Journal of Chromatography B (v.1043, #C)

Bioanalytical method development and validation: Critical concepts and strategies by Mohammad Mahdi Moein; Aziza El Beqqali; Mohamed Abdel-Rehim (3-11).
Bioanalysis is an essential part in drug discovery and development. Bioanalysis is related to the analysis of analytes (drugs, metabolites, biomarkers) in biological samples and it involves several steps from sample collection to sample analysis and data reporting. The first step is sample collection from clinical or preclinical studies; then sending the samples to laboratory for analysis. Second step is sample clean-up (sample preparation) and it is very important step in bioanalysis. In order to reach reliable results, a robust and stable sample preparation method should be applied. The role of sample preparation is to remove interferences from sample matrix and improve analytical system performance. Sample preparation is often labor intensive and time consuming. Last step is the sample analysis and detection. For separation and detection, liquid chromatography-tandem mass spectrometry (LC–MS/MS) is method of choice in bioanalytical laboratories. This is due to high selectivity and high sensitivity of the LC–MS/MS technique. In addition the information about the analyte chemical structure and chemical properties is important to be known before the start of bioanalytical work.This review provides an overview of bioanalytical method development and validation. The main principles of method validation will be discussed. In this review GLP and regulated bioanalysis are described. Commonly used sample preparation techniques will be presented. In addition the role of LC–MS/MS in modern bioanalysis will be discussed. In the present review we have our focus on bioanalysis of small molecules.
Keywords: Bioanalysis; Bioanalytical method development; Sample preparation; Liquid chromatography; Tandem mass spectrometry; Method validation; GLP;

High throughput solid phase microextraction: A new alternative for analysis of cellular lipidome? by Afsoon Pajand Birjandi; Barbara Bojko; Zhibin Ning; Daniel Figeys; Janusz Pawliszyn (12-19).
A new SPME method for untargeted lipidomic study of cell line cultures was proposed for the first time. In this study the feasibility to monitor changes in lipid profile after external stimuli was demonstrated and compared to the conventional Bligh & Dyer method. The human hepatocellular carcinoma (HCC) cell line was used as a model. The obtained results provided a list of up-regulated and down-regulated lipids through a comparison between control (non-stimulated) cells versus the group of cells treated with polyunsaturated fatty acid (20:5). Use of the SPME technique yielded a list of 77 lipid species whose concentrations were recognized to be significantly different between control and treated cells, from which 63 lipids were up-regulated in treated cells. In general, the list was comparable to the peer list obtained by the Bligh & Dyer method. However, more diversity of lipid classes and subclasses such as LPC, sphingomyelins, ceramides, and prenol lipids were observed with the application of the SPME method. Method precision for the SPME approach was within the acceptable analytical range (5–18% RSD) for all detected lipids, which was advantageous over solvent extraction applied. The evaluation of ionization efficiency indicated no matrix effect for the SPME technique, while Bligh & Dyer presented significant ionization suppression for low abundant species such as LysoPC, PG, ceramides, and sphingomyelins, and ionization enhancement for high abundant phospholipids such as PE.
Keywords: Lipidomics; Cell line study; Solid phase microextraction; Automation;

In this work, for the first time, a method has been developed for the determination of AZD6118, a candidate drug, in dog plasma samples. The method is based on microextraction by packed sorbent (MEPS) of the drug prior to liquid chromatography-electrospray ionization tandem mass spectrometry assay. Various important factors affecting MEPS performance were optimized, and under the optimized condition, a linear calibration curve in the concentration range of 20–25,000 nmol L−1 with a coefficient of determination over 0.99 was obtained. The back-calculated values of the calibration points showed good agreement with the theoretical concentrations (coefficients of variation percent between 0.3–3.8). The lower limit of quantification and limit of detection were 20.0 and 2.9 nmol L−1, respectively. The repeatability and accuracy of the method was evaluated by determination of quality control samples at three concentration levels (low, medium and high) using the developed method, and the results (coefficients of variation values were between 1.9% and 3.2%, relative recoveries ranged between 93.5–102.1%) confirm that a powerful method has been developed for the extraction and determination of the investigated drug in dog plasma.
Keywords: Microextraction by packed sorbent; AZD6118; Dog plasma samples; LC-ESI–MS/MS; Validation;

