Journal of Chromatography B (v.879, #19)

Determination of puerarin in rat plasma by rapid resolution liquid chromatography tandem mass spectrometry in positive ionization mode by Cheng-Feng Luo; Mu Yuan; Min-Sheng Chen; Shi-Ming Liu; Bi-Yun Huang; Xia-Wen Liu; Liu Zhu (1497-1501).
A highly sensitive and specific method of rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) in positive ionization mode has been developed and validated for pharmacokinetic study of puerarin in rat plasma. Chromatography was carried out on a Zorbax XDB C18 reversed-phase column using a mobile phase comprising a mixture of methanol and 0.05% acetic acid in water (35:65, v/v) with a flow rate of 0.3 mL/min from 0 min to 5.4 min and then 0.6 mL/min from 5.41 min to 12 min. The mass spectrometer operated in ESI positive ionization mode. Multiple reaction monitoring (MRM) was used to measure puerarin and tectoridin (internal standard). The method was sensitive with a detection limit of 0.33 ng/mL. A good linear response was observed over a range of 10–2000 ng/mL in rat plasma. The inter- and intra-day precision ranged from 2.97% to 7.52% and accuracy from 93.70% to 101.60%. This validated method was applied successfully to a pharmacokinetic study in rat plasma after intravenous administration of puerarin. The main pharmacokinetic parameters were as follows: AUC0→t 45.37 ± 13.19 (mg h/L), AUC0→∞ 47.03 ± 14.78 (mg h/L), MRT 1.03 ± 0.46 (h), T 1/2 1.31 ± 0.31 (h), V ss 0.09 ± 0.02 (L), V z 0.17 ± 0.04 (L), Cl 0.10 ± 0.04 (L/h).
Keywords: Rapid resolution liquid chromatography; Mass spectrometry; Electrospray ionization; Puerarin; Pharmacokinetics; Rats;

Methylmalonic acid quantification in low serum volumes by UPLC–MS/MS by Theresa L. Pedersen; William R. Keyes; Setareh Shahab-Ferdows; Lindsay H. Allen; John W. Newman (1502-1506).
Methylmalonic acid (MMA) is a metabolic intermediate transformed to succinic acid (SA) by a vitamin B12-dependent catalytic step, and is broadly used as a clinical biomarker of functional vitamin B12 status. However, reported methods use between 100 and 1000 μL of serum or plasma making them sub-optimal for sample-limited studies, including those with neonates and infants. LC–MS/MS based protocols to measure MMA as n-butyl esters in the presence of tri-deuterated MMA (MMA-d3) were modified for use with 25 μL of human serum by scaling down sample processing volumes and analysis by UPLC–MS/MS. Plasma-based calibration solutions were found to be unnecessary, and chromatographic resolution and peak shape of SA and MMA was optimized in <4 min with isocratic 53:47 methanol/1.67 mM (pH 6.5) ammonium formate. Additionally, 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) was included as internal standard allowing direct assessment of MMA recovery. Sample concentrations in the low normal range produced a signal:noise of >100:1. MMA intra- and inter-assay variability was under 10%. MMA-d3 surrogate recovery averaged 93 ± 14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles. In conclusion, we report that methylmalonic acid can be measured with 25 μL of serum using water based standards. The assay signal:noise per concentration indicates that the method could perform as implemented with as little as 5 μL of serum. The reported method is applicable for studies of functional B12 status in sample limited experiments including investigations of nutritional status in neonates and in studies where low normal MMA levels are expected.
Keywords: Methylmalonic acid; Vitamin B12 status; UPLC; LC–MS/MS; Nutrition;

An olive (Olea europaea L.) β-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate and using specifically designed sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. The purification was 155 fold with an overall enzyme yield of 54%. The molecular mass of the protein was estimated as ca. 65 kDa. The purified β-glucosidase was effectively active on p-/o-nitrophenyl-β-d-glucopyranosides (p-/o-NPG) with K m values of 2.22 and 14.11 mM and V max values of 370.4 and 48.5 U/mg, respectively. The enzyme was competitively inhibited by δ-gluconolactone and glucose against p-NPG as substrate. The K i and IC50 values of δ-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with K i and IC50 values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified β-glucosidase was investigated. Of the ions tested, only the Fe2+ increased the activity while Cd2+ Pb2+ Cu2+, Ni+, and Ag+ exhibited different levels of inhibitory effects with K i and IC50 values of 4.29 × 10−4 and 0.38 × 10−4, 1.26 × 10−2 and 5.3 × 10−3, 2.26 × 10−4 and 6.1 × 10−4, 1.04 × 10−4 and 0.63 × 10−4, 3.21 × 10−3 and 3.34 × 10−3  mM, respectively.
Keywords: β-Glucosidase; Hydrophobic interaction chromatography; Olive fruit; Purification;

