Comparison of several HPLC methods for the analysis of vitamin C.

Ascorbic acid is a water-soluble vitamin common in food and dietary supplements. A usual problem with ascorbic acid analysis is the lack of stability of its samples and standard solutions owing to oxidation. A procedure to protect ascorbic acid from oxidation using mercaptoethanol is described in this study in connection with the comparison of three HPLC measuring methods. Two reversed-phase columns were evaluated for the separation. One technique uses UV detection, and two others use MS/MS detection. The methods were calibrated for quantitation on different ranges of concentrations. The LC-UV method covers the range 3.9 µg/ml to 500 µg/ml, one LC-MS/MS the range 80 ng/ml to 20 µg/ml, and the other 0.1 ng/ml to 20 µg/ml. As a proof of functionality all three methods were utilized for measuring vitamin C in energy drinks and chews (gummies). The sensitivity of LC-MS/MS methods was not necessary for the analysis of those samples, but the high sensitivity can be beneficial for other types of sample such as environmental or biological, where the levels of ascorbic acid are very low. The study showed that the formation of 2,3-diketogulonic acid is not a likely path for ascorbic acid oxidation following hydrolysis as reported in some studies.

Revisiting the dependence of retention factor on the content of organic component in the mobile phase in reversed-phase HPLC.

In high-performance liquid chromatography, the dependence of retention factor k on volumetric fraction ϕ of organic phase is expressed by log k = F(ϕ) with F(ϕ) obtained by measuring log k at different ϕ values. From F(ϕ), a value kw is calculated by taking ϕ = 0. The equation log k = F(ϕ) is applied for predicting k, and kw is a descriptor of hydrophobic character of solutes and stationary phases. Calculated kw should not depend on the nature of organic component of mobile phase but extrapolation procedure leads to different kw for different organic components. The present study shows that the expression of F(ϕ) changes depending on the range of ϕ and the same function F(ϕ) cannot be used for the full range of ϕ from 0 to 1. Consequently, kw obtained by extrapolation of ϕ to zero is not correct because the expression of F(ϕ) was generated by fitting the data using ϕ with higher values. The present study shows the proper way to obtain the value of kw .

Partial Vapor Pressures, Activity Coefficients, Henry Volatility, and Infinite Dilution Activity Coefficients of Nicotine from Binary Mixtures with Glycerol and with 1,2-Propanediol at 298.15 K.

The equilibrium headspace concentration of nicotine in nitrogen gas was measured by gas chromatography for binary mixtures of nicotine with glycerol and with 1,2-propanediol at temperatures near 298.15 K. The storage temperature ranged from 296.25 to 298.25 K. The nicotine mole fraction ranged from 0.0015 ± 0.00010 to 0.998 ± 0.0016 for the glycerol mixtures and 0.00506 ± 0.000019 to 0.999 ± 0.0038 for the 1,2-propanediol mixtures (k = 2 expanded uncertainty). The headspace concentration was converted to nicotine partial pressure at 298.15 K using the ideal gas law, followed by the Clausius-Clapeyron equation. Both solvent systems had a positive deviation of nicotine partial pressure from ideal behavior, but the deviation of the glycerol mixtures was much greater than that of the 1,2-propanediol mixtures. For mole fractions of about 0.02 or less, the glycerol mixtures had nicotine activity coefficients of 11, while that for the 1,2-propanediol mixtures was 1.5. The Henry's law volatility constant and infinite dilution activity coefficient ± expanded uncertainty for nicotine from glycerol mixtures (51.4 ± 1.8 Pa and 12.4 ± 1.5, respectively) were approximately an order of magnitude greater than those from 1,2-propanediol mixtures (5.26 ± 0.52 Pa and 1.42 ± 0.14, respectively).

Comparison of two methods for ginsenosides quantitation.

