High-performance liquid chromatographic method for the simultaneous determination of the camptothecin derivative irinotecan hydrochloride, CPT-11, and its metabolites SN-38 and SN-38 glucuronide in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT

We established a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the camptothecin (CPT) derivative, irinotecan hydrochloride (CPT-11) and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT. Plasma samples were pretreated with 0.146 M H₃PO₄ to inactivate carboxylesterase and β-glucuronidase in rat plasma, and added with the internal standard solution (0.146 M H₃PO₄ containing 1 μg/ml CPT) and then analyzed. The method was validated for CPT-11 (5 to 25 000 ng/ml), SN-38 (5 to 2500 ng/ml) and SN-38G (2.5 to 500 ng/ml). This method enabled the determination of many samples within a relatively short time with easy sample preparation. It also had four advantages compared with conventional determination methods, i.e. automation of a complicated sample preparation, time-saving by the simultaneous determination of three compounds, the direct determination of SN-38G, and the small amount of plasma required for the determination.     

High-performance liquid chromatographic assay with fluorescence detection for the simultaneous measurement of carboxylate and lactone forms of irinotecan and three metabolites in human plasma

Irinotecan (CPT-11), a camptothecin analog, is metabolized to SN-38, an active topoisomerase I inhibitor, and inactive metabolites, including APC and SN-38 glucuronide (SN-38G). A high-performance liquid chromatographic assay method to simultaneously measure the lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in human plasma was developed. Chromatography was accomplished with a reversed-phase C(8) column and fluorescence detection. A gradient mobile phase system was used. The buffer for mobile phase A consisted of 0.75 M ammonium acetate, 5 mM tetrabutylammonium phosphate (pH 6.0), and acetonitrile (86:14, v/v). The buffer for mobile phase B was identical to mobile phase A with the exception of the concentration (50:50, v/v). Precipitation of plasma proteins was performed with cold methanol. The linear range of detection of the lactone and carboxylate forms of SN-38, SN-38G, and APC was 2-25 ng/ml, and 5-300 ng/ml for CPT-11. The limit of quantitation for the analytes ranged from 0.5 to 5 ng/ml. Analysis of patients' plasma samples obtained before and after CPT-11 administration showed that the assay is suitable for measuring lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in clinical studies.     

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile-50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm x 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9-108.3% for CPT-11 in culture media and 94.3-107.2% for CPT-11 in cell lysates; and 87.7-106.8% for SN-38 in culture media and 90.1-105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.     

Simultaneous determination of the lactone and carboxylate forms of irinotecan (CPT-11) and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in the rat

Irinotecan (CPT-11) and its main metabolite SN-38 are potent anticancer derivatives of camptothecin (CPT), with active lactone and inactive carboxylate forms coexisting. A simple and sensitive HPLC method using the ion-pairing reagent tetrabutylammonium hydrogen sulfate (TBAHS) was developed to simultaneously determine all four analytes in rat plasma samples. Camptothecin (CPT) was used as internal standard. The mobile phase was 0.1M potassium dihydrogen phosphate containing 0.01 M TBAHS (pH 6.4)-acetonitrile (75:25, v/v). Separation of the compounds was carried out on a Hypersil C18 column, monitored at 540 nm (excitation wavelength at 380 nm). All four compounds gave linear response as a function of concentration over 0.01-10 microM. The limit of quantitation in rat plasma was 0.01, 0.008, 0.005 and 0.005 microM for CPT-11 lactone, CPT-11 carboxylate, SN-38 lactone and SN-38 carboxylate, respectively. The method was successfully used in the study on the effect of coadministered thalidomide on the plasma pharmacokinetics of CPT-11 and SN-38 in rats. Coadministered thalidomide (100mg/kg body weight by intraperitoneal injection) significantly increased the AUC(0-10h) values of CPT-11 lactone and CPT-11 carboxylate by 32.6% and 30.3 %, respectively, (P < 0.01), but decreased the values by 19.2% and 32.4% for SN-38 lactone and carboxylate, respectively, (P < 0.05). Accordingly, the value of total body clearance (CL) of CPT-11 lactone was significantly lower in combination group compared to the control (1.329 versus 1.837 L/h/kg, P = 0.0002). Plasma t(1/2beta) values for SN-38 lactone and carboxylate were significantly (P < 0.01) smaller in rats with coadministered thalidomide, as compared to rats receiving CPT-11 alone. Further studies are needed to explore the underlying mechanisms for the observed kinetic interaction between CPT-11 and thalidomide.     

