Protonation-Induced Chirality Drives Separation by Differential Ion Mobility Spectrometry.

Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

[Forecast of the metrological characteristics of the method of the quantitative determination of verapamil in blood in forensic chemical analysis]. / Prognoz metrologicheskikh kharakteristik metodiki kolichestvennogo opredeleniya verapamila v krovi v sudebno-khimicheskom analize.

The aim of the work is to test theoretical prognosis of metrological characteristics of methods of quantitative determination in forensic chemical analysis on the example of method of determination of verapamil content in blood by thin layer chromatography with computer densitometry. The algorithm of prognostic determination of relative error for methods of quantitative analysis in forensic chemical examination using computer program «ChemMetrEvaluation 1.0¼ is offered. The implementation of the algorithm is based on a detailed analysis of error estimation of measurements at the stages of sample preparation, measuring the value of the analytical signal.

Rapid detection and structural characterization of verapamil metabolites in rats by UPLC-MSE and UNIFI platform.

High-resolution mass spectrometry (HRMS) is an important technology for studying biotransformations of drugs in biological systems. In order to process complex HRMS data, bioinformatics, including data-mining techniques for identifying drug metabolites from liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or multistage mass spectrometry (MSn ) datasets as well as elucidating the detected metabolites' structure by spectral interpretation software, are important tools. Data-mining technologies have widely been used in drug metabolite identification, including mass defect filters, product ion filters, neutral-loss filters, control sample comparisons and extracted ion chromatographic analysis. However, the metabolites identified by current different technologies are not the same, indicating the importance of technique integration for efficient and complete identification of metabolic products. In this study, a universal, high-throughput workflow for identifying and verifying metabolites by applying the drug metabolite identification software UNIFI is reported, to study the biotransformation of verapamil in rats. A total of 71 verapamil metabolites were found in rat plasma, urine and faeces, including two metabolites that have not been reported in the literature. Phase I metabolites of verapamil were identified as N-demethylation, O-demethylation, N-dealkylation and oxidation and dehydrogenation metabolites; phase II metabolites were mainly glucuronidation and sulfate conjugates, indicating that UNIFI software could be effective and valuable in identifying drug metabolites.

Analytical techniques for the determination of verapamil in biological samples and dosage forms: an overview.

Verapamil (VER) is a calcium channel blocker that is widely used to treat various cardiovascular diseases and is also effective in migraine prophylaxis. As the therapeutic range of VER is very narrow and toxicity can occur in patients after oral administration, therapeutic drug monitoring is recommended to optimize pharmacotherapy. The choice of an appropriate bioanalytical method for therapeutic drug monitoring of VER in the biological samples is a very important step in achieving fast and reliable results. This review focuses on the various analytical methods reported between 1976 and 2019 for the determination of VER in different biological samples and pharmaceutical dosage forms along with their methodological limitations. This review provides an overview for pharmaceutical industry researchers, clinicians and clinical chemists.

Univariate calibrations with or without chemometric assistance for determination of drugs lacking peak maxima in their zero-order profiles.

Trandolapril has no sharp peak in its zero-order spectrum and therefore, it is difficult to be measured by direct spectrophotometry. In this manuscript, several univariate and multivariate spectrophotometric methods were developed and validated for determination of Trandolapril (TR) and Verapamil (VR) combination. The first method for measuring Trandolapril is Constant Multiplication-Spectrum Subtraction (CM-SS), where Trandolapril was measured at 210 nm in its zero-order curve after elimination of Verapamil spectrum. Second and third methods are two Base Points (2BP) and area under the curve (AUC) to measure Trandolapril concentration without depending on the shoulder peak. The fourth method for Trandolapril is Derivative Subtraction (DS) that utilizes the sharp peak appeared in the first order spectrum of Trandolapril. Verapamil was determined by two methods, Constant Multiplication (CM) and Derivative Subtraction-Constant Multiplication (DS-CM). Also, two multivariate methods were developed for measurement of the mixture, Partial Least Squares (PLS) and Principal Component Regression (PCR). All the developed methods were validated as per ICH guidelines and the results proved that the developed methods are accurate and selective. Moreover, a statistical comparison between the developed methods and a reference method was done. Also, One-way ANOVA statistical test was done between all the proposed univariate and multivariate spectrophotometric methods.

