Development and validation of a high-performance liquid chromatography-mass spectroscopy assay for determination of artesunate and dihydroartemisinin in human plasma.

A sensitive method has been developed and validated for the determination of artesunate and its active metabolite dihydroartemisinin (DHA) in human plasma using artemisinin as an internal standard. Solid phase extraction (SPE) using Oasis HLB extraction cartridges was used for sample preparation and analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ion for artesunate, alpha- and beta-DHA was m/z 221 and for artemisinin was m/z 283. Chromatography was carried out using a Synergi Max-RP, 4 mu, 75 mm x 4.6 mm column using glacial acetic acid 0.1%, acetonitrile and methanol mixture (38:46.5:15.5) as a mobile phase delivered at a flow rate of 0.5 mL/min. The retention times of artesunate, alpha- and beta-DHA and artemisinin were 17.4, 11.8, 18.7 and 13.4 min, respectively, with a total run time of 21 min. The assay was linear over the range 1-3000 ng/mL for artesunate and DHA. The analysis of quality control samples for artesunate 50, 300, 1300 and 2600 ng/mL demonstrated excellent precision with relative standard deviation of 14.3, 11.3, 7.5 and 12.1%, respectively (n=5). Recoveries at concentration of 50, 300, 1300 and 2600 ng/mL were 75, 94.5, 74.3 and 75.5%, respectively; similar results were obtained for precision and recovery of DHA. This liquid chromatography-mass spectroscopy (LC-MS) method for the determination of artesunate and DHA in human plasma has superior specification for sensitivity, sample throughput and robustness than previous methods and can reliably quantitate concentrations of both (artesunate and DHA) compounds as low as 1 ng/mL.

Pharmacokinetic prodrug modeling: in vitro and in vivo kinetics and mechanisms of ancitabine bioconversion to cytarabine.

Conversion rates of the prodrug ancitabine to the antileukemic cytarabine have been measured in vivo (rabbits) and in vitro (in the presence of rabbit blood and human red blood cells, blood, and plasma) using HPLC analyses for the prodrug, drug, and its inactive metabolite, 1-beta-D-arabinosyluracil. These observed pH-dependent in vitro rate constants were consistent with those for chemical hydrolysis determined from controls using Tris buffers. Hydrolysis of ancitabine to cytarabine is chemically, not enzymatically, mediated. The blood concentration-time course for administered compound was described by a two-compartment open model following a rapid intravenous injection of prodrug, drug, or metabolite in each of three rabbits. The in vivo conversion rate constant (kc) following a rapid intravenous prodrug injection was estimated by simultaneous nonlinear regression of ancitabine and cytarabine blood concentration-time courses using equations for two-compartment prodrug and drug with all possible models describing potential conversion sites. The best fit was obtained for the case allowing simultaneous conversion of the prodrug in both central and peripheral compartments to the drug in the central compartment with a common value for kc. The resulting kc value (0.09 h-1, three rabbits) is similar to that for chemical hydrolysis (0.07 h-1) at 38.8 degrees C. Reasons why this agreement is regarded as fortuitous are discussed.

Aqueous conversion kinetics and mechanisms of ancitabine, a prodrug of the antileukemic agent cytarabine.

The kinetics of conversion of the prodrug ancitabine to the anticancer drug cytarabine have been studied in aqueous solutions in the pH range of 1.5-10.7, temperature range of 19.5-80.0 degrees C, ionic strength range of 10(-4) to 1.5, and in the presence of several general-base catalysts. Under all conditions ancitabine was quantitatively converted to cytarabine. The pH-rate profiles were linear with slope = 1 in alkaline pH, becoming pH independent in the region of maximum stability at pH less than or equal to 4, where buffer catalysis was found to be insignificant and kobs approximately equal to (1.12 X 10(11) h-1)-exp [-10121 deg/T]. At 30 degrees C, pH less than or equal to 4, it is calculated that an aqueous ancitabine solution will maintain 90% of its initial concentration for 12 d. A novel method for measuring general-base catalysis in competition with predominating specific-base catalysis and in the presence of secondary salt effects at constant ionic strength was developed. Three mechanisms of hydrolytic prodrug conversion are proposed: nucleophilic hydroxide addition, general base-assisted nucleophilic water attack, and spontaneous water attack.

Theoretical basis for the detection of general-base catalysis in the presence of predominating hydroxide catalysis.

