A multi-technique analytical approach for impurity profiling during synthesis: The case of difluprednate.

A methodology for the qualitative analysis of a mixture of compounds obtained during the synthesis of difluprednate is described herein for the first time. For this scope a multi-technique analytical approach was developed, combining Liquid Chromatography/Mass Spectrometry (LC/MS), Nuclear Magnetic Resonance (NMR) and computational chemistry. Separation of isomers is frequently required for the identification of impurities in active pharmaceutical ingredients (APIs) to assess the impact they may exhibit on public health. During the final step of the difluprednate synthesis apart from the desired product, various by-products may be obtained. Structural analysis of the products using LC/MS and NMR indicated that the steroid difluprednate was obtained along with its acetyl/butyryl regional isomers, whereas the results were further supported by semi-empirical calculations of the MS-derived data. Following the proposed approach, we managed to elucidate the structures of the challenging 11-acetate, 17-butyrate from the 17-acetate, 21-butyrate, 6α,9α-difluoro prednisolone isomers. The approach utilized may be of general applicability for the analysis of impurities in active pharmaceutical ingredients obtained during chemical synthesis.

Application of Ultra-Centrifugation and Bench-Top 19F NMR for Measuring Drug Phase Partitioning for the Ophthalmic Oil-in-Water Emulsion Products.

Generic drug products are expected to have the same active pharmaceutical ingredient (API) (Q1) with the same content (Q2) and microstructure arrangement (Q3) as the innovator product. In complex oil-in-water emulsion drugs, the hydrophobic API is mainly formulated in oil droplets stabilized by surfactant and micelles composed of extra surfactant molecules. The API phase partition in oil and water (mainly micelle) is a critical quality attribute (CQA) of emulsion product in demonstrating physicochemical equivalence using difluprednate (DFPN) emulsion product Durezol® as a model, we developed a novel low-field benchtop NMR method to demonstrate its applicability in measuring DFPN phase partition for ophthalmic oil-in-water emulsion products. Low-field 19F spectra were collected for DFPN in formulation, in water phase and oil phase after separation from ultra-centrifugation. The NMR data showed the mass balance of DFPN before and after phase separation. The average water phase content of different Durezol® lots was 32 ± 3% with 1% variation from method reproducibility test. The partition results were 52 ± 2% for the in-house control products prepared in Q1/Q2 equivalence to Durezol® but by a different process. The significant difference in DFPN-phase partition between Durezol® and the in-house formulation demonstrated manufacture difference readily changed the API partition. The newly developed ultra-centrifugation and 19F NMR by benchtop instrument is a simple, robust, and sensitive analytical method for ophthalmic emulsion drug product development and control.

Quantification of bupivacaine hydrochloride and isoflupredone acetate residues in porcine muscle, beef, milk, egg, shrimp, flatfish, and eel using a simplified extraction method coupled with liquid chromatography-triple quadrupole tandem mass spectrometry.

In this study, a simple analytical approach has been developed and validated for the determination of bupivacaine hydrochloride and isoflupredone acetate residues in porcine muscle, beef, milk, egg, shrimp, flatfish, and eel using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A 0.1% solution of acetic acid in acetonitrile combined with n-hexane was used for deproteinization and defatting of all tested matrices and the target drugs were well separated on a Waters Xbridge™ C18 analytical column using a mobile phase consisting of 0.1% acetic acid (A) and 0.1% solution of acetic acid in methanol (B). The linearity estimated from six-point matrix-matched calibrations was good, with coefficients of determination ≥0.9873. The limits of quantification (LOQs) for bupivacaine hydrochloride and isoflupredone acetate were 1 and 2ngg-1, respectively. Recovery percentages in the ranges of 72.51-112.39% (bupivacaine hydrochloride) and 72.58-114.56% (isoflupredone acetate) were obtained from three different fortification concentrations with relative standard deviations (RSDs) of <15.14%. All samples for the experimental work and method application were collected from the local markets in Seoul, Republic of Korea, and none of them tested positive for the target drugs. In conclusion, a simple method using a 0.1% solution of acetic acid in acetonitrile and n-hexane followed by LC-MS/MS could effectively extract bupivacaine hydrochloride and isoflupredone acetate from porcine muscle, beef, milk, egg, shrimp, flatfish, and eel samples.

