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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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 (R 2 >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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018. Published by Elsevier B.V.)

We have described a continuous flow ATR-FTIR method for measuring some of the Butyrylcholinesterase enzyme kinetics (K m and V max ). 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 V max to be 8.16 × 10 -8  mol/min/ml and K m 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018. Published by Elsevier B.V.)

Naproxen (nap) is belonging to Non-steriodal anti-inflammatory drugs (NSAIDs) group of drugs that characterized by their free carboxylic group. The therapeutic activity of nap is usually accompanied by GI untoward side effects. Recently synthesized naproxen amides of some amino acid esters prodrugs to mask the free carboxylic group were reported. Those prodrugs showed a promising colorectal cancer chemopreventive activity. The current study aims to investigate the fate and hydrolysis of the prodrugs kinetically in different pH conditions, simulated gastric and intestinal fluids with pHs of 1.2, 5.5 and 7.4 in vitro at 37 °C. The effect of enzymes on the hydrolysis of prodrugs was also studied through incubation of these prodrugs at 37 °C in human plasma and rat liver homogenates. The pharmacokinetic parameters of selected prodrugs and the liberated nap were studied after oral and intraperitoneal administration in male wistar rats. The results showed the hydrolysis of naproxen amides of amino acid esters to nap through two steps first by degradation of the ester moiety to form the amide of nap with amino acid and the second was through the degradation of the amide link to liberate nap. The two reactions were followed and studied kinetically where K 1 and K 2 (rate constants of degradation) is reported. The hydrolysis of prodrugs was faster in liver homogenates than in plasma. The relative bioavailability of the liberated nap in vivo was higher in case of prodrug containing ethyl glycinate moiety than that occupied l-valine ethyl ester moiety. Each of nap. prodrugs containing ethyl glycinate and l-valine ethyl ester moieties appears promising in liberating nap, decreasing direct GI side effect and consequently their colorectal cancer chemopreventive activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In recent years, drug conjugates as a prodrug strategy have been widely studied, especially combined with nanotechnology. Disulfide-linked doxorubicin drug-drug conjugate (DOX-S-S-DOX) nanoparticles, have recently been developed as a doxorubicin prodrug nanoparticles with greater anticancer activity and less toxicity than doxorubicin in vivo, while its intracellular kinetics and metabolism is unclear which may provide us with a deeper understanding of its pharmacological mechanism and antitumor effect. Hence, in this study, a rapid and sensitive ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to detect doxorubicin (DOX) activated from DOX-S-S-DOX, as well as the prodrug itself in human breast cancer tumor cells (MCF-7). Sample preparation involved acetonitrile precipitation to extract the analytes simultaneously and bath sonication to remove intercalated DOX from DNA. The calibration range was 3-60ng/mL for DOX and 20-400ng/mL for DOX-S-S-DOX with the correlation coefficients (r 2 )≥0.99, using daunorubicin as internal standard (IS). The inter- and intra-assay precision (relative standard deviation, RSD%) of quality control samples was in the acceptable range (<15%) and relative error (RE%) for accuracy was between -5.35 and 9.18% for all analytes. Recovery (59.28-69.53% for DOX-S-S-DOX and 99.13-100.10% for DOX) and matrix effect (99.69-111.19%) was consistent, precise, and reproducible at different quality control levels in accordance with FDA guidance. Stability studies showed that DOX-S-S-DOX was unstable both during the bench-top and long-term storage, while the stability during sample preparation and LC-MS runtime was suitable for all the analytes. Hence, the samples should be prepared as soon as possible at the time point to prevent the catabolism of DOX-S-S-DOX. The assay was successfully used in the cellular metabolism and pharmacokinetics study of DOX-S-S-DOX and it may give a clue to explore analytical methods of other prodrug forms of DOX., (Copyright © 2017. Published by Elsevier B.V.)

HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro. In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 μM TAF or 1.1 μM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in T-cells of HIV-infected patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We present here a critical review covering conventional analytical tools of recombinant drug analysis and discuss their evolution towards miniaturized systems foreseeing a possible unique recombinant drug-on-a-chip device. Recombinant protein drugs and/or pro-drug analysis require sensitive and reproducible analytical techniques for quality control to ensure safety and efficacy of drugs according to regulatory agencies. The versatility of miniaturized systems combined with their low-cost could become a major trend in recombinant drugs and bioprocess analysis. Miniaturized systems are capable of performing conventional analytical and proteomic tasks, allowing for interfaces with other powerful techniques, such as mass spectrometry. Microdevices can be applied during the different stages of recombinant drug processing, such as gene isolation, DNA amplification, cell culture, protein expression, protein separation, and analysis. In addition, organs-on-chips have appeared as a viable alternative to testing biodrug pharmacokinetics and pharmacodynamics, demonstrating the capabilities of the miniaturized systems. The integration of individual established microfluidic operations and analytical tools in a single device is a challenge to be overcome to achieve a unique recombinant drug-on-a-chip device., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Dabigatran etexilate (DABE) is a low-molecular-weight prodrug that is converted after oral administration to dabigatran (DAB)-a directly acting oral anticoagulant. In this study, an LC-MSMS assay was developed and validated for the determination of DABE, free DAB and its equipotent O-glucuronide conjugates in plasma. Owing to the susceptibility of DABE and DAB to chemical hydrolysis, cleavage of the O-glucuronide moiety was carried out using β-glucuronidase enzyme. Free and total plasma concentrations of DAB were determined in incurred plasma samples before and after enzymatic cleavage (50°C and 3 h), respectively. RP-HPLC separation was carried out using acetonitrile: water (30:70, v/v), adjusted to pH 3.0 using formic acid. Tandem mass spectrometric detection at positive electrospray ionization in the MRM mode was then employed for the determination of DABE and DAB. The analysis was carried out within 5.0 min over a linear concentration range of 1.00-600.00 ng/mL for the prodrug and its active metabolite. Validation was carried out according to FDA guidelines for bioanalytical method. The recoveries were higher than 89.48%, the accuracy was within 98.33-110.12% and the RSD was below 10% for the studied compounds in both incurred plasma and quality control samples. Results of incurred sample re-analysis and incurred sample stability revealed less than 10% variability. This indicated good assay precision and sufficient stability of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for therapeutic drug monitoring and the determination of the pharmacokinetic parameters were demonstrated., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionization (ESI) mode has been validated for the simultaneous determination of lopinavir (LPV) and three prodrugs, succinic acid ester of lopinavir (SLPV), glutaric acid ester of lopinavir (GLPV), and diglycolic acid ester of lopinavir (DLPV) in BeWo cell lysate. A methanolic precipitation was employed after addition of an internal standard ritonavir (RTV), followed by centrifugation, separation of the supernatant liquid, evaporation, and reconstitution. A reversed-phase Phenomenex C18 column was used for separation of lopinavir and three prodrugs with a mobile phase which comprises methanol (solvent A), acetonitrile (solvent B), and 50mM ammonium acetate buffer adjusted to pH 5.5 with acetic acid (solvent C), with isocratic elution (A:B:C=34:34:32, v/v). The detection of target compounds was conducted using multiple reaction monitoring (MRM) with the following transitions of m/z 729→447, 745→563, 743→429, 629→447 and 721→296 to measure SLPV, DLPV, GLPV, LPV and RTV (I.S.), respectively. The calibration ranges were determined using quadratic regression over concentration ranges of 7-743 ng/mL (DLPV), 7.5-745 ng/mL (GLPV), 7-729 ng/mL (SLPV) and 6-189 ng/mL (LPV). The reverse calculated residuals for LPV and these prodrugs were all less than 15% different from nominal (range). The method we established was fully validated for accuracy (≤15% different from nominal) and precision (≤15% RSD). The results proved that the method is accurate and specific, and also this method has been successfully applied to test the uptake of lopinavir and prodrugs into BeWo cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Pomaglumetad methionil (LY2140023) is the prodrug of a novel metabotropic glutamate 2/3 receptor agonist (LY404039) being investigated for the treatment of schizophrenia. Using accelerator mass spectrometry (AMS) and an intravenous (i.v.) radiolabeled tracer approach, the absolute bioavailability of the prodrug and the extent of its conversion to active moiety (LY404039) were estimated at presystemic (intestinal/first pass) and systemic sites after simultaneous oral and i.v. dosing in healthy subjects. The mean absolute bioavailability of prodrug (80 mg oral) was 0.68. On the basis of these data and a previous radiolabeled mass balance study in which no prodrug was recovered in feces, we concluded that 0.32 of the dose is converted to active drug in the intestinal tract. The fraction of prodrug converted to active moiety was approximately 1, indicating complete conversion of the prodrug that reaches the systemic circulation to the active moiety. Prodrug (80 mg oral and 100 μg i.v.) and active moiety (100 μg i.v.) were well tolerated in healthy subjects. Thus, the absolute bioavailability of prodrug LY2140023 and the fraction converted presystemically and systemically to active moiety LY404039 were estimated simultaneously using radiolabeled tracer microdosing and AMS., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Environmental markers of chemical warfare agents (CWAs) comprise millions of chemical structures. The simultaneous detection and identification of these environmental markers poses difficulty due to their diverse chemical properties. In this work, by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF), a generic analytical method for the detection and identification of wide range of environmental markers of CWAs (including precursors, degradation and co-products of nerve agents and sesqui-mustards) in drinking water, was developed. The chromatographic analysis of 55 environmental markers of CWAs including isomeric and isobaric compounds was accomplished within 20 min, using 1.8 μm particle size column. Subsequent identification of the compounds was achieved by the accurate mass measurement of either protonated molecule [M+H](+) or ammonium adduct [M+NH4](+) and fragment ions. Isomeric and isobaric compounds were distinguished by chromatographic retention time, characteristic fragment ions generated by both in-source collision induced dissociation (CID) and CID in the collision cell by MS/MS experiments. The exact mass measurement errors for all ions were observed less than 3 ppm with internal calibration. The method limits of detection (LODs) and limits of quantification (LOQs) were determined in drinking water and found to be 1-50 ng mL(-1) and 5-125 ng mL(-1), respectively. Applicability of the proposed method was proved by determining the environmental markers of CWAs in aqueous samples provided by Organization for the Prohibition of Chemical Weapons during 34th official proficiency test., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Colistin is a last resort's antibacterial treatment in critically ill patients with multi-drug resistant Gram-negative infections. As appropriate colistin exposure is the key for maximizing efficacy while minimizing toxicity, individualized dosing optimization guided by therapeutic drug monitoring is a top clinical priority. Objective of the present work was to develop a rapid and robust HPLC-MS/MS assay for quantification of colistin plasma concentrations. This novel methodology validated according to international standards simultaneously quantifies the microbiologically active compounds colistin A and B, plus the pro-drug colistin methanesulfonate (colistimethate, CMS). 96-well micro-Elution SPE on Oasis Hydrophilic-Lipophilic-Balanced (HLB) followed by direct analysis by Hydrophilic Interaction Liquid Chromatography (HILIC) with Ethylene Bridged Hybrid--BEH--Amide phase column coupled to tandem mass spectrometry allows a high-throughput with no significant matrix effect. The technique is highly sensitive (limit of quantification 0.014 and 0.006 μg/mL for colistin A and B), precise (intra-/inter-assay CV 0.6-8.4%) and accurate (intra-/inter-assay deviation from nominal concentrations -4.4 to +6.3%) over the clinically relevant analytical range 0.05-20 μg/mL. Colistin A and B in plasma and whole blood samples are reliably quantified over 48 h at room temperature and at +4°C (<6% deviation from nominal values) and after three freeze-thaw cycles. Colistimethate acidic hydrolysis (1M H2SO4) to colistin A and B in plasma was completed in vitro after 15 min of sonication while the pro-drug hydrolyzed spontaneously in plasma ex vivo after 4 h at room temperature: this information is of utmost importance for interpretation of analytical results. Quantification is precise and accurate when using serum, citrated or EDTA plasma as biological matrix, while use of heparin plasma is not appropriate. This new analytical technique providing optimized quantification in real-life conditions of the microbiologically active compounds colistin A and B offers a highly efficient tool for routine therapeutic drug monitoring aimed at individualizing drug dosing against life-threatening infections., (Copyright © 2014 Elsevier B.V. All rights reserved.)