Quantitation of dissolved gas content in emulsions and in blood using mass spectrometric detection by Everett Grimley; Nicole Turner; Clayton Newell; Cuthbert Simpkins; Juan Rodriguez (1513-1518).
Quantitation of dissolved gases in blood or in other biological media is essential for understanding the dynamics of metabolic processes. Current detection techniques, while enabling rapid and convenient assessment of dissolved gases, provide only direct information on the partial pressure of gases dissolved in the aqueous fraction of the fluid. The more relevant quantity known as gas content, which refers to the total amount of the gas in all fractions of the sample, can be inferred from those partial pressures, but only indirectly through mathematical modeling. Here we describe a simple mass spectrometric technique for rapid and direct quantitation of gas content for a wide range of gases. The technique is based on a mass spectrometer detector that continuously monitors gases that are rapidly extracted from samples injected into a purge vessel. The accuracy and sample processing speed of the system is demonstrated with experiments that reproduce within minutes literature values for the solubility of various gases in water. The capability of the technique is further demonstrated through accurate determination of O2 content in a lipid emulsion and in whole blood, using as little as 20 μL of sample. The approach to gas content quantitation described here should greatly expand the range of animals and conditions that may be used in studies of metabolic gas exchange, and facilitate the development of artificial oxygen carriers and resuscitation fluids.
Keywords: Dissolved gases; Gas content; Mass spectrometry; Blood; Artificial oxygen carriers; Resuscitation fluids;

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B2), anthocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid–solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1 g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4 h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.
Keywords: Anthocyanins; Biological tissues; Procyanidins; Theobromine; Caffeine; UPLC–MS/MS;

A highly sensitive, rapid LC–APCI–MS method for identification and quantification of mono and disaccharides in simple or complex aqueous phase has been developed. This original method is easy to use, no derivation and no post-column injection are needed. The separation is performed with a hydrophilic amino interaction (HILIC) column allowing high-throughput analysis with analysis times of 15 min for monosaccharides to 22 min for disaccharides. The development of the method carried out with 9 standard saccharides allowed to point out a dynamic range from 0.1–25.6 to 1–256 μg mL−1 depending on the considered sugar. Next, the method was validated on saccharides at known concentrations in water and on 2 real samples: orange juice and aqueous phase obtained after enzymatic hydrolysis of sunflower seeds.
Keywords: Liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry; Aqueous phase analysis; Monosaccharides analysis; Disaccharides analysis;

A reversed-phased liquid chromatography with tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma. Sample preparation involved a liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters X-Terra™ MS C18 column with an isocratic mobile phase consisting of methanol/0.45% formic acid in water (60:40, v/v) running at a flow rate of 0.2 ml/min for 6 min. The lower limits of quantitation (LLOQs) were 5 ng/ml for the total RO4929097 in plasma and 0.5 ng/ml for the unbound drug in phosphate buffer solution (PBS). Calibration curves were linear over RO4929097 concentration range of 5–2000 ng/ml in plasma for the total drug and 0.5–200 ng/ml in PBS for the unbound drug. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the total and unbound plasma pharmacokinetics of RO4929097 after its oral administration in cancer patients.
Keywords: RO4929097; γ-Secretase inhibitor; High performance liquid chromatography; Mass spectrometry; LC–MS/MS; Pharmacokinetics;

Quantification of 4 antidepressants and a metabolite by LC–MS for therapeutic drug monitoring by Eva Choong; Serge Rudaz; Astrid Kottelat; Sophie Haldemann; Davy Guillarme; Jean-Luc Veuthey; Chin B. Eap (1544-1550).
A liquid chromatography method coupled to mass spectrometry was developed for the quantification of bupropion, its metabolite hydroxy-bupropion, moclobemide, reboxetine and trazodone in human plasma. The validation of the analytical procedure was assessed according to Société Française des Sciences et Techniques Pharmaceutiques and the latest Food and Drug Administration guidelines. The sample preparation was performed with 0.5 mL of plasma extracted on a cation-exchange solid phase 96-well plate. The separation was achieved in 14 min on a C18 XBridge column (2.1 mm × 100 mm, 3.5 μm) using a 50 mM ammonium acetate pH 9/acetonitrile mobile phase in gradient mode. The compounds of interest were analysed in the single ion monitoring mode on a single quadrupole mass spectrometer working in positive electrospray ionisation mode. Two ions were selected per molecule to increase the number of identification points and to avoid as much as possible any false positives. Since selectivity is always a critical point for routine therapeutic drug monitoring, more than sixty common comedications for the psychiatric population were tested. For each analyte, the analytical procedure was validated to cover the common range of concentrations measured in plasma samples: 1–400 ng/mL for reboxetine and bupropion, 2–2000 ng/mL for hydroxy-bupropion, moclobemide, and trazodone. For all investigated compounds, reliable performance in terms of accuracy, precision, trueness, recovery, selectivity and stability was obtained. One year after its implementation in a routine process, this method demonstrated a high robustness with accurate values over the wide concentration range commonly observed among a psychiatric population.
Keywords: Method development; Therapeutic drug monitoring; Solid phase extraction; LC–MS; Antidepressant;