One-step extraction of polar drugs from plasma by parallel artificial liquid membrane extraction by Veronika Pilařová; Mumtaz Sultani; Kristine Skoglund Ask; Lucie Nováková; Stig Pedersen-Bjergaard; Astrid Gjelstad (25-32).
The new microextraction technique named parallel artificial liquid membrane extraction (PALME) was introduced as an alternative approach to liquid–liquid extraction of charged analytes from aqueous samples. The concept is based on extraction of analytes across a supported liquid membrane sustained in the pores of a thin polymeric membrane, a well-known extraction principle also used in hollow fiber liquid-phase microextraction (HF-LPME). However, the new PALME technique offers a more user-friendly setup in which the supported liquid membrane is incorporated in a 96 well plate system. Thus, high-throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3–5 μL per sample. With a sample volume of 250 μL and acceptor solution volume of 50 μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC–MS. Hence, the sample preparation is performed in one step. With optimized conditions, the extraction recoveries were in the range 50–89% from human plasma after 45 min extraction. The data from the method evaluation were satisfactory and in line with current guidelines, and revealed an extraction method with substantial potential for high throughput bioanalysis of polar basic drugs.
Keywords: Parallel artificial liquid membrane extraction; PALME; Microextraction; Supported liquid membrane; Carrier-mediated transport;

Microextraction by packed sorbent (MEPS) is a new miniaturized form of solid-phase extraction and it is a green sample pretreatment technology. MEPS has been widely accepted and used by several research groups online or offline as a sample preparation technique before instrument analysis. MEPS reduces the sample handling time and organic solvent consumption. MEPS is suitable for small sample volumes and can easily be connected with different chromatographic techniques without modification. The sorbent bed in MEPS is integrated into a liquid handling syringe that allows for low void volume sample manipulations either manually or in combination with laboratory robotics. MEPS is a simple, fast and robust sample preparation technique with several advantages, miniaturization, automation, fast operation course, on-line coupling with analytical instruments and low-cost operation with less solvent and low sample consumption. Sorbent type, device, and matrix are important factors in MEPS research and applications. The performance of MEPS has recently been illustrated by online with liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry assays for pharmaceutical, environmental, and food analyses. This paper deals with MEPS device-optimized sorbent, sample matrix, and application. The progress and potential development of the technique are also discussed.
Keywords: Microextraction by packed sorbent (MEPS); Sorbent; Sample matrix; Sample pretreatment; Application;

Green approaches in sample preparation of bioanalytical samples prior to chromatographic analysis by Olga Filippou; Dimitrios Bitas; Victoria Samanidou (44-62).
Sample preparation is considered to be the most challenging step of the analytical procedure, since it has an effect on the whole analytical methodology, therefore it contributes significantly to the greenness or lack of it of the entire process.The elimination of the sample treatment steps, pursuing at the same time the reduction of the amount of the sample, strong reductions in consumption of hazardous reagents and energy also maximizing safety for operators and environment, the avoidance of the use of big amount of organic solvents, form the basis for greening sample preparation and analytical methods.In the last decade, the development and utilization of greener and sustainable microextraction techniques is an alternative to classical sample preparation procedures. In this review, the main green microextraction techniques (solid phase microextraction, stir bar sorptive extraction, hollow-fiber liquid phase microextraction, dispersive liquid − liquid microextraction, etc.) will be presented, with special attention to bioanalytical applications of these environment-friendly sample preparation techniques which comply with the green analytical chemistry principles.
Keywords: Green analytical chemistry; Microextraction; Bioanalysis; Bioanalytical samples;

Cannabis is one of the most available and consumed illicit drug in the world and its identification and quantification in biological specimens can be a challenge given its low concentrations in body fluids. The present work describes a fast and fully validated procedure for the simultaneous detection and quantification of ▵9-tetrahydrocannabinol (▵9_THC) and its two main metabolites 11-hydroxy ▵9_tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-▵9- tetrahydrocannbinol (THC-COOH) in plasma samples using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC–MS/MS). A small plasma volume (0.25 mL) pre-diluted (1:20), was extracted with MEPS M1 sorbent as follows: conditioning (4 cycles of 250 μL methanol and 4 cycles of 250 μL 0.1% formic acid in water); sample load (26 cycles of 250 μL); wash (100 μL of 3% acetic acid in water followed by 100 μL 5% methanol in water); and elution (6 cycles of 100 μL of 10% ammonium hydroxide in methanol). The procedure allowed the quantification of all analytes in the range of 0.1–30 ng/mL. Recoveries ranged from 53 to 78% (THC), 57 to 66% (11-OH-THC) and 62 to 65% (THC-COOH), allowing the limits of detection and quantification to be set at 0.1 ng/mL for all compounds. Intra-day precision and accuracy revealed coefficients of variation (CVs) lower than 10% at the studied concentrations, with a mean relative error within ±9%, while inter-day precision and accuracy showed CVs lower than 15% for all analytes at the tested concentrations, with an inaccuracy within ±8%.
Keywords: Cannabis; Plasma; Microextraction by packed sorbent; Gas chromatography–tandem mass spectrometry;

