Liquid chromatography–electrospray ionization mass spectrometry determination of methylphenidate and ritalinic acid in conventional and non-conventional biological matrices

A procedure based on liquid chromatography–electrospray ionization mass spectrometry is described for determination of methylphenidate (MPH) and its principal metabolite ritalinic acid (RA) in plasma, urine, oral fluid and sweat using 3,4-methylendioxypropylamphetamine (MDPA) as internal standard. Aliquots of 100 μL biological fluids and sweat patch were initially treated with acetonitrile, centrifuged, and clear supernatants evaporated and redissolved in 10 mM ammonium acetate. Chromatography was performed on a reversed-phase column using a gradient of 10 mM ammonium acetate and acetonitrile as a mobile phase at a flow rate of 1 mL/min. Separated analytes were confirmed and quantified by positive electrospray ionization mass spectrometry and selected ion monitoring acquisition mode. Limits of quantifications were 1 ng/mL plasma, 1 ng/sweat patch, 0.5 ng/mL oral fluid and urine for MHF; 1 ng/mL plasma and oral fluid, 1 ng/sweat patch, 0.5 ng/mL urine for RA using 100 μL biological fluids or one sweat-patch per assay. Calibration curves were linear over the calibration ranges for both MPH and RA, with r² > 0.99. At three concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 67.9–90.3% for MPH and 36.3–92.4% for RA in the different biological matrices. This method was applied to therapeutic monitoring of MHP and RA in conventional and non-conventional biological matrices from individuals in drug treatment.     

Validation of a liquid chromatography–tandem mass spectrometric assay for the quantitative determination of hydrastine and berberine in human serum

A high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of berberine and hydrastine in human serum, after oral administration of goldenseal (Hydrastis canadensis L.), was developed using simple acetonitrile treatment of serum samples. Noscapine served as the internal standard. Lower limit of quantification for both analytes was 0.1 ng mL⁻¹ using positive ion electrospray tandem mass spectrometry (MS/MS). The intra-day (n = 5) accuracy and precision of the method for hydrastine was 82 ± 8.8%, 97.9 ± 2.4% and 96.2 ± 3.3%, respectively. The inter-day (n = 4) accuracy and precision for hydrastine was 90.0 ± 15.17%, 99.9 ± 7.1% and 98 ± 6.54%, respectively. For berberine quantitation intra-day accuracy and precision was 96.0 ± 8.4%, 92.5 ± 4.7% and 94.4 ± 3.7%, respectively. The respective values for inter-day quantitation were 91.0 ± 8.4%, 94.3 ± 4.7% and 94.4 ± 3.7%. The analytical recovery for hydrastine was 82.4–96.2% and for berberine it was 94.4–96.0%. The analytes and noscapine were stable for 24 h at room temperature (CV 5–10%). Matrix ion effects were studied by post-column infusion of hydrastine and berberine, calculation of calibration curve slope precision was obtained using serum from five different subjects, and by comparison of the response of methanol standards and extracted serum samples. The method was further validated by determination of serum pharmacokinetics of hydrastine and berberine after administration of a single oral dose of goldenseal extract containing 77 mg of hydrastine and 132 mg of berberine.     

Determination of nutlin-3a in murine plasma by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS)

A sensitive and precise LC–ESI-MS/MS method for determination of nutlin-3a in murine plasma using ketoconazole as an internal standard was developed and validated. Plasma nutlin-3a samples were prepared by either a simple protein precipitation (PP) for the high concentration range (10–20,000 ng/mL) or by liquid–liquid extraction (LLE) for the low concentration range (0.25–300 ng/mL). Nutlin-3a and ketoconazole were separated on a modified C18 analytical column (4 μm, 75 mm × 2 mm) with an isocratic mobile phase (acetonitrile/5 mM HCOONH₄ = 70/30, v/v). The retention times of nutlin-3a and ketoconazole were 1.14 and 1.45 min. Detection was achieved by a tandem MS system, monitoring m/z 582/99 and m/z 532/82 for nutlin-3a and ketoconazole, respectively. The PP method was linear in a range of 10–20,000 ng/mL (R² ≥ 0.993) and the LLE method was linear in a range of 0.25–300 ng/mL (R² ≥ 0.992). The mean recoveries for PP and LLE were 24% and 78%, respectively. Within-day and between-day precisions were ≤4.5% for PP and were ≤4.9% for LLE. Within-day and between-day accuracies (% error) ranged from 4.8 to −7.9 for PP, and from −0.2 to −8.4 for LLE. The two extraction methods produced equivalent results, allowing use of both within the same study. This method has been applied to the measurement of nutlin-3a concentrations in murine plasma samples obtained from a preclinical pharmacokinetic study.     

