Journal of Chromatography B (v.986-987, #C)

Recovery and purification of recombinant 503 antigen of Leishmania infantum chagasi using expanded bed adsorption chromatography by Francisco Caninde de Sousa Junior; Michelle Rossana Ferreira Vaz; Carlos Eduardo de Araújo Padilha; Abimaelle Silva Chibério; Daniella Regina Arantes Martins; Gorete Ribeiro de Macedo; Everaldo Silvino dos Santos (1-7).
Visceral leishmaniasis, a disease caused by Leishmania infantum chagasi, represents a major public health problem in many areas of the world. However, there is currently no vaccine for human use. The aim of this work was to purify the 503 antigen of Leishmania i. chagasi directly from unclarified Escherichia coli feedstock through expanded bed adsorption (EBA) chromatography. Batch experiments were performed to optimize the adsorption and elution conditions of the antigen onto a STREAMLINE™ Chelating resin using two central composite rotatable designs (CCRD). The results showed that the optimal binding conditions of the 503 antigen were pH 8.0 in the presence of 2.4 M NaCl. For the elution of the target protein, the optimized conditions included the presence of 600.0 mM imidazole. The adsorption isothermal data of the 503 antigen were fitted to the Langmuir adsorption isotherm. The EBA experiment successfully recovered 59.2% of the 503 antigen from the unclarified E. coli homogenate with a purification factor of 6.0.
Keywords: Expanded bed adsorption; Leishmania infantum chagasi; Recombinant protein purification; Unclarified bacterial homogenate; Visceral leishmaniasis;

An LC–MS/MS/MS GHB quantification method in human serum supported by the adduct formation process with the components of the mobile phase was developed and validated. The continuous infusion of GHB made the identification of a GHB MS3 mass transition possible (185/103/85). A Luna 5 μm C18 (2) 100 Å, 150 mm × 2 mm analytical column and the elution with a programmed flow of the mobile phase consisting of 10% A (H2O/methanol = 95/5, v/v) and 90% B (H2O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid (pH = 3.2) were used. The analytical sample preparation consisted of protein precipitation with 1 mL of the mobile phase B. The calculated limit of detection/quantification was 0.28/0.96 μg/mL, respectively. The study presented demonstrated that the process of analyte adduct formation with the components of the mobile phase can be used successfully to generate MS3 transitions in the case of very small molecules which cannot be detected in this way by the application of conventional analytical strategies.
Keywords: γ-Hydroxybutyrate; GHB; LC–MS/MS/MS; Analyte adduct formation;

Methyl gallate (MG) and pentagalloyl glucopyranose (PGG) are bioactive phenolic compounds that are widely distributed in herbs and plant foods. Their potential activities include anti-oxidant, anti-inflammatory, anti-cancer, anti-bacterial and anti-viral activities. However, knowledge concerning the pharmacokinetic characteristics of MG and PGG is limited. The purpose of this study was to develop a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC–MS/MS) method to simultaneously quantify MG and PGG in rat blood samples. The linear response ranges for MG and PGG were 0.0195–20 and 0.0390–20 μM, respectively. The lower limit of quantification was 0.0195 μM for MG and 0.0390 μM for PGG. The intra- and inter-day variances were less than 15%, and accuracy was within 80–120%. This assay was successfully applied to pharmacokinetic studies in Sprague-Dawley rats after intraperitoneal administration of MG and PGG (20 mg/kg). The values of areas under the blood concentration time curves (AUC0-24h) for MG and PGG were 109.9 ± 73.40 and 38.78 ± 24.53 h* μM, respectively. The maximum blood concentrations (C max) of MG and PGG were 34.72 ± 17.32 and 6.39 ± 4.25 μM, respectively. The time required to reach the maximum concentration (T max) was 0.85 ± 0.70 h for both MG and PGG. The values of the elimination rate constant (K e), elimination half-life (t 1/2), volume of distribution (V d), clearance (Cl) and mean resident time (MRTlast) were 0.056 ± 0.032 h−1, 17.50 ± 12.25 h, 530.95 ± 247.54 L/kg, 159.91 ± 76.05 L/h/kg, 8.71 ± 2.53 h for MG and 0.023 ± 0.012 h−1, 38.66 ± 22.89 h, 7838.89 ± 3474.72 L/kg, 30.98 ± 21.73 L/h/kg, 12.47 ± 2.77 h for PGG, respectively. In conclusion, a UPLC–MS/MS method was fully validated over a wide linear range and used to quantify the levels of MG and PGG in pharmacokinetic studies of MG and PGG in rats. The main advantages of this method are the use of small blood volumes (10 μL), rapid analysis (5 min) and excellent recoveries.
Keywords: LC−MS/MS; Methyl gallate; Pentagalloyl glucopyranose; Pharmacokinetics;

