A sensitive and specific method for the determination of total ribavirin in human red blood cells by liquid chromatography-tandem mass spectrometry

A sensitive and specific method using high-performance liquid chromatography (LC)-tandem mass spectrometry (MS) for the analysis of total ribavirin in human red blood cells (RBC) is developed and validated. The method involves the addition of an internal standard and perchloric acid, the conversion of ribavirin phosphorylated metabolites to ribavirin, purification with a solid-phase exchange cartridge, and LC-MS-MS analysis. The MS-MS is selected to monitor m/z 245-113 for ribavirin and m/z 250-113 for [13C]ribavirin using positive electrospray ionization. The calibration curve is linear over a concentration of 100-10,000 ng/mL with a limit of quantitation of 100 ng/mL. Mean interassay accuracy for quality control (QC) at 100, 1000, and 10,000 ng/mL are 101.8%, 99.4%, and 98.8%, respectively. Mean interassay precision (%CV) for QC at 100, 1000, and 10,000 ng/mL are 5.0%, 5.0%, and 2.5%, respectively. Extractibility of total ribavirin from RBC is confirmed with RBC obtained from a [(14)C]ribavirin-dosed monkey. The method is used to determine the free and total ribavirin concentration in human RBC obtained from hepatitis C patients treated with ribavirin.     

超高效液相色谱-串联质谱法测定鸡肉中的利巴韦林和金刚烷胺

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定鸡肉中利巴韦林和金刚烷胺的分析方法。样品用1%(体积分数)三氯乙酸溶液-乙腈(1:1, v/v)溶液提取,经Supelco LC-SCX固相萃取柱净化后用Acquity UPLC BEH Hillic柱(150 mm×2.1 mm, 1.7 μm)分离,以甲醇和0.1%(体积分数)甲酸作为流动相进行梯度洗脱,电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式进行检测。结果表明,利巴韦林和金刚烷胺在10.0~100.0 μg/L范围内线性关系良好(r²≥0.99)。方法的定量限(信噪比为10)为4.0 μg/kg,在4.0、8.0、20.0 μg/kg添加水平的回收率为78%~102.5%,相对标准偏差(n=6)在2.2%~7.6%之间。该方法快速、灵敏、准确,适合于鸡肉中利巴韦林和金刚烷胺的同时、快速、高灵敏度的分析检测。     

Spectrophotometric determination of azathioprine in pharmaceutical formulations

Four simple and sensitive visible spectrophotometric methods (A–D) have been described for the assay of azathioprine (ATP) either in pure form or in pharmaceutical formulations. Methods A and B are based on the oxidation of ATP with excess N-bromosuccinimide (NBS) or chloramine-T (CAT) and determining the consumed NBS or CAT with a decrease in colour intensity of celestine blue (CB) (method A) or gallocyanine (GC) (method B), respectively. Methods C and D are based on the diazotisation of reduced azathioprine (RATP) with excess nitrous acid and estimating either the consumed nitrous acid (HNO₂) with cresyl fast violet acetate (CFVA) (method C) or by coupling reaction of the diazonium salt formed with N-1-naphthyl ethylene diamine dihydrochloride (NED) (method D). All of the variables have been optimized and the reactions presented. The concentration measurements are reproducible within a relative standard deviation of 1.0%. Recoveries are 99.2–100.3%.     

Determination of niobium and tantalum in binary alloys and zirconium alloys

Recent developments in the metallurgy of niobium, tantalum and zirconium have necessitated provision of analytical procedures for determining niobium and tantalum in the presence of each other and in the presence of zirconium. For this purpose, absorptioinetric procedures based on the formation of yellow coloured complexes, between pyrogallol and niobium or tantalum, have been critically examined. Direct absorptiometric procedures are described, which are suitable for determining niobium or tantalum in the range 2 to 7%; when either of these metals exceeds 7%, differential absorptiometric procedures are recommended. Corrections must lie made for absorption due to the presence of other metals which form complexes with pyrogallol. In tlie determination of niobium or tantalum up to 5%, the precision of the method is about ±0.05%. About 12 determinations can be made in a day, by one analyst.     