The present study provides a comparison of two liquid chromatography-tandem mass spectrometry methods for ginsenosides analysis. The two methods have the same liquid chromatography separation procedure, and both use tandem mass spectrometry detection. However, one method uses multiple reaction monitoring transitions commonly recommended in the literature starting with [M + Na]+ as the molecular ions and with detection of specific fragment ions from the molecules M, while the other is an original method using [M + Cs]+ as molecular ions and Cs+ as fragment ion. The method using [M + Cs]+ as molecular ion has a very high sensitivity allowing the measurement of concentrations in the injecting solutions as low as 4 ng/ml with peaks at this concentration showing signal to noise ratio of 20 or higher. The procedures were utilized for the measurement of eight ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf (S), Rg1, and Rg2), although the method using [M + Cs]+ has the potential for measuring other ginsenosides. As an application, the ginsenosides were measured in several types of ginseng root, several dietary supplements containing ginseng extracts, four energy drinks, and a sample of ashwagandha.

Interconversion of nicotine enantiomers during heating and implications for smoke from combustible cigarettes, heated tobacco products, and electronic cigarettes.

Physiological properties of (R)-nicotine have differences compared with (S)-nicotine, and the subject of (S)- and (R)-nicotine ratio in smoking or vaping related items is of considerable interest. A Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) method for the analysis of (S)- and (R)-nicotine has been developed and applied to samples of nicotine from different sources, nicotine pyrolyzates, several types of tobacco, smoke from combustible cigarettes, smoke from heated tobacco products, e-liquids, and particulate matter obtained from e-cigarettes aerosol. The separation was achieved on a Chiracel OJ-3 column, 250 × 4.6 mm with 3-µm particles using a nonaqueous mobile phase. The detection was performed using atmospheric pressure chemical ionization (APCI) in positive mode. The only transition measured for the analysis of nicotine was 163.1 → 84.0. The method has been summarily validated. For the analysis, the samples of tobacco and smoke from combustible cigarettes were subject to a cleanup procedure using solid phase extraction (SPE). It was demonstrated that nicotine upon heating above 450°C for several minutes starts decomposing, and some formation of (R)-enantiomer from a sample of 99% (S)-nicotine is observed. An analogous process takes place when a 99% (R)-nicotine is heated and forms low levels of (S)-nicotine. This interconversion has the effect of slightly increasing the content of (R)-nicotine in smoke compared with the level in tobacco for combustible cigarettes and for heated tobacco products. The (S)/(R) ratio of nicotine enantiomers in e-liquids was identical with the ratio for the particulate phase of aerosols generated by e-cigarette vaping.

Antioxidant Character and Levels of Polyphenols in Several Tea Samples.

The present study measured the antioxidant properties of 15 commercial tea samples as expressed by the oxygen radical absorbance capacity (ORAC) hydro, ORAC lipo, and ferric reducing antioxidant power (FRAP) indexes. The main antioxidant compounds known to be present in tea are several catechins and catechin gallates, gallic acid, theaflavin and some theaflavin gallates, and theogallin. In this study, only gallic acid and the four most common catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) were analyzed in the tea samples. In addition, caffeine levels were measured. The ORAC and FRAP values for these compounds were also determined. The levels of theaflavin, theaflavin gallates, and theogallin were not measured since these compounds are present at relatively low levels in tea. The ORAC (and FRAP) indexes for each tea sample were also calculated based on the content of individual antioxidant compounds and their ORAC and FRAP indexes. Correlations between the experimental ORAC (and FRAP) and the calculated values were further obtained. The correlations were poor, with R 2 = 0.3657 for ORAC hydro, R 2 = 0.2794 for ORAC lipo, and R 2 = 0.6929 for FRAP. The poor correlation between the overall catechin content and the experimental ORAC values in tea infusions was previously reported in the literature. The present study directly calculated the expected ORAC index from individual antioxidant components and reached the same result of poor correlation. For FRAP values, no comparison was previously reported in the literature. The poor correlations were not well explained, indicating that the cause of the antioxidant character of tea is more complex than simply produced by the main catechins.

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis.

