Journal of Chromatography B (v.930, #C)

Pharmacokinetics of ganoderic acid D and its main metabolite by liquid chromatography–tandem mass spectrometry by Chun-Ru Cheng; Min Yang; Shu-Hong Guan; Xiao-Hui Wu; Xiao-Yan Pang; Yang Wang; Yi Yang; Jie Ding; De-An Guo (1-6).
The present study aims to investigate the pharmacokinetics of ganoderic acid D (GD), a representative active triterpenoid from Ganoderma lucidum. A sensitive and selective liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of the concentrations of GD and its main metabolite (ganoderic acid B) in rat plasma. Following protein precipitation, the analytes were separated on a reversed-phase C18 column. Acetonitrile–water–acetic acid (40:60:0.01) was used at a flow-rate of 0.2 ml/min. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used as the detector and was operated in the negative ion mode. Multiple reaction monitoring using the characteristic transitions was performed to quantify the analytes. The method had a lower limit of quantification of 8.19 ng/ml for GD, and 8.59 ng/ml for ganoderic acid B (GB). The calibration curves were demonstrated to be linear over the concentration range of 8.19–4096 ng/ml and 8.59–2149 ng/ml, respectively. Variations within- and between-batch were less than 6.4% and 4.6%, respectively. The extraction recovery rates ranged from 98.8 to 105.2% and 100.7 to 113.6%, respectively. The validated method was successfully applied to the quantification of GD and GB concentrations in rat plasma after oral administration (or intravenous administration) of GD preparations at a dose of 15 mg/kg. The data showed that the absolute bioavailability increased from 22% to 70% after the GD suspension was changed to GD loaded solid lipid nanoparticles. In the meantime, the C max increased from 107.2 to 1555.6 ng/ml; the t max changed from 2.0 h to 0.3 h. These results are very helpful in the further studies.
Keywords: Ganoderic acid D; Ganoderic acid B; LC/MS/MS; Nanoparticle; Pharmacokinetics; Triterpenoid;

Quantitative analysis of autoinducing peptide I (AIP-I) from Staphylococcus aureus cultures using ultrahigh performance liquid chromatography–high resolving power mass spectrometry by Hiyas A. Junio; Daniel A. Todd; Keivan A. Ettefagh; Brandie M. Ehrmann; Jeffrey S. Kavanaugh; Alexander R. Horswill; Nadja B. Cech (7-12).
Staphylococcus aureus infections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant S. aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25 μM and a linear dynamic range of 2.6 to 63 μM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16 h. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.
Keywords: UHPLC; Staphylococcus aureus; Virulence; AIP-I; Mass spectrometry;

Para-aminobenzamidine linked regenerated cellulose membranes for plasminogen activator purification: Effect of spacer arm length and ligand density by Ezio Fasoli; Yiaslin Ruiz Reyes; Osiris Martinez Guzman; Alexandra Rosado; Vivian Rodriguez Cruz; Amaris Borges; Edmarie Martinez; Vibha Bansal (13-21).
Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: (i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; (ii) the affinity ligand (pABA) density on membrane surface (5–25 nmol/cm2). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi-component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14-atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmoles of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in mono-component system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11 ± 1.0 nmol/cm2) in mono- and multi-component systems for 7- as well as 14-atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.
Keywords: Affinity membrane; Plasminogen activator; Para-aminobenzamidine; Spacer arm; Ligand density;

