Journal of Chromatography B (v.1048, #C)

Evaluation of superficially porous and fully porous columns for analysis of drugs in plasma samples by UHPLC–MS/MS by Vinicius Ricardo Acquaro; Fernando Mauro Lanças; Maria Eugênia Costa Queiroz (1-9).
This work compares the performance of recently introduced C18 superficially porous columns (four columns) and C18 fully porous columns (two columns), with different particle sizes, during the analysis of drugs in plasma samples by MS/MS detection. The following chromatographic parameters were evaluated for all the columns: reduced plate height (h) vs reduced linear velocity (v), impedance vs v, chromatographic run time vs flow rate (mL min−1), backpressure vs flow rate (mL min−1), resolution, peak capacity, asymmetry, and retention factor. By using DAD detection, hydrophobicity, silanol activity, and metal impurities were also assessed for the columns. The columns with charged surface displayed improved chromatographic efficiency for the drugs in their ionized form. The columns with particles smaller than 2 μm (Cortecs 1.6 μm, Acquity 1.7 μm, and Kinetex 1.7 μm) presented higher chromatographic efficiency for the drugs, which were in their partially ionized form. The generated mathematical models were able to predict the backpressure and chromatographic run time at different flow rates for all the columns. Considering the efficiency, impedance, resolution, peak capacity, retention factor, and hydrophobicity, Cortecs 1.6 μm and Acquity 1.7 μm were the columns that given the best performance during analysis of the target drugs in plasma samples.
Keywords: Superficially porous columns; UHPLC–MS/MS; Chromatographic parameters; Drugs; Plasma sample;

Cannabis is used widely in the United States, both recreationally and for medical purposes. Current methods for analysis of cannabinoids in human biological specimens rely on complex extraction process and lengthy analysis time. We established a rapid and simple assay for quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy Δ9-tetrahydrocannabinol (11-OH THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannbinol (THC―COOH) in human plasma by U-HPLC–MS/MS usingΔ9-tetrahydrocannabinol-D3 (THC-D3) as the internal standard. Chromatographic separation was achieved on an Acquity BEH C18 column using a gradient comprising of water (0.1% formic acid) and methanol (0.1% formic acid) over a 6 min run-time. Analytes from 200 μL plasma were extracted using acetonitrile (containing 1% formic acid and THC-D3). Mass spectrometry was performed in positive ionization mode, and total ion chromatogram was used for quantification of analytes. The assay was validated according to guidelines set forth by Food and Drug Administration of the United States. An eight-point calibration curve was fitted with quadratic regression (r2  > 0.99) from 1.56 to 100 ng mL−1 and a lower limit of quantification (LLOQ) of 1.56 ng mL−1 was achieved. Accuracy and precision calculated from six calibration curves was between 85–115% while the mean extraction recovery was >90% for all the analytes. Several plasma phospholipids eluted after the analytes thus did not interfere with the assay. Bench-top, freeze-thaw, auto-sampler and short-term stability ranged from 92.7 to 106.8% of nominal values. Application of the method was evaluated by quantification of analytes in human plasma from six subjects.
Keywords: Cannabinoids; THC; CBD; Plasma; U-HPLC–MS/MS; Protein precipitation;

