Initial Mobile Phase Composition Research Articles

B-194 Development and Validation of Rapid LC-MS/MS Method for Simultaneous Determination of 8-iso-PGF2α and 11-DHTXB2 in Human Urine

Abstract Background Cardiovascular disease (CVD) is one of the leading causes of death globally, the prevalence ofCVD is expected to increase from 36.9% to 40.5% between 2010 and 2030 in the United States.Atherosclerosis (AS) is thought to be the primary cause of cardiovascular diseases (CVDs),however there are several others. About half of Americans between ages 45 and 84 haveatherosclerosis and don’t know it. Eicosanoids (8-iso-PGF2α and 11-Dehydrothromboxane B2) arethe most common biomarkers for predicting atherosclerosis. The objective of this study was todevelop a rapid LC-MS/MS method for simultaneous determination of 8-iso-PGF2α and 11-DehydrothromboxaneB2 (11-DHTXB2) in human urine. Methods One milliliter of human urine spiked with 0.5 µL of each internal standard (1 µg/mL) was mixedwith 1 mL of heptane, 1 mL of ethyl acetate, and 1 mL of Acetonitrile in a glass tube followed by theaddition of 1 mL of pure water. The mixture was vortexed for 1 min and then centrifuged (4000rpm) for f 5 min. The middle organic layer was transferred to another tube and dried undernitrogen and resuspended with 50 µL of the initial mobile phase composition (70:30 v/v H2O:ACN) for the analysis. One milliliter of human urine spiked with 0.5 µL of each internal standard(1 µg/mL) was loaded to a conditioned SPE cartridge, then washed with 1 mL of 10% ACN afterthat analytes eluted with 1 mL of pure ACN at the end eluent is collected, dried and reconstitutedin 50 µL of (70:30 v/v H2O: ACN). LC-MS/MS was performed using AB Sciex 5500 QTRAPtriple quadrupole tandem mass spectrometer coupled to Shimadzu HPLC with 10 minutesgradients for both samples. Results The method was optimized by using standard solutions of 8-iso-PGF2α and 11-dehydrothromboxane B2 and showed separation at 5.27 and 5.45 min respectively. The methodapplied on pooled urine sample from healthy individuals and showed No bias in retention time.Two purification methods were used to extract the two analytes from pooled urine samples, threephase liquid extraction (3PLE) and solid phase extraction (SPE). 3PLE showed a better resultsSPE method in eicosanoids extraction which results in higher intensity signals by 2-fold for 8-iso-PGF2α and 5-fold for 11-DHTXB2.The method was shown to be linear from 0.1 to 5 ng/mL forboth analytes (R2 ≥ 0.98). Conclusion The three-phase liquid extraction method extracted the two analytes better than the solid phaseextraction method, which is the most used purification method for eicosanoids. Furthermore, arapid and simple LC-MS/MS method for simultaneously measuring urinary 8-iso-PGF2α and 11-DHTXB2 was successfully developed.

Determination of Total Radiochemical Purity of [18F]FDG and [18F]FET by High-Performance Liquid Chromatography Avoiding TLC Method

The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET). The separation of [18F]fluoride ions, [18F]FET and [18F]FET intermediate was accomplished on LiChrosper RP-18, 250 × 4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [18F]FDG was performed using LiChrosper 100 NH2, 250 × 4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH2PO4 buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [18F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[19F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [18F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r2 = 0.9981–0.9996), specificity (RS = 3.7–10.2), repeatability (%Area RSD% = 1.2–4.3%) and limit of quantitation (LOQ = 1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.

Closed form approximations to predict retention times and peak widths in gradient elution under conditions of sample volume overload and sample solvent mismatch

Closed form expressions for the prediction of retention times and peak widths for gradient liquid chromatography are particularly useful in understanding, rationalizing and optimizing separations. These expressions are obtained by integrating differential equations, in conjunction with a model of the variation of the retention factor as a function of mobile phase composition. Two of these models, the linear solvent strength (LSS) model and the Neue-Kuss (NK) model are explored in the present work. Here, we expand on these closed form expressions to account for effects of sample volume overload and a mismatch between the sample solvent and the initial mobile phase composition for the gradient. We show that there have been errors in expressions reported in the literature, and we have evaluated the accuracy of the predictions from the closed form expressions reported here using a recently developed liquid chromatography simulator. The expressions assume a constant plate height and consider elution across four zones of the gradient profile – elution in the sample solvent, elution in the initial (isocratic) mobile phase caused by the gradient delay volume, elution during a linear gradient, and elution post-gradient at the final (isocratic) mobile phase composition. The expressions generally give reasonably accurate predictions for retention times and peak widths, except for cases where the solute elutes during transitions between the different zones. The average magnitude of the prediction errors for retention time and peak width relative to simulation were 0.093% and 0.40% for the LSS expressions for ten amphetamine solutes at 36 different separation conditions, and 0.22% and 1.8% for the NK expressions for eight alkylbenzene solutes at 36 different separation conditions, respectively.

