Journal of Chromatography B (v.1009-1010, #C)

Sensitive and rapid HPLC-UV method with back-extraction step for the determination of sildenafil in human plasma by Hamza Al-Hroub; Bayan Alkhawaja; Eman Alkhawaja; Tawfiq Arafat (1-6).
In this work we provided a selective, sensitive and rapid HPLC-UV method for quantification of sildenafil in human plasma. We have adopted a simple liquid–liquid extraction procedure followed a back-extraction in 5% perchloric acid solution. Chromatographic separation was achieved on a BDS C-18Column (150 mm × 4.6 mm, 5 μm) using a mobile phase consisted 63% water, 37% acetonitrile and 0.1% triethylamine (pH 7.7). The analysis was detected at 230 nm. The achieved lower limit of quantification was 2.00 ng/ml. The method showed linear calibration curve over the range of 2.00–200 ng/ml. Intra- and inter day precision (CV%) were less than 6.80 and 5.19%, respectively. Whilst intra- and inter day accuracy% were ranged between (98.3 and 105%) and (99.4 and 103%), respectively. Tests confirmed the stability of sildenafil in plasma at room temperature for 24 h, during three freeze–thaw cycles, after 24 h in autosampler at 10 °C and after 60 days in plasma at −30 °C. The recovery of sildenafil was greater than 78.4%. The described simple UV method achieved very low limit of quantification and by using simple and inexpensive extraction procedure, complete separation was obtained within short run time. Having demonstrated the validity and novelty of our method, thus it is applicable for the clinical and pharmacokinetic studies of sildenafil in human volunteers especially in laboratories in countries where cost of modern techniques and instrumentation is prohibitive.
Keywords: Sildenafil; Back extraction; HPLC-UV; Plasma; Determination;

GSK1278863 is an investigative drug under investigation for treatment of anemia associated with chronic kidney disease. Its metabolism is primarily metabolized by P450 enzymes where 19 unique metabolic species have been identified. These include multiple products of mono-, di-, and tri-oxygenation. Initially, two separate and complex ultra high performance liquid chromatography (UHPLC) reverse phase methodologies were developed, validated and applied to measure parent and various predominant and circulating metabolites in numerous clinical studies. However, 5 of the 6 oxidative metabolites may exist in different stereoisomeric forms, resulting in 14 separate species; therefore a chiral methodology was required to determine which stereoisomeric forms circulated in human. A variety of conventional approaches were explored, where in the end a supercritical fluid chromatography (SFC) method was required to separate this complex mixture of 14 stereoisomeric metabolites; data from these experiments provided important information on which species circulate in human. The details of these methodologies will be discussed herein.
Keywords: GSK1278863; Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS); Supercritical fluid chromatography (SFC); Ultra performance convergence chromatography (UPC2); Stereoisomeric metabolite separation; High performance fraction collector LCJet system;

Comparison of in vitro metabolism and cytotoxicity of capsaicin and dihydrocapsaicin by Mia Halme; Maija Pesonen; Heta Salo; Martin Söderström; Markku Pasanen; Kirsi Vähäkangas; Paula Vanninen (17-24).
Capsaicin and dihydrocapsaicin are the major active components in pepper spray products, which are widely used for law enforcement and self-protection. The use of pepper sprays, due to their irreversible and other health effects has been under a strong debate. In this study, we compared metabolism and cytotoxicity of capsaicin and dihydrocapsaicin using human and pig liver cell fractions and human lung carcinoma cell line (A549) in vitro. Metabolites were screened and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Using liver cell fractions, a novel aliphatic hydroxylated metabolite (m/z 322) was detected to dihydrocapsaicin but no structure was found corresponding to capsaicin. Instead, a novel phase I metabolite of capsaicin, corresponding to the structure of aliphatic demethylation and dehydrogenation (m/z 294) was identified. In addition, two novel conjugates, glycine conjugates (m/z 363 and m/z 365) and bi-glutathione (GSH) conjugates (m/z 902 and m/z 904), were identified for both capsaicin and dihydrocapsaicin. The medium of the exposed A549 cells contained ω-hydroxylated (m/z 322) and alkyl dehydrogenated (m/z 304) forms, as well as a glycine conjugate of capsaicin. As to dihydrocapsaicin, an alkyl dehydrogenated (m/z 306) form, a novel alkyl hydroxylated form, and a novel glycine conjugate were found. In A549 cells, dihydrocapsaicin evoked vacuolization and decreased cell viability more efficiently than capsaicin. Furthermore, both compounds induced p53 protein and G1 phase cell cycle arrest. Usefulness of the found metabolites as biomarkers for capsaicinoid exposures will need further investigations with additional toxicity endpoints.
Keywords: Pepper spray; LC–MS/MS; Cell viability; Immunoblotting; Accurate mass measurement;