Surface-assisted laser desorption/ionization time-of-flight mass spectrometry of small drug molecules and high molecular weight synthetic/biological polymers using electrospun composite nanofibers.

Polyacrylonitrile/Nafion®/carbon nanotube (PAN/Nafion®/CNT) composite nanofibers were prepared using electrospinning. These electrospun nanofibers were studied as possible substrates for surface-assisted laser desorption/ionization (SALDI) and matrix-enhanced surface-assisted laser desorption/ionization time-of-flight mass spectrometry (ME-SALDI/TOF-MS) for the first time in this paper. Electrospinning provides this novel substrate with a uniform morphology and a narrow size distribution, where CNTs were evenly and firmly immobilized on polymeric nanofibers. The results show that PAN/Nafion®/CNT nanofibrous mats are good substrates for the analysis of both small drug molecules and high molecular weight polymers with high sensitivity. Markedly improved reproducibility was observed relative to MALDI. Due to the composite formation between the polymers and the CNTs, no contamination of the carbon nanotubes to the mass spectrometer was observed. Furthermore, electrospun nanofibers used as SALDI substrates greatly extended the duration of ion signals of target analytes compared to the MALDI matrix. The proposed SALDI approach was successfully used to quantify small drug molecules with no interference in the low mass range. The results show that verapamil could be detected with a surface concentration of 220 femtomoles, indicating the high detection sensitivity of this method. Analysis of peptides and proteins with the electrospun composite substrate using matrix assisted-SALDI was improved and a low limit of detection of approximately 6 femtomoles was obtained for IgG. Both SALDI and ME-SALDI analyses displayed high reproducibility with %RSD ≤ 9% for small drug molecules and %RSD ≤ 14% for synthetic polymers and proteins.

Analytical method development and validation for the analysis of verapamil hydrochloride and its related substances by using ultra perfomance liquid chromatography.

A novel, economic, and time-efficient stability-indicating, reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method has been developed for the analysis of verapamil hydrochloride in the presence of both impurities and degradation products generated by forced degradation. When verapamil hydrochloride was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and thermal stress, degradation was observed only in oxidative and base hydrolysis. The drug was found to be stable to other stress conditions. Successful chromatographic separation of the drug from impurities formed during synthesis and from degradation products formed under stress conditions was achieved on a Shimpak XR ODS, 75mm×3.0mm, 1.7µ particle size column, UV detection at 278nm and a gradient elution of ammonium formate, orthophosphoric acid and acetonitrile as mobile phase. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of verapamil hydrochloride. To the best of our knowledge, a validated UPLC method which separates all the sixteen impurities disclosed in this investigation has not been published elsewhere. Total elution time was about 18min which allowed quantification of more than 100 samples per day. The analytical method discussed in British Pharmacopeia was pH sensitive and not compatible to LC-MS analysis but the method reported in this study is not involved any pH adjustment.

Multiple Time-of-Flight/Time-of-Flight Events in a Single Laser Shot for Improved Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry Quantification.

Quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) approaches have historically suffered from poor accuracy and precision mainly due to the nonuniform distribution of matrix and analyte across the target surface, matrix interferences, and ionization suppression. Tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity as well as improve signal-to-noise ratios by eliminating interferences from chemical noise, alleviating some concerns about dynamic range. However, conventional MALDI TOF/TOF modalities typically only scan for a single MS/MS event per laser shot, and multiplex assays require sequential analyses. We describe here new methodology that allows for multiple TOF/TOF fragmentation events to be performed in a single laser shot. This technology allows the reference of analyte intensity to that of the internal standard in each laser shot, even when the analyte and internal standard are quite disparate in m/z, thereby improving quantification while maintaining chemical specificity and duty cycle. In the quantitative analysis of the drug enalapril in pooled human plasma with ramipril as an internal standard, a greater than 4-fold improvement in relative standard deviation (<10%) was observed as well as improved coefficients of determination (R2) and accuracy (>85% quality controls). Using this approach we have also performed simultaneous quantitative analysis of three drugs (promethazine, enalapril, and verapamil) using deuterated analogues of these drugs as internal standards.

Bioconcentration of two basic pharmaceuticals, verapamil and clozapine, in fish.

The present study examined the bioconcentration of 2 basic pharmaceuticals: verapamil (a calcium channel blocker) and clozapine (an antipsychotic compound) in 2 fresh water fishes, fathead minnow and channel catfish. In 4 separate bioconcentration factor (BCF) experiments (2 chemicals × 1 exposure concentration × 2 fishes), fathead minnow and channel catfish were exposed to 190 µg/L and 419 µg/L of verapamil (500 µg/L nominal) or 28.5 µg/L and 40 µg/L of clozapine (50 µg/L nominal), respectively. Bioconcentration factor experiments with fathead consisted of 28 d uptake and 14 d depuration, whereas tests conducted on catfish involved a minimized test design, with 7 d each of uptake and depuration. Fish (n = 4-5) were sampled during exposure and depuration to collect different tissues: muscle, liver, gills, kidneys, heart (verapamil tests only), brain (clozapine tests only), and blood plasma (catfish tests only). Verapamil and clozapine concentrations in various tissues of fathead and catfish were analyzed using liquid chromatography-mass spectrometry. In general, higher accumulation rates of the test compounds were observed in tissues with higher perfusion rates. Accumulation was also high in tissues relevant to pharmacological targets in mammals (i.e. heart in verapamil test and brain in the clozapine test). Tissue-specific BCFs (wet wt basis) for verapamil and clozapine ranged from 0.7 to 75 and from 31 to 1226, respectively. Tissue-specific concentration data were used to examine tissue-blood partition coefficients.

Multi-drug intoxication fatality involving atorvastatin: A case report.

Mixed antihypertensive drug intoxication poses a significant risk for patient mortality. In tandem to antihypertensives, hypolipidemic medicines (especially statins) are often prescribed. Among their well-known adverse effects belongs rhabdomyolysis. We report a case of fatal multi-drug overdose in a 65-year-old female alcoholic. The patient was unconscious at admission. Empty blister packs indicated the abuse of 250 tablets of urapidil, 42 tablets of verapamil/trandolapril, 50 tablets of moxonidin, 80 tablets of atorvastatin and 80 tablets of diacerein. Standard measures (gastric lavage, activated charcoal, mechanical ventilation, massive doses of vasopressors, volume expansion, diuretics and alkalinization) failed to provide sufficient drug elimination and hemodynamic support and the sufferer deceased on the fourth day. Dramatic elevations of serum myoglobin (34,020 µg/L) and creatine kinase (219 µkat/L) were accompanied by rise in cardiac troponin I and creatinine. Gas chromatography revealed ethanol 1.17 g/kg (blood) and 2.81 g/kg (urine). Thin layer chromatography and gas chromatography of gastric content and urine verified verapamil, moxonidin and urapidil fragment (diacerein method was unavailable). Atorvastatin and trandolapril concentrations (LC-MS(n)) equaled 277.7 µg/L and 57.5 µg/L, resp. (serum) and 8.15 µg/L and 602.3 µg/L, resp. (urine). Histology confirmed precipitates of myoglobin with acute necrosis of proximal renal tubules in association with striated muscle rhabdomyolysis and myocardial dystrophy. Cardiogenic-distributive shock in conjunction with acute renal failure due to the combined self-poisoning with vasoactive agents and atorvastatin were determined to be this decedent's immediate cause of death. The manner of death was assigned to be suicidal.

