Synthesis and characterization of a series of N,N'-substituted urea derivatives by using electrospray ionization tandem mass spectrometry: Differentiation of positional isomers.

RATIONALE: Characterization of N,N'-substituted ureas was found to be challenging by nuclear magnetic resonance (NMR) spectroscopy, particularly N-di- and tri-alkylated ureas because of the absence of adjacent protons. In the present study, electrospray ionization tandem mass spectrometry has been used to differentiate positional isomeric pairs and to characterize a series of N,N'-substituted ureas, as these compounds have significant importance for drug discovery. Additionally, urea is an essential functionality in several bioactive compounds as well as a variety of clinically approved therapies. METHODS: High-resolution electrospray ionization tandem mass spectrometry (ESI-HR-MS/MS) has been used to characterize a series of N,N'-substituted urea derivatives and differentiate two pairs of positional isomers. The data was acquired by Xcaliber application in positive ionization mode. RESULTS: ESI-HR-MS/MS spectra of [M + H]+ ions of the positional isomeric urea derivatives 8a and 8b show distinct fragmentation patterns. For example, the MS/MS spectrum of the [M + H]+ ion of isomer 8a displays the abundant fragment ion at m/z 285.1595, which was totally absent in isomer 8b. This would be plausibly formed by the cleavage of the C-N bond of the urea group with the elimination of the isocyanate moiety. In contrast, the MS/MS spectrum of the [M + H]+ ion of isomer 8b shows an intense ion at m/z 311.1389 which is completely absent in isomer 8a which would be formed by the cleavage of the C-N bond attached to the ring nitrogen. Similarly, another pair of positional isomers, 8c and 8d, have been clearly distinguished by their fragmentation behaviour. In addition, a series of N,N'-substituted urea derivatives were studied to investigate the impact of different substitution on the fragmentation behaviour. CONCLUSIONS: The present study demonstrates that ESI-HR-MS/MS can be used to differentiate pairs of N,N'-substituted urea positional isomers and characterize a series of derivatives. It was observed that a characteristic fragment ion was formed by the C-N bond cleavage with the elimination of an isocyanate moiety. The proposed mechanism of fragmentation was supported by the change in the fragmentation pathway upon alkylation of the NH. In order to generalize this fragmentation pattern, a series of N-alkylated ureas was synthesized and studied by MS/MS.

Use of Hybrid Capillary Tube Apparatus on 400 MHz NMR for Quantitation of Crucial Low-Quantity Metabolites Using aSICCO Signal.

Metabolites of new chemical entities can influence safety and efficacy of a molecule and often times need to be quantified in preclinical studies. However, synthetic standards of metabolites are very rarely available in early discovery. Alternate approaches such as biosynthesis need to be explored to generate these metabolites. Assessing the quantity and purity of these small amounts of metabolites with a nondestructive analytical procedure becomes crucial. Quantitative NMR becomes the method of choice for these samples. Recent advances in high-field NMR (>500 MHz) with the use of cryoprobe technology have helped to improve sensitivity for analysis of small microgram quantity of such samples. However, this type of NMR instrumentation is not routinely available in all laboratories. To analyze microgram quantities of metabolites on a routine basis with lower-resolution 400 MHz NMR instrument fitted with a broad band fluorine observe room temperature probe, a novel hybrid capillary tube setup was developed. To quantitate the metabolite in the sample, an artificial signal insertion for calculation of concentration observed (aSICCO) method that introduces an internally calibrated mathematical signal was used after acquiring the NMR spectrum. The linearity of aSICCO signal was established using ibuprofen as a model analyte. The limit of quantification of this procedure was 0.8 mM with 10 K scans that could be improved further with the increase in the number of scans. This procedure was used to quantify three metabolites-phenytoin from fosphenytoin, dextrophan from dextromethorphan, and 4-OH-diclofenac from diclofenac-and is suitable for minibiosynthesis of metabolites from in vitro systems.

