Simultaneous analysis of adrenergic amines and flavonoids in citrus peel jams and fruit juices by liquid chromatography: part 2

Citrus species are rich sources for various bioactive compounds such as flavonoids, adrenergic amines, limonoids, and coumarins. Six adrenergic amines and 20 flavonoids in the jams and juices of Citrus species were analyzed by high-performance liquid chromatography with a C18 reversed-phase column using gradient mobile phase of sodium acetate buffer (pH 5.5) and acetonitrile at a flow rate of 1.0 mL/min with detection at 254, 280, and 330 nm. Synephrine was present in levels from 3.65 to 60.66 mg/L in 48 Citrus juices and 0.018-1.02 mg per serving in 32 Citrus jams. The content of these amines and flavonoids varied to a large extent depending on the type of the Citrus species used. The method has been successfully applied to the determination of adrenergic amines and flavonoids in several samples of Citrus fruit juices and jams.     

Determination of synephrine in bitter orange raw materials, extracts, and dietary supplements by liquid chromatography with ultraviolet detection: single-laboratory validation

A method has been developed to quantify synephrine in bitter orange raw material, extracts, and dietary supplements. Single-laboratory validation has been performed on the method to determine the repeatability, accuracy, selectivity, limit of detection/limit of quantification (LOQ), ruggedness, and linearity for p-synephrine and 5 other biogenic amines: octopamine, phenylephrine (m-synephrine), tyramine, N-methyltyramine, and hordenine, which may be present in bitter orange. p-Synephrine was found to be the primary biogenic amine present in all materials tested, accounting for >80% of the total biogenic amine content in all samples except a finished product. Repeatability precision for synephrine was between 1.48 and 3.55% RSD. Synephrine recovery was between 97.5 and 104%. The minor alkaloids were typically near the LOQ of the method (300-900 microg/g) in the test materials, and between-day precision for the minor compounds was poor because interferences could sometimes be mistakenly identified as one of the minor analytes. Recoveries of the minor components ranged from 99.1 to 103% at approximately 6000 microg/g spike level, to 90.7 to 120% at 300 microg/g spike level.     

Chromatographic and electrophoretic methods for the analysis of phenethylamine [corrected] alkaloids in Citrus aurantium

Citrus aurantium (bitter orange) is a plant belonging to the family Rutaceae, whose fruit extracts have been used recently for the treatment of obesity. The most important biologically active constituents of the C. aurantium fruits are phenethylamine alkaloids (i.e. octopamine, synephrine, tyramine, N-methyltyramine and hordenine). Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is present in the peel and the edible part of Citrus fruit. Of the adrenergic amines of natural origin, synephrine has been found to be the main constituent of C. aurantium fruits and extracts; the other alkaloids are either absent or present in only low concentrations. It is known that synephrine and the other amines found in C. aurantium have adverse effects on the cardiovascular system, owing to adrenergic stimulation. In light of the great commercial proliferation of C. aurantium herbal medicines in recent years, this review provides an overview of various extraction, separation and detection techniques employed for the qualitative and quantitative determination of the alkaloids in C. aurantium and related species. The application of chromatographic and electrophoretic methods for the separation and determination of these active components in C. aurantium plant material and derivatives are described. Since synephrine is a chiral compound, enantioselective chromatographic and electrophoretic techniques for the analysis of synephrine enantiomers in natural products are presented. Furthermore, examples of identification of these active compounds in complex matrices by hyphenated methods, such as gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry, are described. The advantages and limitations of these separation and identification methods are assessed and discussed.     

