Identification of alkyl carbazoles and alkyl benzocarbazoles in Brazilian petroleum derivatives

The present work describes (a) the identification and characterization of an impurity, 2,4,5,7-tetrabromo-6-hydroxy-9-(2,3,4,5-tetrachlorophenyl)-3H-xanthen-3-one (BCPX), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of BCPX contamination in certified lots of these colors. For these purposes, BCPX (a compound not previously reported in the literature) was synthetically prepared. Test portions from 42 certified lots of D&C Red Nos. 27, 28 and 27 lakes were analyzed for BCPX using an HPLC method that included gradient elution and UV-vis photodiode array detection. Those lots were submitted for certification by both domestic (six) and foreign (six) manufacturers during the past 4 years. Of the test portions analyzed, 32 (76.2%) contained BCPX in amounts ranging from 0.01 to 3.21%. The remaining 10 test portions (23.8%) contained no detectable BCPX or less than 0.008%, which is the limit of quantification for the present method. The analyses revealed substantial differences in the level of BCPX across different manufacturers. The wide range of BCPX levels found in the analyzed lots suggests that the presence of BCPX in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process.     

Determination of 1,3-diphenyltriazene and azobenzene in D&C Red No. 33 by solvent extraction and reversed-phase high-performance liquid chromatography

Data are presented for the determination of trace levels of 1,3-diphenyltriazene (DPT) and azobenzene (AB) in D&C Red No. 33. The contaminants are extracted with chloroform from an aqueous solution of the color, and the chloroform is removed under vacuum. The residue is dissolved in acetonitrile and the solution is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with detection at 365 nm. DPT and AB were determined at levels of less than or equal to 439 ppb and less than or equal to 2.2 ppm, respectively, in samples of commercial D&C Red No. 33. Method precision for the DPT determination (five analyses) was 6.8% relative standard deviation. The UV-Vis spectra of the HPLC analytes, obtained by using a rapid-scan diode-array spectrophotometer, were used to confirm the identity of the DPT and AB responses produced during analysis of the D&C Red No. 33 extracts. The method is also useful for the separation of cis- and trans-AB.     

Identification of 2-bromo-3,4,5,6-tetrachloroaniline and its quantification in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction and gas chromatography-mass spectrometry

The present work describes (a) the identification and characterization of a contaminant, 2-bromo-3,4,5,6-tetrachloroaniline (2BTCA), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of 2BTCA contamination in certified lots of these colors. For these purposes, 2BTCA (a compound not previously reported in the literature) and its positional isomer 4-bromo-2,3,5,6-tetrachloroaniline (4BTCA) were synthetically prepared. 4BTCA was used as the internal standard for the quantification of 2BTCA in the colors. Test portions from 35 certified lots of D&C Red Nos. 27 and 28 were analyzed for 2BTCA using a solid-phase microextraction-GC-MS method. Those lots were submitted for certification by both domestic (seven) and foreign (four) manufacturers during the past 4 years. Of the test portions analyzed, 22 (62.9%) contained 2BTCA in amounts ranging from 0.15 to 435.7 ppm with an average value of approximately 131.7 ppm. The remaining 13 (37.1%) test portions contained no detectable 2BTCA or less than 0.01 ppm, which is the limit of quantification of the present method. The analyses revealed substantial differences in the level of 2BTCA across lots from the same manufacturer as well as among different manufacturers. The wide range of 2BTCA levels found in the analyzed lots suggests that the presence of 2BTCA in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process. A direct correlation was observed between the presence of 2BTCA and that of 3,4,5,6-tetrachlorophthalic acid in analyzed batches of D&C Red Nos. 27 and 28. A chemical pathway that could explain the presence of 2BTCA in these color additives, and ways to avoid its formation, are also proposed.     

