A reversed-phase high-performance liquid chromatographic assay for the determination of N-acetylcysteine in aqueous formulations

A stability-indicating assay is described for the determination of N-acetylcysteine in aqueous pharmaceutical formulations. The sample is diluted to an appropriate concentration with dilute aqueous orthophosphoric acid. An aliquot of the solution, containing added l-tyrosine as an internal standard, is chromatographed using a 10-mum C(18) stationary phase with dilute orthophosphoric acid (pH 2.0) containing 0.5% w/v of sodium perchlorate as the mobile phase. The assay, which has a relative standard deviation of about 0.8%, can also be used as a test for related impurities in N-acetylcysteine. It is also suitable for determining the N-acetylcysteine content of the drug substance.     

Simultaneous determination of codeine and ibuprofen in plasma by high-performance liquid chromatography

A procedure is described for the simultaneous determination of codeine and ibuprofen in human plasma following the administration of the two substances in a proposed combination dosage form. The two substances were extracted separately from plasma and then determined together by high-performance liquid chromatography (HPLC) using a fluorescence detector. The codeine was first extracted from alkalinized plasma with hexane-dichloromethane (2:1, v/v) and then washed with sodium hydroxide solution. The ibuprofen was then extracted with hexane from the plasma acidified with sulphuric acid. The organic layers were collected, evaporated to dryness and the reconstituted residue was subjected to HPLC. The detection limit for codeine was 8 microg 1(-1) and for ibuprofen 1 mg 1(-1).     

The determination of D-penicillamine and its disulphide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Reversed-phase ion-pair conditions are used for the determination of D-penicillamine and penicillamine disulphide. Two chromatographic systems were employed, one for penicillamine and the other for penicillamine disulphide. The procedures permit the determination of total penicillamine (protein-bound, free and as disulphides) in whole plasma, and total penicillamine (free and as disulphides) in plasma ultrafiltrate, using an incubation step in the presence of dithiothreitol. Free penicillamine and penicillamine disulphide may be determined independently by direct injection of plasma ultrafiltrate. Both solutes may be measured at an on-column sensitivity of 10 ng, utilizing an electrochemical detector based on a glassy carbon electrode.     

The determination of nadolol in biological samples using high-performance liquid chromatography

A method has been developed for the determination of nadolol in biological samples by reversed-phase high-performance liquid chromatography with fluorimetric detection. The method has been applied to plasma, serum and urine samples, which are prepared by extraction with diethyl ether-dichloromethane (5:2,v/v), evaporation of the organic solvent, and dissolution of the resultant residue in the chromatographic eluent. The sample is then subjected to chromatography on a C(18)-silica column, with an eluent of water-acetonitrile-triethylamine (800:200:1,v/v) adjusted to pH 3.0 with orthophosphoric acid. A single point external standard is used for quantitation. The working ranges were 1-400 ng/ml for plasma/serum, and 0.1-40 mug/ml for urine, although a detection limit of 0.1 ng/ml appears to be readily attainable. The sample size was 0.5 ml, and for both types of sample the method showed good correlation with a previously published fluorimetric method (for plasma, r = 0.9544, n = 70; for urine, r = 0.9919, n = 35).     

Determination of the metabolites of mexiletine in human plasma

A liquid chromatographic method for the determination of mexiletine and unconjugated metabolites in plasma has been developed. A reversed-phase C₁₈ column is used with isocratic elution and either UV or amperometric detection. Sample pretreatment involves double extraction of the metabolites. The method enables the measurement of four mexiletine metabolites at levels as low as 10 ng ml⁻¹, with both precision and accuracy of about 6%. Forty-nine samples from patients receiving mexiletine were analysed. Metabolite VII was found to be the major metabolite (mean concentration 225 ng ml⁻¹), metabolites IX and VI were also found at mean concentrations of 95 and 10 ng ml⁻¹, respectively, whilst metabolite VIII was not detected.     

Chromatographic isolation and estimation of zanthoxylol: an antisickling agent from the roots of Zanthoxylum species

The roots of four species of Zanthoxylum were examined for the presence of zanthoxylol (2-dimethylallyl-4-(3-hydroxy-propyl)phenol) but only Z. zanthoxyloides was found to contain this substance. Purification was effected using Sephadex ionexchange resins; isolation and quantification were performed on reversed-phase HPLC columns. The identity was confirmed by UV, IR, NMR, GC and MS analysis. The zanthoxylol content was calculated as p-hydroxybenzoic acid. The possible chemotaxonomic value of the zanthoxylol content in Zanthoxylum species is speculated.     

Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Montelukast Sodium and Ebastine in Tablet Dosage Form

A rapid and sensitive RP-HPLC method with UV detection (244 nm) for routine analysis of montelukast sodium and ebastine in a pharmaceutical formulation (Ebast-M) was developed. Chromatography was performed with mobile phase containing a mixture of methanol:acetonitrile:ammonium acetate (80:10:10, % v/v/v), pH of mobile phase was adjusted 5.5 using glacial acetic acid and flow rate was 1.2 ml/min. The method was validated for linearity, accuracy, robustness and intermediate precision. The linearity was established over the concentration range of 0.01−0.06 mg/ml for both drugs. The correlation coefficients (r²) for ebastine and montelukast were 0.9989 and 0.9955, respectively. Statistical analysis of the data showed that the method was precise, accurate, reproducible and selective for the analysis of ebastine and montelukast drugs. The method was successfully employed for the determination of ebastine and montelukast in commercially available tablet dosage form.     

手性荧光衍生化反相高效液相法分离DL-异丙肾上腺素

目的探讨R(-)-4-(N,N-Dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidino)-2,1,3-benzoxadiazole(DBD-PyNCS)为手性荧光衍生化试剂,建立DL-异丙肾上腺素对映体的柱前衍生反相高效液相色谱法(RP-HPLC)高灵敏分离及分析的最佳方法。方法分别取DBD-PyNCS乙腈溶液(36mmol/L)、异丙肾上腺素对映体水溶液(1mmol/L)、20%吡啶乙腈溶液各10μL混合于聚丙烯管中,用涡旋搅拌器搅拌1min,在金属恒温仪遮光65℃条件下,反应35min。取衍生产物10μL进样于HPLC中。结果生成的非对映体衍生物(λex=460nm,λem=550nm)在流动相为乙腈-水(35∶65,v/v),流速为1.0mL/min,在Diamon-silTMC18(150mmx4.6mm,i.d.,5μm)色谱柱上分离成两个完全独立的峰,D-异丙肾上腺素、L-异丙肾上腺素的保留时间分别为26min、31min。异丙肾上腺素对映体在3.23×10-4mol/L~2.63×10-3mol/L范围内与峰面积呈良好的线性关系(r=0.9997),峰面积测定结果的相对标准偏差为1.83%(n=7),最低检测限为2.1×10-11mol/L(S/N=3)。结论利用柱前衍生反相高效液相色谱法可以使DL-异丙肾上腺素对映体得到快速、高效的分离。     

Diketopiperazine formation and N-terminal degradation in recombinant human growth hormone

A new degradation process has been identified that occurs in recombinant DNA-derived human growth hormone. Non-enzymatic cyclization of the first two amino acids from the N-terminus and subsequent cleavage results in the formation of a diketopiperazine and a truncated variant of rhGH. The truncated protein was separated using hydrophobic interaction chromatography and identified as desPhe¹Pro²-rhGH using N-terminal sequence analysis, tryptic mapping, and mass spectrometry.     

Development and Validation of RP-HPLC Method for the Determination of Methamphetamine and Propranolol in Tablet Dosage Form

A new isocratic reversed-phase HPLC method with diode-array UV detection was developed and validated for the determination of methamphetamine and propranolol in tablet dosage forms. Chromatography was carried out on an XTerra RP18 (150×4.6 mm, 5 μm) column using 50 mM pyrrolidine (pH 11.5) - acetonitrile (50:50, v/v) as mobile phase at a flow rate of 1 ml/min. Spectrophotometric detection was performed at a wavelength of 214 nm. The linearity was established over the concentration range of 0.075-0.60 mg/ml for both drugs. The correlation coefficients (r(2)) were ≥0.9998 in each case. The relative standard deviation values for intermediate precision studies were <1%. Statistical analysis of the data showed that the method was precise, accurate, reproducible and selective for the analysis of methamphetamine and propranolol drugs. The method was successfully employed for the determination of propranolol and methamphetamine in commercially available tablet dosage form.     

