Normal-phase Liquid Chromatography Research Articles

On-chip integration of normal phase high-performance liquid chromatography and droplet microfluidics introducing ethylene glycol as polar continuous phase for the compartmentalization of n-heptane eluents

We introduce an integrated chip-approach for a postcolumn segmentation of normal phase liquid chromatography. This is achieved by the seamless integration of a chiral NP-chip-HPLC column, a flow-focusing droplet generator, and a segmented flow channel in a single microfluidic glass chip. This allows a continuous segmentation of the eluent into droplets which are picked up and transported via an immiscible continuous phase. The combination of NP-chip-HPLC and droplet microfluidics enables to fractionate and conserve chromatographic runs for further downstream processes at picoliter scale. An essential aspect is the proper choice of the continuous phase concerning polarity, wetting properties and viscosity. For this purpose, ethylene glycol is introduced which facilitates this first combination of normal phase chromatography and droplet microfluidics. By adjusting the flow rates and varying the generator geometry, the size and frequency of the droplets could be precisely controlled.

Tylosin A and desmycosin in honey by salting-out assisted liquid-liquid extraction and aqueous normal phase ultraperformance liquid chromatography-tandem mass spectrometry.

A simple and rapid method was developed for the determination of tylosin A and desmycosin residues in honey. Aliquots of honey samples were dissolved in a concentrated solution of sodium acetate and the target analytes were subsequently extracted with acetonitrile. The resulting organic extract was chromatographed under aqueous normal phase (ANP) LC conditions using a bare silica stationary phase with acidified solutions of ammonium formate in both water and5:95 water: acetonitrile as the mobile phases. Tylosin A and desmycosin residues were measured using MS/MS in the multiple reaction monitoring (MRM) mode. Based on the analysis of replicate honey samples fortified at 5, 20, and 100μgkg-1, the method was found to provide high accuracy and precision with average intraday trueness ranging from 90.2 to 111.2% and standard deviations of less than 7%. For spiked replicates fortified at the limit of quantification (1μgkg-1), the intraday accuracies ranged from 72.0 to 102.7% for tylosin A and from 72.1 to 93.8% for desmycosin, with standard deviations all lower than 12%. Matrix effects were relatively minimal and consistent between honey samples which eliminated the need to perform any additional cleanup of the sample extracts prior to ANP-UPLC-MS/MS analysis. Graphical abstract.

Determination of molecular mass 302 polycyclic aromatic hydrocarbons in Standard Reference Material 1597a by reversed-phase liquid chromatography and constant energy synchronous fluorescence spectroscopy

High-performance liquid chromatography with fluorescence detection is often used for the analysis of polycyclic aromatic hydrocarbons (PAHs) in environmental extracts. Since fluorescence chromatograms are usually recorded at a single set of excitation and emission wavelengths, qualitative parameters for PAH identification are restricted to characteristic retention times. The research presented here explores the use of constant energy synchronous fluorescence spectroscopy (CESFS) for the analysis of co-eluting PAHs with similar chromatographic behaviors under normal and reversed-phase conditions. Twenty-one PAH isomers with molecular mass (MM, g/mol) 302 were studied. Synchronous fluorescence spectra were recorded under stop-flow conditions with the aid of a commercial HPLC system. PAHs were extracted from a coal tar sample (Standard Reference Material 1597a) and fractionated by normal-phase liquid chromatography (NPLC). Qualitative and quantitative analysis of NPLC fractions was carried out via reversed-phase liquid chromatography-CESFS (RPLC-CESFS). Of the 21 isomers investigated in the coal tar sample, 16 were spectrally identified in the absence of matrix interference. Three of the remaining isomers were tentatively identified in the presence of moderate spectral interference. Thirteen isomers were used to investigate the accuracy of the RPLC-CESFS approach. Mass fractions statistically equivalent to the NIST reported values (P = 95%; N1 = N2 = 3) were obtained for ten isomers ranging from 2.04 (dibenzo[a,h]pyrene) to 10.41 mg/kg (naphtho[2,1-a]pyrene). The non-destructive nature of CESFS provides an additional layer of selective and accurate information to the analysis of PAH isomers with MM 302.