Identification of isomers and subsidiary colors in commercial Fast Green FCF (FD&C Green No. 3, Food Green No. 3) by liquid chromatography-mass spectrometry and comparison between amounts of the subsidiary colors by high-performance liquid chromatography and thin-layer chromatography-spectrophotometry

The existence of five of the six expected isomers in commercial Fast Green FCF (G3: Food Green No. 3, FD&C Green No. 3, CAS No. 2353-45-9, C.I. No. 42053), the main product of which is m,m-G3 and the sub-products of which are presumed to be m,p-G3, o,m-G3, p,p-G3, o,p-G3 and o,o-G3, was confirmed using LC/MS, and the levels of the isomers, m,m-G3, m,p-G3, p,p-G3, o,m-G3 and o,p-G3, were determined by analytical HPLC. The existence of seven subsidiary colors that were decomposed from G3 was also confirmed using LC/MS. The levels of the subsidiary colors in ethanol extracts from TLC were determined by HPLC and spectrophotometry, and these results were compared. It was clear that the values determined by TLC-spectrophotometry were higher than those by HPLC. It was recommended that the levels of subsidiary colors in G3 should be determined by HPLC.     

High-performance liquid chromatography with fluorescence detection and ultra-performance liquid chromatography with electrospray tandem mass spectrometry method for the determination of indoleamine neurotransmitters and their metabolites in sea lamprey plasma

We present a comparison of two sensitive methods, HPLC with fluorescence detector (HPLC/FLD) and UPLC with electrospray tandem mass spectrometry (UPLC/MS/MS), for the determination of indoleamine neurotransmitters (NTs) and their metabolites in sea lamprey plasma samples. Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) were also tested for recovery and matrix effect. The recoveries of SPE determined by HPLC/FLD and UPLC/MS/MS ranged from 75 to 123% and 78 to 105%, respectively, while the recoveries of LLE ranged from 45 to 73% and 48 to 75%, respectively. SPE combined with HPLC/FLD and UPLC/MS/MS to determine the target analytes in plasma samples were validated of the sensitivity, reproducibility, accuracy and precision. Both methods exhibited excellent linearity in the range of 0.2–50 ng mL⁻¹ for all analytes. The limits of detection (LOD) varied from 0.04 ng mL⁻¹ to 0.13 ng mL⁻¹ for HPLC/FLD method and 0.003 ng mL⁻¹ to 0.02 ng mL⁻¹ for UPLC/MS/MS method. The inter-day accuracy ranged from 82.5 to 127.0% for HPLC/FLD and 93.0 to 113.0% for UPLC/MS/MS. The inter-day precision ranged from 9.9 to 32.3% for HPLC/FLD and 5.4 to 13.2% for UPLC/MS/MS. These results demonstrated that the values obtained by both methods were within the satisfactory range and the UPLC/MS/MS method provided more accurate and precise measurements than HPLC/FLD method. The comparison is of great importance to determine the available detectors, considering the complexity and expensiveness versus quality parameters. These two methods were applied to the analysis of four important indoleamine neurotransmitter analytes (5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, tryptamine and melatonin) in sea lamprey plasma samples.     

Rapid quantification of hexachlorobenzene in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction and gas chromatography-mass spectrometry

The present paper describes the development of a method for the quantification of hexachlorobenzene (HCB) in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. The method is simple and fast (1 h for each analysis), generates little solvent waste, and does not involve a solid matrix, thus permitting a more efficient extraction than does a previously developed Soxhlet extraction-GC-MS method. Test portions from 30 batches of US-certified color additives D&C Red Nos. 27 and 28 were analyzed for HCB using the new method. Those batches represent domestic (five) and foreign (one) manufacturers that requested certification for the colors during the past four years. All the samples contained HCB, ranging from 0.2 ppm to 244.3 ppm. The analyses revealed significant differences in the levels of HCB across batches from the same manufacturer as well as among different manufacturers. The range of HCB levels found in the analyzed batches (0.2-244.3 ppm) suggest that the contamination with HCB may be decreased by avoiding use of starting material (tetrachlorophthalic anhydride) heavily contaminated with HCB.     

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。     

High-throughput quantification for a drug mixture in rat plasma-a comparison of Ultra Performance liquid chromatography/tandem mass spectrometry with high-performance liquid chromatography/tandem mass spectrometry