Determination of acacetin in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study by Li-hua Fan; Xiaoheng Li; De-yuan Chen; Ning Zhang; Yiyan Wang; Yuanyuan Shan; Yuanyuan Hu; Ren-ai Xu; Jian Jin; Ren-Shan Ge (18-22).
A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of acacetin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 2.0 min and the elution of acacetin was at 0.83 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 285.3 → 242.2 for acacetin and m/z 237.2 → 194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1–1600 ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of acacetin in plasma was in the range of 78.4–85.2%. Intra-day and inter-day precision were both <10.5%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0 mg/kg acacetin in rats.
Keywords: Acacetin; UPLC-MS/MS; Rat plasma; Pharmacokinetics;

A reversed phase liquid chromatography tandem mass spectrometry method was developed and validated for quantification of cardamonin, a potential anticancer chalcone, in rat serum. Curcumin was used as an internal standard. Following liquid–liquid extraction using n-hexane and ethyl acetate (60:40, v/v), the processed samples were chromatographed on a C18 column using acetonitrile and ammonium acetate buffer (0.01 M, pH 4.5) (85:15, v/v) as mobile phase at a flow rate of 0.6 mL min−1. Mass spectrometric detection was performed in the negative electrospray ionization mode by multiple reaction monitoring (m/z 269 → 122 and 367 → 217 for cardamonin and curcumin, respectively). The method was validated in terms of selectivity, accuracy, precision, sensitivity, reproducibility, dilution integrity and stability. The linearity was established in the range of 1–200 ng mL−1 (r  ≥ 0.999). The recovery of cardamonin from spiked serum was always >90%. The intra- and inter-day precision (%RSD) and accuracy (%bias) were well within the acceptable limits. The method was applied for single oral and intravenous dose pharmacokinetics in male and female Sprague Dawley rats. Following oral dose, cardamonin showed peak serum concentration that occurred at ∼2 h with very low bioavailability in both male (0.6%) and female (4.8%) rats. Cardamonin exhibited a significant gender influence on pharmacokinetics and bioavailability in rats.
Keywords: Cardamonin; Pharmacokinetics; Bioavailability; Liquid Chromatography; Tandem mass spectrometry;

Determination of kurarinone in rat plasma by UPLC–MS/MS by Wei-min Zhang; Rui-fang Li; Jian-fei Qiu; Zhi-yin Zhang; Hong-bo Wang; Lu Bian; Jia-hui Lei (31-34).
A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine kurarinone in rat plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 437.0 → 301.2 for kurarinone and m/z 168.1 → 132.1 for IS. The linearity of this method was found to be within the concentration range of 20–2000 ng/mL with a lower limit of quantification of 20 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 94.7–107.2% for kurarinone. The intra- and inter-day precision (RSD%) were less than 8.2% and accuracy (RE%) was within ±9.0%. The recovery ranged from 77.3% to 85.6%. Kurarinone was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of kurarinone in rats.
Keywords: Kurarinone; UPLC–MS/MS; Rat plasma; Pharmacokinetics;