Disposition of isoflupredone acetate in plasma, urine and synovial fluid following intra-articular administration to exercised Thoroughbred horses.

The use of isoflupredone acetate in performance horses and the scarcity of published pharmacokinetic data necessitate further study. The objective of the current study was to describe the plasma pharmacokinetics of isoflupredone acetate as well as time-related urine and synovial fluid concentrations following intra-articular administration to horses. Twelve racing-fit adult Thoroughbred horses received a single intra-articular administration (8 mg) of isoflupredone acetate into the right antebrachiocarpal joint. Blood, urine and synovial fluid samples were collected prior to and at various times up to 28 days post drug administration. All samples were analyzed using liquid chromatography-Mass Spectrometry. Plasma data were analyzed using a population pharmacokinetic compartmental model. Maximum measured plasma isoflupredone concentrations were 1.76 ± 0.526 ng/mL at 4.0 ± 1.31 h and 1.63 ± 0.243 ng/mL at 4.75 ± 0.5 h, respectively, for horses that had synovial fluid collected and for those that did not. The plasma beta half-life was 24.2 h. Isoflupredone concentrations were below the limit of detection in all horses by 48 h and 7 days in plasma and urine, respectively. Isoflupredone was detected in the right antebrachiocarpal and middle carpal joints for 8.38 ± 5.21 and 2.38 ± 0.52 days, respectively. Results of this study provide information that can be used to regulate the use of intra-articular isoflupredone in the horse.

Ocular distribution of difluprednate ophthalmic emulsion 0.05% in rabbits.

PURPOSE: To evaluate ocular distribution and excretion of tritium-labeled difluprednate ((3)H-DFBA) ophthalmic emulsion 0.05% after a single or repeated instillation to pigmented rabbit eyes. METHODS: (3)H-DFBA ophthalmic emulsion 0.05% was instilled in the right eyes of pigmented rabbits, in either a single or repeated quater in die (QID) for 3 days or 7 days) dose of 25 µg/50 µL. The radioactivity in right and left ocular tissues, urine, blood, plasma, and feces were measured with a liquid scintillation counter. Additionally, the distribution of radioactivity around ocular tissues was investigated with autoradiography. RESULTS: After a single instillation, the highest maximum radioactive concentrations were found in the cornea (2,081 ng eq./g), followed by the iris/ciliary body (926 ng eq./g), conjunctiva (330 ng eq./g), anterior retina/choroid (273 ng eq./g), sclera (222 ng eq./g), and aqueous humor (144 ng eq./mL) of treated eyes. The maximum radioactivity concentration of the posterior retina/choroid was 59 ng eq./g, and difluprednate delivered as a topical ophthalmic emulsion reached the back of the eye. However, radioactivity in untreated eyes was very low. Total radioactivity excreted in urine and feces 168 h after a single instillation was 99.5% of the total dose. Radioactivity concentration levels measured 24 h after 28 instillations were no more than twice those measured 24 h after 12 instillations. Radioactive concentrations in ocular and periocular tissues were highest at 0.5 or 1 h after a single instillation, and were mostly eliminated from these tissues by the end of the study. Radioactivity was barely detectable in the blood, with very little accumulation even after multiple doses. CONCLUSIONS: After instillation of (3)H-DFBA ophthalmic emulsion 0.05% in rabbit eyes, radioactivity was distributed at the anterior segment and cleared rapidly. Some radioactivity was detected in the posterior retina/choroid, suggesting that difluprednate and its metabolites reach these tissues. These results suggest that difluprednate delivered as a topical ophthalmic emulsion reached the anterior and posterior segments of the eye quickly and may be a potential treatment for ocular inflammation in these areas.

Analysis of an anti-inflammatory steroidal drug, difluprednate, in aqueous humor by combination of semi-micro HPLC and column switching method.