Quantification of sunitinib in mouse plasma,brain tumor and normal brain using liquid chromatography–electrospray ionization-tandem mass spectrometry and pharmacokinetic application

The primary challenge associated with the development of an assay method for the determination of drug concentrations in relatively small amount of mouse plasma and tissue samples is to improve extraction efficiency and detection sensitivity. In this work, a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method combined with protein precipitation, liquid–liquid extraction and solid-phase extraction techniques was developed for the determination of sunitinib in mouse plasma, brain tumor and normal brain tissue, respectively. The instrument was operated under the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive ion mode. A good linear relationship with coefficients of determination ≥0.99 was achieved over the concentration ranges of 1.37–1000 ng/mL for plasma and 4.12–1000 ng/g for the normal brain and brain tumor. The limits of quantification (LOQs) for sunitinib in mouse plasma, brain tumor and normal brain tissue are 1.37 ng/mL, 4.12 ng/g and 4.12 ng/g, respectively. The reproducibility of the LC–MS/MS method is reliable, with the intra- and inter-day precision being less than 15% and accuracy within ±15%. The established method was successfully applied to the characterization of sunitinib disposition in the brain and brain tumor as well as its systemic pharmacokinetics in a murine orthotopic glioma model.     

Liquid chromatography–tandem mass spectrometric assay for the quantitation in human plasma of the novel indenoisoquinoline topoisomerase I inhibitors,NSC 743400 and NSC 725776

Topoisomerase I (Topo I) is a recognized target for ovarian, lung, and colorectal cancer therapy. The FDA-approved camptothecin (CPT) Topo I inhibitors, topotecan and irinotecan are labile and their effects are rapidly reversible. The indenoisoquinoline topoisomerase I inhibitors, NSC 743400 and NSC 725776, have been developed as a new generation of Topo I inhibitors and are being advanced to clinical evaluation. To support the clinical development of NSC 743400 and NSC 725776, we developed and validated, according to FDA guidelines, LC–MS/MS assays for the sensitive, accurate and precise quantitation of NSC 743400 and NSC 725776 in 0.2 mL human plasma. After ethyl acetate extraction, separation was achieved with a Synergi Polar RP column and a gradient of 0.1% formic acid in acetonitrile:water. NSC 743400 and NSC 725776 eluted at approximately 3 min, and the total run time was 14 min. Detection consisted of electrospray, positive-mode ionization mass spectrometry. Between 3 and 1000 ng/mL, accuracy was 96.9–108.2% for NSC 743400 and 95.1–106.7% for NSC 725776, and precision was <11.4% for NSC 743400 and <5.9% for NSC 725776. Extraction recovery was >80% for both analytes, and ion suppression ranged from −46.7 to 5.7%. The use of isotopically labeled internal standards and a wash phase at the end of the run were necessary to achieve adequate assay performance. Protein binding in human plasma as assessed by equilibrium dialysis showed both indenoisoquinolines to be more than 98% protein bound.     