High-performance liquid chromatographic and subcritical fluid chromatographic separation of α-arylated ß-carboline, N-alkylated tetrahydroisoquinolines and their bioisosteres on polysaccharide-based chiral stationary phases.

New, pharmacologically interesting chiral amino compounds, namely, stereoisomers of α-hydroxynaphthyl-ß-carboline, benz[d]azepine and benz[c]azepine analogs as well as N-α-hydroxynaphthylbenzyl-substituted isoquinolines were enantioseparated by high-performance liquid chromatographic and subcritical fluid chromatographic methods on polysaccharide-based chiral stationary phases. Separation of the stereoisomers was optimized in both subcritical fluid chromatography and normal phase liquid chromatographic modes by investigating the effects of the composition of the bulk solvent, temperature, and the structures of the analytes and selectors. Both normal phase liquid chromatography and subcritical fluid chromatography exhibited satisfactory performance, albeit with somewhat different effectiveness in the separation of the stereoisomers studied. The optimized methods offer the possibility to apply preparative-scale separations thereby enabling further pharmacological investigations of the enantiomers.

Quantification of Vitamins A, E, and K and Carotenoids in Submilliliter Volumes of Human Milk.

Background: Human milk is the optimal nutrition for all newborns in the first 6 months of life. In order to assess the nutritional needs of the breastfed infant, human milk is often characterized for multiple nutrients. Objective: To ensure that we minimize the volume of milk dedicated for research and optimize the number of nutrients characterized, we developed analytical methodologies for the determination of vitamins A (retinol), E (alpha and gamma tocopherol), K (phylloquinone and menaquinone-4), and five carotenoids (β-carotene, lycopene, β-cryptoxanthin, lutein, and zeaxanthin) using <1 mL human milk. Method: Vitamins E and K and carotenoids are simultaneously isolated from 750 μL milk by liquid-liquid extraction (LLE). Tocopherols and carotenoids are determined by normal-phase LC with fluorescence and ultraviolet detection respectively. Vitamin K is analyzed on the same extracts after resuspension and clean-up by reversed phase liquid chromatography coupled to tandem MS. The analysis of vitamin A involves saponification of 200 μL milk followed by LLE and determination by normal-phase LC with UV detection. Results: Full single-laboratory validation at four different concentration levels is presented. Recovery rates were within 90-105% in all except one case (retinol at 1.9 μg/mL, 88% recovery), with RSDs of repeatability and intermediate reproducibility below 10 and 15%, respectively for all the compounds. Conclusions and Highlights: To the best of best knowledge, this is the first report that allows for the characterization and quantification of vitamins A, E, and K and five carotenoids in <1 mL human milk.

A metabolomic study of Asian and American Ginseng based on RRLC-QTOF/MS methods

In order to understand the significant differences in activities and effects between Asian ginseng (ASG) and American ginseng (AMG), it is important to study the correlation between chemical structures and biological activities in the two types of herbs. However, more attention has been paid to the analysis of ginsenosides in previous reports distinguishing between ASG and AMG. There are some other bioactive compounds besides ginsenosides, however, few studies have focused on a systematic comparison of these types of compounds. Two metabolomic methods were developed in this study by qualitative data acquisition using normal phase liquid chromatography mass spectrometry (NPLC-MS) and reverse phase liquid chromatography mass spectrometry (RPLC-MS) respectively, in combination with principal component analysis (PCA). Results show that both NPLC-MS and RPLC-MS-based metabolomic methods are feasible in composition profiling, biomarker screening as well as in discrimination of ASG and AMG. 17 ginsenosides were identified as analytical markers in RPLC-MS-based metabolomic method. In comparison, using NPLC-MS-based method, 5 ginsonosides, two amino acids as well as 1 oligosaccharide were identified as analytical markers. Therefore, RPLC-MS-based metabolomic method exhibits better profiling in ginsenosides, while NPLC-MS-based metabolomic method offers the advantage that multiple active ingredients can be determined simultaneously. The two methods are both helpful in identification of biomarker as well as in discrimination of American ginsengs from Asian ginsengs.

