Methylprednisolone Concentrations in Breast Milk and Serum of Patients with Multiple Sclerosis Treated with IV Pulse Methylprednisolone.

BACKGROUND: It is commonly known that women with multiple sclerosis (MS) have an increased risk for relapses during the post-partum period. High-dose IV methylprednisolone is the first-line treatment for acute relapses. Methylprednisolone is administered to lactating women although there is insufficient data as to the levels of concentration in breast milk and serum, and the calculated steroid exposure to infants. OBJECTIVES: The study aimed to measure the transfer of methylprednisolone into breast milk and the correlation of milk and serum methylprednisolone concentrations in breastfeeding MS patients during and after infusion therapy. METHODS: IV methylprednisolone pulse therapy was given to 12 lactating MS patients. Breast milk and maternal serum samples were obtained; before infusion, 30 minutes into the infusion, at the end of infusion and at the 1st, 2nd, 4th, 8th, 12th and 24th hours subsequently. RESULTS: The highest level of methylprednisolone concentration in breast milk (2.09 µg/ml) and serum (6.09 µg/ml) was detected at the end of the infusion. According to the measurements recorded at the 1st, 2nd, 4th, 8th, 12th, and 24th hours after infusion, the concentrations showed a gradual decrease both breast milk and serum. The milk and serum methylprednisolone concentrations were below detection limits just before infusion and at the 24th hour after infusion. A highly significant correlation was found between breast milk and maternal serum levels. The absolute infant dose was calculated to be 69.50 µg/kg/day and the relative infant dose (RID) was 0.50%. CONCLUSION: Results have shown that the transfer of methylprednisolone into breast milk seems to be low. Although, concentration levels may not seem to pose a threat to the infant, mothers can choose to wait 2 to 4 hours to further limit the level of exposure.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

Stability and Sterility of Extemporaneously Prepared Nonpreserved Cefazolin, Ceftazidime, Vancomycin, Amphotericin B, and Methylprednisolone Eye Drops.

PURPOSE: To determine in-use stability and sterility of fortified cefazolin, ceftazidime, vancomycin, amphotericin B, and methylprednisolone eye drops in a simulated inpatient setting with and without a mobile refrigerated container (MR). METHODS: Each drug was prepared and divided into 4 groups: 1) simulated patient use with the MR group: stored at 4°C and kept in the MR during drug administration, 2) simulated patient use without the MR (NoMR) group: stored at 4°C and no MR, 3) refrigerated control group: stored at 4°C, and 4) room temperature control group: stored at room temperature. Stability and sterility data were evaluated at days 0, 4, 7, 14, 21, and 28. Linear mixed-effects model and survival analysis were performed. RESULTS: Median time to 10% loss of concentration for in-use medications (MR/NoMR groups) was >28/27.9, 22.2/22.2, 19.4/19.4, 10.18/<4, and >28/>28 days for cefazolin, ceftazidime, vancomycin, amphotericin B, and methylprednisolone, respectively. There was no significant difference in the predicted concentration loss per day among all groups for vancomycin and methylprednisolone (all P > 0.05). For the other study medications, all room temperature control groups, the cefazolin NoMR group, and the ceftazidime NoMR group had significantly greater predicted concentration loss per day compared with the refrigerated control groups (all P ≤ 0.02). Culture results were negative for all drugs throughout the study. CONCLUSIONS: The NoMR group showed that the drug significantly degraded rapidly for cefazolin, ceftazidime, and amphotericin B. Implementation of MR could decrease the predicted loss of concentration per day for cefazolin and ceftazidime. In vitro antimicrobial activity and sterility were retained for 28 days.

A novel and innovative hair test to determine glucocorticoid levels in racing camels for use in assessment of doping, health, and disease.

The aim of this project was to develop and validate a new test for the analysis of glucocorticoids in camel hair and to use the new test to analyse hair samples from a variety of camel breeds in sports and racing applications. These findings could be of importance when evaluating racing camels for suspected doping offenses or for injury and disease control. Camel hair samples were collected from 30 non-racing dromedary camels along with 3 racing camels in Al Ain, UAE and were decontaminated, pulverised, sonicated, and extracted prior to analysis. A liquid chromatographic-mass spectrometric method was employed to determine the levels of glucocorticoids in the hair samples. The 4 drugs of interest, namely hydrocortisone, dexamethasone, flumethasone and methylprednisolone, and an internal standard were quantified in camel hair samples. All 4 of the glucocorticoids were detected in camel hair samples with concentrations ranging between 31 and 935 pg/mg for hydrocortisone, 8-59 pg/mg for dexamethasone, 0.7-1034 pg/mg for flumethasone and 5-66 pg/mg for methylprednisolone in non-racing camels. One of the racing camels displayed high concentrations of hydrocortisone (1130 pg/mg), flumethasone (2576 pg/mg), methylprednisone (1156 pg/mg) and dexamethasone (29 pg/mg). The authors believe this is the first report of a test for corticosteroids in camel hair. The new test has been validated according to Food and Drug Administration (FDA) guidelines. This new hair test could be useful for further studies in doping control, toxicological studies, pharmacological studies and other clinical applications in camel health, injury, and disease.

