Analysis of betamethasone, dexamethasone and related compounds by liquid chromatography/electrospray mass spectrometry

A reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) method has been developed to conclusively differentiate the epimers betamethasone and dexamethasone and various esterification products (betamethasone and dexamethasone 21-acetate, betamethasone and dexamethasone 21-phosphate, betamethasone 17-valerate, betamethasone 21-valerate and betamethasone 17,21-dipropionate) in counterfeit drugs. Good separation with baseline resolution of all epimers or isomers was obtained on a Zorbax Eclipse XDB or Luna C8 column, using a step gradient with mobile phases of 0.05 M ammonium acetate and acetonitrile. Betamethasones can also be distinguished by the relative abundance of their m/z 279 ion in the positive electrospray tandem mass spectra. The LC/MS or LC/MS/MS method developed was successfully applied to the analysis of drug product samples, i.e. creams and tablets.     

The Synthesis of 17-Ester of Corticosteroids Protection of 11β-Hydroxyl by the Trimethylsilyl Group

17–Esters of corticosteroids have received considerable attention due to their high topical anti-inflammatory activity. one of the best known is betamethasone 17-valerate which is widely used against a variety of skin diseases². The relatively simple compound, hydrocortisone 17–butyrate (3a)³ proved to be as active topically as the more sophisticated corticosteroids such as triamcinolone acetonide, while its systemic activity is of the same order as that of hydrocortisone⁴.     

Studies on topical antiinflammatory agents. IV. 21-(Alkylthio)acetates and (methylthio)methoxides of corticosteroids

A series of 21-(alkylthio)acetates and 21-(methylthio)methoxides of corticosteroids were synthesized and examined for vasoconstrictive activities. The activities of seven compounds were equal to or greater than that of 9 alpha-fluoro-11 beta,21-dihydroxy-16 beta-methyl-17 alpha-valeryloxy-1,4-pregnadiene-3,20-dione (betamethasone 17-valerate, BV). Among them, betamethasone 21-(methylthio)acetate 17-propanoate (2Ca) was found to have the most potent activity, which is superior to that of BV. A structure-activity relationship study revealed that substitution of the 21-hydroxy group of corticosteroids with the (methylthio)acetate function is a useful approach for obtaining potent activity.     

Studies on topical antiinflammatory agents. I. Synthesis and vasoconstrictive activity of corticosteroid 17-succinyl esters

A series of 17-succinyl derivatives of four corticosteroids was prepared. They were tested for vasoconstrictive activity in humans, using 9 alpha-fluoro-11 beta, 21-dihydroxy-16 beta-methyl-17 alpha-valeryloxy-1,4-pregnadiene-3,20-dione (betamethasone 17-valerate, BV) as a standard. The activities of the 21-chloro 17-methylsuccinate compounds (6A, 6C and 6D) were greater than that of BV. A structure-activity relationship study showed that the activities of the 21-chloro 17-methylsuccinates were more potent than those of the corresponding 21-esters.     

Studies on topical antiinflammatory agents. III. Synthesis of 17 alpha-acyloxy-9 alpha-fluoro-11 beta-hydroxy-16 beta-methyl-1,4-pregnadiene-3,20-dione 21-thio derivatives and related compounds

A series of 21-thio derivatives of 9 alpha-fluoro-11 beta,17 alpha-dihydroxy-16 beta-methyl-1,4-pregnadiene-3, 20-dione 17-esters and related compounds were synthesized and evaluated as topical antiinflammatory agents. These compounds were prepared by the reaction of 9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-16 beta-methyl-1,4-pregnadiene-3, 20-dione (betamethasone, I) 17-ester derivatives and various mercapto compounds. A structure-activity relationship study revealed that the structural combination of a thio group at the 21-position and an ester group at the 17-position contributed to vasoconstrictive activity. Among these compounds, the 21-methylthio 17-propanoate compound (6) was found to have the most potent activity, being more potent than betamethasone 17-valerate (BV).     