Investigation of Unusual N-(Triphenyl-λ5-phosphanylidene) Amide Fragmentation Observed upon MS/MS Collision-Induced Dissociation.

A mechanism of unusual tandem (MS/MS) fragmentation of protonated species of N-(triphenyl-λ5-phosphanylidene) derivatives, [M + H]+ to generate triphenylphosphine oxide (TPPO) within the mass spectrometer has been investigated and reported. Collision-induced dissociation of these molecules resulted in the generation of TPPO as a signature fragment. This fragment suggested the presence of a P-O bond in the structure which was contrary to the structure of the compound identified by nuclear magnetic resonance spectrometry (NMR) and single-crystal X-ray diffractometry (SXRD) techniques with a P═N bond rather than a P-O bond. In order to confirm the generation of the TPPO fragment within the mass spectrometer, 14 different N-(triphenyl-λ5-phosphanylidene) derivatives containing amide, 18O-labeled amide, thiamide, and nonacyl phosphazene derivatives were synthesized and their MS/MS behavior was studied by liquid chromatography-high-resolution mass spectrometry. Fragmentation of these amide derivatives generated TPPO/TPPS or their 18O-labeled analogues as the major fragment in almost all cases under similar MS conditions. Based on the outcome of these experiments, a plausible mechanism for such fragmentation, involving the intramolecular shifting of oxygen from carbon to phosphorus, has been proposed. DFT calculations for the protonated species at B3LYP-D3/6-31+G(d,p) further supported the proposed mechanism involving a four-membered ring, P-O-C-N, as the transition state. Details of this work are presented here.

RP-HPLC separation of interconvertible rotamers of a 5-tetrahydroisoquinolin-6-yl-pyridin-3-yl acetic acid derivative and confirmation by VT NMR and DFT study.

Due to emergence of drug resistance and drug tolerability, there is urgent need for discovery of new chemical entity for the treatment of HIV infection. As a part of in-house small molecule drug discovery program for HIV infection, sodium-2-(tert-butoxy)- 2-(5-(2-(2-chloro-6-methylbenzyl)- 1,2,3,4-tetrahydroisoquinolin-6-yl)- 4-(4,4-dimethylpiperidin-1-yl)- 2,6-dimethylpyridin-3-yl) acetate (SCMTDDA) was prepared as an intermediate for the synthesis of an API, designed as a HIV-1 integrase inhibitor. Initially, the final compound was isolated as a mixture of rotamers. In the current study, we have developed a simple and efficient achiral, reversed phase (RP) HPLC method to separate the interconvertible rotamers of SCMTDDA. The effect of several parameters, including stationary phase, buffer, modifiers and column temperature, were optimized for the chromatographic separation and it was observed that best separation was achieved on a SunFire C18 column using TFA/acetonitrile (ACN) - methanol (MeOH) (1:1 v/v) as the mobile phase at 10 0C. The chromatographic observations were complemented with variable-temperature NMR and energy barrier calculations using density functional theory (DFT).

Development of HPLC-CAD stability indicating assay method for polyethylene glycol-conjugated phospholipid (DMPE-PEG 2000) and identification of its degradation products.

The study introduces first report on a liquid chromatographic method for the quantification of 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (DMPE-PEG 2000), which is an important constituent of lipid-based nanoparticles. It involves an HPLC-CAD stability-indicating assay method development for DMPE-PEG 2000 and structure elucidation of its degradation products. Hypersil Gold™ PFP column (150 mm × 4.6 mm, 3.0 µm) was used to achieve the separation among DMPE-PEG 2000 and its degradation products using 0.0025% formic acid in water: methanol (80:20 v/v) as mobile phase A and methanol: acetonitrile (60:40 v/v) as mobile phase B in a gradient elution mode. The method was validated for precision, linearity, sensitivity, solution stability and robustness. Relative standard deviations for the intra-day precision, inter-day precision and sensitivity were 1.6%, 0.6% and 3.8%, respectively. The method was linear in the range from 210 µg/mL to 390 µg/mL with R2 value of 0.996. Further, the solution stability of DMPE-PEG 2000 was evaluated under different stressed and storage conditions to understand the impact of any excursion to its regular storage temperature of -20 °C. The observed degradation products were identified through liquid chromatography high resolution mass spectrometry and a tentative pathway was proposed for the generation of these degradants.

