Thorough evaluation of the validity of conventional enantio-gas chromatography in the analysis of volatile chiral compounds in mandarin essential oil: A comparative investigation with multidimensional gas chromatography

The present research is focused on the determination of the enantiomeric distribution of chiral compounds, contained in mandarin essential oils, by means of conventional chiral gas chromatography with flame ionization detection (enantio-GC-FID); the results attained were compared with those derived from heart-cutting multidimensional GC-mass spectrometry (MDGC/MS), to evaluate the reliability of the monodimensional technique as a tool for quality control. The Deans-switch MDGC system was equipped with two GC ovens, which were connected via a heated transfer line, a flame ionization detector (FID1) in the first dimension and a quadrupole MS as second-dimension detector. The a priori knowledge of potential co-elutions concerning target compounds (an enantiomer and an interfering compound), when using enantio-GC-FID, could enable the use of corrected enantiomer excess values. Correction factors could be calculated through a preliminary GC-FID analysis (using an apolar column), considering the peak areas of the known interferences. The method used for the calculation of a so-called “coelution correction factor” is described, along with some examples.     

Two-dimensional liquid chromatography coupled with mass spectrometry for the analysis of Lobelia chinensis Lour. using an ESI/APCI multimode ion source

A comprehensive approach for the separation and identification of components in a traditional Chinese medicine Lobelia chinensis Lour. was developed using 2D-HPLC coupled with an online photodiode array (PDA) detector and a mass spectrometer. The extract of L. chinensis Lour. was separated on a CN column in the first-dimensional HPLC, and then each of the collected fractions was further separated on an ODS column followed by an online PDA detector. After separation in the two different chromatographic modes, the eluents were delivered to a quadrupole mass spectrometer equipped with a multimode ion source of an ESI and an atmospheric pressure chemical ionization (ESI/APCI). At least 536 components in L. chinensis Lour. extract were detected and 6 of them were identified as apigenin 7-O-rutinoside, luteolin, lobetyolinin, lobetyolin, diosmin, and linarin, respectively, according to their UV spectrum and mass spectrum. The results demonstrated the powerful resolution, high peak capacity, as well as the identification capability of the 2D-HPLC combined with PDA and ESI/APCI-MS for the analyses of complex samples.     

Simultaneous determination of urinary metabolites of methoxypsoralens in human and Umbelliferae medicines by high-performance liquid chromatography

A high-performance liquid chromatography method has been developed for the determination of coumarins and furocoumarins (psoralens). Nine coumarins and furocoumarins are separated simultaneously on a Hypersil C(8) (25 cm x 4.6-mm i.d.) column with a gradient of methanol and acetonitrile aqueous solution as mobile phase at 1.0 mL/min with two-channel UV-vis absorbance detection. The limits of detection are 0.366, 0.219, 0.317, 0.440, 0.536, 0.300, 0.531, 0.531, 0.237, and 0.280 ng/mL for coumarin, 7-hydroxycoumarin, 7-methoxycoumarin, citropten (5,7-dimethoxycoumarin), 7-ethoxy-4-methylcoumarin, psoralen, xanthotoxin (8-methoxypsoralen), bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), and imperatorin (9-isopenteneoxypsoralen), respectively. Human urine is analyzed 1-6 days after ingestion of the oral Chinese medicines. This lead to the conclusion that the concentration of coumarins and furocoumarins is higher than that of the control urine. The coumarins and furocoumarins are detected at 312 and 249 nm, respectively.     

Optimization of a comprehensive two-dimensional normal-phase and reversed-phase liquid chromatography system

The present investigation is based on the evaluation of the performance of a comprehensive two-dimensional liquid chromatography (LCxLC) system during method optimization. The LCxLC set-up, operated in normal phase (NP) mode (adsorption) in the first dimension (1D) and reversed-phase (RP) mode in the second dimension (2D), is equipped with a 1D microbore silica column and a 2D monolithic C(18) column with a 10-port two position valve as the interface. A photodiode array detector is used after the 2D separation. A possible cause of peak distorsion because of the immiscibility of the mobile phases employed in the two dimensions is resolved. The optimization of the analytical run time and flow rate for both dimensions and the initial gradient in the 2D is carried out with various standard compounds. The potential and versatility of this LCxLC approach is demonstrated through the separation of 11 standard components, most of them allergens. The latter, which are characterized by a scattered distribution on the 2D space plane, underwent separation on both a hydrophobicity and polarity basis.     

