Orthogonal method development using hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography for the determination of pharmaceuticals and impurities

A hydrophilic interaction chromatography (HILIC) method has been developed and validated as a secondary or orthogonal method complementary to a reversed-phase HPLC (RP-HPLC) method for quantitation of a polar active pharmaceutical ingredient and its three degradation products. The HILIC method uses a diol column and a mobile phase consisting of acetonitrile/water and ammonium chloride. The compounds of interest show significant differences in retention behaviors with the two very different chromatographic systems, which are desired in developing orthogonal methods. The HILIC method is validated and has met all validation acceptance criteria for the support of drug development activities.     

Hydrophilic interaction liquid chromatography method for the determination of ascorbic acid

Hydrophilic interaction liquid chromatography (HILIC) method using internal standard for the determination and stability study of ascorbic acid was developed. HILIC method was very fast and simple using the following analytical conditions: ZIC HILIC (150 x 2.1 mm, 3.5 microm) chromatographic column and mobile phase composed of ACN and 50 mM ammonium acetate buffer pH 6.8 (78:22 v/v). Diode array detection was performed and chromatograms were processed at 268 nm, the maximum wavelength of absorbance of ascorbic acid. An extensive stability study of ascorbic acid as a function of various factors including temperature, stabilizing agents, oxygen presence and its concentration in solution was performed in order to gain information about the quantitative influence of individual stability factors. Low temperature and stabilizing agents (o-phosphoric acid and oxalic acid) were found to be key factors enabling substantial enhancement of the stability of ascorbic acid.     

Ultrafast liquid chromatography/tandem mass spectrometry bioanalysis of polar analytes using packed silica columns

Ultrafast liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalysis was demonstrated with the use of packed silica columns operated under elevated flow rates. A special effort has been made to achieve ultrafast analysis without sacrificing chromatographic resolution. Two multiple analyte/metabolites assays, (1) morphine/morphine-6-glucuronide(M6G)/morphine-3-glucuronide(M3G) and (2) midazolam/1'-hydroxymidazolam/4-hydroxymidazolam, were used to demonstrate the speed, sensitivity, peak shape and separation of the ultrafast methods utilizing silica columns. In both methods adequate chromatographic separation was a necessity because quantitation results would be otherwise compromised due to cross interference between different selected reaction monitoring (SRM) transitions. Baseline resolutions between morphine, M6G and M3G in human plasma extracts were achieved within 30 s on a 50 x 3 mm Betasil silica column operated at 4 mL/min of isocratic acetonitrile/water mobile phase. The total injection-to-injection cycle time was 48 s with a simple, single-autosampler/single-column setup, when a Shimadzu SIL-HT autosampler was used. Baseline resolution between 1'-hydroxymidazolam and 4-hydroxymidalolam in monkey plasma extracts was achieved within 33 s using similar conditions. Due to the absence of carry-over in this case, no rinsing of the injection needle was necessary, resulting in a cycle time of only 39 s/sample. These ultrafast methods were successfully used to analyze extracted biological samples and proved to be reproducible, reliable and generated equivalent pharmaco-kinetic (PK) results to those obtained by regular flow LC/MS/MS analysis to support discovery PK studies.     

Hydrophilic interaction chromatography separation mechanisms of tetracyclines on amino-bonded silica column

Effects of mobile-phase variations on the chromatographic separation on amino-bonded silica column in hydrophilic interaction chromatography (HILIC) were investigated for four zwitterionic tetracyclines (TCs): oxytetracycline, doxycycline, chlortetracycline, and tetracycline. A mixed-mode retention mechanism composed of partitioning, adsorption, and ion exchange interactions was proposed for the amino HILIC retention process. Buffer type and pH significantly influenced the retention of TCs, but showed similar separation selectivity for the tested analytes. Experiments varying buffer salt concentration and pH demonstrated the presence of ion exchange interactions in TCs retention. The type and concentration of organic modifier also affected the retention and selectivity of the analytes, providing direct evidence supporting the Alpert retention model for HILIC. The retention time of the analytes increased in the following order of organic modifiers: tetrahydrofuran < methanol < isopropanol < acetonitrile. The linear relationships of logk' versus %water (v/v) curve and logk' versus logarithm of %water (v/v) in the mobile phase indicated that TCs separation on the amino phase was controlled by partitioning and adsorption. The developed method was successfully utilized in the detection of TCs in both river water and wastewater samples using solid-phase extraction (SPE) for sample cleanup.     

