA). The mobile phase,consisting of methylene chloride–methanol–ammoniumthiocyanate (90 : 10 : 0.2 mM, v/v/w) at a flow-rate of400 µl min�1, was directly introduced into the mass spec-trometer. Mass spectral analysis was performed in thepositive ion electrospray ionization mode with the elec-trospray needle voltage set at 4.5 kV. Nitrogen was usedas the sheath gas (70 psi) and the auxiliary gas (25 psi)to assist with nebulization. A 10µl min�1 sheath flowof methanol–water (80 : 20, v/v) containing 2 mM ammo-nium thiocyanate was used as sheath liquid. The interfacecapillary was heated to 200°C to provide optimum desol-vation. The electrospray interface and mass spectrometerparameters were optimized to obtain maximum sensitivityat unit resolution. A multiple reaction monitoring (MRM)experiment was conducted by monitoring the transitionsfrom m/z 356 (Q1) tom/z 295 (Q3) for SC-68328 andfrom m/z 360 (Q1) tom/z 299 (Q3) for the internal stan-dard (IS). Argon was used as the collision gas. A collisionoffset energy of 25 eV and a collision cell pressure of2.4 mTorr.1 TorrD 133.3 Pa/ were used to induce frag-mentation in the collision cell. The tube lens and thecapillary were operated at 78.8 and 9.2 V, respectively.The electron multiplier was set at 1580 V. The scan timewas 1 s and the product ion scan width was 1 u.

Method validation

For method validation, duplicate plasma calibration curvesand four replicates of each QC pool were analyzed onthree separate days. The peak heights generated by theMRM of SC-68328 and its IS were obtained using theQUAN program in the Finnigan ICIS II data system. Peak-height ratios of them/z 356 to 295 and them/z 360 to299 transitions were calculated using an Excel spread-sheet program, and calibration curves were obtained by aweighted (1/concentration) least-squares linear regressionanalysis. Concentrations of SC-68328 in the samples andcontrols were then calculated using the equations from theappropriate calibration curves.

RESULTS AND DISCUSSION

Method development

The chromatographic and ionization difficulties caused bythe high water solubility of the manganese-based SODmimetics, SC-68328 and its IS, were overcome by con-verting the analytes into their bis-isothiocyanate deriva-tives from their corresponding dichloro precursors byreacting them with 200µl of 150 mM ammonium thio-cyanate in 10 mM sodium phosphate buffer (pH 7.05). Thebis-isothiocyanate derivatives were more stable than thedichloro precursors, insoluble in water and easily extractedwith methylene chloride. Aqueous sodium phosphate solu-tion (10 mM, pH 7.05) was used as a buffer because SODmimetics are unstable under acidic conditions. No signalcorresponding to SC-68328 was observed from sampleswhen a saturated ammonium thiocyanate solution was

Copyright 2000 John Wiley & Sons, Ltd. J. Mass Spectrom. 35, 354–360 (2000)

356 J. Y. ZHANG ET AL.

used as the derivatization reagent. This result may becaused by supression of ionization in the ion source ofthe mass spectrometer due to the excessive amount ofammonium thiocyanate present in the samples. Therefore,150 mM ammonium thiocyanate solution was chosen asthe derivatization reagent. The derivatized samples wereextracted with 0.5 ml of methylene chloride. The extrac-tion efficiency of SC-68328 in dog plasma was exam-ined using labeled [14C]SC-68328 at a concentration of1 µg ml�1 in three replicates. After derivatization, extrac-tion and liquid scintillation counting, it was found thatthe extraction efficiency of [14C]SC-68328 was 60.4%.n D 3/ when 10µg ml�1 of the IS was used in plasmasample. In contrast, only 39.4%.n D 3/ of [14C]SC-68328was recovered when 1µg ml�1 of the IS was used. Theresults demonstrated that the addition of 10µg ml�1 of

IS was necessary in order to act as an efficient carrier ofSC-68328 to improve the extraction efficiency.

Neither SC-68328 nor its IS were observed in massspectrometric analysis when the samples were spikedwith EDTA-treated dog plasma. The results indicated thatEDTA cannot be used as an anticoagulant since EDTAwould destroy SOD mimetics by chelating the manganesefrom the drug to form manganese EDTA complexes.

Various normal-phase, reversed-phase and cation-ex-change HPLC columns were evaluated to retain SC-68328. It was found that SC-68328 adhered to mostsilica-based HPLC columns, resulting in very poor recov-ery and a broad peak shape. Therefore, flow injec-tion was chosen in order to eliminate the chromato-graphic difficulties. Mobile phases using methanol andmethylene chloride containing different concentrations of

Figure 2. (a) Full-scan ESI mass spectrum of SC-68328 bis-isothiocyanate derivative and (b) CID mass spectrum of m/z 356.

