Despite decreasing incidence and mortality, gastric cancer remains the second leading cause of cancer-related deaths in the world. Successful management of gastric cancer is hampered by lack of highly sensitive and specific biomarkers especially for early cancer detection. Cell surface proteins that are aberrantly expressed between normal and cancer cells are potentially useful for cancer imaging and therapy due to easy accessibility of these targets. Combining two-phase partition and isobaric tags for relative and absolute quantification methods, we compared the relative expression levels of membrane proteins between noncancer and gastric cancer cells. About 33% of the data set was found to be plasma membrane and associated proteins using this approach (compared to only 11% in whole cell analysis), several of which have never been previously implicated in gastric cancer. Upregulation of SLC3A2 in gastric cancer cells was validated by immunoblotting of a panel of 13 gastric cancer cell lines and immunohistochemistry on tissue microarrays comprising 85 matched pairs of normal and tumor tissues. Immunofluorescence and immunohistochemistry both confirmed the plasma membrane localization of SLC3A2 in gastric cancer cells. The data supported the notion that SLC3A2 is a potential biomarker that could be exploited for molecular imaging-based detection of gastric cancer.

A sample labeled to be a natural herbal supplement for the enhancement of sexual function, was sent to Health Sciences Authority (HSA) of Singapore for testing. An unknown compound was detected and isolated from the product. The structure of the unknown compound was identified using LC-UV, high-resolution MS, ESI-MS/MS, IR, and NMR. The compound was characterized as a phosphodiesterase-5 (PDE-5) inhibitor, benzamidenafil. This is the first report of benzamidenafil, representing a new class of PDE-5 inhibitors, as an adulterant of a dietary supplement.

Two unknown compounds are detected and isolated from health supplements for the enhancement of sexual function. The structures of the unknown compounds are elucidated using high-resolution MS, ESI-MS/MS, NMR, UV and IR. One compound is identified as an analogue of sildenafil in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety, and an ethyl group instead of a methyl group is attached to the piperazinyl nitrogen. Hence, this compound is named thiohomosildenafil. Another compound is also a sildenafil analogue in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety. This compound is named thiosildenafil. Both the two compounds are first detected in health supplements. The UV, IR and completely assigned NMR data of thiohomosildenafil and thiosildenafil are first reported.

An analogue of acetildenafil was detected in an extract of pre-mixed bulk powder. To our knowledge, the powder was destined to be encapsulated and sold as a dietary supplement. The structure was identified by NMR, HR-ESI-MS, ESI-MSn and FTIR analyses. Owing to the inclusion of a hydroxyl group in acetildenafil, the detected compound was called 'hydroxyacetildenafil'. With increasing use of dietary supplements marketed for penile erectile dysfunction, the detection of analogues of sexual performance enhancers is important and timely.