A sensitive high-performance liquid chromatography (HPLC) assay with dual UV detection has been developed and validated for the simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples. Liquid-liquid extraction using hexanes was applied for samples extraction with Levo-Tetrahydropalmatine (L-THP) as the internal standard. Chromatographic separation of the analytes was achieved on a reversed-phase Waters Symmetry® C18 column (150 mm × 4.6 mm, 5 μm). A gradient elution was employed with a mobile phase consisting of 5 mM potassium phosphate containing 0.1% triethylamine (pH = 6.5) (A) and acetonitrile (B) with a flow rate of 1 mL/min. UV detection was employed at 215 nm and 235 nm for the determination of methadone and cocaine, respectively. The calibration curves were linear over the range of 0.05–10 μg/mL for both methadone and cocaine. The assay was validated according to FDA guidelines for bioanalytical method validation and results were satisfactory and met FDA criteria. Inter-day accuracy values of serum and brain samples ranged from 96.97 to 105.59% while intra-day accuracy values ranged from 91.49 to 111.92%. Stability assays showed that both methadone and cocaine were stable during sample storage, preparation, and analytical procedures. The method was successfully used to analyze biological samples obtained from a drug- drug interaction pharmacokinetics (PK) study conducted in rats to investigate the effect of methadone on cocaine PK. Our method not only can be used for bioanalysis of samples obtained from rats but also can potentially be applied to human biological serum samples to monitor compliance to methadone maintenance therapy (MMT) and to detect possible cocaine-methadone co-abuse.
Keywords: HPLC; Methadone; Cocaine; Rats; Serum; Brain;

The purpose of this study was to assess the possibility of using competitive binding experiments with ultrafiltration-HPLC analysis to identify potent xanthine oxidase (XO) inhibitors from the Perilla frutescens extract as an attempt to reduce the number of false positive results. To isolate the enzyme-ligand complex from unbound compounds, the P. frutescens extract was either incubated in the absence of XO, in the presence of XO, or with the active site blocked XO before the ultrafiltration was performed. Allopurinaol was used as the XO active site blocker. The unbound compounds were subjected to HPLC analysis. The degree of total binding (TBD) and degree of specific binding (SBD) of each compound were calculated using the peak areas. TBD represents the binding affinities of compounds from the P. frutescens extract for the XO binding site. SBD represents the XO competitive binding between allopurinol and ligands from the extract samples. Two criteria were applied to select putative targets that could help avoid false positives. These include TBD > 30% and SBD > 10%. Using that approach, kaempferol-3-O-rutinoside, rosmarinic acid, methyl-rosmarinic acid, apigenin, and 4′,5,7-trimethoxyflavone were identified, from total 11 compounds, as potent XO inhibitors. Finally, apigenin, 4′,5,7-trimethoxyflavone, and luteolin were XO inhibitors verified through an XO inhibition assay and structural simulation of the complex. These results showed that the newly developed strategy has the advantage that the number of targets identified via ultrafiltration-HPLC can be narrowed from many false positives. However, not all false positives can be eliminated with this approach. Some potent inhibitors might also be excluded with the use of this method. The limitations of this method are also discussed herein.
Keywords: Ultrafiltration; Competitive binding experiment; False positive; Perilla frutescens; Xanthine oxidase;

Display OmittedThe desoxy phenethylamine analogues in this study represent a combination of alkyl side-chain and cyclic amines (azetidine, pyrrolidine, piperidine and azepane) to yield a set of molecules of identical elemental composition as well as major mass spectral fragment ions (base peaks) of identical elemental composition. These desoxy phenethylamine analogues of the aminoketone designer drug, 3,4-methylenedioxy-pyrrovalerone (MDPV) related to the natural product cathinone were prepared from piperonal (3,4-methylenedioxybenzaldehyde) via the intermediate precursor ketones. The aminoketones and the desoxy phenethylamine regioisomers were each separated in capillary gas chromatography experiments using an Rxi®-17Sil MS stationary phase with the aminoketones showing greater retention than the corresponding desoxyamines.