Evaluation and comparison of three different separation techniques for analysis of retroamide enantiomers and their biological evaluation against h-P2X7 receptor by Davy Baudelet; Christophe Furman; Alina Ghinet; Xavier Dezitter; Sahil Adriouch; Frédéric Capet; Tiphaine Rogez-Florent; Philippe Gautret; Benoit Rigo; Régis Millet; Claude Vaccher; Emmanuelle Lipka (35-43).
The P2X receptors are seven-transmembrane domain G protein-coupled receptors and the 7 subtypes of P2X receptors identified in humans, and named P2X1 to P2X7, are channel receptors whose endogenous ligand is ATP. New antagonists of the P2X7 receptor were developed, since this purinergic receptor was highlighted to be involved in many diseases such as different types of pain, cancer, ischemia, neurodegenerative diseases (including Parkinson's and Alzheimer's diseases) characterized by inflammatory processes. With the aim of evaluate the impact of chirality on the pharmacological activity of a new P2X7R antagonist, a semi-preparative method was developed in supercritical fluid chromatography (SFC). Among four polysaccharide based chiral stationary phases: Chiralcel OD-H and OJ-H and Chiralpak AS-H and AD-H, the last one namely amylose tris (3,5-dimethylphenylcarbamate) with a mobile phase consisted of carbon dioxide–ethanol (80:20, v/v), led to the successful separation of the enantiomers in short run time and with good resolution. Limits of detection and quantification were calculated and were found equal for compound 1, to 1.37 μM and 4.57 μM respectively, for peak 1 and were equal to 1.60 μM and 5.30 μM respectively, for peak 2 at λ  = 210 nm. Before carrying out the pharmacological evaluation of each enantiomer, two complementary methodologies, e.g. liquid chromatography and capillary electrophoresis were performed in parallel to improve the limits of detection and quantification to assess the enantiomeric purity. HPLC using a Chiralpak AD stationary phase led to four times lower limits of detection and quantification with regard to SFC. In the same time, capillary electrophoresis involving dual cyclodextrins system constituted of a SBE-β-CD and a MM-β-CD mixture enhanced the signal-to-noise ratio and led to similar limits of detection and quantification with regard to SFC. No trace of the other enantiomer was found in the isolated one. Biological activities of individual enantiomers were then evaluated and revealed no cytotoxicity against cell lines and a significant difference in terms of their IC50 values with respect to the investigated racemate (6.43 μM): 3.49 μM for the (R)-enantiomer and >10−4  μM for the (S)-enantiomer, for compound 1, showing that, this antagonist activity is stereospecific.
Keywords: Amylose tris (3,5-dimethylphenylcarbamate); Capillary electrophoresis; Dual cyclodextrins system; High performance liquid chromatography; N-[1-(2,4-Dichlorobenzyl)-5-oxopyrrolidin-2-yl]-2-(2,4-dichlorophenyl) acetamide; Supercritical fluid chromatography;

Radix Astragali (Huangqi in Chinese) and Fructus Ligustri Lucidi (Nvzhenzi in Chinese) (2:1, w/w) are combined in an herbal formulation called Zhenqi Fuzheng capsules (ZFCs) for use in China to improve immunity, promote the recovery of normal functions after surgical operations, and as the most important adjuvant therapy in cancer. In this study, the tissue distribution profiles of the six major bio-active constituents (calycosin-7-O-β-D-glucoside, ononin, calycosin, formononetin, astragaloside IV and astragaloside II) were examined after oral administration of ZFCs to rats. All six constituents in each tissue were detected simultaneously using UPLC-ESI-MS, and the concentration of each constituent per gram of each tissue was determined. Quantification was performed using low-energy collision tandem mass spectrometry (CID-MS/MS) in multiple reaction monitoring (MRM) scan mode for the following precursor ion → product ion transitions at m/z 447.21 → 285.30 for calycosin-7-O-β-D-glucoside, m/z 285.29 → 270.38 for calycosin, m/z 431 → 269 for ononin, m/z 269 → 237 for formononetin, m/z 807.40 → 627.50 for astragaloside IV, m/z 849.60 → 669.65 for astragaloside II and m/z 633.18 → 331.18 for the internal standard (hesperidin). The results showed that in general the tissue concentrations for all six constituents were in the following order: spleen > stomach > thymus > lung > liver > kidney > heart > testicle. The high levels in the spleen and thymus indicated that all six compounds accumulated in organs involved in the immune response, consistent with the immunity effects of ZFC. The high levels in the stomach were consistent with the oral administration of ZFC. This study was the first to compare the tissue distribution of calycosin-7-O-β-D-glucoside with that of calycosin or of ononin with that of formononetin in rats. It was also the first study to examine the tissue distribution of astragaloside II, calycosin and formononetin following oral administration of ZFC to rats.
Keywords: UPLC-ESI-MS; CID-MS/MS; MRM; Flavonoids; Saponins; Zhenqi Fuzheng capsule; Tissue distribution;