Tadalafil is the active compound in Cialis®, a prescription drug approved by the US FDA in 2003 for penile erectile dysfunction (ED). With the popularity of herbal remedies and dietary supplements, some food and drug manufacturers have added tadalafil or its modified analogues to herbal products for the treatment of ED.1-3 It is potentially dangerous for patients to unknowingly consume dietary supplements adulterated with tadalafil or its analogues. Thus, identification of tadalafil-related compounds in herbal products is important and urgent. Recent studies have demonstrated the advantages of electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) in the structural characterization of components in herbal complexes.4-7 With ESI-MSn, online identification of unknown modified analogues of tadalafil will be possible if the characteristic fragmentation pathways of this class of compounds are elucidated. Recently, accurate mass spectrometry has been used to elucidate the structures of sildenafil, tadalafil and vardenafil analogues found in products marketed as dietary supplements.8 In the present study, we have investigated the differences in the mass spectra of tadalafil and its analogue. As shown in Fig. 1, the analogue is structurally similar to tadalafil except that the 2-methyl group in tadalafil has been replaced by an amino group, resulting in a [M+H]+ ion at m/z 391 compared with the [M+H]+ of tadalafil at m/z 390. Thus, their fragmentation pathways may be similar and could reasonably be rationalized by comparisons of their multi-stage mass spectra. Chemical structures of tadalafil (RCH3) and its analogue (RNH2). Tadalafil was supplied by Eli Lilly Company (Indianapolis, IN, USA). The analogue of tadalafil was isolated from a herbal product.3 ESI-MS and -MSn analyses of tadalafil and its analogue were performed using an LCQ ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Tadalafil and its analogue were each dissolved in acetonitrile (ACN)/H2O (1:1) at 10 µg/mL. Samples were injected into the spectrometer at a flow rate of 5 µL/min using an external syringe pump. The mass spectrometer parameters were set as follows: sheath gas flow rate, 30 (arbitrary); auxiliary gas flow rate, 0 (arbitrary); ionspray voltage, 5.5 kV; capillary temperature, 200°C; and capillary voltage, 80 V. In the ESI-MS spectra, the source fragmentation was turned on and the collision energy was 25 V. In the ESI-MS/MS spectra, the source fragmentation was turned off and the relative collision energy (RCE) was 27%. In the ESI-MS3 spectra, the RCE ranged from 25% to 40% of the instrument maximum. The [M+H]+ ion was selected as the precursor ion and ESI-MSn spectra were acquired in positive in mode. Figure 2 shows the ESI-MS/MS spectra of tadalafil and its analogue. It is obvious that the [M+H]+ ion and the three MS2 product ions of the analogue (m/z 391, 269, 274 and 363) have a mass shift of 1 m/z unit compared with the corresponding ions of tadalafil. ESI-MS/MS spectra of [M+H]+ of (a) tadalafil and (b) its analogue. Figures 3(a) and 3(b) show the MS3 spectra of ions at m/z 268 of tadalafil and at m/z 269 of its analogue, respectively. Product ions at m/z 268 and 240 are detected for tadalafil whereas ions at m/z 269 and 241 are detected for the analogue. The results suggest that the product ions of tadalafil at m/z 268 and 240 contain the 2-methyl group. It is proposed that the ion at m/z 268 is derived from protonated tadalafil by the loss of the benzodioxole moiety (Scheme 1), a view shared by other researchers.2, 8, 9 The ion at m/z 240 is produced by the loss of carbon monoxide from m/z 268. Similar elimination of one CO molecule from protonated piperazinedione has been previously reported.10 The MS3 spectrum of m/z 268 (Fig. 3) displays three other product ions (m/z 224, 197 and 169). The ion at m/z 224 is probably derived from m/z 240 by the loss of a methane molecule. For the tadalafil analogue, the product ion at m/z 224 is produced by the cleavage of the NN bond and the loss of NH3 from m/z 241. As shown in Fig. 3, the relative abundance of the product ion at m/z 224 of the analogue (Fig. 3(b)) is higher than that of tadalafil (Fig. 3(a)). This may be explained by the easier cleavage of the N–N bond than of the CN bond. The product ion at m/z 197 is derived from m/z 224 by the loss of hydrogen cyanide and the ion at m/z 169 is derived from m/z 197 by the loss of CO. The structures proposed in Scheme 1 are consistent with the accurate mass data reported for tadalafil, its analogue and their product ions.8 (a) MS3 spectrum (390 → 268 →) of tadalafil. (b) MS3 spectrum (391 → 269 →) of the analogue. Proposed mechanisms for the formation of the tadalafil MS2 product ion at m/z 268 and its product ions. As shown in Fig. 4, the product ions at m/z 273, 245 and 217 are detected in the MS3 spectrum of tadalafil whereas product ions at m/z 274, 246, 218 are detected in the MS3 spectrum of the tadalafil analogue, suggesting that the 2-methyl group is still present in the product ions of tadalafil at m/z 273, 245 and 217. The product ion at m/z 257 (Fig. 4(b)) is the base peak in the MS3 spectrum of the analogue. It may be derived from m/z 274 by the loss of NH3, suggesting that the product ion at m/z 274 of the analogue still contains the amino group. However, an ion at m/z 257 is not detected in the MS3 spectrum of tadalafil. This may be due to the greater difficulty in cleaving the CN bond in tadalafil than in cleaving the NN bond in the analogue. Hence, it is proposed that the ion at m/z 273 is derived from protonated tadalafil by the loss of an indole molecule. The ion at m/z 273 further gives rise to product ions at m/z 245 and 217 by sequential losses of CO. (a) MS3 spectrum (390 → 273 →) of tadalafil. (b) MS3 spectrum (391 → 274 →) of the analogue. In addition, the detection of the product ion at m/z 362 of tadalafil and the product ion at m/z 363 of the analogue suggests that the 2-methyl group is present in the product ion at m/z 362, which we proposed to be derived from protonated tadalafil by the loss of a CO molecule. The present study highlights the differences in the mass spectra of tadalafil and its amino analogue. The ESI-MSn data reported in this paper are useful for the detection and differentiation of tadalafil and its analogue in herbal products. The NUS Academic Research Fund (KHL) and a research scholarship (ZP) are acknowledged. Peng Zou*, Peiling Hou*, Sharon Sze-Yin Oh , Min-Yong Low , Hwee-Ling Koh phakohhl@nus.edu.sg*, * Department of Pharmacy, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Centre for Analytical Sciences, Health Sciences Authority, 11 Outram Road, Singapore 169078.

