Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

On-line concentration and determination of all-trans- and 13-cis- retinoic acids in rabbit serum by application of sweeping technique in micellar electrokinetic chromatography

A simple and rapid micellar electrokinetic chromatography (MEKC) method with UV detection was developed for the simultaneous separation and determination of all-trans- and 13-cis-retinoic acids in rabbit serum by on-line sweeping concentration technique. The serum sample was simply deproteinized and centrifuged. Various parameters affecting sample enrichment and separation were systematically investigated. Under optimal conditions, the analytes could be well separated within 17min, and the relative standard deviations (RSD) of migration times and peak areas were less than 3.4%. Compared with the conventional MEKC injection method, the 18- and 19-fold improvements in sensitivity were achieved, respectively. The proposed method has been successfully applied to the determination of all-trans- and 13-cis-retinoic acids in serum samples from rabbits and could be feasible for the further pharmacokinetics study of all-trans-retinoic acid.     

Quantification of retinoid concentrations in human serum and brain tumor tissues

Retinoic acid signaling is essential for central nervous system (CNS) differentiation and appears to be impaired in tumors. Thus far, there are no established methods to quantify relevant retinoids (all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis retinoic acid, and retinol) in human brain tumors. We developed a single step extraction and quantification procedure for polar and apolar retinoids in normal tissue, lipid-rich brain tumor tissues, and serum. This quantification procedure is based on high performance liquid chromatography (HPLC) with diode-array detection (DAD) using all-trans-acitretin as an internal standard and extraction by liquid–liquid partition with ethyl acetate and borate buffer at pH 9. Recovery with this extraction procedure was higher than earlier (two-step) liquid–liquid extraction procedures based on hexane, NaOH, and HCl. The overall quantification procedure was validated according to Food and Drug Administration (FDA) guidelines and fulfilled all criteria of accuracy, precision, selectivity, recovery, and stability. The overall method accuracy varied between −5.6% and +5.4% for serum and −3.8% and +6.2% for tissues, and overall precision ranged from 3.1% to 6.9% for serum and 2.1% to 8.3% for tissues (%CV batch-to-batch). The lower limit of quantification for all compounds in tumor tissue (and serum) was 3.9 ng g⁻¹ (ng mL⁻¹). Using this assay, photodegradation of the retinoids was evaluated and endogenous polar and apolar retinoids were quantified in sera and brain tumor tissues of patients and compared with serum and tonsil tissue concentrations of controls. It may thus serve as a suitable method for the characterization of retinoid uptake and metabolism in the respective compartments.     

Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues.

Two high-performance liquid chromatography (HPLC) procedures, a rapid normal-phase isocratic method for the analysis primarily of retinol and retinoic acid on a 3 mu silica column, and a reversed-phase gradient method for the simultaneous analysis of retinoids and very polar to nonpolar carotenoids on a 3 mu C18 column, are described. The normal-phase isocratic HPLC procedure is rapid (12 min), requires a sample size of 100 microl or less of serum, and is suitable for routine analysis of retinol in any serum, and of retinol and retinoic acid in serum after administration of retinoic acid. The reversed-phase gradient method is suitable for the simultaneous analysis of very polar to nonpolar carotenoids such as epoxy-xanthophylls and xanthophyll esters, along with other carotenoids and retinoids that occur normally in human serum and other plant and animal tissues. A run time of 30-70 min is necessary, depending on the presence or absence of xanthophyll esters in the sample.     

Rapid and sensitive determination of fatty acids in edible oil by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and robust on-line LC/MS method was developed for quantitative determination of linoleic acid,docosahexaenoic acid and docosanoic acid from edible oil samples.The oil samples were dissolved in chloroform-isopropyl alcohol(20:80,v:v)solution and the three fatty acids were separated by HPLC with a C4 column using 10 mmol/L ammonium acetate-isopropyl alcohol-acetonitrile(20:40:40,v:v:v)mobile phase in isocratic elution.Electrospray ionization mass spectrometry with the selected ion recording monitoring was used to detect and quantify the fatty acid.The calibration curves were linear in the range of 10.00–5000 pg/mL for linoleic acid and docosanoic acid,and 1.000–500.0 pg/mL for docosahexaenoic acid.The limit of detection was 2.0 pg/mL for linoleic acid,3.0 pg/mL for docosanoic acid,and 0.20 pg/mL for docosahexaenoic acid.The results showed that the method described in this paper could be utilized for rapid determination of three fatty acids at picogram levels in edible oils.     