Auto-regeneration of anion-trap columns for improved determination of silica by ion chromatography

The goal of this project was to develop an automated method to regenerate the ATC-3 trap columns that are used on the DX-800 on-line ion chromatography silica systems. The old method of regenerating the ATC-3 trap columns was to physically remove the trap columns from the silica system once every 2 weeks and manual regenerate them. A new automated regeneration method was developed by re-plumbing the silica system to allow 300 mM NaOH to run as the eluent. This regenerates the trap column automatically once every 24 h. The data have shown that regenerating the ATC-3 trap columns once per day improves the R.S.D. values for 250 ng/l silica analysis from 26.0 to 8.7%. The length of useful lifetime for the silica concentrator column was increased by an average of 9 months.     

Hydrophilic interaction chromatography for the analysis of aminoglycosides

The effect of mobile-phase constituents (pH and ionic strength) and chromatographic behaviour of ten aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paramomycin, kanamycin A, gentamycin C1, gentamycin C2/C2a, gentamycin C1a and neomycin) in the bare silica, amino, amide and zwitterionic phases of hydrophilic interaction chromatography (HILIC) were studied systematically. Among the stationary phases studied, the zwitterionic phase provided the best separation of aminoglycosides. The effect of pH, ionic concentration and column temperature on retention time, peak shape and sensitivity was studied using a central composite design. pH affected sensitivity of the detection of analytes but not the retention time. High ionic strength in the mobile phase was necessary to control the ionic interactions between ionised aminoglycosides and the hydrophilic phase, thereby influencing peak shape and retention time. Column temperature affected sensitivity of the detection but not the retention time. During method development, crosstalk between the MS/MS channels of the analytes was observed and resolved.     

Direct injection of solid-phase extraction eluents onto silica columns for the analysis of polar compounds isoniazid and cetirizine in plasma using hydrophilic interaction chromatography with tandem mass spectrometry

Isoniazid and cetirizine do not retain well on reversed-phase columns due to their high polarity. Silica columns, when operated under hydrophilic interaction conditions, do provide excellent retention of these compounds. We have developed simple and proof of concept analytical methods for the analysis of isoniazid and cetirizine in animal and human plasma, respectively. Both methods employed the approach of direct injection of solid-phase extraction (SPE) organic eluents onto silica columns for analysis, thus eliminating evaporation and reconstitution steps that are typically needed for reversed-phase liquid chromatographic analysis. Isoniazid was extracted from animal plasma samples using a Waters Oasis HLB 96-well plate and then eluted with acetonitrile, while cetirizine was extracted from human plasma with a Waters MCX mu-Elute plate and then eluted with acetonitrile containing 5% concentrated ammonium hydroxide. The direct injection of the SPE eluent onto the analytical column was necessary since significant loss of isoniazid was found during the evaporation and reconstitution steps. The method for isoniazid also enabled ultra-fast analysis due to the relatively low back-pressure exhibited by silica columns even under high flow conditions. Both methods show good linearity, accuracy and precision covering the range of 10-2000 ng/mL of isoniazid, and 1-1000 ng/mL of cetirizine in plasma. Substantial time savings were realized as a result of both the elimination of the evaporation and reconstitution steps and the fast chromatographic analysis.     

High speed hydrophilic interaction liquid chromatographic method for simultaneous determination of selected pharmaceuticals in wastewater using a cyano-bonded silica column