Determination of IMM-H004, a novel neuroprotective agent, in rat plasma and brain tissue by liquid chromatography–tandem mass spectrometry by Ziqian Zhang; Xiangmeng Wu; Manman Zhao; Yakun Yang; Yan Wang; Jinping Hu; Baolian Wang; Li Sheng; Yan Li (49-55).
A rapid and sensitive liquid chromatography–tandem mass spectrometry method for determination of IMM-H004, a novel neuroprotective agent, in rat plasma and brain was developed. Plasma and brain tissue homogenate samples containing IMM-H004 and propranolol (internal standard, IS) were prepared by using a direct protein precipitation of acetonitrile. Separation was carried out in Zorbax SB-C18 column at a flow rate of 0.3 mL/min utilizing acetonitrile/water as mobile phases which contain 0.5% formic acid (v/v). Triple quadrupole mass spectrometer was used for detection with selective reaction monitoring. The mass transition ion-pairs were 305 → 248 for IMM-H004 and 260 → 183 for IS in positive ion mode. The linear ranges of IMM-H004 were 5–1000 ng/mL in plasma and 1–200 ng/mL in brain tissue homogenate. The intra- and inter-day precisions were within ±14.9% for analyte in both matrices (±17.0% at the lowest limit of quantification level), while the deviation of assay accuracy was within ±12.9%. No obvious matrix effect was observed. The recovery of the analyte was higher than 85.3%. IMM-H004 was stable during the whole analytic process. The method was applied successfully to the plasma and brain pharmacokinetic study of IMM-H004 in rats after a single intravenous administration.
Keywords: IMM-H004; LC–MS/MS; Plasma; Brain; Pharmacokinetic;

Purification of quercetin-3-O-sophoroside and isoquercitrin from Poacynum hendersonii leaves using macroporous resins followed by Sephadex LH-20 column chromatography by Hongjuan Li; Ying Liu; Yuetao Yi; Qin miao; Sujing Liu; Feng Zhao; Wei Cong; Chunhua Wang; Chuanhai Xia (56-63).
In China, Poacynum hendersonii is frequently used as a substitute for Apoacynum venetum L (Luobuma), which is a famous traditional Chinese medicine. Quercetin-3-O-sophoroside and isoquercitrin are two major flavonoids in Poacynum hendersonii leaves. In this work, a suitable method was established for the large-scale preparation of quercetin-3-O-sophoroside (QOS) and isoquercitrin (ISO) from Poacynum hendersonii leaves using macroporous resin combined with Sephadex LH-20 column chromatography. The adsorption/desorption capacities and desorption ratios of six macroporous resins were evaluated using static experiments. The HPD-300 resin had the best adsorption performance because it had the largest surface area, and was selected for further study. Compared with pseudo-first-order and intraparticle diffusion kinetics models, the pseudo-second-order model could better fit the adsorption kinetics of both QOS and ISO on the HPD-300 resin. In addition, the adsorption isotherms of the two compounds on the HPD-300 resin were fitted well to the Langmuir model. Under optimal conditions, the purities of QOS and ISO in the product were increased from 2.16% and 1.26% to 21.34% and 10.70% with recovery yields of 82.1% and 77.3%, respectively. Subsequently, Sephadex LH-20 column chromatography was employed for improving the purities of the two compounds. After separation by Sephadex LH-20 column chromatography, the purities of QOS and ISO achieved 93.5% and 95.6%, respectively.
Keywords: Macroporous resins; Quercetin-3-O-sophoroside; Isoquercitrin; Purification; Poacynum hendersonii;

Praziquantel is the drug of first choice for the control and treatment of all forms of schistosomiasis. Praziquantel is administered as a racemate, including R-enantiomer and S-enantiomer. Among them, R-enantiomer has main contribution to schistosomicidal activity. In this study, a sensitive and rapid liquid chromatography with tandem mass spectrometry was established and validated to determine the concentration of racemate praziquantel and R-enantiomer in rat plasma after oral administration. Chromatographic separation was performed on an Agilent Zorbax SB-C18 column. An entire run time for chromatographic separation was no more than 5 min. The present method for analytes manifested that high sensitivity (the lower limit of quantification was 3.0 ng/mL), satisfactory accuracy (relative error ≤ ± 15%) and precision (relative standard deviation ≤15%) were achieved. There was no obvious matrix effect found. The average recoveries of racemate praziquantel and R-enantiomer were both above 85%. Then, the developed method had a successful application to comparative pharmacokinetic study of racemate praziquantel and R-enantiomer. Meanwhile, the differences in their pharmacokinetic parameters were compared and analyzed. The present quantification method and comparative pharmacokinetic study would provide a useful reference for the drug development of enantiopure schistosomicidal R-enantiomer as a replacement of racemate praziquantel for treatment of schistosomiasis.
Keywords: Liquid chromatography; Tandem mass spectrometry; Pharmacokinetics; Racemate praziquantel; R-enantiomer;

