A Robust HPLC Approach for Quantitation of Camptothecin in Mesoporous Silica Nanoparticles Matrix and in the Presence of Its Degradation Products.

BACKGROUND: Camptothecin is a potent anticancer drug used for the treatment of various cancers. OBJECTIVE: The goal of this research investigation was to develop and validate a new stability-indicating HPLC technique for the quantitative assessment of camptothecin in in-house developed mesoporous silica nanoparticles, a novel nanoformulation matrix for the treatment of cancer. METHOD: The Waters Inertsil® HPLC column (C18) was used for the chromatographic separation, with a flow rate of 1 mL/min, a column oven temperature of 40°C, an injection volume of 10 µL, a detection wavelength of 216 nm, and a 10 min runtime overall. An isocratic blend of phosphate buffer (10 mM, pH7.0) and acetonitrile (60:40, v/v) served as the mobile phase. Various stress conditions including acid, alkali, oxidative, photolytic, thermal, and humidity environments were tested for the quantitative estimation of the camptothecin through the proposed method. RESULTS: The results demonstrated that the proposed method is specific (peak purity ≥0.999), accurate (99.69-100.64% w/w), precise (RSD, % <2.0), and sensitive (LOD-0.17 µg and LOQ-0.56 µg) in accordance with ICH guideline Q2 (R1). Any unidentified degradation products did not interfere with the drug's estimation. Furthermore, the current method of analysis has eliminated any excipient interference from the matrix effect caused by the numerous excipients of the formulation matrix. CONCLUSIONS: To quantify camptothecin for routine assay purposes, this research work offers a novel and straightforward HPLC methodology with optimized chromatographic parameters, contributing to the research and development community while ensuring an appropriate and efficient use of the drug through a variety of nanoformulation for cancer treatment. HIGHLIGHTS: The stability-indicating HPLC method was found to be specific and suitable for routine analysis of camptothecin. The absence of any interference from excipients was confirmed by forced degradation studies.

Biotechnology for micropropagation and camptothecin production in Ophiorrhiza sp.

Camptothecin (CPT) is a monoterpenoid-alkaloid, an anticancer compound from plant. Ever since its discovery in 1996 from the bark of Camptotheca acuminata, various researches have been conducted for enhancing its production. CPT has also been reported in several other species belonging to the plant families Icacinaceae, Rubiaceae, Apocynaceae, Nyssaceae, Betulaceae, Violaceae, Meliaceae, and Gelseminaceae. Out of these, Ophiorrhiza sp. (Rubiaceae) is the next possible candidate for sustainable CPT production after C. acuminata and Nothapodytes nimoonia. Various biotechnological-studies have been conducted on Ophiorrhiza sp. for searching the elite species and the most optimal strategies for CPT production. The genus Ophiorrhiza has been used as medicines for antiviral, antifungal, antimalarial, and anticancer activities. Phytochemical analysis has revealed the presence of alkaloids, flavonoids, triterpenes, and CPT from the plant. Because of the presence of CPT and its herbaceous habit, Ophiorrhiza sp. has now become a hot topic in research area. Currently, for mass production of the elite spp., tissue culture techniques have been implemented. In the past decades, several researchers have contributed on the diversity assessment, phytochemical analysis, mass production, and in vitro production of CPT in Ophiorrhiza sp. In this paper, we review the on the biotechnological strategies, optimal culture medium, micropropagation of Ophiorrhiza sp., effect of PGR on shoot formation, rhizogenesis, callus formation, and enhanced production of CPT for commercial use. KEY POINTS: • Latest literature on in vitro propagation of Ophiorrhiza sp. • Biotechnological production of camptothecin and related compounds • Optimization, elicitation, and transgenic studies in Ophiorrhiza sp.

One-step electrodeposition preparation of polyaniline/f-MWCNTs as electrochemical sensors for detection of 10-hydroxycamptothecine.

