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Analytical Biochemistry 297, 15–24 (2001)doi:10.1006/abio.2001.5317, available online at http://www.idealibrary.com on

High-Performance Liquid Chromatographic Technique forthe Simultaneous Determination of Lactone and HydroxyAcid Forms of Camptothecin and SN-38 in Tissue CultureMedia and Cancer Cells

Gary Boyd, John F. Smyth, Duncan I. Jodrell, and Jeffrey Cummings1

Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital,Edinburgh, EH4 2XU, United Kingdom

Received March 28, 2001; published online August 31, 2001

Analysis of camptothecins in biologic media is ham-pered by chemical hydrolysis of the parent lactone(form I) to an inactive hydroxy acid (form II). A solid-phase extraction (SPE) method utilizing C2-bondedsilica particles (100 mg, 1 ml) is presented for simulta-neous determination of forms I and II of camptothecin(CPT) and SN-38 (active metabolite of clinically usedCPT-11) in culture media and cell lysates. A new HPLCseparation is described that efficiently resolves allfour compounds employing gradient elution with 10mM ammonium acetate, increasing methanol (20–80%over 15 min), and a 15-cm by 3-mm Symmetry Shield(RP8) column. Components were detected by fluores-cence at an excitation wavelength of 380 nm and emis-sion wavelength of 423 nm. Lactones were shown to beunstable at alkaline pH and hydroxy acids unstable atalkaline pH while the following conditions preservedthe chemical equilibrium in specimens: samples kepton ice, final pH of eluates 7.4, autosampler tempera-ture 4°C, and analysis cycle <4 h. Quantitative recov-ry of lactones was achieved from RPMI culture me-ium over a wide concentration range (93.5–111.6% for–400 ng/ml) although greater variability was notedith the hydroxy acids (59.6–110.3%, 1–400 ng/ml).imit of quantitation (precision and accuracy <20%)as 0.2 ng/ml for CPT lactone, 0.5 ng/ml for SN-38

actone, and 2 ng/ml for the two hydroxy acids. Theethod was applied to quantitate the accumulation of

N-38 and CPT (form I and II) in HT29 and HCT116uman colon cancer cells. © 2001 Academic Press

1

To whom reprint requests and correspondence should be ad-dressed. Fax: 144 131 332 8494. E-mail: j.cummings@icrf.icnet.uk.

0003-2697/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

Key Words: camptothecin; SN-38; HPLC; solid-phaseextraction; colon cancer cells.

Originally isolated from the Chinese tree Camptoth-eca acuminata, 20(S) camptothecin (CPT2; see Fig. 1) isa pentacyclic, highly unsaturated natural product mol-ecule related to the indole alkaloids (1). The drug ex-hibits a unique mechanism of action by inhibiting thenuclear enzyme Topoisomerase I (topo I) resulting inthe formation of a ternary complex (the cleavable com-plex) between CPT, topo I, and DNA (2). Structure–activity relationships (SAR) studies have demon-strated a strict requirement for an intact lactonesystem in the E ring of CPT with the 20-hydroxyl groupin the R position for the efficient inhibition of topo I (3).Recent crystallographic studies with a reconstitutedform of human topo I in complex with a 22-base-pairDNA duplex provided the molecular basis for the ear-lier SAR studies, showing that both these functionalgroups are necessary to participate in key hydrogenbonding interactions (4, 5). Initial clinical trials as ananticancer drug were performed with the sodium saltof the lactone ring opened hydrolysis product of CPT(the hydroxy acid form, see Fig. 1), since this product iswater-soluble whereas the lactone is not, but the drugwas demonstrated to be inactive in this formulation(1).

2 Abbreviations used: SPE, solid phase extraction; CPT, campto-thecin; topo I, topoisomerase I; SAR, structure–activity relationship;DMSO, dimethylsulfoxide; EI, electron impact ionization; LOD, limit

of detection; PA, precision and accuracy; LOQ, limit of quantitation;NA, not available; ND, not detected.

15

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a

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16 BOYD ET AL.

