Digital techniques for luminescence detection in liquid chromatography with an intensified linear photodiode array

The use of a rapid-scanning fluorescence detector in liquid chromatography is examined in the context of its potential contribution in pharmaceutical and biomedical analysis. The data generated by an intensified linear photodiode array detector is presented as an isometric plot of (I_em,λ_em,t) at a defined excitation wavelength. Techniques examined for peak homogeneity assessment include: emission spectral normalisation at points through the chromatographic peak profile, second order differentiation (d²I/dt²) and the fluorescence emission ratio chromatogram, generated by calculating the ratio of emission intensifies at two defined emission wavelengths, at all points in the time domain elution profile. These techniques are illustrated with reference to some polynuclear aromatic hydrocarbons and to the β-blocker drug atenolol and its related impurities. Future developments of this new detector technology in LC are also considered.     

二极管阵列检测技术在中药化学分析中的应用

本文简要介绍了二极管阵列检测技术在中药化学分析中的几方面应用实例。峰纯度、色谱峰光谱一致性检测是高效液相色谱分析方法的可靠性和适用性评价的较先进的手段。     

Determination of cortisol and cortisone in urine using high-performance liquid chromatography with UV detection

The application of reversed-phase gradient high-performance liquid chromatography with UV detection to the determination of cortisol and cortisone in 24-h urine samples is described. The method employs Sep-pak C18 cartridges for the part-purification and concentration of the corticosteroids, with sample enrichment at the head of an HPLC pre-column and separation using water/acetonitrile gradient. The internal standard is 6alpha-methylprednisolone. Measurement of both cortisone and cortisol provides further information on adrenocortical function. 24-hour excretion rate data from normal subjects are reported.     

Ion-pairing detection technique in reversed-phase high-performance liquid chromatography of drugs and related compounds

The principles and practice of UV-detection methods based on the ion-pairing technique are discussed with respect to reversed-phase high-performance liquid chromatography. The detection of amino acids, aliphatic alcohols and vitamins is described together with the rationale for optimizing sensitivity of detection in using the ion-pairing technique for pharmaceutical analysis.     

Determination of nefazodone and its metabolises in plasma by high-performance liquid chromatography with coulometric detection

Nefazodone and its metabolites together with the internal standard are extracted from plasma using CN Bond-Elut sorbent columns. Chromatography and detection are performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with a CN column and coulometric end-point detection. The lower limits of quantitation for nefazodone,mCPP, and OH-nefazodone was 0.9, 0.2 and 0.6 ng ml⁻¹ when 1 ml of plasma was extracted. The inter-assay relative standard deviations were less than 6% for nefazodone and its metabolites. Recovery averaged over 80%. Plasma profiles of nefazodone and its metabolises following oral dosing are presented.     

A semi-automated high-performance liquid chromatographic system for the determination of 25-hydroxyvitamin D in human plasma: elimination of interference by barbiturates and use of photodiode array detection

A semi-automated high-performance liquid chromatographic (HPLC) method for the measurement of 25-hydroxyvitamins D₂ and D₃ is described. Plasma was extracted using acetonitrile and a Bond-Elut C₁₈ cartridge system, eluted with methanol and fractionated on Sep-Pak SIL. After formation of isotachysterol isomers straight-phase HPLC was carried out monitoring the mobile phase with a photodiode array detection system. Two internal standards have been used, namely [³H]25-hydroxyvitamin D₃ and 25-hydroxydihydrotachysterol₃ both of which are shown to give satisfactory results. The use of the extraction system described eliminates interference from barbiturates which had previously been reported to interfere. Photodiode array detection allows confirmation of the identity of the analytes.     

Analysis of 5-OH-indoles in human gut biopsy tissues by reversed-phase high-performance liquid chromatography with fluorimetric detection

A method was devised for the rapid simultaneous determination of major indoles in human gut tissues. Analysis with picomol detection limits was done by HPLC on a C₁₈ reversed-phase column with fluorimetric detection at 276/350 nm. This simple method for which there is no necessity of derivatization or purification was validated for routine analysis of small mucosa samples (less than 4 mg fresh weight) obtainable during endoscopy. A comprehensive list of 5-OH-indole compounds in human gut tissue is presented.     