Analysis of chlormequat in human urine as a biomarker of exposure using liquid chromatography triple quadrupole mass spectrometry by Christian H. Lindh; Margareta Littorin; Gunvor Johannesson; Bo A.G. Jönsson (1551-1556).
In this study, a method using liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is described for the analysis of the plant growth regulator chlormequat (CCC) in human urine. Analysis was carried out using selected reaction monitoring (SRM) in the positive ion mode. [2H4] labeled CCC as internal standard (IS) was used for quantification of CCC. The limit of detection (LOD) was determined to 0.1 ng/mL. The method was linear in the range 0.3–800 ng/mL urine and had a within-run precision of 4–9%. The between-run precision was determined at urine levels of 7.0 and 31 ng/mL and found to be 5 and 6% respectively. The reproducibility was 3–6%. To validate CCC as a biomarker of exposure, the method was applied in a human experimental oral exposure to CCC. Two healthy volunteers received 25 μg/kg b.w. CCC in a single oral dose followed by urine sampling for 46 h post-exposure. The CCC was estimated to follow a first order kinetic and a two compartment model with an elimination half-life of 2–3 h and 10–14 h respectively. One hundred 24 h urine samples were collected from non-occupationally exposed individuals in the general population in southern Sweden. All samples had detectable levels above the LOD 0.1 ng/mL urine. The median levels were 4 ng/mL of CCC in unadjusted urine. The levels found in the population samples are several magnitudes lower than those found in the experimental exposure, which corresponds to an oral exposure of 50% of the ADI for CCC.
Keywords: Biomarkers; Urine; LC/MS/MS; Pesticides; Chlormequat; HILIC;

Phospholipase A2 (PLA2) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-derivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA2 and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA2 and PAF-AH determination, the isocratic elution systems CH3OH–H2O (80:20, v/v) and CH3OH–H2O–CH3COOH (60:40:0.2, v/v/v) separated efficiently C12-NBD-FA/C12-NBD-PC and C6-NBD-FA/C6-NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was ∼15 min/injection. The reproducibility, expressed as relative standard deviation, was ≤13% for PLA2 and ≤16% for PAF-AH, respectively. The assay was linear for PLA2 activities extending from 1 pmol up to at least 250 nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca2+ concentrations.
Keywords: Enzymic assay; Phospholipase A2; NBD lipids; HPLC; Fluorescence;

Adrenal steroid profiling, including 17α-OH progesterone (17OHP), 11-deoxycortisol (S), Δ4-androstenedione (Δ4-A) and cortisol (F) in blood spots by tandem mass spectrometry, is used for newborn screening to detect congenital adrenal hyperplasia (CAH). Pre-analytical sample processing is critical for assay specificity and accuracy; however, it is laborious and time-consuming. This study describes the development and validation of a new Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) method for the simultaneous quantification of five steroids: 17OHP, S, Δ4-A, F and cortisone (E) in blood spots from newborns. Whole blood was eluted from a 5.00 mm dried blood spot by an aqueous solution containing the deuterium-labeled internal standards d8-17OHP and d4-cortisol. The steroids extracted from blood spot into aqueous solution were subsequently purified via Extelut mini NT1 column using diethylether. The extracts were evaporated and quantified using LC–MS/MS. The detection limit was 0.25 ng/mL for 17OHP and S, 0.4 ng/mL for Δ4-A and 0.5 ng/mL for F and E. The limit of quantification was 0.5 ng/mL for 17OHP, S and Δ4-A and 1 ng/mL for F and E. Precision for 17OHP, S, Δ4-A at concentrations of 0.5, 2, and 8 ng/mL (n  = 5) in fortified steroid free serum samples was 1.3–3.5% (intra-assay CV) and 7–14.8% (inter-assay CV). Precision for F and E at concentrations of 5 and 20 ng/mL was 1.5–4.8% (intra-assay, CV%) and 6–15% (inter-assay, CV%). Accuracy was calculated at concentrations of 0.5, 2, and 8 ng/mL for 17OHP, S and Δ4-A and ranged from −0.3 to 0.2%, while for F and E it ranged from −3.2 to 0.2%. Relative recoveries at concentration 2 ng/mL and 8 ng/mL for 17OHP, S, Δ4-A and at 5 ng/mL and 20 ng/mL for F and E ranged from 55% to 80%. Reference intervals were estimated for all steroids in newborns (on day 3). The steroid profile assay herein described is sensitive, specific and accurate and involves a simple pre-analytical sample manipulation; it is therefore suitable for routine analysis and provides data for samples within normal range as well as those with elevated levels. For the first time to our knowledge, cortisone levels are reported in dried blood spots from newborns.
Keywords: Tandem mass spectrometry; Blood spots; 17α-OH progesterone; 11-Deoxycortisol; Δ4-Androstenedione; Cortisol; Cortisone;