Authentication of the geographical origin of extra-virgin olive oil of the Arbequina cultivar by chromatographic fingerprinting and chemometrics

This paper proposes to use chromatographic fingerprints coupled to multivariate techniques to authenticate the geographical origin of extra-virgin olive oils (EVOO) of the Arbequina botanical variety. This methodology uses the whole or part of the chromatogram as input data for the classification models but does not identify or quantify the chemicals constituents.Arbequina monovarietal EVOOs from three geographical origins were studied: two from adjacent European Protected Designation of Origin areas, Siurana and Les Garrigues, in Catalonia in the northeast of Spain; and the third from the south of Spain (Andalucia and Murcia). Three chromatographic fingerprints of each sample were obtained by both reverse and normal phase liquid chromatography coupled to charged aerosol detector (HPLC-CAD), and high temperature gas chromatography coupled to flame ionization detector [(HT)GC-FID].Principal component analysis (PCA) was used as exploratory technique and soft independent modelling of class analogy (SIMCA) and partial least square-discriminant analysis (PLS-DA) were used as classification methods. High and low-level data fusion strategies were also applied to improve the classification results obtained when the data acquired from each analytical technique were separately used.The results were best for the PLS-DA model with low-level fusion of two techniques (HT)GC-FID with HPLC-CAD, independently of the phase mode. Sensitivity and specificity were 100% in almost all classes, error was 0% for all classes and an inconclusive ratio of just 4% was obtained for the Les Garrigues class due to double assignations.

Differentiation of avocados according to their botanical variety using liquid chromatographic fingerprinting and multivariate classification tree.

The oil content, composition and marketing threshold value of an avocado depends on the cultivar hence, identifying the cultivar of the avocado fruit is desirable. However, analytical methods have not been reported with this aim. A multivariate classification tree method was proposed to discriminate three commercial botanical varieties of avocado: Hass, Fuerte and Bacon, using high-performance liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). Prior to the chromatographic analysis the avocados were lyophilized and then the oil fraction was extracted using a pressurized liquid extraction system. Normal and reverse phase liquid chromatography were applied in order to obtain the chromatographic fingerprint for each sample. Soft independent modelling of class analogies (SIMCA) and partial least-squares discriminant analysis (PLS-DA) were applied. Classification quality metrics were determined to evaluate the performance of the classification. Several strategies to develop the classification models were employed. Finally, the useful application of 'classification trees' methodology, which has been scarcely applied in the field of analytical food control, was evaluated to perform a multiclass classification. Discrimination of the three botanical varieties was achieved. The best classification was obtained when the PLS-DA is applied on the normal-phase chromatographic fingerprints. Classification trees are showed to be useful tools that provide complementary information to single concatenated models showing different results from the same prediction sample set. © 2019 Society of Chemical Industry.

Chiral high-performance liquid and supercritical fluid chromatographic enantioseparations of limonene-based bicyclic aminoalcohols and aminodiols on polysaccharide-based chiral stationary phases.

