Journal of Chromatography B (v.1001, #C)

A liquid chromatography–tandem mass spectrometry method using electrospray ionization in positive ionization mode was developed for the simultaneous detection of multiple opioid-type drugs in plasma. The presented assay allows the quantitative determination of alfentanil, buprenorphine, codeine, desomorphine, dextromethorphan, dextrorphan, dihydrocodeine, dihydromorphine, ethylmorphine, fentanyl, hydrocodone, hydromorphone, methadone, morphine, naloxone, naltrexone, oxycodone, oxymorphone, pentazocine, pethidine, pholcodine, piritramide, remifentanil, sufentanil, and tramadol as well as the metabolites 6-monoacetylmorphine, bisnortilidine, morphine-3-glucuronide, morphine-6-glucuronide, naltrexol, norbuprenorphine, norfentanyl, norpethidine, nortilidine, and O-desmethyltramadol. Serum and blood samples were purified by solid-phase extraction. The analytes were separated on a phenyl–hexyl (100 mm) column by formic acid/acetonitrile gradient elution using an UPLC 1290 Infinity coupled with a 6490 Triple Quadrupole mass spectrometer. The limits of detection ranged from 0.02 to 0.6 ng/mL and the lower limits of quantification ranged from 0.1 to 2.0 ng/mL. The calibration curves were linear between Calibration Levels 1–6 for all 35 substances. Recovery rates ranged between 51 and 88% for all compounds except alfentanil, bisnortilidine, pethidine, and morphine-3-glucuronide. The matrix effect ranged from 86% for ethylmorphine to 105% for desomorphine. Using the validation procedure proposed by the German Society of Toxicological and Forensic Chemistry, acceptable precision and accuracy data for almost all analytes were obtained. The method was successfully applied to 206 authentic serum samples provided by the palliative and intensive care units of the University Medical Center and the police authorities. Furthermore, a suspected fatal intoxication is demonstrated by an analysis of the sufentanil in post mortem body fluids and tissues.
Keywords: LC–MS/MS; Opioids; Blood level; Solid-phase extraction; Method validation;

LC-ESI–MS/MS method for bioanalytical determination of osteogenic phytoalexin, medicarpin, and its application to preliminary pharmacokinetic studies in rats by Isha Taneja; Kanumuri Siva Rama Raju; Muralikrishna Challagundla; Ashutosh Raghuvanshi; Atul Goel; Muhammad Wahajuddin (9-16).
Medicarpin is the active phytoalexin found in the stem bark of Butea monosperma having potent osteogenic activity. An LC-ESI–MS/MS was developed and validated for quantification of medicarpin in rat plasma using liquid-liquid extraction technique and diethyl ether as the extraction solvent. Medicarpin was separated on RP18 column (4.6 mm × 50 mm, 5.0 μm) using methanol and 10 mM ammonium acetate (pH 4.0) in the ratio of 80:20 (v/v) as mobile phase. The method was linear within the concentration range of 1–500 ng/mL and its sensitivity was 1 ng/mL. The precision value for intra- and inter-day assays and stability assays was within 0.88–14.22% while the accuracy ranged between 87.46–116.0% at all four QC levels. The validated method was successfully applied to study the preclinical pharmacokinetics of medicarpin in rats. Medicarpin showed multiple peak phenomenon upon oral administration. Its oral bioavailability was 17.43%. It was found to be a rapidly absorbed (T max  = 15 min), 81.61% protein bound and pH stable compound. The present study provides important information regarding preliminary pharmacokinetics of medicarpin for its further exploration as a potential therapeutic agent.
Keywords: Bioavailability; Pharmacokinetics; Bioanalytical method validation; Extraction; Recovery; LC–MS/MS;

Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method by Szymon Dziomba; Teresa Łepek; Zbigniew Jaremicz; Maria Łuczkiewicz; Adam Prahl; Piotr Kowalski (17-21).
An electrophoretic method for fast separation of three tropane alkaloids (hyoscyamine, anisodamine and scopolamine) was presented. The substances were complete resolved in less than one minute due to utilization of relatively short capillary (20.2 cm effective length) and high voltage (25 kV). Detector probing frequency was found among the parameters that significantly affected the detection sensitivity. The performed experiments showed insufficient available probing frequency of used commercial spectrophotometric detector according to capillary electrophoresis (CE) separation potential.Under the optimized conditions the background electrolyte (BGE) was composed of 20 mM Tris, 6 mM HCl and 20 mM NaCl (pH 8.50). All analyses were carried out in fused silica capillaries of 50 μm (inner diameter) and 31.2 cm (total capillary length). Samples were injected hydrodynamically (5 s; 3.45 kPa) without any sample preparation step and separation was performed at 25 kV. The elaborated method was applied in plant cultures growth media analysis after incubation with hairy roots of selected Solanaceae species. The performed experiments proved the usefulness of CE in quality control of biotechnological processes.
Keywords: Anisodamine; Biotechnology; Capillary zone electrophoresis; Hyoscyamine; Scopolamine; Tropane alkaloids;