Determination of doxorubicin hydrochloride by visible spectrophotometry

Four simple and sensitive visible spectrophotometric methods (A-D) have been described for the assay of doxorubicin hydrochloride either in pure form or in pharmaceutical formulations. Method A was developed based on oxidation of the drug with Fe(III) to produce Fe(II), which subsequently reacts with 1.10-ortho-phenanthroline to form a red colored complex (lambda(max): 510 nm) at pH 4.6. Method B involves the reduction of Folin-Ciocalteu (F-C) reagent by the drug and the reduced species formed possesses a characteristic intense blue color (lambda(max): 770 nm). In methods C and D. oxidation of the drug with periodate at specified experimental conditions yields formaldehyde and dialdehyde, which in turn react either with 3-methyl-2-benzothiazolinone hydrazone hydrochloride to form an intensely brilliant blue cationic dye (lambda(max): 620-670 nm. method C) or by condensation with phenylhydrazine hydrochloride (PHH) to form orange-red colored product (lambda(max): 510 nm, method D) in the presence of potassium ferricyanide. All of the variables have been optimized and the reaction mechanisms presented. The concentration measurements are reproducible within a relative standard deviation of 1.0%.     

Spectrophotometric methods for the determination of nifurtimox in bulk form and pharmaceutical formulations

Three simple and sensitive methods for the assay of Nifurtimox (NIF) which is an active antitrypanocide were developed. These methods are based on the formation of coloured species by treating either its reduction product with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of ferric chloride (method A) or its hydrolysis product with 2-thiobarbituric acid (TBA) (method B) or by oxidizing it with excess N-bromosuccinamide (NBS) and determining the consumed NBS using p-N-methylaminophenol sulphate (metol)-isonicotinic acid hydrazide (INH) (method C). All variables have been optimized and the reaction mechanisms presented. Regression analysis of Beer's plot showed good correlation in the concentration range of 2.5-10, 2.5-30 and 1.25-7.5 microg/ml for methods A, B and C, respectively. No interference was observed from the additives and the validity of the methods was tested by analysing the tablets. Recoveries were 99.2-100.9%.     

Copper-catalyzed synthesis of unsymmetrical triarylphosphines

Various triarylphosphines have been prepared by coupling diphenylphosphine with aryl iodides with catalytic amounts of CuI in the presence of either K(2)CO(3) or Cs(2)CO(3), in good yields. This method can tolerate a variety of functional groups and does not require the use of expensive additives, or harsh reaction conditions, and is palladium free.     

Application of inorganic oxidants to the spectrophotometric determination of ribavirin in bulk and capsules

Eight spectrophotometric methods for determination of ribavirin have been developed and validated. These methods were based on the oxidation of the drug by different inorganic oxidants: ceric ammonium sulfate, potassium permanganate, ammonium molybdate, ammonium metavanidate, chromium trioxide, potassium dichromate, potassium iodate, and potassium periodate. The oxidation reactions were performed in perchloric acid medium for ceric ammonium sulfate and in sulfuric acid medium for the other reagents. With ceric ammonium sulfate and potassium permanganate, the concentration of ribavirin in its samples was determined by measuring the decrease in the absorption intensity of the colored reagents at 315 and 525 nm, respectively. With the other reagents, the concentration of ribavirin was determined by measuring the intensity of the developed colored reaction products at the wavelengths of maximum absorbance: 675, 780, 595, 595, 475, and 475 nm for reactions with ammonium molybdate, ammonium metavanidate, chromium trioxide, potassium dichromate, potassium iodate, and potassium periodate, respectively. Different variables affecting the reaction conditions were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9984-0.9998) were found between the absorbance readings and the concentrations of ribavirin in the range of 4-1400 microg/mL. The molar absorptivities were correlated with the oxidation potential of the oxidants used. The precision of the methods were satisfactory; the values of relative standard deviation did not exceed 1.64%. The proposed methods were successfully applied to the analysis of ribavirin in pure drug material and capsules with good accuracy and precision; the recovery values were 99.2-101.2 +/- 0.48-1.30%. The results obtained using the proposed spectrophotometric methods were comparable with those obtained with the official method stated in the United States Pharmacopeia.     