High-performance liquid chromatographic determination of irinotecan (CPT-11) and its active metabolite (SN-38) in human plasma

A simplified method for the simultaneous determination of irinotecan (CPT-11, I) and its active metabolite (SN-38, II) in human plasma by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. Following the addition of the internal standard (I.S.) camptothecin, the drugs were extracted from plasma using methanol. The average extraction efficiencies were 87% for I, 90% for II and 90% for the I.S. Chromatography was performed using a TSK gel ODS-80Ts column, monitored at 556 nm (excitation wavelength, 380 nm) and the mobile phase was acetonitrile-50 mM disodium hydrogen phosphate (28:72) containing 5 mM heptanesulphonate (pH 3.0). The linear quantitation ranges for I and II were 30–2000 and 1–30 ng/ml, respectively.     

High-performance liquid chromatographic technique for the simultaneous determination of lactone and hydroxy acid forms of camptothecin and SN-38 in tissue culture media and cancer cells

Analysis of camptothecins in biologic media is hampered by chemical hydrolysis of the parent lactone (form I) to an inactive hydroxy acid (form II). A solid-phase extraction (SPE) method utilizing C2-bonded silica particles (100 mg, 1 ml) is presented for simultaneous determination of forms I and II of camptothecin (CPT) and SN-38 (active metabolite of clinically used CPT-11) in culture media and cell lysates. A new HPLC separation is described that efficiently resolves all four compounds employing gradient elution with 10 mM ammonium acetate, increasing methanol (20-80% over 15 min), and a 15-cm by 3-mm Symmetry Shield (RP8) column. Components were detected by fluorescence at an excitation wavelength of 380 nm and emission wavelength of 423 nm. Lactones were shown to be unstable at alkaline pH and hydroxy acids unstable at alkaline pH while the following conditions preserved the chemical equilibrium in specimens: samples kept on ice, final pH of eluates 7.4, autosampler temperature 4 degrees C, and analysis cycle <4 h. Quantitative recovery of lactones was achieved from RPMI culture medium over a wide concentration range (93.5-111.6% for 1-400 ng/ml) although greater variability was noted with the hydroxy acids (59.6-110.3%, 1-400 ng/ml). Limit of quantitation (precision and accuracy <20%) was 0.2 ng/ml for CPT lactone, 0.5 ng/ml for SN-38 lactone, and 2 ng/ml for the two hydroxy acids. The method was applied to quantitate the accumulation of SN-38 and CPT (form I and II) in HT29 and HCT116 human colon cancer cells.     

High-performance liquid chromatographic determination of furosemide in plasma and urine and its use in bioavailability studies

A sensitive, selective and efficient reversed-phase high-performance liquid chromatographic (HPLC) method is reported for the determination of furosemide in human plasma and urine. The method has a sensitivity limit of 5 ng/ml in plasma, with acceptable within- and between-day reproducibilities and good linearity (r²>0.99) over a concentration range from 0.05 to 2.00 μg/ml. The one-step extract of furosemide and the internal standard (warfarin) from acidified plasma or urine was eluted through a μBondapak C₁₈ column with a mobile phase composed of 0.01 M potassium dihydrogenphosphate and acetonitrile (62:38, v/v) adjusted to pH 3.0. Within-day coefficients of variation (C.V.s) ranged from 1.08 to 8.63% for plasma and from 2.52 to 3.10% for urine, whereas between-day C.V.s ranged from 4.25 to 10.77% for plasma and from 5.15 to 6.81% for urine at three different concentrations. The minimum quantifiable concentration of furosemide was determined to be 5 ng/ml. The HPLC method described has the capability of rapid and reproducible measurement of low levels of furosemide in small amounts of plasma and urine. This method was utilized in bioavailability/pharmacokinetic studies for the routine monitoring of furosemide levels in adults, children and neonate patients.     

High-performance liquid chromatographic determination of oxodipine enantiomers, a new 1,4-dihydropyridine, applied to stereoselectivity studies in man and dog

A specific and reproducible HPLC method using a Chiral-AGP column and UV detection was developed for the evaluation of the pharmacokinetic profile of oxodipine enantiomers in dog and man. Each enantiomer was determined in plasma in the concentration range 1–400 ng/ml using the internal standard calibration method with linear regression analysis. After extraction of oxodipine and the internal standard at alkaline pH with diethyl ether—n-hexane (50:50, v/v), this method permitted the determination of each enantiomer at levels down to 10 ng/ml in dog plasma and 25 ng/ml in human plasma with sufficient accuracy (relative error <11%, n = 6) and precision (coefficient of variation <16%, n = 6). The extracted plasma volume was 500 μl and after evaporation of the organic phase, the dry residue was dissolved in 100 μl of water—2-propanol; an aliquot of 80 μl was injected into the HPLC system.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.     