An HPLC method for the determination of adenosine diphosphate: An important marker of hexokinase activity in metabolic diseases

Hexokinases play a critical role in the cellular uptake and utilization of glucose. As such, they are of fundamental importance to all cells. By catalyzing glucose to produce glucose‐6‐phosphate, hexokinases control the first irreversible step of glucose metabolism and initiate all major pathways of glucose consumption. Our objective was to develop and validate highly sensitive and selective high‐performance liquid chromatography with photodiode array detector (HPLC‐PDA) assays allowing the determination of adenosine diphosphate, which was used for the determination of hexokinase activity. Samples were analyzed by HPLC‐PDA using a C₁₈ analytical column (250 × 4.6 mm) for chromatographic separation. Optimal detection was achieved based on isocratic elution with a mobile phase consisting of a mixture of sodium phosphate monobasic buffer and methanol. This method met all of the requirements of specificity, sensitivity, linearity, precision, accuracy and stability generally accepted in bioanalytical chemistry and was successfully applied to a study of hexokinase activity in an alloxan‐induced diabetic rat model. Determination of hexokinase activity will permit characterization of cellular metabolic state in many diseases, such as cancer and diabetes.     

Isolation and determination of carbendazim residue from wheat grain by matrix solid‐phase dispersion and HPLC

Carbendazim residues were analyzed by column‐switching high‐performance liquid chromatography (HPLC) with photodiode array detection (PDA). The active ingredient was extracted by matrix solid‐phase dispersion (MSPD) from wheat grain on an acidic silica gel column using a methanol‐dichloromethane mixture. The recovery rate for fortified samples was 87.3 ± 3.3% with a standard deviation (SD) of 2.9%. The detection limit was 0.02 μg/mL. The method was applied to the determination of carbendazim residues in wheat grain samples from a treated field.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

高效液相色谱-二极管阵列检测器同时测定化妆品中的九种染料及中间体

建立了用高效液相色谱-二极管阵列检测器(HPLC-PDA)同时检测9种染料及中间体的系统方法。首先采用超声提取的方法处理样品,对提取溶剂和提取时间进行了选择,确定用甲醇-0.01 mol/L 乙酸铵(体积比为2∶1)作提取溶剂,超声提取20 min。然后,采用C18柱,以甲醇-0.01 mol/L乙酸铵(pH 6.2)为流动相梯度洗脱,用PDA检测。以保留时间定性,并以紫外吸收光谱图辅助定性,以外标法定量。定量检测波长为230 nm,15 min内可对9种目标物同时进行测定,且各化合物都达到基线分离(分离度大于1.5)。经测定,该方法的平均回收率(n=8)为81.0%～105.6%,相对标准偏差（RSD）为0.8%～4.9%,检出限(以信噪比为3计)为0.1～2 μg。该方法简单、快速,能有效提取和分离测定化妆品中9种染料及中间体。将该方法用于实际化妆品样品的检测,结果令人满意。     

超高效液相色谱法检测土壤中的多环芳烃

采用二极管阵列(PDA)检测器,建立了超高效液相色谱(UPLC)定性定量分析土壤中16种多环芳烃(PAHs)的方法。并将该方法与传统高效液相色谱(HPLC)的分析性能进行了详细的比较。研究结果表明,采用UPLC法分析16种PAHs具有分析速度快(13.5 min)、检出限低(2～20 pg)、灵敏度高等优点。     

Preparative separation of six coumarins from the pummelo (Citrus maxima (Burm.) Merr. Cv. Shatian Yu) peel by high-speed countercurrent chromatography

In this work, six coumarins, including two new ones, 8-(3-hydroxy-2,2-dimethylpropyl)-7-methoxy-2H-chromen-2-one (2) and 5-[(7′,8′-dihydroxy-3′,8′-dimethyl-2-nonadienyl)oxy] psoralen (4), as well as four known ones, 5-[(6′,7′-dihydroxy-3′,7′-dimethyl-2-octenyl) oxy] psoralen (1), marmin (3), epoxybergamottin (5), and aurapten (6) were successfully separated from the crude extract of pummelo (Citrus maxima (Burm.) Merr. Cv. Shatian Yu) peel by high-speed countercurrent chromatography in a single run with petroleum-ether–ethyl acetate–methanol–water (4:6:6:4, v/v). The structures of these six coumarins were elucidated by ESI-MS, extensive 1D and 2D NMR spectroscopy.     

