A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 µL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 µm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000 ng/mL for daidzein, and 8.0-4000 ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values < 10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

Bioanalysis in the Age of New Drug Modalities.

In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.

Challenges and stepwise fit-for-purpose optimization for bioanalyses of remdesivir metabolites nucleotide monophosphate and triphosphate in mouse tissues using LC-MS/MS.

Remdesivir is a prodrug of the nucleotide analogue and used for COVID-19 treatment. However, the bioanalysis of the active metabolites remdesivir nucleotide triphosphate (RTP) and its precursor remdesivir nucleotide monophosphate (RMP) is very challenging. Herein, we established a novel method to separate RTP and RMP on a BioBasic AX column and quantified them by high-performance liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. Stepwise, we optimized chromatographic retention on an anion exchange column, improved stability in matrix through the addition of 5,5'-dithiobis-(2nitrobenzoic acid) and PhosSTOP EASYpack, and increased recovery by dissociation of tight protein binding with 2 % formic acid aqueous solution. The method allowed lower limit of quantification of 20 nM for RMP and 10 nM for RTP. Method validation demonstrated acceptable accuracy (93.6%-103% for RMP, 94.5%-107% for RTP) and precision (RSD < 11.9 % for RMP, RSD < 11.4 % for RTP), suggesting that it was sensitive and robust for simultaneous quantification of RMP and RTP. The method was successfully applied to analyze RMP and RTP in mouse tissues. In general, the developed method is suitable to monitor RMP and RTP, and provides a useful approach for exploring more detailed effects of remdesivir in treating diseases.

Pró-Fármacos dendriméricos potencialmente ativos em Malária e Tuberculose: Síntese e avaliação da atividade biológica / Dendrimeric prodrugs potentially active in malaria and tuberculosis: Synthesis and evaluation of biological activity

HIV/AIDS, tuberculose, malária e as doenças tropicais negligenciadas representam uma grande preocupação em Saúde em muitas regiões do mundo. Os fármacos disponíveis para o tratamento apresentam diversos problemas, tais como toxicidade e resistência ao parasita. Mesmo com esse triste panorama, o investimento em pesquisa nessa área é, ainda, pouco significativo. Assim, dentre os métodos de modificação molecular para melhorar propriedades farmacêuticas, farmacocinéticas e/ou farmacodinâmica de compostos bioativos destaca-se a latenciação. Já os dendrímeros vêm despertando interesse em aplicações biológicas, principalmente como transportadores de fármacos, além de atuarem como transportadores de genes, imagem em diagnóstico e compostos com ação per se. Face ao exposto e tendo em vista o caráter promissor dos dendrímeros como sistemas de drug delivery, o objetivo deste trabalho foi a síntese de pró-fármacos dendriméricos potencialmente ativos em malária e tuberculose. Os dendrímeros de Bis-MPA (gerações 0, 1 e 2) foram sintetizados pelo grupo do Professor Scott Grayson, da Tulane University (EUA). No Brasil, foram feitas as funcionalizações destes compostos, através do acoplamento do ácido succínico (que funciona como espaçante) e as moléculas ativas. Selecionaram-se as seguintes substâncias: (1) primaquina, com ação antimalárica e (2) isoniazida, de ação nos primeiros estágios da tuberculose. Foram sintetizados os pró-fármacos dendriméricos de isoniazida nas gerações 0 e 1 (G0-Iso e G1-Iso), e primaquina nas gerações 0, 1 e 2 (G0-Pq, G1-Pq e G2Pq). Importante mencionar que os resultados de Ressonância Magnética e Nuclear de 1H e de 13C demostraram as obtenções dos respectivos produtos, porém contendo impurezas. Já a análise do resultado proveniente da espectrometria de massas do composto G0-Iso revelou a presença de um subproduto ciclizado da isonizaida succinoilada (CIso-Suc), o qual pode ser um potencial pró-fármaco ou apresentar atividade per se. Como não se conhece este composto, o laboratório coordenado pela Profas Elizabeth Igne Ferreira e Jeanine Giarolla manifestou interesse em pesquisa-lo, principalmente quanto suas propriedades físico- químicas, bem como quanto à atividade biológica. Assim, utilizando metodologia analítica previamente estabelecida para o G0-Iso, os estudos de estabilidade química da CIso-Suc, em diferentes valores de pH, demonstraram a capacidade da forma ciclizada em se converter no protótipo Iso-Suc, majoritariamente em pH 7,4 e 8,5. Como perspectivas, destaca-se a avaliação da estabilidade enzimática deste potencial derivado. Ressalta-se, ainda, a a avaliação da respectiva atividade antimicobacteriana. Em relação aos pró-fármacos, as necessidades de aprimoramentos das sínteses são, também, evidenciadas. Uma vez sintetizados e caracterizados, estes últimos derivados serão avaliados quanto à atividade biológica. Ademais, estudos computacionais, sobretudo simulações de docking molecular, foram desenvolvidos com intuito de se entender o modo de interação de alguns compostos com alvos biológicos pré-determinados