Determination of sugammadex in human plasma, urine, and dialysate using a high-performance liquid chromatography/tandem mass spectrometry assay by Marcel A.H. de Zwart; Jolanda ten Bruggencate-Broeders; Henk J.M. van Hal; René (H) J.J.J. Megens; Helma (W) L.H. Frasa (1573-1586).
Sugammadex (Bridion®, Merck Sharp & Dohme Corp., Oss, The Netherlands) is a modified γ-cyclodextrin which has the ability to reverse the neuromuscular blockade induced by the steroidal neuromuscular blocking agents rocuronium and vecuronium. The objective of the current study is to describe the bioanalytical methods that have been developed and validated according to US Food and Drug Administration guidelines on bioanalytical method validation, and subsequently applied to determine total sugammadex (i.e., free sugammadex plus sugammadex bound to the neuromuscular blocking agent) in human heparinized plasma, urine and dialysate. Sugammadex was extracted from human plasma and urine using solid phase extraction with Isolute HAX 96-well extraction plates; no extraction was performed on dialysate samples. Samples from plasma, urine, and dialysate were analyzed on a Polaris® C18-A PEEK (polyaryletheretherketone) analytical column (50 mm × 4.6 mm internal diameter, 5 μm) with a linear mobile phase gradient of 0.1% (v/v) formic acid in water:methanol from 70:30 to 20:80. The flow rate was 1 mL/min with a total run time for each injection of 6 min. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ion mode with a turbo ion-spray interface to quantify the concentration of sugammadex. Inter- and intra-assay precision and accuracy were within pre-defined acceptance limits. The presence of rocuronium did not interfere with the assay in plasma, urine or dialysate; similarly, vecuronium did not interfere with the plasma assay (not tested for interference in urine or dialysate). Sugammadex was found to be stable in plasma, urine and dialysate in the short-term at room temperature, in the long-term at −20 °C, and after several freeze/thaw cycles. The validated bioanalytical methods developed here have been successfully applied in a series of clinical studies for the determination of total sugammadex in plasma, urine and dialysate.
Keywords: Sugammadex; Liquid chromatography tandem mass spectrometry (LC–MS/MS); Plasma; Urine; Dialysate; Bridion;

The burgeoning field of oligonucleotide therapeutics is based upon synthetically derived biopolymers comprised of relatively simple RNA and DNA building blocks. Significant gains in knowledge around mechanisms of action (RNA interference, RNA splicing, etc.) and oligonucleotide design (ASO, siRNA, DsiRNA, miRNA, locked nucleic acid, etc.) have been the main drivers of recent investment for this field . As therapeutics, there is currently great interest in oligonucleotides due to the reduced time required to achieve lead molecules and to their potential for treating previously untractable diseases. One of the more challenging areas for the field of oligonucleotide therapeutics is the development of high-quality analysis schemes for the determination of purity in drug substance and product. This, in part, is due to the fact that the synthesis of oligonucleotides results in a significant number of closely related impurities that are not easily removed during purification . As a result, these macromolecules (4000–8000 MW on average, depending on chain length) and their soup of closely related impurities are typically not well resolved from one another via conventional chromatographic approaches. One of the more common chromatographic techniques used for oligonucleotide analysis is reversed phase-ion pairing liquid chromatography (RP-IP). Our research led us to the discovery that the use of multiple ion pairing agents combined in the mobile phase can improve the overall chromatographic resolution and peak shape of the oligonucleotide analytes over the use of a single ion pairing agent alone, resulting in enhanced purity analysis and the opportunity to identify related impurities with greater certainty. In addition, the use of combined ion pairing agents allowed for the development of a “universal” method which has provided superior chromatography for several different oligonucleotide compounds and their related impurities regardless of differences in nucleotide sequence. The RP-IP UPLC method conditions are ESI-MS compatible and have allowed for the mass identification of five positional isomeric impurities chromatographically resolved and present at less than 1% of the nominal parent peak area.
Keywords: Oligonucleotide; UPLC; Purity; siRNA; Reversed-phase; Ion-pairing;

Determination of tylosins A, B, C and D in bee larvae by liquid chromatography coupled to ion trap-tandem mass spectrometry by J. Bernal; Ma.T. Martín; L. Toribio; R. Martín-Hernández; M. Higes; J.L. Bernal; M.J. Nozal (1596-1604).
A LC–MS/MS method has been developed to simultaneously quantify tylosins A, B, C and D in bee larvae, compounds currently used to treat one of the most lethal diseases affecting honey bees around the world, American Foulbrood (AFB). The influence of different aqueous media, temperature and light exposure on the stability of these four compounds was studied. The analytes were extracted from bee larvae with methanol and chromatographic separation was achieved on a Luna C18 (150 × 4.6 mm i.d.) using a ternary gradient composed of a diluted formic acid, methanol and acetonitrile mobile phase. To facilitate sampling, bee larvae were initially dried at 60 °C for 4 h and afterwards, they were diluted to avoid problems of pressure. MSD-Ion Trap detection was employed with electrospray ionization (ESI). The calibration curves were linear over a wide range of concentrations and the method was validated as sensitive, precise and accurate within the limits of quantification (LOQ, 1.4–4.0 ng/g). The validated method was successfully employed to study bee larvae in field tests of bee hives treated with two formulations containing tylosin. In both cases it was evident that the minimal inhibitory concentration (MIC) had been reached.
Keywords: Antibiotics; Tylosin; Stability; Bee larvae; Tandem mass-spectrometry;