In this work, a fast analytical method based on hydrophilic interaction liquid chromatographic-Ultraviolet detection (HILIC-UV) using a short narrow bore cyano-bonded silica column packed with fully porous sub-2?µm particles has been developed for simultaneous determination of eight pharmaceuticals in wastewater. The method involved pre-concentration and clean-up by solid phase extraction using Oasis HLB extraction cartridges. The analytes were separated using a mobile phase consisted of acetonitrile and 5?mM ammonium acetate buffer (95:5?v/v) with a flow rate of 0.6?mL/min. The chromatographic separation was optimized in order to achieve short analysis time and good resolution for all analytes in a single run. Each analyte was detected at its maximum wavelength for higher sensitivity. All analytes could be separated in 5.7?min with resolution ≥2.7. The optimized method was validated based on linearity, precision, detection and quantification limits, selectivity and accuracy. The detection limits of the studied pharmaceuticals ranged from 0.6 to 3?µg/L, while limits of quantification were in the range from 2 to 10?µg/L with UV detection. The developed method is fast, reliable, cost-effective and could be used for the analysis of the studied analytes in other matrices such as food, pharmaceutical preparations and biological fluids.     

Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish

This paper describes a new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC/MS). The high percentage of organic modifier in the mobile phase and the omission of ion-pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on an ionspray ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray triple-quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. MRM experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit for DA in tissue (20 microg/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004 demonstrated for the first time in Italy the presence of DA as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

Development of a supercritical fluid chromatography high-resolution separation method suitable for pharmaceuticals using cyanopropyl silica

A method has been developed for the analysis of a broad spectrum of pharmaceuticals using packed column supercritical fluid chromatography (pSFC) on a cyanopropyl silicagel stationary phase. Five 25 cm x 4.6 mm I.D., 5.0 microm columns were coupled to generate ca. 100000 plates. The selectivity was tuned by varying the nature and concentration of various modifiers and additives in the carbon dioxide mobile phase. It was noted that pressure influences both efficiency and selectivity of the chromatographic process. Final method conditions are: outlet pressure 100 bar, flow 2.0 mL/min, temperature 40 degrees C, organic modifier program from 5% (1 min) to 40% at 2.0%/min, organic modifier composition methanol:acetonitrile in a ratio of 3:1 (variable according to sample composition) with peak symmetry additives trifluoroacetic acid and diisopropylamine both at levels of 0.5%.     

Monolithic silica rod columns for high-efficiency reversed-phase liquid chromatography

Chromatographic properties of a new type of monolithic silica rod columns were examined. Silica rod columns employed for the study were prepared from tetramethoxysilane, modified with octadecylsilyl moieties, and encased in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing. A 25 cm column provided up to 45,000 theoretical plates for aromatic hydrocarbons, or a minimum plate height of about 5.5 μm, at optimum linear velocity of ca. 2.3 mm/s and back pressure of 7.5 MPa in an acetonitrile-water (80/20, v/v) mobile phase at 40°C. The permeability of the column was similar to that of a column packed with 5 μm particles, with K(F) about 2.4×10(-14) m(2) (based on the superficial linear velocity of the mobile phase), while the plate height value equivalent to that of a column packed with 2.5 μm particles. Generation of 80,000-120,000 theoretical plates was feasible with back pressure below 30 MPa by employing two or three 25 cm columns connected in series. The use of the long columns enabled facile generation of large numbers of theoretical plates in comparison with conventional monolithic silica columns or particulate columns. Kinetic plot analysis indicates that the monolithic columns operated at 30 MPa can provide faster separations than a column packed with totally porous 3-μm particles operated at 40 MPa in a range where the number of theoretical plates (N) is greater than 50,000.     

Capillary zone electrophoresis method for the determination of famotidine and related impurities in pharmaceuticals

A simple and rapid capillary zone electrophoresis (CZE) method with UV detection has been developed for the determination of famotidine and its potential impurities in pharmaceutical formulations. The electrophoretic separation of these compounds was performed using a fused silica capillary and 37.5mmolL(-1) phosphate buffer pH 3.5 as the electrolyte. Under the optimised conditions, six impurities could be resolved from the famotidine peak in less than 7min. The calibration curves obtained for the seven compounds were linear over the concentration range investigated (from 1.5 to 78.5microg mL(-1)). The intra- and inter-day relative standard deviations for the migration times and corrected peak areas were less than 2% and 5%, respectively. The detection limits were found to be 0.09microg mL(-1) for famotidine, and from 0.1 to 0.62microg mL(-1) depending on the impurities. The method has been successfully applied to the determination of famotidine in commercial dosage forms.     