Derivative spectrum chromatographic method for the determination of trimethoprim in honey samples using an on-line solid-phase extraction technique

A simple, selective and rapid analytical method for determination of trimethoprim (TMP) in honey samples was developed and validated. This method is based on a SPE technique followed by HPLC with photodiode array detection. After dilution and filtration, aliquots of 500 μL honey samples were directly injected to an on-line SPE HPLC system. TMP was extracted on an RP SPE column, and separated on a hydrophilic interaction chromatography column during HPLC analysis. At the first detection step, the noise level of the photodiode array data was reduced with two-dimensional equalizer filtering, and then the smoothed data were subjected to derivative spectrum chromatography. On the second-derivative chromatogram at 254 nm, the limit of detection and the limit of quantification of TMP in a honey sample were 5 and 10 ng/g, respectively. The proposed method showed high accuracy (60-103%) with adequate sensitivity for TMP monitoring in honey samples.     

Determination of seven compounds in Tang Maikang granule by high-performance liquid chromatography

An accurate and sensitive high-performance liquid chromatography method is developed and applied to the determination of seven compounds in a kind of traditional Chinese medicinal preparation of Tang Maikang Granule. The method is performed on Hypersil C(18) column (250- x 4.6-mm i.d., 5 microm), and different mobile phases and detectors are selected according to the various compounds. For astragaloside IV, an evaporative light scattering detector (ELSD) is used with a gradient of methanol-water at an eluent gas rate of 2.0 mL/min, under a drift tube temperature of 80 degrees C. Formononetin and calycosin are also eluted by a gradient of methanol-water, but a photodiode array (PDA) detector is used at a wavelength of 254 nm for formononetin and calycosin. A PDA detector at a wavelength of 230 nm is used for paeoniflorin, with methanol-water (30:70, v/v) as the mobile phase. For danshensu and protocate chualdehyde, an eluent of methanol-0.5% acetic acid (12:88, v/v) is used, with PDA detection at 280 nm. For berberine, methanol and water containing 0.1% sodium dodecanesulphonate (SDS) and 0.1% phosphorous acid (70:30, v/v) is employed as the mobile phase, also using a PDA detector, but the detection wavelength is 265 nm. The intra- and interrun precision (relative standard deviation) of this method is less than 5% for seven analytes.     

Separation, characterization, and quantitation of process-related substances of the anti-hypertensive drug doxazosin mesylate by reversed-phase LC with PDA and ESI-MS as detectors

A simple and rapid reversed-phase liquid chromatography (LC) method with photodiode array (PDA) and electrospray ionization (ESI)-mass spectrometry (MS) as detectors was developed and validated to separate, identify, and quantitate the related substances of Doxazosin mesylate (DXZN) for monitoring the reactions involved during process development. The high-performance liquid chromatography profiles of related-substances of DXZN are used as fingerprints to follow the procedures used in the manufacturing units. The separation is accomplished on an Inertsil ODS-3 column with acetonitrile-ammonium acetate (10 mM, pH 4.0) as the mobile phase, using a gradient elution mode and monitoring the eluents by a photodiode array detector at 265 nm at ambient temperature. LC-ESI-MS-MS is used to identify the additional impurities formed during the synthesis. The identified impurities were synthesized and characterized by UV, Fourier transform-IR, 1H NMR, and MS data. The detection limits for the impurities are 0.74 - 4.14 x 10(-9) g, and the method is found to be suitable not only for the monitoring of synthetic reactions, but also for quality assurance of DXZN in bulk drugs and formulations.     

Urinary metabolites of nobiletin orally administered to rats

During the course of our systematic investigation of the metabolism of flavonoids, the polymethoxyflavone nobiletin, occurring in the fruits of Citrus depressa, was orally administered to rats. The urinary metabolites were separated and identified by three-dimensional HPLC equipped with a photodiode array detector and the structure was determined by spectroscopic methods to be 4'-hydroxy-3',5,6,7,8-pentamethoxyflavone (1).     