A liquid chromatography/electrospray ionization mass spectrometry (LC-MS/MS) assay for the determination of irinotecan (CPT-11) and its two major metabolites in human liver microsomal incubations and human plasma samples

A sensitive, rapid LC-MS/MS assay has been developed and validated for the simultaneous quantification of CPT-11 and its two principal metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38), and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxy-camptothecin (APC) in human liver microsomal fractions and plasma. The method was linear over the ranges of 1.56-100 ng/mL, 3.13-150 ng/mL, and 0.78-100 ng/mL for CPT-11, SN-38, and APC, respectively. The total run time was 7.0 min. This assay offers advantages in terms of expediency, recovery of analytes, and suitability for the analysis of CPT-11 and its metabolites in various biological fluids.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients

CPT-11 {I; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin} is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C₁₈) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C₁₈ reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml–10 μg/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5–1000 ng/ml) was 13.0% (range 4.9–19.4%) for I and 12.8% (6.7–19.1%) for II; the between-day R.S.D. (5–10 000 ng/ml was 7.9% (5.4–17.5%) for I and 9.7% (3.5–15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m² I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 ± 285 ng/ml (mean ± standard error of the mean). Plasma decay was triphasic with half-lives α, β and γ of 5.4 ± 1.8 min, 2.5 ± 0.5 h and 20.2 ± 4.6 h, respectively. The volume of distribution at steady state was 105 ± 15 l/m², and the total body clearance was 12.5 ± 1.9 l/h · m². The maximum concentrations of the active metabolite II reached 36 ± 11 ng/ml.     

The Study of Polyethyleneglycol-Coated Liposomes Containing CPT-11

Abstract

Polyethyleneglycol (PEG) -coated liposomal CPT-11 (PEG-LCPT(11)) was prepared and its pharmaceutical usefulness was examined. These liposomes, plain liposomal CPT-11 (PLCPT(11)) and PEG-LCPT(11), were composed of dimyristoylphosphatidylcholine, cholesterol, and dimyristoylphosphatidylglycerol (10 : 10 : 6, mol/mol) with or without PEG. The mean particle diameters were both about 1 60 nm. The trapping efficiencies were approximately 90%. In a distribution study, CDFl mice were injected with CPT-11 solution (CPT(11)sol), PLCPT(11) and PEG-LCPT(11) at a dose of 10 mg/kg (i.v.). Concentrations in each tissue of CPT-11 and SN-38, the active metabolite of CPT-11, were determined. After the administration, CPT-11 and SN-38 concentrations in the blood increased by liposomal encapsulation (liposomalization), and the circulation time in the blood was prolonged further by PEG-modification of the liposomes (PEGylation). In the liver, PLCPT(11) was rapidly taken up by the reticuloendothelial system (RES), and the uptake was avoided by PEGylation. Tumor accumulations of CPT-11 and SN-38 were accompanied by an increase in antitumor activity of CPT-11 by liposomalization. Thus, the prolongation of the circulation time in the blood by liposomalization and the avoidance of the RES uptake by PEGylation caused passive targeting of the tumor, with a resulting increase in the antitumor activity of CPT-11.     

Preparation of Irinotecan-Loaded Folate-Targeted Liposome for Tumor Targeting Delivery and Its Antitumor Activity

The purpose of this study was to investigate the in vivo distribution and antitumor activity of irinotecan (camptothecin (CPT)-11)-loaded folate-targeted liposome (F-Lip) in tumor-bearing mice following i.v. administration. Folate–poly(ethylene glycol)–distearoylphosphatidylcholine (FA–PEG–DSPE) was synthesized by amide reaction of DSPE–PEG–NH₂ and FA. F-Lip modified by FA–PEG–DSPE was prepared by an ammonium sulfate gradient. The mean particle size and entrapment efficiency of F-Lip with negative charge were 197.8 ± 4.58 nm and 91.39 ± 2.34 %, respectively. The distributions of CPT-11 and SN-38 in the tumor after i.v. administration of F-Lip, CPT-11-loaded liposomes (C-Lip), and CPT-11 injection (C-Inj) were far greater with the F-Lip group in comparison to the C-Inj and C-Lip, which might contribute to folate-meditated targeting uptake by the folate receptor on the surface of the tumor cells. The uptake of CPT-11 in the liver and rectum for two liposome groups were all markedly increased as compared to the C-Inj. Moreover, F-Lip exhibited a dose-dependent tumor growth inhibition and superior anticancer activity to C-Lip and C-Inj after i.v. administration. It also showed no significant body weight loss and much lower toxicity on the center immune organs. Therefore, F-Lip may be presented as potential candidates for tumor targeting drug delivery.KEY WORDS: cancer targeting, CPT-11, folate, liposomes, SN-38     

High-performance liquid chromatographic analysis for the determination of a novel polymer-bound camptothecin derivative (MAG-camptothecin) and free camptothecin in human plasma

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C₈ analytical column, using a mixture of acetonitrile–25 mM phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.     