Development and validation of LC–MS methods for peptaibol quantification in fungal extracts according to their lengths by Anne-Isaline Van Bohemen; Aurore Zalouk-Vergnoux; Laurence Poirier; Nam Ngoc Phuong; Nicolas Inguimbert; Khoubaib Ben Haj Salah; Nicolas Ruiz; Yves François Pouchus (25-33).
Some terrestrial Trichoderma sp. strains are already used as biological control agents (BCAs). They all produce peptaibols, small antimicrobial peptides which are supposed to play a role in the anti-phytopathogenic activity of Trichoderma sp. Trichoderma strains producing high amounts of peptaibols could represent new potential BCAs. In this context, marine-derived Trichoderma strains from the marine fungal strain collection of the “Mer, Molécules, Santé” (MMS) laboratory were investigated for their peptaibol production. Previously, the quantification of peptaibols was performed using alamethicin, as standard (20-amino acid residues peptaibol). In this study, the development and validation of quantification LC/ESI-TI-MS methods using different standards of peptaibols (11-, 14- and 20-amino acid residues) was performed in order to quantify all of them, in a single analysis, in Trichoderma crude extracts according to their chain length. The developed and validated methods were used to study the peptaibol production kinetic of a marine-derived Trichoderma strain, i.e., Trichoderma longibrachiatum (MMS 151). The results showed the optimal culture time at the 9th day with concentrations reaching 1.4 ± 0.2% and 2.3 ± 0.4% of the fungal biomass respectively for 11- and 20-residue peptaibols. Then, the different peptaibol subgroups produced by 13 Trichoderma strains were quantified. According to their 18-, 19- and 20-residue peptaibol production, three strains referenced as MMS 1541, MMS 639 and MMS 151 seemed to be good candidates as potential new biological control agents with respective production of 0.4, 0.4 and 2.1%.
Keywords: Trichoderma sp; Peptaibols; LC/ESI-IT-MS; Quantification; Validation; Biocontrol;

In this study, ionic liquid-modified silica was used as sorbent for simultaneous extraction and preconcentration of 3-indole butyric acid and 3-indole acetic acid in pea plants. The effect of some parameters such as pH and ionic strength of sample solution, amount of sorbent, flow rate of aqueous sample solution and eluent solution, concentration of eluent solution, and temperature were studied for each hormone solution. Percent extraction of 3-indole butyric acid and 3-indole acetic acid was strongly affected by pH of aqueous sample solution. Ionic strength of aqueous phase and temperature showed no serious effects on extraction efficiency of studied plant hormones. Obtained breakthrough volume was 200 mL for each of studied hormones. Preconcentration factor for spectroscopic and chromatographic determination of studied hormones was 100 and 4.0 × 103 respectively. Each solid sorbent phase was reusable for almost 10 times of extraction/stripping procedure. Relative standard deviations of extraction/stripping processes of 3-indole butyric acid and 3-indole acetic acid were 2.79% and 3.66% respectively. The calculated limit of detections for IBA and IAA were 9.1 × 10−2  mg L−1 and 1.6 × 10−1  mg L−1 respectively.
Keywords: Extraction; HPLC; 3-Indole acetic acid; 3-Indole butyric acid; Ionic liquid-modified silica gel;