Sometimes it is not necessary to separate the individual compounds of a sample to resolve an analytical problem, it is enough to obtain a signal profile of the sample formed by all the components integrating it. Within this strategy, electronic noses based on the direct coupling of a headspace sampler with a mass spectrometer (HS-MS) have been proposed. Nevertheless, this coupling is not suitable for the analysis of non-volatile compounds. In order to propose an alternative to HS-MS determinations for non-volatile compounds, here we present the first ‘proof of concept' use of the direct coupling of microextraction by packed sorbents (MEPS) to a mass spectrometer device using an electron ionization (EI) and a single quadrupole as ionization source and analyzer, respectively. As target compounds, a set of analytes with different physic-chemical properties were evaluated (2-ethyl-1-hexanol, styrene, 2-heptanone, among others). The use of MEPS extraction present many advantages, such as it is fast, simple, easy to automate and requires small volumes of sample and organic solvents. Moreover, MEPS cartridges are re-usable as samples can be extracted more than 100 times using the same syringe. In order to introduce into the system all the elution volume from the MEPS extraction, a programmable temperature vaporizer (PTV) is proposed as the injector device. Results obtained with the proposed methodology (MEPS-PTV/MS) were compared with the ones obtained based on the separative scheme, i.e. using gas chromatography separation (MEPS-PTV-GC/MS), and both methods provided similar results. Limits of detection were found to be between 3.26 and 146.6 μg L−1 in the non-separative scheme and between 0.02 and 1.72 μg L−1 when the separative methodology was used. Repeatability and reproducibility were evaluated with values below 17% in all cases.
Keywords: MEPS-MS; Electron ionization-single quadrupole; Programmable temperature vaporizer; Multivariate calibration;

New materials for sample preparation techniques in bioanalysis by Carlos Eduardo Domingues Nazario; Bruno Henrique Fumes; Meire Ribeiro da Silva; Fernando Mauro Lanças (81-95).
The analysis of biological samples is a complex and difficult task owing to two basic and complementary issues: the high complexity of most biological matrices and the need to determine minute quantities of active substances and contaminants in such complex sample. To succeed in this endeavor samples are usually subject to three steps of a comprehensive analytical methodological approach: sample preparation, analytes isolation (usually utilizing a chromatographic technique) and qualitative/quantitative analysis (usually with the aid of mass spectrometric tools). Owing to the complex nature of bio-samples, and the very low concentration of the target analytes to be determined, selective sample preparation techniques is mandatory in order to overcome the difficulties imposed by these two constraints. During the last decade new chemical synthesis approaches has been developed and optimized, such as sol-gel and molecularly imprinting technologies, allowing the preparation of novel materials for sample preparation including graphene and derivatives, magnetic materials, ionic liquids, molecularly imprinted polymers, and much more. In this contribution we will review these novel techniques and materials, as well as their application to the bioanalysis niche.
Keywords: Sample preparation; Sorbent materials; Sol-gel; Ionic liquids; Graphene; Magnetic materials; Molecularly imprinted polymer; Monolithic; Restricted access material;

Carbonaceous sorbents alongside an optimized magnetic solid phase extraction (MSPE) towards enrichment of crude Paclitaxel extracts from callus cultures of Taxus baccata by Jaber Nasiri; Mohammad Reza Naghavi; Elaheh Motamedi; Houshang Alizadeh; Mohammad Reza Fattahi Moghadam; Mohammad Nabizadeh; Alireza Mashouf (96-106).
Here, a number of synthesized and commercial nano-sorbents including graphene (G), multi-wall carbon nanotube (MWCNT), graphene oxide (GO), reduced graphene oxide (rGO) and magnetic nanoparticles anchored on GO (Fe3O4NPs@GO) followed by graphite (Gt) powder were employed for paclitaxel pre-purification from callus culture-derived extract of Taxus baccata. Based on our preliminary work, rGO and Fe3O4NPs@GO exhibited the maximum potential, not only to remove impurities, but also to ameliorate taxol purity. According to the results of response surface methodology (RSM) developed for the superior nano-sorbent (i.e., Fe3O4NPs@GO), for both proposed quadratic polynomial models, statistically substantial relationships obtained between the actual and predicted values (‎p < 0.0001‎). Furthermore, both agitation power and sorbent dosage compared to the sorption temperature exhibited more efficacies on the two responses of interest [i.e.‎, efficiency of calli-derived pigments removal (ECPR‎) and efficiency of taxol purity (ETP‎)]. Based on the results of simultaneous optimization to attain the highest ratio of ECPR‎ (‎‎‎96.35%) and ETP‎ (‎‎‎30.60‎‎ %), the values of 29.9 g/L, ‎‎‎‎29.6‎‎ °C and ‎‎‎165.5‎‎ rpm were respectively predicted for sorbent dosage, sorption temperature and agitation power, and subsequently certified through experimental rechecking survey. Remarkably, the sorption capacity and magnetism feature of the magnetic nano-sorbent remained nearly constant, even upon nine consecutive treatments. Considering the results as a whole, the combination of magnetic solid-phase extraction (MSPE) using the current Fe3O4NPs@GO nano-sorbent and RSM can be recommended as a simple, cost-effective and fast route for sorbent-assistant pre-purification investigations of paclitaxel.
Keywords: Carbonaceous sorbents; Magnetic solid phase extraction (MSPE); Paclitaxel; Response surface methodology (RSM); Taxus baccata;