Certification of standard reference materials containing bitter orange

A suite of three dietary supplement standard reference materials (SRMs) containing bitter orange has been developed, and the levels of five alkaloids and caffeine have been measured by multiple analytical methods. Synephrine, octopamine, tyramine, N-methyltyramine, hordenine, total alkaloids, and caffeine were determined by as many as six analytical methods, with measurements performed at the National Institute of Standards and Technology and at two collaborating laboratories. The methods offer substantial independence, with two types of extractions, two separation methods, and four detection methods. Excellent agreement was obtained among the measurements, with data reproducibility for most methods and analytes better than 5% relative standard deviation. The bitter-orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rapid simultaneous determination of amines and organic acids in citrus using high-performance liquid chromatography

Rapid analytical method for the simultaneous separation and determination of amines and organic acids is a vital interest for quality control of citrus and their products. In the present study, a simultaneous high performance liquid chromatography (HPLC) method for the rapid separation of three amines and two organic acids was developed. Chromatographic separation of compounds was achieved using Xbridge C₁₈ column at ambient temperature, with an isocratic mobile phase of 3 mM phosphoric acid at a flow rate of 1.0 mL min⁻¹. A photodiode array (PDA) detector was used to monitor the eluent at 223 nm and 254 nm with a total analysis time of 10 min. Extraction of amines and organic acids from citrus juice was optimized. The method was validated by tests of linearity, recovery, precision and ruggedness. The limit of detection (LOD) and limit of quantification (LOQ) for amines and ascorbic acid were determined to be 5 ng and 9.8 ng, respectively. All calibration curves showed good linearity (R² ≥ 0.9999) within the test ranges. The recoveries of the amines and organic acids ranged between 84% and 117%. The identity of each peak was confirmed by mass spectral (MS) analysis. The developed method was successfully applied to analyze the content of amines and organic acids in six different species and two varieties of citrus. Results indicate that mandarin and Marrs sweet orange contain high level of amines, while pummelo and Rio Red grapefruit had high content of ascorbic acid (137-251 μg mL⁻¹) and citric acid (5-22 mg mL⁻¹). Synephrine was the major amine present in Clementine (114 μg mL⁻¹) and Marrs sweet orange (85 μg mL⁻¹). To the best of our knowledge, this is the first report on simultaneous separation and quantification of amines and organic acids in Marrs sweet orange, Meyer lemon, Nova tangerine, Clementine, Ugli tangelo and Wekiwa tangelo.     

禁用偶氮染料检测中假阳性结果的鉴别方法

全面比较了薄层色谱法、握相色谱法、液相色谱法（ＨＰＬＣ）、气相色谱－质谱联用法（ＧＣ／ＭＳ）在禁用偶氮染料检测中的优缺点。分析实际检测中假阳性结果产生的原因,研究了８种致癌芳香胺的１９种异构体在气相色谱和订色谱上的分离情况,提出取ＧＣ／ＭＳ和ＨＰＬＣ互为确证手段的阳性结果鉴别方法,可有效提高定性结果和准确性,避免假阳性结果。     

Sensitive and rapid determination of elemental nanosulfur/sulfur by liquid chromatography

In order to identify the most suitable method for the estimation of nanosulfur for studying its residue dynamics, the present work was taken up. HPLC and GC methods were explored for its analysis. A comparative study of the existing analytical methods for the quality control of nanosulfur was undertaken. UV spectrophotometry and HPLC methods were superior with lower LOD when compared to GC–MS, which was not satisfactory due to breakage of catenated S₂₀ into S₆ and S₈. The method has been validated by analyzing various nanosulfur formulations of known concentrations. The recovery of the UV and HPLC methods ranged from 80.71 to 109.51% and 82.31 to 109.84%, respectively. The LOD of UV, GC–MS, and HPLC is 4, 20, and 1 ppm, respectively. The retention time of sulfur was 13.77 (HPLC), 2.89 (ultra high performance liquid chromatography), and 12.715 + 21.524 min (GC–MS). The method was successfully utilized for estimating sulfur in natural samples such as water from a sulfur hot spring and wastewater. The method has been validated by following the method recommended by the American Society for Testing and Materials. The HPLC method emerged as the best analytical method for the estimation of elemental sulfur.     

Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation

We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, "nuclease P1/HPLC," the DNA is hydrolyzed by nuclease P1 and the resulting 2'-deoxynucleoside 5'-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19-54 microg range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30-40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01+/-.07 (nuclease P1/HPLC), 0.8+/-0.17 (dye binding), and 1.1+/-0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important.     

HPGPC和HPLC测定齐聚物的聚合度分布与平均聚合度

用高效凝胶色谱和反相高效液相色谱测定了聚对苯二甲酸乙二酯齐聚物、聚对苯二甲酸丁二酯齐聚物的聚合度分布与平均聚合度,获得了满意的结果。同时,比较了这两种分析测试方法的优缺点。     

Preparative high-performance liquid chromatographic separation and isolation of bacitracin components and their relationship to microbiological activity.

Bacitracin, a polypeptide antibiotic, is one of the most commonly used antibiotics in the world. The approved method of analysis for bacitracin is microbial. To correlate the microbiological method with a high-performance liquid chromatographic (HPLC) method, bacitracin was chromatographed using HPLC with ultraviolet detection and a YMC basic column. Adequate separation of the isomers was obtained to scale up this procedure to preparative HPLC using a Prep HPLC system and a 250 x 21 mm YMC basic column. The various fractions were separated, isolated and examined for microbial activity. The individual fractions could be precipitated by adding zinc or methylene disalicylic acid and lowering the pH. The crude fractions were recycled to ensure chromatographic purity. The chromatograms can accurately predict (in minutes) the microbiologically determined potency which usually takes 16-24 h to develop. The chromatographic procedure also provides information on the amounts of isomers and degradation products present in the sample, whereas the microbiological assay only provides activities or potencies of the antibiotic. The reported HPLC method also possesses some advantages over some other published HPLC methods in terms of accuracy and time of analysis.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4 degrees C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions

A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C₁₈ column by gradient elution that used mixtures of trifluoroacetic acid (TFA)–acetonitrile and TFA–water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.     

三七素手性拆分方法的研究

使用气相色谱法和高效液相色谱法分离了三七素对映体,并探讨了影响液相色谱法分离效果的因素。结果表明,HPLC法利用手性固定相进行直接拆分,无法实现对映体的完全分离;GC法和HPLC的手性试剂衍生化法均可对三七素对映体进行较好的分离。但GC法由于衍生化过程中副产物的存在,干扰了对映体的准确定量。手性试剂衍生化HPLC法,以邻苯二甲醛、N-酰化-L-半胱氨酸为衍生化试剂,反应得到的三七素对映体的衍生物在ODS柱上分离良好,且方法简单、快速。     

Liquid chromatographic method for the determination of calcium cyanamide using pre-column derivatization.

A specific and stability-indicating high-performance liquid chromatographic (HPLC) method has been developed for the analysis of calcium cyanamide in bulk material and dosage form. Calcium cyanamide in samples was converted into dansyl cyanamide. A muBondapak C18 column was employed for HPLC with 0.01 M sodium phosphate (pH 6.3)-acetonitrile (75:25, v/v) as the mobile phase. The proposed HPLC method was validated for linearity, specificity, accuracy and reproducibility.     

清开灵注射液HPLC/ELSD指纹图谱建立及质量相关性研究

运用高效液相色谱／蒸发光散射检测器(HPLC／ELSD)建立了复方清开灵注射液的指纹图谱。对其中7种有效成分进行了定量测定,方法学考察结果良好;通过指纹图谱相似度评价软件,对10批清开灵注射液的HPLC／ELSD指纹图谱进行了相似度计算,结果表明：10批样品相似度很好。该方法为中药质量控制提供了一种可以同时实现整体定性、指标成分定量且简便易行的方法模式。     

Animal test or chromatography? Validated high-performance liquid chromatographic assay as an alternative to the biological assay for ornipressin

Ornipressin is a peptide drug which is usually assayed by a test on live rats. In order to reduce the animal experiments an alternative method was developed which uses gradient high-performance liquid chromatography (HPLC) on reversed-phase. The HPLC method was validated and shown to be selective and precise. Correlation studies were performed on samples of different dosage strengths and on thermally degraded samples, showing good correlation with the results obtained by the biological assay. The HPLC method was applied on various batches of ornipressin in bulk and in pharmaceutical preparations. HPLC is a rapid and inexpensive method which can replace the animal assay. A new quality control concept is proposed which uses HPLC for the analysis of ornipressin in bulk and in pharmaceutical preparations. With this concept animal testing can be reduced by 90%.     