Display OmittedThe serum phospholipid (PL) profiles of healthy volunteers (HE) and patients with recently diagnosed dengue fever (DF), hepatitis B (HBV), and hepatitis C (HCV) were investigated using liquid chromatography-ion trap-mass spectrometry (LC-IT-MS) and liquid chromatography-triple quad-mass spectrometry (LC-TQ-MS). Major PLs, including lyso-phosphatidylcholins (LPCs), phosphatidylcholins (PCs), phosphatidylinositols (PIs), phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), were characterized in human serum using LC-IT-MS. Thirty-five PLs were quantified using seven non-endogenous odd-carbon PL standards. An MS search protocol for the identification of PLs is described. The analytical method was optimized to achieve maximum recovery and detection. PLs were detected with minimal ionization suppression. The PLs species were characterized on the basis of (i) MS2 peaks due to polar head, (ii) precursor ion or neutral loss scans, (iii) identification of fatty acid, (iv) identification of sn-1 and sn-2 fatty acid. The quantitation data were subjected to principal component analysis (PCA), and a significant difference was observed between the PL profiles of the investigated diseases and those of HE subjects. The significance of the changes in each lipid among the four groups was statistically assessed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple comparison. The serum profiles of 28 PLs were determined to be significantly different and enabled the discrimination between HE individuals and the studied patients. Potentially dysregulated PLs were considered as differentiating biomarkers to diagnose DF, HBV, and HCV.
Keywords: Phospholipids; LC-ESI-MS/MS; Differentiating biomarkers; Dengue fever; Hepatitis B; Hepatitis C;

During the metabolism of different arsenic-containing compounds in human, a variety of metabolites are produced with significantly varying toxicities. Currently available analytical methods can only detect a limited number of human metabolites in biological samples during one run due to their diverse characteristics. In addition, co-elution of species is often unnoticeable with most detection techniques leading to inaccurate metabolic profiles and assessment of toxicity. A high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) method was developed that can identify thirteen common arsenic metabolites possibly present in human with special attention dedicated to thiolated or thiol conjugated arsenicals. The thirteen species included in this study are arsenite (AsIII), arsino-glutathione (As(GS)3), arsenate (AsV), monomethylarsonous acid (MMAIII), monomethylarsino-glutathione (MMAIII(GS) 2), monomethylarsonic acid (MMAV), dimethylarsinous acid (DMAIII (from DMAIIII)), S-(dimethylarsinic)cysteine (DMAIII (Cys)), dimethylarsino-glutathione (DMAIII(GS)), dimethylarsinic acid (DMAV), dimethylmonothioarsinic acid (DMMTAV), dimethyldithioarsinic acid (DMDTAV), dimethylarsinothioyl glutathione (DMMTAV(GS)). The developed method was applied for the analysis of cancer cells that were incubated with darinaparsin (DMAIII(GS)), a novel chemotherapeutic agent for refractory malignancies, and the arsenic metabolic profile obtained was compared to results using a previously developed method. This method provides a useful analytical tool which is much needed in unequivocally identifying the arsenicals formed during the metabolism of environmental arsenic exposure or therapeutic arsenic administration.
Keywords: High performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS); Arsenic speciation; Arsenic metabolism in humans; Dimethylarsinous glutathione;

This paper reports on a method based on magnetic solid phase extraction (MSPE) for the determination of pseudoephedrine. Magnetic nanographene oxide (MNGO) was applied as a new adsorbent for the extraction of pseudoephedrine from urine samples. Synthesis of MNGO was characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). The main factors influencing extraction efficiency, including the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, ionic strength of extraction medium, and agitation rate, were investigated and optimized. Under optimized extraction conditions, a good linearity was observed in the range of 100–2000 ng/mL with a correlation coefficient of 0.9908 (r 2). Limit of detection (LOD) and limit of quantification (LOQ) were 25 and 82.7 ng/mL, respectively. Inter-day and intra-day precision and accuracy were 6.01 and 0.34 (%), and 8.70 and 0.29 (%), respectively. The method was applied for the determination of pseudoephedrine in urine samples of volunteers receiving pseudoephedrine with the recovery of 96.42. It was concluded that the proposed method can be applied in diagnostic clinics.
Keywords: Magnetic solid phase extraction; Graphene oxide; Nanoparticles; Pseudoephedrine; Urine analysis; HPLC;