An efficient molecular docking method for adsorbent screening by Jun Wang; Saihui Zhang; Jing Zhang; Ping Ren; Yingchao Chen; Jihong Li; Wei Wang; Yi Ma; Rongfu Shi; Chunhong Wang; Zhi Yuan (1605-1609).
In this study, with flavonol glycosides (FG) and terpene lactones (TL) in ginkgo biloba extract (GBE) as the targets for separation, we investigated the effectiveness of molecular docking in adsorbent screening. Several polyamine-modified methyl acylate-co-divinylbenzene (MA-co-DVB) adsorbent models were built, and their affinity to rutin, quercetin and ginkgolide B (GB) was evaluated via molecular docking. The model of ethylenediamine-modified adsorbent showed the largest difference in affinity between to GB and to quercetin as well as rutin, and thus this adsorbent could have the best separation performance. The results of the subsequently conducted static adsorption and dynamic adsorption experiments correlated well with docking results. Finally, using ethylenediamine-modified MA-co-DVB adsorbent, nearly complete separation of the FG and TL in GBE was simply achieved by one step of adsorption–desorption. Thus, the reported molecular docking method is expected to be helpful for rapid adsorbent screening.
Keywords: Adsorbent screening; Adsorption separation; Ginkgo biloba extracts; Molecular docking;

A HPLC method for the quantitative determination of N-(2-hydroxy-5-nitrophenylcarbamothioyl)-3,5-dimethylbenzamide in biological samples by Igor Skidan; Jacob Grunwald; Ritesh Thekkedath; Alexei Degterev; Vladimir Torchilin (1610-1616).
A sensitive and simple HPLC method was developed for the determination of a novel compound, a potential anti-cancer drug, N-(2-hydroxy-5-nitrophenylcarbamothioyl)-3,5-dimethylbenzamide (DM-PIT-1), a member of the new structural class of non-phosphoinositide small molecule antagonist of phosphatidylinositol-3,4,5-trisphosphate–pleckstrin-homology domain interactions, in mouse plasma and tumor tissue homogenates. The chromatographic separation of DM-PIT-1 was achieved on C18 column using isocratic elution with acetonitrile–water (70:30) containing 0.1% formic acid (v/v). DM-PIT-1 was detected by UV absorbance at 320 nm and confirmed by LC–MS. The extraction of the DM-PIT-1 from the plasma and tumor tissue with methylene chloride resulted in its high recovery (70–80%). HPLC calibration curves for DM-PIT-1 based on the extracts from the mouse plasma and tumor tissue samples were linear over a broad concentration range of 0.25–20 μg/ml/g, with intra/inter-day accuracy of 95% and the precision of variation below 10%. The limits of detection and quantification were 0.1 ng and 0.2 ng, respectively. The described method was successfully applied to study the pharmacokinetics of the DM-PIT-1 following the parenteral injections of DM-PIT-1 entrapped in 1,2-disteratoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (PEG-PE) micelles.
Keywords: N-(2-hydroxy-5-nitrophenylcarbamothioyl)-3,5-dimethylbenzamide (DM-PIT-1); PEG-PE micelles; HPLC; Pharmacokinetics;

A new surface imprinting technique was reported to synthesize multi-walled carbon nanotubes-molecularly imprinted polymers (MWNTs-MIPs) using erythromycin as the template, acryloyl-β-cyclodextrin (acryloyl-β-CD) and methacrylic acid (MAA) as the binary functional monomers. The MWNTs-MIPs were characterized by transmission electron microscopy (TEM), scanning electron micrograph (SEM) and Fourier transform-infrared spectroscopy (FT-IR). Adsorption experiments indicated the MWNTs-MIPs prepared with acryloyl-β-CD and MAA have high selective for erythromycin. The feasibility of the MWNTs-MIPs as solid-phase extraction (SPE) sorbent was evaluated, and the results showed that it can selectively extract erythromycin from chicken muscle samples with the recoveries ranging from 85.3% to 95.8%. The molecularly imprinted solid-phase extraction (MISPE) method could be applied for preconcentration and purification of erythromycin from chicken muscle samples.
Keywords: Erythromycin; Multi-walled carbon nanotube; High-performance liquid chromatography; β-Cyclodextrin; Surface molecular imprinting;

Simultaneous determination of five flavonoids from Scutellaria Barbata extract in rat plasma by LC–MS/MS and its application to pharmacokinetic study by Rui Shi; Shi Qiao; Deqing Yu; Xiaowei Shi; Man Liu; Xijuan Jiang; Qiao Wang; Lantong Zhang (1625-1632).
A new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of five flavonoids including scutellarin, naringenin, apigenin, luteoline and wogonin in rat plasma using sulfamethalazole as internal standard (IS). Plasma samples were pretreated with liquid–liquid extraction procedure and acid hydrolysis method was used for converting conjugated flavonoids to their respective free forms. The chromatographic separation was performed on a C18 column with a linear gradient elution using a mobile phase consisted of 0.01% acetic acid and methanol. The detection was accomplished by multiple-reaction monitoring (MRM) scanning with electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for scutellarin, naringenin, apigenin, luteoline, wogonin and IS were 461.1/285.1, 271.0/119.0, 269.0/117.0, 285.0/132.9, 283.0/268.0 and 252.0/155.9, respectively. The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.9915. The lower limit of quantitation (LLOQ) of scutellarin was 9.15 ng/mL and other compounds were all less than 2.0 ng/mL. The proposed method showed appropriate accuracy and repeatability and was suitable for pharmacokinetic studies of the five flavonoids after oral administration of Scutellaria Barbata extract.
Keywords: Flavonoids; Conjugated metabolites; Scutellaria Barbata; LC–MS/MS; Pharmacokinetics;

Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification by Cristiana Boi; Simone Dimartino; Stefan Hofer; Jeannie Horak; Sharon Williams; Giulio C. Sarti; Wolfgang Lindner (1633-1640).
Microporous membranes are an attractive alternative to circumvent the typical drawbacks associated to bead-based chromatography. In particular, the present work intends to evaluate different affinity membranes for antibody capture, to be used as an alternative to Protein A resins. To this aim, two Mimetic Ligands™ A2P and B14, were coupled onto different epoxide and azide group activated membrane supports using different spacer arms and immobilization chemistries. The spacer chemistries investigated were 1,2-diaminoethane (2LP), 3,6-dioxa-1,8-octanedithiol (DES) and [1,2,3] triazole (TRZ). These new mimetic membrane materials were investigated by static and by dynamic binding capacity studies, using pure polyclonal human immunoglobulin G (IgG) solutions as well as a real cell culture supernatant containing monoclonal IgG1. The best results were obtained by combining the new B14 ligand with a TRZ-spacer and an improved Epoxy 2 membrane support material. The new B14-TRZ-Epoxy 2 membrane adsorbent provided binding capacities of approximately 3.1 mg/mL, besides (i) a good selectivity towards IgG, (ii) high IgG recoveries of above 90%, (iii) a high Pluronic-F68 tolerance and (iv) no B14-ligand leakage under harsh cleaning-in-place conditions (0.6 M sodium hydroxide). Furthermore, foreseeable improvements in binding capacity will promote the implementation of membrane adsorbers in antibody manufacturing.
Keywords: Antibody purification; A2P; B14; Click-Chemistry; Azide-functionalized membrane; Affinity membranes;

A novel two-dimensional (2D) separation method, which hyphenated micellar electrokinetic capillary chromatography (MEKC) and capillary zone electrophoresis (CZE), was developed for analysis of flavonoids in Leonurus cardiaca. The Leonurus cardiaca sample was separated and purified in first dimension by MEKC. Then only a selected portion of the first dimension separation was transferred into the second dimension by pressure. Finally, the zone of flavonoids was separated by CZE. As the key to successful hyphenation of MEKC and CZE, an analyte focusing by micelle collapse (AFMC) concentration method was employed between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The whole heart-cut 2D separation process can be performed in a conventional CE analyzer. The relative standard deviation of peak height, peak area and migration time were in the range of 2.3–4.2%, 1.5–3.8% and 3.6–5.5%, respectively, and detection limits (S/N = 3) were 15–55 ng/mL. The new methodology was applied with success for the flavonoids separation of Leonurus cardiaca.
Keywords: Two-dimensional separation; Micellar electrokinetic capillary chromatography; Capillary zone electrophoresis; Analyte focusing by micelle collapse;

Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50–5.70%. By using 2-deoxyguanosine as an internal standard, UPLC–MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2′-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC–MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.
Keywords: Global DNA methylation; Human sperm; UPLC–MS/MS;

Development and validation of a LC-ESI–MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics by Hong-Liang Li; Xiao-Jin Peng; Jian-Chang He; En-Fu Feng; Gui-Li Xu; Gao-Xiong Rao (1653-1658).
A new LC-ESI–MS/MS assay method has been developed and validated for the quantification of swertiamarin, a representative bioactive substance of Swertia plants, in rat plasma using gentiopicroside, an analog of swertiamarin on chemical structure and chromatographic action, as the internal standard (IS). The swertiamarin and IS were extracted from rat plasma using solid-phase extraction (SPE) as the sample clean-up procedure, and they were chromatographed on a narrow internal diameter column (Agilent ZORBAX ECLIPSE XDB-C18 100 mm × 2.1 mm, 1.8 μm) with the mobile phase consisting of methanol and water containing 0.1% acetic acid (25:75, v/v) at a flow rate of 0.2 mL/min. The detection was performed on an Agilent G6410B tandem mass spectrometer by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M+CH3COO]  → 179 and m/z 415 [M+CH3COO]  → 179 for swertiamarin and IS, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL within a linear range of 5–1000 ng/mL (n  = 7, r 2  ≥ 0.994), and the limit of detection (LOD) was demonstrated as 1.25 ng/mL (S/N ≥ 3). The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day), accuracy, recovery, freeze/thaw, long-time stability and dilution integrity. This method was successfully applied to determination of the pharmacokinetic properties of swertiamarin in rats after oral administration at a dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration, 1920.1 ng/mL; time to reach maximum plasma concentration, 0.945 h; elimination half-time, 1.10 h; apparent total clearance, 5.638 L/h/kg; and apparent volume of distribution, 9.637 L/kg.
Keywords: Swertiamarin; LC-ESI–MS/MS; Rat plasma; Pharmacokinetics;