A number of analogs of CPT are currently undergo-ing clinical evaluation as potential anticancer drugs (6,7). CPT-11 (Irinotecan, Campto) is a promising, water-soluble analog of CPT with proven clinical activityagainst human colon cancer (8, 9). CPT-11 is a pro-drug for the active, non-water-soluble metaboliteSN-38 (see Fig. 1) produced in vivo by the action of

biquitously distributed carboxylesterases (10–12).N-38 can undergo metabolism by glucuronidation athe free hydroxyl group at C10, which is then elimi-ated by biliary excretion (13–15). At least two furtheretabolites of CPT-11 (referred to as APC and NPC)ave been identified as a result of microsomal metab-lism catalyzed by cytochrome P450 CYP3A (16).In order to study the cellular pharmacology of CPT-

ased compounds meaningfully, it is essential to pre-erve the chemical equilibrium between the lactonend hydroxy acid and quantitate each form separately.he high in vitro potency of camptothecin-related com-ounds (CPT and SN-38) (17, 18) dictates that cellsave to be exposed to low drug concentrations (1 mM) to

FIG. 1. Molecular structures of the lactone (form I) and hydroxycid (form II) forms of camptothecin (CPT) and SN-38.

TAB

Simultaneous High-Performance Liquid Chromatogrof Camptothecin-Ba

Study CompoundIonpair

Extractiontechnique

ivory and Robert, 1994 SN-38 Yes Nob

oos et al., 1996 Topotecan Yes Nob

i and Zhang, 1996 10-OHCPT

Yes Nob

aneda et al., 1997 SN-38 Yes Nob

arner and Burke, 1997 SN-38 Yes Nob

hollet et al., 1998 SN-38 Yes Nob

Kurita and Kaneda, 1999 SN-38 Yes SPEPresent study SN-38

(and CPT)No SPE

a HA, hydroxy acid.b

Plasma protein precipitation method employed.c NA, not available; ND, not detected.

maintain viability during drug transport studies (19).Radiolabeled compounds are thus employed in order toprovide the sensitivity to detect drug intracellularly(19, 20). Though this approach provides adequate sen-sitivity it does not discriminate between the differentchemical forms or detect the presence of further me-tabolites in the case of SN-38. Due to the paucity ofpreviously published HPLC methods sufficiently sen-sitive or relevant to the analysis of cancer cells in vitro(see Table 1), in the present study a new solid phaseextraction (SPE) sample preparation technique is re-ported specifically adapted for the simultaneous deter-mination of the lactone and hydroxy acid forms of CPTand SN-38 in tissue culture media and cancer celllysates/sonicates. A simplified HPLC separation is alsoreported that does not require addition of ion pairingagents or high ionic strength buffers normally neces-sary additions to improve peak symmetry of the nega-tively charged hydroxy acids (Table 1). Extensive sta-bility and validation studies were performed to supportthe new SPE method and identify optimal conditions topreserve the equilibrium between the lactone and hy-droxy acid in biologic specimens during sample prepa-ration.

MATERIALS AND METHODS

Chemicals

CPT (lactone, 95% purity) was obtained from SigmaChemical Co. (Poole, UK). SN-38 lactone was a kindgift of Rhone Poulenc Rorer (Vitry-sur-Seine, France).All other chemicals, reagents, and solvents were thehighest grade available commercially and were used asreceived. Water was deionized in a MilliQ water puri-fication system (Millipore, Watford, UK). Standard lac-tone solutions (100 mg/ml) were made up and diluted in

1

ic Analysis of the Lactone and Hydroxy Acid FormsAnticancer Drugs

LOQ lactone(ng/ml)

LOQ HAa

(ng/ml)Glucuronide

detectedAdaptedfor cells Ref

2 2 No 240.1 0.1 No No 212 2 No No 38

5 5 No No 28NAc NA No No 22NA NA No No 275 NDc Yes No 340.5 2 Yes Yes

LE

aphsed

m

u

17CAMPTOTHECINS ANALYSIS IN CANCER CELLS

dimethylsulfoxide (DMSO) and stored at 24°C for notore than 1 week. Standard hydroxy acid solutions (50

mg/ml) were made up and diluted in 0.13 M Tris buffer(pH 10.8) and were always prepared freshly.

