Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC–QTOF

Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi‐synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA‐tryptophan (MTMSA‐Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA‐phenylalanine (MTMSA‐Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA‐Trp was stable for 30 days at ?80°C and for at least three freeze–thaw cycles. Methanol (?80°C) extraction afforded 92% of MTMSA‐Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.     

Replicates Number for Drug Stability Testing during Bioanalytical Method Validation—An Experimental and Retrospective Approach

Background: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. Methods: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes—tramadol and its major metabolite (O-desmethyl-tramadol)—in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). Results: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85–115% acceptance criteria. Conclusions: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.     

Protection of immobilized sulfhydryl groups against autooxidation by alterations in their microenvironment

Immobilized sulfhydryl groups were prepared by partial thiolation of NH₂-glass beads. The microenvironment of the immobilized SH groups was varied by different chemical modifications of neighboring NH₂ groups. Introduction of a strong charge in the surroundings of immobilized sulfhydryls results in their dramatic stabilization against autooxidation. This effect is due to the salting of O₂ from the surface microlayer of the thiolated beads.     

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex^® C₁₈ column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x²) over a concentration range of 1–1000, 2.5–800, and 5–500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.     

Evidence of covalent interaction of fumaric acid esters with sulfhydryl groups in peptides

Fumaric acid esters, namely dimethylfumarate, have been used for the treatment of psoriasis for many years. Still, their mode of action is not fully clear. Because addition of nucleophiles to the double bonds of fumarates can occur (Michael analogous addition), a study of the interaction of fumarates with cysteine and cysteine-containing peptides possessing nucleophilic sulfhydryl group was carried out. Experiments were performed in aqueous medium at pH 7.4 and at 37 degrees C to simulate physiological conditions. It was proven by mass spectrometric measurements using an ion-trap and time-of-flight instrument that a covalent bond can form between fumarates and the sulfhydryl group of cysteine or cysteinyl residues in peptides. Structures of the interaction products were elucidated by multistage mass spectrometry applying collision-induced dissociation. Higher reactivity of dimethylfumarate in comparison to monomethylfumarate and fumaric acid was observed.     

Effect of quinone derivatives and sulfhydryl compounds on swelling of poly(4-vinylpyridine) gels in organic solvents

Interactions of sulfhydryl compounds and quinone derivatives with poly(4-vinylpyridine) gels have been studied. Adsorption of long-chain sulfhydryl compounds onto gels causes the gel to swell, whereas oxidation of the adsorbed sulfhydryl compound to disulfide by a quinone derivative results in the gel shrinking. The higher the donating ability of the sulfhydryl compound, the stronger its interaction with a quinone derivative and thus the faster the removal from the gels, as revealed by HPLC. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 795–798, 1997     

LC/MS characterization of undesired products formed during iodoacetamide derivatization of sulfhydryl groups of peptides

Many undesired by-products have been noticed during alkylation with iodoacetamide, a widely used derivatization reaction in proteomics for the determination of sulfhydryl groups in peptides and proteins. We report here that iodoacetamide reacts with the N-terminal NH2 and the C-terminal carboxylic acid groups, in addition to the peripheral residues bearing protic functional groups. If sufficient reaction time is given, the N-terminal NH2 group is readily dialkylated by iodoacetamide. In fact, the N-terminal NH2 group reacts even faster than the reactive sites present in residues, such as tyrosine or histidine. LC/MS investigations with certain reactive peptides show that by-products are formed in a relatively short reaction time, even at room temperature. Interestingly, derivatives formed in this way are useful for sequence determination of peptides by MS since the intensities of y' ions are highly suppressed in the spectra of N-terminus mono- and dialkylated peptides, whereas those of b-ions are significantly enhanced. For example, in the spectrum of N,N-dicarboxamidomethyl derivative of Val-Ala-Ala-Phe (VAAF), the y-series ions are virtually absent. On the other hand, when the derivatization takes place at the carboxylic group, the y-series ions are markedly observed in the spectra of these undesired O-carboxamidomethyl derivatives.     