Identification of geographical origins of raw American ginseng and tablets based on stable isotope ratios by Zhihao Tian; Shouying Du; Chunsheng Liu; Renquan Liu; Huanhuan Pang; Tianxuan Duan; Fanyao Kong; Changhua Ma (73-79).
American ginseng (Panax quinquefolius) is a medicine food homology plant, whose origin determines the medicinal and economical values. Therefore, a reliable method for the determination of its geographical origin must be established. An accurate, common and stable method to identify geographical origins of raw American ginseng and tablets was established by isotope ratio mass spectrometry (IRMS). 53 samples from 5 origins were collected and analyzed for isotope ratios of the elements C, H, O and N. The result showed that δ 2H, δ 18O and δ 15N values were different among each geographical origin. The δ 2H, δ 18O and δ 15N values were also used to establish discrimination model of geographical origin. According to verification test, the discrimination model of geographical origin was accurate and stable. The δ 2H, δ 18O and δ15N values were also found no significant difference between raw American ginseng and its tablet, so the discrimination model of geographical origin could also be used to discriminate the geographical origin of American ginseng tablet. In conclusion, the δ 2 H, δ 18 O and δ 15 N values can be used to discriminate the geographical origin of raw American ginseng and its tablet.
Keywords: Raw American ginseng; American ginseng tablet; IRMS; Stable isotope ratios;

The determination of both the water-soluble and lipid-soluble vitamin D compounds in human biological fluids is necessary to illuminate potentially significant biochemical mechanisms. The lack of analytical methods to quantify the water-soluble forms precludes studies on their role and biological functions; currently available liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods are able to determine only a single sulfated form of Vitamin D. We describe here a highly sensitive and specific LC–MS/MS method for the quantification of four sulfated forms of vitamin D: vitamins D2- and D3-sulfate (D2-S and D3-S) and 25-hydroxyvitamin D2- and D3-sulfate (25(OH)D2-S and 25(OH)D3-S). A comparative evaluation showed that the ionization efficiencies of underivatized forms in negative ion mode electrospray ionisation (ESI) are superior to those of the derivatized (using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD)) forms in positive ion mode ESI. Separation was optimised to minimise co-elution with endogenous matrix compounds, thereby reducing ion suppression/enhancement effects. Isotopically labelled analogues of each compound were used as internal standards to correct for ion suppression/enhancement effects. The method was validated and then applied for the analysis of breastmilk and human serum. The detection limits, repeatability standard deviations, and recoveries ranged from 0.20 to 0.28 fmol, 2.8 to 10.2%, and 81.1 to 102%, respectively.
Keywords: Vitamin D sulfate; Water-soluble vitamin D; Breastmilk; Human serum; Electrospray ionisation;

Metabonomic study of the effects of different acupuncture directions on therapeutic efficacy by Liang Ju; Yan Wen; Jia Yin; Shizhe Deng; Jiangang Zheng; Lei Wang; Haoyue Deng; Zhiguo Hou; XiaoFeng Zhao; Si He; Linghui Huang; Chao Zhang; Guang Tian; Zhihong Meng; Yubo Li (87-95).
Posterior circulation ischemia (PCI) is a common clinical ischemic cerebrovascular disease that can endanger the lives of patients in severe cases. Our previous research found that needling the Fengchi (GB20) acupoint presents a significant effect on PCI and that different acupuncture directions can exert different effects. To investigate the biological mechanism of acupuncture directions, rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry-based metabonomic techniques are used to analyze the metabolic profiles of urine samples. The urine samples were obtained from 30 healthy control subjects, 60 PCI patients before and after treatment of different acupuncture directions. Six metabolites, including LPE (22:6), estrone, uric acid, vanillylmandelic acid, N-acetyl-l-tyrosine, and 4-hydroxyphenylacetylglutamine were identified as potential biomarkers of acupuncture treatment of PCI. Acupuncture treatment of PCI patients significantly changed the levels of these potential biomarkers. Moreover, different acupuncture directions showed different effects on the contents of these biomarkers. These results strongly support the belief that acupuncture direction performs an important function in acupuncture intervention. The findings provide new insights into the mechanism of acupuncture treatment and reveal that acupuncture manipulation results in various curative.
Keywords: Metabonomics; Rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry; Acupuncture; Posterior circulation ischemia;