In our work, one-step electro-deposition method was adopted to produce polyaniline (PANI) and functional multiwalled carbon nanotubes (f-MWCNTs) films on glass carbon electrodes, and the modified electrodes were applied as an electrochemical sensor for determination of 10-hydroxycamptothecine (10-HCPT). The f-MWCNTs were handled by ultrasound processing in concentrated oxidizing acid solution, which can obtain a wonderful dissolution in water and attach new functional groups, such as -COOH and -OH. Then, aniline monomer could polymerize on the surface easily. The surface characterization was investigated using various techniques including scanning electron microscope, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction, and electro-catalytic properties were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under optimal conditions, the resulting of PANI/f-MWCNTs sensor showed a wide linear range (3 × 10-9 to 7 × 10-7 mol L-1 ) and a low detection limit (1 × 10-9 mol L-1 ), which is attributing to its large special surface area and good conductivity. Moreover, the modified electrodes are convenient to fabricate, which can be used to detect 10-HCPT in urine samples successfully.

Biosynthesis-inspired mining and identification of untapped alkaloids in Camptotheca acuminate for enzyme discovery using ultra-high performance liquid chromatography coupled with quadrupole-time of flight-mass spectrometry.

Leaves, flowers, fruits and stems (44 sample groups) were collected from mature Camptotheca acuminate during 2017.3-2018.3 and classified by ultra-high performance liquid chromatography coupled with quadrupole-time of flight-mass spectrometry based metabolomics. One hundred metabolites including forty-seven alkaloids, fifteen terpenes, thirty-two polyphenols and six other metabolites were rapidly identified through the in-house database alignment at first glance. Thirty-three alkaloids classified into five groups including camptothecin group (CG1-13), pumiloside group (PG1-5), strictosidinic acid group (SG1-3), vincosamide group (VG1-7), and a new hybrid group, vincosamide-camptothecin group (VC1-5) were mined and further characterized by MS/MS analyses. The identification of two untapped biosynthetic precursors, 2-hydroxypumiloside (PG2) and 16­hydroxy­15, 16-dihydrocamptothecoside (CG3), along with sixteen new alkaloids enables us for a better understanding of camptothecin biogenetic reasoning. The underlying enzymes involved in camptothecin biosynthesis were also proposed according to the guiding metabolic map, thus purposefully mining of enzymes involved in the downstream biosynthetic pathway of camptothecin could be initiated with the help of this map.

The development and validation of an LC-MS/MS method for the quantification of CZ112, a prodrug of 9-Nitrocamptothecin in rat plasma.

9-Nitrocamptothecin-20-O-propionate (CZ112) and 9-Nitrocamptothecin (9NC) are the bioactive derivatives of camptothecin (CPT), an alkaloid isolated from Camptotheca acuminata, and have been confirmed to possess high anti-cancer properties. In the present study, 9NC was identified as the major metabolite of CZ112 in rat plasma through HPLC/photodiode array detection (PDA) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A highly sensitive LC-MS/MS method was developed and validated for the simultaneous analysis of CZ112 and 9NC in rat plasma, and camptothecin-20-O-acetate (CZ44) was used as an internal standard (IS). The calibration curves were linear (r2 > 0.999) over concentrations from 2.5 to 320 ng/mL for both CZ112 and 9NC. The method had an accuracy of 96.7-109.6%, and the intra- and inter-day precision (RSD%) were 10.9% or less for CZ112 and 9NC. The stability data showed no significant degradation occurred under the experimental conditions. This method was successfully applied to the pharmacokinetic study of CZ112 and its metabolite 9NC in rat plasma after intravenous and intragastric administration. The oral bioavailability of CZ112 was 6.2 ±â€¯3.3% (n = 6).

Multifunctional Gold Nanocluster Decorated Metal-Organic Framework for Real-Time Monitoring of Targeted Drug Delivery and Quantitative Evaluation of Cellular Therapeutic Response.