A selective liquid chromatographic–mass spectrometric method (LC–MS/MS) has been established and validated for simultaneous determination of subutinib and its active metabolite in human plasma. Plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on a Wondasil C18 (150 mm × 2.1 mm, 3.5 μm), with methanol–0.2% formic acid solution (73:27, v/v) as mobile phase at flow rate of 0.2 ml/min. The linear range was 0.25–100 ng/mL for subutinib and 0.125–50.0 ng/mL for its active metabolite, with lower limit of quantitation of 0.25 ng/mL and 0.125 ng/mL, respectively. Intra- and inter-run precision were within 7.0 and 13.1%, and the accuracies (relative errors) were < 7.0 and 8.0%, with the extraction recoveries 97.0–101.2% and 93.0–98.1% for the two analytes, respectively. The validated method was successfully applied to a pharmacokinetic study of subutinib and its active metabolite in human after oral administration of subutinib maleate capsules.
Keywords: Subutinib; Active metabolite; LC–MS/MS; Pharmacokinetics;

Quantification of 15 bile acids in lake charr feces by ultra-high performance liquid chromatography–tandem mass spectrometry by Ke Li; Tyler J. Buchinger; Ugo Bussy; Skye D. Fissette; Nicholas S. Johnson; Weiming Li (27-34).
Many fishes are hypothesized to use bile acids (BAs) as chemical cues, yet quantification of BAs in biological samples and the required methods remain limited. Here, we present an UHPLC–MS/MS method for simultaneous, sensitive, and rapid quantification of 15 BAs, including free, taurine, and glycine conjugated BAs, and application of the method to fecal samples from lake charr (Salvelinus namaycush). The analytes were separated on a C18 column with acetonitrile–water (containing 7.5 mM ammonium acetate and 0.1% formic acid) as mobile phase at a flow rate of 0.25 mL/min for 12 min. BAs were monitored with a negative electrospray triple quadrupole mass spectrometer (Xevo TQ-S™). Calibration curves of 15 BAs were linear over the concentration range of 1.00–5,000 ng/mL. Validation revealed that the method was specific, accurate, and precise. The method was applied to quantitative analysis of feces extract of fry lake charr and the food they were eating. The concentrations of analytes CA, TCDCA, TCA, and CDCA were 242.3, 81.2, 60.7, and 36.2 ng/mg, respectively. However, other taurine conjugated BAs, TUDCA, TDCA, and THDCA, were not detected in feces of lake charr. Interestingly, TCA and TCDCA were detected at high concentrations in food pellets, at 71.9 and 38.2 ng/mg, respectively. Application of the method to feces samples from lake charr supported a role of BAs as chemical cues, and will enhance further investigation of BAs as chemical cues in other fish species.

Development and validation of an ultra performance liquid chromatography tandem mass method for sildenafil and N-desmethyl sildenafil plasma determination and quantification by Marco Simiele; Debora Pensi; Daniela Pasero; Francesca Ivaldi; Mauro Rinaldi; Giovanni Di Perri; Vito Marco Ranieri; Antonio D'Avolio (35-40).
Sildenafil is a selective inhibitor of cGMP-specific type 5 phosphodiesterase (PDE5) used for the treatment of masculine erectile dysfunction and Pulmonary Arterial Hypertension (PAH). Sildenafil causes vasodilatation; relax of the smooth muscle and reduction of pulmonary arterial pressure. In the liver cytocrome P450 metabolizes sildenafil into its active metabolite, N-desmethyl sildenafil. The determination of plasma levels of sildenafil and N-desmethyl sildenafil could be useful for therapy optimization and pharmacokinetic studies. We have developed and validated a new method for the quantification of sildenafil and its metabolite in human plasma by rapid protein precipitation extraction, using an UPLC system, coupled with a tandem mass spectrometric detector (UPLC–MS/MS). The calibration range was fitted at least square model (r 2  ≥ 0.999), with an accuracy and an intra- and inter-day RSD% (Relative Standard Deviation), both for sildenafil and N-desmethyl sildenafil, lower than 15%, as required by the FDA guidelines; LLOQ, LLOD, ULOQ were 3.9 ng/mL, 1.95 ng/mL and 1000 ng/mL, respectively, for both analytes. Matrix effect, expressed as mean percent deviation of peak areas, was in the range between 2.6% and 5.8%, lower than 15% as required by guidelines. The mean recovery was 83.2 % for sildenafil and 84.5% for N-desmethyl sildenafil. This method has successfully been applied to a clinical pharmacokinetic study of sildenafil and N-desmethyl sildenafil in patients with PAH undergoing cardiac surgery.
Keywords: Ultra performance liquid chromatography tandem mass; Sildenafil; N-desmethyl sildenafil; Therapeutic drug monitoring; Metabolite;