Studies on topical antiinflammatory agents. II. Synthesis and vasoconstrictive activity of 21-substituted corticosteroids with sulfur-containing moieties

As part of the search for new topical antiinflammatory agents, various 21-substituted corticosteroids having sulfur-containing moieties were prepared and tested for vasoconstrictive activity in humans. A structure-activity relationship study revealed that substitution of the 21-hydroxy group with a lower alkyl-thio group enhanced the activity. The activities of the 21-methylthio (3Ad) and the 21-ethylthio (3Ae) compounds were more potent than that of 9 alpha-fluoro-11 beta,21-dihydroxy-16 beta-methyl-17 alpha-valeroyloxy-1,4- pregnadiene-3,20-dione (betamethasone 17-valerate, BV).     

Studies on topical antiinflammatory agents. V. 17-(Alkylthio)- and methoxyalkanoates of corticosteroids

As part of our search for new topical antiinflammatory agents, a series of corticosteroid 17-(alkylthio)- and methoxyalkanoate derivatives was prepared and tested for vasoconstrictive activities. Several compounds were proved to have activity superior or comparable to that of 9 alpha-fluoro-11 beta,21-dihydroxy-16 beta-methyl-17 alpha-valeryloxy-1,4-pregnadiene-3,20-dione (betamethasone 17-valerate, BV). Among these compounds, 21-chloro-11 beta-hydroxy-17 alpha-(methylthio)acetoxy-4-pregnene-3,20-dione (5Aa) was found to have the most potent activity, being more active than BV. The structure-activity relationships of the series revealed that introduction of a (methylthio)acetate function into the 17-position as well as the 21-position of corticosteroids was effective for enhancing the topical antiinflammatory activity.     

超高效液相色谱法测定美白、祛痘化妆品中4种禁用激素

建立了超高效液相色谱同时测定美白、祛痘化妆品中4种糖皮质激素氢化可的松、曲安奈德醋酸酯、氯倍他索丙酸酯、倍他米松双丙酸酯的方法。样品采用甲醇涡旋超声提取,色谱柱采用Waters ACQUITY UPLC BEH C18柱(100 mm×2.1 mm,1.7μm),流动相为乙腈–水溶液,梯度洗脱,流量为0.5 m L/min,用PDA检测器检测,检测波长为240 nm。方法检出限以3倍空白噪音计,氢化可的松、曲安奈德醋酸酯、氯倍他索丙酸酯、倍他米松双丙酸酯的检出浓度分别为0.12,0.18,0.30,0.30 mg/kg。在质量浓度为1.0~40.0 mg/L时,标准工作曲线相关系数r0.999 8,4种激素的回收率为85.3%~102.8%,测定结果的相对标准偏差为2.1%~5.0%(n=6)。该方法前处理简单,分离效果好,且灵敏度高,速度快,能满足美白、祛痘化妆品中4中糖皮质激素的检测需要。     

Thin Layer Chromatographic Method for Rapid Identification and Quantification of Corticosteroid Sodium Phosphates in Pharmaceutical Preparations

Abstract

A rapid and inexpensive thin layer chromatography (TLC) procedure for the assay of dexamethasone sodium phosphate (DSP) and betamethasone sodium phosphate (BSP) persent in pharmaceutical preparations is described. Free steroids liberated after alkaline phosphatase reaction is isolated by TLC on silica gel layer and estimated after elution with ethanol. Assay of unesterified steroids and identification of the preservatives like methyl or propyl parabenzoates, phenol, benzyl alcohol etc. may also be carried out by this method without any additional cost. Possibility of using this method for other corticosteroid sodium phosphates (CSP) are discussed.     

污水中18种类固醇激素化合物的痕量分析

采用HLB固相萃取柱和超高效液相色谱-串联质谱法(UPLC - MS/MS),在多反应监测(MRM)模式下建立了污水处理厂进出水样中18种类固醇激素(群勃龙、诺龙、雄烯二酮、睾酮、炔诺酮、甲基睾酮、孕酮、康力龙、羟孕酮、泼尼松、氢化泼尼松、六甲强龙、醋酸甲地孕酮、安宫黄体酮、倍他米松、地塞米松、醋酸泼尼松龙和醋酸氢化可...     

