Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Development and validation of a nylon6 nanofibers mat-based SPE coupled with HPLC method for the determination of docetaxel in rabbit plasma and its application to the relative bioavailability study

A simple and sensitive HPLC method was established and validated for the determination of docetaxel (DTX) in rabbit plasma. Biosamples were spiked with paclitaxel (PCX) as an internal standard (I.S.) and pre-treated by solid-phase extraction (SPE). The SPE procedure followed a simple protein digestion was based on nylon6 electrospun nanofibers mats as sorbents. Under optimized conditions, target analytes in 500 μL of plasma sample can be completely extracted by only 2.5 mg nylon6 nanofibers mat and eluted by 100 μL solvent. The HPLC separation was obtained on C18 column and UV detector was used to quantify the target analytes. The extraction recovery was more than 85%; the standard curve was linear over the validated concentrations range of 10–5000 ng/mL and the limit of detection was 2 ng/mL. The inter-day coefficient of variation (CV%) of the calibration standards was below 5.0% and the mean accuracy was in the range of 92.8–113.4%. Moreover, analysing quality control plasma samples in 3 days, the results showed that the method was precise and accurate, for the intra- and inter-day CV% within 10% and the accuracy from 96.0% to 114.0%. The developed and validated method was successfully applied to relative bioavailability study for the preclinical evaluation of a new injectable DTX–sulfobutyl ether beta-cyclodextrin (DTX–SBE-β-CD) inclusion complex freeze-dried powder (test preparation), compared with the reference preparation (DTX injection, Taxotere^®) in healthy rabbits. On the basis of the mean AUC(0–t) and AUC(0–infinity), the relative bioavailability of the test preparation was found to be 113.1%.     

Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation

Despite the fact that the area of glycolysis in Lactococcus lactis has been intensively studied, only a limited number of studies have been focused on the regulation of uptake of glucose itself. Using the tool of the glucostat fed-batch mode of culture, it was demonstrated in our earlier work that the concentration of glucose regulates its uptake rate and that the control of the glycolytic flux resides to a large extent in processes outside the pathway itself, like glucose transport and the ATP consuming reactions, while allosteric properties of key enzymes like phosphofructokinase (PFK) have a significant influence on the control. Extending our work, we report here the results of fermentations with engineered L. lactis strains with altered PFK activity in which the pfkA gene from Aspergillus niger, and its truncated version pfk13 that encodes a shorter PFK1 fragment were cloned. The results in this study suggest that, under the optimum for the microorganism applied microaerobic conditions, the glycolytic capacity of L. lactis was significantly increased in engineered strains with increased PFK activity. The transformant strain in which the truncated pfk13 gene of A. niger was expressed performed more efficiently as it was able to grow successfully in glucostat cultures with 277 mM glucose - while the optimum glucose concentration for the parental strain was 55 mM. The present work demonstrates the direct effect of PFK activity on the glycolytic flux in L. lactis since a twofold increase in specific PFK activity (from 7.1 to 14.5 U/OD₆₀₀) resulted in a proportional increase of the maximum specific rates of glucose uptake (from 0.8 to 1.7 μM s⁻¹ g CDW⁻¹) and lactate formation (from 15 to 22.8 g lactate (g CDW)⁻¹ h⁻¹).     

Sensitive counting of viable Enterobacteriaceae in seawaters and relationship with fecal indicators

A fluorescent in situ hybridization based assay was used to enumerate viable Enterobacteriaceae members in seawaters by solid phase cytometry. The method was specific, highly sensitive (1 cell/100 ml) and allowed the quantification of VNC Enterobacteriaceae cells during an osmotic stress. Investigations on contaminated coastal seawater revealed a strong correlation between Enterobacteriaceae counts and standard fecal indicators.     

A novel analysis method for diterpenoids in rat plasma by liquid chromatography-electrospray ionization mass spectrometry

A sensitive, specific, and rapid liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for analysis of lasiodonin, oridonin, ponicidin, and rabdoternin A in rat plasma using sulfamethoxazole as an internal standard (IS). The plasma samples were pretreated and extracted by liquid-liquid extraction. Chromatographic separation was performed on a C₁₈ column with linear gradient elution using water and methanol, both of which were acidified with 0.1% aqueous formic acid, at a flow rate of 0.8 ml/min. Detection was accomplished by scanning with multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. Higher sensitivity was achieved by setting three scanning periods in a novel detection mode. The optimized mass transition ion pairs (m/z) for quantitation were 365.3/347.3 for lasiodonin and oridonin, 361.2/343.2 for ponicidin, 363.2/283.1 for rabdoternin A, and 254.1/156.0 for IS. The total run time was 13.50 min between injections. The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for all analytes in the rat plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon rubescens extract to rats.     