The purpose of this study is to develop and validate an UPLC-MS/MS method to quantify ethoxzolamide in plasma (EZ) and apply the method to absorption, brain distribution, as well as pharmacokinetic studies. A C18 column was used with 0.1% of formic acid in acetonitrile and 0.1% of formic acid in water as the mobile phases to resolve EZ. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode. The results show that the linear range of EZ is 4.88–10,000.00 nM. The intra-day variance is less than 12.43% and the accuracy is between 88.88 and 108.00%. The inter-day variance is less than 12.87% and accuracy is between 89.27 and 115.89%. Protein precipitation was performed using methanol to extract EZ from plasma and brain tissues. Only 40 μL of plasma is needed for analysis due to the high sensitivity of this method, which could be completed in less than three minutes. This method was used to study the pharmacokinetics of EZ in SD rats, and the transport of EZ in Caco-2 and MDCK-MDR1 overexpressing cell culture models. Our data show that EZ is not a substrate for p-glycoprotein (P-gp) and its entry into the brain may not limited by the blood-brain barrier.
Keywords: Ethoxzolamide; UPLC-MS/MS; Pharmacokinetics; Absorption; CNS distribution;

We present a method for the qualitative and quantitative analysis of felbinac and its major metabolites in human plasma and urine by HPLC–MS/MS and its application. Qualitative analysis through LC–Triple-TOF-MS/MS indicated that oxidization was the main phase-I metabolic pathway of felbinac in human, conjugation with sulfate and glucuronide groups produced at least 7 phase-II metabolites. Quantitative analysis through HPLC–MS/MS in MRM mode was developed and validated for the quantification of felbinac and its major metabolite (4′-hydroxyfelbinac) in human plasma and urine. Linear calibration curves were obtained for felbinac and 4′-hydroxyfelbinac in plasma and urine (r  > 0.996); intra- and inter-day precision values (RSD%) obtained were ranged from 1.13 to 6.49%, and the accuracy were between 95.9% and 108.6% for the two analytes. The pharmacokinetics and excretion analysis showed that the t 1/2 of 4′-hydroxyfelbinac (8.25 ± 4.15 h) is a litter longer than that of felbinac (6.13 ± 2.01 h), but the mean AUC(0–t) value of felbinac was about 20 times higher than that of 4′-hydroxyfelbinac; excretion of felbinac and 4′-hydroxyfelbinac reached their peak values at about 3–6 h after intravenous administration of felbinac trometamol in human.
Keywords: Felbinac; Metabolites; Pharmacokinetics; Excretion; Human; HPLC–MS/MS;