High-Performance Liquid Chromatography

The liquid chromatograph used throughout con-sisted of an Alliance 2690 separations module, a 996photodiode array detector, a 486 fluorescence detector,and a Thermabeam mass detector (particle beam in-terface, electron impact (EI) ionization) all from Wa-ters Ltd. (Watford, UK). Full system control, data col-lection, analysis, and reporting were performed usingMillennium Software (revision 2.21, Waters) operatingon a Pentium processor personal computer. In addi-tion, the Wiley Registry of Mass Spectral data libraryof EI mass spectra containing in excess of 220,000compounds was installed on the personal computer andwas fully searchable through the Millennium softwareinterface. The stationary phase was Symmetry Shield(RP8) 5-mm particle size packed in a 15-cm-long by3-mm-internal-diameter stainless steel analytical col-umn and 2-cm-long by 3.8-mm-diameter stainless steelpre-column (Waters). The mobile phase comprised 10mM ammonium acetate as buffer A and methanol assolvent B. Gradient elution was employed according tothe following linear program: time zero, 20% solvent B;15 min, 80% solvent B; and 17 min, 20% solvent B.Flow rate was 0.35 ml/min, total run time was 23 min,and column temperature was maintained at 40°Cwhile the autosampler was set at 4°C. Mobile phasecomponents were filtered prior to use and vacuum de-gassed in situ during chromatography. Injection vol-

me was 20 or 50 ml. The photodiode array detectorwas set to record signals at 367 nm (CPT and CPT-11)and 380 nm (SN-38) and spectra between 200 and 600nm. The fluorescence detector was set at an excitationwavelength of 380 nm and emission wavelength of 423nm. Interface parameters of the Thermabeam massdetector were as follows: source temperature (ion vol-ume), 200°C; nebulizer temperature, 80°C; expansionregion temperature, 85°C. Ion optics including ion vol-ume (ionization), extractor lens, the prequadrupolemass filter, exit lens, and electron multiplier were op-timized each day after tuning the system with a per-fluorotributylamine calibration gas. Spectra acquisi-tion rate was 1 per second, gain was set at 10, and themass scan range was 100–550.

Sample Preparation

Preparation of cancer cell lysates/sonicates. TheHT29 human colon cancer cell line was obtained fromATCC (Rockville, MD, USA) and the HCT116 human

colon carcinoma cell line was obtained from ECACC

(European Collection of Cell Culture, Salisbury, UK).Cells were cultured in RPMI 1640 tissue culture mediasupplemented with 5% heat-inactivated fetal calf se-rum containing a 1% antibiotic mixture under stan-dard conditions and maintained at 37°C in a humidi-fied atmosphere of 5% CO2 in air. In drug transportstudies, cells were grown in 12.5 cm2 plastic tissueculture flasks (Falcon, 25 ml volume polystyrene tissueculture flask, Becton Dickinson, Franklin Lakes, NJ)at a density of approximately 106 cells per flask in avolume of 4 ml of media. At the start of each incuba-tion, media was removed and replaced with fresh me-dia containing freshly made up SN-38 and CPT at afinal concentration of 1 mM: 392 and 348 ng/ml, respec-tively. At the end of each incubation the 4 ml of mediawas immediately transferred to a plastic bijou samplecontainer and placed in ice. To minimize sample pro-cessing times and delays before HPLC analysis, onlythree replicate flasks were included per time point andtime points were well spaced: 5 min, 1 h, 4 h, 6 h, and24 h.

Cell monolayers were washed twice with 4 ml ofice-cold phosphate-buffered saline, lysed by the addi-tion of 2 ml of ice-cold water and then mechanicallydetached from the plastic by scraping with the additionof a further 1 ml of ice-cold water. Efficacy of the celllysis protocol was confirmed using a particle size ana-lyzer (Model ZM, Coulter Electronics, Luton, England).Finally, the lysed cell suspensions (3 ml) were sub-jected to further disruption by two brief (15 s) periodsof sonication while being maintained at 0°C in ice. Thecell lysates/sonicates were stored in plastic bijou sam-ple containers placed in ice prior to solid phase extrac-tion. Both media and cell samples were processed asquickly as possible to prevent in situ conversion be-tween the two chemical forms of both compounds.