A new chromatographic procedure, based upon chiral ligand-exchange principal, was developed for the resolution of salbutamol enantiomers. The separation was carried out on a C18 column. (l)-Alanine and Cu2+ were applied as chiral resolving agent and complexing ion, respectively. The kind of copper salt had definitive effect on the enantioseparation. Density functional theory (DFT) was used to substantiate the effect of various anions, accompanying Cu2+, on the formation of ternary complexes, assumed to be created during separation process. The DFT results showed that the anion kind had huge effect on the stability difference between two corresponding diastereomeric complexes and their chemical structures. It was shown that the extent of participation of the chiral selector in the ternary diastereomeric complexes formation was managed by the anion kind, affecting thus the enantioseparation efficiency of the developed method. Water/methanol (70:30) mixture containing (l)-alanine–Cu2+ (4:1) was found to be the best mobile phase for salbutamol enantioseparation. In order to analyze sulbutamol enantiomers in plasma samples, racemic salbutamol was first extracted from the samples via nano-sized salbutamol-imprinted polymer and then enantioseparated by the developed method.
Keywords: (l)-Alanine; Chiral separation; Salbutamol; C8 column; Ligand exchange; Nano-sized imprinted polymer;

A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping by Yekaterina Kori; Rekha Patel; Alyssa Neill; Hongcheng Liu (107-113).
Asn deamidation is a common post-translational modification of proteins with significant biological consequences. Asn deamidation can cause changes in structure, stability and function of proteins. LC–MS peptide mapping is the most widely used method to detect and quantify Asn deamidation. However, a significant amount of deamidation can occur during sample preparation for peptide mapping, making it challenging to accurately determine the original level of deamidation. Although several protocols to reduce procedure-induced deamidation have been reported, they either require special procedural steps or are not optimal for maintaining trypsin activity. In the current study, several commonly used buffers that are optimal for trypsin activity were evaluated. The results demonstrated that much lower levels of Asn deamidation artifacts were observed when Tris buffer was used, especially at lower concentrations. The addition of 10% acetonitrile further reduced the levels of Asn deamidation artifacts. The utility of the optimized procedure was demonstrated by the digestion of a recombinant monoclonal antibody. The proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures.
Keywords: Deamidation; Isoaspartate; Mass spectrometry; Peptide mapping; Monoclonal antibody;

Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma by Patricia Giuliani; Mariachiara Zuccarini; Silvana Buccella; Margherita Rossini; Iolanda D’Alimonte; Renata Ciccarelli; Matteo Marzo; Antonio Marzo; Patrizia Di Iorio; Francesco Caciagli (114-121).
Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1 ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13 min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375 nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions.
Keywords: HPLC; UV-Fluorescence detection; Adenine- and guanine-derivatives; Purine nucleoside phosphorylase; Enzyme kinetics; Human plasma;

A sensitive and rapid method for determination of loganin, morroniside, catalpol and acteoside in rat plasma after oral administration of Rehmannia glutinosa Libosch and Cornus officinalis Sieb drug pair based on ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS). Chromatographic separation was achieved using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using gradient mode containing 0.1% formic acid in water and acetonitrile were used as the mobile phase A and B. Loganin, morroniside, catalpol, acteoside and the internal standard (chloramphenicol) were detected by selected reaction monitoring in the negative ion mode with the mass transition of m/z 451.0 → 179.0 (morroniside), m/z 435.0 → 227.0 (loganin), m/z 407.1 → 199.1 (catalpol), m/z 623.2 → 161.0 (acteoside) and m/z 320.8 → 151.9 (chloramphenicol), respectively. All calibration curves showed good linearity (r  > 0.991). The precision was evaluated by intra-day and inter-day assays and the RSD% were all within 9.58%. The recovery ranged from 67.62 to 80.14%. The method was successfully applied to pharmacokinetic study of the analytes in normal and doxorubicin-induced chronic kidney disease rat plasma.
Keywords: UPLC–MS; Loganin; Morroniside; Catalpol; Acteoside; Chronic kidney disease; Rehmannia glutinosa; Cornus officinalis; Pharmacokinetic study;