A rapid screen for four corticosteroids in equine synovial fluid.

Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-articularly administered corticosteroids--betamethasone, methylprednisolone, methylprednisolone acetate and triamcinolone acetonide. Sample preparation consisted of protein precipitation followed by a basified liquid-liquid extraction. LC-MS-MS experiments consisted of a six-min isocratic separation using a Phenomenex Polar-RP stationary phase and a mobile phase consisting of 35% acetonitrile, 5 mM ammonium acetate and 0.1% formic acid in nanopure water. The detection system used was a triple quadrupole mass analyzer with thermospray ionization, and compounds were identified using selective reaction monitoring. The method was validated to the ISO/IEC 17025 standard, and real synovial fluid samples were analyzed to demonstrate the application of the method in an antidoping context. The method was highly selective for the four corticosteroids with limits of detection of 1-3 ng/mL. The extraction efficiency was 50-101%, and the matrix effects were 14-31%. These results indicate that the method is a rapid and sensitive screen for the four corticosteroids in equine synovial fluid, fit for purpose for equine antidoping assays.

Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors.

Pancreatic islets, transplanted into recipients with type 1 diabetes, are exposed to allogenic and auto-immune T-cell responses. We set out to develop an assay to measure these responses using PBMC. Our approach was to prepare spleen extract from the islet donors (allo-antigen) and islet extracts (auto-antigen). To our surprise, we found that spleen extracts potently inhibited the proliferation of human T cells driven by antigen (tetanus toxoid) and mitogen (anti-CD3 mAb, OKT3), whereas extracts prepared from pancreatic islets from the same donor did not suppress T-cell proliferation. Suppression mediated by spleen extracts was unaffected by blocking mAbs against the IL-10R, transforming growth factor-ß or CD152 (CTLA-4). It was also unaffected by denaturing the spleen extracts by heating, exposing to reducing agents or protease digestion. Because deceased organ donors are commonly given the immunosuppressive glucocorticoid methylprednisolone prior to death, we hypothesized that suppression was due to residual methylprednisolone in the spleen extracts. Methylprednisolone could be detected by mass spectrometry in spleen extracts at concentrations that suppress T-cell proliferation. Finally, the glucocorticoid receptor antagonist mifepristone completely reversed the suppression caused by the spleen extracts. We conclude that extracts of human spleen, but not islets, from deceased organ donors contain sufficient residual methylprednisolone to suppress the proliferation of T-cells in vitro.

Pharmaceutical evaluation of steroidal ointments by ATR-IR chemical imaging: distribution of active and inactive pharmaceutical ingredients.

We recently used micro attenuated total reflection infrared (ATR-IR) spectroscopy to conduct imaging analysis of ointments and evaluate the distributions of the active pharmaceutical ingredient (API) and excipients. An alclometasone dipropionate (ALC) ointment was used as a model product. Almeta, a brand-name product, had a domain with absorbance at 1656 cm(-1) attributable to the carbonyl group of ALC, the API. Absorbances at 1040 and 3300 cm(-1) were also noted in this domain, indicating the presence of the solubilizer, propylene glycol. Data also suggested the presence of benzyl alcohol in this domain. More detailed analysis showed the distribution of surfactants and other excipients in the base. Similar results were obtained for Vitra, a generic version of Almeta. Imaging analysis with micro ATR-IR confirmed that both ointments are liquid droplet dispersions with ALC dissolved in propylene glycol and dispersed in a base. However, minor differences in the ingredient distributions of the two ointments were detected and reflect differences in excipient concentrations and type, or manufacturing differences. In summary, we used micro ATR-IR for imaging analysis of an original ointment, Almeta, and its generic form Vitra, and established a method for visually evaluating the distributions of the API and excipients in these ointments.

