HPLC determination of estradiol, its degradation product, and preservatives in new topical formulation Estrogel HBF

This paper deals with the development of a novel method for simultaneous determination of estradiol, its degradation product estrone, and two preservatives, methylparaben and propylparaben, in the topical preparation Estradiol HBF. After optimization of the analytical conditions the method was validated and applied in studies of the stability of the topical preparation Estrogel HBF. Separation of all these compounds was performed on a Supelco Discovery C18 (250 mm×3.0 mm, 5 m) analytical column. A mixture of acetonitrile, methanol, and water (23:24:53 v/v) was chosen as mobile phase. UV absorbance at 225 nm was used for detection and quantitation of analytes. The total analysis time was less than 12 min at a flow rate of 0.9 mL min^–1. All the compounds were isolated from the topical gel by simple extraction with an acetonitrile solution of hydrocortisone, as internal standard, and using sonication and centrifugation thereafter. The supernatant was injected directly on to the analytical column. The recovery of the procedure was from 96.9 to 100.4%. Validation of method according international guidelines was successfully performed.     

Reversed-phase high-performance liquid chromatographic method for the quantification of 5-hydroxymethylfurfural as the major degradation product of glucose in infusion fluids

Analysis of 5-hydroxymethylfurfural (5-HMF) is an important indicator of degradation in glucose infusion fluids. Current pharmacopoeial methods for analysis are insensitive and non-specific. A method is described here, incorporating an internal standard, to give a sensitive, accurate and precise method, suitable for use in the quality control of glucose infusion fluids, and with possible applications for the quantitative determination of 5-HMF in food and drink, bacterial cultures and hydrothermolysed wood.     

HPLC analysis of a syrup containing nimesulide and its hydrolytic degradation product

The present paper deals with the evaluation of nimesulide, 2-phenoxy-4-nitroaniline, the main hydrolytic degradation product of nimesulide, of methylparaben and propylparaben, and eventually of 4-hydroxybenzoic acid by HPLC with UV detection at 254 nm in syrup as a pharmaceutical formulation. HPLC analysis was employed on the reversed phase C18 with methanol and 0.01 M dibasic ammonium phosphate (ρ _r = 60: 40, pH 4.0). Validation was performed using standards and a pharmaceutical preparation containing the compounds described above.     

化妆品中双香豆素和环香豆素的高效液相色谱二极管阵列检测器测定

建立了用高效液相色谱(HPLC)/二极管阵列检测器(DAD)测定化妆品中两种香豆素物质--双香豆素和环香豆素的方法.样品用乙腈-氢氧化钠溶液(0.1 mol/L)(体积比9:1)的提取溶液超声提取,高效液相色谱DAD扫描检测,并在306 nm波长进行分析.用保留时间结合二种香豆素的紫外光谱定性,外标法定量,并且采用液质联用(LC-MS/MS)确证.双香豆素的回收率为96%～105%,精密度RSD为0.51%～2.08%,定量下限为1 ng;环香豆素的回收率为88%～104%,精密度RSD为0.51%～3.36%,定量下限为1 ng.     

GC-MS与HPLC/DAD联用对纺织品中4-氨基偶氮苯的测定

从化学反应条件的控制及气相色谱-质谱(GC-MS)和高效液相色谱/二极管阵列检测器(HPLC/DAD)联用方面,探讨了4-氨基偶氮苯的检测方法。实验表明,在样品中加入氢氧化钠,样品中的4-氨基偶氮苯和新鲜配制的连二亚硫酸钠发生反应,使4-氨基偶氮苯的碳碳双键断裂。释放的4-氨基偶氮苯在氯化钠存在的混合物中与叔丁基甲醚进行液液萃取。取叔丁基甲醚层进行分析,通过GC-MS的保留时间及特征离子碎片并结合HPLC/DAD紫外法对4-氨基偶氮苯进行定性和定量分析。4-氨基偶氮苯在2.5~30 mg/kg范围内呈良好的线性关系,其线性方程为:y=91.23x+5.272,相关系数r=1.000,检出限为3.20 mg/kg,平均回收率不低于90%。     

Determination of azidothymidine and its degradation product thymine in pharmaceutical dosage forms by HPLC and HPTLC densitometry

Summary HPTLC densitometry and HPLC are considered for the determination of azidothymidine and its degradation product thymine in pharmaceutical dosage forms. In HPTLC the substances were separated on silica gel with fluorescence indicator in methanol-chloroform (1090) and methanol-chloroform (1585) systems. Absorbance measurement (detection of reflectance) of the separated substances was carried outin situ at 268 nm using four-level calibration (external standard, linear regression function) in the concentration range of 25–100 ng thymine/spot and using single-level calibration (external standard) at the concentration of 100 ng azidothymidine/spot. HPLC was carried out using RP-18 stationary phase and methanol+aqueous 0.03 mol/l KH₂PO₄ (18+82, v/v) as the mobile phase. The temperature was 50°C and the detection wavelength 266 nm. The detection limit of thymidine was 0.05%. The concentration range for azidothymidine was 0.5–1.5 mg/ml and for thymine 1–40 g/ml (for an injection volume of 10 l). The results were evaluated by linear regression analysis.     

Development and validation of an HPLC method to quantify 3,4‐dideoxyglucosone‐3‐ene in peritoneal dialysis fluids

A method was developed and validated to quantify 3,4‐dideoxyglucosone‐3‐ene in peritoneal dialysis fluids by high‐performance liquid chromatography with UV detection after derivatization with o‐phenylenediamine. The advantages of this method compared with direct HPLC analysis are (i) the possibility of quantifying 3,4‐dideoxyglucosone‐3‐ene simultaneously together with other glucose degradation products, (ii) the compatibility of the method with MS detection for unequivocal identification of the analyte and (iii) a bathochromic shift of the UV absorbance maximum which leads to higher selectivity. The validated method was used to measure 3,4‐dideoxyglucosone‐3‐ene concentrations additionally to the glucose degradation products 3‐deoxyglucosone, methylglyoxal, glyoxal, 5‐hydroxymethylfurfural, 2‐furaldehyde, formaldehyde and acetaldehyde in 19 commercial products for peritoneal dialysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of a new degradation product of the antifouling agent irgarol 1051 in natural samples

A main degradation product of Irgarol [2-(methylthio)-4-(tert-butylamino)-6-(cyclopropylamino)-s-triazine], one of the most widely used compounds in antifouling paints, was detected at trace levels in seawater and sediment samples collected from several marinas on the Mediterranean coast. This degradation product was identified as 2-methylthio-4-tert-butylamino-s-triazine. The unequivocal identification of this compound in seawater samples was carried out by solid-phase extraction (SPE) coupled on-line with liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS). SPE was carried out by passing 150 ml of seawater sample through a cartridge containing a polymeric phase (PLRP-s), with recoveries ranging from 92 to 108% (n=5). Using LC-MS detection in positive ion mode, useful structural information was obtained by increasing the fragmentor voltage, thus permitting the unequivocal identification of this compound in natural samples. Method detection limits were in the range of 0.002 to 0.005 microg/l. Overall, the combination of on-line SPE and LC-APCI-MS represents an important advance in environmental analysis of herbicide degradation products in seawater, since it demonstrates that trace amounts of new polar metabolites may be determined rapidly. This paper reports the LC-MS identification of the main degradation product of Irgarol in seawater and sediment samples.     

Identification and quantification of cotton-bound,cyclic polycarboxylic acids by means of isocratic HPLC

Cotton fabrics are modified by means of polycarboxylic acids (PCA) in combination with an inorganic catalyst in order to impart durable press properties. To evaluate the effectiveness of cyclic PCA, 100% cotton fabrics were treated with 1,2,3,4,5,6-cyclohexanehexacarboxylic acid (CH-HCA), 1,3,5-cyclohexanetricarboxylic acid (CH-TCA), 1,2,3,4-cyclopentanetetracarboxylic acid (CP-TCA), and 1,2,3,4-tetrahydrofurantetracarboxylic acid (THF-TCA) in combination with sodium hypophosphite (SHP) as catalyst. The amount of PCA that reacted with the cellulosic material was determined by means of isocratic HPLC (Aminex HPX-87-H). The results clearly indicate that the cyclic PCA are less effective in respect of durable press performance. CH-TCA does not react with the cellulosic material thus confirming the assumption that the crosslinking reaction between PCA and the cellulose proceeds via a five-membered cyclic anhydride.     

QuEChERS/HPLC/DAD法同时检测果蔬中多种植物激素残留

采用高效液相色谱法,建立了同时分析玉米素(Z)、赤霉酸(GA)、多效唑(PBZ)、4-氟苯氧乙酸(4-FPA)、4-氯苯氧乙酸(4-CPA)、吲哚-3-乙酸(IAA)、吲哚-3-丁酸(IBA)、6-苄氨基嘌呤(6-BA)、脱落酸(ABA)、萘乙酸(NAA)、氯吡脲(CPPU)、2,4-二氯苯氧乙酸(2,4-D)及2,4,5-三氯苯氧乙酸(2,4,5-T)13种植物激素含量的方法。采用含0.5%甲酸的80%乙腈进行提取,分散固相萃取吸附剂(C18和硅藻土)进行净化,选取Waters XBridge C_(18)色谱柱,以乙腈-水为流动相进行梯度洗脱,二极管阵列检测器200~400nm检测,外标法定量。结果表明,13种植物激素在50 min内可实现基线分离,在线性范围内的相关系数(r)为0.992 1~0.999 3;加标回收率为68.4%~95.1%;相对标准偏差(RSD)均小于5%;方法的检出限为0.005~0.020 mg/kg;定量下限为0.01~0.09 mg/kg。该方法前处理操作快速、简便,具有良好的灵敏度、精密度和回收率,适用于果蔬的质量监控。     

Determination of methylparaben,propylparaben, triamcinolone acetonide and its degradation product in a topical cream by RP-HPLC

A novel reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of active component triamcinolone acetonide, its degradation product triamcinolone (occurring in formulation after long-term stability tests) and two preservatives presented in the cream-methylparaben and propylparaben, using hydrocortisone as an internal standard.The chromatographic separation was performed on a Supelco Discovery C18 column; the mobile phase for separation of all compounds consists of a mixture of acetonitrile and water (40:60 v/v). The analysis time was less than 9 min, at a flow rate of 0.6 mL min(-1) and detection at 240 nm. The method was found to be applicable for routine analysis (stability tests, homogeneity) in the pharmaceutical product topical cream Triamcinolon cream 0.1%.     

A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP‐HPLC method

The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital‐pretreated rats and detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3′(R)‐hydroxy‐nodakenetin‐3′‐ol and 3′(S)‐hydroxy‐nodakenetin‐3′‐ol, respectively, on the basis of their ¹H‐NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP‐HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C₁₈ column, a mobile phase with MeOH‐H₂O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat‐inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50–400, 20–120 and 150–24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI. Copyright © 2009 John Wiley & Sons, Ltd.     

Total metal concentrations in serum of dialysis patients and fractionation of Cu, Rb, Al, Fe and Zn in spent continuous ambulatory peritoneal dialysis fluids

Total metal concentrations were determined in the serum of 12 continuous ambulatory peritoneal dialysis (CAPD) patients and in fresh and spent CAPD fluids by electrothermal and flame atomic absorption spectrometry (ETAAS, FAAS). Concentrations of Cu in serum of CAPD patients ranged from 720 to 1780 ng cm⁻³, Rb from 128 to 346 ng cm⁻³, Al from 10 to 72 ng cm⁻³, Fe from 800 to 2300 ng cm⁻³ and Zn from 659 to 1310 ng cm⁻³. The accuracy of the analytical procedure was checked by the analysis of the reference material Seronom™, Trace Elements in Serum. Good agreement between the certified and determined values was obtained for Al, Cu, Fe and Zn. The data on the total metal concentrations in CAPD fluids indicated that during CAPD fluid exchange the losses of Cu from 5.0 to 35 ng cm⁻³, of Rb from 50 to 110 ng cm⁻³ and of Al from 3.0 to 14.0 ng cm⁻³ occurred through the peritoneal membrane. Although fresh CAPD fluids contained traces of Fe (3.0-5.0 ng cm⁻³), the transfer of this element took place through the peritoneal membrane into spent CAPD fluid (13.0-38.0 ng cm⁻³). Zn concentrations were in general lower in spent (20.0-80 ng cm⁻³) than in fresh CAPD fluids (∼100 ng cm⁻³). To follow the mechanisms of the transfer of trace elements through the peritoneal membrane of CAPD patients, fractionation of metals was carried out in spent CAPD fluids by size exclusion chromatography with UV and AAS detection, applying Superdex HR 10/30 column. The chromatographic run was followed at 278 nm and separated metal species also determined ‘off line’ in 1 cm³ fractions by ETAAS or FAAS. From the UV chromatograms and AAS analysis of trace elements in the separated fractions it was demonstrated that Cu, Al, Fe and Zn were bound to proteins and only partially to low molecular weight (LMW) species, while Rb was associated exclusively with LMW species. For characterisation of the high molecular weight (HMW) binding proteins, fractions containing trace elements were subjected to SDS-PAGE electrophoresis. Al and Fe were presumably bound to transferrin, but due to its low concentration in spent CAPD fluids, it was not possible to confirm its presence in the separated fractions. About 10% of Al and 15% of Fe corresponded to LMW species. A fraction of HMW proteins of Cu in spent CAPD fluids was most probably bound to albumin and Zn to albumin and globulins. About 50% of Cu and Zn existed in LMW proteins, while Zn was also found partially in ionic form.     

Validation of a HPLC/MS method for simultaneous quantification of clonidine,morphine and its metabolites in human plasma

A high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of morphine, morphine's major metabolites morphine‐3‐glucuronide and morphine‐6‐glucuronide, and clonidine, to support the pharmacokinetic analysis of an ongoing double‐blinded randomized clinical trial that compares the use of morphine and clonidine in infants diagnosed with neonatal abstinence syndrome. Plasma samples were processed by solid‐phase extraction and separated on an Inertsil ODS‐3 (4 μm) column using an 0.1% formic acid in water–0.1% formic acid in methanol gradient. Detection of the analytes was conducted in the positive multiple reaction monitoring mode. The range of quantitation was 1–1000 ng/mL for morphine, morphine‐3‐glucuronide and morphine‐6‐glucuronide, and 0.25–100 ng/mL for clonidine. Intra‐day and inter‐day accuracy and precision were ≤15% for all analytes across the quantitation range. Extraction recovery rates were ≥94% for morphine, ≥90% for M3G, ≥87% for M6G and ≥ 79% for clonidine. Matrix effect ranged from 85–94% for clonidine to 101–106% for M3G. The method fulfilled all predetermined acceptance criteria and required only 100 μL of starting plasma volume. Furthermore, it was successfully applied to 30 clinical trial plasma samples.     

Identification and analysis of a degradation product of the glucocorticoid desonide in ointment

The major degradation product of desonide in a pharmaceutical ointment formulation has been shown to be identical with the C-17-carboxylic acid obtained on oxidative cleavage of the alpha-ketol group of desonide with alkaline hydrogen peroxide. The pKa value of this acid has been estimated from chromatographic data.     