The detection of general-base catalysis in the presence of predominating specific-base catalysis in aqueous buffer solutions is examined for various relationships between k0cat and k0OH, the bimolecular rate constants for general-base and hydroxide-ion attack. The three experimental variables that affect the detection of buffer-base catalysis are the type of buffer, conjugate-acid concentration, and ionic strength. Various buffers used in pharmaceutical kinetic studies are considered, and it is concluded that buffers with high Ka values favor detection. Additionally, high conjugate-acid concentrations and ionic strengths appear to optimize the detection of general-base catalysis.

The kinetics of the alkaline degradation of daptomycin.

The aqueous degradation of daptomycin, a lipopeptide antibiotic, was investigated as a function of substrate concentration (0.5-10.0 mM), pH (9.0-10.5), buffer concentration (0.06-0.20 M borate, glycinate, or carbonate buffers), temperature (20-50 degrees C), and ionic strength (0.1-0.8). The primary degradation pathway was determined by electrospray-mass spectroscopy (ES-MS), Fourier transform infrared (FTIR), and fluorescence spectroscopy to be hydrolysis of the ester linkage between the C-terminus (kynurenine) and the side chain of the fourth residue (threonine). The reaction was first order with respect to time; however, the reaction order with respect to substrate concentration was <1 at substrate concentrations >1 mM. Insignificant buffer effect was observed. The reaction was subject to specific base catalysis. Activation parameters were E(a) = 13.6 kcal/K.mol, DeltaH++ = 13.0 kcal/K.mol, and DeltaS++ = -19.2 eu. The positive primary salt effect was observed with negative deviation at high concentration of salt. The magnitude of the salt effect depended on salt identities in the order sodium < potassium < calcium chloride.

The degradation pathways of glucagon in acidic solutions.

OBJECTIVE: Glucagon is a 29 amino acid peptide hormone that exhibits degradation via both chemical and physical pathways. The objective of the studies reported herein was to identify the degradation products and scheme for glucagon hydrolysis in acidic solutions. METHODS: Solutions of glucagon in 0.01 N HCl (pH 2.5) were degraded at 60 degrees C for 70 h. One isocratic and two gradient RP-HPLC methods were developed to separate the degradation products. Structure elucidation of the separated peaks was achieved using amino acid sequencing, amino acid analysis, and mass spectrometry. Degradation was carried out in the pH range 1.5-5 to check for changes in degradation scheme with pH. Authentic samples of degradation products were degraded under similar acidic conditions to confirm precursor successor relationships in the degradation scheme. RESULTS: Sixteen major degradation products were isolated and identified. The major pathways of degradation were found to be aspartic acid cleavage at positions 9, 15, and 21 and glutaminyl deamidation at positions 3, 20, and 24. Cleavage occurred on both sides of Asp-15 but only on the C-terminal side of Asp-9 and Asp-21. Deamidation of the Asn residue at position 28 was not detected.

Quantitation of taurolidine decomposition in polymer solutions by chromotropic acid formaldehyde assay method.

A sensitive, selective, and specific assay was needed to study the degradation kinetics of taurolidine and its stabilization by polyvinylpyrrolidone (PVP). The purpose of the present study was to evaluate the usefulness of the chromotropic acid method and other formaldehyde or amine derivatization methods. The methods evaluated included formaldehyde derivatization with chromotropic acid, acetylacetone, 4-amino-5-hydrazino-3-mercapto-1,2,4-triazole, semicarbazide hydrochloride, or 2,4-dinitrophenylhydrazine and taurolidine decomposition product derivatization with dansylchloride or 7-chloro-4-nitrobenz-2-oxa-1,3-diazole chloride. Results indicated that the chromotropic acid method provided sufficient selectivity, reproducibility and sensitivity. It was able to quench taurultam decomposition and avoided PVP interference. The method was optimized by performance based selection of reagent lots, appropriate reagent storage and preparation, and controlled derivatization conditions. In conclusion, the optimized chromotropic method was the most appropriate method for quantitating taurolidine decomposition.

Pharmaceutical container/closure integrity. VI: A report on the utility of liquid tracer methods for evaluating the microbial barrier properties of pharmaceutical packaging.