HIV/AIDS, tuberculosis, malaria and neglected diseases are a major health concern in many regions of the world. The drugs available present various problems, such as toxicity and parasite resistance. Even with this sad outlook, research investment in this area is still insignificant. Among the molecular modification methods to improve the pharmaceutical, pharmacokinetic and/or pharmacodynamic properties we stands out prodrug design. On the other hand, dendrimers are arousing interest in biological applications, mainly as drug carriers, besides gene delivery, diagnostic imaging, as well as acting as compounds with activity per se. Considering that, added to the promising dendrimer drug delivery features, the aim of this study was to synthesize potentially active dendrimer prodrugs in malaria and tuberculosis. Bis-MPA dendrimers (generations 0, 1 and 2) were synthesized by the group of Professor Scott Grayson of Tulane University (USA). Herein in Brazil, the compounds were functionalized by coupling succinic acid (spacer group), as well as the active molecules. We selected the following substances: (1) primaquine, with antimalarial action and (2) isoniazid, acting in the early stages of tuberculosis. Isoniazid dendrimer prodrugs were synthesized generations 0 and 1 (G0-Iso and G1-Iso), and primaquine in generations 0, 1 and 2 (G0-Pq, G1-Pq and G2-Pq). It is important to mention that the results related to Nuclear and Magnetic Resonance 113C showed chemical structures features, however with impurities. Analysis of the mass spectrometry regarding G0-Iso has revealed the presence of a cyclized by-product of succinylated isonized (CIso-Suc), which may be a potential prodrug or may presentactivity itself. Using the analytical methodology performed for G0-Iso, ICso-Suc demonstrated its ability to convert the Iso-Suc prototype at different pH values, especially at pH 7.4 and 8.5. As perspectives, we highlight the determinations of the chemical stability of ICsoSuc at pH 1.5 and 6.0, as well as the evaluation of the enzymatic stability. We will also investigate the respective antimicobacterial activities. Regarding prodrugs, the needs for synthesis enhancements are also necessary. Once synthesized and characterized, these latter derivatives will be evaluated for biological activity. Moreover, computational studies, especially molecular docking simulations, were developed in order to understand the mode of interaction of some compounds with predetermined biological targets

Blood-brain barrier disruption and delivery of irinotecan in a rat model using a clinical transcranial MRI-guided focused ultrasound system.

We investigated controlled blood-brain barrier (BBB) disruption using a low-frequency clinical transcranial MRI-guided focused ultrasound (TcMRgFUS) device and evaluated enhanced delivery of irinotecan chemotherapy to the brain and a rat glioma model. Animals received three weekly sessions of FUS, FUS and 10 mg/kg irinotecan, or irinotecan alone. In each session, four volumetric sonications targeted 36 locations in one hemisphere. With feedback control based on recordings of acoustic emissions, 98% of the sonication targets (1045/1071) reached a pre-defined level of acoustic emission, while the probability of wideband emission (a signature for inertial cavitation) was than 1%. BBB disruption, evaluated by mapping the R1 relaxation rate after administration of an MRI contrast agent, was significantly higher in the sonicated hemisphere (P < 0.01). Histological evaluation found minimal tissue effects. Irinotecan concentrations in the brain were significantly higher (P < 0.001) with BBB disruption, but SN-38 was only detected in <50% of the samples and only with an excessive irinotecan dose. Irinotecan with BBB disruption did not impede tumor growth or increase survival. Overall these results demonstrate safe and controlled BBB disruption with a low-frequency clinical TcMRgFUS device. While irinotecan delivery to the brain was not neurotoxic, it did not improve outcomes in the F98 glioma model.

Characterization of chemical profiles of pH-sensitive cleavable D-gluconhydroximo-1, 5-lactam hydrolysates by LC-MS: A potential agent for promoting tumor-targeted drug delivery.