Determination of amoxycillin in plasma by ion pair column extraction and reversed-phase ion pair high-performance liquid chromatography

A quantitative method is described for the determination of amoxycillin in plasma. The method utilizes ion pair extraction of amoxycillin on disposable columns packed with Baker-10 SPE octadecyl, with tetrabutylammonium ion as the counter ion and methanol as the eluent. Separation and quantitation is performed by reversed-phase ion pair high-performance liquid chromatography (Nucleosil C-18) using the same counter ion and a mobile phase of methanol-phosphate buffer (pH 6.0) (31:69 v/v) with detection at 229 nm.     

Synthesis and biological activity of substance P analogues

The synthesis of the hexapeptide [Glu⁶]SP_6-11 and its glycosylated analogue at the Glu⁶γ-carboxyl position by solution procedures according to several strategies is discussed. The biological activity of SP, [GIu⁶]SP_6-11 (VI) and [Glu(β-d -Glcp)⁶]SP_6-11 (VIII) have been determined and compared to SP by the GPI and RVD assays. The introduction of a β-d -glucopyranosyl moiety at the sixth position of the [Glu⁶]SP_6-11 did not affect to a great extent the in vitro activity pattern of the parent hexapeptide.     

Spontaneous degradation via diketopiperazine formation of peptides containing a tetrahydroisoquinoline-3-carboxylic acid residue in the 2-position of the peptide sequence

The δ-opioid antagonists H-Tyr-Tic-Phe-OH (Tic = tetrahydroisoquinoline-3-carboxylic acid) and H-Tyr-Tic-Phe-Phe-NH₂ were shown to undergo spontaneous Tyr-Tic diketopiperazine formation with concomitant cleavage of the Tic-Phe peptide bond in DMSO but not in aqueous solution at pH 7.7.     

高效液相色谱法在合成多肽分离与纯化中的应用

近年来，多肽药物化学形成并迅速发展成为药物化学的重要分支。而随着分离纯化技术的进展，又大大加快了新活性多肽的发现和合成速度。在多肽合成中．许多杂质显示与产物类似的性质，随着肽链的增长，分离的难度也增大，因此纯化的方法和工艺显得异常重要。本文综述了凝胶过滤色谱、离子交换色谱和反相色谱等高效液相色谱技术在合成多肽分离与纯化中的设计和应用。     

Convergent solid-phase peptide synthesis 12. * Chromatographic techniques for the purification of protected peptide segments

The purification of a range of protected peptide segments has been carried out using modified reversed-phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue. © Munksgaard 1995.     

Stability-indicating determination of adrenaline in injections containing procaine

Adrenaline was determined in injections containing procaine in a 1000-fold excess by reversed-phase high-performance liquid chromatography using UV detection at 205 nm and aqueous sulphuric acid (100 μmol/l) as eluent. The relative standard deviation was 2.1%, and the method was selective in the presence of adrenaline degradation products. Changes of the capacity factor with pH and ionic strength of the eluent were studied, and a simple model is suggested to explain the retention data.     

The combined use of high-performance liquid chromatography and radioimmunoassay for the bioanalysis of nicomorphine and its metabolites

Methods have been developed for the determination of nicomorphine using reversed-phase HPLC with UV detection; for the simultaneous assay of morphine and mononicotinoylmorphine by a coupled normal-phase HPLC-radioimmunoassay method; and for conjugates of morphine and mononicotinoylmorphine by radioimmunoassay. The methods have been evaluated and applied to a pharmacokinetic study of nicomorphine administered intramuscularly.     

A comparison of the release of substance P and some synthetic analogues from micropipettes by microiontophoresis or pressure

The release of substance P (SP) and two analogues by iontophoresis or pressure from microelectrodes was compared. Substance P was released linearly by iontophoresis from electrodes while no release of the analogues was detected. [N-methylphenylalanine8, N-methylglycine9-] SP5-11 (DiMeC7) and [methyl-2-aminoethyl]11 SP (SP-DAE) were released from electrodes by pressure ejection with linear relationships in all cases between pressure and the amounts released. Under the tested experimental conditions, release of substance P by iontophoresis was between 2 and 3 orders of magnitude less than that by pressure over a given time. The release of substance P and the uncharged analogue DiMeC7 by pressure was very similar while release of SP-DAE was one order of magnitude less.