A tadalafil analogue and hydroxyhomosildenafil were isolated from a herbal product marketed for erectile dysfunction. The structure of the tadalafil analogue was elucidated using LC-UV, high resolution MS, ESI-MS/MS, IR, and NMR. The compound was determined to be ( 6R, 12aR)-2-amino-6-( 1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydropyrazino[ 10,20: 1,6] pyrido[ 3,4-b] indole-1,4-dione. This compound should be included as a target compound when screening for adulterants in herbal products. This is the first published paper on a tadalafil analogue and hydroxyhomosildenafil found as adulterants of a herbal product.

A high-performance liquid chromatography–diode array detection (HPLC–DAD) method and a liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) method were developed to screen for the presence of synthetic phosphodiesterase-5 (PDE-5) inhibitors and their analogues, namely sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil and hydroxyhomosildenafil. The methods were applied to pre-market samples submitted to the Health Sciences Authority of Singapore (HSA) for testing. One sample was in the form of capsules while six other samples were pre-mixed bulk powder samples for dietary supplements to be repackaged or formulated into the final dosage forms (usually capsules). Identification of PDE-5 inhibitors and their analogues was achieved by comparing individual peak retention times, UV spectra and mass spectra with those of reference standards. The seven samples were found to contain at least one of the following compounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil and acetildenafil. The five compounds were simultaneously determined by LC–ESI–MS/MS in multiple reactions monitoring (MRM) scan mode. The method has been validated for accuracy, precision, linearity and sensitivity.

Rapid Communications in Mass SpectrometryVolume 18, Issue 24 p. 3142-3147 Letter to the Editor Application of a novel quadrupole linear ion trap mass spectrometer to study the metabolism of 6-aminobutylphthalide in rat brains Jin-ping Qiao, Jin-ping Qiao Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorZeper Abliz, Corresponding Author Zeper Abliz zeper@imm.ac.cn Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.Search for more papers by this authorFeng-ming Chu, Feng-ming Chu Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorPei-ling Hou, Pei-ling Hou Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorFeng Liang, Feng Liang Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorYan Chang, Yan Chang Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorZong-ru Guo, Zong-ru Guo Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this author Jin-ping Qiao, Jin-ping Qiao Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorZeper Abliz, Corresponding Author Zeper Abliz zeper@imm.ac.cn Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.Search for more papers by this authorFeng-ming Chu, Feng-ming Chu Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorPei-ling Hou, Pei-ling Hou Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorFeng Liang, Feng Liang Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorYan Chang, Yan Chang Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this authorZong-ru Guo, Zong-ru Guo Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, ChinaSearch for more papers by this author First published: 24 November 2004 https://doi.org/10.1002/rcm.1717Citations: 7Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume18, Issue2430 December 2004Pages 3142-3147 RelatedInformation