Determination of 2-hydroxyflutamide in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS): Application to a bioequivalence study on Chinese volunteers

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)–ESI-MS/MS method was developed for the determination of 2-hydroxyflutamide in human plasma using tegafur as the internal standard. The plasma sample was pretreated with methanol for protein precipitation and the analytes were separated on an Ultimate C18 column (5 μm, 2.1 mm × 50 mm, MD, USA) with the mobile phase consisted of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a negative multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 290.90–204.8 for 2-hydroxyflutamide and 198.9–128.8 for tegafur. Linear calibration curves were obtained in the concentration range of 1.742–1452 ng/ml with a lower limit of quantification of 1.742 ng/ml. The intra- and inter-batch precision values were less than 8.1% and 5.6%, respectively. The established method was successfully applied to a bioequivalence study of two flutamide preparations (250 mg) in 20 healthy male volunteers.     

Simultaneous quantification of capsaicin and dihydrocapsaicin in rat plasma using HPLC coupled with tandem mass spectrometry

A rapid, simple and sensitive HPLC–ESI–MS/MS method was developed for the simultaneous determination of capsaicin and dihydrocapsaicin in rat plasma. Plasma samples containing capsaicin, dihydrocapsaicin and phenacetin (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analytes and internal standard were separated on a Zorbax SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with mobile phase of acetonitrile/water (55:45, v/v) containing 0.1% formic acid (v/v) at a flow rate of 0.2 mL/min with an operating temperature of 25 °C. Quantification was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source by selected reaction monitoring (SRM) of the transitions at m/z 306–137 for capsaicin, m/z 308–137 for dihydrocapsaicin and m/z 180–110 for the IS. Linear detection responses were obtained for capsaicin and dihydrocapsaicin ranging from 1 to 500 ng/mL and the lower limits of quantitation (LLOQs) for the two compounds were 1 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 9.79% for the two analytes, while the deviations of assay accuracies were within ±10.63%. The average recoveries of the analytes were greater than 89.88%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of capsaicin and dihydrocapsaicin in rats after subcutaneous administration of capsaicin (natural, containing 65% capsaicin and 35% dihydrocapsaicin).     

New method for electroporation of Lactobacillus species grown in high salt

We here describe a new method for electroporation of Lactobacillus species, obligately homofermentative and facultatively heterofermentative, based on the cell-wall weakening resulting from growth in high-salt media. For L. casei, optimum transformation efficiency of up to 10⁵ transformants per microgram of plasmid DNA was achieved following growth in the presence of 0.9 M NaCl. Plasmids of different sizes and replication origins were also similarly transformed. These competent cells could be used either directly or stored frozen, up to 1 month, for future use, with similar efficiency. This protocol was assayed with different Lactobacillus species: L. delbrueckii subsp. lactis, L. paracasei, L. plantarum and L. acidophilus, and it was found that they were transformed with similar efficiency.     

Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15-0.3% and 1.0-5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5-25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populus × canadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Simultaneous determination of phenylethanoid glycosides and aglycones by capillary zone electrophoresis with running buffer modifier

Although the separation efficiency of capillary electrophoresis (CE) is much higher than that of other chromatographic methods, it is sometimes difficult to adequately separate the complex ingredients in biological samples. This article describes how one effective and simple way to develop the separation efficiency in CE is to add some modifiers to the running buffer. The suitable running buffer modifier β-cyclodextrin (β-CD) was explored to fast and completely separate four phenylethanoid glycosides and aglycones (homovanillyl alcohol, hydroxytyrosol, 3,4-dimethoxycinnamic acid, and caffeic acid) in Lamiophlomis rotata (Lr) and Cistanche by capillary zone electrophoresis with ultraviolet (UV) detection. It was found that when β-CD was used as running buffer modifier, a baseline separation of the four analytes could be accomplished in less than 20 min and the detection limits were as low as 10⁻³ mg L⁻¹. Other factors affecting the CE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage, and sample injection time, were investigated extensively. Under the optimal conditions, a successful practical application on the determination of Lr and Cistanche samples confirmed the validity and practicability of this method.     

Influence of Carbohydrate Starvation and Arginine on Culturability and Amino Acid Utilization of Lactococcus lactis subsp. lactis

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.     

Trypanosoma cruzi: multiple nucleoside diphosphate kinase isoforms in a single cell

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml⁻¹. TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.     

Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> Type-1 in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Using Nisin Inducible Controlled Expression (NICE)

Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host’s proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol. Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 ΔhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0556-2) contains supplementary material, which is available to authorized users.     