Rapid and efficient chiral method development for lamivudine and tenofovir disoproxil fumarate fixed dose combination using ultra-high performance supercritical fluid chromatography: A design of experiment approach.

Fixed dose combination (FDC) of tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) is one of the most preferred FDC for the treatment of acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV) infection. To the best of authors' knowledge there are no reported methods for chiral purity estimation of both drugs simultaneously from a FDC. The current study was focused on the development of a single chiral method uisng supercritical fluid chromatography (SFC) for separation of stereoisomers of TDF and 3TC combination employing design of experiment (DoE) approach. Method development was planned in three steps by using different experimental designs for each step. I-optimal, Taguchi orthogonal array and face-centred central composite designs (CCD) were employed for primary parameter selection, secondary parameter screening and final method optimization, respectively. All six stereoisomers were separated in a 10 minute run on Chiralpak IA column with carbon di-oxide /methanol (containing 0.5 % v/v n-butylamine) as mobile phase at 1.5 mL/min in gradient mode. The optimized method was verified for performance through establishing specificity, precision, linearity, accuracy, limit of quantification, and solution stability. Resolution between each isomeric pair was more than 1.5. The method was found to be linear from 1.5 µg/mL to 7.5 µg/mL for 3TC and 7.5 µg/mL to 37.5 µg/mL for TDF stereoisomers. The R2 values for all the linearity curves for undesired isomers were greater than 0.995. The method proved to be rapid, reproducible and efficient to quantify stereoisomers of both drugs in a single run.

Applications of 2, 2, 2 trifluoroethanol as a versatile co-solvent in supercritical fluid chromatography for purification of unstable boronate esters, enhancing throughput, reducing epimerization, and for additive free purifications.

Analysis and purification of boronic acid pinacol esters by RPLC is very challenging due to their degradation in aqueous and alcoholic solvents. These compounds are difficult to purify by SFC too as they are equally sensitive to traditional co-solvents like methanol, ethanol, and 2-propanol. 2,2,2 trifluoroethanol (TFE), which has been reported for the purification of a few alcohol sensitive compounds, was evaluated as a co-solvent in this study for the purification of chiral and achiral boronate esters by SFC. Examples of twelve compounds were presented in this paper where degradation of boronic acid pinacol esters was successfully controlled by replacing methanol with TFE as the co-solvent in SFC. A separate study showed that TFE can also control the epimerization of the enantiomers of 3 substituted 1,4 benzodiazepine analogues during the purification process. In addition to above benefits, 2,2,2trifloroethanol showed improved selectivity and resolution for most of the compounds. With its stronger solvent strength compared to other alcohols, TFE could also be used to reduce the co-solvent percentage needed for elution and to shorten retention time of highly polar samples which did not elute even in 50% of other co-solvents in SFC. A case study of compound B demonstrated that TFE provided a reduced co-solvent percentage and a shorter cycle time with much improved resolution as compared to methanol, thus resulting in higher loading and throughput with reduction of total solvent consumption.

Impact of CO2-solvent separators on the degradation of benzyl-2,3-dihydroxypiperidine-1-carboxylate during preparative supercritical fluid chromatographic (SFC) purification.