First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms

The study of glycoproteins is a rapidly growing field, which is not surprising considering that approximately 70% of human proteins are glycosylated and that numerous biological functions are associated to the glycosylation. In this work, our interest focused on the heterodimeric human Chorionic Gonadotropin (hCG) glycoprotein that is the specific hormone of the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. This protein possesses a very high structural heterogeneity, essentially due to the presence of 8 glycosylation sites, but also other types of post-translational modifications. In this study, for the first time, the potential of hydrophilic interaction liquid chromatography (HILIC) was investigated to separate the intact hCG isoforms. Three different HILIC stationary phases were tested using an hCG-based drug as standard, a recombinant hCG. For each stationary phase, the effect of the initial mobile phase composition based on ACN/H2O mixture, the slope of the gradient, the content and nature of the acidic additive (formic acid and trifluoroacetic acid (TFA)), and the addition of a volatile salt (ammonium formate) on the retention and the resolution were studied. The best HILIC separation was obtained with the amide column and a mobile phase composed of water/ACN containing 0.1% of TFA. The repeatability in terms of retention times and peak areas was then assessed. Finally, the method was applied to the analysis of a second hCG-based drug obtained from urine of pregnant women. Both drugs gave chromatograms with more than 10 peaks. However, they were significantly different, which demonstrated the potential of HILIC method for hCG isoform fingerprinting.

Method development for the certification of a ginsenoside calibration solution via liquid chromatography with absorbance and mass spectrometric detection

The research presented here describes the development of two analytical methods for use in the certification of a ginsenoside calibration solution Standard Reference Material (SRM) 3389 consisting of seven ginsenosides: Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd. The new methods utilized the liquid chromatographic (LC) separation of ginsenoside mixtures with absorbance detection (UV) and mass spectrometry (MS). Ginsenosides Rb3, Rg2, Rg3, Rh1, and Rh2 were evaluated for use as internal standards for LC/MS measurements. The 12 ginsenosides were baseline resolved by gradient elution LC/UV, with an initial mobile phase composition of 22% acetonitrile and 78% water, flow rate of 0.7 mL/min, and column temperature of 25 °C. The work presented here includes a detailed investigation into the optimization of the chromatographic conditions to minimize measurement biases that result from unresolved constituents. Temperature and mobile phase composition are known to play a significant role in column selectivity; however, flow rate is expected to influence primarily the separation efficiency and detection sensitivity. In the current study, column selectivity changed with changes in flow rate and the relative retention of ginsenoside Rg2 and Rh1 changed as the flow rate increased from 0.6 mL/min to 1.0 mL/min.

An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level

For the first time, the human Chorionic Gonadotropin (hCG) hormone at the intact level was characterized by reversed phase liquid chromatography (RPLC) coupled with high resolution mass spectrometry (HRMS). This heterodimeric protein is specific to human pregnancy, consists in an α and a β subunit, so-called hCGα and hCGβ, respectively, and has 8 glycosylation sites leading to a high number of isoforms. First, the LC method was optimized to separate the largest number of isoforms and also to facilitate the MS ionization process and data treatment. The initial mobile phase composition, slope of the gradient, and column temperature were appropriately selected to maximize the number of separated isoforms. Moreover, the MS detection parameters were adjusted to i) promote the efficient transfer of the heaviest ions, ii) avoid or limit the fragmentation of the ions and iii) improve the sensitivity. The repeatability of the final method in terms of retention times and peak areas was assessed. The method was next used to characterize two hCG-based drugs: Ovitrelle® (a recombinant hCG, r-hCG) and Pregnyl® (hCG isolated from urine of pregnant women, u-hCG). After the deconvolution step, the analytical method did not allow to observe the isoforms of the hCGβ. This may be due to its dramatic higher heterogeneity induced by its 6 glycosylation sites and a lack of ionization in the MS source. Nevertheless, the results revealed the presence of more than 30 hCGα isoforms, which differ by their number and their nature in the two drugs. Then, the molecular weights of the N-glycans already described in the literature for hCG were compiled in a database to identify the hCGα glycoforms by mass matching. This strategy was successfully applied for the identification of five glycoforms for both r-hCG and u-hCG. This work demonstrates for the first time the potential of RPLC-HRMS for the identification of the intact hCGα glycoforms.