Colorimetric Microdetermination of some Corticosteroid Drugs Using Indophenol as Chromophoric Reagent

Abstract

A simple, rapid and sensitive method for the determination of betamethasone (I), dexamethasone (II) and hydrocortisone (III), either in the pure form or in pharmaceutical formulations is described. The method is based on the development of a brown product with indophenoi in basic aqueous-ethanolic (50% v/v) medium. The optimum reaction conditions for the charge transfer complex formed were assessed. The absorbance measurements were made at 820, 816 and 822 nm for I, II and III respectively. The calibration graph was linear in the range 1-26, 1-32 and 1-35 μg/ml of I, II and III with slopes of 0.028, 0.021 and 0.024, respectively. For more accurate analysis, Ringbom optimum concentration ranges were 2.5-23.0, 3.0-28.5 and 3.0-33.0 μg/ml for I, II and III, respectively. The precision of the procedure was checked by calculating the relative standard deviation of ten replicate determinations on a sample containing 20 μg/ml for each drug and was found to be 1.67, 1.39 and 1.85% for I, II and III, respectively. Many common excepience and common drugs present in their dosage forms do not interfere, and the tolerable levels were evaluated. Results of analysis of pure drugs and their dosage forms by the proposed method are in good agreement with those of the British Pharmacopoeia 1993 procedure.     

Reliable routine method for the determination of plasma amitriptyline and nortriptyline by gas chromatography

A gas chromatographic method has been developed for the determination of amitriptyline and nortriptyline in plasma. OV-17 is used in a 1 m long packed column, with a flame ionization detector and an electronic integrator. Five internal standards are added. The base-specific extraction procedure and the method of calibrating the chromatograph are described in detail. The accuracy, precision and reliability of the method are demonstrated by the results of nearly 700 determinations of each drug, at concentrations ranging from 5 to 400 ng/ml in the plasma. An interlaboratory comparison with a double radioactive isotope derivative assay for nortriptyline has also shown satisfactory agreement.     

Kinetic spectrophotometric determination of hydrocortisone acetate in a pharmaceutical preparation by use of partial least-squares regression

A kinetic spectrophotometric method for the determination of hydrocortisone acetate based on its condensation with isonicotinic acid hydrazide is proposed. The method is applied to the determination of hydrocortisone acetate in a commercially available pharmaceutical preparation, presented as a pomade, that also contains another corticosteroid and additional active compounds. The operating procedure involves dissolving the pomade in chloroform and the addition of the reagent solution directly to the cuvette, in this way avoiding the previous extraction of analytes from the insoluble pomade matrix required by the alternative HPLC procedure. Calibration is performed by partial least-squares regression, using absorbance or first derivative spectra values recorded each minute during the first 30 min of reaction. Use of first derivative spectra overcomes possible scattered light problems produced by excipients precipitating, and produced slightly better results than absorbance data. The relative standard deviation obtained for 11 replicates analysed on different days was approx. 1.5%. The proposed method improves both accuracy and precision of the classical initial rate method and the precision of the HPLC procedure.     

High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C18 column (Spherisorb, 5 micron) using a mobile phase of 2% glacial acetic acid, 30--35% acetonitrile, 70--65% water with ultraviolet detection (254 nm). The method uses 17 alpha-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 microgram/ml for HC, 0.07 microgram/ml for MP, 0.04 microgram/ml for MPHS, and 0.10 microgram/ml for HCHS, with a detection limit of 0.02 microgram/ml for all four steroids. Calibration curves are linear up to 3 micrograms/ml for MP or MPHS (as equivalent MP) and up to 4 micrograms/ml for HC and 7 micrograms/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate for each steroid is less than 7%. Plasma concentration--time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.     

Determination of betamethasone and triamcinolone acetonide by GC-NCI-MS in excreta of treated animals and development of a fast oxidation procedure for derivatisation of corticosteroids.