A high-throughput, simultaneous analysis of carotenoids, chlorophylls and tocopherol using sub two micron core shell technology columns by Kranthi K. Chebrolu; Gad G. Yousef; Ryan Park; Yoshinori Tanimura; Allan F. Brown (41-48).
A high-throughput, robust and reliable method for simultaneous analysis of five carotenoids, four chlorophylls and one tocopherol was developed for rapid screening large sample populations to facilitate molecular biology and plant breeding. Separation was achieved for 10 known analytes and four unknown carotenoids in a significantly reduced run time of 10 min. Identity of the 10 analytes was confirmed by their UV–Vis absorption spectras. Quantification of tocopherol, carotenoids and chlorophylls was performed at 290 nm, 460 nm and 650 nm respectively. In this report, two sub two micron particle core-shell columns, Kinetex from Phenomenex (1.7 μm particle size, 12% carbon load) and Cortecs from Waters (1.6 μm particle size, 6.6% carbon load) were investigated and their separation efficiencies were evaluated. The peak resolutions were >1.5 for all analytes except for chlorophyll-a′ with Cortecs column. The ruggedness of this method was evaluated in two identical but separate instruments that produced CV < 2 in peak retentions for nine out of 10 analytes separated.
Keywords: Simultaneous analysis; Sub-two micron columns; Core shell technology; Acquity UPLC; Carotenoids; Broccoli;

Simultaneous determination of six bioactive constituents of Guizhi Fuling Capsule in rat plasma by UHPLC–MS/MS: Application to a pharmacokinetic study by Longshan Zhao; Zhili Xiong; Yang Sui; Heyun Zhu; Zhiyu Zhou; Zhenzhong Wang; Yiwu Zhao; Wei Xiao; Jianyang Lin; Kaishun Bi (49-57).
A rapid and selective ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for simultaneous determination of gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol and cinnamic acid, the major bioactive constituents of Guizhi Fuling Capsule in rat plasma using phenacetin as internal standard (IS). The plasma samples were pretreated by protein precipitation with acetonitrile after acidification and separated on a Waters BEH C18 column (50 mm × 2.1 mm, 1.7 μm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.2 mL/min. Mass spectrometric detection was performed on Micromass Quattromicro API mass spectrometer equipped with electrospray ionization source in multiple reaction monitoring (MRM) mode. The intra- and inter-day precisions (as relative standard deviation) were below 14.6% for all analytes, and the accuracies (as relative error) were within ±5.0%. The lower limits of quantification (LLOQ) were 10, 10, 5, 5, 25, 25 ng/mL for gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol and cinnamic acid, respectively. Extraction recovery, matrix effect and stability were satisfactory in rat plasma. This method was fully validated and applied to a pharmacokinetic study of the six bioactive constituents after oral administration of Guizhi Fuling Capsule to rats.
Keywords: Guizhi Fuling Capsule; UHPLC–MS/MS; Pharmacokinetics; Rat plasma;

In the present communication, uniformly sized molecularly imprinted polymer (MIP) as nanospheres were synthesized based on precipitation polymerization using dual-template imprinting approach and used it as sorbent for solid phase extraction of six urinary benzene metabolites (UBMs). This approach in combination with injector port silylation (IPS) has been used for the quantitative determination of these UBMs by gas chromatography-tandem mass spectrometry. The MIP was synthesized by using t,t-muconic acid (t,t-MA) and 1,2,4-trihydroxybenzene (THB) as templates, methacrylic acid (MAA) as a monomer, ethyleneglycoldimethacrylate (EGDMA) as crosslinker, acetonitrile and dimethylsulphoxide as a porogen and azobisisobutyronitrile (AIBN) as an initiator. The factors affecting the performance of polymer and IPS were investigated and optimized for the simultaneous determination of UBMs in urine. Binding study of imprinted and non-imprinted polymer (NIP) shows that, MIP possesses higher affinity in comparison to NIP for these analytes. Under the optimum conditions, the method developed was found to be linear with regression coefficients falls in the range of 0.9721–0.9988 for all the analyzed metabolites. The percent recovery of the metabolites analyzed in urine was found to be in the range of 76–89%, while the limit of detection and limit of quantification were found to be in the range of 0.9–9.1 ng mL−1 and 2.8–27 ng mL−1 respectively. The validated method was successfully applied to the real urine samples collected from different groups (kitchen workers, smokers and petroleum workers) and found that the developed method has been promising applications in the routine analysis of urine samples of benzene exposed population.
Keywords: Dual-template imprinting approach; Molecularly imprinted nanospheres; Precipitation polymerization; Injector-port silylation; Urinary benzene metabolites; Gas chromatography-tandem mass spectrometry;

Satureja hortensis L. and Oliveria decumbens Vent. are known for their diverse effects in drug therapy and traditional medicine. One of the most interesting properties of their essential oils is good antioxidant activity. In this paper, essential oils of aerial parts of S. hortensis L. and O. decumbens Vent. from different regions were obtained by hydrodistillation and were analyzed by gas chromatography–mass spectrometry (GC–MS). Essential oils were tested for their free radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to identify the peaks potentially responsible for the antioxidant activity from chromatographic fingerprints by numerous linear multivariate calibration techniques. Because of its simplicity and high repeatability, orthogonal projection to latent structures (OPLS) model had the best performance in indicating the potential antioxidant compounds in S. hortensis L. and O. decumbens Vent. essential oils. In this study, P-cymene, carvacrol and β-bisabolene for S. hortensis L. and P-cymene, Ç-terpinen, thymol, carvacrol, and 1,3-benzodioxole, 4-methoxy-6-(2-propenyl) for O. decumbens Vent. are suggested as the potentially antioxidant compounds.
Keywords: Chromatographic fingerprints; Linear multivariate calibration; Potentially antioxidant compounds; Satureja hortensis L.; Oliveria decumbens Vent.;