Development of a liquid chromatography–tandem mass spectrometry with pressurized liquid extraction method for the determination of benzimidazole residues in edible tissues by Dongmei Chen; Yanfei Tao; Huahai Zhang; Yuanhu Pan; Zhenli Liu; Lingli Huang; Yulian Wang; Dapeng Peng; Xu Wang; Menghong Dai; Zonghui Yuan (1659-1667).
A confirmatory and quantitative method of liquid chromatography–tandem mass spectrometry (LC–MS/MS) combined with a pressure liquid extraction (PLE) was developed for the determination of 11 benzimidazole and 10 metabolites of albendazole, fenbendazole and mebendazole in the muscles and livers of swine, cattle, sheep and chicken. For sample preparation, we used an automated technique of PLE method. The optimum extraction conditions were obtained using an 11 ml Accelerated Solvent Extraction (ASE) cells, acetonitrile/hexane as the extraction solvent. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 5 mmol l−1 formic ammonium as mobile phase. The analytes were detected in the positive ion multiple reaction monitoring (MRM) mode by the LC–ESI–MS/MS analysis. The recoveries of benzimidazole (BZDs) spiked at the levels of 0.5 μg kg−1 ranged from 70.1% to 92.7%; the between-day relative standard deviations were no more than 10%. The limits of quantification were 0.02–0.5 μg kg−1. The optimized method was successfully applied to monitor real samples containing BZDs, demonstrating the method to be simple, fast, robust and suitable for identification and quantification of BZDs residues in animal products.
Keywords: Benzimidazoles; Liquid chromatography; Mass spectrometry; Pressurized liquid extraction; Residues; Edible tissues;

A sensitive and selective gas chromatographic mass spectrometric method for the determination of midazolam and its biologically active metabolite, 1-hydroxymidazolam, in rabbit plasma has been developed and validated. Sample preparation includes mixed-mode solid-phase extraction and derivatization with silylating reagents. Midazolam-d4 was used as an internal standard for the determination of parent drug and its active metabolite. The instrumentation consisted of a capillary column gas chromatography and a single quadrupole mass spectrometer with a negative chemical ionization. The method was found to be valid in terms of selectivity, linearity, precision, accuracy, and recovery over the concentration range of 2–200 ng/ml and 1–100 ng/ml for midazolam and 1-hydroxymidazolam, respectively. For both analytes, the lower limit of quantification was 2 ng/ml. Midazolam was stable in stock solutions stored three months at −20 °C and in human plasma stored for three months at −80 °C. In addition, no degradation of midazolam was found after three freeze–thaw cycles, in short-term stability at room temperature for 24 h, or in post-preparative stability in the autosampler. The validity of the method was further tested by performing a pharmacokinetic study of sublingual administration of midazolam in rabbits. The method will be used in studies related to a formulation development of novel midazolam formulations for use in paediatric anaesthesia.
Keywords: Midazolam; 1-Hydroxymidazolam; GC–MS; Mixed-mode SPE; Negative chemical ionization; Sublingual administration;

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4′-hydroxytamoxifen, 4-hydroxytamoxifen and 4′-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 μL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4′-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4′-hydroxytamoxifen and N-desmethyl-4′-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4′-hydroxytamoxifen, 4-hydroxytamoxifen and 4′-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.
Keywords: Endoxifen; Liquid chromatography; Mass spectrometry; Metabolites; Tamoxifen;

A sensitive, precise and accurate quantitative LC–MS/MS method for the measurement of naproxen in human plasma was developed and completely validated according to current FDA and EMA guidelines. The new method employs acetonitrile protein precipitation for sample preparation and uses ketoprofen as the internal standard. Suitability of the new assay was assessed in comparison with 36 reported bioanalytical assays and the pharmacokinetic results obtained by the new method were compared to 11 reported studies in humans. The principal advantage of this LC–MS/MS method is the simultaneous achievement of high absolute recovery (90.0 ± 3.6%), acceptable sensitivity (lower limit of quantitation of 0.100 μg/mL), high inter-day precision (CV ≤ 9.4%), high analytical recovery (between 94.4 and 103.1%), and excellent linearity over the concentration range 0.100–50.0 μg/mL (r 2  ≥ 0.998) combined with a short run time of only 2 min.
Keywords: Naproxen; Liquid chromatography; Tandem mass spectrometry; Validation; Pharmacokinetics;