Solid-phase extraction. Biological specimens (1 mlfor media, 3 ml for cell sonicates) were processed bySPE using C2 bonded 40-mm silica particles packed in1-ml-capacity mini-columns containing 100 mg of sor-bent operating under negative pressure (Varian Asso-ciates supplied by Phenomenex, Macclesfield, UK).Mini-columns were activated with 1 ml of methanoland conditioned with 1 ml of 50 mM Tris–HCl (pH 7.4).Samples were then loaded on to the columns, whichwere then washed with 2 ml of 50 mM Tris–HCl (pH7.4). The compounds of interest were finally eluted in400 ml of methanol:1 M ammonium acetate (90:10, v:v).

Stability Studies

Stability of SN-38 and CPT hydroxy acids. Stocksolutions of 50 mg/ml hydroxy acid (nominal concentra-tion) were generated by treatment of the parent druglactones with alkali (0.1 M sodium hydroxide) and

evaporated to dryness under reduced pressure. The

pbH

V

mdtscl4laatarw

wcuTdhtB5

A

d

c

wtc

18 BOYD ET AL.

resulting hydroxy acid was then resuspended in a va-riety of buffers and solvents: methanol; ammonia(30%):methanol (1:20, v:v); 0.13 M Tris–HCl, pH 7.0,and 0.13 M Tris base, pH 10.8. The solutions weretransferred to amber HPLC vials and placed in thedark in the HPLC, set at 20°C. Aliquots (20 ml) of thesesolutions were then injected over a period of up to 24 hfor the determination of percentage lactone and hy-droxy acid content.

A further stability study was performed modeled onthe above protocol where the hydroxy acids were recon-stituted in methanol:1 M ammonium acetate (90:10),the eluting solution from the SPE method in whichsamples were stored for up to 2 h prior to injection oncolumn. These studies were carried out at 4°C as wellas 20°C and were performed in triplicate.

Stability of SN-38 and CPT lactones. The effect ofpH and temperature on SN-38 and CPT lactones wasevaluated in amber autosampler vials as above over24 h. Drug concentrations were 25 mg/ml, the autosam-

ler temperature was set at either 4 or 20°C, anduffer (50 mM) pH was 5.0 (citrate), 7.4, or 9.0 (Tris–Cl).

alidation

Emphasis was placed on the ability of the SPEethod to recover intact both the lactone and the hy-

roxy acid forms of CPT and SN-38 with minimal in-erconversion. Extracted standard curves were con-tructed with the compounds added to RMPI mediaontaining the supplements described above at the fol-owing concentration range of 1, 10, 50, 100, 200, and00 ng/ml. Stock solutions were prepared in DMSO foractones and 0.13 M Tris base, pH 10.8, for hydroxycids. Media were placed in ice prior to addition of drugnd analyzed as quickly as possible. Recovery (extrac-ion efficiency) was calculated for each form against theppropriate nonextracted standard curve. Within-dayecovery (n 5 5) and between-day recovery (n 5 3–5)ere both calculated.Accuracy and precision of the assay were determinedith freshly prepared six-point extracted calibration

urves employing the same range of concentrationstilized above in the recovery studies (1–400 ng/ml).he quality control samples were prepared indepen-ently from separate stock solutions of lactone andydroxy acid and batches of media and extended overhe full concentration range of the calibration curves.etween-day validation runs were performed on up toseparate days.

nalysis of Tissue Culture Media and Cell Sonicates

The cellular accumulation of CPT and SN-38 was

etermined in HT29 and HCT116 cells cultured in 12.5

m2 plates over 24 h after exposure to 1 mM drug, asdescribed under Preparation of Cancer Cell Lysates/Sonicates. Both media and cell samples were analyzedfrom three replicate flasks. Separate 12.5 cm2 flasks

ere utilized to count cells at different time pointshroughout drug treatment and intracellular drug con-entrations were normalized to ng/106 cells.