For the first time a high performance liquid chromatography method with tandem mass spectrometry detection (HPLC-MS/MS) was developed for simultaneous determination of a pro-drug treosulfan (TREO) and its active monoepoxide (S,S-EBDM) in biological matrices. Small volumes of rat plasma (50 μL) and the brain homogenate supernatant (100 μL), equivalent to 0.02 g of brain tissue, were required for the analysis. Protein-free TREO, S,S-EBDM and acetaminophen, internal standard (IS), were isolated from the samples by ultrafiltration. Complete resolution of the analytes and the IS was accomplished on Zorbax Eclipse column using an isocratic elution with a mobile phase composed of ammonium formate - formic acid buffer pH 4.0 and acetonitrile. Detection was performed on a triple-quadrupole MS via multiple-reaction-monitoring following electrospray ionization. The developed method was fully validated according to the current guidelines of the European Medicines Agency. Calibration curves were linear in ranges: TREO 0.2-5720 μM and S,S-EBDM 0.9-175 μM for plasma, and TREO 0.2-29 μM and S,S-EBDM 0.4-44 μM for the brain homogenate supernatant. The limits of quantitation of TREO and S,S-EBDM in the studied matrices were much lower in comparison to the previously used bioanalytical methods. The HPLC-MS/MS method was adequately precise (coefficient of variation≤12.2%), accurate (relative error≤8.6%), and provided no carry-over, acceptable matrix effect as well as dilution integrity. The analytes were stable in acidified plasma and the brain homogenate supernatant samples for 4 h at room temperature, for 4 months at-80°C as well as within two cycles of freezing and thawing, and demonstrated 18-24h autosampler stability. The validated method enabled determination of low concentrations of TREO and S,S-EBDM in incurred brain samples of the rats treated with TREO, which constitutes a novel bioanalytical application., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A simple and sensitive HPLC-MS/MS method was developed and validated for the simultaneous determination of decitabine and valdecitabine in rat plasma. The analytes were separated on a C(18) column (150mm×4.6mm, 3.5μm) and a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was applied for detection. A clean solid-phase extraction procedure with cation exchange cartridge was employed to extract the analytes from rat plasma with high recovery of decitabine (>82%). The calibration curves were linear over a concentration range of 10-10,000ng/mL for decitabine and 5-500ng/mL for valdecitabine. The lower limit of quantitation (LLOQ) of decitabine and valdecitabine was 10 and 5ng/mL, respectively. The intra-day and inter-day precisions were less than 15% and the relative error (RE) was all within ±15%. The validated method was successfully applied to a pharmacokinetics study in rats after either decitabine or valdecitabine orally administrated to the Sprague-Dawley rats., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Validated methods for the determination of CA1, the active agent derived from the prodrug CA1P, in human plasma and urine, and of CA1P and three glucuronides CA1G1, CA1G2 and CA1DG in human urine were developed using LC-MS. Plasma CA1 was extracted using solid phase extraction and validated over the range 5-1000 nM. Urine samples were analysed without extraction, and the assays validated over the range 50-2000 nM (CA1P), 25-2000 nM (CA1), 50-40,000 nM (CA1G1 and CA1G2) and 25-4000 nM (CA1DG). The mean correlation coefficient (r²) was ≥ 0.997 for all assays. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical methods (<15%). Mean recovery of CA1 from plasma was 101%, and 97% from urine. Mean urine recovery of CA1P was 98%, CA1G1 96%, CA1G2 93% and CA1DG 93%. The method was applied to plasma and urine samples from a recently completed clinical trial of the prodrug. Peak plasma concentrations of up to 470 nM CA1 were seen. The majority of drug-related material measured in urine comprised of the two monoglucuronides; CA1 and the diglucuronide were about 10-fold lower. No CA1P was detectable in urine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cefpodoxime proxetil (CFP), an oral third-generation cephalosporin, is a prodrug that is de-esterified in vivo to its active metabolite, cefpodoxime acid (CFA). Therefore, this study aimed to develop a facile and efficient one-pot reaction for selective and sensitive determination of CFA and its prodrug (CFP). The method was based on single-step reaction between CFP or CFA and 1,2-naphthoquinone-4-sulfonate (NQS) as a selective derivatizing reagent in alkaline medium without heating, extraction or reduction steps as usual for NQS derivatization reactions. The fluorescence of the formed NQS-derivative was monitored directly at emission wavelength of 440 nm after excitation at 330 nm. The method can easily be implemented in plating facilities by operators and/or incorporated in on-line derivatization reaction. The correlation coefficients of 0.9991 and 0.9984 were obtained in the concentration ranges of 50-2000 ng mL(-1) for CFA and CFP, respectively. The detection limits were 9.17 and 9.48 ng mL(-1) for CFA and CFP, respectively. The method was validated in accordance with the requirements of ICH guidelines and shown to be suitable for their efficient and sensitive determinations. The developed method was successfully applied for selective determination of CFP in pure form and in pharmaceutical dosage forms as well as CFA in human urine after single dose of CFP without prior need for separation. The method is valuable for quality control laboratories for monitoring of CFP and its active metabolite CFA., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Simmitecan (L-P) is an anticancer ester prodrug, which involves activation to chimmitecan (L-2-Z). In the current study, a liquid chromatography/tandem mass spectrometry-based method was developed for simultaneous determination of L-P and L-2-Z in various plasma samples. Because L-P is rapidly converted to L-2-Z by blood carboxylesterase during and after sampling, which hampers accurate determination of L-P and L-2-Z in the biological samples, different carboxylesterase inhibitors were tested. As a result, bis(4-nitrophenyl)phosphate gave the best results with respect to inhibitory capability, hemolysis, and matrix effects and was used to deactivate blood carboxylesterases when sampling. The plasma samples were precipitated with acetonitrile and the resulting supernatants were separated using a pulse gradient method on a C18 column. Irinotecan and camptothecin were used as internal standards for quantification of L-P and L-2-Z, respectively. Protonated L-P, L-2-Z and their internal standards were generated by electrospray ionization and their precursor-product ion pairs (m/z 599→124, 405→361, 587→195, and 349→305, respectively) were used for measurement. The newly developed bioanalytical assay processed favorable accuracy and precision with lower limits of quantification of 2.1 nM for L-P and 3.4 nM for L-2-Z, and was successfully applied to pharmacokinetic studies in tumor-bearing nude mice, rats, and dogs. There are substantial species differences in the ester activity. The experimental strategies illustrated in our report may be adopted for measurement of other prodrugs (including irinotecan) or drugs subject to ester hydrolysis, as well as their metabolites, in biological matrices., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The partitioning behavior of a series of perhydrocarbon nicotinic acid esters (nicotinates) between aqueous solution and dipalmitoylphosphatidylcholine (DPPC) membrane bilayers is investigated as a function of increasing alkyl chain length. The hydrocarbon nicotinates represent putative prodrugs, derivatives of the polar drug nicotinic acid, whose functionalization provides the hydrophobic character necessary for pulmonary delivery in a hydrophobic, fluorocarbon solvent, such as perfluorooctyl bromide. Independent techniques of differential scanning calorimetry and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy measurements are used to analyze the thermotropic phase behavior and lipid bilayer fluidity as a function of nicotinate concentration. At increasing concentrations of nicotinates over the DPPC mole fraction range examined (X(DPPC)=0.6-1.0), all the nicotinates (ethyl (C2H5); butyl (C4H9); hexyl (C6H13); and octyl (C8H17)) partition into the lipid bilayer at sufficient levels to eliminate the pretransition, and decrease and broaden the gel to fluid phase transition temperature. The concentration at which these effects occur is chain length-dependent; the shortest chain nicotinate, C2H5, elicits the least dramatic response. Similarly, the DPH anisotropy results demonstrate an alteration of the bilayer organization in the liposomes as a consequence of the chain length-dependent partitioning of the nicotinates into DPPC bilayers. The membrane partition coefficients (logarithm values), determined from the depressed bilayer phase transition temperatures, increase from 2.18 for C2H5 to 5.25 for C8H17. The DPPC membrane/water partitioning of the perhydrocarbon nicotinate series correlates with trends in the octanol/water partitioning of these solutes, suggesting that their incorporation into the bilayer is driven by increasing hydrophobicity., (Copyright 2010 Elsevier B.V. All rights reserved.)

A rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2-20,000 ng/mL for didanosine and 4-300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC-MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague-Dawley rats., (2009 Elsevier B.V. All rights reserved.)

Numerous drugs are carboxylic acid derivatives containing amino group, and hydrolysis reaction of these agents often generates toxic amines. Thus, the detection of amine impurity is of great importance in drug quality control of these amino group-containing ester and amide. A capillary electrophoresis method coupled with end-column electrochemiluminescent detection based on tris(2,2'-bipyridyl)ruthenium(II) system was proposed for the analysis of N,N-dimethyl ethanolamine (DMEA, the degradation product of meclophenoxate) in the presence of its precursor. Baseline separation of DMEA and meclophenoxate can be easily achieved under the selected conditions. DMEA can be assayed within 3 min over the concentration range of 5.0x10(-8) to 3.0x10(-6) mol L(-1) with a detection limit of 2.0x10(-8) mol L(-1) at the signal-to-noise ratio of 3. The relative standard deviations of the signal intensity and the migration time were less than 5.3 and 2.5% for a standard sample containing 1.0x10(-7) mol L(-1) DMEA (n=5), respectively. The presented method has been successfully applied for the profiling of DMEA resulting from the hydrolysis of meclophenoxate in commercial formulations. A primary stability investigation of meclophenoxate in aqueous solution was also carried out at different temperatures, and the results showed that the degradation of meclophenoxate accelerated at the higher temperature.

