Thermospray liquid chromatographic-mass spectrometric analysis of anti-AIDS nucleosides: quantification of 2',3'-dideoxycytidine in plasma samples.

Thermospray liquid chromatography-mass spectrometry was investigated as a method for quantification of 2',3'-dideoxycytidine (DDC) from human plasma. A stable isotope analog of DDC ([15N2,2H2]DDC) was used as an internal standard. Selected ion monitoring of the protonated molecular ions for DDC and the internal standard was used to record mass chromatograms. The areas of the peaks in the mass chromatograms were used for quantification. The detection limit of DDC in this assay was 50 pg on-column. The calibration curve was linear over the desired range, 0.25-20 ng/ml. The major advantages of this assay over others are: no derivatization, high sensitivity, high specificity and short assay time.     

Application of high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry for analysis and quality control of Radix Astragali and its preparations

A high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) method has been developed for qualitative and quantitative analysis of isoflavonoids and saponins, as well as for the quality control of Radix Astragali and its preparations. The selectivity, reproducibility and sensitivity are compared with HPLC with diode array detection (DAD) and evaporative light scattering detection (ELSD). Limits of detection and quantification fell in ranges of 9-40 and 23-103 ng/mL for 13 analytes with a injection of 10 microL samples, and all calibration curves showed good linear regression (r(2)>0.9938) within the test range. The assay was successfully utilized to analyze the 13 marker components in 20 samples of Radix Astragali products. The quantitative results demonstrated that samples from different localities and manufacturers showed different quality. Advantages, in comparison with conventional HPLC-DAD-ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements (<3 ppm) along with characteristic retention time, extracted ions chromatograms using a narrow mass window for quantification ensure that the chromatographic peaks are free from background or co-elutes interference, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Radix Astragali matrixes.     

Determination of Propylene Glycol in Low Volume Plasma and Urine Samples of Neonates by LC with Photodiode Array Detection

In order to enhance the sensitivity and to develop a method suitable for quantification of propylene glycol (PG) in low volume neonate plasma and urine samples, several steps in earlier described high performance liquid chromatographic methods were optimised. Chromatographic separation on a reversed-phase column and ultraviolet detection resulted in cleaner chromatograms without interfering compounds. Linearity of the standard curves was validated in the concentration range 0.25?C50 mg L^?1. The lower limit of quantification was 20 times lower than in earlier described methods. Presented method was suitable for quantification of PG concentrations in low volume neonate plasma (15?C46 mg L^?1) and urine samples (20?C175 mg L^?1) enabling us to document very low renal versus non-renal contribution of PG clearance in neonates.     

Evaluation of the ion trap MS performance for quantification of flavonoids and comparison to UV detection

The application of an ion trap mass spectrometer, usually employed for identification, has been here systematically evaluated for quantitative analysis of various conjugated forms of flavonoids and compared with UV quantification. Three MS methods were tested to assess the potential and limits of the ion trap for quantification of flavonoids: full‐scan experiment MS², isolated ion experiment MS, and full‐scan experiment MS. The test was performed using nine reference standards of flavonoids with six different aglycones: luteolin, apigenin, hypolaetin, 4′‐O‐methylhypolaetin, isoscutellarein and 4′‐O‐methylisoscutellarein in the form of 7‐O‐glucosides and diglucosides, mono or diacetylated, isolated from Sideritis scardica. The analytical characteristics of the tested MS methods were shown to be comparable to UV with regards to precision and accuracy, and superior for selectivity and sensitivity especially when using extracted ion chromatograms. Detection limits did not differ significantly between the MS methods but were significantly lower than those obtained with UV detection by one order of magnitude. Another issue addressed by these results was the choice of most suitable standard substances for quantification of flavonoids with various substituents attached when using MS. In UV detection, the nature of the aglycone is crucial for the absorbance properties, and various derivatives can be quantified with the available one with the same aglycone. Here, it was shown that in MS detection, one flavone derivative can be quantified using other available derivatives with similar substitution pattern with regards to attached and acetylated sugars, whereas the nature of the aglycone is not crucial. Copyright © 2012 John Wiley & Sons, Ltd.     

Application of image analysis technique for the determination of thiophanate methyl by thin-layer chromatography

A simple and convenient thin-layer chromatography (TLC) method combined with image analysis technique was developed to determine thiophanate methyl. The detection of pesticide was based on iodine–azide reaction. Digital images of TLC plate chromatograms were analysed using TLSee software, and quantitative analysis was conducted. The linearity (0.3–3.0 µg per spot), sensitivity, accuracy and precision of the system were investigated.     