In this work, eleven compounds were successfully separated from Trollius chinensis Bunge by using a two-step high-speed counter-current chromatography (HSCCC) method. NRTL-SAC (nonrandom two-liquid segment activity coefficient) method, a newly developed solvent system selection strategy, was applied to screening the suitable biphasic liquid systems. Hexane/ethyl acetate/ethanol/water (3:7:3:7, v/v) solvent system was used in the first step, while the hexane/ethyl acetate/methanol/water (1:2:1:2, 1:4:1:4, 1:9:1:9, v/v) systems were employed in the second step. The chemical structures of the separated compounds were identified by UV, high resolution ESI-MS and MS/MS data. The separated compounds are 3,4-dihydroxyphenylethanol (1), vanillic acid (2), orientin (3), vitexin (4), veratric acid (5), 2″-O-(3‴, 4‴-dimethoxybenzoyl) orientin (6), 2″-O-feruloylorientin (7), 2″-O-feruloylvitexin (8), 2″-O-(2‴-methylbutyryl) vitexin (9), 2″-O-(2‴-methylbutyryl) isoswertiajaponin (10), 2″-O-(2‴-methylbutyryl) isoswertisin (11). The results demonstrate that HSCCC is a powerful tool for the separation of compounds from extremely complex samples.
Keywords: HSCCC; Trollius chinensis Bunge; Separation; Flavonoids; Phenolic acids;

The tissue distribution, excretion, and metabolic pathway of 2,2′,4,4′,5-penta-chlorinated diphenylsulfide (CDPS-99) in ICR mice were investigated after oral perfusion at 10 mg/kg body weight (b.w.). Biological samples were extracted and separated and, for the first time, were determined by a novel, sensitive, and specific GC–MS method under the full scan and selected ion monitoring (SIM) modes. The results showed that the concentrations of CDPS-99 in the liver, kidneys, and serum reached a maximum after a one-day exposure and that the CDPS-99 concentration in the liver was the highest (3.43 μg/g). The increase in the concentration of CDPS-99 in muscle, skin, and adipose tissue was slower, and the concentrations of CDPS-99 achieved their highest levels after 3 days of exposure. It was observed that the CDPS-99 concentration in adipose tissue was still very high (0.71 μg/g) after 21 days of exposure, which suggested that CDPS-99 was able to accumulate in adipose tissue. In addition, mouse feces accounted for approximately 75% of the total gavage dose, indicating that CDPS-99 was mainly excreted via mouse feces. Metabolism analysis demonstrated that there were three possible metabolic pathways of CDPS-99 in mice: dechlorination reactions with the formation of tetra-CDPS and hydroxylation and oxidation reactions with the formation of OH-CDPS-99 and chlorinated diphenylsulfone. The present study will help to develop a better understanding of mammalian metabolism of CDPS-99.
Keywords: 2,2′,4,4′,5-penta-chlorinated diphenylsulfide (CDPS-99); Distribution; Excretion; Metabolism;

Display Omitted Rehmannia glutinosa is a widely used traditional Chinese medicine (TCM) in clinical practice to tackle chronic kidney disease for thousands of years. However, the in vivo metabolism of its two major bioactive components (catalpol and acteoside) remains unknown. In this paper, a highly sensitive, rapid and robust ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with MetaboLynx™ software combined with mass defect filtering (MDF) method was established. This validated analysis method was successfully applied to investigate the in vivo metabolic profiles of R. glutinosa extract in normal and chronic kidney disease (CKD) rats. The results showed that a total of 17 metabolites of two parent compounds in normal rats in vivo were tentatively detected and identified according to the characteristics of their protonated ions and relevant literature. While 11 of the metabolites were observed in the CKD rat samples. These metabolites suggested that catalpol was firstly deglycosylated to its aglycone and subsequently to two main metabolites (M1 and M4) by conjugation and hydrogenation respectively and acteoside was mainly metabolized by O-glucuronide conjugation and O-sulphate conjugation. In conclusion, this study showed an insight into the metabolism of R. glutinosa extract in vivo and the proposed metabolic pathways of bioactive components might play a key role in further pharmacokinetic experiments evaluations.
Keywords: Rehmannia glutinosa extract; Catalpol; Acteoside; UPLC-Q/TOFMS; Metabolites;