A complete physicochemical characterisation of MDMA and its synthetic precursors MDA, 3,4-methylenedioxybenzaldehyde (piperonal) and 3,4-methylenedioxy-beta-methyl-beta-nitrostyrene was carried out through voltammetric assays and Raman spectroscopy combined with theoretical (DFT) calculations. The former provided important analytical redox data, concluding that the oxidative mechanism of the N-demethylation of MDMA involves the removal of an electron from the amino-nitrogen atom, leading to the formation of a primary amine and an aldehyde. The vibrational spectroscopic experiments enable to afford a rapid and reliable detection of this type of compounds, since they yield characteristic spectral patterns that lead to an unequivocal identification. Moreover, the rational synthesis of the drug of abuse 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") from one of its most relevant precursors 3,4-methylene-dioxyamphetamine (MDA), is reported. In addition, several approaches for the N-methylation of MDA, a limiting synthetic step, were attempted and the overall yields compared.

Introduction: The properties and potential liabilities of drug candidate are investigated in detailed ADME assays and in toxicity studies, where findings are placed in context of exposure to dosed drug and metabolites. The complex nature of biological samples may necessitate work-up procedures prior to high performance liquid chromatography-mass spectrometric (HPLC-MS) analysis of endogenous or xenobiotic compounds. This concept can readily be applied to biological fluids such as blood or urine, but in localized samples such as organs and tissues potentially important spatial, thus anatomical, information is lost during sample preparation as the result of homogenization and extraction procedures. However, the localization of test article or spatial identification of metabolites may be critical to the understanding of the mechanism of target-organ toxicity and its relevance to clinical safety., Methods: Tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) and ion trap mass spectrometry (MS) with higher order mass spectrometric scanning functions was utilized for localization of dosed drug or metabolite in tissue. Laser capture microscopy (LCM) was used to obtain related samples from tissue for analyses by standard MALDI-MS and HPLC-MS., Results: In a toxicology study, rats were administered with a high dosage of a prodrug for 2 weeks. Birefringent microcrystalline material (10-25 microm) was observed in histopathologic formalin-fixed tissue samples. Direct analysis by IMS provided the identity of material in the microcrystals as circulating active drug while maintaining spatial orientation. Complementary data from visual cross-polarized light microscopy as well as standard MALDI-MS and HPLC-MS experiments on LCM samples validated the qualitative results obtained by IMS. Furthermore, the HPLC-MS analysis on the LCM samples afforded a semi-quantitative assessment of the crystalline material in the tissue samples., Discussion: IMS by MALDI ion trap MS proved sensitive, specific, and highly amenable to the image analysis of traditional small molecule drug candidates directly in tissue.

The present work explains the development and validation of a simple and reliable isomer specific liquid chromatographic method for the quantitative determination of cefpodoxime proxetil (CP) in rat in situ intestinal perfusate samples. Chromatography was carried out by reversed-phase technique on a C-18 column with a mobile phase composed of 20 mM ammonium acetate buffer (pH 5.0) and acetonitrile in the ratio of 62:38 pumped at a flow-rate of 1 ml/min. The detection was carried out at 235 nm and a column temperature of 30 degrees C. The method was evaluated for the various validation parameters, such as linearity, accuracy, precision, LOD, LOQ, specificity, selectivity, and sample stability. The results of intra- and inter-day validation (n = 3) showed the method to be efficient and the same was applied in an in situ permeability study conducted for CP in rats.

PROLI/NO is an agent of structure XN(O)==NONa (X = L-prolyl) whose 2-s half-life for nitric oxide (NO) release at physiological pH makes it an excellent prodrug for localizing NO's therapeutic effects at the site of application, but a difficult one to formulate and certify as pure. Despite its extraordinary thermal and hydrolytic instability, however, PROLI/NO could be formulated as an injectable drug by dissolving it in cold 0.1 M sodium hydroxide containing 5% D-mannitol, then quickly ultrafiltering and lyophilizing it in evacuated septum vials. No evidence for decomposition was seen in the contents of these evacuated vials when stored at -20 degrees C over a 140-day observation period, as judged by quantifying NO release in simulated infusate solutions (10 mM carbonate/bicarbonate, pH 10.5). The only hydrolysis products detected were NO, nitrite ion, proline, and N-nitrosoproline, all products of normal human physiological processes.

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the simultaneous determination of buprenorphine and buprenorphine propionate in human and animal blood. Buprenorphine propionate was also proven to be a prodrug of buprenorphine. It was comprised of only a one-step extraction procedure with ethyl acetate and normal-phase chromatography on a Betasil Silica column. The recoveries of buprenorphine and buprenorphine propionate were above 84%. Calibration graphs were linear for buprenorphine over the concentration range 2-1500 ng/ml and for buprenorphine propionate over the concentration range 20-1500 ng/ml with a coefficient of variation, both within- and between-day, or less than 10% at any level. The limits of quantitation of buprenorphine and buprenorphine propionate in human or animal blood were 2.0 and 20 ng/ml, respectively, based on a single-to-noise ratio of 3. The method has been successfully applied to pharmacokinetic studies of buprenorphine and buprenorphine propionate in rabbits. The results demonstrated that buprenorphine propionate was rapidly and totally converted to its parent drug, buprenorphine, following intravenous administration. Buprenorphine propionate is a prodrug of buprenorphine.

3'-Azido-2',3'-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (I), 5'-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (II), 5'-O-valinyl-3'-azido-2',3'-dideoxyuridine hydrochloride (III) and 5'-O-phenylalanyl-3'-azido-2',3'-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 microM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3'-Azido-2',3'-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 microg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25-100 microg/ml for AZDU and prodrugs II, III, and IV and 0.5-100 microg/ml for prodrug I. The limit of quantitation was 0.25 microg/ml for prodrugs II, III and IV and 0.5 microg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.