RESULTS AND DISCUSSION

High-Performance Liquid Chromatography

Adoption of the Symmetry Shield column facilitatedthe use of a low ionic strength mobile phase whileachieving high efficiency separation of CPT and SN-38lactones and hydroxy acids without peak broadeningand tailing often observed with hydroxy acids (21–23)(see Table 1 and Fig. 2). This enabled the chromato-graphic method to be interfaced with mass spectrome-try in order to validate chemical identity of chromato-

FIG. 2. Reversed-phase HPLC chromatograms of standard solu-tions of the lactone (form I) and hydroxy acid (form II) forms of SN-38(chromatogram A) and camptothecin (CPT, chromatogram B) to-gether with the C10-O-glucuronide of SN-38 (chromatogram C). Con-centrations of each peak were nominally 1 mg/ml for the parent drugsand 0.5 mg/ml for the glucuronide. Chromatographic details arereported under Materials and Methods.

graphic peaks (see Table 2). Electron impact (EI)

cRltlpTst1

ettwsrwnefsasoicisflttt

lp

tw

C

an

19CAMPTOTHECINS ANALYSIS IN CANCER CELLS

ionization spectra were generated for both the lactonesand hydroxy acids of SN-38 and CPT, producing mo-lecular ions for the lactones and interpretable fragmen-tation patterns for all four species. The mass spectra ofthe lactones yielded a match factor of .97% whenompared to those obtained from the Wiley Library.etention times were extremely stable for all four ana-

ytes of interest, varying by less than 1% throughouthe day. Column performance was maintained at thisevel for at least 4 months and column-to-column re-roducibility in retention times varied by less than 2%.he chromatographic separation also efficiently re-olved the C10-O-glucuronide of SN-38; the major me-abolite of SN-38 normally generated in the liver (13–5) (Fig. 2).SN-38 and CPT exhibit different fluorescence prop-

rties with maxima of 370 nm versus 378 nm for exci-ation and 423 nm versus 530 nm for emission, respec-ively (24–26). In order to avoid problems associatedith wavelength changes mid run, such as drift and

piking of the baseline, potentially compromising accu-ate quantitation of peaks (27), a single excitationavelength of 380 nm and emission wavelength of 423m was adopted for both compounds. The choice of themission wavelength of 423 nm, which appears not toavor SN-38, was supported by the fact that its emis-ion spectrum does shift dramatically from 530 nm inqueous solutions to under 450 nm in the nonpolarolvents used in the present study (25). Lactone ringpening does not significantly affect fluorescence max-ma but can affect fluorescence intensity (25). In thease of SN-38 hydroxy acid an increase in fluorescencentensity was observed, whereas a reduction in inten-ity occurred with CPT hydroxy acid, and this is re-ected in different limit of detection (LOD) values forhe two chemical forms. The wavelengths adopted inhe present work thus resulted in high-sensitivity de-

TAB

Chromatographic Character

Compound tR (min) 6 CV

LC/MS of thechromatograph

ic peak

PT-lactone 14.8 6 0.6% M1, 348

CPT-hydroxy acid 10.1 6 1.0% M1-H2O, 348

SN-38-lactone 16.3 6 0.2% M1, 392

SN-38-hydroxy acid 11.4 6 0.7% M1-H2O,392

a LOD, limit of detection at a signal to noise ratio of 3.b LOQ, limit of quantitation at a precision and accuracy of less th

ection of all four species without the need for wave-

ength switching, equaling or in most cases betteringreviously published methods (see Tables 1 and 2).Over the concentration range of interest nonex-

racted and extracted calibration curves were linearith correlation coefficients (r 2) better than 0.998 for

the lactones of CPT and SN-38 (Table 2).