Molecularly imprinted polymers for bioanalytical sample preparation by Mariana Roberto Gama; Carla Beatriz Grespan Bottoli (107-121).
Molecularly imprinted polymers (MIP) are stable polymers with molecular recognition abilities, provided by the presence of a template during their synthesis, and are excellent materials with high selectivity for sample preparation in bioanalytical methods. This short review discusses aspects of MIP preparation and its applications as a sorbent material in pharmaceutical and biomedical analysis. MIP in different extraction configurations, including classical solid-phase extraction, solid-phase microextraction, magnetic molecularly imprinted solid-phase extraction, microextraction by packed sorbent and solid-phase extraction in pipette tips, are used to illustrate the good performance of this type of sorbent for sample preparation procedures of complex matrices, especially prior to bioanalytical approaches.
Keywords: Molecularly imprinted polymers; Sorbents; Biomarkers; Metabolites; Bioanalysis;

Immobilized metal affinity chromatography (IMAC) technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins. In this study, nickel(II)-immobilized carboxyl cotton chelator (CCC-Ni2+) fibers was synthesized by a simple method based on the coordination effect between Ni2+ and carboxyl group. The nickel content of the CCC-Ni2+ fibers was determined to be 5 times larger than that of Ni2+-immobilized sulfhydryl cotton fiber (SCF-Ni2+) fibers developed in our previous work. The prepared CCC-Ni2+ fibers were then applied for the selective and rapid separation of His-tagged protein from escherichia coli (E. coli) cell lysates on the basis of the high affinity of Ni2+ to 6 × His with a lab-in-syringe format. Benefiting from the good biological compatibility and high nickel content, the results showed that CCC-Ni2+ fibers were able to selectively capture His-tagged proteins from complex E. coli cell lysates and exhibited a relatively large adsorption capacity toward His-tagged protein. The recoveries of His-tagged GFP in E. coli cell lysates were in the range of 89.8%-106.7% with the relative standard deviations (RSDs) less than 9.4% (intra-day) and 10.3% (inter-day). Taken together, this efficient approach for the purification of recombinant proteins extends the application of CCC-based fibrous materials in biological analysis.
Keywords: Ni2+-immobilized carboxyl cotton chelator (CCC-Ni2+); Immobilized metal ion affinity chromatography (IMAC); His-tagged; Proteins; E. coli cell lysates;

Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors by Kinga Meller; Paweł Pomastowski; Michał Szumski; Bogusław Buszewski (128-137).
In the present work the preparation of capillary-based microreactors with immobilized trypsin was investigated. The monolithic support was synthesized from 2-hydroxyethyl methacrylate (HEMA) as a functional monomer and N,N′-methylenebis(acrylamide) (MBA) as a hydrophilic crosslinker. Two monomers contents in the polymerization mixture (27% and 35%) at the ratio of HEMA:MBA = 3:2 were tested. The results indicated that the heated mixture of the above monomers and the porogen consisting of 40% 1-decanol, 40% 2-propanol and 20% water was the most appropriate polymerization mixture for preparation of HEMA-MBA copolymer. The elaborated novel procedure of HEMA-MBA hydrophilic monolith preparation allowed for the introduction of higher monomers content compared to earlier literature data. The obtained monolith exhibited lower adsorption of BSA in comparison with the commonly used glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) material. In the second step, the covalent enzyme attachment on the hydroxyl groups via 1,1′-carbonyldiimidazole activation was performed. Two immobilization techniques were tested. The first approach involved direct trypsin attachment to the hydroxyl groups of two-carbons HEMA chain (microreactor T1). In the other strategy, 5-amino-1-pentanol was employed to form a twelve-atom spacer arm (microreactor TS1). The prepared microreactor s activities and specificities were evaluated using low molecular weight trypsin substrate (Nα-benzoyl-l-arginine ethyl ester (BAEE)) and bovine serum albumin (BSA). The chromatographic analysis of the eluates from T1 and TS1 microreactors indicated that the microreactor T1 showed higher activity toward the BAEE than the microreactor TS1. However, the BSA digestion and subsequent MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of the collected eluates resulted in a sequence coverage of 43.9% and 35.7% in the case of TS1 and T1 microreactors, respectively.
Keywords: Trypsin; Immobilized enzyme microreactor; Proteomics; Monolithic bed; 2-Hydroxyethyl methacrylate; N,N′-Methylenebis(acrylamide);