Two analytical methods were developed for quantitative determination of DADLE (H(2)N-Tyr-D-Ala-Gly-Phe-D-Leu-COOH) and its two cyclic prodrugs in rat plasma. For high-performance liquid chromatography with fluorescence detection (LC-FLU), precolumn derivatization of DADLE was accomplished by labeling the N-terminal amino group with the reagent naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN) to form a highly fluorescent 1-cyanobenz[f]isoindole (CBI) derivative. A multi-dimensional LC system was employed to improve selectivity, and solid-phase extraction (SPE) was used for plasma sample preparation. The cyclic prodrugs were converted to DADLE prior to their derivatization. With fluorescence detection after derivatization, the limit of quantitation (LOQ) was 6 ng ml(-1) for the analysis of DADLE, and good linearity was observed up to 6000 ng ml(-1) in rat plasma. Quantitative analysis of DADLE and its cyclic prodrugs was also performed using liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Chromatographic separation was achieved on a C(18) column using gradient elution in a water-acetonitrile system containing 0.1% (v/v) formic acid. The tandem mass spectrometric analysis was performed in the multiple reaction monitoring mode using internal standardization to improve assay precision and accuracy. For plasma sample pretreatment, acetonitrile was added first to precipitate proteins and SPE was used to minimize matrix effects. Using LC-ESI-MS-MS, the LOQ was 0.5 ng ml(-1) for DADLE and 2 to 5 ng ml(-1) for its prodrugs. Good linearity was observed from the LOQ up to 1000 ng ml(-1) for all compounds. For the analysis of DADLE, both analytical methods showed good precision, accuracy and stability. However, for prodrug analysis, LC-FLU showed some sensitivity and accuracy problems, while the LC-ESI-MS-MS method provided consistent and satisfactory results. In conclusion, LC-ESI-MS-MS is the method of choice for the analysis of DADLE and its cyclic prodrugs in rat plasma samples due to its good selectivity, high sensitivity, and fast analysis. Its application was demonstrated through biodisposition and bioconversion studies of the coumarinic acid-based prodrug after intravenous administration in rats., (Copyright 2002 Elsevier Science B.V.)

Allyl 4,6-O-ethylidene-beta-D-glucopyranoside is an intermediate used in the synthesis of etoposide phosphate, a water-soluble derivative of the antineoplastic drug etoposide. In this paper, a method for the determination of allyl 4,6-O-ethylidene-beta-D-glucopyranoside and related glucopyranosides is described. Samples are analyzed by gradient high-performance liquid chromatography using postcolumn derivatization with cuprammonium hydroxide. The method is used to monitor the ethylidenation reaction of allyl beta-D-glucopyranoside with acetal to form allyl 4,6-O-ethylidene-beta-D-glucopyranoside. Samples are chromatographed on an octadecyl-bonded phase column with aqueous acetonitrile. Triethylamine is added to the mobile phase to accelerate mutarotation and suppress anomeric separations. The column effluent is mixed via a postcolumn tee with cuprammonium hydroxide and monitored with ultraviolet detection at 310 nm.

The major objective of this study was to investigate the effects of beta-cyclodextrin (beta-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the solid-state chemical reactivity of the drug, quinapril, when amorphous samples are prepared by colyophilization of quinapril and each of these beta-CDs. For comparison, a physical mixture with beta-CD and colyophilized mixtures with trehalose and dextran were also prepared and subjected to a similar chemical stability test at 80 degrees C followed by HPLC analysis. Significant inhibition of degradation was observed only for colyophilized miscible mixtures with beta-CD and HP-beta-CD at molar ratios in excess of 1:1. Colyophilized mixtures with trehalose and dextran, shown to have phase separated, and the physical mixture with beta-CD exhibited no inhibiting effects. This suggests that specific molecular complexation is responsible for the significant inhibition by the beta-CDs. The tendency of quinapril to form molecular complexes in solution with the beta-CDs was measured by (1)H solution NMR, by estimating complexation constants from the chemical shift of specific groups on quinapril. Supporting evidence for solid-state complexation was provided by FTIR analysis. DSC and TSC measurements indicated that the beta-CDs do not have high enough glass transition temperatures to reduce reactivity by reducing molecular mobility., (Copyright 2002 Wiley-Liss, Inc.)

Objective: The purpose of this study was to evaluate various oral antimicrobial agent levels in tooth extraction sites., Study Design: The concentration of dental alveolar blood in extraction wounds after the oral administration of talampicillin (500 mg), cefaclor (500 mg), cefteram pivoxil (200 mg), cefuroxime axetil (250 mg), cefdinir (200 mg), and ofloxacin (100 mg) was determined in 338 patients and was assessed on the basis of its antimicrobial activity against Streptococcus isolated in odontogenic infections., Results: The percentage of patients whose concentrations exceeded the minimum inhibitory concentration for 90% of Streptococcus was 62.5% to 100% for talampicillin at 30 to 360 minutes, 0% to 12.5% for cefaclor at 30 to 360 minutes, 18.2% to 100% for cefteram pivoxil at 30 to 480 minutes, 50% to 100% for cefuroxime axetil at 30 to 480 minutes, 0% to 50% for cefdinir at 16 to 290 minutes, and 0% to 40% for ofloxacin at 30 to 480 minutes., Conclusion: These results indicate that talampicillin, cefteram pivoxil, and cefuroxime axetil have minimum inhibitory concentration levels for 90% of Streptococcus in tooth sockets.

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC-MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C18 analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25-1,000 ng/ml (r2 > or = 0.996) with accuracy < or =9.8% and precision < or = 13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1,500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1,300 ng/ml for MPA. This HPLC-MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.