A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of cinnamaldehyde, cinnamic acid, and 2-methoxy cinnamic acid in rat whole blood. It was the first time to study the pharmacokinetics of 2-methoxy cinnamic acid in rat whole blood. Samples were processed by a one-step protein precipitation with acetonitrile-37% formaldehyde (90:10, v:v). Chromatographic separation was performed on a Thermo Scientific C18 column (2.1 mm × 50 mm, 1.9 μm) at room temperature. The total run time was 4 min. The detection was accomplished by using positive and negative ion electrospray ionization in multiple reaction monitoring mode. The method was linear for all of the analytes over 1000 times concentration range with correlation coefficients greater than 0.99. The lower limits of quantification (LLOQ) were 0.1 ng/mL for cinnamaldehyde, 5.8 ng/mL for cinnamic acid, and 10 ng/mL for 2-methoxy cinnamic acid, respectively. To our knowledge, this was the first time that the LLOQ for cinnamaldehyde in validated methods for biological samples was as low as 0.1 ng/mL. Intra- and inter-day precision and accuracy were within ±9% for all of the analytes during the assay validation. Assay recoveries were higher than 80% and the matrix effects were minimal. The half-life were 8.7 ± 0.7 h for cinnamaldehyde, 1.0 ± 0.5 h for cinnamic acid, and 1.4 ± 0.4 h for 2-methoxy cinnamic acid, respectively. The validated assay was firstly applied to the simultaneous quantification of cinnamaldehyde, cinnamic acid, and 2-methoxy cinnamic acid, especially for 2-methoxy cinnamic acid in rat whole blood after oral administration of 15 mg/kg essential oil of Cinnamoni Ramulus. It was observed that the C max and AUC of 2-methoxy cinnamic acid (0.01% in essential oil of Cinnamoni Ramulus) were greater than those of cinnamaldehyde (83.49% in essential oil of Cinnamoni Ramulus), which implied that 2-methoxy cinnamic acid might be the major bioactive constitutes in essential oil of Cinnamoni Ramulus.
Keywords: Cinnamomi Ramulus; Cinnamaldehyde; Cinnamic acid; 2-Methoxy cinnamic acid; UHPLC-MS/MS; Pharmacokinetics;

A novel and practical sample pretreatment method based on hollow fiber centrifugal ultrafiltration (HFCF-UF) was developed to determine plasma protein binding by using HPLC. The samples for analyzing unbound and total concentrations could be prepared in parallel simultaneously by the same device. It only required centrifugation for a short time and the filtrate could be injected directly for HPLC analysis without further treatment. Coumarins were selected as the model drugs. Acetone was chosen as the releasing agent to free the binding drug from the drug–protein complex for the total drug concentration determination. Non-specific bindings (NSBs) between the analytes and hollow fiber membrane materials were investigated. The type and volume of protein binding releaser were optimized. Additionally, centrifugal speed and centrifugal time were considered. Under the optimized conditions, the absolute recovery rates of the unbound and total concentrations were in the range of 97.5–100.9% for the three analytes. The limits of detection were in the range of 0.0135–0.0667 μg mL−1. In vitro plasma protein binding of the three coumarins was determined at three concentrations using the validated method and the relative standard deviations (RSDs) were less than 3.4%. Compared with traditional method, the HFCF-UF method is simple to run, no specialized equipment requirement and is a more accurate plasma pretreatment procedure with almost excellent drug–protein binding equilibrium. Therefore, this method can be applied to determine the plasma protein binding in clinical practice. It also provides a reliable alternative for accurate monitoring of unbound or total drug concentration in therapeutic drug monitoring (TDM).
Keywords: Plasma protein binding; Hollow fiber centrifugal ultrafiltration; Acetone; Protein binding displacer; Coumarins; HPLC;

Ionic liquid matrix (ILM) is found to be a very versatile substance for analysis of broad range of organic molecules in matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS) due to good solubility for a variety of analytes, formation of homogenous crystals and high vacuum stability of the matrix. In the present work, an ILM, cyno-4-hydroxycinnamic acid-butylamine (CHCAB) was employed in dispersive liquid–liquid microextraction (DLLME) as sample probe and matrix for extraction and ionization of phospholipids from food samples (soybean) prior to MALDI–MS analysis. With the employed technique, 8–125 fold improvement in signal intensity and limit of detection were achieved for the analysis of phospholipids. The best extraction efficiency of phospholipids in ILM–DLLME was obtained with 5 min extraction time in presence 30 mg/mL CHCAB and 1.2% NaCl using chloroform as an extracting solvent and methanol as a dispersing solvent. Further, the developed ILM–DLLME procedure has been successfully applied for the analysis of phospholipids in soybean samples in MALDI–MS.
Keywords: MALDI–MS; Ionic liquid matrix; Dispersive liquid–liquid microextraction; Phospholipids; Soybean;