Liquid chromatography–tandem mass spectrometry for the determination of paclitaxel in rat plasma after intravenous administration of poly(l-glutamic acid)-alanine-paclitaxel conjugate

A specific and sensitive liquid chromatography–tandem mass spectrometric method for quantitative determination of paclitaxel in rat plasma was developed and validated using docetaxel as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma. The separation of paclitaxel was performed on a C₁₈ column with a mobile phase of acetonitrile:water:formic acid (65:35:0.1, v/v/v) over 5 min. The assay was based on the selected reaction monitoring transitions at m/z of the precursor-product ion transitions m/z 854.2 → 286.1 for paclitaxel and 808.3 → 527.2 for internal standard. The lower limit of quantification was 0.5 ng/mL based on 100 μL of plasma. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 95.4 and 105.4%. The extraction recoveries ranged from 96.7 to 103.7% across the calibration curve range. The method was successfully applied to measurement of low concentrations of paclitaxel or regenerated paclitaxel in plasma after intravenous administration of a single dose (10 mg/kg) of a poly(l-glutamic acid)-alanine-paclitaxel conjugate to rats.     

Qualification and application of a liquid chromatography–tandem mass spectrometric method for the determination of human Aβ1-40 and Aβ1-42 peptides in transgenic mouse plasma using micro-elution solid phase extraction

A liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and applied for the determination of human Aβ1-40 and Aβ1-42 peptides in transgenic mouse plasma to support preclinical pharmacodynamics studies. The method consisted of micro-elution solid phase extraction for sample preparation and LC–MS/MS analysis in the negative ion mode using electrospray ionization for analysis. ¹⁵N₅₃-Aβ1-40 and ¹⁵N₅₅-Aβ1-42 peptides were used as internal standards. A quadratic regression (weighted 1/concentrations), with an equation y = ax ² + bx + c, was used to fit calibration curves over the concentration range of 0.500–100 ng/mL for both Aβ1-40 and Aβ1-42 peptides. For quality control samples at 6.00, 40.0 and 80.0 ng/mL from the qualification experiment, the within-run accuracy ranged from ?2.69 to 0.583 % with precision values ≤8.23 % for Aβ1-40. Within-run accuracy ranged from ?4.83 to 10.1 % with precision values ≤8.87 % for Aβ1-42. Samples from a pharmacodynamics study using Tg2576 transgenic mice were analyzed by this qualified LC–MS/MS method and concentrations were compared to those generated by ELISA. The two methods were shown to be comparable for Aβ1-40 quantification of samples from the Tg2576 amyloid precursor protein transgenic mouse model, but varied slightly for Aβ1-42.     

Simultaneous determination of danshensu,rosmarinic acid,cryptotanshinone, tanshinone IIA,tanshinone I and dihydrotanshinone I by liquid chromatographic–mass spectrometry and the application to pharmacokinetics in rats

A sensitive and specific liquid chromatography–tandem mass spectrometry method (LC–MS) was developed and validated for the separation and simultaneous determination of danshensu, rosmarinic acid and tanshinone compounds including cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA in rat plasma. Chromatographic separation of the analytes was successfully achieved on a C₁₈ column using a mobile phase composed of acetonitrile–water containing 0.5% glacial acetic acid. This method demonstrated good linearity and did not have endogenous material interfering with the active compounds and I.S. peaks. The limit of quantification of danshensu, rosmarinic acid, cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA were 5, 0.75, 0.1, 0.1, 1 and 0.5 ng/mL. The average extraction recoveries of these analytes from rat plasma were all over 60%. The precisions determined from five days were all within 10%. This method has been successfully applied in the simultaneous quantification and the pharmacokinetic studies of these six compounds in animals which were orally administered with danshen preparations.     

Quantitative analysis of a novel HIV fusion inhibitor (sifuvirtide) in HIV infected human plasma using high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive method for measuring sifuvirtide, a novel HIV fusion inhibitor peptide drug in HIV-1⁺ human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. The plasma samples were treated by solvent/detergent (S/D) method to inactivate viral activity before analysis. After protein precipitation sifuvirtide was determined by LC–MS/MS. A structure analog was used as internal standard (IS). The mass spectrometer was operated in positive ion and multiple reaction monitoring mode with transitions m/z 946.3 → 159.0 for sifuvirtide and 951.7 → 159.2 for IS. The intra-day precision ranged from 2.74% to 7.57% with accuracy from 91.63% to 102.53%. The inter-day precision ranged from 2.65% to 3.58% and the accuracy from 95.53% to 105.28%. Stability studies showed that sifuvirtide was stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) was 9.75 ng ml⁻¹. The method was used for analyzing samples from phase IIa clinical study of sifuvirtide in China.     