Food-drug interaction of (-)-epigallocatechin-3-gallate on the pharmacokinetics of irinotecan and the metabolite SN-38

The aim of the present study was to investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on the pharmacokinetics of irinotecan (CPT-11) and its metabolite SN-38. EGCG was potentially used to modulate the ATPase activity of P-glycoprotein (P-gp). Experimental Sprague-Dawley rats were treated with EGCG (20mg/kg, i.v.) 10min before CPT-11 (10mg/kg, i.v.) administration, whereas the control group received CPT-11 (10mg/kg, i.v.) only. The biological samples were prepared by the protein precipitation and detected by HPLC-fluorescence detection which provided a good separation of CPT-11 and SN-38 within 10min. The pharmacokinetic data indicate that the area under the plasma concentration-time curves (AUC) of CPT-11 and SN-38 were increased by 57.7 and 18.3%, and AUC in bile were decreased by 15.8 and 46.8%, respectively, for the group pretreated with EGCG. The blood to bile distribution ratio (AUC(bile)/AUC(blood)) was significantly reduced after group coadministration of EGCG, it can be seen that the bile efflux transport system of CPT-11 and SN-38 may be markedly reduced by the treatment of EGCG which plays the role of P-gp inhibitor. In conclusion, EGCG was found to inhibit the transport of CPT-11 and SN-38 into the biliary elimination and their half-lives in plasma could be substantially prolonged. Based on the food-drug interaction, persons taking daily nutritional supplements should be warned of this interaction possibility.     

Sensitive assay for midazolam and its metabolite 1′-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1′-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 μm capillary C₁₈ column (150 mm×0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer–acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer–acetonitrile, 40:60. The flow-rate of the mobile phase was 16 μl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1′-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1′-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 μg/kg).     

Determination by liquid chromatography with fluorescence detection of total 7-ethyl-10-hydroxy-camptothecin (SN-38) in beagle dog plasma after intravenous administration of liposome-based SN-38 (LE-SN38)

An HPLC- fluorescence method to quantitate total 7-ethyl-10-hydroxy-camptothecin (SN-38) in beagle dog plasma spiked with liposome based formulation of SN-38 (LE-SN38) and using camptothecin (CPT) as the internal standard (I.S.) was developed and validated to support pharmacokinetics/toxicokinetics studies. Sample preparation was done by protein precipitation using acetonitrile with 0.5% acetic acid. The supernatant was evaporated, and reconstituted in acetonitrile-20 mM ammonium acetate, pH 3.5 (20:80, v/v). When injected onto a Zorbax SB-C(18) HPLC column SN-38 as well as I.S. were detected by fluorescence using an excitation at 368 nm and emission at 515 nm. The SN-38 concentrations in samples were calculated from a standard curve of peak area ratios of SN-38 to the I.S. using weighted linear regression. The sensitivity limit for SN-38 was 1.00 ng/ml in beagle dog plasma with a precision (expressed as relative standard deviation) of 12.4% and an accuracy (expressed as analytical recovery) of 104%. The assay was linear within the standard curve range of 1-750 ng/ml. Acceptable precision and accuracy were also obtained for concentrations over the balance of the standard curve range from between-run and within-run calculations.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Determination of irinotecan (CPT-11) and SN-38 in human whole blood and red blood cells by liquid chromatography with fluorescence detection

An analytical method was developed for the anticancer agent irinotecan (CPT-11) and its main metabolite SN-38 in human whole blood and in red blood cells (RBCs). Sample pretreatment involved deproteinization of whole blood or plasma-diluted RBCs isolated by MESED instruments, with a mixture of aqueous perchloric acid and methanol (1:1, v/v). Separation was carried out using isocratic elution on a Hypersil ODS stationary phase, with detection at excitation and emission wavelengths of 355 and 515 nm, respectively. The lower limit of quantitation (LLQ) in blood was established at 5.00 ng/ml for both compounds, with values for within-run precision (WRP) and between-run precision (BRP) of less than 10%. The method is currently being applied to investigate the blood distribution of CPT-11 and SN-38 in cancer patients.     

Trace analysis of SN-38 in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C₁₈ column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10–500 pM (3.9–195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of ‘trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.