Bioassay directed identification of natural aryl hydrocarbon-receptor agonists in marmalade

Citrus fruit and citrus fruit products, like grapefruit, lemon and marmalade were shown to contain aryl hydrocarbon receptor (AhR) agonists, as detected with the DR CALUX bioassay. This is of interest regarding the role of the Ah-receptor pathway in the adverse effects of dioxins, PCBs and other aromatic hydrocarbons. So far it is unclear which compounds in citrus fruit are responsible for the AhR-mediated activity and whether regular exposure to these compounds can cause effects comparable to, e.g. dioxins. The present study aimed at developing a method for identifying unknown Ah-receptor agonists in citrus products based on bioassay directed analysis, using marmalade as a first target. Following extraction with hexane and purification on an aluminium oxide-column, the extract was fractionated by HPLC using a C-18 semi-preparative column. Fractions were extracted, solvent-exchanged into dimethylsulfoxide and subsequently tested with DR CALUX. Extracts were shown to contain primarily coumarins, furocoumarins (FCs) and polymethoxyflavones (PMFs). Identification of fractions most active in the bioassay via LC/MS revealed that bergapten (an FC) is the most important Ah-receptor agonist in marmalade. The approach and method developed resulted in the successful identification of the bioactive component. However, potential pitfalls of the procedure will be discussed.     

反相高效液相色谱与紫外光谱对硝基苯类化合物的联合定性

结合高效液相色谱和紫外光谱各自的优点和信息，建立了有机化合物的高效液相色谱和紫外光谱联合定性方法。在高效液相色谱中，二极管阵列检测器实时在线地提供样品组分的紫外光谱，由此建立紫外光谱谱图库，并发展了谱图库检索算法，同时测定了硝基苯类化合物的反相色谱用热力学参数ａ、ｃ值。结果表明，该法是可行的，满足定性所需的精度。     

Chemical profiling of bioactive constituents in Sarcandra glabra and its preparations using ultra-high-pressure liquid chromatography coupled with LTQ Orbitrap mass spectrometry

In this study, a fast and reliable method based on an ultra‐high‐pressure liquid chromatography coupled with photodiode‐array detection (PDA) and a linear ion trap high‐resolution mass spectrometer (LTQ‐Orbitrap XL) has been developed for the identification of bioactive constituents in the whole plant of Sarcandra glabra and its related four preparations. By accurate mass measurements within 5 ppm error for each molecular ion and subsequent fragment ions in routine analysis, 50 compounds, including organic acids, caffeoyl derivatives, flavonoids, coumarins and terpenoids, were identified or tentatively characterized. Among them, 6,7,8‐trihydroxycoumarin‐O‐rhamnopyranoside (17), (2R)‐naringenin‐6‐C‐B‐D‐glucopyranosyl‐(6→1)‐apiose (25) and (2S)‐naringenin‐6‐C‐B‐D‐glucopyranosyl‐(6→1)‐apiose (27) were tentatively identified as new compounds. Besides, 21 of these compounds were coexisting in four preparations of Sarcandra glabra. Fragmentation behaviors of the four major categories of compounds were also investigated. This established UPLC‐PDA/ESI‐MSⁿ method was reliable and effective for the separation and identification of the major constituents and would be the basis for quality control of Sarcandra glabra and its related preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

Application of high-performance liquid chromatography diode array detection and mass spectrometry to the analysis of characteristic compounds in various essential oils

A high-performance liquid chromatography (HPLC) method was established using an analytical reversed-phase column and gradient elution to achieve chromatographic separation of typical compounds in essential oils. For detection, a diode array detector monitoring different wavelengths simultaneously as well as a mass spectrometer (MS) were used. Atmospheric pressure chemical ionization operating in the positive mode turned out to be a suitable tool to detect volatiles of different chemical classes and to identify them in essential oil matrices. Characteristic fingerprints of eucalyptus, lavender, may chang, pine, rosemary, thyme, and turpentine essential oils monitored at a representative wavelength (220 nm) demonstrated the suitability of HPLC in essential oil analysis. Additional monitoring wavelengths (210, 250, and 280 nm) provided useful information about the identity of the specific component and opened the possibility to differentiate presumably coeluting compounds by means of their distinct absorption behavior. Finally, peak assignment in seven essential oils was performed on the basis of characteristic retention times and UV and MS data of a broad set of reference volatiles.     

Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

高效液相色谱–光电二极管阵列法测定虾青素的含量

建立虾青素含量测定的高效液相色谱–光电二极管阵列法。采用Purospher STAR RP 18(4.6 mm×250mm,5μm)色谱柱,以甲醇–水(体积比为95∶5)为流动相,流速1.0 mL/min,检测波长为482 nm,柱温为30℃,进样量为20μL。在所选定的液相色谱条件下,虾青素主峰与其它杂质峰分离良好,虾青素在0.2~16μg/mL范围内线性良好,线性相关系数r=0.999 9,检出限为0.01μg/mL,测定结果的相对标准偏差为0.42%(n=6),平均回收率为100.4%。该法分析快速准确、灵敏度高、重现性好。