An efficient and novel enantioseparation and determination method was developed to quantify the enantiomers of chiral triazole fungicide triticonazole in fruit, vegetable, and soil samples. Under the optimal chromatographic conditions, the enantiomers of triticonazole were completely enantioseparated on a cellulose tris(3-chloro-4-methyl phenyl carbamate) column with relatively good resolution (Rs  = 14.04). Two cleanup methods were compared to quantify the enantiomers of triticonazole. The modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction procedure was achieved with sufficient recoveries and low detection limits. Good recoveries were obtained for the two enantiomers ranging from 84.1% to 103.2% in the six matrices, and the relative standard deviation values ranged from 1.7% to 8.4%. Under the optimal conditions, the obtained limits of detection (LODs) were in the range of 0.0012–0.0031 mg/kg for the two enantiomers, and the limits of quantification (LOQs) were in the range of 0.0036–0.0091 mg/kg, which were lower than the maximum residue levels established in Japan. In addition, the stereochemical structure of triticonazole enantiomers were determined for the first time using a combination of experimental and predicted electronic circular dichroism (ECD) spectra. The first eluted enantiomer was confirmed to be (+)-(S)-triticonazole. These results indicate that the proposed method is convenient and reliable for the enantioselective detection of triticonazole in authentic samples. The proposed method could be widely applicable for investigating the stereoselective degradation of triticonazole in food and environmental matrices, providing additional information for reliable risk assessment of triazole fungicides.
Keywords: Triticonazole; Chiral column; Enantioseparation; Optical rotation dispersion; Absolute configuration; Solid phase extraction;

Moxifloxacin (MFX) and levofloxacin (LFX), class of fluoroquinolone antibiotics, are the two most prescribed drugs to multidrug resistant tuberculosis (MDR-TB) patients. A single, sensitive and reliable LC-ESI-MS/MS method was developed and validated to simultaneously quantitate the levels of these drugs in human serum where enrofloxacin (EFX) was used as internal standard (IS). Quantification was achieved by multiple reaction monitoring of selected mass transitions from precursor ions to product ions m/z 402.2 → 384.2 for MFX, 362.2 → 318.2 for LFX, and 362.1 → 318.3 for EFX. Calibration curves were plotted using concentrations ranging between 0.23–1000 ng/mL for MFX and 0.13–1000 ng/mL for LFX, and the correlation coefficients (r 2) were in excess of 0.999. Intra- and inert-day accuracy was ranged between 92.1–104% with mean recoveries of 96% and 95.5% for MFX and LFX, respectively and precision was <9% at all quality control concentration levels. Matrix effect analysis showed extraction efficiency of 93.0–94.6% for MFX and 90.9–99.5% for LFX. Application of the developed method to real sample analysis resulted in efficient quantification of MFX and LFX in serum samples obtained from ten MDR-TB patients. The result indicated that the method could be applied as a potential drug monitoring tool to accurately analyze MFX and LFX within a short run time.
Keywords: LC–MS/MS; Levofloxacin; Moxifloxacin; Multi-drug resistant tuberculosis; Serum; Real sample analysis;