Therapeutic drug monitoring is central to optimize therapeutic efficacy and minimize adverse events; however, the pharmacokinetics and pharmacodynamics of most drugs differ greatly among individuals. Evaluation of the therapeutic response is in urgent need to help clinician predict the clinical dose of drug. Herein, we described an analytical assay using gold nanocluster (AuNC)-decorated metal-organic frameworks (MOFs) for targeted drug delivery and monitoring pharmacodynamics, giving attractive options for studying individualized therapy. The camptothecin (Cam) anticancer drug was caged in the NH2-MIL-101(Fe) MOFs. Modification with pegylated folate (FA) for specifically recognizing a FA receptor on the cancer cell membrane significantly improved the delivery efficiency. Intercellular delivery of Cam initiated programmed death of cells and upregulated the apoptosis indicator (caspase-3), which cleaved the peptide linker between AuNCs and MOFs. The quenched fluorescence of AuNCs was then recovered once the peptide was enzymatically cleaved by caspase-3. Real-time monitoring of targeted drug delivery was achieved by imaging of the light-up fluorescence in HepG2 cells, while the amount of caspase-3 could be quantified by detecting Au in the released AuNCs in inductively coupled plasma mass spectrometry (ICP-MS) with a limit of detection (LOD) of 0.12 ng mL-1. Our assay emphasizes the application of multifunctional nanomaterial for therapeutic self-monitoring and quantitative evaluation of therapeutic response, allowing the acceleration of drug evaluation.

Development of an enzyme­linked immunosorbent assay for camptothecin.

The use of camptothecin and its analogues has increased in clinical settings and in agriculture. Therefore, camptothecins and their derivatives, metabolites and degradation products are frequently found in the environment. Therefore, it is important to develop an ELISA for the quantification of camptothecins in human plasma, plants, animal tissues and other matrices. The present study developed a novel competitive indirect ELISA for camptothecin using a monoclonal antibody (MAb). In total, two haptens and various carrier proteins were tested to select the most suitable immunogen for the production of MAbs against camptothecin. Hapten 1 conjugated with keyhole limpet hemocyanin was selected for the preparation of MAb 5A3, and was used to establish a competitive indirect ELISA for camptothecin. A total of three derivatives of camptothecin used in clinical practice were examined. Topotecan showed an IC50 value of 0.68 µg/ml with a detection limit of 0.19 µg/ml, belotecan showed an IC50 value of 0.87 µg/ml with a detection limit of 0.22 µg/ml and irinotecan showed an IC50 value of 2.85 µg/ml with a detection limit of 0.47 µg/ml. The cross­reactivity results suggested that the assay developed in the present study possessed a high sensitivity to camptothecin. Therefore, this immunoassay technique may be suitable for monitoring the levels of camptothecin in compound analysis, clinical applications, and analyses of food and environmental samples.

Expanding the "minimalist" small molecule tagging approach to different bioactive compounds.

"Minimalist" small molecule tagging (MSMT) is a promising approach that easily converts bioactive compounds into affinity-based probes (AfBPs) for proteomic studies. In this work, seven bioactive compounds targeting diversified protein classes were installed with "minimalist" linkers through common reactions to generate the corresponding AfBPs. These probes were evaluated for cell-based protein profiling and target validation. Among them, the entinostat-derived probe EN and the camptothecin-derived probe CA were further utilized in cellular imaging and SILAC-based large-scale target identification. Our extensive studies suggest that the "minimalist" small molecule tagging approach could be expanded to different classes of bioactive compounds for modification into AfBPs as a dual functional tool for both proteomics and cellular imaging.

Practical fluorimetric assay for the detection of anticancer drug SN-38 in human plasma.

The implementation of therapeutic drug monitoring in the routine clinical practice in oncology is mainly limited by the lack of therapeutic indexes for the majority of the anticancer drugs, and by the absence of suitable analytical tools, which can accurately quantify in real time the concentration of the administered drugs and their relevant metabolites in biological fluids. In this work, a simple and efficient fluorimetric determination of SN-38, the active metabolite of the anticancer drug irinotecan, was developed and applied to human plasma samples. The intrinsic fluorescence of SN-38 allowed its quantification in the range 10-500 ng mL-1 with a LOQ of 5.0 ng mL-1 and a LOD of 1.5 ng mL-1. Low interferences due to main metabolites of irinotecan and comedications, commonly associated with administration of irinotecan, were observed. A validation study, according to FDA and EMA guidelines for bioanalytical method validation, was carried out and, finally, blind samples were analyzed in parallel with a HPLC-MS method obtaining an excellent agreement between the two techniques.