Copyright 2000JohnWiley & Sons,Ltd. J. MassSpectrom. 35, 354–360 (2000)

FLOW INJECTION AND MS/MS DETERMINATION OF SC-68328 IN DOG PLASMA 357

Figure 3. (a) Full-scan ESI mass spectrum of [13C4]SC-68328 bis-isothiocyanate derivative and (b) CID mass spectrum of m/z 360.

ammoniumthiocyanatewereevaluatedto achievethebestsensitivity for SC-68328in massspectrometry.An iso-cratic systemconsistingof methylenechloride–methanol(90: 10, v/v) containing0.2 mM ammoniumthiocyanatewas chosen as the mobile phase. The flow-rate was0.4 ml min�1 and20 µl of samplewere injectedinto themassspectrometerfor analysisusing flow injection. TheanalysiswasperformedusingaFinniganTSQ-7000triple-quadrupolemass spectrometerequippedwith an elec-trospray interface. The positive ESI massspectrumofSC-68328showeda significant fragmention at m/z 356[Fig. 2(a)], generatedfrom the loss of HSCN from theprotonatedmolecularspecies.Thecollision-induceddisso-ciation(CID) of m/z 356producedthreeabundantproductions at m/z 297, 295 and 293 correspondingto a loss ofHSCN,HSCNC 2H andHSCNC 4H, respectively,fromm/z 356 [Fig. 2(b)]. Similarly, the IS [13C4]SC-68328showeda significantfragmention at m/z 360 [Fig. 3(a)],

which is four massunits higher than SC-68328becauseit containsfour carbon-13atoms.The CID spectrumofm/z 360 showed the similar three major product ionsat m/z 301, 299 and 297 [Fig. 3(b)]. The ion transi-tions from m/z 356 to 295 and from m/z 360 to 299were used to monitor SC-68328and its stable isotopicIS [13C4]SC-68328,respectively.The MRM ion currentprofilesof extractsfrom blankdogplasma,theblankwiththe IS (10 µg ml�1) and the dog plasmasamplecontain-ing 0.250 µg ml�1 of SC-68328and10 µg ml�1 of theISareshownin Fig. 4(A), (B) and(C), respectively.

It wasalsonotedthat the SC-68328bis-isothiocyanatederivative was unstable under acidic conditions. Thederivative was convertedinto its correspondingacetate,formateor trifluoroacetatederivativewhen1%aceticacid,1% formic acid or 1% trifluoroaceticacid, respectively,was used in the mobile phase.Sheathliquid additionimproved the sensitivity approximatelyfivefold with a

Copyright 2000JohnWiley & Sons,Ltd. J. MassSpectrom. 35, 354–360 (2000)

358 J. Y. ZHANG ET AL.

Figure 4. Representative MRM chromatograms of (A) control plasma, (B) plasma spiked with the internal standard (10 µg ml�1) and(C) plasma spiked with both SC-68328 (0.25 µg ml�1) and the internal standard (10 µg ml�1). Injection volume: 20 µl.

10 µl min�1 sheathflow of methanol–water(80: 20, v/v)containing 2 mM ammoniumthiocyanate.The responsedecreasedwhen the flow-rate of the sheathliquid wasincreasedto over 50 µl min�1.

Initial standardand QC samplesthat werepreparedatroom temperaturewithin 1 h indicatedthat the linearityof the calibration curve and the analytical recoveriesof the QC sampleswere not satisfactory.Therefore,allstandardandQCsampleswerepreparedwith ice-colddogplasmawithin 5 min and were quickly frozen in a dryice–acetonebathandstoredin a�70°C freezeruntil use.The plasmasampleswerethawedin ice-coldwaterusinga microwaveovento achieverapid thawingof theplasmasamples.8 All stepsof the samplepreparationprior toliquid–liquid extractionwereconductedon ice to preventsampledegradation.

Linearity, precisionand accuracy

Calibrationcurvesgeneratedacceptabledataoverthecon-centrationrange0.250–10.00 µg ml�1 SC-68328in dogplasma.A full calibrationcurvecontainingeightstandardsyielded acceptableresultsand was usedfor all calcula-tions. A typical calibrationcurve for SC-68328obtainedusing extracted dog plasma standardsis presentedinFig. 5. The correlationcoefficient of the weightedstan-dard curve was 0.999. The calibration curves obtainedas describedabovewere suitablefor generatingaccept-able data for the concentrationsof SC-68328in the QCsamplesfor the validation.

Assay precision and accuracy were assessedusingthree QC samples at concentrationsof 0.25 µg ml�1

(lower limit of quantitation(LOQ)), 1.00 µg ml�1 (middle

Copyright 2000JohnWiley & Sons,Ltd. J. MassSpectrom. 35, 354–360 (2000)

FLOW INJECTION AND MS/MS DETERMINATION OF SC-68328 IN DOG PLASMA 359

Figure 5. A typical SC-68328 dog plasma calibration curve.