Use of DMPC and DSPC lipids for verapamil and naproxen permeability studies by PAMPA.

Verapamil and naproxen Parallel Artificial Membrane Permeability Assay (PAMPA) permeability was studied using lipids not yet reported for this model in order to facilitate the quantification of drug permeability. These lipids are 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an equimolar mixture of DMPC/DSPC, both in the absence and in the presence of 33.3 mol% of cholesterol. PAMPA drug permeability using the lipids mentioned above was compared with lecithin-PC. The results show that verapamil permeability depends on the kind of lipid used, in the order DMPC > DMPC/DSPC > DSPC. The permeability of the drugs was between 1.3 and 3.5-times larger than those obtained in lecithin-PC for all the concentrations of the drug used. Naproxen shows similar permeability than verapamil; however, the permeability increased with respect to lecithin-PC only when DMPC and DMPC/DSPC were used. This behavior could be explained by a difference between the drug net charge at pH 7.4. On the other hand, in the presence of cholesterol, verapamil permeability increases in all lipid systems; however, the relative verapamil permeability respect to lecithin-PC did not show any significant increase. This result is likely due to the promoting effect of cholesterol, which is not able to compensate for the large increase in verapamil permeability observed in lecithin-PC. With respect to naproxen, its permeability value and relative permeability respect lecithin-PC not always increased in the presence of cholesterol. This result is probably attributed to the negative charge of naproxen rather than its molecular weight. The lipid systems studied have an advantage in drug permeability quantification, which is mainly related to the charge of the molecule and not to its molecular weight or to cholesterol used as an absorption promoter.

Studies on assessment methods of malignant ascites residue and changes of verapamil concentration in intraperitoneal perfusion chemotherapy.

PURPOSE: To establish a simple method for estimating residual peritoneal ascites in order to determine the optimum verapamil (VRP) initial concentration in the intraperitoneal perfusion chemotherapy. METHODS: (1) Pelvic size of adults was assessed by measuring distance from the superior margin of pubic symphysis to the connecting line of two anterior superior spine (SL) and to the midpoint of the line (SM) in 172 adults; (2) 35 postoperative gastric or colon cancer patients with indications for use of preventive intraperitoneal chemotherapy were infused with 1,000-1,250 mL 0.9 % normal saline solution for about 15 min and used for perfusate detection by moving along the midpoint of connecting line of two anterior superior spine after 5 min of infusion; (3) The VRP concentration in ascites was detected by liquid chromatography. RESULTS: The distance between two anterior superior spines for adult were 29.6 ± 2.6 cm and the distance from the superior margin of pubic symphysis to the midpoint between two anterior superior spines was 10.6 ± 1.9 cm. When the total intraperitoneal infusion fluid was 1,000-1,250 mL, it could be detected by B-mode ultrasonic device at 0.1-0.3 cm directly below the midpoint of two anterior superior spines. The VRP reversal concentration of drug resistance could maintain for 90 min when the residual ascites volume was within the range of 1,000-1,250 mL. CONCLUSIONS: Detection of liquid at the position directly below or above the midpoint of two anterior superior spines by B-mode ultrasonic device in patients in erect position could be a simple method for estimation of ascites volume (liquid found at 0.1-0.3 cm directly below the midpoint of two anterior superior spines suggested that ascites volume was smaller than 1,000-1,250 mL). The method could be used for determination of VRP initial concentration for IPC treatment.

Toward single-cell analysis by plume collimation in laser ablation electrospray ionization mass spectrometry.