Currently, controllable linker cleavage at the target site will facilitate the clinical treatment of cancer. Dual-functional prodrugs in combination of carbohydrate as targeting group and pH-sensitive cleavable linker are desired in clinical development. Here, a qualified structure of N-phenylcarbamate-d-gluconhydroximo-1,5-lactam was employed and proved to be a potential candidate prodrug in the drug design. To proof this concept, the possible mechanism of Beckmann rearrangement and the degraded products were confirmed by HPLC and LC-MS under the acid condition mimic lysosome. Hence, the strategy of d-gluconhydroximo-1,5-lactam as a prodrug carrier fabricated with interested drugs will provide a great potential approach for chemotherapy.

Review on Characteristics and Analytical Methods of Tazarotene: An Update.

Tazarotene (TZR) is the first topical receptor-selective retinoid prodrug derived from vitamin A used for management of plaque psoriasis and efficacious in dealing of acne vulgaris, and photo aging. As per US food and drug administration (FDA), 0.1% strength of TZR is permitted for the treatment of acne. This article draws attention to various advanced and conventional analytical methods. The hyphenated and conventional chromatographic techniques such as LC-MS/MS and HPTLC, HPLC respectively. Moreover, spectrophotometric methods like UV/visible spectroscopy also used to quantify TZR as active pharmaceutical ingredient and its formulations, especially in topical preparations. Moreover, the TZR is required alternative methods for routine quality control and to estimate TZR in pharmaceutical dosage form especially in pharmacokinetic studies of topical preparation. This write up focus on critical review of characteristics, uses and the information about the physicochemical, pharmacokinetics properties, mechanisms of action and more emphasis on different analytical methods for estimation of TZR in pharmaceutical formulations.

Analytical method considerations regarding carryover for monophosphate prodrugs for in vivo samples by liquid chromatography-tandem mass spectrometry.

Three different components that impact carryover in a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were evaluated to establish baseline conditions for analyzing in vivo samples for twelve monophosphate prodrug compounds and their corresponding parent compounds. The three components were: wash solvent modifier, column shell material (metal vs. metal free), and tubing composition. These components were tested for their impact on system carryover by using rat plasma extracted samples. It was determined that a wash solution containing hexylamine yielded the lowest average carryover of the solutions tested. In addition, metal free columns and PEEK (poly ether ether ketone) tubing yielded the lowest carryover when compared to metal columns, stainless steel tubing and nickel tubing. These conditions were also tested against the parent molecules for each prodrug in the test set, to ensure that changing the conditions for the prodrugs did not impact the ability to analyze the parent, since there is typically a desire to measure both compounds in study samples. Under all conditions, the carryover of the corresponding parent molecule was not adversely impacted in these studies.

Total drug quantification in prodrugs using an automated elemental analyzer.

Polymeric prodrugs have become an increasingly popular strategy for improving the pharmacokinetic properties of active pharmaceutical ingredients (API). Therefore, identifying a robust method for quantification of the API in these prodrug products is a key part of the drug development process. Current drug quantification methods include hydrolysis followed by reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC)-based molecular weight determination, and mass spectrometry. These methods tend to be time-consuming and often require challenging method development. Here, we present a comparative study highlighting the automated elemental analyzer as a facile approach to drug quantification in this up-and-coming class of therapeutics. A polymeric prodrug using poly(L-lysine succinylated) (PLS) and the drug lamivudine (LAM) was prepared and analyzed using the elemental analyzer in comparison to the traditional approaches of hydrolysis followed by RP-HPLC and SEC using multi-angle light scattering (MALS) detection. The elemental analysis approach showed excellent agreement with the conventional methods but proved much less laborious, highlighting this as a rapid and sensitive analytical method for the quantitative determination of drug loading in polymeric prodrug products.

Analytical methodologies used for the determination of colistin in biological fluids. Is it still a challenge?

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested.

The importance of evaluating the chemical structures and strategies to avoid pitfalls in quantitative bioanalysis.

Quantitative bioanalytical data are crucial in pharmaceutical research and development, allowing project teams to make informed scientific decisions on the progression of candidate molecules to medicines. Many challenges are often encountered during the bioanalysis of drugs in biological matrices which require resolution in a timely manner. In this publication, guidance is provided to bioanalytical scientists on how to identify potential problems before they become an obstacle for the drug development and to share our experiences dealing some of most common problems encountered in the bioanalytical laboratory. Relevant topics in bioanalysis such as stabilization approaches for glucuronides (Acyl and N-); prodrugs (phosphate and esters), amides, amines, N-oxides; bioanalysis of light sensitive molecules, halogenated drugs and lactones are discussed in this publication.

Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for simultaneous quantifications of CZ48, lactone-stabilized camptothecin, and camptothecin and their pharmacokinetic and biliary evaluations in rats.

CZ48, a prodrug of camptothecin (CPT), has a broad spectrum of antitumor activity against various types of human tumors without severe toxicity in preclinical human tumor-xenografted mouse models, which facilitates further preclinical and clinical pharmacokinetic (PK) evaluations of CZ48. In this study, a UHPLC-MS/MS method was developed and validated to simultaneously quantify CZ48 and CPT in rat plasma and bile. Detection was performed using the API 3200 Q Trap triple quadrupole mass spectrometer in a positive ion mode. Chromatographic separation was achieved on Waters ACQUITY UPLC BEH Shield RP18 column with a gradient elution at a flow rate of 0.45 ml/min, using mobile phases of 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). The method was linear at the concentration ranges of 0.98 (LLOQ) -1000 ng/ml of CZ48 and CPT in rat plasma and 3.9 (LLOQ) -1000 ng/ml in bile. Intra- and inter-day accuracy and precision values did not deviate by more than 6.57% and 10.15% for CZ48 and CPT, respectively, in plasma, and 12.09% and 13.48% in bile. Extraction recoveries of CZ48 were 90.18-95.42% from plasma and 86.51 -91.66% from bile. The recoveries of CPT were 91.56-97.06% from plasma and 84.89-89.15% from bile. No significant matrix effects were observed in plasma and bile within 14.00% and 16.19%, respectively. CZ48 and CPT in plasma were stable after extraction process and different storage conditions, including bench-top, processed sample in autosampler, three cycles of freeze and thaw, and long-term (3 month) stability at -80 °C. The application of the validated method was demonstrated by a PK study after an intravenous dose of CZ48 in rats.

A sensitive, high-throughput, and eco-friendly analysis of daidzein and its valine carbamate prodrug in rat plasma by supercritical fluid chromatography with tandem mass spectrometry.

A valine carbamate prodrug of daidzein was synthesized to improve its bioavailability because of the poor solubility and low permeability of daidzein. To evaluate the pharmacokinetic behavior of the prodrug, a sensitive and high-throughput method was developed and validated for the simultaneous determination of daidzein and its prodrug in rat plasma. The samples were extracted by ethyl acetate and then analyzed by a supercritical fluid chromatography with electrospray ionization tandem mass spectrometry method. The separation was achieved by an ACQUITY UPC2 ™ BEH 2-EP column maintained at 40°C using carbon dioxide (≥99.99%) and methanol within 3.0 min by gradient elution. The mass transition ion pairs were m/z 254.8→136.7, 398.0→254.9, and 271.0→91.07 for daidzein, the prodrug, and genistein, respectively. The calibration curves were linear over the concentration ranges of 2-500 (r > 0.997) and 10.0-5000.0 ng/mL (r > 0.996) with lower limits of quantification of 2 and 10 ng/mL for daidzein and the prodrug, respectively. The intra- and interday accuracy and precision were within ±15% for all quality control samples. This developed method enabled high specificity, low cost, low solvent consumption, and a brief analysis time and was successfully applied to a bioavailability evaluation of daidzein and its carbamate prodrug.

Quantification of triacontanol and its PEGylated prodrug in rat plasma by GC-MS/MS: Application to a pre-clinical pharmacokinetic study.

PEGylation techniques have been increasingly employed in drug delivery system and chemical modification of compounds with low aqueous solubility. Triacontanol (TA) is a natural product with several pharmacological activities, but its low aqueous solubility significantly limited its application. PEGylated triacontanol (PEG-TA) was designed as the prodrug to improve the aqueous solubility and pharmacokinetic properties of TA. On the basis of salting-out assisted liquid-liquid extraction (SALLE) and saponification sample preparation procedure, a reliable gas chromatography tandem mass spectrometric (GC-MS/MS) method was developed and validated for the quantification of PEG-TA and its metabolite TA in rat plasma after separation and transformation. Acetonitrile-methanol (9:1, v/v) and ammonium acetate (10 M) were utilized to separate PEG-TA and TA (including conjugated TA with fatty acid). Saponification facilitated the complete conversion of PEG-TA into TA, so PEG-TA could be indirectly quantified. The results revealed that the GC-MS/MS method had excellent selectivity, accuracy and linearity. Calibration curves were linear (R2>0.99) within the range of 20.0-1000.0 ng/mL for TA and 100.0-10,000.0 ng/mL for PEG-TA. The intra- and inter-day precision of quality control samples were within 15%, and their accuracy values varied from 93.54% to 113.38%. This analytical method has been successfully applied to pharmacokinetic study of PEG-TA. This study can facilitate the further exploration and quantification of PEGylated prodrugs.