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds

Organic acids with very low pK_a require extremely low pH conditions to achieve adequate retention in reversed‐phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed‐phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core–shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.     

Hydrophilic interaction chromatography in nonaqueous elution mode for separation of hydrophilic analytes on silica-based packings with noncharged polar bondings

Chromatographic effects of dedicated stationary and mobile phase variations in hydrophilic interaction chromatography (HILIC) were investigated using a set of nucleobases, nucleosides and deoxynucleosides as polar test solutes. Retention and selectivity profiles were comparatively mapped on four in-house developed silica materials modified with short alkyl chains (C4, C5) which carry hydroxyl functionalities (including diol motifs) as well as embedded sulphide or sulphoxide groups. These data were complemented by results obtained with two commercially available diol-type phases and a bare silica column. Besides elucidation of packing-related aspects this work concentrated specifically on extending aqueous HILIC (AQ-HILIC) to nonaqueous polar-organic elution conditions herein termed NA-HILIC. The exchange of the polar modifier water by various alcohols in ACN-rich mobile phases containing 5 mM ammonium acetate decreased the eluotropic strength of the resulting eluents. The gain in retention largely followed the order ethanol (EtOH)>methanol (MeOH)>1,2-ethanediol (Et(OH)2) and was accompanied by distinct effects on chromatographic selectivity. For example, on the most polar home-made packing the purine nucleoside selectivity guanosine/adenosine increased from 2.25 in the AQ-HILIC (kguanosine=8.3) to 7.33 (kguanosine=59) in the NA-HILIC mode when EtOH was employed as NA modifier while this value was 5.84 and 2.93 with MeOH and Et(OH)2, respectively (eluent: 5 mM ammonium acetate in ACN/modifier 90:10 v/v). Besides the type of protic modifier its percentage as well the retention and selectivity effects upon varying the ammonium acetate concentration and column temperature, respectively, were also investigated. Notable inter-column differences were found for all of these elution parameters. A mixed-mode retention model composed of partitioning and adsorption is proposed for both AQ- and NA-HILIC retention processes. The potential of (i) the implementation of novel polar bondings (such as ones containing sulphoxide functionalities) and (ii) the comprehensive exploitation of elution variables (type of protic modifiers, salt, etc.) for providing new selectivity increments to the separation of polar analytes in HILIC is emphasised.     

Ion chromatography for determination of nitrite and nitrate in seawater using monolithic ODS columns

A fast and highly sensitive ion chromatographic method using monolithic ODS columns was developed for the determination of nitrite (NO2-) and nitrate (NO3-) in seawater. Two monolithic ODS columns (50 mm x 4.6 mm i.d. + 100 mm x 4.6 mm i.d.) connected in series were coated and equilibrated with 5 mM cetyltrimethylammonium chloride (CTAC) aqueous solution. The column efficiency with 0.5 M NaCl as the mobile phase did not decrease in spite of the increase in flow rate of the mobile phase. Thus, good chromatograms were obtained within 3 minutes for NO2- and NO3 in artificial seawater without interferences by coexisting ions. The detection limit (S/N = 3) with UV detection at 225 nm was 0.8 and 1.6 microg/L for NO2- and NO3-, respectively. The characteristics of the monolithic CTA(+)-coated ODS columns were discussed. The present method was successfully applied to the fast and sensitive determination of NO2- and NO3- in real seawater samples.     

Rapid determination of bromide in seawater samples by capillary ion chromatography using monolithic silica columns modified with cetyltrimethylammonium ion

Monolithic silica capillary columns dynamically modified with quaternary ammonium ions were evaluated for the determination of bromide in seawater samples. A quaternary ammonium ion such as cetyltrimethylammonium ion was dynamically introduced onto monolithic silica surfaces. The first layer of the modifier was introduced by electrostatic interaction, whereas the second layer was introduced by hydrophobic interaction. The latter layer worked as the anion-exchange sites. The modified monolithic silica capillary columns could be used for rapid separation of inorganic anions. Separation of authentic mixture of five anions was achieved within a few minutes. The addition of small amount of the modifier in the eluent improved the repeatability of the retention time. Seawater samples could be directly injected onto the prepared capillary columns, and bromide could be determined to be 63 mg/L.