A rapid multi-method was developed for the determination of 21 growth promoters from different classes, including gestagens, corticosteroids, RALs, stilbenes, steroids in bovine milk by liquid chromatography–tandem mass spectrometry (LC–MS/MS). All compounds were eluted from the analytical column in less than 8.5 min and were subsequently analyzed with atmospheric pressure chemical ionization (APCI) using both positive and negative mode. Sample preparation included extraction of the compounds with acetonitrile and purification with solid-phase extraction (SPE). The method was validated according to Commission Decision 2002/657/EC, at a validation level of 1 ng/ml. The specificity, accuracy, precision, decision limit (CCα) and the detection capability (CCβ) were satisfactory evaluated. The recoveries ranged from 80.7% to 118.8% and reproducibility represented as coefficient of variance (CV) was from 1.8% to 13.0%. The CCα and CCβ values were in the ranges 0.06–0.10 ng/ml and 0.11–0.17 ng/ml, respectively. The developed method was applied in real samples proving its rapidness and sensitivity for the determination of the 21 growth promoters.
Keywords: Growth promoters; Hormones; Milk; Validation; Liquid chromatography; Mass spectrometry;

Sophisticated network of quorum sensing involves the production of chemical signals which regulate the combined expression of virulence genes and biofilm formation in Pseudomonas aeruginosa. Two well-characterized acyl homoserine lactone based las and rhl systems together with alkyl quinolone based Pseudomonas quinolone signalling (PQS) are fundamental components of this network. Third signalling molecule, 2-heptyl-3-hydroxy-4-quinolone (PQS) is of paramount importance because of its interconnecting role in quorum sensing hierarchy in P. aeruginosa. Accurate detection of PQS molecule is very important to understand the involvement of this system in infection process of P. aeruginosa. In this study, high performance-thin layer chromatography (HP-TLC) method was developed for detection as well as quantification of PQS signal molecules in P. aeruginosa, which combines conventional method like TLC with sophisticated instrumentation. This method was validated using parameters like linearity, accuracy, precision, reproducibility and sensitivity. Intra- and inter-day accuracy and precision values were determined which were found to be within acceptable level and hence showed reproducibility. Measurement of PQS in the range of 0.01 nmol indicated excellent sensitivity of this approach for quantifying PQS molecule. Automated sampling, rapid and simultaneous analysis of large number of samples and minimal errors make this method more suitable for analysis of PQS signalling molecules. Production of PQS was found to be strain dependent since variation in amount of PQS was observed among different P. aeruginosa isolates. Further, PQS production was also dependent on growth phase of P. aeruginosa with maximum production in late stationary phase.
Keywords: Pseudomonas aeruginosa; PQS; Quorum sensing; High performance-thin layer chromatography;

Glycerol is an important compound participating in the lipid metabolism and energy conversion of body. Its level, especially in the blood circulation, has been considered as an index in assessing the triglycerides level, fat mobilization and potential risk of hyperlipidemia. In this gas chromatography mass spectrometry (GC–MS) method, 1,2,3-butanetriol was selected as an internal standard instead of isotope labeled glycerol. The internal standard and sample were derivatized by trimethylsilyl imidazole. The glycerol and internal standard derivatives in the sample were measured by a selected ion monitor mode of GC–MS. The sensitivity and repeatability of this method were examined by analyzing glycerol in eleven different types of biological tissue and fluid samples. The glycerol level in the measured mouse plasma sample was at 11.71 ± 0.48 μg/mL, while it was in a range of 0.15 ± 0.01 (brain) to 0.39 ± 0.02 μg/mg (liver) in the tissue samples. It was 27.06 ± 0.12 and 1.60 ± 0.04 μg/mL in the tested human blood and urine samples, respectively. Also, the glycerol recoveries of all samples were higher than 80% and over 90% for the fluid samples, especially. With the satisfactory repeatability and recovery and non-isotope internal standard, the GC–MS method could be a reliable technique in monitoring the glycerol status of biological samples regardless of whether they are from a study in which isotope labeled glycerol or other stable isotope materials were involved.
Keywords: Glycerol; Triglyceride; Fatty acids; Hyperlipidemia; Lipid;