Simultaneous determination of tapentadol and its carbamate prodrug in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

A prodrug of tapentadol, namely tapentadol carbamate (WWJ01), was synthesized to improve the bioavailability of tapentadol owing to its extensive first-pass metabolism. In this study, a highly rapid and sensitive UPLC-MS/MS method was developed and validated for the simultaneous determination of tapentadol and WWJ01 in rat plasma with fluconazole as an internal standard. The analytes and internal standard were treated by methanol and then separated on a Phenomenex Kinetex® XB-C18 (2.1 × 50 mm × 2.6 µm) column at a flow rate of 0.3 mL/min. The mobile phase comprised methanol and water with a gradient elution. The mass transition ion-pairs were m/z 222.2 → 107.0, m/z 293.2 → 71.9 and m/z 307.1 → 220.0 for tapentadol, WWJ01 and IS, respectively. Excellent linearity was observed over the concentration range of 2-1250 ng/mL (r = 0.995) with a lower limit of quantification of 2 ng/mL for both tapentadol and WWJ01. The intra- and inter-day accuracy and precision for all quality control samples were within ±15%. The validated method was accurate, rapid and reproducible, and was successfully applied to a pharmacokinetic study of tapentadol and WWJ01.

Simultaneous determination of parecoxib and its main metabolites valdecoxib and hydroxylated valdecoxib in mouse plasma with a sensitive LC-MS/MS method to elucidate the decreased drug metabolism of tumor bearing mice.

Parecoxib (PX), a prodrug of valdecoxib (VX), is an injectable selective COX-2 inhibitor, and is recommended for the treatment of cancer pain. PX can be rapidly hydrolyzed into its active metabolite VX, and VX is further metabolized into hydroxylated valdecoxib (OH-VX) by cytochrome P450 enzymes. However, cancer patients have been reported to possess reduced drug metabolism ability, which might cause excessive drug accumulation. Such overdose of PX significantly increased the risk of renal safety and cardiovascular events. Therefore, it is necessary to elucidate the concentration profiles of PX and its metabolites in cancer status. In this study, a sensitive, rapid and specific LC-MS/MS method for quantification of PX, VX and OH-VX in the plasma of tumor bearing mouse was developed and validated. After protein precipitation, all the analytes were separated on an Agilent ZORBAX Extend-C18 HPLC column (2.1 × 100 mm, 3.5 µm) with gradient elution. The analytes were detected by an electrospray negative ionization mass spectrometry in the multiple reaction monitoring mode. The transition m/z 369.0 → 119.0, m/z 312.9 → 117.9, m/z 329.0 → 196.0, and m/z 307.1 → 161.3 were used for monitoring PX, VX, OH-VX and IS respectively. The calibration curves of the analytes showed good linearity over the concentration range of 3-3000 ng/mL for PX and VX, and 3-1000 ng/mL for OH-VX. Intra- and inter-batch accuracies (in terms of relative error, RE < 9.9%) and precisions (in terms of relative standard deviation, RSD < 8.8%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. The method was successfully applied to the pharmacokinetics study of PX in tumor bearing mice, and PX and VX levels were found elevated with the growth of tumor volume, which might increase the risk of drug overdose.

Sensitive analysis and pharmacokinetic study of a novel gemcitabine carbamate prodrug and its active metabolite gemcitabine in rats using LC-ESI-MS/MS.