Gua-Lou-Gui-Zhi decoction (GLGZD) is a classical formula of traditional Chinese medicine, which has been commonly used to treat dysfunction after stroke, epilepsy and spinal cord injury. In this study, a systematic method was established for chemical profiling and quantification analysis of the major constituents in GLGZD. For qualitative analysis, a method of high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (Q-TOF MS) was developed. 106 compounds, including monoterpene glycosides, galloyl glucoses, phenolic acids, flavonoids, gingerols and triterpene saponins were identified or tentatively presumed by comparison with reference standards or literature data. According to the qualitative results, a new quantitative analysis method of ultra-performance liquid chromatography/triple quadrupole mass spectrometry (QqQ-MS) was established. 24 representative compounds were simultaneously detected in 10 batches of GLGZD samples in 7.5 min. The calibration curves for all analytes showed good linearity (r  > 0.9959) within the test ranges. The LODs and the LOQs were less than 30.6 and 70.9 ng/mL, respectively. The RSDs of intra- and inter-day precision, repeatability and stability were below 3.64%, 4.85%, 4.84% and 3.87%, respectively. The overall recoveries ranged from 94.94% to 103.66%, with the RSDs within 5.12%. This study established a high sensitive and efficient method for the integrating quality control, including identification and quantification of Chinese medicinal preparation.
Keywords: Gua-Lou-Gui-Zhi decoction; Chemical profiling and quantification; Quality control; QTOF mass spectrometry; QqQ mass spectrometry;

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and optimized for the simultaneous determination of bisphenol A, 4-t-octylphenol and 4-nonylphenol in human blood serum. For the first time, the electrospray ionization (ESI) parameters of probe position, voltage potential, sheath gas flow rate, auxiliary gas flow rate, and ion transfer tube temperature were thoroughly studied and optimized for each phenol by a univariate approach. As a consequence, low instrumental limits of detection were reported, demonstrating at 0.2 ng/mL (in solvent matrix) excellent injection repeatability (RSD < 14.5%) and a confirmation peak for all target phenols. Extraction and purification of serum was performed by the novel Hybrid Solid Phase Extraction–Precipitation Technology technique (Hybrid SPE-PPT). The limits of detection in human blood serum were 0.80, 1.3 and 1.4 ng/mL for BPA, 4-t-OP and 4-NP, respectively.
Keywords: EDCs; Bisphenol A; 4-t-Octylphenol; 4-Nonylphenol; Hybrid SPE-PPT; Bioanalysis;

Voriconazole is an azole antifungal drug indicated for use in the treatment of invasive aspergillosis. Due to the large intra- and interindividual variation seen in voriconazole pharmacokinetics along with a high probability of drug–drug interactions, therapeutic drug monitoring is of considerable clinical value. As such, we developed and validated a LC–MS/MS assay to quantify serum voriconazole to improve turnaround time and decrease costs.After protein precipitation with D3-voriconazole (deuterated internal standard) in acetonitrile was performed, samples were separated by gradient elution and injected into the mass spectrometer with a total run-time of 4 min per sample. Multiple reaction monitoring was employed using Q1/Q3 transitions of 350/127 and 350/281 for voriconazole and 353/284 and 353/127 for D3-voriconazole.Sample preparation took 30 min for 6 patient samples. The limit of quantitation was 0.1 μg/mL and the linearity ranged from 0.1 μg/mL to 10.0 μg/mL. Extraction recovery was ∼69% and ion suppression ∼13%. Intra- and inter-assay imprecision (%CV) was <5% at the limit of quantitation and <4% through the rest of the linear range. Method comparisons between our assay and two reference laboratory methods, HPLC-UV and LC–MS/MS, revealed mean biases of 11% and 4%, respectively.We have developed an accurate, rapid, and sensitive LC–MS/MS assay for quantification of human serum voriconazole. Our assay reduces current specimen volume requirements, decreases result turnaround time, and saves institutional funds.
Keywords: Voriconazole; LC–MS/MS; Mass spectrometry;