6-Aminobutylphthalide (ABP) is a new drug candidate which is currently being developed for the treatment of cerebral ischemia. The pharmacokinetics and metabolism of ABP were studied using in situ microdialysis sampling in the brains of awake freely-moving rats. Two LC-MS/MS methods were used for the quantitative and qualitative analysis of microdialysate. For comparison and confirmation, brain tissue samples were also analyzed by LC-MS/MS and GC/MS. The results described provide more authentic information in pharmacokinetics and metabolism at the site of action by using the coupling of microdialysis to LC-MS/MS technique than the traditional sampling methods.

目的:观察菊花提取物对大鼠肝微粒体细胞色素P450及其同工酶的影响.方法:大鼠分别灌服给予相当于生药量10g/kg、2g/kg菊花提取物,连续15天,用超速离心法制备肝微粒体,用紫外分光光度法测定细胞色素P450及其同工酶的活性.结果:给药组的P450酶水平和二甲基亚硝胺(NDMA)脱甲基酶活性明显降低,而红霉素脱甲基酶的活性没有明显变化.结论:菊花提取物对大鼠肝细胞色素P450有明显抑制作用,并具有一定的亚族选择性.

AIM:To develop a sensitive and rapid HPLC method for the determination of tanshinone IIA (TS) in rat plasma and to study its pharmacokinetics in rats.METHODS:TS and 4-chlorodiphenyl (internal standard) were extracted from plasma with ethyl acetate. After liquid-liquid extraction, the sample was analyzed by HPLC with YMC C18 column (5 microns, 150 mm x 3.0 mm ID). The mobile phase consisted of acetontrile-water-acetic acid (74:26:1) at the flow rate of 0.3 mL.min-1, the UV detection wave length was 270 nm.RESULTS:The calibration curve was linear (r = 0.9981) in the range from 0.05 to 6.40 mg.L-1. The lowest detectable concentration was 0.05 mg.L-1. The recoveries at the concentration of 0.05, 1.60 and 6.40 mg.L-1 were 98.9%, 102.1% and 100.4%, respectively. The inter- and intra-day RSDs were all less than 5%.CONCLUSION:This method is proved to be rapid, precise and reliable enough to be applied to the pharmacokinetics studies of TS in rats after a single dose of 15 mg.kg-1 by oral administration.

The aim of this study was to determine the effects of the aqueous extract of Salvia miltiorrhiza Bge (danshen in Chinese) on the pharmacokinetics of diazepam and on liver microsomal cytochrome P450 enzyme activity in rats. Rats (n = 5) were pretreated with danshen extract (100 mg kg(-1) per day, p.o.) for 15 consecutive days. Control rats (n = 5) received saline at the same time. Each rat was then administered a single oral dose of 15 mg kg(-1) diazepam. The pharmacokinetic parameters of diazepam were significantly different between the two groups. In the danshen pretreated group, the maximum concentration of diazepam and the area under the plasma concentration-time curve were reduced to about 72.7% and 44.4%, respectively, while the total body clearance was markedly increased by 2-fold. To help explain the results, liver microsomal suspensions were obtained from rats that were randomly divided into the control group (n = 10), and the low- (20 mg kg(-1) for 15 days, p.o., n=10) and high-dose groups (100 mg kg(-1) for 15 days, p.o., n=10) pretreated with danshen extract. Compared with the control rats, the microsomal protein content, cytochrome P450 enzyme level and erythromycin N-demethylase activity of pretreated rats were significantly increased. These results indicate that danshen extract can stimulate the activity of cytochrome P450 isoforms, and changes in the pharmacokinetics of diazepam resulting from danshen extract are related to an increase in metabolic activity of cytochrome P450.