Enantioseparation of limonene-based bicyclic 1,3-aminoalcohols and 1,3,5- and 1,3,6-aminodiols was performed by normal-phase high-performance liquid chromatographic and supercritical fluid chromatographic (SFC) methods on polysaccharide-based chiral stationary phases. The effects of the composition of the mobile phase, the column temperature and the structures of the analytes and chiral selectors on retention and selectivity were investigated by normal-phase LC and SFC technique. Thermodynamic parameters derived from selectivity-temperature-dependence studies were found to be dependent on the chromatographic method applied, the nature of the chiral selector and the structural details of the analytes. Enantiorecognition in most cases was enthalpically driven but an unusual temperature behavior was also observed: decreased retention times were accompanied by improved separation factors with increasing temperature, i.e. some entropically driven separations were also observed. The elution sequence was determined in all cases. The separation of the stereoisomers was optimized in both chromatographic modalities.

Targeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry.

Lipopolysacharride (LPS) forms the outer leaflet of the outer membrane in Gram-negative bacteria and contributes to the permeability barrier and immune response. In this study, we established a method for monitoring the LPS biosynthetic intermediates of the Raetz pathway (lpxA-lpxK) in Escherichia coli. Metabolites from compound-treated cells and genetically-perturbed cells were extracted from whole cells and concentrated by mixed-mode weak anion exchange (WAX) solid-phase extraction (SPE) prior to analysis by normal phase (NP)LC-MS/MS. Data was normalized to cell density and an internal standard prior to comparison against untreated cells in order to determine fold accumulation and depletion for affected metabolites. Using this LC-MS/MS method, we were able to reliably monitor changes in levels of the LPS intermediates in response to compound-treatment and genetic modification. In addition, we found that deletion of periplasmic CDP-diacylglycerol pyrophosphatase dramatically increased levels of the UDP-containing LPS intermediates, suggesting the enzymatic breakdown during sample preparation. This assay allows for probing a key essential pathway in Gram-negative bacteria in an effort to discover antibacterial agents that inhibit enzymes in the LPS biosynthetic pathway.

Exploring the Effect of Buffer Strength on the Retention Time of Weak Acids, Neutral and Weak Bases in Hydrophilic Interaction Liquid Chromatography (HILIC) Mode

Background: Hydrophilic Interaction Liquid Chromatography (HILIC) orthogonal to conventional reversed phase High-Performance Liquid Chromatography (HPLC) mode allowing separation of polar compounds. HILIC has been reported to be an alternative to normal phase liquid chromatography, yet the separation mechanism reported in HILIC is much more complicated than that in normal phase liquid chromatography. Objective: To investigate the effect of water layer thickness on silica gel and the amount of ammonium ions present within the buffer on retention mechanism in hydrophilic interaction chromatography. Methodology: A test system was designed which used weak acids, neutrals and weak bases as probes with three different strengths (5, 10 and 20 mM) of ammonium acetate, ammonium formate and ammonium propionate as the counter-ions to compete with the test probes with ionised silanol groups and water present in the stationary phase. A Kromasil 60-5SIL column (150 mm×4.6 mm×4 μm, pore size 60Å) was used as the stationary phase to perform the study. Results: Retention times were examined for the test probes at 90% acetonitrile (ACN) with 10% of 5, 10 and 20 mM of ammonium acetate, ammonium formate and ammonium propionate. As the buffer strength increases, the thickness of the water layer on the surface of the silica gel increases and also the repulsion between ionized silanol groups and acidic test probes will decrease. On the other hand, such increase in buffer strength will increase the competition between the ammonium ions and basic test probes. In addition, the hydration energy of buffer’s counter ions and hydrophilicity may be important in retention mechanism in HILIC mode. Conclusion: At 20 mM buffer strength acidic probes with low log P values retain more due to reduced repulsion by silanol groups, while basic probes retention time will decrease due to increased competition from ammonium counter ions. However, in 5 mM buffer strength basic probes with low logP value will be retained longer, while acidic probes will be eluted earlier due to the repulsion between ionized acids and ionized silanol groups.