Derivatization, or chemical structure modification, is often used in bioanalysis performed by liquid chromatography technique in order to enhance detectability or to improve the chromatographic performance for the target analytes. The derivatization process is discussed according to the analytical procedure used to achieve the reaction between the reagent and the target compounds (containing hydroxyl, thiol, amino, carbonyl and carboxyl as the main functional groups involved in derivatization). Important procedures for derivatization used in bioanalysis are in situ or based on extraction processes (liquid-liquid, solid-phase and related techniques) applied to the biomatrix. In the review, chiral, isotope-labeling, hydrophobicity-tailored and post-column derivatizations are also included, based on representative publications in the literature during the last two decades. Examples of derivatization reagents and brief reaction conditions are included, together with some bioanalytical applications and performances (chromatographic conditions, detection limit, stability and sample biomatrix).

Does phase ratio in reversed phase high performance liquid chromatography vary with temperature?

Phase ratio Φ for an high performance liquid chromatography (HPLC) column is a parameter defined as the ratio between the volume of the stationary phase Vs and the void volume of the column Vm. Together with the equilibrium constant K of the separation process, phase ratio is part of the retention factor k (k = KΦ). Although a considerable number of studies have been dedicated to the evaluation of Vs and Vm with the goal of obtaining the value for Φ, there are still debatable results regarding the true value of Φ, which for a column with a specific stationary phase may vary with the composition of the mobile phase. One route for the evaluation of the value of Φ uses the measurements of retention factors k on a specific column and mobile phase for two or more hydrocarbons for which the octanol/water partition coefficients log Kow are known. This procedure has been applied in the present study for the evaluation of Φ for three commercially available C18 columns and two mobile phase compositions water/acetonitrile, in the temperature range 20 °C to 50 °C. It was found that phase ratio does change depending on the temperature, its "effective value" decreasing as the temperature increases which is in accordance with the decrease of retention times in reversed phase HPLC when the temperature increases. Besides other factors that may affect the correct calculation of thermodynamic functions, the change of phase ratio with temperature has implications regarding the possibility to calculate the enthalpy and entropy values from van't Hoff plots of the variation of log k as a function of 1/T, even when the retention process is dominated by unique hydrophobic type interactions.

Sources of Nonlinear van't Hoff Temperature Dependence in High-Performance Liquid Chromatography.

In HPLC, the nonlinear behavior of the retention factor k' with temperature (dependence of ln k' on 1/T) can be attributed to the multiple interactions of a unique analyte in the separation process and/or to the existence in solution of multiple forms of the analyte (also leading to different free enthalpies of interaction). In this study, several examples of nonlinear retention-temperature dependence are evaluated for both reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) separations. The potential explanation for nonlinear retention-temperature behavior is evaluated for each example, some caused by multiple interactions in the separation system of a unique analyte and others by multiple forms of the analyte. In cases where the analyte does not have more forms and the separation is based predominantly on one type of interaction (e.g., hydrophobic interaction in RP-HPLC), the dependence is linear, as expected. By studying the changes in the chemical structure of a compound as a function of pH it is possible to decide, in many cases, if a unique form or multiple forms of a compound are present in the solution. The use of this information allows us to determine when the lack of linearity (when present) is caused by multiple interactions in the separation system (for one form of the compound) and when more forms are causing the lack of linearity. The approximation with a quadratic form for the nonlinear dependence has been verified in most cases to be good, and only minor improvements were obtained when using higher polynomial dependencies.

Critical evaluation of several techniques for the analysis of phthalates and terephthalates: Application to liquids used in electronic cigarettes.