A specific and sensitive method for the determination for difluprednate (DFBA) and its metabolite (deacetylated DFBA, DFB) in aqueous humor was developed. DFBA and DFB were initially absorbed on a Pinkerton-type column, then analyzed by high-performance liquid chromatography using a semi-micro column after column switching. Under the optimized conditions, calibration curves for DFBA and DFB showed good linearity over the range of 1.0-50 and 0.5-50 ng/ml, respectively. We applied the method to the analysis of DFBA and DFB in rabbit aqueous humor, and found that DFBA in rabbit aqueous humor 1 h after instillation of 0.002% DFBA ophthalmic emulsion was not detected, but DFB was present at the concentration of 4.3+/-3.1 ng/ml.

Plasma, urine, and synovial fluid disposition of methylprednisolone acetate and isoflupredone acetate after intra-articular administration in horses.

OBJECTIVE--To document plasma, urine, and synovial fluid disposition of 2 common intra-articularly administered steroid preparations, methylprednisolone acetate (MPA) and isoflupredone acetate (IPA). DESIGN--Descriptive investigation. SAMPLE POPULATION--100 mg of MPA or 4 mg of IPA was administered to 2 groups of 4 healthy sound radiographically normal female horses. PROCEDURE--Blood samples were collected at time 0 (before) and 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after administration of the designated steroid. Complete urine collection for measurement of designated steroid was accomplished by use of occluding 28-F balloon catheters. Synovial fluid samples were aseptically aspirated from the injected and contralateral uninjected tarsocrural joint at time 0 and 8, 24, 48, 240, and 672 hours after administration of the designated steroid. All samples were screened by ELISA to detect parent drug or metabolite equivalent, with a sensitivity of 2.5 ng/ml for MPA and 0.1 ng/ml for IPA. If drug was detected by ELISA in the plasma or synovial fluid, the samples were further quantified and specified, using HPLC with a lower limit of quantification (10 ng/ml). RESULTS--Between 2 and 12 hours after administration, plasma contained < 10 ng of MPA or IPA/ml (parent drug or metabolite equivalent), as intermittently detected by ELISA. Parent drug or metabolite equivalent was detected in the urine for 24 and 72 hours after injection of IPA and MPA, respectively. Synovial fluid from the contralateral joint contained no detectable MPA or IPA at any sample collection time. Median half-life for MPA, as detected by HPLC, was 10.3 hours (range, 6.1 to 10.6) in the synovial space. Median half-life for methylprednisolone, as detected by HPLC, was 10.4 (range, 9.9 to 32.1) hours. CONCLUSIONS--Both steroids appeared to be rapidly hydrolyzed to their respective ester forms, as detected by HPLC. The ELISA appeared to be a useful screening tool for detection of corticosteroids in this variety of body fluids.

The identification of related substances in 9 alpha-fluoroprednisolone-21 acetate by means of high-performance liquid chromatography with diode array detector and mass spectrometry.

A method for the analysis and identification of the principal related substances in 9 alpha-fluoroprednisolone acetate is described. This compound has been chosen as a model for the investigation of related substances which can be originated in the general procedure for introducing a fluorine substituent at position 9 of a corticosteroid molecule. HPLC procedures, both in reversed and in normal phase were used; a rapid scanning UV detector which allows direct spectrophotometric data to be obtained on chromatographic peaks, proved to be a tool of great importance. Thus, after reversed-phase chromatographic separation and observation of the UV spectra and their respective second derivatives, it was possible to characterize some of the principal effective and potential related substances such as 9 alpha-fluorohydrocortisone acetate, 9 alpha-bromoprednisolone acetate, 9 beta, 11 beta-epoxyprednisolone acetate and 9(11)-dehydroprednisolone acetate, emerging as chromatographic peaks. Identification of 9 alpha-bromoprednisolone acetate and of 9 alpha-fluorohydrocortisone acetate which proved to be the most significant impurities, was confirmed by means of an exhaustive study of the mass spectra of these substances conveniently isolated by normal-phase HPLC. The chromatographic, spectrophotometric and mass-spectrometric characteristics of the studied compounds are reported.