提高维甲酸毛细管电泳分析灵敏度的微柱固相萃取法研究

本文发展了一种适于复杂样品预处理的微柱固相萃取装置。该装置与毛细管电泳结合用于13-顺-维甲酸和全反式-维甲酸异构体的分析,灵敏度可提高100倍以上。维甲酸异构体的微柱固相萃取过程包括柱老化、上样、洗涤、洗脱以及柱再生等步骤。将该法用于加标血浆样品中的维甲酸分析,得到满意的结果。     

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate-formic acid-water (85:10:15, v/v/v) and n-hexane-ethyl acetate-formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis. Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr = 562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).     

Reversed-phase high-performance liquid chromatographic separation and electrochemical detection of retinol and its isomers

Baseline separation of the isomers of retinol using reversed-phase high-performance liquid chromatography (HPLC) in less than 30 min is presented. A new approach to the detection of retinol using electrochemical detection is developed. The oxidative electrochemistry of retinol is studied at a glassy-carbon electrode using coulometry, ultraviolet-visible spectrophotometry and HPLC. Amperometric detection in HPLC for retinol provided a linear response from 0 to 1.5 micrograms/ml and a detection limit of 4.1 ng/ml. Electrochemical detection was compared to ultraviolet-visible absorbance detection for the determination of retinol in human serum extracts. Good agreement is found for the results obtained with the two detectors.     

Studies on the mechanism of the Carr-Price blue colour reaction

The reaction of retinoids (retinol, retinyl acetate and anhydroretinol) with Br?nsted acids was studied as a model system for the Carr-Price reaction. The anhydroretinylic cation was characterised by VIS and 2D NMR spectroscopy, including an estimate of the charge distribution and region of bond inversion, observed in a mixture of identified E/Z isomers. Products obtained by quenching with NaOMe-MeOH were identified by HPLC and MS. The classical Carr-Price reaction between retinol (vitamin A) and the Lewis acid SbCl(3) in saturated chloroform solution was reinvestigated by VIS, NMR, EPR, dynamic light scattering and chemical quenching. Whereas product instability and failure to provide informative NMR spectra indicated a radical cation, EPR results excluded free-radical species. Dynamic light scattering experiments, in comparison with model systems, revealed strong aggregation for the Carr-Price complex, rationalizing the low stability, NMR problems and dimerisation observed by chemical quenching. The VIS data support structural similarity of the blue Carr-Price product with the delocalized anhydroretinylic cation, and a detailed structure of the antimony complex is evaluated.     

Single-run analysis of isomers of retinoyl-beta-D-glucuronide and retinoic acid by reversed-phase high-performance liquid chromatography

Reversed-phase HPLC methods capable of separating several retinoic acid isomers are generally not designed for simultaneous analysis of isomers of other classes of retinoids. A reversed-phase HPLC method is presented which allows the separation of at least four retinoic acid (RA) isomers (13-cis-RA, 9,13-dicis-RA, 9-cis-RA, all-trans-RA)and of all isomers of retinoyl-beta-D-glucuronide (RAG), which have been observed in vivo (13-cis-RAG, 9-cis-RAG,all-trans-RAG). The recovery of retinoids was generally between 80 and 90%. Intra-day reproducibility (expressed as relative standard deviation) was less than or equal to 7%. As little as 0.25 ng of RA isomers and of all-trans-RAG could be detected. This method allowed the study of the metabolism of 9-cis-retinoids, where isomerization reactions play a predominant role.     