超高效亲水色谱-串联质谱法快速检测鸡肉及其制品中的利巴韦林及其代谢物的总残留量

建立了鸡肉及其制品中利巴韦林和磷酸化利巴韦林代谢物总残留量的亲水作用色谱-串联质谱分析方法(HILIC-MS/MS)。样品采用含1%(v/v)乙酸的乙腈超声提取,于37 ℃条件下以酸性磷酸酶酶解,正己烷除脂后用C₁₈与PSA填料分散固相萃取净化。采用酰胺基超高效亲水作用色谱柱,在乙腈和0.1%甲酸组成的梯度洗脱流动相中分离,在电喷雾正离子电离多反应监测模式下监测,结果表明,样品中利巴韦林残留量在1~200 μg/kg之间时,线性相关系数大于0.999,方法检出限(LOD, S/N≥3)为1 μg/kg,定量限(LOQ, S/N≥10)为5 μg/kg,在3个添加水平下的回收率为67.8%~112.7%,相对标准偏差为6.1%~13.6%。经测定多种实际样品,证明该方法简便、快速、准确,可以满足日常鸡肉及其制品中利巴韦林及其代谢物总残留量的分析要求。     

Rapid and sensitive HPLC-MS/MS method for pharmacokinetic assessment of ribavirin in healthy Chinese

A rapid and sensitive quantitative assay method was developed for determining ribavirin pharmacokinetic in human plasma. The chromatographic separation was achieved within 4.5 min using a SinoChrom ODS-BP column (4.6 x 150 mm, 5 microm) with acetonitrile-water (1 mmol/L ammonium acetate buffer, 0.1% formic acid; 15:85, v/v) at a constant flow rate of 0.8 mL/min. The MRM pairs were m/z 245.2 --> m/z 113.1 for ribavirin and m/z 226.1 --> m/z 152.1 for acyclovir (internal standard), respectively, with dwell times of 200 ms for each transition. The results showed calibration curve for ribavirin was linear over a concentration range of 1-1000 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL ribavirin. Twenty healthy volunteers received a 300 mg oral dose of ribavirin. Blood samples were then collected up to 120 h postdosing. All plasma data were comodeled for ribavirin by using noncompartmental modeling. The single dose of ribavirin was well tolerated and no serious adverse effects occurred. The mean time to maximum concentration was about 1.25 h. The mean maximum concentration of drug in plasma for oral ribavirin was 250 ng/mL. The mean elimination half-life was 43.6 h. The present study describes a simple, specific, sensitive HPLC-MS/MS method for measuring plasma drug concentration and analyzing human pharmacokinetics of ribavirin.     

Determination of ribavirin in human serum and plasma by capillary electrophoresis

The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 microL sample and reconstitution of the dried extract into 100 microL of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 microg/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-alpha-2b for a hepatitis C virus infection.     

超高效液相色谱-串联质谱法分析鸡蛋中利巴韦林及其代谢物残留

建立了超高效液相色谱-串联质谱法同时快速测定鸡蛋中利巴韦林及其两种主要代谢物 TCONH2和RTCOOH 的分析检测方法。样品采用乙腈-水(9∶1, V/ V)提取,乙腈饱和正己烷除脂,C18结合 GCB 进行固相分散萃取除杂,Agilent ZORBAX SB-Aq 色谱柱(100 mm ×3.0 mm,1.8μm)分离,超高效液相色谱-串联质谱测定。结果表明：利巴韦林、TCONH2和 RTCOOH 分别在2.0~200μg/ L,0.5~200μg/ L,5.0~200μg/ L 浓度范围内,线性良好,相关系数 R2>0.99,检出限分别为0.54,0.09和1.54μg/ L,定量限分别为1.79,0.31和5.13μg/ L。在5.0,10.0和50.0μg/ L 加标水平下,利巴韦林和 RTCOOH 回收率分别为96.1%~99.6%和42.9%~58.3%;在0.5,2.0和5.0μg/ L 加标水平下,TCONH2的回收率为75.9%~106.7%,相对标准偏差均为4.2%~12.7%。实际样品测定结果表明,本方法操作简单、快速、准确,能够满足鸡蛋中利巴韦林及其两种主要代谢物的分析检测。     