Instrument parameters controlling retention precision in gradient elution reversed-phase liquid chromatography

The precision of retention time in RPLC is important for compound identification, for setting peak integration time windows and in fundamental studies of retention. In this work, we studied the effect of temperature (T), initial (ϕo) and final mobile phase (ϕf) composition, gradient time (tG), and flow rate (F) on the retention time precision under gradient elution conditions for various types of low MW solutes. We determined the retention factor in pure water (k′w) and the solute-dependent solvent strength (S) parameters of Snyder's linear solvent strength theory (LSST) as a function of temperature for three different groups of solutes. The effect of small changes in the chromatographic variables (T, ϕo, ϕf, tG and F) by use of the LSST gradient retention equation were estimated. Peaks at different positions in the chromatogram have different sensitivities to changes in these instrument parameters. In general, absolute fluctuations in retention time are larger at longer gradient times. Drugs showed less sensitivity to changes in temperature compared to relatively less polar solutes, non-ionogenic solutes. Surprisingly we observed that fluctuations in temperature, mobile phase composition and flow rate had less effect on retention time under gradient conditions as compared to isocratic conditions. Overall temperature and the initial mobile phase composition are the most important variables affecting retention reproducibility in gradient elution chromatography.

Improving peak shapes with counter gradients in two-dimensional high performance liquid chromatography

To achieve the greatest peak capacity in two-dimensional high performance liquid chromatography (2D-HPLC) a gradient should be operated in both separation dimensions. However, it is known that when an injection solvent that is stronger than the initial mobile phase composition is deleterious to peak performance, thus causing problems when cutting a portion from one gradient into another. This was overcome when coupling hydrophilic interaction with reversed phase chromatography by introducing a counter gradient that changed the solvent strength of the second dimension injection. It was found that an injection solvent composition of 20% acetonitrile in water gave acceptable results in one-dimensional simulations with an initial composition of 5% acetonitrile. When this was transferred to a 2D-HPLC separation of standards it was found that a marked improvement in peak shape was gained for the moderately retained analytes (phenol and dimethyl phthalate), some improvement for the weakly retained caffeine and very little change for the strongly retained n-propylbenzene and anthracene which already displayed good chromatographic profiles. This effect was transferred when applied to a 2D-HPLC separation of a coffee extract where the indecipherable retention profile was transformed to a successful application multidimensional chromatography with peaks occupying 71% of the separation space according to the geometric approach to factor analysis.

Development of a new analytical method for the determination of sulfites in fresh meats and shrimps by ion-exchange chromatography with conductivity detection

An accurate and reliable analytical method, based on ion chromatography and suppressed conductivity detection, has been developed and validated for the quantitative determination of sulfites in fresh meats and shrimps. The chromatographic separation was accomplished by using an anion-exchange column eluted with sodium carbonate and sodium hydroxide. The optimized step-change elution, followed by column re-equilibration at the initial mobile phase composition, guaranteed a good resolution even toward endogenous interfering peaks, and an excellent retention time repeatability (1.1%, n = 6). Good results in terms of sample extract stability, recovery efficiency were achieved with an extraction solvent mixture based on sodium hydroxide, fructose and EDTA. The method validation, performed by an in-house model according to Decision 657/2002/EC and Regulation 882/2004/EC, provided excellent results with respect to linearity (correlation coefficient up to 0.9998), limits of detection and quantification (2.7 and 8.2 mg kg −1, respectively, expressed as SO 2), expanded measurement uncertainty (below 10%), recovery values (ranging from 85% to 92%) and repeatability (down to 8%), demonstrating the conformity of the proposed method with the European directives. Finally, by major changes ruggedness studies, the method applicability to the quantitative analysis of cow hamburger, pork and horse sausage, and shrimps was demonstrated.

Response surface methodology and support vector machine for the optimization of separation in linear gradient elution

Experimental design methodology was used to optimize the linear gradient elution chromatography. The effect of initial mobile phase composition (phi(in)), initial isocratic time (t(in)), and gradient time (t(G)) on the retention times of phenyl thiohydantoin aminoacids (PTH-amino acids) was investigated. The experiments were performed according to Box-Behnken experimental design to map the chromatographic response surface. Then multiple linear regression and support vector machine (SVM) methods were used to fit the retention times of solutes. Results shows the SVM models have better ability to predict retention time and used for grid search in factor space. The SVM models were verified, as good agreement was observed between the predicted and experimental values of retention time in the optimal condition.