The use of corticosteroids in combination with other hormonal substances has long been known to result in increased mass gain with bovines. Practice has demonstrated, however, that even the single use of a glucocorticoid may result in growth promoting effects. In addition to the popular dexamethasone, more recently other corticosteroids have also been misused for fattening purposes. The first part of this study deals with the detection of two of them, namely betamethasone and triamcinolone acetonide. Betamethasone was administered orally to a cow at a dose of 50 mg d-1 for 5 d, then later the same cow was injected intramuscularly with a dose of 50 mg of betamethasone dipropionate. Excretion in urine and faeces was followed with both HPLC-enzyme immunoassay and a previously described method based on negative chemical ionization mass spectrometry (NCI-MS) after oxidation. For the triamcinolone acetonide study a cow was treated with 50 mg d-1 of the drug during a 7 d period. Excretion in faeces was followed with GC-NCI-MS. As triamcinolone acetonide is resistant to the previously described oxidation procedure, however, a hydrolysis step had to be introduced prior to oxidation. In addition to this specific modification necessary for triamcinolone acetonide, in a subsequent part of this study the original oxidation procedure with pyridinium chlorochromate was re-investigated especially to shorten the procedure. With the introduction of potassium dichromate the reaction time could be decreased from 3 h to 10 min.     

Simultaneous determination of dexamethasone and betamethasone in pharmaceuticals by reversed-phase HPLC

Chromatographia - An HPLC method for the simultaneous determination of the glucocorticoids betamethasone and dexamethasone is described. The method based on the separation of these compounds using...     

Application of high-performance liquid chromatography to the study of the biological transformation of adiponitrile by nitrile hydratase and amidase.

A procedure for the assay of nitrile hydratase and amidase activities by high-performance liquid chromatography is described. The method can be used to assay the intermediate compounds resulting from the hydrolysis of adiponitrile to adipic acid, and to determine the kinetics of the hydrolysis of these compounds using whole cells and enzyme extracts. The precision of the method makes it suitable for the determination of the enzymic parameters.     

Mechanism of base-catalyzed autooxidation of corticosteroids containing 20-keto-21-hydroxyl side chain

Corticosteroids and related compounds containing the 20-keto-21-hydroxyl side chain such as betamethasone, betamethasone 9,11-epoxide, dexamethasone, and dexamethasone 9,11-epoxide have been found to undergo facile autooxidation on the 1,3-dihydroxyacetone side chain of their D-rings under strong alkaline conditions to yield five main degradants (17-formyloxy-17-acid, 17-acid, 21-aldehyde, 20-hydroxy-21-acid, and 17-ketone). The rate of the autooxidation was correlated with the strength and concentration of the base used in the reaction. A novel mechanism for the observed autooxidation is proposed, in which the facile oxidation of the presumed enolate (resulting from the carbanion at the 21-position) by molecular oxygen is the key step. The proposed autooxidation mechanism, supported by LC-MS isotope experiments using ¹⁸O₂ as the oxidant, can satisfactorily explain the oxidative degradation patterns observed for corticosteroids containing the 20-keto-21-hydroxyl side chain.     

Spectropolarimetric Assay of R(-)-Epinephrine

Abstract

A rapid, simple and specific procedure for the simultaneous assay and determination of enantiomeric purity of pharmaceutical grade 1-epinephrine solution is described. The procedure is faster, more precise and more specific for the levoisomer than the U.S.P. XXI method. It can also be used for 1-epine-phrine inhalation solution and 1-epinephrine injection solution.     

Differential pulse polarographic determination of hydrocortisone in pharmaceutical preparations

Differential pulse polarograms of hydrocortisone recorded from acetate buffer pH 4.6 exhibit a well-defined peak at —1.25 V vs. SCE and the peak current is proportional to the concentration in the range 10^-5–8 × 10^-5 M. The electrode reaction involves one electron and one hydrogen ion. A simple rapid method is proposed for the determination of hydrocortisone in creams and ointments. The procedure does not involve time-consuming extractions, but it is not applicable to samples containing surfactants like polyethyleneglycol which are more strongly adsorbed on the electrode than hydrocortisone. Because degradation in the side chain of hydrocortisone is not detected polarographically, the method is suitable for production control but not for stability tests.