FY363 is a new chemical entity of gemcitabine analog, which has been shown to have a significant inhibitory effect on cell proliferation in a variety of tumor cell lines in vitro. As a carbamate derivative, FY363 would be converted to the active metabolite gemcitabine through enzyme action in vivo. In order to clarify the exposure of FY363 prototype and its metabolite gemcitabine in vivo after administration of FY363, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated to simultaneously determine FY363 and gemcitabine in rat plasma after liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a highly stable polar column of Synergi 4u Polar-RP 80A (4 µm, 4.6 × 250 mm) which has a unique ether - phenyl bonded phase. Gradient elution was accomplished with mobile phase system consisting of 5 mM ammonium formate buffer containing 0.1% formic acid and mixed organic solvents containing methanol-acetonitrile (3:2, v/v). Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r > 0.99) over the established concentration range of 1.0-1000 ng/mL both for FY363 and gemcitabine. The assay was validated to be selective, robust and reproducible. This well validated method was successfully applied to demonstrate the pharmacokinetic behavior and the metabolic transformation of FY363 in rats. Results revealed that about 20% of FY363 were converted into its active metabolite gemcitabine in rats by comparing the exposure of gemcitabine after the FY363 administration with that after direct gemcitabine administration at equimolar dose.

Simultaneous determination of gemcitabine prodrug, gemcitabine and its major metabolite 2', 2'-difluorodeoxyuridine in rat plasma by UFLC-MS/MS.

To improve bioavailability and provide resistance to deamination, an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4-N-carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC-MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid, 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 398.1 → 202.2 (Cbz-dFdC), m/z 264.1 → 112.0 (dFdC), m/z 265.3 → 113.2 (dFdU) and m/z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate. The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC-MS/MS analysis and well-suited to support pharmacokinetic studies.

Quantitation of the anticancer drug abiraterone and its metabolite Δ(4)-abiraterone in human plasma using high-resolution mass spectrometry.

Abiraterone acetate is administered as a prodrug to patients with metastatic, castration-resistant prostate cancer (mCRPC) and is readily metabolized into the potent 17a-hydroxylase/17,20-lyase (CYP17) enzyme inhibitor and androgen receptor inhibitor abiraterone and Δ(4)-abiraterone (D4A), respectively. To investigate pharmacokinetic variability in abiraterone acetate metabolism we developed highly sensitive liquid chromatography/mass spectrometry (LC/MS) assays for the simultaneous quantitation of abiraterone and D4A in human plasma using high-resolution mass spectrometry (HRMS) on an Orbitrap mass spectrometer. This study demonstrates the quantitative performance of HRMS and compares the conventional Parallel Reaction Monitoring (PRM) mode of quantitation with the unconventional Full scan MS mode conducted at high resolution (>70,000 resolution). The use of HRMS for quantitation of abiraterone and D4A yielded assays that were linear over a broad concentration range (0.074-509.6 ng/mL for abiraterone; 0.075-59.93 ng/mL for D4A) in both Full scan MS and PRM modes. The assay precision for abiraterone and D4A was below 5% in PRM mode and 7% in Full scan MS mode. Accuracies fell within 98-107% for abiraterone and 104-112% for D4A in PRM mode, and 96-116% for abiraterone and 96-105% for D4A in Full scan MS mode, each meeting the acceptance criteria of FDA approved guidelines for bioanalytical methods The PRM analysis of abiraterone and D4A provided high specificity and reduced background interference, however the Full scan MS detection at a resolution of 70,000 was advantageous in that it required minimal optimization, was simple to implement, yielded comparable quantitative characteristics to PRM and the data is useful for re-analysis. Use of the assays were demonstrated for quantitation of these metabolites in steady state trough level plasma of seventeen (17) patients with mCRPC, demonstrating the inter-patient variability of up to 10-fold concentration.

Attenuated Total Reflectance Fourier Transformation Infrared spectroscopy fingerprinted online monitoring of the kinetics of circulating Butyrylcholinesterase enzyme during metabolism of bambuterol.

We have described a continuous flow ATR-FTIR method for measuring some of the Butyrylcholinesterase enzyme kinetics (Km and Vmax). This is done by developing a circulating system to be close as much as possible to the human circulation using human serum as a source of the enzyme with adjusted pH, isotonicity and temperature to give the maximum affinity of the enzyme towards its substrate (bambuterol). The experiment was running continuously for 90 min to monitor the production of terbutaline from the zero time of its appearance with a measured spectrum in each minute using ZnSe prism. The method was selective and successful for determination of Vmax to be 8.16 × 10-8 mol/min/ml and Km to be 2.28 × 10-5 mol, showing high affinity of the enzyme towards its prodrug substrate Bambuterol. This study critically probes the quantitative ability of the ATR-FTIR method for terbutaline, which was validated according to ICH guidelines showing high accuracy 100.39% and high selectivity towards the produced terbutaline, as the produced spectrums considered as fingerprint of each compound.