The relationship between a liquid tracer package leak test (Mg ion ingress) and microbial immersion challenge test was demonstrated by direct and indirect correlation techniques. Rubber-stoppered glass vials with micropipette leaks were evaluated by a helium leak rate method, filled with broth, sterilized, and immersed in a bath containing microorganisms (E. coli/B. diminuta) and liquid tracer (Mg ions). After exposure and incubation, each unit was evaluated for liquid tracer ingress by atomic absorption spectrophotometry and for microbial ingress by visual inspection and blood agar streaking. Two hundred and eighty sterile broth-filled test units were challenged with microorganisms and liquid tracer. One hundred and fourteen units showed neither liquid tracer nor microbial ingress. One hundred and eight units were positive for both microbial and liquid tracer ingress. No test units were positive for microbial ingress but not for liquid tracer ingress. Fifty-eight units were positive for liquid tracer ingress but failed to show microbial ingress. Logistical regression was used to demonstrate that the probability of liquid tracer ingress was greater than microbial ingress at all leak sizes. The results indicate that the liquid tracer method studied herein was a useful indicator of the microbial barrier properties of pharmaceutical packaging. Additionally, the results support the contention that liquid penetration of a leak is required for microbial ingress.

Pharmaceutical container/closure integrity. III: Validation of the helium leak rate method for rigid pharmaceutical containers.

Validation of a helium leak rate method for pharmaceutical container/closure integrity quality assurance required the demonstration that this physical testing method was as good or better than microbial immersion challenge testing in detecting potential integrity failures. One lot of rubber-stoppered, broth-filled glass vials also containing defective vials with known leaks were subjected to both helium leak rate and microbial challenge testing. The defective vials were prepared by affixing glass micropipettes (0.1 to 10 microns) into the vial side walls. The validation lot included a 10% seeded defect rate of which about 50% contained leaks with a predicted probability of failing a microbial challenge (> 10%). Helium tracer was placed in the test units by charging them for 4 hours under a 40 psi helium pressure. The critical leak rate after charging was determined to be 10(-7) standard cc/second, and test units with measured leak rates greater than this value were considered helium leak rate failures. Microbial immersion challenge was conducted by exposing the test units in a bath inoculated with 10(9-10) viable E. coli and B. diminuta organisms for 24 hours followed by a 13 day (35 degrees C) incubation. Microbial failures were determined visually. The helium and microbial leak test methods were compared statistically using mean failure rates. The mean helium failure rate was 6.9%, whereas the mean microbial failure rate was 2.8%. The difference between helium and microbial failure rates was significantly greater than zero. Thus, helium leak rate testing was demonstrated to be a suitable pharmaceutical container/closure integrity method for microbial quality assurance of rigid containers.

Pharmaceutical container/closure integrity. I: Mass spectrometry-based helium leak rate detection for rubber-stoppered glass vials.

The development of mass spectrometry-based leak detection for pharmaceutical container integrity was undertaken to provide an alternative to microbial challenge testing. Standard 10-mL vials were modified to contain pinholes (0.5 to 10 microns) by affixing micropipettes with epoxy into 2-mm vial side wall holes. The absolute leak rate was determined using vials that were sealed in a tracer (helium) environment with butyl rubber stoppers and crimps. Alternatively leak rates were determined using vials that were sealed in room air and exposed to tracer under pressure (charging or bombing). Tracer leak rates were measured with mass spectrometry leak rate detectors. The absolute leak rate was correlated the squared nominal leak radius which suggested that the mode of gas flow through the glass pipette leaks was more turbulent than viscous even at low leak rates typically associated with viscous flow. The minimum observed absolute leak rate was about 10(-6.6) std cc/sec and was likely due to helium permeation through the rubber stoppers. Heat-stressed rubber stoppers did not affect the baseline absolute leak rate. Adsorption of helium tracer to the test unit surfaces was found to confound baseline leak rate measurement reliability but was eliminated as a source of variation by exposing the test units to ambient air for > or = 12 hours. The absolute leak rate and the leak rate measured after charging were related in a mathematically predictable way.

Pharmaceutical container/closure integrity. II: The relationship between microbial ingress and helium leak rates in rubber-stoppered glass vials.