Determination of nitrite and nitrate in cerebrospinal fluid by microchip electrophoresis with microsolid phase extraction pre-treatment by Peter Troška; Richard Chudoba; Ladislav Danč; Róbert Bodor; Michal Horčičiak; Eva Tesařová; Marián Masár (41-47).
A new method for the determination of nitrite and nitrate, indicators of various neurological diseases (meningitis, multiple sclerosis, Parkinson's disease) in cerebrospinal fluid (CSF) on an electrophoresis chip was developed. An on-line combination of isotachophoresis (ITP) with capillary electrophoresis (CE) on a poly(methylmethacrylate) chip assembled with coupled separation channels (CC) and contact conductivity detectors was employed. ITP separations performed at low pH (3.6) in the first separation channel enabled a highly selective transfer of the analytes to the second CE stage working under micellar conditions implemented by zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)-propanesulfonate. The proposed method achieved low limits of detection varied from 0.2 to 0.4 μg L−1 when the sample volume injected onto the chip (9.9 μl) was almost the same as the volume of both separation channels. Preferable working conditions on the CC chip (suppressed hydrodynamic and electroosmotic flow) contributed for reproducible migration velocities (intra-day reproducibility up to 2.1% RSD) and determinations of trace concentrations of nitrite and nitrate (intra-day precision up to 3.0% RSD). Huge amount of chloride present in CSF (approx. 4.5 g L−1) was removed from analyzed CSF samples by microsolid phase extraction performed on silver-form resin prior to the ITP-CE analysis. Developed method provided fast (approx. 20 min total analysis time) and reliable determinations of trace nitrite and nitrate and could be fully integrated into the analysis of CSF samples.
Keywords: Nitrite and nitrate; Cerebrospinal fluid; Microchip electrophoresis; Microsolid phase extraction;

On-line screening of matrix metalloproteinase inhibitors by capillary electrophoresis coupled to ESI mass spectrometry by Xu Wang; Zhiying Dou; Yaozuo Yuan; Shuli Man; Kris Wolfs; Erwin Adams; Ann Van Schepdael (48-53).
Capillary electrophoresis (CE) with the use of mass spectrometry (MS) has been considered as a unique tool for microscale enzyme assay and inhibitor screening. In this study, matrix metalloproteinase-9 (MMP-9) was selected as target enzyme due to its important role in tumor invasion and metastasis. In order to define the optimal MS parameters, a two level half fraction factorial experimental design was performed. A background electrolyte consisting of 20 mM ammonium acetate (pH 6.8) and a sheath liquid of water–methanol (50:50, v/v) containing 0.05% formic acid at a flow rate of 4 μl/min were selected. This system was operated in the positive ion mode with a detection-limit of 10 nM for the MMP reaction product and provided 60 folds enhancement of sensitivity by using selected reaction monitoring detection compared with MS full scan mode, which significantly increased the detectability of the system and therefore reduced the enzyme reaction time in both off-line and in-line mode. Both electrophoretically mediated microanalysis and pressure mediated microanalysis combined with MS detection were investigated for MMP inhibitor screening. Good repeatability (RSD of peak area and migration time were lower than 5.0%) and linearity (R 2  > 0.996) were obtained for both in-capillary approaches. Several tetracycline antibiotics and natural products were selected to test the system. The results indicated an agreement on the ranking of inhibitory potency for both in-capillary approaches.
Keywords: Matrix metalloproteinase; Inhibitor screening; CE/MS; On-line; Electrophoretically mediated microanalysis; Pressure mediated microanalysis;