Comprehensive urine screening for drugs and metabolites by LC-HR-MS/MS using Orbitrap technology has been described with precipitation as simple workup. In order to fasten, automate, and/or simplify the workup, on-line extraction by turbulent flow chromatography and a dilute-and-shoot approach were developed and compared. After chromatographic separation within 10 min, the Q-Exactive mass spectrometer was run in full scan mode with positive/negative switching and subsequent data dependent acquisition mode. The workup approaches were validated concerning selectivity, recovery, matrix effects, process efficiency, and limits of identification and detection for typical drug representatives and metabolites. The total workup time for on-line extraction was 6 min, for the dilution approach 3 min. For comparison, the established urine precipitation and evaporation lasted 10 min. The validation results were acceptable. The limits for on-line extraction were comparable with those described for precipitation, but lower than for dilution. Thanks to the high sensitivity of the LC-HR-MS/MS system, all three workup approaches were sufficient for comprehensive urine screening and allowed fast, reliable, and reproducible detection of cardiovascular drugs, drugs of abuse, and other CNS acting drugs after common doses.
Keywords: Sample workup; Orbitrap; High-resolution mass spectrometry; Liquid chromatography–mass spectrometry; Drug screening; Urine;

Sample preparation procedures utilized in microbial metabolomics: An overview by Małgorzata Patejko; Julia Jacyna; Michał J. Markuszewski (150-157).
Bacteria are remarkably diverse in terms of their size, structure and biochemical properties. Due to this fact, it is hard to develop a universal method for handling bacteria cultures during metabolomic analysis. The choice of suitable processing methods constitutes a key element in any analysis, because only appropriate selection of procedures may provide accurate results, leading to reliable conclusions. Because of that, every analytical experiment concerning bacteria requires individually and very carefully planned research methodology. Although every study varies in terms of sample preparation, there are few general steps to follow while planning experiment, like sampling, separation of cells from growth medium, stopping their metabolism and extraction. As a result of extraction, all intracellular metabolites should be washed out from cell environment. What is more, extraction method utilized cannot cause any chemical decomposition or degradation of the metabolome. Furthermore, chosen extraction method should correlate with analytical technique, so it will not disturb or prolong following sample preparation steps. For those reasons, we observe a need to summarize sample preparation procedures currently utilized in microbial metabolomic studies. In the presented overview, papers concerning analysis of extra- and intracellular metabolites, published over the last decade, have been discussed. Presented work gives some basic guidelines that might be useful while planning experiments in microbial metabolomics.
Keywords: Metabolomics; Bacteria; Sample preparation; Intracellular metabolites; Extracellular metabolites;

GC–MS and GC–MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d0-ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl-2H3-4-(isobutyl)phenylacetic acid (d3-ibuprofen) as internal standard. Plasma (10 μL) was diluted with acetate buffer (80 μL, 1 M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2 × 500 μL). After solvent evaporation d0- and d3-ibuprofen were derivatized in anhydrous acetonitrile by using pentafluorobenzyl (PFB) bromide and N,N-diisopropylethylamine as the base catalyst. Under electron-capture negative-ion chemical ionization (ECNICI), the PFB esters of d0- and d3-ibuprofen readily ionize to form their carboxylate anions [M-PFB] at m/z 205 and m/z 208, respectively. Collision-induced dissociation (CID) of m/z 205 and m/z 208 resulted in the formation of the anions at m/z 161 and m/z 164, respectively, due to neutral loss of CO2 (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H from d0-ibuprofen and D from d3-ibuprofen and is minimum at a CE value of 5 eV. Quantitative GC–MS determination was performed by selected-ion monitoring of m/z 205 and m/z 208. Quantitative GC–MS/MS determination was performed by selected-reaction monitoring of the mass transitions m/z 205 to m/z 161 for d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0–1000 μM) d0-ibuprofen added to human plasma was determined with accuracy (recovery, %) and imprecision (relative standard deviation, %) ranging between 93.7 and 110%, and between 0.8 and 4.9%, respectively. GC–MS (y) and GC–MS/MS (x) yielded almost identical results (y  = 4.00 + 0.988x, r 2  = 0.9991). In incubation mixtures of arachidonic acid (10 μM), d3-ibuprofen (10 μM) or d0-ibuprofen (10 μM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d3-ibuprofen and d0-ibuprofen did not change upon incubation at 37 °C up to 60 min. The trough pharmacokinetics of an inhaled arginine-containing ibuprofen preparation in mice was studied after once-daily treatment (0.0, 0.07, 0.4 and 2.5 mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10 μL) and administered dose 24 h after the last drug administration was observed.
Keywords: Collision-induced dissociation; Derivatization; Electron-capture negative-ion chemical ionization; H/D isotope effects; Quantification; Solvent extraction;