Bioanalytical methods for the determination of antipsychotic drugs

Antipsychotic drugs are popular for the treatment of schizophrenia and other psychoses. In general, antipsychotics are administered in oral doses of only a few milligrams per day and they are widely metabolized in the body. Therefore, the concentration of these drugs in plasma is very low (pg-ng/mL levels). In addition, therapeutic drug monitoring (TDM) of antipsychotic drugs has proven to be of notable value for determining poor compliance of patients and addressing the challenges associated with considerable genetic variability in their metabolism. Thus, in order to conduct pharmacology and toxicology studies and clinical TDM of antipsychotics, as well as address the challenges associated with polypharmacy and drug metabolism, highly sensitive, selective and accurate bioanalytical methods are essential. The most recent studies on the determination of antipsychotics will be reviewed, including separation techniques, sample pretreatment, detectors and validation.     

Synthesis and characterization of cationic latex particles bearing sulfhydryl groups and their use in the immobilization of Fab antibody fragments (1)

Emulsion copolymerization of styrene and a cationic monomer vinylbenzyl-isothiouronium chloride (VBIC) with initiation by 2,2 azobis(2-amidinopropane)-dihydrochloride (V-50) gave monodisperse latex particles. After post-stabilization with cationic or non-ionic surfactants, the colloids were diluted in basic buffers with concomitant deprotection of the sulfhydryl groups. Therefore, coupling reactions on the latex beads were possible. Coupling conditions were determined with Ellman's reagent.¹⁴C iodoacetamide and anti C-reactive protein antibody Fab fragments were immobilized and finally, the immunological activity of the sensitized latex was assessed.     

Development and validation of a HPLC‐PDA bioanalytical method for the simultaneous estimation of Aliskiren and Amlodipine in human plasma

A simple, unique and selective HPLC‐PDA method was developed and validated for the simultaneous estimation of aliskiren (ALS) and amlodipine (AML) in human plasma. Extraction of the sample was accomplished by protein precipitation. Plasma proteins were precipitated by employing acetonitrile containing hydrochlorothiazide as internal standard. The compounds were analyzed by HPLC by using PDA detector on a Hibar C₁₈ (250 × 4.6 mm) column with a mobile phase comprising acetonitrile and phosphate buffer (pH 4.2 and 25 mm ; 60:40 v/v) with a flow rate of 0.8 mL/min. Different sample pretreatment techniques were evaluated but protein precipitation was found to be satisfactory, offering good recovery values of 97.11–98.45% for ALS and 97.5–99.12% for AML. The within‐day precisions for ALS were 96.66, 99.16 and 99.41% at 90, 240 and 480 ng/mL, respectively, and for AML they were 97.27, 99.54 and 99.31% at 3.3, 8.8 and 17.6 ng/mL, respectively. The between‐day precisions for ALS were 96.66, 99.16 and 99.41% at 90, 240 and 480 ng/mL, respectively and the between‐day precisions for AML were 98.18, 99.20 and 99.40% at 3.3, 8.8 and 17.6 ng/mL, respectively. The limit of quantitation was 30 and 1.0 ng/mL for ALS and AML respectively. Different constituents of plasma proteins did not interfere with the absolute recovery of ALS and AML. Copyright © 2014 John Wiley & Sons, Ltd.     

A method for functional analysis of organosilicon compounds containing silacyclobutane groups

A method of determining quantitatively silacyclobutane rings in organosilicon compounds is worked out. It is based on addition of bromine or iodine to a Si-C bond in the ring, followed by determination of those elements in the resultant Si-Br or Si-I group. The error in determining bromine or iodine numbers by the method is 0.5–2.0%; the error in determining percentage bromine (iodine) is 0.5–2.0%. A determination takes 3–4 hr. The silacyclobutane group does not react with thiocyanogen, so that it is possible to determine separately multiple bonds and silacyclobutane groups present in one and the same molecule.     

Fast and sensitive HPLC/UV method for cefazolin quantification in plasma and subcutaneous tissue microdialysate of humans and rodents applied to pharmacokinetic studies in obese individuals