Chemical fingerprint and quantitative analysis for the quality evaluation of Vitex negundo seeds by reversed‐phase high‐performance liquid chromatography coupled with hierarchical clustering analysis

A simple and efficient method was developed for the chemical fingerprint analysis and simultaneous determination of four phenylnaphthalene‐type lignans in Vitex negundo seeds using high‐performance liquid chromatography with diode array detection. For fingerprint analysis, 13 V. negundo seed samples were collected from different regions in China, and the fingerprint chromatograms were matched by the computer‐aided Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A). A total of 21 common peaks found in all the chromatograms were used for evaluating the similarity between these samples. Additionally, simultaneous quantification of four major bioactive ingredients was conducted to assess the quality of V. negundo seeds. Our results indicated that the contents of four lignans in V. negundo seeds varied remarkably in herbal samples collected from different regions. Moreover, the hierarchical clustering analysis grouped these 13 samples into three categories, which was consistent with the chemotypes of those chromatograms. The method developed in this study provides a substantial foundation for the establishment of reasonable quality control standards for V. negundo seeds.     

UPLC-UV-MSE analysis for quantification and identification of major carotenoid and chlorophyll species in algae

A fast method for quantification and identification of carotenoid and chlorophyll species utilizing liquid chromatography coupled with UV detection and mass spectrometry has been demonstrated and validated for the analysis of algae samples. This method allows quantification of targeted pigments and identification of unexpected compounds, providing isomers separation, UV detection, accurate mass measurements, and study of fragment ions for structural elucidation in a single run. This is possible using parallel alternating low- and high-energy collision spectral acquisition modes, which provide accurate mass full scan chromatograms and accurate mass high-energy chromatograms. Here, it is shown how this approach can be used to confirm carotenoid and chlorophyll species by identification of key diagnostic fragmentations during high-energy mode. The developed method was successfully applied for the analysis of Dunaliella salina samples during defined red LED lighting growth conditions, identifying 37 pigments including 19 carotenoid species and 18 chlorophyll species, and providing quantification of 7 targeted compounds. Limit of detections for targeted pigments ranged from 0.01?ng/mL for lutein to 0.24?ng/mL for chlorophyll a. Inter-run precision ranged for of 3 to 24 (RSD%) while inter-run inaccuracy ranged from ?17 to 11.

Determination of Piribedil in Human Serum, Urine and Pharmaceutical Dosage Form by LC-DAD

A rapid, selective and sensitive reversed-phase liquid chromatographic (LC) method was developed for the determination of piribedil in human serum, urine and pharmaceutical dosage form. LC analysis was carried out using reversed-phase isocratic elution with a C₁₈ column and a mobile phase of 0.01 M phosphate buffer-acetonitrile (50:50, v/v). The chromatograms showed good resolution and sensitivity with no interference of human serum and urine. Piribedil concentrations were determined using diode array detection at 240 nm. Sildenafil citrate was used as internal standard. The limit of quantification (LOQ) and limit of detection (LOD) concentrations were 107.2 and 321.6 pg mL^?1, 96.6 and 290.4 pg mL^?1, 161.7 and 53.9 pg mL^?1 for urine, serum and pharmaceutical dosage forms, respectively. The method was validated for its linearity, precision and accuracy and applied to the tablets, urine and human serum. In addition, the results were compared to those obtained from UV-spectrophotometry.     

Densitometric high performance thin-layer chromatography identification and quantitative analysis of psychotropic drugs

A thin-layer chromatography (TLC)-densitometry method has been developed to identify and quantify haloperidol, amitriptyline, sulpiride, promazine, fluphenazine, doxepin, diazepam, trifluoperazine, clonazepam, and chlorpromazine in selected psychotropic drugs. Separation was performed on precoated silica gel 60 F254 TLC plates. Chromatograms were developed in various mobile phases, and 8 of 30 tested phases were selected based on spot location and developing time. The identification and quantification were carried out based on ultraviolet densitometric measurements at chosen wavelengths. In addition to retention coefficients, the absorption spectra recorded directly from chromatograms were also used in qualitative analysis. Under established experimental conditions, high sensitivity of the method was achieved. The limit of detection ranged from 0.009 to 0.260 microg, depending on the wavelength selected for measuring. A satisfactory recovery, ranging from 92.99 to 104.70%, was achieved for individual constituents.     