Determination of MRL regulated corticosteroids in liver from various species using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC).

Dexamethasone, betamethasone, prednisolone and methylprednisolone are corticosteroids widely used in animal husbandry. These compounds are licensed for therapy in veterinary practices while their use for growth promoting practices, mainly in combination with other growth promoters, is prohibited within the European Union. In order to protect the consumer, maximum residue limits (MRLs) have been set by the European Community in liver to 2.0 µg kg(-1) (dexamethasone and betamethasone) and 10.0 µg kg(-1) (prednisolone and methylprednisolone) for different species. The major challenges in the analysis of dexamethasone and betamethasone consist in performing an efficient separation of both isomers and in detecting and identifying all the molecules according to the regulatory requirements fixed in Commission Decision 2002/657/EC. In this context, an UHPLC-MS/MS method with a short runtime (7 min) and using the SRM acquisition mode was developed and validated. An efficient selectivity of the sample preparation combined with a high sensitivity of the measurement system allowed identifying and quantifying the four corticosteroids of interest in this complex biological matrix. Signals obtained were found very repeatable, even at very low concentration levels with an unambiguous identification of the compounds. The performance limits of the method have been validated according to the regulatory requirements and the method has been successfully applied to the confirmation of incurred liver samples.

Liquid chromatography-tandem mass spectrometry for the determination of methylprednisolone in rat plasma and liver after intravenous administration of its liver-targeted dextran prodrug.

A specific and sensitive liquid chromatography (LC)-tandem mass spectrometric method for quantitative determination of methylprednisolone (MP) in rat plasma and liver was developed and validated using triamcinolone acetonide as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma and liver. The separation of MP was performed on a C(18) column with a mobile phase of acetonitrile:0.5% formic acid aqueous solution (85:15, v/v) over 4min. The assay was based on the selected reaction monitoring transitions at m/z 375-->161 for MP in plasma, 375-->357 for MP in liver, and 435-->415 for internal standard in both plasma and liver. The lower limit of quantification was 20ng/mL based on 100microL of plasma or liver homogenate. Intra- and inter-day assay variations were

Differential pulse voltammetric determination of methylprednisolone in pharmaceuticals and human biological fluids.

Electrochemical behaviour of methylprednisolone (MP) at the fullerene-C60-modified glassy carbon electrode has been investigated using differential pulse voltammetry. The experimental results suggest that the modified electrode exhibits electrocatalytic effect on the oxidation of MP resulting in a marked enhancement of the peak current response. Under the selected conditions, the oxidation peak current was linearly dependent on the concentration of MP in the range 5.0 nM-1.0 microM with a sensitivity of 0.0107 microA microM(-1). The detection limit was estimated to be 5.6 nM. The electrode showed good sensitivity, selectivity, stability and reproducibility. In addition, the developed method was satisfactorily applied to the determination of MP in pharmaceutical formulations and human serum and urine samples without any necessity for sample treatment or time-consuming extraction steps prior to the analysis. GC-MS method was used to cross-validate the results obtained for the quantitative estimation of MP in biological fluids and the results showed approximately 2% deviation to those obtained using the proposed method.

[Using ultra performance liquid chromatography].

An analytical method based on ultra performance liquid chromatography has been developed for the simultaneous determination of 15 hormones (triamcinolone, hydrocortisone, prednisone, cortisone, 6a-methylprednisolone, betamethasone, dexamethasone, prednisolone acetate, hydrocortisone acetate, estriol, beta-estradiol, estrone, diethylstilbestrol, testosterone and progesterone) in cosmetics. Various cosmetic samples were extracted with methanol under ultrasonication, and then cleaned up by a Oasis HLB solid phase extraction cartridge. The 15 hormones were separated and detected within 6 min on a Waters ACQUITY UPLC BEH C18 column (1.7 microm, 2.1 mm x 50 mm) by gradient elution with acetonitrile and water system. In the range of 1-25 ng, the calibration curves of 15 hormones showed good linearity with the correlation coefficients larger than 0.9995. The mean recoveries at the three spiked levels (2, 10, 20 mg/kg) were 88.2%-102.4% with the relative standard deviations of 1.6%-7.4%.

Rapid multi-residue method for the quantitative determination and confirmation of glucocorticosteroids in bovine milk using liquid chromatography-electrospray ionization-tandem mass spectrometry.