Isolation of mono-caffeoylquinic acids from tobacco waste using continuous resin-based pre-separation and preparative HPLC

Three isomers of mono-caffeoylquinic acid, specifically, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid, were successfully isolated from a crude extract of tobacco (Nicotiana tobaccum L.) wastes using continuous resin-based pre-separation and preparative high-performance liquid chromatography (HPLC). The extract of tobacco wastes was continuously pre-separated by resin-based columns packed with D101 and XAD-4, yielding total mono-caffeoylquinic acids with a purity of 67.71% and a recovery rate of 90.06%. Variables affecting resolution and productivity of three mono-caffeoylquinic acid isomers in preparative HPLC (i.e. mobile-phase composition, pH, flow rate and loading amount) were studied. The optimum chromatographic conditions were determined to be a mobile phase consisting of 15% (v/v) methanol and aqueous acetic acid with a pH of 4.5, a flow rate of 4.0 mL/min, a loading amount of 4 mL and a detection wavelength of 360 nm. From 300 mg of loading sample, 56.3 mg of 3-O-caffeoylquinic acid, 92.8 mg of 5-O-caffeoylquinic acid and 73.1 mg of 4-O-caffeoylquinic acid were obtained in a single run, each with a purity of over 98% by HPLC. The structures of the isolated compounds were elucidated by ESI-MS, (1) H-NMR and (13) C-NMR spectral data.     

Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana)

HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.     

Detecting aristolochic acids in herbal remedies by liquid chromatography/serial mass spectrometry

Targeted liquid chromatography/serial mass spectrometry (LC/MS/MS) analysis, using a quadrupole ion-trap mass spectrometer, permitted the detection of aristolochic acids I and II in crude 70% methanol extracts of multi-component herbal remedies without any clean-up or concentration stages. The best ionisation characteristics were obtained using atmospheric pressure chemical ionisation (APCI) and by including ammonium ions in the mobile phase. Limits of detection for aristolochic acids were influenced by the level of interference created by other components in the sample matrix. They were determined to be between 250 pg and 2.5 ng on-column within a matrix containing compounds extracted from 2 mg of herbal remedy. With a herbal remedy that only permitted the higher limit of detection, this sensitivity was sufficient to detect the aristolochic acids extracted from 0.1% dry weight of Aristolochia manshuriensis included in the preparation.     

Characterization of carboxylic acids in atmospheric aerosols using hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC/ESI-MS/MS) method was developed for determination of selected aliphatic (i.e. malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, maleic, fumaric, glycolic and pyruvic acid), alicyclic (i.e. cis-pinonic and pinic acid) and aromatic (i.e. trimesic, phthalic acid and its isomers) carboxylic acids. Analytes were separated on an amide column using a gradient elution with a 10mM constant ionic strength mobile phase containing acetonitrile and aqueous ammonium acetate buffer (pH 5.0). The influence of the buffer type, pH, polar modifier and temperature on analyte retention under HILIC was studied. Static sonication-assisted solvent extraction was optimized for sample preparation prior to analysis. The recoveries obtained were higher than 90% for most analytes. The method was proven to be sensitive with limits of detection ranged from 0.03 to 16.0 μg/L in selected reaction monitoring mode (SRM). The repeatability and intermediate precision of the method, expressed as RSD (%) of the peak area ratio between analytes and their internal standards were generally lower than 5%. The method was successfully applied for determination of the studied acids in samples of ambient aerosol particles. A big advantage of the new method is also its ability to detect and separate the isobaric compounds of the selected carboxylic acids. Our results demonstrate that the method is specific and sensitive for the determination of a wider range of polar carboxylic acids at low concentrations in complex samples of aerosol particles.     