During a preparative separation of the cis enantiomeric pair of benzyl-2,3-dihydroxypiperidine-1-carboxylate using supercritical-fluid chromatography (SFC) with methanol modifier, significant degradation of the products in the collected fractions was observed when a Waters SFC-350® (Milford, MA, USA) was used, but same was not observed when a Waters SFC-80q® (Milford, MA, USA) was used. Through a systematic investigation, we discovered that the compound degraded over time under an acidic condition created by the formation of methyl carbonic acid from methanol and CO2. The extent of the product degradation was dependent on the time and the concentration of CO2 remained in the product fraction, which was governed by the efficiency of CO2-methanol separation during the fraction collection. Hence, we demonstrated that the different designs of CO2-solvent separator (high pressurized cyclone in Waters SFC-350® and low-pressurized vortexing separator in Waters SFC-80q®®) had a significant impact on the degradation of an acid-sensitive compound. The acidity caused by CO2 in methanol was supported by diminished degradation after a nitrogen purging or after neutralizing the collected fractions with a base. Three different solutions to overcome the degradation problem of the acid sensitive compounds using SFC-350® with the high pressurized separator were investigated and demonstrated. The degraded products were isolated as four enantiomers and their relative stereochemistry were established based on 2D NMR data along with the plausible mechanism of degradation.

Integrating a post-column makeup pump into preparative supercritical fluid chromatography systems to address stability and recovery issues during purifications.

Purification of many pharmaceutical compounds by supercritical fluid chromatography (SFC) has always been challenging because of degradation of compound during the isolation step in the presence of acidic or basic modifiers in the mobile phase. Stability of such acid or base-sensitive compounds could be improved by post-column addition of a solvent containing base or acid modifier as counter ion through a make-up pump respectively to neutralize the compound fraction without affecting the resolution. One such case study has been presented in this work where the stability of a base-sensitive compound was addressed by the addition of acidic co-solvent through the make-up pump. Details of this setup and the investigation of degradation of the in-house base-sensitive compound are discussed in this paper. In addition, poor retentivity and low recovery of many non-polar compounds in SFC eluting under low co-solvent percentage is another major concern. Even though the desired separation could be achieved with low percentage of co-solvent, it's difficult to get the proper recovery after purification due to precipitation of the sample and significant aerosol formation inside the cyclone. We have demonstrated the first-time use of a post-column make-up pump on SFC 350 system to introduce additional solvent prior to cyclone to avoid the precipitation, reduce the aerosol formation and thus improve the recovery of non-polar compounds eluting under less than 10% of co-solvent.

Second-hand exposure to aerosolized intravenous anesthetics propofol and fentanyl may cause sensitization and subsequent opiate addiction among anesthesiologists and surgeons.

We hypothesize that aerosolization of anesthetics administered intravenously to patients in the operating room may be an unintended source of exposure to physicians. This may lead to inadvertent sensitization, which is associated with an increased risk for developing addiction. This may contribute to the over-representation of certain specialties among physicians with addiction. We retrospectively reviewed the de-identified demographic information of all licensed physicians treated for substance abuse in the State of Florida since 1980, to determine if medical specialty was associated with addiction in this group of individuals. Then, to identify the potential for exposure, two mass spectrometry assays were developed to detect two intravenously administered drugs, fentanyl and propofol, in air. Since 1980, 7.6% of licensed Florida physicians underwent treatment for addiction. Addiction in anesthesiologists was higher than expected. Opiate abuse was greater in anesthesiologists and surgeons compared to other specialties. Aerosolized fentanyl was detected in the air of the cardiothoracic operating room, in patients' expiratory circuits, and in the headspace above sharps boxes, but not in adjoining hallways. Aerosolized propofol was detected in the expirations of a patient undergoing transurethral prostatectomy. While access and stress may place anesthesiologists and surgeons at greater risk for substance abuse, an additional risk factor may be unintended occupational exposure to addictive drugs. This report provides preliminary evidence of detection of aerosolized intravenous anesthetics using two newly developed analytical methods. We conclude that the potential exists for chronic exposure to low levels of airborne intravenously administered drugs. Further studies are under way to determine the significance of this exposure.

Mass spectral fragmentation of the intravenous anesthetic propofol and structurally related phenols.