A quantitative Ultra Performance liquid chromatography/tandem mass spectrometry (UPL/MS/MS) protocol was developed for a five-compound mixture in rat plasma. A similar high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) quantification protocol was developed for comparison purposes. Among the five test compounds, three preferred positive electrospray ionization (ESI) and two preferred negative ESI. As a result, both UPLC/MS/MS and HPLC/MS/MS analyses were performed by having the mass spectrometer collecting ESI multiple reaction monitoring (MRM) data in both positive and negative ion modes during a single injection. Peak widths for most standards were 4.8 s for the HPLC analysis and 2.4 s for the UPLC analysis. There were 17 to 20 data points obtained for each of the LC peaks. Compared with the HPLC/MS/MS method, the UPLC/MS/MS method offered 3-fold decrease in retention time, up to 10-fold increase in detected peak height, with 2-fold decrease in peak width. Limits of quantification (LOQs) for both HPLC and UPLC methods were evaluated. For UPLC/MS/MS analysis, a linear range up to four orders of magnitude was obtained with r2 values ranging from 0.991 to 0.998. The LOQs for the five analytes ranged from 0.08 to 9.85 ng/mL. Three levels of quality control (QC) samples were analyzed. For the UPLC/MS/MS protocol, the percent relative standard deviation (RSD%) for low QC (2 ng/mL) ranged from 3.42 to 8.67% (N = 18). The carryover of the UPLC/MS/MS protocol was negligible and the robustness of the UPLC/MS/MS system was evaluated with up to 963 QC injections.     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.     

Development and validation of UPLC tandem mass spectrometry assay for separation of a phase II metabolite of ramipril using actual study samples and its application to a bioequivalence study

In this paper, we present a validated UPLC‐MS/MS assay for determination of ramipril and ramiprilat from human plasma samples. The assay is capable of isolating phase II metabolites (acylglucornides) of ramipril from in vivo study samples which is otherwise not possible using conventional HPLC conditions. Both analytes were extracted from human plasma using solid‐phase extraction technique. Chromatographic separation of analytes and their respective internal standards was carried out using an Acquity UPLC BEH C₁₈ (2.1 × 100 mm), 1.7 µm column followed by mass spectrometric detection using an Waters Quattro Premier XE. The method was validated over the range 0.35–70.0 ng/mL for ramipril and 1.0–40.0 ng/mL for ramiprilat. Copyright © 2010 John Wiley & Sons, Ltd.     

Densitometric thin-layer chromatographic determination of artemisinin and its lipophilic derivatives,artemether and arteether

A thin-layer chromatograpy (TLC) method is developed to analyze artemisinin (AT) and its derivatives, artemether (AM) and arteether (AE), using a silica-gel plate with a mobile phase containing pure chloroform. After development, all products are visualized after dipping in a 4-methoxybenzaldehyde dipping reagent of 1% (v/v) in an acidic solution of sulphuric acid (98%, v/v) and acetic acid (96-98%, v/v) (respectively, 2% and 10%, v/v in alcohol-water, 60:30, v/v), presenting a purple color against a slightly colored background. This TLC system is quantitatively evaluated in terms of stability of the color, precision, accuracy, and calibration. Activation is performed at 110 degrees C. Stability of the color of both analytes is reached after 12 min. Precision, less than 5%, is obtained at two levels. Good linearity is obtained in the range of 0.5-8 micro g for all analytes. Some applications show its utility in the quality control of capsules. The prederivatization technique, applying the described dipping reagent before development, reveals the presence of various reaction products, possibly isomers. These results prove that TLC can be a cheap and easy alternative for the analysis of AT and its lipophilic derivatives, AM and AE, as pure powder and in pharmaceutical-dosage forms.     

偶氮染料刚果红在水中的光催化降解过程

 通过研究在间歇悬浮体系中直接偶氮染料刚果红在水中的光催化降解过程,确定了最佳降解条件. 同时测定了染料溶液的脱色率、COD去除率和矿化率以考察降解过程中脱色与矿化的关系. 另外,对降解过程中溶液pH值的变化及反应的可能中间体进行分析,结果表明: 刚果红染料分子的光催化降解过程分为脱色和矿化两个阶段. 矿化主要发生在脱色结束以后, 并且伴随着脱色溶液的酸化现象. 脱色过程进行较快,完全脱色后形成的中间产物需要较长时间才能被逐步矿化.     

Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.     

高效液相色谱及薄层色谱分析蔗糖脂肪酸酯

采用薄层色谱（ＴＬＣ）扫描法和高效液相色谱法（ＨＰＬＣ）定性及定量地分析了蔗糖脂肪酸酯的组分。这两种方法的测定结果基本一致,而ＨＰＬＣ与ＴＬＣ相比,则更具快速、灵敏、准确性     

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

Abstract

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile?+?methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (R_M⁰/m, logk₀/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.     