Sensitive simultaneous quantitation of testosterone and estradiol in serum by LC–MS/MS without derivatization and comparison with the CDC HoSt program by Ryan C. Schofield; Damodara R. Mendu; Lakshmi V. Ramanathan; Melissa S. Pessin; Dean C. Carlow (70-76).
Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC–MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators.Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes.The lower limits of quantitation (LLOQs) of E2 and Te were 5 pg/mL and 1 ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5–600 pg/mL and 1–1,170 ng/dL for Te. Assay accuracy was determined both by comparison with a LC–MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N = 10) and between-day (N = 20) CVs at concentrations spanning the AMR were less than 7% for both analytes.We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC–MS/MS without chemical derivatization and using commercially available calibrators.
Keywords: Testosterone; Estradiol; Mass spectrometry; Liquid chromatography; Method correlation; CDC HoSt; NIST SRM 971;

Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood by Linda Vårdal; Hilde-Merete Askildsen; Astrid Gjelstad; Elisabeth Leere Øiestad; Hilde Marie Erøy Edvardsen; Stig Pedersen-Bjergaard (77-84).
Parallel artificial liquid membrane extraction (PALME) was combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC–MS) and the potential for screening of new psychoactive substances (NPS) was investigated for the first time. PALME was performed in 96-well format comprising a donor plate, a supported liquid membrane (SLM), and an acceptor plate. Uncharged NPS were extracted from plasma or whole blood, across an organic SLM, and into an aqueous acceptor solution, facilitated by a pH gradient.MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate, 2C-E (2,5-dimethoxy-4-ethylphenethylamine), bromo-dragonfly, and AH-7921 (3,4-dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide) were selected as representative NPS. Optimization of operational parameters was necessary as the NPS were novel to PALME, and because PALME was performed from whole blood for the very first time. In the PALME method developed for plasma, NPS were extracted from a 250 μL alkalized donor solution consisting of 125 μL plasma sample, 115 μL 40 mM NaOH, and 10 μL internal standard. In the PALME method from whole blood, the 250 μL alkalized donor solution consisted of 100 μL whole blood, 50 μL deionized water, 75 μL 80 mM NaOH, and 25 μL internal standard. In both methods, extraction was accomplished across an SLM of 5 μL dodecyl acetate with 1% trioctylamine (w/w), and further into an acidic acceptor solution of 50 μL 20 mM formic acid. The extraction was promoted by agitation at 900 rpm and was carried out for 120 min. Method validation was performed and the following parameters were considered: linearity, limits of quantification (LOQ), intra- and inter-day precision, accuracy, extraction recoveries, carry-over, and matrix effects. The validation results were in accordance with FDA guidelines.
Keywords: Liquid-phase microextraction; Parallel artificial liquid membrane extraction; New psychoactive substances; Plasma samples; Whole blood samples;