Urinary nitrite and malondialdehyde (MDA) are biomarkers of nitrosative and oxidative stress, respectively. At physiological pH values of urine and plasma, nitrite and MDA exist almost entirely in their dissociated forms, i.e., as ONO (ONOH, pK a  = 3.4) and CH(CHO)2 (CH2(CHO)2, pK a  = 4.5). Previously, we reported that nitrite and MDA react with pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone. Here, we report on the simultaneous derivatization of nitrite and MDA and their stable-isotope labeled analogs O15NO (4 μM) and CH2(CDO)2 (1 μM or 10 μM) with PFB-Br (10 μL) to PFBNO2, PFB15NO2, C(PFB)2(CHO)2), C(PFB)2(CDO)2 by heating acetonic urine (urine-acetone, 100:400 μL) for 60 min at 50 °C. After acetone evaporation under a stream of nitrogen, derivatives were extracted with ethyl acetate (1 mL). A 1-μL aliquot of the ethyl acetate phase dried over anhydrous Na2SO4 was injected in the splitless mode for simultaneous GC–MS analysis in the electron capture negative-ion chemical ionization mode. Quantification was performed by selected-ion monitoring (SIM) the anions [M–PFB] m/z 46 for ONO, m/z 47 for O15NO, m/z 251 for C(PFB)(CHO)2, and m/z 253 for C(PFB)(CDO)2. The retention times were 3.18 min for PFB-ONO2/PFB-O15NO2, and 7.13 min for C(PFB)(CHO)2/C(PFB)(CDO)2. Use of CH2(CDO)2 at 1 μM but not at 10 μM was associated with an unknown interference with the C(PFB)2(CDO)2 peak. Endogenous MDA can be quantified using O15NO (4 μM) and CH2(CDO)2 (10 μM) as the internal standards. The method is also useful for the measurement of nitrate and creatinine in addition to nitrite and MDA. Nitrite and MDA were measured by this method in urine of elderly healthy subjects (10 females, 9 males; age, 60–70 years; BMI, 25–30 kg/m2). Creatinine-corrected excretion rates did not differ between males and females for MDA (62.6 [24–137] vs 80.2 [52–118] nmol/mmol, P  = 0.448) and for nitrite (102 [71–174] vs. 278 [110–721] nmol/mmol P  = 0.053). We report for the first time a close correlation (r  = 0.819, P  < 0.0001) between MDA and nitrite in human urine. This correlation is assumed to be due to involvement of myeloperoxidase which catalyzes the formation of hypochlorite (OCl) from chloride and hydrogen peroxide. In turn, hypochlorite reacts both with nitrite and with polyunsaturated fatty acids such as arachidonic acid, with the later reaction generating MDA. The proposed mechanisms are supported by the literature but remain to be fully explored.
Keywords: Derivatization; Malondialdehyde; Myeloperoxidase; Nitrosative stress; Oxidative stress; Pentafluorobenzyl bromide;

A simple, accurate and highly sensitive multiresidues method was developed for the determination of 9 anabolic steroids and 16 β-agonists in milk. Target compounds were extracted and separated by a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method with Primary secondary amine (PSA) and Zinc oxide (ZnO) nanoparticles as adsorbents. The analytes were determined by ultra high performance liquid chromatography coupled to an electro-spray ionization tandem mass spectrometer (UHPLC–MS/MS) operating in negative/positive multiple reaction monitoring mode. The effect of QuEChERS including organic solvent, amount of ZnO nanoparticle was evaluated. Good linearity was obtained for the analytes in the concentration range of 0.1–200 μg/L with a correlation coefficients higher than 0.994. Decision limits (CCα) was from 0.007 μg/kg–0.1 μg/kg, and detection capabilities (CCβ) was in the range of 0.02–0.4 μg/kg. The recoveries of these compounds were between 63% and 126% at three fortified levels. Reproducibility represented by RSD was with 10% or less. The method was successfully applied to screening of real milk samples obtained from local markets and confirmation of the suspected target analytes.
Keywords: Anabolic steroids; β-Agonists; QuEChERS; ZnO nanoparticles; UHPLC–MS–MS;