高效液相色谱荧光检测法测定健康志愿者血浆中氟罗沙星

目的建立测定人血浆中氟罗沙星浓度的高效液相色谱方法,并用于其药代动力学研究。方法采用加替沙星为内标,血浆样品经甲醇沉淀蛋白后直接进样。色谱柱为Diamonsil C18柱,流动相为1%三乙胺-磷酸缓冲液(pH4.8)/乙腈(80/20,V/V),流速1.0mL·min-1。采用荧光检测器,激发波长290nm,发射波长458nm。结果氟罗沙星血浆浓度0.025~8.00μg·L-1范围内线性关系良好(W=1/x,r=0.9999),低、中、高浓度质控样品的日内、日间精密度不超过5.16%,方法精密度99.1%~100.9%,提取回收率86.7%~92.0%。氟罗沙星血浆样品实验条件下均稳定。结论该方法灵敏度高、定量准确,适用于氟罗沙星人体药代动力学研究。     

Analysis of opiates, cocaine and metabolites in urine by high-performance liquid chromatography with diode array detection (HPLC-DAD)

An analytical method is proposed for the simultaneous determination of morphine, codeine, 6-acetyl-morphine (MAM), cocaine, benzoylecgonine (BEG), cocaethylene, methadone and 2-ethylen-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine using high performance liquid chromatography coupled to a diode array detector (HPLC-DAD). The selection of working wavelengths is based on the highest chromatographic response for each component: 233 nm for cocaine, BEG and cocaethylene; 285 nm for morphine, codeine and MAM; and 292 nm for methadone and EDDP. The mobile phase, which is a mixture of acetonitrile and 0.02 M phosphate buffer at pH 6.53, was eluted in gradient mode through an XTerra RP-8 column (250 mm x 4.6 mm i.d., 5 microm particle size). After applying a solid-phase extraction procedure with Bond Elut Certify cartridges, the recoveries obtained were between 60% (EDDP) and 97% (cocaethylene). A good linearity of the method in the 0.1-10 microg mL(-1) range of urinary concentrations was obtained because the coefficient of correlation exceeded 0.99 for each drug. The precision and accuracy were quite good, with values of <7% and within the range +/- 6%, respectively. Finally, the proposed method was applied to 23 urine samples from fatal intoxications related to methadone, heroin and[sol ]or cocaine.     

Fingerprint analysis of fruiting bodies of cultured Cordyceps militaris by high-performance liquid chromatography-photodiode array detection

We have developed and optimized a novel, efficient and accurate fingerprint method using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) for the quality control of cultured Cordyceps militaris (L.) Link. The feasibility and advantages of the used chromatographic fingerprint were verified for the evaluation of cultured C. militaris by systematically comparing chromatograms with a professional analytical software recommended by State Food and Drug Administration (SFDA) of PR China. The results revealed that the chromatographic fingerprint combining similarity evaluation could efficiently identify and distinguish cultured C. militaris from different sources.     

The determination of metoclopramide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Methodology based on reversed-phase ion-pair high-performance liquid chromatography is described for the determination of metoclopramide in plasma. The chromatography was optimized in terms of the peak shape for the drug and its resolution from endogenous plasma components by investigating the effects of quaternary ammonium (competing) ions and alkylsulphate (pairing) ions in an acidic mobile phase containing acetonitrile (20%) and 20 mM acetic acid. Optimum chromatographic conditions were obtained with an ODS-Hypersil column and a mobile phase containing 20% acetonitrile, 20 mM acetic acid, 0.6 mM sodium octylsulphate and 0.5 mM tetrabutylammonium chloride. A simplified method of sample preparation is described in which only 1 ml of plasma is required. The limit of detection (at 310 nm) was 7 ng/ml and no interference from endogenous plasma components or from any drugs commonly used in the treatment of cancer was observed. Consequently the methodology should be applicable to pharmacokinetic studies on metoclopramide, when used clinically to control the gastro-intestinal side-effects of chemotherapy.     

Determination of catecholamines by flow-injection analysis and high-performance liquid chromatography with chemiluminescence detection