A method for semi-preparative isolation of major resveratrol metabolites from human urine after oral intake of a trans-resveratrol-containing dietary supplement was developed. Pretreatment of the urine (6 L) by using solid-phase extraction gave a brown oily residue (9.3 g), which was separated using a combination of normal phase column chromatography and reversed-phase flash column chromatography resulting in fractions containing 1.1 g crude trans-resveratrol-3-O-sulfate (M1), 86 mg of a crude mixture of trans-resveratrol-3,5-O-disulfate (M2) and trans-resveratrol-3,4′-O-disulfate (M3), and 568 mg of a crude mixture of trans-resveratrol-3-O-β-d-glucuronide (M4) and dihydroresveratrol-3-O-β-d-glucuronide (M5). Purification of the crude metabolites was performed by semi-preparative reversed-phase HPLC using a gradient of aqueous ammonium acetate (2.5 mmol/L, pH 6.7)/acetonitrile for purification of M1, M2 and M3 or trifluoroacetic acid in water (pH 2.5)/acetonitrile for purification of M4 and M5. From a part of the crude metabolites (50–75 mg), 47 mg M1 (purity 98.7%), 14 mg M2 (purity 96.1%), 10 mg M3 (purity 96.3%), 38 mg M4 (purity 98.2%) and 18 mg M5 (purity 97.8%) were obtained. The structures of all isolated resveratrol metabolites were elucidated by spectroscopic and spectrometric methods such as 1D and 2D NMR, UV, and LC–MS. This method represents a novel approach to obtain resveratrol metabolites being the first method describing the direct isolation of pure resveratrol metabolites from urine samples in quantities sufficient for full chemical characterization and testing in vitro and in preclinical trials.
Keywords: Resveratrol metabolites; Urine; Solid-phase extraction; Column chromatography; Semi-preparative HPLC; NMR spectroscopy;

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic–electrospray ionization-tandem mass spectrometric method (LC–ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol–formic acid, purified by Hybrid-SPE cartridges and subjected to LC–ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p  < 0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain.
Keywords: Pregnenolone sulphate; Allopregnanolone; Dehydroepiandrosterone; HPLC–ESI-MS/MS; Rat brain tissue;

A method for determination of proline–hydroxyproline dipeptide (PHP) was developed using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). This method resulted in good separation of proline (Pro), hydroxyproline (Hyp), and PHP within 20 min using a mobile phase of 1.2 mM Ba(OH)2  + 1.5 mM Ba(OAc)2. The linear dynamic ranges and their detection limits (S/N  = 3) were 1–100 (r 2  = 0.9990–0.9999) and 0.05–0.3 μM, respectively. Mean recoveries were 91.6–121.3% and 92.2–110.3% for intra- and inter-day assays, respectively. Our HPAEC-PAD method showed clear differences in the corrected PHP levels measured in urine samples from two groups of rats, sham-operated and ovariectomized, without the need for prior acid hydrolysis or sample derivatization.
Keywords: Anion-exchange chromatography; Pulsed amperometric detection; Barium hydroxide; Proline–hydroxyproline; Hydroxyproline;

Development of a simple analytical method for the simultaneous determination of paracetamol, paracetamol-glucuronide and p-aminophenol in river water by Lúcia H.M.L.M. Santos; Paula Paíga; Alberto N. Araújo; Angelina Pena; Cristina Delerue-Matos; M. Conceição B.S.M. Montenegro (75-81).
Paracetamol is among the most worldwide consumed pharmaceuticals. Although its occurrence in the environment is well documented, data about the presence of its metabolites and transformation products is very scarce. The present work describes the development of an analytical method for the simultaneous determination of paracetamol, its principal metabolite (paracetamol-glucuronide) and its main transformation product (p-aminophenol) based on solid phase extraction (SPE) and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). The method was applied to analysis of river waters, showing to be suitable to be used in routine analysis. Different SPE sorbents were compared and the use of two Oasis WAX cartridges in tandem proved to be the most adequate approach for sample clean up and pre-concentration. Under optimized conditions, limits of detection in the range 40–67 ng/L were obtained, as well as mean recoveries between 60 and 110% with relative standard deviations (RSD) below 6%. Finally, the developed SPE-HPLC/DAD method was successfully applied to the analysis of the selected compounds in samples from seven rivers located in the north of Portugal. Nevertheless all the compounds were detected, it was the first time that paracetamol-glucuronide was found in river water at concentrations up to 3.57 μg/L.
Keywords: Paracetamol; Paracetamol-glucuronide; p-Aminophenol; Solid phase extraction; HPLC; River water;