Stability Studies

Incubation of the lactone parent drug of CPT andSN-38 with various different buffers was investigatedwith the aim of generating the hydroxy acid in highyield, following its stability and then eventually study-ing its analytical behavior during sample preparation.Reconstitution of SN-38 in 0.13 M Tris base, pH 10.8,resulted in 93% conversion to the hydroxy acid with nosignificant degradation or interconversion to lactoneover 24 h at 20°C (Fig. 3). Ammonia was less effectivethan Tris buffer in converting the lactone into hydroxyacid, never achieving greater than 90% transforma-tion, although once formed the hydroxy acid was stableover 24 h. Significantly, the hydroxy acid of SN-38 wasnot stable in pure methanol, reverting back to thelactone by up to 75% after 24 h incubation at 20°C. Inprevious HPLC methods where simultaneous determi-nation of lactone and hydroxy acid forms in plasma wasachieved, ice-cold methanol was used as a sample prep-aration technique to precipitate proteins (21, 22, 27,28). Due to the instability of the hydroxy acid in meth-anol, extra care must be observed in order to analyzesamples as quickly as possible, since after only 4 h at20°C as much as 50% of the hydroxy acid content mayartifactually interconvert to lactone. Indeed, the hy-droxy acid of the closely related CPT analog, topotecan,has been shown to be unstable in methanolic plasmaextracts at 4°C requiring storage at 280°C to preservestability (21). Nonetheless, the hydroxy acid was dem-onstrated to be more stable in 90% methanol:10% 1 M

2

cs of the New HPLC Assay

Extracted calibrationcurves (1–400 ng/ml)

LODa

(3:1, S/N)(pg)

LOQb

(PA , 20%)(ng/ml)

y 5 15820x 2 10487 1 0.2r 2 5 0.999y 5 3697x 2 561 4 2r 2 5 0.9823y 5 5105x 2 2425 3 0.5r 2 5 1.000y 5 10361x 2 336213 1 2r 2 5 0.998

20%.

LE

isti

ammonium acetate, the eluting solution used in the

oiw

Evsatte

s ut

20 BOYD ET AL.

present study as the final stage of the sample prepa-ration technique. Here, no significant loss of hydroxyacid occurred after 4 h at 4°C (data not shown).

CPT and SN-38 lactones are highly unstable in aque-us solutions at temperatures above 27°C degradingnto the hydroxy acid via pseudo-first-order kineticsith half lives of less than 1 h at pH 7.4 (29, 30).

FIG. 3. Incubation of 50 mg/ml SN-38 lactone with various differpreserving its stability. At various different time points the amoundescribed under Materials and Methods. The data are presented as thTris buffer, pH 10.8, proved to be the most effective buffer and wavalidation of the new analytical methodology developed.

FIG. 4. Stability of CPT and SN-38 lactones in aqueous media. 25the autosampler at 4°C and the concentration of lactone and hydrox

are presented as the proportion of the total concentration represented bwere stable at 4°C for up to 6 h.

valuation of kinetic variables, in these studies, re-ealed that pH largely determined the equilibrium po-ition of lactone to hydroxy acid with essentially noffect due to concentration. Figure 4 illustrates thathe stability of CPT and SN-38 lactones can be sus-ained for up to 6 h by chilling samples to 4°C or less,ven at physiological pH. Thus, maintaining biological

buffers at 20°C with the aim of generating the hydroxy acid andlactone versus hydroxy acid present was quantitated by HPLC as

ercentage of the total concentration represented by the hydroxy acid.ilized in the present study to generate hydroxy acid standards for

/ml of both compounds were made up fresh in HPLC vials placed incid determined by HPLC at various different time points. The data

entt ofe p

mgy a

y the lactone. At physiological pH it was shown that both lactones

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21CAMPTOTHECINS ANALYSIS IN CANCER CELLS

specimens in ice during sample preparation and stor-ing SPE eluants in autosampler vials at 4°C prior toanalysis is recommended to preserve the ratio of lac-tone to hydroxy acid provided samples are analyzedwithin 4 h.