Liquid Chromatography Techniques in Lipidomics Research

Lipids represent a very diverse group of compounds with a high variety of physicochemical properties determining their functional activities. For omics-wide identification of lipid species from complex biological samples, several crucial analytical steps including extraction, chromatographic separation and mass spectrometry analysis need to be carefully considered and validated. Here we review applications of three main chromatography techniques—reversed phase, normal phase and hydrophilic interaction liquid chromatography—for analysis of complex natural lipidomes aiming to uncover the diversity of lipid species. To correlate lipid separation with their physicochemical properties, lipid chemical space was reconstructed and used to explain principles underlying different chromatographic techniques. Furthermore, examples of available methods for analysis of complex natural lipidomes characterized by high diversity and dynamic range of lipid concentrations are illustrated for adipose tissue lipidomics.

Lipid organization in xerosis: the key of the problem?

Although xerosis is a common skin disorder among the population, there is no invivo global study focusing on xerotic skin. Hence, the objective of this study was to characterize xerotic skin from the surface to the molecular scale with invivo and non-invasive approaches. For this purpose, 15 healthy volunteers with normal skin and 19 healthy volunteers with xerotic skin were selected by a dermatologist, thanks to a visual scorage. Firstly, the skin surface was characterized with biometric measurements. Then, the state of skin dryness was assessed by invivo confocal microscopy. The molecular signature of xerotic skin was then determined by invivo confocal Raman microspectroscopy. Finally, an identification of stratum corneum (SC) lipids was performed using Normal phase liquid chromatography (NP-LC) coupled to two detectors: Corona and High Resolution/Mass Spectroscopy (HR/MS). Results obtained at the skin surface displayed an increase in the transepidermal water loss (TEWL) and a decrease in the hydration rate in xerotic skin. Confocal microscopy revealed an alteration of the cell shape in xerotic skin. Moreover, confocal Raman microspectroscopy demonstrated directly invivo and non-invasively the lack of organization and conformation of lipids in this skin. Finally, HPLC analyses revealed that the three ceramide sub-classes (NdS, NS and EOP) significantly decrease in xerosis. Altogether, these results identify parameters for the characterization of xerotic skin compared to normal. This study highlighted discriminative parameters from the surface to the molecular level invivo and non-invasively between xerotic and normal skins. These results will be useful for the development of new cosmetic active ingredients dedicated to xerotic skin.

Classification of olive oils according to their cultivars based on second-order data using LC-DAD

Second-order data acquired using liquid chromatography coupled to a diode array detector were used to classify extra virgin olive oils samples according to their cultivars. The chromatographic fingerprints from the epoxidised fraction were obtained using normal-phase liquid chromatography. To reduce the data matrices two strategies were employed: (1) multivariate curve resolution-alternating least squares (MCR-ALS) and (2) a new strategy proposed in this work based on the fusion of the mean data profiles in both spectral and time domains. Several conventional chemometric tools were then applied to both raw and reduced data: principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA), soft independent modelling of class analogies (SIMCA) and n-way partial least-squares-discriminant analysis (NPLS-DA). Furthermore, an emergent multivariate classification method known as random forest (RF) has been first applied to second-order data. It was shown that RF is more efficient than conventional tools. Indeed, the obtained sensibility, specificity and accuracy are 1.00, 0.92 and 0.95 respectively; these performance metrics are significantly better than the values found for the other methods.

Desarrollo de un método analítico de preparación de muestras de alimentos para determinar óxidos de colesterol (COP) mediante cromatografía