Ambient ionization methods for mass spectrometry have enabled the in situ and in vivo analysis of biological tissues and cells. When an etched optical fiber is used to deliver laser energy to a sample in laser ablation electrospray ionization (LAESI) mass spectrometry, the analysis of large single cells becomes possible. However, because in this arrangement the ablation plume expands in three dimensions, only a small portion of it is ionized by the electrospray. Here we show that sample ablation within a capillary helps to confine the radial expansion of the plume. Plume collimation, due to the altered expansion dynamics, leads to greater interaction with the electrospray plume resulting in increased ionization efficiency, reduced limit of detection (by a factor of ~13, reaching 600 amol for verapamil), and extended dynamic range (6 orders of magnitude) compared to conventional LAESI. This enhanced sensitivity enables the analysis of a range of metabolites from small cell populations and single cells in the ambient environment. This technique has the potential to be integrated with flow cytometry for high-throughput metabolite analysis of sorted cells.

High-throughput liquid chromatography/mass spectrometry method for the quantitation of small molecules using accurate mass technologies in supporting discovery drug screening.

RATIONALE: Drug discovery samples are routinely analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods on triple quadrupole mass spectrometers employing multiple reaction monitoring (MRM). In order to improve analysis throughput, quantitation of small molecules on a quadrupole time-of-flight (QqTOF) instrument using TOF scan and high-resolution MRM (MRM-HR) modes was evaluated in this study. METHODS: Cassette dosed plasma and brain samples from nine compounds were extracted using a protein precipitation method. Separation was achieved by reversed-phase liquid chromatography. Mass spectrometric analysis was performed using TOF scan and high-resolution MRM approaches on a QqTOF mass spectrometer with turbo-ionspray ionization. Results were compared to those obtained on a triple quadrupole mass spectrometer. RESULTS: The dynamic range varied depending on compounds and instruments and was similar between the MRM on QqQ and full TOF scan mode on QqTOF. Linear or quadratic regression and 1/x(2) weighting were used. Resolution on the QqTOF instrument was around 32000 and mass accuracy was within 4.4 ppm. The MRM-HR method showed better sensitivity compared to the TOF scan method, and was comparable to the MRM on a QqQ mass spectrometer. Assay accuracy was within ±25%. CONCLUSIONS: A TOF scan method allowed the use of the generic method without compound-specific optimization and was an alternative choice for routine high-throughput quantitation of small molecules. The MRM-HR method on the QqTOF showed good sensitivity which was comparable to that obtained by the MRM method on the triple quadrupole mass spectrometer.

Multiplex mass spectrometry imaging for latent fingerprints.

We have previously developed in-parallel data acquisition of orbitrap mass spectrometry (MS) and ion trap MS and/or MS/MS scans for matrix-assisted laser desorption/ionization MS imaging (MSI) to obtain rich chemical information in less data acquisition time. In the present study, we demonstrate a novel application of this multiplex MSI methodology for latent fingerprints. In a single imaging experiment, we could obtain chemical images of various endogenous and exogenous compounds, along with simultaneous MS/MS images of a few selected compounds. This work confirms the usefulness of multiplex MSI to explore chemical markers when the sample specimen is very limited.

Simultaneous detection of nonpolar and polar compounds by heat-assisted laser ablation electrospray ionization mass spectrometry.

A heat-assisted laser ablation electrospray ionization (HA-LAESI) method for the simultaneous mass spectrometric analysis of nonpolar and polar analytes was developed. The sample was introduced using mid-infrared laser ablation of a water-rich target. The ablated analytes were ionized with an electrospray plume, which was intercepted by a heated nitrogen gas jet that enhanced the ionization of analytes of low polarity. The feasibility of HA-LAESI was tested by analyzing, e.g., naphtho[2,3-a]pyrene, cholesterol, tricaprylin, 1,1',2,2'-tetramyristoyl cardiolipin, bradykinin fragment 1-8, and 1-palmitoyl-2-oleoyl-sn-glycerol. HA-LAESI was found better suited for low polarity compounds than conventional LAESI, whereas polar compounds were observed with both techniques. The sensitivity of HA-LAESI for the polar bradykinin fragment 1-8 was slightly lower than observed for LAESI. HA-LAESI showed a linear response for 500 nM to 1.0 mM solutions (n = 11) of verapamil with R(2) = 0.988. HA-LAESI was applied for the direct analysis of tissue samples, e.g., avocado (Persea americana) mesocarp and mouse brain tissue sections. Spectra of the avocado showed abundant triglyceride ion peaks, and the results for the mouse brain sections showed cholesterol as the main species. Conventional LAESI shows significantly lower ionization efficiency for these neutral lipids. HA-LAESI can be applied to the analysis of nonpolar and polar analytes, and it extends the capabilities of conventional LAESI to nonpolar and neutral compounds.