Comparison of sandwich enzyme-linked immunoadsorbent assay and radioimmunoassay for determination of exogenous glucagon-like peptide-1(7–36)amide in plasma

A sensitive sandwich enzyme-linked immunoadsorbent assay (ELISA) for determination of exogenous glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) in plasma samples from pharmacokinetic studies is described. The assay employs an N-terminally directed antibody and a C-terminally directed antibody. The ELISA has a working range from 10 to 500 pmol 1⁻¹, and can be applied to plasma samples from humans, dogs, pigs, minipigs, cats, rabbits, and rats. The assay was compared to a validated radioimmunoassay (RIA), employing an antibody directed against the mid-region of GLP-1. After s.c. administration of GLP-1(7–36)amide, the plasma immunoreactivity of GLP-1 (P-GLP-1-IR) measured by ELISA was markedly lower than P-GLP-1-IR measured by RIA. After HPLC fractionation of plasma samples with subsequent RIA and ELISA analyses of the fractions, this difference was shown to be due to cross reaction with biologically inactive fragments of GLP-1(7–36)amide in the RIA but not in the ELISA.     

Evaluation of antidepressant activity of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol, a β-substituted phenylethylamine in mice

The β-phenylethylamines are known to act as ligands for the trace amine receptors, a novel family of G-protein-coupled receptors. The trace amines are stored and released along with various neurotransmitter agents such as norepinephrine, serotonin, and dopamine and thus work as neuromodulator or neurotransmitter agents. Trace amines are known to play an important role in the pathophysiology of major depression. In our earlier study, we have demonstrated the synthesis of various β-substituted phenylethylamine molecules hypothesized to be effective in various central nervous system disorders. The present study is an attempt to evaluate one of such molecules, 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-yl)-cyclohexanol, in animal models of depression. Various behavioral paradigms of despair such as forced swim and tail-suspension tests were used to assess the antidepressant-like activity. Further, an alteration in the levels of various neurotransmitters (norepinephrine, serotonin, and dopamine) in the mouse brain following 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-yl)-cyclohexanol administration was evaluated. The molecule (4–16 mg/kg., i.p.) dose-dependently inhibited the immobility period in mouse forced swim test, the effect comparable to venlafaxine. The ED50 values of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-yl)-cyclohexanol and venlafaxine in mouse forced swim test were found to be 5.27 [4.38–6.35] mg/kg., i.p and 4.66 [3.48–6.25] mg/kg., i.p., respectively. Further, 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-yl)-cyclohexanol at 4–16 mg/kg., i.p. reversed the immobility period in mouse tail-suspension test. Additionally, the molecule at 8 mg/kg., i.p. reversed reserpine-induced behavioral despair in mouse forced swim test. When administered simultaneously, it (4 and 8 mg/kg., i.p) enhanced the antidepressant activity of sub-effective doses of imipramine (2 mg/kg., i.p.) or fluoxetine (2 mg/kg., i.p.) in the mouse forced swim test. Neurochemical analysis revealed that the molecule at 8 mg/kg., i.p. increased the levels of norepinephrine (21% increase) without affecting serotonin in the mouse brain. However, at higher dose (16 mg/kg., i.p.), it increased the levels of norepinephrine (13% increase), serotonin (37% increase), and dopamine (42% increase). The molecule enhanced the locomotor activity in mice only at higher doses. The molecule, unlike venlafaxine, which potentiated barbiturate-induced hypnosis, was devoid of any sedative activity. In conclusion, 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-yl)-cyclohexanol, possess antidepressant-like activity in animal models of depression by modulating the neurotransmitter levels in the brain. Such an activity might be due to the modulating action of this novel molecule on trace amine receptors. Such a molecule may be the future drugs of choice for the treatment of major depression.     