Carboxylesterase and UDP-glucuronosyltransferases mediated metabolism of irinotecan: In vitro and in vivo insights from quantitative ultra-performance liquid chromatography-mass spectrometry analysis.

Carboxylesterase and UDP-glucuronosyltransferase-mediated metabolism of irinotecan (CPT-11) has long been proposed to be responsible for its anti-tumor activity and toxicity, like delayed-onset diarrhea. However, recent studies failed to gain more comprehensive in vivo and in vitro pharmacokinetic profiles of irinotecan. Herein, we use rat plasma, human liver microsomes and immortalized HepG2 cell as experimental subjects to describe a sensitive and versatile UHPLC-MS/MS method for simultaneously quantifying CPT-11 and its metabolites, including SN-38 and SN-38G. The method was applied to investigate the pharmacokinetic and metabolic behavior of CPT-11 in the biological samples. Calibration curves for all bio-matrices showed acceptable linearity (r2 > 0.99). The intra- and inter-day precisions (RSD, %) were within 15% and the excellent accuracy (RE) was between 2.96 and 14.12%. In addition, the specificity, matrix effect and extraction recovery all met the requirements of biological sample analysis. We successfully applied this method to investigate the pharmacokinetics of irinotecan in various biological samples, mediated by carboxylesterase and UDP-glucuronosyltransferase. This method could be employed in monitoring the metabolic status and clinical efficacy of irinotecan in the future.

Simultaneous separation and analysis of camptothecin alkaloids in real samples by large-volume sample stacking in capillary electrophoresis.

Large-volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether-ß-cyclodextrin and 20 mm ionic liquid 1-ethyl-3-methyllimidazole l-lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 µg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.

One-step targeted accumulation and detection of camptothecin analogues from fruits of Camptotheca acuminata Decne using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry.

Camptothecins, a kind of monoterpene-quinoline alkaloids from Camptotheca acuminata Decne, have long attracted much attention worldwide as an anti-cancer drug. However, there is still a lack of effective methods for the accumulation and discovery of camptothecin analogues from botanic resources for camptothecin-based drug research. This work develops a one-step method for the targeted accumulation, quick detection, and identification of camptothecin analogues from C. acuminata fruit using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (bilayer-SPE-UHPLC-Q-TOF-MS/MS). The bilayer-SPE cartridge, with polyamide (PA) as the upper layer and octadecyl silane (ODS) as the lower layer, was designed for the removal of flavonoid and ellagic acid impurities and the enrichment of camptothecins for further MS analysis. Subsequently, the mass spectrometry fragmentations, especially multistage retro-Diels-Alder cleavage, were summarized based on the MS/MS data of 10 reference camptothecins. The UHPLC-Q-TOF-MS/MS conditions were optimized, and the MS/MS data of the potential camptothecin analogues in the bilayer-SPE enriched fractions were analyzed. A total of 30 camptothecin analogues, including 15 new compounds, were identified from the fruit according the fragmentation pathways of the reference standards. The proposed structure of peak 20 was confirmed using its NMR data through rapid enrichment and purification. Overall, the bilayer-SPE enrichment and reliable mass spectrometry fragmentation in our work could provide an effective and simple method for the exploration of the biosynthesis pathway and metabolomics of camptothecin analogues.

A high-throughput chemical screen identifies novel inhibitors and enhancers of anti-inflammatory functions of the glucocorticoid receptor.