Recently, a fixed dose combination (FDC) of memantine (MM) and donepezil (DPZ) has been approved for the treatment of Alzheimer’s disease (AD). In the present work, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of MM and DPZ was developed and validated in rat plasma over the linearity range of 0.2–400 ng/mL using amantadine (AM) as an internal standard. Both the analytes and IS were extracted using one step liquid–liquid extraction procedure. The analytes were separated on C18 reversed phase column with mobile phase consisting of a mixture of methanol and 10 mM ammonium acetate, pH 5 (92:8 v/v) at a flow rate of 0.7 mL/min. The detection of the analytes was done on triple quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI) and quantified using multiple reaction monitoring (MRM). The method was fully validated in terms of linearity, accuracy, precision, recovery, matrix effect, dilution integrity, carry-over effect and stability. The within- and between-run precisions were <10% and accuracy was all within ±10%. The mean recovery of MM and DPZ was found to be greater than 80%. The % RSD value at higher as well as lower concentration was well within the acceptable range (±15%) in all the stability experiments. The method was successfully applied to the oral pharmacokinetics and drug–drug interaction study of MM and DPZ in male Sprague Dawley (SD) rats.
Keywords: Memantine; Donepezil; Alzheimer’s disease; LC–MS/MS; Pharmacokinetics; Drug interaction;

A new, sensitive and selective liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI-MS/MS) method was developed for the analysis of Phospholipids (PLs) in bio-oils and fats. This analysis employs hydrophilic interaction liquid chromatography–scheduled multiple reaction monitoring (HILIC–sMRM) with a ZIC–cHILIC column. Eight PL class selective internal standards (homologs) were used for the semi-quantification of 14 PL classes for the first time. More than 400 scheduled MRMs were used for the measurement of PLs with a run time of 34 min. The method's performance was evaluated for vegetable oil, animal fat and algae oil. The averaged within-run precision and between-run precision were ≤10% for all of the PL classes that had a direct homologue as an internal standard. The method accuracy was generally within 80–120% for the tested PL analytes in all three sample matrices.
Keywords: Phospholipid; Bio-oil; HILIC; Electrospray; Tandem mass spectrometry;

Isomers 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7–dihydroxy-3,6,8 trimethoxy flavone) (flavone A) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) (flavone B) have recently demonstrated differential antineoplastic activities against pancreatic cancer in vitro. These studies also indicated that these compounds target highly tumorigenic cells while sparing normal cells. The in vivo antitumor activities of these flavones have not been determined, and detection protocols for these compounds are needed to conduct pre-clinical assays following intravenous dosing. Here, we report methods developed using acetonitrile to extract two flavone isomers and corresponding internal standards, celecoxib and diclofenac, from rat plasma. Separation was achieved using a Shimadzu liquid chromatography system with a C18 column and mobile phase acetonitrile/water (60:40 and 70:30 for flavones A and B, respectively) containing 0.2% acetic acid and 0.05% triethylamine at a flow rate of 0.4 mL/min and detection at 245 nm. Calibration curves ranging from 250 to 2500 ng/mL and 2500 to 100,000 ng/mL for both flavones were linear (r 2  ≥  0.99) with the lower limits of quantification being 250 ng/mL. Recovery of concentrations 250, 1000, 2500, 5000, and 100,000 ng/mL ranged from 87 to 116% and 84 to 103% (n  = 3) for flavone A and B, respectively. Stability of both flavones after a freezing/thawing cycle yielded a mean peak ratio ≥0.92 when compared to freshly extracted samples. Intravenous administration of a 20 mg/kg dose in rats yielded half-lives of 83.68 ± 56.61 and 107.45 ± 53.31 min with clearance values of 12.99 ± 13.78 and 80.79 ± 35.06 mL/min/kg for flavones A and B, respectively.
Keywords: 5,7-Dihydroxy-3,6,8 trimethoxy flavone; 3,5-Dihydroxy-6,7,8-trimethoxy flavone; Flavonoids; Cancer; HPLC; Pharmacokinetics;

Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC–MS/MS). A biologically relevant tryptic peptide ‘GDSVVYGLR’ was used as a signature peptide for this method. The optimized method involved immunocapture of the analyte protein using hOPN specific antibodies followed by trypsin digestion to obtain the signature peptide. Analysis was carried out on a Waters IonKey/MS system using a flow rate of 2.5 μL/min. Immunocapture buffer was used as a surrogate matrix for the validation studies. The method was validated over a range of 25–600 ng/mL. Intra-assay and inter-assay accuracies were within ±13%. Intra-assay and inter-assay precision were within 17%. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. Inherent digestion variability i.e., under controlled conditions, was within ±20% with both IS peptides. In digestion variability studies, where trypsin activity was varied (20–180%), the use of the extended SIL peptide as an internal standard limited the variability to within ±30% of the normalized response. Alternatively, when the SIL peptide was used as the internal standard, the variability ranged from −67.4% to 50.6% of the normalized response. The applicability of the validated method was demonstrated by quantification of OPN from plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. The plasma OPN concentrations in healthy individuals ranged from 38 to 85 ng/mL with a mean concentration of 55.4 ± 15.3 ng/mL. A 1.5–12 fold increase in OPN concentrations, ranging from 85 to 637 ng/mL, was seen in breast cancer patient samples.
Keywords: Human osteopontin (hOPN); Stable labeled isotope internal standard (SIL-IS); Extended stable labeled isotope internal standard; Cancer biomarker; Microflow liquid chromatography and tandem mass spectrometry (MFLC–MS/MS); IonKey/MS; Digestion variability;