Dexamethasone, betamethasone and prednisolone are synthetic glucocorticosteroids authorized for therapeutic use in bovine animals within the European Union. Dexamethasone and betamethasone are used mainly for the treatment of metabolic and inflammatory diseases. Prednisolone is used to treat bovine mastitis. Maximum residue limits (MRLs) of 0.3 microg kg(-1) for both dexamethasone and betamethasone and 6.0 microg kg(-1) for prednisolone in bovine milk have been established. 6alpha-Methylprednisolone and flumethasone are not authorized for use in bovine animals and are completely banned in bovine milk. The proposed method is based on deprotenisation of milk using 20% (w/v) trichloroacetic acid. Samples are filtered using glass microfiber filters and subject to clean-up using OASIS HLB solid phase extraction. Separation was achieved on a Hypercarb 100 mm x 2.1 mm x 5 microm column. Mobile phase was: 90/10 acetonitrile/0.1% formic acid in water; flow rate was 600 microL min(-1). The method allowed the rapid identification and confirmation of the five glucocorticosteroids according to the criteria laid down in Commission Decision 2002/657/EC. Matrix calibration curves for all compounds were linear in the interval 0.0 MRL to 2.0 MRL with a correlation coefficient (r(2)) higher than 0.96. Relative recoveries ranged from 97% for betamethasone to 111% for prednisolone. Precision at the MRL ranged from 3.8% for prednisolone to 13.8% for betamethasone. Decision limits, CCalpha, and detection capability, CCbeta have been calculated for all compounds.

Characterization of methylprednisolone esters of hyaluronan in aqueous solution: conformation and aggregation behavior.

Methylprednisolone steroid esters of hyaluronan differing in degree of functionalization and molecular weight were investigated in aqueous solution. Conformation and aggregation phenomena were elucidated by means of circular dichroism, viscometry, rheology, and nuclear magnetic resonance, mainly by (1)H pulsed field gradient (PFG) NMR, which allows the determination of the diffusion coefficient of the species under investigation. The functionalization of hyaluronan with the steroid induces a reduction of the molecular volume, as a consequence of intramolecular hydrophobic interactions. For concentrated samples we have observed the coexistence of unimolecular collapsed chains and of aggregates, the latter disappearing upon dilution. The methylprednisolone ester of lower molecular weight hyaluronan has a larger molecular volume than its higher molecular weight analogue, even though still smaller than the underivatized polymer. This effect can be explained with the reduced flexibility of the polymer backbone probably impairing intramolecular interactions.

Concentration of methylprednisolone in the centrodistal joint after administration of methylprednisolone acetate in the tarsometatarsal joint.

REASONS FOR PERFORMING STUDY: The centrodistal (CD) and tarsometatarsal (TMT) joints are often injected individually with a corticosteroid to resolve lameness caused by osteoarthritis (OA). There are no data available regarding diffusion of methylprednisolone (MP) from the TMT joint to the CD joint. HYPOTHESIS: A therapeutic concentration of MP diffuses into the CD joint after methylprednisolone acetate (MPA) is administered into the TMT joint. OBJECTIVE: To measure the concentration of MP in the CD joint after MPA was administered into the TMT joint. METHODS: MPA was administered into a TMT joint of 16 horses. At different times, the ipsilateral CD joint of these horses was injected with a small amount of saline and recovered saline was measured for concentration of MP using high performance liquid chromatography. RESULTS: Six hours after administration of MPA into the TMT joint, a therapeutic concentration of MP was found in all 10 CD joints sampled at this time. CONCLUSIONS: Horses with pain arising from the distal 2 joints of the hock can be treated by administering MPA into the TMT joint alone. POTENTIAL RELEVANCE: Administering MPA into the TMT joint only, to treat OA of the distal 2 hock joints, reduces the difficulties and risks associated with centesis of the CD joint.

Micellar electrokinetic chromatography for simultaneous determination of six corticosteroids in commercial pharmaceuticals.

We have developed a micellar electrokinetic chromatography (MEKC) method using bile salts for the simultaneous determination of six corticosteroids, including betamethasone, cortisone, prednisolone, 6alpha-methylprednisolone, triamcinolone, and prednisone. The separation was performed using borate buffer containing sodium cholate and sodium deoxycholate. Several parameters were studied, including bile salt concentrations, concentrations and pH of borate buffer, and analytical voltages. In method validation, calibration curves were linear over a range of 10-100 microM for each corticosteroid. The RSD (relative standard deviation) and RE (relative error) were all less than 5% for intra- and interday assays. The limit of detection of each analyte was 5 microM. The recoveries were greater than 95%. Application of this method for quality control of commercial tablets also proved to be feasible. All analytical values fall within the labeled amount of 90-110% for betamethasone and prednisolone, and of the labeled amount of 92.5-107.5% for 6alpha-methylprednisolone, as required by the United State Pharmacopeia 25 (USP 25).