Sample Preparation Technique

Table 3 demonstrates that the SPE technique pre-sented achieved quantitative recovery of the lactones ofboth CPT (global mean over the concentration range of1–400 ng/ml of 99.6 6 10.1%) and SN-38 (global mean,98.0 6 5.9%) from tissue culture media. A greater levelof variability in recovery was observed with the hy-droxy acids and extraction efficiencies were generallyless than 100% with a global mean of 81.0 6 16.9% forCPT and 77.3 6 9.0% for SN-38. The reasons for thisatter result are probably twofold. First, since the hy-roxy acids are considerably more water-soluble thanhe lactones and carry a full negative charge, theiretention on the C2 SPE column may be less complete.econd, stock solutions of the hydroxy acids had to beenerated from the lactone parent drugs and this pro-edure, which is based on an equilibrium process (30),annot easily generate 100% yields. Previous methodshere simultaneous determinations of lactones andydroxy acids of camptothecins have been achieved doot employ an extraction step (21, 22, 27, 28). Onlyrganic solvent extractions or SPE have been employedo selectively recover the lactone from plasma (31, 32).ven here variable recoveries of SN-38 and CPT lac-

ones have been reported (33). However, in the presenttudy where quantitative recovery of the lactones waschieved employing SPE, tissue culture media was in-estigated which only contains 5% serum. Lower ex-raction efficiencies similar to (33) were observed whenhe present SPE method was applied to whole plasmaamples.Analysis of several batches of media samples over a

TAB

Percentage Recovery of Camptothecin (CPT) Lactone and HStandards from RPMI Tissue Culture M

Concentration(ng/ml)

CPT LactoneC

betWithina day Betweenb day

1 87.6 6 9.0 93.5 6 12.6 69.10 102 6 9.4 69.50 99.6 6 10.3 76.

100 90.0 6 4.3 102.3 6 12.4 93.200 100.2 6 7.5 83.400 98.9 6 6.9 99.8 6 7.9 93.

a Data represent the mean 6 standard deviation from n 5 5 replb Data represent the mean 6 standard deviation from n 5 3–5 re

eriod of 2 years confirmed an absence of interfering

eaks. Minor endogenous peaks were detected in cellysate/sonicate samples when operating at high detec-or sensitivity. However, these peaks appeared consis-ently and did not interfere with identification of theour compounds of interest (see Fig. 5).

The SPE method also detected the 10-O-glucuronideetabolite of SN-38 in HT29 human colon cancer cells

ut not HCT116 human colon cancer cells cell (andedia samples) (Fig. 5). Purification and confirmation

f the structure of this metabolite by LC/MS are re-orted separately (Cummings et al., submitted for pub-ication). Based on the known spectral characteristicsf this metabolite (13) and its chromatographic behav-or, it was clear that the SPE method extracted this

etabolite with efficiency comparable to that of theydroxy acid of SN-38.

ethod Validation and Sample Analysis

Precision and accuracy (PA) for CPT lactone nevereviated by more than 7% from the nominal valuesver the concentration of 1–400 ng/ml while PA variedy even less for SN-38 lactone over the concentration of0–400 ng/ml (Table 4). However, there was a signif-cant increase in PA of SN-38 lactone at 1 ng/ml, lim-ting the limit of quantitation (LOQ) for this compoundo 0.5 ng/ml. Acceptable PA (,15%) was recorded withhe hydroxy acids of both CPT and SN-38 over theoncentration range of 10–400 ng/ml, while unaccept-ble results were recorded at 1 ng/ml, limiting the LOQor both these compounds to 2 ng/ml (see Table 2).evertheless, quantitation limits exceeded several pre-iously published methods by one order of magnitudeue to the innovation of the SPE method (see Table 1)24, 28, 34).

Applying the new methodology to the determinationf the cellular accumulation of CPT in HT29 humanolon cancer cells revealed rapid uptake to a peakactone level of 5.61 ng/106 cells measured after 5 min,

3

roxy acid (HA) and SN-38 Lactone and Hydroxy acid (HA)dia after Solid Phase Extraction (SPE)

HAn day

SN-38 LactoneSN-38 HA

between dayWithin day Between day

24.4 72.4 6 1.1 90.9 6 11.2 110.3 6 20.724.3 111.6 6 14.0 60.9 6 1.73.6 97.8 6 1.9 59.6 6 0.219.6 92.0 6 0.30 95.4 6 2.3 59.8 6 0.421.4 96.5 6 2.3 87.2 6 16.47.7 98.9 6 0.74 95.8 6 3.9 86.2 6 14.6

es.cates.