A capillary ion chromatography–mass spectrometry (MS) method was proposed to determine glucosylglycerol (GG), sucrose, and five other carbohydrates. MS conditions and make-up flow parameters were optimized. This method is accurate and sensitive for simultaneous analysis of carbohydrates, with mean correlation coefficients of determination greater than 0.99, relative standard deviation of 0.91–2.81% for eight replicates, and average spiked recoveries of 97.3–104.9%. Limits of detection of sodium adduct were obtained with MS detection in selected ion mode for GG (0.006 mg/L), sucrose (0.02 mg/L), and other carbohydrates (0.03 mg/L). This method was successfully applied to determine GG and sucrose in intracellular extracts of salt-stressed cyanobacteria.
Keywords: Capillary ion chromatography; Mass spectrometry; Glucosylglycerol; sucrose; Cyanobacteria;

Azilsartan medoxomil potassium salt (TAK-491) is an orally administered angiotensin II type 1 receptor blocker for the treatment of hypertension and is an ester-based prodrug that is rapidly hydrolyzed to the pharmacologically active moiety, azilsartan (TAK-536), during absorption. TAK-536 is biotransformed to the 2 metabolites M-I by decarboxylation and M-II by dealkylation. In this study, we developed and validated a LC/MS/MS method which can simultaneously determine 4 analytes, TAK-491, TAK-536, M-I and M-II. The bioanalytical method can be outlined as follows: two structural analogues are used as the internal standards. The analytes and the IS are extracted from human plasma using solid phase extraction. After evaporating, the residue is reconstituted and injected into a LC/MS/MS system with an ESI probe and analyzed in the positive ion mode. Separation is performed through a conventional reversed-phase column with a mobile phase of water/acetonitrile/acetic acid (40:60:0.05, v/v/v) mixture at a flow rate of 0.2 mL/min. The total run time is 8.5 min. The calibration range is 1–2500 ng/mL in human plasma for all the analytes. Instability issues of the prodrug, TAK-491, were overcome and all the validation results met the acceptance criteria in accordance with the regulatory guideline/guidance. As a result of the clinical study, the human PK profiles of TAK-536, M-I and M-II were successfully obtained and also it was confirmed that TAK-491 was below the LLOQ (1 ng/mL) in the human plasma samples.
Keywords: Prodrug; Azilsartan medoxomil; TAK-491; TAK-536; LC/MS/MS; Validation;

A new quantitation method based on a multiple stable isotope dilution assay (SIDA) was developed for polyethylene glycol (PEG) homologues from PEG mixtures with average molecular weights (MW) of 400, 1500, 3000 and 4000 Da in urine. Seven [13C4 2H4] and two [13C8 2H8]PEG homologues were synthesized and served as labelled internal standards for SIDA. PEG oligomers were resolved by reversed phase high performance liquid chromatography (RP-HPLC) coupled to mass spectrometry (MS) in multiple ion (MI) scan modus. Very low limits of detection (LODs) in a range of 0.4–12 ng/mL were achieved for the single homologues. Higher PEG homologues showed increased LODs and LOQs and less effective recovery (77–87%) than PEG with lower molecular masses (95–121%). Precision (relative standard deviation) varied between 3 and 13% and showed no dependence of the chain length. The method was successfully applied to human and mice urine samples. Beside an accurate quantitation of single PEG homologues it was possible to show an alteration in the MW distribution in urine samples compared to the dosed PEG solutions. The highest MW, with which a PEG can pass the intestinal wall (so called “cut off”) for humans appeared to be higher than for mice.
Keywords: Polyethylene glycols; PEG reference compounds; Stable isotope dilution assay; Quantitative NMR; Human and mice urine; Membrane permeability;

An ultrahigh performance liquid chromatography–tandem mass spectrometry method for the identification and quantification of neurotransmitters, metabolites and precursors at different stages in zebrafish life was developed. Betaine, glutamine, glutamic acid, γ-aminobutyric acid, phosphocholine, glycerophosphocholine, cytidine 5′-diphosphocholine, choline, acetylcholine, dopamine, norepinephrine, serotonin, tyrosine, epinephrine, tryptophan, 5-hydroxyindolacetic acid and agmatine were selected as analytes. The method consisted of a simple deproteinization of samples using methanol and formic acid, subsequent injection onto the chromatographic equipment and quantification with a triple quadrupole mass spectrometer detector using an electrospray ionization interface in positive mode. Limits of detection ranged from 0.02 to 11 ng mL−1 and limits of quantification from 0.1 to 38 ng mL−1, depending on the analyte. The method was validated according to US Food and Drugs Administration (FDA) guideline for bioanalytical assays. Precision, expressed as relative standard deviation (%RSD), was lower than 15% in all cases, and the determination coefficient (R 2) was equal or higher than 99.0% with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 85% and 115%. The method was applied to determine of these compounds in zebrafish from early stages of development to adulthood and showed the time-course of neurotransmitters and others neurocompounds through the life cycle. The possibility of measuring up to 17 compounds related with the main neurotransmitter systems in a simple analytical method will complement and reinforce the use of zebrafish in multiple applications in the field of neurosciences. The proposed method will facilitate future studies related with brain development.
Keywords: Neurotransmitters; Zebrafish; Life cycle; Analytical method validation; UHPLC–MS/MS;