Antimicrobial prophylactic dosing of morbidly obese patients may differ from normal weighted individuals owing to alterations in drug tissue distribution. Drug subcutaneous tissue distribution can be investigated by microdialysis patients and animals. The need for cefazolin prophylactic dose adjustment in obese patients remains under discussion. The paper describes the validation of an HPLC‐UV method for cefazolin quantification in plasma and microdialysate samples from clinical and pre‐clinical studies. A C₁₈ column with an isocratic mobile phase was used for drug separation, with detection at 272 nm. Total and unbound cefazolin lower limit of quantitation was 5 μg/mL in human plasma, 2 μg/mL in rat plasma, and 0.5 and 0.025 μg/mL in human and rat microdialysate samples, respectively. The maximum intra‐ and inter‐day imprecisions were 10.7 and 8.1%, respectively. The inaccuracy was <9.7%. The limit of quantitation imprecision and inaccuracy were < 15%. Cefazolin stability in the experimental conditions was confirmed. Cefazolin plasma concentrations and subcutaneous tissue penetration were determined by microdialysis in morbidly obese patients (2 g i.v. bolus) and diet‐induced obese rats (30 mg/kg i.v. bolus) using the method. This method has the main advantages of easy plasma clean‐up and practicability and has proven to be useful in cefazolin clinical and pre‐clinical pharmacokinetic investigations.     

LC–MS/MS method with chemical derivatization for quantitation of L‐threonate in human plasma

An LC–MS/MS‐based bioanalytical method has been developed to measure the concentration of L‐threonate at its endogenous level in human plasma. Following isotope dilution and protein precipitation, the samples were acetylated and chromatographed under reversed‐phase conditions for baseline separation of the derivatized L‐threonate and its stereoisomer D‐erythronate. The method was assessed by a fit‐for‐purpose validation with a calibration range from 100 to 10,000 ng/mL. The intra‐run coefficients of variation (CVs) were <3.6% and the inter‐run CV was 3.2% for the QC samples at endogenous level. At the lower limit of quantitation, the intra‐run CV was 6.1% and the average inaccuracy was ?1.4%. This method provides an efficient and reliable quantitation of L‐threonate and could be useful to certain biomarker investigators.     

Synthesis and extraction properties of oxathiacrown compounds containing benzyl groups

8-Benzyl-1.4-dioxa-7,10-dithiacyclododecane and 11-benzyl-1,4,7-trioxa-10,13-dithiacyclopentadecane were obtained by the interaction of (2,3-dibromo-1-propyl)benzene with 1,8-dimercapto-3,6-dioxaoctane and 1,11-dimercapto-3,6,9-trioxaundecane. The extracting ability of the obtained compounds has been studied in relation to Sr²⁺ and Pb²⁺ ions from aqueous solutions in the presence of anions of various degrees of hardness with determination of the metal content by a radiometric method. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 244–249, February, 2006.     

Synthesis of new synthons for organofluorine compounds from halothane containing sulfur functional groups

To develop new synthons for the syntheses of organofluorine compounds, the treatment of Halothane, 2-bromo-2-chloro-1,1,1-trifluoroethane, (1) with 4-methylbenzenethiol (2) in the presence of sodium hydride gave 1-chloro-2,2,2-trifluoroethyl 4-methylphenyl sulfide (3), which was oxidized with m-chloroperbenzoic acid (m-CPBA) to the corresponding sulfoxide (4) and sulfone (5). Reaction of 3 and 5 with allyltributyltin in the presence of 2,2'-azobis(isobutyronitrile) (AIBN) gave 1-(trifluoromethyl)-3-butenyl compounds (9, 11). Sulfoxide 4 was decomposed in this condition. The treatment of 3 with allyltrimethylsilane in the presence of Lewis acids gave 1-(trifluoromethyl)-3-butenyl compounds (9) in good yield. This result suggests that 4-methylphenylthio substituent stabilizes the alpha-carbocation effectively, though the trifluoromethyl group destabilizes it strongly. Aromatic compounds similarly reacted with 3 in the presence of titanium(IV) chloride to give 2-aryl-1,1,1-trifluoro-2-(4-methylphenylthio)ethanes. Thus, sulfur compounds derived from Halothane were found to be useful new synthons for organofluorine compounds.     

Perspectives on updates,clarifications and controversies in chromatographic assay guidance for bioanalytical method validation from major regulatory agencies and organizations

Bioanalysis, a key supporting function for generating data for pre‐clinical and clinical studies in drug development, is under the regulation of local agencies as well as global organizations to ensure the data integrity and quality in submission. As major regulatory agencies and organizations, the US Food and Drug Administration, the European Medicines Agency and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use have been updating their industry guidance for bioanalytical method validation, to keep up with the development new modalities, technologies and regulations. This article summarizes the recent updates and any clarifications and controversies triggered by those updates. Perspectives and recommendations are given based on our own experience as well as commonly accepted practice in the bioanalytical community.     