This study describes several original methods that were developed with the goal of measuring phthalates and terephthalates. These methods include gas chromatography/mass spectrometry (GC/MS), GC/MS/MS, liquid chromatography with UV detection (LC/UV), LC/MS, and LC/MS/MS. The study compares the advantages and disadvantages of these methods and their applicability to measuring phthalates and terephthalates in the liquids used in electronic cigarettes (e-liquids). The analytes evaluated include eight phthalates and two terephthalates. The phthalates were diethyl, dibutyl, benzyl butyl, diphenyl, bis(2-ethylhexyl), di-n-octyl, diisononyl and diisodecyl. The terephthalates were dimethyl and bis(2-ethylhexyl). Intentionally, no cleanup or concentration step were used in the methods. The methods used two chromatographic standards, dimethyl phthalate-3,4,5,6-d4, and di-(2-ethylhexyl) phthalate-3,4,5,6-d4. All techniques were validated for selectivity/specificity, precision, sensitivity (evaluation of LOD and LOQ), as well as for repeatability and matrix interference. The GC methods were not adequate for the analysis of diphenyl, diisononyl, and diisodecyl phthalates which were not volatile enough to be seen in the conditions used for the GC separation. Also, alcohols should not be used as solvents for the injection of the sample in the GC system to avoid transesterification in the hot injection port. The single quadrupole MS detection in GC offers sensitivities around 1 µg/mL in the e-liquid and was not sensitive enough for the analysis of trace phthalates and terephthalates. Compared to all evaluated methods, the MS/MS detection in GC offered the best sensitivity (below 10 ng/mL in the e-liquid). The LC is adequate for the separation of all the evaluated analytes. However, the UV detection in LC does not offer good sensitivity compared to all the other techniques. The MS detection in LC provides poor sensitivity for terephthalates, but better than the UV for the rest of the analytes. The MS/MS detection for LC offers slightly better sensitivity than the MS detection, but both LC/MS and LC/MS/MS were only able to measure levels above about 100 ng/mL of analytes in the e-liquid. A group of 39 e-liquids were analyzed by three of the evaluated procedures. Benzyl butyl phthalate, bis(2-ethylhexyl) terephthalate, and di-n-octyl phthalate were not detected in the e-liquids. Some of the other evaluated phthalates were present at trace levels in certain e-liquids while most e-liquids did not contain phthalates at detectable levels.

Evaluation of the phase ratio for three C18 high performance liquid chromatographic columns.

For a chromatographic column, phase ratio Φ is defined as the ratio between the volume of the stationary phase Vst and the void volume of the column V0, and it is an important parameter characterizing the HPLC process. Although apparently simple, the evaluation of Φ presents difficulties because there is no sharp boundary between the mobile phase and the stationary phase. In addition, the boundary depends not only on the nature of the stationary phase, but also on the composition of the mobile phase. In spite of its importance, phase ratio is seldom reported for commercially available HPLC columns and the data typically provided by the vendors about the columns do not provide key information that would allow the calculation of Φ based on Vst and V0 values. A different procedure for the evaluation of Φ is based on the following formula: log k'j=a log Kow,j+log Φ, where k'j is the retention factor for a compound j that must be a hydrocarbon, Kow,j is the octanol/water partition coefficient, and a is a proportionality constant. Present study describes the experimental evaluation of Φ based on the measurement of k'j for the compounds in the homologous series between benzene and butylbenzene for three C18 columns: Gemini C18, Luna C18 both with 5 µm particles, and a Chromolith Performance RP-18. The evaluation was performed for two mobile phase systems at different proportions of methanol/water and acetonitrile/water. The octanol/water partition coefficients were obtained from the literature. The results obtained in the study provide further support for the new procedure for the evaluation of phase ratio.

Analysis of four pentacyclic triterpenoid acids in several bioactive botanicals with gas and liquid chromatography and mass spectrometry detection.

Several pentacyclic triterpenoid acids including betulinic, oleanolic, and ursolic acids were reported to have health beneficial properties such as antiviral and anti-inflammatory properties, as well as the capability to inhibit "in vitro" the development of various cancer cell types. For this reason betulinic, oleanolic, and ursolic acids are used as neutraceuticals. For the analysis of the pentacyclic triterpenoid acids in complex plant materials, an improved scheme was developed, involving a qualitative screening using silylation and gas chromatography with mass spectrometry analysis, followed by quantitation using a novel liquid chromatography with tandem mass spectrometry procedure. The use of the two methods provides more reliable information regarding the plant materials with unknown composition. Besides betulinic, oleanolic, and ursolic acids that were analyzed, by this procedure a fourth pentacyclic triterpenoid acid was identified and quantitated that was not previously reported to be present in plants. This acid has been identified as 3ß-3-hydroxy-lupa-18,20(29)-dien-28-oic acid. The newly identified acid has a structure as a derivative of lupane, although lupane with a double bond in the 18-position was not previously reported as present in plants. The new liquid chromatography with tandem mass spectrometry procedure developed for this study offers a very low limit of quantitation, excellent precision, and robustness. Rosemary was found to contain the largest levels of pentacyclic triterpenoid acids among all the analyzed botanicals.