Engineering of Carbon Distribution between Glycolysis and Sugar Nucleotide Biosynthesis in Lactococcus lactis

We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an ~100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.     

A quantitative method to assess muscle tissue oxygenation in vivo by monitoring H nuclear magnetic resonance myoglobin resonances

A nuclear magnetic resonance (NMR) method was implemented to assess in vivo oxygenation levels by a quantitative determination of the ¹H NMR myoglobin (Mb) resonances. The proximal His-F8 N_δH at 70-90 ppm and Val-E11 γCH₃ resonance at -2.8 ppm, reflecting deoxygenated (deoxy-Mb) and oxygenated (met-Mb) states, were alternately recorded. The method was developed in vitro choosing a couple of NMR sequences that could each maximize the signal-to-noise ratio (SNR) while avoiding baseline rolling and suppressing the water signal. Two quantitative calibration methods were implemented for deoxy- and met-Mb samples (0.1-1 mM), respectively. The respective limit of detection (LOD) and limit of quantification (LOQ) were 0.015 and 0.05 mM for met-Mb and 0.013 and 0.042 mM for deoxy-Mb. Sequences and calibration curves were employed in vivo in Arenicola marina to obtain, for the first time, an accurate measurement of oxy- and deoxy-Mb actual concentrations. In Arenicola, the peaks at approximately 87 and -2.7 ppm, reflecting the deoxy- and oxy-Mb states, respectively, were alternately recorded during increasing hypoxia. The deoxy-Mb concentrations were obtained from the calibration curve. The oxy-Mb concentrations were calculated from the calibration of met-Mb because it was proved that oxy- and met-Mb gave the same NMR molar response. From oxy- and deoxy-Mb concentrations, the intracellular oxygen partial pressure (P_iO₂) trend was determined.     

Effects of UV radiation and nitrate limitation on the production of biogenic sulfur compounds by marine phytoplankton

We tested the effects of UV radiation (UVR) and nitrate limitation on the production of dimethylsulfide (DMS), particulate dimethylsulfoniopropionate (DMSPp), and particulate dimethylsulfoxide (DMSOp) in natural seawater from the Gulf of Mexico and in phytoplankton cultures. DMS/Chl a ratios in PAR-only and PAR + UV-exposed seawater were 0.44–2.0 and 0.46–1.9 nmol DMS μg⁻¹ Chl a, respectively, whereas the ratios in cultures of Amphidinium carterae were 1.0–2.2 nmol μg⁻¹ in PAR-exposed samples and 0.91–2.1 nmol μg⁻¹ in PAR + UV-exposed samples. These results suggested that UVR did not substantially affect DMS/Chl a ratios in seawater and A. carterae culture samples. Similarly, UVR had no significant effect on DMSOp/Chl a in seawater samples (0.83–1.6 nmol DMSO μg⁻¹ Chl a for PAR + UV vs. 0.70–1.5 nmol μg⁻¹ for PAR-exposed seawater samples, respectively) or in A. carterae cultures (0.20–1.3 and 0.19–0.88 nmol DMSO μg⁻¹ Chl a in PAR + UV- and PAR-exposed cultures, respectively). In an experiment with the diatom, Thalassiosira oceanica, the culture was grown in high nitrate (30 μM) or low nitrate (6 μM) media and exposed to PAR-only or PAR + UV. The low nitrate, PAR-only samples showed an increase of intracellular dimethylsulfoniopropionate (DMSP) concentration from 2.1 to 15 mmol L⁻¹ in 60 h, but the increase occurred only after cultures reached the stationary phase. Cultures of T. oceanica grown under UVR had lower growth rates than those under PAR-only (μ′ = 0.17 and 0.32 d⁻¹, respectively) and perhaps did not experience severe nitrate limitation even in the low nitrate treatment. These results suggest that the elevated UVR in low nitrate environments could result in reduction of DMSP in some species, whereas DMSP concentrations would not be affected in eutrophic areas.     

Simultaneous quantitative analysis of N-acylethanolamides in clinical samples

A simple and rapid analytical method is described for the simultaneous quantitative analysis of three different N-acylethanolamides in human biological samples: anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The method is based on a new hybrid solid phase extraction-precipitation technology followed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis using d₄-AEA as the internal standard. The method is linear up to 100 ng/ml with a limit of quantitation of 50 pg/ml for AEA and 100 pg/ml for OEA and PEA. Good reproducibility, accuracy, and precision were demonstrated during the method validation. Application of this new methodology to the analysis of clinical study samples is presented.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.