Pentafluorobenzyl bromide (PFB-Br) is a versatile derivatization agent. It is widely used in chromatography and mass spectrometry since several decades. The bromide atom is largely the single leaving group of PFB-Br. It is substituted by wide a spectrum of nucleophiles in aqueous and non-aqueous systems to form electrically neutral, in most organic solvents soluble, generally thermally stable, volatile, strongly electron-capturing and ultraviolet light-absorbing derivatives. Because of these greatly favoured physicochemical properties, PFB-Br emerged an ideal derivatization agent for highly sensitive analysis of endogenous and exogenous substances including various inorganic and organic anions by electron capture detection or after electron-capture negative-ion chemical ionization in GC–MS. The present article attempts an appraisal of the utility of PFB-Br in analytical chemistry. It reviews and discusses papers dealing with the use of PFB-Br as the derivatization reagent in the qualitative and quantitative analysis of endogenous and exogenous inorganic anions in various biological samples, notably plasma, urine and saliva. These analytes include nitrite, nitrate, cyanide and dialkyl organophosphates. Special emphasis is given to mass spectrometry-based approaches and stable-isotope dilution techniques.
Keywords: Biomarkers; Chromatography; Derivatization; Drugs; Mass spectrometry; Sensitivity;

Isolation of atropine and scopolamine from plant material using liquid-liquid extraction and EXtrelut® columns by Paula Śramska; Artur Maciejka; Anna Topolewska; Piotr Stepnowski; Łukasz P. Haliński (202-208).
Tropane alkaloids are toxic secondary metabolites produced by Solanaceae plants. Among them, plants from Datura genus produce significant amounts of scopolamine and hyoscyamine; the latter undergoes racemization to atropine during isolation. Because of their biological importance, toxic properties and commonly reported food and animal feed contamination by different Datura sp. organs, there is a constant need for reliable methods for the analysis of tropane alkaloids in many matrices. In the current study, three extraction and sample-clean up procedures for the determination of scopolamine and atropine in plant material were compared in terms of their effectiveness and repeatability. Standard liquid-liquid extraction (LLE) and EXtrelut® NT 3 columns were used for the sample clean-up.Combined ultrasound-assisted extraction and 24 h static extraction using ethyl acetate, followed by multiple LLE steps was found the most effective separation method among tested. However, absolute extraction recovery was relatively low and reached 45–67% for atropine and 52–73% for scopolamine, depending on the compound concentration. The same method was also the most effective one for the isolation of target compounds from Datura stramonium leaves. EXtrelut® columns, on the other hand, displayed relatively low effectiveness in isolating atropine and scopolamine from such a complex matrix and hence could not be recommended. The most effective method was also applied to the extraction of alkaloids from roots and stems of D. stramonium. Quantitative analyses were performed using validated method based on gas chromatography with flame ionization detector (GC-FID). Based on the results, the importance of the proper selection of internal standards in the analysis of tropane alkaloids was stressed out.
Keywords: Tropane alkaloids; Atropine; Scopolamine; Liquid-liquid extraction; EXtrelut®; Gas chromatography;

A GC–MS method is reported for the quantitative analysis of S-nitrosothiols (RSNO) derived from endogenous low- and high-molecular mass thiols (RSH) including hemoglobin, cysteine, glutathione, N-acetylcysteine, and the exogenous N-acetylcysteine ethyl ester. The method is based on the conversion of RSNO to nitrite by aqueous Na2S (S2−). 15N-Labelled analogs (RS15NO) or 15N-labelled nitrite and nitrate were used as internal standards. The nitrite (14NO2 and 15NO2 ) and nitrate (O14NO2 and O15NO2 anions were derivatised by pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone and their PFB derivatives were separated by gas chromatography. After electron-capture negative-ion chemical ionization, the anions were separated by mass spectrometry and detected by selected-ion monitoring of m/z 46 for 14NO2 , m/z 47 for 15NO2 , m/z 62 for O14NO2 , and m/z 63 for O15NO2 . The expected thionitrites (S14NO and S15NO) were not detected, suggesting that they are intermediates and rapidly exchange their S by O from water, presumably prior to PFB-Br derivatization. The reaction of S2− with RSNO and sodium nitroprusside (SNP) resulted in the formation of nitrite and nitrate as the major and minor reaction products, respectively. The novel Na2S procedure was compared with established procedures based on the use of aqueous HgCl2 or cysteine/Cu2+ reagents to convert the S-nitroso group to nitrite. Our results provide evidence for an equilibrium S-transnitrosylation reaction between S2− with RSNO in buffered solutions of neutral pH. Use of Na2S in molar excess over RSNO shifts this reaction to the right, thus allowing almost complete conversion of RSNO to nitrite and nitrate. The Na2S procedure should be useful for the quantitative determination of RSNO as nitrite and nitrate after PFB-Br derivatization and GC–MS analysis. The Na2S procedure may also contribute to explore the complex reactions of S2− with RSNO, SNP and other NO-containing compounds.
Keywords: Derivatization; Nitric oxide; S-Nitrosothiols; Polysulfides; Sulfide; Thiols;