Enantiomeric Separation of Adrenergic Amines in Citrus Species, Related Genera and Dietary Supplements by Capillary Electrophoresis

Citrus aurantium L. (Rutaceae) fruit extracts have recently been used for weight loss. Among the adrenergic amines the most important active constituent is the sympathomimetic compound synephrine and commercially available extracts are standardized for their content of this active principle. A capillary electrophoresis method was developed for the quantitative and qualitative determination of d-synephrine, l-synephrine, d-octopamine, l-octopamine, tyramine, n-methyl tyramine and hordenine. The electrophoretic separation was performed using a 75 cm × 50 µm ID (66.5 cm effective length) fused silica capillary. The samples were injected by pressure for 5s at 50 mbar and the running voltage was 30 kV at the injector end of the capillary. The method developed was successively applied to the determination of the adrenergic amines in dietary supplements, in various Citrus species including Citrus aurantium, jams and juices. Synephrine was the main component and present in the levels from 0.02–0.17% in various Citrus species and 0.42–69.28 mg in dietary supplements claiming to contain Citrus aurantium. Parameters affecting the resolution between (+) and (−)-enantiomers, such as pH, cyclodextrin concentration, temperature, organic modifier, buffer concentration and capillary dimensions were reported.     

Determination of enantiomers in a synthetic argininal peptide using capillary zone electrophoresis and high-performance liquid chromatography

SCH 201781 is a synthetic argininal peptide containing two chiral centers and an aromatic sulfonamide group. It can exist as four reversible forms, the aldehyde, the hydrate, and two diastereomeric aminals. Capillary zone electrophoresis (CZE) and reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to separate and quantitate the enantiomers in SCH 201781. Comparable results were obtained using both methods. The CZE method uses direct injection, while the HPLC method requires a precolumn derivatization and is more time consuming. The CZE method provides superior sensitivity to the HPLC method. Both methods were shown to be precise and reproducible.     

A Simple HPLC Method for the Quantitation of Molindone in Human Plasma or Serum

Abstract

A simple HPLC method is described for the quantitation of molindone in human serum or plasma using trazadone as an internal standard and UV detection (247 nm). The method was successfully used to determine the steady state levels of molindone in 15 patients receiving the drug. The HPLC results were confirmed by identifying the HPLC peak using LC thermospray mass spectrometry.     

高效液相色谱法测定家蝇细胞色素P450O-脱甲基活性

建立了以乙酸乙酯/正己烷为终止剂(含1% H3PO4)和萃取剂,利用高效液相色谱,ZORBAX Eclipse XDB C18色谱柱,梯度洗脱,在316 nm检测,外标法分析对硝基苯酚的生成量,表征家蝇粗酶液中细胞色素P450催化的对硝基苯甲醚 O-脱甲基活性的新方法.本方法的检出限为0.1 ng; 分析精密度的RSD为1.02%; 0.351, 1.755和8.755 μg对硝基苯酚3个添加水平的回收率为92.34%～87.60%; 回收率的RSD为2.64%～5.90%;对硝基苯酚在9.72～486 ng范围内,线性关系良好, r=0.9995.应用本方法比较了家蝇吡虫啉抗性品系与敏感品系细胞色素P450的活性差异.结果表明,家蝇抗性品系的P450 O-脱甲基活性是敏感品系的3.34倍; 细胞色素P450活性的提高是家蝇对吡虫啉产生抗性的一个重要机制.