Helium leak rate measurements were quantitatively correlated to the probability of microbial ingress for rubber-stoppered glass vials subjected to immersion challenge. Standard 10-mL tubing glass vials were modified by inserting micropipettes of various sizes (0.1 to 10 microns nominal diameter) into a side wall hole and securing them with epoxy. Butyl rubber closures and aluminum crimps were used to seal the vials. The test units were sealed in a helium-filled glove bag, then the absolute helium leak rates were determined. The test units were disassembled, filled with media, resealed, and autoclaved. The test units were thermally treated to eliminate airlocks within the micropipette lumen and establish a liquid path between microbial challenge media and the test units' contents. Microbial challenge was performed by immersing the test units in a 35 degrees C bath containing magnesium ion and 8 to 10 logs of viable P. diminuta and E. coli for 24 hours. The test units were then incubated at 35 degrees C for an additional 13 days. Microbial ingress was detected by turbidity and plating on blood agar. The elimination of airlocks was confirmed by the presence of magnesium ions in the vial contents by atomic absorption spectrometry. A total of 288 vials were subjected to microbial challenge testing. Those test units whose contents failed to show detectable magnesium ions were eliminated from further analysis. At large leak rates, the probability of microbial ingress approached 100% and at very low leak rates microbial ingress rates were 0%. A dramatic increase in microbial failure occurred in the leak rate region 10(-4.5) to 10(-3) std cc/sec, which roughly corresponded to leak diameters ranging from 0.4 to 2 microns. Below a leak rate of 10(-4.5) std cc/sec the microbial failure rate was < 10%. The critical leak rate in our studies, i.e. the value below which microbial ingress cannot occur because the leak is too small, was observed to be between 10(-5) and 10(-5.8) std cc/sec, which corresponds to an approximate leak diameter of 0.2-0.3 micron.

Pharmaceutical container/closure integrity. IV: Development of an indirect correlation between vacuum decay leak measurement and microbial ingress.

The rational development of a physical test method to evaluate the microbial barrier properties of sterile containers necessitates its correlation to microbiological exclusion. This can be accomplished by direct or indirect correlation. In the former, the proposed physical test is directly compared to microbial challenges using appropriate test units that stimulate container leaks at both high and low probabilities of microbial ingress. Previous work has demonstrated the development of a direct correlation using helium leak rate methods and microbial immersion challenges. An indirect correlation can be established by comparing the proposed physical method to well-defined leakage standards that represent various known levels of microbial ingress. Thus the quality assurance properties of a physical test method can be established by comparison to another physical test method that has been previously characterized. This approach has the distinct advantages of being faster, quantitatively rigorous, and less subject to the vicissitudes, of microbial testing. This approach was demonstrated by comparing the helium leak rate method to vacuum decay testing. Additionally it was demonstrated that vacuum decay testing was a fast and reproducible method for detecting leaks of about 1 to 2 mm. Leaks were simulated by affixing micropipettes into glass vials.

Pharmaceutical container/closure integrity. V: An evaluation of the WILCO "LFC" method for leak testing pharmaceutical glass-stoppered vials.

The sensitivity, reliability, reproducibility and ease of use of the WILCO LFC package integrity test method was evaluated by preparing and testing a series of rubber-stoppered glass vials which were modified by affixing a glass micropipette through the vial side wall. The test units contained water, 50% aqueous ethanol, 20% lithium chloride or 20% aqueous glycerol. Leakage measurement obtained by LFC testing were compared to helium leak rate measurements. The LFC methods detected all leak > 0.0014 standard cubic centimeters per second (sccs), which represents a sensitivity about fourteen-fold greater than standard vacuum decay methods. The minimum detectable leak corresponded to a nominal micropipette internal diameter of between 1 and 2 microns. The effective detection range corresponded to a leak size associated with a 40 to 100% probability of microbial ingress based on a previously reported logistical regression model between helium leak rate and microbial immersion. The sensitivity did not vary with solvent or testing duration in range of 5 to 10 seconds. The coefficient of variation was about 3%. The LFC operation was rapid and without apparent mechanical or electronic problems over the two day testing period used in these studies.

The effect of polyvinylpyrrolidine on the stability of taurolidine.