Mercurimetric determination of thiocyanates and isothiocyanates

A method for the estimation of thiocyanates and isothiocyanates based on titration with o-hydroxymercuribenzoate (HMB) is described. The thioeyanates are determined as thiols either by direct titration with HMB in alkaline solution, or by back-titration of the unconsumed HMB. The isothiocyanates are converted by means of dimethylamine into the corresponding thioureas, and titrated with HMB in acid solution. Procedures for the selective determination of thioeyanates and isothiocyanates as well as for aliphatic and aromatic isothiocyanates in the presence of each other have been described.     

Rapid determination of antiviral medication ribavirin in different feedstuffs using a novel magnetic molecularly imprinted polymer coupled with high‐performance liquid chromatography

A novel magnetic molecularly imprinted polymer adsorbing material was successfully synthesized to detect ribavirin in animal feedstuff. Molecularly imprinted polymer was prepared through surface polymerization by using ribavirin as template molecule, methyl methacrylate, and γ‐methacryloxypropyl trimethoxy silane functionalized magnetic mesoporous silica as bifunctional monomers, and ethylene diglycidyl ether as crosslinking agent. The prepared magnetic molecularly imprinted polymer was characterized by scanning electron microscopy and infrared spectroscopy. Static and dynamic adsorption experiments and selective adsorption analysis were performed to evaluate the adsorption and selectivity of magnetic molecularly imprinted polymer. Different experiments were conducted to optimize the magnetic solid‐phase extraction conditions. Under optimal experimental conditions, a magnetic molecularly imprinted solid‐phase extraction coupled with high‐performance liquid chromatography method was successfully developed for ribavirin detection. The established method achieved a satisfactory linear range of 0.20–50 mg/L (R² > 0.99) and a low detection limit (0.081 mg/kg). An average recovery of 92–105% with relative standard deviation of <6.5% was obtained upon the application of the developed method to detect ribavirin in real feedstuff samples. Thus, established method can be used for the rapid and simple separation and detection of added ribavirin in feedstuff.     

Spectrophotometric determination of micro amounts of molybdenum and vanadium by reduction of iron(III) in the presence of ferrozine

Molybdenum and vanadium, in the range 0.1-1 ppm have been determined with a relative precision of 1-2% with iron(III) in the presence of Ferrozine. The separation of molybdenum and vanadium from interfering elements has been achieved by ion-exchange with either cation-exchange RG 50 followed by anion exchanger AG 1-X8, or the exchange-resin NBL 17.     

Bismuth compounds in organic synthesis. A one-pot synthesis of homoallyl ethers and homoallyl acetates from aldehydes catalyzed by bismuth triflate

[reaction: see text] Three one-pot methods for the conversion of aldehydes to homoallyl ethers catalyzed by Bi(OTf)(3).xH(2)O (1 < x < 4) have been developed. The one-pot synthesis of homoallyl ethers can be achieved either by in situ generation of the acetal followed by its reaction with allyltrialkylsilane or by a three-component synthesis in which the aldehyde, trimethylorthoformate or an alkoxytrimethylsilane and allyltrimethylsilane are mixed together in the presence of bismuth triflate (0.1-1.0 mol %). In addition, a three-component synthesis of homoallyl acetates, which is achieved by reacting the aldehyde, acetic anhydride, and allyltrimethylsilane in the presence of bismuth triflate (3.0-5.0 mol %), has been developed. The use of a relatively nontoxic, easy to handle, and inexpensive catalyst adds to the versatility of these methods.     