Quantification of Vincristine and its Major Metabolite in Human Plasma by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

An analytical method using electrospray ionization and high-performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify vincristine and M1, the CYP3A-mediated metabolite of vincristine, in human plasma. Vinblastine (internal standard), vincristine, and M1 in plasma were extracted in methylene chloride after acidification with TCAA. The analytes were separated on an Inertsil ODS-3 C18 column (2.1 x 150 mm) with a 5-mum particle size using a gradient elution with a run time of 20 min. The initial mobile phase composition was 0.2% formic acid/water (80:20, v/v) with a final composition of 0.2% formic acid/water (20:80, v/v). Detection was accomplished with multiple reaction monitoring for vinblastine (m/z 406.3--> 271.7), vincristine (m/z 413.2--> 362.2), and M1 (m/z 397.3 --> 376.2). At three concentrations of vincristine and M1, the inter-day and intra-day accuracy and precision were within the acceptable limits for validation (106.8 +/- 9.6% for intra-day, n = 5 each concentration; 90.9 +/- 10.9% for inter-day, n = 4 each concentration). For both vincristine and M1, the concentration limits of quantification and detection were 12 pg/mL and 6 pg/mL, respectively. Stability studies indicated that 80% of M1 degraded in plasma after 15 hours at room temperature (n = 3, high and low QC concentrations). Therefore, short plasma processing times (<30 min) are recommended. The assay was used successfully to quantify vincristine and M1 in pediatric plasma samples up to 24 hours after vincristine administration. Vincristine and M1 concentrations were within the limits of quantification for all patient plasma samples.

Prediction of the Elution Profile of Aromatic Compounds in RP‐HPLC

An efficient prediction method for the elution profile was used to separate aromatic compounds such as benzene, toluene, chlorobenzene, o‐xylene, and 1,2‐dichlorobenzene by RP‐HPLC. The retention factor and bandwidth were predicted under linear‐gradient condition with the three retention models. The elution profiles were calculated based on the linear and quadratic equations of retention factor, lnk=lnk w +Sϕ, lnk=L+Mϕ+Nϕ2, k=A+B/ϕ, where ϕ was the vol.% of methanol. The elution profiles were calculated by the Gaussian distribution with obtained retention factor and bandwidth. Two kinds of experiments were performed; one is the isocratic runs to estimate the coefficients of three retention models, and the other is the linear gradient runs that were carried out with same initial mobile phase composition (water/methanol=96/4, vol.%), two final mobile phase compositions (water/methanol=24/76 and 40/60, vol.%), and three gradient times (20, 40, and 60 min). The predicted elution profiles by the three retention models and new prediction method have good agreement with experimental data in the employed gradient conditions. The minimum average errors of calculated and experimental results of aromatic compounds were lower than 3.5% by Bi‐poly equation.

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.

Liquid chromatographic method for the analysis of buspirone HCl and its potential impurities.

An accurate, reproducible, and sensitive method for the determination of buspirone HCl and its potential impurities is developed and validated. The validated liquid chromaography method is conducted to meet the Food and Drug Administration/ International Conference on Harmonization requirements for the analysis of buspirone HCI in the presence of its impurities. Five buspirone HCI potential impurities, including 1-(2-pyrimidinyl)-piperazine (I), propargyl chloride (II), 3,3'-tetramethylene glutarimide (III), propargyl glutarimide (IV), and the Mannich base-condensate of I-IV fumarate (V), are separated using a microBondapack C18 column by gradient elution with a flow rate 2.0 mL/min. The initial mobile phase composition is 90:10 (v/v) 10mM KH2PO4 (pH 6.1)-acetonitrile. After a 1-min initial hold, a linear gradient is performed in 26 min to 35:65 (v/v) 10mM KH2PO4 (pH 6.1)-acetonitrile. The samples are detected at 210 and 240 nm using a photo-diode array detector. The linear range of detection for buspirone HCI was between 1.25 ng/microL and 500 ng/microL, with a limit of quantification of 1.25 ng/microL. The linearity, range, peak purity, selectivity, system performance parameters, precision, accuracy, and robustness for all of the impurities were also shown to have acceptable values.

Sensitive assay for midazolam and its metabolite 1′-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1′-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 μm capillary C 18 column (150 mm×0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer–acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer–acetonitrile, 40:60. The flow-rate of the mobile phase was 16 μl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1′-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1′-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 μg/kg).

On-line buffer removal and fraction selection in gradient capillary high-performance liquid chromatography prior to electrospray mass spectrometry of peptides and proteins.

The technique for on-line removal of buffers and for fraction selection to eliminate source clogging and suppression of electrospray ionization by unwanted constituents consisted of: (i) selecting the initial mobile phase composition to retain analytes on top of a capillary high-performance liquid chromatography (HPLC) column; (ii) washing the buffer isocratically to waste, using a two-way micro dump valve; (iii) starting the gradient and switching the dump valve to introduce analytes into the source. Peptide and protein mixtures were prepared in 0.05-0.5 M phosphate and 0.55 mM tris-based buffers, and water. After buffer removal, chromatograms and electrospray spectra were indistinguishable from those of aqueous controls, down to 1 pmol (acidic fibroblast growth factor) consumed, and up to 78 kDa (bovine transferrin) molecular weight. Aided by the dump valve, 100 fmol of angiotensin could be fractionated and identified on the slope of a 10 x 10(6) fmol of leucine enkephalin HPLC peak. On-line buffer removal and fraction selection eliminate the need for additional preparation steps than can lead to excessive sample losses.

Predictive calculation methods for optimization of gradient elution using binary and ternary solvent gradients

Predictive optimization methods in gradient elution liquid chromatography are reviewed. The optimization approach is based on calculations of elution volumes and band widths in various modes of gradient elution liquid chromatography. The calculation makes use of the parameters of retention-mobile phase composition equations determined in a few preliminary isocratic experiments. In liquid chromatography with binary solvent gradients, either a direct calculation method may be used to optimise the gradient profile in order to achieve a desired resolution of a “critical pair” of sampls solutes, or “maps” of the dependence of resolution of the individual pairs of compounds on gradient steepness and initial mobile phase composition can be constructed for an assumed constant gradient volume (time). Calculation methods are also applicable to ternary mobile phase gradients in reversed-phase systems. There, ternary “solvent strength”, “selectivity”, or “combined selectivity-solvent strength” gradients are chosen, depending on the separation problem to be solved. In these systems, construction of resolution “maps” is the method of choice of predictive optimization. The precision of predicted gradient elution data depends on the instrumentation used and on its ability to reproduce accurately the gradient profile programme and on possible deviations of the experimental retention-mobile phase composition plots from the theoretically expected forms of these dependences. The effect of the choice of gradient shape (curvature) and volume on the optimized separation is also addressed.

Method for characterization of selectivity in reversed-phase liquid chromatography : III. Retention behaviour in gradient-elution chromatography:Application to the chromatography of pesticide compounds

The method introduced recently for the characterization and prediction of absolute and relative retention in reversed-phase liquid chromatography under isocratic conditions has been applied to gradient-elution chromatography. The method is based on two indices (one lipophilic, n ce, and the other polar, q i to characterize the behaviour of each solute in methanol-water mobile phases of various compositions. The indices n ce and q i of phenylurea and triazine herbicides were found to be composed of additive contributions of structural elements (functional groups) in the molecules of the solutes. Using the present approach, it is possible to predict retention volumes in gradient-elution chromatography with a precision comparable to those under isocratic conditions ( i.e., less than 10% relative for most compounds tested). Simultaneous adjustment of the initial mobile phase composition and the slope of the linear gradient makes it possible to control to a certain extent the selectivity in gradient-elution chromatography using binary solvent gradients and offers an alternative approach to the use of multi-solvent gradients for selectivity control. This method may be especially useful in optimizing gradient-elution separations of pairs of compounds with positive differences in both indices n ce and q i, Δ n ce > 0 and Δ q > 0. Selection of the optimal gradient profile is illustrated on a practical example of gradient-elution chromatography of phenylurea herbicides.

Multivariate and univariate optimization studies of liquid-chromatographic separation of steroid mixtures.

We developed a high-performance liquid-chromatographic separation of five steroids (estriol, estradiol, cortisol, progesterone, and testosterone), eluting with a water-acetonitrile gradient from a reversed-phase (C18) column. By applying a simplex search algorithm to maximize a chromatographic-response function, we sought to optimize the original conditions of the chromatographic analysis, which did not separate two pairs of overlapping peaks. Our chromatographic-response function incorporated both peak separation and total time of analysis. Three factors were varied simultaneously to maximize this function: flow rate, column temperature, and gradient shape. From the simplex optimization, we selected a flow rate of 1.50 mL/min, a temperature of 52 degrees C, and a linear gradient for our analysis. Subsequent univariate studies of the initial mobile phase composition showed that acetonitrile/water (20/80 by vol) gave an adequate separation.