Development of a capillary electrophoretic method for determination of plasma clearance of iohexol in dogs and cats

Renal function can be monitored by estimation of the glomerular filtration rate (GFR), for example, through measurement of the plasma clearance of a marker that is freely filtrated through the kidney without reabsorption. It has been proposed that iohexol is the most accurate marker for GFR determination in cats and dogs. However, there is a need for a validated capillary electrophoretic method that covers the concentration range for a full curve clearance estimate of iohexol. In the final method, the plasma samples were protein precipitated and the supernatant was analyzed in a background electrolyte containing borate buffer (0.06 m , pH 10.0). The method developed was proved to be linear (concentration range 18– 2900 mg/L) and had a good precision (e.g. 2.3–2.9% at 88 mg/L) and accuracy (e.g. 101–105% at 88 mg/L). Finally, the method was compared with a previously published and validated HPLC‐UV method by parallel analysis of clinical plasma samples from dogs and cats administered Omnipaque®. This comparison showed excellent agreement between the two methods and no proportional or systematic error was observed. The proposed method is simple and has a low cost per sample, which makes it applicable for routine analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Chromatographic method for clobetasol propionate determination in hair follicles and in different skin layers

Clobetasol propionate (CLO) is a potent steroid used for the treatment of several dermatological diseases. Recent studies suggest its additional use in alopecia topical treatment, generating a demand for novel formulations with specific delivery into hair follicles. Hence, a selective analytical method for drug quantification in follicular structures and skin layers is required. For this, a simple HPLC‐UV method was developed. Quantification was performed using a RP‐C₁₈ column (4.6 mm × 15 cm, 5 μm), with a mixture of methanol–acetonitrile–water (50:15:35 v /v) as mobile phase, a flow rate of 1.2 mL/min, oven temperature of 30°C, injection volume of 50 μL and detection at 240 nm. The optimized conditions enabled a 12 min running with CLO elution at 10.1 min and resolution of 2.424 from skin matrix interferences. Validation was performed in accordance with International Conference on Harmonization guidelines and fulfilled the criteria of selectivity, linearity (0.5–15.0 μg/mL), robustness, precision, accuracy and limits of detection and quantification (0.02 and 0.07 μg/mL, respectively). The validated method was successfully applied for CLO quantification following in vitro skin permeation experiments and differential tape‐stripping for hair follicle deposition determination, demonstrating its suitability.     

Synthesis and polycondensation of some new organotellurium compounds containing hydroxymethyl groups

A new series of organotellurium compounds [i.e. 2‐HOCH₂C₆H₄TeBr₃ (1), (2‐HOCH₂C₆H₄)₂TeBr₂ (2), (2‐HOCH₂C₆H₄)₂Te (3), (2‐HOCH₂C₆H₄Te? )₂ (4), 4‐HOCH₂C₆H₄TeBr₃ (5), (4‐HOCH₂C₆H₄)₂TeBr₂ (6), (4‐HOCH₂C₆H₄)₂Te (7), and (4‐HOCH₂C₆H₄Te? )₂ (8)] were prepared by reacting hydroxymethylphenylmercury chlorides with tellurium tetrabromide in dry dioxane. Bis(2‐hydroxymethylphenyl) telluride (3), bis(2‐hydroxymethylphenyl) ditelluride (4) and bis(4‐hydroxymethylphenyl) telluride (7) were polymerized by a solution polycondensation technique with toluene diisocyanate and terephthaloyl chloride, leading to new organic tellurium polyurethanes and polyesters. All the new compounds were characterized by elemental analysis and spectroscopic data. Copyright © 2002 John Wiley & Sons, Ltd.     

含六氟异丙氧基五配位磷化合物的合成及其NMR的研究

最近Schmutzler等人合成了几种含有甲基六氟异丙氧基的五配位磷化合物,通过在不同温度下~(19)FNMR测试以及计算活化能,表明这类化合物在分子内配位基重组过程(intramolecula ligand reorganization)中,各配位基的空间效应是主要因素。我们采用另一路线合成了几种含有苯基六氟异丙氧基的五配位磷化合物,通过NMR的研究,进一步提供了关于这类化合物在假旋转(pseudorotation)时,配位基空间效应的影响。