Propofol (2,6-diisopropyl phenol) is a widely used intravenous anesthetic. To define its pharmacokinetics and pharmacodynamics, methods for its quantitation in biological matrixes have been developed, but its pattern of mass spectral fragmentation is unknown. We found that fragmentation of the [M - H](-) ion (m/z 177) of propofol in both APCI MS/MS and ESI MS/MS involves the stepwise loss of a methyl radical and a hydrogen radical from one isopropyl side chain to give the most intense product ion, [M -H - CH(4)](-), at m/z 161. This two-step process is also the preferred mode of fragmentation for similar branched alkyl substituted phenols. This mode of fragmentation of the [M - H](-) ion is supported by three independent lines of evidence: (1) the presence of the intermediary [M - H - CH(3)](-) radical ion under conditions of reduced collision energy, (2) the determination of the mass of the predominant [M - H - CH(4)](-) product ion by high resolution mass spectrometry, and (3) the pattern of product ions resulting from further fragmentation of the [M - H - CH(4)](-) product ion. Phenols with a single straight chain alkyl substituent, in contrast, undergo beta elimination of the alkyl radical irrespective of the length of the alkyl chain, yielding the most intense product ion at m/z 106. This product ion represents a special case of a stable intermediary radical for the two-step process described for branched side chains, because further elimination of a hydrogen radical from the beta carbon is not possible.

A new method for the quantitation of propofol in human plasma: efficient solid-phase extraction and liquid chromatography/APCI-triple quadrupole mass spectrometry detection.

Propofol (2,6-diisopropyl phenol) is widely used for the induction and maintenance of anesthesia. Analyses of its pharmacokinetics require simple and sensitive methods for quantitation of propofol in human plasma. Previously reported HPLC and GC methods are limited by cumbersome extraction steps. We describe a novel method that combines sample preparation by solid-phase extraction (SPE) with hydrophilic-lipophilic balance cartridges and analysis with a sensitive LC-APCI-triple quadrupole mass spectrometry (MS/MS) method for better quantitation. The absolute recovery of the analyte was greater than 96%. The limit of quantification for propofol in plasma at a signal-to-noise ratio of 10 was 5 ng/ml. The precision of the assay yielded coefficients of variation ranging from 2.9 to 5.3% and an accuracies of 99-105%. Our method advances the quantitative analysis of propofol in human plasma by combining simple, rapid and efficient SPE with specific and sensitive quantitation by HPLC with APCI-MS/MS detection.

Sensitive and cost-effective LC-MS/MS method for quantitation of CVT-6883 in human urine using sodium dodecylbenzenesulfonate additive to eliminate adsorptive losses.

CVT-6883, a novel selective A(2B) adenosine receptor antagonist currently under clinical development, is highly lipophilic and exhibits high affinity for non-specific binding to container surfaces, resulting in very low recovery in urine assays. Our study showed the use of sodium dodecylbenzenesulfonate (SDBS), a low-cost additive, eliminated non-specific binding problems in the analysis of CVT-6883 in human urine without compromising sensitivity. A new sensitive and selective LC-MS/MS method for quantitation of CVT-6883 in the range of 0.200-80.0ng/mL using SDBS additive was therefore developed and validated for the analysis of human urine samples. The recoveries during sample collection, handling and extraction for the analyte and internal standard (d(5)-CVT-6883) were higher than 87%. CVT-6883 was found stable under the following conditions: in extract - at ambient temperature for 3 days, under refrigeration (5 degrees C) for 6 days; in human urine (containing 4mM SDBS) - after three freeze/thaw cycles, at ambient temperature for 26h, under refrigeration (5 degrees C) for 94h, and in a freezer set to -20 degrees C for at least 2 months. The results demonstrated that the validated method is sufficiently sensitive, specific, and cost-effective for the analysis of CVT-6883 in human urine and will provide a powerful tool to support the clinical programs for CVT-6883.