Concentration and clean-up of trace hydrocarbons from atmospheric particulate matter

Summary The aim of this work is to establish the best conditions for concentration and purification steps in the trace analysis of aliphatic and polycyclic aromatic hydrocarbons (PAH) from atmospheric particulate matter by gas chromatography-flame ionisation detection (GC-FID) and high performance liquid chromatography with ultraviolet and fluorescence detection (HPLC-UV-FL). The best results for the more volatile compound were obtained with a combination of rotary evaporation and a stream of nitrogen (near to 100% for aliphatic hydrocarbons and from 70 to 105% for PAH). Two types of solid phase extraction (SPE)cartridges (Supelclean ^tm LC-Silica SPE tubes and Sep-Pak^? Plus silica cartridges) and glass column were examined for the purification and fractionation step. Blank chromatograms of both types of cartridges analysed by GC-FID made this study difficult, because a PSS (programmed split-splitless) injector was employed thereby increasing the sensitivity. This problem was not observed in the HPLC-UV-FL blank chromatograms of these cartridges. Glass columns filled with silica and alumina were chosen because no interference was found in the GC-FID blank chromatograms and the best recoveries in the fractionation of both aliphatic hydrocarbons and PAH were achieved. This is especially important when aliphatic hydrocarbons concentrations are lower than 1 μg mL⁻¹. Finally, the selected conditions were applied to the analysis of hydrocarbons in real atmospheric particulate samples.     

Assessment of metastable atom bombardment (MAB) ionization mass spectrometry for the fast determination of heterocyclic aromatic amines in cooked meat

An investigation of metastable atom bombardment (MAB) ionization mass spectrometry for the fast characterization of mutagenic/carcinogenic heterocyclic aromatic amines (HAAs) formed during heating processes of meats is presented. The aim of our study was to use the selective ionization of MAB to develop a detection method for HAAs in non-purified meat extracts, thus avoiding purification and concentration steps and reducing analysis time. Sample introduction into the MAB ion source was achieved by pyrolysis, allowing the direct and fast insertion of complex food extracts into the mass spectrometer. Analysis conditions were optimized on standard HAAs by using different ionization gases for the MAB process. Metastable nitrogen was selected as the best MAB gas for the analysis of HAAs. Ionization selectivity is shown by the detection of heterocyclic amines in non-purified chicken meat extracts spiked with HAAs. A quantitative approach is also presented by using pyrograms as chromatograms for quantification purposes. HAAs determination using Py-MAB-ToF was finally performed on cooked chicken breast extracts and compared to an LC-APCI-MS/MS method. Although Py-MAB-ToF sensitivity remains to be improved in the present state of development of our prototype device, only 2 h from the cooking were required to obtain quantitative results in good agreement with HAAs concentrations measured by LC-MS/MS in 36 h. Figure Experimental set-up for pyrolysis-MAB-ToF mass spectrometry experiments.     

Comparison of volatile constituents extracted from model grape juice and model wine by stir bar sorptive extraction-gas chromatography-mass spectrometry

A stir bar sorptive extraction (SBSE) method coupled with gas chromatography-mass spectrometry was optimised for the analysis of volatile components of a model wine, based on a previously optimised method used for analysis of the same components in model grape juice. The presence of ethanol in the model wine sample matrix resulted in decreased sensitivity of the method toward most of the volatile constituents. Mean percent relative recoveries and reproducibilities (%CV) were 22.8% and 7.1%, respectively, compared with 28.4% and 8.5% for model grape juice. The mean limit of detection (LoD) ratio (juice:wine) was 0.25. Similar sensitivities for the two sample matrices using this method were achieved by changing the split ratio from 20:1 (grape juice) to 5:1 (wine), giving a mean limit of detection ratio (juice:wine) of 1.0, thus allowing direct comparison of chromatograms of volatile components in the two matrices. This enabled direct comparisons of grape juices and the wines derived from them by alcoholic yeast fermentation. The influence of ethanol concentration in the range 9-15% on method sensitivity is discussed, using an overlay of the total ion chromatograms. The use of a gas saver device for the 5:1 split ratio analysis of desorbed model wine aroma compounds is discussed in terms of preventing extraneous reaction of sorbent and stationary phases with air during analysis.     