Determination of Sudan Dyes and Para Red in Duck Muscle and Egg by UPLC

A simple, sensitive and reliable UPLC method was developed and validated for the simultaneous determination of six Sudan dyes (Sudan Red G, Sudan I, II, III, Sudan Red 7B, Sudan IV) and Para Red in duck muscle and egg samples. Samples were extracted with acetonitrile, then the extract was dried under rotary evaporation and dissolved in acetonitrile/0.1% formic acid (85:15, v/v). Chromatographic separation was achieved on an Acquity UPLC BEH C18 column under gradient conditions. Samples were detected using a diode array detector set to 480 and 500 nm. The method was validated with respect to linearity, accuracy, precision, and specificity.     

Fast simultaneous determination of 14 nucleosides and nucleobases in cultured Cordyceps using ultra-performance liquid chromatography

Determination of nucleosides and their metabolic compounds is important for physiological and pharmacological studies. Herein, a rapid ultra-performance liquid chromatography (UPLC) method was developed for the simultaneous determination of 14 nucleosides and nucleobases, namely adenine, adenosine, cytosine, cytidine, uracil, uridine, guanine, guanosine, hypoxanthin, inosine, thymine, thymidine, 2′-deoxyuridine and cordycepin. The separation was performed on Waters Acquity UPLC system with Acquity UPLC BEH C₁₈ column and gradient elution of 0.5 mM acetic acid and acetonitrile in 5 min. The correlation coefficients of 14 analytes were high (R² > 0.9995) within the test ranges. The LOD and LOQ were lower to 11.9 and 47.0 ng/ml with 1 μl of injection volume, respectively. The overall R.S.D. for intra- and inter-day of 14 analytes were less than 1.8%. The developed method was applied for the analysis of nucleosides and nucleobases in cultured Cordyceps, which also could be used for the fast determination of the analytes in pharmaceutical products and biological fluids.     

Simultaneous quantification of fentanyl,sufentanil, cefazolin,doxapram and keto‐doxapram in plasma using liquid chromatography–tandem mass spectrometry

A simple and specific UPLC–MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto‐doxapram. The internal standard was fentanyl‐d5 for all analytes. Chromatographic separation was achieved with a reversed‐phase Acquity UPLC HSS T3 column with a run‐time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli‐Q ultrapure water or in methanol with a total flow rate of 0.4 mL min⁻¹. A plasma volume of only 50 μL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto‐doxapram, which is useful for research as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run‐time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run.     

Ultra-performance liquid chromatography/mass spectrometry of intact proteins

Given that numerous small molecule applications of ultra-performance liquid chromatography (UPLC) have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of 10 protein standards, the optimized method yielded improved chromatographic resolution, enhanced sensitivity, and a threefold increase in throughput. Subsequent cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality at 1/3 of the original run time.     

A Validated HPLC Method for Simultaneous Determination of 2-Hydroxy-4-methoxybenzaldehyde and 2-Hydroxy-4-methoxybenzoic Acid in Root Organs of Hemidesmus indicus

A reverse phase HPLC method was developed and validated for the simultaneous determination of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in the root extracts of Hemidesmus indicus. A comprehensive validation of the method including sensitivity, linearity, reproducibility, accuracy, limit of detection (LOD) and limit of quantification (LOQ) was conducted using the optimized chromatographic conditions. The method was found to be linear (r > 0.998) in the range of 5–350 μg mL⁻¹ for 2-hydroxy-4-methoxybenzaldehyde and for 2-hydroxy-4-methoxybenzoic acid (r > 0.999) in the range 10–300 μg mL⁻¹. The method was found to be precise with inter-day precision values (% RSD) in the ranges of 0.61–1.76% for 2-hydroxy-4-methoxybenzaldehyde and 1.3–2.8% for 2-hydroxy-4-ethoxybenzoic acid while intra-day precisions (% RSD) of two analytes were in the range of 0.41–1.07 and 0.95–2.5%. The limits of detection (LODs) for 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid were 0.84 and 2.34 μg mL⁻¹. The described method was fast, sensitive and reproducible, and thus well suited for routine analysis of these two compounds from root extracts of H. indicus and other plants.