Las transformaciones surgidas durante los procesos de limpieza de los alimentos grasos para determinar productos de oxidación de colesterol (COP) limitan los resultados reales en un estudio. Por tanto, el objetivo de este estudio fue comparar los COP obtenidos por distintos métodos de extracción: fase sólida (SPE) y cromatografía líquida en fase normal y reversa, comparando sus perfiles de elución. Adicionalmente, se tuvo como objetivo optimizar los procesos de limpieza para reducir las transformaciones de COP. Se usaron muestras de lomo de cerdo crudo, jamón serrano y salmón ahumado. Las transformaciones de COP se determinaron por SPE y GC–FID y se optimizaron los métodos de limpieza con la técnica SPE. Los cartuchos apolares no mostraron transformación de COP durante el tratamiento de muestras y los cambios de pH generaron transformaciones del triol a 6 ceto epóxido. Debido a que la limpieza del cartucho suele ser deficiente y genera coelución de compuestos indeseables (ácidos grasos) implicados en la transformación de epóxidos α y β en triol, estas transformaciones se evitaron con la técnica SPE, uso de cartuchos C18, con ajuste del volumen de elución de MeOH y ajustes del pH. La reducción de los pasos de la limpieza de muestras optimizó el proceso en un 73%.

Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatography

A column-switching system, composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was developed for the analysis of vitamin D in oily and fatty matrices. The SFC with the similar retention behavior as normal-phase liquid chromatography (NPLC), was applied for an on-line clean-up of oily and fatty samples, then followed by separation and detection using a reverse-phase LC-MS/MS. Three SFC columns packed with materials of different functional groups (Silica, NH2, Diol) were compared and the column with diol groups, on which the retention time of vitamin D was the longest, was finally selected for purification of the samples. 100% methanol was chosen to carry vitamin D from the clean-up column to the pre-treatment column. It was also used as the mobile phase for the separation of vitamin D on a reverse phase C18 column. Vitamin D2 and D3 were baseline separated by using this system. The linearity was calculated with a value of coefficient of determination (r2) ≥ 0.998. The linear range is from 20 ng/mL to 200 ng/mL. Two kinds of liquid vitamin D3 supplements (Baby Ddrops and Vitamin AD drops) were directly analyzed using this system without any fussy preparation procedure. The limit of detection (LOD) for vitamin D3 in the two oily samples was estimated to be 10 ng/mL. The relative standard deviations (RSD) of intra- and inter-day precision, repeatability were 1.47%, 2.43% and 1.59% for Baby Ddrops and 5.76%, 8.24% and 5.86% for Vitamin AD drops. The recoveries vary between 84.3% and 102.8% with 7.1% RSD for Baby Ddrops and 90.8–109.6% with 5.83% RSD for Vitamin AD drops, respectively. These results suggest that the method based on the SFC-RPLC/MS column-switching system is simple and suitable for analysis of vitamin D in oily and fatty samples.

Isotope Effects on Hydrogen Bonding and CH/CD−π Interaction

We study the isotope effect by liquid chromatography (LC) with a variety of separation media under reversed and normal phase conditions using the protiated and/or deuterated compounds as the solutes. Results of reversed phase LC (RPLC) suggested that the protiated compounds were more hydrophobic than that of deuterated compounds due to the isotope effect based on the hydrogen bonding between hydrogen atoms of isotopologues and hydroxy groups in the mobile phase. The importance of the hydrogen bonding was also supported by the separation of isotopologues with a silica stationary phase on normal phase LC (NPLC), where the deuterated compounds showed stronger hydrogen bonding to hydroxy groups on silanol. Additionally, we investigated the difference of the strength between CH−π and CD−π interactions. Comparison of free energies of isotopologues by RPLC suggested that the CH−π interaction was slightly stronger than CD−π interaction. Finally, we demonstrated the separation of a few isotopologues on NPLC using ...

Selective labeling for the identification and semi-quantification of lipid aldehydes in food products.