On-line SPE sample treatment as a tool for method automatization and detection limits reduction: Quantification of 25-hydroxyvitamin D3/D2 by Dimitrios Palaiogiannis; Evangelia Bekou; Kalliopi Pazaitou-Panayiotou; Victoria Samanidou; Andreas Tsakalof (219-227).
The development and approbation of new, automated UHPLC-DAD method for the quantification of 25-hydroxyvitamin D3/D2 (25OH-D3/D2) metabolites in plasma/serum for the evaluation of patient’s vitamin D status are presented. The method was developed on the Ultimate 3000 UHPLC dual gradient system supplied with the on-line SPE-concentration column coupled through six port switching valve to analytical column. This configuration and materials selected enable large volume sample injection (500 μL) and on-line sample preconcentration, clean up and subsequent selective metabolites transfer onto the analytical column. The new method abrogates main conventional time consuming and error source off-line steps of analysis and thus simplifies analysis. The large volume injection increases the sensitivity of instrumental analysis by about ten-fold on-line pre-concentration of metabolites. The instrument response is linear (R > 0.99) in the investigated concentration range 10–100 ng mL−1 which covers all the possible vitamin D status from serious deficiency (<12 ng mL−1) to excess. The method detection limits (S/N = 3) are LOD (25OH-D3) = 0.94 ng mL−1 and LOD (25OH-D2) = 2.4 ng mL−1. The method performance was assessed with the use of certified reference samples and perfect agreement between certified and measured values is demonstrated. The method was applied to human samples previously analyzed for total vitamin D by Competitive Protein-binding assay and findings of the two methods are compared.
Keywords: On-line solid phase extraction; Vitamin D; 25-Hydroxy vitamin D3/D2; Large volume injection;

Continous renal replacement therapy (CRRT) is particularly recommended for septic shock patients in intensive care units. The CRRT technique used most frequently is high volume continuous veno-venous haemofiltration. It provides a high rate of clearance of uremic toxins and inflammatory cytokines. However, it should also be taken into account that substances important for homeostasis may be concurrently unintentionally removed. Accordingly, water-soluble vitamins can be removed during continuous renal replacement therapy, and the estimate of the loss is critical to ensure appropriate supplementation. The aim of this work was to develop a simple methodology for a purification step prior to the LC–MS/MS determination of water-soluble vitamins in ultrafiltrate samples. For this purpose, two types of resin and a mix of resins were used as sorbents for the purification step. Moreover, parameters such as the amount of resin and the extraction time were optimized. The LC–MS/MS method was developed and validated for final determination of 11 vitamins. The results demonstrated the high purification capability of DEAE Sephadex resin with recoveries between 65 and 101% for water-soluble vitamins from ultrafiltrate samples. An optimized method was applied to assess the loss of B-group vitamins in patients after 24 h of renal replacement therapy. The loss of vitamins B2, B6 pyridoxamine, B6 pyridoxal, B7, B1, and B5 in ultrafiltrates was similar in all patients. In the native ultrafiltrates, vitamins B6 pyridoxine, B9 and B12 were not detected.
Keywords: Water-soluble vitamins; Ion exchange resins; LC–MS; High volume continuous veno-venous haemofiltration; Ultrafiltrates;

Fast and sensitive HPLC method was developed, optimized and validated for quantification of linezolid (LNZ) in human plasma using guaifenesin as an internal standard (IS). Analyte and IS were extracted from plasma by simple protein precipitation extraction technique using methanol as the precipitating solvent. The pretreated samples were injected in a mobile phase formed of acetonitrile:water:methanol (20:70:10 v/v/v) in an isocratic mode at a flow rate of 1.5 mL/min with UV detection at 251 nm. Separation was done using Aglient ODS C18. The method showed linearity in the range of 0.75–50 μg/mL with correlation coefficients equals to 0.9991. Precision and accuracy were in conformity with the criteria normally accepted in bio-analytical method validation. The RSDs for intra- and inter-day assays were <3.56 and 4.63%, respectively. The intra- and inter-day accuracies were 94.67–98.28% and 91.25–96.18%, respectively. The mean absolute recoveries ranged from 92.56 ± 1.78 to 95.24 ± 2.84. According to stability results, LNZ was stable in human plasma during the storage and analysis. LNZ a pharmacokinetic behavior was studied by applying the proposed analytical method.
Keywords: Linezolid; HPLC; Human plasma; Protein precipitation; Pharmacokinetic study;