High-performance liquid chromatography with both absorbance and fluorescence detection has been applied to the determination of the potential anti-tumour agent combretastatin A-4 and its phosphate ester in murine and human plasma. The presence of different interfering peaks in the two species makes absorbance detection at 295 nm the method of choice for the mouse, and fluorescence detection (295 nm/390 nm) for human plasma. The calibration was linear over the range studied (0.01-50 microM for combretastatin A-4, 0.02-200 microM for combretastatin A-4 phosphate), with quantitation limits of 0.05 microM for both drugs in the mouse, and 0.05 microM and 0.0125 microM for the phosphate ester and free drug, respectively, in human plasma. The method should be useful for pharmacokinetic studies in the forthcoming Phase I clinical trial of combretastatin A-4 phosphate.

A rapid and sensitive method was developed for the simultaneous determination of the new doxorubicin glucuronide prodrug HMR 1826, the parent drug doxorubicin and its metabolites in human lung tissue samples. Homogenization of frozen tissue samples with the micro-dismembrator was followed by a silver nitrate precipitation step. By removing the exceeding silver ions with sodium chloride further purification steps could be omitted. Compounds were separated by isocratic high-performance liquid chromatography on a LiChrospher 100 RP18 column and a mobile phase consisting of citric acid buffer-acetonitrile-methanol-tetrahydrofuran within 30 min and quantified with fluorescence detection. The method showed good recoveries for all compounds (86-99%) and a linear calibration range of 20 ng/g-80 microg/g for doxorubicin and 1-600 microg/g for HMR 1826.

A new ion-pair HPLC method coupled with evaporative light-scattering detection (ELSD) for the simultaneous determination of clodronate and its partial esters has been developed. The simultaneous chromatographic separation was achieved on a reversed-phase C8 column with a gradient system and butylamine as an ion-pair reagent. This method provides good enough reproducibility and sensitivity for in vitro determinations of clodronate and its ester derivatives. The method is applied for hydrolysis studies of clodronate monoesters which have been described as possible prodrugs of clodronate.

L,L-Ethylene dicysteine (EC) is a ligand used in nuclear medicine for the preparation of 99mTc-L,L-EC, a tracer agent for renal function studies. A capillary zone electrophoretic method is described which allows us to monitor the purity of ethylene dicysteine, for which no other suitable method has been reported to date. A fused-silica capillary (L = 70 cm, l = 62 cm, 75 microns i.d.) was used with 10 mM sodium tetraborate, pH 10.0, as the background electrolyte. The voltage applied was 20 kV and the temperature was 25 degrees C. Benzyl alcohol served as the internal standard. The limit of detection was 0.07% and the limit of quantitation was 0.15%.

One possibility to optimize the therapeutic application of dopaminergic compounds with a catechol function is the reversible protection of this moiety using a prodrug approach. Important features in this respect are a proper chemical stability in the gastrointestinal tract, an adequate release rate after arrival in the blood stream or the possibility to cross the blood-brain barrier. A HPLC method was developed to measure the hydrolysis of prodrugs of dopamine and epinine directly. The method is based on reversed-phase separation followed by post-column ion-pair extraction with a fluorescent counter-ion. The separation of di-isobutyryl esters of dopamine and epinine is obtained within 10 min while the more hydrophobic dopaminergic esters, di-benzoyl and di-pivaloyl dopamine, are retained for 30 min. The precision of the assay measuring 160 ng dibudop and 100 ng ibopamine was 1.2 and 1.0%, respectively. The detection limit of all prodrugs tested was approximately 10 ng.

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C18 column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 microg/ml for plasma and 0.03 to 80 microg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 microg/ml for plasma and from 0.02 to 5.0 microg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.

(-)-beta-D-2-Aminopurine dioxolane (APD), (-)-beta-D-2-amino-6-chloropurine dioxolane (ACPD) and dioxolane guanine (DXG) are nucleoside analogues possessing potent activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) in vitro. APD and ACPD are metabolized in vivo to yield DXG. Reversed-phase HPLC analytical methodologies were developed for the simultaneous determination of APD and DXG, and for ACPD and DXG in monkey serum, urine and cerebrospinal fluid (CSF). 2-Fluoro-2',3'-dideoxyinosine (FDDI) served as the internal standard. The extraction recoveries of the nucleoside analogues from serum samples were similar, averaging approximately 90%. The limit of quantitation of the analytical method for serum samples was 0.1 microg/ml for DXG, and 0.25 microg/ml for APD and ACPD. The intra- and inter-day relative standard deviations for each compound at low, medium and high nucleoside concentrations were less than 9.0%. The accuracy of the assay methods was greater than 90% for prodrugs and parent compound. Similar results were observed with urine and CSF samples. Thus, these methods provide sensitive, accurate and reproducible determination of the prodrugs and parent nucleoside in biological samples.

The objective of this research was to develop a rapid, sensitive and reliable method for the separation of phosphonodipeptide prodrugs and parent compounds to facilitate the evaluation of cell permeation using in vitro cell culture models. Separation was accomplished isocratically within 10.0 min using a C18 (150x4.6 mm I.D., 3 microm) reversed-phase column. The mobile phase consisted of 5 mM tetrahexyl ammonium (ion-pair reagent) in 0.02 M phosphate buffer pH 6.5-acetonitrile (48.5:51.5, v/v). The flow-rate was 1.1 ml/min with detection at 221 nm. The standard curves were linear (r2>0.999) over the concentration range 1-100 microM. The method was reliable and reproducible, with the limit of quantitation being 1 microM (25 ng on column).