Rapid, simultaneous determination of isomers of retinal, retinal oxime and retinol by high-performance liquid chromatography

Thirteen geometric isomers of retinol, retinal and retinal oxime were resolved in a single chromatographic run on two 250 X 4 mm 5-micron LiChrosorb Si 60 columns in series, using 11.2% ethyl acetate, 1.4% 1-octanol and 2% dioxane in hexane as the mobile phase. All the 11-cis and all-trans isomers of retinol and retinal oxime were completely resolved from each other and from the 9-cis and 13-cis isomers. This chromatographic procedure should be particularly useful for quantifying geometric isomers of retinoids in methylene chloride-hydroxylamine extracts of biological samples such as ocular tissues.     

Determination of phthalic,terephthalic, and isophthalic acids in the presence of each other by gas chromatography

A procedure was developed for the simultaneous determination of phthalic acid isomers; the procedure includes the preparation of diisoamyl esters by reaction with isoamyl alcohol in a benzene solution in the presence of sulfuric acid with the distillation of an azeotrope mixture of benzene with water, the neutralization of acid with triethylamine, and the separation of esters by gas-liquid chromatography on a steel column (1 m × 3 mm) filled with Chromaton N-AW-HMDS with 5% Apiezon L in the temperature programming mode with flame-ionization detection. The time of separation is 13 min. The time of the sample preparation step is 2–2.5 h. The lower determination limit is 20 ng. The procedure allows the determination of phthalic acids in the concentration range from 0.02 to 2 mg/L in liquid samples and from 0.03 to 3 mg/m³ in air.     

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high‐throughput amino acid analysis, using precolumn derivatization high‐performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI‐MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home‐built auto‐sampler system. Amino acids were derivatized with 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate, and a 3 μm Wakosil‐II 3C8‐100HG column (100 × 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra‐ and inter‐precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of lignans in flaxseed using liquid chromatography with time-of-flight mass spectrometry

A new method of flaxseed-derived lignan determination was developed using HPLC with high-resolution time-of-flight MS (TOF-MS), optimized, and compared to two existing methods (HPLC/MS/MS and GC/MS). The limits of detection (LODs) for HPLC/TOF-MS (0.002–0.043 pg) were comparable with those of the optimized and improved HPLC/MS/MS (0.001–0.015 pg), whereas the LODs for the optimized GC/MS were higher (0.02–3.0 pg, yet lower than reported before). Using the newly developed detection and separation methods, several key flaxseed sample preparation parameters (including extraction, hydrolysis, and sample purification) were evaluated resulting in the development of efficient protocol for lignan quantification from flaxseed hulls and embryos. The results confirmed the importance of quantification of both aglycones and unhydrolyzed glucosides in order to obtain the total lignan estimates.     

Simple reversed-phase high-performance liquid chromatographic method for 13-cis-retinoic acid in serum.

An isocratic reversed-phase high-performance liquid chromatographic method for the analysis of 13-cis-retinoic acid in serum is developed. Sample preparation includes deproteination with acetonitrile-perchloric acid-acetic acid followed by centrifugation. 9-Methylanthracene is used as the internal standard. Chromatographic separation is achieved on a C18 column (Zorbax) using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) eluent containing 0.05% (w/v) sodium hexanesulfonate. The limit of detection is 12 ng/ml in serum, using 0.5 ml samples. Quantitative recoveries and excellent intra-day and inter-day precision are reported.     

Determination of retinoic acid (13-cis- and all-trans-) and aromatic retinoic acid analogs possessing anti-tumor activity, in biological fluids by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic assay was developed for the determination of 13-cis- and all-trans-retinoic acid in blood or urine with an overall recovery of 90 +/- 5.0% and a limit of detection of 10-20 ng/ml of sample. The method provides for rapid and simple quantitation of the compounds using 1 ml of blood. The assay was applied in the determination of blood levels of 13-cis-retinoic acid in the dog following intravenous and oral administration of 9.5 mg/kg and 2.0 mg/kg doses, and in man following a single 100-mg oral dose and following divided daily doses totalling 2 mg per kg of body weight. The assay is also applicable with minor modifications to the determination of a series of aromatic retinoic acid analogs of clinical interest as anti-tumor agents.