Hydrophilic interaction liquid chromatography with tandem mass spectrometric detection applied for analysis of pteridines in two Graphosoma species (Insecta: Heteroptera) by Petr Kozlík; Jan Krajíček; Květa Kalíková; Eva Tesařová; Radomír Čabala; Alice Exnerová; Pavel Štys; Zuzana Bosáková (82-89).
A new separation method involving hydrophilic interaction chromatography with tandem mass spectrometric detection has been developed for the analysis of pteridines, namely biopterin, isoxanthopterin, leucopterin, neopterin, xanthopterin and erythropterin in the cuticle of heteropteran insect species. Two columns, Atlantis HILIC Silica and ZIC®-HILIC were tested for the separation of these pteridines. The effect of organic modifier content, buffer type, concentration and pH in mobile phase on retention and separation behavior of the selected pteridines was studied and the separation mechanism was also investigated. The optimized conditions for the separation of pteridines consisted of ZIC®-HILIC column, mobile phase composed of acetonitrile/5 mM ammonium acetate, pH 6.80, 85/15 (v/v), flow rate 0.5 mL/min and column temperature 30 °C. Detection was performed by tandem mass spectrometry operating in electrospray ionization with Agilent Jet Stream technology using the selected reaction monitoring mode. The optimized method provided a linearity range from 0.3 to 5000 ng/mL (r  > 0.9975) and repeatability with relative standard deviation < 8.09% for all the studied pteridines. The method was applied to the analysis of pteridines in the cuticle of larvae and three adult color forms of Graphosoma lineatum and one form of Graphosoma semipunctatum (Insecta: Hemiptera: Heteroptera: Pentatomidae). The analysis shows that different forms of Graphosoma species can be characterized by different distribution of individual pteridines, which affects the coloration of various forms. Only isoxanthopterin was found in all the five forms tested.
Keywords: Hydrophilic interaction liquid chromatography; Tandem mass spectrometry; Pteridines; Graphosoma; Insecta; Heteroptera;

Aqueous size-exclusion chromatographic method for the quantification of cyanobacterial native glycogen by Yoshihiro Izumi; Shimpei Aikawa; Fumio Matsuda; Tomohisa Hasunuma; Akihiko Kondo (90-97).
Cyanobacterial glycogen has gained interest as a valuable biomass feedstock for biofuel production. However, an ideal method for native glycogen quantification has not been developed. Here, we have proposed a simple methodology that enables the quantitative determination of cyanobacterial glycogen concentration with high repeatability using aqueous size-exclusion chromatography with a differential refractive index detector (SEC/RID). Our SEC/RID system also allows size distributions for native glycogen based on hydrodynamic volumes (V h ), which is proportional to the product of the molecular mass (M) and intrinsic viscosity [η], obtained by universal calibration using linear homopolymers of known M with Mark–Houwink–Sakurada parameters. The universal calibration curve achieved a broad linear range (V h parameter [η]M  = 2 × 102–8 × 108  mL g−1) with a high correlation coefficient (R 2  = 0.9942), because the developed system is equipped with an OHpak SB-806M HQ aqueous column containing four types of polyhydroxy methacrylate-based particles with different particle and pore sizes. Based on the SEC/RID system, response of molecular size distribution of glycogen in microalgae to the cultivation condition was first observed. Our established SEC/RID method has several advantages over conventional techniques, including the simultaneous quantitative and size distribution analyses of glycogen, and represents a potentially useful tool to elucidate the relationship between structural properties and the roles of glycogen in metabolism.
Keywords: Size-exclusion chromatography; Native glycogen; Quantitative analysis; Polyhydroxy methacrylate; Microalgae; Arthrospira platensis;