Understanding the Relationship Between Biotherapeutic Protein Stability and Solid–Liquid Interfacial Shear in Constant Region Mutants of IgG1 and IgG4

Relative stability of therapeutic antibody candidates is currently evaluated primarily through their response to thermal degradation, yet this technique is not always predictive of stability in manufacture, shipping, and storage. A rotating disk shear device is proposed that produces defined shear conditions at a known solid–liquid interface to measure stability in this environment. Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE. Antibodies were ranked for stability based on shear device output (protein decay coefficient, PDC), and compared with accelerated thermal stability data and the melting temperature of the CH2 domain (T_m1) from differential scanning calorimetry to investigate technique complimentarity. Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation. Molecular modeling shows the modifications’ effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability. The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.     

Effect of β-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig

The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood–brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with β-naphthoflavone (βNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood–brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with βNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by βNF treatment. The high expression of metabolic enzymes found in blood–brain interfaces could represent a very important defence toward toxins of CNS.     

Selective inhibition by 4,α-dimethyl-m-tyramine (H77/77) and 4-methyl-α-ethyl-m-tyramine (H75/12) of the monoamine oxidase within serotonergic and noradrenergic neurons in the rat brain

Summary 1. The inhibition of monoamine oxidase (MAO) by 4,-dimethyl-m-tyramine (H77/77) and 4-methyl--ethyl-m-tyramine (H75/12), two amine releasing compounds, within monoaminergic neurons in the rat hypothalamus and striatum in vivo was determined. This was performed by measuring the protection of MAO by the test compound against the irreversible inhibition produced by phenelzine. The MAO activity inside and outside monoaminergic synaptosomes in homogenates of brain tissue was measured in the absence and presence of selective uptake inhibitors at low concentrations of ¹⁴C-labelled 5-hydroxytryptamine, noradrenaline or dopamine. 2. It was found that H77/77 and H75/12 produced a pronounced protection against phenelzine within the serotonergic and noradrenergic neurons, whereas much less effect was observed outside these neurons. 3. It was shown that the protection by H75/12 within the serotonergic neurons was somewhat reduced in 5-HT depleted reserpinized rats and that the protection outside these neurons was abolished. Some of the protection of MAO might therefore have been brought about by 5-HT molecules released by H75/12. 4. The marked inhibition of MAO within serotonergic and noradrenergic neurons was counteracted by amine uptake inhibitors and is accordingly brought about by the high concentrations of the accumulated compounds. 5. In contrast to other neuron selective MAO inhibitors, H75/12 decreased the 5-HT concentration in the hypothalamus showing that the releasing effect dominated over the MAO inhibitory effect. Send offprint requests to S. B. Ross at the above address     

The psycho- and neurotropic profiling of novel 3-(N-R,R′-aminomethyl)-2-methyl-1H-quinolin-4-ones in vivo

The article presents the study of psycho- and neurotropic properties of novel 3-(N-R,R′-aminomethyl)-2-methyl-1H-quinolin-4-ones in vivo. The research was carried out using the open field test, elevated plus maze, rotarod test, tail suspension test, passive avoidance test after scopolamine-induced amnesia and acute normobaric hypoxia with hypercapnia. As a result, two promising substances have been found. According to our results 3-[[(4-methoxyphenyl)amino]methyl]-2-methyl-1H-quinolin-4-one in the dose of 10?mg/kg shows a specific sedative effect and a considerable anti-amnesic activity. The most interesting N-[(2-methyl-4-oxo-1H-quinolin-3-yl)methyl]-N-phenylbenzamide (100?mg/kg) combines a potent anti-anxiety action, the anti-amnesic activity and a considerable antihypoxic effect. They are of interest for further profound studies as promising psychoactive compounds.     

Physical dependence of a dermorphin tetrapeptide analog, [D-Arg2, Sar4]-dermorphin (1–4) in the rat