A semi-automated method for the integrated evaluation of half-life and metabolic soft spots of discovery compounds.

BACKGROUND: An integrated method that provides rates of both parent disappearance and metabolite formation was developed. RESULTS: Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 µM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 µM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 µM samples to UV areas in order to compare relative metabolite concentrations. CONCLUSION: The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.

Infrared laser ablation atmospheric pressure photoionization mass spectrometry.

In this paper we introduce laser ablation atmospheric pressure photoionization (LAAPPI), a novel atmospheric pressure ion source for mass spectrometry. In LAAPPI the analytes are ablated from water-rich solid samples or from aqueous solutions with an infrared (IR) laser running at 2.94 µm wavelength. Approximately 12 mm above the sample surface, the ablation plume is intercepted with an orthogonal hot solvent (e.g., toluene or anisole) jet, which is generated by a heated nebulizer microchip and directed toward the mass spectrometer inlet. The ablated analytes are desolvated and ionized in the gas-phase by atmospheric pressure photoionization using a 10 eV vacuum ultraviolet krypton discharge lamp. The effect of operational parameters and spray solvent on the performance of LAAPPI is studied. LAAPPI offers ~300 µm lateral resolution comparable to, e.g., matrix-assisted laser desorption ionization. In addition to polar compounds, LAAPPI efficiently ionizes neutral and nonpolar compounds. The bioanalytical application of the method is demonstrated by the direct LAAPPI analysis of rat brain tissue sections and sour orange (Citrus aurantium) leaves.

Silica coated paper substrate for paper-spray analysis of therapeutic drugs in dried blood spots.

Paper spray is a newly developed ambient ionization method that has been applied for direct qualitative and quantitative analysis of biological samples. The properties of the paper substrate and spray solution have a significant impact on the release of chemical compounds from complex sample matrices, the diffusion of the analytes through the substrate, and the formation of ions for mass spectrometry analysis. In this study, a commercially available silica-coated paper was explored in an attempt to improve the analysis of therapeutic drugs in dried blood spots (DBS). The dichloromethane/isopropanol solvent has been identified as an optimal spray solvent for the analysis. The comparison was made with paper spray using chromatography paper as substrate with methanol/water as solvent for the analysis of verapamil, citalopram, amitriptyline, lidocaine, and sunitinib in dried blood spots. It has been demonstrated that the efficiency of recovery of the analytes was notably improved with the silica coated paper and the limit of quantitation (LOQ) for the drug analysis was 0.1 ng mL(-1) using a commercial triple quadrupole mass spectrometer. The use of silica paper substrate also resulted in a sensitivity improvement of 5-50-fold in comparison with chromatography papers, including the Whatman ET31 paper used for blood cards. Analysis using a hand-held miniature mass spectrometer Mini 11 gave LOQs of 10-20 ng mL(-1) for the tested drugs, which is sufficient to cover the therapeutic ranges of these drugs.

[Circular dichroism in study of drug chirality].

Verapamyl substance and market verapamyl were investigated with circular dichroism. It was shown that circular dichroism provided rapid and highly efficient determination of the optic isomers and could be recommended as a method for control of drug quality.