Described below is an optimized solid-phase extraction method (SPE) for the simultaneous determination of three hazardous plastic additives. Two high-performance liquid chromatographic–electrospray ionization-mass spectrometric (HPLC–ESI-MS) methods were developed and validated to estimate the melamine (MEL), bis(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA) contents in drinking water, syringes, soft drinks, and dry milk powder. One extraction procedure optimally recovered all three substances from the different matrices. Two extraction columns were combined and included a silica gel LiChroprep RP-2 column (20:1, g/g, top column) and a Sep-Pak with a C18 column (500 mg, bottom column). The analytical column was an Agilent Eclipse XDB-C8 column, 4.6 mm × 150 mm, 5 μm, maintained at 50 ± 2 °C. MEL and DEHP were monitored by positive triple quad mass spectrometry (TQ-MS) using an acidic mobile phase, while BPA was monitored by negative TQ-MS using a mobile phase containing a 0.05% ammonia solution. The general linear range of the three compounds ranged from 12 to 1000 pg/μL in the injected solution (25 μL). The average extraction recoveries were within the range of 83.0–102.5%. Relatively high concentrations of BPA and DEHP were found in the milk powder and sterile syringes.
Keywords: Bisphenol A; Melamine; Bis(2-ethylhexyl) phthalate; LC–MS; Different matrices;

For the first time a simple, selective and sensitive liquid chromatography method was developed and validated for the simultaneous determination of levofloxacin (LEV), pazufloxacin (PAZ), gatifloxacin (GAT), moxifloxacin (MOX) and trovafloxacin (TRO) in human plasma. Samples were pre-treated with acetonitrile for precipitation of plasma proteins followed by evaporation and reconstitution steps. Chromatographic separation of the analytes and norfloxacin, used as internal standard (IS), was performed under gradient elution on a LiChroCART® Purospher Star C18 column (55 mm × 4 mm, 3 μm). The mobile phase comprised a mixture of 0.1% aqueous formic acid adjusted to pH 3.0 with triethylamine, acetonitrile and methanol pumped at a flow rate of 1.0 mL/min. The detector was set at excitation/emission wavelengths of 260/455 nm. Calibration curves were linear (r 2  ≥ 0.9923) in the ranges of 0.005–5 μg/mL for GAT, 0.02–5 μg/mL for LEV, PAZ and MOX and 0.04–5 μg/mL for TRO. The intra and interday precision did not exceed 7.32% and the intra and interday accuracy ranged from −11.73 to 8.92%. The limits of quantification were established at 0.005 μg/mL for GAT, 0.02 μg/mL for LEV, PAZ and MOX and 0.04 μg/mL for TRO. No endogenous or tested exogenous compounds were found to interfere at the retention times of the analytes and IS. Since the proposed method proved to be reliable for the quantitative determination of LEV, PAZ, GAT, MOX and TRO it may be a useful tool for routine analysis and to support clinical pharmacokinetic and toxicological studies involving these antibiotics.
Keywords: Bioanalytical method validation; Fluoroquinolone; HPLC; Human plasma; Protein precipitation; Therapeutic drug monitoring;

The advent of new not yet legally regulated psychoactive substances sold over the Internet has created a challenge for clinical toxicology and drug testing laboratories. The routine use of immunoassay screening may no longer be the optimal solution in many instances since the number of analytes covered is becoming insufficient. The aim of this work was to design, validate and apply a multi-component LC–MS/MS method suitable for screening of a large number of target analytes belonging to the class of new psychoactive substances – legal highs. The analytical method was using a five-fold dilution of urine with internal standard (pethidine-d5) and injection of 2 μL. The chromatographic system was using a 1.7-μm 100 mm × 2.1 mm Ethylene Bridged Hybrid (BEH) C18 column and gradient elution with a flow rate of 600 μL/min. Solvent A consisted of 0.1% formic acid and Solvent B was 100% acetonitrile. The gradient elution application was designed to have a wide polarity coverage with total run time of 4.0 min. The tandem mass spectrometer was using an electrospray interface and operated in positive mode. Selected reaction monitoring of two ion transitions was used for each of 26 analytes. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (0.1–10 μg/mL), acceptable imprecision in quantification (CV < 15%). Some analytes were found not to be stable in urine upon storage. The method was successfully applied in routine drug testing. A total of 87 positive samples with 100 analytical findings were found to contain O-desmethyl-cis-tramadol (mostly without mitragynine), methylenedioxypyrovalerone, 4-fluoroamphetamine, methoxetamine, desoxypipradol, 4-fluoromethcathinone, 5,6-methylenedioxy-2-aminoindane, 4-methylmethcathinone, 3-fluoromethcathinone, 4-hydroxy-N-methyl-N-ethyltryptamine, α-methylamino-butyrophenone and 4-methoxymethcathinone.
Keywords: Urine; Liquid chromatography; Mass spectrometry; Internet drugs; Legal highs; Screening;