In this work we show the potential of using a double stacking procedure based on field enhancement as a means to increase the concentration sensitivity in CZE analysis of human plasma extracted by the supported liquid membrane (SLM) technique. A basic drug, bambuterol, was used as a model substance. The low ionic strength of the SLM extract makes this pretreatment technique compatible with the double stacking sequence. No significant loss of separation performance was observed when 3 microliters of SLM extract was concentrated by the CZE double stacking sequence. Almost no visible difference was seen between the electropherograms after enrichment of a plasma blank and an aqueous blank. Good performance of the whole procedure was demonstrated and detection limits in the low nM range were obtained in spite of the relatively weak UV absorbance of bambuterol. The developed procedure was evaluated for both achiral and chiral separation. In the latter approach chiral selectivity was obtained by adding cyclodextrin to the separation electrolyte.

A sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of a new potent antihypertensive agent, TCV-116, and its two metabolites (M-I and M-II) in human serum or urine. After pre-treatment of the specimens, the analytes were determined using a column switching technique, except for the metabolites in urine which were determined by gradient elution mode HPLC. The quantitation limits for TCV-116, M-I and M-II were all 0.5 ng/ml in serum, and 0.5, 10 and 110 ng/ml in urine, respectively. The methods were applied to clinical trials of TCV-116.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of SK&F 105058 (I), the N-ethyl tricarbamate ester pro-drug of fenoldopam, in dog plasma. Fenoldopam is a selective agonist of peripheral dopaminergic (DA-1) receptors and has been shown to improve blood flow and lower blood pressure. The method involves isolation of I and the internal standard (I.S.) from plasma by solid-phase extraction prior to chromatographic separation on an octyl silica column. Following chromatographic separation, the carbamate esters of I and I.S. were simultaneously hydrolyzed and the liberated alkylamines were derivatized, by mixing the column effluent with an alkaline solution of o-phthaldialdehyde and a thiol, to generate a highly fluorescent isoindole product which was subsequently detected with a fluorometer. Optimization of chromatographic and post-column reaction conditions resulted in an on-column detection limit of 0.4 ng. The recoveries for I and I.S. from plasma were 80.0 +/- 5.0% and 62.0 +/- 4.0%, respectively. The limit of quantification for I using 0.2 ml of plasma was 5 ng/ml. Linear response was observed for concentrations of I ranging from 5 to 2000 ng/ml plasma. The method was suitably specific and sensitive for pharmacokinetic and metabolic studies of I in dogs. The methodology developed should be generally applicable to the determination of carbamate-type pro-drugs in biological media.

Phospholipid micoremulsions have been suggested as a drug-delivery system for hydrophobic compounds. In this study hydrophobicity was achieved by derivatizing with cholesterol. Cholesteryl ibuprofen (3) and cholesteryl flufenamate (4) were synthesized. 3 was isolated as an amorphous, white solid with a melting range of 114-120 degrees C. 4 was isolated as a crystalline, white solid with a melting range of 145-148 degrees C. The proposed structures of 3 and 4 were supported by IR, NMR, MS, and organic microanalysis. Phospholipid:cholesteryl ester microemulsions were prepared by the addition of a 1-propanol solution of the cholesteryl ester, other lipids, and phospholipid to a rapidly mixing KCl/KBr solution. The hydrophobic phase was modified by the addition of cholesteryl oleate or triolein to study the effect of the fluidity of the hydrophobic core on the formation of the microemulsions. The results indicated that a molar ratio of 75:25 and a total lipid concentration of 60 mg/mL consistently gave microemulsions with a mean size of 100-150 nm. In addition, the formation of eutectic mixtures of 3 and 4 with cholesteryl oleate were determined to be 16% (w/w) for 3 and 12% (w/w) for 4; melting points were 35.2 and 45.2 degrees C, respectively. The solubilities of 3 and 4 in triolein were determined to be 13.2% (w/w) and 11.5% (w/w), respectively. Other investigators have shown that if the core of a phospholipid:cholesteryl estermicroemulsion exists in a liquid state at physiologic temperature, the turnover of the cholesteryl esters from these microemulsions occurs at a faster rate.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific method for the determination of erythromycin 2'-ethylsuccinate (EM-ES) in plasma is described. The method involves a liquid-liquid extraction procedure followed by the analysis of extracts using phase-system switching (PSS) continuous-flow fast atom bombardment (CF-FAB) liquid chromatography-mass spectrometry (LC-MS). In PSS EM-ES is enriched after analytical separation on a short trapping column, from which it is desorbed to the LC-MS interface. In this way, favourable mobile phases can be used for the LC separation and for the MS detection. Using the PSS approach a flow-rate reduction from 1.0 ml/min in the LC system to 15 microliters/min going into the mass spectrometer was achieved without splitting. The determination limit for EM-ES was 0.1 microgram/ml.

The antitumour drug 1-beta-D-arabinofuranosylcytosine (ara C) was acylated by means of oleic acid anhydride, resulting in the prodrug N4-oleoyl-ara C. Together with a lipophilic biotin derivative, this lipophilic prodrug was incorporated into the bilayer membrane of unilamellar liposomes prepared by means of the detergent dialysis method. On addition of these biotinylated prodrug-liposomes to an excess of avidin, biotin residues were complexed with avidin. The unreacted avidin was removed by chromatography on the Ultrogel AcA-22 column. The prodrug-liposome-avidin complex was coupled to biotinylated monoclonal antibodies through the free binding sites of the immobilized avidin. Unreacted antibodies were removed by chromatography on an Ultrogel AcA-22 column. In vitro, the liposome-antibody complexes selectively bound to cells which were recognized by the monoclonal antibodies linked to the liposomes. For this reason, a promising strategy towards a specific chemotherapy of cancer is expected.