A sensitive UPLC-MS/MS method was established and validated for the quantitation of celecoxib and its metabolites in rat blood. The analytes were extracted from rat blood samples by a salting-out liquid–liquid extraction method followed by the UPLC chromatography. The mass analysis of effluent was performed on an API 5500 Qtrap mass spectrometer via multiple reactions monitoring (MRM). The linear response ranges were 0.3–20000 nM for celecoxib, and 1.2–20000 nM, 0.3–20000 nM, 2.0–2000 nM, 1.5–6000 nM for its metabolites carboxycelecoxib (M2), hydroxycelecoxib (M3), hydroxycelecoxib glucuronide (M1), and carboxycelecoxib glucuronide (M5), respectively. The inter-day and intra-day accuracies were within 85–115%, and the inter-day and intra-day precision were acceptable (<12%) for all analytes. Recoveries were above 70% and no obvious matrix effects were observed. The validated UPLC-MS/MS method was successfully applied to a pharmacokinetics study of oral celecoxib (20 mg/kg) in Sprague-Dawley rats, and the rat blood concentrations (0–48 h) of celecoxib and two of its metabolites M2 and M3 were successfully determined. Using the same method, we also showed preferential distributions of celecoxib, M2 and M3 in the blood cells as compared to the plasma. In conclusion, our results showed that our validated LC–MS/MS method can be successfully used for the pharmacokinetic studies of celecoxib and that the blood cells are a very important compartment for this drug such that profiles of celecoxib and its metabolites in whole blood will be more comprehensive and accurate representation of their profiles in vivo than the plasma.
Keywords: Celecoxib; Metabolites; Pharmacokinetics; UPLC-MS/MS; Drug distribution;

Quantification of endogenous nucleotides is of interest for investigation of numerous cellular biochemical processes, such as energy metabolism and signal transduction, and may also be applied in cancer and antiretroviral therapies in which nucleoside analogues are used. For these purposes we developed and validated a sensitive and high accuracy ion-pair liquid chromatography tandem mass spectrometry (IP LC–MS/MS) method for simultaneous quantification of eight endogenous nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP) and 2′,2′-difluoro-2′-deoxycytidine triphosphate (dFdCTP), an intracellular metabolite of the nucleoside analogue gemcitabine. The assay was validated using 200 μL aliquots of peripheral blood mononuclear cell (20 × 106  cells/ml, 4 × 106  cells) extracts, pretreated with activated charcoal and spiked with unlabeled nucleotides, deoxynucleotides and dFdCTP. Analytes were extracted by simple precipitation with cold 60% methanol containing isotope labeled internal standards and separated on a porous graphitic carbon column. For method validation, the concentration ranges were: 0.125–20.8 pmol injected for deoxynucleotides, 0.25–312.5 pmol injected for dFdCTP and 5–3200 pmol injected for nucleotides. The highest coefficients of variation (CV) were 12.1% for within run assay and 11.4% for between run assay, both representing the precision at the lowest analyte concentrations. The method was applied to monitor dFdCTP and changes in endogenous nucleotides in patients who were receiving gemcitabine infusions.
Keywords: Nucleotides; Gemcitabine; dFdCTP; PBMC; LC–MS/MS;

In 2013, the World Anti-Doping Agency (WADA) drastically lowered the minimum required performance levels (MRPLs) of most doping substances, demanding a substantial increase in sensitivity of the existing methods. For a number of compounds, conventional electron impact ionization gas chromatography tandem mass spectrometry (GC-EI-MS/MS) is often no longer sufficient to reach these MRPLs and new strategies are required.In this study, the capabilities of positive ion chemical ionization (PICI) GC–MS/MS are investigated for a wide range of drug related compounds of various classes by injection of silylated reference standards. Ammonia as PICI reagent gas had superior characteristics for GC–MS/MS purposes than methane. Compared to GC-EI-MS/MS, PICI (with ammonia as reagent gas) provided more selective ion transitions and consequently, increased sensitivity by an average factor of 50. The maximum increase (by factor of 500–1000) was observed in the analysis of stimulants, namely chlorprenaline, furfenorex and phentermine. In total, improved sensitivity was obtained for 113 out of 120 compounds.A new GC-PICI-MS/MS method has been developed and evaluated for the detection of a wide variety of exogenous doping substances and the quantification of endogenous steroids in urine in compliance with the required MRPLs established by WADA in 2013. The method consists of a hydrolysis and extraction step, followed by derivatization and subsequent 1 μL pulsed splitless injection on GC-PICI-MS/MS (16 min run). The increased sensitivity allows the set up of a balanced screening method that meets the requirements for both quantitative and qualitative compounds: sufficient capacity and resolution in combination with high sensitivity and short analysis time. This resulted in calibration curves with a wide linear range (e.g., 48–9600 ng/mL for androsterone and etiochanolone; all r 2  > 0.99) without compromising the requirements for the qualitative compounds.
Keywords: Chemical ionization; GC–MS/MS; Doping; Sensitivity;