Use of proficiency testing materials for the calculation of detection and quantification limits in the analysis of organochlorine compounds in human serum

A method for the calculation of the limits of detection (LD) and quantification (LQ) for the analysis of organochlorine compounds in serum is described. The method is based on the analysis of proficiency testing materials, an external quality assessment for selected pollutants, and the study of the signal/noise ratio of chromatograms obtained from GC-ECD injection. This method provides representative results for matrix effects, instrumental variability and extraction recoveries in the analysis of serum samples.     

LC Determination of Adulterated Saffron Prepared by Adding Styles Colored with Some Natural Colorants

A high-performance liquid chromatographic method previously developed (Lozano et al. in J Chromatogr A 830:477–483, 1999) for simultaneous detection, identification and quantification of the secondary metabolites in commercial saffron was extended for the detection of adulterated saffron prepared by adding styles colored with the natural colorants extracted from saffron petals, safflower, madder and red beet. The chromatograms of the methanol-water (50%, v/v) extracts of pure and adulterated saffron were obtained at the assayed wavelengths, 402 (or 254), 260 and 535 (or 440) nm and then by applying two-way analysis of variance (ANOVA) to the obtained data the presence of the styles colored with the colorant of safflower (>14.3%), styles colored with the colorant of madder (>9.1%) and styles colored with the colorant of red beet (>14.3%) in saffron were significantly detected. But the detection of adulterated saffron prepared with the colorant of saffron petals was not successful.     

High-performance anion-exchange chromatography combined with intrinsic fluorescence detection to determine erythropoietin in pharmaceutical products

A high-performance anion-exchange chromatography (HPAEC) method was developed for determination of recombinant human erythropoietin (EPO) in pharmaceutical products. A fluorescence detector was added to the HPLC system as intrinsic fluorescence detection compared favourably to UV detection regarding sensitivity and selectivity. The HPLC method has been successfully applied to analyse erythropoietin products even in the presence of albumin as excipient. The intrinsic fluorescence chromatograms of both proteins revealed various peaks attributed to either micro-heterogeneous erythropoietin or albumin variants. The intrinsic fluorescence signal was linear over the range 10-200 microg/ml erythropoietin corresponding to pharmaceutically relevant concentrations. The HPLC method appeared to be a suitable method for differentation between recombinant human erythropoietin epoetin-alpha and -beta as they revealed different intrinsic fluorescence elution profiles. In conclusion, this study contributes to the development of a straightforward physicochemical method for specific quantification of recombinant human erythropoietin in pharmaceutical preparations.     

Differentiation of Lippia gracilis Schauer Genotypes by LC Fingerprint and Chemometrics Analyses

A liquid chromatographic method with photodiode-array detection has been developed for differentiation of seven genotypes of Lippia gracilis Schauer, originated from Sergipe state (named 106 to 110) and from Bahia state (201 and 202), cultivated and collected in the rural campus of the Federal University of Sergipe. For a comparative analysis of the fingerprint chromatograms, chemometric tools were applied with exploratory methods. The genotypes were differentiated and their relationship with original place was established (Sergipe and Bahia states) by principal component analysis of the fingerprint chromatograms of both leaves and stem samples.     

Fully automated reagent peak‐free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column‐switching system and fluorous scavenging derivatization

In this study, we combined a column‐switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak‐free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1‐pyrenemethylamine in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and 1‐hydroxy‐1H‐benzotriazole. Residual excess of the unreacted reagent was tagged with 2‐(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column‐switching system placed in front of an analytical reversed‐phase column. The signal of the fluorous‐tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type‐II model mice and their control.     

High-performance liquid chromatography analysis of ten dyes for control of safety of commercial articles

Two high-performance liquid chromatographic methods, with ultraviolet-visible spectrophotometry detection (HPLC-UV/Vis) and with tandem mass spectrometry triple quadrupole interfaced with positive ion mode electrospray ionization detection (HPLC-ESI+-QqQ-MS/MS), for determination and quantification of ten commercial dyes are proposed for control in commercial products. Multiple peaks observed for some of the studied dyes in HPLC-UV/Vis chromatograms forced to obtain structural information by HPLC-ESI-MS/MS method with scan mode. The quality parameters of the two proposed chromatographic methods were evaluated for different requirements of normative, showing detection (LODs) and quantification (LOQs) limits around 60-890 and 200-2990 microg L(-1) for HPLC-UV/Vis, and 4.54-14.3 and 15.0-47.6 microg L(-1) for HPLC-ESI+-QqQ-MS/MS.