In an aqueous solution, taurolidine reversibly degrades to taurultam, hydroxymethyltaurultam, taurineamide, and formaldehyde. The objectives of this research were to investigate the mechanism of polyvinylpyrrolidine (PVP)-induced taurolidine stabilization by measuring the effect of PVP on the rate and extent of formaldehyde release from taurolidine and its decomposition products, the ionization of taurineamide, and the NMR and FT-IR spectra of taurineamide. PVP was found to a) increase the rate constant for taurultam formation from taurineamide and formaldehyde, b) decrease the apparent pKa of taurineamide, and c) alter the NMR and FT-IR taurineamide spectra. The effect of PVP on the stability of taurolidine was to increase the stability of its primary degradation product, taurultam, which, in turn increased taurolidine stability because taurultam was reversibly formed from taurolidine. Furthermore, the increased stability of taurultam in acidic, aqueous PVP solutions was due to an increased rate of taurultam formation from its primary degradation products: taurineamide and formaldehyde. The rate constant for taurultam formation increased three-fold in the presence of 5% PVP. The rate increase was caused by the interaction of unprotonated taurineamide with PVP. The estimated taurineamide-PVP association constant was 1800 M-1. This interaction decreased the concentration of unreactive/protonated taurineamide while increasing the concentration of unprotonated/associated substrate which was found to be fivefold less reactive than the free/unprotonated amine.

Kinetics of the aspartyl transpeptidation of daptomycin, a novel lipopeptide antibiotic.

Two degradation products of the lipopeptide antibiotic, daptomycin, were identified and the reaction pathway and kinetics were delineated in aqueous solution at 60 degrees C, pH range 3 to 8 and ionic strength 0.01. The degradation products were 1) a succinimido intermediate (anhydro-daptomycin) formed by attack of side-chain carbonyl on the peptide-bond nitrogen in the asp-gly sequence and 2) a beta-asp daptomycin isomer formed by rehydration of the anhydrodaptomycin succinimide. This aspartyl transpeptidation pathway was found to be reversible. Formation of the anhydrodaptomycin from either daptomycin or beta-asp daptomycin was pH dependent but the pH-rate profiles for anhydrodaptomycin formation were not mechanistically interpretable. The pH-rate profiles for the formation of daptomycin or beta-asp daptomycin from the anhydrodaptomycin were linear with slopes = 1, which is consistent with nucleophilic hydroxide ion attack of the succinimido intermediate at either the alpha-carbonyl, giving rise to the beta-asp daptomycin, or the beta-carbonyl, giving rise to daptomycin.

Preformulation studies on the S-isomer of oxybutynin hydrochloride, an Improved Chemical Entity (ICE).

(S)-Oxybutynin HCl (S-OXY) is a white crystalline solid powder with an acicular particle morphology. Differential scanning calorimetry (DSC) thermograms revealed one characteristic endotherm at 116.2 degrees C. On rescanning a sample heated to 120 degrees C, no thermal events were distinguished in the temperature range 25 degrees C to 150 degrees C. Weight loss curves determined by thermogravimetric analysis showed a continuous, gradual weight loss of about 0.15% over the temperature range 30 degrees C to 110 degrees C, followed by a change in slope and more rapid weight loss beginning at 150 degrees C. Observation by hot-stage microscopy confirmed the melting endotherm observed by DSC. Equilibrium moisture uptake studies indicated low water vapor uptake at low relative humidities (<52.8%). At relative humidities of 75.3% and 84.3%, S-OXY first deliquesced and then converted to a lower melting point crystal form. X-ray powder diffraction (XRPD) data supported the DSC findings. S-OXY underwent degradation by ester hydrolysis at alkaline pHs. The kinetics of this reaction were studied at 25 degrees C in carbonate-bicarbonate buffers. Observed rate constants of 0.008 h(-1) and 0.0552 h(-1) were determined at pH 9.69 and 10.25, respectively. The pKa of S-OXY was 7.75. The aqueous solubility of S-OXY was described as a function of pH and the free-base solubility. The mean partition coefficient log P was 3.33 using 1-octanol. The surface tensions of aqueous solutions of S-OXY decreased with increasing concentration, but no concentration-independent region was observed, indicating that S-OXY does notform micelles in aqueous solution. The dissolution rate of S-OXY from a compressed disk in 0.1 N HCl was rapid, whereas it was considerably slower at pH 7.4. Addition of 1% hexadecyltrimethylammonium bromide (CTAB) at pH 7.4 significantly improved the dissolution rate. S-OXY displayed very poor flow properties when compared to standard pharmaceutical excipients. XRPD results indicated that S-OXY exhibited a loss in crystallinity following ball milling. Hiestand tableting indices indicated that S-OXY has good bonding properties andforms strong compacts, but is likely to be susceptible to capping on ejection from the die. This indicated the needfor a plastically deformable excipient such as Avicel PH-101 in tablet formulations.