Measurement of tamsulosin in human serum by liquid chromatography–tandem mass spectrometry by Rita Upreti; Natalie Z.M. Homer; Gregorio Naredo; Diego F. Cobice; Katherine A. Hughes; Laurence H. Stewart; Brian R. Walker; Ruth Andrew (121-128).
A simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100 μL) via liquid–liquid extraction with methyl tert-butyl ether (2 mL) following dilution with 0.1 M ammonium hydroxide (100 μL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis® Express C18 (100 mm × 3 mm, 2.7 μm) column using a gradient elution with mobile phases methanol and 2 mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6 min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409 → 228 and d9-finasteride m/z 382 → 318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10 ppm). The limit of quantitation was 0.2 ng/mL, and the method was validated in the linear range 0.2–50 ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia.
Keywords: Tamsulosin; Finasteride; Liquid chromatography; Mass spectrometry; Serum; Prostate;

There is significant interest in the development of methods to validate novel biomarkers for Alzheimer's disease (AD) diagnosis. Previously, a proteomic panel of cerebrospinal fluid (CSF) biomarker candidates that differentiated AD and non-AD CSF with accuracy higher than 90% was found; information about these CSF proteins can be used to develop multiple reaction monitoring (MRM) based analytical assays, which offer the possibility of quantifying protein expression level changes in samples, as well as, validation among multiple laboratories. Here we report an MRM assay that demonstrates good linearity (average R 2  = 0.969) and reproducibility (average coefficient of variance of 6.93%) for the proposed AD CSF biomarkers. MRM quantification results of Aβ1-40, Aβ1-42, retinol-binding protein and cystatin C correlated well with those from ELISA (average R 2  = 0.974). Analysis shows that 12 out of 16 selected targets exhibit the same trend in protein expression as that in literature.
Keywords: Alzheimer's disease (AD); Biomarker; Cerebrospinal fluid (CSF); Targeted proteomics; Multiple reaction monitoring (MRM);

A simple, selective and robust reverse phase high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous quantitation of pioglitazone (PIO), pioglitazone metabolite M-IV – hydroxypioglitazone (OH-PIO) and metformin (MET) in human plasma using deuterated internal standards (IS) is developed and fully validated as per industrial practices. After acetonitrile-induced protein precipitation of the plasma samples; PIO, OH-PIO, MET and IS were chromatographed on reverse phase column and analyzed in the multiple reaction monitoring in positive ion mode. The ion transitions were monitored at m/z 357.2 → 134.2 for PIO, 373.0 → 150.1 for OH-PIO, 130.2 → 71.0 for MET, 361.1 → 134.2 for PIO-IS and 136.1 → 77.1 for MET-IS. The total chromatographic run time was 4.0 min. A linear response function (r  > 0.998) was established for the range of concentrations 15–2500 ng/mL, 10–1500 ng/mL and 25–3000 ng/mL for PIO, OH-PIO and MET respectively in human plasma. The intra and inter-day precision and accuracy values have met the set acceptance criteria. The method is simple, selective, robust economic and has been applied successfully to more than 2000 plasma samples as part of pharmacokinetic study in humans.
Keywords: Pioglitazone; Hydroxypioglitazone; Metformin; Human plasma; LC–MS/MS; Pharmacokinetics;