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5 64 69 64 64 64 6

similar to the rapid uptake behavior of CPT related

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22 BOYD ET AL.

compounds reported in normal colon cells (35). A valueof 1.57 ng/106 cells was detected at 1 h and this com-pares with a value of 1.23 ng/106 cells reported for the

ptake of CPT in HT29 cells after a 1-h exposure de-ermined utilizing radiolabeled drug (19). After theapid uptake of drug, the intracellular concentration ofPT lactone decreased to a level of 0.84 ng/106 cells at

4 h, which was maintained over 24 h (0.98 ng/106 cells),corresponding to the lactone reaching its equilibriumlevel with the hydroxy acid in tissue culture media(data not shown). Only nominal intracellular concen-trations of hydroxy acid were detected (,0.20 ng/106

cells), despite this form being the major species present

FIG. 5. Analysis of SN-38 cellular accumulation in human colon caby a combination of hypoosmotic shock with ice-cold water and sonidetermined in ng/106 cells were as follows: HCT116 (human colon ca

ydroxy acid, 0.49; HCT116 at 24 h, SN-38 lactone, 1.65, and SN-38 hactone, 3.13, and SN-38 glucuronide, 1.52.

TAB

Between-Day Precision and Accuracy of the

Concentration(ng/ml)

CPT lactonea CPT hydroxy

Accuracy Precision Accuracy P

1 106.4 2.7 133.110 101.3 2.6 106.350 101.6 4.2 88.9

100 97.0 6.8 105.4200 98.7 2.9 92.1400 99.0 0.62 91.6

a Validation runs were performed on up to 5 separate days.

in media after 4 h, confirming its limited ability tocross the biomembrane without facilitated transport(35–37).

In summary, a new SPE sample preparation tech-nique is reported for the simultaneous determinationof the lactone (biologically active) and hydroxy acidforms (inactive) of CPT and SN-38 in tissue culturemedia and cancer cell lysates/sonicates. Sample prep-aration conditions were optimized to preserve thechemical equilibrium of the lactone and hydroxy acidforms of both compounds in biologic specimens. A sim-plified HPLC method is also reported that does notrequire addition of ion pairing agents or high ionic

r cells after treatment with 1 mM drug (392 ng/ml). Cells were lysedion prior to SPE using C2 bonded silica and HPLC. Concentrationsr) cells after 5 min drug incubation, SN-38 lactone, 3.17, and SN-38oxy acid, 1.84; and HT29 (human colon cancer) cells at 5 min, SN-38

4

PLC Assay in RPMI Tissue Culture Media

d SN-38 lactone SN-38 hydroxy acid

sion Accuracy Precision Accuracy Precision

.7 119.9 16.4 155.2 35.1

.8 99.2 2.6 115.1 10.1

.8 98.6 1.1 110.1 6.6

.8 99.7 0.26 104.5 6.9

.3 100.4 0.82 99.7 4.4

.2 101.7 2.2 95.8 3.7

ncecatnce

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192

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23CAMPTOTHECINS ANALYSIS IN CANCER CELLS

strength buffers to achieve high efficiency separation ofthe lactones and hydroxy acids. The SPE methodachieves quantitative recovery of the lactones and ac-ceptable recovery of the hydroxy acids from culturemedia. Good precision and accuracy enabled the limitof quantitation to be set at 0.2 ng/ml for CPT lactone,0.5 ng/ml for SN-38 lactone, and 2 ng/ml for CPT andSN-38 hydroxy acids. The new methodology was ap-plied to determine the accumulation of CPT and SN-38in HT29 and HCT116 human colon cancer cells over24 h after treatment with 1 mM drug.

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