Spectrophotometric Estimation of Nimesulide and its Formulations

 Four simple and sensitive visible spectrophotometric methods (A–D) have been described for the assay of nimesulide (NMD) either in pure form or in pharmaceutical formulations. Methods A and B are based on the oxidative coupling between the reduced product of NMD (RNMD) and p-N,N-dimethyl phenylenediamine dihydrochloride (DMPD) in presence of chloramine – T (CAT) or 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) in presence of ferric chloride (Fe III) to form coloured products with λ_max at 540 nm and 600 nm respectively. Method C is based on the diazotization of RNMD with excess nitrous acid (HNO₂) and estimating the consumed HNO₂ with cresyl fast violet acetate (CFVA). Method D is based on the formation of the coloured charge-transfer complex, when RNMD is treated with metol (p-methyl aminophenol sulphate, PMAP) in presence of potassium dichromate. All variables have been optimized and the reaction sequence is presented. The concentration measurements are reproducible within a relative standard deviation of 1.0%. Recoveries are 98.6–100.2%. Received February 5, 1998. Revision February 20, 1999.     

Indirect Electrochemical Determination of Ribavirin Using Boronic Acid-Diol Recognition on a 3-Aminophenylboronic Acid-Electrochemically Reduced Graphene Oxide Modified Glassy Carbon Electrode (APBA/ERGO/GCE)

A novel method for the indirect electrochemical determination of ribavirin based on boronic acid-diol recognition was developed using the platform as the sensing element. The device was constructed using a 3-aminophenylboronic acid (APBA)-electrochemically reduced graphene oxide (ERGO) modified electrode. When the electrode was immersed in a solution of ribavirin, complexation of boronic acid groups of APBA with ribavirin occurred at the surface of the electrode and simultaneously caused steric hindrance, resulting in a current decrease because the ferricyanide redox probe was unable access the surface. Under the optimized conditions, a linear relationship was obtained between the relative change in current (%Δi) of [Fe(CN)₆]^3?/4?and the concentration of ribavirin at levels from 10.0 to 7.50?×?10²?ng mL^?1. The proposed electrochemical sensor performed with acceptable sensitivity and reproducibility and was successfully used to determine the content of ribavirin in an injection with satisfactory results.     

超高效液相色谱-串联质谱法测定畜禽毛发中利巴韦林及其代谢物

建立了一种测定畜禽毛发中利巴韦林及其代谢物1H-1,2,4-三氮唑-3-甲酰胺(TCONH2)残留的检测方法。1%十二烷基硫酸钠清洗毛发,2%甲酸-甲醇(2:98,V/V)溶液提取,13C-利巴韦林(13CRBV)内标法定量,乙二胺-N-丙基硅烷(PSA)和十八烷基硅烷(C18)基质分散净化,Agilent ZORBAX SB-Aq色谱柱分离,电喷雾串联质谱多反应监测模式测定。利巴韦林和TCONH2在毛发中线性关系良好(R^2>0.99)在2,10,100μg/kg3个添加水平下利巴韦林和代谢物TCONH2的回收率分别为101.5%~108.5%和98.5%~101.5%相对标准偏差均小于7%,检出限和定量限分别为0.2μg/kg和0.5μg/kg。该方法适合于畜禽毛发中违禁抗病毒药物利巴韦林及其代谢物TCONH2的测定。     

Design,Synthesis, and Characterization of Photolabeling Probes for the Study of the Mechanisms of the Antiviral Effects of Ribavirin

Ribavirin, the only small molecule available so far for treating hepatitis‐C‐virus infection, was recently used in an emergency context to treat patients with severe acute respiratory syndrome (SARS) in the early stages of the disease. To study the mechanisms responsible for the antiviral effects of ribavirin by using a photolabeling approach, we designed, synthesized, and characterized the azidotriazole nucleosides 1 and 2 as photolabeling probes of ribavirin. These probes were synthesized either by performing nucleophilic substitution of the corresponding bromotriazole nucleoside with NaN₃ (Scheme 2) or by directly coupling the azidotriazole with the protected ribose sugar (Scheme 4). The azidotriazole nucleosides 1 and 2 showed a fast, clear‐cut photochemical reaction, which suggests that they are promising candidates for use in photolabeling studies.