Free amino acids analysis by liquid chromatography with tandem mass spectrometry in several botanicals with antioxidant character.

A novel method based on liquid chromatography with tandem mass spectrometry for the analysis of 19 amino acids in plant materials is described. For the analysis, the plant material is extracted with 0.1 N hydrochloric acid with internal standards present in the extraction solution. The filtered extracts are injected using no clean-up into the liquid chromatographic system coupled with a triple-quadrupole tandem mass spectrometer with an electrospray ionization source. The analytes are separated using ion pair chromatography on a reversed-phase column. The detection is performed in multiple-reaction monitoring positive-ion mode. Quantitation is obtained using calibrations. The validated procedure has been applied for the analysis of amino acids in 18 samples of plant material including botanicals with antioxidant character. The analysis requires 16 min separation time, has excellent precision and accuracy allowing amino acid analysis in a wide range of concentrations.

Dependence of the distribution constant in liquid-liquid partition equilibria on the van der Waals molecular surface area.

The direct calculation of free energy of interactions between a solute j and two immiscible liquids shows a linear dependence between the (logarithm of) the distribution constant in liquid-liquid partition equilibrium log K(j) and the van der Waals surface area of the solute. The study provides a thermodynamic proof for the formula log K(BA,j) = c1 log K(BC,j) + c2 that describes the linear dependence between (the logarithm of) the distribution constant for a solute j in a solvent system (B/A) and (the logarithm of) the distribution constant for the same solute in a different solvent system (B/C). This relation has been well proven by various experimental studies and it is frequently used in liquid chromatographic separations as well as in liquid-liquid extractions, but was not explained previously based on thermodynamic results. The theory was verified using the prediction of octanol/water distribution constants log K(ow) for a wide range of molecules, including hydrocarbons and compounds with a variety of functional groups. The results have also been verified for the distribution constants in other solvent systems. The expression for the distribution constant obtained in this study also gives a theoretical base for the additive fragment methodology used for the prediction of log K(ow).

Dual analysis of triglycerides from certain common lipids and seed extracts.

A number of reference oils, two commercial oils, and several oil extracts from seeds of Nicotiana species were analyzed for the fatty acid content and also for triglyceride composition. The seed oils were obtained using an accelerated solvent extraction procedure, which was proven to be very efficient and reproducible. The fatty acids were analyzed after the hydrolysis of the oils, using trimethylsilylation and gas chromatography/mass spectrometry (GC/MS) analysis. The levels of sixteen molecular species of triglycerides in the oils were measured after GC separation using MS for identification and flame ionization detection (FID) for quantitation. The results for the fatty acids and those for triglycerides were combined to generate uniform information regarding the composition of the analyzed oils. For a number of oils, the individual triglyceride quantitation and mass spectra were reported for the first time. The study showed that in some cases, oils with similar fatty acid content do not have the same triglycerides profile. The fatty acids and triglycerides profile for selected Nicotiana species were described for the first time in the literature.

Gas chromatography/mass spectrometry versus liquid chromatography/fluorescence detection in the analysis of phenols in mainstream cigarette smoke.

A new gas chromatographic/mass spectrometric (GC/MS) technique for the analysis of hydroxybenzenes (phenols) in mainstream cigarette smoke has been developed. The technique allows the measurement of 24 individual compounds, and the sum of a few other alkyl-dihydroxybenzenes. A critical evaluation is done for the new technique and for an established high-performance liquid chromatographic (HPLC) technique reported in the literature for the analysis of hydroxybenzenes in cigarette smoke, which uses fluorescence detection. Compared with the HPLC procedure, the new technique has similar accuracy, precision, and robustness. However, the GC/MS procedure allows for a larger number of phenols to be analyzed simultaneously, and eliminates any potential interference that may appear in the HPLC method. Using the GC/MS analysis, it was found that besides the main phenols typically measured in mainstream cigarette smoke such as phenol, catechol, hydroquinone, and cresols, many other phenols that are present at lower levels can be quantitated in mainstream cigarette smoke.