Trans-resveratrol (Res) is rapidly metabolized, extensively distributed into various tissues and mainly excreted by urine. The present study aimed to establish a simple LC–MS/MS method to simultaneously quantify Res and its major phase II metabolites (Res-3-O-β-d-glucuronide, R3G; Res-4′-O-β-d-glucuronide, R4′G; Res-resveratrol 3-sulfate, R3S; and Res-4′-sulfate, R4′S), and apply this method to assess their urinary and biliary excretions in rats. A simplified salting-out assisted liquid–liquid extraction (SALLE) strategy was developed to prepare samples with acetonitrile-methanol mixture (8:2, v/v) as extractant and ammonium acetate solution (10 M) as salting-out reagent. The method validation demonstrated an acceptable recovery (>80%), good accuracy (85–115%), low deviation of detection (<15%) and no obvious matrix effect (<20%). Then the validated method was successfully applied to analyze the excretion of Res and its metabolites after intragastric administration of Res at 50 mg/kg in rats. Only a minor proportion of Res (0.51 nmol) and its metabolites (R3S, 35.8 nmol; R4S, 0.25 nmol; R3G, 142.3 nmol; R4′G, 0.19 nmol) were eliminated via bile, while the majority of Res (1670.2 nmol), R3G (14,089.0 nmol) and R3S (2975.6 nmol) were excreted through urine. The major forms found in feces were Res and R3S, which were accumulated up to 241.8 and 250.8 nmol, respectively. In summary, the SALLE technique simplified the samples preparation and could be well popularized, especially for those highly polar compounds in biosamples like urine, bile and feces, where various endogenous substances could significantly affect the extraction recovery and detection response.
Keywords: Trans-resveratrol; Conjugated metabolites; Excretion; LC–MS/MS;

A non-target urinary and serum metabolomics strategy reveals therapeutical mechanism of Radix Astragali on adjuvant-induced arthritis rats by Tengfei Xu; Guifang Feng; Bing Zhao; Jiadi Zhao; Zifeng Pi; Shu Liu; Fengrui Song; Zhiqiang Liu (94-101).
Radix Astragali has been used traditionally in China to treat rheumatoid arthritis (RA) in formulas. In this paper, we conducted a holistic evaluation of Radix Astragali acted on adjuvant-induced arthritis (AIA) rats by urinary and serum metabolomic studies. Histological results and hind paw swelling were used to assess the joint damage, while the levels of IL-1β, TNF-α, SOD and MDA in serum were used to assess inflammation injury and oxidative stress. Metabolomic study and multivariate statistical analyses were used to investigate the differences between different groups. After processing with multivariate statistical analysis, 13 and 21 potential biomarkers were respectively found in urine and serum when Radix Astragali treatment group compared with model group. The main metabolism pathways in which Radix Astragali affected on AIA rats were tryptophan metabolism, phenylalanine metabolism, citrate cycle metabolism, fatty acid metabolism, vitamin B6 metabolism and so on. The present study demonstrates that urinary and serum metabolomics method could be a potentially powerful tool to understand the holistic therapeutic effect and the mechanisms of herb medicines.
Keywords: Mass spectrometry; Radix Astragali; Rheumatoid arthritis; Metabolomics;

Display OmittedThe molecularly imprinted silica monolithic synthesis procedureA novel strategy was presented for the synthesis and application of functionalized silica monolithic as artificial receptor of gallic acid at micro-pipette tip. A sol–gel process was used to prepare the sorbent. In this in-situ polymerization reaction, tetraethyl orthosilicate (TEOS), 3-aminopropyl trimethoxysilane (APTMS), gallic acid and thiourea were used, respectively, as cross-linker, functionalized monomer, template and precursor to make crack-free and non-fragile structure. Such durable and inexpensive in-situ monolithic was successfully employed as useful tool for highly efficient extraction of gallic acid from orange juice samples. The effective parameters in extraction recovery were investigated and optimum conditions were obtained using experimental design methodology. Applying HPLC-UV for separation quantification at optimal conditions, the gallic acid was efficiently extracted without significant matrix interference. Good linearity for gallic acid in the range of 0.02–5.0 mg L−1 with correlation coefficients of R2  > 0.999 revealed well applicability of the method for trace analysis.
Keywords: Molecularly imprinted; Monolithic; Pipette-tip solid phase microextraction; Gallic acid; Experimental design; HPLC-UV;