Determination of CMC of sodium glucocorticides hemisuccinates by CE.

The capillary zone electrophoresis (CZE) method was applied to the determination of the critical micelle concentration (CMC) and the anionic mobilities (mu(e)) for sodium glycocorticides hemisuccinates (Urbason solubile forte 1000, Hydrocortison 100 Rotexmedica, Prednisolut 100) in phosphate solution at pH 7.2. The CZE enables an efficient and very fast determination of parameters for characterizing physicochemical properties of micelles.

A high-performance liquid chromatography/tandem mass spectrometric screening method for eight synthetic corticosteroids in bovine feces and the simultaneous differentiation between dexamethasone and betamethasone.

A screening method was developed to monitor the illegal use of synthetic corticosteroids in cattle. Diethyl ether extracts from spiked feces samples were cleaned-up by solid phase extraction followed by semipreparative reversed-phase chromatography (RPC). The fraction containing the corticosteroids was derivatized with ethoxyamine hydrochloride. The corresponding ethoximes were separated using silica-based C18 RPC and analyzed on-line in an ion trap mass spectrometer using atmospheric pressure positive chemical ionization. Ethoxime derivatives of dexamethasone and betamethasone were baseline resolved, allowing for the simultaneous mass spectrometric differentiation of both epimers in bovine feces by conventional non-chiral chromatography. At the lowest level tested (1 micro g/kg), corticosteroids (except triamcinolone) could be identified in compliance with the recent European criteria for residue identification. The quantitative performance of the method was best at residue levels > or = 2 micro g/kg.

Collision-induced dissociation of corticosteroids in electrospray tandem mass spectrometry and development of a screening method by high performance liquid chromatography/tandem mass spectrometry.

A screening method based on liquid chromatography/electrospray tandem mass spectrometry was developed in order to control the illegal use of corticosteroids as growth promoters in cattle. The objective was the detection of low residue levels of corticosteroids or metabolites in biological matrices. Relative to other studies published on this subject, the present work focused on enhancing specificity and sensitivity. Firstly, fragmentation of corticosteroids by collision-induced dissociation was studied. In positive mode, the losses of H(2)O for each hydroxyl group fixed on the molecule, as well as the loss of HF or HCl for halogenated compounds, were observed. For higher collision energy, fragmentations in the B, C and D rings were induced. The negative mode was found to be more specific, inducing a cleavage of the C(20)-C(21) bond with concomitant loss of formaldehyde (CH(2)O). Secondly, three acquisition methods in the negative mode were studied and evaluated, recorded signals being the parent ion [M + acetate](-) and the two daughter ions, [M - H](-) and [M - H - CH(2)O](-). For dexamethasone, MS/MS instrumental detection limits of fragment ion and neutral loss scans, and of multiple reaction monitoring (MRM), were 250, 20 and 5 pg injected, respectively. The MRM method was then evaluated with the objective of use for the detection of corticosteroid residues in biological samples (urine, hair, muscle) and for a metabolism study.

Simultaneous analysis of dextran-methylprednisolone succinate, methylprednisolone succinate, and methylprednisolone by size-exclusion chromatography.

An analytical HPLC method is reported for simultaneous measurement of low (1.0-100 microg ml(-1)) concentrations of dextran-methylprednisolone succinate (DEX-MPS) and its degradation products methylprednisolone hemisuccinate (MPS) and methylprednisolone (MP). The analytes were detected at 250 nm after resolution using a size exclusion column with a mobile phase of KH2PO4 (10 mM): acetonitrile (3:1) and a flow rate of 1 ml min(-1). The resolution of MP and MPS peaks was substantially affected by the pH of the mobile phase; while MP and MPS co-eluted at pH 3.4, they were baseline-resolved at pH > or = 5. Linear relationships (r > or = 0.997) were found between the detector response and the concentrations of the analytes (1.0-100 microg ml(-1) for MP and MPS and 2.5-100 microg ml(-1) for DEX-MPS). Intra- and inter-run error (< 13%) and precision (CV of < or = 6%) data indicated that the assay could accurately and precisely quantitate all three components in the examined concentration range. The application of the assay to determination of degree of substitution, purity, and stability of DEX-MPS was also demonstrated.