Picrasma quassioides (D. Don) Benn. is used in traditional Chinese medicine for the treatment of inflammation. Characteristic components of the medicinal extract are canthinone alkaloids. In this study, a sensitive and rapid liquid chromatography with tandem mass spectrometry method has been developed for simultaneous quantification of two major canthinone alkaloids, 5-hydroxy-4-methoxycanthin-6-one and 4,5-dimethoxycanthin-6-one, in rat plasma after oral administration of P. quassioides extract (200 mg/kg). The chromatographic separation was performed on a C18 column using acetonitrile–aqueous 0.1% formic acid (90:10, v/v) as the mobile phase. Plasma samples were prepared for analysis using a simple liquid–liquid extraction with ethyl acetate. Analytes were detected using tandem mass spectrometry in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 1.25–900 ng/mL for 5-hydroxy-4-methoxycanthin-6-one and 0.5–800 ng/mL for 4,5-dimethoxycanthin-6-one with satisfactory intra- and inter-day precision, accuracy and recovery. Samples were stable under the conditions tested. The pharmacokinetic profiles of the analytes in rats showed that both canthinones were rapidly absorbed and that 4,5-dimethoxycanthin-6-one was eliminated faster than 5-hydroxy-4-methoxycanthin-6-one.
Keywords: Picrasma quassioides (D. Don) Benn.; Canthinone alkaloids; HPLC–MS/MS; Pharmacokinetics; Method validation;

A liquid chromatography–tandem mass spectrometry method (LC-MS/MS) was developed to quantify ezetimibe (EZM) and its major glucuronide (ezetimibe glucuronide, EZM-G) in human plasma simultaneously. The analytes were purified by solid phase extraction (SPE) without hydrolysis. Separation of the analytes was achieved using acetonitrile–water (0.08% formic acid) (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min on an Agilent Extend C18 column. The analytes were detected by LC-MS/MS using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of m/z 408.4→271.0 and m/z 584.5→271.0 were used to detect EZM and EZM-G, respectively. The analytical method was linear over the concentration range of 0.1–20 ng/mL for EZM and 0.5–200 ng/mL for EZM-G. Within- and between-run precision for EZM was no more than 8.6% and 12.8%; and for EZM-G was no more than 9.0% and 8.7%, respectively. This method was reproducible and reliable, and was successfully used to analyze human plasma samples for application in a bioequivalence study.
Keywords: Ezetimibe; Ezetimibe glucuronide; LC-MS/MS; SPE;

Direct detection of Mycobacterium tuberculosis in sputum using combined solid phase extraction–gas chromatography–mass spectrometry by Ngoc A. Dang; Marta Mourão; Sjoukje Kuijper; Elisabetta Walters; Hans-Gerd Janssen; Arend H.J. Kolk (115-122).
Recently, thermally-assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry (THM–GC–MS) in combination with chemometrics has been used to develop a 20-compound model for fast differentiation of Mycobacterium tuberculosis (MTB) from Non-tuberculous mycobacteria (NTM) in bacterial cultures. This model provided better than 95% accuracy. In our current work a hexane/methanol/water extraction followed by a solid phase extraction (SPE) clean-up procedure was developed for use before THM–GC–MS, to make the test suitable for the identification of mycobacteria in sputum. The 20 biomarker model had to be adapted since many compounds were also found in the sputum of non-tuberculosis patients. An algorithm was established based on tuberculostearic acid, hexacosanoic acid and mycoserosates. The detection limit of the method was approximately 1 × 104 bacteria/mL sputum. Sputum specimens from 32 patients from South Africa who were suspected of having tuberculosis were blindly tested using the new method. Eight of the nine culture-positive sputum specimens were detected by the new SPE–THM–GC–MS method, resulting in a sensitivity of 89%. The specimen that was missed by the new method was also microscopy negative. The specificity of the test was 100%; all 23 microscopy- and culture-negative specimens were correctly identified as negative by SPE–THM–GC–MS.
Keywords: Mycobacterium tuberculosis; Direct detection; Sputum; Solid phase extraction; Gas chromatography–mass spectrometry;

Ceramides are derivatised using 7-(diethylamino)coumarin-3-carbonyl azide; subsequent gradient HPLC separation allows sensitive optical quantification of individual cellular ceramides. Compared to 9-anthracenecarbonyl cyanide (9-anthroyl nitrile) as derivatisation agent, the limit of detection could be improved 415-fold, respectively 10,000-fold (detection limit 0.6 pmol labelled ceramide/sample) when compared to benzoyl chloride-labelling. Acidic or alkaline catalysts are not required, enabling drying and storing of the labelled samples and a free choice of solvents for subsequent HPLC-separation. The quantitative method is characterised by high sensitivity, linearity and robustness in the pico- to nanomolar concentration range and does not require mass-spectrometry for quantification of cellular ceramides.
Keywords: Cellular ceramide quantification; Reversed phase HPLC; Fluorescence labelling;