In this paper, the preparation of new supported ionic liquids (SILs) composed of the N-methylimidazolium cation and the quinoline cation is described. They have been confirmed and evaluated by infrared spectroscopy, elemental analysis and thermogravimetric analysis. Six kinds of different SILs included SiO2·Im+·Cl, SiO2·Im+·BF4 , SiO2·Im+·PF6 , SiO2·Qu+·Cl, SiO2·Qu+·BF4 and SiO2·Qu+·PF6 . The adsorption characteristics of ferulic acid (FA), caffeic acid (CA) and salicylic acid (SA) on SILs were investigated by static adsorption experiments. It was found that SiO2·Qu+·Cl had excellent adsorption and desorption capacity to three tested phenolic compounds. The dynamic adsorption characteristics of FA, CA and SA on SiO2·Qu+·Cl were also studied. The saturated adsorption capacity of FA, CA and SA using SiO2·Qu+·Cl as adsorbent was 64.6 mg/g, 53.2 mg/g and 72.2 mg/g respectively. Using 70% ethanol as eluent, the saturated desorption efficiencies of FA, CA and SA were 97.2%, 90.3% and 96.5% respectively. Thus, SiO2·Qu+·Cl had strong adsorption and separation capacity for FA, CA and SA.
Keywords: Supported ionic liquids; Adsorption; Ferulic acid; Caffeic acid; Salicylic acid;

In the last years, chromatographic supports with amino acids as immobilized ligands (AAILs) were been used successfully for isolation of several biomolecules, such as proteins. In this context and based on specific properties of human soluble cathecol-O-methyltransferase (hSCOMT), we screened and analyzed the effect of experimental conditions, such as pH and ionic strength manipulation for hSCOMT adsorption, over six different AAIL commercial supports. Typically, the proteins adsorption on AAIL chromatographic supports is around their pI. While hSCOMT isoelectric point is around 5.5, this parameter leads us to design new adsorption strategies with several acid buffers for the chromatographic process. In terms of the ionic strength manipulation strategy, the results suggest that the AAILs–hSCOMT interaction is strongly affected by the intrinsic hSCOMT hydrophobic domains. On the other hand, the interaction mechanism of hSCOMT on amino acid resins appears to be highly dependent on the binding pH. Consequently the retention mechanism of the target enzyme on the AAILs can be as either in typical hydrophobic or ionic chromatographic supports, so long as selecting various mobile phases and separation conditions. In spite of these mixed-mode interactions and operation strategies, the elution of interferent's proteins from recombinant host can be achieved only with suitable adjusts in pH mobile phase set point. This lead to a new approach in biochromatographic COMT retention, while possess a higher specificity than other chromatographic methods reported in literature.
Keywords: Amino acids immobilized ligands; Human soluble catechol-O-methyltransferase; Bioaffinity interactions; Mixed-mode; Proteins;

A simple, fast, sensitive and robust method for the determination of the sulfur mustard (SM) exposure biomarker–N 7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (HETEG) was reported using high-performance liquid chromatography–positive electrospray tandem mass spectrometry (HPLC–ESI-MS/MS), working in multiple reaction monitor (MRM) mode. The method provided limit of detection of 0.330 ng/mL and lower limit of quantitation of 0.940 ng/mL. The method was linearly calibrated from 0.940 ng/mL to 587 ng/mL with precisions of 3.5–14.5%, and accuracies of 88–112%. The recovery varied from 102% to 118%. HETEG spiked in DNA hydrolytes isolated from the human whole blood was stable after five freeze/thaw cycles and 35-day frozen at −20 °C. For the exposed biological samples, alkylated DNA was isolated from SM-treated human whole blood, followed by DNA digestion and adducts enrichment, the resulting alkylation base was determined. By the procedure, the HETEG level in DNA hydrolytes isolated from the human whole blood exposure to 312 ng/mL SM was detected successfully.
Keywords: N 7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine; Human whole blood; Determination; Validation; High performance liquid chromatography–tandem mass spectrometry;

Dried blood spot assay for estimation of metronidazole concentrations in rats and its application in single animal drug pharmacokinetic study by Prashant Laxman Kole; Rita Majithia; Thakur Raghu Raj Singh; Martin J. Garland; Katarzyna Migalska; Ryan F. Donnelly; James McElnay (1713-1716).
An HPLC-UV-dried blood spot (DBS) method for the estimation of metronidazole (MTZ) in rat whole blood is reported. Method employs Ahlstrom 226 sample collection paper and DBS samples were prepared by spotting with 30 μl of whole blood (spiked calibration standards/quality control samples/in vivo study samples). A 6 mm disc was punched from each DBS and extraction was carried out using water containing the internal standard (tinidazole). The calibration for MTZ was linear over 2.5–50 μg/ml concentration range. Accuracy (% bias) and precision (expressed as % Coefficient of variation) values for within and between day were <20% at the lower level quality control sample (LQC) and <15% at all other concentrations tested. The limit of quantification (LOQ) of the method was 2.5 μg/ml. The validated method was applied for the analysis of in vivo pharmacokinetic (PK) study samples after intravenous administration of MTZ to a rat. Whole blood PK parameters observed in this study were in compliance with literature based PK parameters. The DBS sampling approach was found to be useful in a single animal pharmacokinetic study.
Keywords: Dried blood spot; Single animal pharmacokinetic study; Ahlstrom 226; Metronidazole;