Glucocorticoids (GCs)-ligands of the glucocorticoid receptor (GR)-are widely used to treat inflammatory diseases, but suffer from significant side effects and poor responsiveness in certain patient populations. Identification of chemical GR modulators may provide insights into the regulatory mechanisms of anti-inflammatory functions of GR and help improve GC-based therapy. Here we report the development and application of a high-throughput screening to identify compounds that either enhance or suppress the anti-inflammatory effect of GR function. Using a cell-based GR activity assay that measures Dexamethasone (Dex)-mediated NF-κB repression, we have screened ~8,000 compounds and identified several compounds that suppressed GR activity, including multiple GSK3ß inhibitors and anti-cancer agent camptothecin. Notably, we also identified two kinase IKK2 inhibitors, including TPCA-1, as GR enhancers that improve the anti-inflammatory effect of GR. In particular, TPCA-1 augmented the activity of Dex in NF-κB repression by attenuating GR down-regulation. Consistent with the observation, siRNA-mediated IKK2 knockdown decreased GR down-regulation and increased GR expression. Together, our results identified chemical compounds as novel modulators of GR and revealed an unexpected role for IKK2 in GR down-regulation. Furthermore, we have established a high-throughput screening platform for discovering GR-modulating compounds that may be repurposed to improve current GC-based therapies.

Ionic Liquid Dispersive Liquid-Liquid Microextraction Method for the Determination of Irinotecan, an Anticancer Drug, in Water and Urine Samples Using UV-Vis Spectrophotometry.

A new, simple, efficient, and environmentally friendly ionic liquid dispersive liquid-liquid microextraction method was developed for the determination of irinotecan, an anticancer drug, in water and urine samples using UV-Vis spectrophotometry. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate was used as the extraction solvent, and ethanol was used as the disperser solvent. The main parameters affecting the extraction efficiency, including sample pH, volume of the ionic liquid, choice of the dispersive solvent and its volume, concentration of NaCl, and extraction and centrifugation times, were investigated and optimized. The effect of interfering species on the recovery of irinotecan was also examined. Under optimal conditions, the LOD (3σ) was 48.7 µg/L without any preconcentration. Because the urine sample was diluted 10-fold, the LOD for urine would be 487 µg/L. However, this could be improved 16-fold if preconcentration using a 40 mL aliquot of the sample is used. The proposed method was successfully applied to the determination of irinotecan in tap water, river water, and urine samples spiked with 10.20 mg/L for the water samples and 8.32 mg/L for the urine sample. The average recovery values of irinotecan determined were 99.1% for tap water, 109.4% for river water, and 96.1% for urine.

Irinotecan binds to the internal cavity of beta-lactoglobulin: A multi-spectroscopic and computational investigation.

The binding of irinotecan, a potent anti cancer drug, to bovine beta-lactoglobulin (ß-LG) was investigated by various spectroscopic techniques including fluorimetry, circular dichroism (CD), UV-vis, and Fourier transform infrared (FT-IR), in 10mM phosphate buffer, pH 7.75, in combination with a molecular docking study. Analysis of the fluorescence quenching data showed that combined static and dynamic quenching occurs, with the predominant contribution of the static mode. Molecular docking results were in full agreement with the results obtained from thermodynamic analysis of the fluorescence data indicating the existence of one binding site for irinotecan in ß-LG structure and revealed the hydrophobic nature of the interaction between irinotecan and the protein. The binding distance between ß-LG and irinotecan, r, was estimated to be 5.74nm based on the Förster's theory of non-radiative energy transfer. The obtained results of near-UV CD and FT-IR experiments suggested the occurrence of partial compactness of the protein structure upon irinotecan binding. Based on the experimental data and the possible binding mode revealed by molecular docking study, we concluded that irinotecan binds to the hydrophobic calyx of ß-LG with induction of some alterations in the secondary and tertiary structure of the protein.

Enhanced production of camptothecin and biological preparation of N 1-acetylkynuramine in Camptotheca acuminata cell suspension cultures.

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ∼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.

A theranostic prodrug based on FRET for real-time drug release monitoring in response to biothiols.