A chemiluminescence (CL) detection of catecholamines [norepinephrine (NE), epinephrine (E), dopamine (DA) and l-dopa (LD)] is described for the flow-injection (FI) and high-performance liquid chromatographic (HPLC) determination of these compounds. The detection method is based on the inhibition effect of catecholamines (CAs) on the CL reaction of luminol with iodine in the alkaline medium. The proposed FI method allows the determination of CAs in pharmaceutical preparations for the purpose of drug quality control. The calibration curves show good linearity in the concentration range of: 1.1–20.0 μg l⁻¹ (NE), 0.5–5.0 μg l⁻¹ (E), 0.6–9.0 μg l⁻¹ (DA) and 0.6–10.0 μg l⁻¹ (LD). The limits of detection (defined as a signal-to-noise ratio of 3) are: 0.34 μg l⁻¹ (NE), 0.15 μg l⁻¹ (E) and 0.18 μg l⁻¹ (DA, LD). The HPLC procedure was successfully applied for the determination of catecholamines (NE, E, DA) in human urine after solid-phase extraction (SPE). In a simple run time CAs can be determined in 20 min. The chromatographic linear ranges are: 5.0–72.0 μg l⁻¹ (NE), 5.0–48.0 μg l⁻¹ (E) and 5.0–96.0 μg l⁻¹ (DA). The limits of detection for three urinary CAs are: 0.71 μg l⁻¹ (NE), 0.26 μg l⁻¹ (E) and 0.73 μg l⁻¹ (DA).     

高效液相色谱法测定痰咳净中咖啡因的含量

本文报道了痰咳净中咖啡因的ＨＰＬＣ测定法。使用Ｚｏｒｂａｘ－Ｃ１８柱，流动相为甲醇－水（１：１），检测波长２５４ｎｍ，非那西丁为内标物。测定中其它组分无干扰，方法简便，重现性好，平均回收率为９９．６％，ＲＳＤ为０．５％。     

Simultaneous determination and validation of antimicrobials in plasma and tissue of actinomycetoma by high-performance liquid chromatography with diode array and fluorescence detection

A simple, precise, and reliable chromatographic method was developed for the simultaneous determination in plasma and infected tissue of five antimicrobials proposed for the treatment of actinomycotic mycetoma: amoxicillin, trimethoprim, linezolid, sulfamethoxazole and garenoxacin. Separation of the analytes was achieved on an Atlantis dC18 column (150 mm × 4.6 mm, ID 5 μm) with a mobile phase composed of acetonitrile and trifluoroacetic acid (ATF) 0.1% (v/v) using a gradient program. The detection was carried out using a diode array detector at 254 nm and in a fluorescence detector at wavelengths of excitation and emission of 292 nm and 392 nm for linezolid and sulfamethoxazole, and 292 nm and 408 nm for garenoxacin, respectively. The intraday precision was in the range of 0.7–15% of relative standard deviations (%R.S.D.) for plasma and 1–18% for tissue. Linearity range was from 2.4 to 20 μg/ml for amoxicillin, 0.3 to 20 μg/ml for trimethoprim, sulfamethoxazole and linezolid, and 0.3 to 10 μg/ml for garenoxacin. Acetonitrile was used to precipitate proteins from plasma. Recoveries in plasma ranged from 71% to 118% and in infected tissue from 78% to 122%. Limits of detection (LODs) were 1.2 and 0.5 μg/ml for amoxicillin in plasma and tissue, respectively and 0.15 and 1.2 μg/ml in plasma and tissue, respectively for the other antimicrobials. The method can be applied for individual or simultaneous determination of the antimicrobials in plasma and tissue of mouse infected with actinomycetoma.     

Determination of gabapentin in human plasma and urine by high-performance liquid chromatography with UV-vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV–vis detection has been developed and validated for the determination of gabapentin (GBP) in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction with C18-cartridge. After the clean up procedure, the samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) with isocratic elution (35:65). Baclofen was used as an internal standard (I.S.). The method developed for GBP was linear over the concentration range of 0.05–5.0 μg/ml and 0.1–10.0 μg/ml for plasma and urine, respectively. The method is precise (relative standard deviation, R.S.D. <4.05%) and accurate (relative mean error, RME <0.15%); mean absolute recoveries were 72.21% for plasma and 72.73% for urine.     

Analysis of tryptophan, tyrosine and related dipeptides in mouse brain by isocratic high-performance liquid chromatography with switchable wavelength fluorescence detection

Tryptophan (Trp) and tyrosine (Tyr) are pharmacologically active compounds which, after administration of adequate doses, are increased in level in the brain, and stimulate neurotransmitter synthesis. Trp and Tyr containing dipeptides were tested as possible substitutes with regard to the effect on precursor level in the brain. Glycyltryptophan, alanyl-tryptophan and glycyl-tyrosine were intravenously applied to young female mice and the brain levels of dipeptides, Trp and Tyr measured 30 min after application. Neurotransmitter precursor levels in the brain increased similarly in all cases. The results suggest that the dipeptides are as effective as the single amino acids and may be superior because of their better solubility.