A simple and efficient method based on ultrafiltration liquid chromatography–mass spectrometry (UFLC–MS) was applied to rapidly screen and identify ligands for lactate dehydrogenase (LDH) from the flowers of Pueraria lobata, and the compounds were assessed for anti-stroke activity using a PC12 cell model. Seven major isoflavones, kakkalide, 3′-hydroxy puerarin, puerarin, puerarin xyloside, tectoridin, tectorigenin, and ononin, were identified as potent LDH inhibitors. A continuous online method, which consisted of microwave-assisted extraction and countercurrent chromatography (MAE-CCC), was newly developed for scaled-up production of these compounds with high purity and efficiency. This novel approach, using UFLC–MS coupled with MAE-CCC and a PC12 cell model, provided a powerful tool for screening, extraction, and separation of LDH inhibitors from complex samples, and a useful platform for the large-scale production of functional food and nutraceutical ingredients.
Keywords: Flowers of Pueraria lobata; Stroke; Lactate dehydrogenase inhibitors; Microwave-assisted extraction; Countercurrent chromatography;

Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) has been widely used to separate and quantify monoclonal antibody (mAb) fragments and impurities. Two fragments with different masses smaller than monomer were observed in the CE-SDS analysis of a recombinant IgG1 mAb treated at 40 °C for 28 days. Reversed phase liquid chromatography mass spectrometry (RP–LC–MS) and trypsin digestion peptide mapping followed by LC–MS/MS analysis were employed to conclusively demonstrate that these antibody fragments were the degradation products derived from a Ser–Ser peptide bond cleavage at the heavy chain complementarity determining region (CDR). The total percentage of the cleaved antibody heavy chain was increased from 1.1% in the control sample to 4.6% in the forced degradation sample stored at 40 °C for 28 days by using CE-SDS analysis. Native size exclusion liquid chromatography (SEC-HPLC) was used to analyze the fragment profile of this treated mAb but did not reveal fragments smaller than the monomer. However, a small fragment was detected using denaturing size exclusion liquid chromatography (dSEC-HPLC) and was confirmed to be the N-terminal fragment of the heavy chain cleaved at the CDR region using RP–LC–MS analysis. These results suggested that this N-terminal fragment may be closely associated with the rest of the cleaved antibody molecule through non-covalent bond interactions. Moreover, when dSEC-HPLC and RP–LC–MS were used to analyze the aggregate and monomer samples individually collected from SEC-HPLC, both samples contained the same amount of the cleaved N-terminal fragment. These results suggested that the CDR region cleaved fragments likely not be involved in aggregates formation of this antibody. This case study reiterated that a combination of orthogonal analytical methods plays a crucial role in comprehensively analyzing and characterizing antibody fragmentation.
Keywords: Monoclonal antibody; Fragmentation; CDR region; Size exclusion chromatography; Capillary electrophoresis; Liquid chromatography mass spectrometry;

Numerous putative post-translational modifications may induce variations of monoclonal antibodies charge distribution that can potentially affect their biological activity. The characterization and the monitoring of these charge variants are critical quality requirements to ensure stability and process consistency. Charge variants are usually characterized by preparative ion exchange chromatography, collection of fractions and subsequent reverse-phase liquid chromatography with mass spectrometry analysis. While this process can be automatized by on-line two-dimensional chromatography, it remains often complex and time consuming. For this reason, a straightforward on-line charge variant analysis method is highly desirable and analytical laboratories are actively pursuing efforts to overcome this challenge.In this study, a mixed mode ion exchange chromatographic method using volatile salts and coupled on-line to native mass spectrometry was developed in association with a middle-up approach for a detailed characterization of monoclonal antibodies charge variants. An aged monoclonal antibody, presenting a complex charge variant profile was successfully investigated by this methodology as a case study. Results demonstrate that deamidation of the heavy chain was the major degradation pathway after long-term storage at 5 °C while oxidation was rather low. The method was also very useful to identify all the clipped forms of the antibody.
Keywords: Mass spectrometry; Ion exchange chromatography; IdeS; Deamidation; Monoclonal antibody; Charge variants;