The clinical applications of tradition prodrug are restricted to a certain extent due to its some defects such as side effects, low selectivity and single function. Herein, we designed a theranostic strategy based on the fluorescence resonance energy transfer (FRET) mechanism. The theranostic prodrug was constructed with an anticancer drug camptothecin (CPT), a cleavable linker based on disulfide bonds (SS) and a fluorophore naphthalimide derivative (NAP). For this drug delivery and reporting system (NAP-SS-CPT), FRET occurs between CPT (energy donor) and NAP (energy receptor); and upon cellular uptake by GSH-overexpressing cancer cells, disulfide bonds could be successfully cleaved by the high concentration of GSH, and FRET process was interrupted to achieve dual fluorescence response and specifically release of CPT. The drug delivery system features some favorable properties, like excellent pH-stability, high selectivity and cytotoxicity towards GSH-overexpressing cells and relatively lower cytotoxicity towards normal cells. Fluorescence analysis reveals that NAP-SS-CPT shows a ratiometric fluorescence signal under excitation at 400nm for quantitative detection of GSH. Intracellular fluorescence imaging studies indicate that the prodrug can be efficiently internalized in HeLa cells and used for real-time monitoring of drug release. Moreover, MTT and flow cytometry assay indicate that NAP-SS-CPT exhibits high pro-apoptotic effect for cancer cells. This strategy may provide a new approach for cancer diagnosis and therapy.

Combined use of ionic liquid and sulfobutylether-ß-cyclodextrin as electrolyte additives for separation and determination of camptothecin alkaloids by CZE.

This work reported that ionic liquid (IL) ([Bmim] [PF6 ]) and sulfobutylether-ß-CD (SBE-ß-CD) were used as electrolyte additives for the separation and determination of camptothecin (CPT) alkaloids by CZE. Separation parameters such as the buffer type, pH, and concentration of the running buffer, the concentration of SBE-ß-CD and IL, temperature, and separation voltage were all investigated in order to achieve the maximum possible resolution. The four analytes were baseline separated within 10 min in capillary at the separation voltage of 15 kV with a running buffer consisting of 20 mM borate buffer, 20 mM IL, and 100 mM SBE-ß-CD at pH 9.0. Under such conditions, good linearity about two orders of magnitudes of peak areas was achieved for the investigated CPT alkaloids with the correlation coefficients ranging from 0.9946 to 0.9985. For all analytes, detection limits (S/N = 3) and quantitation limits (S/N = 10) range from 0.05 to 0.92 µg/mL and 0.17 to 3.06 µg/mL, respectively. The proposed method has not only been successfully applied to the separation and determination of CPT alkaloids but also showed that IL seemed to be a promising additive in CZE separation.

Detection of low-quantity anticancer drugs by surface-enhanced Raman scattering.

Ultrasensitive detection of low-quantity drugs is important for personalized therapeutic approaches in several diseases and, in particular, for cancer treatment. In this field, surface-enhanced Raman scattering (SERS) can be very useful for its ability to precisely identify analytes from their unique vibrational spectra, with very high sensitivity. Here, we report a study about SERS detection of sunitinib, paclitaxel and irinotecan, i.e. three commonly used antineoplastic drugs, and of SN-38, i.e. the metabolite of irinotecan, dissolved in methanol solutions. By using commercial Klarite substrates, we found that sunitinib, irinotecan and SN-38 have detection limits of 20-70 ng, which is below the threshold for applications in cancer therapy. Conversely, the SERS signal was not appreciable with paclitaxel, and this is explained by the absence of optical resonances in the visible range. Overall, our results show that ultrasensitive SERS detection of sunitinib, irinotecan and SN-38 is feasible, encouraging further development of this technology also for other drugs with similar molecular structure especially for those analytes with absorption bands in the visible range.

Development and validation of an UPLC-MS/MS method for the quantification of irinotecan, SN-38 and SN-38 glucuronide in plasma, urine, feces, liver and kidney: Application to a pharmacokinetic study of irinotecan in rats.

The objective of this research is to develop and validate a sensitive and reproducible UPLC-MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88-10000 nM, 39-5000 nM, 48.8-6250 nM and 48.8-6250 nM, respectively (R(2)>0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25-2000 nM, 4.88-1250 nM, 9.8-1250 nM and 9.8-1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC-MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 µl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples.