Studies on steroids. CCXVI. Separation of bile acid 3-glucuronides by high-performance liquid chromatography

The separation of 3-glucuronides of cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate and lithocholate, and their glyco- and tauro-conjugates, has been carried out by high-performance liquid chromatography on a reversed-phase column. The chromatographic behaviour of bile acid 3-glucuronides was dependent on the type of conjugation. An effect of the pH of the mobile phase on the capacity ratio was observed at higher pH for chenodeoxycholate 3-glucuronide, probably owing to steric interaction of the 7 alpha-hydroxy group with the carboxy group in the glucuronyl moiety. Conversion of the alpha-hydroxy function on the steroid nucleus into an oxo group resulted in a 50% decrease in the capacity ratio. Bile acid 3-glucuronides were efficiently separated on Shodex ODS Pak F-411 using three kinds of ammonium phosphate buffer-acetonitrile systems.     

Analysis of etimicin sulfate by liquid chromatography with pulsed amperometric detection

A new method for determination of etimicin's (ETM) purity and content is developed by liquid chromatography (LC) and pulsed amperometric detection (PAD). A reversed-phase ion-pair LC method with pulsed amperometric detection on a gold electrode after post-added NaOH is described. The mobile phase consisted of an aqueous solution containing 0.033 mol L(-1) oxalic acid, 0.012 mol L(-1) heptafluorobutyric acid, and 210 mL L(-1) acetonitrile with pH 3.40 adjusting by dilute NaOH solution. The total analysis time was not more than 30 min. The effects of the different chromatographic parameters on the separation were also investigated. A number of commercial samples of etimicin sulfate were analyzed using this method.     

CCXVII. Separation and characterization of bile acid 3-glucuronides in human urine by high-performance liquid chromatography

The separation and characterization of unconjugated and conjugated bile acid 3-glucuronides in biological fluids without prior deconjugation by high-performance liquid chromatography (HPLC) are described. A urine sample from a patient with obstructive jaundice was passed through a Sep-Pak C18 cartridge and was separated into groups by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20, providing the glucuronide fraction. Subsequent resolution into individual 3-glucuronides was attained by HPLC on muBondapak C18 and Shodex ODS Pak F-411 columns. The 3-glucuronides of cholate, deoxycholate, chenodeoxycholate, glycocholate, glycochenodeoxycholate and taurochenodeoxycholate were identified on the basis of their behaviour in HPLC using mobile phases of different pH. The enzymatic hydrolysis of these glucuronides and derivatization of deconjugated bile acids with 1-anthroyl nitrile followed by chromatographic separation on a Cosmosil 5C18 column with fluorescence detection were carried out for unequivocal characterization. The ratio of unconjugated, glyco- and tauro-conjugated bile acid 3-glucuronides excreted in urine was found to be ca. 2:3:1.     

Pulsed electrochemical detection of orotic acid by an activated potential waveform at a gold working electrode following anion-exchange chromatography

The application of activated pulsed amperometric detection (APAD) for the determination of orotic acid (OrA) in real samples at a gold working electrode in alkaline solutions, in combination with anion-exchange chromatography, is reported. Such an activated potential waveform was designed with an initial step that involves the formation of redox active species (e.g., adsorbed AuOH/AuO), which in turn is halted upon lowering the applied potential at the detection value while the adsorbed gold hydroxide/oxide species are still catalytically active. A direct comparison between the activated potential waveform and the more commonly used pulsed amperometric detection showed roughly a 20-fold increase in sensitivity. The chromatographic separation of OrA was accomplished by using a microbore anion-exchange column eluted with an isocratic mobile phase composed of 100 mM NaOH+40 mM NaNO(3). Orotic acid was determined at the concentration ranges of 0.2-30 microM (r=0.9997) with an absolute detection limit of 80 pg (10 microL injected). The levels of OrA in cows' milk samples evaluated by standard additions, using 5-aminoorotic acid as an internal standard, ranged from 56 to 126 mg/L. Lower levels were found in raw sheep's milk (<20 mg/L). The assay is shown to be very useful in clinical investigations where relatively high levels of OrA in human urine are correlated to metabolic diseases.     

反相高效液相色谱-脉冲电化学检测法测定重组人生长激素中异丙基-β-D-硫代半乳糖苷

采用反相高效液相色谱（RP-HPLC）-脉冲电化学检测（PED）法测定了重组人生长激素（rhGH）中的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）。rhGH样品经超滤离心后,用超纯水提取,经C18色谱柱分离,用脉冲电化学器检测。结果表明,样品中添加0.02~0.10 mg/L的IPTG,其回收率为100%~102%,相对标准偏差（n=3）小于10%。在优化的条件下,IPTG的检出限可达1 μg/L（0.1 pmol,25 μL）。该方法简便高效,具有良好的灵敏度、回收率和重复性。     

高效阴离子交换色谱在易极化阴离子痕量分析中的应用

以45 mmol/L的 NaOH溶液作淋洗液,采用强亲水性的IonPac AS16阴离子交换分析柱和脉冲安培检测器,在15 min内分离检测了Br-,S2O2-3,I-和SCN-4种易极化阴离子,检测限(以3倍信噪比计)分别为0.5,0.2,0.05和2 μg/L (进样25 μL)。痕量离子的标准溶液平行9次进样测定的相对标准偏差为0.8%～3.7%。而在相同的色谱条件下,抑制型电导检测器对上述4种阴离子的检测限分别为1,1,2和10 μg/L,平行9次进样测定的相对标准偏差为0.9%～4.7%。通过实验比较可知,脉冲安培检测器的灵敏度是抑制型电导检测器的2～40倍。在测定上述易极化阴离子时,脉冲安培检测器具有选择性强、精密度好和灵敏度高等优点。     

A direct method for the separation and quantification of bile acid acyl glycosides by high-performance liquid chromatography with an evaporative light scattering detector

A direct method for the separation and quantification of a series of bile acid acyl glycosides using high-performance liquid chromatography coupled to an evaporative light scattering detector (HPLC-ELSD) is described. Complete separation of each of 15 bile acid acyl 24-alpha-glucosides and their 24-beta-anomers and 24-beta-galactosides was achieved by the stepwise gradient elution mode on a C18 column using a mixture of acetonitrile-methanol (8:2, v/v) and 1% aqueous acetic acid as the mobile phase. 24-beta-Galactosides were always eluted faster than the corresponding 24-beta-glucosides, which eluted after the corresponding 24-alpha-anomers. Calibration curves of different 24-beta-galactosides were linear over a range of 0.2-40 nmol of injected amount and the detection limits (S/N > 3) were from 0.08 to 0.1 nmol. The present HPLC-ELSD method may provide an insight into the separation and quantification of the biologically interesting neutral bile acids.     

Analysis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine by ion chromatography and pulsed amperometric detection

A novel high-performance liquid chromatography (HPLC) method is presented for the detection and trace level determination of the tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV). The tripeptide, an intermediate in penicillin production, is derived from fungal fermentation. The technique relies on ion-exchange separation of the tripeptide on an anion-exchange column followed by detection by reduction on a gold electrode using pulsed amperometry. The sensitivity of direct determination of ACV is increased by employing pulsed amperometric detection (PAD) over direct ultraviolet detection. Choice of the working electrode and optimisation of electrode potentials was based on cyclic voltammograms recorded for the tripeptide in the mobile phase. A linear regression equation was obtained over the range 0-100 micrograms ml-1. The detection limit in fermentation broths was found to be 0.1 micrograms ml-1 whereas in buffer the detection limit was found to be 10 ng ml-1. A good correlation coefficient was observed when ACV concentrations determined by ion chromatography-PAD were compared with measurements obtained by pre-column derivatisation with fluoromethylorthochloroformate followed by HPLC separation on a reversed-phase C18 silica column with UV detection. The procedure has been applied to the measurement of natural levels of ACV in fermentation broths of selected strains of Aspergillus nidulans and Penicillin chrysogenum.     

In-line coupling capillary electrochromatography with amperometric detection for analysis of explosive compounds

Amperometric detection at a bare gold electrode has been in-line coupled with capillary electrochromatography (CEC) for analysis of nitroaromatic and nitroamine explosives in contaminated soils and ground water. The CEC column packed with 3 microm C18 particles performed best using a mobile phase containing 70-80% methanol, 30 or 20% water, 5 mM sodium dodecyl sulfate (SDS) and 10mM 2-(N-morpholino)ethanesulfonic acid (MES). In contrast, the separation column packed with 1.5 km C18 particles exhibited the best separation when only 30% methanol was added to a mobile phase containing 70% water, 7 mM SDS, and 10 mM MES. The detection, based on electrochemical reduction of the explosives (-0.7 or -1 V vs. Ag/AgCl, depending upon the level of methanol in the mobile phase), was compatible with such mobile phases. The detection limits for 13 explosives ranged from 100 to 200 ppb, i.e., about twofold better than those obtained with electrokinetic chromatography (EKC)/amperometric detection. From an operational viewpoint, exhaustive column conditioning was a prerequisite and care should be taken to prevent bubble formation and current breakdown during the course of separation. The CEC column equipped with amperometric detection successfully measured explosives in ground water and extracts prepared from contaminated soils and the results obtained agreed well with those of the U.S. Environmental protection Agency (EPA) method.     

离子色谱-柱切换技术同步测定饮料中的三氯蔗糖、葡萄糖、果糖和蔗糖

建立了一种离子色谱-柱切换-安培检测技术同步测定饮料中三氯蔗糖、葡萄糖、果糖和蔗糖的方法。以CarboPac PA10阴离子交换保护柱和分析柱进行分离,用水和250 mmol/L NaOH梯度淋洗,脉冲安培检测。弱保留糖类在25 mmol/L NaOH流动相淋洗下分离检测,通过柱切换将保护柱切换至分析柱后,同时切换至250 mmol/L NaOH淋洗,三氯蔗糖仅通过较短的保护柱分离,4种糖类能够得到同步检测。4种糖类在0.01~20 mg/L范围内有良好的线性关系和较低的检出限,重复性好,样品测定的回收率分别为90.38%~102.88%（三氯蔗糖）、95.56%~102.75%（葡萄糖）、101.66%~114.33%（果糖）和105.03%~106.49%（蔗糖）。该方法可广泛应用于食品中强保留物质的测定。     

Optimizing the integrated pulsed amperometric multicycle step waveform for the determination of tetracyclines

A method of modified integrated pulsed amperometric detection with multicycle step waveform (Multi-IPAD) following high-performance liquid chromatography (HPLC) was applied for the determination of tetracyclines (TCs) including dimethyltetracycline (DMTC), oxytetracycline (OTC) and tetracycline (TC). The key advantages of the Multi-IPAD are the abilities to enhance sensitivity and reproducibility and the ability to keep working electrode clean through the use of a high-frequent waveform alteration in integration step and the use of a cleaning potential, which is quite different from conventional three-step potential waveform. The analyses were carried out using the mobile phase of acetonitrile-water mixture solution (10:90, v/v) containing 1% perchloric acid on a C(18) column at a flow rate of 0.21 mL/min. The IPAD waveform parameters were optimized to maximize the signal-to-noise ratio (S/N) and successfully applied for the sensitive detection of TCs. The detection limits (S/N=3, 20 microL injected) were 0.07 mg/L for DMTC, 0.08 mg/L for OTC and 0.05 mg/L for TC. The peak height relative standard deviations (RSDs) of every compound for replicate injection (n=15) determined were below 4.6%.     

Studies on steroids. CCXIX. Separation and determination of 4-hydroxyoestriol monoglucuronides and monosulphates in biological fluids by high-performance liquid chromatography with electrochemical detection

The separation and determination of 4-hydroxyoestriol monoglucuronides and monosulphates by high-performance liquid chromatography with electrochemical detection on a reversed-phase column has been carried out. The effects of the salt, composition and pH of the mobile phase on the resolution were investigated with a Develosil ODS-5 column. Each group of isomeric monoglucuronides and monosulphates of 4-hydroxyoestriol was efficiently resolved on this column when 0.5% sodium acetate-acetonitrile and 0.5% sodium acetate-tetrahydrofuran-acetonitrile were used as mobile phases, respectively. The use of the present method revealed that 4-hydroxyoestriol orally administered to the rat was excreted as 4-, 3-, 16-glucuronides and 4-sulphate in bile.     

Development of a Liquid Chromatography/Amperometric Detection Method for the Determination of Multiresidue Sulfonamide Antibiotics in Meat‐Based Baby Foods

Sulfonamides were oxidized at a polycrystalline gold electrode in neutral buffered phosphate medium at pH 7 and the electrochemical behavior was investigated using cyclic voltammetry technique. A liquid chromatography methodology based on the C₁₈ reverse phase for the simultaneous separation of nine sulfamides, was studied and optimized. The amperometric detector using a polycrystalline gold substrate as working electrode and operating under pulsed amperometric detection mode (PAD) was tested for the determination of the selected molecules. Under optimal chromatographic and amperometric conditions, the limit of detection of the investigated sulfamides are comprised between 1 µM and 0.05 µM and the dynamic linear range spanned generally over three orders of magnitude. A liquid extraction procedure based on the use of acetonitrile solvent was tested and proposed for the quantitative extraction of sulfonamides from homogenized meat samples. The analytical method was successful tested for the determination of sulfonamides in homogenized meat‐based baby foods with reproducibility and recovery levels ranged between 5.0 % and 7.6 %. and 86 %–106 %, respectively.     

Simultaneous determination of rocuronium and its eight impurities in pharmaceutical preparation using high-performance liquid chromatography with amperometric detection

A sensitive and selective HPLC method with amperometric detection (HPLC-ED) for the determination of rocuronium bromide and its eight impurities has been developed. The analysis was performed on Hypersil 100 Silica column 5 microm (250 mm x 4.6 mm; Thermo Electron). The mobile phase consisting of 4.53 g l(-1) solution of tetramethylammonium hydroxide adjusted to pH 7.4 with 85% phosphoric acid:acetonitrile (1:9), was found the best for the separation and determination of the studied compounds. The chromatograms were recorded over 10 min using the amperometric detection at a potential +0.9 V of the glassy carbon electrode versus the reference electrode Ag/AgCl. The limit of quantitation was 45 ng ml(-1) for rocuronium and from 25 to 750 ng ml(-1) for the examined impurities. The proposed HPLC-ED method was successfully applied to the analysis of rocuronium and its impurities in Esmeron solution for injection.     

Simultaneous determination of 13 carbohydrates using high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry

A simple, accurate, and highly sensitive method was developed for the determination of 13 carbohydrates in polysaccharide of Spirulina platensis based on high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry. Samples were extracted with deionized water using ultrasonic‐assisted extraction, and the ultrasound‐assisted extraction conditions were optimized by Box–Behnken design. Then the extracted polysaccharide was hydrolyzed by adding 1 mol/L trifluoroacetic acid before determination by high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and confirmed by high‐performance anion‐exchange chromatography coupled with mass spectrometry. The high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection method was performed on a CarboPac PA20 column by gradient elution using deionized water, 0.1 mol/L sodium hydroxide solution, and 0.4 mol/L sodium acetate solution. Excellent linearity was observed in the range of 0.05–10 mg/L. The average recoveries ranged from 80.7 to 121.7%. The limits of detection and limits of quantification for 13 carbohydrates were 0.02–0.10 and 0.2–1.2  μg/kg, respectively. The developed method has been successfully applied to ambient samples, and the results indicated that high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry could provide a rapid and accurate method for the simultaneous determination of carbohydrates.     

Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of gentamicin

The official method for the determination of the composition and related substances of gentamicin prescribed by the European Pharmacopoeia (Ph. Eur.) is liquid chromatography combined with pulsed electrochemical detection (LC-PED). However, this method utilizes a polymer stationary phase which shows rather low efficiency towards the separation of the main gentamicin components. Moreover, the mobile phase contains a lot of non volatile salts: sodium sulphate and sodium octanesulphonate. Following a comparative study, the most performant LC-PED method has been evaluated and validated using a reversed phase C18 column (C18, 250 x 4.6mm ID, 110 A, 5 microm) kept at 35 degrees C with a mobile phase containing volatile ion pairing agents: trifluoroacetic acetic acid (TFA) and pentafluoropropionic acid (PFPA). In addition to the selectivity of the main gentamicin components and its related substances, the method is repeatable, linear and proves to be robust. It is also applicable to a wider number of C18 columns.     

液相色谱-脉冲安培电化学法测定硫酸卡那霉素中各组分含量

建立了一种用反相离子对液相色谱(LC)分离,以金电极为工作电极的脉冲安培电化学法(PAD)直接检测硫酸卡那霉素中主要组分及杂质含量的分析方法。流动相为0.033mol/L草酸、0.012mol/L七氟丁酸、105mL/L乙腈,用稀NaOH调节pH至3.4。考察了各色谱参数对分离测定的影响。实验证明,本方法不需要衍生化,可直接检测硫酸卡那霉素。与报道的其它方法相比,本方法不仅能使硫酸卡那霉素中的卡那霉素A、B得到了很好的分离,还分离出了其它一些未能确证的组分。如用质谱法等对未知组分进行确证后,对硫酸卡那霉素原料药、注射液、滴眼液及兽药等实际样品中的卡那霉素各组分进行测定。可望成为一种标准分析方法。     

糖的高效阴离子交换色谱-脉冲安培检测法分析

高效阴离子交换色谱-脉冲安培检测法是新近发展的一种分析糖的有效方法。本文讨论了糖的高效阴离子交换色谱分离和脉冲安培法检测的原理,色谱条件的选择,包括分离糖的固定相、流动相以及检测糖的脉冲电位波形,并对方法的应用进行了较为详细的叙述。     

Highly sensitive liquid chromatographic method combined with online ion suppression to remove interfering anions and mass spectrometry for impurity profiling of paromomycin sulfate

A previously developed high-performance liquid chromatography method combined with pulsed amperometric detection allowed to separate many impurities of paromomycin. However, due to the presence of ion pairing agents and sodium hydroxide in the mobile phase, direct coupling to mass spectrometry for the identification of the chemical structures of the impurities was not an option. Indeed, ion suppression was encountered by trifluoroacetic acid and pentafluoroproponic acid in the mobile phase. A cation self-regenerating suppressor, which was originally designed for increasing analyte conductivity of ammonia and amines analysis in ion chromatography, was coupled between the liquid chromatography and ion trap-time of flight-mass spectrometry and almost all trifluoroacetic acid and pentafluoroproponic acid in the mobile phase was removed. The limit of detection of paromomycin in this integrated system improved significantly to 20 ng/ml (0.4 ng). The chemical structures of 19 impurities were elucidated and seven impurities were reported for the first time.     

Development of capillary electrophoresis methods for quantitative determination of taurine in vehicle system and biological media

CE methods have been developed for the determination of taurine in pharmaceutical formulation (microemulsion) and in biological media such as sweat. The CE system with end-column pulsed amperometric detection has been found to be an interesting method in comparison with UV and fluorescence detection for its simplicity and rapidity. A gold-disk electrode of 100 mm diameter was used as the working electrode. The effects of a field decoupler at the end of the capillary, separation voltage, injection and pressure times were investigated. A detection limit of 4 x 10(-5) mol/L was reached using integrated pulsed amperometric detection, a method successfully applied to taurine analysis of the biological samples such as sweat. For taurine analysis of oil-in-water microemulsion, fluorescence detector was the favored method, the detection limit of which was 4 x 10(-11) mol/L.