Lipid oxidation reactions in foods rich in healthy unsaturated fatty acids result in the formation of a wide range of oxidation products that can have adverse effects on food quality and safety. To improve the understanding of oxidation reactions and methods for their inhibition, detailed information on the type and levels of the oxidation products formed is required. Accurate measurement of lipid oxidation products, especially of the non-volatile aldehyde products, has so far been a challenge due to the low sensitivity and limited specificity of most analytical methods. Here, a novel normal-phase LC method that uses selective labeling of aldehydes and epoxides with 7-(diethylamino)coumarin-3-carbohydrazide (CHH) is described. Labeling of alkanals is quantitative within 10h. For alkenals, conversion is only around 50% at 24h reaction time. Detailed MS identification of all aldehydes and epoxides is possible by using high-resolution MS and data-dependent MS2 acquisition. Fluorescence detection was successfully used to quantify groups of oxidation products. Sensitivity was high enough to allow accurate quantification even in fresh mayonnaises, where levels of around only 0.3g total aldehydes/kg oil were found. Individual species can be quantified by MS if suitable reference standards are available. If no standards can be used, semi-quantification using an average response factor is an option. Clearly, the novel derivatization method is suitable for monitoring secondary lipid oxidation products in the early stages of shelf life. This makes it an important tool for developing improved food products. Graphical abstract Selective labeling of low and high molecular weight lipid oxidation products with 7-(diethylamino) coumarin-3-carbohydrazide for identification and semi-quantification.

Qualitative characterization of three combustion-related standard reference materials for polycyclic aromatic sulfur heterocycles and their alkyl-substituted derivatives via normal-phase liquid chromatography and gas chromatography/mass spectrometry.

The research described here provides the most comprehensive qualitative characterization of three combustion-related standard reference materials (SRMs) for polycyclic aromatic sulfur heterocycles (PASHs) and some alkyl-substituted (alkyl-) derivatives to date: SRM 1597a (coal tar), SRM 1991 (coal tar/petroleum extract), and SRM 1975 (diesel particulate extract). An analytical approach based on gas chromatography/mass spectrometry (GC/MS) is presented for the determination of three-, four-, and five-ring PASH isomers and three- and four-ring alkyl-PASHs in the three SRM samples. The benefit of using a normal-phase liquid chromatography (NPLC) fractionation procedure prior to GC/MS analysis was demonstrated for multiple isomeric PASH groups. Using a semi-preparative aminopropyl (NH2) LC column, the three combustion-related samples were fractionated based on the number of aromatic carbon atoms. The NPLC-GC/MS method presented here allowed for the following identification breakdown: SRM 1597a - 35 PASHs and 59 alkyl-PASHs; SRM 1991-31 PASHs and 58 alkyl-PASHs; and SRM 1975-13 PASHs and 25 alkyl-PASHs. These identifications were based on NPLC retention data, the GC retention times of reference standards, and the predominant molecular ion peak in the mass spectrum. Prior to this study, only 11, 1, and 0 PASHs/alkyl-PASHs had been identified in SRM 1597a, SRM 1991, and SRM 1975, respectively. Graphical abstract NPLC-GC/MS analysis for the three- and four-ring parent PASH isomers in SRM 1597a.

Separation and quantitation of eight isomers in a molecule with three stereogenic centers by normal phase liquid chromatography

A normal phase liquid chromatography method was developed for the separation and detection of eight stereoisomers of the key intermediate, CORE + OMe, having three chiral centers. The stereochemistry of this intermediate dictates the stereochemistry of the active pharmaceutical ingredient generated by an additional six synthetic steps. Multiple columns and mobile phases were screened during the development based on a platform approach. The use of dichloromethane as mobile phase additive and adjustment of flow rate and column temperature contributed in achieving resolution of these eight stereoisomers.The separation and detection of these stereoisomers was achieved using a Chiralcel OD-H, 4.6 × 250 mm, 5 μm dp column with heptane: ethanol: dichloromethane in a ratio of 95:3:2 (v:v:v) as mobile phase at a flow rate of 0.7 mL/min. UV detection was carried out at 245 nm and the column temperature was maintained at 15 °C. The analytical method was phase appropriately validated. The limit of detection and limit of quantification were found to be 0.035 and 0.07 μg, respectively. The newly developed method has been implemented for routine utilization to monitor the chiral control during process development and used as the quality control method for chiral purity of the desired compound.