HPLC and TLC methods for analysis of [18F]FDG and its metabolites from biological samples by Johanna Rokka; Tove J. Grönroos; Tapio Viljanen; Olof Solin; Merja Haaparanta-Solin (140-149).
The most used positron emission tomography (PET) tracer, 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG), is a glucose analogue that is used to measure tissue glucose consumption. Traditionally, the Sokoloff model is the basis for [18F]FDG modeling. According to this model, [18F]FDG is expected to be trapped in a cell in the form of [18F]FDG-6-phosphate ([18F]FDG-6-P). However, several studies have shown that in tissues, [18F]FDG metabolism goes beyond [18F]FDG-6-P. Our aim was to develop radioHPLC and radioTLC methods for analysis of [18F]FDG metabolites from tissue samples. The radioHPLC method uses a sensitive on-line scintillation detector to detect radioactivity, and the radioTLC method employs digital autoradiography to detect the radioactivity distribution on a TLC plate. The HPLC and TLC methods were developed using enzymatically in vitro–produced metabolites of [18F]FDG as reference standards. For this purpose, three [18F]FDG metabolites were synthesized: [18F]FDG-6-P, [18F]FD-PGL, and [18F]FDG-1,6-P2. The two methods were evaluated by analyzing the [18F]FDG metabolic profile from rodent ex vivo tissue homogenates. The HPLC method with an on-line scintillation detector had a wide linearity in a range of 5 Bq–5 kBq (LOD 46 Bq, LOQ 139 Bq) and a good resolution (Rs ≥1.9), and separated [18F]FDG and its metabolites clearly. The TLC method combined with digital autoradiography had a high sensitivity in a wide range of radioactivity (0.1 Bq–2 kBq, LOD 0.24 Bq, LOQ 0.31 Bq), and multiple samples could be analyzed simultaneously. As our test and the method validation with ex vivo samples showed, both methods are useful, and at best they complement each other in analysis of [18F]FDG and its radioactive metabolites from biological samples.
Keywords: [18F]FDG; [18F]FDG-6-P; [18F]FD-PGL; [18F]FDG-1,6-P2; HPLC; TLC;

Development of an indirect competitive immunochromatographic strip test for rapid detection and determination of anticancer drug, harringtonine by Seiichi Sakamoto; Gorawit Yusakul; Poomraphie Nuntawong; Tharita Kitisripanya; Waraporn Putalun; Tomofumi Miyamoto; Hiroyuki Tanaka; Satoshi Morimoto (150-154).
Harringtonine (HT) is a natural compound, which is mainly produced by the genus Cephalotaxus, and has been clinically utilized in China for the treatment of acute leukemia and lymphoma. However, the amounts of HT in the Cephalotaxus species are very small; therefore, plant tissue cultures have been focused upon to enhance HT production. Qualitative/quantitative methods for HT detection are required to screen superior cell lines. We developed a one-step indirect competitive immunochromatographic assay (ICA) using colloidal gold nanoparticles conjugated with highly specific monoclonal antibodies against HT (MAb 1D2) for simple, rapid, and sensitive detection of HT in plant samples. This ICA can be completed in 15 min after dipping the strip into analytes with a limit of detection of ∼313 ng/mL. In developed ICA, fiber pad which is usually used for conventional ICA, was not used to shorten the time for preparing chromatographic strip, resulting in a decrease in the volume of valuable analytes (20 μL). Considering simplicity, rapidity, and sensitivity of the developed ICA, this study could be applied to a fieldwork study for finding new natural resources containing HT.
Keywords: Immunochromatographic strip test; Cephalotaxus alkaloids; Colloidal gold nanoparticles; Harringtonine; Monoclonal antibody against HT (MAb 1D2);