Simultaneous quantification of cardiovascular disease related metabolic risk factors using liquid chromatography tandem mass spectrometry in human serum by Mo Wang; Ruiyue Yang; Jun Dong; Tianjiao Zhang; Siming Wang; Weiyan Zhou; Hongxia Li; Haijian Zhao; Lijiao Zhang; Shu Wang; Chuanbao Zhang; Wenxiang Chen (144-151).
Recent observations from metabonomic studies have consistently found that branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamine (Gln), glutamic acid (Glu), Gln/Glu ratio, carnitine, and several species of acylcarnitines and lysophosphatidylcholines (LPCs) are possible risk factors for metabolic diseases such as diabetes mellitus (DM) and cardiovascular diseases (CVD). We described here a simple and reliable method for simultaneous quantification of these metabolic risk factors by liquid chromatography tandem mass spectrometry (LC–MS/MS). Serum samples were extracted with isopropanol, and the extracted metabolites were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospary ionization (ESI) inpositive ion mode with multiple reaction monitor (MRM) mode. All the metabolites were effectively separated within 5.5 min. Analytical recoveries were in the range of 92.8–106.9%, with an average of 100.6%. The intra- run and total imprecisions for the measurement of these metabolites were 1.2–3.8% and 1.5–7.4%, respectively. Serum concentrations of the metabolites were analyzed in 123 apparently healthy volunteers. Significant associations between the metabolites and traditional CVD risk factors were observed. The newly developed LC–MS/MS method was simple, precise, and accurate and can be used as an efficient tool in CVD research and studies.
Keywords: Amino acids; Carnitine; Acylcarnitines; Lysophosphatidylcholines; Liquid chromatography tandem mass spectrometry; Cardiovascular disease;

Volatiles of five kinds of Chrysanthemum essential oils with different manufactures were characterized by descriptive sensory analysis, gas chromatography–olfactometry (GC–O), gas chromatography-mass spectrometry (GC–MS) and statistics analysis. Six sensory attributes (floral, woody, grassy, fruity, sour and minty) were selected to assess Chrysanthemum essential oils. A total of 38 volatile compounds were detected and quantified using standard substances by GC–O and GC–MS. Terpenes constituted the largest chemical group among the volatiles of the essential oils. Then partial least squares regression (PLSR) was used to elucidate the relationship between sensory attributes and aroma compounds. The result showed that α-pinene, β-thujene, α-terpinolen, β-cubebene, caryophyllene, (Z)β-farnesene, (−)-spathulenol, linalool, camphor, camphene, 4-terpineol, Z-citral and 4-isopropyltoluene were typical aroma compounds covaried with characteristic aroma of Chrysanthemum essential oils.
Keywords: Chrysanthemum essential oil; Sensory evaluation; GC–O; GC–MS; PLSR;

Traditional Chinese medicine (TCM) has been used in clinical practice for thousands of years. Catalpol, an iridoid glucoside, abundantly found in the root of the common used herb medicine Rehmannia glutinosa Libosch, has been reported to show various biological effects and pharmacological activities. After oral administration, the active ingredient might have interactions with the intestinal bacteria, which could help unravel how the medicine was processed in vivo. In this work, different pure bacteria from healthy human feces were isolated and used to bioconvert catalpol. Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC–Q–TOF/MS) technique combined with Metabolynx software was applied to analyze catalpol metabolites. Compared with blank samples, parent compound (M0) and four metabolites (M1–M4) were detected and tentatively identified based on the characteristics of their protonated ions. The metabolites were likely to be: catalpol aglycone (M1), acetylated catalpol (M2), dimethylated and hydroxylated catalpol aglycone (M3), nitrogen-containing catalpol aglycone (M4). M1 and M4 were generated in the majority of the samples like Bacteroides sp. 45. M3 was obtained in several bacterial samples like Enterococcus sp. 8-2 and M2 was detected only in the sample of Enterococcus sp. 43-1. To our knowledge, the metabolic routes and metabolites of catalpol produced by human intestinal bacteria were all firstly reported.
Keywords: Catalpol; Human intestinal bacteria; UPLC–Q–TOF/MS; Metabolites;

This study aims to provide a rapid, sensitive and precise UPLC–MS/MS method for target steroid quantitation in biological matrices. We developed and validated an UPLC–MS/MS method to simultaneously determine 16 steroids in plasma and tissue samples. Ionization sources of Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) were compared in this study by testing their spectrometry performances at the same chromatographic conditions, and the ESI source was found up to five times more sensitive than the APCI. Different sample preparation techniques were investigated for an optimal extraction of steroids from the biological matrices. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 in broad concentration ranges. The limit of detection (LOD) was from 0.003 to 0.1 ng/mL. The method was validated according to FDA guidance and applied to determine steroids in sea lamprey plasma and tissues (fat and testes) by the developed method.
Keywords: Steroids; Sea lamprey; UPLC–MS/MS; SPE; LLE; Plasma; Tissue;