The characteristics of an analog of tetrapeptide dermorphin (H-Tyr-D-Arg-Phe-Sar-OH), [D-Arg², Sar⁴]-dermorphin (1–4) were examined in comparison with morphine by the appearance of typical withdrawal signs upon cessation of administration or treatment with naloxone, an opioid antagonist. The dose of [D-Arg², Sar⁴]-dermorphin (1–4) or morphine in the physical dependence test can be quantified by determining the ED₅₀ to inhibit the tail-flick response to thermal stimuli. Doses from 8 to 64 times the ED₅₀ doses were employed in the subcutaneous injection schedules. The cessation of [D-Arg², Sar⁴]-dermorphin (1–4) or naloxone treatment was largely without effect on body weight, in contrast to a marked loss of weight in morphine-dependent rats. The tetrapeptide failed to substitute for morphine in morphine-dependent rats. The physical dependence of [D-Arg², Sar⁴]-dermorphin (1–4) was revealed by the behavioral signs of withdrawal precipitated by naloxone. However, the scores of lacrimation, diarrhea and urination were much lower in chronically tetrapeptide-treated rats than in morphine-treated rats, though the score of teeth chatter was higher. These findings indicate that [D-Arg², Sar⁴]-dermorphin (1–4) may differ from morphine in physical dependence.     

Brain penetration of methadone (R)- and (S)-enantiomers is greatly increased by P-glycoprotein deficiency in the blood–brain barrier of Abcb1a gene knockout mice

Rationale Methadone maintenance treatment is complicated by the wide variability of efficacy among patients. The large interindividual variability of the plasma concentrations of methadone was previously thought to be responsible for the variable therapeutic efficacy. However, recent studies suggested that methadone may be a substrate of P-glycoprotein (P-gp). Therefore, the function of P-gp in blood–brain barrier (BBB) may affect the concentration of methadone at its site(s) of action in the central nervous system, thereby contributing to its therapeutic efficacy and/or adverse events.Objective To investigate the effect of P-gp on brain penetration of methadone (R)- and (S)-enantiomers and their major oxidative metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP).Methods We compared the tissue distribution of methadone (R)- and (S)-enantiomers and EDDP in the Abcb1a–/– gene knockout mice and the Abcb1a+/+ wild-type mice 1 h following intraperitoneal administration of 15 g Rac-methadone/g mouse.Results Plasma concentrations of (R)- and (S)-methadone were similar between the two animal groups. However, the brain concentrations of (R)- and (S)-methadone in the Abcb1a–/– mice were markedly higher (15- and 23-fold, respectively, P<0.0001) than those of the Abcb1a+/+ wild-type mice. No statistically significant difference was found for other organs between the mutants and controls. No organ difference was found for EDDP between the mutants and controls.Conclusions (R)- and (S)-methadone are substrates of P-gp. The P-gp in BBB greatly limits the brain entry of (R)- and (S)-methadone to their central nervous system acting sites. The interindividual variation in expression of P-gp in BBB may represent a source of variation for the access and effects of methadone in the brain.     

New gamma-hydroxybutyric acid (GHB) biomarkers: Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of GHB amino acid,carnitine, and fatty acid conjugates in urine

Gamma-hydroxybutyric acid (GHB) represents an important drug in clinical and forensic toxicology, particularly in the context of drug-facilitated crimes. Analytically, GHB remains a major challenge given its endogenous occurrence and short detection window. Previous studies identified a number of potential interesting novel conjugates of GHB with carnitine, amino acids (AA, glutamate, glycine, and taurine), or fatty acids. As a basis for comprehensive studies on the suitability of these novel biomarkers, we developed and validated a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method in human urine. Additionally, already known markers 2,4-dihydroxy butyric acid (2,4-DHB), 3,4-DHB, glycolic acid, succinic acid, succinylcarnitine, and GHB glucuronide were included. The method was fully validated according to (inter)national guidelines. Synthetic urine proved suitable as a surrogate matrix for calibration. Matrix effects were observed for all analytes with suppression effects of about 50% at QC LOW, and approximately 20% to 40% at QC HIGH, but with consistent standard deviation of <25% at QC LOW and <15% at QC HIGH, respectively. All analytes showed acceptable intra- and inter-day imprecision of below 20%, except for inter-day variation of GHB taurine and FA conjugates at the lowest QC. Preliminary applicability studies proved the usefulness of the method and pointed towards GHB glycine, followed by other AA conjugates as the most promising candidates to improve GHB detection. FA conjugates were not detected in urine samples yet. The method can be used now for comprehensive sample analysis on (controlled) GHB administration to prove the usefulness of the novel GHB biomarkers.