Fecal SN-38 Content as a Surrogate Predictor of Intestinal SN-38 Exposure and Associated Irinotecan-induced Severe Delayed-Onset Diarrhea by a Novel Use of the Spectrofluorimetric Method.

BACKGROUND: Irinotecan administration can lead to severe delayed-onset diarrhea (SDOD) in clinical practice. Currently, there is no reliable surrogate predictor of intestinal exposure to SN-38 and subsequent diarrhea incidence. METHODS: The relationship between fecal 7-ethyl-10-hydroxycamptothecin (SN-38) content and SDOD was investigated in Fisher 344 rats using a novel spectrofluorimetric method. Additionally, a pharmacokinetic study of irinotecan was performed to evaluate the biodistribution of SN-38 to establish the relationship between tissue and fecal SN-38 exposure. RESULTS: The spectrofluorimetric method was successfully employed to measure fecal SN-38 and CPT-11 content from Day 3 to Day 6 post-irinotecan administration. Only fecal SN-38 content on Day 3 exhibited a significantly positive correlation with SDOD incidence on Days 4 and 5. A cutoff value of SN-38 ≥ 0.066 mg/g in feces was identified, predicting severe diarrhea incidence with 81% accuracy and 80% specificity. The positive correlation between fecal SN-38 content and SN-38 exposure in the ileum on Day 3 was also reflected in the changes of indicators during intestinal injury, such as prostaglandin E2 level and antioxidant activity. CONCLUSION: Fecal SN-38 content proves to be representative of intestinal exposure to SN-38, indicative of intestinal injury, and predictive of SDOD incidence in rats, while the spectrofluorimetric method demonstrates the translational potential.

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.

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.

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.

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.

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.

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.

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.

Distribution and quantification of irinotecan and its active metabolite SN-38 in colon cancer murine model systems using MALDI MSI.

Tissue distribution and quantitative analysis of small molecules is a key to assess the mechanism of drug action and evaluate treatment efficacy. The prodrug irinotecan (CPT-11) is widely used for chemotherapeutic treatment of colorectal cancer. CPT-11 requires conversion into its active metabolite SN-38 to exert the desired pharmacological effect. MALDI-Fourier transform ion cyclotron resonance (FT-ICR) and MALDI-time-of-flight (TOF) mass spectrometry imaging (MSI) were performed for detection of CPT-11 and SN-38 in tissue sections from mice post CPT-11 injection. In-depth information was gained about the distribution and quantity of drug compounds in normal and tumor tissue. The prodrug was metabolized, as proven by the detection of SN-38 in liver, kidney and digestive tract. In tumors from genetic mouse models for colorectal cancer (Apc (1638N/wt) x pvillin-Kras (V12G) ), CPT-11 was detected but not the active metabolite. In order to correlate drug distribution relative to vascularization, MALDI data were superimposed with CD31 (PECAM-1) immunohistochemistry. This analysis indicated that intratumoral access of CPT-11 mainly occurred by extravasation from microvessels. The present study exploits the power of MALDI MSI in drug analysis, and presents a novel approach to monitor drug distribution in relation to vessel functionality in preclinical and clinical research.

Simultaneous determination of camptothecin and 10-hydroxycamptothecine in the Camptotheca acuminate, its medicinal preparation and in rat plasma by liquid chromatography with fluorescence detection.

Camptotheca acuminata Decne is an important medicinal plant that contains various cytotoxic alkaloids, such as camptothecine (CPT) and 10-hydroxycamptothecine (HCPT). A rapid and sensitive liquid chromatography with fluorescence detection (LC-FLD) method for the quantification of CPT and HCPT is described. The separation was carried out on a DL-Cl8 column (4.6 × 150 mm, 5 µm), with the mobile phase of acetonitrile-sodium dihydrogen phosphate buffer (10 mm) using an gradient elution at the flow rate of 0.6 mL/min. The LC-FLD method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The lower detection limits of CPT and HCPT were 0.4 and 0.1 ng/mL, respectively. The precision was <1.58% and the mean recovery of the analytes was 96.0-98.6%. The LC-FLD method was successfully applied to determine CPT and HCPT in real samples including C. acuminate, HCPT injection and rat plasma.

A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies.

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.

Versatile online SPE-HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials.

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide. Sample clean-up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH2 PO4 pH 2.9. Online transfer to an analytical µBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH2 PO4 pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN-38 glucuronide and Irinotecan or 540 nm for SN-38 results in high sensitivity (1-2 pg) and short (∼10 min) run times. The method was used to determine the degree of SN-38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN-38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan-based tumor targeting therapies.

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.

Drug-initiated ring-opening polymerization of O-carboxyanhydrides for the preparation of anticancer drug-poly(O-carboxyanhydride) nanoconjugates.