Photocatalytic removal of tetracycline by a Z-scheme heterojunction of bismuth oxyiodide/exfoliated g-C3N4: performance,mechanism, and degradation pathway

Antibiotics, once being released into the environment, become recalcitrant organic pollutants, which pose a potential risk to ecological balance and human health. In this study, a Z-scheme heterojunction of bismuth oxyiodide (BiOI)/exfoliated g-C₃N₄ (BiOI/ECN hereafter) was synthesized by the combination of thermal exfoliation of g-C₃N₄ and chemical precipitation of BiOI for efficient photocatalytic degradation of tetracycline in aqueous solutions under visible light irradiation. The optimized BiOI/ECN delivered an outstanding degradation rate at circa 0.0705 min^?1, which was 10 times higher than that of the bulk g-C₃N₄. The photocatalytic degradation efficiency of tetracycline remained almost unchanged in a pH range of 3–11, and the BiOI/ECN displayed an excellent photostability upon recycled usage. The photocatalytic mechanism of tetracycline was ascribed to the main reactive oxidation species of photogenerated holes and superoxide radicals. In addition, the possible degradation pathways of tetracycline were investigated by HPLC-MS to identify intermediates. The toxicity of photocatalytic-generated intermediates of tetracycline was found significantly alleviated according to the calculation of quantitative structure–activity relationship prediction. This work not only provides an attractive photocatalyst for the removal of tetracycline but also opens a new avenue for rational design of Z-scheme heterojunction composites for tetracycline degradation.     

Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reverse-phase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm?×?4.6 mm, 5 μm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ?=?214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines. We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.     

Improved RP‐HPLC method for determination of bovine lactoferrin and its proteolytic degradation in simulated gastrointestinal fluids

The objective of this study was to qualitatively and quantitatively evaluate bovine lactoferrin (bLf) and its stability using a rapid RP‐HPLC method. bLf could be rapidly detected within 20 min and quantitated at levels down to 5 µg/mL, and the equation of linearity was y = 86.10x + 178.31 with the correlation coefficient (r²) 0.9997. Quantitative data obtained in the present study proved the improved RP‐HPLC method to be a sensitive and accurate analytical tool for bLf determination. The proteolytic cleavage of bLf in simulated human gastrointestinal fluids was further analyzed by RP‐HPLC, and found to follow pseudo‐first‐order kinetics. The typical equation obtained by pepsin was log₁₀ [A_t]/[A₀] = ?0.03x (r² = 0.85), and log₁₀ [A_t]/[A₀] = ?0.01x (r² = 0.81) for trypsin and chymotrypsin combination. Pepsinolysis of bLf in simulated gastric fluid was relatively fast with the half‐life t_1/2 23.1 min. The digestion of bLf in simulated intestinal fluid was slower with about a 3‐fold increase in half‐life (69.3 min). After the complete proteolysis of bLf, small cleaved peptide fragments were fully separated and identified by RP‐HPLC. The proteolytic study indicated that this validated RP‐HPLC was able to evaluate bLf stability though monitoring the derivatization products. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and quantification of gossypol in cotton by using packed micro-tips columns in combination with HPLC

Self-packed micro-tip columns containing a C₁₈-bonded silica stationary phase, based on the same principles as solid-phase extraction methods, were used to obtain gossypol and related sesquiterpenoid aldehyde-enriched fractions. The enriched metabolite fractions were then analyzed by optimized high-performance liquid chromatography (HPLC) with a C₁₈ column (4.6 mm×25 cm) eluted with the binary mobile phase acetonitrile–0.1% aqueous TFA solution (80:20). This method has proven to be highly reproducible. The precision and accuracy, as %RSD and %RME values, were determined to be less than 15% for the method. The minimum detection limit of gossypol was determined to be 10 ng (absolute gossypol). Absolute recovery was greater than 94% with a standard deviation of ±3.68%. This is a simple, fast, and cost-effective method for isolation, identification, and quantification of gossypol and related secondary metabolites. Comparative analysis of gossypol content was performed on different parts of the cotton plant (seeds, stems and leaves) of two different cultivars of Gossypium hirsutum L. (Acala_1517–70 and OR₁₉). The results indicate that the OR₁₉ cv naturally contains higher gossypol levels than the Acala cv. It was also found that treatment of leaves with a Verticillium dahliae-derived elicitor induced production of deoxyhemigossypol rather than gossypol.