Table 1. Intra-day and inter-day precisionand accuracyof theFI/ESI-MS/MS assayfor SC-68328in dog plasma

Spikedconcentration No. of Precision (RSD (%)) Accuracy (AR (%))(µg ml�1) Day analyses Inter-day Inter-day Inter-day Inter-day

0.25 1 4 13.6 1082 4 11.0 1113 4 13.5 11.8 119 113

1.0 1 4 7.07 84.42 4 2.52 83.23 4 11.5 7.71 85.9 84.5

8.0 1 4 2.02 98.72 4 2.14 99.23 4 4.38 3.46 103 100

100a 1 4 1.58 1102 4 2.12 1053 4 2.89 3.32 103 106

a Twenty-fold dilution of 100 µg ml�1 samples with blank dogplasma.

quality control (MQC)) and 8.00 µg ml�1 (upperqualitycontrol (UQC)), and using a 20-fold dilution sampleatconcentrationof 100 µg ml�1. The data are presentedin Table1. For the inter-day validation, the precision(expressedas relative standarddeviation (RSD)) was11.8%,andtheaccuracy(expressedasanalyticalrecovery(AR)) was113%for LOQ. TheRSDsfor MQC andUQCwere7.71and3.46%,respectively,andtheARswere84.5and100%,respectively.Theupperconcentrationlimit canbe extended,with acceptableprecisionand accuracy,to100 µg ml�1 by utilizing a 20-fold dilution with controldog plasma.The RSD and AR for the dilution QC were3.32and106%,respectively.For the intra-dayvalidation,theRSDswerein therange11.0–13.6%andtheARswerein the range108–119% for LOQ. The RSDs for MQCandUQC werein the ranges2.52–11.5and2.02–4.38%,respectively,and the ARs were in the ranges83.2–85.9and98.7–103%,respectively.TheRSDsandARs for thedilution QCwerein theranges1.58–2.89and103–110%,

respectively.Thesedata indicate that the assaydemon-stratedacceptableinter-day and intra-day precisionandaccuracyduring the validation.

Storagestability

The samplestoragestability at �70°C was evaluatedtodeterminethe storageconditions for dog samples.Dogplasmasamplesspikedwith SC-68328at concentrationsof 0.25,0.3, 1 and8 µg ml�1 wereanalyzedon day one.After storageat�70°C for 32days,thesampleswerethenre-analyzedagainsta freshly madecalibrationcurve.Theresultsindicatedthat SC-68328wasstablefor at least32dayswhen storedfrozenat �70°C. The RSDsand ARson day 32 werein the ranges4.34–18.8and85.3–105%,respectively(Table2).

Application

Four anesthetizedmalemongreldogswereintravenouslyadministereda bolus of 3 mg kg�1 SC-68328followedby an infusion of 3 mg kg�1 h�1 SC-68328for 30 min.Approximately3 ml of blood werecollectedat appropri-ate time intervalsand1 ml of plasmawasharvestedandstoredin a �70°C freezer.The meanplasmaconcentra-tion–time profile of SC-68328is shown in Fig. 6. Themeanmaximum plasmaconcentrationof SC-68328was22.7 µg ml�1 at 5 min post-dose.SC-68328was rapidly

Table 2. Resultsfor fr ozenstoragestability of samplesof SC-68328at −70◦C

Spiked Mean calculatedconcentration concentration Storage No. of RSD AR(µg ml�1) (µg ml�1) (days) replicates (%) (%)

0.25 0.262 32 7 18.8 1050.30 0.266 32 7 15.2 88.51 0.853 32 6 10.6 85.38 7.80 32 6 4.34 97.5

Figure 6. Mean plasma SC-68328 concentration time profilefollowing intravenous administration of 3 mg kg�1 bolus plus3 mg kg�1 h�1 infusion for 30 min to four mongrel dogs. Barsrepresent standard deviations.

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360 J. Y. ZHANG ET AL.

eliminated from plasma after completion of the 30 mininfusion. The plasma concentration of SC-68328 reachedthe assay sensitivity of 0.25 µg ml�1 at ¾90 min post-dose.

CONCLUSIONS

An accurate and reliable FI/ESI-MS/MS assay wasdeveloped using a bis-isothiocyanate derivatization andliquid–liquid extraction technique to decrease polarity

and improve the ionization and detection by ESI-MS/MS.This assay has proven simple, robust and amenableto high-throughput analysis. The assay is the firstreported analytical method to accurately measure unstablemanganese-based SOD mimetics in biological fluids andwas successfully applied to the determination of SC-68328 in dog plasma. During the assay development, aseries of very challenging problems for the manganese-based complex, such as stability and extraction capability,were resolved. Therefore, this assay may provide insightsfor developing analytical methods for other metal- ormanganese-based complexes.

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Copyright 2000 John Wiley & Sons, Ltd. J. Mass Spectrom. 35, 354–360 (2000)