We report a novel synthetic strategy of polymer-drug conjugates for nanoparticulate drug delivery: hydroxyl-containing drug (e.g., camptothecin, paclitaxel, doxorubicin and docetaxel) can initiate controlled polymerization of phenyl O-carboxyanhydride (Phe-OCA) to afford drug-poly(Phe-OCA) conjugated nanoparticles, termed drug-PheLA nanoconjugates (NCs). Our new NCs have well-controlled physicochemical properties, including high drug loading, quantitative drug loading efficiency, controlled particle size with narrow particle size distribution, and sustained drug release profile over days without "burst" release effect as observed in conventional polymer/drug encapsulates. Compared with polylactide NCs, the PheLA NCs have increased noncovalent hydrophobic interchain interactions and thereby result in remarkable stability in human serum with negligible particle aggregation. Such distinctive properties can reduce the premature disassembly of NCs upon dilution in the bloodstream and prolong NCs' in vivo circulation with the enhancement of intratumoral accumulation of NCs, which has a bearing on therapeutic effectiveness.

Development and validation of a microfluidic chip-based nano-liquid chromatography-triple quadrupole tandem mass spectrometry method for a sensitive and reliable quantification of 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma.

There is an increasing need for more sensitive analytical methods in pharmacokinetic studies, for example, for phase 0 clinical trials. A novel HPLC Chip-triple quadrupole mass spectrometer method (HPLC Chip-MS/MS method) for the quantification of 7-ethyl-10-hydroxycamptothecin (SN38) was developed, validated, and employed to the pharmacokinetic analysis of SN38 in ICR mice. Protein precipitation with a ratio of plasma/acetonitrile of 1:10 was chosen as the sample processing method. The nano-electrospray inserted in the microfluidic chip operated in positive mode, and selected reaction monitoring was used for quantification. Our bioanalytical method met all essential validation parameters-selectivity, accuracy, precision, dilution integrity, calibration curve, matrix effect, recovery, and different stability tests (benchtop, freeze-thaw, autosampler stability). The calibration curves (weight 1/x (2)) were linear for the range 50-10,000 pg/mL. Clogging was not observed until the end of the lifetime of the microfluidic chip (350-400 injections), and carryover was practically eliminated through the introduction of a step gradient elution program. After intraperitoneal injection of 0.1 mg/kg irinotecan, SN38 concentration could be measured up to 6 h with accuracy and precision. Thus, we developed a new, very sensitive HPLC Chip-MS/MS method for the determination of plasma SN38 that has been validated in compliance with guidelines from different regulation authorities.

Characterization and quantification of 10-hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography-ion trap mass spectrometry.

A rapid and sensitive method for the identification and quantification of 10-hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MS(n) spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI-MS(n) fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MS(n) in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative-ion mode at a product ion at m/z 363 → 319 by LC-MS. The LC-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC-MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time.

Isolation and characterization of endophytic fungi from Camptotheca acuminata.

In this study, a total of 161 endophytic fungal isolates from Camptotheca acuminata were obtained and classified to 16 taxa according to morphological and molecular analysis. These taxa were composed of 2 frequent genera (Botryosphaeria and Fusarium) and 14 infrequent groups such as Xylaria, Diaporthe, Rhizopus, Epicoccum, and Preussia, demonstrating that fungal endophytes in C. acuminata were highly abundant and diverse. Antimicrobial activity screening using filter-paper diffusion method showed that 47.6 % of the tested isolates had antimicrobial activity against at least one of the test microorganisms. Screening of fungal endophyte-derived camptothecin analogues by TLC and LC-MS/MS³ demonstrated that a strain Botryosphaeria dothidea, X-4 could produce 9-methoxycamptothecin (9-MCPT) when cultured in Sabouraud's dextrose broth for 12 days under shake flask and bench-scale fermention conditions. This work showed that the fungal endophytes from C. acuminata could be an alternative source for the production of 9-MCPT and other natural antimicrobial compounds.

[Determination of 7-ethyl-10-hydroxycamptothecin in microdialysates from rat brain with LC-MS/MS].

OBJECTIVE: To establish a method for determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in microdialysates from rat brain. METHODS: The concentrations of SN-38 were measured by LC-MS/MS method with Agilent Eclipse Plus C18 (2.1 mm ×100 mm, 1.8 µm) reversed phase column using acetonitrile-0.1% methanoic acid as mobile phase with gradient elution at a flow rate of 0.3 ml/min and temperature at 35 degree. Multiple reaction monitoring using the precursor to product ion combinations of m/z 393.1→349.1 was performed to detect SN-38 in microdialysates from rat brain. RESULTS: Blank microdialysate had non-interference. The method was linear over the concentration range of 0.1015-1015 ng/ml (r=0.9995); and the lower limit of quantification (LOQ) was 0.1015 ng/ml. The recovery of assay for SN-38 ranged from 97.54%-100.60%. The intra- and inter-day precision and stability were both well. The concentrations of SN-38 in brain microdialysates presented pharmacokinetics process and achieved the peak after 220 min. CONCLUSION: The fully validated LC-MS/MS analytical method has high specificity and sensibility, which can be used effectively to analyze SN-38 in microdialysates from rat brain.