Determination of zolpidem in human plasma by liquid chromatography–tandem mass spectrometry for clinical application by Ji-Yeong Byeon; Hye-In Lee; Yun-Jeong Lee; Jung-Eun Lee; Se-Hyung Kim; Young-Hoon Kim; Han-Sung Na; Choon-Gon Jang; Seok-Yong Lee (129-134).
Zolpidem (ZPD) is widely described for the short-term treatment of insomnia. We have developed and validated a simple and rapid liquid chromatography analytical method using tandem mass spectrometry (LC–MS/MS) for the quantification of ZPD in human plasma. Using dibucaine as an internal standard (IS), the analyte was extracted with methyl t-butyl ether (MTBE). Chromatographic separation of ZPD was performed on a reversed-phase Luna C18 column (50 mm × 2.0 mm i.d., 5 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.0)–methanol (15:85, v/v) at a flow rate of 250 μm/min. The total run-time was 2.5 min and the retention times of ZPD and IS were 0.66 and 0.74 min, respectively. The mass-to-charge transition monitored for quantification of ZPD and IS was 308.2 → 235.2 and 344.0 → 271.0, respectively. The lower limit of quantification (LLOQ) using 100 μL of human plasma was 0.05 ng/mL and the calibration curves were linear over a range of 0.05–200 ng/mL (r 2  > 0.9964). The mean accuracy and precision for intra- and inter-run validation of ZPD were within acceptable limits. In the present LC–MS/MS method, we showed improved sensitivity for quantification of the ZPD in human plasma using lower volume of plasma compared with previously described analytical methods for ZPD. This validated method was successfully applied to a pharmacokinetic study in humans.
Keywords: Zolpidem; LC–MS/MS; Human plasma; Pharmacokinetics;

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography–mass spectrometry by Simona C. Nicoara; Nicholas W. Turner; David E. Minnikin; Oona Y.-C. Lee; Denise M. O'Sullivan; Ruth McNerney; Reggie Mutetwa; Liz E. Corbett; Geraint H. Morgan (135-142).
A proof of principle gas chromatography–mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36–68% for PDIMs, allowing some superior detection of the target analytes.
Keywords: Cholesterol; Mycobacterium tuberculosis; Solid phase extraction; Molecularly imprinted polymers; Thermochemolysis; GC–MS;

A method of capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection has been applied to detect three major phenylurea herbicides (monuron, monolinuron and diuron) simultaneously. The effects of yam sample preparation, injection voltage and time, detection potential, detection buffer concentration and pH, Ru(bpy)3 2+ concentration, separation buffer type, separation buffer pH and concentration, separation voltage were investigated in detail. Under optimum conditions, a good baseline separation and highly sensitive detection for monuron, monolinuron and diuron were achieved. The ECL intensity (I) was in proportion to three analytes concentration (ρ) in the range of 0.1–10,000 μg/L for monuron (r  ≥ 0.9993), 0.1–18,000 μg/L for monolinuron (r  ≥ 0.9995) and 0.1–20,000 μg/L for diuron (r  ≥ 0.9997). The detection limits for monuron, monolinuron and diuron were 0.05, 0.04 and 0.01 μg/L (S/N  = 3), respectively. The developed method was successfully applied for the analysis of monuron, monolinuron and diuron residues in yam simultaneously. The average recoveries are in the ranges of 90.0–99.2% with relative standard deviations less than 3.2%. The limits of detection of the proposed method were 0.010 μg/kg for monuron, 0.008 μg/kg for monolinuron and diuron in yam.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Monuron; Monolinuron; Diuron; Yam;