Analysis of alpha- and beta-carbolines in mainstream smoke of reference cigarettes by gas chromatography-mass spectrometry.

The levels of 2-amino-9H-pyrido[2,3-b]indole (AalphaC or 2-amino-alpha-carboline), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC or 2-amino-3-methyl-alpha-carboline), 9H-pyrido[3,4-b]indole (norharman), and 1-methyl-pyrido[3,4-b]indole (harman) have been determined in the mainstream smoke condensate from three reference cigarettes, namely Kentucky reference 1R5F, Kentucky reference 2R4F, and CORESTA CM4. The amino-alpha-carbolines, and norharman and harman (beta-carbolines) can be classified as heterocyclic aromatic amines (HAAs) and are listed as biologically active agents in the mainstream smoke of cigarettes. For the analysis, the mainstream smoke condensate from cigarettes was collected on a filter pad, the analytes were isolated using solid-phase extraction (SPE), and quantified without derivatization on a GC-MS. Total amounts of carbolines found in the condensate increased from ultralight 1R5F to full-flavor CM4 cigarettes. The level of harman was about 250 ng/cigarette for the 1R5F cigarette and about 1025 ng/cigarette for the CM4 cigarette. Norharman was typically three times more abundant than harman. The AalphaC was found at about 10 times lower level compared to harman, and MeAalphaC was about 50 times lower than harman. The use of reference cigarettes can provide a common measure for laboratories to assess carboline amounts among cigarette brands.

Gas chromatography-mass spectrometry of carbonyl compounds in cigarette mainstream smoke after derivatization with 2,4-dinitrophenylhydrazine.

An improved gas chromatography-mass spectrometry (GC-MS) method was described for the analysis of carbonyl compounds in cigarette mainstream smoke (CMS) after 2,4-dinitrophenylhydrazine (DNPH) derivatization. Besides formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, methyl ethyl ketone, butyraldehyde, and crotonaldehyde that are routinely analyzed in cigarette smoke, this technique separates and allows the analysis of several C4, C5 and C6 isomeric carbonyl compounds. Differentiation could be made between the linear and branched carbon chain components. In cigarette smoke, the branched chain carbonyls are found at higher level than the linear chain carbonyls. Also, several trace carbonyl compounds such as methoxyacetaldehyde were found for the first time in cigarette smoke. For the analysis, cigarette smoke was collected using DNPH-treated pads, which is a simpler procedure compared to conventional impinger collection. Thermal decomposition of DNPH-carbonyl compounds was minimized by the optimization of the GC conditions. The linear range of the method was significantly improved by using a standard mixture of DNPH-carbonyl compounds instead of individual compounds for calibration. The minimum detectable quantity for the carbonyls ranged from 1.4 to 5.6 microg/cigarette.

The role of the anion in the reaction of reducing sugars with ammonium salts.

Reactions of reducing sugars with ammonia and its compounds are important commercially, particularly in the preparation of flavors and caramel colors. However, such reactions generally produce a complex series of products ranging from simple molecules to complex polymeric materials, particularly since commercial systems generally involve mixtures of sugars as opposed to single sugars. This complexity has made understanding the mechanisms of such reactions difficult. Therefore, investigatory work has generally been focused on model systems. Herein we report one such study with model systems: the effects of the nature of the anion of the reactions of reducing sugars with ammonium salts. D-Glucose was reacted in aqueous solution with each of the following ammonium salts: acetate, bicarbonate, carbonate, chloride, citrate, formate, monohydrogenphosphate (DAP), sulfate, and sulfite. These reactions were carried out in a Parr bomb at 93 degrees C for 2.5 h. The initial pH of the reaction mixtures was adjusted to pH 8.0 at 25 degrees C. The resulting mixtures were analyzed by LC-MS, and the results were analyzed by comparing the product yields and distributions with those obtained with DAP. The major reaction product of interest was 2,6-deoxyfructosazine, as it had been shown to be a marker for the polymeric material formed from such reactions. It was found that ammonium salts of weak acids were much more effective in effecting the desired reactions than were those of strong acids; however, none was as effective as DAP.